### Petition for the Determination of Nonregulated Status for **Insect -**  **Resistant and Herbicide** **- Tolerant**  **4114 Maize** We submit this petition under 7 CFR §340.6 to request that the Administrator, Animal and Plant Health Inspection Service, make a determination that the article should no longer be regulated under 7 CFR §340. Submitting Company: Pioneer Hi - Bred International, Inc. 7100 NW 62nd Avenue PO Box 1014 Johnston, IA 50131 - 1014 Submitted by: Natalie Weber, Registration Manager Pioneer Hi - Bred Intern ational, Inc. DuPont Agricultural Biotechnology DuPont Experimental Station, E353/305C

PO Box 80353 Wilmington DE 19880-0353 Telephone: 302 -695 -8160 Fax: 302 -695 -3075 Prepared by: Natalie Weber, Jennifer Tallman, Tracy Rood, Kent Brink, John Larsen, Sally Catron, Stephanie Kadlicko-Stare, Trevor Jones, Bonnie Hong, Lacy Elmore, and Holly Hettinger

OECD Unique Identifier: DP-ØØ4114

-3 Submission date : August 25, 2011 Revised S ubmission date: November 9, 2011 Revision to the latest date: December 3, 2012

**NO CBI**Pioneer Hi - Bred International, Inc. Page 2 of 25 8

4114 Maize **Table of Contents** List of Tables ................................ ................................ ................................ ................................ ... 4 List of Figures ................................ ................................ ................................ ................................ .. 7 Release of Information ................................ ................................ ................................ ................. 10 Certification ................................ ................................ ................................ ................................ ... 11 Summary ................................ ................................ ................................ ................................ ....... 12 Abbreviations, Acronyms, and Definitions ................................ ................................ ................... 16 **1. Rationale for the Development of 4114 Maize ................................**  **................................ .**  **19** 1-A. Basis for the Request for a Determination of Nonregulated Status under 7 CFR §340.6 .... 19 1- B. Similarity of 4114 Maize to Previously Approved 1507 and 59122 Maize ........................... 19 1-C. Purpose and Need for 4114 Maize ................................ ................................ ....................... 21 1- D. Prior Environmental Release and Submission s to Other Regulatory Age ncies ................... 23 1- E. Maize Crop Cultivation in the U.S. and Usage ................................ ................................ ...... 23 **2. Introduced Trait, Development of the Transgenic Line, and Characterization of Insertion and Expressed Products**  **................................ ................................**  **.................... 2** **8** 2 -A. The Biology of Maize ................................ ................................ ................................ ............ 28 2 - B. Description of DNA Used for Transform ation and Intended Phenotype of Introduced Proteins ................................ ................................ ................................ ...................... 28 2 -C. Description of Transformation, Selection, and Breeding ................................ ..................... 38 2 - D. Mol ecular Characterization of the I nsertion in 4114 Maize ................................ ................ 43 2 - E. Inheritance and Stability of the Inserted DNA ................................ ................................ ...... 75 2 - F. Concentrations of Cry1F, Cry34Ab1, Cry35Ab1, and PAT in 4114 Maize ............................. 77 **3. Potential**  **Allergenicity and Toxicity of 4114 Maize ................................**  **........................... 91**  3 -A. Allergenicity of Maize ................................ ................................ ................................ ........... 91 3 - B. Assessment of the Allergenicity of the Introduced Proteins in 4114 Maize ........................ 92 3 -C. Assessment of the Toxicity of the Protein s Expressed in 4114 Maize ................................ . 94 3 - D. Overall Conclusions on the Potential Allergenicity and Toxicity of 4114 Maize .................. 9 8 **4. Comp** **arative Assessment of 4114 Maize ................................**  **................................ .........**  **100** 4-A. Compositional Assessment ................................ ................................ ................................ . 101 4- B . Agronomic Performance and Field Observations ................................ ............................... 124Pioneer Hi - Bred International, Inc. Page 3 of 25 8

4114 Maize 4-C . Overall Conclusions to Com parative Assessment of 4114 Maize ................................ ...... 141 **5. Potential Environmental Impact of the Intr** **o duction of 4114 Maize**  **................................ 143**  5 -A. Potential for 4114 Maize to Have Altered Disease and Pest Susceptibilities or to Become Weedy or Invasive ................................ ................................ ................................ 143 5 - B. Potential for Gene Flow Between 4114 Maize and Sexually Compatible Wild Relatives ................................ ................................ ................................ .............................. 144 5 -C. Potential Environmental Impact of the Introduced Proteins in 4114 Maize ..................... 148 **6. Overall Conclusions**  **................................ ................................**  **................................ ........**  **16 4**  **Appendix 1. 4114 Maiz e USDA Release Permits and Notifications and Planted Acreage**  **...... 16** **5 Appendix 2.**  **Equivalency of the Proteins Produced in 4114 Maize to T** **hose Produced in 1507 and 59122 Maize**  **................................ ................................**  **........... 16** **8** 2.1 Overview and Summary ................................ ................................ ................................ ....... 168 2.2 Western Blot Analysis of the Introduced Proteins ................................ .............................. 168 2.3 Conclusions ................................ ................................ ................................ .......................... 1 69 2.4 Experimental Methods ................................ ................................ ................................ ......... 1 74 **Appendix 3. Materials and Methods for Genetic Characterization of 4114 Maize**  **................ 17** **7** 3.1. Southern Blot Characterization of 4114 Maize ................................ ................................ ... 17 7 3.2. Test Material ................................ ................................ ................................ ....................... 17 7 3.3. Control Material ................................ ................................ ................................ .................. 17 7 3.4. Reference Material ................................ ................................ ................................ ............. 17 7 3.5. Identification of the 4114 Maize Plants Used for Southern Blot Analysis .......................... 178 3.6. Genomic DNA Extraction ................................ ................................ ................................ .... 179 3.7. Quantification of Genomic DNA ................................ ................................ ......................... 179 3.8. Digestion of DNA for Southern Blot Analyses ................................ ................................ ..... 179 3.9. Electrophoretic Separation and Southern Transfer ................................ ............................ 1 80 3.10. DNA Probe Labeling for Southern Blot Hybridization ................................ ....................... 1 80 3.11. Probe Hybridization and Visualization ................................ ................................ .............. 1 80 3.12. Stripping of Probes and Subsequent Hybridizations ................................ ........................ 18 1 **Appendix 4. Materials and Methods for Segregat** **ion Analysis of Five Generations of 4114 Maize**  **................................ ................................**  **................................ .....................**  **18 2**  4.1. Greenhouse Experimental Design ................................ ................................ ...................... 18 2 4.2. Planting and Leaf Sample Collection ................................ ................................ ................... 18 2 4.3. Genotypic Analysis ................................ ................................ ................................ .............. 18 2 4.4. Phenotypic Analysis ................................ ................................ ................................ ............ 18 3Pioneer Hi - Bred International, Inc. Page 4 of 25 8

4114 Maize 4.5. Statistical Analysis ................................ ................................ ................................ ............... 18 3 **Appendix 5 .**  **Materials and Methods for Determination of Cry1F, Cry34Ab1, Cry35Ab1,**  **and PAT Protein Concentrations ................................**  **................................ .........................**  **1 8** **4** 5.1. Field Trial Experimental Design ................................ ................................ ........................... 184 5.2. Plant Material Collection, Shipping, Processing, and Storage ................................ ............ 184 5.3. Protein Concentration Determination ................................ ................................ ................ 18 6 5.4. Statistical Analysis ................................ ................................ ................................ ............... 188 **Appendix 6. Materials and Methods for Nutrient Composition ................................**  **........... 1** **9 2**  6.1. Field Trial and Experimental Design ................................ ................................ .................... 19 2 6.2. Plant Material Collection, Shipping, Processing, and Storage ................................ ............ 19 2 6.3. Nutrient Composition Analyses ................................ ................................ .......................... 19 3 6.4. Statistical Analyses ................................ ................................ ................................ .............. 19 8 **Appendix 7. Ma** **terials and Methods for Analysis of Agronomic Characteristics and Germination Evaluation**  **................................ ................................**  **................................ ......**  **20 2**  7.1. Germination and Dormancy Evaluation ................................ ................................ .............. 20 2 7.2. Agronomic Characteristics Evaluation ................................ ................................ ................ 205 **Appendix 8**  **. Field Insect a**  **nd Disease Observations ................................**  **............................ 21** **4 References**  **................................ ................................**  **................................ .........................**  **2 4**  **1**

**List of Tables** Table 1. Regulatory History of 1507, 59122, and 1507x59122 Maize in the U.S. ....................... 20 Table 2 . Description of Genetic Elements in the T- DNA Region of PHP27118 ............................ 32 Table 3. Generations and Comparators Used for Analysis of 4114 Maize ................................ .. 41 Table 4. Description of DNA Probes Used for Southern Blot Hybridization ................................ 46 Table 5. Pred icted and Observed Hybridizing Bands on Southern Blots; Bcl I Digest ................. 50 Table 6. Predicted and Observed Hybridizing Bands on Southern Blots; Hin d III Digest ............ 57 Table 7. Summary of Genotypic and Phenotypic Results for Segregating 4114 Maize ............... 76Pioneer Hi - Bred International, Inc. Page 5 of 25 8

4114 Maize **List of Tables (continued)** Table 8. Maize Growth Stage Descriptions ................................ ................................ .................. 78 Table 9. Mean Concentrations of Transgenic Proteins in 4114 Maize ................................ ........ 78 Table 10. Cry1F Concentrations (ng/mg Tissue Dry Weight) ................................ ....................... 81 Table 11. Cry34Ab1 Concentrations (ng/mg Tissu e Dry Weight) ................................ ................ 83 Table 12. Cry35Ab1 Concentrations (ng/mg Tissue Dry Weight) ................................ ................ 85 Table 13. PAT Concentrations (ng/mg Tissue Dry Weig ht) ................................ .......................... 87 Table 14. Summary of 4114 Maize Protein Concentrations as a Ratio of 1507, 59122, and/or 1507x59122 Maize ................................ ................................ ...................... 89 Table 15. Proximates in Maize Grain ................................ ................................ ......................... 104 Table 16. Vitamins in Maize Grain ................................ ................................ ............................. 106 Table 17. Minerals in Maize Grain ................................ ................................ ............................. 108 Table 18. Fatty Acids in Maize Grain ................................ ................................ .......................... 111 Table 19. Total Amino Acids in Maize Grain ................................ ................................ .............. 1 15 Table 20. Key Anti- Nutrients in Maize Grain ................................ ................................ ............. 119 Table 21. Key Secondary Plant Metabolites in Maize Grain ................................ ...................... 120 Table 22. Proximates and Minerals in Maize Forage ................................ ................................ . 122 Table 23. Description of Seed Germination Conditions ................................ ............................ 126 Table 24. Summary of Warm Germination Test Results ................................ ............................ 127 Table 25. Summary of Cold Germination Test Results ................................ .............................. 127 Table 26. Summary of Diurnal Germination Test Results ................................ .......................... 127 Table 27. Tetrazolium Chloride (TZ) Testing of Non -Germinated Seed ................................ ..... 128 Table 28. Site Locations for 2010 Field Trial Experiments ................................ ......................... 131Pioneer Hi - Bred International, Inc. Page 6 of 25 8

4114 Maize **List of Tables (continued)** Table 29. Agronomic Characteristics Measured ................................ ................................ ........ 133 Table 30. Experiment A: Summary of Agronomic Performance of 4114 Maize Across Eleven Locations ................................ ................................ ................................ .............. 135 Table 31. Experiment B: Summary of Agronomic Characteristics of 4114 Maize Across Six Locations ................................ ................................ ................................ .................... 138 Table 32 . 4114 Maize Estimated Margins of Exposure to Non -Target Organisms (Cry1F) ....... 154 Table 33. 4114 Maize Estimated Margins of Exposure to Non -Target Organisms (Cry34/35Ab1) ................................ ................................ ................................ ............................. 15 6 Table 2.1. Primary and Secondary Antibody Descriptions Used for Qualitative Western Blot Analysis ................................ ................................ .............................. 17 6 Table 5.1. Maize Growth Stage Descriptions ................................ ................................ ............. 18 5 Table 6.1. Methods for Compositional Ana lysis of 4114 Maize ................................ ................ 19 3 Table 7. 1. Description of Germination Test Classifications ................................ ....................... 203 Table 7.2 . Maize Growth Stage Descriptions ................................ ................................ ............. 207 Table 8.1. Observation s of Insects Present and Comparison Between 4114 Maize and Control ................................ ................................ .............................. 21 5 Table 8.2. Observati ons of Diseases Present and Comparison Between 4114 Maize and Control ................................ ................................ .............................. 23 1Pioneer Hi - Bred International, Inc. Page 7 of 25 8

4114 Maize **List of Figures** Figure 1. U.S. M aize Usage by Segment, 2010 ................................ ................................ ............. 24 Fig ure 2. U.S . Maize Fed by Ani m al Group, 1993 - 2010 ................................ ............................... 26 Fig ure 3 . Schemati c Diagram of Plasmid PHP27118 ................................ ................................ .... 30 Fi gure 4 . Schematic Diagram of the T- DNA Region from Plasmid PHP27118 ............................. 31 Figure 5 . Deduced Amino Acid Sequence of the Cry1F Protein ................................ ................... 36 Figure 6 . Deduced Amino Acid Sequence of the Cry34Ab1 Protein ................................ ............ 36 Figure 7 . Deduced Amino Acid Sequence of the Cry35A b1 Protein ................................ ........... 37 Figure 8 . Dedu ced Amino Acid Sequence of the PAT Protein ................................ .................... 37 Figure 9 . Sch ematic Diagram of the Development of 4114 Maize ................................ .............. 39 Fig ure 10 . Breeding Diagram for 4114 Maize and Generations Used for Analyses .................... 40 Figure 11. Plasmid Map of PH P27118 ................................ ................................ .......................... 44 Figure 12. Map of PHP27118 T- DNA ................................ ................................ ............................ 45 Figure 13. Map of the Insertion in 4114 Maize ................................ ................................ ............ 47 Figure 14. Southern Blot Analysis of the T3 Generation of 4114 Maize; Bcl I Digested DNA with ubiZM1 Promoter and ubiZM1 Intron Probes ................................ ....... 51 Figure 15. Southern Blot Analysis of the T3 Generation of 4114 Maize; Bcl I Digested DNA with cry1F and ORF25 Terminator Probes ................................ ..................... 52 Figure 16. Southern Blot Analysis of the T3 Generation of 4114 Maize; Bcl I Digested DNA with cry34Ab1 and pin II Terminator Probes ................................ .................. 53 Figure 17. Southern Blot Analysis of the T3 Generation of 4114 Maize; Bcl I Digested DNA with TA Peroxidase Promoter and cry35Ab1 Probes ................................ ..... 54 Figure 18. Southern Blot Analysis of the T3 Generation of 4114 Maize; Bcl I Digested DNA with 35S Promoter, pat, and 35S Terminator Probes ................................ ... 55Pioneer Hi - Bred International, Inc. Page 8 of 25 8

4114 Maize **List of Figures (continued)** Figure 19. Southern Blot Analysis of the T3 Generation of 4114 Maize; Hin d III Digested DNA with ubiZM1 Promoter and ubiZM1 Intron Probes ................................ .. 58 Figure 20. Southern Blot Analysis of the T3 Generation of 4114 Maize; Hin d III Digested DNA with cry1F and ORF25 Terminator Probes ................................ ................ 59 Figure 21. Southern Blot Analysis of the T3 Generation of 4114 Maize; Hin d III Digested DNA with cry34Ab1 and pin II Terminator Probes ................................ ............. 60 Figure 22. Southern Blot Ana lysis of the T3 Generation of 4114 Maize; Hin d III Digested DNA with TA Peroxidase Promoter and cry35Ab1 Probes ................................ 61 Figure 23. Southern Blot Analysis of the T3 Generation of 4114 Maize; Hin d III Digested DNA with 35S Promoter, pat, and 35S Terminator Probes ............................... 62 Figure 24. Southern Blot Analysis of the T2, T3, BC3F1 *3 and BC3F2 *2 Generations of 4114 Maize; Bcl I Digested DNA with cry1F Probe ................................ ................................ ... 64 Figure 25. Southern Blot Analysis of the T2, T3, BC3F1 *3 and BC3F2 *2 Generations of 4114 Maize; Bcl I Digested DNA with cry34Ab1 Probe ................................ ............................ 65 Figure 26. Southern Blot Analysis of the T2, T3, BC3F1 *3 and BC3F2 *2 Generations of 4114 Maize; Bcl I Digested DNA with cry35Ab1 Probe ................................ ............................ 66 Figure 27. Southern Blot Analysis of the T2, T3, BC3F1 *3 and BC3F2 *2 Generations of 4114 Maize; Bcl I Digested DNA with pat Probe ................................ ................................ ...... 67 Figure 28. Southern Blot Analysis of the T2, T3, BC3F1 *3 and BC3F2 *2 Generations of 4114 Maize; Bcl I Digested DNA with LB Probe ................................ ................................ ........ 69 Figure 29. Southern Blot Analysis of the T2, T3, BC3F1 *3 and BC3F2 * 2 Generations of 4114 Maize; Bcl I Digested DNA with spc Probe ................................ ................................ ....... 70 Figure 30. Southern Blot Analysis of the T2, T3, BC3F1 *3 and BC3F2 *2 Generations of 4114 Maize; Bcl I Digested DNA with tet Probe ................................ ................................ ....... 71 Figure 31. Southern Blot Analysis of the T2, T3, BC3F1 *3 and BC3F2 *2 Generations of 4114 Maize; Bcl I Digested DNA with virG Probe ................................ ................................ ..... 72 Figure 32. Southern Blot Analysis of the T2, T3, BC3F1 *3 and BC3F2 *2 Generations of 4114 Maize; Bcl I Digested DNA with RB Probe ................................ ................................ ....... 73Pioneer Hi - Bred International, Inc. Page 9 of 25 8

4114 Maize **List of Figures (continued)** Figure 33. Map of Locations for Agronomic Data Collection for 4114 Maize ........................... 131 Figure 34. Maize Planted Acres by County in the U.S. ................................ ............................... 146 Figure 2.1. Western Blot Comparison of Cry1F Protein ................................ ............................. 1 70 Figure 2.2. Western Blot Comparison of Cry34Ab1 Protein ................................ ...................... 17 1 Figu re 2.3. Western Blot Comparison of Cry35Ab1 Protein ................................ ...................... 17 2 Figure 2.4. Western Blot Comparison of PAT Protein ................................ ............................... 1 7 3Pioneer Hi - Bred International, Inc. Page 10 of 25 8

4114 Maize **Release of Information**  Pioneer Hi - Bred International, Inc. (Pioneer) is submitting the information in this petition for review by USDA as pa rt of the regulatory process. By submitting this information, Pioneer does not authorize its release to any third party except to the extent it is requested under the Freedom of Information Act (FOIA), 5 U.S.C., Section 552; USDA complies with the provisi ons of FOIA and USDA’s implementation regulations (7 CFR Part 1.4); and this informat i on is responsive to the specific request. Except in accordance with the Freedom of Information Act, Pioneer does not authorize the release, publication or other distrib ution of this information (including website posting) without Pioneer’s prior notice or consent.

© **2011 Pioneer Hi -** **Bred, International, A DuPont Business. All Rights Reserved.** This document is protected under copyright law. This document is for use only by USDA-APHIS and its contractors, and only in support of actions requested by Pioneer. Any other use of this material, without prior written consent of Pioneer, is strictly prohibited. By submitting this petition, Pioneer does not grant any party or entity any right or license to the information or intellectual property described in this document.Pioneer Hi - Bred International, Inc. Page 11 of 25 8

4114 Maize

**Certification** The undersigned certifies that, to the best knowledge and belief of the undersigned, this petition includes all info rmation and views on which to base a determination, and that it includes all relevant data and information known to the petitioners, which are favorable and unfavorable to the petition.

Date Natalie Weber, Registration Mana ger Pioneer Hi - Bred International, Inc. DuPont Agricultural Biotechnology DuPont Experimental Station, E353/305C PO Box 80353 Wilmington DE 19880-0353 Telephone: 302 -695 -8160 Fax: 302 -695 -3075Pioneer Hi - Bred International, Inc. Page 12 of 25 8

4114 Maize **Summary** Pioneer Hi - Bred International, Inc. , a DuPont Bu siness (Pioneer) is submitting a Petition for Determination of Nonregulated Status for insect- resistant and herbicide-tolerant maize event DP-ØØ4114

-3, hereafter referred to as 4114 maize. Maize line 4114 was developed by Pioneer. Pioneer requests a dete rmination from USDA - Animal and Plant Health Inspection Service (APHIS) that 4114 maize and any crosses of this line with other nonregulated Zea mays no longer be considered regulated articles under 7 CFR §340. 4114 maize produces the Cry proteins Cry1F, Cry34Ab1, and Cry35Ab1, as well as the herbicidetolerance protein PAT. The Cry1F protein confers resistance to certain lepidopteran pests, including European corn borer ( Ostrinia nubilalis), a major maize pest. This protein and its associated genetic elements are identical to those in DAS-Ø 15 Ø 7- 1 maize (hereafter referred to as 1507 maize ), which was deregulated by USDA, registered by EPA, and reviewed by FDA in 2001. The Cry34Ab1 and Cry35Ab1 proteins together comprise an active binary insecticidal c rystal protein that confers resistance to corn rootworm pests, including western corn rootworm (Diabrotica virgifera virgifera), also a major maize pest. This binary protein and the associated genetic elements are identical to those in DAS-59122 -7 maize ( hereafter referred to as 59122 maize ), which was deregulated by US DA in 2005, registered by EPA since 2005, and reviewed by FDA in 2004. Finally, t he PAT protein confers tolerance to the herbicidal active ingredient glufosinate-ammonium at current labeled rates . This protein is identical to the protein found in a number of approved events across several different crops that are currently in commercial use, including 1507 and 59122 maize; maize containing the PAT protein has been commercially grown in the U.S. since 1996. 1507 maize, 59122 maize, and the breeding stack of the two lines, 1507x59122 maize, were joint ly developed by Pioneer and Dow AgroSciences and are now licensed broadly across the seed industry. I n 2010, commercial products containing 1507x59122 maize were grown on approximately 14 million acres or approximately 16% of U.S. maize acres . 4114 maize is a new transformation event that, if deregulated, will provide an alternative to the breeding stack combination of two previously approved events: 1507 maize, which expresses the Cry1F and PAT proteins, and 59122 maize, which expresses the Cry34Ab1, Cry35Ab1, and PAT proteins . The 1507x59122 maize breeding stack combination was reviewed and registered by EPA in 2005. 4114 maize will provide similar insect resistance and herbicide tolerance to that of 1507x59122 maize. I n 4114 maize, the genes for the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins a re contained on a single transformation construct and have been integrated at a single locus in t he genome; this is in contrast to the 1507x59122 maize where the insertions for the events are located at two unlinked loci. As a new event with all genes located at a single locus, 4114 maize will be bred more efficiently into new product offerings for g rowers that are customized to their local insect protection and agronomic needs. Efficient breeding of multiple traits in single commercial maize products is becoming more important as growers demand more complex products, including multiple modes of acti on for l epidopteran and corn rootworm insect resistance and tolerance to one or more classes of herbicides.Pioneer Hi - Bred International, Inc. Page 13 of 25 8

4114 Maize 4114 maize was developed by Agrobacterium- mediated transformation using the DNA plasmid vector PHP27118. The T- DNA region of PHP27118 contains t he cry1F, cry34Ab1, cry35Ab1, and pat genes . These gene cassettes are identical to those found in 1507 and 59122 maize, based on sequencing of the 4114 maize insertion, and the translated proteins were found to be identical. F urthermore, western blot ana lysis demonstrated similar molecular weight and immunoreactivity of the proteins . Therefore, identity of the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins in 4114 maize to those in 1507 and 59122 maize was confirmed . Molecular characterization of the 4114 maize insertion by Southern blot analysis confirmed that a single, intact copy of the T- DNA of PHP27118 has been inserted into the maize genome and that the insertion is stable during the traditional breeding process. Southern blot analysis verified the absence of plasmid backbone DNA. Segregation analysis of five breeding generations of 4114 maize confirmed the stability and Mendelian inheritance of the insertion. The concentrations of the introduced Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins were me asured in a number of 4114 maize tissues and were compared to the concentrations in the respective 1507, 59122, and/or 1507x59122 maize tissues. In general, t hese comparisons revealed that 4114 maize tissues have similar or lower concentrations of the int roduced proteins than those of 1507, 59122, and/or 1507x59122 maize. Exceptions were Cry1F and Cry34Ab1 concentrations in senescent tissue and also Cry1F concentrations in pollen, where concentrations in 4114 maize were higher. In general, senescent tiss ue can contain a variable degree of extractable protein and likely accounts for the variability in the comparison. Overall, these comparisons indicate that any previously conducted safety studies that used 1507, 59122, and 1507x59122 maize are applicable to 4114 maize. Safety studies that considered exposure to the Cry1F protein from 1507 or 1507x59122 pollen would require the reassessment of results for 4114 maize using the higher concentration. The safety of the introduced proteins in 4114 maize has be en previously evaluated by regulatory agencies in the U.S., as referenced above, and there is a history of safe use and exposure. The Cry1F, Cry34Ab1, and Cry35Ab1 proteins were derived from Bacillus thuringiensis and the PAT protein was derived from Streptomyces viridochromogenes . B. thuringiensis and S. viridochromogenes are naturally occurring soil bacteria and are not pathogenic; therefore, animals and humans are regularly exposed to these organisms and their components without adverse consequences. The potential for allergenicity and toxicity of 4114 maize was evaluated by examining the allergenic potential of maize as a crop and by assessing the allergenic and toxic potential of the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins. M aize is not a comm on allergenic food and the modification in 4114 maize is not expected to alter the allergenic potential of maize. The Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins have been assessed previously for 1507 and 59122 maize and have been determined to be unlikel y to be potential allergens or toxins to humans and animals. P revious assessments of these proteins included bioinformatic analyses, digestibility studies, and acute protein toxicity studies and are relevant for the assessment ofPioneer Hi - Bred International, Inc. Page 14 of 25 8

4114 Maize 4114 maize. U pdated bioi nformatic analyses support the original co nclusions that the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins are unlikely to be allergens or toxins. These data support the conclusion that the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins in 4114 maize are therefore safe for the food and feed supply. Th ese data ha ve been submitted to EPA to confirm the safety of these proteins and will be submitted to the FDA for the ir food and feed safety assessment of 4114 maize. Comprehensive agronomic performance assessments for 4114 maize were conducted in replicated field studies at a total of 17 locations in the U.S. and Canada . These assessments were conducted independently of the intended effects of the introduced traits (i.e., trait efficacy) so that appropriate compa risons would be made to conventional maize and any unintended effects due to the 4114 maize insertion would be evaluated . The following c haracteristics were measured : early population, final population, time to silking, time to pollen shed, pollen viabil ity (measured at six locations), seedling vigor, stalk lodging, root lodging, stay green, disease incidence, insect damage, plant height, ear height, and yield (measured at 11 locations) . Seed germination and dormancy data were also collected in laboratory experiments. Analysis of these data showed no statistically significant differences between 4114 maize and control maize lines, indicating the agronomic comparability of 4114 maize to conventional maize. In addition, 4114 maize has been field tested si nce 2006 in the U.S. and Puerto Rico. A ll releases in the U.S. have occurred under field permits and notifications granted by USDA - APHIS. All field trials of 4114 maize were observed for naturally occurring insects or diseases, and no unexpected differences between 4114 maize and control were observed. Together, t hese data support the conclusion that 4114 maize is unlikely to pose a greater plant pest risk than conventional maize . Extensive nutrient composit ion analyses of grain (60 analytes) and fo rage (nine analytes) were conducted to compare the composition of 4114 maize to that of a control maize line and eight commercial maize varieties. These analyses were used to evaluate any changes in the levels of key nutrients, anti- nutrients an d secondary metabolites. Based on the results of the compositional evaluation, the grain and forage of 4114 maize are comparable to commercially available maize and there would be no significant impact on raw or processed maize commodities . A long with the agronomi c data included in this petition, compositional comparability is a general indicator that 4114 maize will not exhibit unexpected effects with respect to plant pest risk. The potential environmental impact of the introduction of 4114 maize considered t hree primary areas: the potential for 4114 ma ize to become weedy or invasive; the potential for gene flow to sexually compatible wild relatives; and the potential impacts of the introduced Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins. Comparative analyses i ndicated that 4114 maize was comparable to conventional maize with respect to the nutrient composition and agronomic characteristics measured and independently of the introduced traits. In general, m aize does not possess weediness characteristics and is n ot considered a weed y or invasive species. Therefore, 4114 maize does not exhibit any characteristics that would indicate it is any more likely than conventional maize to become a weed or plant pest. The potential for gene flowPioneer Hi - Bred International, Inc. Page 15 of 25 8

4114 Maize examined maize pollination biology and the hybridization potential and geographic overlap of maize wild relatives. W hile maize does possess some pollination characteristics favorable to gene flow, the distribution of wild relative populations are limited in the U.S. and there is l ow fitness or sterility of hybrids; therefore, it is unlikely that the inserted DNA in 4114 maize would be introgressed significantly in to these wild relative populations . As referenced earlier, 4114 maize contains the same introduced proteins as those present in 1507, 59122, and/or 1507x59122 maize and has protein concentrations that are similar to or lower than these lines; therefore, previously conducted environmental safety studies for these lines were relevant for evaluating the environmental impact of 4114 maize cultivation . Based on the expected environmental exposure and the sufficient margin of safety of the proteins from 4114 maize, these environmental studies indicated that there is a low risk of the Cry1F and Cry34/35Ab1 proteins to non -target orders. A review of m ultiple field studies conducted for 1507, 59122, and/or 1507x59122 maize provide support that the abundance of non -target organisms would not be impacted by the cultivation of 4114 maize . In addition , certain threatened and endange red Lepidoptera and Coleoptera are unlikely to be adversely impacted from 4114 maize cultivation based on the lack of habitat overlap. N utritional and toxicological studies on 1507, 59122, and/or 1507x59122 maize also support that 4114 maize will not adve rsely impact non -target vertebrates . Therefore, it is unlikely that 4114 maize will pose a risk to non -target organisms including beneficial, th reatened and endangered species, as well as non -target vertebrates including birds, mammals, and humans . In su pport of this conclusion, 1507 maize has been commercially available in the U.S. since the 2003 growing season and 59122 and 1507x59122 maize ha ve been available in the U.S. since 2006 with no negative safety or environmental effects. Taken together, it i s unlikely that 4114 maize will pose a plant pest risk or impact non -target organisms in the environment. In conclusion, based on the data contained herein, Pioneer requests that APHIS grant the request for a determination of nonregulated status for 4114 maize and any crosses of this line with other nonregulated Zea mays .Pioneer Hi - Bred International, Inc. Page 16 of 25 8

4114 Maize **Abbreviations, Acronyms, and Definitions** ~ approximately %DB percent dry basis 1507 maize maize line containing the DAS-Ø15Ø7

- 1 event 1507x59122 maize maize line containing the breeding stack DAS-Ø15Ø7

- 1xDAS-59122 -7 32D78 Pioneer® commercial hybrid line 3394 Pioneer® commercial hybrid line 33G26 Pioneer® commercial hybrid line 33J24 Pioneer® commercial hybrid line 34A15 Pioneer® commercial hybrid line 34M94 Pioneer® commercial hybrid line 34P88 Pioneer® commercial hybrid line 35K02 Pioneer® commercial hybrid line 35T06 Pioneer® commercial hybrid line 35T36 Pioneer® commercial hybrid line 36M28 Pioneer® commercial hybrid line 37H24 Pioneer® commercial hybrid line 37Y12 Pio neer® commercial hybrid line 38B85 Pioneer® commercial hybrid line 4114 maize maize line containing the DP -ØØ4114

-3 event 59122 maize maize line containing the DAS-59122-7 event AACC American Association of Cereal Chemists ADF acid detergent fiber AOAC Association of Official Analytical Chemists AOSA Association of Official Seed Analysis APHIS Animal and Plant Health Inspection Service of USDA Bcl I restriction enzyme from Bacillus caldolyticus Bt Bacillus thuringiensis bp base pair CaMV cauliflower mosaic virus CCUR Center for Crops Utilization Research CFR Code of Federal Regulations CI confidence interval CMH Cochran - Mantel - Haenszel test cry1F gene encoding the Cry1F protein from Bacillus thuringiensis var. aizawai C ry1F protein en coded by the cry1F gene from Bacillus thuringiensis var. aizawai cry34Ab1 gene encoding the Cry34Ab1 protein from Bacillus thuringiensis strain PS149B1 C ry34Ab1 protein encoded by the cry34Ab1 gene from Bacillus thuringiensis strain PS149B1 cry35Ab1 gen e encoding the Cry35Ab1 protein from Bacillus thuringiensis strain PS149B1 C ry35Ab1 protein encoded by the cry35Ab1 gene from Bacillus thuringiensis strain PS149B1 CS compound symmetry CT threshold cycle df degree s of freedom DI deionized DIG digoxygenin DNA deoxyribonucleic acid DT50 time for 50% dissipationPioneer Hi - Bred International, Inc. Page 17 of 25 8

4114 Maize **Abbreviations, Acronyms, and Definitions (continued)** E. coli Escherichia coli EEC expected environmental concentration E score expectation score ELISA enzyme linked immunosorbent assay EPA Environmental Protection Agency EUP experimental use permit FAME fatty acid methyl ester FARRP Food Allergy Research and Resource Program FDA Food and Drug Administration FDA CFSAN Food and Drug Administration’s Center for Food Safety and Applied Nutrition FDR false discovery rate FOIA Freedom of Information Act GC/FID gas chromatography with flame ionization detection GLMM Generalized Linear Mixed Model GNAT GCN 5 - related N-acetyltransferases Hae III Restriction enzyme from Haemophilus aegyptius HFCS high -fructose corn syrup Hin d III Restriction enzyme from Haemophilus influenzae HPLC high performance liquid chromatography HRP horseradish peroxidase ICP-OES inductively coupled plasma optical emission spectroscopy IgE i mmunoglobulin E kb kilobase pair kDa kilodalton KR Kenward - Roger LB Left Border of the T- DNA LC50 lethal concentration at which 50% of a test population are affected LLOQ lower limit of quantification LS- Mean Least Squares Mean LTP lipid tra nsfer protein ML maximum likelihood MOE margin of exposure MS mass spectrometry NCBI National Center for Biotechnology Information NCGA National Corn Growers Association NDF neutral detergent fiber NOEC no observed effect concentration OD optical density OECD Organisation for Economic Co-operation and Development ORF open reading frame pat gene encoding the PAT ( phosphinothricin acetyltransferase ) protein from Streptomyces viridochromogenes PAT phosphinothricin acetyltransferase protein from Streptomyces viridochromogenes PBST phosphate buffered saline plu s Tween a

-20

a Registered trademark of ICI Americas, Inc.Pioneer Hi - Bred International, Inc. Page 18 of 25 8

4114 Maize **Abbreviations, Acronyms, and Definitions (continued)** PCR polymerase chain reaction PH09B Pioneer proprietary maize inbred PH1B5 Pioneer proprietary maize inbred PHNA R Pioneer proprietary maize inbred PHR03 Pioneer proprietary maize inbred PHTFE Pioneer proprietary maize inbred PHWWE Pioneer proprietary maize inbred; used for initial transformation to produce 4114 maize PIR Protein Information Resource PR pathogen esis- related PRF Protein Research Foundation pin II proteinase inhibitor II RB Right Border of the T- DNA REML Residual Maximum Likelihood SAS Statistical Analysis Software SDS- PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis SGF sim ulated gastric fluid SIF simulated intestinal fluid spc spectinomycin resistance gene ssp. subspecies T- DNA portion of the Agrobacterium transformation plasmid between the Left and Right Borders that is expected to be transferred to the plant tet tetracycline resistance gene TZ tetrazolium chloride ubiZM1 Zea mays polyubiquitin gene UPLC ultra performance liquid chromatography USDA United States Department of Agriculture USDA-APHIS BRS United States Department of Agriculture-Animal and Plant Hea lth Inspection Service, Biotechnology Regulatory Services USDA- ERS Economic Research Service of the U.S. Department of Agriculture USDA- NASS National Agricultural Statistics Service of the U.S. Department of Agriculture USDA- NCRS United States Departmen t of Agriculture - Natural Resources Conservations Service UTR untranslated region UV ultraviolet var. variety wwPDB Worldwide Protein Data Bank * Abbreviations of units of measurement and of physical and chemical quantities are used according to the standard format described in Instructions to Authors in the Journal of Biological Chemistry ( http://www.jbc.org/)Pioneer Hi - Bred International, Inc. Page 19 of 25 8

4114 Maize **1. Rationale for the Development of 4114 Maize** 4114 maize (OECD Unique Identifier DP-ØØ4114

-3 ) is a new event that has been transformed with a single genetic construct containing each of the proteins found in the previously approved maize events, DAS-Ø 15 Ø 7- 1 (1507 maize; Cry1F and PAT proteins) and DAS-59122-7 (59122 maize; Cry34Ab1, Cry35Ab1, and PAT protei ns) and has been developed to provide an alternative to the 1507x59122 maize breeding stack for more complex stack combinations. 4114 maize is not intended to be a stand -alone commercial product and will be combined with other approved events using conven tional breeding to create stacked products with multiple modes of action for control of pest insects and with tolerance to one or more classes of herbicides . As part of a complex breeding stack, 4114 maize will have similar insect resistance and herbicide tolerance benefits as those containing the combination of 1507 and 59122 maize, and will have added breeding advantages over the available 1507x59122 maize . 1507, 59122, and 1507x59122 maize contain familiar traits and are currently licensed b roadly across the seed industry; i n 2010, commercial products containing 1507x59122 maize were grown on approximately 14 million acres or approximately 16% of U.S. maize acres (GfK Kynetec, 2010 ). Maize is the largest crop grown in the U.S. in terms of acreage and n et value. M aize has multiple downstream uses for feed, fuel, and food that are significant for the U.S. and global supply. The introduction of new maize trait offerings that meet grower needs, such as stacked products containing 4114 maize, is critical to help keep pace with i ncreasing maize demand in the U. S . and globally. **1 -** **A. Basis for the Request for a Determination of Nonregulated Status under 7 CFR** §**340.6** The Animal and Plant Health Inspection Service (APHIS) of the U.S. Department of Agriculture (USDA) has responsibility, under the Plant Protection Act (7 U.S.C. 7701-7772) and the Plant Quarantine Act (7 U.S.C. 151- 167), to prevent the introduction or dissemination of plant pests into or within the U.S . P art 340 regulates introduction of organis ms altered or produced through genetic engineering which are plant pests or for which there is a reason to believe are plant pests. The APHIS regulations at 7 CFR § 340.6 provide that an applicant may petition APHIS to evaluate submitted data on the genetically engineered crop to determine that a regulated article does not present a plant pest risk and therefore should no longer be regulated. Pioneer Hi - Bred International, Inc. is submitting data for genetically engineered insect- resistant and herbicide-tol erant 4114 maize and requests a determination from USDA - APHIS that event DP-ØØ4114

-3 and any crosses with other Zea mays no longer be considered regulated articles under 7 CFR §340. **1 -**  **B. Similarity of**  **4114 Maize to Previously Approved 1507 and 59122 Ma ize**  As mentioned earlier, 4114 maize is a new event with all genes at a single locus that will provide an alternative to 1507x59122 maize. 4114 maize contains each of the trait proteins found inPioneer Hi - Bred International, Inc. Page 20 of 25 8

4114 Maize the previously approved 1507 maize ( Cry1F and PAT proteins) and 59122 maize ( Cry34Ab1, Cry35Ab1, and PAT proteins). Both 1507 and 59122 maize and their combination 1507x59122 maize are fully approved by U .S. regulatory a gencies (Table 1). 1507, 59122, and 1507x59122 maize products have been commercially availa ble in the U.S. since 2003, 2006, and 2006, respectively. 4114 maize has the same introduced genetic material and proteins as 1507x59122 maize, and has similar agronomic properties, protein expression, and efficacy to the previously approved events; t herefore, portions of previous regulatory data and analyses of 1507, 59122 and 1507x59122 are relevant to 4114 maize and will be cited in this petition. Furthermore, 1507, 59122, and 1507x59122 maize were jointly developed by Pioneer and Dow AgroSciences; these commercial lines contain familiar traits and are currently licensed broadly across the seed industry. In 2010, commercial products containing 1507x59122 maize were grown on approximately 14 million acres, which represents approximately 16% of U.S. maize acres ( GfK Kynetec, 2010) .

