Dr. Michael Hansen on Detecting
Likely Allergenicity of GE Foods

Science-based Approaches to Assessing Allergenicity of New Proteins in
Genetically Engineered Foods

Presentation to FDA Food Biotechnology Subcommittee,
Food Advisory Committee
College Park, MD

August 14, 2002
Michael Hansen, Ph.D., Research Associate, Consumers Union

Key Points
Amino Acid sequence homology/similarity
6.1. Sequence Homology as Derived from Allergen Databases
Digestive Stability (enzymatic digestion)
Simulated gastric fluid (SGF)
6.4. Pepsin Resistance
Heat stability
Animal models


Thank you for the chance to present the views of Consumers Union, publisher
of Consumer Reports, to this Subcommittee. The Food and Drug
Administration is taking a very positive, important and much-needed step by
undertaking an effort to develop a protocol for assessing the potential
allergenicity of genetically engineered food. Food allergies can be life
threatening for the estimated 2% of adults and 8% of children who suffer
from them. The potential to inadvertently transfer a gene that codes for an
allergen through genetic engineering was demonstrated when Brazil nut genes
were transferred into soybeans by Pioneer Seeds. Pioneer fortunately
conducted tests that determined that an allergen had been inadvertently
transferred, and voluntarily stopped developing the product. However this
case, and the subsequent case of Starlink corn, whose potential
allergenicity was much more difficult to predict, underline the need to have
a sound, consistent, and comprehensive assessment protocol. The FDA
protocol should be one which when scientific data is incomplete errs on the
side of protecting consumer health, and is used by all companies developing
products and by all agencies regulating them.

We think this guidance should be incorporated in the rule on Pre-Market
Notification which FDA has under development. Our comments will focus
primarily on the specifics of what the assessment should contain and how it
should be conducted.

The FDA can profitably draw on the work of several excellent bodies that
have already given considerable thought to the difficult question of
allergenicity assessment. We would like to draw special attention to the
2001 report of the Joint FAO/WHO Joint Expert Consultation on Allergenicity
of Foods Derived from Biotechnology, chaired by Dr. Dean Metcalfe of the
National Institute of Health (FAO/WHO, 2001), to the Annex on Allergenicity
to the Guidelines for Assessment of the Safety of Recombinant DNA Plants,
agreed to last March by the Codex Alimentarius Ad Hoc Committee on Foods
Derived from Biotechnology, and to the work of the Environmental Protection
Agency's (EPA) FIFRA Scientific Advisory Panel (SAP) which was charged with
developing mammalian toxicity assessment guidelines for protein plant
pesticides and with assessing the human safety of Starlink corn (SAP,

Key Points

As an overview, we urge FDA to:

--make this protocol a rule not guidance; it needs to be mandatory and not

--include all allergens, dermal and inhalant as well as food when
determining amino acid sequence homology/similarity between new proteins and
known allergens

--consider all the assessment criteria used by the EPA SAP and/or FAO/WHO
2001 Expert Consultation and/or EPA: amino acid sequence homology,
digestive stability, heat stability, animal models, physical characteristics
(size/molecular weight, probable glycosylation)

-- integrate all criteria into a decision tree since no single criterion is
absolutely predictive as to allergenicity; we suggest use of the FAO/WHO
2001 decision tree, modified where necessary to take account of the fact
that some assessment techniques are much better developed than others. In
general, FDA should require standardized procedures/methodologies for the
use of individual assessment criteria used in the decision tree with a view
to a harmonized application of the decision tree.

--conduct tests for all "newly expressed proteins" (language from Annex 1 of
draft safety assessment guidelines for rDNA plants from the Codex
Alimentarius Ad Hoc Task Force on Foods Derived from Modern Biotechnology);
this means not just the intended transgene product (e.g. protein), but also
includes all the unintended newly expressed proteins (e.g. the process of GE
may turn on genes in a plant/animal that had been previously turned off, or
the transgene protein could interact with the complex metabolic pathway in
the organisms to create a new protein).

--require proteins be tested in purified form and as they exist in the food

--require purified proteins be extracted from the plant and/or animal from
which the food will be derived; FDA should not allow a company to test the
protein as it is expressed in a bacterial or other microbial source if that
is not the form that will be consumed.

We will now comment on several key assessment techniques that we think must
be part of an assessment protocol.

