Page 1 of 268 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-1MIR162-USDA-1 Page 2 of 268 Petition for Determinatio n of Nonregulated Status

The undersigned submits this petition under 7 CFR 340.6 to request that the Administrator
make a determination that the article s hould 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 informati on 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 la w. 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 s ubmitting 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 3 of 268 Release of Information

Syngenta Biotechnology, Incorporated (Syngenta) is submitting the information in this petition for review by the United States Depart ment 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 th at, 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 re lease 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 av ailable to the public as part of the public comment process. Except in acco rdance with the foregoing, Syngenta does not authorize the release, publication, or other distribution of this information (i ncluding internet posting) without prior noti ce and Syngenta consent.MIR162-USDA-1 Page 4 of 268 Petition for Determination of Nonregulated Status
for Insect-Resistant MIR162 Maize

Summary of the Petition

Using the techniques of modern molecula r 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 feed ing. MIR162 maize plants contain the vip3Aa20 gene encoding the Vip3Aa20 protein and the manA gene encoding the enzyme phosphomannose isomerase (PMI). The Vip3Aa 20 protein is a variant of th e 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 tran sferred into the maize genome during transformation. The vip3Aa expression cassette consisted of the vip3Aa coding region regulated by the Z. mays polyubiquitin promoter (ZmUbiInt) a nd cauliflower mosaic virus 35S 3 ′ polyadenylation sequences. The manA expression cassett e consisted of the manA coding region regulated by the ZmUbiInt promoter and the nopaline sy nthase (NOS) polyadenylation sequence.

Southern blot analyses and nucleotide sequenc ing demonstrated that MIR162 maize contains a single intact T-DNA insert in the nuclear maize genome. S outhern 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. Nucl eotide sequencing of
the T-DNA insert in MIR162 maize rev ealed 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 in sert 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 si ngle locus in the maize genome, and indicate stable inheritance of the transgenes. These da ta 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 inMIR162-USDA-1 Page 5 of 268 MIR162 maize pose no risk of ha rm for mammalian species.

Laboratory, greenhouse, growth chamber, an d 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 exposur es to Vip3Aa proteins in a range of nontarget indicator species appropriate for a maize ecosyst em and the highest doses tested represented no adverse effect levels. With one exception, th e levels tested were in excess of expected environmental exposure levels, indicating a lo w probability of harm for nontarget organisms inhabiting maize ecosystems. In one study it was not possible to exceed a theoretical expected environmental concentration; howev er, no adverse effects were observed on this study.

The narrow spectrum of insecticidal activity ob served for Vip3Aa proteins indicates with high certainty that no endangered or threaten ed species other than Lepidoptera would be harmed by contact with Vip3Aa20 via MIR162 maize. Furthermore, there is minimal exposure of endangered Lepidopter a to maize and therefore, cu ltivation of MIR162 maize is unlikely to harm any endangered or th reatened species in the U.S.

Syngenta knows of no study results or observati ons associated with MIR162 maize that are anticipated to result in adverse consequen ces to the quality of the human environment, directly, indirectly, or cumulatively. This incl udes a lack of anticipated adverse effects on endangered species, unique ge ographic areas, critical habita ts, public health and safety (including children and minorities), genetic divers ity of maize, farmer or consumer choice, insect resistance, or the economy, either with in or outside the U.S. MIR162 maize offers growers high efficacy, convenience, and an add itional choice for protection of maize crops from feeding damage caused by lepidopteran pest s. As such, MIR162 maize is expected to produce beneficial effects simila r 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 sele ction pressure for resistance among target pests.

The Animal and Plant Health Inspection Serv ice of the United States Department of Agriculture has responsibility, under the Plan t Protection Act (Title IV Public Law 106-224, 114 Statute 438, 7 U.S.C. §7701-7772) to prevent th e 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 unrestr icted introduction of the ar ticle. 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 6 of 268 Table of Contents Page

Certification..................................................................................................................

............ 2

Release of Information .........................................................................................................

... 3

Summary of the Petition........................................................................................................

.. 4

List of Tables .................................................................................................................

........... 9

List of Figures................................................................................................................

......... 10

I. Rationale for Submission of Request for Deregulation ................................................. 11 I.A. Basis for the Request for a Determination of Nonregulated Status........................... 11 I.B. Rationale for Development of MIR162 Maize .......................................................... 11
I.C. Status with Ot her Regulatory Agencies..................................................................... 13 I.C.1. U.S. Environmental Protection Agency............................................................ 14 I.C.2. U.S. Food and Drug Administration................................................................. 14 I.C.3. Foreign Governments........................................................................................ 14

II. The Maize Family ..........................................................................................................

.. 14 II.A. Maize as a Crop........................................................................................................ 14 II.B. Biology of Maize...................................................................................................... 15
II.C. Weediness of Maize.................................................................................................. 16 II.D. Characteristics of the Recipient Germplasm............................................................ 17

III. Transformation and Development of MIR162 Maize................................................. 18 III.A. Description of the Transformation System ............................................................. 18 III.B. Development of MIR162 Maize ............................................................................. 19
III.C. Production of Test and Control Materials............................................................... 19

IV. Donor Genes and Regulatory Sequences...................................................................... 23 IV.A. Transformation Vector pNOV1300........................................................................ 23
IV.B. Characterization of the T-DNA in MIR162 Maize................................................. 25 IV.B.1. Sequence of the T-DNA................................................................................. 26 IV.B.2. Copy Number of Functional Elements........................................................... 26 IV.B.2.a. Copy Number of Functional Elements: vip3Aa20 Gene ...................... 26 IV.B.2.b. Copy Number of Functional Elements: manA Gene ............................ 30 IV.B.2.c. Copy Number of Functiona l Elements: ZmUbiInt promoter................ 33 IV.B.2.d. Copy Number of Functional Elements: NOS Terminator .................... 36
IV.B.3. Absence of Plasmid Backbone Elements....................................................... 38 IV.C. Stability of the T-DNA Across Generations........................................................... 41
IV.D. Mendelian Inheritance of the T-DNA..................................................................... 44 IV.E. Flanking Sequence Analysis ................................................................................... 45 IV.F. Summary of the Genetic Characterization of MIR162 Maize................................. 46

V. Characterization of the Vip3Aa20 and PMI Proteins .................................................. 47 V.A. Vip3Aa20 Protein – Origin and Function ................................................................ 47
V.A.1. Mechanism of Action a nd Insecticidal Specificity ......................................... 47 V.A.2. Characterization of Vip3Aa20 ........................................................................ 48 V.A.3. Safety Assessment for Vip3Aa20 ................................................................... 50MIR162-USDA-1 Page 7 of 268 V.B. Phosphomannose Isomerase – Origin and Function ................................................ 52
V.B.1. Mechanism of Action ...................................................................................... 52 V.B.2. Characterization of Phosphomannose Isomerase............................................ 53 V.B.3. Safety Assessment for Phosphomannose Isomerase....................................... 53 V.C. Vip3Aa20 and PMI Le vels in MIR162 Maize ......................................................... 54

VI. Phenotypic and Compositional Evaluation.................................................................. 57 VI.A. Phenotypic Assessment of MIR162 Plants............................................................. 57 VI.A.1. Seed Dormancy and Germination.................................................................. 58 VI.A.2. Field Agrono mic Performance....................................................................... 62 VI.A.3. Pollen Viability and Morphology .................................................................. 67 VI.A.4. Seed Dispersal................................................................................................ 69 VI.B. Compositional Assessment of MIR162 Forage and Grain ..................................... 69 VI.C. Summary of Phenotypic and Compositional Evaluations....................................... 73

VII. Environmental Consequences of Introduction........................................................... 73 VII.A. Spectrum of Insecticidal Activity for Vip3Aa Proteins ........................................ 74 VII.B. Activity Against Target Insects ............................................................................. 75 VII.C. Expected Environmenta l Concentrations for Vip3Aa20 ....................................... 77 VII.C.1. Concentrations of Vip3Aa20 in MIR162 Maize .......................................... 77 VII.C.2. Soil Fate of Vip3Aa Proteins........................................................................ 77 VII.C.3. Expected Environmental Con centrations for Nontarget Organisms ............ 78
VII.C.3.a. EEC for Wild Birds .............................................................................. 78
VII.C.3.b. EEC for Wild Mammals....................................................................... 79
VII.C.3.c. EEC for Pollinators............................................................................... 80 VII.C.3.d. EEC for Above-ground Nontarget Arthropods .................................... 80
VII.C.3.e. EEC for Soil-dwelli ng Nontarget Invertebrates ................................... 81 VII.C.3.f. EEC for Aquatic Organisms ................................................................. 82 VII.C.3.g. Summary of Envir onmental Exposure Estimates................................. 83 VII.D. Impact on Nontarget Organisms............................................................................ 84 VII.D.1. Effect of Vip3Aa on Wild Birds................................................................... 85 VII.D.2. Effect of Vip3Aa on Wild Mammals ........................................................... 85 VII.D.3. Effect of Vip3Aa on Pollinators................................................................... 85 VII.D.4. Effect of Vip3Aa on Above-ground Arthropods.......................................... 86 VII.D.5. Effect of Vip3Aa on Soil-dwelling Invertebrates......................................... 87 VII.D.6. Effect of Vip3Aa on Aquatic Organisms ..................................................... 87 VII.E. Toxicity Exposure Ratios for Nontarget Organisms.............................................. 88 VII.F. Safety Assessment for Endangered and Threatened Species................................. 89
VII.G. Conclusion on Environmental Risk....................................................................... 90
VII.H. Gene Flow Assessment.......................................................................................... 90 VII.H.1. Gene Flow to Wild Species .......................................................................... 91
VII.H.2. Potential for Horizontal Gene Transfer ........................................................ 92 VII.I. Impact of MIR162 Introduction on Current Maize Agronomic Practices.............. 92

VIII. Insect Resistance Management .................................................................................. 96

IX. Adverse Consequences of Introduction........................................................................ 97 IX.A. Effects on the Quality of the Human Environment ................................................ 97 IX.B. Context of the Proposed Action.............................................................................. 98MIR162-USDA-1 Page 8 of 268 IX.C. Intensity of the Proposed Action........................................................................... 100 IX.C.1. Impacts That May Be Bo th Beneficial and Adverse.................................... 100
IX.C.2. Effects on Public Heal th or Environmental Safety ...................................... 101 IX.C.3. Unique Characteristic s of the Geographic Area .......................................... 102
IX.C.4. Effects on Human Environment are Unlikely to be Controversial .............. 102 IX.C.5. Potential Unique or Unknown Risks............................................................ 102 IX.C.6. Potential Impacts for Future Considerations................................................ 104 IX.C.7. Potential Cumulative Impacts ...................................................................... 104
IX.C.8. Historical and Cultural Effects..................................................................... 108 IX.C.9. Effects on Endangered Species.................................................................... 108 IX.C.10. Compliance with Law ................................................................................ 108 IX.D. Economic Impacts................................................................................................. 108 IX.D.1. Enhanced Productivity................................................................................. 109 IX.D.2. Increased Competition for Bt Lepidopteran Control Products .................... 110
IX.D.3. Farmer and Consumer Choice ..................................................................... 110 IX.D.4. Effects on the Export Market....................................................................... 111
IX.E. Conclusion............................................................................................................. 112

X. References.................................................................................................................

...... 112

Acknowledgements .............................................................................................................. 124

Key to Abbreviations and Acronyms ................................................................................. 125

Appendix A. USDA Notifications for Field Trials with MIR162 Maize ......................... 127

Appendix B. Genetic Characterization of MIR162 Maize.............................................. 129

Appendix C. Characterization of the Vip3Aa20 and PMI Proteins............................... 136

Appendix D. Quantification of Vip3Aa20 and PMI in MIR162 Maize ......................... 153

Appendix E. Compositional Analysis of MIR162 Forage and Grain............................. 158

Appendix F. Benefit Analysis for MIR162 Maize............................................................ 181MIR162-USDA-1 Page 9 of 268 List of Tables Page

Table 1. Categorization of maize insect pests......................................................................... 12
Table 2. Description of test and control s eed materials used in regulatory studies................ 21
Table 3. Description of genetic elem ents inserted in vector pNOV1300. .............................. 23
Table 4. Expected versus observed hybridization ba nd sizes for probes with vip3Aa19.

...... 29
Table 5. Expected versus observed hybridization ba nd sizes for probes with manA

. ............ 32
Table 6. Expected versus observed hybridization band sizes for probes with ZmUbiInt....... 35
Table 7. Expected versus observed hybridization band sizes for probes with NOS............... 38
Table 8. Expected versus observed band sizes for probes with pNOV1300 backbone.......... 41
Table 9. Expected versus observed hybridization band size with vip3Aa19 probe................ 44

Table 10. Observed versus expected frequencies for vip3Aa20 across generations............... 45

Table 11. Observed versus expected frequencies for manA across generations..................... 45

Table 12. Amino acid differences betw een Vip3Aa1, Vip3Aa19, and Vip3Aa20................. 48
Table 13. Comparative bioactivity of Vi p3Aa proteins from different sources. .................... 50

Table 14. Mean tissue concentrations of Vip3Aa20 in MIR162 plants.................................. 55

Table 15. Mean tissue concentratio ns of PMI in MIR162 plants. .......................................... 56
Table 16. Phenotypic characteristics evaluated for MIR162 maize hybrids. ......................... 57
Table 17. Test, control, and reference seed s used in germination and dormancy test............ 59
Table 18. Summary of seed germination and dormancy frequencies..................................... 61
Table 19. Statistical analys is of proportions for normal germinated seeds. ........................... 62
Table 20. Test and control hybrids planted in field agronomic equivalence trials................. 62
Table 21. Locations and dates of MIR162 agronomic performance trials. ............................ 63
Table 22. Results of agronomic performance trials conducted in 2005. ................................ 65
Table 23. Results of agronomic performance trials conducted in 2006. ................................ 66 Table 24. Pollen viability and diameter measurements. ......................................................... 67
Table 25. Forage and grain components meas ured in MIR162 and conventional maize. ...... 70
Table 26. Statistically significant differen ces between MIR162 and control analytes........... 72
Table 27. Insect species screened for se nsitivity to Vip3Aa protein variants. ....................... 74

Table 28. Tissue concentrations of Vi p3Aa20 used for computation of EECs. ..................... 78

Table 29. Tabulation of EEC and D DD values for nontarget organisms. .............................. 83
Table 30. Vip3Aa test substances used in nontarget organism hazard studies....................... 84
Table 31. Nontarget organism TER values for Vip3Aa proteins............................................ 89

Table 32. Conventional insectic ide usage for control of se lected lepidopteran pests. ........... 94MIR162-USDA-1 Page 10 of 268 List of Figures Page

Figure 1. U.S. maize usage by segment in 2006..................................................................... 15
Figure 2. Geographic distribution of acres planted with maize in 2006................................. 17
Figure 3. Plasmid map for vector pNOV1300........................................................................ 18
Figure 4. Steps in the deve lopment of MIR162 maize. .......................................................... 20
Figure 5. MIR162 pedigree chart............................................................................................ 22
Figure 6. Schematic representation of the MIR162 T-DNA................................................... 25
Figure 7. Restriction sites and probe for vip3Aa20

................................................................. 27
Figure 8. Southern blot anal ysis of MIR162 maize with a vip3Aa19 -specific probe............. 28
Figure 9. Restriction sites and probe for manA

....................................................................... 30
Figure 10. Southern blot anal ysis of MIR162 maize with a manA -specific probe................. 31
Figure 11. Restriction sites and pr obe for the ZmUbiInt promoter. ....................................... 33
Figure 12. Southern analysis of MIR162 maize with ZmUbiInt-specific probe. ................... 34
Figure 13. Restriction sites a nd probe for NOS terminator. ................................................... 36
Figure 14. Southern analysis of MIR162 ma ize with NOS terminator-specific probe........... 37
Figure 15. Restriction s ites and probe for plasmid backbone elements.................................. 39
Figure 16. Southern blot analysis for plasmid backbone sequence. ....................................... 40
Figure 17. Restriction site and probe for vip3Aa19

. ............................................................... 42
Figure 18. Southern blot of BC 1 F 1 , BC 2 F 1 , and BC 4 F 1 generations from MIR162................ 43
Figure 19. Western blot of plant- and microbially produced Vip3Aa proteins. ..................... 49
Figure 20. Photographs of stained pollen co llected from control and MIR162 plants........... 68
Figure 21. MIR162 field efficacy trial results. ....................................................................... 76MIR162-USDA-1 Page 11 of 268 I. Rationale for Submission of Request for Deregulation

The introduction of crops improved through modern biotechnology has been the single most important technological innovation brought to United States (U.S.) agricultu re in the past 25 years. Adoption of genetically engineered crops with insect a nd herbicide tolerance traits has increased dramatically since their commercia l introduction in 1995. Ne t economic benefits
at the farm level have been substantial (Brookes and Barfoo t, 2005). Improved insect and weed control have led to increased crop yiel ds and reductions in c onventional insecticide applications. The continued development and introduction of these traits is expected to benefit both growers and consumers. Tr ansformation event MIR162 maize has been developed by Syngenta to provide growers with maize varieties that ar e resistant to feeding damage caused by a number of significant lepidopteran insect pests. This trait will be offered to growers in combination with other deregulated maize traits ( e.g. herbicide tolerance).

I.A. Basis for the Request for a Determination of Nonregulated Status

The Animal and Plant Health Inspection Service (APHIS) of USDA has responsibility under the Plant Protection Act (Title IV Public Law 106-224, 114 Statute 438, 7 U.S.C. 7701-7772) to prohibit or restrict the im portation, exportation, and interstate movement of plants, plant products, certain biological co ntrol organisms, noxious weeds, and plant pests. APHIS regulations under 7 CFR §340.1 stipulate that a ny organism which has been altered or produced through genetic engineering is consider ed a regulated article if the donor organism, recipient organism, or vector or vector agent belongs to any genera or taxa designated under §340.2 and meets the definition of a plant pest, or is an unclassified organism and/or an organism whose classification is unknown, or a ny product which contains such an organism, or any other organism or produc t altered or produced through genetic engineering which the Administrator determines is a plant pest or has reason to believe is a plant pest. The transformation system used to produce MI R162 maize was derived from the genus Agrobacterium , an organism listed under §340.2. APHIS regulations further state that any person may submit a petition seeking a determination that an article should not be regulated under this regulation. Syngenta herein presen ts data and justification for an APHIS determination of nonregulated st atus for MIR162 maize based on an absence of plant pest
risk.

I.B. Rationale for Development of MIR162 Maize

Maize is susceptible to attack by a variety of insects from the time it is planted until it is consumed as food or feed. Insect pests can be categorized as major and consistent pests, major and sporadic pests, and moderate to minor pests based on annual destructiveness and their geographic distribution. Ta ble 1 categorizes most of the insect pests of maize found in the U.S. The most economically signi ficant insect pests of maize are: Ostrinia nubilalis (European corn borer), Diatraea saccharalis (sugarcane borer), Diatraea grandiosella (southwestern corn borer), Diabrotica spp. (corn rootworm complex), Helicoverpa zea (corn earworm/cotton bollworm), Spodoptera frugiperda (fall armyworm), Agrotis ipsilon (black cutworm), Elasmopalpus lignosellus (lesser cornstalk borer), Rhopalosiphum maidis (corn leaf aphids), and Striacosta albicosta (western bean cutworm). Pests of secondary economicMIR162-USDA-1 Page 12 of 268 importance in maize include both soil-dwelling insects that feed on roots ( e.g., wireworms, billbugs, webworms, white grubs, corn root aphids, the seed corn maggot, grape colaspis and seedcorn beetles) and above-ground insect s that attack the st alk, leaf, and ear ( e.g., cutworms, chinch bugs, grasshoppers, corn flea beetles and Japanese beetles). S. albicosta is a pest of increasing economic importance becaus e of its movement into Iowa, Illinois, and
Indiana.

Table 1. Categorization of maize insect pests. Categorized based on their potential for causing economic losses (modified from Gray and Luckmann, 1994).

Major & Consistent Insect Pests Moderate to Minor Insect Pests (cont.)

Ostrinia nubilalis (European corn borer) Blissus leucopterus (cinch bug)

Helicoverpa zea (corn earworm) Diabrotica undecimpunctata (southern corn rootworm)

Spodoptera frugiperda (fall armyworm) Other cutworms, many species

Diabrotica virgifera virgifera (western corn rootworm) Stenolophus lecontei (seedcorn beetle)

Diabrotica barberi (northern corn rootworm) Delia platura (seedcorn maggot)

Major – Sporadic Insect Pests Oligonychus pratensis (banks grass mite)

Agrotis ipsilon (black cutworm) Tetranychus urticae (two-spotted spider mite)

Rhopalosiphum maidis (corn leaf aphid) Billbugs, many species

Diatraea grandiosella (southwest corn borer) White grubs, many species

Diatraea saccharalis (sugarcane borer) Papaipema nebris (stalk borer)

Elasmopalpus lignosellus (lesser cornstalk borer) Scutigerella immaculate (garden symphylan)

Striacosta albicosta (western bean cutworm) Popillia japonica (Japanese beetle)

Sod webworms, several species

Moderate to Minor Insect Pests Colaspis brunnea (grape colaspis)

Wireworms, many species Thrips, several species

Pseudaletia unipunctata (armyworm) Carpophilus lugubris (dusky sap beetle)

Graphognathus spp. (white-fringed beetles) Stink bugs, several species

Grasshoppers, many species Diatraea crambidoides (southern cornstalk borer)

Chaetocnema pulicaria (corn flea beetle) Anuraphis maidiradicis (corn root aphid)MIR162-USDA-1 Page 13 of 268 In addition to direct damage caused by feeding on plant tissue, insects play an important role in the transmission and dissemination of pat hogenic organisms during maize development. Soil abounds in microorganisms, particularly f ungi, which may infect plant parts injured by soil-dwelling insects. In much of the Corn Belt, pathogenic fungi probably pose more problems in maize production than any other gro up of microorganisms. Primary roots of the seedling and the radical and seminal roots are commonly infected with Fusarium spp. after
the roots have served their function and become senescent. Feeding by Diabrotica
rootworms has been associated with increased frequencies of Fusarium infection (Dicke and Guthrie, 1988); rootworm feeding may also lead to increased incidences of stalk rots. These pathogenic infections can lead to reduced crop quality, harvestability, and yield.

Ear, kernel, and cob rots occur wherever ma ize is grown and can re sult in reduced test weight, poor grain quality, and mycotoxi n contamination of food and feed. Fusarium kernel or ear rot is the most widespread disease of maize ears and is frequently associated with insect feeding damage.

Crop losses attributable to O. nubilalis and Diabrotica infestations have been well characterized and are significant. The introduc tions of transgenic cultivars which encode proteins that are toxic to these species have provided U.S. maize growers with a powerful tool for effectively protecting crop yields. There is not as much quantitative information available on the economic impacts of other major insect pests of maize, specifically H. zea

, S. frugiperda

, A. ipsilon , and S. albicosta . These pests are not as widespread as corn borers and rootworms; however, crop infestations by these leaf and ear-feeding pests can be very costly to growers, as they have the potential to significantly lower gr ain yield and quality. Conventional insecticide applications are an option for reducing feeding damage caused by these insects; however, most grow ers do not treat their crops to control these pests because of
cost and limited effectiveness of the chemi cal agents. Currently available transgenic
varieties are not as efficacious against these lepidopteran in sects as they are against O.
nubilalis . For example, Bt11 maize containing th e Cry1Ab toxin provides only limited or no protection against feeding damage caused by H. zea

, S. frugiperda

, A. ipsilon , and S.
albicosta

.

Transformation event MIR162 maize has been developed by Syngenta to provide U.S. growers with maize hybrids that are resistan t to feeding damage caused by a number of lepidopteran insect pests. MIR162 maize contains a Vip3Aa protein from Bacillus thuringiensis that is highly toxic to H. zea

, S. frugiperda

, A. ipsilon , and S. albicosta larvae.
In combination with an O. nubilalis -protected maize trait, the Vip3Aa protein in MIR162 can provide growers the means of protecting their maize crops from damage caused by a broad range of lepidopteran pests. Commercialization of this new trait has the potential to reduce conventional insecticide use in maize, increa se grower profits, and improve grain quality.

I.C. Status with Other Regulatory Agencies

Syngenta is actively pursuing regulatory appr ovals for MIR162 maize in countries with functioning regulatory systems for genetically modified organisms and that import maize from the U.S. or Canada. Regulatory filings for MIR162 will be made in Mexico, Colombia,MIR162-USDA-1 Page 14 of 268 Japan, South Korea, Taiwan, China, the Phi lippines, Australia and New Zealand, South Africa, the European Union, Russia, and Switzerland.

I.C.1. U.S. Environmental Protection Agency

Substances that are pesticides as defined under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) are subject to regu lation by the Environmen tal Protection Agency (EPA). The Vip3Aa20 protein encoded by the genetic insert in MIR162 maize has insecticidal propertie s and is, therefore, regulated by EPA. Syngenta has obtained an experimental use permit from EPA that allo ws for broad-scale field testing of MIR162 maize; this permit was granted on March 26, 2 007 and is in effect through March 31, 2008. On April 4, 2007 EPA established a temporary exemption from the requirement of a tolerance for Vip3Aa20 residues in maize commod ities, pursuant to §408(d) of the Federal Food, Drug, and Cosmetic Act, 21 U.S.C. §346a (d). Applications to extend both the experimental use permit and temporary tolerance exemption have been submitted to EPA.

On May 17, 2007 Syngenta submitted to EPA an application for regist ration of the plantincorporated protectant encoded by MIR162 ma ize pursuant to FIFRA §3(c)(5) and a petition to establish a permanent exemption from the requirement of a tolerance for Vip3Aa20 residues in food and feed commodities of all crops.

I.C.2. U.S. Food and Drug Administration

MIR162 maize falls within the scope of th e Food and Drug Administ ration’s (FDA) policy statement concerning regulation of food products derived from new plant varieties, including those developed by recombinant DNA techniqu es (FDA, 1992). Syngen ta has initiated a voluntary consultation process with FDA an d has submitted a safety and nutritional
assessment document for MIR162 maize.

I.C.3. Foreign Governments

Syngenta intends to commercialize MIR162 maize in Canada and will seek regulatory approvals to enable this. Syngenta will also seek regulatory approvals to commercialize MIR162 in other countries where armyworm s, cutworms, and ear-feeding lepidopteran species are significant ec onomic pests of maize ( e.g., Argentina and Brazil). Syngenta will also pursue regulatory approvals for im portation of MIR162 maize commodities and processed goods in key export markets for U.S. and Canadian maize growers ( e.g. , Japan, Taiwan, Korea, Philippines, and Mexico).

II. The Maize Family

II.A. Maize as a Crop

Zea mays L. subspecies mays , known as maize throughout the worl d, and as corn in the U.S., is one of the few major crop species indigenous to the Wester n Hemisphere. It has been cultivated in the Americas since early historic times. Field maize is the leading productionMIR162-USDA-1 Page 15 of 268 crop globally, with the 2005/2006 growing season yielding 695 million metric tons of grain (USDA, 2006a). The U.S. accounts for nearly 41% of global maize prod uction. Maize is the largest crop grown in the U.S. in terms of both volume and value. Approximately 78.3 million acres were planted in 2006, yielding 10. 5 billion bushels (267 million metric tons) with a gross crop value of $33.7 billion (USDA, 2007a).

Maize is grown for animal f eed, human food, vegetable oil, high fructose corn syrups (HFCS), starch, fermentation into ethanol, and a multitude of industrial uses. U.S. maize usage by market segment is shown in Figure 1. Maize as a source of fuel ethanol has increased dramatically over the past two years and is expected to continue doing so as the U.S. focuses on utilizing renewable sources of energy. By 2010, U.S. ethanol production could displace the equivalent of 311,000 ba rrels of imported crude oil per day.

Feed/Residual
Export

Ethanol

HFCS

Starch

Sweetners

Cereal/Other

Figure 1. U.S. maize usage by segment in 2006.

The U.S. is by far the world’s largest expor ter of maize, accounting for 68% of global exports. Total U.S. agricultural exports in 2006 were valued at $71 billion, 10% of which was attributable to maize (Brooks, 2007). Agricultural exports generate employment, income, and purchasing power in both farm a nd nonfarm sectors of the economy. Production from almost one-third of U.S. cropland move d into export channels in 2005 and generated $166.1 billion in business activity. Technology advances increase agri cultural pr oductivity and keep domestic growers comp etitive in the global market.

II.B. Biology of Maize

Maize is probably the most studied and best characterized of the crops grown in North America. The biology of maize ( Zea mays L. subspecies mays , corn) has been summarized in a consensus document prepared by the Organisation for Economic Co-operation and Development (OECD, 2003). Other authoritativ e works on the origin of maize (Galinat, 1988; Goodman and Brown, 1988) , its genetics (Coe et al. , 1988), physiology (Hageman and 50.8%

19.1% 18.3% 4.4%

2.4% 2.0% 3.0%MIR162-USDA-1 Page 16 of 268 Lambert, 1988), and breeding (Hallauer et al. , 1988) are also available.

Maize is a large, annual monoecious grass; the duration of its lif e cycle depends on the
cultivar and the environment in which the cultivar is grown. The bulk of maize is grown
between latitudes 30  ̊ and 47  ̊ . Practically no maize is grown where the mean midsummer temperature is < 19  ̊ C or where the average nighttime te mperature during the summer months
falls much below 13  ̊ C. The greatest production occurs where the warmest month isotherms range between 21  ̊ and 27  ̊ C and the freeze-free season last s 120 to 180 days. Maize is grown in areas where annual pr ecipitation ranges from 25 to > 500 cm. Summer rainfall of 15 cm is approximately the lower lim it for maize production without irrigation.

The upper Midwest region of the U.S. provides an ideal combination of temperature, rainfall, and soil type for the cultivation of maize. Iowa, Illinois, Nebraska, Minnesota, Indiana, Ohio, Wisconsin, Missouri, Kansas, and Sout h Dakota are major maiz e growing states.
Production in these ten states accounts for 84 % of total annual production (USDA, 2007a). Figure 2 displays the geographic distributi on of acres planted in 2006 (USDA, 2007b).

Farmers have hundreds of maize varieties from which to choose. Available varieties differ widely in agronomic characteristics, in cluding length of grow ing period. Technology providers continue to develop varieties with desirable traits and increasing yield. Maize yields have increased an average of 3.5 bu/ac per year over the past decade. The average yield reported for the 2006 growing season was 149.1 bu/ac (NCGA, 2007).

The adoption of new maize varieties improve d through biotechnology has added greatly to farm productivity and profits since thei r introduction in 1996 (Fernandez-Cornejo and Caswell, 2006). Varieties containing herbicide- tolerance and insect-pro tection traits have been widely adopted by maize, soybean, an d cotton growers because they protect the inherent yield potential of these crops or typically reduce grower input costs. In 2007,
herbicide tolerant varieties accounted for 91% of soybean acres planted, 70% of cotton acres planted, and 52% of maize acr es planted (USDA, 2007c). Insect-protected varieties
accounted for 59% of cotton and 49% of maize acr es planted. Planting of these varieties has also benefited the environment by displacing conventional pesticid e applications and reducing the production of greenhouse gases. Th e adoption of biotech maize varieties has been estimated to reduce the application of c onventional pesticides by more than 20 million pounds annually (NCGA, 2007).

II.C. Weediness of Maize

Modern-day maize cannot survive outside of cultivation (Gould and Shaw, 1968). Volunteer maize is not found growing in fence rows, ditche s, or roadsides as a weed. Although maize seed from a previous year’s crop can overwin ter and germinate the following year, it cannot persist as a weed. The appearance of maize in soybean fields following a maize crop is a
common occurrence. Manual or chemical meas ures are often applied to remove these volunteers, but the plants that are not re moved do not persist in following years.MIR162-USDA-1 Page 17 of 268 Figure 2. Geographic distribution of acres planted with maize in 2006.

It is difficult for maize to survive as a weed because of past selection in its evolution. In contrast to weedy species, maize has a polystic hous female inflorescence on a stiff central spike enclosed with husks. Consequently, seed dispersal of individual seeds does not occur naturally because of the struct ure of the maize ears. Indi vidual seeds, however, can be dispersed during grain harvest an d transportation. In neither instance does maize become a
troublesome weed. Maize is no t suited to survive without human assistance and is not capable of surviving as a weed.

II.D. Characteristics of the Recipient Germplasm

The recipient germplasm for transformati on to produce MIR162 was Hi-IIxA188. This germplasm is well suited for Type II cultures of maize, has a high transformation frequency, and is easily regenerated from callus. Hi-II is publicly available fr om the Maize Genetics
Stock Center. 1 Inbred line Hi-II, created from a cr oss of line A188 with line B73, was specifically developed for use in embryoni c tissue transformation systems (Armstrong et al.

, 1991). A188 is a publicly available stiff-stal k inbred developed at the University of

1 USDA/ARS. Maize Genetics Cooperation Stock Center , University of Illinois at Urbana, Champaign, Department of Crop Sciences. http://maizecoop.cropsci.uiuc.edu/MIR162-USDA-1 Page 18 of 268 Minnesota. B73 is an elite, publicly availabl e stiff-stalk inbred de veloped at Iowa State University.

III. Transformation and Development of MIR162 Maize

III.A. Description of the Transformation System

Event MIR162 was produced by Agrobacterium tumefaciens -mediated transformation of immature embryos of maize. Agrobacterium -mediated transformation is an efficient method for generating transformants containing simple and low copy number insertions with stable inheritance at a high frequency (Grimsley et al. , 1987; Ishida et al. , 1996). Using this method, DNA within a left border (LB) and ri ght border (RB) of a transformation plasmid,
referred to as the transferred DNA (T-DNA), is integrated into the genome of infected cells, while genetic elements outside of the plasmid borders are not.

MIR162 transformation employed a binary vector system (Hoekema, 1983). Plasmid vector pNOV1300 containing the manA gene from E. coli and maize-optimized coding sequence for vip3Aa19 between the right and left borders was placed into A. tumefaciens strain LBA4404 (licensed from Japan Tob acco, Inc.); see Figure 3.

Figure 3. Plasmid map for vector pNOV1300. 

## pNOV1300 (14,405 bp) vip3Aa19 (2370 bp)

manA (1176 bp) spec (789 bp)

LB (25 bp) RB (25 bp)

iPEPC9 (108 bp)

ZmUbiInt (1993 bp) ZmUbiInt (1993 bp) ColE1ori (807 bp)

35S terminator (70 bp)

NOS terminator (253 bp) cos (432 bp)

Kpn I (4786) Hin dIII (189)

X

ma I (8293)

Eco RV (6965) Sph I (199)

Sph I (2586)

Sph I (4797) Sph I (14,324)

A cc 65I (4782)MIR162-USDA-1 Page 19 of 268 Replication of pNOV1300 in A. tumefaciens was made possible by homologous recombination with an ‘acceptor vector’ carry ing the broad host range replicon origin RK2, which is also known as pSB1 (Komari et al. , 1996). A. tumefaciens strain LBA4404 carries a disarmed Ti plasmid from which the native T-DNA has been removed (Ooms et al. , 1982). The Ti plasmid carries the vir genes which encode proteins that are required for release of the T-DNA from pNOV1300 and its integrati on into the maize nuclear genome.

Immature embryos were excised from 8- to 12 -day old ears of Hi-IIxA188 maize and rinsed with fresh medium in preparation for transf ormation. Embryos were then mixed with a suspension of A. tumefaciens harboring vector pNOV1300. Following a five-minute incubation, excess solution was aspirated and embr yos were transferred to plates containing culture medium. Embryos and the Agrobacterium were incubated at 22 °C for two to three days in the dark. Embryos we re transferred to culture medi um containing ticarcillin and silver nitrate and incubated for ten days . Embryos producing embryogenic callus were transferred to cell culture medium containing mannose. The manA gene encodes the enzyme phosphomannose isomerase (PMI) that served as a selectable marker and enables transformed cells to survive on a mannose substrate (Negrotto et al. , 2000).

Transformed tissue was transf erred to culture medium co ntaining 500 mg/l of the broadspectrum antibiotic cefotoxime to clear the A. tumefaciens ; the transformed tissue was allowed to grow for four months. Regenerated plantlets were tested for the presence of both vip3Aa19 and manA , as well as for the spectinomycin resistance gene ( spec ), by TaqMan polymerase chain reaction (PCR) (Ingham et al. , 2001). Plants that te sted positive for both vip3Aa19 and manA , but negative for spec , were transferred to a greenhouse for growth and propagation.

III.B. Development of MIR162 Maize

Progeny of the origin al transformants (T 0 plants) were field tested for resistance to insect feeding damage and for agronomic performance after introgression of the transgenes into multiple elite lines of maize. Event MIR162 was selected as the lead commercial candidate and placed into regula tory trials. A schematic showing the steps in development of MIR162 maize is shown in Figure 4. All interstate movements and field plantings of MIR162 were conducted under USDA permit or notificati on; these are listed in Appendix A.

III.C. Production of Test and Control Materials

A number of factors ( e.g. , seed availability, trial location, hybrid maturity group) bore on the selection of plant materials for use in field and greenhouse studies with MIR162 maize. With the exception of plant materials utilized in the genetic characteriza tion studies, finished hybrids, produced from converted MIR162 inbreds, were selected for use as test materials in regulatory studies. A converted inbred is a line in which a specific trait(s) has been introgressed and genetically stab ilized through backcrossing, followed by self-pollinations. Parental control hybrids (near-iso genic hybrids) were utilized in all studies except the genetic characterization study. Control plants utilized in the study to characterize the T-DNA were derived from null segregants of a hemizygous MIR162 parent. The goal in producing allMIR162-USDA-1 Page 20 of 268 Figure 4. Steps in the development of MIR162 maize. vip3Aa1 gene from B.thuringiensis strain AB88

Synthesis of vip3Aa19 and manA genes and

assembly of pNOV1300 vector in E. coli

Transformation of Hi-IIxA188 embryos with A. tumefaciens containing plasmid vector pNOV1300

Selection of transformation events on medium containing mannose

Regeneration of transformed cells into maize plants

Evaluation of transformants

for insecticidal efficacy

Introgression of transformation events into elite inbred lines for evaluation of agronomic performance

Selection of MIR162 as lead event

Introgression of MIR162 transgenes into commercial

breeding lines and field Regulatory studies to
assess human and
environmental risksMIR162-USDA-1 Page 21 of 268 control hybrids was to match th eir genetic background with th at of the MIR162 hybrids so
that the effect of the transfor mation event could be assessed in an unbiased manner. Table 2 provides a listing of the seed lo ts used for planting of regulato ry trials. A pedigree chart showing the origin of MIR162 seed lots is shown in Figure 5.

Table 2. Description of test and control seed ma terials used in regulatory studies. The genetic identity is shown for test and cont rol seed materials planted in regulatory trials; generation number in this table is derived from the pedigree chart displayed in Figure 4. The female parent germplasm is stated first in the pedigree for each generation. The parent genotype containing the MIR162 transgenes is accompanied by the generation number in parentheses.

Trial Type Seed Lot
Code Generation MIR162 Genotype Control Genotype

Genetic characterization &
Mendelian inheritance A BC 1 F 1 B9620 B9620

Genetic characterization &
Mendelian inheritance B BC 2 F 1 B9620 B9620

Genetic characterization & Mendelian inheritance; Characterization of plant transgenic protein C BC 4 F 1 B9620 B9620

Germination / dormancy D F 1 NP2391 x NP2222(BC 6 F 4 ) NP2391 x
NP2222

Germination / dormancy E F 1 NP2222(BC 6 F 5 ) x

NP2391 NP2391 x
NP2222

Pollen viability / morphology E F 1 NP2222(BC 6 F 5 ) x

NP2391 NP2222 x
NP2391

2005 agronomic equivalence F F 1 NP2010 x NP2222(BC 6 F 4 ) NP2222 x
NP2010

2006 agronomic equivalence I F 1 NP2673 x NP2171(BC 4 F 5 ) NP2673 x
NP2171

Compositional analysis G & H F 1 & F 2 NP2276(BC 4 F 4 ) x

NP2391 NP2276 x
NP2391

Tissue protein quantification – Hybrid 1 G & H F 1 & F 2 NP2276(BC 4 F 4 ) x

NP2391 NP2276 x
NP2391

Tissue protein quantification – Hybrid 2 J & K F 1 & F 2 NP2153(BC 4 F 4 ) x

NP2391 NP2153 x
NP2391

All test and control seed lots were analyzed by re al time PCR methods for the presence of MIR162 and adventitious transgenic events. All MIR162 seed lots were confirmed to contain the desired gene of interest and pl ant selectable marker based on nucleotide sequence, and the event of inte rest based on the genome-insert junction nucleotide sequence. The analyses did not detect thes e components in control seed lots . All test and control seed lots had no detectable sequences that would be indi cative of other re gulated events underMIR162-USDA-1 Page 22 of 268 Figure 5. MIR162 pedigree chart. Generations in blue and bold type were u sed in regulatory studies; boxed letter code corresponds to specific seed lots used in regulatory studies (see Table 2). T 0

F 1 x NPH8431

F 1 F 1

BC 1 F 1 BC 1 F 1

BC 2 F 1

BC 3 F 1

BC 4 F 1

F 1

BC 1 F 1

BC 4 F 1 x NP2010

BC 4 F 2

BC 4 F 4 BC 4 F 3

F 1 NP2161 x

bc

F 1 BC 2 F 1

bc bc bc bc

BC 1 F 1

BC 2 F 1

BC 3 F 1

BC 4 F 1 bc bc

BC 2 F 1

BC 3 F 1 F 1 bc

BC 1 F 1 x NP2222 B9620 x bc

BC 6 F 1

BC 6 F 2

BC 6 F 4 ⊗

BC 6 F 3 ⊗

BC 6 F 5 F 1
bc

BC 1 F 1

BC 4 F 1
⊗

BC 4 F 2

⊗

BC 4 F 4 ⊗

BC 4 F 3

F 1

x NP2391

⊗ x NP2276

F 1 NP2391 x ⊗ T 0 – original transformant

x – cross bc – backcross

⊗ - self-pollination

F 1 F 2 x NP2391 ⊗ ⊗ ⊗ bc x NP2153 bc bc bc bc

F 1

BC 1 F 1

BC 4 F 1 bc (3 times) bc NP2171 x

F 1 BC 4 F 5 BC 4 F 2 ⊗

⊗

⊗

⊗

x NP2673 x NP2391 ⊗

⊗

F 2 x NP2010

F 1 bc (3 times) bc (3 times) bc (3 times)

BC 4 F 3

BC 4 F 4 B

C A

H D

E F G

I K JMIR162-USDA-1 Page 23 of 268 development at Syngenta, or deregulated even ts for which testing methodology is available.

A number of human and environm ental safety studies employed purified Vip3Aa proteins as the test substances. One of three Vip3Aa va riants was used: Vip3Aa1, the native protein
from B. thuringiensis strain AB88; Vip3Aa19, the variant found in transgenic COT102 cotton and Pacha maize; or Vip3Aa20, the va riant found in MIR162 maize. Vip3Aa1 was
extracted from a culture of B. thuringiensis strain AB88. Vip3Aa 19 and Vip3Aa20 were produced in recombinant cultures of Escherichia coli . The three proteins share >99.7% amino acid sequence homology and have been s hown to be biochemica lly and functionally equivalent.

IV. Donor Genes and Regulatory Sequences

IV.A. Transformati on Vector pNOV1300

MIR162 maize was produced by Agrobacterium tumefaciens -mediated transformation of immature embryos using the plasmid vect or pNOV1300. The T-DNA region between the left and right borders, which included the vip3Aa19 and manA gene expression cassettes, was inserted into the maize genome during transfor mation. The first expression cassette consists
of the vip3Aa19 coding region regulated by the Z. mays polyubiquitin promoter (ZmUbiInt)

and 35S 3 ′ polyadenylation sequences. The second expression cassette consists of the manA coding region regulated by the ZmUbiInt pr omoter and the nopaline synthase (NOS) polyadenylation sequence. A schematic of the vector is shown in Fi gure 3. The size and location of each genetic element in the v ector are shown below in Table 3 below.

Table 3. Description of genetic elements inserted in vector pNOV1300.

Genetic

Element Location in pNOV1300 (bp) Size (bp) Source and Function

RB 1 - 25 25 Right border region of T-DNA from A. tumefaciens nopaline Ti-plasmid (Entrez Accession Number J01826; NCBI, 2007). This region is a short, direct repeat that flanks the T-DNA and is required for transfer of the T-DNA into plant cells (Wang et al ., 1984).

ZmUbiInt 200 - 2192 1993 Promoter region from Z. mays polyubiquitin gene which contains the first intron (Entrez Accession Number S94464; NCBI, 2007). Provides constitutive expression in monocots (Christensen et al ., 1992).

vip3Aa19 2214 - 4583 2370 A variant of the native vip3Aa1 gene (Estruch et al.

, 1996) from B. thuringiensis strain AB88. The vip3Aa19 gene was codon optimized for expression in maize (Murray et al. , 1989). The vip3A19 gene (Entrez Accession Number DQ539887; NCBI, 2007) encodes a Vip3Aa19 protein that has insecticidal activity against many lepidopteran insect pests.
(Continued)MIR162-USDA-1 Page 24 of 268 Table 3 (cont.). Description of genetic elements inserted in vector pNOV1300.

Genetic

Element Location in pNOV1300 (bp) Size (bp) Source and Function

iPEPC9 4600 - 4707 108 Intron #9 from the phosphoenolpyruvate carboxylase gene (Entrez Accession Number X15239; NCBI, 2007)
from Z. mays (Hudspeth and Grula, 1989).

35S 4710 - 4779 70 Terminator sequence from 35S RNA of cauliflower mosaic virus genome (Similar to Entrez Accession Number AF140604; NCBI, 2007). Its function is to provide a polyadenylation sequence (Franck et al ., 1980).

ZmUbiInt 4798 - 6790 1993 Promoter region from Z. mays polyubiquitin gene which contains the first intron (Entrez Accession Number S94464; NCBI, 2007). Provides constitutive expression in monocots (Christensen et al ., 1992).

manA 6803 - 7978 1176 manA gene from E. coli strain K-12 encoding the enzyme phosphomannose isomerase (PMI; Entrez Accession Number M15380; NCBI, 2007). PMI catalyzes the interconversion of mannose-6-phosphate to fructose-6- phosphate (Negrotto et al ., 2000). Used as a selectable marker during transformant selection.

NOS 8039 - 8291 253 Terminator sequence from the nopaline synthase gene of A. tumefaciens (Entrez Accession Number V00087; NCBI, 2007). Its function is to provide a polyadenylation site (Depicker et al ., 1982).

LB 8362 - 8386 25 Left border region of T-DNA from A. tumefaciens nopaline Ti-plasmid (Entrez Accession Number J01825; NCBI, 2006). This region is a short, direct repeat that flanks the T-DNA and is required for the transfer of the T-DNA into the plant cell (Zambryski et al ., 1982).

spec 9562 - 10350 789 Spectinomycin adenylyltransferase aadA gene from E.
coli Tn7 (Entrez Accession Number X03043; NCBI, 2007). Confers resistance to erythromycin, streptomycin, and spectinomycin; used as a b acterial selectable marker (Fling et al ., 1985).

ColE1ori 11549 - 12355 807 Origin of replication that permits replication of plasmid
in E. coli . (similar to Entrez Accession Number V00268: NCBI, 2007) (Itoh and Tomizawa, 1978).

cos 12736 - 13167 432 Cohesive end site that is cut to produce the cohesive, single-stranded extensions located at the ends of the linear DNA molecules of certain phages, such as lambda (Sanger et al ., 1982).MIR162-USDA-1 Page 25 of 268 IV.B. Characterization of the T-DNA in MIR162 Maize

A combination of Southern blot, nucleotide sequence, and Mendelian inheritance analyses were performed to characterize the MIR162 ma ize T-DNA. As described in detail below, data from Southern analyses demonstrated that MIR162 maize contains: i) a single intact insert in the maize genome; ii) single copies of a vip3Aa20 gene and a manA gene; iii) two copies of the ZmUbiInt prom oter; iv) one copy of the NOS terminator; and v) no backbone sequences from transformation plasmid pNOV1300.

The MIR162 T-DNA was found to be stable ove r three breeding generations. Sequence analysis of the entire T-DNA present in MI R162 maize confirmed the intactness of the T-DNA and that the contiguousness of the func tional elements had been maintained. Sequence analysis also revealed tw o single nucleotide changes in the vip3Aa coding sequence contained in the MIR162 maize T-DNA, as compared with the sequence present in the transformation plasmid. One of these mutati ons resulted in a single codon change for the amino acid originally encoded, while the ot her mutation was silent. The new gene incorporated into the MIR162 ma ize genome has been designated vip3Aa20 . See Section
V.A for a discussion of the Vi p3Aa variants. Nucleotide seque nce analysis also confirmed the presence of a single, intact DNA insert in MIR162 maize. A schematic representation of the T-DNA in MIR162 maize is shown in Figure 6.

Figure 6. Schematic representation of the MIR162 T-DNA.
This schematic shows the linear orientation of the genetic elements and restriction
endonuclease sites within the T-DNA.

Statistical analysis of gene segregation patt erns over three generati ons of MIR162 maize confirmed the expected Mendelian inheritance ratio for both the vip3Aa20 and manA genes; this segregation pattern was consistent with the genes being linked at a single locus. The
MIR162 maize DNA insert does not disrupt any known endogenous Z. mays genes. There is a region of homology between the ma ize genomic sequence flanking the 5 ′ region of the MIR162 maize DNA insert and maize transposab le element sequences. There are also regions of homology between the ma ize genomic sequence flanking the 3 ′ region of the MIR162 maize T-DNA and identified maize seque nces. These regions of homology likely do not have any function. Additionally, no novel open reading frames were identified that spanned either the 5 ′ or 3 ′ junction between the MIR162 maize T-DNA and Z. mays genomic iPEPC9 (108 bp)

35S terminator (70 bp) NOS (253 bp) ZmUbiInt (1993 bp) ZmUbiInt (1993 bp) vip3Aa20 (2370 bp) manA (1176 bp) Plant genomic
DNA Plant genomic
DNA Hin dIII (189) Sph I (199) Sph I (2586)

Sph I (4797)

X ma I (8293) A c c 65I (4782)

Kpn I (4786) Eco RV (6965)MIR162-USDA-1 Page 26 of 268 sequence. A detailed descri ption of the materials and methods for the molecular
characterization of MIR162 mai ze can be found in Appendix B.

IV.B.1. Sequence of the T-DNA

Consensus nucleotide sequence data demonstrat ed that the MIR162 T-DNA is intact and that the contiguousness of the functi onal elements within the insert as intended in pNOV1300 has been maintained. Two nucleotide changes we re noted within the coding sequence of the vip3Aa gene in MIR162 maize. The first of th ese nucleotide changes results in a single amino acid change: methionine at positi on 129 of Vip3Aa19 has been substituted by isoleucine (M129I). The second nucleotide change within the vip3Aa coding sequence did not result in an amino acid change. The vip3Aa gene in MIR162 maize was given the designation vip3Aa20 (Entrez Accession Number DQ 539888; NCBI, 2007). The coding sequences of manA , the ZmUbiInt promoters, the 35S te rminator, and the NOS terminator in
MIR162 maize were identical to those in the transformation plasmid pNOV1300. Sequence analysis revealed that some truncation occurred at the right border (RB) and left border (LB) ends of the T-DNA during the transformation pro cess. The entire RB, along with two base pairs of noncoding sequence was truncated, and the entire LB, along w ith 32 base pairs of noncoding sequence, was truncated. These de letions have no appa rent effect on the functionality of the DNA insert as this phenomenon has been previously observed in
transformations with A. tumefaciens (Tinland and Hohn, 1995; Brunaud et al ., 2002; Chilton and Que, 2003).

IV.B.2. Copy Number of Functional Elements

The hybridization data demonstrated that th e T-DNA in MIR162 contains single copies of
the vip3Aa20 and manA genes. As expected, the MIR 162 maize T-DNA contains two copies of the ZmUbiInt promoter, co rresponding to the two copies of the ZmUbiInt promoter present in the transformation plasmid pNOV1 300, and contains a single copy of the NOS terminator. The Southern blot analyses de monstrated that MIR 162 maize contains no pNOV1300 plasmid backbone sequences.

For each Southern blot analyses, there is a map showing the locatio n of the restriction endonuclease digestion sites in rela tion to each element probe used in the analys is. These are found in Figures 7, 9, 11, and 13. The Southern blots are shown in Figures 8, 10, 12, and 14, respectively. In addition, comparisons of th e expected and observed hybridization bands for each Southern blot are displayed in Tables 4, 5, 6, and 7 as an aid to the reader.

IV.B.2.a. Copy Number of Functional Elements: vip3Aa20 Gene

A vip3Aa19 -specific probe was used for the vip3Aa20 Southern blot analyses. The nucleotide sequences of vip3Aa19 and vip3Aa20 are 99.9% identical, differing by only two nucleotides. The two nucleotide changes di d not interfere with the ability of the vip3Aa19 probe to hybridize with the vip3Aa20 coding sequence in MIR162 maize.

A map of the T-DNA region in vector pNOV1300 indicating locations of the vip3Aa19MIR162-USDA-1 Page 27 of 268 coding sequence and the restric tion endonuclease sites targeted in these Southern blot analyses is shown in Figure 7. Three endonucl ease digestion strategies were utilized to confirm the presence, size, intactness, and copy number of the vip3Aa20 coding sequence.

Figure 7. Restriction sites and probe for vip3Aa20

. A schematic of the 8.4 kb T-DNA of pNOV1300 is shown along with the location of the Kpn I, Eco RV, Hin dIII, and Xma I restriction sites and their location relative to the vip3Aa20 coding sequence. The vertical arrows indicate the endonuclease cutting sites and the expected size of the resulting restriction fragments that should be detected when probed with vip3Aa19

.

The results of these analyses are displayed in Figure 8. Table 4 shows a comparison of expected versus observed hybridization band sizes. >7.0 kb

~8.1 kb >4.8 kb

Hin dIII +Xma I Kpn I

Eco RV vip3Aa19 (2370 bp) manA (1176 bp)

RB (25 bp) LB (25 bp) iPEPC9 (108 bp)

vip3Aa19 probe (2370 bp) ZmUbiInt (1993 bp) ZmUbiInt (1993 bp) 35S terminator (70 bp)

NOS (253 bp) Kpn I (4786) Eco RV (6965)

Hind III X ma IMIR162-USDA-1 Page 28 of 268 Figure 8. Southern blot analysis of MIR162 maize with a vip3Aa19 -specific probe

. Maize genomic DNA (7.5 μ g) was subjected to three separate digestions with Kpn I or Eco RV

or Hin dIII plus Xma I restriction endonucleases and subjected to Southern blot analysis with a vip3Aa19 -specific probe (2370 bp).

Lane 1: Molecular weight marker
Lane 2: Blank

Lane 3: BC 4 F 1 generation of MIR162 maize digested with Kpn I
Lane 4: Control from BC 4 F 1 generation maize digested with Kpn I
Lane 5: BC 4 F 1 generation of MIR162 maize digested with Eco RV
Lane 6: Control from BC 4 F 1 generation maize digested with Eco RV
Lane 7: BC 4 F 1 generation of MIR162 maize digested with Hin dIII + Xma I
Lane 8: Control from BC 4 F 1 generation maize digested with Hin dIII + Xma I
Lane 9: Control from BC 4 F 1 generation maize digested with Hin dIII + Xma I plus 20.2 pg of

Hin dIII plus Xma I digested pNOV1300 plasmid.MIR162-USDA-1 Page 29 of 268 Table 4. Expected versus observed hybridization band sizes for probes with vip3Aa19.

Lane # (Figure 8) DNA Restriction Enzyme Probe Expected

Bands Expected
Band Size (kb) Observed
Band Size (kb)

Lane 3 BC 4 F 1 Kpn I vip3Aa19 1 >4.8 8.0

Lane 4 Negative Kpn I vip3Aa19 0 - -

Lane 5 BC 4 F 1 Eco RV vip3Aa19 1 >7.0 14.0

Lane 6 Negative Eco RV vip3Aa19 0 - -

Lane 7 BC 4 F 1 Hin dIII +
Xma I vip3Aa19 1 8.1 8.1

Lane 8 Negative Hin dIII +
Xma I vip3Aa19 0 - -

Positive Control

Lane 9 pNOV1300 Hin dIII +
Xma I vip3Aa19 1 8.1 8.1

Genomic MIR162 maize DNA digested with Kpn I (Figure 8, Lane 3) produced a single hybridization band of approximately 8.0 kb, corresponding to a single copy of vip3Aa20

. Genomic MIR162 maize DNA digested with Eco RV (Lane 5) produced a single hybridization band of approximately 14.0 kb, corresponding to a single copy of vip3Aa20

. Genomic MIR162 maize DNA digested with Hin dIII plus Xma I (Lane 7) produced a single hybridization band of approximately 8.1 kb, corresponding to a single copy of vip3Aa20 and confirming the intactness of the insert. Th e negative control samples for each enzymatic digestion showed no hybridization bands and none were expected ( Kpn I Lane 4, Eco RV Lane 6, and Hin dIII plus Xma I Lane 8). The Hin dIII plus Xma I digestion of the pNOV1300 vector produced the expected hybridization signal at 8.1 kb (positive control, Lane 9).

For Southern blot analyses with the vip3Aa19 -specific probe, each restriction endonuclease digestion resulted in single hybri dization band of the expected size. This demonstrates that
the MIR162 maize T-DNA contains an intact vip3Aa20 coding sequence and the absence of bands other than the three expected indicates that there is only a si ngle copy of the gene
present.MIR162-USDA-1 Page 30 of 268 IV.B.2.b. Copy Number of Functional Elements: manA Gene

A map of the T-DNA region in the MIR 162 maize transformation vector pNOV1300 indicating locations of the manA coding sequence and the re striction endonuclease sites targeted in these Southern blot analyses is show n in Figure 9. Three di gestion strategies were utilized to confirm the presence, size, intactness, and copy number of the manA coding sequence. The results of these analys es are shown in Table 5 and Figure 10.

Figure 9. Restriction sites and probe for manA

. A schematic of the 8.4 kb T-DNA of pNOV1300 is shown along with the location of the Kpn I, Sph I, Hin dIII, and Xma I restriction sites and their location relative to the manA coding sequence. The vertical arrows indicate the endonuclease cutting sites and the expected size of the resulting restriction fragments that should be detected when probed with manA

.

The results of these Southern blot analyses are displayed in Figure 10. Table 5 shows a comparison of expected versus observed hybridization band sizes. Hin dIII vip3Aa19 (2370 bp) manA (1176 bp)

LB ( 25 bp ) RB ( 25 bp ) iPEPC9 ( 108 bp )

manA probe (1176 bp) ZmUbiInt (1993 bp) ZmUbiInt (1993 bp) 35S terminator (70 bp)

NOS (253 bp) Kpn I (4786)

X ma I Sph I (199) Sph I (2586) Sph I ( 4797 )

>3.6 kb

~8.1 kb >3.6 kb

Hin dIII + Xma I Kpn I

Sph IMIR162-USDA-1 Page 31 of 268 Figure 10. Southern blot analysis of MIR162 maize with a manA -specific probe

. Maize genomic DNA (7.5 μ g) was subjected to three separate digestions with Kpn I or Sph I or

Hin dIII plus Xma I restriction endonucleases and subjected to a Southern blot analysis with a

manA -specific probe (1176 bp).

Lane 1: Molecular weight marker
Lane 2: Blank

Lane 3: BC 4 F 1 generation of MIR162 maize digested with Kpn I
Lane 4: Control from BC 4 F 1 generation maize digested with Kpn I
Lane 5: BC4F 1 generation of MIR162 maize digested with Sph I
Lane 6: Control from BC 4 F 1 generation maize digested with Sph I
Lane 7: BC 4 F 1 generation of MIR162 maize digested with Hin dIII + Xma I
Lane 8: Control from BC 4 F 1 generation maize digested with Hin dIII + Xma I
Lane 9: Control from BC 4 F 1 generation maize digested with Hin dIII + Xma I plus 20.2 pg of

Hin dIII + Xma I digested pNOV1300 plasmid.MIR162-USDA-1 Page 32 of 268 Table 5. Expected versus observed hybridization band sizes for probes with manA

.

Lane # (Figure 10) DNA Restriction Enzyme Probe Expected

Bands Expected
Band Size (kb) Observed
Band Size (kb)

Lane 3 BC 4 F 1 Kpn I manA 1 >3.6 4.0

Lane 4 Negative Kpn I manA 0 - -

Lane 5 BC 4 F 1 Sph I manA 1 >3.6 9.0

Lane 6 Negative Sph I manA 0 - -

Lane 7 BC 4 F 1 Hin dIII +
Xma I manA 1 8.1 8.1

Lane 8 Negative Hin dIII +
Xma I manA 0 - -

Positive Control

Lane 9 pNOV1300 Hin dIII +
Xma I manA 1 8.1 8.1

Genomic MIR162 maize DNA digested with Kpn I (Figure 10, Lane 3) produced a single hybridization band of approximately 4.0 kb, corresponding to a single copy of manA

. Genomic MIR162 maize DNA digested with Sph I (Lane 5) produced a single hybridization band of approximately 9.0 kb, corresponding to a single copy of manA . Genomic MIR162 maize DNA digested with Hin dIII + Xma I (Lane 7) produced a single hybridization band of approximately 8.1 kb, corresponding to a single copy of manA and confirming the intactness of the insert. The negative control samples for each enzymatic digestion showed no hybridization bands and none were expected ( Kpn I Lane 4, Sph I Lane 6, and Hin dIII + Xma I Lane 8). The Hin dIII plus Xma I digestion of the pNOV1300 vector produced the expected hybridization band at 8.1 kb (pos itive control, Lane 9).

For the manA -specific probe Southern blot analyses , each restriction endonuclease digestion resulted in a single hybridization band closely matching the expected size for each. This
demonstrates that MIR162 maize contains an intact manA coding sequence and the absence
of bands other than the three e xpected, indicates that there is only a single copy of the gene
present.MIR162-USDA-1 Page 33 of 268 IV.B.2.c. Copy Number of Functional Elements: ZmUbiInt promoter

A map of the T-DNA region in vector pNOV 1300 indicating locations of the ZmUbiInt promoter and the restriction endonuclease site s targeted for use in these Southern blot analyses is shown in Figure 11. Three diges tion strategies were utilized to confirm the presence, size, intactness, and copy number of the ZmUbiInt pr omoter. The results of this analysis are shown in Table 6 and Figure 12.

Figure 11. Restriction sites and probe for the ZmUbiInt promoter. A schematic of the 8.4 kb T-DNA of pNOV1300 is shown along with the location of the KpnI, SphI, HindIII, and XmaI restriction sites and their location relative to the ZmUbiInt promoter. The vertical arrows indicate the endonuclease cutting sites and the expected size of the resulting restriction fragments that should be detected when probed with ZmUbiInt.

Since two copies of the ZmUbiInt promoter are present in the T-DNA region of plasmid pNOV1300 (see Figure 3), two unique bands were expected for the Kpn I and Sph I enzyme digestions, and one band for the Hind III and Xma I digestion. Additionally, ZmUbiInt is an endogenous maize promoter and, thus, the probe will hybridi ze to this endogenous maize

sequence.

The results of these Southern blot analyses are displayed in Figure 12. Table 6 shows a comparison of expected versus observed hybridization band sizes. >4.8 kb

~8.1 kb >3.6 kb

>3.6 kb ~2.4 kb

Hin dIII + Xma I Sph I Kpn I vip3Aa19 (2370 bp) manA (1176 bp)

LB (25 bp) RB (25 bp) iPEPC9 (108 bp)

ZmUbiInt probe (1993 bp) ZmUbiInt probe (1993 bp) ZmUbiInt ( 1993 bp ) ZmUbiInt (1993 bp) 35S terminator ( 70 bp )

NOS ( 253 bp )

Hin dIII (189) Sph I (199) Sph I (2586)

Sph I (4797)

X ma I (8293) Kpn I (4786)MIR162-USDA-1 Page 34 of 268 Figure 12. Southern analysis of MIR162 maize with ZmUbiInt-specific probe. Maize genomic DNA (7.5 μ g) was subjected to three separate digestions with Kpn I or Sph I or

Hin dIII plus Xma I restriction endonucleases and to Southern blot analysis with a ZmUbiInt promoter-specific probe (1993 bp).

