Disclosure to Promote the Right To Information

Whereas the Parliament of India has set out to provide a practical regime of right to information for citizens to secure access to information under the control of public authorities, in order to promote transparency and accountability in the working of every public authority, and whereas the attached publication of the Bureau of Indian Standards is of particular interest to the public, particularly disadvantaged communities and those engaged in the pursuit of education and knowledge, the attached public safety standard is made available to promote the timely dissemination of this information in an accurate manner to the public.

इंटरनेट मानक

“!ान $ एक न' भारत का +नम-ण”Satyanarayan Gangaram Pitroda

“Invent a New India Using Knowledge”

“प0रा1 को छोड न' 5 तरफ”Jawaharlal Nehru

“Step Out From the Old to the New”

“जान1 का अ+धकार, जी1 का अ+धकार”Mazdoor Kisan Shakti Sangathan

“The Right to Information, The Right to Live”

“!ान एक ऐसा खजाना > जो कभी च0राया नहB जा सकता है”Bhartṛhari—Nītiśatakam

“Knowledge is such a treasure which cannot be stolen”

“Invent a New India Using Knowledge”

है”ह”ह

IS 15889 (2010): GUIDELINE FOR THE CONDUCT OF FOOD SAFETYASSESSMENT OF FOODS PRODUCED USING RECOMBINANT-DNAMICRO-ORGANISMS [FAD 23: Biotechnology for Food andAgriculture]

IS 15889 : 2011

© BIS 2011

B U R E A U O F I N D I A N S T A N D A R D SMANAK BHAVAN, 9 BAHADUR SHAH ZAFAR MARG

NEW DELHI 110002

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Indian StandardGUIDELINE FOR THE CONDUCT OF FOOD SAFETY

ASSESSMENT OF FOODS PRODUCED USINGRECOMBINANT-DNA MICRO-ORGANISMS

ICS 67.020

May 2011 Price Group 6

Biotechnology for Food and Agriculture Sectional Committee, FAD 23

FOREWORD

This Indian Standard was adopted by the Bureau of Indian Standards, after the draft finalized by theBiotechnology for Food and Agriculture Sectional Committee had been approved by the Food and AgricultureDivision Council.

A variety of micro-organisms used in food production have a long history of safe use that predates scientificassessment. Few micro-organisms have been assessed scientifically in a manner that would fully characterizeall potential risks associated with the food they are used to produce, including, in some instances, theconsumption of viable micro-organisms. Furthermore, the principles of risk analysis, particularly those forrisk assessment, are primarily intended to apply to discrete chemical entities such as food additives andpesticide residues, or specific chemical or microbial contaminants that have identifiable hazards and risks;they were not originally intended to apply to intentional uses of micro-organisms in food processing or in thefoods transformed by microbial fermentations. The safety assessments that have been conducted havefocused primarily on the absence of properties associated with pathogenicity in these micro-organisms andthe absence of reports of adverse events attributed to ingestion of these micro-organisms, rather than evaluatingthe results of prescribed studies. Further, many foods contain substances that would be considered harmfulif subjected to conventional approaches to safety testing. Thus, a more focused approach is required wherethe safety of a whole food is being considered.

This standard has been aligned with the Guideline for the Conduct of Food Safety Assessment of FoodsProduced Using Recombinant-DNA Micro-organisms (CAC/GL 46-2003) and is identical to it.

This approach is based on the principle that the safety of foods produced using recombinant-DNA micro-organisms, is assessed relative to the conventional counterpart having a history of safe use, not only for thefood produced using a recombinant-DNA micro-organism, but also for the micro-organism itself. This approachtakes both intended and unintended effects into account. Rather than trying to identify every hazard associatedwith a particular food, the intention is to identify new or altered hazards relative to the conventional counterpart.

This standard supports the IS 15887 : 2010 ‘Principles for the risk analysis of foods derived from modernbiotechnology’. It addresses safety and nutritional aspects of foods produced through the actions ofrecombinant-DNA micro-organisms. The recombinant-DNA micro-organisms that are used to produce thesefoods are typically derived using the techniques of modern biotechnology from strains that have a history ofsafe, purposeful use in food production. However, in instances where the recipient strains do not have ahistory of safe use their safety will have to be established. Such food and food ingredients may contain viableor non-viable recombinant-DNA micro-organisms or may be produced by fermentation using recombinant-DNA micro-organisms from which the recombinant-DNA micro-organisms may have been removed.

If a new or altered hazard, nutritional or other food safety concern is identified by the safety assessment, therisk associated with it would first be assessed to determine its relevance to human health. Following thesafety assessment and if necessary further risk assessment, the food or the component of food, such micro-organisms used in production would be subjected to risk management considerations in accordance withIS 15887 before it is considered for commercial distribution.

Clauses 3.1.5 and 3.1.6 refer to the concept of ‘substantial equivalence’ as described in the report of the2000 joint FAO /WHO expert consultations (Document WHO/SDE/PHE/FOS/00.6, WHO, Geneva, 2000).

Report of Joint FAO/WHO Expert Consultation on Allergenicity of Foods Derived from Biotechnology (2001),which includes reference to several decision trees, was used in developing the Annex to these guidelines.Clause A-3.3 of this standard may also require reference to Section 6.4 ‘Pepsin Resistance’ of Report of JointFAO/WHO Expert Consultation, 2001.

While this Guideline is designed for foods produced using recombinant-DNA micro-organisms or theircomponents, the approach described could, in general, be applied to foods derived from micro-organismsthat have been altered by other techniques.

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Indian StandardGUIDELINE FOR THE CONDUCT OF FOOD SAFETY

ASSESSMENT OF FOODS PRODUCED USINGRECOMBINANT-DNA MICRO-ORGANISMS

1 SCOPE

1.1 The standard describes approaches recommendedfor making safety assessments of foods producedusing recombinant-DNA micro-organisms, usingcomparison to a conventional counterpart, andidentifies the data and information that are generallyapplicable to making such assessments.

