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-1 
 MIR162-USDA-1 Page 2 of 268 

Petition for Determination of Nonregulated Status

The undersigned submits this petition under 7 CFR 340.6 to request that the Administrator 

make a determination that the article should not be regulated under 7 CFR Part 340. 

Certification

The undersigned certifies that, to the best knowledge and belief of the undersigned, this 

petition includes all information and views on which to base a determination, and that it 

includes relevant data and information known to the petitioner which are unfavorable to the 

petition. 

 

Petitioner: 

Dennis P. Ward, Ph.D. 

Regulatory Affairs Manager 

Mailing Address: Syngenta Biotechnology, Inc. 

 P.O. Box 12257 

 3054 East Cornwallis Road 

 Research Triangle Park, NC 27709 

 USA 

Telephone: 919.597.3096 

Facsimile: 919.541.8535 

Email: dennis.ward@syngenta.com

© 2007 Syngenta. All Rights Reserved. 

This document is protected under copyright law. This document is for use only by the 

regulatory authority to which this has been submitted by Syngenta, and only in support of 

actions requested by Syngenta. Any other use of this material, without prior written consent 

of Syngenta, is strictly prohibited. By submitting this document, Syngenta does not grant 

any party or entity any right or license to the information or intellectual property described in 

this document. 
 MIR162-USDA-1 Page 3 of 268 

Release of Information

Syngenta Biotechnology, Incorporated (Syngenta) is submitting the information in this 

petition for review by the United States Department of Agriculture (USDA) as part of a 

federal regulatory process. By submitting this information to USDA, Syngenta does not 

authorize its release to any third party. In responding to a request made under the Freedom 

of Information Act (FOIA), 5 U.S.C. §552 and 7 CFR Part 1, covering all or some of the 

information in this petition, Syngenta expects that, in advance of the release of the 

information, USDA will provide Syngenta with a copy of the FOIA request and the material 

proposed to be released, and the opportunity to object to the release of information based on 

appropriate legal grounds (e.g. responsiveness, trade secret, and/or commercial concerns). 

Syngenta understands that a copy of this petition may be made available to the public as part 

of the public comment process. Except in accordance with the foregoing, Syngenta does not 

authorize the release, publication, or other distribution of this information (including internet 

posting) without prior notice and Syngenta consent. 
 MIR162-USDA-1 Page 4 of 268 

Petition for Determination of Nonregulated Status 

for Insect-Resistant MIR162 Maize 

Summary of the Petition

Using the techniques of modern molecular biology, Syngenta has transformed maize (Zea 

mays L.), to produce event MIR162 maize (hereafter ‘MIR162 maize’), a new cultivar that is 

resistant to lepidopteran insect feeding. MIR162 maize plants contain the vip3Aa20 gene 

encoding the Vip3Aa20 protein and the manA gene encoding the enzyme phosphomannose 

isomerase (PMI). The Vip3Aa20 protein is a variant of the native insecticidal Vip3Aa 

protein from Bacillus thuringiensis strain AB88. The Vip3Aa20 protein is insecticidally 

active against a number of significant lepidopteran pests of maize. The manA gene was 

obtained from Escherichia coli strain K-12 and the protein it encodes was utilized as a plant 

selectable marker during development of MIR162. 

MIR162 maize was produced by Agrobacterium tumefaciens-mediated transformation of 

immature maize embryos using plasmid vector pNOV1300. The region between the left and 

right borders of the transformation plasmid included vip3Aa and manA gene expression 

cassettes; this T-DNA was transferred into the maize genome during transformation. The 

vip3Aa expression cassette consisted of the vip3Aa coding region regulated by the Z. mays

polyubiquitin promoter (ZmUbiInt) and cauliflower mosaic virus 35S 3′ polyadenylation 

sequences. The manA expression cassette consisted of the manA coding region regulated by 

the ZmUbiInt promoter and the nopaline synthase (NOS) polyadenylation sequence. 

Southern blot analyses and nucleotide sequencing demonstrated that MIR162 maize contains 

a single intact T-DNA insert in the nuclear maize genome. Southern blot analyses further 

demonstrated that the T-DNA insert contains: i) single copies of a vip3Aa gene and a manA

gene; ii) two copies of the ZmUbiInt promoter; iii) one copy of the NOS terminator; and iv) 

no backbone sequences from transformation plasmid pNOV1300. Nucleotide sequencing of 

the T-DNA insert in MIR162 maize revealed two codon changes within the vip3Aa coding 

sequence relative to the intended vip3Aa sequence; one of these was a silent mutation and the 

other codon change resulted in an amino acid substitution. The vip3Aa gene variant present 

in MIR162 maize has been designated vip3Aa20. Nucleotide sequencing additionally 

determined that the MIR162 maize T-DNA insert did not locate within any known Z. mays

gene. Further, no novel open reading frames were created that spanned either the 5′ or 3′

junctions between the T-DNA and Z. mays genomic sequences. These genetic 

characterization data demonstrate that, apart from the well-characterized change that resulted 

in a single altered amino acid in the vip3Aa coding sequence, there are no unintended 

changes in the MIR162 maize genome as a result of the T-DNA insertion. Observations of 

vip3Aa20 and manA segregation ratios over several generations of MIR162 maize are 

consistent with the genes being linked at a single locus in the maize genome, and indicate 

stable inheritance of the transgenes. These data also indicate that no novel proteins, other 

than Vip3Aa20 and PMI, will be produced in MIR162 maize. 

Based on a well-characterized mode of action, physiochemical properties, and results of 

safety studies, it has been demonstrated that the Vip3Aa20 and PMI proteins present in 
 MIR162-USDA-1 Page 5 of 268 

MIR162 maize pose no risk of harm for mammalian species. 

Laboratory, greenhouse, growth chamber, and field investigations with MIR162 maize 

confirmed that there were no changes in seed, pollen, plant phenotypic, or composition 

parameters suggestive of increased plant pest risk. 

No adverse effects were associated with exposures to Vip3Aa proteins in a range of nontarget 

indicator species appropriate for a maize ecosystem and the highest doses tested represented 

no adverse effect levels. With one exception, the levels tested were in excess of expected 

environmental exposure levels, indicating a low probability of harm for nontarget organisms 

inhabiting maize ecosystems. In one study it was not possible to exceed a theoretical 

expected environmental concentration; however, no adverse effects were observed on this 

study. 

The narrow spectrum of insecticidal activity observed for Vip3Aa proteins indicates with 

high certainty that no endangered or threatened species other than Lepidoptera would be 

harmed by contact with Vip3Aa20 via MIR162 maize. Furthermore, there is minimal 

exposure of endangered Lepidoptera to maize and therefore, cultivation of MIR162 maize is 

unlikely to harm any endangered or threatened species in the U.S. 

Syngenta knows of no study results or observations associated with MIR162 maize that are 

anticipated to result in adverse consequences to the quality of the human environment, 

directly, indirectly, or cumulatively. This includes a lack of anticipated adverse effects on 

endangered species, unique geographic areas, critical habitats, public health and safety 

(including children and minorities), genetic diversity of maize, farmer or consumer choice, 

insect resistance, or the economy, either within or outside the U.S. MIR162 maize offers 

growers high efficacy, convenience, and an additional choice for protection of maize crops 

from feeding damage caused by lepidopteran pests. As such, MIR162 maize is expected to 

produce beneficial effects similar to previously deregulated Bt maize products that are 

commercially available. These benefits include increased competition in the marketplace for 

insect-protected seed products. Moreover, the novel mode of action of the Vip3A20 protein 

is expected to extend the useful life of Bt maize technology for lepidopteran insect control in 

general by reducing the selection pressure for resistance among target pests. 

The Animal and Plant Health Inspection Service of the United States Department of 

Agriculture has responsibility, under the Plant Protection Act (Title IV Public Law 106-224, 

114 Statute 438, 7 U.S.C. §7701-7772) to prevent the introduction and dissemination of plant 

pests into the United States. The APHIS regulation 7 CFR Part 340.6 provides that an 

applicant may petition APHIS to evaluate submitted data to determine that a particular 

regulated article does not present a plant pest risk and should no longer be regulated. If 

APHIS determines that the regulated article does not present a plant pest risk, the petition is 

granted, thereby allowing unrestricted introduction of the article. Syngenta is hereby 

submitting this petition for a determination of nonregulated status for MIR162 maize and 

provides all necessary data upon which to make this determination. 
 MIR162-USDA-1 Page 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 Other 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 Functional 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 and Insecticidal Specificity .........................................47 

V.A.2. Characterization of Vip3Aa20 ........................................................................48 

V.A.3. Safety Assessment for Vip3Aa20 ...................................................................50 
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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 Levels 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 Agronomic 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 Environmental 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 Concentrations 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-dwelling Nontarget Invertebrates ...................................81 

VII.C.3.f. EEC for Aquatic Organisms .................................................................82 

VII.C.3.g. Summary of Environmental 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..............................................................................98 
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IX.C. Intensity of the Proposed Action...........................................................................100 

IX.C.1. Impacts That May Be Both Beneficial and Adverse....................................100 

IX.C.2. Effects on Public Health or Environmental Safety ......................................101 

IX.C.3. Unique Characteristics 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............................................................181
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List of Tables

Page

Table 1. Categorization of maize insect pests.........................................................................12 

Table 2. Description of test and control seed materials used in regulatory studies................21 

Table 3. Description of genetic elements inserted in vector pNOV1300. ..............................23 

Table 4. Expected versus observed hybridization band sizes for probes with vip3Aa19. ......29 

Table 5. Expected versus observed hybridization band 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 between Vip3Aa1, Vip3Aa19, and Vip3Aa20.................48 

Table 13. Comparative bioactivity of Vip3Aa proteins from different sources. ....................50 

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

Table 15. Mean tissue concentrations of PMI in MIR162 plants. ..........................................56 

Table 16. Phenotypic characteristics evaluated for MIR162 maize hybrids. .........................57 

Table 17. Test, control, and reference seeds used in germination and dormancy test............59 

Table 18. Summary of seed germination and dormancy frequencies.....................................61 

Table 19. Statistical analysis 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 measured in MIR162 and conventional maize. ......70 

Table 26. Statistically significant differences between MIR162 and control analytes...........72 

Table 27. Insect species screened for sensitivity to Vip3Aa protein variants. .......................74 

Table 28. Tissue concentrations of Vip3Aa20 used for computation of EECs. .....................78 

Table 29. Tabulation of EEC and DDD 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 insecticide usage for control of selected lepidopteran pests. ...........94 
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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 development 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 analysis of MIR162 maize with a vip3Aa19-specific probe.............28 

Figure 9. Restriction sites and probe for manA.......................................................................30 

Figure 10. Southern blot analysis of MIR162 maize with a manA-specific probe.................31 

Figure 11. Restriction sites and probe for the ZmUbiInt promoter. .......................................33 

Figure 12. Southern analysis of MIR162 maize with ZmUbiInt-specific probe. ...................34 

Figure 13. Restriction sites and probe for NOS terminator. ...................................................36 

Figure 14. Southern analysis of MIR162 maize with NOS terminator-specific probe...........37 

Figure 15. Restriction sites 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 BC1F1, BC2F1, and BC4F1 generations from MIR162................43 

Figure 19. Western blot of plant- and microbially produced Vip3Aa proteins. .....................49 

Figure 20. Photographs of stained pollen collected from control and MIR162 plants...........68 

Figure 21. MIR162 field efficacy trial results. .......................................................................76 

 
 MIR162-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.) agriculture in the past 25 

years. Adoption of genetically engineered crops with insect and herbicide tolerance traits has 

increased dramatically since their commercial introduction in 1995. Net economic benefits 

at the farm level have been substantial (Brookes and Barfoot, 2005). Improved insect and 

weed control have led to increased crop yields and reductions in conventional insecticide 

applications. The continued development and introduction of these traits is expected to 

benefit both growers and consumers. Transformation event MIR162 maize has been 

developed by Syngenta to provide growers with maize varieties that are 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 importation, exportation, and interstate movement of plants, plant 

products, certain biological control organisms, noxious weeds, and plant pests. APHIS 

regulations under 7 CFR §340.1 stipulate that any organism which has been altered or 

produced through genetic engineering is considered 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 any product which contains such an organism, 

or any other organism or product 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 MIR162 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 presents data and justification for an APHIS 

determination of nonregulated status 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. Table 1 categorizes most of the insect pests of maize 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), 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 economic 
 MIR162-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 insects that attack the stalk, leaf, and ear (e.g.,

cutworms, chinch bugs, grasshoppers, corn flea beetles and Japanese beetles). S. albicosta is 

a pest of increasing economic importance because 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 pathogenic organisms during maize development. 

Soil abounds in microorganisms, particularly fungi, 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 group 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 maize is grown and can result in reduced test 

weight, 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 frequently associated with 

insect feeding damage. 

Crop losses attributable to O. nubilalis and Diabrotica infestations have been well 

characterized and are significant. The introductions 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 grain yield and quality. 

Conventional insecticide applications are an option for reducing feeding damage caused by 

these insects; however, most growers do not treat their crops to control these pests because of 

cost and limited effectiveness of the chemical agents. Currently available transgenic 

varieties are not as efficacious against these lepidopteran insects as they are against O. 

nubilalis. For example, Bt11 maize containing the 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 resistant 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, increase grower profits, and improve grain quality. 

I.C. Status with Other Regulatory Agencies 

Syngenta is actively pursuing regulatory approvals 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 Philippines, 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 regulation by the Environmental Protection Agency 

(EPA). The Vip3Aa20 protein encoded by the genetic insert in MIR162 maize has 

insecticidal properties and is, therefore, regulated by EPA. Syngenta has obtained an 

experimental use permit from EPA that allows for broad-scale field testing of MIR162 

maize; this permit was granted on March 26, 2007 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 commodities, 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 registration of the plantincorporated protectant encoded by MIR162 maize 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 the Food and Drug Administration’s (FDA) policy 

statement concerning regulation of food products derived from new plant varieties, including 

those developed by recombinant DNA techniques (FDA, 1992). Syngenta has initiated a 

voluntary consultation process with FDA and 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 armyworms, cutworms, and ear-feeding lepidopteran 

species are significant economic pests of maize (e.g., Argentina and Brazil). Syngenta will 

also pursue regulatory approvals for importation 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 world, and as corn in the U.S., 

is one of the few major crop species indigenous to the Western Hemisphere. It has been 

cultivated in the Americas since early historic times. Field maize is the leading production 
 MIR162-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 production. 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 feed, 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 barrels 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 exporter 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 and nonfarm sectors of the economy. Production 

from almost one-third of U.S. cropland moved into export channels in 2005 and generated 

$166.1 billion in business activity. Technology advances increase agricultural productivity 

and keep domestic growers competitive 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 authoritative 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 life 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 temperature 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 lasts 120 to 180 days. Maize is 

grown in areas where annual precipitation ranges from 25 to > 500 cm. Summer rainfall of 

15 cm is approximately the lower limit 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 South Dakota are major maize growing states. 

Production in these ten states accounts for 84% of total annual production (USDA, 2007a). 

Figure 2 displays the geographic distribution of acres planted in 2006 (USDA, 2007b). 

Farmers have hundreds of maize varieties from which to choose. Available varieties differ 

widely in agronomic characteristics, including length of growing 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 improved through biotechnology has added greatly to 

farm productivity and profits since their introduction in 1996 (Fernandez-Cornejo and 

Caswell, 2006). Varieties containing herbicide-tolerance and insect-protection traits have 

been widely adopted by maize, soybean, and 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 acres planted (USDA, 2007c). Insect-protected varieties 

accounted for 59% of cotton and 49% of maize acres planted. Planting of these varieties has 

also benefited the environment by displacing conventional pesticide applications and 

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). 

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, ditches, or roadsides as a weed. Although maize 

seed from a previous year’s crop can overwinter 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 measures are often applied to remove these 

volunteers, but the plants that are not removed 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 polystichous female inflorescence on a stiff central 

spike enclosed with husks. Consequently, seed dispersal of individual seeds does not occur 

naturally because of the structure of the maize ears. Individual seeds, however, can be 

dispersed during grain harvest and transportation. In neither instance does maize become a 

troublesome weed. Maize is not 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 transformation 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 from the Maize Genetics 

Stock Center.1

 Inbred line Hi-II, created from a cross of line A188 with line B73, was 

specifically developed for use in embryonic tissue transformation systems (Armstrong et al., 

1991). A188 is a publicly available stiff-stalk 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 available stiff-stalk inbred developed 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 right 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 Tobacco, 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)

KpnI (4786)

HindIII (189)

XmaI (8293)

EcoRV (6965)

SphI (199)

SphI (2586)

SphI (4797)

SphI (14,324)

Acc65I (4782)
 MIR162-USDA-1 Page 19 of 268 

Replication of pNOV1300 in A. tumefaciens was made possible by homologous 

recombination with an ‘acceptor vector’ carrying 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 integration 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 transformation. Embryos were then mixed with a 

suspension of A. tumefaciens harboring vector pNOV1300. Following a five-minute 

incubation, excess solution was aspirated and embryos 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 were transferred to culture medium 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 transferred to culture medium containing 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 tested 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 original transformants (T0 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 regulatory 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 notification; 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 characterization 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 stabilized through backcrossing, followed by self-pollinations. 

Parental control hybrids (near-isogenic 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 all 
 MIR162-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 risks 
 MIR162-USDA-1 Page 21 of 268 

control hybrids was to match their genetic background with that of the MIR162 hybrids so 

that the effect of the transformation event could be assessed in an unbiased manner. Table 2 

provides a listing of the seed lots used for planting of regulatory trials. A pedigree chart 

showing the origin of MIR162 seed lots is shown in Figure 5. 

Table 2. Description of test and control seed materials used in regulatory studies. 

The genetic identity is shown for test and control 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 BC1F1 B9620 B9620 

Genetic characterization & 

Mendelian inheritance B BC2F1 B9620 B9620 

Genetic characterization & 

Mendelian inheritance; 

Characterization of plant 

transgenic protein 

C BC4F1 B9620 B9620 

Germination / dormancy D F1

NP2391 x 

NP2222(BC6F4) 

NP2391 x 

NP2222 

Germination / dormancy E F1

NP2222(BC6F5) x 

NP2391 

NP2391 x 

NP2222 

Pollen viability / 

morphology E F1

NP2222(BC6F5) x 

NP2391 

NP2222 x 

NP2391 

2005 agronomic 

equivalence F F1

NP2010 x 

NP2222(BC6F4) 

NP2222 x 

NP2010 

2006 agronomic 

equivalence I F1

NP2673 x 

NP2171(BC4F5) 

NP2673 x 

NP2171 

Compositional analysis G & H F1 & F2

NP2276(BC4F4) x 

NP2391 

NP2276 x 

NP2391 

Tissue protein quantification 

– Hybrid 1 G & H F1 & F2

NP2276(BC4F4) x 

NP2391 

NP2276 x 

NP2391 

Tissue protein quantification 

– Hybrid 2 J & K F1 & F2

NP2153(BC4F4) x 

NP2391 

NP2153 x 

NP2391 

All test and control seed lots were analyzed by real 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 plant selectable marker based on nucleotide 

sequence, and the event of interest based on the genome-insert junction nucleotide sequence. 

The analyses did not detect these components in control seed lots. All test and control seed 

lots had no detectable sequences that would be indicative of other regulated events under
 MIR162-USDA-1 Page 22 of 268 

Figure 5. MIR162 pedigree chart. 

Generations in blue and bold type were used in regulatory studies; boxed letter code 

corresponds to specific seed lots used in regulatory studies (see Table 2). 

T0

F1

x NPH8431 

F1 F1

BC1F1 BC1F1

BC2F1

BC3F1

BC4F1

F1

BC1F1

BC4F1

x NP2010 

BC4F2

BC4F4

BC4F3

F1

NP2161 x 

bc 

F1 BC2F1

bc 

bc 

bc 

bc 

BC1F1

BC2F1

BC3F1

BC4F1

bc 

bc 

BC2F1

BC3F1

F1

bc 

BC1F1

x NP2222 

B9620 x bc 

BC6F1

BC6F2

BC6F4

⊗

BC6F3

⊗

BC6F5

F1

bc 

BC1F1

BC4F1

⊗

BC4F2

⊗

BC4F4

⊗

BC4F3

F1

x NP2391 

⊗

x NP2276 

F1

NP2391 x ⊗

T0 – original transformant 

x – cross 

bc – backcross 

⊗ - self-pollination 

F1 F2

x NP2391 

⊗

⊗

⊗

bc 

x NP2153 

bc 

bc 

bc 

bc 

F1

BC1F1

BC4F1

bc (3 times) 

bc 

NP2171 x 

F1

BC4F5

BC4F2

⊗

⊗

⊗

⊗

x NP2673 x NP2391 

⊗

⊗

F2

x NP2010 

F1

bc (3 times) bc (3 times) bc (3 times) 

BC4F3

BC4F4

B 

C 

A 

H

D 

E 

F G

I K

J
 MIR162-USDA-1 Page 23 of 268 

 development at Syngenta, or deregulated events for which testing methodology is available. 

A number of human and environmental safety studies employed purified Vip3Aa proteins as 

the test substances. One of three Vip3Aa variants 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 variant found in MIR162 maize. Vip3Aa1 was 

extracted from a culture of B. thuringiensis strain AB88. Vip3Aa19 and Vip3Aa20 were 

produced in recombinant cultures of Escherichia coli. The three proteins share >99.7% 

amino acid sequence homology and have been shown to be biochemically and functionally 

equivalent. 

IV. Donor Genes and Regulatory Sequences 

IV.A. Transformation Vector pNOV1300 

MIR162 maize was produced by Agrobacterium tumefaciens-mediated transformation of 

immature embryos using the plasmid vector 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 transformation. 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 promoter and the nopaline synthase (NOS) 

polyadenylation sequence. A schematic of the vector is shown in Figure 3. The size and 

location of each genetic element in the vector 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 bacterial 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 maize 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 promoter; 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 over three breeding generations. Sequence 

analysis of the entire T-DNA present in MIR162 maize confirmed the intactness of the 

T-DNA and that the contiguousness of the functional elements had been maintained. 

Sequence analysis also revealed two 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 mutations resulted in a single codon change for the 

amino acid originally encoded, while the other mutation was silent. The new gene 

incorporated into the MIR162 maize genome has been designated vip3Aa20. See Section 

V.A for a discussion of the Vip3Aa variants. Nucleotide sequence 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 patterns over three generations 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 maize genomic sequence flanking the 5′ region of the 

MIR162 maize DNA insert and maize transposable element sequences. There are also 

regions of homology between the maize genomic sequence flanking the 3′ region of the 

MIR162 maize T-DNA and identified maize sequences. 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) vip3Aa20 ZmUbiInt (1993 bp) (2370 bp) manA (1176 bp) 

Plant 

genomic 

DNA 

Plant 

genomic 

DNA 

HindIII (189)

SphI (199)

SphI (2586)

SphI (4797)

XmaI (8293)

Acc65I (4782)

KpnI (4786)

EcoRV (6965)
 MIR162-USDA-1 Page 26 of 268 

sequence. A detailed description of the materials and methods for the molecular 

characterization of MIR162 maize can be found in Appendix B. 

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

Consensus nucleotide sequence data demonstrated that the MIR162 T-DNA is intact and that 

the contiguousness of the functional elements within the insert as intended in pNOV1300 has 

been maintained. Two nucleotide changes were noted within the coding sequence of the 

vip3Aa gene in MIR162 maize. The first of these nucleotide changes results in a single 

amino acid change: methionine at position 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 DQ539888; NCBI, 2007). The coding 

sequences of manA, the ZmUbiInt promoters, the 35S terminator, 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 process. The entire RB, along with two base 

pairs of noncoding sequence was truncated, and the entire LB, along with 32 base pairs of 

noncoding sequence, was truncated. These deletions have no apparent 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 the T-DNA in MIR162 contains single copies of 

the vip3Aa20 and manA genes. As expected, the MIR162 maize T-DNA contains two copies 

of the ZmUbiInt promoter, corresponding to the two copies of the ZmUbiInt promoter 

present in the transformation plasmid pNOV1300, and contains a single copy of the NOS 

terminator. The Southern blot analyses demonstrated that MIR162 maize contains no 

pNOV1300 plasmid backbone sequences. 

For each Southern blot analyses, there is a map showing the location of the restriction 

endonuclease digestion sites in relation to each element probe used in the analysis. 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 the 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 did 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 vip3Aa19
 MIR162-USDA-1 Page 27 of 268 

coding sequence and the restriction endonuclease sites targeted in these Southern blot 

analyses is shown in Figure 7. Three endonuclease 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 

KpnI, EcoRV, HindIII, and XmaI 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 

HindIII+XmaI 

KpnI 

EcoRV 

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)

KpnI (4786) EcoRV (6965)

HindIII XmaI 
 MIR162-USDA-1 Page 28 of 268 

Figure 8. Southern blot analysis of MIR162 maize with a vip3Aa19-specific probe.

Maize genomic DNA (7.5 μg) was subjected to three separate digestions with KpnI or EcoRV 

or HindIII plus XmaI 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: BC4F1 generation of MIR162 maize digested with KpnI 

Lane 4: Control from BC4F1 generation maize digested with KpnI 

Lane 5: BC4F1 generation of MIR162 maize digested with EcoRV 

Lane 6: Control from BC4F1 generation maize digested with EcoRV 

Lane 7: BC4F1 generation of MIR162 maize digested with HindIII + XmaI 

Lane 8: Control from BC4F1 generation maize digested with HindIII + XmaI 

Lane 9: Control from BC4F1 generation maize digested with HindIII + XmaI plus 20.2 pg of 

HindIII plus XmaI 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 BC4F1 KpnI vip3Aa19 1 >4.8 8.0 

Lane 4 Negative KpnI vip3Aa19 0 - - 

Lane 5 BC4F1 EcoRV vip3Aa19 1 >7.0 14.0 

Lane 6 Negative EcoRV vip3Aa19 0 - - 

Lane 7 BC4F1

HindIII + 

XmaI vip3Aa19 1 8.1 8.1 

Lane 8 Negative HindIII + 

XmaI vip3Aa19 0 - - 

Positive Control 

Lane 9 pNOV1300 HindIII + 

XmaI vip3Aa19 1 8.1 8.1 

Genomic MIR162 maize DNA digested with KpnI (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 EcoRV (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 HindIII plus XmaI (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. The negative control samples for each enzymatic 

digestion showed no hybridization bands and none were expected (KpnI Lane 4, EcoRV 

Lane 6, and HindIII plus XmaI Lane 8). The HindIII plus XmaI 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 hybridization 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 single 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 MIR162 maize transformation vector pNOV1300 

indicating locations of the manA coding sequence and the restriction endonuclease sites 

targeted in these Southern blot analyses is shown in Figure 9. Three digestion strategies were 

utilized to confirm the presence, size, intactness, and copy number of the manA coding 

sequence. The results of these analyses 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 

KpnI, SphI, HindIII, and XmaI 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. 

HindIII 

vip3Aa19 (2370 bp)

manA (1176 bp)

RB (25 bp) LB (25 bp)

iPEPC9 (108 bp)

manA probe (1176 bp)

ZmUbiInt (1993 bp)

ZmUbiInt (1993 bp)

35S terminator (70 bp)

NOS (253 bp)

KpnI (4786)

XmaI 

SphI (199)

SphI (2586) SphI (4797)

>3.6 kb 

~8.1 kb 

>3.6 kb 

HindIII + XmaI 

KpnI 

SphI 
 MIR162-USDA-1 Page 31 of 268 

Figure 10. Southern blot analysis of MIR162 maize with a manA-specific probe.