**Table 1. Re gulatory History of 1507, 59122, and 1507x59122 Maize in the U.S.**  **Product Agency**  **Approval Date a**  1507 maize USDA August 14, 2001 FDA May 18, 2001 b EPA August 10, 2001; expires September 30, 2015 59122 maize USDA October 7, 2005 FDA October 4, 2004 b EPA August 31, 2005; expires September 30, 2015 1507x59122 maize USDA Not Applicable c FDA Not Applicable c EPA October 27, 2005; expires September 30, 2015 a I nformation accessed on U.S. regulatory a gen cy websites (EPA, 2010a; EPA, 2010b; EPA, 2011a; FDA, 2001; FDA, 2004; USDA-APHIS, 2001; USDA-APHIS, 2005). b Indicates completion date of biotechnology consultation. c No review of 1507x59122 maize was required by USDA or FDA as the approved individual events were combined by conventional breeding.Pioneer Hi - Bred International, Inc. Page 21 of 25 8

4114 Maize **1 -** **C . Purpose and Need for 4114 Maize**  Through the use of 4114 maize in new stacked product offerings, there will be similar insect resistance and herbicide tolerance benefits as those of 1507x59122 maize . Additionally, there are added breeding advantages of using a single transformation event in these products over the available 1507x59122 maize breeding stack, as described further below. **Benefits of Insect Resistance and Herbicide Tolerance of 4114 Maiz e**  The use of 4114 maize in commercial products will provide similar insect and weed control benefits to those of 1507x59122 maize. 4 114 maize contains the same Cry1F, Cry34Ab1, and Cry35Ab1 proteins as 1507x59122 maize and will provide growers with a sim ple, inexpensive, highly effective, and environmental ly benign means of controlling l epidopteran and corn rootworm insects, including European corn borer ( Ostrinia nubilalis (Hubner)) and western corn rootworm ( Diabrotica virgifera virgifera) . Both Europe an corn borer and the corn rootworm species complex ( Diabrotica ssp .) are major maize insect pests throughout the U.S.; monetary losses resulting from feeding damage and insect control for each pest exceed $1 billion each year (Gray et al. , 2009; Ostlie et al. , 2002 ). Similar to the benefits of 1507 and 59122 maize, the use of 4114 maize containing the Cry1F, Cry34Ab1, and Cry35Ab1 proteins has the potential to offer effective control of maize insect pests and a reduction in the use of highly toxic agricul tural pesticide chemicals to control these insect pests (EPA, 2010a; EPA, 2010b ). The Cry1F, Cry34Ab1, and Cry35Ab1 proteins also offer crop yield advantage under insect pressure (EPA, 2010a; EPA, 2010b) . I n general, for corn borer- and corn rootworm - protected maize varieties, a 5% yearly average yield advantage compared to conventional maize has been realized in the U.S. since these varieties were cultivated (Brookes and Barfoot, 2009; Brookes and Barfoot, 2010) . In contrast to the chemical pesticides used to control insect pests, the Cry1F, Cry34Ab1, and Cry35Ab1 proteins do not pose risks to humans or to the environment (EPA, 2010a; EPA, 2010b) . As a result, 4114 maize would offer similar benefits as 1507 and 59122 maize -- increased worker safety and grower profits through reductions in the use of a number of highly toxic and expensive chemical pesticide control programs (EPA, 2010a; EPA, 2010b) . I n general, farm income has increased as a result of use of insect- protected maize ( Brookes and Barfoot, 2010 ). In addition, 4114 maize also contains the PAT protein that confers tolerance to the herbicidal active ingredient glufosinate-ammonium at current labeled rates . Glufosinate was registered as an herbicide in 1993 in the U.S. and is currently und er re- review at EPA (CERA, 2002; EPA, 2011b) . G lufosinate-ammonium tolerance will allow growers to proactively manage weed populations and , in a proper herbicide rotation program, delay the development of adverse populations of weeds.Pioneer Hi - Bred International, Inc. Page 22 of 25 8

4114 Maize **Benefits of 4114 M aize as a Replacement Option for 1507x59122**  **Maize** As discussed previously, 4114 maize will provide an alternative to 1507x59122 maize in new complex stacked product offerings. Many maize products will be stacked through traditional breeding with multiple conventional and genetically modified traits to meet evolving grower needs of insect, weed, disease, and abiotic stress management . As a single event replacing a breeding stack with separate unlinked segregating events, 4114 maize will potentially benefit growers by reducing the development time for new products and by increasing product offerings that meet grower and trait durability needs. The option to use 4114 maize, which contain s all genes at a single breeding locus, will reduce the number of breed ing loci over 1507x59122 maize, thus increasing the speed at which new products will be available to growers. This is because the complexity and expense of breeding multiple traits or events into one maize product increases with each breeding locus added. For example, the effort required to breed two transgenic loci into an inbred line is double the effort required for one transgenic locus. F urthermore, each trait locus must be homozygous in an inbred line and as more loci are combined, the proportion of plants that are homozygous for each locus becomes smaller, resulting in more seed discard during the breeding process. With the ability to efficiently create stacked products containing additional traits, both transgenic and native, there is an opportu nity to enhance product durability and to provide custom offerings that better address grower regional needs . G rowers are requiring sophisticated stacked products that include insect resistance (i.e. , Bacillus thuringiensis or Bt traits ) and herbicide tol erance, as well as native traits such as disease tolerance, drought tolerance, and higher yields. For Bt traits, the maize seed industry is transitioning from products with a single mode of action to products with multiple modes of action, in order to extend the durability of the traits that many growers rely on to manage pests. Therefore, it is important that multipl e native and transgenic traits -- including Bt traits with different modes of action -- are bred into a single maize product to efficiently meet the needs of growers. 4 114 maize, as one component of future Bt breeding stack products with multiple modes of action, will provide benefits to growers in the form of multi-trait products with enhanced durability and more diverse germplasm suited for diverse growing regions. 4114 maize is expected to be more efficiently bred into a wide variety of maize genetic backgrounds and with additional traits, thus giving growers more choice in products that offer in - plant insect protection and that are custom ized to their local growing areas and agronomic needs. U ltimately, these offerings will allow growers to continue to increase their overall farm productivity to meet the food security needs of a growing global population.Pioneer Hi - Bred International, Inc. Page 23 of 25 8

4114 Maize **1 -**  **D .**  **Prior Environmental Relea se and**  **Submissions to Other Regulatory Agencies** As a new transformation event, 4114 maize is regulated by the United States Department of Agriculture-Animal and Plant Health and Inspection Service, Biotechnology Regulatory Services (USDA-APHIS BRS) and th e United States Environmental Protection Agency (EPA). 4 114 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. 4114 maize has been extensively field tested in the U.S. and Puerto Rico since 2006 in over 180 separate plantings as authorized by the USDA-APHIS permits and notifications listed in Appendix 1. N o Experimental Use Permit (EUP) a pplications for 4114 maize were submitted to the EPA. 4 114 maize has been grown in small -scale field tests under the 10-acre per pest EUP exemption 40 CFR §172.3(c)(1) . A n application for a seed increase registration for 4114 maize was submitted to EPA on April 18, 2011. Seed increase registrations are used by EPA to authorize breeding and testing of events such as 4114 maize that will not be sold commercially without being stacked with other insect resistant traits . Upon EPA registration , 4114 maize will have county and national acreage limitations imposed by the EPA and will be prohibited from commercial use on its own. F uture commercial breeding stacks of 4114 maize with other EPA- regulated Plant- Incorporated Protectant events are subject to EPA’s regulatory authority and will require full registrations prior to commercialization. A voluntary safety and nutritional assessment of 4114 maize will also be submitted to the FDA’s Center for Food Safety and Applied Nutrition ( FDA CFSAN) in the third quarter of 2011 . Regulatory submissions will be made in key U.S. maize export markets . These countries include Canada, Japan, Mexico, Taiwan, South Korea , and China . A full commercial launch of any products containing 4114 maize will only occur after obtain ing all necessary authorizations in the U.S. and key import countries with functioning regulatory processes . **1 -**  **E. Maize Crop Cultivation in the U.S.**  **and Usage** M aize is the largest crop grown in the U.S. in terms of acreage and net value. Maize has mult iple downstream uses for feed, fuel, and food that are significant for U.S. and global supply. In 2010, over 12 billion bushels of maize were produced in the U.S. from approximately 81 million harvested acres with a crop value of $65.97 billion (NCGA, 201 1 ; USDA- NASS, 2011 ). The U.S. is a major global exporter of maize at approximately 55% of the total trade market (NCGA, 2011). The largest maize U.S. export markets in 2009- 2010 were Japan, Mexico, South Korea, Taiwan, Egypt, and Canada (NCGA, 2011 ) . Ex ports accounted for 14.5% of the maize produced in 2010 (Figure 1; NCGA, 2011 ).Pioneer Hi - Bred International, Inc. Page 24 of 25 8

4114 Maize The introduction of new maize trait offerings that meet grower needs, such as stacked products containing 4114 maize, is critical to help keep pace with increasing maize deman d in the U.S. and globally. As ignificant portion of maize cultivated in the U.S. is genetically modified and contains similar insect resistant traits. I n 2010, 86% of maize grown in the U.S. was genetically modified ; insect resistant varieties accounted for approximately 63% of all maize acreage, which includes the percentage of insect resistant traits as well as stacked varieties ( USDA- NASS, 2010 ). Over the past decade, maize yields and overall production have increased, in part due to improvements in seed varieties and agronomic production practices (NCGA, 2011; USDA- ERS, 2009). Some background on maize processing and use for feed, fuel, and food industries is described below.

**Figure 1. U.S. Maize Usage by Segment, 2010**  HFCS: High -fructose co rn syrup Source: NCGA, 2011Pioneer Hi - Bred International, Inc. Page 25 of 25 8

4114 Maize **Maize Processing**  **for Feed ,**  **Fuel, and Food Uses**  Maize grain requires processing for some downstream uses. Wet and dry milling processes are used to separate grain into components for food, feed , and fuel processing (OECD, 2002). Wet milling starts with softening the kernel in hot water and sulfur dioxide prior to further fractionation and processing (OECD, 2002). Products produced in the wet milling process include germ meal, oil (further processed into margarine, cooking oil, baking and frying fats), corn gluten feed, corn gluten meal, and starch (further processed into ethanol and sweeteners) (OECD, 2002) . There are several means of dry milling maize grain, but by far the most widely used process begins with soaking t he kernel in water to remove the pericarp and germ, followed by drying the remaining grain fraction before additional processing (OECD, 2002). Products produced in the dry milling process include flour, meal, germ meal, oil, beverage and fuel ethanol, distillers dried solubles, flaking grits, hominy feed, and grits (OECD, 2002) . Maize grain may also be cooked in alkali and fine ly ground to produce what is known as masa, which is used for tortillas and snack chips (OECD, 2002). The production of fuel et hanol typically begins with dry milling of maize grain, cooking, saccharification, and fermentation to produce ethanol and the by- product distiller dried grains or solubles (OECD, 2002). **Feed Use of Maize** The largest proportion, 38.7%, of maize produced in the U.S. is used for animal feed (Figure 1; NCGA, 2011). Of the maize grain that is used for feed, the greatest percentage is consumed by beef cattle, followed by poultry, p ork, and dairy cattle (Figure 2; NCGA, 2011 ). An umber of different products from the maize plant and from grain processing may be used as feed. The whole maize plant or its residue from harvesting are frequently used as animal feed. Silage, derived from the above-ground portions of the maize plant, is an important feed ingredie nt for feedlot and dairy cattle and preserves more than 90% of nutrients (OECD, 2002) . In 2009, a pproximately 6% of the U.S. maize crop wa s used for silage ( USDA- NASS, 2010 ). In addition, stalks from harvested maize plants can be grazed by ruminants in t he field (OECD, 2002) . Maize ears, without shelling (i.e. , removing the grain from the cob), can be ground directly for ruminant feed (OECD, 2002). W hen ears are shelled to remove the grain, remnant cobs can also be used in animal feed (OECD, 2002). M ai ze grain can be fed to animals with minimal processing and can be fed whole, rolled, ground, or steam flaked (OECD, 2002) . Rolled orPioneer Hi - Bred International, Inc. Page 26 of 25 8

4114 Maize ground grain is fed to swine and poultry (OECD, 2002). M aize grain added to pet foods is ground, cooked, and pelleted or extruded (OECD, 2002). Processed products from the milling and ethanol fermentation processes are also fed to livestock. A by- product of the wet milling process, corn gluten meal, is fed to ruminants, poultry, and swine (OECD, 2002) . The ethanol fermentation process produces a co- product called distillers dried grains/solubles or corn gluten feed that is used as animal feed to dairy and beef cattle, poultry, and swine ( USDA- ERS, 2009; USDA- ERS, 2010; USDA- NASS, 2007 ).

**Figure 2. U.S. Maize Fed**  **by Animal Group, 1993 -** **201 0**  Source: NCGA, 2011Pioneer Hi - Bred International, Inc. Page 27 of 25 8

4114 Maize **Fuel Use of Maize** Maize is the primary feedstock used to produce ethanol in the U.S.; 36.5% of maize grain produced is fermented into fuel ethanol ( NCGA, 2011 ; USDA- ERS, 2010 ) . I n 2009, ethanol represented 8% of motor vehicle gasoline supplies and is expected to remain below 10% of annual gasoline use by 2015 (USDA- ERS, 2010 ) . **Food Use of Maize** Starch, oil, grits, bran, meal, and flour from maize wet and dry milling are primarily used in foods (OECD, 2 002). Am ajority of starch is converted to sweeteners, such as corn syrup, high fructose corn syrup, maltodextrins, and dextrose, and also fermented into ethanol (OECD, 2002). As ignificant portion of U.S. maize, 3.8%, goes to the production of high -fructose corn syrup as an end product (Figure 1; NCGA, 2011). A pproximately 6.3% (classified in the “other” usage segment) comprises food purposes such as starch, sweeteners, cereal/other, and beverage alcohol (Figure 1; NCGA, 2011). Starch is used for food such as bakery products/mixes, condiments, candies, and prepared (snack, dessert, meat) foods ( CCUR, 2009 ). Sweeteners are used for soft drinks, candies, bakery products/mixes, condiments (jams, jellies, dressings), and prepared foods ( CCUR, 2009 ). Whole maize is consumed as popcorn, sweet corn, and alkali processed grain for tortilla s and snack chips ( CCUR, 2009 ), though these uses comprise a very minor usage segment .Pioneer Hi - Bred International, Inc. Page 28 of 25 8

4114 Maize **2 .**  **Introduced Trait, Development of the Transgenic Line, and Characterization of In sertion**  **and Expressed Products** Characterization of the transgenic crop variety provides additional knowledge about the genetic modification and the trait as a starting point for the safety assessment. K nowledge about the transgenic crop in these areas provides background information for the food, feed, and environmental safety assessments and, if applicable, can also identify certain areas of greater potential risk or concern. This section provides an overall characterization of 4114 maize and includes information about the maize crop, the source of the donor genetic elements, the identity and intended function of the expressed proteins, the development of the 4114 maize line, the structure and genetic stability of the DNA insertion, and the tissue-spec ific concentration of the expressed proteins. The information below provides background for safety assessments in later sections . **2 -** **A. The Biology of**  **Maize** Biology documents on the unmodified plant species have been published by the Organization for Economic Co-operation and Development (OECD) (OECD, 2003). Th e OECD document on maize provides background on the biology of Zea mays including :

 general description, including information on use of maize as a crop plant;  taxonomic status of Zea;  identificat ion methods;  center of origin / diversity and maize diversity;  reproductive biology, including sexual and asexual reproduction;  crosses, including intra - and inter-specific crosses and gene flow; and  agro-ecology, including information about cultivation, volunteers and weediness, soil ecology, and maize- insect interactions. **2 -**  **B. Description**  **of DNA**  **Used for**  **Transformation and**  **Intended Phenotype**  **of Introduced**  **Proteins Identity and Source of Genetic Material in the T** **- DNA**  **Region of PHP27118**  4114 maize was produced by Agrobacterium tumefaciens- mediated transformation with PHP2 7118 ( Figure 3 ), which contains the cry1F , cry34Ab1, cry35Ab1

, and pat expression cassettes (Figure 4 ). A summary of the genetic elements and their position i n the T- DNA region is give n in Table 2 . As described earlier, 4114 maize is a new transformation event that contains the same gene cassettes as in previously registered 1507 and 59122 maize; these cassette comparisons are presente d in Table 2. The first cassette contains a truncated version of the cry1F gene from Bacillus thuringiensis var. aizawai. The expression of the cry1F gene is controlled by the maize polyubiquitin promoterPioneer Hi - Bred International, Inc. Page 29 of 25 8

4114 Maize (Christensen et al., 1992), providing constitutive expression of the Cry1F protein in maize. This region also includes the 5’ untranslated region (UTR) and intron associated with the native polyubiquitin promoter. The terminator for the cry1F gene is the polyadenylation signal from Open Reading Frame 25 (ORF 25) of the Agrobacterium tumefaciens Ti plas mid pTi15955 (Barker et al., 1983). The second cassette contains the cry34Ab1 gene isolated from Bacillus thuringiensis strain PS149B1, which has been codon optimized for expression in plants (Ellis et al., 2002; Herman et al., 2002; Moellenbeck et al., 2001). The expression of the cry34Ab1 gene is controlled by a second copy of the maize polyubiquitin promoter with 5’ UTR and intron (Christensen et al., 1992). The terminator for the cry34Ab1 gene is the 3’ terminator sequence from the proteinase inhib itor II gene of Solanum tuberosum (pin II terminator) (An et al., 1989; Keil et al., 1986). The third gene cassette contains the cry35Ab1 gene, also isolated from Bacillus thuringiensis strain PS149B1, which has been codon optimized for expression in plant (Ellis et al., 2002; Herman et al., 2002; Moellenbeck et al., 2001). The expression of the cry35Ab1 gene is controlled by the Triticum aestivum (wheat) peroxidase promoter and leader sequence (Hertig et al., 1991). The terminator for the cry35Ab1 gene i s a second copy of the pin II terminator (An et al., 1989; Keil et al., 1986). The fourth and final gene cassette contains the phosphinothricin acetyl transferase (pat) gene from Streptomyces viridochromogenes , which has been codon optimized for expression in plants . Expression of the pat gene is controlled by the promoter and terminator regions from the Cauliflower Mosaic Virus (CaMV) 35S transcript (Franck et al., 1980; Odell et al., 1985; Pietrzak et al., 1986).Pioneer Hi - Bred International, Inc. Page 30 of 25 8

4114 Maize

**Figure 3 . Sc** **hematic Diagram of Plas mid PHP27118**  Schematic diagram of plasmid PHP27118 with genetic elements indicated. P lasmid size is 54910 bp. **vir**

**C1 vir**  **C2 vir**  **G vir**  **B cry** **1F**

**cry 34A** **b1 cry**  **35A b1**  **pat**

**spc**

**tet R**  **tet A**  **trf A**  **ctl ubi**  **ZM1 Intron**  **Right Border**

**Left Border**

**ubi ZM1 Promoter** 

**TA Peroxidase**  **Promoter CaMV 35S Promoter**  **colE1 ori** 

**Col E1 ori**  **oriV ORF25 Terminator**  **pin II Terminator** 

**CaMV 35S Terminator ubi**  **ZM1 5 ’**  **UTR cos** 

**cos ubi**  **ZM1 Promoter ubi**  **ZM1 5 ’**  **UTR**

**ubi**

**ZM1 Intron**  **pin II Terminator** 

**oriT**Pioneer Hi - Bred International, Inc. Page 31 of 25 8

4114 Maize

**Figure 4** **. S** **chematic Diagram of the T -**  **DNA R egion from Plasmid PHP27118**  Schematic diagram of the T- DNA indicating the cry1F, cry34Ab1, cry35A b1, and pat genes along with their respective regulatory elements. The size of the T- DNA is 11978 bp. **cry 1F**  **cry 34Ab1** 

**cry**

**35Ab1 pat**  **ubi ZM1**  **Intron ubi**  **ZM 1**  **Intron**

**Right Border Left Border**  **ubi ZM1 Promoter**  **ubi ZM1 Promoter**  **TA Peroxidase**  **Promoter**

**CaMV 35S Promoter ORF25 Terminator**  **pin II Terminator**  **pin II Terminator** 

**CaMV 35S Terminator ubi**  **ZM1 5 ’**  **UTR ubi**  **ZM1 5 ’**  **UTR**Pioneer Hi - Bred International, Inc. Page 32 of 25 8

4114 Maize **Table 2**  **. Descrip tion of Genetic Elements in the T** **- DNA**  **Region of PHP27118**  **Comparison to Approved**  **Event Location on** 

**PHP27118 T** **- DNA**  **(b p**  **position) Geneti**  **c**

**Element Size** 

**(bp) Description**  1 to 25 Right Border 25 T- DNA Right Border region from Ti plasmid of Agrobacterium tumefaciens 26 to 43 Ti Plasmid

Region 18 Non -functional sequence from Ti plasmid of Agrobacterium tumefaciens 44 to 114 Polylinker

Region 71 Region required for cloning genetic elements

cry1F gene cassette in 4114 maize and 1507 maize 115 to 1014 ubiZM1 Promoter 900 Promoter region from Zea mays polyubiquitin gene (Christensen et al. ,

1992) 1015 to 1097 ubiZM1 5’ UTR 83 5’ untranslate d region from Zea mays polyubiquitin gene (Christensen et al. ,

1992) 1098 to 2107 ubiZM1 Intron 1010 Intron region from Zea mays polyubiquitin gene (Christensen et al. ,

1992) 2108 to 2129 Polylinker

Region 22 Region required for cloning genetic elements 2130 to 3947 cry1F Gene 1818 Truncated version of the cry1F gene from Bacillus thuringiensis var. aizawai 3948 to 3992 Polylinker

Region 45 Region required for cloning genetic elements 3993 to 4706 ORF 25

Terminator 714 Terminator sequence from the Agrobacterium tumefaciens pTi15955 ORF 25 (Barker et al. , 1983) 4707 to 4765 Polylinker

Region 59 Region required for cloning genetic elementsPioneer Hi - Bred International, Inc. Page 33 of 25 8

4114 Maize **Table 2 . Descripti**  **on of Genetic Elements in the T -**  **DNA Region**  **of PHP27118 (continued) Comparison**  **to Approve d**  **Event Location on** 

**PHP27118 T** **- DNA**  **(bp position) Genetic** 

**Element Size** 

**(bp) Description** 

cry34Ab1 gene cassette in 4114 maize and 59122 maize 4766 to 5665 ubiZM1 Promoter 900 Promoter region from Zea mays polyubiquitin gene (Christensen et al. , 1992) 566 6 to 5748 ubiZM1 5’ UTR 83 5’ untranslated region from Zea mays polyubiquitin gene (Christensen et al. ,

1992) 5749 to 6758 ubiZM1 Intron 1010 Intron region from Zea mays polyubiquitin gene (Christensen et al. ,

1992) 6759 to 6786 Polylinker

Region 28 Region required for cloning genetic elements

6787 to 7158 cry34Ab1 Gene 372 Codon -optimized version of the cry34Ab1 gene encoding the 14 kDa delta endotoxin parasporal crystal protein from the nonmotile strain PS149B1 of Bacillus thuringiensis (Ellis et al., 2002; Herman et al., 2002; Moellenbeck et al., 2001) 7159 to 7182 Polylinker

Region 24 Region required for cloning genetic elements 7183 to 7492 pin II

Terminator 310 Terminator region from Solanum tuberosum proteinase inhibitor II gene (An et al. , 1989; Keil et al., 1986) 7493 to 7522 Polylinker

Region 30 Region required for cloning genetic elementsPioneer Hi - Bred International, Inc. Page 34 of 25 8

4114 Maize **Table 2 . Descripti**  **on of Genetic Elements in the T -**  **DNA Region**  **of PHP27118 (continued) Comparison**  **to Approved Event**  **Location on**

**PHP27118 T** **- DNA**  **(b p**  **p osition)**  **Genetic**

**Element Size** 

**(bp) Description** 

cry35Ab1 gene cassette in 4114 maize and 59122 maize 7523 to 8820 TA Peroxidase Promoter 1298 Promoter from Triticum aestivum (wheat) peroxidase including leader sequence (Hertig et al. , 1991) 8821 to 8836 Polylinker

Region 16 Region required for cloning genetic elements

8837 to 9988 cry35Ab1 1152 Codon -optimized version of the cry35Ab1 gene encoding a 44 kDa delta -endotoxin parasporal crystal protein from the nonmotile strain PS149B1 of Bacillus

thuringiensis (Ellis et al., 2002; Herman et al., 2002; Moellenbeck et al., 2001) 9989 to 10012 Polylinker

Region 24 Region required for cloning genetic elements 10013 to 10322 pin II

Terminator 310 Terminator region from Solanum tuberosum proteinase inhibitor II gene (An et al. , 1989; Keil et al., 1986) 10323 to 10367 Polylinker

Region 45 Region required for cloning genetic elements

pat gene cassette in 4114 maize, 1507 maize and 59122 maize 10368 to

10897 CaMV

35S Promoter 530 35S promoter from Cauliflower Mosaic Virus (Franck et al. , 1980; Odell et al. , 1985; Pietrzak et al., 1986) 10898 to 10916 Polylinker

Region 19 Region required for cloning genetic elements 10917 to 11468 pat Gene 552 Codon -optimized phosphinothricin acetyltransferase gene from Streptomyces viridochromogenes . 11469 to 11488 Polylinker

Region 20 Region required for cloning genetic elements 11489 to 11680 CaMV35S

Terminator 192 35S terminator from Cauliflower Mosaic Virus (Franck et al. , 1980; Pietrzak et al.,

1986) 11681 to 11874 Polylinker

Region 194 Region required for cloning genetic elements 11875 to 11953 Ti Plasmid

Region 79 Non -functional sequence from Ti plasmid of Agrobacterium tumefaciens 11954 to 11978 Left Border 25 T- DNA Left Border region from Ti plasmid of Agrobacterium tumefaciensPioneer Hi - Bred International, Inc. Page 35 of 25 8

4114 Maize **Activity and Function of Expressed Proteins in 4114 Maize** The insertion of the cry1F gene in 4114 maize confers resistance to plant damage by certain l epidopteran pests. The Cry1F protein is comprised of 605 amino acids and has a m olecular weight of approximately 68 kDa (Figure 5 ). The Cry1F protein expressed in 4114 maize is identical to the one expressed in 1507 maize that has been deregulated by USDA, registered by EPA, and reviewed by FDA in 2001 (Table 1 ) . The cry34Ab1 and cry35Ab1 genes, isolated from the common soil bacterium Bacillus thuringiensis strain PS149B1, produce the proteins Cry34Ab1 and Cry35Ab1. The Cry34Ab1 protein is 123 amino acid residues in length and has a molecular weight of approximately 14 kDa ( Figure 6 ). The full - length Cry35Ab1 protein has a length of 383 amino acids and a molecular weight of approximately 44 kDa ( Figure 7 ). The Cry34Ab1 and Cry35Ab1 proteins together comprise an active insecticidal crystal protein (i.e., the binary Cry34/35Ab1 protein), that confers resistance to certain corn rootworm pests. This binary protein is identical to the one expressed in 59122 maize deregulated by USDA in 2005, registered by EPA since 2005, and reviewed by FDA in 2004 (Table 1 ) . Cry proteins (i.e., d elta -endotoxins), including the Cry1F and Cry34/35Ab1 proteins expressed in 4114 maize, act by selectively binding to specific sites localized on the lining of the midgut of susceptible insect species (de Maagd et al. , 2003; Sc hnepf et al. , 1998) . Followi ng binding, pores are formed that disrupt midgut ion flow, causing gut paralysis and eventual death due to bacterial sepsis (Bravo et al. , 2007) . The Cry1F and Cry34/35Ab1 proteins are lethal only when eaten by the larvae of certain lepidopteran or coleopteran insects, respectively, and their specificity of action can be attributed to the presence of their respective binding sites in the unique environment of the target insect midgut that is required for their activity (Chambers et al. , 199 1; de Maagd et al. , 2003; Ellis et al. , 2002; Hua et al. , 2001; Moellenbe ck et al. , 2001) . There are no binding sites for the delta -endotoxins of B. thuringiensis on the surface of mammalian intestinal cells and the mammalian digestive system environment does not support the steps required for toxicity of these proteins, therefore, livestock animals and humans are not susceptible to these proteins (Siegel, 2001) . The pat gene expresses the phosphinothricin acetyl transferase enzyme (PAT) that confers tolerance to glufosin ate ammonium, the active ingredient in phosphinothricin herbicides. The PAT protein is 183 amino acids residues in length and has a molecular weight of approximately 2 1 kDa ( Figure 8 ). This protein is identical to the protein found in a number of approved events across several different crops that are currently in commercial use, i ncluding 1507 and 59122 maize; maize containing the PAT protein ( e.g., T25) has been commercially grown in the U.S. since 1996. Glufosinate chemically resembles the amino aci d glutamate and acts to inhibit an enzyme, called glutamine synthetase, which is involved in the synthesis of glutamine. Glutamine synthetase is also involved in ammonia detoxification. Due to its similarity to glutamate, glufosinate blocks the activity of glutamine synthetase, resulting in reduced glutamine levelsPioneer Hi - Bred International, Inc. Page 36 of 25 8

4114 Maize and a corresponding increase in concentrations of ammonia in plant tissues, leading to cell membrane disruption and cessation of photosynthesis resulting in plant death. The PAT protein expressed in 4114 maize acetylates glufosinate to N -acetylglufosinate. This action prevents the inhibition of glutamine synthetase and therefore the plant is able to survive applications of herbicides containing glufosinate at current labeled rates .

1 ME NNIQNQCV PYNCLNNPEV EILNEERSTG RLPLDISLSL TRFLLSEFVP 51 GVGVAFGLFD LIWGFITPSD WSLFLLQIEQ LIEQRIETLE RNRAITTLRG 101 LADSYEIYIE ALREWEANPN NAQLREDVRI RFANTDDALI TAINNFTLTS 151 FEIPLLSVYV QAANLHLSLL RDAVSFGQGW GLDIATVNNH YNRLINLIHR 201 YTKHCLDTYN QGLENLRG TN TRQWARFNQF RRDLTLTVLD IVALFPNYDV 251 RTYPIQTSSQ LTREIYTSSV IEDSPVSANI PNGFNRAEFG VRPPHLMDFM 301 NSLFVTAETV RSQTVWGGHL VSSRNTAGNR INFPSYGVFN PGGAIWIADE 351 DPRPFYRTLS DPVFVRGGFG NPHYVLGLRG VAFQQTGTNH TRTFRNSGTI 401 DSLDEIPPQD NSGAPWNDYS HVLNHVTFVR WP GEISGSDS WRAPMFSWTH 451 RSATPTNTID PERITQIPLV KAHTLQSGTT VVRGPGFTGG DILRRTSGGP 501 FAYTIVNING QLPQRYRARI RYASTTNLRI YVTVAGERIF AGQFNKTMDT 551 GDPLTFQSFS YATINTAFTF PMSQSSFTVG ADTFSSGNEV YIDRFELIPV 601 TATLE* **Figure 5. Ded**  **uced Amino Acid Sequence of th e Cry1F Protein**  Deduced amino acid sequence from translation of the cry1F gene from plasmid PHP27118. The Cry1F protein is 605 amino acids in length, has a molecular mass of approximately 68 kDa , and is identical to the one expressed in 1507 maize . The a sterisk (*) indicates the translational stop codon.

1 MSAREVHIDV NNKTGHTLQL EDKTKLDGGR WRTSPTNVAN DQIKTFVAES 51 NGFMTGTEGT IYYSINGEAE ISLYFDNPFA GSNKYDGHSN KSQYEIITQG 101 GSGNQSHVTY TIQTTSSRYG HKS* **Figure 6.**  **De duced Amino Acid Sequence of the Cry34** **Ab1 Protein** Deduced amino acid sequence from translation of the cry34Ab1 gene from plasmid PHP27118. The Cry34Ab1 protein is 123 amino acids in length, has a molecular mass of approximately 14 kDa , and is identical to the one exp ressed in 59122 maize . Th e asterisk (*) indicates the translational stop codon.Pioneer Hi - Bred International, Inc. Page 37 of 25 8

4114 Maize

1 MLDTNKVYEI SNHANGLYAA TYLSLDDSGV SLMNKNDDDI DDYNLKWFLF 51 PIDDDQYIIT SYAANNCKVW NVNNDKINVS TYSSTNSIQK WQIKANGSSY 101 VIQSDNGKVL TAGTGQALGL IRLTDESSNN PNQQWNLTSV QTIQLPQKPI 151 IDTKLKDY PK YSPTGNIDNG TSPQLMGWTL VPCIMVNDPN IDKNTQIKTT 201 PYYILKKYQY WQRAVGSNVA LRPHEKKSYT YEWGTEIDQK TTIINTLGFQ 251 INIDSGMKFD IPEVGGGTDE IKTQLNEELK IEYSHETKIM EKYQEQSEID 301 NPTDQSMNSI GFLTITSLEL YRYNGSEIRI MQIQTSDNDT YNVTSYPNHQ 351 QALLLLTNHS YEEVEEITNI PK STLKKLKK YYF* **Figure 7. D** **educed Amino Acid Sequence of the Cry35Ab1 Protein** Deduced amino acid sequence from translation of the cry35Ab1 gene from plasmid PHP27118. The Cry35Ab1 protein is 383 amino acids in length, has a molecular mass of approximately 44 kDa , and is identical to the one expressed in 59122 maize . The asterisk (*) indicates the translational stop codon.

1 MSPERRPVEI RPATAADMAA VCDIVNHYIE TSTVNFRTEP QTPQEWIDDL 51 ERLQDRYPWL VAEVEGVVAG IAYAGPWKAR NAYDWTVEST VYVSHRHQRL 101 GLGSTLYT HL LKSMEAQGFK SVVAVIGLPN DPSVRLHEAL GYTARGTLRA 151 AGYKHGGWHD VGFWQRDFEL PAPPRPVRPV TQI* **Figure 8. De** **duced Amino Acid Sequence of the PAT Protein** Deduced amino acid sequence from translation of the pat gene from plasmid PHP27118. The PAT protein is 183 amino acids in length and has a molecular mass of approximately 21 kDa. This protein is identical to the protein found in a number of approved events across several different crops that are currently in commercial use. The asterisk (*) indicates the translational stop codon.Pioneer Hi - Bred International, Inc. Page 38 of 25 8

4114 Maize **Equivalency of Expressed Proteins in 4114 Maize and Previously Approved**  **Events** The Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins expressed in 4114 maize are identical to the proteins expressed in previously approved 1507, 59122, and/or 1507x59122 maize. This equivalency was established using the following criteria:

 The same gene expression cassettes (promoters, protein coding sequences, and terminators) were used in 4114 maize as in 1507 and 59122 maize, so all genetic elements are identical (Table 2) .  The DNA insertion in 4114 maize was sequenced and the translated amino acid sequences of the encoded Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins were determined. The translated amino acid sequences were compared and found to be i dentical to the amino acid sequences of those proteins in 1507 and 59122 maize (Figures 5 through 8). Therefore, the proteins in 4114 maize are also expected to be identical to the proteins in the breeding stack of 1507x59122 maize .  Western blot analysis demonstrated the Cry1F, Cry34Ab1, Cry35Ab1, and PAT protei ns in 4114 maize migrate with equivalent molecular weight and similar relative immunoreactivity to the proteins expressed in 1507x59122 maize, indicating eq uivalency (Appendix 2).  Bioequivalency of the expressed Cry1F and Cry34/35Ab1 proteins was established via side- by-side efficacy testi ng of 4114 maize with 1507x59122 maize in the field (data not shown; submitted to EPA) . Therefore, previously submitted safety data for Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins are also applicable for 4114 maize. **2 -** **C . Description of Transformation, Selection, and Breeding**  Pioneer proprietary inbred line PHWWE was transformed with plasmid PHP27118 to produce 4114 maize. Immature embryos of maize (Zea mays L. ) l ine PHWWE were aseptically removed from the developing caryopsis nine to eleven days after pollination and inoculated with Agrobacterium tumefaciens strain LBA4404 containing plasmid PHP27118, essentially as described in Zhao et al., 2001. Agrobacterium tumefaciens strain LBA4404 is a disarmed strain that does not contain tumor- inducing factors, however with the inclusion of plasmid PHP27118 the strain will contain factors (i.e. , the vir genes ) that enable the transfer of the T- DNA region to the inoculated host plant. After three to six days of embryo and Agrobacterium co-cultivation on solid culture medium with no selection, the embryos were then transferred to a medium without herbicide selection but containing carbenicillin for selection against Agrobacterium . After three to five days on this medium, embryos were then transferred to selective medium that was stimulatory to maize somatic embryogenesis and contained bialaphos for selection of cells expressing the pat transgene. The medium also contained carbenicillin to select against any remaining Agrobacterium . After six to eight weeks on the selective medium, healthy, growing calli that demonstrated resistance to bialaphos were identified.Pioneer Hi - Bred International, Inc. Page 39 of 25 8

4114 Maize Plants that were regenerated from transformation and tissue culture (designated T0 plants) were selected for further characterization by molecular analyses, herbicide and insect efficacy, and agronomic evaluations. Refer to Figure 9 for a schematic overview of the transformation and event development process for 4114 maize. The subsequent breeding of 4114 maize proceeded as indicated in Figure 10 to produce specific generations for the characterization and assessments conducted , as well as for the development of commercial lines . Table 3 in dicates the breeding generations used for each of the analyses described in this submission.

