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

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

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

August 14, 2002
Michael Hansen, Ph.D.


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, 2001a,b).

Key Points

As an overview, we urge FDA to:

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

–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

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
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: 58).

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

A number of scientific and other
sources-including the 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-have
agreed that digestive stability (or enzymatic digestion) 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 SIF:

"Simulated gastric fluid

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

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. 9-10).

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 follows:

"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-partial survival of substance when part of a food compared
to testing the pure substance-we feel is applicable. Furthermore, as Dr. Helm
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 ‘Characteristics 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:

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

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-Novartis’ Bt corn (Cry1Ab) and
Monsanto’s Bt cotton (hybrid Cry1Ac/Ab)-the EPA accepted data 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:

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 models:

"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,"-sponsored by
the National Toxicology Program, EPA, FDA and NIH and held in December 2001
in Chapel Hill, North Carolina-was Katherine Sarlo, principal scientist at Proctor
& Gamble. Dr. 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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