1.2 This standard does not address,

a) safety of micro-organisms used inagriculture (for plant protection,biofertilizers, in animal feed or food derivedfrom animals fed the feed, etc);

b) risks related to environmental releases ofrecombinant-DNA micro-organisms used infood production;

c) safety of substances produced by micro-organisms that are used as additives orprocessing aids, including enzymes for usein food production;

d) specific purported health benefits orprobiotic effects that may be attributed tothe use of micro-organisms in food; or

e) issues relating to the safety of foodproduction workers handling recombinant-DNA micro-organisms.

1.3 The criterion for establishing the safety of micro-organisms used in the production of foods wherethere is no history of safe use is beyond the scopeof this standard.

2 DEFINITIONS

For the purpose of this standard, the followingdefinitions shall apply.

2.1 Recombinant-DNA Micro-organism — Bacteria,yeasts or filamentous fungi in which the geneticmaterial has been changed through in vitro nucleicacid techniques including recombinantdeoxyribonucleic acid (DNA) and direct injection ofnucleic acid into cells or organelles.

2.2 Conventional Counterpart — A micro-organism/strain with a known history of safe use in producingand/or processing the food and related to therecombinant-DNA strain. The micro-organism maybe viable in the food or may be removed in processing

or rendered non-viable during processing; or foodproduced using the traditional food productionmicro-organisms for which there is experience ofestablishing safety based on common use in foodproduction.

NOTE — It is recognized that for the foreseeable future,micro-organisms derived from modern biotechnology willnot be used as conventional counterparts.

3 INTRODUCTION TO FOOD SAFETYASSESSMENT

3.1 General

3.1.1 Most foods produced as a result of thepurposeful growth of micro-organisms have theirorigins in antiquity, and have been deemed safe longbefore the emergence of scientific methods forassessing safety. Micro-organisms possessproperties, such as fast growth rates, that enablegenetic modifications, whether employingconventional techniques or modern biotechnology,to be implemented in short time frames. Micro-organisms used in food production derived usingconventional genetic techniques have notcustomarily been systematically subjected toextensive chemical, toxicological, epidemiological, ormedical evaluations prior to marketing. Insteadmicrobiologists, mycologists, and foodtechnologists have evaluated new strains of bacteria,yeasts and filamentous fungi for phenotypiccharacteristics that are useful in relation to foodproduction.

3.1.2 Safety assessments of recombinant-DNAmicro-organisms should document the use of relatedmicro-organisms in foods, the absence of propertiesknown to be characteristic of pathogens in therecombinant-DNA micro-organisms or the recipientstrains used for constructing the recombinant-DNAmicro-organisms, and known adverse eventsinvolving the recipient or related organisms. Inaddition, when a recombinant DNA micro-organismdirectly affects or remains in the food, any effectson the safety of the food should be examined.

3.1.3 The use of animal models for assessingtoxicological effects is a major element in the riskassessment of many compounds, such as pesticides.In most cases, however, the substance to be testedis well characterized, of known purity, of no particular

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nutritional value, and human exposure to it isgenerally low. It is therefore relatively straightforwardto feed such compounds to animals at a range ofdoses some several orders of magnitude greater thanthe expected human exposure levels, in order toidentify any potential adverse health effects ofimportance to humans. In this way, it is possible, inmost cases, to estimate levels of exposure at whichadverse effects are not observed and to set safeintake levels by the application of appropriate safetyfactors.

3.1.4 Animal studies cannot readily be applied totesting the risks associated with whole foods, whichare complex mixtures of compounds, and oftencharacterized by a wide variation in composition andnutritional value. Due to their bulk and effect onsatiety, they can usually only be fed to animals atlow multiples of the amounts that might be presentin the human diet. In addition, a key factor to considerin conducting animal studies on foods is thenutritional value and balance of the diets used, inorder to avoid the induction of adverse effects thatare not related directly to the material itself. Detectingany potential adverse effects and relating theseconclusively to an individual characteristic of thefood can therefore be extremely difficult. If thecharacterization of the food indicates that theavailable data are insufficient for a thorough safetyassessment, properly designed animal studies couldbe requested on the whole food. Anotherconsideration in deciding the need for animal studiesis whether it is appropriate to subject experimentalanimals to such a study if it is unlikely to give rise tomeaningful information.

Animal studies typically employed in toxicologicalevaluations also cannot be readily applied to testingpotential risks associated with ingestion of micro-organisms used for food production. Micro-organisms are living entities, containing complexstructures composed of many biochemicals, andtherefore are not comparable to pure compounds. Insome processed foods, they can survive processingand ingestion and can compete and, in some cases,be retained in the intestinal environment forsignificant periods of time. Appropriate animalstudies should be used to evaluate the safety ofrecombinant-DNA micro-organisms where the donor,or the gene or gene product do not have a history ofsafe use in food, taking into account availableinformation regarding the donor and thecharacterization of the modified genetic material andthe gene product. Further, appropriately designedstudies in animals may be used to assess thenutritional value of the food or the bioavailability ofthe newly expressed substance in the food.

3.1.5 Due to the difficulties of applying traditionaltoxicological testing and risk assessment proceduresto whole foods, a more focused approach is requiredfor the safety assessment of foods produced usingrecombinant-DNA micro-organisms. This has beenaddressed by the development of a multidisciplinaryapproach for assessing safety that takes into accountthe intended effect, the nature of the modificationand detectable unintended changes that may occurin the micro-organism or in its action on the food,using the concept of substantial equivalence.

3.1.6 While the focus of a safety assessment will beon the recombinant-DNA micro-organism, additionalinformation on its interaction with the food matrixshould be taken into consideration when applyingthe concept of substantial equivalence, which is akey step in the safety assessment process. However,the concept of substantial equivalence is not a safetyassessment in itself. Rather it represents the startingpoint that is used to structure the safety assessmentof both a recombinant-DNA micro-organism relativeto its conventional counterpart and the foodproduced using recombinant-DNA micro-organismrelative to its conventional counterpart. This conceptis used to identify for evaluation similarities anddifferences between a recombinant-DNA micro-organisms used in food processing as well as thefood produced using the recombinant-DNA micro-organisms and their respective conventional. It aidsin the identification of potential safety and nutritionalissues and is considered the most appropriatestrategy to date for safety assessment of foodsproduced using recombinant-DNA micro-organisms.The safety assessment carried out in this way doesnot imply absolute safety of the new product; rather,it focuses on assessing the safety of any identifieddifferences so that the safety of the recombinant-DNA micro-organism and the food produced usingrecombinant-DNA micro-organism can be consideredrelative to their respective conventionalcounterparts.