Assembly of plasmid PHP27118 containing cry1F, cry34Ab1, cry35Ab1, and pat gene cassettes in the T- DNA

**↓** Transformation of Pioneer proprietary inbred PHWWE line using Agrobacterium tumefaciens strain LBA4404 with PHP27118 **↓** Selection of transformation events based on tolerance to bialaphos

Regeneration of T0 plants

**↓** Molecular characterization and evaluati on of T0 plants **↓** Nursery evaluation of agronomic performance and insect/herbicide efficacy of subsequent generations **↓** Multiple location herbicide/insect efficacy and agronomic trials **↓** Selection of event DP-ØØ4114

-3 as the commercial candidate **↓** Continued field and laboratory studies to support product registration Continued breeding and testing of 4114 maize for product development **Figure 9 .**  **Schematic Diagram of the Development of**  **4114 M aize** Pioneer Hi - Bred International, Inc. Page 40 of 25 8

4114 Maize

**Figure 10 . Breeding Diagram for 4114 Maize and Generati** **ons Used for Analyses** The breeding steps to produce the generations used for characterization, assessment, and the development of commercial lines are shown schematically . Pioneer proprietary inbred PHWWE was used for transformation to produce 4114 maize . Other Pioneer proprietary inbreds P H1B5, PHR03, PHTFE, PHNAR, and PH09B were used in crossing and backcrossing steps.Pioneer Hi - Bred International, Inc. Page 41 of 25 8

4114 Maize **Table 3 .**  **Generations and Comparators Used for Analysis of 4114 M**  **aize Analysis**  **Generation Comparators**  **Experimental Control and**  **Refer ence Lines**  Genetic Characterization and Absence of Backbone DNA ( Section 2 - D ) T2, T3, BC3F1 *3, BC3F2 *2 Not applicable Pioneer® proprietary maize inbreds PH09B and PHWWE Stability and Inheritance of the DNA Insertion ( Section 2 - E ) F1 *1, BC2F1 *1, BC3F1 *1,

BC2F1 *2, BC3F1 *2 Not applicable Not applicable Concentrations of Cry1F, Cry34Ab1, Cry35Ab1, and PAT ( Section 2 - F) F1 *5 Not applicable a 1507, 59122, and 1507x59122 maize a Compositional Assessment ( Section 4-A) Grain and forage from F1 *5 PHNARxPHTFE Pioneer® hybrids 34M94, 33G26, 33J24, 3394, 38B85, 37Y12, 34A15, and 34P88 Germination and Dormancy (Agronomic Performance) ( Section 4- B ) F1 *5 PHNARxPHTFE Pioneer® hybrids 32D78 and 34P88

Field Trial Evaluations (Agronomic Performance) ( Section 4- B ) F1 *5 PH NARxPHTFE Pioneer® hybrids 34M94, 33G26, 33J24, 3394, 38B85, 37Y12, 34A15, 34P88, 37H24, 36M28, 35T06, 35T36, and 35K02 a To determine actual protein concentrations in 4114 maize, no comparator lines were used. However, comparisons were made to the protein concentrations of 1507, 59122, and 1507x59122 maize lines in the same experiment to determine the applicability of previously conducted safety studies to 4114 maize.Pioneer Hi - Bred International, Inc. Page 42 of 25 8

4114 Maize **Selection of Comparators , Experimental Controls, and Reference Lines**  **for 4114 Mai ze**  For the genetic characterization of the 4114 maize insertion, Pioneer proprietary maize inbreds were used as experimental controls (Table 3 ) . These controls were selected because they represented the genetics of the maize lines that were crossed into the 4114 maize generations analyzed (Figure 10) . Because certain probes were used that contained sequences in the endogenous maize genome, these controls containing the representative genetics eliminated background hybridization not related to the 4114 ma ize insertion. For stability and inheritance of the DNA insertion, no comparators, controls, or reference lines were used or needed to conduct the analysis. For the measurement of concentrations of the expressed proteins in 4114 maize, no comparators were used, however 1507, 59122, and 1507x59122 maize reference lines were used to determine if 4114 maize protein concentrations were comparable to these lines. Th ese comparison s were important to determine if previously conducted safety studies were applica ble to the safety assessment of 4114 maize. In the comparative assessments (i.e., compositional analysis and agronomic performance), a control maize line was used (Table 3 ). For these analyses, the control comparator line had a genetic background approxi mately 99% similar to that of the 4114 maize generation used (i.e., near- isoline), but did not go through the transformation process. In addition, non -transgenic Pioneer commercial maize hybrid li nes, as listed in Table 3, were used to obtain tolerance intervals for compositional and agronomic comparisons . These Pioneer commercial maize hybrids were chosen to re present a wide range of non -genetically modified varieties that would normally be planted commercially. These tolerance intervals represent the normal range of variation of the maize crop for compositional analytes and agronomic characteristics; they help to further determine the comparability of 4114 maize to conventional maize, if any statistical differences were observed.Pioneer Hi - Bred International, Inc. Page 43 of 25 8

4114 Maize **2 -**  **D .**  **Molecular Cha**  **racterization of the Insertion in**  **4114 Maize** Molecular characterization of the inserted DNA evaluates the integrity of the introduced cassettes and provides a confirmation that the elements of the expression cassettes are intact. The inserted DNA is also evaluated over several generations to confirm its stability through t raditional breeding methods. The DNA insertion in 4114 maize was characterized by Southern blot analysis to evaluate the copy number, integrity, and stability of the inserted cry1F, cry34Ab1, cry35Ab1, and pat cassettes. As described earlier in Section 2 -C, 4114 maize was produced by Agrobacteriummediated transformation with plasmid PHP27118. The T- DNA region of PHP27118 contains four cassettes: cry1F, cry34Ab1, cry35Ab1, and pat. Th e cry1F cassette contains the ubiZM1 promoter and intron, cry1F gene, and ORF25 terminator. The cry34Ab1 cassette contains a second copy of the ubiZM1 promoter and intron, cry34Ab1 gene, and pin II terminator. The cry35Ab1 cassette contains the TA peroxid ase promoter, cry35Ab1 gene, and pin II terminator, and the pat cassette contains the CaMV 35S promoter, pat gene, and CaMV 35S terminator. All probes used for the analysis are indicated on the schematic maps of PHP27118 and the PHP27118 T- DNA region (Figu res 11 and 12, respectively) and outlined in Table 4 . P lasmid PHP27118 was used as a positive control for probe hybridization and to verify fragment sizes internal to the T- DNA. The integration pattern of the insertion in 4114 maize was investigated usin g Southern blot analysis with Bcl I digested genomic DNA from individual plants of the T3 generation to determine copy number and Hin d III digested genomic DNA to determine insertion integrity. Copy number and integrity of each genetic element were determ ined using probes specific to each of the genetic elements present in the PHP27118 T- DNA region (Table 4, Figure 12 ). The stability of the insertion was analyzed using Bcl I digested genomic DNA from individual plants of the T2, T3, BC3F1 *3, and BC3F2 *2 generati ons (Section 2; Figure 10 and Table 3 ). P robes specific to each of the gene regions of PHP27118 T- DNA were used to confirm the stability of the insertion (Table 4, Figure 12 ). I n addition, probes to the plasmid backbone region of PHP27118 located outside of the T- DNA region (Table 4, Figure 11 ) were used to show that these regions were not transferred to 4114 maize. Materials and methods for the molecular characterization of 4114 maize are described in Appendix 3. Based on the Southern blot analyses described below, it was determined that a single, intact PHP27118 T- DNA was inserted into the genome of 4114 maize as diagramed in the insertion map (Figure 13 ) and that no region from the backbone of plasmid PHP27118 was inserted. I n addition, these results confirmed the stability of the DNA insertion in 4114 maize across four breeding generations.Pioneer Hi - Bred International, Inc. Page 44 of 25 8

4114 Maize

**Figu re**  **11. Pl**  **asmid Map of PHP27118** Schematic map of PHP27118 indicating Bcl I and Hin d III restriction enzyme sites with base pair positions. The Rig ht Border and Left Border regions flank the T- DNA (Figure 1 2) that is expected to be transferred during Agrobacterium- mediated transformation. P lasmid backbone probe locations are indicated by boxes within the DNA map. **A** : RB probe; **B** : LB probe; **C** : spc probe; **D** : tet probe; **E** : virG probe. PHP2 7118 54910 bp

**vi r**  **C1 vi**  **r C2**  **vi r**  **G vi**  **r B**  **cry 1F** 

**cry 34A** **b1 cr**  **y 35A** **b1 pat** 

**spc**

**te t**  **R tet**  **A trf**  **A ctl**  **ubi ZM**  **1 In** **tro n**  **Ri ght**  **B order** 

**Lef t B**  **or der**  **ubi ZM1 P**  **ro mo** **te r** 

**TA P**  **eroxi dase**  **Pr om** **ot er**  **CaM V**  **35S Pr** **om ot** **er col**  **E1 o**  **ri**

**Col E**  **1 or** **i oriV**  **OR F25**  **Termi nat** **or pi**  **n II**  **Terminator**

**CaM V**  **35S Termi**  **nat or**  **ubi ZM**  **1 5’**  **UT R**  **cos**

**cos**

Hin dIII (78)

Hin dIII (3969)

HindIII (11738)

HindIII (368 77) Hin dIII (47940) Hin dIII (48849) Hin dIII (49970) HindIII (51485) Bcl I (2546)

Bcl I (1 221 2)

Bcl I (18243)

Bcl I (19795) Bcl I (38557) Bcl I (40721 ) Bcl I (43315) Bcl I (448 99) Bcl I (47667) Bcl I (48038 )

Bcl I (50460) **ubi ZM**  **1 Pr** **om oter**  **ubi ZM1 5’U**  **TR**

**ubi**

**ZM 1**  **In tro**  **n pi**  **n II**  **Termi nat** **or**

Bcl I (13742)

**or iT**  B

C

D EPioneer Hi - Bred International, Inc. Page 45 of 25 8

4114 Maize

**F igure**  **12 .**  **Map of PHP27118 T** **- DNA**  Schematic map of T- DNA from PHP27118 indicating Bcl I and Hin d III restriction enzyme sites and the cry1F, cry34Ab1, cry35Ab1 and pat coding and regulatory regions. The T- DNA size i s 11978 bp. The probe locations are indicated by boxes below the DNA map and are identified below: **Number Probe Name**  **1** ubiZM1 promoter **2** ubiZM1 intron **3** cry1F **4** ORF25 terminator **5** cry34Ab1 **6** pin II terminator **7** TA peroxidase promoter **8** cry35Ab1 **9** 35S promoter **10** pat **11** 35S terminator **cry 1F**  **cr y**  **34A b1** 

**cr**

**y 35A** **b1 pat**  **ubi ZM**  **1 In**  **tro n**  **ubi ZM**  **1 In**  **tro n** 

**Ri ght**  **B or** **der Lef**  **t B order**  **ubi ZM**  **1 Pr** **omot er**  **ubi ZM**  **1 Promoter TA**  **P eroxi** **dase Pr** **omot er** 

**CaM V**  **35S Pr** **omot er**  **OR F25**  **Terminator pi**  **n II**  **Terminato r** 

**Ca MV** **3 5S**  **Terminator ubi**  **ZM 1**  **5’ UT**  **R ubi**  **ZM 1**  **5’U TR** 

Bcl I (2546)

HindIII (78) Hin dIII (3969)

HindIII (11738) **pi n**  **II Termi**  **nat or** 

1 2 3 1 5 2 6 7 8 6 9 10 4 11Pioneer Hi - Bred International, Inc. Page 46 of 25 8

4114 Maize **Table 4.**  **Description of DNA Probes Used for Southern Blot Hybridization Probe Name**  **Genetic**

**Element Figure** 

**Probe Position on** 

**PHP27118 T** **- DNA**  **(bp to bp) a**  **Position on**

**PHP27118 (bp to bp)**  **b Length** 

**(bp)** cry1F c cr

y1F gene Figure 12

Probe 3 2133 to 3030

3038 to 3945 2133 to 3030 3038 to 3945 898

908 cry34Ab1 cry34Ab1 gene Figure 12 Probe 5 6817 to 7133 6817 to 7133 317 cry35Ab1 c cry35Ab1 gene Figure 12 Probe 8 8837 to 9303

9408 to 9979 8837 to 9303 9408 to 9979 467

572 pat pat gene Figure 12 Probe 10 10904 to 11451 10904 to 11451 548 ubiZM1 promoter ubiZM1 promoter Figure 12 Probe 1 150 to 1008 (copy 1)

4801 to 5659 (copy 2) 150 to 1008 (copy 1) 4801 to 5659 (copy 2) 859 ubiZM1 intron ubiZM1 5’ UTR

and intron Figure 12

Probe 2 1020 to 2100 (copy 1)

5671 to 6751 (copy 2) 1020 to 2100 (copy 1) 5671 to 6751 (copy 2) 1081 ORF25 terminator ORF25 terminator Figure 12 Probe 4 4003 to 4703 4003 to 4703 701 pin II terminator pin II terminator Figure 12 Probe 6 7235 to 7468 (copy 1)

10065 to 10298 (copy 2) 7235 to 7468 (copy 1) 10065 to 10298 (copy 2) 234 TA peroxidase promoterc TA peroxidase

promoter Figure 12

Probe 7 7523 to 8415

8416 to 8813 7523 to 8415 8416 to 8813 893

398 35S promoter CaMV 35S

promoter Figure 12

Probe 9 10383 to 10900 10383 to 10900 518 35S terminator CaMV 35S

terminator Figure 12

Probe 11 11482 to 11692 11482 to 11692 211 LB Plasmid backbone adjacent to T- DNA Left Border Figure 11

Probe B N/Ad 12003 to 12348 346 spc Spectinomycin

resistance gene Figure 11

Probe C N/Ad 13158 to 13932 775 tetc Tetracycline

resistance gene Figure 11

Probe D N/Ad 19007 to 19545 19651 to 20108 539

458 virG virG gene Figure 11 Probe E N/Ad 39334 to 40077 744 RB Plasmid backbone adjacent to T- DNA Right Border Figure 1 1

Probe A N/Ad 54476 to 54865 390

a The probe position is based on the PHP27118 T- DNA map (Figure 12 ). b The probe position is based on the PHP27118 plasmid map (Figure 11 ). c The cry1F, cry35Ab1, TA peroxidase promoter, and tet probes each are comprised of two non -overlapping labeled fragments that are combined in the respective hybridization solutions. d N/A = Not Applicable; these elements are not present in the PHP27118 T- DNA region.Pioneer Hi - Bred International, Inc. Page 47 of 25 8

4114 Maize

**Figur e**  **13. Ma**  **p of the Insertion in 4114 Maize** Schematic map of the insertion in 4114 maize based on Southern blot analysis. The flanking maize genome is represented by the horizontal dotted line. As ingle, intact copy of the PHP27118 T- DNA integrated into the maize genome. Bcl I and Hin d III restriction sites are i ndicated with the sizes of observed fragments on Southern blots shown below the map in base pairs (bp). The locations of restriction enzyme sites outside the Right and Left Borders are not shown to scale. **cry 1F**  **cry 34A** **b1**

**cry**

**35A b1**  **pat ubi**  **ZM1 In**  **tro n**  **ubi ZM**  **1 In**  **tro n** 

**Ri ght**  **Bo rd**  **er Lef**  **t B order**  **ubi ZM**  **1 Pr** **omot er**  **ubi ZM**  **1 Pr** **om ot** **er TA**  **P eroxi** **dase Pr** **omoter**

**Ca MV** **3 5S**  **Pr omoter**  **OR F25**  **Terminator pi**  **n II**  **Terminator**

**Ca MV** **3 5S**  **Terminator ubi**  **ZM 1**  **5’ UT**  **R ubi**  **ZM1 5’U TR** 

Bcl I

Hin d III Hind III

Hind III **pi n**  **II Ter** **minator**

PHP27118 T- DN A Bcl I ~3 100 bp >8600 bp Hind III 3891 bp 7769 bpPioneer Hi - Bred International, Inc. Page 48 of 25 8

4114 Maize **Copy Number of the DNA Insertion in 4114 Maize**  The restriction enzyme Bcl I has a single recognition site within the PHP27118 T- DNA (Figure 12 ) and provides information about the number of copies of the T- DNA that are integrated in the genome of 4114 maize. H ybridization with probes specific to the genetic elements of the PHP27118 T- DNA would indicate the number of copies of each element found in 4114 maize, as shown by the number of hybridizing bands. The site for Bcl I is located at bp 2546 of the T- DNA (Figure 12 ) and is predicted to yield fragments of greater than approximately 2500 bp and greater than approximately 9400 bp for a single inserted T- DNA (Ta ble 5 ). Th e band of greater than 2500 bp will hybridize to the ubiZM1 promoter and intron and the cry1F hybridization probes (Table 5, Figu re 12 ). P robes specific to the following elements will hybridize to t he fragment of greater than 9400 bp : the ubiZM1 promoter and intron , the TA peroxidase promoter, CaMV 35S promoter, cry1F, cry34Ab1, cry35Ab1, pat, ORF25 terminator, pin II terminator, and CaM V 35S terminator (Figure 12 ). As the Bcl I restriction enzyme site is within the cry1F gene and between the duplicate copies of the ubiZM1 promoter and intron regions, probes for the cry1F gene, the ubiZM1 promoter and ubiZM1 intron will hybridize to both fragments (Table 5, Figure 12 ) . Therefore, the appearance of two bands for these probes indicates a single copy of the T- DNA. A lthough there are two copies of the pin II terminator in the T- DNA, both are located on the same Bcl I fragment and will thus yield a single band (Table 5, Figure 12) . As ingle band for the remaining probes indicates a single copy of those genetic elements. Hybridization of Bcl I -digested genomic DNA from 4114 maize with the ubiZM1 promoter and intron probes resulted in a band of approximately 3100 bp and a band of greater than 8600 bp (Table 5, Figure 14 ). B ased on the expected sizes for the genomic fragments, the band of approximately 3100 bp is derived from the Right Border or 5’ end of the DNA insertion and the band of greater than 8600 bp is the band expected to be greater than 9400 bp and is derived from the Left Border or 3’ end of the 4114 maize insertion. Ab and of approximately 4800 bp was observed in the 4114 maize plants and the PHWWE control maize with both probes (Table 5, Figure 14 ). This band was determined to be due to hybridization of the ubiZM1 promoter and intron probes to the endogenous copy of these elements found in the native maize genome based upon its presence in the non -transformed PHWWE control maize line. As imilar but slightly lower band is also seen in the non -transgenic PH09B control maize line. The presence of the two expected bands with the ubiZM1 promoter and intron probes, and no additional insertion -derived bands, indicates that there is a s ingle copy of the PHP27118 T- DNA in 4114 maize. The Bcl I -digested PHP27118 plasmid lanes did not produce the expected size bands and produced two bands above the 8600 bp marker band ; these bands were characteristic of an undigested plasmid and indicated that the plasmid DNA was likely methylated (Table 5, Figure 14 ). Sensitivity of the Bcl I enzyme to bacterial plasmid DNA methylation is well known and there is a bacterial Dam methylase recognition sequence in the site ( 5’GATC3’ ) (New England Biolabs, 2002). I n all Bcl I sites (recognition sequence 5’TGATCA3’ ) on the plasmid, the central adenine will be methylated, blocking digestion by Bcl I. The PHP27118 plasmid used wasPioneer Hi - Bred International, Inc. Page 49 of 25 8

4114 Maize prepared in a dam + strain of E. coli and thus all Bcl I sites would be methylated and would not digest as expected. Therefore on all Southern blots with Bcl I -digested plasmid, only probe hybridization was confirmed. Hybridization of the Bcl I -digested DNA with the cry1F probe also resulted in the same two bands of approximately 31 00 bp and greater than 8600 bp (Table 5, Figure 15 ). Two bands are expected with the cry1F probe due to the location of the Bcl I restriction site within the cry1F gene (Figure 1 2), and the presence of these two bands indicates there is a single copy of t he cry1F gene in 4114 maize. When hybridized to the ORF25 terminator probe, the same blot resulted in a single band of greater than 8600 bp (Table 5, Figure 15 ). As the ORF25 terminator element is located on the 3’ or Left Border side of the Bcl I site in the PHP27118 T- DNA (Figure 12 ), this demonstrates that the greater than 8600 bp band is indeed associated with the elements on this portion of the T- DNA. The same band of greater than 8600 bp was observed when the Bcl I Southern blot was hybridized with the cry34Ab1 and pin II terminator probes (Table 5, Figure 16 ), the TA peroxidase promoter and cry35Ab1 probes (Table 5, Figure 17 ), and the 35S promoter, pat, and 35S terminator probes (Table 5, Figure 18 ). A lthough there are two copies of the pin II te rminator in the PHP27118 T- DNA, both copies are located on the same Bcl I fragment so that they result in a single hybridizing band on the Southern blot. The presence of single bands with all of these elements confirms that there is a single copy of the P HP27118 T- DNA inserted in 4114 maize. When taken together with the restriction map of the PHP27118 T- DNA (Figure 12 ), the presence of single bands with the ORF25 terminator, cry34Ab1, pin II terminator, TA peroxidase promoter, cry35Ab1, 35S promoter, pat, and 35S terminator probes, along with the expected two bands for the ubiZM1 promoter, ubiZM1 intron , and cry1F probes, demonstrate that there is a single inserted PHP27118 T- DNA in 4114 maize. Additionally, Bcl I digestion of the 4114 maize insertion provides an event-specific hybridization pattern when certain probes are used. I f the hybridization pattern is identical between different plants and generations, this demonstrates the 5’ and 3’ regions of the DNA insertion are stable during the breeding process and is discussed later in this section .Pioneer Hi - Bred International, Inc. Page 50 of 25 8

4114 Maize **Table 5.**  **Predicted and Observed Hybridizing Bands on Southern Blots; Bcl**  **I Digest Probe**  **Figure Predicted Fragment** 

**Size from PHP27118 T** **- DNA (bp)**  **a Predicted Fragment** 

**Size from Plasmid PHP27118 (bp)**  **b Observed**  **Fragment**

**Size in 4114 Maize**  **(bp) c**  ubiZM1 promoter 14 >9400 (border) d, e >2500 (border) d, e 9666 6996 >8600

~3100 ~4800* ubiZM1 intron 14 >9400 (border) d, e >2500 (border) d, e 9666 6996 >8600

~3100 ~4800* cry1F 15, 14 >9400 (border) d, f >2 500 (border) d,f 9666 6996 >8600

~3100 ORF25 terminator 15 >9400 (border) d 9666 >8600 cry34Ab1 16, 23 >9400 (border) d 9666 >8600 pin II terminator 16 >9400 (border) d, g 9666 >8600 TA peroxidase promoter 17 >9400 (border) d 9666 >8600 cry35Ab1 17, 24 >9400 (border) d 9666 >8600 35S promoter 18 >9400 (border) d 9666 >8600 pat 18, 2 5 >9400 (border) d 9666 >8600 35S terminator 18 >9400 (border) d 9666 >8600 LB 2 8 N/A h 1530 (25139) i No hybridization spc 2 9 N/A h 1530 (25139) i No hybridization tet 30 N/A h 1552 (25139) i No hybridization virG 31 N/A h 2164 (11063) i No hybridization RB 3 2 N/A h 6996 (3503) i No hybridization Note: A n asterisk (*) and gray shading indicates the designated band is due to hybridization to endogenous sequences. These bands were iden tified in the maize control lines that were analyzed. a Predicted size for hybridization in genomic DNA samples is based on the map of the T- DNA from PHP27118 (Figure 12 ). Border fragment sizes are rounded to the nearest 100 bp. b Predicted size is based on the plasmid map of PHP27118 (Figure 11 ) and is the size expected for Bcl I digestion of an unmethylated plasmid. The methylated PHP27118 used in this study did not digest with Bcl I and therefore did not yield these fragments. c Observed fragment sizes are approximated from the DIG - labeled DNA Molecular Weight Marker VII fragments on the Southern blots. D ue to the inability to determine exact sizes on the blot, all approximated values are rounded to the nearest 100 bp. d Border fragments are those in w hich one restriction site is in the inserted T- DNA and the other site is located in the flanking genomic DNA, providing a fragment of unique size for a given insertion. Border fragment sizes are rounded to the nearest 100 bp. e There are two copies of the ubiZM1 promoter and intron elements, located on either side of the Bcl I site, so both fragments will hybridize to these probes. f The Bcl I site is located within the cry1F gene, so both fragments hybridize to the cry1F probe. g Both copies of the pin II terminator in the PHP27118 T- DNA are located on the same Bcl I fragment. h N/A=Not Applicable; these elements are not found on the PHP27118 T- DNA. I The fragment size in parenthesis is for digestion of PHP27118 with Hin d III, which was included in Lanes 15 and 16 of certain Bcl I blots to show that the plasmid can be cut by a restriction enzyme that is not sensitive to methylation.Pioneer Hi - Bred International, Inc. Page 51 of 25 8

4114 Maize

**Figure 14.**  **Southern Blot**  **Analysis of**  **the T3**  **Generation of**  **4114 Maize;**  **Bcl I**  **Digested DNA** 

**with ubi** **ZM1 Promoter and**  **ubi ZM1**  **I ntron Probes**  DNA isolated from individual plants of 4114 maize was digested with Bcl I and hybridized to the ubiZM1 promoter and ubiZM1 intron probes. Approximately 3 - 4 μg of genomic DNA was digested and loaded per lane. The gene copy number controls included plasmid PHP27118 at the indicated approximate gene copy number and 4 μg of genomic DNA. S izes of the DIG VII molecular weight markers are indicated adjacent to the blot image in kilobase pairs (kb). **Lane Sample**  **Lane Sample**  1 1 copy PHP27118 + PH09B control 8 1 copy PHP27118 + PH09B control 2 3 copy PHP27118 + PHWWE control 9 3 copy PHP27118 + PHWWE control 3 DIGVII marker 10 DIGVII marker 4 PH09B control 11 PH09B control 5 PHWWE control 12 PHWWE control 6 4114 maize plant 20 (T3 gene ration) 13 4114 maize plant 20 (T3 generation) 7 4114 maize plant 22 (T3 generation) 14 4114 maize plant 22 (T3 generation)

**ubi ZM1**  **P ro**  **mote r**  **ubi ZM1**  **Intron kb**  **8.6 7.4**  **6.1 4.9**  **3.6 2.8** 

**1.95 1.88**  **1.51 1.48**  **1.2 0.99**  **0.72 0.49**  **kb 8.6**  **7.4 6.1**  **4.9 3.6**  **2.8**

**1.95 1.88**  **1.51 1.48**  **1.2 0.99**  **0.72 0.49**  **1 2**  **3 4**  **5 6**  **7 8**  **9 10**  **11 12**  **13 14** Pioneer Hi - Bred International, Inc. Page 52 of 25 8

4114 Maize

**Figure 15.**  **Southern Blot Analysis of the T3**  **Generation of 4114 Maize; Bcl**  **I Digested DNA**

**with cry** **1F and ORF25 Terminator**  **P robes**  DNA isolated from individual plants of 4114 maize was digested with Bcl I and hybridized to the cry1F and ORF25 terminator probes. Approximately 3 - 4 μg of genomic DNA was digested and loaded per lane. The gene copy number controls included plasm id PHP27118 at the indicated approximate gene copy number and 4 μg of genomic DNA. S izes of the DIG VII molecular weight markers are indicated adjacent to the blot image in kilobase pairs (kb). **Lane Sample**  **Lane Sample**  1 1 copy PHP27118 + PH09B control 8 1 copy PHP27118 + PH09B control 2 3 copy PHP27118 + PHWWE control 9 3 copy PHP27118 + PHWWE control 3 DIGVII marker 10 DIGVII marker 4 PH09B control 11 PH09B control 5 PHWWE control 12 PHWWE control 6 4114 maize plant 20 (T3 generation) 13 4114 maize plant 20 (T3 generation) 7 4114 maize plant 22 (T3 generation) 14 4114 maize plant 22 (T3 generation) **cry 1F**  **2 3**  **4 5**  **6 7**  **8 9**  **10 11**  **12 13**  **14 1** 

**kb 8.6**  **7.4 6.1**  **4.9 3.6**  **2.8**

**1.95 1.88**  **1.51 1.48**  **1.2 0.99**  **0.72 0.49**  **kb 8.6**  **7.4 6.1**  **4.9 3.6**  **2.8 1.95**  **1.88 1.51**  **1.48 1.2**  **0.99 0.72**  **0.49 OR** **F2 5**  **Te rmi**  **nator**Pioneer Hi - Bred International, Inc. Page 53 of 25 8

4114 Maize

**Figure 16.**  **Southern Blot Analysis of the T3**  **Generation of 4114 Maize; Bcl**  **I Digested DNA**

**with cry** **34Ab1 and**  **pin II Terminator**  **Probes** DNA i solated from individual plants of 4114 maize was digested with Bcl I and hybridized to the cry34Ab1 and pin II terminator probes. Approximately 3 - 4 μg of genomic DNA was digested and loaded per lane. The gene copy number controls included plasmid PHP271 18 at the indicated approximate gene copy number and 4 μg of genomic DNA. S izes of the DIG VII molecular weight markers are indicated adjacent to the blot image in kilobase pairs (kb). **Lane Sample**  **Lane Sample**  1 1 copy PHP27118 + PH09B control 8 1 copy PHP27118 + PH09B control 2 3 copy PHP27118 + PHWWE control 9 3 copy PHP27118 + PHWWE control 3 DIGVII marker 10 DIGVII marker 4 PH09B control 11 PH09B control 5 PHWWE control 12 PHWWE control 6 4114 maize plant 20 (T3 generation) 13 4114 maize p lant 20 (T3 generation) 7 4114 maize plant 22 (T3 generation) 14 4114 maize plant 22 (T3 generation) **cry 34A** **b1 pi** **n II**  **Te rmi**  **na tor**  **kb 8.6**  **7.4 6.1**  **4.9 3.6**  **2.8**

**1.95 1.88**  **1.51 1.48**  **1.2 0.99**  **0.72 0.49**  **kb 8.6**  **7.4 6.1**  **4.9 3.6**  **2.8 1.95**  **1.88 1.51**  **1.48 1.2**  **0.99 0.72**  **0.49 2**  **3 4**  **5 6**  **7 8**  **9 10**  **11 12**  **13 14**  **1**Pioneer Hi - Bred International, Inc. Page 54 of 25 8

4114 Maize

**Figure 17.**  **Southern Blot Analysis of the T3**  **Generation of 4114 Maize; Bcl**  **I Digested DNA**

**with TA Peroxidase Promoter and**  **cry 35Ab1 Probes**  DNA isolated from individual plants of 4114 maize was digested with Bcl I and hybridized to the TA peroxidase promoter and cry35Ab1 probes. Approximately 3 - 4 μg of genomic DNA was digested and loaded per lane. The gene copy number controls included plasmid PHP2 7118 at the indicated approximate gene copy number and 4 μg of genomic DNA. S izes of the DIG VII molecular weight markers are indicated adjacent to the blot image in kilobase pairs (kb). **Lane Sample**  **Lane Sample**  1 1 copy PHP27118 + PH09B control 8 1 co py PHP27118 + PH09B control 2 3 copy PHP27118 + PHWWE control 9 3 copy PHP27118 + PHWWE control 3 DIGVII marker 10 DIGVII marker 4 PH09B control 11 PH09B control 5 PHWWE control 12 PHWWE control 6 4114 maize plant 20 (T3 generation) 13 4114 maize plant 20 (T3 generation) 7 4114 maize plant 22 (T3 generation) 14 4114 maize plant 22 (T3 generation) **2 3**  **4 5**  **6 7**  **8 9**  **10 11**  **12 13**  **14 1** 

**kb 8.6**  **7.4 6.1**  **4.9 3.6**  **2.8**

**1.95 1.88**  **1.51 1.48**  **1.2 0.99**  **0.72 0.49**  **kb 8.6**  **7.4 6.1**  **4.9 3.6**  **2.8 1.95**  **1.88 1.51**  **1.48 1.2**  **0.99 0.72**  **0.49 TA**  **Per oxida** **se Pr** **om ote**  **r cry** **35A b1** Pioneer Hi - Bred International, Inc. Page 55 of 25 8

4114 Maize

**Figure 18.**  **Southern Blot Analysis of the T3**  **Generation of 4114 Maize; Bcl**  **I Digested DNA**

**with 35S Promoter,**  **pat**

**, and 35S Terminator Probes** DNA isolated from individual plants of 4114 maize was digested with Bcl I and hybridized to the 35S promoter, pat, and 35S Terminator probes. Approximately 3 - 4 μg of genomic DNA was digested and loaded per lane. The gene copy number controls included plasm id PHP27118 at the indicated approximate gene copy number and 4 μg of genomic DNA. S izes of the DIG VII molecular weight markers are indicated adjacent to the blot image in kilobase pairs (kb). **Lane Sample**  **Lane Sample**  1 1 copy PHP27118 + PH09B control 12 PHWWE control 2 3 copy PHP27118 + PHWWE control 13 4114 maize plant 20 (T3 generation) 3 DIGVII marker 14 4114 maize plant 22 (T3 generation) 4 PH09B control 15 1 copy PHP27118 + PH09B control 5 PHWWE control 16 3 copy PHP27118 + PHWWE control 6 4114 maize plant 20 (T3 generation) 17 DIGVII marker 7 4114 maize plant 22 (T3 generation) 18 PH09B control 8 1 copy PHP27118 + PH09B control 19 PHWWE control 9 3 copy PHP27118 + PHWWE control 20 4114 maize plant 20 (T3 generation) 10 DIGVII mar ker 21 4114 maize plant 22 (T3 generation) 11 PH09B control

**2 3**  **4 5**  **6 7**  **8 9**  **10 11**  **12 13**  **14 1** 

**35S Pr** **omote r**  **pat 15**  **16 17**  **18 19**  **20 21** 

**35S Te**  **rmi na** **tor kb**  **8.6 7.4**  **6.1 4.9**  **3.6 2.8** 

**1.95 1.88**  **1.51 1.48**  **1.2 0.99**  **0.72 0.49**  **kb 8.6**  **7.4 6.1**  **4.9 3.6**  **2.8**

**1.95 1.88**  **1.51 1.48**  **1.2 0.99**  **0.72 0.49**  **kb 8.6**  **7.4 6.1**  **4.9 3.6**  **2.8**

**1.95 1.88**  **1.51 1.48**  **1.2 0.99**  **0.72 0.49** Pioneer Hi - Bred International, Inc. Page 56 of 25 8

4114 Maize **Integrity of the DNA Insertion in 4114 Maize**  The restriction enzyme Hin d III was used to confirm the integrity of the PHP27118 T- DNA insertion in 4114 maize. There are three Hin d III sites within the PHP27118 T- DNA: one site at bp 78, one after the cry1F gene at bp 3969, and one at bp 11738 just inside the Left Border of the T- DNA (Figure 12 ). A n internal fragment of 3891 bp is expected to hybridize to the cry1F gene probe (Table 6, Figure 12 ), and another internal fragment of 7769 bp is expected to hybridize to the probes for the ORF25 terminator, cry34Ab1 gene, pin II terminator, cry35Ab1 gene, TA peroxidase promoter, pat gene and the CaMV 35S promoter and terminator regions (Table 6, Figure 12 ). Although there are two copies of the pin II terminator in the PHP27118 T- DNA, both copies are found on the same Hin d III fragment so the probe would hybridize to the single 7769 bp internal fragment . Because the central Hin d III restriction enzym e site is located between the duplicate copies of the ubiZM1 promoter and intron regions, probes for these elements are expected to hybridize to both fragments (Table 6, Figure 12 ). The absence of any insert derived bands other than the expected bands provides a strong indication that the T- DNA is intact and was not truncated upon insertion. The Hin d III plasmid positive control run alongside the 4114 maize DNA samples was used to confirm that the sizes of the observed bands were identical to these intern al fragments. Hybridization of Hin d III -digested genomic DNA from 4114 maize with the ubiZM1 promoter and intron probes resulted in the expected band s of 3891 bp and 7769 bp (Table 6, Figure 19 ). A n additional band of approximately 6400 bp was observed i n the 4114 plants and the PHWWE control maize with both probes (Table 6, Figure 19 ). This band resulted from the hybridization of the ubiZM1 promoter and intron probes to the endogenous copy of these elements in the native maize genome based upon its presence in the PHWWE control maize line. H ybridization with the cry1F probe resulted in the expected band of 3891 bp in the 4114 maize plants (Table 6, Figure 20 ). As expected, t he 7769 bp band was observed in the 4114 maize plants with the ORF25 terminator probe (Table 6, Figure 20 ), with the cry34Ab1 and pin II terminator probes (Table 6, Figure 21 ), the TA peroxidase promoter and cry35Ab1 probes (Table 6, Figure 22 ), and the 35S promoter, pat, and 35S terminator probes (Table 6, Figure 23 ). The presence of the expected size bands that were identical to the internal bands from the PHP27118 plasmid with all of these elements, and no other insertion -derived bands, indicate that the PHP27118 T- DNA inserted intact in 4114 maize and there we re no truncated copie s of the T- DNA.Pioneer Hi - Bred International, Inc. Page 57 of 25 8

4114 Maize **Table 6.**  **Predicted and Observed Hybridizing Bands on Southern Blots; Hin** **d III Digest Probe**  **Figure Predicted Fragment** 

