Section 2.0 2  1 2  Health Effects 2  Comment Excerpts Commenter Name: Dennis Ward Commenter Organization Name: Monsanto Comment Number: 30509B 086000 Excerpt Number: 17 Excerpt Text: Human Health and Food Safety  Reviews by EPA and FDA establish that corn containing Cry3Bb1 protein is safe for consumption by humans and animals.

 The Cry3 class of Bt proteins, including Cry3Bbl that has 85% amino acid similarity to Cry3A in Newleaf potatoes, has an established history of safe consumption by humans.  No adverse effects were observed in mice following exposure to a maximum achievable acute oral dose of Cry3Bb1 protein, a dose which is many orders of magnitude higher than upper bound estimates for human dietary exposure.  Exposure to Cry3Bbl via other routes, such as dermal or inhalation, is considered by EPA to be unlikely because the protein is contained within corn plant cells.  The extremely limited potential for horizontal gene transfer of the intact, functional antibiotic resistance gene nptII from genetically modified corn to bacteria poses minimal risk to the environment and human health.  A 10 base pair sequence of DNA present within the Cry3B1 and nptII transformation cassette was lost upon stabile integration of the cassette into the plant genome and no evidence was found for its incorporation elsewhere in the genome.

EPA Response: EPA agrees with these comments which are reflected in the MON863 Biopesticide Regulatory Action Document ( BRAD) in section IIA and IIB.

Commenter Name: Dennis Ward Commenter Organization Name: Monsanto Comment Number: 30509B 086000 Excerpt Number: 22 Excerpt Text: Comment or Question: There is a lack of acute toxicity data for corn event MON 863, through dermal, inhalation and dietary routes of exposure.

Scientific Background: Cry3Bbl protein resides within plant tissues, thus limiting dermal and inhalation routes of exposure. The EPA has previously stated for other Cry proteins produced in corn that "... exposure via the skin or inhalation is not likely since the CrylAb and Cry1F proteins are contained within corn plant cells which essentially eliminates or reduces exposure routes to negligible." [ Footnote 23: EPA Biopesticides Registration Action Document, Bt Plant Incorporated Protectants, Oct 15, 2001.] There is' no evidence to suggest that Cry proteins should have a greater capacity for contact or respiratory Section 2.0 2  2 sensitization than any other protein found in corn or other food crops.

EPA Response: EPA agrees with these comments which are reflected in the MON863 Biopesticide Regulatory Action Document ( BRAD) in section IIA and IIB.

Commenter Name: Dennis Ward Commenter Organization Name: Monsanto Comment Number: 30509B 086000 Excerpt Number: 23 Excerpt Text: On the other hand, there is the potential for dietary exposure to low levels of Cry3Bb I protein from consumption of corn  rain and processed corn food products; consequently, the safety of Cry3Bb1 protein has been assessed via a high dose acute gavage study in rodents. There was no evidence of toxicity in mice dosed with up to 3,200 mg/ kg Cry3Bbl protein, the highest dose achievable. This exposure level is more than five orders of magnitude higher than upper bound estimates for human dietary consumption. Similar results ' were obtained for the closely related Cry3A protein; no toxicity was observed in mice dosed with up to 5,200 mg/ kg, the highest dose tested.

EPA Response: EPA agrees with these comments which are reflected in the MON863 Biopesticide Regulatory Action Document ( BRAD) in section IIA and IIB.

Commenter Name: Dennis Ward Commenter Organization Name: Monsanto Comment Number: 30509B 106000 Excerpt Number: 13 Excerpt Text: Human Health and Food Safety  Monsanto has utilized current, appropriate and scientifically robust protocols for the simulated gastric digestion and sequence matching tests.

EPA Response: EPA agrees with these comments which are reflected in the MON863 Biopesticide Regulatory Action Document ( BRAD) in section IIA and IIB. Section 2.1 2  3 2.1  Human Health 2.1  Comment Excerpts Commenter Name: Berg, Mark Commenter Organization Name: Foundation E. A. R. T. H. Comment Number: 30509 078000 Excerpt Number: 5 Excerpt Text:

Based on the 1999 Doane information provided by Foundation E. A. R. T. H., corn rootworm insecticide users in the ECI and WCB grew 17.4 million acres of corn in 1999. Almost 60% of those acres were treated with an insecticide to control corn rootworm. The results of this research suggest that the introduction of CRW corn may reduce the acres treated by 44% , representing 4.6 million acres in the first year. Since the current average use rate of corn rootworm is 0.5 pounds of active ingredient per acre, this would result in a reduction of 2.4 million pounds of soil applied insecticide active ingredient.

Commenter Name: Berg, Mark Commenter Organization Name: Foundation E. A. R. T. H. Comment Number: 30509 078000 Excerpt Number: 6 Excerpt Text:

 It is apparent that these producers have concerns with usage of insecticides. While 12% have personally become ill due to exposure to insecticides, 20% claim that a family member or other associate has such an experience.

 By indicating either a 8, 9, or 10, exposure to the insecticide when filling insecticide boxes ( 76%) and exposure of other family members to insecticides ( 72%) surfaced as the primary concerns of growers with current soil applied insecticides  as they are well aware of the potential health hazards associated with these chemicals. About half are extremely concerned about the impact of insecticide on the environment ( 53%) and exposure to livestock ( 47%).

EPA Response: EPA agrees with the commenter that the MON863 corn PIP should alleviate some of the concerns of producers who worry about the effects of the soil insecticides typically employed to control corn rootworm. This should especially be the case for exposure of producers to the soil insecticides whose use may be reduced with this particular PIP.

Commenter Name: Angelika Hilbeck Commenter Organization Name: Greenpeace Comment Number: 30509 113000 Excerpt Number: 11 Excerpt Text: Section 2.1 2  4 Human health assessment We also noted that for the human safety assessment much of the safety conclusions are either based on human safety trials conducted with various old microbial Bt products conducted prior to 1989 or simply on lack of data and evidence.

' The extremely low mammalian toxicity of Bt based insecticide products has been demonstrated in numerous safety studies conducted prior to 1989.' ( Monsanto Document, page 21 of 56)

EPA Response: EPA disagrees with the statement by Greenpeace that the human safety assessment for the MON863 corn PIP is based on " various old microbial Bt products." The safety assessment for MON 863 is based on the data submitted by the company specifically utilizing the proteins expressed in MON863 corn. The tests and their results are found in the appropriate section ( IIB) of the Biopesticide Registration Action Document ( BRAD). The tests are intended to answer questions regarding the potential of the MON863 PIP proteins to display dietary toxicity or potential allergenicity. These tests are based in part on the current guidelines for microbial pesticides and input from Scientific Advisory Panels convened to address issues of protein toxicity and allergenicity. While information about the known previous exposure to the proteins expressed in MON863 corn may be considered as part of the safety assessment, the basis of the assessment continues to be the specific test results and information submitted by the company.

Commenter Name: Angelika Hilbeck Commenter Organization Name: Greenpeace Comment Number: 30509 113000 Excerpt Number: 12 Excerpt Text: ' The conclusion of reasonable certainty of no harm and the resultant tolerance exemptions for this wide array of Bt. mixtures and Cry proteins in food or feed were based on the lack of adverse effects to mammals in numerous toxicological studies. This conclusion is supported by a history of safe use in agriculture for over 40 years ..' ( Monsanto Document, page 21 of 56)

Commenter Name: Charles Benbrook Commenter Organization Name: Union of Concerned Scientists Comment Number: 30509 115000 Excerpt Number: 33 Excerpt Text: There is little known about the degree of similarity or differences between Cry3Bb proteins and other Cry proteins in terms of the health effects of greatest concern, which would include allergenicity and digestive system impacts.

EPA Response: EPA disagrees with the statement of the Union of Concerned Scientists that little is known about the Cry3Bb1 protein's health effects in terms of allergenicity and digestive system impacts. The test results that have been submitted for three variants of the Cry3Bb1 protein expressed in MON863 all have negative results for expected dietary toxicity and potential allergenicity. The results are found in section II. B. 1. of the BRAD. Section 2.1 2  5 Commenter Name: K Kreed Commenter Organization Name: K Kreed Comment Number: 30509B 035000 Excerpt Number: 2 Excerpt Text: I have serious concerns, as the mother of a child with food allergies, regarding the effect of these foods on allergic individuals. There seems to be an increase in the number of children with severe food and respiratory allergies ( and the two are inter related as eating allergenic foods can cause severe respiratory problems) and many are concerned that our food supply is contributing. I did not have, or was not aware that I had any food allergies, until recently when I had a severe reaction, apparently to wheat gluten. It is extremely scary when you or especially your child, has a severe reaction to a substance. Do we know for sure what effects splicing genes from different species will have on allergies? What labeling will be required for these products? I am concerned for example that one who is allergic to tomatoes may eat corn that has been genetically engineered using tomato genes and have a reaction.

