Aventis CropScience
Aventis
EXPERT OPINION CONCERNING
Allergenicity Assessment of Cry9C Bacillus thuringiensis subspecies
tohvorthi Corn Plant Pesticide for FIFRA Scientific Advisory Panel
February 29,2000
Washington, DC
Author
Andrew Cockburn PhD CBiol FIBiol
Registered Toxicologist
Head of Toxicology
Aventis CropScience
(Formerly AgrEvo)
Chesterford Park
Saffron Walden
Essex
CB10 1XL
England
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29 February 2000
Dr Andrew Cockburn
Registered Toxicologist
Dr Andrew Cockburn BSc, PhD, C Biol, FI Biol, is Head of Toxicology UK and Head of UK
Development for Aventis CropScience ("Aventis"), successor in interest to AgrEvo Company
("AgrEvo) based at Chesterford Park, in Essex, United Kingdom. He obtained BSc Hons First
Class in Applied Biology at Brunei University, London in 1968, specialising in biochemistry,
where he also gained his PhD for in vitro studies on the detection of cardiotoxicity. He worked
in the food industry, Rank Hovis McDougall, for two years on the safety of novel foods and
proteins before joining the Pharmaceutical Industry, Beecham, now 8KB, in 1970. As Head of
Toxicology he was involved in the assessment and registration of a wide range of human and
animal medicines and biologicals. In 1987 he moved to his present position in the Agrochemical
Industry as Head of Safety Evaluation with Schering, and was appointed as Head of Toxicology
Worldwide for AgrEvo on formation of the Joint Venture Company with Hoechst. He has been
involved in dealing with Regulatory Authorities worldwide, particularly with regard to
toxicological and safety issues surrounding the registration of new chemical classes including
novel proteins and GM crops. He has been a member of the Executive and Main Committees of
the British Toxicology Society (BTS) and former Chairman of the BAA Health & Safety
Committee. He is currently Chairman of the British Industrial Biological Research Association
(BIBRA) Agriculture Group and a member of the Research Defence Society Council. He has
served on a large number of committees including the 1990 OECD ad hoc meeting in
Washington, on Neurotoxicity Testing. He is presently a member of the Joint Institute of
Biology (IOB)/BTS Register of Toxicologists Committee which considers applications for
membership from suitably qualified professionals in the field. He is a member of the Board of
Trustees for ILSI HESI and a member of the US Society of Toxicology (SOT). He regularly
lectures at the Universities of Cambridge, Surrey, Brunei, Hertfordshire and King's College in
London.
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Contents
1. Executive Summary
2. Introduction
3. Questions posed by EPA for the Peer Review Panel and responses to those questions
4. Safety evaluation philosophy of Cry9C for humans - an Holistic Overview
4.1 General toxicological considerations
4.2 Assessment of potential for allergenicity
4.2.1 Host crop - 'Lea mays
4.2.2 Gene Source of cry9C - Bacillus thuringiensis
4.2.3 The Gene Product - The Cry9C Protein
4.2.4 The Genetically Enhanced Crop - Event CBH-351, Cry9C corn
5. Overall weight of evidence assessment
5.1 General Toxicology
5.2 Potential for Allergenicity
5.3 Exposure for Consumers
5.4 Risk Assessment
6. Substantial equivalence of Cry9C corn to unmodified corn
7. References
8. Appendices (1-3)
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1. Executive Summary
Cry9C corn, also referred to as StarLink™ corn, represents a major advancement in corn
borer control and represents an excellent addition to growers' options that reduces or
eliminates the need for chemical inputs and fits well within an integrated pest
management program.
Cry9C corn hybrids were formed by insertion of the cry9C gene, from Bacillus
thuringiensis subspecies tolworthi. This gene encodes a highly efficacious insecticidal
protein that offers growers superior corn borer control compared with CrylAb and
Cry 1 Ac proteins. While Cry9C shares most of the characteristics of the other Bt proteins
it is of particular value because it targets a completely different binding site in the
insect's gut offering growers new options for insect resistance management.
Having carefully followed and extended the international guidance given for food, feed
and environmental safety assessment of plant pesticides, a large package of studies has
been performed and evaluated from which it has been concluded that Cry9C corn is
substantially equivalent in all respects to the corn currently in commerce. A critical part
of that evaluation involved very detailed consideration of whether the source of the novel
protein, its characteristics and properties, the host plant or the new hybrid might in any
way predispose an allergenic risk for man.
This expert opinion which is backed up by extensive literature surveys, discussions with
international experts in the field of clinical allergy and immunology, and individual and
summary reports (McFarlane, 1998 and Cockburn, 1998) to EPA concludes that
genetically improved Cry9C corn is as safe in all aspects as regular unmodified corn. We
do not believe that concerns over the relative digestibility or thermolability of Cry9C
protein to digestion or heat detracts from the above conclusion or significantly alters the
weight of evidence for reasons articulated in this expert document, and summarized as
follows:
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There are no untoward nutritional, toxicological or wholesomeness findings.
There is no evidence from human worker exposure for up to 5 years of any adverse
response or increase in atopy.
There is no evidence from the animal feed use of Cry9C corn of adverse reactions in
cattle and poultry or the humans consuming the resultant milk, meat and eggs.
There are no indications that Cry9C protein, which shares significant homology with
other Bt proteins, is an allergen.
There is no evidence from human sera studies that Cry9C corn is inherently more
allergenic than standard unmodified corn.
The theoretical maximum daily intake (TMDI) of the Cry9C protein is c.0.003
mg/kg/day for a European and c. 0.005 mg/kg/day for an American. The RfD is
based on the mouse 30-day no-adverse-effect-level of 33.3 mg/kg/day with an
uncertainty factor of xlOO, i.e. 0.3 mg/kg/day. The human dietary exposure is
therefore 60-100-fold lower and utilizes less than 2% of the ADI.
In the absence of any evidence that the Cry9C protein is an allergen, consumption of
Cry9C corn containing very low levels of the novel protein is considered not to pose
an allergenic risk to humans.
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2. Introduction
Bacillus thuringiensis (Bt) proteins have been used for decades as sprayable, microbial
products having a very high order of safety for man, animals and the environment.
No cases of allergenic reaction have been documented despite dermal, oral and inhalation
exposures. A reference to this is made by the Environmental Protection Agency (EPA) in
a Federal Register notice, dated August 16, 1995, (60 FR 42443) (FRL-4971-3).
Similarly, Cry9C corn plants, expressing the Cry9C protein, which have been intensively
researched and developed over the last 5 years and approved for animal food use via
tolerance exemption on May 22, 1998, have not shown any indications of allergenic
reactions in either the workforce involved in field trials, harvesting and processing of
crops, or those including the public, associated with the production and consumption of
milk, poultry, meat and eggs.
Toxicology studies (McClintock et al., 1995), backed up by human experience, mode of
action and receptor-based studies have shown that the safety of Bt proteins to mammals
can be accounted for by their insect-specific binding sites. It is this target species
specificity that creates the ideal non-chemical insecticide. The Cry9C protein is no
exception.
For the environment, Cry9C corn protein poses no foreseeable risk with regard to non-
target organisms, including mammals, birds and non-target insects.
EPA has now reviewed all the data submitted by Aventis CropScience USA LP
("Aventis") successor in interest to AgrEvo USA Company ("AgrEvo") as part of the
process required to consider amendment of 40 CFR 180.1192 to expand the current
exemption from the requirement of tolerance of Bt Cry9C protein for animal feed only to
all food commodities.
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While EPA acknowledges that "none of the data supplied by AgrEvo (now Aventis)
suggests that the Cry9C protein is a food allergen, the Agency is seeking advice to
determine the risk that exists from exposure to this protein based upon two of its
biochemical characteristics (stability to heat and gastric digestion)".
Understandably EPA seeks reassurance by "further data and clarification" to determine
that a reasonable certainty of no harm will result from exposure to this protein and have
posed a number of questions to which answers have been provided.
Aventis has therefore provided data and arguments in this expert document regarding the
relevance of these two physicochemical characteristics in relation to Cry9C to support its
conclusion that Cry9C corn poses no greater allergenic risk to consumers than standard
unmodified corn.
The structure of this report has been arranged for clarity as follows:
Section 3. Response to Questions posed by EPA
Section 4. Safety Evaluation Philosophy for Cry9C
Section 5. Overall weight of evidence assessment
Section 6. Substantial Equivalence of Cry9C corn
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3. Questions Posed by EPA for the Peer Review Panels and Responses to those
Questions
Cry9C - Specific Questions:
1. (part a and b) Q.
Is the brown Norway rat study an animal model of food allergy recognised as valid and useful by
the scientific community? Has the model been validated by recognising known food allergens
and not recognising other food dietary proteins?
A.
No, the Brown Norway rat model has not been internationally validated and is therefore not yet
considered useful by the scientific community. Many attempts have been made to construct and
validate a suitable model for the detection of food allergy in man. These have included the use
of rats, mice etc., yet none have proven reliable or appropriate for man.
