d'mello-food safety contaminants and toxins 2003

Submit a Book Proposal | Write a Review | Inspection Copies | Check Order

Book Details

Contributors

Preface

Glossary

PART I: BIOTOXINS

1 Plant Toxins and Human Health P.S. Spencer and F. Berman

2 Bacterial Pathogens and Toxins in Foodborne Disease E.A. Johnson

3 Shellfish Toxins A. Gago Martínez and J.F. Lawrence

4 Mycotoxins in Cereal Grains, Nuts and Other Plant Products J.P.F. D’Mello

PART II: ANTHROPOGENIC CONTAMINANTS

5 Pesticides: Toxicology and Residues in Food P. Cabras

6 Polychlorinated Biphenyls D.L. Arnold and M. Feeley

7 Dioxins in Milk, Meat, Eggs and Fish H. Fiedler

8 Polycyclic Aromatic Hydrocarbons in Diverse Foods M.D. Guill én and P. Sopelana

Book of the MonthNew Titles Forthcoming TitlesText BooksGuide to OrderingContact Us!Bookshop HomeCABI Publishing

Food Safety: Contaminants and Toxins

Editor: J P F D'Mello, Scottish Agricultural College, Edinburgh, UK Publication Date: April 2003 Number of Pages: 480 Pages Binding: Hardback ISBN: 0851996078 Price: £80.00 (US$145.00)

9 Heavy Metals L. Jorhem

10 Dietary Nitrates, Nitrites and N-nitroso Compounds and Cancer RiskSpecial Emphasis on the Epidemiological Evidence M. Eichholzer and F. Gutzwiller

11 Adverse Reactions to Food Additives R.A. Simon and H. Ishiwata

12 Migration of Compounds from Food Contact Materials and ArticlesJ.H. Petersen

13 Veterinary Products: Residues and Resistant Pathogens J.C. Paige and L. Tollefson

PART III: CASE STUDIES

14 Prion Diseases: Meat Safety and Human Health Implications N. Hunter

15 The Safety Evaluation of Genetically Modified Foods M.J. Gasson

16 Genetically Modified Foods: Potential Human Health Effects A. Pusztai, S. Bardocz and S.W.B. Ewen

17 Radionuclides in Foods: the Post-Chernobyl Evidence J.T. Smith and N.A. Beresford

18 Radionuclides in Foods: American Perspectives E.J. Baratta

PART IV: CONCLUSIONS

19 Widespread and Continuing Concerns over Food Safety J.P.F. D’Mello

Index

Back to Previous Page

© CAB International 2003 Feedback Privacy Policy Licences

Contributors

Arnold, D.L. Toxicology Research Division, Bureau of Chemical Safety, Food Directorate, HealthProducts and Food Branch, Health Canada, Ottawa, Ontario K1A 0L2, Canada

Baratta, E.J. Winchester Engineering and Analytical Center, US Food and Drug Administration,109 Holton Street, Winchester, MA 01890, USA

Bardocz, S. Formerly of The Rowett Research Institute, Aberdeen AB2 9SB, UKBeresford, N.A. Centre for Ecology and Hydrology, Winfrith Technology Centre, Dorchester

DT2 8ZD, UKBerman, F. Center for Research on Occupational and Environmental Toxicology, Oregon Health &

Science University, 3181 SW Sam Jackson Park Road, Portland, Oregon, USACabras, P. Dipartimento di Tossicologia, Università di Cagliari, Viale Diaz 182, 09126 Cagliari, ItalyD’Mello, J.P.F. Formerly of the Crop Science Department, The Scottish Agricultural College, West

Mains Road, Edinburgh EH9 3JG, UKEichholzer, M. Institute of Social and Preventive Medicine, University of Zurich, Sumatrastrasse 30,

CH-8006 Zurich, SwitzerlandEwen, S.W.B. Department of Pathology, University of Aberdeen, Forresterhill, Aberdeen, UKFeeley, M. Chemical Health Hazard Assessment Division, Bureau of Chemical Safety, Food Director-

ate, Health Products and Food Branch, Health Canada, Ottawa, Ontario K1A 0L2, CanadaFiedler, H. Substances Chimiques, UNEP, 11–13 Chemin des Anémones, CH-1219 Chatelaine,

Geneva, SwitzerlandGago Martínez, A. Department of Analytical and Food Chemistry, Faculty of Sciences, University of

Vigo, Campus Universitario, 36200-Vigo, SpainGasson, M.J. Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UKGuillén, M.D. Tecnología de Alimentos, Facultad de Farmacia, Universidad del País Vasco, Paseo de

la Universidad 7, 01006-Vitoria, SpainGutzwiller, F. Institute of Social and Preventive Medicine, University of Zurich, Sumatrastrasse 30,

CH-8006 Zurich, SwitzerlandHunter, H. Neuropathogenesis Unit, Institute for Animal Health, Ogston Building, West Mains

Road, Edinburgh EH9 3JF, UKIshiwata, H. Division of Food Additives, National Institute of Health Sciences, 1-18-1, Kamiyoga,

Setagaya-ku, Tokyo 158-8501, JapanJohnson, E.A. Department of Food Microbiology and Toxicology, Food Research Institute, University

of Wisconsin, Madison, WI 53706, USAJorhem, L. Research and Development Department, National Food Administration, PO Box 622,

SE–751 26 Uppsala, Sweden

vii

20-Feb-03

7Z:\Customer\CABI\A4382 - d’Mello\A4491 - d’Mello #D.vpThursday, February 20, 2003 3:24:22 PM

Color profile: DisabledComposite Default screen

Lawrence, J.F. Food Research Division, Health Canada, Ottawa, Ontario, CanadaPaige, J.C. Division of Epidemiology, DHHS/FDA-CVM, 7500 Standish Place, Rockville, MD 20855,

USAPetersen, J.H. Institute of Food Safety and Nutrition, Danish Veterinary and Food Administration,

Morkhoj Bygade 19, DK 2860 Soborg, DenmarkPusztai, A. Formerly of The Rowett Research Institute, Aberdeen AB2 9SB, UKSimon, R.A. Division of Allergy, Asthma and Immunology, Scripps Clinic, La Jolla, California, USASmith, J.T. Centre for Ecology and Hydrology, Winfrith Technology Centre, Dorchester DT2 8ZD,

UKSopelana, P. Tecnología de Alimentos, Facultad de Farmacia, Universidad del País Vasco, Paseo de la

Universidad 7, 01006-Vitoria, SpainSpencer, P.S. Center for Research on Occupational and Environmental Toxicology, Oregon Health &

Science University, 3181 SW Sam Jackson Park Road, Portland, Oregon, USATollefson, L. Center for Veterinary Medicine, DHHS/FDA-CVM, 7500 Standish Place, Rockville,

MD 20855, USA

20-Feb-03

viii Contributors



Preface

Background

It is perhaps fitting that Food Safety should have its origins in the UK. The idea for this bookwas, indeed, conceived and developed at the height of the various food crises in the UK.However, the primary impetus for this book emerged with the stark realization that some20 years after the initial food scares, college and university undergraduate curricula inagriculture, veterinary medicine and food science have remained quite impervious to foodsafety issues. There is still, unfortunately, the perception that food poisoning is rare and thatdenial and crisis management are effective strategies to restore consumer confidence. Yet wemust appreciate that, in comparison with our predecessors, we live in a highly contaminatedenvironment. There is a need to take stock and address the human health implications of foodcontaminants.

Although recent events may have given the impression of a nation enduring a malaise, theUK has also emerged as a hotbed of dissension regarding other issues such as the attributes andsafety of genetically modified (GM) and organic foods. The current furore in the UK over thesematters undoubtedly has helped in the globalization of food safety concerns in general, andFood Safety has been designed to crystallize the major themes now emerging in Europe, NorthAmerica and Japan.

Policy

If educational policy in undergraduate training is in need of radical change, then current post-graduate research programmes in food safety can best be described as grossly inadequate.There is an urgent need to attract talented science graduates to undertake innovative work thatwill underpin future developments in food safety. Above all, it is critical that an integrated andcoordinated policy is devised and implemented. Thus, there is a long-held philosophy amongacademic and research policy-makers that responsibilities in food production and qualityassurance can be separated. It is often argued that the obligations of food producers cease atthe farm gate. In this philosophy, matters relating to safety of farm produce are assumed to bethe responsibility of a second sector, comprising food processors, manufacturers and retailers.Recent events around the world have served to demonstrate unequivocally the need fora holistic approach in food safety. It is not easy to discern how, for example, pesticide orfertilizer recommendations to arable farmers can be justified solely on agronomic efficacy.

ix

20-Feb-03



Equally, the division of research priorities into ‘strategic’, ‘public good’ and ‘near-market’categories patently has failed as a policy for ensuring that good science is undertaken anddelivered in the interests of food safety. There are now compelling arguments and practicalinstances to show that this policy is discredited. At the very least, these issues are worthdebating in governmental and academic circles.

Content

Food Safety is divided into sections that reflect the major toxins and contaminants in the plantand animal products that constitute our staple diets. The first part includes chapters on plantand microbial toxins that may contribute to common cases of food allergies, intolerance andpoisoning. The second part deals with contaminants arising from anthropogenic activities andenvironmental pollution, while the third part comprises current topics of particular concern infood safety. Specific emphasis is placed on the nature of compounds, distribution of residuesin common foods, uptake, toxicology and regulatory issues. Many food contaminants are nowdefinitively associated with the induction of cancer and with neurotoxic, hepatotoxic andnephrotoxic effects. However, subtle effects of these contaminants on immunocompetenceand endocrine disruption will be more difficult to establish. In the fourth part, a concludingchapter contains a synthesis of the worldwide and continuing concerns over food safety usinginformation from all chapters in the book. Emerging issues and legislation are also addressed,and the chapter ends with a review of research priorities and action points.

Aims

The aims of this book are to provide a scientific documentation of recent advances withguidance on future directions in all matters relating to food safety, and to do this from a globalperspective. As intimated above, this book has been designed to enhance the profile offood safety in college and university curricula. The book should be suitable for final yearundergraduates in agriculture, food science, nutrition, dietetics and veterinary medicine. It isassumed that these readers will have a good working knowledge of organic chemistry andhuman biology. Although the book is structured in a particular way, each chapter is designedto be a self-contained unit to enable readers to make appropriate choices. Ideally, concertedefforts should now be directed at instituting a degree course in food safety, and it is my hopethat this volume will provide the framework for such a course. An additional aim is tostimulate interest among our talented science graduates to become involved in research in allaspects of food safety including analytical methodologies, monitoring and development ofdiagnostics. I firmly believe that only sound scientific training and research will help to allaycurrent apprehension about food safety and ensure consumer protection in the future.

Conclusions

I am delighted to have secured the services of expert authors from the major food safetyagencies, research institutes and universities around the world. All of my authors are activelyinvolved in and committed to innovative work, thus helping to underpin future advances infood safety. I commend their efforts to my readers. I am also pleased to express my gratitude tostaff at CABI Publishing for the encouragement and support they have offered throughout thepreparation of this book. To sum up, I believe that food safety teaching and research are stillundertaken on an ad hoc basis. There is a clear need to formalize these activities into coherent

20-Feb-03

x Preface



education and research programmes. In Food Safety, I have attempted to provide a text andframework to initiate such developments. Sound training and high-quality and sustainedresearch are the best pre-emptive measures at our disposal to restore and perhaps evenenhance consumer confidence in food.

J.P.F. D’Mello

Preface xi

20-Feb-03



Glossary

Introduction

As in most other scientific disciplines, understanding food safety involves an appreciation ofthe particular vocabulary and the technical language that are used to describe the diverseissues that constitute the subject of this book. Although many of the terms and acronyms usedare now in common usage outside the scientific community, it was deemed important toprovide as comprehensive a list as possible to assist those readers who are new to the field offood safety. Further information may be obtained from appropriate scientific dictionaries, inparticular that by Hodgson et al. (1998). In addition, several reports by expert groups containuseful glossaries of terms associated with particular contaminants in food (e.g. Ministry ofAgriculture, Fisheries and Food, 1992a,b,c, 1994; Pennington Group, 1997).

Definition of Terms and Acronyms

Table 1 lists the major terms and acronyms in alphabetical order. Cross-referencing to specificchapters in this volume is also provided to permit a fuller appreciation of the context of usageof selected terms.

xiii

20-Feb-03

Term Meaning

AChEAcute toxicity

Adduct

ADIAFB1, AFB2, AFG1,AFG2

a.i.AnthropogenicASPBA

Acetylcholinesterase (Chapter 1)Severe adverse effects occurring within a relatively short period of exposure to aharmful substance (Chapters 4 and 13)Covalent product of a toxicant or metabolite to large biomolecules such asproteins and DNA (Chapters 4 and 8)Acceptable daily intake (Chapters 5, 11 and 12)Aflatoxin B1, aflatoxin B2, aflatoxin G1 and aflatoxin G2: carcinogenic mycotoxins(Chapter 4)Active ingredient; as used in pesticide formulations (Chapter 5)Arising from human activities, e.g. industry (Chapters 5–13, 17 and 18)Amnesic shellfish poisoning (Chapter 3)Bisphenol A (Chapters 12 and 19)

continued

Table 1. Explanation of major terms and acronyms used in this volume.



20-Feb-03

xiv Glossary

Term Meaning

BADGEBFDGEBHABHTBq

BSE

BWCarcinogenicCASTCEPAChronic toxicity

Cl6DDCl7DDCl8DDCl4DFCl5DFCl6DFCodex AlimentariusCommissionCritical group

CRMsCutting plantDADDTDEFRADEGDEHPDILsDMDNADONDSPECECEHEECELISAEPAEUExternal dose

FAOFB1, FB2, FB3, FB4

FDAFD&CFSAFWGMGMPGRAS

Bisphenol A diglycidylether (Chapter 12)Bisphenol F diglycidylether (Chapter 12)Butylated hydroxyanisole: antioxidant (Chapter 11)Butylated hydroxytoluene: antioxidant (Chapter 11)Becquerel: unit of radioactivity; 1 Bq of a radioactive particle undergoes, onaverage, one radioactive decay per second (Chapter 17)Bovine spongiform encephalopathy, also known as ‘mad cow disease’ (Chapters14 and 19)Body weightCausing cancer (Chapters 6, 7, 10 and 17–19)Council of Agricultural Science and Technology (Chapter 2)Canadian Environmental Protection Act (Chapter 6)Adverse effects resulting from prolonged and repeated exposure to smallquantities of a harmful substance (Chapters 4 and 13)Hexachlorodibenzo-p-dioxin (Chapter 7)Heptachlorodibenzo-p-dioxin (Chapter 7)Octachlorodibenzo-p-dioxin (Chapter 7)Tetrachlorodibenzofuran (Chapter 7)Pentachlorodibenzofuran (Chapter 7)Hexachlorodibenzofuran (Chapter 7)An international body formed by WHO and FAO responsible for establishingstandards for food (Chapters 6, 7, 11 and 13)That part of the population which consumes a particular foodstuff at the highestrate (Chapter 17)Certified reference materials: used in quality assurance (Chapter 9)Premises used for cutting up fresh meat for human consumption (Chapter 19)Domoic acid (Chapter 3)Dichlorodiphenyltrichloroethane (Chapter 5)Department for Environment, Food and Rural Affairs (UK)Diethylene glycol (Chapter 12)Di-(2-ethylhexyl)phthalate (Chapter 12)Derived intervention levels (Chapter 18)Dry matterDeoxyribonucleic acid (Chapter 15)Deoxynivalenol (Chapter 4)Diarrhoetic shellfish poisoning (Chapter 3)European CommissionEuropean Centre for Environment and Health (of WHO; Chapter 7)European Economic CommunityEnzyme-linked immunosorbent assay (Chapters 2 and 3)Environmental Protection Agency (USA) (Chapter 6)European Union (Chapters 3 and 19)The exposure of a person to radioactivity (or other contaminant) from outside thebody, e.g. soil (Chapter 17)Food and Agriculture Organization (of the United Nations)Fumonisins B1, B2, B3 and B4: carcinogenic mycotoxins (Chapter 4)Food and Drug Administration (USA) (Chapters 4, 6 and 19)Food Dye and Coloring (Act) (Chapter 11)Food Standards Agency (UK) (Chapter 19)Fresh weightGenetically modified (Chapters 15 and 16)Good manufacturing practiceGenerally recognized as safe

Table 1. Continued.



Glossary xv

20-Feb-03

Term Meaning

GyhHACCPHalf-life

HAS

HAVHCNHeavy metalsHepatotoxicHPLCIARCID50

Internal dose

IPCSJECFALD50

LOAELLOCMAOMBM

MFOMHSMJMPL

MRL(s)MSGMutagenicNephrotoxicNOAELNOCsNOELOAOECDOPsORsOTAOTM rule

OutbreakPAAPAHsPCBsPCDDsPCDFsPCR

Grays; unit of absorbed radiation energy (Chapter 17)Hour(s)Hazard analysis critical control point (Chapter 2)Time taken for the amount of radioactivity to decrease by one half due tophysical decay (Chapter 17)Hygiene assessment system: used in assessing hygiene standards in licensedslaughterhouses and cutting plants to yield HAS scoresHepatitis A virus (Chapter 19)Hydrogen cyanide (Chapter 1)Collective term for Pb, Hg, Cd and certain other inorganic elements (Chapter 9)Toxic to the liver (Chapter 4)High-performance liquid chromatography (Chapter 3)International Agency for Research on Cancer (Chapters 4, 6, 7 and 10)The dose that infects or causes an infectious or toxic response in 50% of apopulation of test animals in a designated period of time (Chapter 2)The exposure of a person to radioactivity ingested and incorporated in the body(Chapter 17)Intergovernmental Programme for Chemical Safety (Chapter 7)Joint (FAO/WHO) Expert Committee on Food Additives (Chapters 11 and 13)The dose that causes lethality in 50% of a population of test animals in adesignated period of time (Chapters 2 and 5)Lowest observed adverse effect level (Chapter 6)Levels of concern (Chapters 18 and 19)Monoamine oxidase (Chapter 1)Meat and bone meal: now banned as a feedingstuff in EU Member States(Chapters 14 and 19)Mixed-function oxidase (Chapter 8)Meat Hygiene Service (UK; an agency of the FSA) (Chapter 19)MegajouleMaximum permitted level: reference level determined by calculating the meanactivity concentration in a foodstuff which, assuming consumption over a 1-yearperiod, would lead to an acceptably small dose (Chapter 17)Maximum residue limit(s) (Chapters 5, 13 and 19)Monosodium glutamate (Chapter 11)Causing mutations (Chapters 17 and 18)Toxic to the kidney (Chapter 4)No observed adverse effect level (Chapter 6)N-nitroso compounds: includes nitrosamines and nitrosamides (Chapter 10)No observed effect level (Chapter 5)Okadaic acid (Chapter 3)Organization for Economic Cooperation and Development (Chapter 15)Organophosphates (Chapter 5)Odds ratios: used in epidemiological studies (Chapter 10)Ochratoxin A (Chapter 4)Over 30-month rule: in BSE legislation to prevent any OTM cattle (with limitedexceptions) from entering the food chain (Chapter 14)Two or more incidents of disease associated with a common cause (Chapter 19)Primary aromatic amines (Chapter 12)Poly(cyclic) aromatic hydrocarbons (Chapters 8 and 19)Polychlorinated biphenyls (Chapters 6 and 19)Polychlorinated dibenzo-p-dioxins (Chapters 6 and 7)Polychlorinated dibenzofurans (Chapters 6 and 7)Polymerase chain reaction (Chapters 2 and 15)

continued



References

Hodgson, E., Mailman, R.B. and Chambers, J.E. (1998) Dictionary of Toxicology, 2nd edn. MacmillanReference Ltd, London.

Ministry of Agriculture Fisheries and Food (1992a) Report of the working party on pesticide residues:1988–1990. Food Surveillance Paper No. 34. HMSO, London.

Ministry of Agriculture Fisheries and Food (1992b) Nitrate, nitrite and N-nitroso compounds in food. Thethirty-second report of the Steering Group on chemical aspects of food surveillance. Food SurveillancePaper No. 32. HMSO, London.

Ministry of Agriculture Fisheries and Food (1992c) Dioxins in food. The thirty-first report of the SteeringGroup on chemical aspects of food surveillance. Food Surveillance Paper No. 31. HMSO, London.

Ministry of Agriculture Fisheries and Food (1994) Radionuclides in foods. The forty-third report of theSteering Group on chemical aspects of food surveillance. Food Surveillance Paper No. 43. HMSO,London.

Pennington Group (1997) Report on the Circumstances Leading to the 1996 Outbreak with E. coli O157 in CentralScotland, the Implications for Food Safety and the Lessons to be Learned. The Stationery Office, Edinburgh.

20-Feb-03

xvi Glossary

Term Meaning

ProteomesProteomics

PrPPSPPVCRASTReference level

RiskRLsSMLSRM

Sv

2,4,5-TTBHQTCDDTDIsTEFsTEQsTeratogenicTSETWIUNEPvCJDWHO

Total complement of proteins within a cell (Chapter 15)Involves use of two-dimensional gel analysis to separate individual proteinspresent in a particular tissue (Chapter 15)Prion protein (Chapter 14)Paralytic shellfish poisoning (Chapters 3 and 19)Polyvinylchloride (Chapter 12)Radioallergosorbent test (Chapter 16)Level of radioactivity (or other contaminant) in a foodstuff above which someaction must be taken by regulatory authorities (Chapter 17)Probability of ill effects (Chapters 14 and 19)Reporting limits (Chapters 5 and 19)Specific migration limit (Chapter 12)Specified risk material: relates to slaughter procedures and legislation controllingBSE contamination of meat (Chapters 14 and 19)Sievert: unit of absorbed dose equivalent used to estimate radiation risk(Chapter 17)2,4,5-Trichlorophenoxyacetic acidTertiary butylhydroquinone: antioxidant (Chapter 11)Tetrachlorodibenzo-p-dioxin (Chapters 6 and 7)Tolerable daily intakes (Chapters 4, 6, 7 and 12)Toxicity equivalency factors (Chapters 6 and 8)Toxic equivalents (Chapter 6)Causing birth defects (Chapters 4 and 6)Transmissible spongiform encephalopathy (Chapter 14)Tolerable weekly intake (Chapter 7)United Nations Environment Programme (Chapter 7)Variant Creutzfeldt–Jakob disease (Chapter 14)World Health Organization (of United Nations)

Table 1. Continued.



1 Plant Toxins and Human Health

P.S. Spencer* and F. BermanCenter for Research on Occupational and Environmental Toxicology, Oregon Health

& Science University, 3181 SW Sam Jackson Park Road, Portland, Oregon, USA

Introduction

Toxic plants and human illness

Natural substances in plants used for foodimpact the health of the human species. Thischapter focuses on plant products associatedwith acute illness, chronic disease ordevelopmental perturbation. The reader isreferred elsewhere for accounts of the toxiceffects of plants used as over-the-counterherbal medicines (Cupp, 2000). Fungal toxinsare discussed elsewhere in this volume(Chapter 4).

Public comprehension of plant toxicol-ogy is simplistic and naive: a few plants arepoisonous and should not be ingested, but allplants used for food are nutritious and lacktoxic effects. It is understood that certain plantproducts when immature may contain poi-sonous principles, but these are assumed todisappear during maturation. Even pharma-cologically active plants are considered healthpromoting because the chemicals are naturalin origin. The public fails to consider the pres-ence in plants of natural substances with toxicpotential while clamouring for the exclusionof all traces of anthropogenic chemical con-tamination. Demand in affluent countriesfor the freshest fruit and vegetables mini-mizes postharvest chemical breakdown and

therefore tends to magnify the dose of thesenatural toxins.

Those who study chemicals in plants areimpressed with the ingenuity and variety ofsubstances with toxic potential, includingthose that serve to defend against attackby predators. While chemical defence is apresumed function, the actual physiologicalroles of plant chemicals that adverselyimpact human health often are unknown.Individual plants may harbour more thanone category of noxious agent; witness thepresence of a convulsant (β-cyanoalanine)and cyanide-liberating glycosides (vicianin,prunasin) in the common vetch (Vicia sativa)(Roy et al., 1996; Ressler and Tatake, 2001).Reminiscent of the shape, size and colorationof the red lentil (Lens culinaris), this neurotoxicvetch has been marketed profitably tocountries with pervasive poverty (Tate andEnneking, 1992; Tate et al., 1999). Similarly,the neurotoxic grass pea (Lathyrus sativus), thecause of a crippling motor system disease(lathyrism), has been used to adulteratenon-toxic pulses (Dwivedi, 1989). These prac-tices illustrate the importance of protein-richlegumes as a food source and the need fortighter controls on their distribution and use.

The vulnerability of disadvantagedpopulations in poor countries also arisesfrom their tendency of necessity to rely on

©CAB International 2003. Food Safety: Contaminants and Toxins(ed. J.P.F. D’Mello) 1

20-Feb-03 1

* E-mail: [email protected]



monotonous diets derived from cheap, envi-ronmentally tolerant and often potentiallytoxic plants. Drought and flood, but also civildisturbance or war, tend to increase depend-ency on such plants and foster florid diseasetraceable to natural plant toxins. The root cropcassava (Manihot esculenta) is of particularconcern because the tuber and leaves of thishazardous plant feed an estimated 400 millionpeople, half of whom reside in Africa (Roslingand Tylleskär, 2000). Outbreaks of irreversiblecrippling neurological disease among chil-dren and adults hallmark southern Africancommunities that subsist on this plant. Thedrive to expand the production and consump-tion of the carbohydrate-rich but protein-poorcassava tuber must be accompanied byincreased awareness of methods to remove itsnatural toxins; however, this is not likely tohappen, in part because uneducated popula-tions that survive on toxic plants tend to rejectan association with illness. Even acutely poi-sonous species, such as cycads – which kill orparalyse animals after oral ingestion – maybe cherished by communities that have usedsuch plants to survive. Humans often cannotgrasp the notion that disease may evolveand first appear long after ingestion of aplant product that is nourishing in theshort term. The notion that disease mayevolve years or decades after exposure to aplant product is a sophisticated concept thatrequires education to instil. Of course, overtillness is the tip of the iceberg, for populationswith epidemic disease triggered by dietaryreliance on toxic plants often display agradation of clinical manifestations andmay have symptoms that are subclinical onexamination.

Susceptibility to plant chemical toxicityvaries with factors such as the maturity ofthe plant component, soil characteristics andenvironmental conditions; the potency, doseand duration of exposure to the offendingagent(s); differential (target) organ and cellu-lar susceptibility; and factors intrinsic to theaffected subject, notably sex and nutritionalstate. The interaction of these factors deter-mines when disease appears, how severelyindividuals are affected and the potentialfor persistence of or recovery from illness.

Whereas poorly nourished children andadults who subsist on incompletely detoxifiedcassava may suddenly develop cripplingdisease after a few months (Ministry ofHealth, Mozambique, Mantakassa, 1984),others who are exposed to smaller dailydoses seem to experience a slowly develop-ing gait disorder that appears in later years(Osuntokun, 1981).

Precursors or activators

While the bulk of this review is devoted toindividual compounds with potential toxi-city, plants also provide the precursors andactivators of otherwise innocuous substancesthat, if modified, can act as target organtoxins. The toxic substance potentially couldbe formed during postharvest treatment orfood processing, in the gastrointestinal tract,at stages in intermediary metabolism or incells of the target organ itself.

Again, the nervous system is a con-venient tissue with which to consider thisunproven concept, particularly in relation tocertain neurodegenerative disorders, notablyParkinson’s disease (PD). β-Carbolines (BCs)and isoquinolines (IQs), which occur in alarge number of angiosperms, are illustrativeplant neurotoxin precursors. BCs, such asnorharman and harman, are also formedduring the cooking of foods, the elevated tem-peratures promoting a reaction of tryptophanwith aldehyde compounds and subsequentoxidation leading to carboline formation (Col-lins and Neafsey, 2000). N-Methylation of BCsand IQs generates compounds structurallysimilar to the N-methyl-4-phenylpyridiniumcation (MPP+), a proven cause of a PD-like dis-order in humans and animals (Fig. 1.1). WhileBCs and IQs are not taken up by the dopa-mine-containing nigrostriatal neurones thatdegenerate in PD, methylated ionic species(e.g. 2,9-dimethylnorharmanium cation) are,like MPP+, substrates for the dopamine trans-porter of these nerve cells. Phalaris tuberosa, agrass that contains methylated BC-relatedindole alkaloids (gramine, methyltrypta-mine and 5-methoxydimethyltryptamine), is

20-Feb-03 1

2 P.S. Spencer and F. Berman



causally linked to neurological disease incattle and sheep that use these plants forfood.

Methylation of BCs and IQs conceivablymight arise from postharvest seed treatmentwith a methylating agent (e.g. methyl bro-mide), through the action of an endogenousmethyltransferase in animal tissue or, in aspecific unique circumstance, co-exposure toa plant-derived methylating agent, such asmethylazoxymethanol (MAM). MAM is theaglycone of cycasin, a toxic glucoside presentin seed of the false sago palm (Cycas spp.),which through part of the 20th century was asignificant source of food in parts of Oceania.On Guam, where the parkinsonism–dementiaand amyotrophic lateral sclerosis complex hasbeen rampant, there is a remarkably strongcorrelation between the historical incidenceof this disease and the concentration ofcycasin in flour samples used for food bythese communities (Spencer, 2000a). Whethercycasin is the culpable agent or whetherits aglycone MAM methylates a neurotoxicprecursor is unknown.

Nitrogenous Compounds

Plant chemicals with toxic potential can bedivided into those containing nitrogen andthose lacking this element.

Non-protein amino acids

Plants synthesize hundreds of amino acids,but only about 20 are employed in proteins.The balance – amino acids, imino acidsand amides – are secondary metabolites.Non-protein amino acids occur in manyunrelated plant families, but they are particu-larly characteristic of legumes. Several dis-rupt the nervous system, and others damagethe liver, kidney and other organs.

Some of the dicarboxylic plant aminoacids mimic the action of glutamate, theprincipal excitatory neurotransmitter in thehuman central nervous system (CNS). Inculture, micromolar concentrations of these‘excitotoxic’ amino acids trigger the influx of

Plant Toxins and Human Health 3

20-Feb-03 1

Fig. 1.1. Comparison of the structure of three agents that damage nigrostriatal nerve cells in humansand/or laboratory animals. N-Methyl-4-phenylpyridium cation (MPP+) is the metabolite of a street drugcontaminant, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, that produced a parkinsonism-like diseasein addicts. The neurotoxic, MPP+-like 2,9-dimethyl norharmanium ion (2,9-Me2βC+) and 2-methyl-isoquinolinium ion (2-MeIQ+) are generated by N-methylation of plant precursor molecules (Collins andNeafsey, 2000).



sodium and calcium ions into nerve cellsthat are equipped with the appropriateionotropic glutamate receptors. Oedematousswelling and degeneration of nerve cellsfollow. Excitotoxic amino acids includecompounds such as cucurbitine (pumpkinseed), α-amino-β-methylaminopropionicacid, also known as β-N-methylamino-L-alanine (BMAA, false sago palm), and

γ-N-oxalyl-L-α,β-diaminopropanoic acid, alsoknown as β-N-oxalylamino-L-alanine (BOAA)(Liener, 1980) (Fig. 1.2). Since some of theseamino acids chelate metals, it is conceivablethat amino acid levels may reflect soil metalcharacteristics.

BOAA, a pharmacological agonist ofa subclass (AMPA, i.e. α-amino-3-hydroxy-5-methylisoxazole propionic acid)

20-Feb-03 1


Fig. 1.2. Chemical structures of β-N-oxalylamino-L-alanine (BOAA) (Lathyrus sativus), β-N-methylamino-L-alanine (BMAA) (Cycas circinalis), β-aminopropionitrile (BAPN) (Vicia sativa), 2-amino-4-(guanidinooxy)butyric acid (canavanine) (Canavalia ensiformis), hypoglycine A (Blighia sapida), 3,4-dihydroxyphenylalan-ine (DOPA) (Vicia faba), mimosine (Leucaena leucocephala) and djenkolic acid (Pithecolobium lobatum).



of glutamate receptors on the plasma mem-brane of nerve cells, is the active neurotoxicprinciple in the grass pea (L. sativus), pro-longed ingestion of which causes lathyrism(syn.: neurolathyrism) (Spencer, 1995). β-Aminopropionitrile (BAPN), an aminoacid derivative that occurs in Lathyrus spp.as β-(γ-L-glutamyl)-aminopropionitrile, is aninhibitor of lysyl oxidase, an enzyme withan important role in collagen and bonedevelopment. Whereas experimental admin-istration of BAPN to rodents leads to jointdeformities, ligament separation and skeletaldeformities (termed ‘osteolathyrism’), pro-longed treatment of primates with BOAAinduces a neurological disorder (experimentalneurolathyrism) characterized by myoclonicjerks, extensor hindlimb posturing andhindlimb weakness, a model of the early,reversible form of human lathyrism (Roy andSpencer, 1989). This and other human disor-ders that arise from ingestion of toxic aminoacids are described below.

Grass pea and lathyrism

L. sativus is an environmentally tolerant andprotein-rich legume that is eaten on theIndian subcontinent, in northeastern Chinaand the Horn of Africa. Reliance for a fewmonths on a diet of grass pea precipitateslathyrism, a form of spastic paraparesis, char-acterized by weakness, increased muscle toneand hyper-reflexia in the lower extremities.Continued ingestion results in progressivewalking difficulties that eventuate in perma-nent inability to move the legs. Lathyrismaffects all ages, is often seen in several mem-bers of an affected family and sometimes inepidemic form, and usually occurs whenother edible material is scarce or unavailable(Spencer, 1995). Since the late 1980s, therehas been a coordinated worldwide scientificinitiative to control lathyrism through thedevelopment of grass pea strains that containlittle or no BOAA (http://go.to/lathyrus).

Ackee and vomiting sickness

The ackee tree (Blighia sapida) synthesizeswater-soluble toxic amino acids – known ashypoglycin A (hypoglycine) and hypoglycin

B (γ-glutamyl dipeptide) – that causesevere hypoglycaemia and a hepaticencephalopathy comparable with Reye’ssyndrome (Spencer, 2000b). Hypoglycine(Fig. 1.2) and its lower homologue,methylenecyclopropylglycine, are foundtogether in the litchi (Litchi chinensis).

Ackee is a native of west Africa; in the18th century, the plant was imported intothe West Indies, including Jamaica, where thearils of the ripe fruit are used as a staple. ‘Anackee a day keeps the doctor away’, a linefrom a popular Jamaican song, is a sentimentthat conflicts with medical experience. Inges-tion of the arils and seed of unripe fruitscauses violent vomiting, convulsions, comaand death (Meda et al., 1999). In the Caribbeanislands, outbreaks have often been familial,affect poorly nourished children, and occurfrom November to February when matureackees are scarce. Hypoglycine also inducesfetal malformations in rats (Van Veen, 1973).Hypoglycine is metabolized to methylenecyclopropylacetyl-coenzyme-A (CoA), whichblocks the transport of fatty acids, acyl-CoAdehydrogenases and neoglucogenesis. Thiscauses an energy deficit, which is compen-sated by markedly increased carbohydratecatabolism and consequent characteristichypoglycaemia. Both hypoglycaemia andorganic acidaemia are thought to contributeto the toxic effects of hypoglycine (Sheratt,1995).

Canavanine and systemic lupuserythematosus

The arginine analogue L-canavanine,2-amino-4-(guanidinooxy)butyric acid, is atoxic basic amino acid widespread in seed ofLeguminosae. Jackbean (Canavalia ensiformis)and lucerne (Medicago sativa) contain up to15,000 ppm of canavanine. A human subjectdeveloped autoimmune haemolytic anaemiawhile participating in a research studythat required the ingestion of lucerne seeds(Montanaro and Bardana, 1991). Haemato-logical and serological abnormalities similarto those observed in human systemiclupus erythematosus (SLE) developed incynomolgus macaques fed lucerne sprouts(Malinow et al., 1982). Dietary L-canavanine


11-Mar-03 1

21Z:\Customer\CABI\A4382 - d’Mello\A4491 - d’Mello #G.vp11 March 2003 10:33:32


sulphate reactivated the syndrome inmonkeys in which an SLE-like syndromehad been induced previously by the ingestionof these plant materials. Recent work showsthat L-canavanine acts on suppressor-inducerT cells to regulate antibody synthesis. Lym-phocytes of SLE patients are specificallyunresponsive to L-canavanine (Morimotoet al., 1990).

Toxic amino acids and other health conditions

Several other health disorders are recognizedin humans and animals that consume plantscontaining non-protein amino acids (VanVeen, 1973; Liener, 1980). For example, renaldysfunction with haematuria is associatedwith ingestion of seed (djenkol bean) ofthe leguminous tree, Pithecolobium lobatum,which is eaten in certain parts of Sumatraand Thailand (Vachvanichsanong and Lebel,1997). The seed contains 1–4% of djenkolicacid (Fig. 1.2), a sulphur-containing aminoacid that forms needle-like clusters in theurine. Leucaena leucocephala (koa haole inHawaii), another legume that is rarely associ-ated with human illness, contains the toxicagent mimosine (Fig. 1.2) (Van Veen, 1973).Hair loss is the characteristic effect in humansand animals, possibly arising from inhibitionof the conversion of methionine to cysteine, amajor component of hair protein. Mimosineis metabolized to a goitrogenic agent,3,4-dihydroxypyridine.

Amines and monoamine oxidase inhibitors

Biologically active amines with pressor(vasoconstrictive) properties are present ina number of common foods. Pressor aminesof plants include tyramine, tryptamineand substances (serotonin, adrenaline,noradrenaline and dopamine) that serveas chemical neurotransmitters in thehuman CNS. Significant concentrations of3,4-dihydroxyphenylalanine (DOPA) occurin the fava bean (Vicia faba) (Fig. 1.2) (Liener,1980). High levels of pressor amines arefound in pineapple, avocado, walnut, plan-tain and banana, wheat, oats, nuts and

tomatoes. Ingestion of serotonin-rich bananasresults in elevated excretion of adrena-line, noradrenaline, vanillylmandelic acid,metanephrines and 5-hydroxyindolylaceticacid, a measure of circulating serotonin(Heinemann et al., 1981).

Pressor amines and hypertensive crisis

A clinically significant adverse health effectin affluent populations is associated with theingestion of tyramine-rich foods by individu-als using prescribed medications that inhibitmonoamine oxidase (MAO), the liver enzymethat normally deaminates pressor amines(Merriam, 2000). MAO inhibition results inhigh circulating levels of tyramine, whichtriggers the widespread release of the neuro-transmitter noradrenaline. This produces asyndrome characterized by hypertension,headache, diaphoresis, mydriasis, excitationand cardiac arrhythmia. Acute hypertensionhas the potential to eventuate in intracerebralhaemorrhage and myocardial infarction. Thesyndrome may occur for a period of up to 3weeks following drug discontinuation becauseMAO levels recover slowly. Prescriptions forthese drugs should therefore include instruc-tions to avoid tyramine-rich foods, includingaged cheeses, aged meats, herring, concen-trated yeast extracts, sauerkraut, broad beanpods, tap beer and red wine. Beans, wheat,nuts and tomatoes have also been reported totrigger headache in individuals treated withMAO inhibitors (Liener, 1980).

Proteins

Some plants used for food harbour proteinsthat trigger allergic reactions, or bind to cellsand disrupt their function, or disrupt thebreakdown of proteins. These are consideredbelow.

Proteinase inhibitors

Many raw plant products tend to depressthe growth rates of animals, although thesignificance for human health has yet tobe resolved. Reduction of normal growth is

11-Mar-03 1




associated with exposure to heat-resistantproteinase inhibitors that serve as highlyspecific substrates for the respective plantenzymes. These substances are widelydistributed in seeds (legumes), fruits(avocado, peach, plum, tomato and auber-gine), tubers (potato) and vegetative parts(soybean, lucerne, barley, maize and wheat)of dicotyledons and monocotyledons. Beststudied are the inhibitors of serine-typeproteinases, including the soybean trypsininhibitor, the soybean proteinase inihibitor,the potato I and II inhibitor families, thesquash inhibitor family and the α-amylase/trypsin inhibitor family of cereal seeds.Sulphydryl proteinase, acid proteinaseand metalloproteinase inhibitors are alsorecognized (Xavier-Filho and Campos, 1989).

Plant proteinase inhibitors that inhibitthe action of digestive proteinases can pro-duce adverse health effects. Ingestion ofraw soybean reduces proteolysis of dietaryprotein, causes increased secretion of pan-creatic enzymes and impairs body growthof laboratory species. Feeding experimentalanimals on diets containing isolated soy-bean trypsin inhibitors (the Kunitz soybeantrypsin inhibitor (STI) and the Bowman–Birktrypsin–chymotrypsin inhibitor (BBI)) causedinsignificant growth depression in rats andchicks, but induced enlargement of thepancreas in rats, chicks and mice but not inpigs, dogs, calves, monkeys and presumablyhumans (Birk, 1996). Potatoes contain com-pounds that inhibit all of the major pancreaticendo- and exopeptidases of the digestive tractof higher animals (Pearce et al., 1985). Pro-longed feeding of rats and mice with a dietrich in potato and soybean trypsin inhibitorproduced short-term pancreatic hyperplasiain both species and long-term nodularhyperplasia and acinar adenoma in rats(Gumbmann et al., 1989). Where humans fiton the scale of differential susceptibility tothe pre-neoplastic and neoplastic effects ofpotato trypsin inhibitors on the pancreas isunknown.

Lectins

Lectins are heat- and protease-resistantcarbohydrate-binding proteins that bind to

red blood cells and cause haemagglutination.Since lectins are widely distributed in theseeds and vegetative parts of plants,especially Leguminosae and Graminaceae, thehuman gut is regularly exposed to dietarylectins. One study identified over 50 edibleplants with lectin activity, including many infresh (lettuce and fruit) and processed foods(cereals and nuts) (Nachbar and Openheim,1980). Lectins may bind to mannose/galactose (concanavalin A from jackbean),N-acetylglucosamine (potato and wheatgerm lectins) or N-acetylgalactosamine/galactose (ricin and kidney bean lectin). It hasbeen stated that ‘lectins constitute one of themajor antinutritive factors of foods of plantorigin, and their presence in food may havevery serious consequences for growth andhealth’ (Pusztai, 1989).

Extensive experimental animal studieshave been conducted with the lectin ofPhaseolus vulgaris, which comprises 10–15% ofthe total protein content of the red kidneybean (Pusztai, 1989). Inclusion of raw kidneybean in the diet of young and mature ratsresults in rapid weight loss and eventualdeath. Kidney bean lectins are highly resistantto proteolytic breakdown in the gut, and theybind to and inhibit endo- and exopeptidasesthat function in food breakdown. They bindto, perturb and damage intestinal enterocytes,and reduce the absorptive surface of the smallintestine, which undergoes hypertrophy andhyperplasia. The consequent reduction in theabsorption of nutrients from the gut promotesprotein catabolism, decreases stores of subcu-taneous lipid and hepatic glycogen, greatlyamplifies urinary urea, and results in loss ofbody weight. Additional effects include pan-creatic enlargement accompanied by reducedinsulin circulation and involution of thethymus. Lectins are also endocytosed byintestinal cells and enter the circulation boundto unidentified blood cells. Lectins interferewith the gut immune system, and animalsfed kidney beans develop immunoglobulin(Ig)G- and IgE-mediated hypersensitivity tothe specific lectin.

Many other common food plants containheat-labile lectins that compromise intestinalintegrity, interfere with intestinal absorptionand have adverse effects on body growth.


11-Mar-03 1



These include lectins from soybean, teparybean (Phaseolus acutifolius), runner bean(Phaseolus coccineus), lima bean (Phaseoluslunatus), jackbean (Canavalia ensiformis),winged bean (Psophocarpus tetragonolobus),pea (Pisum sativum) and red lentil (Lensculinaris) (Pusztai, 1989). Ewen and Pusztai(1999) recently claimed that diets containinggenetically modified potatoes expressing thesnowdrop (Galanthus nivalis) agglutinin(GNA) had variable effects on different partsof the rat gastrointestinal tract, including aGNA transgene-associated proliferation ofthe gastric mucosa.

LECTINS AND COELIAC SPRUE (GLUTEN

ENTEROPATHY) Intestinal toxicity triggeredby wheat germ agglutinin contaminatinggluten in cereal foods has been implicated inan intestinal malabsorption disorder (coeliacsprue) associated with intolerance to gluten.Severity of gluten enteropathy varies with theextent of the loss of jejunal villi. Coeliac spruehas been considered to have a large geneticcomponent, but the rising age of onset andchanging clinical pattern and prevalence sug-gest that diet or other environmental factorsmake an important contribution to aetiology(Auricchio and Visakorpi, 1992). Changes ininfant feeding practices (doubling or triplingof wheat protein intake) that took place inSweden in the 1980s appear to have playedan important role in an unexpected rise inincidence of coeliac disease (Cavell, 1992).

Children from 6 months to 3 years of agemay have diarrhoea, projectile vomiting and abloated abdomen. Behavioural changes, suchas irritability and restlessness, characterizechildren with coeliac sprue. Speech develop-ment is often markedly impaired, the vocabu-lary limited to a few words, and the intonationis soft and whining. Other signs include foodcraving, retarded growth, weight loss, chronicfatty diarrhoea, abdominal cramping and dis-tension, and myopathy associated with weak-ness and fatigue. Liver, joint, haematological,dental and neuropsychiatric symptoms mayoccur. There may be difficulty in concentra-tion, decreased mental alertness and impairedmemory. The disease is conservatively esti-mated to have a prevalence of 0.1% in Europe(Troncone et al., 1996). Over 75% of patients

with coeliac sprue respond to a gluten-freediet, with symptoms usually improvingwithin weeks. Patients are instructed to avoidfood products prepared from wheat, rye,barley and oats. For affected children, theprovision of a gluten-free diet may result ina marked decrease of neuropsychologicalphenomena (Dohan, 1976).

The basis for the neuropsychiatric mani-festations of coeliac sprue is not understood.One possibility is that neuroactive peptidesproduced during digestion of food proteinscross the defective gut barriers and enter thebrain via the systemic circulation. This ideahas also been advanced to explain theneurobehavioural perturbations of schizo-phrenia, a disorder that has been related tocoeliac disease. Specifically, it has been sug-gested that schizophrenia may be geneticallylinked with coeliac disease, and that cerealgrain proteins may be pathogenic in individu-als with schizophrenia (Dohan, 1969). Arecent report described how a gluten-free dietresulted in the regression of both schizo-phrenic symptoms and an accompanyingfrontal cortex hypoperfusion (demonstratedby single-photon emission computed tomog-raphy) in a 33-year-old patient with coeliacdisease (De Santis et al., 1997). However,studies of small intestine permeability in24 schizophrenic patients failed to revealsignificant differences from normal subjects(Lambert et al., 1989). Attempts to demon-strate links between coeliac disease and child-hood autism have also proved unsuccessful(Pavone et al., 1997).

Nut protein allergens

Proteins in plant products used for food,especially various types of nuts, may elicitan acute-onset, dose-independent, type-1immunological reaction. This involves pro-duction of IgE antibodies directed towardthe plant protein, release of endogenouschemicals from mast cells (e.g. histamine,bradykinin and serotonin) that mediateinflammation, and the rapid development ofanaphylaxis, an illness that can prove fatal.Clinical manifestations include oedema of thelip, urticaria, asthma, hypotension, coma andeven death (Angus, 1998; Taylor et al., 2001).

20-Feb-03 1




Possible sources of contact with nut aller-gens, other than direct ingestion of the plantproduct, include exposure in utero or viabreast milk, or through infant formula andvitamin preparations containing nut oils. Onecohort study found that, by the age of 4 years,approximately 1% of English children aresensitized to peanuts or treenuts (Tariq et al.,1996). Children who suffer from allergicrhinitis or bronchial asthma appear to be atgreatest risk for nut allergy. Exposure to onlytrace amounts of nut protein may be sufficientto trigger an allergic response, and allergyacquired at an early age may persist through-out life (Hourihane, 1998). Fortunately, cross-reactivity among nut proteins is rare, butcross-reactions occur with other allergensin food and other plant materials (latex andgrass). Nut allergy is therefore a significantpublic health problem that will be likely tocontinue to grow in association with increas-ing reliance on legumes as abundant sourcesof cheap protein.

Many allergic responses to nuts are trig-gered by ingestion of peanuts, the shelledcotyledon pairs of the legume Arachis hypogaea(Angus, 1998). The cotyledons are rich inprotein (25–28% by weight), including themajor (Ara h I, Ara h II) and minor (aggluti-nin) peanut allergens. In the early 1990s, anestimated 65–85 severe reactions to peanutsoccurred annually in the UK (Angus, 1998).Reactions to peanut and treenut allergensaccounted for more than 90% of fatalities in amore recent analysis of 32 fatal cases in theUS (Bock et al., 2001). Allergy to crude peanutoil is also reported, but refined peanut oilreportedly appears to be safe for most peoplewho suffer from peanut allergy.

Ingestion of treenuts, the edible kernelsof the seed of several trees, may also causeimmunological illness (Taylor et al., 2001).Sweet almonds (Amygdalus communis) andbitter almonds (Prunus amygdalus) comprise22% protein and contain multiple IgE-bindingproteins that may trigger severe allergicreactions. Brazil nuts (Bertholletia excelsa, B.myrtaceae), ingestion of which has triggeredallergic reactions in children and adults, con-tain 14% protein, including a methionine-richprotein (Ber e 1) that constitutes the majorallergen (Bush and Hefle, 1996). Cashews

(Anarcadium occidentale), like other membersof the Anacardiaceae (mango, poison ivy andpistachio), have caused contact dermatitisand severe anaphylaxis among asthmaticchildren, as have hazelnuts (Corylus avellana)and pecans (Carya illinoinensis). Pistachios(Pistacia vera), which likewise cause allergicreactions in sensitized individuals, have amajor (mol. wt 34,000 Da) and several minorIgE-binding proteins. Pine nuts (Pinus edulis)contain at least three proteins that bindhuman IgE (Koepke et al., 1990). Walnuts(Juglans regia) have also been aetiologicallyimplicated in human anaphylaxis (Angus,1998).

Several nuts and seeds other thantreenuts and legumes (groundnuts) maytrigger allergic reactions. Sunflower seed(Helianthus annuus) has caused anaphylacticreactions after ingestion and dermatitis onskin contact. The seed (and oil) of sesame(Sesam indicum), which reportedly containsnine allergens (Malish et al., 1981), havecaused severe allergic reactions. Ingestion ofcoconut (Cocos nucifera) rarely triggers allergicreactions.

Glycosides

Several plants eaten by humans possess abinary chemical system that presumably isproduced as a means for chemical defence.The two chemical elements are innocuous inisolation and, like nerve agent precursorsdesigned by humans for chemical warfare,lethal when mixed together. One componentconsists of an inactive form of the ultimatetoxic agent, inactive because it is bound tosugar molecules (commonly D-glucose) toform a glycoside (e.g. glucoside) (Fig. 1.3).The second component, which is storedin a separate cellular compartment, is ahydrolytic enzyme (e.g. β-glucosidase) thatis designed to cleave the glycoside andrelease the toxic aglycone. Damage to plantcells brings the β-glycosidase in contact withthe glycoside, thereby releasing the noxiousagent (the aglycone), which itself may bemetabolized to other toxic species. Release ofthe aglycone may occur during insect or


20-Feb-03 1



animal attack, through bruising, during foodpreparation or through the action of intesti-nal microflora. Some aglycones have strongodours, others have marked toxicity andsome are teratogenic. While these propertiesmay be sufficient to ward off attack by manymembers of the animal kingdom, humans are

rarely deterred; in fact, in some cases (e.g.cassava and almonds), the toxic principlesmay be exploited for their bitter taste!

There is a further, largely unrecognizedand uninvestigated potential toxicity of glyco-sides, in particular those that employ glucoseas the carrier for the toxic aglycone. The pan-creas continuously monitors blood glucosebecause the molecule must be available to thenervous system and other organs for normalfunction. Neurones, pancreatic β-islet andother cells are equipped with glucose trans-port systems that shuttle required supplies ofglucose to intracellular sites of metabolismand energy generation. These glucose trans-port systems may be unable to discriminatebetween a glucose molecule and a glucoside.Once inside the cell, the glucoside can becleaved by a β-glucosidase, thereby gene-rating an intracellular biocide with cytotoxicpotential. While little studied, this mechanismmay have importance in populations thathave a high incidence of conditions suchas diabetes mellitus and neurodegenerativedisease (Eizirik et al., 1996).

Four groups of glycosides present in plantproducts ingested by humans are considerednext: (i) fava glycosides, which harbour sub-stances able to cause red blood cell rupture(haemolysis); (ii) thioglycosides (glucosino-lates) in Brassica and other widely consumedvegetables, which liberate odoriferous andthyrotoxic substances; (iii) cyanoglycosides incassava and sorghum, which harbour agentsthat attack the thyroid, brain and, possibly, thepancreas; and (iv) azoxyglucosides of cycads,the aglycone of which is a mutagen, carcino-gen, hepatotoxin and developmental neuro-toxin, with strong epidemiological linkswith amyotrophic lateral sclerosis andparkinsonism–dementia complex (ALS-PDC).Other glycosides (i.e. solanum, isoflavone andβ-sitosterol glycosides) are discussed in latersections in this chapter.

Cycads and neurodegeneration

Studies of aboriginal groups in Australia sug-gest that the poisonous seed of glycoside-containing cycads (e.g. Cycas spp.) havebeen eaten throughout human history.Cycads, the contemporaries of dinosaurs, are

11-Mar-03 1


Fig. 1.3. β-Glucosides that harbour toxicagyclones. Cycasin (methylazoxymethanol-β-glucoside) from Cycas spp. (top), and cyanide-containing linamarin and lotaustralin from Manihotesculenta.



gymnosperms that store the potent alkylatingaglycone methylazoxymethanol (MAM) inthe form of glycosides such as cycasin(MAM-β-D-glucoside) (Fig. 1.3). Australianaborigines have developed elaborate andthorough detoxification methods that consistof crushing, drying, soaking, fermenting andpulverizing cycad seed contents prior tocooking the resulting paste. Similarly, in theRyukyu Islands of Japan, residents haveemployed fermentation processes to renderthe seed and sago (from the inner parts ofthe overground stem) free of cycasin (P.S.Spencer, personal observations). Failure todetoxify cycad materials may result in acuteillness characterized by liver damage, comaand death. In the southern Marianas Islands,notably Guam, cycad seed may be soaked foronly short periods of time and then left to dryin the sun. Flour derived from these incom-pletely detoxified materials contains varyingconcentrations of cycasin and other materialssuch as the neurotoxic amino acid BMAA(Kisby et al., 1992). Epidemiological studieson Guam have shown an exceptionally strongcorrelation between the concentration ofMAM (but not of BMAA) in cycad flour andthe age-adjusted incidence of ALS-PDC inthe Chamorro communities from which theflour was derived (Zhang et al., 1996). Whilea causal relationship between cycad andALS-PDC has yet to be established, it is wellknown that ingestion of cycad leaves (Macro-zamia, Cycas spp.) induces neuromusculardisease in grazing animals. Moreover, MAMperturbs brain development by disruptingcell division and migration, resulting inectopic, multicellular entities that arereminiscent of those seen in Chamorroswith ALS-PDC (Spencer, 2000a). This is akey part of the evidence suggesting thatthis prototypical neurodegenerative disease,which generally appears in the second half oflife, may be acquired in the late pre-natal orearly post-natal period. Given that the brainsof Chamorro people show the hallmarks ofbrain ageing much earlier than those of otherpeople, the general importance of theseobservations in understanding brain ageingcannot be overemphasized. More specifically,it should be noted that cycad stems yield thefinest quality sago, a product of many plants

(notably Metroxylon spp.) imported after theSecond World War from the Dutch EastIndies (Indonesia) that was used to feed Brit-ish and perhaps other schoolchildren who arenow approaching retirement. Additionally,until 1926, a cycad species (Zamia floridana)was harvested and processed in Florida,USA, for the production and regional distri-bution of Florida arrowroot (Spencer, 1990).

Cassava (manioc) and multiorgan disease

Whereas human ingestion of cycad seedis geographically restricted, minimal anddeclining, consumption of another toxicplant, cassava (Manihot esculenta), is wide-spread, massive and steadily rising. A nativeof South America, cassava was probably car-ried by Portuguese explorers in the 16th cen-tury first into Africa and then throughout thediscovered world (Jones, 1959). The introduc-tion of cassava met with widespread accep-tance because its tuber and leaf provide avaluable and reliable source of carbohydrateand protein, respectively. Current estimatescite cassava consumption by 400 millionpeople worldwide, mostly in the tropicsand subtropics, a majority of which lives inAfrica (Rosling and Tylleskär, 2000). Cassavais currently considered to be a valuableexport crop and its penetration now includesEurope and North America.

The root and leaves of cassava harbourlinamarin and lotaustralin (Fig. 1.3), two ofthe more than 50 stable cyanogenic (cyanide-liberating) glucosides that have been isolatedfrom a similar number of plant species, sev-eral of which are used by humans for food(Tewe and Iyayi, 1989). Sweet potato or yam,maize, bamboo, chick pea and sorghum arealso able to liberate hydrogen cyanide. Cas-sava and lima beans, a leguminous speciesthat is widely eaten, are documented causes ofacute cyanide toxicity (Conn, 1973; Roslingand Tylleskär, 2000). Cassava, sorghum andlima bean stand out because they are likely tobe heavily consumed by human populationssubject to nutritional shortage resulting fromwar, civilian disruption or climatic extremes.Since these events occur among populationsthat rarely attract medical and scientific atten-tion, there is little appreciation of the adverse


11-Mar-03 1



health impact associated with cyanogenicplants such as cassava. In brief, reliance oncassava is an established cause of goitre andneurodegeneration, and it may also be anaetiological factor in a tropical form ofdiabetes mellitus (Bokanga et al., 1994).

Cyanogenic plants such as cassavacontain a binary chemical defence systemconsisting of glucosides and an enzymespecific for the β-glucosidic linkage.Degradation of glucoside takes place underenzymatic and base hydrolysis to yieldβ-D-glucopyranose and acetone cyanohydrin(2-hydroxyisobutyronitrile); the latter dissoci-ates to hydrogen cyanide (HCN) under theaction of hydroxynitrile lyase. As with cycadseed on Guam, traditional methods of cassavatuber preparation (soaking, drying andcrushing) may leave residual glycoside orcyanohydrin; hence, ingestion may result inacute HCN intoxication. HCN is absorbedrapidly from the gastrointestinal tract andproduces recognizable effects in both fatal(0.5–3.5 mg kg−1) and non-fatal dosages as aresult of the inhibition of cytochrome oxidase,a key enzyme in energy generation for thebrain. Some plant varieties (‘bitter cassava’)eaten raw induce seizures, coma and death,with the possibility of concomitant brain dam-age expressed in the form of delayed-onsetparkinsonism or dystonia among survivors.Headache and gastrointestinal upset followingestion of the less acutely toxic ‘sweet’varieties. Both sweet and bitter forms areunder widespread cultivation, the latter topromote pest resistance and, after incompletedetoxication, for the quality of their taste.

An important public health problemarises from heavy dietary reliance on incom-pletely detoxified cassava among protein-poor populations, particularly in western andsouthern Africa (Rosling and Tylleskär, 2000).In Nigeria, for example, cassava root (38 mgHCN 100 g−1) is eaten as gari (1.1 mg HCN100 g−1) and purupuru (4–6 mg HCN 100 g−1)in amounts up to 750 g day−1, which corre-spond to 8 mg and 32–48 mg HCN, respec-tively (Osuntokun, 1981). The minimal lethalHCN dose in humans is 35 mg. HCN is metab-olized by reaction with sulphane sulphurto thiocyanate (SCN) through the catalyticaction of rhodanese, an enzyme that is widely

distributed in animal tissues. The thiocyanateion (SCN−) inhibits the uptake of iodine bythe thyroid gland and may cause goitre whenthe iodine content of the diet is low (VanEttenand Wolff, 1973). Higher levels of SCN− inhibitthe formation of thyroxine and relatedcompounds even when the iodine supply ismarginal. In the 1960s, goitre was widespreadin eastern Nigeria where a dry, unfermentedform of cassava formed a major component ofthe diet.

The major concern arising from heavycassava consumption is its effect on thedeveloping and adult nervous system. Whileunproven, there is a strong possibility thatchronic HCN exposure promotes miscarriageand adversely impacts the developing brain.That this concern has neither been discussednor investigated in relation to cassava-consuming populations is shocking. Thereis, however, recognition that cassava depend-ency is associated with neurodegenerativedisease in adults, but this condition appears tobe confined to populations that have protein-poor diets associated with heavy or exclusivedependency on cassava (Rosling andTylleskär, 2000). While SCN− may have a rolein neurotoxicity by increasing binding of glu-tamate to AMPA-type glutamate receptors ontarget neurones, under states of sulphur defi-ciency HCN may be metabolized by a minorpathway to cyanate (OCN−), an establishedcause of peripheral neuropathy in humansand spasticity in primates (Spencer, 1999).Minimally nourished children and womenwho are reliant on poorly detoxified cassavaare prone to be stricken with leg weaknessand spasticity, which may be accompaniedby visual and hearing deficits. Epidemics ofcassava-associated spastic paraparesis (konzoand mantakassa) arising from degenerationof motor nerve cells in the cerebral cortexare reported from cassava-reliant regions ofMozambique, Zaire and the Central AfricanRepublic, among others. Affected subjectsare left with a persistent crippling disease(Rosling and Tylleskär, 2000).

Related to this disorder is a conditiondescribed from west Africa known as tropicalataxic myeloneuropathy, a slowly evolvingillness of adults that affects the brain, spinalcord and peripheral nerves (Osuntokun,

11-Mar-03 1




1981). A similar condition has been reportedamong Senegalese who subsist on a diet ofmillet (sorghum) (Conn, 1973). Also reportedamong elderly Nigerians, but never con-firmed, is a high incidence of a unique neuro-degenerative disorder of the elderly thatconceivably could represent the effects ofprolonged, low-level cassava intoxication.

There are other adverse health effectsthat may be associated with cassava (Bokangaet al., 1994). One is a form of tropical diabetesmellitus (type III) that has been found incassava-consuming populations. While theassociation between cassava and diabeteshas been questioned on the basis of negativeresults in animals chronically treated withcyanide (Soto-Blanco et al., 2001), this doesnot exclude the possibility that cyanogenicglucosides enter and destroy β-islet cells.A second potential adverse health effect ofcassava can be deduced from advances incancer research. Thiocyanate, the principalmetabolite of HCN, is a particularly effectivecatalyst for the formation of carcinogenicnitrosamines through the action of sodiumnitrite on a secondary amine (Archer, 1984).This concern may be relevant to the reporteduse of cassava as a meat extender for use inhamburgers, since meat may be treated withsodium nitrite as a preservative.

Glucosinolates and goitre

The more than 100 known glucosinolatesare sulphur-containing glycosides foundexclusively in cruciferous plants, notably inseed. The highest concentrations are foundin Resedaceae, Capparaceae and Brassicaceae.Species containing glucosinolates includemustard, rape, swede, crambe, kale, turnips,cabbage, cauliflower, broccoli, Brusselssprout and radish; the last five comprise themajor source of glucosinolates in the humandiet. Radishes are an important component ofthe Japanese diet, whereas glucosinolate-richBrussels sprouts contribute heavily to theBritish diet. An estimated 5% of the UK pop-ulation consumes up to more than 300 mgof glucosinolates daily; in 1975, Japan hada daily estimated consumption of approxi-mately 100 mg (radish, daikon, cabbage plusfermented root and leaf vegetables), while

mean daily intake for North Americans isapproximately 15 mg. Boiling reduces andfermentation destroys glucosinolates suchthat, in the UK, mean daily intake calcu-lated for cooked vegetables amounts toapproximately 30 mg (Fenwick et al., 1989).

Glucosinolates make up one componentof a binary chemical system that deliverssubstances with insecticidal propertiesand pungent odours. The second componentis an endogenous enzyme, myrosinase (thio-glucoside glucohydrolase), which is storedin the plant separately from the gluco-sinolates. Bruising, cutting and chewingthe plants activate the chemical defencesystem through enzymatic cleavage of theglucosinolate to yield an unstable aglycone(thiohydroxymate-O-sulphonate). Elimina-tion of sulphur leads to the pH-dependentformation of isothiocyanate, nitrile or thio-cyanate. Glucosinolates and isothiocyanatesprotect against chemical carcinogenesis inrodents although, as noted before, thio-cyanates (which survive after cooking) inthe presence of nitrite would probably favourthe formation of carcinogenic nitrosamines(Archer, 1984).

There is considerable evidence showingthat glucosinolate-rich plant componentshave adverse effects on the health and growthof animals. In several species, rape or crambeseed meal decreases feed intake and growthwhile enlarging the liver, kidney, thyroidand adrenal glands (Verkerk et al., 1998). Forhumans, the principal concern is possibledepression of thyroid function associatedwith glucinosolate derivatives. Thiocyanateand certain isothiocyanates are goitrogenicin states of iodine deficiency. Other metabo-lites, notably S-5-vinyl-oxazolidine-2-thione(goitrin) from rapeseed, interfere with thyrox-ine synthesis and therefore promote goitreirrespective of iodine status. Goitre has beenattributed to the consumption of largeamounts of cabbage or of kale containingthiocyanate, isothiocyanate and goitrin. A1956 survey of children in Tasmania attrib-uted enlarged thyroid glands to the con-sumption of milk from dairy cattle fed kale.However, contemporary surveys in Englandand The Netherlands in areas where cruciferforages were used for dairy cattle gave no


11-Mar-03 1



indication that cow’s milk was goitrogenic(VanEtten and Wolff, 1973). More recently,consumption of goitrogenic substances(cabbage, kale and sulphonamide) was foundto be a major risk factor in a study of thyroidnodules among a population of 430 Serbo-Croatian patients, most of whom were womenwith a mean age of approximately 50 years(Obradovic, 2000).

Fava beans and favism

Vicine and convicine are glycosides primarilyassociated with the fava (broad) bean (V.faba), an important source of protein for pop-ulations in the Mediterranean, North Africa,Middle East and Far East, notably China(Mager et al., 1980; Marquardt, 1989). Thecontent of glycoside, which is highest in theseed, varies by maturity, environmental fac-tors and genetic variation. Cooking has littleeffect on glycoside content. Ingested glyco-sides (vicine and convicine) are hydrolysedby intestinal microflora to the aglyconesdivicine and isouramil, respectively, theapparent causes of a potentially fatal haemo-lytic disorder in susceptible humans knownas favism. The oxidized form of isouramil,which has structural relationships with thepancreatic β-islet toxin alloxan, may havediabetogenic effects (Ashcroft et al., 1986).

Susceptibility to fava beans is associatedwith an inherited X-chromosome-linkedsystemic deficiency of the enzyme glucose-6-phosphate dehydrogenase (G6PD) (EC1.1.1.49). This is one of the most commongenetic polymorphisms in the human popula-tion: over 400 variants have been describedand an estimated 200 million people areaffected worldwide. G6PD is required byred blood cells for the maintenance ofadequate levels of reduced glutathione andnicotinamide adenine dinucleotide phos-phate, which serve as cellular antioxidants.Divicine and isouramil (in addition to anumber of therapeutic drugs, includingcertain antimalarials and antimicrobials)serve as free-radical generators that promoteformation of hydrogen peroxide. In theabsence of adequate antioxidants, peroxideinduces the formation of methaemoglobin,protein cross-linking, loss of red cell shape

and haemolysis. Acute haemolytic anaemiausually occurs in G6PD-deficient children(especially males) within hours of ingestion.Symptoms result from a reduction in theoxygen-carrying capacity of the bloodstream.Mildly affected individuals experiencemalaise, headache, nausea, vomiting, chills,shortness of breath, lumbar pain and fever.Severely affected neonates and children maydevelop jaundice, haemoglobinuria and renalfailure (Luisada, 1941).

The prevalence of G6PD deficiency ishighest among the Kurds, Iraqis, Sardinians,Cypriot Greeks, African-Americans andcertain African populations. A 1979 study inSicily found more than 10% of male subjectswith G6PD deficiency, with most cases offavism arising from ingestion of fresh favabeans but, in addition, cases associated withbreast feeding and pollen inhalation (Schiliroet al., 1979). Temporal changes in favism inci-dence have been reported in Sardinia, whereG6PD screening and health education beganin 1971. In the period 1961–1970, there were508 cases of favism, of which 76% occurredin boys. In contrast, during the period1981–1990, there were 144 cases, of which only52% occurred in boys. The relative increase offavism in girls was attributed to the possiblefailure of the screening method to detect allsubjects with heterozygous G6PD deficiency(Meloni et al., 1992).

Alkaloids

Alkaloids are basic nitrogenous compoundsin which the nitrogen is usually containedwithin a heterocyclic ring system. Some affectthe nervous system, others perturb fetaldevelopment, and pyrrolizidine alkaloidsdamage the liver.

Pyrrolizidine alkaloids

Pyrrolizidine alkaloids (PAs) are found in13 plant families, including Compositae(Asteraceae), Boraginaceae, Leguminosae,Apocyanaceae, Ranunculaceae and Scrophulari-aceae. Their structure is based on two fusedfive-member rings that share a nitrogen

20-Feb-03 1




atom, and they exist in plants either asthe esterified alkaloid, the corresponding N-oxide or both. Human contact occurs throughthe use of various toxic species as herbs,‘health foods’ (Cupp, 2000), food supple-ments, green vegetables and food contami-nants. Comfrey plants (Symphytum spp.),which find use as vegetables and tea, repeat-edly expose consumers to PAs such as inter-medine, lycopsamine, symphytine and others(Bruneton, 1999; Coulombe, 2001), as do anumber of plants (Petasites, Symphytum andTussilago) used by the Japanese in food(Crews, 1998). Humans may also be exposedto PAs from plants visited by honeybees andby herbivores that secrete milk.

PAs are important causes of humanillness and a significant threat to humanhealth, especially in less developed countriessubject to drought and famine (Huxtable,1989; Crews, 1998). Ingested PAs are bio-activated in the liver to form highly reactivedehydroalkaloid pyrroles that alkylate DNA,RNA and proteins. The principal outcome isliver damage in the form of veno-occlusivedisease, hepatic venous thrombosis, ascites,jaundice and, probably, an elevated risk ofliver cancer. Secondary targets of pyrrolesderived from PAs include the lungs, heart,kidney, stomach, reproductive system andbrain (Huxtable, 1989). Monocrotaline ispneumotoxic. Atypically, neurological effects(vertigo, headache, delirium and coma) in theabsence of overt liver toxicity occurred amongUzbeks in the 1950s after consumption ofgrain contaminated with seed of Trichodesmaincanum.

Herbal use of PA-containing legumesof the genus Crotalaria, together with Seneciospp., is held responsible for past outbreaksof veno-occlusive disease and ascites inJamaica (Huxtable, 1989). Seasonal endemicveno-occlusive disease in Madhya Pradesh,India, is attributed to contamination of milletwith Crotalaria nana pods (Krishnamachariet al., 1977). Afghanistan was the setting in1976 for a large outbreak of veno-occlusivedisease resulting from the consumption ofbread prepared from grain contaminated withseed of Heliotropium popovii (Mohabbat et al.,1976). In the 1970s, herbal teas preparedfrom Senecio longilobus caused liver disease,

hepatomegaly, jaundice and fatalities amongAmerican children (Huxtable, 1980).

Solanum alkaloids

The toxic alkaloids of the potato plant(Solanum tuberosum), α-chachonine andα-solanine, are saponin-like alkaloids thatexist in the form of β-D-glycosides (Sharmaand Salunkhe, 1989). These substances inhibitcholinesterase enzymes: butyrylcholinester-ase (BuChE), which is concentrated in liverand lungs and serves as an important defenceagainst toxic substances; and acetylcholines-terase (AChE), which is required to terminatethe transmitter action of acetylcholine at theneuromuscular junction. Since BuChE andAChE hydrolyse and inactivate several ana-esthetic drugs (cocaine, heroin, esmolol andlocal ester anaesthetics) and neuromuscular-blocking agents, ingestion of potatoes mayimpact the metabolism and duration of actionof these substances during and followingsurgery (McGehee et al., 2000).

Changes in the glycoalkaloid contentof fresh and processed potatoes may occurduring storage, under the influence of lightand radiation, following mechanical damageand as a result of food processing (Friedmanand McDonald, 1999). Human toxicity fromingestion of green potatoes with a highsolanum glycoalkaloid content is associatedwith gastric pain, weakness, nausea, vomitingand laboured breathing.

The potential for teratogenic effects hasbeen a significant public health concernin relation to populations consuming largeamounts of potato. The concern arises fromstudies with Syrian hamsters. Animals treatedorally with potato sprouts containing sola-nidine, the common aglycone of α-chaconineand α-solanine, have litters with craniofacialmalformations that result in herniated orexposed brain tissue, defects in the nasalchamber, a single eye and a cleft palate.Salasodine, another teratogenic substance, ispresent in potato cultivars and in related foodplants, namely S. melongena (aubergine) andS. quitoense (Andean naranjilla). A 1972 reportsuggesting that certain birth defects inhumans are caused by ingestion of blightedpotatoes (infested with Phytophthora infestans)


11-Mar-03 1



has not received experimental support (Allenet al., 1977). Note that tomatidine, the aglyconeof the related glycoalkaloid tomatine, lacks ateratogenic property.

Lupin alkaloids

These substances include a large number ofquinolizidine alkaloids with toxic (lupanine> sparteine > lupinine in guinea pigs) orteratogenic (anagyrine) properties found inLupinus spp. Their presence and concentra-tion in the protein-rich seed of these legumesvary with species and environmental factors.Those with high alkaloid content tend to havea bitter taste, are associated with acute toxic-ity in humans and animals and, in the latter,with a congenital skeletal malformationknown as ‘crooked calf disease’ (Keeler,1989). No anagyrine was found in several‘bitter’ and ‘sweet’ selections of lupins usedas human food (Keeler and Gross, 1980). Ithas been suggested that sweet lupin flourmay be used for the improvement of proteinsupply if the alkaloid content does not exceed0.02% and the seed contains no secondaryfungi that cause lupinosis (Gross et al., 1976).Lupin seed flour has been investigated as acomponent of infant formula and bread.Those sensitized to groundnut may haveallergic responses to lupin flour after inges-tion or inhalation exposure (Crespo et al.,2001).

Isoquinoline alkaloids

These alkaloids are said to be more numerousand cover a wider range of structural typesthan those of any other group (Bentley, 1998).Benzyltetrahydroisoquinolines, which areformed from dopamine and phenylacetyl-aldehyde, are pivotal intermediates in themetabolism of isoquinoline alkaloids. Severalisoquinoline alkaloids are active on thenervous system, including tubocurarine(neuromuscular blocker), apomorphine(dopamine D2 receptor agonist), morphine(enkephalin agonist), colchicine (spindleinhibitor and axonal neurotoxin), lycorineand galanthamine (cholinesterase inhibitors).A form of parkinsonism and motor neuronedisease in Guadeloupe, French West Indies,

has been tentatively linked with the use ofherbal teas prepared from Annonaceae (cus-tard apple and paw-paw family) (Caparros-Lefebvre and Elbaz, 1999). These plantscontain tetrahydroisoquinolines (TIQs) suchas reticuline and higenamine, as well asnon-TIQ compounds (acetogenins) that blockmitochondrial respiration (Bruneton, 1999).In addition to these edible tropical fruits,TIQs are found in a variety of widelyconsumed food items of plant (banana) andother origins (Nagatsu, 2000).

Carboline alkaloids

β-Carboline indole alkaloids occur in a num-ber of plants and, together with α-, γ- and δ-carbolines, as pyrolysis-induced tryptophancondensations and rearrangements as a con-sequence of grilling of proteinaceous foods.These compounds form co-mutagenic deriva-tives and also possess neurotoxic activity(Wakabayashi et al., 1997). The β-carbolines ofthe passion flower (Passiflora incarnata), forexample, include harman (motor depressantand convulsant) and its 7-oxygenated deriva-tives harmine and harmaline, both of whichare hallucinogenic. β-Carboline analogues ofMPP+ (Fig. 1.1), such as 2-N-methyl- and2,9-N,N-dimethyl-harminium and harma-linium derivatives, inhibit mitochondrialrespiration and are toxic to dopaminergicneurones (Collins and Neafsey, 2000).

Non-nitrogenous Compounds

Phyto-oestrogens and anti-oestrogens

Many plant species contain active principlesthat act as contraceptives, interceptives,abortifacients, uterine stimulants, antisperm-atogens, spermicides and phyto-oestrogens.Most are beyond the scope of this chapter;however, the phyto-oestrogens are of con-siderable current interest because of theirsignificant presence in plants used for food(Helferich et al., 2001).

Phyto-oestrogens and anti-oestrogenslack the steroid ring structure of oestrogen, themammalian steroid hormone that regulates

20-Feb-03 1




and maintains female sexual characteristics,but they nevertheless have propertiessimilar to the principal human oestrogen17β-oestradiol (Aldridge and Tahourdin,1998). Their ability to disrupt reproductiveperformance has been recognized in sheepgrazing on subterranean clover (Trifoliumsubterraneum) and cattle fed lucerne (M.sativa). Feminization of male animals hasbeen reported following ingestion of phyto-oestrogens during critical periods of develop-ment. This and other health concerns haveresulted in intense scrutiny of the effectsof phyto-oestrogens on reproductive health,development and cancer risk. Vegetariansand certain ethnic groups have the highestexposure to phyto-oestrogens.

Isoflavone glycosides

Isoflavones make up the majority of phyto-oestrogens found in food. These compoundsare linked to a sugar molecule as O-glycosides (genistin, daidzin and glycitin).Hydrolysis to the corresponding biologicallyactive aglycones (genistein, daidzein andglycitein) may occur during fermentationor through the action of microflora in thegut. Soybeans and sprouts are a rich sourceof isoflavones, and soy foods constitutethe main source of phyto-oestrogens in thehuman diet. Infants are exposed through theuse of soy-based infant formula or via breastmilk of mothers who ingest large amountsof soya products. The oestrogenic potential ofgenistein in an in vivo assay has been esti-mated to be two to four orders of magnitudelower than that of 17β-oestradiol.

β-Sitosterol

Plant oils, such as groundnut, sunflower andolive oils, contain the highest concentrationof this major phytosterol of higher plants.β-Sitosterol (BSS), together with its glycoside,β-sitosterolin (BSSG), has been implicatedin the feminization of fish in the vicinity ofpulp mill effluents (Bruneton, 1999). Animalstudies have demonstrated that BSS andBSSG possess anti-inflammatory, antipy-retic, antineoplastic and immune-modulatingproperties. BSS has been used without

adverse health effects for the long-termtreatment of prostatic hypertrophy (Klippelet al., 1997).

Coumestans, lignans and other

Non-glycosidic plant substances with oestro-genic activity include the coumestans (cou-mestrol), found in lucerne, mung bean, cloversprouts, soybeans, lima beans and red beans,and the lignans, precursors of which occurin grains, seeds, berries and nuts (Helferichet al., 2001). Coumestrol is the most potent ofthe phyto-oestrogens, with biological activityrelative to 17β-oestradiol some five timeshigher than that of genistein (Aldridge andTahourdin, 1998). Lignans (enterolactone andenterodiol) form in the gut from plant pre-cursors (matairesinol and secoisolarici-resinol, respectively) (Setchell et al., 1980).Zearalenone, a myco-oestrogenic substanceproduced by Fusarium spp. growing onmouldy maize (Chapter 4), has been impli-cated in fertility problems in pigs and cows.Anti-oestrogenic compounds (indole-3-carbinol) occur in cruciferous vegetables, suchas cabbage, broccoli and Brussels sprouts. Safelevels for human consumption of oestrogenicand anti-oestrogenic compounds have yet tobe established (Helferich et al., 2001).

Ptaquiloside

The ‘fiddleheads’ of bracken fern (Pteridiumaquilinum, P. esculentum) are consumed asgreens and salads in various parts of theworld, such as Japan. The plant contains anumber of toxic substances and, in particular,a potent alkylating glycoside and carcinogenknown as ptaquiloside. Livestock grazing onbracken fern develop bladder cancer, bonemarrow depression, leukaemia, thrombo-cytopenia and a haemorrhagic syndrome.Laboratory rodents fed bracken fern developmalignant tumours of the bladder, lung andintestine, and the milk of cows fed brackenfern is carcinogenic to rats. The high incidenceof oesophageal cancer among the Japanesehas been attributed to dietary use of brackenfern. Since ptaquiloside is heat labile, the


11-Mar-03 1



cooked fronds of bracken fern do not containdetectable amounts of ptaquiloside (Satoet al., 1989).

Alkenylbenzenes

Several spices, essential oils, herbs andcertain vegetables (parsnips, parsley andsesame seed) contain structurally relatedalkenylbenzenes; these form epoxy inter-mediates that develop covalent adducts withguanine and act as weak rodent hepato-carcinogens (Luo and Guenthner, 1996).Alkenylbenzenes present in food includesafrole (1-allyl-3,4-methylenedioxybenzene),a component of sassafras tea, oil of sassafras(Sassafras albidum) and nutmeg (Myristicafragrans). Tarragon, basil and fennel containthe related compound estragole (methyl-chavicol). Isosafrole, a component of theflavourant oil of ylang-ylang (Canangaodorata), and β-asarone, a component of oil ofcalamus (Acorus calamus root), are also rodentcarcinogens (Coulombe, 2001). Anotheralkenylbenzene, myristicin, the major flavourof nutmeg (M. fragrans) and also present inblack pepper, parsley, dill and carrots, isnot thought to be carcinogenic but instead, inlarge quantities, is allegedly hallucinogenic.Piperine, which is responsible for much ofthe pungent flavour of black pepper (Pipernigrum), forms potentially carcinogenic inter-mediates (nitrosamines) in the presence ofnitrite. Capsaicin, the pungent component ofchilli peppers (Capsicum frutescens and others),is questionably a weak carcinogen and betterknown as an experimental neurotoxinselective for substance P-containing nervecells. D-Limonene, a major constituent of oilsobtained from the peel of citrus fruit (orange,lemon and grapefruit), causes renal tumoursin rats but is not considered harmful tohumans (Coulombe, 2001).

Coumarins

Over 1000 coumarins (2H-1-benzopyran-2-ones) have been described, and the simplestamong them are widely distributed among

plants as water-soluble glycosides (Bruneton,1999). Coumarin, first isolated from the tonkabean (Dipteryx odorata), is found in vegetables(cabbage, radish and spinach) and plants usedas flavouring agents or herbs (lavender, sweetwoodruff and sweet clover). Coumarin, usedin human medicine as an anticoagulant, ismetabolized rapidly in the liver to form thehepatotoxin 7-hydroxycoumarin.

Psoralen

Psoralens are straight-chain furanocou-marins that are activated by sunlight tophototoxins with mutagenic and carcino-genic properties. They are found in celery,parsnips, limes, cloves and figs (Coulombe,2001). 8-Methoxypsoralen (xanthotoxin) is acarcinogenic species used in combinationwith ultraviolet (UV) irradiation (PUVA) totreat patients with psoriasis and mycosisfungoides. Long-term PUVA treatment hasbeen associated with squamous-cell carci-noma and melanoma many years after onsetof treatment (Bruneton, 1999).

Conclusions

Several conclusions emerge from the fore-going selected summary of plant toxins.

1. Many plants manufacture, store andrelease chemicals with the potential to causehuman illness.2. These chemicals, some of which may bedesigned for defence, are frequently stored asa binary system. A widely exploited binarysystem employs a glycoside (a sequesteredform of the toxic agent linked to glucose) anda glycosidase (the mechanism by which thetoxic agent is released), which are stored inseparate parts of the plant. Ingestion of theplant product may result in: (i) release ofsequestered toxic substance through theaction of microbial or tissue glycosidases;and/or (ii) uptake of intact glycosides byway of cellular glucose transport systems andsubsequent intracellular enzymatic release ofthe agent at a remote site. Cells with plasma

20-Feb-03 1




membranes rich in glucose transporters(e.g. nerve cells and β-islet pancreatic cells)theoretically are at greatest risk for toxicdamage.3. Humans consume plants that areincompletely detoxified: while they areequipped to detect and reject acutely toxicmaterials, plant products containing levelsof hazardous chemicals that cause delayedillness (months, years or decades) are suffi-ciently palatable to be ingested, especiallyin settings where safer foodstuffs areunavailable.4. Long-term effects of low-level exposureto phytotoxic chemicals are little studied, evenwhen the material is widely ingested, whetherin the setting of poverty or affluence. Forexample, dried roasted Coffea arabica bean isreported to contain atractyloside (17.5–32 mgkg−1), a diterpenoid glycoside that in largerdoses (10- to 20-fold) causes fatal renal proxi-mal tubule necrosis and/or centrilobularhepatic necrosis in humans and animals(Obatomi and Bach, 1998). In general, muchless scientific attention is directed towards thesafety of plant products consumed by popula-tions at greatest risk for plant toxicity, namelyprotein-poor people who subsist on singlestaples.5. In the short term, there is a need toincrease public understanding of the truenature of plant materials, namely that theycontain substances that are beneficial and oth-ers that are hazardous to health; ideally, thelatter should be removed prior to ingestion. Ofspecial concern is the ever widening food useof cassava root, which, when consumed afterincomplete detoxication, is associated with arange of chronic health disorders.6. In the long term, the extraordinary com-plexity of plant chemistry suggests that effortsto develop diets to promote optimal humanhealth and longevity may represent a futilesearch of vast expense. An alternativeapproach would be for humans to expandknowledge of their nutritional and relatedneeds, with the long-term goal of developinga synthetic diet that would support life andmaintain optimal health. Adoption of thisstrategy would reduce and ultimatelyeliminate plant-related human morbidity andmortality; it would also support the species’

ambition to colonize outer space and otherparts of the universe.

Note Added at Proof Stage

Recent studies show that the toxic agentacrylamide is formed in certain plant prod-ucts subjected to high cooking temperaturesassociated with frying, grilling and baking.Formation is proposed to occur through aMaillard reaction between sugars and aminoacids, such as asparagine, methionine andcysteine (Stadler et al., 2002). The highestlevels of acrylamide (> 1000 µg kg−1) arefound in potato crisps; lower levels(150–350 µg kg−1) occur in maize crisps,potato chips, biscuits, toast, cereals and coffeepowder. Cooked foods derived from otherplant (and animal) products are underscrutiny at the time of writing. The estimatedaverage dietary intake is in the order of0.5–1 µg kg−1 body weight day−1, with two- tothreefold higher levels for children.

While acrylamide is known to cause peri-pheral neuropathy following occupationalexposures, with structural damage to both thecentral and peripheral nervous systems, dosesfrom food are not anticipated to be of suffi-cient magnitude to induce comparable changesin the general public. Similar considerationsdiminish concerns in relation to animal stud-ies demonstrating testicular toxicity. How-ever, animal studies demonstrate that acryla-mide, and/or its metabolite glycidamide, isgenotoxic and able to induce somatic andgerm cell damage, with induction of benignand malignant tumours (thyroid, adrenal gland,brain, spinal cord) and heritable damage at thegene and chromosomal level. Since these toxiceffects of acrylamide have no known thresh-old, the substance is classified as a probablehuman carcinogen. Epidemiological studiesof populations with occupational exposureto acrylamide have not revealed increasedoccurrence of cancer, but the detection sensi-tivity of these studies has been low. Researchrecommendations have been made to increaseunderstanding of the public health threatposed by low-level exposure to acrylamidederived from food (WHO/FAO, 2002).


11-Mar-03 1



Acknowledgements

Valerie Palmer, Suzanne Spencer andMeg Heaton are thanked for their helpfulcomments.

References

Aldridge, D. and Tahourdin, C. (1998) Naturaloestrogenic compounds. In: Watson, D.H. (ed.)Natural Toxicants in Food. Sheffield AcademicPress, Sheffield, pp. 54–83.

Allen, J.R., Marlar, R.J., Chesney, C.F., Helgeson,J.P., Kelman, A., Weckel, G., Traisman, E. andWhite, J.W. Jr (1977) Teratogenicity studies onlate blighted potatoes in nonhuman primate.Teratology 15, 17–23.

Angus, F. (1998) Nut allergens. In: Watson, D.H.(ed.) Natural Toxicants in Food. SheffieldAcademic Press, Sheffield, pp. 84–104.

Archer, M.C. (1984) Catalysis and inhibitionof N-nitrosation reactions. IARC ScientificPublications 57, 263–274.

Ashcroft, S.J., Harrison, D.E., Poje, M. and Rocic, B.(1986) Structure–activity relationships ofalloxan-like compounds derived from uricacid. British Journal of Pharmacology 89, 469–472.

Auricchio, S. and Visakorpi, J.K. (1992) Common FoodIntolerances. 1: Epidemiology of Coeliac Disease,Vol. 2. Dynamic Nutrition Research, Karger,Basel.

Bentley, K.W. (1998) The Isoquinoline Alkaloids.Overseas/Harwood, Amsterdam.

Birk, Y. (1996) Protein proteinase inhibitors inlegume seeds – overview. Archivos Latino-americanos de Nutrición 44(4 Supplement 1),26S–30S.

Bock, S.A., Munoz-Furlong, A. and Sampson, H.A.(2001) Fatalities due to anaphylactic reactionsto foods. Journal of Allergy and Clinical Immunol-ogy 107, 191–193.

Bokanga, M., Essers, A.J.A., Poulter, N., Rosling, H.and Tewe, O. (1994) International Workshop ofCassava Safety. Acta Horticulturae 375, 1–416.

Bruneton, J. (1999) Toxic Plants Dangerous toHumans and Animals. Intercept, Andover, UK,pp. 196–203.

Bush, R.K. and Hefle, S.L. (1996) Food allergens.Critical Reviews in Food Science and Nutrition36S, S119–S163.

Caparros-Lefebvre, M. and Elbaz, A. (1999) Possibleassociation of atypical parkinsonism in theFrench West Indies with consumption oftropical plants: a case–control study.

Caribbean Parkinsonism Study Group. Lancet354, 281–286.

Cavell, B. (1992) Increased prevalence of celiacdisease in Sweden: relevance of changes ininfant feeding practices. In: Auricchio, S. andVisakorpi, J.K. (eds) Common Food Intolerances1: Epidemiology of Coeliac Disease, Vol. 2.Dynamic Nutrition Research, Karger, Basel,pp. 71–75.

Collins, M.A. and Neafsey, E.J. (2000) Carbolinesand isoquinolines. In: Spencer, P.S. andSchaumburg, H.H. (eds) Experimental andClinical Neurotoxicology, 2nd edn. OxfordUniversity Press, New York, pp. 304–314.

Conn, E.E. (1973) Cyanogenetic glycosides. In:Toxicants Occurring Naturally in Foods, 2nd edn.National Academy of Sciences, Washington,DC, pp. 199–308.

Coulombe, R.A. Jr (2001) Natural toxins andchemopreventives in plants. In: Helferich, W.and Winter, C.K. (eds) Food Toxicology. CRCPress, Boca Raton, Florida, pp. 137–161.

Crespo, J.F., Rodriguez, J., Vives, R., James,J.M., Reano, M., Daroca, P., Burbano, C.and Muzquiz, M. (2001) OccupationalIgE-mediated allergy after exposure to lupineseed flour. Journal of Allergy and Clinical Immu-nology 108, 295–297.

Crews, C. (1998) Pyrolizidine alkaloids. In: Watson,D.H. (ed.) Natural Toxicants in Food. SheffieldAcademic Press, Sheffield, pp. 11–28.

Cupp, M.L. (2000) Toxicology and Clinical Pharmacol-ogy of Herbal Products. Humana Press, Totowa,New Jersey.

De Santis, A., Addolorato, G., Romito, A., Caputo,S., Giordano, A., Gambassi, G., Taranto, C.,Manna, R. and Gasbarrini, G. (1997) Schizo-phrenic symptoms and SPECT abnormalitiesin a coeliac patient: regression after a gluten-free diet. Journal of Internal Medicine 242,421–423.

Dohan, F.C. (1969) The possible pathogeneticeffect of cereal grains in schizophrenia. ActaNeurologica Scandinavica 31, 195–205.

Dohan, F.C. (1976) Is celiac disease a clue to thepathogenesis of schizophrenia? Mental Hygiene53, 525–539.

Dwivedi, M.P. (1989) Epidemiological aspectsof lathyrism in India – a changing scenario.In: Spencer, P.S. (ed.) The Grass Pea: Threatand Promise. Third World Medical ResearchFoundation, New York, pp. 1–26.

Eizirik, D.L., Spencer, P. and Kisby, G.E. (1996)Potential role of environmental genotoxicagents in diabetes mellitus and neurodegen-erative diseases. Biochemical Pharmacology 51,1585–1591.

20-Feb-03 1




Ewen, S.W. and Pusztai, A. (1999) Effect of dietscontaining genetically modified potatoesexpressing Galanthus nivalis lectin on ratsmall intestine. Lancet 354, 1353–1354.

Fenwick, G.R., Heaney, R.K. and Mawson, R. (1989)Glucosinolates. In: Cheeke, P.R. (ed.) Toxicantsof Plant Origin, Vol. II, Glycosides. CRC Press,Boca Raton, Florida, pp. 1–41.

Friedman, M. and McDonald, G.M. (1999) Post-harvest changes in glycoalkaloid content ofpotatoes. Advances in Experimental Medicine andBiology 459, 121–143.

Gross, R., Morales, E., Gross, U. and von Baer, E.(1976) [Lupin, a contribution to the humanfood supply. 3. Nutritional physiological studywith lupin (Lupinus albus) flour]. Zeitschrift fürErnahrungswissenschaft 15, 391–395 [German].

Gumbmann, M.R., Dugan, G.M., Spangler, W.L.,Baker, E.C. and Rackis, J.J. (1989) Pancreaticresponse in rats and mice to trypsin inhibitorsfrom soy and potato after short- and long-termdietary exposure. Journal of Nutrition 119,1598–1609.

Heinemann, G., Schievelbein, H., Eberhagen, D.and Rahlfs, V. (1981) [The influence ofdifferent diets and smoking on the clinicalchemical diagnosis of pheochromocytoma,neuroblastoma, and carcinoid syndrome]. Kli-nische Wochenschrift 59, 1165–1173 [German].

Helferich, W.G., Allred, C.D. and Young-Hwa, J.(2001) Dietary estrogens and antiestrogens.In: Helferich, W. and Winter, C.K. (eds) FoodToxicology. CRC Press, Boca Raton, Florida,pp. 37–55.

Hourihane, J.O. (1998) Prevalence and severityof food allergy – need for control. Allergy 53(46 Supplement), 84–88.

Huxtable, R.J. (1980) Herbal teas and toxins: novelaspects of pyrrolizidine poisoning in theUnited States. Perspectives in Biology andMedicine 24, 1–14.

Huxtable, R.J. (1989) Human health implications forpyrrolizidine alkaloids and herbs containingthem. In: Cheeke, P.R. (ed.) Toxicants of PlantOrigin, Vol. I, Alkaloids. CRC Press, Boca Raton,Florida, pp. 41–86.

Jones, W.O. (1959) Manioc in Africa. StanfordUniversity Press, Stanford, California.

Keeler, R.F. (1989) Quinolizidine alkaloids in rangeand grain lupins. In: Cheeke, P.R. (ed.) Toxi-cants of Plant Origin, Vol. I, Alkaloids. CRCPress, Boca Raton, Florida, pp. 133–167.

Keeler, R.F. and Gross, R. (1980) The total alkaloidand anagyrine contents of some bitter andsweet selections of lupin species used asfood. Journal of Environmental Pathology andToxicology 3, 333–340.

Kisby, G.E., Ellison, M. and Spencer, P.S. (1992)Content of the neurotoxins cycasin (methyl-azoxymethanol β-D-glucoside) and BMAA(β-N-methylamino-L-alanine) in cycad flourprepared by Guam Chamorros. Neurology 42,1336–1340.

Klippel, K.F., Hiltl, D.M and Schipp, B. (1997) Amulticentric, placebo-controlled, double-blindclinical trial of β-sitosterol (phytosterol) forthe treatment of benign prostatic hyperplasia.German BPH-Phyto Study Group. BritishJournal of Urology 80, 427–432.

Koepke, J.W., Williams, P.B., Osa, S.R., Dolen, W.K.and Selner, J.C. (1990) Anaphylaxis to pinonnuts. Annals of Allergy 65, 473–476.

Krishnamachari, K.A.V.R., Bhat, R.V., Krishnamur-thi, D., Krishnaswamy, K. and Nagararajan, V.(1977) Aetiopathogenesis of endemic ascitesin Sarguja district of Madhya Pradesh. IndianJournal of Medical Research 65, 672–678.

Lambert, M.T., Bjarnason, I., Connelly, J., Crow, T.J.,Johnstone, E.C., Peters, T.J. and Smethurst, P.(1989) Small intestine permeability inschizophrenia. British Journal of Psychiatry155, 619–622.

Liener, I.E. (1980) Toxic Constituents of PlantFoodstuffs. Academic Press, New York.

Luisada, L. (1941) Favism: singular disease affectingchiefly red blood cells. Medicine (Baltimore) 20,229 (cited by Marquardt, 1989).

Luo, G. and Guenthner, T.M. (1996) Covalentbinding to DNA in vitro of 2´,3´-oxides derivedfrom allylbenzene analogs. Drug Metabolismand Disposition 24, 1020–1027.

Mager, J., Chevion, M. and Glaser, G. (1980) Favism.In: Liener, I.E. (ed.) Toxic Constituents of PlantFoodstuffs, 2nd edn. Academic Press, NewYork, pp. 265–294.

Malinow, M.R., Bardana, E.J. Jr, Pirofsky, B., Craig,S. and McLaughlin, P. (1982) Systemic lupuserythematosus-like syndrome in monkeys fedalfalfa sprouts: role of a nonprotein amino acid.Science 216, 415–417.

Malish, D., Glovsky, M.M., Hoffman, D.R.,Ghekiere, L. and Hawkins, J.M. (1981)Anaphylaxis after sesame seed ingestion.Allergy and Clinical Immunology 67, 35–38.

Marquardt, R.R. (1989) Vicine, convicine, and theiraglycones – divicine and isouramil. In:Cheeke, P.R. (ed.) Toxicants of Plant Origin,Vol. II, Glycosides. CRC Press, Boca Raton,Florida, pp. 161–200.

McGehee, D.S., Krasowski, M.D., Fung, D.L.,Wilson, B., Gronert, G.A. and Moss, J. (2000)Cholinesterase inhibition by potato glyco-alkaloids slows mivacurium metabolism.Anesthesiology 93, 510–519.


20-Feb-03 1



Meda, H.A., Diallo, B., Buchet, J.P., Lison, D.,Barennes, H., Ouangre, A., Sanou, M.,Cousens, S., Tall, F. and Van de Perre, P. (1999)Epidemic of fatal encephalopathy in preschoolchildren in Burkina Faso and consumption ofunripe ackee (Blighia sapida) fruit. Lancet 353,536–540.

Meloni, T., Forteleoni, G. and Meloni, G.F. (1992)Marked decline of favism after neonatalglucose-6-phosphate dehydrogenase screen-ing and health education: the northern Sardin-ian experience. Acta Haematologica 87, 29–31.

Merriam, A.E. (2000) Phenelzine and othermonoamine oxidase inhibitors. In: Spencer,P.S. and Schaumburg, H.H. (eds) Experimentaland Clinical Neurotoxicology, 2nd edn. OxfordUniversity Press, New York, pp. 985–987.

Ministry of Health, Mozambique, Mantakassa(1984) An epidemic of spastic paraparesis asso-ciated with chronic cyanide intoxication in acassava staple area of Mozambique. 1. Epide-miology and clinical and laboratory findingsin patients. Ministry of Health, Mozambique.Bulletin of the World Health Organization 62,477–484.

Mohabbat, O., Younos, S.M., Merzad, A.A.,Srivastava, R.N., Sediq, G.G. and Aram, G.N.(1976) An outbreak of hepatic veno-occlusivedisease in north-western Afghanistan. Lancet 2269–271.

Montanaro, A. and Bardana, E.J. Jr (1991)Dietary amino acid-induced systemic lupuserythematosus. Rheumatic Disease Clinics ofNorth America 17, 323–332.

Morimoto, I., Shiozawa, S., Tanaka, Y. and Fujita, T.(1990) L-Canavanine acts on suppressor-inducer T cells to regulate antibody synthesis:lymphocytes of systemic lupus erythematosuspatients are specifically unresponsive toL-canavanine. Clinical Immunology andImmunopathology 55, 97–108.

Nachbar, M.S. and Openheim, J.D. (1980) Lectins inthe United States diet: a survey of lectinsin commonly consumed foods and a reviewof the literature. American Journal of ClinicalNutrition 33, 2338–2345.

Nagatsu, T. (2000) Isoquinoline neurotoxins. In:Storch, A. and Collins, M.C. (eds) NeurotoxicFactors in Parkinson’s Disease and RelatedDisorders. Kluwer Academic, New York,pp. 69–76.

Obatomi, D.K. and Bach, P.H. (1998) Biochemistryand toxicology of the diterpenoid glycosideatractyloside. Food and Chemical Toxicology 36,335–346.

Obradovic, L. (2000) [Thyroid gland nodules regis-tered at the Endocrinology Department of

the Medical Center in Prokuplje]. MedicinskiPregled 53, 64–67 [Serbo-Croatian (Roman)].

Osuntokun, B.O. (1981) Cassava diet, chroniccyanide intoxication and neuropathy in theNigerian Africans. World Review of Nutritionand Diet 36, 141–173.

Pavone, L., Fiumara, A., Bottaro, G., Mazzone, D.and Coleman, M. (1997) Autism and celiacdisease: failure to validate the hypothesis thata link might exist. Biological Psychiatry 42,72–75.

Pearce, G., Seidl, D.S., Jaffe, W.G. and Aizman, A.(1985) Nutritional studies of carboxypeptidaseinhibitor from potato tuber. In: Friedman, M.(ed.) Nutritional and Toxicological Aspects of FoodSafety. Plenum Press, New York, p. 321.

Pusztai, A. (1989) Lectins. In: Cheeke, P.R. (ed.)Toxicants of Plant Origin, Vol. III, Proteins andAmino Acids. CRC Press, Boca Raton, Florida,pp. 29–71.

Ressler, C. and Tatake, J.G. (2001) Vicianin,prunasin, and β-cyanoalanine in commonvetch seed as sources of urinary thiocyanatein the rate. Journal of Agricultural and FoodChemistry 49, 5075–5080.

Rosling, H. and Tylleskär, T. (2000) Cassava.In: Spencer, P.S. and Schaumburg, H.H.(eds) Experimental and Clinical Neurotoxicology,2nd edn. Oxford University Press, New York,pp. 338–343.

Roy, D.N. and Spencer, P.S. (1989) Lathyrogens.In: Cheeke, P.R. (ed.) Toxicants of Plant Origin,Vol. III, Proteins and Amino Acids. CRC Press,Boca Raton, Florida, pp. 169–201.

Roy, D.N., Sabri, M.I., Kayton, R.J. and Spencer, P.S.(1996) β-Cyano-L-alanine toxicity: evidence forthe involvement of an excitotoxic mechanism.Natural Toxins 4, 247–253.

Sato, K., Nagao, T., Matoba, M., Koyama, K., Natori,S., Murakami, T. and Saiki, Y. (1989) Chemicalassay of ptaquiloside, the carcinogen ofPteridium aquilinum, and the distributionof related compounds in the Pteridaceae.Phytochemistry 28, 1606–1611.

Schiliro, G., Russo, A., Curreri, R., Marino, S.,Sciotto, A. and Russo, G. (1979) Glucose-6-phosphate dehydrogenase deficiency inSicily. Incidence, biochemical characteristicsand clinical implications. Clinical Genetics 15,183–188.

Setchell, K.D.R., Lawson, A.M., Mitchell, F.L.,Adlercreutz, H., Kirk, D.N. and Woods, G.F.(1980) Excretion, isolation and structure of anew phenolic constituent of female urine.Nature 287, 738–742.

Sharma, R.P. and Salunkhe, D.K. (1989) Solanumglycoalkaloids. In: Cheeke, P.R. (ed.) Toxicants

20-Feb-03 1




20-Feb-03 1

of Plant Origin, Vol. I, Alkaloids. CRC Press,Boca Raton, Florida, pp. 179–225.

Sherratt, H.S.A. (1995) Vomiting sickness ofJamaica. In: Vinken, P.J. and Bruyn, G.W. (eds)Handbook of Clinical Neurology, Vol. 65, Intoxi-cations of the Nervous System, Pt. II. Elsevier,Amsterdam, pp. 79–113.

Soto-Blanco, B., Sousa, A.B., Manzano, H., Guerra,J.L. and Gorniak, S.L. (2001) Does prolongedcyanide exposure have a diabetogenic effect?Veterinary and Human Toxicology 43, 106–108.

Spencer, P.S. (1990) Are neurotoxins driving uscrazy? Planetary observations on the causes ofneurodegenerative diseases of old age. In: Rus-sell, R.W., Flattau, P.E. and Pope, A.M. (eds)Behavioral Measures of Neurotoxicity. NationalAcademy Press, Washington, DC, pp. 11–36.

Spencer, P.S. (1995) Lathyrism. In: Vinken, P.J. andBruyn, G.W. (eds) Handbook of Clinical Neurol-ogy, Vol. 65, Intoxications of the Nervous System,Pt. II. Elsevier, Amsterdam, pp. 1–20.

Spencer, P.S. (1999) Food toxins, AMPA receptors,and motor neuron diseases. Drug MetabolismReviews 31, 561–587.

Spencer, P.S. (2000a) Cycasin, methylazoxymeth-anol and related compounds. In: Spencer, P.S.and Schaumburg, H.H. (eds) Experimentaland Clinical Neurotoxicology, 2nd edn. OxfordUniversity Press, New York, pp. 436–447.

Spencer, P.S. (2000b) Hypoglycine. In: Spencer, P.S.and Schaumburg, H.H. (eds) Experimentaland Clinical Neurotoxicology, 2nd edn. OxfordUniversity Press, New York, pp. 669–672.

Stadler, R.H., Blank, I., Varga, N., Robert, F., Hau, J.,Guy, P.A., Robert, M.-C. and Riediker, S. (2002)Acrylamide from Maillard reaction products.Nature 419, 449.

Tariq, S.M., Stevens, M., Matthews, S., Ridout, S.,Twiselton, R. and Hide, D.W. (1996) Cohortstudy of peanut and tree nut sensitisationby age of 4 years. British Medical Journal 313,514–517.

Tate, M.E. and Enneking, D. (1992) A mess of redpottage. Nature 359, 357–358.

Tate, M.E., Rathjen, J., Delaere, I. and Enneking, D.(1999) Covert trade in toxic vetch continues.Nature 400, 207.

Taylor, S.L., Hefle, S.L. and Gauger, B.J. (2001) Foodallergies and sensitivities. In: Helferich, W. andWinter, C.K. (eds) Food Toxicology. CRC Press,Boca Raton, Florida, pp. 1–36.

Tewe, O.O. and Iyayi, E.A. (1989) Cyanogenicglycosides. In: Cheeke, P.R. (ed.) Toxicantsof Plant Origin, Vol. II, Glycosides. CRC Press,Boca Raton, Florida, pp. 43–60.

Troncone, R., Greco, L. and Auricchio, S. (1996)Gluten-sensitive enteropathy. Pediatric Clinicsof North America 43, 355–357.

Vachvanichsanong, P. and Lebel, L. (1997) Djenkolbeans as a cause of hematuria in children.Nephron 76, 39–42.

VanEtten, C.H. and Wolff, I.A. (1973) Natural sulfurcompounds. In: Toxicants Occurring Naturallyin Foods, 2nd edn. National Academy ofSciences, Washington, DC, pp. 210–234.

Van Veen, A.G. (1973) Toxic properties ofcertain unusual foods. In: Toxicants Occur-ring Naturally in Foods, 2nd edn. NationalAcademy of Sciences, Washington, DC,pp. 464–475.

Verkerk, R., Dekker, M. and Jongan, W.M.F. (1998)Glucosinolates. In: Watson, D.H. (ed.) NaturalToxicants in Food. Sheffield Academic Press,Sheffield, pp. 29–53.

Wakabayashi, K., Totsuka, Y., Fukutome, K., Oguri,A., Ushiyama, H. and Sugimura, T. (1997)Human exposure to mutagenic/carcinogenicheterocylic amines and comutagenic β-carbolines. Mutation Research 376, 253–259.

WHO/FAO (2002) Health implications of acryla-mide in food. Report of a Joint FAO/WHOConsultation, 25–27 June 2002. Food SafetyProgramme, WHO in collaboration with FAO.Available at: www.who.int/fsf

Xavier-Filho, J. and Campos, F.A.P. (1989)Proteinase inhibitors. In: Cheeke, P.R. (ed.)Toxicants of Plant Origin, Vol. III, Proteins andAmino Acids. CRC Press, Boca Raton, Florida,pp. 1–27.

Zhang, Z.X., Anderson, D.W., Mantel, N. andRoman, G.C. (1996) Motor neuron disease onGuam: geographic and familial occurrence,1956–85. Acta Neurologica Scandinavica 94,51–59.




2 Bacterial Pathogens and Toxins inFoodborne Disease

E.A. Johnson*Department of Food Microbiology and Toxicology, Food Research Institute,

University of Wisconsin, Madison, WI 53706, USA

Introduction

Foodborne disease mediated by pathogenicmicroorganisms or microbial toxins is animportant global public health problem.Foodborne disease has been defined by theWorld Health Organization (WHO) as ‘a dis-ease of infectious or toxic nature caused by,or thought to be caused by, the consumptionof food or water’ (World Health Organiza-tion, 1997). Foodborne disease takes a hugetoll on human health and mortality: in theUSA alone it has been estimated that micro-bial foodborne illnesses number in the mil-lions, causing several thousand deaths, withan economic burden of about $5 billion dol-lars annually (CAST, 1994; Mead et al., 1999).Globally, the WHO has estimated that approx-imately 1.5 billion episodes of diarrhoea andmore than 3 million deaths occur in childrenunder 5 years of age, and a significant pro-portion of these results from consumption offood contaminated with microbial pathogensand toxins (World Health Organization,1997). These estimates of foodborne illnessesare probably 100–300 times less than theactual occurrence for a variety of reasons(Bryan et al., 1997; Lund et al., 2000). Theannual incidence of foodborne illnesses inindustrialized countries has been estimated

to affect 5–10% of the population annually,and in many developing countries theincidence is probably considerably higher.

Foodborne diseases or illnesses arecommonly classified into two main categories:(i) infections commencing within the gastro-intestinal (GI) tract; and (ii) poisonings orintoxications resulting from consumption ofpre-formed toxins in foods. This classification,however, is overly simplistic and does nottake into account the wide spectrum offoodborne illnesses and intoxications, aswell as chronic disease syndromes that candevelop following acute foodborne infections.The classification was expanded to encom-pass five major modes of acute foodborneillness (Granum and Brynestad, 1999): (i)intoxications in which a pre-formed micro-bial toxin in a food is consumed; (ii)toxicoinfections in which a toxin is producedin the intestinal tract in the absence of adher-ence to epithelial cells in the GI tract; (iii)illnesses caused by production of an entero-toxin following adherence of pathogens toepithelial cells in the GI tract but withoutbacterial invasion of intestinal cells; (iv)illnesses caused by bacterial infection of the GItract with mucosal and intestinal cell penetra-tion and usually production of enterotoxin,but in which the infection does not become


20-Feb-03 2




systemic; and (v) systemic infectionsfollowing bacterial GI tract colonization andpenetration through the intestinal barrier.

The production and maintenance of a safefood supply depends on an understanding ofthe biological and virulence properties offood-poisoning organisms. Foods and theirsurroundings can be considered as selectiveenvironments that allow growth or survivalof certain groups of microorganisms. Someof these microorganisms can be beneficial tothe quality and safety of a food, such as manyyeasts and lactic acid bacteria, others areinnocuous, while still others are pathogensand can present safety hazards in foods. Inthis chapter, basic principles of bacterialfoodborne infections and intoxications aredescribed, which is followed by a descriptionof the aetiological agents and toxins, andstrategies for their control in foods.

History of Foodborne Disease andBeginning Concepts

Microorganisms documented to cause foodpoisoning comprise approximately 50 speciesof fungi, bacteria and viruses (reviewed inLund et al., 2000). Recognition of associationsbetween food and disease came about longbefore an understanding of microbiology,and some of the seminal events have beentraced in history to Moses, the Romans, Cato(234–194 BC), Pliny the Elder (AD 23–79), thefall of the Roman Empire, the Dark Ages,17th and 18th century England, and into themodern era (Hutt and Hutt, 1984). Mosesspoke of foods that should not be eaten bythe Israelites because of their propensity tocause illness, and he also provided advice onfood handling practices. Beginning thepre-modern era of epidemiology, the causalassociation of water and disease was realizedin the famous investigations of John Snow,who reported in 1851 that drinking watercould spread cholera, and this in turn ledto filtration methods to eliminate theunknown agent (Hobbs and Gilbert, 1978).The causative agent, Vibrio cholerae, wasnot discovered until the 1880s by RobertKoch. William Budd demonstrated in the

mid-1800s that typhoid fever could bespread by milk. These seminal events clearlyshowed an association of food and waterwith infectious diseases.

Knowledge of the actual microbial causesof foodborne disease began when Pasteur andKoch founded the science of microbiology,allowing microbiologists to isolate, character-ize and systematically describe microorgan-isms associated with spoiled or poisonousfoods (Brock, 1961; Tannahill, 1973). Upuntil this time, the organisms causing mostGI-mediated diseases were of unknownaetiology. The first description of a docu-mented food-poisoning bacterium was in1888 by Gaertner, who isolated a bacterium (aSalmonella species) from meat and the organsof a man who had died from food poisoningafter eating the contaminated food (Hobbsand Gilbert, 1978). Landmark legislation wasdenoted in the USA in the 1906 Pure Food andDrugs Act and its successor the 1938 FederalFood, Drug, and Cosmetic Act (Middlekaufand Shubik, 1989). It has become apparent thatprotecting the safety and wholesomeness offoods is an important discipline fulfilled bylegislators, industry and researchers.

The documented association of micro-organisms and food- and waterborne diseaseformed a conceptual foundation for hygiene,sanitation and food preservation. It alsocontributed significantly to the science ofepidemiology (Evans and Brachman, 1991).Foodborne disease surveillance began in theUSA in the early 1900s as a response to themorbidity and mortality caused by typhoidfever and infantile diarrhoea (Centers forDisease Control and Prevention, 2000). In1939, a public health bacteriological servicewas instituted in Great Britain, and in 1950 thePublic Health Laboratory and the Departmentof Health and Social Security pooled and tabu-lated their reports on food poisoning andthe documented disease agents (Hobbsand Gilbert, 1978). These early surveillancesystems established the foundation for epi-demiological study of foodborne diseases.Guidelines for establishing and evaluatingsurveillance systems and epidemiologicalanalyses have been described (Evans andBrachman, 1991; Bryan et al., 1997; Guzewichet al., 1997; Centers for Disease Control and

20-Feb-03 2

26 E.A. Johnson



Prevention, 2000). The availability of severalwell-designed surveillance studies (Bean andGriffin, 1990; World Health Organization,1997; Mead et al., 1999; Centers for DiseaseControl and Prevention, 2000) has been veryimportant in documenting foodborne diseaseagents and elucidating trends, changingpatterns and discovery of emerging or re-emerging pathogens. Advances in the fieldsof surveillance and epidemiology havedemonstrated the enormous impact thatfoodborne disease has on morbidity, mortal-ity and economic losses throughout the world.In developing countries, foodborne disease isamong the leading causes of morbidity andmortality, particularly among children, andhas been considered to be a leading factorimpeding technological progress (Miller andTaylor, 1989).

Virulence and Foodborne Disease

Of several thousand species of bacteria in themicrobial world (Holt, 1984–1989; Dworkin,1999; Fischetti et al., 2000; Lund et al., 2000;Madigan et al., 2000), only about 40 specieshave been documented to cause foodbornedisease. The main taxonomic groups andgenera of bacterial pathogens are presentedin Fig. 2.1. Similarly, hundreds to thousandsof bacterial species are commonly present inhuman foods, but only a few of these presenta hazard to the consumer. The occurrence infoods of certain pathogens is clearly undesir-able and may render a food unpalatable orunsafe. Bacteria vary tremendously in theirpathogenicity, or their capacity to causedisease.

The quantity of a pathogen or toxin infood required to produce illness is correlatedwith the virulence of the agent. Virulence isa term that describes the infectivity of thepathogen and the severity of the illness thatit produces. Virulence factors are thosephenotypic properties of a pathogen thatwhen lost, for example by mutation, decreasethe pathogenicity but not the viabilityunder laboratory conditions. The phenotypiccharacteristics that determine the pathogenic-ity of microorganisms can be defined by

determining the effects of mutations in certaingenes. For example, mutants of Salmonellatyphimurium impaired in their ability tosurvive within macrophages were no longervirulent when injected intraperitoneally into amouse (cited in Johnson and Pariza, 1989).These mutant bacteria were shown to havemutations in specific genes. The individualgenes and gene products were elucidated,thus defining the specific virulence factors.

The two principal classes of virulencefactors in bacteria are toxins and surface mole-cules, although other classes of moleculescan also affect virulence of many pathogens.These two main classes of virulence factorsare broad and diverse, and among foodbornepathogens they vary greatly in structure andmode of action. The primary extracellularprotein toxins causing true intoxicationsare botulinum and staphylococcal toxins,which vary markedly in properties includingstructure, mechanism, and resistance to heat,acid and proteolytic degradation. Similarly,surface molecules also include a numberof different molecules that provide variousbiological functions such as adherencefactors, capsules that resist phagocytosis andimmune responses, flagella for motility,molecules determining receptor binding andchemotaxis, and so forth. Since virulencefactors are traits that are not required forviability of the pathogen, they may beproduced in a temporal and variable manner,particularly in response to host factors. Theirproduction can vary markedly from strain tostrain, and they may be expressed at certainpoints in the growth cycle or under specificnutritional conditions. Maintenance andexpression of virulence genes depend upon abalanced genome structure in bacterial strainsand species (Relman and Falkow, 2000).Virulence is a highly polygenic property ofbacteria and has evolved to be compatiblewith the overall genome structure and physi-ology of the pathogen. Thus, the insertion ofa virulence gene into most distantly relatednon-pathogenic bacteria would not resultin the formation of an effective pathogen.Pathogens have been found to have a clonalpopulation structure, in which they carryspecific arrays of virulence-associated genes(see Fischetti et al., 2000; Relman and Falkow,

Bacterial Pathogens and Toxins in Foodborne Disease 27

20-Feb-03 2



2000). For example, although many clonallineages of Escherichia coli persist in the humanintestinal tract, only a few lineages such asE. coli O157:H7 are able to cause illness.

A variety of powerful methods usingmolecular biology have become available toidentify virulence genes and their expressionin vitro and in vivo on infection within the host(Relman and Falkow, 2000). Study of viru-lence genes has shown that they frequently are

associated with mobile genetic elements suchas bacteriophages, transposons and plasmids,and may occur in distinct chromosomalregions called pathogenicity islands. Someof these mobile elements carrying virulencegenes can be transferred horizontally to recip-ient bacteria and, in the proper environmentand particularly under genetic selection, canbe maintained in the recipient. An excellentexample of acquisition of traits beneficial to

11-Mar-03 2

28 E.A. Johnson

Fig. 2.1. A taxonomic grouping of the principal foodborne pathogens (expanded from Johnson and Pariza,1989).



pathogenicity is the transfer of genes encod-ing antibiotic resistance, enabling a normallysensitive organism to gain resistance to theanti-microbial agent. These genes can bemaintained in the absence of selection ifthe recipient contains a genome structureand physiology that enable the productiveregulation and expression of the virulence-or resistance-encoding genes. Methodsto identify virulence genes, mechanismsgoverning the expression of virulence factorsand horizontal gene transfer are an extremelyactive area of research, and excellent treatisesare available on the subject for both Gram-negative and Gram-positive bacteria (seeFischetti et al., 2000; Mandell et al., 2000,as examples).

The virulence of a pathogen or toxin,i.e. the infectious dose to cause disease orthe potency of a toxin, often is expressedquantitatively as the ID50 or LD50. Thesevalues represent the dose that infects or causesan infectious or toxic response (e.g. lethality)in 50% of a population of test animals in adesignated period of time. The ID50 and LD50

are chosen to quantify virulence or toxicitybecause of the nature of the dose–response

relationship (Fig. 2.2). The curves in the upperpanel demonstrate that the rate of change inmortality (slope of the curve) as a function ofdose reaches a maximum at the point of about50% survival. Curves with greater slopes givea more accurate estimate of toxin concentra-tion or infectious dose. The sigmoid shape ofthe ID50 or LD50 results primarily from thechance distributions of lethal events in anygiven animal, although the heterogeneity ofthe animal population may also be a factorin certain cases. The type, strain, health andother features of the animal will also influencethe shape of the curve and resulting ID50

or LD50. For these reasons, determining theID50 and LD50 is often the most appropriatemethod for determining the dose required forillness in experimental animals.

Recognition of Pathogenic BacteriaCausing Foodborne Disease

The recognition of a bacterium as an aetio-logical agent of foodborne disease usually isfirst indicated by epidemiological evidence,


11-Mar-03 2

Fig. 2.2. Examples of dose–response curves used to quantitate bacterial virulence and lethality of toxins.The infecting dose is plotted horizontally in logarithmic units. The ID50 or LD50 is calculated by extrapolatingto the dose that causes 50% infection (ID50) or toxicity (usually fatality; LD50). From Wilson and Dick (1983).



in which the occurrence of an illness in ahuman epidemic is examined and foundto correlate with the consumption of a food(Evans and Brachman, 1992). A foodborneillness outbreak occurs when two or morepeople have a similar illness after eating acommon food, and microbiological evidence,as described later in the chapter, implicatesthe food as the vehicle (an exception isbotulism, where one case is considered tobe an outbreak). The epidemiological investi-gation ideally is established by diagnosisof suspected aetiological agents from clinicalsamples and the causative food.

When a connection between a foodconsumption and disease is suggested, theinvestigator tries to satisfy the followingcriteria to demonstrate the microorganism asthe causal agent: (i) the organism is isolatedfrom the food and from the sickened host andcultured on artificial growth media; (ii) theorganism is characterized and shown to beidentical from the two sources; (iii) inocula-tion of the organism to an experimentalanimal model produces a closely similardisease; and (iv) the organism is recoveredfrom the site of infection of the animal andshown to be the same as the pathogen origi-nally inoculated. These criteria, patternedafter the famous Koch’s postulates, can beextremely useful in establishing an unrecog-nized pathogen as the aetiological agent offoodborne disease. Unfortunately, the criteriaoften cannot be satisfied because some organ-isms cannot be grown on artificial culturemedia, a suitable animal model for testing ofpathogenicity is not available, the disease iscaused by more than one pathogen or becausethe specific cause of disease is due toextracellular products of the organism, suchas toxins formed outside the host, ratherthan the organism itself. In practice, manyfoodborne disease outbreaks are diagnosedby first examining the onset time of illness andthe symptoms, and then isolating the likelyaetiological agent(s) or its toxin from the foodand clinical samples (e.g. vomitus, faeces,blood or organs) of the victim(s). The success-ful epidemiological investigation coupledwith the aetiological diagnosis can facilitateboth short- and long-term control measures.

Surveillance and Epidemiology ofFoodborne Disease

Microbial food poisoning is caused by theconsumption of a food that is contaminatedwith harmful levels of pathogenic organismsor microbial toxins. The major taxonomicgroups and genera of bacteria that arespecies that have been documented to causefoodborne disease are portrayed in Fig. 2.1.The recognition of these pathogens has comeabout through collaborative efforts of scien-tists in a variety of disciplines includingepidemiology, public health, microbiology,medicine and others. Surveillance and epi-demiological analysis often initially provideevidence of a causal relationship, and this canlead to isolation and characterization of thesuspected aetiological agent. However, someinfectious agents such as many viruses andparasites as well as prions are difficult orimpossible to culture, and diagnosis willdepend on alternative methods of detection.

Surveys of microbial pathogens and tox-ins transmitted in foods have been publishedin several useful compilations (Bryan, 1982;Bean and Griffin, 1990; World Health Organi-zation, 1997; Petersen and James, 1998; Meadet al., 1999; Centers for Disease Control andPrevention, 2000; Lund et al., 2000). Overall,most of the summaries agree in their conclu-sion that bacterial pathogens are responsiblefor the majority (> 80%) of outbreaks, casesand deaths. Members of the Enterobac-teriaceae, particularly Salmonella serovars,enteropathogenic E. coli and Shigella spp.,and members of the Campylobacteraceae,Campylobacter jejuni and C. coli, are responsi-ble for the majority (> 70%) of foodbornebacterial illnesses. Of secondary importanceare toxicoinfections by Clostridium perfringensand Bacillus cereus, intoxications by staphylo-coccal enterotoxin, B. cereus emetic toxinand botulinum neurotoxin, and infectionsby Vibrio spp., Streptococcus spp. andListeria monocytogenes. Less common food-borne pathogens in US and UK surveysinclude Aeromonas hydrophila, various speciesand strains within genera of the Entero-bacteriaceae (Citrobacter, Edwardsiella, Entero-bacter, Hafnia, Klebsiella, Yersinia and others),

20-Feb-03 2

30 E.A. Johnson



Arcobacter spp., certain Bacillus spp., Brucellaspp. and Mycobacterium spp. The two organ-isms with the highest death to case ratios are L.monocytogenes and Clostridium botulinum, butSalmonella strains (particularly typhoidalserovars and highly virulent non-typhoidalstrains), vibrios, such as Vibrio cholerae O1 andV. vulnificus, and certain other virulent bacte-rial foodborne pathogens can cause deaths.Severe illnesses and fatalities occur mostcommonly in persons with underlyinginfections or diseases, and in individualssuffering from malnutrition or immunedeficiency. Infants and the elderly are alsomore susceptible to foodborne diseases than isthe general population. Recent compilationsindicate that foodborne diseases causeapproximately 76 million illnesses, 325,000hospitalizations and 1800 deaths in the USAeach year (Mead et al., 1999). Of these, 1500deaths have been attributed to Salmonella,L. monocytogenes and Toxoplasma (Mead et al.,1999). Obviously, the magnitude is muchgreater on a global scale, but the actualincidence is difficult to assess because of thelack of surveillance systems and public healthresources in many countries.

Epidemiological investigations in indus-trialized countries have indicated that thespectrum of foodborne disease agents ischanging over time (Altekruse et al., 1997;Mead et al., 1999; Centers for Disease Controland Prevention, 2000). Formerly, the mostcommonly recognized foodborne pathogensor toxins were Salmonella, C. perfringens andstaphylococcal enterotoxin (Bryan, 1982), butthe incidence of the latter two aetiologicalagents in causing disease has decreased overtime in the USA and UK. Certain pathogensincluding antibiotic-resistant Salmonellaserovars (e.g. DT104), E. coli O157:H7, L. mono-cytogenes, parasites such as Giardia, Crypto-sporidium, Cyclospora and Toxoplasma, andhuman enteric viruses (particularly Norwalkvirus) are now among the most frequent food-transmitted pathogens (Mead et al., 1999). Thechanging spectrum is emphasized by the factthat some pathogens of greatest concern,including C. jejuni, E. coli O157:H7, L. mono-cytogenes and Cyclospora cayatenensis, were notrecognized as significant causes of foodborne

illness only 20 years ago (see compilation ofBryan, 1982). It is unknown in the absenceof thorough surveillance if such changes infoodborne disease agents are occurring glob-ally. Primary factors probably contributing tothese paradigm shifts in foodborne diseaseepidemiology are similar to changes in otherinfectious diseases (Box 2.1) (Altekruse et al.,1997; Mossel et al., 1999). It has been empha-sized that foodborne illness is vastly under-reported, not only because many of theillnesses are mild and self-limiting, andthat many illnesses have long incubationtimes and are difficult to associate with foods,but also because a proportion of the illnessesare caused by aetiological agents that cannotbe identified using available methods. Inaddition to the changes in aetiological agents,acute foodborne illnesses are now recognizedfrequently to trigger long-lasting and some-times chronic disease syndromes suchas reactive arthritis, Reiter’s syndromeand Guillain–Barré syndrome (Archer andKvenberg, 1985; Mossel et al., 1999).

Surveillance and epidemiological analy-sis of foodborne disease are limited by severalfactors. Most bacterial foodborne illnessesinvolve sporadic cases and go unnoticed sincethey occur as isolated incidences that often arenot diagnosed and reported to public health


20-Feb-03 2

Box 2.1. Factors contributing to the globalincidence of foodborne disease.

Crowding and poor sanitary conditonsDrought and famineMalnutritionChanging demographics with increasing popula-tions of infants, the elderly and the infirmInadequate public health infrastructureInadequate government involvement andlegislationInadequate pathogen surveillance and reportingsystemsEmerging foodborne pathogensAcquisition of virulence and antibiotic-resistantgenes by non-pathogenic bacteriaAdaptation and enhanced survival of pathogensin foodsLow priority of food safety by certain govern-ments and companiesInadequate education of consumer



authorities. Furthermore, chronic diseasesassociated with ingestion of bacterial patho-gens or toxins are poorly recognized becauseof the long incubation time for the diseaseprocess to occur. Thus, the reported numberof foodborne illnesses reflects a large under-estimation (100- to 300-fold) of the actualoccurrences of the food-mediated illnessesin the human population.

Bacterial Hazards in Foods

The primary aetiological agents of bacterialdisease are presented in Table 2.1, which listsgeographic range and habitats, associatedfoods, and factors affecting transmission ofthe various foodborne pathogens. The table issegregated according to the degree of bacte-rial pathogen or toxic hazard: severe hazards,

20-Feb-03 2

32 E.A. Johnson

Pathogen or toxinGeographical distributionand habitats

Associated foods and conditions contributing tooutbreaks

Severe hazardsClostridium botulinum;botulinum neurotoxin

Salmonella typhi, S.suis, S. paratyphi, S.cholera-suis (typhoidalsalmonellae)Shigella spp.

Vibrio cholerae(serogroup O1)

Vibrio vulnificus

Escherichia coliO157:H7 (EHEC)(enterohaemorrhagic)

Escherichia coli (EIEC)(enteroinvasive)

Listeria monocytogenes

Widespread; distributiondepends on serotype

Widespread; mainly problemin developing countries

Central America, Mexico,North and central Africa,Japan, South-east Asia;host adapted to humansand primatesCoastal countries in SouthAmerica, Central America,endemic in Calcutta,occasionally epidemic inAfrica, southern AsiaCoastal waters, South-eastUSA

Distribution unknown; foundin intestines of dairy cattle;found on dairy farms andcattle ranchesGeographical distributionunknown; found in intestinesof many animalsGeographical distributionlargely unknown; oftenassociated with animals; alsoisolated from silage, soil,other environmental sources

Vegetables, fruits, fermented fish, home-cannedfoods, honey (infant botulism); low acid foods(pH > 4.6); some strains grow under refrigeratedconditions; spores extremely resistant to heatand chemicalsWater, raw meats, raw milk; cells killed bypasteurization and most disinfectants

Water, vegetables, many fruits, salads, raw milk;transmission in most foods is by faecal–oralroute; resistance properties similar to Salmonella

Water, raw shellfish; spread by faecal–oral route,poor sanitation

Raw or poorly cooked shellfish and finfish;halophilic; individuals with underlying diseasessuch as cirrhosis highly susceptible; cases morefrequent in summer monthsUndercooked or raw minced beef; vegetables,fruits; lucerne sprouts; unpasteurized fruitjuices; raw milk, cheese curds; inactivatedby pasteurization and many disinfectantsUnknown, probably spread by faecal–oral route;illness similar to shigellosis (bacillary dysentery)

Raw milk, dairy products made from raw milk,ready-to-eat meats, raw vegetables, raw meat,poultry, fish, smoked fish; minimally processedrefrigerated foods

Brucella spp. Worldwide Associated with raw milk obtained from infectedherds; rare in countries that enforce herd controland adequate pasteurization of milk

Table 2.1. Bacterial pathogens causing foodborne disease in various regions of the world. The agentsare listed according to severity of hazard (modified and expanded from National Research Council,1985).




11-Mar-03 2



Mycobacterium spp. Worldwide Associated with raw milk obtained from infectedherds; rare in countries that enforce herd controland adequate pasteurization of milk

Moderate hazards, potentially extensive spreadSalmonella serovars(non-typhoidal)

MiscellaneousEnterobacteriaceae

Campylobacter jejuni,C. coli

Escherichia coli (EPEC)(enteropathogenic)

Escherichia coli (ETEC)(enterotoxigenic)

Streptococcuspyogenes (group A)

Worldwide; frequentlyassociated with animals,particularly poultry andpigs

Worldwide; frequentlyassociated with animals,but many species also foundfreeliving or associated withplantsProbably worldwide;associated with animals,particularly poultry, but alsocattle, flies, other unknownvectorsUnknown distribution;isolated from humans,cattle, pigs

Probably worldwide;seems more prevalentin developing countries;has caused cruise shipdiarrhoeal episodes

Probably worldwide; mainreservoir is the humanoral–nasal mucosa, alsofound in pus, on skin,freeliving in the environment

Raw meats, poultry, fish and shellfish, raweggs, a variety of other foods wherecontamination with raw animal products canoccur; inactivated by pasteurization and mostdisinfectantsRaw milk, raw meats, shellfish, vegetables, fruits

Poultry, raw milk; readily inactivated bypasteurization and disinfectants

Foodborne outbreaks appear to be rare;frequent cause of infantile diarrhoea,particularly in developing countries; potential foodvectors are raw beef and poultry, butmost outbreaks probably involve faecal–oraltransmissionFrequently causes diarrhoea in infants and also‘traveller’s’ diarrhoea; large infectious doseneeded; food vehicles have included those thatcontact contaminated water such as salads; alsoassociated with unpasteurized milk and dairyproductsVirulent strains cause septic pharyngitis andscarlet fever; also can induce moderate to severeinflammatory responses; like mostGram-positives, streptococci are more resistantthan are Gram-negatives to heat anddisinfectants. Food vehicles have includedsalads, raw milk, ice cream, custards, eggs and avariety of other foods that were allowed to standat warm or ambient temperatures for severalhours; food handlers frequently have pharyngitis

Moderate hazards, limited spreadAeromonas hydrophila

Vibrio cholerae(serogroup non-O1)

Present in freshwaterenvironments andassociated fish, amphibiansand animalsInhabitant of marine andestuarine waters andsediments

Food vehicles have included fish and shellfish;also found in meats and poultry; aetiology notwell understood

Food vehicles are commonly raw, improperlycooked or recontaminated shellfish

continued

Table 2.1. Continued.



moderate hazards with potentially extensivespread, and moderate hazards with limitedspread (National Research Council, 1985).This information should be useful inestablishing microbiological criteria andfor developing HACCP (Hazard Analysisand Critical Control Point) and Food SafetyObjective (FSO) programmes.

The taxonomic and biological character-istics of the various bacterial foodborne patho-gens recently have been reviewed extensivelyin several definitive treatises (Holt, 1986;Blaser et al., 1995; Mossel et al., 1995; Inter-national Commission of the MicrobiologicalSpecifications for Foods, 1996; Collier et al.,1998; Dworkin, 1999; Fischetti et al., 2000;Lund et al., 2000; Mandell et al., 2000; Downesand Ito, 2001). The reader is referred to thesetreatises for biological descriptions of theknown foodborne pathogens. Methods forisolation of bacterial pathogens and determi-nation of toxins have also been published in

excellent manuals and compendia (Food andDrug Administration, 1995; Downes and Ito,2001). These treatises describe necessary sam-pling plans, sample collection and methodsfor analysis of pathogenic bacteria in specificfoods. Other important aspects, including lab-oratory quality assurance, molecular typingand differentiation, and rapid methods, arealso described. Safety guidelines for workingwith pathogens and toxins are also available(Fleming and Hunt, 2000). Physicians’ guide-lines for diagnosis, treatment and reporting offoodborne illnesses were published recently(Centers for Disease Control and Prevention,2001b).

Foods as selective ecological environ-ments influencing bacterial growth and sur-vival have been aptly described (Mossel andIngram, 1955; Mossel et al., 1995; InternationalCommission of the Microbiological Specifica-tions for Foods, 1996). The factors governinggrowth and survival of bacterial pathogens in

11-Mar-03 2

34 E.A. Johnson



Vibrio parahaemolyticus

Yersinia enterocolitica,Y. pseudotuberculosis

Streptococcus spp.(group D)

Staphylococcus aureus

Clostridium perfringens

Bacillus cereus; otherBacillus spp.

Inhabitant of estuarine andmarine environments

Probably worldwide; oftenisolated from pigs, birds,pets

See description for S.pyogenes

Worldwide; diminishing indeveloped countries;commonly associated withpimples, boils on skin,mucous membranes ofhumansWorldwide; diminishing indeveloped countries; sporesare widely distributedthroughout the worldProbably worldwide; sporesresistant to environmentalconditions

Food vehicles are usually raw, improperly cookedor recontaminated shellfish; most outbreaksoccur during summer monthsCommonly associated with meats, especiallypork, also beef, lamb, others; outbreaks haveoccurred in improperly pasteurized milk, tofu;symptoms can mimic appendicitisFood vehicles have included salads, and avariety or foods that were left at ambient or warmtemperatures for several hoursHam, poultry, salads, pastries, other foods thatwere left at ambient or warm temperatures forseveral hours; many strains tolerate highosmotic conditions such as relatively high saltand sugar; outbreaks caused by toxin causingemesisBeef, poultry, casseroles, foods cooked in bulkand cooled insufficiently; can cause fatalities inelderly individuals

Meats, vegetables, casseroles and other foodscooked in bulk and improperly cooled areassociated with diarrhoeal illness; emeticillness nearly always associated with fried riceor rice dishes and less frequently with pastadishes




foods include the physical, chemical andnutritional composition of the food (intrinsicfactors) and factors external to the foods(extrinsic factors) (Mossel and Ingram, 1955;Mossel et al., 1995). The primary intrinsicfactors include hydrogen ion concentration(pH), water activity (aw), redox potential (Eh),nutrients and antimicrobial constituents. Themajor extrinsic parameters include tempera-ture, gaseous atmosphere and relative humid-ity. The effects of intrinsic and extrinsic factorson the growth and survival of foodbornepathogens in a variety of buffers, mediaand foods have been tabulated extensively(Mitscherlich and Marth, 1984; Mossel et al.,1995; International Commission of the Micro-biological Specifications for Foods, 1996). Anunderstanding of the effects of intrinsic andextrinsic parameters on bacterial foodbornepathogens has also facilitated the develop-ment of predictive models for assessmentof growth in various media and foods(summarized in Lund et al., 2000). Intrinsicand extrinsic factors interact in their effects ongrowth and survival of foodborne pathogens,and thus a combination of inhibitory factorsat sublethal concentrations is often morepractical for control of pathogens in foodsthan the use of lethal levels of a single para-meter. Since foods are complex ecosystems, itis often desired or necessary to conduct actualchallenge studies in which the foodbornepathogen is inoculated to the food and growthand survival are monitored. Challenge testsare especially useful in foods that are reformu-lated or processed by newer preservationtechniques (Rahman, 1999; Glass and John-son, 2001).

Foodborne pathogens vary considerablyin their association with certain foods. Inrecent years, some bacterial pathogens havebeen linked to foods, including Campylobacter(milk and poultry), E. coli O157:H7 (groundmeats, unpasteurized apple cider and cheesecurds), Salmonella (eggs, fruits and vegetables),and L. monocytogenes (raw milk, minimallyprocessed and ready-to-eat meats). Severalfoodborne bacterial pathogens such as C.perfringens or B. cereus must grow in foodsto very high numbers (> 108–109) in order toevoke illness, while certain other pathogenssuch as E. coli O157:H7 or Shigella spp. can

evoke illness through ingestion of only a fewcells. The requirement for a high infectivedose implies that the pathogen must eitherbe capable of successfully growing to highnumbers in the food or is introduced in highnumbers to the food by gross contaminationprior to consumption in order to cause illness.In the case of those pathogens causing ill-nesses by only a few cells, limited growth orsurvival of a small number of contaminants issufficient to elicit illness. The infectious dosesof various foodborne pathogens as well as theonset time to illness, clinical symptoms andduration of the illness have been reviewedin several treatises (Bryan, 1982; Blaser et al.,1995; Collier et al., 1998; Fischetti et al., 2000;Lund et al., 2000; Mandell et al., 2000; Centersfor Disease Control and Prevention, 2001a,b)and are summarized briefly in Table 2.2.

General Strategies for PathogenDetection in Foods

The microbiological analysis of foods strivesfor accuracy and reproducibility of pathogennumbers obtained in the food samples. Theaccuracy of the test depends on analysis ofa suitable number of replicate samples andfrom different lots of the test material.Sampling and validation plans for micro-biological testing have been described(National Academy of Sciences, 1985; Mosselet al., 1995; Lund et al., 2000).

The following section describes generalapproaches for the isolation of foodbornepathogens from foods, with an emphasison the Enterobacteriaceae since their isolationhas been studied most extensively and theycause the highest incidence of bacterial-mediated foodborne disease. Other groups offoodborne pathogens (Fig. 2.1 and Table 2.1)are isolated using similar strategies. When iso-lating Enterobacteriaceae and other bacteriafrom most foods or clinical samples (stools,vomitus, occasionally blood or internalorgans), it is usually necessary to use selectivemedia because of the presence of greater num-bers of non-pathogenic flora in the food orclinical samples. Culture media are madeselective by the inclusion of specific inhibitors


20-Feb-03 2



such as antibiotics, which inhibit unwantedbacteria but do not inhibit growth of thepathogen. For example, selective media usedto recover pathogenic Enterobacteriaceaefrom stools or foods are designed to inhibitgrowth of Gram-positive bacteria and to slowthe growth of undesired enterobacteria. Thisis accomplished by taking advantage of theproperty that the enterobacteria are moreresistant than Gram-positive bacteria to inhi-bition by certain dyes (e.g. brilliant green) andsurfactant compounds (e.g. bile salts). Mediadesigned to selectively promote growth of thepathogens greatly facilitate their isolation.Within the enterobacteria, further advantageis taken of the property that the pathogenicgenera, such as Salmonella and Shigella, aremore resistant than non-pathogens to the

metal chelator citrate; therefore, media con-taining both citrate and bile salts (Salmo-nella/Shigella agar) can be used for selectiveisolation of pathogenic species from heavilypopulated samples such as faeces, sewage, ormany raw or minimally processed foods. Itmay be necessary to use non-selective mediain certain analyseswhere cellsmaybe stressedor injured. Recovery of injured cells often ispossible only when the flora in the food orclinical sample is low compared with thetarget organisms. In order to recover certainpathogens that may be present in low num-bers in the stools of carriers, an enrichmentbroth may be used which preferentiallyenhances growth of the pathogens presentrelative to the normal flora. Enrichmentmedia can also facilitate recovery of injured

11-Mar-03 2

36 E.A. Johnson

OrganismIncubationtime

Infectivedose Symptomsa Duration

A. IntoxicationsBacillus cereus (emetic)Clostridium botulinumStaphylococcus aureus

1–6 h12–72 h1–6 h

NA~1 µg100–200 ng

NVNeurologicalNVD

6–24 hWeeks to months8–24 h

B. Toxicoinfections in which the enterotoxin is produced in the intestine without infection of intestinal cellsBacillus cereus (diarrhoeal type)Clostridium perfringens

6–12 h8–16 h

105–107

107–108ADAND (F)

12–24 h16–24 h

C. Infections in which enterotoxins are produced after bacterial adherence to epithelial cells but withoutinvasion into the cells

Aeromonas spp.Escherichia coli

ETEC (ST)ETEC (LT)EHEC (O157:H7)

Vibrio CholeraeVibrio parahaemolyticus

6–48 h

16–48 h16–48 h1–7 days2–5 days3–76 h

103–108

105–108

105–107

10108

105–107

DA (F)

D (AVF)D (AVF)DAB (H)DA (V)DA (NVF)

14–30 h

1–2 days1–3 daysDays–weeks4–6 days3–7 days

D. Infections in which bacterial invasion generally is localized to the epithelial cells and intestinal immunesystem

Campylobacter jejuni/coliSalmonella spp. (non-typhoidal)Shigella spp.Yersinia enterocolitica

3–8 days6–72 days1–7 days3–5 days

≥103

103–106

103–104

103–107

FADBDAF (VH)AFDB (HNV)FDA (VH)

Several days to weeks2–7 daysDays–weeksWeeks

D. Infections that often lead to systemic and organ invasionListeria monocytogenesSalmonella typhiSalmonella paratyphi

Days–weeks10–21 days10–21 days

103–108

1–102

1–102

SystemicSystemicSystemic

WeeksWeeksWeeks

aSymptom abbreviations: A, abdominal pain; H, headache; B, bloody diarrhoea; N, nausea; D, diarrhoea;V, vomiting; F, fever.

Table 2.2. Properties of the primary food poisoning bacteria. Modified from Granum and Brynestad(1999).



or stressed pathogens, which are probablypresent at higher numbers than healthy cellsin many foods, clinical samples and food plantenvironments. Specific media and techniquesfor recovery, enrichment and isolation ofpathogens are described in several technicalmanuals (Holt, 1984–1989; Atlas, 1995; Foodand Drug Administration, 1995; Downes andIto, 2001). The choice of media and conditionsfor recovery and isolation depends onthe samples available for testing and on thepreference and personal experience of theinvestigator. Following the primary isolationand purification of the pathogen generally byisolation of single colonies, further character-ization of the species is performed to differen-tiate the isolate from related bacteria (Holt,1984–1989; Food and Drug Administration,1995; Downes and Ito, 2001). Differentialmedia and test platforms for characterizingpathogens have been designed to discernimportant diagnostic characters. The goal ofthe investigator is to identify the pathogenicorganisms from clinical and food samplesas accurately and rapidly as possible. Theprocedures and interpretation of varioustests are described in diagnostic manuals(Holt, 1984–1989; Food and Drug Administra-tion, 1995; Downes and Ito, 2001).

An important method for character-ization of certain Enterobacteriaceae is

serological analysis of cell surface antigens,which is often used as a final method ofidentification and typing (see Goodfellow andO’Donnell, 1993). Antisera to surface antigenshave been used for identification of manyspecies of bacteria for nearly a century, andit has been useful for strain delineationin several Enterobacteriaceae includingSalmonella and pathogenic E. coli. Threeclasses of surface antigens (H, O, and Vi or K)(Fig. 2.3) have been used as the fundamentalserotyping antigens for Salmonella and E. colibecause of considerable variation in theirstructure, their association with virulenceand their strong antigenicity. Motile speciesof the Enterobacteriaceae possess flagellar(H) antigens, which owe their antigenicity toa heat-labile protein termed flagellin. Certainspecies also contain O antigens, commonlycalled the somatic or cell wall antigen, whichcomprises part of the lipopolysaccharidein the outermost layer of the outer mem-brane. O antigenic analysis by bacterialagglutination separates the genus Salmonellainto more than 1000 distinct serotypes, andE. coli into 173 distinct serotypes. The Oantigens in the Enterobacteriaceae, par-ticularly typhoidal salmonellae, frequentlyare covered by the Vi (capsular virulenceantigen in Salmonella) or the K (capsularantigen in E. coli).


20-Feb-03 2

Fig. 2.3. Designation of the major antigens, O, H and K/Vi, used in serotyping enteric pathogens.



A serotyping scheme for the O, H and Kantigens has been adopted internationally forcharacterization of E. coli. The scheme at pres-ent includes O antigens (1–173), K antigens(1–103) and H antigens (1–56). The number ofpossible combinations of these obviously isenormous. In practice, it is considered neces-sary only to determine the O and H antigens inorder to designate virulent strains of E. coliinvolved in food poisoning. Serotyping isalso useful to distinguish virulent strainsof other pathogenic Enterobacteriaceae, butcertain problems in methodology and inter-pretation of the results can be encountered,and researchers should refer to comprehen-sive treatises (Blaser et al., 1995; Mandell et al.,2000). Analogous strategies are used forcharacterization of surface antigens of severalother genera and species of Gram-negativeand Gram-positive foodborne pathogens(Holt, 1984–1989; Fischetti et al., 2000; Lundet al., 2000).

Rapid Detection of FoodbornePathogens

The diagnosis and prevention of foodbornedisease can be greatly facilitated by rapidmethods that allow identification of patho-gens within a few hours (for recent reviewssee Lund et al., 2000; Downes and Ito, 2001).Methods for rapid isolation and identificationof pathogens or toxins generally are based onnucleic acid or analogous probes that reactwith signature regions in the genetic materialof the organism (DNA or RNA), or onantibodies that can detect specific proteinantigens characteristic of the pathogen (seeLund et al., 2000; Downes and Ito, 2001).Polymerase chain reaction (PCR) has beenused for the detection of signature sequencesof pathogens either following enrichment ordirectly in clinical or food samples. Obstaclesto the use of PCR include inhibition of theamplification reaction by components infoods or clinical samples, and the propertythat PCR can amplify DNA samples presentfree in the food or in dead organisms. Thus,PCR and certain other sensitive and rapidmethods often can only provide presumptive

identification, and confirmation by culturalmethods and phenotypic tests is needed.Antibody-based methods, while theoreticallynot as sensitive as PCR and some otherDNA-based methods, can be useful fordetection of toxins or other protein antigensproduced by the pathogen. Protein detectionoften is performed by enzyme-linkedimmunosorbent assay (ELISA), which allowstesting of multiple samples. Since biologicallyinactive antigens can be detected withantibodies, again the test often is onlypresumptive in its utility.

The field of rapid methods is becomingincreasingly important in food microbiology,and is increasing in sophistication and inno-vation as microbiologists work with molecu-lar biologists and engineers in devising newrapid methods. Newer technologies suchas microfluidics, molecular imprinting andreceptor-based assays currently are beingevaluated as detection methods for bacterialpathogens and toxins. As genomic sequencesof foodborne pathogens increasingly becomeavailable, novel identification methods basedon signature genomic sequences will bedeveloped. Knowledge of genomic sequenceswill also facilitate epidemiological studies ofoutbreaks and tracebacks, since the methodsused could be simpler, more rapid and moreeasily interpreted than currently used meth-ods such as pulsed-field gel electrophoresis(PFGE) (Swaminathan et al., 2001).

Toxins of Foodborne Pathogens

Most bacterial foodborne pathogens producetoxins that are involved in the disease process(Table 2.3). Certain toxins are produced infoods, such as botulinum neurotoxins, staphy-lococcal enterotoxins and B. cereus emetictoxin. Ingestion of these pre-formed toxins issufficient to cause symptoms in the absenceof the producer organism. Since the directingestion can cause symptoms, the onset timecan be quite rapid, typically 1–6 h for staphy-lococcal enterotoxins or B. cereus emetic toxin.The onset of botulism symptoms after inges-tion of botulinum neurotoxin generallyoccurs after 12–36 h, but symptoms have

20-Feb-03 2

38 E.A. Johnson



occurred as early as 6–8 h or as late as 1–2weeks. The longer onset of botulism com-pared with S. aureus and B. cereus emeticintoxications reflects the need for traffickingof the toxin across the intestinal barrier, itstransport in the blood to nerves, the entryprocess into the nerves and its proteolyticaction on neuronal substrates. Secondly,toxins are produced on entrance into the gutwithout establishing infection (C. perfringensenterotoxin and B. cereus diarrhoeal toxin).Thirdly, certain toxins are produced on bind-ing to intestinal cells or during penetrationinto tissues. In the case of enterotoxin forma-tion by C. perfringens and B. cereus, since theseorganisms do not need to establish aninfection to elicit toxin, the incubation timeis generally less than that of infectious patho-gens, and typically symptoms are observedabout 10–18 h after ingestion comparedwith 12–50 h for most infectious pathogens.The properties of toxins from foodbornepathogens are summarized in Table 2.3.

Recognition and Treatment ofFoodborne Illnesses

Guidelines for physicians and public healthworkers for the diagnosis and treatment offoodborne illnesses have been publishedrecently (Centers for Disease Control andPrevention, 2001b). Most patients, but notall, typically present with GI tract symptomssuch as vomiting, diarrhoea and abdominalpain. However, somatic symptoms appar-ently unrelated to GI distress may presentin certain patients, including neurologicalsymptoms in cases of botulism caused byingestion of botulinum toxin. The firstrecognized patient is referred to as theindex case, often with exacerbated symp-toms, which may allow the physician to makean early diagnosis enabling rapid treatmentof other patients and to prevent the illnessfrom spreading.

Several key features can provide clues inelucidating foodborne illness aetiology: the


11-Mar-03 2

Producer organismof toxin Toxin nature

Heat labile (L)/orheat stable (S) Mode of action

Bacillus cereus (emetic)

Bacillus cereus (diarrhoeal)

Clostridium botulinum

Clostridium perfringens

Staphylococcus aureus

Cereulide; small peptide,1.2 kDa

Two–three components;structure not fullycharacterizedPotent neurotoxin (NT);seven serotypes; NT is~ 150 kDa; forms stablecomplex with non-toxicproteins in culture andfoods

Protein toxin of ~35.3 kDa

Proteins; 26–29 kDa;seven serotypes

S

L

L

L

S

Binds to 5-HT3 cells; causesemesis by action on nervusvagusReceptor unknown; causeshaemolysis and/or cytolysis

Binds to gangliosides andputative protein receptor;enters nerve cells byendocytosis and cleavesneuronal proteins involved invesicular trafficking andneurotransmitter releaseBinds to 22 kDa proteins inintestinal cells and causespore formationBinds to TCRVb cells or to Tcells causing emetic or potentsuperantigen responses,respectively

aThe table covers toxins that are pre-formed in foods or elicited in the gut and does not include toxinsthat may be formed during intestinal and/or septic infections.

Table 2.3. Properties of the primary foodborne toxins causing intoxications or toxicoinfectionsa.Modified from Granum and Brynestad (1999).



incubation period; duration of illness; pre-dominant symptoms; and the populationinvolved in the outbreak (Centers for DiseaseControl and Prevention, 2001a,b). Also thehealth care provider should query the indexcase and later cases as to whether the patientshave consumed raw or poorly cooked foodssometimes known to harbour pathogens, suchas eggs, meats, shellfish, unpasteurized milkor juices, fresh produce, home-canned foodsor soft cheeses from unpasteurized milk (seeTable 2.1). Additional questions regardingforeign travel, contact with pets or exotic ani-mals, attendance at picnics or group events,and similar symptoms being experienced bythe patient’s family or close circle can alsoprovide clues as to the aetiology of the illness.Since certain foodborne illnesses involvingneurological symptoms such as botulismand shellfish poisoning can be particularlylife-threatening, a diagnosis should ideallybe made quickly and life support measures(e.g. respiratory assistance, administration ofantitoxin) should be considered.

When a foodborne illness is suspected,appropriate clinical samples including faeces,vomitus (occasionally serum) and likely foodsshould be submitted to state or local healthdepartments for clinical microbiology testing.The public health authorities often can assistin investigation of the epidemiology of theoutbreak, questioning individuals who mayhave eaten the same food or at the samelocation, and collection of suspect foods formicrobial analysis. Rapid identification of anaetiological agent as a cause of foodborne ill-ness can prevent spread of an outbreak. Some-times specimens must be submitted to special-ized laboratories for special testing of agentssuch as botulinum toxin, or rapid diagnosis ofcertain aetiological agents. For example, in theUSA, a specialized and highly competent andexperienced laboratory is responsible for test-ing of all clinical specimens or food samplessuspected of containing botulinum toxin.

Reporting of foodborne illness outbreaksis an important component of an investiga-tion, as it can detect trends in foodborneillnesses and can also lead to the recognitionof previously unrecognized (emerging)

or re-emerging pathogens or toxins. Inthe USA, the local and state healthdepartments are generally responsible forreporting to the Centers for Disease Controland Prevention (CDC) but the physiciansshould also report suspected foodborneillnesses to the local and state health depart-ments. The data are compiled and dissemi-nated to the public through publication andthe Internet (Centers for Disease Control andPrevention, 2001a,b).

The CDC, in cooperation with severalstate health department laboratories, estab-lished and coordinates a national molecularsubtyping network called PulseNet forfoodborne disease surveillance and epi-demiological purposes (Swaminathan et al.,2001). The system uses standardized PFGEto characterize restriction fragment lengthpolymorphisms in DNA extracted fromclinical and food isolates of various patho-gens including E. coli O157:H7, non-typhoidalSalmonella serotypes, L. monocytogenes andShigella. It is anticipated that other bacterial,viral and parasitic organisms will be added tothe system in the near future (Swaminathanet al., 2001). Subtyping has facilitated theidentification of outbreaks and linked theclinical isolates with those in suspect foods,thus providing strong proof for involvementof genetically similar pathogens in foodborneoutbreaks, even when the clinical and foodisolates are from geographically distinctregions. Since in the majority of foodborneillness cases, most ill persons do not recall alikely food or water source for their infection,and foods can be distributed rapidly amongstates and countries, the PulseNet systemhas provided a valuable system to linkfood vehicles and patients in outbreaks andto identify virulent strains in sporadic andisolated clinical cases. Although molecularsubtyping by PulseNet or other methods suchas rDNA analyses are valuable in detectingfoodborne outbreaks and facilitating investi-gation and implementation of public healthprotective measures, the subtyping methodsare an adjunct to and not a replacement formore traditional epidemiological investiga-tions (Swaminathan et al., 2001).

20-Feb-03 2

40 E.A. Johnson



Control of Bacterial Foodborne Diseases

The prevention of bacterial foodborne dis-eases relies on proper handling proceduresof foods, adequate quality and preventiveprogrammes, good sanitation and hygiene,and many other factors (for reviews, seeLund et al., 2000). Raw foods and processedlow-acid (equilibrium pH > 4.6) foods thatdo not reach commercial sterility shouldbe promptly refrigerated to ≤ 5°C (40°F).Foods should be cooked or heated to aninternal temperature of at least 72°C(160°F). Raw milk should be pasteurized.Cross-contamination of raw foods andcooked foods must be avoided by separatingraw and cooked areas in the home, food pro-cessing plants, and in retail and food serviceoperations. Additional precautions includeadequate sewage disposal, prevention ofwater contamination in preharvest andpostharvest foodhandling facilities, and rig-orous avoidance of carriers as food handlers.The eradication of Salmonella from the foodsupply is not likely to occur in the near futurebecause it is extremely difficult to eliminatethe many animal reservoirs of this pathogen.Risk assessment-based approaches such asHACCP and FSO programmes and otherpreventive programmes involving criticalcontrol points and microbiological criteriahave proved useful in control of food-borne disease. Good Manufacturing Practices(GMP), quality systems, and adequatesanitation and hygiene are essential inreducing the incidence of foodborne disease(reviewed in Lund et al., 2000).

The primary goal of food processing is toimprove the microbial safety and quality offoods by destroying pathogenic and spoilagemicroorganisms and associated toxins. Tradi-tionally, the most common method of celland spore inactivation involves thermal pro-cessing. Pasteurization (≥ 70°C for ≥ 15 s) orits equivalent will destroy most vegetativepathogens but not spores of most speciesof food-related bacteria such as C. botulinum,C. perfringens and B. cereus. Temperaturesexceeding 100°C are necessary to inactivatespores (Downes and Ito, 2001), which canbe accomplished in pressurized retorts or

other thermal processing systems. Otherpreservation strategies such as high-pressuretreatment of foods, aseptic processing,electropasteurization, irradiation, UV lightand other technologies are gradually beingevaluated and implemented to enhancequality and safety.

Considerable research has been con-ducted on the inactivation of vegetativepathogens and endospores by traditionaland alternative processes (see Rahman,1999; Lund et al., 2000), but much lessinformation is available on their effectson toxins. Pasteurization will not inactivateheat-resistant toxins including B. cereus emetictoxin and staphylococcal enterotoxins.Botulinum neurotoxins are heat labile andare inactivated rapidly at pasteurizationor boiling temperatures. The enterotoxinsfrom Salmonella, E. coli (LT), Campylobacter,C. perfringens and B. cereus are inactivatedat temperatures exceeding 70°C, while theenterotoxins from E. coli (ST) and Yersiniaenterocolitica are heat resistant to tempera-tures exceeding 100°C. However, it isunlikely that the consumption of enterotoxinsin foods could lead to human illness in theabsence of the toxin-producing pathogen.Research is needed to determine the resistanceof bacterial toxins, particularly staphylococcalenterotoxin, Bacillus toxins and botulinumneurotoxin, to alternative methods of foodprocessing such as pulsed electric fields, highpressure, γ-irradiation and light (Rahman,1999).

Future Issues and Perspectives

Since diarrhoeal diseases are not pleasantand perhaps remind us of our vulnerabilities,the impact of foodborne diseases is probablyunderappreciated (McNeil, 1976), andincreased public health and research effortsare not supported to the extent needed fortheir control. Recommendations have beenmade for improved control of foodbornedisease in the USA (CAST, 1994). Althoughthe recommendations were directed towardscontrol in the USA, they would be applicablefor control of foodborne disease in many


20-Feb-03 2



other countries. The recommendations werecategorized into four areas: (i) goal setting;(ii) research needs; (iii) production control;and (iv) education. In goal setting, it wasemphasized that food safety policy andregulations should be based on risk assess-ment, risk management and risk communica-tion. The risk analysis approach proposedis consistent with those used by the CodexAlimentarius and National Academy ofSciences (National Research Council, 1985).For example, risk analysis can identifythe probability of particular foods trans-mitting foodborne disease, such as Salmonellaenteritidis in raw shell eggs, Vibrio spp. in rawoysters and L. monocytogenes in ready-to-eatmeats. Facets of risk-base analysis include theseverity of the hazards, risks in particularfoods, severity of the disease producedand its consequences, dose response of theaetiological agent and management options.Research needs for decreasing foodbornedisease include expanding epidemiologicaland food safety information to provide morecomplete assessments of the incidence offoodborne disease. As with goal setting, theresults from such an analysis will varydepending on cultural and technologicalpractices of various countries. Anotherresearch area deemed of high priority was tosupport studies of chronic illnesses resultingfrom acute exposure to foodborne agents(Mossel et al., 1999).

Basic and applied research areas ofmicrobiology deemed critical for control offoodborne disease included enhanced knowl-edge in the following areas: (i) microbialecology of pathogenic bacteria in pre- andpostharvest environments; (ii) mechanismsof tolerance of foodborne pathogens to acid,heat, and other processing, sanitation andenvironmental conditions; (iii) mechanismsof virulence in pathogens, genetic transferof virulence determinants and the impact ofenvironmental conditions on expressionof virulence; (iv) development of innovativeprocedures and technologies to eliminate orcontrol pathogens and their toxins in pre- andpostharvest environments; (v) improvementsin strategies and methods to track pathogensin the environment and in epidemiologicalinvestigations; and (vi) development of rapid,

accurate and sensitive methods to detectpathogens from various sources.

In the quickly developing field of rapiddetection, it was realized that genome-baseddetection methods show tremendouspotential for assessment of virulence anddetection of pathogens. Rapid detection couldbe used for online assays of pathogens inprocess flow for monitoring of pathogenlevels in HACCP or other preventive systems.Under the recommendation of productioncontrol, it was emphasized that producersshould adopt effective intervention strategies,and apply control practices from food sourceto consumption. The harmonization of inter-national food safety standards was consid-ered to be of increasing importance taking intoaccount increases in global trading of foodand differences in food safety standardsamong countries. Lastly, education of thepublic and food safety professionals wasconsidered an important area to decreasefoodborne disease. In particular, the educa-tion of high-risk populations regardingfoodborne pathogen risks was considered ahigh priority.

Conclusions

The provision of a nutritious and safe foodsupply is an essential goal of society to ensurethe health and survival of humankindthroughout the world (see Middlekauff andShubik, 1989). Epidemiological evidence hasindicated that certain food-associated bacte-ria and their toxins are the major source ofillness and mortality transmitted by foods.In addition to the importance of pathogenicbacteria in human health, they are alsoimportant in the acceptance of foods. Surveyshave indicated that most consumers are moreconcerned about microbiological hazardsthan any other area, including the presenceof pesticide residues in foods and use of anti-biotics and hormones in animal production(see World Health Organization, 1997; Lundet al., 2000).

Control of microbial foodborne disease isextremely difficult due to a myriad of factors(Box 2.1). Bacterial foodborne pathogens are

20-Feb-03 2

42 E.A. Johnson



constantly changing and elusive to detectionand control, as highlighted by the emergenceof foodborne pathogens such as E. coliO157:H7, antibiotic-resistant Salmonella spp.and L. monocytogenes. The need for maintain-ing a safe food supply in the face of adversitieshas created a need for new technologies suchas biotechnology and advanced preservationmethods to provide a supply of safe andnutritious food. Certain of these novelpreservation technologies (Rahman, 1999) arecurrently being evaluated as potentialadjuncts to or replacements for traditionalmethods of preservation such as thermaltreatments and formulation of foods forsafety. Genomics and proteomics of food-borne bacteria appear to have been largelyneglected with regard to food safety, but thesefields could provide tremendous advances intechnologies to enhance food safety.

Although microbial food safety is a majorpublic health issue of increasing importance,many public health authorities in certaincountries throughout the world do notadequately appreciate its importance forhuman health and economic development(World Health Organization, 1997). Nationaland international programmes frequently areconsidered a low priority within govern-ments. Many countries have not developedlegislation and public health infrastructure tocontrol foodborne disease. Even certain foodcompanies do not consider food safety a highpriority, and legislation is needed to enforcethe production of safe food. On the otherhand, certain multinational companies havetaken a lead role to enhance the safety of thefood supply. Although consumers are integralin the prevention of foodborne disease, manyare unaware of their importance in enhancingfood safety and do not receive adequateeducation to prevent illnesses within thehome or at community events. As emphasizedby the WHO (1997), strategies for decreasingthe incidence of foodborne disease, enhanc-ing human well being, and facilitating tech-nology developments will require a sharedresponsibility among governments, industry,scholarly institutions and consumers toaccomplish these goals.

Acknowledgements

This contribution was supported by a grantfrom the USDA and sponsors of the FoodResearch Institute, University of Wisconsin,Madison, Wisconsin.

References

Altekruse, S.F., Cohen, M.L. and Swerdlow, D.L.(1997) Emerging foodborne diseases. EmergingInfectious Diseases 3, 285–293.

Archer, D.L. and Kvenberg, J.E. (1985) Incidenceand cost of foodborne diarrheal disease in theUnited States. Journal of Food Protection 48,887–894.

Atlas, R.M. (1995) Handbook of Microbiological Mediafor the Examination of Food. CRC Press, BocaRaton, Florida.

Bean, N.H. and Griffin, P.M. (1990) Foodbornedisease outreaks in the United States,1973–1987: pathogens, vehicles, and trends.Journal of Food Protection 53, 804–817.

Blaser, M.J., Smith, P.D., Ravdin, J.I., Greenberg,H.B. and Guerrant, R.L. (eds) (1995) Infectionsof the Gastrointestinal Tract. Raven Press, NewYork.

Brock, T.D. (1961) Milestones in Microbiology.Prentice Hall, Englewood Cliffs, New Jersey.

Bryan, F.L. (1982) Diseases Transmitted by Foods. AClassification and Summary, 2nd edn. Centersfor Disease Control, Atlanta, Georgia.

Bryan, F.L., Guzewich, J.J. and Todd, E.C.D. (1997)Surveillance of foodborne disease II. Summaryand presentation of descriptive data andepidemiologic patterns: their value and limita-tions. Journal of Food Protection 60, 567–578.

CAST (Council of Agricultural Science andTechnology) (1994) Foodborne Pathogens: Risksand Consequences. Task Force Report No. 122,Council of Agricultural Science and Technol-ogy, Ames, Iowa.

Centers for Disease Control and Prevention (CDC)(2000) Surveillance of foodborne disease out-breaks, 1993–1997. Supplemement to Morbidityand Mortality Weekly Reports 49 (No. SS-1).Centers for Disease Control, Atlanta, Georgia.

Centers for Disease Control and Prevention (2001a)Summary of notifiable diseases, United States1999. Morbidity and Mortality Weekly Reports 48.Centers for Disease Control, Atlanta, Georgia.

Centers for Disease Control and Prevention (2001b)Diagnosis and management of foodborne


11-Mar-03 2



illnesses: a primer for physicians. Supplementto Morbidity and Mortality Weekly Reports50 (No. RR-2). Centers for Disease Control,Atlanta, Georgia.

Collier, L., Balows, A. and Sussman, M. (eds) (1998)Topley and Wilson’s Microbiology and MicrobialInfections, 9th edn. Arnold, London, 6 volumes.

Downes, F.P. and Ito, K. (eds) (2001) Compendium ofMethods for the Examination of Foods, 4th edn.American Public Health Association, Wash-ington, DC.

Dworkin, M. (ed.) (1999) The Prokaryotes [ComputerFile]: an Evolving Electronic Resource for theMicrobiogical Community, 1st electronic edn.Springer-Verlag, New York.

Evans, A.S. and Brachman, P.S. (eds) (1991) BacterialInfections of Humans. Epidemiology and Control,2nd edn. Plenum Medical Book Company,New York.

Fischetti, V.A., Novick, R.P., Ferretti, J.J., Portnoy,D.A. and Rood, J.I. (eds) (2000) Gram-positivePathogens. ASM Press, Washington, DC.

Fleming, D.O. and Hunt, D.L. (eds) (2000) BiologicalSafety. Principles and Practices, 3rd edn. ASMPress, Washington, DC.

Food and Drug Administraton (1995) FDA/BAM(Food and Drug Administration/BacteriologicalAnalytical Manual), 8th edn. AOACInternational, Gaithersburg, Maryland.Available via the web at http://www.cfsan.fda.gov/~ebam/bam-toc.html

Glass, K. and Johnson, E.A. (2001) Formulating lowacid foods for safety. In: Juneja, V. and Sofos, J.(eds) Control of Foodborne Microoganisms.Marcel Dekker, New York, pp. 323–350.

Goodfellow, M. and O’Donnell, A.G. (eds) (1993)Handbook of New Bacterial Systematics. Aca-demic Press, Harcourt Brace & Co., London.

Granum, P.E. and Brynestad, S. (1999) Bacterialtoxins as food poisons. In: Alouf, J.E. andFreer, J.H. (eds) The Comprehensive Sourcebookof Bacterial Protein Toxins, 2nd edn. AcademicPress, London, pp. 669–681.

Guzewich, J.J., Bryan, F.L. and Todd, E.C.D. (1997)Surveillance of foodborne disease I. Purposesand types of surveillance systems and net-works. Journal of Food Protection 60, 555–566.

Hobbs, B.C. and Gilbert, R.J. (1978) Food Poisoningand Food Hygiene. Food and Nutrition Press,Westport, Connecticut.

Holt, J.G. (ed.) (1984–1989) Bergey’s Manual ofSystematic Bacteriology. Williams & Wilkins,Baltimore, Maryland, 4 volumes.

Hutt, P.B. and Hutt, P.B. II (1984) A history of gov-ernment regulation and misbranding of food.Food Drug and Cosmetic Law Journal 39, 2–73.

ICMSF (International Commission of the Micro-biological Specifications for Foods of theInternational Union of Biological Societies)(1996) Microoganisms in Foods. Characteristicsof Microbial Pathogens. Blackie Academic &Professional, London.

Johnson, E.A. and Pariza, M.W. (1989) Micro-biological principles for the safety of foods.In: Middlekauf, R.D. and Shubik, P. (eds)International Food Regulation Handbook. Policy.Science. Law. Marcel Dekker, New York,pp. 135–174.

Lund, B.M., Baird-Parker, T.C. and Gould, G.M.(eds) (2000) The Microbiological Safety and Qual-ity of Food, Aspen Publishers, Gaithersburg,Maryland, 2 volumes.

Madigan, M.T., Martinko, J.M. and Parker, J. (2000)Brock Biology of Microorganisms, 9th edn.Prentice Hall, Upper Saddle River, New Jersey.

Mandell, G.L., Bennett, J.E. and Dolin, R. (eds) (2000)Mandell, Douglas, and Bennett’s Principles andPractice of Infectious Disease, 5th edn. ChurchillLivingstone, New York, 2 volumes.

McNeil, W.H. (1976) Plagues and Peoples. DoubledayPublishing, Garden City, New York.

Mead, P.S., Slutsker, L., Dietz, V., McCaig, L.F.,Bresee, J.S., Shapiro, C., Griffin, P.M. andTauxe, R.V. (1999) Food-related illness anddeath in the United States. Emerging InfectiousDiseases 5, 607–625.

Middlekauf, R.D. and Shubik, P. (eds) (1989)International Food Regulation Handbook. Policy.Science. Law. Marcel Dekker, New York.

Miller, S.A. and Taylor, M.R. (1989) Historical devel-opment of food regulation. In: Middlekauff,R.D. and Shubik, P. (eds) International FoodRegulation Handbook. Policy. Science. Law.Marcel Dekker, New York, pp. 7–25.

Mitscherlich, E. and Marth, E.H. (1984) MicrobialSurvival in the Environment: Bacteria andRickettsiae in Human and Animal Health.Springer-Verlag, New York.

Mossel, D.A.A. and Ingram, I. (1955) The physiol-ogy of the microbial spoilage of foods. Journal ofApplied Bacteriology 18, 232–268.

Mossel, D.A.A., Corry, J.E.L., Struigk, C.B. andBaird, R.M. (1995) Essentials of the Microbiologyof Foods. John Wiley & Sons, Chichester, UK.

Mossel, D.A.A., Jansen, J.T. and Struijk, C.B. (1999)Microbiological safety assurance applied tosmaller catering operations world-wide.From angst through ardor to assistance andachievment – the facts. Food Control 10,195–201.

National Academy of Sciences (NAS) (1985) AnEvaluation of the Role of Microbiological Criteria

20-Feb-03 2

44 E.A. Johnson



for Foods and Food Ingredients. NationalAcademy Press, Washington, DC.

National Research Council (1985) An Evaluationof the Role of Microbiological Criteria for Foodsand Ingredients. National Academy Press,Washington, DC.

Petersen, K.E. and James, W.O. (1998) Agents,vehicles, and causal inference in bacterialfoodborne disease outbreaks – 82 reports(1988–1995). Journal of the American VeterinaryMedical Association 212, 1874–1881.

Rahman, M.S. (ed.) (1999) Handbook of Food Preserva-tion. Marcel Dekker, New York.

Relman, D.A. and Falkow, S. (2000) Molecularperspective of microbial pathogenicity. In:Mandell, G.L., Bennett, J.E. and Dolin, R. (eds)Mandell, Douglas, and Bennett’s Principles andPractice of Infectious Disease, 5th edn. ChurchillLivingstone, New York, pp. 2–13.

Smith, J.L. and Fratamico, P.M. (1995) Factorsinvolved in the persistence of food-bornediseases. Journal of Food Protection 58,696–708.

Swaminathan, B., Barrett, T.J., Hunter, S.B., Tauxe,R.V. and the CDC PulseNet Task Force (2001)PulseNet: molecular subtyping network forfoodborne bacterial disease surveillance,United States. Emerging Infectious Diseases 7,382–389.

Tannahill, R. (1973) Food in History. Stein and Day,New York, pp. 344–346.

Wilson, G. and Dick, H.M. (eds) (1983) Topleyand Wilson’s Principles of Bacteriology andImmunology, Vol. 7, 7th edn. Edward Arnold,London.

World Health Organization (WHO) (1997) Foodsafety and foodborne diseases. World HealthStatistics Quarterly, Volume 50.


20-Feb-03 2



3 Shellfish Toxins

A. Gago Martínez1* and J.F. Lawrence21Department of Analytical and Food Chemistry, Faculty of Sciences,

University of Vigo, Campus Universitario, 36200-Vigo, Spain;2Food Research Division, Health Canada, Ottawa, Ontario, Canada

Introduction

Marine phytoplankton is being seriouslyaffected by the presence of certain micro-scopic algae, which are critical food forfilter-feeding bivalve shellfish (mussels,clams scallops, oysters, etc.) as well as larvaeof crustaceans and finfish. The plankton algaeproliferation (‘algal blooms’) is beneficial foraquaculture; however, algal blooms can alsohave negative effects, causing importantsocio-economic damage.

The first written reference to a harmfulalgal bloom could be in the Bible (Exodus 7:20–21): ‘. . . all the waters that were in the riverwere turned to blood, fishes died, Egyptianscould not drink the water of the river’.

One of the first fatal cases of humanpoisoning after eating shellfish contaminatedwith dinoflagellate toxins was reported in1793 (Poison Cove, British Columbia). At thattime, local Indian tribes were not allowedto eat shellfish when the seawater becamephosphorescent due to dinoflagellate blooms;these were related to certain alkaloid toxins,now called paralytic shellfish poisoning (PSP)toxins. Since then, more cases have beenreported and, on a global scale, close to 2000cases of human poisoning by toxins throughfish or shellfish consumption are reportedeach year. For this reason, there is a need to

strictly control the compounds responsible inorder to ensure seafood safety.

Where toxic algal species are present,shellfish can be rendered unfit for humanconsumption. In this way, filter-feeding shell-fish can act as vectors of various seafoodpoisoning syndromes such as PSP, diarrhoeticshellfish poisoning (DSP) and amnesic shell-fish poisoning (ASP) in human consumers.The existence of the phenomenon of toxicphytoplankton blooms has given rise to manyinternational scientific meetings concernedwith the environmental and health impacts ofthese potentially catastrophic incidents.

Paralytic Shellfish Poisoning Toxins

PSP is the neurotoxic syndrome that is theresult of human consumption of contami-nated seafood. Toxins associated with thissyndrome were referred to as saxitoxins sincesaxitoxin initially was thought to be the onlyagent responsible for this contamination.

The incidence of PSP has significantlyincreased since the 1970s, and this poisoningat present is appearing in regions of the worldwhere it has never been known. The poison-ing is sporadic and unpredictable. At present,PSP must be considered as a global problem


11-Mar-03 3




that requires better professional and publicawareness.

This poisoning has long been known tonative Americans (Kao, 1993). Captain GeorgeVancouver aboard the Discovery describedexperiences with PSP intoxications on a tripto British Columbia in 1793. After eatingmussels, some members of his crew weresick with neurotoxic symptoms (Vancouver,1798). Until 1970, about 1600 cases of humanintoxication had been recorded worldwide,especially in North America and Europe(Prakash et al., 1971). Since then, almost 1000cases have been reported, many occurring inregions where PSP had been known (WorldHealth Organization, 1984).

Source organisms

The organisms considered as primarysources of PSP include three morphologicallydistinct genera of dinoflagellates as well asone species of blue-green algae present infreshwater. Aphanizomenon flos-aquae, whichwas long suspected to contain saxitoxin-likecompounds, has been an important tool inthe elucidation of saxitoxin biosynthesis aswell as being responsible for poisoningsoccurring among terrestrial animals drinkingalgal-infested freshwater supplies. Marineanimals were also affected by this poisoning(Carmichael and Falconer, 1993). The linkbetween shellfish toxicity and dinoflagellateswas first established in 1927 after an outbreakof PSP in San Francisco Bay. The toxic

dinoflagellate was assigned to the genusGonyaulax and named G. catenella. Severaldinoflagellates of similar morphology werefound later to be responsible for the PSPtoxicity. These organisms usually have beenassigned to the genus Gonyaulax; taxonomicrevisions have been recently carried outand these dinoflagellates are now consideredas Alexandrium (Balech, 1985). Pyrodiniumbahamense, a dinoflagellate, was also found tobe responsible for a PSP outbreak in PapuaNew Guinea. Figure 3.1 shows some struc-tures of dinoflagellates responsible for PSPtoxicity.

Chemistry

PSP toxins represent a group of highly polarwater-soluble compounds whose structureis shown in Fig. 3.2. More than 20 analoguesof saxitoxin, considered in the past as themain agent responsible for this syndrome,have been reported to occur naturally.The saxitoxin molecule is a tetrahydropurinecomposed of two guanidinium functionsfused together in a stable azaketal linkage.At C-11, saxitoxin possesses a geminaldiol. Traditionally, the PSP toxins, hetero-cyclic guanidines, were divided into threegroups – carbamates, sulphocarbamoyls anddecarbamoyls (Oshima et al., 1989) – with sixtoxins in each. Subsequently, a few deoxy-carbamoyl compounds have been addedto this group. The structural relationshipsamong these compounds suggested the

20-Feb-03 3

48 A. Gago Martínez and J.F. Lawrence

Fig. 3.1. Dinoflagellate species producing PSP toxins: (a) Gymnodinium catenatum; (b) Alexandriumtamarense.



possibility of multiple bioconversions intothese PSP analogues.

