Regulation of toxins in food

Vanessa Steenkamp

29 February 2016

Introduction

• Food safety draws attention in the modern pace of the

world owing to rapid-changing food recipes and food

habits

• Foodborne illnesses associated with pathogens, toxins,

and other contaminants pose serious threat to human

health

• A large amount of money is spent on analyses and

control measures

• Historically learnt that everything is toxic – only dose that separates toxic from non-toxic

• Not true, low doses can be more toxic – effects cannot always be extrapolated from dose

• Regulators put limits on the amounts of potentially toxic substances allowed in food

• Where setting limits is not effective, sufficient information is provided (label) – allergens

Food Safety Legislation

Consumer Protection Act (CPA)

• Consumer rights: – Fair value, good quality and safety

– Disclosure of information: warnings regarding risks/hazards

• Food legislation (FCD Act) – Hygiene requirements for food premises

– Implementation date for food preparation

– Labelling and advertising of foodstuffs

– Trans-fat in foodstuffs

National legislation

Food safety management system

• FDA (Food and Drug Administration) Model Food Code

• Internal and informal safety system audits

• HACCP principles (internationally recognized food

management system)

Principles:

– Conduct a hazard analysis

– Determine the critical control points

– Establish critical limits

– Monitor control of the CCPS

– Establish corrective action

– Verify

– Document

Regulatory bodies

EPA: US Environmental Protection Agency

US Centre for Disease Control and Prevention (CDC)

CODEX (General standards for contaminants and toxins in food and feed)

• Contaminant = “Any substance not intentionally added to food, which is present in such food as a result of the production, manufacture, processing, preparation, treatment, packing, packaging, transport or holding of such food or as a result of environmental contamination”

• Includes: metabolites of microfungi; mycotoxins and phycotoxins; endogenous natural toxicants (*)

• Specific criteria: toxicological information, analytical data, intake data, technological considerations, risk assessment and risk management considerations

Allowable levels

• FOA: Food and Agriculture Organization

• WHO

MAC: Maximum allowable concentration

HRI: Health risk index (weight, amount eating)

Carcinogenic risk: incremental lifetime ca risk

Ca risk = CSF (oral cancer slope factor) x LADD (lifetime average

daily dose)

Tolerable daily intake (TDI) or Acceptable daily intake (ADI)

Chronic daily intake = [ ] x DI/BW (mg/kg/day)

All determine allowable levels for human exposure

Safety testing Guidelines

OECD (Organization for Economic Co-operation and Development) Subacute

Chronic

Reproductive

Multigeneration

Carcinogenicity

Mutagenicity

FDA

• All available laboratory animal studies to determine adequacy and evaluate whether any evidence of toxicological risk (extensive data collection)

• Redbook 2000: General Guidelines for Designing and Conducting Toxicity Studies

Animal studies

• Acute toxicity = single dose (rats/mice) – det. toxic dose

• Repeated-dose toxicity testing = mimic daily exposure (>

1 month)

– Body weight

– Feed consumption

– Blood chemistry

– Organ weights

– Histopathology

Lowest Observed Adverse Effect Level (LOAEL)

No-observed-adverse-effect level (NOAEL)

Animal toxicity

• Reproductive and developmental toxicity (teratology)

- Reproductive performance (mating, fertility, gestation, litter size,

sperm motility)

- Development and viability of pups (incl genital development)

- Organ weights of weaning rats

- Skeletal and visceral malformations

- Histopathology of reproductive tissues

• Two species: rodent (rat/mouse) and non-rodent (rabbit)

Other tests

• Allergenicity: (immunotoxcity)

– radioallergosorbent test in vitro cell-based histamine release

assay

– skin-prick test binds to IgE in serum from subjects

• In vitro cytotoxicity:

– Various LDH; NR; MTT

• Mutagenicity (DNA-damaging effects) i.e. genetic

modification

– Point mutations

– Chromosomal aberrations

– DNA damage/repair

Pesticides, Metals

Microbiological

• An outbreak of foodborne disease is defined as the

occurrence of two or more cases of a similar illness resulting

from ingestion of a common food

• Etiologic agents:

Bacteria

Viruses

Parasites

Viral: Norovirus; Rotavirus

Bacterial: Salmonella; Shinga toxin producing E. coli (STEC)

Parasitic: Cyclospora; Trichinella; Cryptosporidium

Shinga toxin-producing E. coli

• E. coli commensal bacterium

• Inhabits GIT humans and warm-blooded animals

• Not usually cause disease, except immunocompromised

• Indicator organism for faecal contamination

• Some strains acquire virulence factors that enable them to

adapt to new environments e.g. human tissues

• Initiate infections cause serious clinical outcomes

• STEC

STEC (E. coli 0157:H7)

• Important zoonotic foodborne pathogen

• Most overwhelming diarrhoegenic pathotype

• Major health concern

• Diarrhoea

• Haemolytic uraemic syndrome – low platelet count, ARF

• Haemorraghic colitis

• Transmitted by consumption raw/undercooked ground

meat and raw milk (unpasteurized)