As yet there is no single Brown Norway rat allergy model. Until recently BIBRA in the UK was
one of two main groups experimenting with the Brown Norway rat using known protein
allergens. The other group was based at TNO, Nutritional Food Research Institute, in the
Netherlands. The protocols for both models differed significantly, clearly highlighting the
difficulties in trying to design a model that adequately covers all aspects of this complex area in
a species that does not react like man. The two companies have now merged to form one
Contract Research Organisation known as TNO BIBRA. It is anticipated that further work will
be performed to continue to develop this model as with other models currently under
development for the detection of potential food allergens. However, it is generally accepted in
the scientific community that "new, well validated methods with high sensitivity and specificity
cannot be expected in the near future" (Houben et al, 1997). Aventis continues to fully support
such research efforts.
To date, the research work has been directed at the development of the model, and not safety
assessment per se. No animal models of human food allergy have been validated although
efforts continue, mainly in mouse, guinea pig and rat. A validated animal model should
distinguish between food allergens (major and minor) and non-allergenic food proteins,
establishing a linear relationship between known allergenic proteins and IgE reactivity
(allergenic potential). According to BIBRA, all of the proteins tested have produced an IgE
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reaction; e.g. ovalbumin, lactoferrin, bovine serum albumin (BSA), CrylAbS and Cry9C,
(Atkinson et al, 1996). Lactoferrin is not a clinically important allergen, yet it demonstrated the
strongest response. On the other hand, BSA and CrylAbS are proteins with
little or no history as human food allergens, respectively, yet in this model still cause a reaction.
Thus the BIBRA model has not been validated using positive and negative controls.
Additionally the method, by which BIBRA measures responses, using the passive cutaneous
anaphylaxis (PCA) reaction, has a number of problems. First, rats and mice are known to have
an IgG homocytropic antibody response, in addition to any IgE response both of which react by
PCA. Thus at least 48 hours must be allowed following injection of test sera prior to measuring
the response to allow for the IgG antibody to diffuse away. In the research results, a 24-hour
incubation time is utilized, which casts doubt over the specificity of the response to IgE.
Secondly, the skin of mice and rats can respond differently dependent on the location of the
injection, which was not standardized. Third, using the number of responding animals is not a
typical method to analyze immunological reactions. A better method to evaluate responses would
be to titer out each serum (in duplicate or triplicate) and measure the lowest dose mean serum
that causes a reaction, a typical method for human evaluations (Krdppels, 1998). Once antibody
titers are quantitated, comparisons can be made between the allergenicity properties of specific
proteins (See critique of Lehrer, 1998).
The ideal requirements for an appropriate animal model of human food allergenicity would be
expected to stand up to rigorous scientific standards and would inter alia include:
• Sensitization upon oral dietary exposure to the novel protein or food stuff.
• The challenge reactions (clinical signs) showing the involvement of the various
organs and tissues also affected in humans exhibiting the food allergy.
• The animal model showing tolerance to most proteins as in man.
• That a significant IgE antibody response should be measurable to the new protein and
the use of adjuvant is not a pre-requisite.
• Reference, non-allergenic food proteins should be used as negative controls.
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• That the model shows a comparable relative allergenicity ranking to well-
characterised allergens as found in humans.
• That in the animal model the IgE responses to well-known allergenic food products
are directed to the same proteins in the allergenic food as found in sensitive humans.
• That the dietary history and IgE status of the test animals is known for at least 2
generations to prevent assay interference through prior sensitization.
• The model must be easy to conduct and reproducible at any time, at any laboratory in
the world within the same animal species, using standard protocols
and diets, established via a "blinded" ring test among different laboratories.
• International validation should be obtained against agreed criteria e.g. via ICCVAM.
1. (part c) Q.
Is the use of an adjuvant such as carrageenan appropriate to examine a normally functioning
immune system?
A.
No, the use of carageenan in the Brown Norway rat model to stimulate the immune system of the
rat is irrelevant to normal human dietary exposure. Carrageenan itself may be an allergen in
certain individuals.
2. Q.
Does the bioavailability study provide useful information about allergic potential? Can a protein
be a food allergen without being able to cross the GI mucosa? Is gut permeability too variable
within the population to be used as a screening tool?
A.
No, the bioavailability study does not provide useful information about allergenic potential, as it
showed that the amount of Cry9C protein absorbed and detected in the blood (0.0002 to 0.0006%
of total dose) was within the range expected for "normal" dietary proteins of non-allergenic
status (Strobel, 1997; Gardner, 1988).
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It is generally agreed that small amounts of intact proteins from the diet cross the intestinal
mucosa via the sampling cells (M cells) in the Peyer's Patches where they are brought into
contact with migrating lymphocytes. The result may either be the induction of immune tolerance
(typically for most orally introduced proteins) or an allergic reaction, depending on the
solubility, concentration and other features of the antigen encountered. It should be noted that the
human adult immune system is constantly absorbing small but biologically significant amounts
of intact antigens from the intestinal lumen to maintain tolerance (Strobel, 1997). Very few
examples of untoward consequences of this contact are seen. This shows that very few food
proteins elicit an immune response once across the mucosal membrane.
A protein can be a food allergen without crossing the gastrointestinal (GI) mucosa, as in the case
of the so-called oral allergy syndrome (OAS). There is some evidence to suggest that a local
mucosal immune response does occur after oral antigen encounter, however the extent of this
response is under debate (ILSI Europe Concise Monograph Series, 1994). Following
gastrointestinal transfer of proteins, activated lymphocytes within the Peyers Patches migrate via
the lymphatics to the mesenteric lymph nodes where transportation occurs to the systemic blood
circulation. Once in the circulation the potential for a true allergenic response to the food antigen
is more likely. Thus is most cases, crossing the GI mucosa is essential for a true allergenic
response.
Gut permeability is currently considered to be irrelevant as a screening tool. All proteins are
absorbed to some degree under normal circumstances in man and animals as part of the normal
physiological process (Gardner, 1984 and 1987). The typical amount ingested varies between
0.001% and 1% of the total ingested protein (Strobel and Mowat, 1998). For Cry9C in the rat,
dosed at 10,000 times the theoretical maximum daily human exposure on a weight basis, between
0.0002% and 0.0006% Cry9C dose was found in portal blood in cannulated animals. (Noteborn,
1998). In short the absorption of Cry9C for antigen presentation appears to be very low indeed.
The relevance of the process of endocytosis across the M cells of the Peyer's Patches is to permit
direct access of the protein to sub-epithelial lymphocytes for antigen sampling in order to facilitate
the development of immune tolerance. It must be recalled that very few food proteins display
allergenic potential despite being immunogenic. This is because they differ markedly in their
ability to induce IgE and mediate allergic sensitisation.
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Within the population several factors can alter the permeability of the GI tract; e.g. alcoholic
beverages, detergents, gold compounds, chelators, ischemia, radiation and cytotoxic drugs (S.
Gislason, MD at www.nutramed.com/digestion/gastroint.htmn. It should be noted that "controlled"
sampling of antigens is a continuous process making it difficult to set a "base" level.
In conclusion, apart from confirming a very low order of transluminal endocytosis for Cry9C, gut
permeability studies are not considered helpful in isolation for determining allergenic potential as
this characteristic depends on a protein's ability to induce IgE antibody production and binding to
mast cells, which was not the end point of this experiment.
3. Q.
In the case of the Cry9C protein, does the apparent degradation of the 68 kDa protein to a 55
kDa protein Suggest anything regarding the digestibility/allergenicity of this protein.
A.
The in vivo degradation of the Cry9C protein from a 68 to 55 kDa fragment in rat confirms
partial mammalian digestibility of Cry9C. The Cry9C protein (Escherichia coli and B.
thuringiensis derived) has also been shown to be partially digestible in vitro using simulated
gastric fluid (SGF) where 15-25% of the protein was digested (Noteborn, 1998), and more
recent preliminary data from this laboratory has indicated complete digestion of the Cry9C
protein (E. coli derived) under SGF conditions (Aventis, unpublished data, 2000; see Section
4.2.3 and Appendix 2). However, partial digestion does not equate with allergenicity. A relative
lack of digestibility is a feature of some but not all stable proteins, which are not allergens; e.g.
actin and myosin. This is an area that has not been fully explored. The size of a fragment is
irrelevant as a marker for allergenicity, as epitopes as small as 8 amino acids have been shown
to be sufficient to produce an immune response ( Rothbard et al, 1991). In this context
digestibility also has the potential to uncover more epitopes (epitope unmasking) than is the case
for less digestible proteins. It should also be noted that intact proteins which are digestible also
have the potential to cause sensitisation and allergy in the mouth, this is known as oral allergy
syndrome (OAS). Additionally, searching databases of known allergens with amino acid
stretches of Cry9C did not match with any know allergen.
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4. Q.
Does the additional data provided by AgrEvo further reduce or alleviate concern ofCry9C as a
potential allergen or further implicate Cry9C as a potential allergen?