3.2 Unintended Effects

3.2.1 In achieving the objective of conferring aspecific target trait (intended effect) to a micro-organism by the addition, substitution, removal, orrearrangement of defined DNA sequences, includingthose used for the purpose of DNA transfer ormaintenance in the recipient organism, additionaltraits could, in some cases, be acquired or existingtraits could be lost or modified. The potential foroccurrence of unintended effects is not restricted tothe use of in vitro nucleic acid techniques. Rather, itis an inherent and general phenomenon that can alsooccur in the development of strains using traditionalgenetic techniques and procedures, or from exposure

of micro-organisms to intentional or unintendedselective pressures. Unintended effects may bedeleterious, beneficial, or neutral with respect tocompetition with other micro-organisms, ecologicalfitness of the micro-organism, the micro-organism’seffects on humans after ingestion, or the safety offoods produced using the micro-organism.Unintended effects in recombinant-DNA micro-organisms may also arise through intentionalmodification of DNA sequences or they may arisethrough recombination or other natural events in therecombinant-DNA micro-organism. Safetyassessment should include data and information toreduce the possibility that a food derived from arecombinant-DNA micro-organism would have anunexpected, adverse effect on human health.

3.2.2 Unintended effects can result from theinsertion of DNA sequences new to a micro-organisminto the microbial genome; they may be comparedwith those observed following the activity ofnaturally occurring transposable genetic elements.Insertion of DNA may lead to changes in expressionof genes in the genome of the recipient. The insertionof DNA from heterologous sources into a gene mayalso result in the synthesis of a chimeric protein,also referred to as a fusion protein. In addition geneticinstabili ty and its consequences need to beconsidered.

3.2.3 Unintended effects may also result in theformation of new or changed patterns of metabolites.For example, the expression of enzymes at high levelsor the expression of an enzyme new to the organismmay give rise to secondary biochemical effects,changes in the regulation of metabolic pathways, oraltered levels of metabolites.

3.2.4 Unintended effects due to genetic modificationmay be subdivided into two groups: those that couldbe predicted and those that are “unexpected”. Manyunintended effects are largely predictable based onknowledge of the added trait , i ts metabolicconsequences or of the site of insertion. Due to theexpanding knowledge of microbial genomes andphysiology, and the increased specificity in functionof genetic materials introduced through recombinant-DNA techniques compared with other forms ofgenetic manipulation, it may become easier to predictunintended effects of a particular modification.Molecular biological and biochemical techniques canalso be used to analyse changes that occur at thelevel of transcription and translation that could leadto unintended effects.

3.2.5 The safety assessment of foods producedusing recombinant-DNA micro-organisms involvesmethods to identify and detect such unintendedeffects and procedures to evaluate their biological

relevance and potential impact on food safety. Avariety of data and information is necessary to assessunintended effects, because no individual test candetect all possible unintended effects or identify, withcertainty, those relevant to human health. These dataand information, when considered in total, shouldprovide assurance that the food is unlikely to havean adverse effect on human health. The assessmentof unintended effects takes into account thebiochemical, and physiological characteristics of themicro-organism that are typically selected forimproving strains for commercial food or beverageuses. These determinations provide a first screenfor micro-organisms that exhibit unintended traits.Recombinant-DNA micro-organisms that pass thisscreen are subjected to safety assessment asdescribed in 4.

3.3 Framework of Food Safety Assessment

3.3.1 The safety assessment of a food producedusing a recombinant-DNA micro-organism is basedon determining the safety of using the micro-organism, which follows a stepwise process ofaddressing relevant factors that include,

a) description of the recombinant-DNA micro-organism;

b) description of the recipient micro-organismand its use in food production;

c) description of the donor organism(s);

d) description of the genetic modification(s)including vector and construct;

e) characterization of the geneticmodification(s); and

f) safety assessment:

1) expressed substances: assessment ofpotential toxicity and other traits relatedto pathogenicity;

2) compositional analyses of keycomponents;

3) evaluation of metabolites;

4) effects of food processing;

5) assessment of immunological effects;

6) assessment of viability and residenceof micro-organisms in the humangastrointestinal tract;

7) antibiotic resistance and gene transfer;and

8) nutritional modification.

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3.3.2 In certain cases, the characteristics of themicro-organisms and/or the foods produced/processed using these micro-organisms maynecessitate generation of additional data andinformation to address issues that are unique to themicro-organisms and/or food products under review.Experiments intended to develop data for safetyassessments should be designed and conducted inaccordance with sound scientific concepts andprinciples, as well as, where appropriate, goodlaboratory practice. Primary data should be madeavailable to regulatory authorities upon request.Data should be obtained using sound scientificmethods and analysed using appropriate statisticaltechniques. The sensitivity of all analytical methodsshould be documented.

3.3.3 The goal of each safety assessment is toprovide assurance, in the light of the best availablescientific knowledge, that the food will not causeharm when prepared or consumed according to itsintended use, nor should the organism itself causeharm when viable organisms remain in the food.Safety assessments should address the healthaspects for the whole population, including immuno-compromised individuals, infants, and the elderly.The expected endpoint of such an assessment willbe a conclusion regarding whether the new food and/or micro-organisms are as safe as the conventionalcounterparts taking into account dietary impact ofany changes in nutritional content or value. Wherethe micro-organism is likely to be viable uponingestion, its safety should be compared to aconventional counterpart taking into accountresidence of the recombinant-DNA micro-organismin the gastrointestinal tract, and where appropriate,interactions between it and the gastrointestinal floraof mammals (especially humans) and impacts of therecombinant-DNA micro-organism on the immunesystem. In essence, the outcome of the safetyassessment process is to define the product underconsideration in such a way as to enable riskmanagers to determine whether any measures areneeded to protect the health of consumers and if soto make well-informed and appropriate decisions inthis regard.

