On the final afternoon of the workshop "Assessment of the Allergenic Potential
of Genetically Modified Foods," held 10-12 December 2001, in Chapel Hill,
North Carolina, speakers and participants met in breakout groups of 8-12
individuals to discuss key issues in the following areas: use of human clinical
data, animal models to assess food allergy, biomarkers of exposure and effect,
sensitive populations, dose-response assessment, and postmarket surveillance.
Each group was asked to address general questions regarding what can be done
to assess the potential allergenicity of genetically modified (GM) foods and
what is needed to improve this process, as well as questions specific to each
particular topic. In many instances, the discussion topics overlapped such that
a number of topics were addressed by multiple groups, and in some cases their
conclusions differed. Following is a brief summary of the discussions of each
of the six breakout groups regarding our current state of knowledge and what
information is needed to advance the field. Each breakout group contained individuals
with a wide variety of expertise so that the subject material could be covered
fully. The text below is an effort to capture the expertise and opinions of
diverse participants and as detailed in the text below, in some instances consensus
was not achieved.
Use of Human Clinical Data
How important are the following end points in hazard identification
and dose response: immune indicators of sensitization (IgE, skin test positivity),
clinical symptoms from skin, gut, respiratory tract after provocation (DBPCFC),
and anaphylaxis? A clinical syndrome suggestive of an IgE-mediated reaction
(flushing, urticaria, angioedema, wheeze, stridor, abdominal pain, vomiting,
or cardiovascular collapse) after the ingestion of an allergenic food can be
confirmed with a skin prick test (SPT) or serum-specific IgE. However, in the
absence of a clinical history suggestive of allergy, IgE detection, whether
SPT or specific IgE, serves as a good indicator of sensitization but not necessarily
of disease. Conversely, in the clinical setting, the absence of detectable IgE
may be useful at excluding IgE-mediated food allergy. However, this depends
somewhat on the specific antigen and techniques used. It is possible to obtain
positive SPT results in individuals who test negative for serum IgE, as antigen-specific
IgE may be predominantly cell bound when present at low levels. Interpretation
of the usefulness of SPT or food-specific IgE rests with the comparison of SPT/specific
IgE results with the outcome of double-blind, placebo-controlled food challenge
(DBPCFC), which is currently the "gold standard" for determining food allergy.
Approximately 50% of positive SPTs correlate with confirmed DBPCFCs, suggesting
that sensitization to food allergens occurs in the absence of clinical symptoms
(Bock et al. 1977, 1978; Eigenmann and Sampson 1998). The magnitude of the SPT
or specific IgE measurement is useful in predicting the likelihood of clinical
allergy (as confirmed by DBPCFC) but is not useful in predicting severity (Eigenmann
and Sampson 1998; Sampson 2001).
The DBPCFC is an excellent method for confirming suspected allergy. In a controlled
setting with experienced clinicians, the DBPCFC can be safely performed (Bock
et al. 1978, 1988; Watson 1995; Williams and Bock 1999). The rapidity of IgE-mediated
reactions (> 90% within 1 hr) allows the DBPCFC to reproduce objectively
IgE-mediated symptoms resulting from the food administered.
Theoretically, any food containing a protein could elicit an allergic reaction;
however, eight common foods are responsible for > 90% of food allergies.
The remaining 10% of food allergies result from over 150 different proteins
(Hefle et al. 1996). Data are becoming available on threshold doses required
to provoke an allergic reaction in previously sensitized individuals. Recently,
the results of 10 independently conducted clinical challenge studies have been
reported (Taylor et al. 2002). In these 10 well-defined clinical studies involving
peanut, milk, egg, fish, and mustard allergens, 0.25 mg peanut protein (equivalent
to 1 mg whole peanut) was the lowest provoking dose and was therefore considered
to be the lowest observable adverse effect level (LOAEL) for elicitation. Of
the 10 studies, one reported four subjects, from a study cohort of 74, who developed
an allergic response to this LOAEL. In the other nine studies, no other individuals
with this degree of sensitivity were identified. It should also be noted, however,
that the responses in these four individuals were mild and reversed spontaneously
(Taylor et al. 2002).
What end points are appropriate for the premarket assessment of previously
sensitized individuals to source proteins versus the postmarket assessment of
potentially sensitized individuals to source proteins as well as the potential
sensitization and allergenicity of novel proteins? In premarket assessment
of novel proteins for hazard identification, the group fealt tht there was no
role for SPT or measurement of specific IgE, as sensitization would not have
occurred. However, when the protein in question originates from a known allergic
source or a potentially allergic source, clinical testing would be valuable.
Clinical testing could include specific serum testing from well-defined allergic
individuals and/or SPT. SPT may only be necessary in situations where human
serum IgE is detected to the GM protein and the product is to be further developed.
It is expected, however, that in most circumstances where specific serum screening
is positive, there would not be any further development of the product.
In a postmarket assessment, if IgE is detected either serologically or by
skin testing, the relationship between the detected IgE and clinical symptoms
may be confirmed using DBPCFC.
What constitutes "harm" (a term used in standards for food safety evaluation
by both the U.S. Food and Drug Administration and U.S. Environmental Protection
Agency)? Is sensitization harm? What about clinical symptoms? What evidence
links antigen-specific IgE with these responses? Using an accepted legal
definition of harm as "reasonable certainty of no harm," several factors must
be considered. These include at-risk populations, potency and exposure to the
allergen, and background prevalence of food allergy.