Maize genomic DNA (7.5 μg) was subjected to three separate digestions with KpnI or SphI or 

HindIII plus XmaI 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: BC4F1 generation of MIR162 maize digested with KpnI 

Lane 4: Control from BC4F1 generation maize digested with KpnI 

Lane 5: BC4F1 generation of MIR162 maize digested with SphI 

Lane 6: Control from BC4F1 generation maize digested with SphI 

Lane 7: BC4F1 generation of MIR162 maize digested with HindIII + XmaI 

Lane 8: Control from BC4F1 generation maize digested with HindIII + XmaI 

Lane 9: Control from BC4F1 generation maize digested with HindIII + XmaI plus 20.2 pg of 

HindIII + XmaI 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 BC4F1 KpnI manA 1 >3.6 4.0 

Lane 4 Negative KpnI manA 0 - - 

Lane 5 BC4F1 SphI manA 1 >3.6 9.0 

Lane 6 Negative SphI manA 0 - - 

Lane 7 BC4F1

HindIII + 

XmaI manA 1 8.1 8.1 

Lane 8 Negative HindIII + 

XmaI manA 0 - - 

Positive Control 

Lane 9 pNOV1300 HindIII + 

XmaI manA 1 8.1 8.1 

Genomic MIR162 maize DNA digested with KpnI (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 SphI (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 HindIII + XmaI (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 (KpnI Lane 4, SphI Lane 6, and HindIII + XmaI 

Lane 8). The HindIII plus XmaI digestion of the pNOV1300 vector produced the expected 

hybridization band at 8.1 kb (positive 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 expected, 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 pNOV1300 indicating locations of the ZmUbiInt 

promoter and the restriction endonuclease sites targeted for use in these Southern blot 

analyses is shown in Figure 11. Three digestion strategies were utilized to confirm the 

presence, size, intactness, and copy number of the ZmUbiInt promoter. 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 KpnI and SphI enzyme 

digestions, and one band for the HindIII and XmaI digestion. Additionally, ZmUbiInt is an 

endogenous maize promoter and, thus, the probe will hybridize 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 

~2.4 kb >3.6 kb 

HindIII + XmaI 

SphI 

KpnI 

vip3Aa19 (2370 bp) manA (1176 bp)

RB (25 bp) LB (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)

HindIII (189)

SphI (199)

SphI (2586)

SphI (4797)

XmaI (8293)

KpnI (4786)
 MIR162-USDA-1 Page 34 of 268 

Figure 12. Southern analysis of MIR162 maize with ZmUbiInt-specific probe. 

Maize genomic DNA (7.5 μg) was subjected to three separate digestions with KpnI or SphI or 

HindIII plus XmaI 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: BC4F1 generation of MIR162 maize digested with KpnI 

Lane 4: Control from BC4F1 generation maize digested with KpnI 

Lane 5: BC4F1 generation of MIR162 maize digested with SphI 

Lane 6: Control from BC4F1 generation maize digested with SphI 

Lane 7: BC4F1 generation of MIR162 maize digested with HindIII + XmaI 

Lane 8: Control from BC4F1 generation maize digested with HindIII + XmaI 

Lane 9: Control from BC4F1 generation maize digested with HindIII + XmaI plus 20.2 pg of 

HindIII + XmaI 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 BC4F1 KpnI ZmUbiInt 3 

Unknown 

Endogenous 22.0 

Lane 4 Negative KpnI ZmUbiInt 1 Unknown 

Endogenous 22.0 

~2.4 2.4 

>3.6 9.0 Lane 5 BC4F1 SphI ZmUbiInt 3 

Unknown 

Endogenous 2.8 

Lane 6 Negative SphI ZmUbiInt 1 Unknown 

Endogenous 2.8 

8.1 8.1 

Lane 7 BC4F1

HindIII + 

XmaI ZmUbiInt 2 Unknown 

Endogenous 4.0, 5.8 

Lane 8 Negative HindIII + 

XmaI ZmUbiInt 1 Unknown Endogenous 4.0, 5.8 

8.1 8.1 

Lane 9 pNOV1300 HindIII + 

XmaI ZmUbiInt 2 Unknown 

Endogenous 4.0, 5.8 

Genomic MIR162 maize DNA digested with KpnI (Figure 12, Lane 3) produced two 

hybridization bands of approximately 4.0 kb and 8.0 kb, corresponding to the expected size 

of the two copies of the ZmUbiInt 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 negative control (Lane 4). Genomic MIR162 

DNA digested with SphI (Lane 5) produced two unique hybridization signals of 

approximately 2.4 kb and 9.0 kb, corresponding to the expected two copies of the ZmUbiInt 

promoter present in the T-DNA. There was also 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). Genomic MIR162 maize DNA digested with HindIII + XmaI 

(Lane 7) produced a single hybridization signal of approximately 8.1 kb, corresponding to 

the full-length MIR162 T-DNA which contains two copies of the ZmUbiInt promoter, thus 

confirming the intactness of the insert. There were also two hybridization bands of 

approximately 4.0 kb and 5.8 kb present in both MIR162 maize (Lane 7) and the negative 

control (Lane 8) that represent endogenous ZmUbiInt sequence. The negative control 

corresponding to each digest showed no hybridization band other than that observed with 

endogenous ZmUbiInt sequences (KpnI Lane 4, SphI Lane 6, and HindIII + XmaI Lane 8). 

The HindIII + XmaI 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 were 
 MIR162-USDA-1 Page 36 of 268 

observed which represent endogenous ZmUbiInt sequence. 

For the Southern blot analyses with the ZmUbiInt promoter-specific probe, each restriction 

endonuclease digestion resulted in hybridization 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 indicating locations of the NOS terminator and 

the restriction endonuclease sites targeted for these Southern blot analyses is shown in Figure 

13. Three digestion strategies were utilized to confirm the presence, size, 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 

KpnI, SphI, HindIII, and XmaI restriction sites and their location relative to the NOS 

terminator. The vertical arrows indicate the endonuclease cutting sites and the expected size 

of the resulting restriction that should be detected when probed with NOS. 

~8.1 kb 

>3.6 kb 

>3.6 kb 

HindIII + XmaI 

SphI 

KpnI 

vip3Aa19 (2370 bp) manA (1176 bp)

RB (25 bp) LB (25 bp)

iPEPC9 (108 bp)

ZmUbiInt (1993 bp)

ZmUbiInt (1993 bp)

35S terminator (70 bp)

NOS (253 bp)

HindIII (189)

SphI (199)

SphI (2586)

SphI (4797)

XmaI (8293)

KpnI (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 maize with NOS terminator-specific probe. 

Maize genomic DNA (7.5 μg) was subjected to three separate digestions with KpnI or SphI or 

HindIII plus XmaI restriction endonucleases and subjected to Southern blot analysis with a 

NOS-specific probe. 

Lane 1: Molecular weight marker 

Lane 2: Blank 

Lane 3: BC4F1 generation of MIR162 maize digested with KpnI 

Lane 4: Control from BC4F1 generation maize digested with KpnI 

Lane 5: BC4F1 generation of MIR162 maize digested with SphI 

Lane 6: Control from BC4F1 generation maize digested with SphI 

Lane 7: BC4F1 generation of MIR162 maize digested with HindIII + XmaI 

Lane 8: Control from BC4F1 generation maize digested with HindIII + XmaI 

Lane 9: Control from BC4F1 generation maize digested with HindIII + XmaI plus 20.2 pg of 

HindIII + XmaI 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 BC4F1 KpnI NOS 1 >3.6 4.0 

Lane 4 Negative KpnI NOS 0 - - 

Lane 5 BC4F1 SphI NOS 1 >3.6 9.0 

Lane 6 Negative SphI NOS 0 - - 

Lane 7 BC4F1

HindIII + 

XmaI NOS 1 8.1 8.1 

Lane 8 Negative HindIII + 

XmaI NOS 0 - - 

Lane 9 pNOV1300 HindIII + 

XmaI NOS 1 8.1 8.1 

Genomic MIR162 maize DNA digested with KpnI (Figure 14, Lane 3) produced a single 

hybridization band of approximately 4.0 kb, corresponding to a single copy of the NOS 

terminator. Genomic MIR162 maize DNA digested with SphI (Lane 5) produced a single 

hybridization band of approximately 9.0 kb, corresponding to a single copy of the NOS 

terminator. Genomic MIR162 maize DNA digested with HindIII + XmaI (Lane 7) produced 

a single hybridization band of approximately 8.1 kb, corresponding to a single copy of the 

NOS terminator and confirming the intactness of the insert. The negative control 

corresponding to each digest showed no hybridization bands and none were expected (KpnI 

Lane 4, SphI Lane 6 and HindIII + XmaI Lane 8). The HindIII + XmaI digestion of the 

pNOV1300 vector produced the expected 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 contains 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 the 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 vector 
 MIR162-USDA-1 Page 39 of 268 

indicating the region covered by the backbone-specific probe, the location of each backbone 

element, and the restriction endonuclease sites targeted for this Southern blot analyses is 

shown in Figure 15. Three digestion 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 KpnI, EcoRV, 

HindIII, and XmaI 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 KpnI (Lane 3), EcoRV (Lane 5), and HindIII + XmaI (Lane 7). The 

blot was hybridized with a probe covering the entire backbone region 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 HindIII plus XmaI 

digestion of the pNOV1300 vector produced an expected 6.3 kb band (Lane 9). These results 

demonstrate that the MIR162 maize 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)

KpnI (4786)

HindIII (189)

XmaI (8293)

EcoRV (6965)
 MIR162-USDA-1 Page 40 of 268 

Figure 16. Southern blot analysis for plasmid backbone sequence.

Maize genomic DNA (7.5 μg) was subjected to three separate digestions with KpnI or EcoRV 

or HindIII plus XmaI 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: BC4F1 generation of MIR162 maize digested with KpnI 

Lane 4: Control from BC4F1 generation maize digested with KpnI 

Lane 5: BC4F1 generation of MIR162 maize digested with EcoRV 

Lane 6: Control from BC4F1 generation maize digested with EcoRV 

Lane 7: BC4F1 generation of MIR162 maize digested with HindIII + XmaI 

Lane 8: Control from BC4F1 generation maize digested with HindIII + XmaI 

Lane 9: Control from BC4F1 generation maize digested with HindIII + XmaI plus 20.2 pg of 

HindIII + XmaI 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 BC4F1 KpnI Backbone 0 None None 

Lane 4 Negative KpnI Backbone 0 None None 

Lane 5 BC4F1 EcoRV Backbone 0 None None 

Lane 6 Negative EcoRV Backbone 0 None None 

Lane 7 BC4F1

HindIII + 

XmaI Backbone 0 None None 

Lane 8 Negative HindIII + 

XmaI Backbone 0 None None 

Positive Control 

Lane 9 pNOV1300 HindIII + 

XmaI Backbone 1 6.3 6.3 

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

Stability of the T-DNA in MIR162 maize across multiple generations of conventional 

breeding was determined by Southern blot analysis, using backcross generations BC1F1, 

BC2F1, and BC4F1 (see Figure 5). For this analysis, MIR162 maize and negative control 

genomic DNA samples were digested with a restriction enzyme that digests once within the 

DNA insert but not within the functional element being probed. Southern blot analysis was 

performed using a vip3Aa19-specific probe. 

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

coding sequence and the restriction 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 BC1F1, BC2F1, and BC4F1 digested with Acc65I 

produced a single hybridization signal of approximately 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 hybridization (negative segregant of BC4F1; Lane 6). The 

Acc65I digestion of pNOV1300 produced a 14.4 kb band, as expected (positive control; Lane 

7). The hybridization patterns for generations BC1F1, BC2F1, and BC4F1 of MIR162 maize in 
 MIR162-USDA-1 Page 42 of 268 

this Southern blot analysis were identical, demonstrating the stability of the vip3Aa20

cassette over multiple generations. 

Figure 17. Restriction site and probe for vip3Aa19.

The vertical arrow indicates the endonuclease cutting site and the expected band size for the 

resulting restriction fragment that should be detected when probed with vip3Aa19.

These Southern blot analysis data demonstrate that the expected hybridization pattern was 

observed for the vip3Aa20 gene over several generations of MIR162 maize. Therefore, the 

stability of the insert in MIR162 maize during conventional breeding has been established 

over multiple generations. 

vip3Aa19 (2370 bp)

manA (1176 bp)

RB (25 bp) LB (25 bp)

iPEPC9 (108 bp)

vip3Aa19 probe (2370 bp)

NOS (253 bp)

ZmUbiInt (1993 bp)

ZmUbiInt (1993 bp)

35S terminator (70 bp)

Acc65I (4782)

>4.8 kb 

Acc65I 
 MIR162-USDA-1 Page 43 of 268 

Figure 18. Southern blot of BC1F1, BC2F1, and BC4F1 generations from MIR162.

Maize genomic DNA (7.5μg) was digested with Acc65I 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: BC1F1 generation of MIR162 maize digested with Acc65I 

Lane 4: BC2F1 generation of MIR162 maize digested with Acc65I 

Lane 5: BC4F1 generation of MIR162 maize digested with Acc65I 

Lane 6: Control from BC4F1 generation maize digested with Acc65I 

Lane 7: Control from BC4F1 generation maize digested with Acc65I plus 20.2 pg of Acc65I 

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 BC1F1 Acc65I vip3Aa19 1 >4.8 8.0 

Lane 4 BC2F1 Acc65I vip3Aa19 1 >4.8 8.0 

Lane 5 BC4F1 Acc65I vip3Aa19 1 >4.8 8.0 

Lane 6 Negative Acc65I vip3Aa19 0 - - 

Positive Control

Lane 7 pNOV1300 Acc65I 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 the hypothesis that the genes are inherited in 

accordance with the laws of Mendelian genetics. See Table 2 and Figure 5 for identification 

of the three generations used in this analysis. The Chi-square analysis was based on a 

comparison of observed and expected gene segregation ratios from each generation. TaqMan 

PCR analyses were conducted on plants from 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 positive for the two genes were selected for 

backcrossing; thus, the expected inheritance ratio 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 principles. 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. 

BC1F1 BC2F1 BC4F1

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. 

BC1F1 BC2F1 BC4F1

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 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. 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 located 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 homology 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 open reading frames. 

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

Data from Southern blot analyses demonstrated that MIR162 maize contains: i) single copies 

of the vip3Aa20 gene and the manA gene in the T-DNA; ii) two copies of the ZmUbiInt 

promoter within the T-DNA region, in addition 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 plasmid pNOV1300. Additionally, Southern blot 

analyses demonstrated that the MIR162 maize T-DNA was stable over several breeding 

generations. Sequence analysis of the entire 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 nucleotide changes encoded an amino acid change, 

whereby methionine at position 129 has been substituted 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 noncoding sequence, was truncated, and the entire 

left border, along with 32 base pairs of noncoding sequence, was truncated. These deletions 

have no effect on the functionality 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 analyses and DNA sequencing demonstrated 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 consistent with the genes 

being linked at a single locus in the maize genome. 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 the 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 maize have been characterized and tested to 

determine their potential for causing adverse effects 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 properties of the proteins; iv) biological 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 ‘vegetative 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 produced during vegetative bacterial 

growth and are secreted as soluble proteins into the extracellular environment. B. 

thuringiensis cultures continue to produce 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 thermolabile. Vip3Aa1 is the toxin 

designation assigned to the native protein discovered in B. thuringiensis strain AB88. 

Vip3Aa19 and Vip3Aa20 are toxin 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 proliferate in soil. Production of Cry and Vip 

toxins conveys upon the microorganism 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 their insecticidal activity has been studied and 

found to be similar, but not identical, to that which has been previously described for the B. 

thuringiensis Cry proteins. Following ingestion, full-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 plays a role in establishing 

the insecticidal specificity of a given protein for different insect species. As discussed later 

in this petition, the insecticidal activity of Vip3Aa proteins is limited to species within 

selected families of the order Lepidoptera (see Table 27). 

Native Vip3Aa1 is 789 amino acids in length and approximately 89 kDa molecular weight. 

The Vip3Aa20 variant produced in MIR162 maize is also 789 amino acids in length but 

differs from the native amino acid sequence by two amino acids (at positions 129 and 284). 

A Vip3Aa variant is also present in Syngenta cotton transformation 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 information 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 insecticidal 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-length Vip3Aa is proteolytically 

activated to form a 66 kDa active core in midgut extracts of susceptible 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 suggests 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 and environmental toxicology studies with the 

protein, significant quantities of Vip3Aa20 were produced in a recombinant E. coli

fermentation system. Production and extraction of Vip3Aa20 from MIR162 maize is not a 

feasible approach for obtaining the large quantities of the protein needed for these safety 

studies. Biochemical and bioactivity analyses were conducted to establish the equivalence of 

plant- and microbially produced Vip3Aa20; the latter being produced in an E. coli

fermentation system. 

The Vip3Aa20 protein produced in MIR162 maize was characterized by its molecular 

weight, mobility in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 

immunoreactivity, glycosylation status, and amino acid sequence. The amino acid sequence 

of full length Vip3Aa20 was deduced from the nucleotide sequence of the vip3Aa20 gene 

found in MIR162 maize. Peptide mass mapping by matrix-assisted laser desorption 

ionization time of flight mass spectrometry confirmed 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 protein extracts from MIR162 leaf tissue using anti-
 MIR162-USDA-1 Page 49 of 268 

Vip3Aa antibodies revealed an immunoreactive band of approximately 89 kDa molecular 

weight, a size that is consistent with the deduced sequence for full-length protein (see Figure 

19). The plant-produced protein was found not to be glycosylated. A more detailed 

description of the methods and results for the protein characterization 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. The 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 assessment of insecticidal activity, were used to 

compare plant-produced Vip3Aa20 to microbially produced protein. Similar analyses were 

performed to compare the biochemical properties and bioactivity of the Vip3Aa19 and 

Vip3Aa20 variants. 

As shown in lanes 3, 4, and 5 of Figure 19, the electrophoretic mobility, molecular weight, 

and immunoreactivity of microbial Vip3Aa20 and plant Vip3Aa20 are essentially the same. 

N-terminal sequencing and peptide mass mapping of microbial Vip3Aa20 matched the amino 

acid sequence of plant Vip3Aa20. Neither microbial- nor plant-produced Vip3Aa20 was 
 MIR162-USDA-1 Page 50 of 268 

found to be glycosylated. The efficacy and potency 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 lethal concentration (LC50) values for each protein is shown in Table 

13. Although a numeric difference in the computed LC50 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 LC50

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 LC50 

(ng protein/cm2) 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 LC50 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 plant- and microbially produced Vip3Aa20 proteins 

have been verified and it can be concluded that Vip3Aa20 proteins produced in recombinant 

E. coli and MIR162 maize are biochemically and functionally equivalent. Having 

established equivalence of the proteins, it was appropriate to utilize microbially produced 

Vip3Aa20 as a surrogate for the plant-produced 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 amino acid. They are of comparable molecular 

weight, electrophoretic mobility, and immunoreactivity as determined by Western blot 

analysis. Neither protein is glycosylated. As shown in Table 13, a comparison of 

insecticidal activity results shows no biologically significant difference between the 

computed LC50 values for the Vip3Aa20 from MIR162 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 data 
 MIR162-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 commodities of all crops. A 

summary of the pertinent information and toxicology data bearing on the potential risk of 

Vip3Aa20 for humans is presented below: 

• B. thuringiensis microbial insecticides that are commercially 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 mice 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 latest 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 Vip3Aa20, indicating they may be closely related to Vip3Aa20, 

and if any proteins with significant sequence identity to Vip3Aa20 are known to be 

toxins, indicating possible implications for the toxic potential of Vip3Aa20. The 

NCBI Entrez protein database was searched 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 between highly homologous proteins yield 

E-values approaching zero, indicating a very low probability that such matches would 

occur by chance. The probability that amino acid sequence similarities observed in a 

database search occur by chance increases with higher E-values (Ponting, 2001). The 

Vip3Aa20 query sequence showed no significant sequence identity to any proteins 

identified as, or known to be, toxins other than the vegetative insecticidal proteins 

from B. thuringiensis. Vip3Aa20 has no significant sequence homology to known 

mammalian protein toxins. 

• The Vip3Aa20 amino acid sequence was compared to the Syngenta Allergen 

Database (version 4.0) to determine if it has significant sequence identity to proteins 

known or suspected to be allergens, indicating possible implications for the allergenic 

potential of Vip3Aa20. The Syngenta Allergen Database was compiled from entries 

identified as allergens or putative allergens in public protein databases, and was 

supplemented with additional amino acid sequences identified from the scientific 

literature as being known or putative allergens. 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 parameters: 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 sequence were compared 

to protein sequences in the database using the FASTA search algorithm (Pearson and 

Lipman, 1988). Any 80-amino acid peptide of the query sequence having greater 

than 35% amino acid identity to an allergen sequence was identified as having 

significant identity to the allergen sequence in accordance with the recommendations 

of FAO/WHO (2001). The results of this analysis revealed that there was no 

significant identity between any of the sequential 80-amino acid peptides of 

Vip3Aa20 and any entry in the Syngenta Allergen Database. Therefore, Vip3Aa20 

does not share overall sequence homology with any known allergenic protein. 

• The Vip3Aa20 protein sequence was also screened for matches of eight contiguous 

amino acids between the Vip3Aa20 sequence and the allergen sequences in the 

Syngenta Allergen Database to screen for short, local regions of amino acid identity 

that might indicate the presence of common T-cell binding epitopes (Hileman et al., 

2002; Stadler and Stadler, 2003; Silvanovich et al., 2006). There were no matches of 

eight contiguous amino acids between Vip3Aa20 and any entry in the allergen 

database. No significant amino acid sequence identity of Vip3Aa20 to known or 

putative allergenic proteins having implications for allergenic 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 degraded within two minutes in simulated gastric fluid 

containing pepsin. Thus, there will be minimal opportunity for human and domestic 

animal systemic exposure to Vip3Aa20. 

• Vip3Aa20 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 indicate that the Vip3Aa20 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 that catalyzes the reversible conversion of 

mannose 6-phosphate and fructose 6-phosphate and 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 survive and grow on media containing 

mannose as the only or primary energy source. When placed in medium containing 

predominantly or only mannose as the sole sugar source, nontransformed tissue remains 

dormant and becomes outgrown by transformed tissue. Mannose itself is not toxic to plant 
 MIR162-USDA-1 Page 53 of 268 

cells. Inclusion of the manA gene in the T-DNA of transformation plasmid pNOV1300 

allowed for selection of successfully transformed maize plantlets. 

V.B.2. Characterization of Phosphomannose Isomerase 

Western blot analysis was used to characterize the PMI enzyme produced in MIR162 maize. 

In order to conduct animal toxicology studies with the protein, significant quantities of PMI 

were produced in a recombinant E. coli fermentation system. Production and extraction of 

PMI from MIR162 maize is not a feasible approach for obtaining the large quantities of the 

protein needed for these safety studies. Biochemical and bioactivity analyses were 

conducted to establish the equivalence of plant- and microbially produced PMI. 

PMI from MIR162 maize was found to be approximately 42.8 kDa molecular weight which 

matched the predicted molecular weight based 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 acid 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 sources 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 measurements fell within the range of 

historically observed values at Syngenta. 

Based on these results, it can be concluded that PMI from MIR162 maize and PMI from E. 

coli are biochemically and functionally equivalent. A more detailed description of the 

methods and results for this protein characterization work can be found in Appendix C. 

V.B.3. Safety Assessment for 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 consultation. 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 toxicology data bearing on the 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 enzyme is also found in a wide variety 

of other species. Thus, there is a history of safe consumption of PMI proteins in 

human food and animal feed sources. 

• No adverse effects were observed in mice administered 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 administered to the animals. Mice have 

historically been a suitable surrogate species for detecting evidence of toxicity in 

mammals. 
 MIR162-USDA-1 Page 54 of 268 

• The amino acid sequence of PMI has been compared 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 homology 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 significant amino acid sequence identity to 

known or putative allergenic protein sequences that are biologically relevant or that 

have implications for allergenic potential. This sequence comparison was conducted 

following the methodology described above for 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 between PMI and the 

serum from the single individual known to have demonstrated IgE-mediated allergy 

to this specific α-parvalbumin. The patient’s serum 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 for 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 indicate that the PMI protein 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 proteins in plant tissues from two MIR162 hybrids 

were determined using an enzyme-linked immunosorbent 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 fresh-weight and dry-weight basis were 

determined and are reported in Tables 14 and 15, respectively. No meaningful differences 

between the two hybrids were noted in their concentrations of Vip3Aa20 and PMI in each 

tissue type at each growth stage sampled. Therefore, values presented in the tables are means 

of the two hybrids. Vip3Aa20 and PMI concentrations 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 expected 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 tissue and dry weight basis. All ELISA results 

have been corrected for method extraction efficiency. 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 μg/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 μg/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 overnight 
 MIR162-USDA-1 Page 56 of 268 

Table 15. Mean tissue concentrations of PMI in MIR162 plants. 

Concentrations are presented on both a fresh tissue and dry weight basis. All ELISA results 

have been corrected for method extraction efficiency. 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 μg/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 μg/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 overnight 
 MIR162-USDA-1 Page 57 of 268 

VI. Phenotypic and Compositional Evaluation 

Laboratory, greenhouse, growth chamber, and field investigations have been conducted to 

assess the phenotype and biochemical composition of MIR162 maize. The purpose of these 

investigations was to determine if unintended changes occurred in MIR162 maize as a result 

of the transformation process and to determine if any unintended 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 assessing 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 created 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 characteristics could be considered indicative of 

increased weediness potential and plant pest risk. If no significant 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 laboratory, 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 laboratory, 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 (μm) 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 characteristics that can be indicative of plant 

pest potential. Dormancy mechanisms, including 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 germination and dormancy characteristics of MIR162 

seed using a modification of the testing protocol established by the Association of Official 

Seed Analysts 

Seed samples of two MIR162 hybrids, their respective near-isogenic controls, and three 
 MIR162-USDA-1 Page 59 of 268 

conventional hybrids served as test, control, and reference materials 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 MIR162 hybrid #2 corresponds 

to seed lot code ‘E’ as described 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 F1 NP2391 x NP2222(BC6F4) 

Control hybrid #1 F1 NP2391 x NP2222 

MIR162 hybrid #2 F1 NP2222(BC6F5) x NP2391 

Control hybrid #2 F1 NP2222 x NP2391 

Reference hybrid #1 F1 N59-Q9 

Reference hybrid #2 F1 N72-G8 

Reference hybrid #3 F1 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 divided into four replicates of 40 seeds per 

replicate per temperature regime. Six 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 temperature-controlled growth chambers. For the 

alternating temperature regimes, the lower temperature was maintained for 16 hours and the 

higher temperature for eight hours, and then the 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 development 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 visibly deteriorated and soft to the touch) 

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

• hard seed (seed that had not imbibed 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 towel 
 MIR162-USDA-1 Page 60 of 268 

was carefully unrolled and examined four, seven, and 12 days after 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 visibly deteriorated and soft to the touch) 

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

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

At the end of each experiment, all firm swollen 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 swollen’, ‘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 frequency of zero response rates 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 higher temperatures, there was a high frequency of 

100% normal germinated seeds among the replicates. 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 contingency tables and subjected to 

Fisher’s Exact Test. Significance was assigned at p<0.05. Only the results for ‘normal 

germinated’ seeds were statistically analyzed. 

There were no statistically significant differences 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 demonstrates 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 replicates from test, control, and reference hybrids 

evaluated under different temperature regimes. 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 - 1.3 
 MIR162-USDA-1 Page 62 of 268 

Table 19. Statistical analysis of proportions for normal germinated seeds. 

Fisher’s Exact Test was used to compare 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 conducted during the 2005 and 2006 growing seasons to compare a 

range of agronomic characteristics of two MIR162 maize hybrids 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 F1 NP2010 x NP2222(BC6F4) 2005 

Control hybrid F1 NP2222 x NP2010 

MIR162 hybrid F1 NP2673 x NP2171(BC4F5) 2006 

Control hybrid F1 NP2673 x NP2171 

Near-isogenic hybrid pairs were grown in six locations in 2005 and 10 locations in 2006. 

These locations are representative 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 location and trials planted in 2006 included five replicates per 

location. Plot size was 0.02 acres, using two-row 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 across locations using the 

following model: 

Yijk = U + Ti + Lj +B(L)jk + LTij + eijk

Yijk is the observed response for entry i at location j block k; U is the overall mean; Ti is the 

entry effect, Lj is the location effect, B(L)jk is the effect of block within a location; LTij is the 

location-by-entry interaction effect and eijk 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 between 
 MIR162-USDA-1 Page 64 of 268 

the two genotypes was statistically significant 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, the data sets contained too few 

nonzero entries upon which to compute a reasonable estimate of residual error. 