4 GENERAL CONSIDERATIONS

4.1 Description of the Recombinant-DNA Micro-organism

A description of the bacterial, yeast, or fungal strainand the food being presented for safety assessmentshould be provided. This description should besufficient to aid in understanding the nature of theorganism or food produced using the organism beingsubmitted for safety assessment. Recombinant-DNAmicro-organisms used in food production or

contained in food should be conserved as stockcultures with appropriate identification usingmolecular methods, and preferably, in establishedculture collections. This may facilitate the review ofthe original safety assessment. Such stock culturesshould be made available to regulatory authoritiesupon request.

4.2 Description of the Recipient Micro-organismand Its Use in Food Production

4.2.1 A comprehensive description of the recipientmicro-organism or micro-organism subjected to themodification should be provided. Recipient micro-organisms should have a history of safe use in foodproduction or safe consumption in foods. Organismsthat produce toxins, antibiotics or other substancesthat should not be present in food, or that beargenetic elements that could lead to geneticinstability, antibiotic resistance or that are likely tocontain genes conferring functions associated withpathogenicity (that is also known as pathogenicityislands or virulence factors) should not beconsidered for use as recipients. The necessary dataand information should include, but need not berestricted to:

a) identity: scientific name, common name orother name(s) used to reference the micro-organism, strain designation, informationabout the strain and its source, or accessionnumbers or other information from arecognized culture repository from which theorganism or i ts antecedents may beobtained, if applicable, informationsupporting its taxonomical assignment;

b) history of use and cultivation, knowninformation about strain development(including isolation of mutations orantecedent strains used in strainconstruction); in particular, identifying traitsthat may adversely impact human health;

c) information on the recipient micro-organism’s genotype and phenotyperelevant to its safety, including any knowntoxins, antibiotics, antibiotic resistancefactors or other factors related topathogenicity, or immunological impact, andinformation about the genetic stability of themicro-organism;

d) history of safe use in food production orsafe consumption in food; and

e) information on the relevant productionparameters used to culture the recipientmicro-organism.

4.2.2 Relevant phenotypic and genotypicinformation should be provided not only for the

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recipient micro-organism, but also for related speciesand for any extra-chromosomal genetic elements thatcontribute to the functions of the recipient strain,particularly if the related species are used in foodsor involved in pathogenic effects in humans or otheranimals. Information on the genetic stability of therecipient micro-organism should be consideredincluding, as appropriate, the presence of mobileDNA elements, that is, insertion sequences,transposons, plasmids, and prophages.

4.2.3 The history of use may include informationon how the recipient micro-organism is typicallygrown, transported and stored, quality assurancemeasures typically employed, including those toverify strain identity and production specificationsfor micro-organisms and foods, and whether theseorganisms remain viable in the processed food orare removed or rendered non-viable as aconsequence of processing.

4.3 Description of the Donor Organism(s)

Information should be provided on the donororganism(s) and any intermediate organisms, whenapplicable, and, when relevant, related organisms. Itis particularly important to determine if the donor orintermediate organism(s) or other closely relatedspecies naturally exhibit characteristics ofpathogenicity or toxin production, or have othertraits that affect human health. The description ofthe donor or intermediate organism(s) should include:

a) identity: scientific name, common name orother name(s) used to reference theorganism, strain designation, informationabout the strain and its source, or accessionnumbers or other information from arecognized culture repository from which theorganism or i ts antecedents may beobtained, if applicable, and informationsupporting its taxonomic assignment;

b) information about the organism or relatedorganisms that concerns food safety;

c) information on the organism’s genotype andphenotype relevant to its safety including anyknown toxins, antibiotics, antibiotic resistancefactors or other factors related to pathogenicity,or immunological impact; and

d) information on the past and present use, ifany, in the food supply and exposureroute(s) other than intended food use (forexample, possible presence ascontaminants).

4.4 Description of the Genetic Modification(s)Including Vector and Construct

4.4.1 Sufficient information should be provided onthe genetic modification(s) to allow for the

identification of all genetic material potentiallydelivered to or modified in the recipient micro-organism and to provide the necessary informationfor the analysis of the data supporting thecharacterization of the DNA added to, inserted into,modified in, or deleted from the microbial genome.

4.4.2 The description of the strain constructionprocess should include,

a) information on the specific method(s) usedfor genetic modification;

b) information on the DNA used to modify themicro-organism, including the source (forexample plant, microbial, viral, synthetic),identity and expected function in therecombinant-DNA micro-organism, andcopy number for plasmids; and

c) intermediate recipient organisms includingthe organisms (for example other bacteria orfungi) used to produce or process DNA priorto introduction into the final recipientorganism.

4.4.3 Information should be provided on the DNAadded, inserted, deleted, or modified, including,

a) characterization of all genetic componentsincluding marker genes, vector genes,regulatory and other elements affecting thefunction of the DNA;

b) size and identity;c) location and orientation of the sequence in

the final vector/construct; andd) function.

4.5 Characterization of the Genetic Modification(s)

4.5.1 In order to provide clear understanding of theimpact of the genetic modification on the compositionand safety of foods produced using recombinant-DNA micro-organisms, a comprehensive molecularand biochemical characterization of the geneticmodification should be carried out. To facilitate thesafety assessment, the DNA to be inserted shouldbe preferably limited to the sequences necessary toperform the intended functions.

4.5.2 Information should be provided on the DNAmodifications in the recombinant DNA micro-organism; this should include,

a) characterization and description of theadded, inserted, deleted, or otherwisemodified genetic materials, includingplasmids or other carrier DNA used totransfer desired genetic sequences. Thisshould include an analysis of the potentialfor mobilization of any plasmids or othergenetic elements used, the locations of the

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added, inserted, deleted, or otherwisemodified genetic materials (site on achromosomal or extra-chromosomallocation); if located on a multi-copy plasmid,the copy number of the plasmid;

b) number of insertion sites;

c) organization of the modified genetic materialat each insertion site including the copynumber and sequence data of the inserted,modified, or deleted material, plasmids orcarrier DNA used to transfer the desiredgenetic sequences, and the surroundingsequences. This will enable theidentification of any substances expressedas a consequence of the inserted, modifiedor deleted material;

d) identification of any open reading frameswithin inserted DNA, or created by themodifications to contiguous DNA in thechromosome or in a plasmid, including thosethat could result in fusion proteins; and

e) particular reference to any sequences knownto encode, or to influence the expression of,potentially harmful functions.