A.
If a comparison could be drawn with the assessment of standard agrochemical products with
respect to carcinogenic potential, a weight of evidence approach is seen as a very powerful
indirect aid in assessing the carcinogenic potential of an agrochemical for man following
evaluation of data from oncogenicity studies. Likewise here, in the absence of appropriate
methodology, a weight of evidence approach would seem reasonable and appropriate. Under the
current decision tree paradigm for food allergens, only two parameters have been partially
triggered for Cry9C out of a much larger range of parameters. The strong weight of evidence is
against the Cry9C protein having any allergenic potential for man.
Very little research has been conducted into non-stable protein allergens or conversely stable
protein non-allergens. Until better understanding of the complex nature of food proteins and IgE
antibody induction by food proteins is understood, no one parameter can be seen as being
definitive and each weight of evidence consideration should be given equal, not unequal
emphasis. It cannot be concluded that because some food allergens are stable proteins, all stable
proteins are food allergens.
A weight of evidence approach is a logical way to assess Cry9C potential allergenicity
• No allergenic history from the source of the genes (Cry9C - Bacillus thuringiensis) nor any
significant allergenic history from the crop (Zea mays), into which they were introduced.
• No epitope sequence homology - full-length protein or at the 8 amino acid level - to known
allergens.
• Low prevalence in corn grain (0.17% of total protein) - much less than the >1% level cited as
a characteristic of food allergens (soybean p conglycinin 18.5%, glycinin 51%, ovalbumin
(egg) 54% or casein (milk) 80%).
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• No increased allergenic reactions in more than 1900 seed company workers exposed for up to
3 years.
• Molecular weight of the Cry9C protein (c. 70 kDa) is relatively higher than expected for
most allergenic proteins; e.g. 10-40 kDa.
• Post-translational modification, such as glycosylation, has not been demonstrated for the
Cry9C protein as expressed in corn.
• No evidence of immunostimulation observed in acute intravenous mouse study, sub-chronic
30-day mouse or 42-day poultry study. Irritation of the mucosal membranes is often
observed in food allergy reactions; none was observed in either mouse study.
• No increase in the inherent allergenicity of Cry9C corn vs. standard unmodified corn when
evaluated in immune sera from corn sensitive individuals.
• Sequence homology with many other Bt proteins currently in the human food supply.
Overall Protein Allergenicity:
5. Q.
Are the characteristics of heat stability and resistance to digestive enzymes useful criteria to
screen for food allergenicity? Are there known examples of dietary proteins that have these
stability characteristics yet are not allergenic?
A.
The characteristics of heat stability and resistance to digestion are considered to be of equal
weight to other characteristics listed above in the list of parameters which permit a weight of
evidence judgement to be formed regarding potential allergenicity. However, they do not
deserve to be overweighted because stability is by no means characteristic of all food allergens
and the same characteristics are common in food non-allergens. As an example, consider a novel
protein that has the following characteristics: digestible in gastric fluids and thermolabile,
molecular weight of 30 kDa, no allergenic history of the gene sources, protein is expressed in
soybean, no sequence homology to the whole protein but the novel protein does share sequence
homology at the 8 amino acid level with a known peanut allergenic protein. What would be the
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agency decision for this novel protein? Certainly, if stability to digestion were the most
important criteria, then this new protein would be allowed into commerce.
Yes, there are examples of food proteins that are relatively stable to heat and resistant to
digestive enzymes. Actin and myosin are two that most people could name. Thus the current
concern of stability equating to allergenicity is imperfect and is seen only in certain specific
cases. The full "grid" (Table 1. see below) of stable allergenic, non-stable allergenic, stable non-
allergenic, and non-stable non-allergenic proteins needs to be greatly expanded before we can
give greater weight to any one particular correlation. It is clear from even this limited list that
there is no absolute black and white boundary. Moreover when one examines digestion time, for
many proteins it is not an all or none reaction within 15 seconds. Up to 60-80% may degrade
rapidly but often the remaining 20 - 40% may take hours to degrade.
TABLE 1
Stability designation of allergenic and non-allergenic proteins
ALLERGENIC
LESS STABLE PROTEIN
Apple (Mai dl)u"z;
Celery (Bet v 1 like)(1)
Castor Bean (3)
Ragweed(4)
Fish(5)
MORE STABLE PROTEIN
rttr
lVJLVyj.Vl_/ O J. /-VJJJL/JL
Peanut (Ara hi)'
Soybean(6)
Hazelnut(6)
Castor Bean(3)
Tomato (7)
Fish(egGadCl)(5)
Mnrc<=> T?5»Hi«Vi Pf>rr»Yi
NON-ALLERGENIC
Apple(1&2)
Rubisco02
Lipoxygenase(13)
Horse Radish Peroxidase (HRP)
Mothers Colostrum
Potato (9)
Rice <9)*
Glutamate decarboxylase ^10 &' ^
Maize (P-l 00)(11)
Processed food proteins (beef,
poultry, pork)
* Separate from the known allergenic protein
(Protein Allergen in brackets)
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Table References
1. ViethsSetal(1995) 7. Dircks L Ket al (1996)
2. DreborsS&FoucardT(1983) 8. McLean E& Ash R (1996)
3. LehrerSBetal(1981) 9. Astwood JDet al (1997)
4. Marsh DGetal( 1981) 10. Strobel S (1997)
5. Bernhisel-BroadmentJetal(1992) 11. Vantard et al (1994)
6. Astwood JDetal( 1997) 12. Ellis R J (1979)
13. Stedow,NJ(1991)
6. Q.
Does the lack ofamino acid homology offer predictive function for examine the allergenicity of a
new dietary protein, or alternatively does it simply indicate what allergenie population should be
examined to look for possible reactivity?
A.
Yes, although the comparison of amino acid homology can not fully predict whether particular
combinations of amino acids will be allergenic in man following consumption, these
comparisons are a very useful predictive tool. No epitope homology was seen for Cry9C when 8
key databases were searched. Most dietary proteins are unfolded and may even be broken down
into smaller pieces, exposing allergenic epitopes. Homology comparisons, especially at the fine
level of 8 amino acids, can identify closely related allergenic proteins, which allows for direct
testing using sera from sensitive individuals. It should also be understood that these searches do
not take into account the tertiary structure of the protein that could be important in creating a
discontinuous epitope not seen in a linear sequence.
Should an amino acid homology search identify a match to a known allergenic protein, a direct
testing program can be initiated. Working with a clinical immunologist can identify sources of
sera from allergenic individuals, which can be utilized for a more detailed study of cross
reactivity. A large number of people may have to be screened because of the low incidence of
food allergy in the adult population (c. 1-2%).
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7. Q.
There is anecdotal evidence that total dietary exposure to a food correlates to food allergy (i.e.,
prevalence of rice allergy in eastern Asia, fish allergy in Scandinavia, wheat allergy in Europe
and the Americas). Does level of exposure in the diet affect the sensitization phase of food
allergy? Would exposure to a protein as a minor component of direct dietary consumption
lessen the likelihood a protein would become a food allergen? Is there evidence that feed
exposure (i.e. soybean meal as an animal feed) can affect the allergenicity of the resulting meat,
milk or eggs? For example, does the use of soybean meal as animal feed make the resulting
meat, milk or eggs an allergenic risk for a soybean sensitive individual?
A.
Many, although not all major food allergens are present as a relatively high percentage of the
total protein in the plant or foodstuff, e.g. soybean P conglycinin 18.5%, glycinin 51%,
ovalbumin (egg) 54% or casein (milk) 80% (Metcalf, D.D. et al, 1996). It is generally believed
that an individual usually is exposed to a high level of a dietary protein in the process of
sensitization, which may in certain individuals ultimately lead to an allergenic reaction. Expert
opinion is divided upon, when, (in life) how much (exposure level), how often and for what
duration exposure to a particular food protein is needed before the induction of an allergenic
response occurs. Exposure to a protein as a minor component of a diet is the typical route of
exposure of most dietary proteins and man rarely suffers from food allergies (1 - 2% of the adult
population) from the large and diverse group of ingested proteins. This sensitization process
probably varies from individual to individual due to exposure, genetic make-up and possibly as
yet undefined environmental factors and between different populations due to cultural
differences in eating habits within these groups.
There is absolutely no evidence that cattle exposed to Cry9C through feed since approval of
animal food use in 1998, has resulted to increase allergenicity of milk, meat or eggs in man.
In conclusion the lack of any concrete allergenic alert and the very low levels of absorption of
Cry9C coupled with the very low level of Cry9C from dietary intake supports the unlikelihood of
any allergic development in man when considering the exposure situation reflected in the
anecdotal cases cited in the question. This is further reinforced by existing inhalation (a sensitive
route) and dermal exposure by seed workers exposed to Cry9C containing dust and also by lack
of evidence or any untoward effects from human consumption of milk, meat and eggs.
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8. Q.