Results of the 2005 agronomic equivalence trials are presented in Table 22. Evaluation of 

the 2005 trial results revealed only one statistically significant difference between a control 

and MIR162 variate. The average number of germinated 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 biological 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 differences 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% lower 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 represent 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 characteristics 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 representative 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 

analyzed 
 MIR162-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 representative 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 

analyzed 
 MIR162-USDA-1 Page 67 of 268 

VI.A.3. Pollen Viability and Morphology 

Viability and morphology were evaluated to assess the potential impact of transformation on 

pollen characteristics of MIR162 maize. Pollen 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-isogenic 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 from 23°C-28°C and nighttime temperatures 

ranging from 18°C-22°C. Pollen was 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/I2 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. Nonviable 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 significant difference detected between MIR162 and 

control percent viable pollen. In fact, the frequency of nonviable cells averaged 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 (μm) 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 magnification. 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 the MIR162 and control samples. These means 

were compared by a t-test with significance assigned at the p<0.05 level. Photographs of 

representative MIR162 and control 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 μm 

MIR162 Plant 

#88708 80X 

Viable Cell 

Nonviable Cell 

Nonviable Cell 
 MIR162-USDA-1 Page 69 of 268 

VI.A.4. Seed Dispersal 

Dispersal of individual maize seeds does not occur naturally. The kernels are held inside the 

husks of the cob and are too heavy to be wind-blown. There was 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 place 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 and 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 performed 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 analyses 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 corresponding near-isogenic 

control were harvested from six locations in the U.S. during 2005. These locations are 

representative of the major corn 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 agronomic 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 components analyzed are listed in 

Table 25. 

All analyses were conducted using methods published and approved by the Association of 

Analytical Communities (AOAC) International or other industry-standard analytical 

methods. Based on the moisture content of each sample, analyte levels were converted to 

equivalent units of dry weight. 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 subjected to an analysis of variance using the 

model: 

Yijk = U + Ti + Lj + B(L) jk + LTij + eijk

where Yijk is the observed response for genotype i at location j block k, U is the overall mean, 

Ti is the genotype effect, Lj is the location effect, B(L)jk is the effect of block within a 

location, LTij is the location-by-genotype interaction effect and eijk is the residual error. 
 MIR162-USDA-1 Page 70 of 268 

Table 25. Forage and grain components measured 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 B1 (thiamine) 

Moisture Iron Starch Vitamin B2 (riboflavin) 

Neutral detergent fiber 

(NDF) Magnesium Total Dietary Fiber (TDF) Vitamin B3 (niacin) 

Protein Manganese Fatty acids Vitamin B6

(pyridoxine) 

Phosphorus Palmitic acid Vitamin B9 (folic acid) 

 Potassium Stearic acid Vitamin E (αtocopherol) 

 Selenium Oleic acid 

 Sodium Linoleic acid 

 Zinc Linolenic acid 

For each analyte, the statistical significance 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 used to detect potential 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 genotypes averaged across 

locations may not be valid. In such cases, the 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 locations 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 compositional analyses, expressed as mean values across locations and a 

range of individual replicate values, are presented in the tables found in Appendix E (Tables 

E-3 through E-11). Nine components of maize forage were measured; the difference 

between MIR162 and control mean values was found to be statistically significant for one of 

these analytes. Fifty-six components of grain 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 that 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 control mean values. The mean value for 

MIR162 NDF was 11.34% higher than the corresponding control value. This difference is 

considered relatively small and the MIR162 mean falls well within the range of normal 

values reported by ILSI and OECD. No statistically 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 small (< 8%) and the MIR162 mean values were 

well within the ranges of normal values for the control maize. Additionally, the average 

values for all proximates were within the ranges reported by ILSI and OECD. 

Statistically significant differences 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 normal values reported by ILSI and OECD for 

conventional maize. 

Statistically significant differences were noted between MIR162 and control mean levels of 

vitamin A (β-carotene), vitamin B6 (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 observed 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 B9 a statistically 

significant genotype-by-location interaction 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. Individual location means for the two analytes are 

provided in Table E-8 (Appendix E). The vitamin A and vitamin B9 levels at all locations 

were within the ranges reported in the literature. 

There were no significant differences noted for any of the 18 amino acids or anti-nutrients 

measured and all average values were within 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 observed for these fatty 

acids were within the ranges of values observed for the control grain. Furthermore, the
 MIR162-USDA-1 Page 72 of 268 

Table 26. Statistically significant differences between MIR162 and control analytes. 

Summary composition results for the one forage 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 B6

(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 acids 
 MIR162-USDA-1 Page 73 of 268 

average values for all fatty acids were within the range of normal values reported for 

conventional maize by ILSI and OECD. 

Statistically significant differences were noted in the secondary metabolites ferulic acid and 

ρ-coumaric acid. These differences were relatively 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 values reported for conventional maize by ILSI and 

OECD. 

Collectively, the observed differences between MIR162 and control means are considered of 

no biological significance and represent typical random variance. The magnitude of the 

differences was small, all MIR162 values fell within normal ranges for conventional maize, 

and the MIR162 and control data ranges significantly overlapped. MIR162 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. Nevertheless, several features of maize make it unlikely to form selfsustaining weedy populations in agricultural systems: i) it is easily controlled in subsequent 

crops by selective herbicides; ii) seed dispersal is limited because seeds are held inside the 

husks of the cob; and iii) the seeds lack dormancy. 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 uncompetitive against perennial vegetation. The results of 

laboratory, greenhouse, and field studies indicate that MIR162 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 protein that has toxicological activity against a number of 

lepidopteran species which are pests of U.S. agriculture. Studies have been conducted to 

define the spectrum of insecticidal activity for Vip3Aa proteins and to assess the efficacy of 

MIR162 maize hybrids in resisting larval feeding 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 environmental concentrations (EECs) for Vip3Aa20; ii) a 

characterization of potential hazards posed by the protein to nontarget indicator organisms 

appropriate for a maize ecosystem; iii) a comparison of EECs to no observable effect 

concentrations for nontarget organisms; and iv) a specific characterization of endangered 

species risk. 

Given the ubiquitous nature of PMI enzymes in nature and its apparent absence of 

environmental risk, it can reasonably be assumed that the presence of PMI protein in 
 MIR162-USDA-1 Page 74 of 268 

MIR162 will do nothing to alter the ecological balance within maize ecosystems. 

VII.A. Spectrum of Insecticidal Activity for Vip3Aa Proteins 

Syngenta has conducted mortality bioassays with Vip3Aa protein variants 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 order Lepidoptera, yet all lepidopteran species are not 

sensitive to the protein. It is notable that Vip3Aa proteins appear to have no activity against 

O. nubilalis, probably the most significant lepidopteran pest of U.S. maize. Table 27 

provides a listing of insect species 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 currently lists 25 variants of the Vip3Aa 

protein.5

 This narrow spectrum of activity for Vip3Aa proteins is a very positive attribute 

from an ecological perspective; maize hybrids containing a Vip3Aa protein are unlikely to 

pose a risk to nontarget organisms inhabiting maize ecosystems. 

Table 27. Insect species screened for sensitivity 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 sensitivity 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 

Other Lepidoptera Earias vittella Active 

Ephestia kuehniella Active 

Culex pipiens Not Active 

Diptera Drosophila melanogaster Not Active 

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 against 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 logical approach for limiting crop losses attributable to 

these four pests, especially considering the deficiencies of conventional 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 cooperators, and extension agents. Figure 21 

provides a graphic representation of the comparative feeding damage for each of the 
 MIR162-USDA-1 

0

20406080

100120

Bt11 MIR162 Bt11xMIR162 Insecticide

Treatment

% of Control Damage 

Figure 21. MIR162 field efficacy trial results. Comparative feeding damage ratings are from replicated field efficacy trials conducted in 2005 and 2006 with Bt11xMIR162 maize, and a conventional insecticide treatment (Warrior

® Insecticide). Mean damage ratings 

percentage of the damage observed in the untreated control plots. 

® Trademark of a Syngenta group company 
 MIR162-USDA-1 Page 77 of 268 

treatments, expressed as a percentage of damage measured in the control plants, for five of 

the insects evaluated. Trials were placed in locations to take advantage of natural pest 

infestations. In some locations trials were artificially infested to test trait performance under 

high levels of insect pressure. MIR162 maize, Bt11 maize, Bt11xMIR162 maize, 

conventional maize, and conventional maize with an insecticide application were the 

treatments employed in most trials. These trials were conducted under 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 insects. The combined-trait 

Bt11xMIR162 hybrid is very efficacious against all five insects. MIR162 addresses the 

lepidopteran pest-control limitations of existing Cry1Ab-containing maize hybrids. In 

combination with the Bt11 trait, it will provide 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 MIR162 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 risks for endangered species are 

addressed separately in Section VII.F. 

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

The concentrations of Vip3Aa20 were measured by ELISA in several MIR162 maize tissues 

and whole plants at growth stages V9-V12, anthesis, seed maturity and senescence (refer to 

Table 14). The values chosen for computation of EECs are shown in Table 28. These values 

represent the highest mean concentration across locations at any growth stage of MIR162 

hybrid maize. EECs were calculated from fresh weight values. 

VII.C.2. Soil Fate of Vip3Aa Proteins 

Most proteins do not persist or accumulate in the 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 occurring 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 determined by ELISA in tissues of two fieldgrown MIR162 hybrids. All values are reported on a fresh weight basis. 

Tissue Vip3Aa20 (μg/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 the degradability of Vip3Aa proteins in live 

soils. For this investigation, the Vip3Aa19 variant was used. Live soil samples from Brazil 

and the U.S., plus one artificial soil sample, representing four soil textures (clay, sandy clay 

loam, sandy loam, and silt loam) were used to examine the rate of degradation of Vip3Aa19. 

The source of Vip3Aa19 for this study was a concentrated protein extract 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 

μg/g dry weight equivalent soil). This concentration was expected to greatly 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 observed in all soil types; degradation was measured as loss of 

insecticidal activity. The time to 50% dissipation (DT50) was estimated to be between 6.0 

and 12.6 days across soil types and test concentrations. 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 Concentrations 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 run-off or gene flow is unlikely 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 annum 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 kernels and weed seeds (Linz et al., 2003). Therefore, the 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 Vip3Aa20 in MIR162 maize kernels is 29.81μg/g fresh weight 

(Table 28). Exposure to birds may be expressed 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 consuming fresh food were estimated by Crocker 

et al. (2002). Among the seven species 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 grackles, 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 μg Vip3Aa20/g kernels. This yields a realistic EEC for wild 

birds of: 

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

with a DDD for wild birds of: 

0.5 x 0.35 x 29.81 μg Vip3Aa20/g = 5.22 μg 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 corn seeds. Frequently these species remove 

so many seeds that the field 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 rodents. Larger mammals such as Odocoilus virginianus
 MIR162-USDA-1 Page 80 of 268 

(white-tailed deer) and Procyon lotor (raccoons) cause injury to ripening ears. Deer typically 

nip off ear tips, whereas raccoons chew through 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 μg/g fresh weight 

(Table 28). As with birds, exposure to mammals may be expressed more suitably as a DDD 

(calculated by the formula 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 derived from the proportion of maize kernels in the diet of 

rodents feeding in maize growing areas. The proportion of maize kernels in wild rodent diets 

varies greatly according to species (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 μg 

Vip3Aa20/g kernels. This yields a realistic EEC for wild mammals of: 

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

with a DDD for wild mammals of: 

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

VII.C.3.c. EEC for Pollinators 

Honeybees forage for maize pollen and therefore, can be exposed to Vip3Aa20 via MIR162 

pollen (Severson and Perry, 1981). Honeybees can successfully 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 other pollinators is 

computed to be: 

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

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

Nontarget arthropods rarely, if ever, eat leaves of maize. The more likely route of exposure 

to Vip3Aa20 for these species is consumption 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 will vary depending on 

the prey species, its developmental stage, and the concentration of Vip3Aa20 in plant parts 

on which they are feeding. Several studies have examined the concentration of Cry proteins 

in herbivores relative to the concentration of plants on which they are feeding; most tested 
 MIR162-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 oilseed 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 these studies show that herbivores contain lower concentrations of 

B. thuringiensis toxins than the plants on which they are feeding. Sucking insects, such as 

aphids, contain only trace amounts of Cry1Ab 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 concentration 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 transitory; adults contain about half this 

amount and pupae less than 1/40th the concentration in larvae (Obrist et al., 2005). The 

herbivores with the highest concentrations of Cry protein are Tetranychus urticae (spider 

mites); they have been found to contain between 0.7 and 3X the concentration 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 arthropods may be exposed to Vip3Aa20 through 

consumption of these species. 

A precise EEC is difficult to compute given the 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 reasonably 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 drought 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 diet 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 concentrations of toxin compared with 

adult mites (Roy et al., 2002). Hence, 0.2X the leaf concentration of Vip3Aa20 is a 

reasonably conservative EEC. The highest average concentration of Vip3Aa20 in above 

ground tissue of MIR162 plants is 50.41 μg/g leaf fresh weight at seed maturity (Table 28). 

The realistic EEC for above-ground nontarget arthropods is computed to be: 

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

VII.C.3.e. EEC for Soil-dwelling Nontarget Invertebrates 

A realistic EEC for soil-dwelling nontarget arthropods 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 μg/g at seed maturity 

(Table 28). The average planting density of maize is 65,500 plants per hectare and the 
 MIR162-USDA-1 Page 82 of 268 

average fresh weight of a corn plant is 750 g. If MIR162 plants contain an average of 17.35 

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

65,500 plants x 750 g/plant x 17.35 μg Vip3Aa20/g = 8.52x108 μg 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 m3

 of soil per hectare. The average density of soil is 1,500 

kg/m3. Therefore, the MIR162 maize will be incorporated into 1,500 m3/ha x 1,500 kg/m3 = 

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: 

8.52x108 μg Vip3Aa20/ha ÷ 2,250,000 kg soil/ha = 379 μg Vip3Aa20/kg soil 

(or 0.38 μg Vip3Aa20/g soil) 

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

The main route of potential exposure of aquatic organisms to Vip3Aa20 is through MIR162 

pollen deposited in water bodies adjacent to maize fields. Maize produces up to 3.15x1011

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 1.26x108 mg of pollen per hectare. A worst-case assumption for loading of maize 

pollen into a body of water is that it is deposited onto the surface of the water at the same 

density it is produced in the field. For a 1-hectare pond (10,000 m2) that is 2 m deep, the 

pollen concentration would be: 

1.26x108 mg pollen ÷ (10,000 m2 x 2 m) = 6300 mg pollen/m3 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 immediate edge. A reasonably conservative 

extrapolation from this observation is that pollen loading into an adjacent water body is likely 

to be at least 0.05X the density of pollen within the maize field.7

 Estimates of settling and 

degradation of maize pollen in ponds are not available, 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 realistic 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 μg 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 concentration of Vip3Aa20 in MIR162 grain is 29.81 

μg/g. Fish feed is heat-treated during preparation and therefore, it is likely that Vip3Aa20 

will be denatured and lose activity in feed prepared from MIR162 grain. Maize in fish feed 

is unlikely to comprise 100% MIR162 grain. 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 Vip3Aa20 is lost during heat treatment of the 

feed, a realistic EEC for farmed fish is: 

29.81 μg Vip3Aa20/g x 0.25 x 0.40 x 0.5 = 1.49 μg 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 estimating the risks to populations of nontarget 

organisms. Once defined, these EEC or DDD values can be directly compared to Vip3Aa 

exposure or dose levels that cause no adverse effects 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 highest average concentration of Vip3Aa20 in 

applicable MIR162 plant tissues and conservative 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 μg/g kernels 5.22 μg/g bw 

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

Pollinator 14.91 μg/g pollen - 

Above-ground arthropods 10.08 μg/g diet - 

Soil-dwelling invertebrates 0.38 μg/g soil - 

Aquatic organisms 0.0068 μg/l water - 

Farmed fish 1.49 μg/g feed - 
 MIR162-USDA-1 Page 84 of 268 

VII.D. Impact on Nontarget Organisms 

Thirteen species representing groups of nontarget organisms potentially exposed to 

Vip3Aa20 in MIR162 maize were exposed to Vip3Aa variants in laboratory bioassays for the 

purpose of identifying adverse effects and establishing a no observable effect concentration 

(NOEC) or NOEL. Several test substances, containing one of three variants of Vip3Aa, were 

used in these studies. The identity and source 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 that are found in the scientific 

literature. As discussed earlier in this petition (see Section V.A.1), these variants share 

99.7% sequence identity and are functionally equivalent. Therefore, Vip3Aa1 and Vip3Aa19 

are suitable test substance 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 μg/g Daphnia magna 

Plant-incorporated 

Vip3Aa19 Pacha maize pollen 144.8 μg/g 

Colleomegilla 

maculata, 

Chrysoperla carnea

Plant-incorporated 

Vip3Aa19 Pacha maize leaf 3640 μg/g Eisenia foetida 

Plant-incorporated 

Vip3Aa19 Pacha maize leaf 86.7 μg/g Folsomia candida 

Plant-incorporate 

Vip3Aa19 Pacha maize grain 7.10 μg/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 the 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 VipCotTM 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 applicable for predicting hazards and risk for MIR162 

maize because Vip3Aa19 is biochemically and 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 concentration of Vip3Aa protein was confirmed 

in all artificial meat-based diets by Western 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 protein/kg bw, by oral gavage. The effects on 

bird mortality and growth were compared with 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:ADfCD-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 control group of male and female mice for 14 

days after exposure, and many histological and biochemical endpoints were assessed at the 

end of the test. No adverse effects were observed 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 μg/g diet; these concentrations represented approximately 

1, 4 and 10 times the concentration of Vip3Aa20 in MIR162 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 μg 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, near-isogenic maize. Fresh diet was supplied 

daily. The difference in survival of the beetles in the treatment and control groups was not 

statistically significant, and the NOEC was shown to be ≥ 7.24 μg 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 μg Vip3Aa19/g diet. The negative control diet comprised 

50% sucrose solution only. The treatment and control groups were fed fresh diet daily. The 

difference in survival between the treatment and control groups was not statistically 

significant, and the NOEC was shown to be ≥ 7250 μg protein/g diet. 

Second-instar Orius insidiosus (minute pirate bug) were exposed to microbial Vip3Aa19 test 

substance. The test substance was incorporated into an artificial liver-based diet at 7250 μg 

Vip3Aa19/g diet. The negative control liver-based diet was treated with buffer only. The 

treatment and control groups were fed fresh diet 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 μg 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-isogenic 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 μg 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-based diet at 7250 μg 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 control groups was not statistically significant; 

however, the control mortality exceeded the guideline validity criterion of not greater than 

25% (Vogt et al., 2000); the control mortality remained within 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 terminated when the control mortality exceeds 20%; the 

control mortality remained within this guideline up to day 14. At day 14 and day 21, the 

differences between the treatment and negative control groups were not statistically 

significant. Fourteen and 21 days represent significant portions of larval development, and 

therefore, the data can be considered a rigorous assessment of the toxicity of Vip3Aa19 to 

green lacewings. The NOEC was shown to be ≥ 7250 μg protein/g diet 
 MIR162-USDA-1 Page 87 of 268 

VII.D.5. Effect of Vip3Aa on Soil-dwelling Invertebrates 

Adult Eisenia foetida (earthworms) were exposed to Vip3Aa19 protein extracted from Pacha 

maize leaf tissue. The test substance was incorporated into an artificial soil substrate at 3.60 

μg Vip3Aa19/g soil. The negative control was artificial soil treated with a leaf protein test 

substance prepared from nontransgenic, near-isogenic maize. After 14 days, there was 100% 

survival in both the treatment and negative control groups, and there were no statistically 

significant differences in body weight between the groups. The NOEC was shown to be ≥

3.60 μg protein/g soil. 

Juvenile Folsomia candida (collembola) were exposed to Vip3Aa19 via lyophilized Pacha 

maize leaf tissue. The test substance was mixed with an equal weight of yeast to form the 

treatment diet containing 43.2 μg Vip3Aa19/g diet. A negative control diet containing equal 

parts yeast and lyophilized leaves of nontransgenic, near-isogenic maize was also prepared. 

The collembola were provided with fresh diet daily. After 28 days, there were no statistically 

significant differences in survival, or in the number of new juveniles, between the Pacha 

maize leaf treated- and the nontransgenic maize leaf-treated groups. The NOEC was shown 

to be ≥ 43.2 μg 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. Fresh diet was supplied daily. 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 statistically significantly different from that of the control group. The 

NOEC was shown to be ≥ 500 μg 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 water at 120 mg pollen/l, representing 10.1 μg 

Vip3Aa19/l. The negative control was nontransgenic, 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 there was no sign of immobilization or other 

sublethal effects. The NOEC was ≥ 10.1 μg protein/l water. 

Juvenile Ictalurus punctatus (channel catfish) were exposed to Vip3Aa19 via fish feed 

prepared from Pacha maize grain. The feed was formulated to contain the maximum 

proportion of maize grain that provides 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 from nontransgenic, near-isogenic maize. 

Treatment and control groups of catfish were fed the respective feeds three times daily for 30 

days. There were no statistically significant differences in mean wet body weight increase 
 MIR162-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 day of the study. The NOEC was shown to be ≥ 7.1 

μg protein/g feed. 

VII.E. Toxicity Exposure Ratios for Nontarget Organisms 

The EEC and DDD values are compared with results of Vip3Aa hazard studies in order to 

make judgments about risk. The hazard studies identified any potential for Vip3Aa proteins 

to cause adverse effects in representative nontarget 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 attainable. No harmful effect of 

such exposure was observed in any species tested, and the concentration 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 results in the toxicity exposure 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 safety of Vip3Aa20 in MIR162 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 contains 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 concentration of Vip3Aa tested in the studies was 

sufficient to achieve margins of exposure of ≥ 1 for all but one species 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 indication of elevated risk but rather a reflection 

of the fact that the test substance was pollen 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-incorporated 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 contained 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 μg/g bw 5.22 μg/g bw ≥ 77 

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

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

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

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

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

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

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

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

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

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

D. magna ≥ 10.1 μg/l water 0.0068 μg/l water ≥ 1507

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

a - adults 

b - larvae 

VII.F. Safety Assessment for Endangered and Threatened Species 

There is a weight of evidence that at concentrations in MIR162 maize the toxicity of 

Vip3Aa20 will be limited to Lepidoptera. Its receptor-mediated mechanism of action and 

absence of activity in bioassays with multiple species outside of the order Lepidoptera 

support this conclusion. Furthermore, no harmful effects of Vip3Aa proteins have been 

observed in nontarget organism hazard identification studies. These studies used a wide 

range of taxa and at expected environmental concentrations they indicate a lack of risk 

associated with exposure to Vip3Aa20 in MIR162 maize (see Table 31). 

The only endangered or threatened lepidopteran 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 exposure for this species is consumption of 

maize pollen that has settled on the leaves of its larval food plant, the wild lupine (Lupinus 
 MIR162-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 populations are separated from maize fields by at 

least 500 metres, and because maize anthesis usually 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 interest, and there has been concern 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 insecticidal 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 trypsinized Vip3Aa1 did not form 

pores in the midgut of monarchs; pore formation appears to be essential for toxicity and 

occurs in the guts of insects susceptible to Vip3Aa1. These investigators also found no 

mortality of D. plexippus in a surface diet bioassay limit test at 1000 ng/cm2

. MIR162 maize, 

therefore, poses low risk to monarchs because 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 appropriate for a maize ecosystem, and the NOEC or 

NOEL was the highest concentration (or dose) tested in each study. The exposures in all but 

one study were in excess of EEC or DDD levels for the nontarget organism groups 

represented, indicating a low probability of harm to these groups from Vip3Aa20 in MIR162 

maize. 

The very narrow spectrum of insecticidal activity observed for Vip3Aa proteins indicates 

with high certainty that no endangered or threatened species other than Lepidoptera would be 

harmed by contact with Vip3Aa20 via MIR162 maize. There is minimal exposure of 

endangered Lepidoptera to maize and therefore, cultivation of MIR162 maize is unlikely to 

harm any endangered or threatened species in the U.S. 

VII.H. Gene Flow Assessment 

An assessment of the environmental fate of Vip3Aa20 resulting from the cultivation of 

MIR162 maize requires consideration not only of the production and degradation of the 

protein within maize fields, but also the possibility that Vip3Aa20 could persist, or spread 
 MIR162-USDA-1 Page 91 of 268 

from, areas of maize cultivation because of gene flow or the establishment of weedy 

populations of MIR162 maize. It is highly unlikely that Vip3Aa20 will appear 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. 

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 indicative 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 collectively called teosinte. Several 

types of teosinte are classified as subspecies of Zea mays, whereas others are regarded as 

separate species of Zea. Teosinte species are natives of Central America 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 both states. Teosinte species are grown in 

botanical gardens, but as maize pollen is heavy and relatively 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 with 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 useful genes for traits including 

apomixis, pest and disease resistance, and drought tolerance (OECD, 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 sterile. 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. would 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 not 
 MIR162-USDA-1 Page 92 of 268 

hybridize readily with Z. mays, and the probability of backcross or F2 progeny of Tripsacumby-Zea hybrids being produced in the field is negligible. Therefore, Vip3Aa20 is unlikely to 

spread from maize cultivation and persist in the environment as the result 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 field conditions, and only equivocal evidence for 

horizontal gene transfer under laboratory 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 homology between the transgene 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 hence the gene is likely to have low sequence 

homology with genes of soil microbes. Therefore, 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 production 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 production. 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). The total plantings in 2007 are estimated 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 state of the U.S. with the majority concentrated 

in ten states of the upper Midwest: 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 insects were substantial until the introduction of 

crop protection chemicals in the 1960s. Weeds compete 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 addition, a maize crop is susceptible to attack 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 the 19 program states tracked by the National Agricultural 

Statistics Service of USDA, nitrogen was applied to 96% of maize acres 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 sulfur 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 program state maize acres in 2005 (USDA, 2006c). 

Atrazine was applied to 66% of acres, glyphosate was applied to 31% of planted acres, Smetolachlor and acetochlor were each applied 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 insecticide applications, 

microbial insecticide applications, crop rotation, and planting of insect resistant cultivars. 

The major and moderate insect pests of maize 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 practical options for controlling stalk boring 

insects; only about 10% of growers applied insecticides 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 insects would be physically positioned on the plant where they could 

be exposed to an insecticide application. The introduction of the first Bt maize hybrids in 

1996 provided growers 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 

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 growers 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 2003, an estimated 14 

million acres were treated annually with conventional 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 program states were treated with conventional insecticides. 

Tefluthrin, cyfluthrin, and tebupirimphos were the most widely 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-feeding insects. A grower 
 MIR162-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 applications is critical for efficacy. 

Data obtained from the 2005 and 2006 Doane Marketing 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 pesticides; however, three million acres treated 

represents a significant use compared to chemical usage in other crops. 

Table 32. Conventional insecticide usage for control of selected lepidopteran pests. 

Cost and maize acres treated with conventional 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 pathogenic organisms during maize development. 

Soil abounds in microorganisms, particularly fungi, 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 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. Ear, kernel, and 

cob rots occur wherever maize is grown and result in reduced test weight, 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 frequently 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 reformulate field lots because of contamination. These 

pathogenic infections can lead to reduced crop quality, harvestability, and yield. 

MIR162 maize provides excellent protection against feeding damage caused by A. ipsilon, H. 

zea, S. albicosta, and S. frugiperda. For this reason, its introduction will impact in a very 
 MIR162-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 in Table 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 because many of the above-ground feeding insects are shielded 

from contact with the insecticides by virtue of their feeding location on the plant. 

Two years of efficacy field trials, conducted at multiple locations under varying levels of 

insect pressure, have demonstrated the superior leaf, stalk, and ear protection provided by 

MIR162 maize compared to hybrids treated with a conventional insecticide 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 usage, some of which include improper calibration 

and maintenance of application equipment, handling of hazardous chemical insecticides, 

container disposal, chemical misplacement, runoff, and spray drift. 