**Size from PHP27118 T** **- DNA (bp)**  **a Predicted Fragment** 

**Size from Plasmid PHP27118 (bp)**  **b Observed Fragment** 

**Size in 4114 Mai** **ze (bp)**  **c** ubiZM1 promoter 1 9 7769 d 3891 d 7769 3891 7769 e

3891 e ~6400* ubiZM1 intron 1 9 7769 d 3891 d 7769 3891 7769 e

3891 e ~6400* cry1F 20 3891 3891 3891 e ORF25 terminator 20 7769 7769 7769 e cry34Ab1 21 7769 7769 7769 e pin II terminator 21 7769 f 7769 7769 e TA peroxidase promoter 2 2 7769 7769 7769 e cry35Ab1 2 2 7769 7769 7769 e 35S promoter 2 3 7769 7769 7769 e pat 2 3 7769 7769 7769 e 35S terminator 2 3 7769 7769 7769 e Note: A n asterisk (*) and gray shading indicates the designated band is due to hybridization to endogenous sequences. These bands were identified in the maize control lines that were analyzed. a Predicted size for hybridization in genomic DNA samples is based on the map of the T- DNA from PHP27118 (Figure 12 ). b Predicted size is b ased on the plasmid map of PHP27118 (Figure 11 ). c Observed fragment sizes are approximated from the DIG - labeled DNA Molecular Weight Marker VII fragments on the Southern blots. D ue to the inability to determine exact sizes on the blot, all approximated values are rounded to the nearest 100 bp. d There are two copies of the ubiZM1 promoter and intron elements, located on either side of the central Hin d III site, so both fragments will hybridize to these probes. e Size is same as predicted based on equal mi gration as the control plasmid fragment on the Southern blot. f Both copies of the pin II terminator in the PHP27118 T- DNA are located on the same Hin d III fragment.Pioneer Hi - Bred International, Inc. Page 58 of 25 8

4114 Maize

**Figure 19**  **. Southern Blot Analysis of the**  **T3 Generation of 4114 Maize;**  **Hin d**  **III Digeste d DNA** 

**with ubi** **ZM1 Promoter and**  **ubi ZM1**  **Intron Probes** DNA isolated from individual plants of 4114 maize was digested with Hin d III and hybridized to the ubiZM1 promoter and ubiZM1 intron probes. Approximately 3 - 4 μg of genomic DNA was digested and loaded per lane. The gene copy number controls included plasmid PHP27118 at the indicated approximate gene copy number and 4 μg of genomic DNA. S izes of the DIG VII molecular weight markers are indicated adjacent to the blot image in kilobase pairs (kb). **Lane Sample**  **Lane Sample**  1 4114 maize plant 20 (T3 generation) 8 4114 maize plant 20 (T3 generation) 2 4114 maize plant 22 (T3 generation) 9 4114 maize plant 22 (T3 generation) 3 PH09B control 10 PH09B control 4 PHWWE control 11 PHWWE control 5 DIGVII marker 12 DIGVII marker 6 3 copy PHP27118 + PHWWE control 13 3 copy PHP27118 + PHWWE control 7 1 copy PHP27118 + PH09B control 14 1 copy PHP27118 + PH09B control **12 8** 

**ubi ZM1**  **Promote r**  **ubi ZM1**  **Intron 1**  **2 3**  **4 5**  **6 7**  **9 10**  **11 13**  **14**

**kb 8.6**  **7.4 6.1**  **4.9 3.6**  **2.8**

**1.95 1.88**  **1.51 1.48**  **1.2 0.99**  **0.72 0.49**  **kb 8.6**  **7.4 6.1**  **4.9 3.6**  **2.8**

**1.95 1.88**  **1.51 1.48**  **1.2 0.99**  **0.72 0.49** Pioneer Hi - Bred International, Inc. Page 59 of 25 8

4114 Maize

**Figure 20**  **. Southern Blot Analysis of the**  **T3 Generation of 4114 Maize;**  **Hin d III D** **igested DNA**

**with cry** **1F and ORF25 Terminator Probes**  DNA isolated from individual plants of 4114 maize was digested with Hin d III and hybridized to the cry1F and ORF25 terminator probes. Approximately 3 - 4 μg of genomic DNA was digested and loaded per lan e. The gene copy number controls included plasmid PHP27118 at the indicated approximate gene copy number and 4 μg of genomic DNA. S izes of the DIG VII molecular weight markers are indicated adjacent to the blot image in kilobase pairs (kb). **Lane Sample**  **Lane Sample**  1 4114 maize plant 20 (T3 generation) 8 4114 maize plant 20 (T3 generation) 2 4114 maize plant 22 (T3 generation) 9 4114 maize plant 22 (T3 generation) 3 PH09B control 10 PH09B control 4 PHWWE control 11 PHWWE control 5 DIGVII marker 12 DIGVII marker 6 3 copy PHP27118 + PHWWE control 13 3 copy PHP27118 + PHWWE control 7 1 copy PHP27118 + PH09B control 14 1 copy PHP27118 + PH09B control **cry 1F**  **ORF 25**  **Te rmi**  **na tor**  **kb 8.6**  **7.4 6.1**  **4.9 3.6**  **2.8**

**1.95 1.88**  **1.51 1.48**  **1.2 0.99**  **0.72 0.49**  **kb 8.6**  **7.4 6.1**  **4.9 3.6**  **2.8 1.95**  **1.88 1.51**  **1.48 1.2**  **0.99 0.72**  **0.49 12**  **8 1**  **2 3**  **4 5**  **6 7**  **9 10**  **11 13**  **14**Pioneer Hi - Bred International, Inc. Page 60 of 25 8

4114 Maize

**Figure 21**  **. Southern Blot Analysis of the**  **T3 Generation of 4114 Maize;**  **Hin d III Digeste** **d DNA**

**with cry** **34Ab1 and**  **pin II Terminator Probes**  DNA isolated from individual plants of 4114 maize was digested with Hin d III and hybridized to the cry34Ab1 and pin II terminator probes. Approximately 3 - 4 μg of genomic DNA was digested and loaded per lan e. The gene copy number controls included plasmid PHP27118 at the indicated approximate gene copy number and 4 μg of genomic DNA. S izes of the DIG VII molecular weight markers are indicated adjacent to the blot image in kilobase pairs (kb). **Lane Sample**  **Lane Sample**  1 4114 maize plant 20 (T3 generation) 8 4114 maize plant 20 (T3 generation) 2 4114 maize plant 22 (T3 generation) 9 4114 maize plant 22 (T3 generation) 3 PH09B control 10 PH09B control 4 PHWWE control 11 PHWWE control 5 DIGVII marker 12 DIGVII marker 6 3 copy PHP27118 + PHWWE control 13 3 copy PHP27118 + PHWWE control 7 1 copy PHP27118 + PH09B control 14 1 copy PHP27118 + PH09B control **cry 34A** **b1 pin**  **II Te**  **rmi na** **tor 2**  **3 4**  **5 6**  **7 8**  **9 10**  **11 12**  **13 14**  **1**

**kb 8.6**  **7.4 6.1**  **4.9 3.6**  **2.8**

**1.95 1.88**  **1.51 1.48**  **1.2 0.99**  **0.72 0.49**  **kb 8.6**  **7.4 6.1**  **4.9 3.6**  **2.8 1.95**  **1.88 1.51**  **1.48 1.2**  **0.99 0.72**  **0.49**Pioneer Hi - Bred International, Inc. Page 61 of 25 8

4114 Maize

**Figure 22**  **. Southern Blot Analysis of the**  **T3 Generation of 4114 Maize;**  **Hin d III Digested**  **DNA**

**with TA Peroxidase Promoter and**  **cry 35Ab1**  **Probes** DNA isolated from individual plants of 4114 maize was digested with Hin d III and hybridized to the TA peroxidase promoter and cry35Ab1 probes. Approximately 3 - 4 μg of genomic DNA was digested and loa ded per lane. The gene copy number controls included plasmid PHP27118 at the indicated approximate gene copy number and 4 μg of genomic DNA. S izes of the DIG VII molecular weight markers are indicated adjacent to the blot image in kilobase pairs (kb). **N ote:**  Blotches on the TA peroxidase panel are due to non -specific chemiluminescence from the detection process and are not due to hybridization of the probe to maize DNA. **Lane Sample**  **Lane Sample**  1 4114 maize plant 20 (T3 generation) 8 4114 maize plant 20 (T3 generation) 2 4114 maize plant 22 (T3 generation) 9 4114 maize plant 22 (T3 generation) 3 PH09B control 10 PH09B control 4 PHWWE control 11 PHWWE control 5 DIGVII marker 12 DIGVII marker 6 3 copy PHP27118 + PHWWE control 13 3 copy PHP2711 8 + PHWWE control 7 1 copy PHP27118 + PH09B control 14 1 copy PHP27118 + PH09B control

**TA Pe**  **ro xi**  **dase Pr** **om ote**  **r cry** **35A b1**  **2 3**  **4 5**  **6 7**  **8 9**  **10 11**  **12 13**  **14 1** 

**kb 8.6**  **7.4 6.1**  **4.9 3.6**  **2.8**

**1.95 1.88**  **1.51 1.48**  **1.2 0.99**  **0.72 0.49**  **kb 8.6**  **7.4 6.1**  **4.9 3.6**  **2.8**

**1.95 1.88**  **1.51 1.48**  **1.2 0.99**  **0.72 0.49** Pioneer Hi - Bred International, Inc. Page 62 of 25 8

4114 Maize

**Figure 23**  **. Southern Blot Analysis of the**  **T3 Generation of 4114 Maize;**  **Hin d III Digested DNA** 

**with 35S Promoter,**  **pat**

**, and 35S Terminator Probes** DNA isolated from individual plants of 4114 maize was digested with Hin d III and hybridized to the 35S promoter, pat, and 35S Terminator probes. Approximately 3 - 4 μg of genomic DNA was digested and loaded per lane. The gene copy number controls included plasmid PHP2711 8 at the indicated approximate gene copy number and 4 μg of genomic DNA. S izes of the DIG VII molecular weight markers are indicated adjacent to the blot image in kilobase pairs (kb). **Lane Sample**  **Lane Sample**  1 4114 maize plant 20 (T3 generation) 12 DI GVII marker 2 4114 maize plant 22 (T3 generation) 13 3 copy PHP27118 + PHWWE control 3 PH09B control 14 1 copy PHP27118 + PH09B control 4 PHWWE control 15 4114 maize plant 20 (T3 generation) 5 DIGVII marker 16 4114 maize plant 22 (T3 generation) 6 3 copy PHP27118 + PHWWE control 17 PH09B control 7 1 copy PHP27118 + PH09B control 18 PHWWE control 8 4114 maize plant 20 (T3 generation) 19 DIGVII marker 9 4114 maize plant 22 (T3 generation) 20 3 copy PHP27118 + PHWWE control 10 PH09B control 21 1 copy PHP27118 + PH09B control 11 PHWWE control

**35S Promote**  **r pat**  **35S Te**  **rmi nator**  **2 3**  **4 5**  **6 7**  **8 9**  **10 11**  **12 13**  **14 1**  **15 16**  **17 18**  **19 20**  **21**

**kb 8.6**  **7.4 6.1**  **4.9 3.6**  **2.8**

**1.95 1.88**  **1.51 1.48**  **1.2 0.99**  **0.72 0.49**  **kb 8.6**  **7.4 6.1**  **4.9 3.6**  **2.8**

**1.95 1.88**  **1.51 1.48**  **1.2 0.99**  **0.72 0.49**  **kb 8.6**  **7.4 6.1**  **4.9 3.6**  **2.8**

**1.95 1.88**  **1.51 1.48**  **1.2 0.99**  **0.72 0.49** Pioneer Hi - Bred International, Inc. Page 63 of 25 8

4114 Maize **Stability of the Insertion in 4114 Maize**  Stability of the 4114 maize insertion was confirmed by performing Southern blot analysis on Bcl I -digested genomic DNA from individual plants of four gene rations of 4114 maize: T2, T3, BC3F1 * 3, and BC3F2 * 2 . As described earlier, hybridization of Bcl I -digested DNA to the probe for the cry1F gene resulted in an event-specific hybridization pattern consisting of a band of approximately 3100 bp and a band of g reater than 8600 bp (Figure 13 ). This hybridization pattern can be used to demonstrate the stability of the 5’ and 3’ region of the 4114 maize insertion. H ybridizations with the cry34Ab1, cry35Ab1, and pat probes resulted in a single band of greater tha n 8600 bp (Figure 13 ), and can be used to demonstrate the stability of the 3’ region of the insertion . The presence of these event-specific hybridization patterns in each of the four generations with these probes would confirm the stability of the inserti on in 4114 maize during breeding . Hybridization of the Bcl I -digested DNA with the cry1F probe resulted in two bands of approximately 3100 bp and greater than 8600 bp that were consistently observed in each of the four generations, indicating stability of the 5’ and 3’ regions of the 4114 maize insertion (Table 5, Figure 24 ). When hybridized to the cry34Ab1 (Table 5, Figure 25 ), cry35Ab1 (Table 5, Figure 26 ), and pat probes (Table 5, Figure 27 ), a band of greater than 8600 bp was consistently observed in all plants from the four generations. The observation of these expected event specific hybridization patterns with all probes indicate d that the 4114 DNA insertion was stable during the breeding process .Pioneer Hi - Bred International, Inc. Page 64 of 25 8

4114 Maize

**Figure 24** **. Southern Blot Analysis of the**  **T2 ,**  **T3 ,**  **BC3F1** ***** **3 and**  **BC3F2** ***** **2 Generations of 4114** 

**Maize; Bcl**  **I Digested DNA with cry** **1F Probe** DNA isolated from individual plants of 4114 maize was digested with Bcl I and hybridized to the cry1F probe. Approximately 3 - 6 μg of genomic DNA was digested and loa ded per lane. The gene copy number controls included plasmid PHP27118 (digested with Bcl I in Lanes 1 and 2; digested with Hin d III in Lanes 15 and 16) at the indicated approximate gene copy number and 4 μg of genomic DNA. S izes of the DIG VII molecular weight markers are indicated adjacent to the blot image in kilobase pairs (kb). **Lane Sample**  **Lane Sample**  1 1 copy PHP27118 + PH09B control 9 4114 maize plant 37 ( BC3F1 * 3 generation) 2 3 copy PHP27118 + PHWWE control 10 4114 maize plant 39 ( BC3F2 * 2 gen eration) 3 DIGVII marker 11 4114 maize plant 41 ( BC3F2 * 2 generation) 4 4114 maize plant 9 (T2 generation) 12 PH09B control 5 4114 maize plant 12 (T2 generation) 13 PHWWE control 6 4114 maize plant 20 (T3 generation) 14 DIGVII marker 7 4114 maize p lant 22 (T3 generation) 15 3 copy PHP27118 + PH09B control ( Hin d III) 8 4114 maize plant 30 ( BC3F1 * 3 generation) 16 1 copy PHP27118 + PHWWE control ( Hin d III) **2 3**  **4 5**  **6 7**  **8 9**  **10 11**  **12 13**  **14 1**  **15 16** 

**kb 8.6**  **7.4 6.1**  **4.9 3.6**  **2.8**

**1.95 1.88**  **1.51 1.48**  **1.2 0.99**  **0.72 kb**  **8.6 7.4**  **6.1 4.9**  **3.6 2.8** 

**1.95 1.88**  **1.51 1.48**  **1.2 0.99**  **0.72**Pioneer Hi - Bred International, Inc. Page 65 of 25 8

4114 Maize

**Figure 25**  **. Southern Blot Analysis of the**  **T2 ,**  **T3 ,**  **BC3F1** ***** **3 and**  **BC3F2** ***** **2 Generations of 411** **4**

**Maize; Bcl**  **I Digested DNA with cry** **34Ab1 Probe** DNA isolated from individual plants of 4114 maize was digested with Bcl I and hybridized to the cry34Ab1 probe. Approximately 3 - 6 μg of genomic DNA was digested and loaded per lane. The gene copy number controls included plasmid PHP27118 (digested with Bcl I in Lanes 1 and 2; digested with Hin d III in Lanes 15 and 16) at the indicated approximate gene copy number and 4 μg of genomic DNA. Sizes of the DIG VII molecular weight markers are indicated adjacent to the blot image in kilobase pairs (kb). **Lane Sample**  **Lane Sample**  1 1 copy PHP27118 + PH09B control 9 4114 maize plant 37 ( BC3F1 * 3 generation) 2 3 copy PHP27118 + PHWWE control 10 4114 maize plant 39 ( BC3F2 * 2 generation) 3 DIGVII marker 11 4114 m aize plant 41 ( BC3F2 * 2 generation) 4 4114 maize plant 9 (T2 generation) 12 PH09B control 5 4114 maize plant 12 (T2 generation) 13 PHWWE control 6 4114 maize plant 20 (T3 generation) 14 DIGVII marker 7 4114 maize plant 22 (T3 generation) 15 3 copy P HP27118 + PH09B control ( Hin d III) 8 4114 maize plant 30 ( BC3F1 * 3 generation) 16 1 copy PHP27118 + PHWWE control ( Hin d III) **2 3**  **4 5**  **6 7**  **8 9**  **10 11**  **12 13**  **14 1**  **15 16** 

**kb 8.6**  **7.4 6.1**  **4.9 3.6**  **2.8**

**1.95 1.88**  **1.51 1.48**  **1.2 0.99**  **0.72 kb**  **8.6 7.4**  **6.1 4.9**  **3.6 2.8** 

**1.95 1.88**  **1.51 1.48**  **1.2 0.99**  **0.72**Pioneer Hi - Bred International, Inc. Page 66 of 25 8

4114 Maize

**Figure 26**  **. Southern Blot Analysis of the**  **T2 ,**  **T3 ,**  **BC3F1** ***** **3 and**  **BC3F2** ***** **2 Generations of 4114** 

**Maize; Bcl**  **I Digested DNA with cry** **3 5Ab1 Probe**  DNA isolated from individual plants of 4114 maize was digested with Bcl I and hybridized to the cry35Ab1 probe. Approximately 3 - 6 μg of genomic DNA was digested and loaded per lane. The gene copy number controls included plasmid PHP27118 (d igested with Bcl I in Lanes 1 and 2; digested with Hin d III in Lanes 15 and 16) at the indicated approximate gene copy number and 4 μg of genomic DNA. Sizes of the DIG VII molecular weight markers are indicated adjacent to the blot image in kilobase pairs (kb). **Lane Sample**  **Lane Sample**  1 1 copy PHP27118 + PH09B control 9 4114 maize plant 37 ( BC3F1 * 3 generation) 2 3 copy PHP27118 + PHWWE control 10 4114 maize plant 39 ( BC3F2 * 2 generation) 3 DIGVII marker 11 4114 maize plant 41 ( BC3F2 * 2 generation) 4 4114 maize plant 9 (T2 generation) 12 PH09B control 5 4114 maize plant 12 (T2 generation) 13 PHWWE control 6 4114 maize plant 20 (T3 generation) 14 DIGVII marker 7 4114 maize plant 22 (T3 generation) 15 3 copy PHP27118 + PH09B control ( Hin d III) 8 4114 maize plant 27 ( BC3F1 * 3 generation) 16 1 copy PHP27118 + PHWWE control ( Hin d III) **2 3**  **4 5**  **6 7**  **8 9**  **10 11**  **12 13**  **14 1**  **15 16** 

**kb 8.6**  **7.4 6.1**  **4.9 3.6**  **2.8**

**1.95 1.88**  **1.51 1.48**  **1.2 0.99**  **0.72 kb**  **8.6 7.4**  **6.1 4.9**  **3.6 2.8** 

**1.95 1.88**  **1.51 1.48**  **1.2 0.99**  **0.72**Pioneer Hi - Bred International, Inc. Page 67 of 25 8

4114 Maize

**Figure 27**  **. Southern Blot Analysis of the**  **T2 ,**  **T3 ,**  **BC3F1** ***** **3 and**  **BC3F2** ***** **2 Generations of 4114** 

**Maize; Bcl**  **I Digested DNA with pat**  **Probe** DNA isolated from individual p lants of 4114 maize was digested with Bcl I and hybridized to the pat probe. Approximately 3 - 6 μg of genomic DNA was digested and loaded per lane. The gene copy number controls included plasmid PHP27118 (digested with Bcl I in Lanes 1 and 2; digested w ith Hin d III in Lanes 15 and 16) at the indicated approximate gene copy number and 4 μg of genomic DNA. S izes of the DIG VII molecular weight markers are indicated adjacent to the blot image in kilobase pairs (kb). **Lane Sample**  **Lane Sample**  1 1 copy PHP2 7118 + PH09B control 9 4114 maize plant 37 ( BC3F1 * 3 generation) 2 3 copy PHP27118 + PHWWE control 10 4114 maize plant 39 ( BC3F2 * 2 generation) 3 DIGVII marker 11 4114 maize plant 41 ( BC3F2 * 2 generation) 4 4114 maize plant 9 (T2 generation) 12 PH09B control 5 4114 maize plant 12 (T2 generation) 13 PHWWE control 6 4114 maize plant 20 (T3 generation) 14 DIGVII marker 7 4114 maize plant 22 (T3 generation) 15 3 copy PHP27118 + PH09B control ( Hin d III) 8 4114 maize plant 27 ( BC3F1 * 3 generation) 16 1 copy PHP27118 + PHWWE control ( Hin d III) **2 3**  **4 5**  **6 7**  **8 9**  **10 11**  **12 13**  **14 1**  **15 16** 

**kb 8.6**  **7.4 6.1**  **4.9 3.6**  **2.8**

**1.95 1.88**  **1.51 1.48**  **1.2 0.99**  **0.72 kb**  **8.6 7.4**  **6.1 4.9**  **3.6 2.8** 

**1.95 1.88**  **1.51 1.48**  **1.2 0.99**  **0.72**Pioneer Hi - Bred International, Inc. Page 68 of 25 8

4114 Maize **Absence of Plasmid Backbone DNA**  **in 4114 Maize** Four generations of 4114 maize (T2, T3, BC3F1 * 3, and BC3F2 * 2) were analyzed by Southern blot analysis for plasmid sequences from the PHP27118 plasmid backbone. P rob es for genes and regions on the PHP27118 plasmid backbone outside the T- DNA region were used to determine if any plasmid backbone was inserted in 4114 maize during transformation. The spc, tet, and virG probes hybridize to the spectinomycin resistance, tetracycline resistance, and virG genes respectively (Table 4, Figure 11 ). The LB probe hybridizes to the plasmid backbone region outside the Left T- DNA border, and the RB probe hybridizes to the region outside the Right Border of the T- DNA of PHP27118 (Tab le 4, Figure 11 ). These probes would show whether region s of the PHP27118 backbone outside the T- DNA was transferred into 4114 maize. Genomic DNA from the T2, T3, BC3F1 *3, and BC3F2 *2 generations was digested with Bcl I and hybridized to the backbone p robes described above. The LB (Figure 28 ), spc (Figure 2 9 ), tet (Figure 30 ), virG (Figure 31 ), and RB (Figure 3 2 ) probes showed no bands from backbone hybridization in the 4114 maize or control maize plants. This confirmed that no sequence from the PHP27 118 plasmid backbone was inserted during transformation. The plasmid lanes showed hybridization to the undigested plasmid in the Bcl I lanes (Figures 28 through 32, Lanes 1 and 2) and to the expected size bands for digestion with Hin d III (Figures 28 thro ugh 3 2, Lanes 15 and 16), thus demonstrating successful hybridization of the probes to plasmid backbone sequences.Pioneer Hi - Bred International, Inc. Page 69 of 25 8

4114 Maize

**Figure 28**  **. Southern Blot Analysis of the**  **T2 ,**  **T3 ,**  **BC3F1** ***** **3 and**  **BC3F2** ***** **2 Generations of 4114** 

**Maize; Bcl**  **I Digested DNA with LB Probe**  DNA isolated from individual plants of 4114 maize was digested with Bcl I and hybridized to the LB probe. Approximately 3 - 6 μg of genomic DNA was digested and loaded per lane. The gene copy number controls included plasmid PHP27118 (digested with Bcl I in Lanes 1 and 2; digested with Hin d III in Lanes 15 and 16) at the indicated approximate gene copy number and 4 μg of genomic DNA. S izes of the DIG VII molecular weight markers are indicated adjacent to the blot image in kilobase pairs (kb). **Lane Sample**  **L ane**  **Sample** 1 1 copy PHP27118 + PH09B control 9 4114 maize plant 37 ( BC3F1 * 3 generation) 2 3 copy PHP27118 + PHWWE control 10 4114 maize plant 39 ( BC3F2 * 2 generation) 3 DIGVII marker 11 4114 maize plant 41 ( BC3F2 * 2 generation) 4 4114 maize plant 9 (T2 generation) 12 PH09B control 5 4114 maize plant 12 (T2 generation) 13 PHWWE control 6 4114 maize plant 20 (T3 generation) 14 DIGVII marker 7 4114 maize plant 22 (T3 generation) 15 3 copy PHP27118 + PH09B control ( Hin d III) 8 4114 maize plant 30 ( BC3F1 * 3 generation) 16 1 copy PHP27118 + PHWWE control ( Hin d III) **2 3**  **4 5**  **6 7**  **8 9**  **10 11**  **12 13**  **14 1**  **15 16** 

**kb 8.6**  **7.4 6.1**  **4.9 3.6**  **2.8 1.95**  **1.88 1.51**  **1.48 1.2**  **0.99 0.72**  **kb 8.6**  **7.4 6.1**  **4.9 3.6**  **2.8 1.95**  **1.88 1.51**  **1.48 1.2**  **0.99 0.72**  **0.49 0.49** Pioneer Hi - Bred International, Inc. Page 70 of 25 8

4114 Maize

**Figure 2** **9 .**  **Southern Blot Analysis of the T2** **, T3** **, BC3F1**  ***** **3 and**  **BC3F2** ***** **2 Generations of 4114** 

**Maize; Bcl**  **I Digested DNA with spc**  **Probe** DNA isolated from individual plants of 4114 maize was digested with Bcl I and hybridized to the spc probe. Approximately 3 - 6 μg of genomic DNA was digested and loaded per lane. The gene copy number controls included plasmid PHP27118 (digested with Bcl I in Lanes 1 and 2; digested with Hin d III in Lanes 15 and 16) at the indicated approximate gene copy number and 4 μg of genomic DNA. S izes of the DIG VII molecular weight markers are indicated adjacent to the blot image in kilobase pairs (kb). **Lane Sample**  **Lane Sample**  1 1 copy PHP27118 + PH09B control 9 4114 maize plant 37 ( BC3F1 * 3 generation) 2 3 copy PHP27118 + PHWWE control 10 4114 maize plant 39 ( BC3F2 * 2 generation) 3 DIGVII marker 11 4114 maize plant 41 ( BC3F2 * 2 generation) 4 4114 maize plant 9 (T2 generation) 12 PH09B control 5 4114 maize plant 12 (T2 generation) 13 PHWWE control 6 4114 maize plant 20 (T3 generation) 14 DIGVII marker 7 4114 maize plant 22 (T3 generation) 15 3 copy PHP27118 + PH09B control ( Hin d III) 8 4114 maize plant 30 ( BC3F1 * 3 generation) 16 1 copy PHP27118 + PHWW E control ( Hin d III) **2 3**  **4 5**  **6 7**  **8 9**  **10 11**  **12 13**  **14 1**  **15 16** 

**kb 8.6**  **7.4 6.1**  **4.9 3.6**  **2.8 1.95**  **1.88 1.51**  **1.48 1.2**  **0.99 0.72**  **kb 8.6**  **7.4 6.1**  **4.9 3.6**  **2.8**

**1.95 1.88**  **1.51 1.48**  **1.2 0.99**  **0.72**Pioneer Hi - Bred International, Inc. Page 71 of 25 8

4114 Maize

**Figure 30**  **. Southern Blot Analysis of the**  **T2 ,**  **T3 ,**  **BC3F1** ***** **3 and**  **BC3F2** ***** **2 Generations of 4114** 

**Maize; Bcl**  **I Digested DNA with tet**  **Probe** DNA isolated from individual plants of 4114 maize was digested with Bcl I and hybridized to the tet probe. Approximately 3 - 6 μg of genomic DNA was digested and loaded per lane. The gene copy number controls included plasmid PHP27118 (digested with Bcl I in Lanes 1 and 2; digested with Hin d III in Lanes 15 and 16) at the indicated approximate gene copy number and 4 μg of genomic DNA. S izes of the DIG VII molecular weight markers are indicated adjacent to the blot image in kilobase pairs (kb). **Lane Sample**  **Lane Sample**  1 1 copy PHP27118 + PH09B control 9 4114 maize plant 37 ( BC3F1 * 3 generation) 2 3 copy PHP27118 + PHWWE control 10 4114 maize plant 39 ( BC3F2 * 2 generation) 3 DIGVII marker 11 4114 maize plant 41 ( BC3F2 * 2 generation) 4 4114 maize plant 9 (T2 generation) 12 PH09B control 5 4114 maize plant 12 (T2 generation) 13 PHWWE control 6 4114 maize plant 20 (T3 generation) 14 DIGVII marker 7 4114 maize plant 22 (T3 generation) 15 3 copy PHP27118 + PH09B control ( Hin d III) 8 4114 maize plant 30 ( BC3F1 * 3 generation) 16 1 copy PHP27118 + PHWWE control ( Hin d III) **2 3**  **4 5**  **6 7**  **8 9**  **10 11**  **12 13**  **14 1**  **15 16** 

**kb 8.6**  **7.4 6.1**  **4.9 3.6**  **2.8 1.95**  **1.88 1.51**  **1.48 1.2**  **0.99 0.72**  **kb 8.6**  **7.4 6.1**  **4.9 3.6**  **2.8**

**1.95 1.88**  **1.51 1.48**  **1.2 0.99**  **0.72**Pioneer Hi - Bred International, Inc. Page 72 of 25 8

4114 Maize

**Figure 31**  **. Souther** **n Blot Analysis of the T2** **, T3** **, BC3F1**  ***** **3 and**  **BC3F2** ***** **2 Generations of 4114** 

**Maize; Bcl**  **I Digested DNA with vir** **G Probe**  DNA isolated from individual plants of 4114 maize was digested with Bcl I and hybridized to the virG probe. Approximately 3 - 6 μg of genomic DNA was digested and loaded per lane. The gene copy number controls included plasmid PHP27118 (digested with Bcl I in Lanes 1 and 2; digested with Hin d III in Lanes 15 and 16) at the indicated approximate gene copy number and 4 μg of genomic DNA. S izes of the DIG VII molecular weight markers are indicated adjacent to the blot image in kilobase pairs (kb). **Lane Sample**  **Lane Sample**  1 1 copy PHP27118 + PH09B control 9 4114 maize plant 37 ( BC3F1 * 3 generation) 2 3 copy PHP27118 + PHWWE control 10 4114 ma ize plant 39 ( BC3F2 * 2 generation) 3 DIGVII marker 11 4114 maize plant 41 ( BC3F2 * 2 generation) 4 4114 maize plant 9 (T2 generation) 12 PH09B control 5 4114 maize plant 12 (T2 generation) 13 PHWWE control 6 4114 maize plant 20 (T3 generation) 14 DIGV II marker 7 4114 maize plant 22 (T3 generation) 15 3 copy PHP27118 + PH09B control ( Hin d III) 8 4114 maize plant 30 ( BC3F1 * 3 generation) 16 1 copy PHP27118 + PHWWE control ( Hin d III) **2 3**  **4 5**  **6 7**  **8 9**  **10 11**  **12 13**  **14 1**  **15 16** 

**kb 8.6**  **7.4 6.1**  **4.9 3.6**  **2.8 1.95**  **1.88 1.51**  **1.48 1.2**  **0.99 0.72**  **kb 8.6**  **7.4 6.1**  **4.9 3.6**  **2.8**

**1.95 1.88**  **1.51 1.48**  **1.2 0.99**  **0.72**Pioneer Hi - Bred International, Inc. Page 73 of 25 8

4114 Maize

**Figure 32**  **. Southern Blot Analysis of the**  **T2 ,**  **T3 ,**  **BC3F1** ***** **3 and**  **B C3F2**  ***** **2 Generations of 4114** 

**Maize; Bcl**  **I Digested DNA with RB Probe**  DNA isolated from individual plants of 4114 maize was digested with Bcl I and hybridized to the RB probe. Approximately 3 - 6 μg of genomic DNA was digested and loaded per lane. The gene copy number controls included plasmid PHP27118 (digested with Bcl I in Lanes 1 and 2; digested with Hin d III in Lanes 15 and 16) at the indicated approximate gene copy number and 4 μg of genomic DNA. S izes of the DIG VII molecular weight markers are indi cated adjacent to the blot image in kilobase pairs (kb). **Lane Sample**  **Lane Sample**  1 1 copy PHP27118 + PH09B control 9 4114 maize plant 37 ( BC3F1 * 3 generation) 2 3 copy PHP27118 + PHWWE control 10 4114 maize plant 39 ( BC3F2 * 2 generation) 3 DIGVII marker 11 4114 maize plant 41 ( BC3F2 * 2 generation) 4 4114 maize plant 9 (T2 generation) 12 PH09B control 5 4114 maize plant 12 (T2 generation) 13 PHWWE control 6 4114 maize plant 20 (T3 generation) 14 DIGVII marker 7 4114 maize plant 22 (T3 generation) 15 3 copy PHP27118 + PH09B control ( Hin d III) 8 4114 maize plant 30 ( BC3F1 * 3 generation) 16 1 copy PHP27118 + PHWWE control ( Hin d III) **2 3**  **4 5**  **6 7**  **8 9**  **10 11**  **12 13**  **14 1**  **15 16** 

**kb 8.6**  **7.4 6.1**  **4.9 3.6**  **2.8 1.95**  **1.88 1.51**  **1.48 1.2**  **0.99 0.72**  **0.49 kb**  **8.6 7.4**  **6.1 4.9**  **3.6 2.8**  **1.95 1.88**  **1.51 1.48**  **1.2 0.99**  **0.72 0.49** Pioneer Hi - Bred International, Inc. Page 74 of 25 8

4114 Maize **Physical Map of the DNA Insertion**  **in 4114 Maize** Based on the Southern blot analysis, it was determined that a sin gle, intact PHP27118 T- DNA was inserted into the genome of 4114 maize. Ap hysical map of the DNA insertion in 4114 maize showing the restriction enzymes employed in the analysis was developed using these data and is presented in Figure 13 . **Summary and Co nclusions**  Southern blot analysis was conducted on 4114 maize to confirm insertion copy number, integrity, and stability of the insertion. Analysis with Bcl I, examining the sequences flanking the DNA insertion, indicated a single copy of the PHP27118 T- D NA is present in 4114 maize. The Hin d III analysis indicated that the PHP27118 T- DNA had inserted intact in the genome. Analysis with Bcl I demonstrated that the 4114 DNA insertion is stable across the T2, T3, BC3F1 * 3, and BC3F2 * 2 generations and during the traditional breeding process, as identical hybridization pattern s were observed in each of the four generations. In addition , analysis with probes for the regions of PHP27118 outside the T- DNA region demonstrated that no plasmid backbone sequences were incorporated into the 4114 maize genome.Pioneer Hi - Bred International, Inc. Page 75 of 25 8

4114 Maize **2 -**  **E .**  **Inheritance and Stability of the I nserted DNA**  The stability of the inserted DNA during the breeding process is evaluated by examining the inheritance and segregation of the genes and/or traits in multi ple generations. The segregation of these genes or traits as a single unit and as a single genetic locus will confirm that the inserted DNA will be predictably and stably inherited through the commercial breeding process. For 4114 maize, the i nheritance of both the DNA insertion and the herbicide-tolerance phenotype was evaluated to ensure stability of the traits during the plant breeding process and to confirm the traits were inserted at a single genetic locus. For the analyses, segregating generations of 4114 maize ( F1 * 1, BC2F1 * 1, BC3F1 * 1, BC2F1 * 2, an d BC3F1 * 2 gen erations ) were evaluated. The bre eding history of these five generations is shown in the breeding diagram (Section 2, Figure 10 and Table 3 ). These generations were selected for analysis beca use they represent a range of different crossing and backcrossing points in a typical maize breeding program. Chi -square analysis was conducted on the five segregating generations to determine if the observed segregation ratios were consistent with the ex pected ratios. The assay methods and statistical analysis for the trait inheritance data are described in Appendix 4. The presence of the 4114 event insertion was determined by event-specific and cry1F, cry34Ab1, cry35Ab1, and pat gene-specific endpoint PCR analyses performed on leaf punches from seedlings of each generation. The herbicide-tolerance phenotype was determined by treating the plants with glufosinate herbicide and visually evaluating each plant for herbicide injury. A positive plant exhibited no herbicidal injury and a negative plant exhibited severe herbicide injury. Results from the segregation analysis are provided in Table 7 . In every case, a positive plant tested positive for the presence of the 4114 event, the cry1F, cry34Ab1, cry3 5Ab1 and pat genes, and the herbicide-toleran ce phenotype, indicating the 4114 insertion segregated as a unit. To confirm that the inserted DNA and the herbicide-tolerance phenotype segregate according to Mendel’s laws of genetics for a single genetic loc us, chi -square analysis was performed for the PCR and the herbicide-tolerance phenotype data. No statistically significant differences were found between the observed and expected segregation ratios for the F1 * 1, BC2F1 * 1, BC3F1 *1, and BC2F1 *2 generations of 4114 maize (Table 7 ), indicating that these four generations all segregated as expected based on Mendel’s laws. The 4114 insertion segregated as a unit and the observed ratios were indicative of an insertion at a single genetic locus. The observed seg regation ratio of the BC3F1 *2 generation in the original analysis of 99 seedlings was statistically significant (p-value <0.05) compared to the expected 1:1 segregation ratio, with a chi -square value of 5.34 and p-value of 0.0208. Typically a n observed segregation ratio would not be statistically different compared to a 1:1 expected ratio (50:50 based on 100 plants) if it fell in the range of 50:50 ± 9 (ratio of 41:59 or 59:41). The BC3F1 *2 generation observed segregation ratio for the original sample was 38:61, only two plants outside of the acceptablePioneer Hi - Bred International, Inc. Page 76 of 25 8

4114 Maize range. I n order to determine if the statistical difference was a false positive result due to random sampling, additional sampling of 96 seedlings from the same seed source was performed . For the original sample, the chance of a false positive result due to random sampling was 5%; if a statistical difference was found when sampling was repeated, the chance of a false positive result would be gre atly reduced to 5% x 5% = 0.25% . This additional sampling wou ld not only confirm the prior result, but would also increase the statistical power of the BC3F1 *2 generation analysis . Chi -square analysis of the combined data (n=195) for this single seed source (95% confidence) was performed and no statistically sign ificant difference was found between the observed segregation ratio and the expected segregation ratio for the BC3F1 *2 generation (Table 7 ). The significant difference observed in the original BC3F1 *2 generation analysis was therefore likely due to a false positive result associated with random sampling. These results indicate that the inserted DNA and the herbicide-tolerance phenotype in 4114 maize segregate predictably according to Mendel’s laws of genetics and are consistent with the finding that the 4 114 maize insertion is at a single genetic locus. Taken together with the stability results described earlier for the Southern analysis, these results confirm t he stability of the insertion through the breeding process .