Lane 1: Molecular weight marker
Lane 2: Blank

Lane 3: BC 4 F 1 generation of MIR162 maize digested with Kpn I
Lane 4: Control from BC 4 F 1 generation maize digested with Kpn I
Lane 5: BC 4 F 1 generation of MIR162 maize digested with Sph I
Lane 6: Control from BC 4 F 1 generation maize digested with Sph I
Lane 7: BC 4 F 1 generation of MIR162 maize digested with Hin dIII + Xma I
Lane 8: Control from BC 4 F 1 generation maize digested with Hin dIII + Xma I
Lane 9: Control from BC 4 F 1 generation maize digested with Hin dIII + Xma I plus 20.2 pg of

Hin dIII + Xma I digested pNOV1300 plasmid.MIR162-USDA-1 Page 35 of 268 Table 6. Expected versus observed hybridization band sizes for probes with ZmUbiInt.

Lane # (Figure 12) DNA Restriction Enzyme Probe Expected

Bands Expected
Band Size (kb) Observed
Band Size (kb)

>3.6 4.0

>4.8 8.0 Lane 3 BC 4 F 1 Kpn I ZmUbiInt 3 Unknown Endogenous 22.0

Lane 4 Negative Kpn I ZmUbiInt 1 Unknown Endogenous 22.0

~2.4 2.4

>3.6 9.0 Lane 5 BC 4 F 1 Sph I ZmUbiInt 3 Unknown Endogenous 2.8

Lane 6 Negative Sph I ZmUbiInt 1 Unknown Endogenous 2.8

8.1 8.1
Lane 7 BC 4 F 1 Hin dIII +
Xma I ZmUbiInt 2 Unknown Endogenous 4.0, 5.8

Lane 8 Negative Hin dIII +
Xma I ZmUbiInt 1 Unknown Endogenous 4.0, 5.8

8.1 8.1 Lane 9 pNOV1300 Hin dIII +
Xma I ZmUbiInt 2 Unknown Endogenous 4.0, 5.8

Genomic MIR162 maize DNA digested with Kpn I (Figure 12, Lane 3) produced two hybridization bands of approximately 4.0 kb an d 8.0 kb, corresponding to the expected size of the two copies of the Zm UbiInt promoter present in the T-DNA. There was also a hybridization band representing endogenous maize sequence at approximately 22.0 kb present in MIR162 maize (Lane 3) and the ne gative control (Lane 4). Genomic MIR162 DNA digested with Sph I (Lane 5) produced two unique hybridization signals of approximately 2.4 kb and 9.0 kb, corresponding to th e expected two copies of the ZmUbiInt promoter present in the T-DNA. There was al so a hybridization band observed representing endogenous maize sequence at approximately 2.8 kb present in MIR162 maize (Lane 5) and the negative control (Lane 6). Geno mic MIR162 maize DNA digested with Hin dIII + Xma I (Lane 7) produced a single hybridization signa l of approximately 8.1 kb, corresponding to the full-length MIR162 T-DNA which contains tw o copies of the ZmUbiInt promoter, thus confirming the intactness of the insert. Th ere were also two hybridization bands of approximately 4.0 kb and 5.8 kb present in bot h MIR162 maize (Lane 7) and the negative control (Lane 8) that repres ent endogenous ZmUbiInt sequence. The negative control corresponding to each digest showed no hybridization band ot her than that observed with endogenous ZmUbiInt sequences ( Kpn I Lane 4, Sph I Lane 6, and Hin dIII + Xma I Lane 8).
The Hin dIII + Xma I digestion of the pNOV1300 vector produced the expected hybridization signal at 8.1 kb (positive control, Lane 9) . Two hybridization bands of 4.0 and 5.8 wereMIR162-USDA-1 Page 36 of 268 observed which represent endogenous ZmUbiInt sequence.

For the Southern blot analyses with the ZmUb iInt promoter-specific probe, each restriction endonuclease digestion resulted in hybridizat ion bands demonstrating that MIR162 maize contains two intact copies of the ZmUbiInt promoter within the T-DNA and confirmed the known presence of endogenouos maize ZmUbiInt promoter sequence.

IV.B.2.d. Copy Number of Functional Elements: NOS Terminator

A map of the T-DNA region in pNOV1300 indicati ng locations of the NOS terminator and the restriction endonuclease sites targeted for thes e Southern blot analyses is shown in Figure 13. Three digestion strategies were utilized to c onfirm the presence, si ze, intactness, and copy number of the NOS terminator sequence.

Figure 13. Restriction sites and probe for NOS terminator. A schematic of the 8.4 kb T-DNA of pNOV1300 is shown along with the location of the Kpn I, Sph I, Hin dIII, and Xma I restriction sites and their location relative to the NOS
terminator. The vertical arrows indicate th e endonuclease cutting sites and the expected size of the resulting restriction that shoul d be detected when probed with NOS. ~8.1 kb >3.6 kb

>3.6 kb

Hin dIII + Xma I Sph I Kpn I vip3Aa19 (2370 bp) manA (1176 bp)

LB (25 bp) RB (25 bp) iPEPC9 (108 bp)

ZmUbiInt ( 1993 bp ) ZmUbiInt (1993 bp) 35S terminator ( 70 bp )

NOS ( 253 bp )

Hin dIII (189) Sph I (199) Sph I (2586)

Sph I (4797)

X ma I (8293) Kpn I (4786)

NOS probe (253 bp)MIR162-USDA-1 Page 37 of 268 The results of these Southern blot analyses are displayed in Figure 14. Table 7 shows a comparison of expected versus observed hybridization band sizes.

Figure 14. Southern analysis of MIR162 mai ze with NOS terminator-specific probe. Maize genomic DNA (7.5 μ g) was subjected to three separate digestions with Kpn I or Sph I or

Hin dIII plus Xma I restriction endonucleases and subjected to Southern blot analysis with a NOS-specific probe.

Lane 1: Molecular weight marker
Lane 2: Blank
Lane 3: BC 4 F 1 generation of MIR162 maize digested with Kpn I
Lane 4: Control from BC 4 F 1 generation maize digested with Kpn I
Lane 5: BC 4 F 1 generation of MIR162 maize digested with Sph I
Lane 6: Control from BC 4 F 1 generation maize digested with Sph I
Lane 7: BC 4 F 1 generation of MIR162 maize digested with Hin dIII + Xma I
Lane 8: Control from BC 4 F 1 generation maize digested with Hin dIII + Xma I
Lane 9: Control from BC 4 F 1 generation maize digested with Hin dIII + Xma I plus 20.2 pg of

Hin dIII + Xma I digested pNOV1300 plasmid.MIR162-USDA-1 Page 38 of 268 Table 7. Expected versus observed hybridization band sizes for probes with NOS.

Lane # (Figure 14) DNA Restriction Enzyme Probe Expected

Bands Expected
Band Size (kb) Observed
Band Size (kb)

Lane 3 BC 4 F 1 Kpn I NOS 1 >3.6 4.0

Lane 4 Negative Kpn I NOS 0 - -

Lane 5 BC 4 F 1 Sph I NOS 1 >3.6 9.0

Lane 6 Negative Sph I NOS 0 - -

Lane 7 BC 4 F 1 Hin dIII +
Xma I NOS 1 8.1 8.1

Lane 8 Negative Hin dIII +
Xma I NOS 0 - -

Lane 9 pNOV1300 Hin dIII +
Xma I NOS 1 8.1 8.1

Genomic MIR162 maize DNA digested with Kpn I (Figure 14, Lane 3) produced a single hybridization band of approximately 4.0 kb, co rresponding to a single copy of the NOS terminator. Genomic MIR162 maize DNA digested with Sph I (Lane 5) produced a single hybridization band of approximately 9.0 kb, co rresponding to a single copy of the NOS terminator. Genomic MIR162 maize DNA digested with Hin dIII + Xma I (Lane 7) produced a single hybridization band of approximately 8.1 kb, corresponding to a single copy of the NOS terminator and confirming the intactne ss of the insert. The negative control corresponding to each digest showed no hybridization bands and none were expected ( Kpn I Lane 4, Sph I Lane 6 and Hin dIII + Xma I Lane 8). The Hin dIII + Xma I digestion of the pNOV1300 vector produced the ex pected hybridization signal at 8.1 kb (positive control, Lane 9).

For the Southern blot analyses with the NOS terminator-specific probe, each restriction endonuclease digestion resulted in a single hybridization band of the expected size. This
demonstrates that the MIR162 maize T-DNA cont ains an intact NOS terminator sequence,
and the absence of bands other than the three expected indicates that there is only a single copy of the terminator present.

IV.B.3. Absence of Plasmid Backbone Elements

Genomic DNA was digested and probed for th e possible presence of plasmid backbone elements in a Southern blot analysis. None of these backbone elements should have been incorporated into the MIR162 maize genome. A map of the pNOV1300 plasmid vectorMIR162-USDA-1 Page 39 of 268 indicating the region covered by the backbone-sp ecific probe, the location of each backbone element, and the restriction e ndonuclease sites targeted for this Southern blot analyses is shown in Figure 15. Three digest ion strategies were utilized to test for the presence of
backbone elements.

Figure 15. Restriction sites and probe for plasmid backbone elements. A schematic of the pNOV1300 plasmid vector showing the location of the Kpn I, Eco RV,

Hin dIII, and Xma I restriction sites and their location relative to the region outside of the right and left borders of the T-DNA. The backbone-specific probe spans the 6019 base pairs
outside of the borders.

The results of these Southern blot analyses are displayed in Figure 16. Table 8 shows a comparison of expected versus observed hybridization band sizes. Genomic MIR162 maize DNA was digested with Kpn I (Lane 3), Eco RV (Lane 5), and Hin dIII + Xma I (Lane 7). The blot was hybridized with a probe covering th e entire backbone re gion of pNOV1300. No detectable hybridization bands were observed in the MIR162 maize genomic samples (Lanes 3, 5, and 7) or in the negative control samples (Lanes 4, 6, and 8). The Hin dIII plus Xma I digestion of the pNOV1300 vector produced an expected 6.3 kb band (Lan e 9). These results
demonstrate that the MIR162 ma ize T-DNA does not contain any backbone sequences from the pNOV1300 transformation plasmid. 

## pNOV1300 (14,405 bp) vip3Aa19 (2370 bp)

manA (1176 bp) spec (789 bp)

LB (25 bp) RB (25 bp)

iPEPC9 (108 bp)

ZmUbiInt (1993 bp) ZmUbiInt (1993 bp) ColE1ori (807 bp)

35S terminator (70 bp)

NOS terminator (253 bp) cos (432 bp)

Kpn I (4786) Hin dIII (189)

X

ma I (8293)

Eco RV (6965)MIR162-USDA-1 Page 40 of 268 Figure 16. Southern blot analysis for plasmid backbone sequence. Maize genomic DNA (7.5 μ g) was subjected to three separate digestions with Kpn I or Eco RV

or Hin dIII plus Xma I restriction endonucleases and subjected to Southern blot analysis with a pNOV1300 backbone-specific probe (6019 bp).

Lane 1: Molecular weight marker
Lane 2: Blank

Lane 3: BC 4 F 1 generation of MIR162 maize digested with Kpn I
Lane 4: Control from BC 4 F 1 generation maize digested with Kpn I
Lane 5: BC 4 F 1 generation of MIR162 maize digested with Eco RV
Lane 6: Control from BC 4 F 1 generation maize digested with Eco RV
Lane 7: BC 4 F 1 generation of MIR162 maize digested with Hin dIII + Xma I
Lane 8: Control from BC 4 F 1 generation maize digested with Hin dIII + Xma I
Lane 9: Control from BC 4 F 1 generation maize digested with Hin dIII + Xma I plus 20.2 pg of

Hin dIII + Xma I digested pNOV1300 plasmid.MIR162-USDA-1 Page 41 of 268 Table 8. Expected versus observed band sizes for probes with pNOV1300 backbone.

Lane # (Figure 16) DNA Restriction Enzyme Probe Expected

Bands Expected
Band Size (kb) Observed
Band Size (kb)

Lane 3 BC 4 F 1 Kpn I Backbone 0 None None

Lane 4 Negative Kpn I Backbone 0 None None

Lane 5 BC 4 F 1 Eco RV Backbone 0 None None

Lane 6 Negative Eco RV Backbone 0 None None

Lane 7 BC 4 F 1 Hin dIII +
Xma I Backbone 0 None None

Lane 8 Negative Hin dIII +
Xma I Backbone 0 None None

Positive Control

Lane 9 pNOV1300 Hin dIII +
Xma I Backbone 1 6.3 6.3

IV.C. Stability of the T-DNA Across Generations

Stability of the T-DNA in MIR162 maize acr oss multiple generations of conventional breeding was determined by Southern blot analysis, using backcross generations BC 1 F 1 , BC 2 F 1 , and BC 4 F 1 (see Figure 5). For this analysis , MIR162 maize and negative control genomic DNA samples were digested with a rest riction enzyme that di gests once within the
DNA insert but not within the functional elemen t being probed. Southern blot analysis was performed using a vip3Aa19 -specific probe.

A schematic of the T-DNA region in vect or pNOV1300 indicating locations of the vip3Aa19

- coding sequence and the restricti on endonuclease site targeted for this Southern blot analysis is shown in Figure 17.

The results of this Southern blot analysis are displayed in Figure 18. Table 9 shows a comparison of the expected versus observed hybridization band sizes.

Genomic MIR162 maize DNA from BC 1 F 1 , BC 2 F 1 , and BC 4 F 1 digested with Acc 65I produced a single hybridization signal of a pproximately 8.0 kb corresponding to the single copy of the vip3Aa20 gene present in MIR162 maize (Figure 18; Lanes 3, 4, and 5). The negative control showed no hybridi zation (negative segregant of BC 4 F 1 ; Lane 6). The

Acc 65I digestion of pNOV1300 produced a 14.4 kb band, as expected (positive control; Lane 7). The hybridization patterns for generations BC 1 F 1 , BC 2 F 1 , and BC 4 F 1 of MIR162 maize inMIR162-USDA-1 Page 42 of 268 this Southern blot analysis were iden tical, demonstrating the stability of the vip3Aa20 cassette over multiple generations.

Figure 17. Restriction site and probe for vip3Aa19

. The vertical arrow indicates the endonuclease cu tting site and the expected band size for the resulting restriction fragment that should be detected when probed with vip3Aa19

.

These Southern blot analysis data demonstrat e that the expected hybr idization pattern was
observed for the vip3Aa20 gene over several generations of MIR162 maize. Therefore, the stability of the insert in MIR162 maize duri ng conventional breeding has been established over multiple generations. vip3Aa19 (2370 bp) manA (1176 bp)

LB (25 bp) RB (25 bp) iPEPC9 (108 bp)

vip3Aa19 probe (2370 bp) NOS (253 bp)

ZmUbiInt (1993 bp) ZmUbiInt (1993 bp) 35S terminator (70 bp)

A c c 65I (4782 )

>4.8 kb

Acc 65IMIR162-USDA-1 Page 43 of 268 Figure 18. Southern blot of BC 1 F 1 , BC 2 F 1 , and BC 4 F 1 generations from MIR162. Maize genomic DNA (7.5 μ g) was digested with Acc 65I restriction endonuclease and subjected to Southern blot analysis with a vip3Aa19- specific probe (2370 bp).

Lane 1: Molecular weight marker
Lane 2: Blank

Lane 3: BC 1 F 1 generation of MIR162 maize digested with Acc 65I
Lane 4: BC 2 F 1 generation of MIR162 maize digested with Acc 65I
Lane 5: BC 4 F 1 generation of MIR162 maize digested with Acc 65I
Lane 6: Control from BC 4 F 1 generation maize digested with Acc 65I
Lane 7: Control from BC 4 F 1 generation maize digested with Acc 65I plus 20.2 pg of Acc 65I digested pNOV1300 plasmid.MIR162-USDA-1 Page 44 of 268 Table 9. Expected versus observed hybridization band size with vip3Aa19 probe.

Lane # (Figure 18) DNA Restriction Enzyme Probe Expected

Bands Expected
Band Size (kb) Observed
Band Size (kb)

Lane 3 BC 1 F 1 Acc 65I vip3Aa19 1 >4.8 8.0

Lane 4 BC 2 F 1 Acc 65I vip3Aa19 1 >4.8 8.0

Lane 5 BC 4 F 1 Acc 65I vip3Aa19 1 >4.8 8.0

Lane 6 Negative Acc 65I vip3Aa19 0 - -

Positive Control

Lane 7 pNOV1300 Acc 65I vip3Aa19 1 14.4 14.4

IV.D. Mendelian Inheritance of the T-DNA

Chi-square analysis of vip3Aa20 and manA inheritance data over three generations of MIR162 backcrossing was performed to test th e hypothesis that the ge nes are inherited in
accordance with the laws of Mende lian genetics. See Table 2 a nd Figure 5 for identification of the three generations used in this analys is. The Chi-square an alysis was based on a comparison of observed and expected gene segr egation ratios from each generation. TaqMan PCR analyses were conducted on plants fr om three generations of MIR162 maize to determine the number of plants that were positive or negative for both the vip3Aa20 and

manA gene. Only progeny that tested positiv e for the two genes were selected for backcrossing; thus, the expected inheritance rati o for positive to negative plants was 1:1 in each generation.

The expected and observed frequencies of the vip3Aa20 and manA genes for each generation are presented in Tables 10 and 11. The critical value for rejection of the null hypothesis at p<0.05 was 3.84. The Chi-square values for each generation tested were found to be less than 3.84. This analysis demonstrates that both the vip3Aa20 and manA genes are inherited in a predictable manner according to Mendelian pr inciples. These results are consistent with the genetic characterization data, which indicate a stable integration of the T-DNA at a single locus in the genome.MIR162-USDA-1 Page 45 of 268 Table 10. Observed versus expected frequencies for vip3Aa20 across generations. The progeny of three generations were tested for the presence of vip3Aa20 coding sequence by PCR over three backcross generations.

BC 1 F 1 BC 2 F 1 BC 4 F 1 Trait o* e* o* e* o* e*

Positive 21 20.5 45 48.5 148 143.5

Negative 20 20.5 52 48.5 139 143.5

Total 41 41.0 97 97.0 287 287.0

χ

2 value 0.000 0.371 0.223

* o = observed value; e = expected value

χ 2 = ∑ ( | o – e | - 0.5) 2 / e

Table 11. Observed versus expected frequencies for manA across generations. The progeny of three generation were tested for the presence of manA coding sequence by PCR over three backcross generations.

BC 1 F 1 BC 2 F 1 BC 4 F 1 Trait o* e* o* e* o* e*

Positive 21 20.5 45 48.5 148 143.5

Negative 20 20.5 52 48.5 139 143.5

Total 41 41.0 97 97.0 287 287.0

χ

2 value 0.000 0.371 0.223

* o = observed value; e = expected value

χ 2 = ∑ ( | o – e | - 0.5) 2 / e

IV.E. Flanking Sequence Analysis

Nucleotide sequences flanking the 5 ′ and 3 ′ ends of the T-DNA in MIR162 maize were screened for homology with sequences found in public databases. This comparison provides
an indication of whether the MIR162 maize T-DNA inserted in to a known plant functional genetic unit. A sequence similarity analysis was performed using the BLASTN software (Altschul et al. , 1997; version 2.2.6 Apr-9-2003) whic h compared the flanking sequences with nucleotide sequences in the latest version of the Na tional Center for Biotechnology Information nonredundant database. An analysis of the nucleotide junctions between the T-DNA and genomic DNA was also conducted to determine if novel open reading frames were generated at the point of T-DNA insertion (see Appendix B for details).MIR162-USDA-1 Page 46 of 268 The BLASTN analysis of maize genomic sequence flanking the 5 ′ border of the T-DNA showed significant homology with Dissociation1 (Ds1) -related transposable elements. Ds1 is a nonautonomous transposable element that requires an active Activator (

Ac ) element to
become mobile. Previous studies have shown that the excision of Ds elements can extend beyond the element, but the largest amount of surrounding DNA that has been shown to be deleted is 36 bp (Shen et al ., 1992). The region of homology between the 5 ′ flanking sequence and the Ds1 -related elements is locat ed more than 500 bp from the insert sequence. BLASTN analysis of the maize genomic sequence flanking the 3 ′ region of the MIR162
maize DNA insert shows homol ogy to sequence defined as a Z. mays cyclophilin gene, but the region of homology lies outside of the cyclophilin coding region.

Analysis of all six potential reading frames at both the 5 ′ and 3 ′ DNA insert to maize genome junctions did not detect the presence of any functional novel ope n reading frames.

IV.F. Summary of the Genetic Characterization of MIR162 Maize

Data from Southern blot analys es demonstrated that MIR162 maize contains: i) single copies
of the vip3Aa20 gene and the manA gene in the T-DNA; ii) tw o copies of the ZmUbiInt promoter within the T-DNA region, in add ition to endogenous maize ZmUbiInt promoter elsewhere in the genome; iii) a single copy of the NOS terminator in the T-DNA; and iv) no backbone sequences from transformation plas mid pNOV1300. Additionally, Southern blot analyses demonstrated that the MIR162 maize T-DNA was st able over several breeding generations. Sequence analysis of the en tire T-DNA in MIR162 maize confirmed the intactness of the insert and the contiguousness of the functional elements. Sequence analysis also revealed two transformation-induced nucleotide changes in the vip3Aa coding sequence contained in the T-DNA. Only one of the nuc leotide changes encoded an amino acid change, whereby methionine at position 129 has been s ubstituted by isoleucine. Sequence analysis
revealed that some truncation occurred at the RB and LB ends of the T-DNA. The entire right border, along with two base pairs of nonc oding sequence, was truncated, and the entire left border, along with 32 base pairs of noncodi ng sequence, was truncated. These deletions
have no effect on the functiona lity of the DNA insert. A schematic diagram of the experimentally confirmed T-DNA in MIR162 maize is shown in Figure 6.

Data from Southern blot an alyses and DNA sequencing demons trated the presence of a single intact transgene insert in MIR162 maize. Observations of vip3Aa20 and manA segregation ratios over several generations of MIR162 maize are consis tent with the genes being linked at a single locus in the maize ge nome. The MIR162 maize DNA insert did not locate within any known Z. mays gene. There is a region of homology between the maize genomic sequence flanking the 5 ′ region of the MIR162 T-DNA and maize transposable element sequences, but the homologous region is more than 500 bp removed from the insert. There is a region of homology between th e maize genomic sequence flanking the 3 ′ region of
the MIR162 maize T-DNA and the Z. mays cyclophilin gene, but the region of homology lies outside of the cyclophilin coding sequence. Additionally, no novel open reading frames were identified that spanned either the 5 ′ or 3 ′ junctions between the T-DNA and Z. mays genomic

sequences.MIR162-USDA-1 Page 47 of 268 V. Characterization of the Vip3Aa20 and PMI Proteins

The two novel proteins produced in MIR162 mai ze have been characte rized and tested to determine their potential for causing adverse e ffects in mammals. This process included an assessment of: i) the origin and function of each protein; ii) the mode of action of each protein; iii) physiochemical prope rties of the proteins; iv) biol ogical activity of the proteins; v) results of safety studies for each protein; and vi) Vip3Aa20 and PMI concentrations in plant tissues.

V.A. Vip3Aa20 Protein – Origin and Function

Vip3Aa is a class of recently discovered ‘v egetative insecticidal proteins’ produced by B. thuringiensis , a gram positive bacterium (Estruch et al., 1996). Unlike the crystal (Cry) proteins of B. thuringiensis , Vip protein toxins are produc ed during vegetative bacterial growth and are secreted as soluble protei ns into the extracellular environment. B. thuringiensis cultures continue to produ ce Vip proteins during the stationary phase of growth and sporulation. In contrast to the thermostabile nonproteinaceous β -exotoxins secreted by

some B. thuringiensis strains, Vip proteins are ther molabile. Vip3Aa1 is the toxin designation assigned to the native protein discovered in B. thuringiensis strain AB88. Vip3Aa19 and Vip3Aa20 are toxi n designations assigned by the Bacillus thuringiensis Toxin Nomenclature Committee to two engineered variants of the native protein. 2 B. thuringiensis is a scavenger organism that does not readily pr oliferate in soil. Production of Cry and Vip toxins conveys upon the microorgani sm a means of killing insect prey that can then serve as
nutrient sources.

V.A.1. Mechanism of Action and Insecticidal Specificity

The mechanism by which Vip proteins exert thei r insecticidal activity has been studied and found to be similar, but not id entical, to that which has been previously described for the B. thuringiensis Cry proteins. Following ingestion, fu ll-length Vip proteins are proteolytically processed into active fragments of approximately 66 kDa which bind to receptors in the midgut epithelium of susceptible insects. Competitive binding assays have shown that Vip proteins and Cry proteins bind to different receptors (Lee et al. , 2003). Receptor binding is followed by the formation of selective ion channels (pores) in epithelial membranes which leads to cell lysis and death (Yu et al ., 1997). Each of these steps pl ays a role in establishing the insecticidal specificity of a given protein fo r different insect species. As discussed later in this petition, the insecticidal activity of Vip3Aa proteins is limited to species within
selected families of the or der Lepidoptera (see Table 27).

Native Vip3Aa1 is 789 amino acids in length a nd approximately 89 kDa molecular weight. The Vip3Aa20 variant produced in MIR162 mai ze is also 789 amino acids in length but differs from the native amino acid sequence by two amino acids (at po sitions 129 and 284). A Vip3Aa variant is also pres ent in Syngenta cotton transf ormation event COT102; this

2 Bacillus thuringiensis Toxin Nomenclature Committee (2007). http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/MIR162-USDA-1 Page 48 of 268 variant has been assigned the designation Vip3Aa19. Vip3Aa19 differs from the native sequence by one amino acid at position 284. Vip3Aa19 and Vip3Aa20 differ by one amino acid at position 129. Table 12 compares selected sequence in formation for these protein variants. COT102 cotton was deregulated by USDA in 2005.

Table 12. Amino acid differences between Vip3Aa1, Vip3Aa19, and Vip3Aa20.

Vip3Aa Source Toxin Designation Amino
Acids Position
129 a Position
284 a

B. thuringiensis strain AB88 Vip3Aa1 789 M K

COT102 cotton Vip3Aa19 789 M Q

MIR162 maize Vip3Aa20 789 I Q

a – M = methionine; I = isoleucine; K = lysine; Q = glutamine

Based on Syngenta laboratory bioassay results, the amino acid differences between these Vip3Aa variants do not impact in secticidal activity against target insect pests. The single amino acid difference between Vip3Aa19 and Vip3Aa20 (position 129) occurs outside of the protein tryptic core. Lee et al. (2003) have shown that full-le ngth Vip3Aa is proteolytically
activated to form a 66 kDa active core in midgut extracts of sus ceptible insects. N-terminal sequencing of the 66 kDa active core indicates that cleavage occurs at amino acid residue 199 (Estruch and Yu, 2001). This finding sugges ts that amino acid residue 129 does not play a role in defining species specificity of the toxin since it would be lost during proteolytic processing in the insect midgut.

V.A.2. Characterization of Vip3Aa20

A number of analytical methods were used to characterize the Vip3Aa20 protein produced in
MIR162 maize. In order to conduct animal an d environmental toxico logy studies with the protein, significant quantities of Vip3A a20 were produced in a recombinant E. coli fermentation system. Production and extractio n of Vip3Aa20 from MIR162 maize is not a feasible approach for obtaining the large quantiti es of the protein needed for these safety
studies. Biochemical and bioactiv ity analyses were conducted to establish the equivalence of plant- and microbially produced Vip3Aa 20; the latter being produced in an E. coli fermentation system.

The Vip3Aa20 protein produced in MIR162 ma ize was characterized by its molecular weight, mobility in sodium dodecyl sulfate pol yacrylamide gel electrophoresis (SDS-PAGE), immunoreactivity, glycosylation status, and am ino acid sequence. The amino acid sequence of full length Vip3Aa20 was deduced fr om the nucleotide sequence of the vip3Aa20 gene found in MIR162 maize. Peptide mass mappi ng by matrix-assisted laser desorption ionization time of flight mass spectrometry c onfirmed the deduced amino acid sequence of Vip3Aa20. Based on its amino acid sequence, Vip3Aa20 has a predicted molecular weight of 88,647 Da. Western blot analysis of protei n extracts from MIR162 leaf tissue using anti-MIR162-USDA-1 Page 49 of 268 Vip3Aa antibodies revealed an immunoreactive band of appr oximately 89 kDa molecular weight, a size that is consistent with the de duced sequence for full-le ngth protein (see Figure 19). The plant-produced protei n was found not to be glycos ylated. A more detailed description of the methods and results for the protein characte rization analyses can be found in Appendix C.

1 2 3 4 5 67

188

49

38

28

17

14

6

3 62 98

MW [kDa]

Vip3Aa 188

49

38

28

17

14

6

3 62 98 MW [kDa]

Figure 19. Western blot of plant- and microbially produced Vip3Aa proteins. This Western blot shows the immunoreactivity of Vip3Aa19 from E. coli , Vip3Aa20 from E.
coli , and Vip3Aa20 from MIR162 maize. Th e molecular weights of Vip3Aa19 and Vip3Aa20 correspond to approximately 89 kDa.

Lanes 1 and 7: molecular weight standard Lane 2: 10 ng Vip3Aa19 from E. coli Lane 3: 10 ng Vip3Aa20 from E. coli Lane 4: 10 ng Vip3Aa20 from MIR162 maize leaf tissue extract Lane 5: 10 ng immunoaffinity-purified Vip3Aa20 from MIR162 maize leaf tissue Lane 6: protein extract from control maize leaf tissue.

These same analytical methods, plus an assess ment of insecticidal activity, were used to compare plant-produced Vip3Aa20 to microbially produced protei n. Similar analyses were performed to compare the biochemical prope rties and bioactivity of the Vip3Aa19 and Vip3Aa20 variants.

As shown in lanes 3, 4, and 5 of Figure 19, th e electrophoretic mobility, molecular weight, and immunoreactivity of microbi al Vip3Aa20 and plant Vip3Aa20 are essentially the same. N-terminal sequencing and peptide mass mappi ng of microbial Vip3Aa20 matched the amino acid sequence of plan t Vip3Aa20. Neither microbial- nor plant-produced Vip3Aa20 wasMIR162-USDA-1 Page 50 of 268 found to be glycosylated. The efficacy and po tency of the two proteins were found to be similar as measured by an assessment of lethality against first instar S. frugiperda larvae. A comparison of median le thal concentration (LC 50 ) values for each protein is shown in Table 13. Although a numeric diffe rence in the computed LC 50 values is observed, their overlapping confidence intervals indicate that the difference was not biologically significant.

Table 13. Comparative bioactivity of Vip3Aa proteins from different sources. Results of mortality bioassays with first-instar S. frugiperda larvae are presented as LC 50 values with their 95% confidence intervals. In the first set of bioassays, Vip3Aa20 from MIR162 maize leaf tissue was compared to Vip3Aa20 produced in E. coli . In the second set of bioassays, Vip3Aa20 from maize leaf tissue was compared to Vip3Aa19 produced in E.
coli

.

Test Substance LC 50 (ng protein/cm 2 ) 95% Confidence Intervals

Vip3Aa20 – MIR162 leaf extract 318 a 232 – 451

Vip3Aa20 – E. coli 225 155 – 289

Vip3Aa20 – MIR162 leaf extract 154 a 94 – 222

Vip3Aa19 – E. coli 137 82 – 199

a – no significance should be assigned to the apparent difference in LC 50 values from the two assays with MIR162 leaf extract (318 vs 154). The assays were temporally separated,
conducted with different leaf tissue extracts , and different batches of insect larvae.

Based on these results, the identities of plan t- and microbially produced Vip3Aa20 proteins
have been verified and it can be concluded th at Vip3Aa20 proteins produced in recombinant
E. coli and MIR162 maize are biochemically a nd functionally equivalent. Having established equivalence of the proteins, it was appr opriate to utilize microbially produced Vip3Aa20 as a surrogate for the plant-pr oduced protein in toxicology tests.

The biochemical and functional properties of Vip3Aa20 were compared to those of Vip3Aa19 in a separate set of experiments. This comparison was made because the Vip3Aa19 variant has been used as a surrogate test substance in environmental safety studies. These protein variants differ by only one ami no acid. They are of comparable molecular weight, electrophoretic mobility, and immunor eactivity as determined by Western blot analysis. Neither protein is glycosylated. As shown in Table 13, a comparison of insecticidal activity results shows no biol ogically significant difference between the computed LC 50 values for the Vip3Aa20 from MI R162 leaf tissue and Vip3Aa19 from E.
coli

.

V.A.3. Safety Assessment for Vip3Aa20

A detailed assessment of human and animal safety for the Vip3Aa20 protein has been provided to the FDA as part of the MIR162 premarket consultation. A toxicology dataMIR162-USDA-1 Page 51 of 268 package has also been submitted to EPA in support of a petition to exempt Vip3Aa20 residues from the requirement of a tolerance in food and feed commod ities of all crops. A summary of the pertinent information and toxico logy data bearing on th e potential risk of Vip3Aa20 for humans is presented below:

• B. thuringiensis microbial insecticides that are co mmercially available and registered by EPA ( e.g . Dipel ® Biological Insecticide, Javelin ® Biological Insecticide, and
Condor ® Bioinsecticide) have been found to contain Vip3Aa or Vip3Aa-like proteins. 3 Thus, there is a history of human and environmental exposure to Vip3Aa or Vip3Aa-like proteins without corresponding evidence of adverse effects.

• No adverse effects were observed in mi ce administered a single oral dose of Vip3Aa20 at 1250 mg/kg body weight. The no observable effect level (NOEL) was
found to be ≥ 1250 mg/kg, which was the highest dose that could be physically administered to the animals. Mice have historically been a suitable surrogate species for detecting evidence of toxicity in mammals.

• The amino acid sequence of Vip3Aa20 has been systematically compared to the sequence of other proteins in the late st posting of the National Center for Biotechnology Information (NCBI) Entrez Protein Database (NCBI, 2007). This comparison determined if any proteins in the database showed significant amino acid sequence identity to Vip3Aa 20, indicating they may be cl osely related to Vip3Aa20, and if any proteins with si gnificant sequence identity to Vip3Aa20 are known to be toxins, indicating possible implications fo r the toxic potential of Vip3Aa20. The NCBI Entrez protein database was sear ched using the BLASTP program with Vip3Aa20 as the query sequence (Altschul et al., 1997). 4 All database sequences with a conservative Expect value (E-value) of 10 or lower were identified by default by the BLASTP program. Comparisons be tween highly homologous proteins yield E-values approaching zero, indicating a very low probability that such matches would occur by chance. The probability that ami no acid sequence similarities observed in a database search occur by chance increases with higher E-values (Ponting, 2001). The Vip3Aa20 query sequence showed no significa nt sequence identity to any proteins identified as, or known to be, toxins other than the vegetative in secticidal proteins
from B. thuringiensis . Vip3Aa20 has no signifi cant sequence homology to known mammalian protein toxins.

• The Vip3Aa20 amino acid sequence was co mpared to the Syngenta Allergen Database (version 4.0) to dete rmine if it has significant sequence identity to proteins known or suspected to be alle rgens, indicating possible imp lications for the allergenic potential of Vip3Aa20. The Syngenta Allerg en Database was compiled from entries identified as allergens or putative allergens in public protein databases, and was supplemented with additional amino acid sequ ences identified fr om the scientific literature as being known or putative alle rgens. The Syngenta Allergen Database

® DiPel (EPA Reg. No. 275-37) is a trademark of Valent USA Corp; Javelin (EPA Reg. No. 70051-66) and Condor (EPA Reg. No. 70051-78) are trademarks of Certis USA, LLC.

3 Vip3Aa-like indicates proteins contained in the specified products were bound by Vip3Aa antibodies in western blot analyses but differed in molecular weight from Vip3Aa.

4 BLASTP version 2.2.6 was used with the following parame ters: No complexity filter; expect score = 10; word size = 3; gap costs: existence = 11 and extension = 1. The similarity matrix was Blosum 62.MIR162-USDA-1 Page 52 of 268 contained 1414 nonredundant entries at the time this search was performed. Sequential 80-amino acid peptides of the Vip3Aa20 protein seque nce were compared to protein sequences in the database using the FASTA search algorithm (Pearson and Lipman, 1988). Any 80-amino acid peptid e of the query sequence having greater than 35% amino acid identity to an allergen sequence was identified as having significant identity to the allergen sequen ce in accordance with the recommendations of FAO/WHO (2001). The results of this analysis revealed that there was no significant identity between any of th e sequential 80-amino acid peptides of Vip3Aa20 and any entry in the Syngenta Al lergen Database. Therefore, Vip3Aa20 does not share overall sequence homology w ith any known allergenic protein.

• The Vip3Aa20 protein sequence was also sc reened for matches of eight contiguous amino acids between the Vip3Aa20 sequenc e and the allergen sequences in the Syngenta Allergen Database to screen for s hort, local regions of amino acid identity that might indicate the presence of common T-cell binding epitopes (Hileman et al

., 2002; Stadler and Stadle r, 2003; Silvanovich et al ., 2006). There were no matches of eight contiguous amino acids between Vi p3Aa20 and any entry in the allergen database. No significant amino acid se quence identity of Vip3Aa20 to known or putative allergenic proteins having implications for allerg enic potential was identified.

• The mode of insecticidal action for B. thuringiensis Cry and Vip proteins is not operative in mammalian species because they do not possess the epithelial cell receptors for binding of the proteins.

• Vip3Aa20 protein was degrad ed within two minutes in simulated gastric fluid containing pepsin. Thus, th ere will be minimal opportuni ty for human and domestic animal systemic exposure to Vip3Aa20.

• Vip3Aa20 protein degrades upon exposur e to elevated temperatures ( ≥ 65°C); thus, it will degrade at temperatures used in maize cooking and processing.

Collectively, the results of these studies i ndicate that the Vip3Aa 20 protein present in MIR162 maize presents no risk of harm for mammalian species.

V.B. Phosphomannose Isomerase – Origin and Function

MIR162 maize plants express the manA gene from E. coli strain K-12 (Miles and Guest, 1984). This gene encodes the enzyme PMI th at catalyzes the reversible conversion of mannose 6-phosphate and fructose 6-phosphate a nd enables many microorganisms to utilize mannose as a carbon and energy source. Many plant species do not possess genes that encode PMI enzymes.

V.B.1. Mechanism of Action

PMI has utility as a selectable marker for transformation of many plant species (Bojsen et al.

, 1994; Joersbo, et al. , 1998; Negrotto et al. , 2000). Plant cells that have been transformed
with the manA gene encoding PMI are able to su rvive and grow on media containing mannose as the only or primary energy sour ce. When placed in medium containing predominantly or only mannose as the sole sugar source, nontransformed tissue remains dormant and becomes outgrown by transformed ti ssue. Mannose itself is not toxic to plantMIR162-USDA-1 Page 53 of 268 cells. Inclusion of the manA gene in the T-DNA of tr ansformation plasmid pNOV1300
allowed for selection of successfu lly transformed maize plantlets.

V.B.2. Characterization of Phosphomannose Isomerase

Western blot analysis was used to characteri ze the PMI enzyme produced in MIR162 maize. In order to conduct animal toxicology studies with the protein, signifi cant quantities of PMI were produced in a recombinant E. coli fermentation system. Production and extraction of PMI from MIR162 maize is not a feasible appr oach for obtaining the large quantities of the protein needed for these safety studies. Biochemical and bioactivity analyses were
conducted to establish the e quivalence of plant- and microbially produced PMI.

PMI from MIR162 maize was found to be appr oximately 42.8 kDa molecular weight which matched the predicted molecular weight ba sed on the amino acid sequence deduced from having sequenced the manA gene in the T-DNA. The observed molecular weight for PMI produced in the recombinant E. coli system was approximately 44.4 kDa. The microbial protein contained an additional 16 nonfunctional amino acids at the N-terminus which were added for purification purposes (13 amino aci d T7-Tag and three additional amino acids arising from the polylinker). Both proteins reacted with the same anti-PMI antibodies. Specific activity was measured for both source s of the enzyme. PMI extracted from MIR162 maize leaf had a specific activity of 55.5 U/mg and the microbial enzyme had a specific activity of 33.2 U/mg. Both of these unit activity measuremen ts fell within the range of historically observed values at Syngenta.

Based on these results, it can be conclude d that PMI from MIR162 maize and PMI from E.
coli are biochemically and functionally equivale nt. A more detailed description of the methods and results for this protein charac terization work can be found in Appendix C.

V.B.3. Safety Assessment fo r Phosphomannose Isomerase

An assessment of human and animal safety of the PMI protein has been provided to the FDA as part of the MIR162 premarket consultati on. A toxicology data package has been submitted to EPA and supported establishment of an exemption of PMI residues from the requirement of a tolerance in food and feed commodities of all crops (40 CFR §174.527). A summary of the pertinent information and toxico logy data bearing on th e potential risk of PMI for humans is presented below:

• PMI enzymes are ubiquitous in nature . PMI is a functional enzyme in E. coli , a common contaminant of food and feed. The en zyme is also found in a wide variety of other species. Thus, ther e is a history of safe consumption of PMI proteins in
human food and animal feed sources.

• No adverse effects were observed in mice ad ministered a single oral dose of PMI at 3030 mg/kg body weight. The NOEL was found to be ≥ 3030 mg/kg, which was the highest dose that could be physically ad ministered to the animals. Mice have historically been a suitable surrogate speci es for detecting evidence of toxicity in
mammals.MIR162-USDA-1 Page 54 of 268 • The amino acid sequence of PMI has been co mpared to the sequence of other proteins in a comprehensive sequence database. This sequence comparison was conducted following the methodology described above for Vip3Aa20. PMI was found to have no significant sequence homol ogy to known protein toxins.

• PMI is derived from a donor organism ( E. coli ) that is not known to be a source of allergenic proteins and it possesses no signi ficant amino acid sequence identity to known or putative allergenic protein sequences that are biologically relevant or that have implications for allerg enic potential. This se quence comparison was conducted following the methodology described above fo r Vip3Aa20. There was one region of sequence homology of eight contiguous identical amino acids between PMI and a known allergen, α -parvalbumin from Rana species CH2001 (unidentified edible frog) (Hilger et al., 2002). Further investigation using sensitive serum screening methodology (CODEX, 2003) demonstrated no cross-reactivity be tween PMI and the serum from the single indi vidual known to have demonstrated IgE-mediated allergy to this specific α -parvalbumin. The patient’s seru m did not recognize any portion of the PMI protein as an allergenic epitope. Therefore, the sequence identity between

PMI and the α -parvalbumin from Rana species CH2001 is not biologically meaningful and has no implications for the potential allergenicity of PMI.

• PMI protein is degraded within seconds in simulated gastric fluid containing pepsin. Thus, there will be minimal opportunity fo r human or domestic animal systemic exposure to PMI.

• PMI protein degrades upon exposure to elevated temperatures ( ≥ 65°C); thus, it will degrade at temperatures used in maize cooking and processing.

Collectively, the results of these studies indi cate that the PMI prot ein present in MIR162 maize presents no risk of harm for mammalian species.

V.C. Vip3Aa20 and PMI Levels in MIR162 Maize

Concentrations of the Vip3Aa20 and PMI protei ns in plant tissues from two MIR162 hybrids were determined using an enzyme-linked immuno sorbent assay (ELISA). Plant tissues were collected from two locations at four stages of growth: V9-V12, anthesis, seed maturity, and
senescence. The materials and methods for these ELISA experiments are described in Appendix D.

Vip3Aa20 and PMI concentrations on both a fr esh-weight and dry-weight basis were determined and are reported in Tables 14 a nd 15, respectively. No meaningful differences between the two hybrids were no ted in their concentrations of Vip3Aa20 and PMI in each tissue type at each growth stage sampled. Theref ore, values presented in the tables are means of the two hybrids. Vip3Aa20 and PMI concen trations in the conventional maize tissues were less than the limit of detection or limit of quantification.

Vip3Aa20 fresh weight levels in leaf, pith, and whole plant were highest at seed maturity. Levels in root tissue were relatively constant throughout the plant’s life cycle. These freshweight values are used in computing expect ed environmental concentrations for risk assessment purposes (see Section VII.C.3).MIR162-USDA-1 Page 55 of 268 Table 14. Mean tissue concentrations of Vip3Aa20 in MIR162 plants. Concentrations are presented on both a fresh tissu e and dry weight basis. All ELISA results
have been corrected for method extraction effi ciency. Vip3Aa20 concentrations in control plant tissues were all <LOD or <LOQ. a Not all tissues were analyzed at all growth stages.

Tissue Type V9-V12 Anthesis Seed Maturity Senescence

Vip3Aa20 μ g/g fresh weight (Range)

Leaves 17.63 (13.11 – 22.35) 24.44 (21.08 – 29.07) 50.41 (35.85 – 60.92) 13.40 (8.87 – 18.20)

Roots 5.23 (3.99 – 7.07) 4.32 (4.18 – 4.69) 4.81 (2.27 – 6.60) 5.29 (4.61 – 5.82)

Pith N/A 3.54 (3.17 – 4.16) 11.47 (10.21 – 12.75) N/A

Kernels N/A N/A 29.81 (27.78 – 34.13) 28.65 (25.06 – 32.42)

Silk N/A 12.55 (8.05 – 18.91) N/A N/A

Pollen b N/A 43.21 (37.42 – 49.70) N/A N/A

Whole Plants 11.98 (8.96 – 15.39) 12.16 (11.51 – 12.97) 20.84 (15.54 – 25.98) 17.35 (13.24 – 24.07)

Vip3Aa20 μ g/g dry weight (Range)

Leaves 97.26 (76.12 – 119.12) 107.74 (97.10 – 118.80) 121.79 (77.25 – 159.66) 21.32 (12.93 – 30.28)

Roots 31.80 (28.10 – 35.65) 28.34 (26.30 – 30.20) 20.29 (9.87 – 27.48) 21.66 (11.58 – 32.13)

Pith N/A 31.71 (29.43 – 36.18) 58.21 (52.74 – 63.68) N/A

Kernels N/A N/A 43.56 (40.47 – 50.50) 34.25 (30.90 – 37.67)

Silk N/A 97.40 (60.54 – 149.00) N/A N/A

Pollen N/A 47.13 (41.45 – 53.52) N/A N/A

Whole Plants 91.53 (88.68 – 96.51) 67.61 (61.68 – 72.63) 49.04 (34.84 – 63.14) 34.30 (21.12 – 55.17)

a - LOQ = limit of quantification; LOD = limit of detection; N/A = not analyzed

b - pollen was analyzed as received, air-dried overnightMIR162-USDA-1 Page 56 of 268 Table 15. Mean tissue concentrations of PMI in MIR162 plants. Concentrations are presented on both a fresh tissu e and dry weight basis. All ELISA results
have been corrected for method extraction effici ency. PMI concentrations in control plant tissues were all <LOD or <LOQ. a Not all tissues were analyzed at all growth stages.

Tissue Type V9-V12 Anthesis Seed Maturity Senescence

PMI μ g/g fresh weight (Range) b

Leaves 2.02 (1.42 – 3.14) 2.18 (1.69 – 3.06) 2.44 (1.94 – 3.50) < 0.17 (<LOD – <LOQ)

Roots 0.69 (0.43 – 0.90) 0.54 (0.36 – 0.83) 0.48 (0.25 – 0.74) 0.36 (0.19 – 0.49)

Pith N/A 0.23 (0.17 – 0.27) 0.54 (0.47 – 0.62) N/A

Kernels N/A N/A 1.32 (0.92 – 1.71) 0.63 (0.46 –0.79)

Silk N/A 2.68 (1.68 – 3.45) N/A N/A

Pollen c N/A 4.87 (3.45 – 7.07) N/A N/A

Whole Plant 1.15 (0.76 – 1.56) 1.28 (1.18 – 1.32) 1.60 (0.96 – 2.21) 1.22 (1.11 – 1.35)

PMI μ g/g dry weight (Range) b

Leaves 11.12 (8.26 – 16.76) 9.75 (6.92 – 14.68) 5.78 (4.57 – 7.55) < 0.26 (<LOD – <LOQ)

Roots 4.32 (3.17 – 7.08) 3.49 (2.51 – 5.22) 2.00 (1.08 – 3.09) 1.51 (0.47 – 2.53)

Pith N/A 2.02 (1.53 – 2.40) 2.75 (2.36 – 3.19) N/A

Kernels N/A N/A 1.93 (1.33 – 2.54) 0.75 (0.54 –0.97)

Silk N/A 20.70 (12.60 – 27.16) N/A N/A

Pollen N/A 5.30 (3.82 – 7.62) N/A N/A

Whole Plants 8.74 (7.49 – 9.75) 7.10 (6.32 – 7.61) 3.77 (2.16 – 5.37) 2.36 (1.85 – 3.09)

a - LOQ = limit of quantification; LOD = limit of detection; N/A = not analyzed

b - mean values preceded by ‘<’ indicate that the LOQ or LOD was used for some samples in calculating the mean

c - pollen was analyzed as received, air-dried overnightMIR162-USDA-1 Page 57 of 268 VI. Phenotypic and Compositional Evaluation

Laboratory, greenhouse, growth ch amber, and field investigati ons have been conducted to assess the phenotype and biochemical compositi on of MIR162 maize. The purpose of these investigations was to determine if unintende d changes occurred in MIR162 maize as a result of the transformation process and to determine if any unint ended changes conveyed a plant pest risk potential to the new cultivar. For each parameter evaluated in a MIR162 hybrid, a comparable set of data were collected from a genetically matched nontransgenic hybrid.

VI.A. Phenotypic Assessment of MIR162 Plants

A range of phenotypic parameters assessi ng seed germination and dormancy, growth characteristics, reproductive capability, seed dispersal, and interactions with biotic and
abiotic stressors have been examined for MIR162 and control plants. Table 16 provides a listing of the parameters evaluated. The purpose of these evaluations was to ascertain
whether or not the transformation that crea ted MIR162 maize has imparted some phenotypic
characteristic on cultivars that would make it undesirable to growers, for example, a reduction in yield potential, or that could increase the new cultivars’ persistence or result in
invasive characteristics. These latter char acteristics could be c onsidered indicative of increased weediness potential and plant pest ri sk. If no significan t differences are found between MIR162 and control hybrids it can be concluded that the transformation has not increased the new cultivars’ plant pest potential.

Table 16. Phenotypic characteristics evaluated for MIR162 maize hybrids.
Evaluations were conducted in la boratory, greenhouse, growth chamber, or field experiments.

Phenotypic
Characteristic Variable Measured Timing Description

Dormancy /
Germination Dormancy and germination After 4, 7, and 12 days Percent normal germinated, abnormal germinated, dormant, dead, and viable firm swollen seed

Emergence Emerged plants 14 days postplanting Number of emerged plants per plot

Early growth rating Stage V6 Growth rating

Green snapped plants Prior to anthesis Percent of plants per plot broken
due to adverse environmental
conditions

Root lodged plants After anthesis Percent of plants leaning >30° from
vertical

Ear height Stage R2-R6 Height from plant base to node where ear connects to stalk (cm)

Plant height Stage R2-R6 Height from plant base to collar of flag leaf (cm) Vegetative
Growth

Leaf color rating Stage R4-R6 Color rating: 5=same as commercial check, 1=darker, 9=severely
chlorotic (continued)MIR162-USDA-1 Page 58 of 268 Table 16 (cont.). Phenotypic characteristics evaluated for MIR162 maize.
Evaluations were conducted in la boratory, greenhouse, growth chamber, or field experiments.

Phenotypic
Characteristic Variable Measured Timing Description

Late season intactness Prior to harvest Plant integrity above the ear: 1=all plant parts intact at harvest, 9=all plants broken at the ear node prior to
harvest

Stalk lodging Harvest Percent of plants per plot broken
below the ear

Stay green rating Harvest Percent staygreen at harvest as measure of late season plant health Vegetative
Growth

Population count Harvest Extrapolated estimate of plant population per acre Heat units to 50% pollen shed Pollen shed Heat units to 50% pollen shed

Pollen viability Pollen shed Percent viable pollen based on staining characteristics

Pollen morphology Pollen shed Diameter ( μ m) of viable pollen grains
Heat units to 50% silking Silk emergence Heat units to 50% silk emergence

Barren plants Harvest Percent of plants per plot that do not develop an ear

Grain moisture Harvest Percent grain moisture

Test weight Harvest Grain test weight (lb/bu) converted
to 15.5% moisture Reproductive
Growth

Yield Harvest Grain yield (bu/ac) converted to
15.5% moisture

Seed Retention Dropped ears Prior to harvest Percent of plants per plot that dropped a developed ear
Plant-ecological Interactions Disease susceptibility Planting to harvest Observations of disease occurrence

VI.A.1. Seed Dormancy and Germination

Enhanced germination or seed dormancy are char acteristics that can be indicative of plant pest potential. Dormancy mechanisms, incl uding hard seed, vary with species and are generally complex processes. Seed dormancy is not a characteristic of maize. A laboratory study was conducted to evaluate the germinat ion and dormancy characteristics of MIR162 seed using a modification of the testing protoc ol established by the Association of Official Seed Analysts

Seed samples of two MIR162 hybr ids, their respective near-i sogenic controls, and threeMIR162-USDA-1 Page 59 of 268 conventional hybrids served as te st, control, and reference mate rials for the study. Table 17 shows the genotypes of the test, control, and reference hybrids (see also Table 2 and Figure 5). MIR162 hybrid #1 corresponds to seed lot code ‘D’ and MIR 162 hybrid #2 corresponds
to seed lot code ‘E’ as desc ribed in Table 2 and Figure 5.

Table 17. Test, control, and reference seeds used in germination and dormancy test.

Hybrid Designation Seed Generation Lineage or Variety

MIR162 hybrid #1 F 1 NP2391 x NP2222(BC 6 F 4 )

Control hybrid #1 F 1 NP2391 x NP2222

MIR162 hybrid #2 F 1 NP2222(BC 6 F 5 ) x NP2391

Control hybrid #2 F 1 NP2222 x NP2391

Reference hybrid #1 F 1 N59-Q9

Reference hybrid #2 F 1 N72-G8

Reference hybrid #3 F 1 N36-J2

The study design followed that described by AOSA (2005) with the addition of four temperature regimes to assess germination and dormancy characteristics of the maize seed under nonoptimal conditions. Seed lots were di vided into four replicates of 40 seeds per replicate per temperature regime. Si x temperature regimes were utilized:

• constant temperatures: 5°C, 10°C, and 25°C

• alternating temperatures: 5°C /20°C, 10°C /20°C, and 20°C /30°C

Experiments were conducted in unlighted temp erature-controlled growth chambers. For the alternating temperature regimes, the lower te mperature was maintained for 16 hours and the higher temperature for eight hours, and then th e cycle was repeated. The study was initiated by rolling 40 seeds in moistened germination towels and then placing the rolled towels into the growth chambers.

For the tests carried out at 25°C and 20°C /30°C (the standard AOSA temperatures), each germination towel was carefully unrolled and examined four and seven days after study initiation. Each seed was examined and categorized as one of the following:

• normal germinated (seed exhibited normal de velopment of the root and shoot with a

shoot length of at least 0.5 in (1.3 cm))

• abnormal germinated (seed lacked a well- developed root and shoot, or possessed a

hollow coleoptile, or exhibited mechanical damage)

• dead seed (seed not germinated and visibl y deteriorated and soft to the touch)

• firm swollen seed (seed visibly swollen and firm to the touch)

• hard seed (seed that had not imbibe d water and was firm to the touch)

For the tests carried out at 5° C, 10°C, 25°C, 5/20°C, and 10/20°C, each germination towelMIR162-USDA-1 Page 60 of 268 was carefully unrolled and examin ed four, seven, and 12 days af ter study initiation. Each seed was examined and categorized as one of the following:

• normal germinated (seed with a radicle protruding beyond the seed coat)

• abnormal germinated (seed lacked a well- developed root and shoot, or possessed a

hollow coleoptile, or exhibited mechanical damage)

• dead seed (seed not germinated and visibl y deteriorated and soft to the touch)

• firm swollen seed (seed visibly swollen and firm to the touch)

• hard seed (seed that had not imbibe d water and was firm to the touch)

At the end of each experiment, all firm sw ollen and hard seeds were subjected to a tetrazolium test to evaluate their viability ( AOSA, 2000). Seeds subjected to the tetrazolium test were then categorized as ‘viable firm sw ollen’, ‘viable hard’, or ‘dead’ (nonviable firm swollen and nonviable hard).

The results of these germination and dormancy experiments are summarized in Table 18. Inspection of the data revealed a high freque ncy of zero response ra tes for the endpoints being measured. For example, only one of 28 replicates gave a response rate above zero for percent normal germinated at 5°C. At the high er temperatures, there was a high frequency of 100% normal germinated seeds among the replicat es. These data are not considered suitable for analysis of variance because they do not satisfy the assumptions upon which the validity of an analysis of variance depends. Instead, the data for each MIR162 and control hybrid were combined across replicates and assembled into 2x2 continge ncy tables and subjected to
Fisher’s Exact Test. Si gnificance was assigned at p <0.05. Only the results for ‘normal germinated’ seeds were statistically analyzed.

There were no statistically significant differe nces in the proportions of normal germinated MIR162 and control seeds. Table 19 presents the probability values for each of the contingency table analyses. No viable hard seeds were found for any of the hybrids tested under any temperature regime, thus confirming that dormancy is not a normal characteristic
of maize seeds and that MIR162 maize demonstr ates no increase in seed dormancy potential.MIR162-USDA-1 Page 61 of 268 Table 18. Summary of seed germination and dormancy frequencies. Mean germination responses of four replicat es from test, control, and reference hybrids evaluated under different temperature regim es. No significant differences were observed between MIR162 and control proportions of normal germinated seeds ( p<0.05 ).

Temp (°C) Hybrid Genotype Normal
Germinated (%) Abnormal
Germinated (%) Viable Firm Swollen (%) Viable Hard (%) Dead (%)

MIR162 #1 0.0 0.0 95.6 0.0 4.4

Control #1 0.0 0.0 96.3 0.0 3.8

MIR162 #2 0.0 0.0 94.4 0.0 5.6

Control #2 0.6 0.0 96.3 0.0 3.1 5

Reference Range 0.0 – 0.0 0.0 – 0.0 90.6 - 96.3 0.0 3.8 - 9.4

MIR162 #1 98.1 0.0 0.0 0.0 1.9

Control #1 97.5 0.0 2.5 0.0 0

MIR162 #2 94.4 0.0 4.4 0.0 1.3

Control #2 98.8 0.0 0.0 0.0 1.3 10

Reference Range 90.6 - 98.1 0.0 – 0.0 0.0 - 8.6 0.0 - 0.0 0.6 - 1.9

MIR162 #1 97.5 0.0 0.6 0.0 1.9

Control #1 99.4 0.0 0.0 0.0 0.6

MIR162 #2 95.6 0.0 1.9 0.0 2.5

Control #2 98.6 0.0 0.6 0.0 0.6 5/20

Reference Range 91.9 - 100 0.0 - 0.0 0 - 5.6 0.0 - 0.0 0 - 2.5

MIR162 #1 100.0 0.0 0.0 0.0 0.0

Control #1 99.4 0.0 0.0 0.0 0.6

MIR162 #2 97.5 0.0 1.9 0.0 0.6

Control #2 99.4 0.0 0.0 0.0 0.6 10/20

Reference Range 97.5 - 99.4 0.0 - 0.0 0 - 0.6 0.0 – 0.0 0.6 - 1.9

MIR162 #1 100.0 0.0 0.0 0.0 0.0

Control #1 98.1 1.3 0.0 0.0 0.6

MIR162 #2 99.4 0.6 0.0 0.0 0.0

Control #2 96.3 1.9 0.0 0.0 1.9 25

Reference Range 98.1 - 99.4 0 – 1.3 0 - 0.6 0.0 – 0.0 0.0 - 0.6

MIR162 #1 98.1 0.6 0.0 0.0 1.3

Control #1 98.1 0.6 0.0 0.0 1.3

MIR162 #2 96.3 1.9 0.0 0.0 1.9

Control #2 99.4 0.6 0.0 0.0 0.0 20/30

Reference Range 97.5 - 99.4 0.6 - 1.3 0.0 – 0.0 0.0 – 0.0 0.0 - 1.3MIR162-USDA-1 Page 62 of 268 Table 19. Statistical analysis of proportions for normal germinated seeds. Fisher’s Exact Test was used to compar e the proportion of MIR162 and control seeds classified as normally germinated versus not normally germinated. Probability values for each comparison are presented in the table.

p values Temperature (°C) MIR162 #1 vs Control #1 MIR162 #2 vs Control #2

5 1.000 1.000

10 1.000 0.061

5/20 0.371 0.174

10/20 1.000 0.371

25 0.248 0.121

20/30 1.000 0.121

VI.A.2. Field Agronomic Performance

Small field trials were c onducted during the 2005 and 2006 gr owing seasons to compare a range of agronomic characteristics of two MIR162 maize hybrid s to near-isogenic nontransgenic commercial varieties. Table 20 shows the pedigree of the MIR162 and control hybrids planted in these trials (see also Table 2 and Figure 5) . The MIR162 hybrid used in 2005 trials corresponds to seed lot code ‘F’ and the MIR162 hybrid used in 2006 trials corresponds to seed lot code ‘I’ as described in Table 2 and Figure 5.

Table 20. Test and control hybrids planted in field agronomic equivalence trials.

Trial Year Hybrid Designation Seed Generation Lineage or Variety

MIR162 hybrid F 1 NP2010 x NP2222(BC 6 F 4 ) 2005 Control hybrid F 1 NP2222 x NP2010

MIR162 hybrid F 1 NP2673 x NP2171(BC 4 F 5 ) 2006 Control hybrid F 1 NP2673 x NP2171

Near-isogenic hybrid pairs were grown in si x locations in 2005 and 10 locations in 2006. These locations are representativ e of the major maize growing regions of the U.S. and are
listed in Table 21.MIR162-USDA-1 Page 63 of 268 Table 21. Locations and dates of MIR162 agronomic performance trials.

Trial Year City State Planting

Date Harvest

Date APHIS
Notification #

Alleman Iowa June 20 October 26

Bloomington Illinois May 28 October 10

Hudson Illinois May 28 October 19

Mackinaw Illinois May 28 October 19

Seward Nebraska June 15 October 28 2005

Wapella Illinois May 27 October 17 05-062-02n

Bloomington Illinois May 28 October 10

Brookings South Dakota May 31 October 30

El Paso Illinois June 3 October 24

Gaylord Minnesota May 31 October 26

Janesville Wisconsin June 1 October 29

Mackinaw Illinois May 29 October 23

Maxwell Iowa June 6 October 30

Monroeville Indiana May 30 October 28

Sadorus Illinois June 5 October 22 2006

Seward Nebraska June 1 October 7 06-055-08n

Trials were planted in a randomized complete block design. The trials planted in 2005 included four replicates per loca tion and trials planted in 2006 included five replicates per location. Plot size was 0.02 acres, using two-ro w plots, 17.5 ft long, with 30-inch spacing between the rows. Each plot contained approximately 68 plants. The agronomic characteristics assessed and the timing of each assessment are listed in the Table 16 above.

The agronomic data sets were subjected to an analysis of variance ac ross locations using the following model:

Y ijk

= U + T i + L j +B(L) jk

+ LT ij

+ e ijk

Y

ijk

is the observed response for entry i at location j block k ; U is the overall mean; T i is the entry effect, L j is the location effect, B(L) jk

is the effect of block within a location; LT ij

is the location-by-entry inte raction effect and e ijk is the residual error. For each variate, the statistical significance of the genotype effect for each of the hybrids was determined using a standard F-test. An F-test probability of < 0.05 indicates that the difference observed betweenMIR162-USDA-1 Page 64 of 268 the two genotypes was statistically si gnificant at the customary 5% level.

The data for several variates were not subjected to formal statistical analysis because they did not satisfy the assumptions upon which the validity of an analysis of variance depends. In some cases, the data were too discrete to be considered normally distributed, with values taking one of a very limited range of options. In other cases, th e data sets contained too few nonzero entries upon which to compute a r easonable estimate of residual error.

Results of the 2005 agronomic equivalence trials are presented in Table 22. Evaluation of
the 2005 trial results revealed on ly one statistically significant difference between a control and MIR162 variate. The average number of ge rminated plants per plot was slightly higher in the 2005 MIR162 plots compared to control plots; the difference was only 3.2%. This difference is considered to be of no biologi cal significance because the effect was not repeated in the 2006 trials and there was no effect of genotype observed in the seed germination and dormancy study (see Tables 18 and 19). No other differences between MIR162 and control plots were observed for the wide range of phenotypic endpoints
assessed in the 2005 trials.