Toxicology

The in vitro effects of saxitoxins have beenstudied carefully (Kao et al., 1971). Saxitoxinexhibits a relaxant action on vascularsmooth muscle, and the action of the cardiacmuscle is depressed as it also has a physio-logical channel-blocking effect. Guanidiniumgroups are key structural features of toxinsinvolved in blockage of Na+ conductancethrough nerve membranes.

The main symptoms of this intoxicationinclude tingling and numbness of the mouthand lips, appearing shortly after intake of sea-food containing PSP toxins. The symptomsspread to the rest of the face and the neck. Asensation of prickling in the fingers and toes isexperienced, followed by headache and dizzi-ness. In some cases, symptoms of nausea andvomiting can occur in the early stages of thePSP intoxication. In cases of moderate to

severe intoxications, paraesthesia spreadsto the arms and legs. After that, the patientscan speak only incoherently, and a feelingof weakness is also experienced. In addition,respiratory difficulties can appear andpatients with severe intoxication may experi-ence paralysis of muscles and, finally, death asa consequence of the progressive respiratoryproblems (Prakash et al., 1971).

The main source of the intoxication is theconsumption of bivalves, including mussels,clams, oysters, etc. Nevertheless, some otherseafood such as crabs, several fish, etc. canalso be responsible for this poisoning.

These toxins are absorbed rapidly fromthe gastrointestinal tract due to the fact thatthey are positively charged, since the twoguanidinium functions have an alkaline pKa

and are protonated with a net cationic chargein the human body pH of 7.4. The eliminationof PSP toxins takes about 90 min, and clinicalstudies have shown that patients who survivethe first 24 h usually recover with no apparentlate effects (Kao, 1993). In terms of toxicity,sulphocarbamoyl compounds are consideredas the less toxic PSP compounds; nevertheless,

Shellfish Toxins 49

20-Feb-03 3

Fig. 3.2. Chemical structures of PSP toxins.



these compounds can be converted into carba-mates, which are the most toxic PSPs underacidic conditions (Hall et al., 1990). The levelsof PSP toxins reported to cause intoxica-tions vary considerably, possibly due tointerindividual differences in sensitivity aswell as the precision of the methods used forquantification. Prakash defines mild poison-ings in adults at doses of PSP toxins between304 and 4128 µg per person, while severepoisonings are caused by doses between 576and 8272 µg (Prakash et al., 1971). Othersources report mild symptoms from dosesbetween 144 and 1660 µg saxitoxin equiva-lents per person, and fatal intoxications fromdoses between 456 and 12,400 µg saxitoxinequivalents (Acres and Gray, 1978).

There is no specific antidote for PSPtoxins. The clinical management of patientsintoxicated with these toxins is entirely sup-portive; if vomiting does not occur spontane-ously, induced emesis or gastric lavageare required. The toxins can be adsorbedeffectively by activated charcoal.

In moderately severe cases, maintenanceof adequate ventilation is the primary con-cern. Periodic monitoring of blood pH andblood gases to ensure adequate oxygenation isimportant. Because of the toxin interferencewith respiratory functions, the acidosis can-not be compensed by hyperventilation. Fluidtherapy is essential to correct any possibleacidosis and, additionally, the renal excretionof the toxin must be facilitated.

There is no rational basis for the use ofanticholinesterase agents to improve muscu-lar performance, even if the practical effectappears beneficial, since this does not involvea reversal of the sodium channel blockagecaused by these toxins, Similarly, there is norational basis for any beneficial effects of vig-orous exercise; this would only increase theproduction and accumulation of lactate, toadd to the pathophysiological derangement.Since the half-time of elimination of the PSPtoxins from the body is around 90 min, asmentioned above, this should be adequatefor a physiological reduction of the toxinconcentration to harmless levels, except inthose cases where the toxin concentration isvery high or victims have damage to renalfunction.

Regulatory levels

Today, most countries apply a tolerance levelfor PSP toxins at 0.8 mg saxitoxin equivalentskg−1 mussel meat (equivalent to 400 mouseunits). If the consumption of mussels is esti-mated at 100 g, this indicates a safety factorof about 2–4 for the risk of developing mildsymptoms among the most susceptible, and,more importantly, a minimum safety factorof about 6–7 for serious intoxication or death.

Analytical methods

The common method used for the control ofPSP toxin is the mouse bioassay (Associationof Official Analytical Chemists, 1990). Thisbioassay is still the official method in mostcountries. It measures the total toxicity ofshellfish extracts and can monitor shellfishsafety efficiently. This method poses somelimitations regarding its selectivity, sensi-tivity and variability of results, as well asthe constant supply of mice and mainte-nance facilities not available in most analyti-cal chemistry laboratories. High-performanceliquid chromatography (HPLC) is themethod widely used as an alternative tothis mouse bioassay for the detection andquantification of PSP toxins. Fluorescencedetection has been selected as the more sensi-tive approach, and derivatization oxidationreactions are required for converting the PSPtoxins into the corresponding fluorescentanalogues. Post- and pre-column techniqueshave been developed for this purpose, andthe oxidation reaction is based on earlierwork (Bates and Rapoport, 1975) where PSPtoxins were oxidized with peroxide to yieldfluorescent products and the total amountof fluorescence produced was used as anestimate of PSP concentration. It was found,however, that the N-1 hydroxy compoundsare poorly oxidized with peroxide, so the useof this reagent can seriously underestimatethe true PSP concentration in unknownextracts. With the post-column approach,individual PSP analogues are separatedusing gradient elution ion-pair chromato-graphy, the toxins being detected by fluores-cence after conversion to purine derivatives

20-Feb-03 3




with periodate. Periodate was foundto produce fluorescent products with all PSPtoxins. The advantage of using a chemicalmethod is the ability to separate the differentanalogues and quantitate them individually.The post-column method is much moresuited to monitoring PSP contamination onan on-going basis rather than being set upfor determinations on an occasional basis.Oshima et al. (1989) have modified thepost-column methods developed by Sullivanand Iwaoka (1983). The significant changesmade were in the chromatography, usingthree isocratic ion-pair mobile phases insteadof a gradient elution, resulting in a separatedetermination of the three PSP groups oftoxins, as well as in an improvement in thedetection limits for individual toxins becauseof the higher efficiency separations.

The pre-chromatographic mode hasemerged to overcome problems related totime and special equipment required forsetting up the post-column oxidation mode.With this approach, the oxidation reactionis carried out prior to the chromatographicseparation; the oxidation products are thenseparated by HPLC and quantitated directlywith no post-column equipment. The reactionis simple, requiring only peroxide orperiodate at weakly basic pH. The pre-chromatographic oxidation method wasstudied extensively and optimized furtherby Lawrence et al. (1995), who evaluated bothperoxide and periodate under a variety ofreaction conditions. Optimal conditions forthe oxidation reaction have been evaluatedrecently (Gago-Martínez et al., 2001).

Although HPLC techniques are promis-ing, capillary electrophoresis (CE) is emergingas an analytical alternative for such toxins(Piñeiro et al., 1999). HPLC has also been usedcoupled with mass spectrometry (MS). FABionization MS has provided useful dataon a variety of individual PSP analogues.Electrospray MS coupled with CE has alsobeen used for the analysis of PSP toxins (Lockeand Thibault, 1994; Gago-Martínez et al.,1996). These techniques are not particularlysuited for routine analysis, but neverthe-less can offer useful information aboutthe PSP toxins present in contaminatedsamples.

Differences in toxicity between thesulphocarbamoyls and the other groups ofPSP toxins present a problem, since theyundergo hydrolysis under acidic conditions,being transformed into the more toxiccarbamates (Hall et al., 1990). The degree ofhydrolysis depends on the acidity. The acidityapplied in the traditional extraction proce-dure, at about pH 3, is insufficient for totalhydrolysis. Consequently, analysis of PSPtoxins extracted from seafood may underesti-mate the total toxicity if the sulphocarbamoylspresent in the seafood are transformed to agreater degree in the human stomach, and ifthey constitute a significant amount.

Several biochemical assays have alsobeen developed. Among the most interestingare enzyme-linked immunosorbent assay(ELISA) methods. Use of ELISA methods ishampered by the lack of sensitivity towardsmany of the toxins making up the PSP toxincomplex; however, fast screening methods forPSP toxins that show good correlation withthe mouse bioassay are being developed.Some other analytical methods such asthe neuroblastoma assay have also beendeveloped (Gallacher and Birbek, 1992) andapplied for the determination of PSP toxins inPortuguese samples (Alvito, 2001).

The main obstacle to the developmentof analytical methods is associated with theproblem in obtaining standards and referencematerials, although this situation is improv-ing currently.

Diarrhoetic Shellfish Poisoning

DSP is an illness in humans that can occur asa result of consuming shellfish contaminatedwith toxic dinoflagellates. The first evidenceof the presence of this new type of gastro-intestinal illness associated with the con-sumption of mussels that had ingested dino-flagellates was reported in The Netherlandsin the 1960s (Kat, 1979). Another toxic inci-dent due to the consumption of scallops wasreported in Japan in 1976–1977, when a largegroup of people suffered from gastrointesti-nal symptoms. These symptoms have nowbecome typical in cases of intoxication due to

Shellfish Toxins 51

20-Feb-03 3



the consumption of shellfish that havebecome contaminated with okadaic acid(OA) and related compounds (Yasumotoet al., 1978). Although it was believed thatsimilar episodes occurred in Scandinaviaduring the 1960s, it was not until the 1980sthat an episode of DSP was confirmed(Kumagai et al., 1986).

The discovery of this type of shellfishpoisoning is attributed to Yasumoto and hisresearch team. These workers found a closecorrelation between the dinoflagellate Dino-physis fortii and this contamination; conse-quently, the toxin was named dinophysistoxin(DTX) and, because of the diarrhoetic symp-toms, the syndrome was named ‘diarrheticshellfish poisoning’ (Yasumoto et al., 1980).

DSP toxins can be divided into threegroups, OA and its analogues the DTXs,pectenotoxins (PTXs) and yessotoxins (YTXs);these last two groups of toxins initially wereincluded in this group despite having dif-ferent toxicological effects. While PTXs areclearly hepatotoxic, YTXs show a cardiotoxicsymptomatology.

OA and analogues are now consideredas the typical DSP toxins and are widelydistributed. For this reason, the study ofthis group is emphasized in this chapter;nevertheless, a brief description of PTX andYTX will also follow.

PTXs are polyether lactones. PTX-2 isthe only PTX found in phytoplankton, whilea whole range of structurally closely similarcompounds are found in shellfish, probably asa result of transformations. The PTXs have notbeen associated with the typical DSP symp-toms in humans; however, they are acutelytoxic to mice. The mechanism of toxicity ofthe PTXs is not clear. According to Quilliamet al. (2000), PTX-2 seco acids may havecontributed to gastrointestinal symptoms,vomiting or diarrhoea in humans, after con-sumption of a bivalve mollusc in New SouthWales, Australia.

The YTXs are polyethers closely resem-bling the brevetoxins. In addition to YTX, sev-eral derivatives are identified (among others45-OH-YTX, homo-YTX and 45-OH-homo-YTX; Satake et al., 1997). The target organ forYTX is the myocardium, while the smallintestine is unaffected (Murata et al., 1987).

According to recent studies, the oral toxicityof YTX is at least one order of magnitudelower compared with its intraperitoneal (i.p.)toxicity (Aune et al., 2000, oral communica-tion). In contrast to YTX, the desulphatedderivative displays no toxicity in the heartmuscle, but it exerts toxicity in the liver andpancreas at 300 µg kg−1 upon i.p. injection.Mice treated orally with desulphated YTXat 500 µg kg−1 body weight developed fattydegeneration of the liver.

Source organisms

Dinoflagellates belonging to the genusDinophysis were implicated in outbreaksof DSP toxicity The confirmation of theirtoxigenicity has been difficult because of thedifficulty in culturing these dinoflagellates.Prorocentrum lima has also been proved to bea producer of OA and related compounds(Lee et al., 1987).

In outbreaks of DSP in Japan in 1976 and1977, DTX-1 was the major toxin presentin mussels and the causative organism wasDinophysis fortii. DTX-3 was also found as thepredominant toxin in scallops collected in1982; nevertheless, DTX-3 was not foundin Dinophysis species, so the origin of DTX-3was suggested to be in the acylation of DTX-1in the hepatopancreas of scallops (Murataet al., 1982).

From outbreaks of DSP in France, Spain,Portugal, Italy and Sweden, it was reportedthat OA was the major toxin, D. acuminata andD. acuta being the species responsible for thesetoxins. DSP outbreaks in The Netherlandswere mostly due to high concentrationsof Prorocentrum species. Important DSP out-breaks in Norway and Sweden in 1985 and1986 were attributed to the presence ofD. acuta (Aune and Yndestad, 1993). Whilethe dominant toxin in Europe is OA, DTX-1was the major DSP toxin in Japan; this differ-ence is attributed to the presence of differentdinoflagellates in European and Japanesewaters. However, DTX-1 was also the majortoxin found in a toxic episode in Norway in1986 in mussels harvested in Songdal. Incontrast, OA was the main toxin responsible

20-Feb-03 3




for DSP toxicity in another part of Norway; D.acuta and D. norvegica were responsible forthese toxins. Although OA was considered themain toxin responsible for DSP toxicity in Ire-land, DTX-2 has been found together with OAduring routine monitoring for DSP toxins(Hu et al., 1992). This toxin was also found tobe responsible for DSP toxicity in Galicianwaters, being on occasions the predominantDSP toxin in mussels (Gago-Martínez et al.,1996).

DSP outbreaks in America are associatedwith the presence of OA and DTX-1, Proro-centrum spp. being responsible for this toxicprofile in Canada, while Dinophysis species areresponsible for DSP toxicity in the USA andChile. The structures of some species capableof producing DSP toxins are shown in Fig. 3.3.

Chemistry

The DSP toxins, as mentioned above, areconsidered as three main groups of toxins:OA and derivatives (DTXs), PTXs and YTXs.Among all these toxins, OA and the DTXsare most commonly distributed worldwide.The group of OA and DTXs initially wascomposed of OA and DTX-1. A newdinophysistoxin (DTX-2) was isolated inIreland (Hu et al., 1992) and this toxin waslater found in dinoflagellates and mussels

from the Galician Rias (north-west of Spain)(Gago-Martínez et al., 1996). The chemicalstructures of some of these compounds areshown in Fig. 3.4. More DTXs have been dis-covered recently and included in the DSPgroup, such as DTX-3, where saturated orunsaturated fatty acyl groups are attached(Yasumoto et al., 1985). These compoundsalso possess toxic activity, but were onlyfound in shellfish tissues, suggesting a proba-ble metabolic origin. Recently, OA analogueshave been identified in shellfish (DTX-2B andDTX-2C). The first evidence of the existenceof diol esters of OA came from the isolation ofa mixture of such esters from P. lima(Yasumoto et al., 1989). Subsequently, estersof OA such as OA-DE1 have also been iso-lated from many types of Prorocentrum spp.such as P. lima and P. maculosum (Hu et al.,1992). These diol esters were also found inSpanish strains of P. lima (Norte et al., 1994).

A water-soluble DSP toxin that has beennamed DTX-4 has been discovered recently(Hu et al., 1995). This new toxin was discov-ered after observing that the mouse toxicitywas not representative of the concentrationsof known DSP toxins present. Othersulphated esters of OA were isolated fromP. maculosum, namely DTX-5a and DTX-5b,which were found to hydrolyse rapidly to OAby the action of esterases.

The DSP toxins are lipid-soluble long-chain compounds containing cyclic polyether

Shellfish Toxins 53

20-Feb-03 3

Fig. 3.3. Dinoflagellate species producing DSP toxins: (a) Dinophysis spp.; (b) Prorocentrum spp.



rings. They are soluble in acetone, chloroform,methylene chloride, methanol and dimethyl-sulphoxide (DMSO).

Toxicology

The toxic effects exerted by the OA deriva-tives have been well documented since thefirst clinical reports in Japan in 1978, when 42people experienced severe vomiting anddiarrhoea. From the results obtained with themouse bioassay, a correlation between toxic-ity of these toxins in humans and physicaleffects in mice was obtained. The amount oftoxin required to produce illness in humanswas defined by mouse units: 1 mouse unit(MU) is defined as the amount of toxinrequired to cause death to a 20 g mouse overa specific time period (48 h). The amount oftoxin needed to cause mild poisoning in anadult was determined to be 12 MU. In lateryears, more information about the toxiceffects has been reported from a variety ofresearch teams. From these studies, it wasconcluded that DSPs also present chroniceffects; these toxins have been shown to pos-sess the ability to induce tumour promotion(Fujiki and Suganuma, 1999), These toxinshave also been reported to strongly inhibitprotein phosphatases, thereby disruptingnormal eucaryotic cell functions. Concerningthe mechanism of action, OA and DTX-1 are

potent inhibitors of protein phosphatase 1and 2A (PP1 and PP2A, respectively). PP2A isabout 50–100 times more strongly inhibitedthan PP1 by OA/DTX-1 (Fujiki andSuganuma, 1993). There are also some reportson mutagenic and genotoxic effects of OAand DTX-1. According to Aonuma et al.(1991), the mutagenic effects were due toinhibition of protein phosphatases involvedin DNA repair, and not formation of DNAadducts.

The health hazard associated with expo-sure to toxins from the DSP complex is relatedto the toxic effects of the individual com-pounds. DSP symptoms start after intake ofOA or DTXs above 40–50 µg per person(adult). Experience from a whole range of DSPepisodes indicates that the patients recoverafter a few days. Since the effects in questionare diarrhoea, vomiting, headache and gen-eral discomfort, but no serious and irrevers-ible adverse health effects, a lower uncertaintyfactor may be tolerated, compared with toxinsproducing more severe effects. However,human health associated with the chronic tox-icity of DSP as tumour promoters and muta-genic compounds cannot be estimated yet.

Regulatory levels

Today the European Union applies a toler-ance level of 0.16 µg OA equivalents kg−1

20-Feb-03 3


Fig. 3.4. Chemical structure of DSP toxins (okadaic acid group).



mussel meat. Depending upon the amountof shellfish consumed, this indicates a mini-mum safety factor of ≥ 2 before symptomsappear (Aune and Yndestad, 1993).

Depending on the amount of toxiningested, the intensity of the symptoms can bedifferent. Patients intoxicated with DSP toxinsare not usually hospitalized. Intravenousinjection of an electrolyte mixture can be usedfor a fast recovery and the symptoms willdisappear in a few days.

Analytical methods

The most commonly used method for detec-tion of the DSP toxins is the mouse bioassay(Yasumoto et al., 1978). This method hasmany disadvantages; one of the majorobjections is the use of animals for researchpurposes. A lack of selectivity is alsoobserved since other toxins or fatty acidspresent in mussels or seafood can come intothe lipid fraction causing interference, whichmay make difficult the identification of thestudied toxins or cause false-positive results.The mouse bioassay cannot discern reliablybetween different types of toxins but pro-vides information on the overall toxicity pres-ent in samples.

Cytotoxicity assays were developed afterdiscovering that DSP toxins were responsiblefor morphological changes in some cells.These are sensitive, rapid and more ethicallysatisfactory than live animal assays. Effectiveuse was made of the fact that DSP toxinsinhibit protein phosphatases. Assays basedon the inhibitory power of DSP toxins havebeen developed and provide sensitive detec-tion of DSP toxins; however, the response isnon-specific and, like the mouse bioassay,gives information regarding the total toxicity.Inmunoassays can also be used to detect OAand some of its analogues; these assays showpoor reactivity, especially for DTX-3.

Physico-chemical approaches have beendeveloped for a sensitive determination ofDSP toxins. Methods based on HPLC arethe most widely used, coupled with variousdetection modes. Fluorescence detection(FLD) provides a highly sensitive response,

and this alternative has been widely used asa routine monitoring tool. Different derivati-zation reagents have been used for convertingthe DSP analogues into the correspondentfluorescent derivatives by mean of thederivatization of the carboxylic acid moietyof the compounds to form highly fluorescentesters, which are then separated by reverse-phase chromatography. The method whichhas received most attention up to the presentis that developed by Lee et al. (1987).This method uses 9-anthryldiazomethane(ADAM) as a derivatization reagent. A num-ber of modifications of the method of Lee et al.have been reported, especially focused onclean-up improvements. Since the ADAMreagent is relatively expensive and of limitedstability, a method for synthesizing it immedi-ately before use has been described (Quilliamet al., 1998). Other reagents, such as coumarin,luminarine-3, 9-chloromethylanthracene, etc.,have also been evaluated for OA and DTX-1.

The only reports to date on the directdetermination of OA and DTXs in shellfishhave involved HPLC combined with ion-spray MS (Quilliam, 1998). With thisapproach, extracts of shellfish can be analyseddirectly without derivatization and clean-up,resulting in a fast and sensitive technique forthe determination and confirmation of DSPtoxins present in contaminated samples. Thistechnique is also useful to determine DSPtoxins when standards are not available.

Amnesic Shellfish Poisoning

A new type of shellfish intoxication namedASP was first discovered in Prince EdwardIsland, Canada, in 1987, after a seriousoutbreak of shellfish poisoning (Quilliam andWright, 1989).

The ASP toxin domoic acid (DA) origi-nally had been isolated from a red microalgaChondria armata by Japanese researchersstudying the insecticidal properties of algalextracts (Takemoto and Daigo, 1958).

In the Canadian episode, due to the con-sumption of blue mussels, none of the knowntoxins was implicated in this incident andeventually DA was identified as the toxic

Shellfish Toxins 55

20-Feb-03 3



agent (Wright el al., 1989). The toxin waspresent at levels as high as 1 g kg−1 of edibletissue, and this was the first report of DA as ashellfish toxin (Wright and Quilliam, 1995)The victims were reported to have sufferedneurotoxic and gastrointestinal symptomsbut also an acute loss of memory; thesesymptoms were observed within 24 h.

Source organisms

The source of DA in the eastern Canadianincident was the diatom Pseudonitzschiapungens f. multiseries (Subba Rao et al., 1988).Until this toxic event, it was believed thatphycotoxins were only produced bydinoflagellates, and diatoms were notconsidered as a possible source of toxins.Other species belonging to the genusPseudonitzschia, such as P. pseudodelicatissimaand P. australis, were responsible for deathsof pelicans and cormorants in Monterey Bay,California (Fritz et al., 1992), after the inges-tion of anchovies containing domoic acidat concentrations as high as 0.1 g kg−1. Inaddition, DA has also been found in otherbivalve molluscs (scallops, clams, oysters,etc.) as well as gastropods, crabs and lobsters.An example of a species of Pseudonitzschia isshown in Fig. 3.5.

Chemistry

DA is a naturally occurring analogue ofglutamic acid and belongs to the kainod classof compounds, which have been isolated

from marine microalgae. The chemical struc-tures of DA and isomers, which werediscovered after investigations in the red algaChondria armata, are shown in Fig. 3.6. DAseems to be the dominating toxin associatedwith ASP in both plankton and contaminatedshellfish. Some of these isomers such asisodomoic A, C and D or domoilactones Aand B were not even found in shellfish tissueor plankton extracts. DA is a crystallinewater-soluble compound with typical acidicamino acid properties. The structure is clearlypH-dependent, and five protonated forms ofthe toxin are possible.

Toxicology

The toxic effects of DA were establishedafter studies were carried out using mice,rats or monkeys. After i.p. injection inmice, this toxin induces a very peculiarsymptomatology, known as ‘scratchingsyndrome’. The animals scratch theirshoulders using the hind leg, followed byconvulsions and often death. Subtle effectssuch as hypoactivity rigidity, tremors, etc.have also been reported (Tasker et al., 1991).The toxic effects in humans have beenreported in the Canadian incident when107 people had to be hospitalized; 14 of themdisplayed severe neurological poisoningand four among the oldest persons intoxi-cated by the mussels died after 11–24 days.Severe damage to the hippocampus andother parts of the brain was found (Todd,1993). The human symptoms were relatedmainly to gastrointestinal disorders, but

20-Feb-03 3


Fig. 3.5. Species of diatoms producing ASP toxins: Pseudonitzschia spp.



neurological symptoms were also observed,a permanent short-term memory deficitbeing one of the most characteristic symp-toms associated with this intoxication. Thepharmacokinetics and mechanism of actionof DA show that, upon oral exposure, most ofthe toxin is excreted in the faeces of mice andrats. In the bloodstream, DA is cleared veryeasily by the kidneys (Suzuki and Hierlihy,1993).

The mechanism of action of DA is as anagonist of the glutamate receptor (Takemoto,1978). Domoic and kainic acids can beregarded as conformationally restricted formsof glutamic acid, both acting as high-affinityglutamate receptors of the quisqualate type.The glutamate receptor conducts Na+ ionchannels in the postsynaptic membrane sothen DA acts to open the Na+ channels, lead-ing to Na+ influx, inducing depolarization. As

Shellfish Toxins 57

20-Feb-03 3

Fig. 3.6. Chemical structures of domoic acid and isomers.



a result of this, the Ca+ ion influx is increasedand may lead to cell death. DA is a 2–3 timesstronger neuroexcitator than kainic acid, andabout 100 times more potent than glutamate.

Regulatory levels

After the Canadian incident, a safe limit forDA was established; this limit was set at20 mg kg−1 shellfish tissue (Iverson andTruelove, 1994). This level has been adoptedby most countries as the regulatory levelfor this toxin. Recently, the action level forDA in crab viscera has been modified andincreased to 80 mg kg−1. Data reported fromthe Canadian incident estimated that theconcentration of DA in shellfish was in therange of 300–1000 mg kg−1 and the intoxi-cated individuals may have ingested 1–2 mgkg−1 of the toxin.

Consumption of 250 g of mussel meat atmaximum tolerance level will give an intakeof about 0.1 mg DA kg−1 body weight for anadult.

The rates of accumulation of DA in shell-fish and the speed of its elimination vary, bothwithin different species and between differentorgans. In most shellfish, DA accumulates inthe digestive organs.

Analytical methods

DA can be detected by the mouse bioassayfor PSP if the observation time is extendedto more than 4 h. The toxin is detected inmice by means of a unique syndrome, theabove mentioned ‘scratching syndrome’.The success of this biological assay in theCanadian incident was due in part to thehigh levels of toxin present in contaminatedshellfish (300–1000 mg kg−1 tissue). However,the sensitivity of the bioassay is inadequatefor the action level of 20 mg kg−1 tissue estab-lished as the regulatory level. Symptomssuch as scratching are observed in mice withextracts containing more than 40 mg kg−1.

Several alternatives have been developedfor the analysis of ASP toxins. The first chemi-cal approach involves the use of reverse-phase

HPLC analysis with UV detection of theunderivatized compound at its absorptionmaximum of 242 nm (Quilliam et al., 1989a).

Since then, several alternatives usingdifferent extraction procedures or differentdetection methods, such as fluorescenceafter derivatization using different reagents(Pocklington et al., 1990; James et al., 2000),have been developed, all with detection limitsat 1 mg kg−1 or lower. CE, a very promisinganalytical technique, has also been investi-gated and applied to the analysis of DA(Nguyen et al., 1990; Zhao et al., 1997; Piñeiroet al., 1999). Gas chromatography (GC)-MSand liquid chromatography (LC)-MS tech-niques have also been proposed for thedetermination of these compounds. GC-MS isapplicable to concentrations of DA in contam-inated shellfish ranging from 1 to 500 mg kg−1;nevertheless, a derivatization reaction isrequired to convert the ASP compoundsinto the N-trifluoroacetyl-O-silyl derivatives,requiring an intensive clean-up to facilitatethis derivatization (Pleasance et al., 1990).HPLC combined with ion-spray MS has beenshown to be particularly useful for confirma-tion of DA in shellfish (Quilliam et al., 1989b).

Among biochemical assays, severalELISAs have been developed. According toGarthwaite et al. (1998), a robust and highlysensitive ELISA method is now availablewhich should be suitable for routine testingof shellfish for regulatory purposes.

Among all these analytical alternativesfor the control of ASP toxins, HPLC-UV isthe preferred method and has been usedby most regulatory agencies worldwide forpreventing incidents of ASP (Lawrenceet al., 1989, 1991; Quilliam et al., 1989a;Association of Official Analytical Chemists,1991). This method is suitable for detectingcontamination levels greater than 20 mg kg−1,nevertheless, interferences commonly presentin such complex matrices can cause falsepositives with crude extracts. It has beenshown that tryptophan and some of itsderivatives are often present in shellfishtissues, eluting close to DA and isomers, and itis necessary to use efficient clean-up proce-dures to remove such interferences and conse-quently obtain an accurate control of thesetoxic compounds. Although intensive work

20-Feb-03 3




in developing selective clean-up methods, bymeans of solid phase extraction using C18or anion exchange as stationary phases, hasbeen carried out recently, enormous variabil-ity has been found (Piñeiro, 2001). Improve-ments are still required, and thus the use ofconfirmatory techniques such as MS is highlyrecommended to ensure the presence orabsence of ASP toxins in seafood.

New and Emerging Toxins

The progress in the development of newanalytical techniques has led to the dis-covery of new toxins including pinnatoxins,azaspiracids, gymnodimine and spirolides.

Pinnatoxins

Pinnatoxins are a group of potent marinetoxins implicated in human food poisoningresulting from the ingestion of shellfishbelonging to the Pinna genus. This bivalve isa common seafood in China and Japan, andhuman intoxication is a regular occurrence(Twohig, 2001). The symptoms associatedwith this intoxication involve diarrhoea withtypical neurological symptoms. Pinnatoxinsare thought to be Ca2+ activators.

Azaspiracids

A toxic incident occurred in The Netherlandswhere eight people became ill after hav-ing eaten mussels originally from Killaryharbour. Symptoms were typical of DSPincluding nausea, vomiting, diarrhoea andabdominal cramps. High mouse toxicity wasnot proportional to the low levels of OA andDTX-2 found in the same mussels. The struc-ture of the original azaspiracid found inmussels taken from Killary harbour has beendetermined (Satake et al., 1998), and severalisomers described. Toxicological studies ofazaspiracid show that, in addition to causingdamage to the small intestine, the toxin alsocauses damage in both liver and spleen (Itoet al., 2000). Both the target organs and mode

of action of azaspiracid are distinctly differ-ent from those of DSP, PSP and ASP toxins.

Gymnodimine

In 1994, oysters from South Island, NewZealand, were analysed and gave rise tomouse toxicity levels that could not beattributed to known toxins. After multiplechromatographic steps using UV diode arraydetection, and mouse bioassays, the potentcompound responsible was isolated. Thiscompound was named gymnodimine, sincethe causative organism was Gymnodiniumsp. The minimum lethal dose in mice was450 mg kg−1. Mice injected died within5–15 min. Gymnodimine also showed potentichthyotoxicity at levels of 250–500 ppb. Thestructure of gymnodimine was characterizedusing nuclear magnetic resonance.

Spirolides

A family of macrocyclic toxins was isolatedfrom the digestive glands of shellfish whichwere collected from the eastern shore of NovaScotia, Canada. These compounds werenamed spirolides as they possess an unusualseven-membered cyclic imine moiety thatis spiro-linked to a cyclohexene ring. Thepharmacological activity of the spirolidesmay be the activation of Ca channels. Fourspirolides were initially isolated and structur-ally elucidated (Hu et al., 1995). Two minorcomponents were isolated later, which wereinactive in mice and did not possess a cyclicimine moiety, suggesting that this group isessential for pharmacological activity.

Conclusions

A number of seafood toxins are now knownfollowing the development of new analyticalmethods. Most of these toxins are naturallyoccurring substances that can negativelyaffect seafood safety and, consequently,human health at very low levels. Thesearch for sensitive analytical approaches is

Shellfish Toxins 59

20-Feb-03 3



necessary for an accurate risk assessment.Toxicological studies are still required fora better understanding of the toxicity ofthese compounds. The lack of standardsand reference materials clearly compromisesadvances in this area. Nevertheless, interestin the study of these compounds is increasingconsiderably, due to their enormous socio-economic impact. New analytical methodsare being developed by research teamsinvolved in this field, which will result ina better knowledge of the toxic compoundsinvolved in such poisonings, thereby reduc-ing health risks to consumers.

References

Acres, J. and Gray, J. (1978) Paralytic shellfishpoisoning. Canadian Medical Association Journal119, 1195–1197.

Alvito, P. (2001) Determinaçao de toxinas para-lisantes (PSP) de dinoflagelados marinhos ebactérias asociadas. MSc thesis, UniversidadeLisboa, Portugal.

Association of Official Analytical Chemists (1990)Official Methods of Analysis, 15th edn. AOACInternational, Arlington, Virginia, section959.08.

Association of Official Analytical Chemists (1991)AOAC Official Methods of Analysis. AOACInternational, Arlington, Virginia.

Aonuma, S., Ushijima, T., Nakayasu, M., Shima, H.,Sugimura, T. and Nagao, M. (1991) Mutationinduction by okadaic acid, a proteinphosphatase inhibitor in CHL cells, butnot in S. typhimurium. Mutation Research 250,375–381.

Aune, T. and Yndestad, M. (1993) Diarrhetic shell-fish poisoning. In: Falconer, I.R. (ed.) AlgalToxins in Seafood and Drinking Water. AcademicPress, London, pp. 87–104.

Balech, E. (1985) The genus Alexandrium orGonyaulax of the Tamarensis group. In:Anderson, D.M., White, A.W. and Baden, D.G.(eds) Toxic Dinoflagellates, pp. 33–38.

Bates, H.A. and Rapoport, H. (1975) Chemical assayfor saxitoxin, the paralytic shellfish poison.Journal of Agricultural and Food Chemistry 23,237–239.

Carmichael, W.W. and Falconer, I.R. (1993) Diseaserelated to freshwater algal blooms. In: Fal-coner, I.R. (ed.) Algal Toxins in Seafood andDrinking Water. Academic Press, London,pp. 187–209.

Fritz, L., Quilliam, M.A., Wright, J.L.C., Beale, A.M.and Work, T.M. (1992) An outbreak of domoicacid and poisoning attributed to the pennatediatom Pseudonitzschia australis. Journal ofPhycology 28, 439–442.

Fujiki, H. and Suganuma, M. (1993) Tumor promo-tion by inhibitors of protein phosphatases 1and 2A: the okadaic acid class of compounds.Advances in Cancer Research 61, 143–194.

Fujiki, H. and Suganuma, M. (1999) Unique featuresof the okadaic acid activity class of tumourpromoters. Journal of Cancer Research andClinical Oncology 125, 150–155.

Gago-Martínez, A., Rodríguez-Vázquez, J.A.,Quilliam, M.A. and Thibault, P. (1996) Simulta-neous occurrence of diarrhetic and paralyticshellfish poisoning toxins in Spanish musselsin 1993. Natural Toxins 4, 72–79.

Gago-Martínez, A., Aldea, S., Leao, J.M., RodríguezVázquez, J.A., Niedzwiadek, B. and Lawrence,J.F. (2001) Effect of pH on the oxidation ofparalytic shellfish poisoning toxins foranalysis by liquid chromatography. Journalof Chromatography A905, 351–357.

Gallacher, S. and Birbek, T.H. (1992) A tissue cultureassay for direct detection of sodium channelblocking toxins in bacterial culture supernates.FEMS Microbiology Letters 92, 101–108.

Garthwaite, I., Ross, K.M., Miles, C.O., Hanse, R.P.,Foster, D., Wilkins, A.L. and Towers, N.R.(1998) Polyclonal antibodies to domoic acid,and their use in immunoassays for domoic acidin sea water and shellfish. Natural Toxins 6,93–104.

Hall, S., Strichartz, G., Moczydlowski, E.,Ravindran, A. and Reichardt, P.B. (1990)The saxitoxins: sources, chemistry, andpharmacology. In: Hall, S. and Strichartz, G.(eds) Marine Toxins: Origin, Structure andMolecular Pharmacology. American ChemicalSociety Symposium Series, Washington, DC,pp. 29–65.

Hu, T., Marr, J., DeFreitas, A.S.W., Quilliam, M.A.,Walter, J.A., Wright, J.L.C. and Pleasance, S.(1992) New diol esters isolated from culturesof the dinoflagellates Prorocentrum lima andProrocentrum concavum. Journal of NaturalProducts 55, 1631–1637.

Hu, T., Curtis, J.M., Oshima, Y., Quilliam, M.A.,Watson-Wright, W.M. and Wright, J.L. (1995)Spirolides B and D, two novel macrocycles iso-lated from the digestive glands of shellfish.Journal of the Chemical Society, London, ChemicalCommunications, 2159–2161.

Ito, E., Satake, M., Ofuji, K., Kurita, N., McMahon, T.,James, K.J. and Yasumoto, T. (2000) Multipleorgan damage caused by a new toxin

20-Feb-03 3




azaspiracid, isolated from mussels producedin Ireland. Toxicon 38, 917–930.

Iverson, F. and Truelove, J. (1994) Toxicology andseafood toxins: domoic acid. Natural Toxins 2,334–339.

James, K.J., Gillman, M., Lehane, M. and Gago-Martínez, A. (2000) New fluorimetric methodof liquid chomatography for the determinationof the neurotoxin domoic acid in seafood andmarine phytoplankton. Journal of Chromato-graphy A871, 1–6.

Kao, C.Y. (1993) Paralytic shellfish poisoning.In: Falconer, I.R. (ed.) Algal Toxins in Seafoodand Drinking Water. Academic Press, London,pp. 75–86.

Kao, C.Y., Nagasawa, J., Spiegelstein, M.Y. andCha, Y.N. (1971) Vasodilatory effects oftetrodotoxin in the cat. Journal of Pharmacologyand Experimental Therapeutics 178, 110–121.

Kat, M. (1979) The occurrence of Prorocentrumspecies and coincidental gastrointestinalillness of mussel consumers. In: Taylor, D.and Seliger, H.H. (eds) Toxic DinoflagellateBlooms. Elsevier, North-Holland, Amsterdam,pp. 215–220.

Kumagai, M., Yanagi, T., Murata, M., Yasumoto, T.,Kat, M., Lassus, P. and Rodríguez-Vázquez,J.A. (1986) Okadaic acid as the causative toxinof diarrhetic shellfish poisoning in Europe.Agricultural and Biological Chemistry 50,2853–2857.

Lawrence, J.F., Charbonneau, C.F., Menard, C.,Quilliam, M.A. and Sim, P.G. (1989) Liquidchromatographic determination of domoicacid in shellfish products using the AOACparalytic shellfish poison extraction proce-dure. Journal of Chromatography 462, 349–356.

Lawrence, J.F., Charbonneau, C.F. and Menard, C.(1991) Liquid chromatographic determinationof domoic acid in mussels, using AOACparalytic shellfish poison extraction proce-dure: collaborative study. Journal of AOAC(Association of Official Analytical Chemists)International 74, 68–72.

Lawrence, J.F., Menard, C. and Cleroux, C. (1995)Evaluation of prechromatographic oxidationfor liquid chromatographic determination ofparalytic shellfish poisons in shellfish. Journalof AOAC (Association of Official AnalyticalChemists) International 78(2), 514–520.

Lee, J.S., Yanagi, T., Kenma, R. and Yasumoto, T.(1987) Fluorometric determination of diar-rhetic shellfish toxins by high performanceliquid chromatography. Agricultural andBiological Chemistry 51, 877–891.

Locke, S.J. and Thibault, P. (1994) Improvementin detection limits for the determination of

paralytic shellfish poisoning toxins in shell-fish tissues using capillary electrophoresis/electrospray mass spectrometry and discontin-uous buffer systems. Analytical Chemistry 20,3436–3446.

Murata, M., Shimatani, M., Sugitani, H., Oshima, Y.and Yasumoto, T. (1982) Isolation and struc-tural elucidation of the causative toxin of thediarrhetic shellfish poisoning. Bulletin of theJapanese Society of Sciences and Fisheries 48,549–552.

Murata, M., Kumagi, M., Lee, J.S. and Yasumoto, T.(1987) Isolation and structure of yessotoxin,a novel polyether compound implicated indiarrhetic shellfish poisoning. TetrahedronLetters 28, 5869–5872.

Nguyen, A.L., Luong, J.H. and Masson, C. (1990)Capillary electrophoresis for detection andquantitation of domoic acid in mussels.Analytical Letters 23, 1621–1634.

Norte, M., Padilla, A., Fernández, J.J. and Souto,M.L. (1994) Tetrahedron 50, 9175–9180.

Oshima, Y., Sugino, T. and Yasumoto, T. (1989)Latest advances in HPLC analysis ofparalytic shellfish toxins. In: Natori, S.,Hashimoto, K. and Ueno, Y. (eds) Myco-toxins and Phycotoxins. Elsevier, Amsterdam,pp. 319–326.

Piñeiro, N. (2001) Avances en la determinaciónde toxinas amnésicas mediante técnicascromatográficas y electroforéticas. MSc thesis,Universidad de Vigo, Vigo, Spain.

Piñeiro, N., Leao Martins, J.M., Gago-Martínez, A.and Rodríguez-Vázquez, J.A. (1999) Capillaryelectrophoresis with diode array detection: analternative in the analysis of paralytic andamnesic shellfish poisoning toxins. Journal ofChromatography A847, 223–232.

Pleasance, S., Xie, M., Leblanc, Y. and Quilliam,M.A. (1990) Analysis of domoic acid andrelated compounds by mass spectrometryand gas chromatography/mass spectrometryas N-trifluoro acetyl-O-silyl derivatives. Bio-medical and Environmental Mass Spectrometry 19,420–427.

Pocklington, R., Milley, J.E., Bates, S.S., Bird, C.J.,DeFreitas, A.S.W. and Quilliam, M.A. (1990)Trace determination of domoic acid inseawater and phytoplankton by liquidchromatography of the fluorenyl-methoxy-carbonyl (FMOC) derivative. InternationalJournal of Environmental Analytical Chemistry38, 351–368.

Prakash, A., Medcof, J.C. and Tennant, A.D. (1971)Paralytic shellfish poisoning in easternCanada. Bulletin of the Fisheries Research Boardof Canada 177, 1, 871.

Shellfish Toxins 61

20-Feb-03 3



Quilliam, M. (1998) Liquid chromatography-massspectrometry: a universal method for analysisof toxins. In: Reguera, B., Blanco, J., Fernandez,M.L. and Wyatt, T. (eds) Harmful Algae. IOC ofUNESCO, pp. 509–514.

Quilliam, M.A. and Wright, J.L. (1989) Amnesicshellfish poisoning mystery. Analytical Chemis-try 61, 1058a–1060a.

Quilliam, M.A., Sim, P.G., McCulloch, A.W. andMcInnes, A.G. (1989a) High performanceliquid chromatography of domoic acid, amarine neurotoxin, with application to shell-fish and plankton. International Journal ofEnvironmental Analytical Chemistry 36, 139–154.

Quilliam, M.A., Thompson, B.A., Scott, G.J. and Siu,K.W.M. (1989b) Ion spray mass spectrometryof marine neurotoxins. Rapid Communicationsin Mass Spectrometry 3, 145–150.

Quilliam, M.A., Gago-Martínez, A. and Rodríguez-Vázquez, J.A. (1998) Improved method forpreparation and use of 9-anthryldiazomethanefor derivatization of hydroxy carboxylicacids: application to diarrhetic shellfish poi-soning toxins. Journal of Chromatography 807,229–239.

Quilliam, M., Eaglesham, G., Hallegraeff, G.,Quaine, J., Curtis, J., Richard, D. and Nunez, P.(2000) Detection and identification of toxinsassociated with a shellfish poisoning incidentin New South Wales, Australia. In: Inter-national Conference on Harmful Algal Blooms,Tasmania, Abstract, p. 48.

Satake, M., Tubaro, A., Lee, J.S. and Yasumoto, T.(1997) Two new analogues of yessotoxin,homoyessotoxin and 45-hydroxyhomoyesso-toxin, isolated from mussels of the AdriaticSea. Natural Toxins 5, 107–111.

Satake, M., Ofuji, K., Naoki, H., James, K.J.,Furey, A., McMahon, T., Silke, J. andYasumoto, T. (1998) Azaspiracid, a newmarine toxin having unique spiro ringassemblies, isolated from Irish mussels,Mytilus edulis. Journal of the American ChemicalSociety 120, 9967–9968.

Subba Rao, D.V., Quilliam, M.A. and Pocklington,R. (1988) Domoic acid – a neurotoxic aminoacid produced by the marine diatom Nitzschiapungens in culture. Canadian Journal of Fisheriesand Aquatic Sciences 45, 2076–2079.

Sullivan, J.J. and Iwaoka, W.T. (1983) High pressureliquid chromatographic determination oftoxins associated with paralytic shellfishpoisoning. Journal of Association of OfficialAnalytical Chemists 66, 297–303.

Suzuki, C.A.M. and Hierlihy, S.L. (1993) Renal clear-ance of domoic acid in the rat. Food and ChemicalToxicology 31, 710–716.

Takemoto, T. (1978) Isolation and structural identifi-cation of naturally occurring excitatory aminoacids. In: McGeer, E.G., Olney, J.W. andMcGeer, P.L. (eds) Kainic Acid as a Tool inNeurobiology. Raven Press, New York, pp. 1–15.

Takemoto, T. and Daigo, K. (1958) Constituents ofChondria armata. Chemical and PharmaceuticalBulletin 6, 578–580.

Tasker, R.A.R., Connell, B.J. and Strain, S.M. (1991)Pharmacology of systematically administereddomoic acid in mice. Canadian Journal ofPharmacy and Pharmacology 69, 378–382.

Todd, E.C.D. (1993) Domoic acid and amnesicshellfish poisoning – a review. Journal of FoodProtection 56, 69–83.

Twohig, M. (2001) New analytical methods forthe determination of acidic polyether toxinsin shellfish and marine phytoplankton. MScthesis, Cork Institute of Technology, Cork,Ireland.

Vancouver, G. (1798) A Voyage of Discovery to theNorth Pacific Ocean and Around the World, Vol. 2.Robinson, G.G. and Robinson, J. (eds) London,pp. 284–286.

World Health Organization (1984) Aquatic Marineand Freshwater Biotoxins. Environmental HealthCriteria 37. International Programme onChemical Safety, World Health Organization,Geneva.

Wright, J.L.C. and Quilliam, M.A. (1995) Methodsfor domoic acid, the amnesic shellfish poisons.In: Hallegraef, G.M., Anderson, D.M. andCembella, A.D. (eds) Manual on Harmful MarineMicroalgae. IOC Manuals and Guides No. 33,UNESCO.

Wright, J.L.C., Boyd, R.K., Defreitas, A.S.W., Falk,M., Foxall, R.A., Jamieson, W.D., Laycock,M.V., McCulloch, A.W., Mcinnes, A.G.,Odense, P., Pathak, V., Quilliam, M.A.,Ragan, M., Sim, P.G., Thibault, P., Walter, J.A.,Gilgan, M., Richard, D.J.A. and Dewar, D.(1989) Identification of domoic acid, aneuroexcitatory amino acid, in toxic musselsfrom eastern P.E.I. Canadian Journal of Chemis-try 67, 481–490.

Yasumoto, T., Oshima, Y., Sugawara, W., Fukuyo,Y., Oguri, H., Igarashi, T. and Fujita, N.(1978) Identification of Dinophysis fortii asthe causative organism of diarrhetic shell-fish poisoning. Bulletin of the Japanese Societyof Science and Fisheries 44, 1249–1255.

Yasumoto, T., Oshima, Y. and Yamaguchi, M. (1979)Occurrence of a new type of shellfish poison-ing in Japan and chemical properties of thetoxin. In: Taylor, D. and Seliger, H.H. (eds)Toxic Dinoflagellate Blooms. Elsevier, Amster-dam, pp. 495–502.

20-Feb-03 3




20-Feb-03 3

Yasumoto, T., Oshima, Y., Sugawara, W., Fukuyo,Y., Oguri, H., Igarashi, T. and Fujita, N. (1980)Identification of Dinophysis fortii as thecausative organism of diarrhetic shellfishpoisoning. Bulletin of the Japanese Society ofScience and Fisheries 46, 1405–1411.

Yasumoto, T., Murata, M., Oshima, Y., Sano, M.,Matsumoto, G.K. and Clardy, J. (1985) Diarr-hetic shellfish toxins. Tetrahedron 41, 1019–1025.

Yasumoto, T., Murata, M., Lee, S.J. and Torigoe, K.(1989) Polyether toxins produced bydinoflagellates. In: Natori, S., Hashimoto, K.and Ueno, Y. (eds) Mycotoxins and Phycotoxins,88. Elsevier, Amsterdam, pp. 375–382.

Zhao, J.Y., Thibault, P. and Quilliam, M.A. (1997)Analysis of domoic acid and isomers inseafood by capillary electrophoresis. Electro-phoresis 18, 268–276.

Shellfish Toxins 63



4 Mycotoxins in Cereal Grains, Nuts andOther Plant Products

J.P.F. D’Mello*Formerly of The Scottish Agricultural College, West Mains Road, Edinburgh

EH9 3JG, UK

Introduction

Mycotoxins are a diverse and ubiquitousgroup of fungal compounds specificallyassociated with the precipitation of deleteri-ous effects in humans and animals. Viewedglobally, food safety is regularly compro-mised by the presence of mycotoxins occur-ring in cereal grains, nuts, fruit and greencoffee beans. If feeds are contaminated withmycotoxins, associated residues and meta-bolites may appear in animal products. Themycotoxins of major concern in humanhealth emanate from the secondary meta-bolism of Claviceps, Aspergillus, Penicillium,Fusarium and Alternaria genera. Mycotoxinsmay be categorized and, indeed, named onthe basis of their fungal origin. Mycotoxinsmay also be classified on the basis of theirbiosynthetic origin from key primary inter-mediates. Thus, the polyketide mycotoxinsare derived from acetyl coenzyme A, whilethe terpene mycotoxins are synthesized frommevalonic acid. Amino acids are incorpo-rated in the formation of a third group ofmycotoxins comprising cyclic polypeptidesand their derivatives. It is salutary to note,however, that mycotoxin production may bestrain specific. Thus both toxigenic and

atoxigenic strains exist within the Aspergillusflavus species. It is conventional to subdividetoxigenic fungi into ‘field’ (or plant patho-genic) and ‘storage’ (or saprophytic/spoil-age) organisms. Claviceps, Fusarium andAlternaria are classical representatives of fieldfungi, while Aspergillus and Penicilliumexemplify storage organisms. This distinctionis academic since the inoculum forpostharvest spoilage of grain and fruit, forexample, frequently originates from fieldsources such as soil or plant debris. Further-more, mycotoxins from storage fungi fre-quently are detected on grain, nuts andfruit prior to harvest. Mycotoxigenic speciesmay be distinguished further on the basis ofgeographical prevalence, reflecting specificenvironmental requirements for growth andsecondary metabolism. Thus, A. flavus, A.parasiticus and A. ochraceus readily proliferateunder warm, humid conditions, whereasPenicillium expansum and P. verrucosum areessentially temperate fungi. Consequently,the Aspergillus mycotoxins predominate inplant products emanating from the tropicsand other warm regions, while the Penicilliummycotoxins occur widely in temperate foods,particularly cereal grains and infected fruit.Fusarium fungi are more ubiquitous, but even


20-Feb-03 4




this genus contains toxigenic species whichare associated almost exclusively with cerealsfrom warm countries.

The diverse ill effects caused by thesecompounds are incorporated within thegeneric term ‘mycotoxicosis’, including dis-tinct conditions and syndromes which mayadd to or occur concurrently with existingdisorders such as kwashiorkor and gastro-enteritis. In this chapter, the mycotoxins likelyto prejudice human health are reviewed interms of origin and chemical nature, distribu-tion in foods, toxicology and risk manage-ment. Particular emphasis is placed on recentevidence indicating continuing human expo-sure to these fungal toxins.

Origin and Nature of Compounds

The foodborne mycotoxins most frequentlyimplicated in human disorders are presentedin Table 4.1, which also indicates the fungalorigin of these compounds. The pathways ofbiosynthesis are summarized in Table 4.2. Inhistorical terms, the ergot alkaloids, synthe-sized by Claviceps purpurea, have occupied acentral position by virtue of their assumedrole in widespread gangrenous and convul-sive manifestations in Europe during theMiddle Ages. Current concerns relate to theaflatoxins, ochratoxins, fumonisins andpatulin. However, the trichothecenes andzearalenone have emerged recently as global

20-Feb-03 4

66 J.P.F. D’Mello

Mycotoxins Fungal species Foods

Ergot alkaloidsAflatoxinsCyclopiazonic acidOchratoxin A

CitrininPatulinCitreoviridinT-2 toxin (type Atrichothecene)Diacetoxyscirpenol (type Atrichothecene)Deoxynivalenol (type Btrichothecene)Zearalenone

Fumonisins; moniliformin;fusaric acidTenuazonic acid; alternariol;alternariol methyl ether;altenuene

Claviceps purpureaAspergillus flavus; A. parasiticusA. flavusA. ochraceus; Penicilliumviridicatum; P. cyclopiumP. citrinum; P. expansumP. expansumP. citreo-virideFusarium sporotrichioides; F. poae

F. sporotrichioides; F. poae

F. culmorum; F. graminearum

F. culmorum; F. graminearum;F. sporotrichioidesF. moniliforme

Alternaria alternata

Cereal grainsNuts; maize kernels; dried fruitsNutsCereal grains and products; pigproducts; raw coffeeCereal grainsApple productsRiceCereal grains

Cereal grains

Cereal grains

Cereal grains

Maize kernels

Fruit; vegetables; cereal grains

Table 4.1. Principal foodborne mycotoxins of confirmed or potential relevance in human health.

Primary metabolite Pathway Mycotoxins

Acetyl coenzyme A

Mevalonic acid

Amino acids

Polyketide

Isoprenoid

Peptide synthesis

Patulin, citrinin, ochratoxins, zearalenone,moniliformin, aflatoxins, fumonisinsTrichothecenes: deoxynivalenol, nivalenol, T-2 toxin,HT-2 toxin, diacetoxyscirpenolErgot alkaloids

Table 4.2. Biosynthesis of the major foodborne mycotoxins.



contaminants of the major cereal grains, andthe human health implications need to beaddressed.

Ergot alkaloids

The major ergot alkaloids comprise the lyser-gic acid derivatives ergocristine and ergota-mine (Fig. 4.1), although ergosine, ergocor-nine and ergometrine may also occur incontaminated cereal grains (Flannigan, 1991).

Aflatoxins and cyclopiazonic acid

Aflatoxins B1, B2, G1 and G2 (AFB1, AFB2,AFG1 and AFG2) are the secondary productsof A. flavus and A. parasiticus (Smith, 1997). Inaddition, aflatoxin M1 (AFM1) has been iden-tified in the milk of dairy cows and in womenconsuming and metabolizing AFB1 from con-taminated diets. The aflatoxins are a group ofstructurally related fluorescent heterocycliccompounds characterized by dihydrofuranor tetrahydrofuran residues fused to a substi-tuted coumarin moiety. The AFG moleculesdiffer from the AFB structures in possessinga δ-lactone ring in place of a cyclopentenonering. As explained later, the presence of adouble bond in the terminal furan ring ofAFB1 (Fig. 4.2) and AFG1, but not in AFB2

or AFG2, confers distinct biological propertiesto the former two aflatoxins. It is nowgenerally acknowledged that A. flavus onlysynthesizes AFB1 but is also capable of yield-ing cyclopiazonic acid, a mycotoxin recentlyconfirmed as a co-contaminant in a batch ofgroundnuts associated with mass mortalityin turkey poults in 1960. On the other hand,A. parasiticus often produces all fouraflatoxins. However, in both species ofAspergillus, there are strains which arenon-aflatoxigenic. The two species developwhen conditions such as temperature andhumidity/water activity favour their prolif-eration. In the case of A. parasiticus, tempera-tures of 25 to 30°C are optimal for maximiz-ing aflatoxin synthesis. However, bothtemperature and water activity may interactin the promotion of aflatoxin synthesis, and

the risk of contamination is, therefore, muchgreater in foods produced in warm andhumid regions.

Ochratoxins and citrinin

The ochratoxins, produced by several speciesof Aspergillus and Penicillium, are a family ofstructurally related compounds based on anisocoumarin molecule linked to L-phenylala-nine (Abramson, 1997). Ochratoxin A (OTA;Fig. 4.3) and ochratoxin B (OTB) are the onlyforms to occur naturally in contaminated

Mycotoxins in Cereal Grains, Nuts and Other Plant Products 67

20-Feb-03 4

Fig. 4.1. Ergotamine (Moss, 1996; reproducedwith permission from Mycological Research).

Fig. 4.2. Aflatoxin B1 (Moss, 1996; reproducedwith permission from Mycological Research).

Fig. 4.3. Ochratoxin A (Moss, 1996; reproducedwith permission from Mycological Research).



foods and, of the two, OTA is moreubiquitous, often occurring with anotherpentaketide mycotoxin, citrinin, in cerealsand associated products. Citrinin issynthesized by a number of Penicilliumspecies.

Patulin and citreoviridin

Several Penicillium species are also capable ofsynthesizing patulin (Fig. 4.4), a low molecu-lar weight hemiacetal lactone with antibioticproperties. Penicillum expansum is of particu-lar relevance since it is commonly associatedwith storage rot of apples and a wide varietyof other fruits. The occurrence of patulin inapple juice has been attributed to the useof mouldy fruit. Other species of Penicilliumcontaminating rice from Italy, Spain,Thailand, Burma and other countries arenow recognized as producers of anopen-chain nonaketide derivative known ascitreoviridin.

Fusarium mycotoxins

The natural occurrence of mycotoxins fromFusarium species is generally associated withtemperate countries, since many of thesefungi require somewhat lower temperaturesfor growth and mycotoxin production thanthe aflatoxigenic Aspergillus species. How-ever, extensive data exist to indicate theglobal scale of contamination of cereal grains

with a number of Fusarium mycotoxins.Indeed, F. moniliforme and its mycotoxins areassociated primarily with foods from tropicaland subtropical regions. Fusarium species areimportant pathogens of cereal plants, causingdiseases such as fusarium head blight (FHB).The very same species may also synthesize awide range of mycotoxins, of which the mostimportant from the point of view of humanhealth are the trichothecenes, zearalenone,moniliformin and the fumonisins (D’Melloet al., 1997). Following episodes of FHB,residues of these mycotoxins may contami-nate harvested grain. The co-occurrence ofFusarium mycotoxins in cereal grains has nowemerged as an intractable issue with regardto risk assessment and establishment ofregulatory or advisory directives.

Trichothecenes

The trichothecenes comprise four basicgroups, with types A and B representing themost important mycotoxins. Type Atrichothecenes include T-2 toxin, HT-2 toxin,neosolaniol and diacetoxyscirpenol (DAS),while type B trichothecenes includedeoxynivalenol (DON, also known asvomitoxin) and its 3-acetyl and 15-acetylderivatives (3-ADON and 15-ADON, respec-tively), nivalenol (NIV) and fusarenon-X. Alltrichothecenes possess a basic tetracyclicsesquiterpene structure with a 6-memberedoxygen-containing ring and an epoxidegroup. These features are illustrated in thestructure for DON (Fig. 4.5). The synthesisof the two types of trichothecenes appears to

20-Feb-03 4

68 J.P.F. D’Mello

Fig. 4.4. Patulin (Moss, 1996; reproduced withpermission from Mycological Research).

Fig. 4.5. Deoxynivalenol (Moss, 1996; reproducedwith permission from Mycological Research).



be characteristic for a particular Fusariumspecies. Thus, for example, production oftype A trichothecenes predominates in F.sporotrichioides and possibly also F. poae,whereas synthesis of type B trichothecenesoccurs principally in F. culmorum and F.graminearum.

Zearalenone

A common feature of many Fusarium speciesis their ability to synthesize zearalenone(ZEN), and its co-occurrence with certaintrichothecenes raises important issuesregarding additivity and/or synergism in theaetiology of mycotoxicoses in humans. ZEN(also known as F-2 toxin) is a phenolicresorcyclic lactone (Fig. 4.6), which alsooccurs as a hydroxy derivative in the form ofα-zearalenol. The presence of appropriate

reductases in animal tissues implies thatα-zearalenol may be the active form of ZENin animals.

Fumonisins and moniliformin

With respect to the co-occurrence of myco-toxins, the secondary metabolism of F.moniliforme is of particular significance sinceit is capable of producing at least three myco-toxins: the fumonisins, moniliformin andfusarin C. The fumonisins are relativelyrecent additions to the list of mycotoxins, buttheir significance as major contaminants ofmaize has already been established andlinked with the incidence of cancer inhumans. Several structurally related forms offumonisins (FBs) have been characterized,with FB1, FB2 and FB3 occurring regularly inmaize from different geographical sources.FB1 (Fig. 4.7) is 2-amino-12,16-dimethyl-3,5,10,14,15-pentahydroxyicosane with a pro-pane-1,2,3-tricarboxylate substituent at C-14and C-15, whereas FB2 and FB3 are, respec-tively, the C-10 and C-5 deoxy analogues ofFB1. In addition, FB1 is structurally similar tosphinganine and sphingosine, intermediatesin the biosynthesis and degradation ofsphingolipids. Moniliformin occurs as the Naor K salt of 1-hydroxycyclobut-1-ene-3,


20-Feb-03 4

Fig. 4.6. Zearalenone (Moss, 1996; reproducedwith permission from Mycological Research).

Fig. 4.7. Fumonisin B1 (Moss, 1996; reproduced with permission from Mycological Research).



4-dione and, like the fumonisins, has beendetected in maize.

Alternaria mycotoxins

A wide range of Alternaria species are capableof synthesizing mycotoxins of diverse chem-istry. The dibenzo-α-pyrone group includesalternariol, alternariol methyl ether andaltenuene. The nitrogen-containing groupincludes tenuazonic acid and the cyclicpolypeptide tentoxin. In addition, Alternariaspp. produce a number of metabolites ofvaried structure, including altertoxin I, anunusual partially saturated perylene.

Distribution in Foods

The ubiquitous distribution of toxigenicfungi as plant pathogens (e.g. in FHB) and asspoilage organisms implies that contamina-tion of primary and processed foods is almostinevitable when appropriate environmentalor storage conditions prevail. Mycotoxinshave been detected in such diverse commodi-ties as cereal grains, nuts and fruit, often atlevels that exceed legal or advisory limits.Considerable data already exist to demon-strate the global scale of mycotoxin contami-nation of these foods. The evidence hasbeen presented elsewhere (D’Mello andMacdonald, 1998), but there is scope forreviewing more recent data.

Ergot alkaloids

Historically, rye and other cereals intendedfor breadmaking have been linked withergot contamination (Flannigan, 1991). Theincidence of contamination is now consid-ered to be negligible due to surveillance andlegislation as well as the global decline in theproduction of rye. However, some moderncultivars of malting barley appear to be proneto infection with C. purpurea, resulting in therejection of grain for brewing. For example, in1999, large consignments of barley harvested

in Scotland were rejected due to detectablequantities of ergot in the grain. Ergot contam-ination of sorghum grain is an emerging issuein some developing countries, and vigilanceis, therefore, still necessary.

Aflatoxins

Predictably, aflatoxin contamination ofpeanuts continues to attract worldwide atten-tion. However, surveillance has extendedto other foods and products. A selection ofrecent data is presented in Table 4.3. It is clearthat diverse foods contain levels of aflatoxinthat exceed current statutory limits. Theoutstanding feature is the high level of AFB1

contamination of Indonesian maize. Of equalconcern are the relatively high concentrationsin maize-based gruels used as weaning foodfor children in Nigeria (Oyelami et al., 1996).Samples of peanut butter analysed in the UKin 1986 and 1991 showed that ‘crunchy’ typescontinued to contain more aflatoxin than‘smooth’ varieties (Ministry of Agriculture,Fisheries and Food, 1993). The maximumconcentrations of total aflatoxin found in thetwo surveys were similar, at 53 µg kg−1 in asample of crunchy peanut butter obtainedin 1986 and 51 µg kg−1 in a smooth samplecollected in 1991. The UK Committee onToxicity of Chemicals in Food, ConsumerProducts and the Environment (COT)expressed concern that there had been nodecrease in these levels since the previousreport, but anticipated reductions with theimplementation of new regulations (Ministryof Agriculture, Fisheries and Food, 1993).

Reports in 1990 drew attention to afla-toxin contamination of imported pistachionuts. UK surveillance conducted betweenMarch 1990 and April 1991 and between May1991 and April 1992 indicated that 52 and 28%,respectively, of samples exceeded the 4 µgkg−1 statutory limit (total aflatoxins) forfinished products. In addition, 38 and 25%,respectively, exceeded the 10 µg kg−1 limit inproducts destined for further processing.Elsewhere, there are similar reports of con-tamination of pistachio nuts, particularlysmall pistachio ‘scalpers’ in California, which

20-Feb-03 4

70 J.P.F. D’Mello



may contain total aflatoxin concentrations ofup to 149 µg kg−1. In The Netherlands, AFB1

levels as high as 165 µg kg−1 have beenreported for pistachio nuts, with much lowerconcentrations in shells (up to 8 µg kg−1). Inwhole dried figs, UK data (Ministry of Agri-culture, Fisheries and Food, 1993) showed thatbetween December 1988 and April 1992, thepercentage contaminated with aflatoxins(total) at levels above 4 µg kg−1 fell from 26 to16%. However, samples containing up to427 µg kg−1 were found. The incidence ofaflatoxins in fig paste samples above the 4 µgkg−1 level also fell during this period from 50

to 14%. The maximum concentration of totalaflatoxins found in fig paste also declinedfrom 165 to 15 µg kg−1. These findingsattracted comment by COT, who were clearlyconcerned by the high levels of contaminationof pistachio nuts, dried figs and fig pastes butwere satisfied that consignments exceedingthe 10 µg kg−1 limit were refused entry by theUK port health authorities. The results of arecent survey of Egyptian foods indicatedhigh incidence and unacceptable levels ofAFB1 in spices, herbs and medicinal plants. Aswill be seen later, contamination of spices isthe subject of scrutiny by EC authorities, but


20-Feb-03 4

Food Aflatoxin

Incidence ofcontamination

(%)Mean/range

(µg kg−1) Country

Maize

Maize-based gruelsPeanuts

Pistachio nuts

Peanut butter:‘smooth’‘crunchy’

Dried figsFig pasteDate fruits

Spices

TotalTotalB1

B2

B1

B2

TotalB1

B2

G1

G2

TotalB1

B2

G1

G2

TotalTotalB1

B1

TotalTotalB1

B2

G1

G2

TotalTotalTotalB1

G1

B1

19

8156

25

52

28–52

1128

716424

40

170–760–700–6

0–4280–160

0.002–19.70.8–161.6–161.6–81.6–163–48

0.8–10.90.2–1.70.1–21.80.4–4.14.1–224up to 149up to 1650.8–128

4.1–104.1–103.2–161.6–203.2–201.6–201.6–644–227

4.1–16511313325

ZambiaCosta Rica

Indonesiaa

NigeriaBotswana

Japan

UKCalifornia, USAThe NetherlandsJapan

UK

Botswana

UKUKUnited Arab Emirates

Egypt

aSee also Table 4.6.

Table 4.3. Aflatoxin contamination of foods.



there may be a case for surveillance of otherimported foods not currently controlled bylegislation.

Ochratoxin A

Ochratoxin A is ubiquitous in foods (Table4.4; see also D’Mello and Macdonald,1998), occurring principally in cereal grains(Vrabcheva et al., 2000), dried vine fruit(MacDonald et al., 1999) and green coffeebeans (Blanc et al., 1998). The relatively highvalues in Bulgarian cereals were associatedwith grain samples taken from villages with ahigh incidence of Balkan endemic nephro-pathy. A recent study in France indicatedconsistent contamination of cereals andoilseeds with OTA, the values ranging from0.6 to 12.8 µg kg−1 in positive samples. Inthe UK, OTA analyses of dried vine fruitimported from Greece and other countries(Table 4.4) indicated that 88% were contami-nated with levels in the range 0.2–53.6 µgkg−1. The OTA data for green coffee beansshown in Table 4.4 are at the lower end ofa range of other published values, whichincluded a maximum of 360 µg kg−1 (Blancet al., 1998).

Use of contaminated grain in brewingand as animal feed regularly results in transferof residues into beer and offal. Some OTAcontamination of porcine organs has beenreported in a survey conducted in the UK(Ministry of Agriculture, Fisheries and Food,1993). Of 104 samples of kidney, 12% werecontaminated with OTA at 1–5 µg kg−1, while

3% had concentrations of up to 10 µg kg−1.Of the black pudding samples analysed, 13%were contaminated with OTA in the range1–5 µg kg−1.

Citrinin often occurs with OTA in cerealgrains. In naturally contaminated samples ofBulgarian wheat, citrinin levels up to 420 µgkg−1 were detected (Table 4.4).

Patulin

The occurrence of patulin in fruit juice hasbeen a cause of concern in the UK and else-where in Europe (D’Mello and Macdonald,1998). In recent years, there has been amarked increase in the production of cloudyapple juices prepared by pressing the fruitand stabilizing with vitamin C prior topasteurization of the juice. The reduction inprocessing steps, as compared with the pro-cedure for production of clear juices, meansthat patulin losses during fining and filtrationare restricted, with higher residual levelsof the mycotoxin in the cloudy juices. Acomparison of the patulin concentrations inthe two types of juices has been publishedrecently for samples from the UK and Spain.Although the UK data were derived from arelatively small number of samples, it wasapparent that the incidence of patulincontamination was higher in cloudy juices,with a median value of 28 µg kg−1, comparedwith 0–10 µg kg−1 for clear juices. Four cloudysamples had patulin concentrations in excessof 50 µg kg−1, compared with only one of theclear juice samples. In two cloudy samples,

11-Mar-03 4

72 J.P.F. D’Mello

Source Food Ochratoxin A (range/mean) Citrinin

Bulgaria

UK

Greece

Several countriesThailand

WheatOatsBranWheatBarleyOatsCurrantsSultanasRaisinsGreen coffee beans

< 0.5–390.9–140

< 0.5–3.40.30.70.2

< 0.2–54< 0.2–18< 0.2–20

4.1–22.1

< 5–420< 5–230< 5–230

Table 4.4. Ochratoxin A and citrinin contamination of foods (µg kg−1).



patulin concentrations exceeded 151 µg kg−1.In a more extensive study in Spain, patulinwas detected in 82% of commercial samples(assumed to be clear for this comparison).However, 70% of these juices contained levelsof less than 10 µg l−1, although 22% had levelsof 10–50 µg l−1 and, in two samples, concen-trations of 164 and 170 µg l−1 were recorded.On a more reassuring note, patulin wasabsent in all 12 tested samples of apple foodfor children.

Trichothecenes and zearalenone

D’Mello and Macdonald (1998) provided anexhaustive survey of the global contamina-tion of cereal grains with trichothecenes andZEN. Recent data confirm the widespreaddistribution of these mycotoxins, particularlywith respect to DON, NIV and ZEN(Table 4.5). The levels of DON in Polishwheat and maize and in Japanese barley arestriking, but it will be noted that some sam-ples from the USA exceeded advisory limits.

Within-country variation in DON contamina-tion of wheat has also been observed. Highestlevels in the 1991 harvest in the USA wereseen in Missouri, North Dakota and Tennes-see. In the 1993 harvest, 86% of samples fromMinnesota and up to 78% of samples fromNorth and South Dakota had levels in excessof 2 mg kg−1. A comprehensive review oftrichothecene levels in Canadian grain isnow available (Scott, 1997), indicating highervalues for DON in cereal grains (Table 4.5)than those previously reported (see D’Melloand Macdonald, 1998). In Ontario, DON inci-dence was consistently higher for maize thanfor soft wheat over a 15-year period (Scott,1997). Of particular note are the lower levelsof DON in soft spring wheats over this period(Table 4.5). It may be concluded that DON isa frequent contaminant of Canadian cereals.

The predominant feature of ZEN distri-bution in cereal grains is its co-occurrencewith other Fusarium mycotoxins, includingtrichothecenes (Table 4.5). This observation isconsistent with the confirmed production ofZEN by virtually all toxigenic and plantpathogenic species of Fusarium (D’Mello et al.,


20-Feb-03 4

Country Cereal grains DON NIV ZEN

Germany

Poland

FinlandThe Netherlands

NepalJapan

USA

Canada

ArgentinaBrazil

BarleyWheatWheatMaize kernelsOatsWheatBarleyOatsRyeMaizeWheatBarleyWheat (winter), 1991Wheat (spring), 1991Wheat, 1993Barley, 1993Wheat (hard)Wheat (soft, winter)Wheat (soft, spring)MaizeWheatWheat

0.032–0.440.036–0.37

2–404–3201.3–2.6

0.020–0.2310.004–0.1520.056–0.1470.008–0.384

1.2–6.50.029–11.7

61–71< 0.1–4.9< 0.1–0.9< 0.5–18< 0.5–260.01–10.50.01–5.670.01–1.510.02–4.090.10–9.250.47–0.59

0.01

0.007–0.2030.030–0.1450.017–0.0390.010–0.034

0.01–4.414–26

0.16–0.40

0.005–0.0060.005–0.012

0.01–2

0.002–0.1740.004–0.0090.016–0.029

0.011

0.053–0.5111–15

0.04–0.21

Table 4.5. Natural occurrence of deoxynivalenol (DON), nivalenol (NIV) and zearalenone (ZEN) incereal grains (mg kg−1).



1997). The highest values for ZEN in Table 4.5(11 and 15 mg kg−1) relate to two barleysamples from the Fukuoka region of Japan(Yoshizawa, 1997).

Fumonisins

The widespread contamination of maize withfumonisins is unmistakable and likely toremain an issue of overriding concern. Recentsurveillance has confirmed the extensivedistribution of fumonisins, particularly inmaize produced in the tropics (Table 4.6,adapted from D’Mello and Macdonald,1998). In most instances, the predominant

fumonisin is FB1. Outstanding featuresinclude high FB1 concentrations in samplesfrom Thailand (Yamashita et al., 1995), China(Wang et al., 1995a) South Africa (cited byShephard et al., 2000) and Kenya (Kederaet al., 1999). Highest levels of FB2 werereported in Argentinian (Chulze et al., 1996)and South African samples. In the Philip-pines, Thailand and Indonesia, FB1 and FB2

occurred in over 50% of maize samples, whileincidence rates of 82–100% were recorded forsamples from Italy, Portugal, Zambia andBenin. In Honduras, Julian et al. (1995)detected FB1 in all 24 samples of maize tested.In Costa Rica, significant regional differenceswere observed in contamination of maizewith FB1, while in Mexico concern has been

20-Feb-03 4

74 J.P.F. D’Mello

Country FB1 FB2 FB3 Total

BeninBotswanaMozambiqueSouth Africa (Transkei)MalawiZambiaZimbabweTanzaniaKenyaHondurasMexicoArgentinaCosta RicaItalyPortugalUSAVietnamChinaChina

TaiwanThe PhilippinesThailandIndonesiaNepal

n.d.a–2,630,35–255

240–295< 50–46,900n.d.–11520–1,420,55–1,910,

n.d.–160110–12,000,68–6,555,

1,000–1,800b,,,85–8,791,

1,700–4,78010–2,330,90–3,370,

n.d.–350268–1,516,160–25,970,

< 500–8,800c ,< 500–5,700d ,< 500–7,200e ,

,0–1,14857–1,820,63–18,800,

226–1,780,

n.d.–680n.d.–7575–110

< 50–16,300n.d.–30n.d.–290n.d.–620n.d.–60

n.d.–11,300,

n.d.–520n.d.–1,080,

155–401160–6,770,

0–25558–1,21050–1,400

231–556

n.d.–3025–50

n.d.

n.d.–205n.d.

,n.d.–3,537

101–268, 110–4,130

35–305340–395

n.d.–145

,55–2,735n.d.–220

, 85–16,760

,10–2,850,90–4,450

,524–2,185,430–36,870

110–8,400

aNot detectable.bMasa and tortillas.cMaize (corn) meal.dUnfermented batter.eFermented batter.

Table 4.6. Worldwide contamination of maize kernels and products with fumonisins B1, B2 and B3, (µgkg−1). Data for maize products are identified by footnotes.



expressed at the higher levels in masa andtortillas compared with similar productsimported from the USA. The data for maizemeal and batter (Groves et al., 1999) preparedin the Shandong Province of China may alsobe viewed with disquiet. As with ZEN, adisturbing feature is the co-occurrence offumonisins with other mycotoxins.

Co-occurrence

The co-occurrence of several mycotoxins inthe same sample of cereal grains has pro-voked worldwide concern. Of considerablesignificance are consistent reports ofco-occurrence of Fusarium mycotoxins(D’Mello and Macdonald, 1998). In the Lublinregion of south-eastern Poland, type Atrichothecene contamination of barley grainwas linked with the natural incidence of FHB,in which the predominating organism wasF. sporotrichioides (Perkowski et al., 1997). Of24 barley grain samples, 12 were positive forT-2 toxin with a range of 0.02–2.4 mg kg−1.In five of these samples, co-contaminationwith HT-2 toxin occurred with a range of0.01–0.37 mg kg−1. The findings of anotherstudy in Poland indicated that infection withF. graminearum can result in contaminationof cobs simultaneously with DON and15-ADON. Concentrations of DON and15-ADON in Fusarium-damaged kernelsranged from 4 to 320 mg kg−1 (Table 4.5) andfrom 3 to 86 mg kg−1, respectively, but theaxial stems of the cobs were more heavilycontaminated at 9–927 mg kg−1 and 6–606 mgkg−1, respectively. A study of Japanese barleysamples confirmed the co-occurrence of DONwith NIV (Table 4.5; Yoshizawa, 1997). Inaddition, an appreciable number of the barleysamples were found with 3-ADON at levelsof up to 19 mg kg−1. In highly contaminatedgrains, a positive correlation occurredbetween levels of DON and its acetyl deriva-tives. DON levels were always higher thanthose of 3-ADON and 15-ADON, with ratiosranging from 3 to 155. Regional differenceswere also observed in that DON was themajor contaminant in grain from northerndistricts of Japan, whereas in central districts

NIV was the predominant trichothecene.These differences were correlated withchemotype variants of Fusarium species.Furthermore, Yoshizawa (1997) and Laurenet al. (1996) revealed the occurrence ofrelatively high levels of ZEN with DONand NIV in cereal samples from Japanand New Zealand, respectively (Table 4.5). Ofthe 29 cereal samples tested in The Nether-lands, 90 and 79% were positive for DON andNIV, respectively, with 76% containing bothmycotoxins together, while ZEN occurred asa third contaminant, albeit at low levels(Table 4.5).

In China, FB1 and AFB1 co-occurred in85% of maize samples (Wang et al., 1995a),while, in the Philippines, Thailand andIndonesia, FB1 and FB2 co-occurred withaflatoxins in 48% of maize samples. Thesefumonisins also co-occurred with NIVand ZEN (Yamashita et al., 1995). Multiplecontamination of maize with fumonisins,DON, NIV and AFB1 was also observedin north Vietnam (Wang et al., 1995b). Ofadditional concern is the co-occurrence ofFB1, fusaproliferin and beauvericin in Italiansamples of maize.

Uptake and Disposition

The principal route of exposure to myco-toxins in humans is through consumption ofcontaminated diets. Uptake of foodbornemycotoxins is implied from the appearanceof these compounds and associated deriva-tives (e.g. adducts) in body fluids. A widearray of factors may affect absorption. Forexample, FB1 absorption is greater in fastedthan in fed rats, with potentially profoundimplications for undernourished humans.Uptake of mycotoxins may also be affectedby the onset of other conditions such as gas-trointestinal disorders. Mycotoxin form caninfluence both uptake and disposal. Thus,studies with animal models indicate thathydrolysed FB1 is absorbed more readily thanFB1 itself, and urinary excretion is alsogreater.

Mycotoxin metabolism is an importantfeature preceding events such as carcino-


20-Feb-03 4



genesis and hepatotoxicity. The hepaticmetabolism of AFB1 exemplifies the diversereactions involving mixed-function oxidasesand cytosolic enzymes. In the case of AFB1, avariety of metabolites are produced, includ-ing AFM1, aflatoxicol and AFB1-8,9-epoxide.This epoxide form is a key intermediatewhich covalently binds to DNA to initiatecarcinogenesis (Smith, 1997). Alternatively,the epoxide may bind to proteins. Conjuga-tion with glutathione represents a detoxifica-tion route, whereas adduct formation withother proteins results in hepatotoxic effects.The major protein adduct in blood isAFB1–albumin and its level in humans isindicative of exposure to the mycotoxin.

Disposition of mycotoxins occurs primar-ily via the faeces and urine. However, inhumans, considerable quantities of aflatoxinsmust be ingested before they are detected inthe urine. Furthermore, energy–protein mal-nutrition may determine aflatoxin disposition(de Vries et al., 1990). Thus, in children withkwashiorkor, aflatoxins in urine disappeared2 days after rehabilitation on an aflatoxin-freediet, whereas, in those with marasmic kwashi-orkor, excretion continued for up to 4 days. Incontrast, faecal disposition of aflatoxin wasobserved up to the 9th day in kwashiorkor but

had ceased by the 6th day in children withmarasmic kwashiorkor. AFB1 and aflatoxicolwere the most frequently found form of afla-toxin in children with kwashiorkor, whileAFB1 occurred least frequently in stools fromsubjects with marasmic kwashiorkor, whichalso contained no aflatoxicol.

Toxicology

The classical assessment of toxicity of anycompound inevitably centres on the acquisi-tion of LD50 data (D’Mello and Macdonald,1998). These values of acute toxicity aresubject to wide variation, depending, forexample, on age, sex and size of animals.There are also distinct species differences insensitivity to a particular mycotoxin (Table4.7). Thus day-old ducklings are more sus-ceptible to AFB1 than laboratory animals.OTA is also acutely toxic, but its effects(together with that of citrinin) in the kidneyare of greater relevance to human health. Onthe other hand, ZEN is much less toxic butexerts profound effects on mammalianreproduction.

20-Feb-03 4

76 J.P.F. D’Mello

Mycotoxins LD50 data Other properties

Aflatoxin B1

Aflatoxin M1

Ochratoxin A

CitrininDeoxynivalenol

Diacetoxyscirpenol (DAS)and T-2 toxinZearalenone

Fumonisins

Tenuazonic acid

1–17.9 mg kg−1 BW (laboratory animals),0.5 mg kg−1 BW (ducklings)12–16 µg per duckling (newly hatched)

<6 mg kg−1 BW (pigs)

105–112 mg kg−1 BW (mice)70 mg kg−1 (mice)

23 and 5 mg kg−1 BW, respectively (mice)

2–10 g kg−1 BW (rodents)

No data

125–225 mg kg−1 BW (mice)

Hepatotoxin; teratogen;immunotoxin; carcinogenHepatotoxin; nephrotoxin;carcinogenNephrotoxin; teratogen;carcinogen;immunosuppressorNephrotoxinFeed intake inhibitor;teratogen; immunosuppressorInduces oral lesions

Inhibitor of reproduction;endocrine disruptorHepatotoxin; causes lesions inlungs and brainCauses emesis andcirculatory failure

aLD50 = median lethal dose; BW = body weight.

Table 4.7. Deleterious properties of mycotoxins as determined with animal models.a



Human Disorders

Mycotoxins have long been implicated inspecific human conditions (Table 4.8). How-ever, conclusive evidence for such an associa-tion has yet to emerge for several of thesedisorders.

Ergotism

One of the ancient European episodes ofmycotoxicoses in humans relates to ergotism(St Anthony’s Fire) caused by the bioactivealkaloids produced in the sclerotia of C.purpurea. The alkaloids cause constriction ofperipheral blood capillaries leading tooxygen starvation and gangrene of the limbs(Flannigan, 1991). Occasional cases ofergotism still occur to the present day.

Aflatoxicosis

Aflatoxins can induce acute effects inhumans, and field cases continue to occurdespite worldwide awareness of the toxico-logical and health implications. Thus, in 1974,an outbreak of liver disease occurred in Indiafollowing the consumption of mouldy graincontaining aflatoxins (see D’Mello and

Macdonald, 1998). Of 997 subjects, 97 werereported to have died during this episode.Principal pathological features in the liverincluded destruction of centrilobular zones,thickening of central veins and cirrhosis.Chronic aflatoxin exposure may modulateimmune function, thereby increasing sus-ceptibility to infection. Indeed, it has beensuggested that aflatoxins are the major causeof kwashiorkor in children.

Ochratoxicosis

Balkan endemic nephropathy is a chronicdisease occurring in rural populations ofBulgaria, Romania and the former state ofYugoslavia (see D’Mello and Macdonald,1998). In affected subjects, the kidneys aremarkedly reduced in size and, histologically,the disease is characterized by tubular degen-eration, interstitial fibrosis and glomerulardefects. Tubular function is also impaired.The similarities to porcine nephropathyare striking and have led to the conclu-sion that OTA is also the causative agent inBalkan endemic nephropathy. However, theco-occurrence of OTA with citrinin in cerealsfrom Bulgarian villages with a history ofBalkan endemic nephropathy suggests aninteraction between the two mycotoxins inthe aetiology of this condition (Vrabcheva


20-Feb-03 4

Mycotoxin Disease Food/source Countries

Ergot alkaloidsAflatoxins

Cyclopiazonic acidOchratoxins

CitreoviridinT-2 toxinFumonisins

Moniliformin

Ergotism (St Anthony’s Fire)Liver cancer; kwashiorkor,cirrhosis; acute hepatitis;Reye’s syndrome‘Kodua poisoning’Balkan (and possible Tunisian)endemic nephropathy‘Shoshin-kakke’Alimentary toxic aleukiaOesophageal cancerPrimary liver cancer‘Keshan disease’

RyePeanuts; maize

MilletCereal grains

RiceCereal grainsMaizeMaizeMaize

EuropeEast and West Africa;India; Taiwan; Thailand;the PhilippinesIndiaBulgaria; Romania; formerYugoslavia; TunisiaJapanFormer USSRSouth AfricaChinaChina

Table 4.8. Mycotoxins implicated in human disease.



et al., 2000). It will be noted that the twomycotoxins have similar properties in animalmodels (Table 4.7). A possible endemicochratoxin-related nephropathy has alsobeen suggested to occur in Tunisia. Affectedsubjects were classified into those withchronic interstitial nephropathy, chronic glo-merular nephropathy and chronic vascularnephropathy.

Cancer

Current concern over mycotoxins centres ontheir carcinogenic potential in humans. Intropical countries, particularly East and WestAfrica, India, Thailand, the Philippines andChina, aflatoxin exposure is a continuinghealth issue among the indigenous popula-tions. There is now good epidemiologicalevidence linking aflatoxin exposure with theincidence of liver cancer (Smith, 1997). In onestudy, it was possible to demonstrate thatmen were more sensitive than women to thecarcinogenic effects of aflatoxins but that inboth cases there was a linear effect of doseon the development of liver cancer. Theepidemiological data should be inter-preted with caution, as other factors such asmalnutrition and disease may have contrib-uted to the incidence of liver cancer. Further-more, interactions may occur. Thus, Wanget al. (1996) indicated that aflatoxin exposuremay enhance the carcinogenic potential ofhepatitis B virus. The generally acceptedorder of carcinogenicity is AFB1 > AFG1 >AFB2 > AFG2. In toxicological classification,AFB1 has been designated as a group 1 carcin-ogen (i.e. sufficient evidence in humans forcarcinogenicity), whereas AFM1 falls in thegroup 2B category (i.e. probable humancarcinogen).

Epidemiological evidence has alsobeen presented to link human oesopha-geal cancer in South Africa with dietaryexposure to the fumonisins. In addition, ithas been suggested that, in China, fumonisinsmay promote primary liver cancer initiatedby AFB1 and/or hepatitis B virus (Ueno et al.,1997).

Continuing Human Exposure toFoodborne Mycotoxins

Despite enhanced awareness and the adop-tion of legal or advisory guidelines, humanexposure to foodborne mycotoxins continueson a global scale, even in developed countries(see D’Mello and Macdonald, 1998). Recentevidence is summarized in Table 4.9 foraflatoxins and in Table 4.10 for OTA. Thetables are not designed to be exhaustive butrather illustrative of widespread exposure tothese mycotoxins.

The evidence of exposure generally isbased on mycotoxin residues in body fluids,mother’s milk and tissue specimens. In addi-tion, the association between mycotoxin expo-sure and cancer relies on presumptive intakeof contaminated foods, rather than directdeterminations of metabolites or DNAadducts. However, efforts are now focusingon measurements of the major adducts intissues and fluids.

Aflatoxins

The widespread contamination of maizeand peanuts with aflatoxin is reflected in theanalyses of faeces, urine, blood and breastmilk samples of people in different partsof Africa (Table 4.9). In addition to thefour forms of aflatoxin, metabolites such asaflatoxicol, AFM1 and AFM2 may appear inbody fluids and tissues. One study showedwidespread fetal exposure to aflatoxins inEast and West Africa, as demonstrated byanalysis of cord and maternal blood samples(Maxwell, 1998). Aflatoxins were alsodetected in breast milk samples of mothers.Thus, there is widespread pre- and post-natalexposure of infants to aflatoxins, which maypredispose children to infection. Indeed, ahypothesis has been advanced implicatingaflatoxin exposure with the pathogenesis ofkwashiorkor in African children. It will benoted that despite stringent EU regulations,detectable levels of AFB1–albumin adductshave been recorded for individuals in the UK(Table 4.9; Turner et al., 1998). Detection ofadducts is clearly a highly sensitive means of

20-Feb-03 4

78 J.P.F. D’Mello



assessing human exposure to aflatoxins, andit is likely that the UK results will be repli-cated in other developed countries. The UKdata suggest problems with sampling andmonitoring of regulated imported foodstuffs.Alternatively, or in addition, aflatoxin expo-sure may arise from the consumption of otherimported foods not currently controlled bylegislation. Spices, breakfast cereals andethnic foods might fall in this category.

Ochratoxin A

OTA exposure in humans is also widespread,as indicated by analyses of physiological

fluids (Table 4.10). However, geographicaldifferences may exist. Thus it has been con-cluded that levels of exposure are lower inJapan than in Europe (Ueno, 1998; Table4.10). Regional variations are also apparentwithin Europe. In Croatia, the highestblood levels of OTA were reported for inhab-itants living in villages noted for theincidence of endemic nephropathy (Radicet al., 1997). This observation is consistentwith the incidence of Balkan endemicnephropathy in the region. Levels insome Norwegian and Italian breast milksamples give cause for concern in thatthey imply infant OTA exposure exceedingthe tolerable daily intake (TDI) of 5 ngkg−1 bodyweight. In Tunisia, an endemic


20-Feb-03 4

Region/country Basis of evidence Observations

Kenya

East and West Africa

Sierra Leone

Sierra Leone

Nigeria

UK

Worldwide

Analysis of urine and stools

Analysis of body fluids

Analysis of cord blood andmaternal sera

Analysis of serum, urine andbreast milk

Analysis of autopsy kidneysamples

Serum albumin adducts

Hepatocellular carcinoma p53G→T transversions at codon249

Following feeding of aflatoxin-free diet, childrenwith kwashiorkor continued to excrete aflatoxinsin urine for 2 days; but those with marasmusexcreted aflatoxins for up to 4 days. Differencesalso seen in the type of aflatoxins discharged infaecesUp to 7 ng of AFM1 and 65 µg of AFB1 l−1 in cordblood; AFM1 and AFM2 detected at 12–1689 ngl−1 in maternal bloodAflatoxinsa detected in 91% of cord blood and75% of maternal blood samples. Highest valuesin cord blood recorded for AFB1, AFG1, AFG2,AFM1 and AFM2; in maternal blood, AFG2

detected most frequentlyMajor aflatoxins and metabolites detected inserum and urine of children of varying nutritionalstatus; 95% of breast milk samplescontaminated mainly with aflatoxinsa

Concentrations ranged from 6 pg g−1 for AFB2 to42,452 pg g−1 for AFG1 in kidneys of childrenwho died from kwashiorkor; comparable valuesin kidneys of children who died frommiscellaneous diseases were 1843 and23,626 pg g−1, respectively. Aflatoxicol detectedin kidneys from both groupsDetectable levels of AFB1–albumin adductsobserved in the UK populationIncreased proportions of p53 mutations inhepatocellular carcinoma probably attributableto aflatoxin exposure. Further studies requiredto confirm that p53 mutations are the fingerprintof aflatoxin exposure

aSee also Table 4.10.

Table 4.9. Continuing human exposure to aflatoxins.



OTA-related nephropathy is thought tooccur with similarities to the Balkan syn-drome. Three subsets were identified inaffected subjects: those with chronic inter-stitial nephropathy, chronic glomerularnephropathy and chronic vascular nephro-pathy. Patients with chronic interstitialnephropathy had the highest blood OTA lev-els in comparison with the other subgroupsor with the general population. However,even the latter group had overall blood OTAlevels which were in excess of those seen inSweden. In Sierra Leone, monitoring of breastmilk samples showed that only 9% weremycotoxin-free, with 35% containing OTA. Itis clear that infants and mothers in Sierra

Leone are exposed to OTA at levels greaterthan the current allowances of TDI (Table4.10). The urinary excretion of OTB by infantsin Sierra Leone was quantitatively similar tothat of OTA (Jonsyn, 1999).

Other individuals at risk may be patientswith renal disorders. Although OTA andcitrinin are established nephrotoxins, anyassociation with conditions such as the Balkanand Tunisian endemic nephropathies stillremains tentative. Thus, the higher incidenceand concentrations of OTA in blood ofpatients requiring haemodialysis and in thosewith urothelial cancer await elucidation todistinguish between cause and effect (Table4.10; Jimenez et al., 1998; Wafa et al., 1998).

20-Feb-03 4

80 J.P.F. D’Mello

Region/country Basis of evidence Observations

SwedenNorway

UKCroatiaItaly

Hungary

Spain

Egypt

Tunisia

Sierra Leone

Sierra Leone

Canada

Japan

Blood analysisBreast milk analysis

Blood and urine analysisBlood analysisBreast milk analysis

Blood and colostrum analysis

Blood analysis

Blood and urine analysis

Blood analysis

Blood and urine analysis

Breast milk analysis

Blood analysis

Blood analysis

OTA levels below 0.3 µg l−1

OTA detected in 33% of samples at levels of10–130 ng l−1; 12% of samples contained > 40 ngl−1

All blood and 92% of urine samples contained OTAOTA levels 2–50 ng ml−1

Significant exposure of babies to OTA at levelsexceeding tolerable daily intakes estimated fromanimal models52% of random blood samples with 0.2–12.9 ngml−1; 41% of colostrum samples with 0.2–7.3 ngml−1

53% of healthy donors and 78% of patientsundergoing haemodialysis positive for OTA; meanconcentrations 0.7 and 2 ng ml−1, respectivelyOTA levels of 0–10 and 0–8 ng ml−1, respectively,in blood and urine of patients with nephroticsyndrome; OTA levels of 0–3.4 and 0–0.3 ng ml−1,respectively, in urine of potential kidney donors andhealthy volunteersChronic forms of interstitial, glomerular andvascular nephropathy; OTA levels of 25–59 µg l−1

in patients with interstitial nephropathy, 6–18 µg l−1

in other groups and 0.7–7.8 µg l−1 in the generalpopulationOTA detected in 25% of cord blood samples atlevels of 0.2–3.5 ng ml−1; 24% of urine samplescontained OTA; 20% of urine samples containedOTBConfirmed exposure of infants to combinations ofOTA and various aflatoxinsOTA levels 0.6–1.4 ng ml−1 depending ongeographical locationOTA levels 0.004–0.28 ng ml−1

Table 4.10. Continuing human exposure to ochratoxins.



Combinations

The pre-natal and neonatal exposure of chil-dren in Sierra Leone to combinations ofaflatoxins and OTA is noteworthy (Jonsyn,1998). Of 64 cord blood samples analysed,94% contained either OTA, aflatoxins or both(Tables 4.9 and 4.10). It is suggested thatpre-natal exposure to such combinations mayhave resulted in low birth weights andpremature mortality of infants. Continuedexposure post-natally is likely in view of con-tamination of breast milk and cereal grainswith combinations of OTA and aflatoxins. Ofparticular concern, however, is the apparentabsence of direct determinations of humanexposure to combinations of aflatoxins andfumonisins in countries where peanuts andmaize constitute the staple foods (Tables 4.3and 4.6).

Tolerable daily intakes

The foregoing account demonstrates thatmycotoxin intake is inevitable even in coun-tries with stringent regulatory and processcontrols. In instances where there are ade-quate toxicological data, TDI have been esti-mated for humans (Table 4.11). As previouslystated, the actual intakes in many countriesmay exceed the TDI allowances. In the case ofAFB1, the TDI estimates have been based onstudies conducted in certain tropical coun-tries where infection with hepatitis B virus isan additional carcinogenic factor. In coun-tries where this virus is not a major risk, theTDI for AFB1 may be set considerably higher.It will be noted that, for the fumonisin carcin-ogens, TDI limits have yet to be established.

Regulatory Control

The ubiquitous distribution, acute effects andcarcinogenic potential of mycotoxins haveresulted in the imposition or adoption of reg-ulations for maximum permitted levels ofthese contaminants in primary foods andassociated products. Regulations also applyto feedingstuffs in order to reduce transmis-sion of mycotoxins to edible animal products.Van Egmond and Dekker (1995) indicatedthat 90 countries had regulations relating tomaximum permissible levels of mycotoxinsin various commodities. However, 13 coun-tries were known to have no regulations and,for some 50 countries, mostly in Africa, nodata were available. It is unlikely that the sit-uation has changed significantly since 1995.Virtually all developed countries have statu-tory regulations for the aflatoxins and advi-sory directives for a limited number of theother mycotoxins. Of particular concern,however, is the lack of statutory or advisoryregulations for control of fumonisins infoods.

Rationale

With the aflatoxins, the underlying rationaleis based on the need to reduce contaminationto ‘irreducible levels’, defined as the concen-tration which cannot be eliminated froma food without involving the completerejection of the food, thereby severely limit-ing the ultimate availability of major foodsupplies. However, in the evolution of statu-tory regulations for aflatoxins, the guidingprinciple has remained unaltered, which is toreduce contamination to the lowest level thatis ‘technologically achievable’, taking intoaccount advances in analytical methodolo-gies. In the preparation of proposals,comments received through groups such asthe World Trade Organization are taken intoaccount. The resulting regulations, therefore,represent a compromise between avoidanceof international trade disputes with producercountries and maintenance of consumerprotection.


20-Feb-03 4

Mycotoxin TDI

Aflatoxin B1

Ochratoxin ADeoxynivalenol

ZearalenoneFumonisins

0.11–0.19 ng1.5–5 ng1.5 µg (infants)3.0 µg (adults)100 ngInadequate data

Table 4.11. Tolerable daily intakes (TDIs) ofmajor mycotoxins (kg−1 body weight).



Statutory instruments

On a worldwide basis, statutory control onlyexists for the aflatoxins, and current regula-tions reflect evolution over time (see D’Melloand Macdonald, 1998). For example, in theUK, port authorities had applied a 10 µg kg−1

total aflatoxin limit to imported nuts anddried figs. Consignments exceeding thisvalue have been rejected since implementa-tion of regulations in 1988. The statutorylimits were amended and extended in 1992 toreflect recommendations that aflatoxin con-centrations in susceptible commodities bereduced to the lowest level ‘that is techno-logically achievable’, and to take account ofimprovements in analytical methodology.The regulations were extended to driedfig products, which were also considered tobe susceptible to aflatoxin contamination.The limits were reduced to 4 µg kg−1 fornuts, dried figs and their products for saleor for incorporation in any compound foodor for import for direct human consumption;for such imports intended for furtherprocessing before sale or incorporation inany compound food for human consump-tion, the limit for total aflatoxin was set at10 µg kg−1. In instances where aflatoxin levelsbetween 4 and 10 µg kg−1 were found, theimporter was required to give a writtenundertaking to process the batch so that itcomplied with the 4 µg kg−1 limit. Alterna-tively, the consignment could be returned tothe consignor, or used for a purpose otherthan human consumption, or destroyed.Schedules for food sampling and analysis ofaflatoxins were also provided. The latterincluded performance parameters forthe aflatoxin tests. For example, a detectionlimit of ≤ 2 µg kg−1 was set for foodsintended for direct human consumption. Thestatutory instruments also included regula-tions concerning importation procedure,authorized places of entry and duties ofauthorized officers.

In the UK, new regulations for aflatoxinswere introduced on 30 June 1999, bringinginto force an EC regulation setting maximumlimits for the foods most commonly contami-nated with aflatoxins, namely cereals, milk,

nuts, dried fruits and any products derivedfrom these commodities. The new regulationscontain separate maximum limits for AFB1 aswell as total aflatoxins. Higher limits aredesignated for foods which will undergo fur-ther processing. As before, the new regula-tions prescribe methods of sampling andanalysis of aflatoxins for use by law enforce-ment bodies. The 1992 UK regulations foraflatoxins were revoked on introductionof the new measures. A comparison of foodregulations for aflatoxins in force in selectedcountries is presented in Table 4.12.

Since human exposure to aflatoxins isdetermined partly by intake via milk andsince animal health and productivity may becompromised by these compounds, statutoryregulations also apply to feedingstuffs (Table4.13). Higher limits are allowed for animalfeeds than for human foods. It will be notedthat, in parts of Asia, permitted levels ofaflatoxins in human foods (Table 4.12) equalor exceed current norms for animal feeds inEU countries (Table 4.13).

Draft EU regulations for ochratoxin Aand deoxynivalenol

Proposed EU regulations for OTA limits inhuman foods and beverages are at anadvanced stage of preparation. In addition,action levels have been intimated for DON incereals and flour. Measures under discussionwithin European Commission expert com-mittees are presented in Table 4.14. Thereappears to be general agreement for statutorylimits for cereals, but no consensus has yetemerged on the precise levels. Discussionnecessarily has focused on limits for cerealssince these food items account for 50–70% ofOTA intakes in Europe. The position withderived cereal products such as bran war-rants further consideration. Recently, atten-tion has turned to limits for dried vine fruitand spices. It is not clear how the proposedaction levels for DON will be interpreted andused in the absence of sufficient data onwhich to base regulatory limits.

20-Feb-03 4

82 J.P.F. D’Mello




20-Feb-03 4

Aflatoxins: maximum levels

Country Foods B1 B1+B2+G1+G2 M1

European Union

South AfricaTaiwanThailandJapanUSA

Groundnuts, nuts and dried fruit, and processedproducts thereof intended for direct humanconsumption or as an ingredient in foodstuffsGroundnuts to be subjected to sorting or otherphysical treatment before human consumption or useas an ingredient in foodstuffsNuts and dried fruit to be subjected to sorting or otherphysical treatment before human consumption or useas an ingredient in foodstuffsCereals and processed products thereof intended fordirect human consumption or as an ingredient infoodstuffsSpicesa

MilkAll foodsCerealsAll foodsAll foodsAll foodsMilk

2

8

5

2

5

5

10

4

15

10

4

10

105020

20

0.05

0.5

aProposals under consideration.

Table 4.12. Examples of worldwide regulations for control of aflatoxins in human foods (µg kg−1).

Country Aflatoxins FeedingstuffsMaximum

levels Status

Indonesia

European Union

Taiwan

USA

B1+B2+G1+G2

B1

B1+B2+G1+G2

B1+B2+G1+G2

CopraGroundnutSunflower seed mealStraight feedingstuffs except: groundnut,copra, palm kernel, cottonseed, babassu,maize and products derived from theprocessing thereofComplete feedingstuffs for cattle, sheep andgoats (with the exception of completefeedingstuffs for calves, lambs and kids)Complete feedingstuffs for pigs and poultry(except those for young animals)Other complete feedingstuffsFeed, oilseed meals for feed under 4% ofmixed feedCottonseed mealMaize and groundnut products intended forbreeding beef cattle/pigs or mature poultryMaize and groundnut products intended forfinishing beef cattle

1000200905020

50

20

101000

300100

300

Proposal

Statutory

Statutory

Statutory

Table 4.13. Examples of worldwide regulations for aflatoxins in animal feedingstuffs (µg kg−1).



Advisory directives

In several countries, advisory directives existwhich are not enforceable by law. However,the limits suggested have been used to reducehuman exposure to mycotoxins. In theUSA and Canada, the advisory level forDON is 1000 µg kg−1 in finished wheatproducts such as flour and bran. For appleproducts including juice, cider and puree, theadvisory level for patulin in the UK is set at50 ppb.

Methodologies

Specific methodologies are prescribed forthe aflatoxins, OTA and DON. Of particularrelevance are the protocols for the legallycontrolled aflatoxins.

Sampling

Due to the heterogeneous distribution ofmycotoxins in foods, adequate sampling is aprimary consideration. In the official controlof aflatoxins in the EU, samples are takenaccording to prescribed methods. Three typesof samples are identified. An incrementalsample is the quantity of food taken from asingle position in a lot or sublot. An aggre-gate sample represents the combined total ofall the incremental samples taken from the

particular lot. Laboratory samples arederived from the mixed aggregate sample.The number and size of incremental samplesare laid down in the provisions, and specificprotocols are prescribed for nuts and driedfruit, milk and derived products. Treatmentof laboratory samples is also described indetail.

Analytical

Specific methods for the determination ofaflatoxins are not prescribed at the EC level,and laboratories may select any method pro-vided that it is consistent with a number ofcriteria based on recoveries and precisionparameters such as repeatability and repro-ducibility. However, adequate laboratorystandards must be demonstrated throughparticipation in proficiency testing and inter-nal quality control schemes. In practice threeprincipal methods are employed in aflatoxinanalysis worldwide. Thin-layer chromato-graphy (TLC) remains the method of choicein many countries and its efficacy has beenenhanced by new technology including theuse of immunoaffinity columns in clean-upand the application of densitometry forquantification. Other methods include liquidchromatography and enzyme-linked immu-nosorbent assay. For DON, the officialmethods used for regulatory purposes inthe USA and Canada are TLC and gaschromatography.

20-Feb-03 4

84 J.P.F. D’Mello

Suggested/actionlimits

Food/beverages Categories OTA DON

Cereals

Coffee

Dried vine fruitSpicesBeerWine

To be subjected to sorting or other physical treatment prior tohuman consumption or use as an ingredient in foodstuffsCereals and processed products thereof intended for directhuman consumption or use as an ingredient in foodstuffsGreen beansRoasted beans and coffee productsCurrants, raisins and sultanas

5

3

8410100.20.2–1.0

750a

500

aFlour used as raw material in food products; monitoring level for raw cereals.

Table 4.14. Permitted levels (µg kg−1) for ochratoxin A (OTA) in foods and beverages and action levelsfor deoxynivalenol (DON) in cereals: measures under discussion within the European Commission.



Compliance

Surveillance of food consignments at ports ofentry and from retail outlets has resulted in anumber of actions to ensure compliance withdirectives. It is instructive to consider recentmeasures concerning mycotoxin contamina-tion of nuts, fruit and apple juice (Table 4.15).The EC proposed and implemented actionslisted in Table 4.15 were conducted underArticle 10 of the Food Hygiene Directive(93/43/EEC). It is clear that despite increasedawareness and a protracted history, aflatoxincontamination of nuts and products is still

a formidable issue resulting in temporarysuspension of imports into EU MemberStates. Following the EU mission to Iran in1998, improvements in pistachio productionwere noted but it was recommended that thesuspension of imports be extended for anadditional period of 12 months to allow forfurther investigations. The specific points atissue were the development of an effectivetraceability system and improvements insampling methods. It should be noted that, inthe case of the peanut butter contamination(Table 4.15), no action was taken becausesamples were not taken for enforcement


20-Feb-03 4

Commodity Year

Countryinstigatingaction Issue Outcome

Peanuts(Egyptian)

Peanuts(Indian)

Peanut butter(retail own-brandsamples, UK)

Pistachio nuts(Iranian)Dried vine fruits

Apple juice

1999

1999

1994

1997

1997

1998

EU MemberStates

EU MemberStates

UK

EU MemberStatesUK

UK

Unacceptable incidenceand levels of AF.a Noassurances given byEgyptian authorities ofmeasures to reducecontaminationUnacceptable incidenceand levels of AFB1 (up to400 µg kg−1)

Five samples with AFlevels in excess of 4 µgkg−1 (one sample with20 µg AF kg−1)

Unacceptable incidenceand levels of AFB1

High-level consumerscalculated to exceed thetolerable intake of OTAb

Four samples of freshlypressed juices containedpatulin in the range73–171 ppb

Temporary suspension (initially for4 months) of imports into EUstates. EC mission to visit Egypt toconduct further investigations

Proposal for temporary suspensionof imports into EU states notimplemented. Indian authoritiesprovided assurances ofimprovements in productionpractices. Since then no furtherreports of contaminated peanutsfrom IndiaBatch with AF at 20 µg kg−1

withdrawn from sale; no actiontaken over other batches assampling did not comply withofficial regulationsTemporary suspension of importsinto EU countriesIndustry to implement testprocedures for OTA both inproducing countries and on importinto the UK; code of practicesuggested; surveillance to verifyefficacy of new measuresRelevant local authorities informedof results. Producers contacted todiscuss findings and to identifystrategy for reducingcontamination

aAflatoxin.bOchratoxin A.

Table 4.15. Actions resulting from mycotoxin surveillance of foods.



purposes; neither were the samples taken inaccordance with the relevant Regulations.Compliance can also be secured with non-statutory directives. For example, in 1997, itwas calculated that high-level consumers ofdried vine fruits in the UK were at risk dueto potentially widespread contamination ofthese food items with OTA. The dried-fruitindustry has responded with plans, as out-lined in Table 4.15. Patulin contamination ofapple juices in the UK has clearly declinedsince 1995 when 6% of samples containedlevels above the advisory limit. In 1995,action was taken to remove affected batchesfrom sale and to name those brands withunacceptable levels of contamination.

Preventive Strategies

The abiding principle in food safety must bethe prevention of contamination, as curativemethods are of limited efficacy. When fungi-cides are used effectively to control fungaldiseases of crop plants, then the risk may beminimized. However, under certain condi-tions, fungicides may enhance mycotoxinproduction (see D’Mello et al., 1998). In thecase of FHB of cereals, it is generally acceptedthat fungicide control is only partially

effective and the potential exists formycotoxin contamination of harvested grain(Table 4.16). There is growing optimism that,in terms of an environmentally acceptablesolution, plant selection and breeding offerconsiderable potential.

Experimental studies show that breedingmaize plants that are resistant to colonizationand ear rot caused by A. flavus generallyresults in lower contamination of grain withAFB1. Similarly, exploitation of genetic resis-tance to FHB in wheat has been used success-fully to reduce DON levels in the grain. Selec-tion of Chinese cultivars of wheat which areresistant to FHB can also result in lower levelsof DON in kernels compared with those ofgrain from susceptible Canadian cultivars.

Adequate storage of harvested grain,nuts and fruit is fundamental in the preven-tion of mycotoxins from storage fungi. Grainmoisture content and temperature are criticalfactors during storage. In addition, insect androdent invasion should be minimized as thesepests adversely affect the microclimate withingrain silos and also act as important vectorsfor transmission of fungal inoculum.

Prevention of aflatoxin-induced cancersis one strategy which may be advocated forsubjects at particular risk in Africa and Asia.Experimentally, it has been shown that

20-Feb-03 4

86 J.P.F. D’Mello

Class DescriptorExamples offungicides

Trichotheceneb

affected Conditions

IIIA

IIB

IIIA

IIIB

EffectivePartially effective (growth-dependentinhibition; mycotoxin residues possible)

Partially effective (direct inhibition ofmycotoxin synthesis; disease/infection/fungal growth possible)Ineffective

Stimulatory and/or inducing resistance

NoneTebuconazoleThiophanate-methylProchlorazThiabendazoleDicloran

PropiconazoleMorpholinesIprodioneTridemorphDifenoconazoleCarbendazimAzoxystrobin

—DONDON and NIV3-ADONDONDAS

DON3-ADONDONT-2 toxin3-ADONT-2 toxinT-2 toxin, DASand NEO

—Field trialField trialIn vitroField trialIn vitro

Field trialIn vitroField trialIn vitroIn vitroIn vitroIn vitro

aD’Mello et al. (2001).bDON = deoxynivalenol; NIV = nivalenol; 3-ADON = 3-acetyl deoxynivalenol; DAS = diacetoxyscirpenol;NEO = neosolaniol.

Table 4.16. Fungicide efficacy: a tentative classification for trichothecene control.a



antioxidants, when administered duringaflatoxin exposure, significantly reduced theincidence of hepatic cancers in rats. Aflatoxin–DNA adducts formed in the liver werealso reduced substantially by antioxidantprovision. In other studies with rats, anti-oxidants provided protection against freeradical-mediated lipid peroxidation inducedby DON or T-2 toxin. Other rat studies pointto the potential of dithiocarbamates in thechemoprevention of liver carcinogenesisinduced by AFB1. In trials with humansshowing detectable serum aflatoxin–albuminadduct levels, administration of theanti-schistosomal drug oltipraz may bebeneficial. At intermittent high doses, oltiprazinhibited activation of aflatoxin, while atsustained low doses the drug increasedelimination of the mycotoxin as the aflatoxin–mercapturic acid conjugate. At the practicallevel, prevention of aflatoxin-induced livercancer may be feasible through consumptionof brassica vegetables. Thus, rats given adiet with freeze-dried cauliflower showedreduced toxic effects of AFB1. Epidemiologicalevidence strongly indicates that consumptionof brassica vegetables is associated withreductions in the incidence of cancer at severalsites in humans, possibly through provisionof natural sulphur-containing compoundssuch as glucosinolates and S-methylcysteinesulphoxide.

Remedial Measures

Mycotoxin contamination of foods isunavoidable even with implementation ofgood agronomic practices. Once mycotoxincontamination of primary foods hasoccurred, a number of remedial options, ofvarying efficacy, may be considered.

Efficacy of processing technologies

Processing is an acceptable method of reduc-ing mycotoxin contamination, and currentlegislation and advisory directives (Tables4.12 and 4.14) distinguish between foodsintended for direct consumption and those

likely to warrant some kind of treatment.Conventional physical methods range frombasic to sophisticated. For example, sorting ofsusceptible foods such as nuts and cereals hasbeen advocated. It was observed that, whenpistachio nuts were sorted on the basis ofquality, a set of process streams with differ-ing aflatoxin levels were obtained. Theselevels were correlated with preharvest physi-cal damage, such as that caused by hullsplitting and insect invasion. Hull discolor-ation was also linked with high aflatoxincontent. In developing countries, hand sort-ing of visibly diseased maize kernels is aneffective method of reducing exposure tomycotoxins such as DON and fumonisins,but some prior training of personnel may beadvisable. Methods to remove DON fromcontaminated cereal grains primarily dependupon physical separation from the moreheavily contaminated outer layers of the ker-nels. Milling of grain to produce flour andextrusion to yield products for direct con-sumption are other examples with potentialto reduce contamination. The efficacy ofdecontamination varies with the proceduresused, but none of these has been shown to becompletely effective.

Processing of green coffee beans in themanufacture of soluble powder can markedlyreduce final OTA residues. Thus, Blanc et al.(1998) demonstrated that cleaning of beans bydensity segregation and air suction removedsome OTA, but the most significant reductionoccurred during roasting. Soluble coffeepowder contained only 16% of the OTAoriginally present in the beans.

Other treatments are known to beineffective. Thus in the preparation ofmaize-based products, baking or frying haslittle effect on fumonisin contamination.However, fumonisin–sugar molecules mayform during food processing, with the resultthat toxicity may be reduced. Brewing isanother ineffective process for reducingmycotoxin contamination of beer.

Ammoniation is a highly effective com-mercial process for detoxifying aflatoxins inanimal feed. As a result, AFM1 residues inmilk of dairy cows offered feeds decontami-nated in this manner are substantiallyreduced or eliminated altogether.


20-Feb-03 4



Conclusions

Current surveillance indicates unavoidable,widespread and continuing mycotoxin con-tamination of basic plant products, withglobal implications for human health.Concentrations of aflatoxins in maize andpeanut kernels regularly exceed safetythreshold limits. At particular risk areconsumers in warm and humid countrieswhere these foods constitute a signifi-cant proportion of the diet. OTA andtrichothecenes are ubiquitous, occurringprimarily in the major cereal grains. How-ever, use of grain contaminated with OTA inbrewing and as animal feed regularly resultsin transfer of residues into beer and offal.In addition, the occurrence of OTA in driedvine fruits and green coffee beans is anemerging issue currently under review inseveral EU countries. Of considerable con-cern is the widespread contamination ofmaize and associated products withfumonisins. Humans in the tropics andsouthern hemisphere countries, for example,are frequently exposed to various combina-tions of foodborne mycotoxins.

Contamination of foods with the majormycotoxins continues unabated even in spe-cific regions where the incidence of hepato-cellular and oespohageal cancers and nephro-pathy have been linked epidemiologicallywith consumption of, respectively, aflatoxins,fumonisins and ochratoxins. However, thereis evidence of chronic dietary exposure ina wider context, possibly associated withan array of other human disorders. Thus,foodborne aflatoxins may enhance the carci-nogenic potential of hepatitis B virus. It hasalso been proposed that kwashiorkor in Afri-can children may be a primary manifestationof aflatoxicosis. Moreover, it has beensuggested that pre-natal exposure to combi-nations of aflatoxins and ochratoxins resultin low birth weights and premature mortalityof infants in Sierra Leone and elsewhere.Post-natal exposure is inevitable in viewof contamination of breast milk and cerealgrains with combinations of aflatoxins andochratoxins. Of particular concern, however,is the absence of direct assessments of human

exposure to combinations of aflatoxins andfumonisins in countries where peanuts andmaize constitute the staple foods.

In Europe and elsewhere, legal and advi-sory regulations exist with the aim of reducingmycotoxin contamination of foods to the low-est level that is technologically achievable.However, even in these countries, there is evi-dence of general chronic exposure to particu-lar mycotoxins. The detection of specific afla-toxin–albumin adducts in the serum of UKindividuals suggests problems with samplingand monitoring of regulated imported food-stuffs. Alternatively, or in addition, aflatoxinexposure may arise from the consumption ofother imported foods, such as breakfast cere-als and spices, not currently controlled by legi-slation. The occurrence of OTA in the bloodand breast milk of donors in several Europeancountries underlines the need for statutorycontrol with respect to contamination of cere-als, dried vine fruits and coffee. The lack ofany legislative measures for aflatoxins andochratoxins in countries at greatest risk is anissue of considerable concern. As regards thefumonisins, there is an urgent need to intro-duce guidelines for its control in maize-basedfoods and to monitor exposure to this group ofcontaminants in vulnerable populations.

Acknowledgements

This work was partly funded by the ScottishExecutive Rural Affairs Department.

References

Abramson, D. (1997) Toxicants of the genusPenicillium. In: D’Mello, J.P.F. (ed.) Handbook ofPlant and Fungal Toxicants. CRC Press, BocaRaton, Florida, pp. 303–317.

Blanc, M., Pittet, A., Munoz-Box, R. and Viani, R.(1998) Behaviour of ochratoxin A during greencoffee roasting and soluble coffee manufac-ture. Journal of Agricultural and Food Chemistry46, 673–675.

Chulze, S.N., Ramirez, M.L., Farnochi, M.C.,Pascale, M., Visconti, A. and March, G. (1996)Fusarium and fumonisin occurrence inArgentinian corn at different ear maturity

20-Feb-03 4

88 J.P.F. D’Mello



stages. Journal of Agricultural and Food Chemis-try 44, 2797–2801.

de Vries, H.R., Maxwell, S.M. and Hendrickse, R.G.(1990) Aflatoxin excretion in children withkwashiorkor or marasmic kwashiorkor – aclinical investigation. Mycopathologia 110, 1–9.

D’Mello, J.P.F. and Macdonald, A.M.C. (1998) Fun-gal toxins as disease elicitors. In: Rose, J. (ed.)Environmental Toxicology: Current Develop-ments. Gordon and Breach Science Publishers,Amsterdam, pp. 253–289.

D’Mello, J.P.F., Porter, J.K. and Macdonald, A.M.C.(1997) Fusarium mycotoxins. In: D’Mello,J.P.F. (ed.) Handbook of Plant and FungalToxicants. CRC Press, Boca Raton, Florida,pp. 287–301.

D’Mello, J.P.F., Macdonald, A.M.C., Postel, D.,Dijksma, W.P.T. and Dujardin, A. (1998)Pesticide use and mycotoxin production inFusarium and Aspergillus phytopathogens.European Journal of Plant Pathology 104, 741–751.

D’Mello, J.P.F., Macdonald, A.M.C. and Rinna, R.(2001) Effects of azoxystrobin on mycotoxinproduction in a carbendazim-resistant strainof Fusarium sporotrichioides. Phytoparasitica 29,431–441.

Flannigan, B. (1991) Mycotoxins. In: D’Mello, J.P.F.,Duffus, C.M. and Duffus, J.H. (eds) Toxic Sub-stances in Crop Plants. The Royal Society ofChemistry, Cambridge, pp. 226–257.

Groves, F.D., Zhang, L., Ross, P.F., Casper, H.,Norred, W.P. and Fraumeni, J.F. (1999)Fusarium mycotoxins in corn and cornproducts in a high-risk area for gastric cancerin Shandong Province, China. Journal of theAssociation of Official Analytical Chemists Inter-national 82, 657–662.

Jimenez, A.M., Lopez de Cerain, A., Bello, J. andCreppy, E.E. (1998) Exposure to ochratoxin Ain Europe: comparison with a region of north-ern Spain. Journal of Toxicology – Toxin Reviews17, 479–491.

Jonsyn, F.E. (1998) Evidence of an early exposure tocarcinogens and other toxic compounds byneonates in Sierra Leone. Journal of Nutritionaland Environmental Medicine 8, 213–218.

Jonsyn, F.E. (1999) Intake of aflatoxins andochratoxins by infants in Sierra Leone: possibleeffects on the general health of these children.Journal of Nutritional and EnvironmentalMedicine 9, 15–22.

Julian, A.M., Wareing, P.W., Phillips, S.I., Medlock,V.F.P., MacDonald, M.V. and del Rio, L.E.(1995) Fungal contamination and selectedmycotoxins in pre- and post-harvest maize inHonduras. Mycopathologia 129, 5–16.

Kedera, C.J., Plattner, R.D. and Desjardins, A.E.(1999) Incidence of Fusarium spp. and levelsof fumonisin B1 in maize in western Kenya.Applied and Environmental Microbiology 65,41–44.

Lauren, D.R., Jensen, D.J., Smith, W.A., Dow, B.W.and Sayer, S.T. (1996) Mycotoxins in NewZealand maize: a study of some factorsinfluencing contamination levels in grain. NewZealand Journal of Crop Horticultural Science 24,13–20.

MacDonald, S., Wilson, P. and Barnes, K. (1999)Ochratoxin A in dried vine fruit: methoddevelopment and survey. Food Additives andContaminants 16, 253–260.

Maxwell, S.M. (1998) Investigations into thepresence of aflatoxins in human body fluidsand tissues in relation to child health inthe tropics. Annals of Tropical Paediatrics 18,S41–S46.

Ministry of Agriculture, Fisheries and Food (1993)Mycotoxins: Third Report. Food SurveillancePaper No. 36. HMSO, London.

Moss, M. (1996) Mycotoxins. Mycological Research100, 513–523.

Oyelami, O.A., Maxwell, S.M. and Adeoba, E. (1996)Aflatoxins and ochratoxin A in the weaningfood of Nigerian children. Annals of TropicalPaediatrics 16, 137–140.

Perkowski, J., Jelen, H., Kiecana, I. and Golinski, P.(1997) Natural contamination of spring barleywith group A trichothecene mycotoxins insouth-eastern Poland. Food Additives andContaminants 14, 321–325.

Radic, B., Fuchs, R., Peraica, M. and Lucic, A. (1997)Ochratoxin A in human sera in the area withendemic nephropathy in Croatia. ToxicologyLetters 91, 105–109.

Scott, P.M. (1997) Multi-year monitoring ofCanadian grains and grain-based foods fortrichothecenes and zearalenone. Food Additivesand Contaminants 14, 333–339.

Shephard, G.S., Marasas, W.F.O., Leggott, N.L.,Yazdanpanah, H., Rahimian, H. and Safavi, N.(2000) Natural occurrence of fumonisins incorn from Iran. Journal of Agricultural and FoodChemistry 48, 1860–1864.

Smith, J.E. (1997) Aflatoxins. In: D’Mello, J.P.F. (ed.)Handbook of Plant and Fungal Toxicants. CRCPress, Boca Raton, Florida, pp. 269–285.

Turner, P.C., Dingley, K.H. and Garner, C.R. (1998)Detectable levels of serum aflatoxin B1–albumin adducts in the United Kingdompopulation: implications for aflatoxin B1

exposure in the United Kingdom. Cancer Epide-miology, Biomarkers and Prevention 7, 441–447.


20-Feb-03 4



20-Feb-03 4

Ueno, Y. (1998) Residue and risk of ochratoxin A inhuman plasma and beverages in Japan. Myco-toxins 47, 25–32.

Ueno, Y., Iijima, K. and Wang, S.D. (1997)Fumonisins as a possible contributory riskfactor for primary liver cancer: a 3-year studyof corn harvested in Haimen, China, by HPLCand ELISA. Food and Chemical Toxicology 35,1143–1150.

Van Egmond, H. and Dekker, H. (1995) Worldwideregulations for mycotoxins in 1994. NaturalToxins 3, 332–336.

Vrabcheva, T., Usleber, E. and Dietrich, R. (2000)Co-occurrence of ochratoxin A and citrininin cereals from Bulgarian villages with ahistory of Balkan endemic nephropathy.Journal of Agricultural and Food Chemistry 48,2483–2488.

Wafa, E.W., Yahya, R.S., Sobh, M.A. and Creppy,E.E. (1998) Human ochratoxicosis andnephropathy in Egypt: a preliminary study.Human and Experimental Toxicology 17, 124–129.

Wang, D.-S., Liang, Y.-X., Iijima, K., Sugiura, Y.,Tanaka, T., Chen, G., Yu, S.-Z. and Ueno, Y.

(1995a) Co-contamination of mycotoxins incorn harvested in Haimen, a high risk area ofprimary liver cancer in China. Mycotoxins 41,67–70.

Wang, D.-S., Liang, Y.-X., Chau, N.T., Dien, L.D.,Tanaka, T. and Ueno, Y. (1995b) Naturalco-occurrence of Fusarium toxins and aflatoxinB1 in corn for feed in north Vietnam. NaturalToxins 3, 445–449.

Wang, L.-Y., Hatch, M., Levin, B. and Santella, R.M.(1996) Aflatoxin exposure and risk ofhepatocellular carcinoma in Taiwan. Inter-national Journal of Cancer 67, 620–625.

Yamashita, A., Yoshizawa, T., Aiura, Y., Sanchez,P.C., Dizon, E.I., Arim, R.H. and Sardjono(1995) Fusarium mycotoxins (fumonisins,nivalenol and zearalenone) and aflatoxinsin corn from southeast Asia. Bioscience,Biotechnology and Biochemistry 59, 1804–1807.

Yoshizawa, T. (1997) Geographic difference intrichothecene occurrence in Japanese wheatand barley. Bulletin of the Institute of Compre-hensive Agricultural Sciences Kinki University 5,23–30.

90 J.P.F. D’Mello



5 Pesticides: Toxicology and Residuesin Food

P. Cabras*Dipartimento di Tossicologia, Università di Cagliari, Viale Diaz 182,

09126 Cagliari, Italy

Introduction

The term pesticides includes all chemical,natural or synthetic substances used to fightparasites on crops. Though pesticides areused mainly for this purpose, they can also beused to fight the carriers of illnesses such asmalaria, yellow fever, typhoid fever, etc., oreven against domestic insects. About 20%of the production of insecticides is used forthis purpose. The first organic pesticide to beintroduced on the market by Geigy in 1939was dichlorodiphenyltrichloroethane (DDT)as a result of systematic research on its insectkilling activity by the Swiss entomologistPaul Müller. Till then, the substances avail-able to fight crop parasites were very limited,and almost all inorganic, e.g. sulphur(reported by Homer in 1000 BC), arsenic (rec-ommended by Pliny in 50 BC to kill insects)and a few natural insect-killing substances,such as pyrethrum, rotenone and nicotine. Afew catastrophes, such as the destruction of aharvest of potatoes by Phytophthora infestansin Ireland in 1845, causing a million deathsand driving a 1,500,000 people to emigrate,and the destruction of French vineyards bydowny mildew, which was imported fromthe USA in 1878, led to the development ofsignificant research in mineral chemistry for

plant protection. The fungitoxic activity ofcopper was discovered casually by Millardetin 1882. He observed that the rows of grape-vine along roads that were treated withcopper sulphate and lime to discouragetrespassers were protected from Plasmoparaviticola and, based on this information, hedeveloped the Bordeaux mixture, containingcalcium hydroxide and copper sulphate, withwhich he managed to control this pathogen.DDT, on the other hand, was the result of sys-tematic research that opened up a new meth-odology in the research on pesticides. Beforebeing used in agriculture, DDT was appliedextensively against the carriers of diseasesduring and after the Second World War.Diseases such as malaria and typhoid feverwere eliminated in many areas where theyhad been endemic. Paul Müller was awardedthe Nobel Prize for Medicine in 1948 for thisdiscovery, which saved millions of lives.

The extraordinary success of DDT againsta host of insects harmful both to agricultureand to human health led to the developmentof other synthetic products. At present thenumber of compounds marketed around theworld as pesticides is about 1300 (Tomlin,1997). Due to their heterogeneous nature,these compounds are difficult to classify;they are normally classified, according to


20-Feb-03 5




their target, as insecticides, fungicides andherbicides. Other less important typologies(4.7% of the world market in 1998; WoodMackenzie, 1999), such as nematocides,fumigants, growth regulators, etc., comeunder the general classification of ‘others’.The term insecticides also includes acaricides.

Insecticides

The world market for insecticides in 1998 wasUS$6930 million or 23.9% of the total marketvalue (Table 5.1). With 37.1% in the 1960s and1970s, insecticides had the largest marketshare among the pesticides. This was laterreduced progressively to 34.7% in 1980,29.0% in 1990 and 23.9% in 1998 (WoodMackenzie, 1999).

Most insecticides come under one of thefollowing five chemical classes: organochlorinecompounds, organic phosphorus compounds,carbamates, pyrethroids and benzoylureas.The term ‘others’ includes stannic organiccompounds such as fenbutatin oxide, growthregulators such as cyromazine, etc.

As can be seen from the data reported inTable 5.2, from a commercial point of vieworganophosphorus compounds are the most

important class with 37.2% of the market,followed by pyrethroids and carbamates with18.3 and 13.9%, respectively. Organochlorinecompounds and benzoylureas are less impor-tant. The decline in the use of organochlorinecompounds can be attributed to the fact thatsome of them, such as DDT, aldrin, dieldrin,eldrin, etc., have been banned all over theworld, while the benzoylureas have only beenintroduced recently. Fruit and vegetables arethe crops that take up most of the pesticideswith 38.9%, followed by cotton, rice andmaize with 22.8, 16.1 and 9.4%, respectively.Ninety-three per cent of the demand forpesticides for rice is located in Asia, whichaccounts for the fact that Asia has the largestworld consumption of pesticides (Table 5.2).

Organochlorines

DDT is the historic predecessor of syntheticpesticides and organochlorine compounds. Itwas followed in fast succession by other mol-ecules belonging to the same chemical family,such as lindane (1942), aldrin (1948), dieldrin(1949) and endrin (1951). The characteristicsshared by this chemical class of pesticides istheir effectiveness towards numerous insect

20-Feb-03 5

92 P. Cabras

Year 1970 1980 1990 1998

Value ($ billion)Insecticides (%)Fungicides (%)Herbicides (%)Others (%)

2.637.122.234.85.9

11.434.718.841.05.5

26.129.021.044.06.0

29.023.919.551.94.7

Table 5.1. The development of the pesticide market.

Areas % Crops % Classes %

Western EuropeEastern EuropeNorth AmericaFar EastLatin America

Rest of the world

15.83.9

23.929.613.8

13.0

RapeSugarbeetCottonRiceFruit andvegetablesCerealsSoybeanMaize

1.33.4

22.816.138.9

5.32.79.4

OrganophosphatesPyrethroidsCarbamatesOrganochlorinesBenzoylureas

Others

37.218.313.92.52.7

25.4

Table 5.2. World insecticide market in 1998.



species, their high persistence and theirlipophilicity. Though these characteristicswere considered ideal for an insecticide ini-tially, they were soon found to be negativebecause of their persistence in the environ-ment, and their tendency to accumulate in thefood chain. Though not lethal, they directlyor indirectly affected the fertility and repro-duction of many wild species. For this reason,DDT and organochlorine compounds havebeen banned in agriculture since 1973 andheavily limited in the fight against thecarriers of diseases of mankind. Since themid-1980s, the use of DDT has been bannedin agriculture in all countries of the world.

Chlorinated insecticides are a hetero-geneous group of compounds belongingto three different chemical classes: thediphenylethanes, the cyclodienes and thecyclohexanes (Fig. 5.1). The diphenylethanesinclude DDT, dicofol and methoxychlor.

Synthesized DDT was a mixture of theisomers pp´ (75–80%) and op´ (15–20%), and

could contain up to 4% 4,4′-dichlorodiphenylacetic acid (pp´ DDA) as an impurity. The gen-erally accepted main metabolic route includesthree main processes: (i) dehydrochlorinationto 1,1-dichloro-2,2-bis(4-chlorophenyl)ethyl-ene (DDE); (ii) reductive dechlorination to1,1-dichloro-2,2-bis(4-chlorophenyl)ethane(DDD); and (iii) oxidation of DDD to DDA(Fig. 5.2). DDT and its major metabolites DDEand DDD are lipophilic compounds andtend to accumulate in body fats. DDT tendsto degrade very slowly in the environment.A half-life of 4.3–5.3 years has been calculatedin soil (Woodwell et al., 1971) and of15 years in seawater (Edwards, 1973). Themetabolite DDE is also very persistent. InDDT, the substitution of a hydrogen atomin position 1 with a hydroxy group, withformation of dicofol, radically changes thestability of the molecule, which tends todegrade very rapidly, with formation of4,4´-dichlorobenzophenone (Roberts andHutson, 1999). For this reason, dicofol is still

Pesticides: Toxicology and Residues in Food 93

20-Feb-03 5

Fig. 5.1. Structure of organochlorine insecticides.



commonly used as an insecticide in agrariancultures.

Aldrin, which degrades rapidly andforms its epoxide dieldrin by hydroxylation,is very stable in the environment. A half-lifeof 5 years in the soil has been calculated fordieldrin. Endrin is a stereoisomer of dieldrin.They are now used only in a very few specialcases such as the control of termites. Unlikethe other cyclodienes, endosulphan showsmoderate stability; in fruit and vegetables ittends to degrade and form the correspondingsulphate with half-lives mostly rangingbetween 3 and 7 days.

Hexachlorocyclohexane (HCH) mainlycontains four isomers (α, β, γ and δ). Theisomer γ, lindane, which is the active isomer,has been isolated by crystallization from thisproduct. Lindane is the least persistent amongthe organochlorine compounds.

Organophosphates

Organophosphorus insecticides (OPs) werefirst synthesized at Bayer in Germany in1937. Due to their high toxicity, they weredeveloped during the Second World War aschemical weapons. In 1944, the insecticideactivity of parathion, the first marketedorganophosphorus insecticide, was discov-ered. Thanks to its remarkable efficacy, widerange of action and fast degradation in the

environment, it rapidly became widespreadnotwithstanding its high toxicity. The num-ber of OPs registered in various parts of theworld has increased rapidly, and has nowreached about 250. At present, they are themost widely marketed insecticides (37.2%).The general structure of an OP is representedby the following scheme:

where R and R1 are alkyl groups which couldbe bonded directly to phosphorus or throughatoms of S, O and N (Fig. 5.3).

Further in-depth reading on the chemis-try and biochemistry of OPs is available in thebook by Fest and Schmidt (1982).

Carbamates

The first carbamate insecticide, carbaryl, wasdeveloped by Union Carbide in the USAin 1953. Within a few years, a number ofinsecticides of the same class followed. Thesecompounds generally present low toxicityfor mammals, and many of them aresystemic. Thanks to the latter property,insects that develop in the roots can becontrolled. Chemically they are divided intothree classes: N-methylcarbamate (carbaryl),

20-Feb-03 5

94 P. Cabras

Fig. 5.2. Metabolism of DDT.




20-Feb-03 5

Fig. 5.3. Structure of organophosphorus insecticides.



N,N-dimethylcarbamate (pirimicarb) andoxime-carbamate (methomyl) (Fig. 5.4).

Pyrethroids

Pyrethroids are the synthetic analogues ofpyrethins, which are natural constituentsof the flowers of Tanacetum cinerariae-folium.Since the natural insecticide, pyrethrum, isextremely labile to light, it was unsuitablefor use in the field. However, when thestructure of pyrethrin II, the most effectivecomponent of pyrethrum, was modified,and its stability to light improved, thesecompounds became suitable to be used inthe field. The first synthetic pyrethroid,fenvalerate, was put on the market in 1978,

and today the class includes 42 activeingredients. Thanks to the discovery ofthe importance of the stereochemistry ofmolecules for the bioactivity and toxicity inmammals, it became possible to use thesecompounds in the field at very low doses(deltamethrin, which is the isomer 1RcisαS,is used at 12 g ha−1, since the LD50 for the flyis 0.0003 µg). Another very important aspectis their low toxicity in mammals. For thesereasons, after the OPs, pyrethroids are themost widely used insecticides (18.3% of theinsecticide market). They can be grouped intotwo classes containing 3-phenoxybenzylicalcohol (permethrin) and α-cyano-3-phenoxybenzylic alcohol (cypermethrin,deltamethrin) (Fig. 5.5). Since pyrethroidscannot penetrate the plant, their action ismainly by contact, which is favoured by their

20-Feb-03 5

96 P. Cabras

Fig. 5.4. Structure of carbamate insecticides.



liposolubility, which allows them to pene-trate the layer of epicuticular waxes.

Benzoylureas

This class of compounds was discovered bychance in the 1970s. During a programmedsynthesis between dichlobenil derivativesand fenuron, a product without any herbi-cidal activity but with a very high insecticidalactivity was obtained. The first compoundof this class to be out on the market wasdiflubenzuron in 1975. At present, there areten benzoylureas on the market (Fig. 5.6). Themechanism of action of the benzoylureas iscompletely different from that of the otherknown chemical classes. The compounds of

this class act on the formation of chitin,hindering the development of larvae duringmoult (by causing the imperfect formationof the new cuticle) and causing their death.For this reason, they are classified as insectgrowth modulators. These pesticides are notsystemic and they exert their action mainlyby ingestion.

Toxicology

Most insecticides are neurotoxic and act bypoisoning the nervous system of the targetorganisms. Moreover, since they are notselective, they also act on non-target species.The central nervous system (CNS) of insectsis highly developed and not very different


20-Feb-03 5

Fig. 5.5. Structure of pyrethroid insecticides.



from that of mammals. Therefore, chemicalcompounds that act on the nervous systemof insects also have similar effects on man.DDT acts by causing a disturbance in thesodium balance of the nerve membranes.Because of its high persistence in theenvironment, DDT tends to bioaccumulate inthe food chains. In mammals, it undergoesslow biotransformation, forming a verystable metabolite, DDE. Due to its lipophilicproperties, DDT tends to accumulate in lipid-rich tissues (liver, kidney, nervous and fattytissue). In the 1950s and 1960s, when DDTwas used extensively, the accumulated levelsin the fatty tissues were of the order of 5and 15 mg kg−1 for DDT and its metabolites,respectively (Morgan and Roan, 1970).Today, only traces of DDT (2 mg kg−1 ofits metabolites) are observed in humanfatty tissue (Stevens et al., 1993). Studiescarried out on wild species have shown thatorganochlorine insecticides interfere directlyor indirectly with their fertility and reproduc-tion, in particular birds and fish (Stickel, 1968;

Longcore et al., 1971). Among organochlorinepesticides, the most toxic are the cyclodienes,with extremely low acceptable daily intakes(ADIs) (0.0001–0.0002 mg kg−1 body weight(BW), while the least toxic is DDT, withone of the highest ADIs of all insecticides.Cyclodienes are a major hazard to profession-ally exposed individuals, since, unlikeDDT, they are easily absorbed through theskin.

Though very different structurally, phos-phoric and carbamic acid esters have thesame mechanism of action. They inhibit theenzyme acetylcholinesterase, which degradesthe neurotransmitter, acetylcholine, causingthe latter to accumulate, leading to manifesta-tions of intoxication.

In OPs, metabolism is a very importantfactor for toxicity. A prerequisite of its toxicaction is the oxidation of the thionatesto the corresponding phosphates. Thusparathion is oxidized to paraoxon, whichexerts toxic action. The toxicity of organo-phosphorus compounds and carbamates

20-Feb-03 5

98 P. Cabras

Fig. 5.6. Structure of benzoylurea insecticides.



varies depending on their structure (Table5.3). In fact, in the same class, there arehighly toxic compounds (e.g. carbofuranand aldicarb representing the carbamates;parathion and azinphos methyl representingthe OPs), and poorly toxic compounds(carbaryl for the carbamates; malathion forthe OPs).

The mechanism of action of pyrethroidsis different from that of the OPs and carba-mates, but very similar to that of DDT. Theytoo close the sodium channels. Benzoylureasdo not have a toxic mechanism towardsinsects, but they act on them as inhibitorsof the biosynthesis of chitin. They are there-fore poorly toxic towards mammals. Further


20-Feb-03 5

ClassLD50

(mg kg−1 rats)NOEL

(mg kg−1 rats)ADI (mg kg−1

BWb)Toxicityclass

Organochlorine compoundsAldrinDDTDicofolDieldrinEndosulphanEndrinγ-HCH (lindane)

Organophosphorus compoundsAzinphos methylChlorpyrifosDimethoateFenitrothionFenthionMalathionMethamidophosParathionQuinalphosTetrachlorvinphos

CarbamatesCarbarylCarbofuranEthiofencarbMethiocarbPirimicarbPropoxur

PyrethroidsCypermethrinDeltamethrinFenvalerateTau-fluvalinatePermethrin

BenzoylureasDiflubenzuronFlufenoxuronHexaflumuronTeflubenzuronTriflumuron

38–67113–118

57837–87

7010–4088–270

9135–163

387250250

1375–2800202

17504000–5000

8508

2002014750

250–4150135–5000

451261

430–4000

> 4640> 3000> 5000> 5000> 5000

15

15

25

5—5

10< 5100

223

125

20020

33067

250200

7.51

2501

100

4050758

20

0.0001c

0.020.0020.0001c

0.0060.00020.008

0.0050.010.0020.0050.0070.020.0040.004——

0.010.0020.10.0010.020.02

0.050.010.020.010.05

0.02——

0.010.007

IIIII

II

II

IbIIIIIIIIIIIIbIaIIIII

IIIbIIIIIIII

IIIIIIIIII

IIIIIIIIIIIIIII

aTomlin (1997).bBody weight.cAddition of aldrin + dieldrin.

Table 5.3. Mammalian toxicology of insecticides.a



details on the toxicology of insecticides maybe found in Ecobichon (1997).

Fungicides

The expenditure on fungicides in 1998 wasUS$5640 million, or 19.5% of the worldmarket value. This market share has beenconstant since the 1970s, fluctuating around20% (Table 5.1). The most importantmarket is the fruit and vegetable market,which on its own accounts for almost 50%,followed by cereals and rice. Europe is themain consumer of fungicides, since its cropsare mainly fruit and vegetables (Table 5.4).The most widely used synthetic fungicides,apart from the traditional inorganic com-pounds (7.3%), belong to the chemical classesof triazoles (19.5%), dithiocarbamates(14.1%), anilinopyrimidines (8.4%), strobi-lurines (7.4%) and benzimidazoles (5.9%).

Inorganic fungicides

This group of fungicides includes sulphurand copper salts. Sulphur has been used sincethe time of Homer. The sulphurs available onthe market are extremely pure (99.5–100%)since they must be free from selenium,which is harmful to man and animals. Thefungicidal power of sulphur depends ontemperature, the fineness of the particles andrelative humidity. Fungicidal activity startsat 10–12°C with the finest sulphurs and at

18–20°C with the coarser ones, and pro-gressively increases up to 40°C. Their actiondecreases on increasing the humidity. Itshould be mentioned that at high tem-peratures sulphurs are toxic to plants andtherefore applications should be made earlyin the morning in summer.

Copper is included in fungicidal formula-tions as oxychloride (Cu2Cl(OH)3), sulphate(CuSO4·5H2O) or hydroxide (Cu(OH)2). Itscontinued use has led to a significant increasein copper levels in the soil, which have causedecotoxicological problems. Copper is currentlybeing monitored with a view to limiting itsuse.

Dithiocarbamates

Zineb, which appeared on the market in1948, was the first synthetic organiccompound to be used in the control ofcryptogamic diseases. This active ingredi-ent was followed by other compoundsderived from dithiocarbamic acid thatbelong to two groups: the EBDCs(ethylenebisdithiocarbamates) (maneb andmancozeb) and the dialkyl dithiocarbamates(thiram and ziram) (Fig. 5.7). These com-pounds are not systemic and act by leafcontact. One of the degradation products ofthe EBDCs is ethylene thiourea (ETU), apotentially carcinogenic product that formsduring normal storage conditions, especiallywith increased humidity. This product is alsofound in the formulation as an impurity. Inorder to limit its presence as a residue in

20-Feb-03 5

100 P. Cabras



Rest of the world

42.22.8

12.328.111.6

3.0

ColzaSugarbeetCottonRiceFruit andvegetablesCerealsSoybeanMaize

0.70.91.8

16.549.6

27.50.91.8

BenzimidazolesTriazolesSubstituted anilidesOrganophosphorus compoundsMorpholines

StrobilurinesOther systemic compoundsDithiocarbamatesInorganic compoundsOther non-systemic compounds

5.919.58.43.82.4

7.415.214.17.3

16.1

Table 5.4. World fungicide market in 1998.



food, a maximum limit of 0.5% has beenestablished in the technical active ingredientwhen marketed.

Benzimidazoles

The fungicidal activity of benzimidazoleswas first described in 1964 for thiabendazole.Benzimidazoles are systemic fungicides thatpenetrate through the cuticle into the plant,where they exert their fungicidal activity.Benomyl and thiophanate methyl are trans-formed into carbendazim and it is thismetabolite that exerts the fungicidal action.

Dicarboximides

Chlozolinate, iprodione, procymidone andvinclozolin are fungicides that belong tothis chemical class. Procymidone andchlozolinate are systemic, while iprodioneand vinclozolin are mainly contactfungicides with both preventive andcurative activity. They were the mostwidely used fungicides in the 1980s but,when resistance phenomena developed,their efficacy was diminished. With theappearance of the new molecules belongingto the class of the anilinopyrimidines andstrobilurines, their use has been reducedgreatly.


20-Feb-03 5

ClassLD50

(mg kg−1 rats)NOEL

(mg kg−1 rats)ADI (mg kg−1

BWb)Toxicityclassa

DithiocarbamatesManebMancozebThiramZinebZiram

BenzimidazolesBenomylCarbendazimThiabendazoleThiophanate methyl

DicarboxamidesChlozolinateIprodioneProcymidoneVinclozolin

TriazolesBitertanolCyproconazoleHexaconazolePropiconazoleTebuconazole

AnilinopyrimidinesCyprodinilMepanipyrimPyrimethanil

StrobilurinesAzoxystrobinKresoxin-methyl

> 5,000> 5,000> 2,600> 5,200>, 320

> 5,000> 15,000> 3,600

> 5,000> 2,000> 6,800

> 15,000

> 5,000> 1,020> 2,189> 1,517

> 2,000> 5,000> 4,150

> 5,000> 5,000

> 2,250.45—

> 2,501.5——

> 2,500.45—

> 2,540.45

> 2,200.45> 2,150.45> 1,000.45> 2,501.4

> 2,100.45> 2,501.45> 2,502.5> 2,503.6

> 2,503.45> 2,502.45> 2,520.45

> 2,518.45> 2,800.45

0.030.030.010.030.02

0.10.030.1

—0.060.10.01

0.01—

0.0050.02

0.030.0240.2

0.20.4

IIIIIIIIIIIIIII

IIIIIIIII

IIIIIIIIIIII

IIIIIIIIII

IIIIIIIII

——

aTomlin (1997).bAccording to WHO.

Table 5.5. Mammalian toxicology of fungicides.a



20-Feb-03

5

102P.C

abras

118

Z:\Customer\CABI\A4382 - d’Mello\A4491 - d’Mello #D.vp

Thursday, February 20, 2003 3:24:54 PM

Color profile: Disabled

Composite Default screen

Pesticides: Toxicology and Residues in Food

103

20-Feb-03

5

Fig. 5.7. Fungicide structures.

119





Triazoles

Triazoles account for almost 20% of the fungi-cide market. This class includes a numberof compounds (Tomlin, 1997). Triazoles aresystemic fungicides that enter the plant andspread from the site of application tountreated or newly grown areas, uprootingexisting fungi or protecting the plant fromfuture attacks. The mechanism of action ofthese fungicides is due to their ability to inter-fere with the biosynthesis of biosteroids or toinhibit the biosynthesis of ergosterol. Theyare used at very low doses and generallyhave a very low toxicity to mammals.

Anilinopyrimidines

In the early 1990s, three fungicides belongingto the class of the anilinopyrimidines appearedon the market. They were cyprodinil, mepani-pyrim and pyrimethanil. These anilinopyri-midines are systemic fungicides that act onthe biosynthesis of amino acids and key cellenzymes. They are mildly toxic to man.

Strobilurines

Azoxystrobin and kresoxim-methyl belongto the class of strobilurines, whose namecomes from the fact that these molecules weresynthesized as a development of the naturalproduct, strobilurine. This class of com-pounds was put on the market in the 1990sand they are marginally toxic to man.

Toxicology

Most fungicides are minimally toxic tomammals since they have an oral LD50 inrats ranging between 800 and > 15,000 mgkg−1 (Table 5.5). Nevertheless, many givepositive results in current mutagenetictests. Among the first-generation fungicides,a few compounds, such as hexachloroben-zene, a few organomercurial fungicides andpentachlorophenol, caused such large-scale

poisoning and intoxication that they werebanned. In the case of the EBCDs, ETU,a mutagenic, carcinogenic and teratogenicproduct, can be formed from their degrada-tion. Recent studies have not provided proofof the existence of hazards to human health.

Herbicides

At present, herbicides have the largest mar-ket share (Table 5.1). In past years, herbicideuse has increased significantly from 34.8% in1970 to 51.9% in 2001. One of the main rea-sons for this increase is related to developingcountries, where there has been a change tomore intensive production due to a shortageof cheap labour. New chemical compoundshave been developed to fight against a largernumber of weeds. First-generation herbicides(non-systemic) were characterized by a widerange of action, low cost and rather highdoses. The newer products are more selectiveand can be applied in doses of the order of afew tens of grams per hectare. Cereals, rice,maize and soybean are the crops that mostrequire herbicides, with consumptions ofabout 20% for each crop, and it is especiallyin North America that herbicides are usedvery extensively (Table 5.6).

Herbicides can be classified not only ac-cording to their chemical class, but also accord-ing to their selectivity, nature of action andapplication characteristics (region and time).

1. Selectivity. Herbicides that destroy allvegetation are classified as total or non-selective, while those that control some weedswithout damaging other agricultural culturesare defined as selective. Non-selectivepesticides, e.g. paraquat, are used to weedorchards, industrial farmyards, wheel tracks,embankments, etc. 2,4-dichlorophenoxyaceticacid (2,4-D), which is selective, is used withthe Gramineae (wheat, barley, rice, oats) tocontrol infesting annual (papaver, etc.) andperennial (convolvulus, etc.) dicotyledons.2. Nature of action. Contact herbicides exerttheir action only on the part of the plant wherethey have been deposited (e.g. paraquat). Sys-temic herbicides, on the other hand, penetratethe plant and reach regions that are far from

104 P. Cabras

20-Feb-03 5



the point of application. Translocation withinthe plant occurs via the phloem and the xylem.A few herbicides may also be absorbed by theroots.3. Region of application. Herbicides maybe applied to the foliage or to the soil.Foliage-applied herbicides, such as a fews-triazines, normally have low solubility inwater, and are absorbed by the roots andtranslocated into the plant via the xylem.Soil-applied herbicides may be subject todegradation in the soil and only part of theapplied dose may be available to be absorbedby the plant. Foliage-applied herbicides pene-trate the cuticular membrane and translocateinto the plant via the phloem system.4. Timing of application. Application tim-ing normally is correlated to the developmen-tal stage of the culture. We therefore have her-bicides used in pre-sowing, pre-emergence,and post-emergence. Pre-sowing treatmentsare made with non-selective herbicides whenselective elimination of weeds is difficult.Pre-emergence herbicides are used to controlannual weeds whose germination competeswith the culture. Post-emergence compoundsare used to control the weeds that competewith the culture during its development.

The best known and most commonlyused herbicides belong chemically to the

following categories: phenoxy derivatives(phenoxyalkanoic acids), dipyridilic com-pounds, amides, dinitroanilines, ureas, tri-azines, sulphonylureas and amino acidderivatives (Fig. 5.8).

Phenoxyalkanoic acids

The phenoxy derivatives make up a historicalgroup of herbicides, since, with the introduc-tion of MCPA in 1942 followed shortly afterby 2,4-D, they marked the start of the modernpractice of chemical weeding. Chemicallythey are very similar to natural auxins,hormones that regulate the physiologicalprocesses underlying plant growth. By sub-stituting themselves to natural auxins, theyinterfere with plant growth. They are selec-tive, systemic herbicides that, applied to thefoliage in post-emergence, can exert theirherbicidal action at very low doses (0.1%).

Bipyridyls

The herbicidal activities of diquat andparaquat, the two compounds that belong tothis chemical class, were discovered in 1956.


20-Feb-03 5



Rest of the world

22.63.144.012.814.9

2.6

ColzaSugarbeetCottonRiceFruit and vegetablesCerealsSoybeanMaize

3.25.65.6

11.115.0

20.619.119.8

TriazinesAmidesCarbamatesUreasToluidines

HormonesDiazinesDiphenyl ethersSulphonylureasImidazolinonesBipyridylsAmino acid derivativesArylphenoxypropionatesCyclohexanedionesPyridinesBenzonitrilesOthers

7.011.33.88.64.1

2.93.52.27.65.93.5

19.95.11.23.01.5

12.7

Table 5.6. World herbicide market in 1998.



20-Feb-03

5

106P.C

abras

122





Pesticides: Toxicology and Residues in Food

107

20-Feb-03

5

Fig. 5.8. Structure of herbicides.

123





These compounds, chemically ammoniumquaternary salts, are not selective and actrapidly by contact on the green parts of theplants, but not on its ligneous parts. Theherbicidal mechanism of action is theinhibition of chlorophyll photosynthesis.Since these compounds are irreversiblyadsorbed by the colloids of the ground,where they remain sunk in the superficiallayers, they are not biologically active.

Amides

The compounds belonging to this familyare divided into three groups: acetamides(e.g. diphenamide), anilides (e.g. alachlor,propanyl) and benzamides (e.g. isoxaben).They are widely used (11.3%), especially onproducts such as rice, maize, wheat and soy-bean. They generally have antigerminativeactivity, but also act via the roots since theycan also be adsorbed by the young roots.

Dinitroanilines

Trifluralin was the first herbicide of this classto be introduced on the market in 1960, andwas followed by several other compounds.These compounds are applied to the soil,where they inhibit seed germination by rootabsorption and block the development ofyoung plantlets. Since these products areunstable to light and volatile, they have tobe incorporated immediately into thesoil. They are selective herbicides used inpre-emergence.

Ureas

This is one of the chemical classes with thelargest number of marketed compounds(Tomlin, 1997) (e.g. diuron, linuron). Theyhave been on the market since 1950. Sincethese herbicides are absorbed mainly throughthe roots, they generally are administeredto the soil during pre-emergence. Theyare selective systemic herbicides that act

as inhibitors of photosynthesis. Accordingto modern standards, these compounds areused at high doses (0.4–4 kg ha−1).

Triazines

Simazine appeared on the market in 1956and was followed by a number of othercompounds. At present, there are 14 triazineson the market. These compounds are inhibi-tors of photosynthesis that are adsorbed bythe leaves and roots. They are chemicallyvery stable and therefore persist in theenvironment. They are selective for a limitednumber of cultures (maize, sorghum, chard,etc.). Among the best-known compounds isatrazine on account of problems related to thepollution of the water table.

Sulphonylureas

Chlorsolfuron, which appeared on themarket in 1980, was the first herbicide of thisclass. The success of sulphonylureas was dueto their low dose of application (10–20 g ha−1)and reduced toxicity for man and theenvironment. At present, there are 25sulphonylureas on the market. Their mecha-nism of action is the inhibition of thebiosynthesis of essential amino acids. Theyare selective systemic herbicides that can beabsorbed by both foliage and roots.

Amino acid derivatives

This class of compounds (also classified asorganophosphorus) has the largest marketshare at 19.9%. The first herbicide of thisgroup, glyphosate, appeared in 1971 and ischaracterized by high systemicity and awide range of action. It acts by inhibiting thesynthesis of aromatic amino acids. It is alsomarketed as a salt. The great success of thisherbicide is related to its lack of residues inthe soil and its low toxicity.

108 P. Cabras

20-Feb-03 5



Toxicology

Among the pesticides, herbicides aregenerally the least toxic compounds forvertebrates. It has often been said that, sincethe mechanism of action of herbicides is aninteraction with the biochemical processes ofvegetables, they have no toxicity for animals.As can be deduced from the data reportedin Table 5.7, almost all herbicides belongto the toxicological class III (according toWHO). In general, since the main absorptionpathway is the skin, the most widespreadtoxic effects are contact dermatitis. Now thatformulation impurities have been greatly

reduced, especially in very toxic compounds,as the technical product must be more than95% pure, some of their attributed toxiceffects have been removed. A case in pointis the herbicide 2,4,5-trichlorophenoxyaceticacid (2,4,5-T), where the presence of a dioxin(TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin)was the real cause of the toxicity. Paraquat isvery toxic to the lungs, but most intoxicationswith paraquat are due to ingestion of theproduct. Normally, herbicides are adminis-tered on the soil and not directly on the cul-ture; moreover, since they are administeredwhen no edible parts are present, they do notpresent particular contamination problems.


20-Feb-03 5

ClassLD50 (mg kg−1

rats)NOEL (mg kg−1

rats)ADI (mg kg−1

BW)Toxicityclassb

Phenoxyalkanoic acids2,4-DMCPABipyridylsDiquatParaquat

AmidesAlachlorDiphenamidIsoxabenMetoalachlorPropanyl

DinitroanilinesDinitraminePendimethanilTrifluralin

UreasChlortoluronDiuronLinuron

TriazinesAtrazineSimazineTerbutylazine

SulphonylureasChlorsulfuronRimsulfuronTriasulfuron

Amino acid derivativesGlyphosateGlufosinate NH4

639–7641,900–1,160

>, 231>, 157

1,930–1,350> 1,050

> 10,000>, 580> 2,500

> 3,000> 1,250> 5,000

> 5,000> 3,400

1,500–4,000

1,869–3,090> 5,000

1,590–2,000

> 5,545> 5,000> 5,000

> 5,600> 2,000

> 2,005.25> 2,020.25

> 2,000.25> 2,170.25

> 2,002.5> 2,000.25> 2,005.6

—> 2,400.25

> 2,000.252,> 100.25> 2,813.25

> 2,100.25> 2,250.25

—

> 2,010.25> 2,000.5> 2,000.22

> 2,100.25> 2,300.25> 2,032.1

2,> 410.25> 2,002.25

0.3—

0.0020.004

——

0.056—

0.005

——

0.024

—0.0020.008

0.0050.0050.002

0.05—

0.012

1.750.02

IIIII

IIII

IIIIIIIIIIIIII

IIIIIIIII

IIIIIIIII

IIIIIIIII

IIIIIIIII

IIIIII

aTomlin (1997).bAccording to WHO.

Table 5.7. Mammalian toxicology of herbicides.a



One of the main problems related to the useof herbicides is the fact that some moleculesmay percolate into the ground and pollutethe water table.

Formulation of Pesticides

Active ingredients show activity at particu-larly low doses. Therefore, since they haveto be distributed evenly on large areas, andat times in concentrations of a few gramsper hectare, formulations that allow a homo-geneous protection of the vegetable area witha precise amount of parasiticide are used.Dose precision is very important to avoidenvironmental pollution in the event ofoverdosing, and lack of efficacy in the eventof underdosing.

The formulation is made up of the activeingredient added to other compounds todevelop maximum biological activity, alloweasy and safe distribution and give sufficientadhesion to the treated surfaces for the timeneeded to exert its biological activity. Theactive ingredient included in the formulationis of a technical grade. The early pesticideswere of poor purity, e.g. in the synthesisof lindane, only 13% of the γ isomer ofhexachlorocyclohexane, which has insecticideactivity, was obtained, while other isomersthat were environmental pollutants werepresent. The synthesis of malathion, whichhas low toxicity, produced isomalathion as animpurity, which is highly toxic. Its use in 1976in the fight against malaria in Pakistan causeda poisoning epidemic among 7500 workers(Baker et al., 1978). In the production of theherbicide 2,4,5-T, a product contaminated byTCDD was obtained. This compound is highlytoxic for man and other mammals and causesa form of acne in the workers involved in itsproduction (Kimmig and Schultz, 1957). Forthis reason, severe restrictions were imposedon the presence of contaminants more toxicthan the active ingredient generated in theproduction process or as degradation prod-ucts. Today, the technical products of pesti-cides have a content of about 95% and do notcontain any contaminants hazardous to healthor the environment.

The formulations can be considereddistributed in the field in liquid or solid form.Those distributed in liquid form are dilutedin water before use and are subdivided asfollows:

1. Wettable powders, in which a suspensionof the solid phase, finely subdivided anddispersed, is obtained in the liquid phase.2. Emulsifiable concentrates, in which theactive ingredient, which is insoluble in water,is dissolved in an appropriate organic solvent.Thanks to the action of a surfactant, the activeingredient forms an emulsion when added towater at a ratio of 1:1000–10,000.3. Granules, in which the active ingredienttogether with the various adjuvants, all finelyground, are fixed on to round mineralgranules.4. Flowable powders, in which the activeingredient is micronized and added toappropriate adjuvants and water to obtain asmooth flowing paste.5. Microcapsules, in which the active ingredi-ent is enclosed in nylon microcapsules ofa diameter of a few micrometres (7–30)preserved in aqueous suspensions. Thecharacteristic of this formulation is that aftertreatment, once the water has evaporated, theactive ingredient is released progressivelyand flows outwards through the pores of thecapsule walls.

The formulations distributed in solid formare made up of dry powders and must be sub-mitted to forced grinding, since their adhe-siveness is inversely proportional to the parti-cle diameter. These formulations are pumpedforcefully on to the plants by an air jet.

The components of the formulation maybe subdivided as follows:

1. Adhesive agents, made up of carboxy-methylcelluloses and paraffin-type mineraloils, which increase the adhesiveness of theactive ingredients, since adhesiveness is gen-erally insufficient to guarantee an adequatedeposit on the treated vegetable surface.2. Anti-evaporating agents, made up ofmixtures of aliphatic hydrocarbons, used tocontain the fast evaporation of molecules at ahigh vapour tension within limits sufficient toallow adequate biological activity.

110 P. Cabras

20-Feb-03 5



3. Diluents for solids, made up of SiO2,carbonates, silicates, montmorillonites,bentonites, talc, etc., and diluents for liquids,made up of aliphatic and/or aromatichydrocarbons.4. Dispersants, made up of oligo- andpolysaccharides, gelatins, bentonites, etc.,used to avoid the sedimentation of dispersedparticles.5. Penetrants, made up of ethoxylatedamines and fatty acid amines that favourthe penetration of the active ingredient insidethe plant.6. Solvents, mainly made up of xylenes,alkylnaphthalines, cyclohexanones, etc., usedto dissolve solid or liquid active ingredients.These solutions may be absorbed on inertpowders (granular formulations) or dilutedfurther in liquid by adding a surfactant(emulsifiable concentrates).7. Surfactants are subdivided into non-ionicor apolar (alkyl phenols, fatty acids andalcohols condensed with ethylene oxide)and ionic or polar (organic acid salts suchas alkyl- and aryl-sulphonates, dodecylbenzenesulphonates, lauryl sulphonates,etc.). Their function is to reduce the surfacetension of water, thus making the dropletscompletely wettable on all contact surfaces,with regard both to parasites and vegetables(Martelli, 1992).

The main objective of the formulation isto improve the efficacy of the active ingredi-ent, which is obtained with co-formulantsthat prolong its presence on the cultureover time. From the point of view ofpublic health, however, the pesticide residueshould disappear as rapidly as possible.The correct balance between these twocontrasting needs is the aim of adequateformulations.

Registration

A company that intends to market a pesticidein a country must register the product withthe appropriate authorities, before putting iton the market. The authority that issues theauthorization to market a pesticide is usuallythe Ministry of Agriculture, less often the

Ministry of Health, and in some cases astate agency such as the EPA in the USA, orthe BCE in Germany. In order to register aproduct, the company must present studiesshowing that the pesticide is efficient againstthe target parasite and does not cause harm-ful effects on human and animal health oron the environment. The documentation thatevery company must present is establishedby law and must be carried out according tothe criteria of good laboratory practice. Theadoption of guidelines and principles of goodlaboratory practice has allowed proceduresto be standardized and has guaranteed thequality of the data produced. At present,legislation is very similar in developed coun-tries; in Europe, the European Communityis harmonizing the national regulations intoone system. Even in developing countriessimilar laws have been issued, but they areoften not applied due to the lack of the neces-sary competences; moreover, some productsthat have been banned by richer countries arestill marketed in these countries. The studiesthat must be presented by the companiesapplying for registration can be dividedinto the following categories: toxicological,agronomic and environmental.

The toxicological studies include thefollowing: (i) acute toxicity; (ii) short-termtoxicity (at least 90 days); (iii) long-term toxic-ity (2 years); (iv) toxicity on reproduction; and(v) late neurotoxicity. The studies are carriedout for all possible forms of contaminationwith the active ingredient: oral intake, cutane-ous intake or intake by inhalation. Basedon the toxicological data the dose causingno observed effect is determined, the NOEL(no observed effect level), i.e. the dose atwhich no toxic effects are observed in animalstudies. Relating to toxic effects, the NOEL isextrapolated from long-term studies on themost sensitive species and on species similarto man. The ADI is obtained from the NOELby dividing it by a safety factor of between 10and 1000. The factor 100 is generally used. Theaim of this factor is to provide the consumerwith a sufficient safety margin, supposingthat humans are 10 times more sensitive thana laboratory animal and that differences insensitivity within a human population arebetween one and ten. The NOEL and the ADI


20-Feb-03 5



are expressed in mg kg−1 BW day−1. In order tomaintain the concentration of the pesticide inthe food at levels of exposure of the ADI, thetolerance level (TL) is calculated according tothe following formula:

TLADIDFI

=

where DFI = daily food intake (kg).The TL depends on the daily food intake

and will therefore vary according to the diet ineach country. The TL is the maximum residueallowed in the food and serves as a basis toestablish the legal MRL (maximum residuelevel). This is decided on the basis of residuesactually found in the food as a result ofpractical supervised tests. If the amount ofresidue found in these tests is lower than theTL, it will be the limit that will be chosen forlegal purposes; if it is higher, the product willnot be registered. Therefore, the MRL is theresult of toxicological and agronomic studies.Since the quantity of residue in the fooddepends strongly on the number of treatmentsand on the environmental conditions, thequantity of pesticide residue in the food mayvary depending on the existing environmen-tal conditions in the country. This explains

why different MRLs are established in differ-ent countries for the same active ingredient(Table 5.8).

On registering a pesticide, the followingelements are indicated for each active ingredi-ent: culture for which the ingredient is autho-rized, dose, safety interval (days that mustelapse from last treatment to harvest), MRLsand toxicological class. When a pesticide is notauthorized on a culture, the residue must beless than 0.01 mg kg−1, which is the legal zero.This limit is also established for baby foods.

As can be seen in Table 5.8, the differencesbetween the lowest and the highest MRLsfor the same culture may be a factor of 30.This indicates that the MRLs are often verydifferent from the TL, and that to surpass anMRL does not necessarily mean a hazard toman, but simply that the conditions of useprovided for and indicated on the label havenot been met: admitted cultures, doses andsafety interval. Implementation of these prac-tices is known as good agricultural practice.

The toxicological classes are intended toshow the level of danger to the consumer andare based on acute toxicity. The four classes oftoxicity classification by the WHO based onactive ingredients are reported in Table 5.9,

112 P. Cabras

20-Feb-03 5

MRL (mg kg−1)

Country Apple Cucumber Grape Lettuce Strawberry Tomato

BrazilChileGreeceIsraelItalyUSA

1025—103

25

2—30.50.1

15

15253

—3

25

15—2

—2

50

20253

—0.1

25

—253

—3

25

— = not registered.

Table 5.8. National maximum residue limits (MRLs) for folpet.

LD50 for the rat (mg kg−1 BW)

Oral Dermal

Class Solids Liquids Solids Liquids

Extremely hazardousHighly hazardousModerately hazardousSlightly hazardous

IaIbIIIII

≤ 55–50

50–500≥ 501

≤ 2020–200

200–2000≥ 2001

≤ 1010–100

100–1000≥ 1001

≤ 4040–400

400–4000≥ 4001

Table 5.9. WHO toxicity classification.



while Table 5.10 reports the more simplifiedclassification by the European Union based onthe formulations.

The TL and, consequently, the ADI andthe MRL can be changed as further data aboutthe toxicology and residue levels becomeavailable.

In the past, registration used to be foran indefinite period, but it has been decidedrecently to limit its duration (10 years in theEU) and reassess each molecule with updatedtoxicological and environmental studies at thedate of expiry. This new approach of limitingthe registration validity will result in thebanning of a number of pesticides, among themost toxic and with a larger environmentalimpact, from the market. It is expected that notmore than 250 active ingredients, 174 of whichwill be newly introduced, will be on themarket after the year 2005.

Residues in Food

After the pesticides are used to treat cultures,they are deposited on them and, in order tobe marketed, they must be lower than thelegal limit at harvest. From a legal point ofview, by residue we not only mean the activeingredient on the food, but also its metabo-lites and/or degradation products and toxi-cological impurities in the formulation. Theamount of pesticide residues present on fruitand vegetables at harvest depends on the ini-tial deposit and on the residue reduction rate.

Initial deposit

A number of factors determine the level ofthe initial deposit; some depend on the pesti-cide (rate, formulation, application methods),

others on the culture (surface/weight ratio,shape).

Application rate

With first-generation pesticides, the amountof active ingredient used per hectare wasof the order of 1 kg. Subsequently, withsecond-generation pesticides, it was reducedto a few hundred grams, while with last-generation pesticides it is of the order of afew tens of grams. As can be seen from thedata on the residues on grapevine reported inTable 5.11, the lower the dose of application,the smaller the initial deposit (Cabras et al.,1984a,b, 1991). There is a direct proportionbetween dose and residue but only with thesame active ingredient, and not if the activeingredient is different. This depends mainlyon the characteristics of the formulation andon the physical–chemical properties of theactive ingredient.

Formulation

A few systemic pesticides can be adminis-tered to the soil in granular formulation.There they are absorbed by the plantsthrough the roots. This creates a progressiveabsorption and a distribution effect of the


20-Feb-03 5

LD50 oral for the rat (mg kg−1 BW)

Liquids Solids

Extremely hazardousHazardousNoxious

≤ 2525–200

200–2000

≤ 55–50

50–500

Table 5.10. EU toxicity classification.

PesticideDose

(g ha−1)Residues(mg kg−1)

DeltamethrinBenalaxyl

Vinclozolin

12.5200400

10002000

0.130.801.611.372.53

Table 5.11. Pesticide residues (mg kg−1) ongrapevine immediately after treatment at the appli-cation doses recommended by the manufacturersand at double the doses.



active ingredient throughout the plant, withconsequent dilution and the presence ofresidues at low levels. Experiments carriedout with carbofuran on lettuce (Table 5.12)have shown that absorption occurs progres-sively; the residues were still undetectable4 days after administration and reached amaximum value after 11 days, though lowerthan 0.1 mg kg−1 (Cabras et al., 1988).

Application techniques

In the past year, there has been a great deal ofinterest in pesticide application techniques,in particular with low volumes (300 l ha−1).Thanks to the high micronization of dropsobtained with this technique, a greater distri-bution uniformity and smaller losses ofliquid are possible. Experiments carried outon celery (Table 5.13) have shown that onlyby associating this technique with an electro-static system was an increase in the residuedeposit obtained (Cabras et al., 1993).

Influence of cultivar

Since residues are expressed in mg kg−1, thesurface/weight ratio of a fruit will strongly

affect the amount of residue. On treating twocultivars of Yacouti and Koroneik olives, thelatter with very small fruits, therefore with agreater surface/weight ratio, with the sameamount of formulation greater amounts ofresidue were found on the latter cultivar(Table 5.14) (Cabras et al., 1997c).

Shape of the cultivar

In some cultures, such as artichokes, theedible part (the head) is different in shape indifferent cultivars. Since in a few cultivars(e.g. Masedu) the head is shaped like a calyxwith open bracts, the sprayed pesticide maydeposit even inside, while with othercultivars (e.g. Spinoso Sardo) whose externalbracts tend to close the inner parts are pro-tected and the sprayed pesticide does notenter the artichoke. This causes remarkablydifferent deposits among the different culti-vars (Table 5.15). Analogous considerationscan be made for the Roman type of lettuce(calyx-shaped) and the Iceberg lettuce (ball-shaped); it should be remembered that in thiscase, the lettuce is marketed after removingthe outer leaves (Cabras et al., 1988, 1996).

114 P. Cabras

20-Feb-03 5

Days after treatment

Carbofuran

4

< 0.001

11

0.063

18

0.023

32

0.027

aActive ingredient.

Table 5.12. Residues (mg kg−1) on lettuce foliage treated with carbofuran in granules at a dose of750 g a.i.a ha−1

Distribution volume (l ha−1)

Cyromazine

1500

1.01

900

1.35

300

0.95

300 with electrostatic

1.90

Table 5.13. Residues (mg kg−1) of cyromazine on celery after treatment at doses of 270 g a.i. ha−1 withdifferent distribution volumes.

CultivarAzinphos

methyl Diazinon Dimethoate MetidathionParathion

methyl Quinalphos

YacoutiKoroneik

1.823.02

1.343.46

1.604.71

3.014.25

1.404.26

1.843.56

Table 5.14. Pesticide residues (mg kg−1) on the olives of two different cultivars after treatment.



Disappearance rate

Pesticides are mostly lipophilic and exerttheir activity by contact or systemically,depending on whether they penetrate theplant or not. On account of these properties,after treatment, the residues on the surface ofthe plant spread in the epicuticular waxylayer and in the cuticle in the case of contactproducts, while systemic products continueto penetrate inside the plant.

If the residue penetrates inside the plant,it degrades with different mechanisms by wayof its enzymes, while, if it remains on the sur-face layers, it will undergo mainly reductionprocesses related to environmental conditionssuch as washing, evaporation, co-distillationduring evaporation of the water from the fruitor vegetable, and photodegradation. Thesedegradative processes determine a ‘real’decrease of the residue, while during thegrowth phase the increase in the weight of thefruit will produce an ‘apparent’ reduction ofthe residue by way of dilution.

Disappearance of the residue will dependon the combined effect of these factors.The rate of disappearance normally followsfirst-order kinetics. Below is an assessmentof how the single factors may affect thedisappearance of the initial deposit.

Fruit growth

Experiments on peaches have shown that theresidues of two pesticides, fenbutatin oxide

(Cabras et al., 1992) and pirimicarb (Cabraset al., 1995b), diminish after exclusive treat-ment by dilution effect due to fruit growthwhen grown in greenhouses, while in theopen field the reduction by growth isonly about one-third of the initial residue(Table 5.16). The lack of residue degradationin greenhouses has been attributed to the factthat the glass absorbs the radiation thatcauses their photodegradation. The high sta-bility of these compounds will cause residueincreases in the event of repeated treatment.

Crops

Experiments carried out using the sameactive ingredient on different cultures showthat the disappearance rates are different.From the data reported in Table 5.17, itcan be seen that, after 1 week’s treatment,dimethoate disappeared almost completelyon plums (Cabras et al., 1998); on grapes, an80% reduction was observed after 1 week’streatment but the residue was constantduring the following 3 weeks (Cabras et al.,1994). The disappearance rates on apricots,oranges and peaches are similar, with half-lives of the order of 10 days (Cabras et al.,1995a,c, 1997d; Minelli et al., 1996).

Enzymatic degradation

When a pesticide enters the plant, it canbe transformed rapidly by enzymatic action.This is the case of the insecticide ethiofencarb


20-Feb-03 5

Culture Cultivar Shape Dimethoate Parathion Pyrazophos

Artichoke

Lettuce

Masedu

Spinoso Sardo

Roman

Iceberg

2.20

1.40

Chlozolinate6.18

1.75

4.02

1.96

Parathion1.19

0.36

1.16

0.53

Table 5.15. Residues (mg kg−1) on different cultivars of artichokes and lettuce after treatment.



administered on lettuce. Immediately afterthe treatment, when the plant is dry (after∼1 h), besides the active ingredient, signifi-cant amounts of three metabolites, sulph-oxide phenol, sulphoxide and sulphone, wereobserved (Table 5.18). After only 1 day,though present in significant amounts ini-tially, the sulphoxide phenol was completely

degraded. Three days after the treatment, theamount of active ingredient was not determi-nable. At harvest, only the metabolitessulphoxide and sulphone were present. InItaly, a legal limit is established only for theactive ingredient, while in other Europeancountries, such as Spain, the limit includesthe sum of active ingredient and metabolites.

116 P. Cabras

20-Feb-03 5

Fenbutatin oxide Pirimicarb

Days after treatment Weight (g) Residues Days after treatment Weight (g) Residues

Greenhouse08

152228—Field07

142128—

5079

113138156—

456495

124140—

1.801.361.060.580.59

(1.84)a

1.761.120.630.340.22

(0.68)a

048

1426—

037

1421—

2429315292—

516683

107115—

1.311.110.860.590.36

(1.38)a

0.620.470.400.170.10

(0.23)a

a( ) = residues corrected by dilution effect.

Table 5.16. Residues (mg kg−1) of fenbutatin oxide and pirimicarb after treatment on peaches.

Days aftertreatment Apricots Oranges Olives Peaches Plums Grapes

07

14212835

1.510.790.450.220.130.12

0.410.220.170.17——

1.601.080.17———

0.970.310.220.12——

1.080.05n.d.a

———

1.130.210.260.280.28—

an.d. = not detectable.

Table 5.17. Residues (mg kg−1) of dimethoate on different fruits after treatment.

Days aftertreatment

Weight(g) E. sulphoxide

E. sulphoxidephenol E. sulphone Ethiofencarb

0138

9686

136371

9.2112.794.600.71

8.77n.d.a

n.d.n.d.

1.661.730.710.22

6.021.62n.d.n.d.


Table 5.18. Residues (mg kg−1) of ethiofencarb and its metabolites on lettuce after treatment.



Different systems of assessment of residuescould create problems with the circulation ofgoods, since, depending on the system used,the residues could be legal when referring tothe active ingredient alone and illegal whenreferring to the sum of active ingredient andmetabolites (Cabras et al., 1988).

Washing

The removal of the active ingredient from thesurface of the plant by water (rain, washingor irrigation) is not easy to interpret, since theresults obtained in experiments are often con-tradictory. Experiments carried out on toma-toes irrigated by drop or sprayer have notshown significant differences that could becorrelated with the irrigation system (Cabraset al., 1986a). Also, in washing trials withplums, there was no reduction in residuebefore the drying process. In contrast, in wash-ing trials with olives, there was a residuereduction in some cases, while in other casesthe residue was unchanged (Table 5.19). In allcases, however, there were no further residuereductions on submitting the olives to a sec-ond washing that was even longer than thefirst. This shows that residue reduction afterfirst washing is not related to a solubilizat-ion process. These apparently contradictorybehaviours can be explained as follows.