Amino Acid sequence homology/similarity

Although no single criterion has been shown to be absolutely
accurate in predicting the allergenicity of a (novel) protein, perhaps the
most basic criterion that has been employed is the notion that proteins
which are similar in structure (e.g. homologous) to a know allergen will
have a greater likelihood of being an allergen than a protein which has
little or no structural similarity to known allergens. Thus, virtually all
protocols that have been developed to test the allergenicity of genetically
engineered proteins include the comparison of amino acid sequence of novel
(engineered) proteins with those of known allergens (Metcalfe et al., 1996;
NRC, 2000; SAP, 2000a; FAO/WHO, 2001).

Perhaps the first protocol developed to help test for the
possible structural similarity between a novel protein (of unknown
allergenic potential) and a known allergen was contained in decision tree
developed by the industry-funded International Food Biotechnology Council
(IFBC) in conjunction with the Allergy and Immunological Institute of the
International Life Sciences Institute (ILSI) [Metcalfe et al., 1996]. Given
that the 3-dimensional structure of most allergenic epitopes is not known,
the IFBC/ILSI decision tree focused on the amino acid sequence homology of
the newly introduced protein and a data base of known allergens and
recommended that any sequence of eight contiguous amino acids in the test
protein that exactly matches a corresponding sequence in a known allergen,
using a global algorithm that optimizes alignments/matches across the entire
full-length of the protein, should be a cause for concern and should trigger
further investigation. This has been termed the "eight amino acid match
approach" (EAAM-approach). Sequence of identity of less than 8 amino acids
is not considered to raise concerns about potential allergenicity. A
slightly modified version of the IFBC/ILSI decision tree can be seen in
Figure 1.

In the six years since the IFBC/ILSI decision tree approach, a
number of changes or refinements to the approach, based on accumulating
scientific knowledge, have been suggested for detecting structural
similarity between novel proteins and known allergens. Some of the
suggested refinements include: i) allowing for substitution of chemically
similar amino acids in the 8-amino acid sequence (Fuchs and Astwood, 1996;
Gendel, 1998b; SAP, 2000a); ii) using identity of 6 or 4 identical
contiguous amino acids rather than 8 (SAP, 200a; Becker, 2001; FAO/WHO
2001); iii) using local alignments (regions with a high degree of
similarity) rather than the entire protein (e.g. a global alignment) when
comparing unrelated proteins (Gendel 1998b; Becker, 2001; FAO/WHO, 2001);
iv) using 35% overall amino acid homology to a known allergen as an
additional criterion (FAO/WHO 2001); and v) developing databases and methods
to test for conformational or discontinuous epitopes (defined by 3-D
structure rather than simple amino acid sequence) including those caused by
changed glycolysation patterns (SAP 2000a; FAO/WHO, 2001; Becker, 2001).

Most of the above problems/suggested modifications of the
IFBC/ILSI decision tree approach to sequence homology have been succinctly
described by Dr. Wolf-Mienhard Becker in his paper, "Sequence homology and
allergen structure," written for the 2001 Joint WHO/FAO Expert Consultation
(Becker, 2001). Dr. Becker notes that the use of the EAAM-approach "leads
to the insight that conformational epitopes and contiguous parts of these
epitopes after denaturation, and epitopes made up by sugar residues, are not
identifiable by this procedure. Apart from the result [that] identified
linear epitopes of peanut or cod fish only consist of 6 or 4 contiguous
amino acid residues which are essential for IgE binding. Thus the
EAAM-approach would fail. The conclusion from that is that the EAAM-approach
even including only six contiguous amino acids can only identify potential
allergenic components but not rule them out. Since predicting or excluding
allergenicity is a matter of immunology the epitope, the interface between
chemical structure and the immune system, should come into focus. . . .
chemical structure is suitable but the most convincing tools are epitope
receptors such as patients' IgE or monoclonal antibodies to test the
allergenicity of the protein in question in the genetically engineered food.
Since the maturation of the immune system cannot be predicted monitoring
studies of immune responses in consumers should be undertaken after the
genetically engineered food has reached the market" (Becker, 2001: 1).