Integrated pest management (IPM) in agriculture includes insect scouting or monitoring to 

determine pest populations, consideration and application of compatible 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. Planting 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 application 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 scouting and monitoring programs that provide data 

upon which to base crop management 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 attributable to 

S. albicosta were estimated to be 4.8 bu/ac (see Appendix F). Examination of data provided 

by these growers for the past five seasons suggests that yield losses attributable to the two 

pests are increasing. This conclusion is supported by analysis of insecticide use data for 

2005 and 2006 which indicate that economically 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 controlled by MIR162 maize are not as large as those attributable 

to O. nubilalis and Diabrotica rootworms, they are significant when severe infestations 

occur. As the price of maize grain continues to rise, the economic threshold for growers to 
 MIR162-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 protection against insect feeding will also result in a 

corresponding decrease in mycotoxin levels in grain. 

 VIII. Insect Resistance Management 

Current insect resistance management (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 insects to mate with. Matings of resistant and nonresistant adults 

serve to dilute the frequency and establishment of resistance genes in a population. The size 

and configuration of the structured refuge is determined by toxin dose and insect biology. 

The existing EPA policy for resistance management of maize lepidopteran pests is focused 

primarily O. nubilalis, D. grandiosella, and H. zea. Growers planting Cry1Ab- and Cry1Fcontaining maize hybrids in the Corn 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, adjacent 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 because 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 cotton and has the potential to undergo selection pressure from 

feeding on both corn and cotton varieties that express similar Cry proteins. The refuge 

configuration options are the same as in the Corn Belt. 

As MIR162 maize provides no protection 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 the Corn Belt and cotton growing areas. 

A unique benefit that will be offered by Bt11xMIR162 hybrids is that the Cry1Ab and 

Vip3Aa20 proteins are present at levels that have been demonstrated 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 IRM advantages over other 

lepidopteran-control options that do not demonstrably provide a high dose 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 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 

traits for control of these pests because local populations are very unlikely to evolve 

resistance to two proteins that act on independent receptor sites. 

For growers of Bt11xMIR162 maize hybrids, the reduced refuge requirement in cottongrowing regions will translate into a higher proportion of insect-protected maize acres, with a 

proportional increase in all the attendant benefits of the product in these areas. As an added 

advantage, compliance with the refuge requirement for IRM can be predicted to increase 

because Bt maize growers in cotton-growing 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-growing regions can also help meet the current 

high demand for maize grain. 

IX. Adverse Consequences of Introduction 

Syngenta knows of no data or observations that indicate MIR162 maize would adversely 

impact the quality of the human environment, directly, indirectly, or cumulatively. This 

includes a lack of anticipated effects on endangered species, unique geographic areas, critical 

habitats, public health and safety (including children and minorities), genetic diversity of 

maize, farmer or consumer choice, insect resistance or the economy, either within or outside 

the U.S. MIR162 maize offers growers an additional choice for protection of maize crops 

from feeding damage caused by lepidopteran pests. As such, MIR162 is expected to convey 

benefits similar to those associated with previously deregulated Bt maize products that are 

commercially available. Additional benefits will include increased product choice for 

growers, price competition, and 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 MIR162 maize is analyzed within 

the framework of the factors listed by the Council on Environmental Quality (CEQ).10 

IX.A. Effects on the Quality 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 significantly 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 significance of an action is uncertain, agencies may 

use an environmental assessment (EA) to identify, analyze, and evaluate the impacts of the 

 

10 The Council on Environmental Quality coordinates federal environmental efforts and works closely with 

agencies and other White House offices in the development 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 NEPA 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 affects’ 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 effects 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 occur 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 conventional insecticides. These Bt maize cultivars do not present 

plant pest risks. 

Currently available lepidopteran-protected Bt maize cultivars produce insecticidal Cry 

proteins derived from B. thuringiensis. MIR162 maize produces a vegetative insecticidal 

protein that is also derived from B. thuringiensis. Vip and Cry proteins differ in terms of 

their solubility characteristics, yet they act by the same fundamental mechanism of action 

(i.e., they are activated to insecticidal toxins in the insect midgut and form pores in the 

epithelial membranes). The MIR162 technology will provide benefits in the marketplace in 

the form of crop protection against lepidopteran pests that are poorly 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 entering 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-characterized and intensively managed crop. Its 

genetic diversity is carefully maintained and safeguarded by seed producers. The 

modification of maize to produce MIR162 maize will not present a risk of adverse effects for 

endangered, threatened, or other nontarget species. A comprehensive and scientifically based 

IRM plan will be executed to delay the development 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 deregulation is based in the production of an 

intensively managed row crop, maize. 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 

 

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 feed, human food, vegetable oil, high fructose 

corn syrups, starch, fermentation into ethanol, and a multitude of industrial uses. Maize 

cultivars improved through modern biotechnology have contributed significantly 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 significant insect pests of field maize and more so, of sweet maize 

in the U.S. In addition to direct damage caused by feeding on plant tissue, 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 major lepidopteran maize pests that could be 

prevalent in any given year. 

The improved pest-protection 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. Some lepidopteran pests, such as A. ipsilon, can cause 

major yield losses 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 lepidopteran larvae 

cause physiologic yield loss because the stalk damage interferes with nutrient uptake and 

increases susceptibility to plant diseases. They 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 yield and quality, and occasionally result in 

elevated grain fumonisin levels that render the grain unsafe and unusable as food or feed. 

Growers have significant experience with the management of Bt maize cultivars. 

Deregulated Bt maize cultivars that resist 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. maize crop in 2006 consisted 

of Bt varieties (including single and stacked biotech trait products) and 49% in 2007 (USDA, 

2007c). The presence of Bt maize has not adversely affected 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 protect and maintain the genetic purity of 

breeding lines is critical to seed companies and 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 purity 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 for 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 not aware of any studies demonstrating the 

development of insect resistance in the field. Monitoring target pest populations for 

resistance development is an ongoing stewardship 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 guide the analysis (40 CFR §1508.27(b)). These factors “...do not 

constitute categorical rules such that their 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 adverse. 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 such 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 effects on the human environment are highly 

uncertain or involve unique 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 future 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 environment. Significance cannot be avoided 

by terming an action temporary or by breaking it down into small component parts. 

viii. The degree to which the action may adversely affect districts, sites, highways, 

structures, or objects listed in or eligible for listing in the National Register of 

Historic Places or may cause loss or destruction of significant scientific, cultural, or 

historical resources. 

ix. The degree to which the action may adversely 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 federal, state, or local law or requirements 

imposed for the protection of the environment. 

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

The NEPA regulations (40 CFR §1508.27(b)(1)) emphasize that agencies must take into 

account beneficial effects, as well as adverse ones. As set out below, MIR162 maize is 

expected to deliver the same beneficial effects as previously deregulated Bt maize cultivars 
 MIR162-USDA-1 Page 101 of 268 

that are also registered by the EPA as plant-incorporated protectants and which are 

commercially available. These benefits include additional grower choice of technologies, 

increased price competition and extended useful life of Bt maize technology generally. 

These effects do not constitute significant impacts 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 the 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 superior protection of ears from insect feeding 

damage offered by the MIR162 trait will preserve grain yield and quality, and will limit 

fumonisin levels that render the grain unsafe and unusable as food or feed. 

IX.C.2. Effects on Public Health or Environmental Safety 

Previously deregulated Bt maize cultivars have resulted in reduced conventional pesticide 

use, as farmers find the Bt products more effective in mitigating insect feeding damage. It is 

reasonable to expect that deregulation and commercialization of MIR162 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 present 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 sensitive 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 insect midgut. Research demonstrates that this 

specificity limits the proteins’ toxic effect to certain lepidopteran species. A discussion on 

the mechanism of action for Vip3Aa20, its spectrum of activity, and 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 safety of the introduced proteins, Vip3Aa20 and 

PMI, demonstrate that both proteins are nontoxic to mammalian species and are unlikely to 

be food allergens. The Vip3Aa20 protein is considered nontoxic because it does not share 

significant amino acid homology with known protein toxins, is nontoxic to mice at a very 

high dose of 1250 mg Vip3Aa20/kg bw, it is rapidly degraded in simulated mammalian 

gastric fluid, and its insecticidal mode of action 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 food allergen because it is not derived from a 

known source of allergenic proteins, it does not have any significant amino acid sequence 

identity to known allergenic proteins, it is rapidly degraded in simulated mammalian gastric 

fluid, and it is labile upon heating at temperatures of 65oC and above. The PMI protein is 

considered nontoxic because it does not share significant amino acid 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 gastric fluid. PMI is not likely to be a food 

allergen because it is not derived 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 temperatures of 37oC and above. 

A temporary exemption from the requirement of a food tolerance currently exists under 40 

CFR §174.458 for Vip3Aa20 in maize and a permanent exemption from the 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 currently under review at 

EPA. 

IX.C.3. Unique Characteristics of the Geographic Area 

There is no indication that this action would have a significant effect on the unique 

characteristics of any particular geographic area. As with Bt maize cultivars already 

deregulated and commercialized, MIR162 maize is expected to be used throughout maizeproducing areas of the country. 

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

There is no ‘controversy’ as that term is used 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 size, nature, or effect of the major 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 the 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 having 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 resistance may manifest themselves over time, these 

risks are known and there will be preventative measures implemented upon commercialize of 

Bt11xMIR162 maize. As a condition of the 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 development of insect resistance has been documented 

and submitted to EPA as part of the application 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 combined through conventional breeding with Bt11 

maize containing the Cry1Ab protein. The Cry1Ab and Vip3Aa20 proteins are present in 

these hybrids at levels that have been demonstrated to provide 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 ‘high 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 because insect populations are very unlikely 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 undergo 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 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. In its application for EPA registration of Bt11xMIR162, 

Syngenta provides data and rationale to justify reduction of the maize refuge in cottongrowing areas from 50% to 20% of maize acres 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 proportional 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 IRM compliance will likely increase because 

Bt maize growers in cotton-growing regions have heretofore not been 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 advantages 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 instances 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 practices 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 evaluated 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 specific and detailed stewardship program aimed at 

preventing or significantly delaying insect resistance. The program requires that growers 

plant a structured refuge and compliance with 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, observations 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 action 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 environment which results from the incremental 

impact of the action when added to other past, present, and reasonably foreseeable future 

actions regardless of what agency . . . undertakes such other actions” (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 combinations of interactions between this event and 

features of the environment, such as multiple factor interactions between the Bt-based protein 
 MIR162-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 previous deregulations to cause a significant 

impact. The crucial requirement is that the overall 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 other 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, ‘incorporating the effects of other projects into 

the background’ data. See Sections VI and VII of this petition for discussions of field trial 

results for MIR162 that showed no enhanced plant pest potential and no adverse effects for 

nontarget organisms. Accordingly, the data in this petition indicate that the Vip3Aa20 

protein contained in MIR162 maize is not toxic to nontarget organisms 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 effects could only be considered positive. These 

effects include the potential for reduced conventional 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 production, and the deregulation of multiple Bt maize 

cultivars has not significantly affected these processes, even considering 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 hybrid 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 industry 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-pollination occurs. Many 

options are available for this purpose: maintaining isolation distances to prevent pollen 

movement from other maize, planting border or barrier rows to intercept pollen, employing 

natural barriers to pollen movement such as treelines, manual, or mechanical detasseling, 

genetic male sterility, and staggered 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 robustness of these procedures developed for 

maintaining genetic purity in seed production, and the rigor with which they are routinely 

applied in the maize industry, these procedures have effectively managed the introduction of 

transgenic maize into the marketplace and have continued to do so through the introduction 

of successive transgenic maize cultivars. 

In addition to the specialization adopted across the industry to enable hybrid 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 and 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 developed, and in 2006 there were at least 18 seed companies in the 

U.S. specializing in organic maize seed (see Section IX.D.3). Similar to the production of 

conventional inbred and hybrid seed, industry quality standards for these maize products 

have led specialty maize seed producers and growers to employ a variety of techniques to 

ensure that their products are not pollinated by or commingled with other field maize 

varieties. In general, all the management practices used in conventional 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, rigorous quality standards are also required for 

specialty grain production. To meet these standards, specialty grain growers employ a range 

of additional measures to meet purity standards. Most commonly, to prevent crosspollination of specialty maize by commodity field 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 seed 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 seed 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 and 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 field maize (Thomas, 1955; Ziegler, 2000). 

For decades prior to the introduction of transgenic maize cultivars and since their 

introduction, the maize industry has had effective methods and means to maintain product 

segmentation and genetic purity standards. As a result, these widespread practices have 

served to ensure that the broad 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 specialty maize products. 

There is no evidence to suggest that deregulation of MIR162 maize 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_publications/USGC%20Value%20Enhanced%20Corn%20Report%20

2006%20%20(English).pdf 
 MIR162-USDA-1 Page 107 of 268 

point’ that will undermine the effectiveness of these methods. 

The adoption of multiple varieties of transgenic maize has had no significant impact on the 

genetic diversity of cultivated maize or the availability of diverse maize germplasm 

resources, even considering the effects of these transgenic events cumulatively. Genetically 

distinct maize hybrids have always been developed 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 continuous 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 genetic 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, nongovernment organizations and international 

public cooperators, which was established to further identify maize genetic diversity and to 

provide it in useful form in order to broaden the genetic base of this crop (USDA, 1999). 

The germplasm sources being developed through the Germplasm Enhancement of Maize 

program are available free of charge through the extensive national germplasm collections 

and germplasm repository programs for conservation 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 source 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 (Mexico), tropical maize materials at International Institute 

of Tropical Agriculture (Nigeria), and various collections residing within national programs 

of different countries around the world. Specific maize germplasm is also available from 

individual breeders working on maize at public institutions and universities. The 

deregulation of transgenic maize events provides yet another source of genetic diversity that 

can be utilized in the improvement 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 the record, nor is Syngenta aware of any, to 

suggest that MIR162 will act as a ‘tipping point’ that will undermine the effectiveness of 

these methods for maintaining genetic diversity, 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 Syngenta is not aware of any studies showing insect resistance to 

 

19 http://www.ars-grin.gov/npgs/searchgrin.html 
 MIR162-USDA-1 Page 108 of 268 

Bt maize products, despite the introduction of multiple 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 use of mandatory refugia is effective in 

preventing or delaying the development of insect resistance to B. thuringiensis insecticidal 

proteins, even cumulatively after multiple Bt maize cultivar introductions. 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 suggest that MIR162 will act as a 

‘tipping point’ that will cause the previously-effective refuge system to become ineffective. 

IX.C.8. Historical and Cultural Effects 

There is no indication that this 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 estimates of expected environmental concentrations, 

there is an adequate margin of safety for nontarget organisms (see Section VII). 

EPA determined that the only endangered lepidopteran 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 plant, 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 least 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 would violate federal, state, or local law or 

requirements imposed for the protection of the environment, nor is there any evidence on the 

record that would so indicate. 

IX.D. Economic Impacts 

Economic considerations are not explicitly described within the factors listed in 40 CFR 
 MIR162-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 courts in reviewing NEPA compliance. 

Although some maize-growing areas experience significant lepidopteran infestations in most 

years, it is not possible for individual growers to accurately predict whether lepidopteran pest 

pressure will be economically significant in any particular growing season. Growers must 

make seed purchase decisions prior to knowing whether the seed price premium for 

lepidopteran control in a given year will actually 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 benefits 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 pest protection 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 necessarily 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. Such demand will function to 

increase the value of a grower’s investment in any agricultural practice, technology, or 

product, including the MIR162 traits, that increases or preserves yield. 

Another predicted economic benefit for growers 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 commercial availability of 

MIR162 hybrid maize seed 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 

products that offer control of lepidopteran pests. 

IX.D.1. Enhanced Productivity 

Studies to quantify the expected 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 indicate 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 significant lepidopteran 
 MIR162-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 losses 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 lepidopteran larvae cause physiologic yield loss 

because the stalk damage interferes with nutrient uptake and increases susceptibility to plant 

diseases. They can also cause severe stalk lodging and dropped ears, which results in a 

physical yield loss because the ears cannot be mechanically 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 I.B 

and VII.I; EPA, 2001). In the presence of pest pressure, Bt11xMIR162 maize hybrids can be 

expected to preserve yield potential, grain 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 growers and downstream consumers is increased 

competition in the marketplace for pest-control technologies, including hybrid seed from 

multiple providers of lepidopteran-tolerant Bt maize varieties. The commercial availability 

of hybrid maize seed with the 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 that offer control of lepidopteran pests. 

IX.D.3. Farmer and Consumer Choice 

As described above, maize cultivation has long included effective methods of maintaining 

genetic purity, and mechanisms are in place to protect the genetic diversity of maize. 

Specialization of maize cultivation, in hybrid seed production for commodity field maize and 

in production of specialty maizes, has required genetic purity procedures for more than 70 

years, and maize growers have utilized these methods effectively to prevent undesired gene 

flow. Syngenta is aware of no studies showing 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 organic 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.htm 
 MIR162-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 State Supply, Inc., Onalaska, WI 

Gold Country Seeds, Hutchinson, MN 55350 

Golden Grains, Sparta, WI 

Great Harvest Organics, Atlanta, IN 46031 

Lakeland Organics/Bruenner 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 listing 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 that 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. maize export market since Syngenta is actively 

pursuing regulatory approvals 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 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 agreements 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 the 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 extractable 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.ww 
 MIR162-USDA-1 Page 112 of 268 

IX.E. Conclusion 

Syngenta is seeking a determination of deregulated status for MIR162 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 alternatives to the action (42 

U.S.C. 4332). Where the significance of an action is uncertain, agencies may use an EA to 

identify, analyze and evaluate the impacts of the proposed 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 action 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 warranted thereby satisfying the requirements of 

NEPA. 

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Acknowledgements

The authors would like to acknowledge and thank 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 

DT50 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.)

LC50 Median lethal concentration 

LOD Limit of detection 

LOQ Limit of quantification 

MES 2-(N-morpholino)ethanesulfonic acid 

MIR162 Maize insect resistant transformation event encoding a Vip3Aa20 

protein derived from B. thuringiensis 

MW Molecular weight 

NADP β-nicotinamide adenine dinucleotide phosphate 

NADPH β-nicotinamide adenine dinucleotide 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 insecticidal protein from B. thuringiensis 

w/v Weight per volume 

w/w Weight per weight 
 MIR162-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 1999. A listing of these notifications, along with 

a status of the report for each, 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 multiple backcross (BC) generations: BC1F1, BC2F1, 

and BC4F1. Seeds from each of these generations were planted and grown under standard 

greenhouse conditions and then processed 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 individually 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 individually 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 Purification 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 control, 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 plants 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 identify positives and negatives

Leaf tissue from the 10 positive plants 

was pooled into one sampling bag 
 MIR162-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 polyvinylpyrolidone (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 tissue into a fine powder using a mortar and 

pestle under liquid nitrogen. Eight 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 loop, 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 samples were mixed gently by inversion and 

centrifuged for 20 minutes at 2755 x g. The aqueous layer was collected 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 the 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 resuspended in 1000 μl of 1X TE (10 mM Tris, 1 

mM EDTA pH 8.0) overnight at 4oC. 

After complete resuspension, samples were transferred to microcentrifuge tubes and 6 μl of 

Ribonuclease A (10 mg/ml) was added, and the sample was incubated at 37oC for 30 

minutes. Samples were then 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 NH4OAc 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 volume of isopropanol was added and the samples were mixed 

by inversion to precipitate the DNA. The samples were centrifuged for 10 minutes at 19,600 

x g to pellet the DNA. The supernatant was decanted and the pellet was washed twice in 500 

μl of 70% ethanol, spinning for 5 minutes at 19,600 x g after each wash. The supernatant 

was decanted and the pellet air 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 was measured using the PicoGreen QuantiT 

technology with a Turner Biosystems TBS-380 Fluorometer following the manufacturer’s 

instructions. A Lambda DNA standard was used to calibrate the instrument prior to 
 MIR162-USDA-1 Page 132 of 268 

quantitation. 

DNA Insert Sequencing 

The DNA insert was amplified from MIR162 maize DNA derived from a BC4F1 generation 

(see Figure 5) as four overlapping fragments (see Figure B-2 below). PCR amplification was 

carried out using either an Expand High-Fidelity PCR system or PfuUltra Hotstart HighFidelity DNA polymerase. Each PCR product 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 package from the University of Washington 

and was carried out to an error rate of less than 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 performed using the ClustalW program with the 

following parameters: scoring matrix BLOSUM55, 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 molecular biology techniques 

(Chomczynski, 1992). Genomic DNA (7.5 μg) was digested with the appropriate restriction 

enzymes overnight at the optimal temperature for each enzyme. Additional enzyme was 

added to each reaction the following morning, and the reaction was allowed to continue for 

approximately four hours. Digested DNA was loaded onto 1% agarose gels and bands were 

separated electrophoretically in 1X TAE buffer. Following 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 briefly rinsed in 2X SSC. The 

DNA was then crosslinked to the membrane using 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) 35S terminator (70 bp) ZmUbiInt (1993 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-32P via random 

priming using the Megaprime DNA labeling system. For all element-specific probes 5-25 ng 

was used for labeling; 5 ng was used for DNA molecular weight marker probes. 

Unincorporated isotope was removed using the Micro Bio-Spin Chromatography Columns. 

Membranes were incubated in prehybridization solution (PerfectHyb Plus Hybridization 

Buffer), and calf thymus DNA (100 μg/ml) for approximately one hour at 65oC. The 

element-specific and molecular weight marker radiolabeled probes were added to the 

prehybridization solution, and the membranes were incubated for a minimum of three hours. 

Hybridization was carried out at 65°C, followed 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 equivalent based on plasmid size was added to DNA from negative 

control plants. The calculation for the pNOV1300 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)) * μg loaded) * 1x106

 = pg for 1 copy 

Example: 

Z. mays genome size in bp: 2.67x109

Ploidy: 2 

pNOV1300 size in bp: 14405 

μg of digested MIR162 maize DNA loaded for Southern analysis: 7.5 

Calculation for pNOV1300: 

((14405 / (2.67x109 * 2)) * 7.5) * 1x106 = 20.2 pg 

The copy number of the T-DNA functional elements 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 strategies. In the first enzyme 

digestion, MIR162 maize genomic DNA was digested with an enzyme that digests once 

within the DNA insert but not within the functional element being probed. This digest will 

result in a single unique hybridization band for each copy of the functional element present in 

the MIR162 maize genome. In the second enzyme scheme, another enzyme that digests once 

within the DNA insert but not within the functional element being probed was employed. 

This digest will also result in a single unique hybridization band for each copy of the 

functional element present. Additionally, MIR162 maize genomic DNA was digested with 

an enzyme(s) that released a known size fragment. This restriction enzyme strategy gives 

further evidence of copy number and also demonstrates the intactness of the insert. A 

negative control was included in each Southern blot experiment to identify endogenous Z. 
 MIR162-USDA-1 Page 134 of 268 

mays sequences that may hybridize with the element-specific probe. Digested pNOV1300 

plasmid equal to one copy equivalent based on plasmid size was included in each Southern 

blot analysis to demonstrate a positive control for hybridization, as well as the sensitivity of 

the experiment. 

Mendelian Inheritance of DNA Insert 

The initial MIR162 maize plant (T0 generation) was crossed with maize inbred line 

NPH8431, creating the F1 generation. NPH8431 (MIR162 maize) plants from this F1

generation were crossed to the inbred line NP2161 to yield the F1 generation. NP2161 

(MIR162 maize) plants from this F1 generation were backcrossed to the inbred line NP2161 

to yield the BC1F1 generation. MIR162 maize plants from this BC1F1 generation were 

backcrossed again to inbred line B9620 to yield BC2F1 generation. The F1 generation was 

backcrossed two more time with inbred line B9620 to yield a BC4F1 generation (refer to 

Figure 5). Positive segregants were utilized in each backcross. Material from the BC1F1, 

BC2F1, and BC4F1 generations of B9620 was used in the Mendelian inheritance study. 

Individual plants from the BC1F1, BC2F1, and BC4F1 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 hemizygous trait in these populations 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 expected (e) genotypes using Chi-square (X2) 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 sequences 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 Database 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 nonredundant set of reference standards that includes 

chromosomes, complete genomic molecules (organelle genomes, viruses, and plasmids), 

intermediate assembled genomic contigs, curated genomic regions, mRNAs, RNAs, and 

proteins. The nonredundant database does not contain any EST, STS, GSS, or phase 0, 1, or 

2 HTGS sequences. At the time of this analysis, the nonredundant database contained 

4,654,881 unique sequences and was last updated on December 14, 2006. The query 
 MIR162-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 threshold for reporting 

matches against database sequences; the default value is 10, meaning that 10 matches are 

expected to be found merely by chance, according 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 for nucleotides: +1 for a match and –3 for a 

mismatch. Gap penalties: Existence = 5, Extension = 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 extension penalties are chosen empirically. To prevent the 

accumulation of excessive gaps in an alignment, the introduction of a gap causes the 

deduction of a fixed amount from the alignment score. Extension of the gap to 

encompass additional nucleotides is also penalized 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 reading 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 presence of open reading frames using Vector NTI 

(version 9.0) software. An open reading frame was defined in the bioinformatic program as a 

region corresponding to at least 50 amino acids in length initiating with an ATG codon and 

ending with any of the three stop codons: TAA, TAG, or TGA. 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-grown MIR162 maize plants. Leaves were 

collected four to six weeks after plant emergence, frozen at -80 ± 10°C, and subsequently 

ground into fine powder and lyophilized. Lyophilized leaf powder 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 polyethylenimine and centrifuged at approximately 7280 x g for 

20 min at 2 - 8 ̊C. The supernatant was filtered through Miracloth filtration material and fine 

powdered crystalline ammonium sulfate was added to a final concentration of 40% (24.6 g 

ammonium sulfate/100 ml extract). The mixture 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 times (5 - 12 hr each) in dialysis buffer. The 

dialyzed extract was then lyophilized and stored at -20 ± 8 ̊C. 

Two batches of purified MIR162 maize-derived Vip3Aa20 were prepared and designated test 

substances LPMIR162-0105 and LPMIR162-0106 and were used for the Western blot 

analysis and insect bioassays. 

Immuno-affinity Purified Vip3Aa20 from MIR162 Maize Leaf Tissue 

Immuno-affinity-purified Vip3Aa20 was prepared from the ammonium sulfate precipitate as 

described above for test substances LPMIR162-0105 and LPMIR162-0106. The precipitate 

was re-suspended in column equilibration buffer containing 50 mM Na2HCO3 and 100 mM 

NaCl, then centrifuged to remove insoluble debris. 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-Plus centrifugal filters and stored on ice before further use. 

Two batches of immuno-affinity purified MIR162 maize-derived Vip3Aa20 were prepared 

and designated test substances IAPMIR162-0105 and IAPMIR162-0106. These test 

substances were used in the Western blot and peptide mapping analyses. 

Vip3Aa20 from E. coli 

Test substance MIR162VIP3A-0106, containing Vip3Aa20 protein, was prepared by 
 MIR162-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 predicted 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 prepared 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 debris removed by centrifugation. The Vip3Aa20 

present in the supernatant was further purified with anion exchange chromatography using a 

Q Sepharose FF column. Vip3Aa20 was eluted from the column with a NaCl gradient. 

Fractions containing Vip3Aa20 were pooled and dialyzed in 20 mM ammonium bicarbonate 

buffer (pH 10), frozen and lyophilized. The lyophilized Vip3Aa20 sample preparation was 

designated test substance 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 Vip3Aa19, was prepared from pooled batches 

of E. coli cell paste and was determined to contain approximately 92% Vip3Aa19 by weight, 

and was demonstrated to have insecticidal activity against S. frugiperda larvae. 

A listing of the test, control, and reference substances 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. coli
 MIR162-USDA-1 Page 138 of 268 

Methods for Vip3Aa20 Characterization and Equivalence

Total Protein Determination 

Total protein in test substance MIR162VIP3A-0106 was quantified spectrophotometrically 

by determining the absorption at 280 nm (Gill and von Hippel, 1989). The A280 method is 

based upon the absorption of the aromatic amino acids tryptophan and tyrosine at 280 nm. 