**Table 7.**  **Summary of Genotypic and Phenotypic Results for Segregating 4114 Maize**  **4114 Maize**  **Generation a**  **Observed Values b**  **(expected segregation = 1:1) Statistical Analysis**  **Positive Negative**  **Total Chi** **- Square** **c P** **- value**  F1 *1 52 46 98 0.367 0.545 BC2F1 *1 48 52 100 0.160 0.689 BC3F1 *1 47 53 100 0.360 0.549 BC2F1 *2 53 47 100 0.360 0.549 BC3F1 *2 87 108 195 1.62 0.1326 a The BC3F1 *2 generation analysis was conducted during two time points . The first analysis was conducted with 99 seed lings and found to be statistically significant by Chi -square analysis. To increase the statistical power, an additional 96 seed lings from the same seed lot were tested. The combined results of the two time points within the single seed lot are shown and were not statistically different from the expected segregation ratio. b PCR analysis (consisting of event-specific PCR analysis to confirm the presence or absence of maize event 4114, and gene-specific PCR analysis to confirm the presence or absence of the cry1F, cry34Ab1, cry35Ab1, and pat genes) and herbic ide (i.e., glufosinate) tolerance analysis were conducted for each plant in each entry. A ll PCR results matched the corresponding herbicide tolerance result for each plant analyzed. c Degrees of freedom = 1.Pioneer Hi - Bred International, Inc. Page 77 of 25 8

4114 Maize **2 -**  **F. Concentrations of Cry1F, Cry34Ab1, Cry** **35Ab1 ,**  **and PAT in 4114 Maize** Determining the concentrations of the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins in various tissues of 4114 maize is important background information for the conduct of exposure and safety assessments. To determine if previo us safety studies conducted for 1507 (expresses Cry1F and PAT), 59122 (expresses Cry34Ab1, Cry35Ab1, and PAT), or 1507x59122 maize ( expresses Cry1F, Cry34Ab1, Cry35Ab1, and PAT) are applicable to 4114 maize, it is necessary to determine if the concentratio ns of the expressed proteins in 4114 maize are comparable to these commercial lines. In order to determine the concentrations of Cry1F, Cry34Ab1, Cry35Ab1, and PAT and to assess comparability, 4114 maize was grown side- by-side with commercial 1507, 5912 2, and 1507x59122 maize lines at five commercial maize-growing sites in the U.S. and Canada during the 2010 growing season. The sites were located in Iowa (two sites), Illinois, Nebraska, and Ontario, Canada. The F1* 5 generation of seed (Section 2; Figure 10 and Table 3) was used because F1 hybrid seed is representative of seed that growers would plant in commercial maize fields. Four replications (from different plants) per tissue were collected from each site, for a total of n=20 for each tissue and ti me point . Plant tissue samples were collected throughout the growing season at various growth developmental stages (Table 8 ) and processed as described in Appen dix 5. Time points for sampling were chosen to determine the range of protein concentration s t hroughout the growing season and for their relevance to commercial maize production practices. The protein concentrations in R6 tissues (i.e., plant senescence and grain harvest) and R1 pollen values (i.e., at pollen shed) have relevance for the evaluatio n of impact on non -target arthropods in the environment . The R4 stage of the whole plant sample (i.e., forage ) is the stage at which growers harvest plants for silage for animal feed. Grain is normally harvested at the R6 stage of development and is used for food and feed. Concentrations of Cry1F, Cry34Ab1, Cry35Ab1, and PAT protein in plant tissue extracts were measured in the following tissues using specific quantitative enzyme- linked immunosorbent assay (ELISA) methods, as described in Appendix 5:  Lea f— V6, V9, R1, R4, and R6  Root — V6, V9, R1, R4, and R6  Whole Plant (the above-ground portion of the plant, including the ear at R1 and R6 ) — V9, R1, and R6  Pollen — R1  Forage (the above-ground portion of the plant, including the ear) — R4  Grain — R6 The ranges of transgenic protein mean concentrations in leaf, root, and whole- plant tissues over the course of the growing season, as well as the mean concentrations in pollen and root, are summarized in Table 9 for 4114 maize ( data taken from Tables 10 through 13). Th e me anPioneer Hi - Bred International, Inc. Page 78 of 25 8

4114 Maize PAT concentrations in 4114 maize pollen and grain were below the lower limit of quantification (LLOQ) of the ELISA assay .

**Table 8** **. Maize Growth Stage Descriptions**  **Growth Stage** **a Description**  V6 The collar of the sixth leaf becomes visible. V9 The collar of the ninth leaf becomes visible. R1 Silks become visible. R4 The material within the kernel produces a doughy consistency. R6 Typical harvest maturity for grain (regarded as physiological maturity). a Adapted from Ritchie et al., 2005

**Table 9**  **. Mean Concentrations of Transgenic Proteins in 4114 Maize Tissue**  **Cry1F Cry34Ab1**  **Cry35Ab1 PAT**  **Range in ng/mg tissue dry weight**  a Leaf 2.0 - 34 19 - 110 27 - 90 0.52 - 14 Root 3.8 - 5.5 17 - 23 6.4 - 13 0.13 - 0.65 Whole Plant b 4.1 - 12 23 - 52 21 - 76 0.090 - 8.7 Pollen 35 9.2 0.34 <0.28 c Grain 3.3 24 1.1 <0.069 c a Ranges reflect the range of means at different growth stages. b Whole plant ranges include forage. c < Lower Limit of Quantification (LLOQ); indicates the values of the sampl e(s) were detected below the assay LLOQPioneer Hi - Bred International, Inc. Page 79 of 25 8

4114 Maize For each transgenic protein, the concentrations in each 4114, 1507, 59122, and 1507x59122 maize tissue were measured (Tables 10 through 13) . The Cry1F, Cry34Ab1, and Cry35Ab1 protein concentrations in 4114 maize i n each tissue were divided by the respective protein concentrations in 1507, 59122, and/or 1507x59122 maize to provide an “expression ratio” (Table 14) . W hen the expression ratio was close to one, this indicated that 4114 maize had comparable expression to 1507, 59122, and/or 1507x59122 maize. I f the expression ratio was less than one, this indicated that 4114 maize had lower expression. For values greater than one, expression was determined to be higher in 4114 maize. The concentrations of Cry1F protei n in 4114 maize were comparable to or lower than concentrations in 1507 maize and 1507x59122 maize in all tissues measured except for pollen (R1) and leaf (R6) tissues (Table s 10 and 14 ). Concentrations of Cry1F in pollen (R1) and leaf (R6) tissues were h igher in 4114 maize. The concentrations of Cry34Ab1 protein in 4114 maize were comparable to or lower than concentrations in 59122 maize and 1507x59122 maize in all tissues measured except for leaf (R6) tissue (Tables 11 and 14 ). The concentration of Cry34Ab1 in leaf (R6) tissue was higher in 4114 maize. The concentrations of Cry35Ab1 protein in 4114 maize were comparable to or lower than concentrations in 59122 maize and 1507x59122 maize in all tissues measured (Tables 12 and 14 ). The concentrations of PAT protein in 4114 maize were comparable to or greater than concentrations in 1507 maize and were comparable to or lower than concentrations in 59122 maize and 1507x59122 maize, with the exception of leaf (R6) which was higher (Tables 13 and 14 ). It was expected that PAT concentrations in 1507x59122 maize tissues would be greater than PAT concentrations in 4114, 1507, and/or 59122 maize, since 1507x59122 contains two copies of the pat gene (one each from 1507 and 59122 maize) and 4114, 1507, and 59122 ma ize each contain one copy of the pat gene. I n general, PAT protein concentrations in pollen, R4 root tissue, and all R6 tissues (grain, leaf, root, and whole plant) were very low and, in some cases, below the lower limit of quantification. It is noted that, in most tissues and time points, the concentrations of Cry1F, Cry34Ab1, Cry35Ab1 and PAT in 4114 maize differed from 1507, 59122, and/or 1507x59122 maize, even though the same promoter-gene combinations are present in 4114 maize as in 1507 and/or 591 22 maize . Expression differences can likely be attributed to event-to-event variation and are generally considered minor for a biological system ( i.e ., 5.4-fold or less in all cases and often less than 2 -fold). In addition, these expression differences a re not necessarily biol ogically meaningful . For example, as described fully in insect efficacy data submitted to the EPA, despite the fact that concentrations of Cry1F, Cry34Ab1 and Cry35Ab1 are generally lower in 4114 maize than in 1507, 59122 and/or 1507x59122 maize, 4114 maize wa s equivalent to 1507 maize in efficacyPioneer Hi - Bred International, Inc. Page 80 of 25 8

4114 Maize against target lepidopteran pests. 4 114 maize may be slightly less efficacious and less consistent in efficacy against corn rootworm compared to 1507x59122 maize. At one location in 2010 where corn rootworm pressure was high, 4114 maize was less efficacious than 1507x59122 maize. H owever, for the remaining eight location - by-year comparisons, there were no significant differences in corn rootworm efficacy between 4114 and 1507x59122 maize. Therefore, although in many cases the concentrations of Cry1F, Cry34Ab1, and Cry35Ab1 proteins were generally lower in 4114 maize tissues than in 1507, 59122, and/or 1507x59122 maize, there was little to no biological impact in terms of efficacy. As is summarized in Table 14, in most cases the concent rations of Cry1F, Cry34Ab1, Cry35Ab1, and PAT were generally comparable to or lower than 1507, 59122, and/or 1507x59122 maize. The notable exceptions were Cry1F and Cry34Ab1 in late-season R6 leaf tissue, w here concentrations in 4114 maize were 2.3 - to 5.4-fold higher than 1507, 59122, and 1507x59122 maize, and Cry1F in pollen, where concentrations in 4114 maize were 1.4- to 1.5-fold higher than 1507 and 1507x59122 maize. R6 leaf tissue is collected at harvest time, when tissue has begun to senesce to varying degrees, and can be variable in degree of senescence and total extractable protein levels. This may impact the concentrations of tran sgenic proteins reported; R6 leaf tissue ratios were not representat ive of comparisons between 4114 maize and 1507, 59122, and/or 1507x59122 maize in leaf tissues at other time points . In some cases, PAT concentrations were higher in 4114 maize as compared to 1507 maize and were also higher in R6 leaf for similar reasons described above; however, the concentrations of PAT overall were very low or below the lower limit of quantification which likely impacted ratios and the comparisons. The comparisons for all other tissues, with the exception of Cry1F pollen, demonstrate t hat 4114 maize tissues have similar or lower concentrations of Cry1F, Cry34Ab1, Cry35Ab1, and PAT than 1507, 59122, and/or 1507x59122 maize tissues. In general, these comparisons indicate any previously conducted safety studies that used 1507, 59122, and 1507x59122 maize are applicable to 4114 maize. Safety studies that considered exposure to the Cry1F protein from 1507 or 1507x59122 pollen would require the reassessment of results for 4114 maize using the higher concentration.Pioneer Hi - Bred International, Inc. Page 81 of 25 8

4114 Maize **Table 10**  **. Cry1F Concent rations (ng/mg Tissue Dry Weight)**  **Tissue (Growth Stage) 4114 Maize**  **1507 Maize 1507x59122 Maize**  Leaf (V6) Mean a 11 18 18

Range 7.8 - 17 11 - 26 13 - 23 CI b 9.4 - 13 15 - 21 15 - 21 Root (V6) Mean 4.2 5.7 6.3

Range 0.69 - 6.0 1.1 - 7.8 3.3 - 8.7 C I 2.8 - 5.5 4.4 - 7.1 5.0 - 7.6 Leaf (V9) Mean 9.7 13 16

Range 5.3 - 14 0.78 - 20 9.6 - 24 CI 6.8 - 13 10 - 16 13 - 18 Root (V9) Mean 5.0 7.2 6.9

Range 1.3 - 7.5 4.2 - 12 4.2 - 11 CI 3.1 - 6.9 5.3 - 9.1 5.0 - 8.7 Whole Plant (V9) Mean 12 16 16 Range 8.6 - 15 10 - 19 9.6 - 19 CI 10 - 13 14 - 17 15 - 18 Leaf (R1) Mean 13 16 17

Range 7.2 - 28 10 - 23 11 - 26 CI 9.6 - 18 12 - 21 12 - 23 Pollen (R1) Mean 35 23 24

Range 19 - 49 19 - 26 18 - 28 CI 29 - 42 19 – 27 20 - 29 Root (R1) Mean 5. 5 7.0 6.1

Range 3.9 - 7.8 5.1 - 9.9 3.9 - 7.5 CI 4.8 - 6.3 6.1 - 7.9 5.3 - 7.0 Whole Plant (R1) Mean 9.9 14 13 Range 7.8 - 13 10 - 18 9.6 - 15 CI 9.1 - 11 13 - 15 12 - 14 Forage (R4) Mean 7.8 8.4 9.1

Range 5.6 - 11 5.6 - 12 6.0 - 14 CI 6.4 - 9.4 7.0 - 10 7.5 - 11 Leaf (R4) Mean 34 37 37

Range 19 - 56 25 - 49 23 - 58 CI 27 - 42 30 - 47 29 - 46 Root (R4) Mean 3.8 4.4 5.3

Range 2.3 - 5.7 3.0 - 5.7 3.0 - 7.2 CI 2.9 - 4.6 3.6 - 5.3 4.4 - 6.2 Grain (R6) Mean 3.3 3.2 2.8

Range 2.3 - 7.2 1.9 - 5.1 1.7 - 4.5 CI 2.8 - 4.0 2.7 - 3.8 2.4 - 3.4 Leaf (R6) Mean 2.0 0.79 0.37

Range 0.32 - 21 <0.14c

-21 <0.14c

- 16 CI 0.38 - 10 0.15 - 4.1 0.072 - 1.9Pioneer Hi - Bred International, Inc. Page 82 of 25 8

4114 Maize **Table 10**  **. Cry1F Concentrations (ng/mg Tissue Dry Weight) (continued)**  **Tissue (Growth Stage) 4114 Maize**  **1507 Maize 1507x59122 Maize**  Root (R6) Mean 3.8 5.0 4.7

Range 1.4 - 6.3 2.9 - 7.5 2.7 - 8.1 CI 3.1 - 4.8 4.1 - 6.2 3.8 - 5.8 Whole Plant (R6) Mean 4.1 4.9 3.8 Range 2.4 - 9.4 2.2 - 8.4 2.2 - 7.4 CI 3.0 - 5.5 3.6 - 6.6 2.8 - 5.1 a Lea st squares means (estimated from statistical models; see Appendix 5 for additional details) . b Statistical Confidence Interval c < Lower Limit of Quantification (LLOQ); indicates the values of the sample(s) were detected below the assay LLOQPioneer Hi - Bred International, Inc. Page 83 of 25 8

4114 Maize **Table 11**  **. Cr y34Ab1 Concentrations (ng/mg Tissue Dry Weight)**  **Tissue (Growth Stage) 4114 Maize**  **1507x59122 Maize**  **59122 Maize** Leaf (V6) Mean a 21 32 33

Range 17 - 25 25 - 43 24 - 46 CI b 19 - 22 30 - 35 31 - 36 Root (V6) Mean 17 29 26

Range 9.0 - 23 19 - 42 16 - 3 6 CI 13 - 22 24 - 34 22 - 31 Leaf (V9) Mean 26 42 38

Range 22 - 31 32 - 54 23 - 46 CI 23 - 29 37 - 48 33 -43 Root (V9) Mean 21 36 37

Range 13 - 28 30 - 42 23 - 48 CI 18 - 24 31 - 41 31-41 Whole Plant (V9) Mean 23 41 41 Range 18 - 26 26 - 52 2 6 - 54 CI 18 - 28 36 - 46 36 - 45 Leaf (R1) Mean 50 70 75

Range 36 - 84 52 - 110 54 - 110 CI 33 - 67 53 - 87 58 - 92 Pollen (R1) Mean 9.2 46 44

Range 4.7 - 16 35 - 67 36 - 64 CI 7.7 - 11 38 - 55 36 - 53 Root (R1) Mean 19 29 29

Range 8.7 - 30 14 - 45 16 - 39 CI 10 - 28 20 - 38 19 - 38 Whole Plant (R1) Mean 32 56 57 Range 24 - 42 44 - 82 50 - 76 CI 29 - 35 50 - 62 52 - 64 Forage (R4) Mean 52 93 86

Range 36 - 64 68 - 130 56 - 120 CI 46 - 60 81 - 110 75 - 98 Leaf (R4) Mean 110 200 190

Range 66 - 140 150 - 250 84 - 270 CI 84 - 130 180 - 220 160 - 210 Root (R4) Mean 23 35 32

Range 7.5 - 36 18 - 51 12 - 51 CI 17 - 29 29 - 41 26 - 38 Grain (R6) Mean 24 24 24

Range 14 - 39 14 - 42 17 - 33 CI 20 - 28 20 - 29 20 - 29 Leaf (R6) Mean 19 6.0 7.7

Range 4.2 - 66 0.30 - 130 0.90 - 120 CI 4.4 - 82 1.4 - 26 1.8 - 34Pioneer Hi - Bred International, Inc. Page 84 of 25 8

4114 Maize **Table 11. Cry34Ab1**  **Concentrations (ng/mg Tissue Dry Weight) (continued)**  **Tissue (Growth Stage) 4114 Maize**  **1507x59122 Maize**  **59122 Maize** Root (R6) Mean 18 32 37

Ran ge 5.4 - 54 14 - 54 17 - 66 CI 14 - 24 24 - 42 28 - 49 Whole Plant (R6) Mean 36 48 48 Range 20 - 62 26 - 88 28 - 70 CI 29 - 44 39 - 58 40 - 59 a Least squares means (estimated from statistical models; see Appendix 5 for additional details) . b Stat istical Confidence IntervalPioneer Hi - Bred International, Inc. Page 85 of 25 8

4114 Maize **Table 12. Cry35** **Ab1 Concentrations (ng/mg Tissue Dry Weight) Tissue (Growth Stage)**  **4114 Maize 1507x59122**  **Maize 59122 Maize**  Leaf (V6) Mean a 27 33 36

Range 17 - 38 25 - 50 27 - 50 CI b 22 - 33 26 - 41 29 - 44 Root (V6) Mea n 13 18 16

Range 9.0 - 20 14 - 23 11 - 23 CI 11 - 15 15 - 22 14 - 19 Leaf (V9) Mean 33 46 40

Range 28 - 39 35 - 58 35 - 47 CI 30 - 37 42 - 50 37 - 44 Root (V9) Mean 13 17 17

Range 7.8 - 19 13 - 22 9.3 - 27 CI 10 - 17 13 - 21 13 - 21 Whole Pl ant (V9) Mean 76 79 85 Range 58 - 100 52 - 100 66 - 100 CI 67 - 86 70 - 89 76 - 96 Leaf (R1) Mean 68 71 81

Range 43 - 130 56 - 110 66 - 110 CI 56 - 83 58 - 87 67 - 100 Pollen (R1) Mean 0.34 <0.32 c 0.33

Range <0.32 c

- 0.53 <0.32 c <0.32 c

- 0.44 CI NAd NA NA Root (R1) Mean 9.2 10 11

Range 4.2 - 15 6.6 - 16 7.2 - 15 CI 7.2 - 12 7.9 - 13 8.4 - 14 Whole Plant (R1) Mean 66 75 69 Range 44 - 100 58 - 110 54 - 98 CI 57 - 75 66 - 86 60 - 79 Forage (R4) Mean 29 28 30

Range 18 - 52 17 - 42 20 - 52 CI 23 - 35 23 - 34 24 - 36 Leaf (R4) Mean 90 100 100

Range 66 - 110 84 - 130 52 - 130 CI 81 - 99 95 - 110 93 - 110 Root (R4) Mean 6.4 8.0 8.0

Range 2.1 - 12 3.9 - 14 1.1 - 13 CI 3.6 - 9.3 5.1 - 11 5.1 - 11 Grain (R6) Mean 1.1 1.4 1.4

Ra nge 0.54 - 2.3 0.69 - 2.0 0.75 - 2.3 CI 0.84 - 1.4 1.1 - 1.8 1.1 - 1.8 Leaf (R6) Mean 72 72 59

Range 41 - 110 2.0 - 190 2.2 - 130 CI 36 - 108 36 - 110 23 - 96Pioneer Hi - Bred International, Inc. Page 86 of 25 8

4114 Maize **Table 12. Cry35Ab1**  **Concentrations (ng/mg Tissue Dry Weight) (continued)**  **Tissue (Growth S tage)**  **4114 Maize 1507x59122**  **Maize 59122 Maize**  Root (R6) Mean 6.9 8.9 10.0

Range 2.2 - 14 3.3 - 18 3.3 - 17 CI 3.5 - 10 5.5 - 12 6.6 - 13 Whole Plant (R6) Mean 21 31 27 Range 13 - 54 20 - 46 13 - 64 CI 15 - 30 22 - 44 19 - 38 a Least squares mea ns (estimated from statistical models; see Appendix 5 for additional details) . b Statistical Confidence Interval c < Lower Limit of Quantification (LLOQ); indicates the values of the sample(s) were detected below the assay LLOQ d Greater than 80% of sample s were <LLOQ, statistical analysis not available (NA)Pioneer Hi - Bred International, Inc. Page 87 of 25 8

4114 Maize **Table 13**  **. PAT Concentrations (ng/mg Tissue Dry Weight) Tissue**  **(Growth Stage) 4114 Maize**  **1507 Maize 1507x59122**  **Maize 59122 Maize**  Leaf (V6) Mean a 9.0 5.6 16 12

Range 4.2 - 14 3.1 - 10 7.2 - 24 8. 4 - 18 CI b 7.6 - 11 4.7 - 6.6 13 - 18 9.8 - 14 Root (V6) Mean 0.44 0.29 1.7 1.1

Range 0.14 - 0.78 0.075 - 0.45 0.93 - 2.9 0.42 - 2.1 CI 0.32 - 0.62 0.21 - 0.40 1.2 - 2.3 0.78 - 1.5 Leaf (V9) Mean 9.8 6.6 21 12

Range 4.8 - 15 2.9 - 8.4 10 - 32 4.1 - 16 CI 6.7 - 13 3.4 - 9.7 17 - 24 8.8 - 15 Root (V9) Mean 0.65 0.36 2.2 1.5

Range 0.39 - 0.90 0.22 - 0.57 1.1 - 3.0 0.99 - 2.6 CI 0.30 - 1.00 0.013 - 0.71 1.8 - 2.5 1.1 - 1.8 Whole Plant (V9) Mean 8.7 3.6 15 11

Range 6.6 - 11 3.0 - 4.2 11 - 18 8.0 - 15 CI 7.7 - 9.8 3.2 - 4.0 13 - 17 9.6 - 12 Leaf (R1) Mean 14 6.5 27 19

Range 5.0 - 24 2.0 - 9.6 17 - 31 12 - 26 CI 9.9 - 17 2.8 - 10 23 - 31 15 - 22 Pollen (R1) Mean <0.28 c <0.28 c <0.28 c <0.28 c

Range <0.28 c <0.28 c <0.28 c <0.28 c CI NAd NA NA NA Root (R1) Mean 0.44 0.24 1.1 0.98

Range 0.30 - 0.72 0.12 - 0.48 0.60 - 2.3 0.60 - 1.6 CI 0.34 - 0.57 0.18 - 0.31 0.86 - 1.4 0.76 - 1.3 Whole Plant (R1) Mean 4.9 2.4 8.4 6.7

Range 3.2 - 7.4 1.6 - 3.8 5.4 - 14 4.6 - 9.4 CI 4.0 - 5.9 1.9 - 2. 9 6.9 - 10 5.5 - 8.1 Forage (R4) Mean 1.9 0.75 3.5 2.5

Range 1.1 - 2.8 0.54 - 1.2 1.8 - 4.8 1.0 - 4.0 CI 1.4 - 2.4 0.23 - 1.3 3.0 - 4.0 2.0 - 3.0 Leaf (R4) Mean 11 4.2 18 13

Range 5.7 - 20 2.6 - 5.5 9.6 - 26 3.6 - 22 CI 7.5 - 14 0.95 - 7.3 15 - 2 1 10 - 17 Root (R4) Mean 0.16 0.13 0.62 0.33

Range <0.069 c

- 0.39 <0.069 c

- 0.33 0.30 - 1.9 <0.069 c

- 0.87 CI 0.10 - 0.25 0.083 - 0.20 0.40 - 0.95 0.22 - 0.51 Grain (R6) Mean <0.069 c <0.069 c 0.089 0.071

Range <0.069 c <0.069 c <0.069 c

- 0.45 <0.069 c

- 0.10 CI NA NA NA NA Leaf (R6) Mean 0.52 0.25 0.33 0.25

Range <0.14c

- 2.0 <0.14c

- 1.0 <0.14c

- 2.5 <0.14c

- 1.1 CI NA NA NA NAPioneer Hi - Bred International, Inc. Page 88 of 25 8

4114 Maize **Table 13**  **. PAT Concentrations (ng/mg Tissue Dry Weight) (continued)**  **Tissue (Growth Stage)**  **4114 Maize 1507 Maize**  **150 7x59122**  **Maize 59122 Maize**  Root (R6) Mean 0.13 0.13 0.41 0.41

Range <0.069 c

- 0.66 <0.069 c

- 0.39 0.078 - 1.8 0.11 - 1.6 CI 0.084 - 0.21 0.084 - 0.21 0.26 - 0.64 0.26 - 0.64 Whole Plant (R6) Mean 0.090 0.073 0.18 0.13

Range <0.046 c

- 0.76 <0.046 c

- 0.28 <0.046 c

- 1.1 <0.046 c

- 0.64 CI 0.028 - 0.29 0.023 - 0.24 0.058 - 0.58 0.039 - 0.40 a Least squares means (estimated from statistical models; see Appendix 5 for additional details) . b Statistical Confidence Interval c < Lower Limit of Quantificati on (LLOQ); indicates the values of the sample(s) were detected below the assay LLOQ d Greater than 80% of samples were <LLOQ, statistical analysis not available (NA)Pioneer Hi - Bred International, Inc. Page 89 of 25 8

4114 Maize **Table 14** **. Summary of 4114 Maize Protein Concentrations as a Ratio of 1507, 59122, and/o**  **r 1507x59122 Maize**  **Tissue (Growth Stage)**  **Protein Concentration Ratio**

**4114 Maize:1507 Maize**  **4114 Maize:59122 Maize**  **4114 Maize:1507x59122 Maize**  **Cry1F** Leaf (V6) 0.611 0.611 Leaf (V9) 0.746 0.606 Leaf (R1) 0.813 0.765 Leaf (R4) 0.919 0.919 Leaf (R6) 2.53 5.41 Root (V6) 0.737 0.667 Root (V9) 0.694 0.725 Root (R1) 0.786 0.902 Root (R4) 0.864 0.717 Root (R6) 0.760 0.809 Whole Plant (V9) 0.750 0.750 Whole Plant (R1) 0.707 0.762 Whole Plant (R6) 0.837 1.08 Pollen (R1) 1.52 1.46 Forage (R4) 0.929 0.857 Grain (R6) 1.03 1.18 **Cry34Ab1** Leaf (V6) 0.636 0.656 Leaf (V9) 0.667 0.619 Leaf (R1) 0.667 0.714 Leaf (R4) 0.579 0.550 Leaf (R6) 2.32 3.17 Root (V6) 0.654 0.586 Root (V9) 0.568 0.583 Root (R1) 0.655 0.655 Root (R4) 0 .719 0.657 Root (R6) 0.486 0.563 Whole Plant (V9) 0.561 0.561 Whole Plant (R1) 0.561 0.571 Whole Plant (R6) 0.750 0.750 Pollen (R1) 0.209 0.200 Forage (R4) 0.605 0.559 Grain (R6) 1.00 1.00Pioneer Hi - Bred International, Inc. Page 90 of 25 8

4114 Maize **Table 14** **. Summary of 4114 Maize Protein Concen** **trations as a Ratio of 1507, 59122, and/or 1507x59122 Maize**  **(continued) Tissue**  **(Growth Stage) Protein Concentration Ratio** 

**4114 Maize:1507 Maize**  **4114 Maize:59122 Maize**  **4114 Maize:1507x59122 Maize**  **Cry35Ab1** Leaf (V6) 0.750 0.818 Leaf (V9) 0.825 0.717 Leaf (R1) 0.840 0.958 Leaf (R4) 0.900 0.900 Leaf (R6) 1.22 1.00 Root (V6) 0.813 0.722 Root (V9) 0.765 0.765 Root (R1) 0.836 0.920 Root (R4) 0.800 0.800 Root (R6) 0.690 0.775 Whole Plant (V9) 0.894 0.962 Whole Plant (R1) 0.957 0.880 Who le Plant (R6) 0.778 0.677 Pollen (R1) 1.03 1.06 a Forage (R4) 0.967 1.04 Grain (R6) 0.857 0.800 **PAT** Leaf (V6) 1.61 0.750 0.563 Leaf (V9) 1.48 0.817 0.467 Leaf (R1) 2.15 0.737 0.519 Leaf (R4) 2.62 0.846 0.611 Leaf (R6) 2.08 2.08 1.58 Root (V6) 1.52 0.400 0.259 Root (V9) 1.81 0.464 0.295 Root (R1) 1.83 0.449 0.400 Root (R4) 1.23 0.485 0.258 Root (R6) 1.00 0.317 0.317 Whole Plant (V9) 2.42 0.791 0.580 Whole Plant (R1) 2.04 0.731 0.583 Whole Plant (R6) 1.23 0.692 0.500 Pollen (R1) 1.00 b 1.00 b 1.00 b Forage (R4) 2.53 0.760 0.543 Grain (R6) 1.00 b 0.972 0.775 a 1507x59122 m aize mean Cry35Ab1 protein concentration value was < Lower Limit of Quantification (LLOQ); therefore, the LLOQ was used. b Mean PAT concentration values were < LLOQ; therefore, the LLOQ was used.Pioneer Hi - Bred International, Inc. Page 91 of 25 8

4114 Maize **3. Potential**  **Allergenicity and Toxicity of 4114 Maize** The allergenicity and toxicity of 4114 maize was evaluated by examining the allergenic potential of maize as a crop and by assessing the allergenic and toxic potential of t he introduced Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins. The proteins expressed in 4114 maize are identical to those in previously approved events 1507 and 59122 maize, as discussed in Section 1. As describ ed in Section 2, the equivalency of the protei ns expressed in 4114 maize to those expressed in 1507 and 59122 maize was established by sequencing of the protein coding regions of the insertion and by western blot. These analyses demonstrated that the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins introd uced into 4114 maize are equivalent to those proteins expressed in 1507x59122 maize (Section 2 and Appendix 2). The refore, previous safety studies conducted for 1507 and 59122 maize on the purified proteins are relevant for 4114 maize. The Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins were derived from common soil bacteria and have a history of safe use in agricultural crop commodities. These proteins have been present in commercial maize varieties such as 1507, 59122, and 1507x59122 maize since 2003, 2006, and 2006, respectively; these commercial lines contain familiar traits and are currently licensed broadly across the seed industry and are planted on approximately 16% of U.S. maize acres (GfK Kynetec, 2010) . In addition to these varieties the PAT protein has also been present in a number of other commercial crops and commercially planted in the U.S. since 1996 . The Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins were previously determined to have little potential for toxicity or allergenicity based on bi oinformatics analyses as well as toxicity and allergenicity studies. In this section, published or previously submitted information on the allergenicity and toxicity potential of these proteins is summarized and updated bioinformatics analyses on the prote ins are included. **3 -** **A. Allergenicity of Maize**  Although maize is widely grown worldwide with overall production at over 700 million tons per year and is the fourth most consumed food calorically, it is not considered a major allergenic food (Hefle et al., 1996; Moneret-Vautrin et al., 1998). I n a few case studies, allergenic reactions were reported and maize allergens identified. Specifically, the 9 kDa Zea m 14 protein (i.e., the maize lipid transfer protein [LTP]) was identified as the major allergen ic protein in maize ( Fasoli et al., 2009; Pasini et al., 2002; Pastorello et al., 2000; Pastorello et al., 2003 ). LTPs are small proteins that facilitate the transfer of phospholipids and other lipids across membranes. These proteins are widely distributed throughout the plant kingdom and belong to the pathogenesis- related (PR) protein family (Hoffmann -Sommergruber, 2002). The Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins are neither related to LTPs nor involved in lipid transfer across membranes . Because maize is not a common allergenic food , it is not expected that the genetic modification in 4114 maize would alter the allergenic potential of maize.Pioneer Hi - Bred International, Inc. Page 92 of 25 8

4114 Maize **3 -**  **B. Assessment of the Allergenicity of the Introduced Proteins in 4114 Maize**  The allergenic potential of the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins was previously assessed for 1507 and 59122 maize through a “weight-of-evidence” approach and provided support that the proteins were unlikely to be allergens; this evaluation of allergenicity also has relevance for 4114 maize because it contains the same introduced proteins. A weight-ofevidence approach takes into account a variety of relevant factors and experimental observations used to derive an overall assessment of the allergenic potential of the introduced protein, because no single factor has been recognized as the primary indicator for allergenic potential, and no validated animal model that is predictive of al lergenic potential is available (Codex, 2003). The allergenicity potential assessments are therefore based on what is known about food allergens, including the amino acid sequence id entity to known human allergens; physicochemical properties such as thermal stability or stability to pepsin or pancreatin digestion in vitro (Thomas et al. , 2004 ) ; glycosylation status, and history of exposure a nd safety of the gene(s) source. An important part of the weight-of-evidence approach for assessing the potential allergenicity of proteins introduced and expressed in genetically modified plants is an am ino acid sequence comparison of the introduced protein against the sequences of known allergens. This is performed to identify the degree of potential for cross- reactivity between allergenic proteins and the protein of interest. These evaluations employ two techniques; a search for contiguous, identical stretches of eight amino acid residues or longer, and an identity search using the FASTA3 5 alignment algorithm (Pearson and Lipman, 1988) to search for alignments of 80 amino acid residues or longer possessing a 35% sequence identity or greater (Codex, 2003; FAO/WHO, 2001) . I f the introduced protein lacks both potential allergenic epitopes and identity to known allergens, the protein is “not a known allergen and is unlikely to be cross- reactive to known a llergens” (Codex, 2003; Codex, 2009). The allergenic protein sequences used for all compariso ns were from the peer- reviewed database from the Food Allergy Research and Resource Program (FARRP) at the Department of Food Science and Technology at the University of Nebraska at Lincoln (FARRP Release 11 - January 2011; http://www.allergenonline.com), which contains 1491 entries representing both putative and proven food, environmental, and contact allergens as well as proteins implicated in celiac disease. **Source and History of Safe**  **Exposure to the**  **Cry1F, Cry34Ab1, Cry35Ab1 , and PAT Proteins**  The Cry1F, Cry34Ab1, and Cry35Ab1 proteins were derived from Bacillus thuringiensis, which is a naturally-occurring, common bacterium that is not a known mammalian pathogen and is present in soil, dust, insects, and leaves (EPA, 1998; McClintock et al. , 1995; Sc hnepf et al. , 1998). Some strains of B. thuringiensis have been shown to be opportunistic pathogens; however, this pathogenicity was not related to the Cry proteins ( Hernandez et al. , 1999 ). Microbial preparations of B. thuringiensis containing Cry proteins have been used safely asPioneer Hi - Bred International, Inc. Page 93 of 25 8

4114 Maize pesticide sprays for decades, and have been deemed to pose no toxic effects to mammals ( EPA, 1998 ). The PAT protein from Streptomyces viridochromogenes has a safe history of exposure to humans, animals, and the environment. The source organism S. viridochromogenes is widespread in soil and is not associated with human, animal, or plant pathogens (Hérouet et al. , 2005). Related PAT proteins are found in at least six other species of common soil bacteria, of which none have been reported as toxic or allergenic to humans or animals (Bartsch and Tebbe, 1989; Hérouet et al. , 2005; Kutzner, 1981). Furthermore, PAT has been a protein present in commercial maize events, as well as other crops, since 1996. These proteins have also been present in commercial maize varieties such as 1507, 59122, and 1507x59122 maize since 2003, 2006, and 2006, respectively. These commercial lines are current ly licensed broadly across the seed industry; in 2010, commercial products containing 1507x59122 maize were grown on approximately 14 million acres, which represents approximately 16% of U.S. maize acres (GfK Kynetec, 2010) . Because of the broad use of 15 07x59122 maize in commercial planting and lack of reported adverse effects, there is a history of safe use and exposure of these proteins. **The Potential Allergenicity of the Cry1F Protein** Previous work has established that the Cry1F protein does not have properties typically associated with allergens. The Cry1F protein was found to have no sequence homology with known allergens, to possess no N -glycosylation sites, to be rapidly degraded in simulated gastric fluid (SGF ) and simulated intestinal fluid (SI F ), and to be thermolabile ( EPA, 2010a; Ladics et al. , 2006). The bioinformatics analysis was repeated to include protein allergens that have been added to the updated FARRP allergen database since the original analysis was conducted. When the Cry1F protein sequence was used as a query in the search for contiguous, identical stretches of amino acids 8 residues or greater in length , no 8 residue or greater matches to known or putative allergens were returned. Similarly, there were no alignments greater than 35% identity over 80 or greater amino acids. The updated bioinformatics analysis, the history of safe use of Cry1F, and studies demonstrating the lack of potential allergenicity, support the conclusion that the Cry1F protein is unlikely to be an al lergen. **The Potential Allergenicity of the Cry34Ab1 and Cry35Ab1 Proteins**  Previous work has established that the Cry34Ab1 and Cry35Ab1 proteins do not have properties typically associated with allergens. These proteins were found to have no sequence ho mology with known allergens, to possess no N -glycosylation sites, and to be degraded in SGF (EPA, 2010b).Pioneer Hi - Bred International, Inc. Page 94 of 25 8