The focus on epitopes is a crucial one since the immune system
cannot recognize the whole structure of a macromolecule, such as a protein
or glycoprotein, but can only smaller sections called determinants or
epitopes. The caveat to this is that the immunological behavior of an
epitope can be affected by the whole structure of the macromolecule. In
principal, two types of epitopes exist: linear (or continuous) epitopes
based directly on the primary protein structure (e.g. amino acid sequence)
and conformational (or discontinuous) epitopes based on the (3-dimensional)
surface area of a molecule formed by discontinuous sections of the primary
protein structure. Two compartments of the immune system that deal with
epitopes are the B-cells and T-cells. T-cell epitopes are exclusively
linear in nature while B-cells respond to both conformation and linear
epitopes. Many (but not all) classical food allergens tend to contain
linear epitopes while aeroallergens and pollen-related food allergens (those
responsible for "oral allergy syndrome") often contain conformational
epitopes. The EAAM-approach codified in the IFBC/ILSI decision tree focuses
on T-cell epitopes, where 8 amino acids is the minimal size for such
epitopes. However, B-cell epitopes can be smaller and can occur in food
allergens, as Becker notes with the case of certain peanut and cod allergens
(Becker, 2001).

While epitopes are clearly more important than the general amino
acid sequence of a known allergen, very few epitopes have been determined.
Only a small-to-moderate percentage of food allergens have even been
identified. Various protein data bases contain the amino acid sequence of
180 major allergens of which 30 are food allergens of plant origin (Metcalfe
et al., 1996). At the same time, a literature review found more than 150
foods associated with sporadic allergic reactions (Hefle et al., 1996). It
should be noted, though, that roughly 90% of all moderate to severe allergic
reactions to food come from eight types of food sources: peanuts, soybeans,
milk, eggs, fish, crustacea, wheat and tree nuts. The number of identified
epitope sequences for the various food allergens is miniscule compared to
the probable number of epitopes that exist. Indeed, one of the main
suggestions for further work is that "Research is needed to map all the
epitopes of known allergens and to develop monoclonal antibodies against
them" (Becker, 2001: 4). We concur whole-heartedly.

Becker also notes that glycosylation patterns can affect
allergenicity and immunogenicity of a protein. He cites the example of
"a-amylase [where it is known] that this allergen and protein is
glycosylated, when expressed in eucaryotic plants and immunologically active
but not in E. coli" (Becker, 2001: 3). As further noted in the final
report of the FAO/WHO Expert Consultation, "Glycosylation may alter the
epitope structure, either by shielding part of the protein surface
(particularly if the glycosylation is extensive), or by introducing glycan
epitopes. Glycan epitopes are known to be highly cross-reactive" (FAO/WHO,
2001: 11). Since E. coli does not glycosylate proteins, while many plants
and animals do, we feel that all allergy testing of novel proteins be based
on the protein as it is expressed in the organism destined for food and not
on the protein as expressed in a bacterial host such as E. coli, as has
routinely been permitted by the EPA and FDA.

The FAO/WHO Expert Consultation developed a standardized
methodology for determining sequence homology between and introduced protein
and known allergens. It started with the IFBC/ILSI decision tree and
updated that tree on the basis of evolving scientific knowledge in the area.
In contrast to the IFBC/ILSI decision tree, FAO/WHO suggested using identity
of 6 rather than 8 identical contiguous amino acids as a criterion for
further concern and using local alignments rather than global alignments
when comparing unrelated proteins. They also suggested additional criteria
such as a 35% overall amino acid sequence homology as a cause for further
concern and the development of databases and methods to test for
discontinuous epitopes including those changed by glycosylation patterns.
FAO/WHO recommended the following standardized methodology for determining
sequence homology:

6.1. Sequence Homology as Derived from Allergen Databases

The commonly used protein databases (PIR, SwissProt and TrEMBL) contain the
amino acid sequence of most allergens for which this information is known.
However, these databases are currently not fully up-to-date. A specialized
allergen database is under construction.

Suggested procedure on how to determine the percent amino acid identity
between the expressed protein and known allergens.

Step 1: obtain the amino acid sequence of all allergens in the protein
databases . . . in FASTA-format (using the amino acids from the mature
protein only, disregarding the leader sequences, if any). Let this be data
set (1).

Step2: prepare a complete set of 80-amino acid length sequences derived from
the expressed protein (again disregarding the leader sequence, if any). Let
this be data set (2).

Step 3: go to EMBL internet address: http://www2.ebi.ac.uk
<http://www2.ebi.ac.uk/> and compare each of the sequences of the data set
(2) with all sequences of data set (1), using the FASTA program on the web
site for alignment with the default settings for gap penalty and width.