Is it feasible to monitor changes in the incidence of human food allergy to stable proteins? How
quickly are newly introduced food allergens typically identified after they become part of the
human diet?
A.
There is no current model or system set up that routinely monitors a population for allergenicity
or increased allergenicity to newly introduced proteins. Kiwi fruit, for example, was identified as
an allergen late in 1981, shortly after its introduction as a new fruit. It should be noted that the
prevalence of kiwi fruit allergy is low in food allergenic individuals. Most systems are
anecdotal at best in the first instance and subsequently proven clinically. Overall the dietary
exposure to Cry9C will be so low and the weight of evidence components so generally
reassuring from what we know about Cry proteins in general, that there are no grounds to
consider that Cry9C corn will be any more allergenic than standard unmodified corn.
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4. Safety Evaluation Philosophy for Humans - a Holistic Overview
In this assessment Aventis has taken a very broad, yet detailed, approach to support its
conclusion of the safety of Cry9C corn. Aventis has therefore consulted (1) a wide
variety of national, international and supranational regulatory guidelines for the safety
evaluation of genetically enhanced foods, food ingredients and plant pesticides, (2)
biotechnology guidelines for the development of novel therapeutics, (3) nutritional
publications, (4) papers on food processing and protein modification (5) papers on novel
food testing and (6) multiple papers on or related to the allergenicity of foods.
This information has been underpinned with toxicological principles for hazard
identification with a particular emphasis on the assessment of any potential for
allergenicity. To this end Aventis has not only consulted publications in this arena but
also many of the key workers and academics in the field of allergology. Aventis has
sponsored experimental research to investigate the feasibility of developing models for
the detection or prediction of food allergy and participated in International Trade
Association meetings and European and American projects to debate and progress the
science in this dynamic and complex area.
A weight of evidence approach in conjunction with a decision tree approach espoused in
working groups sponsored several years ago by OECD (OECD (1995)) and WHO (WHO
(1995)) is currently the agreed method of assessment for novel proteins in the absence of
harmonized guidelines. Further efforts have been co-ordinated by the Allergy and
Immunology Institute of the International Life Sciences Institute (ILSI) and the
International Food Biotechnology Council (Metcalfe et al, 1996).
However, it is now generally recognized that the science has moved on and the early
decision trees, which have to date served us well, now need to be revisited.
By following an overarching approach to safety at one level and focussing down on
hazard identification through specific studies at the other, Aventis concludes that, when
exposure estimates are factored in, there exists a very high quality approach and
19
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data set for reassuring the EPA concerning the safety (risk assessment) of Cry9C for
humans.
For brevity and clarity, much of the detail and scientific background relating to the
following argumentation is referred to in an earlier expert report (Cockburn, 1998), and
will therefore not be repeated here.
This document will focus on the assessment of any potential "risk that may exist from
exposure to this protein based upon two of its biochemical characteristics (stability to
heat and gastric digestion)" (EPA OPP Biopesticides Cry9C Peer Review -
www.epa.gov/biopesticides/cry9c/cry9c-peer_review.htm).
4.1. General toxicological considerations
A case by case approach must be adopted when assessing the safety, specifically food
safety of genetically enhanced crops. Such an approach is developed to confirm whether
or not a new food (or food component) is substantially equivalent to an existing food (or
food component), in which case it can be treated in the same manner with respect to
safety.
To this end a holistic approach has been devised and utilized by Aventis. Figure 1 below:
Figure 1
lf^j|^¥§°Spr'sc!s?1 HSi^K^^i&tl
Recipient Gene Construct Protein Structure
History of use Insert Process Mode of action
Toxins/allergens Source of genes Specificity
Toxicity/lrritancy
Bioavailability
Allergenicity
I,G|tfe©rop - ili]<3 Event?
Wholesomeness
Nutritional Equivalence
Toxins/allergens
Stability
TMDI
20
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For the Cry9C protein and the genetically enhamnced crop, a testing strategy involving
both in vitro and in vivo studies has been developed. This program of work has
investigated such key areas as toxicity, irritancy, allergenicity, bioavailability,
nutritional/anutrient impact, wholesomeness, mode of action, target species specificity,
genetic stability/gene transfer and human experience. By gathering data from these areas
and factoring it with critical exposure information, such as consumption patterns and
existing knowledge of human tolerance to corn, a very low hazard and hence risk has
been established (McFarlane, 1998).
Cry9C corn may therefore be considered to be substantially equivalent to the existing
unmodified corn and is considered safe lexicologically for human food use.
4.2. Assessment of potential for allergenicitv
Background Orientation
Food allergy is a very important issue. It is thought to affect 1-2% of the adult population
and perhaps 4-5% of infants and children.
Prevalence is very dependent upon genetic pre-disposition, age, geographical location,
eating as well as social habits. The increasing availability of potent allergenic foods,
such as exotic fruits, for example kiwi and mangoes, has brought an inward migration of
food allergies, whereas the familiar cow's milk allergy seems to be decreasing from its
peak at the beginning of the 20th century, possibly in conjunction with the renewed
popularity of breast feeding. Similarly the trend to modern-day food processing has
spread potent allergens from peanut and soybean into a vastly increased variety of food
products.
Food allergies tend to reduce in infants and children as they grow up, but may
spontaneously appear and disappear in adulthood, (Bruijnzeel-Koomen et al (1995) and
Sampson, 1996). There are currently no validated models (in silico, in vitro or in vivo)
for the accurate prediction of whether a given protein possesses the necessary
characteristics, many of which are still under debate, to elicit clinical signs of food
21
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allergenicity in man. Moreover, the immunogenicity of a food protein may well not
correlate with its allergenicity, which requires specific IgE antibody reaction. The
situation is further complicated as a food protein causing profound allergic symptoms in
one person is often without any perceptible symptomatic effect in another, for example,
peanut allergy.
The reason that most people do not suffer with food allergies, albeit exposed to broadly
similar patterns of food proteins, is that oral immunological tolerance
(hyporesponsiveness) is believed to be developed to the vast majority of proteins during
our lifelong exposure to common daily diets. In operational terms, food induced
allergenic diseases represent a breakdown or failure of tolerance either during induction
or during maintenance (Strobel, 1997). This may occur as a consequence of biparental
genetic predisposition to atopy, or for example, inter-current inflammatory reactions in
the gut interfering with normal antigen processing.
Food allergy (hypersensitivity) may thus be considered to be an abnormal reaction of the
immune system to food proteins or glycoproteins. While literally any food has the
potential to cause an allergic reaction under the right conditions, such effects are more
prevalent with proteins from certain sources of food, the most common being the so-
called "Big Eight" namely fish, shellfish, milk, eggs, legumes (peanut and soy), tree nuts,
wheat and fruits (e.g. banana and kiwi) (James and Burks, 1995), these accounting for
90% of the reactions to foods. The "Second Eight" includes sesame seeds, sunflower
seeds, cottonseeds, poppy seeds, molluscs, beans (other than green beans), peas and
lentils. Overall, some 170 foods have now been implicated. The resulting human
response is mediated by the antibody IgE and can lead to minor symptoms of discomfort
through a spectrum of untoward effects or even, very rarely, anaphylaxis. The process
leading to symptoms of food allergenicity is described in more detail in Appendix 1.
The challenge to develop a robust and reliable model, in vitro, in vivo or in silico, for the
prediction of human food allergy is enormous recognizing the multifactorial components
already alluded to. Attempts have so far failed or have been only partially successful.
Ultimately, it is the interaction between genotype, phenotype, dietary content and
consumption level that will predispose to a food allergy in a sensitive individual.
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With regard to the Cry9C protein and the assessment of allergenicity potential,
comparisons have been made with a number of key characteristics of known allergens,
which have been probed and assessed. Without some qualifications there are a number of
flaws when this approach is used in isolation and these will be discussed. Having looked
at the overall weight of evidence, particular attention will then be paid to the two
characteristics that are of concern to EPA, heat and digestive stability.
Domains considered for evaluating allergenicity potential of Cry9C
4.2.1 The Host Crop - Zeamays
There are very few, if any, scientifically rigorous published reports of confirmed human
allergenicity to corn. Compared to other cereals, corn lacks a reputation for food
allergenicity and indeed toxicity.
4.2.2 The Gene Source of cry9C - Bacillus thurigiensis subsp. tolworthi
The cry9C gene was isolated from Bacillus thuringiensis subspecies tolworthi. Bacillus
thuringiensis cultures have been used on a wide scale for over 30 years for insect control.
Spraying has been highly efficacious and numerous reports have been published
recording the extensive safety-in-use of the products. Indeed, EPA has stated that, "there
is no evidence of any substantial human or environmental safety concerns related to Bt
sprayables" (US EPA, 1988). Thus while not explicit, it may safely be assumed from
their duration and extent of use that Bt proteins are not known to come from an allergenic
source. This implication is confirmed by EPA, who have stated that "Since the
introduction of microbial formulations containing Cry proteins in 1961, no reports of
allergy have occurred" (Astwood et al, 1977; US EPA, 1995).
