Aventis CropScience
Allergenicity Assessment of Cry9C Bacillus thuringiensis subspecies
 tohvorthi Corn Plant Pesticide for FIFRA Scientific Advisory Panel

                      February 29,2000
                      Washington, DC

Andrew Cockburn PhD CBiol FIBiol
Registered Toxicologist
Head of Toxicology
Aventis CropScience
(Formerly AgrEvo)
Chesterford Park
Saffron Walden
CB10 1XL

                                                         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

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)

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

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.

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

       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.

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

3.     Questions Posed by EPA for the Peer Review Panels and Responses to those

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?
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

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.

       •  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?
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?
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).


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

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.

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.
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.

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?
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

•   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%).

•  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?
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

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
                          LESS STABLE PROTEIN
Apple (Mai dl)u"z;
Celery (Bet v 1 like)(1)
Castor Bean (3)
                           MORE STABLE PROTEIN
lVJLVyj.Vl_/ O J. /-VJJJL/JL
Peanut (Ara hi)'
Castor Bean(3)
Tomato (7)
Mnrc<=> T?5»Hi«Vi Pf>rr»Yi
                           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)                      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?
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%).

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?
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.

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?
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.

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

       data set for reassuring the EPA concerning  the safety (risk assessment) of Cry9C for

       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 -
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
       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




I,G|tfe©rop - ili]<3 Event?


Nutritional Equivalence




       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

       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

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.

       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).

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

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".


                                    TABLE 1

Table to demonstrate lack of de facto relationship between stability and allergenicity
                        LESS STABLE PROTEIN
Apple (Maldl)u"zr
Celery (Bet v 1 like)(I)
Castor Bean(3)
                          MORE STABLE PROTEIN
Peanut (Ara hi) UJ
Castor Bean(3)
Tomato (7)
                        Apple'1 &2)
                          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)

.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,

       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

       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.

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.

.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

       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

       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.

      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,

          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.

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
            No history of
                          No history of
No epitope
                                                     Partial digestibility/
                                                       Full digestibility
                                                     Partial heat stability
                                                      MW at upper limit
                                                       for allergenicity
                                                     systemic bioavaibility
Low prevalence <.5% TP
                                                                     not glycoslylated
                                                                      No increase in
                                                                   inherent allergenicity
                                                                        of corn
                                                                    No worker adverse
                                                                   Safe animal feed use
                                                                     for nearly 2 years

       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,

       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

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

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.

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:

       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

   -   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

       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

       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.

Maize flour *
Corn flour*
Maize/Corn oil
Total TMDI
(mg/person x day
Total TMDI
(mg/kg x day)

(g/person x

USA daily
(mean) (g/person
x day)

x day)
x day)
      *  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.

      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

7. References

Astwood J D, Leach J N & Fuchs R L (1996); Stability of food allergens to digestion in
vitro. Nature Biotechnology, 14. 1269-1273.

Astwood J D, Fuchs R L & Lavrik P B (1997);  Food biotechnology and genetic
engineering. In Food Allergy: Adverse reactions to foods and food additives 2" Edition,
Chap. 4, 65-92; Eds. Metcalf DD, Sampson HA & Simon RA.

BernhiscI-Broadbent J, Strause D & Sampson H A (1992); Fish hypersensitivity II:
Clinical relevance of altered fish allergenicity caused by various preparation methods.  J.
Allergy Clin. Immunol., 90, (4), Part 1, 622-629.

Board of Trustees (ed) (1995);  Simulated Gastric Fluid, TS., pp 2053 in The United States
Pharmacopeia 23. The National Formulary 18. United States Pharmacopeial Convention,
Inc.jRrockville MD.

Boisen S & Eggum B O (1991). Critical evaluation of in vitro methods for estimating
digestibility in simple-stomach animals. Nutr. Res. Rev., 4, 141-62.

Bruijnzeel-Koomen C, Ortolan! C, Aus K, Bindslev_Jenson C, Bjorkst&i B, Moneret-
Vautrin D & Wurrich B (1995);  Adverse reactions to food. Allergy; 50:623-635.

Cockburn A (1998); An Expert Assessment of the Allergenic Potential of StarLink™ Com.
AgrEvo Report Reference.

Cheftel J C (1979); Proteins and amino acids. In: Nutritional and Safety
Aspects of Food Processing (Tannenbau, S R Ed) pp 153-215. Marcel Dekkar, NY.

Dircks L K, Vancanneyt G & McCormick S (1996); Biochemical characterisation and
baculovirus expression of the pectate lyase-like LAT56 and LAT59 pollen proteins of
tomato. Plant physiol. Biochem, 34 (4), 509-520.

Drebors S & Foucard T (1983); Allergy to ale, carrot and potato in children with birch pollen
allergy. Allergy, 38, 167.

Ensminger A H, Ensminger M E, Konlande J E & Robson J R K (1994);  Foods & Nutrition
Encyclopaedia Vol 1, p. 595, CRC Press.