Cross-reactivity between the expressed protein and a known allergen (as can
be found in the protein databases) has to be considered where there is: 1)
more than 35% identity in the amino acid sequence of the expressed protein
(i.e. without the leader sequence, if any), using a window of 80 amino acids
and a suitable gap penalty (using Clustal-type alignment programs or
equivalent alignment programs) or: 2) identity of 6 contiguous amino acids.

If any of the identity scores equals or exceeds 35%, this is considered to
indicate significant homology within the context of this assessment
approach. The use of amino acid sequence homologies to identify prospective
cross-reacting allergens in genetically-modified foods has been discussed in
more detail elsewhere (Gendel, 1998a, Gendel, 1998b).

Structural similarity with known allergens may still be important if
significant amino acid identity is found, but it is below 35%. In this case
significant cross-reactivity is unlikely. However, some families of
structurally related proteins are known to contain several allergens. Some
examples are: lipocalins, non-specific lipid transfer proteins, napins (2S
albumins from seeds), parvalbumins.

If the expressed protein belongs to such a family, it may be considered to
have a higher probability to be an allergenic protein. . . . Since identity
of 6 contiguous amino acids has an appreciable risk of occurring by chance,
verification of potential crossreactivity is warranted when criterion (1) is
negative, but criterion (2) is positive. In this situation suitable
antibodies (from human or animal source) have to be tested to substantiate
the potential for crossreactivity" (FAO/WHO, 2001: 10-11).

The report of the FAO/WHO Expert Consultation makes a reference
to the work of Dr. Steven Gendel, chief of FDA's Biotechnology Studies
Branch. In a pair of papers Dr. Gendel discusses the various databases of
allergens and how to use them to determine sequence similarity between an
expressed protein and known allergen (Gendel, 1998a, b). Dr. Gendel argues
persuasively for use of local algorithms rather than global algorithms when
assessing allergenicity of novel proteins because most novel proteins are
not evolutionarily related. As he points out, "sequence algorithms can be
divided into global algorithms that optimize alignments across the entire
length of the sequences involved and local algorithms that attempt to
optimize alignments only with regions of high similarity. Global alignment
algorithms are of greatest utility when the sequences involved are related.
Allergenicity assessment involves sequence alignments between proteins that
are not evolutionarily related. Therefore, it is likely that local
alignment will be more useful" (Gendel, 1998b: 50). Gendel tests this
assumption with known allergens and finds that the local alignment works
best. The original IFBC/ILSI decision tree used a global alignment
algorithm. Local alignment algorithms include the FASTA and BLAST program,
which give similar results (Gendel, 1998b); FAO/WHO recommends use of the
FASTA program. Gendel notes that "Although it is likely that immunological
cross-reactivity requires extensive sequence similarity, absolute identity
may not be necessary (for example, see Elsayed et al., 1982)" (Gendel,
1998b: 57). He then goes on to develop a "biochemical similarity matrix"
which "divides the amino acids into six classes based on biochemical
characteristics (i.e., hydrophilic acid amino acids, hydrophilic basic amino
acids, etc.). . . Alignment of members of the same class is scored as a
mismatch. The realignment was confined to a region of 15 to 20 amino acids
in each case to preserve the previously located identities" (Gendel, 1998b:

Using this methodology, Gendel finds significant sequence
homology between b-lactoglobulin (major milk allergen) and Cry3A (found in
Bt potatoes) and between Cry1Ab or Cry1Ac and vitellogenin (egg allergen).
He concludes, "although it is clear that some amino acid residues are
critical for specific binding, some conservative substitutions may not
affect allegenicity. Therefore, it may be prudent to treat sequence matches
with a high degree of identity that occur within regions of similarity as
significant even if the identity does not extend for eight or more amino
acids. For example, the similarity between Cry1A(b) and vitellogenin might
be sufficient to warrant additional evaluation" (Gendel, 1998b: 60).

In sum, we urge FDA to follow the protocol laid out by FAO/WHO as
slightly modified by Dr. Gendel (e.g. allow chemically similar amino acid
residues to be used when determining short sequence similarity/identity for
the contiguous amino acid sequences). We also agree with the EPA SAP,
FAO/WHO and Dr. Becker that developing databases and methods (such as
monoclonal antibodies using animal and/or human materials) to test for
conformational or discontinuous epitopes including those caused by changed
glycolysation patterns is of key importance and urge FDA to try and
encourage studies in these areas.