23
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4.2.3 The Gene Product - The Cry9C Protein
. 1 Stability to digestion
Results are drawn from several experiments
In the original study plant derived (corn) and bacterial produced (E. coli and Bacillus
thuringiensis supspecies tolwothii) Cry9C were tested for in vitro digestion in simulated
gastric fluid (SGF) conditions for different time periods (up to a maximum of 4 hours)
(Perferoen, 1997). Digestion was scored by Western Blot Analysis. No degradation of
the protein was observed irrespective of the source.
In a subsequent study Cry9C protein, purified either from E. coli or Bt tolworthi, was
studied under SGF or simulated intestinal fluid (SIF) for periods up to 2 hours (Noteborn,
1998). Digestion was scored by scanning densitometry of silver-stained SDS-PAGE gels
and by Western Blot analysis. The author concluded that "The recombinant Lys mutant
Cry9C protein (source not quoted) hardly degraded in SGF " as determined by
scanning densitometer. This was confirmed by Western Blot analysis. Examination of
the data reveals that "hardly degraded" accounted for 12 - 25% digestion of the Cry9C
from time points 2 minutes to 120 minutes. It is also worth noting that a relatively
digestible protein (CrylAbS) was still 21% native (undigested) protein at 30 minutes
(compared with 78% Cry9C), and that all 4 reference proteins used (CrylAbS, CrylllB,
NPTII and PAT) were still intact at the first sampling time (designated 0 minutes). A
further 5 minutes in SFG were required to result in 100% digestibility of CrylllB, NPTII
and PAT).
Further studies using a Bt tolworthi source of Cry9C (Appendix 2) have shown that a
simulated intestinal medium of either trypsin, subtilisin or pancreatin readily digest the
Cry9C protein to a 55 kDa product. The above findings are consistent with the partial
digestion of the 68 kDa protein to a 55k Da protein in the rat bioavailability study,
(Noteborn et al, 1998). Perhaps most interestingly the use of proteinase K, papain and
bromolain each individually led to the complete digestion of the Cry9C protein.
Most recently further investigations using Cry9C from E. coli showed complete digestion
of the protein in pepsin (SGF) and digestion to a 20 kDa fragment in the pepsin buffer
24
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alone (acid buffer). This was assayed on SDS-PAGE gels and confirmed by Western Blot
analysis. Again trypsin digestion resulted in a 55 kDa product (as before in this lab) as
did pancreatin. This work continues in order to confirm this apparent digestion
It has been stated that the ability of food allergens to reach and to cross the mucosal
membrane of the intestine are prerequisites to allergenicity (Fuchs and Astwood, 1996).
The macromolecular exclusion by the epithelial barrier is now a rather ancient concept
(Seifert et al, 1974, 1997; Gardner, 1988; Teichberg, 1988). Moreover it has been stated
that common food allergens are stable in simulated gastrointestinal fluid for 60 minutes
or more, whereas food proteins considered not to possess allergenic potential are digested
very rapidly (within 15 seconds) (Astwood, 1996). Regrettably the situation is nowhere
near as straightforward as these references imply and the reader is reminded that the
situation may well be "grey" and not "black and white". Protein digestibility clearly is a
continuum not a bimodal distribution as will now be discussed
Nature seldom builds absolute compartments, and whereas much time and effort has been
spent characterizing the physicochemical attributes of known major allergens, very little
time, has been spent looking at the characteristics of the vastly greater number of food
proteins known not to be allergenic. When this is done the simplistic "straight-jacket"
that stability alone is a prognostic indicator of potential allergenicity is overturned. Table
1 shows from the literature that food allergens can be either more stable or less stable (or
anywhere along this line of digestibility) and that vice versa food non-allergens such as
plant and animal structural proteins may be stable (or digestible) too. It is therefore
important to look at the negative correlations as well as the positive ones. As can be
seen, a stable protein is by no means an automatic high risk as an allergen. As Houben et
al (1997) have stated:
"Unfortunately insufficient information is available on possible
differences in susceptibility to acid-denaturation and gastrointestinal
digestion between strongly allergenic food proteins and proteins that
possess weak or virtually no allergenic potential. Therefore evaluation
of acid-stability and digestibility of food proteins will, in most cases, not
yet provide sufficient information regarding their allergenic potential
upon ingestion, and additional research is need in this respect".
25
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TABLE 1
Table to demonstrate lack of de facto relationship between stability and allergenicity
ALLERGENIC
LESS STABLE PROTEIN
Apple (Maldl)u"zr
Celery (Bet v 1 like)(I)
Castor Bean(3)
Ragweed(4)
Fish(5)
MORE STABLE PROTEIN
Peanut (Ara hi) UJ
Soybean(6)
Hazelnut(6)
Castor Bean(3)
Tomato (7)
Fish(egGadCl)(5)
NON-ALLERGENIC
Apple'1 &2)
Rubisco(12
Lipoxygenase'13)
Horse Radish Peroxidase (HRP)
Mothers Colostrum
Potato (9)
Rice (9)*
Glutamate decarboxylase(10& '^
Maize (P-l 00)(11)
Processed food proteins (beef,
poultry, pork)
* Separate from the known allergenic protein
(Protein Allergen in brackets)
Table References
1. ViethsSetal(1995)
2. Drebors S & Foucard T (1983)
3. LehrerSBetal(1981)
4. Marsh DG et al (1981)
5. Bernhisel-Broadment J et al (1992)
6. Astwood JDetal(1997)
7. DircksLKetal(1996)
8. McLean E& Ash R (1996)
9. Astwood JDetal( 1997)
10. Strobel S (1997)
11. Vantardetal(1994)
12. Ellis RJ (1979)
13. Stedow,NJ(1991)
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.2 Bioavailability
The bioavailability of the Cry9C protein was examined in a single dose gavage study in
the rat where blood samples were removed over an 8-hour period via a cannula inserted
into the hepatic portal vein. Both ELISA detection (antibody system) and Western Blots
(also antibody system) assayed for the potential presence of Cry9C (Noteborn et al,
1998).
The authors concluded that very small traces of Cry9C-like material were detected in the
blood at the top dose. They reported that of the orally administered 298 mg/kg body
weight dose (i.e. top dose), between 0.0002-0.0006% was absorbed. (It should be noted
that values obtained by this method were either on or below the reported limit of
detection (LOD) of assay). The identity of this Cry9C-like material could not be
confirmed by Western Blot.
This result indicates a lack of any significant uptake of immunoreactive species of
Cry9C. The small level at the LOD is in line with that typically seen for proteins (Strobel
and Mowat, 1998),
.3 Stability to processing (heat)
Some food allergens have been shown to be resistant to degradation by high temperatures
(Astwood et al, 1997) but the specific parameters, how hot for how long, have not been
delineated.
The Cry9C protein was shown to be stable to temperatures up to 90°C for 10 minutes
without altering the toxicity to the target insect (Peferoen, 1997). Conflicting results
have been obtained with regard to stability to processing. In one study it was shown that
the Cry9C protein was detectable in processed grain (Shillito, 1998) whilst in a second
study no Cry9C protein was detectable by protein-specific ELISA analysis of catfish
pellets processed from corn kernels containing the Cry9C protein (Macintosh, 1997).
Stability to heat is not considered to be of any greater importance than any of the other
characteristics already listed and should not be considered in isolation.
27
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It is worth noting that there are numerous proteins in food plants that are both heat stable
and resistant to gastric digestion, very few of which are food allergens. In fact most
known food allergens (there being some 170) have not been tested against these
parameters (Fu and Abbot, 1998). For this reason there is not a clear-cut correlation at
this stage and these parameters should therefore not be overweighted.
Evaluation of Homology to Known Protein Allergens and Toxins (Epitope Searching)
The central issue is one of defining and characterizing protein allergenicity. While many
food allergens are proteins, not all proteins are allergens. Thus, very few proteins display
food allergenic potential despite the potential of being immunogenic. This is because
while all proteins have the potential to be recognized as foreign, they differ very
markedly in their ability to induce IgE-mediated allergenic sensitization.
One practical way to gain reassurance is to compare the amino acid sequence of the novel
protein with sequence data for major food allergens which have been published in public
domain genetic databases (King et al, 1994). Although the distinction between allergenic
and non-allergenic T-cell epitopes remains unclear, the optimal number of amino acids
needed to elicit an immunological response appears to be between 8 and 12 amino acids
(Rothbard and Gefler, 1991). No matches were found when a sequential series of 8
amino acids of the Cry9C protein were compared with the Swiss Prot, PIR, HIVAA,
Genbank, EMBL, PRF, DDBI & PDB database of know allergens (Perferoen, 1997;
Macintosh, 1997a). From this, the important conclusions may be drawn that the cry9C
gene, (1) does not encode any known allergen and (2) does not share immunologically or
lexicologically significant sequences with known allergens or toxins (US EPA, 1988;
Astwood etal, 1997; US EPA, 1995).