Development of clinical symptoms unequivocally constitutes harm, but development
of sensitization does not. Not all individuals who have IgE against a specific
protein will develop clinical symptoms (Sarlo and Kirchner 2002). However, IgE-mediated
allergic symptoms and signs will not occur in the absence of sensitization;
therefore, sensitization leads to the potential for harm. In other words, if
the sensitization can be detected and if the exposure is removed prior to the
occurrence of clinical symptoms and signs, harm may not result. There is evidence
to suggest that sensitization to respiratory allergens in the occupational setting
may occur at lower levels of exposure than doses capable of eliciting systemic
hypersensitivity, and that reduction of exposure can also remove the chances
for harm (Schweigert et al. 2000). However, respiratory tract sensitization
may not predict the risk of allergic sensitization from oral ingestion in the
case of foods. For example, baker's asthma is a common occupational disease,
yet very few bakers have allergic responses after eating wheat bread, even though
many have wheat protein-specific IgE (Smith et al. 2000). Additionally,
it has been observed that some IgE antibodies are not clinically relevant and
will not result in clinical sequelae (Aalberse et al. 2001).
As discussed above, the positive predictive value of antigen-specific IgE,
whether SPT or serum measurements, varies with the food in question and the
disease prevalence. In general, the positive predictive value of an SPT ranges
from 25 to 75%.
What kinds of tests can be done in the clinical setting, and should
they be conducted prior to approval or in the postmarket evaluation?
If the gene is derived from a source known to be allergenic, in vitro
studies such as immunoblots should precede clinical studies. If IgE binding
by the protein in question is detected, further development should only proceed
with caution. Clinical studies, including specific serum screening, and/or SPTs
should be performed prior to marketing. Specific serum screening should be performed
with serum obtained from well-defined clinical populations with allergies to
the source protein. The feasibility of this step depends on the availability
of banks that contain sera from well-defined, clinically allergic individuals.
SPTs can also be performed to correlate with the outcome of the specific screening,
but this may not be necessary unless serum data banks are inadequate or unavailable.
If the gene is derived from a source not known to be allergenic, no clinical
tests are likely to be relevant in an unexposed population. However, as recommended
by the Food and Agriculture Organization/World Health Organization Expert Consultation
(FAO/WHO 2001), targeted serum screening for cross-reactivity with sera from
patients allergic to proteins broadly related to the source gene could be conducted.
If cross-reactivity is found, there may be a role for skin testing of individuals
allergic to the related source protein with the novel protein. In this scenario,
it may also be important to use appropriate nonclinical tests, such as animal
models with appropriate positive and negative controls, in conjunction with
human clinical trials. The ethical and technical constraints of conducting human
trials, and the necessity of doing so, is a subject that requires considerable
attention. However, these types of studies may be necessary for the validation
of animal models as predictors of human allergic disease.
Postmarket assessment of novel foods. Clinical testing with
specific IgE should be developed and employed. In addition, SPT with purified
protein would be useful to detect IgE in vivo. Positive tests should
be followed by DBPCFC, under appropriate clinical observation, to correlate
demonstrable IgE with clinical symptoms. The availability of purified protein
may limit the development of specific IgE tests.
What evidence, if any, is there for cross-reactivity between different
food allergens and between respiratory allergens and food allergens? Within
food families, there is considerable in vitro cross-reactivity. However,
this frequently does not correlate with clinical cross-reactivity. Bock and
Atkins (1990) described the results of 480 food challenges where < 1% of
the children demonstrated clinical allergy to more than one member of a food
family. Similarly, Bernhisel-Broadbent and Sampson (1989) demonstrated that
in 41 children with two or more positive SPTs to legumes, only two of these
children had positive challenges to more than one legume. Similar data exist
for cereal grains, nuts, and fish (Bernhisel-Broadbent et al. 1992; Bock and
Atkins 1989; Jones et al. 1995). Although in vitro cross-reactivity greatly
exceeds clinical cross-reactivity, there is significant variability between
patients and their reactivity to members within food families (Bernhisel-Broadbent
et al. 1989).
Cross-reactivity can also be demonstrated between foods and aeroallergens,
where pollens may cross-react with fruits, vegetables, or nuts. Examples include
birch pollen that cross-reacts with apples, pears, and cherries, and ragweed
pollen that cross-reacts with gourds.
The potential utility of targeted serum screening is well described in the
FAO/WHO Expert Consultation Report (FAO/WHO 2001), but widespread use would
require standardized panels of food and inhalant IgE obtained from individuals
with well-characterized allergies. Although targeted serum screening may be
an appropriate approach to the detection of potential allergens, many of the
tools needed to routinely conduct this type of testing are not widely available
(e.g., pooled banks of sera from allergic individuals), and both development
and validation of the associated tests would be necessary prior to their use
for risk assessment. Some IgE epitopes may not be clinically relevant, and such
information should be considered when serum screening is used.
Animal Models to Assess Food Allergy
What attributes should an animal model have to be a reasonable method
for safety evaluation of allergic potential? This group defined allergy
(as opposed to immunogenicity) as the adverse health effects that may result
from the stimulation of a specific immune response. For the purposes of this
discussion the focus was on IgE-mediated responses induced against dietary proteins
and resulting in food allergy and excluded such reactions as celiac disease.
However, it is important to note that not all IgE responses are harmful (e.g.,
protection against roundworm infection). Models should be able to distinguish
between immunogenicity (IgG, IgM, IgA, and cellular immune responses) and allergenicity
(IgE). The participants felt that to meet this criterion an animal model would
likely be genetically predisposed to have an atopic or T-helper cell type 2
(Th2) phenotype involving a skewed response toward the production of IgE antibodies
and Th2 cytokines [interleukin (IL)-4, IL-5, IL-6, IL-10, and IL-13].