Digestive Stability (enzymatic digestion)

A number of scientific and other sources Environmental Protection Agency,
FIFRA's Science Advisory Panel (SAP), the International Life Sciences
Institute (ILSI), the FAO/WHO Expert Consultation on Allergenicity of Foods
Derived from Biotechnology and Codex Alimentarius' Ad Hoc Task Force
on Foods Derived from Modern Biotechnology of new protein produced
in foods developed via bioengineering should be a
criterion that is assessed. A number of these sources agree that in order
for the criterion of digestive stability to be used, standardized methods
need to be developed so that any laboratory can repeat them. As Drs. Steve
Taylor and Samuel Lehrer pointed out in an early paper in this area,
"Although the assessment of the resistance to hydrolysis of proteins could
offer valuable information regarding the potential allergenicity of specific
proteins, a rigorous protocol for such experiments has not been established.
Ideally, this protocol would mimic digestive proteolysis and included tests
on the isolated protein and the protein in the appropriate food matrix"
(Taylor and Lehrer, 1996: ).

All sources quoted above agree that assessing digestive
stability should involve simulating the environment of the human digestive
system. One can either simulate the environment of the stomach, via
simulated gastic fluid (SGF), or simulate the environment of the intestine,
via simulated intestinal fluid (SIF). Most of the authors prefer the use of
SGF. However, some note that if significant amounts of the undegraded or
protein fragments survive SGF, then SIF testing should ensue (Helms, 2001).
There has also been debate about the protocol for developing SGF. One of
the first studies that demonstrated a link between allergenicity of a
protein and resistance to digestion used the United States Pharmacopiea
(USP) protocol for SGF (Astwood et al., 1996). However, the USP protocol
for SGF has been criticized for not being sufficiently physiological in
nature (Helms, 2001). Since the publication of the Astwood et al. paper in
1996, there have been a number of scientific meetings, symposia and papers
that have further discussed protocols (or the need for them) for testing
digestive stability; these are reviewed by Dr. Ricki Helm, of the Arkansas
Children's Hospital Reseach Institute, in his paper "Stability of Known
Allergens (Digestive and Heat Stability)" written for the FAO/WHO expert
consultation. In this paper, Dr. Helms, after reviewing the scientific work
in this area, makes the following recommendations for protocols for SGF and

Simulated gastric fluid (SGF)

1-Standardized source materials and pH ranges.

a. Pepsin should be from a reliable source and enzymatic activity should
be expressed in arbitrary units prior to assessment of novel protein
degradation. For this, the method used by Ryle (6) could be applied, i.e.,
enzymatic activity based upon measuring TCA precipitable hemoglogin after
hydrolysis for 10 min.

b. A standardized enzyme/protein ratio should be established.

c. Bovine serum albumin should be used as a digestible protein.

d. Peanut allergens (and/or a stable protein readily available in pure
form) should be used as a non-digestible protein.

e. The novel protein should be assessed in enriched or pure form, both
recombinant and natural sources. If the matrix is to be assessed,
assessment should be from both the natural and transgenic form.

f. The effects of pH determinations should be made at 1.0, 1.5, 2.0,
4.0 and 6.0 due to the pH variation in the stomach following a meal.

g. Sampling of digestion should be taken at the following time points, 0,
15, and 30 seconds; and 1, 2, 4, 8, 15 and 60 minutes.

h. A scale in arbitrary units should be established using the digestible
and non-digestible proteins to characterize the novel protein.

i. Reasonable criteria of digestibility for acceptance should be
determined. (This could be based upon the data being collected by members
testing the protocol recommended by the ILSI/HESI working subgroup).

j. All analyses should be made at 37°C.

2-Standardized analytical methods for determining degree of degradation.

a. Column chromatography (e.g., HPLC) should be used to assess the
degree of degradation.

b. B. SDS-PAGE analysis, both denaturing and non-denaturing conditions,
should be standardized according to the following criteria.

i. A common gel system should be used, e.g., Novex system.

ii. 10-20% acrylamide gradient gels

iii. A sensitive staining method should be used (Silver stain or colloidal

c. Immunoblot analysis.

i. A standardized blotting system should be used, e.g., Novex sytem.

ii. Both polyclonal and monoclonal antibody assessments should be used to
determine degree of degradation.

d. Data should be provided in publishable format.

Simulated intestinal fluid (SGF)[sic; should be SIF] This assay should only
be used if there are considerable amounts of undegraded or protein fragments
identified in the SGF. A gastroenterologist should be consulted for best
physiologic conditions. Pancreatin sources are too variable, therefore a
standardized mixture of enzymes should be used.

