Biosafety Issues

8/13/2019 Biosafety Issues

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Biosafety Issuesof

Genetically Modified Food

Dr. Debasis Pattanayak

Principal Scientist

National Research Centre on Plant Biotechnology

New Delhi


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Risk

Assessment

*science-based

Risk

Management

*policy-based

Risk

Communication

*interactive

exchange

Risk analysis is composedof three distinct activities

Risk assessment

Risk communication Risk management

Although there is inevitableoverlap, these activities are

separate

Risk analysis


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Risk

Management

*policy-based

RiskCommunication

*interactive

exchange

Risk

Assessment

*science-based

A scientific processused toidentify hazards, the likelihood ofoccurrence, and the impacts onhuman and animal health and onthe environment

Identification of possible riskmitigation measures

Evidence-based and scientificallydefensible

Requires diversecollection ofexpertiseand a significantknowledge base

Harmonizedwith internationally

acceptable approaches

Risk assessment


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Science-based

Realm of Bio-Politics

Risk = Exposure X Hazard

Hypothetical risk:the hazard has

not occurred BUT there is a scientific

basis to support its existence

Probabilistic risk:the hazard exists

AND has occurred at least once

Speculative risk:the hazard has not

occurred AND there is NO scientific

line of reasoning to suggest it will

Types of risks


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Risk

Assessment

*science-based

Risk

Communication

*interactive

exchange

Risk

Management

*policy-based

Decision-making

Implementation of riskmitigationmeasures

Balancebetween safetyand non-safety

considerations (e.g.,socio-economic, cultural,and political)

Consideration of benefits

as well as risks

Risk management


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Risk

Assessment

*science-based

Risk

Management

*policy-based

Risk

Communication

*interactiveexchange

A (non-technical) dialogue about

risks, real and perceived

Goal is to reduce the discrepancy

between real risks and perceived

risks

Effective risk communicators are

trusted and credible

Risk communication fails when

there is an information vacuum

between scientific assessment of

risk and the publics perception of

risk

Risk communication

How to balance public good vs. theindividual rights of developers,

farmers, producers, andconsumers?


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Historically, our beliefs about the safetyof foods havebeen based almost entirely on tradition and culturalexperience

In practice, very few of the foods we eat today have beensubject to any toxicological studies and yet they aregenerally accepted as safe

Even foods that contain toxins or anti-nutrients orallergens have been considered safe through a long

history of use Consider potatoes, tomatoes, peanuts, eggs, milk

products, wheat products, strawberries and otherfruits, fish, shellfish, etc

Traditional views of food safety


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It is impossible to prove that something is safe

The best that can be demonstrated is the absence of

evidence of the production of harm

Present and future safety can only be judged on the

basis of past experience, an absence of evidence of

harm is the only evidence we can ever expect for the

absence of harm

Can safety be proven ?


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At the heart of the risk assessment process is the

principle that GM foods CANbe compared with traditional

counterparts that have an established history of safe use

This comparison can be based on an examination of thesame types of risk factors for both (e.g.toxins, potential

allergens, key nutrients, anti-nutrients).

This comparative approach, whereby the food being

assessed is compared with one that has an acceptedlevel of safety, is often expressed in the concept of

Substantial Equivalence.

The comparative approach


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Genetic modification does not result in food which is inherently less

safe than that produced by conventional means

Risk associated characteristics assessed for GM food are equivalent

to the same characteristics for conventional counterpart

Variance of these characteristics must fall within the natural range

demonstrated for conventional counterpart

Substantially equivalent does not mean that two products are

identical, but that one can be substituted for the other without

adversely affecting the health and/or nutritional status of the

consumer

Substantial equivalence should be considered as the starting point

only, useful for identifying the defined differences

Key points


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Direct consequences

Nutritional, toxic, orallergenic effectsdue to the novelgene products

Purposefully alteredlevels of existinggene products (e.g.use of anti-sensestrategies todecrease

expression levels ofspecific proteinsFlavr Savr tomato)

Indirect consequences

Effects of new gene products, oraltered levels of existing geneproducts, on plant metabolism

Unanticipated effects of geneticmanipulation such as:

gene silencing,

interruption of codingsequences, or

altered regulation of genes, or

effects related to somaclonalvariation arising from tissueculture regeneration of plants

Statistically significant differences should be

assessed for their biological significance

The safety assessment process


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Availability of data on compositional properties

Availability of data on natural variations Availability of analytical methodsparticularly for

detecting unintended effects (e.g.- Low glutelin

transgenic rice and Golden rice)

Limitations ..