4.5.3 Information should be provided on anyexpressed substances in the recombinant-DNAmicro-organism; this should include,

a) the gene product(s) (for example a proteinor an untranslated RNA) or otherinformation such as analysis of transcriptsor expression products to identify any newsubstances that may be present in the food;

b) the gene product’s function;c) the phenotypic description of the new

trait(s);d) the level and site of expression (intracellular,

periplasmic — for Gram-negative bacteria,organellar — in eukaryotic micro-organisms,secreted) in the micro-organism of theexpressed gene product(s), and, whenapplicable, the levels of its metabolites inthe organism;

e) the amount of the inserted gene product(s)if the function of the expressed sequence(s)/gene(s) is to alter the level of a specificendogenous mRNA or protein; and

f) the absence of a gene product, or alterationsin metabolites related to gene products, ifapplicable to the intended function(s) of thegenetic modification(s).

4.5.4 In addition, information should be provided,

a) to demonstrate whether the arrangement ofthe modified genetic material has beenconserved or whether significantrearrangements have occurred afterintroduction to the cell and propagation ofthe recombinant strain to the extent neededfor its use(s) in food production, includingthose that may occur during its storageaccording to current techniques;NOTE — Microbial genomes are more fluid thanthose of higher eukaryotes; that is, the organismsgrow faster, adapt of changing environments, andare more prone to change. Chromosomalrearrangements are common. The general geneticplastici ty of micro-organisms may affectrecombinant DNA in micro-organisms and mustbe considered in evaluating the stabil i ty ofrecombinant DNA micro-organisms.

b) to demonstrate whether deliberatemodifications made to the amino acidsequence of the expressed protein resultin changes in i ts post-translationalmodification or affect sites critical for itsstructure or function;

c) to demonstrate whether the intended effectof the modification has been achieved andthat all expressed traits are expressed andinherited in a manner that is stable for theextent of propagation needed for its use(s)in food production and is consistent withlaws of inheritance. It may be necessary toexamine the inheritance of the inserted ormodified DNA or the expression of thecorresponding RNA if the phenotypiccharacteristics cannot be measured directly;NOTE — Modified strains should be maintainedin a manner to enable verification of the geneticstability.

d) to demonstrate whether the newly expressedtrait(s) is expressed as expected and targetedto the appropriate cellular location or issecreted in a manner and at levels that isconsistent with the associated regulatorysequences driving the expression of thecorresponding gene;

e) to indicate whether there is any evidence tosuggest that one or more genes in therecipient micro-organism has been affectedby the modifications or the genetic exchangeprocess; and

f) to confirm the identity and expressionpattern of any new fusion proteins.

4.6 Safety Assessment

4.6.0 The safety assessment of the modified micro-organism should be performed on a case by case

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basis depending on the nature and extent of theintroduced changes. Conventional toxicologystudies may not be considered necessary where thesubstance or a closely related substance has, takinginto account its function and exposure, beenconsumed safely in food. In other cases, the use ofappropriate conventional toxicology or other studieson the new substance may be necessary. Effects ofthe recombinant-DNA micro-organism on the foodmatrix should be considered as well . If thecharacterization of the food indicates that theavailable data are insufficient for a thorough safetyassessment, properly designed animal or in vitrostudies with the recombinant-DNA micro-organismand/or the food produced using it could beconsidered necessary.

4.6.1 Expressed Substances: Assessment ofPotential Toxicity and Other Traits Related toPathogenicity

4.6.1.1 When a substance is new to foods or foodprocessing, the use of conventional toxicologystudies or other applicable studies on the newsubstance will be necessary. This may require theisolation of the new substance from the recombinant-DNA micro-organism, the food product if thesubstance is secreted, or, if necessary, the synthesisor production of the substance from an alternativesource, in which case the material should be shownto be structurally, functionally, and biochemicallyequivalent to that produced in the recombinant-DNAmicro-organism. Information on the anticipatedexposure of consumers to the substance, thepotential intake and dietary impact of the substanceshould be provided.

4.6.1.2 The safety assessment of the expressedsubstance should take into account its function andconcentration in the food. The number of viablemicro-organisms remaining in the food should be alsodetermined and compared to a conventionalcounterpart. All quantitative measurements shouldbe analysed using appropriate statistical techniques.Current dietary exposure and possible effects onpopulation sub-groups should also be considered.

4.6.1.3 In the case of proteins, the assessment ofpotential toxicity should take into account thestructure and function of the protein and shouldfocus on amino acid sequence similarity between theprotein and known protein toxins and anti-nutrients(for example, protease inhibitors, siderophores) aswell as stability to heat or processing and todegradation in appropriate representative gastric andintestinal model systems. Appropriate oral toxicitystudies may be carried out in cases where the proteinis present in the food, but is not closely similar toproteins that have been safely consumed in food,

and has not previously been consumed safely infood, and taking into account its biological functionin micro-organisms where known.

4.6.1.4 Potential toxicity of non-protein substancesthat have not been safely consumed in food shouldbe assessed in a case-by-case basis depending onthe identity, concentration, and biological functionof the substance and dietary exposure. The type ofstudies to be performed may include evaluations ofmetabolism, toxicokinetics, chronic toxicity/carcinogenicity, impact on reproductive function,and teratogenicity.

4.6.1.5 The newly expressed or altered propertiesshould be shown to be unrelated to anycharacteristics of donor organisms that could beharmful to human health. Information should beprovided to ensure that genes coding for knowntoxins or anti-nutrients present in the donororganisms are not transferred to recombinant-DNAmicro-organisms that do not normally express thosetoxic or anti-nutritious characteristics.

4.6.1.6 Additional in vivo or in vitro studies maybe needed on a case-by-case basis to assess thetoxicity of expressed substances, taking into accountthe potential accumulation of any substances, toxicmetabolites or antibiotics that might result from thegenetic modification.