The extinction coefficient at 280 nm varies with the abundance of these amino acids in the 

target protein. A Genesys 6 spectrophotometer was used to measure absorption of 

MIR162VIP3A-0106 at 280 nm and the extinction coefficient of Vip3Aa20 was calculated 

with Vector NTI software version 9. The absorbance at 280 nm was multiplied by the 

correlation factor for the extinction coefficient, to give an approximate total protein 

concentration. 

Total protein in test substances LPMIR162-0105, LPMIR162-0106 and control substance 

LPMIR162-0105C was quantified using the BCA method (Hill and Straka, 1988), using 

bovine serum albumin (BSA) as the protein standard. 

Densitometry 

For purity determination, aliquots of MIR162VIP3A-0106 (ranging from 1 to 20 μg 

Vip3Aa20 per lane) were subjected to SDS-PAGE using NuPAGE 4-12% Bis-Tris 

polyacrylamide gradient gel and MES running buffer. After electrophoretic separation, 

protein bands were visualized with Coomassie staining and the distribution of the visible 

protein bands was estimated by densitometric analysis 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 Vip3Aa20 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 instructions. Creatinase, a 

nonglycosylated protein, was included 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 membrane, glycan moieties were oxidized using 

periodate, labeled with digoxigenin, and detected with an anti-digoxigenin antibody coupled 

to alkaline phosphatase. 

N-Terminal Amino Acid Sequence Analysis 

For N-terminal amino acid sequence analysis, test substance MIR162VIP3A-0106 was 

subjected to SDS-PAGE followed by electro-blotting 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 Sequencing Services for N-terminal amino acid 
 MIR162-USDA-1 Page 139 of 268 

sequence analysis. ProSeq's methodology was developed specifically for proteins 

immobilized on PVDF membrane and optimized 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 MIR162VIP3A-0106 was determined using 

sandwich ELISA (Tijssen, 1985). Control substance LPMIR162-0105C was used as a 

negative control in the ELISA analysis. 

96-well Nunc MaxiSorp plates were coated overnight 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 chloride (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 containing phosphate buffered 

saline plus 0.05% Tween 20, 1% BSA and 0.02% sodium azide (pH 7.4), for at least 45 min. 

The plates were then washed again as described 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 antibody 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 phosphatase antibody, diluted to 1 μg per ml in 

ELISA diluent, for 1 h at 37 ± 1°C. The plates were washed again and incubated in 

phosphatase substrate solution at room temperature 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-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 

The integrity of Vip3Aa protein in test substances LPMIR162-0105, LPMIR162-0106, 

MIR162VIP3A-0106, and VIP3A-0204 was investigated using Western blot analysis. 

Aliquots containing 10 ng Vip3Aa20 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 buffer. Based on total soluble protein, an 

equivalent amount (containing approximately 676 μg of total soluble protein) of control 

substance LPMIR162-0105C was also included in the analysis as a negative control. After 

electroblotting, the membrane was incubated with goat anti-Vip3Aa immuno-affinity purified 

polyclonal antibodies. Donkey anti-goat IgG linked to alkaline phosphatase diluted 1:3,000 

was used as the secondary antibody to bind to the primary antibody and visualized by 

development with alkaline phosphatase substrate 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 substances LPMIR162-0106, LPMIR162-0105C and MIR162VIP3A-0106 

and tested concurrently. Also a comparison 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 insect feeding assays using freshly hatched firstinstar S. frugiperda. The bioassays were conducted in Costar 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/cm2 diet surface. Each treatment consisted of 24 

individual wells containing one larva/well. Insect diet treated with MilliQ purified 

(deionized) water alone, insect diet treated with buffer alone, and insect diet treated with 

control substance LPMIR162-0105C at approximately 4910 μg total protein (an equivalent 

amount of total protein as applied in the highest test concentration of LPMIR162-0106), were 

used as negative controls. The wells were covered with silicone stoppers and maintained at 

room temperature under ambient laboratory conditions. Mortality was assessed after 168 

hours and is reported for each treatment as the percent mortality. 

Peptide Mass Mapping Analysis 

Aliquots, containing 1 μg Vip3Aa20 from either test substance IAPMIR162-0106 or test 

substance MIR162VIP3A-0106, were subjected to SDS-PAGE using a 4 - 12% Bis-Tris 

polyacrylamide gradient gel and MES running buffer. The gel was stained with Coomassie 

and then prepared for peptide mass mapping analysis. Vip3Aa20 bands were excised from 

the SDS-gel, the protein was reduced, alkylated with iodoacetamide, and digested with 

trypsin. The mass analysis of the Vip3Aa20-derived peptides was performed on a 

quadrupole time-of-flight mass spectrometer, fitted with an EPCAS upgrade and connected 

to a capillary HPLC instrument. Peptide masses were identified by matching the detected 

peptide masses to a protein database using Mascot software. 

Statistical Analysis 

The LC50 and 95% confidence intervals determined in the S. frugiperda larval bioassays were 

computed by means of a probit analysis. 

Results for Vip3Aa20 Characterization 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 Vip3Aa20 represented approximately 90.3% 

of the total protein in MIR162VIP3A-0106 (Figure 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

188

49

38

28

17

14

6

3

62

98

MW

[kDa]

188

49

38

28

17

14

6

3

62

98

MW

[kDa]

2 45 6 7 8 9 3

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 Vip3Aa20 were visible upon DIG Glycan analysis 

(Figure C-2), providing evidence that, as expected, the E. coli-produced Vip3Aa20 is not 

glycosylated. By comparison, 25 ng of the positive control protein, transferrin, 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 equivalents/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 microbial test substance 
 MIR162-USDA-1 Page 142 of 268 

MIR162VIP3A-0106 was loaded in concentrations of approximately 1000 and 2000 ng/lane. 

Thus, the 1.25 ng glycan detection limit corresponds to approximately 0.0625% by weight 

(1.25 ng/2000 ng), or approximately 0.34 glucose equivalents/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 extracted from MIR162 maize (IAPMIR162-0105) 

has also been found not to be glycosylated (data not shown). 

1 2 3 4 5 67

188

49

38

28

17

14

6

3

62

98

MW

[kDa]

Transferrin Vip3Aa20

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 sequences for the Vip3Aa20 in E. coli produced test substance 

MIR162VIP3A-0106 confirmed the predicted amino 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 derived protein could not be determined due to 

technical difficulties (most likely N-terminal blockage of the protein). Instead, the sequence 

of plant-derived Vip3Aa20 protein was determined by peptide mass mapping (described 

below). 

Peptide Mass Mapping Analysis 

The analyses of the plant- and microbially-derived Vip3Aa20 by peptide mass mapping 

covered approximately 40% and 45%, respectively, of the predicted Vip3Aa20 amino acid 

sequence. Furthermore, the identified peptides were representative of regions throughout the 

sequence of the Vip3Aa20 protein (including peptides 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 test substances VIP3A-0204 and MIR162VIP3A-0106 

as well as the plant-derived test substances (LPMIR162-0105 and IAPMIR162-0105) 

revealed dominant immunoreactive bands corresponding to the predicted molecular weight of 

approximately 89 kDa (see Figure C-3). Control substance LPMIR162-0105C showed, as 

expected, no positive response with the anti-Vip3Aa antibody in the Western blot analysis 

(Figure C-3, lane 6). Additional, 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 likely 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

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 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. The 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 microbial- and plant-derived Vip3Aa20 are shown in 

Table C-4; computed LC50 estimates are shown in Table C-5. Test substances 

MIR162VIP3A-0106 and LPMIR162-0106 showed comparable responses in the S. 

frugiperda bioassays, with estimated LC50s of 225 ng Vip3Aa20/cm2

 (95% confidence 

interval = 155 - 289 ng/cm2 diet surface) and 318 ng Vip3Aa20/cm2 (95% confidence 

interval = 232 - 451) after 168 hours, respectively. 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-test comparing the two LC50 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 LC50s 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 bioassays 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). 

Concentration Mortality at 168 hours [%] 

[ng Vip3Aa20/cm2 ]

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 presented in Table C-5. Test substances LPMIR162-0105 and 

VIP3A-0204 showed comparable responses in the S. frugiperda bioassays, with estimated 

LC50 values of 154 ng Vip3Aa20/cm2

 (95% confidence interval = 94 - 222 ng/cm2 diet 

surface) and 137 ng Vip3Aa19/cm2 (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 observed in the negative control using insect 

diet treated with water (0%) and 12% mortality 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 Vip3Aa proteins from different sources.

Results of mortality bioassays with first instar S. frugiperda larvae are presented as LC50

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 tissue was compared to Vip3Aa19 from E. coli. 

Test Substance LC50 

(ng protein/cm2) 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(LC50) a Variance of log(LC50) 

MIR162VIP3A-0106 2.3530 0.00385 

LPMIR162-0106 2.5019 0.00492 

Comparison 

Difference 

in log(LC50) b

Variance of 

Difference Diff /SED P-value Significance 

0.1489 0.00877 1.58981 0.112 nonsignficant 

a - LC50 values are presented in Table B-5. 

b

 - Log(LC50) values and their respective variances were taken from SAS output. 
 MIR162-USDA-1 Page 147 of 268 

Table C-7. Insect mortality results for bioassays 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). 

Concentration Mortality at 120 hours [%] 

[ng Vip3Aa/cm2 ]

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-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 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 and 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 μM 

leupeptin. The mixture was homogenized using a Tomtec Autogizer and placed on ice for 30 

min with an additional 3 ml PMI extraction buffer. The supernatant was collected after 

centrifugation for 15 min at 12,000 x g in a Sorvall RC-5B Refrigerated Superspeed 
 MIR162-USDA-1 Page 148 of 268 

Centrifuge at 2-8oC. The supernatant was then concentrated using Centricon 20-Plus 

centrifugal filters by centrifugation for 25 minutes at 2,600 x g using a Sorvall RC-3B 

Refrigerated Centrifuge at 2-8oC. The concentrated supernatant was stored on ice before 

further use. The test substance derived from MIR162 leaf tissue 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 protein as encoded by this 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 extraction and differential 

ammonium sulfate concentration, followed by hydrophobic interaction chromatography on 

either Phenyl Sepharose or Toyopearl Ether 650M and finally subjected to ion exchange 

chromatography on Q Sepharose. Pooling of the appropriate fractions was performed prior 

to dialysis into 50 mM NH4HCO3, pH 8.0, followed by lyophilization. The lyophilized 

products from two preparations were pooled and designated test substance PMI-0198. PMI 

has been determined to represent approximately 61% of the test substance by weight. The 

PMI protein in test substance PMI-0198 is 407 amino acids in length and 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 leaf 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 PMI using sandwich ELISA (Tijssen, 1985). Nunc MaxiSorp 

plates were coated overnight at 2-8°C with polyclonal goat antibody generated against PMI 

(diluted to 3 μg per ml in borate buffered saline, 100 mM boric acid, 25 mM sodium borate, 

75 mM sodium chloride). Plates were washed five times with ELISA wash solution (100 

mM Tris, 0.5% Tween 20, 0.2% sodium azide) using an automatic plate washer. The plates 

were then blocked with ELISA diluent (phosphate buffered saline + 0.05% Tween 20, 1% 

BSA, 0.02% sodium azide, pH 7.4) for at least 45 minutes at room temperature. Plates were 

washed five times with wash solution and samples of each leaf extract preparation and PMI 
 MIR162-USDA-1 Page 149 of 268 

standard with appropriate dilutions 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 rabbit antibody generated against 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 and then washed five times prior to the addition of 100 μl of 

donkey anti-rabbit alkaline phosphatase 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 substrate solution (two tablets per 20 ml H2O) was added. 

Color was allowed to develop for approximately 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, containing 10 ng PMI, respectively, was subjected 

to SDS-PAGE using a NuPAGE 4-12% Bis-Tris polyacrylamide 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 solution. The Western blot was 

examined for the presence of intact immunoreactive PMI (approximately 42.8 kDa for the 

plant expressed PMI and approximately 44.4 kDa 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 isomerase, and 2 U glucose 6-phosphate dehydrogenase in 

a total volume of 1 ml. PMI activity was measured 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 isomerase, G6PDH = Glucose 6-phosphate dehydrogenase

The specific activity of PMI in test substances MIR162(+) and PMI-0198 was determined 

using the molar extinction coefficient of NADPH (6.2 x 106 cm

2

/Mol). One unit of PMI 
 MIR162-USDA-1 Page 150 of 268 

activity is defined as 1 μmol of NADP reduced 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 Characterization and Equivalence 

Molecular Weight Determination 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 PMI protein in PMI-0198 (Figure C-4; lane 4) 

showed one major band, with a slightly lower mobility than the plant expressed PMI protein, 

as expected from its slightly higher predicted molecular weight 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 (Figure C-4, lane 4). Since this 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 PMI 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 MIR162 maize leaf extract was 55.5 ± 4.18 U/mg PMI 

(Table C-8). PMI extracted from E. coli had a mean specific activity of 33.2 ± 2.37 U/mg 

PMI. Historical activity measurements for maize PMI proteins and test substance PMI-0198, 

as determined at the Syngenta Biotechnology laboratory 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 activity levels measured in previous Syngenta 

studies. Considering the expected levels of variability in the measurements it is concluded 

the PMI extracted from MIR162 maize 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

188

49

38

28

17

14

6

3

62

98

MW

[kDa]

PMI

Figure C-4. Western blot of PMI Protein from 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 from MIR162(-) maize leaf extract 

Lane 3: 10 ng PMI from MIR162(+) 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 

MIR162(+) 557 57.9 

557 57.9 

55.5 4.18 

4500 35.8 

PMI-0198 4500 31.4 

4500 32.3 

33.2 2.37 

ND 

MIR162(-) ND 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.coliderived 

Range 

[U/mg PMI] 

March 2004 43.45-66.27a 35.85-49.27 

April 2005 81.6-112.6a 52.9 

September 

2006 50.7-57.9b 31.4-35.8 

a

 - Extracted from other transgenic maize events containing PMI 

b

 - Extracted from MIR162 maize 
 MIR162-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 control hybrid equivalents (see Table 2 and Figure 5) were 

planted at two locations in 2005 under USDA notification 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 following to local agronomic practices for the 2005 

growing season. 

Ten plants of each MIR162 hybrid, plus two plants 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. The 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 (including roots) were harvested and shipped 

overnight on ice packs to Syngenta Biotechnology, Inc. Upon receipt, five plants were 

separated into parts consisting of all the leaves, roots, pith, silk, or kernels. 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 pollen) were ground to a fine powder in the 

presence of dry ice. Each ground sample was thoroughly mixed to ensure homogeneity. The 

powdered samples were weighed, lypophilized, and weighed again. The percent dry weight 

for each sample was computed based on the difference in pre- and post-lyophilization 

weights. Equal amounts of lyophilized transgenic 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 tissue samples (except pollen) were prepared for Vip3Aa20 and 

PMI analysis. At least three replicate subsamples 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 leupeptin, pH 9.5), and homogenized 

using an Autogizer homogenizer (6 cycles, setting 4). An additional 3 ml of extraction buffer 

was added, and samples were vortexed briefly 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 air-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 vortexed briefly. After centrifugation for 15 

minutes at 10,000 x g at 4oC, the supernatants were used for Vip3Aa20 and PMI 

quantification by ELISA. 

Vip3Aa20 Quantification 

Tissue sample extracts were quantitatively analyzed for Vip3Aa20 by ELISA using 

immunoaffinity-purified goat anti-Vip3Aa and Protein A-purified rabbit anti-Vip3Aa 

polyclonal antibodies. Microbially produced Vip3Aa20 was used to produce the standard 

curves for all measurements except extraction efficiency where microbially produced 

Vip3Aa19 was used. 

PMI Quantification 

Tissue sample extracts were quantitatively analyzed for PMI by ELISA using 

immunoaffinity-purified goat anti-PMI and Protein A-purified rabbit anti-PMI polyclonal 

antibodies. Microbially produced PMI was used to produce the standard curves for all 

measurements. 
 MIR162-USDA-1 Page 155 of 268 

Limit of Quantification and Limit of Detection Determination 

The limit of quantification (LOQ) of the ELISAs was estimated based on the lowest 

concentration of pure protein of interest (POI) lying on the linear portion of the standard 

curve and the dilution factor (DF; based on optical density 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) = lowest concentration on the linear 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 (μg/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/Aa 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/Aa 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 stage 
 MIR162-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 concentration 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 (μg/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/Aa 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 stage 
 MIR162-USDA-1 Page 157 of 268 

Extraction Efficiency for Vip3Aa20 and PMI 

Extraction efficiency measurements were performed to estimate the relative amount of 

Vip3Aa20 and PMI extracted during routine procedures, compared to that which remained 

associated with the post-extraction solids. Tissues were extracted in triplicate as described 

above. The insoluble material was then collected and re-extracted 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, expressed as ng POI/ml, was used to estimate the 

extraction efficiency for the sample. Percent extraction efficiency was calculated as follows: 

[ng POI/ml 1st Extraction ÷ (ng POI/ml 1st extraction + 2nd extraction + 3rd 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 weight = 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 were 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 with a MIR162 hybrid and genetically matched 

nontransgenic hybrid in a randomized complete block design, at multiple locations, with 

three replicated plots per location, and were managed following 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 the 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 plants of each hybrid was harvested at dough 

stage (R4), the stage at which silage would typically be prepared, from each of the three 

replicated plots at each location. For each hybrid, 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 Laboratories, 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 ears collected from 15 plants of each hybrid grown in each of 
 MIR162-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 ambient 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. Samples were stored frozen at -20±10 ̊C until they were 

analyzed. 

Compositional Analysis 

Selection of analytes for measurement in forage 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 calcium and phosphorus (Table 25). Grain was 

analyzed for major constituents (proximates, including starch, ADF, NDF and TDF), 

minerals, amino acids, fatty acids, vitamins, selected anti-nutrients, and secondary 

metabolites (Table 25). 

All compositional analyses were conducted by Covance Laboratories, Inc. using methods 

published and approved by AOAC International, or other industry-standard analytical 

methods (described below). Based on the moisture content of each sample, analyte levels 

were converted to equivalent units of dry weight. 

Statistical Analysis 

An F-test was also used to assess the significance of the location by genotype interaction. An 

F-test probability of <0.05 suggests that the effect of the genotype was not consistent across 

locations and that the comparison 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 locations in both the MIR162 and the nontransgenic 

hybrids. Also included are the F-test probabilities for both the genotype comparisons and the 

location by genotype interactions. F-test probabilities 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 separate table (Table E-8). 

Moisture levels in grain were not subjected to analysis of variance since the moisture analysis 

was performed on grain that had been mechanically 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 Standards for Compositional Analysis

2-Furaldehyde (Furfural) 

Grain samples were extracted with 4% trichloroacetic 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-violet quantitation. The quantitation limit for 
 MIR162-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 furfural 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 and washed with an acidic boiling detergent 

solution that dissolved the protein, carbohydrate, and ash. An acetone wash removed the fats 

and pigments. Lignocellulose fraction was collected on the frit and determined 

gravimetrically. The limit of quantitation 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 glutamic 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 methionine, Total cystine (including cysteine) 

The sample was assayed by three methods to obtain 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 hydrolyzed, the individual amino acids were then quantitated using 

an automated amino acid analyzer. The limit of quantitation for each amino acid was 0.100 

mg/g. 

Reference Standards: 

Beckman K18, 2.5 μmol/mL per constituent (except cystine 1.25 μmol/mL), 

Lot Number S504255 

Sigma L-Tryptophan, >99% (used as 100%), Lot Number 063K0382 
 MIR162-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, 18th 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 nonvolatile matter remaining was quantitated gravimetrically and 

calculated to determine percent ash. The limit 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, 18th Edition. AOAC International, Gaithersburg, Maryland. 

Beta Carotene (Vitamin A) 

The sample was extracted using alcohol and hexane. 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, 18th Edition. AOAC International, Gaithersburg, Maryland. 

Carbohydrates 

The total carbohydrate level was calculated by difference using 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 Agriculture, Washington, DC. 
 MIR162-USDA-1 Page 162 of 268 

Total Fat by Acid Hydrolysis 

The sample was hydrolyzed with hydrochloric acid at an elevated 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. The 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, 18th 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:methanol. The resulting 

methyl esters were extracted with heptane containing an internal standard. The methyl esters 

of the fatty acids were analyzed by gas chromatography using external standards for 

quantitation. The limit of quantitation was 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, used as 100%, Lot Number 035K1392 

Reference: 

AOCS (1997). Method 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 phosphate buffer with the addition of ascorbic 

acid to protect the folic acid during autoclaving. Following hydrolysis by autoclaving, the 

sample was treated with a chicken-pancreas enzyme and incubated approximately 18 hours to 

liberate the bound folic acid. The amount of folic acid was determined by comparing the 

growth response of the sample, using the bacteria Lactobacillus casei, with the growth 

response of a folic acid standard. This response 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, 18th 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 ashed 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 amount 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 standard solutions. The reference 

standards and limits of quantification for each mineral are listed in Table E-2. 

References: 

Dahlquist, R. L. and J. W. Knoll (1978). Inductively Coupled Plasma-Atomic Emission 

spectrometry: Analysis of biological materials 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, 18th 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 converted 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, 18th Edition. AOAC International, Gaithersburg, Maryland. 

Neutral Detergent Fiber, Enzyme Method 

The sample was placed in a fritted vessel and washed with a neutral 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. The 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 ultrasonication, 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 chromatography with ultra-violet quantitation. The 

limit of quantitation for the p-coumaric acid and 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-Coumaric 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 silica based anion exchange (SAX) column. Sample analysis 

was done on a macroporous polymer HPLC column PRP-1, 5μm (150 x 4.1mm) and a 

refractive index detector. The limit of quantitation 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 quantitation of phytic acid and some inositol 

phosphates in foods: problem 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 was 
 MIR162-USDA-1 Page 166 of 268 

calculated and converted to protein using the factor 6.25. The 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, 18th 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 pyridine, 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 quantitation (LOQ): The acceptable range for an 8/5 

dilution was 0.100-1.800%. 

Reference Standard: 

Sigma, D(+)-Raffinose Pentahyrate, 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 chromatography 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 was wet-ashed with nitric acid (HNO3

) using open or closed 

vessel microwave digestion. Samples that were readily soluble in water were diluted with 

HNO3 and analyzed straight. The amount of each element was determined using mass 

spectrometry by comparing the counts generated by the unknowns to those generated by 

standard solutions of known concentration. The limit of quantitation for 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 Inductively 
 MIR162-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 Analysis 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 product proportional to glucose concentration. The 

glucose concentration was quantitated 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 (μg/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, 18th 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-performance 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. Schreurs (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-tocopherol in animal feedstuffs. Journal of the 

Association of Official Analytical Chemists 63(6):1258-1261. 

Total Dietary Fiber 

Duplicate samples were gelatinized with alpha-amylase and digested 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 was 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 limit of quantitation for this 

study was 1.0%. 

Reference: 

AOAC International (2005). Method 985.29. In Official Methods of Analysis of AOAC 

International, 18th Edition. AOAC International, Gaithersburg, Maryland. 

Trypsin Inhibitor 

The sample was ground and/or defatted with petroleum 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 allowed to react for 10 minutes at 37°C. After 

10 minutes, the reaction was halted by the addition 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 limit of quantification 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 B1 (Thiamine) 

The sample was autoclaved under weak acid conditions to extract the thiamine. The 

resulting solution was incubated with a buffered enzyme solution to release any bound 

thiamine. The solution was purified on an ion-exchange column. An aliquot was taken and 

reacted with potassium ferricyanide to convert thiamin to thiochrome. The thiochrome was 
 MIR162-USDA-1 Page 169 of 268 

extracted into isobutyl alcohol and read on a fluorometer against 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, 18th Edition. AOAC International, Gaithersburg, 

Maryland. 

Vitamin B2 (Riboflavin) 

The sample was hydrolyzed with dilute hydrochloric acid (HCl) and the pH was adjusted to 

remove interferences. The amount of riboflavin was determined by comparing the growth 

response of the sample, using the bacteria Lactobacillus casei, with the growth response of 

multi-point riboflavin standard curve. This growth response was measured turbidimetrically. 

The limit of quantification for 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, 18th Edition. AOAC International, Gaithersburg, Maryland. 

Vitamin B3 (Niacin) 

The sample was hydrolyzed with sulfuric acid and the pH was adjusted to remove 

interferences. The amount of niacin was determined 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 turbidimetrically. 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, 18th Edition. AOAC International, Gaithersburg, Maryland. 

Vitamin B6 (Pyridoxine) 

The sample was hydrolyzed with dilute sulfuric acid in the autoclave and the pH was 

adjusted to remove interferences. The amount of B6 was determined by comparing the 

growth response of the sample, using the yeast Saccharromyces carlsbergenesis, with the 
 MIR162-USDA-1 Page 170 of 268 

growth response of a B6 standard. This response was measured turbidimetrically. The limit 

of quantification for this 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, 18th 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 through E-11. Results are presented 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 with 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 interaction 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 interaction revealed two 

analytes for which there was a statistically significant interaction (p<0.05), vitamin A (βcarotene) and vitamin B9 (folic acid). For these two analytes, individual location mean 

values are presented in Table E-8. 
 MIR162-USDA-1 

Table E-3. Proximate composition of maize forage from a MIR162 hybrid and a control hybrid. For comparison, ILS

database and OECD literature values are also shown. All units as described in column headings except F-test values. 

Treatment Moisture (% fw) Protein (% dw) Total Fat (% dw) Ash (% dw) Carbohydrates (% dw) A

(% 

X ̄ 71.2 7.2 1.5 4.1 87.3 2

MIR162 

range 66.2-77.2 3.1-10.1 0.8-1.9 3.1-5.8 82.9-90.7 23.6

X ̄ 70.5 7.3 1.6 4.0 87.1 2

Control 

range 64.1-75.8 3.4-8.9 0.4-2.3 2.9-5.7 83.6-91.7 23.3

% differencea 0.99 -1.37 -6.25 2.50 0.23 -2

p-value for genotypeb 0.184 0.711 0.421 0.796 0.694 0.

p-value for location 

x genotype interaction 0.730 0.645 0.100 0.948 0.973 0.

X ̄ 70.2 7.78 2.039 4.628 85.6 27

ILSI (2006) range 49.1 - 81.3 3.14 - 11.57 0.296 - 4.570 1.527 - 9.638 76.4 - 92.1 16.13 

N 945 945 921 945 945 9

OECD (2002) range 62 - 78 4.7 - 9.2 1.5 - 3.2 2.9 - 5.7 25.6 

a – [ (MIR162 - control)/control] x 100 b - statistically significant F-test (p<0.05) indicated in bold type. 
 MIR162-USDA-1 

Table E-4. Calcium and phosphorus composition of maize forage from a MIR162 hybrid and a control hybrid. FoCrop Composition database and OECD values are also shown. All units as described in column headings, except F-te

noted. 

Treatment Calcium (mg/kg dw) Phosphorus (mg/kg dw) 

X ̄ 2106 1997 

MIR162 

range 1720-2930 1270-2240 

X ̄ 2039 2079 

Control 

range 1440-2620 1760-2560 

% difference a 3.29 -3.94 

p-value for genotype 0.484 0.153 

p-value for location 

 by genotype interaction 0.944 0.066 

X ̄ 2028.6 2066.1 

ILSI (2006) range 713.9 - 5767.9 936.2 - 3704.1 

N 481 481 

OECD (2002) b range 0.15 - 0.31% dw 0.20 - 0.27% dw 

a – [(MIR162 - control)/control] x 100 b - for conversion, 1% = 10,000 mg/kg. 
 MIR162-USDA-1 

Table E-5. Proximate composition of maize grain from a MIR162 hybrid and a control hybrid. For comparison, ILS

database and OECD literature values are also shown. All units as described in column headings except F-test values. 