1-A minimal composition to that of physiological state, i.e., pancreatic
drainage fluid of animal to enzyme mixtures in test sample, should be used.

a. Homogenous sources of
trypsin/amylase/lipase/elastase/chymotrypsin are recommended from reliable
sources (Worthington). (This will be difficult to manage, as sources may be
limited and purity questionable).

2-Standards and conditions for SGF should be applied" (Helms, 2001: pp.

The paper by Dr. Helm (Helm, 2001) served as a starting point
for discussion of the Joint FAO/WHO Expert Consultation on Allergenicity of
Foods Derived from Biotechnology. The final report of the Expert
Consultation recommended a slightly modified version of Dr. Helm's protocol
(for example, rather than test the protein at a range of pHs to simulate the
stomach at various times after feeding, the FAO/WHO Expert Consultation
recommends testing only at pH 2.0), but it contained far more specific
details about what the protocol should contain. Their recommendation

6.4. Pepsin Resistance

Purified of enriched expressed protein (non-heated and non-processed)
should be subjected to pepsin degradation conditions using Standard
Operating Procedures and Good Laboratory Practices (SOP/GLP). In addition,
the expressed protein should be assessed in its principle edible form under
identical pepsin degradation conditions to those used to examine the
expressed protein. Both known non-allergenic (soybean lipoxygenase, potato
acid phosphatase or equivalent) and allergenic (milk beta lactoglobulin,
soybean trypsin inhibitor or equivalent) food proteins should be included as
comparators to determine the relative degree of the expressed pepsin
resistance. The protein concentrations should be assessed using a
colorimetric assay (e.g., Bicinchoninic acid assay (BCA), Bradford Protein
Assay, or equivalent protein assay) with bovine serum albumin (BSA) as a
standard. Pepsin proteolytic activity should be assessed (Ryle).
Enzyme/protein mixtures should be prepared using 500mg of protein in 200mL
of 0.32% pepsin (w/v) in 30mM/L NaCl, pH 2.0, and maintained in a shaking
37°C water bath for 60 minutes. Individual 500 microgram aliquots of
pepsin/protein solution should be exposed for periods of 0, 15, 30 seconds
and 1, 2, 4, 8, 15, and 60 minutes, at which time each aliquot should be
neutralized with an appropriate buffer. Neutralized protein solutions
should be mixed with SDS-PAGE sample loading buffer with and without
reducing agent (DTT or 2-ME) and heated for 5 minutes at 90°C. Samples
containing 5mg/cm gel of protein should be evaluated using 10-20% gradient
Tricine SDS-PAGE gels or equivalent gel system under both non-reducing and
reducing electrophoretic conditions. Protein in the gels should be
visualized by silver or colloidal gold staining procedures. Evidence of
intact expressed protein and/or intact fragments greater than 3.5 kDa would
suggest a potential allergenic protein. Evidence of protein fragments less
than 3.5 kDa would not necessarily raise issues of protein allergenicity and
the data should be taken into consideration with other decision tree
criteria. For detection of expressed protein in an edible food source, a
polyclonal IgG immunoblot analysis should be performed according to the
laboratory procedures. The immunoblot analysis should be compared to the
silver or colloidal gold stained SDS-PAGE gel and reflect the stained
pattern of the expressed protein run under identical conditions" (FAO/WHO,
2001: 12-13).

One significant extension of Dr. Helm's protocol that the
FAO/WHO Expert Consultation included was the notion that "the expressed
protein should be assessed in its principle edible form under identical
pepsin degradation conditions to those used to examine the expressed
protein" (FAO/WHO, 2001: 12). CU absolutely agrees that the expressed form
of the protein should be assessed both in purified form and as part of the
food that it occurs in. The reason for this is that the food matrix can act
as a buffer allowing the expressed protein to survive digestion. There are
many examples of this. For instance, a number of growth hormones in milk,
such as insulin-like growth factor-1 (IGF-1) or epidermal growth factor
(EGF), are protected from digestion by the presence of casein (Kimura et
al., 1997; Playford et al., 1993; Xian et al., 1995). One study with IGF-1
found that 9% survived digestion when fed in pure form to rats; in the
presence of casein, 67% survived digestion (Kimura et al., 1997). More
recently, a study involving transgenic soy or corn DNA found that while 80%
of the naked DNA was degraded in gastric simulations, none of the transgene
DNA was digested when it was part of the food stuff: "The data showed that
80% of the transgene in naked soya DNA was degraded in the gastric
simulations, while no degradation of the transgene contained within GM soya
and maize were observed in these acidic conditions" (Martin-Orue et al.,
2002: 533). While we realize that DNA is not a protein, the general
phenomenon testing the pure substance pointed out in his paper for the
FAO/WHO Expert Consultation, recent industry and scientific thinking
in this area concur: "The working committee on the OCharacteristics of
Protein Food Allergens' held by ISLI/HESI following the symposium
established the following criteria be taken into consideration. . . .
3-Deliver: Consideration should be given to
how the material will be introduced into the diet. Assessment of
allergenicity should be based on the matrix/matrices that the novel protein
would be introduced into the diet" (Helm, 2001: 6).