At the time of treatment, since there couldbe dust on the fruits, the active ingredient willdeposit both on the waxy layer of the fruitsurface and on the grains of dust. The pesti-cide deposited on the waxy layer will tend tospread over the layer and over the underlyingcuticle, and will therefore be protected fromthe action of water. Since the dust is removedfrom the fruit during washing, the greaterthe amount of residue bonded to the dustthe greater the residue removal. Therefore, ifthere is no dust on the fruit at the time of treat-ment or at harvest because it has been washedaway by the rain, as must have happened tosamples 5 and 6 in Table 5.19, washing will notremove any dust and therefore there will beno related residue reduction (Cabras et al.,1997c).

Residues in Processing of Foods

Some foods, such as olive oil and wine, arethe result of food transformation processes;others, such as dried fruit, undergo a concen-tration process by removal of water. Since onaverage 1 l of wine is obtained from 1.5 kg ofgrapes, 1 l of olive oil from 5 kg of olives, and1 kg of dried prunes from 3 kg of plums, if thetechnological process of transformation did


20-Feb-03 5

Solubility inwater (mg l−1)

Residues (mg kg−1)

Sample

Pesticides Treatmenta 1 2 3 4 5 6

Azinphos methyl

Diazinone

Dimethoate

Metidathion

Parathion methyl

Quinalphos

28

60

23,300

200

55

18

CWCWCWCWCWCW

3.021.853.462.294.714.024.253.594.263.033.562.38

2.732.492.631.723.432.473.812.884.584.671.901.70

2.151.401.741.732.351.702.892.512.291.511.751.28

2.121.281.530.912.301.982.632.362.292.361.460.81

1.010.921.461.530.910.852.512.551.691.710.880.93

0.720.791.151.270.760.821.671.741.351.401.061.09

aC = control, W = samples washed in water.

Table 5.19. Effect of washing on residues in olives.



not cause a reduction in residues, the finalproducts would contain a higher residue con-centration factor than the initial fruits. Anumber of studies have been carried out toassess the incidence of the technological pro-cess of transformation on the residue content.

Dried fruit

Some fruits, such as apricots and plums, areconsumed both fresh and dried. The indus-trial drying process is carried out in ovenswith programmes that reach temperaturesof 95°C for plums and 100°C for apricots. Indrying experiments with industrial processescarried out on these fruits, it was shown thatthere is a change in residue after dryingpeculiar to each active ingredient (Cabraset al., 1997d, 1998). From the data reported in

Table 5.20, it can be seen that the residuedimethoate was unchanged. Therefore, con-sidering a concentration factor of 5.3, theresidue was reduced by this factor.

Analogous considerations can be madefor ziram, while fenitrothion and vinclozolin,on apricots and plums, respectively, are com-pletely removed during the drying process.

Olive oil

Olive oil is obtained by pressing the fruit.Therefore, by pressing olives with a yield inoil of between 14 and 16%, 6–7 kg of fruit willbe needed to obtain 1 l of oil. From experi-ments carried out with olives with thesecharacteristics aimed at assessing the amountof residues transferred from the olives to theoil, the results reported in Table 5.21 were

118 P. Cabras

20-Feb-03 5

Fruit Weight (g) Dimethoate Fenitrothion Ziram

ApricotsFreshDriedRehydrated

PlumsFreshDriedRehydrated

468.6

10.5

32.19.1

10.2

0.120.140.09

———

0.03n.d.a

n.d.

0.14n.d.n.d.

0.120.270.22

———


Table 5.20. Changes in the residues (mg kg−1) of some pesticides during the drying process of apricotsand plums.

Pesticide Olive Yield % OilConcentration

factor in oil

Azinphos methyl

Diazinon

Dimethoate

Metidathion

Parathion methyl

Quinalphos

1.030.691.110.351.080.173.011.280.610.190.680.20

161616141616151616141614

3.101.623.781.950.24n.d.a

3.372.911.332.130.80

3.02.33.45.60.2202.32.64.87.03.14.0


Table 5.21. Residues (mg kg−1) of a few insecticides on olives and olive oil.



obtained for a number of pesticides (Cabraset al., 1997c).

From the data reported in Table 5.21,it can be seen that the residues are alwaysgreater in the oil than in the olives, except fordimethoate, which, due to its high solubility,tends to be distributed preferably in thevegetable water.

The amount of residue present in the oilis also a function of the residue in the fruit;generally speaking, the concentration factor inthe oil is greater for smaller concentrations. Inthe case of parathion methyl, at the lowestconcentration, the residue is completely trans-ferred from the olive to the oil. With the otherinsecticides, on average, about 50% of theresidue passes from the olive to the oil.

Wine

To obtain 1 l of wine, an average of about1.5 kg of fruit is needed. This means that if the

entire residue present in the grapes passedinto the wine, we would always have residueincreases in the wine. From the data reportedin Table 5.22, which were obtained from anumber of experiments (Cabras et al., 1986b,1997a,b; Farris et al., 1992; Cabras andAngioni, 2000), it can be seen that in the trans-formation from grapes to wine, each pesticidehas its own peculiar behaviour. Dimethoate,fenthion, metalaxyl and pyrimethanil do notundergo significant reductions, while withthe other compounds there is a residuedecrease that can be complete for somepesticides. In no case were higher residuesfound in the wine than in the grapes.

Must clarification by centrifugation oftencauses a marked residue decrease; this showsthat the residues tend to be adsorbed on thesolid fraction of the must. For this reason, byfermenting ‘clean’ musts, wines with fewerresidues are obtained.

The examples reported above show thatthe quantity of residues present in foodsdepends on a number of variables. Since no


20-Feb-03 5

Must Wine

Pesticide Grapes Not centrifuged Centrifuged Without maceration With maceration

AzoxystrobinBenalaxylCyprodinil

Dimethoate

FenthionFolpetFluazinamFludioxonil

Kresoxim methylIprodioneMetalaxyl

Parathion methylPyrimethanil

QuinalphosTebuconazole

Vinclozolin

0.190.895.541.031.130.280.281.081.211.860.780.153.00—

1.090.561.621.110.183.160.424.300.80

0.130.434.010.360.900.150.221.110.301.790.390.131.401.691.040.261.661.030.063.130.201.500.06

0.130.240.18n.d.a

0.910.150.20n.d.0.081.20n.d.0.050.80——

0.251.290.940.021.35n.d.0.200.03

0.130.360.700.180.920.140.21n.d.n.d.0.710.230.180.601.30—

0.211.041.02n.d.0.960.160.100.01

0.090.120.740.210.900.140.24n.d.n.d.0.50n.d.0.09———

0.211.561.01n.d.0.980.22—


Table 5.22. Residues (mg kg−1) of fungicides in grapes, must and wine.



mathematical models can foretell their behav-iour a priori, it is always necessary to carry outexperiments in real conditions to evaluate theamount of residues at harvest and, on thisbasis, to indicate legal limits.

Monitoring Programmes on PesticideResidues in Food

As already discussed, each country estab-lishes a maximum residue limit (MRL) foreach pesticide for the cultures for which it hasbeen authorized. If this limit is exceeded or ifthis active ingredient is used on cultures thathave not been authorized, the obtained foodsare considered irregular and therefore notmarketable. Though the MRL is not a toxico-logical limit, exceeding it means that thepesticides are not being used correctly and,by comparing the values with the ADI, thetoxicological risk to the consumer can beassessed. For this reason, in the most devel-oped countries, programmes for the monitor-ing of pesticide residues have been promotedby official agencies for the past few years. The

results obtained in European Union countriesin 1998 and in the USA in 1999 are reported inTable 5.23. Those referring to the EuropeanUnion are focused on fruit, vegetables andcereals, while the American results alsoinclude fish, milk and dairy products, andeggs. The US data are also available onthe Internet at the following website:www.cfsan.fda.gov

The number of samples analysed inEurope is remarkable and indicates the specialattention paid to food health problems thesedays. The results obtained indicate that thenumber of irregular samples is small both inthe EU and in the USA. Moreover, on compar-ing these data with those of the two previousyears, it can be seen that the values are notsignificantly different. It should be mentionedthat the data obtained in the different coun-tries are not comparable since the nationalMRLs differ both in entity and in number ofcultures for which each pesticide is registered.Moreover, when planning samplings, thecultures are usually chosen according to thenational diet and the pesticides to be analysed,and selected according to their use, the fre-quency observed in previous years and the

120 P. Cabras

20-Feb-03 5

Pesticides Sampleswithout

residues (%)

Samples withresidues

< MRL (%)

Samples withresidues

> MRL (%)Countries Samples (n) Analysed (n) Found (n)

BelgiumDenmarkGermanyGreeceSpainFranceIrelandItalyLuxemburgNetherlandsAustriaPortugalFinlandSwedenUKEUUSADomesticImported

1,9472,1646,6961,1643,2024,058,33298,779,32304,976,3322,34552,359

34,999,3976

41,336

3,4266,012

122131—93

169224——94

27583

100173—

151147

400400

4676—41—

106——31

108412897—5163

——

65.569.161.676.561.840.243.367.867.556.155.961.554.265.357.360.8

60.264.8

28.328.334.319.336.353.353.331.329.338.341.335.343.333.340.336.3

39.331.3

6.52.94.44.52.26.84.01.23.55.93.13.52.82.03.03.2

0.83.9

Table 5.23. Results of the monitoring programmes for pesticide residues in 15 countries of the EU in1998 and of the USA in 1999.



danger they represent. In addition, there aretechnical problems such as the analyticalmethods used and the analytical expertise ofthe laboratory.

Besides the official checks, many otherstructures (producers’ cooperatives, agricul-tural farms, large distribution chains) carryout checks on their products before puttingthem on the market. In 1999 in Italy, forexample, the National Residue Observatory,a private organization that collects unofficialcontrol data every year, published theresults of a survey of 18,972 samples, 51.4%of which were without residues, 46.7% withregular residues and 1.9% with irregularresidues. The data referred to an analysisof 132 types of food with 276 active ingredi-ents, 137 of which had left determinableresidues.

Thanks to these checks, it is possible todetermine for each country the pesticides thatare used mainly in each culture, their levelsand those that most often exceed the MRL.Moreover, the data obtained are used as abasis for subsequent checks with particularattention to pesticides that are most fre-quently used and are the most hazardous forthe consumers’ health.

Such frequent pesticide checks in food area good tool to calculate the consumers’ realexposure to toxic compounds.

Risk Assessment for PesticideResidues in Food

Risk assessment is an estimate of the likeli-hood of harmful effects on the health of thepopulation as a result of exposure. The bestguarantee that the exposure to pesticide resi-dues will be contained within safety limitsis obtained from dietary ingestion studies.Using the data from toxicological studies, itis possible to assess the quantity of pesticide,in reference to body weight, that may beingested in a lifetime without appreciablerisks to one’s health. This quantity is the ADIvalue. If the quantity of pesticide ingesteddaily is lower than the ADI value, theprobability of harmful effects for health theo-retically is zero. Risk assessment due to the

presence of pesticide residues is subdividedinto the following phases:

• pesticide residue estimate• national diet estimate• dietary pesticide exposure

Residue estimate

Exposure estimates may be carried out ontheoretical and analytical data. The theoreti-cal data are used in the pesticide registrationprocess. In this case, it is assumed that theresidues are present in the food at the maxi-mum legal limit (MRL). In fact, these valuesare difficult to reach even in extreme con-ditions when the maximum doses are usedin the treatment, with the largest number ofapplications and the shortest time intervalsbetween treatment and harvest. If the amountof residue ingested with this theoretical valuein the foods making up the diet and for whichan authorization is requested exceeds theADI, the product may not be registered.

In fact, the residues present in the cul-tures for which they are authorized are oftenvery far from these limit values, because theyare not always used, because a large numberof treatments is rarely carried out and becausethe time interval between the treatment andthe harvest is often longer than the preharvestinterval.

A true assessment of the residues in thefoods may be carried out by analysingthe samples when they are marketed. Thanksto monitoring programmes, it is possible toknow the residue level of each pesticide indifferent foods. The larger the number ofsamples analysed, the better the knowledgeof their pollution level and, therefore, themore reliable the risk assessment.

National diet estimate

Data on food consumption are essential foran assessment of the risk related to foodsafety. Food consumption varies from coun-try to country and often also within the samecountry. For this reason, each country must


20-Feb-03 5



assess its own standard diet taking intoaccount the food habits of the differentcategories of people by age, sex, place, etc.Particular attention must be paid to sensitivegroups such as the newly born. The foodconsumption indices that are used are many,namely: mean daily consumption, size ofportion and the average consumption of thepopulation. Generally these data are easilyavailable since there are institutions inter-ested in the national food diet in everydeveloped country. For an assessment at theworld level, the data contained in the FAO‘Food Balance Sheets’ are the most reliablesource.

Dietary pesticide exposure

Risk assessment relating to pesticide residuesin food has been tackled by the CodexAlimentarius with the special JointFAO/WHO Expert Committee on PesticideResidues (JMPR) made up of groups of inde-pendent experts. This commission carriesout toxicological assessments on pesticides,estimating an ADI value, and proposingMRLs and models to be used to assess thepopulation exposure. The most realisticassessments may be made at the nationallevel since they are based on the most reliabledata of food consumption. In order to assess

the dietary pesticide exposure, the residuesof each pesticide are multiplied by foodconsumption. These data are expressed in µgkg−1 BW day−1, and make up the daily intake(NEDI = national estimated daily intake). Thedata are then looked at in conjunction withthe values of the ADI. The risk to humanhealth starts when the NEDI/ADI % ratio isgreater than 100. In calculating the residueintake, account must be taken of factors thatmay alter their concentration in the actualfood intake, such as the part of the agri-cultural product that is actually eaten (e.g.oranges without peel), the effects of process-ing the raw product (e.g. wheat → flour), ortransforming it (e.g. olives → oil), and thoseof cooking or preparing it (e.g. potatoes →chips). These correction factors are obtainedfrom literature data on each specific activeingredient. In calculating exposure, animportant problem is that of samples thatcontain residues lower than the limit of detec-tion (LOD) and therefore considered absent.In this case, using data from samples of anunknown treatment history, as in the caseof official monitoring, we assign zero or weassign the value of ½ LOD to a certainpercentage and zero to the remaining part. Asan example, we report some exposure datarelating to Italian consumers calculated in1999 by the National Residue Observatory(Table 5.24). These data have been chosenfrom those of the highest residue intake

122 P. Cabras

20-Feb-03 5

Pesticide ADIa (µg kg−1 BWc) NEDIb (µg kg−1 BWc) NEDI/ADI %

AcephateAzinphos methylBuprofezinChlorthalonilChlorpyrifos methylCyprodinilDimethoateOmethoateParathion methylPyrimethanilQuinalphosTeflubenzuronVinclozolin

30.35.3

10.330.310.330.32.30.33.3

170.330.310.310.3

0.01290.04030.00320.03740.02900.03310.02040.00670.01870.09500.00120.00410.0222

< 0.04< 0.8< 0.03< 0.1< 0.3< 0.1< 1.0< 2.2< 0.6< 0.06< 0.01< 0.04< 0.2

aAcceptable daily intake.bNational estimated daily intake.cBody weight.

Table 5.24. Exposure of the average Italian consumer to pesticides in 1999.



and show that dietary residues are somuch less than the safety threshold(NEDI/ADI% = 100) that even the mostrestrictive assessments that take into accountparticularly sensitive groups (newborns, ado-lescents, the elderly, etc.) would lead to 100%safe results. The data on monitoring carriedout in other countries (Table 5.23) show thateven here assessments of the exposure topesticide residues in food are not verydifferent from those made in Italy.

Conclusions

Pesticide toxicology and residues in foodand wine have been reviewed in the contextof human exposure to specific insecticides,fungicides and herbicides. Factors affectingresidues in fruit and vegetables depend uponinitial deposits and disappearance rates.Risk assessments carried out on selectedpesticides show that human exposure is wellbelow safety threshold values for consumersin Italy. In the UK, results of a 2001 surveyof milk, honey, canned salmon, kiwi fruit,grapes, lemons, breakfast cereals and otherfoods have just been published by thePesticide Residues Committee. The resultsindicated that 29% of the 450 samples testedcontained residues of pesticides, with about10% of all samples containing multiple resi-dues. The health implications of multiplepesticide residues and the significance ofany interactions with other types of foodcontaminants remain unresolved.

References

Baker, E.L. Jr, Warren, M. and Zack, M. (1978)Epidemic malathion poisoning in PakistanMalaria workers. Lancet 1, 31–34.

Cabras, P. and Angioni, A. (2000) Pesticide residuesin grapes, wine, and their processing products.Journal of Agricultural and Food Chemistry 48,967–973.

Cabras, P., Meloni, M. and Pirisi, F.M. (1984a)Evoluzione dei residui di Deltamethrinnell’uva e durante il processo di vinificazione.La Difesa delle Piante 3, 139–144.

Cabras, P., Meloni, M. and Pirisi, F.M. (1984b)Persistenza del vinclozolin su vite: esperienzacondotta in Sardegna. Atti GiornateFitopatologiche 1984 2, 31–40.

Cabras, P., Manca, M.R., Meloni, M., Pirisi, F.M.,Cabitza, F. and Cubeddu, M. (1986a)Persistenza di alcuni insetticidi ed acaricidi supomodoro da industria irrigato con diversisistemi. Proc. Giornate Fitopatologiche 3,363–372.

Cabras, P., Meloni, M., Pirisi, F.M. and Lalli, M.G.(1986b) Riduzione di alcuni fungicidi duranteil processo di vinificazione. Enotecnico 12,1219–1222.

Cabras, P., Meloni, M., Manca, M.R., Pirisi, F.M.,Cabitza, F. and Cubeddu, M. (1988) Pesticideresidues in lettuce. I. Influence of the cultivar.Journal of Agricultural and Food Chemistry 36,92–95.

Cabras, P., Spanedda, L., Maxia, L. and Cabitza, F.(1990) Residui di Ciromazina e del suometabolita Melammina nol sedano. Rivistadella Societá Italiana di Scienze Alimentan 19,55–57.

Cabras, P., Porcu, M., Spanedda, L. and Cabitza, F.(1991) The fate of the fungicide benalaxyl fromvine to wine. Italian Journal of Food Science 3,181–186.

Cabras, P., Melis, M., Tuberoso, C., Falqui, D. andPala, M. (1992) HPLC determination offenbutatin oxide and its persistence in peachesand nectarines. Journal of Agricultural and FoodChemistry 40, 901–903.

Cabras, P., Lalli, M.G., Melis, M., Spanedda, L.,Cabitza, F. and Cubeddu, M. (1993) The depo-sition and persistence of Cyromazine in celeryin relation to the methods of application.In: Proceedings of the IX Symposium of PesticideChemistry. Piacenza, Italy, pp. 545–551.

Cabras, P., Garau, V.L., Melis, M., Pirisi, F.M.,Cubeddu, M. and Cabitza, F. (1994) Residui diDimetoate e chlorpirifos nell’uva e nel vino.Proc. Giornate Fitopatologiche 1, 27–32.

Cabras, P., Garau, V.L., Melis, M., Pirisi, F.M.,Spanedda, L., Cubeddu, M. and Cabitza, F.(1995a) Persistence of some organophos-phorous insecticides in orange fruits. ItalianJournal of Food Science 7, 291–298.

Cabras, P., Melis, M., Spanedda, L., Cubeddu, M.and Cabitza, F. (1995b) Persistence ofpirimicarb in peaches and nectarines. Journal ofAgricultural and Food Chemistry 43, 2279–2282.

Cabras, P., Garau, V.L., Pirisi, F.M., Spanedda, L.,Cubeddu, M. and Cabitza, F. (1995c) The fateof some insecticides from vine to wine. Journalof Agricultural and Food Chemistry 43,2613–2615.


20-Feb-03 5



Cabras, P., Angioni, A., Garau, V.L., Melis, M.,Pirisi, F.M., Cabitza, F., Cubeddu, M. andMinelli, E.V. (1996) Pesticide residues inartichoke. Effect of different head shape.Journal of Environmental Science and Health, PartB 31, 1189–1199.

Cabras, P., Angioni, A., Garau, V.L., Melis, M.,Pirisi, F.M., Farris, G., Sotgiu, C. and Minelli,E.V. (1997a) Persistence and metabolism offolpet in grapes and wine. Journal ofAgricultural and Food Chemistry 45, 476–479.

Cabras, P., Angioni, A., Garau, V.L., Melis, M.,Pirisi, F.M., Minelli, E.V., Cabitza, F. andCubeddu, M. (1997b) Fate of some new fungi-cides (cyprodinil, fludioxonil, pyrimethaniland tebuconazole) from vine to wine.Journal of Agricultural and Food Chemistry45, 2708–2710.

Cabras, P., Angioni, A., Garau, V.L., Melis, M.,Pirisi, F.M., Karim, M. and Minelli, E.V. (1997c)Persistence of insecticide residues in olivesand olive oil. Journal of Agricultural and FoodChemistry 45, 2244–2247.

Cabras, P., Angioni, A., Garau, V.L., Minelli, E.V.,Cabitza, F. and Cubeddu, M. (1997d) Residuesof some pesticides in fresh and dried apricots.Journal of Agricultural and Food Chemistry 45,3221–3222.

Cabras, P., Angioni, A., Garau, V.L., Minelli, E.V.,Cabitza, F. and Cubeddu, M. (1998) Pesticideresidues in plums from field treatment todrying processing. Italian Journal of Food Science10, 81–85.

Ecobichon, D.J. (1997) Toxic effect of pesticides. In:Casarett and Doull (eds) Toxicology, 5th edn.McGraw-Hill, New York.

Edwards, C.A. (1973) Persistent Pesticides in theEnvironment. CRC Press, Boca Raton, Florida.

Farris, G.A., Cabras, P. and Spanedda, L. (1992)Pesticide residues in food processing. ItalianJournal of Food Science 4, 149–169.

Fest, C. and Schmidt, K.J. (1982) The Chemistry ofOrganophosphorus Pesticides. Springer-Verlag,Heidelberg, Germany.

Kimmig, J. and Schultz, K.H. (1957) Occupationalacne caused by chlorinated aromatic cyclicethers. Dermatologica 115, 540–546.

Longcore, J.R., Samson, F.B. and Whillttendale, T.W.(1971) DDE thins eggshells and lowersreproductive success of captive black ducks.Bulletin of Environmental Contamination andToxicology 6, 485–490.

Martelli, R. (1992) Pesticide formulation. InformatoreFitopatologico 42, 7–12.

Minelli, E.V., Angioni, A., Cabras, P., Garau, V.L.,Pirisi, F.M., Cubeddu, M. and Cabitza, F. (1996)Persistence of some pesticides in peach fruit.Italian Journal of Food Science 8, 57–62.

Morgan, D.P. and Roan, C.C. (1970) Chlorinatedhydrocarbon pesticide residues in humantissues. Archives of Environmental Health 20,452–457.

Roberts, T. and Hutson, D. (1999) Metabolic Pathwaysof Agrochemicals. The Royal Society ofChemistry, Cambridge, UK.

Stevens, M.F., Ebell, G.F. and Psaila-Savona, P.(1993) Organochlorine pesticides in WesternAustralia nursing mothers. Medical Journal ofAustralia 158, 238–241.

Stickel, L.F. (1968) Organochlorine Pesticides inEnvironment. United States Department ofthe Interior, Fish and Wildlife Service. SpecialScientific Report – Wildlife no. 119, Washing-ton, DC.

Tomlin, C.D.S. (ed.) (1997) The Pesticide Manual,11th edn. British Crop Protection Council,Farnham, UK.

Wood McKenzie (1999) Agrochemical Product Service.Deutsche Bank AG, Edinburgh, UK.

Woodwell, G.M., Craig, P.P. and Johnson, H.A.(1971) DDT in the biosphere: where does it go?Science 174, 1101.

20-Feb-03 5

124 P. Cabras



6 Polychlorinated Biphenyls

D.L. Arnold1* and M. Feeley21Toxicology Research Division and 2Chemical Health Hazard Assessment Division,

Bureau of Chemical Safety, Health Products and Food Branch, Health Canada,Ottawa, Ontario K1A 0L2, Canada

Introduction

Polychlorinated biphenyls (PCBs) aresynthetic chemical mixtures that theoreticallycould contain up to 209 chlorinated con-geners of the biphenyl moiety (Ballschmiterand Zell, 1980). While about 130 congenershave been identified in commercial products(Fig. 6.1), most commercial PCB mixturesonly contain 50–90 different congeners(Nicholson and Landrigan, 1994). Due totheir physical and chemical properties, PCBshad a multitude of industrial applications:dielectric fluids in capacitors and trans-formers, heat transfer agents, plasticizersin paints, flame retardants, pesticide extend-ers, adhesives, coatings, cutting oils andhydraulic lubricants, and inclusion in inks,carbonless copy paper, sealants and caulkingcompounds (Safe, 1994; Eisler and Belisle,1996). Generally, mixtures of PCB congenerswere marketed based on their percentage ofchlorine. For example, Monsanto ChemicalCompany sold the following PCB mixtures:Aroclor 1221, 1232, 1242, 1248, 1254, 1260 and1268. The 12 indicated that the mixture was abiphenyl and the last two digits indicated thepercentage of chlorine by weight (i.e. 21, 32,42%, etc.). One exception to this generalitywas Aroclor 1016, a distillation product of

Aroclor 1242, containing 41% chlorine byweight but with only 1% of the congenerscontaining five or more chlorine atoms.Other companies marketed their PCBmixtures under such trade names asClophens (Bayer, Germany), Delor, Deloreneand Hydelor (Chemko, Czechoslovakia),Fenclors and Apirolio (Caffaro, Italy),Kanechlors (Kanegafuchi Chemical Co.,Japan), Orophene (Deutsche Soda Werkrn-VEB, Germany), Phenochlor and Pyralène(Prodelec, France), Santotherm (Mitsubishi-Monsanto Co., Japan) and Soval (Sovol,USSR) (De Voogt and Brinkman, 1989). Thenumbering system for the latter mixturesindicates an approximation of the meannumber of chlorine atoms per congener.For example, Clophen A60, PhenochlorDP6 and Kanechlor 600 all have an averageof six chlorine atoms per molecule, i.e. 59%chlorine by weight. The congener composi-tion of commercial PCB mixtures varies frombatch to batch since the extent of theirchlorination ranges from 21 to 68% (w/w).Consequently, commercial PCBs are notsold based upon composition per se buton the batch’s physical properties. Inaddition, the composition and amount ofimpurities also vary from batch to batch andamong manufacturers. For example, most


20-Feb-03 6

* E-mail: Doug_Arnold@hc_sc.gc.ca.



commercial PCBs, except Aroclor 1016, con-tain polychlorinated dibenzofurans (PCDFs;Fig. 6.2), and some commercial mixtures maycontain polychlorinated naphthalenes andpolychlorinated quaterphenyls (PCQs; IARC,1978; De Voogt and Brinkman, 1989; Nichol-son and Landrigan, 1994; ATSDR, 2000).

From a historical perspective, PCBs werefirst synthesized by Griefs in 1867. MonsantoChemical Company did not start manu-facturing PCBs in the USA until 1929, whilecommercial production of PCBs in Japandid not start until 1954 (IARC, 1978). Themanufacture of specific Aroclors by Monsantotended to occur during specific periods. Forexample, Aroclor 1254 and 1260 were pre-dominantly used prior to 1950, while Aroclor1242 was the dominant mixture in the 1950sand 1960s. Starting in 1971, Aroclor 1016replaced Aroclor 1242, and the sale of PCBs inthe USA was limited to capacitor and trans-former manufacturers, with Monsanto volun-tarily limiting production of Aroclors to those

containing less than 60% chlorine. In 1974,most domestic uses for PCBs were restrictedto closed applications. In 1976, all new usagesof PCBs were banned, and the first effluentstandards for PCBs were issued by the USEnvironmental Protection Agency (EPA) in1977. Manufacturing and import limitationswere issued in 1979, and subsequent amend-ments to this regulation banned the pro-duction of PCBs in the USA (Nicholson andLandrigan, 1994; Eisler and Belisle, 1996;Danse et al., 1997; ATSDR, 2000).

In an attempt to conceptualize the extentto which PCBs were manufactured/used andtheir potential for contamination of theenvironment, Kannan (2000) reported thatproduction of PCBs by industrialized westernnations totalled an estimated 1,054,800 t,while the former USSR produced another100,000 t. Kannan also reported that a totalof 370,000 t may have escaped into theenvironment while the remainder is stillin use, primarily in electrical equipment.

20-Feb-03 6

126 D.L. Arnold and M. Feeley

Fig. 6.1. Polychlorinated biphenyls (PCBs), x, y ≤ 5.

Fig. 6.2. Polychlorinated dibenzofurans (PCDFs), x ≤ 8.



The physical and chemical propertieswhich made PCBs such a useful industrialcommodity have resulted in their contaminat-ing every component of the global ecosystemas winds and water currents have dispersedPCBs to parts of the globe where they havenever been used (Macdonald et al., 2000). It hasbeen reported that biphenyls with one or nochlorine atoms remain in the atmosphere,while those with one to four chlorine atomsmigrate towards the polar latitudes; thosewith four to eight chlorine atoms remainin the mid-latitudes, and those with eight ornine chlorine atoms remain close to the sourceof contamination (Wania and Mackay, 1996).Over time, the more stable PCB congeners,generally those with a greater degree of chlori-nation, have found their way into the foodchain. Whether such contamination has healthimplications for humans is still a debatableissue, since the potential health effects of PCBscannot easily be distinguished from those ofother environmentally persistent anthropo-genic chemicals (Danse et al., 1997; Johnsonet al., 1998).

Simplistically, there are three generalgroupings into which humans can be placedregarding their exposure to PCBs. There arethose who were exposed to PCBs in an indus-trial setting, where dermal absorption and/orinhalation were the major routes of exposure.A second grouping includes the peoplein Japan and Taiwan who ingested rice oilthat was inadvertently contaminated withPCBs which were being used as a heat transferfluid. The Japanese accident occurred in 1968and became known as the Yusho (‘oil disease’in Japanese) incident, affecting about1800 people (Kuratsune and Shapiro, 1984;Kuratsune et al., 1996), while the Taiwanaccident occurred in 1979 and became knownas the Yu-Cheng (‘oil disease’ in Chinese)incident, affecting approximately 2000people (Kuratsune and Shapiro, 1984). Whilethe heat transfer fluid originally consisted ofKanechlor 400, which contained 48% chlorineby weight, heating the Kanechlor underreduced pressure resulted in the loss of someof the lower chlorinated congeners as well asthe conversion of other congeners into PCDFsand PCQs (Masuda, 1996). The latter two enti-ties are generally considered to be more toxic

than PCBs (Danse et al., 1997; Longneckeret al., 1997). The third exposure groupcomprises the rest of the world population,who are primarily exposed to PCBs via theirdiet, although some additional exposurevia drinking water and inhalation occurs.This simplistic grouping does not recognizevarious subpopulations who may be at riskfor higher exposure to PCBs: for example, rec-reational fishers and hunters who consumecontamined fish and game; native popula-tions who are subsistence hunters and fishers;breast-fed infants whose mothers consumesignificant amounts of PCB-contaminatedfish and/or wild game; farm families whosefood was exposed to PCB-contaminated siloswhen PCBs were used as a silo sealant; thoseliving in proximity to waste storage or dis-posal sites; and other analogous populations(Kimbrough, 1995; Johnson et al., 1998). As thetheme of this book is food safety, the emphasisof the following discussion will be on theingestion of PCBs via food, which is the mostimportant route of human exposure (Hu andBunce, 1999). Dermal and inhalation exposureto PCBs are only of importance in theworkplace (Nicholson and Landrigan, 1994).

Nature of PCBs – Chemical andPhysical Properties

PCBs are thermally stable; they are resistantto acids, bases and oxidation; at room tem-perature, they have a low volatility, whichincreases dramatically with small increasesin temperature; they have a high dielectricconstant; and they are practically fire resis-tant because of their high flash point(170–380°C). PCB vapours, while heavierthan air, are not explosive. They have lowelectrical conductivity, high thermal conduc-tivity and they are resistant to thermal degra-dation. Consequently, PCBs are inert, beingstable to hydrolysis and oxidation by condi-tions encountered during industrial use.

While individual PCB congeners arecolourless crystals when isolated in pure formby recrystallization, commercial PCB mix-tures can range in colour from clear, throughlight yellow to dark brown. Their physical

Polychlorinated Biphenyls 127

20-Feb-03 6



state can range from an oil to a viscous liquidor a sticky resin; however, they do not crystal-lize, even at low temperatures. PCBs arerelatively insoluble in water, with the morehighly chlorinated congeners being the leastsoluble, but they are soluble in oils, non-polarorganic solvents and biological lipids (IARC,1978; Eisler and Belisle, 1996; ATSDR, 2000).

Low levels of PCBs can be found through-out the ecosystem. As they are no longer man-ufactured in significant quantities, PCBs arecontinually redistributing among environ-mental compartments, i.e. soil, water, sedi-ments and air. The fate of PCBs in aquaticsystems and soil depends upon their sorptionand retention, both of which are markedlyinfluenced by the number of chlorine atoms.Generally, the greater the number of chlorineatoms, the greater the retention. In an aqueousenvironment and on soil surfaces, PCBs canevaporate and return to earth via rain or snowor by settling on dust particles. The majorsource of PCBs in surface water is from atmo-spheric deposition. As PCBs absorb stronglyto soil particles, significant leaching from soiland translocation to ground water or plantsis unlikely. While there is no known abioticprocess that will significantly degrade PCBscontaminating soil, photodegradation onsoil surfaces may occur. Aerobic and anaero-bic biodegradation are the major degradationprocesses, but they occur very slowly. Aerobicdegradation of PCB congeners dependsupon such factors as initial concentration,moisture, temperature (warmer temperaturesenhance degradation), inhibitory compounds(e.g. chlorobenzoates) and the availability ofsuch bacterial nutrients as carbon sources (e.g.acetate); however, biodegradation is slowedin soils with a high organic carbon con-tent. Interestingly, anaerobic biodegradationappears to have a greater effect on the morehighly chlorinated congeners while aerobicbiodegradation is more effective on the lowerchlorinated congeners (ATSDR, 2000).

Generally, biological entities do notmetabolize the more highly chlorinated PCBcongeners nor are they readily excreted.Since PCBs are soluble in body lipids, abiological system’s inability to excrete PCBsto any meaningful extent, save for theirsecretion/excretion in breast milk (Feeley and

Brouwer, 2000), results in PCBs being biomag-nified in the food chain. Bioaccumulation/biomagnification of PCBs is largely depend-ent upon a congener’s octanol–water parti-tioning coefficient (Eisler and Belisle, 1996).


As an anthropogenic chemical, PCBs havecontaminated the environment solely as aconsequence of human activity. While theirmanufacture and use in new products areminuscule on a worldwide basis, PCBs con-tinue to redistribute themselves among envi-ronmental compartments. In addition, somePCBs are still released into the environmentfrom waste sites, incineration, leakage fromelectrical equipment, improper disposal,spills and leachates from sewage sludge.However, it should be noted that the levelsof PCBs in all environmental compartmentsappear to have decreased significantly inrecent years (Duarte-Davidson and Jones,1994; Johnson et al., 1998; ATSDR, 2000;Dougherty et al., 2000).

Any attempt to longitudinally study andquantitate the contamination of human foodsby PCBs is difficult because the instrumenta-tion has changed dramatically since PCBswere first detected in the food chain in 1966(Danse et al., 1997). Initially, PCB analyses con-sisted of comparing the PCB chromatographicpattern in the sample of interest with that ofvarious commercial mixtures. The chromato-graphic patterns found when North Americanfood samples from the 1960s and 1970s wereanalysed for PCBs were similar to those ofAroclor 1254 and 1260 (Zitko et al., 1972; Veith,1975; Walker, 1976; Veith et al., 1981), althoughcontaminant patterns resembling those ofAroclor 1242 and 1016 were also reported(Veith et al., 1981). In recent years, conge-ner-specific analyses have been undertaken.McFarland and Clarke (1989) have sug-gested that approximately half of the 209potential PCB congeners account for nearlyall of the environmental contamination attrib-uted to PCBs. Even fewer congeners are bothenvironmentally prevalent and potentiallytoxic. Using the criteria of potential toxicity,

20-Feb-03 6




environmental prevalence and relativeabundance in animal tissues, McFarland andClarke concluded that only 36 congeners wereof environmental concern, and 25 of thesecongeners accounted for 50–75% of all the PCBcongeners found in tissue samples from fish,invertebrates, birds and mammals.

Concurrent with the advances in analyti-cal technology, there was an interest in under-taking toxicological testing with specific PCBcongeners. It was found that there was amarked difference among PCB congenersregarding their effects in various in vivo andin vitro toxicity assays, with the least potentcongeners being those that had a non-planarstearic configuration. As biphenyl rings canrotate at the 1,1′ positions, they can exist in aplanar orientation. When chlorine substitu-tion occurs at the meta and para ring positions,but not at the ortho position, the rotationalbarriers are lowered sufficiently for a smallnumber of the meta- and para-substitutedcongeners to assume a planar configuration.These congeners (i.e. 77, 81, 126, 169; number-ing system of Ballschmiter and Zell, 1980) arereferred to using such terms as non-ortho,planar or ‘dioxin-like’ congeners (Fig. 6.3) dueto their stearic configuration and toxicologicalproperties being analogous to those oftetrachlorodibenzo-p-dioxin (TCDD). Othercongeners (i.e. 105, 114, 118, 123, 157, 157, 167and 189) having only one chlorine atom at theortho position can also exist in a planar confor-mation but, while these mono-ortho congenerscan have a dioxin-like configuration, they areless toxic than the non-ortho PCBs (Kannan,2000).

Human populations are exposed toPCBs primarily via the consumption offish, meat and poultry, although fish hasbeen the major source of PCB exposure inthe USA for the past 25 years (ATSDR,2000). Dougherty et al. (2000) also reportedthat fish consumption, particularly saltwaterfish, accounts for a majority of the PCBsingested by Americans, including childrenaged 1–5 years. Freshwater fish and shellfishwere also significant contributors to PCBingestion, while milk and beef contributedless that 5% to total average exposure. Themost commonly occurring congeners in fishare 95, 101, 110, 118 (pentachlorobiphenyls),138, 153 (hexachlorobiphenyls) and 180(heptachlorobiphenyl). Due to their low prob-ability for degradation, these congeners arethe major contaminants in most biologicaltissues (ATSDR, 2000).

For the contemporary UK population,Duarte-Davidson and Jones (1994) estimatedthat 97% of their total PCB exposure camefrom food, 3.4% from air and 0.04% fromwater. The contributions from fish, milk anddairy products; vegetables; meat; and animalfat were estimated to account for 32, 26, 18and 16% of the exposure, respectively. Vegeta-bles accounted for the majority of the lowerchlorinated congeners, while meat, dairy andfish contained most of the higher chlorinatedcongeners. For example, vegetables accoun-ted for 78% of the total dietary content ofcongener 28 (trichlorobiphenyl) and 0.2% ofcongener 180 (heptachlorobiphenyl), whilefreshwater fish accounted for 1.2% of thedietary content of congener 28 and 27% of


20-Feb-03 6

Fig. 6.3. Polychlorinated dibenzodioxins (PCDDs), x ≤ 8.



congener 180. The authors estimated that theaverage daily PCB exposure in the UK was0.53 µg per person.

The ingestion of PCBs has only been stud-ied in a few other countries, and the findingsare thought to reflect that particular country’sdietary habits. For example, 69% of the PCBsingested by Vietnamese individuals was fromcereals and vegetables. In India, 70% of thePCBs consumed was from cereals, vegetablesand dairy products. The primary source ofdietary PCBs in Germany and The Nether-lands was dairy products; in Canada it wasmeat; in Finland, the Nordic countries andJapan it was fish. It should also be noted thatthe Vietnamese, Indian and Dutch data werebased on raw foodstuffs; it appears that theresults for Germany, Finland and the Nordiccountries were also for uncooked food items;and the Japanese data were based on cookedfoods. It is known that the PCB concentrationsin foodstuffs decrease during cooking. Whilecooking alters the PCB concentration in food,the values obtained from dietary surveys ofthis type are also affected by how the survey-ors chose to handle the data regarding eachindividual’s diet, whether there were or werenot any data for certain food products (bread,preserves, fruit, beverages, etc.) and whethervalues which were below the detection limitswere assumed to contain some (i.e. 0.5 of thedetection limit) or no PCBs (Ahlborg et al.,1992; Duarte-Davidson and Jones, 1994;Boersma et al., 2000).

Absorption, Metabolism and Excretion

Studies with laboratory animals have foundthat gastrointestinal absorption of commer-cial PCB mixtures, as well as that of individ-ual PCB congeners, has often exceeded 90%(Arnold et al., 1993; ATSDR, 2000). Gastro-intestinal absorption has been found to occuron a congener-specific basis by passive diffu-sion, but absorption is enhanced by increasedring chlorination and when the concentrationof PCBs in the gut contents is much greaterthan the concentration in serum lipids. How-ever, it does appear that, similarly to fatsand other fat-soluble chemicals, PCBs are

absorbed from the gut via the lymphaticcirculatory system. PCBs in human plasmaare found predominantly attached to thelipoprotein fraction. As the more chlorinatedPCB congeners are lipophilic, they tend toaccumulate in adipose and lipid-rich tissues,which can then be transferred via breast milkto the nursing infant. It is well known thatPCB congeners cross the placental barrier andaccumulate in fetal tissue, but the tissuelevels of PCBs in the fetus are usually lowerthan those of its mother at parturition. Thisobservation has been attributed to the lowerconcentration of lipids in cord blood whenthe comparison is done on a whole-weightbasis; but, when compared on a lipid basis,the difference is not appreciably large(Ahlborg et al., 1992; Danse et al., 1997;ATSDR, 2000; Feeley and Brouwer, 2000).There are several factors that affect themother’s accumulation of PCBs: her age;number of pregnancies and lactations; placeof residence/exposure to PCBs; and changesin her weight during pregnancy. Whilelactation is a major route of PCB excretion,i.e. approximately 20% of the mother’s bodycontent of PCBs (Duarte-Davidson and Jones,1994), current data suggest that age may bemore of a factor than the number of deliveriesregarding the amount of PCBs accumulatedby the mother. While most studies havefound that breast milk from women living inindustrial areas has greater amounts of PCBsthan from those living in rural areas, breastmilk from native women living in ArcticQuébec contained even greater amounts ofPCBs (Dewailly et al., 1992; ATSDR, 2000). Ithas been estimated that if an infant is breastfed for 6 months, the child will accumulate6.8–12% of his/her lifetime body burdenof PCBs (Kimbrough, 1995; Patandin et al.,1999). In addition to the obvious exposure ofdeveloping infants to PCBs in utero and viabreast milk while nursing, there are a numberof other intrinsic factors which differentiatechildren and adults regarding their exposureto and accumulation of PCBs; for a detaileddiscussion, see ATSDR (2000).

As PCBs are persistent and biodegradeslowly, age and the concentration of PCBs inbiological systems are highly correlated.However, the concentration/pattern of PCB

20-Feb-03 6




congeners accumulated within the various tis-sues from the same individual are often dis-similar. For humans, when the concentrationof PCBs is determined on a lipid weight basis,the highest concentrations are usually foundin adipose (omental/subcutaneous fat), skinand liver tissue, while brain tissue containedthe least amount of PCBs (Ahlborg et al., 1992;ATSDR, 2000).

Metabolism is not a prerequisite forPCBs to exert many of their biochemical andtoxicological effects (Safe, 1992, 1994; ATSDR,2000), but there are exceptions to this general-ity (Sipes and Schnellmann, 1987; Ahlborget al., 1994; Safe, 1994; Koga and Yoshimura,1996). Therefore, the metabolism of PCBsgenerally represents a detoxification process,with the retention or accumulation of conge-ners being correlated with its biological stabil-ity (ATSDR, 2000). Generally, the presence offewer chlorine atoms on the biphenyl rings,coupled with the lack of one or more chlorineatoms at the para position, appears to facilitatemetabolism and excretion (Kimbrough, 1995).The elimination of PCB congeners is largelydependent upon its metabolism, generally toa more polar compound; however, excretionof unmetabolized congeners does occur to alimited degree. Since PCBs are a mixtureof congeners that have different stearic con-figurations, they are metabolized via severalenzymatic pathways whose activity is mark-edly different among species. In general, how-ever, PCBs are metabolized poorly and areeliminated slowly (Sipes and Schnellmann,1987; Ahlborg et al., 1992; Koga and Yoshi-mura, 1996; ATSDR, 2000).

Commercial PCB mixtures are capableof inducing microsomal cytochrome P450

(CYP)-dependent monooxygenases, or phaseI enzymes, in a variety of species. This inturn increases the oxidative metabolism/biotransformation of some PCB congenersin addition to a diverse group of exogenousand endogenous aromatic ring substrates(Safe, 1994). The CYP enzymes frequentlyare termed ‘hepatic drug-metabolizingenzymes’. These enzymes frequently arecharacterized in comparison with the two‘classic’ CYP inducers phenobarbital (PB) and3-methylcholanthrene (MC). As commercialPCBs induce enzymes which possess catalytic

properties similar to PB and MC, they arereferred to as a mixed-type inducer. Variousinducers catalyse the insertion of oxygenin different locations on the biphenyl ringto form reactive arene oxide intermediates,which are often conjugated with such endo-genous substrates as glutathione, glutamicacid or sulphate (McFarland and Clarke,1989). PCBs also induce some of the enzymesassociated with the latter conjugations,which are referred to as phase II enzymesand include such enzymes as glutathioneS-transferase, epoxide hydrolase and glucuro-nosyl transferases (Safe, 1994). Consequently,the major PCB metabolites are hydroxylatedmoieties, although some methyl sulphonyland methyl ether metabolites have beenreported (Hu and Bunce, 1999). Many of thesereactions increase the polarity of the PCB con-geners to facilitate their elimination, primarilyvia the bile and faeces. However, some of thehydroxylated and sulphonated metabolitesalso have toxicological effects (Sipes andSchnellmann, 1987; Safe, 1994). It should alsobe noted that some of the lower chlorinatedcongeners may be excreted via the urine, butthis is often highly species dependent(Ahlborg et al., 1992, 1994).

Further work with commercial PCBmixtures in rodent models has found thatthe mixed-type inducers induce both thePB-induced CYP isozymes of 2A1, 2B1, 2B2,and the MC-induced CYP isozymes of2A1, 1A1 and 1A2 (Safe, 1994). The meta-bolism of PCB congeners has been found tobe isozyme specific, being governed by thelocation of the chlorine substitution onthe biphenyl rings. For example, the CYP1A isozymes are induced by MC-like inducersand preferentially oxidize co-planar (non-ortho) congeners which also have a chlorinesubstitution at the para position of the leastchlorinated ring, i.e. congeners 77, 81, 126 and169. However, congener 81 is the least activeof these four congeners and it also exhibitsPB-type induction. The CYP 2B isozymesare induced by and preferentially oxidizeortho-substituted, non-planar PCBs at an openmeta position. As the CYP 1A and CYP 2Bisozymes can be induced by MC and PB,respectively, co-planar PCBs are oftenreferred to as MC-type inducers and ortho-


20-Feb-03 6



substituted PCBs are referred to as PB-typeinducers (Hu and Bunce, 1999), while all of themono-ortho (i.e. 105, 114, 118, 123, 156, 157, 167and 189) and several of the di-ortho congeners(i.e. 128, 138, 158, 166, 168 and 170) have bothMC- and PB-like inducing properties (Safeet al., 1985; Ahlborg et al., 1992). Some conge-ners can induce the CYP 3A and 4A familyof isozymes, but the structure–activity rela-tionships for these isozymes are less wellcharacterized. However, it appears that thehigher chlorinated PCBs that are biologicallypersistent congeners are those that haveadjacent meta- and para-unsubstituted carbons(ATSDR, 2000).

The mechanism by which the planar andmono-ortho planar PCBs exert their toxico-logical effects is in large part linked to theiraffinity for the cytosol aryl hydrocarbonreceptor (AhR). Congeners that bind to theAhR are referred to as AhR agonists. While anumber of PCB congeners are PB-type induc-ers, congeners that are AhR agonists result inan MC-type induction of CYP isozymes. Thechemical having the greatest affinity for theAhR is 2,3,7,8-TCDD. Once a ligand, such asa mono-ortho planar PCB congener, entersthe cell via passive diffusion through the cellmembrane and binds with the AhR, the result-ing complex undergoes transformation andthen nuclear translocation, where it binds to aspecific genomic sequence prior to the induc-tion of gene transcription (Safe, 1994). Oncethe ligand complex has been formed, twodifferent toxicological pathways have beenidentified. For more details, see Hu and Bunce(1999). The best-characterized interactionthat is mediated directly by the AhR is theinduction of CYP 1A1.

Moore and Peterson (1996) have pointedout that a majority of PCBs apparently haveno toxicological effect upon mammalian sys-tems, and that much of PCBs’ toxicity is attrib-utable primarily to 13 congeners which haveTCDD-like toxicological effects, i.e. AhRagonists. Three of the congeners have no orthochlorines (i.e. the co-planar congeners 77, 126and 169) due to their stearic configuration,while another co-planar congener, number 81,has comparable activity regarding its abilityto induce microsomal enzymes (Safe, 1994).Eight have a single ortho chlorine (105, 114,

118, 123, 156, 157, 167 and 189) and two conge-ners have two ortho chlorines (congeners 170and 180). While there are other di-ortho conge-ners which are AhR agonists, they have beenfound to be less toxic (Ahlborg et al., 1994).

Toxicity and Clinical Effects

Any discussion of the toxicological effects ofPCBs, like that of their metabolism, is compli-cated by the fact that PCBs are a mixture ofcongeners whose mechanism of action isrelatively well known for those congenersthat are AhR agonists versus a less certainmechanism for the remaining congeners thathave toxicological effects. In addition, whencommercial mixtures of PCBs are tested fortoxicity in laboratory animals, they containvarying amounts of contaminants, whichmay have an unquantifiable role in the toxi-cological outcomes. Only recently have someindividual PCB congeners become availablefor in vitro and in vivo toxicological evalua-tion. While the results from such studies willbe illuminating for the congeners in question,such studies are generally poor models forhuman exposure since humans are exposedto a variety of PCB congeners concurrentlywith several different environmental contam-inants which have toxicological properties oftheir own. Unfortunately, sufficient data donot exist to indicate whether such exposurescenarios result in additive, synergistic orantagonistic effects. Similarly, epidemio-logical studies have limitations. For example,it is impossible to find a cohort to serve asthe ‘control’ group that would not havea background level of PCBs in their tissuesand body fluids. In addition, such studiesare also plagued by the presence of other per-sistent environmental contaminants (methylmercury, pesticides, etc.), and various life-style considerations which have been shownto impact health (alcohol, smoking, etc.) andthe parameter being evaluated (i.e. intellec-tual development, which is affected by envi-ronmental, social, economic and geneticfactors; reproduction, which is affected by themother’s age and length of pregnancy). Whilemathematical procedures which are deemed

20-Feb-03 6




to be valid by the scientific community areavailable for ‘controlling’ such circum-stances, the findings of any epidemiologystudy must be viewed with a degreeof caution (Kimbrough, 1995; Seegal, 1996;Danse et al., 1997). In addition, there arevery limited data available regarding howadverse health and/or toxicological effectsare impacted by multiple chemical exposures(Johnson et al., 1998).

McFarland and Clarke (1989) concludedthat the toxic potential of PCB congeners iscorrelated with its ability to induce CYPenzymes, and they have suggested threegroupings. Specifically, congeners that dem-onstrate MC-type and mixed-type inductionhave the greatest potential toxicity. A largergroup of congeners has PB-like inductioncapabilities with less potential toxicity, whileweak inducers and non-inducers have theleast potential toxicity. While Moore andPeterson (1996) have concluded that PCBcongeners with PB-like CYP effects are notovertly toxic, they do point out that such con-geners have the potential to disrupt endocrinehomoeostasis by accelerating the metabolismof endogenous steroids.

Laboratory studies

An acute effect reported in most laboratoryanimal studies in which relatively high dos-ages of PCBs were administered was a wast-ing syndrome, which resulted in non-thriftyanimals continuing to eat and drink whilelosing weight and subsequently dying(McConnell, 1989; Ahlborg et al., 1992). Whilecommercial PCB mixtures have been found toelicit a broad range of toxic responses, theirpotential to elicit such a response dependsupon such factors as: (i) the mixtures’ chlor-ine and contaminant content; (ii) the speciesand strain of laboratory animal; (iii) theanimal’s age and sex; and (iv) the route andduration of administration (Safe, 1994).

Reproductive effects

In a study where rhesus monkeys (Macacamulatta) ingested Aroclor 1248, some of the

reported toxicological effects included de-creased birth weights and other developmen-tal effects. However, the results were con-founded due to maternal toxicity. In anotherstudy, a dose level of approximately 0.04 mgof Aroclor 1016 kg−1 body weight (BW) day−1

produced decreased birth weights in rhesusmonkeys. Further complicating the interpre-tation of these studies was the fact that themonkey chow was found to be contaminatedwith polybrominated biphenyls (Kimbrough,1995). In a subsequent study, where Aroclor1254 was fed to female rhesus monkeys, therewas a statistically significant (P = 0.017)decrease in the conception rate and a sig-nificant (P = 0.04) increasing trend in fetalmortality with increasing dose. The lowestdosed group ingested 5 µg kg−1 BW day−1,and this dose was not considered to be ano-effect level (Arnold et al., 1995). Repro-ductive aberrations have also been foundfor mink ingesting approximately 0.4 mg kg−1

BW day−1 (Ahlborg et al., 1992).

Teratogenic effects

While not as thoroughly studied, teratogeniceffects have been found for mice and chickembryos (Ahlborg et al., 1992).

Endocrine system

Several commercial PCB mixtures havebeen found to be oestrogenic, while co-planarcongeners that are AhR agonists have beenfound to be anti-oestrogenic. Research withindividual congeners has found that con-geners 1, 4, 18, 21, 48, 52, 61, 75, 101, 136 and155 have oestrogenic activity in a variety ofin vivo and in vitro systems, while congener153 is oestrogenic at intermediate levels butnot at high or low dosages. It should be notedthat none of these congeners are AhRagonists, and it has been demonstrated thatthe congeners with the strongest bondingto the oestrogen receptor have at least twoortho chlorines (Moore and Peterson, 1996). Inaddition, hydroxylated PCB congeners havealso been found to have oestrogenic activity(Sipes and Schnellmann, 1987).

Commercial PCB mixtures are known toreduce plasma thyroxine (T4) concentrations,


20-Feb-03 6



increase circulating thyroid-stimulating hor-mone (TSH) levels, and alter thyroid histolog-ical features, but little is known as to whetherthese effects arise due to AhR binding. Whilethe AhR agonist congeners 77, 126 and 169,and the mono-ortho congeners of 118 and156 have all been found to reduce plasma T4

levels, congener 28, a PB-type inducer, doesnot. These observations suggest that PCBsmay affect T4 levels via AhR agonist andAhR-independent mechanisms (Moore andPeterson, 1996; Seegal, 1996).

Neurological development

Commercial PCB mixtures have been foundto cause alterations in active avoidancelearning and retention of a visual discrimina-tion task when rats were exposed pre-natally,but no detectable behavioural changes werefound when rats were exposed to the samePCB mixture post-natally (Ahlborg et al.,1992; Safe, 1994). Behavioural testing withrhesus monkeys whose dams ingestedAroclor 1016 or 1248 showed hyperactivity,retarded learning ability and significantalterations in cognitive behaviour. Many ofthese changes were long lasting and possiblypermanent (Ahlborg et al., 1992; Seegal, 1996).While most of the data have indicated thatthe observed behavioural effects were associ-ated primarily with pre-natal exposure, sig-nificant behaviour alterations have also beenobserved when non-human primates wereexposed to PCB congeners post-natally. Itwas concluded that the structure of the PCBcongener (i.e. ortho-substituted vs. co-planar)and the animal’s age when it was exposed tothe PCBs influence the toxicological response(Seegal, 1996). In addition, commercial PCBmixtures also cause regional alterations inneurotransmitter levels in the brains of somelaboratory animals (Ahlborg et al., 1992; Safe,1994; Moore and Peterson, 1996).

Immunological changes

Data are available from a variety of studieswherein commercial PCB mixtures have beenadministered orally to laboratory animals,and the elicited immunological changes varyamong commercial PCB mixtures and among

species (ATSDR, 2000). It has been observedin mice that the order of potency regardingthe Aroclor-induced immunotoxicity wasAroclor 1260 > 1254 > 1248 > 1242 > 1016 >1232 (Safe, 1994).

The findings with non-human primatestend to be emphasized by some agencies sincethe monkey appears to be a sensitive species,and has biological and phylogenetic similari-ties to humans (ATSDR, 2000). In this regard,changes in the immune function of adultfemale rhesus monkeys and their offspringhave been reported when they were exposedto Aroclor 1254 dosages as low as 5 µg kg−1

BW day−1. The suppression of the antibodyresponse to sheep red blood cells (SRBCs) wasthe parameter most consistently affected inboth groups of monkeys. However, the signif-icance and/or relevance of these data havebeen viewed quite differently (Kimbrough,1995; ATSDR, 2000).

Carcinogenicity studies

Several chronic bioassays had reportedtumours of the liver when PCBs containing60% chlorine were fed to rats (Ahlborg et al.,1992). In one study, diets containing 100 ppmClophen A60 or Clophen A30 resulted inincidence rates of 61 and 3% hepatocellularcarcinomas, while the incidence rate in thecontrols was 2%. In another study, Aroclor1260 was chronically fed to male and femaleSprague–Dawley rats. The histopathologicalevaluation revealed that the incidence ofhepatocellular adenocarcinomas and trabe-cular carcinomas was 51 and 40%, whilein males the incidence was 4 and 0%, respec-tively. When male and female Fischer F344rats were fed Aroclor 1254, the incidence ofgastric intestinal metaplasia and adenocarci-noma was similar in both sexes (Safe, 1994).Thus the carcinogenic potential of commer-cial PCBs is dependent upon its composition,and the sex and strain of the rats being tested.Following a review of such data, it wasdecided to revise the criteria for the classifica-tion system used for various liver lesions. Therevised classification reaffirmed that chronicdietary exposure of rats to PCBs containing60% chlorine did result in the development ofbenign and malignant liver lesions. However,

20-Feb-03 6




chronic exposure of rats to PCB formulationscontaining 54 or 42% chlorine did not resultin a statistically significant increase in benignor malignant liver tumours (Kimbrough,1995). A subsequent comprehensive chronictoxicity and carcinogenicity study withAroclors 1016, 1242, 1254 and 1260, usingdose levels of 25–200 ppm, resulted in ahighly sex-dependent increase in the inci-dence of hepatocellular neoplasms. For themales, only those in the 100 ppm Aroclor1260 group, the highest dose group for thismixture, had a significant increase in thenumber of liver neoplasms. There was alsoa slight non-dose-related increase in theincidence of thyroid gland follicular celladenomas for males receiving diets contain-ing Aroclor 1242, 1254 and 1260. A signifi-cant and generally dose-related increase inthe incidence of hepatic adenomas wasfound for the females in all treatmentsexcept for the 50 ppm group receivingAroclor 1016 in their diet. The magnitudeof the increase was greatest for Aroclor1254 > 1260 ≈ 1242 > 1016. No increase in thy-roid neoplasms was found for the females(Mayes et al., 1998). While the latter studysuggests that a greater range of chlorinatedbiphenyls may be able to induce a carcino-genic response in laboratory rats, it stillsupports the conclusion that different PCBmixtures do not have equal potency regard-ing their ability to cause cancer (Kimbrough,1995).

While a variety of commercial PCB mix-tures have been found to induce carcinogenicresponses in laboratory rats, a substantialamount of evidence is available to suggest thatPCB mixtures are not complete carcinogens,but may only be tumour promoters (Ahlborget al., 1992; Safe, 1994). Simplistically, thecarcinogenic process can be thought of asencompassing two steps: tumour initiationand tumour promotion. Tumour initiationinvolves the interaction of the chemical withDNA resulting in a critical DNA lesion, whichwill evolve into a tumour given sufficienttime. Such chemicals are referred to as com-plete carcinogens or genotoxic carcinogens.Some chemicals have the ability to ‘promote’an initiated DNA lesion but are incapableof producing tumours by themselves. These

chemicals have been referred to as promotersor epigenetic carcinogens.

Many of the clinical findings attributedto PCBs resemble those of a vitamin Adeficiency, and it is known that several PCBmixtures reduce the storage levels of vitaminA in several species (Ahlborg et al., 1992).In a review of the toxicity induced by com-mercial PCB mixtures, Safe (1994) concludedthat the PCB-induced lethality was notdependent solely upon the mixture’s degreeof chlorination. Since the toxicity of PCBswas due to the individual congeners con-tained in a mixture, it was possible that oneor more structural subclasses of congenerswere responsible for the toxicities elicited byPCB mixtures. He concluded that there wasno consistent structure-dependent effect thatwas responsible the specific types of toxico-logical responses observed.

Epidemiology studies – non-cancerousoutcomes

Dermatological

Occupational exposure to PCBs appears to berelated to hyperpigmentation in addition tochloracne. In the Yusho and Yu-Cheng inci-dents, chloracne and hyperpigmentation ofthe skin, gingiva and nails were frequentlyobserved and, while these lesions have dimin-ished in the intervening years, they were stillevident 10–14 years later (Masuda, 1996; Guoet al., 1999). Ocular manifestations such ashypersecretion and swelling of the sebaceousglands of the eyelids were also common(Kimbrough, 1995; Longnecker et al., 1997).

Reproduction

Findings show that women in a number ofPCB exposure situations above backgroundlevels of PCBs – such as industrial, Yusho andYu-Cheng, sport fish consumption and nativepopulations – have given birth to childrenwith slightly lower birth weights or shorterbirth body lengths and/or smaller head cir-cumferences. In some situations, the lowerbirth weights were partially attributable to


20-Feb-03 6



a shorter gestation period. However, therewere a number of methodological problemswith these studies: many did not control forinfluencing factors; there was concern asto the presence of other persistent con-taminants; and there was some question asto what PCB standard was used for theanalyses. The findings were not consistentamong these studies and, therefore, havebeen deemed by some to be inconclusive withregard to the effect of PCBs per se. However,PCB exposure has not been found to affectthe rates of spontaneous abortions or still-births (Ahlborg et al., 1992; Kimbrough, 1995;Longnecker et al., 1997; Johnson et al., 1998;Yu et al., 2000).

Neurological development

Data from three different types of studies,one with a population cross-section in NorthCarolina and in The Netherlands; anotherwith mothers who were frequent consumersof Lake Michigan fish; and a third withmothers who ingested contaminated riceoil in Japan or Taiwan have suggested thatpre-natal exposure to PCBs and other per-sistent toxic substances may be adverselyaffecting the neurological development ofchildren (Chen and Hsu, 1994; Johnson et al.,1998; ATSDR, 2000; Boersma et al., 2000).While the Japanese and Taiwanese mothersshowed signs of toxicity which were attrib-uted primarily to the PCB contaminants, themothers in the other three studies didnot exhibit signs of toxicity. However, likethe Japanese and Taiwanese incidents, thefindings from the Dutch study potentiallywere compromised regarding the effectsof PCBs per se due to the presence of dioxin.In the North Carolina study, the mothers’PCB and DDE (1,1-dichloro-2,2-bis(p-phenyl)ethylene) exposure was limited to back-ground levels of both entities. Whilesome early neurodevelopment deficits werereported for the most highly exposed mem-bers of this population, the deficits were notapparent when the infants were 3, 4 or 5 yearsof age. These results suggest that in uteroexposure to PCBs is potentially more deleteri-ous to an infant than exposure via breast milk(Seegal, 1996). When the Dutch cohorts were

2 weeks of age, an adverse effect of PCBs,polychlorinated dibenzodioxins (PCDDs)and PCDFs on neurological performance wasevident; at 3.5 years of age, they showed anadverse effect of pre-natal PCB exposure oncognitive, but not neurological development.However, at 18 months of age, scores forcognitive development were not related topre- or post-natal exposure to PCBs. In theLake Michigan study, a number of effects onthe developing nervous system and deficitsin intellectual performance were found dur-ing the first testing period and were alsoapparent at subsequent evaluations, but therewas no strong correlation between maternalPCB consumption and lower infant birthweights. Some reviewers have concludedthat the levels of PCB exposure for themothers in the Lake Michigan and NorthCarolina studies were within the range ofPCB blood levels reported for the entireNorth American population and, therefore,have questioned whether the Lake Michiganfindings should be attributed to PCBs per sesince there is no conclusive evidence that thePCB levels in the general population haveresulted in intellectual deterioration in chil-dren exposed to PCBs in utero. It has also beenreasoned that, since the findings from theLake Michigan and North Carolina studieswere dissimilar, it is possible that some otherchemical entity may be responsible for theLake Michigan findings (Ahlborg et al., 1992;Safe, 1994; Danse et al., 1997; Longneckeret al., 1997; Boersma et al., 2000).

In the Yusho and Yu-Cheng incidents,slow nerve conduction, especially of the sen-sory nerves, was documented in many cases(Kuratsune and Shapiro, 1984). In addition,infants of the exposed mothers exhibited arange of neurobehavioural deficits whichpersisted for several years (Chen and Hsu,1994), but this may be due to the presenceof PCDFs and PCQs as contaminants in thePCBs. The fact that the PCB blood levels forJapanese and Taiwanese capacitor workerswere greater than those determined for theYusho and Yu-Cheng victims is cited assupport for the effects of PCDFs and PCQs(Danse et al., 1997; Johnson et al., 1998).

In summary, there appears to be a diver-gence of opinion as to the weight of the

20-Feb-03 6




evidence regarding the neurodevelopmenteffects on cognitive impairment associatedwith PCBs, dioxins and other persistent toxicsubstances in all of the cohorts, except forthe Yu-Cheng children (Chen and Hsu, 1994;Danse et al., 1997; Johnson et al., 1998; ATSDR,2000). In this group, there is evidence for ashift downward in the IQ distribution curvewhich does not appear to be reversible (Chenand Hsu, 1994; Johnson et al., 1998).

Liver abnormalities

Studies with industrially exposed personnel,while having shortcomings analogous tothe reproduction studies, suggested thatPCB exposure can lead to increased levelsof some hepatic enzymes, but the resultswere not uniform nor have there beenany reports of increased incidents of livercirrhosis (Kimbrough, 1995; Longnecker et al.,1997). However, the Yu-Cheng victims haveexperienced a substantial elevation in themortality rates for cirrhosis and chronic liverdisease (Yu et al., 1997).

Thyroid effects

For a cohort of industrially exposed men, norelationship was found between PCB expo-sure and thyroid hormone levels. In the Dutchstudy, background levels of PCBs in breastmilk were associated with lower maternaltri-iodothyronine (T3) and T4 levels, butboth levels were within normal limits; theirinfants, however, had higher plasma levels ofTSH but lower plasma levels of T3 and T4. Theinvestigators concluded that elevated levelsof dioxins and PCBs can alter thyroid status.This group has also reported contradictoryfindings among subsets as to whether anassociation between altered thyroid statusand decreased neurological optimality scoresexists. Consequently, the pathophysiologicalmeaning of these observations currently isuncertain (Seegal, 1996; Longnecker et al.,1997; Johnson et al., 1998; Feeley and Brouwer,2000). In a 14-year follow-up of the Yu-Chengpatients, who were at least 30 years old at thetime of the follow-up, an increased incidenceof goitre was reported for both the men andthe women (Guo et al., 1999).

Immunological effects

Several studies were evaluated and thefindings were inconsistent and may beconfounded by the presence of dioxin(Kimbrough, 1995; Longnecker et al., 1997).Immune changes were demonstrated inthe Yusho and Yu-Cheng populations,but some were reversible. It was concludedthat 16 years after the Yusho incident, thechildren who were exposed in utero did nothave suppressed immunity. In the Dutchstudy, PCBs and dioxin were found toinfluence fetal and neonatal immune sys-tems, but this was not reflected in anincreased incidence of respiratory symptoms(Johnson et al., 1998; Yu et al., 1998).

Respiratory function

There have been suggestions that industrialPCB exposure may be associated withchronic bronchitis, upper respiratory irrita-tion, abnormal forced vital capacity, etc.,but the findings were deemed to be inconclu-sive due to the study’s many confoundingfactors (Kimbrough, 1995). Some Yushoand Yu-Cheng patients suffered froma chronic bronchitis-like syndrome forseveral years, marked by a large amountof expectorant during the early stages.Pathophysiological findings revealed thatthe disease was localized in the small air-ways. However, this bronchitis-like syn-drome has not been detected in studies withpeople who have been exposed to dioxin(Ahlborg et al., 1992).

Miscellaneous health effects

In one study, there was an associationbetween high levels of PCB-contaminated-fish consumption and increased bloodpressure, but this finding has not been repli-cated and the study had several confoundingfactors (Kimbrough, 1995; Longnecker et al.,1997; Johnson et al., 1998).

In conclusion, many of the responsesobserved regarding PCB exposure, particu-larly for industrial exposure, were reversible,and often there was not a significant correla-tion between response and PCB levels in fat


20-Feb-03 6



and blood. For the Yu-Cheng and Yusho inci-dents, the symptomatology included severeand persistent chloracne, dark brown pig-mentation of nails, skin thickening, a varietyof ocular problems and numerous subjectivecomplaints. The offspring, particularly thoseof the Yu-Cheng mothers, were smaller in stat-ure, were found to have a modest learningdeficit and displayed many of the toxic symp-toms observed in their mothers. However,it is generally agreed that these symptomswere not due to PCBs per se, but were moreattributable to the contaminants in the PCBsused to cool the rice oil (Safe, 1994; Feeley andBrouwer, 2000).

Epidemiology studies – cancerous outcomes

Most of the studies have examined industrialexposure to PCBs or there has been anattempt to correlate PCB blood levels withvarious types of cancer. A number of short-comings can be found with these studies,including the number of cohorts, the limitedlength of follow-up, the minimal time ofexposure (i.e. 1 day to 6 months), the variablelevel of exposure, which has limited theirusefulness, and the possibly that the PCBmixtures were contaminated. However, inseveral studies, there were increased inci-dences of specific cancers such as liver andbiliary tract but there were no consistentincreases in one or more types of cancer.Therefore, no conclusive evidence of a linkbetween PCB exposure and a human cancerrisk has been found for industrial exposure(Ahlborg et al., 1992; Safe, 1994; Kimbrough,1995; Danse et al., 1997; Longnecker et al.,1997).

The data from the Yusho study haveshown a significant increase in the incidenceof death attributable to cancer of the liver andrespiratory system in males but not females(Ahlborg et al., 1992). Thirteen years after theYu-Cheng incident, a substantial increase inthe mortality rate for chronic liver disease andcirrhosis was evident. However, the mortalityrate from malignant neoplasms was not sig-nificantly different from that of the generalpopulation (Yu et al., 1997).

Risk Assessment Strategy

While toxicological effects of commercialmixtures of PCBs can be studied in variousin vivo and in vitro assays, the data from suchstudies have significant shortcomings withregard to their risk assessment value in theregulatory context. Such shortcomings relateto the fact that humans are not exposed tocommercial PCB mixtures per se or to singlecongeners, but to a variety of PCB congenersand other environmental pollutants, some ofwhich may be structurally related to PCBs,such as PCDDs and PCDFs. The pragmaticapproach developed to deal with this sce-nario, known as toxicity equivalency factors(TEFs), occurred in the 1980s and was ini-tially used to assess the risks associated withemissions of PCDDs and PCDFs formed dur-ing high-temperature incineration of variouswastes. PCDDs and PCDFs are also producedas by-products during various industrialchlorination processes, during the smeltingof metallic ores and during pulp and paperproduction (Safe, 1994; Kimbrough, 1995).

TEFs were developed to assess thepotency of various polyhalogenated aromatichydrocarbons against that of 2,3,7,8-TCDD,the most toxic of the dibenzo-p-dioxincongeners. Simplistically, TEFs can be deter-mined for any in vivo or in vitro test/assay,but the relative rankings among tests andassays may not always be similar as theymay be species specific and/or affected bypharmacokinetics and exposure time. Gen-erally, TEFs can be developed for such in vivoassays as enzyme induction, thymic atrophy,body weight gain, teratogenicity/develop-mental toxicity, immunotoxicity, carcinoge-nicity and lethality, as well as for a host ofin vitro assays. While 2,3,7,8-TCDD has beenassigned a TEF value of 1, it has been foundthat test congeners have a relative potencythat was 1–5 orders of magnitude less thanthat of 2,3,7,8-TCDD (Clemons et al., 1997; Vanden Berg et al., 1998, 2000). However, it shouldbe noted that the TEFs are only estimates of acongener’s relative potency.

In the first decade after the TEF conceptwas introduced, several TEF schemes weredeveloped which led to a number of criticisms

20-Feb-03 6




before a harmonized approach was devel-oped (Kimbrough, 1995; Van den Berget al., 2000). For a compound to have aharmonized TEF value, there was agreementthat it must be structurally similar to PCDDsand PCDFs; it must bind to the AhR; it mustelicit AhR-mediated biochemical and toxicresponses; it must be environmentally persis-tent; and it must accumulate in the food chain.When deriving a TEF value, in vivo studieswould be given more weight than in vitrostudies, with chronic in vivo studies beinggiven more weight than subchronic > sub-acute > acute, and the AhR toxic responseswould receive more weight than biochemicalresponses. Enhanced acceptance of the TEFconcept, and the resulting TEF values, isattributable to the finding that TEF valuesthat were based on AhR-mediated responseswere generally additive. However, thereare still three major criticisms of the TEFapproach: (i) non-additive interactions whenmixtures of dioxin-like and non-dioxin-like congeners are tested; (ii) differences inspecies’ responsiveness; and (iii) differencesin the shapes of the dose–response curvesamong AhR agonists. Even with theseacknowledged shortcomings, it has beensuggested that the use of TEFs is pragmati-cally the most feasible approach for humanrisk assessment. However, their use severelyunderestimates the risk to humans exposedto PCBs, PCDDs and PCDFs since onlydioxin-like congeners are included in the TEFcalculation (Van den Berg et al., 1998, 2000).

After the TEF values have beendetermined, they can be combined with thechemical residue data for the calculation oftoxic equivalents (TEQs) in an environmentalsample, animal tissues, food, soil, etc. TEQconcentrations for samples are calculatedusing the following equation:

TEQ PCDD TEF PCDF

TEF [PCB TEF

= n1

i in3

[ ] [

]i i in× + ∑∑

× + ∑ ×2

i]

In short, this equation allows one to cal-culate TEQs for complex mixtures of chemi-cals for which TEFs are known, thereby reduc-ing a complex mixture of congeners to a singlevalue which represents the amount of TCDD

equivalents in the sample. Recently, agree-ment has been reached whereby TEQs can becalculated not only for samples containingPCDDs, PCDFs and planar PCBs but also fora large number of other halogenated com-pounds that meet the criteria for inclusion inthe TEF concept; this could enhance the use-fulness of TEQs derived for environmentalsamples (Safe, 1994; Van den Berg et al., 1998).

Risk Management Issues

While production of PCBs may have startedin 1929, it has been estimated that, inrecent years, over 1 million kg of PCBs haveentered the environment annually fromworldwide mobile reserves, due to accidentalrelease, leaching/volatilization from hazard-ous waste sites, illegal dumping, etc. PCB riskmanagement plans, initially developed by anumber of industrialized countries, includedpolicies designed to prevent future releasesof PCBs into the environment (Kannan, 2000).For example, the European Union CouncilDirective 96/59/EC outlined steps to controlthe release of PCBs into the environment byoutlining measures designed to address thedisposal of PCBs and the decontamina-tion/disposal of equipment known to containPCBs. The ultimate goal was the completecessation of further environmental contami-nation by PCBs. As an initial step, the direc-tive required EC member states to compileinventories of all equipment containing morethan 5 dm3 of PCBs. (It appeared as if the ini-tial endeavours associated with this directivewere to decontaminate and properly disposeof the larger PCB ‘reservoirs’ without concernfor the PCB concentration per se.) The inven-tories were to be completed by 1999 withplanned regular updates. Once the inventor-ies were completed, the objective was tohave all inventories, depending on thepercentage PCB content, decontaminatedand/or disposed of by 2010. For example,decontamination of electrical equipmentcontaining PCBs is aimed at reducing thePCB concentrations to less than 0.05% byweight, with the ultimate goal of havinga concentration of less than 0.005%. Recent


20-Feb-03 6



amendments to this directive, suggestedas part of the final draft of the StockholmConvention on Persistent Organic Pollutants(2001), would permit PCB-containing equip-ment to remain in use until 2025 but no laterthan 2028. Also, additional effort should bemade to identify and inventory other articlescontaining greater than 0.005% PCBs.

Similar PCB management/disposaloptions have been developed in the USA. Themanufacture of PCBs has been prohibitedunder the Toxic Substances Control Actsince 1977, and any material containinggreater than 50 ppm PCBs is considered to behazardous waste and treated accordingly. Theuse of products, such as hydraulic fluids,paper products, etc., which contain less than50 ppm PCBs is still allowed provided theEPA (2000) has determined that the productsin question do not present an environmentalor human health risk. Additional regulationsdeal with the proper storage of PCBs alongwith the import and export of PCBs fordisposal.

Until 1977, PCBs were imported into Can-ada mainly for use in electrical transformersand capacitors. As part of the ChlorobiphenylRegulations under the Canadian Environ-mental Protection Act (CEPA, 2000), a varietyof guidelines have been promulgated todeal with the use, inventory, transportation,storage and disposal of PCBs. Based on a com-prehensive survey, approximately 16 kt ofPCBs were estimated to have been dispersedinto the environment. National inventoriesof PCBs-in-use and PCB-containing materialsin regulated storage facilities have been con-ducted on an annual basis in Canada since1989. There currently are over 130 kt of PCBwaste in regulated storage facilities. Proposeddraft amendments to the ChlorobiphenylRegulations include the following require-ments: the use of PCBs in any equipmentbe discontinued by 2010; any PCB materialcurrently in storage be disposed of by 2015;and the environmental release of any liquidcontaining PCBs be restricted when theconcentration is greater than 0.1 ppb foraqueous mixtures and 400 ppb for oils andnon-aqueous liquids.

Regulations enacted to control the furtherreleases of PCBs from closed or partially

closed applications as well as remediation ofidentified hazardous waste sites have beenshown to reduce the levels of PCBs in the envi-ronment. For example, in the North AmericanGreat Lakes ecosystem, concentrations ofPCBs found in predatory fish species, suchas lake trout, have declined from mid-1970values of 8 ppm to less than 1 ppm by 1994(Scheider et al., 1998). However, PCBs stillaccount for 47% of the fish consumptionadvisories issued in Canadian waters; theseadvisories are issued by various levels ofgovernment to sport anglers warning themnot to consume specific species of fish fromspecific waters due to contamination with var-ious persistent chemicals. Further indicationsof the overall decline of PCBs in the environ-ment can be found in human specimens – forexample, PCB residues in breast milk. From1972 to 1992, the average concentration ofPCBs in breast milk samples from Swedishwomen declined by approximately 70% (from1.09 to 0.324 ppm) (Norén and Meironyté,2000), while similar declines have beenobserved in Canadian breast milk samplesfrom 1982 to 1992 (from 0.68 to 0.21 ppm)(Newsome et al., 1995).

With a number of persistent organic con-taminants known to be present in the foodsupply, such as PCBs, government agenciesattempt to monitor their presence in foodsdirectly so as to limit the population’sexposure to them. They conduct nationaldietary monitoring surveys to identify thosefood commodities that contain the greatestconcentrations of persistent contaminantsso that changes over time can be monitoredand future data collections prioritized.Congener-specific PCB analysis of 138 pre-pared food composites, collected from acrossCanada between 1992 and 1996, estimatedthat the average person consumed 342 ngPCBs day−1 (Newsome et al., 1998) comparedwith a 1980 estimate of 3.9 µg day−1. Thedairy (40%), meats (26%) and fish (16%) foodgroups combined accounted for approxi-mately 80% of the total ingestion. In compari-son, recent food surveys from the UK haveestimated their dietary PCB ingestion atapproximately 512 ng day−1, with the dairy,meat and fish composites accounting for 32, 16and 26% of the total exposure, respectively

20-Feb-03 6




(Duarte-Davidson and Jones, 1994). The aver-age dietary intake of PCBs in the UK for 1982was estimated at 1.0 µg day−1, indicatinga decrease of 66% between 1982 and 1992(MAFF, 1997). Total diet studies from the USAindicated that between 1991 and 1997, themean daily intake of PCBs by all segments ofthe population ranged from approximately10 ng for infants to 324 ng for adults (ATSDR,2000). From the available data, with the excep-tion of infants and children up to 2 yearsof age, PCB ingestion by all other segments ofthe population declined by over 50% between1991 and 1997.

Risk assessment

International perspectives

The importance of a safe food supply fromthe perspective of health and internationaltrade was well recognized prior to the UnitedNations establishing the Food and Agri-culture Organization (FAO) in 1945, whoseinitial mandate was to improve nutritionalstandards and agricultural productivity. TheFAO combined with the World HealthOrganization (WHO) in 1962 to form theFAO/WHO Food Standards Programme,and designated the Codex AlimentariusCommission (CAC) as the authoritative bodyresponsible for establishing internationalfood standards designed to protect consum-ers from unsafe food. The subsidiary of theCAC given the responsibility for developingguidelines and standards related to foodcontaminants was the Codex Committee onFood Additives and Contaminants (CCFAC),which, in turn, is supported by the riskassessment activities of the FAO/WHOJoint Expert Committee on Food Additives(JECFA). The task of JECFA is to provide rec-ommendations regarding the maximum tol-erable intake of specific contaminants; theseserve as the basis for any related guidelinedecisions by CCFAC. Prior to the ratificationof any contaminant guideline by CCFAC andits recognition by the World Trade Organiza-tion, member countries are consulted at leasttwice during the development process in

accordance with the step-wise procedureoutlined in the CAC General Standard forContaminants and Toxins in Foods (GSCTF)(FAO/WHO, 1995). After ratification, it isthen the responsibility of each membercountry to introduce the ratified guidelineinto their national legislation. While the useof Codex standards is still at the discretion ofindividual countries, the Agreement on theApplication of Sanitary and PhytosanitaryMeasures (SPS Agreement) of 1995 formallyrecognizes these guidelines as the interna-tional standard and provides legal ‘encour-agement’ for their use. Currently, CAC has165 member nations, representing more than98% of the world’s population. Additionaldetails regarding the international aspectsof the development of food contaminantstandards can be found in a recent publica-tion by Rees and Watson (2000).

The WHO previously has conductedformal reviews of PCBs that resulted in pub-lished Environmental Health Criteria docu-ments in 1976 and 1993, and JECFA initiallyconsidered PCBs in 1989. The final conclusionreached by the latter group was that it wouldnot be possible to suggest a precise tolerableintake level for PCBs regarding human con-sumption; however, attempts should be madeto set guidelines or standards for those nutri-tionally essential food commodities in whichPCBs occur, such as fish, milk, meat and dairyproducts (WHO, 1990). The Committee didstate, however, that dietary intake levels ofPCBs up to 0.2 µg kg−1 BW day−1 ‘did notinvolve any long term hazard’. The Com-mittee also reported that, although breast-fedinfants may ingest levels of PCBs up to 12.0 µgkg−1 BW day−1, they considered that theknown benefits of breast feeding outweighedany potential health hazard associated withPCB ingestion. Since that time, a discussionpaper on dioxins and PCBs was presented byThe Netherlands to the 27th session of CCFACin 1995 (as outlined in the GSCTF guideline forthe development of a Codex standard). Thediscussion paper put forward the opinion thatthere were potential health concerns related tothe dietary intake of PCBs and that, in certaininstances, international trade had alreadybeen affected by PCB-contaminated food-stuffs. The paper included recommendations


20-Feb-03 6



that CCFAC should develop a maximumlevel guideline for PCBs in foods involvedin international trade and that JECFAshould maintain PCBs on its priority assess-ment list.

Risk assessment process

Assessing the potential risk to human healthposed by environmental contaminants suchas PCBs initially involves the identificationof the hazardous substances, followed bythe description or definition of the riskassociated with any potential exposures tothe hazardous substances. Identificationusually is accomplished using in vivo and/orin vitro toxicity assays and/or epidemiologi-cal findings.

Hazard identification involves determin-ing whether an agent or chemical causes toxiceffects, the nature of these effects and whetherthe effects are likely to occur in humans, i.e.potential relevance to human health. The sec-ond stage, exposure assessment, is the processof actually measuring or estimating the inten-sity, frequency and duration of human expo-sure to the agent in question. For persistentorganic contaminants such as PCBs, where themajority of the exposure will be from the diet,a number of countries have participated inthe WHO Global Environment MonitoringSystem (GEMS) – Food Contamination Moni-toring and Assessment Programme, whichwas established in 1976 (Weigert et al., 1997).The third stage, hazard characterization ordose–response analysis, generates estimatesof a no observable adverse effect level(NOAEL) and the lowest observed adverseeffect level (LOAEL) doses, usually fromin vivo toxicology data. This stage can alsoinvolve deriving the best mechanism/proce-dure by which to extrapolate experimentalfindings to humans. The final stage, riskcharacterization, compares the exposure datawith the dose–response analysis in order todevelop risk estimations of an adverse healtheffect for a particular exposure. Variousmodels of this risk assessment paradigm areemployed by international (World HealthOrganization) and national (Health Canada

(CEPA, 2000); US Environmental ProtectionAgency (EPA, 2000)) regulatory agencies.

Through the process of scientific evalua-tion of all pertinent toxicological data,dose–response relationships for contaminat-ing agents/chemicals can be established, i.e.identifying those exposure levels known tocause and, more importantly, not to causetoxic effects. The latter values commonly arereferred to as the NOAELs and are defined asthe dose at which no biologically significantadverse effects are observed in the studypopulation compared with the controls. Whileepidemiological studies with documentedexposure assessments are preferred, experi-mental animal bioassays with internationallyaccepted study protocols are generally usedfor identification of NOAELs. While animalbioassays have certain advantages such ascontrolled exposures and the thoroughquantification of toxic responses, the resultsrequire extrapolation to humans. This extrap-olation process is an inexact exercise at best,due to such things as pharmacokinetic/toxicokinetic differences between species; theuse of high dosages in animal bioassaysversus the low dosage human exposures;the species-specific mechanism of actions; thedifficulty in ascertaining the dose–responsecurve, etc. Typically, regulatory agencies haveemployed uncertainty or safety factors tocompensate or adjust for the known physio-logical and biological differences betweenexperimental animals and humans. Initially,the US Food and Drug Administration (FDA)suggested, as a default, that a 100-fold safetyfactor be applied to NOAELs derived fromchronic animal bioassays – other than chroniccancer bioassays – to estimate a safe exposurelevel for food additives which could be pres-ent in the diet (Lehman and Fitzhugh, 1954).The defined rationale for the 100-fold safetyfactor is that humans are potentially tenfoldmore sensitive to the toxic effects of chemicalscompared with experimental animals, anda tenfold difference in human susceptibilityexists within a population, i.e. 10 × 10 = 100(Waltner-Toews and McEwen, 1994). Based,in part, on human responses to a wide rangeof environmental contaminants, it has beenestimated that this tenfold human variabilityfactor would provide protection for up to

11-Mar-03 6




95% of a population (Calabrese, 1985). Avariety of additional factors have been consid-ered regarding extrapolation methodologies,including the overall adequacy of the scien-tific database and the severity of the toxico-logical end point (Vermeire et al., 1998).Using such additional information, regula-tory agencies have been known to deviatefrom the standard 100-fold uncertainty factor,especially in the risk assessment for foodcontaminants. Conversely, the lack of soundepidemiological studies and/or deficienciesin the experimental animal database haveresulted in higher degrees of uncertainty andforced risk assessors to adopt a more conser-vative approach, i.e. the use of an uncertaintyor ‘safety’ factor greater than 100-fold.

After identification of the appropriateNOAELs, based on the available toxicologicaldata, and after deciding on the appropriateextrapolation factor, exposure regulations,such as tolerable daily or weekly intakes(TDIs/TWIs), can be set. However, the termi-nology used to describe essentially safe expo-sure levels can vary among organizations; forexample, WHO and Health Canada use TDIs,the EPA uses oral reference dose (RfD), whilethe US Department of Human Health Services(of which the FDA is a component) uses mini-mal risk level. Regardless of the nomenclatureused, these intakes, when expressed on a bodyweight basis averaged over an entire humanlifetime, are thought to represent an exposurelevel that is without appreciable risk of anadverse effect to human health.

A major part of the overall hazard charac-terization process includes the determinationof the genotoxic/carcinogenic potential ofthe chemical. For chemicals thought to begenotoxic carcinogens, a more conservativerisk assessment approach is taken since anyexposure to the chemical is considered to be apotential risk to human health. This approachis referred to as the non-threshold concept andits tenet is that exposure to as little as one mol-ecule of the chemical poses a risk to health.Therefore, no safety or uncertainty factor isusually given for a genotoxic carcinogen. Asanalytical technology has not progressed tothe point where a single molecule of anundesirable chemical (i.e. a genotoxic carcino-gen) can be analysed for in a foodstuff, the

exposure level is defined alternatively asthe average daily dose during a lifetime thatwould be associated with a negligible or back-ground cancer risk, i.e. one additional cancerper 105–107 lifetimes. This mathematicallyextrapolated value is derived from the avail-able scientific data via the use of probabilisticmodels, such as the linearized multistagemodel or the Moolgavkar–Venzon–Knudsonmodel. Both models provide estimates of apotential cancer risk in the low or environ-mental dose range as compared with the highdose experimental animal studies. Currently,the International Agency for Research on Can-cer (IARC, 1979) places chemicals or agentswhich may cause cancer in humans into twogroups: in group 1, the agent is carcinogenic tohumans; in group 2A, the agent is probablycarcinogenic to humans; and, in group 2B,the agent is possibly carcinogenic to humans.To date, the data supporting IARC’s classifica-tion of an agent or chemical as a group 1 or2 human carcinogen have been obtainedsolely from occupational exposure studies.Conversely, IARC has not found sufficientevidence to conclude that environmentalexposure to any chemical or agent on thegroup 1 or 2 list has been associated with anyincrease in human cancers.

The final step in the process involves arisk management strategy. These decisionsare implemented when the intake of a par-ticular contaminant exceeds the TDI. Such ascenario would depend on the duration ofexposure, i.e. how often the TDI is or maybe exceeded during the average lifespan; thenature and severity of the known toxicologicaleffects in humans; and the known benefitsassociated with the exposure venues, i.e.breast feeding, social/cultural and nutritionalaspects of foods. A more complete descrip-tion of the risk assessment process and themajor uncertainties involved with it can befound in a National Research Council publica-tion (1994).

Risk assessment – PCBs

Initial risk assessments for PCBs were under-taken after the 1968 rice oil poisoning episode


20-Feb-03 6



in Japan and after it was known that PCBswere widely prevalent in the environmentas well as in the food supply to the extentthat PCBs were found in human breastmilk samples throughout the world. TheYusho incident originally was attributedto the rice oil being contaminated with PCBs,but subsequent analysis of the rice oil indi-cated the presence of substantial amounts ofPCBs’ thermal degradation products, whichwere either known or subsequently foundto be more toxic than PCBs. A variety ofexperimental studies conducted with rodentsand non-human primates, using commercialPCB mixtures, have determined that PCBscan: cause immune system effects; functionas endocrine disruptors; induce adverseneurobehavioural and developmental effects,especially in infants; and cause cancer. Anyattempt to extrapolate these experimentalresults with commercial PCBs to humansshould consider a variety of issues, including:

• Commercial PCBs were manufactured byvarious techniques to a specific weightper cent of chlorine and were known to besubject to lot-to-lot variability, especiallyfor Aroclors 1248 and 1254. In addition,certain production techniques for PCBsresulted in higher concentrations ofdioxin-like PCB congeners and con-taminating dibenzofurans (Frame, 1999).

For example, a number of adverseeffects induced when rhesus monkeyswere chronically exposed to Aroclor1254 were similar to effects seen whennon-human primates had been fed dietscontaining low levels of TCDD. Theseeffects included ocular and dermato-logical lesions, nail bed deformities,reduced fecundability and increasedfetal mortality. On the basis of therelative concentrations of PCB congenerswith dioxin-like activity, and the knownlevels of dibenzofuran contaminantsin the Aroclor mixtures, an estimatedTCDD toxic equivalent (TEQ) dose cal-culated for rhesus monkeys consuming80 µg Aroclor 1254 kg−1 BW day−1 couldbe as high as 2400 pg TCDD TEQ kg−1

BW day−1. For comparison purposes,rhesus monkeys exposed to doses of

750 pg TCDD kg−1 BW day−1 or greaterwere found to have reduced fertility,increased incidence of absorptions/resorptions and overall lower repro-ductive success (Bowman et al., 1989).In addition, rhesus monkeys chronicallyexposed to TCDD at dosages as low as150 pg kg−1 BW day−1 have also beenfound to have an increased frequencyand severity of endometriosis, a diseasethought to be associated with immuno-suppression. Subsequent immunologicaltesting of these same monkeys 3 yearsafter cessation of their TCDD exposurerevealed only a decreased mixed lym-phocyte response. Offspring of thesesame animals did, however, exhibit anincreased antibody response to T-cell-dependent tetanus toxoid immunization(Hong et al., 1989).

• Once released into the environment,commercial PCBs will be altered in termsof their congener pattern due to variousphysical, chemical and biological trans-formation processes. This environmental‘weathering’ of commercial PCBs resultsin the contamination of biota, humanfoodstuffs, etc. with congener patternsthat are not representative of any com-mercial PCB (Schwartz et al., 1987;Draper et al., 1991).

Significant differences exist in thenumber of PCB congeners detected insurveys of Canadian foodstuffs andbreast milk when compared with thecongener content of Aroclor 1254 (Fig.6.4). Whereas the congeners indicated inFig. 6.4 accounted for 83–93% of the PCBcongeners present in the food samples,they account for only 49% of the conge-ners in Aroclor 1254.

• Reductive dechlorination of commercialPCBs by anaerobic sediment bacteriachanges the congener proportions rela-tive to the congener composition ofcommercial mixtures, while concur-rently altering aspects of their toxico-logical effects (Mousa et al., 1998). Forexample, exposure of pregnant rats toa reconstituted PCB mixture based onhuman breast milk was more effectiveat altering behaviour and endocrine-

20-Feb-03 6




related functions in the offspring than anequivalent dose of Aroclor 1254 (Hanyet al., 1999).

• Occupational exposure to commercialPCBs results in the bioaccumulation of aPCB congener profile different from thatfound in the general population, and thecongeners present due to the industrialexposure constitute a higher percentageof the total PCB body burden (Kannanet al., 1994).

• The percentage contribution that anyPCB congener would make to the totalPCB body burden depends on the majorsource of PCB exposure. For example,Great Lakes fish eaters have elevatedcord blood levels of those PCB congenersfound in fish when compared with thecord blood congener content of thegeneral population (Stewart et al., 1999).Certain PCB congeners, particularlythose that bioaccumulate in fish and thatare not readily excreted by humansconsuming such fish, have been shownto contribute a greater percentage to thetotal PCB content found in humanplasma samples as a consequence of theamount of contaminated fish consumed(Asplund et al., 1994).

A number of epidemiological studies,other than the Japanese and Taiwanese rice oilpoisonings, are also available for PCB riskassessment consideration. In the Lake Michi-gan and North Carolina studies initiated inthe late 1970s, subtle developmental andneurobehavioural deficits were observed ininfants born to women with breast milk PCBconcentrations of 1.25–1.7 µg g−1 lipid. A con-servative estimate of the human PCB intakerequired to achieve these breast milk levelswould be 2 µg day−1, compared with an aver-age consumption of approximately 0.34 µgday−1 for that era (Tilson et al., 1990). However,high-end consumers from the 1992 UK totaldiet study were estimated to be ingesting upto 1.9 µg PCB day−1, which could result in PCBtissue burdens and breast milk levels near thepotential effect level for subtle behaviouraland developmental deficits. In the DutchPCB/dioxin study, pre-natal PCB exposurewas negatively associated with overallcognitive abilities of 42-month-old children(Patandin et al., 1999). At maternal plasmaPCB levels of ≥ 3.0 µg PCBs l−1, or approxi-mately 0.89 µg g−1 lipid, children scored lowerin a series of tests designed to assess intel-lectual functioning when compared withchildren with pre-natal exposure of < 1.5 µg


20-Feb-03 6

Fig. 6.4. PCB congener distribution.



PCB l−1 plasma or 0.45 µg g−1 lipid, althoughall of the scores were within the normalrange for the Dutch population. Approxi-mately 16% of the total study population of415 mother–infant pairs had maternal PCBlevels ≥ 3.0 µg PCB l−1, which, by the previousestimates, would be achieved followingchronic ingestion of 1.5 µg PCB day−1. Whilethe exact significance of these observationsis unknown, available evidence does suggestthat, for certain subpopulations, PCB expo-sure via the mother’s diet can be sufficientto induce subtle developmental alterations intheir pre-natally exposed infants. In almostall epidemiological studies to date involvingdietary sources of PCBs, maternal body bur-dens (i.e. pre-natal exposure of the developingfetus) and not lactational exposures have beenassociated with the observed effects.

Legislation/regulatory issues

PCBs, based on their resistance to degrada-tion and metabolism, have been shown tobioaccumulate readily in all ecosystemtrophic levels, leading to human exposuresprimarily from food consumption. In addi-tion, due to their vapour pressures andpartitioning coefficients, PCBs are subject tolong-range atmospheric transport, resultingin global redistribution from areas of past orcurrent use to colder climates (cold condensa-tion). While the control measures developedby a variety of nations to prevent furtherenvironmental releases have been partiallysuccessful, further emphasis should beplaced on reducing the PCB dietary intake ofpotentially susceptible populations, particu-larly high-end consumers of fatty foods andwomen of reproductive age. This can beaccomplished most effectively by identifica-tion and remediation of open hazardouswaste sites and closer scrutiny of renderingpractices used in the production of animalfeeds, as illustrated by the 1999 BelgianPCB/dioxin incident. In the latter episode,40–50 kg of PCB-contaminated mineral oil,originating from a waste recycling centre,was inadvertently mixed with renderingfat delivered to ten feed plants. The result

was that approximately 500 t of animal feedwas produced, containing elevated concen-trations of PCBs and dioxins, which was usedby over 1800 farms (Van Larebeke et al., 2001).Following disclosure, the initial public healthresponse was to remove from the market allpoultry and associated products as well as allmeat with a fat content greater than 25%. Asthe original source of the PCBs was thoughtto be discarded transformers, proper inven-tory control could have prevented further useof their contents.

International harmonization of PCB riskassessment activities, including the develop-ment of food tolerances through the CodexAlimentarius Commission, would assist theoverall legislative process.

National perspectives

While there is currently a lack of internation-ally recognized food standards for PCB con-tamination in foods, the lack of such stan-dards does not preclude individual countriesfrom developing their own national riskassessment guidelines in an attempt to pro-vide adequate human health protection forits citizens. A variety of food commodity-specific PCB guidelines have been developedin Canada under the authority of the CanadaFood and Drug Regulations, which state that‘. . . no person shall sell an article of food thathas in or upon it any poisonous or harmfulsubstance’ (Part 1, Section 4(a)). The USA hasalso developed tolerances for PCBs in foodsunder provisions of their Federal Food, Drugand Cosmetic Act, which deals with the inter-state commerce of foods and to what extentthey can be contaminated/adulterated with-out any adverse effect upon human health(Sections 402(a)(1) and 402 (1)(2)). Thesetolerances also take into consideration theextent to which contamination/adulterationcannot be avoided even when goodmanufacturing practices are employed.

US Food and Drug Administration

When the FDA initially started to formu-late its regulatory response regarding PCB

20-Feb-03 6




contamination of foodstuffs, it was knownthat there was widespread contamination offreshwater fish and there had been reportedincidences of contaminated cattle feed result-ing in PCB residues being detected in dairyproducts. The FDA’s response involvedthe development of a total dietary tolerableintake value, and guidelines or action levelsfor specific food commodities (Table 6.1).Food commodities found to exceed the Table6.1 values could theoretically be excludedfrom the retail market.

In 1968, the Yusho incident occurredwhen rice oil became accidentally contami-nated with Kanechlor 400. By the end of 1982,1788 Yusho patients were identified as exhib-iting such symptoms of poisoning as abnor-mal skin pigmentation, dermatological effectsand neurological complaints (Masuda, 1985).Initial analysis of the contaminated riceoil indicated PCB contamination at levelsof 2000–3000 ppm. It was determined subse-quently that an average estimated cumulativedose of 2000 mg of PCBs was required beforedisease symptoms were observed. Therefore,this dose could be regarded as a LOAEL. Atenfold safety factor was used to estimate aNOAEL of 200 mg; this was deemed to bedivisible by 1000, the number of days that thevictims were exposed to the contaminated riceoil, to determine a crudely estimated averagedaily intake of PCBs (200 µg). Consequently,an adult should not ingest more than 200 µgof PCBs day−1 or approximately 3 µg kg−1

BW day−1 for the average 65 kg adult (i.e.200 µg 65 kg−1 ≈3 µg kg−1 BW day−1). It was

also realized that infants/young childrencould represent a more sensitive subpopulat-ion; therefore, in a similar manner, the lowestminimal cumulative PCB dose associatedwith toxic effects in infants was determined tobe 500 mg. The estimated tolerable daily expo-sure value was calculated not to exceed 1 µgof PCBs kg−1 BW day−1 (Cordle and Kolbye,1979). These values were supported by reportsby the Michigan Department of Public Healthin 1983, which indicated that consumers ofLake Michigan fish could be ingesting up to4 µg PCBs kg−1 BW day−1 with no apparentadverse health effects noted (Boyer et al.,1991). Additional experimental results avail-able at the time with the commercial PCBthought to most closely resemble the chro-matographic pattern of PCB congeners foundin food residues, i.e. Aroclor 1254, did notsuggest a cancer risk at doses up to 100 ppm inthe diet (~7 mg kg−1 BW day−1).

Health Canada

An analysis of breast milk samples fromacross Canada during the early 1970s indi-cated that PCBs were detectable in almost allof the samples. Partially in response to thehuman health concerns associated with thesefindings, a toxicological evaluation for PCBswas conducted. Examination of the experi-mental results with Aroclors 1242, 1254 and1260 using rodents and dogs indicated adietary NOEL (no observed effect level)of 10 ppm following chronic exposure which,on a body weight basis, was approximately


20-Feb-03 6

Tolerance or maximum residue limit(mg kg−1)

Type of food US FDA Health Canada

Milk (fat basis)Manufactured dairy products (fat basis)Poultry (fat basis)EggsMeat, beef (fat basis)Fish and shellfish (edible portion)Infant and junior foods

1.51.53.00.3—2.00.2

0.20.20.50.10.22.0—

aAdapted from D’Itri and Kamrin (1983).

Table 6.1. Canadian and US food tolerances for PCBs.a



0.5 and 0.25 mg kg−1 BW day−1, respectively.Application of a 100-fold safety factorresulted in a tolerable exposure range of2.5–5.0 µg kg−1 BW day−1, which was similarto the values derived by the FDA as a conse-quence of their safety evaluation using theYusho data.

Subsequent re-analysis of the contami-nated Yusho rice oil revealed the presenceof other related halogenated aromatic con-taminants such as PCQs and PCDFs atconcentrations of approximately 866 and5 ppm, respectively. This ratio of PCQs/PCDFs (866/5) was deemed to be a some-what unique toxic mixture since it wasapproximately 100-fold greater than theratio of PCQs/PCDFs found in the originalKanechlor 400. This ratio also suggested thatdibenzofurans were one of the principalaetiological agents responsible for Yusho(Masuda and Yoshimura, 1984). Conse-quently, Health Canada decided not to baseits PCB hazard characterization solely onhuman data. At the time, additional labora-tory experiments were being conducted withnon-human primates – rhesus monkeys, aspecies which appeared to be appreciablymore sensitive to the toxic effects of a vari-ety of halogenated aromatics. In these studies,the rhesus monkeys were fed diets containing2.5 or 5.0 ppm Aroclor 1248, which, on a bodyweight basis, were calculated to be approxi-mately 100 to 200 µg kg−1 BW day−1. The inges-tion of Aroclor 1248 resulted in a varietyof reproduction-related effects, i.e. reducedfecundability, increased spontaneous abor-tion rates and reduced birth weights (Allenet al., 1979). Based on these data, the initialrecommendation that PCB-contaminatedfoodstuffs should not result in the ingestionof more than 5 µg kg−1 BW day−1 wasrevised to a temporary tolerable exposurelevel of 1 µg kg−1 BW day−1 (i.e. the 2.5 ppmdietary level in the monkey study – deemed tobe the LOAEL – plus the 100-fold ‘safety’ fac-tor) (Grant, 1983). Following the compilationof detailed Canada-wide market basketsurveys for PCB contamination, a variety ofmaximum residue limits was established forCanadian food commodities, taking intoconsideration the above tolerable exposurelevel (Table 6.1).

Conclusions

PCBs are persistent pollutants which werenot readily degraded during their industrialapplications. They are not readily degradedonce they have contaminated the environ-ment nor are they readily metabolized inbiological systems. Due to their persistence,PCBs have contaminated all components ofthe global ecosystem and are readily foundin areas of the globe where they were neverused. However, the level of PCBs in the eco-system has decreased dramatically in the lasttwo decades due to legislation which hasbanned their manufacture and mandatedtheir safe disposal.

PCBs have been found to result in amyriad of toxicological effects, in a varietyof in vivo and in vitro systems, but theirpotential health implications for humans areless clear due to various factors that havecompromised the interpretation of epidemio-logical studies. While the individual epi-demiological study findings, using cohortsexposed to background levels of PCBs, are notalways persuasive regarding the effect ofPCBs upon human health, when the data areviewed as a whole, there is a suggestion thatsome subpopulations may be experiencingsubtle health effects from the ingestion ofPCBs. Therefore, further research into theeffective and efficient disposal of equipmentcontaining PCBs and the effective clean-up ofdump sites appears warranted if the potentialhealth threat from chronic exposure to PCBs isto be minimized further.

References

Ahlborg, U.G., Hanberg, A. and Kenne, K.(1992) Risk Assessment of Polychlorinated Biphen-yls (PCBs). Institute of Environmental Medi-cine, Karolinska Institutet, Stockholm,Sweden.

Ahlborg, U.G., Becking, G.C., Birnbaum, L.S.,Brouwer, A., Derks, H.J.G.M., Feeley, M.,Golor, G., Hanberg, A., Larsen, J.C., Liem,A.K.D., Safe, S.H., Schlatter, C., Wærn, F.,Younes, M. and Yrjänheikki, E. (1994)Toxic equivalency factors for dioxin-likePCBs. Report on a WHO-ECEH and IPCS

20-Feb-03 6




consultation, December 1993. Chemosphere 28,1049–1067.

Allen, J.R., Barsotti, D.A., Lambrecht, L.K. and vanMiller, J.P. (1979) Reproductive effects ofhalogenated aromatic hydrocarbons on non-human primates. Annals of the New York Acad-emy of Sciences 320, 419–425.

Arnold, D.L., Bryce, F., Karpinski, K., Mes, J., Fernie,S., Tryphonas, H., Truelove, J., McGuire, P.F.,Burns, D., Tanner, J.R., Stapley, R., Zawidzka,Z.Z. and Basford, D. (1993) Toxicological con-sequences of Aroclor 1254 ingestion by femalerhesus (Macaca mulatta) monkeys. Part 1B.Prebreeding phase: clinical and analytical lab-oratory findings. Food and Chemical Toxicology31, 811–824.

Arnold, D.L., Bryce, F., McGuire, P.F., Stapley, R.,Tanner, J.R., Wrenshall, E., Mes, J., Fernie, S.,Tryphonas, H., Hayward, S. and Malcolm, S.(1995) Toxicological consequences of Aroclor1254 ingestion by female rhesus (Macacamulatta) monkeys. Part 2. Reproduction andinfant findings. Food and Chemical Toxicology 33,457–474.

Asplund, L., Svensson, B.G., Nilsson, A., Eriksson,U., Jansson, B., Jensen, S., Wideqvist, U. andSkerfving, S. (1994) Polychlorinated biphenyls,1,1,1-trichloro–2,2-bis(p-chlorophenyl) ethane(p,p´-DDT) and 1,1-dichloro–2,2-bis(p-chlorophenyl)-ethylene (p,p´-DDE) in humanplasma related to fish consumption. Archives ofEnvironmental Health 49, 477–486.

ATSDR, Agency for Toxic Substances and DiseaseRegistry (2000) Toxicological Profile for Poly-chlorinated Biphenyls (Update). US Departmentof Health and Human Services, Public HealthService, Washington, DC.

Ballschmiter, K. and Zell, M. (1980) Analysis ofpolychlorinated biphenyls (PCB) by glasscapillary gas chromatography: composition oftechnical Aroclor- and Clophen-PCB mixtures.Fresenius Zeitschrift für Analytische Chemie 302,20–31.

Boersma, E.R., Lanting, C.I., Patandin, S., Weisglas-Kuperus, N., Touwen, B.C.L. and Sauer, P.J.(2000) Effect of perinatal exposure to back-ground levels of PCBs and dioxins on thechild’s PCB body burden and on neurologiccognitive development during the first 42months of life. In: Aggett, P.J. and Kuiper, H.A.(eds) Risk Assessment in the Food Chain ofChildren. Lippincott Williams and Wilkins,Philadelphia, Pennsylvania, pp. 47–60.

Bowman, R.E., Schantz, S.L., Weerasinghe, N.C.A.,Gross, M.L. and Barsotti, D.A. (1989) Chronicdietary intake of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) at 5 or 25 parts per trillion in

the monkey: TCDD kinetics and dose–effectestimate of reproductive toxicity. Chemosphere18, 243–252.

Boyer, I.J., Kokoski, C.J. and Bolger, P.M. (1991) Roleof FDA in establishing tolerable levels fordioxin and PCBs in aquatic organisms. Journalof Toxicology and Environmental Health 33,93–101.

Calabrese, E.J. (1985) Uncertainty factors andinterindividual variation. Regulatory Toxicol-ogy and Pharmacology 5, 190–196.

CEPA, Canadian Environmental Protection Act(2000) See website under EC Pollution andToxics PCB page: www.ec.gc.ca/pcb/eng/index.html

Chen, Y.-J. and Hsu, C.-C. (1994) Effects of prena-tal exposure to PCBs on the neurologicalfunction of children: a neuropsychologicaland neurophysiological study. DevelopmentalMedicine and Child Neurology 36, 312–320.

Clemons, J.H., Dixon, D.G. and Bols, N.C. (1997)Derivations of 2,3,7,8-TCDD toxic equivalentfactors (TEFs) for selected dioxins, furans andPCBs with rainbow trout and rat liver cells andthe influence of exposure time. Chemosphere 34,1105–1119.

Cordle, F. and Kolbye, A.C. (1979) Food safety andpublic health. Interaction of science and law inthe federal regulatory process. Cancer 43,2143–2150.

Danse, I.R., Jaeger, R.J., Kava, R., Kroger, M.,London, W.M., Lu, F.C., Maickel, R.P.,McKetta, J.J., Newell, G.W., Shindell, S., Stare,F.J. and Whelan, E.M. (1997) Review: positionpaper of the American Council on Science andHealth: public health concerns about environ-mental polychlorinated biphenyls (PCBs).Ecotoxicology and Environmental Safety 38,71–84.

De Voogt, P. and Brinkman, U.A.T. (1989) Produc-tion, properties and usage of polychlorinatedbiphenyls. In: Kimbrough, R.D. and Jensen,A.A. (eds) Halogenated Biphenyls, Terphenyls,Naphthalenes, Dibenzodioxins and Related Prod-ucts. Elsevier Science Publishers, Amsterdam,pp. 3–45.

Dewailly, E., Nantel, A., Bruneau, S., Laliberté, C.,Ferron, L. and Gingras, S. (1992) Breast milkcontamination by PCDDs, PCDFs and PCBs inArctic Québec: a preliminary assessment.Chemosphere 25, 1245–1249.

D’Itri, F.M. and Kamrin, M.A. (1983) PCBs: Humanand Environmental Health Hazards. ButterworthPublishers, Boston, Massachusetts.

Dougherty, C.P., Holtz, S.H., Reinert, J.C.,Panyacosit, L., Axelrad, D.A. and Woodruff,T.J. (2000) Dietary exposures to food


20-Feb-03 6



contaminants across the United States.Environmental Research A 84, 170–185.

Draper, W.M., Wijekoon, D. and Stephens, R.D.(1991) Specification and quantification ofAroclors in hazardous wastes based on PCBcongener data. Chemosphere 22, 147–165.

Duarte-Davidson, R. and Jones, K.C. (1994)Polychlorinated biphenyls (PCBs) in the UKpopulation: estimated intake, exposure andbody burden. Science of the Total Environment151, 131–152.

Eisler, R. and Belisle, A.A. (1996) Planar PCB Hazardsto Fish, Wildlife, and Invertebrates: a SynopticReview: Contaminant Hazard Reviews, Report 31.US Department of the Interior, Washington,DC.

EPA (2000) For US Environmental Protection Agencyreview document see EPA’s website at: irptc.unep.ch/pops/indxhtms/cspcb01–07.html

FAO/WHO (1995) Preamble to the Codex GeneralStandard for Contaminants and Toxins inFoods. ALINORM 95/12A, Appendix IV.Twenty-eighth session of CCFAC, Rome.

Feeley, M. and Brouwer, A. (2000) Health risks toinfants from exposure to PCBs, PCDDs andPCDFs. Food Additives and Contaminants 17,325–333.

Frame, G.M. (1999) Improve procedure for singleDB-XLB column GC-MIS-SIM quantitation ofPCB congener distributions and characterisa-tion of two different preparations sold as‘Aroclor 1254’. Journal of High ResolutionChromatography 22, 533–540.

Grant, D.L. (1983) Regulation of PCBs in Canada.In: D’Itri, F.M. and Kamrin, M.A. (eds)PCBs: Human and Environmental Hazards.Butterworth Publishing, Boston, Massachu-setts, pp. 383–392.

Guo, Y.L., Yu, M.-L., Hsu, C.-C. and Rogan, W.J.(1999) Chloracne, goiter, arthritis, and anemiaafter polychlorinated biphenyl poisoning:14-year follow-up of the Taiwan Yu Chengcohort. Environmental Health Perspectives 107,715–719.

Hany, J., Lilienthal, H., Sarasin, A., Roth-Harer, A.,Fastabend, A., Dunemann, L., Lichtensteiger,W. and Winneke, G. (1999) Developmentalexposure of rats to a reconstituted PCB mixtureor Aroclor 1254: effects on organ weights,aromatase activity, sex hormone levels, andsweet preference behaviour. Toxicology andApplied Pharmacology 158, 231–243.

Hong, R., Taylor, K. and Abonour, R (1989) Immuneabnormalities associated with chronic TCDDexposure in Rhesus. Chemosphere 18, 313–320.

Hu, K. and Bunce, N.J. (1999) Metabolism ofpolychlorinated dibenzo-p-dioxins and related

dioxin-like compounds. Journal of Toxicologyand Environmental Health, Part B 2, 183–210.

IARC, International Agency for Research on Cancer(1978) Monographs on the Evaluation of theCarcinogenic Risk of Chemicals to Humans,Polychlorinated Biphenyls and PolybrominatedBiphenyls, Vol. 18. IARC, Lyon.

IARC, International Agency for Research on Cancer(1979) Monographs on the Evaluation of the Carci-nogenic Risk of Chemicals to Humans, Suppl. 1,Chemicals and Industrial Processes Associatedwith Cancer in Humans, Vol. 1–20. IARC, Lyon.

Johnson, B.L., Hicks, H.E., Jones, D.E., Cibulas, W.,Wargo, A. and De Rosa, C.T. (1998) Publichealth implications of persistent toxic sub-stances in the Great Lakes and St. Lawrencebasin. Journal of Great Lakes Research 24,698–722.

Kannan, N. (2000) Non- and mono-ortho chlori-nated biphenyls. In: Hutzinger, O. (ed.) TheHandbook of Environmental Chemistry, Vol. 3,Anthropogenic Compounds, Part K. Springer-Verlag, Berlin, pp. 127–156.

Kannan, N., Schulz-Bull, D.E., Petrick, G., Duinker,J.C., Macht-Hausmann, T. and Wasserman, O.(1994) Toxic chlorobiphenyls in adipose tissueand whole blood of an occupationally/accidentally exposed man and the generalpopulation. Archives of Environmental Health49, 375–383.

Kimbrough, R.D. (1995) Polychlorinated biphenyls(PCBs) and human health: an update. CriticalReviews in Toxicology 25, 133–163.

Koga, N. and Yoshimura, H. (1996) Metabolism ofPCBs and related compounds, and their toxic-ity. In: Kuratsune, M., Yoshimura, M., Hori, Y.,Okumura, M. and Masuda, Y. (eds) Yusho: aHuman Disaster Caused by PCBs and RelatedCompounds. Kyushu University Press,Fukuoka, Japan, pp. 105–120.

Kuratsune, M. and Shapiro, R.E. (1984) PCB Poison-ing in Japan and Taiwan. Alan R. Liss, New York.

Kuratsune, M., Yoshimura, H., Hori, Y., Okumura,M. and Masuda, Y. (eds) (1996) Yusho: a HumanDisaster Caused by PCBs and Related Compounds.Kyushu University Press, Fukuoka, Japan.

Lehman, A.J. and Fitzhugh, O.G. (1954) 100-foldmargin of safety. Association of Food and DrugOfficials, US Quarterly Bulletin 18, 33–35.

Longnecker, M.P., Rogan, W.J. and Lucier, G. (1997)The human health effects of DDT (dichloro-diphenyl-trichloromethane) and PCBs (poly-chlorinated biphenyls) and an overview oforganochlorines in public health. AnnualReview of Public Health 18, 211–244.

Macdonald, R.W., Barrie, L.A., Bidleman, T.F.,Diamond, M.L., Gregor, D.J., Semkin, R.G.,

11-Mar-03 6




Strachan, W.M.J., Li, Y.F., Wania, F., Alaee, M.,Alexeeva, L.B., Backus, S.M., Bailey, R.,Bewers, J.M., Gobeil, C., Halsall, C.J., Harner,T., Hoff, J.T., Jantunen, L.M.M., Lockhart,W.L., Mackay, D., Muir, D.C.G., Pudykiewicz,J., Reimer, K.J., Smith, J.N., Stern, G.A.,Schroeder, W.H., Wagemann, R. and Yunker,M.B. (2000) Contaminants in the CanadianArctic: 5 years of progress in understandingsources, occurrence and pathways. Science ofthe Total Environment 254, 93–234.

MAFF, Ministry of Agriculture Fisheries and Food(1997) Dioxins and Polychlorinated Biphenylsin Food and Human Milk. Food SurveillanceInformation Sheet No. 105. Library andInformation Services, London.

Masuda, Y. (1985) Health status of Japanese and Tai-wanese after exposure to contaminated rice oil.Environmental Health Perspectives 60, 321–325.

Masuda, Y. (1996) Casual agents in Yusho. In:Kuratsune, M., Yoshimura, H., Hori, Y.,Okumura, M. and Masuda, Y. (eds) Yusho:a Human Disaster Caused by PCBs and RelatedCompounds. Kyushu University Press,Fukuoka, Japan, pp. 49–80.

Masuda, Y. and Yoshimura, H. (1984) Poly-chlorinated biphenyls and dibenzofurans inpatients with Yusho and their toxicologicalsignificance. A review. American Journal ofIndustrial Medicine 5, 31–44.

Mayes, B.A., McConnell, E.E., Neal, B.H., Brunner,M.J., Hamilton, S.B., Sullivan, T.M., Peters,A.C., Ryan, M.J., Toft, J.D., Singer, A.W.,Brown, J.F. Jr, Menton, R.G. and Moore, J.A.(1998) Comparative carcinogenicity inSprague–Dawley rats of the polychlorinatedbiphenyl mixtures Aroclors 1016, 1242, 1254,and 1260. Toxicological Sciences 41, 62–76.

McConnell, E.E. (1989) Acute and chronic toxicityand carcinogenesis in animals. In: Kimbrough,R.D. and Jensen, A.A. (eds) Halogenated Biphen-yls, Terphenyls, Naphthalene, Dibenzodioxins andRelated Products. Elsevier Science Publishers,Amsterdam, pp. 161–193.

McFarland, V.A. and Clarke, J.U. (1989) Environ-mental occurrence, abundance, and potentialtoxicity of polychlorinated biphenyl conge-ners: considerations for a congener-specificanalysis. Environmental Health Perspective 81,225–239.

Moore, R.W. and Peterson, R.E. (1996) Reproductiveand developmental toxicity of polychlorinatedbiphenyls: to what extent are the effectsof aryl hydrocarbon receptor-independent?Comments on Toxicology 5, 347–365.

Mousa, M.A., Ganey, P.E., Quensen, J.F. III,Madhukar, B.V., Chou, K., Giesy, J.P., Fischer,

L.J. and Boyd, S.A. (1998) Altered biologicactivities of commercial polychlorinatedbiphenyl mixtures after microbial reductivedechlorination. Enviromental Health Perspec-tives 106, 1409–1418.

National Research Council (1994) Science and Judge-ment in Risk Assessment. National AcademyPress, Washington, DC.

Newsome, W.H., Davies, D. and Doucet, J. (1995)PCB and organochlorine pesticides inCanadian human milk – 1992. Chemosphere30, 2143–2153.

Newsome, W.H., Davies, D. and Sun, W.F. (1998)Residues of polychlorinated biphenyls (PCB)in fatty foods of the Canadian diet. FoodAdditives and Contaminants 15, 19–29.

Nicholson, W.J. and Landrigan, P.J. (1994) Humanhealth effects of polychlorinated biphenyls. In:Schecter, A. (ed.) Dioxins and Health. PlenumPress, New York, pp. 487–524.

Norén, K. and Meironyté, D. (2000) Certain organo-chlorine and organobromine contaminants inSwedish human milk in perspective of past20–30 years. Chemosphere 40, 1111–1123.

Patandin, S., Dagnelie, P.C., Mulder, P.G.H., deCoul, E.O., van der Veen, J.E., Weisglas-Kuperus, N. and Sauer, P.J.J. (1999) Dietaryexposure to polychlorinated biphenyls anddioxins from infancy until adulthood: acomparison between breast-feeding, toddler,and long-term exposure. Environmental HealthPerspectives 107, 45–51.

Rees, N. and Watson, D.W. (2000) InternationalStandards for Food Safety. Aspen Publications,Gaithersburg, Maryland.

Safe, S. (1992) Toxicology, structure–functionrelationship, and human and environmentalhealth impacts of polychlorinated biphenyls:progress and problems. Environmental HealthPerspectives 100, 259–268.

Safe, S.H. (1994) Polychlorinated biphenyls (PCBs):environmental impact, biochemical and toxicresponses, and implications for risk assess-ment. Critical Reviews in Toxicology 24, 87–149.

Safe, S., Bandiera, S., Sawyer, T., Robertson, L.,Safe, L., Parkinson, A., Thomas, P.E., Ryan,D.E., Reik, L.M., Levin, W., Denomme, M.A.and Fujita, T. (1985) PCBs: structure–functionrelationships and mechanism of action.Environmental Health Perspectives 60, 47–56.

Scheider, W.A., Cox, C., Hayton, A., Hitchin, G. andVaillancourt, A. (1998) Current status andtemporal trends in concentrations of persistenttoxic substances in sport fish and juvenileforage fish in the Canadian waters of theGreat Lakes. Environmental Monitoring andAssessment 53, 57–76.


11-Mar-03 6



Schwartz, T.R., Stalling, D.L. and Rice, C.L. (1987)Are PCB residues adequately described byAroclor mixture equivalents? Isomer-specificprincipal component analysis of such residuesin fish and turtles. Environmental Science andTechnology 21, 72–76.

Seegal, R.F. (1996) Epidemiological and laboratoryevidence of PCB-induced neurotoxicity. Criti-cal Reviews in Toxicology 26, 709–737.

Sipes, I.G. and Schnellmann, R.G. (1987) Biotrans-formation of PCBs: metabolic pathways andmechanisms. In: Safe, S. and Hutzinger, O.(eds) Polychlorinated Biphenyls (PCBs):Mammalian and Environmental Toxicologies.Springer-Verlag, London, pp. 98–110.

Stewart, P., Darvill, T., Lonky, E., Reihman, J.,Pagano, J. and Bush, B. (1999) Assessmentof prenatal exposure to PCBs from maternalconsumption of Great Lakes fish: an analysis ofPCB pattern and concentration. EnvironmentalResearch, Section A, 80, S87–96.

Stockholm Convention on Persistent OrganicPollutants (2001) See the UNEP website at:www.chem.unep.ch/pops

Tilson, H.A., Jacobson, J.L. and Rogan, W.J.(1990) Polychlorinated biphenyls and thedeveloping nervous system: cross-speciescomparisons. Neurotoxicology and Teratology 12,239–248.

Van den Berg, M., Birnbaum, L., Bosveld, A.T.C.,Brunström, B., Cook, P., Feeley, M., Giesy, J.P.,Hanberg, A., Hasegawa, R., Kennedy, S.W.,Kubiak, T., Larsen, J.C., van Leeuwen, F.X.R.,Liem, A.K.D., Nolt, C., Peterson, R.E.,Poellinger, L., Safe, S., Schrenk, D., Tillitt, D.,Tysklind, M., Younes, M., Wærn, F. andZacharewski, T. (1998) Toxic equivalencyfactors (TEFs) for PCBs, PCDDs, PCDFs forhumans and wildlife. Environmental HealthPerspectives 106, 775–792.

Van den Berg, M., Peterson, R.E. and Schrenk, D.(2000) Human risk assessment and TEFs. FoodAdditives and Contaminants 17, 347–358.

Van Larebeke, N., Hens, L., Schepens, P., Covaci, A.,Baeyens, J., Everaert, K., Bernheim, J.L.,Vlietinck, R. and de Poorter, G. (2001) The Bel-gian PB and dioxin incident of January–June1999: exposure data and potential impact onhealth. Environmental Health Perspectives 109,265–273.

Veith, G.D. (1975) Baseline concentrations ofpolychlorinated biphenyls and DDT in Lake

Michigan fish. Pesticide Monitoring Journal 9,921–929.

Veith, G.D., Kuehl, D.W., Leonard, E.N., Welch, K.and Pratt, G. (1981) Fish, wildlife, and estuar-ies. Pesticide Monitoring Journal 15, 1–8.

Vermeire, T.G., Stevensen, H., Pieters, M.N.,Rennen, M., Slob, W. and Hakkert, B.C. (1998)Assessment Factors for Human Health RiskAssessment: a Discussion Paper. RIVM Reportno. 620110007. RIVM, Bilthoven.

Walker, C.R. (1976) Polychlorinated biphenylcompounds (PCB’s) and fishery resources.Fisheries 1, 19–22.

Waltner-Toews, D. and McEwen, S.A. (1994)Chemical residues in foods of animal origin:overview and risk assessment. PreventativeVeterinary Medicine 20, 161–178.

Wania, F. and Mackay, D. (1996) Tracking anddistribution of persistent organic pollutants.Environmental Science and Technology 30,390A–396A.

Weigert, P., Gilbert, J., Patey, A.L., Key, P.E., Wood,R. and Barylko, P.N. (1997) Analytical qualityassurance for the WHO GEMS/Food-EUROprogram – results of 1993/94 laboratory profi-ciency testing. Food Additives and Contaminants14, 399–410.

WHO (1990) Evaluations of certain food additivesand contaminants. Thirty-fifth report ofJECFA. WHO Technical Report Series 789.Geneva.

Yu, M.-L., Guo, Y.L, Hsu, C.-C. and Rogan, W.J.(1997) Increased mortality from chronic liverdisease and cirrhosis 13 years after the Taiwan‘Yucheng’ (‘oil disease’) incident. AmericanJournal of Industrial Medicine 31, 172–175.

Yu, M.-L., Hsin, J.-W., Hsu, C.-C., Chan, W.-C. andGu, Y.L. (1998) The immunologic evaluation ofthe Yu cheng children. Chemosphere 37, 9–12.

Yu, M.-L., Guo, Y.L., Hsu, C.-C. and Rogan, W.J.(2000) Menstruation and reproduction inwomen with polychlorinated biphenyl (PCB)poisoning: long-term follow-up interviewsof the women from the Taiwan Yuchengcohort. International Journal of Epidemiology 29,672–677.

Zitko, V., Hutzinger, O. and Choi, P.M.K. (1972)Contamination of the Bay of Fundy–Gulf ofMaine area with polychlorinated biphenyls,polychlorinated terphenyls, chlorinateddibenzodioxins, and dibenzofurans. Environ-mental Health Perspectives 1, 47–50.

11-Mar-03 6




7 Dioxins in Milk, Meat, Eggs and Fish

H. Fiedler*United Nations Environment Programme, 11–13, Chemin des Anémones,

CH-1219 Chatelaine, Geneva, Switzerland

Introduction

Contamination of food with chemicals playsan important role especially for persistentand bioaccumulating substances wheredietary intake is the major pathway ofexposure for humans. For the generalpopulation and some compounds, such aspolychlorinated dibenzo-p-dioxins andpolychlorinated dibenzofurans (PCDDs/PCDFs), ingestion of food accounts forapproximately 95% of the body burden. Toguarantee safe and high quality food forhuman consumption, international regula-tion such as the Codex Alimentarius of theWorld Health Organization (WHO) andthe Food and Agriculture Organization(FAO) has been established. This food code isfollowed in terms of harmonizing nationalfood regulation, food additives, hygiene andprocessing as well as facilitating internationaltrade (Codex Alimentarius, n.d.).

The occurrence of unintentional contami-nation with chemicals but also with bacteriaand viruses needs special attention and sur-veillance to protect humans from consumingunsafe food. Within the chemical contami-nants, a major concern is associated withdioxins and furans for several reasons: someof the PCDD/PCDF congeners are highlytoxic, they are persistent and bioaccumulate

in the food chain and thus can cause chroniceffects due to long-term low exposure and,finally, dioxins and furans have been associ-ated with accidents and severe foodcontaminations.

Nature of the Compounds

Dioxins (PCDDs) and furans (PCDFs) are twogroups of planar, tricyclic ethers which haveup to eight chlorine atoms attached at carbonatoms 1–4 and 6–9. In total, there are 75 possi-ble PCDD congeners and 135 possible PCDFcongeners, giving a total of 210 congeners(see Chapter 6). PCDDs and PCDFs are gen-erally very insoluble in water, are lipophilicand are persistent. Dioxins and furans havenever been produced intentionally but areunwanted by-products of many chemicalindustrial processes and of all combustionprocesses. The sources and activities that leadto the formation of PCDDs/PCDFs, and sub-sequently to the release of these contaminantsinto air and water, with products and resi-dues, have been subject to intensive research,and today the most important dioxin sourcesseem to be identified. In the past, thechemical industry, with its production oforganochlorine chemicals, was the major


20-Feb-03 7




source of PCDDs/PCDFs: chemicals withhigh concentrations of dioxins and furanswere pentachlorophenol (PCP), 2,4,5-trichloroacetic acid (2,4,5-T), polychlorinatedbiphenyls (PCBs; note that they containPCDFs only, not PCDDs) (Fiedler et al., 1990).In 1977, PCDDs/PCDFs were identified inthe emissions of a municipal waste incinera-tor in Amsterdam (Olie et al., 1977) and in1980 the trace chemistry of fire was estab-lished, which states that, in thermal processesand in the presence of organic carbon, oxygenand chlorine, dioxins and furans can beformed (Bumb et al., 1980). Today, in industri-alized countries, the major sources of dioxinand furan release are combustion processes.Among these sources are the incineration ofmunicipal and hospital waste, the productionof iron and steel and other non-ferrousmetals, e.g. copper, aluminium, lead andzinc (especially in recycling processes), andall types of uncontrolled burning, e.g. landfillfires, trash burning on soil, forest and bushfires (especially when chlorinated herbicideshave been applied).

Lastly, natural formation of PCDDs/PCDFs has been shown on different occasions.Peroxidases are capable of synthesizingPCDDs/PCDFs from precursors such aschlorophenols. The formation of especially.Cl7DD and Cl8DD during the composting pro-cess has been proven where it was found thatthe international toxic equivalent (I-TEQ)increases by about 1–2 parts per trillion (ppt)during the composting process. Recent stud-ies provide a strong indication that PCDDs/PCDFs may have been present in the environ-ment for considerably longer than the onset ofthe chlorine industry, and that they may beformed through non-anthropogenic activities.High concentrations of mainly PCDDs werefound in mined ball clay from the USA,kaolinitic clay from Germany, deep soilsamples from Great Britain, in dated marinesediment cores from Queensland/Australiaand in man-made lake sediment cores fromMississippi, USA. Typical for all samples isthe almost total absence of PCDFs and thenearly identical congener/isomer distribu-tion throughout all geographies.

Almost all possible 210 congeners arereleased from anthropogenic sources and,

due to chemical, physical and biologicalstability and long-range transport, areubiquitous and have been detected in allenvironmental compartments. Due to the per-sistence of the 2,3,7,8-substituted congenersand the lipophilicity of these compounds,PCDDs/PCDFs accumulate in fatty tissuesand in carbon-rich matrices such as soils andsediments.

National release inventories

With this mandate to facilitate a conventionon reduction and elimination of releases ofpersistent organic pollutants (POPs), UNEPChemicals will ‘. . . assist countries in theidentification of national sources of dioxin/furan releases by promoting access to theinformation on available sources of dioxins/furans . . .’. Table 7.1 summarizes initialfindings obtained from national inventoriesof releases of dioxins and furans, whichhave been compiled by the United NationsEnvironment Programme (UNEP) in 1999and have been updated since then. Theupdated UNEP report for a reference yeararound 1995 would estimate annual releasesto air of approximately 13,000 g I-TEQ year−1

from about 20 countries. This amount isbased on best estimates from most countriesand the lower bound emission for the rest ofthe countries. The upper estimate would bearound 30,000 g I-TEQ year−1 and would alsoinclude another 2400 g I-TEQ in preliminaryestimates from US sources, which have beenaddressed only recently (US-EPA, 2000a).The PCDD/PCDF releases into air per yearand country are shown in Table 7.1. It shouldalso be noted that, for example, Japanupdates its inventory on an annual basisand for its last reporting year estimatedmuch lower emissions, namely 2260–2440 gTEQ year−1 for 1999 coming down from6301–6370 g TEQ year−1 in 1997 (Environ-ment Agency Japan, 2000).

Most data are available for industrial-ized countries from Western Europe andNorth America. From Asia, there is only aninventory for Japan and an additional esti-mate of 22 g I-TEQ year−1 for emissions from

20-Feb-03 7

154 H. Fiedler



waste incinerators in the Republic of Korea.From the southern hemisphere, only Austra-lia and New Zealand have estimated annualemissions. From Africa, Central and SouthAmerica and the rest of Asia there are no dataat all. At present, the geographical coverageis not sufficient to estimate global emissionsof PCDDs/PCDFs. Further, the presentinventories do not cover all known sourcesof dioxins and furans. There are severalefforts underway to identify dioxin sourcesin parts of the world where, so far, there isno information available. Existing inventorieswill be updated, as it is obvious that countrieshave initiated measures to reduce emissionsof dioxins and furans.

Environmental concentrations,fate and transport

Many data are available for PCDD/PCDFconcentrations in soils, sediments and air.Biomonitors, such as vegetation or cows’milk, have been applied successfully to

identify or monitor ambient air concentra-tions in the neighbourhood of potentialpoint sources, although a linear correlationbetween PCDD/PCDF concentrations in veg-etation and air samples cannot be established.Due to public concern regarding dioxinsand furans, many studies have been aimed atidentifying potential ‘hotspots’ of contamina-tion. As a result, the overall presentation ofdata is often biased towards contaminatedsamples and higher concentrations, ratherthan baseline information.

When evaluating concentrations ofPCDDs/PCDFs in the environment, it shouldbe taken into account that some matrices aresensitive to short-term inputs, e.g. ambient airor short-lived vegetation, whereas othermatrices, such as sediments and soils, arerelatively insensitive to temporal variation.Further important factors for the interpre-tation of results are season (e.g. in winterPCDD/PCDF concentrations in air may behigher by a factor of ten on a toxic equivalent(TEQ) basis than in summer), length of thesampling or exposure (e.g. a few hours vs.weeks), location (e.g. urban vs. rural), the

Dioxins in Milk, Meat, Eggs and Fish 155

20-Feb-03 7

Country Emission (g TEQ year−1) Reference year Sourcea

AustriaAustraliaBelgiumCanadaCroatiaDenmarkFinlandFranceGermanyHungaryJapan

The NetherlandsNew ZealandNorwaySlovak RepublicSwedenSwitzerlandUKUSAGlobal flux

2,529.152,150–2,300

2,661.152,164.152,595.519–170

98.3–198.2,873–2,737

2,327.152,112–8,4366,301–6,370

(2,260–2,440)2,486.1514–51

2, 59.152,542.1522–88

2,181.152,569–1,099

2,501.1512,655–25,945

1994199819951999

~19971998/99~19971998199419951997

(1999)1991

~199719941993199519951995

1112

311114

15111116

aSource: 1 = Fiedler (1999); 2 = Environment Canada (2001); 3 = Hansen (2001); 4 = EnvironmentAgency Japan (2000); 5 = Buckland et al. (2000); 6 = US-EPA (2000a).

Table 7.1. National dioxin and furan inventories: PCDD/PCDF emissions to air (UNEP, 1999, updated).



sampling method (e.g. high volume samplingvs. particulate deposition), sampling depth(e.g. surface vs. core), etc.

Soils are natural sinks for persistent andlipophilic compounds such as PCDDs/PCDFs, which adsorb to the organic carbon ofthe soil and, once adsorbed, remain relativelyimmobile. Soil is a typical accumulatingmatrix with a long memory; in other words,dioxin inputs received in the past will remainand, due to the very long half-lives ofPCDDs/PCDFs in soils, there is hardly anyclearance. Soils can receive inputs of environ-mental pollutants via different pathways, ofwhich the most important are: atmosphericdeposition, application of sewage sludge orcomposts, spills, and erosion from nearbycontaminated areas. Sediments are the ulti-mate sink for PCDDs/PCDFs (and other per-sistent and lipophilic organic substances). Aswith soils, sediment samples are accumulat-ing matrices for lipophilic substances and canreceive inputs via different pathways: atmos-pheric deposition, industrial and domesticeffluents, stormwater, spills, etc. Today,PCDDs/PCDFs can be detected ubiquitouslyand have been measured in the Arctic, wherealmost no dioxin sources are present. Itbecame clear that the lipophilic pollutants,such as PCDDs/PCDFs, at the North and theSouth Poles originated from lower (warmer)latitudes. Emission of most PCDDs/PCDFsfrom combustion sources into the atmo-sphere occurs in the moderate climate zones;PCDDs/PCDFs then undergo long-rangetransport towards the North Pole, condens-ing in the cooler zones when the tempera-tures drop. This process of alternatingre-volatilization and condensing, also named

the ‘grasshopper effect’, can carry pollutantsthousands of kilometres in a few days. Thus,the air is an important transport medium forPCDDs/PCDFs. An indirect method of deter-mining ambient air concentrations is the useof biomonitors, such as vegetation. The outerwaxy surfaces of pine needles, kale or grassabsorb atmospheric lipophilic pollutants andserve as an excellent monitoring system forPCDDs/PCDFs (Buckley-Golder et al., 1999,Task 2).

In most countries, a broad range ofPCDD/PCDF concentrations has beendetected in all media. Table 7.2 presents therange of reported background concentrationsand maximum concentrations measured incontaminated locations from European coun-tries. As illustrated in Table 7.2, the lowestconcentrations for all matrices are below 1 ngI-TEQ kg−1 dry matter (DM) and the highestbackground values are around 100 ng I-TEQkg−1 DM. At contaminated locations, mea-sured concentrations in soils range from sev-eral hundred to around 100,000 ng I-TEQ kg−1

DM (Finland, sites contaminated with woodpreservatives; and The Netherlands, close toa scrap car and scrap wire incinerator) andin sediments up to 80,000 ng I-TEQ kg−1 DM(Finland, downstream from a wood preserva-tive-producing site). The extremely high con-centration of 14,800 fg I-TEQ−3 was measuredin 1992/93 at the Pontyfelin House site, in thePanteg area of Pontypool in South Wales,which is very close (~150 m) to an industrialwaste incinerator (Buckley-Golder et al., 1999,Task 2).

Fish and shellfish frequently havebeen used as biomonitors for the aquatic envi-ronment as they are highly bioaccumulative

20-Feb-03 7

156 H. Fiedler

Environmental matrixMeasured range

backgroundMaximum concentration at

contaminated sites Units

SoilSedimentAir (ambient)

(deposition)Sewage sludge

Spruce/pine needles(biomonitors)

< 1–100< 1–200< 1–100s< 1–100s< 1–200

(average 10–40)0.3–1.9

100s–100,000100s–80,000

14,800

1,200

50–100

ng I-TEQ kg−1 DMng I-TEQ kg−1 DMfg I-TEQ m−3

pg I-TEQ m−2 day−1

ng I-TEQ kg−1 DM

ng I-TEQ kg−1 DM

Table 7.2. Concentrations of PCDDs/PCDFs measured in EU Member States.



for PCDDs/PCDFs, and concentrations ofseveral hundred pg TEQ g−1 fat have beendetected. These concentrations are muchhigher than those found in terrestrial animals,such as cattle, pigs or chickens. Top-predators, such as sea eagles or guillemots,also showed high concentrations of PCDDs/PCDFs: as an example, 830–66,000 pg TEQ g−1

fat were found in Finnish white-tailed seaeagles (Buckley-Golder et al., 1999, Task 2).

Understanding of the environmentalfate of PCDDs/PCDFs is fundamental to eval-uating human exposure. Although the TEQapproach was developed and proven as ahelpful tool for risk assessment, input data formodels and exposure assessment have to becongener specific.

Knowledge of the numerical values ofcertain parameters characterizing the proper-ties of individual PCDDs/PCDFs is necessaryin order to predict the behaviour of the mix-tures found in the environment. The physicaland chemical properties, which are measuresof or control the behaviour of dioxins are:

• their low vapour pressure (rangingfrom 4.0 × 10−8 mmHg for 2,3,7,8-Cl4DFto 8.2 × 10−13 mmHg for Cl8DD);

• their extremely low solubility inwater (ranging from 419 ng l−1 for2,3,7,8-Cl4DF, 7.9 and 19.3 ng l−1 for2,3,7,8-Cl4DD to 0.074 ng l−1);

• their solubility in organic/fatty matrices(log Kow range from 5.6 for Cl4DF and6.1/7.1 for Cl4DD to 8.2 for Cl8DD);

• their preference for binding to organicmatter in soil and sediments (log Koc

values for 2,3,7,8-Cl4DD were between6.4 and 7.6).

The processes by which PCDDs/PCDFsmove through the environment are reason-ably well known. PCDDs/PCDFs are multi-media pollutants and, once released to theenvironment, become distributed betweenenvironmental compartments (Buckley-Golder et al., 1999, Task 3).

PCDDs/PCDDFs are semi-volatile com-pounds and, in the atmosphere, can existin both the gaseous phase and bound toparticles, depending upon the congenerand the environmental conditions. Especiallyduring the warmer (in the northern

hemisphere, summer) months, the lowerchlorinated PCDD/PCDF congeners tendto be found predominantly in the vapourphase. PCDD/PCDF in the vapour phase canundergo photochemical transformation, witha dechlorination process leading to more toxiccongeners if octa- and heptachlorinated con-geners degrade to tetra- and pentachlorinatedand finally to non-toxic compounds with onlythree or fewer chlorine atoms. PCDDs/PCDFsattached to particulate matter seem to beresistant to degradation.

In the terrestrial food chain (air → grass→ cattle → milk/meat → man), PCDDs/PCDFs can be deposited on plant surfaces viawet deposition, via dry deposition of chemi-cals bound to atmospheric particles or viadiffusive transport of gaseous chemicals inthe air to the plant surfaces. Each of theseprocesses is governed by a different set ofplant properties, environmental parametersand atmospheric concentrations. Investiga-tions with native grassland cultures showedthat dry gaseous deposition played the domi-nant role for the accumulation of the lowerchlorinated PCDDs/PCDFs, whereas dry par-ticle-bound deposition played an importantrole in the uptake of the PCDDs/PCDFs withsix and more chlorine atoms. There wasalso some evidence indicating an input ofthe higher chlorinated PCDD/PCDF fromwet deposition (Welsch-Pausch et al., 1995).Levels in, for example, grass reflect recentexposure to PCDDs/PCDFs, as vegetation isonly exposed for a relatively short time, withnew growth replacing old and crops beingharvested. For agricultural leaf crops, themain source of contamination is directdeposition from the atmosphere and soilsplash. Root uptake and translocation ofdioxin contamination into the crop has beenconfirmed for courgette and cucumber only.Grazing animals are exposed to dioxins byingesting contaminated pasture crops, andPCDDs/PCDFs are found to accumulateprimarily in the fatty tissues and milk.

For agricultural soils, an additionalsource of PCDD/PCDF can be the applicationof sewage sludge. Small amounts of PCDDs/PCDFs deposited on to soil can be returnedto the atmosphere by the resuspension of pre-viously deposited material or revolatilization


20-Feb-03 7



of the less chlorinated congeners. Becauseof their chemical characteristics and verylow solubility, PCDDs/PCDFs accumulate inmost soil types, with very little water leachingand negligible degradation of the 2,3,7,8-substituted PCDD/PCDF congeners.

PCDDs/PCDFs partition quickly toorganic matter and so accumulate in sedi-ments. They accumulate in aquatic fauna asa result of the ingestion of contaminatedorganic matter. The concentration of PCDDs/PCDFs in fish tissue is found to increase up thefood web (biomagnification) as a result of theprogressive ingestion of contaminated prey.

Carry-over rates: from environment to food

The transfer of dioxins from grass into cattlehas been studied, and carry-over rates havebeen determined. In general, carry-over ratesdecrease with increasing degree of chlorina-tion of the chemical, indicating that absorp-tion through the gut also decreases. Thisdecrease in absorption is attributed to thegreater hydrophobicity of the higher chlori-nated PCDDs/PCDFs, which inhibits theirtransport across aqueous films in the diges-tive tract of the cow.

In studies conducted at backgroundconcentrations, the highest transfer was deter-mined for two lower chlorinated dibenzo-p-dioxins and one dibenzofuran, namely 2,3,7,8-Cl4DD (2,3,7,8- tetrachlorodibenzo-p-dioxin),1,2,3,7,8-Cl5DD (1,2,3,7,8-pentachlorodibenzo-p-dioxin), and 2,3,4,7,8-Cl5DF (2,3,4,7,8-penta-chlorodibenzofuran). For these three conge-ners about 30–40% are transferred from feedto cow’s milk. About 20% are transferred forthe 2,3,7,8-substituted Cl6DD (hexachlorodi-benzo-p-dioxin) and Cl6DF (hexachlorodi-benzofuran) homologues. For the hepta- andoctachlorinated PCDDs and PCDFs, not morethan 4% of the ingested congeners find theirway into the milk. Although highly depend-ent on the characteristics of each congener, theoverall transfer on a TEQ basis is about 30%;in other words: about 30% of the most toxicPCDD/PCDF congeners which are ingestedby the cow are excreted via the milk(Welsch-Pausch and McLachlan, 1998).


The largest database on dioxin concentrationsin food exists for some European countries,and the major findings are discussed in thisfollowing section. From North America,especially from the USA, the database ondioxin concentrations in food is small com-pared with the European database (US-EPA,2000b). The Organochlorine Programme inNew Zealand found very low concentra-tions of PCDDs/PCDFs in the foodstuffs(NZ, 1998). Concentrations of PCDD/PCDFranged from 0.072 to 0.57 pg I-TEQ g−1 fatfor meats and meat products; 0.056–0.26 pgI-TEQ g−1 fat for dairy products, 0.41–1.82 pgI-TEQ g−1 fat for fish, and 0.12 and 0.29 pgI-TEQ g−1 fat for eggs and poultry, respec-tively. Cereal products and bread werebetween 0.19 and 0.66 pg I-TEQ g−1 fat (allnumbers include half of the detection limitfor non-quantifiable congeners when calcu-lating the TEQ)

In 2000, a database with informationon concentrations of PCDDs, PCDFs and/ordioxin-like PCBs (polychlorinated biphenyls)in food products and human milk was estab-lished and evaluated. The samples originatedfrom rural and industrial sites in ten EU Mem-ber States and were collected between 1982and 1999. Due to the high demands on dioxinand furan analyses, broad field surveys basedon a large number of samples are rare. Never-theless, the current database can be con-sidered relatively complete for PCDDs andPCDFs, but rather incomplete for dioxin-likePCBs.

With respect to dioxin contamination,highest relevance is for foods of animalorigin, where in principle only 2,3,7,8-substituted congeners are found. These arethe most toxic and most persistent. Foods ofplant origin normally have lower concentra-tions of dioxins and furans but, for example,grass plays an important role as feedstufffor cattle, sheep, etc., and the contaminationin the grass translocates into the animal andits products, e.g. meat, milk. Humans andbreast-fed infants are the last steps in thefood chain and thus have the highestconcentrations.

20-Feb-03 7

158 H. Fiedler



A survey of European food data can besummarized as follows:

• The national average concentration ofPCDDs/PCDFs in eggs, fats, oils, meat(and its products) and milk (and itsproducts) is generally less than 1 pgI-TEQ g−1 fat, with an upper limit of2–3 pg I-TEQ g−1 fat.

• PCDDs/PCDFs in fish ranged from0.25 pg I-TEQ g−1 fresh weight (FW) upto 10–20 pg I-TEQ g−1 FW.

• Concentrations in fruits, vegetables andcereals were generally close to the limitsof quantification.

• Concentrations in meat and meat pro-ducts and fish and fish products seem tovary with the organ analysed, e.g. higherconcentrations on a fat basis in liverthan in adipose tissue. Further, thereis a difference between animal species,e.g. lower concentrations on a fat basis inpork than in beef, poultry or mutton.

• Decreasing trends in the concentration ofPCDDs and PCDFs in foods, especiallyin consumer milk and some types ofmeat, have been determined in a fewcountries. However, the available infor-mation is insufficient and too incompleteto draw a general conclusion on tempo-ral trends for other types of foods.

• Although the data on concentrationsof dioxin-like PCBs in foods are scarce,the available information indicates thatthese PCB congeners may add one totwo times of the PCDD/PCDF TEQ. Inparticular, PCB congeners 126 and 118may contribute much more strongly tothe total TEQ content of foods than dothe PCDDs and PCDFs.

• The largest database exists for PCDDsand PCDFs in human milk, and for somecountries strong downward trends havebeen observed. Since 1995, the nationalaverage concentrations have rangedbetween 8 and 16 pg I-TEQ g−1 fat.Although the database is incomplete,results from the years 1990–1994 indicatethat, on a TEQ basis, PCBs can accountfor the same to up to three times theconcentration of the PCDDs and PCDFs(7–29 pg TEQ g−1 fat).

Milk and milk products

Analysis of dioxins in cow’s milk has beenperformed since 1986. As dairy products arethe main contributor to the human dioxinburden and cow’s milk also serves as abiomonitor, the database for milk samplesis large. When comparing dioxin concentra-tions in cow’s milk, seasonal variations of upto 25% can occur due to changes in animalfeeding stuffs. The differences between cer-tain regions can be even higher. In the late1980s, contamination of cow’s milk withdioxins by chlorine-bleached cardboard con-tainers was established. After elimination ofelemental chlorine in the bleaching process,the PCDD/PCDF levels in cow’s milk wereno longer influenced by cardboard contain-ers. At the end of 1997, increasing levelsof dioxins in cow’s milk were detected inBaden-Württemberg (Germany). Finally,contaminated citrus pulp, a component offeedingstuff imported from Brazil, wasfound to be the cause of elevated dioxinconcentrations (Malisch, 1998a,b). Thiscontamination was found in other federalLänder of Germany and later other Europeancountries as well.

The most recent surveys show nationalaverage concentrations in the range of0.3–2.1 pg I-TEQ g−1 for PCDDs/PCDFs and0.2–1.8 pg PCB TEQ g−1 fat for dioxin-likePCBs. To explain the concentrations ofPCDDs/PCDFs/PCBs in milk and milk prod-ucts, several factors have to be considered:obviously, deposition of dioxins and relatedcompounds emitted from either point ordiffuse sources on pasture as well as con-taminations present in animal feedstuffs areimportant routes of exposure for cattle. Dueto stringent enforcement of limit values, thenational average concentrations of dioxins indairy products have decreased over the lastdecade in many European countries.

Meat and meat products

PCDD/PCDF concentrations in foodstuffs ofanimal origin depend on the animal. Thus,distinctions have to be made between


20-Feb-03 7



different types of meat. The lowest levelswere found in pork. As sausages mainly con-tain pork, their dioxin concentrations are sim-ilar but tend to be slightly higher than thoseof pure pork due to the addition of beef andliver and occasionally through the smokingprocess.

In general, beef, veal, poultry and mixedmeat have quite similar concentrations ofPCDDs/PCDFs, in the range of 0.5–0.7 pgI-TEQ g−1 fat. The mean for pork is lower andaround 0.3 pg I-TEQ g−1 fat. Game meat andliver had significantly higher dioxin concen-trations than the other meat subgroups (SCF,2000).

Eggs

The most recent surveys on concentrations ofPCDDs/PCDFs in eggs gave mean concen-trations between 0.5 and 2.7 pg I-TEQ g−1 fatwith an overall mean around 1 pg I-TEQ g−1

fat (SCF, 2000). Older studies tend to givea similar picture, suggesting that concen-trations have not changed substantially.German studies have shown that the PCDD/PCDF levels depend on the type of thechicken’s housing (cage, ground or field).Higher concentrations were found for eggsfrom hens which can take up dioxins fromsoil. Lower levels were detected in eggs fromhens housed in elevated wire cages. Recentresults indicate that these dependencies aredecreasing. From the limited information onPCBs, it can be assumed that the contributionof the PCBs to a total TEQ is in the same rangeas that of PCDDs/PCDFs (SCF, 2000).

Fish and fish products

PCDD/PCDF concentrations in fish arehighly variable. It is problematic to generaterepresentative data on dioxin levels in fish, asa lot of fish species and fishing grounds exist.In this context, a recent representative studyincluding 184 samples of fish and fishproducts is of great importance. Samplingwas based on the real intake according tothe share of different species and fishing

grounds. Because of large differences in thefat content of fish, levels on a whole weightbasis were preferred.

Wild fish and farmed freshwater fish hadmean concentrations of around 10 pg I-TEQg−1 fat for PCDDs/PCDFs and 30 pg PCB-TEQg−1 fat for co-planar and mono-ortho PCBs.The threefold higher mean PCB concentrationreflects the fact that PCB levels are constantlyhigher than the combined PCDD/PCDFlevels. It should be noted that PCDD/PCDFconcentrations range over three orders ofmagnitude and PCB concentrations over twoorders of magnitude (SCF, 2000).

Fruit and vegetables

There are no recent data for dioxin concen-trations in foods of plant origin. Productsof vegetable origin, such as cereals with lessthan 2% fat, fruit and vegetables, had verysimilar contamination levels, with a meanconcentration of around 0.02–0.3 pg I-TEQg−1 on a whole food basis (SCF, 2000).

Human milk

A large database exists for dioxin concen-trations in human milk; the national averageconcentrations of PCDDs, PCDFs anddioxin-like PCBs, expressed in I-TEQ andPCB-TEQ, respectively, are presented inTable 7.3. For the period 1995–1999, the cur-rent database shows national average con-centrations between 8 and 16 pg I-TEQ g−1 fat.For the period before 1995, the national aver-ages ranged between 10 and 34 pg I-TEQ g−1

fat. Generally, the data demonstrate a down-ward trend for human milk concentrations ofPCDDs/PCDFs. Most complete time trendscan be established for Germany based onmore than 1732 individual samples collectedin various German ‘Bundesländer’ during1985–1998. The German database showsa 60% decline in the average as well as inthe highest PCDD/PCDF levels found inhuman milk between the late 1980s and1998. The database is too incomplete to draw

11-Mar-03 7

160 H. Fiedler



conclusions about the TEQ contribution ofdioxin-like PCBs.

Food consumption data

The consumption data from the participatingcountries are generally produced from stud-ies performed rather recently. The surveymethods differ, including consumptionrecord studies (2–28 days) as well as 24 hrecall, household budget and food frequencyquestionnaire studies. The study populationswere generally adults (from teenagers tothe elderly), but the UK and Germany havealso studied separate groups of consumers,including breast-fed infants, toddlers, school-children and adults. The food consumptiondata reveal variations between countries inconsumption of different food groups, a mir-ror of the country-specific food traditions andhabits.

Dietary intakes

Based on the data collected on PCDD,PCDDF and PCB concentrations in food,mean dietary intakes can be calculated bymultiplying the average concentrations byaverage consumption of major food groups.In EU Member States, and for the period after1995, the average dietary intakes of PCDDsand PCDFs ranged between 29 and 97 pg

I-TEQ day−1, which on a body weight (BW)basis corresponds to 0.4–1.5 pg I-TEQ kg−1

BW day−1. Surveys of chemical analyses offoods collected in the 1970s and 1980s gavemuch higher estimates, ranging from 127to 314 pg I-TEQ day−1, corresponding to1.7–5.2 pg I-TEQ kg−1 BW day−1. The 95percentile (or 97.5 percentile) intake, basedon data from The Netherlands and the UK,was two to three times the mean intake.

The intake of co-planar and mono-orthoPCBs would add another 48–110 pg PCB-TEQday−1 (= 0.8–1.8 pg PCB-TEQ kg−1 BW day−1).Whereas the contribution of PCBs to the totalTEQ equals the intake of PCDDs and PCDFsin countries such as Finland, The Netherlands,Sweden and the UK, studies in Norwayshowed that the contribution from dioxin-likePCBs is up to four times the TEQ contributionof the PCDDs and PCDFs. Thus, averagehuman daily intake of PCDDs, PCDFs anddioxin-like PCBs in European countries hasbeen estimated to be 1.2–3.0 pg WHO-TEQkg−1 BW day−1. More than 90% of the humanexposure derives from food. Foodstuffs ofanimal origin normally contribute to morethan 80% of the overall exposure (SCF, 2000).

In European countries, milk and dairyproducts are the main contributors to theaverage daily intake of PCDDs and PCDFs onan I-TEQ basis with 16–39%, meat and meatproducts are second (6–32%) and fish and fishproducts are third (2–63%). Other products,mainly of plant origin, such as vegetables andcereals, contributed 6–45% in those countries


11-Mar-03 7

TEQPCDD/PCDF TEQPCB

Country < 1990 1990–1994 1995–1999 < 1990 1990–1994 1995–1999

BelgiumGermanyDenmarkFinlandFranceItalyThe NetherlandsNorway

Range of means

30.718.120.0

25.034.2

18–34

24.820.616.713.2

23.510.413.3

10–25

13.8

16.4

7.908–16

25.3

25

6.63

18.012.0

20.929.119.47–29 No data

Table 7.3. National average concentrations of dioxins and related PCBs (in pg TEQ g−1 fat) inrepresentative human milk samples.



for which data were available. The total intakeof I-TEQ differed from country to country.Reasons for these differences may result fromdifferent food consumption habits but alsofrom applied sampling strategy and the largevariations in concentrations of dioxin-relatedsubstances in some of the food groups (e.g.vegetables and fruits, eggs and fish).

It is well known that during thebreast-feeding period, on a body weight basis,the intake of PCDDs and PCDFs is 1–2 ordersof magnitude higher than the average adultintake. A few countries (i.e. Finland, Ger-many, The Netherlands, Sweden and theUK) reported clear downward trends for theexposure of the general population to dioxinsand furans and, for Germany (see Table 7.4),Finland, The Netherlands and Sweden, thisdecline is also noted for concentrations inhuman milk.

Although different dietary habits makedirect comparison of results from differentcountries difficult, the daily intakes of PCDDsand PCDFs by males living in New Zealandare consistently lower than those of othercountries. The intakes are also below theWHO-recommended tolerable daily intake(TDI) of 1–4 pg TEQ kg−1 BW day−1. Thedietary intake estimated for an 80 kg adultmale consuming a median energy (10.8 MJday−1) diet was 14.5 pg I-TEQ day−1 (equiva-lent to 0.18 pg I-TEQ kg−1 BW day−1) and anadditional 12.2 pg TEQ day−1 (= 0.15 pg TEQkg−1 BW day−1) for dioxin-like PCBs. Dietaryintakes estimated for a 70 kg adolescent male

consuming a high energy (21.5 MJ day−1) dietwere 30.6 pg I-TEQ day−1 (= 0.44 pg I-TEQkg−1 BW day−1) and 22.7 pg PCB-TEQ day−1

(= 0.32 pg TEQ kg−1 BW day−1) (NZ, 1998).

Food and feedingstuff-related accidents

In the past, high exposures occurred throughaccidents. Well-known examples are the con-tamination of edible rice oils, such as theYusho in Japan in 1968 and the Yu-Cheng inTaiwan in 1978. In these cases, PCBs fromhydraulic oils leaked into edible oils, whichwere sold and consumed by thousands ofpeople. Severe toxic effects were detected inboth populations due to high levels of PCDFsand PCBs (Needham, 1993; Guo et al., 1994;Masuda, 1994).

Each year from 1997 to 1999, cases ofdioxin (and PCB) contamination of animalfeeds and foods occurred. Among these arethe dioxin contamination of citrus pulp pellets(an ingredient for feeding stuffs) from Brazilin the years 1997–1998, the contaminationof animal feeds with PCBs and dioxins inBelgium in spring of 1999, and the dioxin con-tamination of kaolinitic clay (a feed additive)from some mines in the USA and Germany.In each of these cases, preventive measureswere taken to avoid a further distributionof contaminated products and to protectthe consumer against foods with elevatedlevels.

11-Mar-03 7

162 H. Fiedler

Age(months) Year

Mean intake(pg TEQ day−1)

Mean intake(pg TEQ BW day−1) Comments

12346567–94444

199819981998199819981998199819981986–1990199219941996

291338360370369271180108879604502402

7068625748382413

135937762

Fully breast-fedFully breast-fedFully breast-fedFully breast-fedFully breast-fedPartly breast-fedPartly breast-fedPartly breast-fedFully breast-fedFully breast-fedFully breast-fedFully breast-fed

Table 7.4. Dietary intake of PCDDs/PCDFs of breast-fed infants in Germany.



The citrus pulp pellet contamination

From mid-1997 until March 1998, on average,twice the concentrations of PCDDs/PCDFs incow’s milk were detected by German FoodControl laboratories: starting from a levelof about 0.6 pg I-TEQ g−1 fat in summer1997, the average concentration increased to1.41 pg I-TEQ g−1 fat in different regions ofGermany in February 1998. The highest valuewas 7.86 pg I-TEQ g−1 fat and thus exceededthe concentration of 5 pg I-TEQ g−1 fat,the maximum permissible concentration toplace milk products on the German market.Although this observation was made inGermany first, later the same observationwas found in the 12 Member States of the EU.Whereas feedingstuff samples typically hadconcentrations in the range from 100 to300 pg I-TEQ kg−1, a compound feed formilk production, which had been found attwo different dairy farms, had about 1800 pgI-TEQ kg−1. It affected the level of dioxins andfurans in cow’s milk, beef and veal (Malisch,1998a,b).

The contamination was traced back tocitrus pulp pellets imported from Brazil andused in compound feed for ruminants all overEurope. The Brazilian citrus pellet productionhad been contaminated by dioxin-containinglime, which was a by-product from a chemicalfactory. The lime apparently was used for feedproduction against the advice of the supplier,who believed it was for construction. As men-tioned earlier, this contamination may havehad an impact on the general level of dioxinexposure of the European population and aslight increase in the dioxin content in breastmilk and tissue.

The Belgian chicken accident

In March 1999, serious animal health prob-lems in poultry production were discoveredin Belgium. There was a marked reduction inegg hatchability and an increased mortalityof chickens. At the end of May, analysisof feedingstuff samples, hens and breedingeggs showed high levels of dioxins andfurans. The first analyses showed dioxinconcentrations 1000 times above backgroundlevel; the contamination dropped by more

than 100 times from February to March 1999(Table 7.5). The contamination seems to havebeen caused by the discharge of about 25 l ofPCB transformer oil into a waste collectionunit for animal fats recycled into animals feedcontaminating 107 t of fat. From this, about90 t of fat was used for production offeedstuff for poultry, and the remaining fatwas used for production of milk and meat. Atthe beginning of October 1999, the number ofaffected or suspected farms was 505 poultryfarms, 1625 pig farms and 411 cattle farms.The estimated costs for Belgium in con-nection with the dioxin food contaminationis about US$1 billion; indirect costs areestimated to be three times higher. A correctwaste disposal of the 25 l of transformer oilwould have cost about US$1000. Thoughthe Belgian dioxin contamination had a majoreffect on the Belgian food production econ-omy, it gave only a short-term peak exposureto dioxins and furans for humans, whichcannot be detected in the general population.

The kaolinitic (ball) clay case

In 1999, a dioxin contamination of poultryand mink was traced back to the use ofkaolinitic clay as an anti-caking agent inpoultry feed and in mineral feed for mink.The origin of the contamination was tracedback to a ball clay mine in Germany. Similarexamples were found in the USA, wherecatfish and beef had been impacted throughthe use of ball clay in animal feedingstuffproduction.

The Brandenburg case

Repeated detection of elevated dioxin levelsin eggs produced in the German state ofBrandenburg was identified in 1999 when, inan open system, grass meal (for feedingstuff


20-Feb-03 7

Concentration (pg WHO-TEQ kg−1)

Poultry feedPoultry fatEgg fat

811,000775266

1,009713

Table 7.5. Belgian dioxin accident – PCDD/PCDF concentrations in feedingstuff and food.



production) was dried by burning wood asthe fuel. All types of wood were burned,including waste wood with chemical contam-ination from former painting or use of woodpreservatives.

The choline chloride case

In the year 2000, a dioxin contaminationin choline chloride pre-mixtures for feed-stuff was detected in Germany. The originalcholine chloride (= vitamin K) from a Belgianproducer was not contaminated but theSpanish feedstuff pre-mix producer who soldthe pre-mix to Germany had added pinesawdust to the product as a carrier. This pinesawdust was heavily contaminated withdioxin-containing pentachlorophenol.

Uptake and Human Exposure,Maternal Transmission

For humans (and animals), the major uptakeof dioxins and furans is via ingestion. The2,3,7,8-substituted congeners have long half-lives, generally of the order of years, and thiscauses these compounds to bioaccumulate.Metabolism is almost negligible and, to calcu-late body burdens, intake is the parameterfor countermeasures. Protection of the fetusis of particular concern when precautionaryactions are to be taken.

For the general population, the majorpathway of exposure to PCDDs, PCDFs andPCBs is through food. More than 90% ofhuman exposure occurs via the diet, withfoods of animal origin usually being the pre-dominant sources. Contamination of food iscaused primarily by deposition of emissionsfrom combustion sources such as wasteincineration, the metal industry, energyproduction or household heating, and subse-quent accumulation in the food chain. Due totheir lipophilicity, PCDDs, PCDFs and PCBsare associated with fat. Contamination of foodmay also occur through contaminated feed,improper application of sewage sludge, flood-ing of pastures, waste effluents and certaintypes of food processing or packaging (SCF,2000).

Some subpopulations may have higherexposure to dioxins, furans and PCBs asa result of particular consumption habits,e.g. nursing infants and subsistence fishermenliving close to contaminated waters.


Toxic effects in laboratory animals

The extraordinary potency of 2,3,7,8-TCDD(tetrachlorodibenzo-p-dioxin) and related2,3,7,8-substituted PCDDs and PCDFs hasbeen demonstrated in many animal species.They elicit a broad spectrum of responses inexperimental animals such as: liver damage(hepatoxicity); suppression of the immunesystem (immunotoxicity); formation anddevelopment of cancers (carcinogenesis);abnormalities in fetal development (teratoge-nicity); developmental and reproductive tox-icity; skin defects (dermal toxicity); diverseeffects on hormones and growth factors; andinduction of metabolizing enzyme activities(which increases the risk of metabolizingprecursor chemicals to produce others whichare more biologically active).

It is generally believed that 2,3,7,8-substituted PCDDs and PCDFs exhibit thesame pattern of toxicity. The toxic responsesare initiated at the cellular level, by the bind-ing of PCDDs/PCDFs to a specific proteinin the cytoplasm of the body cells, thearyl hydrocarbon receptor (AhR). The 2,3,7,8-substituted PCDDs/PCDFs bind to the AhRand induce CYP1A1 (cytochrome P450 1A1)and CYP1A2 (cytochrome P450 1A2) geneexpression. The binding to the AhR consti-tutes a first and necessary step to initiatethe toxic and biochemical effects of dioxins,although it is not sufficient alone to explainthe full toxic effects. This mechanism of actionof 2,3,7,8- Cl4DD parallels in many ways thatof the steroid hormones, which have a broadspectrum of effects throughout the body andwhere the effects are caused primarily by theparent compound. However, TCDDs andsteroid hormone receptors (e.g. oestrogen,androgen, glucocorticoid, thyroid hormone,vitamin D3 and retinoic acid receptors) do

20-Feb-03 7

164 H. Fiedler



not belong to the same family. AhR-bindingaffinities of 2,3,7,8-Cl4DF, 1,2,3,7,8-Cl5DFand 2,3,4,7,8-Cl5DF are of the same orderof magnitude as observed for 2,3,7,8-TCDD.With increasing chlorination, receptor-binding affinity decreases. The induction ofthe cytochrome P450 1A1 enzyme is frequentlyused as a convenient biomarker for PCDDs/PCDFs and other dioxin-like compounds.

Cancer promotion

2,3,7,8-TCDD is a multisite carcinogen in ani-mals as well as in humans. TCDD causes livertumours in animals at lower concentrationsthan any other man-made chemical. Dioxinsare not genotoxic (i.e. do not initiate cancerdevelopment), but 2,3,7,8-TCDD and otherdioxins and furans are strong promoters oftumour development. TCDD interferes withseveral functions that probably influence thetumour promotion process, such as growthfactors, hormone systems, oxidative damage,intercellular communication, cell prolifera-tion (division and growth), apoptosis (celldeath), immune surveillance and cytotoxicity(cellular toxicity).

In all mammalian species tested so far,lethal doses of 2,3,7,8-TCDD result in delayeddeath preceded by excessive body weightloss (‘wasting’). Other signs of 2,3,7,8-TCDDintoxication include thymic atrophy, hyper-trophy/hyperplasia of hepatic, gastrointesti-nal, urogenital and cutaneous epithelia, atro-phy of the gonads, subcutaneous oedemaand systemic haemorrhage. The lethal doseof 2,3,7,8-TCDD varies more than 5000-foldbetween the guinea-pig (LD50 = 1 µg kg−1 BW),the most sensitive, and the hamster, the leastsensitive species.

In tissue culture, 2,3,7,8-TCDD affectsgrowth and differentiation of keratinocytes,hepatocytes and cells derived from othertarget organs. Toxicity of 2,3,7,8-TCDD segre-gates with the AhR, and relative toxicity ofother PCDD congeners is associated with theirability to bind to this receptor. PCDDs causesuppression of both cell-mediated andhumoral immunity in several species at lowdoses. PCDDs have the potential to suppressresistance to bacterial, viral and parasiticchallenges in mice.

Kinetics

In most vertebrate species, the 2,3,7,8-substi-tuted PCDD and PCDF congeners are pre-dominantly retained; in other words, if chlo-rine atoms are present on all 2,3,7,8 positions,the biotransformation rate of PCDDs/ PCDFsis strongly reduced, resulting in significantbioaccumulation. In most species the liverand adipose tissue are the major storage sites.Although the parent PCDD/ PCDF conge-ners cause the biological effects, biotrans-formation to more polar metabolites shouldbe considered to be a detoxification process.Oxidation by cytochrome P450 primarily occursat the 4 and 6 positions in the molecule, andthe presence of chlorine atoms at these posi-tions reduces metabolism more than substitu-tion at the 1 and 9 positions. The half-lives ofespecially the PCDFs in humans are muchlonger than those in experimental animals.

2,3,7,8-TCDD is both a developmentaland a reproductive toxicant in experimentalanimals. The developing embryo/fetusappears to display enhanced sensitivity to theadverse effects of PCDDs. Perturbations of thereproductive system in adult animals requireovertly toxic doses. In contrast, effects on thedeveloping organism occur at doses morethan 100 times lower that those required in themother. Sensitive targets include the develop-ing reproductive, nervous and immunesystems. Perturbation of multiple hormonalsystems and their metabolism due to PCDDexposure may play a role in these events.

One effect that has been observedrecently is the altered sex ratio (increasedfemales) seen in the 6 years after the accidentin Seveso, Italy. Particularly intriguing in thislatest evaluation is the observation that expo-sure before and during puberty is linked tothis sex ratio effect. Other sites have beenexamined for the effect of TCDD exposure onsex ratio with mixed results, but with smallernumbers of offspring (US-EPA, 2000c).

Toxic effects in humans

In humans, effects associated with exposureto dioxins are observed mainly in accidental


20-Feb-03 7



and occupational exposure situations. Anumber of cancer locations, as well as totalcancer, have been associated with exposureto dioxins (mostly TCDD). In addition, anincreased prevalence of diabetes andincreased mortality due to diabetes andcardiovascular diseases have been reported.In children exposed to dioxins and/or PCBsin the womb, effects on neurodevelopmentand neurobehaviour (object learning) andeffects on thyroid hormone status havebeen observed at exposures at or nearbackground levels. At higher exposures,children exposed transplacentally to PCBsand PCDFs show skin defects, develop-mental delays, low birth weight, behaviourdisorders, a decrease in penile length atpuberty, reduced height among girls atpuberty and hearing loss. It is not totallyclear to what extent dioxin-like compoundsare responsible for these effects, when con-sidering the complex chemical mixturesto which human individuals are exposed.However, it has been recognized that subtleeffects might already be occurring in thegeneral population in developed countries,at current background levels of exposureto dioxins and dioxin-like compounds and,due to the high levels of persistence of thedioxin-like compounds, the concentrations inthe environment, as well as in food, will onlydecrease slowly.

There are a number of cohorts with highexposure to PCDDs/PCDFs (and PCBs), e.g.NIOSH (National Institute of OccupationalSafety and Health, USA) and Boehringer occu-pational studies, veterans of Operation RanchHand in Vietnam, residents of Seveso, etc. TheNIOSH population who were highly exposedfor more than 1 year, and with a 20 yearlatency period, had an increase of all cancers;the Ranch Hand population showed anincrease in diabetes with increasing dioxinlevels (no other effects seen); Seveso residentshad high levels of dioxin and, although thenumber of births was relatively low for 7 yearspost-exposure, there were significantly moregirls born than boys (change in normal sexratio). From these results obtained in high-exposure groups, it seems unlikely thatclinically observable health effects will be

found in the general adult population(Büchert et al., 2001).

The PCDD/PCDF pattern in humansmay yield information as to different sources.Also, people from certain geographicalregions may have specific patterns becauseof predominant exposures from differentsources, e.g. Europeans have higher 2,3,4,7,8-Cl5DF concentrations compared with USresidents (Büchert et al., 2001).

For humans, chronic effects are of greaterconcern than acute toxicity. Amongst themost sensitive end points are reproductive,developmental, immunotoxic and neurotoxiceffects. One of the 17 so-called toxic conge-ners, 2,3,7,8-TCDD, is the most toxic syntheticchemical (LD50 for guinea-pigs = 1 µg kg−1

BW day−1).Human exposure to 2,3,7,8-TCDD or

other PCDD congeners due to industrial oraccidental exposure has been associated withchloracne and alterations in liver enzymelevels in both children and adults. Changes inthe immune system and glucose metabolismhave also been observed in adults. Infantsexposed to PCDDs and PCDFs through breastmilk exhibit alterations in thyroid hormonelevels and possible neurobehavioural andneurological deficits.

Carcinogenicity

Four epidemiological studies of high-exposure industrial cohorts in Germany,The Netherlands and the USA found anincrease in overall cancer mortality. Overall,the strongest evidence for the carcinogenicityof 2,3,7,8-TCDD is for all cancers com-bined, rather than for any specific site. Therelative risk for all cancers combined in themost highly exposed and longer latencysubcohorts is 1.4.

In these cohorts, the blood lipid2,3,7,8-TCDD levels estimated to the last timeof exposure were 2000 ng kg−1 (mean) (up to32,000 ng kg−1) in the US cohort, 1434 ng kg−1

geometric mean (range, 301–3683 ng kg−1)among accident workers in the Dutch cohort,1008 ng kg−1 geometric mean in the group

20-Feb-03 7

166 H. Fiedler



of workers with severe chloracne in theBASF accident cohort in Germany, and upto 2252 kg−1 in the Boehringer cohort inGermany. These calculated blood 2,3,7,8-TCDD levels in workers at time of exposurewere in the same range as the estimated bloodlevels in a 2-year rat carcinogenicity study.In rats exposed to 100 ng kg−1 BW 2,3,7,8-TCDD day−1, hepatocellular carcinomas andsquamous cell carcinomas of the lung wereobserved. Estimated blood levels were5000–10,000 ng kg−1 2,3,7,8-TCDD. In thesame study, in rats exposed to 10 ng kg−1 BW2,3,7,8-TCDD day−1, hepatocellular nodulesand focal alveolar hyperplasia were observed.Estimated blood levels were 1500–2000 ngkg−1 2,3,7,8-TCDD. These results indicate par-allel tumorigenic responses to high exposureto 2,3,7,8-TCDD in both humans and rats.

In view of the results mentioned above, itshould be noted that the present backgroundlevels of 2,3,7,8-TCDD in human populations(2–3 ng kg−1) are 100–1000 times lower thanthose observed in this rat carcinogenicitystudy. Evaluation of the relationship betweenthe magnitude of the exposure in experimen-tal systems and the magnitude of the response(i.e. dose–response relationships) does notpermit conclusions to be drawn on the humanhealth risks from background exposures to2,3,7,8-TCDD (IARC, 1997).

A Working Group for IARC (Interna-tional Agency for Research on Cancer, Lyon,France) classified 2,3,7,8-TCDD as beingcarcinogenic to humans (IARC, 1997). Inmaking this overall evaluation, the workinggroup took into consideration the followingsupporting evidence:

1. 2,3,7,8-TCDD is a multisite carcinogen inexperimental animals that has been shownby several lines of evidence to act through amechanism involving the AhR.2. This receptor is highly conserved in anevolutionary sense and functions the sameway in humans as in experimental animals.3. Tissue concentrations are similar both inheavily exposed human populations in whichan increased overall cancer risk was observedand in rats exposed to carcinogenic dosageregimens in bioassays.

Other PCDDs and non-chlorinateddibenzo-p-dioxin are not classifiable as totheir carcinogenicity in humans.

The IARC concluded that there is inade-quate evidence in humans for the carcinoge-nicity of PCDFs. There is inadequate evidencein experimental animals for the carcinogeni-city of 2,3,7,8-Cl4DF. There is limited evidencein experimental animals for the carcinogeni-city of 2,3,4,7,8-Cl5DF and 1,2,3,4,7,8-Cl6DF.The overall evaluation states that PCDFs arenot classifiable as to their carcinogenicity inhumans (group 3).

In its recent dioxin reassessment, theUS-EPA basically follows the IARC classifica-tions (US-EPA, 2000c) and concludes that ‘un-der EPA’s current approach, TCDD is bestcharacterized as a “human carcinogen”’. Thismeans that, based on the weight of all of theevidence (human, animal, mode of action),TCDD meets the stringent criteria that allowEPA and the scientific community to accept acausal relationship between TCDD exposureand cancer hazard. The guidance suggeststhat ‘human carcinogen’ is an appropriatedescriptor of carcinogenic potential whenthere is an absence of conclusive epidemiolog-ical evidence to clearly establish a cause andeffect relationship between human exposureand cancer, but there are compelling carcino-genicity data in animals and mechanisticinformation in animals and humans demon-strating similar modes of carcinogenic action.The ‘human carcinogen’ descriptor is sug-gested for TCDD because all of the followingconditions are met. Occupational epidemio-logical studies show an association betweenTCDD exposure and increases in cancer at allsites, in lung cancer, and perhaps at othersites, but the data are insufficient on their ownto demonstrate a causal association. There isextensive carcinogenicity in both sexes ofmultiple species of animals at multiple sites(IARC, 1997).