Friedman M, Gumbmann M R & Masters P M (1984); Protein-alkali reactions: chemistry,
toxicology,and nutritional consequences. In: Nutritional and Toxicological Aspects of Food
Safety (Friedman M ed) pp 367-412. Plenum Press, NY.

Fu T J & Abbot UR (1998);  Stability of food allergens to digestion and acid hydrolysis in.
comparison with proteins of unproven allergenicity. Poster Presentation, American Chem. Soc.
Annual Meeting, Boston, MA.

Fuchs RL & Astwood J D (1996);  Allergenicity assessment of foods derived from genetically
modified plants. Food Technology, 83-88.

Gardner M L G (1988); Gastrointestinal absorption of intact proteins. Ann. Rev. Nutr. 8, 329.

Houben G F, Knipples L MJ & Penninks A H (1997); Food Allergy:  Predictive testing of
food products. Env. Tox & Pharm. 4, 127-135

James J M & Burks A W (1995);  Foods Immun. Allergy Clin. NA, 13, 477-488.

King T P, Hoffman D, Lowenstein H, Marsh D G, Platt-Mills T A E & Thomas W (1994);
Allergen Nomenclature, Inf Arch Allergy Immunol. 105, 224-233.

Lambert B, Buysse L, Decock C, Jansens S, Piens C, Saey B, Seurinck J, Van Audenhove
K, Van Rie, Van Vliet A & Peferoen M (1996); A Bacillus thuringiensis insecticidal crystal
protein with a high activity against members of the family Noctuidae, Applied and
Environmental Microbiology, 62, 80-86.

Leeson S (1998); The effect of corn hybrid CBH351 on the growth of male broiler chickens,
University of Guelph, Ontario, Canada Report A67407.

Lehrer S B, Taylor J & Salvaggio J E (1981); Castor bean allergens: evidence for distinct
heat-labile and stable entities. Int. Archs. Allergy Al. Immun. 65, 69-75.

Lehrer S B (1997) Investigation of allergens in wild-type and transgenic corn, report A57777,
MRID# 44384405.

Mauron J(1984); Effect of processing on nutritive value of food protein. In: Handbook of
nutritive Value of Processed Foods. Ed. M Recheigl. CRC Press, Florida, pp 429-71.

Macintosh S C (1997); Test substance characterization report for catfish study: determination of
Cry9C and PAT protein, AgrEvo Report A67494, MRID#44384301.

Macintosh S C (1997a); Amino acid sequence homology search with the corn expressed
truncated Cry9C protein. PGS (America) Inc. On behalf of PGS NV, Gent, Belgium, AgrEvo
Report A59939, MRID # 44258109.

Macintosh SC (1998); Occupational exposure of StarLink™ corn: Garst Seeds, 1996-1998,
AgrEvo Report COO 1748, MRID # 44714003.

Marsh D G (1981) Norman P S, Roebber M & Lichtenstein L M; Studies on allergoids from
naturally occurring allergens. J. Allergy Clin. Immunol.,  68, 449-459.

McLean E & Ash R (1996);  The time course of aearance and net accumulation of horse radish
peroxidase (HRP) presented orally to juvenile carp Cyprius carpio. Comp. Biochem. Physiol
84A, 687-690.

McClintock J T, Schaffer C R & Sjoblad R D (1995);  A comparative review of the
mammalian toxicity of Bacillus thuringiensis-based pesticides Pestic. Sci. 45:95-105).

McFarlane M (1998);  Safety Assessment of StarLink™ Corn for Human Food Use.

Metcalf D D (1985); Food Allergens; In Clin. Rev. Allergy 3:331-349.

Metcalf D , Astwood J D, Townsend R, Sampson H A, Taylor S L & Fuchs R L (1996);
Assessment of the allergenic potential of foods derived from genetically engineered crop plants.
Grit. Rev. Food Sci. Nut. 5165-5186.

Metcalf D (1997) Food allergy in adults. In Food Allergy: Adverse reactions to food and feed
additives, 183-191, Blackwell.

Noteborn H P M (1998);  Assessment of the stability to digestion and bioavailability of the LYS
mutant Cry9C protein from Bacillus thuringiensis serovar tolworthi (RIKILT). AgrEvo Report
C002137, MRID#44734305.

Noteborn H P M (1998);  Mouse short-term (30-day) dietary toxiciry study with the protein
Cry9C. RIKILT, Netherlands, AgrEvo Report C002138, MRID#44734303.

Opstvedt J, Miller R, Hardy R W & Spinelli J (1984); Heat induced changes in sulfhydryl
groups and disulfide bonds in fish protein and their effect on protein and amino acid digestibility
in rainbow trout (Salmo gairdneri). J. Agric. Food Chem., 32, 929-35

Opstvedt J (1988); Influence of drying and smoking on protein quality. In: .fish Smoking and
Drying, Ed. J R Burt, Elsevier Applied Science, NY, pp 23-40

Organisation for Economic Co-operation & Development (OECD) (1995); Environmental
Health & Safety Dividison, Workshop on Food Safety Evaluations, Paris.