Commenter Name: K Kreed Commenter Organization Name: K Kreed Comment Number: 30509B 035000 Excerpt Number: 3 Excerpt Text: As far as suggested research, I would like to state that allergies may take several years to develop to a substance. Many people who acquire pets have no allergy to the animals until they have had them for several years, then severe allergies can " suddenly" develop. Therefore short duration tests to determine whether people can tolerate genetically foods may not be accurate. Corn is a staple food, difficult to avoid in our diets, so I would urge you to move slowly in approving substances affecting the entire population.

EPA Response: EPA sympathizes with the commenter who has a child with a food allergy and suffers herself with gluten sensitivity. One of the difficulties with food allergy is that it may appear over time and there is no effective way to predict when or if one will develop an allergic sensitivity to a normally tolerated food protein. As the commenter indicates allergies develop over time by a process of sensitization then appear suddenly. The sensitization process is probably the least understood of the multiple steps involved in allergenicity. Unlike other areas of concern for pesticide dietary exposure, food allergy does not have a validated animal model test system that can predict a protein's allergenic potential. However, EPA believes that utilizing the best scientific methods currently available, the PIP proteins expressed in MON863 corn have a reasonable certainty of causing no harm including dietary toxicity and potential allergenicity. The basis of the assessment are the specific test results and information submitted by the company about the PIP proteins found in MON863. The results are found in section II. B. 1. IIB of the BRAD and suggest that the PIP proteins do not present a potential allergenicity concern.

Commenter Name: K Kreed Commenter Organization Name: K Kreed Comment Number: 30509B 035000 Excerpt Number: 4 Section 2.1 2  6 Excerpt Text: I would also urge requiring labeling of ALL products utilizing genetic engineering, so that the millions of people with allergies can avoid these products if they wish.

EPA Response: EPA is responsible for the safety assessment of the PIP protein expressed in the MON 863 corn product. EPA does not have regulatory authority to require labeling of food products. Regulatory authority for food labeling rests with the Food and Drug Administration ( FDA). Currently, FDA has determined that it is not necessary to label foods containing GMO constituents, http:// www. cfsan. fda. gov/~ dms/ biolabgu. html.

Commenter Name: Paul Mitchell Commenter Organization Name: Texas A& M University  Department of Agriculture Economics Comment Number: 30509B 082000 Excerpt Number: 5 Excerpt Text: Lastly, I believe that the risks associated with the registration and ensuing ( widespread) adoption of corn event MON 863 are small compared to the costs of not doing so. Experience with the various corn Bt events for corn borer control have been acceptable. No environmental or human health problems have been identified at this time, though many have been looking for such problems. No human allergic reactions have been identified or other adverse human or animal health impacts.

EPA Response: EPA agrees with the commenter as to the current experience with corn borer PIP products. EPA agrees with these comments which are reflected in the MON863 Biopesticide Regulatory Action Document ( BRAD) in section IIA and IIB.

Commenter Name: Dennis Ward Commenter Organization Name: Monsanto Comment Number: 30509B 086000 Excerpt Number: 7 Excerpt Text: Comment or Question: Dietary exposure of Cry3Bb1 in corn event MON 863 exceeds that of other food crops treated with microbial products and may pose a greater risk of allergenic and digestive system impacts.

Scientific Background: Cry3Bb1 has been evaluated using the best available hazard assessment methodology. The Cry3Bb1 gene was obtained from a microbial source that has no history of allergenicity. The Cry3Bb1 protein lacks sequence similarity to known allergens, it is rapidly digested using recommended assays, and is found at levels that are orders of magnitude lower than levels of allergens commonly found in food. The EPA has granted a tolerance exemption for the production of Cry3Bb I protein in corn plants ( 40 CFR 180.1214). Consequently, any residues of Cry3Bb1 protein that may be present in corn grain are considered safe for human consumption. Also, a recently completed consultation with FDA established that corn varieties containing event MON 863 are not materially different in composition, safety and other relevant parameters from corn currently on the market. Section 2.1 2  7 EPA has granted tolerance exemptions for other Cry proteins that either may be introduced into food crops through genetic engineering, or applied topically to plant surfaces as a component of Bt microbial pesticide formulations. EPA completed a comprehensive review of safety studies carried out on Bt microbial formulations that have been registered for several decades. Based on their review of the data, EPA concluded that "... toxicology studies submitted to the U. S. Environmental Protection Agency to support the registration of B thuringiensis subspecies have failed to show any significant adverse effects in body weight gain, clinical observations or upon necropsy." [ Footnote 20: McClintock, J. T., C. R. Schaffer and R. D. Sjoblad ( 1995). A comparative review of the mammalian toxicity of Bacillus thuringiensis  based pesticides. Pestic. Sci. 45: 95 105.] As stated in a World Health Organization International Program on Chemical Safety Environmental Health Criteria monograph on Bt, "... owing to their specific mode of action, Bt products are unlikely to pose any hazard to humans or other vertebrates..." and " Bt has not been documented to cause ally adverse effects on human health when present in drinking water or food." [ Footnote 21: WHO ( 2000). International Program on Chemical Safety ( IPCS) Environmental Health Criteria 217: Bacillus thuringiensis. http:// www. who. int/ pcs/ docs/ ehc 217. html]

Comment or Question: Human exposure to Cry3Bb has been limited since no foliar Cry3Bb product has achieved significant market share in any crop and breakdown in sunlight quickly removes residues before products reach customers. Furthermore, human exposure to the Cry3A protein expressed in NewLeaf potatoes was limited due to its small market share.

Scientific Background: There is a history of safe consumption for Cry3A protein and other related Cry3 class proteins, stemming from the safe use and consumption of NewLeaf potatoes. Approximately 71,000 acres of NewLeaf varieties were planted in North America while the product was on the market. Thus, there has been a history of safe consumption for the Cry3A protein from its consumption as a component of NewLeaf potatoes. The Cry3A protein is functionally and structurally related ( 85% amino acid sequence homology) to Cry3Bb protein. [ Footnote 22: Galitsky et al. ( 2001). Acta Crystallographica D57: 1101 1109.] Assuming a mean concentration of 1 ug/ gram of Cry3A protein in potato tubers, and a lack of degradation of Cry3A protein during processing of the NewLeaf potato into human food, the approximate Cry3A protein consumption is 150 ug/ day following consumption of a 150 gram potato. While this may be a conservative estimate of exposure, it is a reasonable basis for establishing a history of safe consumption for Cry3 proteins of which the Cry3Bb1 protein is a member.

Commenter Name: ICGA Commenter Organization Name: ICGA Comment Number: 30509B 093000 Excerpt Number: 7 Excerpt Text: In addition to benefits for agriculture, there are benefits for the consumer as well. A recent report from the American Phytopathological Society ( APS) outlines the benefits of Bt corn to both human health and the environment. According to Gary Munkvold, plant pathologist at Iowa State University and an APS member, the innovative Bt technology has a distinct health benefit of discouraging the buildup of mycotoxins in corn. Mycotoxin build up is directly related to certain fungal plant diseases, which can be increased by insect damage in crops. Some of these toxins, such as furnonisin, can be fatal to horses and pigs and are probable human carcinogens. Lower mycotoxin concentrations clearly represent a benefit to the public.. Section 2.1 2  8 These studies conducted by Dr Munkvold looked at Bt to control the European corn borer. However, corn rootworm puts similar stress on the corn plant, making it more susceptible to invasion of molds and mycotoxins. We expect that rootworm. Bt would also reduce the mycotoxins in corn.

EPA Response: EPA does not agree with the commenter that MON863' s corn rootworm control can be expected to reduce mycotoxins. While there is good evidence that the PIPs in corn that control the European corn borer have been responsible for reducing fumonisin levels in corn, similar reductions of other mycotoxins such as aflatoxin in corn have not been realized. It could reasonably be expected that if insect damage was the only means of entry for these fungi, similar reductions in all mycotoxins could be seen not only in corn but also cotton. It is always hoped that reduced colonization of mycotoxin producing fungi will result with corn rootworm control but no claims for such an effect have been made by the company nor has data been presented to indicate mycotoxin reductions. EPA expects that this will be an area of active research once commercial release and cultivation has occurred. However, the reduction of mycotoxin levels has not been a consideration for the safety review of MON863 corn.

Commenter Name: Dennis Ward Commenter Organization Name: Monsanto Comment Number: 30509B 106000 Excerpt Number: 8 Excerpt Text: Comment or Question: Simulated gastric digestion and sequence comparison studies to determine the potential allergenicity or toxicity of Cry3Bb1 should be repeated using currently recommended protocols.

Scientific Background: Monsanto has utilized current, appropriate and scientifically robust protocols for the simulated gastric digestion and sequence matching studies.

