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Monsanto Is Submitting the Information in This Petition for Review by the USDA As Part of the Regulatory Process

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Monsanto Is Submitting the Information in This Petition for Review by the USDA As Part of the Regulatory Process CBI Deleted Version Release of Information Monsanto is submitting the information in this petition for review by the USDA as part of the regulatory process. By submitting this information, Monsanto does not authorize its release to any third party. In the event the USDA receives a Freedom of Information Act request, pursuant to 5 U.S.C., § 552 and 7 CFR Part 1, covering all or some of this information, Monsanto expects that, in advance of the release of the document(s), USDA will provide Monsanto with a copy of the material proposed to be released and the opportunity to object to the release of any information based on appropriate legal grounds, e.g., responsiveness, confidentiality, and/or competitive concerns. Monsanto expects that no information that has been identified as CBI (confidential business information), will be provided to any third party. Monsanto understands that a CBI- deleted copy of this information may be made available to the public in a reading room and by individual request, as part of a public comment period. Except in accordance with the foregoing, Monsanto does not authorize the release, publication or other distribution of this information (including website posting) without Monsanto's prior notice and consent. © 2006 Monsanto Company. All Rights Reserved. This document is protected under copyright law. This document is for use only by the regulatory authority to which this has been submitted by Monsanto Company, and only in support of actions requested by Monsanto Company. Any other use of this material, without prior written consent of Monsanto, is strictly prohibited. By submitting this document, Monsanto does not grant any party or entity any right or license to the information or intellectual property described in this document. Monsanto Company 06-CR-166U Page 2 of 262 CBI Deleted Version Petition for the Determination of Non-regulated Status for MON 89034 Summary The Animal and Plant Health Inspection Service (APHIS) of the U.S. Department of Agriculture (USDA) has responsibility under the Plant Protection Act (Title IV Pub. L. 106-224, 114 Stat. 438, 7 U.S.C. § 7701-7772) and the Plant Quarantine Act (7 U.S.C. § 151-167) to prevent the introduction and dissemination of plant pests into the United States. The APHIS regulation 7 CFR Part 340.6 provides that an applicant may petition APHIS to evaluate submitted data to determine that a particular regulated article does not present a plant pest risk and should no longer be regulated. If APHIS determines that the regulated article does not present a plant pest risk, the petition is granted, thereby allowing unrestricted introduction of the article. Monsanto Company is submitting this request to APHIS for a determination of non- regulated status for the new biotechnology-derived corn product, MON 89034, any progeny derived from crosses between MON 89034 and conventional corn, and any progeny derived from crosses of MON 89034 with other biotechnology-derived corn which has been granted non-regulated status under 7 CFR Part 340. Corn (Zea mays L.) is the largest crop grown in the U.S. in terms of acreage planted and net value. Insect pests in the corn fields, if not properly controlled, significantly reduce corn yields and grain quality. In 1997, Monsanto commercialized a biotechnology- derived corn product, YieldGard® Corn Borer corn (i.e., MON 810) which contains the Bacillus thuringiensis (Bt) cry1Ab gene. The expression of Cry1Ab protein in corn plants provides effective protection against damage caused by lepidopteran insect pests, such as European corn borer. Since the launch of MON 810, several other Bt corn products have also been commercialized in the U.S., including YieldGard® Rootworm corn which expresses Cry3Bb1 protein that confers protection against coleopteran pests. In 2005, approximately 35% of the total U.S. corn acreage was planted with corn seed possessing insect resistance trait. Monsanto has recently developed MON 89034 as a second generation insect protection corn product to provide enhanced benefits for the control of lepidopteran insect pests. MON 89034 produces the Cry1A.105 and Cry2Ab2 proteins derived from Bacillus thuringiensis, which are active against lepidopteran insect pests. Compared to MON 810, MON 89034 will better serve corn growers’ need for controlling a wider spectrum of lepidopteran pests and help assure the durability of Bt corn. MON 89034 provides outstanding control of Ostrinia species such as European corn borer (ECB) and Asian corn borer, and Diatraea species such as southwestern corn borer (SWCB) and sugarcane borer. Control of these insects provided by MON 89034 is comparable to MON 810. MON 89034 also provides a high level control of fall armyworm (FAW) throughout the season, whereas MON 810 principally controls fall armyworm larvae during vegetative growth stage. Furthermore, MON 89034 provides significantly