Results of the 2006 agronomic equivalence trials are presented in Table 23. Evaluation of
the 2006 trial results revealed two statistically significant di fferences between MIR162 and control mean values. Grain moisture at harvest was 3.8% lower from the MIR162 plots and grain test weight was 1.4% lo wer from the MIR162 plots. Both of these differences are of very small magnitude and are inconsequential. There was no difference in yield observed between the MIR162 and control plots and there was no effect observed on either variate in the 2005 trials. These differences represen t random variation and are of no biological significance.

The results of these phenotypic assessments indicate that MIR162 maize is not phenotypically different from conventional maize with respect to char acteristics that would increase its weediness potential.MIR162-USDA-1 Page 65 of 268 Table 22. Results of agronomic performance trials conducted in 2005. Trials were planted with a mid-maturity MIR162 hybrid and a near-isogenic control. Trials were conducted at multiple locations representa tive of the major maize-growing areas of the U.S. Mean values across locations are presented in the table for each variate measured. Not
all traits were evaluated at all locations.

Trait Locations Control

X  ̄ MIR162

X  ̄ Δ p -value Significance a

Emerged plants (#) 6 62 64 2.0 0.04 *

Early growth rating 5 4.1 4.3 0.2 NA

Green snapped plants (%) 1 0 0 0 NA

Late root lodging (%) 6 4.0 2.0 -2.0 NA

Ear height (cm) 6 97.0 96.0 -1.0 0.62 NS

Plant height (cm) 6 228.0 227.0 -1.0 0.82 NS

Leaf color rating 5 5.0 5.0 0 NA

Late season intactness rating 4 3.1 3.3 0.2 NA

Stalk lodging (%) 6 0.7 0.9 0.2 NA

Stay green rating (%) 3 67.0 66.0 -1.0 0.66 NS

Plant population at harvest (plants/ac) 6 30,617 30,887 270 0.29 NS

Heat units to 50% pollen shed 5 1,378.0 1,386.0 8.0 0.45 NS

Heat units to 50% silking 5 1,428.0 1,438.0 10.0 0.18 NS

Barren plants (%) 6 0 0 0 NA

Grain moisture at harvest (%) 6 20.0 20.0 0.0 0.38 NS

Test weight (lb/bu) 6 56.7 55.8 -0.9 0.19 NS

Yield (bu/ac) 6 175.5 184.9 9.4 0.07 NS

Dropped ears 2 0 0 0 NA

Grey leaf spot rating 4 4.9 6.3 1.4 NA

a * - difference is significant; NS – difference is not significant; NA – data not statistically

analyzedMIR162-USDA-1 Page 66 of 268 Table 23. Results of agronomic performance trials conducted in 2006. Trials were planted with a mid-maturity MIR162 hybrid and a near-isogenic control. Trials were conducted at multiple locations representa tive of the major maize-growing areas of the U.S. Mean values across locations are presented in the table for each variate measured. Not
all traits were evaluated at all locations.

Trait Locations Control

X  ̄ MIR162

X  ̄ Δ p -value Significance a

Emerged plants (#) 8 62.1 63.5 1.4 0.13 NS

Early growth rating 8 3.1 3.0 -0.1 NA

Late root lodging (%) 7 0.4 0.2 -0.2 NA

Ear height (cm) 9 105.9 106.0 0.1 0.96 NS

Plant height (cm) 9 236.8 236.3 -0.5 0.82 NS

Late season intactness rating 9 4.9 5.4 0.5 NA

Stalk lodging (%) 10 2.4 3.5 1.2 0.21 NS

Plant population at harvest (plants/ac) 10 31,005.4 31,383.8 378.4 0.24 NS

Heat units to 50% pollen shed 8 1,242.1 1,245.9 3.8 0.32 NS

Heat units to 50% silking 8 1,254.2 1,262.8 8.6 0.07 NS

Barren plants (%) 5 0.1 0.1 0 NA

Grain moisture at harvest (%) 10 18.3 17.6 -0.7 0.0001 *

Test weight (lb/bu) 8 57.4 56.6 -0.8 0.03 *

Yield (bu/ac) 10 176.4 178.4 2.2 0.51 NS

Dropped ears 2 0.3 0.5 0.2 NA

Grey leaf spot rating 4 3.7 3.9 0.2 NA

a * - difference is significant; NS – difference is not significant; NA – data not statistically

analyzedMIR162-USDA-1 Page 67 of 268 VI.A.3. Pollen Viability and Morphology

Viability and morphology were ev aluated to assess the potentia l impact of transformation on pollen characteristics of MIR162 maize. Polle n morphology and viability were investigated by microscopically examining pollen grains that had been fixed and stained according to the method described by Pedersen et al. (2004).

Twenty MIR162 hybrid plants and 20 near-is ogenic control plants were grown in an environmentally controlled greenhouse. The greenhouse operated on a 16 hr/8 hr light/dark cycle with daytime temperatures ranging fr om 23°C-28°C and nighttime temperatures ranging from 18°C-22°C. Pollen wa s collected from each plant 69 or 70 days after planting. Pollen samples were immediately fixed in a 70 % (v/v) ethanol solution and refrigerated. Samples were stained with a small volume of a KI/I 2 solution and examined by light microscopy. Viability determinations were made at 50X magnification. Starch contained in the viable pollen cells readily bound to the iodine stain. Nonvi able cells with little or no starch content were weakly stained and readily identifiable. Percent viability was determined by examining a minimum of 100 pollen cells per sample. Mean percent viable pollen was determined for the MIR162 and control samples. These means were compared by a t-test with significance assigned at the standard p< 0.05 level. The results of this analysis are presented in Table 24. There was no signifi cant difference detected between MIR162 and control percent viable pollen. In fact, the fre quency of nonviable cells av eraged less than 1% in both MIR162 and control samples.

Table 24. Pollen viability and diameter measurements. Mean (X  ̄ ) percent pollen viability and mean diameter measurements are presented with their respective standard error of the mean (SEM ). The control and MIR162 means were compared by a t-test; no significant differences were detected.

Variable Control

X  ̄ (SEM) MIR162

X  ̄ (SEM)

Pollen viability (%) 99.71 (0.133) 99.74 (0.094)

Pollen diameter ( μ m) 96.8 (0.734) 97.2 (0.583)

Cell morphology and the dimension of stained pollen samples from five MIR162 and five control plants were examined at 80X magni fication. Morphology was assessed by a visual
examination of all cells in the field of view. Pollen diameter was measured on ten cells per sample. Mean diameter was computed for th e MIR162 and control sa mples. These means were compared by a t-test with significance assigned at the p< 0.05 level. Photographs of representative MIR162 and cont rol pollen samples are shown in Figure 20. Cell diameter statistics are presented in Table 24. There were no readily discernible differences in MIR162 and control pollen morphology. No significant difference in average cell diameter was detected between MIR162 and control pollen samples.MIR162-USDA-1 Page 68 of 268 Figure 20. Photographs of stained pollen collected from control and MIR162 plants. Magnification was set at 80X. Control Plant
#88755 80X

100 μ m

MIR162 Plant

#88708 80X Viable Cell

Nonviable Cell

Nonviable CellMIR162-USDA-1 Page 69 of 268 VI.A.4. Seed Dispersal

Dispersal of individual maize seeds does not occu r naturally. The kernels are held inside the
husks of the cob and are too he avy to be wind-blown. There wa s no significant difference in the propensity of MIR162 maize to drop ears than that of conventional maize (see Tables 22 and 23). Dispersal of individual kernels does, however, take plac e as a result of mechanical harvesting and transportation. In this regard, MIR162 maize kernels would be no different
than conventional maize kernels. Since maize seeds lack dormancy, those that are dispersed outside of cultivated fields would germinate an d the young plants could be exposed to harsh
winter conditions and die.

VI.B. Compositional Assessment of MIR162 Forage and Grain

Compositional analyses of MIR162 maize were pe rformed in order to identify any changes in
nutrient or anti-nutrient content of the new crop in the context of its use as food or feed and to assess its biochemical equivalence and familiarity to conventional maize. This assessment was undertaken by performing quantitative an alyses of 65 components of MIR162 hybrid maize forage and grain. An identical set of analyses was performed on a nontransgenic control variety.

Forage and grain from a MIR162 transgenic hybrid and its corresp onding near-isogenic
control were harvested from six locations in the U.S. during 2005. These locations are representative of the major co rn growing regions of the U.S. At each location, the hybrids were planted in a randomized complete block design, with three replicates for each genotype. All plots were managed according to local agr onomic practices for the respective regions. Plants were self-pollinated by hand and the developing ears were bagged to avoid crosspollination.

The analytes measured in this study were selected based on recommendations of the Organisation for Economic Co -operation and Development (OECD, 2002) for comparative assessment of composition of new varieties of maize. The com ponents analyzed are listed in
Table 25.

All analyses were conducted using methods pu blished and approved by the Association of Analytical Communities (AOAC) International or other indus try-standard analytical
methods. Based on the moisture content of eac h sample, analyte levels were converted to equivalent units of dry wei ght. A detailed description of the study design and methodology for the compositional analyses can be found in Appendix E.

The analytical results for each component were su bjected to an analysis of variance using the
model:

Y ijk

= U + T i + L j + B(L) jk + LT ij

+ e ijk

where Y ijk

is the observed response for genotype i at location j block k

, U is the overall mean,

T

i is the genotype effect, L j is the location effect, B(L) jk

is the effect of block within a location, LT ij

is the location-by-genotype interaction effect and e ijk is the residual error.MIR162-USDA-1 Page 70 of 268 Table 25. Forage and grain components measur ed in MIR162 and conventional maize.

Forage Grain

Minerals Amino acids (18) Proximates Secondary
Metabolites

Calcium Anti-nutrients Acid detergent fiber Ferulic acid

Phosphorus Phytic acid Ash ρ -Coumaric acid

Proximates Raffinose Carbohydrates Furfural

Acid detergent fiber (ADF) Trypsin inhibitor Fat Inositol

Ash Minerals Moisture Vitamins

Carbohydrates Calcium Neutral detergent fiber Vitamin A ( β -carotene)

Fat Copper Protein Vitamin B 1 (thiamine)

Moisture Iron Starch Vitamin B 2 (riboflavin)

Neutral detergent fiber (NDF) Magnesium Total Dietary Fiber (TDF) Vitamin B 3 (niacin)

Protein Manganese Fatty acids Vitamin B 6 (pyridoxine)

Phosphorus Palmitic acid Vitamin B 9 (folic acid)

Potassium Stearic acid Vitamin E ( α

tocopherol)

Selenium Oleic acid

Sodium Linoleic acid

Zinc Linolenic acid

For each analyte, the statistical significan ce of the difference between the MIR162 and control mean values ( i.e., the genotype effect) was determined using a standard F-test. An

F-test p< 0.05 indicates that the difference between the genotypes was statistically significant
at the 5% level. An F-test was also us ed to detect potenti al location-by-genotype interactions. In this case a significant outcome ( p< 0.05) indicates that the effect of genotype was not consistent across locations, and that the comparison of genot ypes averaged across locations may not be valid. In such cases, th e average analyte levels measured at each location were compared to ranges of normal values reported in the literature. For all analytes measured, the average levels across location s were compared to the range of natural variation, as reported in the International Life Sciences Institute crop composition database (ILSI, 2006) and the OECD consensus document on compositional considerations for maize (OECD, 2002).MIR162-USDA-1 Page 71 of 268 The results of these compositiona l analyses, expressed as mean values across locations and a range of individual replicate va lues, are presented in the tabl es found in Appendix E (Tables E-3 through E-11). Nine components of ma ize forage were measured; the difference
between MIR162 and control mean values was f ound to be statistically significant for one of these analytes. Fifty-six components of gr ain were measured; the difference between
MIR162 and control mean values was found to be statistically significant for 13 of these analytes. The results for these 14 analytes th at had a statistically significant outcome for genotype effect are presented in Table 26.

The forage compositional analyses for proximates and minerals revealed a single statistically significant difference between MIR162 and cont rol mean values. The mean value for MIR162 NDF was 11.34% higher than the corres ponding control value. This difference is considered relatively small a nd the MIR162 mean falls well within the ra nge of normal values reported by ILSI and OECD. No st atistically significant genotype by location interactions were noted for the forage compositional analyses.

Compositional analyses of grain revealed no statistically significant differences between
MIR162 and control means for 43 of the 56 analytes examined in across-location comparisons. Statistically significant differences were noted for levels of the proximates ash, NDF, and starch. These differences were sma ll (< 8%) and the MIR162 mean values were well within the ranges of normal values for th e control maize. Additionally, the average values for all proximates were within the ranges reported by ILSI and OECD.

Statistically significant differen ces were noted for three grain minerals: calcium, iron, and phosphorus. The differences observed were small, less than 8%, and the mean MIR162 values were each within the ranges of nor mal values reported by ILSI and OECD for
conventional maize.

Statistically significant differences were noted between MIR162 and cont rol mean levels of vitamin A ( β -carotene), vitamin B 6 (pyridoxine), and vitamin E ( α -tocopherol). These differences were small (< 7%) and the mean values observed for these vitamins in MIR162 grain were well within the range of values obs erved for the control grain. Additionally, the
MIR162 means for all vitamins fell within the normal range of values reported for conventional maize by ILSI and OECD . For vitamin A and vitamin B 9 a statistically significant genotype-by-location in teraction was noted, which suggests that the effect of genotype was not consistent across locations, hence, the comparison of genotypes averaged across locations may not be valid. Individu al location means for the two analytes are provided in Table E-8 (Appendix E). The vitamin A and vitamin B 9 levels at all locations were within the ranges reported in the literature.

There were no significant differenc es noted for any of the 18 am ino acids or anti-nutrients measured and all average values were with in the ranges reported by ILSI and OECD.

Statistically significant differences were noted for linoleic and linolenic fatty acids. These differences were very small (< 4%) and the MIR162 mean values obs erved for these fatty acids were within the ranges of values observe d for the control grain. Furthermore, theMIR162-USDA-1 Page 72 of 268 Table 26. Statistically significant differences between MIR162 and control analytes. Summary composition results for the one fora ge analyte and 13 grain analytes that had statistically significant ( p< 0.05) differences between MIR162 and control maize across location mean values. All values reported are on a dry weight (dw) basis.

Analyte MIR162 X  ̄

(Range) Control X  ̄

(Range) % Δ a ILSI X  ̄

(Range) Literature Range b

Proximates

NDF (%) 43.2 (35.1 – 56.1) 38.8 (32.13 – 46.9) 11.34 41.51 (20.29 – 63.71) 40 – 48.2

Grain

Ash (%) 1.4 (1.1 - 1.6) 1.3 (1.1 – 1.5) 7.69 1.439 (0.616 - 6.282) 1.1 – 3.9

NDF (%) 11.7 (10.1 – 13.0) 11.1 (9.5 – 12.8) 5.41 11.23 (5.59 – 22.64) 8.3 – 11.9

Starch (%) 63.1 (54.8 – 68.1) 64.9 (60.6 – 69.2) -2.77 57.7 (26.5 – 73.8)

Calcium (mg/kg) 38.1 (29.4 – 47.2) 35.3 (25.7 – 44.0) 7.93 46.4 (12.7 – 208.4) 3 – 100 g /100 g

Iron (mg/kg) 20.2 (17.3 – 22.9) 19.2 (15.7 – 22.5) 5.21 21.81 (10.42 – 49.07) 0.1 – 10 g /100 g

Phosphorus (mg/kg) 3173 (2810 - 3550) 3073 (2710 - 3400) 3.25 3273.5 (1470.0 –

5330.0) 234 – 750 mg /100 g

Vitamin A (mg/100 g) 0.277 (0.241 – 0.316) 0.294 (0.244 – 0.358) -5.78 0.684 (0.019 – 4.681)

Vitamin B 6 (mg/100 g) 0.565 (0.434 – 0.694) 0.605 (0.486 – 0.738) -6.61 0.644 (0.368 – 1.132) 4.6 – 9.6

Vitamin E (mg/g) 0.0125 (0.0097 –

0.0154) 0.0132 (0.0110 –

0.0154) -5.44 0.0103 (0.0015 –

0.0687)

18:2 Linoleic acid (% total FA) c 56.99 (55.86 – 59.74) 57.36 (56.26 – 59.47) -0.65 57.60 (36.2 – 66.5) 0.67 – 2.81%
dw

18:3 Linolenic acid (% total FA) c 1.81 (1.72 – 1.89) 1.75 (1.64 – 1.86) 3.43 1.20 (0.57 – 2.25) 0.03 – 0.10%
dw

Ferulic acid (mg/kg) 2682 (2490 - 2980) 2453 (2010 - 2760) 9.33 2201.1 (291.9 – 3885.8) 200 – 3000

ρ -Coumaric acid (mg/kg) 179 (148 - 202) 157 (137 - 179) 14.01 218.4 (53.4 – 576.2) 3 – 300

a – % difference: [ (MIR162 – control)/control ] x 100

b – literature values in comparable units of measure not available for some analytes

c – FA = fatty acidsMIR162-USDA-1 Page 73 of 268 average values for all fatty acids were with in the range of normal values reported for conventional maize by ILSI and OECD.

Statistically significant differen ces were noted in the secondary metabolites ferulic acid and

ρ -coumaric acid. These differences were rela tively small (< 15%) and the MIR162 mean values for these secondary metabolites were within the ranges of values observed for the control grain. Additionally, the mean values for all MIR162 secondary metabolites and antinutrients were within the normal range of valu es reported for conventional maize by ILSI and
OECD.

Collectively, the observed differences between MIR162 and control means are considered of no biological significance and re present typical random varian ce. The magnitude of the differences was small, all MIR162 values fell within normal ranges for conventional maize, and the MIR162 and control data ranges signifi cantly overlapped. MIR 162 is therefore, not compositionally different than conventional maize.

VI.C. Summary of Phenotypic and Compositional Evaluations

Maize has lost the ability to survive without cultivation (OECD, 2003). It can overwinter and germinate in a subsequent crop as a volunteer weed; for example, maize is a common volunteer in soybeans. Neverthele ss, several features of maize make it unlikely to form selfsustaining weedy populations in ag ricultural systems: i) it is easily controlled in subsequent crops by selective herbicides; ii) seed dispersa l is limited because seeds are held inside the husks of the cob; and iii) the seeds lack dormanc y. Maize does not persist in habitats outside agriculture because, in addition to the features listed above, it requires disturbed ground to germinate and it is very uncom petitive against perennial ve getation. The results of laboratory, greenhouse, and fiel d studies indicate that MIR 162 maize is no different than conventional maize with regard to phenotypic and compositional properties that bear on weediness potential for the new cultivar.

VII. Environmental Consequences of Introduction

MIR162 maize contains a novel pr otein that has toxi cological activity ag ainst a number of lepidopteran species which are pests of U.S. agriculture. Studies have been conducted to define the spectrum of insecticidal activity fo r Vip3Aa proteins and to assess the efficacy of MIR162 maize hybrids in resisting larval fe eding damage. An assessment of risk for nontarget organisms and endangered species that might be exposed to the Vip3Aa20 protein in MIR162 maize has been performed. This risk assessment is composed of multiple parts: i) a determination of expected environmenta l concentrations (EECs) for Vip3Aa20; ii) a characterization of potential h azards posed by the protein to nontarget indicator organisms appropriate for a maize ecosystem; iii) a co mparison of EECs to no observable effect concentrations for nontarget organisms; and iv ) a specific characte rization of endangered species risk.

Given the ubiquitous nature of PMI enzymes in nature and its a pparent absence of environmental risk, it can reasonably be assu med that the presence of PMI protein inMIR162-USDA-1 Page 74 of 268 MIR162 will do nothing to alter the ecologi cal balance within maize ecosystems.

VII.A. Spectrum of Insecticidal Activity for Vip3Aa Proteins

Syngenta has conducted mortality bi oassays with Vip3Aa protein vari ants in a range of insect species to identify those that are susceptible to the toxin. The results of these bioassays and the results of assays reported in the scientific literature demonstrate that activity of Vip3Aa proteins is limited to species within the orde r Lepidoptera, yet all lepi dopteran species are not sensitive to the protein. It is notable that Vi p3Aa proteins appear to have no activity against
O. nubilalis , probably the most significant lepidopter an pest of U.S. maize. Table 27 provides a listing of insect sp ecies that have been found by Syngenta, or reported in the literature, as being sensitive to one or more Vip3Aa protein variants. The Bacillus thuringiensis Toxin Nomenclature Committee curren tly lists 25 variants of the Vip3Aa protein. 5 This narrow spectrum of activity for Vi p3Aa proteins is a very positive attribute from an ecological perspective; maize hybrid s containing a Vip3Aa protein are unlikely to pose a risk to nontarget organism s inhabiting maize ecosystems.

Table 27. Insect species screened for sens itivity to Vip3Aa protein variants.Mortality was assessed in standard diet-surface or diet-incorporation bioassays. Treatmentrelated mortality was the criterion used for evidence of sensitivity.

Order: Family Genus: Species Activity

Agrotis ipsilon Active

Helicoverpa zea Active

Helicoverpa armigera Active

Helicoverpa punctifera Active

Heliothis virescens Active

Spodotera exigua Active

Spodoptera frugiperda Active

Spodoptera litura Active

Striacosta albicosta Active Lepidoptera: Noctuidae

Trichoplusia ni Active

Lepidoptera: Gelechidae Phthorimea operculella Active

Lepidoptera: Sphingidae Manduca sexta Active

Chilo partellus Active Lepidoptera: Pyralidae Ostrinia nubilalis Not Active

Lepidoptera: Plutellidae Plutella xylostella Active

(continued)

5 http://www.lifesci.sussex.ac.uk/ home/Neil_Crickmore/Bt/vip.html (August 25, 2007).MIR162-USDA-1 Page 75 of 268 Table 27 (cont.). Insect species screened for sensitiv ity to Vip3Aa protein variants.Mortality was assessed in standard diet-surface or diet-incorporation bioassays. Treatmentrelated mortality was the criterion used for evidence of sensitivity.

Order: Family Genus: Species Activity

Lepidoptera: Danaidae Danaus plexippus Not Active

Lepidoptera: Pieridae Pieris brassicae Not Active

Bombyx mori Not Active

Earias vittella Active Other Lepidoptera

Ephestia kuehniella Active

Culex pipiens Not Active

Drosophila melanogaster Not Active Diptera

Culex quinquefasciatus Not Active

Coleomegilla maculata Not Active

Diabrotica virgifera virgifera Not Active

Leptinotarsa decemlineata Not Active

Tenebrio molitor Not Active

Anthonomus grandis grandis Not Active Coleoptera

Aleochara bilineata Not Active

Hymenoptera Apis mellifera Not Active

Hemiptera: Anthocoridae Orius insidious Not Active

Thysanoptera Frankliniella occidentalis Not Active

Isotomidae Folsomia candida Not Active

Neuroptera Chrysoperla carnea Not Active

VII.B. Activity Against Target Insects

As shown in Table 27, Vip3Aa has activity agains t several of the major lepidopteran pests of maize, specifically: A. ipsilon

, H. zea

, S. albicosta , and S. frugiperda . Expressing a vip3Aa gene at adequate levels in maize is a logica l approach for limiting crop losses attributable to these four pests, especially considering the defici encies of conventiona l insecticides in controlling these pests (see discussion in Section VII.I). Thus, MIR162 maize was developed. In hybrid offerings to growers, the MIR162 trait will be combined through traditional breeding with other insect protection traits ( e.g. , Bt11xMIR162 maize).

Trait efficacy trials with MIR162 maize hybrids were conducted at multiple locations in 2005 and 2006 by Syngenta entomologists, academic c ooperators, and extension agents. Figure 21 provides a graphic representation of the co mparative feeding damage for each of theMIR162-USDA-1 Page 76 of 268020406080100120Bt11 MIR162 Bt11xMIR162 InsecticideTreatment

% of Control DamageO. nubilalisS. frugiperdaH. zeaA. ipsilonS. albicostaFigure 21.MIR162 field efficacy trial results.Comparative feeding damage ratings are from replicated field efficacy trials conducted in 2005 and 2006 with Bt11, MIR162, andBt11xMIR162 maize, and a conventional insecticide treatment (Warrior®Insecticide). Mean damage ratings are expressed as apercentage of the damage observed in the untreated control plots.®Trademark of a Syngenta group companyMIR162-USDA-1 Page 77 of 268 treatments, expressed as a percentage of damage measured in the contro l plants, for five of the insects evaluated. Trials were placed in locations to take advantage of natural pest
infestations. In some locations trials were arti ficially infested to test trait performance under high levels of insect pressure. MIR 162 maize, Bt11 maize, Bt11xMIR162 maize, conventional maize, and conventional maize w ith an insecticide ap plication were the treatments employed in most trials. These trials were conducted unde r USDA notifications
05-062-02n and 06-055-08n.

MIR162 alone has no activity against O. nubilalis but is efficacious in limiting feeding damage caused by the other four insect pests. Whereas Bt11 is highly efficacious against O.
nubilalis , it has limited or no activity against the other four in sects. The combined-trait Bt11xMIR162 hybrid is very efficacious against all five insects. MIR162 addresses the lepidopteran pest-control limitations of ex isting Cry1Ab-containing maize hybrids. In combination with the Bt11 trait, it will prov ide growers with excellent control of the following significant lepidopteran insect pests of maize (not all efficacy data shown): A. ipsilon

, D. crambidoides

, D. grandiosella

, D. saccharalis

, H. zea

, O. nubilalis

, P. nebris

, S. exigua

, S. frugiperda , and S. albicosta

.

VII.C. Expected Environmental Concentrations for Vip3Aa20

Data on the concentration of Vip3Aa20 in MIR 162 maize are used to make estimates of the EEC for Vip3Aa20 in groups of organisms potentially exposed via MIR162 maize. Eestimates of exposure based on conservative assumptions about the dilution of Vip3Aa20 in prey, in soil, and in water have been made . These EECs are suitable for protecting populations of nontarget organisms. Potential exposures and ri sks for endangered species are addressed separately in Section VII.F.

VII.C.1. Concentrations of Vip3Aa20 in MIR162 Maize

The concentrations of Vip3Aa20 were measur ed by ELISA in several MIR162 maize tissues and whole plants at growth stages V9-V12, anth esis, seed maturity and senescence (refer to Table 14). The values chosen for computation of EECs are show n in Table 28. These values represent the highest mean con centration across locations at any growth stage of MIR162 hybrid maize. EECs were calculat ed from fresh weight values.

VII.C.2. Soil Fate of Vip3Aa Proteins

Most proteins do not persist or accumulate in th e soil because they are inherently degradable in soils that have healthy microbial populations ( e.g. , Burns, 1982; Marx et al ., 2005). Multiple investigations have demonstrated that Cry proteins from B. thuringiensis are rapidly degraded in a variety of soil types and that the proteins do not accumulate (EPA, 2001; Head et al., 2002; Dubelman et al., 2005). Vip proteins are similar to Cry proteins in that they are also found in naturally occurri ng soil bacteria and commercial microbial insecticides (De Maagd et al ., 2003). There is no evidence that they accumulate in soil or are protected from the activity of proteases in soil.MIR162-USDA-1 Page 78 of 268 Table 28. Tissue concentrations of Vip3Aa20 used for computation of EECs. Highest mean concentrations of Vip3Aa20 as de termined by ELISA in tissues of two fieldgrown MIR162 hybrids. All values are reported on a fresh weight basis.

Tissue Vip3Aa20 ( μ g/g) Growth stage

Leaves 50.41 Seed maturity

Kernels 29.81 Seed maturity

Roots 5.29 Senescence

Pollen 43.21 Anthesis

Whole plant 17.35 Seed maturity

A laboratory study was conducted to determine th e degradability of Vip3 Aa proteins in live soils. For this investigation, the Vip3Aa19 vari ant was used. Live soil samples from Brazil and the U.S., plus one artificial soil sample, re presenting four soil textures (clay, sandy clay loam, sandy loam, and silt loam) we re used to examine the rate of degradation of Vip3Aa19. The source of Vip3Aa19 for this study was a c oncentrated protein ex tract from lyophilized leaf tissue of transgenic event Pacha maize. The protein was tested at two concentrations, 4 mg/g and 16 mg/g dry weight-equivalent soil (corresponding to approximately 14 and 58

μ g/g dry weight equivalent soil). This concentr ation was expected to gr eatly exceed levels of Vip3Aa protein entering the soil as a result of Vip3Aa maize cultivation. 6 A rapid decline in the levels of Vip3Aa19 was observe d in all soil types; degrada tion was measured as loss of insecticidal activity. The time to 50% dissipation (DT 50 ) was estimated to be between 6.0 and 12.6 days across soil types and test concentrat ions. The results of this study show that Vip3Aa19 is inherently degradable in healthy soils and indicate it is likely to be degraded rapidly in the field.

VII.C.3. Expected Environmental Con centrations for Nontarget Organisms

This section provides estimates of realistic environmental concentrations of Vip3Aa20 for multiple classes of nontarget organisms that may be exposed via cultivation of MIR162 maize. Exposure through soil ru n-off or gene flow is unlik ely for the reasons stated elsewhere in the petition (see Sections VII.C. 2 and VII.H) and is not considered. The approach to estimating environmental exposures for Vip3Aa20 is similar to that previously described for corn rootworm-protected maize event MIR604 (Raybould et al. , 2007).

VII.C.3.a. EEC for Wild Birds

Birds rarely feed on leaf tissue of maize; however, birds such as Corvus brachyrhynchos (crows), Quiscalus quiscula (grackles), and Grus canadensis (sandhill cranes) uproot sprouting corn to feed on the germinating kernels ( e.g ., Steffey et al ., 1999; Blackwell et al

.,

6 MIR162 maize was not the lead line under development.MIR162-USDA-1 Page 79 of 268 2001; Sterner et al ., 2003). Agelaius phoeniceus (red-winged blackbirds) and common grackles destroy over 360,000 metric tons per annu m of ripening field corn in the U.S. and Canada. Blackbirds typically slit open husks with their bills and puncture kernels in the milk stage (Steffey et al ., 1999). Blackbirds are also common in corn stubble where they forage for spilled corn kernel s and weed seeds (Linz et al ., 2003). Therefore, th e concentration of Vip3Aa20 in kernels was used to estimate the exposure of wild birds to Vip3Aa20 via
cultivation of MIR162 maize.

The mean concentration of Vip3Aa 20 in MIR162 maize kernels is 29.81 μ g/g fresh weight (Table 28). Exposure to birds may be expr essed more suitably as a daily dietary dose (DDD), which is computed by a simple formula:

C bw FIR DDD × =

FIR = daily food intake; bw = body weight; C = concentration of Vip3Aa20 in food

FIR/bw ratios for cereal seed-eating birds consum ing fresh food were estimated by Crocker
et al . (2002). Among the seven spec ies represented, values range from 0.11 for the pheasant ( Phasianus colchicus ) to 0.35 for the tree sparrow ( Passer montanus ). These species represent a range of mean body weights; 22 g for the sparrow to 953 g for the pheasant (heavier species have lower FIR/bw ratios). Higher FIR/bw ratios give higher DDDs and therefore, more conservative estimates of risk.

Wild birds are unlikely to consume a diet of 100% maize kernels. A more realistic estimate of exposure can be derived from the proportion of maize kernels in the diet of birds feeding in maize-growing areas. Studies by McNichol et al . (1979) and Homan et al . (1994) of the diets of red-winged blackbirds and common gr ackles, respectively, showed that maize kernels comprise up to 50% of their diet. An EEC can be calculated based on a diet of 50% maize kernels containing 29.81 μ g Vip3Aa20/g kernels. This yi elds a realistic EEC for wild
birds of:

0.5 x 29.81 μ g Vip3Aa20/g = 14.91 μ g Vip3Aa20/g kernels

with a DDD for wild birds of:

0.5 x 0.35 x 29.81 μ g Vip3Aa20/g = 5.22 μ g Vip3Aa20/g bw

VII.C.3.b. EEC for Wild Mammals

The main route of exposure of wild mammals to Vip3Aa20 in MIR162 maize is consumption
of kernels. Rodents such as Spermophilus tridecemlineatus (thirteen-lined ground squirrels), Peromysus maniculatus (deer mice), Mus domesticus (house mice), and Microtus spp. (prairie and meadow voles) feed on germinating co rn seeds. Frequently these species remove so many seeds that the fiel d needs to be replanted. Marmota monax (woodchucks) also feed on sprouting corn seed, but because they feed along the edges of fields, they usually cause less serious damage than other rode nts. Larger mammals such as Odocoilus virginianusMIR162-USDA-1 Page 80 of 268 (white-tailed deer) and Procyon lotor (raccoons) cause injury to ri pening ears. Deer typically nip off ear tips, whereas raccoons chew throu gh husks. In some areas these species are hunted specifically to reduce damage to cornfields (Steffey et al ., 1999).

The mean concentration of Vip3Aa20 in MIR162 maize seeds is 29.81 μ g/g fresh weight (Table 28). As with birds, exposure to ma mmals may be expressed more suitably as a DDD (calculated by the form ula above). Crocker et al. (2002) has estimated the ratio of daily food intake and body weight ( FIR/bw ) for several rodent species. The values for the Micromys

minutus (harvest mouse) and the Apodemus sylvaticus (wood mouse) consuming cereal seeds are 0.33 and 0.28, respectively. Higher FIR/bw ratios give higher DDDs, and therefore, more
conservative estimates of risk.

Wild mammals are unlikely to consume a diet of 100% maize kernels. A more realistic estimate of exposure can be de rived from the proportion of ma ize kernels in the diet of rodents feeding in maize growing areas. The pr oportion of maize kernels in wild rodent diets varies greatly according to sp ecies (Houtcooper, 1978; Ellis et al ., 1998), but can be up to
73%. An EEC can be calculated based on a diet of 73% maize kernels containing 29.81 μ g Vip3Aa20/g kernels. This yields a realistic EEC for wild mammals of:

0.73 x 29.81 μ g Vip3Aa20/g = 21.76 μ g Vip3Aa20/g kernels

with a DDD for wild mammals of:

0.73 x 0.33 x 29.8 μ g Vip3Aa20/g = 7.18 μ g Vip3Aa20/g bw

VII.C.3.c. EEC for Pollinators

Honeybees forage for maize pollen and th erefore, can be exposed to Vip3Aa20 via MIR162 pollen (Severson and Perry, 1981 ). Honeybees can successf ully rear young on a diet of 100% maize pollen; however, it is unlikely that maize pollen regularly comprises more than 50% of their diet (Babendreier et al ., 2004). Assuming a diet of 50% MIR162 pollen at the mean concentration of Vip3Aa20, a realistic EEC for honeybees and ot her pollinators is computed to be:

0.5 x 29.81 μ g Vip3Aa20/g = 14.91 μ g Vip3Aa20/g pollen

VII.C.3.d. EEC for Above-ground Nontarget Arthropods

Nontarget arthropods rarely, if ev er, eat leaves of maize. Th e more likely route of exposure to Vip3Aa20 for these species is consump tion of prey that have fed on maize ( e.g. , Harwood
et al. , 2005), or consumption of pollen if prey are scarce (Coll and Guershon, 2002).

The concentration of Vip3Aa20 in the prey of nontarget arthropods wi ll vary depending on the prey species, its developmental stage, a nd the concentration of Vip3Aa20 in plant parts on which they are feeding. Seve ral studies have examined the concentration of Cry proteins
in herbivores relative to the concentration of plants on which they are feeding; most testedMIR162-USDA-1 Page 81 of 268 the concentration of Cry1Ab in herbivores feeding on Bt maize and others have examined herbivores feeding on cotton and oi lseed rape containing Cry1Ac (Head et al ., 2001; Raps et al. , 2001; Dutton et al ., 2002; Obrist et al ., 2005, 2006a, 2006b; Torres et al ., 2006; Howald
et al ., 2003).

In general, the results of thes e studies show that herbivores contain lower concentrations of B. thuringiensis toxins than the plants on which they ar e feeding. Sucking insects, such as aphids, contain only trace amounts of Cr y1Ab when feeding on Cry1Ab maize (Head et al

., 2001; Raps et al ., 2001; Dutton et al ., 2002; Obrist et al ., 2006a). Lepidopteran larvae
contain between 0.1 and 0.25X the concentratio n of Cry1Ab in Cry1Ab maize on which they are feeding (Raps et al ., 2001; Dutton et al ., 2002; Obrist et al ., 2006b), and similar results were obtained by Torres et al . (2006) with Spodoptera exigua feeding on cotton containing Cry1Ac. Frankliniella tenuicornis (thrips) contain up to 0.35X the concentration of Cry1Ab in Cry1Ab-maize, although this concentration is tran sitory; adults contain about half this amount and pupae less than 1/40 th the concentration in larvae (Obrist et al ., 2005). The herbivores with the highest conc entrations of Cry protein are Tetranychus urticae (spider mites); they have been found to contain betw een 0.7 and 3X the concen tration of Cry1Ab in Cry1Ab maize (Dutton et al ., 2002; Obrist et al ., 2006a,b). All of these pests are found in maize, and therefore, nontarget arthr opods may be exposed to Vip3Aa20 through consumption of these species.

A precise EEC is difficult to compute given th e variety of food that nontarget arthropods are likely to consume. Setting the EEC at 0.2X the overall mean leaf concentration at the highest expressing developmental stage seems reasonab ly conservative as many lepidopteran larvae contain less than this amount, and aphids and lepidopteran eggs contain considerably less. Spider mites may contain higher concentrations of Vip3Aa20 than leaf tissue, and serious outbreaks of spider mites can occur in maize, particularly under drough t conditions (Holtzer
et al ., 1988). However, most predators in maize fields are generalist feeders that do not depend on a single pest species as a food source (Steffey et al ., 1999); therefore, nontarget arthropods are highly unlikely to consume a di et comprising solely spider mites. Possible exceptions are the specialist spider mite predators Stethorus spp. (Coccinellidae). S. punctillum is found in maize ( e.g. , Obrist et al. , 2006a); however, this species preferentially eats spider mite eggs, which are likely to contain low concentra tions of toxin compared with adult mites (Roy et al ., 2002). Hence, 0.2X the leaf c oncentration of Vip3Aa20 is a reasonably conservative EEC. The highest average concentr ation of Vip3Aa20 in above ground tissue of MIR162 plants is 50.41 μ g/g leaf fresh weight at s eed maturity (Table 28). The realistic EEC for above-ground nontar get arthropods is computed to be:

0.2 x 50.41 μ g Vip3Aa20/g = 10.08 μ g Vip3Aa20/g diet

VII.C.3.e. EEC for Soil-dwe lling Nontarget Invertebrates

A realistic EEC for soil-dwelling nontarget arth ropods can be calculated as the concentration of Vip3Aa20 in soil following incorporation of maize plants into soil post-harvest. The average concentration of Vip3Aa20 in MIR162 whole plants is 17.35 μ g/g at seed maturity (Table 28). The average planting density of maize is 65,500 plants per hectare and theMIR162-USDA-1 Page 82 of 268 average fresh weight of a corn plant is 750 g. If MIR162 plants contai n an average of 17.35

μ g Vip3Aa20/g), one hectare of MIR162 maize contains:

65,500 plants x 750 g/plant x 17.35 μ g Vip3Aa20/g = 8.52x10 8 μ g Vip3Aa20.

If the maize is ploughed into the top 15 cm of soil, the Vip3Aa20 will be incorporated into 100 m x 100 m x 0.15 m = 1,500 m 3 of soil per hectare. The av erage density of soil is 1,500 kg/m 3 . Therefore, the MIR162 maize will be incorporated into 1,500 m 3 /ha x 1,500 kg/m 3 = 2,250,000 kg soil per hectare. Dividing the amount of Vip3Aa20 per hectare by the mass of soil per hectare gives the realistic EEC for soil organisms of:

8.52x10 8 μ g Vip3Aa20/ha ÷ 2,250,000 kg soil/ha = 379 μ g Vip3Aa20/kg soil (or 0.38 μ g Vip3Aa20/g soil)

VII.C.3.f. EEC for Aquatic Organisms

The main route of potential exposure of aqua tic organisms to Vip3A a20 is through MIR162 pollen deposited in water bodies adjacent to maize fields. Maize produces up to 3.15x10 11 pollen grains per hectare during anthesis (Westgate et al ., 2003); 1 mg of maize pollen contains approximately 2,500 pollen grains (Depuis et al ., 1987). Therefore, maize produces up to 1.26x10 8 mg of pollen per hectare. A worst- case assumption for loading of maize pollen into a body of water is that it is depos ited onto the surface of the water at the same density it is produced in the fi eld. For a 1-hectare pond (10,000 m 2 ) that is 2 m deep, the pollen concentration would be:

1.26x10 8 mg pollen ÷ (10,000 m 2 x 2 m) = 6300 mg pollen/m 3 water (or 6.3 mg pollen/l water)

Deposition of maize pollen falls dramatically from the edge of a maize field; for example,
Pleasants et al . (2001) found that pollen deposition 5 m from the edge of the field was approximately one-tenth of that at the i mmediate edge. A reasonably conservative extrapolation from this observation is that polle n loading into an adjacent water body is likely
to be at least 0.05X th e density of pollen within the maize field. 7 Estimates of settling and degradation of maize pollen in ponds are not av ailable, but reduction of the maximum pollen density by 0.5 seems a conservative assumption. Using the mean pollen concentration of 43.21 μg Vip3Aa20/g, an EEC for aquatic organisms exposed via pollen is calculated by multiplying the concentration of Vip3Aa20 in MIR162 pollen, the density of pollen under worst-case conditions, 0.05 to account for rea listic pollen loading, and 0.5 to account for degradation and settling:

43.21 μg Vip3Aa20/g MIR162 pollen x 0.0063 g MIR162 pollen/l x 0.05 x 0.5

= 0.0068 μ g Vip3Aa20/l water

About 25% maize grain by weight is typical in the feed of farm-raised fish (National

7 The assumption is that under worst-case conditions, a 100 m x 100 m field deposits pollen into a 1 ha pond; under more realistic conditions, only a 5 m x 100 m strip of maize deposits pollen into the pond.MIR162-USDA-1 Page 83 of 268 Research Council, 1983). The mean concentr ation of Vip3Aa20 in MIR162 grain is 29.81

μ g/g. Fish feed is heat-treated during preparat ion and therefore, it is likely that Vip3Aa20
will be denatured and lose activit y in feed prepared from MIR 162 grain. Maize in fish feed is unlikely to comprise 100% MIR162 grai n. Insect-protected maize comprised approximately 40% of maize acres planted in the United States in 2006 (Doane Marketing Research, 2006a). If MIR162 maize were to achieve a 50% market share of the insectprotected maize market and assuming that no Vi p3Aa20 is lost during heat treatment of the feed, a realistic EEC for farmed fish is:

29.81 μ g Vip3Aa20/g x 0.25 x 0.40 x 0.5 = 1.49 μ g Vip3Aa20/g feed

VII.C.3.g. Summary of Environmental Exposure Estimates

Realistic EECs have been computed for groups of nontarget organisms that will be potentially exposed to Vip3Aa20 via cultivation of MIR162 maize. These EEC values are compiled in Table 29 and are appropriate for es timating the risks to populations of nontarget organisms. Once defined, these EEC or DDD va lues can be directly compared to Vip3Aa exposure or dose levels that cause no adverse e ffects in test species. This comparison results in a toxicology exposure ratio (TER).

It is highly unlikely that nontarget organisms will be exposed to Vip3Aa20 in environments outside of cultivated maize. Maize pollen does not drift great distances nor are maize seeds wind-borne. The probability of spread of Vip3Aa20 outside maize cultivation through volunteers and self-sustaining feral populations of MIR162 maize is also very low.

Table 29. Tabulation of EEC and DDD values for nontarget organisms. Each EEC value presented is based on the hi ghest average concentration of Vip3Aa20 in applicable MIR162 plant tissues and conservativ e estimates for consumption of these tissues by nontarget organisms. DDD values are computed where appropriate.

Nontarget Organism Group Expected
Environmental Concentration (EEC) Daily Dietary Dose (DDD)

Birds 14.91 μ g/g kernels 5.22 μ g/g bw

Wild mammals 21.76 μ g/g kernels 7.18 μ g/g bw

Pollinator 14.91 μ g/g pollen -

Above-ground arthropods 10.08 μ g/g diet -

Soil-dwelling invertebrates 0.38 μ g/g soil -

Aquatic organisms 0.0068 μ g/l water -

Farmed fish 1.49 μ g/g feed -MIR162-USDA-1 Page 84 of 268 VII.D. Impact on Nontarget Organisms

Thirteen species representing groups of nontarget organisms pot entially exposed to Vip3Aa20 in MIR162 maize were exposed to Vip3A a variants in laborato ry bioassays for the purpose of identifying adverse effects and establ ishing a no observable effect concentration (NOEC) or NOEL. Several test substances, cont aining one of three variants of Vip3Aa, were used in these studies. The identity and sour ce of Vip3Aa variant used in each study are listed
in Table 30. The reason that variants other than Vip3Aa20 have been used for studies supporting this MIR162 petition for deregulation is that Syngenta has had other transgenic crops under development that have incorporated the Vip3Aa19 variant. 8 Also, this petition is relying on nontarget organism bioassay results for Vip3Aa1 th at are found in the scientific literature. As discussed earlier in this petiti on (see Section V.A.1), these variants share 99.7% sequence identity and are functionally equivalent. Ther efore, Vip3Aa1 and Vip3Aa19
are suitable test substanc e surrogates for Vip3Aa20 in nontarget organism bioassays.

Table 30 . Vip3Aa test substances used in nontarget organism hazard studies.

Test Substance Source a Concentration of Vip3Aa Species Tested

Vip3Aa1 purified protein Recombinant E. coli 200 mg/g Colinus virginianus

Plant-incorporated Vip3Aa19 Pacha maize pollen 83.8 μ g/g Daphnia magna

Plant-incorporated Vip3Aa19 Pacha maize pollen 144.8 μ g/g Colleomegilla maculata, Chrysoperla carnea Plant-incorporated Vip3Aa19 Pacha maize leaf 3640 μ g/g Eisenia foetida

Plant-incorporated Vip3Aa19 Pacha maize leaf 86.7 μ g/g Folsomia candida

Plant-incorporate Vip3Aa19 Pacha maize grain 7.10 μ g/g Ictaluris punctatus

Vip3Aa19 purified protein Recombinant E. coli 631 mg/g Orius insidiosus Chrysoperla carnea
Vip3Aa19 purified protein Recombinant E. coli 897 mg/g Coccinella
septempunctata
Vip3Aa20 purified protein Recombinant E. coli 840 mg/g Apis mellifera
Aleochara bilineata

a – Pacha is transgenic maize producing th e Vip3Aa19 variant which is no longer under development by Syngenta.

Pacha maize was a predecessor Vip3Aa19 maize cultivar under development by Syngenta.

8 COT102 is a component event of VipCot TM cotton currently under review by the EPA. COT102 cotton has been deregulated by USDA.MIR162-USDA-1 Page 85 of 268 Its development was discontinued for agronomic performance reasons; it was replaced in commercial development by MIR162 maize. Many of the nontarget organism bioassays
described below utilized Pacha maize tissues as test substances, specifically pollen, leaf, and grain. Data from these bioassays are applicab le for predicting hazards and risk for MIR162 maize because Vip3Aa19 is biochemically an d functionally equivalent to the Vip3Aa20 protein in the corresponding tissues of MIR162 maize.

All nontarget organism studies, except the Colinus virginianus feeding study, employed an appropriate positive control substance to validate the bioassay methodology. Expected responses for test organisms exposed to the positive control substance were observed in all studies (data not shown). The presence and co ncentration of Vip3Aa protein was confirmed in all artificial meat-based diets by West ern blot analysis, ELISA, and bioassay.

VII.D.1. Effect of Vip3Aa on Wild Birds

Five male and five female juvenile Colinus virginianus (bobwhite quail) were exposed to a single dose of microbial Vip3Aa1 at 400 mg pr otein/kg bw, by oral ga vage. The effects on bird mortality and growth were compared wi th a control group for 14 days after exposure, and histological endpoints were assessed at the end of the test . No adverse effects were observed in the treatment or control groups , and the Vip3Aa1 NOEL was shown to be ≥ 400 mg protein/kg bw.

VII.D.2. Effect of Vip3Aa on Wild Mammals

Mus musculus strain Alpk:AD f CD-1 (white laboratory mouse) males and females (five per sex) were exposed to the highest feasible single dose of the microbial Vip3Aa20 test substance, corresponding to 1250 mg protein/kg bw, by oral gavage. The effects on mortality and growth were compared with a co ntrol group of male and female mice for 14 days after exposure, and many histological and biochemical e ndpoints were assessed at the
end of the test. No adverse effects were obs erved in the treatment or control groups, and
NOEL was shown to be ≥ 1250 mg protein/kg bw.

VII.D.3. Effect of Vip3Aa on Pollinators

Apis mellifera (honeybees) were exposed to microbial Vip3Aa20 following the protocol of
Oomen et al . (1992). Vip3Aa20 was incorporated into a diet of 50% sucrose solution at concentrations of 50, 200 and 500 μ g/g diet; these concentrations represented approximately 1, 4 and 10 times the concen tration of Vip3Aa20 in MIR 162 pollen, respectively. The negative control was 50% sucrose solution in buffer. Worker bees collected the sucrose solutions and fed them to brood for 24 days. There were no statistically significant differences in 24-day survival between brood in hives exposed to the Vip3Aa20-treated diet and brood in hives exposed to the negative control diet at any developmental stage. Therefore, the NOEC to bee brood exposed in-hive via a sugar solution diet was shown to be

≥ 500 μ g protein/g diet.MIR162-USDA-1 Page 86 of 268 VII.D.4. Effect of Vip3Aa on Above-ground Arthropods

Eight- to nine-day old adult Coleomegilla maculata (pink spotted ladybird beetle) were exposed to Vip3Aa19 via Pacha maize pollen for 21 days. The test substance was incorporated into an artificial diet at 5% weight by weight (w/w). The negative control diet comprised 5% w/w pollen from nontransgenic, ne ar-isogenic maize. Fr esh diet was supplied daily. The difference in survival of the beet les in the treatment and control groups was not statistically significant, a nd the NOEC was shown to be ≥ 7.24 μ g protein/g diet.

Three- to seven-day old adult Coccinella septempunctata (seven-spot ladybird beetle) were exposed to microbial Vip3Aa19 test substance. The test substance was incorporated into a
50% sucrose solution diet at 7250 μ g Vip3Aa19/g diet. The nega tive control diet comprised 50% sucrose solution only. The treatment and co ntrol groups were fed fresh diet daily. The
difference in survival between the treatmen t and control groups was not statistically significant, and the NOEC was shown to be ≥ 7250 μ g protein/g diet.

Second-instar Orius insidiosus (minute pirate bug) were expo sed to microbial Vip3Aa19 test substance. The test substance was incorporated into an artificial liv er-based diet at 7250 μ g Vip3Aa19/g diet. The negative co ntrol liver-based diet was tr eated with buffer only. The treatment and control groups were fed fresh di et daily for 21 days or until they became
adults. The difference in survival between the treatment and control groups was not statistically significant. The NOEC was shown to be ≥ 7250 μ g protein/g diet.

Adult Chrysoperla carnea (green lacewings) were exposed to Vip3Aa19 via Pacha maize pollen for 13 days. The pollen was incorporated into an artificial diet, comprising dry milk, sugar, brewer's yeast, and de-ionized water, at 15% w/w. The corresponding negative control
diet contained 15% w/w pollen from nontransgenic, near-iso genic maize. The treatment and control groups were provided with fresh diet daily. The difference in survival between the treatment and control groups was not statistically significant, and the NOEC was shown to be

≥ 21.7 μ g protein/g diet.

Two- to three-day old C. carnea larvae were exposed to microbial Vip3Aa19 test substance. The test substance was incorporated into an artificial meat-b ased diet at 7250 μ g Vip3Aa19/g diet. The negative control was diet treated with buffer only. The treatment and control groups were fed fresh diet daily for 30 days or until adult emergence. After 30 days, the difference between the treatment and negative c ontrol groups was not sta tistically significant; however, the control mortality exceeded the guid eline validity criterion of not greater than 25% (Vogt et al ., 2000); the control mortality remained w ithin the guideline validity criterion up to day 21. The OPPTS Guideline 885.4340 (EPA, 1996) for testing of microbial pesticides states that tests should be terminat ed when the control mortality exceeds 20%; the control mortality remained w ithin this guideline up to day 14. At day 14 and day 21, the differences between the treatment and nega tive control groups were not statistically significant. Fourteen and 21 da ys represent significant portions of larval development, and therefore, the data can be considered a rigor ous assessment of the toxicity of Vip3Aa19 to green lacewings. The NOEC was shown to be ≥ 7250 μ g protein/g dietMIR162-USDA-1 Page 87 of 268 VII.D.5. Effect of Vip3Aa on Soil-dwelling Invertebrates

Adult Eisenia foetida (earthworms) were exposed to Vi p3Aa19 protein extracted from Pacha
maize leaf tissue. The test substance was inco rporated into an artificial soil substrate at 3.60

μ g Vip3Aa19/g soil. The negative control was artificial soil treated with a leaf protein test substance prepared from nontransgenic, near-i sogenic maize. After 14 days, there was 100% survival in both the treatment and negative co ntrol groups, and there were no statistically significant differences in body weight between the groups. The NOEC was shown to be ≥
3.60 μ g protein/g soil.

Juvenile Folsomia candida (collembola) were exposed to Vip3Aa19 via lyophilized Pacha
maize leaf tissue. The test substance was mixe d with an equal weight of yeast to form the treatment diet containing 43.2 μ g Vip3Aa19/g diet. A negative control diet containing equal parts yeast and lyophilized leaves of nontransgenic, near-isogeni c maize was also prepared. The collembola were provided with fresh diet da ily. After 28 days, ther e were no statistically significant differences in surviv al, or in the number of new juveniles, between the Pacha maize leaf treated- and the nontransgenic mai ze leaf-treated groups. The NOEC was shown
to be ≥ 43.2 μ g protein/g diet.

Adult Aleochara bilineata (rove beetles) were exposed to microbial Vip3Aa20 test substance following the protocol of Grimm et al . (2000). The treatment group was fed a meat-based diet containing 500 μg Vip3Aa20/ g diet, which was intended to represent approximately 10 times the concentration of Vip3Aa20 in leaves of MIR162 maize. The negative control group was fed diet treated with buffer only. Fr esh diet was supplied da ily. After 35 days, beetles were allowed to parasitize pupae of Delia antique (onion fly). The number of adult offspring of the beetles exposed to the test diets was monitored until the average emergence fell below two beetles per day. The number of beetles that emerged from the Vip3Aa20- treated group was not statistica lly significantly different from that of the control group. The
NOEC was shown to be ≥ 500 μ g protein/g diet.

VII.D.6. Effect of Vip3Aa on Aquatic Organisms

Neonate Daphnia magna (water fleas) were exposed to Vip3Aa19 via Pacha maize pollen. The test substance was suspended in wa ter at 120 mg pollen/l, representing 10.1 μ g Vip3Aa19/l. The negative control was nontrans genic, near-isogenic maize pollen suspended in water at 120 mg/l. A water-only control of maize pollen was also included. After 48 hours, survival was 100% in each group, and ther e was no sign of imm obilization or other
sublethal effects. The NOEC was ≥ 10.1 μ g protein/l water.

Juvenile Ictalurus punctatus (channel catfish) were exposed to Vip3Aa19 via fish feed prepared from Pacha maize grain. The f eed was formulated to contain the maximum proportion of maize grain that pr ovides a nutritious diet and was made using a cold-pelleting technique, which minimizes heating that could denature the protein. Control fish feed was prepared in the same manner using grain fr om nontransgenic, near-isogenic maize. Treatment and control groups of catfish were fed the respective feeds th ree times daily for 30 days. There were no statistically significant differences in mean we t body weight increaseMIR162-USDA-1 Page 88 of 268 between catfish exposed to the Vip3Aa19 diet and fish exposed to the control diet. No abnormalities were noted among the fish during the study except for one thin individual in the control group that died on the last da y of the study. The NOEC was shown to be ≥ 7.1

μ g protein/g feed.

VII.E. Toxicity Exposure Ratios for Nontarget Organisms

The EEC and DDD values are compared with resu lts of Vip3Aa hazard studies in order to make judgments about risk. Th e hazard studies identified an y potential for Vip3Aa proteins to cause adverse effects in representative nonta rget organism species applicable for a maize ecosystem. In each study, a representative species was exposed to high concentrations or doses of Vip3Aa; typically this exposure was the highest attain able. No harmful effect of such exposure was observed in any species test ed, and the concentratio n of Vip3Aa in each study can be interpreted as the minimum value of the NOEC or NOEL. Computing a ratio of
the NOEC to the EEC or the NOEL to the DDD resu lts in the toxicity ex posure ratio (TER). This ratio is also known as the ‘margin of exposure’ or ‘margin of safety’. A TER ≥ 1.0 provides a degree of confidence that the absence of adverse effects in these hazard studies with indicator species is predictive of the sa fety of Vip3Aa20 in MI R162 maize for nontarget organisms in general. The larger the TER the higher the degree of confidence in the safety judgment or the lower the risk. Table 31 c ontains the NOEC or NOEL, and corresponding TER values for each indicator test species.

No adverse effects were observed in any study that exposed representative nontarget organisms to Vip3Aa proteins. The concentr ation of Vip3Aa tested in the studies was sufficient to achieve margins of exposure of ≥ 1 for all but one spec ies based on realistic
EEC values. In the case of the C. maculata where the TER was ≥ 0.7, this lower TER value for the pink-spotted ladybird beetle is not an i ndication of elevated risk but rather a reflection of the fact that the test substance was polle n and there were limitations on the fraction of pollen that could comprise the diet utilized. Additionally, the study conducted with a second ladybird beetle species ( C. septempunctata ) provided a TER of ≥ 719. Collectively, these TER values indicate that plant-incorporat ed Vip3Aa20 will not be harmful to nontarget organisms likely to be found in a maize ecosystem.MIR162-USDA-1 Page 89 of 268 Table 31. Nontarget organism TER values for Vip3Aa proteins. TER values are computed as the ratio of NOEC : EEC (or NOEL : DDD) and are based on estimates of exposure to Vip3Aa20 containe d in MIR162 maize. All NOEC and NOEL values are shown as greater than or equal to ( ≥ ) the specified value because no adverse effect was observed in any species tested at the maximum exposure tested.

Test Species NOEC or NOEL EEC or DDD TER

C. virginianus ≥ 400 μ g/g bw 5.22 μ g/g bw ≥ 77

M. musculus ≥ 1250 μ g/g bw 7.18 μ g/g bw ≥ 174

A. mellifera ≥ 500 μ g/g diet 14.91 μ g/g pollen ≥ 34

C. maculata ≥ 7.24 μ g/g diet 10.08 μ g/g diet ≥ 0.7

C. septempunctata ≥ 7250 μ g/g diet 10.08 μ g/g diet ≥ 719

O. insidiosus ≥ 7250 μ g/g diet 10.08 μ g/g diet ≥ 719

C. carnea a ≥ 21.7 μ g/g diet 10.08 μ g/g diet ≥ 2

C. carnea b ≥ 7250 μ g/g diet 10.08 μ g/g diet ≥ 719

E. foetida ≥ 3.6 μ g/g soil 0.38 μ g/g soil ≥ 9

F. candida ≥ 43.2 μ g/g diet 0.38 μ g/g soil ≥ 114

A. bilineata ≥ 500 μ g/g soil 0.38 μ g/g soil ≥ 1316

D. magna ≥ 10.1 μ g/l water 0.0068 μ g/l water ≥ 1507

I. punctatus ≥ 7.1 μ g/g diet 1.49 μ g/g diet ≥ 4

a - adults
b - larvae

VII.F. Safety Assessment for Endangered and Threatened Species

There is a weight of evidence that at con centrations in MIR162 ma ize the toxicity of Vip3Aa20 will be limited to Lepidoptera. Its receptor-mediated mechanism of action and absence of activity in bioassays with multip le species outside of the order Lepidoptera support this conclusion. Furthermore, no harm ful effects of Vip3Aa proteins have been observed in nontarget organism hazard identifica tion studies. These studies used a wide range of taxa and at expected environmental c oncentrations they indicate a lack of risk associated with exposure to Vip3Aa 20 in MIR162 maize (see Table 31).

The only endangered or threatened lepidopter an species with potential for exposure to insecticidal proteins in maize is the Karner blue butterfly ( Lycaeides melissa samuelis ) (EPA, 2001; USFWS, 2007). The potential route of e xposure for this species is consumption of maize pollen that has settled on the leaves of its larval food plant, the wild lupine ( LupinusMIR162-USDA-1 Page 90 of 268 perennis ). Peterson et al . (2006) determined that exposure of Karner blue larvae to maize pollen was minimal because most lupine populati ons are separated from maize fields by at least 500 metres, and because maize anthesis us ually occurs after Karner blue larvae have finished feeding.

The restriction of toxicity of Vip3Aa20 to Lepidoptera, and the minimal exposure of endangered Lepidoptera to maize, indicates that Vip3Aa20 in MIR162 maize is expected to have no harmful effects on any endangered or threatened species in the U.S.

Although not an endangered or threatened species, Danaus plexippus (monarch butterfly) is a species of high conservation inte rest, and there has been concer n that it may be harmed by consuming pollen from transgenic insect-protected maize. The monarch is susceptible to Cry1Ab (Hellmich et al ., 2001), the commonest inse cticidal protein in transgenic maize. However, the distribution of the monarch’s food plant ( Asclepias syriaca - common milkweed), its pattern of migration, and the timing of maize anthesis means that very few monarchs are exposed to harmful concentrations of Cry1Ab (Sears et al ., 2001).

The exposure assessments used to assess the risks of maize containing Cry1Ab to monarchs
are also valid for MIR162 maize. In addition, it has been shown that monarchs are not susceptible to Vip3Aa1. Lee et al . (2003) showed that trypsi nized Vip3Aa1 did not form pores in the midgut of monarchs; pore formati on appears to be essential for toxicity and occurs in the guts of insect s susceptible to Vip3Aa1. These investigators also found no mortality of D. plexippus in a surface diet bioassay limit test at 1000 ng/cm 2 . MIR162 maize, therefore, poses low risk to monarchs b ecause of minimal hazard of Vip3Aa20 and low exposure to Vip3Aa20-containing pollen.

VII.G. Conclusion on Environmental Risk

No adverse effects were associated with exposures to Vip3Aa1, Vip3Aa19, or Vip3Aa20 proteins in a range of indicator species appr opriate for a maize ecosystem, and the NOEC or NOEL was the highest concentration (or dose) te sted in each study. The exposures in all but one study were in excess of EEC or DDD le vels for the nontarget organism groups represented, indicating a low probability of ha rm to these groups from Vip3Aa20 in MIR162
maize.

The very narrow spectrum of insecticidal acti vity observed for Vip3A a proteins indicates with high certainty that no endangered or threat ened species other than Lepidoptera would be harmed by contact with Vip3Aa20 via MIR162 maize. There is minimal exposure of endangered Lepidoptera to maize and therefore, cultivation of MIR162 maize is unlikely to harm any endangered or threaten ed species in the U.S.

VII.H. Gene Flow Assessment

An assessment of the environmental fate of Vip3Aa20 resulting from the cultivation of MIR162 maize requires consider ation not only of the produc tion and degradation of the protein within maize fields, but also the possibility that Vip3 Aa20 could persist, or spreadMIR162-USDA-1 Page 91 of 268 from, areas of maize cultivat ion because of gene flow or the establishment of weedy populations of MIR162 maize. It is hi ghly unlikely that Vip3Aa20 will appear in
environments outside of cultivated maize. Ma ize pollen does not drift great distances nor are maize seeds wind-borne. The probability of spread of Vip3Aa20 outside maize cultivation through volunteers and self-sustaini ng feral populations of MIR162 maize is also very low.

As described in Section VI above, MIR162 is not phenotypically or compositionally different from conventional maize other than for its ability to resist insect feeding damage. Thus, MIR162 maize has not acquired any properties i ndicative of increased weediness potential.
The likelihood that the vip3Aa20 gene will move to other species as a result of either vertical or horizontal gene transfer is also extremely low.

VII.H.1. Gene Flow to Wild Species

Z. mays L. subsp. mays hybridizes with a group of taxa co llectively called te osinte. Several types of teosinte are cla ssified as subspecies of Zea mays , whereas others are regarded as separate species of Zea . Teosinte species are natives of Central Americ a and have co-existed with cultivated maize for several thousand years. They have remained genetically distinct from cultivated varieties despite occasional introgression (Baltazar et al ., 2005). Teosinte species are not natives of the U.S., but isolated populations have been recorded in Florida and Texas, the former a possible remnant of the use of annual teosinte as a forage grass. These populations are apparently now extinct in bot h states. Teosinte species are grown in botanical gardens, but as maize pollen is heavy and relativ ely short-lived ( e.g. , EPA, 2001; Byrne and Fromherz, 2003; Devos et al ., 2005), fertilization of these plants with pollen from MIR162 maize is extremely unlikely.