As a matter of validation, the vast majority of pepsin digestion assays ( i. e., the current database of information) have been performed at pH 1.2 as outlined in the U. S. Pharmacopeia. [ Footnote 10: The United States Pharmacopeia 23, The National Formulary 18, Rockville, MD: United States Pharmacopial Convention Inc, 1995: 2053.] It was deemed prudent to perform this assay at pH 1.2 to facilitate a direct comparison to data generated under the exact same conditions for previously tested allergens and nonallergens. [ Footnote 11: Astwood, J. D., Leach, J and Fuchs, R. L. 1996. Stability of food allergens to digestion in vitro. Nature Biotechnology 14: 1269 73] [ Footnote 12: de Val, G., Boihom, C. Y., Lozano, R. M., Buchanan, B. B., Ermel, R. W., Lee, Y M., and Frick, O. L. 1999. Thioredoxon treatment increases digestibility and lowers allerenicity of milk. J. of Allergy and Clinical Immunology 103: 690 7]

Regarding sequence matching tests, an evaluation of a protein for clinical cross reactivity is best accomplished using a FASTA [ Footnote 13: Algorithm used to find local high scoring alignments between a pair of protein or nucleotide sequences.] sequence comparison that has been shown to be the most predictive of a potential allergen. [ Footnote 14: Hileman, R., Silvanovich, A., Hoodman, R., Rice, R., Holleschak, G., Astwood, J., and Hefle, S. 2002. Bioinformatic methods for allergenicity assessment using a comprehensive allergen database. International Archives of Allergy and Immunolology ( In Press/ August)] An additional search for matches of eight amino acids provides a margin of safety when assessing the potential allergenicity of a protein, but a six amino acid search window produces many Section 2.1 2  9 random, irrelevant matches.

Commenter Name: N/ A Commenter Organization Name: Center for Science in the Public Interest Comment Number: OPP02 0028 Excerpt Number: 3 Excerpt Text: 1. Monsanto has not evaluated potential allergenicity or toxicity of Cry3Bb1 with the most appropriate methods for determining digestive stability. The protocols used to determine in vitro gastric stability of CryBb1 were not the protocols international experts agree are most useful. Monsanto should repeat gastric stability tests at pH 2.0, and follow other guidelines elaborated by Food and Agriculture Organization/ World Health Organization ( FAO/ WHO) of the United Nations conducting those tests ( 1).

Commenter Name: N/ A Commenter Organization Name: Center for Science in the Public Interest Comment Number: OPP02 0028 Excerpt Number: 14 Excerpt Text: 1. Human Dietary Safety Several safety tests performed by Monsanto did not use protocols that could best assure detection of potential allergenicity or toxicity. In particular, Monsanto incorrectly performed in vitro simulated gastric digestion tests ( hereafter " gastric stability") and sequence comparisons of Cry3Bb1 with allergens and toxins. EPA should require Monsanto to repeat the in vitro digestibility. and sequence matching tests using currently recommended protocols.

A. In Vitro Digestibility Studies Simulated gastric stability is important because stability is a characteristic common to many food allergens and anti nutrients, especially the most prevalent and serious food allergens. By contrast, lack of stability means that intact protein is not exposed to the intestines where it can be absorbed and act systemically. Therefore, gastric stability helps predict the likelihood of allergenicity or toxicity. While Monsanto's tests show that the bacterially produced MON 863 does not possess gastric stability, those tests were not performed with the most reliable protocols. Monsanto notes that its gastric stability study was performed using the protocols described by Astwood et al. in 1996 ( 2). In 2001, however, the FAO/ WHO convened an international conference of experts to reconsider genetically engineered food allergy assessment, including gastric stability assays ( 1). That conference recommended that pH 2.0 be used rather than pH 1.2 used in the Astwood et al. protocols ( 2), since the former is closer to the pH of the stomach when it contains food, while the latter is more indicative of " fasting" pH. That difference in pH can be critical in determining gastric stability. Studies with previous Cry proteins have found different stabilities associated with differences in pH ( 3,4,5).

While several aspects of the FAO/ WHO gastric stability protocols have not been validated against known food allergens or non allergens, the rationale for the pH change is sound. Therefore, EPA should require Section 2.1 2  10 Monsanto to repeat the gastric stability assay using pH 2.0 and should also consider requiring the use of other aspects of the updated FAO/ WHO protocols.

EPA Response: EPA disagrees with CSPI that the company failed to utilize the appropriate protocols that would best assure detection of potential allergencity or toxicity. The basis of the toxicity assessment are the acute oral toxicity test in a rodent species done with a high dose of purified protein and a full length amino acid homology comparison of the introduced proteins with known protein toxins. For the allergenicity assessment, a number of biochemical tests are utilized: two amino acid homology comparisons for the entire sequence and also a stepwise series of 8 amino acid segments, a heat stability test, a test for protein stability to digestive enzymes and an examination of the plant expressed protein for potential posttranslational modifications. These results are discussed in sections IIA and IIB of the BRAD and EPA still believes these data demonstrate a reasonable certainty that the aggregate exposure of the PIP proteins will cause no harm.

The specific comment by CSPI suggests that the FAO/ WHO conference held in Rome, Italy February 2001 is the definitive guidance on the method for assessing allergenicity of a novel protein. In general, CODEX is considered the body that sets international safety standards and a subsequent meeting of an Ad hoc CODEX meeting of food allergy experts held in Vancouver, Canada, September 2001 considered the FAO/ WHO recommendations. The outcome of that CODEX meeting was that, while the FAO/ WHO recommendations should be considered in a safety assessment, there were still questions relating to what appropriate and valid methods were to be used for both amino acid sequence homology and pepsin resistance ( in vitro digestibility). The ambiguity of the CODEX guidance and the demonstrated false positive correlations of corn proteins with allergens arising with the use of shorter than eight amino acid segments ( Hileman et al., 2002) suggest that it is still reasonable to utilize the 8 amino acid segment stepwise analysis until further refinement of the homology assessment with shorter segments is obtained.

The pH of the pepsin resistance ( in vitro digestibility) assay is another question raised by CSPI. The commenter suggests that the pH utilized in the assay should be 2.0 rather than 1.2 to better mimic the pH of the stomach after ingestion of a meal. EPA has stressed that the pepsin resistance assay is a test for a biochemical characteristic, not intended to mimic a physiological process like gastric digestion or imply anything about toxicity. While further information from the scientific community are expected to clarify the range of conditions affecting the assay such as pH, enzyme and substrate concentrations, EPA believes the current information is still useful for making a safety determination. Section 2.2 2  11 2.2  Molecular Biology/ Product Characterization 2.2  Comment Excerpts Commenter Name: Angelika Hilbeck Commenter Organization Name: Greenpeace Comment Number: 30509 113000 Excerpt Number: 1 Excerpt Text:  Molecular description of expressed Bt toxin and its mode of action Although not explicitly stated in the document, probably the 74 kDa size Cry3B protein is expressed in the transgenic plants. This is the natural size of the protein in which it is active. Hence, unlike for Cry1, no inactive large protoxin forms exist and, therefore, microbial derived proteins are at least in a comparable biochemical state as plant produced proteins.

EPA Response: EPA agrees with this comment that the Cry3Bb1 protein is expressed as a 74kD toxin and that unlike the Cry1 family, activity is not dependent on removal of a large portion of a protoxin molecule.

Commenter Name: Charles Benbrook Commenter Organization Name: Union of Concerned Scientists Comment Number: 30509 115000 Excerpt Number: 8 Excerpt Text: The MON 863 event was created using gene gun technology with a 4,691 base pair ZMIR13Lconstruct. This construct incorporates the NPTII neomycin phosphotransferase II marker gene directly adjacent to the cauliflower mosaic virus 35S promoter. Many questions have been raised about the wisdom of using antibiotic resistance marker genes in the development of GMO crop cultivars. There is a global consensus that use of such selectable marker genes may be risky, are no longer necessary, and hence should be phased out.

Commenter Name: Charles Benbrook Commenter Organization Name: Union of Concerned Scientists Comment Number: 30509 115000 Excerpt Number: 9 Excerpt Text: While concerns have been raised before regarding human dietary exposure to NPTII antibiotic marker gene proteins in GMO foods, data in the public arena suggest that the levels of NPTII expressed in edible portions of previously approved Bt corn plants are very low. The Monsanto submission reports higher expression levels of NPTII proteins than earlier Bt corn events.

EPA Response: EPA disagrees with the UCS commenter who believes there is an unreasonable risk associated with the use of the antibiotic resistance gene neomycin phosphotransfersase II ( NPTII) as a selectable marker, especially in terms of horizontal gene transfer. EPA first assessed the safety of the NPTII protein as a selectable Section 2.2 2  12 marker and published a tolerance exemption in 1994 for this protein ( 40 CFR 180.1134). The basis for the tolerance determination was the lack of oral toxicity and absence of similarity to known allergens. EPA holds that the same information on dietary safety still suffices to support the tolerance exemption for NPTII. Further, EPA is not currently aware of a demonstrated " global consensus" on antibiotic selectable marker genes, especially nptII.