Models for safety assessment should show antigen (usually protein)-specific
IgE antibodies. There was some discussion regarding pure carbohydrates as allergens,
recognizing that many allergens are glycosylated proteins. It was also noted
that lipids or oils in highly allergenic foods such as peanuts and tree nuts
might influence immune responses. For all participants in this group, the ability
to demonstrate antigen-specific IgE antibodies mediated by mast cells/basophils
was the critical factor in the utility of any model. Additional markers such
as cytokine or chemokine profiles could be used in conjunction with IgE.
Animal models should focus on hazard identification concerning potential allergenicity
of proteins. As responses measured within the models may be used to assess the
potency of specific allergens, validation of the models might include the determination
of potency of known allergens. A second focus of animal models could then be
the assessment of potency of novel allergens, which may be of use in risk assessment.
Although dog and swine models are useful for studying mechanisms of food allergy,
as they have clinical manifestations similar to those seen in humans, they may
be less practical than the use of rodent models to study hazard identification.
Similarly, while adjuvants may be used to enhance allergic responses for the
study of mechanisms of food allergy and pathology, the use of adjuvants in models
meant for hazard identification should be considered with caution. It is possible
that the use of adjuvants could confer on nonallergic proteins the ability to
cause sensitization, thus creating false positives. However, few data are available
to substantiate this hypothesis.
Finally, there must be a great deal of confidence on the level of false negatives
in any model, as false negatives might lead to an inappropriate conclusion that
a novel food is safe.
What characteristics must the administered allergen have to represent
accurately the risk of effects in humans? Safety evaluation of foods
derived from GM crops is necessary to ensure that the novel food is as safe
as the conventional food. Given this goal, the protein being tested should be
identical, at least as is practically possible, to the protein expressed in
the plant. For testing purposes, the novel protein is usually expressed in a
vector such as Escherichia coli or yeast. It must be determined in advance
if molecular differences exist between proteins expressed in the new crop or
end product and those expressed in the vector. An additional consideration is
whether the introduced gene results in the expression or upregulation or alteration
of endogenous genes (thus potentially coding for allergenic proteins) or results
in changes in posttranscriptional processing of endogenous proteins with the
potential of creating new allergens. Finally, the matrix within which the protein
will appear needs to be considered. Is it appropriate to test purified proteins,
or should they be evaluated along with other food components as they are seen
in the digestive tract?
What are appropriate positive and negative controls? Positive
and negative allergens should be used to evaluate and validate any model. There
was some controversy among the participants regarding the use of only pure proteins
or even pure epitopes, such as arachis hypogaea 2 (Ara h2) from peanut, as the
sole positive versus the use of crude protein extracts. Such extracts would
more closely mimic human exposure by preserving glycosylation and/or binding
to natural adjuvants found in the crop of interest. Positive controls should
be antigens positive in humans, such as peanut Ara h2 and peanut lectin, brazil
nut 2S-albumin, ovomucoid, ovalbumin, and ß-lactoglobulin. Positive allergens
should induce at least a moderate response and dose-response relationships
should be demonstrable. Negative controls should be proteins that the human
population is widely exposed to but seldom lead to allergenic responses such
as rubisco or corn phosphoenolpyruvate-carboxylase. Ideally, both positive
and negative controls should be easily stored, relatively stable, and reasonably
affordable.
What questions can be potentially addressed using animal models? In
addition to questions regarding the sensitizing potential and relative potency
of individual allergens, animal models may be used to complement or provide
corroborative information for other testing methods. For example, there is considerable
debate about how much sequence homology is required with a known allergen in
order for a novel protein to be allergenic and about the relevance of stability
in pepsin as a marker for allergenicity. Animal models could be used to examine
the properties of foods that have some sequence homology or pepsin stability
to determine if the novel protein is an allergen, and if so, what its relative
potency would be. In vivo models could also be used to determine dose-response
relationships and whether there are thresholds below which allergenic proteins
have no clinical effects. In the detergent industry, exposure levels have been
reduced and managed so that between 0 and 3% of the workforce become sensitized
in a given year (Sarlo and Kirchner 2002; Schweigert et al. 2000). Under these
conditions of exposure, clinical disease has been virtually eliminated, indicating
that, at least for aeroallergens, there is a threshold for sensitization that
may be different than that for elicitation. Determination of acceptable limits
for both sensitization and elicitation of clinical symptoms may have important
implications for risk evaluation and management.
Animal models can also be useful tools to determine basic information on the
mechanisms of the allergic response. The specific properties that make a protein
an allergen and how tolerance is induced are of critical importance in understanding
allergic responses. In vivo models may elucidate how specific proteins
could be made to induce tolerance rather than sensitization. In addition, these
types of models could provide data on cross-reactivity between specific allergens.
External factors such as environmental tobacco smoke, particulate materials,
and infectious agents may act as adjuvants to enhance sensitization. These types
of effects are best examined using in vivo models.
How can data generated in animal studies be used in the safety evaluation
process? Results of appropriate animal studies pertaining to hazard
identification and potency evaluation can make a significant contribution to
the safety evaluation process. These types of models can be used to screen proteins
in conjunction with information from stability and in vitro studies.