Species of the genus Tripsacum are considered close relatives of Zea species and some theories postulate that a Tripsacum species may be a progenitor of domesticated corn via hybridization and introgression w ith teosinte ( e.g ., Poggio et al ., 2005). There are sixteen species of Tripsacum worldwide, of which three occur in the U.S.: T. dactyloides , a widespread forage grass; T. floridanum

, known from southern Florida; and T. lanceolatum

, which is present in Arizona and possibly New Mexico (EPA, 2001).

Maize breeders view Tripsacum as a potential source of us eful genes for traits including apomixis, pest and disease resistance, and drought tolerance (OEC D, 2003). Therefore,
substantial effort has been made to obtain and characterize maize-by- Tripsacum hybrids. Hybrids between maize and Tripsacum species are difficult to obtain outside the laboratory or greenhouse, and are often ster ile. Only one record exists of an open-pollinated hybrid
between Zea and Tripsacum , which involved species native to Guatemala. After consultation with experts on improvement of forage grasses, the EPA (2001) concluded that the chance of natural introgression of genes from maize to Tripsacum was ‘extremely remote’ and that no other species in the continental U.S. woul d interbreed with commercial maize.

These observations indicate a very low probability for transfer of the vip3Aa20 gene from MIR162 maize to wild relatives in the U.S. Species of Zea other than maize are not recorded outside botanical gardens in the U.S. Tripsacum dactyloides is widespread, but does notMIR162-USDA-1 Page 92 of 268 hybridize readily with Z. mays , and the probability of backcross or F 2 progeny of Tripsacum

by- Zea hybrids being produced in the field is negligible. Theref ore, Vip3Aa20 is unlikely to spread from maize cultivation and persist in th e environment as the re sult of vertical gene
flow.

VII.H.2. Potential for Horizontal Gene Transfer

An extensive review of information relevant to the potential risks of horizontal gene transfer
for Bt crops to soil microbes was conducted as part of the 2001 reregistration of Bt -based plant-incorporated protectants (EPA, 2001). Studies reviewed by the EPA showed no evidence for horizontal gene transfer under fiel d conditions, and only equivocal evidence for horizontal gene transfer under la boratory conditions designed to maximize the recovery of
transformants. Conner et al . (2003) also reviewed the literature and found very few examples where horizontal gene transfer had been demonstrated convincingly, and these cases relied on artificially high sequence homol ogy between the transgen e and the potential recipient organism ( e.g ., de Vries et al ., 2001). The codons in the vip3Aa20 gene are optimized for expression in plants, and henc e the gene is likely to have low sequence homology with genes of soil microbes. Th erefore, horizontal gene transfer of vip3Aa20 from MIR162 maize to soil microbes is highly unlikely. If transfer did take place, expression of vip3Aa20 is improbable. In addition to the fact that codon use vip3Aa20 is optimized for expression in plants, rather than microbes, the maize ubiquitin promoter is unlikely to function in microbes. The probability for spread of vip3Aa20 outside maize cultivation by horizontal gene transfer is negligible.

VII.I. Impact of MIR162 Introduction on Current Maize Agronomic Practices

Field maize is the leading pr oduction crop globally, with the 2005/2006 growing season yielding 695 million metric tons of grain (US DA, 2006a). The U.S. accounts for nearly 41% of global maize production. Mai ze is the largest crop grown in the U.S. in terms of both volume and value. Approximately 78.3 million acres were planted in 2006, yielding 10.5 billion bushels (267 million metric tons) with a gross crop value of $33.7 billion (USDA, 2007a). The total plantings in 2007 are estimat ed at 92.9 million acres, a 19% increase over 2006 plantings (USDA, 2007c). This increase has been driven by the demand for fuel ethanol. Field corn is planted in almost every st ate of the U.S. with the majority concentrated in ten states of the upper Mi dwest: Illinois, Iowa, Indiana, Kansas, Minnesota, Missouri, Nebraska, Ohio, South Dakota, and Wisconsin . The average annual yield from 2006 was 149.1 bu/ac (USDA, 2006b).

Yield losses due to weeds, diseases, and ins ects were substantial until the introduction of crop protection chemicals in the 1960s. Weeds co mpete with crops for light, nutrients, water, and other growth factors. If weeds are le ft uncontrolled, maize si mply cannot be grown successfully. Estimates of maize yield loss caused by pathogens have ranged from 2 to 17% (Smith and White, 1988). In a ddition, a maize crop is susceptible to att ack by a variety of insects from the time it is planted until it is consumed as food or feed. As a result, maize crops are intensively managed.MIR162-USDA-1 Page 93 of 268 For the 2005 crop year in th e 19 program states tracked by the National Agricultural Statistics Service of USDA, nitrogen was app lied to 96% of maize ac res at a rate of 138 lb/ac, phosphate was applied to 81% of maize acres at a rate of 58 lb/ac, potash was applied to 84% of acres at a rate of 65 lb/ac, and sulf ur was applied to 13% of acres at a rate of 12 lb/ac (USDA, 2006c). The introduction of MIR162 maize is not expected to alter fertilizer application practices.

Weeds are controlled to varying degrees by crop rotation, tillage, and herbicide applications. Herbicides were applied to 97% of the progr am state maize acres in 2005 (USDA, 2006c). Atrazine was applied to 66% of acres, glyphos ate was applied to 31% of planted acres, S

metolachlor and acetochlor were each applie d to 23% of planted maize acres. The introduction of MIR162 maize is not expected to alter maize weed control practices.

Insect control options available to growers include conventional inse cticide applications, microbial insecticide applications, crop rotation, and planting of insect resistant cultivars. The major and moderate insect pests of mai ze are described earlier in this petition (see Section I.B and Table 1). The most widespread and damaging insects of maize in the U.S.
Corn Belt have been O. nubilalis and Diabrotica species. Before the introduction of Bt maize hybrids ten years ago, growers had few pr actical options for controlling stalk boring insects; only about 10% of growers applied ins ecticides for control of corn borers. As a result, most growers incurred significant annual yield losses because most fields were not treated and chemical applications were not always effective. Timing of insecticide applications had to be nearly perfect because there was only a very short period of time (two to six days) that these insect s would be physically positioned on the plant where they could be exposed to an insecticide applic ation. The introduction of the first Bt maize hybrids in 1996 provided growers with an effectiv e means of limiting damage caused by O. nubilalis

. By plant-incorporating the insec ticide, exposure of th e insect to the toxin was guaranteed. In 2006, 42% of maize acres were planted with Bt corn borer-protected hybrids (Doane Marketing Research, 2006a). These hybrids express either a cry1Ab or cry1F gene from B. thuringiensis , both of which encode proteins that are highly toxic to O. nubilalis

.

More options have been available to grow ers for mitigating damage caused by corn rootworms. In many maize growing regions crop rotation has been effective in limiting
Diabrotica populations because it breaks the life cycle of the insect. There have also been many effective conventional insecticide products available to growers for control of these pests. Prior to the introduction of rootworm-protected Bt varieties in 2 003, an estimated 14 million acres were treated annually with conventiona l insecticides to control corn rootworms. This equated to more than 7.7 million pounds of insecticide active ingredient being applied annually in maize fields for the control of Diabrotica species (Ward et al. , 2005). Control of
Diabrotica rootworms accounted for the largest single use of insecticides in the U.S. In 2005, 23% of maize acres in the pr ogram states were treated with conventional insecticides. Tefluthrin, cyfluthrin, and te bupirimphos were the most wide ly applied active ingredients (USDA, 2006c).

Controlling above-ground insects presents a challenge for maize growers. The majority of
maize fields are not treated for control of leaf -, stalk-, and ear-feedi ng insects. A growerMIR162-USDA-1 Page 94 of 268 decision not to treat is generally not reversible because of the feeding location of the pests (in the soil, under the leaf, inside the stalk, or in the ear); the pests are shielded from aerial chemical applications. Timing of a pplications is critical for efficacy.

Data obtained from the 2005 and 2006 Doane Market ing Research AgroTrak studies indicate that growers are currently treating approximately three million acres a year with conventional
insecticides for control of H. zea

, A. ipsilon

, S. albicosta , and S. frugiperda (see Table 32). Compared to the total number of maize acres planted annually in the U.S., this represents a relatively small use of conventional pestic ides; however, three million acres treated represents a significant use compared to chemical usage in other crops.

Table 32 . Conventional insecticide usage for c ontrol of selected lepidopteran pests.
Cost and maize acres treated with conventi onal insecticides during 2005 and 2006 for the
control of S. frugiperda

, A. ipsilon

, S. albicosta , and H. zea (Doane Marketing Research, 2005 and 2006b).

Acres Treated Grower Cost ($)

Pest 2005 2006 2005 2006

S. frugiperda 0 20,441 0 86,550

A. ipsilon 2,721,543 3,064,137 19,457,090 22,328,818

S. albicosta 44,410 72,373 363,801 343,552

H. zea 99,620 161,002 825,654 848,502

Totals 2,865,573 3,317,953 $20,646,545 $23,607,422

In addition to direct damage caused by feeding on plant tissue, insects play an important role in the transmission and dissemination of pat hogenic organisms during maize development. Soil abounds in microorganisms, particularly f ungi, which may infect plant parts injured by soil-dwelling insects. In much of the Corn Belt, pathogenic fungi probably pose more problems in corn production than any other group of organisms. Primary roots of the seedling and the radical and seminal roots are commonly infected with Fusarium spp. after they have served their function an d become senescent. Feeding by Diabrotica rootworms has been associated with increased frequencies of Fusarium infection (Dicke and Guthrie, 1988); rootworm feeding may also lead to increase d incidences of stalk ro ts. Ear, kernel, and cob rots occur wherever maize is grown and re sult in reduced test we ight, poor grain quality, and mycotoxin contamination of food and feed. Fusarium kernel or ear rot is the most widespread disease of maize ears and is frequen tly associated with insect feeding damage. Mycotoxin contamination of maize grain presents a potential threat to livestock health and it is occasionally necessary to reject or reformulat e field lots because of contamination. These pathogenic infections can lead to reduced crop quality, harvestability, and yield.

MIR162 maize provides excellent protect ion against feeding damage caused by A. ipsilon

, H.

zea , S. albicosta , and S. frugiperda . For this reason, its introduction will impact in a veryMIR162-USDA-1 Page 95 of 268 positive way current maize insect control practices. This product has the potential to displace
all of the conventional insecticide applications listed inTable 32. Control of these aboveground insect pests is currently challenging for growers. Conventional insecticide applications are costly and intensive scouting of fields is required to identify the appropriate timing for applications. Growers only have a very narrow time window during which insecticides can be applied b ecause many of the above-ground feeding insects are shielded
from contact with the insect icides by virtue of their f eeding location on the plant.

Two years of efficacy field trials, conducted at multiple locations under varying levels of insect pressure, have demonstr ated the superior leaf, stalk, and ear protection provided by MIR162 maize compared to hybrids treated with a conventional insect icide product (see Figure 21). Vip3Aa20 possesses a number of unique properties that conventional insecticides do not. The protein is efficacious via a mode of action that is selective to lepidopteran insects. The protein is expressed throughout all tissues of the maize plant. This ensures protection where it is needed and eliminates the risk of insecticide failures associated with timing of applications or unfavorable environmental conditions. Furthermore, the delivery of Vip3Aa20 in the maize seed and its production in plants eliminates many risks associated with conventional insecticide usag e, some of which incl ude improper calibration and maintenance of application equipment, handling of hazardous ch emical insecticides, container disposal, chemical mispl acement, runoff, and spray drift.

Integrated pest management (IPM) in agricult ure includes insect scouting or monitoring to determine pest populations, consideration an d application of comp atible alternative biological, cultural, mechanical and chemical controls, and the establishment of action thresholds for agricultural inputs. The delivery of pest management interventions on target and on time is a key to successful IPM. Pl anting of MIR162 hybrids provides much greater accuracy of application compared to chemical treatments due to the localization of Vip3Aa20 within the plant tissues. Timing of applica tion is not a factor with MIR162 hybrids since Vip3Aa20 is present in the plant throughout the growing season. Planting of MIR162 hybrids is compatible with current insect scout ing and monitoring programs that provide data upon which to base crop manageme nt decisions. The product is also fully compatible with cultural control measures such as crop rotation. MIR162 fits seamlessly into the concept of integrated pest management for maize.

From data collected in a telephone survey of 150 maize growers in 12 states, average yield
losses in 2006 attributable to H. zea were estimated to be 4.9 bu/ ac and losses attr ibutable to
S. albicosta were estimated to be 4.8 bu/ac (see A ppendix F). Examination of data provided by these growers for the past five seasons sugge sts that yield losses attributable to the two pests are increasing. This conclusion is suppor ted by analysis of insecticide use data for
2005 and 2006 which indicate that economica lly significant infestations of H. zea and S.
albicosta are on the rise in the Corn Belt and Great Plains. Additionally, there is evidence that populations of S. albicosta are spreading eastward and will have the potential to cause greater harm in critical maize-producing states . While economic losses attributable to the insects that are effectively cont rolled by MIR162 maize are not as large as those attributable

to O. nubilalis and Diabrotica rootworms, they are significan t when severe infestations occur. As the price of maize grain continues to rise, the economic th reshold for growers toMIR162-USDA-1 Page 96 of 268 respond to infestations of A. ipsilon

, H. zea

, S. albicosta , or S. frugiperda will fall. Even relatively small reductions in crop yield (< 5% ) will result in a significant economic loss for

growers.

In a combined-trait hybrid offering with Bt11 maize (Bt11xMIR162 maize), 9 the crop will be protected from the damage and yield losses attributable to all of the economically significant lepidopteran insect pests. This superior protec tion against insect feeding will also result in a corresponding decrease in mycotoxin levels in grain.

VIII. Insect Resistance Management

Current insect resistance mana gement (IRM) strategies for Bt maize products are centered around the planting of a structured refuge that can provide a source of susceptible adult
insects for rare resistant insect s to mate with. Ma tings of resistant an d nonresistant adults
serve to dilute the fre quency and establishment of resistance genes in a population. The size and configuration of the struct ured refuge is determined by toxin dose and insect biology. The existing EPA policy for resistance manageme nt of maize lepidopter an pests is focused primarily O. nubilalis

, D. grandiosella , and H. zea . Growers planting Cry1Ab- and Cry1Fcontaining maize hybrids in the Co rn Belt are required to plant a 20% structured refuge of nonlepidopteran-protected maize. The refuge can be configured as strips within or surrounding a field or as a block within, ad jacent to, or up to 1⁄2 mile away from the Bt maize field. For maize planted in cotton-growing areas of the south, the refuge must be at least 50% of a grower’s total planted areas becau se Cry1Ab- and Cry1F-containing hybrids do not deliver a high dose against H. zea and are present in both corn and cotton varieties. H. zea is a pest of both maize and cott on and has the potential to unde rgo selection pressure from feeding on both corn and cotton varieties that express simila r Cry proteins. The refuge configuration options are the same as in the Corn Belt.

As MIR162 maize provides no protecti on against feeding damage caused by O. nubilalis it will not likely be offered to growers as a stand- alone trait. Instead, it will be commercialized as a combined-trait hybrid with Syngenta’s Bt11 maize event. Together the two traits have been shown to deliver a high dose against O. nubilalis and H. zea , as well as against S. frugiperda . Syngenta has submitted an IRM plan for Bt11xMIR162 maize that requires growers to plant a 20% structured refuge that can be planted as strips within or surrounding
the Bt maize field or as a block within, adjacent to, or up to 1⁄2 a mile away. The proposed refuge requirements are the same in th e Corn Belt and cotton growing areas.

A unique benefit that will be offered by Bt 11xMIR162 hybrids is that the Cry1Ab and Vip3Aa20 proteins are present at levels that have been dem onstrated to provide high-dose
control of O. nubilalis, H. zea and S. frugiperda , thus minimizing the risk of resistance developing in these species . Bt11xMIR162 hybrids offer IR M advantages over other lepidopteran-control options that do not demons trably provide a high dos e against the target pests. Moreover, Vip3Aa20 operates by a mode of action different from that of Cry1Ab or Cry1F and targets a unique binding site(s) in susceptible larvae. The available data support a

9 An application for registration of the plant-incorporated protectants in Bt11xMIR162 maize is currently under review by the U.S. EPA.MIR162-USDA-1 Page 97 of 268 conclusion that Vip3Aa20 shows no potential fo r cross-resistance with Cry proteins (Lee et al. , 2003). Thus, for H. zea and S. frugiperda , which are sensitive to both Cry1Ab and Vip3Aa20, Bt11xMIR162 maize is predicted to significantly extend the durability of both traits for control of these pests because lo cal populations are very unlikely to evolve resistance to two proteins that act on independent receptor sites.

For growers of Bt11xMIR162 maize hybrids, th e reduced refuge requirement in cottongrowing regions will translate in to a higher proportion of insect-protected maize acres, with a proportional increase in all the atte ndant benefits of the product in these areas. As an added advantage, compliance with the refuge require ment for IRM can be predicted to increase
because Bt maize growers in cotton-grow ing regions have heretofore not been able to fully experience the benefits enjoyed by Bt maize growers in other regions of the U.S. The potential for increased maize acres in cotton-gr owing regions can also help meet the current high demand for maize grain.

IX. Adverse Consequences of Introduction

Syngenta knows of no data or observations th at indicate MIR162 ma ize would adversely impact the quality of the human environment, directly, indirectly, or cumulatively. This includes a lack of anticipated effects on endangered species, uni que geographic areas, critical habitats, public health and safety (including children and minorities), genetic diversity of maize, farmer or consumer choice, insect resi stance or the economy, either within or outside the U.S. MIR162 maize offers growers an ad ditional choice for protection of maize crops from feeding damage caused by lepidopteran pest s. As such, MIR162 is expected to convey benefits similar to those associated with previously deregulated Bt maize products that are commercially available. Additional benefi ts will include increased product choice for growers, price competition, a nd extended useful life of Bt maize technology generally.

This section of the petition addresses potential beneficial and adverse consequences associated with deregulation of MIR162 maize and demonstrates that the introduction of MIR162 maize will only affect the quality of the human environment in a positive way. The direct, indirect, and cumulative impact of deregulating of MIR 162 maize is analyzed within the framework of the factors listed by the Council on Environmental Quality (CEQ). 10

IX.A. Effects on the Qualit y of the Human Environment

The National Environmental Policy Act (NEPA), 42 U.S.C. §4321 et seq ., requires that agencies undertaking a major federal action sign ificantly affecting the quality of the human environment provide a detailed statement of the environmental impact of the proposed action, any adverse environmental effects that cannot be avoided, and alternatives to the action (42 U.S.C. §4332 (C)). Where the significa nce of an action is un certain, agencies may use an environmental assessment (EA) to identif y, analyze, and evaluate the impacts of the

10 The Council on Environmental Quality coordinates fe deral environmental efforts and works closely with agencies and other White House offices in the developm ent of environmental policies and initiatives. Congress established the Council on Environmental Quality within the Executive Office of the President as part of the National Environmental Policy Act of 1969.MIR162-USDA-1 Page 98 of 268 proposed action. The EA will satisfy a NE PA obligation where it provides sufficient evidence and analysis to support a finding of no significant impact (FONSI) for the proposed
federal action.

The test of whether an action ‘significantly a ffects’ the environment requires considerations of both context and intensity (40 CFR §1508.27). The term ‘context’ refers to the setting within which the proposed action takes place. 11 The term ‘intensity’ refers to the severity of the impact and includes factors such as e ffects on human health, cumulative impacts, and effects on endangered species.

Data and information submitted by Syngenta in support of this petition for deregulation of
MIR162 maize is sufficient under NEPA for APHIS to prepare an EA in support of a FONSI. The context within which the deregulation of MIR162 maize will oc cur is one in which multiple lepidopteran-protected Bt maize cultivars are currently in use, and have been for many years. These cultivars provide a valuable tool for growers to protect their crops from feeding damage caused by lepidopteran insects that infest maize and that have developed
resistance to a number of conve ntional insecticides. These Bt maize cultivars do not present plant pest risks.

Currently available lepidopteran-protected Bt maize cultivars produc e insecticidal Cry proteins derived from B. thuringiensis . MIR162 maize produces a vegetative insecticidal protein that is al so derived from B. thuringiensis . Vip and Cry proteins differ in terms of their solubility characteristics, yet they act by the same fundamenta l mechanism of action ( i.e. , they are activated to insecticidal toxins in the inse ct midgut and form pores in the epithelial membranes). The MIR162 technology will provide benefits in the marketplace in the form of crop protection against lepidopter an pests that are poor ly or only partially controlled by existing Bt maize cultivars. Its introduction will bring market diversification with no corresponding plant pest or environmental risks.

These same facts demonstrate the low intensity of this action. The cumulative impacts of an additional lepidopteran-protected maize product en tering the market will only be beneficial.
These benefits will come in the form of reduced insecticide usage, increased crop yields, and marketplace competition. Maize is a well-char acterized and intensively managed crop. Its genetic diversity is carefully maintained and safeguarded by seed producers. The modification of maize to produce MIR162 maize w ill not present a risk of adverse effects for endangered, threatened, or other nontarget specie s. A comprehensive and scientifically based IRM plan will be executed to delay the deve lopment of insect resistance to the MIR162 technology. The following sections delineate the evidence supporting these conclusions.

IX.B. Context of the Proposed Action

The context or setting of the proposed dere gulation is based in the production of an intensively managed row crop, maize. Maize is th e largest crop grown in the U.S. in terms of both volume and value. Approximately 78.3 million acres were planted in 2006, yielding 10.5 billion bushels (267 million metric tons) with a gross crop value of $33.7 billion

11 See Coliseum Square Association v. Jackson, 465 F.3d 215, 239-41 (5th Cir. 2006).MIR162-USDA-1 Page 99 of 268 (USDA, 2007a). Maize is grown for animal fe ed, human food, vegetable oil, high fructose corn syrups, starch, fermentation into ethanol, and a multitude of industrial uses. Maize cultivars improved through mode rn biotechnology have contri buted significan tly to this value. Improved insect control has led to increased crop yields and reductions in conventional pesticide use (EPA, 2001; Marra et al. , 2002).

Lepidopteran insects are significan t insect pests of field maize and more so, of sweet maize in the U.S. In addition to direct damage cau sed by feeding on plant tissu e, these insects play an important role in the transmission and dissemination of pathogenic organisms during maize development. Syngenta plans to market the MIR162 trait in a combined-trait hybrid
with the insecticidal trait in Bt11 maize. The broad efficacy of the Bt11xMIR162 product will allow maize growers across wide geographies to benefit from routinely planting Bt11xMIR162 hybrids to control all of the majo r lepidopteran maize pests that could be prevalent in any given year.

The improved pest-protection profile of Bt 11xMIR162 maize can be expected to translate into correspondingly higher overall economic bene fits to growers, consumers, and other
downstream users of mai ze products. Some lepidopteran pests, such as A. ipsilon , can cause major yield losses due to cutting of an entire sta nd of maize in a field. If the crop is left untreated, the grower is sometimes forced to repl ant an entire field. Su ch catastrophic losses will be prevented by planting Bt11xMIR162 maize. Other, stalk-boring lepidopteran larvae cause physiologic yield loss because the stalk da mage interferes with nutrient uptake and increases susceptibility to plant diseases. Th ey can also cause severe stalk lodging, which results in a physical yield loss because the ears cannot be mechanically harvested by the combine. Ear-feeding pests reduce grain yiel d and quality, and occasionally result in elevated grain fumonisin levels that render the grain unsafe and unusable as food or feed.

Growers have significant experi ence with the management of Bt maize cultivars. Deregulated Bt maize cultivars that re sist lepidopteran feeding have been commercially available for more than a decade, and more recent Bt maize cultivars offer corn rootworm resistance, as well. USDA statistics show that 40% of the U.S. mai ze crop in 2006 consisted
of Bt varieties (including single and stacked biotech trait pro ducts) and 49% in 2007 (USDA, 2007c). The presence of Bt maize has not adversely a ffected genetic diversity.

The importance of maize as a food crop, and its dependence on human management, has produced a long history of great care to protect germplasm lines of maize. Decades prior to the introduction of transgenic maize cultivars, the maize seed industry developed effective methods for maintaining product segmentation and genetic purity standards. Specialty maizes, for example, were successfully isolated for years and continue to be grown today, even with transgenic maize being widely adopted in the U.S. Moreover, with respect to both conventional and transgenic maize, the ability to prot ect and maintain the genetic purity of breeding lines is critical to seed companies an d developers of new varieties such as those containing event MIR162. Consequently, seed companies routinely apply rigorous breeding techniques, including physical and temporal isolation, that have proven effective in maintaining the genetic pur ity of breeding lines.MIR162-USDA-1 Page 100 of 268 The EPA registrations for MIR162 and all other Bt maize cultivars require refugia and related measures that have been used successfully fo r several years, as evidenced by the fact that
there have been no documented instances of confirmed insect resistance to Bt maize having developed in the field, and Syngenta is no t aware of any studies demonstrating the development of insect resistance in the fiel d. Monitoring target pest populations for resistance development is an ongoing stewar dship requirement imposed by EPA for all Bt
maize cultivars.

IX.C. Intensity of the Proposed Action

With regard to the intensity element of the ‘significance’ determination, CEQ regulations provide ten factors to guid e the analysis (40 CFR §1508.27(b )). These factors “...do not constitute categorical rules such that thei r presence or absence means an impact is per se significant.” The ten CEQ intensisty factors are as follows:

i. Impacts that may be both beneficial and adve rse. A significant effect may exist even if the federal agency believes that, on balance, the effect will be beneficial.
ii. The degree to which the proposed action affects public health or safety.
iii. Unique characteristics of the geographic area su ch as proximity to historic or cultural resources, park lands, prime farmlands, wetlands, wild and scenic rivers, or ecologically critical areas.
iv. The degree to which the effects on the quality of the human environment are likely to be highly controversial.

v. The degree to which the possible eff ects on the human environment are highly uncertain or involve uniq ue or unknown risks.
vi. The degree to which the action may establish a precedent for future actions with significant effects or represents a decision in principle about a fu ture consideration.
vii. Whether the action is related to other actions with individually insignificant but cumulatively significant impacts. Significance exists if it is reasonable to anticipate a cumulatively significant impact on the envi ronment. Significance cannot be avoided by terming an action temporary or by break ing it down into small component parts.
viii. The degree to which the action may advers ely affect districts, sites, highways, structures, or objects listed in or eligib le for listing in the National Register of Historic Places or may cause loss or destruc tion of significant scientific, cultural, or
historical resources.
ix. The degree to which the action may advers ely affect an endangered or threatened species or its habitat that has been determined to be critical under the Endangered Species Act of 1973.

x. Whether the action threatens a violation of fe deral, state, or local law or requirements imposed for the protectio n of the environment.

IX.C.1. Impacts That May Be Both Beneficial and Adverse

The NEPA regulations (40 CFR §1508.27(b)(1)) em phasize that agencies must take into account beneficial effects, as well as adverse ones. As se t out below, MIR162 maize is expected to deliver the same benefici al effects as previously deregulated Bt maize cultivarsMIR162-USDA-1 Page 101 of 268 that are also registered by the EPA as pl ant-incorporated protectants and which are commercially available. These benefits incl ude additional grower choice of technologies, increased price competition an d extended useful life of Bt maize technology generally. These effects do not constitute significant im pacts on the human environment because they merely provide incremental benefits over the status quo and do not constitute an adverse impact on the human environment.

USDA has already deregulated multiple lepidopteran-protected maize cultivars, each containing proteins derived from B. thuringiensis , which limit the destructive feeding damage caused by insects such as O. nubilalis

, D. grandiosella

, A. ipsilon

, H. zea, S. albicosta, and S. frugiperda , the same insects targeted by th e Vip3Aa20 protein contained in
MIR162 maize. 12 These Bt maize cultivars have reduced the use of conventional pesticides
since their market introduction and this has been viewed as a very favorable benefit to the
environment. The commercial introduction of MIR162 maize is expected to reduce conventional insecticide use further. The supe rior protection of ears from insect feeding damage offered by the MIR162 trait will pres erve grain yield and quality, and will limit fumonisin levels that render the grai n unsafe and unusable as food or feed.

IX.C.2. Effects on Public Heal th or Environmental Safety

Previously deregulated Bt maize cultivars have resulted in reduced conventional pesticide use, as farmers find the Bt products more effective in mitigati ng insect feeding damage. It is reasonable to expect that deregulation and co mmercialization of MIR 162 maize will result in
further reductions in the use of conventional pesticides. This reduction in conventional pesticide use would diminish the environmental risks of insect control, as the chemical alternatives to MIR162 presen t well-characterized risks to humans and other wildlife, whereas Vip3Aa20 presents no such risk. Substantial data support a conclusion that Vip3Aa20 toxicity will be limited to sensitiv e lepidopteran species that are sufficiently exposed to the protein.

The toxicity of insecticidal B. thuringiensis proteins such as Vip3a20 depends on their binding to specific receptors present in the inse ct midgut. Research demonstrates that this specificity limits the proteins ’ toxic effect to certain lepi dopteran species. A discussion on the mechanism of action for Vip3A a20, its spectrum of activity, a nd its lack of toxicity for nonlepidopteran species is presented in Section VII of this petition.

Health and safety studies have been conducted with the novel proteins contained in MIR162 maize. A comprehensive assessment of the sa fety of the introduced proteins, Vip3Aa20 and PMI, demonstrate that both pr oteins are nontoxic to mammalian species and are unlikely to be food allergens. The Vip3Aa20 protein is considered nontoxic beca use it does not share significant amino acid homology with known protei n toxins, is nontoxic to mice at a very high dose of 1250 mg Vip3Aa20/kg bw, it is ra pidly degraded in simulated mammalian gastric fluid, and its insecticidal mode of ac tion for Vip3Aa20 is not relevant to mammals.

12 See determinations of nonregulated status: Monsanto maize line MON 810, 61 Federal Register 10720 (March 15, 1996); Pioneer/Dow maize line 1507, 66 Federal Register 42624 (August 14, 2001); Northrup King maize line Bt11, 61 Federal Register 2789 (January 29, 1996).MIR162-USDA-1 Page 102 of 268 Furthermore, Vip3Aa20 is not likely to be a fo od allergen because it is not derived from a known source of allergenic proteins, it does not have any significan t amino acid sequence identity to known allergenic proteins, it is rapidly degraded in si mulated mammalian gastric fluid, and it is labile upon heat ing at temperatures of 65oC and above. The PMI protein is
considered nontoxic because it does not shar e significant amino aci d homology with known protein toxins, it is nontoxic to mice at a very high dose of 3030 mg PMI/kg bw, and it is rapidly degraded in simulated mammalian gastri c fluid. PMI is not li kely to be a food allergen because it is not deri ved from a known source of allergenic proteins, it does not have any significant amino acid sequence identity to known allergenic proteins with implications for its allergenic potential, it is rapidly degraded in simulated mammalian gastric fluid, and it is labile upon heating at te mperatures of 37oC and above.

A temporary exemption from the requirement of a food tolerance curre ntly exists under 40 CFR §174.458 for Vip3Aa20 in maize and a perman ent exemption from th e requirement of a tolerance exists under 40 CFR §180.1252 for PMI in all plants. A petition for establishment of a permanent tolerance exemption for Vip3Aa20 in all plants is curre ntly under review at

EPA.

IX.C.3. Unique Characterist ics of the Geographic Area

There is no indication that this action w ould have a significant effect on the unique characteristics of any particul ar geographic area. As with Bt maize cultivars already deregulated and commercialize d, MIR162 maize is expected to be used throughout maizeproducing areas of the country.

IX.C.4. Effects on Human Environmen t are Unlikely to be Controversial

There is no ‘controversy’ as that term is us ed in the NEPA context regarding the use of transgenic Bt maize varieties. “The term ‘controversial’ refers to the existence of a substantial dispute ... as to the si ze, nature, or effect of the majo r federal action rather than to the existence of opposition to a use.” 13 The experience with existing Bt maize and cotton cultivars that are used for limiting lepidopteran insect feeding damage demonstates that there is no controversy regarding the use of MIR162 maize. This petition contains substantial evidence demonstrating why th e deregulation of MIR162 maize would have only beneficial effects on the quality of the human environment. Additionally, there is no scientific evidence that contradicts the data submitted in this petition.

IX.C.5. Potential Unique or Unknown Risks

Syngenta’s petition has the advantage of ha ving been preceded by the deregulation of multiple Bt maize cultivars. 12 Experience with maize cultivars producing Cry1Ab and Cry1F proteins from B. thuringiensis serves as a guide to the expected effects of MIR162 maize producing the Vip3Aa20 protein. Moreover, there is a history of safe use for B. thuringiensis proteins because microbial Bt pesticides have been used for decades and

13 See Heartwood, Inc. v. U.S. Forest Service, 380, F.3d 428, 8th Circuit 2004.MIR162-USDA-1 Page 103 of 268 transgenic Bt maize and cotton have been grown commercially for more than 10 years. 14 Therefore, the likely effects of Bt maize are well known.

To the extent that risks such as insect resi stance may manifest them selves over time, these risks are known and there will be preventativ e measures implemented upon commercialize of
Bt11xMIR162 maize. As a condition of th e EPA registration for Bt11xMIR162 maize, Syngenta will be required to implement an IRM plan. This is not a hypothetical solution to the potential development of insect resistance nor is it a response that might be implemented
at an uncertain future date. A detailed IRM plan that includes a refuge and other requirements designed to prevent the developmen t of insect resistance has been documented and submitted to EPA as part of the applic ation for a FIFRA Section 3 registration of
Bt11xMIR162 maize. 15

A significant and unique benefit offered by MIR162 relates to its distinct advantages in the area of resistance management when combin ed through conventiona l breeding with Bt11 maize containing the Cry1Ab protein. The Cr y1Ab and Vip3Aa20 proteins are present in these hybrids at levels that ha ve been demonstrated to prov ide a high-dose for control of O. nubilalis, H. zea and S. frugiperda , thus minimizing the risk of resistance developing in these species. Bt11xMIR162 hybrids will offer IRM advantages in comparison to other control options that do not demonstrably provide a ‘hig h dose’ against the target pests. Moreover, Vip3Aa20 operates by a mode of action different from that of Cry1Ab or Cry1F and targets a different binding site in susceptible larvae. The available data support a conclusion that Vip3Aa20 shows no potential for cross-resistance with Cry proteins (Lee et al. , 2003). Thus,
for H. zea and S. frugiperda , which are sensitive to both Cry1Ab and Vip3Aa20, Bt11xMIR162 maize is predicted to significantly extend the durability of both proteins for control of these pests be cause insect populations are very unli kely to evolve resistance to two proteins that act by independent modes of action.

The possibility of resistance development in H. zea is of particular concern, as it is also a pest of cotton and has the potential to unde rgo selection pressure from both Bt maize and Bt cotton cultivars that express similar cry genes, where the two crops are grown in the same geographies. The principal reason that the EP A requires growers in cotton-growing areas to plant 50% of their maize acres to non- Bt maize hybrids concerns th e potential for resistance
evolution in H. zea populations. In its application for EPA regi stration of Bt11xMIR162, Syngenta provides data and rationale to just ify reduction of the maize refuge in cottongrowing areas from 50% to 20% of maize acr es planted. For growers of Bt11xMIR162 maize hybrids, the reduced refuge requirement in cotton-growing regions will translate into a higher proportion of insect-protected maize acres, with a proportiona l increase in all the attendant benefits of the product in these areas. If EPA grants the reduction in refuge for Bt11xMIR162 it will have the advantage that IR M compliance will likely increase because

Bt maize growers in cotton-growing regions have heretofore not b een able to fully experience the benefits enjoyed by Bt maize growers in other regions of the U.S. No other Bt product offers comparable IRM adva ntages of Bt11xMIR162 maize.

14 See http://www.aphis.usda.gov/brs/not_reg.html for listing of deregulated Bt cultivars dating from 1994.

15 MIR162 will be offered to growers as a combined trait hybrid with the Bt11 trait; it will not be offered for sale to growers as a stand-alone trait.MIR162-USDA-1 Page 104 of 268 The IRM program is itself one component of an overall package of integrated crop management techniques, which include crop rotation, maintenance of refuge quality, and education as to the proper use of insecticides in refugia in order for them to serve as an effective source of susceptible adults to mate with any resistant adults surviving in the Bt
maize field. The effectiveness of IRM plans is not hypothetical. Refugia have been required
for all other commercial Bt maize cultivars and have been deployed over the past decade.
The fact that there have been no documented inst ances of confirmed insect resistance in the
field to Bt maize cultivars is evidence of the success of these IRM plans.

The IRM plan will be implemented as part of a product stewardship program. The specific refuge requirements and other stewardship prac tices to be used are set out in detail in a stewardship agreement. Growers will not have a choice whether to follow these procedures when choosing to grow hybrids containing the MIR162 and Bt11 traits; they will be contractually bound to follow the procedures, by means of the stewardship agreement, and their compliance will be monitored and enforced according to a fully documented compliance program, which is reviewed and ev aluated by EPA. Syngenta will communicate these requirements to growers using a wide -ranging grower education campaign. The stewardship program also requires resistance monitoring, a remedial action plan to be implemented in the event of unexpected levels of lepidopteran pest damage, and an annual IRM plan review.

Thus, Syngenta is obligated to implement speci fic and detailed stewards hip program aimed at preventing or significantly delaying insect re sistance. The program requires that growers plant a structured refuge and compliance w ith this requirement will be monitored and enforced. Data collected to date for existing Bt maize cultivars indicate that this type of stewardship program has been effective in preserving the effectiveness of the Bt technology. Thus, the absence of significant uncertainty is demonstrated by large volumes of data and analysis indicating predicted effects, observa tions of the effects of similar products over previous years, and plans in place to account for any deviation from expected effects.

IX.C.6. Potential Impacts for Future Considerations

By its terms, this petition for deregulation applies only to MIR162 maize. There is no
indication that APHIS intends to use this ac tion as a decision in principle about future deregulations. Nor is there any suggestion that another federal action would be effectively
decided as a result of an APHIS conclusion with regard to MIR162.

IX.C.7. Potential Cumulative Impacts

A cumulative impact is “...the impact on the e nvironment which results from the incremental impact of the action when added to other past , present, and reasonably foreseeable future actions regardless of what agency . . . unde rtakes such other act ions” (40 CFR §1508.7). Cumulative impact refers to the combination of the effect of the proposed action ‘with the effects of other federal actions’, not combina tions of interactions between this event and features of the environment, such as multiple factor interactions between the Bt -based proteinMIR162-USDA-1 Page 105 of 268 and other chemicals in a given Bt maize variety. 16

APHIS has previously made determinations of nonregulated status as to other Bt maize
cultivars. 12 Data and analysis submitted in this petition demonstrate that MIR162 is not a ‘tipping point’ that will combine with these pr evious deregulations to cause a significant impact. The crucial requirement is that the overa ll analysis take into account the effects of other actions in assessing the predicted effects of the action at issue: “It makes sense to consider the ‘incremental impact’ of a project for possible cumulative effects by incorporating the effects of ot her projects into the background ‘data base’ of the project at issue, rather than by restating the results of the prior studies.” 17 Studies and testing that support Syngenta’s petition took place in an environment in which numerous cultivars of Bt maize are widely used (USDA, 2007c). Thus, ‘inc orporating the effects of other projects into the background’ data. See Sections VI and VII of this petition for disc ussions of field trial results for MIR162 that showed no enhanced pl ant pest potential and no adverse effects for nontarget organisms. Accordingly, the data in this petition indicate that the Vip3Aa20 protein contained in MIR162 mai ze is not toxic to nontarget or ganisms and that the protein
will not accumulate in the environment. To the extent that there may be other cumulative effects on the human environment, such effect s could only be considered positive. These effects include the potential for reduced convent ional insecticide use, reduced emissions of greenhouse gases, improved maize grain quality, and for economic gain accruing to U.S.

growers.

Maintaining genetic purity has been a feature of maize cultivation for decades as part of hybrid seed and specialty maize produc tion, and the deregul ation of multiple Bt maize cultivars has not significantly a ffected these processes, even c onsidering the effects of these transgenic cultivars cumulatively. Since the introduction of hybrid maize in the 1930s, maize production has required separation of inbred parent and hybr id seed production activities from the production of grain. This has been necessary to maintain the genetic purity of inbred lines and guarantee the quality of hybrid seed sold to growers.

Standards for genetic purity and seed quality are largely set by industr y associations, state, national, and international institutions. Many of these standards were developed decades prior to the advent of transgenic maize. A basic requirement for maintaining genetic purity in seed production fields is ensuring that only intended cross-poll ination occurs. Many options are available for this purpose: main taining isolation distances to prevent pollen movement from other maize, planting border or barrier rows to inte rcept pollen, employing natural barriers to po llen movement such as treelines, manual, or mechanical detasseling, genetic male sterility, and stagge red planting dates. In addition to these practices, a series of widely-used standard operating procedures ensure that the genetic purity and identity of seed is maintained in all phases of production, from planting to harvest, processing, storage and

16 See TOMAC v. Norton, 433 F.3d 852, 864 (D.C. Circuit. 2006) . “Appellant appears to misunderstand the function of a cumulative impacts analysis. TOMAC construes the requirement to mean that BIA was required to consider the ‘cumulative impact of all the casino's expected impacts when added together.” This is not correct. The ‘cumulative’ impacts to which the regulation refers are those outside of the project in question; it
is a measurement of the effect of the current project along with any other past , present, or likely future actions.”

17 See Coalition on Sensible Transp., 826 F.2d at 70.MIR162-USDA-1 Page 106 of 268 sale (Wych, 1988). The ubiquity and robustn ess of these procedures developed for maintaining genetic purity in seed production, a nd the rigor with whic h they are routinely applied in the maize industry, th ese procedures have effectivel y managed the introduction of transgenic maize into the marketplace and ha ve continued to do so through the introduction of successive transgenic maize cultivars.

In addition to the specializati on adopted across the i ndustry to enable hybr id seed production for commodity field maize, about 5% of U. S. maize production has been composed of various specialty maize varieties. These include popcorn, waxy (high amylopectin) maize, high oil maize, high protein a nd modified protein maize, sweet corn, white corn, blue corn, Indian corn, and high amylose corn. 18 More recently, niche markets for organic maize products have also been develope d, and in 2006 there were at le ast 18 seed companies in the U.S. specializing in organic maize seed (see Se ction IX.D.3). Similar to the production of conventional inbred and hybrid seed, industry quality standa rds for these maize products have led specialty maize seed producers and growers to employ a vari ety of techniques to ensure that their products are not pollinated by or commingled with other field maize varieties. In general, all the management pr actices used in conventio nal seed production to ensure quality standards are also employed in, and are sufficient to meet standards for, the production of specialty maize seed.

In contrast to commodity field maize, rigor ous quality standards are also required for specialty grain production. To m eet these standards, specialt y grain growers employ a rangeof additional measures to meet purity standards. Most commonly, to prevent crosspollination of specialty maize by commodity fiel d maize, growers employ isolation distances, border or barrier rows, natural barriers, and in some cases, plantings staggered to ensure specialty maize is not flowering at the same time as nearby commodity field maize.

The same isolation standards that apply to s eed production fields may result in much higher purity in hybrid grain production fields; a hybrid grain field produces a pollen load 10 to 100 times greater than that of a single-cross s eed production field. In addition, the timing of pollen shed in a hybrid field is usually highly synchronized with silk emergence. The large pollen load and synchronous timing of pollen a nd silks serve to greatly limit pollen mixing from outside sources. In addition, since the 1950s a gametophytic self-incompatability system ( GaI s ) has been available, and is widely used in popcorn production, to prevent cross-pollination of specialty maize by fi eld maize (Thomas, 1955; Ziegler, 2000).

For decades prior to the intr oduction of transgenic maize cultivars and since their introduction, the maize industry has had eff ective methods and means to maintain product segmentation and genetic purity standards. As a result, these widespread practices have
served to ensure that the broa d adoption of transgenic maize in the U.S. (including the sale and cultivation of multiple Bt maize varieties for more than a decade) has had no significant impact, even in the aggregate, on the production of maize seed and special ty maize products. There is no evidence to suggest that deregulation of MIR162 ma ize will act as a ‘tipping

18 See U.S. Grains Council, Value Enhanced Corns Report 2005/2006, available at: http://www.grains.org/galleries/technical_publica tions/USGC%20Value%20Enhanced%20Corn%20Report%20 2006%20%20(English).pdfMIR162-USDA-1 Page 107 of 268 point’ that will undermine the effectiveness of these methods.

The adoption of multiple varieties of transgen ic maize has had no significant impact on the genetic diversity of cultivated maize or th e availability of diverse maize germplasm resources, even considering the effects of these transgenic events cumulatively. Genetically distinct maize hybrids have always been de veloped for various geographies and purposes, and are continually improved by plant breeding. This has in no way been altered by the introduction of transgenic maize; transgenes are simply introgressed into these breeding programs, and have not interfered with the cont inuous improvement of the base genetics that underlie the performance of modern maize hybrids.

Futhermore, the adoption of transgenic maize cultivars was preceded by worldwide efforts to identify and preserve sources of maize gene tic diversity, and to make these resources available for utilization by public and private maize breeders. Among these efforts are the Germplasm Enhancement of Maize program, a cooperative effort undertaken by USDA, public, and private plant sector breeders, non government organizations and international public cooperators, which was established to fu rther identify maize gene tic diversity and to provide it in useful form in order to broade n the genetic base of this crop (USDA, 1999). The germplasm sources being developed thro ugh the Germplasm Enhancement of Maize program are available free of charge through the extensive national germplasm collections and germplasm repository programs for cons ervation of maize genetic diversity.

Maize collections sponsored by the National Plant Germplasm System are located in Ames, Iowa (North Central Regional Plant Introduction Station) and at the Maize Genetics Stock Center in Urbana, Illinois. According to the Germplasm Resources Information Network, there are four maize species in the national collection: Zea diploperennis, Z. luxurians

, Z.

mays and Z. perennis

. Z. mays , which is the primary genetic s ource of cultivated maize, has 19,384 accessions. 19 At Urbana, 5101 accessions are available, primarily Z. mays . Other public maize germplasm resource centers include world collections at the International Maize and Wheat Improvement Center (M exico), tropical maize materials at International Institute of Tropical Agriculture (Nigeria ), and various collections re siding within national programs
of different countries around th e world. Specific maize germpl asm is also available from individual breeders working on maize at public instituti ons and universities. The deregulation of transgenic maize events provide s yet another source of genetic diversity that can be utilized in the improve ment of maize performance.

Thus, observation of the cumulative effects of numerous other transgenic maize products indicates that the genetic diversity of maize has been maintained in coexistence with
conventional maize. There is no evidence on th e record, nor is Synge nta aware of any, to suggest that MIR162 will act as a ‘tipping point’ that will undermine the effectiveness of these methods for maintaining genetic diversit y, and which have been successful through the previous deregulation of Bt cultivars.

There have been no documented instances of confirmed insect resistance to Bt maize having developed in the field, and Synge nta is not aware of any studies showing insect resistance to

19 http://www.ars-grin.gov/npgs/searchgrin.htmlMIR162-USDA-1 Page 108 of 268 Bt maize products, despite the introduction of mu ltiple previous cultivars over the past
decade. All commercialized Bt maize cultivars are subject to mandatory refuge requirements. The fact that there have been no documented instances of confirmed insect
resistance to Bt maize in the field indicates that the us e of mandatory refugi a is effective in preventing or delaying the development of insect resistance to B. thuringiensis insecticidal proteins, even cumulatively after multiple Bt maize cultivar introduc tions. A description of IRM program and supporting data and studies is provided in Section VIII. There is no evidence on the record, nor is Syngenta aware of any, to sugge st that MIR162 will act as a ‘tipping point’ that will cause th e previously-effective refuge system to become ineffective.

IX.C.8. Historical and Cultural Effects

There is no indication that th is action would adversely affect districts, sites, highways, structures, or objects listed in or eligible for listing in the National Register of Historic Places or that it may cause loss or destruction of significant scientific, cultural, or historical

resources.

IX.C.9. Effects on Endangered Species

There is a weight of evidence that at the concentrations of Vip3Aa20 in MIR162 maize adverse effects will be limited to lepidopteran species. No harmful effects of Vip3Aa proteins have been observed in any nontarget organism study. These studies used a wide range of taxa, and even at conservative estimat es of expected environmental concentrations, there is an adequate margin of safety for nontarget organisms (see Section VII).

EPA determined that the only endangered le pidopteran with potential for exposure to insecticidal proteins in maize is the Karner blue butterfly ( Lycaeides melissa samuelis ) (US EPA, 2001). The potential route of exposure is consumption of maize pollen that has settled on the leaves of its food pl ant, the wild lupine ( Lupinus perennis ). Peterson et al . (2006) determined that exposure of the Karner blue to maize pollen was minimal because most lupine populations are separated from maize fields by at leas t 500 metres, and because maize anthesis usually occurs after the Karner blue has finished feeding.

The restriction of toxicity of Vip3Aa20 to Lepidoptera, and the minimal exposure of engendered Lepidoptera to maize, indicates that planting of MIR162 maize is expected to have no harmful effects on any endangered or threatened species in the U.S.

IX.C.10. Compliance with Law

There has been no indication that this action w ould violate federal, state, or local law or requirements imposed for the protection of the e nvironment, nor is there any evidence on the
record that would so indicate.

IX.D. Economic Impacts

Economic considerations are not explicitly de scribed within the factors listed in 40 CFRMIR162-USDA-1 Page 109 of 268 §1508.27. However, economic impacts do relate to the significance of the requested action and have been considered by some c ourts in reviewing NEPA compliance.

Although some maize-growing areas experience significant lepidopter an infestations in most years, it is not possible for individual growers to accurately predict wh ether lepidopteran pest pressure will be economically significant in any particular growing season. Growers must make seed purchase decisions prior to kno wing whether the seed price premium for lepidopteran control in a given year will actuall y be recouped as higher crop yields when compared to the seed cost for unprotected hybrids or the costs of other control measures that might be applied. Nevertheless, the acres planted to Bt maize have continued to increase steadily since the first Bt maize hybrids were introduced in the U.S. in 1996. This is a testament to the actual yield-preserving benefits that Bt maize growers have experienced over the long term, and to the value of the built-in ‘insurance’ against a potential pest outbreak that could otherwise result in high economic losses. For many growers, the broad lepidopteran control offered by Bt11xMIR162 hybrids will represent a higher insurance value than currently available Bt maize cultivars. Additionally, Bt11xMIR162 hybrids will offer unsurpassed convenience to growers by reducing the need to scout fields for pest pressure or to apply other control measures for lepidopteran larvae.

The continued success of Bt maize hybrids in the marketplace since their initial introduction
in 1996 attests to their economic be nefits for growers. Although it is difficult at this time to accurately predict the magnitude of economic benefits that growers of MIR162 maize hybrids in the marketplace, the improved pestprotection profile of Bt11xMIR162 maize can be expected to translate into correspondingly higher overall economic benefits to growers, consumers, and other downstream users of maize products. The magnitude of these
economic benefits will necessari ly depend upon the seed price premium paid for the pestcontrol traits, the level of local pest pressure , and the value of the crop. Commodity prices for maize grain have dramatically increased recently due to high demand for fuel ethanol, and sustained demand is predicted for the comi ng years. Such demand will function to increase the value of a grower’s investment in any agricultural practice, technology, or product, including the MIR162 traits, th at increases or preserves yield.

Another predicted economic benefit for grow ers and downstream consumers is increased competition in the marketplace for pest-control products, including hybrid seed from multiple providers of lepidopteran-tolerant Bt maize varieties. The co mmercial availability of MIR162 hybrid maize seed will represent a signif icant new pest control option and tool for growers. Increased grower choice can be expect ed to exert downward pr essure on the cost of products that offer contro l of lepidopteran pests.

IX.D.1. Enhanced Productivity

Studies to quantify the expected yield a dvantage of Bt11xMIR162 maize across multiple geographies, environmental conditions, croppi ng practices and pest pressure are ongoing.
The data available from a limited set of trials indicate that, in the abse nce of pest pressure, Bt11xMIR162 hybrids will provide the same yield as their nontransgenic counterparts. It is expected that, as a result of superior and season-long control of si gnificant lepidopteranMIR162-USDA-1 Page 110 of 268 pests, Bt11xMIR162 hybrids will demonstrate significant yield advantages under both low and high insect pressure.

Some lepidopteran pests, such as A. ipsilon , can cause major yield loss es due to cutting of an
entire stand of maize in a field. If the crop is left untreated, the grower is sometimes forced to replant an entire field. Such catastrophic losses will be prevented by planting Bt11xMIR162 maize. Other, stalk-boring lepidopt eran larvae cause phys iologic yield loss because the stalk damage interferes with nutrien t uptake and increases susceptibility to plant diseases. They can also cause severe stal k lodging and dropped ears , which results in a physical yield loss because the ears cannot be m echanically harvested by the combine. Earfeeding pests reduce grain yield and quality, and occasionally result in elevated grain
fumonisin levels that render the grain unsafe and unus able as food or feed (see Sections I.B and VII.I; EPA, 2001). In the presence of pest pressure, Bt11xMIR162 maize hybrids can be expected to preserve yield potential, grai n quality, and silage quality by minimizing the damage that could otherwise be caused by lepidopteran insects.

IX.D.2. Increased Competition for Bt Lepidopteran Control Products

Another predicted economic benefit for grow ers and downstream consumers is increased competition in the marketplace for pest-contro l technologies, including hybrid seed from multiple providers of lepidopteran-tolerant Bt maize varieties. The commercial availability of hybrid maize seed with th e MIR162 trait will represent a significant new pest control option and tool for growers. Increased grower choice can be expected to exert downward pressure on the cost of other products th at offer control of lepidopteran pests.

IX.D.3. Farmer and Consumer Choice

As described above, maize cultivation has l ong included effective methods of maintaining genetic purity, and mechanisms are in place to protect the genetic diversity of maize. Specialization of maize cultiv ation, in hybrid seed production for commodity field maize and in production of specialty maizes, has require d genetic purity procedures for more than 70 years, and maize growers have utilized these methods effectiv ely to prevent undesired gene flow. Syngenta is aware of no studies show ing that these methods have been any less effective at preventing gene flow from transgenic maize varieties, or that specialty maizes, such as popcorn, have become less available since transgenic maize has come into general

use.

Thus, despite the introduction and adoption of transgenic maize cultivars over the past decade, including multiple varieties of Bt maize, specialty and orga nic maize remains readily available. In 2006, there were at least 18 seed companies in the U.S. specializing in organic
maize seed: 20

Albert Lea Seed House, Albert Lea, MN The American Organic Seed Co., Warren IL

20 http://www.mosesorganic.org/umord/suppliers.htm#seeds and http://www.cropsci.ncsu.edu/organicgrains/production/seedsuppliers.htmMIR162-USDA-1 Page 111 of 268 Ames Seed Farms, Kelley, IA Brown Seed Farms, Inc., Bay City, WI Brunner Seed, Durand, WI Doebler's, Inc., Callaway, VA Falk's Seed Farm, Murdock, MN
Foundation Direct Seeds/Mid Stat e Supply, Inc., Onalaska, WI Gold Country Seeds, Hutchinson, MN 55350 Golden Grains, Sparta, WI Great Harvest Organics, Atlanta, IN 46031 Lakeland Organics/Bruenne r Seeds, Colfax, WI Lancaster Ag Products, Bird-in-Hand , PA Lawler Farm Center, Lawler, IA Merit Seeds, Berlin, OH Prairie Gold Seeds, Mankato, MN Prairie Hybrids Seeds, Deer Gove, IL Welter Seed and Honey Co., Onslow, IA

See also U.S. Grains Council website for a listi ng of large suppliers of organic seed as of

2005. 21 There is no indication on the record that MIR162 will alter the coexistence between organic/specialty maize and the widespread use of transgenic maize varieties. Accordingly,
there is no reason to believe th at MIR162 will in any way limit farmer or consumer choice.

IX.D.4. Effects on the Export Market

There should be no effects on the U.S. mai ze export market since Syngenta is actively pursuing regulatory approvals for MIR162 mai ze in countries with functioning regulatory systems for genetically modified organisms and that import maize from the U.S. or Canada. Regulatory filings for MIR162 maize are in process for Colombia, Japan, South Korea, Taiwan, China, the Philippines, Australia and New Zealand, South Africa, the European Union, Russia, and Switzerland.

Syngenta’s stewardship agreemen ts with growers will include a term requiring growers to divert this product away from export markets ( i.e. channeling) where the grain has not yet received regulatory approval for import. Syngenta will communicate these requirements to growers using a wide-ranging grower education campaign ( e.g. , grower Stewardship Guide). As noted in the context of the IRM program , these procedures are not hypothetical.

The ability to channel particular types of maize for particular uses, such as the export market, is demonstrated by the continuing success of th e specialty maize market . Use of identity preservation measures has enabled growers to maintain a wide variety of specialized maize products, including white food maize, waxy maize, hard endosperm maize, high oil maize, nutritionally enhanced maize, high extractabl e starch maize, nonGMO maize, and organic maize (U.S. Grains Council, 2006). Channeling programs are well established for separating
each of these maize varieties. As set out above , these practices have continued successfully long after the introduction of numerous varieties of transgenic maize.

21 http://www.grains.org/index.wwMIR162-USDA-1 Page 112 of 268 IX.E. Conclusion

Syngenta is seeking a determination of dere gulated status for MI R162 maize under APHIS regulation 7 CFR §340.6. NEPA (42 U.S.C. 4321 et seq .) requires agencies undertaking such actions to provide a detailed statement of the environmental impact of the proposed action, any adverse environmental effects that cannot be avoided, and alternativ es to the action (42 U.S.C. 4332). Where the signif icance of an action is uncertai n, agencies may use an EA to identify, analyze and evaluate the impacts of the pr oposed action. The EA will satisfy the NEPA obligation where it provides sufficient evidence and analysis to support a FONSI. Factors to assess significance of a proposed acti on as listed by the CEQ are addressed in this petition. Based on the analysis of data and information provided in this petition, Syngenta
believes an EA is sufficient to evaluate the impact of deregulation of MIR162 and that a conclusion of no significant impact is warran ted thereby satisfying the requirements of

NEPA.

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Acknowledgements

The authors would like to acknowledge and th ank Demetra Vlachos, Nykoll Long, Cheryl Stacy, Karen Launis, Kim Hill, and David O’Reilly for their thorough review of, and editorial contributions to, this petition.MIR162-USDA-1 Page 125 of 268 Key to Abbreviations and Acronyms

ADF Acid detergent fiber

AOAC Association of Analytical Communities

APHIS Animal and Plant Health Inspection Service

bc Backcross

bp Base pair

BSA Bovine serum albumin

bw Body weight

CFR U.S. Code of Federal Regulations

Cry Crystal protein from B. thuringiensis

DDD Daily dietary dose

DF Dilution factor

DT 50 Time to 50% dissipation

DTT Dithiotheitol

dw Dry weight

EA Environmental assessment

EDTA Ethylenediamine tetraacetic acid

EEC Expected environmental concentration

ELISA Enzyme linked immunosorbent assay

EPA United States Environmental Protection Agency

FA Fatty acids

FDA United States Food and Drug Administration

FFDCA Federal Food, Drug and Cosmetic Act

FIFRA Federal Insecticide, Fungicide and Rodenticide Act

FONSI Finding of no significant impact

fw Fresh weight

HFCS High fructose corn syrup

HPLC High performance liquid chromatography

ILSI International Life Sciences Institute

IPM Integrated pest management

IRM Insect resistance management

kb Kilobase

LB Left border

(Continued)MIR162-USDA-1 Page 126 of 268 Key to Abbreviations and Acronyms (cont.)

LC 50 Median lethal concentration

LOD Limit of detection

LOQ Limit of quantification

MES 2-(N-morpholino)ethanesulfonic acid

MIR162 Maize insect resistant transfor mation event encoding a Vip3Aa20 protein derived from B. thuringiensis MW Molecular weight

NADP β -nicotinamide adenine dinucleotide phosphate

NADPH β -nicotinamide adenine dinuc leotide phosphate reduced

NCBI National Center for Biotechnology Information

NDF Neutral detergent fiber

NEPA National Environmental Policy Act

NOEC No observable effect concentration

NOEL No observable effect level

OD Optical density

OECD Organisation for Economic Co-operation and Development

PBS Phosphate buffered saline

PCR Polymerase chain reaction

PMI Phosphomannose isomerase

PVDF Polyvinylidene diflouride

RB Right border

SDS PAGE Sodium dodecylsulfate polyacrylamide gel electrophoresis

TDF Total dietary fiber

TER Toxicology exposure ratio

T-DNA Transferred DNA

Tris Tris(hydroxymethyl)aminomethane

U.S. United States

U.S.C. United States Code

USDA U.S. Department of Agriculture

v/v Volume per volume

Vip Vegetative insect icidal protein from B. thuringiensis

w/v Weight per volume

w/w Weight per weightMIR162-USDA-1 Page 127 of 268 Appendix A. USDA Notifications for Field Trials with MIR162 Maize

Field trials with maize transformation event MIR162 have been conducted in the U.S. under USDA release permits or notifications since 199 9. A listing of these not ifications, along with a status of the report for eac h, is provided in Table A.1.

Table A.1. List of field release permits and notifications that MIR162 maize have been planted under.

Reference # Effective Dates Release Sites (State) a Report Status

99-032-02r 03/31/99-03/31/00 IL Submitted

00-024-02r 4/30/00-04/30/01 HI Submitted

01-022-07r/m 4/30/01-4/30/02 AR, FL, ID, IL, MN,

PR Submitted

02-022-01r/m 4/10/02-4/10/03 IL, MN, MS Submitted

02-022-02r/m 4/30/02-4/30/03 HI Submitted

03-021-01r/m 4/30/03-4/30/04 AZ, CA, FL, IA, IL, KS, MN, MO, MS, NC, NE, PR, TX, WI Submitted

04-072-06n 4/26/04-4/26/05 FL, HI, IA, IL, MN, PR, WI Submitted

04-203-05n 8/24/04-8/24/05 PR Submitted

05-062-02n 4/25/05-4/25/06 AR, CA, FL, HI, IA, IL, IN, KS, KY, LA, MD, MN, MO, MS, NC, NE, NY, OH, PA, PR, TX, VA, WI Submitted

05-104-09n 5/12/05-5/12/06 HI Submitted

05-117-07n 5/10/05-5/10/06 OH Submitted

05-255-03n 11/9/05-11/9/06 HI Submitted

06-055-08n 4/27/06-4/27/07 CA, CO, FL, HI, IA, IL, IN, KS, LA, MN, MS, NE, NY, OH, PR, SD, TX, WI Submitted

06-059-07n 4/27/06-4/27/07 IL Submitted

(Continued)MIR162-USDA-1 Page 128 of 268 Table A.1 (cont.). List of field release permits and notifications that MIR162 maize have been planted under.

Reference # Effective Dates Release Sites (State) Report Status

06-284-101n 11/14/06- 11/14/07 PR Trials in progress

06-284-102n 11/13/06- 11/13/07 HI Trials in progress

07-032-104n 3/19/07-3/ 19/08 FL, GA, ID, MN Trials in progress

07-043-109n 4/5/07-4/5/08 AR, CO, DE, FL, GA, HI, ID, IL, IN, IA, KS,

KY, LA, ME, MD, MN, MO, NE, NC, OH, PR, SD, TX, VA,

WI Trials in progress

07-050-101n 4/5/07-4/5/08 HI, IA, IL, MN, NE, PR, TN, WI Trials in progress

07-166-101n 7/16/07 -7/16/08 IA Trials in progress

a – Includes only states where plantings took place.MIR162-USDA-1 Page 129 of 268 Appendix B. Genetic Characterization of MIR162 Maize

Materials and Methods

Plant Material

MIR162 maize seeds were obtained for mu ltiple backcross (BC) generations: BC 1 F 1 , BC 2 F 1 , and BC 4 F 1 . Seeds from each of these generations were planted and grown under standard greenhouse conditions and then pr ocessed to extract genomic DNA. See Table 2 and Figure 5 for the pedigree of each generation.

Plant Processing: From Plants to DNA

All plants grown in the greenhouse were indivi dually sampled and analyzed by TaqMan PCR for the presence of the vip3Aa and manA genes. Leaf tissue was collected from the plants of each generation, pooled, and DNA was extracted and quantified for use in Southern blot analyses. See schematic in Figure B-1.

