AgrEvo Response to EPA Background Document

Information provided for informational purposes only

Note: This information is provided for reference purposes only. Although the information provided here was accurate and current when first created, it is now outdated.

 

Note: The applicant was provided with a copy of the EPA Background document for comment. Their response is provided here as additional input for those seeking to comment

 

Dr. Janet L. Andersen, Director
U.S. Environmental Protection Agency
Office of Pesticide Programs
Biopesticides and Pollution Prevention Division (7511C)
1921 Jefferson Davis Highway
Arlington, VA 22202

December 14, 1999

Re: Comments regarding the "Cry9C Food Allergenicity Assessment Background Document", the DERs cited in that document, and the July 15, 1999 Memorandum "Review of Additional Data from AgrEvo USA on the Cry9C Protein Expressed in Corn."

EPA Registration Number 45639-221

 

Dear Dr. Andersen,

AgrEvo appreciates the opportunity to comment on the "Cry9C Food Allergenicity Assessment Background Document", the EPA Data Evaluation Reviews (DERs) cited in that document, and the July 15, 1999 Memorandum "Review of Additional Data from AgrEvo USA on the Cry9C Protein Expressed in Corn." The cry9C gene, from Bacillus thuringiensis subspecies tolworthi, encodes a highly efficacious insecticidal protein active against the European corn borer. The Cry9C protein offers an alternative mode of action to other proteins currently on the market, creating value in insect resistance management and in a multiple toxin deployment (pyramiding) strategy.

In this letter our approach to assessing the safety of the Cry9C protein will be described along with specific comments for the DERs cited in the "Cry9C Food Allergenicity Assessment Background Document" and the July 15, 1999 Memorandum.

In evaluating the safety of the Cry9C protein AgrEvo followed and extended both the US and the international guidance given for food and feed safety assessment. A large number of studies have been performed and evaluated, from which it has been concluded that Cry9C field corn (to be referred to as Cry9C corn in the remainder of this letter) is substantially equivalent to other corn currently in commerce. An important part of that evaluation was to determine whether the source of the novel protein, its characteristics and properties, or the host plant itself, might in any way predispose an allergenic risk in humans.

There are currently no validated models (in silico, in vitro or in vivo) for the prediction of whether a given protein possesses the necessary characteristics to elicit clinical symptoms of food allergenicity in humans. It is estimated that some 1 to 2 percent of the adult human population may suffer from food allergies, albeit with a variety of different food allergen reactions and sensitivities. It is very important to note that the allergenicity of a food protein does not always correlate with its immunogenicity. Food sensitivity is therefore a rare phenomenon in susceptible individuals, often has a geographic association, may be episodic, and is largely successfully controlled by avoidance. Simple consideration of these and other aspects has led to the conclusion among experts that, while animal models provide important information towards understanding the mechanisms of allergenicity, with today’s technology it is virtually impossible to predict from any single test, with any certainty, the allergenic potential for specific proteins to affect humans.

As a consequence, strategies have been developed which have sought to make an indirect determination of allergenic potential by considering the weight of evidence, in order to support the decision making process concerning such allergenic potential. AgrEvo has utilized, with the help of allergenicity experts, a decision-tree approach to assemble a weight of evidence assessment, which has led us to conclude that the Cry9C protein is unlikely to pose an allergenic risk to humans.

Assessment of the potential for allergenicity to the Cry9C protein and corn containing the Cry9C protein was made by systematically evaluating eight key aspects. The conclusions from these eight evaluations are presented below.

    The source of the cry9C gene - The gene is derived from a source with no allergenic history, Bacillus thuringiensis subspecies tolworthi. The status of wild-type corn (host plant) from the perspective of food allergenicity - Corn has no known history of food allergy. The insertion of the transgene - To provide practical scientific evidence that the process of inserting the new genes has not altered the intrinsic allergenic status of the host plant, a panel of sera from suspected corn-reactive subjects was screened with corn seed extracts from Cry9C corn. The evidence indicated that the insertion did not alter the intrinsic allergenic status of the corn (MRID# 44384405). Homology of the Cry9C protein to known allergens - The Cry9C protein shares no epitope homology with any known allergens, either when examined at the whole protein level or when examined with the more sensitive analysis done at the level of all possible eight amino acid sequences (MRID# 44258109 and 44384404). Protein stability - Some stability for the Cry9C protein was seen in simulated gastric fluids for a period of up to 4 hours (MRID# 44258108). However, the in vivo bioavailability study (MRID# 44734305) showed degradation of the Cry9C protein. It seems probable that the use of in vitro models that are sterile with respect to the normal gut flora as well as to other physiological processes, may be an inexact model to represent the more complex in-life gastrointestinal environment of animals and man. This information should also be considered in context with the knowledge that there are food allergens which are unstable under these conditions and that there are many stable proteins which are not allergens. Results with the Cry9C protein show that its stability is temperature and process dependent. The protein has been found to be relatively stable to heat, but results from different studies are in conflict on this point, as Cry9C protein was found in processed grain in one study (MRID# 44734304), but not found in catfish pellets processed from the Cry9C corn in another study (MRID# 44384301) which used a higher temperature and pressure. It has also been demonstrated that the Cry9C protein degrades in soil, with a half life of about three days (MRID# 44161701). Prevalence of the Cry9C protein in feed made from corn - The Cry9C protein is present at a low level in the kernels of corn, at approximately 0.17% of the total protein (MRID# 44734304). The majority of significant food allergens are present at a relatively high percentage of the total protein in the plant or foodstuff from which allergic reactions occur. Molecular weight of the Cry9C protein - The Cry9C protein has a molecular weight (68.7 kDa) close to the top end of the range for allergens (10-70 kDa), as referred to in literature about food allergens. The majority of food allergens fall within the range of 10-40 kDa. Glycosylation of the Cry9C protein - The Cry9C protein does not appear to be glycosylated (MRID# 44384401).