Peferoen M (1997); In vitro digestibility and heat stability of the endotoxin Cry9C protein
sequence. PGS NV, Gent, Belgium.  AgrEvo Report A59938, MRID#44258108.

Perferoen M (1997); Food allergenicity amino acid sequence homology. PGS NV, Gent,
Belgium. AgrEvo Report A67499, MRID # 44384404.

Rich N, Satterlee D L & Smith L J (1990); A comparison of in vivo apparent protein
digestibility in man and rat to in vitro protein digestibility as determined using human and ran
pancreatins and commercially available proteases.  Nutr. Rep. In., 21. 2, 285-300

Rothbard J D & Gefler M L (1991); Interactions between immunogenic peoptides and
MHC proteins. Ann. Rev. Immunol, 9, 527-565.

Sampson H A (1996);  Diseases of the gastrointestinal tract of children caused by immune
reactions to foods.  In Highlights in Food Allergy.  Wuthrich B, Ortolani C (eds). Monogr
Allergy, Basel, Karger; 32: 36-48.

Seifert J, Ring J & Brendel W (1974); Prolongation of skin allografts after oral application
of ALS in rats. Nature 249, 776.

Seifert J, Ring J, Steininger J & Brendel W (1977);  Influence of the immune response on
the absorption of protein from the gut. Nutr. Metab. 21 (suppl. 1), 256.

Shiltito R (1998); Determination of the stability of PAT and Cry9C protein in processed
grain of transgenic field corn hi fractionated agricultural commodities, ARC, AgrEvo USA,
Report A59927, MRID# 44384301.

Strobel S (1997); Oral Tolerance: hi Food Allergy by Metcalfe, Sampson & Sinon, 2nd
Edition, Blackwell Press, 107-135.

Strobel S & McL Mow at A (1998);  Immune responses to dietary allergens and tolerance.
Immunology today, 19. No 4. 173 et seq.

Schwass, D £ & Finley J W (1984);  Heat and alkaline damage to proteins, racemization and
lysinoalanine formation. J. Agric. Food Chem. 32: 1377-1382.

Taylor S (1997);  Food Allergens. Structure and Immunologic Properties. Ann. Allergy, 59,

Teichberg, S (1988); Intestinal absorption of potentially antigenic macromolecules. Clin.
Pediatr. 5, 81.

USDA (1994-1996); Food Survey Research Group, Agricultural Research Service (ARS).
Nationwide Food Consumption Survey: Continuing Surveys of Food Intakes by individuals
1994-96. Dataset.

USDA (1999); Agricultural Research Service.  USDA Nutrient Database for Standard
Reference, Release 13. Nutrient Data Laboratory Home Page.

US EPA, (1988); Guidance for the re-registration of pesticide products containing Bacillus
thuringiensis as the active ingredient; US Dept of Commerce, National Technical Information
Service, Springfield, VA, 22161, Document # PB, 89-164198.

US EPA, (1995); Plant Pesticide Bacillus thuringiensis Cry III. A delta endotoxin and the
genetic material necessary for its production,  tolerance examption. Fed. Regist., 60, 21725-

US EPA (1998); Bacillus thuringiensis subspecies tolworthi Cry9C Protein and the genetic
material necessary for its production in corn,  tolerance exemption. Fed. Regist. 1998, 63, 28258-

Vantard M, Per C, Fellous A, Schellenbaum P & Lambert A-M (1994); Characterisation of
a 100-kDa heat-stable microtumbule-associated protein from higher plants. Eur. J. Biochem, 220,

Vieths S, Aule H, Becker W M & Buschmann L (1995) Cite Vide, Chap. 10;  Characterisation
of labile and stable allergens in foods of plant origin, 130-149, Food Allergies & Intolerances ;
Deutsche Forschungsgemeinschaft Symposium, Germany.

WHO (1995);  Allocation of the principles of substantial equivalence to the safety evaluation of
foods or food components from plants derived by modern biotechnology - Geneva, WHO Food
Safety Unit, 1-80.

Wuthrich B, Stager J & Johannson SCO (1990); Allergy 45, 566.

Yunginger J W (1990);  Classical Food Allergens. Allergy Proceedings, H., 7-9.
Zapf R (1998); TMDI calculation for StarLink   hybrid maize. Unpublished AgrEvo

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
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.

Appendix 2
Further Investigation of the Digestibility of the Cry9C Protein by a Selection of Protolytic
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,

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.

                                        Table 1

Proteinase K
||Myes jQ5fi|iffYr >/ ^?U
Bovine Pancreas
Subtilisin Carlsberg
Porcine Mucosa
Tritirachium Album
Pineapple stem
Porcine pancreas
Bovine Pancreas
Porcine Intestinal
Porcine stomach
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
No noticeable digestion was

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.

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).

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.

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

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.