TaqMan PCR

All plants were indi vidually analyzed using TaqMan PCR (Ingham et al. , 2001) for the presence of the vip3Aa and manA genes. For each individual plant, DNA was isolated from leaf discs using the Wizard Genomic DNA Puri fication kit. MIR162 maize plants were confirmed positive for both the vip3Aa and manA genes, while absence of these genes was confirmed for the negative segregant plants. All plants were TaqMan PCR positive for the assay’s internal contro l, the endogenous maize adh1 gene, as expected. See Table B-1 for primers used in the detection of vip3Aa, manA, and adh1 . The cycling parameters for this reaction were as follows: 95oC for 5 minutes, 40 cycles of 95oC for 15 seconds, and 60oC for
1 minute.

Table B-1. Primers and probes used for detection of the vip3Aa

, manA , and adh1

.

Gene Forward Primer 5’ to 3’ Reverse Primer 5’ to 3’ Probe 5’ to 3’

vip3Aa CACCTTCAGCAACCCGAACTA GCTTAGCCTCCACGATCATCTT GTCCTCGTCGCTGCCCTTCACCT

manA CCGGGTGAATCAGCGTTT GCCGTGGCCTTTGACAGT TGCCGCCAACGAATCACCGG

adh1 GAACGTGTGTTGGGTTTGCAT TGCAGCCTAACCATGCGCAGGGTA TCCAGCAATCCTTGCACCTT

Plant Pooling for Genomic DNA Extraction

Based upon the TaqMan results, ten positive pl ants and ten negative segregant plants (hereafter negative control) were selected to be used for molecular characterization. Leaf tissue from the ten positive plants was pooled into one sample bag and stored at -80oC. This process was repeated for the ten negative control plants.MIR162-USDA-1 Page 130 of 268 Figure B-1. Schematic representation of the procedure for selection and processing of plant samples for DNA extraction. Genomic DNA Extraction 10 positive plants were chosen based on TaqMan results Plants were grown under standard greenhouse conditions

10 negative segregant control plants were chosen based on TaqMan results

8 g of leaf tissue was ground into a fine powder in liquid nitrogen using a mortar and pestle

Purified DNA (This material was used in all Southern and sequence analyses in this report.) 8 g of leaf tissue was ground into a fine powder in liquid nitrogen using a mortar and pestle Leaf tissue from the 10 negative plants was pooled into one sampling bag

Purified DNA (This material was used in all Southern and sequence analyses in this report.) All plants were individually analyzed using TaqMan PCR to identif y positives and negatives

Leaf tissue from the 10 positive plants

was pooled into one sampling bagMIR162-USDA-1 Page 131 of 268 Genomic DNA Extraction

Genomic DNA used for blot Southern analyses was isolated from the pooled leaf tissue using
a method from Thomas et al . (1993).

The following buffers were used for genomic DNA extraction:

Extraction Buffer A: 0.25 M NaCl, 0.2 M Tris pH 8.0, 50 mM EDTA, 0.1% v/v 2- mercaptoethanol, 2.5% w/v pol yvinylpyrolidone (PVP-40)

Extraction Buffer B: 0.5 M NaCl, 0.2 M Tris pH 8.0, 50 mM EDTA, 1% v/v 2- mercaptoethanol, 2.5% PVP-40, 3% sarkosyl, 20% ethanol

DNA was extracted by first grinding the leaf ti ssue into a fine powder using a mortar and pestle under liquid nitrogen. Ei ght grams of the positive plant tissue and eight grams of the negative control plant tissue were ground and placed into separate 50 ml conical tubes. A 25
ml volume of Extraction Buffer A was added to each tube, and samples were gently mixed and centrifuged for 10 minutes at 2755 x g. The supernatant was discarded and 6 mls of Extraction Buffer B was added to each sample . The samples were mixed and incubated for 30-60 minutes at 65oC. Using a sterile l oop, the samples were mixed once during the incubation period. After the incubation, an equal volume of chloroform/isoamyl alcohol (24:1) was added to each sample. The sa mples were mixed gently by inversion and centrifuged for 20 minutes at 2755 x g . The aqueous layer was co llected in a clean 50 ml conical tube, and a 0.1 volume of 3M NaOAc pH5.2 was added and mixed. Next, a 0.7 volume of 100% isopropanol was added, and th e samples were mixed by inversion and centrifuged for 5 minutes at 2755 x g . The aqueous layer was decanted and the pellet was allowed to air dry briefly. The pellet was re suspended in 1000 μl of 1X TE (10 mM Tris, 1 mM EDTA pH 8.0) overn ight at 4oC.

After complete resuspension, samples were transf erred to microcentrifuge tubes and 6 μl of Ribonuclease A (10 mg/ml) was added, and the sample was incubate d at 37oC for 30 minutes. Samples were th en centrifuged at 19,600 x g for 10 minutes. The supernatant was removed and placed into a new tube, and a 0.5 volume of 7.5M NH 4 OAc was added. The tubes were centrifuged for 10 minutes at 19,600 x g . The supernatant was removed and placed in a new tube. A 0.7 vol ume of isopropanol was added and the samples were mixed by inversion to precipitate the DNA. The sa mples were centrifuged for 10 minutes at 19,600

x g to pellet the DNA. The supernatant was deca nted and the pellet was washed twice in 500 μl of 70% ethanol, spinni ng for 5 minutes at 19,600 x g after each wash. The supernatant was decanted and the pellet ai r dried briefly. Finally, the pellet was resuspended in approximately 500 μl of 1X TE at 65oC. DNA was stored at 4oC.

DNA Quantification

The concentration of each DNA sample wa s measured using the PicoGreen QuantiT technology with a Turner Bios ystems TBS-380 Fluorometer following the manufacturer’s
instructions. A Lambda DNA standard was us ed to calibrate the instrument prior toMIR162-USDA-1 Page 132 of 268 quantitation.

DNA Insert Sequencing

The DNA insert was amplified from MIR162 maize DNA derived from a BC 4 F 1 generation (see Figure 5) as four overlapping fragments (see Figure B-2 below). PCR amplification was carried out using either an Expa nd High-Fidelity PCR system or Pfu Ultra Hotstart HighFidelity DNA polymerase. Each PCR produc t was cloned into either a pCR-XL-TOPO vector or a pCR-BluntII-TOPO vector, and three clones for each PCR product were sequenced. Sequencing was carried out using an ABI3730XL analyzer with ABI BigDye 1.1 or Big Dye 3.1 dGTP (for GC-rich templates) chemistry. The sequence analysis was performed using the Phred, Phrap, and Consed p ackage from the University of Washington
and was carried out to an e rror rate of less th an 1 in 10,000 bases (Ewing and Green, 1998). The final consensus sequence of the DNA was determined by combining sequence data from
the three individual clones from each of the four PCR fragments to generate one consensus sequence. Sequence alignment was performe d using the ClustalW program with the following parameters: scoring matrix BLOS UM55, gap opening penalty 15, gap extension penalty 6.66 (Thompson et al. , 1994).

Figure B-2. Location of PCR-amplified fragments from MIR162 maize to determine insert

sequence.

Southern Blot Analysis

Southern blot analyses were performed using standard molecu lar biology techniques (Chomczynski, 1992). Genomic DNA (7.5 μg) was di gested with the appr opriate restriction enzymes overnight at the optimal temperature for each enzyme. Additional enzyme was added to each reaction the following morning, a nd the reaction was allowed to continue for approximately four hours. Digested DNA was loaded onto 1% agarose gels and bands were separated electrophoretica lly in 1X TAE buffer. Followi ng a 10 minute depurination in 0.25 N HCl, DNA was denatured in 0.5 M NaOH and 1.5 M NaCl for 30 minutes. The DNA was
then transferred to a Zeta-Probe GT membrane via alkaline transfer for one hour using a Boekel/Appligene Vacuum Blotter. The membranes were briefl y rinsed in 2X SSC. The
DNA was then crosslinked to the membrane us ing a Stratalinker UV Crosslinker with the vip3Aa20 (2370 bp) manA (1176 bp)

iPEPC9 (108 bp)

Fragment 1

Fragment 2 Fragment 3

Fragment 4 ZmUbiInt (1993 bp) ZmUbiInt (1993 bp) 35S terminator (70 bp) NOS (253 bp)MIR162-USDA-1 Page 133 of 268 ‘auto crosslink’ setting.

Element-specific, full-length PCR-generated probes were labeled with dCTP- 32 P via random priming using the Megaprime DNA labeling system. For all element-spec ific probes 5-25 ng was used for labeling; 5 ng was used fo r DNA molecular weight marker probes. Unincorporated isotope was removed using th e Micro Bio-Spin Chromatography Columns. Membranes were incubated in prehybridizati on solution (PerfectHyb Plus Hybridization Buffer), and calf thymus DNA (100 μg/ml) for approximately one hour at 65oC. The element-specific and molecular weight mark er radiolabeled probes were added to the prehybridization solution, and th e membranes were incubated fo r a minimum of three hours. Hybridization was carried out at 65°C, followe d by multiple washes in 2X SSC, 0.1% SDS and 0.1X SSC, 0.1% SDS. The membranes were then subjected to autoradiography.

One positive control was included in each Southern blot: a pNOV1300 plasmid control representing one copy equiva lent based on plasmid size wa s added to DNA from negative control plants. The calculation for the pNOV 1300 plasmid is shown below (Table B-2).

Table B-2. Formula used to determine one copy equivalent based on plasmid size.

((Plasmid size/(Genome size * Ploidy)) * μ g loaded) * 1x10 6 = pg for 1 copy

Example:

Z. mays genome size in bp: 2.67x10 9

Ploidy: 2

pNOV1300 size in bp: 14405

μ g of digested MIR162 maize DNA lo aded for Southern analysis: 7.5

Calculation for pNOV1300:

((14405 / (2.67x10 9 * 2)) * 7.5) * 1x10 6 = 20.2 pg

The copy number of the T-DNA functional elem ents present in the MIR162 maize genome was determined by Southern blot analyses. For each functional element of MIR162 maize, genomic DNA was digested using three restriction enzyme strate gies. In the first enzyme digestion, MIR162 maize genomic DNA was digest ed with an enzyme that digests once
within the DNA insert but not w ithin the functional element bei ng probed. This digest will result in a single unique hybridi zation band for each c opy of the functional element present in the MIR162 maize genome. In the second enzy me scheme, another enzy me that digests once
within the DNA insert but not within the f unctional element being probed was employed. This digest will also result in a single un ique hybridization band for each copy of the functional element present. Additionally, MI R162 maize genomic DNA was digested with an enzyme(s) that released a known size fragme nt. This restriction enzyme strategy gives further evidence of copy number and also dem onstrates the intactness of the insert. A negative control was included in each Southern blot experi ment to identify endogenous Z.MIR162-USDA-1 Page 134 of 268 mays sequences that may hybridize with the el ement-specific probe. Digested pNOV1300 plasmid equal to one copy equiva lent based on plasmid size was included in each Southern blot analysis to demonstrate a positive control fo r hybridization, as well as the sensitivity of the experiment.

Mendelian Inheritance of DNA Insert

The initial MIR162 maize plant (T 0 generation) was crossed with maize inbred line NPH8431, creating the F 1 generation. NPH8431 (MIR162 ma ize) plants from this F 1 generation were crossed to the in bred line NP2161 to yield the F 1 generation. NP2161 (MIR162 maize) plants from this F 1 generation were backcrosse d to the inbred line NP2161 to yield the BC 1 F 1 generation. MIR162 maize plants from this BC 1 F 1 generation were backcrossed again to inbred line B9620 to yield BC 2 F 1 generation. The F 1 generation was backcrossed two more time with inbred line B9620 to yield a BC 4 F 1 generation (refer to Figure 5). Positive segregants were utilized in each backcross. Material from the BC 1 F 1 , BC 2 F 1 , and BC 4 F 1 generations of B9620 was used in the Mendelian inheritance study.

Individual plants from the BC 1 F 1 , BC 2 F 1 , and BC 4 F 1 generations were assayed by TaqMan PCR for the presence of the vip3Aa and manA genes. The expected Mendelian inheritance ratio of positive to negative plants for a hemi zygous trait in these p opulations was 1:1. Genotypic data (Tables 10 and 11) were used to assess the goodness-of-fit for the ratio of observed (o) genotypes to the ratio of e xpected (e) genotypes using Chi-square (X 2 ) analysis
with Yates correction factor:

χ 2 = ∑ ( | o – e | - 0.5) 2 / e

Flanking Sequence

Nucleotide sequences flanking the 5’ and 3’ ends of the T-DNA in MIR162 maize were screened for homology with se quences found in public databases. The purpose of this comparison was to provide an indication of whether the MIR162 maize T-DNA inserted into a known plant functional genetic unit. A sequence similarity analysis was performed using the BLASTN software (Altschul et al. , 1997; version 2.2.6 Apr-9- 2003) which compared the flanking sequences with nucleotide sequences in the latest version of the National Center for Biotechnology Information nonredundant database.

The nonredundant database contains all sequences from the National Institutes of Health genetic sequence database (GenBank), RefSeq Nucleotides, the European Molecular Biology Laboratory (EMBL), the DNA Data base of Japan (DDBJ) and sequences derived from the three-dimensional structure (PDB ) database. Whereas GenBank is an archival repository of all sequences, the RefSeq database is a nonredu ndant set of reference standards that includes chromosomes, complete genomic molecules (o rganelle genomes, viruses, and plasmids), intermediate assembled genomic contigs, cu rated genomic regions, mRNAs, RNAs, and proteins. The nonredundant database does not c ontain any EST, STS, GSS, or phase 0, 1, or 2 HTGS sequences. At the time of this an alysis, the nonredundant database contained 4,654,881 unique sequences and was last upda ted on December 14, 2006. The queryMIR162-USDA-1 Page 135 of 268 sequence was not filtered for low complexity. Parameters for the BLASTN analysis were as
follows:

• Expect = 10. The Expect value (E-value) is the significance th reshold for reporting matches against database sequences; the defa ult value is 10, meaning that 10 matches are expected to be found merely by chance, accordi ng to the stochastic model of Karlin and Altschul (1990). Those results for which the Expect value is close to 0 will be unlikely to have occurred at random and, therefore, will be statistically significant hits. Those hits with E-values close to 10 will have happened randomly and are, therefore, not statistically significant.

• The scoring scheme (bits) used is the default fo r nucleotides: +1 fo r a match and –3 for a mismatch. Gap penalties: Existence = 5, Exte nsion = 2. A gap is a space introduced into an alignment to compensate for insertions and deletions in one sequence relative to another. Gap existence and ex tension penalties are chosen empirically. To prevent the accumulation of excessive gaps in an alignm ent, the introduction of a gap causes the deduction of a fixed amount from the alignm ent score. Extension of the gap to encompass additional nucleotides is also pe nalized in determining the score of an alignment. The resultant score is derived from the number of identical matches between the query sequence and the database entry, with higher scores indicating greater homology between the two sequences.

To ascertain if any potential novel open readi ng frames were generated at the point of
insertion in the Z. mays genome, the junctions between genomic sequences and the MIR162 maize DNA insert were examined for the pres ence of open reading frames using Vector NTI (version 9.0) software. An open reading frame was defined in th e bioinformatic program as a region corresponding to at least 50 amino acids in length initi ating with an ATG codon and ending with any of the three st op codons: TAA, TAG, or T GA. All six possible reading frames at both the 5’ and 3’ regions were examined using the above criteria.MIR162-USDA-1 Page 136 of 268 Appendix C. Characterization of the Vip3Aa20 and PMI Proteins

Vip3Aa Test, Control, and Reference Materials

Vip3Aa20 Extracted from MIR162 Maize Leaf Tissue

Maize leaf tissue was used as a source of MIR162 maize-expressed Vip3a20. Leaf tissue samples were harvested from greenhouse-gr own MIR162 maize plan ts. Leaves were
collected four to six weeks af ter plant emergence, frozen at -80 ± 10°C, and subsequently ground into fine powder and lyophi lized. Lyophilized leaf powd er was suspended in Vip3Aa extraction buffer containing 50 mM Tris-HCl (pH 9.5), 2 mM EDTA, 100 mM NaCl, 2 mM DTT and 1 μM leupeptin. The mixture was homogenized in a pre-cooled CB6 Waring industrial blender equipped with a double blade. The homogenate was then filtered through eight layers of cheesecloth and the collected solids were retained and extracted again in half of the original extraction volume. Both extracts were pooled, adjusted to a final concentration of 0.075% in polye thylenimine and centrifuged at approximately 7280 x g for
20 min at 2 - 8  ̊ C. The supernatant was f iltered through Miracloth filt ration material and fine powdered crystalline ammonium sulfate was added to a final concentration of 40% (24.6 g ammonium sulfate/100 ml extract). The mixt ure was slowly stirred overnight at 2 - 8  ̊ C and then centrifuged at approximately 7280 x g for 20 min at 2 - 8  ̊ C. The supernatant was discarded and the resultant precipitates were stored at 2 - 8  ̊ C. The precipitate was resuspended in dialysis buffer containing 50 mM ammonium carbonate (pH 9.5), centrifuged to remove any insoluble debris and dialyzed 3 ti mes (5 - 12 hr each) in dialysis buffer. The dialyzed extract was then lyophi lized and stored at -20 ± 8  ̊ C.

Two batches of purified MIR162 maize-derived Vip3Aa20 were pr epared and designated test
substances LPMIR162-0105 and LPMIR162-0106 a nd were used for the Western blot analysis and insect bioassays.

Immuno-affinity Purified Vip3Aa20 from MIR162 Maize Leaf Tissue

Immuno-affinity-purified Vip3Aa 20 was prepared from the amm onium sulfate precipitate as
described above for test substances LP MIR162-0105 and LPMIR162-0106. The precipitate was re-suspended in column equilibration buffer containing 50 mM Na 2 HCO 3 and 100 mM NaCl, then centrifuged to remove insoluble debr is. The supernatant was dialyzed three times (5 - 12 hr each) in column equilibration buffer, and subsequently loaded onto an equilibrated immuno-affinity column which had rabbit anti-Vip3Aa antibody bound to the matrix. Vip3Aa20 was eluted with 100 mM CAPS buffer (pH 11), neutralized, concentrated with ultra-filtration using Centricon 20-Pl us centrifugal filters and stored on ice before further use. Two batches of immuno-affinity purified MI R162 maize-derived Vip3Aa20 were prepared and designated test substances IA PMIR162-0105 and IAPMIR162-0106. These test
substances were used in the Wester n blot and peptide mapping analyses.

Vip3Aa20 from E. coli

Test substance MIR162VIP3A-0106, containi ng Vip3Aa20 protein, was prepared byMIR162-USDA-1 Page 137 of 268 expressing a synthetic vip3Aa20 gene in an E. coli over-expression system. The Vip3Aa20 in test substance MIR162VIP3A-0106 is predicte d to be identical in amino acid sequence to Vip3Aa20 expressed in MIR162 maize. The synthetic vip3Aa20 gene was cloned into the inducible, over-expression vector pET-24a in E. coli strain BL21DE3pLysS.

Test substance MIR162VIP3A-0106 was pr epared from pooled batches of E. coli cell paste, was lyophilized, and stored at -20 ± 8°C. Purification was accomplished as follows: E. coli cell pellets were ruptured and the cell debr is removed by centrifugation. The Vip3Aa20 present in the supernatant was further purified with anion ex change chromatography using a Q Sepharose FF column. Vip3Aa20 was eluted from the column with a NaCl gradient. Fractions containing Vip3Aa20 were pooled an d dialyzed in 20 mM ammonium bicarbonate buffer (pH 10), frozen and lyophilized. The lyophilized Vip3Aa20 sample preparation was designated test subs tance MIR162VIP3A-0106.

Control Substance – Maize Leaf Extract

Control substance LPMIR162-0105C was prepared from leaf material derived from MIR162 null segregant plants in a similar manner as described above for Vip3Aa20 extracted from
MIR162 maize leaf tissue. Since its preparation, this control substance has been stored at -20

± 8  ̊ C.

Vip3Aa19 from E. coli - Reference Substance

Reference substance VIP3A-0204, containing Vi p3Aa19, was prepared from pooled batches
of E. coli cell paste and was determined to cont ain approximately 92% Vip3Aa19 by weight,
and was demonstrated to have insecticidal activity against S. frugiperda larvae.

A listing of the test, control, and reference substa nces used in these characterization studies is
shown in Table C-1.

Table C-1. Identification and source of test, control, and reference substances.

Test Substance ID Vip3Aa Variant Source

LPMIR162-0105 Vip3Aa20 Extract of MIR162 leaf tissue

LPMIR162-0106 Vip3Aa20 Extract of MIR162 leaf tissue

IAPMIR162-0105 Vip3Aa20 Immuno-affinity purified extract of
MIR162 leaf tissue

IAPMIR162-0106 Vip3Aa20 Immuno-affinity purified extract of
MIR162 leaf tissue

MIR162VIP3A-0106 Vip3Aa20 E. coli

LPMIR162-0105C No Vip3Aa protein Control maize leaf extract

VIP3A-0204 Vip3Aa19 E. coliMIR162-USDA-1 Page 138 of 268 Methods for Vip3Aa20 Characterization and Equivalence

Total Protein Determination

Total protein in test subs tance MIR162VIP3A-0106 was quan tified spectrophotometrically by determining the absorption at 280 nm (Gill and von Hippel, 1989). The A280 method is based upon the absorption of the aromatic ami no acids tryptophan and tyrosine at 280 nm.
The extinction coefficient at 280 nm varies with the abundance of thes e amino acids in the target protein. A Genesys 6 spectrophotometer was used to measure absorption of
MIR162VIP3A-0106 at 280 nm and the extincti on coefficient of Vip3Aa20 was calculated
with Vector NTI software version 9. Th e absorbance at 280 nm was multiplied by the
correlation factor for the extinction coefficien t, to give an approximate total protein
concentration.

Total protein in test substances LPMI R162-0105, LPMIR162-0106 and control substance LPMIR162-0105C was quantified using the BC A method (Hill and Straka, 1988), using bovine serum albumin (BSA) as the protein standard.

Densitometry

For purity determination, aliquots of MI R162VIP3A-0106 (ranging from 1 to 20 μg Vip3Aa20 per lane) were subjec ted to SDS-PAGE using NuPAGE 4-12% Bis-Tris polyacrylamide gradient gel and MES running bu ffer. After electrop horetic separation, protein bands were visualized with Coomassi e staining and the distribution of the visible protein bands was estimated by densitometric an alysis using a Personal Densitometer SI and ImageQuant analysis software for Windows.

Glycosylation Analysis

Vip3Aa20 from MIR162 maize was previously determined to show no evidence of glycosylation. To determine whether Vip3A a20 in test substance MIR162VIP3A-0106 was glycosylated, 1 and 2 μg samples of the insecticidal protein were analyzed using the DIG Glycan Detection Kit, in accordance with the manufacturer’s instruct ions. Creatinase, a nonglycosylated protein, was incl uded as a negative control. Transferrin, a glycosylated protein, was used as a positive control. Samples were separated by SDS-PAGE using
NuPAGE 4-12% Bis-Tris polyacrylamide gradient gel and MES running buffer, and electroblotted to PVDF membrane. While on the me mbrane, glycan moieties were oxidized using periodate, labeled with digoxigenin, and detect ed with an anti-digox igenin antibody coupled to alkaline phosphatase.

N-Terminal Amino Acid Sequence Analysis

For N-terminal amino acid sequence analys is, test substance MIR162VIP3A-0106 was subjected to SDS-PAGE followed by electro-blot ting of the protein to a PVDF membrane. After staining the membrane with Amido black, the corresponding Vip3Aa20 band was excised and sent to Proseq, Inc. Protein Se quencing Services for N-terminal amino acidMIR162-USDA-1 Page 139 of 268 sequence analysis. ProSeq's methodology wa s developed specifically for proteins immobilized on PVDF membrane and optimi zed for automated Edman-based chemistry (Brauer et al., 1984).

Vip3Aa20 Quantification

The concentration of Vip3Aa20 in test substances LPMIR162-0105, LPMIR162-0106, IAPMIR162-0105, IAPMIR162-0106 and MIR162V IP3A-0106 was determined using sandwich ELISA (Tijssen, 1985). Control su bstance LPMIR162-0105C was used as a negative control in the ELISA analysis.

96-well Nunc MaxiSorp plates were coated overni ght and incubated at 2 - 8°C with goat antiVip3Aa immuno-affinity purified polyclonal antibodies in BBS containing 100 mM boric acid, 25 mM sodium borate, 75 mM sodium chlori de (pH 8.5; 100 μl/well) . The plates were
then washed five times with ELISA wash buffer containing 10 mM Tris, 0.05% Tween 20, 0.02% sodium azide (pH 8.0), and incubated in ELISA diluent contai ning phosphate buffered saline plus 0.05% Tween 20, 1% BSA and 0.02% sodi um azide (pH 7.4), for at least 45 min. The plates were then washed again as desc ribed above and incubated with the Vip3Aa20 sample solutions (100 μl/well) for 1.5 hr at 2 - 8°C followed by 30 min at room temperature. After washing, the plates were incubated with rabbit anti-Vip3Aa antib ody diluted in ELISA diluent for 1 h at 37 ± 1°C. The plates were washed and subsequently incubated with donkey anti-rabbit IgG conjugated with alkaline phosphat ase antibody, diluted to 1 μg per ml in ELISA diluent, for 1 h at 37 ± 1°C. The pl ates were washed again and incubated in phosphatase substrate solution at room temperat ure for 30 min to allow color development. The reaction was stopped by addition of 3 N sodium hydroxide and absorbance was
measured at 405 nm with a Tecan Sunrise multi-we ll plate reader. The results were analyzed using the DeltaSoft Curve fitti ng software program. The four parameters algorithm was used
to generate a curve.

Molecular Weight Determination and Immunoreactivity Analysis

The integrity of Vip3Aa protein in test substances LPMIR162-0105, LPMIR162-0106, MIR162VIP3A-0106, and VIP3A-0204 was investig ated using Western blot analysis. Aliquots containing 10 ng Vip3Aa 20 prepared in 4X LDS NuPAGE Sample Buffer from the described test substances were subjected to SDS-PAGE using NuPAGE 4-12% Bis-Tris polyacrylamide gradient gel and MES running bu ffer. Based on total soluble protein, an equivalent amount (containing a pproximately 676 μg of total soluble protein) of control
substance LPMIR162-0105C was also included in th e analysis as a negative control. After electroblotting, the membrane was incubated wi th goat anti-Vip3Aa immuno-affinity purified polyclonal antibodies. Donkey an ti-goat IgG linked to alkalin e phosphatase diluted 1:3,000 was used as the secondary antibody to bind to the primary antibody and visualized by development with alkaline phosphatase substrat e solution. The Western blot was examined for the presence of intact immunoreactive Vip3Aa20 and other immunoreactive Vip3Aa polypeptides. SeeBlue Plus2 molecular weight standard was used to establish approximate molecular weights.MIR162-USDA-1 Page 140 of 268 Insecticidal Activity

Test solutions for the bioassay were prepared in 50 mM Tris-HCl (pH 9.5) containing 2 mM
EDTA from test substa nces LPMIR162-0106, LPMIR162- 0105C and MIR162VIP3A-0106 and tested concurrently. Also a comparis on of Vip3Aa20 bioactivity (LPMIR162-0105) to Vip3Aa19 bioactivity (VIP3A-0204) was conducted in separate, but similar insect bioassays. Bioactivity of the proteins was assessed in inse ct feeding assays using freshly hatched firstinstar S. frugiperda. The bioassays were conducted in Co star 24-well plates. Each well contained 800 μl insect diet overlayed with 50 μl test solution, containing concentrations ranging from 7.5 to 825 ng Vip3Aa/cm 2 diet surface. Each tr eatment consisted of 24 individual wells containing one larva/well. Insect diet treated with MilliQ purified (deionized) water alone, insect diet treated w ith buffer alone, and insect diet treated with
control substance LPMIR 162-0105C at approximately 4910 μg total protein (an equivalent amount of total protein as applied in the highe st test concentration of LPMIR162-0106), were used as negative controls. The wells were c overed with silicone stoppers and maintained at room temperature under ambient laboratory co nditions. Mortality was assessed after 168 hours and is reported for each treat ment as the percent mortality.

Peptide Mass Mapping Analysis

Aliquots, containing 1 μ g Vip3Aa20 from either test su bstance IAPMIR162-0106 or test substance MIR162VIP3A-0106, were subjected to SDS-PAGE using a 4 - 12% Bis-Tris polyacrylamide gradient gel a nd MES running buffer. The gel was stained with Coomassie and then prepared for peptide mass mapping an alysis. Vip3Aa20 bands were excised from the SDS-gel, the protein was reduced, alkyla ted with iodoacetamide, and digested with trypsin. The mass analysis of the Vip3A a20-derived peptides was performed on a quadrupole time-of-flight mass spectrometer, fi tted with an EPCAS upgrade and connected to a capillary HPLC instrument. Peptide masses were identified by matching the detected peptide masses to a protein data base using Mascot software.

Statistical Analysis

The LC 50 and 95% confidence intervals determined in the S. frugiperda larval bioassays were computed by means of a probit analysis.

Results for Vip3Aa20 Charac terization and Equivalence

Vip3Aa20 Quantitation and Purity Determination

MIR162VIP3A-0106 was determined to contain 93.0% protein as measured by absorption at 280 nm. Densitometric analysis indicated that Vi p3Aa20 represented approximately 90.3% of the total protein in MIR162VIP3A-0106 (Fi gure C-1). The overall purity of the test substance was therefore, determined to be 84.0% Vip3Aa20 w/w (Table C-2).MIR162-USDA-1 Page 141 of 268 1

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188

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3 62 98 MW [kDa] 3 2 45 6 7 8 9

Vip3Aa20

Figure C-1. Purity determination of Vip3Aa20 from E. coli (MIR162VIP3A-0106) by SDSpolyacrylamide gel electrophoresis. The molecular weight of Vip3Aa20 corresponds to approximately 89 kDa.

Lanes 1 and 9: Molecular weight standard SeeBlue Plus2 Lanes 2 to 8: 1, 2, 4, 8, 10, 15, 20 μg Vip3Aa20 (MIR162VIP3A-0106)

Table C-2. Characterization of Vip3Aa20 extracted from E. coli (MIR162VIP3A-0106).

Total protein [g protein/g extract] Densitometric Analysis [% Vip3Aa20/total protein] Purity [% Vip3Aa20/extract]

0.93 (93.0%) 90.3 84.0

Glycosylation Analysis

No bands representing glycosylated Vip3Aa 20 were visible upon DIG Glycan analysis (Figure C-2), providing evid ence that, as expected, the E. coli -produced Vip3Aa20 is not glycosylated. By comparison, 25 ng of the pos itive control protein, tr ansferrin, generated a clearly visible band (Figure C- 2, lane 3). Transferrin has a molecular weight of approximately 80,000 and contains approximately 5% glycan moieties by weight. This corresponds to approximately 25 glucose equivale nts/molecule (the molecular weight of the bound glucose moiety was calculated to be 162). Out of the 25 ng of transferrin loaded on the gel lane, 1.25 ng could be attributed to glycan moieties. Therefore, 1.25 ng represents the approximate limit of detection for this system . Vip3Aa20 from micr obial test substanceMIR162-USDA-1 Page 142 of 268 MIR162VIP3A-0106 was loaded in concentratio ns of approximately 1000 and 2000 ng/lane. Thus, the 1.25 ng glycan detection limit corr esponds to approximately 0.0625% by weight (1.25 ng/2000 ng), or approxim ately 0.34 glucose equivale nts/Vip3Aa20 molecule. Therefore, the results of the DIG Glycan analysis indicate that either Vip3Aa20 is not glycosylated, or that glycan moieties occur at a frequency of less than one glucose equivalent per molecule of Vip3Aa20. Vip3Aa20 ex tracted from MIR162 maize (IAPMIR162-0105)
has also been found not to be glycosylated (data not shown).

1 2 3 4 5 67

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Vip3Aa20 Transferrin

Figure C-2. Glycosylation analysis of Vip3Aa20 produced in recombinant E. coli (MIR162VIP3A-0106). Vip3Aa20 from E. coli was analyzed for the presence of glycosyl residues using the DIG Glycan Detection Kit. The molecular weight of Vip3Aa20 corresponds to approximately 89 kDa.

Lanes 1, 2 and 3: 100, 50, 25 ng transferrin (positive control) Lane 4: 2 μg creatinase (negative control) Lane 5: molecular weight standard SeeBlue Plus2 Lanes 6 and 7: 1 and 2 μg Vip3Aa20 (MIR162VIP3A-0106)MIR162-USDA-1 Page 143 of 268 N-Terminal Amino Acid Sequence Analysis

The N-terminal amino acid se quences for the Vip3Aa20 in E. coli produced test substance MIR162VIP3A-0106 confirmed the predicted ami no acid sequence for the protein, as shown
below in Table C-3.

Table C-3. N-terminal sequence of Vip3Aa20 produced in recombinant E. coli

.

Predicted Amino Acid Sequence MNKNNTKLSTRA

Vip3Aa20 from MIR162VIP3A-0106 MNKNNTKLSTRA

The N-terminal sequence of the plant derive d protein could not be determined due to technical difficulties (most likely N-terminal bl ockage of the protein). Instead, the sequence of plant-derived Vip3Aa20 pr otein was determined by peptide mass mapping (described below).

Peptide Mass Mapping Analysis

The analyses of the plant- and microbia lly-derived Vip3Aa20 by peptide mass mapping covered approximately 40% and 45%, respecti vely, of the predicted Vip3Aa20 amino acid sequence. Furthermore, the iden tified peptides were representa tive of regions throughout the sequence of the Vip3Aa20 protein (including pep tides close to the N- and C-termini) and therefore, strongly support the identity of the Vip3Aa20 proteins produced in MIR162 maize
and in E. coli

.

Molecular Weight Determination and Immunoreactivity Analysis

Western blot analysis of the microbial te st substances VIP3A- 0204 and MIR162VIP3A-0106 as well as the plant-derived test s ubstances (LPMIR162- 0105 and IAPMIR162-0105)
revealed dominant immunoreactive bands corres ponding to the predicted molecular weight of approximately 89 kDa (see Figure C-3). C ontrol substance LPMIR162-0105C showed, as expected, no positive response with the anti-V ip3Aa antibody in the We stern blot analysis (Figure C-3, lane 6). Add itional, immunoreactive protein bands of higher mobility (lower molecular weight) than intact Vip3Aa20 were also visible in the Western blot analysis (Figure C-3, Lanes 3 to 5); these additional bands most lik ely represents degradation products of the Vip3Aa20 protein in samples MIR162VIP3A-0106, LPMIR162-0105, and

IAPMIR162-0105.MIR162-USDA-1 Page 144 of 268 1 2 3 4 5 67

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Vip3Aa 188

49

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Figure C-3. Western blot of plant and microbially-produced Vip3Aa proteins. This Western blot shows the immunoreactivity of Vip3Aa19 from E. coli , Vip3Aa20 from E.
coli , and Vip3Aa20 from MIR162 maize. Th e molecular weights of Vip3Aa19 and Vip3Aa20 correspond to approximately 89 kDa. Lanes 1 and 7: molecular weight standard Lane 2: 10 ng Vip3Aa19 from E. coli Lane 3: 10 ng Vip3Aa20 from E. coli Lane 4: 10 ng Vip3Aa20 from MIR162 maize leaf tissue extract Lane 5: 10 ng immunoaffinity-purified Vip3Aa20 from MIR162 maize leaf tissue Lane 6: protein extract from control maize leaf tissue.

Insecticidal Activity

The results of the insect bioassays with micr obial- and plant-derived Vip3Aa20 are shown in Table C-4; computed LC 50 estimates are shown in Table C-5. Test substances MIR162VIP3A-0106 and LPMIR162-0106 showed comparable responses in the S. frugiperda bioassays, with estimated LC 50 s of 225 ng Vip3Aa20/cm 2 (95% confidence interval = 155 - 289 ng/cm 2 diet surface) and 318 ng Vip3Aa20/cm2 (95% confidence interval = 232 - 451) after 168 hours, respec tively. By comparison, no mortality was observed in the negative control using insect diet treated with water (0%) or insect diet treated with buffer (0%). Furthermore, control substance LPMIR162-0105C showed low mortality (4%). The t-te st comparing the two LC 50 values resulted in a p-value of 0.112. Given that the p-value is greater than 0.05 it can be concluded that there is no significant
difference between the LC 50 s for the Vip3Aa20 proteins from both sources as shown in Table
C-6.MIR162-USDA-1 Page 145 of 268 Table C-4. Insect mortality results for bio assays with Vip3Aa20 from recombinant E. coli (MIR162VIP3A-0106) and Vip3Aa20 from MIR162 maize (LPMIR162-0106) in diet surface bioassays with first-instar S. frugiperda (N=24).

Mortality at 168 hours [%] Concentration [ng Vip3Aa20/cm 2 ] a MIR162VIP3A-0106 LPMIR162-0106

7.5 8 0

15 0 0

30 8 4

60 13 4

120 17 42

275 71 33

550 83 58

825 100 88

Water control b 0

Buffer control c 0

LPMIR162-0105C d 4

a - Insect diet treated with Vip3Aa20 from E. coli or Vip3Aa20 from MIR162 leaf tissue dissolved in 50 mM Tris-HCl (pH 9.5), 2 mM EDTA (50 μl/dish).

b - Water control diet: Insect diet treated with water (50 μl/dish).

c - Buffer control diet: Insect diet treated with 50 mM Tris-HCl (pH 9.5), 2 mM EDTA (50 μl/dish).

d - Insect diet treated with control maize leaf extract in 50 mM Tris-HCl (pH 9.5), 2 mM
EDTA.

The computed LC50 estimates for the insect bioassays with plant- derived Vip3Aa20 and E.
coli -derived Vip3Aa19 are presen ted in Table C-5. Test substances LPMIR162-0105 and VIP3A-0204 showed comparable responses in the S. frugiperda bioassays, with estimated
LC 50 values of 154 ng Vip3Aa20/cm 2 (95% confidence interval = 94 - 222 ng/cm 2 diet surface) and 137 ng Vip3Aa19/cm 2 (95% confidence interval = 82 - 199) after 120 hours, respectively. Table C-7 displays the mortality results by individual test concentration and treatment. By comparison, no mortality was obs erved in the negative control using insect
diet treated with wate r (0%) and 12% mortalit y was observed in the group exposed to diet treated with buffer. Furthermore, control substance LPMIR162-0105C showed, as expected, low mortality (4%).MIR162-USDA-1 Page 146 of 268 Table C-5. Comparative bioactivity of Vi p3Aa proteins from different sources. Results of mortality bioassays with first instar S. frugiperda larvae are presented as LC 50 values with their 95% confidence intervals. In the first set of bioassays, Vip3Aa20 from
MIR162 maize leaf tissue was compared to Vip3Aa20 from E. coli . In the second set of bioassays, Vip3Aa20 from maize leaf tis sue was compared to Vip3Aa19 from E. coli

.

Test Substance LC 50 (ng protein/cm 2 ) 95% Confidence Intervals

Vip3Aa20 – MIR162 leaf extract 318 232 – 451

Vip3Aa20 – E. coli 225 155 – 289

Vip3Aa20 – MIR162 leaf extract 154 94 – 222

Vip3Aa19 – E. coli 137 82 – 199

Table C-6. Statistical comparison of the estimated LC50 values for Vip3Aa20 from recombinant E. coli (MIR162VIP3A-0106) and Vip3Aa20 from MIR162 maize leaf (LPMIR162-0106). LC50 estimates statistically compared by t-test.

Sample Log(LC 50 ) a Variance of log(LC 50 )

MIR162VIP3A-0106 2.3530 0.00385

LPMIR162-0106 2.5019 0.00492

Comparison

Difference in log(LC 50 ) b Variance of
Difference Diff /SED P-value Significance

0.1489 0.00877 1.58981 0.112 nonsignficant

a
- LC 50 values are presented in Table B-5.

b - Log(LC 50 ) values and their respective vari ances were taken from SAS output.MIR162-USDA-1 Page 147 of 268 Table C-7. Insect mortality results for bio assays with Vip3Aa19 from recombinant E. coli (VIP3A-0204) and Vip3Aa20 extracted from MIR162 leaf tissue in diet surface bioassays
with first-instar S. frugiperda (N=24).

Mortality at 120 hours [%] Concentration [ng Vip3Aa/cm 2 ] a VIP3A-0204 LPMIR162-0105

7.5 8 8

15 4 0

30 17 21

60 38 38

120 54 46

275 75 58

550 83 92

825 92 92

Water control b 0

Buffer control c 12

LPMIR162-0105C d 4

a - Insect diet treated with Vip3Aa19 from E. coli or Vip3Aa20 from MIR162 leaf tissue
extract was dissolved in 50 mM Tris-H Cl (pH 9.5), 2 mM EDTA (50 μl/dish).

b - Water control diet: Insect diet treated with water (50 μl/dish).

c - Buffer control diet: Insect diet treated with 50 mM Tris-HCl (pH 9.5), 2 mM EDTA (50 μl/dish).

d - Insect diet treated with protein extract from control leaf tissue (LPMIR162-0105C) in 50 mM Tris-HCl (pH 9.5), 2 mM EDTA.

PMI Test and Control Substances

Leaf Tissue Extractions from MIR162 and Control Maize

Leaf tissue samples used in this study came from greenhouse-grown MIR162 and nearisogenic, nontransgenic maize hybrids. Leaves were collected four to six weeks after the plants emerged, frozen at -80°C ± 10°C an d subsequently ground into fine powder and lyophilized. Lyophilized leaf powder was re -suspended in 3 ml PMI extraction buffer containing 50 mM Tris-HCl, pH 9.5, 2 mM EDTA, 100 mM NaCl, 2 mM DTT and 1 μ M leupeptin. The mixture was homogenized using a Tomtec Autogizer and placed on ice for 30
min with an additional 3 ml PMI extraction bu ffer. The supernatant was collected after centrifugation for 15 min at 12,000 x g in a Sorvall RC-5B Refrigerated SuperspeedMIR162-USDA-1 Page 148 of 268 Centrifuge at 2-8oC. The supernatant was then concentr ated using Centricon 20-Plus centrifugal filters by centrifugation for 25 minutes at 2,600 x g using a Sorvall RC-3B Refrigerated Centrifuge at 2-8o C. The concentrated supernat ant was stored on ice before
further use. The test substa nce derived from MIR162 leaf ti ssue was designated MIR162(+). The PMI protein in test substance MIR162(+) is 391 amino acids in length and has an expected molecular weight of approximately 42.8 kDa. The nontransgenic control leaf extract, designated MIR162(-), was produced in the same manner as test substance MIR162(+).

PMI from E. coli

The manA gene from E. coli was cloned into an inducible pET-3a over-expression vector in
E. coli strain BLRDE3. The PMI prot ein as encoded by th is vector is identical in amino acid sequence to that encoded by the vector used to produce MIR162 maize, with the exception of an additional, nonfunctional 16 amino acids at the N-terminus (13 amino acid T7-Tag and three additional amino acids arising from the polylinker), which was added for purification purposes. PMI was purified from the E. coli expression system by extr action and differential
ammonium sulfate concentra tion, followed by hydrophobic inte raction chromatography on either Phenyl Sepharose or Toyopearl Ether 6 50M and finally subjected to ion exchange chromatography on Q Sepharose. Pooling of th e appropriate fractions was performed prior to dialysis into 50 mM NH 4 HCO 3 , pH 8.0, followed by lyophili zation. The lyophilized products from two preparations were pooled and designated test substance PMI-0198. PMI has been determined to repres ent approximately 61% of the te st substance by weight. The PMI protein in test substan ce PMI-0198 is 407 ami no acids in length a nd has an expected molecular weight of approximately 44.4 kDa. This material has been stored as a lyophilized powder, desiccated at -20 ± 8°C.

Methods for PMI Characterization and Equivalence

Total Protein Determination

Total protein in each of the le af extracts was quantified with a Coomassie protein assay using BSA as the protein standard (Bradford, 1976).

PMI Quantification

The leaf extracts, test substance MIR162( +) and control substance MIR162(-), were quantitatively analyzed for PM I using sandwich ELISA (Tijsse n, 1985). Nunc MaxiSorp plates were coated overnight at 2-8°C with polyclonal goat an tibody generated against PMI (diluted to 3 μg per ml in borate buffered sa line, 100 mM boric acid, 25 mM sodium borate, 75 mM sodium chloride). Plates were wash ed five times with ELISA wash solution (100 mM Tris, 0.5% Tween 20, 0.2% sodium azide) usi ng an automatic plate washer. The plates were then blocked with ELISA diluent (pho sphate buffered saline + 0.05% Tween 20, 1% BSA, 0.02% sodium azide, pH 7.4) fo r at least 45 minutes at room temperature. Plates were washed five times with wash solution and samp les of each leaf extrac t preparation and PMIMIR162-USDA-1 Page 149 of 268 standard with appropriate dilu tions prepared in ELISA diluent were applied (100 μl per well).

Following incubation for approximately 1.5 hours at ambient temperature, the plates were washed five times and 100 μl of polyclonal ra bbit antibody generated agai nst PMI (diluted to 3 μg per ml in ELISA diluent) was added to each well. The plates were incubated for approximately 1 hour at 37± 2oC an d then washed five times prio r to the addition of 100 μl of donkey anti-rabbit alkaline phosphata se antibody, diluted to 1 μg per ml in ELISA diluent. After incubation for approximately 1 hour at 37± 2oC, the plates were washed five times and 100 μl per well of phosphatase substrat e solution (two tablets per 20 ml H 2 O) was added. Color was allowed to develop for approximate ly 30 minutes at room temperature and the reaction stopped by the addition of 3N NaOH (100 μl per well). Absorbance at 405 nm was measured using a Tecan Sunrise multi-well plate reader. The results were analyzed using the DeltaSoft Curve Fitting software program. The four parameters algorithm was used to
generate a curve.

Molecular Weight Determination and Immunoreactivity Analysis

An aliquot of MIR162(+) and PMI-0198, contai ning 10 ng PMI, respectively, was subjected to SDS-PAGE using a NuPAGE 4-12% Bis-Tris polyacrylamid e gradient gel. For the negative control leaf extract, approximately 58 μg of total soluble protein was loaded per lane on the gel. After electroblotting, the PVDF membrane was probed with polyclonal goat antibody generated against PMI purified from E. coli . Donkey anti-goat IgG linked to alkaline phosphatase was used to bind to the primary antibody and visualized by development with an alkaline phosphatase substrate solu tion. The Western blot was examined for the presence of intact imm unoreactive PMI (approximately 42.8 kDa for the plant expressed PMI and approximately 44.4 kD a for the microbially expressed PMI) and other immunoreactive PMI polypeptides.

Enzymatic Activity Determination

The enzymatic activity of PMI was measured in triplicate in a coupled enzyme activity assay (as shown in the diagram below) effectively described in Gill et al. (1986). Briefly, the reaction assay was started by adding 4 or 10 μl of MIR162(+), MIR162(-), or PMI-0198 to an assay mixture consisting of 50 mM Tris -HCl (pH 7.0), 0.8 mM NADP, 1.6 mM mannose6-phosphate, 2 U phosphoglucose is omerase, and 2 U glucose 6- phosphate dehydro genase in a total volume of 1 ml. PMI activity was m easured by monitoring the formation of NADPH at 340 nm on a Thermo Spectronic Genesys6 spectrophotometer at room temperature.

Mannose 6-P Fructose 6-P Glucose 6-P + NADP 6-Gluconolactone + NADPH PMI PGI G6PDH

PMI = Phosphomannose isomerase, PGI = Phosphoglucose isomera se, G6PDH = Glucose 6- phosphat e dehydrog enase

The specific activity of PMI in test substa nces MIR162(+) and PMI-0198 was determined using the molar extinction co efficient of NADPH (6.2 x 10 6 cm 2 /Mol). One unit of PMIMIR162-USDA-1 Page 150 of 268 activity is defined as 1 μmol of NADP redu ced to NADPH per min. The concentration of PMI added to the assay was also determined.

Statistical Analysis

ELISA values were analyzed using the DeltaSoft Curve Fitting software program. Mean
values and standard deviations for the PMI enzyme assay were calculated using Microsoft
Excel.

Results for PMI Characteri zation and Equivalence

Molecular Weight Determin ation and Immunoreactivity

A single immunoreactive PMI band corresponding to the anticipated molecular weight of approximately 42.8 kDa was found in the MIR162- derived plant extract, MIR162(+) (Figure C-4; lane 3). The microbially-produced PM I protein in PMI-0198 (F igure C-4; lane 4) showed one major band, with a s lightly lower mobility than th e plant expressed PMI protein, as expected from its slightly higher predicted molecular we ight of approximately 44.4 kDa (resulting from the additional 16 amino acids at the N-terminus). The Western blot analysis
of test substance PMI-0198 also revealed another immunoreactive protein band with a molecular weight of approximately 90 kDa (Figur e C-4, lane 4). Since th is protein also cross reacted with the anti-PMI antibody and the fact that this band showed mobility of a protein corresponding to that of a molecular weight twice that of PMI ( i.e. , 2 x 44.4 kDA = 89 kDA), this protein band most likely represents a dimer of the PM I protein. No immunoreactive
band was found in the extract of control leaf tissue, MIR162(-) (Figure C-4; lane 2).

PMI Enzymatic Activity

The mean PMI specific activity of the MIR 162 maize leaf extract was 55.5 ± 4.18 U/mg PMI (Table C-8). PMI extracted from E. coli had a mean specific ac tivity of 33.2 ± 2.37 U/mg PMI. Historical activity meas urements for maize PMI proteins and test substance PMI-0198, as determined at the Syngenta Biotechnology la boratory are in the ranges of 44 – 113 U/mg maize PMI and 31 – 53 U/mg microbial PMI (see Table C-9). PMI activity measurements in this study are not materially different from ac tivity levels measured in previous Syngenta studies. Considering the expected levels of va riability in the measurements it is concluded
the PMI extracted from MIR162 mai ze has similar activity to PMI in E. coli test substance PMI-0198. In addition, the control maize leaf extract showed no PMI enzymatic activity.MIR162-USDA-1 Page 151 of 268 1 2 3 4

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Figure C-4. Western blot of PMI Protein fro m MIR162 maize leaf tissue and from E. coli (PMI-0198). The molecular weights of the plant-produced PMI and the E. coli -produced PMI correspond to approximately 42.8 kDa and 44.4 kDa, respectively.

Lane 1: Molecular weight standard SeeBlue Plus2, 8 μl Lane 2: 58 μg total protein fr om MIR162(-) maize leaf extract Lane 3: 10 ng PMI from MI R162(+) maize leaf extract Lane 4: 10 ng PMI from E. coli ( PMI-0198)MIR162-USDA-1 Page 152 of 268 Table C-8. Enzymatic activity comparison of PMI from MIR162 maize leaf tissue, PMI
from E. coli (PMI-0198), and from an extract of control maize leaf tissue.

Sample PMI Conc. [ng PMI/ml] Specific Activity [U/mg PMI] a Mean [U/mg PMI] SD [U/mg PMI] c

557 50.7

557 57.9 MIR162(+)

557 57.9 55.5 4.18

4500 35.8

4500 31.4 PMI-0198

4500 32.3 33.2 2.37

ND

ND MIR162(-) ND b

ND ND ND

a - one unit (U) of PMI activity is defined as 1 μmol of NADP reduced to NADPH per min.

b - ND = not detectable

c - SD = standard deviation

Table C-9. Enzymatic activity determinations for PMI from maize and E. coli as determined in previously conducted Syngenta studies.

Date of Analysis Maize-derived Range [U/mg PMI] E.coli

derived Range [U/mg PMI]

March 2004 43.45-66.27 a 35.85-49.27

April 2005 81.6-112.6 a 52.9

September

2006 50.7-57.9 b 31.4-35.8

a - Extracted from other transgenic maize events containing PMI

b - Extracted from MIR162 maizeMIR162-USDA-1 Page 153 of 268 Appendix D. Quantification of Vip3Aa20 and PMI in MIR162 Maize

Materials and Methods

Source of Plants for Evaluation of Vip3Aa20 and PMI Concentrations

Two MIR162 maize hybrids, designated Hybrid 1 and Hybrid 2, and their corresponding near-isogenic, nontransgenic c ontrol hybrid equivalents (see Table 2 and Figure 5) were planted at two locations in 2005 under USDA notif ication 05-062-02n. The trial with Hybrid 1 was planted in Bloomington, Illinois, and the trial with Hybrid 2 was planted in York, Nebraska. The trials were managed followi ng to local agronomic practices for the 2005 growing season.

Ten plants of each MIR162 hybrid, plus two pl ants from each of the corresponding control hybrids, were harvested at each of four developmental stages (see Table D-1). Five each of the MIR162 hybrids, plus one plant from each of the corresponding control hybrids, were kept as whole-plant samples for analysis. Th e remaining plants from each sampling interval were separated into distinct plant parts for analysis (see Table D-1).

Table D-1. Growth stages selected for collection of whole plants and specific plant tissues.

Sampling Interval

(Growth Stage) Tissues Sampled

V9 – V12 (~ 8 weeks after planting) leaf, root, whole plant

Anthesis leaf, root, silk, pith, pollen, whole plant

Seed maturity (black layer) leaf, root, kernels, pith, whole plant

Senescence (~ 23 weeks after planting) leaf, root, kernels, whole plant

At each sampling interval, whole plants (inc luding roots) were harvested and shipped overnight on ice packs to Syngenta Biotechno logy, Inc. Upon receipt, five plants were separated into parts consisting of all the leaves, roots, pith, silk, or ke rnels. After weighing, all samples were stored at –80 ± 10oC until the tissue was processed further.

Pollen Collection

At anthesis, pollen was collected and pooled from at least 10 plants of the same genotype, sieved at the collection facility to remove nonpollen debris ( e.g., anthers and aphids), airdried overnight, and stored frozen at –80 ± 10oC . Prior to analysis, the pollen was sieved again through a 250 μm mesh screen.MIR162-USDA-1 Page 154 of 268 Plant Tissue Processing

Whole plants and individual parts (except po llen) were ground to a fine powder in the presence of dry ice. Each ground sample wa s thoroughly mixed to ensure homogeneity. The powdered samples were weighe d, lypophilized, and weighed agai n. The percent dry weight for each sample was computed based on the difference in pre- and post-lyophilization weights. Equal amounts of lyophilized transg enic samples were pooled to create one composite sample for each tissue type from each hybrid at each sampling stage. Lyophilized samples were stored at –80 ± 10oC.

Tissue Extraction

Extracts of the lyophilized ti ssue samples (except pollen) we re prepared for Vip3Aa20 and PMI analysis. At least three replicate subsampl es of each composite transgenic tissue sample were analyzed to determine the variability of extraction. For each subsample analyzed, an aliquot of approximately 0.1 g of the powdered lyophilized material was transferred into a 15-ml polypropylene tube, suspended in 3 ml extraction buffer (500 mM Tris, 20 mM EDTA, 1 M NaCl, 1 mM AEBSF, 2 mM DTT, 1 μM leupepti n, pH 9.5), and homogenized using an Autogizer homogenizer (6 cycles, settin g 4). An additional 3 ml of extraction buffer was added, and samples were vortexed briefl y and incubated on wet ice for 30 minutes. After centrifugation for 15 minutes at 10,000 x g at 4oC, the supernatants were used for Vip3Aa20 and PMI quantification by ELISA.

Pollen extracts were prepared by suspending ai r-dried pollen 1:30 (w/v) in extraction buffer and incubating the samples on ice for 30 minutes . The pollen suspensions were disrupted by three passages through a French pressure cell at 15,000 psi. An additional 3 ml of buffer was added to each sample and the samples were vo rtexed briefly. After centrifugation for 15 minutes at 10,000 x g at 4oC, the supernatants we re used for Vip3Aa20 and PMI quantification by ELISA.

Vip3Aa20 Quantification

Tissue sample extracts were quantitativel y analyzed for Vip3Aa20 by ELISA using immunoaffinity-purified goat anti-Vip3Aa a nd Protein A-purified rabbit anti-Vip3Aa polyclonal antibodies. Microbially produced Vip3Aa20 was used to produce the standard curves for all measurements except extract ion efficiency where microbially produced Vip3Aa19 was used.

PMI Quantification

Tissue sample extracts were quantitat ively analyzed for PMI by ELISA using immunoaffinity-purified goat anti-PMI and Prot ein A-purified rabb it anti-PMI polyclonal antibodies. Microbially produ ced PMI was used to produce the standard curves for all

measurements.MIR162-USDA-1 Page 155 of 268 Limit of Quantification and Li mit of Detection Determination

The limit of quantification (LOQ) of the ELISAs was estimated based on the lowest concentration of pure protein of interest (P OI) lying on the linear portion of the standard curve and the dilution factor (D F; based on optical de nsity values from dilutions of negative control tissue extracts). LOQ values are shown in Table D-2 and were calculated as follows:

LOQ (μg POI/g dry-weight) = lo west concentration on the li near portion of standard curve (ng POI/ml) × DF × volume of buffer used in extraction (ml) ÷ g of tissue
extracted ÷ 1000

Table D-2. LOQs for Vip3Aa20 and PMI in MIR162 tissues. Values have not been corrected for extraction efficiency.

Limits of Quantification ( μ g/g dw)

Tissue V9 - V12 Anthesis Seed Maturity Senescence

Vip3Aa20

Leaf 0.22 0.27 0.33 0.24

Root 0.22 0.27 0.23 0.24

Pith N/A a 0.24 0.21 N/A

Silk N/A 0.24 N/A N/A

Kernels N/A N/A 0.24 0.30

Pollen N/A 0.35 N/A N/A

Whole Plants 0.22 0.24 0.24 0.21

PMI

Leaves 0.22 0.29 0.22 0.26

Roots 0.21 0.21 0.21 0.23

Pith N/A a 0.24 0.21 N/A

Silk N/A 0.31 N/A N/A

Kernels N/A N/A 0.21 0.26

Pollen N/A 0.35 N/A N/A

Whole Plants 0.41 0.40 0.26 0.27

a - N/A = not analyzed at this stageMIR162-USDA-1 Page 156 of 268 The limit of detection (LOD) of the ELISAs was estimated based on the mean optical density (OD) + 2 standard deviations of the lowest co ncentration of pure POI used in the standard curve and the dilution factor (DF; based on OD values from dilutions of negative control tissue extracts). LOD values are shown in Table D-3 and were calculated as follows:

LOD (μg POI/g dw) = mean OD + 2 S.D. of lowest concentration used in the standard curve (expressed as ng POI/ml) × DF × volume of buffer used in extraction (ml) ÷ g of tissue extracted ÷ 1000

Table D-3. LODs for Vip3Aa20 and PMI in MIR162 tissues. Values have not been corrected for extraction efficiency.

Limits of Detection ( μ g/g dw)

Tissue V9 - V12 Anthesis Seed Maturity Senescence

Vip3Aa20

Leaves 0.030 0.030 0.030 0.039

Roots 0.030 0.030 0.029 0.045

Pith N/A a 0.030 0.030 N/A

Silk N/A 0.030 N/A N/A

Kernels N/A N/A 0.030 0.030

Pollen N/A 0.035 N/A N/A

Whole Plants 0.030 0.030 0.030 0.030

PMI

Leaves 0.066 0.049 0.064 0.046

Roots 0.041 0.041 0.041 0.032

Pith N/A 0.042 0.048 N/A

Silk N/A 0.057 N/A N/A

Kernels N/A N/A 0.063 0.056

Pollen N/A 0.112 N/A N/A

Whole Plants 0.070 0.069 0.090 0.109

a - N/A = not analyzed at this stageMIR162-USDA-1 Page 157 of 268 Extraction Efficiency for Vip3Aa20 and PMI

Extraction efficiency measurements were pe rformed to estimate the relative amount of Vip3Aa20 and PMI extracted during routine pro cedures, compared to that which remained associated with the post-extraction solids. Tissu es were extracted in triplicate as described
above. The insoluble material was then co llected and re-extract ed twice more, while retaining the supernatant for analysis each time. If an extract was either <LOQ or <LOD, the LOQ or LOD value for the sample, expresse d as ng POI/ml, was used to estimate the extraction efficiency for the sample. Percent ex traction efficiency was calculated as follows:

[ng POI/ml 1 st Extraction ÷ (ng POI/ml 1 st extraction + 2 nd extraction + 3 rd extraction)] x 100 = % extraction efficiency

ELISA values were corrected for extraction efficiency as follows:

μg POI/gdw (uncorrected) ÷ % extraction efficiency = μg POI/gdw (corrected)

μg POI/gdw (corrected) × % dry we ight = μg POI/gfw (corrected)

Statistical Methods

The mean and standard deviation of replicate samples were calculated using Microsoft Excel.MIR162-USDA-1 Page 158 of 268 Appendix E. Compositional Analysis of MIR162 Forage and Grain

Study Design

Forage and grain for compositional analysis we re harvested from multiple locations planted
in U.S. in 2005. The locations chosen were representative of major maize producing regions in the country. Trials were planted w ith a MIR162 hybrid and genetically matched nontransgenic hybrid in a randomized complete block design, at multiple locations, with three replicated plots per lo cation, and were managed followi ng local agronomic practices. See Table 2 and Figure 5 for a description of the hybrids used in this study. The plants were self-pollinated by hand and th e developing ears were bagged to avoid cross-pollination. Eight locations were planted for this study to insure that grain and forage from six locations
could be harvested in the event of loss due to adverse environmental conditions (early freeze, drought, etc. ). Six locations that produced sufficient grain and forage were selected for this study (see Table E-1).

Table E-1. Locations selected for planting and harvest of forage and grain.

ID Location

Location 1 Stanton, Minnesota

Location 2 Janesville, Wisconsin

Location 3 Alleman, Iowa

Location 5 Seward, Nebraska

Location 6 Bloomington, Illinois

Location 8 Bondville, Illinois

Forage Sampling and Processing

The entire above-ground portion from five pl ants of each hybrid was harvested at dough stage (R4), the stage at which silage would typically be prep ared, from each of the three replicated plots at each location. For each hybr id, plants were pooled to create a composite sample for each plot, then ground with a chipper-shredder. A subsample from each composite sample was shipped overnight on ice packs to Syngenta Crop Protection, Inc. (Greensboro, NC USA). Samples were stored frozen at -20±10  ̊ C, then finely ground and shipped on dry ice to Covance La boratories, Inc. (Madison, WI USA) where they were stored
frozen at -20±10  ̊ C until they were analyzed.

Grain Sampling and Processing

Ears were harvested after physiological maturity (R6) at 18-24% moisture content, and then mechanically dried to approximately 10-13% moisture content. Each grain sample represented grain shelled from ear s collected from 15 plants of each hybrid grown in each ofMIR162-USDA-1 Page 159 of 268 the three replicated plots, at each location. A subsample of approximately 500 g of grain from each replicate plot was shipped at ambi ent temperature to Syngenta Crop Protection,
Inc. where it was stored frozen at -20±10  ̊ C, then finely ground and shipped on dry ice to Covance Laboratories, Inc. Sample s were stored frozen at -20±10  ̊ C until they were analyzed.

Compositional Analysis

Selection of analytes for measurement in fora ge and grain was based on recommendations of the OECD for comparative assessment of composition of new varieties of maize. Forage was analyzed for proximates and the minerals ca lcium and phosphorus (Table 25). Grain was analyzed for major constituents (proxima tes, including starc h, ADF, NDF and TDF), minerals, amino acids, fatty acids, vitamins , selected anti-nutri ents, and secondary metabolites (Table 25).

All compositional analyses were conducted by Covance Laborat ories, Inc. using methods published and approved by AOAC In ternational, or other indus try-standard analytical methods (described below). Based on the mois ture content of each sample, analyte levels were converted to equivale nt units of dry weight.

Statistical Analysis

An F-test was also used to assess the significan ce of the location by genotype interaction. An F-test probability of <0.05 sugge sts that the effect of the genot ype was not consistent across locations and that the compar ison of genotypes averaged across locations may not be valid.

The analyte composition tables for forage and grain (Tables E-3 through E-11) include the overall averages of each analyte across locat ions in both the MIR162 and the nontransgenic hybrids. Also included are the F-test probabili ties for both the genotype comparisons and the location by genotype interactions. F-test proba bilities that were statistically significant ( p<0.05 ) are indicated in bold type. For analytes showing statistically significant location by genotype interactions, the means for both the MIR162 and the nontransgenic hybrid at each location are provided in a se parate table (Table E-8).