• 20-50% illnesses in USA due to STEC (37,000)

• ICU and dialysis still deaths US ? developing countries

Detection

• Time consuming and laborious

• Requires sophisticated instruments

• Trained personnel

• Expensive reagents

• Conventional methods:

- microscopy-based methods: long incubation

- nucleic acid-based: polymerase chain reaction, molecular id (serogrouping)

- immunoassays: target specific proteins, molecular fingerprint of pathogen

• Nanomaterial-based sensors (“flash” enrichment to decrease time-to-test result)

Pesticides (Glycophosphate)

• Broad-spectrum herbicide, used on food crops (Roundup)

• ADI differs from country to country (0.3 mg/kg bw/d Europe; 1.75 mg/kg bw/d USA)

• Calculation based on lowest dose considered non-toxic in animal feeding trials by industry

• Industry toxicity studies are out of date

• May be endocrine disruptors i.e. long term studies conducted on wrong animal age group (adults, should be foetal)

• Formulations sold contain additives which are toxic – only individual compounds tested

• Formulations tested only one month without blood parameters

• WHO has reclassified it as probable carcinogen in 2015

Glycophosphate

• SA has been found in bread and maize meal

• Used to spray GM crops

• MRLs (max residue limits) established by UN Food and

Agriculture Organization and WHO Codex Committee

on Pesticide Residues

• Analysis done in France

• Not done in South Africa

Allergens

• 10% of world’s population suffer from some food allergy

• Higher % children than adults are allergic

• Seafood and nut allergies most prevalent

• Mild discomfort to anaphylactic shock and death

• Common allergens: egg, cow’s milk, fish, peanuts,

soybeans, some cereals, crustaceans

• Skin prick test carried out at clinics

• Info included on label

Mycotoxins

• Abiotic hazards produced by fungi that can grow on crops

• Enter food chain: directly consumed or used as livestock feed

• Metabolism may result in its accumulation in organs and tissues

• WHO ~25% cereals in world contaminated by mycotoxins

• Nuts, spices, fruits also at risk

• Mycotoxin production may occur pre-harvest, harvest and drying (improper), storage

• Influenced by water activity in stored products

Most prevalent groups

• Aflatoxins (Aspergillus spp.) AFB1 most toxic, stable,

resist processes like roasting, baking, cooking (nuts,

bakery products)

• Ochratoxin A (Aspergillus and Penicillium) most toxic,

structurally similar to amino acid phenylalanine (inhibit

protein synthesis, mitochondrial poison) >250ºC destroy

• Trichothecenes: immune-depressants

• Zearalenone: estrogenic activity

• Fumonisins B1 (most important) and B2 [in maize]

• Emerging mycotoxins: fusaproliferin, moniliformin, beauvericin,

enniatins produced by Fusarium spp

Aflatoxins

• Carcinogenic, target liver and genotoxic

• Acute toxicity: high [ ] in food

• Chronic toxicity: most common

• Toxicity depends on age, species, gender, nutritional

status, dose, length of exposure

• Animals see decrease in productive parameters

• Humans chronic exposure

– Liver cancer

– Effects on reproductive system (testicular development; sperm)

– Effects on immune system (reduced immunity)

– Encephalopathy with fatty degeneration of viscera (Reye’s)

Detection

• Hazard identification, hazard characterization

• No harmonization on best methodologies

• Consumption data taken from national dietary surveys

• Enzyme immunoassays

• Chromatographic methods: TLC, HPLC, GC linked to

MS

• Except for ELISA, methods requires clean up and

extraction: LLE, SPE

Maximum levels for metals in foodstuffs

(4 common heavy metals)

Foodstuff Cadmium (Cd) Maximum level (mg/kg or mg/L)

Lead (Pb) Maximum level (mg/kg or mg/L)

Arsenic (As) Maximum level (mg/kg or mg/L)

Mercury (Hg) Maximum level (mg/kg or mg/L)

Cereals, pulses and legumes

0.1 0.2 - -

Meat and processed meat

0.05 0.1 1.0 -

Fish and processed fish

0.5 0.5 3.0 0.5

Natural mineral water

0.003 0.01 0.01 0.001

Vegetable and fruit juices

- 0.3 0.2 -

Metals

• Cd: chronic potent nephrotoxin

class one carcinogen

intake via Cd containing food

• Pb: inorganic form carcinogenic

CNS effects in children

• Cd + Pb: atmospheric deposition

• Cd application of phosphate fertilizers

• No correlation soil and plant heavy metal [ ]

- Depends soil pH

- Acidic soil wheat accumulates more Cd

- Organic carbon content, clay content, cation exchange capacity, oxide content of Al, Fe, Mn

Arsenic (As)

Inorganic (iAs) Organic

(consumption of contaminated seafood (fish, prawns)

food, water)

Low dose, long term = “Arsenicosis”