4114 Maize The bioinformatics analysis was repeated to include protein allergens that have been added to the updated FARRP allergen database since the origina l analyses were conducted. When the Cry34Ab1 and Cry35Ab1 protein sequences were used as a query in the search for contiguous, identical stretches of eight amino acids residues or greater in length , no eight residue or greater matches to known or putative allergens were returned. Similarly, there were no alignments that were greater than 35% identity over 80 or greater amino acids. The updated bioinformatics analysis, in addition to the history of safe use of the Cry34Ab1 and Cry35Ab1 proteins and studie s demonstrating the lack of potential allergenicity, support the conclusion that these proteins are unlikely to be allergens. **The Pote ntial Allergenicity of the**  **PAT Protein** Previous work has established that the PAT protein does not have properties typically associated with allergens ( Hérouet et al. , 2005). The PAT protein was found to have no sequence homology with known allergens, to possess no N -glycosylation sites, to be rapidly degraded in SGF and SIF, and to be thermolabile ( Hérouet et al. , 2005). The bioinformatics analysis of the PAT protein was repeated to take into account protein allergens that have been added to the updated FARRP allergen database since the original work took place. When the PAT protein sequence was used as a query in the search for contiguous, identical stretches of eight amino acids residues or greater in length , no eight contiguous amino acid residue or greater matches to known or putative allergens were returned. Similarly, t he re were no alignments that were greater th an 35% identity over 80 or greater amino acids. The updated bioinformatics analysis, in addition to the history of safe use of PAT protein and studies demonstrating the lack of potential allergenicity support the conclusion that the PAT protein is unlikely to be an allergen. **3 -** **C. Assessment of the Toxicity of the Proteins Expressed in 4114 Maize**  The potential toxicity of the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins was previously assessed for 1507 and 59122 maize by acute toxicity studies and by bi oinformatic comparison of the protein sequences to publicly available protein sequences . These assessments provided evidence that the proteins are unlikely to be toxic and are also relevant for the assessment of the proteins in 4114 maize . As described e arlier, the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins expressed in 4114 maize have been present in commercial maize lines such as 1507, 59122, and 1507x59122 maize for a number of years and represent a sizable portion of current maize acreage . To date, no known adverse reactions to these prote i ns have been reported. Updated bioinformatics analyses were conducted on the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins to include proteins that have been added to public datasets since the original analysesPioneer Hi - Bred International, Inc. Page 95 of 25 8

4114 Maize we re conducted. The amino acid sequences of each of the proteins were compared with publicly available protein sequences. P roteins most similar to those produced in 4114 maize were manually inspected to identify any that could be potentially toxic to human s or animals. A close match could be an indicator of toxicological potential of these proteins. To search for potential similarity to known toxins, the proteins were queried using the BLASTP 2.2.13 algorithm against Release 183.0 (4/15/11) of the NCBI E ntrez Protein dataset, which incorporates non - redundant entries from all GenBank and RefSeq nucleotide translations along with protein sequences from SWISS- PROT (http://www.expasy.org/sprot/), PIR (Protein Information Resource; http://pir.georgetown.edu/), PRF (Protein Research Foundation; http://www.prf.or.jp/index-e.html), and wwPDB (Worldwide Protein Data Bank; http://www.wwpdb.org/) . The scoring matrix used was the default (BLOSUM62), low complexity filtering was tu rned off, and the number of alignments returned was set to the maximum value of 2000. One of the most important parameters to monitor when performing similarity searches is the expectation score, or E score. This score represents the probability that a p articular alignment is due to random chance and can be used to evaluate the significance of an alignment. The calculated E score depends on the overall length of the aligned sequences (including inserted gaps), the number of identical and conserved residu es within the alignment, and the size of the database ( Baxevanis, 2005 ; Pearson and Lipman, 1988 ) . W hen examining an alignment between two protein sequences, a very small E score is more likely to reflect a true similarity, while a large E score is more l ikely to be produced by chance and therefore less biologically relevant. A cutoff expectation ( E) value of 1.0 was used to generate biologically meaningful similarity between the protein of interest and proteins in the NCBI datasets. A lthough a statistically significant sequence similarity generally requires a match with an expectation value less than 0.01, a cutoff of E < 1.0 insures that proteins with even limited similarity will not be overlooked in the search (Pearson, 2000). **The Potential Toxicity of the**  **Cry1F Protein** Previous work has established that the Cry1F protein does not have properties typically associated with toxins. Cry1F was found to lack sequence homology with known toxins and was not found to be acutely toxic in mice (EPA, 2010a) . In the updated bioinformatics analysis, t he Cry1F protein sequence returned 544 protein accessions with an E score less than 1.0. S ix accessions possessed virtually complete identity ( E = 0); the top scoring alignment was to the originally isolated Cry1 F protein (Chambers et al. , 1991) at 99% identity while the remaining 5 accessions were to closely related Cry1F- like proteins from Bacillus thuringiensis . Of the remaining 538 alignments returned, 465 are from the soil bacterium Bacillus thuringiensis, i ncluding 19 accessions classified as hypothetical or unnamed. These proteins are known by a variety of descriptions (parasporal crystal protein,Pioneer Hi - Bred International, Inc. Page 96 of 25 8

4114 Maize crystal protein, insecticidal protein, pesticid al crystal protein, insecticidal delta -endotoxin, crystalline e ntomocidal protoxin, crystal protein, insecticidal toxin, and delta -endotoxin, among others), and represent a class of highly specific insect toxins with a well characterized mode of action that are considered safe for humans and animals (Bravo et al., 2007). Twenty- nine additional matches are to synthetic constructs, consisting primarily of transformation vectors containing intact, truncated, or chimeric versions of the Cry proteins mentioned above. The remaining 44 accessions represent Cry- related prote in sequences from other Bacillus families, or translations of nucleotide sequences generated during genome sequencing projects. None of the protein sequences returned by the BLASTP search identified safety concerns that might arise from the expression of Cry1F in genetically modified plants. The updated bioinformatics analysis, in addition to the history of safe use of Cry1F and studies demonstrating the lack of potential toxicity, support the conclusion that the Cry1F protein is unlikely to be a toxin to humans or animals . **The Potential Toxicity of the Cry34Ab1 and Cry35Ab1 Proteins** Previous work has established that the Cry34Ab1 and Cry35Ab1 proteins do not have properties typically associated with toxins. The Cry34Ab1 and Cry35Ab1 proteins were foun d to not be acutely toxic in mice ( EPA, 2010b; Juberg et al. , 2009 ). F urthermore, earlier submitted bioinformatic analysis showed the proteins did not share sequence homology with any known protein toxins (EPA, 2010b) . In the updated bioinformatics analysis, t he Cry34Ab 1 protein sequence returned 41 protein accessions with an expectation ( E) score less than 1.0. The top 16 accessions are derived from Bacillus thuringiensis ( Bt) . The two highest scoring accessions had identities of 100%; one is listed as a “13.6 kDa insecticidal crystal protein” while the other is annotated as a “Cry34Ab 1- like” protein. The fourteen other accession s possess a variety of descriptions (crystal protein, insecticidal crystal protein, Cry34Ab1- like protein, among others), and represent a class of highly specific insect toxins with a well characterized mode of action that are considered safe for humans and animals (Bravo et al. , 2007). The next highest scoring sequence (31% sequence identity) is to a recently added (March 20 11) putative protein translation from the Streptomyces whole genome sequencing project (RefSeq accession ZP_08234223) that is described as an “aegerolysin”; this sequence has a significant match to the Pfam aegerolysin domain (PF06355) (NCBI, 2011). Seque nces from these types of projects are not peer reviewed for annotation accuracy; while they may possess domain similarities to certain classes of proteins, they often have unconfirmed function (NCBI, 2009; NCBI, 2011). In addition, this match is not surprising given that three Cry34Ab1- like protein sequences were used as a scaffold to define the Pfam aegerolysin domain (NCBI, 2011). Aegerolysins were originally isolated from edible mushrooms and are associated with primordial and fruiting body formation; these proteins have also been isolated from bacteria and plants (Berne et al. , 2002; Berne et al. , 2009). O nly certain fungal aegerolysins have beenPioneer Hi - Bred International, Inc. Page 97 of 25 8

4114 Maize shown to have hemolytic properties (Berne et al. , 2002). W hile proteins may be described as aegerolysins and share the aegerolysin domain, many of these proteins possess diverse biological functions unrelated to hemolytic properties ( Berne et al. , 2009). The few known hemolytic fungal aegerolysins include Aa - Pri1 from Agrocybe aegerita ( i.e., edible poplar or chestnut mushroom; Fernandez and Labarère, 1997), pleurotolysin A and ostreolysin from Pleurotus ostreatus (i.e., edible oyster mushroom; Berne et al., 2005; Sakurai et al., 2004 ), and Asp- hemolysin from Aspergillus fumigatus (Ebina et al. , 1994). AC LUSTALW alignment was used to further compare the similarity of Cry34Ab1 and the ZP_08234223 accession with these sequences. Both Cry34Ab1 and the ZP_08234223 accession show very limited identity (13 -20%) to any of these known fungal toxins, while the toxi n sequences are more homologous to each other (41-80% identity). F urthermore, none of these known fungal toxins appeared in the Cry34Ab1 BLAST search. Therefore, it is unlikely that Cry34Ab1 possesses the toxic properties of the known fungal aegerolysins . Of the remaining 24 sequences, 10 represent insecticidal crystal proteins from non -Bt species, while 12 others are annotated as hypothetical or predicted proteins derived from the translations of nucleotide sequences generated during genome sequencing p rojects. The two remaining sequences are low significan ce matches, one annotated as a beta - N acetylglucosaminidase and the other a lysophospholipase Plb1. None of the protein sequences returned by the BLASTP search identified safety concerns that might a rise from the expression of Cry34Ab 1 protein in genetically modified plants. The Cry35Ab1 protein sequence returned 45 protein accessions with an expectation ( E) score less than 1.0. N ine accessions possessed an E score of 0; all 9 were derived from Bacillus thuringiensis. The top scoring alignment, a “43.8 kDa insecticidal crystal protein”, displayed 100% identity over the entire length of the Cry35Ab 1 sequence. There were an additional seven accessions from Bacillus thuringiensis, and 19 accessions di splaying similarity to the mosquitocidal Bin toxins from Lysinibacillus sphaericus ( synonym: Bacillus sphaericus) and Bacillus cereus (Baumann et al. , 1988; Baumann et al. , 1991; reviewed by Federici et al. , 2003). Of the remaining 10 sequences, six seque nces of low significance ( E=0.053 -0.45) represent translations of nucleotide sequences generated during genome sequencing projects and four accessions are described as hypothetical proteins. None of the protein sequences returned by the BLASTP search identified safety concerns that might arise from the expression of the Cry35Ab1 protein in genetically modified plants. The updated bioinformatics analysis, in addition to the history of safe use of the Cry34Ab1 and Cry35Ab1 proteins and studies demonstrating the lack of potential toxicity, support the conclusion that these proteins are unlikely to be toxins to humans or animals . **The Potential Toxicity of the PAT Protein**  Previous work has established that the PAT protein does not have properties typically associated with toxins. PAT was found to lack sequence homology with known toxins and were found not to be acutely toxic in mice ( Hérouet et al. , 2005) .Pioneer Hi - Bred International, Inc. Page 98 of 25 8

4114 Maize In the updated bioinformatics analysis, t he PAT protein sequence returned 1752 protein accessions wit h an expectation ( E) score less than 1.0. The highest scoring alignments ( E = 1 x 10- 105 and 1 x 10-88, respectively) were attributed to PAT proteins from Streptomyces viridochromogenes (100% identity over the entire 183 amino acid length of the query seq uence) and Streptomyces hygroscopicus (84% identity to the query sequence). Both of these proteins have undergone extensive safety evaluations and have been deemed safe for expression in transgenic plants (Hérouet et al. , 2005). Seven other accessions re presented sequences derived from these two bacterial strains. Twelve of the alignments were to synthetic constructs, consisting primarily of transformation vectors containing portions of the PAT proteins mentioned above. Of the remaining 1731 sequences, 518 were annotated as phosphinothricin acetyltransferases . This bacterial enzyme deactivates the non -selective herbicide glufosinate, which is a potent inhibitor of glutamine synthesis (Dröge- Laser et al. , 1994) and is widely used for the generation of glufosinate-tolerant transgenic plants. Another 861 alignments are with sequences characterized as known or putative acetyltransferases, N -acetyltransferases, or GCN5 or GNAT acetyltransferases, based upon the possession of an acetyltransferase domain present in the PAT query sequence . O ne hundred and thirty-seven other accessions were classified as acetyltransferase related sequences including sortases, acyltransferases, antibiotic or toxin resistance proteins, ribosomal - protein -alanine acetyltransferases, or histone acetyltransferases. Ten other accessions were annotated as ArsR family transcriptional regulators, containing both the Arsenical Resistance Operon Repressor domain as well as the acetyltransferase domain. Fourteen accessions described as possessing a range of functionalities were returned; all of these sequences contained additional domains responsible for their annotations. Lastly, 191 of the accessions returned were classified as hypothetical, predicted, putative, unnamed, or unknown proteins. None of the protein sequences returned by the BLASTP search identified any safety concerns from the expression of PAT protein in genetically modified plants. The updated bioinformatics analysis, in addition to the history of safe use of the PAT protein and studies demonstrating the lack of potential toxicity, support the conclusion that this protein is unlikely to be a toxin. **3 -**  **D. Overall Conclusions on the**  **Potential Allergenicity and Toxicity of 4114 Maize**  The potential for allergenicity and toxicity of 4114 maize was evaluated by examining the allergenic potential of maize as a crop and by assessing the allergenic and toxic potential of the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins. Maize is not a common allergenic food and the modification i n 4114 maize is not expected to alter the allergenic potential of maize. The Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins have been assessed previously for 1507 and 59122 maize and have been determined to be unlikely to be potential allergens or toxins to humans and animals. Previous assessments of these proteins included bioinformatic analyses, digestibility, N -glycosylation studies, acute protein toxicity studies, and in some cases, heat stability studies; these studies are relevant for the assessment of the proteins in 4114 maize.Pioneer Hi - Bred International, Inc. Page 99 of 25 8

4114 Maize Updated bioinformatic analyses support the original conclusions that the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins are unlikely to be allergens or toxins. Additionally, 1507x59122 maize has been commercially available in th e U.S. since the 2006 growing season with no reports of allergenicity or toxicity. These data support the conclusion that the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins in 4114 maize are unlikely to be potential allergens or toxins to humans and animals, and therefore support the food and feed safety of these proteins. These data have been provided to EPA in support of the safety of these proteins and also will be submitted to the FDA for the food and feed safety assessment of 4114 maize.Pioneer Hi - Bred International, Inc. Page 100 of 25 8

4114 Maize **4. Comparati** **ve Assessment of 4114 Maize** In order to assess the safety of 4114 maize relative to conventional maize, a comparative assessment was conducted that was designed to be independent of the intended effects of the insect- resistant and herbicide-tolerant traits . The comparative assessment uses non transformed (i.e., conventional ) comparators for compositional and agronomic comparisons and will establish the safety of 4114 maize relative to varieties that have a history of safe use in the environment and as foo d and feed . The objective is to determine if the modified organism presents any new or greater risks relative to its comparator, or whether it can be used interchangeably with its comparator without negatively affecting human and animal health and the e nvironment in which it is grown . The compositional and agronomic analyses will highlight any differences between 4114 maize and its comparators in order to determine if 4114 maize is as safe as other lines that have a history of safe use. The studies were designed to eliminate the intended effects of the introduced traits (i.e., trait efficacy) so that appropriate comparisons would be able to be made between 4114 maize and conventional maize and unintended effects due to the 4114 maize insertion would also be evaluated . Therefore, the receiving environment was managed to minimize insect and weed pressure in the evaluations. To ensure an accurate comparative evaluation of the transgenic plant, a proper selection of comparator plants is important. As descri bed ear lier in Section 2, non -transgenic near- isoline maize lines were selected as comparators for 4114 maize in these analyses, since they had a similar genetic background (i.e. , approximately 99% similar). Statistical comparisons were made to these comp arators to determine if there were any significant differences. I n order to determine if any observed statistical differences were indicative of differences with conventional maize varieties, a statistical tolerance interval was established from non transgenic commercial maize lines for the compositional and agronomic comparisons. These commercial lines are typical of those grown in maize-growing regions; represent a wide range of varieties that would normally be planted commercially; and represent the no rmal range of variation of the maize crop. In addition, for the compositional assessment, publicly available information was gathered on the range of natural variation of maize analyte concentrations (i.e., literature range). If the measured values of 41 14 maize fell within the statistical tolerance interval or the literature range, then these measured values would be considered comparable to conventional maize. The compositional and agronomic comparative assessments of 4114 maize are discussed further below. These analyses indicated that 4114 maize was comparable to conventional maize with respect to the compositional analytes and agronomic characteristics measured and independently of the intended effects of the insect- resistant and herbicide-tolerant traits . Overall, these analyses indicate that 4114 maize is as safe as conventional maize varieties and does not pose a greater risk than conventional maize varieties in food, feed, and the environment .Pioneer Hi - Bred International, Inc. Page 101 of 25 8

4114 Maize **4 -** **A .**  **Compositional Assessment** Compositional anal ysis of 4114 maize grain and forage was used to evaluate any changes in the levels of key nutrients, anti - nutrients, and secondary metabolites compared to the non transformed, near- isoline control. For this analysis, grain was selected because of its use in both food and feed , and forage was selected for its use as feed . These analyses provide d an indication whether 4114 maize is as nutritious as conventional maize. The U.S. FDA will review the details of the compositional analyses as a component of the food and feed safety assessment of 4114 maize. Comprehensive compositional analyses were performed on grain and forage from 4114 maize and a near- isoline control grown in 2010 at six field locations in the U.S. and Canada ( see Section 4- B; Table 28; Experiment B ). These sites are representative of commercial maizegrowing areas. The F1* 5 generation of seed (Section 2; Figure 10 and Table 3 ) was planted because F1 hybrid seed is representative of seed that growers would plant in commercial maize fields. The near- isoline control plants had a genetic background approximately 9 9% similar to that of the 4114 maize generation used, but did not go through the transformation process. Although 4114 maize contains traits that are efficacious against certain lepi dopteran and corn rootworm target pests and glufosinate-ammonium tolerance, each field trial site was managed to maintain a relatively insect-free and weed -free environment. Evaluation in this environment ensured that compositional characteristics measure d would be independent of the intended effects of the insect- resistant and herbicide-tolerant traits; therefore, these evaluations would appropriately compare 4114 maize to conventional maize and also assess unintended effects of the transgene. Compositio n was not impacted by the intended effects of the traits and therefore could be used to assess comparability of 4114 maize to conventional maize. Each location utilized a randomized complete block design containing four blocks. E ach block contained 4114 maize and the control maize planted in two- row plots. One forage sample (subsample of 3 whole plants) was collected at the R4 growth stage from 4114 maize and the control maize from each of the four blocks. Forage samples were obtained by cutting the aerial portion of the plants from the root system approximately 1 inch (2.5 cm) above the soil surface after secondary or tertiary ears with exposed silks were removed. The plants (including the primary ear) were then cut into sections approximately 3 inc hes (7.5 cm) or less, and approximately 1/3 of the total sample was collected in a pre- labeled plastic- lined cloth bag. One grain sample (equal to five pooled ears) was collected at the R6 growth stage from 4114 maize and the control maize from each of th e four blocks. A ll samples were collected from impartially selected, healthy individual plants that had previously been self- pollinated. Forage and grain were also collected from non - modified commercial maize hybrids (reference hybrids) in two separate experiments. P ioneer® hybrids 34M94, 33G26, 33J24, and 3394 were analyzed in 2003 at six field locations in maize-growing areas of North America (Bagley, IA; York, NE; Chula, GA; New Holland, OH; Larned, KS and Hereford, PA). Pioneer® hybrids 38B85, 37Y12, 34A15, and 34P88 were analyzed in 2007 at six field locations in maize-growing areas of NorthPioneer Hi - Bred International, Inc. Page 102 of 25 8

4114 Maize America (Tallahassee, FL; York, NE; Germansville, PA; Richland, IA; Larned, KS and Branchton, Ontario ). These Pioneer commercial products were chosen to represent a wide range of non genetically modified varieties that would normally be planted commercially. For the reference hybrid trials, e ach location utilized a randomized complete block design containing three blocks. E ach block contained two- row plots. Procedures for planting, harvesting, processing, and compositional analysis of the reference hybrid trials were similar to those employed for the trials containing near- isoline control and 4114 maize. Composition data collected from the reference hybrid s was used to help determine the normal variation for the measured analytes in commercial maize. The analytes for compositional assessment were selected considering the OECD consensus document on compositional considerations for new varieties of maize (OE CD, 2002). Compositional analyses of grain samples included protein, fat, acid detergent fiber (ADF), neutral detergent fiber (NDF), ash, carbohydrates, fatty acids, amino acids, vitamins and minerals, key anti - nutrients, a nd key secondary metabolites . Compositional analyses of forage samples included protein, fat, ADF, NDF, ash, carbohydrates, calcium, and phosphorus. Statistical analysis of nutrient composition data was conducted to test for differences in the analyte mean values between 4114 maize an d the control. ( For details of the statistical methodology, refer to Appendix 6). W he n numerous analytes are being evaluated on the same samples, controlling false positive outcomes is important. Since the introduction of the false discovery rate (FDR) approach in the mid - 1990’s, it has been widely employed across a number of scientific disciplines, including genomics, ecology, medicine, plant breeding, epidemiology, dairy science, and signal/image processing ( e.g., Pawitan et al., 2005; Spelman and Bove nhuis, 1998). A false positive outcome occurs when an analyte mean of the transgenic line is deemed significantly different from the analyte mean of the control line, when in fact the two means are not different. I f one uses a 5% type I error rate for ea ch analyte, then the number of false positives increases as the number of analytes increase. I n order to help manage the false positive rate, the false discovery rate (FDR) method of Benjamini and Hochberg was applied to account for making multiple comparisons ( Benjamini and Hochberg, 1995; Westfall et al., 1999). P-values were adjusted accordingly, resulting in the false positive rate being held to 5%. Both adjusted and non -adjusted P-values are reported for the analyses that follow . A significant difference between the mean of 4114 maize and that of the control line was established with an FDR-adjusted P-value <0.05. Using the data obtained from the reference hybrids, a statistical tolerance interval was calculated to contain, with 95% confidence, 99% of the values contained in the population of commercial maize. This statistical tolerance interval and the combined range of values for each analyte from the published literature, where available, provided further context for interpretation of the compos ition results for 4114 maize. 4 114 maize analyte ranges that fell within the tolerance interval and/or combined literature range for that analyte were considered to be within the range of normal variability of commercial maize hybrids.Pioneer Hi - Bred International, Inc. Page 103 of 25 8

4114 Maize **Key Nutrients in M aize Grain**  As desc ribed in Section 1- E, mai ze grain is used in the U.S. for food, feed, and fuel. Approximately 38.7% of maize grown in the U.S. is used domestically for animal feed (primarily beef, poultry, pork and dairy) because of its high nutrient value and relative low cost (NCGA, 2011). A pproximately 10.1% of maize grain is used for food products, with 3.8% going to high fructose corn syrup, and 6.3% comprising beverage alcohol, cereal, other sweeteners, and starch (NCGA, 2011). As ignificant portion of maize is used for fuel ethanol (36.5%) with the remainder being exported (14.5%) (NCGA, 2011). **Proximates in Maize Grain** Proximates were analyzed in 4114 maize and near- isoline control grain. Results are shown in Table 15 . N o statistically sign ificant differences were observed between the 4114 maize and control mean values for crude protein, crude fat, crude fiber, acid detergent fiber (ADF), neutral detergent fiber (NDF), and carbohydrates (adjusted P-values > 0.05). Astatistically significan t difference was observed between the 4114 maize and control mean values for ash, however all of the individual values were within the tolerance interval. In conclusion, analysis of proximates in maize grain demonstrates that 4114 maize is comparable to n ear- isoline and reference maize hybrids.Pioneer Hi - Bred International, Inc. Page 104 of 25 8

4114 Maize **Table 15 . P**  **roximates in Maize Grain Analyte (% Dry Weight)**  **Control (n=24)**  **4114 maize**

**(n=24) Tolerance** 

**Interval Literature** 

**Range a** 

Crude Protein Mean b 10.2 10.4 6.59 - 13.5 6.00 - 17.3 Range c 8.92 - 11.4 7.32 - 11.7 CI d 9.54 - 10.7 9.78 - 10.9 Adjusted P-Value e 0.789 P-Value f 0.455 Crude Fat Mean 4.81 4.68 1.45 - 5.75 2.47 - 5.90

Range 4.28 - 5.73 3.88 - 5.67 CI 4.47 - 5.17 4.35 - 5.04 Adjusted P-Value 0.679 P-Value 0.177

C rude Fiber Mean 2.57 2.48 0.941 - 3.73 0.490 - 5.50 Range 1.36 - 3.38 1.34 - 3.16 CI 2.35 - 2.80 2.27 - 2.70 Adjusted P-Value 0.789 P-Value 0.520

ADF Mean 3.83 4.01 1.43 - 5.73 1.82 - 11.3

Range 3.24 - 4.48 3.53 - 5.16 CI 3.56 - 4 .12 3.72 - 4.32 Adjusted P-Value 0.679 P-Value 0.0965

NDF Mean 10.4 10.4 5.75 - 20.6 5.59 - 22.6

Range 9.39 - 11.3 9.58 - 11.2 CI 9.97 - 10.8 9.93 - 10.8 Adjusted P-Value 0.908 P-Value 0.824 Ash Mean 1.33 1.44 0.531 - 2.16 0.616 - 6.28 Range 1.12 - 1.52 1.30 - 1.60 CI 1.29 - 1.38 1.39 - 1.48 Adjusted P-Value 0.0498 g P-Value 0.00298 Carbohydrates Mean 83.7 83.6 80.3 - 89.7 77.4 - 89.5 Range 82.5 - 84.9 82.2 - 85.7 CI 83.2 - 84.2 83.1 - 84.1 Adju sted P-Value 0.908 P-Value 0.758 a Literature ranges are taken from published literature for maize (Codex, 1996; Codex, 2005; ILSI, 2006; OECD, 2002; Watson, 1982; Watson, 1987). b Least squares mean c Range denotes the lowest and highest in dividual value across locations. d 95% Confidence Interval e False Discovery Rate (FDR) adjusted P-value f Non -adjusted P-value g Statistically significant difference, adjusted P-value < 0.05Pioneer Hi - Bred International, Inc. Page 105 of 25 8

4114 Maize **Vitamins and Minerals in Maize Grain** Maize is a nutriti onal source of vitamins and minerals for both humans and animals; therefore vitamins and minerals were measured in 4114 maize and near- isoline control grain. Based on OECD guidance, the following vitamins were analyzed: beta -carotene, vitamin B1 (thiamin ), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B6 (pyridoxine), vitamin B9 (folic acid ), and vitamin E ( α-tocopherol) (OECD, 2002). The following minerals were also analyzed, based on OECD guidance: calcium, copper, iron, magnesium, phosphorus, potassium, sodium and zinc (OECD, 2002). Vitamin results are shown in Table 16 an d mineral results are show n in Table 17 . N o statistically significant differences were observed between the 4114 maize and control mean values for any of the vitamins an alyzed (adjusted P-values were > 0.05). For the minerals, there were no statistical differences for all of the analytes except phosphorus and potassium, however all individual data points were within the respective tolerance intervals. In conclusion, vitamin and mineral analyses of maize grain demonstrate that 4 114 maize is comparable to near- isoline and reference maize hybrids.Pioneer Hi - Bred International, Inc. Page 106 of 25 8

4114 Maize **Table 16 . Vitamin**  **s in Maize Grain**

**Analyte (**  **mg/kg Dry Weight )**  **Control (n=24)**  **4114 maize**

**(n=24) Tolerance** 

**Interval Literature** 

**Range a** 

Beta -Carotene Mean b 16.4 18.1 0 - 68.3 0.190 - 46.8 Range c 7.27 - 26.2 11.7 - 26.4 CI d 12.4 - 21.7 13. 7 - 24.0 Adjusted P-Value e 0.679 P-Value f 0.267 Vitamin B1 (Thiamine) Mean 1.97 2.20 0.414 - 6.64 1.26 - 40.0 Range <1.80g

- 2.92 <1.80g

- 3.28 CI 1.44 - 2.70 1.62 - 2.99 Adjusted P-Value 0.679 P-Value 0.168 Vitamin B2 (Ri boflavin) Mean <0.900g <0.900g NC h 0.250 - 5.60 Range <0.900g <0.900g CI NAi NAi Adjusted P-Value NAi P-Value NAi Vitamin B3 (Niacin) Mean 13.9 13.8 0 - 51.7 9.30 - 70.0

Range 10.6 - 18.0 11.7 - 16.6 CI 12.8 - 15.1 12.7 - 15.0 Adjusted P-Value 0.908 P-Value 0.786 Vitamin B6 (Pyridoxine) Mean 4.68 4.29 1.83 - 11.1 3.68 - 11.3

Range 2.35 - 6.88 2.50 - 6.36 CI 3.60 - 5.90 3.26 - 5.46 Adjusted P-Value 0.772 P-Value 0.404 Vitamin B9 ( Folic Acid ) Mean 0. 872 0.792 0 - 2.30 0.147 – 683 Range 0.181 - 1.91 0.0369 - 1.55 CI 0.656 - 1.09 0.577 - 1.01 Adjusted P-Value 0.789 P-Value 0.523Pioneer Hi - Bred International, Inc. Page 107 of 25 8

4114 Maize **Table 16 . Vitamins in Maize Grain (continued)** 

**Analyte (**  **mg/kg Dry Weight )**  **Control (n=24)**  **4114 maize**

**(n=24) Tolerance** 

**Interval Literature** 

**Range a**  Vitamin E ( α-Tocopherol ) Mean 5.47 5.68 2.18 - 28.2 1.50 - 68.7

Range 2.09 - 13.3 2.14 - 14.8 CI 3.06 - 9.75 3.18 - 10.1 Adjusted P-Value 0.908 P-Value 0.672 a Literature ranges are taken from published literature for maize (Codex, 1996; Codex, 2005; ILSI, 2006; OECD, 2002; Watson, 1982; Watson , 1987). b Least squares mean c Range denotes the lowest and highest individual value across locations d 95% Confidence Interval e False Discovery Rate (FDR) adjusted P-value f Non -adjusted P-value g <Lower limit of quantification (LLOQ); indicates that sample value(s) were detected below the assay LLOQ. Sample results which were below the LLOQ were assigned a value equal to the LLOQ for statistical analysis h Tolerance interval not calculated ( NC) i Statistical analysis not available (NA)Pioneer Hi - Bred International, Inc. Page 108 of 25 8

4114 Maize **Table 17 . M**  **inerals in Maize Grain Analyte (% Dry Weight)**  **Control (n=24)**  **4114 maize**

**(n=24) Tolerance** 

**Interval Literature** 

**Range a** 

Calcium Mean b 0.00325 0.00335 0.00127 - 0.00902 0.00127 -

0.100 Range c 0.00270 - 0.00399 0.00251 - 0.00448 CI d 0.00288 - 0.00367 0.00296 - 0.00378 Adjusted P-Value e 0.679 P-Value f 0.308 Copper Mean 0.0000645 0.0000653 0 - 0.000662 0.0000730 -

0.00185 Range <0.0000625 g

-

0.0000869 <0.0000625 g

-

0.0000876 CI NA h NA h Adjusted P-Value NA h P-Value NA h

Iron Mean 0.00168 0.00166 0.000857 - 0.00269 0.000100 -

0.0100 Range 0.00146 - 0.00228 0.00147 - 0.00210 CI 0.00156 - 0.00181 0.00155 - 0.00179 Adjusted P-Value 0.908 P-Value 0.775 Magnesium Mean 0.133 0.135 0.0381 - 0.195 0.0594 - 1.00 Range 0.103 - 0.155 0.112 - 0.157 CI 0.121 - 0.146 0.123 - 0.149 Adjusted P-Value 0.679 P-Value 0.251 Phosphorus Mean 0.289 0.311 0.127 - 0.472 0.147 - 0.750 Range 0.258 - 0.334 0.296 - 0.330 CI 0.278 - 0.301 0.298 - 0.324 Adjusted P-Value 0.0115 i P-Value 0.000353

Potassium Mean 0.387 0.416 0.194 - 0.687 0.181 - 0.720 Range 0.343 - 0.441 0.385 - 0.478 CI 0.373 - 0.401 0.401 - 0.431 Adjusted P-Value 0 .0498 i P-Value 0.00306

Sodium Mean 0.0000582 0.0000628 0 - 0.00207 0 - 0.150

Range <0.0000625 g

-

0.000489 <0.0000625 g

-

0.000171 CI 0.0000409 -

0.0000828 0.0000447 -

0.0000882 Adjusted P-Value 0.908 P-Value 0.714Pioneer Hi - Bred International, Inc. Page 109 of 25 8

4114 Maize **Table 17 . Mi**  **nerals in Maize Grain (continued)**  **Analyte (% Dry Weight) Control**  **(n=24) 4114 maize** 

**(n=24) Tolerance** 

**Interval Literature** 

**Range a** 

Zinc Mean 0.00174 0.00174 0.00104 - 0.00271 0.000650 -

0.00372 Range 0.00150 - 0.00203 0.00149 - 0.00201 CI 0.00162 - 0. 00188 0.00161 - 0.00188 Adjusted P-Value 0.946 P-Value 0.919 a Literature ranges are taken from published literature for maize (Codex, 1996; Codex, 2005; ILSI, 2006; OECD, 2002; Watson, 1982; Watson , 1987). b Least squares mean c Range d enotes the lowest and highest individual value across locations. d 95% Confidence Interval e False Discovery Rate (FDR) adjusted P-value f Non -adjusted P-value g <Lower limit of quantification (LLOQ); indicates that sample value(s) were detected bel ow the assay LLOQ. Sample results which were below the LLOQ were assigned a value equal to the LLOQ for statistical analysis. h Statistical analysis not available (NA) i Statistically significant difference, adjusted P-value < 0.05Pioneer Hi - Bred International, Inc. Page 110 of 25 8

4114 Maize **Fatty Acids in Maiz e Grain**  The oil in maize consists mostly of five major fatty acids: p almitic (C16:0), stearic (C18:0), oleic (C18:1), linoleic (C18:2), and linolenic (C18:3) (OECD, 2002). Other fatty acids are present, but usually fall below 1% of total fatty acids (OE CD, 2002). The complete fatty acid profile was measured in seed samples derived from 4114 maize, and compared with corresponding values from samples of control maize. Results of the fatty acid analysis are sh own in Table 18 . The concentrations of the following fatty acids were below the lower limit of quantification for both 4114 maize and control and are not reported in Table 18 : Caprylic Acid (C8:0); Capric Acid (C10:0); Lauric Acid (C12:0); Myristic Acid (C14:0); Myrisoleic Acid (C14:1); Pentadecanoi c Acid (C15:0); Pentadecenoic Acid (C15:1); Heptadecenoic Acid (C17:1); γ- Linolenic Acid (C18:3); Nonadecanoic Acid (C19:0); Eicosatrienoic Acid (C20:3); Arachidonic Acid (C20:4); Heneicosanoic Acid (C21:0); Erucic Acid (C22:1); and Tricosanoic Acid (C23:0). A sm all but statistically significant difference (adjusted P-value was < 0.05) was observed in the concentration of e icosenoic a cid between 4114 maize and control samples, however all individual data points for 4114 maize were within the tolerance interval. No statistically significant diffe rences were observed between the 4114 maize and near- isoline control for any of the other fatty acid mean values (adjusted P-values were > 0.05). In conclusion, fatty acid analysis of maize grain demonstrates that 4114 maize is comparable to near- isoline and reference maize hybrids.Pioneer Hi - Bred International, Inc. Page 111 of 25 8

4114 Maize **Table 18 .**  **Fatty Acids in Maize Grain Analyte (% Total Fatty Acids)**  **a Control**  **(n=24) 4114 maize** 

**(n=24) Tolerance** 

**Interval Literature** 

**Range b** 

Palmitic Acid (C16:0) Mean c 14.1 14.2 5.51 - 18.4 7.00 - 20.7

Range d 13.2 - 15.6 13.1 - 16.2 CI e 13.6 - 14.5 13.7 - 14.7 Adjusted P-Value f 0.789 P-Value g 0.518 Palmitoleic Acid (C16:1) Mean 0.124 0.139 0 - 0.337 0 - 1.00

Range 0.0948 - 0.144 0.125 - 0.156 CI NA h NA Adjusted P-Value NA P-Value NA He ptadecanoic Acid (C17:0) Mean 0.0940 0.100 0 - 0.189 0 - 0. 111 Range 0.0940 - 0.0940 0.100 - 0.100 CI NA NA Adjusted P-Value NA P-Value NA Heptadecadienoic Acid (C17:2) Mean 0.307 0 NC i NRj Range 0.307 - 0.307 0 – 0 CI NA NA Adjusted P-Value NA P-Value NA Stearic Acid (C18:0) Mean 1.60 1.60 0.566 - 4.67 0 - 4.00

Range 1.45 - 2.07 1.37 - 2.14 CI 1.46 - 1.75 1.47 - 1.75 Adjusted P-Value 0.908 P-Value 0.810 Oleic Acid (C18:1) Mean 22.6 22.1 10.4 - 65.6 17.4 - 50.0 Range 20.7 - 25.8 20.3 - 23.8 CI 21.5 - 23.8 21.0 - 23.2 Adjusted P-Value 0.0817 P-Value 0.00629 Linoleic Acid (C18:2) Mean 60.1 60.5 30.4 - 81.7 34.0 - 70.0

Range 54.9 - 62.9 56.3 - 63.5 CI 58.4 - 61.8 58.8 - 62.2 Adjusted P-Value 0.679 P-Value 0.203 (9,15) Isomer of Linoleic Acid (C18:2) Mean 0 0.249 0 - 6.92 NR

Range 0 - 0 0.240 - 0.258 CI NA NA Adjusted P-Value NA P-Value NAPioneer Hi - Bred International, Inc. Page 112 of 25 8

4114 Maize **Table 18 .**  **Fatty Acids in Maize Grain (continued)**  **Analy te (% Total Fatty Acids)**  **a Control**  **(n=24) 4114 maize** 

**(n=24) Tolerance** 

**Interval Literature** 

**Range b**  Linolenic Acid (C18:3) Mean 0.787 0.770 0 - 3.34 0 - 2.25

Range 0.410 - 1.22 0.305 - 1.12 CI 0.576 - 1.08 0.563 - 1.05 Adjusted P-Value 0.908 P-Value 0.697 Arachidic Acid (C20:0) Mean 0.392 0.394 0.159 - 0.849 0 - 2.00 Range 0.343 - 0.505 0.347 - 0.481 CI 0.359 - 0.428 0.361 - 0.430 Adjusted P-Value 0.789 P-Value 0.518 Eicosenoic Acid (C20:1) Mean 0.235 0.265 0.213 - 0.370 0 - 1.92 Range 0.204 - 0.255 0.229 - 0.336 CI 0.224 - 0.246 0.253 - 0.278 Adjusted P-Value 0.00472 k P-Value <0.0001 Eicosadienoic Acid (C20:2) Mean 0.275 0 0 - 0.351 l 0 - 0.533