improved protection from Monsanto Company 06-CR-166U Page 4 of 262 CBI Deleted Version damage caused by corn earworm (CEW) than MON 810. In addition to the wider spectrum of insect control, the combination of the Cry1A.105 and Cry2Ab2 insecticidal proteins in a single plant, MON 89034, provides a much more effective insect resistance management (IRM) tool. The results of mathematical modeling indicate that biotechnology-derived plants expressing two Cry proteins will have significantly greater durability than plants producing either of the single proteins if the cross-resistance between the Cry proteins is low and the mortality of susceptible insects caused by each of the individual proteins is at least 90%. Comparative biophysical studies indicate that the Cry1A.105 and Cry2Ab2 proteins have important differences in their mode of action, specifically in the way in which they bind to the lepidopteran midgut. Therefore, the probability of cross-resistance between these two proteins is low. Furthermore, in vitro and in planta studies with Cry1A.105 and Cry2Ab2 demonstrate that both proteins are highly active against the primary lepidopteran pests of corn (ECB, SWCB, CEW, and FAW), particularly ECB, achieving close to or greater than the critical 95% level of control in all cases. With these properties, MON 89034 should be durable with a significantly smaller structured refuge than is necessary for Bt corn products producing a single insecticidal protein. The data and information presented in this application demonstrate that MON 89034 is not likely to pose an increased plant pest potential or to have an adverse environmental impact compared to conventional corn. This conclusion is based on multiple lines of evidence. The first is the detailed molecular characterization of the inserted DNA. Results confirm the insertion of a single functional copy of cry1A.105 and cry2Ab2 expression cassettes at a single locus within the corn genome. The second is a detailed biochemical characterization of the Cry1A.105 and Cry2Ab2 proteins produced in MON 89034. In addition, the data demonstrate that the Cry1A.105 and Cry2Ab2 proteins produced in MON 89034 are equivalent to the respective Cry1A.105 and Cry2Ab2 proteins produced by recombinant strains of Escherichia coli, which were used in the various safety assessment studies. The third line of evidence is an assessment of the toxicity and allergenicity potential of the Cry1A.105 and Cry2Ab2 proteins based on extensive information collected and studies performed on the two proteins. The results demonstrate with reasonable certainty that the Cry1A.105 and Cry2Ab2 proteins are unlikely to be allergens or toxins. The fourth line of evidence is the compositional and nutritional assessment which confirms that MON 89034 grain and forage are compositionally equivalent to and as safe as those of conventional corn. The fifth line of evidence is the extensive evaluation of the MON 89034 phenotypic and agronomic characteristics and ecological interactions, which demonstrates that MON 89034 is not likely to have an increased plant pest potential compared to the conventional corn. Finally, an assessment on the potential impact on non-target organisms (NTO) and endangered species concludes that MON 89034 is unlikely to have adverse effects on these organisms under the conditions of use. MON 89034 was produced by Agrobacterium-mediated transformation of corn with PV- ZMIR245, which is a binary vector containing 2T-DNAs. The first T-DNA, designated as T-DNA I, contains the cry1A.105 and the cry2Ab2 expression cassettes. The second T-DNA, designated as T-DNA II, contains the nptII (neomycin phosphotransferase II) Monsanto Company 06-CR-166U Page 5 of 262 CBI Deleted Version expression cassette. During transformation, both T-DNAs were inserted into the genome. The nptII gene was used as the selectable marker which was needed for selection of the transformed cells. Once the transgenic cells were identified, the selectable marker gene was no longer needed. Therefore, traditional breeding was used to isolate plants that only contain the cry1A.105 and cry2Ab2 expression cassettes (T-DNA I) and do not contain the nptII expression cassette (T-DNA II), thereby, producing marker-free corn MON 89034. Molecular characterization of MON 89034 by Southern blot analyses demonstrated that the DNA inserted into the corn genome is present at a single locus and contains one functional copy of the cry1A.105 and the cry2Ab2 expression cassettes. All genetic elements are present in the inserted DNA as expected with the exception that the e35S promoter, which regulates expression of the cry1A.105 gene, has been modified and that the Right Border sequence present in PV-ZMIR245 was replaced by a Left Border sequence in MON 89034. No backbone plasmid DNA or nptII sequences were detected. PCR and DNA sequence analyses provided the complete DNA sequence of the insert and confirmed the organization of the elements within the insert. The stability of the integrated DNA was demonstrated by the fact that the Southern blot fingerprint of MON 89034 was maintained for seven generations tested in the breeding history.
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