Treatment Moisturea (% fw) Protein (% dw) Fat (% dw) Ash (% dw) Carbohydrates (% dw) ADF (% dw) NDF (% dw) 

X ̄ 10.3 9.8 3.79 1.4 85.0 5.0 11.7 

MIR162 

range 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 

X ̄ 10.5 9.6 3.78 1.3 85.3 4.6 11.1 

Control 

range 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 

% difference b -1.91 2.08 0.26 7.69 -0.36 8.70 5.41 

p-value for genotypec 0.103 0.904 0.012 0.062 0.154 0.016 

p-value for location 

 x genotype interaction 0.832 0.538 0.381 0.696 0.309 0.703 

X ̄ 11.3 10.30 3.555 1.439 84.6 4.05 11.23 

range 6.1 - 40.5 6.15 - 17.26 1.742 - 5.823 0.616 - 6.282 77.4 - 89.5 1.82 - 11.34 5.59 - 22.64 

ILSI (2006) 

N 1434 1434 1174 1410 1410 1350 1349 

OECD (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 

a - moisture levels in grain not subject to analysis of variance as grain was mechanically dried after harvest b – [(MIR162 - control)/control) x 100 c - statistically significant F-test (p<0.05) indicated in bold type
 MIR162-USDA-1 

Table E-6. Mineral composition of maize grain from a MIR162 hybrid and a control hybrid. For comparison, ILSdatabase and OECD values are also shown. All units as described in column headings, except F-test values and where 

Treatment 

Calcium 

(mg/kg 

dw) 

Copper 

(mg/kg 

dw) 

Iron 

(mg/kg 

dw) 

Magnesium

(mg/kg dw) 

Manganese

(mg/kg dw)

Phosphorus

(mg/kg dw) 

Potassium

(mg/kg 

dw) 

Seleniu

(mg/kg 

X ̄ 38.1 1.3 20.2 1252 6.3 3173 3352 N/A 

MIR162 

range 29.4-47.2 0.96-1.95 17.3-22.9 1090-1480 4.14-7.97 2810-3550 3160-3710 <LOQ 

0.41

X ̄ 35.3 1.2 19.2 1218 6.1 3073 3357 N/A 

Control 

range 25.7-44.0 1.00-1.58 15.7-22.5 960-1470 4.59-8.01 2710-3400 2950-3660 <LOQ 

0.53

% difference c 7.93 8.33 5.21 2.70 3.28 3.25 -0.15 N/A 

p-value for genotype d 0.002 0.073 0.011 0.102 0.078 0.030 0.916 

p-value for location 

x genotype interaction 0.148 0.360 0.685 0.288 0.697 0.136 0.291 

X ̄ 46.4 1.75 21.81 1193.8 6.18 3273.5 3842 0.2 

range 

12.7 - 

208.4 

0.73 - 

18.5 

10.42 - 

49.07 

594.0 - 

1940.0 1.69 - 14.30 1470.0 - 5330.0 1810.0 - 6030.0 0.05 - 0

ILSI (2006) 

N 1344 1249 1255 1257 1256 1349 1257 89 

OECD (2002) e range 3 -100 0.09 - 1.0 0.1 - 10 82 - 1000 234 - 750 320 - 720 0.001 - 

a - where some values were <LOQ, statistical comparison was not possible and only the range is shown b - LOQ for selenium 0.056-0.057 mg/kg dw, LOQ for sodium 110-114 mg/kg dw c – [(MIR162 - control)/control] x 100 d - statistically significant F-test (p<0.05) indicated in bold type e – units of measure for OECD values: mg/100 g dw; for conversion, 1 mg/100g = 10 mg/kg 
 MIR162-USDA-1 

Table E-7. Vitamin analysis of maize grain from a MIR162 hybrid and a control hybrid. For comparison, ILSI 

database and OECD values are also shown. All units as described in column headings, except F-test values and where 

Treatment 

Vitamin A a

ß-Carotene 

(mg/100g dw) 

Vitamin B1

Thiamine 

(mg/100g dw) 

Vitamin B2

Riboflavin 

(mg/100g dw) 

Vitamin B3

Niacin 

(mg/100g dw) 

Vitamin B6

Pyridoxine 

(mg/100g dw) 

Vita

Foli

(mg/1

MIR162 

X ̄ 0.277 0.393 0.190 2.37 0.565 0

 range 0.241-0.316 0.358-0.433 0.112-0.238 2.11-2.83 0.434-0.694 0.02

Control 

X ̄ 0.294 0.392 0.180 2.47 0.605 0

 range 0.244-0.358 0.339-0.443 0.144-0.226 2.03-3.15 0.486-0.738 0.02

% difference b -5.78 0.26 5.56 -4.04 -6.61 

p-value for genotype c 0.001 0.983 0.314 0.093 0.002 0

p-value for location 

by genotype Interaction c 0.048 0.952 0.278 0.639 0.765 0

X ̄ 0.684 0.530 0.125 2.376 0.644 0.

ILSI (2006) Range 0.019 - 4.681 0.126 - 4.000 0.050 - 0.236 1.037 - 4.694 0.368 - 1.132 0.0147 

 N 276 894 704 415 415 8

OECD (2002) d Range 2.3 - 8.6 0.25 - 5.6 9.3 – 70 4.6 - 9.6 

a - for direct comparison to literature values, original units of RE/g dw were converted to units of mg/100 g dw based o

carotene. Institute for Laboratory Animal Research (1995). b – [(MIR162 - control)/control] x 100 

c - statistically significant F-test (p<0.05) indicated in bold type d – units of measure for OECD values: mg/kg dw; for conversion, 1 mg/100g = 10 mg/kg 
 MIR162-USDA-1 

Table E-8. Location means for ß-carotene and folic acid in maize grain from a MIR162 hybrid and a control hybrid. 

Treatment 

Vitamin A a

ß-Carotene 

(mg/100g dw) 

Vitamin B9

Folic Acid 

(mg/100g dw) 

Treatment 

Vitamin A 

ß-Carote

(mg/100g 

MIR162 0.283 0.027 MIR162 0.266 

Location 1 

Control 0.296 0.028 

Location 5 

Control 0.286 

MIR162 0.304 0.032 MIR162 0.292 

Location 2 

Control 0.352 0.029 

Location 6 

Control 0.291 

MIR162 0.272 0.027 MIR162 0.243 

Location 3 

Control 0.280 0.024 

Location 8 

Control 0.260 

X ̄ 0.684 0.0651 

ILSI (2006) range 0.019 - 4.681 0.0147 - 0.1464 

N 276 895 

a - for direct comparison to literature values, original units of RE/g dw were converted to units of mg/100 g dw based o

carotene. Institute for Laboratory Animal Research (1995). 
 MIR162-USDA-1 

Table E-9. Amino acid composition of maize grain from a MIR162 hybrid and a control hybrid. For comparison, ILSdatabase and OECD values are also shown. All units as described in column headings, except F-test values and where 

Treatment 

Aspartic 

Acid 

(mg/g dw)

Threonine

(mg/g dw)

Serine 

(mg/g dw)

Glutamic 

Acid 

(mg/g dw) 

Proline 

(mg/g dw) 

Glycine 

(mg/g dw)

Alanine 

(mg/g dw) 

X ̄ 6.66 3.55 5.21 19.54 9.12 3.84 7.70 

MIR162 

range 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 

X ̄ 6.54 3.47 5.11 19.16 8.96 3.79 7.55 

Control 

range 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 

% difference a 1.83 2.31 1.96 1.98 1.79 1.32 1.99 

p-value for genotype 0.171 0.059 0.149 0.129 0.119 0.269 0.135 

p-value for location 

x genotype interaction 0.500 0.699 0.307 0.443 0.233 0.265 0.557 

X ̄ 6.88 3.75 5.12 20.09 9.51 3.85 7.90 

range 

3.35 - 

12.08 ILSI (2006) 2.24 - 6.66 2.35 - 7.69 9.65 - 35.36 4.62 - 16.32 1.84 - 5.39 4.39 - 13.93 

N 1350 1350 1350 1350 1350 1350 1350 

OECD (2002) b range 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 

a – [(MIR162 - control)/control] x 100 (continued) b - unit of measure for OECD values: % dw; for conversion, 1% = 10 mg/g
 MIR162-USDA-1 

Table E-9 (cont). Amino acid composition of maize grain from a MIR162 hybrid and a control hybrid. For comComposition database and OECD values are also shown. All units as described in column headings, except F-test valu

Treatment Methionine (mg/g dw) Isoleucine (mg/g dw) Leucine (mg/g dw) Tyrosine a (mg/g dw) Phenyl- alanine (mg/g dw) Lysine (mg/g dw) Histidine (mg/g dw) 

X ̄ 2.15 3.38 12.85 3.42 5.09 3.05 2.87 

MIR162 

range 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 

X ̄ 2.10 3.31 12.57 3.35 4.99 2.96 2.85 

Control 

range 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 

% difference b 2.38 2.11 2.23 2.09 2.00 3.04 0.70 

p-value for genotype 0.177 0.113 0.115 0.201 0.128 0.095 0.438 

p-value for location 

by genotype interaction 0.459 0.372 0.561 0.694 0.483 0.256 0.195 

X ̄ 2.09 3.68 13.41 3.36 5.25 3.15 2.96 

range 1.24 - 4.68 1.79 - 6.92 6.42 - 24.92 1.03 - 6.42 2.44 - 9.30 1.72 - 6.68 1.37 - 4.34 

ILSI (2006) 

N 1350 1350 1350 1350 1350 1350 1350 

OECD (2002) c range 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 

a – one transgenic value was an outlier and was excluded from analysis b – [(MIR162 - control)/control] x 100 c – units of measure for OECD values: % dw; 1% = 10 mg/g 
 MIR162-USDA-1 

Table E-10. Fatty acid (FA) composition of maize grain from a MIR162 hybrid and a control hybrid.q For com

Composition database and OECD values are also shown. All units as described in column headings, except F-test valu

Treatment 16:0 Palmitic (% of total FA) 18:0 Stearic (% of total FA) 18:1 Oleic (% of total FA) 18:2 Linoleic (% of total FA) 

X ̄ 12.78 1.84 25.49 56.99 

MIR162 

range 12.25-13.09 1.56-1.99 22.67-26.57 55.86-59.74 

X ̄ 12.69 1.88 25.22 57.36 

Control 

range 12.29-13.12 1.625-2.07 23.38-26.77 56.26-59.47 

% difference b 0.71 -2.13 1.07 -0.65 

p-value for genotype c 0.275 0.051 0.057 0.026 

p-value for location 

By genotype interaction 0.554 0.934 0.154 0.444 

X ̄ 11.50 1.82 25.8 57.60 

ILSI (2006) range 7.94 - 20.71 1.02 - 3.40 17.4 - 40.2 36.2 - 66.5 

N 1344 1344 1344 1344 

OECD (2002) d range 0.29 - 0.79 0.04 - 0.17 0.70 - 1.39 0.67 - 2.81 

a - five most abundant fatty acids (FA) in maize grain. b – [(MIR162 - control)/control] x 100 c - statistically significant F-test (p<0.05) indicated in bold type d – units of measure for OECD values: % dw 
 MIR162-USDA-1 

Table E-11. Secondary metabolite and anti-nutrient analysis of maize grain from a MIR162 hybrid and a control hybr

ILSI Crop Composition database and OECD values are also shown. All units as described in column headings, exce

where noted. 

Treatment Ferulic Acid (mg/kg dw) 

p-Coumaric 

Acid

(mg/kg dw)

Inositol 

(ppm) 

Phytic 

Acid 

(% dw) 

Trypsin 

Inhibitor 

(TIU/mg dw)

Furf

(mg/

X ̄ 2682 179 2957 0.745 2.82 N

MIR162 

range 2490-2980 148-202 2410-3530 0.621-0.871 2.27-3.72 <L

<L

X ̄ 2453 157 2792 0.727 2.92 N

Control 

range 2010-2760 137-179 2180-3610 0.593-0.919 2.38-3.48 <L

<L

% difference c 9.34 14.01 5.91 2.48 -3.42 N

p-value for genotype d <0.001 <0.001 0.196 0.334 0.416 N

p-value for location 

x genotype interaction 0.436 0.073 0.459 0.589 0.671 N

X ̄ 2201.1 218.4 1331.5 0.745 2.73 3

range 291.9 - 3885.8 53.4 - 576.2 89.0 - 3765.4 0.111 - 1.570 ILSI (2006) 1.09 - 7.18 3.000 

e

N 817 817 504 1196 696 

OECD (2002) f range 0.02 - 0.3% dw 0.003 - 0.03% dw 0.45 - 1.0 <0.0

a - 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% dw c – [(MIR162 - control)/control) x 100 d - statistically significant F-test probability (<0.05) indicated in bold type e - below LOQ values are not included. f – For conversion of ferulic acid, 0.02% dw = 200 mg/kg; of 

p-coumeric acid, 0.003% dw = 30 mg/kg; of furfural, 1pp
 MIR162-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, Bt11xMIR162, and Bt11xMIR162xMIR604 Maize”. 

This document was written for the U.S. EPA application for registration of MIR162 maize 

and it describes in some detail the agronomic, environmental, and economic benefits of the 

MIR162 maize technology. Such a benefit assessment addresses the need of the EPA 

Administrator to make a finding that registration of a new pesticide (plant-incorporated 

protectant) is in the public’s interest. The benefits characterized herein are equally applicable 

to addressing USDA’s obligations 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 87 
Statement 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 United States Environmental Protection Agency 

specifically under provisions contained 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 requirements 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 87 
Statement Concerning Good Laboratory Practices

This assessment was not conducted in compliance 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 87 
Table 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 87 
A. 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 87 
Summary

Syngenta is seeking registration for a new plant-incorporated protectant, the Vip3Aa20 

derived from Bacillus thuringiensis, as produced in maize transformation event MIR162. 

Syngenta is also seeking registration for two combined trait maize cultivars containing 

MIR162 and two other registered plant-incorporated 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 Bt11xMIR162xMIR604. 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 against lepidopteran insect feeding damage when compared to 

the protection provided by conventional insecticides 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 the potential to provide U.S. 

agriculture with an economic benefit exceeding $371 million annually at product maturity. 

The introduction of the MIR162 trait in combination with Bt11 also has the potential to 

replace many conventional insecticide applications, reduce greenhouse gas emissions, and 

reduce mycotoxin contamination of livestock feed. There will also be IRM benefits 

stemming from the introduction of these combined 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 suppressed by Cry1Ab. Data has been 

developed showing that Bt11xMIR162 is high dose against these two pests; accordingly a 

reduction from the 50% structured refuge requirement 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 growers is predicted to offer crop yield 

advantages and important new options for control 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 offers health and 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. 

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 87 
I. Introduction 

At the time of application for a §3 pesticide registration under the Federal Insecticide, 

Fungicide, and Rodenticide Act (FIFRA), it is often not possible to determine if the 

registration will be granted under special circumstances. Since their introduction, plantincorporated protectant registrations have only been granted under special circumstances. 

FIFRA authorizes the Administrator of the Environmental Protection Agency (EPA) to 

conditionally register a pesticide 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 pesticide during such period will not cause any 

unreasonable adverse effect on human health or the 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.) agriculture in the past 25 

years. Adoption of genetically engineered crops with insect and herbicide tolerance traits has 

increased dramatically since their commercial introduction in 1995. Net economic benefits 

at the farm level have been substantial, exceeding a cumulative $27 billion (Brookes and 

Barfoot, 2005). Improved insect and weed control have led to increased crop yields and 

reductions in conventional pesticide applications (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 Biotechnology, Inc. to 

provide U.S. growers with maize hybrids that are resistant to feeding damage caused by a 

number of significant lepidopteran insect pests. This assessment will characterize 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 active 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 Western Hemisphere. It has been cultivated in 

the Americas since early historic times. Field 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 and 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 87 
Maize is grown for animal feed, 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 exporter 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% 

19.1% 50.8%

18.3% 

Figure 1. U.S. maize usage by segment, 2006 (Baker and Allen, 2007).

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 and nonfarm sectors of the economy. Production from

almost one-third of U.S. cropland moved into export channels in 2005 and generated $166.1 

billion in business activity. Technology advances increase agricultural productivity and keep 

domestic growers competitive in the global market. 

B. Maize Agronomics 

Z. mays is a large, annual monoecious grass; the duration of its life cycle depends on the 

cultivar and the environment in which the cultivar is grown. The bulk of maize is produced 

between latitudes 30 ̊ and 47 ̊. Practically no maize is grown where the mean midsummer

temperature is < 19 ̊C or where the average nighttime temperature 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 lasts 120 to 180 days. Maize is 

grown in areas where annual precipitation ranges from 25 to > 500 cm. Summer rainfall of 

15 cm is approximately the lower limit for maize production without irrigation. 

SSB-518-07 Page 8 of 87
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 South Dakota are major maize growing states. 

Production in these ten states accounts for 84% of total annual production (USDA, 2007a). 

Figure 2 displays the geographic distribution 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 which to choose. Available hybrids differ 

widely in agronomic characteristics, including length of growing period. Technology 

providers continue to develop varieties with desirable traits and increasing yield. Maize 

yields have increased an average of 3.5 bushels per year over the past decade. The average 

yield reported for the 2006 growing season 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 (Fernandez-Cornejo and Caswell, 

2006). Varieties containing herbicide-tolerance and insect-protection traits have been widely 

adopted by maize, soybean, and cotton growers because they protect the inherent yield 

SSB-518-07 Page 9 of 87 
potential of these crops or typically reduce grower input costs. In 2006, herbicide tolerant 

varieties accounted for 89% of soybeans planted, 65% of cotton planted, and 36% of maize 

planted (USDA, 2006b). Insect-protected varieties accounted for 52% of cotton and 40% of 

maize acres planted. Planting of these varieties has also benefited the environment by 

displacing conventional pesticide applications and 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 insects were huge until the introduction of crop 

sect pests can be categorized as major and 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 maize is grown and result in reduced test weight, 

Y

protection chemicals in the 1960s. Weeds compete 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 addition, a maize crop is susceptible to attack 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 destructiveness and their geographic distribution. Table 

1 categorizes most of the insect pests of maize 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-dwelling insects that feed 

on roots or other subterranean tissue (e.g., wireworms, billbugs, webworms, white grubs, 

corn root aphids, the seed corn maggot, grape colaspis and seedcorn beetles) and 

aboveground insects that attack the stalk, leaf, and ear (e.g., cutworms, corn leaf aphids, 

chinch bugs, grasshoppers, corn flea beetles and Japanese beetles). Striacosta albicosta

(western bean cutworm) is a pest of increasing economic importance because of its 

movement into Iowa, Illinois, and Indiana. 

In

in the transmission and dissemination of pathogenic organisms during maize development. 

Soil abounds in microorganisms, particularly fungi, 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 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. 

E

poor grain quality, and mycotoxin contamination of food and feed. Fusarium kernel or ear 

SSB-518-07 Page 10 of 87 
SSB-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.) 

Northern corn rootworm (Diabrotica barberi) Cinch bug (Blissus leucopterus) 

Western corn rootworm (Diabrotica virgifera 

virgifera) 

Southern corn rootworm (Diabrotica 

undecimpunctata) 

European corn borer (Ostrinia nubilalis) Other cutworms, many species 

Corn earworm (Helicoverpa zea) Seedcorn beetle (Stenolophus lecontei) 

Fall armyworm (Spodoptera frugiperda) Seedcorn maggot (Delia platura) 

Banks grass mite (Oligonychus pratensis) 

Major - Sporadic Insect Pests Two-spotted spider mite (Tetranychus urticae) 

Black cutworm (Agrotis ipsilon) Billbugs, many species 

Corn leaf aphid (Rhopalosiphum maidis) White grubs, many species 

Southwest corn borer (Diatraea grandiosella) Stalk borer (Papaipema nebris) 

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 brunnea) 

Moderate to M or Insect Pests in Thrips, several species 

Wireworms, many species Dusky sap beetle (Carpophilus lugubris) 

Armyworm (Pseudaletia unipunctata) Stink bugs, several species 

White-fringed beetles (Graphognathus spp.) Southern cornstalk borer (Diatraea 

crambidoides) 

Grasshoppers, many species Corn root aphid (Anuraphis maidiradicis) 

Corn flea beetle (Chaetocnema pulicaria) 

Crop losses attributable to O. nubilalis and Diabrotica infestations have been well 

characterized. However, there is surprisingly little quantitative information available on the 

economic impacts of the other major insect pests of maize, H. zea, S. frugiperda, A. ipsilon, 

and S. albicosta. These pests are not as widely spread 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 geographic distribution of these four 

insect pests in the continental U.S. As described in Appendix 1, there is compelling evidence
SSB-518-07 

Figure 3. U.S. geographic distribution of the maize insect pests H. zea (CEW), S. frugiperda (FAW), A. ipsilon (BCW

(WBCW). Two regions are shown for WBCW occurrence, one where its presence presents an economic problem other where it is spreading into. 

BCW 

CEW & FAW 

WBCW 

Economic

WBCW 

Spreading
that 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 regions of highest maize production 

 in Figure 2. 

icoverpa zea (Corn Earworm) 

verpa zea, formerly Heliothis zea, is native to the Americas 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 clipped as larvae tunnel 

he ear. The ear will h

plete kernel set if the silks are 

yed before pollination 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 growers in 1995 attributable 

 infestations totaled $510,000 in combined chemical 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 

ing

tes

 the

 a 

t

anno

plie

s c

ap

SSB-518-07 Page 13 of 87 
2. 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 

 weed species. Damage caused by A. ipsilon larvae is 

articularly severe in rotated maize that is planted 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 weed hosts are killed 

with cultivation or herbicides, the larvae 

begin feeding on maize. They are 

nocturnal feeders, cutting 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 surface 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 insecticide 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 are numerous university 

c

feeds on many hosts, including several

p

A. ipsilon larvae also cause economic damage to vegetables, cotton, tobacco, and turf grasses. 

A. ipsilon does not overwinter in the Midwest; moths migrate from the coastal areas of the 

Gulf of Mexico in the early spring. Females prefer to deposit eggs 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 87 
extension reports documenting its spread into Illinois and Indiana (see Figure 3). 

ther yellow tissue. Larvae then move to feeding 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 larva per ear can

larvae are not cannibalistic. One larva per ear r

have shown that even one larva per ear on a field 3.7 bu/ac 

(Cook, 2004). In years of severe infestation with m

ear’s kernels can be damaged. This 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 the larvae feed on the flag 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 reduce yield by 

ultiple larvae per ear, 50 to 60% of an 

rectly to significant reductions in yield and 

e risk of major infestations. Insecticide 

applications can be effective but timing of application is critical. Once the larvae have 

entered the silks and are inside the husk, insecticide applications are ineffective and larvae 

will feed until mature. Few cultural controls are effective against this pest. 

4. Spodoptera frugiperda (Fall Armyworm) 

S. frugiperda is native to tropical regions of North and South America. In the U.S. it 

overwinters in Florida and Texas and the moths can fly great distances. The insect has been 

found in virtually all states east of the Rocky Mountains. As a pest of maize, it is typically 

only a problem in the southeastern states. 

SSB-518-07 Page 15 of 87 
Maize, 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 yield; 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 through six instars and feed on foliage. Young larvae initially 

consume leaf tissue from one side, leaving the opposite epidermal layer intact. Feeding by 

second and third-generation larvae results in holes 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 

la r in the season. Control of armyworms 

with conventional insecticides 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 

insecticide app

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 

c maize leaf (top) and whorl feeding damage 

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 controlling stalk boring insects; only about 10% of 

growers applied insecticides for control of

SSB-518-07 Page 16 of 87 
annual 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. 

wers 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 2003 an estimated 14 

ventional insecticides to control corn rootworms. 

ds of insecticide active ingredient being applied 

abrotica species (Ward et al., 2005). Control of 

est single use of insecticides in the U.S. 

ontrolling aboveground insects presents a challenge for maize growers. The majority of 

eaf, stalk, and ear feeding 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 AgroTrak studies indicate that growers are 

n acres 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 prote

 A. ipsilon, frugiperda, and S. albicosta. A 

has been incorporated into the genome of MIR162 

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 insects would be physically 

positioned on the plant where they could be exposed to an insecticide application. The 

introduction of the first Bt maize hybrids in 1996 provided growers 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 introduction 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 planted 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 highly toxic to numerous

H. zea, S.

 

lepidopteran pests of maize, including:

maize-optimized variant of the gene 

maize and encodes a protein assigned the following 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 87 
Table 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. albicosta 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 locations 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 maize with 

ing feeding 

. saccharalis (sugarcane borer), H. zea (corn 

borer), P. nebris (common stalk borer), S. exigua

yworm), and S. albicosta (western bean cutworm). 

an insecticide application were the treatments employed in most trials. Figure 8 presents a 

graphic representation of the comparative feeding 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

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 insects. The combined-trait 

hybrid, Bt11xMIR162, is very efficacious against all five insects. MIR162 addresses the 

lepidopteran pest control limitations of Syngenta'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 Bt11xMIR162 hybrids with commercially available 

lepidopteran-protected Bt varieties, specifically YieldGard CB (MON 810) and Herculex I

(TC1507), were not possible due to intellectual 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 information 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 87 
0

20406080

100120

Bt11

%of Control Damage 

MIR162

Trea

Bt11xMIR162

nt

Insecticide

tme

 

Fig ra cond wit 11, MIR16 con m ngs s 

plo

ure 8. Feeding damage 

ventional insecticide treat

ts. 

tings from field efficacy trials 

ent (Warrior

ucted in 2005 and 2006 

are expressed as a percen

h Bt

tage of the da

2, B

e ob

® Insecticide). Rati mag 

mark of a Syngenta group co ® Trade mpany 

SSB-518-07 
Other than its resistance to lepidopteran insect feeding, MIR162 maize hybrids are 

phenotypically equivalent to conventional maize hybrids. The presence of the MIR162 

transgenes will protect the inherent yield potential 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 planted 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 between MIR162 and conventional hybrids.

 

Table 3. Yield results from agronomic equivalency 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 least 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 significant; * = means significantly different (p<0.05)

SSB-518-07 Page 20 of 87 
In 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 potentially 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 properties of the Vip3Aa20 protein (Graser and 

Stacy, 2006). The results of these studies demonstrate that no adverse effects were observed 

 mice exposed to a maximum attainable oral dose, that the protein is rapidly degraded in a 

gas an 

tox al. 

A r 

Vip3Aa20 when used as a plant-incorporated protectant 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 results of these studies has been p red by 

Raybould (2007a). 

This failure to elicit adverse effects in nontarget species is not surprising given the known 

mode of insecticidal action for 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 produced during vegetative 

bacterial growth and are secreted as soluble into the extracellular environment. B. 

th tures continu produce Vip

de nd sporulation like 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 which 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 specificity of a given protein for different insect 

sp

Syn a range of insect 

ec and the results of 

rted in the scientific literature clearly demonstrate that activity of Vip3Aa proteins 

is limited to species within order Lepidoptera, yet all lepidopteran species are not sensitive to 

in

tric matrix, that the protein does not share sequence homology with known mammali

ins and allergens, and it does not possess properties suggestive of food allergen potenti

temporary exemption from the requirement of a tolerance has been established fo

c

n developed

162 maize. No harmful effects have been observed 

ich in 

tal co preh

vironmental repa

um o

rotein

or V

mber of inse e na

 gram terium

sis, Vip pro

proteins

uringiensis cul e to protein 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

stion, 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 bioassays with Vip3Aa protein variants in

sp ies to identify those that are susceptible. The results of these bioassays

assays repo

SSB-518-07 Page 21 of 87 
the protein. Table 4 provides a listing of insect species that have been found by Syngenta or 

reported in the literature as being sensitive to one or more Vip3Aa protein variants. 