It is also of importance to note that Cry9C shares some 50% amino acid homology with
CrylAb, a widely used Cry protein with no known/reported allergenic history.
Additionally, like Cry9C, CrylAb does not show any homology, even at the 8 amino acid
level, with any known allergens or toxins.
28
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.5 Molecular Weight
According to the literature, molecular weight (MW) may be a possible characteristic
for weight of evidence consideration. The Cry9C protein has a MW of 68.7 kDa.
Other Cry proteins expressed in plants, CrylA and Cry3A, are similar in molecular
size (about 70 kDa) as the Cry9C protein. Reference to the literature for the major
food allergens has shown that almost all fall within the range of 10 - 40 kDa
(Metcalfe D, 1985, 1997).
The upper limits on the MW of a food allergen may be dictated by the constraints of
intestinal Peyer's patch antigen sampling M cell permeability to macromolecules. It
has been proposed that proteins about 70 kDa and larger are less likely to be
efficiently absorbed by the Gastro-intestinal Lymphoid Tissue (GALT) (Taylor S,
1997). Cry9C is at 68.7 kDa, the very top of the range, a further factor indicating the
unlikelihood of the Cry9C protein being a potential food allergen.
.6 Post-translational Modification : Glycosylation
Protein glycosylation is widely found with known protein allergens (Metcalfe D D,
1997). Cry9C protein extracted from plants has not been found in glycosylated form
(Lambert B et al, 1996).
4.2.4 The Genetically Modified Crop - Event CBH-351, StarLink™ Com
. 1 Prevalence
Most major food allergens are present as a relatively high percentage of the total
protein in the plant or foodstuff, e.g. soyabean P conglycinin 18.5%, glycinin 51%,
ovalbumin (egg) 54% or casern (milk) 80% (Yunginger J W, 1990).
In contrast the Cry9C protein is typically found at c. 0.17% or less total protein in the
com kernals, and this coupled with some evidence for heat lability and in vitro and in
vivo digestibility (Macintosh SC, 1997, Noteborn H P J M, 1998 and Aventis
unpublished data, 2000), indicates that some reduction in Cry9C protein content is
likely to occur before it reaches the GALT. However, as mentioned previously, this is
not considered to be a critical factor, as in the absence of any observed epitopic
sequences from homology
29
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searching, Cry9C should not be considered in any different light to other stable plant or
indeed animal proteins, which also lack any known history of allergenicity.
.2 Could insertion of the transgene have modified the allergenic status of corn?
To provide practical scientific evidence that the insertion process of the new genes does
not alter the intrinsic allergenic status of standard, unmodified corn, a panel of sera from
suspected corn-reactive subjects was screened with corn seed extracts from Cry9C corn.
Twenty-one sera samples were assayed for specific IgE antibody to aqueous standard,
unmodified or genetically enhanced corn extracts by radio-allergo-sorbent test (RAST).
Comparison of the results were remarkably similar confirming that the genetic
engineering process per se caused no apparent difference in the allergenic status ofCry9C
corn compared to standard unmodified corn (Lehrer, 1997).
.3 Supportive evidence for a lack of allergenic potential for Cry9C corn.
Repeated exposure is normally necessary in the context of food allergy, for the
occurrence first of immunization followed subsequently by an allergic reaction if
sensitization has taken place. It, therefore, is helpful to evaluate all cases of repeated
exposure to determine if any possible evidence.of hypersensitivity to Cry9C protein or
Cry9C corn has been seen. This information would be classed as supportive rather than
prima facia. Nevertheless, it is an important factor in an overall safety assessment,
especially when extensive human exposure has already occurred over the last several
years. The following is a list of work to support the lack of allergenic potential of Cry9C
corn.
A. Mouse 30-day repeat dose study of Cry9C in drinking water (Noteborn , 1998)
No evidence of any untoward signs were seen in clinical conditions, haematological or
blood biochemical analysis (including total protein and albumen levels) or
histopathological (including the reticuloendothelial (RE) elements of bone marrow,
jejunum with Peyer's Patches, mesenteric lymph nodes, spleen and thymus) parameters.
30
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B. Poultry wholesomeness feeding study of Cry9C corn (Leeson, 1998).
Cry9C corn versus standard, unmodified conr did not show any adverse effects on
feeding, growth or clinical condition when fed to groups of male broiler chickens for
up to 42 days. The test diet was deemed as wholesome as the unmodified diet.
C. Approved animal feed use of Cry9C corn (US EPA, 1998)
No untoward findings have been reported in almost 2 years of the use of this feedstuff
in North America.
D. Human experience with workers for Garst Seeds, exposed to Cry9C corn (Macintosh,
1998).
Garst has been actively working with Cry9C corn since 1996 and no unusual
reactions have been described in normal or pre-existing atopic personnel involved in
the field, harvesting or processing of Cry9C corn. In consequence, there is no
evidence that respiratory exposure (generally recognized to be a more sensitive route
for hypersensitization) has led to any unusual reactions (including allergic response or
cross-reactivity) amongst 1990 respondents canvassed by Dr. Alan Hawkins,
Research Director of Garst.
Results Not Taken Into Account Due To Experimental Invalidity
The Brown Norway Rat: Research into New Methods for Allergenicity Assessment
There were a number of experimental, methodological and control shortcomings that render the
data uninterpretable, particularly as a consequence of an apparent response from the corn portion
of the extracts themselves as well as contamination of the controls. The data has been peer
reviewed by external sources who derived the same conclusion that the study is flawed,
Cockburn (1998) and Section 3 above, Question No. 1.
31
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5.
Overall Weight of Evidence Assessment.
Data Interpretation
5.1 General Toxicology
From a broad range of studies, well beyond those mandated, it may be concluded that
Cry9C corn and the Cry9C protein are fully wholesome and non-toxic to laboratory
animals and human food producing animals. Because of the insect specific binding site
these results may be safely extrapolated to man.
5.2 Allergenic Potential - Evaluation and Discussion
The following conclusions may be distilled from Section 3 above.
Figure 2
Summary of Weight of Evidence Evaluation for Allergenicity
P
Event*
No history of
allergenicity
No history of
allergenicity
No epitope
homology
Partial digestibility/
Full digestibility
Partial heat stability
MW at upper limit
for allergenicity
Negligible
systemic bioavaibility
Low prevalence <.5% TP
not glycoslylated
No increase in
inherent allergenicity
of corn
No worker adverse
incidents
Safe animal feed use
for nearly 2 years
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As demonstrated from the results summarized here, there is a strong overall weight of
evidence from the results summarized above, that:
(i) Cry9C corn containing the novel insecticidal protein Cry9C, does not pose an
allergenic risk to man; and
(ii) The Cry9C protein per se is not an allergen.
The key issue relates to what "Weighting" should be given to the finding of partial
digestibility and heat stability for Cry9C? These are the principal findings of concern to
EPA. As might be expected, they have been considered in depth by Aventis and
regulatory authorities have been consulted in line with the published WHO and OECD
decision trees. The case is presented herein.
5.2.1 Protein digestibility
It is first necessary to consider the whole process of gastrointestinal digestion.
While protein digestion is initiated in the stomach, most digestion and absorption occurs
in the small intestine. Numerous pancreatic and intestinal enzymes split proteins into
peptones, polypeptides, and finally their constituent amino acids, which are subsequently
absorbed. In humans, it has been estimated that 50% of the digestive protein comes from
the diet, 25% from the proteins in the digestive fluids, and the .remaining 25% from
sloughed cells of the gastrointestinal tract. The rate of turnover of mucosal intestinal
cells is extremely rapid, 1-3 days, thereby giving an excellent source of recyclable
protein. Overall about 92% of the dietary protein is digested. The digestibility of
vegetable protein is 80-85%, while that of animal protein is about 97%. (Ensminger et al,
1994).
It is commonly assumed either (a) that dietary proteins are digested completely to free
amino acids within the lumen of the gastrointestinal tract before absorption occurs, or (b)
that only trace amounts of macromolecular fragments enter the circulation and that these
are of absolutely no nutritional, physiological or clinical relevance. The first of these
assumptions is blatantly untrue. It is now known that intestinal peptide transport is a
33
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major process, with the terminal stages of protein digestion occurring intracellularly after
transport of peptides into the mucosal absorptive cells. Also, there now is irrefutable
evidence that small amounts of intact peptides and proteins do enter the circulation under
normal circumstances (Gardner, 1988 & Strobel, 1997).
The presentation of intact protein and peptides to the GALT for absorption is critical to
the essential development of oral tolerance to foods and food proteins via the presence of
immunologically relevant proteins in the circulation. Simplistically therefore, complete
hydrolysis of an allergenic protein to its constituent amino acids would be predicted to
destroy the IgE binding capability.