Risk Assessment

First risk assessments only focused on themost toxic congener, 2,3,7,8-TCDD. Soon it


20-Feb-03 7



was recognized, though, that all PCDDs/PCDFs substituted at least in positions 2, 3, 7or 8 are highly toxic and thus major contri-butors to the overall toxicity of the dioxinmixture. In addition, despite the complexcomposition of many PCDD/PCDF-containing ‘sources’, only congeners withsubstitutions in the lateral positions of thearomatic ring, namely the carbon atoms 2, 3,7 and 8, persist in the environment andaccumulate in food chains.

For regulatory purposes so-called toxic-ity equivalency factors (TEFs) have beendeveloped for risk assessment of complexmixtures of PCDDs/PCDFs (NATO/CCMS,1988). The TEFs are based on acute toxicityvalues from in vivo and in vitro studies. Thisapproach is based on the evidence that thereis a common, receptor-mediated mechanismof action for these compounds. Althoughthe scientific basis cannot be considered assolid, the TEF approach has been adopted asan administrative tool by many agencies andallows conversion of quantitative analyticaldata for individual PCDD/PCDF congenersinto a single TEQ. As TEFs are interim valuesand administrative tools, they are based on

the present state of knowledge and shouldbe revised as new data become available.Today’s most commonly applied TEFswere established by a NATO/CCMS WorkingGroup on Dioxins and Related Compoundsas international toxicity equivalency factors(I-TEFs) (NATO/CCMS, 1988). However,in 1997, a WHO/IPCS (World Health Organ-ization/Intergovernmental Programme onChemical Safety) working group re-evaluatedthe I-TEFs and established a scheme, whichbesides human and mammalian TEFs, alsoestablished TEFs for birds and fish (Table 7.6).The same expert group also assessed thedioxin-like toxicity of PCB and assigned TEFvalues for 12 co-planar and mono-ortho-substituted PCB congeners (see Table 7.7)(WHO, 1997).

It should be noted that most existinglegislation and most assessments still usethe I-TEF scheme. However, the recentlyagreed Stockholm Convention on POPs (forreference see UNEP, 2001) refers to the com-bined WHO-TEFs as the starting point as areference.

Different international expert groupshave performed health risk assessment of

11-Mar-03 7

168 H. Fiedler

WHO-TEF

Congener I-TEF Humans/mammals Fish Birds

2,3,7,8-Cl4DD1,2,3,7,8-Cl5DD1,2,3,4,7,8-Cl6DD1,2,3,7,8,9-Cl6DD1,2,3,6,7,8-Cl6DD1,2,3,4,6,7,8-Cl7DDCl8DD2,3,7,8-Cl4DF1,2,3,7,8-Cl5DF2,3,4,7,8-Cl5DF1,2,3,4,7,8-Cl6DF1,2,3,7,8,9-Cl6DF1,2,3,6,7,8-Cl6DF2,3,4,6,7,8-Cl6DF1,2,3,4,6,7,8-Cl7DF1,2,3,4,7,8,9-Cl7DFCl8DF

10.50.10.10.10.010.0010.10.050.50.10.10.10.10.010.010.001

110.10.10.10.010.00010.10.050.50.10.10.10.10.010.010.0001

110.50.010.010.001

—0.050.050.50.10.10.10.10.010.010.0001

110.050.010.1

< 0.001—

10.110.10.10.10.10.010.010.0001

For all non-2,3,7,8-substituted congeners, no TEF has been assigned.

Table 7.6. International toxicity equivalency factors (I-TEFs) for PCDDs/PCDFs (NATO/CCMS, 1988)and WHO-TEFs for PCDDs/PCDFs (WHO, 1997).



dioxins and related compounds. A Nordicexpert group (for Scandinavian countries)proposed a TDI for 2,3,7,8-TCDD and struc-turally similar chlorinated PCDDs and PCDFsof 5 pg kg−1 BW, based on experimentalstudies on cancer, reproduction andimmunotoxicity. A first WHO meeting in 1990established a TDI of 10 pg kg−1 BW for 2,3,7,8-TCDD, based on liver toxicity, reproductiveeffects and immunotoxicity, and making useof kinetic data in humans and experimentalanimals. Since then, new epidemiological andtoxicological data have emerged, in particularwith respect to neurodevelopmental andendocrinological effects. In May 1998, a jointWHO–European Centre for Environmentand Health (ECEH) and IPCS expert groupre-evaluated the old TDI and came up with anew TDI (which is a range) of 1–4 pg TEQ kg−1

BW, which includes all 2,3,7,8-substitutedPCDDs and PCDFs as well as dioxin-like PCBs(for reference, see the 12 PCBs in Table 7.7).The TDI is based on the most sensitive adverseeffects, especially hormonal, reproductiveand developmental effects, which occur atlow doses in animal studies, e.g. in ratsand monkeys at body burdens in the range of10–50 ng kg−1 BW. Human daily intakes corre-sponding to body burdens similar to thoseassociated with adverse effects in animalswere estimated to be in the range of 10–40 pgkg−1 BW day−1. The 1998 WHO-TDI does notapply an uncertainty factor to account forinterspecies differences in toxicokinetics sincebody burdens have been used to scale doses

across species. However, the estimatedhuman intake was based on lowest observedadverse effect levels (LOAELs) and not on noobserved adverse effect levels (NOAELs). Formany end points, humans might be less sensi-tive than animals; uncertainty still remainsregarding animal to human extrapolations.Further, differences between animals andhumans exist in the half-lives for the differentPCDD/PCDF congeners. To account for allthese uncertainties, a composite uncertaintyfactor of 10 was recommended. As subtleeffects might already be occurring in thegeneral population in developed countriesat current background levels of exposure todioxins and related compounds, the WHOexpert group recommended that every effortshould be made to reduce exposure to below1 pg TEQ kg−1 BW day−1 (WHO, 1998).

In November 2000, the Scientific Com-mittee on Food (SCF) for the European Com-mission recommended a temporary tolerableweekly intake (t-TWI) of 7 pg 2,3,7,8-TCDDkg−1 BW using the body weight approach. Itwas also concluded that the TEQ approachshould be applied to include all 2,3,7,8-substituted PCDDs/PCDFs and dioxin-likePCBs. Thus, the t-TWI of 7 pg TEQ kg−1 BWday−1 is applicable for these compounds(seven PCDDs, ten PCDFs and 12 PCBs). Thet-TWI is based on the most sensitive endpoints from animal studies, e.g. develop-mental and reproductive effects in ratsand monkeys and endometriosis in monkeys(SCF, 2000).


20-Feb-03 7

Congener Humans/mammals Fish Birds

3,4,4´,5-TCB (81)3,3´,4,4´-TCB (77)3,3´,4,4´,5-PeCB (126)3,3´,4,4´,5,5´-HxCB (169)2,3,3´,4,4´-PeCB (105)2,3,4,4´,5-PeCB (114)2,3´,4,4´,5-PeCB (118)2´,3,4,4´,5-PeCB (123)2,3,3´,4,4´,5-HxCB (156)2,3,3´,4,4´,5´-HxCB (157)2,3´,4,4´,5,5´-HxCB (167)2,3,3´,4,4´,5,5´-HpCB (189)

0.00010.00010.10.010.00010.00050.00010.00010.00050.00050.000010.0001

0.00050.00010.0050.00005

< 0.000005< 0.000005< 0.000005< 0.000005< 0.000005< 0.000005< 0.000005< 0.000005

0.10.050.10.0010.00010.00010.000010.000010.00010.00010.000010.00001

Table 7.7. TEFs for PCBs (WHO, 1997).



When compared with adults, breast-fedinfants are exposed to higher intakes ofPCDDs, PCDFs and PCBs on a body weightbasis, although for a limited time only.Despite the higher exposure to contaminants,the WHO, like other agencies noted the bene-ficial effects associated with breast feedingand therefore promote and support breastfeeding. Further, the subtle effects detected ininfants were associated with transplacentalrather than lactational exposure (WHO, 1998).

Risk Management

As PCDDs and PCDFs have never beenproduced intentionally, their production anduse cannot be regulated by chemical legisla-tion and a prohibition of production. Indirectmeasures have to be taken by, for example,banning production and use of chemicals thatare known to be contaminated with PCDDs/PCDFs and taking measures to reduce emis-sions into the environment from knownsources of dioxins and furans (see nextsection).

The SCF concluded that, although dioxinsource reduction has been accomplishedsuccessfully in many European countries, aconsiderable proportion of the Europeanpopulation still exceeds the t-TWI. There-fore, further measures are needed to limitenvironmental releases of PCDDs/PCDFsand dioxin-like compounds (SCF, 2000).

The recent incidents of food and feedcontamination have shown that presentregulation is non-existent or inadequate, anda root cause analysis is required to developappropriate monitoring, prevention andmanagement. Setting feed and food limitsalone will not prevent further accidentsand there is no way to exclude the possibilityof similar incidents occuring in the futureunless specific measures are taken. However,regulatory levels would build the legalbasis at least to eliminate products withextraordinary contamination levels from themarket.

Monitoring of the animal feed productionchain could mitigate impacts and identify

causes. In contrast to former dioxin cases,which mainly originated from high emissionsof individual sources, recent incidents havebeen caused by entry of contaminants moredirectly into the human food chain. Dealingwith these accidents, there are mainly threedistinct objectives to address. These requiredifferent approaches for assessment, preven-tion, monitoring and regulatory response(Büchert et al., 2001):

• identification and response to an emer-gency situation of an acute contamina-tion (e.g. the Belgian case);

• identification and seizure of productswith exceptionally high levels (e.g. thecitrus pellet, choline chloride andBrandenburg cases) which can evenaffect the general population if usedto a large extent in the feed and foodchains;

• measures aiming to reduce exposure ofthe general population by ceasing useof feed ingredients that are more highlycontaminated than comparable compo-nents (e.g. fish meal and fish oil from thenorthern hemisphere).

Each case should be addressed carefully andit should be recognized that solutions for onecase will not necessarily prove effective forothers.

Legislation/Regulatory Issues

Several countries have taken action toreduce exposure to dioxins and furans and,in many places, especially in industrializedcountries of the northern hemisphere, envi-ronmental concentrations of PCDDs/PCDFsare decreasing. Legislation includes estab-lishment of limit values for stack emissions,e.g. for waste incinerators and otherindustrial plants, limit values for pulp milleffluents, limit values for sewage sludgespread on agricultural land or guidelines, forexample, for soil uses. In Europe, emissionslimits for incineration processes are usuallyset on the basis of stack gas converted tonormal temperature and pressure (273 K,

20-Feb-03 7

170 H. Fiedler



101.3 kPa), dry gas, and expressed at 11%oxygen. In the USA, the convention oftenuses a reference oxygen level of 7% andtemperature of 298 K. These differences canbe very important, e.g. the emission limit fora European incinerator of 0.1 ng I-TEQ Nm−3

(per normal cubic metre) dry gas at 11% oxy-gen is equivalent to approximately 0.13 ngI-TEQ dscm−1 (per dry standard cubic metre)at 7% oxygen as specified under the USregulation.

Within the food and feedingstuffregulations, the only recommended limitvalues exist for dairy products. To keep theagricultural food chain free of dioxins andfurans, an EU Directive sets a maximumtolerance level of 500 pg WHO-TEQ kg−1 forcitrus pellets and for lime used as additivein animal feed production. The same limitalso applies to the maximum limit for dioxincontent of 500 pg WHO-TEQ kg−1 for mostadditives belonging to the group ‘binders,anti-caking agents and coagulants’. Lastly,as a secondary measure, the use of ‘wood,sawdust and other materials derived fromwood treated with wood protection products’is prohibited in compound feedingstuffs(see Table 7.8).

Many countries have established guide-line values for various foods or food catego-ries. Table 7.9 shows present regulationsfor PCDDs/PCDFs and, for comparison andcompleteness, for PCBs in foods for Europeancountries.

Conclusions

Many actions taken since the late 1980s haveresulted in a reduction of the daily intakeof dioxins and furans for many Europeancountries. However, recent accidents haveshown the vulnerability of the food chain tocontamination with these compounds. Thereare strong indications that the citrus pelletcontamination has reversed the formerdownward trend in body burden on a broadbasis all over Europe. Special attention hasto be paid to the high intake of dioxins andfurans for infants, which is still in a range thatposes risk to the developing organism.

Exposure issues relating to dioxins (andother contaminants) should not be consideredin isolation. As shown in Scandinavia, theFinnish population is eating more fish, whichhas contributed to an important improve-ment in cardiovascular disease prevention,although this may seem inadvisable from aPCDD/PCDF/PCB exposure point of view asthese fish can be highly contaminated withdioxins and other lipophilic contaminants.The relative risk is important and needs to beconsidered, and knee-jerk reactions should beavoided. Also, reduction of dioxin emissionsshould be seen in context and must occurtogether with the controls of other pollutantsand contaminants, whether chemical, resi-dues or pathological. Lessons learned in thisfield must be used to improve understandingin other fields, and vice versa.


20-Feb-03 7

EU Directive Description

98/60/EC Citrus pulp pellets asfeedingstuffs (Amendment to 74/63/EEC)2439/1999/EC and 739/2000/EC

91/516/EC

Sets an upper-bound detection limit of 500 pg I-TEQ kg−1;in force since 1 July 1998Maximum limit for dioxin content of 500 pg WHO-TEQkg−1 for most additives belonging to the group ‘binders,anti-caking agents and coagulants’ (applies from 1 March2000; to be re-examined before October 2000)The use of ‘wood, sawdust and other materials derivedfrom wood treated with wood protection products’a isprohibited in compound feedingstuffs

aWood preservatives may contain high concentrations of PCDDs/PCDFs, e.g. PCPs, otherchlorophenols, chlorobenzenes.

Table 7.8. EU directives addressing PCDD/PCDF in feedingstuffs.



20-Feb-03 7

172 H. Fiedler

Foodstuffs of animal origin

Country PCDDs and PCDFs PCBs

Austria

Belgium

DenmarkFinlandFranceGermany

GreeceIrelandItaly

Luxemburg

NorwayPortugalSpain

Sweden

The Netherlands

UK

Provisional limits WHO-TEQ(PCDD/PCDF) g−1 fat: pork 2, milk 3,poultry and eggs 5 and beef 6 pgMilk, bovine, poultry, animal fats andoils, eggs and derived products, if> 2% fat: 5 pg WHO-TEQ(PCDD/PCDF) g−1 fatPork and derived products, if > 2% fat:3 pg WHO-TEQ (PCDD/PCDF) g−1 fat

No national limitsNo national limitsMilk and dairy products: 5 pg g−1 fatRecommendations for milk and dairyproducts in pg I-TEQ g−1 milk fat:• < 0.9 (desirable target)• 3.0 (identification of sources;

measures to reduce input;recommendations for land use;recommendation to stop directsupply of milk products toconsumers)

• > 5.0 (ban on trade of contaminatedmilk products)

No national limitsInternational normsNo national limits

Recommended: pork 2, beef 6, poultry5, milk 3 and eggs 5 pg g−1 fatNo national limitsNo national limitsLevels > 5 pg g−1 fat are considered asnon-acceptable in dairy productsNo national limits

Dairy products and foods with milk ordairy product as ingredients: 6 pg TEQg−1 fat

Guideline for cows’ milk: 0.66 ngWHO-TEQ kg−1 whole milk (16.6 ngWHO-TEQ kg−1 fat)

For the sum of PCBs 28, 52, 101, 118,138, 153 and 180Milk and derived products, if > 2% fat:100 ng g−1 fatBovine, pork, poultry, animal fats and oils,eggs and derived products, if > 2% fat:200 ng g−1 fatNo national limitsNo national limitsNo national limitsCongener-specific limits for PCBs 28, 52,101, 138, 153 and 180 in foods of animalorigin: 0.008–0.6 mg kg−1 fat or wholeweight basis

No national limitsInternational normsAction level for the sum of tri- tooctachlorobiphenyls in various foods ofanimal origin (excluding freshwater andmarine fish and derived products): 100 ngg−1 fat

No national limitsNo national limitsNo national limits

PCB 153: meat products > 10% fat: 0.1,milk and milk products > 2% fat: 0.02, andeggs: 0.1 mg kg−1 fatMeat products < 10% fat: 0.01, milk andmilk products < 2% fat: 0.001, and fish:0.1 mg kg−1 wet weightCongener-specific limits for PCBs 28, 52,101, 118, 138, 153 and 180 in foods ofanimal origin: 0.02–2 mg kg−1 fat (for fishmg kg−1 wet weight)

Table 7.9. Guidelines and maximum levels for concentrations of PCDDs, PCDFs and PCBs in foods inEuropean countries.



References

Büchert, A., Cederberg, T., Dyke, P., Fiedler, H.,Fürst, P., Hanberg, A., Hosseinpour, J.,Hutzinger, O., Kuenen, J.G., Malisch, R.,Needham, L.L., Olie, K., Päpke, O., RiveraAranda, J., Thanner, G., Umlauf, G.,Vartiainen, T. and van Holst, C. (2001) ESFworkshop on dioxin contamination in food.Environmental Science and Pollution Research 8,84–88.

Buckland, S.J., Ellis, H.K. and Dyke, P.H. (2000) NewZealand Inventory of Dioxin Emissions to Air,Land and Water, and Reservoir Sources. Organo-chlorines Programme, Ministry for the Envi-ronment, Wellington, New Zealand.

Buckley-Golder, G., Coleman, P., Davies, M., King,K., Petersen, A., Watterson, J., Woodfield, M.,Fiedler, H. and Hanberg, A. (1999) Compilationof EU Dioxin Exposure and Health Data. Reportproduced for European Commission DGEnvironment and UK Department of theEnvironment Transport and the Regions(DETR). Full report at: europa.eu.int/comm/environment/dioxin/download. htm

Bumb, R.R., Crummett, W.B., Artie, S.S., Gledhill,J.R., Hummel, R.H., Kagel, R.O., Lamparski,L.L., Luoma, E.V., Miller, D.L., Nestrick, T.J.,Shadoff, L.A., Stehl, R.H. and Woods, J.S.(1980) Trace chemistries of fire: a source ofchlorinated dioxins. Science 210, 385–390.

Codex Alimentarius (n.d.) For information, seewebsite at: www.fao.org/

Environment Agency Japan (2000) Results pre-sented by S. Sakai ‘Formation Mechanism andEmission Reduction of PCDDs in MunicipalWaste Incinerators’ at UNEP Workshop onTraining and Management of Dioxins, Furans,and PCBs. Seoul, Republic of Korea, 24–28July 2000. Available at: www.chem.unep.ch/pops/newlayout/prodocas.htm

Environment Canada (2001) Inventory of Releases –Updated Edition. Prepared by EnvironmentCanada, February.

Fiedler, H. (1999) Dioxin and Furan Inventories –National and Regional Emissions of PCDD/PCDF.Report by UNEP Chemicals, Geneva, Switzer-land. May.

Fiedler, H., Hutzinger, O. and Timms, C. (1990)Dioxins: sources of environmental load andhuman exposure. Toxicology and EnvironmentalChemistry 29, 157–234.

Guo, Y.L., Ryan, J.J., Lau, B.P.Y., Hsu, M.M. andHsu, C.-C. (1994) Blood serum levels of PCDFsand PCBs in Yucheng women 14 years after

exposure to a toxic rice oil. Organohalogen Com-pounds 21, 509–512.

Hansen, E. (2001) Substance Flow Analysis for Dioxinsin Denmark. COWI, Environmental Project No.570 2000, Miljøprojekt, Copenhagen.

IARC (1997) Polychlorinated dibenzo-para-dioxinsand polychlorinated dibenzofurans. IARCMonographs on the Evaluation of CarcinogenicRisks to Humans, Vol. 69. IARC, Lyon, France.

Malisch, R. (1998a) Update of PCDD/PCDF-intakefrom food in Germany. Chemosphere 37,1687–1698.

Malisch, R. (1998b) Increase of PCDD/F-contamination of milk and butter in Ger-many by use of contaminated citrus pulps ascomponent in feed. Organohalogen Compounds38, 65–70.

Masuda, Y. (1994) Approach to risk assessment ofchlorinated dioxins from Yusho PCB poison-ing. Organohalogen Compounds 21, 1–10.

NATO/CCMS (1988) International Toxicity Equiva-lency Factor (I-TEF) Method of Risk Assessmentfor Complex Mixtures of Dioxins and RelatedCompounds. Pilot Study on International Infor-mation Exchange on Dioxins and RelatedCompounds, Report Number 176, August1988, North Atlantic Treaty Organization,Committee on Challenges of Modern Society,Brussels.

Needham, L.L. (1993) Historical perspective onYu-Cheng incident. Organohalogen Compounds14, 231–233.

NZ (1998) Organochlorines Programme – Con-centrations of PCDDs, PCDFs and PCBs inRetail Foods and an Assessment of DietaryIntake for New Zealanders. Ministry for the Envi-ronment, Wellington.

Olie, K., Vermeulen, P.L. and Hutzinger, O.(1977) Chlorodibenzo-p-dioxins and chloro-dibenzofurans are trace components of fly ashand flue gas of some municipal waste incinera-tors in the Netherlands. Chemosphere 6,445–459.

SCF (2000) Opinion of the SCF on the Risk Assessment ofDioxins and Dioxin-like PCBs in Food. Adoptedon 22 November 2000. European Commis-sion, Health & Consumer Protection Director-ate-General, Scientific Committee on Food.SCF/CS/CNTM/DIOXIN/8 Final.

UNEP (1999) Dioxin and furan inventories –national and regional emissions of PCDD/PCDF. Report by UNEP Chemicals, Geneva.

UNEP (2001) The text of the Stockholm Con-vention on POPs can be found at:http://www.chem.unep.ch/pops

US-EPA (2000a) Exposure and Human Health Reas-sessment of 2,3,7,8-Tetrachlorodibenzo-p-dioxin


20-Feb-03 7



(TCDD) and Related Compounds. Part I: Esti-mating Exposure to Dioxin-Like Compounds –Volume 2: Sources of Dioxin-Like Compoundsin the United States. Draft Final Report,EPA/600/P–00/001Bb. Washington, DC. Seewebsite at: www.epa.gov/ncea/pdfs/dioxin/dioxreass.htm

US-EPA (2000b) Report on the Peer Review of theDioxin Reassessment Documents: Toxicity Equiv-alency Factors for Dioxin and Related Compounds(Chapter 9) and Integrated Risk CharacterizationDocument – Final Report. Prepared by EasternResearch Group, Inc., Washington, DC.

US-EPA (2000c) Exposure and Human Health Reassess-ment of 2,3,7,8-Tetrachlorodibenzo-p-dioxin(TCDD) and Related Compounds. Part II: HealthAssessment of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (PCDD) and Related Compounds. DraftFinal Report, Chapters 1–7, EPA/600/P–00/001Be. Washington, DC. See website at:

w w w . e p a . g o v / n c e a / p d f s / d i o x i n /dioxreass.htm

Welsch-Pausch, K. and McLachlan, M.S. (1998)Fate of airborne polychlorinated dibenzo-p-dioxins and dibenzofurans in an agriculturalecosystem. Environmental Pollution 102,129–137.

Welsch-Pausch, K., McLachlan, M.S. and Umlauf,G. (1995) Determination of the principal path-ways of polychlorinated dibenzo-p-dioxinsand dibenzofurans to Lolium multiflorum(Welsh rye grass). Environmental Science andTechnology 29, 1090–1098.

WHO (1997) WHO Toxic Equivalency Factors (TEFs)for Dioxin-like Compounds for Humans and Wild-life. 15–18 June 1997, Stockholm, Sweden.

WHO (1998) Executive Summary – Assessment ofthe Health Risk of Dioxins: Re-evaluation of theTolerable Daily Intake (TDI). WHO Consulta-tion, 25–29 May 1998, Geneva.

20-Feb-03 7

174 H. Fiedler



8 Polycyclic Aromatic Hydrocarbons inDiverse Foods

M.D. Guillén* and P. SopelanaTecnología de Alimentos, Facultad de Farmacia, Universidad del País Vasco,

Paseo de la Universidad 7, 01006-Vitoria, Spain

Introduction

Polycyclic aromatic hydrocarbons (PAHs)are widespread environmental contaminantswhich represent a very important groupof carcinogens or co-carcinogens. They arefound in coal, asphaltic rocks and petro-leum, and are also formed by the incom-plete combustion of organic matter, thatis to say by the incomplete combustion ofsome of the above materials, as well as thatof proteins, lipids and carbohydrates. Inaddition, it has been suggested that thesecompounds could be synthesized during themetabolic processes of plants, seaweed andbacteria.

Due to industrial and engine combustionemissions and other processes, these com-pounds contaminate air, water and soil, andso are passed on to foods; they can also begenerated during incorrect food processingand cooking. As a result, they are present inboth unprocessed and processed foods.

The relationship between exposure tocombustion emissions and carcinogenicity inhumans has been known for a long time. Itwas noticed by Pott in 1775 with regard to skincancer in chimney sweeps. Afterwards, boththe observation of a higher frequency of can-cer in human groups whose diets were rich

in smoked foods and studies showing thecarcinogenicity of some PAHs in animalswere the starting point for many studiesof these compounds, some aspects of whichhave been reviewed (Howard and Fazio, 1980;Guillén, 1994; Shaw and Connell, 1994;Guillén et al., 1997).

Nature of Polycyclic AromaticHydrocarbons

PAHs are a very numerous group ofcompounds formed by fused aromatic ringsmade up of carbon and hydrogen atoms,the most simple of which is naphthalene. Thenumber of PAHs is very large and, further-more, these compounds can either bepartially hydrogenated or have alkyl sub-stituents. There are other compounds withfused aromatic rings in the molecule, whichalso include heteroatoms and other func-tional groups, such as amine, phenol or nitrogroups; these latter, together with PAHs,constitute a wider group named polycyclicaromatic compounds.

PAHs have been classified in two classes:peri- and cata-condensed. Peri-condensedPAHs can be defined as those systems whose


20-Feb-03 8




graphs, or lines which connect the ringcentres, form cycles; these can be subdividedfurther into two classes: alternants, which areformed exclusively by six-membered rings,and non-alternants, which include somefive-membered rings. Cata-condensed PAHscan be defined as those systems whose graphsdo not form cycles, and can be classifiedfurther as branched or not branched, theformer being thermodynamically more stableand chemically less reactive than non-branched systems of the same size. Cata-condensed PAHs are always alternantsystems. Figure 8.1 shows some examples.

In PAH topology, some regions andcarbon atom positions have been relatedto biological activity: the K region definedas the external corner of a phenanthrenicmoiety; the L region consisting of a pairof opposed open anthracenic point atoms;the ‘bay’ region defined as an open inner

corner of a phenanthrenic moiety; the dis-tal bay region also known as the M region;and the peri position, which correspondsto the carbon atom opposite the bayregion and adjacent to the angular ring.Figure 8.2 shows these regions and theperi position in the benz(a)anthracenemolecule.

Table 8.1 names and gives formulae,structures, molecular weights, boilingpoints and some other properties such aswater solubility and octanol/water partitioncoefficient of some of the PAHs most fre-quently studied in foods. All these com-pounds are solid at room temperature;their boiling points are high and their volatil-ity is low. They are lipophilic, so their watersolubility is low and their octanol/waterpartition coefficients are fairly high; these twolatter properties have been related to theirbiological activity.

20-Feb-03 8

176 M.D. Guillén and P. Sopelana

Fig. 8.1. Different types of structures in PAH.

Fig. 8.2. Regions related to biological activity.



Polycyclic Aromatic Hydrocarbons 177

20-Feb-03 8

Nomenclature Formula Structure MWa BPb (°C) WSc (µg l−1) PCowd

Naphthalene

Acenaphthylene

Phenanthrene

Anthracene

Fluoranthene

Pyrene

Benzo(ghi)fluoranthene

Cyclopenta(cd)pyrene

Benz(a)anthracene

7,12-Dimethylbenz(a)anthracene

Chrysene

Triphenylene

Naphthacene

C10H8

C12H8

C14H10

C14H10

C16H10

C16H10

C18H10

C18H10

C18H12

C20H16

C18H12

C18H12

C18H12

128

152

178

178

202

202

226

226

228

256

228

228

228

218

270

338

340

383

393

432

439

435

441

439

450

31,700.0

16,100.0

1,290.0

73.0

260.0

135.0

14.0

61.0

2.0

43.0

0.6

3.37

3.92

3.24

4.54

5.22

5.18

5.91

6.00

5.61

5.49

5.76

continued

Table 8.1. Some of the PAHs most frequently studied in foods: structure, molecular weight and otherproperties.



20-Feb-03 8



Benzo(b)fluoranthene

Benzo(j)fluoranthene

Benzo(k)fluoranthene

Benzo(e)pyrene

Benzo(a)pyrene

Perylene

Indeno(1,2,3-cd)pyrene

Dibenz(a,c)anthracene

Dibenz(a,h)anthracene

Dibenz(a,j)anthracene

Pentacene

Picene

Benzo(ghi)perylene

Anthanthrene

C20H12

C20H12

C20H12

C20H12

C20H12

C20H12

C22H12

C22H14

C22H14

C22H14

C22H14

C22H14

C22H12

C22H12

252

252

252

252

252

252

276

278

278

278

278

278

276

276

481

480

481

493

496

495

536

524

519

547

1.5

2.5

0.8

4.0

4.0

0.4

1.6

0.6

12.0

0.3

5.8–6.1

6.12

6.0–6.8

6.04

6.58

6.50

6.50




Distribution of PAHs in Foods

Before beginning a description of the resultsobtained by several authors concerningdistribution of PAHs in foods, some aspectsshould be considered. First, PAHs aregenerated as complex mixtures, so the pres-ence of only one PAH in contaminated foodsis not common. However, in some studiesof the ocurrence of PAHs in foods, only theconcentration of benzo(a)pyrene has beendetermined, because this compound isconsidered an indicator of other PAHs.Others study only those PAHs whoseanalysis is recommended by internationalorganizations such as WHO (World HealthOrganization) or EPA (the US EnvironmentalProtection Agency); other studies concern allthose PAHs found in the food sample.

Secondly, it should be pointed out thatthe methodology used for the extraction,clean-up, separation, identification and quan-tification of PAHs in food samples decisivelyinfluences the results obtained. For thisreason, all data and comments on distributionof PAHs in foods should be seen in the contextof each study.

The ocurrence of PAHs in foods of vege-table origin is due basically to environmentalcontamination, especially of air and soil. In

fruits and vegetables, the concentrations ofPAHs detected vary considerably dependingon the food surface/weight ratio, time ofexposure, proximity to contamination sourceand level of contamination in the air. Fruitsand vegetables growing in regions free ofcontamination are also free of PAHs. Further-more, grilled vegetables show higher concen-trations than raw vegetables (Tateno et al.,1990). Table 8.2 gives the concentrations ofPAHs found in lettuce growing at differentdistances from a highway, as well as in rawand grilled vegetables.

Detected PAH levels are not high inraw cereals and beans, and contamination isdue basically to aerial deposition (Jones et al.,1989); this is in agreement with the ocurrenceof PAHs in higher concentrations in branthan in flour (Dennis et al., 1991). Dryingtechniques used in some countries for cerealpreservation, such as direct combustion gasheating, can increase their PAH concentra-tions. Likewise, cereal and bean smoking ortoasting also contribute to the level of PAHs(Klein et al., 1993). Table 8.2 gives concentra-tions found in raw wheat grains, white flourand bran, as well as in raw and toasted coffeebean samples.

Raw sugarcane does not contain PAHsbut, in some countries, sugarcane plantations


11-Mar-03 8


Coronene

Dibenzo(a,e)pyrene

Dibenzo(a,h)pyrene

Dibenzo(a,i)pyrene

Dibenzo(a,l)pyrene

C24H12

C24H14

C24H14

C24H14

C24H14

300

302

302

302

302

525 0.1 6.75

aMolecular weight; bboiling point; cwater solubility; doctanol/water partition coefficient.Data are taken from Mackay and Shiu (1977), Bjorseth (1983) and Dabestani and Ivanov (1999).




are usually set alight before harvesting, con-taminating the sugarcane, then the unrefinedsugar, and so the sugarcane spirits (Serra et al.,1995). Sugar refining may contribute to avoid-ing this contamination. The contaminationlevel in nuts, roots and tubers is low (Denniset al., 1991).

Although there are hardly any studiesof PAH contamination in oilseeds and olives(Dennis et al., 1991), oils from different vegeta-ble sources such as virgin and refined oliveoil, sunflower, soybean, maize, coconut, rape-seed, cotton, groundnut, grapeseed, rice, palmand palm kernel oils, cocoa butter, as well asother commodities derived from vegetable oilsuch as margarines, cream substitutes andsome infant formulae powders have been

widely studied. The presence of these contam-inants in oils, discarding the biosyntheticroute, can be attributed both to environmentalcontamination, basically from the air, and tocontamination during processing. Oilseedsare sometimes dried directly by combustiongases, which causes contamination; this is thecase with copra and grapeseeds. In addition,the possibility of contamination by PAHscontained in the organic solvent used in theoil extraction process has been commented on.The way to reduce the PAH level in thesefoods is by means of the refining process,especially if activated charcoal is used in thebleaching step. Table 8.3 gives the resultsof several oil studies, and great variationsin PAH content in the different samples

20-Feb-03 8


Compound L50S L12S WGUK WFUK BUK RCG TCG RVJ TVJ

PhenanthreneAnthracene1-Methylphenanthrene2-Methylphenanthrene9-MethylanthraceneFluoranthenePyreneBenzo(ghi)fluorantheneCyclopenta(cd)pyreneBenz(a)anthraceneChryseneBenzo(b)fluorantheneBenzo(k)fluorantheneBenzo(e)pyreneBenzo(a)pyrenePeryleneIndeno(1,2,3-cd)pyreneDibenz(a,c)anthraceneDibenz(a,h)anthraceneBenzo(ghi)peryleneCoronene

5.00.20.60.7–

5.35.8––

0.93.3c

0.60.4e

0.80.50.00.6––

0.5–

7.50.31.61.6–9.1

10.4––4.67.1c

7.36.1e

6.76.21.78.3––

10.8–

–––––

0.60.40.00.10.20.8c

0.6d

0.30.3–

0.3–

0.00.30.1

–––––

0.20.5––

0.10.10.00.10.20.1n.d.0.1–

0.00.1–

–––––

0.70.1––

0.70.80.30.50.40.4–

1.1–

0.10.5–

–––––

8.08.1––

0.31.8c

1.9d

0.80.90.30.50.1f

0.6–

–––––

14.316.3

––

1.22.6c

1.4d

0.60.80.20.40.0f

0.6–

2.20.10.0–

n.d.1.30.3––

0.0––

0.00.20.10.0–

n.d.n.d.–

0.2

3.90.1

n.d.b

–0.00.80.6––

0.4––

0.00.20.71.3–

1.60.1–

5.0

aL50S, lettuce grown at 50 m from a Swedish highway (µg kg−1 fresh weight) (Larsson and Sahlberg,1981); L12S, lettuce grown at 12 m from a Swedish highway (µg kg−1 fresh weight) (Larsson andSahlberg, 1981); WGUK, wheat grain from Broadbalk (UK) (Jones et al., 1989); WFUK, wheat flour (Denniset al., 1991); BUK, bran (Dennis et al., 1991); RCG, raw coffee (Klein et al., 1993); TCG, toasted coffee(Klein et al., 1993); RVJ, raw vegetables (Tateno et al., 1990); TVJ, toasted vegetables (Tateno et al.,1990).bn.d., not detectedcConcentration of chrysene + triphenylene.dConcentration of benzo(b)fluoranthene + benzo(j)fluoranthene + benzo(k)fluoranthene.eConcentration of benzo(j)fluoranthene + benzo(k)fluoranthene.fConcentration of dibenz(a,c)anthracene + dibenz(a,h)anthracene.

Table 8.2. PAH concentrations (µg kg−1 dry weight) in several foods of vegetable origin.a



have been observed. The high PAH concen-trations in some oil samples is a causefor concern because vegetable oils andfats are ingredients in a great number ofmanufactured foods.

Another group of foods of concerncomes from the aquatic environment. Studieson the ocurrence of PAHs in oysters, mussels,fish, shellfish and other marine organismssuch as seals and sea lions have beenmade, and great variations have also beenfound, due both to the level of contaminationwhere these organisms grow and to theirability to metabolize PAHs. Molluscs and fishaccumulate light PAHs to a similar degree;however, heavy PAHs seem to accumulatemore in molluscs than in fish. These facts canbe observed in Table 8.4, which gives PAHcontent data for oyster (Sanders, 1995) and

fish (Vassilaros et al., 1982) samples, comingfrom two very differently contaminatedplaces, as well as of mussels and fish, comingfrom the same place (Baumard et al., 1998).The known ability of some seafoods toaccumulate PAHs is why the concentrationof PAHs in these organisms has been con-sidered as an indicator of the contaminationof their habitat. However, these same organ-isms can release, in a short period of time,their accumulated PAHs if they are trans-ferred to clean water; this fact should betaken into account for lowering their PAHlevels. In addition, processing and cookingtechniques such as smoking and grillingcan contribute to increasing the PAH levelsof these commodities. Table 8.4 also givesPAH concentrations of fresh and smoked fishsamples.


20-Feb-03 8

Compound VO O S So G CC RC Mc

AcenaphthylenePhenanthreneAnthracene1-Methylphenanthrene2-Methylphenanthrene2-Methylanthracene4,5-MethylphenanthreneFluoranthenePyrene1-MethylpyreneBenz(a)anthraceneChryseneBenzo(b)fluorantheneBenzo(k)fluorantheneBenzo(e)pyreneBenzo(a)pyrenePeryleneIndeno(1,2,3-cd)pyreneDibenz(a,c)anthraceneBenzo(ghi)peryleneCoronene

–15.30.9––––4.25.0–0.20.50.10.1–0.0–0.2–0.0–

–4.7–0.91.0––0.42.1–

2.8b

––––

0.0f

0.6f

–

4.42.30.0––––

6.75.0–

3.11.7c

2.22.0e

4.11.50.61.30.0f

1.70.3

0.92.22.1––––8.92.6–

21.917.3c

24.827.6e

25.228.410.022.84.7f

16.92.1

–––––––

17.17.2–

78.563.3c

85.398.8e

87.6105.736.280.612.9f

65.77.4

–970.0200.0120.0140.060.059.0

520.0440.038.076.0

120.0c

55.0–

20.022.06.39.8–9.6–

–2.80.32.51.70.61.5

18.020.03.61.34.1c

0.7–0.40.2

< 0.1< 0.1

–< 0.1

–

–6.00.91.81.30.3–9.0

15.02.9

21.0b

4.5d

1.82.20.60.70.2f

0.70.2

aVO, virgin olive oil (Moret et al., 1997); O, olive oil (Hopia et al., 1986); S, refined sunflower oil(Kolarovic and Traitler, 1982); So, refined soybean oil (Kolarovic and Traitler, 1982); G, refinedgroundnut oil (Kolarovic and Traitler, 1982); CC, crude coconut oil (Larsson et al., 1987); RC, refinedcoconut oil (Larsson et al., 1987); Mc, cooking margarine (Hopia et al., 1986).bConcentration of benz(a)anthracene + chrysene + triphenylene.cConcentration of chrysene + triphenylene.dConcentration of benzo(b)fluoranthene + benzo(j)fluoranthene + benzo(k)fluoranthene.eConcentration of benzo(j)fluoranthene + benzo(k)fluoranthene.fConcentration of dibenz(a,c)anthracene + dibenz(a,h)anthracene.

Table 8.3. PAH concentration (µg kg−1) in several vegetable oil and fat samples.a



Drinking water may also be contami-nated. Atmospheric pollution contaminatesthe surface of open-air supplies and the runofffrom waste deposits may contaminate groundwater; in addition, the use of tar-coated waterpipes increases PAH levels in water. Levels ofbenzo(a)pyrene up to 1 µg kg−1 have beendetected in tap water (IARC, 1983).

Foods of animal origin such as meat, fatand lard, milk, butter, cheese and eggs gener-ally do not contain high levels of PAHs. How-ever, recent studies of animal products fromcontaminated zones show significant levels(Husain et al., 1997); this has been detectedespecially in egg and milk samples, as can beobserved in Table 8.5. In addition, some smok-ing processes, some cooking procedures andsome types of heat sources used for cookingcontribute to PAH levels in foods. Examples

of PAH levels in raw and barbecued beef(Lodovici et al., 1995) and in frankfurtersgrilled on a log fire or fried in a pan (Larssonet al., 1983) are given in Table 8.5.

Finally, manufactured foods made up ofseveral ingredients have a PAH content whichis a function of the PAH content of each ingre-dient as well as of the processes involved intheir manufacture.

Estimations of PAH intake from food,carried out in several countries such as Aus-tria, Germany, Italy, The Netherlands, the UK,Sweden and the USA, range from 0.1 to 1.6 µgof benzo(a)pyrene per person per day. ThePAH intake and the foods that mainly contrib-ute to this depend on the eating habits of thecountry, on the environmental contaminationof the region in which the foods are produced,on the techniques used for food preserving

20-Feb-03 8


Compound OMP OOC2 CFBR CFBL MFB FFB FF SF

MethylnaphthaleneDimethylnaphthaleneAcenaphthylenePhenanthreneAnthraceneFluoranthenePyreneBenz(a)anthraceneChryseneTriphenyleneBenzo(b)fluorantheneBenzo(k)fluorantheneBenzo(j)fluorantheneBenzo(e)pyreneBenzo(a)pyrenePeryleneIndeno(1,2,3-cd)pyreneDibenzanthraceneBenzo(ghi)perylene

–––

76.030.0

680.0407.0228.0261.0

–53.0

161.0––

31.0–––

41.0

–––

18.06.0

32.0n.d.n.d.n.d.–

n.d.n.d.––

n.d.–––

n.d.

6.0100.0270.0

2700.0–

1800.01500.0

22.0–––––14.07.08.0

–––

5.017.0n.d.b

2.0–4.04.0

n.d.6.0––––

n.d.n.d.n.d.–––

–––

10.41.15.74.71.85.4c

2.8d

2.20.71.01.20.1e

1.8

–––

19.20.8

25.835.10.52.1c

1.7d

0.81.30.00.00.0e

0.0

–––

n.d.–

18.883.00.4

175.0–

11.50.3––

44.0––4.9

149.0

–––

61.8–

20.7117.0

4.5290.0

–30.01.0––

48.0––8.0

201.0

aOMP, oyster, Crassostrea virginica, from MP-Murrells Inlet (South Carolina) (Sanders, 1995); OOC2,oyster, Crassostrea virginica, from OC2-Murrells Inlet (South Carolina) (Sanders, 1995); CFBR, catfish,Ictalurus nebulosus, from Black River (Ohio) (Vassilaros et al., 1982); CFBL, catfish, I. nebulosus, fromBuckeye Lake (Vassilaros et al., 1982); MFB, mussel, Mytills galloprovincialis, from Fort Brescou (France)(Baumard et al., 1998); FFB, fish, Serranus scriba, from Fort Brescou (France) (Baumard et al., 1998);FF, fresh fish, Pseudotolithus elongatus, from Lagos (Nigeria) (Akpan et al., 1994); SF, smoked fish,Pseudotolithus elongatus, from Lagos (Nigeria) (Akpan et al., 1994).bn.d. = not detected.cConcentration of chrysene + triphenylene.dConcentration of benzo(b)fluoranthene + benzo(j)fluoranthene + benzo(k) fluoranthene.eConcentration of dibenz(a,c)anthracene + dibenz(a,h)anthracene.

Table 8.4. PAH concentrations (µg kg−1 dry weight) in oyster, mussel, and fresh and smoked fishsamples.a



and processing, and finally on the cookingmethods. In spite of the differences found,there is general agreement that food isan important source of PAH exposure inhumans.

Uptake and Metabolism

Once PAHs have entered the body orally,they reach the intestine, where they can beabsorbed and distributed to other organsthrough enterohepatic circulation. Foodcomponents may alter the uptake of PAHs,either enhancing or reducing their absorptionand, as a result, potentiating or inhibitingtheir toxic effects. According to Stavric and

Klassen (1994), the adsorption of PAHsto some components of diet, such as thecarbon present in certain processed foods,can reduce their availability for absorption.These authors also observed that food poly-phenols such as quercetin and chlorogenicacid produce a reduction in the absorptionof benzo(a)pyrene and its metabolites,although to a lesser extent than carbon. Theformation of complexes with some food com-ponents can also lead to a reduction in thebioavailability of some PAHs ingested withfood. The solubility of PAHs in food ingestedalso plays an important role in their absorp-tion. Water, in which benzo(a)pyrene andother PAHs are not soluble, may reduce thetransfer to the intestinal mucosa, whereas‘oily’ foods, in which PAHs are soluble,


20-Feb-03 8

Compound E1 E2 M1 M2 B1 B2 FLF FFP

PhenanthreneAnthracene2-Methylphenanthrene2-Methylanthracene1-Methylphenanthrene9-MethylanthraceneFluoranthenePyrene1-MethylpyreneBenz(a)anthraceneChryseneTriphenyleneBenzo(b)fluorantheneBenzo(k)fluorantheneBenzo(j)fluorantheneBenzo(e)pyreneBenzo(a)pyrenePeryleneIndeno(1,2,3-cd)pyreneDibenz(a,h)anthraceneBenzo(ghi)peryleneAnthanthrene

––––––

0.1n.d.–

0.0n.d.–

0.40.0––

0.0––

n.d.n.d.–

18.529.7

––––1.25.5–4.55.6–3.54.5––7.5–8.74.71.2–

––––––

0.10.0–

0.0n.d.–

0.00.0–

n.d.0.0–

n.d.n.d.0.0–

3.00.5––––3.4

35.5–2.48.6–3.1

n.d.–

n.d.1.5–

n.d.n.d.n.d.–

––––––

1.00.0–

2.2n.d.–

0.60.1––

0.6––

1.00.0–

––––––

10.81.3–0.5

24.7–1.20.6––1.4––1.50.0–

168.035.415.27.3

14.42.1

119.0127.016.244.544.1c

29.841.9d

21.854.27.9

41.43.5e

35.514.9

4.50.71.10.10.7

n.d.b

1.91.8n.d.0.30.6c

n.d.n.d.d

n.d.0.1n.d.n.d.n.d.e

n.d.n.d.

aE1, egg samples studied by Lodovici et al. (1995); E2, egg samples studied by Husain et al. (1997) (datain µg kg−1 wet weight); M1, milk sample studied by Dennis et al. (1983); M2, cow milk sample studied byHusain et al. (1997) (data in µg kg−1 wet weight); B1, beef meat (Lodovici et al., 1995); B2 barbecued beefmeat (Lodovici et al., 1995); FLF, frankfurters grilled on a log fire (Larsson et al., 1983); FFP, frankfurtersgrilled in frying pan (Larsson et al., 1983).bn.d. = not detected.cConcentration of chrysene + triphenylene.dConcentration of benzo(j)fluoranthene + benzo(k)fluoranthene.eConcentration of dibenz(a,c)anthracene + dibenz(a,h)anthracene.

Table 8.5. PAH concentrations (µg kg−1 dry weight) in several foods of animal origin.a



facilitate this transfer (Stavric and Klassen,1994). It is clear from these findings thatthe uptake of PAHs from diet is influencedmarkedly by the composition of foods withwhich they are ingested. This could helpexplain the difficulty in establishing a corre-lation between the presence of PAHs in thediet and the development of cancer, sincethere could be food components exertingsome protective effect.

It is known that PAHs undergo metabolictransformation in the organism, which canresult either in the formation of active metabo-lites that can finally form covalent adductswith DNA, or in the formation of productswhich will be excreted further. Given thatadduct formation is considered the initialevent in chemical carcinogenesis, the forma-tion of active metabolites is considered tobe closely related to the carcinogenicity ofPAHs. As an example of the metabolic trans-formations undergone by PAHs, a model ofthe metabolic path of benzo(a)pyrene, includ-ing both activation and detoxification routes,is shown in Fig. 8.3. One of the most widelyaccepted approaches to explain the PAHbiotransformation process begins with acytochrome P450-mediated epoxidation of themolecule (see Fig. 8.3). This epoxidation iscatalysed by an enzyme complex calledmixed-function oxidase (MFO), which islocated in the endoplasmic reticulum ormicrosomal fraction. The second step involvesa hydroxylation process with the formationof diols, and is catalysed by a hydrase, theso-called epoxidohydrase (EH), which isclosely linked to the MFO enzyme complex.The enzyme complex including the hydraseis often referred to as an aryl-hydrocarbonhydroxylase (AHH). The diols formed canbe converted further into dihydrodiolepoxides. From a chemical and biologicalpoint of view, the dihydrodiol epoxides(especially those formed in the bay region) arevery reactive because they can attack criticalnucleophilic sites in DNA, either directly inan SN2 reaction or after forming a carbocationin an SN1 reaction (Guillén et al., 1997). Never-theless, the intermediate diols can alsoundergo a detoxification process by conjugat-ing with glucuronic acid or glutathione, lead-ing to conjugated metabolites, which can be

excreted by renal or biliary channels. It isworth noting that some authors (Jacob et al.,1995) make a distinction between phase Imetabolism, which includes the steps leadingto the formation of trans-dihydrodiols (diols),and phase II metabolism, which refers to thefurther reactions of the phase I metabolites.Metabolites of PAHs with two and three ringsare excreted preferentially in the urine, whilehigher molecular metabolites are released inthe faeces.

Ingested PAHs potentially can be metab-olized by the gut microflora, by the intestinalwall and by the liver. The intestinal epithe-lium contains all the enzymes which havebeen identified as being involved in activationand detoxification of PAHs, although theseactivities are generally much lower than in theliver (Benford and Bridges, 1985). Moreover,the low levels of inducible P450 isozymes in theintestinal tract could influence the occasionaldevelopment of tumours in the small andlarge intestine as a consequence of the inges-tion of PAH-containing food (Stavric andKlassen, 1994). Nevertheless, the resultingbiological activity of ingested PAHs is deter-mined not only by their degree of absorptionand metabolization, but also by the presenceof compounds which can act as inducers,promoters or inhibitors of the PAH metabo-lism by acting on enzymatic factors. Thus, theactivity of intestinal enzymes that metabolizePAHs into ultimate carcinogens may beinduced by drugs, certain vegetables, envi-ronmental pollutants such as polychlorinatedbiphenyls, and gastric hormones (Benfordand Bridges, 1985). The activity of thecytochrome P450-dependent-mono-oxygenas-es can also be induced by the PAHs them-selves. Among PAHs, benzo(a)pyrene and3-methylcholanthrene are the most studiedinducer agents. There are other PAHs which,despite their inability as inducer agents, canplay a role in carcinogenesis as promoters ofthe process initiated by other compounds(Jacob, 1996). There are also some dietary fac-tors such as certain flavones present in vegeta-bles which can promote or activate certaincytochrome P450-dependent reactions, includ-ing benzo(a)pyrene hydroxylation, in bothliver and intestine. On the contrary, food com-ponents such as antioxidants, certain flavones

20-Feb-03 8




PolycyclicA

romatic H

ydrocarbons185

20-Feb-03

8

Fig. 8.3. Metabolic path of benzo(a)pyrene and possible effects.

201





and vitamins A, C and E may inhibit PAHmetabolism (Benford and Bridges, 1985). Itshould be noted that some substances havebeen considered to act as both inducers andinhibitors.

It must be taken into account that mostof this information comes from experimentswith animals and with a limited number ofPAHs. Therefore, the extrapolation of theresults to different species, including humans,or from one compound to another, can some-times lead to erroneous conclusions and pre-dictions. Jacob et al. (1995), in an experimentwith embryonic epithelial lung cells from rats,hamsters and humans, found qualitative andeven quantitative similarities in the patternof primary (or phase I) metabolites in thecases of pyrene, benzo(a)pyrene, chryseneand anthanthrene, but significant differencesin the phase II metabolism. In another study,the same author (Jacob, 1996) obtained signifi-cant differences in the phase I metabolismof benz(a)anthracene with liver microsomesfrom human, rats, dogs, mice and rabbits.Considerable interindividual variations existin the metabolism and excretion of PAHs,regardless of the administration route con-sidered. Although the mechanism for such avariable response is unclear, it may be due tointerindividual differences in constitutive orinduced physiological mechanisms such asdigestion, absorption, metabolism or excre-tion. Consequently, each subject exhibitsan individual and invariant PAH metaboliteprofile, with a certain ratio of carcinogenic andnon-carcinogenic PAH metabolites, whichmay indicate an individual equipment ofPAH-metabolizing enzymes and a potentialpredisposition for cancer risk.

Toxicity and Carcinogenicity

PAH exposure is associated with manyadverse effects in experimental animals,including reproductive toxicity, cardiovascu-lar toxicity, bone marrow toxicity, immunesystem suppression and liver toxicity (Collinset al., 1998). In addition, teratogenic, muta-genic and carcinogenic properties havebeen reported for many PAHs (IARC, 1973,

1983). Some general considerations mustbe taken into account when evaluating thetoxicity and biological effects of PAHs: (i)studies have been carried out on animals, socare must be taken before extrapolation tohumans; (ii) most of the experiments involveadministration of PAHs by routes whichare not oral ingestion, considered the bestway to predict the possible effects of dietaryPAHs; and (iii) there are studies of thebiological effects of PAHs whose resultsare ambiguous or inconclusive, so furtherinvestigations would be necessary to reachdefinitive conclusions.

Non-carcinogenic effects

There are few studies regarding the effectsof oral exposure to PAHs. The results fromNousiainen et al. (1984) do not reveal acutetoxic effects in rats given 50 or 150 mgbenzo(a)pyrene kg−1 day−1 by gavage for 4consecutive days, except for alterations ofthe enzyme activity of the gastrointestinalmucosa and induction of hepatic carboxyl-esterase activity, which cannot be consideredadverse effects per se.

Reproductive toxicity of PAHs has beenreported. In general, the reproductive effectsof benzo(a)pyrene, the best documented PAH,include resorptions, malformations, stillbirthsand decreased fertility in the progeny. This isbecause active metabolites can cross the pla-centa and reach the fetuses of orally exposedanimals. The inducibility of the cytochromeP450 system of the animals has been shownto be related directly to the embryotoxicityof PAHs. The dose orally administered alsoseems to have an effect on the embryotoxiceffects observed. Thus, in a group of pregnantCD-1 mice treated with benzo(a)pyrene dur-ing gestation, a marked reduction in theviability of litters from individuals exposedto the highest dose was found (Mackenzieand Angevine, 1981). An embryotoxic effect inrats has also been observed for dibenz(a,h)-anthracene when given in high doses (IARC,1983). More recently, an ovotoxic effecthas been reported for 9,10-dimethylbenz(a)-anthracene, 3-methylcholanthrene and benzo-

20-Feb-03 8




(a)pyrene. These compounds administeredintraperitoneally produced the destruction ofprimordial follicles in mice and rats whengiven in repeated low doses, so they might berelated to the early menopause seen in womenexposed to cigarette smoke (Borman et al.,2000).

Immunotoxic effects have also beenfound in rats after oral administration ofbenzo(a)pyrene, affecting bone marrow, thy-mus, spleen and lymph nodes. Davila et al.(1996) examined the toxic effects of nine differ-ent PAHs on human peripheral blood T-cellmitogenesis. They found that benzo(a)pyrene,3-methylcholanthrene and 7,12-dimethylbenz-(a)anthracene were highly immunotoxic in thehuman system, while dibenz(a,c)anthraceneand dibenz(a,h)anthracene were of intermedi-ate toxicity, 9,10-dimethylanthracene, benzo-(e)pyrene and benz(a)anthracene were mildlyimmunotoxic, and anthracene had no measur-able toxicity at the concentrations tested.

Genotoxicity

Many PAHs show mutagenic activity to Sal-monella typhimurium and even to mammaliancells in vitro in the presence of an exogenousmetabolic system (IARC, 1973, 1983),although this activity is not always related tothe production of tumours. However, it mustbe noticed that some PAHs which have beenfound to be mutagenic are also active as initi-ators in the mouse skin initiation–promotionassay, so their influence cannot be ruled outwhen evaluating the biological effects ofmixtures of PAHs. Fluoranthene or coronenecan be cited as examples.

In addition to mutagenic activity, somePAHs can also induce unscheduled DNAsynthesis, sister chromatid exchange, mor-phological transformation or chromosomalaberrations in mammalian cells either inculture or in vivo (IARC, 1973, 1983).

Carcinogenicity

In spite of the variety of the toxic effectsrelated to PAHs, the one of most concern is

cancer. Many PAHs have been shown tobe carcinogenic to experimental animals bydifferent administration routes but, as men-tioned previously, there are not many studiesconcerning oral administration. Most of theselatter have been carried out with benz(a)-anthracene, dibenz(a,h)anthracene and benzo--(a)pyrene, resulting in hepatomas, lung ade-nomas, squamous papillomas, forestomachpapillomas and carcinomas, and stomachtumours. There are several factors whichhave an influence on the effects observed.These factors can be external, such as the doseof the PAHs administered, the administrationroute, the vehicle supporting the PAHs,the presence of several PAHs and the fre-quency of exposure, or individual, such asage, sex, genetic factors and nutritional sta-tus. The dose administered can determine theextent of the carcinogenic effects observed.Goldstein et al. (1998), in a 2-year feedingexperiment with mice, observed that animalsgiven 17.5 µg benzo(a)pyrene day−1 did notdevelop tumours, whereas doses of 350 µgbenzo(a)pyrene day−1 produced forestomach,oesophagus and tongue tumours. The admin-istration route can also lead to differencesin the carcinogenic response of experimentalanimals. The response of rats or hamsters tobenzo(a)pyrene administered orally is small,even though they rapidly develop skintumours after skin application. It has alsobeen observed that when benzo(a)pyrene isadministered to rats by gavage in a specificsolution, a higher tumorigenic response isobserved than when benzo(a)pyrene is givenwith the diet. This could be a result of theprotective effect exerted by some compo-nents of the diet which can interact withthe absorption and metabolism of PAHs. Itmust be noticed that there are some PAHswhich, in spite of their inability to producetumours per se, contribute to increasing theincidence of some types of tumours producedby complete carcinogenes such as benzo-(a)pyrene when administered with them.These compounds include benzo(ghi)-perylene, fluoranthene and pyrene (IARC,1973, 1983). These two latter deserve specialattention for the purposes of risk assessment,because of their wide distribution in the envi-ronment and in foods. Some studies have


20-Feb-03 8



revealed that the frequency of administrationalso has an influence on the toxic effect of acertain PAH. Qing et al. (1997) split a 1 mgdosage of dimethylbenz(a)anthracene givenonce a week for 6 weeks into five daily dosesof 200 µg given intragastrically to femaleSENCAR mice each week for 6 weeks, andfound that the toxicity was higher. It mustalso be said that oral administration of PAHsto young individuals can result in a higherincidence of tumours than treatment at otherages, showing the greater sensitivity of infantanimals to carcinogens compared with adultsof the same species (Lijinski, 1991).

To get an overall view of the carcinoge-nicity of PAHs, it can be said that, in general,the hydrocarbons with fewer than four fusedrings are non-carcinogenic, although thereare some methyl derivatives such as 9,10-dimethylanthracene and 1,2,3,4-tetramethyl-phenanthrene which are carcinogens ofmoderate potency. The compounds contain-ing four fused rings are non-carcinogenic(triphenylene, naphthacene and pyrene) orweakly carcinogenic (benz(a)anthracene andchrysene). However, as with the three-ringcompounds, methyl substitution in some ofthese compounds gives rise to hydrocarbonsof very great carcinogenic potency, althoughall monomethyl compounds are not equal andthe carcinogenicity depends to a large extenton the position of substitution in the molecule,leading to products as lacking in carcinogenicactivity as the parent hydrocarbon or to com-pounds of great carcinogenic potency. As faras the five-ring compounds are concerned,these exhibit a varying carcinogenic potency,ranging from the non-carcinogenic picene,pentacene, perylene and benzo(e)pyrene tothe potent benzo(a)pyrene and dibenz(a,h)-anthracene, including also weak carcinogens,such as dibenz(a,c)anthracene. Most of thesix-ring hydrocarbons examined are carcino-genic, although there are some of them, suchas benzo(ghi)perylene, which are non-carcinogenic. Coronene, a seven-ring hydro-carbon, is also non-carcinogenic (Lijinsky,1991). The great differences observed amongPAHs in relation to their carcinogenic activitycan be explained because some characteristicsare necessary both in the parent PAHs and intheir metabolites to form adducts with DNA,

which, as has been mentioned, represents theinitial event in chemical carcinogenesis.

One of the first mechanisms proposedfor the formation of active intermediatesinvolved the formation of simple K regionepoxides (see Fig. 8.3). However, it was laterrecognized that nucleic acid adducts formedwith K region epoxides were not identified inthose formed in tissues treated with the parentPAHs (Shaw and Connell, 1994). Further-more, Jacob (1996) pointed out that themetabolic activation at the K region mainlyresults in non-toxic and non-carcinogenicmetabolites and hence plays a role in detoxifi-cation. It is now admitted that PAHs areactivated mainly by the formation of vicinaldiol-epoxides and that, in most cases, thediol-epoxides are formed adjacent to a bayregion (see Fig. 8.3). The existence of a bayregion in the molecule has been considered formany authors as a prerequisite for carcino-genic activity, because the bay region-deriveddihydrodiolepoxides exhibit the most pro-nounced tendency to form carbonium ionsand turn out to be the most reactive. (The bayregion activation mechanism of benzo(a)-pyrene is marked in Fig. 8.3 by means of dot-ted lines.) However, this characteristic alonedoes not predict the carcinogenicity of a PAH.It has been found that methyl substitution atthe peri position reduces carcinogenic activityby blocking distal bay region diol formation(Loew et al., 1985). The other main region ofactivity in PAH molecules which can also playa role in the carcinogenicity of some PAHs isthe L region, which features localization of πelectrons across para positions, e.g. the 7,12positions of benz(a)anthracene. The presenceof an L region in a PAH was recognized asbeing responsible for the absence of carcino-genic activity in certain PAHs. However,substitution at this region can enhance thecarcinogenicity of the unsubstituted PAH,such as in the case of 7,12-dimethylbenz-(a)anthracene, which is a potent carcinogencompared with benz(a)anthracene (Shaw andConnell, 1994). Nevertheless, there are manyother factors such as metabolic, stereochem-ical and conformational factors, as well as thebiological reactivity of the metabolites,which contribute to the marked differencesin tumorigenicity of various PAHs. As an

20-Feb-03 8




example, in the case of picene, quantummechanical calculations predicted its carcino-genicity, but none has been detected in severalanimal studies. This absence could resultfrom the inability of microsomal enzymesto transform its M region dihydrodiol todihydrodiol bay region epoxides in sufficientamounts to initiate carcinogenesis (Platt et al.,1988).

In spite of the role of PAH–DNA adductsin carcinogenesis, it must be pointed out thatthe formation of PAH–DNA adducts doesnot necessarily imply the development oftumours, since, as shown in Fig. 8.3, the dam-aged template can be repaired before cell rep-lication has occurred (Shaw and Connell, 1994).

Despite the fact that the bay region theoryexplains the carcinogenicity of many PAHs,there are others which, although tumorigenic,do not have bay regions or it has been shownthat they are not activated via a bay regionepoxide. Thus, Cavalieri and Rogan (1985)suggested an alternative hypothesis to explainactivation of PAHs, based on a one-electronoxidation that yields a reactive radical cationintermediate which acts as an ultimatecarcinogen. Flesher and Myers (1991) alsodeveloped some rules of molecular geometryfor predicting the carcinogenic activity ofunsubstituted PAHs. Other approaches havecorrelated carcinogenicity with a superde-localizability index, which represents thepotential reactivity at the bond adjacent to thebay region in the dihydrodiol intermediate(Berger et al., 1978).

The degree of carcinogenic activity ofPAHs has been expressed by different codesor indexes. The IARC (International Agencyfor Research on Cancer) has defined catego-ries which refer only to the strength of the evi-dence that an exposure is carcinogenic, andnot to the extent of its carcinogenic activity(potency) nor to the mechanism involved.Therefore, this is a classification which maychange as new information becomes avail-able. The evidence relevant to carcinogenicityis classified into four categories: sufficientevidence of carcinogenicity (SE), limitedevidence of carcinogenicity (LE), inadequateevidence of carcinogenicity (IE) and evidencesuggesting lack of carcinogenicity (LC). Eachcategory is defined in a different way

depending on whether the studies are carriedout in humans or experimental animals.Taking into account not only the strength ofthe evidence derived from studies in humans,but also studies in experimental animals andother relevant data, the IARC categorize car-cinogens in five groups, as indicated in Table8.6: 1 (carcinogenic to humans), 2A (probablycarcinogenic to humans), 2B (possibly carci-nogenic to humans), 3 (unclassifiable as tocarcinogenicity to humans) and 4 (probablynot carcinogenic to humans). Group 1 is usedwhen there is SE in humans, or when there isless than SE in humans but SE in experimentalanimals, with strong evidence in exposedhumans that the agent acts through a relevantmechanism of carcinogenicity. Group 2A isused when there is LE in humans and SEin experimental animals, or when there is IEin humans and SE in experimental animalsand strong evidence that the carcinogenesis ismediated by a mechanism that also operatesin humans. Group 2B is used when there is LEin humans and less than SE in experimentalanimals, or when there is IE in humans butSE in experimental animals. Group 3 is usedmost commonly when there is IE in humansand IE or LE in experimental animals, orwhen there is IE in humans but SE inexperimental animals, with strong evidencethat the mechanism of carcinogenicity inexperimental animals does not operate inhumans. Finally, group 4 is used when there isLC in humans and in experimental animals. Itis worth pointing out that there is no PAHincluded in group 1.

As well as the classification of thecarcinogenic activity of PAHs given by theIARC, there are other ways to express thedegree of carcinogenicity of the differentPAHs, such as the Badger index, in whichcarcinogenicity ranges from (−) to (++++),the carcinogenic scale proposed by Cavalieriand co-workers, who characterize carcinoge-nicity from (−) to (+++++) (Cavalieri et al.,1983), or the less commonly reported Iballindex I (Braga et al., 1999). This latter is propor-tional to the fraction of subject animals thatshow a carcinogenic response divided bythe mean latent period. Carcinogenicity dataaccording to the scale of Cavalieri andco-workers and to the Iball index for a group


20-Feb-03 8



of PAHs are presented in Table 8.6. In addi-tion, it is worth mentioning some parametersrelated to the carcinogenicity of PAHs which

express carcinogenic potencies relative to thatof benzo(a)pyrene; these are relative potencies(RPs) and toxicity equivalency factors (TEFs)

12-Mar-03 8


Compound Carcinogenicity RPs TEFs TEFs PEFs

NaphthalenePhenanthreneAnthraceneFluoranthenePyreneBenz(a)anthraceneChryseneTriphenylene1-Methylchrysene2-Methylchrysene3-Methylchrysene4-Methylchrysene5-Methylchrysene6-Methylchrysene7,12 Dimethylbenz(a)anthraceneBenzo(b)fluorantheneBenzo(j)fluorantheneBenzo(k)fluorantheneBenzo(a)pyreneBenzo(e)pyrenePeryleneDibenz(a,c)anthraceneDibenz(a,h)anthraceneDibenz(a,j)anthraceneIndeno(1,2,3-cd)pyreneBenzo(ghi)peryleneAnthanthreneCyclopenta(cd)pyreneCoroneneDibenzo(a,e)pyreneDibenzo(a,h)pyreneDibenzo(a,i)pyreneDibenzo(a,l)pyrene

n.d.a,b

IELCNENESELEIEIELELELESELEn.d.

SESESESEIEIELESELESEIELELEIESESESESE

n.d.c

3333

2A333333

2B3

n.d.

2B2B2B2A333

2A3

2B3333

2B2B2B2B

–d

––

n.d.–±±–

n.d.n.d.n.d.n.d.+++

±+++++

n.d.n.d.n.d.

++++––+

++++

n.d.–±––

+++++++++++

+++++

n.d.e

00n.d.n.d.n.d.070500n.d.n.d.n.d.n.d.n.d.n.d.n.d.

n.d.n.d.n.d.7202n.d.032604n.d.n.d.n.d.n.d.n.d.50687433

n.d.f

n.d.n.d.n.d.0.0810.1450.0044n.d.n.d.n.d.n.d.n.d.n.d.n.d.n.d.

0.1410.0610.0661.00.004n.d.n.d.1.11n.d.0.2320.0220.3200.023n.d.n.d.n.d.n.d.n.d.

0.001g

0.0010.010.0010.0010.10.01n.d.n.d.n.d.n.d.n.d.n.d.n.d.n.d.

0.1n.d.0.11.0n.d.n.d.n.d.5.0n.d.0.10.01n.d.n.d.n.d.n.d.n.d.n.d.n.d.

n.d.h

n.d.n.d.n.d.n.d.0.0140.026n.d.n.d.n.d.n.d.n.d.n.d.n.d.n.d.

0.11n.d.0.0371.0n.d.n.d.n.d.0.89n.d.0.0670.012n.d.n.d.n.d.n.d.n.d.n.d.n.d.

n.d.i

n.d.n.d.n.d.n.d.0.10.01

n.d.n.d.n.d.n.d.n.d.1.0

n.d.n.d.

0.10.10.11.0n.d.n.d.n.d.n.d.n.d.0.1

n.d.n.d.n.d.n.d.1.0

101010

an.d. no data.bIARC (1973, 1983), evidence of carcinogenicity in experimental animals (SE: sufficient evidence ofcarcinogenicity; LE: limited evidence; IE: inadequate evidence; LC: evidence suggesting lack ofcarcinogenicity; NE: no evidence that it is carcinogenic per se to experimental animals).cIARC, overall evaluation (1: carcinogenic to humans; 2A: probably carcinogenic to humans; 2B: possiblycarcinogenic to humans; 3: unclassifiable as to carcinogenicity to humans; 4: probably not carcinogenicto humans).dData from Cavalieri et al. (1983): extremely active, +++++ ; very active, ++++ ; active, +++; moderatelyactive, ++ ; weakly active, +; very weakly active, ±; inactive, –.eIball index.fRP, relative carcinogenic potencies. Adapted from Krewski et al. (1989) (Collins et al., 1991).gFrom Nisbet and LaGoy (1992) (Collins et al., 1998).hFrom Muller et al. (1997) (Thomson and Muller, 1998).i From the Office of Environmental Health Hazard Assessment (OEHHA) (Collins et al., 1998).

Table 8.6. Carcinogenicity and other parameters related to carcinogenicity of PAHs.



or potency equivalency factors (PEFs). Theseparameters have been established to assign acarcinogenic activity to PAHs with unknowncancer potency values, and they are usedto estimate quantitatively the cancer riskassociated with exposure to PAHs. Someauthors (Collins et al., 1998) suggest the useof PEFs instead of TEFs, since nearly all PEFsfor PAHs are based on cancer bioassay infor-mation and they do not take into accountother studies such as acute toxicity deter-minations, structure–activity relationshipsor short-term tests such as AHH induction.TEFs or PEFs are usually indexed at incre-ments of a factor of 10. Data relative to the RP,TEFs and PEFs of a group of PAHs can be seenin Table 8.6.

Risk Assessment

According to the EPA, risk assessment is theprocess that scientists and governmentofficials use to estimate the increased risk ofhealth problems in people who are exposedto different amounts of toxic substances.The assessment of human health effectsassociated with exposure to chemical carcino-gens is normally performed in four stages:(i) hazard identification (what healthproblems are caused by the toxic substance);(ii) exposure assessment (how much entersthe body); (iii) dose–response assessment(the health problems at different exposures);and (iv) risk characterization (the extrarisk to health). Combining the results of theexposure assessment and the dose–responseassessment gives an estimate of the increased

lifetime risk of cancer for an individualexposed to the maximum predicted long-term concentration.

Figure 8.4 shows the main monitoringlevels in the risk assessment of PAHs. The firstapproach to evaluating the risk associatedwith dietary exposure to PAHs is based onthe measurement of PAH levels in foodstuffs,since high concentrations of PAHs in ingestedfood will probably result in adverse effectsfor human health. However, this analysis ofexternal exposure, although very useful, doesnot take into account either the absorption,metabolism, distribution or excretion mecha-nisms of PAHs or interindividual differencesrelative to these mechanisms, which clearlydetermine the final effects of PAHs. Bio-markers can be used as a means of obtaininginformation on an individual’s internalexposure to a xenobiotic or on the actual orpotential effects of that exposure. Biomarkersare parameters that can be evaluated quantita-tively, semi-quantitatively or qualitativelyin body fluids, cells or tissues. In general,biomarkers can be classified as: biomarkersof exposure, which reflect dose of toxic agents;biomarkers of effect, which indicate bio-logical response to exposure with potentialtoxicological implications; and biomarkersof susceptibility, which provide informationabout the intrinsic sensitivity of an individualto the toxic agent. As examples of the differenttypes of biomarkers, PAH metabolites inurine, DNA and protein adducts, mutationsor chromosomal aberrations can be cited, andthey provide information about differentstages of the process comprised betweenexposure to PAHs and development ofmalignant effects.


20-Feb-03 8

Fig. 8.4. Different levels of monitoring for risk assessment.



Biomarkers for PAHs

The most widely used biomarkers for dietaryexposure to PAHs are measurement ofPAH–DNA adducts, generally in nucleatedwhite blood cells, and PAH metabolitesexcreted in urine, but protein adducts alsoappear as a promising tool to assess PAHexposure. Table 8.7 summarizes the main fea-tures of the biomarkers most used for PAHs.

Among all the PAH metabolites studied,1-hydroxypyrene (1-OHP), which can bedetermined by high-performance liquid chro-matography (HPLC) with fluorescence detec-tion (FL) or by gas chromatography–massspectrometry (GC–MS), has been consideredas the preferred biomarker for routine assess-ment of exposure to PAHs. It must be noticedthat, although 1-OHP is used preferentially asan indicator for PAH exposure at workplaces,it can also be used for exposure to dietaryPAHs. Thus, increased levels of urinaryexcreted 1-OHP have been observed inhumans after consumption of grilled meat(Van Maanen et al., 1994). Moreover, a sig-nificant correlation between urinary 1-OHPand peripheral blood PAH–DNA adducts hasbeen found in a study of dietary exposure toPAHs (Kang et al., 1995). Neverthelss, in spiteof the sensitivity and ease of measurementof 1-OHP, a potential disadvantage of urinebiomarkers is that, in general, they only reflectrecent exposure. Besides, effective biologicalmonitoring based on the determination of1-OHP requires an understanding of its excre-tion kinetics and it must be used for exposure

assessment of homogeneous groups, sincethe relative proportion of pyrene in complexmixtures of PAHs can vary among differentsources.

PAH–DNA adducts provide informationon the molecular or biologically effective doseof PAHs reaching a critical target, which canbe considered as an integration of internalexposure and metabolism. Many investiga-tions have been focused on the study of therelationship between PAH exposure and for-mation of DNA adducts, but contradictoryresults have been obtained. Thus, the resultsfrom studies in which dietary exposure toPAHs has been examined, reveal that theconcentration of PAH–DNA adducts in bloodcells after controlled consumption of char-grilled beef increased only in some subjects(Kang et al., 1995). On the contrary, adose-dependent response to PAH ingestionwas observed in a feeding study carried outby Van Maanen et al. (1994). The discrepancybetween results could be due to the greatvariability observed in the response of differ-ent individuals, to some difficulties in DNAadduct measurement by agent-specific imm-unoassays or by non-agent specific 32P-post-labelling assay, and to the kinetics of forma-tion and elimination of PAH–DNA adducts.The level of DNA adducts correlates directlywith the concentration of the carcinogen inthe diet when the rate of adduct formation iscompensated by the rate of adduct removal(steady state), whereas this correlation failsif adduct measurement is made after thisperiod. Finally, it must be noticed that the

20-Feb-03 8


Biomarker Characteristics Determination

1-Hydroxypyrene

DNA adducts

Protein adducts

Effective as biomarker of PAH recent exposureSensitive and easy to determineCertain correlation with DNA adductsValid for homogeneous groups of exposureNo clear relationship with cancer riskEffective as biomarker of internal exposureLimited usefulness as biomarker of effectNo clear relationship with tumour inductionAffected by a great variabilityEffective to assess longer exposureGreat accessibility and stabilityNo clear relationship with cancer risk

HPLC-FLGC–MS

32P-post-labellingImmunochemical methods

GC–MSImmunoassaysHPLC-UV/FL

Table 8.7. Main characteristics of the most-used biomarkers for PAHs.



persistence of biomarkers measured in bloodcells, such as PAH–DNA adducts, is deter-mined by the lifetime of the cell types chosenfor their analysis.

It can be said that, currently, the presenceof a DNA adduct in human tissue indicatesthat exposure has occurred and, in some cases,how much exposure there has been. However,an association between adduct formation andcancer risk has not yet been shown for PAHs.An attempt to correlate tumour induction andadduct formation was made by Goldstein et al.(1998), who directly compared both para-meters in mice given benzo(a)pyrene and coaltars, either orally or intraperitoneally. DNAadducts were found in both tumours andtumour-free tissue, but neither quantitationof total DNA adducts nor quantitation ofthe DNA adducts formed by benzo(a)pyrenecould predict the development of tumours.Moreover, the data indicated significantdifferences for tumour induction by benzo(a)-pyrene compared with coal tars.

At present, no clear and accepted modelsof risk assessment on the basis of DNA adductlevels are available. However, it must be saidthat the relationship between DNA adductformation and human cancer has been eluci-dated in the case of tobacco smoking and lungcancer (Poirier et al., 2000).

The formation of adducts of PAHs withproteins, generally haemoglobin and serumalbumin, is considered to be a valuable surro-gate for DNA adduct formation, since manychemical carcinogens bind to both DNA andprotein in blood with similar dose–responsekinetics (Poirier et al., 2000). The main reasonsfor the use of protein adducts in biochemicaleffect monitoring are their relative easyaccessibility of target tissues and their relativestability in comparison with DNA adducts,which constantly undergo repair. Becauseof this stability, protein adducts tend torepresent exposure over the life of the tissuemonitored (Shaw and Connell, 1994). Thelevel of protein adducts, which can bedetermined by GC–MS, immunoassays orHPLC with UV or FL detection, has beenfound to be directly proportional to the dailycarcinogen dose at steady state and is typicallylinear over a large dose range (Poirier et al.,2000).

Protein adducts have been employedas biomarkers for many human exposuresincluding tobacco-related, workplace andmedicinal (psoriasis) PAHs (Poirier et al.,2000). However, there are few studies onthe correlation between protein adductsand cancer risk and, besides, protein adductsare less well accepted than DNA adducts asindicators of carcinogenic potential.

It seems that, in the future, approaches tocancer risk assessment will take into accountnot only a single biomarker, but the results ofa battery of biomarker tests, including DNAadduct and protein adduct analyses.

Quantitative Risk Assessment

There are very few studies in which aquantitative risk assessment from exposureto dietary PAHs has been achieved, and theyare based on an extrapolation of the cancerpotency of a single PAH, benzo(a)pyrene,from animal studies to humans. This can beexplained by the lack of information relativeto the carcinogenicity of PAHs in humans.The wide range and the high variability ofthe data used in the risk assessment of PAHsfrom dietary sources result in different cancerpotency estimates, which makes it difficult tomake an accurate estimation of cancer risk.The largest data set currently available isthat obtained by Neal and Rigdon (1967) in adietary study with mice. However, althoughthese data have been used together withthose of Thyssen et al. (1981) by the EPA forrisk assessment, their use for quantitativelow-dose extrapolation results in some uncer-tainty in the determination of the cancerpotency factor, which makes it difficult toobtain a reliable quantitative risk assessment(Collins et al., 1991). More recently, humancancer potency figures for oral exposure toPAHs have been derived from inhalationpotencies of coke oven emissions (Thomsonand Muller, 1998), assuming that the relativepotency of PAHs by oral and inhalationroutes in humans and rodents is similar.

One proposed method for establishingquantitative risk estimates for PAH mixturesis based on the use of the TEFs or PEFs


20-Feb-03 8



mentioned above. In a TEF approach forPAHs, overall potency of the mixture isexpressed relative to benzo(a)pyrene, andcontributions of individual carcinogenicPAHs are taken to be additive (Goldstein et al.,1998). No risk assessments for oral exposure toPAHs have been reported using this method-ology, except for that of Thomson and Muller(1998). These authors tried to estimate the can-cer risk from dietary sources of PAHs by usingfour different approaches. Two methods werebased on the sum of risk from individualPAHs, using cancer potency values fromrodent studies or from human inhalation data.The other two used benzo(a)pyrene as a surro-gate, either representing a proportion of therisk from the total mixture, or being assigned apotency representative of the PAH mixture asa whole. The differences among the resultsmay be due to the assumptions that eachapproach involves, to the number of PAHsincluded or to the data used to estimate cancerpotencies. These authors also suggest that anassessment based on benzo(a)pyrene as a sur-rogate for the potency of the PAH fraction ofan orally administered mixture is more realis-tic than an assessment based on summing therisk from a limited group of identified PAHs.However, there are other authors (Goldsteinet al., 1998) who have questioned both theaccuracy of risk assessment based onbenzo(a)pyrene and the use of TEFs.

Risk Management

Risk management takes the information gen-erated in the risk assessment and translates itinto a policy decision. It must be pointed outthat, to the best of our knowledge, no riskmanagement of the ingestion of PAH-containing foods exists. Instead, consideringthe adverse effects associated with exposureto these compounds, some measures havebeen suggested to reduce the intake of PAHs.The first goal in reducing the levels ofingested PAHs would be the reduction ofenvironmental PAH pollution, which isresponsible for the contamination of manyfood sources such as vegetables or marineorganisms. Efforts should be made to avoid

PAH contamination of foods during theirprocessing and cooking. To this end, somemeasures can be implemented, involvingsome of the most contaminated foodstuffs.The contamination of vegetable oils can bereduced by avoiding the contamination ofseeds during processes such as drying, andby removing PAHs during refinement.Deodorizing removes some of the lightPAHs, whereas the use of activated carbonduring the bleaching step can have asignificant effect on heavy PAH reduction.Contamination of oils and fats can also beavoided by purifying extraction solvents byfiltration through silica gel, or by usingnon-hydrocarbon solvents. With regard tosmoked foods, a rigorous control of thesmoking process and the use of liquid smokescould give rise to lower PAH concentrationsin the products. To reduce the PAH contentof grilled or broiled meat and fish, the useof foods with lower proportions of fat andthe control of the cooking temperature arerecommended. The contamination of grilledfood with PAHs can also be minimized byusing charcoal as fuel, by avoiding openflames and by special grill constructions thatprevent the fat from dripping on to the heatsource. In the case of vegetables, washingthem in water before an adequate processingcan help reduce the levels of environmentalPAHs, especially those of the heavy PAHs(Larsson and Sahlberg, 1981). However,despite all the measures suggested, it is clearthat, at the moment, the environmental PAHload constitutes an important source ofcontamination for some raw materials usedin the food industry. Therefore, effortsshould be made to avoid ingredients fromvery polluted areas.

Legislation

The most outstanding feature concerning theregulation of PAHs in foods is the lack ofmeasures to limit or avoid the presence ofthese compounds, shown to be detrimentalfor human health. In fact, few countries haveestablished a limit for PAHs in foodstuffs.Germany has limited the benzo(a)pyrene

11-Mar-03 8




content of smoked meat to 1 µg kg−1. Thislimit was adopted subsequently in Austriaand Poland. In 1988, the European Unionestablished a maximum limit of 0.03 µgkg−1 of benzo(a)pyrene in food as a result ofthe use of smoke flavourings. In Finland,smoking additives must have a benzo(a)-pyrene concentration lower than 30 µgkg−1, and their incorporation into foods islimited to 0.5 g kg−1. The use of liquid smokesin foods traditionally subjected to smokinghas been authorized by the FAO/WHOJoint Committee on Food Additives, pro-vided that the benzo(a)pyrene content doesnot exceed 10 µg kg−1. In Germany, the Ger-man Society for Fat Science (DGF) has pro-posed a value of 5 µg kg−1 as the limit valuefor heavy PAHs and a value of 25 µg kg−1 forthe sum of both light and heavy PAHs inrefined fats and oils. The ‘Czech guidelinesfor additives and contaminants in foods’proposal from the Czech Republic includeslimiting values of 2 µg kg−1 for each PAHenumerated (no light PAHs are involved)and 20 µg kg−1 for total PAHs in oils, fats andoil products. Recently, the Spanish govern-ment has established limiting values of2 µg kg−1 for each heavy PAH enumeratedand 5 µg kg−1 for total heavy PAHs in olivepomace oil. However, no legislation existsregarding either benzo(a)pyrene or otherPAH levels in other types of food.

Conclusions

PAHs constitute a group of environmentalcontaminants including compounds with dif-ferent degrees of carcinogenicity. They arealso widespread in foods as a result of bothenvironmental pollution and inadequate pro-cessing and cooking. Since toxic and carcino-genic effects after oral administration of someof these compounds have been demonstratedin experimental animals, their presence infoods should be avoided and controlled.Consequently, the use of reliable methodsthat allow the accurate determination ofPAHs in foodstuffs as a first estimate ofhuman exposure is encouraged. Moreover,it would be valuable to develop screening

methods usable in routine analysis whichcould help estimate the levels of PAHs in dif-ferent foodstuffs. The measurement of certainparameters, known as biomarkers, in bodyfluids, cells or tissues could also constitutea very useful tool for assessing exposure toPAHs or even for identifying early biologicaleffects which can lead further to the develop-ment of cancer; however, much work remainsto be done before establishing clear correla-tions between biomarkers and cancer risk.Although some attempts have been made toestimate the risk derived from ingestion ofPAHs, the difficulty in evaluating dietaryPAH intake and the lack of accurate dataregarding carcinogenic potency of PAHs byoral exposure make it difficult to estimatereliably the cancer risk of ingested PAHs.Finally, even though epidemiological studiespoint to the contribution of PAHs to humancancer, legal dispositions concerning the reg-ulation of PAHs in foods are very scarce.Besides, they refer only to very concretegroups of foods and to a single PAH,benzo(a)pyrene, although there are otherPAHs with equal or greater carcinogenicpotential. Consequently, considerable effortsmust be made to avoid PAH contaminationof foods, to ensure that regulatory directivesare adhered to and human health effects areminimized.

References

Akpan, V., Lodovici, M. and Dolara, P. (1994)Polycyclic aromatic hydrocarbons in fresh andsmoked fish samples from three Nigeriancities. Bulletin of Environmental Contaminationand Toxicology 53, 246–253.

Baumard, P., Budzinski, H., Garrigues, P.,Sorbe, J.C., Burgeot, T. and Bellocq, J. (1998)Concentrations of PAHs (polycyclic aromatichydrocarbons) in various marine organisms inrelation to those in sediments and to trophiclevel. Marine Pollution Bulletin 36, 951–960.

Benford, D.J. and Bridges, J.W. (1985) Carcinogenicpolycyclic aromatic hydrocarbons in food. In:Gibson, G.G. and Walker, R. (eds) Food Toxicol-ogy – Real or Imaginary Problems? Taylor andFrancis, London, pp. 152–166.

Berger, G.D., Smith, I.A., Seybold, P.G. andServe, M.P. (1978) Correlation of an electronic


20-Feb-03 8



reactivity index with carcinogenicity in poly-cyclic aromatic hydrocarbons. TetrahedronLetters 3, 231–234.

Bjorseth, A. (1983) Handbook of Polycyclic AromaticHydrocarbons. Marcel Dekker, New Yorkpp. 709–718.

Borman, S.M., Christian, P.J., Sipes, I.G. and Hoyer,P.B. (2000) Ovotoxicity in female Fischer ratsand B6 mice induced by low-dose exposureto three polycyclic aromatic hydrocarbons:comparison through calculation of an ovotoxicindex. Toxicology and Applied Pharmacology 167,191–198.

Braga, R.S., Barone, P.M.V.B. and Galvao, D.S.(1999) Identifying carcinogenic activity ofmethylated polycyclic aromatic hydrocarbons(PAHs). Journal of Molecular Structure(Theochem) 464, 257–266.

Cavalieri, E. and Rogan, E. (1985) Role of radical cat-ions in aromatic hydrocarbon carcinogenesis.Environmental Health Perspectives 64, 69–84.

Cavalieri, E.L., Rogan, E.G., Roth, R.W., Saugier,R.K. and Hakam, A. (1983) The relationshipbetween ionization potential and horseradishperoxidase/hydrogen peroxide-catalyzedbindings of aromatic hydrocarbons to DNA.Chemico-Biological Interactions 47, 87–109.

Collins, J.F., Brown, J.P., Dawson, S.V. and Marty,M.A. (1991) Risk assessment for benzo(a)pyrene. Regulatory Toxicology and Pharmacology13, 170–184.

Collins, J.F., Brown, J.P., Alexeeff, G.V. and Salmon,A.G. (1998) Potency equivalency factors forsome polycyclic aromatic hydrocarbons andpolycyclic aromatic hydrocarbon derivatives.Regulatory Toxicology and Pharmacology 28,45–54.

Dabestani, R. and Ivanov, I.N. (1999) A compila-tion of physical, spectroscopic and photo-physical properties of polycyclic aromatichydrocarbons. Photochemistry and Photobiology70, 10–34.

Davila, D.R., Romero, D.L. and Burchiel, S.W. (1996)Human T cells are highly sensitive to suppres-sion of mitogenesis by polycyclic aromatichydrocarbons and this effect is differentiallyreversed by α-naphtoflavone. Toxicology andApplied Pharmacology 139, 333–341.

Dennis, M.J., Massey, R.C., McWeeny, D.J. andKnowles, M.E. (1983) Analysis of polycyclicaromatic hydrocarbons in UK total diets. Foodand Chemical Toxicology 21, 569–574.

Dennis, M.J., Massey, R.C., Cripps, G., Venn, I.,Howarth, N. and Lee, G. (1991) Factors affect-ing the polycyclic aromatic hydrocarbon con-tent of cereals, fats and other food products.Food Additives and Contaminants 8, 517–530.

Flesher, J.W. and Myers, S.R. (1991) Rules of mol-ecular geometry for predicting carcinogenicactivity of unsubstituted polynuclear aromatichydrocarbons. Teratogenesis, Carcinogenesis,and Mutagenesis 11, 41–54.

Goldstein, L.S., Weyand, E.H., Safe, S., Steinberg,M., Culp, S.J., Gaylor, D.W., Beland, F.A. andRodriguez, L.V. (1998) Tumours and DNAadducts in mice exposed to benzo(a)pyreneand coal tars: implications for risk assess-ment. Environmental Health Perspectives 106,1325–1330.

Guillén, M.D. (1994) Polycyclic aromatic com-pounds: extraction and determination infood. Food Additives and Contaminants 11,669–684.

Guillén, M.D., Sopelana, P. and Partearroyo, M.A.(1997) Food as a source of polycyclic aromaticcarcinogens. Reviews on Environmental Health12, 133–146.

Hopia, A., Pyysalo, H. and Wickström, K. (1986)Margarines, butter and vegetable oils assources of polycyclic aromatic hydrocarbons.Journal of the American Oil Chemists’ Society 63,889–893.

Howard, J.W. and Fazio, T. (1980) Review ofpolycyclic aromatic hydrocarbons in foods.Journal of the Association of Official AnalyticalChemists 63, 1077–1104.

Husain, A., Naeemi, E., Dashti, B., Al-Omirah, H.and Al-Zenki, S. (1997) Polycyclic aromatichydrocarbons in food products originatingfrom locally reared animals in Kuwait. FoodAdditives and Contaminants 14, 295–299.

IARC (1973) Certain polycyclic aromatic hydro-carbons and heterocyclic compounds. In: IARCMonographs on the Evaluation of CarcinogenicRisk of Chemicals to Humans, Vol. 3. IARC, Lyon,France.

IARC (1983) Polynuclear aromatic compounds, part1: chemical, environmental and experimentaldata. In: IARC Monographs on the Evaluationof Carcinogenic Risk of Chemicals to Humans,Vol. 32. IARC, Lyon, France.

Jacob, J. (1996) The significance of polycyclic aro-matic hydrocarbons as environmental carcino-gens. Pure and Applied Chemistry 68, 301–308.

Jacob, J., Grimmer, G., Emura, M., Raab, G., Knebel,J.W. and Aufderheide, M. (1995) Metabolismof polycyclic aromatic hydrocarbons in fetalhuman, rat and hamster epithelial lung cells.Experimental and Toxicologic Pathology 47,428–431.

Jones, K.C., Grimmer, G., Jacob, J. and Johnston, A.E.(1989) Changes in the polynuclear aromatichydrocarbon content of wheat grain and pas-ture grassland over the last century from one

20-Feb-03 8




site in the U.K. The Science of the Total Environ-ment 78, 117–130.

Kang, D.H., Rothman, N., Poirier, M.C., Greenberg,A., Hsu, C.H., Schwartz, B.S., Baser, M.E.,Groopman, J.D., Weston, A. and Strickland,P.T. (1995) Interindividual differences in theconcentration of 1-hydroxypyrene-glucuron-ide in urine and polycyclic aromatic hydrocar-bon–DNA adducts in peripheral white bloodcells after charbroiled beef consumption.Carcinogenesis 16, 1079–1085.

Klein, H., Speer, K. and Schmidt, E.H.F. (1993)Polycyclic aromatic hydrocarbons in raw cof-fee and roasted coffee. Bundesgesundheitsblatt36, 98–100.

Kolarovic, L. and Traitler, H. (1982) Determinationof polycyclic aromatic hydrocarbons in vegeta-ble oils by caffeine complexation and glass cap-illary gas chromatography. Journal of Chroma-tography 237, 263–272.

Larsson, B.K. and Sahlberg, G. (1981) Polycyclicaromatic hydrocarbons in lettuce. Influenceof a highway and an aluminium smelter. In:Cooke, M., Dennis, A.J. and Fisher, G.L. (eds)Polynuclear Aromatic Hydrocarbons: Sixth Inter-national Symposium on Physical and BiologicalChemistry. Springer, New York, pp. 417–426.

Larsson, B.K., Sahlberg, G., Eriksson, A.T. and Busk,L.A. (1983) Polycyclic aromatic hydrocarbonsin grilled food. Journal of Agricultural and FoodChemistry 31, 867–873.

Larsson, B.K., Eriksson, A.T. and Cervenka, M.(1987) Polycyclic aromatic hydrocarbons incrude and deodorized vegetable oils. Jour-nal of the American Oil Chemists’ Society 64,365–370.

Lijinski, W. (1991) The formation and occurrenceof polynuclear aromatic hydrocarbons associ-ated with food. Mutation Research 259, 251–261.

Lodovici, M., Dolara, P., Casalani, C., Ciappellana,S. and Testolin, G. (1995) Polycyclic aro-matic hydrocarbon contamination in theItalian diet. Food Additives and Contaminants12, 703–713.

Loew, G.H., Poulsen, M., Kirkjian, E., Ferrell, J.,Sudhindra, B.S. and Rebagliati, M. (1985) Com-puter-assisted mechanistic structure–activitystudies: applications to diverse classes ofchemical carcinogens. Environmental HealthPerspectives 61, 69–96.

Mackay, D. and Shiu, W.Y. (1977) Aqueous solubil-ity of polycyclic aromatic hydrocarbons.Journal of Chemical and Engineering Data 22,399–402.

Mackenzie, K.M. and Angevine, D.M. (1981) Infer-tility in mice exposed in utero to benzo(a)pyrene. Biology of Reproduction 24, 183–191.

Moret, S., Piani, B., Bortolomeazzi, R. and Conte,L.S. (1997) HPLC determination of polycyclicaromatic hydrocarbons in olive oils. Zeitschriftfür Lebensmittel Untersuchung und Forschung A205, 116–120.

Neal, J. and Rigdon, R.H. (1967) Gastric tumors inmice fed benzo(a)pyrene: a quantative study.Texas Reports on Biology and Medicine 25,553–577.

Nousiainen, U., Torronen, R. and Hanninen, O.(1984) Differential induction of variouscarboxylesterases by certain polycyclic aro-matic hydrocarbons in the rat. Toxicology 32,243–251.

Platt, K.E., Petrovic, P., Seidel, A., Beermann, D. andOesch, F. (1988) Microsomal metabolismof picene. Chemico-Biological Interactions 66,157–175.

Poirier, M.C., Santella, R.M. and Weston, A. (2000)Carcinogen macromolecular adducts and theirmeasurement. Carcinogenesis 21, 353–359.

Pott, P. (1775) Chirurgical Observations Relative toCataract, the Polypus of the Nose, the Cancer ofScrotum, the Different Kinds of Ruptures andthe Mortification of the Toes and Feet. Hawess,Clarke & Collins, London.

Qing, W.G., Conti, C.J., LaBate, M., Johnston, D.,Slaga, T.J. and MacLeod, M.C. (1997) Inductionof mammary cancer and lymphoma by multi-ple, low oral doses of 7,12-dimethylbenz-(a)anthracene in SENCAR mice. Carcinogenesis18, 553–559.

Sanders, M. (1995) Distribution of polycyclicaromatic hydrocarbons in oyster (Crassostreavirginica) and surface sediment from two estu-aries in South Carolina. Archives of Environmen-tal Contamination and Toxicology 28, 397–405.

Serra, G., Pupin, A. and Toledo, M.C. (1995)Preliminary studies on the contamination ofsugarcane and its by-products by polycyclicaromatic hydrocarbons. Boletim da SociedadeBrasileira de Ciencia e Tecnologia de Alimentos 29,107–203.

Shaw, G.R. and Connell, D.W. (1994) Prediction andmonitoring of the carcinogenicity of polycyclicaromatic compounds (PACs). Reviews ofEnvironmental Contamination and Toxicology135, 1–62.

Stavric, B. and Klassen, R. (1994) Dietary effectson the uptake of benzo(a)pyrene. Food andChemical Toxicology 32, 727–734.

Tateno, T., Nagumo, Y. and Suenaga, S. (1990)Polycyclic aromatic hydrocarbons producedfrom grilled vegetables. Journal of Food HygienicSociety of Japan 31, 271–276.

Thomson, B. and Muller, P. (1998) Approaches tothe estimation of cancer risk from ingested


20-Feb-03 8



20-Feb-03 8


PAH. Polycyclic Aromatic Compounds 12,249–260.

Thyssen, J., Althoff, J., Kimmerle, G. andMohr, U. (1981) Inhalation studies withbenzo(a)pyrene in Syrian golden hamsters.Journal of the National Cancer Institute 66,575–577.

Van Maanen, J.M.S., Moonen, E.J.C., Maas, L.M.,Kleinjans, J.C.S. and van Schooten, F.J.

(1994) Formation of aromatic DNA adductsin white blood cells in relation to urinary excre-tion of 1-hydroxypyrene during consumptionof grilled meat. Carcinogenesis 15, 2263–2268.

Vassilaros, D.L., Stoker, P.W., Booth, G.M. andLee, M.L. (1982) Capillary gas chromato-graphic determination of polycyclic aromaticcompounds in vertebrate fish tissue. AnalyticalChemistry 54, 106–112.



9 Heavy Metals

L. Jorhem*Research and Development Department, National Food Administration,

PO Box 622, SE-751 26 Uppsala, Sweden

Introduction

Definition

Heavy metal: a metal or alloy with a densityhigher than 4.5–5.0 kg dm−3.

Heavy metals include metals that areessential as well as toxic. In the public mind,however, heavy metals usually cover allmetals that have been connected to negativeproperties in some way, even including alu-minium (2.7 kg dm−3). Arsenic is qualified byits density (5.7 kg dm−3), but is by definitionnot a metal.

Nature of the compounds

In Table 9.1 are described some of the morecommonly used physical properties that areimportant in the categorization of metals andother elements.

The metals that usually first come tomind when heavy metals are mentioned arelead, cadmium and mercury, all well knowndue to their documented toxic effects. Twoother commonly encountered heavy metalsare chromium and nickel, which are not toxicin the concentrations normally found in foodbut are used in vast quantities, not least inequipment coming into contact with food.

Another concept that needs to bementioned is trace metals, which many peopleuse as a synonym for heavy metals. Trace hasbeen defined in the analytical nomenclatureas the range 10−4–10−2 parts per million(ppm = mg kg−1). In up-to-date nomenclature,the term trace is not mentioned, but popularexpressions die hard. There is thus someconfusion in terms, since heavy metals mostlyoccur at trace, or microtrace, concentrations infoodstuffs.

Analytical Quality Assurance

‘It is easy to make an analysis, but difficult toget the right result!’

A tremendous amount of data on heavymetals in foodstuffs have been publishedsince the 1950s. Only quite recently has theimportance of analytical quality assurance(AQA) procedures in analytical chemistrybeen realized. The sad implication of this isthat most of the data for heavy metals infoods at trace or ultra-trace levels publishedprior to the early 1980s are highly unreliable.This is not to say that everything publishedlater is reliable (or, for that matter, thateverything published earlier is unreliable).However, the probability for reliability hasincreased. This is, of course, very fine, but


20-Feb-03 9




how can I, ‘the consumer’ of results, elucidatewhat is reliable or not? It is not always possi-ble, but there are several means available tohelp evaluate the reliability of an analyticalsurvey.

When it comes to assessing food safetyfrom the point of view of heavy metal content,we must keep in mind that the analysis ofheavy metals at the concentration levelscommonly found in foods is a fairly difficultanalysis, with several traps on the way.All current results are relative, a result of acomparison, which may go wrong. Althoughresearchers are working on absolute methods,it will probably be a long while before anypractical results are available.

‘How certain is uncertainty?’

A result, as it comes out of an analyticalinstrument, may have a virtually infinitenumber of decimals, which to the unsuspect-ing eye may give the impression of absolutereliability, but this is an illusion. Assume thatyou for some reason decide to reanalyse asample and you get a result quite differentfrom the first. How do you know which isright? Or are both wrong? Maybe both arecorrect! In order to determine this, we needto know the measurement uncertainty (MU)for the results. The MU is the sum of alluncertainties introduced during the analyti-cal process, and can be rather large at lowconcentrations. A result of, for example, Pb of0.025 mg kg−1 may have an MU of ±0.010 mgkg−1, which means that the ‘true’ value can beanywhere between 0.015 and 0.035 mg kg−1.In light of this, a large number of decimalsdoes not make much sense.

‘Analytical error’

Two of the main reasons for analyticalunreliability in heavy metal analysis are theneglected importance of contamination andanalytical interferences, which are not to beconfused with MU.

Very few foods are totally free fromheavy metals, although they may not bedetectable. They are usually occurring eithernaturally or through contamination. Analyti-cal blanks, carried out appropriately, wouldindicate the level of contamination during theanalytical steps prior to the determination.Interferences can often be corrected for if theyare known to occur. In light of this, it is quiteeasy to understand that past results are notalways what they seem to be. A third reasonis over-reliance on recovery tests. Recoveriesare mostly useful in analyses involving anextraction step, or other procedures in whichyou might expect to lose analyte prior todetermination. In the analysis of heavy met-als, full recovery (allowing for measurementuncertainty) is, generally, expected. A fourthreason is simply analytical carelessness orincompetence.

The two main pillars of quality assurance

Certified reference materials

Certified reference materials (CRMs) arematerials that provide you with the possibil-ity to check your performance against a sam-ple with a ‘known’ quantity of the analyte ofinterest. When it comes to heavy metals, the‘known’ level is, of course, only the bestestimate available with all its shortcomings.

20-Feb-03 9

200 L. Jorhem

MetalAtomicnumber

Density(kg dm−3)

Meltingpoint (°C)

Boilingpoint (°C)

Cadmium (Cd)Chromium (Cr)Mercury (Hg, hydrargyrum)Nickel (Ni)Lead (Pb, plumbum)

4824802882

8.657.2

13.558.9

11.35

3211857− 39

1453327.5

7652672357

27321740

Table 9.1. Physical properties of the five heavy metals described in this chapter.



CRMs are used to evaluate the performanceover time, as well as the result of a specificdetermination. Many aspects of the use ofCRMs are discussed in a book by Stoeppleret al. (2001).

Proficiency testing

Proficiency testing (PT) is probably the onlyobjective way to assess the analysis (method)as carried out in a specific laboratory at aspecific time. The result of the participationin a PT programme is invariably worse thanthe result of a CRM, probably because the‘assigned’ value is unknown at the time ofanalysis. In combination, PT and CRMs arethe backbone of quality assurance (QA) inheavy metal, or trace element, analysis.

‘Analysts are only human’

It is natural that an analyst who authors apaper focuses on his assets and suppresseshis shortcomings. It is consequently impor-tant that the ‘consumer’ of published datais to some degree able to ‘read between thelines’. If what you are looking for, in terms ofAQA, in the report is not mentioned, it hasprobably not been done. If a specific itemis given more room than seems justified, itmight be hiding something else.

Effects of QA

The general effects of QA procedures maynot always be visible. When it comes to heavymetals at trace levels, however, it may showup when publications with different degreesof QA are compared (Engman and Jorhem,1998) and can be summarized as follows:

1. Papers with satisfactory QA, describingthe content of, for example, Cd or Pb at tracelevels in uncontaminated foods, usually findthe results within a limited range.2. Papers with unsatisfactory QA, describ-ing the content of, for example, Cd or Pb inuncontaminated foods, often find the results

spreading over two to three orders ofmagnitude.

ISO-Guide 17025 (ISO/IEC, 1999) pro-vides a good picture of what is required for asatisfactory QA procedure.

Cadmium

A soft silvery metal that can easily be cut witha knife. It was discovered in 1817 in Germanyby F. Stromeyer.

The most well known event of toxiceffects of Cd on man is probably the ‘Itai-itai’disease (‘ouch-ouch’ disease). In a districtof Japan, after the Second World War andup to the early 1970s, it was found that manypeople suffered from a disease that, undermuch pain and suffering, resulted in severebone deformation and, in many cases, death.It was found to be the result of river waterbeing polluted by Cd-containing waste frommining activities. The river water was usedfor irrigation of rice fields, which resultedin Cd-contaminated rice, often with Cd levelsbetween 0.5 and 1 mg kg−1. The consumers,women in particular, then suffered fromdecalcification of the skeleton (osteomalacia),which led to skeletal deformation andfrequent bone fractures. Even the slightestexertion, such as coughing, could result in, forexample, broken ribs (Friberg et al., 1974).

Uses

One of its main uses is in Ni–Cd batteries.Another common use has been for surfaceplating. Red–yellow Cd pigments are used inpaints and on ceramics. Cd has also beenused as a stabilizer in plastics.

Distribution in foods

The Cd content can vary drastically betweendifferent food products, from less than 0.001to 100 mg kg−1. Most of the more commonlyconsumed products contain low levels ofCd. Muscle tissues from most animals,

Heavy Metals 201

20-Feb-03 9



including fish, contain levels below 0.01 mgkg−1. A notable exception is horse meat,which generally contains levels in excess of0.1 mg kg−1. Kidney from older domestic ani-mals is the tissue in which the highest levelsare found. Levels approaching 100 mg kg−1

have been detected in crab hepatopancreas.In liver and kidney of game animals andhorses, the Cd level is often so high that theirconsumption should be severely restricted.

In vegetable foods the Cd level normallydoes not vary that much, and seldom exceeds0.05 mg kg−1. However, there are somenotable exceptions. Certain seeds, for examplesunflower and flax, often have Cd levelsapproaching 0.5 mg kg−1. Very high levelshave been found in mushrooms from theAgaricus genus (e.g. Agaricus augustus), inwhich levels of 10–20 mg kg−1 are notuncommon.

The levels in wheat, rice and potatoesare of particular interest. The Cd levels areusually not very high (≤ 0.05 mg kg−1).However, being basic foodstuffs, consumedin large quantities, their influence on the totalintake is considerable. Table 9.2 indicates thenormal levels for most types of food. Higherlevels than those indicated may, however, befound occasionally.

Normal intake levels

As evident from the different levels encoun-tered in foodstuffs, the intake of Cd mayvary considerably depending on eatinghabits. Apparently, most people, regardlessof nationality, have eating habits that seldominclude items with abnormal Cd levels, as canbe seen in Table 9.3. The two most commonmethods used to establish the actual intake

20-Feb-03 9

202 L. Jorhem

Foodstuffmg kg−1 fresh

weight

Meat (excluding horse), including most fishes, dairy productsMost vegetables, certain fish (e.g. herring), rye flour, oats, eggsWheat flour, potatoes, certain vegetables (e.g. spinach, carrots)Wild mushrooms, horse meat, wheat branLiver and kidney from domestic animals, sunflower and flax seedsMussels and oystersCertain mushrooms (e.g. Agaricus augustus), horse kidney, crab hepatopancreas

≤ 0.005≤ 0.025≤ 0.05≤ 0.2≤ 0.5≤ 1

≥ 10

Table 9.2. Examples of Cd levels in uncontaminated foodstuffs (based on results from Jorhem andSundström, 1993, 1995; Marro, 1996; Hardy, 1998; Ysart et al., 1999, 2000).

Adults Adult females Adult males Reference

Australia 1994 (MB)a

Australia 1996 (MB)Belgium 1992 (DD)b

Germany 1991 (MB)Germany 1996 (DD)Japan 1992 (DD)Spain (MB)Sweden 1987 (MB)

Sweden 1996 (DD)UK 1994 (MB)UK 1997 (MB)

0.023

0.024

0.0140.011

0.0130.021

0.0110.0070.027

0.010

0.0190.027

0.0140.009

0.012

Marro (1996)Hardy (1998)Cauwenbergh et al. (2000)Müller et al. (1998)Seifert and Anke (1999a)Tsuda et al. (1995)Cuadrado et al. (1995)Becker and Kumpulainen(1991)Vahter et al. (1996)Ysart et al. (1999)Ysart et al. (2000)

aMB, market basket study.bDD, duplicate diet study.

Table 9.3. Average daily intake of Cd (mg) through the diet in certain countries.



of food components are duplicate diet(DD) studies, in which duplicate portionsof everything ingested are collected, andmarket basket (MB) studies, in which fooditems are collected, e.g. in relation to nationalconsumption statistics.

When the measured levels are below thelimit of detection (or quantification), this givesrise to problems in estimating the intake.Depending on the method used, the intakemay be under- or overestimated. It is thusrather difficult to compare results fromreports and publications, since they are notalways that explicit on the background to theirfigures.

In the UK, the Cd intake has been moni-tored through MB surveys since 1976 (Ysartet al., 2000). They show a slight tendency todecrease, but more data are probably neededto verify this (Table 9.4).

Uptake and metabolism in humans

The Cd uptake by adults is in the order of5%, and is stored primarily in the kidneys.Several studies have shown that factors suchas the dose, the composition of the foodand the individual’s nutritional status mayinfluence the uptake rate of ingested Cd (Fox,1988; Sandström, 1988; Andersen et al., 1992).Iron deficiency is one factor which enhancesCd uptake. There are also indications that asudden exposure to a foodstuff with a highconcentration may result in a higher uptakeof Cd than a lower level of exposure overtime (Lind et al., 1997).

Toxicity and clinical effects

Cd has no known function in the humanmetabolism and, since its damaging effectsare well documented, it is desirable to keep

the intake as low as possible. Examples ofacute effects are vomiting and diarrhoea. Oneof its chronic effects is slight kidney damage,which results in low molecular proteins inthe urine (proteinuria). This may be followedby severe kidney damage (uraemia, possiblylethal), osteomalacia and osteoporosis.

Risk assessment

Better diagnostic procedures have led to theconclusion that kidney damage may occurat much lower intake levels than was earlierthought. A consequence of this is that thedifference between normal intake levels andintake where negative effects may start tooccur is without a safety margin. A Belgiansurvey (Buchet et al., 1990) has shown that asmuch as 10% of the general, non-smoking,population has an internal dose of Cd suffi-cient to cause slight renal dysfunction.

An international expert group (WHO,1993a) has agreed on a provisional tolerableweekly intake (PTWI) for Cd of 0.007 mg kg−1

body weight (BW). For an adult weighing70 kg, this means a PTWI of 0.490 mg of Cdper week.

Risk management

The rationale for the maximum limits(MLs) laid down by the EU working group(Table 9.5) states that

Cadmium may accumulate in the humanbody and may induce kidney dysfunction,skeletal damage and reproductive deficien-cies. It cannot be excluded that it acts as ahuman carcinogen. The SCF (ScientificCommittee for Food) recommended in itsopinion of 2 June 1995 greater efforts toreduce dietary exposure of cadmium since

Heavy Metals 203

20-Feb-03 9

Year of study 1976 1978 1980 1982 1984 1986 1988 1991 1994 1997

Intake of Cd 0.02 0.02 0.026 0.018 0.019 0.017 0.019 0.018 0.014 0.012

Table 9.4. Comparison of population dietary exposure results for cadmium from the UK Total DietStudies 1976–1997 (Ysart et al., 2000). Average daily intake in mg.



foodstuffs are the main source of humanintake of cadmium. Therefore, maximumlevels should be set as low as reasonablyachievable (EC, 2001).

Legislation/intake recommendations

The European Union has reached an agree-ment to set MLs for Cd in certain foods. Theseare binding for the Member States after 22April 2002 and will replace existing nation-ally set limits. The MLs are summarized inTable 9.5.

Conclusions

Cd constitutes a serious risk for, primarily,kidney damage, even at today’s level ofexposure. The intake via food must thusbe kept as low as possible, and hopefully belowered even further. It is therefore impor-tant to reduce the distribution of Cd into theenvironment, from where it may find its wayinto the food chain.

Chromium

Bluish-white, hard, brittle, lustrous and resis-tant to corrosion, it was discovered in 1797 inFrance (Paris) by Nicolas-Louis Vauquelin.

Uses

Its main uses are as a plating metal and as analloy in stainless steel. It is also often used intanning of hides.


The Cr content of foods varies considerably,with most major foods at the low end ofthe spectrum (Table 9.6). It is probable thata substantial part of Cr present in foods is dueto contamination during the various stepsof production. Cr is a major componentof stainless steel, which, in the form of,for example, knives, benches, tanks and

20-Feb-03 9

204 L. Jorhem

ProductMaximum level

(mg kg−1 wet weight)

Meat of bovine animals, sheep, pig and poultry. Muscle meat of most fish.Vegetables and fruits, excluding leafy vegetables, fresh herbs, all fungi, stemvegetables, root vegetables and potatoesCereals, excluding bran, germ, wheat grain and rice. Muscle meat of wedge sole,eel, European anchovy, louvar or luvar, horse mackerel or scad, grey mullet,common two-banded seabream, European pilchard or sardineMeat of horse. Bran, germ, wheat grain and rice. Soybeans. Leafy vegetables,fresh herbs, celeriac and all cultivated fungiLiver of cattle, sheep, pig and poultry. Crustaceans, excluding brown meat of crabKidney of cattle, sheep, pig and poultry. Bivalve molluscs. Cephalopods (withoutviscera)

0.05

0.1

0.2

0.51.0

Table 9.5. Maximum levels of Cd in certain foodstuffs (EC, 2001). All references in the official list areomitted.

Foodstuffmg kg−1 fresh

weight

Meat, fish, milk and milk products, vegetables, fruits and berries, cereals, cattleliver and kidneyBeans, lentils, seeds, blue poppy seeds, pig liver and kidney, wild mushroomsDark chocolate – cocoa, white poppy seeds, buckwheat, sugar

≤ 0.02

≤ 0.10.1–5

Table 9.6. Examples of Cr levels in uncontaminated foodstuffs (based on results from, for example,Anderson et al. 1992; Jorhem and Sundström, 1993; Hardy, 1998; Ysart et al., 1999, 2000).



machinery, frequently comes into contactwith food during processing. Another sourcefor Cr is the stainless steel utensils used forcooking in the household, especially in cook-ing of acidic foods. In older papers, meat isoften cited as a good source for Cr, but thisis probably due to contamination, or othertypes of interferences during the analysis.Table 9.6 exemplifies the Cr level normallyfound in food in newer studies.

A seldom recognized source for Cr in thediet is food preserved in tin cans. The tin plateundergoes a passivation treatment using, forexample, sodium dichromate in order toimprove resistance to oxidation and lacqueradherence. A survey of Cr in canned fruitand vegetables showed a median Cr level of0.06 mg kg−1 in products from unlacqueredcans, whereas the median level in correspond-ing products in cans with a lacquered inside,as well as fresh products, was 0.01 mg kg−1

(Jorhem and Slorach, 1987).


There are several studies, both MB and DD,available on the daily intake of Cr (Table 9.7).With the exception of the UK studies(Ysart et al., 1999, 2000), the levels are quiteconsistent at ≤ 0.05 mg day−1. The reason forthe higher levels in the UK studies probablystems from the fact that those foods areprepared for consumption, which means thatthe foods have been exposed to the kind ofcontamination normally encountered duringpreparation. Thus, they probably give a truerpicture of the actual intake than the otherstudies. For presumably the same reason,

some of the UK foods in Table 9.6 exceed theexemplified levels.


Cr in foods is present mostly in its trivalentform (Cr3+), which plays an important rolein the metabolism of sugar, and functionsthrough insulin in maintaining normal glu-cose tolerance. Less insulin is required in thepresence of optimal amounts of biologicallyactive Cr (Anderson, 1992). Body uptakeof Cr3+ is estimated to be in the order of0.5%. The hexavalent form (Cr6+) is moretoxic and may give rise to, for example,allergy and contact eczema, and may alsobe carcinogenic. This form is normally notfound in foods, but may be present inwater. Most analytical methods for Cr infoods do not distinguish between the twoforms; only the total is therefore usuallyknown.


Negative effects are due mainly to occupa-tional exposure, and not via food. Drinkingwater contaminated with Cr6+, however,has been known to produce gastrointestinalsymptoms (e.g. abdominal pain, vomitingand diarrhoea). Hexavalent Cr is also aller-genic, i.e. allergenic contact eczema mayresult from contact with Cr-containing prod-ucts. There is today no information availableindicating that intake of Cr via the diet

Heavy Metals 205

20-Feb-03 9


USA (MB)a

Austria (DD)c

Belgium 1992 (DD)Sweden 1988 (DD)UK 1994 (MB)UK 1997 (MB)

0.053

0.300.10

0.012b

0.031

0.020

0.019b

0.038Anderson et al. (1992)Wilplinger et al. (1996)Cauwenbergh et al. (1996)Jorhem et al. (1998)Ysart et al. (1999)Ysart et al. (2000)

aMB, market basket study.bMean level 1000 kcal−1.cDD, duplicate diet study.

Table 9.7. Average daily intake of Cr (mg) through the diet in certain countries.



should cause, or have a negative effect onpeople having, Cr allergy (NAS, 2001).

Risk assessment

There seems to be little risk of overexposureto Cr through the diet, including drinkingwater. Risks are related mainly to contactwith products containing Cr, e.g. phosphatedetergents and tanned leather. Metallic Cr,e.g. Cr-plated surfaces and stainless steel, isnot known to give rise to contact eczema.

The US National Academy of Science hasconcluded that an acceptable intake (AI) of Crfor women 19–50 years of age is 25 µg day−1,and for men of the same age 35 µg day−1. A tol-erable upper intake level was not established,since few serious adverse effects have beenassociated with excess intake from food (NAS,2001).

Risk management

Intake recommendations

As Cr is not known to give any toxic effectsat the concentrations found in normal food-stuffs, no maximum levels are laid down.As it is, to some degree, essential, there aresome requirements on intake. In the UK, theCommittee on Medical Aspects of FoodPolicy has recommended that chromiumintakes should be above 25 µg day−1 foradults and between 0.1 and 1.0 µg kg−1 BWday−1 for children and adolescents (COMA,1991).

Conclusions

Cr is occurring at low concentrations in mostfoods, although the concentration may varyconsiderably. It plays a part in the metabo-lism of glucose. The intake via (unprocessed)food is in some countries so low that intakerecommendations are barely met. Low levelcontamination of food during processing andcooking is probably a considerable source forCr in the diet.

Mercury

Silver-white; liquid at room temperature;stable in air, water and alkali. Mercury wasone of the earliest metals known to man. Ithas been used in medicine and in cosmeticsfor millennia.

During the 20th century there wereseveral major catastrophes of Hg poisoningthrough contaminated food, of which‘Minamata’ probably is one of the more wellknown. This was caused by consumption offish and shellfish contaminated by wastewater containing Hg from chemical plantsin the Minamata bay area in Japan. Methylmercury was formed from inorganic Hg andaccumulated in fish, which in turn poisonedthe consumers. A large number of people diedof the effects (Fujiki, 1972). In Iraq in 1972,over 6000 people were poisoned after eatingbread made from wheat treated with methylmercury. More than 400 people died (Bakiret al., 1973).

Uses

Although debated, it is still used in dentalamalgam as well as in batteries. A largebut decreasing use is as a catalyst inindustrial processes. Another decreasing useis in electric switches in instruments and inthermometers.


Hg is not widely distributed in uncontami-nated foodstuffs. The main source is fish, ofwhich the predatory species, e.g. pike andswordfish, who are at the top of the marinefood chain, have the highest levels, whereasherring, for example, usually have levelsat the lower end (Table 9.8). Hg occurspredominantly (in the order of 50–80%) asmethyl mercury in fish.


The great difference between the three fishsurveys in Table 9.8 is probably due to

20-Feb-03 9

206 L. Jorhem



differences in fish species. As can be seenfrom Table 9.9, the daily intake of Hg via thediet is rather similar in all studies, independ-ent of country. This would indicate that largepredatory fish are not a staple food evenin Japan, where consumption of marineproducts is high.


Inorganic Hg in food is absorbed up toapproximately 10%, whereas methyl mercuryis absorbed efficiently to nearly 100%. Asmethyl mercury is both stable and lipophilic,it can penetrate cell membranes as well as theblood–brain barrier and be absorbed in thebrain, where it can cause severe damage. Itcan also pass the placenta and be taken up bythe fetus and affect the development of thenervous system. Children exposed to methylmercury prior to birth may thus experiencenegative effects on their mental development(EHC, 1990).


Hg is a toxic metal with no known functionin human metabolism. Inorganic Hg givesrise to a number of both acute and chronicsymptoms. Some acute symptoms are: thirst;metallic taste; inflammation of the mouth, thelining of the stomach and the lining ofthe colon; nausea; abdominal pain; tenesmus(a continual inclination to evacuate thebowels or bladder, accompanied by a painfulstraining); and kidney degeneration. Somechronic symptoms are: excessive salivation;loosened teeth and inflammation of thegums; nervousness and irritability; tremors;and slurred speech. Symptoms from inor-ganic Hg, however, are not likely to occurthrough intake via food.

As previously described, methyl mercuryis a highly toxic substance that has caused alot of injury. Some of its early symptoms arefatigue, paraesthesia (sensation of prickling,burning, etc. on the skin) in, for example,the tongue and extremities, and headache.Later symptoms are insomnia, depression,

Heavy Metals 207

20-Feb-03 9

Foodstuff mg kg−1 fresh weight

Fruit, vegetables, dairy products, beveragesMeat and meat products, offal, eggs, cerealsFish Spain: Cuadrado et al. (1995)

Sweden: Ohlin (1993)UK: Ysart et al. (1999, 2000)

≤ 0.001≤ 0.01

0.13–0.19a

0.02–1.50.054 and 0.043

aRecalculated from dry weight.

Table 9.8. Examples of Hg levels in uncontaminated foodstuffs (based on results from, for example,Ohlin, 1993; Cuadrado et al., 1995; Ysart et al., 1999, 2000).


Sweden 1987 (MB)a

Japan 1992 (DD)b

Japan 1992 (MB)Spain (MB)Australia 1994 (MB)Australia 1996 (MB)UK 1994 (MB)UK 1997 (MB)

0.0018

0.006

0.0050.003

0.00990.0035

0.0130.014

0.0160.018

Becker andKumpulainen (1991)Tsuda et al. (1995)Tsuda et al. (1995)Cuadrado et al. (1995)Marro (1996)Hardy (1998)Ysart et al. (1999)Ysart et al. (2000)


Table 9.9. Average daily intake of Cr (mg) through the diet in certain countries.



spasticity, constricted visual field, blurredspeech, paralysis and fetal neurodevelop-mental effects.

Risk assessment

An international expert group (WHO, 1993a)have decided on a PTWI for total Hgof 0.005 mg kg−1 BW, which is equal to amaximum intake of 0.350 mg of Hg week−1

for a person weighing 70 kg. In addition, only0.0033 mg kg−1 BW may be present as methylmercury (= 0.23 mg−1 week for a 70 kg per-son). It was noted furthermore that pregnantwomen and nursing mothers were likely tobe at greater risk for the negative effects ofmethyl mercury (WHO, 2000).

Risk management

Foodstuffs other than fish are generally verylow in Hg and pose no threat to the healthof the general population. The EC workinggroup that established the MLs in Table 9.9has given a good assessment of the risk.

Methyl mercury may induce alterations inthe normal development of the brain ofinfants and at higher levels may induceneurological changes in adults. Mercurycontaminates mostly fish and fishery prod-ucts. To protect public health, maximumlevels of mercury in fishery products are laiddown by Commission Decision 93/351/EEC.The levels should be as low as reasonablyachievable, taking into account that for phys-iological reasons certain species concentratemercury more easily in their tissues thanothers (EC, 2001).


The EC has reached an agreement on MLs forHg in fish and fishery products, which will bebinding for the Member States after 22 April2002 and will replace existing national limits.These MLs are summarized in Table 9.10.

Conclusions

Food in general contains very low levels ofHg and it is no threat to human health. Certainpredatory fish may contain very high levelsand could constitute a health risk. For thesespecies, an ML of 1.0 mg kg−1 has been estab-lished. Women should limit their consump-tion of fish during pregnancy and lactation.

Nickel

A silver-white, lustrous, ductile, corrosion-resistant metal. It was discovered in 1751 inSweden by Axel Fredrik Cronstedt.

Uses

Its main use is as an alloy in stainless steeland coins. It is also used for metal plating andin batteries. Another important area of use isas a catalyst in chemical processes.


Ni can be found in virtually every food-stuff. The lowest levels are usually found

20-Feb-03 9

208 L. Jorhem



Fishery products, except those listed belowExamples of species excepted from above:Anglerfish, Atlantic catfish, bass, eel, halibut, marlin, pike,rays, shark (all species), sturgeon, swordfish, tuna

0.51.0

Table 9.10. Maximum levels of Hg in foodstuffs (EC, 2001). All references in the official list areomitted.



in animal products and milk. Cereals,fruit and berries have intermediate levels,and high levels are found, for example,in cocoa. More information is found inTable 9.11. The number of reliable surveysof Ni in foods and diets is, however, ratherlimited.


Much of the Ni content in diets probablystems from contamination during processingand preparation. It is therefore rather surpris-ing to see the very high degree of agreementbetween the different types of study aswell as within and between countries(Table 9.12).

Electric water heaters may constitute aconsiderable source of Ni in the diet, a sourcemore or less unlikely to be included in intakestudies. Studies in Denmark and Sweden haveshown that heaters with Ni-plated or stainlesssteel elements can give hot water with up to1 mg Ni l−1, although the variation is consider-able. (Pedersén and Petersén, 1995; Jorhemet al., 1997).


Absorption of Ni from food is estimated to bein the order of 0.7%, if ingested together withfood, whereas Ni in beverages is absorbedmore efficiently, especially if ingested on anempty stomach. Ni has been shown to beessential in animal studies, but not in thehuman metabolism (NAS, 2001).


Toxic effects are due mainly to occupationalor accidental exposure and not via food.Accidental ingestion of Ni salts has resultedin nausea, diarrhoea and vomiting. The mostpronounced negative effect of Ni is its strongallergenic properties. It is estimated thatapproximately 10% of all women and 1% ofmen in Denmark and Sweden develop aller-gic reactions to Ni-containing items such asjewellery, coins and metal buttons (Jorhemet al., 1996). Eczema usually develops on skinthat is directly exposed to Ni-containingobjects. There are, however, people who

Heavy Metals 209

20-Feb-03 9


Meat, fish, milk, liver and kidneyFruit and vegetables, cerealsMilk chocolate, berries, wild mushroomsCocoa – dark chocolate, buckwheat, lentils, seeds, beans, nuts

≤ 0.02≤ 0.1≤ 11–5

Table 9.11. Examples of Ni in uncontaminated foodstuffs (based on results from, for example Jorhemand Sundström, 1993; Ysart et al., 1999, 2000).


Sweden 1987 (MB)a

Sweden 1988 (DD)b

Germany 1992 (DD)Germany 1996 (DD)UK 1994 (MB)UK 1997 (MB)

0.082

0.130.12

0.110.140.090

0.170.097

Becker and Kumpulainen(1991)Jorhem et al. (1998)Seifert and Anke (1999b)Seifert and Anke (1999b)Ysart et al. (1999)Ysart et al. (2000)


Table 9.12. Average daily intake of Ni (mg) through the diet in certain countries.



develop eczema or blisters on non-exposedskin. For this group of people, it has beensuggested that intake of Ni via food can havean enhancing effect (Veien and Menné, 1990).

Risk assessment

The risk of consuming foods with toxic levelsof Ni seems highly improbable. A majorsource of Ni for some people could be waterheaters. Beverages from such heaters can havehigh levels of Ni. As such beverages often areconsumed on an empty stomach, which pro-motes Ni absorption, they could constitute arisk for enhanced problems for people withgrave Ni allergy (Jorhem et al., 1996).

As nickel is not known to be toxic at theconcentrations found in normal foodstuffs, nomaximum levels are laid down. The WHO hasset a tolerable daily intake (TDI) of 0.005 mgkg−1 BW, which corresponds to 0.35 mg day−1

for a person weighing 70 kg (WHO, 1993b).The US National Academy of Science (NAS,2001) has, for certain population groups,established an upper intake level (UL) whichis ‘the highest level of daily nutrient intakethat is likely to pose no risk of adverse healtheffects for almost all individuals’. For adoles-cents and during pregnancy and lactation, theUL is set at 1.0 mg day−1 (of soluble Ni salts).

Conclusions

The Ni level in food constitutes no healthrisk to the general population. Groups withsevere Ni allergy may be helped by selectingfood with low Ni levels. The major sourceof Ni in the diet is probably contaminationduring processing and, for some people,through the use of water heaters. People withserious Ni allergy may benefit from avoidingfoods with high Ni levels.

Lead

Soft, malleable, dark greyish metal, whichhas been known by man for more than 6000

years. Pb and Pb compounds have foundwide use over the millennia. Pots and pans ofPb and pewter were popular for cookingin Roman times. Grape syrup boiled down ina lead pot acquired a nice sweet taste (leadacetate), which made it useful for sweeteningof sour wines. It was also an excellentpreservative. It has been used in medicinesince ancient times, e.g. Pb acetate againstdiarrhoea and even as tooth fillings. DifferentPb compounds have been used as cosmetics,e.g. galena (lead sulphate) as eye make-up.Pb poisonings of epidemic proportions havebeen reported repeatedly during the lastmillennium, e.g. in 1738 in Devon throughPb-contaminated cider. In the USA, vastnumbers of inner city children were identi-fied as lead poisoned into the 1980s. Onesource was identified as the ingestion ofPb-containing paint flakes. Ceramics withpoorly applied Pb glazing have always been a‘reliable’ source for Pb intake (Gilfillan, 1965;Lin-Fu, 1980; Nriagu, 1983; Wooley, 1984).The introduction of tetramethyl- and tetra-ethyl lead as an anti-knocking agent inpetrol during the 1920s assisted in spreadingenormous quantities of Pb into the environ-ment and to man, through contaminated foodand (urban) air.

Pb compounds have also been used foroutright adulteration of foodstuffs: curry byaddition of lead chromate (yellow), butter andsugar with ‘white lead’, and paprika powderwith ‘red lead’.

Uses

Pb is used in car batteries, pewter, as a stabi-lizer in plastics, colour pigments, porcelainglazes and for radiation protection.


Pb can be detected in most foods, butthere are only a few foods that naturallycontain high levels (Table 9.13). Many ofthe most consumed foods, such as meat,potatoes and milk, have levels below, oreven far below, 0.01 mg kg−1. Wine

20-Feb-03 9

210 L. Jorhem



usually has Pb levels below 0.05 mg kg−1,but can reach higher levels, mostly inwine from ‘old’ wine countries or in vintagewine.

During the last two decades two parallelevents have led to a considerable decrease inthe intake of Pb via food.

1. Welding of the side seam of tin canshas gradually replaced the Pb-soldered sideseams during the last two decades. The impactof the introduction of this new productiontechnique cannot be overemphasized. Theearlier type of tin can with Pb-soldered sideseams resulted in foodstuffs often containingmore than 0.1 mg of Pb kg−1, not seldomexceeding 0.5 mg kg−1. This lead source isnow, fortunately, history. In cans with weldedside seams, the lead content is not very dif-ferent from that in the corresponding freshfood. Foodstuffs in cans with an unlacqueredinner surface may, however, still have aslight increase in Pb due to contaminationfrom the exposed tin layer (Jorhem andSlorach, 1987).2. The reduction/elimination of petrol withadded tetraethyl or tetramethyl has resultedin a much reduced level of Pb contaminationof, in particular, vegetables. The effect of thereduced exposure to Pb via food is clearly visi-ble in Table 9.14, which is based on the resultsfrom the UK Total Diet Studies 1976–1997(Ysart et al., 2000).


The present-day intake levels in the UK arevery similar to what has been found inseveral other countries from the late 1980sand onwards (Table 9.15).


The uptake of Pb from food by adults is inthe order of 10%, whereas children may havean uptake of up to 50%. Most of the Pb isaccumulated in the skeleton. Pb can pass theplacenta barrier and the blood–brain barrierin children.


Pb has no known function in human metabo-lism. Some of its negative effects have beenknown for millennia. Some acute effectsare headache, irritability and colic (gripes).Pb displays several chronic effects, suchas colic, constipation, anaemia, pallor, palsy,disturbed reproduction, fetal neurodevelop-mental effects and reduced learning capacityin children. Experiments on mice have shownthat Pb poisoning may have a negative effecton female reproduction for three generations(Wide, 1985).

Heavy Metals 211

20-Feb-03 9


Meat including most fish, milk, eggs, potatoesMost vegetables, wheat and rye flour, oats, wineLeafy vegetables, liver, kidney from domestic animalsMost wild mushrooms, musselsCertain wild mushrooms, liver from game animals

≤ 0.01≤ 0.05≤ 0.2≤ 0.5≤ 1

Table 9.13. Examples of Pb levels in uncontaminated foodstuffs (based on results from, for example,Jorhem and Sundström, 1993; Marro, 1996; Hardy, 1998; Ysart et al., 1999, 2000).

Year of study 1976 1978 1980 1982 1984 1986 1988 1991 1994 1997

Intake level 0.11 0.11 0.12 0.069 0.065 0.06 0.06 0.028 0.024 0.026

Table 9.14. Comparison of population dietary exposure results for lead from the UK Total Diet Studies1976–1997 (Ysart et al., 2000). Average daily intake in mg.



Risk assessment

Intake of Pb via food should be kept as lowas possible. A PTWI for Pb (0.025 mg kg−1

BW) has been decided by an internationalexpert group. This is equal to 1.75 mg of Pbweek−1 for a person weighing 70 kg (WHO,1993a).

Risk management

The banning of Pb additives in petrol and thephasing out of tin cans with Pb-soldered sideseams have, as mentioned, radically reducedthe Pb burden in man as well as the

environment. Although several sources forPb have been drastically reduced/eliminatedover the last decades, risks still remain. Thishas been nicely described by the EC workinggroup who established the MLs in Table 9.16:

Lead absorption may constitute a seriousrisk to public health. Lead may inducereduced cognitive development and intellec-tual performance in children and increasedblood pressure and cardiovascular diseasesin adults. Over the past decade the levels infood decreased significantly due to theawareness of lead being a health problem,source-related efforts to reduce the emissionof lead and improvements in quality assur-ance of chemical analysis. The SCF concludedin its opinion of 19 June 1992 that the mean

212 L. Jorhem

20-Feb-03 9


Australia 1994 (MB)a

Australia 1996 (MB)Germany 1996 (DD)b

Spain (MB)Sweden 1987 (MB)

Sweden 1988 (DD)UK 1994 (MB)UK 1997 (MB)

0.18

0.0230.024

0.0210.0300.019

0.026

0.0270.0380.019

0.017

Marro (1996)Hardy (1998)Seifert and Anke (2000)Cuadrado et al. (1995)Becker and Kumpulainen(1991)Vahter et al. (1990)Ysart et al. (1999)Ysart et al. (2000)


Table 9.15. Average daily intake of Pb (in mg) through the diet in certain countries.



Cow’s milk, infant formulaeFruit juices, concentrated fruit juices (for direct consumption)and fruit nectarsMeat of bovine animals, sheep, pig and poultry; vegetables,excluding brassica, leafy vegetables, fresh herbs and all fungi.In the case of potatoes, the maximum level applies to peeledpotatoes. Fruits, excluding berries and small fruits, fats and oilsMuscle meat of most fish, berries and small fruits, cereals,legumes and pulses, winesBrassica, leafy vegetables and all cultivated fungiMuscle meat of wedge sole, eel, spotted seabass, horsemackerel or scad, grey mullet, common two-banded seabream,grunt, European pilchard or sardineEdible offal of cattle, sheep, pig and poultry; crustaceans,excluding brown meat of crabBivalve molluscs, cephalopods (without viscera)

0.020.05

0.1

0.2

0.30.4

0.5

1.0

Table 9.16. Maximum levels of Pb in foodstuffs (EC, 2001). All references in the official list are omitted.



level of lead in foodstuffs does not seem to bea cause of alarm, however, longer term actionshould follow with the objective of furtherlowering the mean levels of lead in food-stuffs. Therefore, the maximum levelsshould be as low as reasonably achievable(EC, 2001).

Another amelioration, perhaps small butwith a great symbolic value, is the banning ofPb seals on wine bottles, which came intoeffect in the early 1990s.


The EU has reached an agreement on MLsfor Pb in certain foods. These will be bindingfor the Member States after 22 April 2002and will replace existing national limits(Table 9.16).

Conclusions

The contamination of food with Pb has beendrastically reduced over the last decadesthrough source-related actions, such as thephasing out of organic Pb compounds inpetrol, introduction of Pb-free tin cans andprohibiting Pb seals on wine bottles. This has,in turn, led to a radically decreased intake ofPb via food. Although no immediate dangercan be seen, there are continuous on-goingefforts to reduce further the intake of Pb viafood.

References

Andersen, O., Nielsen, J.B. and Nordberg, G.F.(1992) Factors affecting the intestinal uptake ofcadmium from the diet. In: Nordberg, G.F.,Herber, R.F.M. and Alessio Lyon, L. (eds)Cadmium in the Human Environment: Toxicityand Carcinogenicity. International Agencyfor Research on Cancer, Lyon, France,pp. 173–187.

Anderson, R.A. (1992) Chromium, glucose toler-ance, and diabetes. Biological Trace ElementResearch 32, 19–24.

Anderson, R.A., Bryden, N.A. and Polansky, M.M.(1992) Dietary chromium intake – freelychosen diets, institutional diets and individual

foods. Biological Trace Element Research 32,117–121.

Bakir, F., Damluji, L., Amin-Zaki, M., Murthada, A.,Khalidi, A., Al-Ravi, N.Y., Tikriti, S., Dhahir,H.I., Clarkson, T.W., Smith, J.C. and Doherty,R.A. (1973) Methylmercury poisoning in Iraq.Science 181, 230–241.

Becker, W. and Kumpulainen, J. (1991) Contentsof essential and toxic mineral elements inSwedish market basket diets in 1987. BritishJournal of Nutrition 66, 151–160.

Buchet, J.P., Lauwerys, R., Roels, H., Bernard, A.,Bruaux, P., Claeys, F., Ducoffre, G., de Plaen,P., Staessen, J., Amery, A., Lijnen, P., Thijs, L.,Rondia, D., Sartor, F., Saint Remy, A. andNick, L. (1990) Renal effects of cadmium bodyburden of the general population. Lancet 336,699–702.

Cauwenbergh, R., Hendrix, P., Robberecht, H.and Deelstra, H.A. (1996) Daily dietarychromium intake in Belgium, using duplicateportion sampling. Zeitschrift für LebensmittelUntersuchung und Forschung 203, 203–206.

Cauwenbergh, R., Bosscher, D., Robberecht, H. andDeelstra, H.A. (2000) Daily dietary cadmiumintake in Belgium using duplicate portion sam-pling. European Food Research and Technology212, 13–16.

COMA (Committee on Medical Aspects of FoodPolicy) (1991) Dietary Reference Values for FoodEnergy and Nutrients in the United Kingdom.Department of Health, HSMO, London.

Cuadrado, C., Kumpulainen, J. and Moreiras, C.(1995) Lead, cadmium and mercury in averageSpanish market basket diets from Galicia,Valencia, Andalucía and Madrid. FoodAdditives and Contaminants 12, 107–118.

EC (European Commission) No. 466/2001 of 8March (2001) Setting maximum levels of cer-tain contaminants in foodstuffs. Official Journalof the European Communities L 77, 16.3.2001.

EHC (1990) Environmental Health Criteria 101.Mercury. International Programme on ChemicalSafety. World Health Organization, Geneva.

Engman, J. and Jorhem, L. (1998) Toxic and essentialelements in fish from Nordic waters, withthe results put in a quality perspective. FoodAdditives and Contaminants 15, 884–892.

Fox, S.M.R. (1988) Nutritional factors that mayinfluence bioavailability of cadmium. Journal ofEnvironmental Quality 17, 175–180.

Friberg, L., Piscator, M., Norberg, G.F. andKjellström, T. (eds) (1974) Cadmium in theEnvironment, 2nd edn. CRC Press, Cleveland,Ohio.

Fujiki, M. (1972) The transitional condition ofMinamata bay and the neighbouring sea

Heavy Metals 213

20-Feb-03 9



polluted by factory wastewater containingmercury. In: Proceedings of the 6th Inter-national Water Pollution Conference. Jerusalem,pp. 902–917.

Gilfillan, S.G. (1965) Lead poisoning and the fallof Rome. Journal of Occupational Medicine 7,53–60.

Hardy, B. (1998) The 1996 Australian Market BasketSurvey. Australia New Zealand Food Author-ity, Canberra, Australia.

ISO/IEC (1999) General requirements for the com-petence of testing and calibration laboratories.International Standard ISO/IEC 17025.

Jorhem, L. and Slorach, S. (1987) Lead, chromium,tin, iron and cadmium in foods in welded cans.Food Additives and Contaminants 4, 309–316.

Jorhem, L. and Sundström, B. (1993) Levels of lead,cadmium, zinc, copper, nickel, chromium,manganese and cobalt in foods on the Swedishmarket, 1983–1990. Journal of Food Compositionand Analysis 6, 223–241.

Jorhem, L. and Sundström, B. (1995) Levels of sometrace elements in edible fungi. Zeitschrift fürLebensmittel Untersuchung und Forschung 201,311–316.

Jorhem, L., Svensson, K., Thuvander, A., WicklundGlynn, A. and Petersson Grawé, K. (1996)Nickel in foodstuffs and nickel allergy (inSwedish). SLV-Rapport 8/96. National FoodAdministration, Uppsala, Sweden.

Jorhem, L., Bergmark, A., Sundström, B. andEngman, J. (1997) Dissolution of metalsfrom materials in contact with foodstuffs(in Swedish). SLV-Rapport 4/97. National FoodAdministration, Uppsala, Sweden.

Jorhem, L., Becker, W. and Slorach, S. (1998) Intakeof 17 elements by Swedish women, determinedby a 24-hour duplicate portion study. Journal ofFood Composition and Analysis 11, 32–46.

Lind, Y., Engman, J., Jorhem, L. and Glynn, A.W.(1997) Cadmium accumulation in liver andkidney of mice to the same weekly cadmiumdose continuously or once a week. Food andChemical Toxicology 35, 891–895.

Lin-Fu, J.S. (1980) Lead poisoning and undue leadexposure in children: history and currentstatus. In: Needleman, H.L. (ed.) Low Level LeadExposure: the Clinical Implications of CurrentResearch. Raven Press, New York, pp. 5–16.

Marro, N. (1996) The 1994 Australian Market BasketSurvey. Australia New Zealand Food Author-ity, Canberra, Australia.

Müller, M., Anke, M., Illing-Günther, H. andThiel, K. (1998) Oral cadmium exposureof adults in Germany. 2: Market basket calcu-lations. Food Additives and Contaminants 15,135–141.

NAS (National Academy of Sciences) Food andNutrition Board (2001) Intakes (DRI) andRecommended Dietary Allowances. Dietary Refer-ence Intakes for Vitamin A, Vitamin K, Arsenic,Boron, Chromium, Copper, Iodine, Manganese,Molybdenum, Nickel, Silicon, Vanadium and Zinc.National Academy Press, Washington, DC.

Nriagu, J.U. (1983) Saturnine gout among Romanaristocrats. New England Journal of Medicine 308,660–663.

Ohlin, B. (1993) The mercury level in fish from retailshops (in Swedish). Vår Föda 45, 390–397.

Pedersén, G.A. and Petersén, J. (1995) Undersökelseaf nikkel, chrom og blyavgivelse frael-koge-kander samt kartläggning avmetalavgivelse fra kaffemaskiner (in Danish).Rapport ILF 1995.1. Levnedsmiddelstyrelsen,Copenhagen.

Sandström, B.M. (1988) Factors influencing theuptake of trace elements from the digestivetract. Proceedings of the Nutrition Society 47,161–167.

Seifert, M. and Anke, M. (1999a). Daily intake ofcadmium in Germany in 1996 determined withthe duplicate portion technique. Journal of Traceand Microprobe Techniques 17, 101–109.

Seifert, M. and Anke, M. (1999b) Alimentary nickelintake of adults in Germany. Trace Elements andElectrolytes 19, 17–21.

Seifert, M. and Anke, M. (2000) Alimentarylead intake of adults in Thuringia/Germanydetermined with the duplicate portiontechnique. Chemosphere 41, 1037–1043.

Stoeppler, M., Wolf, W.R. and Jenks, P. (2001) Refer-ence Materials for Chemical Analysis. Wiley-VCHVerlag GmbH, Weinstein, Germany.

Tsuda, T., Inoue, T., Kojima, M. and Aoki, S. (1995)Market basket duplicate portion estimation ofdietary intakes of cadmium, mercury, arsenic,copper, manganese and zinc by Japaneseadults. Journal of the Association of OfficialAnalytical Chemists International 78, 1363–1368.

Vahter, M., Berglund, M., Friberg, L., Jorhem, L.,Lind, B., Slorach, S. and Åkesson, A. (1990)Dietary intake of lead and cadmium inSweden. Vår Föda 42, Supplement 2.

Vahter, M., Berglund, M., Nermell, B. and Åkesson,A. (1996) Bioavailability of cadmium fromshellfish and mixed diet in women. Toxicologyand Applied Pharmacology 136, 332–341.

Veien, N.S. and Menné, T. (1990) Nickel contactallergy and a nickel-restricted diet. Seminars inDermatology 9, 197–205.

WHO (1993a) Evaluation of Certain Food Additivesand Contaminants. WHO Technical ReportSeries, No. 837. World Health Organization,Geneva.

20-Feb-03 9

214 L. Jorhem



WHO (1993b) Guidelines for Drinking Water, 2ndedn, Vol. 1, Recommendations. World HealthOrganization, Geneva.

WHO (2000) Safety Evaluation of Certain Food Addi-tives and Contaminants. WHO Food AdditivesSeries 44. World Health Organization, Geneva.

Wide, M. (1985) Lead exposure on critical days offetal life affects fertility in the female mouse.Teratology 32, 375–380.

Wilplinger, M., Shoensleben, I. and Pfannhauser, W.(1996) Versorgungszustand der Österreichermit dem Spurenelement Chrom. Zeitschrift fürLebensmittel-Untersuchung und Forschung 203,207–209.

Wooley, D.E. (1984) A perspective of lead poisoningin antiquity and the present. Neurotoxicology 5,353–362.

Ysart, G., Miller, P., Crews, H., Robb, P., Baxter, M.,de L’Argy, C., Lofthouse, S., Sargent, C. andHarrison, N. (1999) Dietary exposure estimatesof 30 elements from the UK total diet study.Food Additives and Contaminants 16, 391–403.

Ysart, G., Miller, P., Croasdale, M., Crews, H., Robb,P., Baxter, M., de L’Argy, C. and Harrison, N.(2000) 1997 UK total diet study: aluminium,arsenic, cadmium, chromium, copper, lead,mercury, nickel, selenium, tin and zinc. FoodAdditives and Contaminants 17, 775–786.

Heavy Metals 215

20-Feb-03 9



10 Dietary Nitrates, Nitrites and N-nitrosoCompounds and Cancer Risk with SpecialEmphasis on the Epidemiological Evidence

M. Eichholzer* and F. GutzwillerInstitute of Social and Preventive Medicine, University of Zurich, Sumatrastrasse 30,

CH-8006 Zurich, Switzerland

Introduction

The present chapter is an update of ourreview article published in 1998 (Eichholzerand Gutzwiller, 1998) on the epidemiologicalevidence relating estimated dietary intake ofN-nitroso compounds (NOCs), nitrates andnitrites and the human risk of cancer of vari-ous sites. Thus, this chapter is not intended toassess health risks other than cancer, suchas nitrite-induced methaemoglobinaemia ininfants. Nor does it intend to be a comprehen-sive survey on subjects such as distribution infoods, uptake and metabolism, and animaltoxicity of nitrates, nitrites and N-nitrosocompounds. Rather, its main purpose is tohighlight information which may be ofrelevance of human cancer risk assessmentof these components in the diet. No attemptis made to consider non-dietary routes ofexposure (e.g. inhalation).

Nature of Nitrate, Nitrite and N-nitrosoCompounds

Nitrate is usually referred to as the NO3− ion

or as sodium nitrate (NaNO3); similarly,

nitrite is expressed as the NO2− ion or

sodium nitrite (NaNO2). N-nitroso com-pounds can be divided into two categories:the class of nitrosamines (e.g. N-nitrosodime-thylamine (NDMA)) and the class ofnitrosamide-type compounds includingN-nitrosoureas, N-nitrosocarbamates andN-nitrosoguanidines. Compounds of bothgroups differ considerably in chemical for-mation and biological effectiveness (Councilof Europe, 1995).


Vegetables usually contribute 75–80% of thetotal daily intake of nitrate, with high levelsin lettuce, spinach, celery, beetroot, turnipgreens, etc. The nitrate concentration ofdrinking water, another contributor to thetotal exposure to nitrate, varies widelydepending on the source (high concentra-tions in private water supplies), season andproximity to arable land. Nitrates and nitritesare widely used in the production of curedmeat products and added as preservatives tofish in some countries. Nitrites are also foundnaturally in some grains and vegetables.


20-Feb-03 10




Many NOCs have been detected in foods, butonly NDMA is well studied; thus, more isknown, for example about the sources ofhuman exposure to nitrosamines than tonitrosamides. NDMA has been found in vari-ous processed meats (salted, cured or smoked,such as bacon) and fish, and in beer, etc.Overall, nitrate, nitrite and NOC concentra-tions in food products can vary widely for thesame food or for drinking water from differ-ent sites. Furthermore, nitrosation may occur,before food intake, between nitrite which hasbeen formed from nitrate by contaminatingbacteria and amines and amides present inthe same food. In assessing the health risk toman from dietary exposure to nitrate, nitriteand NOCs it is therefore important to recog-nize that the analysis of the exposure is par-ticularly complex. In addition, endogenousformation of NOCs and their precursors,may be an important source of exposure(see below) (Ikins et al., 1986; Walker, 1990;Gangolli et al., 1994; Council of Europe, 1995).

Uptake and Metabolism inHumans/Animal Models and

Maternal Transmission

Dietary nitrate is absorbed from the proximalsmall intestine into the plasma. About 25% ofthe ingested nitrate is secreted in the salivaand, of this, approximately 20% is convertedto nitrite in the mouth by nitrate-reducingbacteria. Nitrate and nitrite can also beformed endogenously in enzymatic reactionsmediated by bacteria, macrophages and neu-trophils. It has been postulated that in thestomach nitrite might nitrosate, for example,secondary amines ingested in food to formnitrosamines. Such endogenous formation ofNOCs has been demonstrated in experi-mental animals and in humans; it occursby nitrosation of amines or amides via theiracid- or bacterial (gastric hypochlorhydria)catalysed reaction with nitrite, or by reactionwith products of nitric oxide generatedduring inflammation and infection. Reducingsubstances, such as ascorbic acid, activelysecreted by the gastric mucosa and present invegetables, etc., on the other hand prevent

nitrosation. Thus humans are exposed topre-formed NOCs and to NOCs producedin vivo, but it is very difficult to estimate thedaily amount formed endogenously. In rela-tion to maternal transmission, it has, forexample, been observed that ethyl urea andnitrite fed to pregnant rats caused formationof ethyl nitrosourea brain tumours in all theiroffspring (Preston-Martin et al., 1982; Bartschet al., 1988, 1992; Bartsch, 1991; Gangolli et al.,1994; Mirvish, 1995; Hecht, 1997; Lee et al.,1997; Hill, 1999; McKnight et al., 1999).


The carcinogenicity of nitrate, nitrite andNOCs has been reviewed extensively andupdated continually in the light of new dataemerging from ongoing research on theseimportant chemicals, and this chapter is notintended to be a comprehensive survey onthe carcinogenic effects of these compounds.Nitrate in itself is not carcinogenic. There isalso no (or controversial) evidence of directnitrite (other than through nitrosamineformation) carcinogenicity in animals. On thecontrary, various NOCs have been found tobe carcinogenic to multiple organs in at least40 animal species, including higher primates(Bogovski and Bogovski, 1981). The cellularand molecular changes induced by someNOCs in animals have been shown to be verysimilar to those in human tissues (Bartsch,1991). Distinct organ specificity is an impor-tant characteristic of NOCs (Gangolli et al.,1994; Council of Europe, 1995; McKnightet al., 1999).

Risk Assessment in Epidemiology

Ecological studies are typically a startingpoint for more detailed and better controlledepidemiological research, i.e. analytical epi-demiological studies such as case–control,cohort and intervention studies. Evidencefrom ecological studies cannot, in isolation,amount to more than a possible causal rela-tionship. Greater reliance can be placed onaggregate evidence from individually based

20-Feb-03 10

218 M. Eichholzer and F. Gutzwiller



case–control and cohort studies. Althoughcase–control studies are of shorter durationand less expensive than cohort studies, riskestimates may be distorted by selection andrecall bias. Cohort studies are less susceptibleto such bias, as information is collated beforea disease develops. Both types of studies are,however, prone to confounding, in that thevariables analysed are merely surrogatesfor the actual (but unmeasured) active agent.In randomized, placebo-controlled, inter-vention trials, subjects are allocated atrandom to either active treatment or placebo,and this type of study design avoids knownand unknown confounding. When the resultsof case–control and cohort studies are repeat-edly consistent, this strengthens the case forcausal links. Large, well-designed controlledtrials may produce strong and consistent con-clusions, but they are not feasible in the pres-ent context. Data from experimental studiesin animal models, on the contrary, may rein-force human evidence, but, in isolation, are oflimited evidence (Hennekens and Buring,1987; World Cancer Research Fund andAmerican Institute for Cancer Research,1997). Despite extensive information regard-ing carcinogenicity of NOCs to animals, therehave been few analytical epidemiologicalstudies investigating the risk in humans,and what is available is mainly limited to eco-logical and case–control studies. The presentchapter is an update of our review(Eichholzer and Gutzwiller, 1998) on the epi-demiological evidence (excluding ecologicalstudies) relating estimated dietary intakeof NOCs, nitrates and nitrites (and someexamples of individual foods rich in thesesubstances when no data on NOCs, nitrateand nitrite are available) and the risk of brain,stomach, oesophageal and nasopharyngealcancers. The main emphasis is given to braintumours, as most of the newer data arerelated to this cancer site. For a numberof additional cancer sites such as leukaemia,non-Hodgkin’s lymphoma, renal cell andtesticular cancers and cancer of the bladderand colon, single studies exist showingassociations with NOCs and precursors(Foster et al., 1997; Moller, 1997; Yuan et al.,1998; Law et al., 1999; Mohsen et al., 1999;Roberts-Thomson et al., 1999). These cancer

sites as well as cancer of the oesophagus willnot be discussed in the present chapter, thelatter due to the fact that not enough newdata have been published since we reviewedthe evidence in 1998.

Brain Tumours

Incidence rates and pathogenesis

Astrocytoma, medulloblastoma, ependy-moma, glioblastoma and meningioma are themost common types of brain tumours. Theage curve of these tumours shows a peakduring the first decade of life followed bypeaks in adults, except for medulloblastoma,which is rarely observed in adults, andmeningioma, which is less prevalent in chil-dren than in adults. Brain tumours accountfor about one in five childhood cancers. Thehighest rates of tumours of the central ner-vous system are observed in Israeli femalesand in the female population of Iceland.Intermediate rates are seen in most westerncountries. Rates in Asian populations arelowest. In children, the highest rates areobserved in Nordic countries. Very little isknown about the aetiology of brain tumours.Inherited syndromes that predispose to braintumour development such as neurofibroma-tosis are present in fewer than 5% of patients.Ionizing radiation, the only established envi-ronmental cause, similarly accounts for nomore than a few per cent of cases (Higginsonet al., 1992; Preston-Martin et al., 1996). Onepostulated risk factor that has been the sub-ject of investigation is exposure to NOCs andprecursor nitrates and nitrites, some of whichare nervous system carcinogens in animals,especially when exposure occurs transpla-centally (see above). The hypothesis has beenexpanded recently to include adults as wellas childhood brain cancer.

Epidemiological evidence

Under the auspices of the SEARCH pro-gramme of the International Agency forResearch on Cancer (IARC), a series of multi-

Dietary Nitrogenous Compounds and Cancer Risk 219

20-Feb-03 10



centred international coordinated case–control studies was initiated to evaluate, interalia, the roles of NOCs, their precursors andmodulators of their metabolism in the occur-rence of childhood and adult brain tumours.Common methods of exposure and specific-ity of diagnosis will allow pooling of the dataof all studies. This increases statistical power,and analysis by histological subtypes will bepossible (Giles et al., 1994; McCredie et al.,1994).

Diet during pregnancy and risk of childhoodbrain tumours

The observation that various NOCs arepotent nervous system carcinogens, particu-larly when animals are exposed transplac-entally, prompted Preston-Martin et al. (1982)to propose that pre-natal and early exposuresmight be related to childhood brain tumoursin humans. Since 1982, 11 case–control stud-ies of childhood brain tumours and maternaldiet during pregnancy have focused onaspects of diet related to the hypothesis thattransplacental exposure to NOCs increasesthe risk of brain tumours in childhood. Threestudies also considered dietary intake of chil-dren (Howe et al., 1989; Sarasua and Savitz,1994; Lubin et al., 2000). The methods and theresults of these studies are described in Table10.1. Most studies investigated all childhoodbrain tumours combined, despite the fact thatdifferent brain tumours may have differentaetiologies. Furthermore, in most studies,consumption of cured meat was used as acrude indicator of NOCs and nitrite expo-sure; in five studies, intake of nitrates, nitritesand/or NOCs was estimated. In Los AngelesCounty, Preston-Martin and co-workers(1982) questioned mothers of 209 young braintumour patients and mothers of 209 popula-tion-based controls about experiences ofpossible aetiological relevance which theyhad during pregnancy, including frequencyof consumption of cured meats. Results sug-gested an aetiological role for cured meats(odds ratios (ORs) = 1.2 for moderate, 2.3 forhigh vs. low intake; P trend = 0.008) andother NOC-containing substances in child-hood brain tumours. In a small Canadiancase–control study (Howe et al., 1989)

comparing the children’s intake of curedmeats prior to diagnosis, no significant asso-ciation was observed. Beer consumption dur-ing pregnancy, on the other hand, increasedthe risk of childhood brain tumour signifi-cantly. Two other rather small studies, i.e.surveys with a low power to detect existingassociations, generally observed no associa-tions with cured meat consumption in preg-nancy (Cordier et al., 1994; Sarasua andSavitz, 1994), but in the study by Sarasua andSavitz (1994) an increased risk for consump-tion of hot dogs during pregnancy (OR = 2.3(95% confidence interval (CI) = 1.0–5.4)) andchildhood (OR = 2.1 (95% CI = 0.7–6.1)) wasfound. A slightly increased risk was alsoobserved with consumption of ham, sausageand bacon by the child. Non-significant posi-tive and negative associations, respectively,were observed by Cordier et al. (1994) withintake of nitrate and nitrite. Despite the smallnumber of cases, in the Australian case–control study by McCredie et al. (1994), therisk of childhood brain tumours rose sig-nificantly with reported increasing consump-tion, during pregnancy, of cured meats. Thesame was true for the much larger studiesof Preston-Martin et al. (1996) and Schymuraet al. (1996). In the former study, nitrite fromcured meat, but neither total nitrite nor nitritefrom vegetables, was related to brain cancerrisk. A recently published survey carriedout in Israel (Lubin et al., 2000) observedno association between intake of nitrate andnitrite during pregnancy or childhood andrisk of brain tumour.

Few studies have concentrated on asingle type of brain tumour in children.Gestational and familial risk factors wereinvestigated for their association withastrocytoma in a case–control study of 163pairs performed in Pennsylvania, New Jerseyand Delaware (Kuijten et al., 1990). A signifi-cant trend showing more frequent consump-tion of cured meats in mothers of astrocytomacases compared with control mothers wasobserved. However, the association was onlysignificant among more highly educatedmothers (OR = 6.8 (95% CI = 1.8–26.3)).Conversely, a study by Bunin and co-workers(1993) showed no elevated risk with frequentmaternal consumption of cured meats,

11-Mar-03 10




Dietary

Nitrogenous C

ompounds and C

ancer Risk

221

11-Mar-03

10

Reference andnumber of cases Brain tumour

Dietary variable(intake duringpregnancy) Comparison Association OR (95% CI)

Adjusted/matched for Population

Preston-Martin et al.(1982)n = 209Howe et al. (1989)n = 74

Kuijten et al. (1990)n = 163

Bunin et al. (1993)n = 166

Bunin et al. (1994)n = 155

Cordier et al. (1994)n = 75

McCredie et al.(1994) n = 82

‘Brain tumours’

‘Brain tumours’

Astrocytoma

Primitiveneuroectodermaltumour

Astrocytic glioma

‘Brain tumours’

Tumour of brainor cranial nerves

Cured meats

Cured meats (child)

Beer (pregnancy)Cured meatMothers

highly educatedless educated

NitrateNitrateNitrosaminesCured meatsCured meatsNitriteNitrateDimethylnitrosamineAll cured meats

hamother

NitriteNitrateCured meats

High vs. lower

> 1 × week−1 vs. ≤ 1 ×week−1

Ever vs. neverYes vs. noFrequency

highhigh

Quartile 4 vs. quartile 1


≥ 1 × week−1 vs. < 1 ×week−1

Quartile 4 vs. quartile 1Quartile 4 vs. quartile 1Quartile 4 vs. quartile 1

↑

NS

↑NS

↑NSNSNSNSNSNSNSNSNSNS

NSNSNSNS↑

2.3; P trend 0.008

1.13 (0.55–2.31)

3.53 (1.16–10.8)1.9 (0.9–4.2);P trend 0.046.8 (1.8–26.3)1.2 (0.4–3.8)0.54 (–)1.06 (–)1.55 (–)1.10 (0.60–2.03)1.7 (0.8–3.4)1.3 (0.7–2.6)0.7 (0.3–1.4)0.8 (0.4–1.8)0.7 (0.2–3.0)

0.9 (0.2–3.4)0.7 (0.3–1.6)0.4 (0.1–1.4)1.5 (0.5–4.6)2.5 (1.1–5.7)

A–D

A, B, E, F

A, C, E, Ra

A, C, R, S

A, C, D, R

A, B, E, G, H

B, E, H, K, J, L

Los AngelesCounty, USA,children < 25 yearsSouthern Ontario,Canada, children≤ 19 yearsChildren < 15 yearsold in New Jersey,Delaware andPennsylvania, USAUSA and Canada,children < 6 years

USA and Canada,children < 6 years

Ile de France,children ≤ 15 years

New South Wales,Australia, children≤ 14 years

continued

Table 10.1. Case–control studies on dietary intake of nitrates, nitrites and N-nitroso compounds (or the corresponding foods) during pregnancy and the risk ofbrain tumours in children.

237

Z:\Customer\CABI\A4382 - d’Mello\A4491 - d’Mello #G.vp

11 March 2003 12:35:00



12-Mar-03

10

222M

.Eichholzerand F.G

utzwiller

Reference andnumber of cases Brain tumour

Dietary variable(intake duringpregnancy) Comparison Association OR (95% CI)


Sarasua and Savitz(1994)n = 45

Preston-Martin et al.(1996)n = 540

Schymura et al.(1996)n = 338Lubin et al. (2000)n = 300

‘Brain tumour’

‘Brain tumour’

‘Brain tumour’

‘Brain tumour’

During pregnancyham, bacon,sausage, hotdogs, lunch meats

Child up to diagnosisham, bacon,sausage, hotdogs, lunch meats

Cured meatsNitrite total

from cured meatfrom vegetables

Hot dogs‘Other’ cured meat

During pregnancynitratenitrite

Child lifenitratenitrite

≥ 1 week−1 vs. < 1 ×week−1

> 0 week−1 vs. 0 week−1

≥ 1 week−1 vs. < 1 ×week−1

≥ 1 week−1 vs. < 1 ×week−1

≥ 1 week−1 vs. < 1 ×week−1

≥ 1 week−1 vs. < 1 ×week−1

> Daily vs. neverQuartile 4 vs. quartile 1Quartile 4 vs. quartile 1Quartile 4 vs. quartile 1Once week−1 vs. less2–3 × week−1 vs. lessOnce week−1 vs. less

Intermediate vs. low

NS

(↑)↓

NS

NS

NS

↑NS↑NS(↑)↑↑

NSNS

NSNS

1.0 (0.5–2.1)

2.3 (1.0–5.4)0.4 (0.2–0.8)

1.4 (0.6–3.1)

2.1 (0.7–6.1)

0.6 (0.3–1.4)

2.1 (1.3–3.2)1.1 (0.79–1.50)1.9 (1.3–2.6)0.98 (0.71–1.3)1.33 (1.00–1.76)2.01 (1.10–3.63)6.04 (1.89–19.31)

1.10 (0.83–1.46)0.97 (0.74–1.27)

1.07 (0.81–1.40)0.91 (0.66–1.25)

B, D, E, F, I

A–F

A–C, M

A, B, N–Q

Denver,Colorado, USA,children < 14years

19 counties, USWest Coast,children < 20yearsNew York, USA,children

Israel, children< 18 years

CI, confidence intervals; NS, not statistically significant; ↑, statistically significant direct association; (↑), lower 95% CI = 1; ↓, statistically significant inverseassociation; A, birth year; B, sex; C, race; D, socio-economic status; E, age at diagnosis; F, residence; G, maternal age; H, maternal education; I, other types ofmeat (charcoal grilled foods, hamburgers, lunch meats and each other); J, mother’s body mass index just before pregnancy; K, vegetables; L, fruit; M, potentialconfounders; N, country of birth; O, energy intake; P, vitamin C; Q, each other; R, telephone exchange; S, food components and supplements.a‘Controls were pair matched to cases for telephone exchange, i.e. a series of telephone numbers was formed by retaining the area code exchange, and nexttwo digits of the phone number and randomly generating the final two digits’ (Kuitjen et al., 1990).


238

Z:\Customer\CABI\A4382 - d’Mello\A4491 - d’Mello #G.vp

12 March 2003 15:58:04



nitrates (OR = 0.54) or nitrites and primitiveneuroectodermal tumour in children, but anon-significant increased risk of 1.5 forhigh intake of nitrosamines. A parallel studyof astrocytic glioma in children (155 case–control pairs) was conducted by the sameinvestigators and interviewers using theidentical questionnaire (Bunin et al., 1994).Non-significant elevated risks between curedmeat and nitrite consumption during preg-nancy and risk of astrocytic glioma wereshown, but high vs. low intake of dimethyl-nitrosamines was associated with an OR of 0.8(95% CI = 0.4–1.6).

Adult brain tumours

More recently, the NOCs hypothesis has beenexpanded to include adult as well as child-hood brain cancer. The methods and theresults of these studies are described in Table10.2. Burch et al. (1987) studied 215 adultmales (25–80 years of age) and an equal num-ber of hospital-based controls. The studyincluded many dead cases. Thus, the qualityof dietary data was poor because of thelarge number of proxy respondents. Theinvestigators observed elevated risks forreported use of spring water (OR = 4.33(95% CI = 1.24–15.2)) and wine consumption(OR = 2.14 (95% CI = 1.28–3.60)) (ever vs.never) for brain tumours in general.Although wine and spring water consump-tion is consistent with a role for NOCs in theaetiology of brain tumours, for several otherfactors related to this hypothesis (e.g. con-sumption of processed meat and fish prod-ucts), no significant association was found.Preston-Martin et al. (1989) studied employ-ment histories and other potential risk factorsof 272 men aged 25–69 years with a primarybrain tumour first diagnosed during 1980–1984 in Los Angeles County. Separate analy-ses were carried our for 202 glioma pairsand 70 meningioma pairs. No significantdirect association between NOC-rich beer,wine and hard liquor consumption, and riskof gliomas or meningiomas in males wasobserved, but there was a significantinverse association of glioma with beerconsumption and a non- significant increasedrisk with hard liquor. A small Swedish

case–control study (Ahlbom et al., 1986)found a marginally significant OR of 2.1 (95%CI = 1.0–4.4) for the consumption of bacon orsmoked ham, and non-significant elevatedORs for the consumption of smoked sausageor fish when astrocytoma cases were com-pared with community controls. Deletingproxy data from the analysis of this studydid not affect the ORs. A German case–con-trol study (Boeing et al., 1993) revealed anincreased glioma risk associated with theconsumption of various NOCs (NDMA,N-nitrosopyrrolidine and N-nitrosopiperi-dine), but no association with endogenousN-nitrosation, i.e. consumption of nitrate ornitrite, was observed. In an Australian study,risk of glioma or meningioma in adults wasdecreased with consumption of beer, wine orspirits; for wine, these inverse associationswere statistically significant (Ryan et al.,1992). In a population-based case–controlstudy in Melbourne (Giles et al., 1994)comprising 416 gliomas, a significantlyelevated OR in men and a non-significantOR in women were associated with the intakeof NDMA. In women but not in men, theintake of nitrate was significantly inverselyassociated with gliomas. In men but notin women, the intake of nitrite showed anon-significant increased risk. A case–controlstudy in Los Angeles County (Blowers et al.,1997) of 94 women with gliomas foundthat risk increased with increasing consump-tion of cured meats and fish (OR = 1.7(95% CI = 0.8–3.8)), most notably of bacon(OR = 6.6 (95% CI = 1.9–22.5)), and estimatednitrite intake from cured meats (OR = 2.1(95% CI = 1.0–4.6)) but not significantlywith nitrite intake from all foods (OR = 1.4(95% CI = 0.6–3.5)). In a case–control study inIsrael, intake of NOCs was associated with anincreased risk of meningiomas (OR = 1.98(95% CI = 0.97–4.05)) but not gliomas(OR = 0.79 (95% CI = 0.32–1.96)) in adultsaged 18–75 years (Kaplan et al., 1997). Inthe case–control study by Lee et al. (1997), forboth men and women, glioma cases weremore likely than controls to be categor-ized as having a high risk diet (highconsumption of cured foods and low con-sumption of fruits and vegetables rich invitamin C, or high consumption of nitrite


20-Feb-03 10



20-Feb-03

10

224M

.Eichholzerand F.G

utzwiller

Reference andnumber of cases Brain tumour Dietary variable Comparison Association OR (95% CI)


Ahlbom et al. (1986)n = 78(population control)Burch et al. (1987)n = 215

Preston-Martin et al.(1989)n = 272

Ryan et al. (1992)n = 170

Boeing et al. (1993)n = 115

Astrocytomas

‘Brain tumour’

Gliomas (G)Meningiomas(M)

Gliomas (G)Meningiomas(M)

Gliomas

Bacon, smoked ham,smoked sausage,smoked fishSpring waterWine

Beer

Wine

Hard liquor

GliomasBeerWineSpirits

MeningiomasBeerWineSpirits

NitrateNitriteNDMANPYRNPIP

≥ 1 × week−1 vs. < 1 ×week−1

Ever vs. neverEver vs. never

> 1 × month−1 vs. less

Yes vs. no

Tertile 3 vs. tertile 1

(↑)NSNS↑↑

NS↓NSNSNSNS

NS↓NS

NS↓NSNSNS↑↑↑

2.1 (1.0–4.4)1.7 (0.9–3.1)1.5 (0.4–5.6)4.33 (1.24–15.2)2.14 (1.28–3.60)

G: 0.7 (0.5–1.2)M: 0.4 (0.1–0.9)G: 0.7 (0.5–1.1)M: 0.7 (0.3–1.4)G: 1.3 (0.8–1.9)M: 0.7 (0.3–1.4)0.77 (0.47–1.27)0.58 (0.38–0.91)0.78 (0.49–1.24)

0.51 (0.25–1.06)0.54 (0.30–0.97)0.66 (0.35–1.27)

0.9 (0.5–1.5)1.1 (0.6–2.0)2.8 (1.5–5.3)3.4 (1.8–6.4)2.7 (1.4–5.2)

A–C

A–E

A, C, F

A–C

A–C, G, H

Stockholm,Sweden, adults20–75 yearsSouthernOntario, Canada,adults 25–80yearsLos AngelesCounty, USA,men 25–69years

Adelaide,Australia, adults25–74 years

Germany, adults25–75 years

Table 10.2. Case–control studies on dietary intake of nitrates, nitrites and N-nitroso compounds (or the corresponding foods) and the risk of brain tumours inadults.

240





Dietary

Nitrogenous C

ompounds and C

ancer Risk

225

20-Feb-03

10

Giles et al. (1994)n = 416

Blowers et al. (1997)n = 94

Kaplan et al. (1997)n = 139Lee et al. (1997)n = 434

Gliomas

Gliomas

GliomasMeningiomasGliomas

MalesNitrateNitriteNDMA

FemalesNitrateNitriteNDMA

NitriteAll foodsCured meats

Nitrate

NOCs

Cured foods plusvitamin C-rich fruitand vegetables

Nitrite and vitamin C

Tertile 3 vs. tertile 1


≥ 1.06 vs. ≤ 0.75 µg1000 calories−1

High and low vs. low andhigh intake

menwomen

High and low vs. low andhigh intake

menwomen

NSNS↑

↓NSNS

NS(↑)NS

NSNS

↑NS

↑NS

1.13 (0.68–1.86)1.58 (0.96–2.58)1.78 (1.12–2.84)

0.53 (0.28–0.96)0.98 (0.55–1.72)1.45 (0.78–2.68)1.4 (0.6–3.5)2.1 (1.0–4.6)0.7 (0.2–1.8)

0.79 (0.32–1.96)1.98 (0.97–4.05)

2.0 (1.2–3.5)1.5 (0.8–2.7)

2.1 (1.1–3.8)1.5 (0.7–3.1)

A–C, G, H

A, C, F, I, J

A, B, F, K

A, B, F, L, M

Melbourne,Australia, adults20–70 years

Los AngelesCounty,California, USA,women 25–74yearsIsrael, adults18–75 yearsSan FranciscoBay Area,California, USA,adults ≥ 20 years

Cl, confidence intervals; NS, not statistically significant; ↑, statistically significant direct association; (↑), lower 95% CI = 1; ↓ statistically significant inverseassociation; A, birth year; B, sex; C, residence; D, marital status; E, date of diagnosis or death; F, race; G, alcohol consumption; H, smoking; I, body massindex; J, total grams of food; K, total energy intake; L, income; M, education; NMDA, N-nitrosodimethylamine; NPYR, N–nitrosopyrrolidine; NPIP,N–nitrosopiperidine.

241





and low vitamin C intake), although the asso-ciations were stronger and statistically signif-icant only in men.

Conclusions

In summary, the general impression from11 case–control studies on childhood braintumours and maternal diet during pregnancyis that mothers of cases were more likely thancontrols to consume cured meats. There isalso limited evidence that consumption ofcured meat in childhood increased risk ofbrain tumours in children, and all threestudies on adult brain tumours showed anincreased risk with intake of some sort ofcured meats. Intravenous administration ofNOCs induced gliomas in experimental ani-mals. Pregnant rats fed nitrites plus amidesproduced offspring at increased risk of braintumours, with the effect being suppressed byvitamins C and E, which interfered with invivo nitrosation reactions. It has been hypoth-esized that nitrite levels of cured meats in thestomach may be highly concentrated in therelative absence of vitamins. Conversely,nitrite formed in the saliva from nitratein vegetables may be considerably morediluted, and nitrosation inhibitors are pres-ent. This hypothesis would explain why, fornitrate and nitrite intake overall, no clear riskpattern emerged in the discussed studies andwhy in two studies nitrate/nitrite from curedmeat, but not total nitrate/nitrite, was relatedto brain tumour risk. A causal associationbetween cured meat and (childhood) braintumours cannot be concluded on the basis ofthe available data. Alternative explanations,such as chance findings by multiple statisticalcomparisons and effects of bias and confoun-ding, are possible. The potential for recallbias is a particular concern, given the wide-spread perception that at least some curedmeats are unhealthy. Parents of childrenwho developed brain cancer may over-report(or report more accurately than controls)their consumption of foods that are believedto be undesirable. The causes of brain cancersare not well understood, so few knownrisk factors could be considered confounders.Nevertheless, it is possible that additional

(dietary) factors, such as vegetable andfruit intake or some component of curedmeats other than pre-formed NOCs and NOCprecursors (e.g. heterocyclic amines), could atleast in part be responsible for the observedpositive association between cured meatand brain tumours. The present data, on theother hand, do not allow us to rule out thepossibility that cured meat consumptionmay increase risk of (childhood) brain cancer.Cohort studies, which limit recall bias andconsider potential confounders in the analy-ses, are needed to evaluate the effect of totalas well as specific cured meat intake on braintumour risk (Hennekens and Buring, 1987;Preston-Martin et al., 1996; Blowers et al.,1997; Lee et al., 1997; Bunin, 1998; Blot et al.,1999).

Stomach Cancer


Stomach cancer is the second most commonincident cancer and cause of cancer mortalitythroughout the world, with a distinct geo-graphical pattern. The highest incidence ratesare found in Japan, South America and east-ern Asia; intermediate rates are found, forexample, in Switzerland and France; and lowrates are found in North America, Canadaand Greece. The decline of stomach cancerrates over the past decades, most of allin developed countries, and the results ofmigrant studies suggest a predominantaetiological role for external environmentalfactors generally believed to be dietary. Dietshigh in vegetables and fruits and low in saltand the use of refrigeration are consideredto be the most effective means of preventingstomach cancer. An important non-dietaryrisk factor for stomach cancer is infectionwith the Helicobacter pylori bacterium. Otherpotential risk factors such as high consump-tion of grilled and barbecued meat and fishand cured meats are discussed. The stomachis an established site for NOC carcinogenesisin animals (Higginson et al., 1992; World Can-cer Research Fund and American Institute forCancer Research, 1997).