What are the limitations in current animal models, and how can we improve
them? Validation studies are needed to assess whether animal models
can accurately predict allergenicity in humans. Although allergic responses
in animals and humans share many common mechanisms, there are clearly some cross-species
differences such as in reaginic antibodies and complement components that induce
anaphylaxis. Allergy or atopy in both humans and animals depends on genetic
factors that differ between individuals and between species. Introduction of
novel genes may alter the metabolism or expression of endogenous proteins that
may differ across species. Because of genetic differences across species, the
ability to sensitize or alter endogenous protein expression may not readily
be captured in some models. In addition to species differences, there are questions
regarding differences in responses to specific allergens between rodent strains.
It is unlikely that a single animal model will be sufficient to address all
the issues concerning prediction of allergenicity to humans. Rather, some species
(such as rodents) that are less expensive and technically challenging may provide
information on hazard identification and statistical validity of nonallergenicity.
Other species, for example, dogs and swine, that more closely follow the clinical
symptoms in the atopic human population may provide important mechanistic information.
Although reagents to evaluate allergic responses, such as cytokines, chemokines,
and monoclonal antibodies to cell surface markers, are readily available for
rodents, they are only now being developed for dogs and swine. It is clear that
we must evaluate the currently available animal models, validate their utility
to predict human responses, and decide how best to integrate one, some, or none
of them into a coherent strategy for safety assessment.
Biomarkers of Exposure and Effect
The appropriateness of measuring antigen-specific IgE and other potential
biomarkers of allergy and exposure was considered for both premarket and postmarket
evaluations of the safety of GM foods. Factors were identified that were deemed
important to the development and validation of the recommended analytical procedures.
What biomarkers are available that can be applied to safety evaluation
and risk assessment of novel foods? Is IgE sufficient? When a gene is
transferred into a food crop, using the techniques of biotechnology, the newly
expressed protein should be evaluated regarding its potential allergenicity
(Metcalfe et al. 1996). If the gene donor organism is known to be allergenic,
or if the amino acid sequence of the introduced protein is similar to a known
allergen, the potential allergenicity is most appropriately evaluated by testing
for antigen-specific reactions in those individuals with documented allergies
to the donor or to the homologous allergen. Definitive proof that a protein
is a food allergen would be the observation of a clinical reaction within a
short time after ingestion of the food, which is most appropriately performed
in a DBPCFC (Sampson 1997). Because of practical and safety considerations,
a biomarker of potential effect, such as measurement of antigen-specific serum
IgE, or positive skin test results may be more appropriate in the premarket
phase of testing. However, these methods typically have modest to significant
levels of false-positive and false-negative results, which may be minimized
by proper assay design and reagent selection (Bindslev-Jensen and Poulsen 1997).
A variety of antigen-specific IgE immunoassays have been used for preliminary
diagnosis of food allergy. The same type of tests could also be used to monitor
potential allergic reactions once the product is in the marketplace.
The potential utility of other biomarkers was discussed, including Th2-cell
cytokine expression or proliferation, serum basophil or mast cell protease levels,
and serum histamine levels. There is little evidence to support the use of these
as biomarkers of allergic responses to dietary proteins. Measurements of other
antibody isotypes including IgG4, IgA, or IgM were similarly dismissed as not
being specific markers of allergic reactions.
In addition to the need for an antigen-specific IgE assay, the utility of
measuring antigen-specific IgG was considered. Opinions were mixed regarding
the usefulness of the expected data except in the case of negative IgE test
results. In that case a positive IgG response demonstrates that individuals
were exposed to the introduced protein and that this protein was immunogenic.
If they do not have protein-specific IgE or IgG, it is difficult to ascertain
whether they were exposed or are just immunologically unresponsive to the protein.
Measurement of IgG levels in IgE-positive individuals is not likely to provide
additional information on exposure or responsiveness.
What parameters are important in assays used to measure biomarkers of
exposure and effect? Protein standards. For all assays discussed,
the protein used as the test reagent and/or standard must be appropriate and
representative of the material that will be in the food supply. Further, because
the introduced protein in the currently approved GM products are expressed at
low levels (< 1 ppm to ~ 400 ppm fresh weight), considerable time and effort
are required to produce gram quantities of purified protein needed for use in
assays and as a standard for the immunoassays. Because the development of GM
plants suitable for commercial production is complex and time consuming, a recombinant
protein produced from bacteria, yeast, plant, or animal cell culture system
will typically be used. However, both the recombinant protein produced in the
plant and in the heterologous organism will need to be characterized to demonstrate
equivalence.
Because the epitopes recognized by some antibodies are formed by noncontiguous
amino acids in close proximity because of the secondary structural folding of
the antigen, optimal binding may require native conformation of the protein
used in the immunoassays. However, a number of the most important allergenic
epitopes found in food allergens involve only the primary structure and may
require a denatured form for optimal detection. There is no single correct answer
for all proteins regarding the question of which form is the correct one to
use in evaluating IgE binding. Further, it is technically impossible to evaluate
the absolute secondary, tertiary, and quaternary structure of all proteins in
complex mixtures found in foods. However, as most proteins purified from either
microbial sources or plants will be present as a population of native and denatured
forms, antibodies from exposed individuals or immunized animals are expected
to bind to a substantial fraction of either form.
With regard to the traceability of the introduced protein in foods, some proteins
are enzymatically cleaved in the plant or during processing. Detection of fragments
of the protein in processed foods may therefore not be possible unless immunoassays
are developed with antibodies that recognize different segments of the protein.
It is therefore recommended that the epitopes recognized by the antibodies used
in these assays be mapped in order for the limitations of the assay to be understood.
Traceability is important for exposure assessment in the case of a protein determined
to be allergenic or if certain types of postmarket surveillance were performed.