Recent work by Fu and Abbot (1998) in this area attempts to understand the relationship
between protein functionality, stability to digestion and acid hydrolysis. In particular this
work concentrates on how the relative stability of known food allergens compares with
functionally similar non-allergenic proteins. According to Fu and Abbot (1998), "it is not
clear, for example, if a food allergen which acts as a storage protein is more stable than a
non-allergenic storage protein".
The results of Fu and Abbot (1998) have shown that "most of the storage proteins, plant
lectins, and contractile proteins tested, irrespective of their allergenicity, were very stable
in the acidic salt solution (more than 120 minutes). The stability of the allergens to
digestion in the simulated gastric fluid varied greatly (ranging from 30 seconds to 60
minutes) and there was not a clear relationship between protein function and digestion
stability".
This work further casts real doubt on the criterion of stability per se as a reliable indicator
for potential allergenicity. It seems very probable that the characteristics of known
protein food allergens were noted for the purpose of characterization, while the converse,
to define the characteristics of non-allergens in foods has not had the same impetus.
In conclusion, the ability of intact protein (and indeed peptides), which may contain
epitopic sequences, to reach the GALT is not only scientifically established but essential
for the development of oral tolerance.
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5.2.2 Heat stability
Heat stability has been added as an alert for precautionary reasons as in the case of
digestibility - see above. However, it is again not a definitive characteristic of
allergenicity as there are well known thermolabile as well as thermostable allergens, for
example, in celery roots (Wuthrich et al, 1990).
Argumentation Against the Overweighting of Digestibility and Heat Lability in Isolation in
the Assessment of Allergenicitv
• There are no direct methods to assess novel proteins for potential allergenicity and no single
factor described in a weight of evidence approach is predictive. This underlines the value of
a weight of evidence approach.
• Logically and in the absence of any published work to the contrary, each weight of evidence
factor should be equally weighted. (Anecdotally there is some evidence that the allergenic
history/heritage of the host crop and/or the gene source are the 2 main determinants for
assessing a transgenic crop's potential for allergenicity).
• It is implied that in vitro digestibility studies predict the fate of proteins in the human
digestive system. The gut is more sophisticated than the US Pharmacopeal Model for
Digestibility (Board of Trustees, 1995). Many elements influence the ultimate
immunogenicity or allergenicity of food proteins. These include peristalsis, churning,
linearity of the digestive system with changing pH conditions, a range of digestive enzymes,
bile acids, transit time, the flora with additional metabolic and digestive capability, as well as
the intestinal barrier function, permeability and absorption (Boisen and Eggum, 1991).
Existing data with Cry9C from the rat bioavailability study show partial digestion from the
68 kDa to 55 kDa protein c.f. an apparent lack of in vitro digestibility. This is highly
relevant because Rich et al (1990) have shown excellent correlation between rat and man in
their ability to digest protein in vivo.
• It has been stated by some that stable proteins have an increased chance of reaching the
intestine, where many food allergens elicit their response. There are several issues here:
35
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This finding is not unique to stable proteins. Strobel and Mowat (1998) have
shown that a relatively fixed amount of all proteins reach the intestine for antigen
sampling.
- The normal healthy baseline response for animals and man exposed to protein
antigens in the diet is the induction of immune tolerance.
- Proteins do not fall into two neat compartments, stable and unstable. Stability
depends on protein folding, bonding, conformational characteristics, enzyme
cleavage sites, disulphide bonding, and amino acid content. There are relatively
more stable or less stable proteins!
- Not all stable proteins are allergens.
- Many unstable proteins are allergens due to the "unmasking" of epitopic sites
during the process of digestion.
- Because of lack of scientific interest, little work has been performed on relatively
stable proteins with no history of allergenicity (e.g. zein, actin and myosin) from
beef and chicken.
Proteins which are unstable before food processing often become more stable afterwards.
For example Opstvedt et al (1984) found a linear decrease in the content of-SH
(sulfhydryl) groups and a concomitant increase in the content of S-S bonds when rainbow
trout was heated at increasing temperatures from 50 to 115°C. The impact of disulphide
bond formation on protein utilization is not fully known, but some experimental data
indicate that it may reduce protein digestibility. Mauron (1984) reported that protein
digestibility was reduced as a result of complex chemical (crosslinking) reactions such as
protein interactions or protein-fat interactions when food was broiled at high
temperatures. Also, Opstvedt (1988) reported that smoking conditions (time,
temperature, compounds of wood smoke) reduced protein digestibility.
Additionally, during processing of foods, protein sources are treated with heat, oxidizing
agents (such as hydrogen peroxide), organic solvents, alkalis, and acids for a variety of
36
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reasons such as to sterilize/pasteurize, to improve flavor, texture, and other functional
properties, to deactivate antinutritional factors and to prepare concentrated protein
products (Cheftel, 1979; Friedman et al, 1984; Schwass and Finley 1984). These
processing treatments may cause the formation of Maillard compounds, oxidized forms
of sulfur amino acids, D-amino acids, and cross-linked peptide chains (such as
lysinoalanine and lanthionine), resulting in lower amino acid bioavailability and protein
quality.
In consequence, protein stability/digestibility in the raw agricultural commodity may well
be irrelevant to the typical consumer, because of changes during processing or cooking.
In conclusion, the overall results lead only to the conclusion of possibly limited
digestibility and thermolability for Cry9C not indigestibility or heat stability.
Irregardless, these two characteristics are not predictive of a food allergen.
5.3 Exposure for Consumers
There are no known reports of adverse incidents to man exposed to the Cry9C protein in
seed, plant or bacterial from occupational exposure (Macintosh 1998).
Since the protein is not commercially available in the diet there is no data to examine.
However, a calculation of the Theoretical Maximum Daily Intake (TMDI) for the Cry9C
protein has been determined using various worst case scenarios, since at this stage in
development, it is not finally decided which crops/commodities will have the Cry9C
protein inserted into them. This calculation serves to give an idea of the possible
exposure/consumption of the protein in the normal European and American diets. This
takes note of the very low prevalence of Cry9C in corn.
37
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Commodity
Maize flour *
Corn flour*
Maize/Corn oil
Sweetcorn
Bran
Popcorn
Total TMDI
(mg/person x day
Total TMDI
(mg/kg x day)
Cry9C
Protein
mg/kg
8.4
10.8
<0.0005**
12.3
32.6
12.3
European
daily
consumption
(g/person x
day)
8.8
N/A
1.3
8.3
N/A
N/A
USA daily
consumption
(mean) (g/person
x day)
N/A
14.5
2.0
9.6
0.07
1.5
European
TMDI
(mg/person
x day)
0.074
N/A
N/A
0.102
N/A
N/A
0.176
0.0029
USA TMDI
(mg/person
x day)
N/A
0.155
N/A
0.118
0.002
0.018
0.293
0.0049
* Definition of flour between USA and Europe varies. Includes cornmeal in the USA.
** Limit of quantitation in oil.
The calculation was based on the possible content of the Cry9C protein in various
commodities and on the maximum measured levels of Cry9C in processed grain (Shillito,
1998). USDA CSFII mean consumption data (USDA, 1994-1996) was used to calculate
the American TMDI. As there is no protein in corn syrup (USDA, 1999) no value is
included for this matrix. Further details on the European TMDI is available (Zapf, 1998).
An acceptable daily intake (ADI) or reference dose (RfD) figure of 0.3 mg/kg/day was
derived from the No-observed-adverse-effect-level (NOAEL) of 33.3 mg/kg/day in the
mouse 30-day study by the addition of an uncertainty factor (UF) of 100 (10 for inter-
species differences x 10 for inter-animal variation). It is clear that in addition to this 100-
fold UF, consumption by Europeans is a further 100-fold lower than this ADI figure and
for Americans is 60-fold lower. Thus, huge margins (up to x 10,000 (Europe) and x
6,000 (USA)) of safety (or MOE) exist in relation to the NOAEL of 33.3 mg/kg/day in
the mouse 30-day study even when using worst case assumptions of total crop treated.
5.4 Risk Assessment Summary
There are no untoward nutritional, toxicological or wholesomeness findings.
There is no evidence from human worker exposure for up to 5 years of any adverse
response or increase in atopy.
38
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There is no evidence from the animal feed use of Cry9C corn of adverse reactions in
cattle and poultry or the humans consuming the resultant milk, meat and eggs.
There are no indications that Cry9C protein, which shares significant homology with
other Bt proteins, is an allergen.
There is no evidence from human sera studies that Cry9C corn is inherently more
allergenic than standard unmodified corn.
The theoretical maximum daily intake (TMDI) of the Cry9C protein is c.0.003 mg/kg/day
for a European and c. 0.005 mg/kg/day for an American. The RfD is based on the mouse
30-day no-adverse-effect-level of 33.3 mg/kg/day with an uncertainty factor of xlOO, i.e.
0.3 mg/kg/day. The human dietary exposure is therefore 60 - 100-fold lower and utilises
less than 2% of the ADI.
In the absence of any evidence that the Cry9C protein is an allergen, consumption of
Cry9C corn containing very low levels of the novel protein is considered not to pose an
allergenic risk to humans.