Commenter Name: Charles Benbrook Commenter Organization Name: Union of Concerned Scientists Comment Number: 30509 115000 Excerpt Number: 10 Excerpt Text: 4. Missing DNA Warrants Further Study In the study entitled " Molecular Analysis of Corn Event MON 863" ( MRID # 451568 01), Cavato et al. describe the techniques used to confirm that just one copy of the transgene cassette was expressed and that expression is stable in subsequent generations of plants bred from the MON 863 line.

The ZMIR13L vector used in engineering MON 863 corn contains two major sections. The first contains the Cauliflower mosaic 35S promoter and the NPTII selectable marker gene, flanked by the NOS 3' regulatory nopaline synthase gene of Agrobacterium tumefaciens T DNA. The purpose of this regulatory gene is to end transcription and direct polyadenylation. Molecular analysis of the MON 863 event suggests that just one copy of this sequence has been expressed and that expression is stable.

The second section within the ZMIR13L plasmid contains the Cry3Bb1.11098 gene. It is flanked to the left by

 An activating sequence ( 4 AS1);  " wt CAB," a 5' untranslated leader of the wheat chlorophyll a/ b protein; and  An intron from the rice actin gene.

To the right, the Cry3Bb1.11098 gene is flanked by " tahsp17," which is described in the submission as a " 3' nontranslated region on the coding sequence for wheat heat shock protein 17.3 which ends transcription and directs polyadenylation." This second regulatory sequence is coupled to a HindIII restriction endonuclease " that cuts DNA at specific locations," as well as a MluI restriction endonuclease, also incorporated to assist in the proper placement and truncation of the transgene within the corn genome.

The Cavato et al. report states that

" Genomic flanking sequence indicates that while the HindIII site at the 3' end of PV ZMIR13L is missing, the entire tahsp 17 3' polyadenylation sequence is present in event MON 863."

The " missing" DNA is approximately 10 base pairs and includes the HindIII site and MluI half site. The authors stress that despite the missing DNA, the Cry3Bb gene and its regulatory sequence are properly and stably expressed in the MON 863 event. The authors do not explain, however, what may have happened to the 10 base pair segment of DNA containing the two restriction endonucleases. Presumably, Monsanto explored whether this missing DNA lodged somewhere else in the transformed corn lines. We would expect Section 2.2 2  13 that the company has developed probes capable of detecting these restriction endonucleases. The lack of further explanation of what they found in their search for the missing DNA raises important questions.

Possible implications have been suggested in research by Nielsen and colleagues. Their work focused on the impacts of transgenic sugar beets on bacterial gene flow in the soil. The transgene used to develop the modified sugar beets included several of the same promoter and marker genes as the Cry 3Bb transgene. They found in their work that the " presence in transgenic plants of various prokaryotic markers and vector sequences may facilitate additive insertion of foreign genetic material into bacterial hosts after homologybased heteroduplex formation" ( Nielsen et al., 2000).

The restriction enzymes missing in Cry 3Bb transformed corn lines could trigger unexpected and possibly detrimental pleiotrophic effects, depending on whether and where they are lodged in the genome of various corn hybrids including the MON 863 transgene. Monsanto must develop a mechanism to reliably determine the fate of this missing DNA and make it widely and freely available. Lacking such a probe and ongoing scrutiny of all the hybrids into which the MON 863 event is placed, the U. S. corn industry will be vulnerable to a loss in consumer confidence and export sales if, a few years down the road, it is discovered that this missing DNA had, in certain varieties under certain circumstances, been expressed in a way leading to detectable changes in corn grain or forage composition or product performance.

EPA Response: EPA disagrees with the UCS commenter that more effort should be made to account for the loss of the 10 base pair section of DNA not found in the MON863 corn integration. The integrated genes function as expected. It is highly unlikely that the 10 base pair DNA section, which basically codes for restriction enzyme recognition sites, would insert elsewhere, would be detectable if inserted elsewhere or would have a significant effect that would not be discovered by a comparison of the agronomic performance or nutritional characteristics of the MON863 corn plant compared to traditional corn cultivars. As none of these features have been altered except for the intentionally introduced traits, further consideration of the 10 base pair of lost DNA is not necessary.

Commenter Name: Charles Benbrook Commenter Organization Name: Union of Concerned Scientists Comment Number: 30509 115000 Excerpt Number: 14 Excerpt Text: In addition, we fully expect that Monsanto also intends to incorporate the Cry 3Bb gene in corn varieties engineered to be Roundup Ready via modification of the EPSPS gene, a critical regulatory protein within the shikimate pathway of all plants. The aromatic amino acids are synthesized within this pathway and play multiple, critical roles in plant growth and development and plant defense responses to pest attack and abiotic stress.

Commenter Name: Charles Benbrook Commenter Organization Name: Union of Concerned Scientists Comment Number: 30509 115000 Excerpt Number: 15 Excerpt Text: Section 2.2 2  14 Recent evidence has emerged that in some Roundup Ready plants, changes in the levels of expression of phenylalanine ( Padgett et al., 1995), or other aromatic amino acids or their secondary products ( Sidhu et al., 2000), may be impairing plant defense mechanisms and retarding or weakening the systemic acquired resistance ( SAR) response of soybean and corn plants ( see Benbrook, 2001).

In corn varieties with stacked Bt and Roundup Ready genes, it is probable that different combinations of pest and abiotic stresses will trigger a wide array of unusual protein expression patterns. Some of these combinations will surely impact product performance and the efficacy of resistance management plans, while others may pose allergenicity or other food safety risks.

Commenter Name: Charles Benbrook Commenter Organization Name: Union of Concerned Scientists Comment Number: 30509 115000 Excerpt Number: 16 Excerpt Text: For this reason, EPA must make it clear in response to this and other applications that additional data must be submitted on each proposed stacked gene product sufficient to settle uncertainty regarding the unique risks posed when a given variety is genetically engineered in two or more ways, especially when the modifications target different physiological processes and pathways that are known to interact, as the case with Bt and Roundup Ready corn varieties.

EPA Response: The comments concerning potential adverse physiological effects of transformations conferring tolerance to glyphosate are outside the scope of this regulatory action. In the future, should EPA be presented with a registration application for a product expressing both pesticidal and other traits, EPA will continue to coordinate its review of the pesticidal traits of such plant with USDA APHIS' review of non pesticidal traits.

Commenter Name: Charles Benbrook Commenter Organization Name: Union of Concerned Scientists Comment Number: 30509 115000 Excerpt Number: 35 Excerpt Text: Nielsen and colleagues point out in a recent article in Applied and Environmental Microbiology that

" Of the 15 different resistance genes incorporated into plants, several encode resistance to clinically used antibiotics.... Recently, uptake of transgenic plantharbored DNA fragments by bacteria based on restoration of a partially deleted ( 10 or 317 bp internal deletion) bacterial kanamycin ( KM) resistance gene ( nptII) after recombination with transgenic plant inserted homologues was demonstrated" ( Nielsen et al., 2000).

Recent evidence documenting how freely bacteria can exchange genes has rekindled concerns over the emergence of new strains of antibiotic resistant bacteria as a result of use of antibiotic selectable marker genes. The Cry 3Bb gene cassette contains the 0.79 Kb nptII gene from a transposon isolated from E. coli. It confers resistance to kanamycin and neomycin. [ Footnote 2: Kanamycin and neomycin are antibiotics used to control disease causing bacteria ( Berkow, 1992). FDA has approved a food additive petition from Section 2.2 2  15 Calgene, Inc., ( now owned by Monsanto) for use of the nptII gene in genetically engineered crops.

EPA Response: EPA disagrees with the UCS commenter on the issue of gene transfer form genetically engineered plants to environmental bacteria. There have been a number of scientific questions raised about the use of antibiotic resistance markers in genetically engineered plants since the initial approvals which contained antibiotic marker genes. Little of the scientific data generated on this topic demonstrates a risk scenario that would translate into a detectable adverse effect in the environment. The major concern has been the possibility that the presence of the NPTII gene in genetically engineered plants may lead to an increase in the level of antibiotic resistance in bacteria, particularly clinically important bacteria. The commenter cites several scientific papers ( refs.?) that purport to demonstrate the horizontal gene transfer of the NPTII gene from genetically engineered plant DNA by transformation into a soil bacterium. What is actually demonstrated is the reversal of a deletion mutation by homologous recombination. The soil bacterium had an endogenous NPTII gene which was inactivated by a deletion mutation of 312 base pairs, a removal of some of the gene to render the gene product inactive. The researchers were able to show that high concentrations of purified DNA isolated from genetically engineered sugar beet was able through transformation to repair the existing inactivated NPTII gene and restore the NPTII kanamycin resistance phenotype in the recipient bacterium.

While this thought provoking work is able to demonstrate the previously theoretical possibility of horizontal gene transfer from plant DNA to a bacterium, the results do not change EPA's safety findings. The experiments were done in a sterile soil system, with high concentrations of purified plant DNA unlikely to be found in nature, with a naturally transformation competent bacterium that had a pre existing NPTII gene. These conditions describe several very low probability contingencies that we expect would probably have to coincide for this situation to occur in nature. Horizontal gene transfer is theoretically possible and has been shown to occur in these highly manipulated systems but given the low levels detected in these experimental systems it remains extremely unlikely such transfer would occur in nature and that such a transfer would result in any impact on relatively high background kanamycin/ neomycin antibiotic resistance in the bacterial microflora.