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While each product is evaluated on a case-by-case basis, generally the evaluation focuseson these key areas: Host and Donor Organisms

Provide knowledge on natural range and variation of key

components, potential toxicity and allergenicity of donor geneproducts

Molecular Characterization Description of the donor and host organisms, modification

process, genetic stability, expressed proteins

Nutritional and Compositional Analysis Altered levels of naturally occurring toxins, nutrients, and anti-

nutrients

Toxicology Potentialtoxicity of novel protein

Allergenic Potential Possibility that introduced protein(s) will result in allergic reaction

Food safety assessment:

What do evaluators typically look at?


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Knowledge on Host and Donor Organisms


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Host = organism that was transformed

Context wherein introduced trait is expressed

Cognizance of normal range in variation of hosts

traits under different growing environments,

seasons, developmental phases, geneticbackgrounds, etc.

- Green potatoes: high concentrations of the glycoalkaloids solanine

- Tomatoes: high concentrations of glycoalkaloids -tomatine, which

decline dramatically with ripening

Knowledge of the host organism


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Knowledge of the properties of the final edible product is

important in establishing appropriate comparator(s)

Refined, bleached oil, which is free of DNA and protein,

is the only product of oilseed rape (canola) consumed

by humansfrom a risk assessment perspective, howrelevant is it to consider levels of expression of

introduced proteins?

Cyanogenic glycosides in cassava can release high

concentrations of HCN. They must be removed byleaching and/or cooking in order to prevent toxicity

Knowledge on food processing


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Usefulness of substantial equivalence for assessing risks

depends on a thorough knowledge of the host organism

and the counterpart foods that are used as the basis for

comparisonwith the GM food

Natural range and variation of key:

Nutrients

Vitamins, cofactors, essential amino acids

Toxicants Glycoalkaloids, glucosinolates, cucurbiticin

Anti-nutrients

Soybean trypsin inhibitor

Allergens

Knowledge of the host organism


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Provides important information on the

potential for toxicity, pathogenicity, or

allergenicity

Introduced proteins from known sources of

allergens must be considered potential

allergens until proven otherwise

2S storage protein from Brazil nut that was

introduced into soybeanwas suspected to be

a potential allergen at the development phase

and this was confirmed in subsequent testing

Knowledge of the donor organism


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Required information

Taxonomic classification, common name,

scientific name

Information on known pathogenicity,

toxicity, alergenicity Previous use/presence of organism (or

specific products) in food or feed

(contaminant, infectious agent, aditive,

etc.)

Gene(s) donated

Knowledge of the donor organism


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Gene / Sequence Mode of Action / Toxicity / Allergenicitycry1AB (Bacillus

thuringiensis subsp.

kurstaki)native gene

(3468 nucleotides, 1156

amino acids)

Affects the larvae of susceptible insect species by

binding to specific receptors on the plasma

membrane of mid-gut epithelial cells, resulting in

the formation of pores and ion equilibration. The

gut becomes immobilized, the larva stops feeding,

gut pH drops, and bacteria invade causing lethal

septicemia.

While target insects are susceptible, there is no

evidence of toxic effects on non-target beneficial

insects, mammals, fish, or birds given the

equivalent of 10 g/g (body wt) of Bt protein.

Enhanced, duplicated

CaMV 35S promoter,

hsp70intron, NOS 3

terminator

Regulatory sequences, no expression products.

None of these sequence are known to have any

pathogenic or harmful effects.

Knowledge of the donor gene


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Gene / Sequence Mode of Action / Toxicity / AllergenicityEPSPS (Zea mays)native

gene

The native maize EPSPS encoding gene was cloned,

modified by site-directed mutagenesis, and reintroduced

into embryogenic maize cells by microparticle

bombardment (biolistic transformation).

The amino acid sequence of the modified EPSPS proteindiffers from the wild-type sequence by two amino acids

(99.3% sequence identity).