20-Feb-03 10




Dietary nitrate and risk of stomach cancer

Of six case–control studies that esti-mated dietary intake of nitrate and its associ-ation with stomach cancer risk (for refer-ences, see Eichholzer and Gutzwiller, 1998),all showed a decreased risk with high vs. lowconsumption, one of them when adjustedfor age, gender, vitamin C, β-carotene andα-tocopherol moving close to unity (Hanssonet al., 1994). Three of the inverse associationswere statistically significant (Risch et al., 1985;González et al., 1994; La Vecchia et al., 1994).These findings might result from the fact thatvegetables, the main source of nitrate, mightthemselves or some of their constituents pro-tect against gastric cancer (Block et al., 1992).Risch et al. (1985) adjusted their analysesonly for total food consumption (in grams)and ethnicity. González et al. (1994) and LaVecchia et al. (1994) adjusted only for totalenergy. The two existing cohort studies donot support a positive association betweendietary intake of nitrate and stomach cancerincidence. In the study by van Loon et al.(1998), the relative risk of the highest versusthe lowest quintile of intake from food was0.80 (95% CI = 0.47–1.37) (adjusted for age,sex, smoking, education, coffee consumption,intake of vitamin C and β-carotene, familyhistory of stomach cancer, prevalence ofstomach disorders, and use of refrigeratorand freezer). Similarly, in a Finnish cohortstudy, a non-significant inverse trend(P = 0.09) between dietary nitrate intake andrisk of stomach cancer was observed (relativerisk (RR) highest vs. lowest quartile = 0.56(95% CI = 0.27–1.18); adjusted for age, sex,municipality, smoking and energy intake)(Knekt et al., 1999). Despite the fact that, incontrast to vegetables, drinking water doesnot contain protective substances, in the studyby van Loon et al. (1998) nitrate intake fromdrinking water was associated with a slightlyreduced RR of 0.88 (95% CI = 0.59–1.32)(adjusted for the variables mentioned above).Similarly, Rademacher et al. (1992) found noassociation between nitrate levels in water(central or private water sources) and cancer

risk in a case–control study of Wisconsinresidents. This may be due to the fact that theplace of residence listed on the death certifi-cate (hospitals or nursing homes excluded)was assumed to be the source of the subjects’nitrate exposure via drinking water for atleast 20 years prior to death. Conversely,Boeing et al. (1991) did report in a Germancase–control study a significantly elevatedrisk for users of well water compared withusers of central water supplies at some timeduring a subject’s life (OR = 2.26 (95%CI = 1.19–4.28)). These results were adjustedonly for smoking of meat at home, years ofrefrigerator use, age, sex and hospital. Nitratecontent of drinking water was not measured,but analyses from other countries haveshown that private water sources cancontain considerable amounts of nitrate. Ina newer case–control study from Taiwan,nitrate content of drinking water was sig-nificantly associated with an increased riskof stomach cancer mortality (OR = 1.14 (95%CI = 1.04–1.25)) for those with nitrate levelshigher than 0.45 mg NO3-N l−1, when theresults were adjusted for urbanization levelof residence, sex, year of birth, year of deathand calcium and magnesium levels in drink-ing water (Yang et al., 1998).

Dietary nitrite and risk of stomach cancer

Of seven case–control studies that estimatedthe intake of nitrite (de Stefani et al., 1998;Eichholzer and Gutzwiller, 1998), fiveshowed an increased risk of stomach cancer.In the above-mentioned study of Risch et al.(1985), the positive association was statisti-cally significant (OR = 2.61 (95% CI = 1.61–4.22); adjusted for dietary fibre, chocolate,carbohydrates, no refrigerator, total food con-sumption and ethnicity). The same held truefor a case–control study conducted in theGreater Milan area (La Vecchia et al., 1997)when the interaction between methionineand nitrites was considered. Comparedwith subjects with low methionine andlow nitrite intake, the OR was 2.45 (95%CI = 1.9–3.2) in the high methionine and highnitrite stratum. Conversely, in a case–controlstudy in Uruguay (de Stefani et al., 1998), the


20-Feb-03 10



highest quartile of nitrite consumption wasassociated with a significantly decreased riskof stomach cancer when compared withthe lowest quartile (OR = 0.55 (95%CI = 0.48–0.62); adjusted for age, sex, resi-dence, urban/rural status, smoking, alcoholand ‘mate’ consumption). In one of the twoexisting cohort studies, multivariate analysis(including the variables mentioned above)revealed an RR of the highest versus thelowest quintile of nitrite intake of 1.44 (95%CI = 0.95–2.18) (van Loon et al., 1998). In thementioned Finnish cohort study by Knektet al. (1999), a slightly decreased risk of stom-ach cancer was observed in those with intakeof nitrite in the highest quartile comparedwith those in the lowest (RR = 0.71 (95%CI = 0.28–1.78); adjusted for age, sex, munici-pality, smoking and energy intake).

Dietary NOCs and risk of stomach cancer

Of five case–control studies that estimatedNOC intake, four showed a statisticallyincreased risk with high intake of NDMA(Eichholzer and Gutzwiller, 1998). In theFrench case–control study conducted byPobel et al. (1995), the OR for the third versusthe first tertile of intake was 7.00 (95%CI = 1.85–26.46; adjusted for age, sex, occupa-tion and total energy intake). Only dietaryexposure to NDMA was assessed, although itmay not be representative of the whole groupof pre-formed nitrosamines in food. In thestudy by González et al. (1994), it was sug-gested that high consumption of a protectivefactor, such as vitamin C, neutralizes theincreased risk related to the consumptionof pre-formed nitrosamines (OR = 2.09 in thehighest quartile, adjusted for total energy).In the study by La Vecchia et al. (1995) themultivariate OR for the highest NDMAintake tertile was 1.37 (95% CI = 1.1–1.7)including age, sex, education, family historyof gastric cancer, combined food score index,and intake of β-carotene, vitamin C and totalenergy, nitrite and nitrate. No information onH. pylori in cases and controls was available,although H. pylori antibody prevalence hasnot been shown to correspond to high riskareas of gastric cancer in Italy. In a morerecent case–control study in Uruguay (de

Stefani et al., 1998), NDMA intake was associ-ated with an increased risk of gastric cancer,with an OR = 3.6 (95% CI = 2.4–5.5) for thehighest category of exposure. The dose–response pattern was highly significant. Jointexposure to NDMA and heterocyclic amines(2-amino-1-methyl-6-phenylimidazo(4,5-b)-pyridine (PhIP)) displayed independenteffects by both chemicals, and their inter-action followed a multiplicative model withan elevated OR of 12.7 (95% CI = 7.7–21.2).When nutrients and related chemicals(methionine, nitrite, NDMA, PhIP, vitamin Cand β-carotene) were in the same modelsimultaneously, NDMA and PhIP were bothassociated with significantly elevated ORs.The only existing cohort study (Knekt et al.,1999) revealed a non-significant decreasedrisk of stomach cancer in those with a dietaryintake of NDMA in the highest quartile(RR = 0.75 (95% CI = 0.37–1.51)).

Conclusions

In summary, the toxicological data unequivo-cally show that pre-formed NOCs cause car-cinoma in animals. Four of five case–controlstudies that estimated NOC intake in humansshowed a statistically significant increasedrisk of stomach cancer with high intakeof NDMA. The only cohort study found aslightly decreased risk with high NDMAintake. Humans are exposed to pre-formedNOCs and to NOCs produced in vivo. Dietarynitrites and nitrates have been suggested tobe precursors of endogenous synthesis ofNOCs, and by this to increase human cancerrisk. In five of seven case–control studies onnitrite and gastric cancer risk, a positive asso-ciation was reported. In one of these studies,the association was statistically significant;conversely, another case–control studyshowed a significant decreased risk withhigh nitrite intake. One of two cohort studiesfound a non-significant increased, the othera slightly reduced stomach cancer risk withhigh intake of nitrite. In six case–control andtwo cohort studies, inverse associations werereported between nitrate intake and gastriccancer risk; in three studies, these resultswere statistically significant. These findingsmight result from the fact that vegetables –

20-Feb-03 10




the main source of nitrate – also contain pro-tective factors such as vitamin C. Basedon results from ecological studies, thehypothesis of an increased risk of stomachcancer with high intake of nitrate from drink-ing water (does not contain protectivefactors?) was postulated. So far, only threecase–control and one cohort study haveevaluated this hypothesis. Two of the case–control studies showed significant increasedrisks. Misclassification or low levels ofexposure (van Loon et al., 1998) could haveinfluenced the negative findings of theother studies. In addition, Yang et al. (1998)observed in their case–control study inverseassociations between calcium and magne-sium concentrations in drinking water andstomach cancer, i.e. drinking water could alsocontain protective factors. Overall, the associ-ation between nitrate in drinking water andstomach cancer risk should be evaluatedin additional case–control and cohort studieswith special emphasis on accurate estimationof exposure.

Nasopharyngeal Cancer


Nasopharyngeal carcinoma (NPC) is rarein most countries, including the USA andWestern Europe. NPC occurs in an endemicform in Chinese people in South-east Asia,Arabs in North Africa and in AlaskanInuits of mongoloid origin. Known andsuspected causes are genetic factors, Epstein–Barr virus (EBV), inhaled substances, smok-ing and diet, especially Cantonese saltedfish during childhood (Higginson et al., 1992;Vokes et al., 1997; World Cancer ResearchFund and American Institute for CancerResearch, 1997).


Several case–control studies (for referencessee Eichholzer and Gutzwiller, 1998) insouthern China, Malaysia, Hong Kongand Thailand demonstrated an association

between the consumption of salted fish,especially during weaning, and the risk ofNPC. Ning et al. (1990) observed in a case–control study performed in a low risk regionfor NCP that exposure to salted fish (evervs. never) was significantly associated withan increased risk of NPC (OR = 2.2 (95%CI = 1.3–3.7)). Controls were matched tocases by age, sex and race. The followingfour characteristics of exposure to salted fishindependently contributed to the increasedrisk: earlier age at first exposure, increasingduration and frequency of consumption andsteaming fish rather than frying, grilling orboiling it. Results were not adjusted for otherrisk factors. In a separate analysis, a signifi-cant increased risk was observed for the con-sumption of salted shrimp paste and saltedfish when adjusted for each other and forcarrot consumption, but not for infectionwith EBV and other factors. The case–controlstudy of Zheng et al. (1994) (88 NPC cases,176 age-, sex- and neighbourhood-matchedcontrols) was conducted in Zangwu County,Guangxi, China. The multivariate analysis(including use of wood fuel, consumptionof herbal tea and a socio-demographic score)found a significantly increased risk (OR = 3.8(95% CI = 1.5–9.8)) for the consumption ofsalted fish in rice porridge before the ageof 2 years. These results may be affected byrecall bias, as subjects provided data on theirdiet from almost 30 years previously. Addi-tionally, Sriamporn et al. (1992) conducted acase–control study with data from 120 NPCcases and the same number of hospital-, age-and sex-matched controls in North-east Thai-land, a region which shows an intermediaterisk for this neoplasm. The consumption ofsea-salted fish at least once a week versusnever in adult life was a significant risk factorfor nasopharyngeal cancer (OR = 2.5 (95%CI = 1.2–5.2); adjusted for alcohol, cigaretteconsumption, occupation, education andarea of residence). Again, EBV infection asa potential confounder was not assessed.Similarly, in a recent case–control study inShanghai, a region with intermediate risk forNPC (Yuan et al., 2000), adults who ate saltedfish at least once a week had an 82% increasein risk of NPC, relative to those who atesalted fish less than once a month (P = 0.07).


20-Feb-03 10



As already mentioned, rates of NPCcomparable with those in South-east Asiahave been reported in Inuit populations inCanada, Alaska and Greenland and in theArabs of northern Africa. Cantonese Chinese,Maghrebian Arabs and Inuits were comparedin anthropological studies by Hubert et al.(1993). It should be noted that, for example,the diet of Maghrebian Arabs is very differentfrom that of the Chinese, and does not includesalted fish. The conclusion of Hubert’s studywas that traditional preserved food prepara-tions could be the common factors linkingthese groups. Laboratory analyses of foodsamples of South China, Macao, Tunisia andGreenland revealed, among other things, thepresence of volatile nitrosamines (Poirier et al.,1987). In a third step of the study by Hubertet al. (1993), case–control studies in Tunisiaand China tested the hypotheses based onthese data. The results suggested that theconsumption in early youth of salted andpreserved foods other than salted fish, forexample, fermented fish sauce, salted shrimppaste, mouldy bean curd and two kinds ofpreserved plums, was also associated with anincreased risk of NPC (Eichholzer andGutzwiller, 1998). Correspondingly, a morerecent case–control study in Nagaland, India,revealed a direct association of NPC withconsumption of smoked meat (adjustedOR = 10.8 (95% CI = 3.0–39.0)) (Chelleng et al.,2000). Similarly, in the case–control study byYuan et al. (2000), in addition to salted fish(see above), subjects in the highest quartile ofintake of protein-containing preserved foodscompared with those with low intake (firstquartile) also experienced a statistically signif-icant 78% increase in risk of NPC (OR = 1.78(95% CI = 1.37–2.31)). When the joint effect ofpreserved food and oranges/tangerines onrisk of NPC was examined, subjects in thehighest tertile of preserved food and the low-est tertile of orange/tangerine intake had athreefold increase in risk (95% CI = 2.08–4.91)compared with those in the lowest tertileof preserved food and the highest tertile oforange/tangerine intake. In a case–controlstudy in Malaysian Chinese (Armstrong et al.,1998), consumption of four salted preservedfoods (fish, leafy vegetables, egg and root),fresh pork/beef organ meats, and beer and

liquor 5 years prior to diagnosis exhibitedsignificant positive associations with NPCrisk. The associations were less strong fordietary intake at age 10 years. In addition,in a case–control study in the USA, wherethe annual incidence of the disease is low,risk of non-keratinizing and undifferentiatedtumours of the nasopharynx was increasedin frequent consumers of preserved meats(including bacon, hot dogs and sausage),which contain high levels of added nitrites(RR: highest vs. lowest quartile 4.59 (95%CI = 0.78– 27.01); P trend 0.04). For squamouscell carcinoma, the corresponding RR was 1.15(95% CI = 0.46–2.87; P trend 0.58). The resultsindicate that future studies should considerthe effects of dietary risk factors on the riskof specific histological subsets of NPC, andnot assume that the disease is aetiologicallyhomogeneous (Farrow et al., 1998). Overall,studies have not estimated exposure to NOCsdirectly. In a recent case–control study inTaiwan (Ward et al., 2000), intake of nitro-samines and nitrite (based on 66 foods) as anadult was not associated with risk of NPC.High intake of nitrosamines and nitrites (fromfoods other than soy products, which containinhibitors of nitrosation) during childhoodand weaning were associated with signifi-cantly increased risks of NPC.

Conclusions

In high risk areas such as China, studieson NPC found elevated risks with higherconsumption of salted fish, particularlyduring childhood. Correspondingly, in 1997,the World Cancer Research Fund and theAmerican Institute of Cancer Research con-sidered the overall evidence that diets highin Cantonese-style salted fish increase therisk of NPC as convincing. Salted fish has ahigh level of secondary amines. These aminesare believed to interact with nitrite salts usedas preservatives, leading to the formation ofNOCs, which are possibly organotrophic forthe nasopharynx (World Cancer ResearchFund and American Institute of CancerResearch, 1997). This has been demonstratedin vivo by Yu et al. (1989), who induced malig-nant nasal cavity tumours in rats fed saltedfish. In areas with food habits very different

20-Feb-03 10




from those of Chinese people, such as thoseof Arabs in North Africa, or of the low risk USpopulation, other (traditionally) preservedfoods (meats, etc.) with high content ofNOCs may be of importance in the aetiol-ogy of NPC. As already discussed forbrain tumours, for the observed associationbetween preserved foods and NPC, alterna-tive explanations, such as confounding, arepossible. For example, the preserved meatsmost commonly consumed in the US diet,including bacon, hot dogs and sausage, areoften grilled or pan-fried, processes thatresult in the formation of heterocyclic amines;thus, the increased risk of NPC associatedwith these foods may result not from theirnitrite or nitrosamine content, but from themethods used for cooking them. So far, withfew exceptions, studies have not estimatedexposure to NOCs from the whole dietdirectly. This should be done in future stud-ies by simultaneously adjusting for dietarynitrosation inhibitors in the analyses.

Summary and Overall Conclusions

NOCs are potent carcinogens in animalstudies. Many cancer sites are suspected tobe related to NOCs in humans, but for mostcancer locations only a few epidemiologicalstudies exist. So far, high consumption ofcured meats and salted fish was associatedwith increased risk of brain tumours andNPC. Exogenous and endogenous exposureto NOCs is suspected to be the causal link,but dietary intake of NOCs and precursornitrates and nitrites has not yet been studiedadequately for these cancer sites. For stomachcancer, four of six epidemiological studiesshowed a significant increased risk withhigh intake of NDMA. For nitrite, the positiveassociation was weaker and, for nitrate fromvegetables (the main contributor to total dailyintake), a rather consistent inverse associa-tion was observed. Overall, a causal relation-ship between dietary NOC, nitrite and nitratecannot be concluded or excluded on the basisof the available data. Alternative explana-tions, such as effects of bias and confounding,are possible. The present studies are mainly

of case–control design, thus particularlyprone to recall and misclassification bias.Furthermore, it is possible that other (dietary)factors such as intake of vegetables, fruit andnitrosation inhibitors, or some componentof cured meat and salted fish other thanpre-formed NOCs and NOC precursors, forexample, heterocyclic amines, could at leastin part be responsible for the observed associ-ations. Cohort studies, which limit recall biasand consider potential confounders in theanalyses, are needed to evaluate properly theeffect of dietary NOC, nitrite and nitrate onhuman cancer risk. Meanwhile, present legalmeasures to limit overall dietary NOC expo-sure, and exposure to nitrites and nitrates asfood additives, are reasonable. As high intakeof vegetables and fruit is promoted for cancerprevention (World Cancer Research Fundand American Institute for Cancer Research,1997), restrictive legal nitrate levels in vegeta-bles, on the other hand, may be counteractive.Drinking water, another dietary contributorof nitrate, does not contain nitrosation inhibi-tors; thus its cancer risk should be evaluatedseparately.

References

Ahlbom, A., Lindberg Navier, I., Norell, S., Olin, R.and Spännare, B. (1986) Nonoccupational riskindicators for astrocytomas in adults. AmericanJournal of Epidemiology 124, 334–337.

Armstrong, R.W., Imrey, P.B., Lye, M.S.,Armstrong, M.J., Yu, M.C. and Sani, S. (1998)Nasopharyngeal carcinoma in Malaysian Chi-nese: salted fish and other dietary exposures.International Journal of Cancer 77, 228–235.

Bartsch, H. (1991) N-nitroso compounds and humancancer: where do we stand? In: O’Neill, I.K.,Chen, J. and Bartsch, H. (eds) Relevance toHuman Cancer of N-Nitroso Compounds, TobaccoSmoke and Mycotoxins. IARC Scientific Publica-tion no. 105. IARC, Lyon, France, pp. 1–10.

Bartsch, H., Ohshima, H. and Pignatelli, B. (1988)Inhibitors of endogenous nitrosation: mecha-nisms and implications in human cancerprevention. Mutation Research 1202, 307–324.

Bartsch, H., Ohshima, H., Pignatelli, B. and Calmels,S. (1992) Endogenously formed N-nitrosocompounds and nitrosating agents in humancancer etiology. Pharmacogenetics 2, 272–277.


20-Feb-03 10



Block, G., Patterson, B. and Subar, A. (1992) Fruit,vegetables, and cancer prevention: a reviewof the epidemiological evidence. Nutrition andCancer 18, 1–29.

Blot, W.J., Henderson, B.E. and Boice, J.D. (1999)Childhood cancer in relation to cured meatintake: review of the epidemiological evi-dence. Nutrition and Cancer 34, 111–118.

Blowers, L., Preston-Martin, S. and Mack, W.J.(1997) Dietary and other lifestyle factors ofwomen with brain gliomas in Los AngelesCounty (California, USA). Cancer Causes andControl 8, 5–12.

Boeing, H., Frentzel-Beyme, R., Berger, M., Berndt,V., Göres, W., Körner, M., Lohmeier, R.,Menarcher, A., Männl, H.F.K., Meinhardt, M.,Müller, R., Ostermeier, H., Paul, F.,Schwemmle, K., Wagner, K.H. andWahrendorf, J. (1991) Case–control studyon stomach cancer in Germany. InternationalJournal of Cancer 47, 858–864.

Boeing, H., Schlehofer, B., Blettner, M. andWahrendorf, J. (1993) Dietary carcinogensand the risk for glioma and meningiomain Germany. International Journal of Cancer53, 561–565.

Bogovski, P. and Bogovski, S. (1981) Animal speciesin which N-nitroso compounds induce cancer.International Journal of Cancer 27, 471–474.

Bunin, G.R. (1998) Maternal diet during pregnancyand risk of brain tumors in children. Interna-tional Journal of Cancer 11 (Supplement), 23–25.

Bunin, G.R., Kuijten, R.R., Buckley, J.D., Rorke, K.B.and Meadows, A.T. (1993) Relation betweenmaternal diet and subsequent primitiveneuroectodermal brain tumors in youngchildren. New England Journal of Medicine 329,536–541.

Bunin, G.R., Kuijten, R.R., Boesel, C.P., Buckley, J.D.and Meadows, A.T. (1994) Maternal diet andrisk of astrocytic glioma in children: a reportfrom the Children’s Cancer Group (UnitedStates and Canada). Cancer Causes and Control5, 177–187.

Burch, J.D., Craib, K.J.P., Choi, B.C.K., Miller, A.B.,Risch, H.A. and Howe, G.R. (1987) An explor-atory case–control study of brain tumors inadults. Journal of National Cancer Institute 78,601–609.

Chelleng, P.K., Narain, K., Das, H.K., Chetia, M.and Mahanta, J. (2000) Risk factors for cancernasopharynx: a case–control study fromNagaland, India. National Medical Journal ofIndia 13, 6–8.

Cordier, S., Iglesias, M.J., Le Goaster, C., Guyot,M.M., Mandeveau, L. and Heman, D. (1994)Incidence and risk factors for childhood brain

tumors in the Ile de France. International Journalof Cancer 59, 776–782.

Council of Europe (1995) Health Aspects of Nitratesand its Metabolites (Particularly Nitrite). Proceed-ings of an International Workshop. Council ofEurope Press, Bilthoven, The Netherlands.

de Stefani, E., Boffetta, P., Mendilaharsu, M.,Carzoglio, J. and Deneo-Pellegrini, H. (1998)Dietary nitrosamines, heterocyclic amines,and risk of gastric cancer: a case–control studyin Uruguay. Nutrition and Cancer 30, 158–162.

Eichholzer, M. and Gutzwiller, F. (1998) Dietarynitrates, nitrites, and N-nitroso compoundsand cancer risk: a review of the epidemiologicevidence. Nutrition Reviews 56, 95–105.

Farrow, D.C., Vaughan, T.L., Berwick, M., Lynch,C.F., Swansan, G.M. and Lyon, J.L. (1998) Dietand nasopharyngeal cancer in a low-riskpopulation. International Journal of Cancer 78,675–679.

Foster, A.M., Prentice, A.G., Copplestone, J.A.,Cartwright, R.A. and Ricketts, C. (1997) Thedistribution of leukaemia in associationwith domestic water quality in South WestEngland. European Journal of Cancer Prevention6, 11–19.

Gangolli, S.D., van den Brandt, P.A., Feron, V.J.,Janzowsky, C., Koeman, J.H., Speijers, G.J.A.,Spiegelhalder, B., Walker, R. and Wishnok, J.S.(1994) Nitrate, nitrite and N-nitroso com-pounds. European Journal of Pharmacology,Environmental Toxicology and PharmacologySection 292, 1–38.

Giles, G.G., McNeil, J.J., Donnan, G., Webley, C.,Staples, M.P., Ireland, P.D., Hurley, S.F. andSalzberg, M. (1994) Dietary factors and the riskof glioma in adults: results of a case–controlstudy in Melbourne, Australia. InternationalJournal of Cancer 59, 357–362.

González, C.A., Riboli, E., Badosa, J., Batiste, E.,Cardona, T., Pita, S., Sanz, J.M., Torrent, M.and Agudo, A. (1994) Nutritional factors andgastric cancer in Spain. American Journal ofEpidemiology 139, 466–473.

Hansson, L.E., Nyrén, O., Bergström, R., Wolk, A.,Lindgren, A., Baron, J. and Adami, H.O.(1994) Nutrients and gastric cancer risk. Apopulation-based case–control study inSweden. International Journal of Cancer 57,638–644.

Hecht, S.S. (1997) Approaches to cancer preventionbased on an understanding of N-nitrosaminecarcinogenesis. Proceedings of the Society forExperimental Biology and Medicine 216, 181–191.

Hennekens, C.H. and Buring, J.E. (1987) Epidemiol-ogy in Medicine. Little, Brown and Company,Boston.


20-Feb-03 10



Higginson, J., Muir, C.S. and Munoz, N. (1992)Human Cancer: Epidemiology and EnvironmentalCauses. Cambridge Monographs on CancerResearch. Cambridge University Press,Cambridge.

Hill, M.J. (1999) Nitrate toxicity: myth or reality?Invited commentary. British Journal of Nutrition81, 343–344.

Howe, G.R., Burch, J.D., Chiarelli, A.M., Risch, H.A.and Choi, B.C.K. (1989) An exploratorycase–control study of brain tumors in children.Cancer Research 49, 4349–4352.

Hubert, A., Jeannel, D., Tuppin, P. and de Thé, G.(1993) Anthropology and epidemiology: apluridisciplinary approach of environmentalfactors of nasopharyngeal carcinoma. In:Tursz, T., Pagano, J.S., Ablashi, G., de Thé, G.,Lenoir, G. and Pearson, G.R. (eds) TheAssociation of Epstein–Barr Virus and Diseases.John Libbey, Colloque INSERM, Vol. 225,Montrouge, pp. 775–788.

Ikins, W.G., Gray, J.I., Mandagere, A.K., Booren,A.M., Pearson, A.M. and Stachiw, M.A. (1986)N-Nitrosamine formation in fried bacon pro-cessed with liquid smoke preparations. Journalof Agriculture and Food Chemistry 34, 980–985.

Kaplan, S., Novikov, I. and Modan, B. (1997) Nutri-tional factors in the etiology of brain tumors.American Journal of Epidemiology 146, 832–841.

Knekt, P., Järvinen, R., Dich, J. and Hakulinen, T.(1999) Risk of colorectal and other gastro-intestinal cancers after exposure to nitrate,nitrite and N-nitroso compounds: a follow-upstudy. International Journal of Cancer 80,852–856.

Kuijten, R.R., Bunin, G.R., Nass, C.C. and Meadows,A.T. (1990) Gestational and familial risk factorsfor childhood astrocytoma: results of acase–control study. Cancer Research 50,2608–2612.

La Vecchia, C., Ferraroni, M., D’Avanzo, B., Decarli,B. and Franceschi, S. (1994) Selectedmicronutrient intake and the risk of gastriccancer. Cancer Epidemiology, Biomarkers andPrevention 3, 393–398.

La Vecchia, C., D’Avanzo, B., Airoldi, L., Airoldi, L.,Braga, C. and Decarli, A. (1995) Nitrosamineintake and gastric cancer risk. European Journalof Cancer Prevention 4, 469–474.

La Vecchia, C., Negri, E., Franceschi, S. and Decarli,A. (1997) Case–control study on influenceof methionine, nitrite, and salt on gastriccarcinogenesis in Northern Italy. Nutrition andCancer 27, 65–68.

Law, G., Parslow, R., McKinney, P. and Cartwright,R. (1999) Non-Hodgkin’s lymphoma andnitrate in drinking water: a study in Yorkshire,

United Kingdom. Journal of Epidemiology andCommunity Health 53, 383–384.

Lee, M., Wrensch, M. and Miike, R. (1997) Dietaryand tobacco risk factors for adult onset gliomain the San Francisco Bay Area (California,USA). Cancer Causes and Control 8, 13–24.

Lubin, F., Farbstein, H, Chetrit, A., Farbstein, M.,Freedman, L. and Alfandary, E. (2000) The roleof nutritional habits during gestation andchild life in pediatric brain tumor etiology.International Journal of Cancer 86, 139–143.

McCredie, M., Maisonneuve, P. and Boyle, P. (1994)Antenatal risk factors for malignant braintumours in New South Wales children. Inter-national Journal of Cancer 56, 6–10.

McKnight, G.M., Duncan, C.W., Leifert, C. andGolden, M.H. (1999) Dietary nitrate in man:friend or foe? British Journal of Nutrition 81,349–358.

Mirvish, S.S. (1995) Role of N-nitroso compounds(NOC) and N-nitrosation in etiology of gastric,esophageal and bladder cancer and contri-bution to cancer of known exposure to NOC.Cancer Letters 93, 17–48.

Mohsen, M., Hassan, A., El-Sewedy, S., Aboul-Azm,T., Magagnotti, C. and Fanelli, R. (1999)Biomonitoring of N-nitroso compounds,nitrite and nitrate in the urine of Egyptian blad-der cancer patients with or without Schistosomahaematobium infection. International Journal ofCancer 82, 789–794.

Moller, H. (1997) Work in agriculture, childhoodresidence, nitrate exposure, and testicularcancer risk: a case–control study in Denmark.Cancer Epidemiology, Biomarkers and Prevention6, 141–144.

Ning, J.P., Yu, M.C., Wang, Q.S. and Henderson,B.E. (1990) Consumption of salted fishand other risk factors for nasopharyngealcarcinoma (NPC) in Tianjin, a low-risk regionfor NPC in the People’s Republic of China.Journal of the National Cancer Institute 82,291–296.

Pobel, D., Riboli, E., Cornée, J., Hemon, B. andGuyader, M. (1995) Nitrosamine, nitrateand nitrite in relation to gastric cancer: acase-control study in Marseille, France.European Journal of Epidemiology 11, 67–73.

Poirier, S., Ohshima, H., de Thé, G., Hubert, A.,Bourgade, M.C. and Bartsch, H. (1987)Volatile nitrosamine levels in common foodsfrom Tunisia, South China and Greenland,high-risk areas for nasopharyngeal carcinoma(NPC). International Journal of Cancer 39,293–296.

Preston-Martin, S., Yu, M.C., Benton, B. andHenderson, B.E. (1982) N-Nitroso compounds


20-Feb-03 10



and childhood brain tumors: a case–controlstudy. Cancer Research 42, 5240–5245.

Preston-Martin, S., Mack, W. and Henderson,B.E. (1989) Risk factors for gliomas andmengingiomas in males in Los AngelesCounty. Cancer Research 49, 6137–6143.

Preston-Martin, S., Pogoda, J.M., Mueller, B.A.,Holly, E.A., Lijinsky, W. and Davis, R.L. (1996)Maternal consumption of cured meats andvitamins in relation to pediatric brain tumors.Cancer Epidemiology, Biomarkers and Prevention5, 599–605.

Rademacher, J.J., Young, T.B. and Kanarek, M.S.(1992) Gastric cancer mortality and nitratelevels in Wisconsin drinking water. Archives ofEnvironmental Health 47, 292–294.

Risch, H.A., Jain, M., Choi, N.W., Fodor, J.G., Pfeifer,C.J., Howe, G.R., Harrison, L.W., Craib, K.J.P.and Miller, A.B. (1985) Dietary factors and theincidence of cancer of the stomach. AmericanJournal of Epidemiology 122, 947–959.

Roberts-Thomson, I.C., Butler, W.J. and Ryan, P.(1999) Meat, metabolic genotypes and risk ofcolorectal cancer. European Journal of CancerPrevention 8, 207–211.

Ryan, P., Lee, M.W., North, J.B. and McMichael, A.J.(1992) Risk factors for tumors of the brainand meninges: results from the Adelaideadult brain tumor study. International Journal ofCancer 51, 20–27.

Sarasua, S. and Savitz, D.A. (1994) Cured andbroiled meat consumption in relation tochildhood cancer: Denver, Colorado (UnitedStates). Cancer Causes and Control 5, 141–148.

Schymura, M.J., Zheng, D. and Baptiste, M.S. (1996)A case–control study of childhood braintumors and maternal life-style. AmericanJournal of Epidemiology S8, 143 (abstract).

Sriamporn, S., Vatanasapt, V., Pisani, P.,Yongchaiyudha, S. and Rungpitarangsri, V.(1992) Environmental risk factors for naso-pharyngeal carcinoma: a case–control studyin Northeastern Thailand. Cancer Epidemiology,Biomarkers and Prevention 1, 345–348.

van Loon, A.J.M., Botterweck, A.A.M., Goldbohm,R.A., Brants, H.A.M., van Klaveren, J.D. andvan den Brandt, P.A. (1998) Intake of nitrate

and nitrite and the risk of gastric cancer: a pro-spective cohort study. British Journal of Cancer78, 129–135.

Vokes, E.E., Liebowitz, D.N. and Weichselbaum,R.R. (1997) Nasopharyngeal carcinoma. Lancet350, 1087–1091.

Walker, R. (1990) Nitrates, nitrites and N-nitrosocompounds: a review of the occurrence in foodand diet and the toxicological implications.Food Additives and Contaminants 7, 717–768.

Ward, M.H., Pan, W.H., Cheng, Y.J., Li, F.H.,Brinton, L.A., Chen, C.J., Hsu, M.M.,Chen, I.H., Levine, P.H., Yang, C.S. andHildesheim, A. (2000) Dietary exposure tonitrite and nitrosamines and risk of nasopha-ryngeal carcinoma in Taiwan. InternationalJournal of Cancer 86, 603–609.

World Cancer Research Fund and AmericanInstitute for Cancer Research (1997) Food,Nutrition and the Prevention of Cancer: a GlobalPerspective. American Institute for CancerResearch, Washington, DC.

Yang, C.Y., Cheng, M.F., Tsai, S.S. and Hsieh, Y.L.(1998) Calcium, magnesium, and nitrate indrinking water and gastric cancer mortality.Japanese Journal of Cancer Research 89,124–130.

Yu, M.C., Nichols, P.W., Zou, X.N., Estes, J. andHenderson, B.E. (1989) Induction of malignantnasal cavity tumours in Wistar rats fed Chinesesalted fish. British Journal of Cancer 60, 198–201.

Yuan, J., Gago-Dominguez, M., Castelo, J.,Hankin, J.H., Ross, R.K. and Yu, M.C. (1998)Cruciferous vegetables in relation to renal cellcarcinoma. International Journal of Cancer 77,211–216.

Yuan, J.M., Wang, X.L., Xiang, Y.B., Gao, Y.T.,Ross, R.K. and Yu, M.C. (2000) Preserved foodsin relation to risk of nasopharyngeal carcinomain Shanghai, China. International Journal ofCancer 85, 358–363.

Zheng, Y.M., Tuppin, P., Hubert, A., Jeannel, D.,Pau Y.J., Zeng, Y. and de Thé, G. (1994)Environmental and dietary risk factors fornasopharyngeal carcinoma: a case–controlstudy in Zangwu County, Guangxi, China.British Journal of Cancer 69, 508–514.

20-Feb-03 10




11 Adverse Reactions to Food Additives

R.A. Simon1* and H. Ishiwata21Division of Allergy, Asthma and Immunology, Scripps Clinic, La Jolla, California, USA;

2Division of Food Additives, National Institute of Health Sciences, Tokyo, Japan

Introduction

Food additives are different from other com-pounds shown in other chapters in view ofthe fact that food additives are added inten-tionally with some purpose, whereas othercompounds described in other chapters suchas polychlorinated biphenyls, dioxins andpolycyclicaromatic hydrocarbons occur ascontaminants. The Codex Alimentarius bythe Joint FAO/WHO Codex AlimentariusCommission (1991) defined food additives asfollows.

‘Food additive’ means any substance notnormally consumed as a food by itself andnot normally used as a typical ingredient ofthe food, whether or not it has nutritive value,the intentional addition of which to foodfor a technological (including organoleptic)purpose in the manufacture, processing,preparation, treatment, packing, packaging,transport or holding of such food results,or may be reasonably expected to result(directly or indirectly) in it or its by-productsbecoming a component of or otherwiseaffecting the characteristics of such foods.

The term does not include ‘contaminants’ orsubstances added to food for maintaining orimproving nutritional qualities. The defini-tions of food additives, however, differ withcountries. Some countries allow nutrients asfood additives. Postharvest pesticides are

allowed as food additives in some countries,but some other countries categorize themas residual pesticides. A food act in eachcountry takes precedence over the CodexAlimentarius.

Estimates are that 2000–20,000 agents areadded to the food that we consume (Collins-Williams, 1983). These include preservatives,stabilizers, conditioners, thickeners, colour-ings, flavourings, sweeteners and antioxi-dants (Box 11.1). Despite such enormousexposure to these agents, only a small numberhave been associated with hypersensitivityreactions. In a questionnaire study of UShouseholds (Altman, 1996), self-reportedadverse reactions to food additives in familymembers ranged from 1.2% for food dyes andcolourings to 2.7% for monosodium gluta-mate (MSG). Because the perceived adversereactions to food additives were not verifiedby appropriate diagnostic challenge proce-dures, the true frequency of food additivereactions in the general population remainslargely unknown. In a Dutch study thatstarted with a survey of 1483 Dutch adultsand proceeded through clinical challengetrials, only three individuals were identifiedwith food additive sensitivities (NiestijlJansen et al., 1993), which amounts to 0.2%of the population. In a large study of foodadditive-induced sensitivities that startedwith a survey of 4274 Danish schoolchildren


20-Feb-03 11




and proceeded through clinical trials, anintolerance to food additives confirmed bydouble-blind challenge occurred in 2% of thechildren selected from the survey on the basisof atopic history but only in 0.13% of the entiresurveyed population (Fuglsang et al., 1993,1994). Young et al. (1987) evaluated the preva-lence of sensitivities to food additives among aBritish population using a combination of asurvey questionnaire given to 18,582 individ-uals and a series of mixed additive challengesconducted at home with self-reporting ofsymptoms. They estimated the prevalenceof adverse reactions to food additives as0.01–0.23% (Young et al., 1987). Thus, food

additive-induced sensitivities seem to occurrarely in the overall population.

For years, some investigators have sug-gested that a significant number of patientswith asthma or chronic urticaria and angio-oedema have symptoms caused by theingestion of food additives. Despite manystudies that have attempted to establishthe prevalence of reactions to additives inpatients, the true incidence of reactionsremains unknown. This is due primarily to thelack of properly controlled studies. Althoughmany anecdotal reports exist, rigorouslycontrolled studies are rarely found in thisarea of clinical investigation.

Box 11.2 lists the additives that have beenreported to be most commonly associatedwith adverse reactions. Figure 11.1 illustratesthe chemical structure of selected additives.A common chemical structure does not linkthese compounds together into a single mol-ecular configuration. These agents will be

20-Feb-03 11

236 R.A. Simon and H. Ishiwata

Box 11.1. Common food additives.

AntioxidantsButylated hydroxyanisole (BHA)Butylated hydroxytoluene (BHT)

Extraction solventsDichloromethane (methylene chloride)Trichloroethylene (TCE)

Flavouring agentsTrans-anetholeBenzyl acetate(+)-Carvone and (−)-carvoneEthylmethylphenolglycidate

Food colour: FD&C dyesTartrazine (FD&C yellow no. 5)Erythrosine (FD&C red no. 3)Indigotin (FD&C blue no. 2)

PreservativesBenzoatesSulphites

Sweetening agentsAspartameHydrogenated glucose syrupSaccharin

Thickening agentsEthyl celluloseKaraya gumTragacanth gum

Miscellaneous food additivesAmmonium phosphate, monobasic(monoammonium orthophosphate)Insoluble polyvinylpyrrolidone or polyvinylpolypyrrolidone (PVPP)Polyvinylpyrrolidone (PVP) (Polyvidone)Potassium bromateL-(+) Tartaric acid, ammonium, calciumand magnesium salts

Box 11.2. Additives most commonly associ-ated with adverse reactions.

FD&C dyesAzo dyes

Tartrazine (FD&C yellow no. 5)Ponceau (FD&C red no. 4)Sunset yellow (FD&C yellow no. 6)Amaranth (FD&C red no. 5)

Non-azo dyesBrilliant blue (FD&C blue no. 1)Erythrosine (FD&C red no. 3)Indigotin (FD&C blue no. 2)

Parabens/benzoatesParahydroxy benzoic acid

Methyl parabenEthyl parabenButyl paraben

Sodium benzoateHydroxy benzoic acid

Butylated hydroxyanisole (BHA)Butylated hydroxytoluene (BHT)NitratesNitritesMonosodium glutamate (MSG)Sulphites

Sulphur dioxideSodium sulphite, potassium sulphite,bisulphite, metabisulphite



Adverse Reactions to Food Additives 237

20-Feb-03 11

Fig. 11.1. Chemical structures of selected additives.



discussed individually as they relate tourticaria and angio-oedema, anaphylaxis oranaphylactoid reactions, and asthma.

General Considerations and Descriptionsof General Food and Drug Additives

Food colours

Both synthetic and natural colours are usedfor food colouring. Synthetic food colours,coal-tar colours, need a certification by theofficial chemical examination. Many coun-tries allow 10–15 synthetic food colours, butallowable colours differ in every country.Some food colours are prepared as alu-minium lakes. Caramel, carrot carotene,turmeric, annatto extract, etc. are naturalcolours. Food colour changes easily by heat-ing or oxidation, and thus food colours areused to compensate for colours of food. Foodcolours are also used to sharpen the con-sumer’s appetite.

Dyes

Dyes approved by the Food Dye andColoring Act (FD&C) are coal tar derivatives,the best known of which is tartrazine (FD&Cyellow no. 5). All dyes contain aromatic rings.In addition to tartrazine, the azo dyes(containing N:N-linkages) include ponceau(FD&C red no. 4) and sunset yellow (FD&Cyellow no. 6). Amaranth (FD&C red no. 5)was banned from use in the USA in 1975because of claims of carcinogenicity. Non-azodyes include brilliant blue (FD&C blue no. 1),erythrosine (FD&C red no. 3) and indigotin(FD&C blue no. 2).

Tartrazine (CI food yellow 4, FD&C yellowno. 5, EEC no. E102)

The structural formula is given in Fig. 11.1(a).Molecular weight: 534.37 Da. Description:light orange powder or granules. The λmaximum of 10 mg l−1 in 0.15% ammonium

acetate solution is 426–430 nm. Functionaluse: food colour, a mono azo colour. Naturaloccurrence: not known. Use: one of mostwidely used food colours in the world. Usedalone or with other food colours in softdrinks, sweets, salted vegetables, jam, con-fections and a variety of processed foods.Acceptable daily intake (ADI) 0–7.5 mg kg−1

body weight (BW).

Erythrosin (CI food red 14, FD&C red no. 3,EEC no. E127)

The structural formula is given in Fig. 11.1(b).Molecular weight: 897.88 Da. Description: redpowder or granules. The λ maximum of 3 mgl−1 in 0.15% ammonium acetate solutionis 524–528 nm. Functional use: food colour,a xanthene colour. Natural occurrence: notknown. Use: used alone or with other foodcolours in canned fruit cocktail, cannedcherry, ice cream, sherbets and confections.This colour precipitates in acidic conditionsand thus is not used in acidic beverages ordrops. ADI: 0–0.1 mg kg−1 BW.

Brilliant blue FCF (CI food blue 2, FD&C blueno. 1, EEC no. E133)

The structural formula is given in Fig. 11.1(c).Molecular weight: 792.86 Da. Description:blue powder or granules. The λ maximum of5 mg l−1 in 0.15% ammonium acetate solutionis 628–632 nm. Functional use: food colour,a triphenylmethane colour. Natural occur-rence: not known. Use: used alone or withother food colours in confections, soft drinks,sweets and bakery products. Stable to light,heat, salts and acids. Usable in acidic bever-ages or drops. ADI: 0–12.5 mg kg−1 BW.

Sulphites/sulphur dioxide

In AD 79, the respiratory death of Pliny theElder was attributed to the sulphur dioxide(SO2)-rich gases emanating from the eruptionof Mount Vesuvius. Sulphur oxides, includ-ing SO2 and particulate sulphuric acid(H2SO4), are generated from the combustion

20-Feb-03 11




of sulphur-containing fossil fuels and maybe significant aeropollutants for asthmaticpersons (Boushey, 1982a; Sheppard, 1988).Plumes of exhaust fumes in the vicinity ofpower plants and smelters reach concen-tration peaks of 0.5–1 ppm SO2 for 5–10 minintervals and yet do not exceed the NationalAmbient Air Quality standard of 0.14 ppmaverage for 24 h (Balmes et al., 1987).

Sulphur dioxide gas inhalation chal-lenges in asthmatic subjects induce broncho-constriction dependent on the concentrationof SO2 (Shepphard et al., 1980; Fine et al.,1987), the minute ventilation at which it isinhaled (Linn et al., 1987) and the severity ofasthma as measured by non-specific bronchialhyper-reactivity to histamine or methacholine(Sher and Schwartz, 1985). The responseoccurs more readily with oral than withnasal breathing (Bethel et al., 1983a) and ismitigated during breathing at high tempera-ture, high humidity as compared withlow temperature, low humidity conditions(Linn et al., 1985). The maximal broncho-constrictor response occurs over 5–10 minand, if not sufficient to produce symptomsnecessitating bronchodilator treatment, itdoes not progress over ensuing hours ofcontinued exposure. Eucapnic hyperven-tilation or moderate exercise while breathingSO2 at 0.5–1 ppm has produced clinicallysignificant bronchoconstriction in mostasthmatics studied (Bethel et al., 1983a,b; Linnet al., 1983, 1985; Balmes et al., 1987; Fine et al.,1987); at 0.25 ppm, the effect is small (Bethelet al., 1985).

Likewise, sulphuric acid aerosols at aconcentration of 1000 µg m−3, the thresholdlimit value of the Environmental ProtectionAgency for occupational exposure, producesignificant declines of forced expiratoryvolume in 1 s (FEV1) in asthmatic subjects(Utell et al., 1983), whereas aerosols at 100 µgm−3, the ‘worst case’ for ambient exposure,do so only in asthmatic subjects duringexercise (Koenig et al., 1983; Utell et al.,1991; Hanley et al., 1992). The prevalenceof persistent cough and phlegm is signifi-cantly higher among adults living in aUtah community near a smelter, withexposure at the 100 µg m−3 mean annual level,

than among subjects in communities withexposure at one-third of that level or less(Chapman et al., 1985).

The effects, if any, on the induction ormaintenance of bronchial hyper-reactivity oflesser concentrations of SO2 and sulphuricacid particles – for example, the annual aver-ages in urban southern California are in therange of 0.001–0.01 ppm SO2 – singly or incombination with other aeropollutants orother factors associated with asthma, such asatopic state and viral respiratory infection,have not yet been clearly elucidated.

The term sulphiting agents is used todescribe SO2 and several inorganic sulphitesthat may be added to foods, beverages andpharmaceuticals. Until recently, SO2 and fivesulphite salts (sodium sulphite SO3, sodium orpotassium bisulphite HSO3, or metabisulphiteS2O5) have been listed in the Code of FederalRegulations (1984) as ‘generally recognized assafe’ (GRAS) with the provision that they arenot be used on foods considered to be a sourceof thiamine (vitamin B1).

SO2 is a non-flammable, colourless gasthat readily dissolves in water and undergoeshydration to form sulphurous acid, whichthen dissociates to bisulphite and sulphite(Schroeter, 1966). Under physiological condi-tions at pH 7.4, sulphite is the predominantchemical species. However, in acid solution(e.g. the gastric lumen with pH near 1),sulphite undergoes proton association to formbisulphite and sulphurous acid. The latterdehydrates once again to form SO2 (Schroeter,1966). In addition to acid pH, the generation ofSO2 is also enhanced by heat.

Because of this interchangeability, sul-phite concentrations in food may be expressedas parts per million (ppm) of SO2 or, viceversa, SO2 content can be expressed as SO2

equivalent (SDE) or milligrams of sulphite.The conversion is ppm of SO2 = mg of sulphiteper kg of food.

Sulphites are highly reactive and com-bine with a number of biological compoundsincluding carbohydrates and pyridinonucleo-tides (Schroeter, 1966). Sulphites also reactwith disulphide bonds present in proteins(Cecil, 1963). Therefore, when identifyingsulphite (or SO2) concentrations in foods,


20-Feb-03 11



results will be expressed as free or bound SO2.The bound SO2 is usually reported as totalSO2, although, even with the harshest ofchemical extractions, all potential SO2 formsprobably cannot be measured. The impor-tance of bound sulphites as causing sensitivityreactions is difficult to evaluate.

Use of sulphites in foods and beverages

Sulphiting agents are still widely used inthe food and beverage industry (Box 11.3).Sulphites can inhibit a number of enzymatic

reactions, for example polyphenoloxidase,ascorbate oxidase, lipoxygenase and per-oxidase (Cecil, 1963). Although themechanism of action is unknown, sulphiteinhibition of polyphenoloxidase is importantin the control of enzymatic browning. Thiswas the primary reason for adding sulphitesto items in salad bars, such as lettuce,avocados and guacamole, as well as to cutpotatoes and apples, fresh mushrooms andtable grapes (Komanowsky et al., 1970;Ponting et al., 1971; Nelson, 1983; Taylor andBush, 1983). The enzyme tyrosinase, a type of

20-Feb-03 11


Box 11.3. Sulphite-containing foods and drugs.

Foods

High contentDried fruit (excluding dark

raisins and prunes)Lemon juice (non-frozen)Lime juice (non-frozen)WineMolassesSauerkraut juiceGrape juice (white, white

sparkling, pink sparkling,red sparkling)

Moderate contentDried potatoesWine vinegarGravies, saucesFruit toppingMaraschino cherriesPectinShrimp (fresh)SauerkrautPickled peppersPickled cocktail onionsPickles/relishes

Low content (< 10 ppm)a

Corn starchDry maizeFrozen potatoesMaple syrupImported jams and jelliesFresh mushroomsMalt vinegarDried codCanned potatoesBeerDry soup mixSoft drinksInstant teaPizza dough (frozen)Pie doughSugar (especially beet sugar)GelatinCoconutFresh fruit saladDomestic jams and jelliesCrackersCookiesGrapesHigh fructose corn syrup

Drugs

Sulphite-preserved inhalantsSulphite-preserved subcutaneousinjectants

Sulphite-preserved intravenousinjectants

0.5–1.50.3–10

0.3–10

High rareb

Very low

Low

aFoods with low sulphite content have not been implicated in inducing reactions in sulphite-sensitiveindividuals.bRare bronchoconstriction but (?) no bronchodilation.



polyphenoloxidase, catalyses tyrosine oxida-tion, which leads to black spot formationon shrimp. This oxidative reaction (notinfection) can be prevented by sulphites.

Sulphites can also be used as inhibitorsof non-enzymatic browning in wines, driedfruits, dehydrated vegetables (especiallypotatoes), vinegar, white grape juice, coconut,and pectin (Taylor and Bush, 1983). Thechemistry of these reactions is complex andbeyond the scope of this chapter, but has beenreviewed by McWheeney et al. (1974).

The antimicrobial actions of sulphitesare useful as sanitizing agents for food con-tainers and fermentation equipment becausesulphites reduce or prevent microbial spoil-age of food (e.g. table grapes), and act asselective inhibitors of undesirable organismsduring fermentation.

The antioxidative effects of sulphitesserve a major function in the brewing process,where oxidative changes impede develop-ment of the beer’s flavour (Roberts andMcWheeney, 1972). The ability of sulphites tobreak disulphide bonds in the gluten reactionof dough accounts for their widespread use(although in minimal residual quantities)as dough conditioners for biscuits, cookies,crackers, frozen pizza dough and pie crusts(McWheeney et al., 1974). In the productionof maraschino cherries, sulphites are used tobleach the fruit before injecting red dye.

Regulator restrictions

After the discovery of sulphite-sensitive asth-matic persons (Stevenson and Simon, 1981),the Food and Drug Administration (FDA),the Bureau of Alcohol, Tobacco and Firearms(BATF) and the Environmental ProtectionAgency (EPA) moved to regulate the usesof sulphites in 1986. The FDA required thedeclaration of sulphites on the food labelswhen sulphite residues exceeded 10 ppm.The BATF followed suit with wines. The FDAbanned the use of sulphites from fresh fruitsand vegetables other than potatoes. This banaffected the practice of sulphiting lettuce,cut fruits, guacamole, mushrooms and manyother foods, and in particular the once com-mon practice of sulphiting fresh fruits andvegetables in salad bars. The FDA also

moved to ban sulphites from fresh, pre-peeled potatoes. The EPA required thatimported table grapes be detained at theirport of entry until sulphite residues can nolonger be detected. The FDA has also enacteda regulation specifying the allowable residuelevels for sulphites in shrimp. Foods thatcurrently contain sulphites are listed inBox 11.3. Foods with low levels of sulphitescontain ≤ 10 ppm SO2 and have not beenassociated with producing reactions insulphite-sensitive subjects.

Mechanisms of sulphite sensitivity

The mechanisms of sensitivity reactions tosulphiting agents are unknown. Dependingon the route of exposure, a number of mecha-nisms have been postulated. It is known thatasthmatic subjects, upon inhalation of lessthan 1.0 ppm of SO2, develop broncho-constriction (Boushey, 1982b). Fine et al. (1987)demonstrated that bronchoconstriction deve-loped in asthmatic subjects who inhaled SO2

and bisulphite (HSO3−) but not sulphite

(SO3−). Alteration of airway pH was not a

cause of bronchoconstriction. Thus, depend-ing upon pH and the ionic species, asthmaticsdevelop bronchoconstriction after exposureto certain forms of sulphite. It is also recog-nized that some asthmatic individualsrespond to either oral or inhalation challengewith sulphite, but that inhalation is more aptto induce bronchoconstriction (Schwartz andChester, 1984). Variability in the response tosulphites in capsule and acidic solutions,administered via the oral route, has also beenobserved (Lee et al., 1986). The same individ-uals may not always develop broncho-constriction when challenged on repeatedoccasions with sulphites. The following rep-resent further attempts to understand morefully the variables and reasons for thisinconsistent response.

INHALATION DURING SWALLOWING Deloheryet al. (1984) studied ten sulphite-sensitiveasthmatic subjects. All subjects reacted to achallenge with acidic metabisulphite solutionwhen it was administered as a mouthwashor swallowed, but not when it was instilledthrough a nasogastric tube. Furthermore,


20-Feb-03 11



these same individuals did not respond withchanges in pulmonary function when theyheld their breath while swallowing the solu-tion. Ten non-sulphite-sensitive asthmaticsubjects did not react to sulphites whenadministered as a mouthwash or swallowingchallenge. Researchers therefore hypothe-sized that some individuals respond tosulphites during oral challenges because ofinhalation of SO2 during the swallowingprocess.

LINKAGE OF SULPHITE SENSITIVITY WITH AIRWAY

HYPER-REACTIVITY Asthmatic persons areknown to respond to various stimuli (airwayirritants) at concentrations lower than normalindividuals (i.e. to have airway hyper-responsiveness); therefore, attempts havebeen made to link sulphite sensitivity withairway responsiveness as measured duringhistamine and methacholine inhalation chal-lenges. Australian investigators were unableto demonstrate a relationship between thedegree of airway responsiveness to inhaledhistamine and the presence of sulphite sensi-tivity (Delohery et al., 1984). Taylor et al. (1997)attempted to induce sulphite sensitivity ina group of 16 asthmatic subjects. Theyfirst established the provocative dose ofmethacholine producing a 20% decrease inFEV1 (PD20). Then the researchers used asulphite bronchial/oral challenge using anacidic sulphite solution to determine the pres-ence of sulphite sensitivity, and three of the 16subjects reacted to the sulphiting agent with a20% decrease in FEV1. One week later, thepatients underwent bronchial challenge withan antigen to which they were known to besensitive. They returned 24 h later for a repeatmethacholine challenge. This was followed24 h later by a second sulphite challenge. Afterantigen challenge, only one additional subjectshowed a response to sulphiting agent thathad not been present before the antigen chal-lenge, and there was no significant increase inairway response to methacholine. Therefore,these investigators were unable to inducesulphite sensitivity by exacerbating airwayhyper-reactivity.

CHOLINERGIC REFLEX Because SO2 may pro-duce bronchoconstriction through cholinergic

reflex mechanisms, the effect of atropine andother anticholinergic agents has been studied(Simon et al., 1984a; Simon and Stevenson,1991; Taylor et al., 1997). Pre-inhalation ofatropine blocked the airway response to oralchallenge with sulphiting agents in threeof five subjects and partially inhibited theresponse in two others. Doxepin, whichpossesses anticholinergic as well as antihista-minic properties, was protective in three offive individuals undergoing oral challengewith sulphites.

POSSIBLE IgE-MEDIATED REACTIONS Some in-vestigators have attempted to identify animmunological basis for these reactions.Positive patch tests with sulphites suggesteda delayed hypersensitivity mechanism inpatients with contact dermatitis (Epstein,1970). The presence of precipitating antibod-ies to sulphites (Prenner and Stevens, 1976) oralterations in complement activity (Twarogand Leung, 1982) have not been detected. Amore likely explanation would be the pres-ence of an IgE-mediated response in selectedsubjects. Prenner and Stevens (1976) observeda positive skin scratch test to an aqueoussolution of sodium bisulphite at 10 mg ml−1

in their patient who experienced laryngealoedema after sulphite challenge. This patientalso exhibited a dramatic response withintradermal testing at the same concentration.Three non-sensitive control subjects hadnegative skin tests to sulphites. Of the fiveasthmatic subjects studied by Stevenson andSimon (1981), none showed positive skin teststo sulphites. However, the patient of Twarogand Leung (1982), who experienced ana-phylaxis after sulphite exposure, showed apositive intradermal skin test response to anaqueous solution of bisulphite at 0.1 mg ml−1.Control subjects were found to have negativeskin tests with 1.0 mg ml−1 of this solution.Meggs et al. (1985) reported that a patientdeveloped wheezing when skin-tested withsodium bisulphite at 100 µg ml−1. Yang et al.(1986) identified two asthmatic subjects witheither positive prick or intradermal skintests to sulphites, as well as one subject withurticaria and one with anaphylaxis, who alsowas found to have positive intradermal teststo sulphites. Boxer et al. (1988) reported two

20-Feb-03 11




additional cases with positive skin testsand oral challenges to sulphiting agents thatinduced bronchoconstriction. Selner et al.(1987) reported positive intradermal and skinpuncture tests with 0.1 and 10 mg ml−1 potas-sium metabisulphite solutions, respectively,in a sulphite-sensitive asthmatic subject. Twonon-sensitive control subjects had negativeskins tests. Simon and Wasserman (1986) alsoreported two sulphite-sensitive asthmaticswith positive intradermal skin tests tobisulphites at a concentration of 10 mg ml−1,a concentration that did not produce wheal-and-flare cutaneous responses in controls.These observations are consistent with an IgE-mediated mechanism, with sulphites acting aschemical haptens.

Further evidence for an IgE-mediatedmechanism is supported by passive transferexperiments (Prausnitz–Küstner transfer).Several investigators have successfully trans-ferred skin-test reactivity to non-sensitivesubjects with sera from sulphite-sensitiveindividuals (Prenner and Stevens, 1976;Simon and Wasserman, 1986; Yang et al.,1986). Skin sensitizing activity was abolishedby heating the sera to 56°C for 30 min (Simonand Wasserman, 1986). Others have not beensuccessful in repeating these experiments(Epstein, 1970). These data suggest thepresence of a serum factor, presumablyIgE, but, to date, specific IgE antibodies tosulphiting agents or sulphiting agents conju-gated to human serum albumin have not beendemonstrated successfully (Meggs et al., 1985;Boxer et al., 1988). That sulphiting agentscould by themselves stimulate direct media-tor release from mast cells or basophils inthe absence of IgE has also been considered.Histamine release from mixed peripheralblood leucocytes could not be demon-strated in the five subjects studied byStevenson and Simon (1981), but none of thefive had positive cutaneous wheal-and-flareresponses to sulphites. Simon and Wasserman(1986) also found inconsistencies in leucocytehistamine release from peripheral bloodleucocytes from a patient whose skin tests tosulphites were positive. In contrast, Twarogand Leung (1982) found that 20% of the totalbasophil histamine was released in the patientthey studied using concentrations of 10−3–10−7

M sodium bisulphite. Cells from controlsubjects did not release histamine. Moreover,the histamine release was enhanced by pre-incubating the patient’s serum with sodiumbisulphite.

Similarly, inconsistencies in the measure-ment of mast cell or basophil mediators in theperipheral blood of challenged patients havebeen reported. No rise in plasma histaminelevels were observed in patients experiencinghypotension and gastrointestinal responseduring sulphite challenges (Delohery et al.,1984). Likewise, Altman et al. (1985) failedto observe changes in serum neutrophilchemotactic activity in sulphite-sensitiveindividuals during sulphite challenges. Incontrast, Meggs et al. (1985) observed a sig-nificant rise in plasma histamine levels in twoof seven subjects with systemic mastocytosisundergoing sulphite challenges (Stevensonand Simon, 1981). However, no clinicalresponse was observed. In an asthmaticsubject, whose skin tests to sulphites werepositive, the plasma histamine level tripledduring the time of the asthmatic responseto sulphite challenge. Four subjects withasthma or rhinitis, attributed to sulphiteexposure, when challenged intranasallywith 5 mg of potassium metabisulphitein distilled water, demonstrated increasedhistamine levels in nasal lavage fluid7.5 min after challenge (Ortolani et al., 1987).In control subjects with chronic rhinitis,similar results were also obtained, althoughthe level of histamine in nasal secretionswas generally less than in the patients withsulphite sensitivity.

Indirect evidence for mast cell mediatorsplaying a role in the production of bron-choconstriction resulting from sulphitingagents has been reported. Freedman (1980)mentions that inhaled sodium cromolynprevented the asthmatic response to acidicsolutions of sulphite. Simon and Stevenson(1997) found that inhaled cromolyn inhibitedsulphite-induced asthma in four of six sub-jects and partially inhibited the response intwo other subjects undergoing oral challengewith sulphites. Schwartz (1986) reported thatoral cromolyn at a dose of 200 mg blocked anasthmatic response to an oral sulphite chal-lenge in one subject.


20-Feb-03 11



SULPHITE OXIDASE DEFICIENCY It has beenproposed that a deficiency in the enzyme thatmetabolizes sulphite to sulphate (sulphiteoxidase) may be responsible for some adversereactions to sulphites. (Simon, 1986; Simonand Stevenson, 1997). Six subjects, found tobe sulphite-sensitive by oral provocativechallenge exhibited less sulphite oxidaseactivity in skin fibroblasts when comparedwith normal control subjects. The majorsource of sulphite oxidase activity in humans,however, is in the liver.

Preservatives

Preservatives are used for protectionfrom food poisoning by the prevention ofputrefaction and deterioration with micro-organisms, and for the improvement ofthe shelf-life of processed foods. Someorganic acids and their salts or esters, plantextracts and some proteins are used as pre-servatives. The use of preservatives is limitedby regulations in most cases. Compounds toadjust pH or water activity are not calledpreservatives.

Benzoic acid

The structural formula is given in Fig. 11.1(d).C6H5-COOH. Molecular weight: 122.12 Da.Description: white laminar crystals or nee-dles. It is odourless or has a slight odourof benzaldehyde. Solubility of benzoic acidin water is 0.29% at 20°C. Calcium, potassiumand sodium salts are also used as preserva-tives. Functional use: an acid-type antimi-crobial preservative, a growth inhibitor formould, yeast and bacteria. The minimuminhibition concentrations on the growth ofAspergillus orizae are 1/8000 at pH 3.0, 1/2000at pH 4.5 and 1/500 or less at pH 6.0 (Suzukiet al., 1999). The same tendency is observedin other moulds, bacteria and yeast. Naturaloccurrence: raspberries, plums, anise, tea, etc.(Juhlin, 1977; Williams, 1978). Use: marga-rine, baked goods, beverages, soy sauce, etc.,at the concentration 0.6–2.5 g kg−1. The allow-able concentration depends on foods. ADI:0–5 mg kg−1 BW.

Parabens (parahydroxy benzoic acid esters)

The structural formula is HO-(C6H4)-COO-R,where R: alkyl group (Fig. 11.1(e)). Methyl,ethyl, propyl or butyl esters are cited in theCompendium of Food Additive Specifica-tions (Joint FAO/WHO Expert Committee onFood Additives (JECFA), 1992). In additionto the above esters, isopropyl, isobutyl andheptyl eaters are also used as preservatives.Molecular weight: 152.15 Da for the methylester, 166.18 Da for the ethyl ester, 180.21 Dafor the propyl ester and 194.23 Da for thebutyl ester. Description: almost odourless,small, colourless crystals or a white, crystal-line powder. Functional use: neutral-typeantimicrobial preservative for moulds andyeast. The mixture of esters is used for adjust-ing the solubility. The solubility of methyl,ethyl, propyl and butyl esters in 100 ml ofwater at 25°C is 0.25, 0.17, 0.05 and 0.02 g,respectively. Inhibition concentrations on thegrowth of moulds are 100–160 mg l−1 (Suzukiet al., 1999). Natural occurrence: methyl-paraben is known as a pheromone producedby the queen honey bee (Barbier et al., 1960),and is present in royal jelly at a concentrationof 15–30 mg kg−1 (Ishiwata and Yamada,2000). Use: beverages (0.001–0.003%), bakedgoods (0.003–0.01%), sweets (0.003–0.01%),jams and preserves (0.1%). Parabens are alsoused in cosmetics and medicines as preserva-tives. ADI: 0–10 mg kg−1 BW. Benzoic acid,sodium benzoate, methylparaben, propyl-paraben and heptylparaben are approvedas food and drug additives by the US FDAand have been assigned a GRAS status(Jacobsen, 1997).

Sorbic acid

Structural formula: H3C-CH=CH-CH=CH-COOH (Fig. 11.1(f)). Molecular weight:112.12 Da. Description: colourless needles orwhite free-flowing powders, having a slightcharacteristic odour. Solubility in water is0.25% at 30°C, and that of potassium sorbateis 58.2% at 20°C. Functional use: an acid-typeantimicrobial preservative having a widespectrum for microorganisms, a fungistaticagent and a growth inhibitor for moulds,yeast and aerobic bacteria. Inhibition

20-Feb-03 11




concentrations on the growth of mouldsand yeast are 1/4000–1/16,000 at pH 3.0,1/2000–1/4000 at pH 4.5 and 1/500–1/2000at pH 5.5 (Suzuki et al., 1999). Natural occur-rence: may be obtained from berries of themountain ash (Budavari et al., 1996), but noother natural occurrence is known. Use:sorbic acid and its calcium, magnesium,potassium or sodium salt is used for manykinds of foods such as beverages, cheese,baked goods, cakes, fish and meat products.Concentrations in foods range from 0.03 to3 g kg−1. ADI: 0–25 mg kg−1 BW.

Monosodium glutamate

Glutamic acid is a non-essential, dicarboxylicamino acid that constitutes 20% of dietaryprotein. Glutamate appears naturally in somefoods in significant amounts, e.g. 100 g ofCamembert cheese contains as much as 1 g ofMSG; however, the greatest exposure to MSGoccurs after it is added to foods as a flavourenhancer. The sodium salt of glutamic acid(MSG) is added to a wide variety of foodsby manufacturers, restaurant chefs and indi-viduals. A Japanese chemist established 90years ago that MSG was responsible for theflavour-enhancing properties of the seaweedLaminaria japonica (traditionally used in Japa-nese cooking) (Marshall, 1948). MSG is addedroutinely to Chinese, Japanese and otherSouth-east Asian foods and soups. Up to 6 gof MSG may be ingested in a highly seasonedChinese meal. A single bowl of wonton soupcan contain 2.5 g of MSG. MSG is also oneof Kentucky Fried Chicken’s secret herbs andspices for its fried chicken. MSG is foundin most manufactured meat and chickenproducts, particularly soup stocks and theincreasingly popular diet foods (lean/light/low-calorie/low-fat/low-cholesterol).

MSG currently remains among theadditives listed by the FDA as GRAS. Thefact that MSG is added to a particular foodis usually displayed on the label. However,the amount of MSG added is seldom revealed.MSG may also appear on a label as ‘hydro-lysed vegetable protein’. The flavour-enhancing properties of MSG stem from its

excitatory (depolarizing) action on sensorytaste receptors. Current research suggests thatadverse reactions are linked to the ingestionof large amounts of MSG, which are rapidlyabsorbed, particularly when ingested in solu-tion and on an empty stomach (Allen, 1991).

Chinese restaurant syndrome

Chinese restaurant syndrome (CRS) was firstdescribed in 1968 by a Chinese physician,Dr Robert Homan Kwok, in the New EnglandJournal of Medicine (Kwok, 1968). This syn-drome, occurring within hours of a Chineserestaurant meal, is characterized by head-ache, a burning sensation along the back ofthe neck, chest tightness, nausea and sweat-ing. In 1969, Schaumburg et al. reported thefirst formal study of the effects of MSG inhumans. These investigators suggested thatMSG elicits three categories of symptoms: aburning sensation, facial pressure and chestpain. Headache was considered to be a con-sistent complaint in only a minority of indi-viduals. Symptoms appeared in susceptibleindividuals only if the meal contained freeMSG and was ingested on an empty stomach.Such individuals responded to 3 g or less offree MSG, an amount found to be present in a200 ml serving of wonton soup in one NewYork restaurant.

Schaumburg et al. (1969) determined thatthe systemic reactions in their subjects werecaused by L-glutamate. The intensity andduration of symptoms were related to thedosage of MSG. The onset of symptoms wasusually 15–25 min after ingestion. After intra-venous administration, the first symptomsappeared in 17–20 s, with a threshold con-centration for inducing minimum symptomsranging from 25 to 125 mg. In two subjects,intravenous injection of MSG into the fore-arm, while the circulation was occluded by anaxillary cuff, produced a burning sensationover the entire arm. The burning sensationwas felt over the chest and neck 17 s afterthe cuff was removed. Schaumburg et al.concluded that this burning sensation was aperipheral neuroexcitatory phenomenon andwas not due to central nervous system stimu-lation. Ghadimi et al. (1971) reported similarstudies and showed that onset and severity of


20-Feb-03 11



symptoms are both dose related. Subsequentstudies of the CRS by Kenny and Tidball(1972) and Reif-Lehrer (1977) suggest that theprevalence of the CRS in the population eatingin Chinese restaurants is about 30%.

Sweeteners

Both synthetic and natural sweeteners areallowed as food additives. Sugar alcoholssuch as xylitol and D-sorbitol are naturallyoccurring sweeteners. Artificial sweetenerssuch as saccharin, acesulphame potassium,and sucralose are non-calorie sweeteners.

Saccharin

The structural formula is given in Fig. 11.1(g).Molecular weight: 183.19 Da. Description:white crystals or a white, crystalline powder,odourless or with a faint aromatic odourhaving a sweet taste even in very dilute solu-tions with about 500 times the sweetnessof sugar. Slightly soluble in water. Sodiumsaccharin is freely soluble in water. Func-tional use: non-calorie sweetening agent.Natural occurrence: not known. Use: usedalone or with other sweeteners. The sodiumsalt is one of the widely used sweetenersadded in dentifrices and lipsticks. Freesaccharin is effective in keeping the tastein chewing gum (not more than 0.05 g kg−1

in Japan) because of its insolubility in water.The allowable limit of sodium saccharin inJapan is: vinegar pickles 2.0 g kg−1, soft drinks0.30 g kg−1, jam 0.20 g kg−1 and confectionsand sweets 0.10 g kg−1. ADI: 0–2.5 mg kg−1

BW (tentative).

Acesulphame potassium

The structural formula is given in Fig. 11.1(h).Molecular weight: 201.14 Da. Description:odourless, white crystalline powder havingan intensely sweet taste, with about 200 timesthe sweetness of sugar. Functional use: non-calorie sweetening agent. Natural occur-rence: not known. Use: confections, chewinggum, jam, wines, soft drinks, fermented milk,tabletop sweetener. ADI: 0–15 mg kg−1 BW.

Aspartame (L-α-aspartyl-L-phenylalaninemethyl ester)

The structural formula is given in Fig. 11.1(i).Molecular weight: 294.31 Da. Description:white, odourless, crystalline powder, havinga strong sweet taste, with about 200 times thesweetness of sugar. A dipeptide. Functionaluse: sweetening agent. Natural occurrence:not known. Use: tabletop sweetener, sweets,soft drinks, chewing gum, salted vegetables,etc. Decomposition rate at 80°C for 2 h is 3%at pH 3.0 and 4.0, but increases to 92.5% atPH 6.5, The major decomposition product isdiketopiperidine. ADI: 0–40 mg kg−1 BW.

Aspartame was discovered serendipit-ously in 1965 by a chemist seeking to findan inhibitor of gastrin which might functionas an antiulcer agent (Mazur, 1984). In 1973,G.D. Searle petitioned the FDA for approvalto market aspartame as a sweetener (UnitedStates General Accounting Office/HRD,1987). In 1974, aspartame was approvedfor use in dry foods. In December 1975,the FDA held the approval for market-ing aspartame because of concern overproblems noted in studies by Searl Labora-tories (Chicago, Illinois) and because ofallegations that aspartame was unsafe andcould cause mental retardation and endocrinedysfunction. In July 1981, the FDA Com-missioner reapproved aspartame as a foodadditive, and marketing was initiated thatsame year. In July 1983, aspartame wasapproved for use in carbonated beverages(Garriga and Metcalfe, 1988).

Antioxidants

These are substances to protect foods fromdeterioration by the oxidation of fats, oils andsome other food components. Antioxidantssuch as BHA, BHT and TBHQ (tertiarybutylhydroquinone) inhibit the formation ofperoxides of fats and oils causing foodpoisoning, and inhibit the browning of cutvegetables and fruits by the oxidation ofpolyphenols. Ascorbic acid (vitamin C, watersoluble) and tocopherol (vitamin E, oil solu-ble) are known as natural antioxidants.

11-Mar-03 11




Butylated hydroxyanisole (BHA)

The structural formula is given in Fig. 11.1(j).Molecular weight: 180.25 Da. Description:white or slightly yellow crystals or waxy sol-id, with a faint characteristic odour. Insolublein water. Functional use: antioxidant. Naturaloccurrence: not known. Use: fat, oil and but-ter, dried fish, salted fish, mashed potato, fro-zen marine products, cereals, dry yeast, driedvegetables, processed meat, etc., at the con-centration of 0.001–0.02%. BHA is sometimesused in combination with BHT. The limit isthe total amount with BHT. The antioxidativeactivity of 3-isomer is 1.5–2 times higher thanthat of the 2-isomer. Recent BHA consists ofthe 3-isomer. ADI: 0–0.5 mg kg−1 BW.

Butylated hydroxytoluene (BHT)

The structural formula is given in Fig. 11.1(k).Molecular weight: 220.36 Da. Description:white, crystalline or flaked solid, odourless orhaving a characteristic faint aromatic odour.Functional use: antioxidant. Natural occur-rence: not known. Use: same as BHA, or usedin a wider variety of foods. Used with otherantioxidants such as vitamin C or citric acid.BHT is also used as an antioxidant for plasticsand petroleum products. ADI: 0–0.3 mg kg−1

BW.BHA and BHT are commonly used in

cereal and other grain products. They weredeveloped originally as antioxidants for pet-roleum and rubber products, but were discov-ered to be effective in preventing oxidation ofanimal fatty acids in the mid-1950s (Babich,1982). BHT has been promoted by some asan anticancer, antiageing substance and asa treatment for genital herpes (Pearson andShaw, 1984). In addition to such claims beingunsubstantiated, legitimate toxic side effects,including severe gastrointestinal and neuro-logical toxicities, have been reported afteringestion of standard and suggested doses(Grogan, 1986; Sklian and Goldstone, 1986).

Colour fixatives

Nitrates and nitrites are used as colour fixa-tives. The sodium and potassium salts are

common for colour fixatives. Nitrate isreduced to nitrite by bacteria in foods, andreacts with myoglobin to form nitrosomyo-globin, which has a stable pink colour. Nitriteinhibits the growth of bacteria, and thereforeis also used as a preservative. Nitrates andnitrites are widely used as preservatives.However, their popularity as additives stemsfrom their ability to add flavouring andcolouring. These agents are added to pro-cessed meats (e.g. frankfurters, salamis).

Sodium nitrate

Structural formula: NaNO3. Molecular weight:84.99 Da. Description: clear, colourless, odour-less, transparent crystals, or white granulesor powder, and deliquescent in moist air.Functional use: colour fixative, antimicrobialagent, preservative. Natural occurrence: invegetables (Walker, 1990) at concentrationsof more than 1000 mg kg−1 as a natural com-ponent of plants. Nitrate is reduced to nitriteby microorganisms or chemically, and thenitrite formed acts as a colour fixative.Use: cured meats, meat products, dried fish,cheese. ADI: 0–3.7 mg kg−1 BW as nitrate.

Sodium nitrite

Structural formula: NaNO2. Molecularweight: 69.00 Da. Description: white orslightly yellow, hygroscopic and deliques-cent granules, powder, or opaque, fusedmasses of sticks. Functional use: colourfixative, antimicrobial agent, preservative,flavour enhancer. Natural occurrence: vege-tables (0–6 mg kg−1) (Walker, 1990); somesalted vegetables contain up to 50 mg kg−1

as nitrite (Suzuki et al., 1999). About 5% ofingested nitrate is reduced to nitrite in humansaliva (Walker, 1990). Use: cured meats, meatproducts, dried fish, cheese. Effective toinhibit the growth of Clostridium botulinum.ADI: 0–0.06 mg kg−1 BW as nitrite.

Acidulants

All acidulants used as food additives areknown as natural components in plants or


20-Feb-03 11



animal bodies. Acidulants are used for someother purposes such as flavour enhance-ment, controlling the pH of food, preventinggrowth of microorganisms and antioxidationby trapping metals, in addition to their majorrole. The following compounds are used asacidulants: acetic acid, adipic acid, citric acid,fumaric acid, lactic acid, malic acid, phospho-ric acid, succinic acid, tartaric acid, etc. Thesodium, potassium and calcium salts are alsoused for the above purposes.

Acetic acid

Structural formula: CH3COOH. Molecularweight: 60.05 Da. Description: clear, colour-less liquid having a pungent characteristicodour, miscible with water and alcohol.Functional use: acidifier, flavouring agent,pH control agent. Natural occurrence: vine-gar, fermented foods, fruits. Use: acetic acidis the principal component of vinegar.Diluted acetic acid (4–5%) is used as vinegarmixing with sugar, sweetener and aminoacids. Acetic acid (vinegar) is also employedin preparing salad dressings, sauce, mayon-naise, pickles, ketchups, syrups and cheese.ADI: not limited.

Citric acid

Structural formula: CH2COOH-HO-C-COOH-CH2COOH. Molecular weight: 192.13Da. Description: white or colourless, odour-less crystalline solid, having a strongly acidtaste. The anhydrous and monohydrateforms are listed. Functional use: acidifier,antioxidant, synergist, sequestrant, flavour-ing agent. Natural occurrence: citrus fruits.Use: soft drinks (0.1–0.3%), juice, jelly, jam,sweets (1%). ADI: not limited.

Lactic acid

Structural formula: CH3CH(OH)COOH.Molecular weight: 90.08 Da. Description:colourless or yellowish, nearly odourless,syrupy liquid with an acid taste, consisting ofa mixture of lactic acid and lactic acid lactate.It is obtained by the lactic fermentation ofsugars or is prepared synthetically. Commonproducts of commerce are 50–90% solutions.

Functional use: acidifier. Natural occurrence:lactic fermented milk, muscle. Use: 0.05–0.2%in soft drinks, sweets, jam, sherbet, etc. as anacidifier, with expecting preservative effect.ADI: not limited.

Distribution in foods and daily intake

ADIs of food additives have been establishedby the Joint FAO/WHO Expert Committeeon Food Additives (JECFA) (1996) to ensurethat consumers can always confidentlychoose healthy and enjoyable diets from asafe and varied food supply. The use of manyfood additives is regulated by food acts orfood sanitation law. Some food additives areallowed for use only in limited foods, andsome food additives are limited by the con-centrations that may be used. The determina-tion of food additives in foods and the esti-mation of the daily intake, especially incomparison with the ADI, are very importantto ensure public health.

Food additives are not always used inallowable foods or up to the allowable limits.Some food additives decompose graduallyprior to consumption. The allowable limitdiffers depending on the type of foods. Theconcentrations of some food additives infoods in Japan were determined in 1995(Ishiwata et al., 1995). Preservatives, benzoicacid, dehydroacetic acid, p-hydroxybenzoicacid esters and sorbic acid, were detected in16,660 (14.9%) of the total of 112,131 allowableand non-permissible food samples. The aver-age concentration of sorbic acid in foods was14.1% of the allowable limits.

The JECFA (1999) assessed (first draft)the daily intake of benzoic acid, BHA, BHT,sulphites and TBHQ, and concluded that theestimates of national mean intake by consum-ers of these food additives were unlikely toexceed the ADI. In general, the daily intake offood additives is estimated by the poundagemethod (production or used amount), marketbasket method (total diet study), individualfood analysis method, etc. Every methodincludes both overestimation and under-estimation factors. For example, in the case ofpoundage method, it is possible to estimate

11-Mar-03 11




overall intake of food additives, and thelabour and cost for analysis of food additivesare relatively low compared with other meth-ods. However, natural sources such as organicacid or inorganic compounds are not includedin the estimated intakes. A market basketmethod is suitable to estimate the total intakeof some food additives in foods, both inten-tionally added and naturally contained, butthere are labour and analysis costs involved.The individual food analysis method utilizesanalytical results in published papers or offi-cial inspection for the estimation of the dailyintake. Therefore, daily intakes are estimatedbased on a huge number of analytical results,but food additives for which the daily intakecan be estimated are limited. Estimated dailyintakes of some food additives are shown inTable 11.1 together with the ADI.

No daily intake of food additives exceptthat of nitrate exceeded the ADI. Nitrateintake is close to or above the ADI. The majorfunctional use of nitrate is as a preservativeand a colour fixative for meat products. Thedaily intake of nitrate was 0.73 mg per person(Ito, 2000) from the food category of meat and

fish products or 0.62 mg per person from meatproducts (Ishiwata et al., 2000). The dailyintake of nitrate in Japan estimated by a mar-ket basket method was 232 mg per person in1996 (Ito, 2000) and 189 mg per person in 1999(Yamada, 2000), and most (~90% or more)nitrate came from unprocessed foods such asfruits and vegetables (Ito, 2000). When thebody weight of adults was assumed to be50 kg, the daily intake was at or above theADI. The JECFA (1995) evaluated the intakeof nitrate from vegetables as ‘the Committeeconsidered it inappropriate to compare expo-sure to nitrate from vegetables directly withthe ADI and hence to derive limits for nitratein vegetables directly from it’.

Urticaria, Angio-oedema, Anaphylaxisand Additives

In 1959, Lockey first reported three patientswith a history of urticaria after the ingestionof tablets containing tartrazine. One patientwas in the middle of treatment for a skin


20-Feb-03 11

Daily intake (mg per person)

PoundageBased on the

inspection results

Total diet(marketbasket)

Food additiveADI

(mg kg−1 body weight)UKa

1984–1986Japanb

1995Japanc

1996Finlandd

1980Japane

1994–1995

Benzoic acidp-Hydroxy benzoicacid esterSorbic acidNitrateSulphur dioxideBHABHTSodium saccharin

0–5g0–10f

0–25. 0–3.7

. g0–0.7g

. 0–0.5

. 0–0.3. g0–5.0h

48.90.1

29.41.3

18.40.40.22.8

4.050.38

33.90.198.460.0090.262.27

111.1

261.41.50.110.227.6

400.18

376.44.00.176.6

2.40.124

27.5232

0.0880.0020.0660.416

aMinistry of Agriculture, Fisheries and Food, UK (1993).bFujii (1996).cIshiwata et al. (2000); Yamada et al. (2000).dPenttilä et al. (1988).eIto (2000).fGroup ADI of methyl, ethyl and propyl esters.gGroup ADI of sulphur dioxide and sulphites.hGroup ADI of saccharin and its salts.

Table 11.1. Estimated daily intake of some food additives and comparison with ADI.



eruption caused by another agent. The othertwo subjects were challenged with tartrazinesublingually in an open manner. One ‘re-acted’ (no further description offered). Theother had only mild complaints localized tothe mouth.

The incidence of reactions to anyadditives, including tartrazine, in patientswith chronic urticaria and angio-oedema isunknown. This is not because of inadequatenumbers of studies, but rather because of thelack of properly and vigorously controlledstudies and inherent problems in challengingpatients with chronic urticaria.

Food additive challenge studies inurticaria patients

Design considerations

PATIENT SELECTION Selection of patients forstudy may include: (i) all available patientswith chronic urticaria (or only those withchronic idiopathic urticaria); (ii) only thosewith histories suggestive of food additive-provoked urticaria; or (iii) those whoseurticaria improved after a diet free of com-monly implicated additives. Depending uponthe group selected and the challenge process,different percentages of so-called positivereactors have been reported. These variables,often omitted or poorly stated in reports andstudies, add more confusion to the alreadydifficult task of comparing results betweendifferent studies.

ACTIVITY OF URTICARIA AT THE TIME OF STUDY

The relative degree of activity or inactivityof urticaria or angio-oedema at the time ofchallenge appears to determine the ability ofthe skin to respond with cutaneous reactionsduring subsequent additive challenges.Patients with active urticaria are more likelyto develop further urticarial activity, whilechallenges performed upon patients whoseurticaria is in remission are more likely toyield negative results. In the study of Lumryet al. (1982), only one of 15 patients whoseurticaria was in remission experienced a reac-tion to acetylsalicylic acid (ASA), whereasseven of ten patients whose urticaria was

active at the time of challenge reacted toASA. These challenges were conducted usingobjective reaction criteria, and reactions werecompared with a baseline observation periodin the same patient.

MEDICATIONS In several studies, referenceis not made as to whether medications, partic-ularly antihistamines, are continued or with-held during challenges. However, there areimportant caveats to bear in mind when inter-preting challenge studies that mention detailsof withdrawing medications: (i) discontinua-tion of antihistamines immediately before orwithin 24 h of challenge is likely to inducefalse-positive reactions; (ii) continuation ofantihistamines during challenges may blocksome of the milder additive-induced cutane-ous responses and therefore promote false-negative results; and (iii) subjects are alsomore likely to experience breakthroughurticaria the longer the interval from the lastantihistamine dose to the test substance asso-ciated with a ‘positive challenge’. This phe-nomenon is accentuated if placebo controlsare given first, before additive challenges, andin closest proximity to the protective effect ofthe last antihistamine tablet.

REACTION CRITERIA In most studies, a periodof baseline observation for comparison withreaction data was never made. Most challengestudies reported loosely defined and subjec-tive criteria for identifying urticarial response.The reaction criteria simply consisted of ‘clearsigns of urticaria developing within 24 h’. Thestudies by Stevenson et al. (1986) and Lumryet al. (1982) represent the only reportedchallenge studies which utilized an objectivesystem of scoring urticarial responses.

PLACEBOS The importance of placebo-controlled studies in additive challengescannot be overemphasized. Studies that didnot utilize placebo controls are useless inspecifically linking urticarial responses to achallenge substance. There are a surprisingnumber of published studies of additivechallenges that never employed placebo con-trols. Even in most placebo-controlled studies,the placebo was usually the first challengesubstance, followed by ASA and then an

20-Feb-03 11




additive. Thus, a spontaneous flare ofurticaria was least likely to coincide withthe first placebo challenge, particularly ifantihistamine was last ingested just beforebeginning challenges. The use of multiple pla-cebos, including randomization of placebos,enhances the design of placebo-controlledchallenges and eliminates the bias of firstchallenge placebo alone.

CONTROLS Among the most important fea-tures of any food additive challenge protocolis a double-blind challenge. Because exacerba-tions of urticaria may be stress provoked, it isnecessary to blind the study subjects. Further-more, nurses and physicians can transmitunspoken signals of concern and apprehen-sion when the ‘real’ test substance is adminis-tered. In addition, it is important to eliminateobserver bias whenever possible becausepositive responses always consist of theappearance of hives or hives in greaternumbers than were observed at baseline.

DOSES Box 11.4 lists maximum doses forcommon additives implicated in adversereactions. Starting doses should be individu-alized depending upon the estimated amountingested at the time of the reported reaction/severity of reaction/level of sensitivity. Dosesusually double between initial and maximumdoses.

Multiple additive challenges inchronic urticaria

EXAMPLES OF STUDIES WITH LESS STRINGENT DESIGN

CRITERIA One of the earliest open additivechallenge studies in chronic urticaria patientswas reported by Doeglas (1975). He found thatseven of 23 patients (30.4%) reacted to

tartrazine and ‘four or five’ (17.4 or22.7%) reacted to sodium benzoate. Placebo-controlled challenges were not performed.Thune and Granholt (1975) reported that 20of 96 patients reacted to tartrazine, 13 of 86reacted to sunset yellow, five of seven reactedto parabens, and six of 47 reacted to BHA/BHT. Furthermore, in their total group of 100patients with chronic idiopathic urticaria,62 reacted to at least one of the 22 differentadditives used in challenges. Because noneof the challenges were placebo controlled,conclusions about the specificity of reactions,linked to a particular additive, are difficult tosupport.

In a study of 330 patients with recurrenturticaria, Juhlin (1981) performed single-blindchallenges using multiple additives and onlya single placebo, which was always givenfirst, preceding the additive challenges.He found one or more positive reactionsin 31% of patients challenged. Reactioncriteria were subjective. Reactions werejudged to be ‘uncertain’ in 33% of patientsbecause, as the author stated, ‘judgingwhether a reaction is positive or negative isnot always easy’. Furthermore, if patients‘reacted’ to the lactose placebo, a wheatstarch placebo was then used in re-testing,presumably because the author assumed thatthe original burst of urticaria was due to theplacebo. Questionable reactors were re-testedand, if the repeat test was positive, it wasassumed that the first test was positive; thesame logic applied for re-testing with negativeresponse.

Supramaniam and Warner (1986) repor-ted that 24 out of 43 children reacted to oneor more additives in their double-blind chal-lenge study. However, a baseline observationperiod was not used, and only one placebowas interspersed among the nine differentadditives used as challenge substances.Furthermore, whether antihistamines werewithheld prior to or during challenges wasnot mentioned.

In 1985, Genton et al. performed single-blind additive challenges on 17 patientswith chronic urticaria and/or angio-oedemaof unknown type. All medications were alsodiscontinued at the beginning of the diet,and patients were subjected to a 14-day


11-Mar-03 11

Box 11.4. Suggested maximum doses for addi-tives used in challenge protocols.

TartrazineSulphitesMSGAspartameParabens/benzoatesBHA/BHT

50 mg200 mg

5 g150 mg100 mg100 mg



elimination diet (free of food additives)before any challenges. Of the 17 patients,15 developed urticaria after at least one ofthe six additives used for challenge in thisstudy.

EXAMPLES OF STUDIES WITH BETTER DESIGN AND

REACTION CRITERIA Ortolani et al. (1988)studied 396 patients with recurrent chronicurticaria and angio-oedema as a follow-up to astudy performed in 1984 (Ortolani et al., 1984).Double-blind, placebo-controlled, oral foodprovocation challenges were performed onpatients who had significant remissions intheir urticaria while following an eliminationdiet. Medications were discontinued duringchallenges, but the timing of discontinuationof medications was not mentioned. Basedon history alone, 179 patients were consideredfor, but only 135 patients participated in, anelimination diet for suspected food or foodadditive intolerance. Only eight out of 87patients that reported significant improve-ment on the 2-week elimination diet hadpositive challenges to foods. Of the 79 patientswith negative food challenge, 72 underwentdouble-blind, placebo-controlled, oral foodadditive provocation challenges. Twelvepatients had positive challenges to one ormore additives; many of these patientswere reported to have reacted to two or threeof the test additives. Five of 16 patients withpositive ASA challenges had positive addi-tive challenges, four of these to sodiumsalicylate. The similarity of chemical struc-tures between ASA and sodium salicylatesupports the concept of cross-reactivitybetween ASA and sodium salicylate; how-ever, the doses used (> 400 mg) in the sodiumsalicylate challenge far exceeded that encoun-tered in conventional diets and thereforehad little to do with native dietary exposure.Considering that the proposed mechanismsfor reactions to additives such as tartrazine,sodium benzoate and sulphites are so differ-ent, the meaning of ‘positive challenges’ inthis study is unclear. Furthermore, although apatient’s history is important to the consider-ation of food sensitivity, it is usually a poorindicator of a possible additive hypersensitiv-ity, since patients are usually not aware of all

the additives they consume daily and arealways reporting urticarial flare-ups inrelation to external events and ingestions.Elimination of more than 50% of the originalstudy population may have been proper forfood sensitivity determinations, but it was notjustified for selection of patients for additivechallenges.

Hannuksela and Lahti (1986) challenged44 chronic urticaria patients with severalfood additives, including sodium metabi-sulphite, BHA/BHT, β-carotene and benzoicacid, in a prospective, double-blind, placebo-controlled study. Only one of the 44 patientshad a positive challenge (to benzoic acid).However, one of 44 patients also reactedto a placebo challenge. All medicationswere discontinued 72 h before the firstchallenge and during the study. Patientswere not following an additive-free dietbefore the challenges. The challenge doseof metabisulphite was quite low (only 9 mg).Similarly, Kellett et al. (1984) noted thatapproximately 10% of 44 chronic idiopathicurticaria patients reacted to benzoates and/ortartrazine, but 10% of the same subjects alsoreacted to placebos.

STUDIES OF ELIMINATION DIETS An alternativeway to investigate urticaria which is pre-sumed to be secondary to food additives isto eliminate all additives from the diet andobserve a decrease in hives. Unfortunately,blind or placebo-controlled studies of thistype have not been reported in the literature.In uncontrolled studies, Ros et al. (1976)reported an additive-free diet to be ‘com-pletely helpful’ in 24% of patients with chronicurticaria. Another 57% of patients were ‘muchimproved’, and 19% were ‘slightly better’ orexperienced ‘no change’ in their urticaria.Rudzki et al. (1980) reported that 50 of 158patients responded to a diet free of salicylates,benzoates and azo dyes. These studies didnot address the question as to which, if any,additives had been inducing urticaria.

In another study, Gibson and Clancy(1980) found that 54 of 76 patients who under-went a 2-week additive-free diet ‘responded’.Using the same study population, they thenchallenged the responders with individual

20-Feb-03 11




additives. Although the challenges wereplacebo controlled, the placebo was alwaysgiven first. Furthermore, no mention wasmade as to whether the challenges wereblinded. A diet free of the offending additivewas then continued for 6–18 months, followedby a repeat challenge. All three patients whoinitially experienced a positive challenge aftertartrazine did not develop urticaria uponrechallenge with tartrazine 6–18 months later.One of the four patients with initially positivebenzoate challenges also experienced a nega-tive challenge upon re-exposure 6–18 monthslater. Therefore, despite a dietary eliminationapproach, the incidence of additive-inducedurticaria continues to be elusive.

Reports of additive sensitivity using singleor limited challenges

TARTRAZINE (FD&C YELLOW NO. 5) Even theincidence of reactions to tartrazine, the mostcommonly implicated additive causing reac-tions in patients with urticaria, is not known.In a double-blind, placebo-controlled study,three of 38 patients with chronic urticaria(8%) reacted to tartrazine (Gibson and Clancy,1980). All three patients were probablysensitive to aspirin; however, the details ofthe challenge protocols were not presentedand the challenge dose of tartrazine was only0.22 mg. The choice of challenge dose wasbased on the quantity of tartrazine addedto pharmaceutical tablets. Much greateramounts of tartrazine are found in foods anddrinks (25–50 mg).

Settipane and Pudupakkam (1975) alsoreport tartrazine sensitivity in some patientswith urticaria who were also sensitive to aspi-rin. However, in a single-blind study of theincidence of aspirin sensitivity in chronic idio-pathic urticaria at Scripps Clinic, we adminis-tered 25 and 50 mg doses of tartrazine (up to atotal dose of 75 mg in most patients duringone challenge day), and only one of 24 patientsreacted with urticaria. This single suspectedtartrazine reactor was then rechallengedusing a double-blind, placebo-controlledtartrazine challenge and again developedurticaria after 25 mg of tartrazine (Stevensonet al., 1986). This patient did not experienceany reaction to aspirin (receiving a total of

975 mg, with 650 mg as a final single dose)and gave an excellent history of reported urti-caria after exposure to tartrazine in her dietpreviously.

SUNSET YELLOW (FD&C YELLOW NO. 6) A singlecase report described a 43-year-old physicianwith acute episodes of severe abdominal painand hives believed to be secondary to yellowdye no. 6. Despite ongoing ingestion of thisdye, the subject experienced only four isolatedepisodes of hives in 2 years. Two challenges,one single and the other double-blind,provoked ‘reactions’. The single-blindchallenge was associated with both abdom-inal pain and urticaria. However, thedouble-blind challenge was only associatedwith pain and not with urticaria (Gross et al.,1989).

SULPHITES In 1976, Prenner and Stevensreported the occurrence of an anaphylacticreaction after the ingestion of food sprayedwith sodium bisulphite. The patient, a50-year-old male, experienced generalizedurticaria and pruritis, swelling of the tongue,difficulty with swallowing and tightness inhis chest within minutes after eating lunchat a restaurant. He responded promptlyto treatment with subcutaneous adrenaline.Subsequently, a prick test to sulphite as wellas an intradermal test were significantlypositive (with negative controls). The authorswere able to demonstrate passive transfer,via Prausnitz–Küstner (P–K) testing, to anon-atopic human recipient.

In 1980, Clayton and Busse described anon-atopic female who developed generalizedurticaria that progressed to life-threateninganaphylaxis within 15 min of drinking wine.Her symptoms were not reproduced by inges-tion of other alcoholic beverages. In retrospect,this may have been a case of sulphite-provoked urticaria and anaphylaxis.

Habenicht et al. (1983) described twopatients with several episodes of urticaria andangio-oedema following restaurant meals.Only one of these patients underwent asingle-blind oral challenge with potassiummetabisulphite; generalized urticarial lesionsdeveloped in this patient within 15 min ofthe 25 mg challenge dose. However, a placebo


20-Feb-03 11



challenge was not performed. Avoidance ofpotential sulphite sources apparently hasled to resolution of this patient’s recurrentsymptoms.

Schwartz reported two patients withrestaurant-related symptoms who underwentoral metabisulphite challenges (Schwartz,1983). Both patients had symptoms includingweakness, a feeling of dissociation from body,dizziness, nausea, chest tightness and possi-ble hives temporally related to ingestion ofsalads. Upon challenge, both patients experi-enced abdominal distress, dizziness, border-line hypotension and bradycardia. Thesesigns and symptoms were more consistentwith vasovagal reactions than anaphylaxis.In a 1985 report, Schwartz and Sher (1985b)described a patient who received less than2 ml of procaine (Novocaine®) with adrena-line subcutaneously. Within several minutes,she developed a sense of flushing, warmthand pruritis followed by scattered urticaria,dyspnoea and a sense of anxiety. Skin tests,using various local anaesthetics and sulphite,were negative. She developed ‘a sense offullness in her head, nasal congestion anda pruritic erythematous blotchy eruption’30 min after a single-blind oral dose of 10 mgof sodium bisulphite. Respiratory symptomsdid not develop and pulmonary functiontest results remained normal. This patientwent on to tolerate the same local anaestheticswithout adrenaline. It is critical to notethat this patient did not describe a historyof food-related symptoms. Furthermore, theusual dose of aqueous adrenaline containsonly 0.3 mg of sulphite, and local anaestheticscontain only up to 2 mg ml−1 of sulphite.Therefore, usual doses of such anaesthetics,even in the most sensitive individuals,would not be expected to provoke reactions.The mechanism of this patient’s ‘reaction’cannot be linked confidently to sulphiteand was probably a vasomotor responsesecondary to anxiety and/or to the effects ofadrenaline.

There are now two publications demon-strating the inability of investigators toprovoke reactions to sulphites in patientswith idiopathic anaphylaxis, some withhistories of restaurant-associated symptoms(Sonin and Patterson, 1985; Kulczycki, 1986).

In a study describing food skin testing in102 patients with idiopathic anaphylaxis,only one patient was found to have cutaneoussensitivity to metabisulphite (Stricker et al.,1986). In addition, we have performedsulphite ingestion challenges in 25 patientswith chronic idiopathic urticaria and angio-oedema without encountering any reactions.Therefore, except for the reports by Prennerand Stevens (1976) and Yang et al. (1986),no other studies using properly controlledchallenges confirm sulphite-induced urti-caria, angio-oedema and/or anaphylaxis.Yang et al. (1986) described one patient witha history of sulphite-provoked anaphylaxis.A borderline intradermal skin test wasdemonstrated, as was a positive single-blind oral provocation challenge to 5 mgof potassium metabisulphite. This patient’scutaneous reactivity was also transferredpassively via the P–K reaction. However,these investigators were unable to elicit posi-tive challenges in nine patients with historiesof hives after eating restaurant food. Inconclusion, IgE-mediated immediate hyper-sensitivity reactions to sulphites (possiblyvia a hapten recognition) appear to be extra-ordinarily rare, if they exist at all, in inducingurticaria and anaphylaxis. In the over-whelming majority of cases, the mechanismof sulphite-provoked urticaria, angio-oedemaand anaphylaxis (or anaphylactoid reactions)remains an enigma.

BENZOATES AND PARABENS In the literature,we are aware of a total of two reports (threecases) of apparent IgE-mediated, paraben-induced urticaria and angio-oedema (Aldreteand Johnson, 1969; Nagel et al., 1977).Parabens in pharmaceutical preservativeswere the presumed source of these additives.All three patients had positive skin tests toparabens, but not to the drugs themselves,which were free of paraben preservatives.These patients were able to tolerate oralbenzoates in their diet without reaction.Michels et al. (1991) reported the case of ateenager who had experienced several food-associated reactions in which sodium benzo-ate seemed to be the common factor. One ofthese episodes involved flush, angio-oedema,dyspnoea and severe hypotension. An oral

20-Feb-03 11




challenge with 20 mg of sodium benzoateproduced itching and urticaria.

MONOSODIUM GLUTAMATE A 1987 letter inthe Lancet (Squire, 1987) described a50-year-old man with recurrent angio-oedema of the face and extremities which wasrelated by history to ingestion of a soup whichcontained MSG. A single-blind, placebo-controlled challenge with the soup baseresulted in ‘a sensation of imminent swelling’within a few hours, but visible angio-oedemaappeared 24 h post-challenge. In a gradedchallenge, angio-oedema occurred 16 hafter challenge with 250 mg of MSG alone.Avoidance of MSG led to an extendedremission. Details of the challenge werenot reported, nor was there any mention ofwhether medications were withheld duringchallenges.

ASPARTAME Two cases of aspartame-provoked urticaria and angio-oedema havebeen reported (Kulczycki, 1986). In bothindividuals, hives began after aspartame wasapproved as a sweetener in carbonated bever-ages in 1983. Both patients reported the onsetof urticaria within 1 h of ingesting aspartame-sweetened soft drinks. Double-blind, placebo-controlled challenges reproduced urticariawith doses of aspartame (25–75 mg) belowthe amount contained in a typical 12-ouncecan (100–150 mg). Despite the widespread useof aspartame in diet drinks and elsewhere,other reports have not followed these initialfindings. Even an attempt to recruit patientsbelieving themselves to be sensitive toaspartame did not yield additional subjectsfor challenge studies (Nagel et al., 1977). Inthis study, 12 subjects with urticaria, butwithout a history of aspartame-associatedurticaria, were challenged with aspartameand none experienced a reproducible adversereaction.

BHA/BHT Roed-Petersen and Hjorth (1976)found four patients with eczematous dermati-tis who had positive patch tests to BHA andBHT. Dietary avoidance of the antioxidantsresulted in remissions in two patients.When challenged with ingestion of 10–40 mgBHA or BHT, both patients experienced

exacerbations of their dermatitis. Osmundsen(1980) reported a case of contact urticaria,apparently due to BHT contained in plasticfolders; the patient had positive wheal-and-flare responses to 1% BHA and BHT inethanol. A case of acute urticarial vasculitisrelated to BHT in chewing gum has also beenreported (Moneret-Vautrin et al., 1986).

Two patients with chronic idiopathicurticaria, in whom remissions were achievedwhile following dye and preservative elimi-nation diets, had exacerbations of their urti-caria when challenged under double-blind,placebo-controlled conditions with BHA andBHT (Goodman et al., 1990). After eliminationof BHA and BHT from their diets, the patientswere observed to have marked abatement ofthe frequency, severity and duration of theirurticaria.

Natural food colourants

Many natural colourants are allowed foruse in foods, including annatto, carmine,carotene, turmeric, paprika, beet extract andgrape skin extract. These types of colourantsare not used to any extent in pharmaceuticalapplications. Several studies have reportedpositive reactions after challenges withnatural colours (Mikkelsen et al., 1978; Juhlin,1981; Fuglsang et al., 1994) or mixtures of nat-ural and synthetic colours (Veien et al., 1987).The natural colourants involved in thesechallenges were annatto, betanin, curcumin,turmeric, β-carotene, canthaxanthin and beetextract. The adverse reactions were asthma,urticaria, atopic dermatitis, colic and vomit-ing. Of course, no one colour can be identifiedas the causative factor when challenges areconducted with mixtures.

Annatto

Annatto is obtained as an extract from theseeds of the fruit of the Central and SouthAmerican tree, Bixa orellana. Bixin, the princi-pal pigment in annatto, is a carotenoid. Theextracts are red in colour, but annatto is oftenused to impart an orange or deep-yellowcolour to the finished food.


11-Mar-03 11



Nish et al. (1991) reported a case of a pos-sible IgE-mediated allergic reaction to annattoextract. The patient experienced angio-oedema, urticaria and severe hypotensionwithin 20 min of ingesting a breakfast cerealcontaining annatto. The patient had a stronglypositive skin test to annatto extract, and anIgE-binding protein was identified throughsodium dodecyl sulphate–polyacrylamide gelelectrophoresis (SDS–PAGE) with immuno-blotting. Because annatto extract is derivedfrom a seed, the presence of proteins in theextracts is likely. IgE-mediated allergies toannatto proteins are possible, although this isthe only reported case in the medical litera-ture. Young et al. (1987) estimated the preva-lence of annatto sensitivity at 0.01–0.07%.

Carmine

Carmine and cochineal extract are derivedfrom dried female insects of the speciesDactylopius coccus, which lives as a parasiteon the prickly pear cactus. An aqueousalcoholic extract of the dried insects is madeand concentrated, by removal of the alcohol,to obtain the colour additive, cochinealextract. The colouring principle of cochinealextract is carminic acid. Carmine is thealuminium or calcium–aluminium lake of thecolouring principles, primarily carminic acid,obtained by aqueous extraction of cochineal.Carmine and cochineal extract have a redcolour.

Carmine is widely used in cosmetics, butonly a few cases of dermatological reactionshave been attributed to it (Sarkany et al., 1961;Kagi et al., 1994). Park (1981) reported a caseof severe anaphylactic shock possibly linkedto the cutaneous use of carmine. A soldierinvolved in a casualty simulation drill wassmeared with a make-up stick to simulateburns. An immediate anaphylactic responseensued, characterized by severe hypotensionand tachycardia. Unfortunately, no follow-upwas done on this patient to confirm the role ofcarmine in this case.

Two individuals with carmine-associatedoccupational asthma also reacted to oralchallenges with carmine solution (Burge et al.,1979). One responded with asthma and gas-trointestinal upset after challenge with 1 ml of

cochineal extract diluted in 100 ml of water.The other experienced asthma after drinkingCampari, a beverage that contains carmine.Kagi et al. (1994) described an individual withanaphylaxis characterized by rhinitis, asthma,urticaria and multiple gastrointestinalcomplaints after ingestion of Campari-orange. This individual had positive skinprick tests to the Campari beverage, carmineand carmine-containing cosmetics, indicatinga possible IgE-mediated reaction. Anothercase of probable IgE-mediated allergy tocarmine has been described in an individualwho reacted with urticaria, angio-oedemaand asthma after ingestion of a carmine-containing yoghurt (Beaudouin et al., 1995).A histamine-release assay using the patient’sbasophils was also positive, another indica-tion of an IgE-medicated reaction (Beaudouinet al., 1995). Because carmine is obtained froman extract of insect bodies, it might containproteins and could elicit IgE-mediated reac-tions in rare cases such as these.

Summary

Tartrazine and other dyes, benzoates andparabens occasionally may aggravate buthave not been reported as the cause ofchronic urticaria. Parabens have been shownto provoke (rarely) anaphylaxis whengiven parenterally. Sulphites, although notimplicated in chronic urticaria, occasionallyhave been reported to provoke anaphylaxis.MSG has been reported to provoke angio-oedema in a single case report. Aspartame,BHA and BHT have been shown to be thecause of isolated cases of chronic idiopathicurticaria. Nitrates and nitrites have not beenassociated with urticaria, angio-oedema oranaphylaxis.

Asthma and Additives

Design considerations for challenge studies

General considerations

A screening challenge should be conductedin a single-blind, open fashion. Because so

11-Mar-03 11




few patients are sensitive to additives, single-blind challenges simplify the procedure.More importantly, such a challenge fulfilsthe important safety requirement of individ-ualizing doses. At each successive challengestep in the protocol, the doses increase two-fold. If a patient has a 10–15% drop in FEV1

after a particular dose, one may wish to add adose at half the usual increment of increase.This would not be possible with a double-blind challenge protocol.

Other safety factors include performingthe challenge when the patient’s asthma isstable (FEV1 value of at least 1.5 l and 70% ofpredicted or prior best values). At times, espe-cially in patients most likely to be sulphite-sensitive (e.g. chronic asthmatic patients arecorticosteroid dependent), these patients mayrequire a burst of corticosteroids to stabilizetheir asthma activity (as in aspirin-sensitivesubjects). Challenges routinely begin in themorning and can take place in the physician’soffice if one is prepared to treat rapidlymoderate to severe asthma with inhaledbronchodilators. Using the recommendedprotocol and individualized doses, we havenot encountered severe reactions during hun-dreds of sulphite challenges, and all patients’bronchoconstriction could be reversed rap-idly with albuterol or metaproterenol admin-istered with a hand-held nebulizer.

On the day of challenge, patients shouldwithhold their inhaled and oral β2 agonistsand inhaled anticholinergics, and anti-histamines and cromolyn should be withheldfor 24 h before the challenge. We continuetheophylline at therapeutic levels andcontinue (even increase) inhaled and oralsteroids. In our experience, theophylline andsteroids do not interfere with sulphite (oraspirin) challenges (Stevenson and Simon,1981; Pleskow et al., 1983; Simon andStevenson, 1987). Although a recent studysuggests that corticosteroids may increase thethreshold reaction dose (Nizankowska andSzczeklik, 1989), withholding theophyllineand steroids may lead to a false-positivechallenge in an unstable asthmatic patient.To control this variable, and to establishstability of their bronchial airways, werecommend that patients undergo a placebo

challenge for a length of time equal to that ofthe sulphite challenge.

Pulmonary function is measured beforechallenge and before the next dose or sooner ifsymptoms occur. A 20% drop in FEV1 valuefrom the baseline is considered a positivechallenge.

Sulphite challenge

Specific considerations

Since the FDA regulation on sulphite usage inthe late 1980s, the indications for challengeswith SO2, sulphite and sulphurous acidparticles have become limited to scientificinvestigation and very occasional clinicalpurposes.

Challenges for investigation of the envi-ronmental and occupational hazards of SO2

and sulphuric acid exposures include epide-miological studies and studies of asthmaticpopulations aimed at better drawing the linesfor regulating emissions of these chemicals(Boushey, 1982a). Likewise, scientific investi-gations aimed at better understanding thebronchial hyper-reactivity associated withasthma may employ SO2 or sulphite chal-lenges akin to histamine, methacholine, exer-cise, water, osmolar, allergen, mediator andpharmacological challenges outlined in otherchapters in this volume.

For clinical purposes, when an asthmaticpatient recognizes reactivity to SO2/sulphite,as is most likely to occur with exposure-ingestion of dried fruits or wine, he or she canavoid these sulphite sources and is protectedfurther by regulations of the FDA requiringlabelling of processed food containing morethan 10 ppm SO2 equivalents. If there issuspicion, particularly if there is uncertaintyregarding reaction to dried fruits, wine,processed potatoes or shrimp, or when anasthmatic patient must be reassured that he orshe is not relevantly sensitive to sulphites, oralchallenge with the suspected food or beverage(Taylor et al., 1988) or capsule doses of sulphiteunder single-blind and, if the result is appar-ently positive, then double-blind conditionsare appropriate.


20-Feb-03 11



At Scripps Clinic, we use the followingprotocol for sulphite capsule challenges:patients are challenged at a time when theirasthma is in remission as documented byFEV1 greater than 70% of the predicted orbest previously observed value and at least1.5 l absolute. Inhaled cromolyn (Koeniget al., 1988) and anticholinergic (Simon et al.,1984b), antihistamine (Simon et al., 1982) andadrenergic bronchodilator medications arewithheld on the day of challenge; othermedications are continued. Open oral chal-lenges with capsule doses of 5, 25, 50, 100 and200 mg of potassium metabisulphite at 30-minintervals are administered, and spirometricmeasurements are performed before eachdose. If there is a fall of FEV1 of 20% ormore, the challenge is suspended, andobstruction is reversed with inhalations ofadrenergic bronchodilator. Apparent reac-tions need to be verified with double-blind,placebo-controlled challenges.

Before concluding that a patient issulphite sensitive, one should repeat the chal-lenge in a double-blind, placebo-controlledmanner, starting with the patient’s previouslyestablished provoking dose and using at leasttwo other placebo challenges. For suggestedchallenge doses for common food additives,see Box 11.4.

Specific additives and asthma

Tartrazine

Data on the incidence of reactivity toadditives in patients with asthma are, onthe whole, only slightly better than thosefor patients with urticaria. The additive mostfrequently implicated in provoking asthmaticreactions has been tartrazine. Critical reviewof the medical literature, however, suggeststhat sensitivity to tartrazine in patientswith asthma is distinctly unusual, if itexists at all (Simon, 1984; Stevenson, 1991).In 1958, Speer stated that agents used inartificial colouring were the cause of asthmain sick children; however, the authorpresented no details about how this con-clusion was reached. In 1967, Chafee and

Settipane reported a patient with severeasthma who experienced angio-oedema afteraspirin ingestion and severe attacks ofasthma shortly after ingesting a number ofunrelated drugs. After approximately 2 yearsof such reactions and a great deal of investi-gative activity, the attacks disappearedwhen benzoates and tartrazine wereeliminated from this patient’s food and medi-cation. During eight double-blind, placebo-controlled challenges with various dyes,significant symptoms (tickling of throat,tight cough and wheeze) occurred onlyafter receiving tartrazine. Unfortunately, nopulmonary function studies were conductedand the double-blind challenge for tartrazinewas not repeated.

In their classic monograph on aspirinintolerance, Samter and Beers (1968) dis-cussed the fact that benzoates and tartrazinewere commonly used in the food ingested bytheir aspirin-sensitive individuals. In theirfirst report, 80 patients with asthma were chal-lenged with unknown doses of tartrazine andthree ‘reacted’ (Samter and Beers, 1967). How-ever, essential information concerning with-holding or continuing medications, use ofplacebo controls, criteria for positive reaction,etc. were not provided. Juhlin et al. (1972)reported that seven of eight patients withasthma who were sensitive to aspirin alsoreacted to tartrazine. However, the investiga-tor’s criteria for a positive reaction were sub-jective, and details of the placebo challengeswere not discussed. As the studies of Steniusand Lemola (1976) point out, such details areimportant. Their protocol called for withhold-ing bronchodilators on the day of challenge,then giving a placebo first, followed by aspirinand finally tartrazine. All these challengestook place on the same day; therefore, anypatient requiring bronchodilators would beleast likely to ‘react’ to placebo and morelikely to ‘react’ to aspirin. Finally, as the daywore on and any possible bronchodilatoreffects wore off, patients’ bronchial treesbecame most likely to constrict, in the absenceof bronchodilator, or ‘react’ to tartrazine. Itis also unclear from these studies what hap-pened to the patients who ‘reacted’ to aspirin.That is, when exactly was the tartrazinechallenge performed in the sequence of

20-Feb-03 11




challenges? Was it performed after treatmentfor the aspirin reaction, after the aspirin reac-tion spontaneously resolved, in the middle ofan unresolved aspirin reaction or on anotherday? In view of any of these uncertainties,how would one interpret a ‘reaction’ totartrazine? Finally, the criteria for a positivereaction was a 20% fall in peak expiratoryflow. Their data were also reported inexactly:‘. . . about 25% of 140 asthmatics were aspi-rin-sensitive and 20% tartrazine-sensitive.’

In another study without placebo con-trols, Freedman (1977) challenged 14 of 30patients with asthma who gave a history ofasthma after ingestion of orange-coloureddrinks. Only one patient experienced a‘reaction’ to tartrazine; her maximal fall inFEV1 was only 14% after ingestion of a 20 mgdose (apparently the criterion for a reactionwas a 14% decline in FEV1 values). In a morerecent study by Rosenhall (1982), 2.3% of 542patients with asthma had a ‘definitely’ posi-tive response to tartrazine, and another 6%had a ‘probably’ positive response. Someproblems with this study included single-blind challenges and the fact that placebostudies were conducted only if challengesto other substances were ‘difficult to inter-pret’. Furthermore, non-respiratory tractchanges, including cutaneous responsessuch as urticaria and gastrointestinal com-plaints such as vomiting or diarrhoea, wereincluded as criteria for a positive response inthese asthmatic subjects.

A decade earlier, one of the few double-blind, placebo-controlled challenges withtartrazine was performed in 38 patients with ahistory of aspirin-provoked asthma (Settipaneand Pudupakkam, 1975). Although only0.44 mg tartrazine was used as the highestprovoking dose, three of 38 patients werefound to be responsive to tartrazine (experi-encing a > 20% fall in vital capacity, FEV1 andexpiratory flow rates).

Spector et al. (1979) performed placebo-controlled aspirin and tartrazine challenges inmore than 200 patients. Of 230 patients, 44 hadpositive reactions to aspirin (an incidence ofalmost 20%). Of 277 patients, 11 reacted totartrazine (FEV1 falls of > 20%), an incidenceof less than 4% in the population studied;however, five of these 11 patients did not

undergo placebo challenges. All 11 patientswho were reported to have reacted totartrazine also had a reaction to aspirin duringanother challenge. In other words, tartrazinesensitivity was not observed in patientswith asthma who were not sensitive to aspirin.One could extrapolate from these data that15–25% of ASA-sensitive asthmatic patientsare also sensitive to tartrazine. Yet, in double-blind, placebo-controlled challenges of 45patients who had a history of moderatelysevere asthma (one-half of whom also hadnasal polyps and up to 45% of whom weresensitive to aspirin), Weber et al. (1979) didnot find any who were sensitive to tartrazinein doses up to 20 mg. Along these lines,Vedanthan et al. (1977) conducted tartrazinechallenges in 54 children (aged 10–17 years)with asthma and found none who weresensitive to tartrazine. Five of the 54 childrenwere sensitive to aspirin during challengesconducted at another time.

Tarlo and Broder (1982) performeddouble-blind ingestion challenges withtartrazine, benzoate and aspirin. Of the28 subjects, only one responded to tartrazine(15 mg producing a 20.4% drop in FEV1)and one to benzoate (25 mg provoking a29% drop). Neither of these patients wasfound to be aspirin sensitive during chal-lenges with this drug, and neither respondedto dietary elimination of the two suspectedadditives.

In 1985, Genton et al. reported challengeresults with additives, including tartrazine,in 17 asthmatic subjects. Attempts were notmade to mask the flavour or colour of theagents tested. β Agonists were withheld andonly a single placebo was administered,versus multiple doses of other substances. Apositive challenge was defined as a 20% dropin peak flow rates up to 8 h after a challenge.Even with this protocol, only one subject‘reacted’ to tartrazine.

For more than 20 years, investigators atScripps Clinic have been studying aspirin-sensitive asthma. One should note thattartrazine is not a cyclo-oxygenase inhibitor(Gerber et al., 1979) and therefore would notbe expected to cross-react with aspirin, asdo non-steroidal anti-inflammatory drugs,in such patients. In any case, we performed


20-Feb-03 11



tartrazine challenges before aspirin chal-lenges as a routine procedure in morethan 150 single-blind, placebo-controlledchallenges (with 25 and 50 mg of tartrazine)in our aspirin-sensitive asthmatic population.In this single-blind screening study, weidentified six patients whose FEV1 declinedafter tartrazine (Stevenson, 1991). In five ofsix patients, rechallenge with tartrazine usingdouble-blind, placebo-controlled challengesequences was negative. One patient whoexperienced a decline in FEV1 valuesduring single-blind tartrazine challengesmoved out of town, and we have beenunable to rechallenge this patient in adouble-blind, placebo-controlled fashion.We remain sceptical that tartrazine-inducedasthma attacks even exist. Certainly, evidencelinking tartrazine to aspirin sensitivity has notbeen forthcoming.

Other dyes

Reactions to non-azo dyes and azo dyes otherthan tartrazine are reported far less com-monly than those to tartrazine, even in thosestudies that reported tartrazine sensitivity.Therefore, these agents will not be discussedfurther.

Sulphites

In 1973, Kochen described a child whodeveloped acute bronchospasm after openingcellophane packages and ingesting driedfruits treated with SO2. Although the termsulphite was not mentioned, this reportmay in fact be the first example of sulphiteinhalation-induced asthma. The first casereport actually specifying sulphite was in1976 by Prenner and Stevens. They describeda non-asthmatic but atopic individual witha history of hay fever. After a sulphite-containing restaurant meal, the subject devel-oped generalized urticaria, angio-oedemaand possibly laryngeal oedema. During anunlinked oral challenge with a 10 mg sulphitecapsule, the patient developed itching, coughand tightness in the throat. The challengewas considered positive and was discon-tinued without producing urticaria, angio-

oedema or laryngeal oedema. Pulmonaryfunction tests to determine whether therewas an asthmatic response were notperformed.

In 1977, Freedman, in the UK, noted thatmany asthmatic patients gave histories ofreacting to citrus drinks. These drinks containtartrazine (FD&C yellow no. 5) and benzoateas well as SO2. Freedman’s ‘sulphur dioxidechallenges’ were performed by subjectsingesting solutions into which sulphiteshad been dissolved. Some of the asthmaticsubjects did have wheezing after SO2

challenges. However, the challenges werenot placebo controlled and the amount of SO2

inhaled is not clear. The author considered afall in FEV1 of as little as 12% to be a positivereaction.

In 1981, Stevenson and Simon firstreported five adult asthmatic patients with ahistory of severe restaurant-provoked asthmaand even anaphylaxis who underwentsingle-blind, placebo-controlled capsule chal-lenges. A 20% or greater fall in FEV1 10–20 minafter ingesting capsules containing 5–50 mgof potassium metabisulphite (K2S2O5) wasreproduced in all five patients during single-blind oral challenges (Stevenson and Simon,1981). Simultaneously, Baker et al. (1981)reported asthmatic reactions to sulphitescontained in pharmaceutical products. In1982, Twarog and Leung observed an 18-year-old asthmatic subject with a historyof recurring asthma attacks after restaurantmeals. In addition, on two separate occasionswhile hospitalized for non-asthma-relatedproblems, the patient received Bronkosol®and experienced severe attacks of asthmaresulting in respiratory arrest. This patientalso developed an equally severe reactionafter receiving intravenous metoclopramide(Reglan®). Comparison of the constituentsof these two agents revealed bisulphite asthe only common substance. A similar patientwith a paradoxical bronchospastic reactionfollowing Bronkosol® inhalation wasreported by Koepke et al. (1984). Genton et al.(1985) found four of 17 adult asthmaticswho reacted to high concentrations of acidicsolutions of sulphites. Also in 1985, in a letter,a patient was described who experienced epi-sodes of bronchoconstriction after application

20-Feb-03 11




of an eye solution containing sulphite preser-vatives (Schwartz and Sher, 1985a).

Clinical characteristics of sulphite-sensitiveasthmatic individuals

Patient profile

The typical clinical features of sulphite-sensitive asthmatic individuals were firstdescribed in 1981 (Stevenson andSimon, 1981). All have chronic asthma,usually are corticosteroid dependent andare provoked by multiple other factors (e.g.upper respiratory infections, irritants andexercise). The irritant effects of smog, pre-sumably on afferent receptors in the trachea,are particularly troublesome for these indi-viduals. Characteristically, the most severecorticosteroid-dependent, sulphite-sensitivesubjects did not have asthma until their firstisolated sulphite reactions. Within months,they progressed from asymptomatic tochronic asthma with corticosteroid depend-ency. This indicates that any member ofthe population is at risk for sulphite sensitiv-ity. The typical sulphite-sensitive asthmaticis usually non-atopic with chronic vasomo-tor-type rhinosinusitis. These individuals aredifferentiated from aspirin-sensitive patientsbecause they lack nasal polyps and eosino-philia. Sinus X-rays are abnormal in a highpercentage of both aspirin-sensitive andsulphite-sensitive asthmatics.

Cross-sensitivity

Studies by Stevenson and Simon in 15sulphite-sensitive asthmatics had shownthat none reacted to aspirin during oral chal-lenges and, vice versa, none of a group of15 aspirin-sensitive asthmatic subjects hadpositive oral sulphite challenges (Simon andStevenson, 1987). Moreover, careful review ofthe medical literature does not confirm thataspirin and sulphite sensitivity co-exist in thesame individual. One report described dualpositive challenges in some patients withchronic asthma (Kochen, 1973). However, thedetails of the challenges and the clinical

characteristics of the patients were not avail-able in the report. One sulphite-sensitiveasthmatic patient originally described asaspirin sensitive (Koepke et al., 1984) wassubsequently rechallenged with both aspirinand sulphite at Scripps Clinic and found tohave only sulphite sensitivity (Simon, 1985).None of our sulphite-sensitive asthmaticpatients have experienced positive challengesto tartrazine (50 mg) or MSG (2500 mg), norhave they manifested IgE-mediated sensitiv-ity with food antigens by skin testing.

Although Baker et al. (1981) describedsulphite-sensitive patients who also had sen-sitivity to aspirin and, in some cases, otheradditives, such as MSG and benzoates, thesedata were largely historical and not confirmedby challenges. In addition, the dose of sulphiteused during their challenges was 500 mg. Wenow recognize that this dosage is excessive.Finally, one report described an individualwith a history of MSG sensitivity who reactedto both MSG and sulphite (50 mg in solution)in a double-blind, placebo-controlled chal-lenge (Koepke and Selner, 1986).

Prevalence

The prevalence of adverse reactions tosulphiting agents is not known despiteattempts to establish the prevalence ofsulphite sensitivity in asthmatic subjects.Because of the nature of the populations stud-ied and the challenge methods employed,the incidence can only be estimated. Simonet al. (1982), in a preliminary study, examinedthe prevalence of sensitivity to ingestedmetabisulphite in a group of 61 adultasthmatic subjects. None gave a historyof sulphite sensitivity. Challenges were con-ducted with potassium metabisulphitecapsules and solutions. Positive responseswere confirmed by a placebo-controlledchallenge. Of 61 patients, five (8.2%) had a25% or greater decline in FEV1 values duringsulphite challenge. Koepke and Selner (1982)conducted open challenges with sodiummetabisulphite in 15 adults with a history ofasthma after ingestion of sulphited foods andbeverages. One of 15 (7%) had a 28% declinein FEV1. A confirmatory challenge was notconducted. In a larger study by Buckley et al.


20-Feb-03 11



(1985), 134 patients underwent single-blindchallenges with potassium metabisulphitecapsules. Of these, 4.6% were reported tohave sulphite sensitivity. In these threepreliminary studies, the patient populationsconsisted of asthmatics with corticosteroiddependency who were being evaluated atan allergy referral centre. Thus, the preva-lence, based on these observations, is proba-bly not applicable to the asthma populationas a whole, because corticosteroid depend-ency is one of the clinical characteristics ofsulphite-sensitive asthmatics.

In the largest study reported to date, Bushet al. (1986) conducted capsule and neutralsolution sulphite challenges in 203 adultasthmatic subjects. Patients were not selectedfor a history of sulphite sensitivity. Of thesepatients, 120 were not receiving cortico-steroids, while 83 were corticosteroid depend-ent. Of the non-corticosteroid-dependentgroup, only one experienced a 20% or greaterdecline in FEV1 values after single-blind andconfirmatory double-blind challenges. Theresponse rate in the corticosteroid-dependentasthmatic group was higher (8.4%). Theprevalence in the asthmatic population as awhole was less than 3.9%, and corticosteroid-dependent asthmatic patients appeared to bemost at risk for sulphite sensitivity.

Although corticosteroid-dependent asth-matic individuals have the highest incidenceof positive sulphite challenge results, they arenot the only group at risk. Notable in the his-tory of sulphite-sensitive asthmatic patients isthat originally they were not corticosteroiddependent or even asthmatic while under-going their initial sulphite reactions. In fact,most did not have asthma when they beganhaving severe restaurant-provoked broncho-spastic reactions. Only later did they developchronic asthma that became corticosteroiddependent. Therefore, the population atrisk can be an early asthmatic or pre-asthmatic individual, indistinguishablefrom the general population. Fortunately,based upon studies and reports, the numberof these patients is small (Simon andStevenson, 1997).

The incidence of sulphite sensitivity inthe paediatric population is also unknown. Asnoted earlier, the first case of sulphite-induced

bronchospasm was in a child (Kochen, 1973).Since then, there have been two other iso-lated case reports of alleged sulphite-sensitivechildren (Sher and Schwartz, 1985; Wolfand Nicklas, 1985). However, these reportsdid not include properly designed andblinded challenges. Whether any of thesethree patients are, in fact, sulphite sensitiveremains in question. In one study ofchronic asthmatic children, almost two-thirds were reported to be sulphite sensitiveafter open ingestion challenge (Towns andMellis, 1984). Challenges did not includecontrols, and the children reacted only to largedoses of sulphite solutions. These childrenmay have reacted to the higher levels of SO2

generated in solution which are beingswallowed. Whatever the prevalence, investi-gators generally agree that sulphite reactionshave decreased markedly since 1986 whenthe FDA banned the use of sulphites in freshfood and required labelling for other sourcesof sulphites.

Benzoates and parabens

The next most commonly reported additivesthat might cause bronchospasm in patientswith asthma are the parabens. In Freedman’s(1977) study, four of 14 patients with a historyof sensitivity to orange drinks had positivebronchospastic reactions to sodium benzoatein uncontrolled challenges. Maximum de-creases in FEV1 values ranged from 23 to 33%between 10 and 30 min after 20–100 mgsodium benzoate. Samter and Beers (1968), asnoted earlier for tartrazine, reported thatsodium benzoate was a commonly used pre-servative in the foods which their aspirin-sensitive patients reacted to by history. InRosenhall’s (1982) study, despite poorlydesigned challenges, only one of 504 patientsreacted to a dose of sodium benzoate(< 100 mg). Weber et al. (1979) found onlyone of 43 patients with a positive reactionto 250 mg of sodium benzoate or hydroxy-benzoic acid in double-blind studies. Further-more, when this patient was rechallenged2 years later, he did not react to the sameprovoking dose of sodium benzoate.

20-Feb-03 11




The study of Genton et al. (1985) alsoexamined asthmatic reactions to sodiumbenzoate and only found one of 17 subjectswho was reported to have ‘reacted’.

The only authors to report a double-blind, placebo-controlled, benzoate-inducedreaction were Tarlo and Broder (1982). Onceagain, their patient was described as beingaspirin insensitive. Additionally, no improve-ment was noted in this patient’s asthma whenbenzoate was removed from the diet.

Monosodium glutamate

The initial report of MSG-provoked asthmadescribed two patients with delayed asthmaticreactions (12 h post-ingestion) (Allen andBaker, 1981b); however, a subsequent reportdescribed four other patients with a history ofCRS and asthma in the fourth patient experi-encing a respiratory arrest within 3 h of theChinese meal (Allen and Baker, 1981a). Chal-lenges performed open or single-blind withchanges in peak respiratory flow rates wereused to confirm positive reactions. In addi-tion, three of 12 asthmatics without a historyof Chinese restaurant-provoked asthma hadpositive challenges to MSG (all late). Withsingle-blind, placebo-controlled screeningchallenges, 100 subjects with asthma (30subjects with a history of Chinese restaurantasthma attacks; 70 patients with a negativehistory) were challenged with 2.5 g of MSG.No patient had a significant fall in FEV1 valueor the development of asthma symptomsduring the MSG challenge. The mean changein FEV1 with MSG challenge was no differentfrom that of placebo challenge. A case des-cribing MSG-provoked asthma has beenreported. This individual had positivedouble-blind, placebo-controlled MSG andsulphite challenges (Koepke and Selner,1986). Not surprisingly, there are two reportsinvolving small numbers of mild asthmatics,without a suggestive history, who were notfound to react to MSG during oral challenges(Schwartzstein et al., 1987; Germand et al.,1991). We have not seen a positive early or

late reaction to MSG in our Scripps Clinicasthma population (Woessner et al., 1999).

BHA/BHT

In 1973, Fisherman and Cohen reportedseven patients with either asthma or rhinitiswho were said to be intolerant to BHAand BHT. These patients were identifiedby a doubling of their earlobe bleedingtimes. Clinical details, or the reasonwhy BHA or BHT was ever suspectedto cause difficulty, were not given. Ratio-nale for the reported effect on the bleedingtime was not given. The next year, perform-ing a similar study, Cloninger and Novey(1974) refuted these findings (Fisherman andCohen, 1976).

Other chemicals

Benzalkonium chloride

Paradoxical responses to nebulized ipatro-pium bromide (Beasley et al., 1987) andbeclomethasone dipropionate (Clark, 1986)led to the discovery that the antibacterialpreservative benzalkonium chloride causesbronchoconstriction in about 60% of asth-matic subjects; the characteristics of theresponses – rapid onset with slow recoveryover 60 min, and inhibition by cromolyn butnot ipatropium – suggest a mechanism ofaction via release of mediators (Zhang et al.,1990). The benzalkonium chloride concentra-tion in commercial nebulizer solutions hasbeen reduced so that only the rare patientwith apparent immunological sensitivity willnow react (Ponder and Wray, 1993).

Spearmint

The flavours spearmint (Mentha spicata),peppermint (Mentha piperita) and menthol(Mentha labiateae), used in chewing gumand toothpaste, have been confirmed bychallenges in two cases to have triggered


20-Feb-03 11



asthma (Spurlock and Dailey, 1990; Subizaet al., 1992).

Summary

Carefully designed, well-controlled studieshave failed to confirm tartrazine (or otherazo and non-azo dye)-provoked asthmaticreactions. Sulphites, on the other hand, haveclearly been shown to produce serious,even life-threatening, asthmatic reactions byseveral proposed mechanisms. Approxi-mately 3–5% of asthmatic patients aresulphite sensitive, most of whom react byinhaling SO2 generated when sulphites areplaced in solution. When the FDA bannedsulphites added to fresh foods in 1986, thefrequency, and therefore importance, of thisproblem was greatly diminished. Benzoatesand parabens have not been shown con-clusively to be a significant problem for asth-matics, even those who are aspirin sensitive.MSG may occasionally produce asthmaticreactions but certainly does not present aproblem to the vast majority of asthmatics.BHA and BHT have not been shown to pro-duce asthmatic problems.

Benzalkonium chloride frequently cancause bronchoconstriction when inhaled byasthmatic subjects but the concentration nowused is so low that only individuals withimmediate hypersensitivity will react; fortu-nately, such patients are rare. Recently, spear-mint flavouring has been shown to triggerasthma in two cases.

References

Aldrete, J.A. and Johnson, D.A. (1969) Allergyto local anaesthetics. Journal of the AmericanMedical Association 207, 356–357.

Allen, D.A. (1991) Monosodium glutamate. In:Metcalf, D.D., Sampson, H.A. and Simon, R.A.(eds) Adverse Reactions to Food and Food Addi-tives. Blackwell Scientific, Boston, pp. 261–266.

Allen, D.H. and Baker, G.H. (1981a) Asthma andMSG. Medical Journal of Australia 2, 576.

Allen, D.H. and Baker, G.J. (1981b) Chinese restau-rant asthma. New England Journal of Medicine305, 1154–1155.

Altman, D.R. (1996) Public perception of foodallergy. Journal of Allergy and Clinical Immunol-ogy 97, 1247–1251.

Altman, L.C., Sprenger, J.D. and Ayars, G.H. (1985)Neutrophil chemotactic activity (NCA) insulphite-sensitive patients. Annals of Allergy55, 234 (abstract).

Babich, H. (1982) Butylated hydroxytoluene (BHT):a review. Environmental Research 29, 1–29.

Baker, G.J., Collette, P. and Allen, D.H. (1981)Bronchospasm induced by metabisulphite-containing foods and drugs. Medical Journal ofAustralia 2, 614.

Balmes, J.R., Fine, J.M. and Sheppard, D. (1987)Symptomatic bronchoconstriction aftershort term inhalation of sulphur dioxide.American Review of Respiratory Diseases 136,1117–1121.

Barbier, M., Lederer, E., Reichstein, T. andSchindler, O. (1960) [Separation of the acidcomponents of extracts from queen bees (Apismellifica L.); isolation of the pheromonedesignated as queen substance.] HelveticaChimica Acta 60, 1682–1689 (in German).

Beasley, C.R.W., Rafferty, P. and Holgate, S.T. (1987)Bronchoconstrictor properties of preserva-tives in ipratropium bromide (Atrovent)nebuliser solution. British Medical Journal 294,1197–1198.

Beaudouin, E., Kanny, G., Lambert, H. et al. (1995)Food anaphylaxis following ingestion ofcarmine. Annals of Allergy, Asthma, andImmunology 74, 427–430.

Bethel, R.A., Erle, D.J. and Epstein, J. (1983a) Effectof exercise rate and route of inhalation onsulphur dioxide-induced bronchoconstrictionin asthmatic subjects. American Review ofRespiratory Diseases 128, 592–596.

Bethel, R.A., Epstein, J. and Sheppard, D. (1983b)Sulphur dioxide-induced bronchoconstrictionin freely breathing, exercising, asthmaticsubjects. American Review of Respiratory Diseases128, 987–990.

Bethel, R.A., Sheppard, D. and Geffroy, B.(1985) Effect of 0.25 ppm sulphur dioxideon airway resistance in freely breath-ing, heavily exercising, asthmatic subjects.American Review of Respiratory Diseases 131,659–661.

Boushey, H. (1982a) Asthma, sulphur dioxide andthe Clean Air Act. Medical Staff Conference,Univerity of California, San Francicso. WesternJournal of Medicine 136, 129–135.

Boushey, H.A. (1982b) Bronchial hyperreactivityto sulphur dioxide: physiologic and politicalimplications. Journal of Allergy and ClinicalImmunology 69, 335–338.

20-Feb-03 11




Boxer, M.B., Bush, R.K., Harris, K.E., Patterson, R.,Pruzansky, J.J. and Yang, W.H. (1988) Thelaboratory evaluation of IgE antibody tometabisulphites in patients skin test positive tometabisulphite. Journal of Allergy and ClinicalImmunology 82, 622–626.

Buckley, R., Saltzman, H.A. and Sieker, H.O. (1985)The prevalence and degree of sensitivityto ingested sulphites. Journal of Allergy andClinical Immunology 77, 144 (abstract).

Budavari, S., O’Neil, M.J., Smith, A., Heckelman,P.E. and Kinneary, J.F. (eds) (1996) Sorbic acid.In: The Merck Index. Merck & Co., WhitehouseStation, New Jersey, p. 1489.

Burge, P.S., O’Brien, I.M., Harries, M.G. et al. (1979)Occupational asthma due to inhaled carmine.Clinical Allergy 9, 185–189.

Bush, R.K., Taylor, S.L., Holden, K., Nordlee, J.A.and Busse, W.W. (1986) The prevalence ofsensitivity to sulphiting agents in asthmaticpatients. American Journal of Medicine 81,816–820.

Cecil, R. (1963) Intramolecular bonds in proteins. I.The role of sulphur in proteins. In: Neurath, H.(ed.) The Proteins, Vol. I. Academic Press, NewYork, p. 379.

Chafee, S.H. and Settipane, G.A. (1967) Asthmacaused by FD&C approved diet. Journal ofAllergy 40, 65–72.

Chapman, R.S., Calafiore, D.C. and Hassiblad, V.(1985) Prevalence of persistent cough andphlegm in young adults in relation to long-term ambient sulphur oxide exposure. Ameri-can Review of Respiratory Diseases 132, 261–267.

Clark, R.J. (1986) Exacerbation of asthma afternebulised beclomethasone diproprionate.Lancet 574.

Clayton, D.E. and Busse, W. (1980) Anaphylaxis towine. Clinical Allergy 10, 341–343.

Cloninger, P. and Novey, H.S. (1974) The acuteeffects of butylated hydroxyanisole ingestionin asthma and rhinitis of unknown etiology.Annals of Allergy 32, 131–133.

Code of Federal Regulations (1984) CFR, 1984 Code ofFederal Regulations Title 21: Food and Drugs.Office of the Federal Register, General ServiceAdministration, Washington, DC.

Collins-Williams, C. (1983) Intolerance to additives.Annals of Allergy 51, 315–316.

Delohery, J., Simmul, R., Castle, W.D. and Allen,D.H. (1984) The relationship of inhaledsulphur dioxide reactivity to ingested metabi-sulphite sensitivity in patients with asthma.American Review of Respiratory Diseases 130,1027–1032.

Doeglas, H.M. (1975) Reactions to aspirin and foodadditives in patients with chronic urticaria,

including the physical urticarias. British Journalof Dermatology 93, 135–144.

Epstein, E. (1970) Sodium bisulphite. Contact Derma-titis Newsletter 7, 115.

Fine, J.M., Gordon, T. and Sheppard, D. (1987) Theroles of pH and ionic species in sulphurdioxide- and sulphite-induced bronchocon-striction. American Review of RespiratoryDiseases 136, 1122–1126.

Fisherman, E.W. and Cohen, G.N. (1973) Chemicalintolerance to butylated hydroxyanisole(BHA) and butylated hydroxytoluene (BHT)and vascular response as an indicator andmonitor of drug tolerance. Annals of Allergy 31,126–133.

Fisherman, E.W. and Cohen, G.N. (1976) Recurringand chronic urticaria: identification of etiolo-gies. Annals of Allergy 36, 400–409.

Freedman, B.J. (1977) Asthma induced by sulphurdioxide, benzoate and tartrazine contained inorange drinks. Clinical Allergy 7, 407–415.

Freedman, B.J. (1980) Sulphur dioxide in foods andbeverages: its use as a preservative and itseffect on asthma. British Journal of the DiseasedChest 74, 128–134.

Fuglsang, G., Madsen, C., Saval, P. et al. (1993) Prev-alence of intolerance to food additives amongDanish school children. Pediatric Allergy andImmunology 4, 123–129.

Fuglsang, G., Madsen, C., Halken, S. et al. (1994)Adverse reactions to food additives in childrenwith atopic symptoms. Allergy 49, 31–37.

Fujii, M. (1996) Estimated daily intake of food addi-tives. In: Report of the Health Science ResearchGrants, Ministry of Health and Welfare. Ministryof Health and Welfare, Tokyo, pp. 6–33.

Garriga, M.M. and Metcalfe, D.D. (1988) Aspartameintolerance. Annals of Allergy 61, 63–69.

Genton, C., Frei, P.C. and Pecond, A. (1985) Value oforal provocation tests to aspirin and food addi-tives in the routine investigation of asthma andchronic urticaria. Journal of Allergy and ClinicalImmunology 76, 40–45.

Gerber, J.G., Payne, N.A., Oelz, O., Nies, A.S. andOates, J.A. (1979) Tartrazine and the prosta-glandin system. Journal of Allergy and ClinicalImmunology 63, 289–295.

Germand, P., Cohen, S.G., Hahn, B. and Metcalfe,D.D. (1991) An evaluation of clinical reactionsto monosodium glutamate (MSG) in asthmat-ics using a blinded placebo-controlled chal-lenge. Journal of Allergy and Clinical Immunology87, 177 (abstract).

Ghadimi, H., Kumar, S. and Abaci, F. (1971) Studieson monosodium glutamate ingestion. 1. Bio-chemical explanation of the Chinese restaurantsyndrome. Biochemical Medicine 5, 447–456.


20-Feb-03 11



Gibson, A. and Clancy, R. (1980) Management ofchronic idiopathic urticaria by the identifica-tion and exclusion of dietary factors. ClinicalAllergy 10, 699–704.

Goodman, P.L., McDannell, J.T., Nelson, N.S.,Vaughan, T.R. and Weber, R.W. (1990) Chronicurticaria exacerbated by the antioxidantfood preservatives butylated hydroxyanisole(BHA) and butylated hydroxytoluene (BHT).Journal of Allergy and Clinical Immunology 86,570–575.

Grogan, W.A. (1986) Toxicity from BHT ingestion(correspondence). Western Journal of Medicine145, 245–246.

Gross, P.A., Lance, K., Whitlock, R.J. and Blume, R.S.(1989) Additive allergy: allergic gastroenteritisdue to yellow dye #6. Annals of Internal Medi-cine 111, 87–88.

Grunbaum, A. (1985) Explications and implicationsof the placebo concept. In: White, L., Tursky, B.and Schwarz, G.E. (eds) Placebo: Theory,Research, and Mechanisms. Guilford, New York,pp. 37–58.

Habenicht, H.A., Preuss, L. and Lovell, R.G. (1983)Sensitivity to ingested metabisulphites: causeof bronchospasm and urticaria. Immunologyand Allergy Practice 5, 243–245.

Hanley, Q.S., Koenig, J.Q., Larson, T.V., Anderson,T.L., Van Belle, G., Rebolledo, V., Covert, D.S.and Pierson, W.E. (1992) Response of youngasthmatic patients to inhaled sulphuric acid.American Review of Respiratory Diseases 145,326–331.

Hannuksela, M. and Lahti, A. (1986) Peroralchallenge tests with food additives in urticariaand atopic dermatitis. International Journal ofDermatology 25(3), 178–180.

Ishiwata, H. and Yamada, T. (2000) Estimation offood additive concentrations in foods and theirdaily intake based on official inspection infiscal year 1996. Food Sanitation Research 50,7–34.

Ishiwata, H., Takeda, Y., Yamada, T., Watanabe, Y.,Hosogai, T., Ito, S., Sakurai, H., Aoki, G. andUshiama, N. (1995) Determination and confir-mation of methyl p-hydroxybenzoate in royaljelly and other foods produced by the honeybee. Food Additives and Contaminants 12,281–285.

Ishiwata, H., Sugita, T., Kawasaki, Y., Takeda, Y.,Yamada, T., Nishijima, M. and Fukasawa, Y.(2000) Estimation of inorganic food additive(nitrite, nitrate, and sulphur dioxide) concen-trations in foods and their daily intake basedon official inspection results in Japan in fiscalyear 1996. Journal of Food Hygienic Society ofJapan 41, 79–85.

Ito, Y. (2000) Present state and the investigationof daily intakes of food additiaves in Japan(1976–1996). Food Sanitation Research 50,89–125.

Jacobsen, D.W. (1997) Adverse reactions tobenzoates and parabens. In: Metcalfe, D.D.,Sampson, H.A. and Simon, R.A. (eds) FoodAllergy: Adverse Reactions to Foods and FoodAdditives, 2nd edn. Blackwell Scientific,Boston, pp. 375–386.

Joint FAO/WHO Codex Alimentarius Commission(1991) Codex Alimentarius. Food and Agri-culture Organization of the United Nations,Rome, p. 11.

Joint FAO/WHO Expert Committee on FoodAdditives (JECFA) (1992) Compendium of FoodAdditive Specifications. Food and AgricultureOrganization of the United Nations, Rome.

Joint FAO/WHO Expert Committee on FoodAdditives (JECFA) (1995) Nitrate and Nitrite.WHO Technical Report Series. World HealthOrganization, Geneva, pp. 29–35.

Joint FAO/WHO Expert Committee on FoodAdditives (JECFA) (1996) Toxicological Evalua-tion of Certain Food Additives and Contaminantsin Foods. WHO Food Additive Series. Inter-national Programme on Chemical Safety,Geneva.

Joint FAO/WHO Expert Committee on FoodAdditives (JECFA) (1999) Assessment of Intakeof Specific Food Additives. InternationalProgramme on Chemical Safety, Geneva,pp. 403–459.

Juhlin, L. (1977) Intolerance to food additives.In: Marzulli, F.M. and Maibach, H.I. (eds)Advances in Modern Toxicology, Vol. 4.Dermatotoxicology and Pharmacology. JohnWiley, New York, pp. 455–463.

Juhlin, L. (1981) Recurrent urticaria: clinicalinvestigation of 330 patients. British Journalof Dermatology 104, 369–381.

Juhlin, L., Michaelsson, G. and Zetterstrom, O.(1972) Urticaria and asthma induced by foodand drug additives in patients with aspirinsensitivity. Journal of Allergy and Clinical Immu-nology 50, 92–98.

Kagi, M.K., Wuthrich, B. and Johansson, S.G.O.(1994) Campari-orange anaphylaxis due tocarmine allergy. Lancet 344, 60–61.

Kellett, J.K., August, P.J. and Beck, M.H. (1984)Double-blind challenge tests with foodadditives in chronic urticaria. British Journalof Dermatology 111 (Supplement), 32.

Kenny, R.A. and Tidball, C.S. (1972) Humansusceptibility to oral monosodium glutamate.American Journal of Clinical Nutrition 25,140–146.

20-Feb-03 11




Kochen, J. (1973) Sulphur dioxide, a respiratory tractirritant, even if ingested (letter). Pediatrics 52,145.

Koenig, J.Q., Pierson, W.E. and Horike, M. (1983)The effect of inhaled sulphuric acid onpulmonary function in adolescent asthmatics.American Review of Respiratory Diseases 128,221–225.

Koenig, J.Q., Marshall, S.G. and Van Belle, G. (1988)Therapeutic range cromolyn dose – responseinhibition and complete obliteration of SO2-induced bronchoconstriction in atopic adoles-cents. Journal of Allergy and Clinical Immunology81, 897–901.

Koepke, J.W. and Selner, J.C. (1982) Sulphur dioxidesensitivity. Annals of Allergy 48, 258 (abstract).

Koepke, J.W. and Selner, J.C. (1986) Combinedmonosodium glutamate (MSG) and metabisul-phite (MBS)-induced asthma. Journal of Allergyand Clinical Immunology 77, 158 (abstract).

Koepke, J.W., Christopher, K.L., Chai, H. and Selner,J.C. (1984) Dose-dependent bronchospasmfrom sulphites in isoetharine. Journal of theAmerican Medical Association 251, 2982.

Komanowsky, M., Talley, F.B. and Eskew, R.K.(1970) Air drying of cultivated mushrooms.Food Technology 24, 80.

Kulczycki, A. (1986) Aspartame-induced urticaria.Annals of Internal Medicine 104, 207–208.

Kwok, R.H.M. (1968) Chinese restaurant syn-drome. New England Journal of Medicine 278,796.

Lee, R.J., Braman, S.S. and Settipane, G.A. (1986)Reproducibility of metabisulphite challenge.Journal of Allergy and Clinical Immunology 77,157 (abstract).

Linn, W.S., Venet, T.G. and Shamoo, D.A. (1983)Repiratory effects of sulphur dioxide inheavily exercising asthmatics. American Reviewof Respiratory Diseases 127, 278–283.

Linn, W.S., Shamoo, D.A. and Anderson, K.R. (1985)Effects of heat and humidity on the response ofexercising athmatics to sulphur dioxide expo-sure. American Review of Respiratory Diseases131, 221–225.

Linn, W.S., Avol, E.L. and Peng, R.C. (1987) Repli-cated dose–response study of sulphur dioxideeffects in normal, atopic and asthmatic volun-teers. American Review of Respiratory Diseases136, 1127–1134.

Lockey, S.D. (1959) Allergic reactions due to FD&Cyellow no. 5 tartrazine, an aniline dye used asa coloring and identifying agent in varioussteroids. Annals of Allergy 17, 719–721.

Lumry, W.R., Mathison, D.A., Stevenson, D.D. andCurd, J.C. (1982) Aspirin in chronic urticariaand/or angioedema: studies of sensitivity and

desensitization. Journal of Allergy and ClinicalImmunology Supplement 69, 135.

Marshall, A.E. (1948) Monosodium Glutamate: a Sym-posium. Quartermaster Food and ContainerInstitute for the Armed Forces and Associates,Chicago, Illinois.

Mazur, R.H. (1984) Discovery of aspartame. In:Stegnik, L.D. and Filer, L.J. Jr (eds) Aspartame:Physiology and Biochemistry. Marcel Dekker,New York, pp. 3–10.

McWheeney, D.J., Knowles, M.E. and Hearne, J.F.(1974) The chemistry of non-enzymic brown-ing in foods and its control by sulphites. Journalof the Science of Food and Agriculture 25, 735.

Meggs, W.J., Atkins, F.M., Wright, R., Fishman, M.,Kaliner, M.A. and Metcalfe, D.D. (1985) Failureof sulphites to produce clinical responses inpatients with systemic mastocytosis or recur-rent anaphylaxis: results of a single-blindstudy. Journal of Allergy and Clinical Immunol-ogy 76, 840–846.

Michels, A., Vandermoten, G., Duchateau, J. et al.(1991) Anaphylaxis with sodium benzoate.Lancet 337, 1424–1425.

Mikkelsen, H., Larsen, J.C. and Tarding, F. (1978)Hypersensitivity reactions to food colourswith special reference to the natural colourannatto extract (butter colour). Archives ofToxicology Supplement 1, 141–143.

Ministry of Agriculture, Fisheries and Food, UK(1993) Appendix VII: Estimates of per capitaintakes of food additives and the extent towhich they are used. In: Dietary Intake of FoodAdditives in the UK: Initial Surveillance. HMSO,London, pp. 42–47.

Moneret-Vautrin, D.A., Faure, G. and Bene, M.C.(1986) Chewing-gum preservative-inducedtoxidermic vasculitis. Allergy 41, 546–548.

Nagel, J.E., Fuscaldo, J.T. and Fireman, P. (1977)Paraben allergy. Journal of the American MedicalAssociation 237, 1594–1595.

Nelson, K.E. (1983) Effects of in-package sulphurdioxide generators, package liners andtemperature on decay and dessication oftable grapes. American Journal of Enology andViticulture 34, 10.

Niestijl Jansen, J.J., Kardinall, A.F.M., Huijbers, G.et al. (1993) Prevalence of food allergy andintolerance in the adult Dutch population.Journal of Allergy and Clinical Immunology 4,123–129.

Nish, W.A., Whisman, B.A., Goetz, D.W. et al. (1991)Anaphylaxis to annatto dye: a case report.Annals of Allergy 66, 129–131.

Nizankowska, E. and Szczeklik, A. (1989)Glucocorticoids attenuate aspirin-precipitatedadverse reactions in aspirin-intolerant patients


12-Mar-03 11



with asthma. Annals of Allergy 63, 159(abstract).

Ortolani, C., Pastorello, E., Luraghi, M.T.,Della-Torre, F., Bellani, M. and Zanussi, C.(1984) Diagnosis of intolerance to foodadditives. Annals of Allergy 53, 587–591.

Ortolani, C., Mirone, C. and Fontana, A. (1987)Study of mediators of anaphylaxis in nasalwash fluids after aspirin and sodium meta-bisulphite nasal provocation in intolerantrhinitis patients. Annals of Allergy 59, 106–112.

Ortolani, C., Pastorello, E., Fontana, A., Gerosa, S.,Ispano, M., Pravettoni, V., Rotondo, F., Mirone,C. and Zanussi, C. (1988) Chemicals and drugsas triggers of food-associated disorder. Annalsof Allergy 60, 358–366.

Osmundsen, P.E. (1980) Contact urticaria fromnickel and plastic additives (butylhydroxy-toluene, oleylamide). Contact Dermatitis 6,452–454.

Park, G.R. (1981) Anaphylactic shock resulting fromcasualty simulation. Journal of the Royal ArmyMedical Corps 127, 85–86.

Pearson, D. and Shaw, S. (1984) The Life ExtensionCompanion. Warner Books, New York.

Penttilä, P.-L., Salminen, S. and Niemi, E.Z. (1988)Estimates on the intake of food additivesin Finland. Zeitschrift für Lebensmittel-Untersuchung und-Forschung 186, 11–15.

Pleskow, W.W., Stevenson, D.D. and Mathison,D.A. (1983) Aspirin sensitivity rhinosinusitis/asthma: spectrum of adverse reactions. Journalof Allergy and Clinical Immunology 71, 574(abstract).

Ponder, R.D. and Wray, B.B. (1993) A case report:sensitivity to benzalkonium chloride. Journal ofAsthma 30, 229–231.

Ponting, J.D., Jackson, R. and Watters, G. (1971)Refrigerated apple slices: effects of pH,sulphites and calcium on texture. Journal ofFood Science 36, 349.

Prenner, B.M. and Stevens, J.J. (1976) Anaphylaxisafter ingestion of sodium bisulphite. Annals ofAllergy 37, 180–182.

Reif-Lehrer, L. (1977) Adverse reactions in humansthought to be related to ingestion of elevatedlevels of MSG. Federal Proceedings 36,1617–1623.

Roberts, A.C. and McWheeney, D.J. (1972) The usesof sulphur dioxide in the food industry – areview. Journal of Food Technology 7, 221.

Roed-Petersen, J. and Hjorth, N. (1976) Contact der-matitis from antioxidants: hidden sensitizersin topical medications and foods. BritishJournal of Dermatology 94, 233–241.

Ros, A.M., Juhlin, L. and Michaelsson, G. (1976)A follow-up study of patients with recurrent

urticaria and hypersensitivity to aspirin,benzoates and azo dyes. British Journal ofDermatology 95, 19–24.

Rosenhall, L. (1982) Evaluation of intolerance toanalgesics, preservatives and food colorantswith challenge tests. European Journal ofRespiratory Diseases 63, 410–419.

Rudzki, E., Czubalski, K. and Grzywa, Z. (1980)Detection of urticaria with food additiveintolerance by means of diet. Dermatologica161, 57–62.

Samter, M. and Beers, R.F. (1967) Concerning thenature of the intolerance to aspirin. Journal ofAllergy 40, 281.

Samter, M. and Beers, R.F. (1968) Intolerance toaspirin: clinical studies and considerations ofits pathogenesis. Annals of Internal Medicine 68,975–983.

Sarkany, I., Meara, R.H. and Everall, J. (1961)Cheilitis due to carmine in lip salve. Trans-actions, Annual Report of the St John’s HospitalDermatology Society 46, 39–40.

Schaumburg, H.H., Byck, R., Gerstl, R. andMarshman, J.H. (1969) Monosodiumglutamate: its pharmacology and role inthe Chinese-restaurant syndrome. Science163, 826–828.

Schroeter, L.C. (1966) Sulphur Dioxide: Applications inFoods, Beverages and Pharmaceuticals. PergamonPress, New York.

Schwartz, H.J. (1983) Sensitivity to ingested metabi-sulphite: variations in clinical presentation.Journal of Allergy and Clinical Immunology 71,487–489.

Schwartz, H.J. (1986) Observations on the useof oral sodium cromoglycate in a sulphite-sensitive asthmatic subject. Annals of Allergy57, 36–37.

Schwartz, H.J. and Chester, E. (1984) Bronchospasticresponses to aerosolized metabisulphite inasthmatic subjects: potential mechanisms andclinical implications. Journal of Allergy andClinical Immunology 74, 511–513.

Schwartz, H. and Sher, T.H. (1985a) Bisulphite intol-erance manifest as bronchospasm followingtopical dipirefrin hydrochloride therapy forglaucoma (letter). Archives of Ophthalmology103, 14–15.

Schwartz, H.J. and Sher, T.H. (1985b) Bisulphitesensitivity manifesting as allergy to localdental anesthesia. Journal of Allergy and ClinicalImmunology 75, 525–527.

Schwartzstein, R.M., Weinberger, S.E., Weiss, J.W.and Drazen, J.M. (1987) Airway effects ofmonosodium glutamate in subjects withchronic stable asthma. Journal of Asthma 24,167–172.

11-Mar-03 11




Selner, J., Bush, R. and Nordlee, J. (1987) Skin reac-tivity to sulphite and sensitivity to sulphitedfoods in a sulphite-sensitive asthmatic. Journalof Allergy and Clinical Immunology 79, 241(abstract).

Settipane, G.A. and Pudupakkam, R.K. (1975) Aspi-rin intolerance III: subtypes, familial occur-rence and cross-reactivity with tartrazine.Journal of Allergy and Clinical Immunology 56,215–221.

Shepphard, D. (1988) Sulphur dioxide and asthma –a double-edged sword? Journal of Allergy andClinical Immunology 82, 961–964.

Shepphard, D., Wong, W.S. and Uehara, C.F. (1980)Lower threshold and greater bronchomotorresponsiveness of asthmatic subjects to sul-phur dioxide. American Review of RespiratoryDiseases 122, 873.

Sher, T.H. and Schwartz, H.J. (1985) Bisulphitesensitivity manifesting as an allergic reactionto aerosol therapy. Annals of Allergy 54, 224.

Simon, R.A. (1984) Adverse reactions to drug addi-tives. Journal of Allergy and Clinical Immunology74, 623–630.

Simon, R.A. (1985) Reactivity to inhaled Bronkosol(isoetharine) in sulphite sensitive asthmatics.Journal of Allergy and Clinical Immunology 75,145 (abstract).

Simon, R.A. (1986) Sulphite sensitivity. Annals ofAllergy 56, 281–288.

Simon, R.A. and Stevenson, D.D. (1987) Lack ofcross sensitivity between aspirin and sulphitein sensitive asthmatics. Journal of Allergy andClinical Immunology 79, 257 (abstract).

Simon, R.A. and Stevenson, D.D. (1997) Adversereactions to sulphites. In: Middleton, E., Reed,C. and Ellis, E. (eds) Allergy, Principles and Prac-tice, 2nd edn. Mosby, St Louis, Missouri.

Simon, R.A. and Wasserman, S.I. (1986) IgE-mediated sulphite-sensitive asthma. Journalof Allergy and Clinical Immunology 77, 157(abstract).

Simon, R.A., Green, L. and Stevenson, D.D. (1982)The incidence of metabisulphite sensitivity inan asthmatic population. Journal of Allergy andClinical Immunology 69, 118 (abstract).

Simon, R., Goldfarb, G. and Jacobsen, D. (1984a)Blocking studies in sulphite-sensitive asthmat-ics (SSA). Journal of Allergy and ClinicalImmunology 73, 136 (abstract).

Simon, R.A., Goldfarb, G. and Jacobsen, D.W.(1984b) Blocking studies in sulphite-sensitiveasthmatics. Journal of Allergy and ClinicalImmunology 73, 136.

Sklian, D.M. and Goldstone, J. (1986) Toxicityof butylated hydroxytoluene (letter). NewEngland Journal of Medicine 34, 648–649.

Sonin, L. and Patterson, R. (1985) Metabisulphitechallenge in patients with idiopathicanaphylaxis. Journal of Allergy and ClinicalImmunology 75, 67–69.

Spector, S.L., Wangaard, C.H. and Farr, R.S.(1979) Aspirin and concomitant idiosyncrasiesin adult asthmatic patient. Journal of Allergy andClinical Immunology 64, 500–506.

Speer, K. (1958) The Management of Childhood Asthma.Charles C. Thomas, Springfield, Ohio.

Spurlock, B.W. and Dailey, T.M. (1990) Shortnessof (fresh) breath-toothpaste-induced broncho-spasm (letter). New England Journal of Medicine323, 1845–1846.

Squire, E.N. Jr (1987) Angio-oedema andmonosodium glutamate. Lancet 1, 988.

Stenius, B.S.M. and Lemola, M. (1976) Hyper-sensitivity to acetylsalicylic acid (ASA) andtartrazine in patients with asthma. ClinicalAllergy 6, 119–129.

Stevenson, D.D. (1991) Tartrazine, azo and nonazodyes. In: Metcalfe, D.D., Sampson, H.A.and Simon, R.A. (eds) Food Allergy: AdverseReactions to Foods and Food Additives. BlackwellScientific, Boston, pp. 365–373.

Stevenson, D.D. and Simon, R.A. (1981) Sensitivityto ingested metabisulphites in asthmatic sub-jects. Journal of Allergy and Clinical Immunology68, 26–32.

Stevenson, D.D., Simon, R.A., Lumry, W.R. andMathison, D.A. (1986) Adverse reactions totartrazine. Journal of Allergy and ClinicalImmunology 78, 182–191.

Stricker, W.E., Anorve-Lopez, E. and Reed, C.E.(1986) Food skin testing in patients withidiopathic anaphylaxis. Journal of Allergy andClinical Immunology 77, 516–519.

Subiza, J., Subiza, J.L., Valdivieso, R., Escribano,P.M., Garcia, R., Jerez, M. and Subiza, E. (1992)Toothpaste flavor-induced asthma. Journal ofAllergy and Clinical Immunology 90, 1004–1006.

Supramaniam, G. and Warner, J.O. (1986) Artificialfood additive intolerance in patients withangio-oedema and urticaria. Lancet 2, 907–909.

Suzuki, I., Nojima, S., Tanimura, E. (1999) Com-mentary of the 7th Edition of Japan’s Specificationsand Standards for Food Additives. HirokawaPublishing Co., D-40-D-47 Tokyo.

Tarlo, S.M. and Broder, I. (1982) Tartrazineand benzoate challenge and dietary avoid-ance in chronic asthma. Clinical Allergy 12,303–312.

Taylor, S.L. and Bush, R.K. (1983) Sulphites: aTechnical and Scientific Review. InternationalFood Additives Council, Washington, DC.

Taylor, S.L., Bush, R.K., Selner, J.C., Nordlee, J.A.,Weiner, M.B., Holden, K., Koepke, J. and


11-Mar-03 11



11-Mar-03 11

Busse, W.W. (1988) Sensitivity to sulphitedfoods among sulphite-sensitive subjects withasthma. Journal of Allergy and Clinical Immunol-ogy 81, 1159–1167.

Taylor, S.L., Bush, R.K. and Nordlee, J.A. (1997)Sulphites. In: Metcalfe, D.D., Sampson, H.A.and Simon, R.A. (eds) Food Allergy: AdverseReactions to Foods and Food Additives. BlackwellScientific, Boston, pp. 339–357.

Thune, P. and Granholt, A. (1975) Provocation testswith antiphlogistic and food additives inrecurrent urticaria. Dermatologica 151, 360–367.

Towns, S.J. and Mellis, C.M. (1984) Role ofacetylsalicylic acid and sodium metabisul-phite in chronic childhood asthma. Pediatrics73, 631.

Twarog, F.J. and Leung, D.Y.M. (1982) Anaphylaxisto a component of isoetharine (sodiumbisulphite). Journal of the American MedicalAssociation 248, 2030–2032.

United States General Accounting Office/HRD(1987) Food and Drug Administration’s Approvalof Aspartame. June. Washington, DC.

Utell, M.J., Morrow, P.E. and Speers, D.M. (1983)Airway response to sulphite and sulphuricacid aerosols in asthmatics. American Reviewof Respiratory Diseases 128, 444–450.

Utell, M.J., Frampton, M.W. and Morrow, P.E.(1991) Air pollution and asthma: clinicalstudies with sulphuric acid aerosols. AllergyProceedings 12, 385–388.

Vedanthan, P.K., Menon, M.M., Bell, T.D. andBergin, D. (1977) Aspirin and tartrazine oralchallenge: incidence of adverse response inchronic childhood asthma. Journal of Allergyand Clinical Immunology 60, 8–13.

Veien, N.K., Hattel, T., Justesen, O. et al. (1987)Oral challenge with food additives. ContactDermatitis 17, 100–103.

Walker, R. (1990) Nitrates, nitrites and N-nitrosocompounds: a review of the occurrence in foodand diet and the toxicological implications.Food Additives and Contaminants 7, 717–768.

Weber, R.W., Hoffman, M., Raine, D.A. and Nelson,H.S. (1979) Incidence of bronchoconstrictiondue to aspirin, azo dyes, non-azo dyes, andpreservatives in a population of perennialasthmatics. Journal of Allergy and ClinicalImmunology 64, 32–37.

Williams, A.E. (1978) Benzoic acid. In: Kirk–OthmerEncyclopedia of Chemical Technology. Wiley-Interscience, New York, pp. 778–792.

Woessner, K.M., Simon, R.A. and Stevenson, D.D.(1999) Monosodium glutamate sensitivity inasthma. Journal of Allergy and Clinical Immunol-ogy 104, 305–310.

Wolf, S.I. and Nicklas, R.A. (1985) Sulphite sensitiv-ity in a seven year old child. Annals of Allergy54, 420.

Yamada, T. (2000) Report of the Health ScienceResearch Grants. Ministry of Health andWelfare, Tokyo.

Yang, W.H., Purchase, E.C.R. and Rivington, R.N.(1986) Positive skin tests and Prausnitz–Kustner reactions in metabisulphite-sensitivesubjects. Journal of Allergy and Clinical Immunol-ogy 78, 443–449.

Young, E., Patel, S., Stoneham, M., Rona, R. andWilkinson, J.D. (1987) The prevalence of reac-tion to food additives in a survey population.Journal of the Royal College of Physicians of London21, 241–247.

Zhang, Y.G., Wright, W.J., Tam, W.K.,Nguyen-Dang, T.H., Salome, C.M. andWoolcock, A.J. (1990) Effect of inhaledpreservatives on asthmatic subjects. II.Benzalkonium chloride. American Review ofRespiratory Diseases 141, 1405–1408.




12 Migration of Compounds from FoodContact Materials and Articles

J.H. Petersen*Institute of Food Safety and Nutrition, Danish Veterinary and Food Administration,

Soborg, Denmark

Introduction

Food comes into contact with a variety ofmaterials and articles during the productionprocess, packaging and storage, and in itsfinal preparation as a meal in the consumer’skitchen. Practical examples of contact materi-als used in the food industry are metal tankswith or without epoxy coatings, and tubesof rubber or plastic. Typical materials usedin large volumes for retail packaging areglass, paper, lacquered metal and plastics.In the consumer’s kitchen, a broad selectionof household equipment made from plastics,metal coated to various degrees, rubber andlacquered wood is used.

It is clear that extensive use of efficientfood packaging materials is indispensable,especially in today’s western lifestyle. Duringthe last few decades there has been an increas-ing demand for retail packagings containingsmall portions of food which can be kept forlong periods. The basic quality of a packagingmaterial is its efficiency in containing the foodand in being a barrier against external micro-biological and chemical contaminants. Fur-ther, the packaging has to market the product,be convenient to use and provide essentialinformation of nutritional value, food addi-tives and the price of the product.

In the case of ready-cooked foods, weexpect that a plastic packaging material withall the qualities mentioned above, but with aweight of only a few grams, can be used in hotfill packing of a portion of food under asepticconditions and maintain an efficient barrierfor 6 months in the freezer at −18°C and fur-ther during a short heating period to about100°C in the microwave oven or in boilingwater. The packaging industry has developedquite complex materials for such purposes,although these qualities are not visible to theconsumer. In many instances, they consist ofmultilayered structures built up from severaltypes of polymers, adhesives, lacquers andprinting inks, as well as a mixture of additivesnecessary to stabilize the plastic during stor-age and in the production process.

Consumers are dependent on the safetyof these materials and it is no wonder ifthey become a little nervous when news-paper headlines connect a human health riskto the amount of a certain invisible chemicalsubstance migrating from food contact mate-rials. In the past, there have been severalexamples of materials showing too high alevel of migration of some compound.However, in many instances, such ‘foodpackaging scandals’ reflect that the toxicproperties of the compound have not been


20-Feb-03 12




related to the actual human consumption. Aproper risk assessment requires knowledge ofboth toxicity and intake. Because of the wide-spread use of these materials, everybody canagree that it is essential to limit the migrationof chemical substances to the food to a safelevel.

During the 1980s, the European Commis-sion introduced a series of technical directiveson how to control migration, and in 1989fundamental requirements concerning theinertness and stability of the finished foodcontact materials were laid down in theso-called framework directive. A detailedregulation of food contact plastics can beexpected to be completed in the early 2000sand can be foreseen to cover more than 1000allowed substances, which have all beenthrough a toxicological evaluation. However,there still remains work for the regulatorsfor the next decades. Important food contactmaterials such as paper and cardboard,surface coatings, printing inks, rubber, corkand others still need to be regulated in detail.

The food packaging industry tradi-tionally has been quite reluctant to provideinformation about the composition of theirproducts, and only for the last decades has itbeen fully recognized by the main producersthat the performance of their product consti-tutes an integrated part of the final safety levelof the food product used for human con-sumption. For the analytical chemist, controlof migration from food contact materials istherefore a challenging area since it is often

not known what substances to look for and inwhich concentrations.

Nature of Materials and Compounds

Food contact materials are produced frommany different types of materials, rangingfrom mixtures of anthropogenic substancesbased on mineral oil to slightly modified nat-ural materials. Some of the more importanttypes of materials are listed in Table 12.1.

It is obvious from Table 12.1 that the bulkof materials used in the production of foodcontact materials are anthropogenic or naturalorganic macromolecular substances as wellas common inorganic materials. However,such substances constitute only the back-bone of the material, which must be modifiedfurther depending on the purpose of its practi-cal use. Taking plastics as a first example, thefinal composition of a packaging material canbe made up potentially from thousands ofindividual starting materials and additives.Beside the monomers themselves, othergroups of functional compounds are neces-sary either in the production of the polymer, inthe conversion of the plastic material or for thefinal performance of the material. Importantgroups of such compounds are listed inTable 12.2.

When discussing a potential risk ofmigration of plastic constituents to the food,the compounds of interest are mainly theadditives or their breakdown products, which

20-Feb-03 12

272 J.H. Petersen

Material Common starting materials Typical area of use

Plastics

Paper and cardboard

MetalsGlass

Rubbers

Lacquers and coatings

Natural or, more commonly, syntheticmonomers converted to polymersPulp obtained from plant fibres orrecycled paper and cardboardSteel, aluminium, tinSilica from sand or quartz andcarbonates of alkali metalsCross-linked natural rubber andpolymers based on syntheticmonomersA diverse group including waxes,polymers, additives, silicones andothers

Almost all types of food contactmaterialsBags, cartons, grease-resistant paper,kitchen rollsCans, household utensils, tubes, tanksBottles, glass containers

Stoppers, tubes, teats

Surface treatment of many foodcontact materials

Table 12.1. Important types of materials used in food contact materials.



are of sufficiently low molecular weight tomove by diffusion in the polymer network.For the widely used polyolefins, polyethyleneand polypropylene, the additives constituteonly a few per cent of the total weight of theplastic. This is in strong contrast to a materialsuch as plasticized polyvinylchloride (PVC)where up to 50% of the final material can beadditives, mainly plasticizers.

When chemicals of reasonable purityare used in production, the full compositionof an anthropogenic material such as aplastic is well known. In many cases, thetoxicology of the pure compounds and ofteneven of their foreseeable reaction/breakdownproducts has been evaluated by internationalexpert panels. Together, the responsible pro-ducer of the polymer, the converter and theend user of such a plastic material in principlehave all the necessary information to ensurethat the material is safe in its end use.

The situation is somewhat different whenwe look at another group of food contactmaterials such as paper and cardboard. Thematerials are made from renewable resourcesand some people might consider them saferthan plastics because of their ‘natural’ origin.In general, however, the amount of chemicals

used as processing aids during their pro-duction and as additives in the final productis significant.

The primary raw material used in theproduction is plant fibres of natural originfrom wood. Compared with, for examplepolyethylene, the fibres do not have a verywell defined polymer backbone since thecomposition can differ between differenttypes of wood. The main components ofthe fibres are cellulose, a linear polymer builtup from glucose units (∼50%), hemicellulose,which is the polymer of a mixture of polysac-charides (∼10%), and lignin, a branched alkylaromatic polymer, constituting the rest.

The first step in the production of paperand cardboard is the pulping process wherethe fibres are obtained from chips of wood andseparated. Depending on the required qualityof the paper, the fraction containing ligninsand hemicelluloses can be removed partly ortotally. The classical methods to obtain a pulpare either by mechanical treatment of thechips of wood or by cooking combined with achemical treatment (the sulphite and the sul-phate methods). In the mechanical method, apulp with many broken fibres is obtained, butin a process with a high yield. In the chemical

Migration of Compounds from Food Contact Materials 273

20-Feb-03 12

Stage ofproduction Function Type of compounds

Examples of specificchemicals used

Polymerization ofthe monomers

Converting thepolymers to a foodcontact material

Protection ofpolymers fromdeterioration

Control of the polymerizationprocess

Processing of additives

Expanding the materialPlasticizing

LaminatingMaterial performance

Capture of UV photons

Capture of free radicals

Initiators/catalysts

Inhibitors/retardantsLubricants

Heat stabilizers

Foaming agentsPlasticizers

AdhesivesAnti-dew treatmentAntistatic agentsColourants/fillersPhotostabilizers

Antioxidants

Peroxides, alkyl-lithium

Substituted phenolsSterically hindered phenols,aromatic amines

Calcium–zinc–carboxylic acidcomplexPentane, carbon dioxideDialkyl esters of phthalic,citric and adipic acidsEpoxidized soybean oilIsocyanatesGlycerol stearateEthoxylated fatty aminesTitanium dioxideAlkyl-substitutedo-hydroxybenzophenones

Sterically hindered phenols

Table 12.2. Chemicals used in different stages in the production of plastic packaging.



methods, long fibres of the cellulose fractionare obtained, but about 50% of the raw mate-rial, lignin and hemicellulose, is excluded.However, in general, a modern paper milluses a combination of mechanical and chemi-cal treatment to obtain higher yields and areasonable strength of the final product.

For many packaging applications, it isrequired that the colour of the paper andcardboard is white, and for that reason thepulp is bleached. The aromatic chromophoresof the lignin are responsible for the deviationsfrom white since the natural colour of cellu-lose and hemicellulose is indeed white. Thepreferred method today is using peroxides,which, in contrast to the more classicalmethods involving, for example, chlorinebleaching, does not lead to significant lossin the yield. In some final applications, acertain proportion of recycled fibres are used,alone or in a mixture with virgin fibres.This requires repulping of the used paperand a series of cleaning steps to removeas much as possible of the fraction of fibreswhich become too small during the repeatedrecycling process, as well as to removeadditives, printing inks and other potentialcontaminants.

After the pulping and bleaching process,the fibres must be formed for their final use asa food packaging material. This includes theaddition of several types of compounds asfillers, colourants, pigments, sizing agentsand adhesives, which are all used to improvethe functional and visual properties of thefinal product. Further, in paper and cardboardproduction, different chemicals are usedas processing aids in order to avoid theformation of foam and the growth of micro-organisms, or as dispersion agents used toensure a good distribution of added resins,etc. Finally, a barrier layer of wax or plasticscan be applied on the surface.

More complex food contact materialsare being developed nowadays for whichit can be quite difficult for the controllingauthorities to foresee their composition. Acurrent trend is towards development of‘active and intelligent food packaging materi-als’, i.e. packaging materials which – besideprotecting the food ‘as usual’ – can monitor,control or even react to phenomena taking

place inside the packaging. The differenttypes of active packaging have been catego-rized into four groups with regard to theirmode of functioning. One group of packagingmaterials includes ingredients, ‘scavengers’,which are added for the purpose of absorbing,removing and eliminating substances such asoxygen, ethylene, moisture or taint from theinterior of a food packaging with the intentionof extending the shelf-life of the foodstuff.Activated charcoal is an example of such acompound, which has been used to removeethylene in fruit packagings. A second groupof packaging contains or produces substances,‘emitters’, which are meant to migrate into thefood itself or into the packaging headspace inorder to produce an effect in the food itself.Sulphite-containing sachets emitting sulphurdioxide in packagings for fresh grapes is atypical example of an emitter. The third groupof active packagings include devices, ‘indica-tors’, which are able to give informationabout the food product itself or the storageconditions of the packaging. For perishableproducts packed in a modified atmospherefree of oxygen, leak indicators containing anoxygen-sensitive redox dye (such as methy-lene blue) formulated as a tablet or label canbe a useful tool to indicate possible spoilageof the foodstuff when a leak occurs. Afourth group includes other categories ofactive packaging, printed electronic circuits,susceptor packaging for popcorn and pizzabeing examples. In all probability, new typesof active packaging will be seen in the nearfuture (Fabech et al., 2000).

The examples above are only used toexemplify the variety of chemicals used inordinary food contact materials, some ofwhich could potentially migrate to the foodand potentially be harmful to humans. Com-pounds of low and high molecular weight andreactive and non-reactive species are amongthem; foreseeable as well as not-foreseeablemigrants are undoubtedly among them. Atpresent, to some extent, the consumer has torely on a responsible industry, which takescare that the products they sell are safe in use.The next step is to have suitable regulationsto define the desired safety standard andsuitable regulations against which to hold theindustry standards.

20-Feb-03 12

274 J.H. Petersen



Legislation

The EU legislation in the field of ‘materialsand articles intended to come into contactwith foodstuffs’ is expressed in general termsin the Framework Directive 89/109/EEC andis more detailed in specific directives (EEC,1989). National legislation exists in severalcountries, often in quite general terms, thelegislation in Germany (BgVV, 2002), TheNetherlands (Warenwet) (SDU, 2002) and theUSA (Code of Federal Regulation) (FDA,2002) being exceptions containing details thatare useful to know when it is necessary toassess materials not covered by specific EUdirectives. However, in Europe, the joint anddeveloping EU legislation on food contactmaterials and articles together with resolu-tions and guidelines from the Council ofEurope (COE, 2002) is at present the frame ofreference, also taking into consideration thatsome harmonization with the US legislationtakes place. In this context, only the EUlegislation will be discussed further.