Table 4. Insect species that have been 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 

Lepidoptera: Noctuidae Heliothis virescens Active 

Spodotera exigua Active 

Spodoptera frugiperda Active 

Spodoptera litura Active 

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 

Lepidoptera: Danaidae Danaus plexippus Not Active 

Lepidoptera: Pieridae Pieris brassicae Not Active 

Bombyx mori Not Active 

Other Lepidoptera Earias vittella Active 

Ephestia kuehniella Active 

Culex pipiens Not Active 

Diptera Drosophila melanogaster Not Active 

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 87 
SSB-518-07 Page 23 of 87 

Table 4 (cont.). Insect species that have been 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 

Hymenoptera Apis mellifera N ot Active

Hemiptera: Anthocoridae Orius insidious N e ot Activ

Thysanoptera Frankliniella occidentalis N e ot Activ

Isotomidae Folsomia candida N e ot Activ

Neuroptera Chrysoperla carnea N e 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 registration must c interest. 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 riority 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 Use Pesticide d environ concerns. 

o MIR162 Maize

Vip3Aa20 protein e elements of the

n event MIR162 can be presumed 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

lic 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 make 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 

se 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 listed in Table 5. The m

s due to human an

 these in

mental risk 
Table 5.

zea, S. 

precautio

 Most commonly used 

bicosta, or S. frugiper

ary text from each prod

conven

a. 

ct lab

tional insecticide products (excluding seed treatm

ctive ingredients contained in each product,

l are shown. 

ents) used in maize for cont

al d A E se classification, Sig

n u e

PA u

Product Name Active Ingredient Signal rd Wo / Use Classification 

Asana XL Insect 8 e e: re 

sfenvalerate WARNING. Restricted Us Highly toxic to bees. Ext mely toxic to fish an

icide .4% 

A Gra 2 te 0 cyflu e: xic to fish and ife

.0% .1% bupirimfos thrin ztec 2.1% nular Insectide WARNING. Restricted Us To wildl

Baythroid 2 

yrethroid 

Emu

P Insec 2 yflu ely toxic to f nd 

thirn DANGER. Restricted Use: Highly toxic to bees. lsifiable ticide Extrem ish a 

5% c

B L In cyf e: remely toxic to fish an

luthrin WARNING. Restricted Us Highly toxic to bees. Ext 

aythroid X secticide 12.7%

Brigade 2EC Capture 2EC 

Ins

Ins

25.1% bife e: remely an

ecticide/Miticide & 

ecticide/Miticide 

nthrin WARNING. Restricted Us Highly toxic to bees. Ext toxic to fish 

Chlorpyrifos 4E 40.7% chl e: xic 

 t to orpyrifos WARNING. Restricted Us mammals, and birds. Highly To oxic to f beesish, . aquatic inve

AG Insecticide 

C Ins 16.6% delt 

amethrin DANGER. Restricted Use: Highly toxic to bees. Extremely toxic to fish and 

Decis 1.5E ecticide E Gran 1.15% bife : em nthrin CAUTION. Restricted Use Highly toxic to bees. Extr ely toxic to fish an

mpower2 ular Insecticide 

E uraly ntrust Nat te Insect Control 80% spinosad CAUTION. General Use: Toxic to aquatic i tebrate

nver 

Fanfare 2EC I Nem nsecticide/ 25.1% bin e: Extremely toxic to fish an

fenthrin WARNING. Restricted Us Highly toxic to bees. 

aticide 

Furadan 4F Inse 44% carbo Poisono 

hly fuan DANGER. Restricted Use: birds and other wildlife. Hig us i toxic to bees. f swallowed or 

cticide 

cide 3.75 zeta-c 11.25% bi ypermethrin : Extrem fenthrin CAUTION. Restricted Use Highly toxic to bees. ely toxic to fish an

Hero InsectiIntrepid 2F Insecticide 22.6% methoxyfenozide CAUTION. General Use: Hazardous to sensitive aquat

SSB-518-07 
SSB-518-07 

Table 5 (cont.). Most commonly used conventional insecticide products (excluding seed treatments) used in mai

ipsilon, H. zea, S. albicosta, or S. frugiperda. Active ingredients contained in each product, EPA use classification, S

precautionary text from each product label are shown. 

Product Name Active Ingredient Signal Word / Use Classification 

Lambda-T 11.4% λ-cyhalothrin WARNING. Restricted Use: Extremely toxic to fish an

Highly toxic to bees. 

Lannate SP Insecticide 90% methomyl DANGER. Restricted Use: Fatal if swallowed. Toxic t

invertebrates, and mammals. Highly toxic to bees. 

Lorsban 4E Insecticide 44.9% chlorpyrifos WARNING. General Use: Toxic to fish, aquatic invert

and birds. Highly toxic to bees.

Lorsban 75WG Insecticide 75% chlorpyrifos WARNING. General Use: Toxic to fish, aquatic invert

and birds. Highly toxic to bees. 

Mocap 15% Granular Nematicide-Insecticide 15% ethoprop DANGER. Restricted Use: Poisonous if swallowed. T

organisms and wildlife. 

Mustang Insecticide 17.1% zeta- cypermethrin WARNING. Restricted Use: Extremely toxic to fish an

Highly toxic to bees. 

Pounce 25 WP Insecticide 25% permethrin WARNING. Restricted Use: Extremely toxic to fish an

Highly toxic to bees.

Regent 4 SC Insecticide 39.4% fipronil WARNING. Restricted Use: Toxic to birds, fish, and aSevin brand 4F Carbaryl Insecticide 43% carbaryl CAUTION. General Use: Extremely toxic to aquatic an

invertebrates. Highly toxic to bees. 

Silencer Insecticide 11.4% λ-cyhalothrin WARNING. Restricted Use: Extremely toxic to fish an

Highly toxic to bees. 

Success Naturalyte Insect Control 22.8% spinosad CAUTION. General Use: Toxic to aquatic invertebrateWarrior with Zeon Technology Insecticide 11.4% λ-cyhalothrin WARNING. Restricted Use: Extremely toxic to fish an

Highly toxic to bees. 
The Restricted Use classification, imposed due to adverse environmental effects under 

no se practi (40 CF 5 1(a)), l s us f the hemicals 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 adverse effects on the environme Th ification is 

an indication of EPA concern about 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 insects 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 aboveground maize insect pests is challenging for 

gr on ional i tic a c are c ensive 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 insecticides 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

stici

ten

d fo

cor

pti

ing 

otice

its, 

 there

d pe

 risky

cludin

istere

ese fa

 in the

R 1

d sp

itho

ispla

ol o

Vi

blic

c In

ing co

d co

need 

s or 

alth o

nomic

ticide

 as the

ic inte

2.17

cial

t th

 the

e th

lepi

Aa2

nter

res

nditio

sts. E

for th

none

r the 

 bene

s and

y rela

rest. 

imit

otec

kee

ricti

f th

 ma

n pe

icid

g 

egist

olic

cide 

nmen

rom t

r av

Vip3A

the 

ive 

ing 

ns E

se pr

ts of

 pro

atio

 (EP

mical 

alternatives; (2) the new pesticide is 

t than

he use

ailable

a20 m

e o

g n

uip

 re

 ha

uct

aize

in encoded in MIR162 m

indi

, 19

tha

 cur

 of t

 no

aiz

se c

 fo

to

 fo

term

nt. 

sed 

that

gre

ates

ot bein

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w chem

mical 

arly de

safe

duce

he s

ed that use of the

is c

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ent,

The

g m

ere

ic

tec

mo

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 o

ale 

lass

s co

 spe

 Ag

et w

d pesti

al exce

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nstrate

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s. 

l to d

 contr

rated

 of pu

 Publi

regard

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 is a 

sticide

 to he

g eco

d pes

ctors,

 publ

ecor

e re

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pter

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t. 

ind

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e new che

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quir

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. B

cates 

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t is n

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 whe

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 sev

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for 

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 fall

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 As th

nd to

tively 

grower

ilal

 pri

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s. A

s an

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tati

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ddit

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maiz

ns o

onally, th

roti

gra

A. 

s in

a ro

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psil

rop

two

tinu

n, H. zea

yield (

cur.

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n o

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 MIR

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 the

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SSB-518-07 Page 26 of 87 
2. Composition Factors 

The composition of MIR162 maize is fundamentally different from the composition of 

conventional insecticide end-use products. The active ingredient, Vip3Aa20, is plantincorporated. It is safer than all currently registered conventional maize insecticide products. 

This characteristic of the product virtually eliminates the occupational and environmental 

risks currently associated with the application of chemical controls 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 expressed throughout all tissues of the maize plant. This ensures 

rotection where it is needed and eliminates the risk of insecticide failures associated with 

ation and application of compatible 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 disposal of millions of pounds of hazardous 

chemicals annually and to eliminate the greenhouse gas emissions associated with these 

activities. These product characteristics support a conclusion that registration 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 product 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 leaf, stalk, and ear protection 

provided by MIR162 maize compared to hybrids treated with a conventional insecticide 

product. Vip3Aa20 possesses a number 

d

insects. The prot

p

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 usage, some of which include improper calibration and 

maintenance of application equipment, handling of hazardous chemical insecticides, 

container disposal, chemical misplacement, runoff, and spray drift. 

Integrated pest management (IPM) in agriculture 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. Planting 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 application 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 scouting and monitoring programs that provide data 

upon which to base crop management decisions. The product is also fully compatible with 

cultural control measu

SSB-518-07 Page 27 of 87 
integrated pest management for maize. Superior 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 pests minimizes risk for nontarget organisms. 

dentification studies has been conducted with nontarget indicator species, 

cluding many species that are part of the maize 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 insecticide 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 studies failed to provide any evidence of 

Vip3Aa20-induced adverse effects. The protein is rapidly degraded in mammalian digestive 

systems and it bears no amino acid sequence similarities to known toxins and allergens. 

Since the insecticidal protein is plant-incorporated, 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 concentrations (Raybould, 2007a). 

M

discrete risks to a multit

a

insects are of concern to growers, consumers, 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 quality, 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 occasionally necessary to reject or reformulate 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 MIR162 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 undertaken 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 considered the potential economic effects of MIR162 

introduction on the market for existing insect-protection trait technologies. Second, they 

commissioned a grower survey to assess willingness to adopt the new technology. Lastly, 

they estimated the spatial distribution of the costs of control for H. zea and S. albicosta and 

how these costs might change in future years. A full report for this economic study is 

SSB-518-07 Page 28 of 87 
contained in Addendix 1. This analysis only examined the potential benefits associated with 

improved control of H. zea and S. albicosta. 

Following general economic principles, the introduction of a new technology will have an 

ffect on the market for existing technologies that is beneficial to users of either technology. 

 be 4.8 bu/ac. Examination 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 registration of MIR162 

 in the public interest. 

oncurrent with the application for a FIFRA §3 registration of the Vip3Aa20 plant-

 maize, Syngenta 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 attributable 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 significant 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 measures decreases. 

The grower survey results indicate that 70% of respondents 

if

after introduction. 

A potential economic benefit for maize growers 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 information 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 provides 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 passed along to consumers in the form of lower 

commodity prices. These substantial economic benefits 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’, that possess the combined insect-control 

characteristics of Bt11 and MIR162. Hybrids that contain multiple transgenic traits are often 

referred to as ‘stacked’ hybrids or hybrids containing ‘pyramided’ 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 87 
Reg. No. 67979-1; OECD ID SYN-BTØ11-1). At that time, the Agency found that its 

registration was in the public interest. Upon extension of the 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 typically depends on careful scouting of fields for 

re

Action Document for Bt crops (EPA, 2001) and reflects the 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 restate the benefits of the traits conferred by Bt11 

and MIR162 maize individually, some specific 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 produce 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. The broad efficacy of the product will allow maize 

growers across wide geographies to benefit from routinely planting Bt11xMIR162 hybrids to 

control the majority of lepidopteran maize pests that could be prevalent in any given year. In 

addition to the insect-control advantages of the product, the herbicide tolerance conferred by 

the phosphinothricin acetyltransferase protein in Bt11xMIR162 maize will provide growers 

with the option of using glufosinate 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 insecticide 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 inconvenience, 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, where they are inaccessible to foliar insecticide 

applications. Even with carefully timed applications, under typical use conditions the 

 

1

 The registration for the plant-incorporated pesticide active 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 87 
efficacy of foliar or soil-applied insecticide products cannot achieve the consistent level of 

season-long control that is possible using the technology represented by Bt11xMIR162 

maize. 

Maize hybrids expressing Cry1F are commercially available and offer a measure of broadspectrum lepidopteran control. In 2006, Cry1F hybrids accounted for 7.1% of maize acres 

planted in the U.S. (Doane, 2006a). In contrast, 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 that 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 benefits of stacking the Cry1Ab 

lectively, the results of field efficacy trials 

emonstrate that Bt11xMIR162 maize will be protected 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 predict that, as the benefits of broad-lepidopteran 

control are more widely recognized and available to growers, the relative proportion of acres 

planted to such hybrids will change. While it has not been possible to conduct direct side-byside efficacy comparisons of Cry1F and Bt11xMIR162 hybr

e

available Bt hybrids or conventional inse

B

resistance management criteria for ‘high dose’ (see V.B.5 below and Kurtz et al., 2007a), 

whereas Cry1F hybrids provide only ‘suppression’ 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 several 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 same 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 indicate 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 significant lepidopteran 

pests, Bt11xMIR162 hybrids will demonstrate significant yield advantages under both low 

Some lepidopteran pests, such as A. ipsilon, can cause major yield losses due to cutting of an 

SSB-518-07 Page 31 of 87 
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 lepidopteran larvae cause physiologic yield loss 

ecause the stalk damage interferes with nutrient uptake and increases susceptibility to plant 

 in Bt11 maize 

0 CFR §174.511 and 40 CFR §174.522, respectively). The selectable marker in MIR162 

empt from tolerances 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 maize do not interact in a manner that results in toxicity to 

he combined mammalian and environmental safety profile of Bt11xMIR162 indicates that 

the introduction of the first (Cry1Ab) Bt maize hybrids in 1996 and 

000, 3.9 million fewer acres of insecticide applications were made to maize for control of 

b

diseases. They can also cause severe stalk lodging and dropped ears, which results in a 

physical yield loss because the ears cannot be mechanically 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 presence of pest pressure, Bt11xMIR162 maize 

hybrids can be expected to preserve yield potential, grain quality, and silage quality by 

minimizing the damage that could otherwise be caused by lepidopteran insects. 

3. Risk Factors 

Syngenta has provided data to the Agency supporting the mammalian safety of the transgenic 

proteins produced in Bt11xMIR162 maize. Permanent exemptions from the requirement of a 

tolerance in all crops exist for both Cry1Ab and 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 Bt11xMIR162 maize that might trigger a concern for 

mammalian s

sp

nontarget insects (Raybould, 2007b). A FIFRA Scientific Advisory Panel concluded that Bt

insect toxins that are individually nontoxic to mammals do not pose a mammalian safety 

concern when present in combination (FIFRA SAP, 2004). 

Despite the broad-spectrum lepidopteran activity of Bt11xMIR162 maize, the environmental 

safety assessment supporting this product (Raybould, 2007b) indicates that it will pose no 

significant risk to nontarget organisms. 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 significant risks. Accordingly, it offers health and environmental 

advantages over current chemical alternatives 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 87 
Vip3Aa20 protein in MIR162 maize is expected to allow maize growers to further reduce 

their use of insecticides for lepidopteran control. Significantly, many of the conventional 

insecticides currently registered for lepidopteran 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 allow 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 agricultural workers, and will reduce the impact of 

insecticide use on wildlife and the environment. 

An 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 lepidopteran 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 measures for lepidopteran larvae. 

tely predict 

e magnitude of economic benefits that growers of Bt11xMIR162 maize will realize beyond 

level of fumonisi

g

levels of fumonisin. This is an indirect benefit of protecting maize ears from feeding damage 

by lepidopteran pests. The additional control of ear-feeding pests, particularly H. zea and S. 

albicosta, that will be provided by Bt11xMIR162 maize will likely further reduce mycotoxin 

contamination in grain. 

4. Economic Factors 

A

years, it is not possible for individual growers to accurately predict whether lepidopteran pest 

pressure will be economically significant in any particular growing season. Growers must 

make seed purchase decisions prior to knowing whether the seed price premium for 

lepidopteran control in a given year will actually 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 growers 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 Bt11 (EPA, 2001; Van Duyn, 

2005) or MIR162 maize (see Appendix 1). Nevertheless, the improved pest protection 

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 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 87 
of 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 investment 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 proteins 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 resistance developing in these species. 

t11xMIR162 hybrids offer IRM advantages in comparison to other control options that do 

n the report by Kurtz et al. (2007a), 

yngenta provides data and rationale to justify reduction of the maize refuge in cottonp

Another predicted economic benefit for growers 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 control of lepidopteran pests. 

5. Other Factors 

A unique benefit offered by Bt11xMIR162 hybrids relates to their distinct advantages in the 

area of insect resistance management (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’ 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 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 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 resistance 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 undergo selection pressure from 

both Bt maize and Bt cotton varieties that express similar Cry proteins, where the two crops 

are grown in the same geographies. The principal 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 acres for growers of Bt11xMIR162 maize. No 

other Bt product offers comparable IRM advantages in maize.2

For growers of Bt11xMIR162 maize hybrids, the reduced refuge requirement in cottongrowing regions will translate into a higher proportion of insect-protected maize acres, with a 

proportional increase in all the attendant benefits of the product in these areas. As an added 

advantage, compliance with the refuge requirement 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 87 
because Bt maize growers in cotton-growing 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-growing 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 competition in the marketplace for pest-control 

1xMIR162 maize offers health and environmental safety advantages over 

onventional insecticides, as well as insect resistance management benefits that will help to 

eferenced or described in previous sections of this document. The 

gnificant benefits of MIR604 maize as a stand-alone product for rootworm control have 

he benefits offered by the component traits in Bt11xMIR162xMIR604 maize will not be 

The information presented herein 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 lepidopteran pests as well as for growers who use other options for 

lepidopteran control. Another economic benefit

a

traits. Use of Bt1

c

preserve the durability of this and other Bt-based products for lepidopteran control. 

VI. Public Interest Factors Applicable to Bt11xMIR162xMIR604 Maize 

Concurrent with the application for a FIFRA §3 registration of MIR162 maize and 

Bt11xMIR162 maize, Syngenta is submitting an application for registration of a new product 

that contains the combined active ingredients in Bt11 maize, MIR162 maize, and MIR604 

maize (EPA Reg. No. 67979-5; OECD ID SYN-IR6Ø4-5). The transgenes in Bt11, MIR162, 

and MIR604 maize have been combined by traditional breeding methods to create maize 

hybrids, designated herein as ‘Bt11xMIR162xMIR604 maize’, that express 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-incorporated protectants in Bt11 maize and MIR604 maize have been 

combined by traditional breeding in Bt11xMIR604 maize, for combined control of certain 

lepidopteran and rootworm (coleopteran) pests. The plant-incorporated protectants in 

Bt11xMIR604 maize were approved by the EPA in 2007 (EPA Reg. No. 67979-8) and the 

benefits of Bt11xMIR604 maize have been described by Steffens and Tinsworth (2006). 

T

restated here. This section will focus on the specific characteristics of 

Bt11xMIR162xMIR604 maize and the benefits it affords by combining multiple insecticidal 

traits in a single product. 

SSB-518-07 Page 35 of 87 
A. Product Description and Intended Use 

Bt11xMIR162xMIR604 maize plants combine the transgenic traits of Bt11, MIR162, and 

MIR604 maize and thereby produce a total of six transgenic proteins: the B. thuringiensiserived insecticidal proteins Cry1Ab, Vip3Aa20 and modified Cry3A (mCry3A). 

rowers 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 hybrids 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 proteins. 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 from routinely planting Bt11xMIR162xMIR604 maize hybrids to 

control the majority of lepidopteran and rootworm pests that might 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 Bt11xMIR162xMIR604 maize will provide growers with the 

option of using labeled glufosinate products for weed control. 

B. Factors Affecting a Public Interest Finding 

1

T

II.D and IV.A. The alternative methods of maize rootworm control have been summarized 

separately (EPA, 2007; Steiner et al., 2004), and include soil-applied insecticides, seed 

treatments, and transgenic maize hybrids 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 pests controlled by Bt11xMIR162 maize (see Section 

V.B), the mCry3A protein in Bt11xMIR162xMIR604 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 major 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 87 
reports of efficacy studies in O. nubilalis (White 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 previously described for Bt11 maize (EPA, 2001), 

IR162 maize (see Section IV.B.5), MIR604 maize (EPA, 2007; Steiner et al., 2004), and 

rowers better 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 Bt11xMIR162xMIR604 maize that might trigger a 

 the environmental safety assessment supporting this product 

aybould, 2007b) indicates that it will pose no significant risk to nontarget organisms. This 

S

substantiate the predicted efficacy of combining multiple insecticidal traits in 

Bt11xMIR162xMIR604 maize hybrids. Therefore, it is reasonable to assume that growers 

will realize the cumulative benefits of all three insecticidal traits in this product. 

Studies to quantify the yield advantage of Bt11xMIR162xMIR604 maize across multiple 

geographies, environmental conditions, cropping practices and pest pressure are ongoing. 

Data available from a limited set of trials indicate 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 advantages under pest pressure. 

3. Risk Factors 

The health and e

M

Bt11xMIR162 maize (see Section V.B.3) will also apply, in combination, to 

Bt11xMIR162xMIR604 maize. Therefore, this product offers significant 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 hybrids will further help the EPA achieve its 

risk-reduction objectives, and will help g

th

Syngenta has provided data to the Agency supporting 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 described in Secti

The available data furthe

b

concern for mammalian safety. The proteins are individually not toxic to mammals and the 

insect-specific toxins in Bt11xMIR162xMIR604 maize do not interact in a manner that 

results in toxicity to nontarget insects (Raybould, 2007c). A FIFRA Scientific Advisory 

Panel also concluded that Bt insect toxins that are individually nontoxic to mammals do not 

pose a mammalian safety concern when present in combination (FIFRA SAP, 2004). 

Despite the broad-spectrum lepidopteran activity and coleopteran activity of 

Bt11xMIR162xMIR604 maize,

(R

assessment also considered the potential for effects on endangered species and for synergistic 

effects of the Cry1Ab, Vip3Aa20 and mCry3A proteins on nontarget insects. 

SSB-518-07 Page 37 of 87 
The combined mammalian and environmental safety profile of Bt11xMIR162xMIR604 

maize indicates that the product will pose no significant safety risks. Accordingly, it offers 

significant health and environmental advantages over current chemical alternatives for 

control of lepidopteran and rootworm pests. For maize growers who currently rely upon 

onventional insecticide applications for lepidopteran and rootworm control, 

he broad efficacy of Bt11xMIR162xMIR604 hybrids will provide ‘insurance’ for growers 

y multiple pests that might otherwise 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-control profile of Bt11xMIR162xMIR604 

aize can be expected to translate into correspondingly 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 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 agricultural workers, and will reduce 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 users of maize products. The magnitude of these 

economic benefits will necessarily 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 growers and downstream consumers is increased 

competition in the marketplace for pest-control products, including hybrid seed from multiple 

providers of lepidopteran-active and or rootworm-active transgenic varieties. The 

commercial availability of Bt11xMIR162xMIR604 hybrid maize seed will represent a 

significant new pest control option and tool available 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 87 
maize 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. Bt11xMIR162xMIR604 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). The 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 Science Society of America and Soil Science 

resistance management benefits described above (Section V.B.) for Bt11xMIR162 maize will 

also apply to Bt11xMIR162xMIR604 maize. Accordingly, a 20% non-Bt maize refuge in 

cotton-growing regions will be justified. The stacking of three insecticidal proteins in this 

product is not expected to increase selection pressure for cross-resistance 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 rootworm pests, introduction of this product is 

expected to help extend the durability of other commercially available rootworm-protected Bt

maize products. 

6. Public Interest Find

The information summarized herein supports 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 pests, 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, 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 

Baker, A. and E. Allen (2007). Feed outlook. Supplies lowered for feed grains and 

roughage. Electronic outlook report from the Economic Research Service, United States 

Department of Agriculture. FDS-07a, January 17, 2007. Accessed on May 17, 2007: 

http://usda.mannlib.cornell.edu/usda/ers/FDS//2000s/2007/FDS-01-17-2007.pdf 

Brookes, G. and P. Barfoot (2005). GM crops: 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.cornell.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/insects/western_bean_cutworm/index.html 

D

Corn and Cor

Society of America, Madison, Wisconsin. 

SSB-518-07 Page 39 of 87 
Doane (2005). 2005 AgroTrak. The US agrochemical usage study. A Doane Marketing 

Research syndicated tracking study. St. Louis, Missouri. 

Doane (2006a). US seed corn TraitTrak study. A Doane Marketing Research syndicated 

tracking study. St. Louis, Missouri. 

Doane (2006b). 2006 AgroTrak. The US agrochemical usage study. A Doane Marketing 

Research syndicated tracking study. St. Louis, Missouri. 

Draper, 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 Registration Action Document - Bacillus thuringiensis plantincorporated protectants. Accessed May 17, 2007: 

http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm

EPA (2005) Updated Biopesticide Registration Action Document (BRAD) for Bacillus 

thuringiensis Cry1F corn. Accessed May 17, 2007: 

www.epa.gov/pesticides/biopesticides/ingredients/tech_docs/brad_006481.pdf

EPA (2007) Biopesticides Registration Action Document - Modified 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/biopesticides/ingredients/tech_docs/brad_006509.pdf. 

Estruch, J., G. Warren, M. Mullins, G. Nye, J. Craig, and M. Koziel (1996). Vip3A, a novel 

ctivities 

against lepidopteran insects. Proceedings of the National Academy of Sciences 93:5389-

ernandez-Cornejo, J. and M. Caswell (2006). The first decade of genetically engineered 

Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of a

5394. MRID 468808-04. 

F

crops in the United States. Economic Research 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 health risk, ecological risk, and insect 

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 microbially-produced Vip3A test 

substance MIR162VIP3A-0106 and comparison with Vip3A expressed in event MIR162-

derived maize (corn). Unpublished Syngen

SSB-518-07 Page 40 of 87 
Gray, M. and W. Luckmann (1994). Integrating the cropping system for corn insect pest 

management. Pp 507-541. In Introduction To Insect Pest Management. Third 

Edition. Metcalf, R. L. and W. H. Luckmann (eds.). John Wiley & Sons, Inc., New 

arper, B. (2006). Vip3A as expressed in event 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 from insect damage and costs of control in 

York, New York. 

H

sequence homology with known toxins. Unpublished Syngenta report, No. SSB-112-06. 

MRID 468648-08 

Harper, B. (2006). Vip3A as expressed in event 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 insects. Accessed May 17, 2007: 

http://www.bugwood.org/sl95/field_corn.html

Kurtz, R., A. McCaffery and E. Dunder (2007a). Insect resistance management 

-

Kur ent 

considerations for Bt11 x MIR162 x MIR604 corn. Unpublished Syngenta report, No. 

Lee

tative insecticidal protein Vip3A differs from that of Cry1Ab 

δ-endotoxin. Applied and Environmental Microbiology 69(8):4648-4657. MRID 

Ma fs of transgenic field crops: An 

3-50. 

CGA (2007). 2007 World of corn. An annual 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, and J. Chen (2003). The mode of action of the 

Bacillus thuringiensis vege

468808-01. 

rra, M., P. Pardey, and J. Alston (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 87 
Raybould, A. (2007a). The environmental fate of Vip3Aa20 in MIR162 maize: Expected 

Raybould, A. (2007b). The environmental fate and potency of the insecticidal proteins 

Cry1Ab and Vip3Aa20 in Bt11 x MIR162 stacked maize hybrids. Unpublished Syngenta 

Raybould, A. (2007c). The environmental fate and potency of the insecticidal proteins 

Cry1Ab, Vip3Aa20 and mCry3A in Bt11 x MIR162 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. Unpublished Syngenta report, No. SSB-002-07. 

Ste

tration of the plant-incorporated protectants Cry1Ab and modified Cry3A proteins, 

as expressed in stacked Bt11 x MIR604 corn. Unpublished Syngenta report, No. SSBSteiner, H., M. Kostiuk, and R. Tinsworth (2004). Amended public interest document in 

support of registration of the plant-incorporated protectant, modified Cry3A protein, as 

US

f Agriculture. December 2006. Accessed on May 17, 2007: 

http://www.fas.usda.gov/wap/circular/2006/06-12/toc.html 

US icultural Statistics Service, United 

States Department of Agriculture. Accessed on May 17, 2007: 

USDA (2007a). Crop production 2006 summary. National 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 research on the benefit of Yieldgard ECB® Bt Corn in 

environmental concentrations, margins of exposure in nontarget organism hazard studies 

and endangered species assessment. Unpublished 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 Science Society of America and Soil Science Society of 

America, Madison, Wisconsin. 