Additional supporting evidence for a lack of allergenic potential for Cry9C corn is made by evaluating the cases of repeated exposure to the Cry9C protein and to Cry9C corn. Repeated exposure is generally required before a food allergy develops from first immunization by the allergen, followed subsequently by an allergenic reaction if sensitization has taken place. This supportive information about exposure is important in an overall safety assessment, especially when one considers that extensive human exposure to the Cry9C protein and to Cry9C corn has already occurred over the last 5 years. The following is a list of work examining exposure to date to Cry9C protein in order to support the finding of a lack of allergenic potential of Cry9C corn:

    In 1998 the USEPA granted a feed tolerance exemption for Bacillus thuringiensis subspecies tolworthi Cry9C protein and the genetic material necessary for its production in corn. (Federal Register 63: 28258–28261, 1998). No adverse findings are known to have occurred regarding the use of this feed stuff in the United States. Human experience, by workers for Garst Seeds who have been exposed to Cry9C corn (MRID# 44714003) - Garst Seeds employees have been actively working with Cry9C corn since 1995. Although no clinical evaluations were performed, no unusual reactions have been described in normal or pre-existing atopic personnel involved in the field, harvesting or processing activities for Cry9C corn. Of additional consequence worth noting, there is no evidence that the respiratory exposure to Cry9C corn in a corn seed production environment (generally recognized to be a more sensitive route for hypersensitisation) has led to any unusual reactions (including allergic responses or cross-reactivity) among 1,990 worker respondents that were canvassed by Garst on this topic. Mouse 30-day repeat dose study of Cry9C in drinking water (MRID# 44734303) - No evidence of any adverse signs were seen in clinical condition, haematological, blood biochemical (including total protein and albumen levels) or histopathological (including the reticuloendothelial (RE) elements of bone marrow, jejunum with Peyers patches, mesenteric lymph nodes, spleen and thymus) parameters measured in the study. The gastric lining is often the first indicator of an allergic response, yet no inflammation of the oral or gastric lining was detected. (AgrEvo recognizes that this study was not originally designed to assess allergenicity and that this lack of allergenic response is indirect evidence.) Poultry wholesomeness feeding study of Cry9C corn (MRID# 44734306) - No abnormalities were detected in a 6 week feeding study, which evaluates a period of rapid growth and weight gain, and which is therefore susceptible to very subtle dietary effects. (AgrEvo recognizes that this study was not originally designed to assess allergenicity and that the lack of allergenicity response is indirect evidence.)

 

When the eight key aspects outlined above were evaluated for evidence of potential allergenicity, and when these aspects are put together with the supportive exposure information given above, the weight of evidence supports a lack of allergenicity and a lack of allergenic potential for the Cry9C protein.


The following Table contains AgrEvo’s comments to the EPA DERs and the July 15, 1999 Memorandum for Cry9C corn.
MRID # Title EPA Rating AgrEvo Response

44714001

 Safety Assessment of StarLink corn, genetically modified corn containing the truncated Bt insecticidal protein Cry9C, for human food use Supplemental This is a summary of the data presented as the agency states.

44734301

 Bt Cry9C protein: Investigative study of the potential for binding to mouse intestinal brush membrane vesicles Supplemental; Upgradable In reviewing the report and the supporting information, AgrEvo has found that the blot is not available to be re-photographed. However, this binding data is not critical for assessing potential allergenicity.