Reduced (trivalent) iAs

Oxidized (pentavalent) iAs

• Low to moderate levels exposure (drinking water): skin lesions,

circulatory disorders, neurological complications, hepatic and renal

dysfunction

• Chronic exposure: malignant tumours

• Affects all cellular processes and organ functions in body

• In utero exposure: can induce epigenetic effects and disease

susceptibility in later stages of life

accumulates in tissue and body fluids, skin,

lungs, liver, kidneys

As – developmental neurotoxicity in children

Systematic review: 24 studies

Low-level As exposure (<100 µg/L) in drinking water

Children 5-11 years

Biomarkers As in urine or blood

• Problems:

inaccuracies of biomarkers of iAs

insufficient adjustment for nutritional deficiencies

presence of other neurotoxicants

• Malnutrition (folate and protein deficiency) affect neurodevelopment and As toxicity

Tsuji et al. Toxicology 2015;337:91-107

Arsenic exposure

Abdul et al., Environmental Toxicology and Pharmacology, 2015;40: 828–846

Combination of metals

Abdul et al., Environmental Toxicology and Pharmacology, 2015;40: 828–846

Detection

• Inductively coupled plasma mass spectrometry

instrumentation

Ion chromatography-ICP MS (IC-ICP MS)

Laser ablation ICP-MS (Pb, Cd)

• Graphite furnace atomic absorption spectrometry (GF-AAS)

• Lack standardized analytical methods

• Unavailability of certified reference materials for As in food

matrices

Genetically Modified (GM) Foods

• GM plant and derived food and feed have been

modified through insertion of single or a few genes

which express traits – herbicide tolerance, insect

resistance

• Improve agronomic properties e.g. drought resistance,

salt tolerance

• Enhance nutritional properties

• Health benefits

Plant/species Altered characteristic Transgene/Mechanism

Canola Increased vitamin E Y-Tocopherol methyl transferase

Rice Increased iron Ferritin, metallothionein, phytase

Soybean Increased isoflavones Isoflavone synthase

EFSA GMO Panel Working Group Food Chem Toxicol 2008;46:S2-S70

GM vs non-GM

• Non-GM safe to eat because of history

• GM assumed that same traits except newly introduced

• OECD: food is safe if “there is reasonable certainty that

no harm will result from its consumption under

anticipated conditions of use”

• GM tomatoes

• GM potatoes and rice No abnormalities

European Food Safety Authority (EFSA)

Stepwise procedure for safety assessment of GM food

Background knowledge is required on:

Parental plant (history of safe use, phenotype, chemical

composition)

Transformation process (source of transferred gene(s), DNA

construct, consequences of DNA insertion)

Newly expressed proteins and other constituents (potential

toxicity or allergenicity)

GM plant (ability to transfer genetic material to other organisms)

Anticipated intake/extent of use

Nutritional properties

Food processing characteristics

GM food assessments

• Safety: focused on presence and characteristics of

newly expressed proteins and other new constituents

• Toxicology: done using standardized toxicological

methodology designed for the assessment of defined

chemical substances

• OECD Guidelines for Testing of Chemicals

• European Commission Directive on dangerous

substances

• According to Good Laboratory Practice principles

• Similarities and differences: macro-nutrients, micro-

nutrients, inherent toxins, allergens, anti-nutrients

Irradiated foods

• Studies evaluated: outcome accepted; accepted with reservation, rejected

• Rejected:

Radiation dose not reported

Number animals per group not reported

Number of animals per group was small (<5)

Study conducted without controls fed non-irradiated diet

Diet fed was nutritionally inadequate

Lab in violation of GLP

• Animal data + chemical studies + in vitro studies = weight of evidence equation

• Comparative studies establish Margin-of-Safety for toxicity and nutritional adequacy

Problems with whole foods (vs single

compound) in in vitro tests

• Cannot be assessed at high concentrations

• Usually incompatable with test system (not soluble in

matrix appropriate for in vitro test

• Certain constituents may interfere with test system

• Poor bioavailability of bioactive compounds due to

matrix structure

• Interaction between different bioactive compounds

New prospects

• Recent developments in molecular biology and analytical chemistry

• New opportunities to evaluate effects of chemicals on food and diet

• Transcriptomics (transcript profiling)

• Proteonomics (protein profiling)

• Metabolomics (metabolite profiling)

• “omics” technologies applied to toxicology = toxicogenomics – Provide better understanding of mechanism of action of

chemicals and contribute to development of alternatives to animal testing

Permits measurement of thousands of variables

simultaneously

Requirements

• Identify and incorporate variability in human exposure and

vulnerability into health assessments, so that all people are

better protected

• Aware of potential impacts of exposure to multiple toxicants

i.e. cumulative risk

• Not assume that low level exposure is risk-free = some level

of risk (especially as exposed to multiple stressors)

• Significant improvements in both testing and risk assessment

are needed to protect people from toxicants

Testing facilities

• Animal testing NWU accredited laboratory

• SABS food laboratories

Chemical

Microbiological

Physical tests

• More specifically: (Food Safety System Certification 2200)

– Mineral and trace elements

– Mycotoxins

– Pesticide residues

– Veterinary drug residues

– Microbiological testing

Shortly ….

• SaRChi chair in Sanitary and Phytosanitary

Risk analysis

– Food safety

– Veterinary health

– Plant health

Thank you