No reports of allergenicity, pathogenicity, or acute toxicity

for EPSPS proteins.

Rice actin I promoter andintron sequences; Rubisco

derived chloroplast transit

peptide sequences (maize

and sunflower); nos3

polyadenylation signal fromA.

tumefaciens

Promoter and terminator sequences do not encode proteinexpression products. Chloroplast transit peptide sequence

is plant derived and not known to have any pathogenic or

harmful effects.

Knowledge of the donor gene


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Molecular Characterization


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It is very important that a rigorous molecular

characterization of each transgenic plant

submitted for review be completed, it is equally

important to recognize the limitations of thisapproach in predicting the safety of a novel food.

The molecular characterization of transgenic

plants often receives a disproportionate amount

of attention from regulators relative to the

information it imparts in terms of food, feed or

environmental safety.

Significance of molecular characterization


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Maybe able to address issues related to

positional effects, pleiotropic effects, and gene

silencing

Provides information on the composition and

integrity of the inserted DNA

Ensures that the developer has appropriately

characterized the genetic modification

Utility of molecular characterization


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In the absence of other empirical data (phenotypic

characteristics) the molecular characterization is unlikely

to predict unforeseen effects on the concentrations of key

nutrients, anti-nutrients, or endogenous toxins

There is, for example, no correlation between copy

number of the inserted DNA and safety

B. napusevents 23-198, 23-18: developed by inserting

a thioesterase gene from Lauris nobilis(California bay

tree) in order to increase levels of lauric acid.

The original transformant was estimated to contain 15

copies of the inserted gene

Limitations


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1. The transformation system Agrobacterium-mediated

Protoplast system

Microparticle bombardment

2. Molecular characterization of the inserted DNA

Insert number

Insert composition

3. Genetic stability of the introduced trait

Segregation analysis

Integron stability

Three aspects to consider


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Common soil bacterium responsible for causing crowngall disease in susceptible plants

Incorporation of a region of transfer DNA (T-DNA) from alarge (150-250 kb) circular Ti (tumour inducing) plasmid

Replace the phytohormone biosynthetic genes within theT-DNA with genes of interest

A. tumefaciensnaturally infects only dicots but methodshave been developed for monocots such as rice, banana,maize, wheat, and sugarcane

Ag robacter ium tumefaciens


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A. tumefacienstransformation is generally characterized

by:

Low transgene copy number

Minimal rearrangements

Higher transformation efficiency than direct DNA

delivery techniques such as microparticle

bombardment



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Up until 1995, it was generally assumed that only

sequences between the left and right borders of the T-

DNA were incorporated into the host genome, but this is

not always the case

Inserted sequences may be coupled with the right or left

border sequences, or as an independent unit unlinked

from the T-DNA



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Differential expression of transgenes has been attributed

to:

copy number - the number of transgene copies

integrated into the host genome

positional effects - the position of the T-DNA

integration site in the host genome may affect the level

of expression

variable arrangements of transgene sequences in the

host genome e.g.multiple copies in direct or inverted

repeats



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The food safety dossier should include:

A summary table of all the genetic elements within theplasmid including coding and non-coding regions.

For each element include:

Citations where sequence was described, isolatedand characterized

Portion and size of the insert

Location, order and orientation of the vector

Function in the host plant The donor organism

Information for the risk assessor


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A detailed map of the plasmid with the

location of the sequences described in the

summary table should be provided for use

in the analysis of data supporting the

characterization of the host plant.

The map should include relevant restriction

enzyme sites, locations of primers used forPCR, and any regions used as probes.

Information on the plasmid map


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Refers to the number of sites where the transgenic

element is incorporated into the host genome.

This is deduced by digesting genomic DNA with a

restriction enzyme that does not cut within the transgenicelement followed by Southern blot analysis with a probe

specific to one or more of the introduced genes.

More than one band = more than one insertion site.

This should be repeated with at least one other restriction

enzyme to confirm the number of inserts.