In conclusion, we urge that FDA require companies to follow the
protocol as laid out in FAO/WHO Expert Consultation, which we described
above. If there are to be deviations from this protocol, companies should
be required to give a scientific justification for such deviations. In
particular, we feel the FDA should not allow the companies to simply use USP
protocol for SGF. Furthermore, FDA should not allow the companies to simply
test the protein at pH 1.2 (as per the USP protocol). If a company wants to
test the protein at pH 1.2, the FDA should also require higher pHs as well,
including, at least, pH 2.0.

Second, we feel the FDA should require the company to test the
protein in both the purified expressed form as well as in the form in which
it occurs in food, e.g. as part of the food matrix. For the purified
expressed form, we feel that the company should extract the protein from the
transgenic material that is intended to be commercialized and not use a form
of the protein that is extracted from a bacterial or other microbial source.

Finally, if a significant portion of the expressed protein does
survive digestion in SGF, we recommend that it be tested further in SIF,
using the protocol laid out by Dr. Helm.

Heat stability

Both allergy scientists as well as the Environmental Protection
Agency (EPA) consider stability of a protein to heat to be a characteristic
property of food allergens (Sampson, 1999; EPA, 2001; Helm, 2001;
and Taylor and Hefle, 2001). During the Bt crop reregistration process,
EPA vaguely adopted heat stability as a criterion for potentially allergenicity
for the Bt Cry endotoxins, stating that a characteristic "considered as an
indication of possible relation to a food allergen are [is] a protein's
ability to withstand heat or the conditions of food processing" (EPA, 2001b:
IIB2). However, EPA has neither strictly required nor even suggested a test
protocol for such data. Indeed, for a couple of Bt crops (Cry1Ab) and
Monsanto's Bt cotton (hybrid Cry1Ac/Ab) that processed corn or
cottonseed meal were inactive in an insect bioassay.

Monsanto submitted a more formal heat stability study for a relatively new
Bt corn variety (containing Cry1F rather than the usual Cry1Ab), but the
methodology was flawed. The study's main methodological flaw consisted of
the sole end-point (e.g., measure of degradation) being "growth inhibition
of neonate tobacco budworm larvae" following "application of treated Cry1F
to the surface of an insect diet" (EPA 2001b: 10). Such a study implicitly
assumes that the insecticidal mode of action correlates with allergenicity
and that loss of insecticidal action means no allergenicity. There is no
scientific justification for such an assumption. Theoretically, a protein
could be allergenic and have insecticidal activity; loss of that activity
does not imply loss of allergenicity. As has been noted by a number of
scientists, degraded proteins or protein fragments can still elicit an
allergenic response even though the protein is functionally inactive; a
perfect example is the major milk allergen b-lactoglobulin (Haddad et al.,

In contrast to the EPA's lack of a consistent protocol, Dr. Helm
has developed a science-based protocol as part of the paper on the topic
that he wrote for the 2001 FAO/WHO Expert Consultation: "Heat Stability:
The definition of heat stability should be standardized using the following
criteria. 1-Heat treatment of the novel protein, native and recombinant,
should be for 5 minutes at 90°C. 2-Assessment of stability by a combination
of molecular sieving using HPLC and standardized SDS-PAGE analysis (both
native and denaturing/reducing gels). See SDS-PAGE protocol below" [see the
section on digestive stability, above for this protocol] (Helm, 2001: 8-9).

We urge that the FDA require data on heat stability and use the
science-based protocol as outline by Dr. Helm (Helm, 2001). We would
suggest the following additions/explanations to the protocol. The
recombinant protein should be tested in both purified form and as part of
the food in which it occurs. The purified form of the protein should be
extracted from the engineered organism (usually plant) that will make up the
food; the company should not be permitted to use a bacterial or other
microbial source to produce the recombinant protein. Also, the engineered
protein should be added to a food matrix/matrices, preferably to the matrix
in which it will occur.