Commenter Name: Charles Benbrook Commenter Organization Name: Union of Concerned Scientists Comment Number: 30509 115000 Excerpt Number: 36 Excerpt Text: When the first Bt transgenic plant applications were reviewed, there was little evidence that antibiotic marker genes might trigger resistance problems. Now the transfer of antibiotic marker genes in plant cells to soil bacteria has been demonstrated ( Gerbhard and Smalla, 1998). In a key article in the journal Applied and Environmental Microbiology, Gebhard and Smalla hypothesize that:

"  the introduction of bacterial genes into the plant genome leads to a higher probability of gene transfer from plants to bacteria due to the presence of homologous sequences. However, until now, there has been a lack of clear experimental evidence that successful gene transfer from plants to bacteria can occur at all." ( Gebhard and Smalla, 1998).

Commenter Name: Charles Benbrook Section 2.2 2  16 Commenter Organization Name: Union of Concerned Scientists Comment Number: 30509 115000 Excerpt Number: 38 Excerpt Text: Also contrary to conventional wisdom, genes that confer antibiotic resistance have been shown to move readily between people and from bacteria in the gut of farm animals to people ( Tschape, 1994). A team in Denmark has shown that indigenous soil bacteria can serve as a sink for plasmid borne antibiotic resistance traits from E. coli entering agricultural soils from animal manure or other wastes ( Sorensen et al., 1999).

Until recently it was also thought that foreign DNA, such as the gene constructs in Cry 3Bb corn, would pass through the mammalian digestive without being activated and without consequence. The transfer of foreign DNA in food into the blood and organ systems in mice has now been demonstrated ( Schubbert et al., 1997). In addition some of the transgenic DNA was found to covalently bind to mouse DNA, in effect becoming a part of the mouse genome. The authors modestly summed up this article by stating  " The medical and evolutionary implications of these observations may be considerable" ( Schubbert et al., 1997).

EPA Response: EPA disagrees with the assertion that mammals effectively take up and incorporate DNA from the digestive system. While recent experimental evidence has indicated that certain DNA vaccine therapies may have promise, none have been developed that could encompass the scenario provided by consuming genetically engineered foods. Moreover, there is no indication that consumption of traditional foods has resulted in transfer and incorporation of any traits from food plants. The scientific article cited by the commenter showed that there was a transient detection of the M13 phage DNA in mouse organs and blood when introduced by the oral route. However, it is unclear that the claimed incorporation of the M13 Phage DNA into mouse organs and tissues is a real phenomenon let alone a stable incorporation of foreign DNA. These results are controversial and have not been repeated or confirmed by another laboratory. It is unlikely that the amount of DNA typically associated with food consumption represents a DNA exposure similar to the very high concentrations described and employed in the articles.

Commenter Name: Dennis Ward Commenter Organization Name: Monsanto Comment Number: 30509B 086000 Excerpt Number: 9 Excerpt Text: Comment or Question: In the safety assessment, it is inappropriate to compare the Cry3Bb1 protein produced in corn event MON 863 to the Cry3Bb protein present in the commercial microbial Bt pesticide trademarked Raven. This product contains a longer Cry3Bb protein than produced in MON 863.

Scientific Background: The Cry3Bb1 protein that is produced in corn plants is structurally identical to that produced in Raven ( differing by only seven amino acids). Therefore, the comparison is relevant.

Comment or Question: Molecular analysis of MON 863 reveals a " missing" 10 base pair sequence that could have unpredictable effects. Furthermore, there is a lack of publicly available data and methods to determine whether the " missing" DNA may have inserted into another location of the genome. Section 2.2 2  17 Scientific Background: A 10 base pair DNA sequence present within the transformation cassette was lost upon stable integration of the cassette into the plant genome. Southern blot analyses using probe combinations spanning the deleted 10 base pair region did not detect any integration of the 10 base pair segment elsewhere in the genome. The 10 base pair DNA segment most likely was deleted during transformation and degraded by intracellular nucleases. Such deletions commonly occur as a consequence of DNA repair that takes place at the integration site, as well as in regions of natural strand breakage and repair.

Commenter Name: Dennis Ward Commenter Organization Name: Monsanto Comment Number: 30509B 086000 Excerpt Number: 19 Excerpt Text: Comment or Question: Adaptation of CRW populations to the Cry3Bb1 protein expressed in MON 863 could limit the longevity of future biopesticide formulations of Cry3Bb, other Cry3Bb events or Bt events with a different mode of action.

Scientific Background: The Cry3Bb protein has never been used in any formulated Bt microbial based product to control CRW larvae for two important reasons: delivery and timing. CRW larvae feed on corn roots over a largely unpredictable period of three to eight weeks, and the Bt insecticidal protein must be ingested to ellicit a pesticidal physiological response. Thus, in order for CRW larvae to come into contact with a microbial Bt formulation, it would need to be applied so as to constantly cover the root tissue and remain active for a prolonged period of time. Neither of these issues can be addressed with current Bt microbial formulation technology. Thus, it is reasonable to assume that use of MON 863 will not pose an unreasonable risk to a future, hypothetical Bt microbial based product for the control of CRW. Studies also indicate that there is little cross resistance between Bt plant and foliar sprays with similar proteins because of the presence of other critical ingredients, such as spores, in the spray formulations.

Corn event MON 863 produces the Cry3Bbl protein at levels sufficient to minimize CRW larval root damage. Regulatory approval and commercial use of this technology will require the deployment of a scientifically sound and feasible IRM plan that suppresses the emergence of insect resistance in targeted pest populations. In the event that future Cry3Bbl events are introduced, EPA will require and determine the acceptability of appropriate IRM plans for all products.

Event MON 863 represents a negligible risk to other Bt events that work via a different mode of action. It is well understood that Bt proteins having similar modes of action are at a greater risk of exhibiting crossresistance Conversely, Bt proteins that differ in their mode of action represent a minimal risk of crossresistance It is for this reason that EPA and the scientific community recommend that developers of Bt crops develop and express Bt proteins with differing modes of action as a means to further reduce the likelihood of resistance development in pest populations.

The generally recognized mode of action of Bt proteins has several distinct steps after ingestion by the insect. These steps include: solubilization, proteolytic stability, binding to the insect midgut epithelium, formation of ion channels in the insect midgut cells, and finally analysis of these cells. Each of these steps is governed by the sequence dependent structure of the protein and the insect specific midgut environment. Section 2.2 2  18 Thus, the more two proteins differ structurally, the more likely they are to differ in their mode of action and would exhibit differing mechanisms of resistance. The sequence dissimilarity between two Bt proteins is an empirical measure of their likelihood to act by different modes of action. Recently Cry34Abl and Cry35Ab1 have been listed as new classes of novel Cry proteins, which require them to share less than 45% amino acid identity with any other known Cry protein ( e. g., Cry3Bbl). The Cry34 and Cry35 proteins expressed in CRW protected events under development by Dow AgroSciences and Pioneer Hy Bred International function as a binary protein active against CRW larvae. These proteins not only are very different from Cry3Bbl in amino acid sequence, but both proteins are absolutely required for CRW activity. Due to these differences in protein sequence and mode of action, it is unlikely that Cry3Bb1 and the new Cry34/ Cry35 binary protein have similar modes of action.

Commenter Name: Dennis Ward Commenter Organization Name: Monsanto Comment Number: 30509B 086000 Excerpt Number: 20 Excerpt Text: Comment or Question: The presence of the nptII antibiotic resistance marker ( ARM) gene in MON 863 may pose a risk to human and animal health through horizontal gene transfer ( HGT) from genetically modified MON 863 corn to bacteria either in the soil environment, or in the mammalian digestive tract. The potential transfer of these genes to bacteria has been suggested as an important mechanism for the spread of antibiotic resistance in bacteria, which could impede medical treatment of humans or animals, since the ARM used in the transgenic CRW plant encodes resistance to antibiotics ( kanamycin/ neomycin) which are still in use in some clinical applications.

Scientific Background: Researchers have never detected an HGT event with the transfer of an intact gene under field conditions. Under laboratory conditions, the frequency of HGT with segments of DNA serving as model plant genes is extremely low. More importantly, the genes of interest in MON 863 corn ( Cry3Bbl and npt11) possess no harmful characteristics to the environment compared to the background of genes and gene products already found in soil or water borne microorganisms. Finally, the Cry3Bb1 and nptII genes, and their homologues, are naturally present in soil dwelling microbes. For example, manure has been shown to contain high numbers of bacteria harboring the nptII gene. [ Footnote 12: Nielsen, K. M., A. M. Bones, K. Smalla and J. D. can Elsas ( 1998). Horizontal gene transfer from transgenic plants to terrestrial bacteria  a rare event? FEMS Microbiol Reviews 22: 79 103.] Since manure is frequently used as a nitrogen source in agricultural fields, the potential addition of ARMs to soil microbes via an unlikely, low frequency HGT event is trivial compared to the population of antibiotic resistant microbes already present in soil environments.