The inserted DNA: insert number


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Gene A Gene B

BamHI

Inserted Element

Genomic DNABamHI digest

32P-labelled cDNA probe

Southern

blot

Should yield a

single band if

one insert

The inserted DNA: insert number

3.5 kb

109 135 136 146 147 150 151 152 154 155 156 157 NT PC


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This is not the same as the number of insertion sites

Digestion with one or more (i.e. can be single or doubledigests) restriction enzymes that either do not cut withinthe transgenic element, or cut only once but not within the

sequence complementary to the hybridization probe This should yield one band per inserted element.

Usually this is done with more than one restrictionenzyme, or combination of enzymes.

Transgene copy number


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Gene A Gene B

EcoRI

Inserted Element

32P-labelled cDNA probe

Genomic

DNA

EcoRI digest Southern

blotA

A

B

B

XhoI

XhoI digest Southern

blot

A singleband from

each

digestion

indicates a

single copy

Transgene copy number

G I t it


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The goal is to determine if the gene(s) of interest are intact, or

whether there have been truncations/deletions

Digest genomic DNA with restriction enzymes to isolate the

gene of interest

Hybridize with a gene-specific probe. Resolved band should be the same size as that isolated from

the plasmid.

Alternatively, PCR with 5- and 3-terminal specific primers in

order to amplify a fragment of the same size as the insertedgene

Gene Integrity

G ti t bilit


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For each novel trait, the pattern and stability of inheritance

must be demonstrated as well as the level of expression of the

trait.

Serological techniques are generally used to measure trait

expression either qualitatively [e.g., Western immunoblotting,enzyme linked immunosorbent assay (ELISA), etc.] or

quantitatively (e.g., ELISA, radioimmunoassay, etc.).

If the new trait is one that does not result in the expression of a

new or modified protein (e.g., transgenic plants containing

inserted antisense sequences) then its inheritance will have tobe determined by examining the DNA insert directly or by

measuring RNA transcript production.

Genetic stability

P tt f i


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How, when, and where

Based on the promoter used (constitutive, tissue-specific,

inducible, etc), is the novel protein expressed in the

expected tissues and under the expected conditions?

Presence or absence and amounts of expressed protein

should be determined for a range of plant tissues (e.g.

roots, leaves, seeds, pollen)

And, from a food safety perspective, levels of expression

of the protein(s) in the edible portionsof the plant arecritical

Patterns of expression


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Nutritional assessment

Ai f t iti l t


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For GM plants that were not developed to have

intentionally altered nutritional value, the aim of

the nutritional evaluation is to investigate

whether there have been unintentional changesin levels of key nutrients, toxicants, allergens and

anti-nutrients

Aim of nutritional assessment

Bi il bilit


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In addition, in foods modified to increase nutrient

levels, bioavailability needs to be substantiated

Both in vitroand in vivostudies may be used for

the purpose

Bioavailability

U i t d d ff t


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Can arise in several ways:

Insertional inactivationdisruption or alteration of the

expression of a normal gene

Alteration of metabolite poolsexpression of the new

gene could alter the availability of amino acids, or

divert substrates from other metabolic pathways

The expressed protein, or altered levels of other

proteins, could have anti-nutritional effects

None of these possibilities are unique to transgenic

plants, they can also occur with traditional breeding, and

in fact may be less frequent in transgenic plants because

only a few genes are transferred during the genetic

modification

Unintended effects


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Single compound analysis reveals secondary changesonly by chance or if anticipated

e.g., looking for changes in levels of glycoalkaloids

Profiling methods allow for the simultaneous screening ofmany compounds without prior identification

Protein profiles2D gel electrophoresis

Chemical fingerprintschromatography/NMR/MS

mRNA profiles to look for altered gene expressionDNA microarray technology, has been used to

examine green vs. ripe tomatoes

Detecting unintended effects


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None of the profiling methods is sufficiently welladvanced to be used on a routine basis for identifyingunintended consequences of genetic modification

Much to be done on standardization and validation ofmeasurements, sample collection

In many cases the natural range of variation due toabiotic factors, plant growth stage, disease status, etc

may overwhelm any differences due to geneticmodification

Bioinformatics challenge to handle the very large datasets generated, particularly from microarray testing

Limitation of profiling

Traditional compositional analysis


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Protein

Fat

Fibre

Starch Amino acid composition

Fatty acid composition

Ash

Sugars

Calcium

Phosphorus

Traditional compositional analysis

Context of nutrient changes


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Changes in the nutrient levels become

significant when they differ markedly from

literature values and also:

The relative contribution of the nutrient to thetotal intake (including other sources) is high

The range of consumption of the grain or

food/feed in the specific population is high The food is intended for specific groups such

as pregnant/lactating women, children and the

elderly.