Moisture levels in grain were not subjected to analysis of variance sinc e the moisture analysis was performed on grain that had been mechanica lly dried, thus altering the original moisture content of the harvested grain. Mechanical drying after harvest is a standard agronomic practice for improving storage characteristics of maize grain.

Analytical Methods and Reference St andards for Compositional Analysis

2-Furaldehyde (Furfural)

Grain samples were extracted with 4% trichloroa cetic acid in MilliQ water, centrifuged and filtered. The level of furfural in the extract was determined by reverse phase high performance liquid chromatography with ultra-vi olet quantitation. The quantitation limit forMIR162-USDA-1 Page 160 of 268 this study was calculated to be 0.500 ppm.

Reference Standard: ACROS 2-Furaldehyde, 99%, Lot Number A018806701

Reference: Albala-Hurtado S., M. T. Veciana-Nogues, M. Izquierdo-Pulido, and M. C. Vidal-Carou (1997). Determination of free and total furfur al compounds in infant milk formulas by highperformance liquid chromatography. Journal of Agricultural and Food Chemistry 45 :2128-
2133.

Acid Detergent Fiber

The sample was placed in a fritted vessel an d washed with an acidic boiling detergent solution that dissolved the protein, carbohydrate, and ash. An acetone wash removed the fats and pigments. Lignocellulose fraction wa s collected on the frit and determined gravimetrically. The limit of qua ntitation for this study was 0.1%.

Reference: USDA (1970). Forage fiber analyses. In Agriculture Handbook No. 379 , United States Department of Agriculture, Washington, DC.

Amino Acids

Total aspartic acid (including asparagine) Total threonine Total serine Total glut amic acid (including glutamine) Total praline Total glycine
Total alanine Total valine
Total isoleucine Total leucine Total tyrosine Total phenylalanine Total histidine Total lysine Total arginine Total tryptophan Sulfur-containing amino acids: Total methi onine, Total cystine (including cysteine)

The sample was assayed by three methods to ob tain the full profile. Tryptophan required a base hydrolysis with sodium hydroxide. The sulfur containing amino acids required an oxidation with performic acid prior to hydrolysis with hydrochloric acid. Analysis of the samples for the remaining amino acids was accomplished through direct acid hydrolysis with hydrochloric acid. Once hydroly zed, the individual amino acids were then quantitated using an automated amino acid analyzer. The lim it of quantitation for each amino acid was 0.100 mg/g.

Reference Standards: Beckman K18, 2.5 μ mol/mL per constituent (except cystine 1.25 μ mol/mL),
Lot Number S504255 Sigma L-Tryptophan, >99% (used as 100%), Lot Number 063K0382MIR162-USDA-1 Page 161 of 268 Fluka L-Cysteic Acid Monohydrate, 99.9% (used as 100%), Lot Number 1157629 Sigma L-Methionine Sulfone, >99% (used as 100%), Lot Number 012H3349

Reference: AOAC International (2005). Method 982.30. In Official Methods of Analysis of AOAC International, 18 th Edition. AOAC International, Gaithersburg, Maryland.

Ash

The sample was placed in an electric furnace at 550 ° C and ignited to drive off all volatile organic matter. The nonvolat ile matter remaining was quant itated gravimetrically and calculated to determine percent ash. The lim it of quantitation for all samples in this study
was 0.1%.

Reference: AOAC International (2005). Method 923.03. In Official Methods of Analysis of AOAC International, 18 th Edition. AOAC International, Gaithersburg, Maryland.

Beta Carotene (Vitamin A)

The sample was extracted using alcohol and hexa ne. The extracts were injected on a reverse phase HPLC system and compared to a standard curve. The limit of quantitation for Beta Carotene in this study was approximately 0.0333 RE/g.

Reference Standard: Sigma, Beta Carotene, 102%, Lot Number 104K2514

References: AOAC International (2005). Method 941.15. In Official Methods of Analysis of AOAC International, 18 th Edition. AOAC International, Ga ithersburg, Maryland.

Carbohydrates

The total carbohydrate leve l was calculated by difference usin g the fresh weight-derived data and the following equation:

% carbohydrates = 100 % - (% protein + % fat + % moisture + % ash)

The limit of quantitation for this study was 0.1%.

Reference: USDA (1973). Energy value of foods. Pp 2-11. In Agriculture Handbook No. 74 . United States Department of Ag riculture, Washington, DC.MIR162-USDA-1 Page 162 of 268 Total Fat by Acid Hydrolysis

The sample was hydrolyzed with hy drochloric acid at an elevat ed temperature. The fat was extracted using ether and hexane . The extract was washed with a dilute alkali solution and filtered through a sodium sulfate column. Th e extract was then evaporated, dried, and weighed. The limit of quantitation for all samples in this study was 0.1%.

Reference: AOAC International (2005). Methods 922.06 and 954.02. In Official Methods of Analysis of AOAC International, 18 th Edition. AOAC International, Gaithersburg, Maryland.

Fatty Acids

The lipid was extracted and saponified with 0.5 N sodium hydroxide in methanol. The saponification mixture was methylated with 14% boron trifluoride:meth anol. The resulting methyl esters were extracted wi th heptane containing an internal standard. The methyl esters of the fatty acids were analyzed by gas ch romatography using external standards for quantitation. The limit of quantitation wa s for each individual fatty acid was 0.004%.

Reference Standards: Nu Chek Prep GLC Reference Standard Hazleton No. 1, used as 100%,
Lot Number AU22-P Nu Chek Prep GLC Reference Standard Hazleton No. 2, used as 100%,
Lot Number M13-0 Nu Chek Prep GLC Reference Standard Hazleton No. 3, used as 100%,
Lot Number MA13-0 Nu Chek Prep GLC Reference Standard Hazleton No. 4, used as 100%,
Lot Number JA13-P Nu Chek Prep Methyl Gamma Linolenate, used as 100%,
Lot Number U-63M-J1-P Sigma Methyl Tridecanoate, us ed as 100%, Lot Number 035K1392

Reference: AOCS (1997). Me thod Ce 1-62. In Official Methods and Recommended Practices of the AOCS, 5th Edition. American Oil Chemists’ Society, Champaign, Illinois.

Folic acid

The sample was hydrolyzed in a potassium phos phate buffer with the addition of ascorbic acid to protect the folic acid during autoclavin g. Following hydrolysis by autoclaving, the sample was treated with a chicken-pancreas enzyme and incubated approximately 18 hours to
liberate the bound folic acid. Th e amount of folic acid was de termined by comparing the growth response of the sample, using the bacteria Lactobacillus casei , with the growth response of a folic acid standard. This res ponse was measured turbidimetrically. The limit of quantitation for this study was 0.00600 mg/100g.MIR162-USDA-1 Page 163 of 268 Reference Standard: USP Folic acid, 98%, Lot Number P

References: AOAC International (2005). Methods 960.46 and 992.05. In Official Methods of Analysis of AOAC International, 18 th Edition. AOAC International, Gaithersburg, Maryland.

Infant Formula Council (1973). Section C-2. In Methods of Analysis for Infant Formulas. Infant Formula Council, Atlanta, Georgia.

Minerals by ICP Emission Spectrometry

Calcium Copper Iron Magnesium Manganese Phosphorus
Potassium Sodium Zinc

The sample was dried, precharred, and ashe d overnight in a muffle set to maintain 500 ° C. The ashed sample was treated with hydrochloric acid, taken to dryness, and put into a solution of 5% hydrochloric acid. The am ount of each element was determined at appropriate wavelengths by comparing the emission of the unknown sample, measured by the inductively coupled plasma, with the emission of the standa rd solutions. The reference standards and limits of quantification fo r each mineral are listed in Table E-2.

References: Dahlquist, R. L. and J. W. Knoll (1978). Inductively Coupled Plas ma-Atomic Emission spectrometry: Analysis of biological material s and soils for major, trace, and ultra trace
elements. Applied Spectroscopy 32 :1-29.

AOAC International (2005). Methods 984.27 and 985.01. In Official Methods of Analysis of AOAC International, 18 th Edition. AOAC International, Gaithersburg, Maryland.MIR162-USDA-1 Page 164 of 268 Table E-2. Inorganic Ventures/IV Labs reference standards and limits of quantification.

Mineral Lot Numbers Concentration (ppm) Limit of Quantification (ppm)*

Calcium Y-MEB198136, Y-MEB198138 200, 1000 20.0

Copper Y-MEB198136, Y-MEB198137 2, 10 0.50

Iron Y-MEB198136, Y-MEB198139 10, 50 2.00

Magnesium Y-MEB198136, Y-MEB198137 50, 250 20.0

Manganese Y-MEB198136, Y-MEB198137 2, 10 0.30

Phosphorus Y-MEB198136, Y-MEB198138 200, 1000 20.0

Potassium Y-MEB198136, Y-MEB198138 200, 1000 100

Sodium Y-MEB198136, Y-MEB198138 200, 1000 100

Zinc Y-MEB198136, Y-MEB198137 10, 50 0.40

* Calculated on a fresh weight basis.

Moisture

The sample was dried in a vacuum oven at approximately 100 ° C to a constant weight. The moisture weight loss was determined and co nverted to percent moisture. The limit of quantitation for this study was 0.1%.

Reference: AOAC International (2005). Methods 926.08 and 925.09. In Official Methods of Analysis of AOAC International, 18 th Edition. AOAC International, Gaithersburg, Maryland.

Neutral Detergent Fiber, Enzyme Method

The sample was placed in a fritted vessel a nd washed with a neut ral boiling detergent solution that dissolved the protein, carbohydrate, enzyme, and ash. An acetone wash removed the fats and pigments. Hemicellulose, cellulose, and lignin fractions were collected on the frit and determined gravimetrically. Th e limit of quantitation for this study was 0.1%.

References: AACC (1998). Method 32.20. In Approved Methods of the American Association of Cereal Chemists, 9th Edition

.MIR162-USDA-1 Page 165 of 268 USDA (1970). Forage fiber analyses. In Agriculture Handbook No.379. United States Department of Agriculture, Washington, DC.

p -Coumaric Acid and Ferulic Acid

The sample was extracted with methanol using ultrasonicatio n, hydrolyzed using 4 N sodium hydroxide, buffered using acetic acid/sodium hydroxide, acidified with 3 N hydrochloric acid, and filtered. The levels of p-coumaric and ferulic acids in the extract were determined by reverse phase high performance liquid chromat ography with ultra-violet quantitation. The limit of quantitation for the p-coumaric acid a nd ferulic acid assays were approximately 50.0

ppm.

Reference Standards: Acros, 4-Hydroxy-3-methoxycinnamic Acid (Ferulic Acid), 99.9%, Lot Number

A014010401 Acros, p-Hydroxycinnamic Acid (p-Cou maric Acid), 97.9%, Lot Number A018661301

Reference: Hagerman, A. E. and R. L. Nicholson (1982) . High-performance liquid chromatographic determination of hydroxycinnamic acids in maize mesocotyl. Journal of Agricultural and Food Chemistry 30 (6):1098-1102.

Phytic Acid

The sample was extracted using 0.5M HCl with ultrasonication. Purification and
concentration was done on a sili ca based anion exchange (SAX) column. Sample analysis was done on a macroporous polym er HPLC column PRP-1, 5 μ m (150 x 4.1mm) and a refractive index detector. The limit of quan titation for this study was approximately 0.100%.

Reference Standard: Aldrich, Phytic Acid, Dodecasodium Salt Hydrate, 95%, Lot Number 1913EC

References: Lehrfeld, J. (1989). High-performance liquid chromatography analysis of phytic acid on a ph-stable, macroporous polymer column. Cereal Chemistry 66 (6):510-515.

Lehrfeld, J. (1994). HPLC separation and qua ntitation of phytic acid and some inositol phosphates in foods: probl em and solutions. Journal of Agricultural Food Chemistry

42 :2726-2731.

Protein

Nitrogenous compounds in the sample were reduced in the presence of boiling sulfuric acid and a mercury catalyst mixture to form ammonia. The acid digest was made alkaline. The
ammonia was distilled and then titrated with a standard acid. The percent nitrogen wasMIR162-USDA-1 Page 166 of 268 calculated and converted to pr otein using the factor 6.25. Th e limit of quantitation for this study was 0.1%.

References: AOAC International (2005). Methods 955.04 and 979.09. In Official Methods of Analysis of AOAC International, 18 th Edition. AOAC International, Gaithersburg, Maryland.

Bradstreet, R. B. (1965). The Kjeldahl Method for Organic Nitrogen. Academic Press, New York, New York.

Kalthoff, I. M., and E. B. Sandell (1948). Quantitative Inorganic Analysis. MacMillan, New York, New York.

Raffinose

The sample was extracted with deionized water and the extract treated with a hydroxylamine hydrochloride solution in pyr idine, containing phenyl- β -D-glucoside as an internal standard. The resulting oximes were converted to silyl derivatives by treatment with hexamethyldisilazane and trifluoracetic acid and analyzed by gas chromatography using a flame ionization detector. The limit of quantit ation (LOQ): The acceptable range for an 8/5
dilution was 0.100-1.800%.

Reference Standard: Sigma, D(+)-Raffinose Pentahyr ate, 99%, Lot Number 073K0938

References: Mason, B. S., and H. T. Slover (1971). A gas chromatographic method for the determination of sugars in foods. Journal of Agricultural and Food Chemistry 19 (3):551-554.

Brobst, K. M. (1972). Gas-liquid chroma tography of trimethylsilyl derivatives. In Methods in Carbohydrate Chemistry, Volume 6. Academic Press, New York, New York.

Selenium by ICP-Mass Spectrometry

An appropriately sized sample wa s wet-ashed with nitric acid (HNO 3 ) using open or closed vessel microwave digestion. Samples that were readily soluble in water were diluted with
HNO 3 and analyzed straight. The amount of each element was determined using mass spectrometry by comparing the counts genera ted by the unknowns to those generated by
standard solutions of known c oncentration. The lim it of quantitation fo r this study was 50 ppb.

Reference Standard: SPEX, Selenium, 100 mg/L, Lot Number 8-175VY

References: EPA Method 200.8, Determination of Trace Elements in Waters and Wastes by InductivelyMIR162-USDA-1 Page 167 of 268 Coupled Plasma-Mass Spectrometry

Cabrera et al. (1994). Determination of levels of lead contamination in food and feed crops. Journal of AOAC International 77 (5):1249-1252.

Methods for Chemical Anal ysis of Water and Wastes , Metals 1-19 and Method 239

Starch

The sample was extracted with alcohol to remove carbohydrates other than starch i.e. sugars. Then it was hydrolyzed into glucose with alpha -amylase and amyloglucosidase. Glucose was oxidized with glucose oxidase to form peroxide, which reacted with a dye in the presence of peroxidase to give a stable colored produc t proportional to glucos e concentration. The glucose concentration was quantita ted by a spectrophotometer at 540 nm. Percent starch was then calculated from the glucose concentration. The limit of quantification for this study was 0.05%.

Calculations:

% starch = glucose concentration from curve ( μ g/mL) x dilution factor x initial volume (mL) x 0.9 sample weight (g) x 10,000

Reference Standard: Sigma, D(+)-Glucose, 99.9% Lot Number 123K0095

Reference: AOAC International (2005). Method 996.11. In Official Methods of Analysis of AOAC International, 18 th Edition. AOAC International, Gaithersburg, Maryland.

Tocopherols, Total

The product was saponified to break down any fat and release any vitamin E. The saponified mixture was dissolved directly in hexane and then quantified directly by high-performance liquid chromatography on a silica column. The limit of quantitation for this study was approximately 0.500 mg/100g.

Reference Standards: USP, Alpha Tocopherol, 100%, Lot Number M Matreya, Beta Tocopherol, 50 mg/mL, Lot Number 22077 Sigma, Delta Tocopherol, 98%, Lot Number 072K1326 Sigma, Gamma Tocopherol, 99%, Lot Number 095K1062

Reference: Cort, W. M., T. S. Vincente, E. H. Waysek, and B. D. Williams (1983). Vitamin E content of feedstuffs determined by high-performa nce liquid chromatographic fluorescence. Journal of Agricultural Food Chemistry 31 :1330-1333.MIR162-USDA-1 Page 168 of 268 Speek, A. J., J. Schijver, and W. H. P. Schreu rs (1985). Vitamin E composition of some seed oils as determined by high-performance liquid chromatography with fluorometric quantitation. Journal of Food Science 50 (1):121-124.

McMurray, C. H., W. J. Blanchflower, and D. A. Rice (1980). Influence of extraction techniques on determination of alpha-t ocopherol in animal feedstuffs. Journal of the Association of Official Analytical Chemists 63 (6):1258-1261.

Total Dietary Fiber

Duplicate samples were gelatini zed with alpha-amylase and dige sted with enzymes to break down starch and protein. Ethanol was added to each sample to precipitate the soluble fiber. The samples were filtered, and the residue wa s rinsed with ethanol and acetone to remove starch and protein degradation products and moisture. Protein content was determined for one of the duplicates; ash content was determined for the other. The total dietary fiber in the sample was calculated using the protein and ash values. The li mit of quantitation for this study was 1.0%.

Reference: AOAC International (2005). Method 985.29. In Official Methods of Analysis of AOAC International, 18 th Edition. AOAC International, Gaithersburg, Maryland.

Trypsin Inhibitor

The sample was ground and/or defatted with pe troleum ether, if necessary. A sample of matrix was extracted for 3 hours with 0.01 N sodium hydroxide. Varying aliquots of the sample suspension were exposed to a known amount of trypsin and benzoy1-DL-arginine-pnitroanalide hydrochloride. The sample was al lowed to react for 10 minutes at 37°C. After 10 minutes, the reaction was halted by the additio n of acetic acid. The solution was filtered or centrifuged and the absorbance was determined at 410 nm. Trypsin inhibitor activity (TIU) was determined by photometrically measuring the inhibition of trypsin’s reaction with benzoyl-DL-arginine- p -nitroanalide hydrochloride. The lim it of quantificati on for this study was 1.00 Trypsin Inhibitor Unit/mg.

Reference: AOCS (1997). Method Ba 12-75. In Official Methods and Recommended Practices of the American Oil Chemists’ Society, 5th Edition. American Oil Chemists’ Society, Champaign, Illinois.

Vitamin B 1 (Thiamine)

The sample was autoclaved under weak acid conditions to extract the thiamine. The resulting solution was incubated with a bu ffered enzyme solution to release any bound thiamine. The solution was purified on an ion- exchange column. An aliquot was taken and reacted with potassium ferricyan ide to convert thiami n to thiochrome. The thiochrome wasMIR162-USDA-1 Page 169 of 268 extracted into isobutyl alcohol and read on a fluorometer ag ainst a known standard. The limit of quantification for this study was 0.01 mg/100g.

Reference Standard: USP, Thiamine, 97.22% after correction for degree of hydration, Lot Number O

Reference: AOAC International (2005). Methods 942.23, 953.17, and 957.17. In Official Methods of Analysis of AOAC International, 18 th Edition. AOAC International, Gaithersburg, Maryland.

Vitamin B 2 (Riboflavin)

The sample was hydrolyzed with dilute hydrochlor ic acid (HCl) and the pH was adjusted to
remove interferences. The am ount of riboflavin was determin ed by comparing the growth response of the sample, using the bacteria Lactobacillus casei , with the grow th response of multi-point riboflavin standard curve. This gr owth response was measured turbidimetrically. The limit of quantification fo r this study was 0.0200 mg/100g.

Reference Standard: USP Riboflavin, 100%, Lot Number N0C021

Reference: AOAC International (2005). Method 940.33. In Official Methods of Analysis of AOAC International, 18 th Edition. AOAC International, Gaithersburg, Maryland.

Vitamin B 3 (Niacin)

The sample was hydrolyzed with sulfuric ac id and the pH was adjusted to remove
interferences. The amount of niacin was de termined by comparing the growth response of the sample, using the bacteria Lactobacillus plantarum, with the growth response of a niacin standard. This response was measured turbidim etrically. The limit of quantification for this study was 0.03 mg/100g.

Reference Standard: USP, Niacin, 100%, Lot Number H2C121

Reference: AOAC International (2005). Method 944.13. In Official Methods of Analysis of AOAC International, 18 th Edition . AOAC International, Gaithersburg, Maryland.

Vitamin B 6 (Pyridoxine)

The sample was hydrolyzed with dilute sulf uric acid in the autoclave and the pH was adjusted to remove interf erences. The amount of B 6 was determined by comparing the growth response of the sample, using the yeast Saccharromyces carlsbergenesis, with theMIR162-USDA-1 Page 170 of 268 growth response of a B 6 standard. This response was meas ured turbidimetrically. The limit of quantification for th is study was 0.00700 mg/100g.

Reference Standard: USP Pyridoxine, 100%, Lot Number P

Reference: AOAC International (2005). Method 961.15. In Official Methods of Analysis of AOAC International, 18 th Edition. AOAC International, Gaithersburg, Maryland.

Results

Results of the forage analyses are presented in Tables E-3 and E-4. Results of grain analyses are presented in Tables E-5 th rough E-11. Results are presen ted as the mean values of analyte results across the six locations. The range of individual plot values for both the MIR162 and control genotypes are shown, along w ith the computed difference between the mean values (expressed as a percentage of the control). The results of the across location statistical comparison and the test for potential genotype by location inte raction are presented as their respective p -values. Significance was assigned at the standard level of 5%.

The results of the statistical analysis for genotype by location inte raction revealed two analytes for which there was a stat istically significant interaction ( p <0.05), vitamin A ( β

carotene) and vitamin B 9 (folic acid). For these two analytes, individual location mean values are presented in Table E-8.MIR162-USDA-1 Page 171 of 268Table E-3.Proximate composition of maize foragefrom a MIR162 hybrid and a control hybrid. For comparison, ILSI Crop Compositiondatabase and OECD literature values are also shown. All units as described in column headings except F-test values.TreatmentMoisture(% fw)Protein(% dw)Total Fat(% dw)Ash(% dw)Carbohydrates(% dw)ADF(% dw)NDF(% dw)X ̄71.2 7.2 1.5 4.1 87.3 28.2 43.2MIR162range 66.2-77.2 3.1-10.1 0.8-1.9 3.1-5.8 82.9-90.7 23.6-34.2 35.1-56.1X ̄70.5 7.3 1.6 4.0 87.1 28.8 38.8Controlrange 64.1-75.8 3.4-8.9 0.4-2.3 2.9-5.7 83.6-91.7 23.3-34.8 32.1-46.9% differencea0.99 -1.37 -6.25 2.50 0.23 -2.10 11.34p-value for genotypeb0.184 0.711 0.421 0.796 0.694 0.6350.010p-value for locationx genotype interaction0.730 0.645 0.100 0.948 0.973 0.687 0.286X ̄70.2 7.78 2.039 4.628 85.6 27.00 41.51range 49.1 - 81.3 3.14 - 11.57 0.296 - 4.570 1.527 - 9.638 76.4 - 92.1 16.13 - 47.39 20.29 - 63.71ILSI (2006)N 945 945 921 945 945 945 945OECD (2002) range 62 - 78 4.7 - 9.2 1.5 - 3.2 2.9 - 5.7 25.6 - 34 40 - 48.2a– [ (MIR162 - control)/control] x 100b- statistically significant F-test (p<0.05) indicated in bold type.MIR162-USDA-1 Page 172 of 268Table E-4.Calcium and phosphorus composition ofmaize forage from a MIR162 hybrid and acontrol hybrid. For comparison, ILSICrop Composition database and OECD values are also shown. All units as described in column headings, except F-test values andwherenoted.TreatmentCalcium(mg/kg dw)Phosphorus(mg/kg dw)X ̄2106 1997MIR162range 1720-2930 1270-2240X ̄2039 2079Controlrange 1440-2620 1760-2560% differencea3.29 -3.94p-value for genotype 0.484 0.153p-value for locationby genotype interaction0.944 0.066X ̄2028.6 2066.1range 713.9 - 5767.9 936.2 - 3704.1ILSI (2006)N 481 481OECD (2002)brange 0.15 - 0.31% dw 0.20 - 0.27% dwa– [(MIR162 - control)/control] x 100b- for conversion, 1% = 10,000 mg/kg.MIR162-USDA-1 Page 173 of 268Table E-5.Proximate composition of maize grain from a MIR162 hybridand a control hybrid. For comparison, ILSI Crop Compositiondatabase and OECD literature values are also shown. All units as described in column headings except F-test values.TreatmentMoisturea(% fw)Protein(% dw)Fat(% dw)Ash(% dw)Carbohydrates(% dw)ADF(% dw)NDF(% dw)TDF(% dw)Starch(% dw)X ̄10.3 9.8 3.79 1.4 85.0 5.0 11.7 16.8 63.1MIR162range 9.5-11.5 7.5-11.2 3.3-4.6 1.1-1.6 83.2-87.1 3.3-7.0 10.1-13.0 14.1-19.4 54.8-68.1X ̄10.5 9.6 3.78 1.3 85.3 4.6 11.1 16.3 64.9Controlrange 9.4-12.0 7.1-11.0 3.0-4.4 1.1-1.5 83.3-88.1 3.3-6.2 9.5-12.8 14.3-17.8 60.6-69.2% differenceb-1.91 2.08 0.26 7.69 -0.36 8.70 5.41 3.18 -2.77p-value for genotypec0.103 0.9040.0120.062 0.1540.0160.0920.015p-value for locationx genotype interaction0.832 0.538 0.381 0.696 0.309 0.703 0.653 0.455X ̄11.3 10.30 3.555 1.439 84.6 4.05 11.23 16.43 57.7range 6.1 - 40.56.15 -17.261.742 -5.8230.616 -6.28277.4 - 89.51.82 -11.345.59 -22.648.85 -35.3126.5 - 73.8ILSI (2006)N 1434 1434 1174 1410 1410 1350 1349 397 168OECD (2002) range 7.0 - 23 6 - 12.7 3.1 - 5.8 1.1 - 3.9 82.2 - 82.9 3.0 - 4.3 8.3 - 11.9 11.1a- moisture levels in grain not subject to analysisof variance as grain was mechanically dried after harvestb– [(MIR162 - control)/control) x 100c- statistically significant F-test (p<0.05) indicated in bold typeMIR162-USDA-1 Page 174 of 268Table E-6.Mineral composition of maize grain from a MIR162 hybrid and a control hybrid. For comparison, ILSI Crop Compositiondatabase and OECD values are also shown. All units as described in column headings, exceptF-test values and where noted.TreatmentCalcium(mg/kgdw)Copper(mg/kgdw)Iron(mg/kgdw)Magnesium(mg/kg dw)Manganese(mg/kg dw)Phosphorus(mg/kg dw)Potassium(mg/kgdw)Seleniuma,b(mg/kg dw)Sodiuma,b(mg/kgdw)Zinc(mg/kgdw)X ̄38.1 1.3 20.2 1252 6.3 3173 3352 N/A N/A 21.7MIR162range 29.4-47.2 0.96-1.95 17.3-22.9 1090-1480 4.14-7.97 2810-3550 3160-3710<LOQ -0.414<LOQ -<LOQ18.8-24.3X ̄35.3 1.2 19.2 1218 6.1 3073 3357 N/A N/A 21.5Controlrange 25.7-44.0 1.00-1.58 15.7-22.5 960-1470 4.59-8.01 2710-3400 2950-3660<LOQ -0.531<LOQ -<LOQ19.2-23.8% differencec7.93 8.33 5.21 2.70 3.28 3.25 -0.15 N/A N/A 0.93p-value for genotyped0.0020.0730.0110.102 0.0780.0300.916 0.606p-value for locationx genotype interaction0.148 0.360 0.685 0.288 0.697 0.136 0.291 0.326X ̄46.4 1.75 21.81 1193.8 6.18 3273.5 3842 0.2 31.75 21.6range12.7 -208.40.73 -18.510.42 -49.07594.0 -1940.01.69 - 14.301470.0 -5330.01810.0 -6030.00.05 - 0.750.17 -731.546.5 - 37.2ILSI (2006)N 1344 1249 1255 1257 1256 1349 1257 89 223 1257OECD (2002)erange 3 -100 0.09 - 1.0 0.1 - 10 82 - 1000 234 - 750 320 - 720 0.001 - 0.1 0 - 150 1.2 - 3.0a- where some values were <LOQ, statistical comparison was not possible and only the range is shownb- LOQ for selenium 0.056-0.057 mg/kg dw, LOQ for sodium 110-114 mg/kg dwc– [(MIR162 - control)/control] x 100d- statistically significant F-test (p<0.05) indicated in bold typee– units of measure for OECD values: mg/100 g dw; for conversion, 1 mg/100g = 10 mg/kgMIR162-USDA-1 Page 175 of 268Table E-7.Vitamin analysis of maize grain from a MIR162 hybrid and a control hybrid. For comparison, ILSI Crop Compositiondatabase and OECD values are also shown. All units as described in column headings, exceptF-test values and where noted.TreatmentVitamin Aaß-Carotene(mg/100g dw)Vitamin B1Thiamine(mg/100g dw)Vitamin B2Riboflavin(mg/100g dw)Vitamin B3Niacin(mg/100g dw)Vitamin B6Pyridoxine(mg/100g dw)Vitamin B9Folic Acid(mg/100g dw)Vitamin Eα-Tocopherol(mg/g dw)MIR162X ̄0.277 0.393 0.190 2.37 0.565 0.028 0.0125range 0.241-0.316 0.358-0.433 0.112-0.2382.11-2.83 0.434-0.694 0.021-0.034 0.0097-0.0154ControlX ̄0.294 0.392 0.180 2.47 0.605 0.028 0.0132range 0.244-0.358 0.339-0.443 0.144-0.226 2.03-3.15 0.486-0.738 0.024-0.033 1.10-1.54% differenceb-5.78 0.26 5.56 -4.04 -6.61 0 -5.30p-value for genotypec0.0010.983 0.314 0.0930.0020.4710.016p-value for locationby genotype Interactionc0.0480.952 0.278 0.639 0.7650.0360.917X ̄0.684 0.530 0.125 2.376 0.644 0.0651 0.0103ILSI (2006) Range 0.019 - 4.681 0.126 - 4.000 0.050 - 0.236 1.037 - 4.694 0.368 - 1.132 0.0147 - 0.1464 0.0015 - 0.0687N 276 894 704 415 415 895 863OECD (2002)dRange 2.3 - 8.6 0.25 - 5.6 9.3 – 70 4.6 - 9.6a- for direct comparison to literature values, original units ofRE/g dw were converted to units of mg/100 g dw based on 1 RE =6 μg betacarotene. Institute for Laboratory Animal Research (1995).b– [(MIR162 - control)/control] x 100c- statistically significant F-test (p<0.05) indicated in bold typed– units of measure for OECD values: mg/kg dw; for conversion, 1 mg/100g = 10 mg/kgMIR162-USDA-1 Page 176 of 268Table E-8.Location means for ß-carotene and folic acid in maize grain from a MIR162 hybrid and a control hybrid.TreatmentVitamin Aaß-Carotene(mg/100g dw)Vitamin B9Folic Acid(mg/100g dw)TreatmentVitamin Aaß-Carotene(mg/100g dw)Vitamin B9Folic Acid(mg/100g dw)MIR162 0.283 0.027 MIR162 0.266 0.029Location 1Control 0.296 0.028Location 5Control 0.286 0.032MIR162 0.304 0.032 MIR162 0.292 0.024Location 2Control 0.352 0.029Location 6Control 0.291 0.025MIR162 0.272 0.027 MIR162 0.243 0.025Location 3Control 0.280 0.024Location 8Control 0.260 0.029X ̄0.684 0.0651range 0.019 - 4.681 0.0147 - 0.1464ILSI (2006)N 276 895a- for direct comparison to literature values, original units ofRE/g dw were converted to units of mg/100 g dw based on 1 RE =6 μg betacarotene. Institute for Laboratory Animal Research (1995).MIR162-USDA-1 Page 177 of 268Table E-9.Amino acid composition of maize grain from a MIR162 hybridand a control hybrid. For comparison, ILSI Crop Compositiondatabase and OECD values are also shown. All units as described in column headings, exceptF-test values and where noted.TreatmentAsparticAcid(mg/g dw)Threonine(mg/g dw)Serine(mg/g dw)GlutamicAcid(mg/g dw)Proline(mg/g dw)Glycine(mg/g dw)Alanine(mg/g dw)Cysteine(mg/g dw)Valine(mg/g dw)X ̄6.66 3.55 5.21 19.54 9.12 3.84 7.70 2.31 4.81MIR162range 5.29-7.72 2.83-4.06 3.95-6.06 14.0-23.3 6.79-10.8 3.26-4.27 5.59-9.17 1.96-2.62 3.78-5.61X ̄6.54 3.47 5.11 19.16 8.96 3.79 7.55 2.29 4.74Controlrange 4.85-7.45 2.64-3.96 3.68-5.84 13.2-22.5 6.51-10.3 3.13-4.10 5.24-8.89 1.96-2.65 3.52-5.37% differencea1.83 2.31 1.96 1.98 1.79 1.32 1.99 0.87 1.48p-value for genotype 0.171 0.059 0.149 0.129 0.119 0.269 0.135 0.545 0.247p-value for locationx genotype interaction0.500 0.699 0.307 0.443 0.233 0.265 0.557 0.301 0.329X ̄6.88 3.75 5.12 20.09 9.51 3.85 7.90 2.21 4.90range3.35 -12.082.24 - 6.66 2.35 - 7.69 9.65 - 35.36 4.62 - 16.32 1.84 - 5.39 4.39 - 13.93 1.25 - 5.14 2.66 - 8.55ILSI (2006)N 1350 1350 1350 1350 1350 1350 1350 1350 1350OECD (2002)brange 0.48 - 0.85 0.27 - 0.58 0.35 - 0.91 1.25 - 2.58 0.63 - 1.36 0.26 - 0.49 0.56 - 1.04 0.08 - 0.32 0.21 - 0.85a– [(MIR162 - control)/control] x 100 (continued)b- unit of measure for OECD values: % dw; for conversion, 1% = 10 mg/gMIR162-USDA-1 Page 178 of 268Table E-9 (cont).Amino acid composition of maize grain from a MIR162 hybrid and a control hybrid. For comparison, ILSI CropComposition database and OECD values are also shown. All unitsas described in column headings, except F-test values and wherenoted.TreatmentMethionine(mg/g dw)Isoleucine(mg/g dw)Leucine(mg/g dw)Tyrosinea(mg/g dw)Phenyl-alanine(mg/g dw)Lysine(mg/g dw)Histidine(mg/g dw)Arginine(mg/g dw)Tryptophan(mg/g dw)X ̄2.15 3.38 12.85 3.42 5.09 3.05 2.87 4.77 0.570MIR162range 1.76-2.54 2.55-4.00 8.86-15.6 2.58-4.09 3.70-6.04 2.52-3.44 2.28-3.26 3.89-5.30 0.453-0.645X ̄2.10 3.31 12.57 3.35 4.99 2.96 2.85 4.68 0.562Controlrange 1.71-2.42 2.35-3.85 8.28-15.1 2.35-3.86 3.43-5.84 2.47-3.29 2.20-3.14 3.64-5.27 0.479-0.636% differenceb2.38 2.11 2.23 2.09 2.00 3.04 0.70 1.92 1.42p-value for genotype 0.177 0.113 0.115 0.201 0.128 0.095 0.438 0.276 0.349p-value for locationby genotype interaction0.459 0.372 0.561 0.694 0.483 0.256 0.195 0.157 0.090X ̄2.09 3.68 13.41 3.36 5.25 3.15 2.96 4.33 0.627range 1.24 - 4.68 1.79 - 6.926.42 -24.921.03 - 6.42 2.44 - 9.30 1.72 - 6.68 1.37 - 4.34 1.19 - 6.390.271 –2.150ILSI (2006)N 1350 1350 1350 1350 1350 1350 1350 1350 1350OECD (2002)crange 0.1 - 0.46 0.22 - 0.71 0.79 - 2.41 0.12 - 0.79 0.29 - 0.64 0.05 - 0.55 0.15 - 0.38 0.22 - 0.64 0.04 – 0.13a– one transgenic value was an outlier and was excluded from analysisb– [(MIR162 - control)/control] x 100c– units of measure for OECD values: % dw; 1% = 10 mg/gMIR162-USDA-1 Page 179 of 268Table E-10.Fatty acid (FA)composition of maize grain from a MIR162 hybrid and a control hybrid.qFor comparison, ILSI CropComposition database and OECD values are also shown. All unitsas described in column headings, except F-test values and wherenoted.Treatment16:0 Palmitic(% of total FA)18:0 Stearic(% of total FA)18:1 Oleic(% of total FA)18:2 Linoleic(% of total FA)18:3 Linolenic(% of total FA)X ̄12.78 1.84 25.49 56.99 1.81MIR162range 12.25-13.09 1.56-1.99 22.67-26.57 55.86-59.74 1.72-1.89X ̄12.69 1.88 25.22 57.36 1.75Controlrange 12.29-13.12 1.625-2.07 23.38-26.77 56.26-59.47 1.64-1.86% differenceb0.71 -2.13 1.07 -0.65 3.43p-value for genotypec0.275 0.051 0.0570.026 <0.001p-value for locationBy genotype interaction0.554 0.934 0.154 0.444 0.163X ̄11.50 1.82 25.8 57.60 1.20range 7.94 - 20.71 1.02 - 3.40 17.4 - 40.2 36.2 - 66.5 0.57 - 2.25ILSI (2006)N 1344 1344 1344 1344 1344OECD (2002)drange 0.29 - 0.79 0.04 - 0.17 0.70 - 1.39 0.67 - 2.81 0.03 - 0.10a- five most abundant fatty acids (FA) in maize grain.b– [(MIR162 - control)/control] x 100c- statistically significant F-test (p<0.05) indicated in bold typed– units of measure for OECD values: % dwMIR162-USDA-1 Page 180 of 268Table E-11.Secondary metabolite and anti-nutrient analysis of maize grain from a MIR162 hybridand a control hybrid. For comparison,ILSI Crop Composition database and OECD values are also shown. All units as describedin column headings, except F-test valuesandwhere noted.TreatmentFerulic Acid(mg/kg dw)p-CoumaricAcid(mg/kg dw)Inositol(ppm)PhyticAcid(% dw)TrypsinInhibitor(TIU/mg dw)Furfurala,b(mg/kg dw)Raffinosea,b(% dw)X ̄2682 179 2957 0.745 2.82 N/A N/AMIR162range 2490-2980 148-202 2410-3530 0.621-0.871 2.27-3.72<LOQ -<LOQ<LOQ - 0.116X ̄2453 157 2792 0.727 2.92 N/A N/AControlrange 2010-2760 137-179 2180-3610 0.593-0.919 2.38-3.48<LOQ -<LOQ<LOQ - 0.148% differencec9.34 14.01 5.91 2.48 -3.42 N/A N/Ap-value for genotyped<0.001 <0.0010.196 0.334 0.416 N/A N/Ap-value for locationx genotype interaction0.436 0.073 0.459 0.589 0.671 N/A N/AX ̄2201.1 218.4 1331.5 0.745 2.73 3.697 0.132range 291.9 - 3885.8 53.4 - 576.2 89.0 - 3765.40.111 -1.5701.09 - 7.18 3.000 - 6.340 0.020 - 0.320ILSI (2006)eN 817 817 504 1196 696 14 701OECD (2002)frange 0.02 - 0.3% dw 0.003 - 0.03% dw 0.45 - 1.0 <0.01 ppm 0.21 - 0.31a- where some values were <LOQ, statistical comparison was not possible so only the range is shown.b- LOQ for furfural 0.55-0.57 mg/kg dw; LOQ for raffinose 0.11-0.15% dwc– [(MIR162 - control)/control) x 100d- statistically significant F-test probability (<0.05) indicated in bold typee- below LOQ values are not included.f– For conversion of ferulic acid, 0.02% dw = 200 mg/kg; ofp-coumeric acid, 0.003% dw = 30 mg/kg; of furfural, 1ppm = 1 mg/kgMIR162-USDA-1 Page 181 of 268 Appendix F. Benefit Analysis for MIR162 Maize

The document contained within this Appendix is entitled “Public Interest Assessment Supporting Registration of MIR162, Bt 11xMIR162, and Bt11xMIR162xMIR604 Maize”.
This document was written for the U.S. EPA application for regist ration of MIR162 maize and it describes in some detail the agronomic, environmental, and economic benefits of the MIR162 maize technology. Such a benefit as sessment addresses the need of the EPA Administrator to make a finding that registra tion of a new pesticide (plant-incorporated protectant) is in the public’s interest. The bene fits characterized herein are equally applicable to addressing USDA’s obligati ons under NEPA, 42 U.S.C. §§ 4321 et seq

.

The attached report is unaltered from the version submitted to EPA. As such, it has its own pagination, table of contents, and references. It comprises pages 182- 268 of this petition.Title

Public Interest Assessment Supporting Registrations of
MIR162, Bt11xMIR162, and Bt11xMIR162xMIR604 Maize

Data Requirement

Federal Register 51 :7626 (1986)

Authors

Dennis P. Ward, Ph.D.
Demetra Vlachos

Syngenta Biotechnology, Inc. Research Triangle Park, NC 27709

Date

May 17, 2007

Submitter

Syngenta Seeds, Inc. – Field Crops
P.O. Box 12257
3054 East Cornwallis Road Research Triangle Park, NC 27709

Report ID

SSB-518-07

Page 1 of 87Statement of No Data Confidentiality Claim

No claim of confidentiality is being made for information in this application on the basis of its falling within the scope of FIFRA §10(d)(1)(A), (B), or (C).

Syngenta submits this material to the Unite d States Environmental Protection Agency specifically under provisions co ntained in FIFRA, as amended, and consents to use and disclosure of this material by EPA according to FIFRA. In submitting this material to EPA according to method and format requirement s contained in PR Notice 86-5 and 40 CFR §158.33, Syngenta does not waive any protection of rights involving this material that would have been claimed by the company if this material had not been submitted to the EPA.

Company: Syngenta Seeds, Inc.

Agent: Dennis P. Ward, Ph.D. Regulatory Affairs Manager

Date: May 17, 2007

SSB-518-07 Page 2 of 87Statement Concerning Good Laboratory Practices

This assessment was not conducted in comp liance with the Good Laboratory Practice Standards as set forth in 40 CFR §160.

Study Director: There was no GLP Study Director for this investigation

Submitter: Dennis P. Ward, Ph.D. Regulatory Affairs Manager

Date: May 17, 2007

SSB-518-07 Page 3 of 87Table of Contents

Page No.

Statement of No Data Confidentiality Claim .......................................................................2

Statement Concerning Good Laboratory Practices ..............................................................3

Summary ..............................................................................................................................6

I. Introduction ...................................................................................................................7

II. Overview of Maize ......................................................................................................7 A. Importance of Maize to the U.S. Economy .............................................................7 B. Maize Agronomics ...................................................................................................8

C. Pests of Maize ........................................................................................................10

1. Helicoverpa zea (Corn Earworm) .......................................................................13

2. Agrotis ipsilon (Black Cutworm) ........................................................................14

3. Striacosta albicosta (Western Bean Cutworm) ...................................................14

4. Spodoptera frugiperda (Fall Armyworm) ...........................................................15

D. Current Insect Control Practices............................................................................16

III. Characteristics of MIR162 Maize ............................................................................17

IV. Public Interest Factors Applicable to MIR162 Maize ............................................23
A. Presumption of Public Interest..............................................................................23
B. Factors Affecting a Public Interest Finding ..........................................................26

1. Need Factors ......................................................................................................26
2. Composition Factors ..........................................................................................27
3. Usage Factors .....................................................................................................27

4. Performance Factors ...........................................................................................27

5. Risk Factors ........................................................................................................28

6. Economic Factors ...............................................................................................28

V. Public Interest Factors Applicable to Bt11xMIR162 Maize ....................................29
A. Product Description and Intended Use ..................................................................30
B. Factors Affecting a Public Interest Finding ...........................................................30

1. Need Factors........................................................................................................30

2. Performance Factors ............................................................................................31

3. Risk Factors .........................................................................................................32

4. Economic Factors ................................................................................................33

5. Other Factors .......................................................................................................34
6. Public Interest Finding for Bt11xMIR162 ..........................................................35

VI. Public Interest Factors Applicable to Bt11xMIR162xMIR604 Maize ....................35

SSB-518-07 Page 4 of 87A. Product Description and Intended Use ..................................................................36
B. Factors Affecting a Public Interest Finding ...........................................................36

1. Need Factors........................................................................................................36

2. Performance Factors ............................................................................................36

3. Risk Factors .........................................................................................................37

4. Economic Factors ................................................................................................38

5. Other Factors .......................................................................................................38
6. Public Interest Finding for Bt11xMIR162xMIR604 Maize ................................39

VII. References ...............................................................................................................39

Appendix 1 .........................................................................................................................44

SSB-518-07 Page 5 of 87Summary

Syngenta is seeking registration for a new pl ant-incorporated protectant, the Vip3Aa20
derived from Bacillus thuringiensis , as produced in maize tran sformation event MIR162. Syngenta is also seeking regi stration for two combined tra it maize cultivars containing MIR162 and two other registered plant-incorp orated protectants, Cry1Ab in Bt11 maize and mCry3A in MIR604 maize. The first combined trait product will be a breeding cross of MIR162 and Bt11, designated Bt11xMIR162, and the second will be a breeding cross of MIR162, Bt11, and MIR604, designated Bt11xMI R162xMIR604. Data has been developed by Syngenta demonstrating that issuance of each of these registrations will be in the public
interest.

Field efficacy trials demonstrate that MIR162 maize and Bt11xMIR162 maize hybrids provide improved protection agai nst lepidopteran insect feedi ng damage when compared to the protection provided by conventional insectic ides or Bt11 maize alone. This improved product efficacy is expected to translate into increased maize grain yield and quality. In a time of rising demand for maize grain, the MIR162 trait has th e potential to provide U.S. agriculture with an economic benefit exceed ing $371 million annually at product maturity.
The introduction of the MIR162 trait in comb ination with Bt11 also has the pot ential to replace many conventional ins ecticide applications, reduce greenhouse gas emissions, and reduce mycotoxin contamination of livestock feed. There will also be IRM benefits stemming from the introduction of these comb ined trait hybrids. The Vip3Aa20 protein contained in MIR162 maize brings a second mode of action against Helicoverpa zea and Spodoptera frugiperda , two pests that are only suppre ssed by Cry1Ab. Data has been developed showing that Bt11xMIR162 is high dos e against these two pests; accordingly a reduction from the 50% structured refuge require ment in the South is warranted. This will greatly benefit maize growers in the affected counties of the South as it will allow them to protect more of their maize acres against feeding damage from lepidopteran pests.

Adoption of Bt11xMIR162xMIR604 hybrids by grower s is predicted to offer crop yield advantages and important new options for contro l of virtually all the major insect pests of maize, all built into a single seed product. The availability of a new product for lepidopteran and rootworm control will provide choices for growers in the marketplace, and lead to increased price competition for traits, which will benefit growers and others in the maize
value chain. Bt11xMIR162xMIR604 maize also o ffers health and environmental safety advantages over the use of conven tional insecticides, as well as insect resi stance management benefits that will preserve the durability of this and other Bt -based products.

Collectively, the information presented in this document convincingly supports a public interest finding for registration of the plant-incorporated protectants in MIR162, Bt11xMIR162, and Bt11xMIR162xMIR604 maize.

SSB-518-07 Page 6 of 87I. Introduction

At the time of application for a §3 pestic ide registration under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), it is often not po ssible to determine if the registration will be granted under special ci rcumstances. Since their introduction, plantincorporated protectant regist rations have only b een granted under special circumstances.
FIFRA authorizes the Administrator of the Environmental Protection Agency (EPA) to conditionally register a pesticid e containing a new active ingredient for a period sufficient to generate and submit additional data. Conditional registrations are granted if the Administrator determines that use of the pest icide during such period will not cause any
unreasonable adverse effect on human health or th e environment and that use of the pesticide is in the public interest.

The introduction of crops improved through modern biotechnology has been the single most important technological innovation brought to United States (U.S.) agricultu re in the past 25 years. Adoption of genetically engineered crops with insect a nd herbicide tolerance traits has increased dramatically since their commercia l introduction in 1995. Ne t economic benefits at the farm level have been substantial, exceeding a cumulative $27 billion (Brookes and Barfoot, 2005). Improved insect and weed cont rol have led to increased crop yields and reductions in conventional pes ticide applicat ions (Marra et al. , 2002). The continued development and introduction of these traits is expected to benefit both growers and

consumers.

Maize transformation event MIR162 has been developed by Syngenta Bi otechnology, Inc. to provide U.S. growers with maize hybrids that are resistant to feed ing damage caused by a number of significant lepidopter an insect pests. This as sessment will ch aracterize the benefits of MIR162 maize (field corn) hybrids and combined trait Bt11xMIR162 and Bt11xMIR162xMIR604 hybrids, and will show how these benefits support a determination that their registration their insecticidal act ive ingredients is in the public interest.

II. Overview of Maize

Zea mays Linnaeus, known as maize throughout the world, and as corn in the U.S., is one of the few major crop species indigenous to the West ern Hemisphere. It has been cultivated in the Americas since early historic times. Fiel d maize is the leading production crop globally, with the 2005/2006 growing season yielding 695 million metric tons of grain (USDA, 2006a).

A. Importance of Maize to the U.S. Economy

The U.S. accounts for nearly 41% of global maize production. Maize is the largest crop grown in the U.S. in terms of both volume a nd value. Approximately 78.3 million acres were planted in 2006. In the same year, 70.6 million acres were harvested for grain, yielding 10.5 billion bushels (267 million metric tons) with a gross crop value of $33.71 billion (USDA, 2007a).

SSB-518-07 Page 7 of 87Maize is grown for animal f eed, human food, vegetable oil, high fructose corn syrups (HFCS), starch, fermentation into ethanol, and a multitude of industrial uses. U.S. maize usage by market segment is shown in Figure 1. Maize as a source of fuel ethanol has increased dramatically over the past two years and is expected to continue doing so as the U.S. continues to focus on finding renewable sources of energy. By 2010, U.S. ethanol production could displace the equivalent of 311,000 barrels of imported crude oil per day. The U.S. is by far the world’s largest expor ter of maize, accounting for 68% of global exports. Domestic maize production falls on the positive side of the U.S. trade balance sheet.

Feed/Residual

Export

Ethanol

HFCS

Starch

Sweetners

Cereal/Other 4.4%

2.4% 2.0% 3.0%

50.8% 19.1% 18.3%

Figure 1. U. S. maize usag e by segment, 200 6 (Baker and Allen, 20 07).

Total U.S. agricultural exports in 2006 were valued at $ 71 billion, 10% of which was
attributable to m a ize (Brooks, 2007). Agricult ural exports generate employm e nt, incom e , and purchasing power in both farm and nonfar m

sectors of the economy. Production fro m alm o st one-third of U.S. cropland m o ved into export channels in 2005 and generated $166.1 billion in business activity. T echnology advances increase agri cultural productivity and keep
dom e stic growers com p etitive in the global m a rket.

B. Maiz e Agronomics

Z. mays is a large, ann u al m onoecious grass; th e duration of its life cycle depends on the
cultivar and the environ m ent in whi c h the cultivar is grown. The bulk of m a ize is produced
between latitudes 30  ̊ and 47  ̊ . Practically no maize is grow n where the m ean m i dsumm e r
tem p erature is < 19  ̊ C or where the average nigh ttim e te m p erature during the summ er months
f a lls m u ch below 13  ̊ C. The greatest production occurs where the warm est m onth isotherm s range between 21  ̊ and 27  ̊ C and th e freeze-free season last s 120 to 1 80 days. Maize is grown in areas where annual pr ecipitation ranges from 25 to > 500 c m . Summ er rainfall of

15 cm is approxim a tely the lower lim it for m a ize production without irrigation.

SSB-5 18 -07 Pag e 8 of 8 7The upper Midwest region of the U.S. provides an ideal combination of temperature, rainfall, and soil type for the cultivation of maize. Iowa, Illinois, Nebraska, Minnesota, Indiana, Ohio, Wisconsin, Missouri, Kansas, and Sout h Dakota are major maiz e growing states.
Production in these ten states accounts for 84 % of total annual production (USDA, 2007a). Figure 2 displays the geographic di stribution of acres planted in 2005.

Figure 2. Geographic distribution by county of acres planted with maize in 2005 (source USDA/NASS Charts & Maps).

Farmers have hundreds of maize hybrids from wh ich to choose. Available hybrids differ widely in agronomic characteristics, in cluding length of grow ing period. Technology providers continue to develop varieties with desirable traits and increasing yield. Maize yields have increased an aver age of 3.5 bushels per year over the past decade. The average yield reported for the 2006 growing s eason was 149.1 bushels/acre (NCGA, 2007).

The adoption of new varieties improved through biotechnology has added greatly to farm productivity and profits since their introduction in 1996 (Fer nandez-Cornejo and Caswell, 2006). Varieties containing herbic ide-tolerance and inse ct-protection traits have been widely adopted by maize, soybean, and cotton grower s because they protect the inherent yield

SSB-518-07 Page 9 of 87potential of these crops or typi cally reduce grower input costs. In 2006, herbicide tolerant varieties accounted for 89% of soybeans plante d, 65% of cotton planted, and 36% of maize planted (USDA, 2006b). Insect-p rotected varieties accounted for 52% of cotton and 40% of maize acres planted. Planting of these varie ties has also benefited the environment by displacing conventional pestic ide applications a nd reducing the production of greenhouse gases. The adoption of biotech maize varieties has been estimated to reduce the application of conventional pesticides by more than 20 million pounds annually (NCGA, 2007).

C . Pests of Maize

ield losses due to weeds, diseases, and ins ects were huge until th e introduction of crop

sect pests can be categorized as major a nd consistent pests, major and sporadic, and

addition to direct damage caused by feeding on plant tissue, insects play an important role

ar, kernel, and cob rots occur wherever mai ze is grown and result in reduced test weight, Y protection chemicals in the 1960s. Weeds compet e with crops for light, nutrients, water, and other growth factors. If weeds are left uncontrolled, maize simply cannot be grown successfully. Estimates of maize yield loss caused by pathogens have ranged from 2 to 17% (Smith and White, 1988). In a ddition, a maize crop is susceptible to att ack by a variety of insects from the time it is planted until it is consumed as food or feed.

In
moderate to minor based on annual destructiven ess and their geographic distribution. Table 1 categorizes most of the insect pests of ma ize found in the U.S. The most economically significant insect pests of maize are: Ostrinia nubilalis (European corn borer), Diatraea
saccharalis (sugarcane borer), Diatraea grandiosella (southwestern corn borer), Elasmopalpus lignosellus (lesser cornstalk borer), Helicoverpa zea (corn earworm/cotton bollworm), Agrotis ipsilon (black cutworm), Spodoptera frugiperda (fall armyworm), Rhopalosiphum maidis (corn leaf aphids), and Diabrotica spp. (corn rootworm complex). Pests of secondary economic importance in maize include both soil-dwell ing insects that feed on roots or other subterranean tissue ( e.g., wireworms, billbugs, webworms, white grubs, corn root aphids, the seed corn maggot, gr ape colaspis and seedcorn beetles) and aboveground insects that attack the stalk, leaf, and ear ( e.g., cutworms, corn leaf aphids, chinch bugs, grasshoppers, corn fl ea beetles and Japanese beetles). Striacosta albicosta (western bean cutworm) is a pest of in creasing economic importance because of its movement into Iowa, Illinois, and Indiana.

In in the transmission and dissemination of pat hogenic organisms during maize development. Soil abounds in microorganisms, particularly f ungi, which may infect plant parts injured by soil-dwelling insects. In much of the Corn Belt, pathogenic fungi probably pose more problems in corn production than any other group of organisms. Primary roots of the seedling and the radical and seminal roots are commonly infected with Fusarium spp. after they have served their function an d become senescent. Feeding by Diabrotica rootworms has been associated with increased frequencies of Fusarium infection (Dicke and Guthrie, 1988); rootworm feeding may also lead to in creased incidences of stalk rots. These pathogenic infections can lead to reduced crop quality, harvestability, and yield.

E poor grain quality, and mycotoxin c ontamination of food and feed. Fusarium kernel or ear

SSB-518-07 Page 10 of 87SSB-518-07 Page 11 of 87 rot is the most widespread disease of maize ears and is frequently associated with insect feeding damage.

Table 1. Categorization of maize insect pests based on their potential for causing economic losses (modified from Gray and Luckmann, 1994).

Major & Consistent Insect Pests Moderate to Minor Insect Pests (cont.)

Northe eri ) Cin rn corn rootworm ( Diabrotica barb ch bug ( Blissus leucopterus )

Western corn rootworm ( Diabrotica virgifera virgifera ) Southern corn rootworm ( Diabro tica undecimpunctata )

European c orn borer ( Ostrinia nubilalis ) any species Other cutworms, m

Corn earworm ( Helicoverpa zea ) Seedcorn beetle ( Stenolophus le contei )

Fall armyworm ( Spodoptera frugip erda ) Seedcorn maggot ( Delia platura )

Banks grass mite ( Oligonychus pra tensis )

Major - Sporadic Insect Pests ae ) Two-spotted spider mite ( Tetranychus urtic

Black cu tworm ( Agrotis ipsilon ) Billbugs, many species

Corn leaf aphid ( Rhopalosiphum maidis ) ies White grubs, many spec

Southwest corn borer ( Diatraea grandiose lla ) ris ) Stalk borer ( Papaipema neb

Sugarcane borer ( Diatraea saccharalis ) Garden symphylan ( Scutigerella immaculata )

Lesser cornstalk borer ( Elasmopalpus lignosellus ) Japanese beetle ( Popillia japonica )

Western bean cutworm ( Srtiacosta albicosta ) Sod webworms, several species

Grape colaspis ( Colaspis brunne a )

Moderate to M or Insect Pests in Thrips, several species

Wirew philus lugubris ) orms, many species Dusky sap beetle ( Carpo

Armyworm ( Pseudaletia un ipunctata ) Stink bugs, several species

White-fringed beetles ( Graphognathus spp. ) iatraea Southern cornstalk borer ( D
crambidoides )

Grasshoppers, many species Corn root aphid (

Anuraphis maidiradicis )

Corn flea beetle ( Chaetocnem a pulicaria )

rop losses attributable to O. nubilalis and Diabrotica infestations have been well C characterized. However, there is surprisingly little quantitative information available on the economic impacts of the other ma jor insect pests of maize, H. zea

, S. frugiperda

, A. ipsilon

, and S. albicosta . These pests are not as widely sp read as corn borers and rootworms; however, they can be very costly to growers as they have the potential to significantly lower grain yield and quality. Figure 3 shows the approximate geog raphic distribution of these four insect pests in the continental U.S. As described in Appendix 1, there is compelling evidenceSSB-518-07Page 12 of 87Figure 3.U.S. geographic distribution of the maize insect pestsH. zea(CEW),S. frugiperda(FAW),A. ipsilon(BCW), andS. albicosta(WBCW). Two regions are shown for WBCW occurrence, one where its presence presents an economic problem for growers and theother where it is spreading into.BCWCEW & FAWWBCWEconomicWBCWSpreadingthat one of these pests, S. albicosta , is spreading eastward at an alarming rate. Note the p of S. albicosta occurrence in Figure 3 with the re gions of highest maize production in Figure 2.

icoverpa zea (Corn Earworm)

verpa zea , formerly Heliothis zea , is native to the Ameri cas and occurs wherever is grown. In its larval stage i a major economic pest of maize. Larvae feed on , tassels, silk, and ears; refer to Figure 4. Whorl feeding appears as ragged holes in the . Earworm larvae prefer to feed on developing ears during the silking period.
are often cli pped as larvae tunnel
he ear. The ear will h plete kernel set if the silks are yed before polli nation is complete. l feeding on kernels causes the most cant damage. Larvae feed on the nd ears, leav ir waste behind.
ehavior crea highly favorable
nvironment for the invasion of ary pests and for the production of oxins. Ear molds developing in the ay cause toxicity
ms in livestock.

gh H. zea can be very damaging to , injury is so evenly distributed and
l is so difficult that farmers lly accept the loss. Chemical l of earworms is prohibitively sive except to protect sweet maize.

l is difficult because eggs are laid erging silks; a pro ective insecticide g of the silk t be maintained sprays are d every two to

ays.

y conducted by Hudson and All 35,000 acres of field maize in

were identified as having H. Figure 4. H. zea larva feeding on maize silks (top) and ear (bottom).

tations at levels warranting control. Yield loss was 1.5 bu/ac on chemically treated 5.6 bu/ac on untreated acres. Losses to Georgia maize grower s in 1995 attributable infestations totaled $510,000 in combined ch emical costs and yield loss. Losses in
ibutable to H. zea were even higher, totaling $872,000. overla
shown

1. Hel

Helico
maize
leaves
leaves
Silks
into t
incom
destro

Larva signifi
silks a
This b
microe
second
mycot damaged kernels m proble

Althou
maize

contro

genera
contro

expen Contro

on em coatin
unless
three d

In a stud (1995), Georgia

zea infes
acres and

to H. zea
1996 attr t is

ave an

in g
te s the

a

t

anno plie s c

ap

SSB-518-07 Page 13 of 872. Agrotis ipsilon (Black Cutworm)

A. ipsilon (formerly Agrotis ypsilon ) is by far the most destructive species of the cutworm

omplex in maize (see Figure 5). A. ipsilon occurs throughout North and South America. It

w eed species. Damage caused by A. ipsilon larvae is articularly severe in rotated maize that is pl anted late and in maize planted in flood plains.

Figure 5. A. ipsilon larva (top) and cut eeds and debris in fields that have not
been tilled. When w eed hosts are killed with cultivation or herbicides, the larvae begin feeding on maize. They are nocturnal feeders, cut ting seedlings at or just below the soil surface. Older instars can then tunnel into the growing stalk.

Plants in the one- to four-leaf development stage are particularly susceptible to cutting and the extent of yield reduction is dependent on where the plant is severed. Maize seedlings cut below the soil surface or larger plants that have been tunneled usually do not recover. Seedlings cut
above the soil surfac e often recover if moisture is adequate, but they will not yield as well. While some plants cut at the soil surface produce ears, there is a significant yield loss. Plants cut below the soil surface do not produce ears.

Tillage, removal of winter and early spring vegetation, and insectic ide applications at planting can be used to prevent cutworm damage. However, when outbreaks occur

injury should be conducted as soon as maize -emergent insecticide application when ≥ 3% cut

. albicosta (formerly Loxagrotis albicosta and Richia albicosta ), has been categorized as a pest of moderate significance in the western Corn Belt. Over the past several years it has
become established in Iowa and continues to move east. There ar e numerous university c feeds on many hosts, including several

p A. ipsilon larvae also cause economic damage to vege tables, cotton, tobacco, and turf grasses.

A. ipsilon does not overwinter in the Midwest; moth s migrate from the coastal areas of the Gulf of Mexico in the early spring. Fema les prefer to deposit e ggs on densely growing

maize stalk (bottom). w

insecticides. Scouting for cutworm feeding plants emerge from the soil. A rescue post , they are difficult to control even with

plants are found is necessary to prevent devastating crop losses.

3. Striacosta albicosta (Western Bean Cutworm)

S

SSB-518-07 Page 14 of 87extension reports documenting its spre ad in to Illinois and Indiana (see Figure 3).

ther yellow tissue. Larvae then move to f eeding on emerging silks. As both larvae and

rvae feeding on ears.

Figure 6. S. albicosta larval feeding damage cause

An infestation of more than one la rva per ear can larvae are not cannibalistic. One larva per ear r have shown that even one larva per ear on a fi eld 3.7 bu/ac (Cook, 2004). In years of severe infestation wi th m ear’s kernels can be damaged. Th is contributes di grain quality.

Scouting is essential for effectively reducing th One generation of S. albicosta occurs each year. Moth emergence begins in July and after mating, females lay eggs on available host plants such as field maize, sweet maize, or dry beans. In maize, females lay eggs primarily on the upper surface of leaves. After hatching, larvae move to more protected sites on the host plant and progress through five instars. Feeding on maize occurs in the whorls where th e larvae feed on the fl ag leaf, tassel, and

o maize develop, they begin feeding on ear tips. Fusarium and other secondary pests typically follow kernel injury. Figure 6 shows the nature and extent of damage that can be caused by
la

d in maize ears.

occur because, unlike H. zea

, S. albicosta arely causes economic injury, but studies
-wide basis can redu ce yield by ultiple larvae per ear, 50 to 60% of an

rec tly to significant redu ctions in yield and

e risk of major infestations. Insecticide
ication is critical. Once the larvae have applications can be effective but timing of appl
entered the silks and are inside the husk, insecticide applicati ons are ineffective and larvae
will feed until mature. Few cultural cont rols are effective against this pest.