4.6.2 Compositional Analysis of Key Components

Key nutrients or key anti-nutrients are thosecomponents in a particular food that may have asubstantial impact in the overall diet. They may bemajor nutritional constituents (fats, proteins,carbohydrates), enzyme inhibitors as anti-nutrients,or minor compounds (minerals, vitamins). Keytoxicants are those toxicologically significantcompounds known to be produced by the micro-organism, such as those compounds whose toxicpotency and level may be significant to health. Micro-organisms traditionally used in food processing arenot usually known to produce such compoundsunder production conditions. Analysis ofconcentrations of key components (key nutrients,key anti-nutrients and key toxicants) of foodsproduced by recombinant-DNA micro-organismsshould be compared with an equivalent analysis of aconventional counterpart produced under the sameconditions. The statistical significance of anyobserved differences should be assessed in thecontext of the range of natural variations for thatparameter to determine its biological significance.Ideally, the comparator(s) used in this assessmentshould be food produced using the near isogenicparent strain. The purpose of this comparison, inconjunction with an exposure assessment as

necessary, is to establish that substances that canaffect the safety of the food have not been altered ina manner that would have an adverse impact onhuman health.

4.6.3 Evaluation of Metabolites

4.6.3.1 Some recombinant-DNA micro-organismsmay be modified in a manner that could result in newor altered levels of various metabolites in foodsproduced using these organisms. Where alteredmetabolite levels are identified in foods,consideration should be given to the potentialimpacts on human health using conventionalprocedures for establishing the safety of suchmetabolites (for example procedures for assessingthe human safety of chemicals in foods).

4.6.3.2 New or altered levels of metabolitesproduced by a recombinant-DNA micro-organismmay change the population of micro-organisms inmixed culture, potentially increasing the risk forgrowth of harmful organisms or accumulation ofharmful substances. Possible effects of geneticmodification of a micro-organism on other micro-organisms should be assessed when a mixed cultureof micro-organisms is used for food processing, suchas for production of natural cheese, miso, soy sauce,etc.

4.6.4 Effects of Food Processing

The potential effects of food processing, includinghome preparation, on foods produced usingrecombinant-DNA micro-organisms should also beconsidered. For example, alterations could occur inthe heat stability of an endogenous toxicant or thebioavailability of an important nutrient afterprocessing. Information should therefore beprovided describing the processing conditions usedin the production of a food. For example, in the caseof yoghurt, information should be provided on thegrowth of the organism and culture conditions.

4.6.5 Assessment of Immunological Effects

4.6.5.1 When the protein(s) resulting from aninserted gene is present in the food, it should beassessed for its potential to cause allergy. Thelikelihood that individuals may already be sensitiveto the protein and whether a protein new to the foodsupply will induce allergic reactions should beconsidered. A detailed presentation of issues to beconsidered is presented in Annex A.

4.6.5.2 Genes derived from known allergenic sourcesshould be assumed to encode an allergen and beavoided unless scientific evidence demonstratesotherwise. The transfer of genes from organismsknown to elicit gluten-sensitive enteropathy insensitive individuals should be avoided unless it is

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documented that the transferred gene does not codefor an allergen or for a protein involved in gluten-sensitive enteropathy.

4.6.5.3 Recombinant-DNA micro-organisms thatremain viable in foods may interact with the immunesystem in the gastrointestinal tract. Closerexamination of these interactions will depend on thetypes of differences between the recombinant-DNAmicro-organism and its conventional counterpart.

4.6.6 Assessment of Viability and Residence ofMicro-organisms in the Human GastrointestinalTract

4.6.6.1 In some foods produced using recombinant-DNA micro-organisms, ingestion of these micro-organisms and their residence may have an impacton the human intestinal tract. The need for furthertesting of such micro-organisms should be based onthe presence of their conventional counterpart infoods, and the nature of the intended and unintendedeffects of genetic modifications. If processing of thefinal food product eliminates viable micro-organisms(by heat treatment in baking bread, for example), or ifaccumulations of end-products toxic to the micro-organism (such as alcohol or acids) eliminate viability,then viability and residence of micro-organisms inthe alimentary system need no examination.

NOTE — Permanent life-long colonization by ingestedmicro-organisms is rare. Some orally administered micro-organisms have been recovered in faeces or in the colonicmucosa weeks after feeding ceased. Whether thegenetically modified micro-organism is established in thegastrointestinal tract or not, the possibility remains thatit might influence the microflora or the mammalian host.

4.6.6.2 For applications in which recombinant-DNAmicro-organisms used in production remain viable inthe final food product (for example, organisms insome dairy products), it may be desirable todemonstrate the viability (or residence time) of themicro-organism alone and within the respective foodmatrix in the digestive tract and the impact on theintestinal microflora in appropriate systems. Thenature of intended and unintended effects of geneticmodification and the degree of differences from theconventional counterpart will determine the extentof such testing.

4.6.7 Antibiotic Resistance and Gene Transfer

4.6.7.1 In general, traditional strains of micro-organisms developed for food processing uses havenot been assessed for antibiotic resistance. Manymicro-organisms used in food production possessintrinsic resistance to specific antibiotics. Suchproperties need not exclude such strains fromconsideration as recipients in constructingrecombinant-DNA micro-organisms. However,strains in which antibiotic resistance is encoded by

transmissible genetic elements should not be usedwhere such strains or these genetic elements arepresent in the final food. Any indication of thepresence of plasmids, transposons, and integronscontaining such resistance genes should bespecifically addressed.

4.6.7.2 Alternative technologies, demonstrated tobe safe, that do not rely on antibiotic resistancemarker genes in viable micro-organisms present infoods should be used for selection purposes inrecombinant-DNA micro-organisms. In general, useof antibiotic resistance markers for constructingintermediate strains should pose no significanthazards that would exclude the use of the ultimatestrains in food production, provided that theantibiotic resistance marker genes have been removedfrom the final construct.

4.6.7.3 Transfer of plasmids and genes between theresident intestinal microflora and ingestedrecombinant-DNA micro-organisms may occur. Thepossibility and consequences of gene transfer fromrecombinant-DNA micro-organisms and foodproducts produced by recombinant-DNA micro-organisms to gut micro-organisms or human cellsshould also be considered. Transferred DNA wouldbe unlikely to be maintained in the absence ofselective pressure. Nevertheless, the possibility ofsuch events cannot be completely discounted.