Horizontal gene transfer between bacteria has occurred widely, primarily through the transfer of plasmids between members of various species, and has led to the rapid spread of antibiotic resistance genes in clinical environments. [ Footnote 13: Davidson, J. ( 1999). Genetic exchange between bacteria in the environment. Plasmid 42: 73 91.] Bacteria that are already resistant to both kanamycin and neornycin are quite common in the environment and there is no data to support the suggestion that HGT could increase the risk of resistance in humans or livestock. Furthermore, the clinical use of kanamycin and neomycin is limited by human toxicity and the availability of less toxic, more effective antibiotics. Substantial research efforts have tried but failed to demonstrate that ARM genes may be transferred from plants to bacteria. Section 2.2 2  19 Commenter Name: Dennis Ward Commenter Organization Name: Monsanto Comment Number: 30509B 086000 Excerpt Number: 21 Excerpt Text: Bacteria from soils at field sites have not been able to accept and incorporate transgenic plant DNA in controlled experiments. [ Footnote 14: Gebhard, F. and K. Smalla ( 1998). Transformation of Acinetobacter sp. Strain BD413 by transgenic sugar beet DNA. Appl. Environ. 64: 1550 1554.] [ Footnote 15: Paget, E, M. Lebrun, G. Freyssinet and P. Simonet ( 1998). The fate of recombinant plant DNA in soil. Eur. J. Soil Biol. 34: 81 844.] Moreover, attempts to demonstrate in vitro transformation. of naturally competent bacteria to antibiotic resistant bacteria using genomic DNA from transgenic potato or sugar beet plants carrying an npt1I transgene were unsuccessful, [ Footnote 16: Nielsen, K. M., F. Gebhard, K. Smalla, A. M. Bones and J. D. van Elsas ( 1997). Evaluation of possible horizontal gene transfer from transgenic plants to the soil bacterium Acinetobacter calcoaceticus BD413. Theor. Appl. Genet. 95: 815 821.] as were attempts to transform the bacterial pathogen Erwinia chrysanthemi with DNA from transgenic potatoes carrying an ampicillin resistance gene. [ Footnote 17: Schluter, K., J. Futterer and I. Potrykus ( 1995). Horizontal gene transfer from a transgenic potato line to a bacterial pathogen ( Erwinia chrysanthemi) occurs if at all at an extremely low frequency. Biotechnology 13: 1094 1098.] The highest potential transformation frequencies in these experiments were in the range of 10 13, 10 17 per recipient bacteria.

In contrast, in vitro HGT of plant transgene sequences to a model soil bacterium has been demonstrated under laboratory conditions when sequence homology to the plant transgene was purposefully engineered into the recipient bacterium. These studies utilized the naturally transformable soil bacterium Acinetobacter strain BD413 that carried a plasmid carrying a deletion derivative of the nptII gene with either a 10 bp or 317 bp internal deletion as the recipient which could be rescued after transformation with purified leaf DNA from transgenic plants carrying an nptII transgene [ canola, potato, tobacco, tomato and sugar beet] [ Footnote 18: de Vries, J. and W. Wackernagel ( 1998). Detection of nptII ( kanamycin resistance) genes in genomes of transgenic plants by marker rescue transformation. Mol. Gen. Genet. 257: 606 613.] Similar rescue experiments were also performed in sterile soil microcosms using a plasmidborne 317 nptII deletion and purified transgenic sugar beet DNA as gene donors. [ Footnote 19: Nielsen, K. M., J. D. van Elsas and K. Smalla ( 2000). Transformation of Acinetobacter sp. Strain BD413( pFG4? npII) with transgenic plant DNA in soil microcosm and effects of kanamycin on selection of transformants. Appl. Environ. Microbiol. 66: 1237 1242.] Transformation frequencies of 10 9 to 10 10 were obtained in these experiments. The use of nonsterile soil decreased the transformation frequency to undetectable limits. Additionally, the plasmid borne kanamycin resistance phenotype was unstable when incubated in the microcosm for days without selection. Consequently, while HGT has been demonstrated at very low levels in laboratory model systems with engineered bacteria, the HGT event was not detectable in a nonsterile soil microcosm and unstable under sterile laboratory conditions without artificial selection.

Commenter Name: N/ A Commenter Organization Name: Center for Science in the Public Interest Comment Number: OPP02 0028 Excerpt Number: 4 Excerpt Text: 2. Monsanto did not adequately compare the amino acid sequence of Cry3Bb1 with known allergen protein Section 2.2 2  20 sequences. Current protocols recommend testing for homology of six identical contiguous amino acids as a possible indicator of allergenicity. Monsanto should repeat their analysis since international experts ( 1) now agree that Monsanto's use of eight contiguous amino acid homology may miss shorter matches that indicate allergenicity.

Commenter Name: N/ A Commenter Organization Name: Center for Science in the Public Interest Comment Number: OPP02 0028 Excerpt Number: 15 Excerpt Text: B. Protein Sequence Comparisons Between MON 863 and Known Allergens Monsanto searched protein sequence databases for matches of eight or more contiguous amino acids to indicate possible allergenicity, while the recent FAO/ WHO protocols ( 1) call for determining sequence matches of six contiguous amino acids. Thus, Monsanto's test might not detect similarity between MON 863 and a known allergen.

Monsanto argues against using less than eight contiguous amino acids to determine potential allergenicity. Monsanto acknowledges that cross reactive allergy epitopes as short as five amino acids have been found in other proteins, but that they do not bind antibody with affinity as high as the original allergen. Thus Monsanto suggests that antibody affinity is an adequate measure of allergenicity, and that even if Cry3Bb I had six consecutive amino acids in common with an allergen epitope, that would not be enough to cause an allergic reaction.

However, Monsanto does not give adequate support to the use of affinity as an accurate predictor of clinical symptoms. In fact, in some cases of oral allergy syndrome where there is evidence that the cross reacting pollen allergen is the sensitizing agent, the homologous cross reacting food allergen can cause clinically significant symptoms, despite having no more than six identical or similar contiguous amino acids in common with the pollen ( 6,7). In addition, only a relatively small number of cross reacting epitopes have been mapped, so it is premature to dismiss conclusions about the clinical importance of matches at a six amino acid level based on Monsanto's limited examples.

Monsanto mentions concern that matches of six amino acids may occur randomly at relatively high rate. It is possible that a six ( or eight) amino acid match could occur with a known allergen that does not correspond to an IgE epitope, and therefore is not directly involved in the allergic response. In addition, because relatively few allergen epitopes have been determined, sequence data may not be sufficient to determine the allergenic importance of such a match. The FAO/ WHO protocol ( 1) recognizes that possibility, but also recognizes that it can be addressed with an assay using pooled serum from individuals known to be allergic to the sequence matched allergen. EPA should require Monsanto to repeat the search for protein sequence matching at a six amino acid level, which can be done easily since the databases are already available.

EPA Response: EPA disagrees with the CPSI commenter about the lack of validity for the amino acid comparison of the Section 2.2 2  21 PIP expressed proteins. For the allergenicity assessment, two amino acid homology comparisons for the entire sequence and a stepwise series of 8 amino acid segment comparisons are performed by computer analysis. These results are discussed in sections IIA and IIB of the BRAD and EPA still believes these data demonstrate a reasonable certainty that the aggregate exposure of the PIP proteins will cause no harm including potential allergy concerns.

The specific comment by CSPI suggests that the FAO/ WHO conference held in Rome, Italy February 2001 is the definitive guidance on the method for assessing allergenicity of a novel protein. In general, CODEX is considered the body that sets international safety standards and a subsequent meeting of an Ad hoc CODEX meeting of food allergy experts held in Vancouver, Canada, September 2001 considered the FAO/ WHO recommendations. The outcome of that CODEX meeting was that, while the FAO/ WHO recommendations should be considered in a safety assessment, there were still questions relating to what appropriate and valid methods were to be used for amino acid sequence homology assessments. The ambiguity of the CODEX guidance and the demonstrated false positive correlations of corn proteins with allergens arising with the use of shorter than eight amino acid segments ( Hileman et al., 2002) suggest that it is still reasonable to utilize the 8 amino acid segment stepwise analysis until further refinement of the homology assessment with shorter segments is obtained. Section 2.3 2  22 2.3  Data Needs and Methods 2.3  Comment Excerpts Commenter Name: Angelika Hilbeck Commenter Organization Name: Greenpeace Comment Number: 30509 113000 Excerpt Number: 12 Excerpt Text: ' The conclusion of reasonable certainty of no harm and the resultant tolerance expemptions for this wide array of B. t. mixtures and Cry proteins in food or feed were based on the lack of adverse effects to mammals in numerous toxicological studies. This conclusion is supported by a history of safe use in agriculture for over 40 years ..' ( Monsanto Document, page 21 of 56)

Commenter Name: Angelika Hilbeck Commenter Organization Name: Greenpeace Comment Number: 30509 113000 Excerpt Number: 13 Excerpt Text: It must be understood that much of the ' history of safe use in agriculture for over 40 years..' is not based on data but on lack of data. But lack of data does not mean lack of evidence. It must further be understood that the ' safe use' refers to the use of microbial Bt pesticides that were not used widely in industrial agriculture and differ in a number of important aspects from current transgenic Bt plants: significantly extended temporal exposure, degree of exposure ( concentration, types of proteins in all types of foods), biochemical state of Bt protein. etc. As new and different products come on the market and enter the human food chain also interaction effects of combined consumption of several different Bt toxins must be included in human safety tests.