Range 0.275 - 0.275 0 – 0 CI NA NA Adjusted P-Value NA P-Value NA Behenic Acid (C22:0) Mean 0 0.291 0 - 0.566 0 - 0.500

Range 0 - 0 0.291 - 0.291 CI NA NA Adjusted P-Value NA P-Value NAPioneer Hi - Bred International, Inc. Page 113 of 25 8

4114 Maize **Table 18 .**  **Fatty Acids in Maize Grain (continued)**  **Analyte (% Total Fatty Acids) a**  **Co ntrol**  **(n=24) 4114 maize** 

**(n=24) Tolerance** 

**Interval Literature** 

**Range b**  Lignoceric Acid (C24:0) Mean 0.240 0.233 0 - 0.675 0 - 0.500

Range 0.185 - 0.318 0.211 - 0.276 CI 0.205 - 0.280 0.200 - 0.273 Adjusted P-Value 0.908 P-Value 0.796 a The concentrations of the following fatty acids were below the lower limit of quantification for both 4114 maize and control and are not reported here: Caprylic Acid (C8:0); Capric Acid (C10:0); Lauric Acid (C12:0); Myristic Acid (C14:0); Myrisoleic Acid ( C14:1); Pentadecanoic Acid (C15:0); Pentadecenoic Acid (C15:1); Heptadecenoic Acid (C17:1); γ- Linolenic Acid (C18:3); Nonadecanoic Acid (C19:0); Eicosatrienoic Acid (C20:3); Arachidonic Acid (C20:4); Heneicosanoic Acid (C21:0); Erucic Acid (C22:1); and Tricosanoic Acid (C23:0). b Literature ranges are taken from published literature for maize (Codex, 1996; Codex, 2005; ILSI, 2006; OECD, 2002; Watson, 1982; Watson , 1987). c Least squares mean d Range denotes the lowest and highest individual value across locations. e 95% Confidence Interval f False Discovery Rate (FDR) adjusted P-value g Non -adjusted P-value h Statistical analysis not available (NA) i A tolerance interval could not be calculated (NC) j Analyte ranges were not reported (NR) in th e published literature references k Statistically significant difference, adjusted P-value < 0.05 l No tolerance interval could be calculated for this analyte, and the listed interval is the min/max range.Pioneer Hi - Bred International, Inc. Page 114 of 25 8

4114 Maize **Total Amino Acids in Maize Grain** Maize grain is generally a good source of essential and non -essential amino acids for most domestic animal species. Total levels of 18 amino acids were measured in 4114 maize and near- isoline control grain. Results are show n in Table 19 . No statistically significant differences were observed between the 4114 maize and near- isoline control maize for any of the amino acid mean values (adjusted P-values were > 0.05). In conclusion, total amino acid analysis of maize grain demonstrates that 4114 maize is comparable to near- isoline and reference maize hybrids.Pioneer Hi - Bred International, Inc. Page 115 of 25 8

4114 Maize **Table 19 . T** **otal Amino Acids in Maize Grain Analyte (% Dry Weight)**  **Control (n=24)**  **4114 maize**

**(n=24) Tolerance** 

**Interval Literature** 

**Range a** 

Alanine Mean b 0.739 0.757 0.491 - 1.09 0.439 - 1.39 Range c 0.604 - 0 .840 0.460 - 0.886 CI d 0.676 - 0.797 0.695 - 0.813 Adjusted P-Value e 0.772 P-Value f 0.398 Arginine Mean 0.434 0.438 0.253 - 0.551 0.119 -

0.640 Range 0.362 - 0.492 0.364 - 0.514 CI 0.408 - 0.463 0.411 - 0.466 Adjusted P-Value 0.908 P-Value 0.707 Aspartic Acid Mean 0.680 0.701 0.442 - 0.947 0.335 -

1.21 Range 0.562 - 0.751 0.460 - 0.813 CI 0.634 - 0.724 0.657 - 0.743 Adjusted P-Value 0.679 P-Value 0.260 Cystine Mean 0.193 0.194 0.136 - 0.418 0.0800 -

0.514 Range 0.157 - 0.253 0.144 - 0.252 CI 0.168 - 0.220 0.169 - 0.222 Adjusted P-Value 0.946 P-Value 0.931 Glutamic Acid Mean 1.93 1.96 1.17 - 2.88 0.965 - 3.54 Range 1.59 - 2.24 1.21 - 2.31 CI 1.78 - 2.07 1.82 - 2.10 Adjuste d P-Value 0.789 P-Value 0.546 Glycine Mean 0.372 0.377 0.249 - 0.485 0.184 -

0.539 Range 0.318 - 0.442 0.305 - 0.445 CI 0.346 - 0.400 0.351 - 0.406 Adjusted P-Value 0.789 P-Value 0.461 Histidine Mean 0.293 0.303 0.180 - 0.362 0.137 -

0.434 Range 0.257 - 0.342 0.230 - 0.426 CI 0.271 - 0.317 0.280 - 0.328 Adjusted P-Value 0.679 P-Value 0.274 Isoleucine Mean 0.339 0.349 0.143 - 0.587 0.179 -

0.710 Range 0.288 - 0.388 0.229 - 0.408 CI 0.312 - 0.364 0.323 - 0.374 Adjusted P-Value 0.679 P-Value 0.251Pioneer Hi - Bred International, Inc. Page 116 of 25 8

4114 Maize **Table 19 . T** **otal Amino Acids in Maize Grain (continued)**  **Analyte (% Dry Weight) Control**  **(n=24) 4114 maize** 

**(n=24) Tolerance** 

**Interval Literature** 

**Range a** 

Leucine Mean 1.26 1.31 0.659 - 1.95 0.642 - 2. 49

Range 1.02 - 1.49 0.775 - 1.56 CI 1.13 - 1.37 1.19 - 1.42 Adjusted P-Value 0.679 P-Value 0.206 Lysine Mean 0.293 0.288 0.112 - 0.551 0.0500 -

0.668 Range 0.224 - 0.349 0.213 - 0.350 CI 0.256 - 0.335 0.252 - 0.330 Adjusted P-Value 0.789 P-Value 0.474 Methionine Mean 0.179 0.182 0.0724 - 0.490 0.100 - 0.468 Range 0.144 - 0.231 0.136 - 0.230 CI 0.156 - 0.206 0.158 - 0.209 Adjusted P-Value 0.916 P-Value 0.846 Phenylalanine Mean 0.523 0.554 0.298 - 0 .693 0.244 - 0.930 Range 0.443 - 0.611 0.370 - 0.666 CI 0.474 - 0.568 0.508 - 0.596 Adjusted P-Value 0.679 P-Value 0.121 Proline Mean 0.899 0.926 0.454 - 1.64 0.462 - 1.63

Range 0.763 - 1.01 0.624 - 1.11 CI 0.828 - 0.964 0.858 - 0 .990 Adjusted P-Value 0.679 P-Value 0.235

Serine Mean 0.500 0.518 0.266 - 0.683 0.235 - 0.910 Range 0.421 - 0.554 0.352 - 0.745 CI 0.459 - 0.545 0.475 - 0.564 Adjusted P-Value 0.679 P-Value 0.308 Threonine Mean 0.361 0.373 0.176 - 0.578 0.224 - 0.666 Range 0.318 - 0.402 0.284 - 0.438 CI 0.341 - 0.381 0.354 - 0.392 Adjusted P-Value 0.679 P-Value 0.165 Tryptophan Mean 0.0634 0.0639 0.00876 - 0.127 0.0271 -

0.215 Range 0.0367 - 0.0726 0.0377 - 0.0773 CI 0.0563 - 0.0692 0.0569 - 0.0696 Adjusted P-Value 0.908 P-Value 0.806Pioneer Hi - Bred International, Inc. Page 117 of 25 8

4114 Maize **Table 19 . To** **tal Amino Acids in Maize Grain (continued)**  **Analyte (% Dry Weight) Control**  **(n=24) 4114 maize** 

**(n=24) Tolerance** 

**Interval Literature** 

**Range a**  Tyrosine Mean 0.263 0.257 0.0707 - 0.505 0.103 - 0.790 Range 0.199 - 0.333 0.163 - 0.367 CI 0.240 - 0.289 0.234 - 0.282 Adjusted P-Value 0.792 P-Value 0.561 Valine Mean 0.460 0.473 0.159 - 0.749 0.210 - 0.855 Range 0.393 - 0.507 0.334 - 0.542 CI 0. 428 - 0.488 0.443 - 0.500 Adjusted P-Value 0.679 P-Value 0.187 a Literature ranges are taken from published literature for maize (Codex, 1996; Codex, 2005; ILSI, 2006; OECD, 2002; Watson, 1982; Watson , 1987). b Least squares mean c Range denotes the lowest and highest individual value across locations. d 95% Confidence Interval e False Discovery Rate (FDR) adjusted P-value f Non -adjusted P-valuePioneer Hi - Bred International, Inc. Page 118 of 25 8

4114 Maize **Key Anti -**  **nutrients in Maize Grain** Maize grain contains several key anti - nutrients: raffinose, phytic acid, and trypsin inhibitor (OECD, 2002). Raffinose is a low molecular weight oligosaccharide that is non -digestible, creating flatulence from consumption, but typically can be removed through processing of maize (OECD, 2002). In additio n, phytic acid chelates mineral nutrients including calcium, magnesium, potassium, iron and zinc, rendering them unavailable to monogastric animals. Trypsin inhibitor can interfere with the digestion of proteins, resulting in decreased animal growth. Levels of key anti- nutrients were measured in 4114 maize and near- isoline control grain. Results are shown in Table 20 . N o statistically significant differences were observed between the mean values for 4114 maize and near- isoline control for any of the ant i - nutrient measured (adjusted P-values were > 0.05). In conclusion, anti - nutrient analysis of maize grain demonstrates that 4 114 maize is comparable to near- isoline control maize and reference maize hybrids.Pioneer Hi - Bred International, Inc. Page 119 of 25 8

4114 Maize **Table 20 . Key A** **nti -**  **N utrients in Maize Grain** 

**Analyte (% Dry Weight or as Indicated)**  **Control (n=24)**  **4114 maize**

**(n=24) Tolerance** 

**Interval Literature** 

**Range a**  Phytic Acid Mean b 0.998 1.02 0.418 - 1.41 0.111 - 1.57

Range c 0.842 - 1.17 0.827 - 1.52 CI d 0.917 - 1.09 0.941 - 1.11 Adjusted P-Valu e e 0.789 P-Value f 0.542

Raffinose Mean 0.0823 0.108 0 - 0.398 0.0200 - 0.320

Range <0.0800g

-

0.210 <0.0800g

-

0.236 CI 0.0503 - 0.134 0.0670 - 0.173 Adjusted P-Value 0.679 P-Value 0.0826 Trypsin Inhibitor (TIU/mg) h Mean 2.67 2.58 1.60 - 4.89 1.09 - 7.18

Range 1.45 - 4.68 1.70 - 3.48 CI 2.24 - 3.18 2.16 - 3.07 Adjusted P-Value 0.908 P-Value 0.694 a Literature ranges are taken from published literature for maize (Codex, 1996; Codex, 2005; ILSI, 2006; OECD, 2002; Watson, 1982; Watson , 1987). b Least squares mean c Range denotes the lowest and highest individual value across locations. d 95% Confidence Interval e False Discovery Rate (FDR) adjusted P-value f Non -adjusted P-value g <Lower limit of quant ification (LLOQ); indicates that sample value(s) were detected below the assay LLOQ. Sample results which were below the LLOQ were assigned a value equal to the LLOQ for statistical analysis. h Abbreviation: TIU = trypsin inhibitor unitsPioneer Hi - Bred International, Inc. Page 120 of 25 8

4114 Maize **Key Secondary Plant Metabolites in Maize Grain**  Secondary plant metabolites are neither nutrients nor anti - nutrients, but can be analyzed as indicators of the absence of unintended effects of the genetic modification on metabolism (OECD, 2002). Characteristic plant metabolites in maize are the phenolic acids: ferulic acid , furfural, and p-coumaric acid. Ferulic acid , furfural, and p-coumaric acid were measured in 4114 maize and near- isoline control grain. Results are show n in Table 21 . No statistically significant differences were observed between the mean values for 4114 maize and near- isoline control for any of the secondary plant metabolites measured (adjusted P-values were > 0.05). In conclusion, metabolite analysis of maize grain demonstrates that 4114 maize i s comparable to near- isoline control maize and reference maize hybrids. **Table 21 . Key Secondary Plant Meta**  **bolites in Maize Grain**

**Analyte (% Dry Weight)**  **Control (n=24)**  **4114 maize**

**(n=24) Tolerance** 

**Interval Literature** 

**Range a** 

p-Coumaric Acid Mean b 0.0183 0.0186 0.000341 - 0.0387 0.003 - 0.0576 Range c 0.0120 - 0.0276 0.0151 - 0.0249 CI d 0.0159 - 0.0210 0.0162 - 0.0214 Adjusted P-Value e 0.789 P-Value f 0.546 Furfural Mean <0.000100 g <0.000100 g NC h 0 - 0.000634

Range <0.000100 g <0.000100 g CI NA NA Adjusted P-Value NA P-Value NA

Ferulic Acid Mean 0.255 0.266 0.0553 - 0.309 0.0200 - 0.389

Range 0.199 - 0.299 0.227 - 0.292 CI 0.243 - 0.268 0.253 - 0.280 Adjusted P-Value 0.679 P-Value 0.231 a Literature ranges are taken from published literature for maize (Codex, 1996; Codex, 2005; ILSI, 2006; OECD, 2002; Watson, 1982; Watson , 1987). b Least squares mean c Range denotes the lowest and highest individual value across sites. d 95% Confidence Interval e False Discovery Rate (FDR) adjusted P-value f Non -adjusted P-value g <Lower Limit of Quantification (LLOQ); Indicates that the values of the sample or samples were detected below the assay’s LLOQ. Sample results which were below the LLOQ were assi gned a value equal to the LLOQ for statistical analysis. h A tolerance interval could not be calculated (NC).Pioneer Hi - Bred International, Inc. Page 121 of 25 8

4114 Maize **Key Nutrients in Maize Forage** Maize silage is an important feed ingredient for feedlot cattle and dairy cattle. I n the U.S., approximately 10% of the maize crop is harvested as forage. The whole maize plant contains about one and one- half times the nutrients of the grain, and the ensiling process preserves more than 90% of the nutrients ( OECD, 2002). **Proximates and Minerals in Maize Forage** C rude p rotein, crude fat, crude fiber, ADF, NDF, ash, carbohydrates, and the minerals calcium and phosphorus were measured in 4114 maize and near- isoline control forage. Results are shown in Table 22 . For each analyte measured, there was no statistical difference between 4114 maize and the near- isoline control maize (adjusted P-values were > 0.05). In conclusion, proximate and mineral analysis of maize forage demonstrated that 4114 maize forage is comparable to near- isoline and reference maize forage.Pioneer Hi - Bred International, Inc. Page 1 22 of 25 8

4114 Maize **T able 22. Pr** **oximates and Minerals in Maize Forage Analyte (% Dry Weight)**  **Control (n=24)**  **4114 maize**

**(n=24) Tolerance** 

**Interval Literature** 

**Range a** 

Crude Protein Mean b 8.93 9.10 3.35 - 14.6 3.14 - 15.9

Range c 5.82 - 11.0 5.72 - 11.0 CI d 7.52 - 10.0 7.7 5 - 10.1 Adjusted P-Value e 0.679 P-Value f 0.297 Crude Fat Mean 3.30 3.26 1.17 - 3.98 0.296 - 6.70

Range 1.97 - 4.89 1.95 - 5.60 CI 2.63 - 4.15 2.60 - 4.10 Adjusted P-Value 0.938 P-Value 0.881 Crude Fiber Mean 21.8 21.2 13. 4 - 32.6 19.0 - 62.8

Range 15.6 - 27.6 17.6 - 25.1 CI 19.9 - 23.9 19.3 - 23.3 Adjusted P-Value 0.789 P-Value 0.524

ADF Mean 28.8 27.6 17.8 - 42.0 16.1 - 47.4

Range 21.7 - 38.2 20.1 - 34.6 CI 25.3 - 32.9 24.2 - 31.4 Adjusted P-Value 0.679 P-Value 0.215

NDF Mean 47.4 47.5 30.8 - 71.4 20.3 - 63.7

Range 39.5 - 57.4 41.2 - 52.8 CI 44.0 - 51.1 44.1 - 51.2 Adjusted P-Value 0.966 P-Value 0.966 Ash Mean 4.98 4.87 0.773 - 8.52 1.30 - 10.5

Range 3.27 - 7.09 3.50 - 6.36 CI 4.19 - 5.92 4.10 - 5.79 Adjusted P-Value 0.760 P-Value 0.374 Carbohydrates Mean 82.8 82.7 76.7 - 90.9 76.4 - 92.1 Range 78.7 - 87.6 80.1 - 86.6 CI 81.2 - 84.4 81.2 - 84.3 Adjusted P-Value 0.939 P-Value 0.896

Calcium Mean 0.259 0.216 0 - 0.563 0.0714 - 0.600

Range 0.143 - 0.654 0.0878 - 0.375 CI 0.195 - 0.344 0.162 - 0.287 Adjusted P-Value 0.679 P-Value 0.156Pioneer Hi - Bred International, Inc. Page 123 of 25 8

4114 Maize **Table 22. Pr oximates and Minerals in Maize Forage**  **(continued) Analyte (% Dry**  **Weight) Control**  **(n=24) 4114 maize** 

**(n=24) Tolerance** 

**Interval Literature** 

**Range a**  Phosphorus Mean 0.274 0.289 0.0198 - 0.465 0.0936 - 0.550

Range 0.191 - 0.369 0.213 - 0.367 CI 0.243 - 0.308 0.257 - 0.326 Adjusted P-Value 0.679 P-Value 0.115 a Literature ranges are taken from published literature for maize (ILSI, 2006; Watson, 1982). b Least squares mean c Range denotes the lowest and highest individual value across locations. d 95% Confidence Interval e False Discovery Rate (FDR ) adjusted P-value f Non -adjusted P-value

**Conclusions on Compositional Assessment of 4114 maize** Extensive nutritional compositional analyses of grain and forage were conducted to evaluate the composition of 4114 maize as co mpared to a non -transgeni c near- isoline control. Compositional analysis of 4114 maize was used to evaluate any changes in the levels of key nutrients, anti- nutrients or secondary metabolites. Compositional analyses of grain included crude protein, crude fat, crude fiber, acid detergent fiber (ADF), neutral detergent fiber (NDF), ash, carbohydrates, fatty acids, total amino acids, key anti - nutrients, and key secondary metabolites. Compositional analyses of forage included crude protein, crude fat, crude fiber, ADF, NDF, ash, carb ohydrates, calcium, and phosphorus. In total, data from 69 different analyt ical components ( 60 in grain, nine in forage) were analyzed . While statistical differences were observed in some of these analytes between 4114 maize and its near- isoline control, all values for 4114 maize were within the ranges for the commercial maize lines (i.e. , tolerance intervals) or within published literature ranges for maize. Therefore, based on these analyses, the grain and forage of 4114 maize are considered to be compa rable to conventional maize with respect to nutrient composition. F urthermore, the presence of 4114 maize would not be expected to impact raw or processed maize commodities. In conclusion, 4114 maize is comparable to conventional maize with respect to nutrient composition.Pioneer Hi - Bred International, Inc. Page 124 of 25 8

4114 Maize **4 -**  **B . Agronomic Performance and**  **Field Observations**  Agronomic evaluations were conducted on 4114 maize and conventional and/or control maize. These evaluations form the basis to determine whether 4114 maize is agronomically comparabl e to conventional maize and they provide an appropriate scientific determination whether 4114 maize is no more likely to pose a plant pest risk when compared to conventional maize. The agronomic evaluations were based on both laboratory experiments and re plicated, multi-site field trials conducted by agronomists and scientists who are considered experts in the production and evaluation of maize. To evaluate the agronomic characteristics of 4114 maize, data were collected on representative characteristics that influence reproduction, crop survival, and potential weediness. I n each of these assessments, 41 14 maize was compared to a near- isoline control (i.e., approximately 99% similar) that did not go through the transformation process. I n each experiment, the agronomic characteristics of 4114 maize were comparable to the control or commercial comparators. Field observations were made to assess responses of 4114 maize and control maize to naturally occurring insects and diseases in a wide variety of enviro nmental conditions. In each case, 4114 maize demonstrated no unexpected responses to insects or diseases as compared to the control plants. Based on the analyses described below, 4114 maize is comparable to conventional maize and will not pose a greater plant pest risk or increased weed potential when compared to conventional maize. **Germination and Dormancy Evaluations** In order to evaluate germination and dormancy, seeds from the F1 *5 generation (Section 2; Figure 10 and Table 3 ) of 4114 maize were tested for germination assays under warm, cold, and diurnal conditio ns (Table 2 3 ). The F1* 5 generation of seed was used because F1 hybrid seed is representative of seed that growers would plant in commercial maize fields. An ear- isoline control was used for comparison. I n addition, two commercial maize lines, Pioneer® hybrids 32D78 and 34P88, were evaluated in the study to establish a reference range for germination and dormancy evaluations but were not included in the statistical analysis. This reference range provided context for any statistical differences observed in the comparisons; if the values for 4114 maize fell within this reference range, it indicated that 4114 maize was comparable to conventional maize lines. Each germination test contained eigh t replicates of 50 seeds each of 4114 maize, near- isoline control, and two commercial lines. The “Rules for Testing Seeds”, published by the Association of Official Seed Analysis, were used as guidelines for the germination methods and interpretation of results (AOSA, 2007) . E ach replicate was placed between sheets of moist germination toweling, rolled up, and placed in a growth chamber set to the appropriate testPioneer Hi - Bred International, Inc. Page 125 of 25 8

4114 Maize conditions as s pecified in Table 23 . At the end of each germination test, each seed was defined as either germinated or non -germinated. Ag iven seed was classified as germinated if some or all of the essential structures necessary to produce a normal plant under favorable conditions had emerged, or non -germinated if none of these structures had emerged. G ermination rates were reported as a percentage of germinating seed as follows: (number of germinated seeds/total seeds)*100. The results are presented in Tables 24, 25, and 26 . Non -germinated seed were further defined into three categories:  hard (i.e., did not absorb water);  imbibed (i.e., absorbed water but did not show signs of growth during the germination test); or  dead (i.e., absorbed water, did not show signs of growth during the germination test, and displayed distinct signs of decay such as an extremely soft interior that did not hold shape under gentle pressure). Non -germinated seeds that were classified as hard or imbibed were further evaluated for potential dormancy using a standard Tetrazolium Chloride (TZ) test (AOSA, 2005). The TZ test was developed as a color test to evaluate seed viability. The color phase of the test occurs when the colorless testing solution consisting of water and 2,3,5 -triphenyltetrazolium chloride is added to the seed. O nce this solution penetrates i nto living cells, the TZ solution is reduced to a reddish (pink), water- insoluble compound. The absence or presence of variation in color characteristics within the tissues allows for recognition and location of living tissues within the embryo structure. Dead seed were excluded from the TZ test, due to the possibility of a false positive result from bacterial growth. The counts for each class of non -germinated seed and the corresponding TZ test result are presented in Table 27 . I n non -germinated hard seed, potential dormancy may be indicated if the seed is viab le (i.e., p ositive TZ test) ; however, seed dormancy is not commonly observed in maize . Germination rates in 4114 maize under warm, cold , and diurnal growing conditions were comparable to those of control maize under corresponding growing conditions. N o potentially dormant seed were identified using the TZ test. The data provided here support the conclusion that 4114 maize is comparable to conventional maize with respect to germination and potent ial dormancy.Pioneer Hi - Bred International, Inc. Page 126 of 25 8

4114 Maize **Table 23 . Description of Seed Germination Conditions**  Warm germination test  Simulated optimal maize growth conditions  Continuous setting of 25°C and 90% relative humidity for 10 days  Germinated seed counted after 10 days Cold germination test  Simulated early spring planting conditions in Midwestern U.S.  Continuous setting of 10°C and 90% relative humidity for 10 days, followed by three days at a continuous setting of 25°C and 90% relative humidity  Germinated seed counted after 13 days Diurnal germination test  Simulated daily weather conditions in Midwestern U.S.  Cyclical setting of 10°C and 90% relative humidity for 16 hours and then 25°C and 90% relative humidity for 8 hours, repeated daily for 10 days  Germinated seed counted after 10 daysPioneer Hi - Bred International, Inc. Page 127 of 25 8

4114 Maize **Table 24 . Summary of Warm Germination Test Results**  **Statistic 4114** 

**Maize Control** 

**Maize Reference** 

**Range** Frequency 396/400 395/399 96.0% - 100% Mean 99.0% 99.0% Range 96.0% -

100% 96.0% - 100% P-value a 1.00 a P-value was determined using Fi sher’s exact test for germination frequencies.

**Table 25 . Summary of Cold Germination Test Results**  **Statistic 4114** 

**Maize Control** 

**Maize Reference** 

**Range** Frequency 371/398 383/399 86.0% - 98.0% Mean 93.2% 96.0% Range 86.0% -

98.0% 91.8% - 100% LS- Mea n 93.2% 96.0% SEM 1.26% 0.982 % P-value a 0.0865 a P-value was determined using GLMM - based statistical test for mean germination rates.

**Table 26 . Summary of Diurnal Germination Test Results**  **Statistic 4114 Maize**  **Control Maize**  **Reference**

**Range** Fre quency 395/400 395/399 96.0% - 100% Mean 98.8% 99.0% Range 94.0% - 100% 96.0% - 100% P-value a 1.00 a P-value was determined using Fisher’s exact test for germination frequenciesPioneer Hi - Bred International, Inc. Page 128 of 25 8

4114 Maize **Table 27 . Tetrazolium Chloride (TZ) Testing of Non** **- Germinated Seed** 

**Germination Test**  **Maize Line Total Non** **** **Germinated Seed TZ Test of Non**  **- Germinated Seed**  **Non****** **Germinated Seed Not**  **Tested with TZ (** **i.e. Dead Seed)**  **Result: Viable Result:** 

**Non -** **Viable Hard**  **Imbibed Dead**  **Hard Seed Imbibed**  **Seed Hard or** 

**Imbibed Seed** Warm 4 114 Maize 0 4 0 0 0 4 0

Control Maize 0 3 1 0 0 3 1 32D78 (Reference) 0 1 0 0 0 1 0 34P88 (Reference) 0 6 0 0 0 6 0 Cold 4114 Maize 0 27 0 0 7 20 0

Control Maize 0 16 0 0 1 15 0 32D78 (Reference) 0 31 0 0 1 30 0 34P88 (Reference) 0 15 0 0 2 13 0 Diurnal 4114 Maize 0 5 0 0 2 3 0

Control Maize 0 4 0 0 4 0 0 32D78 (Reference) 0 1 0 0 0 1 0 34P88 (Reference) 0 5 0 0 1 4 0Pioneer Hi - Bred International, Inc. Page 129 of 25 8

4114 Maize **Field Trial Evaluations** Agronomic data were collected from the F1* 5 generation of 4114 maize and near- isoline control maize within two experiments (denoted A and B) that were conducted at 17 total field locations in 2010 (Table 28; Figure 33 ). For sites listed in the same location ( e.g., sites 3 and 12; Table 28 ), t he fi eld trials were conducted independently. The F1 * 5 generation of seed (Section 2; Figure 10 and Table 3 ) wa s used because F1 hybrid seed is representative of seed that growers would plant in commercial maize fields. The trial locations provided a range of environmental and agronomic conditions represen tative of the major maize-growing regions of the U.S. and Canada, where commercial production of 4 114 maize is expected. Agronomic parameters observed for Experiments A and B are provided in Table 29 . The data collected were identical for both experiments, except yield was only collected in Experiment A (11 locations) and pollen viability was only collected in Experiment B (6 locations) (Table 29 ). Details of the methods used are presented in Appendix 7. Although 4114 maize contains traits that are efficacious against certain lepidopteran and corn rootworm target pests and glufosinate-ammonium tolerance, efficacy of the traits was not assessed in these agronomic evaluations. As with the compositional assessments, each field trial site was managed to m aintain a relatively insect-free and weed -free environment in order to ensure that agronomic characteristics measured would be independent of the intended effects of the insect- resistant and herbicide-tolerant traits; therefore, these evaluations would app ropriately compare 4114 maize to conventional maize and also assess unintended effects of the transgene. For example, yield or insect damage scores were not impacted by the intended effects of the traits and therefore could be used to assess comparability of 4114 maize to conventional maize. Agronomic data were also collected from non - modified commercial maize hybrids (reference hybrids) in three separate experiments. Pioneer® hybrids 34M94, 33G26, 33J24, and 3394 were analyzed in 2003 at six field locations in maize-growing areas of North America (Bagley, IA; York, NE; Chula, GA; New Holland, OH; Larned, KS and Hereford, PA). Pioneer® hybrids 38B85, 37Y12, 34A15, and 34P88 were analyzed in 2007 at six field locations in maize-growing areas of North Am erica (Tallahassee, FL; York, NE; Germansville, PA; Richland, IA; Larned, KS and Branchton, Ontario). Pioneer® hybrids 37H24, 36M28, 35T06, 35T36, 35K02, and 34P88 were analyzed in 2009 at four field locations in the maize-growing areas of Chile. These P ioneer commercial products were chosen to represent a wide range of non -genetically modified varieties that would normally be planted commercially. For the reference hybrid trials, each location utilized a randomized complete block design containing th ree blocks. E ach block contained two- row plots. P rocedures for planting and collecting data from the reference hybrid trials were similar to those employed for the trials containing near- isoline control and 4114 maize. Agronomic data collected from the reference hybrids was used to help determine the normal range of variation for the agronomic characteristics in commercial maize.Pioneer Hi - Bred International, Inc. Page 130 of 25 8

4114 Maize A statistical analysis of agronomic data was conducted to test for differences in the mean values between the 4114 maize and the near- isoline control (see Appendix 7 for statistical model). W hen numerous comparisons are being made, it is important to control the rate of false- positive results . S ince the introduction of the false discovery rate (FDR) approach in the mid - 1990’s, it has been widely employed across a number of scientific disciplines, including genomics, ecology, medicine, plant breeding, epidemiology, dairy science, and signal/image processing ( e.g., Pawitan et al., 2005; Spelman and Bovenhuis, 1998). A false positive result occurs when two means are deemed significantly different when, in fact, they are not. I f one uses a 5% type I error rate for each agronomic characteristic measured, then the number of false positives increases as the number of characteristics increase. In order to help manage the false positive rate, the FDR method of Benjamini and Hochberg was applied to account for making multiple comparisons (Benjamini and Hochberg, 1995; Westfall et al., 1999). P-values were adjusted accordingly. This resulted in the false positive rate being held to 5%. Both adjusted and unadjusted P-values are provided for the agronomic data. I n each experiment, a significant difference between the mean of 4114 maize and that of the control line was established with a n FDA-adjusted P-value of less than 0.05 . Using the data obtained from the reference hybrids, a statistical tolerance interval was calculated to contain, with 95% confidence, 99% of the values contained in the population of commercial maize. This statist ical tolerance interval provided further context for interpretation of the agronomic results for 4114 maize. The agronomic characteristic measurements for 4114 maize that fell within the tolerance interval for that characteristic were considered to be wit hin the range of normal variability of commercial maize hybrids.Pioneer Hi - Bred International, Inc. Page 131 of 25 8

4114 Maize **Table 28 . Site Locations for 2010 Field Trial Experiments**  **Map Site Number**  **Country Location** 

**Experiment A: 2010 Field Trials** 1

U.S. Sheridan, IN 2 Deerfield, MI 3 Richland, IA 4 Ro chelle, IL 5 Bagley, IA 6 Seymour, IL 7 York, NE 8 Carlyle, IL 9 Wyoming, IL 10 Atlantic, IA 11 Geneva, MN **Experiment B: 2010 Field Trials** 12 U.S. Richland, IA 13 Wyoming, IL 14 Geneva, MN 15 York, NE 16 Canada Branchton, ON 17 Th orndale, ON

**1 2**  **3, 12**  **5 6**  **7, 15**  **8** Canada

Un ited States **9, 13**  **10 11,**  **14 4**  **16 17** 

**Figure 33 . Map of Locations for Agronomic Data Collection for 4114 Maize** Pioneer Hi - Bred International, Inc. Page 132 of 25 8

4114 Maize **Experiment A**  contains data from 11 field site locations planted in the U.S. during the 2010 growing se ason (Table 28, Figure 33 ). The purpose of Experiment A was to evaluate and compare the agronomic characteristics and yield of 4114 maize with a near- isoline control. Each site consisted of a randomized complete block design with four blocks, with each block containing 4114 maize and the control maize. Procedure s employed to control the introduction of experimental bias in this study were as follows: non -systematic selection of trial areas and plot areas within each site, randomization of the maize entries within each block, and uniform maintenance treatments across each plot area. The following agronomic characteristics were evaluated in all four blocks: early population, seedling vigor, time to silking, time to pollen shed, plant height, ear height, stalk lodging, root lodging, final population, stay green, disease incidence, insect damage, and yield. D escriptions of the characteristics and their measurement are fou nd in Table 29 . The results of Experiment A are summa rized in Table 30 . No statistically significant differences were observed between 4114 maize and control maize values for all characteristics measured. Based on the data obtained in this study, agronomic characteristics and yield were comparable between 4114 maize and the control maize.Pioneer Hi - Bred International, Inc. Page 133 of 25 8

4114 Maize **Table 29 .**  **Agronomic Characteristics Measured General**  **Characteristic Characteristic** 

**Measured Evaluation** 

**Timing a**  **Data Description Scale**  Germination/ Emergence Early Population V2 -V4 Number of plants emerged per plot Actual count per plot Seedling Vigor V2 -V4 Visual estimate of average vigor of emerged plants per

plot From 1 -9, where

1=short plants with small leaves, and 9=tall plants with large leaves

Vegetative Parameters Plant Height R4 Height from the soil surface to the tip of the tassel Height in cm Ear Height R4 Height from the soil surface to the base of the primary

ear Height in cm Stalk Lodging R6 Visual estimate of percent of plants in the plot with stalks broken below the primary ear 0 to 100%

Root Lodging R6 Visual estimate of percent of plants in the plot leaning approximately 30° or more in the first 2 feet (0.6m) above the soil surface 0 to 100% Final Population R6 The number of plants remaining per plot Actual count per plot

Stay Green R6 Overall plant health Ranging from 1 -9 where 1=no visible green tissue; 5=approximately 50% gree n tissue remaining; 9=very green approximately 90% or greater green tissue remainingPioneer Hi - Bred International, Inc. Page 134 of 25 8

4114 Maize **Table 29 .**  **Agronomic Characteristics Measured (continued)**  **General Characteristic**  **Characteristic**

**Measured Evaluation** 

**Timing a**  **Data Description Scale** 

Reproductive Parameters Time to Silking Approximately 50% silking From the time of planting

until approximately 50% of the plants have emerged

silks Number of

Accumulated Heat Units (AH U) Time to Pollen Shed Approximately

50% pollen shed From the time of planting

until approximately 50% of the plants have tassels shedding pollen Number of Accumulated Heat Units (AH U) Yield Approximately

R6 Harvest weight per area

adjusted to 13% moisture content Bushels per acre

Pollen Viability b During pollen

shed Pollen Shape Percentage of pollen grains with collapsed walls at 0, 30, 60, and 120 minutes Pollen Color Percentage of

pollen grains with intense yellow color at 0, 30, 60, and 120 minutes

Pest Response Disease

Incidence Approximately

R6 Visual estimate of foliar

disease in cidence Ranging from 1 -9 where 1=poor disease resistance or high infection; 9=best disease resistance or low infection Insect Damage Approximately

R6 Visual estimate of insect

damage Ranging from 1 -9

where 1=poor insect resistance or high damage; 9=best i nsect resistance or low damage a Refer to Ritchie et al., 2005 for a description of maize growth stages. b Pollen viability has been correlated to pollen shape and color (Luna et al. , 2001)Pioneer Hi - Bred International, Inc. Page 135 of 25 8

4114 Maize **Table 30 . Exper** **iment A: Summary of Agronomic Performance of 4 114 Maize Across Eleven**  **Locations Agronomic Trait** **a Control Maize**  **4114 Maize Tolerance Interval**  **b** Early Population (Count) Mean c 56 56 41 - 60

Range d 37 - 60 40 - 60 CI e 53 - 58 53 - 58 Adjusted P-Value f 0.823 P-Value g 0.690 Final Populatio n (Count) Mean 54 55 34 - 60

Range 39 - 60 38 - 63 CI 51 - 57 51 - 57 Adjusted P-Value 0.823 P-Value 0.823 Time to Silking (Accumulated Heat Units) Mean 1370 1370 841 - 1790

Range 1020 - 1590 1020 - 1640 CI 1270 - 1480 1270 - 1480 Adjusted P-Value 0.823 P-Value 0.563 Time to Pollen Shed (Accumulated Heat Units) Mean 1410 1410 855 - 1810

Range 1100 - 1620 1100 - 1670 CI 1310 - 1500 1310 - 1500 Adjusted P-Value 0.823 P-Value 0.759 Seedling Vigor (1-9 Scale) Mean 7 7 3 - 9

Range 4 - 9 4 - 9 CI 6 - 8 6 - 8 Adjusted P-Value 0.823 P-Value 0.614 Stalk Lodging ( %) Mean 2 2 0 - 20 i

Range 0 - 20 0 - 20 CI NA h NA Adjusted P-Value 0.823 P-Value 0.386 Root Lodging ( %) Mean 6 6 0 - 8 i

Range 0 - 45 0 - 45 CI NA NA Adjusted P-Value 0.823 P-Value 0.485 Stay Green (1-9 Scale) Mean 5 5 1 - 9

Range 1 - 9 1 - 9 CI 3 - 6 3 - 6 Adjusted P-Value 0.823 P-Value 0.121Pioneer Hi - Bred International, Inc. Page 136 of 25 8

4114 Maize **Table 30 .**  **Experiment A: Summary of Agronomic Performanc e of 4114 Maize Across Eleven**  **Locations (continued)**  **Agronomic Trait Control Maize**  **4114 Maize Tolerance Interval**  Disease Incidence (1-9 Scale) Mean 7 7 1 – 9