Immunoassays. Direct enzyme-linked immunosorbent assay (ELISA), ELISA
inhibition, direct radioallergosorbent test (RAST), RAST inhibition, Western
blot or Western inhibition, or similar assay can be used to measure antigen-specific
serum IgE. ELISA and RAST assays provide quantitative data of antibody binding
to the solid-phase antigen. These assays have a qualitative measurement of the
antibody-epitope interaction as well when dose responses and the slope
of the dose-response curves are compared, although the inhibition assays
are most appropriate for that evaluation. Western blot assays are rarely used
for quantitative measurement but are quite useful to determine antibody specificity
and, if appropriate, to evaluate the ability of the antibody to recognize fragments
of the protein. General considerations for each assay were discussed in varying
detail, with most emphasis placed on direct ELISA assays.
Appropriate positive and negative controls must be chosen to verify the specificity
of binding and detection as well as day-to-day assay reliability. Licensed diagnostic
allergy tests are required to have appropriate positive controls (NCCLS 1997).
However, a direct positive control for antigen-specific human IgE is only available
if there are individuals identified who have the specific allergy and who have
significantly high titers of antigen-specific IgE in their sera. That is not
likely to be the case for novel proteins, as they are intentionally chosen to
avoid known allergens. Therefore alternative indirect controls must be used.
Animal sera may be used to test for the presence of the antigen in the assay.
Further, a different allergen-specific IgE assay and sera may be used to test
the specificity and sensitivity of detection reagents to allow evaluation of
the test articles and reagents. Antigen-specific human IgG may be assayed to
evaluate whether an individual has been exposed to the antigen and whether the
test protein is appropriately immunogenic. If there are no positive human sera,
animal sera from specifically sensitized animals may be used to evaluate the
protein antigen and test system.
Selection of subjects for human studies. Premarket testing may be used
to determine whether allergic subjects who have been previously exposed to a
protein have developed IgE that binds to the novel protein. Subjects will normally
be consenting adults who have been identified through clinical allergy practices
or by specific searches or surveys. These individuals are evaluated by careful
interviews and screening of serum reactivity. The history of exposure of the
serum donors must be clearly defined and documented. Individuals who have been
occupationally exposed to the novel protein may be appropriate subjects for
premarket testing if exposure is clearly defined and documented. Even if specific
IgE were detected in serum, definitive proof of allergy to the novel protein
can be achieved only if the subjects also have clear clinical histories consistent
with the specific allergy and if controlled challenge tests are positive. However,
allergic responses after occupational exposure, which is typically through inhalation,
may not be predictive of responses that may result from oral exposure to a novel
protein. For example, baker's asthma is a common occupational disease, yet very
few bakers have allergic responses after eating wheat bread, even though many
bakers have wheat protein-specific IgE. Identifying a sufficient number
of appropriate subjects may be another hurdle when designing human studies.
Aside from the major allergens, the prevalence of allergy to any one complex
allergen may be significantly less than 1 in 10,000 randomly chosen individuals.
Reactions to a single protein may be one-third that number. In practice, it
is very difficult to identify 10 soybean-allergic subjects to provide sera to
perform simple screening assays, even though allergy to soy is thought to be
common (Goodman RE. Personal communication).
Postmarket testing could be performed if there are reports of allergic reactions
to specific foods produced from the commodity containing the GM product. Alternatively,
new patients with allergies to appropriate foods may be asked to participate
in a screen to specifically evaluate IgE binding to the introduced protein.
As indicated above, it is unlikely that screening of a randomly chosen population
of consumers would be productive in identifying allergies to a specific protein.
In postmarket testing, antigen-specific IgG may be measured to evaluate exposure
of the subjects and immunogenicity of the protein but should not be considered
as a marker of potential allergenicity, as antigen-specific IgG is not considered
sufficiently robust or predictive for the diagnosis of allergies. Further, the
only two plausible mechanisms of action by which IgG binding would cause immediate
allergic disease are through either complement activation, which may enhance
the IgE effect in mast cells, or receptor-mediated IgG activation of mast cells,
for which the evidence in human allergic reactions is quite weak, although some
studies demonstrate activity in rodents (Siraganian 1997). Although there have
been occasional demonstrations of antigen-specific IgGs that may enhance IgE
binding and allergic responses (Denepoux et al. 2000), other clear studies provide
evidence that IgG antibodies are important in blocking IgE reactions after allergen-specific
immunotherapy (van Neerven et al. 1999). Therefore, measurement of antigen-specific
IgG has not been found to correlate with clinical allergy.
Testing of sera from human subjects should always be done with careful attention
to ethical standards. These include obtaining informed consent from subjects
and the right of the subject to access the results from their own sera.
How should biomarkers of exposure and effect be validated? Validation
must include tests to verify the specificity of binding, and measurement of
the dose-response characteristics of the antibodies and protein similar
to what would be required for a clinical diagnostic assay (NCCLS 1997). Assay
performance should be verified in an independent laboratory. Positive cutoff
values must be established that would indicate probable clinical significance,
not just a given statistical level above background. Significance scores for
commercially available in vitro allergy tests such as the Unicap or CapRAST
system (Pharmacia Diagnostics, Uppsala, Sweden) (Sampson 2001) may serve as
a model for these tests. Although levels should not be set to the level found
to be 95% predictive of clinical symptoms, the cutoff should be high enough
to minimize false-positive reports. There are many examples in the literature
of positive IgE binding to proteins that do not seem to cause clinical reactions
(e.g., Fujita et al. 2001). Sera from many individuals without clinically identifiable
allergic reactions to foods will contain some low-level binding to common food
proteins. The IgE in that case is typically much less abundant than in those
with clinical symptoms, or may have significantly lower affinity for binding.