The Framework Directive

The Framework Directive applies to materi-als and articles which, in their finished state,are intended to be brought into contact withfoodstuffs intended for human consumption.The basic principles of the EU Treaty requirethe Member States to ensure not only freemovement of the goods within the internalmarket, but also a high level of protection ofpublic health. To fulfil the second aim, article2 of the directive 89/109/EEC sets the follow-ing standard for such materials:

Materials and articles must be manu-factured in compliance with good manu-facturing practice so that, under theirnormal and foreseeable conditions of use,they do not transfer their constituents tofoodstuffs in quantities, which could:• endanger human health,• bring about an unacceptable change

in the composition of the foodstuffsor

• deterioration in the organolepticcharacteristics thereof.

For food contact materials, which are notalready in contact with food when they aresold, the directive specifies labelling require-ments. A ‘glass and fork’ symbol, introducedby directive 80/590/EEC, can be used toindicate that a material is suitable for suchuse (EEC, 1980).

It is of paramount importance to note thatthe responsibility for ensuring compliancewith legislation lies with the manufacturer,importer and retailer since no system ofgovernmental approval of food contactmaterials exists.

These general rules apply to all types offood contact materials with a few exceptions:‘antiques, fixed water supply equipment andcovering or coating substances which form apart of the foodstuff and may be consumed’.In the Framework Directive, it is decidedfurther that specific directives should coverall kinds of food contact materials suchas plastics, cellulose regenerates, paper andcardboard, rubbers, silicones, etc.

It is a difficult task to produce detailedlegislation acceptable for an innovativeindustry as well as lining up preciserestrictions, which enable the controllingauthorities to control the legislative measuresensuring consumer safety according to theabove standard. For that reason, it hasbeen a long process to develop and agreeupon, first, the principles in the frameworkdirective and, secondly, the principles for theregulation of specific types of food contactmaterials.

So far, the specific legislation on foodcontact materials is based on a principle ofpositive lists of compounds which can be usedin the production of such materials and whichhave been evaluated individually by thetoxicologists of the EU Scientific Committeefor Food (SCF). At present, due to the heavyworkload connected with these evaluations,only one single material based on organicpolymers – cellulose regenerates (cellophane)– can be considered fully regulated. Thedirectives on regenerated cellulose film(93/10/EEC and 93/111/EC) include suchpositive lists of individual compounds as wellas some compositional limits in the material.Moreover, some specific limits for constitu-ents which may be transferred into the food,


20-Feb-03 12



specific migration limits, are given in thesedirectives.

The directives on plastic materialsand articles

The completion of specific directives regulat-ing plastics has been on the agenda for a longtime, and it seems likely that plastics will befully regulated in the year 2004.

The plastics directive

The plastics directive (2002/72/EC) was firstadopted in 1990, and a series of amendmentshas followed, mainly adding compoundsto the positive lists of the annexes, whenthey have been toxicologically assessed andspecific migration restrictions are laid down(EC, 2002). The new codified directive setsa limit for the maximum amount of plasticconstituents allowed to migrate to the food-stuff – the so-called overall migration limit.The limit can be expressed either as 60 mgkg−1 foodstuffs or as 10 mg dm−2 of plasticsurface area, and can be considered as ageneral hygienic limit independent of thetoxicity of the compounds.

The eighth amendment is expected to beadopted in 2004 and, by then, a full positivelist of all main compounds which can beused legally in the production of food contactplastics will exist. To many of the monomersand starting substances on the list, a specificmigration limit (SML) or a maximum resid-ual quantity in the material (Qm) has beenprescribed. SML is expressed in mg kg−1 offood or in mg dm−2 of surface area, whereasQm is expressed in mg or µg kg−1 of plastic.QmA restrictions were introduced recently(1999/91/EEC) and they are expressed as mgor µg 6 dm−2 of surface area.

Technical directives about migration testing

Fundamental agreements about how to testthe materials with respect to exposure condi-tions were laid down already in directive82/711/EEC with further amendments indirectives 93/8/EEC and 97/48/EC (EEC,

1982). The most important message here isthat the quantity of compounds migratingfrom a food contact material to a foodstuffis dependent on the duration and the temper-ature applied in the period where contactoccurs between the foodstuff and the plastic.Table 12.3 shows how the directives translatea situation in practical life into conventionallyagreed test conditions.

When selecting the test conditions fromTable 12.3, one should consider the worst fore-seeable conditions of use for the material inpractical applications.

In 1985, it was agreed further that, insteadof measuring the migration to the actualfoodstuffs, it could be a more practical andstandardizable approach to use food simu-lants (EEC, 1985). The Member States agreedupon the four different food simulants shownin Table 12.4.

Directive 85/572/EEC also contains a listof all types of foodstuffs and the convention-ally agreed food simulant(s) assigned to eachtype. Further, except for pure fats and oils,a reduction factor of from 2 to 5 is assignedto each type of fatty foodstuff. The result ofthe migration test must be divided by thisreduction factor to compensate for the high

20-Feb-03 12

276 J.H. Petersen

Conditions of contact inactual use Test condition

Contact time (t)≤ 0.5 h0.5 h < t ≤ 1 h1 h < t ≤ 2 h2 h < t ≤ 24 h> 24Contact temperature (T)< 5°C5°C < T ≤ 20°C20°C < T ≤ 40°C40°C < T ≤ 70°C70°C < T ≤ 100°C

100°C < T ≤ 121°C121°C < T ≤ 130°C130°C < T ≤ 150°C150°C < T ≤ 175°C

Test time0.5 h1 h2 h24 h10 daysTest temperature5°C20°C40°C70°C100°C or refluxtemperature121°C130°C150°C175°C

Table 12.3. Time and temperature conditions formigration testing (from 82/711/ECC withamendments).



extraction potential of pure olive oil comparedwith that of most other fatty foodstuffs.

The materials should be tested accordingto the worst conditions, which can be foreseenin practical use. When a producer of a foodcontact material sells a product, it has to belabelled with possible restrictions in its usewith respect to contact time, contact tempera-ture and types of foodstuff. When no restric-tions are given, the product must be able towithstand a 4 h migration test with the foodsimulants 3% acetic acid and 10% ethanol at100°C and a 2 h migration test with olive oilat 175°C. However, for some products, it isevident, even without labelling, that they areintended for use at ambient temperature. Insuch cases, a migration test at 40°C for 10 dayswith the food simulants is a suitable standardtest.

For practical reasons, it is impossible tomeasure the sum of all migrating species, theoverall migration, except in a food simulant.However, for specific compounds, the actualconcentration in a foodstuff measured duringrealistic circumstances will always overrule ameasurement in a food simulant.

Analytical methods

The first directives in this area (78/142/EEC,80/766/EEC and 81/432/EEC) set a Qmrestriction of vinyl chloride monomer (VCM)at 1 mg kg−1 in the plastic material andan SML in food of 10 µg kg−1. They furtherspecify in detail the laboratory methods that

have to be used for this purpose. The devel-opment and validation of the analyticalmethods were organized by the EU Commis-sion, giving rise to a heavy workload. Later,it was recognized that laboratory methodsquickly become obsolete and outdated,and for that reason the EU Commissionnow cooperates with and sustains the Euro-pean Standardization Organization (CEN) indeveloping analytical methods in support ofthe directives. Most of the work takes place inTechnical Committee 194, Scientific Commit-tee 1, called ‘General chemical methods oftests for materials intended to come intocontact with food’.

The standard EN(V) 1186 contains allmethods for the determination of overallmigration, and some methods for measure-ment of specific migration can be found inEN(V) 13130 (CEN, 2002).

Toxicological Evaluations are the Basisfor the EU Legislation

The compounds in the positive lists inthe plastics directive are often connected to aQm or SML restriction. These restrictions arebased on the systematic toxicological evalua-tions made for all compounds and performedby the EU SCF. In this section, a short descrip-tion of the general requirements for toxico-logical studies to be supplied by a petitionerwill be given. Also, it will be summarizedhow the toxicologist uses the results of thesestudies for evaluations which at a later stagemay be used by the legislators to lay down amigration limit or a compositional limit.

Toxicological data required

A precondition for even considering a newcompound to be included on the positive listis that it is well characterized with respect toits general physical and chemical propertiesand that migration data are presented for thecompound itself and its eventual transforma-tion or reaction products. The core set of toxi-cological tests that have to be carried out isshown in Table 12.5. As a general principle,


20-Feb-03 12

Food simulant Area of use

Distilled water3% Acetic acid in water

10% (15%) Ethanol inwaterOlive oilWhen test with oil istechnically inapplicable,use substitute test withisooctane, 95% ethanoland modifiedpolyphenylene oxide

Aqueous foodstuffsAqueous and aceticfoodstuffsAqueous and ethanolicfoodstuffsFatty foodstuffs(Reduction factorsapplicable)

Table 12.4. Food simulants (from directive85/572/EEC).



the greater the extent of migration into food,the more toxicological information will berequired (EU Commission, 2002a).

If the studies mentioned in Table 12.5 orprior knowledge indicates that other relevantbiological effects may occur, additional stud-ies may be required.

Evaluations by the EU ScientificCommittee for Food

Based on the toxicological data presented, theSCF will evaluate a given compound. Whenworking on a compound for which anacceptable daily intake (ADI), a tolerabledaily intake (TDI) or equivalent has alreadybeen established by other relevant authori-ties, the job finishes there. However, on mostoccasions, the purpose will be to establish aTDI value. The first step in this process is toidentify the critical effect of the compound,in principle on the human organism but, inpractice, on rodents. The next step is to findthe highest concentration of the compoundwhich does not give rise to any negativeimpact on the most sensitive part of theorganism – the no-effect level expressed inamount of compound per kg body weight perday. From here, the toxicologist normally willuse a safety factor of 10 to account for the

differences between humans and rodentsmultiplied by another factor of 10 toaccount for the differences between humans.Although deviations from this procedure canoccur, an overall safety factor of 100 will beused most often to obtain a TDI value.

Some of the compounds used in the pro-duction of polymers are reactive species thatcan have a negative impact on human health,some of them even being carcinogenic. Forsuch compounds, a TDI cannot be established,and in general they are allocated restrictionssuch as not being detectable in the polymer orin food/food simulant. Other reasons not toestablish a TDI value for some compounds canbe that they are self-limiting because of theirorganoleptic properties or because the migra-tion limit is set very low (< 0.05 mg kg−1) andthe compound is used only in small quantities.

A summary of the evaluations given bythe SCF can be found in the so-called ‘SynopticDocument’, which is updated regularly andcan be reached by the Internet (EU Commis-sion, 2002b).

The EU Commission lays downthe restrictions

For seriously harmful compounds, the EUCommission will lay down the restriction so

20-Feb-03 12

278 J.H. Petersen

Migration level observed (in mg kg−1 food or food simulant)

Type of test < 0.05 0.05–5 > 5

Mutagenicity studies:gene mutations in bacteriachromosomal aberrations inmammalian cellsgene mutations in mammaliancells

Always required

Absence of potential forbioaccumulation

Always required

90-day oral studyStudies on absorbtion, distribution,metabolism and excretionData on reproductionData on teratogenicityData on long-termtoxicity/carcinogenicity

Not required but SMLrestriction < 0.05 mgkg−1 or equivalent willbe laid down

Under certaincircumstances, notall tests may berequired

Always required. Ifany test is omitted, itmust be justified byproviding appropriatereasons

Table 12.5. General requirements for toxicological studies.



that it is as low as possible in the food contactmaterial or as low as possible in the food. Aconsideration, though, is to ensure that thisdetection limit is enforceable, i.e. it can bemeasured with a sufficient certainty in thelaboratories of the Member States.

For less harmful compounds, the Com-mission in most cases uses a conventional pro-cedure when transforming the SCF opinion toQm or SML values. Some main conventionsare the following:

• The standard reference person has abody weight of 60 kg.

• The standard reference person eats 1 kgof packaged food every day.

• The 1 kg of food is contained in a cube.• The surface area of the cube is

6 × 1 dm × 1 dm = 6 dm2.

These conventions are certainly not veryprecise, but are quite practical for calculationpurposes. Not many adult EU citizens have abody weight of 60 kg, and the weight of mostchildren is less. Also, people do not only eataround 1 kg of food per day, they also drinkseveral litres of liquid every day – some fromplastic bottles. However, the 60 kg personeating 1 kg of food per day is closely related tothe overall migration limit of 60 mg kg−1 foodsimulant. When a given compound has a TDIvalue of 1 mg kg−1 body weight day−1 orabove, no specific migration limit is neededsince the compound will be regulatedsufficiently by the overall migration limit. Inconsequence, a TDI value of 0.5 mg kg−1 bodyweight day−1 will result in a specific migrationlimit of 30 mg kg−1 food.

The convention of 1 kg of food beingpacked in 6 dm2 of plastic is also far fromreality since it is more likely to be more thandouble that. However, the fixed surface tovolume ratio allows for an easy transfor-mation of SML values in the foodstuff toa migration limit from the surface of apackaging material, in this case by division bya factor of 6. In general, the analytical chemistin migration testing uses a sample size of 1 or2 dm2.

It can always be discussed as to whichsafety factors have to be used in riskmanagement. Above, some arguments aregiven in one direction. An argument in the

opposite direction can also be mentioned:most probably several different types of food-stuffs will be packed in several different typesof packaging materials and the same migrat-ing species probably will not be present in allmaterials.

Case Studies where Migration wasFound to be (Too) High

Considering the multifarious areas of use andthe different conditions of use with respect tocontact time, temperature and character ofthe foodstuffs, surprisingly few food contactmaterials and articles give rise to seriousproblems. However, from time to time, newproblems appear, either because nobodyhad even thought of a certain food contactmaterial application as a potential problembefore or because new knowledge aboutindividual compounds tells us that we haveto be especially aware that they do notmigrate to the food. Below, a few examplesfrom history will be treated in a little moredetail, but many other cases can be found inthe scientific literature.

Lead – an ancient useful but toxic metal

Migration of toxic compounds from food con-tact materials has been well known from farback in history. Migration of this heavy metalfrom installations made of lead to the waterin the aqueducts used to supply ancientRome with drinking water has been blamedfor the fall of the Roman Empire (Waldronand Stöfen, 1974). The reason for these accu-sations is that we now know that excessiveintake of lead, especially when it concernsinfants, among other negative effects, canlead to mental retardation. Nevertheless,new cases of excessive and potentiallyharmful migration of lead to foodstuffs havecontinued to emerge during the last decades,for example, from:

• lead in the solder used for soldered cansleached to the foodstuff inside (Jorhemet al., 1995);


20-Feb-03 12



• stoppers made from lead being used forwine bottles liberated lead to the wine(Smart et al., 1990);

• the bearings used in household blendersgave off lead to the foodstuff (Rasmus-sen, 1984).

Occasionally, it is still possible to findin the market-place food contact materialsmade from glass, ceramics or metal whichgive off lead, especially to more acid types offoodstuffs.

In all examples listed above, it waspossible to find alternative solutions bysubstitution. Today, cans are made withoutsoldering, stoppers for wine bottles are madefrom plastics, and lead is avoided in alloysand glaze used for food contact purposes. Toproduce food contact materials free from leadis not a major problem for the responsiblemanufacturer. Unfortunately, not all manu-facturers of ceramics, for example, are suffi-ciently aware of the regulation in this field orthey simply do not act in a responsible way.

Lacquers in cans: another potential sourceof migration problems

As mentioned above, migration of lead fromsoldered cans has been a substantial problem.Today, few cans are soldered and almost alltypes of cans are lined with internal lacquers.The prevalent types of lacquers for this pur-pose are the bisphenol A-based epoxy resins,which are used mainly to cover the bottomand the cylindrical part of the can, and theorganosol lacquers, a dispersion of PVC,which are used especially for the ‘easy open’type of lids. The idea of applying lacquers onthe inner surface of a can is to protect thepacked foodstuff inside the can with an inertmaterial. Both types of lacquers have goodproduct resistance and, whereas the epoxylacquers are rather brittle, the organosols area heavier and more suitable flexible supportfor the stamped lid.

In recent years, some concern has beenexpressed that migration of the monomerbisphenol A (Fig. 12.1), a compound sus-pected to exhibit oestrogenic activity, mayoccur from the epoxy coatings to the food as

well as from polycarbonate plastics. How-ever, several investigations have shown thatonly very limited amounts of bisphenol Amigrate compared with the current specificEU migration limit of 3 mg kg−1 food(Mountfort et al., 1997; Pedersen, 1998; FoodStandards Agency, 2001). At present, theepoxy coatings and polycarbonate plasticsare therefore considered quite safe in usewhen produced and applied in agreementwith good manufacturing practice.

A series of investigations since 1995 haveconcentrated on problems with migration ofbisphenol A diglycidylether (BADGE) and itsreaction products from organosol lacquers. In1995, BADGE occurred on the EU positivelist of monomers and starting substances witha very low SML of 0.02 mg kg−1 food (theanalytical detection limit). The reason for thislow SML was that BADGE seemed to be muta-genic by in vitro testing. When BADGE is usedas a starting substance in the production ofpolymers, e.g. epoxy resins (Fig. 12.1), it is nota problem to keep migration below this limit.Therefore, nobody bothered to perform morecostly toxicological tests of this compound.

In organosol lacquers, BADGE is not usedas a starting substance, but as a heat stabilizer.It is added to the lacquer to stabilize the PVC,which can produce hydrochloric acid whenthe lacquer is heated for curing purposes andwhen the food can is sterilized after filling.Free hydrochloric acid in a metal can will giverise to corrosion problems, but BADGE willact as a scavenger of hydrochloric acid andneutralize it. Unfortunately, BADGE, as wellas its chlorinated reaction products (Fig. 12.1),moves rather freely by diffusion in the thinlayer of cured PVC and migrates easily tothe packed foodstuff. If the food is aqueous,BADGE will react with water to produce itshydrolysis products (Fig. 12.1). No toxicologi-cal information about the reaction productswas available in the mid-1990s.

During 1996–1997, several enforcementlaboratories in Europe analysed samples ofcanned food for BADGE. In these investiga-tions, from 3 to 17% of the samples containedBADGE (without reaction products) inamounts above 1 mg kg−1 (van Lierob, 1998).This was certainly not acceptable and, in thefollowing years the migration decreased since

20-Feb-03 12

280 J.H. Petersen



the industry became aware of the importanceof adding a more appropriate amount ofBADGE to the organosol lacquer and beingmore careful in the curing process. Somecan manufacturers reacted by changing toproducts based on bisphenol F diglycidyl-ether (BFDGE), which is chemically less welldefined and for which even less toxicologicalinformation exists (Grob et al., 1999).

One simple solution has been proposed:remove cans with ‘easy open’ lids from themarket and the use of organosol lacquerscould be completely avoided in cans for food-stuffs. In fact, most European consumers onlyeat canned foods a few times a week, and theyhave not yet forgotten how to use a canopener. However, this proposal was neitherpopular nor accepted by the food and canning

industry, which instead has delivered resultsof new toxicological studies of BADGE to theEU Commission. In vivo studies of BADGEand its main breakdown products did notshow any sign of mutagenic activity. There-fore, in 2001, a directive (EC/61/2001) settinga migration limit for the sum of BADGE andits reaction products of 1 mg kg−1 food wasadopted (EC, 2001b).

Polyvinylchloride plastics – useful but asource of migration problems

Besides being the main ingredient in organo-sol lacquers, PVC is an important linearhalogen-containing polymer, which is used


11-Mar-03 12

Fig. 12.1. Structural formulae and examples of important reactions of the monomer bisphenol A to: theplastic polymer polycarbonate; a bisphenol A-epoxy resin; and bisphenol A diglycidylether (BADGE).Further, examples of a hydrolysis product (BADGE.H2O) and a reaction product with HCl (BADGE.HCl) areshown. Also BADGE.2H2O and BADGE.2HCl (not shown) are, together with BADGE, covered by the EUmigration limit.



for many different purposes, including foodcontact materials. It was among the first plas-tics to be produced and it is still produced inhuge amounts. The polymer can be mouldedin almost any form, and at ambient tempera-ture it is hard in its pure form. By addingplasticizers in increasing amounts, the hardmaterial gradually becomes more rubberyand flexible. Soft plasticized PVC occasion-ally contains as much as 50% plasticizer.

Residual amount of the monomer in PVC

In the middle of the 1970s, it became clearthat among workers involved in the product-ion of PVC there was an abnormally high fre-quency of liver angiosarcoma, a rare form ofcancer (Moore, 1975). The immediate reasonfor this was found to be a high concentrationof VCM in the indoor air in factories produc-ing PVC. It was also revealed that significantconcentrations of VCM (e.g. 100 mg kg−1

plastic) occurred in finished PVC products,such as bottles and foils used for packagingof foodstuffs, and that migration of VCM toPVC-packed foodstuffs, such as oil, butter andliquid foods, could be measured. This wascertainly not acceptable, and over a period ofa few years the plastics industry succeeded inreducing the residual VCM in the polymer tomuch lower levels by improved fabricationtechniques. The present EU limit is 1 mgVCM kg−1 plastics. It has been estimatedthat the maximum intake per person of VCMfrom food in the UK was reduced from 1.3 µgto 0.02 µg day−1 as a result of the loweredVCM level in PVC (Ministry of Agriculture,Fisheries and Food, 1978).

In the same period, the monomers ofother widely used food contact plasticsattracted attention because they showed simi-lar adverse effects on human health. It wasdiscovered that frequently used acrylonitrileco-polymers such as acrylonitrile/butadiene/styrene (ABS) plastics contained rather highresidual levels of the monomer acrylonitrile.However, in parallel with the example above,industry succeeded in lowering the levels ofmonomers by modifications of the productionmethods. As a result, the estimated maximumlikely daily intake per person was reducedfrom 2.5 to 0.2 µg. Moreover, the residual

amount of monomers in polystyrene andpolyvinylidene chloride plastics was investi-gated but the level was found to be sufficientlylow and safe (Ministry of Agriculture, Fish-eries and Food, 1989).

Plasticizers in foods

The most frequently used plasticizers forPVC are the phthalates. Due to the wide-spread use of plasticized PVC for a vastnumber of technical purposes and for somefood contact materials, the phthalates areproduced in huge amounts. The productionin Western Europe has been estimated to beclose to 1 Mt year−1 (European Council forPlasticisers and Intermediates, 2002) and theworld production to be several million tonsper year (WHO, 1992). Phthalates such asdi-(2-ethylhexyl)phthalate (DEHP) can befound in air, water and soil, and they arepresent in low concentrations in homes, theirsurroundings and the environment. Theyshow some persistence in the environment,but not to a degree comparable with classicalpersistent contaminants such as polychlori-nated biphenyls (PCBs), an industrial chemi-cal (Chapter 6). A main factor determiningthe universal presence of phthalates mustbe considered to be the continuing large andrather constant production.

The migration of phthalates from packag-ing materials containing these compoundsto fatty foodstuffs is a well-known source offood contamination. Today, the intended useof phthalates in food packaging materials isless widespread since these plasticizers orthe PVC has been substituted with othercompounds or types of plastic. However, pro-cessing equipment such as plasticized tubing,surface coatings, gaskets and gloves used inthe food industry are other potential sourcesof food contamination. Examples of morediffuse sources of phthalate contaminationwith possible implications for foodstuffs areatmospheric deposition on crops, waste watercontaining phthalates flowing into streams,and vinyl floorings in industry and privatehomes that may release phthalates during use.

In recent years, the intakes of phthalateplasticizers have attracted some attentionbecause of their possible negative impact on

11-Mar-03 12

282 J.H. Petersen



male reproduction. Milk and milk productscan be consumed in rather high quantities bychildren, which can be considered to be agroup particularly susceptible to this possiblenegative impact. In Table 12.6, a selection ofpublished data in this area are shown.

Several remarkable observations can bedrawn from Table 12.6. First of all, it seemsthat even milk obtained by milking cowsby hand contains phthalates in measurableamounts. This is a little surprising since thephthalate esters are expected to hydrolyseto the mono-esters and alcohols on theirway through the digestive tract before beingabsorbed through the intestinal wall andfinally excreted by the urine, mainly asglucuronide conjugates.

A second observation is a tendency forthe phthalate concentration to be correlatedto the fat content of the milk product. A sig-nificant example is the DEHP concentrationin Norwegian milk products, which all comesfrom the same investigation by Sharman et al.(1994). While skimmed milk (1% fat) con-tained 0.05 mg DEHP kg−1, the full milk (3%fat) contained 0.11–0.13 mg kg−1 and cream(35% fat) contained 1.06–1.67 mg kg−1.

A third observation is to be found bycomparing the different DEHP concentrationsin hand- and machine-milked Norwegianmilks found by Castle et al. (1990). A signifi-cant DEHP migration from the plasticizedPVC milking tubes takes place, and this can beseen by comparing the concentration in thehand-milked product with the concentrationin milk from the collection tank. The figurescan be compared further with those of Norwe-gian retail full milk, which has approximatelythe same percentage of fat. Again a significantincrease takes place, probably due to furthermigration to the milk of plasticizers from foodcontact materials such as rubber tubes usedduring transport to the dairy and possiblyfrom gaskets in dairy equipment.

A fourth observation can be based on theCanadian investigation by Page and Lacroix(1995). Whereas in most investigations DEHPis the most dominant plasticizer presentin milk and milk products, these authorsfound rather high concentrations of dibutyl-phthalate (DBP) and butylbenzylphthalate(BBP) in butter. The origin of these plasticizers

was traced to the printing inks and wash coatused in the production of aluminium/paperlaminates used as packaging material.

A final remark must be made regardingthe column ‘Total phthalates’, where thereported data are based on an analyticalmethod developed by the Ministry of Agri-culture, Fisheries and Food in the UK. Inthis method, all phthalates are converted todimethylphthalate and determined as a sum.In this way, the phthalates from complextechnical mixtures of dioctyl-, dinonyl- anddidodecyl phthalates can be determined evenwhen the concentrations of the individualcompounds are below the limit of determina-tion. A full explanation of the reasons for thehigh results found by this method has not yetbeen published.

Migration of isocyanates and theirhydrolysis products

Flexible plastic laminates are used exten-sively for the packaging of foodstuffs,especially for products with a long shelf-lifeor products that need to be conserved ina modified atmosphere – an atmosphere dif-ferent from the surroundings. Even thoughthese films might seem very thin, they areoften manufactured from many layers ofvarious polymers. In some cases, suchmultiple layer materials can be produced by aco-extrusion process, whereby heating alonejoins the layers. In other cases, the materialsare more incompatible, and it is necessary tojoin them with adhesives.

The adhesives used are often made frommonomers of aromatic isocyanates andpolyols called polyurethane in their poly-merized (cured) state. It is of paramountimportance that this polymerization processis allowed to proceed to completion. This isdone by allowing enough time at a suitabletemperature for the polyurethane to form acoherent network, bound to the other layers inthe plastic laminate. If residues of isocyanatemolecules are still present when the laminatecomes into contact with the foodstuff,isocyanates could migrate to the food. Ifisocyanates come into contact with water,


20-Feb-03 12



20-Feb-03

12

284J.H

. Petersen

Concentration (mg kg−1)

Product and countryDibutylphthalate(DBP)

Butylbenzylphthalate(BBP)

Di-(2-ethylhexyl)-phthalate (DEHP)

Totalphthalates Notes References

Raw milkGermanyGermanyNorwayNorway

Retail milksNorway, 1% fatCanada, 2% fatNorway, 3% fatUK, whole milkItaly, mixed

Canada, 3.3% fatDenmark, 3.6% fatUK mixed (evaporated

including cream)Switzerland, mixed

0.0290.034

n.d.a

0.07

n.d.

0.003

0.02

n.d.

n.d.

0.002

0.1300.120< 0.005–0.010.03–0.08

0.050.040.11–0.130.0350.21

0.1< 0.05–0.10.3

0.015

< 0.04–0.6

0.36–1.0

0.5

Hand milking, 1 sampleMachine milking, 1 sampleHand milking, 3 samplesMachine milking (fromcollection tank), 2 samples

Total-diet samples

Mean of positive samples(the half of a total of 50samples)Total-diet samplesSamples from 15 dairiesDifferent types/total dietDiBPb 0.002; DOPc 2.6Different types/total diet

Gruber et al. (1998)Gruber et al. (1998)Castle et al. (1990)Castle et al. (1990)

Sharman et al. (1994)Page and Lacroix (1995)Sharman et al. (1994)Castle et al. (1990)Cocchieri (1986)

Page and Lacroix (1995)Petersen (1991)Ministry of Agriculture,Fisheries and Food (1996)Kuchen et al. (1999)

Table 12.6. Phthalates in milk, cream, butter and cheese: selected literature data.

300





Migration of C

ompounds from

Food Contact M

aterials285

20-Feb-03

12

CreamCanada, 17% fatSwitzerland, mixedNorway, 35% fat

ButterCanada, 80% fat

UKSwitzerland, mixed

CheeseUKCanada, cheddar-type

Canada, processedcheese

ItalySwitzerland, mixed

n.d.n.d.

1.5

0.19

n.d.

n.d.

0.840.30

n.d.

0.64

1.6

n.d.

1.20.251.06–1.67

3.4

2.5–7.41.2

0.24–16.82.2

1.1

1.081.2

2.9–5.1

4.8–56.6

2.4–112

Different creams (total diet)

Four samples in paper/alufoil (total diet)10 different brandsMany types mixed(total diet)

25 cheeses, many imported4 samples in plastic(total diet)3 samples, 17.7% fat(total diet)20 samples, mean valueMany types mixed(total diet)

Page and Lacroix (1995)Kuchen et al. (1999)Sharman et al. (1994)

Page and Lacroix (1995)

Sharman et al. (1994)Kuchen et al. (1999)

Sharman et al. (1994)Page and Lacroix (1995)

Page and Lacroix (1995)

Cocchieri (1986)Kuchen et al. (1999)

aNot detected.bDi-iso-butylphthalate.cDi-octylphthalate.

301





which constitutes a substantial part of mostfoodstuffs, primary aromatic amines (PAAs)can be formed (Fig. 12.2).

A selection of isocyanates appears onthe positive list of monomers and startingmaterials of the plastics directive. Since thecompounds are very reactive and potentiallyharmful to health, there is imposed the limita-tion that the sum of isocyanates remainingin the plastic when it comes into contactwith food may not exceed 1 mg kg−1 plastic(expressed as units active isocyanate groups,NCO). As the similarly toxic PAAs, formedfrom the allowed isocyanates, are not nor-mally used in the production of plastic, mostof the PAAs do not appear on the positive lists.However, in the sixth amendment to the plas-tics directive adopted in 2001, it was decidedthat plastics should not release PAAs in mea-surable quantities (EC, 2001a). In practice, thepermitted limit is set at 20 µg PAA kg−1 foodsimulant, which is the estimated analyticalquantification limit, including the analyticaltolerance, using an agreed analytical method.Such a method is currently under develop-ment by the CEN (2002).

During 2000 and 2001, there were severalattempts, mentioned in the European news-papers, to throw suspicion on the flexiblepackaging industry for selling not fully curedplastic laminates. There certainly could besome economic interest for the industry inselling their products as soon as possible afterlamination of the plastic instead of keepingthem for the full curing period. However, veryfew reliable data on residual amounts ofisocyanate in plastics and of PAA migrationinto food simulants have been published tillnow. Surprisingly, the industry does notseem very interested in publishing data fromtheir internal quality control. However, the

European lamination industry has publisheddemanding standards for in-house control,which seem to be followed by responsiblemanufacturers.

The enforcement laboratories havedifficulties in performing an efficient control.It is a troublesome task to obtain samples bysurprise in a laminating industry immediatelyafter a roll of laminate can be considered readyfor sale. Careful planning of sampling andlogistics is required in order to take a few2 dm2 test samples from a roll containinghundreds or thousands of square metres,to transport them rapidly to the analyticallaboratory under conditions which do notaccelerate the curing process and to performthe test analyses immediately after (Trier andPetersen, 2001).

At the time of writing, several Europeanenforcement laboratories are working onthese problems and undoubtedly results fromsuch investigations will be published soon.

A Systematic Testing Scheme to EnsureCompliance with Legislation

Generally speaking, the chemist only findsthe chemicals he or she is looking for. Inmany cases, a laboratory implements ananalytical method to look for a specificcompound in a specific sample matrix deter-mined from the start. Sometimes, luckily, thechemist at the end of the scheduled projectslooks at other sample matrices because he orshe is curious. One striking example of thisis the ‘Austrian wine scandal’, where it wasdiscovered that diethylene glycol (DEG) wasadded to wine to ‘soften’ its taste. SeveralEuropean control laboratories implemented

20-Feb-03 12

286 J.H. Petersen

Fig. 12.2. Reaction of 2,4-toluene-di-isocyanate with water to produce the primary aromatic amine2,4-toluene-diamine.



the analytical method and analysed a largenumber of wines. When the wine situationwas under control, some of the chemistsbegan to analyse foodstuffs packed inregenerated cellulose film containing DEGas a stabilizing agent and found very highamounts of this agent in foods such as fudges,toffees and cakes (Vaz et al., 1986).

At present, the single compound strategyis also the concept mainly used in the methodsdeveloped by the CEN. However, severalattempts to find more suitable systematicapproaches to identifying potential migrantsfrom a food contact material have provedsuccessful. An example is the proceduredeveloped by the Food Inspection Service ofThe Netherlands (van Lierob, 1997), whichhas also been adopted by other enforcementand industrial laboratories:

1. Identify the polymer(s) using infraredspectroscopy.2. Extract a small amount of the materialwith diethyl ether.3. Add internal standards in knownamounts.4. Inject the extract on a gas chromatograph(GC) with a mass selective detector (MSD).5. Identify the eluting compounds usingdedicated digitalized libraries of mass spectraand GC retention times.6. Perform a semiquantitative determina-tion of the individual compounds identified.7. Compare the results with specific migra-tion limits.8. If necessary, perform a specific migrationtest using agreed test conditions.

This procedure was developed furtherby a group of European laboratories takingadvantage of supplementary techniques, suchas headspace GC for the identification ofvolatiles, nuclear magnetic resonance, liquidchromatography with UV detection and GCwith infrared detection for the identificationof potential non-volatile migrants. It wasdemonstrated further that mutagenicity test-ing of sample extracts could be a possible toolto ensure that unidentified reaction productsand impurities with high toxicity did notoccur (Feigenbaum et al., 2002). It seemslogical to perform mutagenicity testing ofsuch extracts of the final food contact

materials since it is a basic requirement thatsuch tests have to be performed for individualcompounds on the positive list in the plasticsdirective. Further, liquid chromatographywith mass spectrometric detection hasbecome more common as a routine instru-mentation in analytical chemical laboratoriesand this method will be a suitable supplementto the above-mentioned equipment whenidentifying the more polar migration species.

The situation is somewhat different whenit comes to materials and articles not yetcovered by specific directives. As long aswe are speaking about materials based onpurely synthetic materials made from indi-vidually evaluated compounds, proceduressuch as these mentioned above could possiblybe applicable. However, when it comes tomaterials of natural origin or recycled materi-als where the starting material is less welldefined, it would be helpful to have othertools, at least for research purposes.

As a complementation to the chemicalanalysis, the idea of using a battery of in vitrotests has been used to investigate a seriesof toxicological effects in extracts of paperof different qualities, containing virgin fibresalone as well as a mixtures of virgin andrecycled fibres and further recycled fibres,which has been de-inked or not. The biologi-cal testing included a cytotoxicity test usinghuman fibroblasts, a yeast oestrogen assay,the chemical-activated luciferase expressionassay (CALUX) Ah receptor assay (sensitiveto dioxin-like compounds) and the AmesSalmonella assay (mutagenicity test). To someextent, there was a correlation between thecontaminant levels found in the chemicalanalysis and the biological responses. How-ever, especially in extracts of recycled paper,the response in the toxicological tests remainsto be explained by identified contaminants(Binderup et al., 2002).

Possible Future Instruments inRisk Management

The detailed legislation on food contactplastics has been developed through the lastdecade and the plastics industry is now faced


11-Mar-03 12



with customer demands for compliance test-ing and the enforcements laboratories arefaced with the responsibility to check thein-house control of the industry. The largenumber of individual restrictions in the formof SMLs or a maximum allowed concen-tration in the material (Qm and QmA), incombination with actual conditions of use(exposure time and temperature) and therelevant food simulant(s), gives rise to anenormous number of compliance tests.

However, not everything needs to betested using chemical analysis. Sometimes,analysis can be avoided by using a simplecalculation as shown in Fig. 12.3.

Unfortunately, in a significant number ofcases, the calculation produces a need for amigration test to be performed. For this rea-son, the sixth amendment to the plastics direc-tive opens up the possibility for the plasticsindustry to use the results from applicationof a scientifically recognized mathematicalmodel as documentation for compliance withlegislation. Such models, which are basicallybuilt on Fick’s second law of diffusion, havebeen developed, refined and validated, andby now cover homogeneous materials made

from the more common polymers. Insteadof considering 100% migration in Fig. 12.3, amore realistic situation can be calculated bythe model.

A series of other hot topics with possibleimplications for the food contact materialslegislation are to be discussed in the next fewyears in the European Union.

• The concept of a threshold of regulation,or in other words the idea that a com-pound, known or unknown, present at aconcentration below a certain limit in thefood or in the food simulant needs nei-ther to be identified nor to be quantified.This concept presents an attractive anda quite relaxing offer to the analyticalchemist working with extracts of foodcontact materials in an enforcementlaboratory, but it is not yet accepted bytoxicologists.

• The concept of a food consumption factoris mainly being marketed by the plasticsindustry. The idea behind the food con-sumption factor is that if, for example,nobody eats more than 200 g of fat eachday, why should we then consider an

20-Feb-03 12

288 J.H. Petersen

Fig. 12.3. Compliance testing by calculations assuming 100% migration.



SML related to a human intake of 1 kgfood every day for migrants only solublein the fatty food simulant? If this conceptis accepted, many compounds havingSMLs > 12 mg kg−1 food would be cov-ered by the overall migration limit of60 mg kg−1 food. In this way, a substan-tial number of measurements of specificmigration could be saved. An argumentagainst this concept is that, if one funda-mental convention is changed, then onehas to examine more closely all otherconventionally used safety factors. Oneexample is: we drink about 2 l per day,and some of the drinks come in plasticbottles.

• The concept of a packaging use factoris driven by the same idea of saving asubstantial number of specific migrationtests. The fundamental idea is that somepositive list compounds are only usedfor certain types of polymers. When atype of polymer only covers, for exam-ple, 10% of the market, then a ‘reductionfactor’ of 10 could be used for thesecompounds. A logical objection to thisconcept is that nobody knows exactlyhow many per cent of the market a givenpolymer covers. Further, who is goingto change the reduction factors whensuddenly in one country all milk bottlesmade of glass are replaced by bottlesmade of polycarbonate?

• The concept of a functional barrier hasbeen discussed for many years and hasbeen defined by the Council of Europein the following words:

The functional barrier is any integral layerwhich under its normal and foreseeableconditions of use reduces all possiblematerial transfers (permeation and migra-tion) from any layer beyond the barrier intofood to a toxicologically and organolepticallyinsignificant and to a technologicallyunavoidable level.

There are, for the time being, no agreedmethods to test a barrier material forfunctional properties (COE, 2002). Anagreement on what performance sucha barrier must have is needed whenrecycled materials, for which no detailed

knowledge of their composition exists,are being use in food contact materials.

Conclusions

Food contact materials and articles in thou-sands of elaborate forms made from a broadselection of different materials are more thanever an integral part of today’s lifestyle. Thelarge majority of materials have useful prop-erties and are safe in use. However, many ofthe starting compounds used in the produc-tion of the materials are potentially harmful,and new applications are developed con-stantly. Therefore, there is a continuing needfor the authorities to encourage the industryto act in a responsible manner.

It is of importance to note that the respon-sibility for ensuring compliance with legis-lation lies with the manufacturer, importerand retailer, since no system of governmentalapproval of food contact materials exists. Theindustry must have reliable in-house controls,and the enforcement authorities should regu-larly perform a check of the in-house controlsas well as supplementary independent analyt-ical control. As is the case for all food productsin the food industry, at least the consumersand the enforcement authorities should haveeasy and full access to all information aboutall compounds used from production of thepolymer to the finished material or article.

Until now, only a few materials are fullycovered by detailed regulations, and it mustbe recommended that frequently used materi-als such as paper and cardboard, coatings,printing inks, lacquers and rubbers arecovered in the not too distant future. Also, anagreement needs to be reached concerningmaterials made from recycled fibres or plas-tics. Should such materials of partly unknowncomposition be allowed in direct contact withfoods? If yes, under what conditions?

Acknowledgements

I would like to thank Torsten Berg andKirsten H. Lund for their help andencouragement.


20-Feb-03 12



References

BgVV (2002) Kunststoffe im Lebensmittelverkehr,Empfehlungen des Bundesgesundheitsamtes,Carl Heymanns Verlag, Köln, Germany. Avail-able at: www.bgvv.de/

Binderup, M.L., Petersen, G.A., Vinggaard, A.M.,Rasmussen, E.S., Rosenquist, H. andCederberg, T. (2002) Toxicity testing andmicrobiological tests of recycled paper forfood contact. Food Additives and Contaminants,Supplement 19, 13–28.

Castle, L., Gilbert, J. and Eklund, T. (1990) Migrationof plasticizer from poly(vinylchloride) milktubing. Food Additives and Contaminants 7,591–596.

CEN (2002) EN(V)1186 and EN(V)13130: Materialsand Articles in Contact with Food-stuffs – Plastics.European Committee for Standardization,Brussels.

Cocchieri, R.A. (1986) Occurrence of phthalateesters in Italian packaged foods. Journal of FoodProtection 49, 265–266.

COE (2002) Guidelines and resolutions fromCouncil of Europe. Available at: www.coe.fr/soc-sp/sante/pack/pres.htm

EC (2001a) Directive 2001/62/EC ‘CommissionDirective of 9 August 2001 amending Directive90/128/EEC relating to plastic materialsand articles intended to come into contactwith foodstuffs’. Official Journal of the EuropeanCommunities L 221, 18 (17.8.2001).

EC (2001b) Directive 2001/61/EC ‘CommissionDirective of 8 August 2001 on the use of certainepoxy derivatives in materials and articlesintended to come into contact with foodstuffs’.Official Journal of the European Communities L215, 26 (9.8.2001).

EC (2002) Directive 2002/72/EC ‘CommissionDirective of 6 August 2002 relating toplastic materials and articles intended tocome into contact with foodstuffs’. OfficialJournal of the Euopean Communities L 220,18 (15.8.2002).

EEC (1980) Directive 80/590/EEC ‘Council Direc-tive of 9 June 1980 determining the symbolthat may accompany materials and articlesintended to come into contact with foodstuffs’.Official Journal of the European Communities, L151, 21 (19.6.1980).

EEC (1982) Directive 82/711/EEC ‘Council Direc-tive of 18 October 1982 laying down the basicrules necessary for testing migration of theconstituents of plastic materials and articlesintended to come into contact with foodstuffs’.Official Journal of the European Communities L

297, 26 (23.10.1982). (Amendments 93/8/EECand 97/48/EC.)

EEC (1985) Directive 85/572/EEC ‘Council Direc-tive of 19 December 1985 laying down the list ofsimulants to be used for testing migration ofconstituents of plastic materials and articlesintended to come into contact with foodstuffs’.Official Journal of the European Communities L372, 14 (31.12.1985).

EEC (1989) Directive 89/109/EEC ‘CouncilDirective of 21 December 1988 on theapproximation of the laws of the MemberStates relating to materials and articlesintended to come into contact with foodstuffs’.Official Journal of the European Communities L 40,38 (11.2.89).

EU Commission (2002a) Current edition of Notefor Guidance of Petitioner when Presenting anApplication for Assessment of a Substance to beUsed in Food Contact Materials Prior to itsAuthorisation. DG SANCO, EuropeanCommission, Brussels. Available at: cpf.jrc.it/webpack/

EU Commission (2002b) Current edition of SynopticDocument – Draft of Provisional List of Monomersand Additives Used in the Manufacture of Plasticsand Coatings Intended to Come into Contactwith Foodstuffs. Scientific Committee for Food,European Commission, Brussels. Available at:cpf.jrc.it/webpack/

European Council for Plasticisers and Intermediates(2002) Homepage at: www.ecpi.org

Fabech, B., Hellstrøm, T., Henrysdotter, G.,Hjulmand-Lassen, M., Nilsson, J., Rüdinger,L., Sipiläinen-Malm, T., Solli, E., Svensson, K.,Thorkelsson, Á.E. and Tuomaala, V. (2000)Active and Intelligent Food Packaging – A NordicReport on the Legislative Aspects. TemaNord2000:584, Nordic Council of Ministers,Copenhagen.

FDA (2002) Food, Drug and Cosmetic Act. Availableat: vm.cfsan.fda.gov/

Feigenbaum, A., Scholler, D., Bouquant, J.,Brigot, G., Ferrier, D., Franz, R., Lillemark, L.,Riquet, A.M., Petersen, J.H., van Lierob, B.and Yagoubi, N. (2002) Safety and qualityof food contact materials. Part I: Evaluationof analytical strategies to introduce migrationtesting into good manufacturing practice. FoodAdditives and Contaminants 19, 184–201.

Food Standards Agency (2001) Survey of Bisphenolsin Canned Foods of 19 March 2001. FSA Surveil-lance Information Sheet no. 13/2001, FoodStandards Agency Library, Aviation House,London.

Grob, K., Spinner, C., Brunner, M. and Etter, R.(1999) The migration from the internal coatings

20-Feb-03 12

290 J.H. Petersen



20-Feb-03 12

of food cans; summary of findings and call formore effective regulation of polymers in con-tact with foods: a review. Food Additives andContaminants 16, 579–590.

Gruber, L., Wolz, G. and Piringer, O. (1998)Untersuchung von Phthalaten in Baby-Nahrung. Deutsche Lebensmittel-Rundschau 94,177–179.

Jorhem, L., Sundström, B. and Engman, J. (1995)Lead and tin in tin cans. Vår Fφda 47(3), 23–29(in Swedish with English summary).

Kuchen, A., Müller, F., Farine, M., Zimmermann, H.,Blaser, O. and Wüthrich, C. (1999) Die mittleretägliche Aufnahme von Pesticiden undanderen Fremdstoffen über die Nahrung in derSchweiz. Mitteilungen aus Lebensmittelunter-suchung und Hygiene 90, 78–107.

Ministry of Agriculture, Fisheries and Food (1978)Survey of Vinyl Chloride Content of PolyvinylChloride for Food Contact and of Foods. FoodSurveillance Paper no. 2, HMSO, London.

Ministry of Agriculture, Fisheries and Food (1989)Migration of Substances from Food ContactMaterials into Food. Food Surveillance Paperno. 26, HMSO, London.

Ministry of Agriculture, Fisheries and Food (1996)Phthalates in Food. Food Surveillance Infor-mation Sheet no. 82, Food Safety Directorate,Ministry of Agriculture, Fisheries and Food,London.

Moore, J.W. (1975) The vinyl chloride story.Chemistry 48(6), 12–16.

Mountfort, K.A., Kelly, J., Jickells, S.M. and Castle,L. (1997) Investigations into the potentialdegradation of polycarbonate baby bottlesduring sterilisation with consequent release ofbisphenol A. Food Additives and Contaminants14, 737–740.

Page, P.B. and Lacroix, G.M. (1995) The occurrenceof phthalate ester and di-2-ethylhexyl adipateplasticizers in Canadian packaging and foodsampled in 1985–89: a survey. Food Additivesand Contaminants 12, 129–151.

Pedersen, G.A. (1998) Migration of bisphenol A andbisphenol A diglycidyl ether (BADGE) into

canned food, in ‘lacquers in cans’. In: TemaNord594. Nordic Council of Ministers, Copenhagen,pp. 80–83.

Petersen, J.H. (1991) Survey of di-(2-ethylhexyl)phthalate plasticiser contamina-tion of retail Danish milks. Food Additives andContaminants 8 supplement, 701–706.

Rasmussen, G. (1984) Release of Metals (Lead, Cad-mium, Copper, Zinc, Nickel and Chromium) fromKitchen Utensils. Publication no. 88, NationalFood Agency of Denmark (in Danish withEnglish summary).

SDU (2002) Warenwet, Dutch legislation: ISBN 90 1206791 X, SDU Uitgeverij, Koninginnegracht,The Hague.

Sharman, M., Read, W.A., Castle, L. and Gilbert, J.(1994) Levels of di-(ethylhexyl)phthalate andtotal phthalate esters in milk, cream, butterand cheese. Food Additives and Contaminants 11,375–385.

Smart, G.A., Pickford, C.J. and Sherlock, J.C.(1990) Lead in alcoholic beverages: a secondsurvey. Food Additives and Contaminants 7,93–99.

Trier, X.T. and Petersen, J.H. (2001) Migrationof Primary Aromatic Amines from LaminatedPlastics: a Pilot Enforcement Campaign. Report2001.12, Danish Veterinary and FoodAdministation, Søborg, Denmark.

van Lierob, B. (1997) Enforcement of food packaginglegislation. Food Additives and Contaminants 14,555–560.

van Lierob, B. (1998) Investigations in theNetherlands, Switzerland and Austria, in‘Lacquers in cans’. In: TemaNord 594.Nordic Council of Ministers, Copenhagen,pp. 53–74.

Vaz, R., Sandberg, E. and Larsson, B. (1986) Migra-tion af glycoler från cellofanförpakninger tilllivsmedel. Vår Föda 2, 148–152.

Waldron, H.A. and Stöfen, D. (1974) Sub-clinical LeadPoisoning. Academic Press, London.

WHO (1992) Diethylhexyl Phthalate, EnvironmentalHealth Criteria, 131. World Health Organiza-tion, Geneva.




13 Veterinary Products: Residues andResistant Pathogens

J.C. Paige1* and L. Tollefson21Division of Epidemiology and 2Center for Veterinary Medicine, DHHS/FDA-CVM,

7519 Standish Place, Rockville, MD 20855, USA

Introduction

As we enter the 21st century, food safetyfaces a rapidly changing paradigm. As aresult of the projected global demand foranimal protein due to the increase in popula-tions and expanding international travel andtrade, consumers are shifting from a regionalcommerce to a global environment, andemerging infections and foodborne illnesswill continue to have a major impact.

Food safety encompasses food science,which involves applying scientific methodsto the study of foods. Food science relies onvarious disciplines such as chemistry, engi-neering, microbiology, molecular biology,veterinary medicine, epidemiology and pub-lic health. This chapter is intended to focus onthe scientific aspects of food safety and, indoing so, we have selected several significantveterinary compounds that have broadinternational importance, from an economicand public health perspective. They includethe following: (i) penicillin; (ii) tetracycline;(iii) sulphamethazine; (iv) fluoroquinolones;(v) clenbuterol; and (vi) hormones.

In order to address these veterinaryproducts adequately, they will be discussedfrom epidemiology, toxicology/pharmacol-ogy and risk assessment perspectives. A

discussion of public health surveillance as atool and its application to food safety willbe presented. Included in this discussion willbe the significance of existing databases tomonitor antimicrobial residues and changesin susceptibility to antimicrobial drugs. Thesetools enhance food safety efforts, by collatingdata that will, upon analysis, provide infor-mation for making future policy decisions.

The emergence and spread of anti-microbial resistance as a result of the use offluoroquinolones in food animals is exam-ined. Against a backdrop of antimicrobialresistance and a global perspective, drugresidues remain a key factor that is oftenmisunderstood and omitted when food safetyand public health concerns are considered.Epidemiological data for the drugs penicillinand clenbuterol will be presented to demon-strate clearly existing evidence of acuteand chronic health consequences as a resultof residues of the parent compound or itsmetabolites in edible tissues.

Toxic effects may be produced by acuteand/or chronic exposure to chemical agents.Acute exposure is defined as a single exposureor multiple exposures occurring within ashort time (≤ 24 h). Chronic effects occurwhen the agent or its metabolites accumulatein the biological system. This occurs when


20-Feb-03 13




absorption exceeds metabolism and/or excre-tion, when the antimicrobial agent producesirreversible toxic effects or when there isinsufficient time for the system to recoverfrom the toxic effect within the exposurefrequency interval.

Because of a lack of information regard-ing the temporal characterization of exposure,it is not always easy to classify all adverseeffects reported into acute or chronic catego-ries without some overlap. These factors areimportant as you read the toxicity and clinicalaspects for each drug.

Veterinary compounds are used exten-sively in food-producing animals for thera-peutic effects, prevention of infectiousdiseases, growth promotion and the promo-tion of feed efficiency. All veterinary productsapproved for use in developed countries mustmeet stringent standards for safety, efficacyand quality. In considering veterinary prod-ucts for use in food animals, sound scientificevidence must be demonstrated duringthe drug approval process (see Table 13.1).

These agriculture compounds are regulatedextensively by the appropriate agency in eachcountry.

It is critical that the prudent use ofantimicrobials is emphasized not only tominimize the antimicrobial resistance, butalso to ensure the continued efficacy andavailability of antimicrobial products for usein food animals and to minimize drugresidues and their impact on human health(Table 13.2). These aspects will be discussed inthis chapter.

Risk assessment assists in the setting ofpriorities and tactics for national surveillanceactivities. It also assists in surveillance design.Surveillance is used to improve risk assess-ment and risk management decisions. Publichealth surveillance and risk assessment willbe discussed from a food safety perspective.The authors believe that implementation ofvarious on-farm safeguards, such as adequaterecord keeping, animal identification, herdmanagement practices and promoting theprudent and judicious use of antimicrobials,

20-Feb-03 13

294 J.C. Paige and L. Tollefson

Animal drug application Risk assessment Risk management

Laboratory and field studies(observation studies);epidemiological evidence

Toxicology studiesa

basic toxicology studiesmetabolism studiestoxicology testing in laboratoryanimals: genetic toxicity, acutetoxicity, subchronic toxicity, 2–3generation reproductive study,carcinogenic studies if genetictoxicity tests are positiveresidue depletion studiesanalytical methodology

Estimated exposuresCharacteristics of populationAge, sex

Hazard identificationDoes the drug cause an

adverse effect, i.e. residue,hypersensitivity?

Dose–response assessmentThe NOEL is determined from

toxicology testingExtrapolation of high to low

dose and from animal to man

ADI NOELsafety factor

=

Exposure assessmentComparative metabolism study

in the toxicology species.A model for human exposure,i.e. number of grams of meatADI and MRL

Risk characterization

Development of regulatoryoptions, i.e. tolerances,slaughter withdrawal or milkwithheld, time to safe level

Evaluate public health,economic, social, politicalconsequences of theregulatory options andharmonization issues

Agency decision and actionreject approvalapprove with conditionswithdrawal time for drugban for specific specieschanges in manufacturing,labelling, etc.

aBasic toxicology studies: genetic tests, 90 day feeding studies, two-generation reproductive studies witha teratology component.NOEL, no observed effect level; ADI, acceptable daily intake; MRL, maximum residue limit.

Table 13.1. An assessment of hazard and risk management in the drug approval process.



can do much to prolong the lifespan of anti-microbials in animals, while at the same timeensuring that significant human antimicrobialtherapies are not compromised or adversehealth effects (Table 13.2), attributable toresidues in animals, do not occur as a resultof using antimicrobials in food animals.

Antimicrobial Agents

The proper clinical use of an antimicrobialagent requires an understanding of theinter-relationships among the pathogen, thehost animal and the drug. The followingantimicrobial agents, penicillin, tetracycline,sulphamethazine and fluoroquinolones, willbe discussed in terms of pharmacokinetics/pharmacodynamics, food safety, toxicity andclinical aspects.

Antimicrobial drugs exhibit selectivetoxicity in view of their harmful effects to themicroorganism without being harmful tothe animal or human host. Antimicrobials atthe appropriate concentration will demon-strate bacteriostatic or bactericidal effectsagainst the microbial organism.

Antimicrobial mechanisms for the abovedrugs are as follows:

• Inhibition of the cell wall synthesis:penicillin.

• Inhibition of nucleic acid synthesis:sulphonamides.

• Inhibition of protein synthesis:tetracycline.

• Mutations in the topisomerase genes,decreased permeability of the bacterialcell wall and energy-dependent effluxpumps: fluoroquinolones (Gootz andBright, 1996).

Penicillin

Pharmacokinetics/pharmacodynamics

The pharmacokinetic properties of a com-pound include the route of administration,rate of absorption, rate of distribution, vol-ume of distribution, protein-binding capacityof the drug, and the route and rate ofelimination, all of which influence the dosingregimen (Davis, 1995; Craig, 1998).

Pharmacodynamics is the study ofthe biochemical and physiological effectsof drugs and their mechanisms of action.Pharmacodynamic properties include theconcentration versus time in the tissue andother body fluids, toxicological effects andantimicrobial effect at the site of the infection(this interaction is shown in Fig. 13.1).

A drug is broadly defined as any chemi-cal substance that affects living processes.A therapeutic agent may be defined as adrug which, when given in proper dosages,will produce a beneficial pharmacological orchemotherapeutic (antimicrobial) effect. Theprocess by which a veterinary compoundbrings about change in some pre-existingphysiological function or biochemical processof the living organism is the action of the drug(Vaden et al., 1995; Benet et al., 1996). Thepart of the body in which the drug acts andthereby initiates the series of biochemical andphysiological changes that are characteristicof the drug is referred to as the site ofaction, while the mechanism by which a druginitiates the series of events considered as aneffect is its mechanism of action. Hypothetically,the mechanism of action of most drugsinvolves chemical interaction between thedrug and a macromolecular tissue constituentcalled a receptor with which a drug interactsto produce its characteristic biological effect.

Penicillin interferes with the develop-ment of bacterial cell walls by interfering with

Veterinary Products: Residues and Resistant Pathogens 295

20-Feb-03 13

Drug(s) Adverse health effects

Penicillin

TetracyclineSulphamethazineFluoroquinolones

β-Agonist/clenbuterolChloramphenicolAminoglycosides

NeomycinGentamicin

Rash, urticaria andpossible anaphylaxisBlood dyscrasiaThyroid hyperplasiaAntibiotic resistance infoodborne pathogensVentricular fibrillationBlood dyscrasia

NephrotoxicNephrotoxic

Table 13.2. Identifiable risk factors for specificdrug exposures.



transpeptidase enzymes, thereby affectinggrowing cells. The effect of the penicillinis generally characterized as bactericidal tocertain organisms during their growth phase.

Distribution in food

The penicillins gained wide agricultural usein the 1940s. Penicillin is approved for usein food-producing animals in most countriesas a production agent in cattle, sheep andswine, and as a therapeutic agent for thetreatment of bacterial pneumonia in cattleand sheep, erysipelas in swine and stranglesin horses. Penicillin is a potent antibacterialcompound that is effective against a varietyof Gram-positive and Gram-negative organ-isms with relatively low to no toxicity inanimals.

The penicillins are organic acids thatare generally available as the sodium orpotassium salt of the free acid. In the drycrystalline form, penicillins are stable butlose their activity rapidly when dissolved.Penicillins have short half-lives (0.5–1.2 h)(Sundlof, 1994) in all species of domesticanimals. After absorption, the penicillinsare widely distributed in the extracellularfluids of the body, but they cross biological

membranes poorly because they are ionizedand poorly lipid soluble.

Diffusion of penicillin into the tissues andfluids occurs as long as the unbound plasmaconcentration exceeds that of the tissue andfluids. Tissue residues of penicillin in slaugh-ter animals are considered a public healthhazard because of the potential for hypersen-sitivity reactions in people. Based on availabledata, the Joint Food and Agriculture Organi-zation (FAO)/World Health Organization(WHO) Expert Committee on Food Additives(JECFA), with approval by the Codex Ali-mentarius Commission (Codex), establishedthe maximum residue limits (MRLs) formuscle, liver and kidney in cattle, pigs andchickens at 50 mg kg−1 and 4 µg l−1 for milk.

Toxicity and clinical aspects

ACUTE An assessment of toxicity has manyfacets related to the characteristics and con-ditions of exposure. Generally, scientists areconcerned with the frequency of exposure,the route by which exposure occurs and thedose. With acute exposure, the dose is usuallydelivered in a single event and absorptionis rapid. Acute exposure effects are definedas those that occur or develop rapidly after

20-Feb-03 13


Fig. 13.1. Pharmacokinetic/pharmacodynamic interactions (Craig, 1998).



administration of chemical substances. Theability of penicillin to provoke hypersen-sitivity reactions in animals and humans isa problem that requires consideration. Theprevalence of such reactions is a function ofboth route of administration and formulationof the drug. In addition, changes in the intesti-nal flora induced by penicillin administrationcan contribute to diarrhoea (Kidd, 1994).

Hypersensitivity, damaging immunolog-ical reactions, may manifest itself in manyways, from life-threatening anaphylactic reac-tions to lesser reactions, such as rashes (Table13.2). Reportedly, there are documentedadverse reactions in persons consuming foodscontaminated with drug residues. The mostfrequently cited reactions are allergic reactionsto the β-lactam drug penicillin (Riviere, 1992;Sundlof, 1994; Paige et al., 1999b). Other drugsincluding aminoglycosides, sulphonamidesand, in a few cases, tetracycline, may causeallergic reactions in sensitive persons. Report-edly, both epidemiological and experimentaldata indicate that levels of penicillin as low as5–10 IU are sufficient to produce allergic reac-tions in previously sensitized individuals(Sundlof, 1994). Adverse human reactionsare manifested as severe swelling of the skin,serum sickness, shock and less serious reac-tions such as skin rashes, asthma and fever.Since these reactions are not dose related, it ishighly probable that individuals allergic tothe drugs enumerated above, when exposedvia the food, could suffer an allergic reaction.Possibly, the reason that few cases are docu-mented is that many cases might be maskedby other health conditions, especially inelderly populations, as well as problems withunder-ascertainment and under-reporting.

For penicillin, the incidence of deathdue to anaphylaxis is 0.02 per 1000 coursesof therapy (an undetermined number of dosesused in treatment of bacterial infections).

CHRONIC A malfunction of the mecha-nisms for terminating action of the agent is aprimary cause of toxicity. Normal doses arethereby present for a longer period of time inthe animal and repeated doses are likely toresult in accumulation of the drug to toxiclevels. The end result can be an accumulationin the kidney and, in some cases, death.

In most countries, no residue of ananimal drug is permitted in foods intendedfor human consumption if the animal drug orits residue is found to induce cancer wheningested by man or animals. Most residuesof veterinary compounds or chemicals occurin food at such low concentrations that theyrarely pose a chronic or long-term healthhazard to consumers. The pre-approval pro-cess, with its human food safety evaluationand tolerance-setting procedures, represents avery strong safeguard against these hazards.This process will be discussed later in thischapter. However, chronic or long-termeffects such as carcinogenesis as a resultof exposure to drug residues in food are par-ticularly difficult to detect and are certainlyunder-ascertained as well as under-reported(Paige et al., 1999b).

Tetracycline


Tetracycline is produced semi-syntheticallyfrom chlortetracycline. Chlortetracyclineand oxytetracycline are elaborated by Strepto-myces aureofaciens and Streptomyces rimosus,respectively.

Shortly after its introduction in 1948,tetracycline was determined to have a broadrange of antimicrobial activity againstGram-positive and Gram-negative bacteria.However, resistance has reduced its useful-ness over the last decade. Tetracycline is abacteriostatic antibiotic that inhibits bacterialprotein synthesis, with its site of action beingthe bacterial ribosome. Tetracycline enters thebacterial cell through passive diffusion, or byan active transport system that pumps thetetracycline through the inner cytoplasmicmembrane.

Bacteria resistant to one tetracyclinefrequently exhibit resistance to the others(chlortetracycline and oxytetracycline). Thetetracyclines are bound to plasma proteinsto various degrees. The approximate valuesare tetracycline, about 65%, and oxytetracy-cline, 20–40% (Prescott and Baggot, 1993;Benet et al., 1996).


20-Feb-03 13



Oxytetracycline and tetracycline areincompletely absorbed in man and, after asingle oral dose, peak plasma concentrationsare attained in 2–4 h. They have half-lives inthe range of 6–12 h, and they are frequentlyadministered two to four times daily.

The long-acting formulation of oxytetra-cycline available for intramuscular (i.m.)administration in food animals exhibits theirlong-acting effect because of both the highdosage used and the prolonged drugpersistence at the site of i.m. injection.

All of the tetracyclines are concentratedin the liver and excreted, by the bile, into theintestine, where they are partially reabsorbed.Because of their enterohepatic circulation,they may be present in the blood for along time after therapy is discontinued.Tetracyclines account for a relatively highfrequency of illegal residues found in animaltissue.

The evaluation of chlortetracycline, oxy-tetracycline and tetracycline by the JECFA ledto the following recommendations regardingMRLs. These are expressed as parent drug,singly or in combination: 100 µg kg−1 inmuscle; 300 µg kg−1 in liver; and 600 µg kg−1

in kidney of cattle, pigs, sheep and poultry;100 µg l−1 in cattle and sheep milk; and 200 µgkg−1 in eggs (Goodman Gilman et al., 1991;Sundlof, 1994; Roestel, 1998).


Systemic availability of tetracyclines can varywidely among the different oral preparations.The absorption of tetracycline is decreased bythe presence of food in the stomach. In adultruminants, it is believed that oral dosing withtetracycline interferes with the fermentationprocess in the rumen. Because of the entero-hepatic circulation, the tetracycline may bepresent in the body for a long time aftercessation of therapy. This has been attributedto high tissue residue levels.


ACUTE From a pharmacological viewpoint,the tetracyclines are relatively safe. However,adverse events, as a result of the irritant natureof the drug, include nausea, vomiting and

diarrhoea after oral administration and tissuedamage at the injection site. Fatal anaphylaxishas been reported occasionally in dogs admin-istered intravenous (i.v.) injections. Unlessadministered slowly, i.v. injection of a tetracy-cline is likely to cause an animal to collapse(Prescott and Baggot, 1993).

Tetracycline may produce gastrointesti-nal irritation to varying degrees in someindividuals. Although rare, various skinreactions, including rashes and urticaria, mayfollow the use of tetracycline. Angio-oedemaand anaphylaxis are among the more severeallergic responses. Tetracycline undergoesenterohepatic circulation, with much of thecompound excreted in bile being reabsorbedfrom the intestines. This process contributes tothe half-life of 6–10 h, which is long for drugsthat are eliminated mainly by renal excretion.

CHRONIC Hepatotoxicity may result fromaccumulation of tetracyclines when they arenot eliminated quickly enough by the kidneysor by administration of frequent and/or largedoses above recommended therapeutic dos-ages. Tooth moulting/discoloration occurswhen tetracyclines are administrated duringpregancy.

The toxicology of tetracycline has failedto show any evidence of carcinogenicity inrats fed 0, 12,500 and 25,000 ppm of tetracy-cline for 2 years. However, liver and kidneytoxicity have been reported. Most toxic andclinical side effects of tetracycline are due tothe direct toxicity of the drug or to alterationsof microbial flora.

Sulphamethazine


The sulphonamides represent one of theoldest groups of antimicrobial compoundsin veterinary use today. Sulphanilamide,an amide of sulphanilic acid, was the firstsulphonamide used clinically. Sulphona-mides have been used in clinical veterinarymedicine for nearly 50 years, and wide-spread resistance has developed againstmost of these compounds. The emergence of

20-Feb-03 13




resistance has limited their use. In addition tomicrobial resistance, there is concern regard-ing possible carcinogenicity of some sulphon-amides in laboratory animals (Littlefield et al.,1989).

Many derivatives of sulphanilamide withdiffering pharmacokinetics and antimicrobialspectra are used in veterinary medicine. Themost commonly used veterinary compoundsinclude sulphamethazine, sulphadimethox-ine, sulphadiazine, sulphanilamide andsulphamethoxazole. Sulphonamides are usedprimarily to treat microbial infections of theurinary, gastrointestinal and central nervoussystems. Sulphonamides are often given incombination with each other to obtain highblood or urine levels. The antimicrobial effectis additive. Sulphonamides act by interferingwith the normal production of RNA,protein synthesis and microbial replicationmechanisms.

Sulphonamides have a broad spec-trum of activity, affecting Gram-positive,Gram-negative and many protozoal organ-isms. Sulphonamides are bacteriostatic ratherthan bactericidal. Therefore, the cellular andhumoral defence mechanisms of the hostare essential for the final eradication of theinfection.


Sulphonamides are distributed throughoutall tissues of the body. They are majorcontributors to antimicrobial residues. Sulph-onamides are eliminated from the bodypartly as the unchanged drug and partly asmetabolic products. Clinical application ofsulphonamides in swine and cows has led tothe presence of sulphamethazine residues inthese livestock classes.

Sulphonamides in general are absorbedrapidly from the gastrointestinal tract whenadministered orally (Prescott et al., 2000). Sul-phonamides are classified as short, intermedi-ate or long acting according to the plasmaconcentration and time profile, which is essen-tially the rapidity with which compounds areabsorbed and excreted (see Table 13.3).

Sulphonamides are considered short act-ing if blood concentration levels remain higherthan 50 g ml−1 for less than 12 h after a singletherapeutic dose, intermediate if these plasmalevels are obtained between 12 and 24 h, andlong acting if obtained 24 h after dosing.Acetylation occurring in the liver and lungs isthe major pathway by which sulphonamidesare metabolized in most species. All sulpho-namides, with the exception of the entericcompounds, are excreted by the kidneys asparent compounds or as metabolites by wayof glomerular filtration. Ruminants metabo-lize sulphonamides by acetylation pathways,and apparently sulphonamides are the majorurinary metabolites in cattle, sheep and swine.There are additional metabolic pathways,beyond the scope of this chapter, but theyall display metabolites of reduced therapeuticactivity and may be therapeutically inactive.


ACUTE Sulphonamides may produce avariety of side effects either of an allergicnature or by direct toxicity (Table 13.2). In asmall population of humans, sulphonamidetherapy has been known to produce idio-syncratic drug reactions (unpredictablerare events dependent upon the individualresponse to the drug). These reactions mayinclude drug fever and urticaria. Thesereactions are usually reversible in nature.


20-Feb-03 13

Short-actingsulphonamides

Intermediate-actingsulphonamides

Long-actingsulphonamides

Entericsulphonamidea

SulphamethazolSulphathiazoleTrisulphapyrimidine

SulphamimethoxineSulphamethoxazoleSulphamethazineSulphadiazine

SulphamethazineSulphadimethoxine

Sulphaquinoxaline

aThis class is not absorbed (or minimally absorbed) from the gastrointestinal tract after oral adminstrationbut acts locally within the lumen of the gastrointestinal tract (Spoo and Riviere, 1995).

Table 13.3. Classification of sulphonamides based on plasma concentration vs. time profiles.



Aplastic anaemia and thrombocytopeniahave been reported as being induced by drugtherapy with trimethoprim–sulphadiazine.

Acute toxic effects, although rare, aremost commonly associated with overdosageor too rapid rates of i.v. drug administration.For example, dogs receiving large doses ofsulphanilamide (1 g kg−1 of body weight(BW)) have exhibited increased salivation,vomiting, diarrhoea, hyperpnoea, excitement,muscular weakness, ataxia and spasticrigidity of the limbs.

In cats given large doses of sulphanila-mide, spasticity of the limbs and dyspnoeahave been observed. Oral doses of 300–500 mgkg−1 of sulphamethoxypyridazine producedtransient toxic symptoms in pigs (Booth,1977).

CHRONIC Sulphonamide drugs are com-monly administered in a wide variety ofdosage forms including i.v. solutions, oralboluses, sustained-release boluses (whichcontribute to many of the residue problems)and crystalline powder.

Although relatively safe compounds,disorders of the haematopoietic systemhave been observed following the use ofsulphonamide drugs for the treatment ofdiseases in animals. Transient agranulocyto-sis and mild haemolytic anaemia and vesiclehaemorrhage have been associated withtreatment in calves and mink, respectively.

The Food and Drug Administration(FDA) is concerned about thyroid toxicity(Table 13.2) as the major human safetyconcern associated with this drug. In chronicfeeding studies conducted in 1989–1990 at theNational Center for Toxicological Research(NCTR), high doses of sulphamethazine inthe diet were associated with significantincreases of thyroid follicular cell adenomasin both male and female B6C3F1 mice andof thyroid follicular cell adenomas andadenocarcinomas in male and female F344rats (Littlefield et al., 1989, 1990). These neo-plastic lesions in rats appeared only in the4800 ppm dose group.

These findings resulted in agencyconcern regarding the continued use of

sulphamethazine. The primary focus waswhether the Delaney Clause was beingcompromised. The Delaney Clause under theUS Federal Food, Drug, and Cosmetic Act(FFDCA) prohibits the use of veterinarycompounds found to induce cancer wheningested or found to induce cancer in manor animals. However, FDA scientists dur-ing this time felt that thyroid tumoursproduced in rodents ingesting high dosesof sulphamethazine were attributable toenzyme inhibition resulting in elevatedlevels of the thyroid-stimulating hormones(TSH) that do not occur at low doses ofsulphamethazine.

Fluoroquinolones


Fluoroquinolones are one of the most valu-able antimicrobial drug classes available totreat human infections because of their broadspectrum of activity, pharmacodynamics,safety and ease of administration. This classof drugs is effective against a wide rangeof human infections, including those thatare resistant to other drugs, and has beenparticularly important in the treatment offoodborne bacterial diseases (Petruccelli et al.,1992; Moellering, 1993; Hooper, 2000).

Selective pressure exerted by fluoro-quinolone use is the driving force behind thedissemination of resistance determinants tofluoroquinolone compounds. The associationbetween fluoroquinolone use and resistancehas been documented in various settings(McGowan, 1983; Anon., 1995; Piddock,1996).

Fluoroquinolones are totally syntheticantimicrobials, with no known natural ana-logues. Therefore, resistance to the fluoro-quinolones is due only to use of the drug.Resistance to any of the fluoroquinolonesconfers resistance to all members of the drugclass (Acar and Goldstein, 1977; Piddock,1998). The fluoroquinolone drugs used inhuman and veterinary medicines are eitheridentical or very similar (Kidd, 1994; Acar and

11-Mar-03 13




Goldstein, 1997). The primary targets offluoroquinolones are DNA topoisomerase II(DNA gyrase) and DNA topoisomerase IV(Hooper and Wolfson, 1993; Ascar andGoldstein, 1997). Topoisomerase is an essen-tial enzyme found in all cells; topoisomerase IIand IV catalyse negative supercoiling of DNA,a vital process in cellular metabolism (Drlicaand Zhao, 1997).

Fluoroquinolone resistance is mediatedby several mechanisms: (i) target mutationsin the topoisomerase genes; (ii) decreased per-meability of the bacterial cell wall; and (iii)energy-dependent efflux pumps. Resistancedue to a decreased drug influx is generallylow level resistance. Each of the three fluoro-quinolone resistance mechanisms can occursimultaneously within the same cell, leadingto very high resistance levels. Frequent expo-sure to the drug is likely to lead to higherlevels of resistance (Piddock, 1999).

Food safety concern

In industrialized countries, the majorfoodborne pathogens, Campylobacter andSalmonella, are transferred infrequently fromperson to person (Tauxe, 1992; Angulo et al.,1998). In these countries, epidemiologicaldata have demonstrated that the primarysource of antibiotic-resistant foodborne infec-tions in humans is the acquisition of resistantbacteria from animals via food (Harris et al.,1986; Tauxe, 1992; Threlfall, et al., 1996). Evi-dence for the transfer of resistant pathogensfrom animal-derived food to humans comesfrom several different types of foodborne dis-ease follow-up studies, including laboratorysurveillance, molecular subtyping, outbreakinvestigations and examination of infectiousdose and carriage rates (Holmberg et al., 1984;Tacket et al., 1985; Spika et al., 1987; Smithet al., 1999, 2000).

When antimicrobial drugs such asthe fluoroquinolones are administered tofood-producing animals, they promote theemergence of resistance in bacteria that maynot be pathogenic to the animal but are patho-genic to humans (Endtz et al., 1991; Bates et al.,1994; Piddock, 1996; WHO, 1997, 1998; Glynn

et al., 1998). For example, Salmonella andCampylobacter are ubiquitous and can exist inthe intestinal flora of various food-producinganimals without causing disease. However,these bacteria can cause severe, even fatal,foodborne illness in humans. If using anantimicrobial in a food-producing animalcauses resistance to occur in such bacteria andthe resistant bacteria cause an illness in a con-sumer, who needs treatment, that treatmentmay be compromised.

Reports from the scientific and publichealth communities have identified a relation-ship between the approval of fluoroquino-lones for therapeutic use in food-producinganimals and the development of fluoroquino-lone resistance in Campylobacter in animalsand humans. The approval and use of thesedrugs in food-producing animals in TheNetherlands (Endtz et al., 1991; Jacobs-Reitsma et al., 1994), the UK (Piddock, 1995),Spain (Velazquez et al., 1995; Perez-Talleroet al., 1997) and the USA (Smith et al., 1999;Rossiter et al., 2000) temporally preceded anincrease in resistance in Campylobacter isolatesfrom treated animals and ill humans.

In The Netherlands, Campylobacter iso-lates from humans and poultry wereexamined for resistance to the human fluoro-quinolone ciprofloxacin between the years1982 and 1989 to determine the influence oflicensing of enrofloxacin for veterinary use in1987 (Endtz et al., 1991). Initially, none of theCampylobacter isolates from either human orpoultry sources was resistant to ciprofloxacin.From 1987 to 1988, resistance levels rose to8% in humans and 8.4% in poultry, and in1989 fluoroquinolone resistance among theCampylobacter isolates was 11% in humans and14% in poultry (Endtz et al., 1991).

Despite several restrictions placed onthe use of poultry fluoroquinolone productsin the USA, fluoroquinolone-resistant Campy-lobacter has been isolated repeatedly fromdomestic retail chicken products sampled.From January to June 1999, public health labo-ratories in Georgia, Maryland and Minnesota,under the direction of the Centers for DiseaseControl and Prevention (CDC), tested 180chickens with 23 distinct brand names that


11-Mar-03 13



were purchased from 25 grocery stores.Campylobacter were isolated from 80 (44%) ofthe chickens. Nineteen (24%) of the sampleshad Campylobacter isolates resistant to fluoro-quinolones and 25 (32%) were resistant tonalidixic acid, a quinolone antimicrobial drugthat serves as a precursor to fluoroquinoloneresistance development (Rossiter et al., 2000).These retail chicken findings are consistentwith those from an earlier, independent studyin the USA conducted by the MinnesotaDepartment of Health.

Researchers at the Minnesota Depart-ment of Health studied quinolone resistanceamong Minnesota residents, and evaluatedchicken as the source of the resistance. Theyfound that the proportion of quinolone-resistant Campylobacter jejuni isolates fromhumans increased from 1.3% in 1992 to 10.2%in 1998 (Smith et al., 1999). The proportion ofresistant C. jejuni collected from all reportedcases of illness increased only slightly from1992 to 1994. Researchers found that subse-quent increases, between 1996 and 1998, werepredominantly associated with foreign travel.More importantly, the percentage of resistantinfections that were acquired domesticallyalso increased from 0.3 to 4.2% between 1996and 1998 (Smith et al., 1999).

As part of the study, the MinnesotaDepartment of Health, in cooperation withthe Minnesota Department of Agriculture,collected 20 different brands of retail chickenproducts from 18 markets in the Twin Citiesmetropolitan area in 1997. Campylobacter wereisolated from 88% (80/91) of the samples; 20%of these were resistant to fluoroquinolones.The products with resistant strains had beenprocessed in five states (Smith et al., 1999).

Molecular subtyping revealed a strongassociation between resistant C. jejuni strainsfrom chicken products and C. jejuni strainsfrom the domestically acquired humancases of campylobacteriosis. The study usedpolymerase chain reaction with restrictionlength polymorphism flagellin gene typing toidentify strains of fluoroquinolone-resistantC. jejuni among isolates from the domesticallyacquired human cases and locally availableretail chicken products. The investigatorsattributed the 1996–1998 increase in resistant

domestic cases among humans to poultrytreated with fluoroquinolones, which wereapproved in the latter part of 1995 (Smith et al.,1999, 2000).

To assist in establishing the magnitude ofthe human health impact of fluoroquinoloneuse in animals, the US FDA contracted witha risk analyst to develop a risk model. Therisk assessment was intended to estimatethe extent of the risk to human health fromresistant Campylobacter pathogens attributedto the use of fluoroquinolones in chickens inthe USA. The risk assessment addressed thatportion of the risk that was quantifiable,which is that related to the consumption ofchicken. The unquantifiable portion, thatportion due to the spread of the pathogenfrom chicken to other foods through con-tamination during food preparation or fromsecondary spread to other animals, was notconsidered in the risk assessment.

An assumption made in the riskassessment was that the presence offluoroquinolone-resistant Campylobacter onchicken carcasses results from the use offluoroquinolones in chickens. This does notmean that every chicken carrying resistantCampylobacter had to have been treated with afluoroquinolone. Resistant organisms couldhave been acquired from a contaminatedenvironment due to fluoroquinolone drug usein a previous flock, through contact withother chickens during transportation to theslaughter plant and ante-mortem processing,or through contamination of other foods in thehome by raw chicken meat.

The number of Campylobacter culture-confirmed human cases in the US populationwas used to estimate the total burden ofcampylobacteriosis, which was 1.7 millioncases of campylobacteriosis with a 90% confi-dence distribution of 1.1–2.7 million cases for1999 (US Food and Drug Administration,2000).

The model also estimates the numberof fluoroquinolone-resistant Campylobactercases attributable to chickens. This estimateexcludes travellers to countries outside theUSA, those patients who were prescribed afluoroquinolone prior to stool culture, andthose patients who were unsure of the timing

11-Mar-03 13




of their treatment in relation to stool culture.For 1999, the mean number of the domesti-cally acquired fluoroquinolone-resistantCampylobacter cases attributable to chickensis approximately 190,000 (US Food andDrug Administration, 2000). For 1999, theestimated mean number of people infectedwith fluoroquinolone-resistant Campylobacterfrom consuming or handling chicken and whosubsequently received a fluoroquinolone astherapy is approximately 11,500 (US Food andDrug Administration, 2000). These peoplereceived less effective or ineffective therapyfor their infections, resulting in adverse healtheffects. The adverse health effects also have anegative impact on productivity in termsof lost working days and increased cost ofmedical care.


ACUTE Fluoroquinolones are considered tobe relatively safe antimicrobial drugs and,when administered at the therapeutic doses,toxic effects are mild and limited. The mostcommon complaints are nausea, vomiting,diarrhoea and other upper gastrointestinaldiscomfort, headache and dizziness (Prescottet al., 2000).

Fluoroquinolone use in dogs has beenassociated with canine toxic shock syndromecaused by Streptococcus canis.

CHRONIC At very high doses (50 mg kg−1),the fluoroquinolones have been associatedwith arthropathies in young experimentalanimals. Photosensitivity in humans occa-sionally has been associated with the useof fluoroquinolones. Renal toxicities rangingfrom acute renal failure to mild interstitialinflammation of the kidney tubular wallshave been reported in humans. Reportedly,most cases of renal toxicity have beenassociated with the use of fluoroquinolonesat doses greater than the therapeutic range.

Most adverse effects with fluoroquino-lones are associated with the administrationof higher doses. Because of cartilage erosion,fluoroquinolones are not recommended foruse in young, growing animals. It is hardto classify these events as acute or chronic,

as cartilage damage can occur as quicklyas 12–24 h after a single large dose of aquinolone.

Production Drugs

Clenbuterol


The nervous system is divided into the cen-tral nervous system (CNS) and the peripheralnervous system, which is divided furtherinto the somatic and the autonomic nervoussystems. The autonomic nervous systemis separated into two main divisions, thesympathetic and the parasympathetic. Theautonomic nervous system is an importantpart of the complex machinery by which thebody maintains its internal environmentconstant. These aspects are important andhave direct relevance to the clinical use andapplication of the drug clenbuterol.

Amine compounds that cause physiolog-ical responses similar to those evoked by theendogenous adrenergic mediators adrenalineand noradrenaline are called adrenergicdrugs. The pharmacological effects of theseamines are to mimic the sympathetic nervoussystem, thereby resulting in activation ofadrenergic receptors of effector cells. Mostadrenergic drugs affect both α and β receptors(Levine, 1983; Adams, 1995).

One of the most popular illegal drugsused in food-producing animals in the 1990swas the β-agonist compound clenbuterol. Theβ-agonists are a class of compounds that haveprofound pharmacological and physiologicaleffects. These compounds evoke specificresponses in a variety of tissues by bind-ing with affinity and high specificity toβ-adrenergic receptors. The β-adrenergicreceptors are classified as either β1 or β2,based on their pharmacological response.Most tissue possesses both receptors invarying proportions (Levine, 1983; Adams,1995). β-Agonists are members of a pharma-cological class of drugs that have demon-strated effectiveness as bronchodilators andgrowth-promoting and repartitioning agents


20-Feb-03 13



in many species, including cattle, sheep, pigs,poultry and man.


Clenbuterol is a member of a class ofdrugs known as β2-adrenergic agonists. It isapproved in the UK for therapeutic use incattle and horses. It is approved in the USAas a bronchodilator for treating chronicrespiratory disease (heaves) in horses. It hasbeen used illegally in Europe and the USA bysome livestock producers to increase carcassleanness. When used illegally, it is generallyused in the feed.

Animal drugs are regulated on the basisof the total residue present in an edible tissuebecause of the possibility that metabolitesmight also cause residues.

Studies were conducted to determinethe distribution and the concentration ofparent clenbuterol in tissues 48 h after dosing.A single oral dose of [14C]clenbuterol HCl(1.59 µCi mg−1, 3 mg kg−1 BW) was adminis-tered to Holstein calves weighing 74–96 kg.This concentration is greater than thatrequired for therapeutic purposes (0.8 µg kg−1

BW) or growth-promoting purposes (Smithand Paulson, 1997).

Residues of clenbuterol remaining inedible tissues of animals are composed ofparent clenbuterol, metabolites and perhapsbound residues. Human effects after illegaluse of clenbuterol are attributable to parentclenbuterol remaining in edible tissues. How-ever, the role of clenbuterol metabolitesremains unknown.

Studies were done to examine thepharmacokinetics of clenbuterol after effec-tive anabolic dosages of 5 µg kg−1 BW giventwice daily for 3 weeks (Heinrich et al., 1991)(note: the anabolic dosage in such studies isgenerally 5–10 times higher than that requiredfor therapeutic purposes). The analysis wasdone at 0, 3.5 and 14 days after withdrawal.The data revealed that clenbuterol concentra-tions in the lung dropped from a mean of 76 toless than 0.8 ng g−1 after 14 days, and the liverconcentrations decreased from 46 to 0.6 ng g−1.The highest levels were always found in theeye: 118 ng g−1, which dropped to 15 ng g−1

after 14 days (Heinrich et al., 1991). The peak

absorption of clenbuterol is rapid andmeasured as between 2 and 7 h.


Clenbuterol is one of a few residue-producing animal drugs that has been shownto cause an immediate health concern inconsumers. Residues of β-agonists in animaltissues used for food constitute a potentiallyserious human health risk. Many harmfuleffects to humans have been demonstratedfor these drugs due to their bronchodilatoreffects, muscle tremors and tachycardia.Meat from clenbuterol-treated animals hasbeen found to cause illness and even death inhumans. The acute toxic effects are clear andpredictable from the pharmacological modeof action of β-agonist compounds. It is feltthat residues of clenbuterol remaining in theedible tissues of animals are composed ofparent clenbuterol, metabolites and perhapsbound residues.

The main indications in human medicinefor clenbuterol are for treatment of chronicobstructive airway disease such as asthmaand chronic obstructive bronchitis. Like somebronchodilators, the drug expands the tiny airpassage in the lungs and lets air flow morefreely.

Clenbuterol has been implicated in sev-eral outbreaks of foodborne illness in Europe.In 1990, a Spanish epidemiologist reportedthat 135 people became ill after consumingbeef liver containing clenbuterol residues(Martineaz-Knavery, 1990; Pulce et al., 1991).Clinical symptoms consisted of muscle trem-ors, heart palpitations, nervousness, generalmyalgia, fever, nausea, chills and vomiting.These symptoms are of particular concernbecause toxicity can appear suddenly follow-ing the consumption of clenbuterol residues(Table 13.2).

A second episode of clenbuterol foodpoisoning was reported in France in 1991 andaffected 22 people in eight different familieswho consumed bovine liver. The patientssuffered tachycardia and muscle tremors for2 or 3 days. The infective dose was calculatedas 1–2 µg kg−1 day−1 (Salleras et al., 1995). In1994, calf liver was again implicated in a caseinvolving 16 people in Italy. The significant

20-Feb-03 13




aspect of these cases is that veal liver was themain source of exposure, although a recentresponse implicated veal muscle (Sporanoet al., 1998) (see Table 13.4 for a list ofclenbuterol cases).

In 1996, 62 persons in Italy were involvedin an outbreak and sought help at the emer-gency room in two hospitals. Tremor, tachy-cardia, palpitations and nervousness were thepredominant symptoms, lasting between 10and 48 h. The unique aspect of this episode offoodborne poisoning by clenbuterol is thatclinical symptoms appeared after consump-tion of non-organ meat. All of these peoplepurchased beef meat from a common sourceand reported consuming the meat from10–30 min and up to 2–3 h before symptomsappeared. It was concluded that therapeuticdosages (0.8 µg of clenbuterol kg−1 BW) wereingested by patients that ate 20 g of meat;however, a normal meal is 100 g of meat (fivetimes the therapeutic dose), 4.0 µg of drugkg−1 BW; it has been established that cardio-vascular signs appear at this level (Salleraset al., 1995; Sporano et al., 1998; Paige et al.,1999b).

ACUTE Clenbuterol has high β2-agonistactivity and relatively less β1-agonist activity.There are indications that acute heart rateincreases but usually for less than 2 min.This brief tachycardia may be mediatedreflexly due to transient β2 vasodilation andhypotension (Adams, 1995).

CHRONIC Chronic use can result in refrac-toriness due to down-regulation (i.e. reducednumbers) of β receptors and can cause β1 sideeffects at high doses.

Hormones

This section discusses the FDA’s healthrisk assessment procedures for residues ofhormones and the European CommunityDirectives prohibiting the use of anabolicagents for growth promotion.

There are six hormonal agents in theUSA approved for growth promotion pur-poses. The natural hormones supplied exo-genously for growth promotion purposesare 17β-oestradiol, testosterone and proges-terone. The approved synthetic hormonesare trenbolone acetate, melengestrol acetate(MGA) and zeranol; these compounds mimicthe actions of testosterone, progesterone and17β-oestradiol, respectively. With the excep-tion of MGA, the hormones are approvedeither alone or in combination as componentsof an ear implant. The sixth hormone, MGA,is approved only as a feed additive andalso functions to suppress oestrus in feed-lotheifers. Meat from animals treated withany one of the six agents for growth promo-tion purposes cannot be imported intothe European Community (EC) (Leighton,1999).


20-Feb-03 13

Year Case history

1990

1991

19941995

1996

1998

Spanish epidemiologist reported 135 people became ill consuming beef livercontaining 0.16–0.30 ppm residuesReport in France of 22 affected people in eight different families that consumedcalf liver. Contained 0.375–0.500 µg g−1

Reported illness in 127 people in Spain, calf liver implicatedClenbuterol (0.5 ppm) was isolated in beef fillet and rump steaks associated withan outbreak involving 16 people in Italy62 persons in Italy sought help at the emergency room in two hospitals.Consumption of non-organ meat. All cases purchased meat from a commonsource. Treatment doses = 0.8 mg (in 20 g of meat) 5 × treatment dose = 4.0 mg= cardiovascular signsFoodborne illness in China, where 9/14 reported illness from consuminglung soup from pig lung tissue. Reportedly pig given clenbuterol for weightgain

Table 13.4. Foodborne outbreaks associated with clenbuterol.



Whereas the FDA sets food safetystandards for animal drugs within the USA,international food safety standards are estab-lished by Codex. In 1987, JECFA conducted asafety assessment of five hormones used forgrowth promotion purposes in cattle. JECFAfunctions as a scientific advisory body of theCodex. The purpose of JECFA’s safety assess-ment was to establish a safe level of residuesof hormones (an acceptable daily intake, orADI) in animals treated with these agents.Their recommendations subsequently wereadopted by Codex, which is responsible forthe execution of the Joint FAO/WHO FoodStandards Programme (Leighton, 1999).

Naturally occurring hormones

Oestradiol, progesterone and testosteroneare naturally occurring hormones producedthroughout the lifetime of every man, womanand child, and are required for the properphysiological functioning and maturationof every mammal. Because these compoundsare naturally occurring and identical in manand in food-producing animals, the con-sumer is exposed throughout his/her lifetimeto rather large quantities of these hormonesthrough his/her own daily production andto much lesser quantities from food fromunmedicated animals. All products marketedin the USA for livestock are granulated aspellets and all are designed to deliver thehormones at a constant sustained rate wheninjected subcutaneously under the skin ofthe animal’s ear. Numerous scientific studieshave demonstrated that, when these drugsare used in accordance with good husbandrypractices, concentrations of the hormones inmeat remain within the normal physiologicalrange that has been established for untreatedanimals of the same age and sex. Because ofthe slow release of the hormone and becausethe half-life of these endogenous hormonesis extremely short (10 min), no pre-slaughterwithdrawal time is necessary to protectpublic health (Leighton, 1999). Therefore,consumers will not be at risk by eating meatfrom animals treated with these compoundssince the amount of added hormones isnegligible compared with the consumer’sown daily production rate.

Synthetic hormones

Unlike naturally occurring hormones, thereare no daily production rates from the syn-thetic compounds trenbolone acetate, MGAand zeranol. These compounds are not meta-bolized as quickly as the naturally occurringsteroids. Therefore, the FDA requiredextensive toxicological testing in animals todetermine a safe level in meat for these com-pounds. Furthermore, the FDA has requiredthat the manufacturers demonstrate that theamount of hormone left in the meat aftertreatment is below this safe level (Food andDrug Administration, 1966; Code of FederalRegulation, 1999).

As with antimicrobials and antibioticdrugs, the Center for Veterinary Medicine(CVM), in assessing the safety of any product,reviews information on the specific productin question under conditions of use. Aspreviously stated, an assessment of risk isincorporated into the approval process and apost-approval monitoring programme alongwith risk management tools.

The Use of Epidemiological Methodsin Food Safety

Epidemiological concepts and methods canbe used easily with other scientific disciplinesto study food safety. Diseases, residuesand chemical hazards in populations arenot randomly distributed. The methods ofepidemiology are used to describe how age,time trends, geographical trends, husbandrypractices, lack of prudent drug use and othervariables affect the distribution of foodborneillness, residues, resistance and other foodsafety hazards. Epidemiology can be usedto investigate many different types ofhealth outcomes where the causes are eitherunknown or poorly understood. The specificaims and objectives are to describe, explainand predict events, for the purpose ofimplementing some type of intervention.

Surveillance is the systematic collectionof data pertaining to the occurrence of specificevents, the analysis and interpretation of thesedata and the dissemination to those who need

20-Feb-03 13




to know (Lwanga, 1978; Friis and Sellers,1996; Gordis, 2000). Surveillance systemsprovide the means for collecting, testingand monitoring trends in the prevalence offoodborne pathogens and the antimicrobialresistance profiles of these bacteria. Well-designed surveillance systems for anti-microbial resistance can identify outbreaksof foodborne illness and epidemics ofmulti-drug-resistant pathogens in animalsthat have the potential to become foodbornepathogens.

Risk has been termed the probability ofinjury, disease or death under specific circum-stances. Risk may be expressed in quantitativeor qualitative terms. In the former, it takesvalues from 0 to 1 (probability that harm willnot occur to probability that it will). In thelatter, risk can be described as high, low ortrivial.

Hazards are evaluated in terms ofacceptable levels of risk. Safety in its commonusage means without risk; thus, the safety ofchemical/drug residues in food is a conditionof exposure under which there is relativecertainty that no harm will result in theexposed population. This premise is basedon the fact that inherent in the drug approvalprocess are the elements of risk assessment.

In determining the impact of any chemi-cal or drug on human health, two distinct ele-ments have been identified, risk assessmentand risk management. These two aspects willbe discussed as they pertain to food safety.

Assessment of hazard and risk

In today’s environment, the public, media,politicians and our global partners aredemanding that our food supplies are freeof risk. While scientists have disagreementsabout the nature and magnitude of food-related risk, there is general agreement onwhat constitutes food-related risks and theirrelative importance.

Pre-market testing requirements andtolerance setting by regulatory authoritiesand strict monitoring of residue levels havehelped to ensure that risk from residues infood remains low. Tolerance setting involves

utilizing risk assessment and the conceptsof hazard identification, dose–responseassessment, exposure assessment and riskcharacterization in the approval processto ensure food safety for those drugsapproved for food-producing animals (seeTable 13.1).

A tolerance represents the maximal levelor concentration in or on animal feed ingredi-ents or animal tissues at the time of slaughter(e.g. the tolerance for penicillin is 0.05 ppmmg−1 kg−1 (Freidlander et al., 1999)). A viola-tive residue is the occurrence of a drugor chemical residue above the tolerance levelfound in edible tissue, fat, kidney, liver,muscle, meat byproducts or skin of a food-producing animal.

The human food safety evaluation of anew animal drug involves an independentevaluation of its residue chemistry character-istics and its toxicology. Information gener-ated from the toxicology review is usedto establish the safe concentrations for totalresidues in the edible tissues of food-producing species. Information obtainedfrom the residue chemistry review is usedto establish the tolerance and set withdrawaltimes for the new animal drug. The pri-mary objective of these activities is to ensurethat potentially harmful residues of thedrug will not occur in food derived fromtreated animals (Freidlander et al., 1999) (seeTable 13.1).

The initial assessment involves hazardidentification, the examination of the struc-ture of the compound for potential biologicalactivity or carcinogenicity based on availableinformation about the veterinary compoundsor its chemical class.

The first step in assigning a safe concen-tration for a residue is to determine the ADI.The ADI is calculated using data generatedfrom the toxicology studies. To calculate theADI, a no observable effect level (NOEL) isselected from one of the oral toxicity studies.Depending on which toxicity study is used, asafety factor of 100 or 1000 is applied to thecalculation.

ADI = NOEL/safety factor

A safe concentration for total residues inmeat is then calculated:


20-Feb-03 13



Safe concentrationADI ( g kg day kg

g consu= ×− −µ 1 1 60)

med day −1

In the USA, the safe concentration ofresidues is based on the assumption thatedible muscle and organ tissues comprise500 g of a 1500 g solid food diet and that organmeats and fat are consumed in lesser quanti-ties than muscle. The 500 g of edible muscleand organ tissue consumption values aregiven as follows: muscle, 300 g consumed;liver, 100 g consumed; kidney, 50 g con-sumed; and fat, 50 g consumed. Milk andegg products are regulated as independentcommodities as they are not considered ascomponents of the 500 g of muscle and organtissues (Freidlander et al., 1999).

General residue chemistry requirementsinclude a total residue depletion and metabo-lism study, a comparative metabolism studybetween the target species and the laboratorytoxicological species. If results indicate acomparable metabolism in both species, thenthe human population would be exposed tothe same residues when consuming meats,milk or eggs derived from the treated targetspecies. Any metabolite identified as beingof toxicological concern will undergo furthertoxicological examination. Further chemistryrequirements include the development of avalidation or determinative method, a confir-matory analytical method for identification,a cold residue depletion study to set thetolerance for the marker residue in the targettissue, and a marker residue depletion study.

The drug sponsors of veterinary com-pounds are required further to measure thedepletion of total residues of toxicologicalconcern from the edible tissues until theirconcentration is at or below the safe concen-tration. The tissue or organ that requires thelongest time to deplete to the safe concen-tration is referred to as the target tissue. Amarker residue is selected from the targettissue; this is the parent compound or one ofthe metabolites. The marker residue will existin a known relationship to the total residue inthat tissue (e.g. 60% of total, 85% of total, etc.)(Freidlander et al., 1999). The concentrationof the marker residue in the target tissuewhen the total residue has depleted to thesafe concentration is called the tolerance.

The withdrawal period is the amount oftime it takes for the marker residue to depleteto the tolerance in the target tissue.

Public health surveillance and its role

Database systems are diverse and unique incapturing large amounts of data that are reli-able and can be analysed to allow the scientistultimately to generate information useful inhypothesis testing and in facilitating epide-miological or laboratory research. Data cap-tured in these databases can serve a functionin public health surveillance, which involvesthe collection, analysis and interpretation ofdata on the frequency of occurrence and dis-tribution of health events in the population.

One of the major steps in surveillanceis the timely dissemination of information toothers in the public health systems that needto know. Four such databases in the USA andone in Denmark, and their relevance to foodsafety initiatives, are discussed in this section.

A final link in the surveillance chain is theapplication of these data from these systemsto prevention and control. In a larger sense,surveillance is concerned with the ongoingsystematic collection, analysis and interpreta-tion of health data essential to the planning,implementation and evaluation of publichealth practice.

FoodNet

In the early 1990s, the National Academyof Science Institute of Medicine publisheda report emphasizing the ongoing threat ofemerging infectious diseases. The CDC deve-loped a strategy to respond to this threat. Themain feature of this strategy was to establishthe Emerging Infections Program (EIP) inseven sites across the USA (California, Con-necticut, Georgia, New York, Maryland, Min-nesota and Oregon). The goals of the EIP net-work are to improve national surveillance fornew and emerging diseases, conduct appliedepidemiological and laboratory research,develop prevention and control measures,and strengthen the national public healthinfrastructure (Institute of Medicine, 1992).

20-Feb-03 13




The CDC/ US Department of Agriculture(USDA)/FDA Foodborne Disease ActiveSurveillance Network (FoodNet) is thefoodborne disease component of CDC’s EIP. Itis a collaborative project of the CDC, FDA andUSDA with the aim of collecting more preciseinformation on the incidence of foodbornedisease in the USA. FoodNet works withstate and local health departments to provideactive surveillance for foodborne diseases andrelated epidemiological studies designed tohelp public health officials better understandthe epidemiology of foodborne diseases in theUSA (Foodborne Disease Active SurveillanceNetwork, 1998–1999).

NARMS

There is increasing concern that the use ofantibiotics in food animals may lead to devel-opment of resistant strains of bacteria thatcould cause illness in people. The increase inresistance has led to re-examining the rolethat antimicrobial drugs used in food-producing animals play in the emergence ofantimicrobial drug-resistant bacteria.

For public health reasons, there is aneed to monitor carefully the development ofresistance or changes in susceptibility toantibiotics. In order to accomplish this task,the USA developed a system modelled afterthe Danish Integrated Antimicrobial Resis-tance Monitoring and Research Programme(DANMAP) to identify when foodbornebacteria that cause disease in humans beginto develop resistance to antimicrobials usedin food animals. This programme is calledthe National Antimicrobial Resistance Moni-toring System–Enteric Bacteria (NARMS–EB)(Tollefson et al., 1998). The programme alertspublic health agencies to changes in suscepti-bilities of enteric organisms (intestinal) fromboth animal and human sources to severalantimicrobials. The food animal specimensare gathered from healthy farm animals,animal clinical isolates and carcasses offood animals at slaughter. The human originsamples are submitted by 17 state and localDepartments of Health and are tested atthe National Center for Infectious Diseases,(CDC), in Atlanta, Georgia. NARMS tests non-typhoid Salmonella, Shigella (human isolates

only), Escherichia coli, Campylobacter, entero-cocci and Salmonella typhi (human isolates only)for susceptibility to a variety of different anti-biotics. Data can be compared from previousyears to look for evidence of changes in resis-tance of the organisms to these drugs. Data areavailable on the FDA Center for VeterinaryMedicine home page at www.fda.gov/cvm.

These data will prove useful for publichealth officials, regulatory agencies, responsi-ble animal producers, drug manufacturersand veterinarians in developing mechanismsfor the prudent use of antibiotics and for theprotection of public health by ensuring thatsignificant antimicrobial therapies are notlost due to the use of antimicrobials infood-producing animals.

RVIS/TRIMS

Numerous surveys conducted during the1980s indicated that the consumer was con-cerned about the potential risk of chemicalresidues in foods of animal origin. It wasthought that residual chemicals in the edibletissues of food animals over the establishedtolerance level posed greater risk than otherhazards associated with foods.

In response to consumer concerns in theUSA, the USDA, the Food Safety InspectionService (FSIS) and the FDA created the Resi-due Violation Information System (RVIS) toshare pertinent data for regulatory enforce-ment on a regular and open basis. For thebenefit of others associated with chemicalresidue control in food animals and animalproducts, helpful features of this system areelaborated here. The RVIS has proven to be anexcellent tool for supporting residue controlmeasures in meat and poultry because itallows exchange of information amongparticipating agencies regarding regulatoryenforcement.

The RVIS database is a nationwide,interagency computer information systemdesigned to share pertinent data for regula-tory enforcement on an open and regular basis(Paige et al., 1999a). The system operates 24 ha day to provide information on residue viola-tions in livestock and poultry slaughtered inthe USA. It also includes residue testing datafor processed eggs.


20-Feb-03 13



The Tissue Residue Information Manage-ment System (TRIMS) was developed in 1988,and is now linked with the RVIS database. Thedescriptive data collected by TRIMS can beused to generate hypotheses relative to identi-fying risk factors for drug residues. Data fromthis database have been used to write articlesthat have appeared in veterinary journals andagricultural trade journals (Paige et al., 1999a;Sundlof et al., 2000). Information has proveduseful in determining whether patterns ofviolation can be related to specific slaughterclass pairs and pharmacological drug classesand, if so, what preventive measures can beimplemented.

DANMAP

One such programme that has gained inter-national recognition in this area is DANMAP,which was set up in 1995 (Danish Inte-grated Antimicrobial Resistance Monitoringand Research Programme, 1997). Specifically,DANMAP monitors antimicrobial resistancein bacteria from food animals, foods andhumans and it has been designed to providea basis for comparison of the occurrenceof resistance in these three reservoirs. Inaddition, DANMAP reports data on theconsumption of antimicrobials in animalsand in humans, and on associations betweenthe use of antimicrobials and trends inantimicrobial resistance.

Consumption data include the use ofantimicrobials in food animals, includingtherapeutic use, growth promotion use anduse as a coccidiostat. For humans, consump-tion consists of a comparison of consumptionwithin Denmark and an analysis of the trendsover time. The resistance data are from threesources matched with the three reservoirs.The pathogens consist of zoonotic bacteria,indicator bacteria (E. coli and Enterococcusfaecium/Enterococcus faecalis) and bacteriaisolated from diagnostic laboratories.

Conclusion

Regulatory agencies are engaging in riskassessment as a mechanism for makingpolicy decisions on food safety. Since

antimicrobial resistance and residues repre-sent a global problem, we must engage ininternational harmonization as the emer-gence of resistance to antibiotics has gainedglobal prominence. We have tried to empha-size that to confront food safety issues ofresistance and residues requires a scientificand public health strategy. As indicated, suchsafety assessment criteria are inherent in theveterinary drug approval process.

The presence of a strong science-basedapproach by regulatory agencies will do muchto assure our consumers both domesticallyand internationally that they are receiving asafe animal product. We must continue toencourage veterinarians to engage in prudentdrug usage, and to improve farm manage-ment practices, which are viewed as promis-ing ways of preventing foodborne illness. Inaddition, there is also a need to continue toimprove surveillance and applied research toelucidate the mechanism of new control andpreventive methods.

References

Acar, J.F. and Goldstein, F.W. (1997) Trends in bacte-rial resistance to fluoroquinolones. ClinicalInfectious Diseases 24 (Supplement 11), 67–73.

Adams, R.H. (1995) Adrenergic agonist and antago-nist. In: Adams, R.H. (ed.) Veterinary Pharma-cology and Therapeutics, 7th edn. Iowa StateUniversity Press, Ames, Iowa, pp. 87–113.

Angulo, F.J., Tauxe, R.V. and Cohen, M.L. (1998) Theorigins and consequences of antimicrobial-resistant nontyphoidal Salmonella: implica-tions for use of fluoroquinolones in foodanimals. In: Use of Quinolones in Food Animalsand Potential Impact on Human Health. Reportof a WHO meeting, WHO/EMC/ZDI/98.12,Geneva, Switzerland, 2–5 June, pp. 205–219.

Anon. (1995) Report of the American Society forMicrobiology Task Force on Antibiotic Resistance.The American Society for Microbiology, Publicand Scientific Affairs Board, Washington, DC,16 March 1995.

Baquero, F., Martinez-Beltran, J. and Loza, E. (1991)A review of antibiotic resistance patternsof Streptococcus pneumoniae in Europe.Journal of Antimicrobial Chemotherapy Supple-ment C, 31–38.

Bates, J., Jordens, J. and Griffiths, D. (1994)Farm animals as a putative reservoir for

20-Feb-03 13




vancomycin-resistant enterococcal infection inman. Journal of Antimicrobial Chemotherapy 34,507–516.

Benet, L.Z., Kroetz, D.I. and Sheiner, L.B. (1996)Phamacokinetics: the dynamics of drugabsorption distribution, and elimination.In: Hardman, J.G., Goodman Gilman, A. andLimbird, L.E. (eds) Goodman and Gilman’s ThePharmacological Basis of Therapeutics, 9th edn.McGraw Hill, St Louis, Missouri, pp. 3–28.

Booth, N.H. (1977) Drugs and chemical residues inthe edible tissues of animals. In: Jones, L.M.,Booth, N.H. and McDonald, L.E. (eds) Veteri-nary Pharmacology and Therapeutics, 4th edn.Iowa State University Press, Ames, Iowa, pp.1299–1338.

Code of Federal Regulation (CFR) Title 21, Parts 522,556, and 558 (1999) Approved Hormones. Super-intendent of Documents, US GovernmentPrinting Office, Washington, DC.

Craig, W.A. (1998) Pharmacokinetic/pharmacody-namic parameters: rationale for antimicrobialdosing of mice and men. Clinical InfectiousDiseases 26, 1–12.

Danish Integrated Antimicrobial Resistance Moni-toring and Research Programme (DANMAP)(1997) DANMAP 1997. Danish Ministry ofHealth and the Danish Ministry of Food,Agriculture of Fisheries, Copenhagen.

Davis, L.E. (1995) Veterinary pharmacology: anintroduction to the discipline. In: Adams, H.R.(ed.) Veterinary Pharmacology and Therapeutics,7th edn. Iowa State University Press, Ames,Iowa, pp. 3–9.

Drlica, K. and Zhao, X. (1997) DNA gyrase, topiso-merase IV, and the 4-quinolones. Microbiologyand Molecular Biology Review 61, 377–392.

Endtz, H.P., Rvijs, G.J., van Klingeren, B., Jansen,W.H., van der Reyden, T. and Mouton, R.P.(1991) Quinolone resistance in Campylobacterisolated from man and poultry following theintroduction of fluoroquinolones in veterinarymedicine. Journal of Antimicrobial Chemotherapy27, 199–208.

Food and Drug Administration, Center for Veteri-nary Medicine (1966) The Use of Hormonesfor Growth Promotion in Food-producing Ani-mals. Information for Consumers, May. USGovernment Printing Office, Washington, DC.

Foodborne Disease Active Surveillance Network,Population Survey Atlas of Exposures(1998–1999) FoodNet. DHHS/CDC/NCID/Division of Bacterial and Mycotic Diseases,Foodborne, and Diarrheal Diseases Branch,Atlanta, Georgia.

Freidlander, L., Brynes, S.D. and Fernandez, A.H.(1999) The human safety evaluation of new

animal drugs. Veterinary Clinics of NorthAmerica: Food Animal Practice 15, 1–11.

Friis, R.H. and Sellers, T.A. (1996) Epidemiologyfor Public Practice. Aspen Publications,Gaithersburg, Maryland.

Glynn, M.K., Bopp, C., Dewitt, W., Dabney, P.,Mokhtar, M. and Angulo, F.J. (1998)Emergence of multidrug-resistant Salmonellaenterica serotype typhimurium DT104 infectionsin the United States. New England Journal ofMedicine 338, 1333–1338.

Goodman Gilman, A., Rall, T.W., Nies, A.S.and Taylor P. (eds) (1991) Goodman andGilman’s The Pharmacological Basis of Thera-peutics, 8th edn. Pergamon Press, New York,pp. 1117–1125.

Gootz, T.D. and Bright, K.E. (1996) Fluoroquinoloneantibacterials: SAR, mechanism of action,resistance, and clinical aspects. MedicinalResearch Reviews 16, 433–486.

Gordis, L. (2000) Epidemiology, 2nd edn. W.B.Saunders Company, Philadelphia.

Harris, N., Weiss, N. and Nolan, C. (1986) Therole of poultry and meats in the etiologyof Campylobacter jejuni/coli enteritis. AmericanJournal of Public Health 76, 407–411.

Heinrich, H., Meyers, D. and Rinke, L.M. (1991) ThePharmacokinetics and Residues of Clenbuterol inVeal Calves. Technischee Universitat Munchen,Munich, Germany.

Holmberg, S.D., Osterholm, M.T., Senger, K.A. andCohen, M.L. (1984) Drug-resistant Salmonellafrom animals fed antimicrobials. New EnglandJournal of Medicine 311, 617–622.

Hooper, D.C. (2000) New uses for new and oldquinolones and the challenge of resistance.Clinical Infections Diseases 30, 243–254.

Hooper, D.C. and Wolfson, J.S. (1993) Mechanismsof quinolone action and bacteria killing. In:Hooper, D.C. and Wolfson, J.S. (eds) QuinoloneAntimicrobial Agents, 2nd edn. AmericanSociety for Microbiology, Washington, DC,pp. 53–75.

Institute of Medicine (1992) Emerging Infections:Microbial Threats to Health in the United States.National Academy Press, Washington, DC.

Jacobs-Reitsma, W.F., Kan, C.A. and Boulder, N.M.(1994) The induction of quinolone resistancein Campylobacter in broilers by quinolonetreatment. Letters in Applied Microbiology19, 228–231.

Kidd, A.R.M. (1994) The Potential Risk of Effectsof Antimicrobial Residues on Human Gastro-intestinal Microflora. Fédération Européen de laSanté Animale (FEDESA), Brussels, Belgium.

Leighton, J.K. (1999) Center for Veterinary Medi-cine’s perspective on the beef hormone case. In:


20-Feb-03 13



Veterinary Clinics of North America: ChemicalFoodBorne Hazards and Their Control, Vol. 15.W.B. Saunders and Company, Philadelphia,pp. 167–195.

Levine, R.R. (1983) Pharmacology: Drug Actions andReactions, 3rd edn. Little Brown and Company,Boston, Massachusetts.

Littlefield, N.A., Gaylor, D.W., Blackwell, B.N.and Allen, R.R. (1989) Chronic toxicity/carcinogenicity studies of sulphamethazinein B6C3F1 mice. Food Chemical Toxicology 27,455–463.

Littlefield, N.A., Sheldon, W.G., Allen, R. andGaylor, D.W. (1990) Chronic toxicity/carcino-genicity studies of sulphamethazine in Fischer344/N rats: two generation exposure. FoodChemical Toxicology 28, 1157–1167.

Lwanga, S. (1978) Statistical principles of monitor-ing and surveillance in public health. Bulletin ofthe World Health Organization 56, 713–722.

Martineaz-Knavery, J.F. (1990) Food poisoningrelated to consumption of illicit β-agonist inliver (letter). Lancet 336, 1311.

McGowan, J.E. (1983) Antimicrobial resistance inhospital organisms and its relation to antibioticuse. Reviews of Infectious Diseases 5, 1033–1048.

Moellering, R.C. (1993) Quinolone antimicrobialagents: overview and conclusions. In: Hooper,D.C. and Wolfson, J.S. (eds) Quinolone Anti-microbial Agents, 2nd edn. American Society forMicrobiology, Washington, DC, pp. 527–535.

Paige, J.C., Chaudry, M.H. and Pell, F.M. (1999a)Federal surveillance of veterinary drugs andchemical residues with recent data. VeterinaryClinics of North America: Food Animal Practice 15,45–61.

Paige, J.C., Tollefson, L. and Miller, M.A. (1999b)Health implications of residues of veterinarydrugs and chemicals in animal tissues. Veteri-nary Clinics of North America: Food AnimalPractice 5, 31–43.

Perez-Tallero, E., Otero, F., Lopez-Lopategui, C.Montes, M., Garcia-Arenzana, J.M. andGomariz, M. (1997) High prevalence ofciprofloxacin resistant Campylobacter jejuni/coliin Spain. In: Program and Abstracts of the 37thInterscience Conference on Antimicrobial Agentsand Chemotherapy. American Society ofMicrobiology, Washington, DC, AbstractC–21, p. 49.

Petruccelli, B.P., Murphy, G.S., Sanchez, J.L., Walz,S., DeFraites, R., Gelnett, J., Haberberger, R.L.,Echeverria, P. and Taylor, D.N. (1992)Treatment of traveler’s diarrhea with cipro-floxacin and loperamide. Journal of InfectiousDiseases 165, 557–560.

Piddock, L.J.V. (1995) Quinolone resistance andCampylobacter spp. Journal of AntimicrobialChemotherapy 36, 891–898.

Piddock, L.J.V. (1996) Does the use of antimicrobialagents in veterinary medicine and animalhusbandry select for antibiotic resistantbacteria that infect man and compromiseantimicrobial chemotherapy? Journal ofAntimicrobial Chemotherapy 38, 1–3.

Piddock, L.J.V. (1998) Role of mutation in thegyrA and parC genes of nalidixic acid-resistantSalmonella serotypes isolated from animals inthe United Kingdom. Journal of AntimicrobialChemotherapy 41, 635–641.

Piddock, L.J.V. (1999) Mechanisms of fluoro-quinolone resistance: an update 1994–1998.Drugs 58 (Supplement 2), 17–18.

Prescott, J.F. and Baggot, J.D. (1993) Tetracycline.In: Prescott, J.F. and Baggot, J.D. (eds)Antimicrobial Therapy in Veterinary Medicine,2nd edn. Iowa State University Press, Ames,Iowa, pp. 215–228.

Prescott, J.F., Baggot, J.D. and Walker, R.D. (eds)(2000) Antimicrobial Therapy in VeterinaryMedicine, 3rd edn. Iowa State UniversityPress, Ames, Iowa.

Pulce, C., Lamaison, O., Keck, G., Bostvironnois, C.,Nicholas, J. and Descotes, J. (1991) Collectivehuman food poisonings by clenbuterolresidues in veal livers. Veterinary and HumanToxicology 33, 480–481.

Riviere, J.E. (1992) Practical aspects of the pharma-cologic of antimicrobial drug residues in foodanimals. Agri-Practice 13, 11–16.

Roestel, B. (1998) Chlortetracycline, Oxytetracyclineand Tetracycline. Agence National du Médica-ment Vétérinaire, CNEVA, Faugères, France.

Rossiter, S., Joyce, K., Ray, M., Benson, J.,Mackinson, C., Gregg, C., Sullivan, M.,Vought, K., Leano, F., Besser, J., Marano, N.and Angulo, F. (2000) High prevalenceof antimicrobial-resistant, including fluoro-quinolone-resistant, Campylobacter on chickenin U.S. grocery stores. In: 100th Annual Meetingof the American Society for Microbiology, LosAngeles, 24 May, poster C296.

Salleras, L., Domiquez, A., Mata, E., Taberner, J.L.,Moro, I. and Salva, P. (1995) Epidemiologicstudy of an outbreak of clenbuterol poisoningin Catalonia, Spain. Public Health Reports 110,339–342.

Smith, D.J. and Paulson, G.D. (1997) Distribution,elimination, and residues of [14C]clenbuterolHCl in Holstein calves, ARS BiosciencesResearch Lab, Fargo, ND. Journal of AnimalScience 75, 454–461.

20-Feb-03 13




Smith, K., Besser, J., Hedberg, C., Leanoo, F.T.,Bender, J.B., Wicklund, J.H., Johnson, B.P.,Moore, K.A. and Osterholm, M. (1999)The epidemiology of quinolone-resistantCampylobacter infections in Minnesota,1992–1998. New England Journal of Medicine 340,1525–1532.

Smith, K., Bender, J. and Osterholm, M. (2000)Antimicrobial resistance in animals and rele-vance to human infections. In: Namchamkin, I.and Blaser, M. (eds) Campylobacter, 2nd edn.ASM Press, Washington, DC, pp. 483–495.

Spika, J.S., Waterman, S.H., Soo Hoo, G.W., St Louis,M.E., Pacer, R.E., James, S.M., Bissett, M.L.,Mayer, L.W., Chiu, J.Y., Hall, B., Greene, K.,Potter, M.E., Cohen, M.L. and Blake, P.A. (1987)Chloramphenicol-resistant Salmonella newporttraced through hamburger to dairy farms. NewEngland Journal of Medicine 316, 565–570.

Spoo, J.W. and Riviere, J.E. (1995) Sulfonamides. In:Adams, H.R. (ed.) Veterinary Pharmacology andTherapeutics, 7th edn. Iowa State UniversityPress, Ames, Iowa, pp. 753–773.

Sporano, V., Grasso, L., Esposito, M., Oliviero, G.,Brambilla, G. and Loizzo, A. (1998) Clenbu-terol residues in non-liver containing meat as acause of collective food poisoning. Veterinaryand Human Toxicology 40, 141–143.

Sundlof, S.F. (1994) Human risks associated withdrug residues in animal derived foods. Journalof Agromedicine 1, 5–22.

Sundlof, S.F., Fernandez, A.H. and Paige, J.C. (2000)Antimicrobial drug residues in food-producing animals. In: Prescott, J.F., Baggot,J.D. and Walker, R. (eds) Antimicrobial Therapyin Veterinary Medicine, 3rd edn. Iowa StateUniversity Press, Ames, Iowa, pp. 744–759.

Tacket, C.O., Dominguez, L.B., Fisher, H.J.and Cohen, M.L. (1985) An outbreak ofmultiple-drug-resistant Salmonella enteritis

from raw milk. Journal of the American MedicalAssociation 253, 2058–2060.

Tauxe, R.V. (1992) Epidemiology of Campylobacterjejuni infections in the United States and otherindustrial nations. In: Nachamkin, I., Blaser,M.J. and Tompkins, L.S. (eds) Campylobacterjejuni: Current and Future Trends. AmericanSociety for Microbiology, Washington, DC,pp. 9–12.

Threlfall, E., Frost, J., Ward, L. and Rowe, B. (1996)Increasing spectrum of resistance in multiresistant Salmonella typhimurium. Lancet 347,1053–1054.

Tollefson, L., Angulo, F.J. and Fedorka-Cray, P.J.(1998) National surveillance for antibioticresistance in zoonotic enteric pathogens.Veterinary Clinics of North America: Food AnimalPractice 14, 141–150.

US Food and Drug Administration (2000) TheHuman Health Impact from Fluoroquinolone-resistant Campylobacter. FDA Center forVeterinary Medicine, Rockville, Maryland.

Vaden, S.L., Riviere, J.E. and Penicilloina, N.D.(1995) Related β-lactam antibiotics. In: Adams,H.R. (ed.) Veterinary Pharmacology and Thera-peutics, 7th edn. Iowa State University Press,Ames, Iowa, pp. 774–783.

Velazquez, J.B., Jimenez, A., Chomon, B. and Villa,T.G. (1995) Incidence and transmission ofantibiotic resistance in Campylobacter jejuniand Campylobacter coli. Journal of AntimicrobialChemotherapy 35, 173–178.

World Health Organization (1997) The MedicalImpact of the Use of Antimicrobials in Food Ani-mals. Report of a WHO meeting, WHO/EMC/ZOO/97.4, Berlin.

World Health Organization (1998) Use of Quinolonesin Food Animals and Potential Impact on HumanHealth. Report of a WHO meeting, WHO/EMC/ZDI/98.10, Geneva.


20-Feb-03 13



14 Prion Diseases: Meat Safety and HumanHealth Implications

N. Hunter*Neuropathogenesis Unit, Institute for Animal Health, West Mains Road,

Edinburgh EH9 3JF, UK

Introduction

Prion diseases are rare in human beings;however, they are now often the subjectof front page newspaper headlines and havehad a profound effect on international trade,the food industry, pharmaceuticals andhygiene regulations in hospitals. This groupof diseases, also known as the transmissiblespongiform encephalopathies (TSEs), includesscrapie in sheep and goats, chronic wastingdisease (CWD) of deer and Creutzfeldt–Jakobdisease (CJD) and its bovine spongiformencephalopathy (BSE)-related version (variantCJD (vCJD)) in humans (Table 14.1). There isaround one new case of CJD per million ofthe world population per year, but vCJD hasbeen reported in just over 100 cases to date,the vast majority in the UK. TSEs are trans-missible between individuals, either by directinjection – deliberate in the case of laboratoryanimals or accidental in the case of iatrogenicinfection of humans – or by as yet unknownroutes in the natural infections in animals. Itis thought that the vCJD agent is ingestedwith food (possibly meat); however, it is notat all clear how the ‘normal’ or sporadic formof CJD is acquired. TSEs have no cure and arecharacterized by the presence of an abnormalform (PrPSC) of a membrane protein (PrPC) in

infected organs of the body. PrPSC is consid-ered by many to be itself the infectious TSEagent and is known as the prion protein.Other researchers remain unconvinced ofthis hypothesis, or feel it remains unproven;however, at the very least, PrPSC is a reliablemarker for the presence of TSE infectivity.

PrP Protein and the Nature ofthe TSE Agent

The normal form of the PrP protein (PrPC) isattached to neuronal cell surfaces via ananchor made up of sugar molecules. Themain differences between the two isoforms ofPrP are listed in Table 14.2. The function ofPrPC is not known; however, there are severalintriguing hints that it may have a role in theactivation of T cells in the immune system(Mabbott et al., 1997), in the electrophysiol-ogy of nerve cells (Collinge et al., 1994;Manson et al., 1995) and in the maintenanceof sleep continuity (Tobler et al., 1997). Theaggregated form of PrP (PrPSC) is partiallyresistant to proteases and is very closely asso-ciated with infectivity. In one of the majorhypotheses on the nature of the TSE agent,PrPSC is itself the infecting entity agent or


20-Feb-03 14




prion. In this ‘protein only’ theory, PrPSC,arising from an infection or from a mutantPrP gene, acts as a catalyst in the conversionof endogenous PrPC into yet more PrPSC, thuseither destroying the normal function of theprotein or poisoning the nerve cells andresulting in degenerative disease (Prusineret al., 1990). Natural scrapie in sheep tends tobe familial in appearance and has been saidto result from a recessive gene, the proteinproduct of which causes disease (Parry,1984); however, as described later, this hypo-thesis has now been discounted. Around 15%of the human TSEs also show a familial pat-tern with a dominant pattern of inheritance(Brown et al., 1987), and are considered to begenetic diseases (resulting directly from a

mutation). However, genetic disease or not,once a TSE does occur, it is often transmis-sible experimentally to laboratory animals,not the case with simple genetic diseases,for example thalassaemia (Rund et al.,1991), and so additional explanations fordisease spreading mechanisms are required.

Because of the heretical nature of the ‘pro-tein only’ hypothesis, in that it invokes aninfecting agent carrying genetic informationembedded in protein and not in DNA or RNA,the prion theory (Prusiner, 1982) was difficultfor many to accept (Chesebro, 1998). TSEshave been shown to exist in many differentstrains, and so one of the main opposingideas involves PrPSC protein as part of a two-component structure (the virino), including a

20-Feb-03 14

316 N. Hunter

Human diseases Acronym Types Aetiology

Creutzfeldt–Jakob disease

Gerstmann–Straussler–Scheinker syndromeFatal familial insomniaKuruVariant Creutzfeldt–Jakobdisease

CJD

GSS

FFI–vCJD

SporadicFamilialIatrogenic

Familial

FamilialAcquiredAcquired

UnknownLinked to PrP gene mutationsContamination during surgery or of growthhormoneLinked to PrP gene mutation, e.g. codon 102

Linked to PrP gene mutation, e.g. codon 178Associated with funeral rites?Diet, related to BSE

Animal diseases Acronym Types Aetiology

Scrapie

Chronic wasting disease

Transmissible mink encephalopathyBovine spongiform encaphalopathyFeline spongiform encephalopathySpongiform encephalopathies

Sheep, goats

Deer

Farmed minkCattleCatsZoo animals

–

CWD

TMEBSEFSESE

Natural

Natural

AcquiredAcquiredAcquiredAcquired

Infection, unknownmode of transmissionInfection, unknownmode of transmissionContaminated feedContaminated feedstuffDiet, related to BSEDiet, related to BSE

Table 14.1. Transmissible spongiform encephalopathies (prion diseases).

Characteristic PrPC PrPSC

Action of proteinase K enzyme (PK)Molecular weightMolecular weight after PK treatmentDetergentPresent in normal brain?Present in TSE brain?Infectivity

Degraded33–35 kDaDegradedSolubleYesYesDoes not co-purify

Partially resistant33–35 kDa27–30 kDaInsolubleNoYesDoes co-purify

Table 14.2. Differences between PrPC and PrPSC.



nucleic acid which specifies strain informa-tion (Farquhar et al., 1998). The virino hypoth-esis is still tenable as the prion hypothesisremains to be completely proven.

Strains of TSEs

The relatively conventional concept ofstrains, biologically similar to strains ofviruses, has also been applied to the manyidentifiable types of TSEs. For example,around 20 strains of scrapie have been iso-lated from natural sheep scrapie followinginjection into laboratory rodents, where theyproduce precise incubation periods andbrain region pathology in affected animals(Dickinson and Meikle, 1971; Bruce et al.,1991). TSE strain characteristics have beenused most recently to great effect in the iden-tification of BSE-like infections of mammalsother than cattle (Bruce et al., 1994), particu-larly vCJD in humans (Bruce et al., 1997).vCJD transmits very easily to mice, produc-ing a BSE-like pattern of incubation periodsand pathology, and is clearly different fromsporadic CJD, which does not transmit atall well to mice (Bruce et al., 1997). Distinctstrains of natural scrapie may also exist insheep (Hunter et al., 1997b). Strains of TSEsare explained, depending on the hypothesisadopted, in terms of the necessity for ascrapie-specific informational molecule inaddition to PrP (the virino hypothesis), or asthe result of reproducible three-dimensionalstructures of the PrPSC protein molecule(the prion hypothesis), which would act asstrain-related templates for the conversion ofPrPC molecules to the aberrant way of foldingtypical of PrPSC. There is evidence fromtest-tube experiments that such a PrPC/PrPSC

conversion, promoted by added PrPSC fromvarious TSE sources, can happen in astrain-specific manner (Caughey et al., 1998).Alternative means of holding informationwithin protein molecules could be related tothe degree of glycosylation (complex sugarside chains) of PrPSC molecules. These, asrevealed by antibody staining (Westernblots), form characteristic three-bandedpatterns which in some cases can be related

to the strain of the infection (Collinge et al.,1996b) (Fig. 14.1).

Genetics of Prion Diseases

In humans, occurrence of disease has beenlinked to the precise sequence of amino acidsspecified in an individual’s PrP gene. Insheep, goats, deer and mice, PrP amino acidcodon variation has also been linked to TSEdisease and to the length of the incubationperiod, but cattle have shown no such associ-ation with BSE. Mouse genetics will not bediscussed here as extensive reviews are avail-able elsewhere and mice do not represent asubstantial food source for humans!

Genetics of human TSEs

Each species in which linkage has beendemonstrated between PrP genotype andTSE has had its own set of disease-linkedamino acids. Humans are no exception,although CJD and Gerstmann–Straussler–Scheinker syndrome (GSS), which aredistinguishable on clinical and pathologicalcriteria, are each linked to several different

Prion Diseases: Meat Safety and Human Health 317

20-Feb-03 14

Fig. 14.1. Patterns of PrPSC protein on Westernblots. Lanes 1 and 2 are prepared from sheepinfected with different sources of natural scrapie;lanes 3 and 4 are from sheep infected experimen-tally with BSE.



PrP gene sequence changes (polymorphismsor mutations) in different affected families(Table 14.3). The sporadic forms of CJD inhumans are not linked to any mutations ofthe PrP gene and, instead, a codon 129 poly-morphism, methionine (M) or valine (V), isassociated with differences in susceptibilityto disease in that homozygous individuals(either MM or VV) are over-represented inCJD cases and heterozygosity (MV) seems toconfer some protection (Palmer et al., 1991).At the time of writing, all reported vCJDcases are of MM PrP genotype (Collingeet al., 1996a), which occurs in around 37% ofCaucasian populations.

Other forms of TSEs in humans appear tobe genetic diseases, resulting directly from amutation in the PrP gene, for example oneform of GSS which is linked to a codon 102proline to leucine mutation (Hsiao et al., 1989).This mutation, when introduced into themouse PrP gene in transgenic (Tg) mice andexpressed at extremely high levels, resultedin a spontaneous scrapie-like disease which,although no PrPSC was detectable by standardmethods, transmitted infection to hamstersand other Tg mice and not to normal mice(Hsiao et al., 1994). This experiment supportsthe ‘protein only’ hypothesis because appar-ently the only requirement for disease todevelop is a single amino acid mutation.However, the interpretation of the results hasbeen disputed on the grounds of the lack ofPrPSC, the odd transmission characteristicsand the high levels of expression needed tosee the effect – single-copy transgenes do notmake the mice ill (Chesebro, 1998). It is knownthat high levels of protein produced fromnormal PrP genes can also result in illness inTg mice (Westaway et al., 1994) and so PrPpoisoning is a possibility.

There are several other human PrP genemutations associated with disease, and vari-ous forms of human PrP protein expressedby naturally occurring mutant genes havebeen studied in cell lines in culture and havebeen found to be both abnormally processed,for example not appearing on the cellmembrane, and to acquire characteristics ofthe disease-associated PrPSC protein isoform(Lehmann and Harris, 1996; Daude et al.,1997). This suggests that mutations in the PrPgene may cause illness directly through loss offunction of the PrP protein by misprocessingor that the mutant protein forms deposits andpoisons the surrounding cells. In the familial,or genetic, forms of human TSEs, there isthought therefore to be no need to look for asource of disease other than the aberrant PrPgene itself; however, with sporadic CJD, thereis no easy explanation related to genetics,and an environmental source of infection,for example contaminated foodstuffs, mayeventually be found to be a risk factor.

Genetics of sheep TSEs

The means by which scrapie transmitsbetween sheep is not well understood,although there is good evidence that themost common means of entering the bodyis by the oral route (Hadlow et al., 1982;Van Keulen et al., 1999). Studies of naturalscrapie in sheep have confirmed the impor-tance of three amino acid codons in thesheep PrP gene (136, 154 and 171) (Belt et al.,1995; Clouscard et al., 1995; Hunter et al.,1996). (A diagram of the sheep PrP genestructure (similar in all species) is shown inFig. 14.2 and sheep genotypes are usually

20-Feb-03 14

318 N. Hunter

Disease Amino acid number Change Codon 129a

CJD

FFIGSS

178200178102

Aspartic acid → asparagineGlutamic acid → lysineAspartic acid → asparagineProline → leucine

ValineMethionineMethionineMethionine

aCodon 129 polymorphism: the amino acid at this position influences disease type that occurs incombination with mutation.FFI, fatal familial insomnia.

Table 14.3. Examples of human PrP gene mutations associated with familial TSEs.



represented giving each of the three codonsin turn for each allele in turn.) The most resis-tant genotype is ARR/ARR and the most sus-ceptible is VRQ/VRQ, with a range of othergenotypes of varying degree of disease risk inbetween these two extremes (Dawson et al.,1998). Not all sheep breeds are the same,however, as Suffolk sheep have a very simplelinkage with disease: this breed does nothave the VRQ allele and scrapie occurs inARQ/ARQ sheep. Other breeds with theadditional VRQ allele, such as Cheviots andSwaledales, are much more complex and,when the highly susceptible VRQ/VRQgenotype occurs, as it is almost always inscrapie-affected sheep, it has been suggestedthat scrapie may be simply a genetic disease(Ridley and Baker, 1995). However healthyVRQ/VRQ animals can live to more than 8years of age, well past the usual age-at-deathfrom scrapie (2–4 years) (Foster et al., 1996a),and susceptible sheep genotypes are easilyfound in countries that are free of scrapieclinical cases (Hunter et al., 1997a). Thegenetic disease hypothesis seems less likely,therefore, than a disease process whichinvolves an infecting agent (prion or virino)causing disease only in susceptible sheep.However, in order to eliminate TSE diseasesfrom sheep in countries where scrapie isendemic, breeding for resistant genotypes

currently is being promoted, rather thanelimination of infection – the route used withcattle BSE.

A great deal of information on the detailsof PrP genotype linkage with disease insheep has come from experimental infectionsof sheep with scrapie or with BSE undercontrolled conditions (Goldmann et al., 1991a,1994). The genotypes of sheep targeted byBSE are quite distinct, with the shortestincubation periods in ARQ/ARQ sheepand longer incubation periods in ARQ/ARRsheep. Although some sources of experi-mental scrapie (e.g. CH1641) also target thesegenotypes, SSBP/1 is different and affectsCheviot sheep encoding the VRQ allele(Goldmann et al., 1994). It is possible, there-fore, that there are also various types or strainsof natural scrapie which target either particu-lar sheep breeds and/or different PrP codons.The best way to investigate this at the momentis by passage of natural scrapie into a panelof mouse strains where characteristics ofincubation periods and the brain areas whichbecome damaged give distinct profiles orpatterns (Bruce et al., 1994). It is also possiblethat scrapie strains may produce PrPSC

proteins with distinct patterns on Westernblots – a method currently under investigationfor strain typing in several laboratoriesthroughout the world (e.g. Hope et al., 1999).


20-Feb-03 14

Fig. 14.2. Diagrammatic representation of the structure of the sheep PrP gene with the positions of thethree disease-linked amino acids. Black boxes represent the protein-coding region of the gene.



Genetics of BSE in cattle

It is generally accepted that BSE in cattle wasthe result of feeding of infected meat andbone meal-derived protein supplements.Several other captive animals succumbed toBSE-like infections (Bruce et al., 1994), includ-ing domestic cats, although the domestic doghas remained unaffected for reasons whichare not understood. When BSE was foundin cattle, the cattle PrP gene was searchedfor markers of resistance or susceptibility todisease similar to those that had been foundin mice, sheep and humans. The cattle gene,however, is remarkably invariant comparedwith that of sheep and humans and has so farshown no linkage with disease (Goldmannet al., 1991b; Hunter et al., 1994). Cattle there-fore appear to be unusual in not (so far)demonstrating a PrP-related link with TSEincidence. It may be that all cattle would besusceptible to BSE if they received a highenough dose of infection, but clearly breed-ing for resistance to BSE in cattle at themoment is not an available option. Thebest method for control is to eliminate theinfection from cattle feed both in the UK andin other countries throughout the world.

Distribution of Infectivity inBody Tissues

Methods of detection

It is important to establish which tissues ofTSE-affected animals or people are actuallyinfected because, particularly with referenceto BSE, this dictates which tissues or productscan be safely eaten or used in pharma-ceuticals. Demonstration of infection bymeans of mouse transmission studies takes along time (up to 2 years) but is, when posi-tive, unambiguous – there is infection presentand it will transmit to another individual.Negative results, when no disease is trans-mitted to the mice, could mean absence ofinfection but could also mean that there issimply too low a level (titre) of infection tobe detected within the test animals’ lifespan,or that the sensitivity of the test is not high

enough. The latter point relates to what isknown as the species barrier, which occursoften (but not always) when a TSE is trans-mitted from one species to another (Bruceet al., 1994). Because the species of animal ischanged, the TSE infection seems to have towork harder to produce disease, giving longincubation periods and/or lower detectabletitre. When cattle BSE is titred in cattle, it ispossible to detect 500-fold greater levels ofinfection than in mice (Wells et al., 1998).However despite this problem, the mouseassays have been of immense importance andare still used as the main means of demon-strating infection, largely because of the factthat BSE gives a clear ‘fingerprint’ of lengthsof incubation period and patterns of damageproduced in mouse brain tissue, and allowsthe positive identification of the BSE agentin cattle and other species – most notably inhumans affected by vCJD (Bruce et al., 1997).Transgenic mice, which are created in labora-tories and produce the PrP proteins of otherspecies, are being developed in the hope ofimproving the mouse assay system.

The other means of demonstration of thepresence of infection is by detection of PrPSC

protein either by extraction and reaction withanti-PrP antibodies (Western blotting) (Hopeet al., 1999) or by antibody detection withinsections of tissue examined under the micro-scope – immunohistochemistry (Van Keulenet al., 1999). Using PrPSC as a marker ofinfection is an excellent alternative to mousebioassay, although it is subject to the samepotential problems: a positive result is clear,but a negative result may simply mean thatthe detection limit of the method is notsensitive enough. There are examples ofinfection detected in mice without con-comitant detection of PrPSC, however, and soit is always advisable to retain some caution inthe interpretation of results (Lasmezas et al.,1997; Manson et al., 1999).

Species used in food production:sheep and cattle

Using bioassay in mice, natural scrapie infec-tion has been studied in diseased animals and

20-Feb-03 14

320 N. Hunter



also during the preclinical phase of the dis-ease during which animals appear perfectlyhealthy. In one study during developmentof scrapie, infection was detected first (at 10months of age) in sheep gut and lymphoidtissue, then much later in the central nervoussystem (CNS) and brain, the latter of whichhas greatest amounts of infectivity as theclinical signs develop and the animalbecomes ill. In this study, no infection wasfound in milk, udder or muscle (Hadlowet al., 1982). However, in a more recent report,peripheral nerves in scrapie sheep musclewere found to be infected (Groschup et al.,1996). Sheep experimentally infected withBSE have infection present in both brain andspleen (Foster et al., 1996b), suggesting that,should BSE infect sheep naturally, it wouldhave a similar distribution throughout thebody to that found with scrapie. Using bloodtransfusion between sheep (no species bar-rier), BSE infection has also been detected inblood removed from experimentally infectedanimals during the preclinical phase ofdisease (Houston et al., 2000). In contrast, cat-tle naturally infected with BSE have shownsigns of infection only in the brain and spinalcord, and even experimentally infected cattlehave only shown additional sites of infectionin part of the intestine (distal ileum) (Wellset al., 1998) and, specifically, milk from BSEcattle was also negative (Taylor et al., 1995).

Using PrPSC as a biochemical marker forinfectivity, signs of infection can be detectedin sheep tonsil as early as 3 months of age inanimals destined to develop scrapie at about2 years of age (Schreuder et al., 1998). PrPSC isfound throughout the body of scrapie sheep,with some tissues, such as the liver, beingrelatively spared and others having higheramounts (spleen, parts of the intestine); how-ever, up to and during the clinical phase,PrPSC is found in increasing, and very muchhigher, amounts in brain and CNS tissues(Van Keulen et al., 1996). PrPSC has also beenfound in placental tissue, although anyinvolvement of placenta in natural transmis-sion of scrapie from mother to offspring maynot be in utero but could result from ingestionof discarded placental tissue by the ewe her-self or by other sheep. In sheep experimentallyinfected with BSE, lymphoid tissues show

signs of infection early in the incubationperiod (Jeffrey et al., 2001) and, by terminalstages of disease, PrPSC deposits are foundwidespread throughout the body (Foster et al.,2001b). In BSE-affected cattle, it is more diffi-cult to find evidence of PrPSC outside the CNS(Wells et al., 1998). Clearly the pathogenesis ofBSE in cattle is different from that in sheep,with more limited involvement of non-CNStissues. It follows from this, therefore, thatin thinking about the risks of infection ofhumans with BSE, high risk cattle tissues areliable to be fewer in number and easier to elim-inate from the food chain than would be thecase if BSE in sheep were to become endemic.

Although there is no evidence for theoccurrence of BSE in the USA, another relateddisease does occur: chronic wasting disease(CWD) of mule deer and elk (Williams andYoung, 1992; Spraker et al., 1997). This priondisease occurs in both farmed and wildpopulations. Although there are precautionsin place which test the brain tissue of huntedanimals for the presence of PrPSC protein(Laplanche et al., 1999), there are concernsabout the occurrence of CJD in a small numberof individuals known to have consumed wildvenison. Recent epidemiological studies havejudged that there was no link between the twodiseases but, if more CJD cases occur in hunt-ers, there may be a change of opinion. There is,at the time of writing, no published evidencefrom outside North America for CWDoccurrence on venison farms or in the wild.

Humans

In sporadic CJD cases, signs of infection arenot widespread throughout the human body;however, from early studies of vCJD, it wasapparent that this new disease was behavingdifferently. PrPSC was detected in tonsils of anumber of vCJD victims and in the appendixtissue of another case (Hill et al., 1997, 1999).Further studies have since been set up to findout which tissues represent a risk of infectionfor humans and for contamination of surgicalinstruments. Infectivity was found in mousebioassays of vCJD brain, as expected, but alsoin tonsil and spleen at levels between 100


20-Feb-03 14



and 1000 times lower than in brain (Bruceet al., 2001). Blood fractions (buffy coat andplasma) were negative. In addition, PrPSC hasalso been found in lymph nodes, retina andoptic nerve, and at low levels in one vCJDcase in the rectum, adrenal gland and thymus– with obvious implications for contamina-tion of surgical instruments. Other vCJD tis-sues tested for PrPSC were negative, includingblood buffy coat preparations (Wadsworthet al., 2001).

How are TSEs Contracted?

The route of transmission of natural scrapiein sheep is not known with certainty but,because of the early involvement of the ali-mentary tract (PrPSC staining), the oral routeis implicated (Van Keulen et al., 1995, 1996,1999). Other routes of infection throughwounds on the skin or in the mouth arealso possible. Infection may be picked upfrom pasture contaminated with infectedplacental tissue, or by simple contact withother infected animals. BSE in cattle ismost likely to have been spread by oralinfection through eating contaminated meatand bone meal (MBM) (Wilesmith et al., 1991)although in this case the infection seems toby-pass the peripheral tissues and travelstraight to the brain.

In humans, the route of infection insporadic CJD is not known. Epidemiologicalstudies sometimes show connections withdiet or lifestyle, but these have been thought tobe artefactual due to the relatively smallnumbers of affected individuals (Wientjenset al., 1996). One study suggested surgery wasa risk factor (Kmietowicz, 1999), and there areclear instances of iatrogenic infection follow-ing surgical procedures (Shimizu et al., 1999)or following injection with contaminatedgrowth hormone used to treat undersizedchildren (d’Aignaux et al., 1999). There areforms of CJD which appear to be genetic in ori-gin and are linked to the occurrence of specificmutations of the PrP gene (Ghetti et al., 1995).If this view is correct, no further route need besought for the familial human TSEs; however,it has been shown beyond any doubt that

the vCJD infectious agent is identical to BSE(Bruce et al., 1997) and it has been assumedthat the disease is picked up via the oral routeand eating BSE-infected cattle products.

As it is not known how many peopleare already infected with vCJD and quietlyincubating the disease, it is impossible tojudge the risk to others from potentiallycontaminated blood products. Sporadic andgenetic forms of CJD are not thought to haveinfectivity in peripheral tissues, and there isno epidemiological evidence linking bloodtransfusion with incidence of sporadic CJD.As vCJD involves infection of peripheraltissues and to deal with the potential risk, UKblood supplies are depleted of white bloodcells and new US Food and Drug Administra-tion rules will forbid donations from anyonewho spent 3 months in Britain from 1980 to1996 or those who have spent 5 years ormore in France since 1980. The rules will alsoban donations from anyone who received ablood transfusion in Britain since 1980 andfrom American military personnel who spent6 months or more on a European base from1980 to 1996, when British beef was sold tobases there (Cimon, 2001). Only time will tell ifthese precautions are justified.

Risk Factors

There have been more than 177,000 cases ofBSE confirmed in cattle in Great Britain sincethe outbreak started in the late 1980s, peakingin 1992 with 36,682 recorded cases. Between1991 and 1995, there were more than 10,000cases per year; however, in 2000, the numbershad dropped to 1270. Other countries are alsoaffected, although at a much lower rate:Northern Ireland (total to December 2000 =1810, peaking in 1993 with 459); Republicof Ireland (total = 567, peaking in 2000 with132); France (total = 233, with 153 in 2000);Portugal (total = 489, peaking in 1999 with159) and Switzerland (total = 364, peaking in1995 with 68) (data from the UK Departmentfor the Environment, Food and Rural Affairs(DEFRA)). Many, but by no means all, casesin countries outside the UK are in cattleimported from the UK. Some animals are

20-Feb-03 14

322 N. Hunter



home bred, but it should be remembered thatMBM has been exported to many countriesthroughout the world by UK manufacturersand may have caused disease by ingestion.

It is assumed that the source of BSEinfection in humans is through consumptionof infected meat products. In examining theeating patterns of vCJD patients, however,no obvious common factor emerges whichmakes these people different from the rest ofthe UK population. The highest risk factorfor development of vCJD has been said simplyto be residence in the UK. However, as theoutbreak of vCJD continues, intriguing con-nections are being made. Sporadic CJD has auniform distribution related to local popula-tion levels (Cousens et al., 1997), but there is aslightly higher risk of contracting vCJD innorthern parts of the UK, where consumptionof meat products, rather than beef itself, isthought to be more common (Cousens et al.,2001). Meat products (pies, sausages, etc.)may have contained infected cattle CNS tis-sues but it remains difficult to demonstratethat vCJD patients’ diets were much differentfrom those of non-affected individuals withinthe same family. One small cluster of vCJDcases in England has been attributed to localbutchery practices, which involved splittingof cattle heads (Anon., 2001) and potentialcontamination of meat with brain tissue.

It is known that sheep can be infectedwith BSE experimentally by consumption ofas little as 0.5 g of BSE cattle brain (Foster et al.,1993). As UK sheep were also fed supplementscontaining MBM, although in greatly reducedamounts compared with cattle, it is a theoreti-cal possibility that sheep were also infectedwith BSE. Various studies are under way tolook for signs of BSE infection in sheep byidentification of the characterisitic BSE incu-bation period pattern in mouse transmissionexperiments and by looking in scrapie casesfor PrPSC protein which seems to adopt a par-ticular form associated with BSE. In order toincrease the chances of finding BSE should itexist in sheep, and since in experimental stud-ies using BSE infection the sheep which becomesick with the shortest incubation period are ofARQ/ARQ PrP genotype (Foster et al., 2001a),ARQ/ARQ sheep with clinical TSE signs havebeen selected for further study. Natural

scrapie can also occur in ARQ/ARQ sheep,however, and, at the time of writing, no firmevidence of BSE has been found in sheep.

Clinical Symptoms and Signs

Symptoms in humans

Sporadic CJD patients have rapidly advanc-ing dementia with development of move-ment disorders, difficulty in walking, corticalblindness and mutism, with median survivaltime between onset of symptoms and deathbeing 4.5 months. In contrast, vCJD has alonger clinical phase (median survival time~10 months longer than with sporadic CJD).Patients with vCJD tend to be younger onaverage than sporadic CJD cases and havepsychiatric symptoms of withdrawal anddepression. Early symptoms of vCJD alsoinclude painful and persistently cold feet andlegs, and neurological signs do not developuntil later in the clinical course, when move-ment disorders, complete dependence andmutism begin and progress until death. Theneuropathology of vCJD is also distinct fromthat of sporadic CJD in that the formerexhibits very large plaques, or deposits,of PrP protein surrounded by vacuoles(so-called ‘florid’ plaques) in the cerebral andcerebellar cortex of the brain (Stewart andIronside, 1998).

Signs in sheep

Clinical signs of scrapie in sheep (Dickinson,1976) start with mildly impaired socialbehaviour followed by locomotor incoordi-nation or ataxia with trembling. Pruritis andwool loss can result from the animal attempt-ing to relieve what seems to be an intenseitching by scratching against fence posts orby biting the affected area; however, theseclinical signs are highly variable and can lastfrom 2 weeks to 6 months. Lesions in thebrain include neuronal degeneration withthe formation of vacuoles (holes), prolifera-tion of astroglial cells but no demyelinationor other overt inflammatory responses. These


20-Feb-03 14



features develop in the later stages of theincubation period and it is very difficult todetect (by histopathology) those animals withscrapie which are not yet visibly affected bythe disease.

In experimental studies of BSE in sheep,the clinical signs are very similar to thoseof scrapie (Foster et al., 2001a), and in a fieldsituation it is expected that, should BSEhave infected sheep, it would be impossible touse these to differentiate scrapie (presumed tobe non-pathogenic to humans) from BSE.

Treatments

There are as yet no approved treatmentswhich cure TSE diseases. There are possiblecandidates amongst drugs which prolongthe incubation period in animal studies, forexample pentosan polysulphate (PS), whichif administered 7 h after injection with ME7scrapie prolonged the incubation period inmice by up to 66% (Farquhar et al., 1999). Thisis not a cure, but is one example of currentstudies which are aimed at understandinghow therapeutics might work. It has alsobeen suggested that anti-PrP antibodiescould prevent the conversion of PrPC to PrPSC

in the body of affected individuals as itseems to do in cell culture (Peretz et al., 2001).An interesting recent development involvestrials in humans clinically affected withTSE disease of the anti-malarial drugquinacrine, not used widely in the UK sincethe 1970s, and the related chlorpromazine,which carry additional worries of side effects.At present, the only real defence against thesediseases in humans is avoidance of infection,and legislators have been busy trying toensure that infection is removed from thehuman food chain.

Legislation and Regulatory Issues

The first laws designed to prevent the spreadof BSE in cattle by banning the use of feedingruminant tissues to ruminants in the UKcame into force in 1988, and the rules havecontinued to be tightened since then. The

European Commission took the first steps inlimiting cattle exports from the UK in 1989,which culminated in a complete ban in 1996.In 1989, there was also the banning in humanfood produced in England and Wales of cattletissues expected (from previous studies insheep scrapie) to be infectious. The samelegislation was applied in Scotland andNorthern Ireland in the following year. For2 years, from late 1997, meat on the bone wasalso banned from human consumption.

It was clear, however, that the 1988 rumi-nant feed ban had not been completely effec-tive as cattle born after the ban also developedBSE; however, greater control over abattoirsand rendering plants has resulted in a con-tinual drop in numbers of BSE cases in theUK. It is disturbing that the very stringentrules imposed after the announcement of theoccurrence of vCJD in 1996 do not seem to betotally effective, as animals born after thatdate are now showing signs of BSE, albeit invery low numbers. Laws relating to the age ofanimals allowed into the human food chain(1996) mean that UK cattle over 30 monthscannot be used as a source of meat or mech-anically recovered meat (MRM). MBM andruminant-derived products have been subjectto a plethora of regulations in the UK and inEurope about what they can contain and whatthey can be used for. For example, in 1998, thesale of MBM derived from mammalian tissueswas prohibited for use as a fertilizer on agri-cultural land, and the use of certain ruminanttissues in cosmetics was banned in the UK in1997. As sheep also became suspect in the BSEaudit trail, heads of sheep and goats were pro-hibited for human consumption in the UKin 1996. However, attempts by the EuropeanCommission to agree on legislation to controlthe use of high risk tissues in food and foodproducts have been greatly hampered by therelative difficulty of removal of the spinal cordfrom sheep and goats compared with themuch larger cattle. It also appears that, if BSEdoes occur in small ruminants, it may be morewidespread in body tissues than it is in cattle.Since the introduction of a reliable means ofcattle identification by ear tagging and indi-vidual cattle passports with a tracing system,coupled with all of the other control measuresin place in the UK, the export market is

20-Feb-03 14

324 N. Hunter



gradually opening up again for cattle andmeat products (data from DEFRA, UK).

Conclusions

It is too soon to predict accurately the finalnumbers of people who will become affectedby vCJD; however, it is likely that themeasures in place to protect humans andanimals will mean that, if an epidemic ofvCJD does occur, its time span will belimited. Intense efforts are also being madeto understand the disease, how it spreadsand how it can be treated or prevented in sus-ceptible individuals. It seems, however, thata great deal of the dangerous BSE-infectedMBM was exported from the UK to otherparts of the world and may have been fedthere to indigenous ruminants. Organiza-tions such as the World Health Organization,the European Union, the Office Internation-ale d’Epizootiques and the World TradeOrganization have been trying to raise theissue of the dangers of BSE in regions outsideEurope and the USA, and it is to be hopedthat BSE and vCJD will not become majorproblems for the rest of the world population.

References

Anon. (2001) vCJD: SEAC endorses ‘plausibleexplanation’ for Leicestershire cluster.Veterinary Record 148, 23.

Belt, P.B.G.M., Muileman, I.H., Schreuder, B.E.C.,Bos-de Ruijter, J., Gielkens, A.L.J. and Smits,M.A. (1995) Identification of five allelic vari-ants of the sheep PrP gene and their associationwith natural scrapie. Journal of General Virology76, 509–517.

Brown, P., Cathala, F., Raubertas, R.F., Gajdusek,D.C. and Castaigne, P. (1987) The epidemiol-ogy of Creutzfeldt–Jakob disease: conclusionof a 15-year investigation in France and reviewof the world literature. Neurology 37, 895–904.

Bruce, M.E., McConnell, I., Fraser, H. andDickinson, A.G. (1991) The disease charac-teristics of different strains of scrapie in Sinccongenic mouse lines: implications for thenature of the agent and host control ofpathogenesis. Journal of General Virology 72,595–603.

Bruce, M., Chree, A., McConnell, I., Foster, J.,Pearson, G. and Fraser, H. (1994) Transmissionof bovine spongiform encephalopathy andscrapie to mice – strain variation and thespecies barrier. Philosophical Transactions of theRoyal Society of London Series B BiologicalSciences 343, 405–411.

Bruce, M.E., Will, R.G., Ironside, J.W., McConnell, I.,Drummond, D., Suttie, A., McCardle, L.,Chree, A., Hope, J., Birkett, C., Cousens, S.,Fraser, H. and Bostock, C.J. (1997) Transmis-sions to mice indicate that ‘new variant’ CJD iscaused by the BSE agent. Nature 389, 488–501.

Bruce, M.E., McConnell, I., Will, R.G. and Ironside,J.W. (2001) Detection of variant Creutzfeldt–Jakob disease infectivity in extraneural tissues.Lancet 358, 208–209.

Caughey, B., Raymond, G.J. and Bessen, R.A. (1998)Strain-dependent differences in beta-sheetconformations of abnormal prion protein.Journal of Biological Chemistry 273, 32230–32235.

Chesebro, B. (1998) BSE and prions: uncertaintiesabout the agent. Science 279, 42–43.

Cimon, M. (2001) New blood donor rules target‘mad cow’ risk; to protect the nation’s supply,the FDA tightens donation limits on peoplewho have stayed in Europe. Los Angeles Times,28 August.

Clouscard, C., Beaudry, P., Elsen, J.M., Milan, D.,Dussaucy, M., Bounneau, C., Schelcher, F.,Chatelain, J., Launay, J.M. and Laplanche, J.L.(1995) Different allelic effects of the codons 136and 171 of the prion protein gene in sheep withnatural scrapie. Journal of General Virology 76,2097–2101.

Collinge, J., Whittington, M.A., Sidle, K.C.L., Smith,C.J., Palmer, M.S., Clarke, A.R. and Jefferys,J.G.R. (1994) Prion protein is necessary fornormal synaptic function. Nature 370, 295–297.

Collinge, J., Beck, J., Campbell, T., Estibeiro, K. andWill, R. (1996a) Prion protein gene analysis innew variant cases of CJD. Lancet 348, 56.

Collinge, J., Sidle, K., Meads, J., Ironside, J. and Hill,A. (1996b) Molecular analysis of prion strainvariation and the etiology of new variant CJD.Nature 383, 685–690.

Cousens, S.N., Zeidler, M., Esmonde, T.F., DeSilva,R., Wilesmith, J.W., Smith, P.G. and Will, R.G.(1997) Sporadic Creutzfeldt–Jakob disease inthe United Kingdom: analysis of epidemio-logical surveillance data for 1970–96. BritishMedical Journal 315, 389–395.

Cousens, S., Smith, P.G., Ward, H., Everington, D.,Knight, R.S.G., Zeidler, M., Stewart, G. andSmith-Bathgate, E.A.B. (2001) Distribution ofvariant Creutzfeldt–Jakob disease in GreatBritain, 1994–2000. Lancet 357, 1002–1007.


20-Feb-03 14



d’Aignaux, J.H., Costagliola, D., Maccario, J.,Villemeur, T.B., de Brandel, J.P., Deslys, J.P.,Hauw, J.J., Chaussain, J.L., Agid, Y. andDormont, D. (1999) Incubation period ofCreutzfeldt–Jakob disease in human growthhormone recipients in France. Neurology 53,1197–1201.

Daude, N., Lehmann, S. and Harris, D.A. (1997)Identification of intermediate steps in theconversion of a mutant prion protein to ascrapie-like form in cultured cells. Journal ofBiological Chemistry 272, 11604–11612.

Dawson, M., Hoinville, L.J., Hosie, B.D. and Hunter,N. (1998) Guidance on the use of PrP genotyp-ing as an aid to the control of clinical scrapie.Veterinary Record 142, 623–625.

Dickinson, A.G. (1976) Scrapie in sheep and goats.In: Kimberlin, R. (ed.) Slow Virus Diseases ofAnimals and Man. North-Holland, Amsterdam,pp. 209–241.

Dickinson, A.G. and Meikle, V.M. (1971) Host-genotype and agent effects in scrapie incuba-tion: change in allelic interaction with differentstrains of agent. Molecular and General Genetics112, 73–79.

Farquhar, C., Somerville, R. and Bruce, M. (1998)Straining the prion hypothesis. Nature 391,345–346.

Farquhar, C., Dickinson, A. and Bruce, M.(1999) Prophylactic potential of pentosanpolysulphate in transmissible spongiformencephalopathies. Lancet 353, 117.

Foster, J.D., Hope, J. and Fraser, H. (1993) Transmis-sion of bovine spongiform encephalopathy tosheep and goats. Veterinary Record 133, 339–341.

Foster, J., Hunter, N., Williams, A., Mylne, M.,McKelvey, W., Hope, J., Fraser, H. and Bostock,C. (1996a) Observations on the transmission ofscrapie in experiments using embryo transfer.Veterinary Record 138, 559–562.

Foster, J.D., Bruce, M., McConnell, I., Chree, A. andFraser, H. (1996b) Detection of BSE infectivityin brain and spleen of experimentally infectedsheep. Veterinary Record 138, 546–548.

Foster, J.D., Parnham, D., Chong, A., Goldmann, W.and Hunter, N. (2001a) Clinical signs, histo-pathology and genetics of experimental trans-mission of BSE and natural scrapie to sheepand goats. Veterinary Record 148, 165–171.

Foster, J.D., Parnham, D.W., Hunter, N. and Bruce,M. (2001b) Distribution of the prion protein insheep terminally affected with BSE followingexperimental oral transmission. Journal ofGeneral Virology 82, 2319–2326.

Ghetti, B., Dlouhy, S.R., Giaccone, G., Bugiani, O.,Frangione, B., Farlow, M.R. and Tagliavini, F.(1995) Gerstmann Straussler Scheinker disease

and the Indiana kindred. Brain Pathology 5,61–75.

Goldmann, W., Hunter, N., Benson, G., Foster, J.D.and Hope, J. (1991a) Different scrapie-associated fibril proteins (PrP) are encodedby lines of sheep selected for different allelesof the Sip gene. Journal of General Virology 72,2411–2417.

Goldmann, W., Hunter, N., Martin, T., Dawson, M.and Hope, J. (1991b) Different forms of thebovine PrP gene have five or six copies ofa short, G–C-rich element within the proteincoding exon. Journal of General Virology 72,201–204.

Goldmann, W., Hunter, N., Smith, G., Foster, J. andHope, J. (1994) PrP genotype and agent effectsin scrapie – change in allelic interaction withdifferent isolates of agent in sheep, a naturalhost of scrapie. Journal of General Virology 75,989–995.

Groschup, M.H., Weiland, F., Straub, O.C. andPfaff, E. (1996) Detection of scrapie agent inthe perpheral nervous system of a diseasedsheep. Neurobiology of Disease 3, 191–195.

Hadlow, W.J., Kennedy, R.C. and Race, R.E.(1982) Natural infection of Suffolk sheepwith scrapie virus. Journal of Infectious Diseases146, 657–664.

Hill, A.F., Zeidler, M., Ironside, J. and Collinge, J.(1997) Diagnosis of new variant Creutzfeldt–Jakob disease by tonsil biopsy. Lancet 349,99–100.

Hill, A., Butterworth, J., Joiner, S., Jackson, G.,Rosser, M., Thomas, D., Frosh, A., Tolley, N.,Bell, J.E., Spencer, M., King, A., Al-Sarraj, A.,Ironside, J., Lantos, P. and Collinge, J. (1999)Investigation of variant Creutzfeldt–Jakobdisease and other human prion diseases withtonsil biopsy samples. Lancet 353, 183–189.

Hope, J., Wood, S.C.E.R., Birkett, C.R., Chong, A.,Bruce, M.E., Cairns, D., Goldmann, W.,Hunter, N. and Bostock, C.J. (1999) Molecularanalysis of ovine prion protein identifiessimilarities between BSE and an experimentalisolate of natural scrapie, CH1641. Journal ofGeneral Virology 80, 1–4.

Houston, F., Foster, J.D., Chong, A., Hunter, N.and Bostock, C.J. (2000) Transmission of BSEby blood transfusion in sheep. Lancet 356,999–1000.

Hsiao, K., Baker, H.F., Crow, T.J., Poulter, M., Owen,F., Terwilliger, J.D., Westaway, D., Ott, J. andPrusiner, S.B. (1989) Linkage of a prion proteinmissense variant to Gerstmann Srausslersyndrome. Nature 338, 342–345.

Hsiao, K.K., Groth, D., Scott, M., Yang, S.L., Serban,H., Raff, D., Foster, D., Torchia, M., Dearmond,

20-Feb-03 14

326 N. Hunter



S.J. and Prusiner, S.B. (1994) Serial trans-mission in rodents of neurodegeneration fromtransgenic mice expressing mutant prionprotein. Proceedings of the National Academyof Sciences of the USA 91, 9126–9130.

Hunter, N., Goldmann, W., Smith, G. and Hope, J.(1994) Frequencies of PrP gene variants inhealthy cattle and cattle with BSE in Scotland.Veterinary Record 135, 400–403.

Hunter, N., Foster, J., Goldmann, W., Stear, M.,Hope, J. and Bostock, C. (1996) Natural scrapiein a closed flock of Cheviot sheep occurs only inspecific PrP genotypes. Archives of Virology 141,809–824.

Hunter, N., Cairns, D., Foster, J., Smith, G.,Goldmann, W. and Donnelly, K. (1997a) Isscrapie a genetic disease? Evidence fromscrapie-free countries. Nature 386, 137.

Hunter, N., Goldmann, W., Foster, J., Cairns, D.and Smith, G. (1997b) Natural scrapie andPrP genotype: case–control studies in Britishsheep. Veterinary Record 141, 137–140.

Jeffrey, M., Ryder, S., Martin, S., Hawkins, S.A.C.,Terry, L., Berthelin-Baker, C. and Bellworthy,S.J. (2001) Oral inoculation of sheep with theagent of bovine spongiform encephalopathy(BSE). 1. Onset and distribution of disease-specific PrP accumulation in brain andviscera. Journal of Comparative Pathology 124,280–289.

Kmietowicz, Z. (1999) Surgery increases risk ofsporadic CJD. British Medical Journal 318. 625.

Laplanche, J.-L., Hunter, N., Shinagawas, M. andWilliams, E. (1999) Scrapie, chronic wastingdisease and transmissible mink enceph-alopathy. In: Prusiner, S.B. (ed.) Prion Biologyand Diseases. Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, New York,pp. 393–429.

Lasmezas, C., Deslys, J.-P., Robain, O., Jaegly, A.,Beringue, V., Peyrin, J.-M., Fournier, J.-G.,Hauw, J.-J., Rossier, J. and Dormont, D. (1997)Transmission of the BSE agent to mice in theabsence of detectable abnormal prion protein.Science 275, 402–405.

Lehmann, S. and Harris, D.A. (1996) Mutant andinfectious prion proteins display commonbiochemical properties in cultured cells.Journal of Biological Chemistry 271, 1633-1637.

Mabbott, N., Brown, K.L. and Bruce, M. (1997) Tlymphocyte activation and the cellular formof the prion protein, PrPc. Biochemical SocietyTransactions 25, 307s.

Manson, J.C., Hope, J., Clarke, A., Johnston, A.,Black, C. and MacLeod, N. (1995) PrP genedosage and long term potentiation. Neurode-generation 4, 113–115.

Manson, J.C., Jamieson, E., Baybutt, H., Tuzi, N.L.,Barron, R., McConnell, I., Somerville, R., Iron-side, J., Will, R., Sy, M.-S., Melton, D.W., Hope,J. and Bostock, C.J. (1999) A single amino acidalteration (101 L) introduced into murine PrPdramatically alters incubation time of trans-missible spongiform encephalopathy. EMBOJournal 18, 6855–6844.

Palmer, M.S., Dryden, A.J., Hughes, J.T. andCollinge, J. (1991) Homozygous prion proteingenotype predisposes to sporadic Creutzfeldt–Jakob disease. Nature 352, 340–342.

Parry, H. (1984) Scrapie. Academic Press, London.Peretz, D., Williamson, R.A., Kaneko, K., Vergara, J.,

Leclerc, E., Schmitt-Ulms, G., Mehlhorn, I.R.,Legname, G., Wormald, M.R., Rudd, P.M.,Dwek, R.A., Burton, D.R. and Prusiner, S.B.(2001) Antibodies inhibit prion propagationand clear cell cultures of prion infectivity.Nature 412, 739–743.

Prusiner, S.B. (1982) Novel proteinaceousinfectious particles cause scrapie. Science 216,136–144.

Prusiner, S.B., Scott, M., Foster, D., Pan, K.-M.,Groth, D., Mirenda, C., Torchia, M., Yang, S.-L.,Serban, D., Carlson, G.A., Hoppe, P.C.,Westaway, D. and DeArmond, S.J. (1990)Transgenetic studies implicate interactionsbetween homologous PrP isoforms in scrapieprion replication. Cell 63, 673–686.

Ridley, R.M. and Baker, H.F. (1995) The mythof maternal transmission of spongiformencephalopathy. British Medical Journal 311,1071–1075.

Rund, D., Cohen, T., Filon, D., Dowling, C.E.,Warren, T.C., Barak, I., Rachmilewitz, E.,Kazazian, E. and Oppenheim, A. (1991)Evolution of a genetic disease in an ethnic iso-late: β-thalassemia in the Jews of Kurdistan.Proceedings of the National Academy of Sciencesof the USA 88, 310–314.

Schreuder, B.E.C., van Keulen, L.J.M., Vromans,M.E.W., Langeveld, J.P.M. and Smits, M.A.(1998) Tonsillar biopsy and PrPSc detection inthe preclinical diagnosis of scrapie. VeterinaryRecord 142, 564–568.

Shimizu, S., Hoshi, K., Muramoto, T., Homma, M.,Ironside, J.W., Kuzuhara, S., Sato, T.,Yamamoto, T. and Kitamoto, T. (1999)Creutzfeldt–Jakob disease with florid-typeplaques after cadaveric dura mater grafting.Archives of Neurology 56, 357–362.

Spraker, T.R., Miller, M.W., Williams, E.S., Getzy,D.M., Adrian, W.J., Schoonveld, G.G.,Spowart, R.A., Orourke, K.I., Miller, J.M. andMerz, P.A. (1997) Spongiform encephalopathyin free-ranging mule deer (Odocoileus


20-Feb-03 14



20-Feb-03 14

hemionus), white-tailed deer (Odocoileusvirginianus) and Rocky Mountain elk (Cervuselaphus nelsoni) in northcentral Colorado.Journal of Wildlife Diseases 33, 1–6.

Stewart, G.E. and Ironside, J.W. (1998) New variantCreutzfeldt–Jakob disease. Current Opinion inNeurology 11, 259–262.

Taylor, D.M., Ferguson, C.E., Bostock, C.J. andDawson, M. (1995) Absence of disease inmice receiving milk from cows with bovinespongiform encephalopathy. Veterinary Record136, 592.

Tobler, I., Gaus, S.E., Deboer, T., Achermann, P.,Fischer, M., Rulicke, T., Moser, M., Oesch, B.,McBride, P.A. and Manson, J.C. (1997) Alteredcircadian activity rhythms and sleep in micedevoid of prion protein. Journal of Neurosciences17, 1869–1879.

Van Keulen, L.J.M., Schreuder, B.E.C., Meloen, R.H.,Poelenvandenberg, M., Mooijharkes, G.,Vromans, M.E.W. and Langeveld, J.P.M. (1995)Immunohistochemical detection and localiza-tion of prion protein in brain-tissue of sheepwith natural scrapie. Veterinary Pathology 32,299–308.

Van Keulen, L., Schreuder, B., Meloen, R., Mooij-Harkes, G., Vromans, M. and Longeveld, J.(1996) Immunohistochemical detection ofprion protein in lymphoid tissues of sheepwith natural scrapie. Journal of Clinical Micro-biology 34, 1228–1231.

Van Keulen, L.J.M., Schreuder, B.E.C., Vromans,M.E.W., Langeveld, J.P.M. and Smits, M.A.(1999) Scrapie-associated prion protein in thegastrointestinal tract of sheep with natural

scrapie. Journal of Comparative Pathology 121,55–63.

Wadsworth, J.D.F., Joiner, S., Hill, A.F., Campbell,T.A., Desbrusiais, M., Luthert, P.J. andCollinge, J. (2001) Tissue distribution ofprotease resistant prion protein in variantCreutzfeldt–Jacob disease using a highlysensitive immunoblotting assay. Lancet 358,171–180.

Wells, G.A.H., Hawkins, S.A.C., Green, R.B., Austin,A.R., Dexter, I., Spencer, Y.I., Chaplin, M.J.,Stack, M.J. and Dawson, M. (1998) Preliminaryobservations on the pathogenesis of experi-mental bovine spongiform encephalopathy(BSE): an update. Veterinary Record 142,103–106.

Westaway, D., Dearmond, S., Cayetanocanlas, J.,Grothe, D., Foster, D., Yang, S.L., Torchia, M.,Carlson, G. and Prusiner, S. (1994) Degenera-tion of skeletal-muscle, peripheral-nerves, andthe CNS in transgenic mice overexpressingwild-type prion proteins. Cell 76, 117–129.

Wientjens, D.P.W.M., Davanipour, Z., Hofman, A.,Kondo, K., Matthews, W.B., Will, R.G. andVanduijn, C.M. (1996) Risk factors forCreutzfeldt–Jakob disease – a reanalysis ofcase–control studies. Neurology 46, 1287–1291.

Wilesmith, J.W., Ryan, J.B.M. and Atkinson, M.J.(1991) Bovine spongiform encephalopathy –epidemiologic studies on the origin. VeterinaryRecord 128, 199–203.

Williams, E.S. and Young, S. (1992) Spongiformencephalopathies of Cervidae. Reviews inScience and Technology, Office InternationaleEpizootique 11, 551–567.

328 N. Hunter



15 The Safety Evaluation of GeneticallyModified Foods

M.J. Gasson*Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK

Introduction

The use of gene technology in food produc-tion is the cause of significant controversythat has been fuelled by the activities ofvarious pressure groups and by the nature ofsome media reporting. This heightened con-cern has led to a complex situation where thediscussion of genetically modified (GM) foodraises a whole series of social issues that gowell beyond the strictly scientific assessmentof safety. This chapter is intended to high-light those safety issues that are addressedwhen an objective scientific evaluation of GMfoods is undertaken. Discussion is focused onhealth risks to the consumer, and issues thatare associated with agricultural practice arenot considered. In addition, the frequentlyraised question of benefit versus risk and thesubtleties of risk perception are not covered.In this regard, the Agriculture and Environ-ment Biotechnology Commission Report Cropson Trial (AECB, 2001) provides a useful per-spective on the ethical and social impact ofand public attitudes towards GM technology.

The safety assessment of GM foodaddresses a series of well established andinternationally accepted questions. However,there are differences in implementation, mostnotably between Europe and the USA. InEurope, GM food legislation is dominated by

EC Regulation 258/97 on ‘Novel foods andingredients’. This legislation (EC, 1997a)demands a formal process of pre-marketapproval that draws on the opinions ofindependent scientific committees in eachMember State. In practice, the competentauthority of the country where a product isfirst intended to be marketed undertakes theinitial safety assessment. All other EuropeanMember States then have an opportunity tocomment on the initial opinion. Where safetyconcerns are raised, scientific evaluation ispassed on centrally to the Scientific Commit-tee for Food. Some novel foodstuffs, includ-ing GM soybean derivatives, were alreadymarketed in Europe before this legislationcame into force, but they were subject to ear-lier evaluation by national safety committees,notably by the Advisory Committee on NovelFoods and Processes (ACNFP) in the UK(ACNFP, 1994).

In the USA, the Food and Drug Adminis-tration (FDA) holds the prime responsibilityfor GM food safety under the Federal Food,Drug and Cosmetic Act. The FDA policy onfoods developed by biotechnology is outlinedin a policy statement made in 1992 (FDA,1992). While US regulatory involvement isless hands-on, the scientific principles usedfor safety evaluation have much in commonwith those of the EU. A recent review of the



regulatory process in the USA has led to arequirement for data submission 120 daysprior to the marketing of bioengineered foods(FDA, 2001a), and updated extended guide-lines have been announced (FDA, 2001b).

Application of GM Technology in Food

GM technology is applied to food in a widevariety of different ways. Food productioninvolves the use of many ingredients,additives and enzyme preparations thatare derived from a diversity of sources.These include microorganisms that are oftenexploited as ‘cell factories’ for the productionof food ingredients and processing aids.Microorganisms used in this way are oftensubject to genetic improvement, but they areused to produce purified food componentsthat are often nature identical. Calf chymosin,the active ingredient of rennet, is used exten-sively by the dairy industry in cheese makingand provides a good example of a GM cellfactory application. A gene for the chymosinenzyme has been introduced into the yeastKluveromyces lactis, the filamentous fungusAspergillus niger and the bacterium Escherichiacoli, and its expression in these heterologoushosts provides an alternative source to tradi-tional rennet extracted from the stomachs ofslaughtered calves. This represents one of theearliest commercial applications of GM foodtechnology and it has been subject to detailedsafety evaluation both in the USA and inEurope.

Another example of a microbial cellfactory that currently is awaiting EU safetyapproval is the use of a GM strain of Bacillussubtilis for the manufacture of the vitaminriboflavin (ACNFP, 1997). This is distinct inthat the vitamin is a metabolite rather than thedirect protein product of the introduced gene,and it is representative of another genericapplication of GM technology in metabolicengineering.

Traditional biotechnology includes theexploitation of microorganisms for food fer-mentation, and this has evolved into a majorsector of the food industry. This covers the useof yeasts in brewing and bread making, and

the use of lactic acid bacteria in dairying andthe production of fermented vegetables andmeats. GM technology has been used for thedevelopment of improved strains of thesefood fermentation microorganisms. Examplesinclude a bread-making strain of Sac-charomyces cerevisiae with improved maltosemetabolism and a brewing strain of thesame species that expresses an amylase genederived from another closely related Saccharo-myces species. In both cases, safety evaluationhas been undertaken by the ACNFP (ACNFP,1994), but food products based on the useof these GM yeast strains have never beenmarketed in Europe. Because of the manu-facturing methods used for bread and beer,the derived food products would not containviable GM yeast cells or, in the case of alive beer, only very low numbers. In contrast,some dairy products, such as yoghurt, containhigh numbers of viable lactic acid bacteriaand, as a consequence, viable GM derivativesof these microorganisms would also be pres-ent. These distinctions are important for safetyevaluation, and the particular issues raised byviable GM microorganisms in food have beenthe subject of detailed consideration by avariety of organizations including ILSI (1999)and, most recently, FAO/WHO (2001a).

Crop plants account for the vast majorityof current GM food applications. GM plantmaterial might be marketed as an intact food,as in the case of fresh fruits, or it might besubject to processing, as in the case of cannedtomato paste. The safety evaluations of bothfresh GM tomato fruit and canned GM tomatopaste have been undertaken (ACNFP, 1995,1996). Genetic modification involved theintroduction of an ‘antisense’ or ‘sense’ genefor the natural tomato pectinase enzyme.Expression of the engineered gene causesreduced pectinase activity during the fruitripening process with consequent reducedtexture change. This lessens mechanical dam-age to the ripe fruit and reduces the associatedspoilage. In addition, altered processing char-acteristics improve the yield of tomato paste,with associated economic benefits.