44734302

 Bt Cry9C protein mouse acute intravenous toxicity study (152A-13) Acceptable No additional comment

44734303

 Mouse short-term (30-Day) dietary toxicity study with the protein Cry9C Acceptable No additional comment

44734304

 Determination of the stability of PAT and Cry9C proteins in processed grain of transgenic field corn in fractionated agricultural commodities Supplemental; Upgradable The presence of the Cry9C and PAT proteins in the control samples could never be adequately explained, despite AgrEvo’s rigorous efforts to do so no unexpected pollen flow, no mixing of grain prior to or after shipment to the processing facility nor inadvertent contamination during the processing was able to be isolated as the reason. Therefore, AgrEvo elected to repeat the study from grain collected in 1998. In the repeat study, the PAT and Cry9C proteins were NOT detected in the control samples and the proteins were detected in the same fractions and at the same levels as found in the initial study (PAT = 1.4 to 70 mg/g, Cry9C = 0.14 to 41 mg/g).
44714002 Development of new methods for safety evaluation of transgenic food crops Supplemental The materials and methods for the Brown Norway Rat (BNR) study are detailed in the references cited on page 17 of the final report. To address some specific points; 1) The challenge antigen iv injection was made 24 hours after the intradermal serum injections; 2) Test substance characterization reports for the batches will be provided; 3) The BNRs were obtained from Bantin and Kingman (Hull, Humberside, UK), the Sprague-Dawley rats from Harlan Olac, 1976 (Bicester, UK). "Animals received a nutritionally adequate diet, either Rat and Mouse No. 1 expanded...Both food and tap water were freely available."; 4) The number of S-D rats used for each sera analysis was not identified; 5) No data on individual animal responses were received; 6) The original gel for the Western blot is not available as the gel is destroyed after the bands are transferred to the nitrocellulose for the Western analysis. AgrEvo also asked for additional information from BIBRA for items 3), 4) and 5), but was unable to obtain any further information.
July 15, 1999 Memo Review of additional data from AgrEvo USA on the Cry9C protein expressed in corn Not applicable The Brown Norway Rat study indicated that the Cry9C, Cry1Ab, lactoferrin (not considered of clinical importance in milk allergenic reactions) and other proteins were recognized as food allergens by sensitized test animals orally challenged with that same protein.

44714003

 Occupational exposure of StarLink corn: Garst Seeds, 1996-1998 Acceptable No additional comment

44734305

 Assessment of the stability to digestion and bioavailability of the LYS mutant Cry9C protein from Bacillus thuringiensis serovar tolworthi Acceptable

No additional comment

 
The following data were submitted in support of the currently approved feed use only exemption from the requirement of a tolerance.
MRID # Title EPA Rating AgrEvo Response
44258108 \In Vitro Digestibility Acceptable No additional comment
44258109 Amino acid sequence homology search with the corn-expressed truncated Cry9C protein sequence Supplemental; BPPD has not yet determined how to judge amino acid sequence homology for risk assessment purposes. This study at the whole protein level has been deemed supplemental by the Agency, yet allergenicity experts place a high level of importance on this sequence homology study. The lack of any sequence homology to known allergens greatly reduces concerns about potential allergenicity for the Cry9C protein.
44384404 Cry9C Bt insecticidal protein identification of sequence homology with allergens by searching protein databanks Supplemental; BPPD has not yet determined how to judge amino acid sequence homology for risk assessment purposes. This study at the eight amino acid level has been deemed supplemental by the Agency, yet allergenicity experts place a high level of importance on this sequence homology study. The lack of any sequence homology to known allergens greatly reduces concerns about potential allergenicity for the Cry9C protein.
44258107 An acute oral toxicity study in mice with Cry9C protein as purified from Bt Cry9C.PGS2 (152A-10) Acceptable No additional comment
44384405 Investigation of allergens in wild-type and transgenic corn Supplemental This study on the inherent allergenicity of StarLink™ corn should be included in the overall safety assessment despite the comment by the agency that the "study does not address the potential for inducing food allergy from a novel protein…". AgrEvo believes the study does demonstrate that the gene insertion has not triggered the host plant to elicit an allergenic response.
44384301 Test substance characterization report for catfish study: determination of Cry9C and PAT protein No rating by EPA As stated by the reviewer: "The study is scientifically sound and no difference was observed between the processed and control replicates. Cry9C activity was shown to be destroyed following the extrusion procedure utilized in a typical fish food manufacturing process." Therefore, this study sheds light on the ability for processing steps to destroy the Cry9C protein as pointed out in the Safety Assessment (MRID # 447140-01).
44734306 The effect of corn hybrid CBH351 on the growth of male broiler chickens Not rated by EPA No measurable differences or deleterious effects were measured for chicks fed non-transgenic corn versus those fed Cry9C-containing corn in a full lifecycle study of 42 days. Although this wholesomeness study was not performed from start to finish under strict GLP conditions, the protocol was written, approved and followed using GLP standards during the entire feeding phase. Careful records were kept and a final report including all raw data was provided to AgrEvo and submitted to EPA.

 

This summary and data review concludes AgrEvo’s overview regarding the approach used to assess the food safety of the Cry9C protein and addresses some specific comments about the DERs cited in the "Cry9C Food Allergenicity Assessment Background Document." Thank you for the opportunity to comment on these documents.

 

Sincerely,

signed

Sally Van Wert, Ph.D.

Manager, Regulatory Affairs – Biotechnology, AgrEvo USA