' Extensive review of these studies and a determination that proteins act almost exclusively through acute mechanisms, resulted in the focusing of testing requirements for microbial derived products to include acute oral, pulmonary and intravenous toxicity studies...' ( Monsanto Document, pages 21 22 of 56)

However, although based primarily on previous studies conducted with various, very different products in the past, nowhere in the Monsanto Document those studies are mentioned that did find evidence for potential human health effects. For example, a comparatively recent EPA funded study published in Environmental Health Perspectives in 1999 on ' Immune responses in farm workers after exposure to Bacillus thuringiensis pesticides' reports on evidence for potential allergic reactions to one of the microbial Bt pesticides ( Bernstein et al. 1999). Two of 123 farm workers showed a positive skin prick test to the Bt spore extract containing the Bt protein. Although the authors do conclude that because of this low number of workers sensitized through respiratory exposure it is not likely that consumers would develop allergic reactions after oral exposure. But it does demonstrate that new and modern testing procedures are available for human safety studies and that occasional allergic reactions cannot be ruled out once and for all with every Bt containing product. Certainly such recent studies should be cited and discussed in an application package deriving much of the safety conclusion from the published literature instead of conducted own experiments and data. Section 2.3 2  23 We feel that the approach used in the Monsanto Document to arrive at human safety conclusions is unsatisfying for a comprehensive human health assessment. In particular by learning from the StarLink contamination story, it is clear that these products will reach virtually everybody of the society in some form or another in many products for a long time to come. In our opinion, this would merit an extensive and transparent peer review by competent independent scientists of all human safety studies conducted in the past with Bt products, microbial and transgenic, and establish to what degree which of those can be bridged for assessment of current transgenic plants and which cannot be used as evidence and where new human safety studies must be initiated.

EPA Response: First, EPA must point out that the Agency has not based its regulatory determination on this PIP on claims of years of experience with microbial biopesticides utilizing Bacillus thuringiensis. Second, EPA disagrees that the dietary exposure for microbial products is practically nil and that the scientific literature indicates that Cry Proteins have induced positive immune responses. The data that supports the safety determination is specific to the PIP proteins expressed in MON 863 corn. These are toxicity tests done with high doses of purified protein as well as biochemical tests done to ascertain protein characteristics. These results are discussed in sections IIA and IIB of the BRAD. EPA believes these data stand alone to demonstrate a reasonable certainty that the aggregate exposure of the PIP proteins will cause no harm.

Additional data when available can add to the information on dietary safety such as suggestions that there has been a history of dietary exposure and safe use of these proteins in agriculture due to their expression in microbial biopesticides utilizing Bacillus thuringiensis. While definitely different in the nature of exposure, microbial pesticides expressing similar Cry proteins do have significant dietary exposure. There has been signifcant adoption of microbial biopesticides utilizing Bacillus thuringiensis in not only organic agriculture but also conventional vegetable production. This vegetable use would also be expected to result in potential dietary exposure due to uses which allow application up until harvest and use on crops that are harvested over a period of time such as tomatoes or cole crops. EPA agrees that there has probably been much less exposure to the Cry3 family of Bacillus thuringiensis toxins active against coleopterans than the lepidopteran active toxins.

The literature cited by the commenter ( Bernstein et al, 1999) actually gives an indication that the Cry proteins themselves are not responsible for the skin prick positive test in agricultural workers exposed to pesticides containing Bacillus thuringiensis. It is important to note that none of the workers examined in the study reported a clinical allergic reaction to the Bacillus thuringiensis products. In addition, the spore extract preparations that tested positive by skin prick were further purified for the actual crystal protein component. This crystal protein component was shown by the authors to not induce a positive skin prick response. Moreover, skin prick tests are notably inaccurate giving a positive result with an approximately 50% correlation to actual clinical hypersensitivity. Negative skin tests are more indicative of clinical outcome.

Commenter Name: Angelika Hilbeck Commenter Organization Name: Greenpeace Comment Number: 30509 113000Part2 Excerpt Number: 4 Section 2.3 2  24 Excerpt Text: Future releases of transgenic plants In the future, it is expected that transgenic plants containing genes that code for so called ' input' traits, i. e., traits that confer agronomically important characteristics such as insect resistance will be succeeded by a new generation of transgenic plants expressing so called ' output' traits, i. e., traits that confer ' quality' characters such as altered oil, starch, protein compositions of food and feed crops. Additionally, much research is currently invested into the development of transgenic plants and also transgenic animals producing various pharmaceuticals, vaccines, vitamins, enzymes for medical and/ or industrial purposes. Field releases of such transgenic plants for research purposes are being carried out for several years now. However, testing the environmental impact of, for example, vaccine producing transgenic plants or animals will be quite challenging, in particular, since these novel ' output' traits may well be expressed in addition to the ' old' ' input' traits. In order to be able to address these increasingly complex issues in a responsible manner, it is crucial that effective pre release safety testing, hazard identification procedures, risk assessment and management strategies, and sensitive and robust post release monitoring schemes are established well before commercialisation of such potent novel plants.

EPA Response: EPA agrees with the Greenpeace commenter that all genetically engineered plants should be subject to a thorough review for environmental impact as required by federal law. In the future, as has been done for all genetically engineered plants products to date, EPA looks forward to coordinating its environmental impact assessment of PIP expressing plants, including those with output traits, with USDA APHIS.

Commenter Name: Charles Benbrook Commenter Organization Name: Union of Concerned Scientists Comment Number: 30509 115000 Excerpt Number: 32 Excerpt Text: 3. Data Needed to Assess Human Health Risks Are Inadequate Bt foliar insecticides have been used safely for more than two decades. Bt's safety record played a major role in convincing the EPA that only a cursory assessment of human and mammalian health risks from dietary ingestion of truncated Bt endotoxins in plant tissues was required prior to approval of today's Cry1 and Cry2 Bt. k transgenic plant varieties.

Monsanto's current application for Cry3Bb transgenic corn rests heavily upon traditional toxicity studies that were originally submitted to the agency by Ecogen to support registration of Raven, a foliar Bt product containing Cry3Bb1, the endotoxin from Bt tenebrionis ( Btt). Btt studies on file are routine toxicological feeding experiments and were not designed to detect mammalian health hazards. In addition, the Ecogen studies were carried out with Btt derived from bacteria via fermentation, which produces a larger molecule and is different from the truncated form of protein expressed in Cry3Bb transgenic corn plants.

In the summary of the registration application, Monsanto states that the safety of corn containing Cry3Bb proteins to mammals rests on:

 Multiple studies showing very high NOELS (" No Observable Effect Levels") in acute feeding studies with Section 2.3 2  25 a variety of Cry proteins in a number of species.

 Similarity of Cry3Bb proteins to other Cry proteins and Bt insecticides, especially Raven, which contains a mixture of two Cry3 proteins and Cry1Ac.

 A 40 year history of safe use of Bt insecticides and Cry proteins.

There are indeed very wide margins of safety in terms of acute poisoning when mammals ingest Cry proteins. Acute toxicity should still be investigated through dermal, inhalation and dietary routes of exposure, given the high expression levels in MON 863 transformed corn. Allergenicity should also be explored much more thoroughly. The submission refers to a review of a database of known allergens, which produced no evidence of a match.

Commenter Name: Charles Benbrook Commenter Organization Name: Union of Concerned Scientists Comment Number: 30509 115000 Excerpt Number: 33 Excerpt Text: There is little known about the degree of similarity or differences between Cry3Bb proteins and other Cry proteins in terms of the health effects of greatest concern, which would include allergenicity and digestive system impacts.

Commenter Name: Charles Benbrook Commenter Organization Name: Union of Concerned Scientists Comment Number: 30509 115000 Excerpt Number: 34 Excerpt Text: The third point is also not reassuring. The American population has not been extensively exposed to any Cry proteins over 40 years. Exposure has been almost nonexistent to Cry3 proteins. No foliar Bt insecticide containing Bt tenebrionis has achieved significant market share in any crop. In addition, there is very little if any dietary exposure to residues left after foliar Bt applications. Because Bt insecticides break down quickly when exposed to sunlight ( within 48 hours in most cases), residues are long gone by the time the produce reaches consumers.

