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MIR162 USDA Petition Petition for Determination of Nonregulated Status for Insect-Resistant MIR162 Maize OECD Unique Identifier: SYN-IR162-4 Submitted By: Dennis P. Ward, Ph.D. Syngenta Biotechnology, Inc. P.O. Box 12257 3054 E. Cornwallis Road Research Triangle Park, NC 27709 Submission Date: August 31, 2007 Authors: Dennis P. Ward and Scott A. Huber Syngenta Petition ID: MIR162-USDA-1 Page 1 of 268 Petition for Determination of Nonregulated Status The undersigned submits this petition under 7 CFR 340.6 to request that the Administrator make a determination that the article should not be regulated under 7 CFR Part 340. Certification The undersigned certifies that, to the best knowledge and belief of the undersigned, this petition includes all information and views on which to base a determination, and that it includes relevant data and information known to the petitioner which are unfavorable to the petition. Petitioner: Dennis P. Ward, Ph.D. Regulatory Affairs Manager Mailing Address: Syngenta Biotechnology, Inc. P.O. Box 12257 3054 East Cornwallis Road Research Triangle Park, NC 27709 USA Telephone: 919.597.3096 Facsimile: 919.541.8535 Email: dennis.ward@syngenta.com © 2007 Syngenta. 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 Syngenta, and only in support of actions requested by Syngenta. Any other use of this material, without prior written consent of Syngenta, is strictly prohibited. By submitting this document, Syngenta does not grant any party or entity any right or license to the information or intellectual property described in this document. MIR162-USDA-1 Page 2 of 268 Release of Information Syngenta Biotechnology, Incorporated (Syngenta) is submitting the information in this petition for review by the United States Department of Agriculture (USDA) as part of a federal regulatory process. By submitting this information to USDA, Syngenta does not authorize its release to any third party. In responding to a request made under the Freedom of Information Act (FOIA), 5 U.S.C. §552 and 7 CFR Part 1, covering all or some of the information in this petition, Syngenta expects that, in advance of the release of the information, USDA will provide Syngenta with a copy of the FOIA request and the material proposed to be released, and the opportunity to object to the release of information based on appropriate legal grounds (e.g. responsiveness, trade secret, and/or commercial concerns). Syngenta understands that a copy of this petition may be made available to the public as part of the public comment process. Except in accordance with the foregoing, Syngenta does not authorize the release, publication, or other distribution of this information (including internet posting) without prior notice and Syngenta consent. MIR162-USDA-1 Page 3 of 268 Petition for Determination of Nonregulated Status for Insect-Resistant MIR162 Maize Summary of the Petition Using the techniques of modern molecular biology, Syngenta has transformed maize (Zea mays L.), to produce event MIR162 maize (hereafter ‘MIR162 maize’), a new cultivar that is resistant to lepidopteran insect feeding. MIR162 maize plants contain the vip3Aa20 gene encoding the Vip3Aa20 protein and the manA gene encoding the enzyme phosphomannose isomerase (PMI). The Vip3Aa20 protein is a variant of the native insecticidal Vip3Aa protein from Bacillus thuringiensis strain AB88. The Vip3Aa20 protein is insecticidally active against a number of significant lepidopteran pests of maize. The manA gene was obtained from Escherichia coli strain K-12 and the protein it encodes was utilized as a plant selectable marker during development of MIR162. MIR162 maize was produced by Agrobacterium tumefaciens-mediated transformation of immature maize embryos using plasmid vector pNOV1300. The region between the left and right borders of the transformation plasmid included vip3Aa and manA gene expression cassettes; this T-DNA was transferred into the maize genome during transformation. The vip3Aa expression cassette consisted of the vip3Aa coding region regulated by the Z. mays polyubiquitin promoter (ZmUbiInt) and cauliflower mosaic virus 35S 3′ polyadenylation sequences. The manA expression cassette consisted of the manA coding region regulated by the ZmUbiInt promoter and the nopaline synthase (NOS) polyadenylation sequence. Southern blot analyses and nucleotide sequencing demonstrated that MIR162 maize contains a single intact T-DNA insert in the nuclear maize genome. Southern blot analyses further demonstrated that the T-DNA insert contains: i) single copies of a vip3Aa gene and a manA