4

. Spodoptera frugiperda (Fall Armyworm)

S . frugiperda is native to tropical regions of Nort h and South America. In the U.S. it
overwinters in Florida a nd Texas and the moths can fly great distances. The insect has been found in virtually all states east of the Rocky M ountains. As a pest of maize, it is typically only a problem in the southeastern states.

SSB-518-07 Page 15 of 87M aize, sorghum, and other grassy plants are preferred hosts for S. frugiperda . The number

nal to loss of leaf ass but varies with plant development stage. Early whorl-stage feeding produces the least impact on yi eld; feeding at late whorl stage is very damaging to crop yield. Marenco et al. (1992) observed that mean densities of larvae per plant during late whorl stage could reduce yield by 5 to
20%. Larvae can also burrow into ears causing kernel loss and diminished grain quality.

Early planting is an effective cultural practice in some regions for limiting S. frugiperda damage. An early harvest allows many maize ears to escape the hig

te

lications will likely change

(bottom).

corn borers. As a result, they incurred significant of generations per year varies with latitude; up to four generations are common in southern states. The larvae progress th rough six instars and feed on fo liage. Young larvae initially consume leaf tissue from one side, leaving th e opposite epidermal layer intact. Feeding by second and third-generation larvae results in hole s in leaves. Feeding in the whorl of maize produces a characteristic row of perforations in the leaves (see Figure 7). Older larvae cause extensive defoliation, often leaving only the ribs and stalks of a plant or a very ragged, torn

appearance.

Yield loss is proportio

m

her armyworm densities that develop
r in the season. Control of armyworms la
with conventional insect icides is typically not economical, unless infestation is severe and the larvae are feeding on parts of the plant that would bring them into
contact with the insecticide. The economic feasibility of conventional nsecticide app i as the price of maize grain increases. Insecticides are most commonly applied in sweet corn production as S. frugiperda is the economically most damaging pest to

rop yield and quality. Figure 7. S. frugiperda larva feeding on maize leaf (top) and whorl feeding damage c

D. Current Insect Control Practices

The most widespread and damaging insects of maize in the U.S. Corn Belt have been O.
nubilalis and Diabrotica species. Before the introduction of Bt maize hybrids ten years ago growers had few practical options for controll ing stalk boring insects; only about 10% of growers applied insecticides for control of

SSB-518-07 Page 16 of 87a nnual yield losses because most fields were not treated and chemical applications were not

from B. thuringiensis , both of which are highly toxic to O. nubilalis and D.
randiosella

.

w ers for mitigating damage caused by corn

ns crop rotation has been effective in limiting e life cycle of the insect. There have also been
ducts available to growers for control of these -protected Bt varieties in 2 003 an estimated 14
ventiona l insecticides to control corn rootworms. ds of insecticide active ingredient being applied
abrotica species (Ward et al. , 2005). Control of
est si ngle use of insecticides in the U.S.

ontrolling aboveground insects pr esents a challenge for maize growers. The majority of eaf, stalk, and ear feedi ng insects. A grower
le because of the feeding location of the pests (in

in

the ear); the pests are shielded from aerial

s is critical for them to be effective.

006 Agro Trak studies indicate that growers are

n acre s a year with conventional insecticides for S. frugiperda (see Table 2). Compared to the ally this represents a relatively small use of acres treated represents a significant use

ium -mediated transformation using elements of a
the vip3Aa1 uringi getative insecticidal pr ote A. ipsilon

, frugiperda , and S. albicosta . A
has been incorporated into the genome of MIR162 always effective. Timing of insecticide applic ations had to be nearly perfect because there was only a very short period of time (two to si x days) that these ins ects would be physically positioned on the plant where they could be expos ed to an insecticide application. The
introduction of the first Bt maize hybrids in 1996 provided gr owers with an effective means of limiting damage caused by O. nubilalis . By plant-incorporating the insecticide, exposure of the insect to the toxin was guaranteed. In 2006, 42% of maize acres were planted with Bt corn borer-protected hybrids (Doane, 2006a ). These hybrids express either a cry1Ab or cry1F gene

g

More options have been available to gro rootworms. In many maize growing regio
Diabrotica populations because it breaks th many effective conventional insecticide pro pests. Prior to the intro duction of rootworm million acres were treated annually with con This equated to more than 7.7 million poun annually in maize fields for the control of Di
Diabrotica rootworms accounted for the larg

C
maize fields are not treated for control of l decision not to treat is generally not reversib the soil, under the leaf, inside the stalk, or chemical applications. Timing of application

Data obtained from the Doane 2005 and 2 currently treating approximately three millio
control of H. zea

, A. ipsilon

, S. albicosta , and total number of maize acres pl anted annu conventional pesticides; however, three million compared to chemical usage in other crops.

III. Characteristics of MIR162 Maize

MIR162 maize was produced by Agrobacter vector (pNOV1300) containing a variant of
The vip3Aa1 gene was isolated from B. th This gene encodes a ve gene from Bacillus thuringiensis.

ensis strain AB88 (Estruch et al. , 1996). in (Vip) that is hi ghly toxic to numerous
H. zea

, S. lepidopteran pests of maize, including:

aize-optimized variant of the gene m maize and encodes a protein assigned the fo llowing toxin designation: Vip3Aa20. MIR162
maize also contains the manA gene from Escherichia coli which encodes the selectable marker enzyme, phosphomannose isomerase.

SSB-518-07 Page 17 of 87Table 2. Conventional insecticide usage in maize production during 2005 and 2006 for the
control of S. frugiperda

, A. ipsilon

, S. albicosta , and H. zea (Doane, 2005 and 2006b).

2005 2006

Pest Acres Treated Grower Cost ($) Acres Treated Grower Cost ($)

S. frugiperda 0 0 20,441 86,550

A. ipsilon 2,721,543 19,457,090 3,064,137 22,328,818

S. albicost a 44,410 363,801 72,373 343,552

H. zea 99,620 825,654 161,002 848,502

Totals 2,865,573 $20,646,545 3,317,953 $23,607,422

Syngenta conducted field trials at multiple lo cations in 2005 and 2006 to assess the trait efficacy of MIR162 maize hybrids (Huber et al. , 2007; White et al. , 2007a; White et al.

, 2007b). Trials were placed in locations to take advantage of natural pest infestations. In some locations trials were artificially infested to test trait performance at high levels of insect pressure. MIR162, Bt11, Bt11xMIR162, conventional maize, and conventional ma ize with

ing feeding

. saccharalis (sugarcane borer), H. zea (corn borer), P. nebris (common stalk borer), S. exigua yworm), and S. albicosta (western bean cutworm). a n insecticide application were the treatments employed in most trials. Figure 8 presents a graphic representation of the comparative fe eding damage for each of the treatments, expressed as a percentage of damage measured in the control plants, for five of the insects
evaluated.

MIR162 alone has no activity against O. nubilalis but is very efficacious in limit
d amage caused by the other four insect pests. Whereas Bt11 is highly efficacious against O.
nubilalis , it has limited or no activity against the other four in sects. The combined-trait hybrid, Bt11xMIR162, is very efficacious agains t all five insects. MIR162 addresses the lepidopteran pest control limitations of Syngent a's Bt11 product. In combination with Bt11, it will provide growers with excellent control of the following significant lepidopteran insect pests of maize: A. ipsilon (black cutworm), D. crambidoides (southern cornstalk borer), D. grandiosella (southwestern corn borer), D earworm), O. nubilalis (European corn (beet armyworm), S. frugiperda (fall arm

Comparative efficacy trials of Bt11xMIR 162 hybrids with commercially available lepidopteran-protected Bt varieties, specifically YieldGard CB (MON 810) and Herculex I (TC1507), were not possible due to intellectu al property constraints. However, it is reasonable to assume that MON 810 hybrids have the same spectrum of activity limitations that Bt11 hybrids do and, therefore, the Bt11xMIR162 product will provide superior protection against A. ipsilon

, D. saccharalis

, H. zea

, P. nebris

, S. exigua

, S. frugiperda , and
S. albicosta . Publicly available inform ation on the performance of Herculex I hybrids suggests that Bt11xMIR162 hybrids may outperform them in controlling H. zea and S.
albicosta feeding damage.

SSB-518-07 Page 18 of 87020406080100120Bt11

%of Control DamageEuropean Co rrn BoreFallArmywormMIR162TreaBt11xMIR162ntInsecticidetmeCorn orEarwmBlack orCutwmWestern Bea ormFigracondwit 11, MIR16 tandconmngss d he contplon Cutwarolure 8.Feeding damageventional insecticide treatts.tings from field efficacy trialsent (Warrioructed in2005 and 2006are expressed as a percenh Bttage of the da2, Be ob11xMIR162,in t®Insecticide). Ratimagervemark of a Syngenta group co®TradempanySSB-518-07Page 19 of 87Other than its resistance to lepidopteran insect feeding, MIR 162 maize hybrids are phenotypically equivalent to conventional maize hybrids. The presence of the MIR162 transgenes will protect the inherent yield potentia l of varieties that contain them 005 and 2006, Syngenta conducted phenotypic equivalency studies at 16 locations to compare the agronomic performance of two MIR162 hybrids to the performance of two near-isogenic conventional hybrids. These trials were plante d in a randomized complete block design with either four or five replications per location. The trial results are presented in Table 3 and demonstrate no difference in yield be tween MIR162 and conventional hybrids.

Table 3. Yield results from agronomic equiva lency studies conducted with two MIR162 hybrids in 2005 and 2006. Average yield (bu/ ac) at each location and across locations was subjected to an analysis of variance and l east significant difference test. Statistical significance was assigned at p<0.05

. . In 2

Significance a Location MIR162 Yield Control Yield Difference

2005 Trials

Allen, IA 166.3 145.1 21.2 NS

Seward, NE 152.4 158.4 -6.0 NS

Hudson, IL 196.6 185.9 10.7 NS

Bloomington, IL 202.1 200.3 1.8 NS

Wapella, IL 204.7 187.4 17.3 NS

Mackinaw, IL 187.0 175.8 11.2 NS

Average 184.9 175.5 9.4 NS

2006 Trials

Brookings, SD 152.2 155.4 -3.2 NS

Gaylord, MN 204.6 206.7 -2.1 NS

Janesville, WI 155.4 153.6 1.8 NS

Maxwell, IA 153.6 159.7 -6.1 NS

Monroeville, IN 156.3 164.6 -8.3 NS

Seward, NE 194.1 178.2 15.9 NS

El Paso, IL 200.3 193.4 6.9 NS

Bloomington, IL 201.7 187.8 13.9 *

Sadorus, IL 174.8 183.1 -8.3 NS

Mackinaw, IL 190.6 181.7 8.9 NS

Average 178.4 176.4 2.0 NS

a - NS = means not statistically signifi cant; * = means significantly different ( p<0.05 )

SSB-518-07 Page 20 of 87In addition to evaluating the performance characteristics of MIR162 maize, Syngenta has examined its potential human and environmental impacts. The standard battery of studies to identify hazards for mammalian species potentia lly exposed to plant- incorporated Vip3Aa20 has been conducted. These Vip3Aa20 studies include an acute oral toxicity study with mice (Draper, 2007), in vitro digestive fate assays (Stacy, 2007a; Stacy, 2007b), a comparison of amino acid sequence to that of known toxins and allergens (Harper, 2006a; Harper, 2006b), and a characterization of the biochemical prop erties of the Vip3Aa20 protein (Graser and Stacy, 2006). The results of thes e studies demonstrate that no adverse effects w ere observed

mice exposed to a maximum attainable oral do se, that the protein is rapidly degraded in a

gas an
tox al.

A r Vip3Aa20 when used as a plant-incorporated pr otectant in the food and feed commodities of

orn (40 CFR §174.458).

A comprehensive set of data has bee to support an environmental safety assessment for Vip3Aa proteins and MIR in studies with a range of nontarget organisms, wh most cases were exposed to levels of Vip3Aa protein above expected environmen ncentrations. A com ensive en assessment describing the resu lts of these studies has been p red by Raybould (2007a).

This failure to elicit adverse effects in nont arget species is not surprising given the known
mode of insecticidal action fo r Vip proteins and the narrow spectr f activity f ip3Aa proteins. Vip3Aa20 is a me of a class cticidal p s that ar turally produced by B. thuringiensis , a positive bac commonly found in soil. Unlike the crystal proteins (Cry) of B. thuringien teins are pr oduced during vegetative bacterial growth and are secreted as soluble into the extracellular environment. B.
th tures continu produce Vip
de nd sporulation lik e the th abile non inaceous otoxin

se B. thuringien ins, Vip pro e thermolabile.

Th by which Vip proteins exert thei ticidal act has been ed and
been found to be similar to that which has been previously described for the B. thuringiensis
C Following inge ull-length Vip proteins are proteolytically processed to

ac a kDa whic h o receptor e midgut epithelium of insects. Comp a e shown t Vip protei

pr fferent receptors (Lee et al. , 2 Receptor g is follow fo selective ion cha (pores) in e l membranes. Each of pl establishing the ticidal speci ficity of a given protein for different insect

sp

Syn a range of insect

ec and the results of
rted in the scientific literature cl early demonstrate that ac tivity of Vip3Aa proteins is limited to species within order Lepidoptera, yet all lepidopteran species are not sensitive to in tric matrix, that the pr otein does not share sequence homology with known mammali ins and allergens, and it does not possess proper ties suggestive of food allergen potenti temporary exemption from the requirement of a tolerance has been established fo

c

n deve loped
162 maize. No harmful effects have been observed
ich in
tal co preh
vironmental repa

um o rotein or V

e na mber of inse

gram terium

sis , Vip pro proteins uringiensis cul e to prot ein during the stationary phase of velopment a . Un ermost prote β -ex creted by some sis stra teins ar

e mechanism r insec ivity studi

ry proteins. tive fragments of approxim st ion, f tely 65 bind t s in th susceptible etitive binding ass ys hav hat ns and Cry
oteins bind to di 003). bindin ed by the
rmation of ays a role in nnels
insec pithelia these steps

ecies.

genta has conducted mortality bi oassays with Vip3Aa protein vari ants in ies to identify those that are susceptible. Th e results of these bioassays sp

assays repo

SSB-518-07 Page 21 of 87the protein. Table 4 provides a listing of inse ct species that have b een found by Syngenta or reported in the literature as being sensitiv e to one or more Vip3Aa protein variants.

Table 4. Insect species that have b een screened for sensitivity to Vip3Aa protein variants in diet-surface or diet-incorporation bioassays. Activity was determined as evidence of treatment-related mortality.

Order: Family Genus: Species Activity

Agrotis ipsilon Active

Helicoverpa zea Active

Helicoverpa armigera Active

Helicoverpa punctifera Active

Heliothis virescens Active Lepidoptera: Noctuidae

Spodotera exigua Active

Spodoptera frugiperda Active

Spodoptera litura Active

Trichoplusia ni Active

Lepidoptera: Ge lechidae Phthorimea operculella Active

Lepidoptera: Sphingidae Manduca sexta Active

Chilo partellus Active Lepidoptera: Pyralidae Ostrinia nubilalis Not Active

Lepidoptera: Plutellidae Plutella xylostella Active

Lepidoptera: Danaidae Danaus plexippus Not Active

Lepidoptera: Pieridae Pieris brassicae Not Active

Bombyx mori N ot Active

Earias vittella Active Other Lepidoptera

Ephestia kuehniella Active

Culex pipiens Not Active

Drosophila melanogaster Not Active Diptera

Culex quinquefasciatus Not Active

Coleomegilla maculata Not Active

Diabrotica virgifera virgifera Not Active

Leptinotarsa decemlineata Not Active

Tenebrio molitor Not Active

Anthonomus grandis grandis Not Active Coleoptera

Aleochara bilineata Not Active

SSB-518-07 Page 22 of 87SSB-518-07 Page 23 of 87 Table 4 (cont.). Insect species that have been screen ed for sensitivity to Vip3Aa protein variants in diet-surface or diet-incorporation bioassays. Activity was determined as evidence of treatment related mortality.

Order: Family Genus: Species Activity

Hymen N optera Apis mellifera ot Active

Hemiptera: Anthocoridae N e Orius insidious ot Activ

Thysanoptera Fran alis N e kliniella occident ot Activ

Isotomidae N e Folsomia candida ot Activ

Neuroptera N e Chrysoperla carnea ot Activ

IV. Public Interest Factors Applicable t

Registration of the B. thuringiensis ncoded by pNOV1300 vector in maize transformatio public interest because it meets criteria for a de in the EPA policy val or de pestici of a new pesticide during the period of its conditional registra tion must c in terest. M tors can be considered in determining whether this pub t criterion h n satisfied. Registration presume t ent for another pesticide that is of continuing Maize va aining the

MIR16 erous conve insecticides
that ar farmers, and th human an ental risk
factors. Ad 2 hybrids

compa n of conventio extract an economic benefit through increased

attract

A. Presumption of Public Interest

In cer umstances, EPA is empowered umptio of a new pesticide is in the public interest. In th t nee stantiate a public interest finding. EPA has define r iority and if active ingredient addresses one or more of the ies for a presum registration will be in the public inter alifies mption of public interest is “...replacement for conti ern to the Agency”.

The most commonly used conventional insectic ms, cutworms, and armyworms in maize a ajority of secticides
are classified as Restricted Us e Pesticide d environ concerns. o MIR162 Maize

Vip3Aa20 protein e elements of the

n even t MIR162 can be presum ed to be in the

conditional registration as lineated notice regarding appro de products (EPA, 1986). This criterion st nial of applications for conditional registration of
ates that use be in the publi any fac
li c interes as bee of a new pesticide is d to be in the public interes

concern to EPA. if it is a replacem
rieties cont 2 trait have the potential to replace the use of the num ntional e of concern to EPA, e public due to d environm ditionally, the safety, convenience, red to the applicatio and simplicity of plan
nal insecticides, along with the opportunity to ting MIR16

crop yield, are expected to m ake this product ive to growers.

tain circ to make a pres n that use ese instances, the applican d three uses of high p d not sub a new pesticide

s e uses, it qualif ption that its
est. One of the uses that qu

a of for a presu

nother pesticide that is nuing conc

ides registered for control of earwor
re li e m sted in Table 5. Th
s due to human an these in
mental riskTable 5.zea,S.precautioMost commonly usedbicosta, orS. frugiperary text from each prodconvena.ct labtional insecticide products (excluding seed treatmctive ingredients contained in each product,l are shown.ents) used in maize for control ofpsilon,aldAE se classification, Signal word, and keynu eA. iH.PA uProduct NameActive IngredientSignal rdWo/ Use ClassificationAsana XL Insect8 ee:reati rates.sfenvalerateWARNING. Restricted UsHighly toxic to bees.Extmelytoxic tofishandaquc invertebicide.4%A Gra2 te0 cyflue:xic to fish and ife..0%.1%bupirimfosthrinWARNING. Restricted Usztec 2.1%nular InsectideTowildlBaythroid 2yrethroidEmuP Insec2 yfluely toxic to f nd a ic tes.thirnDANGER. Restricted Use:Highly toxic to bees.lsifiableticideExtremish aquatinvertebra5% cB L Incyfe:remely toxic to fish and aquatiluthrinWARNING. Restricted UsHighly toxic to bees.Extc invertebrates.aythroid Xsecticide12.7%Brigade 2ECCapture 2ECInsIns25.1% bifee:remelyand aquatiecticide/Miticide&ecticide/MiticidenthrinWARNING. Restricted UsHighly toxic to bees.Exttoxic to fishc invertebrates.Chlorpyrifos 4E40.7% chle:xicates, st toorpyrifosWARNING. Restricted Usmammals, and birds. HighlyTooxicto fbeesish,.aquatic invertebrmallAG InsecticideC Ins16.6% deltic iamethrinDANGER. Restricted Use:Highly toxic to bees.Extremely toxicto fish and aquatnvertebrates.Decis 1.5EecticideE Gran1.15% bife:emnthrinCAUTION. Restricted UseHighly toxic to bees.Extrelytoxic tofishand aquatic invertebrates.mpower2ular InsecticideE uraly80% spinoToxic to aquatic i tebrates. Toxic tosadCAUTION. General Use:ntrust Natte Insect Controlnverbees.Fanfare 2ECnsecticide/I Nem25.1% bine:Extremely toxic to fish and aquatifenthrinWARNING. Restricted UsHighly toxic to bees.c invertebrates.aticideFuradan 4F Inse44% carboPoisonod.hlyfuanDANGER. Restricted Use:birds and other wildlife. Higus itoxic to bees.f swallowedor inhaleToxic to fish,cticidecide3.75 zeta-c11.25% bi:ExtremypermethrinfenthrinCAUTION. Restricted UseHighly toxic to bees.elytoxic tofishand aquatic invertebrates.Hero InsectiIntrepid 2F Inse22.6% metHazardous to sensitive aquatic invertebrhoxyfenozideCAUTION. General Use:cticideates.SSB-518-07Page 24 of 87SSB-518-07Page 25 of 87Table 5 (cont.).Most commonly used conventional insecticide products (excluding seed treatments) used in maize for control ofA.ipsilon,H. zea,S. albicosta, orS. frugiperda. Active ingredients contained in each product,EPA use classification, Signal word, and keyprecautionary text from each product label are shown.Product NameActive IngredientSignal Word / Use ClassificationLambda-T11.4%λ-cyhalothrinWARNING. Restricted Use:Extremely toxic to fishand aquatic invertebrates.Highly toxic to bees.Lannate SP Insecticide90% methomylDANGER. Restricted Use:Fatal if swallowed. Toxic to fish, aquaticinvertebrates, and mammals. Highly toxic to bees.Lorsban 4E Insecticide44.9% chlorpyrifosWARNING. General Use:Toxic to fish, aquatic invertebrates, small mammals,and birds. Highly toxic to bees.Lorsban 75WG Insecticide75% chlorpyrifosl Use:Toxic to fish, aquatic invertebrates, small mammals,WARNING. Generaand birds. Highly toxic to bees.Mocap 15%GranularNematicide-Insecticide15% ethopropDANGER. Restricted Use:Poisonous if swallowed. Toxic to aquaticorganisms and wildlife.Mustang Insecticide17.1% zeta-cypermethrinWARNING. Restricted Use:Extremely toxic to fishand aquatic inverteHighly toxic to bees.brates.Pounce 25 WP Insecticide25% permethrinWARNING. Restricted Use:Extremely toxic to fish and aquatic invertebrates.Highly toxic to bees.Regent 4 SC Insecticide39.4% fipronilWARNING. Restricted Use:Toxic to birds, fish, and aquatic invertebrates.Sevin brand 4F CarbarylInsecticide43% carbarylCAUTION. General Use:Extremely toxic toaquatic and estuarineinvertebrates. Highly toxic to bees.Silencer Insecticide11.4%λ-cyhalothrinWARNING. Restricted Use:Extremely toxic to fishand aquatic invertebrates.Highly toxic to bees.Success Naturalyte Insect Control22.8% spinosadCAUTION. General Use:Toxic to aquatic invertebrates. Toxic to bees.Warrior with Zeon TechnologyInsecticide11.4%λ-cyhalothrinWARNING. Restricted Use:Extremely toxic to fishand aquatic invertebrates.Highly toxic to bees.The Restricted Use classifi cation, imposed due to advers e environmental effects under no se practi (40 CF 5 1(a)), l s us f the hem icals to certified pesticide applicators who have received special trainin eeded r ndling and application of these products. Personal pr t eq ment re ccupational ex , special li an e r d p are ed r t and use of
Re d Use Pe de W u es st o PA e in products may cause unreasonable adve rse effects on the environme Th ification is
an indication of EPA concern abou t sa o e od

M has th tia c e f ny of the Restricted Use Pesticides that are cu being r f do a s m a on nsideration alo e pla po p3 0 st al te aize is

en o a pr on i es

B. rs Af a te t F in

The 1986 poli r r ns EPA will consider a variety of factors pertaining to the need for a new pesticide ac di cifically its
co ive be f ri y A : “ ency must de e that (1) ith other cu registere co ively less cides; or (3 nefits (in ed those of ative reg es.” A
consideration of th s that its reg on will be

1. Need Factors

While econom s at ut t e inse cts that are effectively controlled by MIR162 ma ot as large as those attributable to O b i d b c o rms, they
are nt n e f io c e c e i n es to rise, the economic s g er r o s o f i o

, S.
alb r S g d ill . n a l c < 10%) will result in a significant economic s i ere is evidence that populations of S. albicosta are spreading eastward and will have the potential to cause greater ha ical maize producing states.

As described in Section II.D, control of a boveground maize insect pe sts is challenging for gr on ional i tic a c are c ensiv e scouting of fields is required to identify the appropriate tim p i ro ave a very

na b p ca m the abovegro in sects a h d t with the insec ticides by virtue of their fee atio n p . n d i i 162 will pr wers wi m e ti n li o gnificant insect pests of maiz rmal u

posure stricte

IR162 rrently ne, th
titled t

Facto

mparat termin rrently
mparat ) the be
altern

istrati

ize are n significa

icosta , o

rm in crit

owers. C

rrow time window during which insecticides can

und feed ding loc ovide gro ces

labe
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its,
there d pe risky
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it ho

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ricti

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a20 m e o

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in encoded in MIR162 m

indi

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tha

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SSB-518-07 Page 26 of 872. Composition Factors

The composition of MIR162 maize is fundament ally different from the composition of conventional insecticide end-use products. The active ingredient , Vip3Aa20, is plantincorporated. It is safer than all currently registered conven tional maize insecticide products. This characteristic of the pr oduct virtually eliminates the occupational and environmental risks currently associated with the application of chemical cont rols for maize insect pests. egistration of this product also provides EPA with an opportunity to reduce the

of unique properties that conventional insecticides o not. The protein is efficacious via a mode of action that is selective to lepidopteran ein is expr essed throughout all tissues of the maize plant. This ensures
rotection where it is needed and eliminates the risk of insecticide failures associated with

ation an d application of comp atible alternative iological, cultural, mechanical and chemical controls, and the establishment of action

res such as crop rotation. MIR162 fits seamlessly into the concept of R manufacture, transportation, storage, and di sposal of millions of pounds of hazardous chemicals annually and to eliminate the greenhouse gas emissions associated with these activities. These product char acteristics support a c onclusion that regist ration of MIR162 is in the public interest.

3. Usage Factors

The safety, convenience, and simplicity of planting MIR162 hybrids compared to the application of conventional insecticides, along with the opportunity to extract an economic benefit through increased crop yield, are expected to make this pr oduct attractive to growers.

4. Performance Factors

Two years of extensive efficacy field trials , conducted at multiple locations under varying levels of insect pressure, have demonstrated the superior l eaf, stalk, and ear protection provided by MIR162 maize compared to hybrid s treated with a conv entional insecticide product. Vip3Aa20 possesses a number
d insects. The prot

p timing of applications or unfavor able environmental conditions. Furthermore, the delivery of Vip3Aa20 in the maize seed and its production in plants eliminates many risks associated
with conventional insecticid e usage, some of which include improper calibration and maintenance of application equipment, ha ndling of hazardous chem ical insecticides, container disposal, chemical mispl acement, runoff, and spray drift.

Integrated pest management (IPM) in agricult ure includes insect scouting or monitoring to determine pest populations, consider
b thresholds for agricultural inputs. The delivery of pest management interventions on target and on time is a key to successful IPM. Pl anting of MIR162 hybrids provides much greater accuracy of application compared to chemical treatments due to the localization of Vip3Aa20 within the plant tissues. Timing of applica tion is not a factor with MIR162 hybrids since Vip3Aa20 is present in the plant throughout the growing season. Planting of MIR162 hybrids is compatible with current insect scout ing and monitoring programs that provide data upon which to base crop manageme nt decisions. The product is also fully compatible with
cultural control measu

SSB-518-07 Page 27 of 87in tegrated pest management for maize. Supe rior protection of crop yield and a seamless fit
e that registration of MIR162 maize is in the public interest.

he selectivity of Vip3Aa20 for lepidopteran pe sts minimizes risk for nontarget organisms.
dentification studies has been conducted with nontarget indicator species, cluding many species that are part of the mai ze ecosystem. No adverse effects attributable

ost currently registered maize insecticides exhibit a broad spectrum of activity and present
ude of nontarget terrestrial and aquatic species when they are pplied. The conventional inse cticide products currently used for control of aboveground

o reduce applications of onventional insecticides and improve grain quality by reducing mycotoxin levels. These with IPM programs indicat

5. Risk Factors

A standard battery of mammalian toxicity st udies failed to provide any evidence of Vip3Aa20-induced adverse effects. The protei n is rapidly degraded in mammalian digestive systems and it bears no amino acid sequence sim ilarities to known toxins and allergens. Since the insecticidal protein is plant-inco rporated, the opportunity for exposure when handling and planting seed is minimal. Planting of MIR162 hybrids will essentially eliminate the occupational health risks currently associated with chemical controls for leaf and ear feeding insect pests.

T
A series of hazard i
in to Vip3Aa proteins were observed in these studies, even at exposure levels exceeding expected environmental con centrations (Raybould, 2007a).

M
discrete risks to a multit

a insects are of concern to growers, consumer s, and the EPA for environmental reasons.

Fusarium ear rot is the most common ear rot disease in the Midwest and is closely associated with insect feeding damage to maize ears. Although the disease does not cause significant yield loss, it reduces grain qual ity, and increases the fungi that can produce mycotoxins, such as fumonisins. Mycotoxin contamination of maize grain presents a potential threat to
livestock health and it is occasio nally necessary to reject or re formulate field lots because of contamination. Due to the superior protection from insect ear feeding damage that will be afforded by planting MIR162 hybrids there is a potential health benefit for the livestock industry resulting from reduced mycotoxin levels in livestock feed.

Thus, the introduction of MIR 162 technology has the potential t

c facts indicate that registration of MIR162 is in the public interest.

6. Economic Factors

At the request of Syngenta a study was unde rtaken by agricultural economists at North Carolina State University to develop an estimate of the value to U.S. farmers of the MIR162 maize trait technology. First they consider ed the potential economic effects of MIR162 introduction on the market for existing insect-p rotection trait technol ogies. Second, they commissioned a grower survey to assess wil lingness to adopt the new technology. Lastly, they estimated the spatial distribu tion of the costs of control for H. zea and S. albicosta and how these costs might change in future year s. A full report for this economic study is

SSB-518-07 Page 28 of 87contained in Addendix 1. This an alysis only examined the potenti al benefits associated with improved control of H. zea and S. albicosta

.

Following genera l economic principles, the in troduction of a new tech nology will have an

ffect on the market for existing technologies that is beneficial to users of either technology.

be 4.8 bu/ac. Exam ination of data provided by these growers r the past five seasons suggests that yield losses attributable to the two pests are increasing.

would purchase MIR162 hybrids they were available and would plant them on an average of 500 acres per farm four years

three rgest maize producing states (IA, IL, and NE). An upper-bound estimate is all that was

t re gistration of MIR162 in the public interest.

oncurrent with the application for a FI FRA §3 registration of the Vip3Aa20 plant- maize, S yngenta is also submitting an application for gistration of a new product that contains the combined plant- incorporated protectant active e This will come in the form of downward pressure on prices of the competing technologies. This is beneficial to growers because prices of maize traits will tend to remain lower and
more stable in the future than would otherwise be the case.

From data collected in a telephone survey of 150 maize growers in 12 states, average yield
losses in 2006 attributable to H. zea were estimated to be 4.9 bu/ ac and losses attr ibutable to
S. albicosta were estimated to
fo This conclusion is supported by analysis of insecticide use data for 2005 and 2006 which indicate that economically si gnificant infestations of H. zea and S. albicosta are on the rise in the Corn Belt and Great Plains. As the price of maize grain increases, the amount of feeding damage needed to exceed an economic threshold for applying corrective m easures decreases. The grower survey results indicate that 70% of respondents
if
after introduction.

A potential economic benefit for maize grow ers from the commercial introduction of MIR162 hybrids has been computed in the form of an upper-bound estimate for the
la able to be computed because county-specific info rmation is not available on infestation levels of the insects being investigated. Providing growers with a means to effectively control H.

zea and S. albicosta in these three states alone provide s an economic benefit of up to $371 million annually. This is an upper-bound estimate on value available to growers; it assumes a 100% market share for MIR162 hybrids and does not take into account potential price responses for competitive products. Ultimately, some portion of the economic gain derived by growers using this new technology will be pass ed along to consumers in the form of lower commodity prices. These substantial economic be nefits indicate tha
is

V. Public Interest Factors Applicable to Bt11xMIR162 Maize

C incorporated protectant in MIR162

re ingredients in Bt11 maize and MIR162 maize. The transgenic traits in Bt11 and MIR162 maize have been combined by traditional breeding methods to create maize hybrids, designated herein as ‘Bt11xMIR162 maize’, th at possess the combined insect-control characteristics of Bt11 and MIR162. Hybrids that contain multiple transgenic traits are often referred to as ‘stacked’ hybrid s or hybrids containing ‘pyram ided’ traits and genes.

The Cry1Ab active ingredient in Bt11 maize was first registered by the EPA in 1996 (EPA

SSB-518-07 Page 29 of 87Reg. No. 67979-1; OECD ID SY N-BTØ11-1). At that time, the Agency found that its registration was in the public interest. Upon extension of th e Bt11 registration in 2001, EPA

iterated its public interest finding, which is summarized in the Biopesticide Registration

he benefits of both Bt11 and MIR162 will be reflected in Bt11xMIR162 combined-trait

m B. thuringienses

.

lthough conventional chemical insecticides are available and sometimes applied to age (see Section II.D and PA, 2001), the efficacy of such products typica lly depends on careful scouting of fields for re Action Document for Bt crops (EPA, 2001) and reflects th e first few years of grower experience and continued research on the benefits of Bt11 maize. 1 Therefore, the various environmental, health, agricultural, and economic benefits of Bt11 maize will not be restated
here.

T maize hybrids. While it is not necessary to rest ate the benefits of the traits conferred by Bt11 and MIR162 maize individually, some specifi c and unique benefits that result from combining the traits in a single product are described below.

A. Product Description and Intended Use

Bt11 maize plants produce a Cry1Ab protein from B. thuringiensis for control of certain lepidopteran maize pests. The plants also produce phosphinothricin acetyltransferase, an enzyme that was used as a selectable marker in transformation and which confers tolerance of the plants to glufosinate herbicides. MIR162 maize plants produ ce a variant of the Vip3Aa protein fro

Bt11xMIR162 hybrids will provide growers with protection and convenience for the control of a wide spectrum of lepidopteran pests. Th e broad efficacy of the product will allow maize growers across wide geographies to benefit from routinely planti ng Bt11xMIR162 hybrids to control the majority of lepidopter an maize pests that could be pr evalent in any gi ven year. In addition to the insect-control advantages of th e product, the herbicide tolerance conferred by the phosphinothricin acetyltransferase protei n in Bt11xMIR162 maize will provide growers with the option of using glufosin ate products for weed control.

B. Factors Affecting a Public Interest Finding

1. Need Factors

A minimize the yield losses associated with lepidopteran pest dam

E the presence of larvae and on timing of insect icide applications to achieve economically
beneficial levels of control. Many maize growers typically do not treat their fields to control lepidopteran infestations for a number of reasons, including inco nvenience, lack of awareness of the yield losses being incurred, and the costs and hazards associated with insecticide applications. After initially feeding on external plant tissues, several lepidopteran maize pests bore into the stalk or ear, wher e they are inaccessible to foliar insecticide applications. Even with carefully timed a pplications, under typical use conditions the

1 The registration for the plant-incorporated pesticide ac tive ingredient in Syngenta’s Bt11 maize is scheduled to expire on October 15, 2008. Prior to this expiration date, Syngenta will request an amendment to extend the registration of Bt11 maize.

SSB-518-07 Page 30 of 87efficacy of foliar or soil-applied insecticide pr oducts cannot achieve the consistent level of season-long control that is possible usi ng the technology represented by Bt11xMIR162
maize.

Maize hybrids expressing Cry1F are commercia lly available and offer a measure of broadspectrum lepidopteran control. In 2006, Cry1F hybrids accounted for 7.1% of maize acres plante d in the U.S. (Doane, 2006a). In cont rast, 34.9% of acres in 2006 were planted to

ry1Ab-containing hybrids (event Bt11 or MON 810), which control a narrower spectrum of

ids, Bt11xMIR162 hybrids are xpected to provide a level of broad lepidopteran control th at is unsurpassed by currently cticide products. For H. zea , in particular, t11xMIR162 hybrids have been shown to provide excellent control that meets EPA insect

t11 maize provides control of O. nubilalis and D. grandiosella , two key pests of U.S. maize

at a level that will outperform urrent technologies. Figure 8 illustrates the performance be nefits of stack ing the Cry1Ab lectively, the resu lts of field efficacy trials emonstrate that Bt11xMIR162 maize will be pr otected from feeding damage caused by the

ests: O. nubilalis

, D. grandiosella

, D. crambidoides

, H. zea

, S. frugiperda,
. nebris

, D. saccharalis

, A. ipsilon

, S. albicosta , and S. exigua (Huber et al. , 2007; White et

and high pest pressure. C lepidopteran pests. It is reasonable to predic t that, as the benefits of broad-lepidopteran control are more widely recogni zed and available to growers, the relative proportion of acres planted to such hybrids will change. While it ha s not been possible to conduct direct side-byside efficacy comparisons of Cry1F and Bt11xMIR162 hybr

e available Bt hybrids or conventional in se B resistance management criteria for ‘high dose’ (see V.B.5 below and Kurtz et al. , 2007a), whereas Cry1F hybrids provide only ‘suppr ession’ of this pest (EPA, 2005).

2. Performance Factors

B (see Table 1). Additionally, Bt11 maize provides some control or suppression of H. zea

, S. frugiperda

, D. crambidoides

, P. nebris , and D. saccharalis . The activity spectrum and efficacy of MIR162 maize for control of severa l lepidopteran maize pests are described in
Section III.

Bt11xMIR162 maize will combine the efficacy of Bt11 maize and MIR162 maize to provide broad-spectrum control of major U.S. lepidopteran maize pests

c and Vip3Aa20 proteins in the sa me hybrid. Col
d following insect p

P
al. , 2007a, 2007b, 2007c, 2007d, 2007e). As MIR162 maize does not control O. nubilalis , a significant maize pest in many areas of the U.S ., it will not likely be marketed as a standalone product.

Studies to quantify the yield advantage of Bt11xMIR162 maize across multiple geographies, environmental conditions, cropping practices and pest pressure are ongoing. The data
available from a limited set of trials indicat e that, in the absence of pest pressure, Bt11xMIR162 hybrids will provide the same yield as their nontransgenic counterparts. It is expected that, as a result of superior and season-long control of si gnificant lepidopteran pests, Bt11xMIR162 hybrids will demonstrate significant yield advantages under both low

Some lepidopteran pests, such as A. ipsilon , can cause major yield loss es due to cutting of an

SSB-518-07 Page 31 of 87entire stand of maize in a field. If the crop is left untreated, the grower is sometimes forced to replant an entire field. Such catastrophic losses will be prevented by planting

Bt11xM IR162 maize. Other, stalk-boring lepidopt eran larvae cause phys iologic yield loss

ecause the stalk damage interferes with nutrien t uptake and increases susceptibility to plant

in Bt11 maize 0 CFR §174.511 and 40 CFR §174.522, respectively). The selectable ma rker in MIR162 empt from tole rances in all crops (40 CFR §174.527) and a mporary tolerance exemption for Vip3Aa20 in MIR162 maize is currently in place (40

afety. The proteins are individually not toxic to mammals and the lepidopteranecific toxins in Bt11xMIR162 ma ize do not interact in a manner that results in toxicity to

he combined mammalian and environmental sa fety profile of Bt11xM IR162 indicates that

the intr oduction of the first (Cry1Ab) Bt maize hybrids in 1996 and 000, 3.9 million fewer acres of ins ecticide applications were ma de to maize for control of b diseases. They can also cause severe stal k lodging and dropped ears , which results in a physical yield loss because the ears cannot be m echanically harvested by the combine. Earfeeding pests reduce grain yield and quality, and occasionally result in elevated grain fumonisin levels that render the grain unsafe and unusable as food or feed (see Sections IV.B.5 and V.B.3 and EPA, 2001). In the pr esence of pest pressure, Bt11xMIR162 maize hybrids can be expected to preserve yield po tential, grain quality, and silage quality by minimizing the damage that could otherw ise be caused by lepidopteran insects.

3. Risk Factors

Syngenta has provided data to the Agency suppor ting the mammalian safety of the transgenic proteins produced in Bt11xMIR162 maize. Perm anent exemptions from the requirement of a tolerance in all crops exist for both Cry1Ab an d the marker protein produced (4 maize is permanently ex

te CFR §174.528).

The available data further support a conclusion that there will be no harmful interactions between the transgenic proteins in Bt11xMIR 162 maize that might trigger a concern for
mammalian s

sp nontarget insects (Raybould, 2007b). A FIFRA Sc ientific Advisory Panel concluded that Bt
insect toxins that are indi vidually nontoxic to mammals do not pose a mammalian safety concern when present in combination (FIFRA SAP, 2004).

Despite the broad-spectrum lepidopteran activ ity of Bt11xMIR162 maize, the environmental safety assessment supporting this product (Raybould, 2007b) indicates that it will pose no significant risk to nontarget organi sms. The environmental safety assessment also considered the potential for effects on endangered species and for synergistic effects of the Cry1Ab and Vip3Aa20 proteins on nontarget Lepidoptera and other nontarget species.

T the product will pose no significan t risks. Accordingly, it offers health and environmental advantages over current chemical alternativ es for control of lepidopteran pests.

EPA estimated that, prior to the adoption of Bt maize hybrids, an annual average of 6.3
million acres were treated with conventional insecticides for control of maize-boring lepidopteran pests (primarily O. nubilalis ) in the U.S. (EPA, 2001). The Agency further
estimated that between

2 the same pests. As detailed in Section III, the additional control spectrum provided by the

SSB-518-07 Page 32 of 87Vip3Aa20 protein in MIR162 maize is expected to allow maize growers to further reduce their use of insecticides for lepidopteran c ontrol. Significantly, many of the conventional
insecticides curre ntly registered for le pidopteran control in maize are Restricted Use Pesticides (see Table 5).

For maize growers who currently rely upon conventional insecticide applications for lepidopteran control, Bt11xMIR162 maize will a llow them to significantly reduce, if not eliminate, the need to apply chemical controls for these pests. This will represent both a reduced health and safety risk for agricultu ral workers, and will reduce the impact of
insecticide use on wildlife and the environment.

A n additional food and feed safety benefit of Bt11xMIR162 is its potential to reduce the n, a harmful fungal toxin, in maize grain. As summarized by EPA (2001),
rain from Bt maize hybrids (including Bt11 maize) is associated with significantly reduced

lthough some maize-growing areas experience significant lepidopter an infestations in most

. in 1996. This is a testament to e actual yield-preserving benefits that Bt maize growers have experienced over the long

or pest pressure or apply other control measur es for lepidopteran larvae.

tely predict e magnitude of economic benefits that gr owers of Bt11xMIR162 maiz e will realize beyond level of fumonisi

g levels of fumonisin. This is an indirect bene fit of protecting maize ears from feeding damage by lepidopteran pests. The additional cont rol of ear-feeding pe sts, particularly H. zea and S.
albicosta , that will be provided by Bt11xMIR162 mai ze will likely further reduce mycotoxin contamination in grain.

4. Economic Factors

A years, it is not possible for individual growers to accurately predict wh ether lepidopteran pest pressure will be economically significant in any particular growing season. Growers must make seed purchase decisions prior to kno wing whether the seed price premium for lepidopteran control in a given year will actuall y be recouped as higher crop yields when compared to the seed cost for unprotected hybrids or the costs of other control measures that might be applied. Nevertheless, the acres planted to Bt maize have continued to increase steadily since the first Bt hybrids were introduced in the U.S
th term, and to the value of the built-in ‘insurance’ against the occasional severe pest outbreak that could otherwise result in high economic losses. For many growers, the broad lepidopteran control offered by Bt11xMIR162 hybrids will represent a higher insurance value than currently available Bt products. Additionally, Bt11xMIR162 hybrids will offer unsurpassed convenience to grower s by reducing the need to scout fields f

to

The continued success of Bt maize hybrids in the marketplace since their initial introduction
attests to their economic benefits to growers. It is difficult at this time to accura
th the previously described benefits expected from growing either Bt 11 (EPA, 2001; Van Duyn, 2005) or MIR162 maize (see Appendix 1). Ne vertheless, the improved pest protection profile of Bt11xMIR162 maize ca n be expected to translat e into correspondingly higher overall economic benefits to growers, cons umers, and other downstream users of maize products. The magnitude of these economic be nefits will necessarily depend upon the seed price premium paid for the pest-control traits, the level of local pest pressure, and the value

SSB-518-07 Page 33 of 87of the crop. As discussed in Section II .A, commodity prices for maize grain have dramatically increased recently due to high demand for fuel ethanol production, and sustained demand is predicted for the coming years. Such demand will function
to increase the value of a grower’s investme nt in any agricultural practice, technology, or

roduct, including the Bt11xMIR162 traits, that increases or preserves yield.

pest control option and tool for growers. creased grower choice can be expected to exert downward pressure on the cost of all

d Vip3Aa20 pr oteins are present in these hybrids at levels that have een demonstrated to provide a high-dose for control of O. nubilalis, H. zea and S. izing the risk of resist ance developing in these species. t11xMIR162 hybrids offer IRM advantages in comp arison to other contro l options that do

n th e report by Kurtz et al. (2007a), yngenta provides data and rationale to just ify reduction of the maize refuge in cottonp

Another predicted economic benefit for grow ers and downstream consumers is increased competition in the marketplace for pest-control products, including hybrid seed from multiple providers of lepidopteran-tolerant Bt varieties. The commercial availability of Bt11xMIR162 hybrid maize seed will represent a significant new

In products that offer contro l of lepidopteran pests.

5. Other Factors

A unique benefit offered by Bt11xMIR162 hybrids rela tes to their distinct advantages in the
area of insect resistance ma nagement (IRM). As fully detailed in the report by Kurtz et al. (2007a), the Cry1Ab an
b frugiperda , thus minim

B not demonstrably provide a ‘high dose’ agai nst the target pests. Moreover, Vip3Aa20 operates by a mode of action different from th at of Cry1Ab or Cry1F and targets a unique binding site(s) in suscep tible larvae. The avai lable data support a conclusion that Vip3Aa20 shows no potential for cross-resi stance with Cry proteins (Lee et al. , 2003). Thus, for H. zea
and S. frugiperda , which are sensitive to both Cr y1Ab and Vip3Aa20, Bt11xMIR162 maize is predicted to significantly extend the durability of both traits for control of these pests because local populations are very unlikely to evolve resistance to two proteins that act on independent target sites.

The possibility of resi stance development in H. zea has been of particular concern to the EPA, as it is also a pest of cotton and has the potential to u ndergo selection pressure from
both Bt maize and Bt cotton varieties that express simila r Cry proteins, where the two crops are grown in the same geographies. The prin cipal reason that the EPA requires growers in cotton-growing areas to plant 50% of their maize acres to non- Bt maize hybrids concerns the potential for resistance evolution in H. zea populations. I
S growing areas from 50% to 20% of maize acr es for growers of Bt11xMIR162 maize. No
other Bt product offers comparable IRM advantages in maize. 2

For growers of Bt11xMIR162 maize hybrids, th e reduced refuge requirement in cottongrowing regions will translate in to a higher proportion of insect-protected maize acres, with a proportional increase in all the atte ndant benefits of the product in these areas. As an added advantage, compliance with the refuge require ment for IRM can be predicted to increase

2 Syngenta’s Cry1Ab+Vip3Aa19 stacked cotton product, VipCotTM cotton, also has similar IRM advantages for the same reasons. An application for FIFRA §3 registration of VipCot cotton is currently under review.

SSB-518-07 Page 34 of 87because Bt maize growers in cotton-grow ing regions have heretofore not been able to fully experience the benefits enjoyed by Bt maize growers in other regions of the U.S. The potential for increased maize acres in cotton-gr owing regions can also help meet the current high demand for maize grain.

6 . Public Interest Finding for Bt11xMIR162

for growers and downstream consumers is

nticipated in the form of increased price co mpetition in the market place for pest-control
1xMIR162 maize offers health and environmental safety advantages over onventional insecticides, as well as insect resi stance management benefits that will help to

eferenced or described in previous sect ions of this document. The gnificant benefits of MIR604 maize as a stand-alone product for rootworm control have

he benefits offered by the component trai ts in Bt11xMIR162xMIR604 maize will not be The information presented here in supports a conclusion that registration of Bt11xMIR162 maize is in the public interest. Use of Bt11xMIR162 hybrids by U.S. maize growers is predicted to offer crop yield advantages both for growers who do not currently use insecticides to control lepidopt eran pests as well as for gr owers who use other options for lepidopteran control. Another economic bene fit

a traits. Use of Bt1

c preserve the durability of this and other Bt -based products for lepi dopteran control.

VI. Public Interest Factors App licable to Bt11xMIR162xMIR604 Maize

Concurrent with the applic ation for a FIFRA §3 regist ration of MIR162 maize and Bt11xMIR162 maize, Syngenta is submitting an a pplication for registration of a new product that contains the combined active ingredie nts in Bt11 maize, MIR162 maize, and MIR604 maize (EPA Reg. No. 67979-5; OECD ID SYN-IR 6Ø4-5). The transgenes in Bt11, MIR162, and MIR604 maize have been combined by tr aditional breeding methods to create maize hybrids, designated herein as ‘Bt11xMIR162xM IR604 maize’, that ex press the combined insect-control traits of Bt11, MIR162, and MIR604 maize.

The characteristics and benefits of Bt11xMIR162 maize, and the component traits from Bt11 and MIR162 maize, are r
si been summarized by EPA (2007) and described in detail by Syngenta in a previous regulatory submission (Steiner et al. , 2004).

Additionally, the plant-incorpor ated protectants in Bt11 mai ze and MIR604 maize have been combined by traditional breeding in Bt11xMIR 604 maize, for combined control of certain lepidopteran and rootworm (coleopteran) pests. The plant-incorporated protectants in Bt11xMIR604 maize were approved by the EP A in 2007 (EPA Reg. No. 67979-8) and the
benefits of Bt11xMIR604 maize have been desc ribed by Steffens and Tinsworth (2006).

T restated here. This section will focus on the specific characteristics of
Bt11xMIR162xMIR604 maize and the benefits it a ffords by combining multiple insecticidal traits in a single product.

SSB-518-07 Page 35 of 87A. Product Description and Intended Use

Bt11xMIR162xMIR604 maize plants combine the transgenic traits of Bt11, MIR162, and MIR604 maize and thereby pr oduce a total of six transgenic proteins: the B. thuringiensis

erived insecticidal proteins Cry1Ab, Vip3Aa20 and modified Cry3A (mCry3A).

rower s with performance and convenience in e control of a wide spectrum of lepidopteran pests and important Diabrotica rootworm

. Need Factors

he available alternative methods of lepidopteran control have been summarized in Sections

lthough other stacked transgenic maize hybr ids offering combined lepidopteran and

ferings.

), iabrotica longicornis barberi (northern corn rootworm), and Diabrotica virgifera zeae

7c).

Combining Cry1Ab, Vip3Aa20, and mCry3A traits in a single maize hybrid retains the insect control efficacy of the individual prot eins. Accompanying the present submission are d Bt11xMIR162xMIR604 hybrids will provide g
th pests of maize. The broad efficacy of the product will allow growers across wide geographies to benefit fr om routinely planting Bt 11xMIR162xMIR604 maize hybrids to control the majority of lepidopter an and rootworm pests that mi ght be prevalent in any given year. In addition to the insect control advantages of the product, the herbicide tolerance conferred by the PAT protein in Bt11xMIR162xM IR604 maize will provide growers with the option of using labeled glufosin ate products for weed control.

B. Factors Affecting a Public Interest Finding

1

T
II.D and IV.A. The alternative methods of ma ize rootworm control have been summarized separately (EPA, 2007; Steiner et al. , 2004), and include soil-appl ied insecticides, seed treatments, and transgenic maize hybr ids expressing coleopteran-specific cry genes. Bt11xMIR162xMIR604 hybrids are expected to deliver superior control of lepidopteran pests in comparison to alternative control methods, and rootworm control that is equal to or superior to that of alternative products.

A coleopteran control are currently available in the U.S., direct efficacy comparisons with Bt11xMIR162xMIR604 hybrids have not been possible. It is expected that Bt11xMIR162xMIR604 hybrids will provide unsurpassed control of target pests. Their excellent broad-lepidopteran control, particularly for H. zea and S. albicosta , can potentially result in better performance than competitor of

2. Performance Factors

In addition to the multiple lepidopteran pest s controlled by Bt11xMIR162 maize (see Section V.B), the mCry3A protein in Bt11xMIR162xMIR 604 maize will provide protection against feeding damage caused by larvae of Diabrotica virgifera virgifera (western corn rootworm

D (Mexican corn rootworm), all of which are majo r coleopteran pests of maize in the U.S. The mCry3A protein has a relatively narrow spectrum of activity among coleopteran species, and
has been demonstrated to have no adverse affects on nontarget beetles or other nontarget arthropods (Raybould, 200

SSB-518-07 Page 36 of 87reports of efficacy studies in O. nubilalis (W hite et al. , 2006f), H. zea (White et al. , 2006g), . frugiperda (White et al. , 2006h) and D. virgifera virgifera (White et al. , 2006i) that

nvironmental benefits prev iously described for Bt11 maize (EPA, 2001), IR162 maize (see Section IV.B.5), MIR604 maize (EPA, 2007; Steiner et al. , 2004), and

rowers be tter protect the safety of their workers and
e environment.

on V.B.

r support a conclusion that there will be no harmful interactions etween the transgenic proteins in Bt 11xMIR162xMIR604 maize that might trigger a

the environmental safety assessment supporting this product aybould, 2007b) indicates that it will pose no signi ficant risk to nontarget organisms. This S substantiate the predicted efficacy of co mbining multiple insecticidal traits in Bt11xMIR162xMIR604 maize hybrids. Therefore, it is reasonable to assume that growers
will realize the cumulative bene fits of all three insecticid al traits in this product.

Studies to quantify the yield advantag e of Bt11xMIR162xMIR604 maize across multiple geographies, environmental conditions, croppi ng practices and pest pressure are ongoing.
Data available from a limited set of trials in dicate that, in the absence of pest pressure, Bt11xMIR162xMIR604 hybrids will provide the same yield as their nontransgenic counterparts. It is expected that, as a result of superior and season-long control of multiple significant lepidopteran and coleopteran pests, Bt11xMIR162xMIR604 hybrids will demonstrate significant yield adva ntages under pest pressure.

3. Risk Factors

The health and e

M Bt11xMIR162 maize (see Section V.B.3) w ill also apply, in combination, to Bt11xMIR162xMIR604 maize. Therefore, this pr oduct offers significa nt advantages over other pest control methods, as it will allow U.S. farmers to reduce their use of soil-applied
insecticides for rootworm control. The EPA has been concerned for several years about the hazard and risk profiles of many of soil-applied rootworm insecticides (EPA, 2007). The availability of Bt11xMIR162xMIR604 maize hybrid s will further help the EPA achieve its risk-reduction objectives, and will help g
th

Syngenta has provided data to the Agency suppor ting the mammalian safety of the transgenic proteins produced in Bt11xMIR162xMIR604 maize. The mCry3A protein in Bt11xMIR162xMIR604 maize is permanently exempt from food and feed tolerances in all corn (40 CFR §174.505). The tolerance exemptions supporting the other transgenic proteins
in Bt11xMIR162xMIR604 maize are desc ribed in Secti

The available data furthe
b concern for mammalian safety. The proteins ar e individually not toxi c to mammals and the insect-specific toxins in Bt11xMIR162xMIR604 maize do not interact in a manner that results in toxicity to nontar get insects (Raybould, 2007c). A FIFRA Scientific Advisory
Panel also concluded that Bt insect toxins that are indivi dually nontoxic to mammals do not pose a mammalian safety concern when present in combination (FIFRA SAP, 2004).

Despite the broad-spectrum lepidopteran activity and coleopt eran activity of Bt11xMIR162xMIR604 maize, (R assessment also considered the potential for e ffects on endangered species and for synergistic effects of the Cry1Ab, Vip3Aa20 and mCr y3A proteins on nontarget insects.

SSB-518-07 Page 37 of 87The combined mammalian and environmen tal safety profile of Bt11xMIR162xMIR604 maize indicates that the product will pose no signi ficant safety risks. Accordingly, it offers significant health and environmental advantag es over current chemical alternatives for control of lepidopteran and rootworm pests. For maize growers who current ly rely upon

onventional insecticide a pplications for lepidopteran and rootworm control,

he broad efficacy of Bt11xMIR162xMIR604 hybrid s will provide ‘insurance’ for growers y multiple pests that might ot herwise cause significant economic loss in any iven year. The same broad efficacy will provide convenience for growers, as they will be

4). However, the improved pest-c ontrol profile of Bt11xMIR162xMIR604 aize can be expected to translate into corr espondingly higher overall economic benefits to

h demand will operate to crease the value of a grower’s investment in any agricultural practice, technology, or maize

choice an be expected to exert downward pressure on the cost of products that offer control of

162xMIR604 c Bt11xMIR162xMIR604 maize will allow them to sign ificantly reduce, if not eliminate, the need to apply chemical controls for these pests. This will re present both a reduced health and safety risk for agricultural workers, and will re duce the impact of insecticide use on wildlife
and the environment.

4. Economic Factors

T against damage b

g able to eliminate the need to apply both a soil insecticide for control of Diabrotica
rootworms and A. ipsilon , and a foliar insecticide later in the season for foliar insects. It will
also reduce or eliminate their need to scout fields for pest pressure.

The increasing acreage planted with Bt maize hybrids for lepidopteran and rootworm control, attests to their economic benefits to growers. It is difficult at this time to accurately predict the magnitude of economic benefits that growers of Bt11xMIR162xMIR604 maize will realize beyond those previously described benefits expected from growing either Bt11 (EPA, 2001; Van Duyn, 2005), MIR162 maize (see Section IV.B.6), or MIR604 maize (EPA, 2007;
Steiner et al. , 200

m growers, consumers, and other downstream user s of maize products. The magnitude of these
economic benefits will necessari ly depend upon the seed price premium paid for the pestcontrol traits, the level of local pest pressure , and the value of the crop. Commodity prices for maize grain have dramatically increased recently due to high demand for fuel ethanol, and sustained demand is predicted for the coming years. Suc
in traits that increase or preserve yield.

Another predicted economic benefit for grow ers and downstream consumers is increased competition in the marketplace for pest-control products, including hybrid seed from multiple providers of lepidopteran-activ e and or rootworm-active tr ansgenic varieties. The commercial availability of Bt11xMIR162xMIR604 hybrid maize seed will represent a significant new pest control option and tool avai lable to growers. Increased grower

c lepidopteran and rootworm pests.

5. Other Factors

A detailed insect resistance management plan and discussion for Bt11xMIR

SSB-518-07 Page 38 of 87m aize is provided in an accompanying data volume (Kurtz et al. , 2007b). The same insect

ing for Bt11xMIR162xMIR604 Maize

ich will benefit growers
nd others in the maize value chain. Bt 11xMIR162xMIR604 maize also offers health and

rooks, N. (2007). U.S. agricultural trade update. Economic Research Service, United

icke, F. F. and W. D. Guthrie (1988). Th e most important corn insects. Pp 767-867. In n Improvement. Third Edition. Sprague, G. F. and J. W. Dudley (eds.). American Society of Agronomy, Crop Scienc e Society of America and Soil Science resistance management benefits described above (Section V.B.) for Bt11xMIR162 maize will also apply to Bt11xMIR162xMIR604 ma ize. Accordingly, a 20% non- Bt maize refuge in cotton-growing regions will be justified. The stack ing of three insecticidal proteins in this product is not expected to increase selection pressure for cross-resi stance among local pest populations, owing to the different modes-of -action and target sites for the Cry1Ab, Vip3Aa20, and mCry3A proteins. Because the mCry3A trait in Bt11xMIR162xMIR604 maize has good efficacy against its target root worm pests, introduction of this product is expected to help exte nd the durability of othe r commercially available rootworm-protected Bt maize products.

6. Public Interest Find

The information summarized herein suppor ts a conclusion that registration of Bt11xMIR162xMIR604 maize is in the public interest. Use of Bt11xMIR162xMIR604 hybrids by U.S. maize growers is predicted to offer crop yield advantages and important new options for control of major pest s, all built into a single seed product. The availability of a new product for lepidopteran and rootworm control will provid e choices for growers in the marketplace, and lead to increased price comp etition for traits, wh

a environmental safety advantages over the use of conventional insecticides, as well as insect resistance management benefits that will preserve the durability of this and other Bt -based products.

VII. References

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Brookes, G. and P. Barfoot (2005). GM cr ops: The global economic and environmental impact – The first nine years 1996-2004. AgbioForum 8 :187-196.

B States Department of Agriculture. Report FAU-122, February 15, 2007. Accessed on May 17, 2007: http://usda.mannlib.corne ll.edu/usda/current/FAU/FAU-02-15-2007.pdf

Cook, K. (2004). Western bean cutworm ( Loxagrotis albicosta Smith). Insect fact sheet. University of Illinois integrated pest management bulletin. Accessed May 17, 2007: http://ipm.uiuc.edu/fieldcrops/ins ects/western_bean_cutworm/index.html

D
Corn and Cor

Society of America, Madison, Wisconsin.

SSB-518-07 Page 39 of 87Doane (2005). 2005 AgroTrak. The US agro chemical usage study. A Doane Marketing Research syndicated tracking st udy. St. Louis, Missouri.

Doane (2006a). US seed corn TraitTrak st udy. A Doane Marketi ng Research syndicated tracking study. St. Louis, Missouri.

Doane (2006b). 2006 AgroTrak. The US ag rochemical usage study. A Doane Marketing Research syndicated tracking st udy. St. Louis, Missouri.

D raper, C. (2007). MIRVIP3A-0106 single dose oral toxicity study in mice. Unpublished Syngenta report, No. AM7543-REG.

EPA (1986). Conditional registration of new pesticides. Federal Register 51 :7628.

EPA (2001) Biopesticides Regi stration Action Document - Bacillus thuringiensis plantincorporated protectant s. Accessed May 17, 2007: http://www.epa.gov/oppbppd1/biope sticides/pips/bt_brad.htm

EPA (2005) Updated Biopesticide Regi stration Action Document (BRAD) for Bacillus thuringiensis Cry1F corn. Accessed May 17, 2007: www.epa.gov/pesticides/biopesticid es/ingredients/t ech_docs/brad_006481.pdf

E PA (2007) Biopesticides Registration Action Document - Modi fied Cry3A protein and the al necessary for its production (via elements of pZM26) in event MIR604 corn. Accessed May 17, 2007: genetic materi

http://www.epa.gov/pesticides/biopes ticides/ingredients/ tech_docs/brad_006509.pdf

.

ruch, J., G. Warren, M. Mullins, G. Nye, J. Craig, and M. Koziel (1996). Vip3A, a no Est vel
ctivities against lepidopteran insects. Proceedings of the National Academy of Sciences 93 :5389-

ernandez-Cornejo, J. and M. Caswell (2006). The first decade of ge netically engineered Bacillus thuringiensis vegetative insecticidal protein w ith a wide spectrum of a

5394. MRID 468808-04.

F crops in the United States. Economic Resear ch Service, United States Department of Agriculture. April 2006. Accessed on May 17, 2007: http://ers.usda.gov/publications/eib11/eib11.pdf

FIFRA SAP (2004). Minutes of the FIFRA Scientific Advisory Panel meeting held June 8- 10, 2004: Product characterization, human hea lth risk, ecological risk, and in sect May 17, 2007: http://www.epa.gov/scipoly/sap/ meetings/2004/index.htm#june.

Gra

ta report, No. SSB-023-06 resistance management for Bacillus thuringiensis ( Bt ) cotton. Accessed

ser, G. and C. Stacy (2006) . Characterization of micr obially-produced Vip3A test substance MIR162VIP3A-0106 and comparison with Vip3A expressed in event MIR162- derived maize (corn). Unpublishe d Syngen

SSB-518-07 Page 40 of 87Gray, M. and W. Luckmann (1994). Integrati ng the cropping system fo r corn insect pest management. Pp 507-541. In Introduction To Insec t Pest Management. Third Edition. Metcalf, R. L. and W. H. Luckma nn (eds.). John Wiley & Sons, Inc., New

arper, B. (2006). Vip3A as expressed in ev ent MIR162 maize: Assessment of amino acid

ens. Unpublished Syngenta report, No. SSB-115-

06. MRID 468648-09

uber, S., J. White, S. Mroczkiewicz, and D. Ward (2007). Insecticidal efficacy field
genta

udson, R. and J. All (1995). Summary losses fr om insect damage and costs of control in

York, New York.