Animal models

Both the EPA's FIFRA Scientific Advisory Panel (SAP), which
looked at StarLink corn, and the FAO/WHO Expert Consultation recommended the
use of animal models. The FIFRA SAP investigating StarLink considered
immunological response in the brown Norway rat (BNR) and bioavailability of
the protein in bloodstream of BNR as criteria suggesting of allergic
potential of the Cry9C protein although they stated that these two assays
had not used a standardized methodology (SAP, 2000b).

The FAO/WHO Expert Consultation had this to say about animal

6.5. Animal Models

For additional assessment of the potential allergenicity of expressed
proteins, informative data can be generated using animal models in
development. A number of animal models may be considered to assess on a
relative scale the potential allergenicity using oral sensitization routes
with the Brown Norway rat model (Knippels et al., 1998) or intraperitoneal
administration in murine models (Dearman et al., 2000) or other relevant
animal models. Results should be presented in characteristic Th1/Th2
antibody (isotype) profiles for assessing the potential
immunogenic/allergenic activity. The different routes of administration in
animal models (oral versus intraperitoneal) may not give the same results.
Therefore, selection of one route of administration is not meant to exclude
other routes of sensitization. It is recommended to consider the results
from two sensitisation routes in the same or different animal species.

It is recommended that the potential allergenicity be ranked against well
known strong and weak food allergens and non-allergenic proteins in the
animal model. As additional information becomes available with respect to
animal models, protocols may need to be modified to give optimal conditions
for assessing protein allergenicity.

Although the present animal models provide additional information on
potential allergenicity of novel proteins, they do not reflect all aspects
of IgE-mediated food allergies in humans" (FAO/WHO, 2001: 13).

While there is some question as to the reliability/applicability
of use of animal models for predicting food allergy, animal models have been
used quite successfully in predicting/evaluating inhalant allergens. One of
the speakers at a conference titled "Assessment of the Allergic Potential of
Genetically Modified Foods," EPA, FDA and NIH and held in December
2001 in Chapel Hill, North Carolina Sarlo gave a talk at the meeting about
how useful rodent models have been over the years in testing for allergic
reactions to enzymes used in their detergents. According to Dr. Sarlo, when
P&G first started using enzymes in their detergents in the mid-1960s, many
workers in their plants developed allergies to the enzymes. In the intervening
decades, P&G developed accurate rodent models using certain strains of
guinea pigs and mice. Certain strains of guinea pigs developed an IgG response
to the enzymes that caused allergic reactions in some workers, while certain
strains of mice showed both IgG and IgE responses to the enzymes. The strains of mice and
guinea pigs used were ones in which there was a correlation between the
responses of the animals and the responses of the workers. Over the years,
the used of these particular animal models, combined with medical
surveillance of the workers and modification of the environment to
dramatically reduce the problem. In sum, the experience of P&G definitely
demonstrates that animal models are both useful and provide predictable
responses as to how humans respond to allergens. She suggested that a
similar approach be used to investigate potential allergenicity of
genetically engineered foods (Anonymous, 2002).

We feel that the experience of P&G definitely shows that animal
models can be successfully used to predict allergenicity of proteins. We
therefore recommend that FDA urge companies to conduct animal studies,
utilizing either the protocol as laid out by FAO/WHO or the protocol
developed by P&G. In this regard, perhaps the same strains of guinea pigs
and mice that were successful surrogates for humans when prediciting
inhalant allergenicity of proteins may be successfully used to predict food
allergenicity. Indeed, we suggest that FDA begin such research with these
strains of guinea pigs and mice.


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Astwood, J.D., Leach, J.N. and R.L. Fuchs. 1996. Stability of food
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Becker, W. M. 2001. Topic 4: Sequence Homology and Allergen Structure.
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Allergenicity of Foods Derived from Biotechnology, January 22-25, 2001.
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Dearman, R.J., Caddick, H., Basketter, D.A. and I. Kimber. 2000. Divergent
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EPA. 2001b. Biopesticide Registration Action Document: Bacillus
thuringiensis Cry1F Corn, August 2001, EPA.

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Gendel, S.M. 1998a. Sequence databases for assessing the potential
allergenicity of proteins used in transgenic foods. Advances in Food and
Nutrition Research, 42: 63-92.

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