Context of nutrient changes


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Assessment for Toxicity

Focus on the defined difference


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The principal focus of toxicity evaluation is/are the protein

expression product(s) of the inserted gene(s)

The inserted DNA, in itself, does not pose a food safety

concern

Normal dietary intakes of DNA/RNA vary widely but

are generally in the range of 0.11.0 g/day

Any concerns over the presence of novel DNA in the

diet must consider that this DNA would represent


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Introduced genes are often derived from microorganisms that do not

have a history of significant consumption by humans

Bt proteins (Cry1Ab, Cry1Ac, Cry3A) from Bacillus

thuringiensis

PAT enzyme from Streptomyces viridochromogenes, S.hygroscopicus

EPSPS fromA. tumefaciens

However, in some cases the protein products of introduced genes

have been consumed in significant amounts

Viral coat proteins from transgenic potatoes, papaya and squash

Isolated plant genes that have been modified by in vitrosite-

directed mutagenesis and re-inserted (e.g.mEPSPS in GA21

maize)

Reason for concern

Weight of evidence approach in evaluating potential


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Gene source

Associated with known toxins?

Relationship with substances having a known history of safeconsumption/exposure

If Yes, then acute toxicity testing or additional testing not

necessary Amino acid sequence comparison

Any relationship with known protein toxinssimilar parametersas for homology searches of allergen databases

In vitrodigestibility models

Stability to pepsin digestionif yes, then possible toxin Heat stability

Stability to heat inactivationif yes, then possible toxin

Weight of evidence approach in evaluating potential

toxicity of transgenic protein

Assessment strategy


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Fully characterizing a novel (transgenic) proteinrequires examining:

Physiochemical properties

Digestive fate Biologic and/or enzymatic activity

Animal feeding trials (acute oral toxicity)

Allergenicity testing

Assessment strategy

Protein toxins


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Generally, novel proteins, like other dietary proteins, havea predictable metabolic fate upon ingestionthey are

broken down to their constitutive amino acids under

digestive conditions

Proteins that behave this way, or are inactivated by heat

(as in processing) are unlikely to exert adverse affects

Protein toxins (and allergens) tend to be resistant todigestionthis is a necessary but not a sufficient

condition, as in the absence of other toxicological

evidence there is no consensus on resistant proteins

being a higher risk

Protein toxins

In v i t ro studies


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Digestibility assaysincubating purified protein with

simulated gastric and intestinal fluids and determining the

extent of degradation over time

This can be followed either by SDS-PAGE or by

measuring biological and/or enzymatic activity ofsamples following incubation with simulated digestive

fluids

Heat inactivation assays

Amino acid sequence comparisons

Lack of homology with known toxins can be used as

additional evidence for low potential for toxicity

In v i t rostudies

Unintended effects


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A related issue is the possibility of unintentionally

modifying metabolic pathways as a consequence

of gene insertion, thus affecting concentrations of

endogenous toxicants

Since the toxic potential of endogenous toxins is

already known, toxicological assessment is not

required but determination of their levels is

necessary as part of the compositional/nutritional

analysis

Unintended effects

A i l t di


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Commonly used for the safety assessment of many

compounds, including pesticides, pharmaceuticals,

industrial chemicals and food additives

Works well for substances of known purity, no

nutritional value, low potential for human exposuretherefore easy to test at high multiples of anticipated

exposure and determine no observed effect levels

(NOELs)and thus establish safe upper limits (e.g.

acceptable daily intakes)

Problematic with whole foods but can be used to

investigate acute oral toxicity of purified proteins

Animal studies


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Assessment for Allergenicity

Adverse reactions to food


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Non toxic

Immune mediated

(food allergy)

Non immune mediated

(food intolerance)