Some cross-reactivity is due to the binding of carbohydrate-specific IgE that
is of questionable clinical significance (Aalberse et al. 2001). A positive
in vitro IgE reaction is not necessarily proof that a protein would cause
clinical allergies but would indicate the need to test by clinical methods for
definitive proof.
Sensitive Populations
What do we currently know about sensitive populations; what populations
do we think are most at risk? Why? Children are more susceptible to
food allergies than adults and therefore are most in need of protection from
food allergies (reviewed in Sampson 1997). In particular, because allergies
have a genetic component, children of atopic parents might be at even higher
risk. The age of the child also probably plays a role, with younger children
and infants being more susceptible. The higher susceptibility of children in
general, and younger children specifically, may be due to the immature immune
system not being able to develop tolerance, to higher gut permeability in the
infant, and/or to higher dietary exposure, for example, from milk- and soy-based
formulas (Sampson 1997). It has been suggested that the type and patterns of
exposure to specific allergens may be an important factor in the increased prevalence
of food allergy in infants (Zeiger 2000). In addition, children who have preexisting
food allergies are more likely to develop allergic reactions to other foods
introduced into their diets. Finally, the issue of exposures to unexpected sources
of allergenic proteins, such as exposure via milk from soy-fed cattle ("food
chain proteins"), needs to be considered.
Atopic adolescents and adults represent other potentially sensitive groups.
As there is evidence that people with atopic dermatitis are more likely to develop
allergies from exposure by other routes (e.g., aeroallergens; Burks et al. 1988),
and allergic dermatitis is easily observed, it is possible that individuals
with atopic dermatitis might serve as sentinels for food allergies to novel
food products.
Workers who process GM food represent a fourth potentially sensitive population.
Data support the idea that exposure to aerosols of food products could lead
to food allergy (Leser et al. 2001; Roberts et al. 2002). Sensitization might
occur via dermal, respiratory, or gastrointestinal exposure. This population,
having a known exposure, might also be useful from a research standpoint.
Is in utero exposure or exposure via breast milk
something we need to be concerned about? In utero exposure and
exposure via breast milk are important considerations. There is suggestive evidence
that children can become sensitized via breast milk to food allergens consumed
by the mother (Frank et al. 1999; Hourihane et al. 1996; Vadas et al. 2001).
Studies have found food antigens present in uterine fluid. Likewise, specific
IgE has also been found in cord blood. It is less clear if there are windows
of vulnerability to sensitization during in utero development.
Is there any evidence for infections or concurrent exposures to agents
with adjuvant effects influencing the development of food allergies? Although
the role of infections in development of food allergy is an important issue,
definitive data on this question are lacking.
There are data suggesting that cryptochrome-1 Ac toxin from Bacillus thuringiensis
is a highly potent systemic and mucosal adjuvant (Vazquez et al. 1999) and that
phylogenetically distant Cry toxins may bind to the same receptors (Shinkawa
et al. 1999). However, the question of whether GM foods or existing food proteins
could have an adjuvant effect, thereby increasing the allergenicity of other
foods, is still controversial.
Are the strategies currently proposed for safety evaluation (risk assessment)
adequate to protect sensitive populations? The group agreed that no
realistic strategy has been proposed to protect the general population, including
sensitive populations. Overall, the procedures represented within the individual
steps in the decision tree need to be better validated. The use of human serum
for screening needs validation. Although targeted serum screening tests may
be valuable, efforts need to be made to identify appropriate individuals to
provide serum. In addition, the use of postmarket surveillance challenge testing
(e.g., DBPCFC) could be a valuable and safe part of the evaluation process with
proper study design. The use of SPTs and DBPCFCs for evaluating novel food allergens
was a controversial topic. However, the group agreed that the use of postmarket
surveillance skin testing (for detection of sensitization) and challenge testing
(e.g., DBPCFC for establishing reactivity) presents minimal safety concerns
if done properly, and therefore the benefits outweigh the risks.
The value of animal testing in evaluating safety is also an important consideration.
However, currently it is difficult to extrapolate animal results to human, and
more research is needed to overcome this obstacle.
The group agreed that developing standards for safety that protect the public,
are acceptable to the public, and are not too restrictive is an important but
difficult goal, and that it will be impossible to achieve zero risk. The concept
of substantial equivalence has been applied to other areas of risk assessment,
and the group agreed that it could be applied to the area of food allergy. Novel
proteins could be evaluated against a panel of known food allergens of varying
potency as a framework.
Models of Dose Response
Evidence for thresholds for sensitization and/or allergic reactions were presented
and discussed. The need for dose-response information in the regulatory
decision-making process was considered for evaluating the safety of GM crops.
Additionally, the current tools available for developing dose-response
information as well as factors such as immunologic tolerance and sensitization
doses (e.g., number, dose, route, etc.) were discussed.