H sequence homology with known toxins. Unpubl ished Syngenta report , No. SSB-112-06.
MRID 468648-08

Harper, B. (2006). Vip3A as expressed in ev ent MIR162 maize: Assessment of amino acid sequence homology with known allerg

H
evaluations with MIR162 maize hybrids in 2005 and 2006. Unpublished Syn report, No. SSB-522-07.

H Georgia 1995. VI. Field corn in sects. Accessed May 17, 2007: http://www.bugwood.org/sl95/field_corn.html

K

urtz, R., A. McCaffery and E. Dunder (2007a). Insect resistance management

-

Kur ent considerations for Bt11 x MIR162 x MIR604 corn. Unpublished S yngenta report, No.

Lee tative insecticidal protein Vi p3A differs from that of Cry1Ab
δ -endotoxin. Applied and Environmental Microbiology 69 (8):4648-4657. MRID

Ma fs of tr ansgenic field crops: An
3-50.

CGA (2007). 2007 World of corn. An annua l report published by the National Corn

considerations for Bt11 x MIR162 maize. Unpublished Syngenta report, No. SSB-519

07.

tz, R., A. McCaffery and E. Dunder (2007b). Insect resistance managem

SSB-508-06.

, M., F. Walters, H. Hart, N. Palekar, a nd J. Chen (2003). The mode of action of the Bacillus thuringiensis vege

468808-01.

rra, M., P. Pardey, and J. Al ston (2002). The payof
assessment of the evidence. AgBioForum 5 (2):4

Marenco, R., R. Foster, and C. Sanchez (1992). Sweet corn response to fall armyworm (Lepidoptera: Noctuidae) damage during vegetative growth. Journal of Economic Entomology 85: 1285-1292.

N Grower’s Association. Accessed on May 17, 2007: http://www.ncga.com/WorldOfCorn/main/WorldOfCorn2007.pdf

SSB-518-07 Page 41 of 87R aybould, A. (2007a). The enviro nmental fate of Vip3Aa20 in MIR162 maize: Expected

Raybould, A. (2007b). The environmental fate a nd potency of the insecticidal proteins Cry1Ab and Vip3Aa20 in Bt11 x MIR162 stacked maize hybrids. U npublished Syngenta

Raybould, A. (2007c). The environmental fate and potency of the in secticidal proteins Cry1Ab, Vip3Aa20 and mCry3A in Bt11 x MI R162 x MIR604 stacked maize hybrids.

Sm Diseases of corn. Pp 687-766. In Corn and Corn Improvement. Third Edition. Sprague, G. F. and J. W. Dudley (eds.). American

tacy, C. (2007a). In vitro digestibility of Vip3Aa20 under simulated mammalian gastric

Sta 20 (MIR162VIP3A-0106) under simulated ammalian intestinal conditions. Unpub lished Syngenta report, No. SSB-002-07.

Ste tration of the plant-inco rporated protectant s Cry1Ab and modified Cry3A proteins, as expressed in stacked Bt11 x MIR604 co rn. Unpublished Synge nta report, No. SSBSteiner, H., M. Kostiuk, and R. Tinsworth (2004). Amended public interest document in support of registration of the pl ant-incorporated protectant, modified Cry3A protein, as

US f Agriculture. December 2006. Accessed on May 17, 2007: http://www.fas.usda.gov/wap/ci rcular/2006/06-12/toc.html

US icultural Statisti cs Service, United States Department of Agricu lture. Accessed on May 17, 2007:

USDA (2007a). Crop production 2006 summary. Na tional Agricultural Statistics Service, United States Department of Agriculture. Report Cr Pr 201(07), January 2007. Accessed

dSu/CropProdSu-01-12-2007.pdf

an Duyn, J. W. (2005). Continuing resear ch on the benefit of Yieldgard ECB ® Bt Corn in environmental concentrations, margins of e xposure in nontarget organism hazard studies and endangered species assessment. U npublished Syngenta report, No. SSB-523-07.

report, No. SSB-524-07.

Unpublished Syngenta report, No. SSB-525-07.

ith, D. R. and D. G. White (1988).

Society of Agronomy, Crop Sc ience Society of America and Soil Science Society of America, Madison, Wisconsin.

S conditions. Unpublished Synge nta report, No. SSB-038-06.

cy, C. (2007b). In vitro digestibility of Vip3Aa

m

ffens, J. and R. Tinsworth (2006). Amen ded public interest document in support of regis

513-06. MRID 468548-01.

expressed in event MIR604 corn. U npublished Syngenta report. MRID 463021-01.

DA (2006a). World agricultu ral production. Foreign Agricultu ral Service, United States Department o

DA (2006b). Acreage, June 30, 2006. Nationa l Agr

http://usda.mannlib.cornell.edu/usda /nass/Acre//2000s/2006/Acre-06-30-2006.pdf

on May 17, 2007: http://usda.mannlib.cornell.edu/usda/cur rent/CropPro

V

SSB-518-07 Page 42 of 87North Carolina. Accessed May 17, 2007: http://www.ces.ncsu.edu/plymouth/pubs/ent/ecb05.html

.

ard, D., T. DeGooyer, T. Vaughn, G. Head, M. McKee, J. Astwood, and J. Perhsing.

nd Management. Vidal, S., U. Kuhlmann, and C. Edwards (eds.). CAB International, Wallingford, Oxfordshire.

Wh genta report, No. SSB-502-07.

hite, J., J. Sagers and M. Meghji (2007c). Insecticidal efficacy of a Bt11 x MIR162 x

rt, No.
SSB-514-07.

Wh

hed Syngenta

d. Unpublished

o. SSB-515-07. W (2005). Genetically enhanced maize as a potential management option for corn
rootworm: YieldGard ® rootworm maize case study. Pp 239-262. In Western Corn Rootworm: Ecology a

ite, J., M. Meehan, and M. Meghji (2007a). Corn earworm tolera nce of a MIR162 maize hybrid: 2006 field trial results. Unp ublished Syn

White, J., J. Sagers, M. Meeh an, and M. Meghji (2007b). Fall armyworm tolerance of a MIR162 maize hybrid: 2006 fiel d trial results. Unpublished Syngenta report, No. SSB503-07.

W GA21 stacked field maize hybrid against Europe an corn borer in the field. Unpublished Syngenta report, No. SSB-512-07.

White, J., J. Sagers and M. Meghji (2007d). Insecticidal efficacy of a Bt11 x MIR162 x GA21 maize) hybrid agai nst corn earworm in the field. Unpublished Synge nta repo

ite, J., J. Sagers and M. Meghji (2007e). Insecticidal efficacy of a Bt11 x MIR162 x GA21 maize hybrid against fall armyworm in the field. Unpublished Syngenta report,
No. SSB-516-07.

White, J., J. Sagers, and M. Meghji (2007f). Insecticidal efficacy of a Bt11 x MIR162 x MIR604 x GA21 maize hybrid agai nst European corn borer in the field. Unpublished Syngenta report, No. SSB-509-07.

White, J., J. Sagers, and M. Meghji (2007g). Insecticidal efficacy of a Bt11 x MIR162 x MIR604 x GA21 maize hybrid against corn ea rworm in the field. Unpublis report, No. SSB-510-07.

White, J., J. Sagers, and M. Meghji (2007h). Insecticidal efficacy of a Bt11 x MIR162 x MIR604 x GA21 maize hybrid against fall armyworm in the fiel Syngenta report, No. SSB-511-07.

White, J., J. Sagers, and M. Meghji (2007i). Western corn rootworm tolerance of Bt11 x MIR162 x MIR604 x GA21 stacked field corn (maize) hybrids in the field. Unpublished Syngenta report, N

SSB-518-07 Page 43 of 87Appendix 1

The Economic Implications of th e Introduction of Syngenta’s

Michele C. Marra, Ph.D.

Raleigh, North Carolina USA Final Report for a Study Entitled:

MIR162 Corn Trait for U.S. Farmers

Nicolas E. Piggott, Ph.D.

Barry Goodwin, Ph.D.

Agri-Analytics, Inc. / North Carolina State University

SSB-518-07 Page 44 of 87Agri-Analytics, Inc. 1



## The Economic Implications of the Introduction of

Submitted to:
Dr. Dennis Ward Syngenta Corporation
3054 East Cornwallis Road earch Triangle Park, NC 27709

Submitted by: Agri-Analytics, Inc. 6708 Virgilia Ct. Raleigh, NC 27616

Ph: 919-637-1976 Email:michele@agr i-analytics.com

Prepared by: Dr. Michele C. Marra Dr. Nicholas E. Piggott Dr. Barry Goodwin

Date Submitted: May 14, 2007 

## Syngenta’s MIR162 Corn Trait for U.S. Farmers

Res

SSB-518-07 Page 45 of 87Agri-Analytics, Inc. 2

EXECUTIVE SUMMARY

This purpose of this study was to examine the available data to attempt to estimate the potential gain at the fa rm and state levels of the commercial introduction of Syngenta Corporation’s MI R162 corn trait. We use seve ral sources of information First, we examine the introduction of a new technology that is a substitute for an existing techno this analysis s
introduction of a of a new substitute not only puts downward pressure on all substitute technology prices in the market, but also dampens technol ogy supplies’ ability to raise prices in
the future.

The second part of the study consists of an examination of the spatial the two insects, the west ern bean cutworm and the corn earwor MIR162 trait will provide superior control relative to current B Using Doane’s Agro-Trak data on acres trea ted for them that pressure from these in sects is likely to sprea
This will increase the value of t he MIR162 trait in

Third, we present the re sults of a grower survey that we designed and commissioned expressly for this study in whic h we asked corn grower area of the High Plains and the Corn Belt about their experienc bean cutworm and the corn earworm over the 2002-2006 time-period. The respondents were asked about the insect densit ies, treatments, tr yield losses on their farms from the two insects of interest . Grow a description of the MIR162 trait and then asked adoption intentions, once the trait is comme rcia positive, with 70% of the respondents indicating that they would try hybrids containing the MIR162 trait and, after three years, would probably plant those hybrids on a significant porti on of their corn acreage.

Lastly, we calculated the total potential gain from t he com MIR162 in three states, Nebraska, Iowa, and Illinois, using inform the analyses above. We find that an upper bound estimate of value of the introduction of MIR162 per year in the thr ee states is more t han $300 million. Given that this estimate is an upper bound estima te as of today because we had no information with which to exclude corn acreage not affected by the insects. At the same time, because of the lack of sufficient data to estimate th states, the estimate may be representativ e of the total regional value of the introduction of MIR162 today. .

logy using the economic t heory of supply and demand. The results of
ho w an unambiguously positive gain to technology buyers of the
new substitute into the ma rket. The results show that introduction

distribution of m, for which the
t traits on the market. in 2005 and 2006, we show d geographically in the near future. future years.

s in a 12-state
e with the western

eatment costs, and

e rs also were read several questions regarding their lized. The responses were quite

merc ial introduction of ation generated by

e value in other

SSB-518-07 Page 46 of 87Agri-Analytics, Inc. 3

TABLE OF CONTENTS

Page

EXECUTIVE SUMMARY ......................................................................................2

INTRODUCTION ..................................................................................................5

THE ECONOMICS OF SUBSTITUTES AND COMPETITION ..............................5

E XISTANCE OF A N EW T ECHNOLOGY ...................................................................6

A DOPTION OF A N EW T ECHNOLOGY (A C HANGE IN D EMAND ) ................................8

ENEFITS B TO FARMERS FROM SUPERIOR CONTROL OF CORN

.........................11

PPENDIX B ......................................................................................................39 EARWORM AND WESTERN BEAN CUTWORM: INFORMATION FROM
SECONDARY SOURCES ..................................................................................10

BENEFITS TO FARMERS FROM SUPERIOR CONTROL OF CORN
EARWORM AND WESTERN BEAN CUTWORM: INFORMATION FROM
A GROWER SURVEY ........................................................................................10

D ESCRIPTIVE STATISTICS

........................................................
P EST P RESSURE AND M EAN Y IELD L OSSES O VER T IME .....................................12

MEASURES AND PREDICTIONS OF PEST PRESSURE FROM DOANE’S
AGRO-TRAK SURVEY DATA ............................................................................19

R ESPONDENT ’ S MIR162 A DOPTION I NTENTIONS ...............................................28

POTENTIAL FARM-LEVEL GAIN FROM THE INTRODUCTION OF
MIR162-TRAITED HYBRIDS .............................................................................29

REFERENCES ...................................................................................................31

APPENDIX A ......................................................................................................32

A

SSB-518-07 Page 47 of 87Agri-Analytics, Inc. 4

LIST ES OF TABLES AND FIGUR

Page

Table 1. Descriptive statistics of survey respondents .................................12 Table 2. Pest pressure as reported by survey respondents by insect, state and y ear ...............................................................................14 Table 3. Reported yield losses by insect, state and year ...........................15 Table 4. Reported percent willing to adopt and average increase in adoption acreage bet ween 2007 and 2010 ...............................28 Table 5. Components needed to ca lculate gain from MIR162 for
selected states fo r 2005 and 200 6 ................................ ...............26

...............41
........42

......7

year ........16

bean cutwo rm ...............................................................................24 igure 10. Spatially smoothed costs ($/harvested acre) for
western bean cu tworm .................................................................25 Figure 11. Actual treatment costs ($/harvested acre ) for corn earworm ........26 Figure 12. Spatially smoothed costs ($/harvested acre) for

corn ear worm ...............................................................................27 Table 6. Gain from MIR162 for se lected states, value of production and percentage gain for 2005 and 2006 .......................................30
Table B.1 Yield loss data by state and year ..................................................40 Table B.2 Regression re sults for I llinois

......................................... Table B.3 Regression re sults for Io wa .................................................. Table B.4 Regression resu lts for Nebr aska ..................................................43

Figure 1. The effect of a new te chnology on the elasticity of demand for an existing technology

......................................................... Figure 2. The effect of a new te chnology on the elasticity of demand for an existing technology ...............................................................9 Figure 3. Number of respondents by state in survey sample .......................11 Figure 4. Nebraska average reported yield losses by insect and Figure 5. Iowa average reported yield losses by insect and year ................17 Figure 6. Illinois average reported yiel d losses by inse ct and year ..............18 Figure 7. Actual treatment costs ($/harvested acre) for all four pests ..........22 Figure 8. Spatially smoothed treatment costs ($ harvested acre)
for all four pests ............................................................................23 Figure 9. Actual treatment costs ($/harvested acre) for western

F

SSB-518-07 Page 48 of 87Agri-Analytics, Inc. 5

The Economic f Syngenta’s
MIR162 Corn Trait for U.S. Farmers

troduction

Syngenta

re

rn ( He rm ( Striacosta
lbicosta ) (W g ven that it is a
iable substit r mpany and
erculex ®

co

e al

e troduction of any new technology on the mark et for existing technol ogies. We then iscuss the results of a grower survey th at we developed for this study that is willingness

te and predict
al

in of related good
d is, if the price of

e holson). The related

arketplace. First, the

r e existing technologies that is beneficial to the technology users. Second, as oon as some adoption of the new technol ogy begins to take place, downward ressure on the prices of the competing te chnologies will be created. Each of these phenomena will be discussed in detail below. Implications of the Introduction o

In

A new genetically-engineered corn trait (MIR162) has been developed by Corp. that is expected to prov ide excellent control of seve ral insect species that a not controlled well with existing technology. Chief among these insects are the co

earworm licoverpa zea ) (CEW) and the western bean cutwo

a BC). Farmers should benefit any where MIR162 is introduced (alon with traits for corn borer and corn rootworm, when appropriate), gi

v ute for the existing technologi es on the market today. The two majo existing technologies are YieldGard ® corn traits from Monsanto Co
H rn traits from Dow AgroSciences.

The purpose of this study is to develop an estimate of the value to farmers of th
introduction of the MIR162 corn trait tec hnology. We approach th is task in sever ways. First we present a theoretical di scussion of the economic effects of th
in
d designed to ascertain the insect pressure , current control measures, and to adopt the new technology. Finally, using Doane’s Agro-Trak data, we estima the spatial distribution of the costs of control for th e CEW and WBC changes in that spatial distribut ion in the future, so that we can estimate the potenti
total value to farmers of MIR162 fo r a few years after its introduction.

The Economics of Substitutes and Competition

In economic terms, goods ar e deemed to be related in consumption if a change the price of one good affects demand for t he other good. One type is a substitute. Two goods are substitu tes if a change in the demand for one goo changes the price of the other good in the opposite direction. That one of a pair of substitute goods increases due to an increase in demand, then th

3 demand for and, thus, the price of the other good will fall (Nic goods in question here are corn trait technologi es grown by farmers in the U.S.

The existence of viable substitu tes creates competition in t he m
mere existence of a new substitute technol ogy will have an effect on the market fo
th

s
p

3 The other types of related goods are ca lled complements, where a change in the price of one good changes the price of t he other in the same direction.

SSB-518-07 Page 49 of 87Agri-Analytics, Inc. 6

SSB-518-07 Page 50 of 87 Existence of a New Tec hnology

strates one effect of the introduction of a new, substitute chnology into the market for an existing technology. The graph on the left hand

owers) at each price per unit. The igher (lower) the price per unit, the smaller (larger) is the quantity dem anded. The

as value to the orn growers. The fewer t he constraints on choice, the better off is the consumer.

rinciple involved is called “Le Chatelier’s Principle”, and its intuitive appeal is easily

e uantity demanded will be larger, given th e more elastic demand schedule, D 1

,

uld lose more market share than if Consider Figure 1, which illu

te side depicts the supply and demand condition s for the existing technology. Originally, before the introduc tion of the new technology, the supply schedule of the existing technology is shown by S e 0 and the demand schedule is shown by D e 0

. The subscript e represents the existing te chnology and the superscript 0 represents time, with 0 denoting time before the introduction of the substi tute. The supply schedule shows the quantity that would be produced at each price per unit. The higher (lower) the price, the more (less) the producers of the tec hnology are willing to produce and sell. The demand schedu le shows the quantity demanded by consumers of the existing technology (cor n gr h intersection of the supply and demand schedules gives the market price, P e 0 and quantity sold, Q e 0 of the existing technology before the new technology is introduced.

Now, suppose a new, substitute technolog y is introduced into the market. The supply schedule for this new technology, S n 0

, is shown in the right hand side graph in Figure 1. Notice that we assume in Figure 1 that there is, as yet, no demand for the new technology; it has just been introduced. Even so, the new technology begins to
affect the market for the ex isting technology because now a new choice exists in the market place. In other words, corn gr owers’ technology choice has become less
constrained . This flexibility (relaxing of constraint s), in and of itself, h

c Although the proof of this assertion is bey ond the scope of this report, the economic

p understood (See, for example, Silberberg, 1990, for a mathematical proof and theoretical explanation).

The effect of the new choice in the technol ogy market is illustrated by a rotation of the demand schedule of the existing technol ogy on the left hand side graph in Figure
1 so that it is “flatter” or “more elastic” than before. Now, supp ose the producers of the existing technology want to raise its price from P e 0 to P e 1 . The decrease in th

q e caused by the existence of the substitu te. The decrease in the quantity demanded for the given price change would be from Q e 0 to Q e 1 before the introduction of the new technology (based on the original de mand schedule). However, the decrease in the quantity demanded after the introducti on of the new technology would be greater, from Q e 0 to Q e 1* . This means that, if t he producers of the existing technology decide to raise its price, they wotics, Inc.7SSB-518-07Page 51 of 87Figure 1: The Effect of a New Technology on theElasticity of Demand for an Existing TechnologyPrice/UnitgyPrice/UnitExisting TechnoloNew TechnologyQuantitySn0Se0De0De1Qe0QuantityQe1Qe1*Pe01Agri-AnalyPeAgri-Analy tics, Inc.

SSB-518-07 Page 52 o the new technology did not exist and, thus they would be more reluctant to do so. This is beneficial to corn growers in that pr ices of corn traited technologies will tend
to remain lower and more stable in the future than otherwise would be the case.

U Adoption of a New Technology (A Change in Demand) 8

f 87 Consider now what happens to market prices and quant ities sold when some growers begin to adopt the new technology (a positive level of demand develops). This is shown in Figure 2. Notice that we now have introduced a demand schedule for the new technology, D n . The initial market price of the new technology is P n and the initial quantity sold is Q n . The initial adoption process of the new technology,
because it is a viable substitute for the existing technology, results in a lower quantity demanded for the existing technology at every price and shifts the (already more elastic) demand schedule for the existing technology from D e 1 to D e 2

. The new equilibrium price for the existing technology has fallen to P e 2 , and the existing technology loses market share to the new technology ( Q n 0

= Q e 2 - Q e 0 ). This benefits corn growers because the price of the existing technology is now lower on every unit
sold

.

Note that we have shown the initial market price for the new technology to be lower than that of the existing technology. T he effect on the existing technology would be qualitatively the same if the new equilibrium price for the new technology were higher than that of the existing technol ogy as long as some adoption of it takes place. A higher market price for the new technology would simply mean that many growers judge the new technology to be somewhat superior to the existing technology and so are willing to pay a higher price for it. In other words, the two technologies depicted in Figur es 1 and 2 need not be perfect substitutes for each other; just substitutes. The more closely related the existing and new technologies are, the stronger will be the effect, however.

The above discussion is necessarily stylized so that the two different effects—the
effect of the introduction of an additional c hoice into the market and the effect of adoption of the new technology—could be explai ned. In reality, these two effects would be expected to take place at about t he same time. That is, it would not be necessary to distinguish the s eparate changes in the demand curve D e 0 to D e 1 to D

B e

PB

2

P instead it would be D e 0 to D e 2

.

The conclusion that can be drawn from this section is that farmers are unambiguously better off when a substitute te chnology is introduced into the market, whether the number of origi nal technologies in the market is one or ten or whether the initial market price for the new technology is the same or different than the price of the existing technology.tics, Inc.9SSB-518-07Page 53 of 87hoFigure 2: Te Effect of a NewTechnology on theElasticityof Demand for an Existing TechnlogyPrice/UnitgyPrice/UnExisting TechnoloitNew TechnologyntityQuaSe0De0De1Qe0Pe0Pe1Qe2Qe1Qe1*Sn0De2Pe2DnQn0Pn0Quantity0Agri-AnalyAgri-Analytics, Inc. 10

Benefits to Farmers from Superior Control of Corn Earworm and Western Bean Cutworm: Information from Secondary Sources

Both the CEW and the WBC feed on the r eproductive parts of the corn plant, especially on developing corn kernels. Recommended management of CEW in field corn is limited to planting resistant hybr ids (hybrids with very tight husks around the ear) and altering planting dates to avoid peak infestations. Chemical control of CEW in field corn is typically not profitable (Cook and Weinzierl), and losses are typically about 2.5% annually, with bigger losses usua lly expected in the South due to multigenerational effects. These losses amount to about 3.75 bushels per acre at an average corn yield of 150 bushels per acre and, at corn pric es around $3.50 is equal to a loss of about $13.13 per acre.

On the other hand, conventional management of the WBC is restri cted to chemical pest control and scouting, along with fairly pr ecise timing of insecticide applications, to achieve acceptable control. Estimates suggest that an average of one WBC larvae per ear can result in losses of four bushels per acre (Pea irs). This would amount to a loss of about $14 per acre with $3.50 corn.

Benefits to Farmers from Superior Control of Corn Earworm and Western
Bean Cutworm: Information from a Grower Survey

A survey of 150 corn growers was conduct ed in early 2007 to collect information about the incidence of the CEW and WBC in the Midwest and High Plains over the past five years. Growers we re randomly selected across th e area of the High Plains and the Corn Belt. The 12 states involv ed in the survey were Colorado, South Dakota, Nebraska, Kansas, Minnesota, Iowa, Missouri, Wisconsin, Illinois, Indiana, Ohio, and Kentucky. Questions were asked about pest infestation, yield losses, treatment for each pest and treatment cost s. Then a description of MIR162 was given to each respondent, and questions were asked about growers’ interest in the new technology. This section contains th e survey results. The survey questionnaire can be found in Appendix A.

Figure 3 shows the number of respondents in the survey from each state. Given that the number of respondents in many of the states is t oo low to be useful from a statistical standpoint, we decided to concent rate on the states of Nebraska, Iowa,
and Illinois for the economic ana lysis of the survey data. These states each have a larger number of respondents with 27 in N ebraska, 32 in Iowa, and 31 in Illinois, respectively.

SSB-518-07 Page 54 of 87Agri-Analytics, Inc. 11

Figure 3: Number of respondents by state in survey sample

WISCONSIN

7 MINNESOTA

8

SOUTH DAKOTA

11

COLORADO

3 OHIO

4

KANSAS

6 MISSOURI

7 INDIANA

11 NEBRASKA

27

ILLINOIS

31 IOWA

32

KENTUCKY

3

Descriptive Statistics

In this section, we report the results for a ll the states in the survey for completeness, even though some of the number s of respondents at the state level are too small for any type of statistical inference. Table 1 describes the farm and farmer characteristics of the survey respondents. Their total crop acres range from 914 in Wisconsin to over 3,000 in Kentucky, wit h an average over all respondents of about 1,400 acres. Of those, t he acres planted to corn t ended to be about one-third to one-half of their total acres, with an average of 740 corn ac res planted in 2007. Of the corn acres planted, the p ercent of t hose planted to any type of Bt corn ranges

om less than 20% to almost 80%, with an average of about 70%. About 70% of

st all of the respondents had at least a high school education (years’ schooling = 12) and most of them had some post-secondary education, ranging from an average of 12.5 years of schooling in Ohio to an average of 18 years of schooling in Colorado. fr those who planted Bt corn hy brids did so, in part, to control the CEW or the WBC, with a range from 25% in Ohio to 100% in Missouri.

Survey respondents’ average years of exper ience operating a farm is about 30 years, with a range of just over 20 years in Colorado to just over 33 years in South
Dakota. Almo

SSB-518-07 Page 55 of 87Agri-Analytics, Inc. 12

Table 1. Descriptive stat istics of survey respondents

S tate N 07 Crop

Acres 07 Corn

Acres 07 Bt

Corn

Acres 07 Pct. Bt

Corn

Acres Bt Planted to

Control CEW or

WBC Operator

Years' Exp. Operator

Years'

School

C

OLORADO 3 1,065.00 803.33 225.00 32.62 0.67 20.67 18.00

LINOIS 31 1,193.39 674.74 445.65 66.37 0.71 30.90 13.87 DIANA 11 1,483.64 786.36 302.73 45.14 0.64 28.36 12.91

WA 32 1,379.41 819.66 632.66 78.12 0.72 31.84 13.56 ANSAS 6 1,616.67 561.67 355.00 61.00 0.83 27.50 14.17 ENTUCKY 3 3,033.33 1423.33 366.67 19.44 0.67 24.00 13.33 INNESOTA 8 1,368.75 712.50 555.00 79.19 0.88 31.63 13.38 ISSOURI 7 1,260.43 565.71 416.29 73.27 1.00 30.29 13.71
EBRASKA 27 1,441.11 726.67 480.56 64.88 0.67 30.63 14.26

HIO 4 950.00 440.00 275.00 64.35 0.25 29.75 12.50 OUTH DAKOTA 11 2,057.27 894.55 324.55 48.62 0.64 33.18 14.00
ISCONSIN 7 914.00 628.57 527.86 75.13 0.57 32.14 14.29

ll States 150 1,406.41 740.84 508.61 69.49 0.70 30.59 13.85 IL

IN

IO

K

K

M

M

N

O

S

W

A

est Pressure and Mean Yield Losses Over Time P

ll of the survey respondents were asked to estimate the intensity of the pest

ressure for each insect by the measure of number of larvae per corn plant. Table 2 hows their responses by state, year, and in sect. Overall in the region, the number f CEW larvae per plant was relatively cons tant over the period 2002-2006 at about .25 larvae per plant. On the other hand, t he overall WBC pressure as reported by
ese farmers increased over time from about 0.19 larvae per plant in 2002 to about later in the time period. These results are consistent with xpectations.

n Iowa and the easternmost is
linois.

analyses for each of the th ree states can be seen in Appendix B. otice that, when a linear coefficient was not statistically signific antly different from zero in the first regression for a state, we used the average yield loss over time as representative of the expected loss in any year. A

p

s

o 0
th 0.26 larvae per plant

e

Perhaps a better way to measure the pest pre ssure is by looking at the respondents’ observed yield losses from the two insects in corn. Respondents were asked to give the best estimate of their per acre yiel d losses from the CEW and from the WBC over the 2002-2006 time period. Table 3 sh ows average reported yield losses for all the states in the sample. The overall averages for the sample indicate that yield losses from CEW may have been increasing slight ly in the region over the past five years, but a significant amount of variability exists across states. There seems to be an upward trend in the overall average yield losses from WBC in the sample region over the past five years. To explore t hese trends further, we examine more closely three states, the westernmost being Nebraska, the
Il

Figures 4, 5 and 6 show these results for Nebraska, Iowa, and Illinois based on statistical analysis of the responses. As mentioned above, these are the only states for which there are enough observations for r easonable statistical reliability. Linear regressions were fitted to each series of yield loss to determine if a trend exists. The full regression
N

SSB-518-07 Page 56 of 87Agri-Analy tics, Inc. 13

SSB-518-07 Page 57 of 87 In Nebraska the CEW seems to be causi me. The estimated increase is about 1.04 bu./acre/ year, and the predicted yield loss for 2007 from CEW in Nebraska is 4.82 bu./acre. The yield losses caused by the WBC are highly variable over the period and no trend could be det ected. This is probably because the WBC moved into Nebraska some time ago, and no upward trend in infestations or yield loss would be expected there. The mean yield loss over the period for the WBC in Nebraska is about 4.89 bu./acre/year.

The increasing trend for yield losses from the CEW is also evident in Iowa. The estimated increase in yield losses from the CEW is about 0.85 bu./acre/year, with the prediction for 2007 of about 3.97 bu./acre. A signifi cant upward trend in yield losses from the WBC is observed in Iowa, as well, at about 0. 68 bu./acre/year, with an expected loss in 2007 of 5.51 bu./acre. This is indicative of t he fact that the WBC

spreading into Iowa and so the yield lo sses could be expected to become larger

by the reported acres treat ed in Doane’s Agro-Trak data for Illinois nd by Pope (2007). ng increasing yield loss over ti

is
over time.

The results for Illinois also show an increase in yield losses due to the corn earworm. The upward trend is estimat ed to be about 1.33 bu./acre/year, with a predicted loss of about 6.12 bu ./acre in 2007. The results for the WBC for Illinois show significant yield losses, as well. No significant trend was discerned, but the average loss per acre per year is estima ted to be 4.54, about one-third bushel lower than the average yield loss due to WBC in Nebr aska over the period. Although this finding is somewhat surprising, since the WB C is thought to be moving west to east, it is supported

aAgri-Analytics, Inc.14d by survey respondentsTable 2. Pest pressure as reportebey insect, state and yarState06 CEWlarvae/cornplant05 CEWlarvae/cornplant04 CEWlarvae/cornplant03 CEWlarvae/cornplant02 CEWlarvae/cornplant06larvapl0.03 0.030.33 0.520.40 0.550.09 0.350.38 0.290.10 0.050.19 0.030.25 0.000.29 0.33. .0.11 0.090.07 0.060.25 0.26WBCe/coantWe/anWBCe/cornantNumber of Obs.COLORADO 3 0.03 0.26 0.030.10.18ILLINOIS31 0.35 0.26 0.350.0.09INDIANA11 0.51 0.35 0.480.10.10IOWA32 0.25 0.25 0.150.0.40KANSAS6 0.30 0.07 0.060.10.10KENTUCKY 3 0.50 0.10 0.10.MINNESOTA 8 0.23 0.03 0.03.MISSOURI 7 0.12 0.12 0.20.NEBRASKA 27 0.18 0.22 0.290.0.10OHIO4 0.53 0.50 .0..SOUTH DAKOTA 11 0.11 0.31 0.07.WISCONSIN 7 0.10 0.29 0.080..All States 150 0.28 0.24 0.220.0.19rn05 WBClarvae/cornplant04larvapl6 0.1725 0.461 0.0831 0.380 0.10. .. 0.05. .28 0.1810 .. 0.0533 .26 0.26BCcornt03 WBClarvae/cornplant02larvapl0.16 0.180.23 0.090.06 0.100.35 0.290.07 0.10. .. 0.05. 0.000.28 0.25. .0.25 0.15. .0.24 0.17SSB-518-07Page 58 of 87Agri-Analytics, Inc.15Table 3. Reported yield losses by insect, state and yearStateN06 CEWyield loss05 CEWyield loss04 CEWyield loss03 CEWyield loss02 CEWyield loss06 WBCyield loss05 WBCyield loss04 WBCyield loss03 WBCyield loss02 WBCyield lossCOLORADO 3 1.00 1.50 1.00 35.00 1.00 1.00 2.33 2.00 2.00 2.00ILLINOIS 31 5.13 4.38 4.92 6.18 2.75 2.88 3.86 6.50 5.80 3.67INDIANA 11 5.61 5.17 6.33 6.00 3.00 6.67 5.17 7.00 7.00 7.00IOWA32 2.79 3.71 4.00 3.50 4.00 4.46 4.11 4.56 2.25 2.00KANSAS 6 3.33 3.00 1.50 2.76 3.20 3.00 3.00 1.50 3.00 3.00KENTUCKY 3 5.00 2.50 6.00 2.00 2.50 . . . . .MINNESOTA 8 2.33 1.50 2.67 1.60 1.50 . 2.00 . 2.00 .MISSOURI 7 1.00 1.00 5.25 7.50 0.00 . . . 0.00 .NEBRASKA 27 3.73 3.89 3.50 3.25 2.38 4.75 7.36 3.50 6.83 2.00OHIO4 11.00 3.00 . . . 20.00 . . . .SOUTH DAKOTA 11 1.33 2.83 1.75 2.50 1.70 . 10.00 2.50 1.50 .WISCONSIN 7 25.13 4.30 5.08 1.08 0.00 11.50 . . . .All States 150 4.90 3.72 3.98 4.50 2.30 4.82 5.07 4.63 3.88 3.00SSB-518-07Page 59 of 87Agri-Analytics, Inc.16Figure 4: Nebraska Average Reported Yield Losses by Insect and Yeary = 0.3339x + 2.34610.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0Yield Loss 2002 Yield Loss 2004Yield Loss 2005 Yield Loss 2006Year

Yield Losses (Bu/acre)Corn EarwormWestern Bean Cutwormy = 4.8893SSB-518-07Page 60 of 87Agri-Analytics, Inc.17Figure 5: Iowa Average Reported Yield Losses by Insect and Yeary = 0.6784x + 1.44180.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0Yield Loss 2002 Yield Loss 2004Yield Loss 2005 Yield Loss 2006Year

Yield Losses (Bu/acre)Corn EarwormWestern Bean Cutwormy=3.600SSB-518-07Page 61 of 87tics, Inc.18SSB-518-07Page 62 of 87Figure 6: Illinois Average ReportYield Losses by Insect and YearedAgri-Analy0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0Yield Loss2002Yield Loss2003Yield Loss2004Yield Loss2005Yield Loss2006Year

Yield Losses (Bu/acre)Corn EarwormWestern Bean Cutwormy = 4.540y=4.670Agri-Analytics, Inc. 19

Measures and Predictions of Pest Pressu re from Doane’s Agro-Trak Survey
Data

An important component of our analysis involved the dev elopment of models that could be used to predict the one-year-ahead le vels of insect pressures at various locations in the region of interest. To do so, we used a simple model of expenditures on treatm ent options for insects in the re levant states included in the survey. These states included Colorado, I llinois, Indiana, Iowa, Kansas, Kentucky, Minnesota, Missouri, Nebraska, Ohio, Sout h Dakota, and Wisconsin. The source for pesticide expenditures data wa s Doane’s Agro-Trak “Treat ment for Selected Corn Insect-Pests”, which was supplied to us by Syngenta Corp. The Doane’s data report expenditures by pesticide br and and the total expenditures for all brands as well as the total number of acres treated. They also report treatment expenditures and number of acres treated by insect (fall armyworm, bla ck cutworm, western bean cutworm, and corn earworm). We can, therefore, isolate expenditures for the insects of interest in this study and calculate the average per-acre expenditures for each insect. These data are reporte d at the Crop Reporting Dis trict (CRD) level. Data were available for the 2005 and 2006 crop y ears. We also collected total (all practices) corn for grain acreage at the CRD level from the National Agricultural Statistics Service (NASS) website for t he 2005 and 2006 crop years. Using the total expenditures on treatment (Grower $) and the total le vel of harvested acreage in each CRD, we calculated a measure of expenditures per harvested corn acre. 4 We inflated the 2005 totals using the Consumer Pr ice Index to place a ll expenditures on a 2006 equivalent basis. Our analysis is con ducted at the county level, but assumes that the CRD-level average expenditures pe r acre apply to all counties within each CR

Our modeling goal was to us e 2005 and 2006 data to predict spatial dispersion in the future—in the current 2007 year. To do so, we adopted a simple set of steps inv g extrapolation of each county’s ex penditures to 2007 and then spatially smoothing the resulting predi ctions across space. Spec ifically, expenditures per acre for 2007 were predicted by adding the difference between the 2006 and 2005
levels to the 2006 level. In cases w here expenditures declined from 2005 to 2006, 2007 expenditures were constrained to be non-negative. 5

We then undertook spatial smoothing techniques to generate a smooth response D.

olvin

4 We use harvested rather than planted ac reage for two reasons. First, harvest acreage statistics from NASS tend to be mo re accurate than planted acreage. Second, we are assuming that abandoned acreage (which is defined by the difference between planted and harvested acreage) is not likely to have received

treatment.

5 This extrapolation process could have resulted in negative estimates of expenditures in cases w here the drop in expenditures from 2005 to 2006 was greater than the 2006 level. Thus, we constrained the 2007 estimates to be nonnegative.

SSB-518-07 Page 63 of 87Agri-Analytics, Inc. 20

surface that could be used to predict tr eatment costs over space and thus to
simula te the spatial spread of the pest thre at. We considered a number of different

patial smoothing techniques, including inverse distance weighting, global

he distribution of actual treatment costs for all insect pests tracked by Doane is

hat the patterns are very imilar but reflect a wider potential fo r pest pressures—the effect of having

We considered spatial predict ion for two specific pests of interest—the Western s polynomial smoothing, local polynomial smoothing, and radial basis function smoothing. 6 On the basis of superior fit (in terms of minimal root mean squared error) and the reasonableness of the fit, we chose local polynomial interpolation methods to fit the response surface. In par ticular, we used a first-order (linear) local smoothing function. 7 For any CRDs (counties) that did not have re ported treatment costs for a particular pest but did have treatment costs r eported for some other pest,
the treatment costs were assumed to be ze ro for the specific pest of interest. 8

The intuition of our smoothi ng methods merits additional discussion. The basic approach involves taking weighted averages of values across space to generate predictions. This has two implications. First, high values that neighbor low values tend to be smoothed downward. Likewise, low values that neighbor high values will tend to have a higher prediction than the act ual value. An example can be used to illustrate the intuition. C onsider a county with costs of $0.50/acre that neighbors a county with zero expenditure. The smoothi ng methods could result in predicted expenditures of $0.40 in the county with positive costs and $0.10 in the neighboring county that actually had no co sts. The idea is to use information from nearby counties to predict the values. The area considered for smoothing (which involves both interpolation and extrapolation in the x- and y-dimensions) is defined by a box constructed by the most extreme lati tude and longitude values of the county centroids for districts surveyed by Doane. This implied a rectangular area that roughly encompassed the north central part of the U.S. (see Figure 7 below).

T illustrated below in Figure 7. High levels of expenditures, indicating significant pest pressures, are apparent in several regions—e specially in the lower Corn Belt, Upper Ohio, and South Dakota areas. It is import ant to again point out that our analysis is confined only to those states targeted by the survey—the absence of treatment expenditures in other states does not necessarily imply that they were zero. Figure 8 presents the spatially smoot hed predictions of treatment costs that were derived from the local polynomial interpolation tec hniques. Note t

s infestations in neighboring areas.

Bean Cutworm and the Corn Earworm. Fi gure 9 presents actual treatment costs

6 Spatial smoothing was accomplis hed using the ArcMap package.

7 Higher ord ered polynomials sometimes ac hieved lower RMSE values, but often

bean cutworm were zero. resulted in negative predicted values.

8 For example, if treatment costs were r eported for black cutworm in a county but nothing was reported for west ern bean cutworm, we assumed that treatment costs
for western

SSB-518-07 Page 64 of 87Agri-Analy tics, Inc. 21

SSB-518-07 Page 65 of 87 and Figure 10 presents the spatially sm oothed values for the Western Bean Cutworm. Localized infestations are appar ent in the Corn Belt and Great Plains. Figures 11 and 12 present actual and s patially-smoothed values for treatment expenditures on the Corn Earw orm. Again, the infestati ons are largely localized but appear to be much more widespread than was the case for the cutworm. The smoothed values encompass a wi der area of infestation, again reflecting the spatial smoothing used to predict treatment costs.

Based on these modeling efforts, it c an be seen from Figures 10 and 12 that the Western Bean Cutworm and the Corn Earw orm are becoming more widesprea d in he Corn Belt and the Great Plains. Because the existing insect resistant corn traits t have no or only moderate activity against these pests and that the cost of scouting and treating them in a timely manner with chemical pesticides is significant or even impractical, it follows t hat the MIR162 corn trait wil l provide additional yield protection for an increasing number of co rn growers. The additional value to growers of hybrids with the MIR162 trait, assuming t hose hybrids are priced competitively, likely will be substantial.Agri-Analytics, Inc.22Figure 7. Actual Treatment Costs ($/harvested acre) for All Four PestsCOST 0 - 00. 0042995595- 0. 085617189862873 -0. 356592581828731 -0. 853472575905659 -11. 6704965070. 0856313131 - 0. 17747038920. 190210. 3721837634 - 0. 63889512430. 642820. 8590703006 - 1. 36490422541. 42569SSB-518-07Page 66 of 87Agri-Analytics, Inc.23FigurePests8. Spatially SmoothedTreatment Costs ($/harvestedacre) for All Fourpredi ct ed 0. 0000000 - 0.0721639 0. 0727597 - 0. 20462070. 2048581 - 0. 36272140. 3632305 - 0. 54135910. 5417020 - 0. 73869880. 7387832 - 0. 99772270. 9984210 - 2. 19474102. 1968913 - 12. 5381956SSB-518-07Page 67 of 87Agri-Analytics, Inc.24Figure 9. Actual Treatment Costs ($/harvested acre) for Western Bean CutwormCOST 00. 0107894381 0. 04921974520. 1516375079 0. 1774703892SSB-518-07Page 68 of 87Agri-Analytics, Inc.25Figuretworm10. Spatially Smoothed Treatment Costs ($/harvested acre) for Western Bean Cupredi ct ed 0. 00000000 - 0. 00000000 0. 00000840 - 0. 000178190. 00018299 - 0. 00069484 0. 00069683 - 0. 001909460. 00191861 - 0. 00493967 0. 00495576 - 0. 008663980. 00873603 - 0. 02349767 0. 02394071 - 0. 10829261SSB-518-07Page 69 of 87Agri-Analytics, Inc.26Figure 11. Actual Treatment Costs($/harvested acre) for Corn EarwormCOST 00. 0389944819 0. 0572607983 0. 08563131310. 0885097625 0. 1153105096 0. 3413251029 0. 4213760832SSB-518-07Page 70 of 87tics, Inc.27SSB-518-07Page 71 of 87Figureworm12. Spatially Smoothed Treatment Costs ($/harvested acre) for Corn Earpredi ct ed 0. 00000000 - 0.00000000 0. 00001795 - 0. 000573030. 00058543 - 0. 002164680. 00220720 - 0. 005511560. 00560493 - 0. 012780330. 01287585 - 0. 027191530. 02747421 - 0. 054391110. 05504717 - 0. 57128119Agri-AnalyAgri-Analytics, Inc. 28

Respondents’ MIR 162 Adoption Intentions

Survey respondents were also given a brief description of the new MIR 162 trait (see Appendix B). After they were read the description twice, they were asked a set of questions about their attitudes and int entions about adopting hybrids with the MIR162 trait. An average of 70 percent of respondents said they would try the MIR162-traited hybrids after listening to its description. The next two questions
asked were about the acres of MIR162-trait ed hybrids they intended to plant in 2007, if it were available and, if it performed as described, how many acres they intended to plant in 2010. Table 4 repor ts the state-level average answers to those questions.

Table 4: Reported percent willing to adopt and average increase in adoption acreage between 2007 and 2010

2 510.00 25.50 484.50
WISCO 6 628.33 153.33 475.00
ILLINO 27 474.52 117.96 356.56
IOWA 27 469.70 115.30 354.41

INDIAN 11 431.82 83.64 348.18
COLO 2 425.00 105.00 320.00
SOUTH KOTA 9 511.11 219.44 291.67

NEBR 20 528.50 243.75 284.75

MINNE A 8 461.88 180.63 281.25
MISSO 6 441.67 206.67 235.00
KANS 5 403.00 170.00 233.00
OHIO 3 305.00 140.00 165.00
ALL S S 126 478.80 152.41 326.39

Notes: N deno umber of individuals sampled in each state which comprise this average. The entire sample ded 150 individuals but 24 of these oberservations were discarded due to nonsensical

respon Average Average Average

State N MIR162 Acres in 2010 MIR162 Acres 2007 Increased Adoption [A] [B] [A]-[B]

KENTU CKY

NSIN

IS

A

RADO

DA

ASKA

SOT

URI

AS

TATE

tes n

inclu

ses.

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SSB-518-07 Page 73 of 87 Potential Farm-Level Gain from the In troduction of MIR162-traited Hy brids

The potential gain at the stat e level is calculated as:

Total Corn Acres in State i x Percent Acres Infested with Insect j [(Percent
Acres Treated for Insect j x Treatment Cost per Acre)

+ (Percent Acres Not Treated x Value of Yield Loss per acre from Insect j )]

We do not have information that would shed light on the percent of acres in a state
th at has been infested with the insects. Therefore, our ca lculations here are to be considered to be an upper bound particular, infiltrates the Co me more reflective of the state-level gain in those states. We have made the ca lculation for only the three states that had a sufficient number of respons es for statistical reliability. Table 5
shows the values of the co mponents used in the calculati on of the potential state level gains. The potential gain in Nebraska is calculated to be about $84 million per year, or about 2.7% of the total value of corn production, for the western bean cutworm and about $646,000 per year, or 0.03% of the total value of the corn crop,
for the corn earworm if the MIR162 trait is adopted by all farmers in the state. The gain in Iowa is estimated to be about $1.7 million for the WBC, or 0.03% of the value of the corn crop and $23 million, or 0.55% of the crop value. In Illinois the total potential gain is about $137 milli on per year, which is 3.2% of the value of the corn crop, for the WBC and about $124 million per year from the corn earworm, which is
about 2.9% of the value of the corn crop.

The upper bound total potential gain in Nebraska, Iowa, and Illinois is estimated to be $222 million per year from the adoption of MIR162 on all corn acres in those
states from control of west ern bean cutworm. This gain is estimated to be about $149 million for the control of corn earworm. The total potential gain in the three states is estimated to be $371 million per year from control of bot h pests. As the western bean cutworm moves east and populat ions of the corn earworm rise, the potential gain from adoption of MIR162-traited hybrids will increase in the Corn Belt.

All three of our measures of the value of the introduction of MIR162-traited corn hybrids were positive and, where measured empirically, subs tantial. These results lend strong support for the trait’s succe ss and the potential value to farmers. to the potential state-level gain. As the WBC, in rn Belt, our estimates would beco30of 87tics, Inc.SSB-518-07Page 74Table 5: Components needed to calculate gain from MIR162 for selected states for 2005 and 2006YearILEBRASKAILLINOAre$2005 23.24 84.42 3.17 8,500014$65, 452 429$02006 37.22 23.66 5.06 8,1000$0.02, 886 644 84,437Aver. 30.23 54.04 4.128,300,0$7.83, 16 037 92,2182005 2.16 25.65 22.33 8,500,0$0.$1 01 7930,7332006 24.50 37.78 5.00 8,1000$0.$ 883 48,872Aver. 13.33 31.71 13.67 8,300,0$0.50 8389,802% Acres TreatednenGain from M 2eester an orC][A]arw[A*C+(1-A)* B%NEIL IANEILAcres,000 12,100,,000 11,300,000 11,700,000 12,100,,000 11,300,000 11,700,Total CorW[B]IANE$/ac00 12,800,000 $5.47 $00 12,600,000 $6.6900 12,700,000 $6.0800 12,800,000 $7.0300 12,600,000 $0.0000 12,700,000$3.52AcresTreamstern Bean Cutworm[Corn EarwormIANEIL IANEIL IANBu/ac$/bu.80 $0.00 4.38 3.89 0.00$1.92 $2.08 $1.9400 $5.63 5.13 3.73 0.00$3.15 $3.35 $3.15 $140 $2.81 4.75 3.81 0.00 $2.54 $2.72 $2.55 $00 $15.45 0.00 7.36 0.00$1.92 $2.08 $1.9400 $4.04 0.00 4.75 0.00$3.15 $3.35 $3.1500 $9.74 0.00 6.06 0.00 $2.54 $2.72 $2.55t CostYield LossCorn Price[D][E]IS,830,628,229,937,529,733IR16CutwormD*E]*IOW$3,5$1,7$44,15$2,5$23,34m608,275,942,,293,$646,W$166,$107,9 $137,7 $137,0 $111,9 $124,n BeCorn ETable 6: Gain frov2 006Year NEm MIR162for selected states,alue of production, and percentage gain for005and 2ILIA66,429,83007,644,628 $3,584,437,037,229 $1,792,27,793,937 $44,150,711,883,529 $2,548,84,838,733 $23,349,8om MIR162$NE IL IA NE$2,439 $3,554 $4,195 2.7%$3,711 $6,088 $6,458 2.8%$3,075 $4,821 $5,327 2.7%$2,439 $3,554 $4,195 0.1%$3,711 $6,088 $6,458 0.0%$3,075 $4,821 $5,327 0.0%Value of Production% ofern Bean CutwormCorn Earworm$ millionsIL2005 $65,6 1$04. %2006 $102,2 1371. %Aver. $83,9 1183. %2005 $1,2 13333. %20061721. %Aver. $6 12022. %frVWest%IA0.00.10.01.10.00.57%8%2%9%8%9%alue08,452 $75,886 $42,169 $93,017 $$0 $46,509 $GainAgri-AnalyAgri-Analytics, Inc. 31

References

Cook, K. and R. Weinzierl. 2004. Cor rworm (Heliothis zea). IPM integrated

est management. University of Illinois rbana-Champaign. Av ailable online at: /insects/corn_earworm/index.html n Ea

at U p http://ipm.uiuc.edu/fieldcrops
Visited 4/18/07.

Nicholson,
Hinsd

P and Dry Beans. Colorado State

at: W. 1978. Microeconomic Theory: Basic Principles and Extensions. ale, Illinois: The Dryden Press.

eairs, F. 2006. Western Bean Cutworm haracteristics and Management in Corn
Extension – Horticulture No . 5.538. Available online http://www.ext.colostate.edu/Pubs/ins 5538.html : C

ect/0

anagement in 2006. Integrated Crop

e University. Available at ipm/icm/2007/2-12/wbc.html Visited 4/18/07.

Pope, R. 2007. Western bean cutworm m Management IC-498(1), Iowa Stat http://www.ipm.iastate.edu/

Silberberg, E. 1990. The St
York: Mc ructure of Economics: A Mat hematical Approach. New Graw-Hill, Inc.

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APPENDIX A

IONNAIRE THE SURVEY QUEST

SSB-518-07 Page 76 of 87Agri-Analytics, Inc. 33

MIR162 Value Questionnaire

Hello, my name is __________ _ with Doane Marketing Rese arch, Inc. We are conducting a survey for researchers at a major land grant university. Insert usual
introduction and offer.

A.1 Are you the primary decision maker for corn seed and insecticides in your farming operation?

Yes _____ No _____

Yes, go to A.2. If No, ask to speak to the principal decision maker.

A.2 Approximately how many acres of corn do you intend to plant this year in
2007? If

Acres (if “0”) thank and terminate

A.3 Have you or your scouts observ ed any Corn Earworm or Western Bean Cutworm larvae in your corn in the past five years? These pests may also be called earworms, head worms, or tomato fruit worms..

Yes _____ No _____ If Yes, continue with Q1. If No, thank you and terminate.

I would like to ask you a few questions about your experience with Corn Earworms
and Western Bean Cutworms in corn and about a new technology geared toward controlling these pests effectively.

Q.1. How many years out of the last five years, 2006 back to 2002 have you found Corn Earworm larvae on your corn?

_______years If greater than Zero, answer Q.2 through Q.6 If Zero, skip to Q.7

Note to enumerator: Please get the re spondent to answer Q.2. through Q.9. based on the number of years reported in Q.1.

SSB-518-07 Page 77 of 87Agri-Analytics, Inc. 34

Q1A. In which of the follow worm larvae in your corn?

2003

please estimate your Corn Earworm infestation based on 1 larvae for
lants?

corn plants

please estimate your Corn Earw orm infestation based on how many larvae er corn plant you experienced? rn plant ing years did you find Corn Ear

2006
2005
2004

2002

Q.2. In (year), every how many corn p

2006,1 larvae per every _____

Interviewer note: If responde nt can not tell you the infestation this way ask: In (year),

p 2006, _______larvae per co

Q.3. Please give me your best estimate of about how much yield loss in bushels per acre you experienced, from Corn Earworm in (year) whether or not you applied an
insecticide for control?.

2006, __ _________ bushels per acre

to cont rol Corn Earworm in (year)?

es ________ No _______

.5.What is the product name of the in secticide you applied to control Corn Q.4. Did you apply an insecticide

Y If Yes, go to Q.5. If No, skip to Q.10.

Q Earworm, in (year)?

2006 Insecticide ________

Q.6..What rate in o unces, pints quarts, or pounds per acre did you use for this pplication of (product)? a 2006 , ounces per acre______ or Pints per acre________ or Quarts per acre ______ or ounds per acre ______

.7. Excluding application costs, how much did you pay per acre, per application for roduct) to control Corn Earworm?
006 P

Q (p
2 Insecticide Cost ________ $ per acre

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Q.8. How many total applicatio ns of (product) did apply to control Corn Earworm in (y ear)? r of applications of (product)______

ects, if any were you al so trying to control with this application of

2006, 2006, numbe

Q.9. What other ins (product) in (year)?

other insects

__

________________
epeat questions 2 through 9 for each y ear the respondent had a Corn Earworm

estions a bout your experience with Western Bean

____years If greater than Zero, answer Q.2 through Q.9 Zero skip to Q.10

Q.9. based the number of years reported in Q.1.

A. lowing years di d you find Western Bean Cutworm in your
n?

2004

2002

ima te your Western Bean Cutworm infestation based on 1

ry _____ corn plants

tervie r note: If responde nt can not tell you the infestation this way ask: In

? rn plant ________________

______________

R infestation in question 1A.

Now I’d like to ask you a few qu Cutworm infestations of your corn fields.

Q.1. How many years out of the last five years, 2006 back to 2002 have you found Western bean cutworm larvae on your corn?

___

If ,

Note to enumerator: Please get the re spondent to answer Q.2. through

on

Q1 In which of the fol

cor

2006
2005

2003

Q.2. In (year), please est larvae for every how many corn plants?

2006,1 larvae per eve

In we (year), please estimate your Western B ean Cutworm infestation based on how many larvae per corn plant you experienced 2006, _______larvae per co

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Q.3. Please give me your best estimate of about how much yield loss in bushels per

acre you experienced, from Western Bean Cutworm in (year) whether or not you

.4. Did you apply an insecticide to control Western Bean Cutworm in (year)? applied an insecticide for control?.

2006, ___________ bushels per acre

Q

Yes ________ No _______ If Yes, go to Q.5. If No, skip to Q.10.

Q.5.What is the product name of the insectic ide you applied to control Western Bean Cutworm, in (year)?

2006 Insecticide ________

Q .6..What rate in ounces, pints quarts, or pounds per acre did you use for this application of (product)?
2006 , ounces per acre______ o r

___

rm?
006 Pints per acre________ or Quarts per acre ______ or Pounds per acre ___

Q.7. Excluding application costs, how much did you pay per acre, per application for (product) to control Western Bean Cutwo
2 Insecticide Cost ________ $ per acre

ow many total applications of (produc t) did apply to control Western Bean utworm in (year)? r of applications of (product)______

ects, if any were you al so trying to control with this application of

2006, Q.8. H
C 2006, numbe

Q.9. What other ins (product) in (year)?

other insects

________________
epeat questions 2 through 9 for each ye ar the respondent had a Western Bean ________________

________________

R Cutworm infestation in question 1A.

SSB-518-07 Page 80 of 87Agri-Analytics, Inc. 37

Q.15. Did you plant any Bt varieties to control Corn Earworm or Western Bean
Cutworm in 2006?

Yes ____ No _______

__ If Yes, Q.16

u plan tin 2006 and which of the pests we’ve discussed

re y ? d ______________________ 16. a.2. Insects

_______________
be to you a new corn technology and then ask you a few
uestions about it.

new Bt corn variety is being developed t hat provides superior control of Corn

ty will be offer ed in combination with European corn borer
ide tolerance traits.

s vailable to you today at a competitive price ologie s that are similar, but do not control effectively for orn E worm and Western Bean Cutworm, would you try it on your farm?

, skip to 19.

8. This year, in 2007, on how many of y our corn acres would you be likely to try

d to the te chnology three years from now?

farm in 2007?

________ acres

t (acres) of corn in 2007? If No, skip to Q.17

Q.16. What Bt brand did y o we ou trying to control
16.a.1. Bt br an

_

Now I want to descri

q

A Earworm and Western Bean Cutworm compared with Bt varieties that are currently
available. This new varie resistance, corn rootworm resistan ce, and herbic

Q.17. If thi new corn tec hnology were a compared to other techn C ar

Yes _______ No _______ If No

Q.1 this new technology?

________ acres

Q.19. If the technology works as advertised, on how many of your corn acres do you think would be plante

________ acres

Q.20. How many total crop acres will you

Earlier, you told us you intend to plan

SSB-518-07 Page 81 of 87Agri-Analytics, Inc. 38

Q .22. On how many of thes e ___ 2007 corn acres do you intend to plant with seed

________ acres

operated a farm?

_______ years

rmal schooling have you had?

_ years that has been engineered to be resistant to insects?

Q.23. How many years have you

Q.24. How many years of fo

______

SSB-518-07 Page 82 of 87Agri-Analytics, Inc. 39

APPENDIX B

REGRESSION RESULTS

SSB-518-07 Page 83 of 87Agri-Analytics, Inc. 40

Table B.1 Yield loss data by state and year

Insect N Mean N Mean N Mean N Mean

Year

2002 35 2.30 4 2.75 2 4.00 8 2.38

2003 50 4.50 11 6.18 2 3.50 12 3.25

2004 65 3.98 12 4.92 11 4.00 12 3

2005 77 3.72 .50

14 3.71 18 3.89

2006 95 4.90 21 2.79 20 3.73

2002 11 3.00 3 67 3 2.00 1 2.00

2003 20 3.88 5 5.80 4 2.25 3 6.83

2004 31 4.63 10 6.50 8 4.56 4 3.50

2005 36 5.07 7 3.86 9 4.11 11 7.36

2006 41 4.82 8 2.88 13 4.46 10 4.75 Source: Grower survey. C Earworm bus els/acre/year All States ILLINOIS IOWA NEBRASKA

orn h

16 4.38

20 5.13

Western Bean Cutworm

3.

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Table B.2 Regression results for Illinois

Regression Statistics Multiple R 0.3700 R Square 0.1369 Adjusted R Squar e -0.1508
Standard Error 1.3492
Observations 5

Coefficients Standard Error t Stat Intercept 3.7867 1.4150 2.6761

t 0.2943 0.4266 0.6899

Corn Earworm - Regression 2 Regression Statistics Multiple R 0.0000 R Square 0.0000 Adjusted R Squar e -0.2500
Standard Error 1.2577

Observations 5.0000

Coefficients Standard Error t Stat Intercept 4.6697 0.5624 8.3025

Western Bean Cutworm - Regression 1 Regression Statistics Multiple R 0.3631 R Square 0.1318 Adjusted R Squar e -0.1575

Standard Error 1.6520
Observations 5

Coefficients Standard Error t Stat Intercept 5.5976 1.7326 3.2308

t -0.3526 0.5224 -0.6750

Western Bean Cutworm - Regression 2 Regression Statistics Multiple R 0.0000 R Square 0.0000 Adjusted R Squar e -0.2500
Standard Error 1.5355

Observations 5.0000

Coefficients Standard Error t Stat Intercept 4.5398 0.6867 6.6112 Source: Grower survey. Corn Earworm - Regression 1

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Table B.3 Regression results for Iowa

Corn Earworm - Regression 1 Regression Statistics Multiple R 0.6981 R Square 0.4873

A djusted R Squar e 0.3164
Standard Error 0.4147
Observations 5

Coefficients Standard Error t Stat Intercept 4.2643 0.4349 9.8053

t -0.2214 0.1311 -1.6887

Corn Earworm - Regression 2 Regression Statistics Multiple R 0.0000 R Square 0.0000

A djusted R Squar e -0.2500
Standard Error 0.5015
Observations 5

Coefficients Standard Error t Stat Intercept 3.6000 0.2243 16.0506

Western Bean Cutworm - Regression 1 Regression Statistics Multiple R 0.8590 R Square 0.7380

A djusted R Squar e 0.6506
Standard Error 0.7381

Coefficients Standard Error t Stat Intercept 1.4418 0.7741 1.8625

t 0.6784 0.2334 2.9067 Source: Grower survey

SSB-518-07 Page 86 of 87Agri-Analytics, Inc. 43

Table B.4 Regression results for Nebraska

Corn Earworm - Regression 1 Regression Statistics Multiple R 0.8879 R Square 0.7883

A djusted R Squar e 0.7178
Standard Error 0.3159

Observations 5

Coefficients Standard Error t Stat Intercept 2.3461 0.3313 7.0820

t 0.3339 0.0999 3.3427

Western Bean Cutworm - Regression 1 Regression Statistics Multiple R 0.4243 R Square 0.1800

A djusted R Squar e -0.0933
Standard Error 2.3497
Observations 5

Coefficients Standard Error t Stat Intercept 3.0803 2.4643 1.2499

t 0.6030 0.7430 0.8116

Western Bean Cutworm - Regression 2 Regression Statistics Multiple R 0.0000 R Square 0.0000

A djusted R Squar e -0.2500

Standard Error 2.2472

Coefficients Standard Error t Stat Intercept 4.8894 1.0050 4.8652 Source: Grower Survey

SSB-518-07 Page 87 of 87Courtesy Copy:

Supplemental Information for the
Petition for Determination of Nonregulated Status
for Insect-Resistant MIR162 Maize [OECD Unique Identifier: SYN-IR162-4]

USDA Petition 07-253-01p

CBI DELETED

Submitted By:

Dennis P. Ward, Ph.D. Syngenta Biotechnology, Inc.
P.O. Box 12257
3054 E. Cornwallis Road Research Triangle Park, NC 27709 dennis.ward@syngenta.com
Tel. 919.225.5699

Submission Date:

November 12, 2007

Syngenta Document ID:

MIR162-USDA-2

Syngenta MIR162-USDA-2 Page 1 of 3Syngenta Biotechnology is submitting this Courtesy Copy of MIR162 maize and Vip3Aa protein technical reports to aid USDA scient ists in their review of our petition for a determination of nonregulated status fo r insect-resistant MIR162 maize: USDA petition #07-253-01p.

Claim of Data Confidentiality

Syngenta is claiming these technical reports as Confidential Business Information. These reports contain technical informa tion that could be of value to our competitors. These same technical reports have been s ubmitted to the U.S. Environmental Protection Agency as part of the MIR162 application for registration and are afforded confidential business information
status under FIFRA.

Report: Author & Title Section Number

CBI deleted 1

CBI deleted 2

CBI deleted 3

CBI deleted 4

CBI deleted 5

CBI deleted 6

CBI deleted 7

Syngenta MIR162-USDA-2 Page 2 of 3Report: Author & Title Section Number

CBI deleted 8

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Syngenta MIR162-USDA-2 Page 3 of 3