4.6.7.4 In order to minimize the possibility of genetransfer, the following steps should be considered:

a) Chromosomal integration of the insertedgenetic material may be preferable tolocalization on a plasmid;

b) Where the recombinant-DNA micro-organism will remain viable in thegastrointestinal tract, genes should beavoided in the genetic construct that couldprovide a selective advantage to recipientorganisms to which the genetic material isunintentionally transferred; and

c) Sequences that mediate integration into othergenomes should be avoided in constructingthe introduced genetic material.

4.6.8 Nutritional Modification

4.6.8.1 The assessment of possible compositionalchanges to key nutrients, which should be conductedfor all foods produced using recombinant-DNAmicro-organisms, has already been addressedunder 4.6.2. If such nutritional modifications havebeen implemented, the food should be subjected toadditional testing to assess the consequences of thechanges and whether the nutrient intakes are likelyto be altered by the introduction of such foods intothe food supply.

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4.6.8.2 Information about the known patterns of useand consumption of a food and its derivatives shouldbe used to estimate the likely intake of the foodproduced using the recombinant-DNA micro-organism. The expected intake of the food should beused to assess the nutritional implications of thealtered nutrient profile both at customary and maximallevels of consumption. Basing the estimate on thehighest likely consumption provides assurance thatthe potential for any undesirable nutritional effectswill be detected. Attention should be paid to theparticular physiological characteristics and metabolicrequirements of specific population groups such asinfants, children, pregnant and lactating women, theelderly and those with chronic diseases orcompromised immune systems. Based on theanalysis of nutritional impacts and the dietary needsof specific population subgroups, additionalnutritional assessments may be necessary. It is alsoimportant to ascertain to what extent the modifiednutrient is bioavailable and remains stable with time,processing, and storage.

4.6.8.3 The use of modern biotechnology to changenutrient levels in foods produced using micro-organisms could result in broad changes to thenutrient profile. The intended modification in themicro-organism could alter the overall nutrient profileof the product, which, in turn, could affect thenutritional status of individuals consuming the food.The impact of changes that could affect the overallnutrient profile should be determined.

4.6.8.4 When the modification results in a foodproduct with a composition that is significantlydifferent from its conventional counterpart, it maybe appropriate to use additional conventional foodsor food components (that is, foods whose nutritionalcomposition is closer to that of the food producedusing the recombinant-DNA micro-organism) asappropriate comparators to assess the nutritionalimpact of the food.

4.6.8.5 Some foods may require additional testing.For example, animal-feeding studies may bewarranted for foods produced using recombinant-DNA micro-organisms if changes in thebioavailability of nutrients are expected or if thecomposition is not comparable to conventionalfoods. Also, foods designed for health benefits, mayrequire an assessment beyond the scope of thesestandards such as specific nutritional, toxicologicalor other appropriate studies. If the characterizationof the food indicates that the available data areinsufficient for a thorough safety assessment,properly designed animal studies could be requestedon the whole food.

ANNEX A(Clause 4.6.5.1)

ASSESSMENT OF POSSIBLE ALLERGENICITY

A-1 INTRODUCTION

A-1.1 All newly expressed proteins produced byrecombinant-DNA micro-organisms that could bepresent in the final food should be assessed for theirpotential to cause allergic reactions. This shouldinclude consideration of whether a newly expressedprotein is one to which certain individuals mayalready be sensitive as well as whether a protein newto the food supply is likely to induce allergic reactionsin some individuals.

NOTE — This assessment strategy is not applicable forassessing whether newly expressed proteins are capable ofinducing gluten-sensitive or other enteropathies. The issueof enteropathies is already addressed in 4.6.5.2 of thisstandard. In addition, the strategy is not applicable to theevaluation of foods where gene products are down regulatedfor hypoallergenic purposes.

A-1.2 At present, there is no definitive test that canbe relied upon to predict allergic response in humansto a newly expressed protein, therefore, it isrecommended that an integrated, stepwise, case bycase approach, as described below, be used in theassessment of possible allergenicity of newlyexpressed proteins. This approach takes into accountthe evidence derived from several types ofinformation and data since no single criterion issufficiently predictive.

A-1.3 The endpoint of the assessment is aconclusion as to the likelihood of the protein being afood allergen.

A-2 ASSESSMENT STRATEGY

A-2.1 The initial steps in assessing possibleallergenicity of any newly expressed proteins are thedetermination of: the source of the introducedprotein; any significant similarity between the aminoacid sequence of the protein and that of knownallergens; and its structural properties, including but

not limited to, its susceptibility to enzymaticdegradation, heat stability and/or, acid and enzymatictreatment.

A-2.2 As there is no single test that can predict thelikely human IgE response to oral exposure, the firststep to characterize newly expressed proteins shouldbe the comparison of the amino acid sequence andcertain physicochemical characteristics of the newlyexpressed protein with those of established allergensin a weight of evidence approach. This will requirethe isolation of any newly expressed proteinsproduced by recombinant-DNA micro-organisms, orthe synthesis or production of the substance froman alternative source, in which case the materialshould be shown to be structurally, functionally andbiochemically equivalent to that produced byrecombinant-DNA micro-organisms. Particularattention should be given to the choice of theexpression host, since post-translationalmodifications allowed by different hosts (that is,eukaryotic versus prokaryotic systems) may have animpact on the allergenic potential of the protein.

A-2.3 It is important to establish whether the sourceis known to cause allergic reactions. Genes derivedfrom known allergenic sources should be assumedto encode an allergen unless scientific evidencedemonstrates otherwise.

A-3 INITIAL ASSESSMENT

A-3.1 Source of the Protein

As part of the data supporting the safety of foodsproduced using recombinant-DNA micro-organisms,information should describe any reports ofallergenicity associated with the donor organism.Allergenic sources of genes would be defined asthose organisms for which reasonable evidence ofIgE mediated oral, respiratory or contact allergy is

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5 REVIEW OF SAFETY ASSESSMENTS

The goal of the safety assessment is a conclusion asto whether the food produced using a recombinant-DNA micro-organism is as safe as the conventionalcounterpart taking into account dietary impact of any

changes in nutritional content or value. Nevertheless,the safety assessment should be reviewed in the lightof new scientific information that calls into questionthe conclusions of the original safety assessment.