Commodity crops, such as soybean, areused for the production of a range of purifiedor semi-purified derivatives that are added toprocessed foods. The extraction of oils, flours

330 M.J. Gasson

and other food ingredients from commoditycrops has implications for safety evaluation inthat transgene DNA and expressed proteinsare subject to varying degrees of inactivationand removal. In addition, it is pertinent thatthese derivatives are used extensively in pro-cessed foods, making consumer exposurevery widespread.

In many examples of GM food, the tech-nology has been used to improve agriculturalperformance. The most widespread applica-tions are the introduction of genes that confertolerance to otherwise non-specific herbicidesand the gene for a Bacillus thuringiensis insecti-cidal protein that provides in-built protectionfrom pest damage. While GM food technol-ogy currently is dominated by these ‘first-generation’ agronomic applications, there isa considerable effort devoted to ‘second-generation’ traits that are intended to benefitthe food manufacturer or the consumer.Examples of second-generation traits includeimprovements to seed storage proteins, oilcontent and starch, the removal of allergensand the fortification of micronutrients andantioxidants. A good example is the develop-ment of GM yellow rice in which three genesfor the biosynthesis of β-carotene were intro-duced into a conventional rice cultivar. Asβ-carotene is the provitamin for vitamin A,this has the potential to address a seriousnutrient deficiency that causes blindness inmany children in developing countries.

This very brief overview of GM foodapplications inevitably will be incomplete, butcritically it serves to emphasize the diversityof GM technology uses in food. It establishesthe need for case-by-case consideration ofsafety, and this is already well established asa key guiding principle in safety assessment.It is particularly relevant that purificationand processing, such as heat treatment,may inactivate or remove introduced foreigngenes and their expressed proteins, withobvious implications for safety assessment.Some examples of the diversity of GM foodapplications that have been the subject ofsafety evaluation are listed in Box 15.1, and amore complete position is readily availablefrom the EC release, Facts on GMOs in the EU(EC, 2000).

GM Technology

The in vitro manipulation of DNA is commonto all applications of GM technology. Foreach target material, there is a need to use aspecific technology for DNA delivery and toensure its subsequent maintenance. This canvary depending on the target species that isthe subject of genetic modification and it hasimplications for safety evaluation. One of thecentral safety assessment criteria is an analy-sis of the DNA that has been introduced aswell as the methods that have been employedduring transgene delivery. In bacteria, genetechnology is generally more advanced andcontrollable than is the case for plants. Therelatively small genome size and the avail-ability of an increasing number of wholebacterial genome sequences provide avaluable pool of detailed information. Animportant advantage is the ease with whichhomologous recombination can be used tofacilitate the directed delivery of transgenesto specific genome sites. In general, it is pos-sible to devise food-compatible genetic sys-tems for the exploitation of GM technology,using both plasmid-based and chromosomeintegration systems.

In contrast, plants suffer from the factthat the exploitation of homologous recombi-nation remains a challenging objective. Gen-erally, DNA delivery does not involve controlover the genome site into which a transgene isintegrated and, in many cases, the deliveredDNA becomes rearranged during geneticmodification. Transgene delivery in plantsinvolves three technologies: protoplast trans-formation; microparticle bombardment orbiolistics; and Agrobacterium binary vectors.The latter two processes are the most widelyused. As its name suggests, microparticlebombardment involves the penetration ofplant tissue by tungsten particles coated withtransgene DNA. Agrobacterium technologyexploits the disease features of the pathogenAgrobacterium tumefaciens, which has evolveda natural process for the delivery of bacterialgenes to the plant genome. Using a processrelated to bacterial conjugation, a specific tractof Agrobacterium DNA is transferred to theplant genome, where its expression leads to

Safety Evaluation of Genetically Modified Foods 331

332 M.J. Gasson

Box 15.1. Examples of the safety assessment of GM foods in Europe.a

Approvals made by ACNFP prior to Regulation (EC) 258/97Calf chymosin expressed in the microorganisms Aspergillus niger, Kluveromyces lactis and Escherichiacoli. (Widely used as a milk-clotting agent in cheese manufacture.)Baker’s yeast Saccharomyces cerevisiae expressing maltose permease. (Approved but nevercommercialized.)Brewer’s yeast expressing an amylase gene to facilitate starch breakdown with improved fermentationand carbohydrate conversion. (Approved but never commercialized.)Paste prepared from Zeneca tomato engineered for reduced expression of the polygalacturonidase geneand altered ripening characteristics giving improved yields and reduced spoilage. (Product marketedsuccessfully by two UK supermarkets but now withdrawn.)Processed derivatives of Monsanto GM soybeans engineered for tolerance to the glyphosate herbicideRoundup. (Imported and used widely in processed foods.)Processed derivatives of Ciba Geigy GM maize engineered to produce Bacillus thuringiensis insecticidalprotein.Some examples of applications pending approval under Regulation (EC) 258/97AgrEvo UK processed oil derived from oilseed rape (TOPAS 19/2) tolerant to glufosinate ammoniumherbicide.Flour, gluten, semolina, starch, glucose and oil derived from Monsanto maize (MON810) expressing theB. thuringiensis insecticidal protein gene crylA(b).Starch, oil, heat-processed and fermented products derived from AgrEvo maize (T25) tolerant toglufosinate ammonium herbicide.Food and food ingredients produced from Pioneer maize (MON809) expressing the B. thuringiensis genecryIA(b).Processed oil from Plant Genetic Systems male-sterile (DBN230-0028) and fertility restorer(DBN212-0005) oilseed rape lines.Riboflavin produced by Hoffman La Roche GM Bacillus subtilis.Some examples of food-relevant approvals under Directive 90/220/EECPlant Genetic Systems male-sterile swede rape resistant to glufosinate ammonium herbicide. (Used forbreeding activities.)Monsanto soybeans tolerant to glyphosate herbicide. (Approved for import and processing.)Bejo-Zaden BV chicory male-sterile and tolerant to glufosinate ammonium herbicide. (Used for breed-ing activities.)AgrEvo swede rape tolerant to glufosinate ammonium herbicide. (Approved for import and processing.)AgrEvo maize (T25) tolerant to glufosinate ammonium herbicide. (Approved for import and processing.)Novartis maize (Bt-11) tolerant to glufosinate ammonium herbicide and expressing the B. thuringiensisinsecticidal protein gene crylA(b). (Approved for import and processing.)Some examples of applications pending approval under Directive 90/220/EECDLF-Trifolium, Monsanto and Danisco Seed fodder-beet tolerant to glyphosate herbicide.Zeneca processing tomato engineered for reduced expression of the polygalacturonidase gene.Amylogene potato with altered starch composition.Novartis maize (Bt-11) tolerant to glufosinate ammonium herbicide and expressing the B. thuringiensisinsecticidal protein gene crylA(b) for cultivation.Pioneer maize (T25 + MON810) tolerant to glufosinate ammonium herbicide and expressing the B.thuringiensis insecticidal protein gene crylA(b).Monsanto maize (GA21) tolerant to glyphosate herbicide.

a Across Europe, the approval of GM food is controlled by EC Regulation 258/97 on ‘Novel foodsand novel food ingredients’. At present, all applications under this Regulation are pending. Prior tothis Regulation, the ACNFP made several approvals of GM foods for commercialization within the UK.Deliberate releases into the environment, including the agricultural use of GM crops, are controlled ata European level by Directive 90/220/EEC. The table gives examples of these approvals and pendingapplications. A more complete data set is available in the EC release Facts on GMOs in the EU (EC,2000).

the development of a gall. Substrates thatsupport the growth of Agrobacterium areproduced within the gall. By disarmingthis disease process, it is possible to engineerAgrobacterium vectors that will carry traitgenes into GM plants. In addition to theintegration of trait genes within the plantchromosomes, it is possible to integratetransgenes within the genomes of plantchloroplasts. Because these organelles havegenetic features that are related to those ofthe prokaryotic bacteria, this involves theuse of homologous recombination to ‘place’transgenes in a predetermined location.A diagrammatic overview of the varioustechniques used in the construction ofGM plants is included in Fig. 15.1.

An important part of the safety evalua-tion process is the provision of molecular datathat demonstrate the nature of foreign DNAthat has been inserted as a result of geneticmodification. The method used to transforma GM plant has a bearing on this. It iswell established that biolistic delivery oftenleads to extensive structural rearrangement ofthe integrated DNA. In some cases, this isso extensive that it is extremely difficult tounravel its DNA sequence. This has led toa preference for the use of Agrobacteriumdelivery, which can also cause the integrationof multiple DNA copies but is less proneto causing structural rearrangement of thetransgeneic DNA.

One limitation of Agrobacterium hadbeen its restriction to dicotyledonous plants,but recent developments have extendedits use to also include monocotyledonousplants such as rice and maize (Ishida et al.,1996).

Another distinct issue that has beenof concern to regulatory authorities is therealization that small fragments of transgeneDNA can sometimes be integrated at second-ary genome sites following biolistic delivery.Recently, this was found to have taken placein the GM soybean line that is widely usedcommercially. Careful analysis of this specificcase eliminated any safety concerns, but thisobservation does serve to emphasize theneed to investigate unintended secondaryintegration of potentially small fragments oftransgenic DNA.

Further options that influence safety andcontainment are:

• The use of a vector designed to delivertransgenic DNA into the plantchloroplast genome by homologousrecombination provides controlledintegration at a known site. In addition,the lack of chloroplasts in pollen pro-vides environmental containment oftransgenes.

• The separation of trait and plant selec-tion genes on distinct DNA fragmentsfacilitates their unlinked integration inthe plant genome. Alternatively, theplant selection gene can be flanked bysequences that are recognized by site-specific recombinases (e.g. cre/lox). Inboth cases, elimination of the selectiongene is possible using conventional plantcrosses.

• The elimination of unnecessary DNAthat was used during bacterial stagesis very straightforward, but this hasnot always been undertaken, leadingto problems with antibiotic resistancegenes and more complex rearrangementof trait DNA.

• The marker elimination process des-cribed in Fig. 15.1(b) is realized byconventional crosses that facilitate thesegregation of trait and selectiongenes. For site-specific recombination,the recombinase gene is introduced froma separate GM plant to effect markerdeletion, and this gene can be removedby a subsequent conventional cross.

The Use of Selectable Marker Genes

In order to effect the introduction and expres-sion of transgenic DNA, there is a universalneed to use some form of selection to dif-ferentiate the transformed cells. This usuallyinvolves the use of a selectable marker genethat may be physically linked to the chosentrait genes. For the primary transformation ofGM plants, antibiotic resistance genes haveoften been exploited as convenient selectionmarkers. The general importance of antibiot-ics for human and veterinary medicine and


the high profile of microbial drug resistancehave made this practice controversial. Thenature of the selection system used in GMOconstruction is a particular focus of safetyassessment.

The nptII gene, originally derived from E.coli transposon Tn5, is used frequently as aplant selection marker, and to this end it has

been equipped with a plant-specific promoterto facilitate its expression in plant cells. A com-prehensive argument about the safety of nptIIused as a plant-selectable marker has beendeveloped, and this was first formally pre-sented by Calgene (1990) and accepted by theUS FDA and other regulatory bodies. A sig-nificant factor is the limited importance of

334 M.J. Gasson

kanamycin and neomycin in the treatment ofbacterial infections in humans mainly as aconsequence of their relative toxicity. In addi-tion, it is recognized that antibiotic resistanceis already widespread in bacteria, and raregene transfer from a GM food source is

unlikely to be of practical consequence (Napet al., 1992).

Alternative selection markers that avoidthe use of antibiotic resistance genes arebecoming available, and mechanisms, suchas the cre/lox system (Dale and Ow, 1991),


Fig. 15.1. Overview of techniques used in the construction of GM plants; adapted from Gasson and Burke(2001).

have been developed to facilitate the removalof selection markers after GM plant construc-tion. Recently, Novartis Seeds announcedtheir intention to phase out the use ofantibiotic resistance genes. Their ‘Positech’marker system uses a selection for growthon mannose and relies on a gene forphosphomannose isomerase (Anon., 2000).Other approaches include the use of co-transformation of trait and selection genesfollowed by segregation of the latter. Thishas been effective in both Agrobacterium trans-formation (Komari et al., 1996) and biolistictransformation (ACNFP, 1996). Figure 15.1includes a schematic representation of thesealternative selection methods and strategies toeliminate selection markers from the final GMplant constructs. Despite these developments,GM plant material carrying antibiotic resis-tance genes continues to be put forward forconsideration by regulatory authorities whoneed to assess safety on the basis of objectivescientific criteria.

In contrast to nptII, several GM plantshave been developed in which other antibioticresistance genes have been introduced. Inthese cases, the antibiotic resistance geneshave not been engineered as plant selectionmarkers and they retain their original bacte-rial promoter. Genes in this category includebla, aad and nptIII, which confer resistance toampicillin, streptomycin/spectinomycin andamikacin, respectively. All of these antibioticshave greater use in clinical medicine thankanamycin and neomycin. The most commonreason for the presence of these genes in GMplants is that the trait gene was first engi-neered using a bacterial vector and E. coli clon-ing techniques where these bacterial selectionmarkers would be of value. Subsequently, thecomplete bacterial vector has been deliveredto the GM plant without first removing thesenow redundant DNA sequences. The bacterialmarker genes are not directly selectable inplants, and there is no good reason for theirpresence in GM material destined for use asfood. Despite this, there are scientifically validarguments that they do not pose a safetyhazard. Most persuasive is the fact that drugresistance is already widespread in bacteria asa direct consequence of the very extensive use

of antibiotics in human and veterinary medi-cine and as animal growth promoters. Also,the process of gene transfer from a GM plantto a microorganism is likely to be a very rareevent, and this is discussed in more detailbelow. This issue has been controversial forregulatory authorities, with somewhat differ-ent views emerging on the use of antibioticresistance genes in GM plants. The ACNFPhas adopted a cautious position (ACNFP,1995) and, in general, the inclusion of anti-biotic resistance genes in GM plants is widelydiscouraged.

The Safety Assessment Process

Despite differences in safety administration,notably between the USA and Europe, thescientific issues that are addressed during thesafety assessment of GM food are very con-sistent. Details have been published by boththe FDA (1992) and the EC (1997b), and anoverview of the main features is presented inBox 15.2. A key principle is that an integrated,stepwise and case-by-case approach isrequired. Safety assessment is aided by theuse of decision trees that give guidance on thespecific points that need to be addressed foran individual case. Substantial equivalenceplays a key role in identifying differencesbetween a GM food and its conventionalcounterpart, and these become a focus forfurther consideration. The role of substantialequivalence in safety assessment is discussedin detail below. The information that isrequired for the safety assessment includes:details of the genetic modification; the stabil-ity of the modification and potential for itstransfer; protein expression and its effecton function, allergy and toxicity; potentialsecondary effects; composition; intendeduses and effects of cooking and processing;and potential intake and dietary impact.

The Role of Substantial Equivalence

The concept of substantial equivalence playsan important role in the safety evaluation of

336 M.J. Gasson

GM food. First, it is important to stress thatthis concept is not in itself a safety assessmentprocess and, in particular, it is not intended toidentify hazard. Rather, it acts to identify keyissues that require detailed safety evaluation.Substantial equivalence was developedunder the guidance of the World HealthOrganization (WHO), the Organization forEconomic Cooperation and Development(OECD) and the Food and Agriculture Orga-nization of the United Nations (FAO) follow-ing on from the realization that conventionalapproaches to safety assessment, as usedfor pharmaceutical products, have seriouslimitations (FAO/WHO, 1991, 1996; OECD,1993). These limitations also explain why, incontrast to pharmaceuticals, animal feedingexperiments are less prominent in the safetyevaluation of novel foods. The generalapproach to safety testing using animal feed-ing involves the consumption of increasedamounts of the test substance until anadverse effect is detected. The sheer bulk ofmany whole foods prevents this increasedexposure, and data interpretation is com-promised by the fact that foods are complexmixtures of many different chemicals.Another critical point is that food contributesto nutrition. Toxicology testing using animalsdepends on the establishment of optimalnutrition to provide a controlled backgroundagainst which to evaluate any effect fromthe fed test substance. In the case of wholeGM food material, it is self-evident that anyobserved negative effect is as likely to arisefrom disturbed nutrition as it is to be caused

by novel technology. These limitations ofconventional animal testing approaches forthe safety evaluation of biotechnology prod-ucts were emphasized during the safetyassessment of other novel technologies: foodirradiation and mycoprotein. Thus there isa well-established scientific basis for seekingnew approaches to the safety evaluation ofnovel foods.

The substantial equivalence conceptrecognizes that many people have eatenconventional foods over a very long periodof time and this establishes an accepted levelof safety. Genetic modification involves theintroduction of only small changes, and thusa comparative approach can be used to revealany intended and unintended differencesbetween GM material and its conventionalcounterpart. The OECD (1993) describedsubstantial equivalence as embodying ‘theidea that existing organisms used as food,or as a source of food, can be used as thebasis for comparison when assessing thesafety of human consumption of a food orfood component that has been modifiedor is new’. By concentrating on the safetyassessment of the differences between a GMderivative and its conventional counterpart,it can be concluded that the establishedand accepted safety of the conventional foodhas not been compromised. The comparativeapproach involves the evaluation of a largebody of phenotypic data that includeagronomic traits and details of chemicalcomposition. Typically, the latter includesfats, proteins, solvent-extracted hydrophilic


Box 15.2. Overview of GM food safety assessment.

Major features of safety evaluation:

• Use of an integrated, stepwise and case-by-case approach• Intended and unintended differences between GM and conventional counterpart identified using the

substantial equivalence approach

Specific data required for:

• Details of the genetic modification• Stability of the modification and possibility of transfer of the modified genetic material• Potential secondary effects of the genetic modification• Composition of the GM food or food ingredient• Intended use and effects of processing or cooking• Potential intake and dietary impact

matter, fatty acids, amino acids, micro-nutrients, antinutrients, crude fibre, ash andmoisture content. Particular attention is givento any known toxins or allergens.

Substantial equivalence has been debatedwidely in recent years, and it has attractedsome vigorous criticism (Millstone et al., 1998;RSC, 2000) and equally robust defence (Burke,1999; Kearns and Mayers, 1999; Trewavasand Leaver, 1999). Recently, the FAO/WHO(2000) report, Safety Aspects of Genetically Mod-ified Foods of Plant Origin, addressed criticismof the application of the concept of substantialequivalence and reaffirmed its usefulness. Inparticular, it emphasized that the determina-tion of substantial equivalence is not in itselfan end point but rather the starting pointfor safety evaluation. The substantial equiva-lence concept is likely to be challenged furtheras genetic manipulation technology advances.The stacking of multiple traits and theengineering of some second-generation traitsthat may deliberately alter metabolic fluxare examples of some areas where theapplication of substantial equivalence mayprove complex.

One major concern is the capacity ofsubstantial equivalence to reveal unintendedconsequences of genetic modification. In thisregard, it is especially pertinent that conven-tional breeding is as likely as GM technologyto generate unintended effects. Conventionalbreeding can exploit techniques, such asmutagenesis and induced polyploidy throughthe use of colchicines, that might be expectedto lead to unpredictable genetic changes. It iscurious that there is relatively little concernwith respect to this very similar risk issue.Overall, the controversy surrounding sub-stantial equivalence serves to highlight thefact that its role in safety evaluation often ismisunderstood. Also, it is clear that substan-tial equivalence does indeed have limitations.

A current area of research activityconcerns the possibility of exploiting newmethodologies such as molecular profilingtechniques to provide a more detailedanalytical comparison. Currently, substantialequivalence involves an analysis of com-position and phenotypic parameters thatis undertaken with a targeted approach. Incontrast, molecular profiling is non-targeted

and more holistic. Molecular profiling encom-passes three distinct technologies: metabolicprofiling; proteomics; and DNA microarrays.These technologies interrogate sequentialsteps in the expression of genes throughmRNA, proteins and metabolism, as isillustrated in Fig. 15.2.

Metabolite profiling exploits a varietyof chemometric technologies to gather grossdata on the distribution of individual meta-bolites. Critically this does not involvethe prior selection of individual targetmolecules for quantification. In particular,gas chromatography–mass spectroscopy(GC–MS), nuclear magnetic resonance (NMR)and high performance liquid chromato-graphy (HPLC) techniques are capable ofdetecting, resolving and quantifying a widerange of compounds in a single sample.

Proteomics involves the use of two-dimensional gel analysis to separate individ-ual proteins that are present in a particulartissue. An example of this technique appliedto four different conventional varieties oftomato fruit is given in Fig. 15.3. While theseprofiling techniques are intended to revealdifferences using a holistic analysis, there is aneed to follow up with an assessment of thesafety implications for any differences that arefound. Proteomics has a valuable dimensionin this regard in that it is possible to identifyindividual protein spots by relating them tothe genes by which they were encoded. Thisrelies on the availability of genome sequencedata and thus the ease of application varies forindividual species. The identification processinvolves the use of MALDI-TOF mass spec-trometry to determine the mass of proteinfragments generated by specific proteolyticcleavage. Because the cleavage pattern ispredictable from the amino acid sequenceof a given protein, identification is possible.Alternatively Q-TOF mass spectrometry canbe used to gain sequence data directly froma protein spot, and this can be matched toinformation available in sequence databases.

In our own laboratory, this latterapproach has been used successfully to iden-tify an unknown protein that appeared in aGM tobacco plant. In this case, an experimen-tal transgenic line with a very severe morpho-logical abnormality was being studied. The

338 M.J. Gasson

gross proteome was remarkably similar tothat of a normal non-GM syngenic plant buttwo new protein spots were detected in theGM plants. These were both identified asthe same plant lectin, suggesting that a plantdefence mechanism had been stimulated inresponse to the genetic modification. The factthat one protein generated two protein spotsis important as it emphasizes the complexitythat can arise where post-translational modifi-cation of a protein takes place. Proteomics is apowerful technique that couples gel electro-phoresis with mass spectrometry and permitsthe visualization and possible identificationof several thousand proteins representing thedetectable part of the cell’s total complementof proteins (the proteome). The technical limi-tation is associated with the relatively slowand demanding gel electrophoresis process,but alternatives to this are currently beingdeveloped.

DNA microarrays with many thousandsof gene sequences arrayed on nylon or glass

substrates permit simultaneous examinationof the steady-state levels of the cell’s mRNAs(the transcriptome). Differential labelling ofmRNA extracted from a GM plant and itsconventional counterpart with fluorescentdyes, and their hybridization to a DNAmicroarray generates data on the relativeabundance of individual mRNA moleculesfor each individual gene. This approachdepends on the availability of sequencedata with which to design the probes used inmicroarrays and it gives instant data on theidentity of any differences that are revealed.

Molecular profiling techniques generatea very large volume of data, but the pointof interest is detecting biological componentsthat are changed in the GM food. Determiningthe significance of the changes and their rele-vance to safety assessment is difficult withouta background context based on the naturalvariation in the levels of the various biologicalcomponents in conventional varieties. Whilethe analytical power of molecular profiling


Fig. 15.2. Targets for molecular profiling.

approaches is without question, it remains tobe seen how useful they will be in practice forsafety evaluation. The new techniques willneed to be validated and, in addition, thesignificance of any observed differences willneed to be determined. Clearly, gene expres-sion under normal circumstances is dynamic,and any differences seen in a GM derivativewill need to be considered against this back-ground. The potential of molecular profilingwas highlighted by the Royal Society ofCanada (RSC, 2000) and has been debated byFAO/WHO (2000). In Europe, a significantprogramme of research supported by the ECand the UK Food Standards Agency is underway with the intention of investigating thepotential of molecular profiling and the rela-tive merits of the different techniques in thecontext of safety evaluation.

The Impact of the Introduced Trait

Clearly, in most cases, genetic modificationinvolves the introduction of a new intendedtrait to a conventional food material. In thecontext of substantial equivalence, it is thuslikely that an intended difference will bepresent at least at the level of the primaryfood material. This last point is significantbecause the processing of a food materialmay, to a greater or lesser extent, eliminatethe difference. A good example is purifiedoils prepared from GM plants such as oilseedrape or maize where it is difficult to differen-tiate these products from their conventionalcounterparts.

The introduced trait is often readilyamenable to safety assessment using conven-tional toxicology approaches. For example,

340 M.J. Gasson

Fig. 15.3. Examples of proteomic analysis of tomato fruits.

the insecticidal protein produced by Bacillusthuringiensis has been evaluated extensivelyin isolation from GM plants that have beentransformed with its gene. One frequentlyexpressed concern is that expression of thesame gene in bacteria and a GM plant maygenerate different protein products and thisneeds to be addressed. An obvious possibilityis that glycosylation may occur in plants whenit is not found in bacteria.

In addition to potential toxicity or otherphysiological effect, there is a need to assessthe potential that a newly expressed proteinmight be allergenic. This involves an assess-ment of the stability of the protein and data-base searching to determine homology withknown allergens. Known allergens share cer-tain common properties that include resis-tance to heat, stomach acid and degradationby gastrointestinal tract enzymes, all of whichcan be assessed.

FAO/WHO have undertaken severalrecent reviews of GM food safety assessmentthat have addressed the question of allergenicpotential, and this has culminated in thereport of an expert evaluation that tookplace very recently in Rome during 2001(FAO/WHO, 2001b). The FAO/WHO reportfrom the Rome meeting updated a widelyused decision tree that was designed to guidethe assessment of allergenic potential, and thisnew version is reproduced as Fig. 15.4. Thismost recent report drew a distinction betweenexpressed proteins that were derived fromsources with known problems of allergenicityand sources with no known allergenicity.In the former case, initial investigation isrecommended to be based on an analysis ofsequence homology to known allergens pres-ent in the source material. A negative result isfollowed up by immunoassays to investigatepossible immunoglobulin E (IgE) binding andpossibly in vivo studies using patients allergicto the source food. It was recognized that theuse of human in vivo methods would raiseethical issues and that their use would need tobe considered on a case-by-case basis. Wherethe expressed protein is derived from a sourcewith no known allergenicity, the initial inves-tigation is also focused on database searches.A negative result is followed by targeted

serum screening using samples containinghigh levels of IgE antibodies broadly relatedto the gene source. A positive result wouldsuggest that the protein was potentially aller-genic. Following a negative result, furtherstudies of pepsin resistance and the use ofsuitable animal models are recommended.The FAO/WHO consultation emphasized theimportance of maintaining and constantlyupdating an allergen database. Also it recog-nized that animal models had not beenevaluated for all food allergens but sufficientscientific evidence was available to suggestthat animal models will contribute valuableinformation regarding the allergenicity offoods derived from biotechnology.

Fate of Consumed DNA

Following the consumption of GM food, thefate of any novel introduced genetic materialis pertinent. Concern extends to the possibleuptake by host cells and by microorganismsthat inhabit the gastrointestinal tract. Thegeneral sensitivity of consumed DNA toinactivation and degradation provides one ofthe best established barriers to the transfer oftransgenes. The enzyme deoxyribonucleaseI produced by the salivary glands, pancreasand small intestine is a potent degradativeenzyme, and the low pH of the stomachremoves adenine and guanine residues fromDNA, thereby eliminating its biologicalactivity (Beever and Kemp, 2000).

Several recent studies add to our under-standing of DNA survival following its con-sumption. An in vivo study by Mercer et al.(1999) investigated the effect of human salivaon DNA survival using competitive polymer-ase chain reaction (PCR) and tested biologicalactivity by measuring transformation into thenaturally competent oral bacterium Strepto-coccus gordonii. Despite evidence of DNAdegradation, sufficient biologically activeDNA survived exposure to saliva to generatetransformants, albeit at a reduced frequency.Duggan et al. (2000) investigated DNA degra-dation by ovine saliva and ovine rumen fluid,measuring biological activity using E. coli


transformation. PCR amplification of DNAwas possible for 30 min after exposure torumen fluid, but transforming ability waslost within 1 min. In contrast, the ability totransform E. coli was retained even after 24 hexposure to saliva.

These studies suggest that, althoughdegraded, DNA remains available for trans-formation in the oral cavity. In contrast, itis inactivated rapidly and loses biologicalactivity further down the gastrointestinaltract. Chambers et al. (2000) investigated the

342 M.J. Gasson

Fig. 15.4. FAO/WHO decision tree guiding the assessment of allergenicity.

fate of the pUC18 ampicillin resistance genein vivo using chicken feeding experiments,and their findings reinforce this view. Bothbacteria carrying pUC18 and transgenic maizecarrying the bla gene were studied. PCR–restriction fragment length polymorphism(RFLP) was used to differentiate the test blagene from naturally occuring bla genes thatmay have been present in the gastrointestinaltract microflora. The pUC18 gene lacks a PstIsite that is present in the natural gene. Whilethe maize-derived gene could be detectedin the crops of experimental chickens, it didnot persist further down the intestinal tract.In contrast, bacteria carrying pUC18 providedprotection, allowing bla gene detectionthroughout the intestinal tract.

A somewhat different picture hasemerged from the work of Schubbert et al.(1994, 1997). These authors fed mice withbacteriophage M13mp18 DNA, and the fate ofthis foreign DNA in the animals was followedby several methods. Fragments of M13mp18DNA were detected in the contents of thesmall intestine, the caecum, the large intestine,the faeces and in blood. It was calculatedthat 2–4% of orally administered DNA wasdetected in the gastrointestinal tract and0.1–0.01% was retrieved from blood.M13mp18 DNA fragments were traced byPCR to peripheral leucocytes and located byfluorescence in situ hybridization (FISH) inabout 1 of 1000 white cells between 2 and 8 hafter feeding and in spleen or liver cells up to24 h after feeding. M13mp18 DNA could betraced by FISH in the columnar epithelialcells, in the leucocytes, in Peyer’s patches ofthe caecum wall, in liver cells, and in B cells,T cells and macrophages from spleen. Thesefindings suggest transport of foreign DNAthrough the intestinal wall and Peyer’spatches to peripheral blood leucocytes andinto several organs. Upon extended feeding,M13mp18 DNA could be cloned from totalspleen DNA into a λ vector. Schubbert et al.(1998) extended this study and obtained simi-lar results using a plasmid expressing the genefor green fluorescent protein. The broad con-clusion from this work is that consumed DNAmay survive, cross the gut epithelium, enterthe bloodstream and interact with mamma-lian cells. However, the significance of these

results has been questioned. The experimentswere artificial in the sense that mice wereexposed to large amounts of pure prokaryoticDNA. Beever and Kemp (2000) questioned thefact that the DNA used in these experimentswas unmethylated and contained sequenceslikely to cause up-regulation of inflammatorycell activity and to stimulate a significantimmune response. It is possible that thisinduced response contributed to the detectionof DNA in white blood cells.

Overall, there is substantial evidence tosuggest that DNA degradation is very effi-cient in the gastrointestinal tract but somemay remain biologically active in the mouthfor sufficient time to be taken up by naturallycompetent oral bacteria. The implicationsof the observations made by Doeffler andcolleagues are less clear, but suggest that,despite its hostile environment, some DNAmay survive in the gastrointestinal tract.

DNA Transfer from GM Plant Materialto Bacteria

Concern about gene transfer from GM plantmaterial into microorganisms needs to berelated to the nature of the genes that areinvolved. As already discussed, the useof antibiotic resistance genes has been a par-ticular concern. The most likely mechanismby which microorganisms might acquiretransgene DNA is by its release from GMfood and its subsequent uptake by naturallycompetent bacteria. This is a real possibilityin the oral cavity, as has been demonstratedby the work of Mercer et al. (1999) that isdescribed above. In general, the status of bac-terial gene transfer by natural genetic trans-formation has been reviewed extensivelyby Lorenz and Wackernagel (1994). A fewstudies have investigated DNA transfer fromGM plant material to microorganisms, andthese studies suggest that such an eventwould be extremely rare.

Schluter et al. (1995) used a modelsystem based on the plant pathogenic speciesErwinia chrysanthemi. A transgenic potato car-rying an integrated pBR322 plasmid wasused, and the plant pathogenic property of


Erwinia provided an intimate associationbetween the plant material and the potentialbacterial recipient. Erwinia causes soft rot bylysing plant tissues, and it is able to supportthe replication of the pBR322 plasmid and theexpression of its antibiotic resistance genes.Evidence for plant to bacterium transfer wasnot detected. Related in vitro experimentswere undertaken to provide quantitative dataon gene transfer. This was estimated to havea maximum probability of 5.8 × 10−14 for anexperiment using 0.9 g of potato tuber and6.4 × 108 bacteria.

DeVries and Wackernagel (1998) usednaturally competent Acinetobacter andfocused on the plant selection marker derivedfrom the nptII kanamycin resistance gene. Inthese experiments, the recipient Acinetobacterstrain carried an inactive homologue of thenptII gene that was under the control of a bac-terial promoter, and this strategy increasedthe likelihood of detecting a positive result.In this case, transforming DNA did notneed to be capable of autonomous replicationor illegitimate recombination to be detected.Homologous recombination between theplant-derived nptII gene and the mutant resi-dent gene would repair the defect in the lattergene, leading to the recovery of kanamycin-resistant transformants by marker rescue.This event was detected readily at a frequencyof 0.9 × 10−4 per nptII gene. However, whenartificial homology between the transgeneand the recipient genome was absent, trans-formation frequency fell below the 1.3 × 10−13

limit of detection. The marker rescue data sug-gest an efficient mechanism for DNA transferin which as few as 2.5 × 103 transgenic potatocells could generate a transformant, and res-cue of the kanamycin resistance marker waseffective even in the presence of a more than a6 × 106-fold excess of plant DNA. It is impor-tant to emphasize that this process dependson the provision of artificial homology andrepresents marker rescue rather than therecovery of unique DNA from the transgenicplant. However, it clearly demonstrates thatnaturally competent bacteria are very effec-tive at taking up transgene DNA and, in theevent of DNA homology being available,

transformation is likely to take place. Gebhardand Smalla (1998) obtained similar resultsusing marker rescue by Acinetobacter andDNA from GM sugarbeet.

As has been emphasized by Nielsen et al.(1998), selective pressure is of critical impor-tance in assessing the consequences of genetransfer. The occurrence of a gene transferevent in itself is unlikely to be of any great sig-nificance unless it led to selective advantagein the recipient. Conversely, the existence ofselective advantage could make even a veryrare genetic event important.

Post-market Monitoring

Post-market monitoring of GM foods isconsidered by many to be an importantcomponent of safety assurance. While thisa desirable objective, it is far from easy torealize. It is difficult to gather data on whichmembers of the population were eating par-ticular GM foods. A proposal in the UK to usesupermarket ‘loyalty’ cards was rejected ongrounds of the invasion of privacy. Also, GMfoods, if they are present in the diet of anypopulation, will be derived from such a rangeof crops and processes and present in such alarge range of foods that it will be extremelydifficult, if not impossible, to correlate con-sumption of any particular GM food to anyrecognizable syndrome. Experience in theUSA, where a large number of people havebeen eating products containing GM soyaand GM maize for a number of years withoutany ill effects, suggests that any effects willbe very small. Despite the difficulties, post-market monitoring continues to be evaluatedas a potentially valuable approach. Indeedthe recent FDA/WHO report on allergenicityrecommended:

Post-market surveillance is a valuable toolin the monitoring of adverse effects andlong-term sequelae of foods derivedfrom biotechnology and the Consultationrecognized that the feasibility of certainaspects of its implementation wouldneed further investigation. (FAO/WHO,2001b)

344 M.J. Gasson

Conclusions

GM food includes a range of distinct appli-cations of modern biotechnology, rangingfrom cell factories for the production ofnature-identical ingredients through to theprovision of GM fruits to be eaten fresh andunprocessed. The safety evaluation of GMfood involves well-established proceduresthat have been developed extensively overthe past decade, and these procedures aresubject to ongoing review and updating.There is broad consensus on the relevant riskissues, and structured approaches have beendesigned to focus on those issues that arerelevant to individual cases. To date, theseprocesses have proved effective in dealingwith GM food innovations but, as morecomplex trait development is undertaken,it will be important that safety assessmentcontinues to meet the challenge.

References

ACNFP (1994) Advisory Committee on Novel Foodsand Processes Annual Report 1993. Ministry ofAgriculture, Fisheries and Food/Departmentof Health, London, Crown Copyright PB1856.




AECB (2001) Crops on Trial. Report of the Agri-culture and Environment Biotechnology Com-mission. DTI Publication 5650/2K/08/01/NP.URN 01/1083. Available at: www.dti.gov.uk

Anon. (2000) Novartis pins hopes for GM seeds onnew marker system. Nature 406, 924.

Beever, D.E. and Kemp, C.F. (2000) Safety issuesassociated with the DNA in animal feedderived from genetically modified crops. Areview of scientific and regulatory procedures.Nutrition Abstracts and Reviews. Series B:Livestock Feeds and Feeding 70, 175–182.

Burke, D.C. (1999) No GM conspiracy. Nature 401,640–641.

Calgene Inc. (1990) KanR Gene: Safety and Use inthe Production of Genetically Engineered Plants.Request for an Advisory Opinion. Washington,DC, FDA Docket Number 90A–00416.

Chambers, P.A., Duggan, P.S., Heritage, J. andForbes, J.M. (2000) Survival of DNA fromfeedingstuffs in the gastrointestinal tract ofchickens (in press).

Dale, E.C. and Ow, D.W. (1991) Gene transferwith subsequent removal of the selectiongene from the host genome. Proceedings of theNational Academy of Sciences of the USA 88,10558–10562.

DeVries, J. and Wackernagel, W. (1998) Detection ofnptII (kanamycin resistance) genes in genomesof transgenic plants by marker-rescue trans-formation. Molecular and General Genetics 257,606–613.

Duggan, P.S., Chambers, P.A., Heritage, J. andForbes, J.M. (2000) Survival of free DNAencoding antibiotic resistance from transgenicmaize and the transformation activity ofDNA in ovine saliva, ovine rumen fluid andsilage effluent. FEMS Microbiology Letters 191,71–77.

EC (1997a) Regulation (EC) No 258/97 of theEuropean Parliament and of the Council of27 January 1997 concerning novel foods andnovel food ingredients. Available at: www.biosafety. ihe.be/GB/Dir .Eur.GB/FF/258_97.html

EC (1997b) Recommendations concerning thescientific aspects of the information necessaryto support applications for the placing on themarket of novel foods and novel food ingredi-ents. Official Journal of the European CommunitiesL253, 1–36.

EC (2000) Facts on GMOs in the EU. Available at:europa.eu.int/comm/dgs/health_consumer/library/press/press63_en.pdf

FAO/WHO (1991) Strategies for Assessing the Safetyof Foods Produced by Biotechnology. Report of ajoint FAO/WHO consultation, Geneva.

FAO/WHO (1996) Biotechnology and Food Safety.Report of a joint FAO/WHO consultation,Geneva.

FAO/WHO (2000) Safety Aspects of GeneticallyModified Foods of Plant Origin. Report of a jointFAO/WHO consultation, Geneva. Availableat: www.who.int/fsf

FAO/WHO (2001a) Safety Assessment ofFoods Derived from Genetically ModifiedMicro-organisms. Report of a joint FAO/WHOconsultation, Geneva.


FAO/WHO (2001b) Evaluation of Allergenicity ofGenetically Modified Foods. Report of a jointFAO/WHO consultation, Rome.

FDA (1992) FDA’s policy for foods developedby biotechnology. Available at: vm.cfsan.fda.gov/~comm/afdo2k.html

FDA (2001a) Premarket notice concerningbioengineered foods. Available at: vm.cfsan.fda.gov/~Ird/fr010118.html

FDA (2001b) FDA announces proposal and draftguidance for food developed through bio-technology. Available at: www.cfsan.fda.gov/~Ird/hhbioen3.html

Gasson, M.J. and Burke, D.B. (2001) Scientificperspectives on regulating the safety ofgenetically modified foods. Nature Reviews inGenetics 2, 217–222.

Gebhard, F. and Smalla, K. (1998) Transformation ofAcinetobacter sp. strain BD413 by transgenicsugar beet DNA. Applied and EnvironmentalMicrobiology 64, 1550–1554.

ILSI (1999) Safety Assessment of Viable GeneticallyModified Micro-organisms Used in Food. ILSIPress, Brussels.

Ishida, Y., Saito, H., Ohta, S., Hiei, Y., Komari, T. andKumashiro, T. (1996) High efficiency trans-formation of maize (Zea mays L.) mediated byAgrobacterium tumefaciens. Nature Biotechnology14, 745–750.

Kearns, P. and Mayers, P. (1999) Substantialequivalence is a useful tool. Nature 401, 640.

Komari, T., Hiei, Y., Saito, Y., Murai, N. andKumashiro, T. (1996) Vectors carrying twoseparate T-DNAs for co-transformation ofhigher plants mediated by Agrobacteriumtumefaciens and segregation of the trans-formants freed from selection marker. PlantJournal 10, 165–174.

Lorenz, M.G. and Wackernagel, W. (1994) Bacterialgene transfer by natural genetic transfor-mation in the environment. MicrobiologicalReviews 58, 563–602.

Mercer, D.K., Scott, K.P., Bruce-Johnson, W.A.,Glover, L.A. and Flint, H.J. (1999) Fate of freeDNA and transformation of the oral bacteriumStreptococcus gordonii DL1 by plasmid DNA

in human saliva. Applied and EnvironmentalMicrobiology 65, 6–10.

Millstone, E., Brunner, E. and Mayer, S. (1998)Beyond ‘substantial equivalence’. Nature401, 525–526.

Nap, J.P., Bijovoet, J. and Stiekma, W.J. (1992)Biosafety of kanamycin-resistant transgenicplants. Transgenic Research 1, 239–249.

Nielsen, K.M., Bones, A.M., Smalla, K. andVan Elsas, J.D. (1998) Horizontal gene transferfrom transgenic plants to terrestrial bacteria –a rare event? FEMS Microbiology Reviews 22,79–103.

OECD (1993) Safety Evaluation of Foods Produced byModern Biotechnology – Concepts and Principles.OECD, Paris.

RSC (2000) The Royal Society of Canada Expert panelon the future of food biotechnology.Available at: www.rsc.ca/foodbiotechnology/GmreportEN.pdf

Schluter, K., Futterer, J. and Potrykus, I. (1995)‘Horizontal’ gene transfer from a trans-genic potato line to a bacterial pathogen(Erwinia chysanthemi) occurs – if at all – at anextremely low frequency. Biotechnology 13,1094–1098.

Schubbert, R., Lettmann, C. and Doerfler, W. (1994)Ingested foreign (phage M13) DNA survivestransiently in the gastrointestinal tract andenters the bloodstream of mice. Molecular andGeneral Genetics 242, 495–504.

Schubbert, R., Renz, D., Schmitz, B. and Doerfler, W.(1997) Foreign (M13) DNA ingested by micereaches peripheral leukocytes, spleen, andliver via the intestinal wall mucosa and can becovalently linked to mouse DNA. Proceedingsof the National Academy of Sciences of the USA 94,961–966.

Schubbert, R., Hohlweg, U., Renz, D. and Doerfler,W. (1998) On the fate of orally ingested foreignDNA in mice: chromosomal association andplacental transmission to the fetus. Molecularand General Genetics 259, 569–576.

Trewavas, T. and Leaver, C.J. (1999) Conventionalcrops are the test of GM prejudice. Nature 401,640.

346 M.J. Gasson

16 Genetically Modified Foods: PotentialHuman Health Effects

A. Pusztai,1* S. Bardocz1 and S.W.B. Ewen21Formerly of The Rowett Research Institute, Aberdeen, UK; 2Department of

Pathology, University of Aberdeen, Forresterhill, Aberdeen, UK

Introduction

The scope of this review is restricted todata-based considerations about the safety ofgenetically modified (GM) foods of plant ori-gin for health. No opinions unless supportedby experimental results will be discussed.The emphasis will be on papers published inpeer-reviewed journals. A few articles will bementioned from non-peer-reviewed journalsbut only if they influenced the developmentof science-based ideas for the regulatoryprocess. Environmental issues will not bedealt with.

Safety evaluation of whole foods derivedfrom crops with considerable natural varia-bility is more difficult than that of a singlechemical, pharmaceutical or food additive, ordefined mixtures of them. Published results oftests for toxicity and nutritional wholesome-ness of complex foodstuffs are therefore fewand far between. A recent comment in Sciencedescribed this in its title: ‘Health risks ofgenetically modified foods: many opinionsbut few data’ (Domingo, 2000). Even a cursorylook at the list of references of a recent majorreview on food safety issues (Kuiper et al.,2001) showed that most of the publicationsreferred to were non-peer-reviewed institu-tional opinions or envisaged future scientific

and methodological developments for safetyassessments, but were short on actual pub-lished scientific papers on which a reliabledatabase of safety could be founded. Judgingby the absence of published data in peer-reviewed scientific literature, apparently nohuman clinical trials with GM food have everbeen conducted. Most attempts to establishthe safety of GM food have been indirect. Atbest, inferences have been drawn from animaltrials, but the preferred approach is to usecompositional comparisons between the GMfoodstuff and its traditional counterpart. Ifthese results show no significant differences,the two foodstuffs are ‘substantially equiva-lent’, meaning that the GM food is as safe asthe non-GM food. Thus, as the regulation isalmost exclusively dependent on ‘substantialequivalence’, the published results of GMfood analyses and inferences drawn fromthem for health will be examined critically inthis review.

In genetic modification, the intendedgene is incorporated into the genome of a cropusing a vector containing several other genes,including, as a minimum, viral promoters,transcription terminators, antibiotic resis-tance marker genes and reporter genes.Although in GM food safety the role of theintended gene is very important, the potential



effects of these other genes need also to betaken into account because other parts of theconstruct or the insertion of the vector couldcontribute substantially to the overall effect(Ewen and Pusztai, 1999a). There is in factsome evidence that some of the other genes ofthe vector may have an effect on safety. This isparticularly so as it is now known that DNAdoes not always break down in the alimentarytract (Schubbert et al., 1994, 1998; Hohlwegand Doerfler, 2001). This opens up the pos-sibility that the antibiotic resistance markergene, in addition to others, may be taken up bybacteria in the digestive tract and contributeto the spreading of antibiotic resistance viahuman gut bacteria. In this context, one poten-tially important observation was that a sub-stantial proportion (6–25%) of a geneticallyengineered plasmid survived a 1-h exposureto human saliva (Mercer et al., 1999). Partiallydegraded plasmid DNA also successfullytransformed Streptococcus gordonii, a bacte-rium that normally lives in the human mouth.Saliva also contains factors which increase theability of bacteria to become transformed bynaked DNA. Therefore, the prospect of theuptake of undegraded or partially degradedvector genes, including the antibiotic resis-tance gene, will have to be seriously consid-ered. However, the main concern in GM foodsafety is what are the direct effects of theexpression of the main intended gene afterits insertion into the plant genome via a geneconstruct. An additional concern is that thismay also cause significant, indirect and unin-tended effects on the expression and function-ality of the plant’s own genes. The number ofcopies of the construct inserted and their loca-tion in the plant genome (pleiotropic effect)are of particular importance in this respect,with the possibility that many unexpectedchanges may occur. This possibility is in factgenerally accepted, and the inadequacy ofthe currently used methods to detect themis frequently acknowledged (Kuiper et al.,2001). Pleiotropic effects always occur withboth conventional cross-breeding and geneticengineering, and their unwanted conse-quences usually are eliminated by empiricallyselecting for the desired trait and discardingthe potentially harmful ones. Some of thesechanges are unpredictable and therefore we

can only compare the known properties andconstituents but cannot look for, or evenless analyse, unknown components. Thisimposes limitations on our selection criteria.Reliance based solely on chemical analysisof macro/micronutrients and known toxinsis at best inadequate and, at worst, danger-ous. More sophisticated analytical methodsneed to be devised, such as mRNAfingerprinting, proteomics and secondarymetabolite profiling (Kuiper et al., 1999).However, and most importantly, there isan urgent need to develop comprehensivetoxicological/nutritional methods to screenfor the unintended potentially deleteriousconsequences for human/animal healthof genetic manipulation to pinpoint theproblems in advance of the incorporation ofthe GM foodstuff into the food chain (Ewenand Pusztai, 1999b). Although some limitedanimal tests have been done, only a few ofthese have been published. However, datafrom some of these studies recently have beenplaced on the Internet. Although they werenot peer reviewed, they were incorporatedinto this review because of their potentialimportance for other scientists.

Non-peer-reviewed Safety Tests onCommercial GM Crops in the

Public Domain

FLAVR SAVR tomatoes

The first example of official safety evalu-ations of a GM crop, Calgene’s FLAVRSAVR tomato, including a 28-day rat feed-ing trial, was commissioned by Calgene forthe Food and Drug Administration (FDA)before its general release. Although thedetails of this study have never been pub-lished properly, because this work had suchan extraordinarily major effect and influ-enced GM food regulation in the USA andelsewhere, there is a compelling need to ana-lyse the methods used and the conclusionsreached. Fortunately, as a result of a courtcase in the USA (Alliance for Bio-Integrityet al. vs. Shalala et al.), most data in the FDA’sfiles are now on the Internet in the public

348 A. Pusztai et al.

domain and can therefore be evaluated(Alliance for Bio-Integrity, 1998).

This GM tomato study shows most ofthe problems which may be encountered inGM food safety evaluation, particularly if, likethe tomato, they are fruits rather than food-stuffs and their protein and energy contentsare insufficient for supporting the growth ofyoung animals. The methods used and resultsobtained in this study are important not onlyfor their own sake but also for their influenceon the process of regulation.

Substantial equivalence

As ‘substantial equivalence’ features soprominently in GM food regulation (Kuiperet al., 2001), including in this GM tomatostudy, there is a need to look more closelyat this concept. This issue has been dealt within some depth by a recent article (Millstoneet al., 1999) in which the problems with thisconcept were highlighted, such as that ‘sub-stantial equivalence’ has never been definedproperly and that there are no legally bindingrules on how to establish it in practice.

Differences in growth conditions canhave a serious impact on composition and,therefore, in the absence of specification of theorigin and the conditions of cultivation ofthe different GM and non-GM samples, strictscientific comparisons cannot be made. Theseare not valid unless the parent line is grownside by side with the GM line. Comparisonswith historical or literary values have onlylimited scientific validity.

‘Substantial equivalence’ is a crude, non-scientific concept. It provides a loophole forthe GM biotechnology companies not to carryout nutritional and toxicological animal teststo establish whether the biological effect ofthe GM crop-based foodstuff is substantiallyequivalent to that of its non-GM counterpart.It therefore allows them to claim that there isno need for biological testing because the GMcrops are similar to their conventional coun-terpart, while on the other hand, because theycontain novel genes from other organism(s),they are patentable. However, unintentionaland unpredictable changes can occur in plantsbecause of the incorporation and positioningof the vector in the plant genome. It cannot

therefore be known which of the hundreds ofcomponents of the GM crop may carry toxicor allergenic properties. As most of theseare unknown, by definition, they cannotbe included in analytical comparisons.Determination of the amounts of protein,carbohydrates, fats and other nutrients canonly be a starting point. The consump-tion of minor and unexpected constituentsof potentially high biological activity mayhave considerable and disproportionatelylarge effects on the digestive tract. Theirpresence, therefore, can only be revealed fromanimal studies, and this makes it imperativethat these are performed with a flawlessdesign and experimentation.

The FLAVR SAVR tomato was pro-duced by ‘antisense’ GM technology. As partof its safety evaluation, it was subjectedto compositional analysis for total protein,vitamins and minerals to establish whetherany unexpected changes in gross fruit compo-sition had arisen as a result of the integrationof the FLAVR SAVR and kanr genes intothe tomato genome. It was claimed that nosignificant changes were found and that thecontents of potentially toxic glycoalkaloids,particularly tomatine, and to a lesser extentsolanine and chaconine, were also similar(Redenbaugh et al., 1992) and therefore thisGM tomato was substantially equivalent toother non-GM tomato lines. However, to sup-plement these, several feeding studies werealso performed by commercial laboratories atthe request of the FDA.

Acute toxicity

First, range-finding, limit acute oral toxicitytests of the processed tomatoes in rats werecarried out by the IIT Research Institute of theLife Sciences Department (Chicago, Illinois,USA). A single dose of the homogenates pre-pared from about 80 g of various GM andcontrol tomatoes, respectively, was adminis-tered (15 ml kg−1) by gavage to groups ofHarlan Sprague–Dawley rats (five male orfemale rats per group) fed ad libitum on ratchow for 14 days to establish whether theGM tomatoes were toxic or not. As claimed,no test substance-related mortalities occurredand increases in mean body weights were not

GM Foods: Potential Human Health Effects 349

significantly different between GM andcontrol groups. However, as the range of therats’ starting weights was unacceptably wide(female rats weighed 131–186 g (± 18%) andmale rats 159–254 g (± 23%)), in such a short(14 day) study with five rats per group,it would have been difficult for significantdifferences to develop. For comparison, onlya few per cent variation in starting weightsis permitted in papers published in high-quality nutritional journals. Thus, the poordesign of this feeding study largely invali-dated the conclusions that GM tomatoeswere not toxic. To supplement these, threemore rat feedings studies of similar designwere carried out by International Researchand Development Corporation (Mattawan,Michigan, USA).

Twenty-eight-day toxicology/histology study

Of the three studies, the most complete setof data is available for the second. In this,four groups of rats (20 males and 20 femalesper group) fed standard rat chow for 28 dayswere gavaged twice daily with homogenizedtomatoes (15 ml kg−1). Two groups weregiven GM tomatoes, CR3–613 or CR3–623(CR3–623 is the commercial FLAVR SAVRtomato). There were two control groups, oneof which was gavaged with the parent CR3tomato homogenates and a second controlgroup in which the rats were gavaged withwater even though the relevance of thisgroup is somewhat questionable. At therequest of Calgene, an expert panel wasretained (ENVIRON Corp., Arlington, Vir-ginia, USA) to evaluate the data. They con-cluded that gavaging rats with GM tomatopurée resulted in no significant changes inbody weight, food consumption and clinicalchemistry or haematology parameters incomparison with control tomatoes. However,there was a possible treatment-relatedincrease in glandular stomach erosion/necrosis in four out of 20 female rats but nonein the controls or in male rats at the end of the28-day feeding period. The number of fourfemale rats was increased to seven when thehistology slides were re-scored by PATHCO,

an independent pathology working group.This prompted a repeat study in which thedose of the tomato purée was increased bytwofold. Unfortunately, in this study, someof the CR3 control and CR3–623 GM tomatolines were grown at different locations andharvested at different times from those in thesecond experiment. However, this was notregarded as important by the expert paneleven though, when the same tomatoes wereused as in the second experiment, the resultsappeared to show similar tendencies; two outof the 15 females developed stomach glandu-lar erosions with the GM tomatoes, whilenone were found in the control females.However, in a not clearly understandableway, the ENVIRON panel concluded that thelesion of glandular erosion was not related tothe administration of GM tomatoes. Accord-ing to them, such lesions occur spontaneouslyin animals that are stressed or given muco-lytic agents, when food is restricted or whenanimals are restrained in cages, even thoughthese parameters have not been investigatedsystematically. Moreover, none of these cir-cumstances applied, since tomatoes containno mucolytic agents, food was provided adlibitum and the rats were not restrained. Itwas also suggested that, because the lesionswere possibly of short duration, they wereincidental, not related to the test materialand would have healed spontaneously.Unfortunately, none of these assumptionswas confirmed by further experimentation asno samples other than those at the end of the28-day experiment were taken to probe intothe timing and reversibility of the incidenceof the stomach lesions. Clearly, the resultsof these three studies should have promptedmore experimentation to investigate in moredetail the effect of GM tomatoes on stomachhistology and, what is even more important,these studies should have been extended toinclude the possible effects of GM tomatoeson both the small and large intestines.

The red or dark red pin-point lesionspresent in the stomach of female rats whichwere described as necrosis would be termed‘erosion’ in human pathology, which mayhave sequelae, such as life-endangering


haemorrhage. Erosions cannot be termed‘mild’, as unpredictable haemorrhage canoccur in the elderly human, particularly onlow dosage aspirin to prevent thromboticevents, and synergy with transgenic tomatoesmay occur. The assumption that the lesionsare related to stress does not explain the lowincidence in other groups, particularly in thesecond study. The relevance and significanceof gastric erosions in the human may bea matter of life and death in the older agegroups. It has been implied that pathologistsin general might not report such a lesion but,in the present era of vexatious litigation, men-tion would have been made in any humanpathologist’s report to avoid an accusationof negligence. This may not be required in vet-erinary pathology but these rat studies weredone with humans in mind and therefore thepathology findings must be put in this humancontext. It is probably true to suggest thatthese lesions are of short duration, but theserious nature of erosive lesions should notbe trivialized. This is the more serious becauseseven out of 40 rats eating GM tomatoes diedwithin 2 weeks. The nature of these deathswas not specified and the evidence that theywere not related to the ingestion of transgenictomatoes was inconclusive.

In a further development, the ScientificCommittee on Food of the European Commis-sion Directorate C (2000) concurred with theconclusion reached in the US Food and DrugAdministration (1994) memorandum. In theiropinion, although the results showed an unex-plained disparity, they were not supportiveof a substance-related effect of the FLAVRSAVR tomato. However, it is likely thatthe EU Committee may not have seen allthe primary data and their opinion wouldtherefore have been based on incompleteevidence. It is also regrettable that, byascribing the gastric erosions in rats to ‘anartefact of gavage studies’, the EU Committeehas in fact labelled the scientists carrying outthe work as incompetent. As these erosionswere found at the end of a 28-day study dur-ing which 160 rats were gavaged twice dailywith tomatoes, it is unlikely that even poorlytrained workers would not have become more

competent, so as to avoid causing such ananomaly.

Effects on body weight, food intakeand organ weights

The conclusion of the ENVIRON panel thatfeeding rats on GM tomatoes (CR3–623)for 28 days had no effect on weight gain,feed intake and organ weights could not bejustified because the starting weights of therats were so widely different – a range of130–258 g (± 33%) for males and 114–175 g(± 21%) for females – that finding significantdifferences in weight gain, feed intake andorgan weights was not likely. Indeed, weightgains varied between wide limits (102–230 gfor males and 46–127 g for females) in 28days. Even under these conditions, althoughthe average starting weight of the male ratsgavaged with CR3–623 GM tomatoes wasthe highest (148.1 g), their final weight(316.5 g) was the lowest. Accordingly, therats gavaged with GM tomatoes grew theleast of the four groups of rats. The feedintake of the different groups also variedbetween wide limits; 133–203 g for males and102–153 g for females. Not surprisingly, thefeed conversion efficiency (weight gain/totalfeed intake) of female rats on GM tomatoes(0.152) was significantly (P < 0.05) less thanthat (0.167) obtained for female rats oncontrol non-GM CR3 tomatoes.

The large range of starting weight differ-ences also excluded the possibility of findingsignificant differences in the organ weightsof the four groups of rats. The standarddeviations of mean values were very large, insome instances more than 20%. It is the moreremarkable that, even under these conditions,some differences in organ weights werefound, including the testes for males andthe thyroid/parathyroid for females. Findingno significant differences in biochemical,haematology and ophthalmology parametersbetween GM and non-GM tomatoes was notunexpected either, because of the large initialbody weight differences.

Overall, it is regrettable that these rattoxicological feeding studies were poorly


designed, as a great deal of effort, work andmoney must have been spent on them andso much rested on the outcome. The FDA’sconclusion that FLAVR SAVR presentedno more dangers to consumers than ordinarytomatoes does not therefore appear to rest ongood science and evidence which could standup to critical examination. Rather tellingly,the results of these studies have never beenpublished in peer-reviewed journals. Thestudy as described not only raises questionsabout the design, methods and conclusionsfor this study but also whether they couldhave any general validity for other GM foods.In this light, it is the more surprising thatafter these studies the FDA has required nonutritional/toxicological testing of other GMfoods.

Aventis’s Chardon LL herbicide-resistantGM maize

Due to the UK government’s attempt to placeChardon LL seed on the National List, a partof the supporting evidence submitted byAventis contained data on the composition oftwo lines of seed to establish their substantialequivalence to the conventional parent maizeline. The evidence also included the results ofa 14-day rat feeding study. All this is to befound in a file deposited by the Ministry ofAgriculture, Fisheries and Food (MAFF) withthe British Library (British Library File, 1997).

Compositional analysis

In the absence of specifying the originand conditions of cultivation of the differentGM and non-GM samples, strict scientificcomparisons could not be made betweenthem. However, even under these conditions,the composition of T14 and T25 GM maizeexpressing phosphinothricin acetyltrans-ferase enzyme (PAT-PROTEIN) showedmany statistically significant differences infat and carbohydrate contents in comparisonwith non-GM grain samples, and fat, proteinand fibre between silage samples from GMand non-GM maize. Thus, the conclusionthat GM maize is not ‘materially different’

from current commercial varieties cannot beregarded as valid.

Repeated dose oral toxicity (14-day feeding)study in rats

The rationale for this study was to assess thecumulative toxicity of PAT-PROTEIN givento rats in their diet for 14 days and to providea rational basis for toxicological risk assess-ment in man. Although testing of the PAT-PROTEIN can be commended, this study wasno substitute for the nutritional testing of theentire GM plant, seeds, vegetative parts andsilage in all target animal species. Withoutthese, the potentially harmful, unintendedand unpredictable effects of the gene transfer,other components of the vector and geneinsertion (positioning effect) cannot beestablished or excluded.

Unfortunately, as the design of the exper-iment was faulty, it is difficult to draw validconclusions from a feeding study, carried outwith five rats per group, in which the startingweight of the rats varied by more than ±20%(53–82 g for males and 50–74 for females)rather than the usual ±2%. For any differencesto reach significance, they needed to exceed± 20%, and to achieve this in a 14-day studywould have required catastrophic experimen-tal conditions. The five rats per group werenot housed singly and therefore their individ-ual feed intakes could not be monitored eventhough the huge differences in the startingweights should have led to major differencesin the feed intakes of the individual rats.Moreover, the group feed intakes were notmeasured continuously. There were fourgroups of rats (five male/female rats pergroup) in the experiment. However, rats ingroup 1 were fed a different diet (full ratchow) from the other three groups and there-fore group 1 was not appropriate for (stat-istical) comparisons. The diet of the secondgroup contained 5 g kg−1 and the third grouphad 50 g kg−1 PAT-PROTEIN mixed in with45 and 0 g kg−1, respectively, of commercial(SOJAMIN, KLIBA Muhlen AG) low soybeanprotein diet (11% raw protein). The diet of thefourth group contained 50 g kg−1 SOJAMINbut no PAT-PROTEIN. Thus, for statisticalanalysis, the second and third groups ought to


have been compared with rats in the fourthgroup. Curiously, although the main targetorgan of the PAT-PROTEIN fed to rats wasthe digestive tract (and pancreas), the weightsof these were not measured. This is a majorexperimental design fault.

The starting weight and the feed intake ofthe third group (high PAT-PROTEIN) werethe highest, but they ended up with the lowestfinal body weight. This indicated an elevatedmetabolic activity, probably induced by thePAT-PROTEIN. Our analysis of variance(ANOVA) shows that the weight gain forboth male and female rats on the high PAT-PROTEIN diet (group 3: 65.2 and 43.6 g formales and females, respectively) was sig-nificantly (P < 0.05) less than that of eitherthe fourth group (control: 72.8 and 48.8 g formales and females, respectively) or group 2(low PAT-PROTEIN diet: 73.4 and 44.4 gfor males and females, respectively). As PAT-PROTEIN reduced feed conversion efficiency,it is potentially harmful. The conclusion that‘there were no differences which could beattributed to treatment with the test article’was therefore not valid. Similarly, that ‘therewere no changes on … clinical biochemistryand urine analysis after 14 days’ is not valideither as the authors’ own results describeddifferences between the groups in glucose,cholesterol, triglyceride and phospholipidlevels, indicating an increased metabolicfunctional load in the rats. It is unexplainedwhy these differences were dismissed bythe authors as incidental and unrelated tothe treatment. Our ANOVA analysis revealedthat the urine output in rats on the high PAT-PROTEIN diet was significantly (P < 0.05)reduced, indicating treatment-related effects(urine output of 5.4 and 4.4 ml for males andfemales in group 3 vs. 7.1 and 6.5 ml for malesand females, respectively, in control group 4).

The large differences in the startingweight of the rats probably prevented find-ing significant differences in organ weights.However, even under these conditions, ratsfed the high level PAT-PROTEIN diet (thirdgroup) had the lowest liver, thymus andspleen weights of all groups (even thoughthe differences with controls were notsignificant). This is of particular importancebecause the macroscopic findings indicated

thymus foci in 20–40% of the animals fed dietscontaining the PAT-PROTEIN.

In conclusion, the design and executionof this feeding study were poor and, contraryto the authors’ conclusions, the resultsindicated treatment-related effects inducedby PAT-PROTEIN (of unspecified origin).The results therefore could not be takenas evidence that the transfer of its gene intomaize represented no risk for the rat and,by inference, for humans, particularly as nogut histology studies have been completedso far. Finally, a recent publication (Chiteret al., 2000) showed that DNA survived inintact form or slightly fragmented unless theGM maize was heat processed extensively.Therefore, the possibility exists that withunderprocessed maize products humans andanimals might be exposed to the DNA used inthe genetic engineering.

Compositional Studies Published inPeer-reviewed Journals

Herbicide-resistant soybean

Befitting its importance in both human andanimal nutrition, a great deal of attention hasbeen given to the compositional analysis ofherbicide-resistant and other GM soybeans.Several publications appeared in nutritionaland other journals demonstrating the compos-itional ‘substantial equivalence’ of GM andnon-GM soya. Thus, it was claimed that themacronutrient composition of glyphosate-tolerant soybean (GTS) seeds resulting fromthe transformation of conventional soybeanwith a gene encoding 5-enolpyruvylshikimate-3-phosphate synthase from Agrobacterium,to make the soya herbicide resistant, wasequivalent to that of conventional soybeans.This applied equally to GTS unsprayed withglyphosate (Padgette et al., 1996) or sprayedwith this herbicide (Taylor et al., 1999). It wasclaimed that the results of proximate chemi-cal analyses of the contents of crude protein,oil, ash, fibre, carbohydrates and amino acidsof solvent-extracted and toasted or untoastedsoybean meals of unsprayed GTS and controlsoybean had shown that all these lines were


substantially equivalent (Padgette et al.,1996). Similar findings were described forsprayed GTS (Taylor et al., 1999). Althoughthis appeared to be true for most macro-nutrients, several significant differencesbetween GM and control lines, such as inash, fat and carbohydrate contents, were alsofound (Padgette et al., 1996). However, thesewere not regarded as having biologicalsignificance.

A closer inspection of the data in thepapers, however, revealed that the statisticalcomparison of the macronutrients of GMand non-GM lines was not scientifically valid.Instead of comparing their amounts in asufficiently large number of samples of eachindividual GTS with its appropriate individ-ual parent line grown side by side at thesame location and harvested at the same timeto establish whether they were ‘substantiallyequivalent’, what the authors compared wasa large number of different samples from dif-ferent locations and harvest times. As growthconditions have a major influence on seedcomposition, the range of the amounts of con-stituents in the different samples, regardlessof whether they were GM or non-GM, wasso great (±10% or more) that the chancesof finding statistically significant differenceswere unreal. It is possible that from a practicalpoint of view the variation in protein concen-tration of samples of the three lines of between36.8 and 45% would fall into the normal rangeof agronomic variability of soybeans andtherefore may not be of major concern foragronomists. However, this comparison is notstrict enough to establish whether the geneticmodification introduced any unintendedcompositional changes. What is remarkableis that, even with this approach, many sig-nificant changes in macronutrient levels werefound. Thus, the claim of ‘substantial equiva-lence’ of GTS lines with non-GM soybean isnot supported by rigorous scientific evidence.

The potential importance in humanhealth of natural isoflavones, such as genistein,daidzein and coumestrol present in soybeans,is generally recognized. It was, therefore, ofconsiderable interest whether any changesoccurred in these components as a resultof genetic modification. Here the publishedevidence is controversial. Thus, while in

some studies no meaningful differences werereported (Padgette et al., 1996; Taylor et al.,1999), an independent study claimed that GMsoya samples had consistently contained sig-nificantly fewer isoflavones than the parentcultivars (Lappe et al., 1999). In one respect, allauthors agreed, i.e. that the isoflavone contentof soybean seeds showed considerable vari-ability between sites and was dependent onagronomic conditions. However, Lappe et al.(1999) went further and claimed that, whilethe variability of the GM samples was indeedconsiderable, conventional soybeans showedless variation in isoflavone content. As theisoflavone content of soybeans might affecthuman health, there needs to be more aware-ness of potential health problems due tothis variability. While the precise details ofthe changes in isoflavone content on geneticmodification will have to be established inthe future, to ensure clinical consistency, theorigin and the actual phyto-oestrogen levelsin soybean may need to be standardized.

In the study by Padgette et al. (1996),no significant differences were found in thelevels of antinutrients, such as trypsin inhibi-tors, lectin and oligosaccharide flatulencefactors, between solvent-extracted, toasted oruntoasted GM and non-GM soybean seeds.However, the comparisons were made bythe same method as for macronutrients andtherefore the large range of natural variabilityexcluded the possibility of finding significantdifferences. Interestingly, in single soybeanmeal samples of each of the two GTS andparent lines, the trypsin inhibitor (also a majorallergen in soybean) content was substantiallyhigher, by almost 30%, in one of the two GTSlines, with a smaller increase in the other. Notrypsin inhibitor analyses were performedon the protein isolate or protein concentratesamples originating from the meal samples.Although there were other compositionaldifferences in these processed soybeanproducts, it is difficult to decide from singledeterminations whether these were signifi-cantly different or not.

In conclusion, there is insufficientevidence to date to decide whether the com-position of GM and conventional soybeansis equivalent or not. In fact, some data,particularly those for phyto-oestrogens, were


significantly different. Furthermore, becausenot strictly comparable compositional datawere used, the case for equivalence was notproperly established. There is therefore anobvious need for further more critical studies.

GM potatoes

Brief references to GM potatoes, particularlythose expressing Bacillus thuringiensis (Bt)toxin, can be found in non-peer-reviewedbook chapters or other articles. In mostinstances, these contain no data and aretherefore of little scientific value. There aretwo exceptions, one of which is an article onthe safety assessment of GM potatoes expres-sing the soybean glycinin gene (Hashimotoet al., 1999a). However, it is not quite clearwhat the authors wanted to achieve because,at the expression level of glycinin in potatoesof between 12 and 31 mg g−1 total solubleprotein, no significant improvements in theprotein content or amino acid profile couldhave been expected. Indeed, the results in thepaper demonstrated that the total proteincontent of the GM potatoes appeared to besignificantly less than that of the control lineand that no improvement in the essentialamino acid profile was achieved either. Thereappeared to be substantial differences insome vitamins between GM and control lines,and the amounts of both solanine andchaconine increased in the GM lines. It is,therefore, not quite clear why it was claimedby the authors that their GM lines wereequivalent to the parent line and could be uti-lized as safely. The other more recent studyis a conventional compositional analysis ofsome macro- and micronutrients of tubersfrom insect- and virus-resistant potato plants(Rogan et al., 2000) performed by methodswhich currently are accepted by most novelfood regulatory bodies. Although theseshowed some significant differences in anumber of tuber constituents, in the absenceof toxicological/nutritional animal studiesit is difficult to ascertain whether these dif-ferences could have any biological effectson humans/ animals, particularly as theseconventional analyses could not have

revealed the development of any unknownpossible toxic/antinutritive components.Additionally, known antinutrients, such aslectins or enzyme inhibitors, were notincluded in the analysis.

GM rice

GM rice lines expressing the soybean glyciningene have been developed (Momma et al.,1999) by a method similar to that used for GMpotatoes. The glycinin expression level wasbetween 40 and 50 mg glycinin g−1 total riceprotein. The GM rice was claimed to contain20% more protein, but its moisture contentwas less than that of the parent line. How-ever, from the paper, it is not quite clearwhether the increased protein content wasdue to the decreased moisture content of theseeds because it was not specified whetherthe values were expressed for air-dried or fullydried seeds. Thus, most of the arguments inthe discussion of whether the higher proteinlevel was due to the positioning effect of geneinsertion or metabolic interference will haveto await clarification by further work.

GM cotton

Several lines of GM cotton plants havebeen developed using the gene encodingan insecticidal protein from B. thuringiensissubsp. kurstaki. These had increased protec-tion against the major lepidopteran insectpests of cotton. As cottonseed is an importantsource of oil for human consumption, andcottonseed and processed cottonseed mealfor animal feed, extensive analytical workhas been done to establish whether the GMlines were ‘substantially equivalent’ to con-ventional lines (Berberich et al., 1996). Thelevels of protein, fat, carbohydrate, moisture,ash, amino acids and fatty acids in the insect-protected lines were claimed to be compara-ble with those found in commercial varieties.Moreover, the levels of antinutrients suchas gossypol, cyclopropenoid fatty acids andaflatoxin were similar to or less than those inconventional seeds. Thus, the GM varieties


were suggested to be equivalent to conven-tional seeds and just as nutritious. However,the statistics used by the authors were identi-cal to those used with glyphosate-resistantsoya and therefore could be similarly criti-cized. Although the content of known con-stituents fell in between the wide range ofvalues of commercial conventional lines, thisdid not mean that they were compositionallyequivalent, particularly as environmentalstress could have major and unpredictableeffects on antinutrient and toxin levels(Novak and Haslberger, 2000). Thus, withoutanimal experimentation, this approach couldnot reveal whether any new and unknowntoxins/allergens had been created or not.

GM maize

A glyphosate-tolerant (Roundup Ready)maize line GA21 has recently been devel-oped. It was claimed (Sidhu et al., 2000) that,except for a few minor differences, which theauthors think are unlikely to be of biologicalsignificance, the results of compositionalanalyses of proximate, fibre, amino acid, fattyacid and mineral contents of the grain, andproximate, fibre and mineral contents offorage collected from 16 field sites over twogrowing seasons showed that control andGM lines were comparable. The comparisonwas carried out by a method similar to thatdescribed for GTS soya (Padgette et al., 1996)and this may therefore not be scientificallyrigorous enough for the establishment ofsubstantial equivalence.

Nutritional/Toxicological StudiesPublished in Peer-reviewed Journals

Herbicide-resistant soybean

As part of a safety assessment of GTS, thefeeding value, wholesomeness (Hammondet al., 1996) and possible toxicity (Harrisonet al., 1996) of two major GM lines of GTSwere compared with those of the parent line.Processed GTS meal was included in thediets of rats, broiler chickens, catfish and

dairy cows at the same concentrations asin commercial non-GM soybean rations. Ratsand dairy cows were fed these diets for4, broilers for 6 and catfish for 10 weeks. Itwas claimed that in rats, catfish and broilersthe growth and feed conversion efficiency,in catfish the fillet composition, in broilersthe breast muscle and fat pad weights, andin dairy cows milk production and composi-tion, rumen fermentation and digestibilitieswere similar for both GTS and parental lines.According to the authors, these resultsconfirmed that the GTS and parental lineshad similar feeding values.

Rat studies

A critical evaluation of the rat study washampered by the lack of adequate primaryindividual data in the paper. Thus, there wasno full description of the rat diet. It appearsthat the total protein content of the diets wasadjusted to 247 g kg−1 diet to be isonitro-genous with Purina Laboratory Rat Chowby the addition of 24.8 g of GTS and parentsoybean meals, respectively (~10% protein),to a base diet. All comparisons were madewith rats fed commercial Purina Chow.The protein concentration in these diets was,however, appreciably higher than the usual10–16% crude protein and exceeded theprotein requirements of the rat. This extraprotein potentially could have masked anypossible transgene product effects, particu-larly with the raw unprocessed soybean dietsin which the GM meals were incorporatedonly at the level of 50 or 100 g kg−1 of the diet.Thus, these meals only replaced 8.5 and 17%,respectively, of the total protein of 247 g kg−1

diet. In other words, the GM soybean proteinin these meals was diluted by other dietaryproteins by 12- and 6-fold, respectively,producing another possible masking effect.The composition of the control Purina Chowdiet in the ground raw soybean feeding studywas not described. This is important becausethe identity of the raw control soybeansincluded in the Purina Chow control diet wasnot specified.

In the feeding study, four groups of rats(ten males and ten females in each group)singly housed were fed diets containing the


parental line or the GTS lines (40-3-2 or61-67-1) for 28 days. No individual values (ortheir ranges) for feed intake or body weightwere given. The bar diagrams of the combinedbody weight of rats at the end of each weekof the 4-week experiment were rather unin-formative. However, it was observed by theauthors that the Purina Chow-fed male ratsgrew significantly better than the three experi-mental groups fed toasted soybeans (includ-ing the parental line). This was attributed tobetter commercial processing. However, thebar diagrams also indicated that the growthin the group fed with one of the GTS lines(61-67-1) was probably equal to that of thePurina Chow-fed control and, therefore, byinference, these rats also grew significantlybetter than the other two experimental lines(the GTS line 40-3-2 and the parental line).This again underlined the importance ofgiving individual data in papers, withoutwhich it is difficult to assess the results.Similarly, there were no individual data fororgan weights, such as liver, kidneys andtestes. However, it was claimed that thekidney weights of the raw GTS line-fed (andparental control?) male rats were significantlyhigher than those of the controls, while thetestes of the parental line-fed rats were signifi-cantly enlarged. According to the authors, asthese differences were neither dose relatednor only shown by the parental line, they werenot caused by genetic modification. Rathercuriously, the weights of the stomach andintestines, the main target organ in any nutri-tional testing, were not recorded. Observa-tions were not recorded on other organs, andno histology appears to have been done onthese tissues either. The only tissue which wassubjected to microscopy was the pancreas, butthe description of the findings was qualitative.Only minimal to mild lesions were foundand these were claimed to be common toall groups. However, under these conditions,this was not surprising because no pancreatichypertrophy was found. This was probablydue to the effect of the unusually high dietaryprotein concentration, which, as the authorspointed out, masked and/or diluted thebiological effect of the trypsin inhibitors. Thisis of particular concern because the trypsininhibitor content of GTS lines in unprocessed

soybean was significantly higher than in thecontrol line (Padgette et al., 1996).

It is regrettable that the design of thisimportant rat feeding study had such unfortu-nate omissions. It is of particular concern thatno histology was apparently carried out ongut tissue. Thus, more critical work is neededto decide whether the feeding value of GMand non-GM soybeans is equal or not.

Chicken study

The broiler chicken feeding study’s experi-mental design closely followed that of com-mercial practice and therefore the resultsshould only be indicative of the commercialfeeding and production value of the varioussoybean lines. As the data were pooled fromall birds fed on the same diet, it is not easy tosee what, if anything, was the significance ofthe small differences found in the study, suchas the slightly lower body weights, breast andfat pad weights obtained with the GTS lines(particularly with GTS 40-3-2) for the utiliza-tion of GM soybean. It would have beenbetter to measure the nutritional performanceof individual birds (or small groups) fed ondifferent diets and then compare them afterstatistical analysis. In the absence of this, wehave to rely on the authors’ conclusion thatthe design of the experiment gave the upperlimit of differences in weight gain of 3.5% andgain/feed ratio of 2% and that the GTS linesvs. parental line were within this limit. Thus,with this restriction, the feeding value of theGTS lines for broilers was practically equal tothat of the parental soybean line.

Catfish experiment

Catfish are excellent and highly sensitiveindicators for the feeding value of diets. Itwas obvious from the results that, similar tothe findings with rats, one of the GTS lines,61-67-1, was superior to the other lines (GTS40-3-2 and the parental line) in most respects.Thus, fish on GTS line 61-67-1 ate more, hadbetter weight gain and gain/feed ratio andweighed more at the end of the 10-weekstudy than the others, even though the com-position of the fillets from these fish wasnot significantly different. This significant


difference in performance must, therefore,indicate that genetic modification may notbe as reproducible as it has been claimedand that the feeding value and metaboliceffects of GM and parent lines are not always‘substantially equivalent’.

Study on lactating cows

Milk production and composition and per-formance data in the lactating cow studyshowed some significant differences betweencows fed diets containing the different linesof soybean, indicating a lack of ‘substantialequivalence’. In view of these differences,even though we may not at present know alltheir biological/nutritional consequences, itmay be difficult to maintain the view that thefeeding value of the GTS and parent lines isequal, and further work is needed to establishwhether the GTS lines are safe or not forhumans/animals.

Testing of E. coli recombinant gene product

Extensive studies have been carried out toascertain the safety of the gene product,5-enolpyruvylshikimate-3-phosphate syn-thase (CP4 EPSPS), which renders thesoybeans glyphosate resistant (Harrison et al.,1996). Unfortunately, there are some flawsin these experiments, the most important ofwhich is that in the acute gavage studies theauthors did not use the enzyme isolated fromGTS lines but instead that from Escherichiacoli. Although they were at pains to show thatthe EPSPS enzyme samples from the twosources were similar in lack of glycosylation,molecular size, reaction with a polyclonalanti-EPSPS antibody and enzyme assays,these methods do not have sufficient powerto show unequivocally whether they wereidentical. The authors themselves pointedout that post-translational modification ofthe completed polypeptide chains emergingfrom the ribosomes may be done differentlyin two such evolutionarily distinct life formsas higher plants and prokaryotic bacteria.Amidation, acetylation and proteolytic pro-cessing can have such major effects onthe conformation of the protein as to makethese gene products behave differently in the

digestive system. Thus, the use of the E. colirecombinant protein for the acute micegavage studies may invalidate the authors’conclusion that the gene product from soy-bean did not have any toxic effects. Thesestudies must be re-done with the gene prod-uct isolated from the transgenic plant beforethe results could be accepted. In any case, insuch gavage studies, young, rapidly growinganimals must be used to show any distincteffect on growth. As all animal weights wereunchanged in the experiment, the test systemused could not have detected any effectunless the consequences of the gavaginghad been disastrous. Feeding studies withthe gene product in young rapidly growingrodents should be the preferred method forthe demonstration of the deleterious effects.

The other flaw in the experimental designwas the reliance on an in vitro simulatedgastric/intestinal digestion assay, which wasalso carried out with the E. coli recombinantgene product. To obtain physiologically validresults, it would have been necessary to usethe gene product isolated from GM soybeanin an in vivo assay in the rat (or other suitableanimals; see Rubio et al., 1994) or a full feedingtrial. Thus, it has been shown before that thekidney bean (Phaseolus vulgaris) α-amylaseinhibitor is fairly stable to proteolytic degra-dation in the rat gut (Pusztai et al., 1995, 1999),but, when its gene was expressed in peas(Pisum sativum), it was rapidly digested andinactivated in the rat stomach/small intestinein vivo (Pusztai et al., 1999). This may havecontributed to the safety of GM peas forrats and, by inference, possibly for othermonogastric mammals. Thus, in vitro diges-tion assays may have little relevance to thesafety of GM food crops.

In a separate feeding study (Teshimaet al., 2000), the possible harmful effectsof toasted glyphosate-resistant GM soybeanwere investigated at 300 g kg−1 inclusion levelin the diet of rats and mice. After feedingthese animals for 15 weeks, no significantdifferences in nutritional performance, organdevelopment, histopathology of the thymus,liver, spleen, mesenteric lymph nodes, Peyer’spatches and small intestine, and the pro-duction of IgE and IgG humoral antibodiesbetween GM and non-GM line diets were


found. However, as rats grew less than30 g and mice not at all in 15 weeks, theconditions were so unphysiological that novalid conclusions could be drawn from theseexperiments.

GM maize

In a major commercial-scale broiler chickenfeeding study with rations containing trans-genic Event 176-derived Bt maize involving1280 birds (Brake and Vlachos, 1998), it wasclaimed that no statistically significant dif-ferences in survival or bird weights betweenbirds fed diets containing GM maize, Event176, or an isogenic parent maize line werefound. Indeed, birds fed GM maize rationsappeared to have significantly better feedconversion ratios and an improved yieldof breast muscle. However, the authorscautioned against the conclusion that thisenhanced performance could be attributedto the Bt maize per se. It is possible that theresults might have been due to slight differ-ences in the overall composition of the diets.This is reasonable considering the length ofthis study and possible problems of consis-tent diet preparation on a commercial scale.Minor differences in composition such asthe slightly lower protein content of the GMmaize and fat contents of the diets magnifiedto the scale of this trial make the results morerelevant to commercial than to academicscientific studies.

In a poultry feeding study, it was claimedthat the GA21 Roundup Ready maize-baseddiets gave similar performance data ingrowth, feed efficiency and fat pad weightsto diets containing the parental control line(Sidhu et al., 2000). However, this and a similarstudy carried out in Germany with a maizeline expressing PAT-PROTEIN (Flachowskyand Aulrich, 2001) were commercial produc-tion experiments and made little contributionto scientific safety assessment.

In a separate study, maize was geneti-cally modified by the transfer of the gene ofegg white avidin to make the seed resistant tostorage insect pests (Kramer et al., 2000). It wasalso claimed that this GM maize was safe

for mice as apparently, when, instead of abalanced diet, they were fed solely on thiscrop, the mice suffered no ill effects. However,the mice used in the experiment were adultswhich did not grow at all, and therefore theconclusion that the GM maize was safe is, atbest, premature.

GM peas

Diets containing transgenic peas expressingthe transgene for insecticidal bean α-amylaseinhibitor (~3 g kg−1 peas) at two differentinclusion levels in the diet, 300 or 650 g kg−1,were subjected to nutritional evaluation withrats in a 10-day feeding trial (Pusztai et al.,1999). The nutritional performance of ratsfed GM pea diets was compared with thoseobtained with rats pair-fed iso-proteinicand iso-energetic diets containing parent-linepeas and also lactalbumin diets spiked withisolated bean and pea α-amylase inhibitors,respectively. At 300 g kg−1, but not at 650 gkg−1 inclusion level, the nutritional value ofdiets containing transgenic or parent peaswas not significantly different. Even at650 g kg−1, the difference was small, mainlybecause the transgenically expressed pearecombinant α-amylase inhibitor was quickly(in < 10 min) degraded in the rat digestivetract and therefore its antinutritive effect wasabolished. In contrast, spiking the parentalline pea diet with the stable bean α-amylaseinhibitor reduced its nutritional value(Pusztai et al., 1995, 1999).

In this study, unfortunately, no gut his-tology was done or lymphocyte responsive-ness measured, and therefore one had to relyon the evaluation of nutritional parametersthat are inherently less sensitive in orderto find possible differences in metabolicresponses between GM and conventionalfood components. Although there were signif-icant differences in the development of someorgans, mainly the caecum and pancreas,most organ weights were remarkably similar.At the end of the study, cautious optimismwas expressed that GM peas could be usedin the diets of farm animals, particularly atthe low/moderate levels recommended in


commercial practice and if the progress of theanimals was monitored carefully. However,this relatively short feeding study withmodest objectives cannot at this stage be takenas proof of the safety of GM peas for humanconsumption. There is a need to carry outfurther and more specific risk assessment test-ing procedures, which must be designed anddeveloped with human consumers in mind.It also has to be kept in mind that only oneparticular line of GM peas was tested in whichthe endogenous antinutrient levels wereselected to be similar to those of the parentpeas. In some other GM lines, however, lectinlevels could vary, up or down, by a factor offour. Moreover, in some field pea cultivars,such as Laura, the concentration of trypsininhibitor increased by about 24% and thechymotrypsin by 100%, while the haemag-glutinating activity decreased by a factor offour in the GM line compared with its parent(A. Pusztai, unpublished). This strengthensthe argument that, in the safety assessment ofGM crops, many lines should be included andthat, from the results of a single GM line, noblanket approval should be given to otherlines developed, even if in the transformationthe same vector was used and carried out atthe same time.

GM potatoes

There have been four independent studies ondifferent GM potatoes.

Glycinin-expressing potatoes

The safety of transgenic potatoes expressingthe soybean glycinin gene was evaluated in ashort (4-week) rat feeding study (Hashimotoet al., 1999b). With an interesting experimen-tal design, control rats and the experimentalgroups were fed the same control commercialdiet. However, the rats were also dailyforce-fed by gavage with 2 g of respectivepotato lines kg−1 body weight. The potatoesused were a parental control line and twotransformed lines, one with the glycinin geneand another one with a designed glyciningene (coding for four additional methionines

in the gene product), respectively. However,there were a number of problems with thisstudy. Thus, although no difference ingrowth, feed intake, blood cell count, bloodcomposition and internal organ weightsbetween the groups was found, the uncer-tainty as to whether the animals were fedwith raw or boiled/baked potatoes leaves aquestion mark over the interpretation of theresults.

Bt toxin potatoes

An interesting, mainly histology, study wascarried out on the ileum of mice fed withpotatoes transformed with a B. thuringiensisvar. kurstaki CryI toxin gene. As a control, theeffect of the toxin itself was also investigated(Fares and El-Sayed, 1998). It was shown thatboth the delta-endotoxin and, to a lesserextent, the Bt potato caused villus epithelialcell hypertrophy and multinucleation, dis-rupted microvilli caused mitochondrialdegeneration, increased numbers of lyso-somes and autophagic vacuoles, andincreased activation of crypt Paneth cells. Asa result, it was recommended that ‘thoroughtests of these new types of genetically engi-neered crops must be made to avoid the risksbefore marketing’. Unfortunately, some flawsin the experimental design detract from thestrength of the conclusions. The most impor-tant of these was that, apart from indicatingthat the gene used in the transformationwas the CryI gene from B. thuringiensis var.kurstaki, there was no description of the Btpotatoes. The gene expression level in theGM potato was not given and it was not clearwhether the potatoes in the diet were cookedor raw. Moreover, the amount of the Bt toxinused for supplementing the potatoes withinthe control potato diet was not specifiedeither. This made it impossible to make aquantitative comparison of the effects onthe ileum of the Bt potato with those of thespiked control potato diets. The assumptionthat the ileum is the most important absorp-tive part of the rodent small intestine couldalso be argued against, because 90% of allnutrient absorption in fact occurs in thejejunum. As this was an electron microscopystudy, the fixation of the ileal samples was


not done on well-oriented sections but onchopped up fine tissue pieces, and importantdetail of villus organization was thereforelost. Finally, the delta-endotoxin-inducedhyperplastic changes on ileal villi shouldhave been demonstrated by measuring cellproliferation and mitotic rates in ileal (andjejunal) crypts rather than on the villi. How-ever, despite these shortcomings, this studyhas established once and for all that, in con-trast to general belief, exposure of the mousegut (ileum) to the CryI gene product hascaused profound hypertrophic and hyper-plastic changes in cells of the gut absorptiveepithelium and can lead to mucosal sensitiza-tion (Vazquez Padron et al., 1999, 2000b). Thisshows up the fallacy of drawing comfortingconclusions from in vitro simulated gut prot-eolysis tests. Clearly, concerns about the pos-sible biological consequences of exposure toGM food should be addressed under in vivoconditions because, even if an E. coli productbreaks down in vitro, this does not necessarilymean that the same gene product expressedin the transgenic crop should also breakdown.

GNA GM potatoes

Some of the results of rat feeding studieswith GM potatoes expressing the snowdrop(Galanthus nivalis) bulb lectin (GNA) genewere similar to the results of Fares andEl-Sayed (1998). A part of this work con-cerning the effect of GNA GM potatoes onthe histology of different compartments ofthe rat gut was published (Ewen and Pusztai,1999a). Although this peer-reviewed scien-tific paper was criticized by some, mostof the criticisms were unpublished personalopinions. Moreover, most of the publishedcriticisms (e.g. Kuiper et al., 1999) wereanswered adequately (Ewen and Pusztai,1999b). Some selected results of the nutri-tional/metabolic studies were, against thewishes of the authors, placed on the websiteof The Rowett Research Institute (www.rri.sari.ac.uk), where most of the work was done(Bucksburn, Aberdeen, UK). However, soas not to jeopardize their eventual properpublication, these results will only bementioned briefly.

Young, rapidly growing rats (startingweight of 84 ± 1 g) were strictly pair-fed oniso-proteinic (60 g total protein kg−1 diet, mostof which was from potatoes) and iso-caloricdiets (in contrast to that described in Kuiperet al., 2001) supplemented with vitamins andminerals for 10 days. The test diets containedeither raw or boiled GM potatoes. The controldiets contained the same amount of parental-line potatoes (raw or boiled) alone or supple-mented with GNA at the same concentrationas expressed in the GM potatoes. A positivecontrol group of rats was also included in theexperiment, and these were fed a lactalbumin-based high quality control diet to checkfor any potential problems in rat behaviourand experimental conditions. As part of thenutritional/metabolic evaluation, samplesof stomach, jejunum, ileum, caecum andcolon were taken, fixed and stained withhaematoxylin and eosin for full quantitativehistological evaluation (Figs 16.1, 16.2 and16.3) or reacted with GNA antibody and sub-sequently stained using a PAP (peroxidase–antiperoxidase) method to establish whetherany GNA was bound to the epithelial surface(Fig. 16.4). By measuring the mucosal thick-ness of the stomach and the crypt length of theintestines (Ewen and Pusztai, 1999a), it wasshown that proliferation in the gastric mucosawas in part caused by GNA, the gene product.However, the growth-promoting stimulus onthe small intestine of diets containing GMpotatoes leading to crypt enlargement and apart of the stomach enlargement was not aGNA effect. As shown before and confirmedhere, there was a slight binding of GNA to thesmall intestinal epithelium (Fig. 16.4). How-ever, GNA is not a mitotic lectin and thereforeit did not induce hyperplastic growth in thistissue (Pusztai et al., 1990). Accordingly, thejejunal growth was probably due to someas yet unknown effects of other parts of thegenetic construct used for the transformationor the genetic transformation itself. Hyper-plasia was shown previously by measuringthe increase in crypt length (Ewen andPusztai, 1999a). However, similar resultswere obtained by measuring the increasein crypt cell numbers (Table 16.1) and cryptmitotic figures (not fully significant) in thejejunum of GM potato-fed rats (Table 16.2).


The results suggested that it is possible thatcrypt hyperplasia and an increase in epithelialT lymphocyte infiltration observed with GMpotatoes might also happen with other GMplants that had been developed using thesame or similar genetic vectors and methodof insertion. It is therefore imperative thatthe effects on the gut structure and metabo-lism of all GM crops should be investigatedthoroughly as part of the regulatory processbefore their release into the human food chain.

Potatoes expressing cationic peptide chimeras

Desiree and Russet Burbank potatoes expres-sing N-terminally modified cecropin–melittincationic peptide chimeras and control linepotatoes fed to mice caused severe weightloss. The animals did not grow even aftersupplementing these potatoes with rodentlaboratory chow. According to the authors(Osusky et al., 2000), mice fed with tubersfrom transgenic potatoes were as healthy andvital (sic) as those from the control group,and their faecal pellets were comparable.

However, the severe weight loss seriouslycalled into question the value of the results ofthis poorly designed feeding experiment.

GM tomatoes

Finally, an important study will have to bedescribed even though it was not publishedin a peer-reviewed journal, but the ideasand experiments described had some influ-ence on the development of GM regulation(Noteborn et al., 1995). Thus, a new labora-tory GM tomato line was developed using theB. thuringiensis crystal protein CRYIA(b) genebut, instead of the cauliflower mosaic virus35 S promoter (CaMV 35 S), which is usedin practically all first-generation GM crops, apotentially safer plant promoter was used.Although with this the expression level of theBt toxin was only about 1/20th of that foundwith CaMV 35 S, this might be improvedupon in future. In contrast with most otherstudies with GM crops, there was a


Fig. 16.1. Comparison of the stomach mucosa of rats fed with raw GM potato diet (b) shows markedthickening due to hypertrophy of mucosal cells in comparison with that of rats given the parental line (a)(bar = 100 µm).


Fig. 16.2. Histology of the jejunum and ileum of rats fed raw GM or parent potato diets. Jejunal cryptlength and cells exhibit marked enlargement after feeding rats GM potato diets for 10 days (b) incomparison with those of rats given parental line potato diets (a). The villus length is similar in both, butintraepithelial lymphocyte cell counts appear to be increased on the GM potato diet. In the ileum, bothcrypts and villi of rats on GM potato diets are elongated (d) in comparison with parent potato-fed rats (c)(bar = 100 µm).


Fig. 16.3. The mucosa of the caecum demonstrates little change. Differences between GM-fed (b) andparent line potato-fed rats (a) are slight, while the colonic mucosa is moderately thickened in GM-fed rats(d) compared with that of rats given the parental line (c) (bar = 50 µm).

commendable attempt to measure the bind-ing of the gene product to the rat gut surfacein vivo rather than using spurious argumentsas to why the gene product should not bind.

Although no in vivo binding was found, thisshould not detract from the significance ofthis initiative because, due to the lack ofavailability of sufficient quantities of Bt toxin


Fig. 16.4. Immunocytochemistry of the jejunum of rats fed raw GM potato diets for 10 days (a) showsmoderate binding of GNA to villus tips (bar = 50 µm). Similar binding of GNA to jejunal villus tips isfound in rats given parent potato diet supplemented with GNA in amounts equivalent to that expressed inthe GM potato (b) (bar = 100 µm). Sections were first treated with anti-GNA rabbit antibody (diluted 1/100),followed by visualization with PAP. Note the strong antibody reactivity of feed particles in the sections.

Diet Parent

Parentvs. parent +

GNA (P)Parent +

GNA

Parent vs.transgenic

(P) Transgenic

Parent +GNA vs.

transgenic(P)

RawBoiledRaw vs. boiled (P)

15.9 (0.5)17.8 (1.1)0.006

0.0370.466

17.0 (0.7)18.2 (0.2)0.003

0.0000.749

20.3 (0.8)18.2 (1.2)0.003

0.0060.769

aThe number of nuclei were counted sequentially on well-oriented haematoxylin and eosin paraffinsections (4 µm). Values represent means (SD) for six rats per treatment; ten crypts per rat were counted.Differences between treatments are significant when P < 0.05 (Student’s t test). The effect of boiling(P = 0.759) is not significant, while that of GNA added or as transgene product (P = 0.019) and the effectof transformation (P = 0.000) are highly significant. The interactions between GNA and cooking(P = 0.043) and between transformation and cooking (P = 0.018) are also significant (multivariantanalysis with Tukey’s test).

Table 16.1. Number of crypt cells in the jejunum of rats fed various potato diets.a

isolated from GM tomatoes, an E. coli recom-binant and potentially less stable form of thegene product was used in the experiment,and its possible degradation in the gut mayhave accounted for the lack of binding. How-ever, Bt toxin was shown byimmunocytochemistry to bind to gut sec-tions, including the caecum and colon, fromhumans and rhesus monkeys in vitro. Unfor-tunately, their short-term toxicity testing inmice (and rabbits) and the in vitro simulatedproteolysis assays were also carried out withthe E. coli recombinant gene product andtherefore their conclusions of finding no toxiceffects may not be valid. Commendably, theauthors carried out a 91-day feeding studywith rats using freeze-dried GM vs. parentline tomatoes, which were included at a 10%level in the diets, but no differences in foodintake or body and organ weights werefound. However, because the Bt toxin expres-sion level in the tomatoes was low, the dailyintake of the gene product(s) by the rats wasalso low. Moreover, as the daily input oftomato proteins was only about 5–6% of thetotal dietary protein intake of the rats, it wassomewhat optimistic to expect any significantchanges in these nutritional parameters. Tohave any reasonable chances to show upsmall differences in the nutritional value ofGM vs. parent line crops, it would have beenimportant to use as high a protein concentra-tion as possible such as that in the 110-dayGM potato feeding study carried out at TheRowett Institute, in which the GM protein inthe diet was diluted only twofold by otherdietary proteins, and this allowed the sig-nificant differences in the growth rates ofrats fed on baked GM potato diets vs. parentpotato diets to show up. In fact, to equalizethe growth rates of the rats on the GMpotatoes to that of the controls, the GM

diet had to be supplemented with an extra12 g lactalbumin kg−1 diet, and this extraprotein gave a quantitative measure of thedifference of the nutritional value betweenGM and non-GM potatoes. Even at thesesimilar growth rates, the weights of someof the rats’ vital organs, such as the gut andparticularly the small intestine, the liver andkidneys, were still significantly different.

There were other omissions in the Bttomato study, the most important of whichwas that no Bt toxin survival was measuredin the gut lumen and no gut histology wasdone to see if there was any Bt toxin bindingto or possible structural changes in the gutepithelium or whether lymphocyte infiltra-tion occurred. This omission is particularlyimportant because later studies showed thatthe similar Bt toxin Cry1Ac could bind togut epithelial cells in mice (Vazquez Padronet al., 2000a,b) and induce mucosal antigenicsensitization (Vazquez Padron et al., 1999,2000a,b). The allergenic potential of Bt toma-toes was not investigated either. However,despite some of its shortcomings, this studyshowed many novel and commendable fea-tures, which, after some improvements, may,hopefully, be incorporated into the generalGM food testing procedures.

Allergenicity

One of the major health concerns with GMfood is its potential to increase allergies inthe human population through the foodchain. The possibility of fatal anaphylaxis insensitized individuals after their unwittingexposure to allergenic proteins in unlabelledGM foodstuffs is a real danger. When agene is transferred from a source of knownallergenic potential, the assessment of theallergenicity of the GM crop is relativelystraightforward. This can be done usingin vitro tests with sera from individualssensitized to the allergen from the originalsource. Similarly, it is relatively easy to assessthe effect of genetic engineering on endoge-nous allergens in crops with some evidenceof allergenicity. With tests such as the radio-allergosorbent test (RAST), RAST inhibition


Diet Parent Parent + GNA Transgenic

RawBoiled

4857

4956

7557

aMitotic cells were expressed per 100 crypts.

Table 16.2. Mitotic numbers per 100 crypts inthe jejunum of rats fed potato diets.a

and immunoblotting, the allergenic potentialof the GM crop is easily measured. There arenow several examples for these, such asthe demonstration of the allergenicity of theBrazil nut 2S seed storage protein in trans-genic soybean (Nordlee et al., 1996) or thecodfish allergy in potatoes genetically engi-neered with cod protein genes to make thepotatoes tolerate cold storage (Bindslev-Jensen and Poulsen, 1997). The claim that inglyphosate-tolerant soybean the introductionof the herbicide resistance gene does notaffect the allergenicity of the soy endogenousallergens is also a good example (Burks andFuchs, 1995). Having shown in a surveillanceprogramme of farm workers before and afterexposure to B. thuringiensis pesticide spraysthat some developed skin sensitization andIgE antibodies to the Bt spore extract and thattwo of them had a positive skin-prick test, itmay now be possible to test for the aller-genicity of Bt toxins engineered into variouscrops (Bernstein et al., 1999). This is all themore important because the Cry1Ac toxinhas now been shown to be a potent oralimmunogen and adjuvant (Vazquez Padronet al., 1999, 2000a,b).

It is much more difficult to assess theallergenicity of GM foods when the gene istransferred from a plant whose allergenicpotential is unknown. Moreover, it is alsopossible that, as a result of the gene transfer orvector insertion, a new allergen is developedor the expression level of a minor allergen isincreased in the GM crop. The gene productcan also have an allergenic adjuvant effect on afood component previously of low allergenicpotential, or some component in the GMfood may have an adjuvant effect on theallergenicity of the transgene product. Unfor-tunately, while there are good animal modelsfor nutritional/toxicological testing, no satis-factory animal models have been developedso far for allergenicity testing (Helm andBurks, 2000). For the time being, only indirectmethods are available for the assessment ofthe allergenic potential of GM foods derivedfrom sources of unknown allergenicity. Thereare a number of recommended approaches tobe followed. A useful preliminary step is toestablish if there are any sequence homologiesin the transgenic protein to any of the about

200 known allergens. If there are, in vitro testsfor IgE reactivity need to be performed. It isthought that the peptide length in the trans-genic protein which is optimally needed forbinding B-cell epitopes requires the presenceof at least eight contiguous identical or similaramino acids. However, the amino acids inthe allergenic epitopes are rarely contiguous.Moreover, the absence of a positive reactionin in vitro testing does not guarantee thatthe transferred protein is not an allergen.In a decision-tree type of indirect approach,the next step is to consider the molecularsize, glycosylation, stability, solubility andisoelectric point of the transgenic protein andcompare them with those of known allergens(O’Neil et al., 1998). Unfortunately, in moststudies to date, the all-important stability ofthe transgenic protein to gut proteolysis isestablished in an in vitro simulated gastric/intestinal system (Astwood et al., 1996;Metcalfe et al., 1996), and this is fundamentallyflawed. The results, therefore, are at best mis-leading and at worst erroneous. Reliance onthe concept that most allergens are abundantproteins is probably also misleading because,for example, Gad c1, the major allergenin codfish, is not a predominant protein(Bindslev-Jensen and Poulsen, 1997).

When the gene responsible for the aller-genicity of a crop is known, its cloning andsequencing open the way for its reductionby antisense RNA strategy. Thus, in rice,the low molecular weight α-amylase/trypsininhibitors are major allergens. A part of thegenomic sequence encoding this protein in anantisense direction was constructed betweenthe promoter of the rice allergen gene andits waxy terminator, and this was introducedinto rice protoplasts. The allergenicity of theregenerated plants was significantly less thanthat of parental wild-type rice (Nakamura andMatsuda, 1996).

In conclusion, allergenicity testingappears to be one of the Achilles heels of GMfood safety. It is clear that, if and when it isknown that the protein gene is derived from asource with a history of allergenicity, there is areasonable certainty that the GM crop will beallergenic. Unfortunately, the reverse is nottrue: the use of a gene from something that isnot allergenic will not guarantee that the GM


crop will not possess allergenicity. In theabsence of new and reliable methods forallergenicity testing, particularly the lack ofgood animal models, at present it is almostimpossible to establish definitely whether anew GM crop is allergenic or not in advance ofits release into the human/animal food/feedchain.

Conclusions

One has to agree with the opinion expressedin Science (Domingo, 2000) that there aremany opinions but very few data on thepotential health risks of GM foods, eventhough research to exclude such risks shouldhave been carried out before the GM cropswere introduced into the food chain. Ourpresent database is therefore woefullyinadequate. This is clearly seen from a closerscrutiny of the reference lists of recentreviews which contain only a handful of toxi-cological/nutritional and immune studiesof GM food crops published in peer-reviewedscience journals (Ruibal-Mendieta and Lints,1998; Betz et al., 2000; Kuiper et al., 2001;Pusztai, 2001). Moreover, the scientificquality of even what is published is, in mostinstances, not up to the standards that oughtto be expected. In this review, data pub-lished in peer-reviewed and some non-peer-reviewed journals have been examined indetail. However, as our future is claimed tobe dependent on the success or failure of thepromise of genetic modification deliveringGM foods which will be wholesome, plentifuland, most importantly, safe for us all, theemphasis was on strict but fair criticism.

From the results, the conclusion seemsinescapable that the present crude methodof genetic modification has not deliveredGM crops that are predictably safe andwholesome. The promise of a superior secondgeneration of GM crops is still in the future.It is possible that some of the first generationof GM crops may superficially satisfy somecommercial end points, such as their usein broiler chicken production. However, weneed to consider that these GM feed ration-fedanimals eventually will be consumed by

humans, and there is absolutely nothingknown about the potential hazards (if any) forhuman health of this indirect exposure to GMfood. Furthermore, the examples in the papershighlighted some differences even betweensuch crude things as macronutrient composi-tion of GM and conventional lines. It is arguedby some that these differences have littlebiological meaning. However, it was clear thatmost GM and parental line crops would argu-ably fall short of the definition of ‘substantialequivalence’. This crude, poorly defined andunscientific concept outlived its possible pre-vious usefulness. There is an urgent needto come up with novel scientific method-ologies to probe into the compositional,nutritional/toxicological and metabolic dif-ferences between GM and conventional cropsif we want to put this technology on a properscientific foundation and also to allay the fearsof the general public. We need more scienceand not less. For proper safety assessment, ourfirst concern ought to be to establish on acase-by-case basis the impact of componentsof GM foods on the digestive system,its structure and metabolism, because theway our body will respond to GM foods willbe predetermined at this level. According tothe Royal Society (1999), we need ‘to refine theexperimental design of the research done todate’. New ideas were also advocated in theLancet debate (Ewen and Pusztai, 1999b;Kuiper et al., 1999) and at the OECDConference in Edinburgh in February 2000.

Recommendations

Main tasks and methods for safetyassessment of GM crops

1. For compositional analysis and compari-son, the parent and transformed lines must begrown under identical conditions, treated andharvested the same way. In addition to pro-teins, starch, lipids, etc. of the parent and GMlines, their contents of bioactive componentsshould also be compared by novel methods(proteomics, fingerprinting, etc.).2. The stability to degradation by acid orpepsin or other proteases/hydrolases of GM


products, foreign DNA, including the geneconstruct, promoter, antibiotic resistancemarker gene, etc., has to be established in thestomach and intestines of model animalsin vivo. With GM lectins, including Bt toxin,the presence/absence of their epithelialbinding should also be demonstrated byimmunohistology.3. The biological, immunological, hormonalproperties and allergenicity of GM productsmust be established with the GM productisolated from the GM crop, and not withrecombinants from E. coli, as these two mayhave substantially different properties.4. As GM food is unlikely to be highlypoisonous, ‘toxicity’ is an unhelpful conceptand difficult to assay. In contrast, nutritionalstudies in which GM crop-based diets are fedto young growing animals should reveal theirpossible harmful effects on metabolism, organdevelopment, immune/endocrine systemsand gut flora, which together determine thesafety of the GM crop and the development ofthe young into healthy adults.5. For animal testing iso-proteinic and iso-energetic diets need to be formulated in whichmost of the dietary protein is derived from theGM crop. The composition of the control dietsshould be the same as the GM diet but contain-ing the parent line with or without supple-mentation with the isolated gene product atthe same level as expressed in the GM line.Groups of animals (five or more per group), ofsimilar weight, should be pair-fed in short-and long-term experiments. Urine and faecalsamples should be collected for the determi-nation of net protein utilization (NPU), nitro-gen balance and feed utilization ratios. Bloodsamples should be taken before, during andat the end of the experiments for immunestudies (i.e. lymphocyte proliferation assay,Elispot), hormone assays (insulin, cholecysto-kinin, etc.) and for the determination of otherblood constituents. The animals are to beweighed daily and any abnormalitiesobserved. After killing the animals, theirbodies should be dissected, the gut rinsedand its contents saved for further studies(enzymes, GM products, DNA). Sectionsshould be taken for histology, and the wet anddry weights of organs recorded and analysed.

Evaluation

With suitable statistical analyses (ANOVA,multiple comparisons and/or multivariateanalysis), the significance of differences, ifany, in the parameters should be established.

• If differences between animals fed GMand parent line diets indicate that thegenetic modification must have had asignificant effect on utilization andnutritional value, the GM crop cannotbe accepted for inclusion in the human/animal diet.

• If, similarly to the GM diet, the parentline diet spiked with the gene productshows differences, the use of this gene inGM food/feed is not acceptable.

• If negative effects are not observed withthe parent line diet containing the iso-lated gene product, it is likely thatthe harm is caused by the use of the par-ticular construct or by an unwanted orunforeseen effect of the gene insertion onthe genome.

Animal testing is but a first step andnot a substitute for human studies. If thereis no indication of harm to the animals, theresults will have to be validated with humanvolunteers in clinical double-blind, placebo-controlled drug-type tests. Such studies mayhave to go on for considerable lengths of time.It must also be kept in mind that any potentialharm with GM food may be most acute inthe young, elderly and sick, particularly thosesuffering from HIV, hepatitis or other viraldiseases. Many people suffer from allergiesand other disorders of the gastrointestinaltract, and for these the consumption of GMfood may have unforeseen consequences andsome of these may be irreversible. Thus, forthese, the clear labelling of GM food must bemade mandatory.

There is a compelling need to developfurther the concepts of biological testing,particularly for potential long-term effects.Since the GM potato work with male ratsshowed abnormalities in the development oftheir sexual organs, it is imperative that simi-lar experiments should be done with femalerats to be followed by studies of the effects on


reproductive performance of rats (or otheranimals) reared and maintained on GM vs.non-GM diets for several generations.

If there is a general willingness to fundresearch along these or similar lines and theregulators accept the concept of biological/toxicological testing transparently and inclu-sively, the methods are available for the workto start. Following this route, publishing theresults and consulting the public will ensurethat a technology which promised safe andplentiful food will deliver it for us all, and weare confident that if people see that everythinghas been done to establish its safety they willaccept it willingly.

References

Alliance for Bio-Integrity Website (1998) www.bio-integrity.org including Calgene FLAVR SAVRtomato report, pp. 1–604; InternationalResearch and Development Corp. first testreport, pp. 1736–1738; conclusions of theexpert panel regarding the safety of the FLAVRSAVR tomato, ENVIRON, Arlington, Vir-ginia, pp. 2355–2382; four week oral(intubation) toxicity study in rats by IRDC, pp.2895–3000.

Astwood, J.D., Leach, J.N. and Fuchs, R.L. (1996)Stability of food allergens to digestion in vitro.Nature Biotechnology 14, 1269–1273.

Berberich, S.A., Ream, J.E, Jackson, T.L., Wood, R.,Stipanovic, R., Harvey, P., Patzer, S. andFuchs, R.L. (1996) The composition of insect-protected cottonseed is equivalent to that ofconventional cottonseed. Journal of AgriculturalFood Chemistry 44, 365–371.

Bernstein, I.L., Bernstein, J.A., Miller, M., Tierzieva,S., Bernstein, D.I., Lummus, Z., Selgrade, M.K.,Doerfler, D.L. and Seligy, V.L. (1999) Immuneresponses in farm workers after exposure toBacillus thuringiensis pesticides. EnvironmentalHealth Perspectives 107, 575–582.

Betz, F.S., Hammond, B.C. and Fuchs, R.L. (2000)Safety and advantages of Bacillus thuringiensis-protected plants to control pests. RegulatoryToxicology and Pharmacology 32, 156–173.

Bindslev-Jensen, C. and Poulsen, L.K. (1997) Haz-ards of unintentional/intentional introductionof allergens into foods. Allergy 52, 1184–1186.

Brake, J. and Vlachos, D. (1998) Evaluation oftransgenic Event 176 ‘Bt’ corn in broilerchicken. Poultry Science 77, 648–653.

British Library File (1997) Public reference ‘BL SUP1113’ Chardon Hearing Documents, No.10. Available at: www.maff.gov.uk/planth/pvs/chardon/index.htm

Burks, A.W. and Fuchs, R.L. (1995) Assessmentof the endogenous allergens in glyphosate-tolerant and commercial soybean varieties.Journal of Allergy and Clinical Immunology 96,1008–1010.

Chiter, A., Forbes, J.M. and Blair, G.E. (2000) DNAstability in plant tissues: implications forthe possible transfer of genes from geneticallymodified food. FEBS Letters 24098, 1–5.

Domingo, J.L. (2000) Health risks of geneticallymodified foods: many opinions but few data.Science 288, 1748–1749.

European Commission Directorate C (2000) Opin-ion of the Scientific Committee on Food on ‘TheEvaluation of Toxicological Information Rela-ted to the Safety Assessment of GeneticallyModified Tomatoes’. CS/NF/TOM/8 ADD 1REV 3 Final. European Commission, Brussels.

Ewen, S.W.B. and Pusztai, A. (1999a) Effects ofdiets containing genetically modified potatoesexpressing Galanthus nivalis lectin on rat smallintestine. Lancet 354, 1353–1354.

Ewen, S.W.B. and Pusztai, A. (1999b) Authors’reply. Lancet 354, 1727–1728.

Fares, N.H. and El-Sayed, A.K. (1998) Fine structuralchanges in the ileum of mice fed on delta-endotoxin-treated potatoes and transgenicpotatoes. Natural Toxins 6, 219–233.

Flachowsky, G. and Aulrich, K. (2001) Nutritionalassessment of feeds from genetically modifiedorganism. Journal of Animal and Feed Sciences 10,Supplement 1, 181–194.

Hammond, B.G., Vicini, J.L., Hartnell, G.F., Naylor,M.W., Knight, C.D., Robinson, E.H., Fuchs,R.L. and Padgette, S.R. (1996) The feedingvalue of soybeans fed to rats, chickens, catfishand dairy cattle is not altered by geneticincorporation of glyphosate tolerance. Journalof Nutrition 126, 717–727.

Harrison, L.A., Bailey, M.R., Naylor, M.W., Ream,J.E., Hammond, B.G., Nida, D.L., Burnette,B.L., Nickson, T.E., Mitsky, T.A., Taylor, M.L,Fuchs, R.L. and Padgette, S.R. (1996) Theexpressed protein in glyphosate-tolerant soy-bean, 5-enolpyruvylshikimate-3-phosphatesynthase from Agrobacterium sp. strain CP4, israpidly digested in vitro and is not toxic toacutely gavaged mice. Journal of Nutrition 126,728–740.

Hashimoto, W., Momma, K., Katsube, T., Ohkawa,Y., Ishige, T., Kito, M., Utsumi, S. and Murata,K. (1999a) Safety assessment of geneticallyengineered potatoes with designed soybean


glycinin: compositional analyses of the potatotubers and digestibility of the newly expressedprotein in transgenic potatoes. Journal of Scienceof Food and Agriculture 79, 1607–1612.

Hashimoto, W., Momma, K., Yoon, H.J., Ozawa, S.,Ohkawa, Y., Ishige, T., Kito, M., Utsumi, S.and Murata, K. (1999b) Safety assessment oftransgenic potatoes with soybean glycinin byfeeding studies in rats. Bioscience BiotechnologyBiochemistry 63, 1942–1946.

Helm, R.M. and Burks, A.W. (2000) Mechanisms offood allergy. Current Opinion in Immunology 12,647–653.

Hohlweg, U. and Doerfler, W. (2001) On the fate ofplant and other foreign genes upon the uptakein food or after intramuscular injection in mice.Molecular and General Genetics 265, 225–233.

Kramer, K.J., Morgan, T.D., Throne, J.E., Dowell,F.E., Bailey, M. and Howard, J.A. (2000) Trans-genic avidin maize is resistant to storage insectpests. Nature Biotechnology 18, 670–674.

Kuiper, H.A., Noteborn, H.P.J.M. and Peijnenburg,A.A.C.M. (1999) Adequacy of methods for test-ing the safety of genetically modified foods.Lancet 354, 1315–1316.

Kuiper, A.H., Kleter, G.A., Noteborn, H.P.J.M. andKok, E.J. (2001) Assessment of the food safetyissues related to genetically modified foods.Plant Journal 27, 503–528.

Lappe, M.A., Bailey, E.B., Childress, C. and Setchell,K.D.R. (1999) Alterations in clinically impor-tant phyto-oestrogens in genetically modified,herbicide-tolerant soybeans. Journal of MedicalFood 1, 241–145.

Mercer, D.K., Scott, K.P., Bruce-Johnson, W.A.,Glover, L.A. and Flint, H.J. (1999) Fate of freeDNA and transformation of oral bacteriumStreptococcus gordonii DL1 plasmid DNA inhuman saliva. Applied and EnvironmentalMicrobiology 65, 6–10.

Metcalfe, D.D., Astwood, J.D., Townsend, R.,Sampson, H.A., Taylor, S.L. and Fuchs, R.L.(1996) Assessment of the allergenic potentialof foods derived from genetically engineeredcrop plants. Critical Reviews in Food Science andNutrition 36 (Supplement), S165–S186.

Millstone, E., Brunner, E. and Mayer, S. (1999)Beyond substantial equivalence. Nature 401,525–526.

Momma, K., Hashimoto, W., Ozawa, S., Kawai, S.,Katsube, T., Takaiwa, F., Kito, M., Utsumi, S.and Murata, K. (1999) Quality and safetyevaluation of genetically engineered rice withsoybean glycinin: analyses of the grain compo-sition and digestibility of glycinin in transgenicrice. Bioscience Biotechnology Biochemistry 63,314–318.

Nakamura, R. and Matsuda, T. (1996) Rice allergenicprotein and molecular–genetic approach forhypoallergenic rice. Bioscience BiotechnologyBiochemistry 60, 1215–1221.

Nordlee, J.A., Taylor, S.L., Townsend, J.A. andThomas, L.A. (1996) Identification of a Brazilnut allergen in transgenic soybean. NewEngland Journal of Medicine 334, 688–692.

Noteborn, H.P.J.M., Bienenmann-Ploum, M.E.,van den Berg, J.H.J., Alink, G.M., Zolla, L.,Raynaerts, A., Pensa, M. and Kuiper, H.A.(1995) Safety assessment of the Bacillus thurin-giensis insecticidal crystal protein CRYIA(b)expressed in transgenic tomatoes. In: Engel,K.H., Takeoka, G.R. and Teranishi, R. (eds)ACS Symposium Series 605 Genetically ModifiedFoods – Safety Issues. American ChemicalSociety, Washington, DC, pp. 135–147.

Novak, W.K. and Haslberger, A.G. (2000) Substan-tial equivalence of antinutrients and inherentplant toxins in genetically modified novelfoods. Food and Chemical Toxicology 38, 473–483.

O’Neil, C., Reese, G. and Lehrer, S.B. (1998) Aller-genic potential of recombinant food proteins.Allergy and Clinical Immunology International 10,5–9.

Osusky, M., Zhoul, G., Osuska, L., Hancock, R.E.,Kay, W.W. and Misra, S. (2000) Transgenicplants expressing cationic peptide chimerasexhibit broad-spectrum resistance to phytopa-thogens. Nature Biotechnology 18, 1162–1166.

Padgette, S.R., Taylor, N.B., Nida, D.L., Bailey, M.R.,MacDonald, J., Holden, L.R. and Fuchs, R.L.(1996) The composition of glyphosate-tolerantsoybean seeds is equivalent to that of con-ventional soybeans. Journal of Nutrition 126,702–716.

Pusztai, A. (2001) Genetically modified foods:are they a risk to human/animal health?Available at: www.actionbioscience.org/biotech/pusztai.html

Pusztai, A., Ewen, S.W.B., Grant, G., Peumans, W.J.,van Damme, E.J.M., Rubio, L. and Bardocz, S.(1990) Relationship between survival andbinding of plant lectins during small intestinalpassage and their effectiveness as growthfactors. Digestion, 46 (Supplement 2), 308–316.

Pusztai, A., Grant, G., Duguid, T., Brown, D.S.,Peumans, W.J., Van Damme, E.J.M. andBardocz, S. (1995) Inhibition of starch digestionby α-amylase inhibitor reduces the efficiencyof utilization of dietary proteins and lipids andretards the growth of rats. Journal of Nutrition125, 1554–1562.

Pusztai, A., Grant, G., Bardocz, S., Alonso, R.,Chrispeels, M.J., Schroeder, H.E., Tabe, L.M.and Higgins, T.J.V. (1999) Expression of


the insecticidal bean alpha-amylase inhibitortransgene has minimal detrimental effecton the nutritional value of peas fed to ratsat 30% of the diet. Journal of Nutrition 129,1597–1603.

Redenbaugh, K., Hatt, W., Martineau, B., Kramer,M., Sheehy, R., Sanders, R., Houck, C. andEmlay, D. (1992) Safety Assessment of GeneticallyEngineered Fruits and Vegetables: a Case Study ofthe FLAVR SAVR Tomato. CRC Press Inc.,Boca Raton, Florida.

Rogan, G.J., Bookout, J.T., Duncan, D.R., Fuchs, R.L.,Lavrik, P.B., Love, S.L., Mueth, M., Olson, T.,Owens, E.D., Raymond, P.J. and Zalewski, J.(2000) Compositional analysis of tubers frominsect and virus resistant potato plants. Journalof Agricultural and Food Chemistry 48,5936–5945.

Royal Society (1999) Review of Data on PossibleToxicity of GM Potatoes. Royal Society London.

Rubio, L.A., Grant, G., Caballé, C., Martinez-Aragon, A. and Pusztai, A. (1994) High in vivo(rat) digestibility of faba bean (Vicia faba), lupin(Lupinus angustifolius) and soya bean (Glycinemax) soluble globulins. Journal of Food Scienceand Agriculture 66, 289–292.

Ruibal-Mendieta, N.L. and Lints, F.A. (1998) Noveland transgenic food crops: overview ofscientific versus public perception. TransgenicResearch 7, 379–386.

Schubbert, R., Lettmann, C. and Doerfler, W. (1994)Ingested foreign (phage M13) DNA survivestransiently in the gastrointestinal tract andenters the blood stream of mice. Molecular andGeneral Genetics 242, 495–504.

Schubbert, R., Hohlweg, U., Renz, D. and Doerfler,W. (1998) On the fate of orally ingested foreignDNA in mice: chromosomal association andplacental transmission in the fetus. Molecularand General Genetics 259, 569–576.

Sidhu, R.S., Hammond, B.G., Fuchs, R.L., Mutz, J.N.,Holden, L.R., George, B. and Olson, T. (2000)Glyphosate-tolerant corn: the composition and

feeding value of grain from glyphosate toler-ant corn is equivalent to that of conventionalcorn (Zea mays L.). Journal of Agricultural andFood Chemistry 48, 2305–2312.

Taylor, N.B., Fuchs, R.L., MacDonald, J.,Shariff, A.B. and Padgette, S.R. (1999)Compositional analysis of glyphosate-tolerantsoybeans treated with glyphosate. Journal ofAgriculture and Food Chemistry 47, 4469–4473.

Teshima, R., Akiyama, H., Okunuki, H., Sakushima,J., Goda, Y., Onodera, H., Sawada, J. andToyoda, M. (2000) Effect of GM and non-GMsoybeans on the immune system of BN rats andB10A mice. Journal of the Food Hygiene Society ofJapan 41, 188–193.

Vazquez Padron, R.I., Moreno Fierros, L., NeriBazan, L., De la Riva, G.A. and Lopez Revilla,R. (1999) Intragastric and intraperitonealadministration of Cry1Ac protoxin from Bacil-lus thuringiensis induces systemic and mucosalantibody responses in mice. Life Sciences 64,1897–1912.

Vazquez Padron, R.I., Moreno-Fierros, L.,Neri-Bazan, L., Martinez-Gil, A.F., de la Riva,G.A. and Lopez Revilla, R. (2000a) Character-ization of the mucosal and sytemic immuneresponse induced by Cry1Ac protein fromBacillus thuringiensis HD 73 in mice. BrazilianJournal of Medical and Biological Research 33,147–155.

Vazquez Padron, R.I., Gonzalez Cabrera, J., GarciaTovar, C., Neri Bazan, L., Lopez Revilla, R.,Hernandez, M., Morena Fierros, L. and De laRiva, G.A. (2000b) Cry1Ac protoxin fromBacillus thuringiensis sp. kurstaki HD73 binds tosurface proteins in the mouse small intestine.Biochemical and Biophysical Research Communi-cations 271, 54–58.

US Food and Drug Administration (1994)Memorandum dated 17 May. Summary ofconsultation with Calgene Inc., concerningFLAVR SAVR tomatoes. Available at:www.bio-integrity.org


17 Radionuclides in Foods: thePost-Chernobyl Evidence

J.T. Smith* and N.A. BeresfordCentre for Ecology and Hydrology, Winfrith Technology Centre, Dorchester, UK

Introduction

The Chernobyl accident in April 1986, theworst nuclear accident in history, hadfar-ranging and long-term implications forthe safety of the food chain. The explosionand subsequent fire at the nuclear powerstation in Ukraine spread volatile radioactiveelements (e.g. radioiodine, 131I, and radio-caesium, 137Cs) over large areas of theformer Soviet Union (fSU) and parts ofWestern Europe. The pattern of depositionof radioactivity (Fig. 17.1) was complex; inWestern Europe, the highest deposition ofradiocaesium isotopes occurred in areaswhere rainfall intercepted the radioactiveplume as it dispersed. Less volatile elementssuch as isotopes of strontium and plutoniumwere deposited principally within 30 km ofthe reactor, in the form of small particles ofradioactive fuel (‘hot particles’).

Radiologically important isotopesand half-lives

The importance of different radioactiveelements varies with time after a nuclearaccident because of differences in theirphysical half-lives. The radioactive half-life isdefined as the time taken for one half of a

given amount of a radioactive element todecay radioactively. For example, 2.6 × 1017

becquerels (Bq) of 131I were emitted fromChernobyl. 131I has a physical half-life of 8.05days (Table 17.1) so, after 8.05 days (onehalf-life), 1.3 × 1017 Bq of 131I remained in theenvironment and, after 32.2 days (four half-lives), this was reduced to 0.1625 × 1017 Bq(½ × ½ × ½ × ½ = 1

16 of the original amount).Initially, the radiation dose to humans

was primarily from 131I (with contributionsfrom other short-lived isotopes) in the firstfew weeks after the accident. Overmonths–decades after the accident, longerlived isotopes, primarily 137Cs with contribu-tions from 134Cs and 90Sr, formed the majorpart of the dose. Over hundreds to thousandsof years, Pu isotopes (with ingrowth of 241Am)will form the major part of the dose as the 137Csdecays away (Fig. 17.2). In this chapter, wewill concentrate primarily on the mid- tolong-term consequences, and therefore on137Cs, which has been the focus of the majorityof scientific and regulatory attention follow-ing the Chernobyl accident.

Summary of contamination of the food chain

Initial concerns over safety of the food chainwere due primarily to short-lived 131I in



milk and fresh vegetables (Tsyb et al., 1996;Drozdovitch et al., 1997). Radioiodine isaccumulated in the thyroid, a problemexacerbated by low natural levels of (stable)iodine in the diet of the affected regionsof the fSU. Iodine deficiency was relativelycommon in these regions, so accumulationof the radioactive form was believed to havebeen extremely high. The primary, well-documented, health effect arising from theChernobyl accident has been the develop-ment of thyroid cancers by children in thefSU, largely as a consequence of 131I ingestionvia contaminated milk (e.g. Demidchik et al.,

1996). At the present time, statistically sig-nificant increases in the incidence of otherradiation-induced cancers have not beenobserved as a result of the accident(UNSCEAR, 2000).

Following the first few weeks afterthe accident, physical decay had reducedactivity concentrations of 131I (and many othershort-half-life isotopes) to insignificant levels,as shown in Fig. 17.2. In the medium term afterthe accident, 137Cs made up the major compo-nent of radiation doses to humans. Significantcontributions to dose were made by theshorter half-life 134Cs and also by 90Sr. The γ

374 J.T. Smith and N.A. Beresford

Fig. 17.1. Extent of lands within the former Soviet Union in 1999 contaminated as a consequence of theChernobyl accident. Cross-hatched areas denote areas of abandoned land. 137Cs deposition is interpolatedfrom contours presented by de Cort et al. (1998).

radiation (see Table 17.2 for a description of α,β and γ radiation) emitted by radiocaesiumpresents a potential hazard from deposits onthe ground (external dose) as well as fromingestion of contaminated foodstuffs (internaldose). 90Sr is a β emitter and presents littleexternal irradiation risk, with the exceptionof direct contact between contaminatedmaterials and the skin.

Within the most affected areas of thefSU, approximately 150,000 people were evac-uated from land with 137Cs deposition greater

than 1480 kBq m−2, to reduce the potentialrisk from external and internal exposure byradiocaesium. The 1480 kBq m−2 evacuationlevel was set in order to ensure that a person’slifetime radiation dose from Chernobylradiocaesium was less than 350 millisieverts(mSv). For comparison, the average lifetimedose from all radiation sources (the majoritybeing natural background radiation and frommedical uses of radiation) to people in Europeis approximately 300 mSv (from data inde Cort et al., 1998), although there is

Post-Chernobyl Evidence of Radionuclides in Foods 375

Radionuclide Physical half-lifePercentage of reactor

core released (%) Amount released (Bq)

89Sr90Sr95Zr103Ru106Ru131I134Cs137Cs140Ba144Ce238Pu239Pu240Pu241Pu242Cm

53 days28.8 years65.5 days39.5 days368 days8.05 days2.1 years30.2 years12.8 days284 days87.7 years2.4 × 104 years6.5 × 103 years14.4 years164 days

4.04.03.22.92.9

2010135.62.833333

8.0 × 1016

8.0 × 1015

1.4 × 1017

1.2 × 1017

5.8 × 1016

2.6 × 1017

1.9 × 1016

3.8 × 1016

1.6 × 1017

9.0 × 1016

3.0 × 1013

2.6 × 1013

3.6 × 1013

5.1 × 1015

7.8 × 1014

Table 17.1. Physical half-lives and amounts released of some radiologically important radionuclidesfrom Chernobyl (adapted from IAEA, 1991). Radionuclides of primary radiological concern (over differenttime scales) after the accident are in bold.

Fig. 17.2. Change in amounts of some released radionuclides over time after Chernobyl due to radioactivedecay. The increase in 241Am activity concentration is due to ingrowth from 241Pu.

considerable variation in this value because ofvarying environmental factors. The presentextent of evacuated areas within the fSUis shown in Fig. 17.1 (Beresford and Wright,1999). While there are no plans to reuse muchof this area in the foreseeable future, peopleare ‘illegally’ living within many of the areas(including the 30 km exclusion zone surroun-ding the Chernobyl nuclear power plant) andproducing their own food.

Contamination of the food chain spreadmuch wider than the evacuated areas, affect-ing many areas of Belarus, Ukraine and Euro-pean Russia. Foodstuffs were contaminated insome areas of relatively low 137Cs depositionas a result of high accumulation from certainsoil types. For instance, Beresford and Wright(1999) report that, in areas of soddy podzolicsoils, a 137Cs deposition in the range of140–500 kBq m−2 will result in an annualingested dose of 1 mSv. In areas of peaty soilthis level of dose is reached at 137Cs deposi-tions as low as 7–50 kBq m−2 (see Fig. 17.1 fordeposition within the fSU). Parts of WesternEurope were also affected, with advice not toconsume fresh vegetables being given in, forinstance, parts of Italy and Germany. Mostaffected were the Scandinavian countries,where activity concentrations in reindeer,goat milk, sheep, game animals and fresh-water fish were above intervention levels. Aswith sheep in some upland areas of the UK,some parts of Scandinavia are still subject torestrictions (Howard et al., 2001).

Previous nuclear accidents

The Chernobyl accident was on a muchgreater scale than previous accidental

releases of radioactivity to the environment.The largest nuclear accident prior to Cherno-byl was the explosion in 1957 of a high-levelwaste tank at the Mayak plutonium pro-duction and reprocessing facility in Siberia.Releases of a mixture of radionuclides,including long-lived 90Sr (physical half-life28 years) resulted in evacuation and removalfrom agricultural production of an area ofapproximately 1000 km2. By 1997, 82% of thisland had been reclaimed (Joint Norwegian–Russian Expert Group, 1997).

Following the 1957 fire at the Windscalenuclear reactor in the UK, a ban on the con-sumption of milk because of high 131I activityconcentrations was implemented over an areaextending to a maximum of 518 km2 (Jacksonand Jones, 1991). It is probable that, at presentday intervention levels, temporary precau-tionary bans on foodstuffs, including meatand milk, would also have been implementedas a consequence of radiocaesium contamina-tion. This may also be the case for some foodproducts in some areas as a consequence offallout from the atmospheric nuclear weaponstesting era (predominantly 1952–1962). Theaccident at Three Mile Island in the USAdid not result in significant contamination ofthe environment and food chain; the highestactivity concentration in a food productdetermined in a sample of goat milk was only1.5 Bq l−1 of 131I, collected 2 km from the site(Katherine, 1984).

Studies Prior to the Chernobyl Accident

Prior to Chernobyl, much was already knownabout the movement of radionuclides (radio-caesium and radiostrontium in particular) in


Radiation type Description Stopped by:Approximate relative

biological effectivenessa

α particleβ particleγ ray

Helium nucleusElectronElectromagnetic wave

Air or outer layers of skinA few millimetres of aluminiumA few centimetres of lead

2011

aRelative biological effectiveness is used to convert radiation energy absorbed by the body to radiationdose: for a given absorbed energy, α radiation is estimated to be 20 times more damaging than β or γradiation.

Table 17.2. Characteristics of some radioactive emissions.

the environment. Studies had been carriedout on the environmental and food chaintransfer of global fallout from the above-ground nuclear weapons tests of the 1950sand 1960s. Laboratory studies had shownthat the sorption of 137Cs by soils and sedi-ments is dominated by specific sorption tocertain sites on illitic clay minerals (Jacobsand Tamura, 1960). This sorption was knownto have a slow kinetic component, in which137Cs is transferred to less available sites inthe mineral lattice (Evans et al., 1983), a pro-cess often referred to as ‘fixation’. Caesiumcompetes for binding sites on the clay min-eral lattice with similarly sized K+ and NH4

+

ions. Thus, a common measure of the ‘avail-ability’ or ‘exchangeability’ of radiocaesiumsorption to soils and sediments is to carry outan extraction with ammonium acetate orammonium chloride solution. Coughtrey andThorne (1983) report measurements by Evansand Dekker (1966) that a large proportion(~ 85%) of radiocaesium in soils was in fixedform, with only 15% exchangeable with amolar solution of ammonium acetate, thoughthis fraction varied considerably with soiltype. Environmental studies of global falloutfrom nuclear weapons testing showed thattransfers of radiocaesium to milk were muchhigher in the Faroe Islands than in Denmark.This was attributed to greater availability of137Cs in the organic soils of the Faroe Islandsthan in more mineral soils in Denmark(Aarkrog, 1979).

Environmental variability in 137Cs bind-ing to soil (and consequent uptake by plants)was therefore linked to the clay mineral(specifically illitic clays) content of soils. Asecond important influence on 137Cs transfersthrough the food chain was shown to be thepotassium content of soils (and, in aquaticsystems, the potassium content of lake or riverwater). Because of its chemical similarity topotassium, an important nutrient, caesiumis accumulated in biota through the samemechanisms as potassium. Thus, by a dilutioneffect, in aquatic systems, concentration fac-tors of 137Cs in fish were known to be inverselyrelated to the K+ concentration of the sur-rounding water (Fleishman, 1973; Blaylock,1982). In terrestrial systems, addition of fer-tilizer containing K+ was shown to decrease

137Cs uptake by plants in some soils, thoughthe effectiveness of application was reducedby the potential for K+ ions to de-sorb 137Csfrom the soil binding sites (Coughtrey andThorne, 1983).

For a given activity concentration ofradiocaesium in soil (Bq kg−1), therefore, itwas known that bioaccumulation factors ofradiocaesium could be very high, but thisaccumulation was extremely variable ‘show-ing a range covering four orders of mag-nitude’ (Coughtrey and Thorne, 1983). Asdiscussed above, uptake was known to beinfluenced by a number of environmental fac-tors, but was expected to be highest in soils oflow nutrient status (low K+) and low clay min-eral content. These effects were illustrated bydata collected by the United Nations ScientificCommittee on the Effects of Atomic Radiation(UNSCEAR) on the transfers of radiocaesiumfrom nuclear weapons test fallout to humans.Whole-body radiocaesium activity concentra-tions were approximately two orders of mag-nitude greater in people living in ‘marginal’arctic and subarctic environments with nutri-ent-poor soils (e.g. Finnish reindeer herders,populations in northern Russia and Alaska)than in populations whose diet originatedpredominantly from intensive agriculture(UNSCEAR, 1977). This higher whole-bodyradiocaesium activity concentration waslargely the result of the consumption of foodswith especially high radiocaesium transfers(e.g. reindeer meat).

Radiostrontium exists in solution incationic form (as Sr2+), and its chemical andbiological behaviour is similar to that ofcalcium. In most circumstances, the environ-mental transfer of radiostrontium was shownto be determined by the Ca2+ status of a sys-tem. In contrast to radiocaesium, radiostron-tium is believed to be predominantly sorbedreversibly to ion-exchange sites in soils (e.g.Tikhomirov and Sanzharova, 1978, quoted inCoughtrey and Thorne, 1983). Transfers tobiota are less variable than for 137Cs, and bio-accumulation factors are often significantlylower. Uptake by plants is related to the Caconcentration, with the Sr/Ca ratio in plantsbeing approximately equal to that in soil(Tikhomirov and Sanzharova, 1978, quotedin Coughtrey and Thorne, 1983). In aquatic


environments, 90Sr accumulation in fish isin inverse proportion to the calcium con-centration of the water (Blaylock, 1982).Similarly, 90Sr absorption in the animal andsubsequent transfer to animal-derived foodproducts was shown to be inversely propor-tional to dietary calcium intake, with Sr/Caratios in tissues and milk being lower thanthose in the diet (e.g. the ratio of Sr/Cain milk to Sr/Ca in the diet is ~0.1 formammals) (Comar et al., 1966; Beresford et al.,1998). An important feature of transfers of Srto animals and humans is its incorporationinto bone, and hence its long retention time inthe body.

Radioactivity in Foodstuffs afterChernobyl

Reference levels of radioactivity in foodstuffs

A reference level is a level of radioactivity ina foodstuff at which some action must betaken by regulatory authorities. Exceedingthe reference level can result in a ban or limiton consumption of the product, but, whereappropriate, may simply indicate the needfor further investigation. Reference levelsvary between countries and according tocircumstance. In part, reference levels are setsuch that the overall ingested dose resultingfrom consumption of foodstuffs at this levelis below a certain limit. For example, theymay be higher in the event of a short-term,temporary increase in levels of radioactivityin food than in the case of long-term

contamination. They may also vary accordingto the number of foodstuffs contaminated: if anumber of foodstuffs, forming an importantpart of the diet, are being considered, thereference level may be lower than that for thecase in which a single, less important productis considered.

An example of a reference level used inthe European Community (EC) (EURATOMCouncil Regulations Nos 3958/87, 994/89,2218/89 and 770/90) is the maximum per-mitted level (MPL), as shown in Table 17.3.Reference levels are usually determined bycalculating the mean activity concentrationin foodstuffs which, assuming consumptionover a 1-year period, would lead to a radiationdose deemed to present a negligible risk. Thedose is determined by the radioactivity in aproduct and its consumption rate; thus, MPLsmay be affected by the rate of consumption ofa foodstuff. For example, minor foodstuffssuch as herbs and spices have higher MPLs(by a factor of 10) than the values shown inTable 17.3, which assume high consumptionrates and apply to major foodstuffs such asdairy produce, potatoes and beef. The MPLsin force in the EC assume that the averageactivity concentration of a person’s total dietis a fraction (10%) of the contamination ofcertain individual foodstuffs. This assump-tion is considered to be appropriate since thetotal diet, particularly in Western Europe, is acombination of foodstuffs of different typesoriginating from very disperse areas.

The MPLs for 137Cs activity concentra-tions in foodstuffs in the fSU countries areshown in Table 17.4. These are generally lowerthan the EC levels, partly because there are


Radioactivity in food in Bq kg−1

or Bq l−1 Baby foods Dairy produceOther foods (except

minor foodstuffs) Liquid foodstuffs

Isotopes of strontium, notably90SrIsotopes of iodine, notably131Iα-emitting isotopes of Pu andtrans-Pu elements, e.g. 239Pu,241AmAll other nuclides of half-life> 10 days, notably 134Cs, 137Cs

75

1501

400

125

50020

1000

750

200080

1250

125

50020

1000

Table 17.3. Agreed maximum permitted levels of radionuclides in foods in place in the EuropeanCommunity.

multiple exposure pathways in the Chernobylaffected areas and diets tend to be much morelocalized in the fSU countries than in the EC.In addition, limits in the fSU countries havegenerally decreased with time after the acci-dent. In the first year after the accident, forpragmatic reasons, limits were higher than atpresent. It has been argued that current limitsin the fSU are too restrictive, leading to imple-mentation of food bans and expensive reme-dial measures where actual radiation risks arelow.

Iodine-131

Ingestion of milk and leafy vegetables wasbelieved to have formed the major pathwayfor radioiodine dose to the thyroid followingthe Chernobyl accident (IAEA, 1991). Theshort half-life of 131I (8.05 days) means thatthere are few available measurements infoodstuffs. In the UK, the maximum reportedlevel of 131I in cow milk was 371 Bq l−1 (mea-sured in Cumbria on 4 May 1986), althoughhigher levels were recorded in goat (1040 Bql−1 measured in Cumbria on 9 May 1986) andsheep milk (2095 Bq l−1 measured in Surrey 4May 1996). The maximum permitted level of131I in dairy produce in the UK is 500 Bq kg−1

(data from UK Ministry of Agriculture, Fish-eries and Food monitoring). Kryshev (1994)presents measurements of 131I in fish in theKiev reservoir shortly after Chernobyl. Activ-ity concentrations in fish flesh declined

rapidly by physical decay from around6000 Bq kg−1 on 1 May 1986 to 50 Bq kg−1 on20 June 1986.

Caesium-134, 137

The transfer of radionuclides to foodstuffs iscommonly estimated using an aggregatedtransfer factor (Tag, m2 kg−1):

Tag

Concentration of radionuclidein foodstuff, Bq

=kg

Density of depositedradioactivity, Bq m

m kg1

2

−

−2

−1

Since the transfer of radionuclides to food-stuffs is dependent on a number of factors(including transfer through the soil, soil com-position and chemistry, time after fallout),the Tag is clearly a simplified concept. It is,however, a valuable tool for estimatingactivity concentrations in foodstuffs frommaps of contamination density. In addition,some models (e.g. Yatsalo et al., 1997) canimprove estimates of the Tag by accountingfor changes over time and using soil-specificTag values. More complex models utilize soilproperty data, such as percentage clay, potas-sium content and percentage organic matter,to estimate radiocaesium transfer to foodcrops and grass (Crout et al., 1999).

As could have been predicted from workcarried out before the Chernobyl accident,foodstuffs from intensively managed systems


Belarus Russia Ukraine

Fresh milkButterCheeseBeefPorkChickenBreadPotatoesOther vegetablesCereals and legumesCultivated berriesOther fresh fruitBaby food

111185

60037037074

10010010010010037

370370370740740740370370370370370370185

370

740740740370590590

590185

Table 17.4. Intervention limits for the 137Cs activity concentration (Bq kg−1) in foodstuffs within Belarus,Russia and the Ukraine as in place in 1999.

(in the fSU, these took the form of collectivefarms) had lower radiocaesium concentra-tions than those arising from semi-natural andnatural systems. In recent years, interventionlimits (Table 17.4) have generally not beenexceeded in intensively produced foodstuffs(i.e. the output of collective farms) (Firsakovaet al., 1996). In Belarus, for example, approxi-mately 525 kt of milk and 21 kt of meat wereabove the intervention limit during 1986: by1994, this had dropped to 12.4 and 0.003 kt,respectively (Firsakova et al., 1996). Reduc-tions in the amounts of animal-derived food-stuffs exceeding intervention limits haveoccurred through natural declines in 137Csbioavailability (see below) as well as by theuse of soil-based countermeasures. In thecase of meat, reductions were brought aboutmainly by the selective feeding of uncontami-nated fodder or transportation of animalsto less contaminated areas during the finalfattening stage prior to slaughter.

The Chernobyl accident highlighted thepotential importance of foods not derivedfrom intensive agriculture in the transfer ofradioactivity to people. The rural populationswithin the fSU produce, or gather, much oftheir own diet. Many families own a dairy cowand keep pigs and poultry. Vegetables andfruit are produced in the garden or on nearbyland allocated to villagers by the collective.There is a common tradition of collectingedible fungi and berries from forests (oftentermed forest gifts) and, in some areas, fishfrom lakes or rivers. Therefore, intensiveagricultural products are often of littleimportance to the diet. Radiologically, thislocal food source is important as thetransfer of radiocaesium to wild foods(such as fungi, ericaceous berries and somespecies of freshwater fish) is often muchgreater than that to other foodstuffs. Inaddition, effective ecological half-lives(Teff, the time taken for the amount of radio-activity in a foodstuff to reduce by one half byphysical decay and environmental processes(see below)) are often much longer than inagricultural systems. Also, privately ownedcows often graze poor quality pastures, forestclearings or meadows along watercourses.The soils in such pastures allow a com-paratively high root uptake of radiocaesium.

Consequently, a greater proportion of pri-vately produced milk has 137Cs activityconcentrations in excess of national inter-vention limits than milk produced on collec-tive farms. Within Belarus, there were 73villages in which privately produced milkhad 137Cs activity concentrations in excess of111 Bq kg−1 between 1995 and 1998 (Beresfordand Wright, 1999). (Since the villages repre-sent measurement centres, it is likely that thetotal number of villages producing milk inexcess of 111 Bq kg−1 was greater than 73.)90Sr activity concentrations in the milk of pri-vately owned cattle also exceed interventionlimits within Belarus (Rolevich et al., 1996).The contribution of different foodstuffs toradiocaesium in the diet is illustrated inTable 17.5, using data from a village in theUkraine (Beresford and Wright, 1999).

In rural settlements surveyed withinBelarus, Russia and the Ukraine, 40–75% ofinterviewees consumed wild fungi, 60–70%forest berries and 20–40% fish from locallakes (Strand et al., 1996). Shutov et al. (1996)estimated that fungi and berries could con-tribute up to 60–70% of dietary 137Cs intakeof those adults within Russia. Indeed, withinthe rural population in the affected parts ofRussia, a mean increase in the whole-bodyradiocaesium activity of 60–70% in autumn asa result of fungi consumption has been noted(Skuterud et al., 1997). Similar correlationswere reported between the consumption ofboth forest berries and freshwater fish caughtin local lakes and 137Cs whole-body measure-ments in two Russian settlements (Strandet al., 1996). Table 17.6 illustrates the dif-ferences in 137Cs Tags between differentfoodstuffs and environments.

INFLUENCE OF STABLE POTASSIUM ON 137CS UPTAKE

BY THE FOOD CHAIN Post-Chernobyl stud-ies confirmed previous work which hadshown the influence of potassium on radio-caesium uptake by the food chain. Uptake ofradiocaesium by plants is inversely relatedto the potassium concentration of the soilinterstitial water. One of the most commonlyused countermeasures to reduce 137Cs infoodstuffs in the fSU has been to increaserates of fertilizer application with potassiumbased-fertilizers. However, the current poor


economic climate has led to a reduction in therates of fertilizer use within contaminatedareas. Alexakhin et al. (1996) report that theK2O fertilizer application rate in the Bryansk

oblast of Russia decreased from 81 kg ha−1 in1991 to 18 kg ha−1 in 1993, and that there was aconsequent threefold increase in the transferof 137Cs to cereals and potatoes.


Foodstuff

137Cs activity concentrationBq kg−1

Consumption ratekg day−1 fresh weight

137Cs intake rateBq day−1

Milk

Meat

Potatoes

Other vegetables

Fungi

Berries

Fish

Bread

Total daily intake

87

58

27

23

215

456

55

22

79

1.00

0.10

0.70

0.20

0.02

0.009

0.02

0.50

2.72

8722–900

5.81.8–22

196.5–45

4.51.3–18

430–759

4.10–141.1

0.3–1511

7.0–28215

52–1390

aData from Beresford and Wright (1999).

Table 17.5. Example of consumption rates of different foodstuffs and the contribution of each foodstuffto the daily 137Cs intake, as determined during June/July 1997 in Milyach, Ukraine.a Results arepresented as median values, and the range in intake rates is indicated in italics.

Predicted 137Cs in foodstuff at differentcontamination densities

Foodstuff Transfer factor (Tag) m2 kg−1 2 × 104 Bq m−2 1 × 106 Bq m−2

Milk, Dubrovitsa,Ukraine 1994/95a

Sheep meat, Norway,1993b

Cow meat,recommendedc

Wild mushroom Suillusluteus, 1994, Belarusd

Berries, Vacciniummyrtillus, 1989–1994,Belarusd

Predatory fish, range oflakes, 1993–1997e

1.5 × 10−4 Gley soil3.7 × 10−3 Peat soil42.7 × 10−3

6 × 10−3

41.7 × 10−3

7.7 × 10−3

1.0 × 10−3 Low transfer50 × 10−3 High transfer

3 Bq kg−1

74 Bq kg−1

854 Bq kg−1

5.1 Bq kg−1

834 Bq kg−1

154 Bq kg−1

20 Bq kg−1

1,000 Bq kg−1

150 Bq kg−1

3,700 Bq kg−1

42,700 Bq kg−1

256 Bq kg−1

41,700 Bq kg−1

7,700 Bq kg−1

500 Bq kg−1

50,000 Bq kg−1

aHoward et al. (1996); bDahlgaard (1994); cIAEA (1994); dKenigsberg et al. (1996); eSmith et al. (2000a).

Table 17.6. 137Cs transfer factors of different foodstuffs. Measured Tag values typically vary by severalorders of magnitude and the values given here are for illustrative purposes only. The higher contamina-tion density is illustrative of land in the 0.55–1.5 MBq m−2 contaminated zone in the fSU, the lower isillustrative of the most contaminated areas in Western Europe.

In agreement with studies of weaponstest fallout, inverse relationships wereobserved between the concentration factor(CF) of 137Cs in fish and the K+ concentration oflake waters (Smith et al., 2000a). Thus, it wasfound that fish in lakes in agricultural areas(where potassium concentrations tend to behigher as a result of runoff of fertilizers) tendto be significantly less contaminated thanthose in semi-natural areas, which have lowerpotassium concentrations. Post-Chernobylresearch has demonstrated, however, thatdietary potassium has little influence on thetransfer of radiocaesium to mammals.

Strontium-90

Though information on 90Sr contaminationfrom Chernobyl is relatively sparse, resultsindicate much lower activity concentrationsin foodstuffs than for 137Cs (IAEA, 1991). Thedata indicate 137Cs/90Sr ratios in foodstuffsof between 10/1 and 100/1, as a result ofrelatively lower releases of 90Sr and loweraccumulation by biota. For example, milksamples from the Ovruch region with1068 Bq l−1 137Cs contained only 5 Bq l−1 90Sr(IAEA, 1991). In the Chernobyl cooling pond,90Sr activity concentrations were around2 kBq kg−1 in fish during 1986, compared witharound 100 kBq kg−1 for 137Cs (Kryshev,1994). It is likely that 137Cs/90Sr ratios willtend to be lower closer to Chernobyl thanin samples from areas further away sinceless volatile 90Sr was associated with fuelparticles deposited mainly within the 30 kmzone. Weathering of such ‘hot particles’ hasincreased in 90Sr in vegetation over time afterthe accident in some parts of the exclusionzone (Kashparov et al., 1999).

Time-dependent Transfer ofRadiocaesium to Foodstuffs

During the years after Chernobyl, thebioavailability and environmental mobility ofradiocaesium declined markedly, resulting inlarge changes in contamination of foodstuffs,vegetation and surface waters. Laboratorystudies on the sorption of radiocaesium by

soils and sediments quantified the selectivebinding of Cs to specific sorption sites(‘frayed edge sites’, FES) on illitic clay miner-als (Cremers et al., 1988). On these sorptionsites, radiocaesium is available for ionexchange with ions that have a similarhydrated radius, specifically potassium andammonium. Over time, however, radio-caesium slowly diffuses into the illite lattice(Comans and Hockley, 1992) becomingunavailable for direct ion exchange, a processcommonly known as ‘fixation’.

During the weeks to months after a fall-out event, radiocaesium activity concentra-tions in both vegetation and surface waters aredetermined by short-term processes. Activityconcentrations in plants are determined by theinterception and washoff rates of the initialfallout, as well as uptake by the roots (e.g.Fesenko et al., 1997). Similarly, in rivers andlakes, activity concentrations are high initiallyas a result of direct deposition to the watersurface and rapid runoff of 137Cs before itis sorbed by catchment soils (Monte, 1995;Smith et al., 1997). Activity concentrationsthen decline over a period of weeks to monthsas a result of reduced runoff from catchmentsand, for lakes, loss of 137Cs through the out-flow and deposition to bottom sediments.

On long time scales (years), the processeswhich determine radiocaesium transfers toand from many different ecosystem compo-nents (e.g. between plants and animals) arefast in comparison with the slow decline inradiocaesium availability in soil (Fig. 17.3).Thus, the change in radiocaesium activityconcentration in the main environmentalcompartments should be controlled by slowchanges in its soil–soil-solution partitioning.To test this hypothesis, Smith et al. (1999)analysed many long-term field studies oftemporal changes in radiocaesium in threedifferent ecosystem components: vegetation,surface waters (dissolved phase) and milkfollowing the Chernobyl accident (examplesshown in Fig. 17.4(a)).

Long-term rates of change in the radio-nuclide content of vegetation and biota aredescribed by assuming an exponential declinein radioactivity concentration, C:

C = C(0)e−λt


Values of λ include a physical decay compo-nent (137Cs decay constant: 0.023 year−1). Therate of decline, λ (year−1), is often quoted asan effective ecological half-life (the time takenfor the amount of radioactivity in a foodstuffto decline by one half), Teff (years), where Teff

= ln(2)/λ.Figure 17.4(b) shows a histogram of

measured Teff values in the three differentecosystem components, vegetation, milk andwater. Combining results for all three ecosys-tem components gives a mean Teff = 1.7 years,with 91% of all measurements falling withinthe range 1–4 years.

Clearly, there is natural variation in Teff

values, which may be linked to soil character-istics. However, to our knowledge, there iscurrently no systematic way of predictingsuch variation. Nevertheless, the range inrates of decline observed (Teff = 1–4 years) isrelatively small: a value of Teff = 2 years willadequately describe most cases.

However, as shown in Fig. 17.4(c), recentdata (Jonsson et al., 1999; Smith et al., 2000b)show a two-component exponential decline(C = C1e−k

1t + C2e−k

2t) in 137Cs activity concen-

trations in surface water, terrestrial vegetationand fish. The observations show that theeffective ecological half-life in young fish,water and terrestrial vegetation has increased

from 1–4 years during the first 5 years afterChernobyl to 6–30 years in recent years. Fromthe observed persisting mobility of radio-caesium in the environment, and particularlythe increase of Teff towards the physical decayrate of 137Cs (t½ = 30.2 years), it was conclu-ded (Smith et al., 2000b) that the sorption–desorption process of radiocaesium in soilsand sediments is tending towards a reversiblesteady state.

Predicting future 137Cs contamination

The continuing mobility of 137Cs in theenvironment means that foodstuffs willremain contaminated for much longer thanwas expected initially. In the UK, restrictionson the sale and slaughter of sheep arecurrently in place on 389 upland farms(~232,000 sheep), on which some sheep have137Cs activity concentrations above the UKlimit for the entry of meat into the food chain(1000 Bq kg−1). During studies conducted onthree restricted farms between 1991 and 1993,the maximum 137Cs level in sheep meat was1870 Bq kg−1. Assuming that this is typical ofrestricted farms within the UK, and using therates of long-term decline estimated above,


Fig. 17.3. Schematic diagram indicating time scales, τ, of release of radiocaesium from soils to terrestrialand aquatic ecosystems during the years after a fallout event (adapted from Smith et al., 1999). The timescale of ‘fixation’ in soils is significantly longer than rates of retention and release of radioactivity in theother parts of the ecosystem. Thus, in the long term, changes in the soil–soil-water partitioning controlchanges in radioactivity concentration in surface waters, vegetation, etc.

384J.T.Sm

ith andN

.A. B

eresford

Fig. 17.4. (a) Examples of changes in 137Cs activity concentration in different ecosystem components after Chernobyl (Smith et al., 1999). (b) Frequency distributionof effective ecological half-lives in different ecological components during the first 5 years after Chernobyl (Smith et al., 1999). (c) Long-term changes in 137Cs inbrown trout, Norway (Jonsson et al., 1999), perch, terrestrial vegetation and water, UK (Smith et al., 2000b). The decline in 137Cs in immature fish, water andvegetation during the first 5 years has a Teff of 1–4 years as a result of ‘fixation’. The dotted lines demonstrate a hypothetical continuation of irreversible fixation.Fits of the two exponential models to the data, indicating reversible ‘fixation’, are shown as solid lines. Long-term declines in the UK and Norwegian systems aresimilar (Teff 6–30 years).

restrictions may need to remain in place onsome farms for a total of 30 years after theChernobyl accident; similar predictions forthe continuing need for restrictions have alsorecently been made by Nisbet and Woodman(1999). The predicted time period for res-trictions is more than 100 times longer thanwas expected initially. In some areas of thefSU, consumption of forest berries, fungi andfish (current 137Cs content 10–100 kBq kg−1),which contributes significantly to people’sradiation exposure, will need to be restrictedfor at least a further 50 years (Beresford et al.,2001).

Risk Assessment

A full description of the methods of radia-tion risk assessment is beyond the scope ofthis chapter. Radiation risk assessment is acomplex subject, and the following is onlya brief outline of the principles of assessmentof risks from ingestion of radioactivity infoodstuffs.

The amount of radiation absorbed in thebody is measured in grays (Gy), where onegray equals one joule of radiation energyabsorbed per kilogram. This absorbed energycan be estimated quite precisely using modelsfor the transfer of ionizing radiation throughtissues. The damage that the absorbed radia-tion does to people is much more difficultto quantify, particularly at the relatively lowdoses associated with environmental contam-ination. There are, however, a number of epi-demiological studies relating absorbed doseto risk of adverse health effects (primarilycancer risk).

Because different types of radiation dam-age cells in different ways, radiation risk ismeasured in dose equivalents, sieverts (Sv),rather than in total energy absorbed. For β andγ radiation, 1 Gy of absorbed energy results ina dose equivalent of approximately 1 Sv. Forthe (potentially) more damaging α particles,1 Gy of absorbed energy results in a doseequivalent of approximately 20 Sv (see Table17.2). The dose equivalent (Sv) is estimatedfrom a concentration of radioactivity in afoodstuff (in becquerels) using an ingestion

dose coefficient (Sv Bq−1). Ingestion dose coef-ficients are calculated from models to estimatethe absorbed radiation energy by the bodyfrom ingestion of radioactivity (ICRP, 1990).Such models account for the energy and typeof emitted particle, the extent of absorptionof the radionuclide by the body, and its distri-bution and retention time in the body. Dosecoefficients are determined for different ages(e.g. infants, children and adults), their valuesgenerally decreasing with age.

Using epidemiological studies, primarilyof survivors of the Hiroshima and Nagasakiatomic bombs, radiation protection agencieshave estimated the lifetime fatal cancer risk topeople from exposure to ionizing radiation.Current risk estimates (ICRP, 1991) predict a5% lifetime risk of fatal cancer per sievert ofdose to each individual of a population. Thismeans that, if a population is exposed to radia-tion leading to a dose equivalent of 1 Sv toeach person, these risk estimates predict a5% increase in the number of fatal cancersin that population. These cancers do notoccur immediately, but may arise some timeafter exposure. Since dose rates to mostindividuals after Chernobyl were generallymuch lower than 1 Sv (so predicted excesscancer risk is significantly less than 5% formost exposed populations), increases in fatalcancers can be expected to be difficult to detectagainst an existing cancer mortality rate ofaround 20% in populations in industrializedcountries.

The fatal cancer risk from consuming‘contaminated’ foodstuffs is calculated byestimating the total amount of ingested radio-activity. For example, if lamb of 137Cs activityconcentration 1000 Bq kg−1 is consumed at theUK average adult consumption rate of 8 kgover 1 year (NRPB, 1996), the total activityof 137Cs ingested is 8000 Bq during the year.The dose arising from this radioactivity iscalculated by multiplying the total becquerelsingested by the ingestion dose coefficient. For137Cs in, for example, adults, the ingestiondose coefficient is 1.3 × 10−8 Sv Bq−1 (ICRP,1990), which results in an estimated dose of1.44 × 10−4 Sv for an intake of 8000 Bq year−1.According to current risk estimates (5% persievert for an exposed population, for chronic,low dose radiation), this is approximately


equivalent to a probability of fatal cancer of7.2 × 10−6 or 1 in 140,000.

In such radiation risk assessments, it iscurrent practice to assume, as we have doneabove, that even very low dose radiation car-ries with it an associated cancer risk, thoughthis is a matter of some debate within theradiation protection community. Epidemio-logical studies have not shown clear evidenceof increased cancer risk at very low dosesand low dose rates (~100 mSv or less). Thelarge-scale studies required to show the verysmall increase in cancers expected haveshown no evidence of an increase, or haveproved inconclusive, largely because of themany confounding factors associated withsuch large-scale studies. Indeed, some studiesof populations exposed to varying levelsof natural radioactivity have observed adecrease in cancer rates with increased expo-sure to radiation. Thus the risks from lowdoses (and low dose rates) of radiation (lessthan ~100 mSv) could perhaps best bedescribed as theoretical, but, for radiationprotection purposes, it is conservativelyassumed that every exposure to ionizingradiation carries a potential risk.

Risk Management: Countermeasures

Because many of the radionuclides releasedby Chernobyl were short lived, radiationdoses were greatest in the few days after theaccident. Thus the most effective measure toreduce radiation doses was the rapid evacua-tion of the population from the immediatearea. Long-term bans on consumption of con-taminated produce were also put in place,and these were believed to have reducedingestion dose rates substantially. Such bans,however, are not always adhered to becauseof the difficulty of obtaining good, radio-actively ‘clean’ produce in the affected areasof the fSU. Thus, in many areas, consumptionof privately produced milk and meat aboveintervention levels and of ‘wild’ foods(mushrooms, berries, freshwater fish) stillcontribute significantly to radiation doses.

Radiation doses from 131I were reducedby bans on consumption of milk in Russia, but

to a lesser extent in Belarus. After the 1957accident at Windscale, a ban on the consump-tion of milk was implemented, which avertedbetween 55 and 75% of the potential maxi-mum 131I dose to a child’s thyroid (Jacksonand Jones, 1991) in the area covered by theban.

After Chernobyl, distribution of iodinetablets was not widespread and could havesignificantly reduced doses to the thyroid.However, a World Health Organization/European Commission working group con-cluded that, for adults over 40 years old livingwell away from the site of an accident, con-trols on food would be a more appropriatemeasure to reduce radioiodine ingestion ratesand hence doses to the thyroid (Rubery andSmales, 1990). This is because there are mem-bers of the population who are sensitive tolarge intakes of stable iodine, so the potentialrisks of stable iodine prophylaxis can begreater than the benefit of reduced radio-iodine burdens.

Restrictions on foodstuffs were alsoimplemented in Western Europe, as a result of137Cs contamination of the food chain. Theseincluded controls on the sale and slaughter ofmore than 4 million sheep on 8914 holdings inthe UK. Restrictions were also put in place inparts of Scandinavia because of high radio-caesium activity concentrations in many foodproducts including goat milk, reindeer, sheepmeat and game animals. Consumption offreshwater fish declined by up to 50% in themore contaminated areas of Sweden, and thesale of freshwater fish to the general publicwas prohibited in these areas (Brittain et al.,1991).

A requirement to respond to theChernobyl accident provided a considerableimpetus to developing effective counter-measures (Howard and Desmet, 1993).Reductions in the amounts of animal-derivedfoodstuffs exceeding intervention limits havebeen brought about by natural declines in137Cs bioavailability (see above) as well as bythe use of soil-based countermeasures suchas deep ploughing (to reduce activity concen-trations in the surface soil layers) or additionof potassium fertilizers. In the case of meat,reductions were also brought about bythe selective feeding of uncontaminated


fodder or transportation of animals to lesscontaminated areas during the final fatteningstage prior to slaughter. Radiocaesium in milkand meat can also be reduced by addingchemicals which bind radiocaesium (hexa-cyanoferrates or ‘Prussian blue’) to the diet ofgrazing animals. The hexacyanoferrate reactswith consumed radiocaesium in the intestineto form a complex that is eliminated in thedung instead of passing into the animal’sbloodstream. In Western Europe, much ofthe long-term problem after the Chernobylaccident has been connected with free-ranging or wild animals. To cope with this,delivery systems which do not need dailyaccess to animals were developed in which thehexacyanoferrate binder is incorporated intodifferent matrixes, including rumen-dwellingboli and salt licks (Hove, 1993).

Though many countermeasures havebeen used to reduce radioactivity afterChernobyl, there has also been an increasedawareness that it is not only the effectivenessof a countermeasure that has to be consideredbut also issues of feasibility, cost effectiveness,side effects and public acceptance (Voigt et al.,2000).

Conclusions

Chernobyl was one of the worst industrialaccidents in history, causing potential risks tothe safety of the food chain on a continentalscale. Thousands of square kilometres of landin Belarus, Russia and Ukraine still cannotbe used for agricultural production and havebeen defined as areas of strict radiation con-trol. Populations of whole towns and villageshave been relocated, creating hundreds ofthousands of ‘Chernobyl refugees’.

Scientific studies on Chernobyl radio-activity in the food chain have establishedwhich areas and foodstuffs are most affected,as well as identifying many of the environ-mental factors which determine food chaincontamination. It is now known that the con-tamination of foodstuffs (particluarly ‘wildfoods’) by 137Cs will remain a problem for 50years or more in the affected regions of thefSU. In spite of the extensive environmental

contamination, however, current radiationdoses to the Chernobyl-affected populationsare low. Indeed, the social and economic con-sequences of the accident may well outweighthe direct health effects of the radiation. Inaddition to the social and economic conse-quences of evacuating large areas of land, amajor problem in dealing with the long-termeffects of the accident is the public’s over-estimation of radiation risk. An InternationalAtomic Energy Authority (IAEA) study foundthe psychological effects of Chernobyl to be‘wholly disproportionate to the biologicalsignificance of the radiation’ (IAEA, 1991).Populations in the contaminated areas havebeen shown to have higher levels of stress,worse perceived health and greater use ofmedical facilities (e.g. number of doctor visits)than similar unaffected populations. Thechallenge for scientists and decision makersnow is to determine how best to manage thecontaminated areas in the decades to come. Akey element of this management strategy isan information and education programme forthe people who have to live with Chernobyl’sradioactive legacy.

References

Aarkrog, A. (1979) Environmental Studies onRadioecological Sensitivity and Variability withSpecial Emphasis on the Fallout Nuclides Sr-90 andCs-137. Risφ National Laboratory Report, Risφ,Denmark.

Alexakhin, R., Firsakova, A., Rauret, G., Arkhipov,N., Vandecasteele, C.M., Ivanov, Y., Fesenko,S. and Sanzharova, N. (1996) Fluxes ofradionuclides in agricultural environments:main results and still unsolved problems. In:Karaoglou, A., Desmet, G., Kelly, G.N. andMenzel, H.G. (eds) The Radiological Conse-quences of the Chernobyl Accident. EuropeanCommission, Brussels, pp. 39–47.

Beresford, N.A. and Wright, S.M. (eds) (1999)Self-help Countermeasure Strategies for Popula-tions Living Within Contaminated Areas of theFormer Soviet Union and an Assessment of LandCurrently Removed from Agricultural Usage.Institute of Terrestrial Ecology, Grange-over-Sands, UK.

Beresford, N.A., Mayes, R.W., Hansen, H.S., Crout,N.M.J., Hove, K. and Howard, B.J. (1998)


Generic relationship between calcium intakeand radiostrontium transfer to milk of dairyruminants. Radiation Environmental Biophysics37, 129–131.

Beresford, N.A., Voigt, G., Wright, S.M., Howard,B.J., Barnett, C.L., Prister, B., Balonov, M.,Ratnikov, A., Travnikova, I., Gillett, A.G.,Mehli, H., Skuterud, L., Lepicard, S.,Semiochkina, N., Perepeliantnikova, L.,Goncharova, N. and Arkhipov, A.N. (2001)Self-help countermeasure strategies for popu-lations living within contaminated areas ofBelarus, Russia and the Ukraine. Journal ofEnvironmental Radioactivity 56, 215–239.

Blaylock, B.G. (1982) Radionuclide data bases avail-able for bioaccumulation factors for freshwaterbiota. Nuclear Safety 23, 427–438.

Brittain, J.E., Storruste, A. and Larsen, E. (1991)Radiocaesium in brown trout (Salmo trutta)from a subalpine lake ecosystem after theChernobyl reactor accident. Journal of Environ-mental Radioactivity 14, 181–191.

Comans, R.N.J. and Hockley, D.E. (1992) Kineticsof cesium sorption on illite. Geochimica etCosmochimica Acta 56, 1157–1164.

Comar, C.L., Wasserman, R.H. and Lengemann,F.W. (1966) Effect of dietary calcium on secre-tion of strontium into milk. Health Physics12, 1–6.

Coughtrey, P.J. and Thorne, M.C. (1983)Radionuclide Distribution and Transport inTerrestrial and Aquatic Systems. A Critical Reviewof Data, Vol. 1. A.A. Balkema, Rotterdam.

Cremers, A., Elsen, A., De Preter, P. and Maes, A.(1988) Quantitative analysis of radiocaesiumretention in soils. Nature 335, 247–249.

Crout, N., Gillett, A., Absalom, J. and Young, S.(1999) SAVE-IT Spatial and Dynamic Predictionof Radiocaesium Transfer to Food Products. Part 3:Model Description. University of Nottingham,Nottingham, UK.

Dahlgaard, H. (1994) Nordic Radioecology: the Trans-fer of Radionuclides Through Nordic Ecosystems toMan. Elsevier, Amsterdam.

de Cort, M., Dubois, G., Fridman, S.D.,Germenchuk, M.G., Izrael, Y.A., Janssens, A.,Jones, A.R., Kelly, G.N., Kvasnikova, E.V.,Matveenko, I.I., Nazavov, I.M., Pokumeiko,Y.M., Sitak, V.A., Stukin, E.D., Tabachny, L.Y.,Tsatavov, Y.S. and Avdyushin, S.T. (1998)Atlas of Caesium Depositon on Europe Afterthe Chernobyl Accident. EUR 16737, CEC,Luxemburg.

Demidchik, E.P., Drobyshevskaya, I.M., Cherstvoy,E.D., Astakhova, L.N., Okeanov, A.E.,Vorontsova, T.V. and Germenchuk, M. (1996)Thyroid cancer in children in Belarus.

In: Karaoglou, A., Desmet, G., Kelly, G.N.and Menzel, H.G. (eds) The RadiologicalConsequences of the Chernobyl Accident.European Commission, Brussels, pp. 677–682.

Drozdovitch, V.V., Goulko, G.M., Minenko, V.F.,Paretzke, H.G., Voigt, G. and Kenigsberg, Y.I.(1997) Thyroid dose reconstruction for thepopulation of Belarus after the Chernobylaccident. Radiation and EnvironmentalBiophysics 36, 17–23.

Evans, D.W., Alberts, J.J. and Clark, R.A. (1983)Reversible ion-exchange fixation of cesium-137 leading to mobilization from reservoirsediments. Geochimica et Cosmochimica Acta 47,1041–1049.

Fesenko, S.V., Spiridonov, S.I., Sanzharova, N.I. andAlexakhin, R.M. (1997) Dynamics of 137Csbioavailability in a soil–plant system in areas ofthe Chernobyl nuclear power plant accidentzone with a different physico-chemical compo-sition of radioactive fallout. Journal of Environ-mental Radioactivity 34, 287–313.

Firsakova, S., Hove, K., Alexakhin, R., Prister, B.,Arkhipov, N. and Bogdanov, G. (1996)Countermeasures implemented in intensiveagriculture. In: Karaoglou, A., Desmet, G.,Kelly, G.N. and Menzel, H.G. (eds) TheRadiological Consequences of the ChernobylAccident. European Commission, Brussels,pp. 379–387.

Fleishman, D.G. (1973) Radioecology of marineplants and animals. In: Klechkovskii, V.M.,Polikarpov, G.G. and Aleksakhin, R.M. (eds)Radioecology. John Wiley & Sons, New York,pp. 347–370.

Hove, K. (1993) Chemical methods for reduction ofthe transfer of radionuclides to farm animals insemi-natural environments. Science of the TotalEnvironment 137, 235–248.

Howard, B.J. and Desmet, G.M. (1993) REACT:relative effectiveness of agricultural counter-measure techniques. Science of the TotalEnvironment 137, 11–12.

Howard, B.J., Hove, K., Prister, B., Ratnikov, A.,Travnikova, I., Averin, V., Pronevitch, V.,Strand, P., Bogdanov, G. and Sobelev, A. (1996)Fluxes of radiocaesium to milk and appro-priate countermeasures. In: Karaoglou, A.,Desmet, G., Kelly, G.N. and Menzel, H.G. (eds)The Radiological Consequences of the ChernobylAccident. European Commission, Brussels,pp. 349–362.

Howard, B.J., Beresford, N.A. and Voigt, G. (2001)Countermeasures for animal products: areview of effectiveness and potential useful-ness after an accident. Journal of EnvironmentalRadioactivity 56, 115–137.


IAEA (1991) The International Chernobyl ProjectTechnical Report. International Atomic EnergyAgency, Vienna.

IAEA (1994) Handbook of Transfer Parameter Valuesfor the Prediction of Radionuclide Transfer inTemperate Environments. Technical ReportSeries No. 364. International Atomic EnergyAgency, Vienna.

ICRP (1990) Age-dependent Doses to Members ofthe Public from Intake of Radionuclides: Part 1.International Commission on RadiationProtection Publication 56, Pergamon Press,Oxford.

ICRP (1991) Recommendations of the InternationalCommission on Radiological Protection. Inter-national Commission on Radiation ProtectionPublication 60, Pergamon Press, Oxford.

Jackson, D. and Jones, S.R. (1991) Reappraisal ofenvironmental countermeasures to protectmembers of the public following theWindscale nuclear reactor accident 1957.In: Comparative Assessment of the EnvironmentalImpact of Radionuclides Released During ThreeMajor Nuclear Accidents: Kyshtym, Windscaleand Chernobyl. 1957, EUR 13574, CEC, Luxem-burg, pp. 1015–1055.

Jacobs, D.G. and Tamura, T. (1960) The mechanismof ion fixation using radio-isotope techniques.In: Proceedings of the 7th International Congress ofSoil Science, Madison, Wisconsin, pp. 206–214.

Joint Norwegian–Russian Expert Group (1997)Sources Contributing to Radioactive Contamina-tion of the Techa River and Areas Surrounding theMAYAK Production Association, Urals, Russia.Norwegian Radiation Protection Authority,Oslo.

Jonsson, B., Forseth, T. and Ugedal, O. (1999)Chernobyl radioactivity persists in fish. Nature400, 417.

Kashparov, V.A., Oughton, D.H., Zvarich, S.I.,Protsak, V.P. and Levchuk, S.E. (1999) Kineticsof fuel particle weathering and 90Sr mobility inthe Chernobyl 30 km exclusion zone. HealthPhysics 76, 251–299.

Katherine, R.L. (1984) Radioactivity in the Environ-ment: Sources, Distribution and Surveillance.Harwood Academic Publishers, Chur, UK.

Kenigsberg, J., Belli, M., Tikhomirov, F., Buglova, E.,Shevchuk, V., Renaud, P., Maubert, H., Bruk,G. and Shutov, V. (1996) Exposures from con-sumption of forest produce. In: Karaoglou, A.,Desmet, G., Kelly, G.N. and Menzel, H.G. (eds)The Radiological Consequences of the ChernobylAccident. European Commission, Brussels,pp. 271–281.

Kryshev, I.I. (1994) Radioactive contamination ofaquatic ecosystems following the Chernobyl

accident. Journal of Environmental Radioactivity27, 207–219.

Monte, L. (1995) Evaluation of radionuclide transferfunctions from drainage basins of freshwatersystems. Journal of Environmental Radioactivity26, 71–82.

Nisbet, A.F. and Woodman, R.F. (1999) Options forthe Management of Chernobyl-restricted Areasin England and Wales. NRPB-R305, NationalRadiological Protection Board, Chilton, UK.

NRPB (1996) Generalised Habit Data for RadiologicalAssessments. Memorandum of the NationalRadiological Protection Board NRPB-M636,Chilton, UK.

Rolevich, I.V., Kenik, I.A., Babosov, E.M. andLych, G.M. (1996) Report for Belarus. In: OneDecade after Chernobyl: Summing up theConsequences of the Accident. IAEA, Vienna,pp. 411–428.

Rubery, E. and Smales, E. (eds) (1990) IodineProphylaxis Following Nuclear Accidents. Pro-ceedings of a joint WHO/CEC workshop July1988. Pergamon Press, Oxford.

Shutov, V.N., Bruk, G.Y., Basalaeva, L.N.,Vasilevitskiy, V.A., Ivanova, N.P. and Kaplun,I.S. (1996) The role of mushrooms and berriesin the formulation of internal exposure dosesto the population of Russia after the Chernobylaccident. Radiation Protection Dosimetry 67,55–64.

Skuterud, L., Balanov, M., Travnikova, I., Strand, P.and Howard, B.J. (1997) Contribution of fungito radiocaesium intake of rural populations inRussia. Science of the Total Environment 193,237–242.

Smith, J.T., Leonard, D.R.P., Hilton, J. and Appleby,P.G. (1997) Towards a generalised model forthe primary and secondary contaminationof lakes by Chernobyl derived radiocaesium.Health Physics 72, 880–892.

Smith, J.T., Fesenko, S.V., Howard, B.J., Horrill,A.D., Sanzharova, N.I., Alexakhin, R.M., Elder,D.G. and Naylor, C. (1999) Temporal change infallout 137Cs in terrestrial and aquatic systems:a whole-ecosystem approach. EnvironmentalScience and Technology 33, 49–54.

Smith, J.T., Kudelsky, A.V., Ryabov, I.N. andHadderingh, R.H. (2000a) Radiocaesium con-centration factors of Chernobyl-contaminatedfish: a study of the influence of potassium and‘blind’ testing of a previously developedmodel. Journal of Environmental Radioactivity48, 359–369.