6. Substantial equivalence of Cry9C corn to Standard Unmodified Corn
The overall weight of evidence taken in conjunction with the lack of any adverse findings
from extensive animal feed use over the last 2 years indicates that there is a reasonable
certainty that the calculated very low direct dietary exposure to Cry9C corn will not pose
an allergenic risk to humans.
Cry9C corn is therefore considered to be substantially equivalent to standard unmodified
corn.
39
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8. Appendices
Appendix 1
The Process Leading to a Food Allergenic Response
The IgE antibody is produced by white cells known as Beta (P) lymphocytes or p cells.
Another type of lymphocyte, the "helper" T cell, is also involved in the process. It is a
secretion of this cell — interleukin 4 (IL-4) — which influences P cells to produce IgE rather
than another class of antibody.
The IgE antibody secreted by p cells is specific for a given antigen and circulates throughout
the body. On part of IgE has a particular affinity for receptors on the surface of mast cells
found in body tissues. Mast cells bind the tail of IgE antibodies, which thereby sensitise
those cells to specific antigens. If, in a subsequent encounter with the sensitised mast cell,
the antigen forms a bridge between two adjacent IgE antibodies, this acts as the signal for the
release of a variety of substances which either have been stored in granules in the mast cell
(for example, histamine) or are newly synthesised at the cell surface (such as prostaglandin
DZ and leukotriene C
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small areas of lymphocyte-rich tissue, and many individual lymphocytes are also scattered
along the lining layers of the intestine where they can migrate to areas of inflammation.
Scattered over the surface of Peyer's patches are specialised cells (M cells) through which
samples of the antigens present in the intestinal lumen penetrate into the lymphoid tissues of
Peyer's patches. Lymphocytes primed to produce antibodies, by contact with an antigen
through the M cells of the Peyer's patches, eventually reach the local lymph nodes and the
circulating blood. From there they seed themselves back to the wall of the intestine in many
more places, and are also spread to other mucosal sites, notably in the lung airways. In this
way, the local effects of an immune response in the intestine can be spread to all the mucosal
surfaces of the body means of re-circulation and distant seeding of T and P lymphocytes. It is
in part the variability of this mechanism, from individual to individual and from one
challenge (exposure) to the next, that results in the differing presentation of food allergies.
48
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Appendix 2
Further Investigation of the Digestibility of the Cry9C Protein by a Selection of Protolytic
Enzymes
This further investigation was carried out to examine the stability of Cry9C (a 68.7kDa protein
from Bacillus thuringiensis subsp. tolworthi (Bt)) to digestion using a variety of proteolytic
enzymes. The enzymes used were obtained from both mammalian and non-mammalian sources,
chosen to reflect the wide variety of enzymes that Cry9C could potentially be exposed to. This
study was designed to highlight potential modes of digestion of Cry9C for future, more detailed,
examinations.
Each enzyme was individually incubated with Bt Cry9C for 3hours at the optimal condition for
each protease based on the US Pharmacopeal Model (Board of Trustees, 1995). Negative
controls of the Cry9C protein and the enzymes were also incubated and digestion of bovine
serum albumin (BSA) used as a positive control. Analysis of the incubated samples was
performed by sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE) with
either silver or Coomassie blue staining. The following results (Table 1) were obtained for
individual incubations.
49
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Table 1
Trypsin
Subtilisin
Pancreatin
Proteinase K
Papain
Bromelain
Elastase
Chymotrypsin
Peptidase
Pepsin
||Myes jQ5fi|iffYr >/ ^?U
Bovine Pancreas
Subtilisin Carlsberg
Porcine Mucosa
Tritirachium Album
Papaya
Pineapple stem
Porcine pancreas
Bovine Pancreas
Porcine Intestinal
Mucosa
Porcine stomach
mucosa
mOTts/C6r||nenfst^ ^r., -^T^
s/sT ;:fc ^. j; ,i
The Cry9C 69 kDa protein
appeared to be partially digested to
a 55k Da protein. In pancreatin the
digest product was most probably
due to the presence of trypsin.
Cry9C appeared to be extensively
digested.
No noticeable digestion was
observed.
Conclusions
1. Cry9C appears to be readily digested to a 55 kDa protein when incubated with trypsin,
Subtilisin and pancreatin.
2. Cry9C appears to be extensively (in most cases completely) digested by papain, bromelain
and proteinase K.
3. The purity and age of the batch of the Cry9C used in these digestion studies was called into
question and as a result of this another study was done with a new and purer batch of protein
from E. coli.
50
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In a second investigative study, Cry9C from E. coll showed complete digestion of the protein in
pepsin (SGF) and partial digestion to a 20 kDa fragment in pepsin buffer (acid buffer) only.
These findings were confirmed both by SDS-PAGE gel electrophoresis following Commassie
Blue staining and by Western Blot analysis using antibodies raised to the Cry9C protein.
Additionally, trypsin and pancreatin digested the 68.7 kDa protein to 55 kDa and papain
produced complete digestion (as seen in previous experiment).
This work indicates that the Cry9C protein (from either bacterial source) can be digested in
several conditions. Repeat experiments using both the new and old batches of the Cry9C used in
these experiments are planned to confirm the results and the reproducibility of the study design
(under full GLP conditions).
51
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Appendix 3
The Agronomic Benefits of Cry9C Corn Hybrids in the US
Bt Cry9C corn hybrids benefit US corn growers by providing sustainable farmer
profitability in a product that is safe to people, safe to non-target plants and animals, and
safe to the environment.
Hybrid corn plants expressing the Cry9C protein have been sold commercially in the United
States since their approval for sale and use in May of 1998. These hybrids constitute a safe,
economical, and environmentally favorable product that gives growers in the United States
improved yields and profitability.
Cry9C corn hybrids allow growers to eliminate application of synthetic chemical insecticides
that would otherwise be needed to protect their crops against European corn borers and
Southwestern corn borers. Damage from these insects is commonly believed to cause an average
of one billion dollars damage or more per year, when averaged across years. Numerous
university research trials have demonstrated not only that Cry9C corn hybrids provide control of
these key insect pests, but they also deliver a more timely, more complete, and more effective
control of these pests than can generally be achieved by chemical sprays. In addition to control
of corn borers, Cry9C corn is a unique product offering among Bt corn products that also
provides activity against Black cutworm, and thereby also helps to eliminate chemical treatments
for control of this soil insect.
The gene that codes for the Cry9C protein was isolated from a common soil bacteria, a strain of
Bacillus thuringiensis subsp. iolworthi. As described above, the Cry9C corn controls many of
the same corn pests as other known Bt-based products, including both sprayable Bt formulations,
such as DiPel™, and also other transgenic Bt corn products that contain a CrylA protein. Since
Cry9C is a Bt protein, it exhibits the favorable safety attributes that have made Bt products the
insecticides of choice for organic farmers for decades. And, since these Bt proteins are narrow in
their spectrum of activity, with control of only a limited number of lepidopteran species, and
with no activity against other non-target species, they represent a specific and targeted insect
control option.
52
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The CrylA and the Cry9C proteins kill insects in the same basic way, by destroying the integrity
of the insect's midgut. Though this basic mechanism to achieve activity is the same for both
proteins, these two classes of proteins do differ by binding to different midgut binding sites.
Since evidence from the field indicates that binding site changes are the most frequent basis for
insect resistance, Cry9C corn and its novel binding site offers a new option to corn growers for
insect resistance management, by offering an alternative site of action.
Concern about resistance development to Bt-proteins by corn borers has been growing because
the acreage of Bt corn has continued to increase since 1996, and because prior to 1998 when
registration was granted for Cry9C corn, only a single type of Bt protein, CrylA, was the "active
ingredient" used in all Bt corn. The same CrylA Bt proteins are also components in most
sprayable Bt formulations used commercially in other crops, in forestry and in horticulture. If
insect resistance would develop to CrylA proteins, either from Bt corn or from sprays, the
Cry9C corn would still be effective, providing excellent insect control. Therefore, use of Cry9C
corn is expected to sustain and possibly increase the longevity both for Bt sprays and for Bt-
based crop systems.
The Cry9C corn hybrids can be deployed in a variety of ways to reduce the likelihood of
resistance development, and to thereby improve the sustainability of planting Bt corn. Initially,
different Bt proteins may be rotated from one year to the next to eliminate insects that are
potentially resistant. Ultimately, the.goal is to stack two or more different Bt proteins, with
different binding sites, into the same hybrid corn plants. This approach vastly reduces the
potential for resistance development, and at the same time should allow growers to plant smaller
refuge sizes. Both of these outcomes provide significant direct and long-term value to the corn
farmers.
Elite hybrid corn varieties expressing the Cry9C protein are currently available from several
leading seed companies. The granting of a tolerance exemption for food uses with the Cry9C
protein will support a wider use of the Cry9C corn hybrids, and extend the benefits that this
product provides to a broader array of corn growers across the US.
53
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