Table
1 |
What is the evidence that there are thresholds for sensitization and/or
allergic reactions? Data from food-challenge studies in humans were
provided for a number of the known allergenic foods (e.g., peanut, shrimp, milk,
egg, soybean, tree nuts) that demonstrated the existence of a threshold level
for eliciting an allergic reaction to either the food or the purified allergenic
food protein (Table 1). For example, Hourihane et al. (1997) and Wensing et
al. (2001) both reported a threshold of 100 µg total peanut protein or
6 µg of the peanut allergen Ara h2, and a no-observable effect level (NOEL)
of between 30 and 50 µg peanut protein or 2-3 µg of Ara h2 in
patients with peanut allergy, using DBPCFCs. From this study, the authors concluded
that the threshold dose of peanut protein needed to elicit a response in a group
of individuals allergic to peanuts varies. In patients with egg allergy, Moneret-Vautrin
et al. (1998) reported a threshold of < 100,000 µg egg and 54,000 µg
ovalbumin and a NOEL of 5,000-10,000 µg egg or 5,400 µg ovalbumin,
whereas Sicherer et al. (2000) reported a threshold of 100,000 µg egg and
54,000 µg ovalbumin and a NOEL of 100,000 µg of egg (i.e., the first
dose tested). These data are important for establishing the relative risk associated
with a potential food allergen. In contrast to the elicitation phase of allergic
reactions, only limited data are available in humans regarding food allergens
and thresholds for sensitization. Furthermore, there may be populations or subpopulations
of individuals (e.g., based on age, ethnicity) that are more sensitive to the
induction or elicitation phase of protein exposure. In addition, limited data
are available from industrial exposures to proteins (i.e., the detergent industry)
regarding thresholds for sensitization and/or elicitation for aeroallergens
that may be relevant (Schweigert et al. 2000). Thresholds for sensitization
have also been reported in various animal models for both dermal (Basketter
et al. 1997, 1999; Kimber et al. 1999) and respiratory (Hillebrand et al. 1987;
Karol 1983) sensitizers.
Is there a need for dose-response information, or is hazard identification
alone adequate to warrant regulatory action? The consensus was that
dose-response information was needed, particularly for setting relative
risk. However, current regulatory action regarding the potential allergenicity
of foods derived from GM plants is driven predominately by hazard identification.
For example, the food allergy decision tree developed by FAO/WHO (1996, 2001)
involves determining the source of the gene(s) (is the source of the gene allergenic?),
evaluating the physicochemical characteristics of the protein (amino acid sequence
homology to known allergens; pepsin resistance) (Taylor and Lehrer 1996), conducting
specific and targeted serum screening, and using validated animal models. If
one or more of the latter evaluations is positive, the protein is labeled as
likely allergenic and removed from further consideration. Therefore, the relative
risk associated with such a protein is usually not considered.
What is the role of immunologic tolerance in establishing dose-response
information? Currently, little is known regarding the mechanisms of
immunologic tolerance. The group concurred that the dose-response curve
begins once tolerance is broken or fails to be established. Furthermore, because
tolerance to a particular protein can be induced by both low and high doses,
it was agreed that a wide range of doses should be used to maximize the chance
of falling within the responsive range of the dose-response curve.
What tools are available to develop dose-response information?
The current tools available to develop dose-response information
include animal models and human food-challenge studies (Table 1). For both technical
and ethical reasons, it would be very difficult to conduct human food-challenge
or sensitization studies with novel proteins. Therefore, it was the opinion
of the group that a validated animal model(s) that assesses the allergenic potential
of proteins currently represents our best tool for establishing dose-response
information. As with humans, the genetic predisposition of a particular animal
strain may influence the degree of allergenicity observed with a particular
protein. Thus, it is important to ensure that haplotypic differences do not
result in misleading results in animal models. In initial validation studies
multiple strains of the same species could be evaluated to determine those strains
most sensitive and specific in discriminating between both known allergenic
and nonallergenic proteins.
Is one large sensitization dose the same as many small sensitizing doses?
The group agreed that one large sensitization dose is not the same as
many small sensitizing doses. Among the factors that need to be considered are
the age at which the sensitization dose(s) occurs, the time between sensitizing
doses, and the route(s) of administration of the sensitizing dose. For example,
Woolhiser et al. (2000), using natural rubber latex proteins, reported differences
in latex-specific IgE immunoblot profiles and pulmonary function between four
different sensitization routes. These authors concluded that the sensitization
route of exposure might partially determine the primary allergen(s) and the
clinical symptoms of the allergic response.
Postmarket Surveillance
What tools and strategies are currently available for postmarket surveillance,
and what can we learn from them? The feasibility of a range of tools
and strategies that could be applied to postmarket surveillance of foods produced
through biotechnology and their role in providing information for safety assessment
were considered. Such tools and strategies included the collection and analysis
of case reports of adverse reactions; conduct of classical epidemiology studies
such as cohort, case-control, ecologic, and cross-sectional studies (telephone
surveys, food frequency questionnaires); and clinical trials (DBPCFCs). The
identification of highly exposed populations in the context of specific foods
(e.g., occupational groups, populations with limited variety in the diet such
as children, infants, ethnic populations) was considered helpful for the conduct
of epidemiologic studies. Marketbasket surveys were discussed as a means of
assessing exposure to specific types of food and would reflect regional food
preferences. It was suggested that modern technologies such as grocery-scanning
devices could assist in data collection. However, exposure to GM foods would
be difficult to establish in the absence of specific labeling.
The most useful tool was considered to be the development of an adverse event
reporting system that would allow the systematic collection of case-report data
on adverse reactions to foods. This offered the possibility of identifying sensitized
individuals. In the United States, such data are currently collected through
state health departments, emergency rooms, and self-reporting. The existing
infrastructure limits the capability for collecting such information. In addition,
a reliable method for detection of recombinant proteins in food products is
essential to establish that exposure to a protein that is the suspected cause
of an adverse reaction did in fact occur. It is important to note that this
kind of system is most useful in identifying individuals sensitized to specific
food items rather than for assessing the potential for GM food-induced
allergy.