Smith, J.T., Comans, R.N.J., Beresford, N.A., Wright,S.M., Howard, B.J. and Camplin, W.C. (2000b)Chernobyl’s legacy in food and water. Nature405, 141.



Strand, P., Howard, B.J. and Averin, V. (eds) (1996)Transfer of Radionuclides to Animals, Their Com-parative Importance Under Different AgriculturalEcosystems and Appropriate Counteremeasures.Experimental collaboration project No. 9. FinalReport. European Commission, Belarus, theRussian Federation, Ukraine. EUR 16539EN.European Commission, Luxemburg.

Tsyb, A.F., Parshkov, E.M., Shakhtarin, V.V.,Stepanenko, V.F., Skvortsov, V.F. andChebotareva, I.V. (1996) Thyroid cancer inchildren and adolescents of Bryansk andKaluga regions. In: Karaoglou, A., Desmet,G., Kelly, G.N. and Menzel, H.G. (eds) TheRadiological Consequences of the ChernobylAccident. European Commission, Brussels,pp. 691–697.

UNSCEAR (1977) Sources, Effects and Risks of IonizingRadiation, Report to the General Assembly,

with Annexes. United Nations Publication,New York.

UNSCEAR (2000) Sources, Effects and Risks of IonizingRadiation. Report to the General Assembly,with Annexes. United Nations Publication,New York.

Voigt, G., Eged, K., Hilton, J., Howard, B.J., Kris, Z.,Nisbet, A.F., Oughton, D.H., Rafferty, B., Salt,C.A., Smith, J.T. and Vandenhove, H. (2000)A wider perspective on the selection of coun-termeasures. Radiation Protection Dosimetry 92,45–48.

Yatsalo, B., Mirzeabassov, O., Okhrimenko, I.,Pichugina, I. and Kulagin, B. (1997) PRANA –decision support system for assessment ofcountermeasure strategy in the long-termperiod of liquidation of the consequences ofa nuclear accident (agrosphere). RadiationProtection Dosimetry 73, 291–294.

18 Radionuclides in Foods: AmericanPerspectives

E.J. Baratta*US Food and Drug Administration, 109 Holton Street, Winchester, MA 01890, USA

Introduction

The US Food and Drug Administration(FDA) has the responsibility for the whole-someness of the food supply in the USA.In 1961, the FDA initiated a programmefor monitoring the radionuclides in foodsin the teenage diet (FDA, 1963; Laug et al.,1963). This was in response to the above-ground weapons testing. At the same time,the US Public Health Service (PHS) insti-tuted a nationwide pasteurized milk net-work (PMN); in cooperation with the FDA(Roecklin et al., 1970), milk samples werecollected from at least one of the largest citiesin each state.

These samples were forwarded to oneof three PHS laboratories for analysis. Theradionuclides of interest were 131I, 137Cs, 140La,90Sr and 89Sr. Various food products werecollected as they were harvested. The PHSalso instituted a food programme (Roecklinet al., 1970) called the Institutional DietProgram (IDP) for collecting samples fromvarious institutions of teenage diets, similarto the FDA programme, except that in thePHS study the total diet sample was homoge-nized, rather than analysing each component.With the signing of the US–Soviet above-

ground weapons testing ban treaty, theseprogrammes were changed.

The FDA had stopped sampling andanalysing the foods in the teenage diet in1969 to avoid duplication. In 1973, the FDAdecided to resume its programme, due to agovernmental reorganization. Two of the PHSlaboratories had been transferred to the Envi-ronmental Protection Agency (EPA) and wereno longer analysing for radioactivity in food(Anderson and Nelson, 1962). The PMN wasput on a standby and EPA in 1973 cancelledthe IDP.This new FDA programme includedthe teenage and infant diet sample and wasresponsible for analysing radioactivity infood (Food and Drug Administration, 1973).This Teenage and Infant Diet Program wasalso a part of its Total Diet Program Study,which analyses for various other components.The radionuclides in the food programmealso included imported foods that haddomestic status. In addition, the FDA wouldbe able maintain a capability for analysingradionuclides in food samples in the eventof a release from a nuclear accident. Suchincidents occurred, the first in 1979, whenthere was an accident at the Three MileIsland Nuclear Power Plant near Harrisburg,Pennsylvania. Later, in 1986, there was an


11-Mar-03 18




accident at the Chernobyl nuclear powerplant near Kiev in the Ukraine, USSR.(see Smith and Beresford, Chapter 17, thisvolume).

Nature of Radionuclides of Interest

The primary radionuclides found in foodsand milk were the short-lived fission prod-ucts 89Sr, 131I, 140Ba and 140La. In addition,there were several longer-lived fission prod-ucts such as 137Cs and 90Sr. 134Cs was found inproducts from the Chernobyl incident. Thefood products contained other short-livedand longer-lived fission products such as65Zn, 95Zr, 95Nb, 103Ru, 106Ru, 140Ce and 141Ce.These radionuclides were not found inmilk. The milk and milk products that wereproduced from the cow contained only theformer, as the cow’s digestive system dis-criminated against the other radionuclides.

Background – nature of radionuclides

The modern epoch in physics may be saidto have begun with the discovery of X-raysby Roentgen in 1895. This was followed bythe discovery of radioactivity by Becquerelin 1896. These two discoveries led to otherdevelopments in the understanding ofnuclear structure, reactions and the varioustypes of particles involved. All these investi-gations finally culminated in the discovery of‘fission’, which resulted in the developmentof atomic energy, in both peace and war.

Atomic structure

All matter is made up of elements. The small-est part of an element is the atom. The size ofa hydrogen atom, which is the smallest, isabout 1.5 × 10−8 cm while its weight is about1.67 × 10−24 g. In comparison, a uranium atomwould weigh about 4.0 × 10−22 g. The atomitself consists of a central nucleus surroundedby a cloud of electrons ranging in numberfrom one for hydrogen to 92 for uranium.These electrons are said to move in orbitsaround the central nucleus. The size of the

nucleus is considerably smaller than that ofthe atom, the size of the hydrogen nucleusbeing 1.4 × 10−13 cm. The nucleus itself con-sists of protons, which carry a unit positivecharge ‘e’ (e = 1.6 × 10−17 coulombs), andneutrons, which carry no charge. The mass ofthe neutron is only slightly greater than thatof the proton. The electron carries a unit neg-ative charge and has a mass approximately1/1840 of that of a proton or a neutron.Hence, most of the mass of the atom is carriedin the nucleus. It is also known that most ofthe energy of the atom is also stored in thenucleus.

Elements and isotopes

All the elements of the periodic table aremade up of protons and neutrons in thenucleus and electrons orbiting around it. Thetotal charge carried by the nucleus is equal tothe number of protons in it. This number iscalled the ‘atomic number’ and is characteris-tic of each element. Since the atom as a wholeis electrically neutral, there will be as manyelectrons around the nucleus as there areprotons in it. The number of protons plusneutrons gives the ‘mass number’ of theatom. Thus, the mass number of carbon, 6

12 C,is 12 and the atomic number is 6. If two nucleihave the same number of protons in thenucleus, but different number of neutrons,they are called isotopes. For example, 4

7 Beand 4

10 Be are isotopes of beryllium havingthe same number of protons and the numberof neutrons being three and six, respectively.Isotopes have the same chemical properties,which depend on the number of electrons inthe orbits around the nucleus, but differentphysical properties, which depend on thecentral nucleus.

Radioactivity

As mentioned before, it was noted early inthis century that certain substances are radio-active, i.e. they spontaneously emit differenttypes of radiations and transform into otherelements depending on the emitted radia-tions. This process, which was first noted in

11-Mar-03 18

392 E.J. Baratta



the heavy elements such as uranium, radium,etc., was found to obey an exponential decaylaw, i.e. the amount of radiation emitteddecreases with time in such a manner thatthe period for reduction to half the initialvalue is constant. This period is called the‘half-life’ and is characteristic of the con-cerned isotope. The various types ofradiations emitted in radioactive decay aredescribed in subsequent sections.

α Radiation

This is emitted generally by the heavyelements such as uranium, thorium, radium,etc. They are identical to the nucleus of thehelium atom, carrying two protons and twoneutrons. Hence, their mass is about fourtimes that of the nucleus of the hydrogenatom (one proton only) and the charge is +2e,i.e. twice that of the hydrogen nucleus. The αradiation is usually highly energetic, havingenergies in the range of 4–9 million electronvolts (1 MeV = 1.602 × 10−6 ergs). In view oftheir heavy mass and charge, α particles arehighly ionizing and are easily absorbed.Hence, they can travel only a few centimetresor so in air. They cannot penetrate beyond theskin and are harmful only when they enterthe body organs either by inhalation or byingestion of contaminated food.

β Radiation

This type of radiation consists of particlesidentical to electrons. They are emitted inradioactive decay with various energies rang-ing from nearly zero to a maximum energycharacteristic of the decaying radioactiveisotope. Hence, their energy spectrum differsfrom that of α rays, which have discreteenergies. The β rays can travel a few feet in airbut cannot penetrate much beyond the depthof the skin of a person. Hence, like the α rays,they are harmful only when they are insidethe body. From outside, they can cause onlyskin burns. β Rays are absorbed in an approx-imately exponential manner, i.e. they have acharacteristic thickness in the particular man-ner that will reduce the radiation intensity tohalf the initial value. This value depends on

the atomic number of the material, its densityand the maximum energy of the β particles.

γ Radiation

Gamma radiation is electromagnetic radia-tion similar to light but of much higherenergy. The wavelength of γ rays is muchshorter than that of visible light. The energyof the electromagnetic radiation or photon isgiven as W = hν, where h is the Planck’s con-stant, ν (nu) is the frequency, equal to cλ−1,c being the speed of light and λ (lambda)the wavelength of the radiation. γ Rays areemitted in radioactive decay along with α orβ radiations. Like α rays, γ rays have discreteenergies. They are absorbed exponentially inmaterials but, in view of their great penetrat-ing power, only thick blocks of concrete, leador other high-density materials can reducetheir intensity to a small value. The γ rays candeliver a whole-body dose from either out-side or inside the body due to their high pen-etrating power. The element does not changedue to γ radiation, but will change dependingon the accompanying α or β radiation.

Distribution of Radionuclides in theFood Chain

Man’s use of nuclear energy involves theproduction of artificial radionuclides, whichpresent a potential public health problemwith regard to the contamination of theterrestrial food chain. The release of theseradionuclides has not introduced to ourenvironment a new pollutant, but ratherhas increased an old pollutant in terms ofradiation exposure. The problem then is amatter of evaluating the extent of man-maderadioactivity in the environment and itssubsequent health significance. At present,fallout from past nuclear weapons tests isthe most widespread source of artificialradionuclide contamination in the terrestrialenvironment. With limited nuclear testing,other sources such as nuclear reactor opera-tions, nuclear fuel reprocessing, wastedisposal, and medical and industrial usesof radioisotopes will be receiving more

Radionuclides in Foods: American Perspectives 393

11-Mar-03 18



emphasis as significant sources of environ-mental contamination.

The food chain

The pathways which radionuclides follow inmoving from their origin to man constitutethe food chain. Radioactive materials areremoved from the atmosphere by meteoro-logical processes, primarily precipitation. Ingeneral, the most serious food chain contami-nation problem arises from direct depositionof radioactive materials on animal feed cropsor on food crops directly consumed by man.Following this initial deposition, various pro-cesses may remove the radioactive materials,such as being washed off by rain or blown offby wind. The extent of this removal is a func-tion of many physical and biological parame-ters. Man’s intake of radioactive material mayoccur from contaminated food crops, fromcontaminated meat and meat products andfrom contaminated milk or milk products.The inhalation route (atmosphere directlyto man) may be important under special cir-cumstances and is not discussed here. Therelative importance of the various pathwaysof intake will depend on many factors,among which are the physical half-livesof the radionuclides, the rate and routeby which they pass through the food chainand the dietary habits of the population. Theimmediate and generally most significantpathway is pasture–cow–milk–man for themore significant radionuclides up to approxi-mately 100 days following deposition (onetime event). Plant losses are such that, afterthis time period, adsorption by plants is themost significant pathway for the longer-livedradionuclides. The final step in the food chain(uptake by man) primarily depends on thechemical characteristics of the radionuclideand the metabolism of the concentratingorgan.

As an example of radionuclide behaviourin the terrestrial food chain, the pathway ofthe fission product 90Sr may be cited. It hasbeen shown that the grazing animal popula-tion readily transfers this nuclide to man fromatmospheric nuclear testing by deposition on

the earth’s surface, with subsequent plantuptake and transference to milk.

In considering the relationships betweenthe food chain and any particular radio-nuclide, it becomes necessary first to considerthe source of the contaminant. The sourceshould be considered as it relates to the physi-cal and chemical state of the radionuclide,since these properties relate to the degree ofmovement in the food chain. Fission productsfrom an atmospheric burst would not beexpected to exist in the same physical andchemical state as those from a reactor incident,fuel reprocessing plant or waste treatmentfacility. Even among the latter operations, awide variety of physical and chemical states ofthe fission products would be expected

Studies of the movement and effects ofradionuclides in the food chain are of valuebecause they:

• make possible the prediction of therelationships between the kinds of radio-activity in the various steps within thefood chain and the resulting levels inthe human population;

• provide a means for evaluating waysby which the levels in the humanpopulation may be minimized;

• provide background information forsetting up environmental samplingprogrammes and for interpreting thedata obtained;

• make available animal data, whichmay be useful for estimating behaviourof radionuclides in humans;

• make possible predictions of the possi-ble effects of environmental contamina-tion, especially in the grazing animalpopulation.

Radioactive materials from atmosphericreleases are deposited on the earth’s surfaceby precipitation or direct deposition. Althoughthe discussion of possible fallout patterns isbeyond the scope of this chapter, it should bestated that fallout distribution depends onmany parameters. For nuclear weapons test-ing, these include meteorological conditions,fission yield, type of explosion (i.e. ground,water or air) and geographical location. In thecase of a nuclear incident, such as an airrelease from a nuclear reactor, the factors of

11-Mar-03 18

394 E.J. Baratta



major concern would be the existing fissionproduct inventory, the micrometeorologicalconditions and the nature of the surroundingterrain.

Contamination of animals andanimal products

Certain radionuclides are readily transferredto the human population via domestic graz-ing animals, which are effective collectors ofcontamination from various vegetative forms(Eisenbud, 1973). There are many factorswhich affect the degree to which animals arecontaminated. The most important include:

• pasture type used for grazing;• extent of barn feeding (purchased and

stored), miscellaneous feeding practices– age of food and supplemental feedinguse of purchased feed, etc.;

• water source; and• animal housing – degree of sheltering

animals from surface contaminants.

Because of the many variables involved,the degree of animal and animal productcontamination may be quite variable, evenfrom apparently similar sources.

Of particular interest in this pathwayof the food chain are the relationships thatexist between the quantities of radionuclidesingested by the animal and the subsequentquantities which are deposited in the tissuesand secretions that serve as human food. Tostudy these relationships requires knowledgeof the metabolic characteristics of the animaland the particular radionuclide. Only thoseradionuclides which enter the food chain ata significant rate and quantity will be ofimportance to man. These radionuclides mustalso possess characteristics that allow for theircontinued movement through the chain.

Radionuclide Metabolism

Classically, metabolism refers to the bio-logical processes whereby complex cellularelements are synthesized.

General considerations involved inradionuclide metabolism

For practical purposes, only the gastrointesti-nal mode of entry of nuclides into the com-partments of a biological system is important(Thompson, 1960; National Academy ofSciences–National Research Council, 1961).In special situations, the pulmonary and skinroutes may be important in permitting assim-ilation of the nuclides. The intravenous routeof entry is artificial and only important asan experimental tool. However, it must benoted that it simulates the situation once anuclide is absorbed into the bloodstream.This method to some extent by-passes theuncertainties involved in a study with naturalroutes of assimilation.

The gastrointestinal system is probablythe most important route of entry for solu-ble forms of nuclides. Insoluble forms willbe dependent on the degree of solubilityand remain in the intestine, with this organreceiving the bulk of the radiation exposure.α Emitters will dissipate about 1% of theirenergy in the tissues of the gastrointestinaltract. A much greater percentage of theenergy from β emitters will be absorbed anddissipated within the gastrointestinal tract.Insoluble γ emitters will effectively radiate theintestine; however, the rest of the body willalso be exposed. Pertinent examples of themore important fission products are given in(Table 18.1).

Metabolic classification of radionuclides

No completely satisfactory classification ofnuclides is possible. One approach is togroup the nuclides according to theirpositions in the periodic table. It wouldbe expected that nuclides of the same groupwould behave similarly because of their simi-lar chemical properties. The contamination ofthe terrestrial food chain from the varioususes of nuclear energy constitutes a potentialhealth problem to man. To cope with thisproblem requires a consideration of the phys-ical, chemical and biological characteristicsof the radio-contaminants and the terrestrial


11-Mar-03 18



food chain. The methods of fission productdeposition along with the mechanisms of soil,plant and animal contamination need to befully understood. With the above consider-ations in mind, the particular radionuclidesof health importance to man in the terrestrialenvironmental can be identified and theirmetabolic relationships studied. By samplingthe appropriate media, a comparison of thequantities of these artificially produced fis-sion products with the naturally occurringradionuclides can be made, and then anassessment of the degree of environmentalcontamination can be obtained.

Milk and milk products contain naturallyoccurring chemicals such as sodium, calcium,strontium and potassium. The latter chemicalhas a natural occurring radionuclide 40K. Itsabundance is 0.118% in nature. The half-life is1.26 × 109 years. It decays by β emission (89%)and electron capture (11%). It also decays by γrays (11%), with an energy of 1.460 MeV. Thisis readily detectable in milk. The metabolismof the cow is such that the long-lived radio-nuclides found in milk are 137Cs, 134Cs(notably from Chernobyl) and 90Sr. Short-lived radionuclides include 131I, 140Ba, 140Laand 89Sr. The latter radionuclides are usuallyfound in fresh fission products. Table 18.2lists these radionuclides by emission decay,half-life and energy. Food productsmay contain these radionuclides and otherfission products. These are listed in Table18.3.

Metabolism of radiation in man and animals

Many radionuclides are produced by man’suse of nuclear energy. Of these, only a limitednumber are important as sources of internalradiation to the human body. These specificradio-contaminants can reach man by way ofthe terrestrial food chain. This discussion hasa twofold purpose: (i) to describe the charac-teristics that determine the environmentalsignificance of a radionuclide; and (ii) to pres-ent a detailed description of the food chainbehaviour of several of these radionuclides ofenvironmental significance.

Characteristics of radionuclides ofenvironmental importance

In order for a radionuclide to be a significantenvironmental contaminant, it must possesscertain characteristics. These characteristicsmust be such that they allow the nuclide tomove from its point of origin through thefood chain, and still remain a possible healthhazard to man. In general, the radionuclidesof environmental significance are those manuses which are readily taken up by plantsand animals. They are either isotopes ofelements important in metabolism or closelysimilar to them. For example, the alkali metal137Cs is metabolically similar to potassium.It is readily absorbed and circulates freelythroughout the body, irradiating all tissues.The fission product 131I, an isotope of the

11-Mar-03 18

396 E.J. Baratta

Chemicalcharacter

Isotopes (pairs) important onaccount of abundance and half-life

Gastrointestinal uptake from GI tract to

Total body Critical organ

HalogensOxygenated anionsAlkali metalsAlkaline earths

Rare earths

Noble metals

131I, 133I, 135I132Te, 132I137Cs, 137Ba89Sr, 90Sr, 90Y140Ba, 140La91Y, 95Zr, 95Nb141Ce, 144Ce, 144Pr143Pr, 147Nd, 147Pm103Ru, 106Ru, 106Rh

1.00.251.00.30.05

10−4..

0.03

0.3–

0.40.20.04

3 × 10−5

4 × 10−6

Thyroid–MuscleBoneBoneBone, liver

Bone, liver

aFood and Agriculture Organization (1960) and Interlaboratory Technical Advisory Committee (1965).bIsotope pairs are classed according to the chemical and biological characteristics of the parent.

Table 18.1. Fission products of biological importance.a,b



essential element iodine, concentrates inthe thyroid gland. 90Sr and 226Ra are alkalineearths similar to calcium and follow it to thebone. 14C and 3H, isotopes of two very essen-tial elements, are distributed throughout allliving tissues. The metabolic processes of allplants and animals are similar; radionuclideswhich concentrate in animal tissues are usu-ally those that pass most readily through thefood chain.

SPECIFIC CHARACTERISTICS The relative im-portance of individual radionuclides dependson many factors. Among the most importantfactors, the following can be cited: the mag-nitude of the hazard from radionuclide

deposition in the body is dependent on thetype and energy of radiation. For the deposi-tion of the same activity of a radionuclide ina given organ, the hazard from the type ofradiation would be α>β>γ. The hazard fromthe given emitter would also increase withincreasing energy of emission.

CHARACTERISTICS BASED ON PHYSICAL, CHEMICAL

AND BIOLOGICAL PROPERTIES An arbitraryseparation of the above-listed characteristicsinto physical and biochemical categories canbe made for discussion purposes. Develop-ments in this area are concerned with theretention of the liberated radon. Recent inves-tigation indicates that, in long-standing cases


11-Mar-03 18

Radioisotopeb 235U (%) 238U (%) Type of radiation Physical half-life GI absorption (%)

90Sr/90Y137Cs147Pm144Ce106Ru/106Rh95Zr89Sr103Ru95Nb141Ce140Ba/140La131T

5.16.22.95.00.56.34.63.46.36.06.13.1

3.26.2

4.52.75.72.0

5.7

ββ, γββ, γβ, γβ, γββ, γβ, γβ, γβ, γβ, γ

28 years29 years2.6 years285 days1.0 years65 days51 days39.7 days35 days33 days12.8 days8.04 days

30100

0.010.010.030.01

300.030.010.015

100

aFood and Agriculture Organization (1960) and Katcoff (1958, 1960).bIsotope pairs are classed according to the chemical and biological characteristics of the parent.

Table 18.2. Characteristics of the more important fission products of food chain significance.a

Chemical character Isotope Fission abundance (%) Half-life

HalogensOxygenated anionsAlkali metalsAlkaline earths

Rare earths

Noble metals

131I, 133I, 135I132Te, 132I137Cs, 137Ba89Sr, 90Sr, 90Y140Ba, 140La91Y, 95Zr, 95Nb141Ce, 144Ce, 144Pr

143Pr, 147Nd, 147Pm

103Rh, 106Ru, 106Rh

3.1, 6.3, 6.04.06.24.6, 5.1

–c, 6.36.0, 5.0

–c, 2.9, 2.7

3.4, 0.5

8.1 days, 22 h, 6.7 h7.8, 2.4 h37 years, 2.6 months51 days, 26 years, 61 h12.8 days, 40 h57 days, 65 days, 35 days33 days, 290 days,17.5 months13.7 days, 11.6 days,3.7 years40 days, 1.0 year, 30 s

Atom yield > 0.03%; half-life > 10 h.aFood and Agriculture Organization (1960) and Katcoff (1960).bFission abundance values are approximately the same for 233U, 235U and 239Pu.cData lacking on abundance.

Table 18.3. Fission products important due to fission abundance and half-life.a,b



of radium poisoning, an average of 70% of theradons produced are exhaled.

1. Chemical properties. As examples, ele-ments of group VII, fluorine, chlorine, bro-mine, iodine and astatine, have strikinglydifferent modes of metabolism. Fluorine isdeposited in bone; chlorine and bromine arefairly equally extensive within the extracell-ular fluid space; and iodine is concentrated inthe thyroid gland. Astatine is also localizedin the thyroid gland.2. Physical properties. The fission processgives rise to a mixture of radionuclides witha wide range of half-lives. Each of thesenuclides is produced in a certain proportion(abundance), which is dependent on the fis-sion materials and the energy of the fissioningneutrons. The abundance of the various nuc-lides produced has been found to be approxi-mately the same for the different fissionablematerials. Table 18.1 presents the mostimportant fission products, based on fissionabundance and half-life, which are of immedi-ate concern in environmental contamination.High fission abundance and a moderateto long half-life when considering parent–daughter relationships characterize thesenuclides. Many of these radioisotopes cansubsequently be eliminated from food chainconsideration because they are not presentsignificantly long enough to be a long-termhealth hazard. The more important nuclideswill be those which are formed in highabundance, with moderate to long half-lives and which are isotopes of, or chemicallysimilar to, essential elements.3. Biochemical properties. The chemical andbiological properties of the various radio-nuclides greatly affect their ability to movethrough the food chain. Table 18.2 presentsthe fission products of biological importancegrouped according to similar chemical charac-teristics, and shows the relative uptake bythe total body and critical organ from thegastrointestinal tract. From Table 18.2, it isseen that the important fission productsare those which comprise the rare earths,the zirconium–niobium isotopes, the noblemetals, particularly ruthenium and rhodium,the isotopes of iodine, the alkali metal caesiumand the alkaline earths, especially strontium

and barium. Examining the fractional uptakefrom the gut of these various groups of iso-topes biologically can give some indication oftheir relative importance. This leaves the alka-line earths strontium and barium, the alkalimetal caesium and the iodine isotopes asnuclides of primary importance. Dependingon the particular situation, these nuclideswill assume a greater or lesser degree ofimportance in the food chain. Additionalbiological factors which aid in accessing thepotential health hazard from the particularnuclide include: (i) the quantity deposited andthe residence time of the nuclide in the criticalorgan; and (ii) the essentialness or indispens-ability of the critical organ to the organism.

Specific radionuclides

The fission products which enter the environ-ment from fallout or from various nuclearfacilities include more than 30 radioactiveisotopes. From the above discussion, it isevident that all of these radionuclides are notequally harmful to the human population.Intensive study of fission product behaviourin the food chain has revealed that 89Sr, 90Sr,140Ba, 131I and 137Cs are the radionuclidesof major concern. 90Sr and 137Cs are radio-nuclides of long physical half-life and areconsidered long-term hazards. 89Sr, 140Ba and131I, due to their shorter physical half-livesare only short-term hazards. This discussionwill deal primarily with the environmentalbehaviour of 90Sr, 131I and 137Cs.

89Sr and 90Sr

The radionuclides of major importance in thealkaline earth series are 89Sr, 90Sr and 140Ba.Like calcium, these alkaline earth radionuc-lides are deposited in large amounts in theskeleton (Food and Agriculture Organiza-tion, 1960, 1964; Comar et al., 1961; Comar,1963; Russell, 1963; Comar and Bronner, 1964;Kahn et al., 1965). All three radionuclidesare produced in relatively large abundanceduring nuclear fission (see Table 18.2) andassume a greater or lesser degree of impor-tance in food chain contamination depending

11-Mar-03 18

398 E.J. Baratta



on the time period considered after thecontaminating event. 89Sr and 90Sr behave thesame chemically so that the food chainbehaviour of one also applies to the other. Asan example of the behaviour of a particularradionuclide in the food chain, the movementof 90Sr in the pathway from atmosphere tosoil to plant to animal to milk to man canbe cited. As a given quantity of 90Sr movesthrough this pathway, the various environ-mental components, including man, tend todiscriminate or reduce the quantity, whichis finally available for bone deposition inman. The 90Sr is deposited on the soil andeffectively diluted with soil constituentsbefore being assimilated by the plant. Thegrazing animal receives the contaminantthrough ingestion of various vegetativeforms and reduces the quantity of 90Srexcreted into the milk by inherent discrimi-natory processes. The remaining 90Sr is thenavailable for deposition in man.

Radioiodine

In nuclear fission, a number of radioisotopesof iodine are formed. Among the most promi-nent are 131I (half-life 8.1 days), 132I (half-life2.2 h) and 133I (half-life 21 h). In fresh fissionproducts, the shorter-lived radioiodine iso-topes initially will make the major exposure

contribution because of their greater abun-dance. However, in older fission products (ofthe order of a few days), the shorter-livediodine isotopes will have decayed, and 131Iwill be the radionuclide of major concern.Thus, the study of the environmental behav-iour of radioiodine has been concentrated on131I, which has a relatively long half-life com-pared with the other iodine isotopes. Becauseof the relatively short half-life 131I, the soilpathway is not important. Since 131I is only ofmajor concern for relatively short periods oftime following the deposition of fresh fissionproducts, it is only necessary to considerdietary foods that reach man shortly aftercontamination. Milk is the primary exampleof such a food. Experimental studies anddietary surveys have indicated that milk isthe only food product that contributes asignificant amount of 131I to the human diet(Comar and Bronner, 1964; Food and Agri-culture Organization, 1964). Thus the mostimportant pathway for 131I is atmosphere–plants–animals–milk–man. It is possible thatthe pathway atmosphere–plants–man couldresult in ingestion of significant amounts of131I from unwashed fruits and vegetables thathave been exposed to surface contamination.However, exposure by this route would mostprobably be significant only on a local orindividual basis (Table 18.4).


11-Mar-03 18

Age

Thyroidweight

(g)

% Uptakein

thyroid

% Uptakeg−1 of

thyroid

Fresh milkconsumption

(l day−1)

(% 131I g−1

thyroid) ×(l milk day−1)

Fetus12 weeks12–15 weeks15–32 weeks

Person0–6 months6–12 months1–2 years2–5 years5–10 years10–15 years15–20 years20–30 years> 30 years

22

2–53–55–1010–1515–20

2020

0

303030303030303030

00.1–11–5

151512

10–66–33–2

2–1.51.51.5

11

0–0.50.50.50.50.70.70.60.30.2

00.1–11–5

0–7.57.56

5–34–22–11

0.40.3

aComar et al. (1961).

Table 18.4. Relative biological availability of 131I to individuals.a



Radiocaesium

In contrast to 90Sr, 137Cs does not readilyfollow the soil uptake route (Food and Agri-culture Organization, 1964). This nuclide isfixed in forms largely unavailable to plants asa result of entrapment in the lattice structureof certain clays. Plant contamination, there-fore, occurs primarily by direct deposition.Since 137Cs is capable of concentrating in softtissues (e.g. muscle), the step from animal toanimal products (other than dairy products)assumes importance for this radionuclide.A long-term genetic dose is thus possiblefrom ingestion of foods contaminated with137Cs. Since a significant quantity of 137Csis excreted into animal’s milk, the path-way atmosphere–plants–animals–milk–manis most important for diets containing aver-age amounts of milk. In general, the biologi-cal significance of 137Cs is somewhat less thanthat of 90Sr because of its shorter effectivehalf-life in the body.

Natural radioactivity

There are a number of naturally occurringradionuclides in the biosphere. They enterand are transferred through the food chainto varying extents. A major portion of thework with natural occurring radionuclideshas been undertaken in order to establishbaseline levels in the environment. Suchlevels have been used for comparison inassessing the degree of contamination of theenvironment with artificial radioactivity.

The most important naturally occurringradionuclides belong to the uranium, thoriumand actinium series. The actinium series isthe least abundant of the three. These naturalseries are composed of a number of theheavier elements of varying half-lives thatexhibit extremely complex decay schemes.Studies of the transfer of these radionuclidesto man via the food chain involves measuringthe activity levels in plants which are con-sumed directly by man, and plants such asgrasses which form the principal food ofanimals, which in turn become the principalfood of man. Dietary surveys indicate thatthere is a wide range of natural activities invegetation and there appears to be no simple

correlation with the activities found in the soil,which have a much smaller range. Also, thereis not much information available concerningthe discrimination factors for soil–food andman–food processes. It appears that plantsand animals absorb the majority of theseradionuclides to only a very small extentwhen compared with the most pertinentartificial radioisotopes. Few quantitative dataare available on absorption phenomena. Thegreatest attention has been paid to radiumsince this element appears to be the principalone absorbed by plants. However, dietarysurveys have indicated that the occurrence ofthis element in the majority of foods is wellbelow that of man-made 90Sr.

The following naturally occurring radio-nuclides are present in a singular form in theenvironment: rubidium, lanthanum, samar-ium and ruthenium. Little information is avai-lable on plant and animal absorption of thesenatural radionuclides. They are known to bepresent in the environment at considerablylower concentrations than the most abundantnaturally occurring radioisotope, 40K. Consid-eration of several of their characteristics suchas their chemical nature, the behaviour of theirman-made counterparts and their essential-ness to plants would tend to indicate thatthese radionuclides are little absorbed byplants. The remaining fission products,activation products and naturally occurringradionuclides that are found in the terrestrialenvironment are of lesser hazard to man asa result of their limited movement in thefood chain. However, depending on specialcircumstances some of these radionuclidesmay assume a greater importance thannormally expected.

Monitoring

Surveillance

The Total Diet Program was reinitiated in1973 (Food and Drug Administration, 1973).This programme was necessary to:

• monitor the foodstuffs in the USA. It waspart of an overall programme to ensure

11-Mar-03 18

400 E.J. Baratta



that the food products were safe andwholesome; and

• maintain a capability in the event that:(i) above-ground testing was initiated;(ii) leaks from around nuclear facilitiesoccurred; (iii) a nuclear accidentoccurred; and (iv) there was increaseduse by medical facilities and increasedcommercial radioactive materials.

The data collected for this programmecan be found in the reports that have beengenerated in the literature (Simpson et al.,1974, 1977, 1981; Tanner and Baratta, 1980;Strobe et al., 1985; Cunningham et al., 1989,1994; Caper and Cunningham, 2000). The datahave shown that the radioactive content ofthe food grown and consumed in the USAmeets the requirements of the Federal Radia-tion Council (FRC) as promulgated in 1961(Table 18.5). These results are also below therequirements of newer regulations issued in1998 (US Department of Health and HumanServices, 1998).

This capability was soon to be put to use.In 1979, an incident occurred at the reactor atThree Mile Island (Unit II) in Pennsylvania,resulting in some releases of radioactivegases. However, the containment vessel wasnot breached. This resulted in the samplingand analyses of thousands of samples duringthe first month. These were mainly milk andproduce samples from the farms and dairies in

the immediate area. Also included were food-stuffs processed and manufactured from thatarea. Sampling from that area continued foranother year at a less vigorous pace. The onlyradionuclide found was 131I, and the concen-trations were in range II of the FRC’s radiationprotection guidelines (FRC, 1961). Intake inthis range calls for active surveillance androutine control. The limits for range II are0.37–3.7 Bq l−1 (Table 18.5). This radionuclide(131I) was only detected during the first week.The limit of detection for this method at the 2sigma level is ± 0.37 Bq l−1. The 90Sr content inall the samples tested was similar to its contentdue to the worldwide fallout. No 89Sr wasdetected, which confirmed this.

The second occasion that required exten-sive sampling and analyses was when anincident occurred at the Chernobyl nuclearpower plant in the Ukraine, of the then USSR.The radionuclides from the incident atChernobyl did not increase the dietary intakeof foods grown and processed in the USA.However, the foods imported into the USAdid contain ‘fallout’. In the early period, themajor radionuclide of concern was iodine.Several mushroom samples and soft cheesesamples were detained as they exceed theUS Department of Agriculture (USDA)–FDAlevels of concern (FDA, 1986) (Table 18.6).Other samples, particularly spices and herbs,contained high amounts of fission products.Later, samples of cheese, apple juice and pasta


11-Mar-03 18

Radionuclide TargetRPG dose

(mSv year−1) Ic II III

131I137Csd

90SrTritium (3H)d

ThyroidWhole bodyBoneWhole body

5.71.75.71.7

0–0.370–54.70–0.740–7,400

0.37–3.754–540

0.74–7.47,400–74,000

3.7–37.7540–5,4007.4–74.7

74,000–740,000

aDerived concentrations were calculated on the basis of an average contaminated food intake of 1 kgday−1 (includes water and other beverages).bFederal Radiation Council (1965).cRange I requires no specific action; range II requires surveillance and routine control of upward trendstowards range III; range III requires surveillance and controls to reduce exposure to range II (1); therange II–range III transition corresponds to the RPG dose 1 Bq = approximately 27 pCi.d137Cs and tritium were not considered by the FRC. The ranges were derived by using the radionuclideconcentrations in water, tabulated by the National Council on Radiation Protection for occupationalexposure, × 1/30 to apply to the general population.

Table 18.5. Radiation protection guides (RPGs) and derived concentration action ranges (Bq kg−1)a forselected radionuclides for the general population.b



were detained due to their exceeding the lev-els of concern (Cunningham et al., 1989, 1994).

Other concerns have been raised over theyears, and the FDA has been called upon toparticipate in them. One of these concerns wasthe ‘dumping’ of low-level radioactive wastein various harbours. The dumping had ceasedin 1970. Samples were collected in 1980–1982,in cooperation with the EPA. Samplescollected at that time were analysed for thevarious radionuclides such as 137Cs and 90Sr.No activity above background was detected.Later, there was concern that 239/240Pu was oneof the radionuclides ‘dumped’ in these areas.Again, extensive sampling in cooperationwith the EPA was conducted. Results of thisstudy showed that the concentrations werebelow background levels or what would beexpected from the 239/240Pu fallout from theprevious above-ground testing in the 1960s(Baratta, 1995).

Methodology

The methodology used in the analyses ofthese radionuclides has been developed andtested previously by the standard settingsocieties, such as the AOAC International(2000) Official Methods and the APHA–AWWA–WEF (1998) Standard Methods, orhave been published in the open literature(Baratta and Reavy, 1969; Baratta, 1992, 1994,1998). The FDA has maintained a qualityassurance programme, using the EPA (nowdiscontinued) for its quality control samples.The results of this programme showed thatthe analytical data produced are within the

expected acceptability for control (Baratta,1993).

Toxicity and Effects

Amount deposited and retained in thecritical organ

The most hazardous irradiation situation res-ults from radionuclides that are concentratedby essential organs of the body in relativelylarge quantities and are retained in theseorgans for long periods of time. The deposi-tion of 90Sr, a bone-seeker of long biologicalhalf-life, is an example of such a situation.

Type and energy of radiation emitted

The magnitude of the hazard from radio-nuclide deposition in the body is dependenton the type and energy of radiation. For thedeposition of the same activity of a radio-nuclide in a given organ, the hazard fromthe type of radiation would be α > β > γ. Thehazard from the given emitter would alsoincrease with increasing energy of emission.

Physical properties

The fission process gives rise to a mixture ofradionuclides with a wide range of half-lives.Each of these nuclides is produced in acertain proportion (abundance), which isdependent on the fissioning material and theenergy of the fissioning neutrons. The abun-dance of the various nuclides produced hasbeen found to be approximately the same forthe different fissionable materials. Table 18.3presents the most important fission productsbased on fission abundance and half-life,which are of immediate concern in environ-mental contamination. One fission productof biological importance is 90Sr. It has beenestimated that all detonations by the USA, theUK and the USSR until the latter part of 1958produced approximately 7,000,000 curies ofthis radionuclide.

11-Mar-03 18

402 E.J. Baratta

Radionuclide Bq kg−1

131I Infant foods134Csb Other foods137Cs All foods

56300370

aFood and Drug Administration (1986).bRefers to the combination of both radionuclides –134Cs and 137Cs – as they appear in Table 18.7.

Table 18.6. Post-Chernobyl ‘action levels’applied by the US FDA–USDA as of December1986 for radionuclides in imported foods.a



Risk Assessments

Philosophy of radiation protection

The setting and execution of guidelines forradiation protection are based on an under-lying philosophy in which two factors areof prime importance. First is the assumptionthat radiation effects follow a linear or non-threshold dose–response relationship. Thereis convincing evidence, particularly in so faras the genetic effects of radiation are con-cerned, that there exists a non-threshold phe-nomenon. Although positive proof is lackingthus far, it has been deemed prudent to adoptthis more conservative hypothesis in settingprotection standards for large numbers ofpeople. According to the non-threshold con-cept, there is no radiation dose so small that itdoes not involve some degree of risk. Thenon-threshold relationship, therefore, impliesthat there is no radiation protection guide-line, no matter how low, which can ensureabsolute safety to every individual in a largepopulation receiving the guideline dosage.However, since the magnitude of the risk isproportional to the dose received, untowardeffects would become manifest at very lowdose levels only if extremely large numbersof exposed individuals were observed.

The radiation protection guide (RPG)(FRC, 1965) may be defined as the radiationdose which should not be exceeded withoutcareful consideration of the reasons for sodoing. In light of the non-threshold phenome-non, every effort should be made to encouragethe maintenance of radiation exposures asfar below the guide as practicable. Methodsof estimating guides are experiments, whichhave contributed greatly to the study ofthe effects of radiation. From this combinedknowledge and from an understanding ofthe relative biological damage producedby various types of radiation, protectionguides for whole-body exposure and forvarious organs have been recommended.These guides, of course, represent doses farbelow those at which any effects have thusfar been observed.

Basis for radiation protection guides

Establishment of ‘safe’ levels of a long-termradiation dose requires knowledge of thecause–effect relationship between radiationdose and biological damage. Such damagemay appear many years after initial exposureand is usually indistinguishable from thenormal diseases and impairments of man.Information accumulated on this subject is,therefore, difficult to evaluate and is oftencontroversial. Nevertheless, observationsinvolving man and animal life have resultedin the accumulation of significant data.

Factors influencing radioactivityconcentration guides

The radioactivity concentration guide (RCG)is the concentration of radioactivity in theenvironment that is determined to resultin whole-body or organ doses equal to theRPG. In calculating RCG values for a givenradionuclide, the following factors must betaken into consideration.

INITIAL BODY UPTAKE Large fractions ofsome elements are absorbed when taken intothe body. In the case of certain other elements,only small fractions are absorbed in passagethrough the gastrointestinal tract. Therefore,the greater retention would increase thehazard from the first group as comparedwith the second, other factors being equal.

When radionuclides are inhaled, unlessinformation specific to the radionuclide isavailable, it is assumed, in the case of solublecompounds, that 25% is retained in the lowerrespiratory tract. From here, the nuclidesmove into the bloodstream and a portion ofeach is deposited in its critical tissue within afew days. Approximately 50% is held in theupper respiratory tract and swallowed. In thecase of insoluble compounds, it is assumedthat 12% is retained in the lower respiratorytract, which is usually taken as a critical organ.The remainder is eliminated by exhalationand swallowing.


11-Mar-03 18



FRACTION RETAINED IN THE BODY The rate ofelimination from the blood and tissues ofthe body varies considerably for differentelements or compounds. The time requiredfor one-half of the original quantity of radio-active material to be removed from the bodyby biological processes is called the biologicalhalf-life.

Some materials in the bloodstream areeliminated rapidly from the body, whereaslarge fractions of others remain in the bodyorgans. For example, radium, plutonium andstrontium are deposited in the bone where therate of turnover is very slow, i.e. the biologicalhalf-life is many years. Radioisotopes of theseelements are much more hazardous thanthose of carbon, sodium, sulphur and thosethat have biological half-lives of a few days orweeks. The principal biological methods ofelimination of radionuclides from the bodyare the urine, faeces, exhalation and perspira-tion. Usually, elimination is much more rapidbefore the radionuclide is translocated fromthe body to a more permanent area, such asthe bone. This time is usually from a few daysto a few weeks. After the initial period,the elimination rate becomes more nearlyexponential, and the application of the term‘biological half-life’ has more meaning.

Regulatory Issues

The US FRC issued its Report No. 2 that setup the RPGs in September 1961 (FRC, 1961).These guides were set up to ensure that thepublic in the USA would be protected fromthe radioactive ‘fallout’ (Table 18.5). Duringthe ‘fallout’ from Chernobyl, imported foodswere regulated using a guide approved byboth the USDA and the FDA. These wereknown as the USDA–FDA ‘levels of concern’(Food and Drug Administration, 1986) (Table18.6). They were primarily for 131I, 137Cs and134Cs. In 1998, the FDA published in theUS Federal Register new guides: AccidentalRadioactive Contamination of Human Food andAnimal Feeds: Recommendations for State andLocal Agencies (US Department of Health andHuman Services, FDA, CDHR, 1998). Thisreplaces the previous US Federal RadiationCouncil Report No. 2. (FRC, 1961, 1965). The

USDA–FDA ‘levels of concern’, became the‘derived intervention levels’ or ‘DILs’.The values remain the same. Table 18.7 showsthe new levels recommended in the Acciden-tal Radioactive Contamination of Human Foodand Animal Feeds: Recommendation for State andLocal Agencies (US Department of Health andHuman Services, FDA, CDHR, 1998).

The previous guidelines (FRC, 1965) werepredicated upon the more or less constantrelease of low level radioactivity into the envi-ronment from the routine uses of radiation,and assumes continuous radionuclide intakeby the population. Control for the populationis based on the source of the release. There arecases, however, in which the contaminationof the environment might be accidental orunforeseen, producing contamination that istransient and not likely to recur; these mightinclude, for example, reactor incidents thatresult in relatively high but temporary localradioactivity levels. In cases of this kind, the‘contaminating event’ would not occur on aregular basis, and control of the populationmight base protective action upon limiting orchanging the uptake of certain contaminatedfoods. However, the impact of such measures

11-Mar-03 18

404 E.J. Baratta

Radionuclide group Bq kg−1

90Sr131I134Cs + 137Cs238Pu + 239Pu + 241Am103Ru + 106Ru

16017012002[C3/6800 + C6/450] < 1c

aThe DIL for each radionuclide group (except for103Ru + 106Ru) is applied independently. Applicableto foods as prepared for consumption (for dried orconcentrated food products, such as powderedmilk or concentrated juices, adjust by a factorappropriate to reconstituted product). For spices,which are consumed in small quantities, use adilution factor of 10.bUS Department of Health and Human Services(1998).cDue to the large differences in DILs for 103Ru and106Ru, their individual concentrations are dividedby their respective DILs and then summed. Thesum must be less than 1. The C3 and C6 must bethe concentrations at the time of measurement.

Table 18.7. Recommended derived interventionlevel (DIL)a or criterion for each radionuclidegroup.b



upon the community which they are designedto benefit requires careful consideration byresponsible authorities to ensure that the ben-efit of the action taken is not outweighed by itsother effects. To deal with this kind of situa-tion, the FRC, in its Report No. 5, introducedthe protective action guide (PAG) concept.The PAG is defined as the projected absorbeddose to individuals in the general populationthat would warrant protective action follow-ing a contaminating event. It is assumed thatthe corresponding projected dose to a suitablesample of the exposed population would beone-third of the PAG. These guidelines have,thus far, been established for 131I, 90Sr and137Cs. It should be noted that a decision to takeaction to limit a community’s intake of animportant basic food item containing radio-nuclides involves balancing of the health ben-efits to be attained against undesirable featuresof the protective action, such as disruption ofdietary habits and nutritional needs. It follows,then, that control actions could be employedat dose levels above or below the PAG,depending upon the degree of total impactthat the action has upon the community.

Based on current information, RPGs forradiation doses have been recommended.These levels are subject to modification asmore knowledge is gained. In view of theirstatus and the possibility that any radiationdose may be damaging, it is well to recall thedefinition of the RPG as that radiation dosewhich should not be exceeded without carefulconsideration of the reasons for doing so. Everyeffort should be made to encourage the main-tenance of radiation doses as far below theRPG as practical. Table 18.5 summarizes theprevious recommendations of the FRC. Table18.6 gives examples of the post-Chernobyl‘action levels’ as applied by various countries.Table 18.7 is an example of the present recom-mended DIL or criterion for each radionuclidegroup (US Department of Health and HumanServices, FDA, CDHR, 1998).

Conclusions

The US FDA has been monitoring radio-nuclides in foods since 1961. During this

period, it has found that the food supply inthe USA has met the criteria as set by the FRC(FRC, 1961) and the levels of concern (Foodand Drug Administration, 1986) followingthe incident at the Chernobyl reactor. Theincident at Three Mile Island showed thatwhat little activity that was released was wellwithin the FRC guidelines. The results ofthis monitoring have been reported in theliterature as have other data concerningpossible contamination (Simpson et al., 1974,1977, 1981; Tanner and Baratta, 1980; Strobe,et al., 1985; Cunningham et al., 1989, 1994;Baratta, 1992; Caper and Cunningham, 2000).The foods imported after the Chernobylincident that exceeded the levels of concernwere detained. The results of the seafoodtested from the former low level dump siteswere found to be near or at background levels(Baratta, 1995).

References

Anderson, E.C. and Nelson, D.J. (1962)Surveillance for radiological contaminationin foods. American Journal of Public Health 52,1391–1400.

AOAC International (2000) Official Methods ofAnalyses of the AOAC: Radioactivity, 17thedn. AOAC International, Gaithersburg,Maryland.

APHA–AWWA–WEF (1998) Standard Methodsfor the Examination of Water and Waste Water,20th edn. American Public Health Association,Washington, DC.

Baratta, E.J. (1992) The FDA’s program for monitor-ing radionuclides in food. Transactions of theAmerican Nuclear Society 6, 139–140.

Baratta, E.J. (1993) FDA quality assurance for radio-activity in foods and radiopharmaceuticals.Fresenius’ Journal of Analytical Chemistry 345,152–155.

Baratta, E.J. (1994) Manual of Food Quality Control 16.Radionuclides in Food. FAO Food and NutritionPaper 14/16, Rome.

Baratta, E.J. (1995) Determination of plutonium-239-240 in fish in low-level radioactive oceanwaste dump sites. Journal of Radioanalytical andNuclear Chemistry 194, 157–162.

Baratta, E.J. (1998) Methodology for the US Foodand Drug Administration’s radionuclides infoods program. Journal of Radioanalytical andNuclear Chemistry 236, 139–144.


11-Mar-03 18



Baratta, E.J. and Reavey, T.C. (1969) Rapiddetermination of strontium-90 in tissues,food, biota and other environmental media bytributi-phosphate. Journal of Agricultural andFood Chemistry 17, 1337–1339.

Caper, G.C. and Cunningham, W.C. (2000) Elementand radionuclide concentrations in foods: FDATotal Diet Study 1991–1999. Journal of theAssociation of Official Analytical Chemists 83,157–177.

Comar, C.L. (1963) Factors influencing the bio-logical availability of fallout radionuclidesfor animals and man. Federal Proceedings 22,1402–1409.

Comar, C.L. and Bronner, F. (1964) MineralMetabolism. Academic Press, New York,pp. 523–572.

Comar, C.L., Wassermann, R.H. and Twardock,O.R. (1961) Secretion of strontium and calciuminto milk. Health Physics 7, 69–80.

Cunningham, W.C., Strobe, W.B. and Baratta, E.J.(1989) Radionuclides in domestic andimported foods in the United States,1983–1986. Journal of the Association of OfficialAnalytical Chemists 72, 15–18.

Cunningham, W.C., Anderson, D.L. andBaratta, E.J. (1994) Radionuclides in domesticand imported foods in the United States,1987–1992. Journal of the Association of OfficialAnalytical Chemists 77, 1422–1427.

Eisenbud, M. (ed.) (1973) Environmental Radio-activity. Academic Press, New York.

Federal Radiation Council (1961) BackgroundMaterial for the Development of RadiationProtection Standards. Federal Radiation CouncilReport No. 2. US Government Printing Office,Superintendent of Documents, Washington,DC.

Federal Radiation Council (1965) BackgroundMaterial for the Development of RadiationProtection Standards. Federal Radiation CouncilReport No. 7. US Government Printing Office,Superintendent of Documents, Washington,DC.

Food and Agriculture Organization of the UnitedNations (1960) Radioactive Materials in Food andAgriculture. FAO Atomic Energy Series No. 2,Rome.

Food and Agriculture Organization of the UnitedNations (1964) Agricultural and Public HealthAspects of Radioactive Contamination in Normaland Emergency Situations. FAO Atomic EnergySeries No. 5, Rome.

Food and Drug Administration (1963) Teenage DietSurvey. Radiological Health Data Reports 4. FDA,Rockville, Maryland, pp. 18–22.

Food and Drug Administration (1973) Total DietStudies, Radionuclides in Food. FDA ComplianceProgram. F-10 7320.08A. FDA, Rockville,Maryland.

Food and Drug Administration (1986) ImportedFoods. Compliance Program. FDA, Rockville,Maryland.

Interlaboratory Technical Advisory Committee(ITAC), Subcommittee on Surveillance (1965)Routine Surveillance of Radioactivity AroundNuclear Facilities. Division of RadiologicalHealth, US Public Health Service, Departmentof Health, Education, and Welfare, Rockville,Maryland.

Kahn, B., Jones, J.R., Porter, C.R. and Straub, C.P.(1965) Transfer of radiostrontium from cow’sfeed to milk. Journal of Dairy Science 48,1023–1030.

Katcoff, S. (1958) Fission yields from thorium,uranium and plutonium. Nucleonics 16, 78–85.

Katcoff, S. (1960) Fission-product yields fromneutron-induced fission. Nucleonics 18,201–208.

Laug, E.P., Mikalis, A., Billinger, H.M., Dimitroff,J.M., Deutsch, W.J., Duffy, D., Pillsbury, H.E.,Loy, H.W. and Mills, P.A. (1963) Total DietStudy. Journal of the Association of AgriculturalChemistry 46, 749–767.

National Academy of Sciences–National ResearchCouncil (1961) Internal Emitters. PublicationNo. 88. National Academy Press, Washington,DC.

Roecklin, P.D., Smedely, C.E. and Simpson, R.E.(1970) Strontium-90 and cesium-137 in totaldiet samples. Radiological Heath Data Reports 11,47–64.

Russell, S.R. (1963) The extent and consequences ofthe uptake by plants of radioactive nuclides.Annual Review of Plant Physiology 14, 271–294.

Simpson, R.E., Baratta, E.J. and Jelinek, C.F. (1974)Radionuclides in foods: monitoring program.Radiation Data and Reports 15, 647–655.

Simpson, R.E., Baratta, E.J. and Jelinek, C.E. (1977)Radionuclides in foods. Journal of theAssociation of Official Analytical Chemists 60,1364–1368.

Simpson, R.E., Shuman, F., Baratta, E.J. andTanner, J.T. (1981) Survey of radionuclidesin food, 1961–77. Health Physics 40, 529–534.

Strobe, W.B., Jelinek, J.C. and Baratta, E.J. (1985)Survey of radionuclides in foods, 1978–1982.Health Physics 49, 731–735.

Tanner, J.T. and Baratta, E.J. (1980) Radionuclidesin foods. In: Goss, B.L. (ed.) Factors AffectingPower Plant Waste Heat Utilization. PergamonPress, Elmswood, New York, pp. 140–151.

11-Mar-03 18

406 E.J. Baratta



Thompson, R.C. (1960) Vertebrate radiobiology:metabolism of internal emitters. Annual Reviewof Nuclear Science 10, 531–560.

US Department of Health and Human Services,FDA, CDHR (1998) Accidental RadioactiveContamination of Human Food and Animal Feeds:Recommendations for State and Local Agencies.FDA, Rockville, Maryland.


11-Mar-03 18



19 Widespread and Continuing Concernsover Food Safety

J.P.F. D’Mello*Formerly of the Scottish Agricultural College, West Mains Road,

Edinburgh EH9 3JG, UK

Introduction

Even the most perfunctory perusal of thisbook will lead to the conclusion that, not-withstanding the best efforts of establishedand new government agencies, food contami-nation is likely to be a continuing issue for theforeseeable future. Despite measures imple-mented in many countries, the general con-sensus is that there is very little scope forcomplacency. In the short term, every nationwill be faced with events that underminepublic confidence in a wide range of fooditems. The recent past has provided us withan unremitting catalogue of food scaresarising from decades of pollution, carelessderegulation and under-funding of servicesin monitoring, research and education. Theproblems associated with negligence on thisscale will not disappear immediately.

Detailed examination of this book willhighlight on-going and widespread contami-nation of the principal foods that make up ourdiets. A number of deleterious constituentsare intrinsic to particular foods, for exampleallergens in cereals and nuts. Other contami-nants occur as a result of environmental pollu-tion, while others arise from poor standardsof hygiene. Microbial contamination hasemerged as a particularly intractable problem

both in terms of range of foods affected and inthe diversity of causal organisms. Escherichiacoli, Salmonella spp., Campylobacter spp. andListeria monocytogenes are of particular con-cern in food safety. Theoretically, the risksassociated with microbial contaminantsshould be relatively easy to minimize, but inpractice this is proving to be more difficult.The spectre of antibiotic resistance has alsoemerged from recent studies. Thus, Walshet al. (2001) demonstrated that the overall inci-dence of antibiotic resistance in Listeria speciesis low. However, they noted a continuing pat-tern of the emergence of strains of Listeriaspecies isolated from foods that are resistantto one or more of the common antibiotics usedto treat listeriosis in humans. Resistance ofCampylobacter species and E. coli O157 toantimicrobial agents has also been reported(Aquino et al., 2002; Schroeder et al., 2002).This chapter is designed to highlight thosefoods that present a risk now and are likely todo so in the foreseeable future. Food legisla-tion will also be considered from a holisticpoint of view, with the aim of suggestingfurther statutory controls over pollution andspecific contaminants. The chapter includes areview of recent alerts and cases of contraven-tion of legislation, and ends with a summaryof future research priorities in key areas of


11-Mar-03 19




food safety. The research programmes sug-gested are not comprehensive by any meansand the reader is referred to individual chap-ters within this book and to specialist docu-ments published by government epartmentsand food agencies (e.g. Ministry of Agricul-ture, Fisheries and Food, 1992a,b,c; Food Stan-dards Agency, 2000a,b, 2001a,b,c,d,e,f,g,h),particularly in Europe and North America.

Statistics

Recent statistics reinforce widespread andcontinuing disquiet over food safety. Thissection is not comprehensive due to limita-tions of space. Nevertheless, the scale ofthe problem is encapsulated in the findingsof Mead et al. (1999). They estimated thateach year 76 million cases of food poisoningoccur in the USA, with 325,000 individualsrequiring hospitalization and 5000 fatalities.Nearly 2.4 million cases were caused byCampylobacter species and 1.4 million cases bynon-typhoidal serovars of Salmonella. In May2000, 1485 cases of Campylobacter poisoningwere recorded in the UK, coinciding withthe barbecue season. In the 1996 outbreak ofE. coli O157 contamination in Scotland, 496cases were recorded, including 18 deaths.This level of mortality was the second highestin the world for an epidemic caused by E. coliO157. In Japan, around 1000 outbreaks of

food poisoning involving 30,000–35,000 indi-viduals have been reported to occur annually(Suzuki et al., 2002). In Canada and the USA,major outbreaks of illness have been linkedwith the consumption of vegetables contami-nated with L. monocytogenes (cited by Li et al.,2002). Kingsley and Richards (2001) statedthat 1.4 million cases of hepatitis A virus(HAV)-mediated illness occur each year on aworldwide scale. Later in this chapter, refer-ence will be made to a number of food alertsand to successful actions directed at individ-uals and small retail businesses. Many of thefood alerts were associated with undeclaredpotential allergens in bread and cakes.Thus the full spectrum of staple foods hasbeen linked with specific contaminants andhuman disorders. It is, therefore, appropriateto review the risks presented by particularfoods in different countries.

Cereals and Nuts

The principal contaminants of cereals, breadand nuts are presented in Table 19.1. Cerealsand nuts constitute a particular risk toconsumers in Africa, Asia and possiblySouth America by virtue of mycotoxin con-tamination of these foods. The aflatoxins andfumonisins have been linked with the inci-dence of specific types of cancer (D’Mello,Chapter 4, this volume). The export of such

11-Mar-03 19

410 J.P.F. D’Mello

Food Contaminant/toxicant Comments/status Further information

CerealsMaizeWheatWheat

Bread

NutsPeanuts

Pistachio

Aflatoxins; fumonisinsAllergens (gliadins)Deoxynivalenol

Pesticide residues above‘reporting limits’

AflatoxinsAllergens

Aflatoxins

On-going in Africa and AsiaCoeliacs at riskCanada and the USA; subject toadvisory directivesIn decline

On-going in producer countriesPrecipitating life-threatening andother conditions in susceptibleindividuals; requires constantvigilanceOn-going in producer countries;currently under surveillance

Chapter 4D’Mello (1991)Chapter 4

Ministry of Agriculture,Fisheries and Food(1992a)

Chapter 4Sampson and Burks(1996)

Chapter 4

Table 19.1. Principal contaminants/toxicants of cereals, bread and nuts.



foods also presents potential risks to consum-ers in the developed world, to the extent thatlegislation is in place or proposed for thesemycotoxins. In the UK, a recent survey foundaflatoxin B1 levels above regulatory limitsin 13% of nuts and nut products (FoodStandards Agency, 2002). In addition, certainindividuals are at risk from allergensoccurring in wheat and peanuts. Allergensin peanuts can cause life-threatening condi-tions (Spencer and Berman, Chapter 1, thisvolume) in susceptible people. Avoidance ofpeanuts is rendered more difficult by theirubiquitous distribution in manufacturedfoods, including biscuits, cakes, sauces andcomplete meals.

In a survey by the Ministry of Agri-culture, Fisheries and Food (1992a), samplesof bread were found to contain pesticides at orabove the reporting limits (RLs). However,incidence declined from 32% in 1988 to 22%in 1990. In addition, the pattern of residueshad changed, with less malathion and moreprimiphos-methyl.

Vegetables and Fruit

The contaminants of vegetables and fruitlisted in Table 19.2 include nitrates, pesticides,arsenic and L. monocytogenes. The levels ofnitrate in food and water have elicited consid-erable concern among medical practitionersand specialists (Eichholzer and Gutzwiller,

Chapter 10, this volume). In some samples,nitrate concentrations in lettuce, celery andbeetroot may exceed 1000 mg kg−1. However,in a report by the UK Ministry of Agriculture,Fisheries and Food (1992b), it was concludedthat only vegetarians might be at risk andthat surveillance of their nitrate intakesshould continue. Nevertheless, it is prudentto control nitrate exposure in the general pop-ulation by reducing nitrogenous pollution.

Pesticide residues (Cabras, Chapter 5,this volume) in certain vegetables and fruitcontinue to be a problem in many countries.Recent surveillance results in the UK indicatea distinct lack of progress in reducing levelsof pesticide residues in imported yams(Table 19.2). The levels were in excess of legalmaximum residue limits (MRLs). However,the levels of contamination were deemed notto be a risk to consumers; MRLs are not safetystandards. In the UK, 69% of lettuce samplesrecently were reported to contain pesticideresidues. In three samples, residues exceededthe legal MRLs, while two samples containednon-approved pesticide residues. Celery,grapes, oranges and apples have all beenfound to contain multiple residues ofpesticides (Table 19.2). Regarding grapes,the Pesticide Residue Committee in the UKrecently reported that 67% of samples con-tained pesticides, 29% being contaminatedwith multiple residues. As indicated later inthis chapter, specific food alerts have beenissued on human health risks presented bypesticides in vegetables and fruit.

Widespread and Continuing Concerns over Food Safety 411

11-Mar-03 19

Vegetables/fruit Contaminant Data source

Lettuce, celery andbeetrootLettuce

Lettuce (also celery andtomatoes)CelerySpinachYams

Grapes, oranges andapples

Nitrates

Pesticide residues in excess of maximumresidue limits (MRLs) in UK-grown andimported samplesListeria monocytogenes

Multiple residues of pesticidesArsenicPesticide residues in excess of MRLs inyams imported into the UKMultiple residues of pesticides

Ministry of Agriculture, Fisheriesand Food (1992b)Food Standards Agency (2001a)

Li et al. (2002)

Food Standards Agency (2000b)Munoz et al. (2002)Food Standards Agency (2001e)

Food Standards Agency (2000b,2001c)

Table 19.2. Principal contaminants in fresh vegetables and fruit.



Li et al. (2002) cited two outbreaks oflisteriosis, one in Canada and the other in theUSA, that were both linked with contamina-tion of vegetables with L. monocytogenes. Theuse of farmyard manure has raised questionsabout the microbiological safety of vegetables,particularly those grown under organic con-ditions. The potential for the transfer of bacte-ria from contaminated manure and irrigationwater to vegetables is no longer a theoreticalissue in the light of evidence presentedby Solomon et al. (2002). Sagoo et al. (2001)conducted a microbiological examinationof ready-to-eat organic vegetables obtainedfrom retail outlets in the UK. They demon-strated the absence of L. monocytogenes, Salmo-nella, Campylobacter and E. coli O157 in theirsamples, and implied that overall agricul-tural, hygiene, harvesting and productionpractices were satisfactory. The situation inmany other countries where organic farmingsystems have been adopted needs to beassessed individually. However, it is reas-suring that McGee et al. (2001) were able todemonstrate that, although E. coli O157 cansurvive in cattle slurry for an extended periodof time, a substantial decline in numbers of thepathogen occurs during storage. Assumingthe same effect is true for other pathogens,the application of animal wastes in organicfarming may not be a major factor affectingthe safety of vegetables, as is sometimesimplied. However, a watching brief should bemaintained, as the use of microbiologicallycontaminated animal wastes is associatedwith some risks.

In parts of Chile, geological factors meanthat consumers are exposed to above-averagelevels of arsenic. Concentrations in soil andaquifers of up to 1099 mg kg−1 and 11 mg l−1,respectively, have been reported (Munozet al., 2002). Not unexpectedly, vegetables andfruit grown in these areas can become contam-inated with arsenic. In the study of Munozet al. (2002), highest levels (up to 0.6 mg kg−1

fresh weight (FW)) were recorded for spinach.The level allowed by Chilean legislation is1 mg kg−1, but other countries, for examplePoland, require levels to be considerablylower (0.2 mg kg−1).

Despite the foregoing, the value ofvegetables and fruit as part of a balanced diet

must always be stressed. Vegetables and fruitare an important source of nutrients and fibre.Significantly, secondary compounds presentparticularly in leafy vegetables may offerprotection against certain types of cancersand asthma.

Seafood

Estuarine and marine pollution has becomea major environmental issue with conse-quences for the safety of seafood. In additionto the compounds listed in Table 19.3, thereare many reports of increased levels of con-taminants arising from authorized dischargesfrom radiochemical plants and other sourcesaround the world (Smith and Beresford,Chapter 17, and Baratta, Chapter 18, thisvolume). For example, recent studies havehighlighted the unexpectedly high levels oftritium in fish and shellfish from Cardiff bayand the River Taff. It has been suggested thatthese elevated concentrations have not com-promised food safety even for local seafoodconsumers (Food Standards Agency, 2001g).Nevertheless, a watching brief must be main-tained particularly around major nuclearestablishments in Europe and America.

Despite the Minamata episode of the1960s in Japan, mercury contamination ofseafood continues to present multiple risksto consumers on a wider geographical scale.Thus, Dickman et al. (1998) observed a linkbetween mercury in fish and subfertility inHong Kong men. In the Faroe Islands, mer-cury exposure occurs through consumption ofwhale meat. Children with pre-natal exposureto this element have been observed to displaycognitive deficits (Grandjean et al., 1997).

Major concerns continue to arise frommicrobial sources in seafood (Table 19.3; seealso Gago Martínez and Lawrence, Chapter 3,this volume). Contamination of shellfish withHAV is a significant risk to seafood consumersin Europe, the USA and China. Coastal watersin the USA are classified as ‘approved’, ‘condi-tional’, ‘restricted’ or ‘prohibited’ for shellfishharvesting, depending on levels of pollution.Similarly, in England and Wales, a revisedclassification of bivalve mollusc harvesting

11-Mar-03 19




has been introduced in response to E. coli con-tamination (Food Standards Agency, 2001f).Three categories have been introduced: inClass A areas, molluscs may be harvested fordirect human consumption, whereas, in ClassB and Class C areas, shellfish are classified asfit for human consumption only after certainrelaying procedures have been followed.

Vibrio parahaemolyticus (serotype O3:K6)recently has been associated with foodborneillness in South-east Asia. This pathogenspread to Taiwan, Laos, Japan, Thailand,Korea and even the USA between 1997 and1998. Hara-Kudo et al. (2001) detected thebacteria in a variety of seafoods, particularlyclams and mackerel.

Meat

The safety of meat has been brought intosharp focus in the aftermath of the bovinespongiform encephalopathy (BSE) crisis inEurope and specifically in the UK. It would,therefore, be easy to assume that the BSEagent has been a major determinant of meatsafety. Such a view would be quite erroneousas the major contaminants have been, andcontinue to be, of bacterial origin (Table 19.4;see also Johnson, Chapter 2, this volume). It isreassuring, therefore, to note that the Food

Standards Agency survey recently indicatedthat the incidence of Salmonella contamina-tion in retail chickens in the UK dropped tothe lowest level ever recorded. Further infor-mation is available at the Food StandardsAgency website (www.foodstandards.gov.uk). In addition, a study of a variety ofretail meats obtained from the greater Wash-ington DC area of the USA (Zhao et al., 2001)showed that only 3% of samples were con-taminated with Salmonella. Despite the fore-going, it should be noted that other bacterialcontaminants continue to represent a majorpublic health hazard, with worldwideimpact. Thus, in the UK, there is still exten-sive contamination of poultry meat withCampylobacter. In the greater Washington, DCarea of the USA, 71 and 14%, respectively,of retail chicken and turkey samplesobtained for the study by Zhao et al. (2001)were contaminated with Campylobacter, and91% of stores visited had Campylobacter-contaminated chicken. The incidence of E. coliin that study was 39% for chicken, 12% forturkey, 16% for pork and 19% for beef.Furthermore, other studies point to thepresence of both Salmonella and Campylobacteron the external and internal surfaces ofmaterial used as packaging of retail chickens(Harrison et al., 2001).

According to Bolton et al. (2002), E. coliO157:H7 contamination of beefburgers is


11-Mar-03 19

Food Contaminant Comments/status Further information

Fish

Shellfish

Mercury

Arsenic

PCBs

Dioxins

Vibrio parahaemolyticus

Diarrhoetic shellfish poisonsHepatitis A virus (HAV)

E. coli

Contamination continues despitethe Minamata episode of the 1950sMay be affected by cookingtemperaturesIn farmed salmon

In farmed salmon

Serotype O3:K6 associated withrecent outbreaksSpecific compounds identifiedApproximately 1.4 million cases ofHAV-induced illness occur globally

Revised harvesting classificationissued by Food Standards Agency

Dickman et al. (1998)

Devesa et al. (2001)

Food Standards Agency(2001b,h)Food Standards Agency(2001b,h)Hara-Kudo et al. (2001)

Chapter 3Kingsley and Richards(2001); Seymour andAppleton (2001)Food Standards Agency(2001f)

PCBs, polychlorinated biphenyls.

Table 19.3. Principal contaminants in seafood.



significant, accounting for 50% of outbreaksassociated with this pathogen in the USAduring the period 1982–1994. Their stud-ies, moreover, indicated that the currentmethods available for controlling the patho-gen during production of beefburgers areineffective.

Eggs

The recent loss of confidence in food safetycan, arguably, be traced to the ‘Salmonella-in-eggs crisis’ in the UK (Table 19.4).Stringent controls over breeding and layingflocks together with the introduction of

11-Mar-03 19


Food Contaminant Comments/status Further information

MeatBeef

Beef/lambBeef/lamb

Lamb

Poultry

Pork

Beef, lamb,poultry andpork

Eggs

Milk

Cheese

BSE agent

E. coliVeterinary residues

Radionuclides

Salmonella

Campylobacter

Undisclosed

Salmonella

Ochratoxin A

Dioxins

Salmonella


Mycobacterium aviumsubspeciesparatuberculosisDioxins

Aflatoxins

Radionuclides


In decline, but incidence ofvCJD in humans set to risein the medium termOn-goingOn-going on a worldwidescaleOn-going on a regionalscaleOn-going on a worldwidescaleOn-going on a worldwidescaleIllegal diversion of unfitpoultry meat into the foodchain in the UK; possiblyworldwideOn-going worldwide; newtracing methods indevelopmentOn-going on a regionalscaleAssociated with a specificcontamination incident inBelgiumOn-going on a worldwidescaleOn-going on a worldwidescalePossible link with Crohn’sdisease in humans

On-going on a regionalscale

On-going on a regionalscaleOn-going on a regionalscale

On-going on a worldwidescale

Chapter 14

Chapter 2Chapter 13

Chapters 17 and 18

Chapter 2

Chapters 2 and 13

Food Standards Agency(2001f)

Giovannacci et al. (2001)

Chapter 4

Food Safety InformationBulletin (1999)

Chapter 2

Chapter 2

Food Standards Agency(2001d)

Chapter 7; Ministry ofAgriculture, Fisheries andFood (1992c)Chapter 4

Chapters 17 and 18; Ministryof Agriculture, Fisheries andFood (1994)Chapter 2

vCJD, variant Creutzfeldt–Jakob disease.

Table 19.4. Principal contaminants of meat, eggs and dairy products.



vaccination against Salmonella enteritidis havenow resulted in reduced contamination ofeggs in the UK and elsewhere. This declinehas correlated with reduced incidence ofhuman salmonellosis. However, in a secondreport on Salmonella in eggs, the AdvisoryCommittee on the Microbiological Safety ofFood (2001) warns against complacency sincethere may be other Salmonella serotypes capa-ble of infecting hens and thus contaminatingeggs. Furthermore, that Committee madea number of specific recommendations onfuture policy. For example, it was suggestedthat ‘use by’ dates would elicit greater healthbenefits to consumers than ‘sell by’ or ‘usebefore’ dates. This recommendation is basedon the conclusion that safety with respectto Salmonella-contaminated eggs decreasesmarkedly some 21–28 days after lay dueto yolk membrane disintegration. Imple-mentation of this recommendation would,however, require permission via revised EUregulations.

Milk and Dairy Products

Milk and dairy products are subject to con-tamination with a wide variety of organisms(Johnson, Chapter 2, this volume) and othersubstances (Table 19.4). For example, L. mono-cytogenes is a regular contaminant of dairyfoods (Kalmokoff et al., 2001), and the organ-ism is capable of adhering to many materialsin food-processing plants, including dairies(Beresford et al., 2001). The recent observationthat Mycobacterium avium subspecies para-tuberculosis can survive pasteurization ofmilk has renewed interest in a possible linkbetween the bacterium and Crohn’s diseasein humans. However, various food agenciesstress that people should continue to consumemilk and other dairy products. Analyses offarm-gate milk in the UK between 1988 and1989 showed low levels of aflatoxin M1 con-tamination, but more than 50% of milk sam-ples in Tanzania were found to contain themycotoxin (D’Mello and Macdonald, 1998).

Levels of dioxins (Fiedler, Chapter 7, thisvolume) in UK milk supplies were observedto be higher in samples obtained from

farms near to urban and industrial sites thanin those from farms in rural areas (Ministryof Agriculture, Fisheries and Food, 1992c).The UK national milk supply has alsobeen affected by contamination withradionuclides arising from the testing ofnuclear weapons and from the Chernobyldischarge in 1986 (Ministry of Agriculture,Fisheries and Food, 1994).

Supplements and Additives

Dietary supplements and additives arewidely used for a variety of purposes (Simonand Ishiwata, Chapter 11, this volume). Con-troversy over the use of these compoundscontinues even with permitted products.Thus, although aspartame has been approvedin the UK since 1983, the Food and AdvisoryCommittee has advised the Food StandardsAgency to review recently published evi-dence to allay persistent consumer appre-hension about the safety of this artificialsweetener. There currently is considerabledebate about the safety of foods for children,with at least one press report suggesting thelabelling of some items with a health warningon the basis of additions of dyes, artificialflavourings and flavour enhancers. Similarly,vitamin C, at levels corresponding to200 mg day−1, has been linked to cellulardamage and cancer under in vitro conditions.While the human health implications ofrelatively high doses have still to be eluci-dated, current advice is that all vitamin Crequirements can be satisfied with a balanceddiet containing five portions of fruit andvegetables per day.

The addition of salt and sugar to manu-factured foods currently is under scrutiny inmany of the developed countries followingmedical and dental advice. Reduction of saltintake is perceived as a vital step towardscontrol of hypertension and cardiac disease. Itis reassuring to observe that in the UK saltlevels in bread have declined by 21% since1998 (Food Standards Agency, 2001h). Thereis, however, an urgent need to reduce levelsof salt and indeed sugar in snacks andconfectionery.


11-Mar-03 19



Other Food Products

A list of contaminants in miscellaneousfood items is presented in Table 19.5. Thislist is not an exhaustive compilation but isindicative of recent concerns in the UK. Foodsafety agencies elsewhere should be ableto provide comparable information moreattuned to local food safety issues. In thecases of mycotoxin contamination of coffee,spices and dried vine fruits, remedial actioncurrently is being implemented. Pettersonet al. (1991) commented on the conflictingevidence concerning the pharmacologicaland physiological effects of caffeine and dis-counted the potential teratogenic effects inhumans on the basis of the large dosesrequired to elicit such responses. However,current advice (Food Standards Agency,2001g) is that pregnant women shouldrestrict their caffeine intake to 300 mg day−1

as a prudent measure to avoid negativeeffects such as low birth weights and sponta-neous abortion. Another group at risk arecigarette smokers, who tend to consumemore caffeine than non-smokers and meta-bolize the alkaloid at a faster rate.

The UK Food Standards Agency suggeststhat consumers may continue to eat Chinesefoods despite the occurrence of cancer-causing chloropropanols in soy sauce samplesstocked by specialist retail outlets. Thecompounds investigated included 1,3-dichlo-ropropanol (1,3-DCP) and 3-monochloro-propane-1,2-diol (3-MCPD). The final advicewas based on results indicating that soy sauceproducts sold in major retail chains were safe

in this respect. Levels of bisphenol A(Petersen, Chapter 12, this volume) in somecanned vegetables were also deemed not topose significant risks to human health.

In Finland, the National Food Agency hasprovided specific advice on contaminants inhoney, olive oil pomace, Hypericum, raspber-ries and imported noodles. In the USA, allergyalerts have been issued by the Food and DrugsAdministration over undeclared ingredientsin cookies, peanut butter bars and low-fatmilk products. Further details of theseand other food alerts are provided in a latersection.

Food Allergy

Allergic responses to certain foods (Spencerand Berman, Chapter 1, this volume) havebeen well documented both in anecdotalaccounts and in scientific investigations(Sampson and Burks, 1996). A true allergicresponse reflects a hypersensitivity to foodor particular components that evokes animmunological reaction in a subject. Whena food elicits a response that is not clearlylinked with an immune reaction, an alter-native term, ‘food intolerance’, is invokedto characterize the resulting adverse effects.Lactose intolerance is one example of sucha syndrome. In addition, many individualsmay react adversely to the various proteinsin milk. The proteins of eggs have also beenimplicated in allergic reactions, with childrenbeing particularly susceptible.

11-Mar-03 19


Food products Contaminant Data source

Soy sauceSpicesCanned vegetablesOlive pomace oilTurkish helva (halva)Dried vine fruitsCoffee

ChloropropanolsAflatoxins and ochratoxin A (OTA)Bisphenol ABenzo(a)pyreneSalmonellaOTAOTA; caffeine

Food Standards Agency (2001d)Chapter 4Food Standards Agency websitea

Food Standards Agency websitea

Food Standards Agency (2001e)Chapter 4Chapter 4; Food StandardsAgency (2001g)

awww.food.gov.uk

Table 19.5. Contaminants in miscellaneous food products available in Britain.



Effects of food allergens range frommild reversible symptoms to life-threateninganaphylaxis in sensitive individuals. Adversereactions to peanuts exemplify the fullspectrum of such effects, requiring totalavoidance by individuals displaying theanaphylactic symptoms. Fruit and vegetablesalso contain allergens, often mediated incross-reactions with pollen components.Thus, a dual and complex allergy may be iden-tified in this type of syndrome (Vieths, 1997).

Special labelling of foods is required toprotect consumers, particularly those sus-ceptible to peanut allergies. The warning‘may contain nuts’ is a common feature ofall types of manufactured foods. Consumerssensitive to other food components arerequired to check the declared list of ingredi-ents. A later section in this chapter givesexamples of particular batches of foods thathave been withdrawn from sale followingallergy alerts.

Processing and Packaging

Processing and packaging are critical compo-nents of any strategy to ensure that foodsare safe for the consumer. Regrettably, unlessstringent methods are adopted, processingand packaging are attended with potentialrisks. In addition, hygiene conditions andpractices in the slaughterhouse will havea significant impact on the safety of meat.Thus, contamination of broiler meat withCampylobacter may occur at slaughterthrough contact with excreta (Heuer et al.,2001). The efficacy of removal of specifiedrisk material, as required by law, will deter-mine the safety of beef and lamb.

Examples of industrial processing tech-niques include:

• cooking• pasteurization• freezing• dehydration• curing• use of preservatives• irradiation• packaging

Contamination of foods during processingoccurs on a disturbing scale and with regularfrequency. For example, L. monocytogenesis often isolated not only from dairy andother food products but also fromfood-processing plants (Kalmokoff et al.,2001). Taormina and Benchat (2002) com-mented on the survival of L. monocytogenesin commercial food-processing equipmentcleaning solutions. They advised that elimi-nation of this organism could be achievedby the use of alkaline cleaning solutionsand appropriate sanitizers. Chasseignauxet al. (2001) isolated L. monocytogenes fromraw meat and associated products in poultry-and pork-processing plants in France.

Thermal methods

Adequate cooking is an obvious and effectivemethod of overcoming the potential effectsof bacterial contamination, and new technol-ogies are being developed for this purpose(Robey et al., 2001). However, improper stor-age and handling subsequently can and oftendoes negate the effects of cooking. The classi-cal example is the storage of cooked meatunder uncooked meat in commercial anddomestic refrigerators. In the case of greencoffee (Table 19.5), roasting conditionsduring processing determine the efficacy ofreduction of ochratoxin A. Van der Stegenet al. (2001) commented on variable reduc-tions ranging from 69 to 96% in differentstudies.

It is maintained that thermal proceduresare not efficient for the complete inactivationof shellfish toxins and that alternative technol-ogies need to be developed. Gonzalez et al.(2002) reported the basis of a new methodinvolving the use of supercritical fluids. Thethermostability of staphylococcal enterotoxinA has also been reported. Suzuki et al. (2002)suggest alternative strategies for food hygienemanagement based on the use of an electro-lysed anodic solution.

Baking of bread may have differentialeffects on allergenic potential. Simonato et al.(2001) showed that some wheat allergens, for


11-Mar-03 19



example the 16 kDa protein, can be denaturedafter baking, but others can be made moreresistant to digestive processes. Conse-quently, they may interact with the gutmucosa in an immunologically activemode. These findings also imply that currentdiagnostic tests for identifying wheat allergiesmay be flawed.

Seafood contaminated with certain spe-cies of arsenic may constitute potentiallygreater risks to human health after cooking.Thus, Devesa et al. (2001) concluded thatarsenobetaine in seafood may undergothermal conversion to more toxic species.

Food-processing methods such as grill-ing and smoking may induce formationof polyaromatic hydrocarbons (PAHs), asdescribed by Guillén and Sopelana in Chapter8 (this volume). Smoke from cooking oils mayalso cause adverse effects by virtue of theproduction of PAHs. Chen and Chen (2001)summarized the evidence for these effects andcompared the mutagenic potential of smokesfrom different cooking oils. It is salutary torecall that cooking meat under grills and onbarbecues is attended by additional risks inthat harmful bacteria may persist in uncookedportions or through inadvertent contact withraw meat.

Irradiation

Irradiation is now permissible for the pro-cessing of certain foods intended for directhuman consumption since this method iseffective against common food spoilagemicrobes (WHO, 1994). However, organo-leptic changes undermine its efficacy forparticular food items unless microbial loadsare low, as demonstrated by studies withsaffron (Crocus sativus L.; Zareena et al., 2001).

Freezing

Freezing has long been used as a methodof food preservation, and storage of foodsat −20°C in household appliances is nowcommon practice. However, it is assumedthat freezing kills all microbial pathogens.

The work of Moorhead and Dykes (2002) dis-pels that notion with respect to Campylobacter,which is able to survive both the freezingprocess and subsequent frozen storage. Theauthors conclude that current methods offrozen meat preservation do not add a sig-nificant margin of safety as regards thispathogen.

Curing

N-Nitroso compounds (NOCs; Eichholzerand Gutzwiller, Chapter 10, this volume)have been implicated in the aetiology of gas-tric cancer in humans. Exposure results fromthe intake of foods cured with nitrate andnitrite and by the smoking and chewing oftobacco products. Processed foods that maybe relevant in this respect are frankfurters,dried salted fish and certain soy sauces.Haorah et al. (2001) reported that 90% ofNOCs and 75% of NOC precursors in frank-furters were not present in fresh meat. It wassuggested that most of the precursors aroseduring processing or storage, while mostNOCs were generated by reaction with theadded nitrite. As regards other forms of cur-ing, it should be noted that cold smoked fishproducts are likely to be contaminated withL. monocytogenes, a pathogen that appears tosurvive this type of processing. In a surveyconducted by the Finnish National FoodAgency, 15–20% of vacuum-packed coldsmoked fish products were contaminatedwith this pathogen during 1997–1998,although low levels were found in 2000.

Effects of Environmental Pollution

Enhanced levels of environmental pollutionhave undermined food safety; this is self-evident and indeed has formed a majortheme in this book. The association betweennuclear incidents and food radioactivity iswell documented and constitutes the basisof two chapters in this book (Smith andBeresford, Chapter 17, and Baratta, Chapter18). The importance of this link has beenacknowledged in a document entitled

11-Mar-03 19




Radioactivity in Food and the Environment, 2000(Centre for Environment, Fisheries andAquaculture Science, 2001). The report wasissued on behalf of the Food StandardsAgency and the Scottish Environment Protec-tion Agency in the UK. Perusal of Chapters 17and 18 will show the global extent to whichbasic food supplies have been, and continueto be, contaminated with radionuclides.Levels in meat and seafood have been high-lighted despite the claim that contaminationhas not compromised human health. Never-theless, the authors of Chapters 17 and 18will agree that monitoring of seafood andagricultural produce must continue, particu-larly around major nuclear establishments inEurope, North America and Japan.

Of equal concern are the humanhealth implications of estuarine and coastalpollution caused by discharges of sewage.Bacterial and viral contaminants arising fromsuch sources accumulate in shellfish and con-stitute a serious health hazard to consumers(Table 19.3).

Consumer concern has also emergedover persistent organic pollutants such aspolychlorinated biphenyls (PCBs) and dioxins(Arnold and Feeley, Chapter 6, and Fiedler,Chapter 7, this volume). Highest concentra-tions occur in fatty foods such as meat and oilyfish. In the UK, dietary exposure has fallen by75% since the 1980s, with concomitantreductions in health risks associated withthese compounds. It has been stated that, atthe levels occurring in UK foods, PCBs anddioxins are unlikely to be associated withadverse effects even in young children (FoodStandards Agency, 2001h). The perceptionelsewhere appears to be quite different. Thus,Patandin et al. (1999) concluded that maternalbody burden of PCBs should be reducedas in utero exposure may affect subsequentcognitive performance of pre-school children.

Of the inorganic elements, mercury con-tinues to present a risk worldwide (Jorhem,Chapter 9, this volume), particularly withrespect to contamination of seafood (Table19.3). The detrimental human health effectsof mercury may be compounded by exposureto other environmental contaminants includ-ing PCBs and organochlorine pesticides(Grandjean et al., 1997).

Water quality

The quality and safety of water used fordirect consumption or for irrigation of foodcrops are very much determined by prevail-ing levels of pollution. The levels of lead andnitrates have been a source of concern formany years. In addition, the occurrence ofpathogenic enteric microorganisms derivedfrom sewage pollution in water is an emerg-ing issue. Outbreaks of enteric virus illnessin humans have been associated with wateron many occasions arising from pipeline fail-ures, pollution of wells and contamination ofmunicipal supplies with sewage (Wyn-Jonesand Sellwood, 2001).

Food Labelling

It is axiomatic that food labelling is animportant way to keep consumers informedof exactly what they are eating. Indeed, label-ling regulations exist in the EU for foodsdestined for the ultimate consumer or fordelivery to catering establishments (seefollowing section and Joint Food Safety andStandards Group, 1998).

In the case of hypersensitive individuals,accurate labelling is an imperative componentof their efforts to avoid food allergies. Conse-quently, the use of terms such as ‘spices’,‘colourings’ and ‘flavourings’ is not helpfulto such individuals, and food manufacturersand retailers must be encouraged to provideprecise details if inadvertent intake of aller-gens is to be avoided. Equally, the appearanceof terms such as ‘may contain nuts’ on thelabels of a large number of packaged foodsconsiderably reduces choice for hyper-sensitive individuals.

Although labelling of manufactured andpackaged foods is regulated and activelyencouraged by the major food agenciesaround the world, attention currently isbeing focused on misleading labels. It isargued that terms such as ‘farmhouse’,‘country style’, ‘fresh’ and ‘natural’ have noplace on food labels. Such terms are notdefined by law and if used without duecare can baffle and mislead the consumer.


11-Mar-03 19



Certainly such terms have no place in anystrategy designed to uphold food safety. Itis clear that more stringent enforcement offood labelling guidelines and regulations isneeded.

Legislation

General

Elaborate directives and statutory instru-ments exist to underpin food legislation inthe EU. Extensive regulations have also beendeveloped in other countries including, forexample, Canada and the USA. It is notintended to review all such legislation here.In any event, relevant legislation pertainingto other countries will be found in variouschapters in this book. The following accountshould illustrate that within the UK in partic-ular and the EU in general there is now aframework that could well serve as a modelfor other countries where food legislationis still rudimentary or non-existent. As willbe seen shortly, comprehensive legislationwas required to reduce BSE contaminationof beef supplies in Britain. It is temptingto suggest that a similar network of regu-lations in the UK and elsewhere might berequired to bring about more satisfactorycontrol of bacterial contaminants. The inci-dence of Listeria, E. coli and Campylobacter inmeat and dairy products continues unabatedand on a worldwide scale (Table 19.4). Anylegislation in this respect, however, willdepend upon achieving global consensuson issues such as sampling, methodology,monitoring and enforcement.

Food labelling

Current EU directives are incorporated intolegislation of Member States. In the UK, theFood Labelling Regulations 1996 implementthese directives. The main provisions ofthe Regulations are that foods intended fordirect consumption or for delivery to cateringestablishments must be labelled with specificdetails as follows.

• Name of the food.

• List of ingredients.• Appropriate durability indication, e.g.

‘best before’ or ‘use by’ dates. The latterterm applies to foods which in the micro-biological context would be consideredas being highly perishable and, in con-sequence, after a short period of time,likely to constitute an immediate healthhazard.

• Any special conditions for storage oruse.

• Name or business name and an addressor registered office of the manufactureror packer or of a seller established withinthe EU.

In certain circumstances, additional regula-tions apply, e.g. origin of the food, if failureto give such information might misleadconsumers, and instructions for use if, inthe absence of such information, consumersmight encounter difficulties in makingappropriate use of the food.

Contaminants

Legislation is now in place for the majorfood contaminants, but there are wide inter-national differences in the scope and detail ofthe regulations. These discrepancies explainthe occurrence of unregulated and bannedpesticides in foods imported into the EU. In alarge number of developing countries, foodsafety legislation is virtually non-existentand, furthermore, commands low priorityin government business. As pointed out byD’Mello in Chapter 4 (this volume), some 13countries have no regulations for mycotoxinsin foods and, for about 50 countries, mostlyin Africa, no data on levels of thesecontaminants are available. For all countries,a particular concern is the lack of statutoryor advisory regulations for control offumonisins in foods (Chapter 4).

For particular foods, there may be con-sumer safety implications following exportof unregulated commodities to countrieswith more comprehensive legislation. A goodexample is the use of banned substances suchas specified pesticides or hormone growthpromoters in agriculture when the endproducts are intended for export to Europe

11-Mar-03 19




and North America where stringent foodsafety regulations are in place.

Relevant food legislation issues areaddressed in detail in several chapters of thisbook. However, it is instructive to illustratethe range of legislation at the disposal of foodsafety agencies in the UK and in other parts ofthe EU. These regulations should provide auseful framework for those countries wherefood safety legislation is still in its infancy. Thelist in Table 19.6 is not designed to be exhaus-tive but rather indicative of the wide arrayof general and specific provisions in existingand new legislation. Many of the specific

regulations have come into force in the post-BSE era. The information in Table 19.6 hasbeen adapted from a most useful book byJukes (1993). However, this list has beenupdated to include new legislation publishedby the Food Standards Agency (FSA) in itswebsite (see Table 19.6). It will be seen that, inaddition to the general provisions of the FoodSafety Act (1990), more specific regulationsexist, particularly for meat and eggs. In addi-tion, there are specific regulations for process-ing and packaging, labelling, additives andnamed contaminants. One cannot helpbut enquire why, in the face of such


11-Mar-03 19

Category Legislation

General

Hygiene and health

Medical foodsAdditives

Food Safety Act (1990)Definition of foodRendering food injurious to healthSelling food not complying with food safety requirementsInspection and seizure of suspected foodImprovement noticesProhibition ordersFalse description

Food Safety (General Food Hygiene) Regulations 1995Food Safety (Temperature Control) Regulations 1995Fresh Meat (Hygiene and Inspection) Regulations 1992Fresh Meat (Beef Control) (No. 2) (Amendment) (England) Regulations 2000Meat Products (Hygiene) Regulations 1994Minced Meat and Meat Preparations (Hygiene) Regulations 1995Poultry Meat (Hygiene) Regulations 1976Poultry Meat, Farmed Game Bird Meat and Rabbit Meat (Hygiene and Inspection)(Amendment) (England) Regulations 2001Ungraded Eggs (Hygiene) Regulations 1990Egg Products Regulations 1993Eggs (Marketing Standards) Regulations 1995Salmonella in poultry: Processed Animal Protein Order 1989; Poultry BreedingFlocks and Hatcheries Order 1993; Zoonoses Order 1989Dairy Products (Hygiene) Regulations 1995Specified Risk Material (Amendment) 2000: implementation of EU Decision2000/418/ECFood Safety (Fishery Products) Regulations 1992Food Safety (Live Bivalve Molluscs and Other Shellfish) Regulations1990Imported Food Regulations 1997Medical Foods (England) Regulations 2000Antioxidants in Food Regulations 1978Colours in Food Regulations 1995Emulsifiers and Stabilizers in Food Regulations 1989Flavourings in Food Regulations 1992Mineral Hydrocarbons in Food Regulations 1966

continued

Table 19.6. A summary of food legislation in the UK and EU: a selection of provisions, specificregulations, orders and amendments for particular categories relevant to food safety.a



comprehensive legislation, food safety contin-ues to be a major source of concern in theperception of European consumers.

Food Safety Act 1990 (Great Britain)

The main provisions of this Act came intoforce on 1 January 1991. The Act relates toGreat Britain and provides the basic frame-work for all its food legislation (Table 19.7).

Key provisions

The key provisions of the Food Safety Act1990 are divided into four parts.

1. Part I: preliminary. This part contains adefinition of ‘food’ and other basic termsused in the Act such as ‘food business’, ‘foodpremises’ and ‘food source’. This part alsosets out presumptions applying to foodand food ingredients. For example, foodcommonly used for human consumptionfound in certain food premises is presumed tobe intended for sale.

2. Part II: main provisions. This part describesthe offence of rendering food injurious tohealth and defines the offence of selling orpossessing for sale food that does not complywith food safety requirements. This is foodthat has been rendered harmful to health,which is unfit for human consumption oris so contaminated as to be unfit for humanconsumption. General enforcement provi-sions confer enforcement officers with powersto inspect food and to seize and detainfood suspected of not complying with foodsafety requirements. Other sections of theAct provide for improvement notices to beissued following infringements of foodhygiene or food-processing regulations.The provision of specific prohibition ordersallows courts, authorized officers andMinisters to take appropriate action whenfood safety is at risk.3. Part III: administration and enforcement.This part describes who may enter premises toenforce the Act and explains what they can dowhile on the premises. A section makes it anoffence to obstruct a person enforcing the Act.Time limits for prosecutions and penalties andmodes of trial are also set out in this part.

11-Mar-03 19


Category Legislation

Contaminants

Processing andpackaging

Labelling

Food protection inan emergency

Aflatoxins in Nuts, Nut Products, Dried Figs and Dried Fig Products Regulations1992Arsenic in Food Regulations 1959Animals and Animal Products (Examination for Residues and Maximum ResidueLimits) Regulations 1997Lead in Food Regulations 1979Pesticides (Maximum Residue Levels in Food) Regulations 1988Tin in Food Regulations 1992Quick-frozen Foodstuffs Regulations 1990Food (Control of Irradiation) Regulations 1990Food Irradiation Provisions (England) Regulations 2000Materials and Articles in Contact with Food Regulations 1987Plastic Materials and Articles in Contact with Food Regulations 1992Plastic Materials and Articles in Contact with Food (Amendment) (England)Regulations 2000Food Labelling Regulations 1996: implement EU directive relating to the labelling,presentation and advertising of foodstuffs for sale to the ultimate consumerFood Additives Labelling Regulations 1992Food and Environment Protection Act 1985

aCompiled from Jukes (1993); Joint Food Safety and Standards Group (1998); Food Safety Agencywebsite (www.foodstandards.gov.uk/regulations.htm).




4. Part IV: miscellaneous and supplemental.Sections in this part provide for the Act toapply to the Crown and to Crown premisessubject to special arrangements and certainexemptions. Another section amends theWater Act 1989 to extend its controls on thequality of water used for domestic purposes tocover water used in food production.

Statutory instruments

A number of statutory instruments imple-menting the Food Safety Act 1990 have comeinto force. The Joint Food Safety and Stan-dards Group (1998) lists these in some detail(Table 19.7). Some of the instruments relateto England and Wales while others applyto the whole of Great Britain. The repeal or

revocation of outdated legislation is providedin certain instruments.

Included in this list are The Food (Sam-pling and Qualifications) Regulations 1990,which set out the procedures to be followed byenforcement officers when obtaining samplesfor analysis or microbiological examination.The Food Premises (Registration) Regulations1991 provide for the registration of foodpremises by food authorities. In addition,the instruments provide emergency powersfor government Ministers, and set out qualifi-cation requirements for Public Analysts andFood Examiners. The emergency provisionswere invoked recently in connection withdioxin contamination of Belgian foods(Fiedler, Chapter 7, this volume). The Food(Animals and Animal Products from Belgium)


11-Mar-03 19

Instruments Provisions

The Food Safety Act 1990 (Commencement(C40) No. 1) Order 1990The Food (Sampling and Qualifications)Regulations 1990

The Food Safety Act 1990 (ConsequentialModifications) (England and Wales) Order1990Detention of Food (Prescribed Forms)Regulations 1990Food Safety (Improvement and ProhibitionPrescribed Forms) Regulations 1991

The Food Premises (Registration)Regulations 1991The Food Safety Act 1990 (CommencementNo. 3) Order 1992

The Food Safety Act 1990 (ConsequentialModifications) (Local Enactments) Order1992

The Food Premises (Registrations)(Amendment) Regulations 1993

The Food Premises (Registrations)(Amendment) Regulations 1997

New powers for Ministers to use in emergencies

Procedures to be followed by enforcement officers whentaking samples for analysis or microbiologicalexamination. Qualification requirements for PublicAnalysts and Food ExaminersContinuation of milk and dairies legislation by amendmentof previous regulations

Prescription of forms of notice which may be issued forthe detention of food under the 1990 ActForms of notice which may be issued in connection withimprovement notices, prohibition orders or emergencyprohibition notices or orders under the respectiveSections of the 1990 ActRegistration of food premises (including vehicles andother movable structures) by food authoritiesBrings into force Section 59(4) of the Act which repealsprevious legislation about the registration of foodhandlers and food businessesRepeal or revocation of certain parts of local law relatingto registration of food premises. These provisions are nolonger necessary as The Food Premises (Registration)Regulations 1991 have been implementedExemption of childminders caring for no more than sixchildren from the requirement to register their premisesas a food businessExemption for people who prepare food at home for salein WI Country Markets Ltd from the requirement toregister their premises as a food business

aCompiled from the Joint Food Safety and Standards Group (1998).

Table 19.7. A selection of the statutory instruments implementing the Food Safety Act 1990 in GreatBritain.a



(Emergency Control) (England and Wales)(No. 2) Order 1999 came into force on 18August 1999.

Codes of practice for local authorities

Codes of practice issued under Section 40of the Food Safety Act 1990 are not legis-lation but documents issued by Ministersfor the guidance of food authorities, andthe provisions can be enforced by directionand court order. The Joint Food Safety andStandards Group (1998) listed some 20 codesof practice for local authorities in GreatBritain. A selection of these is presented inTable 19.8.

BSE control

Stringent regulations are enforced in Europeto control BSE contamination of meat(Hunter, Chapter 14, this volume). Thestatutory instruments are detailed inTable 19.9 to illustrate the diverse and formi-dable array of legislation required to contendwith a single food poisoning issue. There isgrowing optimism that the BSE legislationhas had the desired effect in terms of reducedprion protein contamination of UK beef.These regulations also provide an insightinto the protracted time scale required for thestatutory control of BSE in British herds.

In Britain, compliance with these andother meat hygiene regulations at abattoirsand cutting plants is the responsibility of

the Meat Hygiene Service (MHS). Cattle andsheep carcasses are examined for specifiedrisk material (SRM), which includes the intes-tines and additionally, in cattle over 6 monthsof age, head, thymus, spleen and spinal cord.Any SRM is removed at abattoirs and cuttingplants, stained and disposed of under thesupervision of MHS technicians. Disposalinvolves sealing the SRM in impervious con-tainers before being transported to rendererswhere it is destroyed. However, as pointedout in Chapter 14, it is more difficult to removespinal cord from sheep and goats comparedwith cattle. The BSE control legislationincludes the ‘over 30 month’ rule, whichmeans that cattle over this age are excludedfrom the human food chain. Another featureof BSE legislation forbids the use of mam-malian meat and bone meal as animal feed.It is not yet possible to quantify the role ofmechanically recovered meat in human foodson the spread of variant Creutzfeldt–Jakobdisease (vCJD). The use of such material hasbeen prohibited since 1995. Of particularconcern to those involved in food safety is thecontinued use of meat and bone meal as acomponent of animal feeds in many non-EUcountries.

Compliance and enforcement

Statutory regulations are not meaningfulunless they are complied with and enforcedby the relevant authorities. In the UK, muchemphasis has been placed on enforcement

11-Mar-03 19


Responsibility for enforcement of the Food Safety Act 1990Inspection, detention and seizure of suspect foodThe use of improvement noticesProhibition proceduresFood hygiene inspectionsEnforcement of temperature control requirements of food hygiene regulationsEnforcement of the Food Premises (Registration) RegulationsQuick-frozen foodstuffs: enforcement of temperature monitoring and temperature measurementEnforcement of the Food Safety (Fishery Products) Regulations 1992Enforcement of the Meat Products (Hygiene) Regulations 1994Enforcement of the Dairy Products (Hygiene) Regulations 1995Exchange of information between Member States of the EU on routine food control matters


Table 19.8. A selection of codes of practice for local authorities in Great Britain.a



of the BSE legislation. However, BSE controlfailures have been reported in the UK. Forexample, for the period January to October2001, 20 cases were recorded of beef importsbeing contaminated with traces of spinalcord. Some of these cases related to importsfrom The Netherlands. Exports of beef andrelated products are subject to stringent

control under the provision of the Bovinesand Bovine Products (Trade) Regulations of1999; compliance is monitored by regularchecks at ports. A number of alleged SRMoffences have been considered for possibleprosecution.

As previously indicated, the spread ofmicrobial contamination of foods should, in


11-Mar-03 19

Instrument Details

Zoonoses Order 1988, under the AnimalHealth Act 1981

Bovine Spongiform Encephalopathy(Miscellaneous Amendments) Order 1994,under the Animal Health Act 1981Fertilisers (Mammalian Meat and BoneMeal) Regulations 1996, under theAgriculture Act 1970Fresh Meat (Beef Controls) No. 2Regulations 1996, under the Food SafetyAct 1990

Beef Bones Regulations 1997, under theFood Safety Act 1990

Specified Risk Material Order 1997, underthe Animal Health Act 1981

Specified Risk Material Regulations 1997,under the Food Safety Act 1990

Specified Risk Material (Amendment)Regulations 1997, under the Food SafetyAct 1990

Bovines and Bovine Products (Trade)Regulations 1998

Order designated BSE as a zoonosis, enabling powersunder the Act to be used to reduce any human health riskfrom BSEOrder banned the use of mammalian protein in ruminantfeedingstuffs

Regulations prohibited the use of meat and bone meal asor in fertilizer applied to agricultural land

Prohibited the sale for human consumption of meat fromany bovine animal slaughtered after 28 March 1996 inwhich, at the time of slaughter, there were more than twopermanent incisors erupted, unless there was documentaryevidence to prove that the animal was no more than 2years and 6 months oldRequired all beef from animals aged over 6 months to bedeboned before sale to consumers; prohibited these bonesfrom being sold for human consumption or to be used in thepreparation of foodIntroduced controls on the import of specified risk material(SRM) and certain food products and feedingstuffscontaining SRMExtended controls on the handling and permitted use ofSRM from cattle, sheep and goats; extended existingcontrols on heads of sheep and goat SRM to include theremoval of spleen of all sheep and goats and the spinalcord and tonsils of those over 12 months old or with one ormore permanent incisors erupted through the gumAmended the Specified Risk Material Regulations 1997 toclarify that the requirement for removal of spinal cord fromsheep and goat carcasses aged over 12 months doesnot apply to carcasses of animals slaughtered before 1January 1998Implemented fully in Great Britain (GB) the requirements ofEU Commission Decision 98/256/EC, which replacedprevious Decision on emergency measures to protectagainst BSE; continued to prohibit the dispatch from GB tothird countries and Member States of bovine animals andembryos and meat and other products derived from bovineanimals slaughtered in GB


Table 19.9. BSE legislation as enforced in Great Britain. The following list is a selection of the statutoryinstruments that make direct and indirect provisions for food safety. Indirect provisions relate to animalfeed and fertilizer legislation.a



theory at least, be relatively easy to minimizeproviding basic hygiene measures areadopted at abbatoirs, cutting plants andcatering establishments. In addition, there arestringent regulations pertaining to cattleidentification, bovine and bovine products(trade), animal by-products, SRM and tuber-culosis in the UK and elsewhere in the EU.Notwithstanding, there are many actions,both successful and pending, which havebeen taken by the Food Standards Agency andother authorities in the UK. Similar actions arebeing pursued by food agencies in other coun-tries. In the UK, most actions have relatedto individual farmers, small slaughterhousesand retail outlets (Table 19.10). The infringe-ments relate to quite basic deficiencies ingood practice. At the heart of the matter arequestions such as communication of relevantlegislation to trade personnel and training ofstaff. One is also bound to enquire whethermore inspections might lead to an increasednumber of actions.

In the USA, the Food and Drug Adminis-tration (FDA) publish updated lists of ‘Recalland Safety Alerts’ (Tables 19.11 and 19.12).Recalls are actions taken by food businesses towithdraw products from the market. Recallsmay be conducted voluntarily by the busi-ness, if, for example, internal audits demon-strate the need for such action. In addition,recalls may be at FDA request or by FDA orderunder statutory enforcement. Three classes ofrecall are defined and implemented by theFDA (Table 19.11). Class I food recalls forthe 60 days to 6 December 2001 are listed inTable 19.12. In addition, individual states mayissue separate recalls and alerts relevant tolocal food issues (Table 19.12). Food alertspublished by the National Food Agency inFinland are listed in Table 19.13. All actionsand alerts summarized in Tables 19.10–19.13confirm parallel research and surveillancestudies (Chasseignaux et al., 2001; Harrisonet al., 2001; Zhao et al., 2001) showing thatmicrobial contamination of foods continuesto be a worldwide health issue. In the UK,recent recalls have included ‘customer returnnotices’ for tinned hot dog sausages. Noreasons were given for these recalls, whichwere issued to affirm the manufacturer’s

‘commitment to the highest standards of foodsafety and quality’.

Food alerts may also be highly focused,concentrating on a particular group of con-taminants, for example pesticides (Cabras,Chapter 5, this volume) or veterinary residues(Paige and Tollefson, Chapter 13, thisvolume). Table 19.14 contains recent alertsfor pesticide contamination of foods in theEU. In many cases, levels were deemed topresent no unacceptable risk to consumers.In a few instances, however, levels wereconsidered to exceed acute reference dosesfor both adults and children. In February2002, an alert was issued by the EU concerningthe presence of veterinary residues and othersubstances in meat, fish and shellfish fromChina, leading to suspension of imports. Thisaction was taken after an inspection visit toChina in November 2001.

Monitoring and Surveillance

The need for continued and increasedmonitoring of food for contaminants is atheme that appears in all chapters of thisbook. It is axiomatic that a sound monitoringand surveillance service will help to restoreconsumer confidence in food safety. Thus,although human salmonellosis in the UKand elsewhere has declined as a conse-quence of vaccination of commercial flocksagainst S. enteritidis, surveillance must con-tinue since there may be other Salmonellaserotypes capable of infecting hens andthus contaminating eggs. At the local level,it is worthy of note that Salmonella was linkedwith deaths and infection of a number ofpatients at a Glasgow hospital in January2002.

Recent actions in the UK have demon-strated the value of monitoring and surveil-lance in that a number of food items andproducts have been identified as sourcesof potentially harmful contaminants (Tables19.1–19.5). High-profile court cases haveresulted in convictions for illegal diversion ofunfit poultry meat into the food chain (Table19.4). An action plan has been proposedto include the introduction of staining

11-Mar-03 19




‘high-risk’ poultry meat destined for pet foodand development of a code of practice on thehandling and disposal of animal by-productsby the meat trade.

Current surveillance indicates unavoid-able, widespread and continuing mycotoxincontamination of basic plant products,with global implications for human health

(D’Mello, Chapter 4, this volume). Forexample, concentrations of aflatoxins inmaize and peanut kernels regularly exceedsafety threshold limits, and monitoring mustbe given high priority, particularly in thedeveloping countries of Africa and Asia.Furthermore, monitoring must continue incountries importing such commodities.


11-Mar-03 19

Body Reasons for action/statement Outcome

Food StandardsAgency (FSA)

Department forEnvironment, Foodand Rural Affairs(DEFRA)FSA

FSA

FSA

FSA

Ministry of Agri-culture, Fisheriesand Food (MAFF)b

MAFF

MAFF

Meat HygieneService

Local authority

Failure to ensure that pig carcasses were refrigerated ator below 7°C as required by the Fresh Meat (Hygiene andInspection) Regulations 1995Export of minced meat derived from bovine animalsintended for human consumption produced in unapprovedpremises in the UK: an offence under the Minced Meatand Meat Preparations (Hygiene) Regulations 1995Failure to establish a staff training programme and tocomply with requirement to cleanse and disinfectequipmentUse of exposed wooden pallets in a meat preparationarea, in breach of the Fresh Meat (Hygiene andInspection) Regulations 1995Failure to ensure that cleaning products were used insuch a manner as not to affect the fitness of any freshmeatBeef from a calf whose mother contracted BSE illegallyentered human food chain; statement made in January2002

Cutting fresh poultry at unlicensed premises in breach ofthe Poultry Meat, Farmed Game Bird Meat and Rabbit(Hygiene and Inspection) Regulations 1995. Alsobreaking an order banning the proprietor from managinga food business. Ban imposed under the Food SafetyAct 1990Failure to comply with Improvement Notices served underthe Food Safety Act 1990

Failure to stain specified risk material (SRM) as requiredby the Specified Risk Material Regulations 1997Failure of cutting premises to operate within the FreshMeat (Hygiene and Inspection) Regulations 1995 (asamended)Failure to store SRM in an impervious container asrequired by the Specified Risk MaterialRegulations 1997

Fine plus payment oflegal costs


Imprisonment



No action taken; riskthat meat containedBSE assumed to belowImprisonment

Voluntary surrenderand subsequentrevocation of licencefor premisesCommunity service

Licence revoked


aCompiled from UK Meat Hygiene Enforcement Reports and BSE Enforcement Bulletins; exact issuenumbers and other details not disclosed here in the interest of confidentiality.bNow DEFRA.

Table 19.10. Reasons for and outcomes of recent actions/statements by various authorities in the UKagainst individuals and companies or their directors.a



Food safety agencies

The food safety agencies in Europe and NorthAmerica play a major role in monitoring andsurveillance of food for contaminants. It isright that this book should contain chapterswritten by several authors emanating from

these agencies. Thus, the FDA of the USA,the Food Directorate, Health Canada, theSwedish National Food Administration andthe National Food Agency of Denmark are allrepresented in this book. In the UK, the FSAwas recently set up in response to growingconcerns over BSE contamination of beef and

11-Mar-03 19


Classification Definition

Class I recall

Class II recall

Class III recall

Market withdrawal

Circumstances in which there is a reasonable probability that the use of orexposure to a violative product will cause serious adverse health effects or deathCircumstances in which the use of or exposure to a violative product may causetemporary or medically reversible adverse health effects or in which theprobability of serious adverse health effects is remoteSituation in which the use of or exposure to a violative product is not likely tocause harm to consumersThis occurs when a product represents a minor violation that would not be subjectto FDA legal action

aCompiled from the FDA website: www.fda.gov/opacom/7alerts.html

Table 19.11. Classification of recalls issued by the Food and Drug Administration (FDA) in the USA.a

Date of issue orpreparation ofproduct Alerts

FDA6 December6 December4 December30 November30 November27 November31 October

26 October26 October15 October15 OctoberAlaska19–20 October

2 August

30 July to 3 August30 July to 5 SeptemberNot specified

Crystallized ginger: undeclared sulphitesAllergy alert due to undeclared peanuts and eggs in soft cookiesAllergy alert following undeclared dairy ingredient in cinnamon biscuitsAllergy alert following undeclared peanut butter in cakeAllergy alert following undeclared eggs in raisin cakeAllergy alert following undeclared pasteurized egg yolks in milk productAllergy alert following undeclared milk in assorted chocolates, assortedchocolate-covered nuts and peanut butter barsAllergy alert following undeclared egg in breadAllergy alert following undeclared sulphur dioxide ingredients in cake mixAllergy alert following undeclared whey in margarineAllergy alert following undeclared walnuts in cake

Cheese voluntarily recalled due to potential contamination with ListeriamonocytogenesTurkey ham products recalled due to possible contamination withL. monocytogenesChicken products recalled due to possible under-processingTurkey products incorrectly labelledChicken product recalled due to potential contamination with L. monocytogenes

aCompiled from following websites for, respectively, the FDA and Division of Environmental Health ofthe State of Alaska: www.fda.gov/opacom/7alerts.html and www.state.ak.us/dec/deh/sanitat/sanalert.htm

Table 19.12. Safety alerts issued in 2001 by the Food and Drug Administration (FDA) of the USAa andby the state of Alaska. The FDA list is mainly Class I recalls relating to food safety.



the incidence of vCJD in humans. It remainsto be seen whether the FSA will be proactiverather than merely reactive to the issues of

the day. It will need to take initiatives oversuch topics as the safety of genetically modi-fied (GM) foods (Gasson, Chapter 15, and


11-Mar-03 19

Food or supplement Reasons/status

Honey

Olive oil and pomace oliveoil (imported)Hypericum (St John’s wort)

Raspberries (imported)Noodles (imported)Butter

Fish products

Clostridium botulinum spores; honey not recommended for children under 1year of age; a multiplex PCR assay for detection of the pathogen has justbeen published by Finnish authors (Lindstrom et al., 2001)Unacceptably high concentrations of benzo(a)pyrene, above Finnish limit of2 µg kg−1

Sales ban on Hypericum preparations as foodstuffs due to adverseinteractions with several important medicines; reclassified as herbalremedies regulated under the Medicines Act of FinlandCalicivirus contamination from waterExcessive concentrations of chloropropanol (3-MCPD)Listeria monocytogenes isolated from both butter and patients; 25 cases oflisteriosis recorded in outbreakL. monocytogenes occurred in 15–20% of vacuum-packed cold smokedproducts during 1997–1998; low levels of contamination in 2000

aCompiled from the Finnish National Food Agency website: www.elintarvikevirasto.fi/english/tiedotteet/tiedotteet/press0900.htmlPCR, polymerase chain reaction; 3-MCPD, 3-monochloropropane-1,2-diol.

Table 19.13. Recent food safety alerts in Finland.a

Food Origin Pesticides

Kiwi fruitGrapefruitGrapes (table)

ApplesOrangesRaisinsMushrooms (shiitake)Chilli peppersMintChillies (red, kibbled) andchilli powder (hot, red)Curry powderTeaTea (jasmine)Milk (curdled, buffalo)

GreeceTurkeyGreece

IndiaFranceGreeceSpainChinaThailandEgyptIndia

IndiaChinaChinaRomania

MethidathionChlorpyrifos; parathion-methylb

Fenpropathrin, fludioxonil, penconazole, pyrimethanil,pirimifos-methylTriazophosc

Dicofol, chlorpyrifos, parathion-methyl and dimethoateParathion-methyld

FenthionFormaldehydee

MethamidophosChlorpyrifos, chlorpyrifos-methyl and malathionEthion, triazophos, cypermethrin and chlorpyrifos

Cypermethrin, fenvalerate and phosphamidonPirimicarb, fenvalerateBuprofezin, fenpropathrin and DDTOrganochlorine pesticides

aCompiled from www.pesticides.gov.uk/citizen/residues/other/other_residues.htmb‘Possibly in excess of acute reference dose and chronic dietary parameters for toddlers.’c‘Exposures above the acute reference dose for both adults and toddlers.’d‘Possibly in excess of acute reference dose and chronic dietary parameters for toddlers and infants.’e‘An unacceptable risk to consumers.’DDT, dichlorodiphenyltrichloroethane.

Table 19.14. European Union food alerts relating to pesticide contamination. A summary of selectedstatements issued during 2001.a The UK Pesticides Safety Directorate attached commentsb–e to specificalerts.



Pusztai et al., Chapter 16, this volume), attrib-utes of organic foods and the rising tide offood-related allergy and intolerance in theUK. In the developing countries of Africa,Asia and South America, where there isan overwhelming need for monitoring andsurveillance, it is regrettable that no agenciesexist to uphold food safety on behalf ofconsumers.

Research and Development

The message in this book overwhelminglyunderlines the need for further research invirtually every aspect of food safety. Indeed,the need for scientific research was a primarystimulus in the production of this book.Research proposals will be found in virtuallyevery chapter in the book. Neither is thereany shortage of research recommendationsfrom expert groups. Table 19.15 contains a

selection of proposals from just threeWorking Groups in the UK. There are recom-mendations for other contaminants and othercountries. All Working Groups recognize thatfinancial resources are not infinite and thatthe burden of allocation of funds, rightly, isthe responsibility of the research councils.Any implementation will depend uponcurrent research programmes and competingpriorities within other scientific disciplines.

With regard to bacterial and viral con-taminants of food, concerted research shouldfocus on virulence factors in pathogenicstrains. Work in this area has already begunon certain aspects of virulence in Salmonella(Cano et al., 2002), but there is a need for simi-lar investigations on Campylobacter, E. coliO157:H7 and HAV. Similarly, the concept thatenterococcal cells communicate with eachother to coordinate toxin production (Dunny,2002) is worth investigating in other microbesof relevance in food safety. Investigationsof cell–cell signalling mechanisms should

11-Mar-03 19


Contaminant Recommendations

E. coli O157a

Salmonellab

Radioactivityc

Development of DNA-based methods to identify accurately the different strains of theorganismAsymptomatic excretion of the organism in animals and humansBacterial load of individual animals and humansInteractions with other enteric organisms, e.g. Campylobacter speciesGrowth of Salmonella species in egg contentsContamination of eggs in egg-packing plantsVirulence and pathogenicityDetection and differentiation of strainsContamination of the farm environment as a source of SalmonellaEgg washing and subsequent growth of SalmonellaConsumer behaviourEvaluation of the significance of potential sources of radionuclide contamination in thefood chainPathways in routine surveillanceMore sensitive and efficient methodologies for measurement of radionuclidesin foodImproved methods of data handling and processing

aPennington Group (1997); this report followed an investigation into the E. coli outbreak of 1996 inCentral Scotland.bAdvisory Committee on the Microbiological Safety of Food (2001); this report focused on Salmonella ineggs.cCentre for Environment, Fisheries and Aquaculture Science (2001); this report was compiled on behalfof the UK Food Standards Agency and the Scottish Environment Protection Agency.

Table 19.15. Examples of Working Group recommendations for research on three regular contami-nants of foods.



provide insights into pathogenicity and treat-ment strategies. The application of moleculartechniques in the elucidation of virulencefactors and pathogenicity is likely to becrucial. There is an undisputed need for thedevelopment of appropriate tools for moni-toring food contaminants. Polymerase chainreaction (PCR) methods are likely to formthe basis of several diagnostic kits for thedetection and identification of food pathogens(Lindstrom et al., 2001), but there is scope formuch wider application of such techniques forother food contaminants.

It is unlikely that conventional toxicologi-cal assays will be adequate to evaluate fullythe attributes and safety of GM (Chapters 15and 16) and organic foods. Innovative meth-odologies and technologies will be required todiscern even the most subtle effects of theseand other foods on, for example, immuno-competence, cancer initiation and promotion,brain function and behavioural responses. Con-sumer concern over the safety of GM foodscontinues unabated, and the observations ofQuist and Chapela (2001) indicating that trans-genic DNA has introgressed into traditionalmaize landraces in Mexico is likely to fueladditional debate among environmentalists.

Levels of individual pesticides in com-mon foods are generally below the MRLs andRLs. However, there is at present no wayto predict the human health implications ofcombinations of different pesticides and otherenvironmental contaminants occurring in ourfood. The extent to which these contaminantsmay induce additive or synergistic effects willrequire a level of innovation that does notexist at the present time.

Issues associated with BSE contamina-tion of meat, in all its facets, will continueto demand research inputs for many yearsto come. One particular conundrum requir-ing immediate resolution is whether sheepin the national UK flock are carriers ofBSE. Scientists still cannot give an unequivo-cal answer to this issue although they main-tain that the risk is ‘theoretical’ in the absenceof confirmed cases of BSE in the nationalflock. While this is essentially a monitoringissue, a research programme will be neededif the results confirm the presence of BSE insheep or if the findings are inconclusive. In

any case, the problem cannot be resolvedwithout the development of a screeningprocedure capable of distinguishing betweenBSE and scrapie.

With regard to allergens in nuts (Spencerand Berman, Chapter 1, this volume), thenature of the protein causing life-threateningconditions in certain individuals needs tobe characterized fully in terms of the maineffects as well as cross-reactivity with otherfood components and with other factors.Food allergies and intolerance have been onthe increase in recent years with respect tothe incidence and severity of reactions, andthe reasons underlying both aspects need tobe elucidated.

There undoubtedly is an urgent needfor radical thinking and development ofinnovative technologies in all aspects offood safety. Suzuki et al. (2002) reported thepotential application of electrolysed anodicsolutions in the inactivation of staphylococcalenterotoxin A. Silva et al. (2002) suggested thepotential of bacteriocins as food preserva-tives, while Gonzalez et al. (2002) describedthe use of supercritical fluids to inactivateshellfish toxins. Much more work is requiredto develop these and other technologies and toexamine their potential for use with a widerange of food matrices.

Consumer Advice

The purpose of this book is to provide a scien-tific discourse on all matters relating to foodsafety. However, in the interest of complete-ness, some basic consumer advice might beappropriate here. The various food agenciesaround the developed world provide acontinuous stream of advice in the formof nutritional information, ‘best practice’ andalerts. However, it is clear that the messageis not reaching butchers, caterers and con-sumers, as demonstrated by the significantincreases in food poisoning incidents. Themedia have an important role to play in thisrespect in the various food programmes, butsafety in the handling of particular foods,such as uncooked meats, is rarely given anyattention; indeed, ‘best practice’ guidelines


11-Mar-03 19



often are blatantly contravened in populartelevision broadcasts.

Bacterial and viral contaminants

Basic hygiene guidelines must be imple-mented if the spread of food poisoning frombacterial and viral sources is to be avoided.The following protocols should be noted.

• Hands should be scrubbed and washedafter visiting washrooms and before han-dling food; this procedure should also befollowed after handling uncooked meatand seafoods.

• Cooking utensils, knives and cuttingboards should be washed thoroughlybefore and after use.

• Catering staff should not handle food ifthey have recently contracted specifiedenteric illnesses, particularly if they areinvolved in distributions to vulnerablegroups in day-care centres for youngchildren and in nursing homes andhospitals for the elderly.

• Raw meats and seafoods should be keptapart from other foods; in refrigerators,uncooked meat and seafood should beplaced on the bottom shelf.

• Frozen meat should be thawed thor-oughly before cooking. The appropriatetemperature and duration of cookingshould also be ensured.

Fungal and plant toxins

For these toxins, the scope for consumeradvice is limited due to the diversity andstability of compounds.

• Consumers should discard visiblymouldy foods; nuts should only bepurchased from reputable sources, suchas supermarkets, that implement highstandards of quality control.

• Individuals with specific allergiesshould avoid peanuts and other nutsor their derivatives in food.

• Raw kidney beans and the like should besoaked in water overnight before boiling

vigorously for at least 20 min; use of aslow cooker is not recommended for thispurpose.

• Green potatoes should always be dis-carded to reduce glycoalkaloid intake.

• Pregnant women should limit theircaffeine intake to less than four averagecups of coffee, or six average cups of tea,or eight cans of regular cola drink perday.

Environmental chemicals

Consumers are restricted in terms of theprotocols that they might adopt to reduceintake of the major chemical pollutants.However, the following points may be noted.

• Vegetables and fruit should be washedthoroughly before cooking or eating toreduce intake of pesticides.

• For the same reason, root vegetablesshould, where possible, be peeled toremove potential contaminants such asorganophosphate pesticide residues inthe epidermal layer.

• Fish from contaminated lakes and riversshould not be eaten.

Nutritional attributes

Providing basic protocols are adopted, partic-ularly with respect to personal hygiene andadequate preparation of foods, vegetables,fruit, poultry meat and seafood should forma valuable part of a balanced diet. The lowfat attributes of chicken, turkey and fishare well recognized. Significant benefits alsoresult from the consumption of fatty fishcontaining particular fatty acids that mayhelp in the prevention of heart disease. Inaddition to providing fibre, vegetables mayplay an important role in the preventionof certain cancers. Both vegetables and fruitare excellent sources of vitamins and arerecommended in place of the equivalentpure supplements. It is therefore an issue ofconsiderable concern that in the developedcountries much of this advice is ignored.

11-Mar-03 19




Food and Cancer

Different foods have been implicated inboth the induction and prevention of cancer.Foods contaminated with N-nitrosoamines,PCBs and dioxins have all been associatedwith the precipitation of cancer, and theepidemiological evidence seems convincing.However, momentum is gathering over thebenefits of various foods in the prevention ofcancer. The importance of this role may begauged by the publication of four papers in asingle recent issue of the Journal of Nutrition(Table 19.16). Tomatoes, green leafy vegeta-bles, certain mushrooms and soya and wheyproteins are all attributed with compoundsthat offer protection. It should be emphasizedthat the list in Table 19.16 is not comprehen-sive and other putative anticancer agents arethought to occur in vegetables. These includelycopene, phyto-oestrogens, glucosinolatesand S-methylcysteine sulphoxide amongmany others (see Kinghorn and Kennelly,1997).

Action Points

Unless remedial action is implementedurgently and on an international scale, con-sumers can look forward to a period of sus-tained food safety scares over many years.There are few quick-fix options. The follow-ing list is not exhaustive but should help inkick-starting the process of food safetyimprovements.

• Improved standards of hygiene must beimplemented and monitored regularly

in all cutting plants, butchers, cateringestablishments, hospitals and homes forthe elderly.

• Steps must be taken to reduce the micro-bial risks associated with poultry andseafood since, in unadulterated forms,these foods can constitute a valuablecomponent of reduced-fat diets.

• It is necessary to undertake a detailedreview of the justification for and needfor pesticides and fertilizers in agri-culture and horticulture.

• Although levels of individual pesticidesare generally below the MRLs and RLs,there is at present no way to predictthe human health implications of thecombined effects of several pesticidesand other environmental contaminantsoccurring in our food. The extent towhich these contaminants may induceadditive or synergistic effects willrequire the development of novel andmore sophisticated methodologies asconventional assays have failed toprovide satisfactory clues.

• The use of additives in foods andbeverages must be reduced to thevery minimum. In this regard, a culturechange may be necessary among foodtechnologists and manufacturers asconsumers begin to demand that foodought to be a source of nutrients (and, ofcourse, pleasure) and not a vehicle foradministration of chemicals and otherman-made molecules.

• The use of antibiotics, banned drugs andother compounds in animal productionmust cease and concerted action must betaken on a worldwide basis.


11-Mar-03 19

Vegetables Cancer types Putative anti-cancer agents Reference

CruciferousvegetablesLeafy greenvegetables andedible brown algaeWhite buttonmushroomsSoya

Oestrogen-related,e.g. cervical cancerProstate cancer

Oestrogen-related,e.g. breast cancerLiver and breast cancer

Indole-3-carbinol

Neoxanthin and fucoxanthin

Various phytochemicals inhibitingaromatase activityProteins (and phyto-oestrogens)

Chen et al. (2001)

Kotake-Nara et al.(2001)

Grube et al. (2001)

Rowlands et al. (2001)

Table 19.16. Vegetables and cancer prevention as exemplified in four papers published in a singleissue of the Journal of Nutrition.



• Improved standards of hygiene must beimplemented on livestock farms. Muchstill needs to be accomplished in thisrespect even in developed countries.Antibiotics and probiotics should not beused as substitutes for good hygiene.

• The use of catering waste and animalby-products as livestock feed must notbe permitted.

• The use of meat and bone meal as animalfeed in non-EU countries should bediscontinued as soon as possible.

• Steps must be taken to reduce envir-onmental pollution, particularly inagriculture.

• All governments must invest in educa-tion and training, research and monitor-ing services. In the long run, it would becheaper to underpin these services thanto pay compensation to consumers. Careof consumers affected by food poisoningand loss of productivity in the workplacecaused by such illness would add to thefinancial burden.

• Efforts must be redoubled to ensure thatthose involved in monitoring have theappropriate tools. In this respect, thedevelopment of diagnostics must begiven the highest priority.

• All the above action points should beaccompanied by measures to de-politicize food safety issues. Politicianspatently have failed to allay consumerfears in all the recent food scares inEurope, and there is little reason tobelieve that they have learned from pastmistakes. In this book, we have been atpains to emphasize that food safety issuescan only be addressed by underpinningfood policy with appropriate scientificprocesses and sound legislation.

References

Advisory Committee on the Microbiological Safetyof Food (2001) Second Report on Salmonella inEggs. HMSO, Norwich.

Aquino, M.H.C., Filgueira, A.L.L., Ferriera, M.C.S.,Oliveira, S.S., Bastos, M.C. and Tibana, A. (2002)Antimicrobial resistance and plasmid profilesof Campylobacter jejuni and Campylobacter coli

from human and animal sources. Letters inApplied Microbiology 34, 149–153.

Beresford, M.R., Andrew, P.W. and Shama,G. (2001) Listeria monocytogenes adheres tomany materials in food-processing environ-ments. Journal of Applied Microbiology 90,1000–1005.

Bolton, D.J., Catarame, T., Byrne, C., Sheridan, J.J.,McDowell, D.A. and Blair, I.S. (2002) Theineffectiveness of organic acids, freezing andpulsed electric fields to control Escherichia coliO157:H7 in beef burgers. Letters in AppliedMicrobiology 34, 149–153.

Cano, D.A., Pucciarelli, M.G., Portillo, F.G. andCasadesus, J. (2002) Role of the RecBCD recom-bination pathway in Salmonella virulence.Journal of Bacteriology 184, 592–595.

Centre for Environment, Fisheries and AquacultureScience (2001) Radioactivity in Food and the Envi-ronment, 2000. Crown Copyright, London.

Chasseignaux, E., Toquin, M.T., Ragimbeau, C.,Salvat, G., Colin, P. and Ermel, G. (2001) Molec-ular epidemiology of Listeria monocytogenesisolates collected from the environment, rawmeat and raw products in two poultry- andpork-processing plants. Journal of AppliedMicrobiology 91, 888–899.

Chen, B.H. and Chen, Y.C. (2001) Formation ofpolycyclic aromatic hydrocarbons in thesmoke from heated model lipids and foodlipids. Journal of Agricultural and Food Chemistry49, 5238–5243.

Chen, D.Z., Qi, M., Auborn, K.J. and Carter,T.H. (2001) Indole-3-carbinol and diindolyl-methane induce apoptosis of human cervicalcancer cells and in murine HPV16-transgenicpreneoplastic cervical epithelium. Journal ofNutrition 131, 3294–3302.

Devesa, V., Martinez, A., Suner, M.A., Velez, D.,Almeda, C. and Montoro, R. (2001) Effect ofcooking temperatures on chemical changesin species of organic arsenic in seafood.Journal of Agricultural and Food Chemistry 49,2272–2276.

Dickman, M.D., Leung, C.K.M. and Leong, M.K.H.(1998) Hong Kong male subfertility links tomercury in human hair and fish. Science of theTotal Environment 214, 165–174.

D’Mello, J.P.F. (1991) Antigenic proteins. In:D’Mello, J.P.F., Duffus, C.M and Duffus, J.H.(eds) Toxic Substances in Crop Plants. RoyalSociety of Chemistry, Cambridge, pp. 107–125.

D’Mello, J.P.F. and Macdonald, A.M.C. (1998)Fungal toxins as disease elicitors. In: Rose,J. (ed.) Environmental Toxicology: CurrentDevelopments. Gordon and Breach SciencePublishers, Amsterdam, pp. 253–289.

11-Mar-03 19




Dunny, G.M. (2002) Group effort in toxin synthesis.Nature 415, 33–34.

Food Safety Information Bulletin (1999) Ministry ofAgriculture, Fisheries and Food No. 112.London.

Food Standards Agency (2000a) Food StandardsAgency News No. 2. London.

Food Standards Agency (2000b) Food StandardsAgency News No. 3. London.

Food Standards Agency (2001a) Food StandardsAgency News No. 4. London.

Food Standards Agency (2001b) Food StandardsAgency News No. 5. London.

Food Standards Agency (2001c) Food StandardsAgency News No. 7. London.

Food Standards Agency (2001d) Food StandardsAgency News No. 10. London.

Food Standards Agency (2001e) Food StandardsAgency News No. 11. London.

Food Standards Agency (2001f) Food StandardsAgency News No. 12. London.

Food Standards Agency (2001g) Food StandardsAgency News No. 13. London.

Food Standards Agency (2001h) Food StandardsAgency News No. 14. London.

Food Standards Agency (2002) Food Survey Informa-tion Sheet No. 21/02. London.

Giovannacci, I., Queguiner, S., Ragimbeau, C.,Salvat, G., Vendeuvre, J.L., Carlier, V. andErmel, G. (2001) Tracing of Salmonella spp.in two pork slaughter and cutting plantsusing serotyping and macrorestrictiongenoyping. Journal of Applied Microbiology 90,131–147.

Gonzalez, J.C., Fontal, O.I., Vieytes, M.R., Vieites,J.M. and Botana, L.M. (2002) Basis for a newprocedure to eliminate diarrheic shellfishtoxins from a contaminated matrix. Journal ofAgriculture and Food Chemistry 50, 400–405.

Grandjean, P., Weihe, P., Ahite, R.F., Debes, F.,Araki, S., Yokoyama, K., Murata, K., Sorensen,N., Dahl, R. and Jorgensen, J. (1997) Cognitivedeficit in 7-year-old children with prenatalexposure to methylmercury. Neurotoxicologyand Teratology 19, 417–428.

Grube, B.J., Eng, E.T., Kao, Y.C., Kwon, A. andChen, S. (2001) White button mushroomphytochemicals inhibit aromatase activityand breast cancer cell proliferation. Journal ofNutrition 131, 3288–3293.

Haorah, J., Zhou, L., Wang, X., Xu, G. and Mirvish,S.S. (2001) Determination of total N-nitrosocompounds and their precursors in frank-furters, fresh meat, dried salted fish, sauces,tobacco and tobacco smoke particulates.Journal of Agricultural and Food Chemistry 49,6068–6078.

Hara-Kudo, Y., Nishina, T., Nakawaga, H.,Konuma, H., Hasegawa, J. and Kumagai, S.(2001) Improved method for detection of Vibrioparahaemolyticus in seafood. Applied and Envi-ronmental Microbiology 67, 5819–5823.

Harrison, W.A., Griffith, C.J., Tennant, D. andPeters, A.C. (2001) Incidence of Campylobacterand Salmonella isolated from retail chicken andassociated packaging in South Wales. Letters inApplied Microbiology 33, 450–454.

Heuer, O.E., Pedersen, K., Andersen, J.S. andMadsen, M. (2001) Prevalence and anti-microbial susceptibility of thermophilicCampylobacter in organic and conventionalbroiler flocks. Letters in Applied Microbiology33, 269–274.

Joint Food Safety and Standards Group (1998) FoodLaw. Stationery Office Publications Centre,London.

Jukes, D.J. (1993) Food Legislation of the UK: a ConciseGuide. Butterworth Heinemann, Oxford.

Kalmokoff, M.L., Austin, J.W., Wan, X.-D., Sanders,G., Banerjee, S. and Farber, J.M. (2001) Adsorp-tion, attachment and biofilm formation amongisolates of Listeria monocytogenes using modelconditions. Journal of Applied Microbiology 91,725–734.

Kinghorn, A.D. and Kennelly, E.J. (1997) Medicinalapplications of plant toxicants. In: D’Mello,J.P.F. (ed.) Handbook of Plant and FungalToxicants. CRC Press, Boca Raton, Florida,pp. 255–268.

Kingsley, D.H. and Richards, G.P. (2001) Rapidand efficient extraction method for reversetranscription–PCR detection of hepatitisA and Norwalk-like viruses in shellfish.Applied and Environmental Microbiology 67,4152–4157.

Kotake-Nara, E., Kushiro, M., Zhang, H.,Sugawara, T., Miyashita, K. and Nagao, A.(2001) Carotenoids affect proliferation ofhuman prostate cancer cells. Journal ofNutrition 131, 3303–3306.

Li, Y., Brackett, R.E., Chen, J. and Beuchat, L.R.(2002) Mild heat treatment of lettuce enhancesgrowth of Listeria monocytogenes during subse-quent storage at 5°C or 15°C. Journal of AppliedMicrobiology 92, 269–275.

Lindstrom, M., Keto, R., Markulla, A., Nevas, M.,Hielm, S. and Korkeala, H. (2001) MultiplexPCR assay for detection and identification ofClostridium botulinum types A, B, E and F infood and faecal material. Applied and Environ-mental Microbiology 67, 5694–5699.

McGee, P., Bolton, D.J., Sheridan, J.J., Earley, B. andLeonard, N. (2001) The survival of Escherichiacoli O157:H7 in slurry from cattle fed different


11-Mar-03 19



diets. Letters in Applied Microbiology 32,152–155.

Mead, P.S., Slutsker, L., Dietz, L.F., McGaig, J.S.,Bresee, J.S., Shapiro, C., Griffin, P.M. andTauxe, R.V. (1999) Food-related illness anddeath in the United States. Emergency andInfectious Diseases 5, 607–625.

Ministry of Agriculture, Fisheries and Food (1992a)Report of the Working Party on Pesticide Residues:1988–90. Food Surveillance Paper No. 34. HMSO,London.

Ministry of Agriculture, Fisheries and Food (1992b)Nitrate, Nitrite and N-nitroso Compounds in Food.The Thirty-second Report of the Steering Groupon Chemical Aspects of Food Surveillance. FoodSurveillance Paper No. 32. HMSO, London.

Ministry of Agriculture, Fisheries and Food (1992c)Dioxins in Food. The Thirty-first Report ofthe Steering Group on Chemical Aspects of FoodSurveillance. Food Surveillance Paper No. 31.HMSO, London.

Ministry of Agriculture, Fisheries and Food (1994)Radionuclides in Foods. The Forty-third Report ofthe Steering Group on Chemical Aspects of FoodSurveillance. Food Surveillance Paper No. 43.HMSO, London.

Moorhead, S.M. and Dykes, G.A. (2002) Survival ofCampylobacter jejuni on beef trimmings duringfreezing and frozen storage. Letters in AppliedMicrobiology 34, 72–76.

Munoz, O., Diaz, O.P., Leyton, I., Nunez, N.,Devesa, V., Suner, M.A., Velez, D. andMontoro, R. (2002) Vegetables collected in thecultivated Andean area of Northern Chile:total and inorganic arsenic contents in rawvegetables. Journal of Agricultural and FoodChemistry 50, 642–647.

Patandin, S., Lanting, C.I., Mulder, P.G.H., Boersma,E.R., Sauer, P.J.J. and Weisglas-Kuperus, N.(1999) Effects of environmental exposure topolychlorinated biphenyls and dioxins oncognitive abilities in Dutch children at 42months of age. Journal of Pediatrics 134, 33–41.

Pennington Group (1997) Report on the CircumstancesLeading to the 1996 Outbreak with E. coli O157 inCentral Scotland, the Implications for Food Safetyand the Lessons to be Learned. Stationery Office,Edinburgh.

Petterson, D.S., Harris, D.J. and Allen, D.G. (1991)Alkaloids. In: D’Mello, J.P.F., Duffus, C.Mand Duffus, J.H. (eds) Toxic Substances inCrop Plants. Royal Society of Chemistry,Cambridge, pp. 148–179.

Quist, D. and Chapela, I.H. (2001) Transgenic DNAintrogressed into traditional maize landracesin Oaxaca, Mexico. Nature 414, 541–543.

Robey, M., Benito, A., Hutson, R.H., Pascual, C.,Park, S.F. and Mackey, B.M. (2001) Variationin resistance to high hydrostatic pressure andrpoS heterogeneity in natural isolates ofEscherichia coli O157:H7. Applied and Environ-mental Microbiology 67, 4901–4907.

Rowlands, J.C., He, L., Hakkak, R., Ronis, M.J.J. andBadger, T.M. (2001) Soy and whey proteinsdowngrade DMBA-induced liver and mam-mary gland CYP1 expression in female rats.Journal of Nutrition 131, 3281–3287.

Sagoo, S.K., Little, C.L. and Mitchell, R.T. (2001) Themicrobiological examination of ready-to-eatorganic vegetables from retail establishementsin the United Kingdom. Letters in AppliedMicrobiology 33, 434–439.

Sampson, H. and Burks, A.W. (1996) Mechanisms offood allergy. Annual Review of Nutrition 16,161–177.

Schroeder, C.M., Zhao, C., DebRoy, C., Torcolini, J.,Zhao, S., White, D.G., Wagner, D.D.,McDermott, P.F., Walker, R.D. and Meng, J.(2002) Antimicrobial resistance of Escherichiacoli O157 isolated from humans, cattle, swineand food. Applied and Environmental Micro-biology 68, 576–581.

Seymour, I.J. and Appleton, H. (2001) Foodborneviruses and fresh produce. Journal of AppliedMicrobiology 91, 759–773.

Silva, J., Carvalho, A.S., Teixeira, P. and Gibbs, P.A.(2002) Bacteriocin production by spray-driedlactic acid bacteria. Letters in Applied Micro-biology 34, 77–81.

Simonato, B., Pasini, G., Giannattasio, M., Peruffo,A.D.B., De Lazzari, F. and Curioni, A. (2001)Food allergy to wheat products: the effect ofbread baking and in vitro digestion on wheatallergenic proteins. A study with bread,dough, crumb and crust. Journal of Agriculturaland Food Chemistry 49, 5668–5673.

Solomon, E.B., Yaron, S. and Matthews, K.R. (2002)Transmission of Escherichia coli O157:H7 fromcontaminated manure and irrigation water tolettuce plant tissue and its subsequent internal-ization. Applied and Environmental Microbiology68, 397–400.

Suzuki, T., Itakura, J., Watanabe, M., Ohta, M.,Sato, Y. and Yamaya, Y. (2002) Inactivationof staphylococcal enterotoxin-A with anelectrolyzed anodic solution. Journal ofAgriculture and Food Chemistry 50, 230–234.

Taormina, P.J. and Benchat, L.R. (2002) Survivalof Listeria monocytogenes in commercial food-processing equipment cleaning solutions andsubsequent sensitivity to sanitizers and heat.Journal of Applied Microbiology 92, 71–80.

11-Mar-03 19




Van der Stegen, G.H.D., Essens, P.J.M. and vander Lijn, J. (2001) Effect of roasting condi-tions on reduction of ochratoxin A in coffee.Journal of Agricultural and Food Chemistry 49,4713–4715.

Vieths, S. (1997) Allergens in fruits and vegetables.In: D’Mello, J.P.F. (ed.) Handbook of Plant andFungal Toxicants. CRC Press, Boca Raton,Florida, pp. 157–174.

Walsh, D., Duffy, G., Sheridan, J.J., Blair, I.S. andMcDowell, D.A. (2001) Antibiotic resistanceamong Listeria, including Listeria monocyto-genes, in retail foods. Journal of Applied Micro-biology 90, 517–522.

WHO (1994) Safety and Nutritional Adequacy ofIrradiated Foods. World Health Organization,Geneva.

Wyn-Jones, A.P. and Sellwood, J. (2001) Entericviruses in the aquatic environment. Journal ofApplied Microbiology 91, 945–962.

Zareena, A.V., Variyar, P.S., Gholap, A.S.,Bongirwar, D.R. and Wani, A.M. (2001) Chemi-cal investigation of gamma-irradiated saffron(Crocus sativus L.). Journal of Agricultural andFood Chemistry 49, 687–691.

Zhao, C., Ge, B., De Villena, J., Sudler, R., Yeh,E., Zhao, S., White, D.G., Wagner, D. andMeng, J. (2001) Prevalence of Campylobacterspp., Escherichia coli and Salmonella serovarsin retail chicken, turkey, pork and beeffrom the Greater Washington D.C. area.Applied and Environmental Microbiology 67,5431–5436.


11-Mar-03 19



Index

additivesacidulants 247–248

acetic acid 248citric acid 248lactic acid 248

antioxidants 246–247butylated hydroxy anisole (BHA) 247butylated hydroxy toluene (BHT) 247

and asthma 256–264benzoates 262–263butylated hydroxy anisole (BHA) 263butylated hydroxy toluene (BHT) 263dyes 260monosodium glutamate (MSG) 263other chemicals 263sulphite challenge 257–258sulphites 260–262tartrazine 258–260

colourants 255–256annatto 255–256carmine 256

colour fixatives 247sodium nitrate 247sodium nitrite 247

distribution in foods 248–249benzoic acid 248sorbic acid 248

dyes 238brilliant blue 238erythrosin 238tartrazine 238

food colours 238intake 248–249

acceptable daily intakes (ADIs) 248–249monosodium glutamate (MSG) 245–246

Chinese restaurant syndrome 245–246preservatives 244–245

benzoic acid 244

parabens 244sorbic acid 244–245

regulations 421salt 415structures 237sugar 415sulphites 238–244, 253–254

in foods and beverages 240–241mechanisms of sensitivity 241restrictions 241and urticaria 253–254

sweeteners 246acesulphame potassium 246aspartame 246, 255, 415saccharin 246

and urticaria 249–256acetylsalicylic acid 250, 252aspartame 255benzoates 254butylated hydroxy anisole (BHA) 251, 252,

255butylated hydroxy toluene (BHT) 251, 252,

255design considerations 250–251maximum doses 251monosodium glutamate (MSG) 255parabens 254patients 249–255sodium benzoate 251sulphites 253–254sunset yellow 253tartrazine 253

aflatoxin(s)B1 (AFB1) 67, 70–71, 83B2 (AFB2) 67, 71, 83combinations 81co-occurrence 75exposure 78–79

439

11-Mar-03



aflatoxin(s) continuedG1 (AFG1) 67, 71, 83G2 (AFG2) 67, 71, 83in maize 70–71, 410in nuts 70–71, 410, 422regulations 82–83, 85–86

compliance 85–86remedial measures 87tolerable daily intakes (TDIs) 81toxicology 76, 77

Agaricus augustuscadmium 202

AgrobacteriumGM technology 331, 333, 334, 336, 353

algaetoxins 47–60

alkaloids 14–16, 66, 67, 70allergenic potential 341–342, 367, 368allergens

alerts 428in GM foods 366–368and human health 8–9in nuts 8–9, 428–429

allergieschromium 205, 206consumer advice 432eggs 428genetically modified (GM) foods 341–342,

366–368nickel 210peanuts 8–9, 417, 428, 432

amnesic shellfish poisoning (ASP) 55–59antibiotic

resistance 348, 409antimicrobial agents

fluoroquinolones 300–303Campylobacter 301–303food safety concerns 301–303

penicillin 295–297sulphamethazine 298–300tetracycline 297–298

antinutrients 356, 357, 360, 361antioxidants

butylated hydroxy anisole (BHA) 247, 251,252, 255, 263

butylated hydroxy toluene (BHT) 247, 251,252, 255, 263

apples/apple productspatulin 66, 68, 72, 73, 85, 86

apricotspesticide residues 118

arsenicregulations 412, 422in spinach 412

artichokesresidues

pesticides 114–115

aspartame 246, 255, 415Aspergillus spp.

mycotoxins 66, 67, 70–72, 75–76, 77, 78–82,83, 85, 87

asthma 256–264

Bacillus subtilis 330Bacillus thuringiensis (Bt) 331, 332, 341, 355, 360bacteria 25–43, 301–303, 331, 332, 341, 355, 360,

409, 410, 413, 414, 417, 418beef

bovine spongiform encephalopathy (BSE)legislation 424–425

contaminants (overview) 413–414dioxins 160polycyclic aromatic hydrocarbons

(PAHs) 182, 183benzoates 254, 262bisphenol A (BA) 280–281, 416bisphenol A diglycidylether (BADGE) 280–281bisphenol F diglycidylether (BFDGE) 281bovine spongiform encephalopathy (BSE)

control 424–425detection 320genetics 320legislation 324, 424–425‘over 30 month’ rule 424research programmes 431risk factors 322–323in sheep 324specified risk material (SRM) 424–426tissue distribution 321transmission 322

brain tumours 219–226bread 410, 415, 417Bt maize 359Bt potato 360–361Bt tomato 366butter

phthalates 285butylated hydroxyanisole (BHA) 247, 251, 252,

255, 263butylated hydroxytoluene (BHT) 247, 251, 252,

255, 263

cadmiumclinical effects 203distribution in foods 201–202, 204intakes 202–203maximum levels 204pollution 201risk assessment 203risk management 203–204toxicity 203uptake and metabolism 203

11-Mar-03 20

440 Index



uses 201caffeine 416, 432Campylobacter spp.

in chicken 414, 417fluoroquinolone resistance 301–303in food poisoning 410foodborne disease 30, 31, 33, 35in meat 413–414, 417in milk 33survival 418

cancer 17, 77, 78, 80, 134–135, 165, 187–191,226–230, 231, 385–386, 418, 433

cassavaand multiorgan disease 11–13

cattle productsbovine spongiform encephalopathy

(BSE)-infected 322celery

pesticides 114, 411cereal

grains 65–88, 179, 180, 410–411aflatoxins 66, 67, 71, 410citrinin 66, 67, 68contaminants (overview) 410–411deoxynivalenol (DON) 73, 82, 84ergot alkaloids 66, 67, 70fumonisins 74–75mycotoxins 66–75, 77, 83, 84, 87ochratoxin A (OTA) 66, 67, 68, 72, 82, 84polychlorinated biphenyls (PCBs) 130polycyclic aromatic hydrocarbons (PAHs)

179, 180trichothecenes 66, 68–69, 73zearalenone 66, 73–74

chaconine 15, 349, 355cheese

Listeria monocytogenes 414phthalates 285radionuclides 401

chickencontaminants (overview) 413–414fluoroquinolone-resistant Campylobacter

jejuni 301–303chloropropanols 416chocolate

chromium 204nickel 209

chromiumclinical effects 205–206distribution in foods 204–205intakes 205, 206risk assessment 206risk management 206in sugar metabolism 205toxicity 205uptake and metabolism 205uses 204

Claviceps purpurea 66, 67, 70clenbuterol

distribution in foods 304health concerns 304–305outbreaks of foodborne illness 304–305pharmacokinetics/pharmacodynamics

303–304toxicity 304–305

acute 305chronic 305

coffeecaffeine 416, 432ochratoxin A (OTA) 66, 72, 84, 416polycyclic aromatic hydrocarbons (PAHs)

180constructs 334, 336, 348, 369control

bacterial foodborne diseases 41mycotoxin contamination 81–87

creamphthalates 285

137Csfoods 378, 380, 381, 382–385, 392, 396, 400,

404intakes 381meat 379, 381, 383milk 379, 380, 381, 383, 384, 396mobility 382, 383prediction of contamination 383–385risk assessment 385transfer to foods 382–385

deoxynivalenol (DON)cereal grains

incidence 73, 75draft regulations 82, 84tolerable daily intakes (TDIs) 81toxicology 76

deoxyribonucleic acid (DNA)constructs 334digestive fate 341–343and fluoroquinolones 301manipulation 331transgenic 331, 333

diarrhoeacauses 33, 36, 54, 203, 205, 210, 297, 303

cadmium 203chromium 205fluoroquinolones 303foodborne bacteria 33, 36nickel 210penicillin 297shellfish toxins 54

dichloro diphenyl trichloroethane (DDT) 91–94dioxins see polychlorinated dibenzo-p-dioxins

(PCDDs)

Index 441

11-Mar-03 20



domoic acid (DA) 55–58dried fruit

ochratoxin A (OTA) 72, 84pesticide residues 118

egg(s)bacteria

Salmonella serovars 33, 414–415Streptococcus pyogenes 33

contaminants (overview) 414–415dioxins 160, 163, 164polycyclic aromatic hydrocarbons (PAHs)

183proteins 416

enforcementfood safety regulations 424–426

ergot alkaloids 67Escherichia coli

E. coli O157 28, 30, 31, 32, 33, 35, 36, 37, 38,40, 410, 412

foodborne disease 28, 30, 31, 32, 35, 36, 37,38, 40, 410

in meat 32, 33research 430

feedingstuffs 82, 83, 162–164, 171, 324, 424–425fish

Clostridium botulinum 32137Cs 381, 383, 384dioxins 158, 159, 160, 162, 170, 171Listeria monocytogenes 32mercury 206–207, 208, 412–413polychlorinated biphenyls (PCBs) 129, 145,

413polycyclic aromatic hydrocarbons (PAHs)

181–182principal contaminants (overview)

412–413Salmonella serovars 33salted 229, 230

cancer risk 229, 230smoked 224see also shellfish

fluoroquinolonesfood safety concerns 301–303

resistant bacteria 301–303pharmacokinetics/pharmacodynamics

300–301toxicity 303

Food Safety Act 1990 (Great Britain) 421–424fruit(s)

allergens 417dioxins 160pesticide residues 114, 115–119, 411, 429see also dried fruit

fumonisinscancer 77, 78co-occurrence 75general aspects 66, 69, 76, 77maize 74–75, 410

fungifoodborne 66, 67, 68–70

Aspergillus spp. 66, 67, 86Claviceps purpurea 66, 67, 70Fusarium spp. 66, 68–70, 73, 75Penicillium spp. 66, 67

fungicidesanilinopyrimidines 103, 104benzimidazoles 101–102dicarboximides 101–102dithiocarbamates 100–102efficacy

mycotoxin control 86inorganic 100residues 119

grapes 119wine 119–120

strobilurines 103, 104toxicology 101, 104triazoles 102, 104

Fusarium spp.mycotoxins 66, 68–70, 73–74, 75, 76, 81, 82,

84, 86co-occurrence 75deoxynivalenol (DON) 66, 68, 73, 75, 76, 81,

82, 84, 86fumonisins 66, 69, 74–75, 78headblight 68, 86moniliformin 69–70toxicology 76–77trichothecenes 66, 68–69, 73zearalenone 66, 69, 73–74, 75, 76, 81

genemarker 348recombinant product 358, 359, 366reporter 347

genetically modified (GM) foodsallergenicity 341–342, 366–368evaluation 369–370fate of consumed DNA 341–343gene transfer from plants to bacteria 343–344generic applications 330–331

β-carotene in rice 331herbicide tolerance 331fermentation microorganisms 330rennet production 330riboflavin manufacture 330tomato fruit 330

human health effects 347–370impact of introduced trait 340–341

11-Mar-03 20

442 Index



maize 352–353, 356, 359monitoring 344peas 359–360potatoes 355, 360–365, 367, 369

Bt potato 360–361GNA potato 361–365

recommendations 368–369rice 355safety assessment 332, 336, 337, 430, 431selectable marker genes 333–336soybean 353, 354, 355, 356–359substantial equivalence 336–340, 349,

353–356, 368technology 331–333tomato 348–352, 362–366

grapesresidues

fungicides 119pesticides 411, 429

Gymnodinium catenatum 48

heavy metalsanalysis 199–201

certified reference materials (CRMs) 200effects of quality assurance 201error 200proficiency testing 201uncertainty 200

cadmium 201–204clinical effects 203distribution in foods 201–202, 204intakes 202–203maximum levels 204risk assessment 203risk management 203–204toxicity 203uptake and metabolism 203uses 201

chromium 204–206clinical effects 205–206distribution in foods 204–205intakes 205, 206risk assessment 206risk management 206in sugar metabolism 205toxicity 205uptake and metabolism 205uses 204

definition 199‘itai-itai’ disease 201lead 210–213

clinical effects 211distribution in foods 210–211, 213intakes 211–212legislation 213risk assessment 212

risk management 212–213toxicity 211uptake and metabolism 211uses 210

mercury 206–208, 412–413clinical effects 206–208distribution in foods 206–207, 412–413exposure 412intakes 207–208legislation 208risk assessment 208risk management 208toxicity 207–208uptake and metabolism 207uses 206

in mushrooms 202nature of compounds 199nickel 208–210

clinical effects 209–210distribution in foods 208–209intakes 209risk assessment 210toxicity 209–210uptake and metabolism 209uses 208

properties 200quality assurance 199–201

hepatitis A virus (HAV) 410, 412–413herbicide(s)

amides 107, 108amino acid derivatives 106, 108bipyridyls 105, 106classification 104–105dinitroanilines 106, 108phenoxyalkanoic acids 105, 106resistant maize 352–353resistant soybean 353–355, 356–359sulphonylureas 107, 108toxicology 109–110triazines 107, 108ureas 107, 108

hormonesnaturally occurring 305–306synthetic 306

human milkaflatoxins 79dioxins 160, 161, 170ochratoxin A 80polychlorinated biphenyls (PCBs) 144, 145,

161

131I 374, 375, 376, 378, 379, 392, 396, 397, 399, 401,404

insecticides 92–100benzoylureas 97, 98carbamates 94, 96

Index 443

11-Mar-03 20



insecticides continuedorganochlorines 92–94organophosphates 94, 95pyrethroids 96–97toxicology 97–100

intakesacceptable daily intakes (ADIs)

additives 248–249food contact compounds 278pesticides 98, 99, 101, 109, 112, 113, 120, 122veterinary residues 307, 308

cadmium 202–203chromium 205, 206cured meats 220–223, 225, 226dietary 161–163, 169dioxins 161–162, 169lead 211–212mercury 207–208nickel 209in pregnancy 220, 221, 222, 223

cured meats 220–223nitrates 221, 222nitrites 221, 222, 223nitrosoamines 221, 223

tolerable daily intakes (TDIs)dioxins 169food contact compounds 278–279mycotoxins 81polychlorinated biphenyls (PCBs) 143

international toxic equivalent (I-TEQ) 154–163,168, 169, 171–172

international toxicity equivalency factors (I-TEF)168, 169

intolerancefood 416lactose 416

labellingfood 419–420regulations 419–420

lacquers 272, 280–281lead

clinical effects 211contact with food 279–280distribution in foods 210–211, 213intakes 211–212legislation 213risk assessment 212risk management 212–213toxicity 211uptake and metabolism 211uses 210

lectinsactivity 7in cereal foods 8and coeliac sprue 8

effects 7–8haemagglutination 7in kidney beans 7in potatoes (GM) 361–365

legislationcompliance

compounds from food contact materials286–287

food safety regulations 424–426, 427food

additives 421bovine spongiform encephalopathy (BSE)

control 424–425compliance 85–86, 286–287, 424–426, 427contact compounds and materials 275–277contaminants 420–422dioxins 170–172directives 420enforcement 424–426general 420–422health 421hygiene 421labelling 420–422lead 212–213medical 421mercury 208mycotoxins 82–83, 84polychlorinated biphenyls (PCBs) 139–140,

146–148polycyclic aromatic hydrocarbons (PAHs)

194–195provisions 421–423radionuclides 404–405

lettuceListeria monocytogenes 411residues

nitrates 217pesticides 114–115, 411polycyclic aromatic hydrocarbons (PAHs)

179, 180Listeria monocytogenes

in cheese 414and foodborne disease 30, 31, 36, 40, 410in meat 32, 417in milk 32, 414in salads 32in seafood 32survival 417, 418in vegetables 411–412

maizegenetically modified (GM) 352–353, 356,

359analysis 352poultry study 359toxicity study in rats 352–353

11-Mar-03 20

444 Index



mycotoxinsaflatoxins 71, 410deoxynivalenol (DON) 73fumonisins 74, 410multiple contaminants 75

materialscontact with food 272–277

directives 275–277glass 272lacquers and coatings 272, 280–281legislation 275–277metals 272paper and cardboard 272, 273–274plastics 272–273, 281–286rubber 272simulants 276–277

maximum permitted levels (MPLs)radionuclides 378, 379

baby foods 378, 404dairy produce 378, 379

maximum residue limits (MRLs)penicillin 296pesticides 112, 113, 120, 121tetracycline 298

meatbacteria 32, 33, 34, 413–414, 417

Campylobacter spp. 413–414, 417Escherichia coli 32, 33, 413–414Listeria monocytogenes 32, 417Salmonella serovars 33, 413–414Yersinia spp. 34

beefcontaminants (overview) 414bovine spongiform encephalopathy (BSE)

414, 424–425, 431dioxins 160legislation 424–425

Campylobacter 413–414chicken 413–414

contaminants (overview) 413–414137Cs 379, 381, 383cured 217, 218, 220–223, 225, 226, 247

consumption 220–223, 225, 226nitrates 217, 247nitrites 217, 247N-nitrosodimethylamine (NDMA) 218

dioxins 158, 159, 160, 172, 414Escherichia coli 32, 33, 413–414lamb 41mechanically recovered

safety 424nitrates 217, 247nitrites 217, 247nitrosoamines 217, 218polychlorinated biphenyls (PCBs) 129polycyclic aromatic hydrocarbons (PAHs)

183

porkcontaminants (overview) 414dioxins 172

radionuclides 377, 379, 380, 381, 383, 414, 419safety 413–414, 424Salmonella 413–414storage 417turkey 413

mercuryclinical effects 207–208distribution in foods 206–207, 412–413exposure 412intakes 207–208legislation 208methyl form 206, 207, 208Minamata poisoning 206, 412risk assessment 208risk management 208toxicity 207–208uptake and metabolism 207uses 206

metals 199–213, 272migration

of food contact elements and compounds279–286

bisphenol A (BA) 280–281bisphenol A diglycidylether (BADGE)

280–281bisphenol F diglycidylether (BFDGE) 281isocyanates 283, 286lacquers 280–281lead 279–280phthalates 282–285plasticizers 282polyvinylchloride (PVC) 281–283primary aromatic amines 286vinylchloride monomer 277, 282

milkaflatoxins 67, 79, 81, 87, 414, 415bacteria

Brucella spp. 32Campylobacter jejuni 33Listeria monocytogenes 32Mycobacterium spp. 33Salmonella 32Shigella spp. 32

contaminants (overview) 414, 415cows

aflatoxin M1 67, 87dioxins 158, 159, 161, 172

137Cs 379, 380, 381, 383, 384, 396dioxins 158, 159, 161, 172, 414, 415human

dioxins 160, 161, 170mycotoxins 79, 80polychlorinated biphenyls (PCBs) 144, 145,

161

Index 445

11-Mar-03 20



milk continuedListeria monocytogenes 414, 415Mycobacterium spp. 33, 414, 415ochratoxin A (OTA) 80, 81pasteurization 415, 417phthalates 283–284polycyclic aromatic hydrocarbons (PAHs)

183radioiodine 379, 396, 399radionuclides 374, 379, 380, 383, 384, 392,

396, 399, 414, 415131I 374, 379, 396, 399

molecular profiling 338–340monitoring

actions 426–430Department of Environment, Food and Rural

Affairs (DEFRA) (UK) 427food contaminants 426–430Food Safety Agency (FSA) (UK) 427Meat Hygiene Service (UK) 427pesticides 120–121polycyclic aromatic hydrocarbons (PAHs)

191–194radionuclides 400–405resistant bacteria 309, 310

monosodium glutamate (MSG) 245–246, 255,263

mushroomscadmium 202radionuclides 401

mutationsgene 316, 318

mycotoxinsaflatoxins 66, 67, 70–72, 75–76, 77, 78–79,

81–82, 83, 85, 87of Alternaria spp. 70of Aspergillus spp. 66, 67, 70–72, 75–76, 77, 78,

79, 82, 83, 85, 87citreoviridin 68citrinin 67, 68, 72cyclopiazonic acid 67deoxynivalenol (DON) 73, 75, 76, 81, 82,

84distribution in foods 70–75, 410, 416, 422

aflatoxins 70–72, 410, 422co-occurrence 75ergot alkaloids 70fumonisins 74–75ochratoxin A (OTA) 72, 416patulin 72–73trichothecenes 66, 73zearalenone 73–74

ergot alkaloids 66, 67, 70of Fusarium spp. 66, 68–70

fumonisins 69–70, 74–75, 76, 77, 78moniliformin 69–70trichothecenes 66, 68–69, 73

zearalenone 66, 69, 73–74, 75, 76, 81human disorders 77–78

aflatoxicosis 77cancer 77, 78ergotism 77ochratoxicosis 77–78

human exposure 78–81aflatoxins 78–79combinations 81ochratoxin A (OTA) 79–81tolerable daily intakes (TDIs) 81

nature of compounds 66–70ochratoxin 66, 67, 72, 76, 77, 79–81, 416origin 66–70patulin 66, 68, 72–73, 85, 86of Penicillium spp. 66, 67, 68, 72–73, 79–81preventive strategies 86–87regulatory control 81–86

advisory directives 84compliance 85–86draft EU regulations 82, 84methodologies 84rationale 81sampling 84statutory instruments 82

remedial measures 87processing technologies 87

surveillance 85–86toxicology 76–77uptake and disposition 75–76zearalenone 73–74, 75, 76, 81

nickelclinical effects 209–210distribution in foods 208–209intakes 209risk assessment 210toxicity 209–210uptake and metabolism 209uses 208

nitratesand brain tumours 219–226cancer risk 227in cured meat 217, 247uptake 218

nitritesand brain tumours 219–226cancer risk 227–228in cured meat 217, 247human exposure 217, 418uptake 218

N-nitroso compounds (NOCs)cancer risk 217–231human exposure 217, 418risk assessment 218–219toxicity 218

11-Mar-03 20

446 Index



and tumours 220–226uptake 218

N-nitrosodimethylamine (NDMA)cancer risk 223, 225, 228, 231in fish 218processed meat 218

non-protein amino acids 4–6nuts

allergens 8–9, 410, 411, 431mycotoxins 66, 67, 70–71, 77–78, 82–83, 85,

87, 88aflatoxins 66, 67, 70–71, 78, 82, 83, 85, 87,

88, 410–411peanuts 9, 67, 70, 71, 77, 78, 81, 83, 85,

410–411pistachio 85, 87, 410–411

ochratoxin A (OTA)distribution in foods 72, 416human exposure 79–81ochratoxicosis 77–78regulations 82, 84surveillance

actions 85, 86tolerable daily intakes (TDIs) 81toxicology 76

okadaic acid (OA) 52–55olive oil

pesticide residues 118–119pomace oil 416safety alert 429

olivespesticide residues 114, 116, 117, 118

packaging 271, 273–274, 417, 422paper 272, 273–274pasteurization 415, 417patulin 66, 68, 72–73, 85, 86peaches

pesticide residues 115, 116peanuts

aflatoxins 67, 70, 71, 77, 78, 81, 83, 85,410–411

allergens 9, 410, 411peas

genetically modified (GM) 359–360inhibitors 359–360

pectenotoxins (PTXs) 52, 53penicillin

distribution in food 296pharmacokinetics/pharmacodynamics

295–296toxicity 296–297

acute 296–297chronic 297

Penicillium spp.mycotoxins 66, 67, 68, 72–73, 85, 86

pesticidesfood alerts 429formulation 110–111

liquids 110solids 110–111

fruit 114, 115–119, 429fungicides 100–104

anilinopyrimidines 103, 104benzimidazoles 101–102dicarboximides 101–102dithiocarbamates 100–102inorganic 100strobilurines 103, 104toxicology 101, 104triazoles 102, 104

herbicides 104–110amides 107, 108amino acid derivatives 106, 108bipyridyls 105, 106classification 104–105dinitroanilines 106, 108phenoxyalkanoic acids 105, 106sulphonylureas 107, 108toxicology 109–110triazines 107, 108ureas 107, 108

insecticides 92–100benzoylureas 97, 98carbamates 94, 96organochlorines 92–94organophosphates 94, 95pyrethroids 96–97toxicology 97–100

monitoring 120–121EU 120maximum residue limits (MRLs) 120, 121USA 120

processing effects 117–120dried fruit 118grapes and must 119olive oil 118–119wine 119–120

registration 111–113acceptable daily intakes (ADIs) 112, 113maximum residue limits (MRLs) 112, 113

residues in food 113–120, 411, 429, 431, 433disappearance rate 115–117initial deposit 113–115

risk assessment 121–123dietary pesticide exposure 122–123national diet estimate 121–122residue estimate 121

phthalates 282–285pistachio nuts

aflatoxins 71, 85

Index 447

11-Mar-03 20



plantalkaloids 14–16

carboline 16isoquinoline 16lupin 16potato 15pyrrolizidine 14–15solanum 15–16

allergens 8–9glycosides 9–14

in cassava 11–13and multiorgan disease 11–13convicine 14in cycads 10–11cycasin 10–11in fava beans 14and favism 14glucosinolates 13–14and goitre 12, 13–14and haemolytic anaemia 14and hydrogen cyanide (HCN) 11–13linamarin 10–11lotaustralin 10and neurodegeneration 10–11, 12–13and tropical diabetes mellitus 13vicine 14

lectins 7–8neurotoxins 2–3

precursors 2–3non-protein amino acids 3–6proteinase inhibitors 6–7proteins 6–9

plasticizers 282plastics

food contact 272–273, 276–277directives 276–277

plumspesticide residues 118

pollutionand food safety 418–419

lead 210, 211polycyclic aromatic hydrocarbons (PAHs)

194seafood 412–413

water quality 419polychlorinated biphenyls (PCBs)

absorption, metabolism and excretion130–132

and carcinogenesis 134–135, 138chemical structure 126clinical effects 135–138congeners 127–128, 129, 131, 132, 133, 134,

144, 145, 159, 168distribution in foods 128–130exposure 127, 129–130, 135–138, 143, 144,

145, 147, 148food tolerances 147

guidelines 172human milk 137, 145, 161legislation 139–140, 146–148

national perspectives 146–148lowest observed adverse effect level (LOAEL)

142, 147, 148no observable adverse effect level (NOAEL)

142, 143, 147properties 127–128risk assessment 138–139, 141–146

international perspectives 141–142process 142–143toxic equivalents (TEQs) 139toxicity equivalency factors (TEFs) 138–139

risk management 139–141toxicity and clinical effects 132–138

epidemiology studies, cancerous outcomes138

epidemiology studies, non-cancerousoutcomes 135–138

laboratory studies 133–135mechanisms 132

polychlorinated dibenzofurans (PCDFs) seepolychlorinated dibenzo-p-dioxins(PCDDs)

polychlorinated dibenzo-p-dioxins (PCDDs)cancer promotion 165carcinogenicity 166–167clinical effects 164–168distribution in foods 158–164

accidental contamination 162–164dietary intakes 161–162eggs 160, 163, 164fish 158, 159, 160, 162, 170, 171food consumption data 161fruit 160human milk 160, 161, 170meat 158, 159–160, 172, 414milk 158, 159, 161, 172, 414, 415milk products 159seafood 413vegetables 160

environmental impact 155–158exposure 153, 155, 157, 161, 162, 164, 166,

170, 171intakes 162kinetics 165legislation 170–172maternal transmission 164nature of compounds 153–154release inventories 154–155risk assessment 167–170risk management 170sources 153–158toxicity 164–170transfer to food 158uptake 164

11-Mar-03 20

448 Index



polycyclic aromatic hydrocarbons (PAHs)benzo(a)anthracene 176, 177, 180–183, 187,

188, 190benzo(a)pyrene 178, 180–183, 184–186, 188,

190, 416distribution in foods 179–183

beef 182, 183eggs 183fish 181, 182frankfurters 183meat 183methodology 179milk 183mixtures 179oils and fats 180, 181origin 179seafood 181–182vegetables 179–180

legislation 194–195metabolism 183–186

adducts 184, 185, 188benzo(a)pyrene 184–185conjugates 185mixed function oxidases 184, 185phase I 184, 186phase II 184, 186

nature of compounds 175–179characteristics 176classification 175–176, 189–190formula 176–179nomenclature 176–179properties 176–179regions of biological activity 176structures 176–179

risk assessment 191–194biomarkers 191–193DNA adducts 192–1931-hydroxypyrene 192monitoring 191–194

risk management 194toxicity 186–191

activation 188, 189bay region theory 188–189benzo(a)anthracene 186, 188, 190benzo(a)pyrene 187–188, 190carcinogenicity 187–191DNA adducts 188, 189, 191dose effects 187epoxides 185, 188experimental 187–191genotoxicity 187immune system 187mechanisms 188molecular geometry 189non-carcinogenic effects 186–187nucleic acid adducts 188potency equivalency factors (PEFs) 191

reproduction 186, 187toxicity equivalency factors (TEFs) 190–191tumorigenicity 187–188

uptake 183–184polyvinylchloride (PVC) 273, 280, 281–282potatoes

alkaloids 15–16genetically modified (GM) 355, 360–365, 366,

367, 369and Bt toxin 360–361chimeras 365glycinin-expressing 360lectin-expressing 361–365

preservativesbenzoic acid 244nitrates and nitrites 217parabens 244sorbic acid 244–245

prion diseasesagent 315–317clinical symptoms 323–324

humans 323sheep 323–324

distribution of infectivity 320–322cattle 320–321humans 321–322sheep 320–321

genetics 317–320bovine spongiform encephalopathy (BSE)

320human transmissible spongiform

encephalopathies (TSEs) 317–318sheep transmissible spongiform

encephalopathies (TSEs) 318–319legislation 324, 424–426risk factors 322–323transmissible spongiform encephalopathies

(TSEs) 315, 316, 317–319bovine spongiform encephalopathy (BSE)

315, 316strains 317variant Creutzfeldt–Jakob disease (vCJD)

315, 316transmission 322treatments 324

processingammoniation 87cereals 87coffee 87food

curing 217, 218, 220–223, 225, 226, 418freezing 418grilling 418irradiation 418pasteurization 415, 417smoking 224, 418thermal methods 417–418

Index 449

11-Mar-03 20



processing continuedand pesticide residues 117–120

Prorocentrum spp. 52–53proteinase inhibitors

in plants 6–7, 354, 357distribution 7potato 7serine-type 7soybean 354, 357

proteinsplants 6–9, 354, 357

allergens 8–9lectins 7–8proteinase inhibitors 6–7, 354, 357

see also prion proteinsproteomics 338–340, 348

radionuclidesatomic structure 392and cancer 385–386characteristics 396–398classification 395–396critical organs 396137Cs 377, 379–387, 396, 400, 401, 404decay 375elements and isotopes 392elimination 403–404entry routes 395, 396fission products 394, 396, 397in food chain 380–382, 393–395in foods 373–374, 376–387, 393–395, 419

animal products 379, 381, 395factors 395

genetic effects 403half-life 373, 375, 396, 397, 398, 399, 400131I 374, 379, 392, 396, 397, 399, 401, 404isotopes 373maximum permitted levels (MPLs) 378, 379

baby foods 378dairy produce 378, 379

in meat 377, 379, 380, 383, 414, 419metabolism 395–400

general 395metabolic classification 395–396of radiation 396–398of specific radionuclides 398–400

in milk 379, 380, 384, 392, 396, 399, 414monitoring 400–402

methodology 402surveillance 400–402

nature 392–393pathways 394–395post-Chernobyl studies 378–387pre-Chernobyl studies 376–378properties 398, 402

radioactivity 378–382, 392–393in foods 378–382radiation 393

β radiation 375, 376, 393, 395, 396, 397,402

γ radiation 375, 376, 393, 395, 396, 397, 402reference levels 378–382regulatory issues 404–405release 375, 394, 401research 430risk assessment 385–386, 403–405

initial body uptake 403philosophy 403radiation protection 402–403radiation protection guides 403–405retention in body 403–404

risk management 386–387seafood 419soil contamination 377, 382, 38390Sr 378, 382, 392, 394, 396, 398–399, 401, 402,

404tine-dependent contamination 382–385toxicity and effects 402transfer to food 382–383uptake 380, 395, 396, 397, 399, 400, 401, 403

ricegenetically modified (GM) 355

saladsbacteria 32, 34

Salmonella serovarseggs 33, 414–415foodborne disease 30, 31, 32, 33, 35, 36, 37,

40, 41, 42, 410helva 416meat 33, 413–414milk 32regulations 421research 430seafood 33serotypes 415

saxitoxins 47–49seafood

arsenic 418bacteria

Clostridium botulinum 32Listeria monocytogenes 32Salmonella serovars 33Vibrio cholerae 32, 33Vibrio parahaemolyticus 34

contaminants (overview) 412–413dioxins 413fish 129, 145, 181–182, 206–207, 224, 412–413,

418smoked 224

11-Mar-03 20

450 Index



mercury 206–207, 412–413microbial contamination 412–413and pollution 412–413polychlorinated biphenyls (PCBs) 129, 145,

413polycyclic aromatic hydrocarbons (PAHs)

181–182radionuclides 419shellfish 47–60, 412–413Vibrio parahaemolyticus 413

solanine 15, 349, 350, 356sorbic acid 244–245, 248soybeans

genetically modified (GM) 353–355, 356–359broiler chicken study 357catfish experiment 357–358composition 353–354cow study 358gene product safety 358–359rat studies 356–357statistical comparison 354

89Sr 391, 392, 396, 397, 398–39990Sr 378, 382, 392, 394, 396, 397, 398–399, 401, 402,

404substantial equivalence

concerns 338, 347, 349, 353–356, 368molecular profiling 339–340proteomics 338–340in safety evaluation of GM foods 336–340,

349, 353–356, 368sulphamethazine 298–300sulphites 238–244, 253–254, 257–258, 260–262sulphonamides 298–300sweeteners 246, 415

tartrazine 238, 253, 258–260tetracycline 297–298tomato

genetically modified (GM) 348–352, 365–366safety evaluation 348–352stomach lesions 350–351substantial equivalence 349

proteomic analysis 338, 340transmissible spongiform encephalopathies (TSEs)

animal diseases 316clinical symptoms 323–324genetics 317–320

cattle 320humans 317–318sheep 318–319

human diseases 316, 317–318, 321–324risk factors 322–323strains 317tissue distribution 320–322transmission 322

treatments 324tumours 185, 187–188, 219–226

vegetablesand cancer prevention 433contaminants

in beetroot 411cadmium 202canned 416celery 114, 411dioxins 160lettuce 114–115, 116, 217, 411nitrates 217, 411pesticides 114–115, 116, 411polychlorinated biphenyls (PCBs) 129, 130polycyclic aromatic hydrocarbons (PAHs)

179–180in spinach 411, 412in yams 411

organic 412veterinary products

antimicrobial agents 295–303food safety concerns 301–303fluoroquinolones 300–303penicillin 295–297sulphamethazine 298–300tetracycline 297–298

distribution in foods 296, 298, 299, 304, 414epidemiological methods 306–310

acceptable daily intakes (ADIs) 307–308antimicrobial resistance 309, 310drug-resistant bacteria 309FoodNet 308–309residue violation 309surveillance 308–310tolerance 307

hazards 293–295, 307–308production drugs 303–306

clenbuterol 303–305hormones 305–306

risk assessment 294, 307–308risk factors 295risk management 294

Vibrio parahaemolyticus 34, 413viruses

foodborne 410, 413

water quality 217, 419wheat

citrinin 72deoxynivalenol (DON) 73, 86ochratoxin A (OTA) 72processing 87trichothecenes 73, 86

Index 451

11-Mar-03 20



winefungicide residues 119

yessotoxins (YTXs) 52, 53

zearalenoneas co-contaminant 75foods 73–74tolerable daily intakes (TDIs) 81toxicology 76

11-Mar-03 20

452 Index