Enzymatic

Pharmacological

UndefinedIgE Non IgE

Toxic



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Adverse reactions to an otherwise harmless food or food component

that involve an abnormal response of the bodys immune system

IgE-mediated responses, immediate hypersensitivity responses

usually within minutes or a few hours after ingestion

Non IgE-mediated immune reactions (IgG, IgA) or cell-mediated

immunity (celiac disease, cows milk, egg, soy sensitivity) in the

latter case, symptoms usually occur >8 hrs after ingestion

Food allergies

IgE-mediated

Anaphylactic shock, gastro-intestinal disorders, urticaria(hives), diarrhea, vomiting, abdominal pain

Cell-mediated reactions

Small intestine inflammation, malabsorption, diarrhea, bonepain

Most common food allergens


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More than 170 foods cause food allergies

Eight foods are responsible for >= 90% of all allergic

reactions

Cows milk

Eggs

Fish

Crustaceans

Peanuts Soybeans

Tree nuts

Wheat

Most common food allergens

Properties of allergens


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Virtually all allergens are proteins BUT not allproteins are food allergens

Foods contain tens of thousands of proteins -

very few are allergens Most allergens are stable to digestion and

processing

Major allergens tend to be abundant proteins

Many, but not all food allergens have beencloned and characterized (The Codex AlimentariusCommission)

Properties of allergens

Analysis for allergenicity


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Source of protein Allergic sourcesreasonable evidence of IgE mediated oral,

respiratory, or contact allergy

Amino acid sequence homology

>=35% identity in window of 80 amino acids

Contiguous epitope size should be scientifically justifiable Pepsin resistance

Strongly recommended, although other digestive models could beused with justification

Specific serum screening

If allergic source or sequence homology with allergens If protein from allergenic source, negative result here may still

require additional studiesskin prick test, etc

Absolute exposure to the novel protein and the effects of relevantfood processing will contribute to the overall conclusion aboutpotential allergenicity.

Analysis for allergenicity

Pepsin digestibility model


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Protein

Stability (minutes)

Whole Protein FragmentsEgg Allergens

ovalbumin 60 -

ovomucoid 8 -

conalbumin 0 15

Soybean Allergensbeta-conglycinin (beta subunit) 60 -

beta-conglycinin (alpha subunit) 2 60

soy lectin 15 -

Gly m1 0.5 8

Mustard Allergens

Sin a1 60 -

Peanut Allergens

Ara h2 60 -

peanut lectin 8 -




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Protein

Stability (minutes)

Whole Protein Fragments

Common Plant Proteins

glycoate reductase (spinach) 0.25 (15 sec) -

Rubisco LSU (spinach leaf) 0 (< 15 sec) -

PEP carboxylase 0 (< 15 sec) -

lipoxygenase (soybean seed) 0 (< 15 sec) -

acid phosphatase (potato) 0 (< 15 sec) -

phosphofructose kinase (potato) 0 (< 15 sec) -

Transgenic Proteins

Cry1A 0.5 (30 sec) -

Cry2 0 (< 15 sec) -

Cry3 0 (< 15 sec) -

CP4 EPSPS 0 (< 15 sec) -

mEPSPS 0 (< 15 sec) -

GOX 0 (< 15 sec) -


Assessment strategy


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Source of the introduced protein

Amino acid sequence homology

Pepsin resistance

Specific serum screening

Absolute exposure

Totality of assessments (weight of evidence) provides

reasonable assurance that foods will not be renderednewly allergenic

Assessment strategy

Brazil nut allergen in soybean


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2S methionine-rich albumin was

transferred from the Brazil nut to soybean

as a means of enhancing poultry feed

2S albumin is a major Brazil nut allergen

Because allergic response was an

anticipated possibility, transgenic soybean

extracts were tested on patients allergic toBrazil nuts using skin-prick assays during

the development phase of the project

Brazil nut allergen in soybean

Skin prick testing Brazil Nut


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Skin prick testing Brazil Nut

Conclusions on allergenicity test


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GM foods that are to be commercialized should beassessed for allergenicity except where there is a history

of safe consumption e.g., viral coat proteins

Considering the source, amino acid sequence homology,

and pepsin digestibility are the basic tools Serum screening (specific and/or targeted) may not be

warranted in most casesstill may need human studies

(skin prick test)

Further studies are needed to determine thresholds, ifany, for sensitization

In the future, animal models could be a valuable tool for

predicting allergenicity, but currently they are inadequate

g y

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