The only other possible route of exposure since 1997 would be transgenic potatoes containing Cry3A proteins ( New Leaf potatoes), but they have never accounted for more than a few percent of national potato acreage.

Commenter Name: Dennis Ward Commenter Organization Name: Monsanto Comment Number: 30509B 086000 Excerpt Number: 9 Excerpt Text: Comment or Question: In the safety assessment, it is inappropriate to compare the Cry3Bb1 protein produced in corn event MON 863 to the Cry3Bb protein present in the commercial microbial Bt pesticide Section 2.3 2  26 trademarked Raven. This product contains a longer Cry3Bb protein than produced in MON 863.

Scientific Background: The Cry3Bb1 protein that is produced in corn plants is structurally identical to that produced in Raven ( differing by only seven amino acids). Therefore, the comparison is relevant.

Comment or Question: Molecular analysis of MON 863 reveals a " missing" 10 base pair sequence that could have unpredictable effects. Furthermore, there is a lack of publicly available data and methods to determine whether the " missing" DNA may have inserted into another location of the genome.

Scientific Background: A 10 base pair DNA sequence present within the transformation cassette was lost upon stable integration of the cassette into the plant genome. Southern blot analyses using probe combinations spanning the deleted 10 base pair region did not detect any integration of the 10 base pair segment elsewhere in the genome. The 10 base pair DNA segment most likely was deleted during transformation and degraded by intracellular nucleases. Such deletions commonly occur as a consequence of DNA repair that takes place at the integration site, as well as in regions of natural strand breakage and repair.

EPA Response: EPA disagrees with the Union of Concerned Scientists commenter who claims that EPA relied on the traditional toxicity studies originally submitted by Ecogen for the Raven Microbial Product, a Bacillus thuringiensis strain that expresses the Cry3Bb1 toxin for the safety assessment of the PIP in MON863 corn. The protein that was used as test substance in the toxicity tests was the same protein, Cry3Bb1.11981 ( Q349R), as expressed in MON863. The Cry3Bb1.11981 ( Q349R) protein was produced in a Bacillus thuringiensis strain but was shown to be equivalent to the Cry3Bb1 protein in corn. This information is also presented in the MON863 BRAD in Sections II A and II B.

This MON863 corn plant expressed protein differed from the native Cry3Bb1 protein found in Bacillus thuringiensis var. kumamotoensis by seven amino acids but was not truncated. The Cry3 proteins are not like the lepidopteran active toxins which are usually expressed as an inactive protoxin that is subsequently degraded or enzymatically cleaved in the insect midgut. The Cry3 proteins are expressed in an active form. Some confusion may have arisen from the citation of Ecogen data for justifying the Experimental Use Permit for the Cry 3Bb1 expressing corn varieties. Section 2.4 2  27 2.4  Expression Levels 2.4  Comment Excerpts Commenter Name: Charles Benbrook Commenter Organization Name: Union of Concerned Scientists Comment Number: 30509 115000 Excerpt Number: 1 Excerpt Text: This application seeks registration of Cry3Bb protein for " all corn lines and varieties," including field corn, sweet corn and popcorn. The later two crops are grown for direct human consumption. Because of the much higher expression levels of this protein in edible grain tissues relative to earlier Bt corn events, the EPA must proceed more cautiously and thoroughly in its scientific assessment of the human allergenicity and food safety of this technology, given that people will be exposed to much greater quantities of Cry proteins than arising from any previously reviewed/ approved Bt corn technology.

EPA Response: EPA disagrees with the UCS commenter about the validity of the safety assessment for the PIP proteins expressed in MON863. The toxicity tests are done with purified protein at levels much higher than those expressed in the plant. EPA believes that the lack of toxicity at these exaggerated levels also covers the possibility of higher expression levels in most PIP expressing plants. See BRAD Section II. B.

Commenter Name: Charles Benbrook Commenter Organization Name: Union of Concerned Scientists Comment Number: 30509 115000 Excerpt Number: 9 Excerpt Text: While concerns have been raised before regarding human dietary exposure to NPTII antibiotic marker gene proteins in GMO foods, data in the public arena suggest that the levels of NPTII expressed in edible portions of previously approved Bt corn plants are very low. The Monsanto submission reports higher expression levels of NPTII proteins than earlier Bt corn events.

EPA Response: The levels of NPTII protein in other approved corn varieties is indeed very low as stated by the UCS commenter since none of those corn PIPs approved by EPA contain the NPTII marker gene. The level of NPTII protein reported in MON863 corn young leaves and forage was similar or lower than levels reported in other PIP products expressing NPTII. The NPTII protein level reported for corn grain, the most likely human dietary exposure, is below the limit of detection for MON 863 corn. This means that no NPTII protein could be found by the assay which detected NPTII as low as 0.76 micrograms/ gram fresh weight tissue. While EPA has made the determination that the aggregate exposure to NPTII as a marker gene is safe by issuing a tolerance exemption ( 40 CFR 180.1134), the data presented by the company indicates that probably no exposure to the NPTII will occur through consumption of MON863 corn.

Commenter Name: Rissler Section 2.4 2  28 Commenter Organization Name: UCS Comment Number: 30509B 089000 Excerpt Number: 5 Excerpt Text: A. High Expression Levels Pose Serious Risk Concerns. [ Footnote 11: See Appendix A for additional comments on expression level.]

Cry 3Bb expression levels in virtually all MON 863 corn tissues are several fold higher than the corresponding levels of other Bt endotoxins in today's corn hybrids engineered for control of the ECB. The Cry 3Bb level in leaf tissue is 8 times higher than the level of Cry 1Ab in MON 810 transformed corn and 24.5 times higher than Bt11 Cry 1Ab ( see ratios in bottom five lines in Table 2).

In pollen, the level of Cry 3Bb is a remarkable 5,391 times higher then the level of Cry 1Ac in DeKalb/ Mon transformed corn and 1,240 times the level of Cry 1Ab in the most widely planted Bt ECB corn variety, MON 810.

Table 2 reports what is known about expression levels in different tissues for Cry 3Bb transformed corn, as well as most Bt ECB hybrids. These data lead to two conclusions with important implications for risk. First, Cry 3Bb expression levels in leaf tissue, pollen, grain, and in the whole plant exceed the levels in roots during most of the growing season. Significantly, the level in grain exceeds the level in roots almost threefold at harvest time. Early in the season when levels need to be highest in roots, levels in leaf tissue are 40 percent higher than in the roots. Levels in pollen exceed root levels, as well.

The high level of Cry 3Bb protein in MON 863 corn is not the only expression related issue that warrants careful review by EPA. As discussed in more detail in subsequent sections:

 MON 863 corn does not deliver a " high dose" for corn rootworm resistance management, despite relatively high expression levels in several tissues.

 High levels are expressed in tissues where the toxin is not needed for pest control.

 Cry 3Bb toxins continue to be produced by the plant for at least two months longer than needed for rootworm control, thereby exposing nontarget organisms to unnecessary risks while also needlessly taxing the plant.

 Essentially no data are available on the quantity of root exudates entering the soil, information that is essential to fully assess the risks posed by expression levels in the roots themselves.

EPA Response: Toxicity tests are done with purified protein at levels much higher than those expressed in the plant. EPA believes that the lack of toxicity at these exaggerated levels also covers the possibility of higher expression levels in most PIP expressing plants. See BRAD Section II. B.

The above concerns were addressed by the maximum hazard dose ( limit dose) toxicity testing performed on representative beneficials from several taxa ( USEPA. 2002. MON 863 BRAD Sections C. I. B. 3. a. iv and Section 2.4 2  29 C. I. B). Testing at the maximum hazard dose level takes into account the variability in Cry Bb1 protein levels in Bt corn fields. The toxicity of the Cry3Bb1 protein has been evaluated following challenge of several species of invertebrates including: adult and larval honey bees, a parasitic hymenopteran ( Nasonia), green lacewings, lady beetles, collembola, monarch butterfly, and earthworms ( USEPA. 2002. MON 863 BRAD Sections C. I. B. 3. a. iv and C. I. B). Reproductive and developmental observations were also made on collembola, honeybee and lady beetle larva maturation studies ( USEPA. 2002. MON 863 BRAD Sections C. I. B. 3. a. iv and C. I. B). Insect host range bioassay and field survey data were used to assess the hazard to additional species not directly tested in the laboratory.

Because the Agency believes that additional studies would be useful in completing the database for long term effects assessment, it is requesting additional supplementary studies regarding Cry3Bb1 protein degradation and persistence in various agricultural soils.

The Agency agrees that a high dose/ refuge model is not appropriate for MON 863 corn since there is not a " high dose" of Cry3Bb1 expressed ( see BRAD Dose Section pp. IID 4 to IID 5). The IRM strategy currently recommended is based on conservative parameters and current knowledge of corn rootworm biology ( see BRAD Pest Biology Section pp. IID 2 to IID 4 and Simulation Models of Resistance Section pp IID 5 to IID 8).