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available. Knowledge of the source of the introducedprotein allows the identification of tools and relevantdata to be considered in the allergenicity assessment.These include: the availability of sera for screeningpurposes; documented type, severity and frequencyof allergic reactions; structural characteristics andamino acid sequence; physicochemical andimmunological properties (when available) of knownallergenic proteins from that source.

A-3.2 Amino Acid Sequence Homology

A-3.2.1 The purpose of a sequence homologycomparison is to assess the extent to which a newlyexpressed protein is similar in structure to a knownallergen. This information may suggest whether thatprotein has an allergenic potential. Sequencehomology searches comparing the structure of allnewly expressed proteins with all known allergensshould be done. Searches should be conducted usingvarious algorithms such as FASTA or BLASTP topredict overall structural similarities. Strategies suchas stepwise contiguous identical amino acid segmentsearches may also be performed for identifyingsequences that may represent linear epitopes. Thesize of the contiguous amino acid search should bebased on a scientifically justified rationale in orderto minimize the potential for false negative or falsepositive results. Validated search and evaluationprocedures should be used in order to producebiologically meaningful results.

NOTE — It is suggested to move from 8 to 6 identicalamino acid segments in searches. The smaller the peptidesequence used in the stepwise comparison, the greater thelikelihood of identifying false positives, inversely, thelarger the peptide sequence used, the greater the likelihoodof false negatives, thereby reducing the utility of thecomparison.

A-3.2.2 IgE cross-reactivity between the newlyexpressed protein and a known allergen should beconsidered a possibility when there is more than 35%identity in a segment of 80 or more amino acids orother scientifically justified criteria. All theinformation resulting from the sequence homologycomparison between the newly expressed protein andknown allergens should be reported to allow a case-by-case scientifically based evaluation.

A-3.2.3 Sequence homology searches have certainlimitations. In particular, comparisons are limited tothe sequences of known allergens in publiclyavailable databases and the scientific literature.There are also limitations in the ability of suchcomparisons to detect non-contiguous epitopescapable of binding themselves specifically with IgEantibodies.

A-3.2.4 A negative sequence homology resultindicates that a newly expressed protein is not aknown allergen and is unlikely to be cross-reactive

to known allergens. A result indicating absence ofsignificant sequence homology should beconsidered along with the other data outlined underthis strategy in assessing the allergenic potential ofnewly expressed proteins. Further studies should beconducted as appropriate. A positive sequencehomology result indicates that the newly expressedprotein is likely to be allergenic. If the product is tobe considered further, it should be assessed usingserum from individuals sensitized to the identifiedallergenic source.

A-3.3 Pepsin Resistance

A-3.3.1 Resistance to pepsin digestion has beenobserved in several food allergens; thus a correlationexists between resistance to digestion by pepsin andallergenic potential. Therefore, the resistance of aprotein to degradation in the presence of pepsinunder appropriate conditions indicates that furtheranalysis should be conducted to determine thelikelihood of the newly expressed protein beingallergenic. The establishment of a consistent andwell-validated pepsin degradation protocol mayenhance the utility of this method. However, it shouldbe taken into account that a lack of resistance topepsin does not exclude that the newly expressedprotein can be a relevant allergen.

A-3.3.2 Although the pepsin resistance protocol isstrongly recommended, it is recognized that otherenzyme susceptibility protocols exist. Alternativeprotocols may be used where adequate justificationis provided.

A-4 SPECIFIC SERUM SCREENING

A-4.1 For those proteins that originate from asource known to be allergenic, or have sequencehomology with a known allergen, testing inimmunological assays should be performed wheresera are available. Sera from individuals with aclinically validated allergy to the source of the proteincan be used to test the specific binding to IgE classantibodies of the protein in in vitro assays. A criticalissue for testing will be the availability of humansera from sufficient numbers of individuals. Aminimum of 8 relevant sera is required to achieve a99 percent certainty that the new protein is not anallergen in the case of a major allergen. Similarly, aminimum of 24 relevant sera is required to achievethe same level of certainty in the case of a minorallergen. It is recognized that these quantities of seramay not be available for testing purposes. In addition,the quality of the sera and the assay procedure needto be standardized to produce a valid test result. Forproteins from sources not known to be allergenic,and which do not exhibit sequence homology to aknown allergen, targeted serum screening may beconsidered where such tests are available.

A-4.2 In the case of a newly expressed proteinderived from a known allergenic source, a negativeresult in in vitro immunoassays may not beconsidered sufficient, but should prompt additionaltesting, such as the possible use of skin test and exvivo protocols (Ex vivo procedure is the testing forallergenicity using cells or tissue culture from allergichuman subjects). A positive result in such testswould indicate to a potential allergen.

A-5 OTHER CONSIDERATIONS

A-5.1 The absolute exposure to the newly expressedprotein and the effects of relevant food processingwill contribute toward an overall conclusion aboutthe potential for human health risk. In this regard,the nature of the food product intended forconsumption should be taken into consideration in

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determining the types of processing which would beapplied and its effects on the presence of the proteinin the final food product.

A-5.2 As scientific knowledge and technologyevolves, other methods and tools may be consideredin assessing the allergenicity potential of newlyexpressed proteins as part of the assessmentstrategy. These methods should be scientificallysound and may include targeted serum screening(that is the assessment of binding to IgE in sera ofindividuals with clinically validated allergicresponses to broadly-related categories of foods);the development of international serum banks; useof animal models; and examination of newlyexpressed proteins for T-cell epitopes and structuralmotifs associated with allergens.

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Review of Indian Standards

Amendments are issued to standards as the need arises on the basis of comments. Standards are also reviewedperiodically; a standard along with amendments is reaffirmed when such review indicates that no changes areneeded; if the review indicates that changes are needed, it is taken up for revision. Users of Indian Standardsshould ascertain that they are in possession of the latest amendments or edition by referring to the latest issue of‘BIS Catalogue’ and ‘Standards: Monthly Additions’.

This Indian Standard has been developed from Doc No. : FAD 23 (1794).

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