S

conditions. Unpublished Syngenta report, No. SSB-038-06. 

cy, C. (2007b). In vitro digestibility of Vip3Aa

m

ffens, J. and R. Tinsworth (2006). Amended public interest document in support of 

regis

513-06. MRID 468548-01. 

expressed in event MIR604 corn. Unpublished Syngenta report. MRID 463021-01. 

DA (2006a). World agricultural production. Foreign Agricultural Service, United States 

Department o

DA (2006b). Acreage, June 30, 2006. National 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/current/CropPro

V

SSB-518-07 Page 42 of 87 
North 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 tolerance of a MIR162 maize 

hybrid: 2006 field trial results. Unpublished Syn

White, J., J. Sagers, M. Meehan, and M. Meghji (2007b). Fall armyworm tolerance of a 

MIR162 maize hybrid: 2006 field trial results. Unpublished Syngenta report, No. SSB503-07. 

W

GA21 stacked field maize hybrid against European 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 against corn earworm in the field. Unpublished Syngenta 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 against 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 earworm 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 87 
Appendix 1

The Economic Implications of the 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 87 
Agri-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@agri-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 87 
Agri-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 farm and state levels of the commercial introduction of

Syngenta Corporation’s MIR162 corn trait. We use several 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 technology 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 western bean cutworm and the corn earwor

MIR162 trait will provide superior control relative to current B

Using Doane’s Agro-Trak data on acres treated for them 

that pressure from these insects is likely to sprea

This will increase the value of the MIR162 trait in 

Third, we present the results of a grower survey that we designed and 

commissioned expressly for this study in which 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 densities, 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 commercia

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 portion of their corn acreage. 

Lastly, we calculated the total potential gain from the 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 three states is more than $300 million. Given

that this estimate is an upper bound estimate 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 representative of the total regional value of the 

introduction of MIR162 today. 

. 

logy using the economic theory of supply and demand. The results of 

how an unambiguously positive gain to technology buyers of the 

new substitute into the market. 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 

ers also were read 

 several questions regarding their 

lized. The responses were quite

mercial introduction of 

ation generated by

e value in other 

SSB-518-07 Page 46 of 87 
Agri-Analytics, Inc. 3 

TABLE OF CONTENTS 

 Page

EXECUTIVE SUMMARY ......................................................................................2 

INTRODUCTION ..................................................................................................5 

THE ECONOMICS OF SUBSTITUTES AND COMPETITION ..............................5 

 EXISTANCE OF A NEW TECHNOLOGY ...................................................................6 

 ADOPTION OF A NEW TECHNOLOGY (A CHANGE IN DEMAND) ................................8 

BENEFITS 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 

DESCRIPTIVE STATISTICS ........................................................

 PEST PRESSURE AND MEAN YIELD LOSSES OVER TIME .....................................12 

MEASURES AND PREDICTIONS OF PEST PRESSURE FROM DOANE’S 

AGRO-TRAK SURVEY DATA ............................................................................19 

 RESPONDENT’S MIR162 ADOPTION INTENTIONS ...............................................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 87 
Agri-Analytics, Inc. 4 

LIST OF TABLES AND FIGURES 

Page

Table 1. Descriptive statistics of survey respondents .................................12 

Table 2. Pest pressure as reported by survey respondents by insect, 

state and year ...............................................................................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 between 2007 and 2010 ...............................28 

Table 5. Components needed to calculate gain from MIR162 for 

selected states for 2005 and 2006 ...............................................26 

...............41 

........42 

......7 

year ........16 

bean cutworm ...............................................................................24 

igure 10. Spatially smoothed costs ($/harvested acre) for 

western bean cutworm .................................................................25 

Figure 11. Actual treatment costs ($/harvested acre) for corn earworm ........26 

Figure 12. Spatially smoothed costs ($/harvested acre) for 

 corn earworm ...............................................................................27 

Table 6. Gain from MIR162 for selected 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 results for Illinois .........................................

Table B.3 Regression results for Iowa ..................................................

Table B.4 Regression results for Nebraska ..................................................43 

Figure 1. The effect of a new technology on the elasticity of demand 

for an existing technology .........................................................

Figure 2. The effect of a new technology 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 yield losses by insect 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 87 
Agri-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 market for existing technologies. We then 

iscuss the results of a grower survey that 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 technology begins to take place, downward 

ressure on the prices of the competing technologies 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 provide excellent control of several 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 anywhere MIR162 is introduced (alon

with traits for corn borer and corn rootworm, when appropriate), gi

v ute for the existing technologies 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 technology. We approach this task in sever

ways. First we present a theoretical discussion 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 the CEW and WBC 

changes in that spatial distribution in the future, so that we can estimate the potenti

total value to farmers of MIR162 for a few years after its introduction. 

The Economics of Substitutes and Competition 

In economic terms, goods are deemed to be related in consumption if a change 

the price of one good affects demand for the other good. One type

is a substitute. Two goods are substitutes 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 technologies grown by farmers in the U.S. 

The existence of viable substitutes creates competition in the m

mere existence of a new substitute technology will have an effect on the market fo

th

s

p

3 The other types of related goods are called complements, where a change in the 

price of one good changes the price of the other in the same direction. 

SSB-518-07 Page 49 of 87 
Agri-Analytics, Inc. 6 

SSB-518-07 Page 50 of 87 

Existence of a New Technology

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 demanded. The 

as value to the 

orn growers. The fewer the 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 the 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 conditions for the existing technology. 

Originally, before the introduction of the new technology, the supply schedule of the 

existing technology is shown by Se

0

 and the demand schedule is shown by De

0

. The 

subscript e represents the existing technology and the superscript 0 represents time, 

with 0 denoting time before the introduction of the substitute. 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 technology are willing to 

produce and sell. The demand schedule shows the quantity demanded by 

consumers of the existing technology (corn gr

h

intersection of the supply and demand schedules gives the market price, Pe

0

 and 

quantity sold, Qe

0

 of the existing technology before the new technology is introduced. 

Now, suppose a new, substitute technology is introduced into the market. The 

supply schedule for this new technology, Sn

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 existing technology because now a new choice exists in the 

market place. In other words, corn growers’ technology choice has become less 

constrained. This flexibility (relaxing of constraints), in and of itself, h

c

Although the proof of this assertion is beyond 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 technology market is illustrated by a rotation of 

the demand schedule of the existing technology on the left hand side graph in Figure 

1 so that it is “flatter” or “more elastic” than before. Now, suppose the producers of 

the existing technology want to raise its price from Pe

0

 to Pe

1

. The decrease in th

q e

caused by the existence of the substitute. The decrease in the quantity demanded 

for the given price change would be from Qe

0

 to Qe

1

 before the introduction of the 

new technology (based on the original demand schedule). However, the decrease 

in the quantity demanded after the introduction of the new technology would be 

greater, from Qe

0

 to Qe

1*. This means that, if the producers of the existing 

technology decide to raise its price, they wo
tics, Inc. 

SSB-518-07 

Figure 1: The Effect of a New Technology on the Elasticity of Demand for an Existing Tec

Price/Unit 

gy 

Price/Unit 

Existing Technolo New Techno

Sn0

Se0

De0

De1

Qe0 Qe Quantity 1 Qe1*

Pe0

1

Agri-Analy

Pe
Agri-Analytics, 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 prices of corn traited technologies will tend 

to remain lower and more stable in the future than otherwise would be the case. 

UAdoption of a New Technology (A Change in Demand)

8 

f 87 

Consider now what happens to market prices and quantities 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, Dn. The initial market price of the new technology is Pn and 

the initial quantity sold is Qn. 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 De

1

 to De

2

. The new 

equilibrium price for the existing technology has fallen to Pe

2

, and the existing 

technology loses market share to the new technology (Qn

0

=Qe

2

- Qe

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. The 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 technology 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 Figures 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 choice into the market and the effect of 

adoption of the new technology—could be explained. In reality, these two effects 

would be expected to take place at about the same time. That is, it would not be 

necessary to distinguish the separate changes in the demand curve De

0

 to De

1 to DB

ePB

2

P

instead it would be De

0 to De2

. 

The conclusion that can be drawn from this section is that farmers are 

unambiguously better off when a substitute technology is introduced into the market, 

whether the number of original 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. 

SSB-518-07 

Figure 2: The Effect of a New Technology on the Elasticity of Demand for an Existing Tec

Price/Unit 

gy 

Price/Un

Existing Technolo

it 

New Techno

Se0

De0

De1

Qe0

Pe0Pe1

Qe2 Qe1 Qe1*

Sn0

De2

Pe2

Qn0

Pn0

Quantity 

Agri-Analy
Agri-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 reproductive parts of the corn plant, 

especially on developing corn kernels. Recommended management of CEW in field 

corn is limited to planting resistant hybrids (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 usually 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 prices around $3.50 is equal 

to a loss of about $13.13 per acre. 

On the other hand, conventional management of the WBC is restricted to chemical 

pest control and scouting, along with fairly precise 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 (Peairs). 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 conducted 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 were randomly selected across the area of the High Plains 

and the Corn Belt. The 12 states involved 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 costs. Then a description of MIR162 was 

given to each respondent, and questions were asked about growers’ interest in the 

new technology. This section contains the 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 too low to be useful from a 

statistical standpoint, we decided to concentrate on the states of Nebraska, Iowa, 

and Illinois for the economic analysis of the survey data. These states each have a 

larger number of respondents with 27 in Nebraska, 32 in Iowa, and 31 in Illinois, 

respectively. 

SSB-518-07 Page 54 of 87 
Agri-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 all the states in the survey for completeness, 

even though some of the numbers 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, with an average over all respondents of about 

1,400 acres. Of those, the acres planted to corn tended to be about one-third to 

one-half of their total acres, with an average of 740 corn acres planted in 2007. Of 

the corn acres planted, the percent of those 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 hybrids 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 experience 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 87 
Agri-Analytics, Inc. 12 

Table 1. Descriptive statistics of survey respondents 

State 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

COLORADO 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

Pest Pressure and Mean Yield Losses Over Time

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 insect. Overall in the region, the number 

f CEW larvae per plant was relatively constant over the period 2002-2006 at about 

.25 larvae per plant. On the other hand, the 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 three states can be seen in Appendix B. 

otice that, when a linear coefficient was not statistically significantly 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 pressure 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 yield losses from the CEW and from the WBC 

over the 2002-2006 time period. Table 3 shows 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 slightly 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 these 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 reasonable 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 87 
Agri-Analytics, 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 detected. 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 significant 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 the fact that the WBC 

 spreading into Iowa and so the yield losses could be expected to become larger 

by the reported acres treated 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 estimated 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 estimated to be 4.54, about one-third bushel lower 

than the average yield loss due to WBC in Nebraska over the period. Although this 

finding is somewhat surprising, since the WBC is thought to be moving west to east, 

it is supported 

a
Agri-Analytics, Inc. 

Table 2. Pest pressure as reported by survey respondents by insect, state and yea

State

06 CEW 

larvae/corn 

plant

05 CEW 

larvae/corn 

plant

04 CEW 

larvae/corn 

plant

03 CEW 

larvae/corn 

plant

02 CEW 

larvae/corn 

plant

06 

larva

pl

0.03 0.03

0.33 0.52

0.40 0.55

0.09 0.35

0.38 0.290.10 0.050.19 0.030.25 0.000.29 0.33

. .

0.11 0.090.07 0.06

0.25 0.26

WBC

e/co

ant

W

e/

an

Number of Obs.

COLORADO 3 0.03 0.26 0.03 0.1 

ILLINOIS 31 0.35 0.26 0.35 0. 

INDIANA 11 0.51 0.35 0.48 0.1 

IOWA 32 0.25 0.25 0.15 0. 

KANSAS 6 0.30 0.07 0.06 0.1 KENTUCKY 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 0.29 0. 

OHIO 4 0.53 0.50 . 0. SOUTH DAKOTA 11 0.11 0.31 0.07 WISCONSIN 7 0.10 0.29 0.08 0. 

All States 150 0.28 0.24 0.22 0. 

rn 

05 WBC 

larvae/corn 

plant

04 

larva

pl

6 0.17

25 0.46

1 0.08

31 0.38

0 0.10

. .. 0.05. .

28 0.18

10 .

. 0.05

33 .

26 0.26

B

co

t

0

0

0

0

00

0

0

SSB-518-07 
Agri-Analytics, Inc. 

Table 3. Reported yield losses by insect, state and year 

State N

06 CEW 

yield loss

05 CEW 

yield loss

04 CEW 

yield loss

03 CEW 

yield loss

02 CEW 

yield loss

06 WBC 

yield loss

05 WBC 

yield loss

04 WBC 

yield loss

03 W

yield 

COLORADO 3 1.00 1.50 1.00 35.00 1.00 1.00 2.33 2.00 ILLINOIS 31 5.13 4.38 4.92 6.18 2.75 2.88 3.86 6.50 INDIANA 11 5.61 5.17 6.33 6.00 3.00 6.67 5.17 7.00 IOWA 32 2.79 3.71 4.00 3.50 4.00 4.46 4.11 4.56 KANSAS 6 3.33 3.00 1.50 2.76 3.20 3.00 3.00 1.50 KENTUCKY 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 . MISSOURI 7 1.00 1.00 5.25 7.50 0.00 . . . NEBRASKA 27 3.73 3.89 3.50 3.25 2.38 4.75 7.36 3.50 OHIO 4 11.00 3.00 . . . 20.00 . . SOUTH DAKOTA 11 1.33 2.83 1.75 2.50 1.70 . 10.00 2.50 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 

SSB-518-07 
Agri-Analytics, Inc. 

Figure 4: Nebraska Average Reported Yield Losses by Insect and Year 

y = 0.3339x + 2.3461

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0

Yield Loss 2002 Yield Loss 2004 Yield Loss 2005 Yield Loss 2006

Year

Yield Losses (Bu/acre) Corn Earwo

Western Bea

y = 4.8893

SSB-518-07 
Agri-Analytics, Inc. 

Figure 5: Iowa Average Reported Yield Losses by Insect and Year 

y = 0.6784x + 1.4418

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0

Yield Loss 2002 Yield Loss 2004 Yield Loss 2005 Yield Loss 2006

Year

Yield Losses (Bu/acre) Corn Earw

Western B

y=3.600

SSB-518-07 
tics, Inc. 

SSB-518-07 

Figure 6: Illinois Average Reported Yield Losses by Insect and Year 

Agri-Analy

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0

Yield Loss

2002

Yield Loss

2003

Yield Loss

2004

Yield Loss

2005

Yield Loss

2006

Year

Yield Losses (Bu/acre) Corn Ea

Western 

y = 4.540 y=4.670
Agri-Analytics, Inc. 19 

Measures and Predictions of Pest Pressure from Doane’s Agro-Trak Survey 

Data 

An important component of our analysis involved the development of models that 

could be used to predict the one-year-ahead levels of insect pressures at various 

locations in the region of interest. To do so, we used a simple model of 

expenditures on treatment options for insects in the relevant states included in the 

survey. These states included Colorado, Illinois, Indiana, Iowa, Kansas, Kentucky, 

Minnesota, Missouri, Nebraska, Ohio, South Dakota, and Wisconsin. The source for 

pesticide expenditures data was Doane’s Agro-Trak “Treatment for Selected Corn 

Insect-Pests”, which was supplied to us by Syngenta Corp. The Doane’s data report 

expenditures by pesticide brand 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, black 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 reported at the Crop Reporting District (CRD) level. Data 

were available for the 2005 and 2006 crop years. We also collected total (all 

practices) corn for grain acreage at the CRD level from the National Agricultural 

Statistics Service (NASS) website for the 2005 and 2006 crop years. Using the total 

expenditures on treatment (Grower $) and the total level 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 Price Index to place all expenditures on 

a 2006 equivalent basis. Our analysis is conducted at the county level, but assumes 

that the CRD-level average expenditures per acre apply to all counties within each 

CR

Our modeling goal was to use 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 expenditures to 2007 and then spatially 

smoothing the resulting predictions across space. Specifically, expenditures per 

acre for 2007 were predicted by adding the difference between the 2006 and 2005 

levels to the 2006 level. In cases where 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 acreage for two reasons. First, harvest 

acreage statistics from NASS tend to be more 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 where 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 87 
Agri-Analytics, Inc. 20 

surface that could be used to predict treatment costs over space and thus to 

simulate the spatial spread of the pest threat. 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 for pest pressures—the effect of having 

We considered spatial prediction 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 particular, we used a first-order (linear) local 

smoothing function.7

 For any CRDs (counties) that did not have reported treatment 

costs for a particular pest but did have treatment costs reported for some other pest, 

the treatment costs were assumed to be zero for the specific pest of interest.8 

The intuition of our smoothing 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 actual value. An example can be used to 

illustrate the intuition. Consider a county with costs of $0.50/acre that neighbors a 

county with zero expenditure. The smoothing 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 costs. 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 latitude 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—especially in the lower Corn Belt, Upper 

Ohio, and South Dakota areas. It is important 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 smoothed predictions of treatment costs that were derived 

from the local polynomial interpolation techniques. Note t

s

infestations in neighboring areas. 

Bean Cutworm and the Corn Earworm. Figure 9 presents actual treatment costs 

 

6

 Spatial smoothing was accomplished using the ArcMap package. 

7

 Higher ordered polynomials sometimes achieved lower RMSE values, but often 

bean cutworm were zero. 

resulted in negative predicted values. 

8

 For example, if treatment costs were reported for black cutworm in a county but 

nothing was reported for western bean cutworm, we assumed that treatment costs 

for western 

SSB-518-07 Page 64 of 87 
Agri-Analytics, Inc. 21 

SSB-518-07 Page 65 of 87 

and Figure 10 presents the spatially smoothed values for the Western Bean 

Cutworm. Localized infestations are apparent in the Corn Belt and Great Plains. 

Figures 11 and 12 present actual and spatially-smoothed values for treatment 

expenditures on the Corn Earworm. Again, the infestations are largely localized but 

appear to be much more widespread than was the case for the cutworm. The 

smoothed values encompass a wider area of infestation, again reflecting the spatial 

smoothing used to predict treatment costs. 

Based on these modeling efforts, it can be seen from Figures 10 and 12 that the 

Western Bean Cutworm and the Corn Earworm are becoming more widespread in 

the Corn Belt and the Great Plains. Because the existing insect resistant corn traits 

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 that the MIR162 corn trait will provide additional yield 

protection for an increasing number of corn growers. The additional value to 

growers of hybrids with the MIR162 trait, assuming those hybrids are priced 

competitively, likely will be substantial. 
Agri-Analytics, Inc. 

Figure 7. Actual Treatment Costs ($/harvested acre) for All Four Pests 

COST 0 - 0 0. 0042995595 - 0. 0856171898

62873 - 0. 3565925818

28731 - 0. 8534725759

05659 - 11. 670496507

0. 0856313131 - 0. 1774703892 0. 190210. 3721837634 - 0. 6388951243 0. 642820. 8590703006 - 1. 3649042254 1. 42569

SSB-518-07 
Agri-Analytics, Inc. 

Figure 8. Spatially Smoothed Treatment Costs ($/harvested acre) for All Four Pest

predi ct ed 0. 0000000 - 0. 0721639 0. 0727597 - 0. 2046207

0. 2048581 - 0. 3627214 0. 3632305 - 0. 54135910. 5417020 - 0. 7386988 0. 7387832 - 0. 99772270. 9984210 - 2. 1947410 2. 1968913 - 12. 5381956

SSB-518-07 
Agri-Analytics, Inc. 

Figure 9. Actual Treatment Costs ($/harvested acre) for Western Bean Cutworm 

COST 0 0. 0107894381 0. 0492197452

0. 1516375079 0. 1774703892

SSB-518-07 
Agri-Analytics, Inc. 

Figure 10. Spatially Smoothed Treatment Costs ($/harvested acre) for Western Bean Cu

predi ct ed 0. 00000000 - 0. 00000000 0. 00000840 - 0. 00017819

0. 00018299 - 0. 00069484 0. 00069683 - 0. 001909460. 00191861 - 0. 00493967 0. 00495576 - 0. 008663980. 00873603 - 0. 02349767 0. 02394071 - 0. 10829261

SSB-518-07 
Agri-Analytics, Inc. 

Figure 11. Actual Treatment Costs ($/harvested acre) for Corn Earworm 

COST 0 0. 0389944819 0. 0572607983 0. 085631

0. 0885097625 0. 1153105096 0. 3413251029 0. 421376

SSB-518-07 
tics, Inc. 

SSB-518-07 

Figure 12. Spatially Smoothed Treatment Costs ($/harvested acre) for Corn Earwor

predi ct ed 0. 00000000 - 0. 00000000 0. 00001795 - 0. 00057303

0. 00058543 - 0. 00216468 0. 00220720 - 0. 005511560. 00560493 - 0. 01278033 0. 01287585 - 0. 027191530. 02747421 - 0. 05439111 0. 05504717 - 0. 57128119

Agri-Analy
Agri-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 intentions 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-traited 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 reports 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]

KENTUCKY

NSIN

IS

A

RADO

 DA

ASKA

SOT

URI

AS

TATE

tes n

 inclu

ses.

SSB-518-07 Page 72 of 87 
Agri-Analytics, Inc. 29 

SSB-518-07 Page 73 of 87 

Potential Farm-Level Gain from the Introduction of MIR162-traited Hybrids 

The potential gain at the state level is calculated as: 

Total Corn Acres in Statei x Percent Acres Infested with Insectj [(Percent 

Acres Treated for Insectj 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 

that has been infested with the insects. Therefore, our calculations 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 calculation for only the three 

states that had a sufficient number of responses for statistical reliability. Table 5 

shows the values of the components used in the calculation 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 million 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 western 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 both pests. As the 

western bean cutworm moves east and populations 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, substantial. These results 

lend strong support for the trait’s success and the potential value to farmers. 

to the potential state-level gain. As the WBC, in 

rn Belt, our estimates would beco
tics, Inc. 

SSB-518-07 

Table 5: Components needed to calculate gain from MIR162 for selected states for 2005 a

Year IL EBR

re 

2005 23.24 84.42 3.17 8,500 0 14 $65, 2006 37.22 23.66 5.06 8,100 0 $0. 02, 

Aver. 30.23 54.04 4.12 8,300, 0 $7. 83, 

2005 2.16 25.65 22.33 8,500, 0 $0. $1 

2006 24.50 37.78 5.00 8,100 0 $0. 

Aver. 13.33 31.71 13.67 8,300, 0 $0. 

% Acres Treated n en 

e 

[A] C]

%

NE IL IA NE IL

Acres

,000 12,100,,000 11,300,

000 11,700,

000 12,100,

,000 11,300,

000 11,700,

Total Cor

W

[B]

IA NE

$/ac

00 12,800,000 $5.47 $00 12,600,000 $6.69

00 12,700,000 $6.08

00 12,800,000 $7.0300 12,600,000 $0.0000 12,700,000 $3.52

Acres Tream

stern Bean Cutworm

[

Corn Earworm

IA NE IL IA NE IL IA N

Bu/ac $/bu

.80 $0.00 4.38 3.89 0.00 $1.92 $2.08 $1.94

00 $5.63 5.13 3.73 0.00 $3.15 $3.35 $3.15 $1

40 $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 $3.15

00 $9.74 0.00 6.06 0.00 $2.54 $2.72 $2.55

t Cost Yield Loss Corn Price

[D] [E]

62

9

,2

$6

Table 6: Gain fro v 2 

Year NE

m MIR162 for selected states, alue of production, and percentage gain for 0

IL IA

66,429,83007,644,628 $3,584,4

37,037,229 $1,792,2

7,793,937 $44,150,7

11,883,529 $2,548,8

4,838,733 $23,349,8

om 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 % of 

ern Bean Cutworm

Corn Earworm

$ millions

IL

2005 $65,6 1 $0 4. 

2006 $102,2 1 37 1. Aver. $83,9 1 18 3. 2005 $1,2 13 33 3. 2006 1 72 1. 

Aver. $6 12 02 2. 

 fr V

West

%

7

8

2

9

89

a

08,452 $75,886 $

42,169 $

93,017 $

$0 $

46,509 $

Gain

Agri-Analy
Agri-Analytics, Inc. 31 

References 

Cook, K. and R. Weinzierl. 2004. Cor rworm (Heliothis zea). IPM integrated 

est management. University of Illinois rbana-Champaign. Available online at: 

/insects/corn_earworm/index.html

n Ea

p at U

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 Mathematical Approach. New 

Graw-Hill, Inc. 

SSB-518-07 Page 75 of 87 
Agri-Analytics, Inc. 32 

APPENDIX A

THE SURVEY QUESTIONNAIRE 

SSB-518-07 Page 76 of 87 
Agri-Analytics, Inc. 33 

MIR162 Value Questionnaire 

Hello, my name is ___________ with Doane Marketing Research, 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 observed 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 respondent to answer Q.2. through Q.9. based 

on the number of years reported in Q.1. 

SSB-518-07 Page 77 of 87 
Agri-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 Earworm 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 respondent 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 control Corn Earworm in (year)? 

 

es ________ No _______ 

.5.What is the product name of the insecticide 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 ounces, pints quarts, or pounds per acre did you use for this 

application of (product)? 

 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 

SSB-518-07 Page 78 of 87 
Agri-Analytics, Inc. 35 

Q.8. How many total applications of (product) did apply to control Corn Earworm in 

(year)? 

r of applications of (product)______ 

ects, if any were you also 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 year the respondent had a Corn Earworm 

estions about 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 did you find Western Bean Cutworm in your 

n? 

2004 

2002 

imate your Western Bean Cutworm infestation based on 1 

ry _____ corn plants 

tervie r note: If respondent 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 respondent 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 Bean Cutworm infestation based on how many 

larvae per corn plant you experienced

 2006, _______larvae per co

SSB-518-07 Page 79 of 87 
Agri-Analytics, Inc. 36 

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 insecticide 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______ or 

___ 

 

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 (product) did apply to control Western Bean 

utworm in (year)? 

r of applications of (product)______ 

ects, if any were you also 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 year the respondent had a Western Bean 

 ________________ 

 ________________ 

R

Cutworm infestation in question 1A. 

SSB-518-07 Page 80 of 87 
Agri-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 that provides superior control of Corn 

ty will be offered in combination with European corn borer 

ide tolerance traits. 

s vailable to you today at a competitive price 

ologies 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 your corn acres would you be likely to try 

d to the technology 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 yo

we ou trying to control

 16.a.1. Bt bran

_

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 resistance, and herbic

 Q.17. If thi new corn technology 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 87 
Agri-Analytics, Inc. 38 

Q.22. On how many of these ___ 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 87 
Agri-Analytics, Inc. 39 

APPENDIX B

REGRESSION RESULTS 

SSB-518-07 Page 83 of 87 
Agri-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.

SSB-518-07 Page 84 of 87 
Agri-Analytics, Inc. 41 

Table B.2 Regression results for Illinois 

Regression Statistics

Multiple R 0.3700

R Square 0.1369

Adjusted R Square -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 Square -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 Square -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 Square -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

SSB-518-07 Page 85 of 87 
Agri-Analytics, Inc. 42 

Table B.3 Regression results for Iowa 

Corn Earworm - Regression 1

Regression Statistics

Multiple R 0.6981

R Square 0.4873

Adjusted R Square 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

Adjusted R Square -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

Adjusted R Square 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 87 
Agri-Analytics, Inc. 43 

Table B.4 Regression results for Nebraska 

Corn Earworm - Regression 1

Regression Statistics

Multiple R 0.8879

R Square 0.7883

Adjusted R Square 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

Adjusted R Square -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

Adjusted R Square -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 87 
Courtesy 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 3 
Syngenta Biotechnology is submitting this Courtesy Copy of MIR162 maize and Vip3Aa 

protein technical reports to aid USDA scientists in their review of our petition for a 

determination of nonregulated status for 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 information that could be of value to our competitors. These same 

technical reports have been submitted 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 3 
Report: Author & Title Section Number 

CBI deleted 8 

CBI deleted 9 

CBI deleted 10 

CBI deleted 11 

CBI deleted 12 

CBI deleted 13 

CBI deleted 14 

Syngenta MIR162-USDA-2 Page 3 of 3