gene; ii) two copies of the ZmUbiInt promoter; iii) one copy of the NOS terminator; and iv) no backbone sequences from transformation plasmid pNOV1300. Nucleotide sequencing of the T-DNA insert in MIR162 maize revealed two codon changes within the vip3Aa coding sequence relative to the intended vip3Aa sequence; one of these was a silent mutation and the other codon change resulted in an amino acid substitution. The vip3Aa gene variant present in MIR162 maize has been designated vip3Aa20. Nucleotide sequencing additionally determined that the MIR162 maize T-DNA insert did not locate within any known Z. mays gene. Further, no novel open reading frames were created that spanned either the 5′ or 3′ junctions between the T-DNA and Z. mays genomic sequences. These genetic characterization data demonstrate that, apart from the well-characterized change that resulted in a single altered amino acid in the vip3Aa coding sequence, there are no unintended changes in the MIR162 maize genome as a result of the T-DNA insertion. Observations of vip3Aa20 and manA segregation ratios over several generations of MIR162 maize are consistent with the genes being linked at a single locus in the maize genome, and indicate stable inheritance of the transgenes. These data also indicate that no novel proteins, other than Vip3Aa20 and PMI, will be produced in MIR162 maize. Based on a well-characterized mode of action, physiochemical properties, and results of safety studies, it has been demonstrated that the Vip3Aa20 and PMI proteins present in MIR162-USDA-1 Page 4 of 268 MIR162 maize pose no risk of harm for mammalian species. Laboratory, greenhouse, growth chamber, and field investigations with MIR162 maize confirmed that there were no changes in seed, pollen, plant phenotypic, or composition parameters suggestive of increased plant pest risk. No adverse effects were associated with exposures to Vip3Aa proteins in a range of nontarget indicator species appropriate for a maize ecosystem and the highest doses tested represented no adverse effect levels. With one exception, the levels tested were in excess of expected environmental exposure levels, indicating a low probability of harm for nontarget organisms inhabiting maize ecosystems. In one study it was not possible to exceed a theoretical expected environmental concentration; however, no adverse effects were observed on this study. The narrow spectrum of insecticidal activity observed for Vip3Aa proteins indicates with high certainty that no endangered or threatened species other than Lepidoptera would be harmed by contact with Vip3Aa20 via MIR162 maize. Furthermore, there is minimal exposure of endangered Lepidoptera to maize and therefore, cultivation of MIR162 maize is unlikely to harm any endangered or threatened species in the U.S. Syngenta knows of no study results or observations associated with MIR162 maize that are anticipated to result in adverse consequences to the quality of the human environment, directly, indirectly, or cumulatively. This includes a lack of anticipated adverse effects on endangered species, unique geographic areas, critical habitats, public health and safety (including children and minorities), genetic diversity of maize, farmer or consumer choice, insect resistance, or the economy, either within or outside the U.S. MIR162 maize offers growers high efficacy, convenience, and an additional choice for protection of maize crops from feeding damage caused by lepidopteran pests. As such, MIR162 maize is expected to produce beneficial effects similar to previously deregulated Bt maize products that are commercially available. These benefits include increased competition in the marketplace for insect-protected seed products. Moreover, the novel mode of action of the Vip3A20 protein is expected to extend the useful life of Bt maize technology for lepidopteran insect control in general by reducing the selection pressure for resistance among target pests. The Animal and Plant Health Inspection Service of the United States Department of Agriculture has responsibility, under the Plant Protection Act (Title IV Public Law 106-224, 114 Statute 438, 7 U.S.C. §7701-7772) 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. Syngenta is hereby submitting this petition for a determination of nonregulated status for MIR162 maize and provides all necessary data upon which to make this determination. MIR162-USDA-1 Page 5 of 268 Table of Contents Page Certification..............................................................................................................................2
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