For data on adverse food reactions collected from postmarket surveillance
to be meaningful, they must be compared against baseline data collected prior
to the introduction of the food under surveillance. It was suggested that such
data could be collected from pilot surveillance projects to reflect regional
food preferences across the country.
DBPCFCs were considered essential as a postmarket tool for the validation
of causation between a specific food ingredient and an adverse reaction. Such
studies were not considered useful screening tools to establish the potential
of adverse food responses related to a particular food. The limitations and
issues surrounding the use of such methods were discussed. These include difficulties
associated with proving the negative (how many people would need to be tested)
and ethical issues (e.g., the potential for induction of sensitivity in previously
unexposed individuals or induction of life-threatening allergic response in
the process of screening).
When should postmarket surveillance be done? The issue of whether
the conduct of postmarket surveillance should be routine for foods produced
through biotechnology or should be triggered by a specific event in relation
to a particular food (as in the case of StarLink corn) was discussed by the
breakout group. Participants with concerns about the uncertainties inherent
in the current procedure for allergenicity assessment of recombinant proteins
favored routine monitoring. In contrast, a number of participants were confident
that the present assessment process was adequate to identify novel proteins
with allergenic potential and supported the use of postmarket surveillance only
when triggered by a specific event.
What tools are needed to improve our ability to do postmarket surveillance
and interpret results? The most useful tool was considered to be the
collection of case reports of adverse responses to food. Modification of existing
adverse events reporting systems was suggested for this purpose. Among the features
of such a system that needed to be determined were a) establishing who
would be responsible for notifying, b) who should be notified, and c)
who would be responsible for following up the case reports to collect necessary
information. In terms of the current infrastructure available for the collection
of these kinds of data, two scenarios were envisioned: improvements to the current
system to capture data that would assist assessment of foods produced through
biotechnology, or developing an entirely new system for collection and analysis
of data. Creation of a nonprofit industry association group such as the Drug
Safety Alliance that would be provided with access to the desired information
was suggested.
Several members of the group felt that exposure assessment would be assisted
by labeling foods produced through new technologies, but this clearly was an
area in which consensus was not reached. Difficulties were acknowledged in using
such labeling in products containing commodity crops in which mixing of harvested
crops from different suppliers is a typical practice. Foods in which identity
preservation is part of the marketing strategy, as in cases of nutritional modification
or allergen reduction of products, were considered to be more amenable to labeling.
The lessons of previous experience, (e.g., from events where products not
accepted for human consumption were found in consumer products) were considered
useful in terms of guiding development of postmarket strategies. Such strategies
would benefit from the adoption of a multidisciplinary approach.
Overall Summary
Each breakout group addressed specific issues in the areas of use of human
clinical data, animal models to assess food allergy, biomarkers of exposure
and effect, sensitive populations, dose-response assessment, and postmarket
surveillance. These groups discussed the adequacy of the current tools available
for assessment of the allergenic potential of novel foods and described how
these tools need to be improved for better safety assessment. In several instances
the groups reached different conclusions or could not agree on the utility of
specific tools, such as the use of human sera for hazard identification or labeling
of foods containing GM crops. Each group was asked to identify specific areas
in which data gaps should be the focus of future efforts. These research needs
are discussed in detail in the article by Selgrade et al. (2003).
| Breakout Groups
and Participants |
| Use of Human Clinical Data |
| Charlotte Madsen, Chair, Danish Veterinary and Food Administration |
| Rhoda Kagan, Rapporteur, McGill University |
| James Astwood |
| Brian Buckley |
| Lynn Goldman |
| Michael Hansen |
| Samuel Lehrer |
| Kathleen McAveny |
| Katherine Sarlo |
| Animal Models to Assess Food Allergy |
| Oscar Frick, Chair, University of California, San Francisco |
| Henk van Loveren, Rapporteur, National Institute of Public Health and
the Environment |
| Gary Bannon |
| Bruce Hammerberg |
| Daniel Jones |
| Michael Holsapple |
| Ian Kimber |
| Robert Luebke |
| Dean Metcalfe |
| Christopher Portier |
| Carolyn Van Pelt |
| Biomarkers of Exposure and Effect |
| Genevieve Bondy, Chair, Health Canada |
| Richard Goodman, Rapporteur, Monsanto |
| Raymond Biagini |
| Rebecca Dearman |
| Tong-Jen Fu |
| Steven Gendel |
| Dennis Hinton |
| Richard Raybourne |
| Sensitive Populations |
| Mel Dong, Chair, U.S. Food and Drug Administration |
| James Mastin, Rapporteur, National Institute of Environmental Health Sciences |
| Leonard Bernstein |
| Daniel Goldstein |
| John Kough |
| Joan Rothenberg |
| Hugh Sampson |
| MaryJane Selgrade |
| Deborah Sheely |
| Models of Dose Response |
| Robert Frederick, Chair, U.S. Environmental Protection Agency |
| Gregory Ladics, Rapporteur, DuPont |
| Jonathan Allen |
| Bob Buchanan |
| Barbara Henry |
| Andre Penninks |
| Marsha Ward |
| James Weaver |
| Kimber White |
| Postmarket Surveillance |
| Fujio Kayama, Chair, JICHI Medical School, Japan |
| Elizabeth Vavasour, Rapporteur, Health Canada |
| Dori Germolec |
| Val Giddings |
| Rebecca Goldburg |
| Ricki Helm |
| Thomas Hoban |
| Juliana Jones |
| Kim Waddell |
| Michael Woolhiser |
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Last Updated: June 17, 2003