Range 5 - 9 5 - 9 CI 7 - 8 7 - 8 Adjusted P-Value 0.823 P-Value 0.749 Insect Dama ge (1-9 Scale) Mean 8 8 2 – 9

Range 6 - 9 5 - 9 CI NA NA Adjusted P-Value 0.823 P-Value 0.792 Plant Height (cm) Mean 292 294 112 – 389

Range 232 - 323 234 - 325 CI 277 - 307 279 - 309 Adjusted P-Value 0.823 P-Value 0.196 Ear Height (cm) Mean 121 120 39 – 167

Range 80 - 162 71 - 153 CI 106 - 136 105 - 135 Adjusted P-Value 0.823 P-Value 0.449 Yield (bushels per acre) Mean 174 176 NCj

Range 68 - 257 105 - 240 CI 149 - 199 151 - 201 Adjusted P-Value 0.82 3 P-Value 0.632 a Refer to Table 29 for descriptions of each agronomic characteristic measured. b Tolerance Intervals were calculated from the non - modified commercial maize lines evaluated. These intervals contain 99% of the values with 95% confidence level unless otherwise noted. c Least squares mean d Range denotes the lowest and highest individual value across sites. e 95% Confidence Interval f False Discovery Rate (FDR) adjusted P-value g Non -adjusted P-value h Statistical analysi s was not available (NA) i No tolerance interval could be calculated; these values are minimum/maximum ranges j No tolerance interval could be calculated (NC)Pioneer Hi - Bred International, Inc. Page 137 of 25 8

4114 Maize **Experiment B**  contains data from six field site locations planted in the U.S. and Canada du ring the 2010 growing seaso n (Table 28, Figure 3 3 ). Th e purpose of Experiment B was to evaluate and compare the agronomic and pollen characteristics of 4114 maize with a near- isoline control. Each site included a randomized block design containing four b locks, with each block containing a plot of 4114 maize and one plot of control maize. Procedures employed to control the introduction of experimental bias in this study were as follows: non -systematic selection of trial areas and plot areas within each site, randomization of the maize entries within each block, and uniform maintenance treatments across each plot area. The following agronomic characteristics were measured: early population, final population, time to silking, time to pollen shed, pollen via bility, seedling vigor, stalk lodging, root lodging, stay green, disease incidence, insect damage, plant height, and ear height. Descriptions of the characteristics and their measurement are found in Table 29 . Results of Experiment B are summ arized in Table 31 . N o statistically significant differences were observed between 4114 maize and co ntrol maize values for all characteristics measured. Based on the data obtained in this study, agronomic characteristics and yield were comparable between 4114 maize and the control maize.Pioneer Hi - Bred International, Inc. Page 138 of 25 8

4114 Maize **Table 31**  **. E** **xperiment B:**  **Summary of**  **Agronomic Characteristics**  **of 4114**  **Maize Across**  **Six Locations**  **Agronomic Characteristic a**  **Control Maize 4114 Maize**  **Tolerance Interval b**  Early Population (count) Mean c 57 58 41 – 60 Range d 50 - 59 42 - 60 CI e 53 - 59 55 - 60 Adjusted P-Value f 0.354 P-Value g 0.0596 Final Population (count) Mean 55 57 34 – 60 Range 38 - 59 46 - 60 CI 49 - 58 52 - 59 Adjusted P-Value 0.225 P-Value 0.0125 Time to Silking (accumulated hea t units) Mean 1380 1390 841 – 1790 Range 1300 - 1490 1300 - 1590 CI 1320 - 1450 1320 - 1450 Adjusted P-Value 0.494 P-Value 0.367 Time to Pollen Shed (accumulated heat units) Mean 1420 1430 855 – 1810 Range 1350 - 1510 1350 - 1620 CI 135 0 - 1480 1360 - 1490 Adjusted P-Value 0.494 P-Value 0.384 Pollen Viability – Shape h ( % of collapsed pollen ) 0 minutes Mean 3 2 0 – 65 Range 0 - 80 0 - 70 CI 0 - 13 0 - 12 Adjusted P-Value 0.440 P-Value 0.244 Pollen Viability - Shap e ( % of collapsed pollen ) 30 minutes Mean 67 55 0 – 100 Range 15 - 100 20 - 90 CI 32 - 94 21 - 87 Adjusted P-Value 0.354 P-Value 0.0786 Pollen Viability - Shape ( % of collapsed pollen ) 60 minutes Mean 88 91 4 – 100 Range 40 - 100 50 - 100 CI 63 - 100 67 - 100 Adjusted P-Value 0.494 P-Value 0.348 Pollen Viability - Shape ( % of collapsed pollen ) 120 minutes Mean 98 97 37 – 100 Range 85 - 100 85 - 100 CI NAi NA Adjusted P-Value 0.602 P-Value 0.502Pioneer Hi - Bred International, Inc. Page 139 of 25 8

4114 Maize **Table 31**  **. E** **xperi ment**  **B: Summary**  **of Agronomic**  **Characteri stics**  **of 4114**  **Maize Across**  **Six L** **ocations (continued)**  **Agronomic Characteristic Control Maize**  **4114 Maize Tolerance Interval**  Pollen Viability - Color h ( % of intense yellow pollen ) 0 minutes Mean 2 2 0 - 84 Range 0 – 80 0 - 70 CI 0 – 12 0 - 10 Adjusted P-Value 0.376 P-Value 0.123 Pollen Viability - Color ( % of intense yellow pollen ) 30 minutes Mean 57 50 0 - 100 Range 15 - 100 20 - 90 CI 22 - 89 16 - 84 Adjusted P-Value 0.440 P-Value 0.235 Pollen Viability - Color ( % of intense yellow pollen ) 60 minutes Mean 82 86 14 - 100 Range 40 - 100 55 - 100 CI 50 - 99 56 - 100 Adjusted P-Value 0.376 P-Value 0.155 Pollen Viability - Color ( % of intense yellow pollen ) 120 minutes Mean 95 9 7 56 - 100 Range 85 - 100 85 - 100 CI NA NA Adjusted P-Value 0.376 P-Value 0.151 Seedling Vigor (1-9 scale) Mean 8 7 3 - 9 Range 6 - 9 5 - 9 CI NA NA Adjusted P-Value 0.354 P-Value 0.0522 Stalk Lodging (%) Mean 0 0 0 - 20j

Range 0 - 4 0 - 5 CI NA NA Adjusted P-Value NA P-Value NA Root Lodging (%) Mean 0 0 0 - 8j

Range 0 - 2 0 - 1 CI NA NA Adjusted P-Value NA P-Value NA Stay Green (1-9 scale) Mean 4 4 1 - 9 Range 1 - 7 1 - 7 CI 2 - 6 3 - 6 Adjusted P-Value 0.735 P-Value 0.729Pioneer Hi - Bred International, Inc. Page 140 of 25 8

4114 Maize **Table 31**  **. Experiment**  **B: Summary**  **of Agronomic**  **Characteri stics**  **of 4114**  **Maize Across**  **Six L** **ocations (continued)**  **Agronomic Characteristic Control Maize**  **4114 Maize Tolerance Interval**  Disease Incidence (1-9 scale) Mean 8 8 1 – 9 Range 5 - 9 5 - 9 CI NA NA Adjusted P-Value 0.735 P-Value 0.735 Insect Damage (1-9 scale) Mean 8 8 2 – 9 Range 7 - 9 7 - 9 CI NA NA Adjusted P-Value 0.735 P-Value 0.731 Plant Height (cm) Mean 285 287 112 – 389

Range 256 - 324 265 - 313 CI 268 - 302 270 - 304 Adjusted P-Value 0.494 P-Value 0.333 Ear Height (cm) Mean 113 115 39 – 167

Range 82 - 150 79 - 145 CI 95 - 130 97 - 132 Adjusted P-Value 0.376 P-Value 0.167 a Refe r to Table 29 for descriptions of each agronomic characteristic measured. b Tolerance Intervals were calculated from the non - modified commercial maize lines evaluated. These intervals contain 99% of the values with 95% confidence level unless otherwise noted. c Least squares mean d Range denotes the lowest and highest individual value across sites. e 95% Confidence Interval f False Discovery Rate (FDR) adjusted P-value g Non -adjusted P-value h Pollen viability has been correlated to pollen shape and colo r (Luna et al. , 2001) i Statistical analysis was not available (NA) j No tolerance interval could be calculated; these values are minimum/maximum rangesPioneer Hi - Bred International, Inc. Page 141 of 25 8

4114 Maize **Field Insect and Disease Observations**  As discussed in Section 1, 4114 maize has been field tested in the U.S. and Puerto Rico since 2006, as authorized by USDA-APHIS permits and notifications. For each trial, a survey of the naturally occurring insects and diseases and any unexpected differences in the response of 4114 maize as compared to the cont rol line (near- isoline and/or conventional maize lines) were recorded by experienced plant breeders and field staff at least every four weeks . A summary of these surveys for each trial and any differences seen between 4114 maize and control lines are presented in Appendix 8. These observations provide a means to determine if 4114 maize will respond differently from conventional maize lines to insects or disease s in the environment. In every case, 4114 maize did not exhibit any unexpected responses to nat urally occurring insects or diseases . These results support the conclusion that 4114 maize is comparable to control maize lines with similar genetics or to conventional maize lines with respect to insect or disease response. **Conclusions on Agronomic Pe rformance**  **and Field Observations** 4114 maize was observed in laboratory experiments and at 17 field locations in the U.S. and Canada to measure agronomic parameters. These experiments and field studies evaluate the characteristics of maize over a broad ra nge of environmental conditions that represent regions where 4114 maize will be grown. The agronomic parameters measured are characteristic traits for reproduction, survival, and potential weediness. The agronomic data showed no significant differences between 4114 maize and control maize (near- isoline controls and/or commercial maize lines) with respect to early population, vegetative growth, reproductive parameters, yield , and pest responses . These data support the conclusion that 4114 maize is agronom ically comparable to conventional maize. O bservations from U.S. and U.S. territory field trials showed no unexpected differences in the response of 4114 maize and control maize to naturally occurring insects and diseases. These results support the conclu sion that 4114 maize is comparable to control maize lines with similar genetics and/or to conventional maize lines. Based on these analyses, 4114 maize is comparable to conventional maize and will not pose a greater plant pest risk or increased weed poten tial than conventional maize. **4 -** **C . Overall Conclusions to Comparative Assessment of 4114 Maize**  A comparative assessm ent was conducted to determine if 4114 maize would present any new or greater risks relative to maize varieties that have a history of sa fe use in the environment and as food and feed. This comparative assessment included compositional and agronomic analyses with appropriate comparators to determine comparability to conventional maize. These comparative analyses were designed to eliminate the effects of the introduced insect-Pioneer Hi - Bred International, Inc. Page 142 of 25 8

4114 Maize resistant and herbicide-tolerant traits in 4114 maize, so that an appropriate comparison to conventional maize could be made and any unintended effects that may be due to the 4114 maize insertion could be evaluated. Extensive nutrient composition analyses of grain and forage were conducted to compare the composition of 4114 maize to controls. These analyses were used to evaluate any changes in the levels of key nutrients, anti- nutrients and secondary metabolites. Based on the results of the compositional evaluation, the grain and forage of 4114 maize are as safe as commercially available maize and there would be no significant impact on raw or processed maize commodities. A comprehensive agronomic evaluation for 4114 maize was conducted . The agronomic evaluations were based on both laboratory experiments and replicated, multi-site field trials. D ata were collected on representative characteristics that influence reproduction, crop survival, and potential weediness. Field observations included observed responses of 4114 maize and control mai ze to naturally occurring insects and diseases in a wide variety of environmental conditions. These data support the conclusion that, independent of the introduced insectresista nt and herbicide-tolerant traits, 4114 maize is comparable to conventional maize and is unlikely to pose a greater plant pest risk than conventional maize. In conclusion, t hese analyses indicated that 4114 maize was comparable to conventional maize with respect to the compositional analytes and agronomic characteristics measured. 4114 maize differs from conventional maize only in its herbicide tolerance and its efficacy against certain lepidopteran and corn rootworm pests. Overall, these comparative assessments indicate that 4114 maize is as safe as conventional maize varieties and does not pose a greater risk than conventional maize varieties in food, feed, and the environment.Pioneer Hi - Bred International, Inc. Page 143 of 25 8

4114 Maize **5. Potential Environmental Impact of the Introduction of 4114 Maize** The potential environmental impact of a transgenic plant need s to be considered in the context of the characteristics of the recipient crop, the introduced trait, and the environment in which it will be introduced (OECD, 1993). Knowledge in each of these area s will provide background on which a risk or safety assessment can be made about the environmental release of the transgenic plant (OECD, 1993). I n particular, weediness, gene transfer or flow, and trait effects are particular issues that may be relevant to evaluating the new transgenic line and its safety (OECD, 1993). In order to evaluate the potential environmental impact of the introduction of 4114 maize, the potential for 4114 maize to become weedy or invasive, the potential for gene flow to sexually compatible wild relatives, and the potential impacts of the introduced proteins ( Cry1F, Cry34Ab1, Cry35Ab1, and PAT) were considered. As described further below, in each case, it is not expected that 4114 maize will adversely impact the environment with respect to these considerations. **5 -** **A. P** **otential for**  **4114 Maize**  **to Have**  **Altered Disease**  **and Unintended**  **Pest Susceptibilities**  **or to Become Weedy or Invasive** In evaluating the potential for 4114 maize to become more weedy or invasive than conventional maize, general maize biology was considered. M aize is a cultivated annual plant that generally cannot survive temperatures below freezing and is typically grown in temperate regions (OECD, 2003). M aize is not classified as a weed, is not on the U.S. federal or state noxious weed lists, and possesses few characteristics of notably successful weeds (Baker, 1974; Keeler, 1989; USDA- NRCS, 2011). Therefore, the natural characteristics of maize do not indicate a high potential for weediness or invasiveness. A com parative assessment of 4114 maize was conducted to determine if the DNA insertion altered the nutritive or agronomic characteristics of maize. Compositional and agronomic comparison data were collected on 4114 maize in multiple location field trials as described in Section 4; these analyses were designed to be independent of the introduced traits so that an appropriate comparison to conventional maize could be made and any unintended effects of the 4114 maize insertion could be assessed . These analyses sh owed that 4114 maize was comparable to conventional maize in composition, except for the introduced proteins, and was comparable in agronomics. In the agronomic analyses, a number of characteristics were measured, including certain ones that may be indica tive of weediness : germination and emergence (germination rate, early population , seedling vigor); reproductive (time to silking, time to pollen shed, pollen viability, yield) ; vegetative (final population stalk lodging, root lodging, stay green, plant height, ear height); and pest response (disease incidence, insect damage). C haracteristics related to seed germination, seed production, reproductive time and vegetative competitiveness have been identified with successful weeds (Baker, 1974); changes to these parameters relative to the conventional variety could indicate a change in the potential weediness of a crop . 4114 maize was comparable to conventional maize in each ofPioneer Hi - Bred International, Inc. Page 144 of 25 8

4114 Maize these characteristics, indicating that 4114 maize is unlikely to become more weedy or invasive than conventional maize. In addition, 4114 maize has been field tested since 2006 in multiple locations that provide a range of environmental conditions and also include regions representative of maize cultivation in the U.S. These fields were frequently monitored by expert growers for the incidence of diseases and insects and the effect of these on 4114 maize and control plants. In all cases, no unexpected differences were observed between 4114 maize and the control comparators. In summary, 4114 maize is unlikely to become more weedy or invasive than conventional maize when cultivated. Compositional and agronomic comparisons indicate no unexpected effects of the presence of the introduced proteins that alter the nutritional composition an d weediness potential of maize. No unexpected differences were detected between 4114 maize and control maize in response to insects and diseases . Furthermore, the expression of the introduced proteins (Cry1F, Cry34Ab1, Cry35Ab1, PAT) is unlikely to incre ase the potential of 4114 maize to become weedy. **5 -**  **B. Potential for Gene Flow Between 4114 Maize and Sexually Compatible Wild Relatives** The potential for gene flow between a transgenic crop and its sexually compatible wild relatives is assessed throug h several factors. O ne factor includes the potential for pollen flow and outcrossing to occur significantly outside the cultivated field. Other factors include the overlap of the wild relative geographic distribution with the region of transgenic crop cu ltivation and the possibility of genetic compatibility between the crop and the relative. F inally, to determine the potential for widespread introgression of the trait into wild relative populations, whether the trait itself alters weediness characteristi cs and whether the wild relative is a noxious weed is considered. 4114 maize will be cultivated similarly to other commercial and conventional maize varieties; therefore, it is appropriate to examine maize pollination biology, regions of maize cultivation in the U.S. and the geographic distribution of sexually compatible wild relatives in order to determine the potential for gene flow. The regions of maize cultivation in the U.S. and the genetic compatibility and geographic distribution of sexually compat ible wild relatives of maize, within the genera Zea and Tripsacum, are discussed further below. Based on this information, there is low potential for gene flow between 4114 maize and its wild relatives of the genera Zea and Tripsacum in the U.S. The pote ntial for the insertion in 4114 maize to become widespread in wild relative populations is also unlikely. The insertion, as discussed in Section 5-A, does not make 4114 maize more weedy than conventional cultivated maize; furthermore, none of the sexually compatible wild relatives are listed as noxious weeds.Pioneer Hi - Bred International, Inc. Page 145 of 25 8

4114 Maize **Pollination Biology o f Maize and Impact on Gene Flow**  Maize is almost entirely cross-fertilizing and its pollen is typically wind dispersed (OECD, 2003); millions of pollen grains are produced per plant (Jarosz et al. , 2003). D espite pollination characteristics that are favorable for pollen flow, other factors make it highly unlikely that viable maize pollen will travel significantly outside of the cultivated field. P ollen viability is reduced in a matter of hours under high temperature and low humidity (Aylor, 2004). Studies also indicate that the majority of maize pollen is unlikely to be dispersed significant distances outside the originating field (Jarosz et al. , 2003). Numerous studies show the majority (84-92%) of pollen grains travel less than five meters (Pleasants et al. , 2001), with nearly all (>99.75%) pollen traveling less than 100 meters (Byrne and Fromherz, 2003; Matsuo et al. , 2004; Sears and Stanley- Horn, 2000). Therefore, the potential of cross- pollination between cultivated maize and its wild relatives will be highest where the wild relatives grow near or adjacent to areas of cultivation. Therefore, the geographic range of wild relatives and the regions of maize cultivation are one critical factor in determining the potential for gene flow. **Regions o f Maize Cultivation in the U.S.**  Field maize is a major crop worldwide, but represents the largest crop grown in the U.S. I t is grown in most states, with production concentrated i n the Heartland region (including Illinois, Iowa, Indiana, eastern portions of South Dakota and Nebraska, western Kentucky and Ohio, and the northern two-thirds of Missouri). Iowa and Illinois are the top maize- producing states and typically account for sl ightly more than one-third of the U.S. crop (USDA- ERS, 2009) . Figure 34 indicates acres planted in the U.S. by county (USDA- NASS, 2011). Additional maize varieties include popcorn and sweet corn, both of which are minor crops compared to field maize (OEC D, 2002). W hile the range of cultivation of popcorn and sweet maize include the entire U.S., in total all acreage represents less than 1% of the acreage of field corn in 2007 (USDA- NASS, 2009).Pioneer Hi - Bred International, Inc. Page 146 of 25 8

4114 Maize

**Figure 34** **. Maiz e Plan** **ted Acres by County in the U.S.** Source: USDA- NASS, 2011Pioneer Hi - Bred International, Inc. Page 147 of 25 8

4114 Maize **Taxonomic Classification of Maize and Related Wild Relatives** Taxonomically, maize (Zea mays L.) is a member of the Maydeae tribe of the grass family, Poaceae (OECD, 2003) . Teosinte, within the genus Zea, and the genus Tripsacum are the closest relatives to maize taxonomically. The genus Tripsacum is also included in the Maydeae tribe (OECD, 2003). A nnual teosintes are grouped into the species Zea mays, although there is some dispute of this classification based on characteristics that prevent a high degree of introgression (OECD, 2003). Annual teosintes have been further classified into the subspecies Zea mays ssp. mexicana and Zea mays ssp. parviglumis (OECD, 2003) . In contrast, perennial teosintes are classified as different sp ecies altogether: Zea perennis and Zea diploperennis (OECD, 2003). Both annual and perennial teosintes are considered the closest wild relatives of cultivated maize (OECD, 2003). P erennial plants of the genus Tripsacum are considered the next closest re latives of maize (OECD, 2003). Neither the Zea genus nor the Tripsacum genus are listed as noxious weeds on the federal or state noxious weed lists (USDA- NRCS, 2011). **Potential for Gene Flow with the Genus Zea**  Both annual and perennial teosintes are nor mally confined to the tropical and subtropical regions of Mexico, Honduras, Guatemala, and Nicaragua (Iltis, 2011). In the U.S., sparsely dispersed introduced populations of annual teosintes Zea mexicana (synonym: Zea mays ssp. mexicana) and Zea mays ssp. parviglumis have been reported in Florida, Maryland, and Alabama (USDA, 2011). A lso, an isolated population of Zea perennis (perennial teosinte) has been introduced in South Carolina (USDA, 2011). W hile maize can hybridize with these species under natural conditions, there is incompatibility between some maize populations and certain types of teosinte that results in low fitness of some hybrids and prevents a high rate of introgression (OECD, 2003). Together with the very limited geographic range of the teosinte population in the U.S., the probability of gene flow from cultivated maize fields to these wild relatives is very low . **Potential for Gene Flow with the Genus Tripsacum**  Plants of the genus Tripsacum are mostly found in Mexico, Central, and Sou th America (OECD, 2003). Three of these species ( T. dactyloides, T. floridanum, and T. lanceolatum ) exist as native species populations in the continental U.S.; and two species ( T. fasciculatum and T. latifolium ) were introduced in Puerto Rico (USDA, 2011 ). T. dactyloides occurs throughout the eastern half of the U.S. T. lanceolatum occurs in Arizona and New Mexico (USDA, 2011) and T. floridanum is native to southern Florida (USDA, 2011). Although it is extremely difficult, Tripsacum species ( T. dactyloides, T. floridanum, and T. lanceolatum ) can be crossed with maize; however, hybrids have a high degree of sterility and are genetically unstable (OECD, 2003). S uccessful crosses of maize with Tripsacum species have been made experimentally, however such crosses are not known to occur in the wild (OECD, 2003). Therefore, gene flow between cultivated maize and relatives of the genus Tripsacum is highly unlikely.Pioneer Hi - Bred International, Inc. Page 148 of 25 8

4114 Maize **Conclusions on the Potential for Gene Flow between 4114 Maize and Wild Relatives** The potenti al for gene flow between maize and relatives of the genera Zea and Tripsacum is very low. W hile wild native or introduced populations of these genera occur where maize is cultivated, limited geographic range and low fitness or sterility of hybrids prevent successful gene flow. F urthermore, none of these wild relatives are considered to be noxious weeds and 4114 maize does not exhibit greater potential for weediness as determined from agronomic comparisons to conventional maize. Therefore, any incidental gene flow between 4114 maize and its wild relatives would not transform maize wild relatives into more weedy species, nor would the introduced trait be introgressed widely in wild relative populations. **5 -** **C. Potential Environmental Impact of the Introduc ed Proteins in 4114 Maize**  4114 maize is comparable in agronomic characteristics and compositional characteristics to conventional maize except for the presence of the introduced proteins, Cry1F, Cry34Ab1, Cry35Ab1, and PAT. Therefore, one aspect of assessing the potential for the environmental impact of 4114 maize cultivation is to consider the potential risk of the introduced proteins to non -target organisms; this risk is quantified by examining the environmental exposure and the potential hazard of the introduced proteins to non -target organisms. As described earlier, the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins in 4114 maize are identical to those in previously approved 1507 and/or 59122 maize, which USDA and EPA determined would not adversely impa ct the environment (EPA, 2010a; EPA, 2010b; Hunst and Rood, 2004; Shanahan and Stauffer, 2000; USDA-APHIS, 2001; USDA-APHIS, 2005). The impact of the PAT protein from 4114 maize on the environment is expected to be negligible. The PAT protein is not toxic or allergenic and its food, feed, and environmental safety is well known and documented (CERA, 2011; Hérouet et al. , 2005; OECD, 1999). Furthermore, the PAT protein was evaluated for 1507 and 59122 maize and is not expected to have any significant impact on non -target organisms and threatened and endangered species (USDA-APHIS, 2001; USDA-APHIS, 2005). The Cry1F and the binary Cry34Ab1 and Cry35Ab1 proteins (i.e., the Cry34/35Ab1 protein) are known to have insecticidal activity against certain specific lepidopteran species and corn rootworm species, respectively. The specificity of this activity to target insects has been well established, and there is low risk of hazard to non -target orders, avian species, and mammals, as determined previously by U SDA and EPA for 1507 and 59122 maize and by EPA for 1507x59122 maize (EPA, 2010a; EPA, 2010b; Hunst and Rood, 2004; Shanahan and Stauffer, 2000; USDA-APHIS, 2001; USDA-APHIS, 2005). W ith the exception of Cry1F protein concentrations in pollen, the concent rations of Cry1F and Cry34/35Ab1 in 4114 maize tissues are generally similar to or lower than 1507, 59122, and 1507x59122 maize, as shown in Section 2 - F . Therefore, the previously conducted environmental safety studies for 1507, 59122 and 1507x59122 maize are relevant for evaluating the environmental impact of 4114 maize. The concentration of Cry1F protein in 4114 maize pollen is approximately 1.5 times that of 1507 or 1507x59122 maize. Despite this increase, the laboratory tests still indicate a suffici ent marginPioneer Hi - Bred International, Inc. Page 149 of 25 8

4114 Maize of safety for the expected environmental exposure to the Cry1F protein from 4114 maize pollen. Similar to the previous conclusions for 1507 and 59122 maize, the cultivation of 4114 maize is unlikely to have adverse effects on non -target arthropods, including threatened and endangered arthropods, and non -target vertebrates, including birds, mammal, and humans. I n support of this conclusion, 1507x59122 maize has been commercially available in the U.S. since the 2006 growing season with no negative safety or environmental effects. **Environmental Exposure and Environmental Persistence** One aspect of evaluating the potential environmental impact of the introduced proteins considers the probability and degree to which non -target organisms will be exposed to the proteins. O rganisms can be exposed to the proteins in several ways. A nimals and insects can feed on different parts of the plant and could be exposed to the proteins directly. A lso, as the plant tissue residue degrades in the agricultural fie ld, soil dwelling organisms may be exposed to these proteins if they persist in soil and aquatic organisms could be exposed from water run off. P redatory insects may also be exposed to the Cry1F and Cry34/35Ab1 proteins from prey insects that feed on 4114 maize (i.e. , tri -trophic interactions), if these proteins accumulate in the prey insects. For soil dwelling or aquatic organisms, protein exposure is highly dependent on protein degradation and dissipation in soils. Most Cry proteins do not persist or accumulate in soil over time (Clark et al. , 2005; Icoz and Stotzky, 2008). S imilarly, the soil dissipation of Cry1F and Cry34/35Ab1 proteins is very rapid. I n data previously provided for 1507 and 59122 maize, the time required for 50% of the Cry1F prote in activity to dissipate in soil (DT50) is 3.13 days and the DT50 for Cry34/35Ab1 proteins is approximately 3.2 days (Hunst and Rood, 2004; Shanahan and Stauffer, 2000; USDA-APHIS, 2001; USDA-APHIS, 2005). Later published work concluded that the Cry1F protein activity dissipated even more quickly in soil; it took less than one day for 50% of the activity to dissipate (Herman et al. , 2001). Additionally, no Cry1F protein was detected in soil sampled from fields following three years of successive cultivati on of 1507 maize (Shan et al. , 2008). Lack of accumulation in soil would limit the amount of protein present in field water run off. Because of the rapid dissipation of activity in soil and the low potential for the proteins to accumulate in soil, exposure to Cry1F and Cry34/35Ab1 from soil would not be significant, nor would these proteins accumulate in aquatic habitats. For protein exposure through tri-trophic interactions, the degree of persistence and accumulation of these proteins in the prey insect (i.e. , bioaccumulate) will limit overall exposure to predatory insects. P roteins, in general, do not have the chemical characteristics needed to accumulate (Spacie et al., 1995; USGS, 2010). Lack of accumulation and short halflife has been confirmed for certain Cry proteins in prey species (Li and Romeis, 2010; Lundgren and Wiedenmann, 2005; Meissle and Romeis, 2009; Obrist et al. , 2005; Romeis and Meissle, 2011), there have been no studies using validated and robust ELISA or western blot methods that ha ve indicated bioaccumulation of Cry proteins in prey foods . Exposure to Cry proteins viaPioneer Hi - Bred International, Inc. Page 150 of 25 8

4114 Maize prey is a less significant route of exposure than direct feeding on plant material ( e.g. , pollen). Therefore, predatory insects are unlikely to be significantly exposed to Cry1F or Cry34/Cry35Ab1 proteins through tri -tropic interactions . Potential environmental exposure of the introduced proteins can be estimated to determine the significance of laboratory safety testing conducted. The mean concentrations of the Cry 1F, Cry34Ab1, and Cry35Ab1 proteins were measured in 4114 maize plant tissues (Section 2 - F) and were used to determine or estimate an expected environmental concentration (EEC) . In certain instances, the protein concentration in a particular tissue was used directly to determine the EEC, depending on the feeding habits or the preferred habitat of the non -target organism. I n other cases, estimations were made to determine the EEC. E EC can be estimated by considering the protein concentrations in the plant tissue consumed or contacted and the amount of plant matter present in the environment. Certain factors can limit environmental exposure and can be used to refine estimations. D egree of pollen dispersal and deposition were considered for pollen consumpt ion by certain non -target organisms. For EEC in soil and aquatic environments, field planting density and residual field plant matter were used to guide numerical estimates. As general consideration, the lack of overlap between geographic range or prefer red habitat of the wildlife species to regions of maize cultivation would further limit potential environmental exposure . In summary, non -target organisms can be exposed to proteins from 4114 maize through direct consumption of plant tissue . I n general , indirect exposure via soil and via tri-trophic interactions is not expected to be significant. In order to determine the significance of laboratory safety testing on non -target organisms, the potential environmental exposure for 4114 maize was estimated from the mean Cry1F and Cry34/35Ab1 protein concentrations. Potential environmental exposure was considered in determining the potential impact on non target organisms.Pioneer Hi - Bred International, Inc. Page 151 of 25 8

4114 Maize **Potential Hazard of the Introduced Proteins**  **on Non** **- Target Organisms**  In order to determine the potential hazard of the Cry1F and Cry34/35Ab1 proteins may have on non -target organisms, early-tier laboratory studies using purified protein were conducted on representative species of interest and surrogate species . These species were sele cted to provide an evaluation of the effects of Cry1F and Cry34/35Ab1 on different families and orders of organisms. S urrogate species are typically selected because they are amenable to the laboratory setting, are environmentally sensitive and representative of the agroecosystem, and can be use d to predict potential impacts on related non -target organisms, including beneficial, threatened, or endangered species (Romeis et al. , 2011) . Early-tier testing is typically done to simulate higher than expected environmental exposure, typically 10-fold (Rose, 2007). The 10-fold margin of exposure also accounts for interspecies variability (Romeis et al. , 2011). The EPA has concluded that if there is no significant hazard (i.e. , greater than 50% adverse effect) observed at these unrealistically high laboratory exposures, then the risk of hazard at environmentally relevant concentrations is very low (Ro se, 2007). O nly when the early-tier hazard study detects an effect, should the next tier study be triggered (Ro meis et al. , 2008). Early-tier laboratory testing was conducted for previously approved 1507 and 59122 maize (EPA, 2010a; EPA, 2010b; Hunst and Rood, 2004; Shanahan and Stauffer, 2000; USDA-APHIS, 2001; USDA-APHIS, 2005). These studies include representative and surrogate species in order to predict the potential impacts of the Cry1F and Cry34/35Ab1 proteins on non -target organisms in the environment. These studies were considered in determining the potential hazard of the proteins and were used to deter mine the overall potential impact of 4114 maize on non -target organisms in the environment . **Potential Impact of the Cry1F and Cry34/35Ab1 Proteins on Non -** **Target Organisms** As determined for previously approved events 1507 and 59122 maize, the Cry1F and Cry34/35Ab1 proteins target certain specific lepidopteran species and corn rootworm species, respectively. I n addition, the lack of adverse effects of these proteins on non -target organisms was confirmed through laboratory assays and field assessments. Based on these safety studies for 1507 and 59122 maize, the Cry1F and Cry34/35Ab1 proteins are not expected to adversely affect other invertebrates and all vertebrate organisms, including non -target birds, mammals, and humans, because of the high specificity of these insecticidal proteins to certain insect orders. Many of the previously conducted safety studies for 1507 and 59122 maize are relevant for evaluating the environmental impact of 4114 maize, because the concentrations of Cry1F and Cry34/35Ab1 in 4114 maize tissues are generally similar to or lower than 1507, 59122, and 1507x59122 maize. One exception is the slight increase in Cry1F levels in pollen. Despite this increase, the laboratory tests still indicate a sufficient margin of safety for expe cted environmental concentration (EEC) to the Cry1F protein from 4114 maize pollen. Therefore, asPioneer Hi - Bred International, Inc. Page 152 of 25 8

4114 Maize with 1507 and 59122 maize, the margin of safety of the Cry1F and Cry34/35Ab1 proteins indicated that 4114 maize would not adversely affect non -target organis ms and the environment. An overview of specificity of the proteins and an overview of the potential impact of the proteins on non -target organisms is discussed further below. The safety studies conducted on the purified Cry1F and Cry34/35Ab1 proteins were used to assess the potential hazard on a number of representative non -target species (Tables 32 and 33 ) . The potential exposure to the proteins was calculated by estimating the EEC. Margins of exposure for 4114 maize were calculated by dividing the no observable effect concentration (NOEC) or the lethal concentration (LC50) from the previously conducted safety studies for 1507 and 59122 maize by the EEC of each protein in 4114 maize. Both the potential hazard and the potential exposure were used to con clude the overall potential impact the proteins from 4114 maize would have on non -target organisms. **Specificity to Target Insect Pests** The insecticidal Cry1F and Cry34/35Ab1 proteins have been assessed by laboratory studies to evaluate the specificity of these proteins to target insects. In general, only insect species within a given order and closely related families are susceptible to a given insecticidal protein. Extensive studies have determined that Cry1F has specificity for certain lepidopteran target pests. C ry1F is EPA labeled for protection against European corn borer ( Ostrinia nubilalis), fall armyworm (Spodoptera frugiperda), corn earworm ( Helicoverpa zea), western bean cut worm (Striacosta albicosta Smith), black cutworm (Agrotis ipsilon ), lesser corn stalk borer ( Elasmopalpus lignosellus), southwestern corn borer (Diatraea grandiosella), and sugarcane borer ( Diatraea saccharalis) (EPA, 2011). Similarly, studies have confirmed the specificity of Cry34Ab1 and Cry35Ab1 for certain corn rootworm target pests. C ry34/35Ab1 is EPA labeled for protection against northern corn rootworm ( Diabrotica barberi), western corn rootworm ( Diabrotica virgifera virgifera ), and Mexican corn rootworm ( Diabrotica virgifera zeae) (EPA, 2011). C ry34/35Ab1 also h as activity for southern corn rootworm ( Diabrotica undecimpunctata howardi) as determined by use in bioassays (EPA, 2010b). Along with the non -target assessments described below, the specificity of the Cry1F and Cry34/35Ab1 proteins to the lepidopteran in sects and corn rootworm species, respectively, provides support that these proteins are unlikely to pose a risk to unrelated non -target organisms in the environment.Pioneer Hi - Bred International, Inc. Page 153 of 25 8

4114 Maize **Potential Impact on Soil Dwelling Non -**  **Target Organisms** The potential hazard of the Cry1 F and Cry34/35Ab1 proteins on two representative soil dwelling species, springtail and earthworm, was previously assessed for 1507 and 59122 maize though laboratory assays that used purified protein (EPA, 2010a; EPA, 2010b; Hunst and Rood, 2004; Shanahan a nd Stauffer, 2000; USDA-APHIS, 2001; USDA-APHIS, 2005). The results from the laboratory assays are given in Tables 32 and 33 . Based on the calculation used by Hunst and Rood (2004) for 59122 maize, the EEC of the proteins in soil from 4114 maize was esti mated based on factors such as planting density, dry weight of a maize plant, and mean protein concentrations in senescent (R6) maize tissue (Tables 32 and 33 ). N one of the soil dwelling species tested showed significant adverse effects from the proteins and the margins of exposure were greater than 22 -fold (Tables 32 and 33, Representative Soil Dwelling Species ). Furthermore, as described earlier, soil dwelling non -target organisms are unlikely to be exposed to significant concentrations of the Cry1F and Cry34/35Ab1 proteins in soil , as realistic environmental exposures are expected to be significantly lower based on protein degradation and lack of accumulation in soil. Taking together the potential protein hazard and potential exposure in soil, soil d welling non target invertebrates are unlikely to be impacted by the Cry1F and Cry34/35Ab1 proteins at the expected environmental concentrations for 4114 maize.Pioneer Hi - Bred International, Inc. Page 154 of 25 8

4114 Maize **Table 32**  **. 4114 Maize Estimated Margins of Exposure to Non -** **Target Organisms (Cry1F) Organism:**  **Common Name**  **Species (Order)**  **Cry1F Test**

**Material Expected** 

**Environmental Concentration**  **(EEC) from 4114 Maize**  **Results Margin of**  **Exposure (MOE)**  **a** Representative Soil Dwelling Species Springtail Folsomia candida (Collembola) Purified protein in yeast diet 0.04 1 mg/kg b LC 50 and NOEC >12.5 mg/kg diet >300 Earthworm Eisenia fetida (Haplotaxida) Purified protein in soil 0.041 mg/kg b LC 50 and NOEC >2.5 mg/kg dry soil >60 Representative Coleopteran Species Ladybird beetle Hippodamia convergens (Coleoptera) Family: Coccinellidae Purified protein in honey diet 35 μg/g Cry1F in

pollen c LC 50 >480 ppm

diet >13 Representative Lepidopteran Species Monarch butterfly Danaus plexippus (Lepidoptera) Purified protein in artificial diet 0.5 ng/mg Cry1F

from pollen depositi on on milkweed host plant d LC 50 > 10 ppm

(10,000 ng/mL); NOEC < 10 ppm 20

Representative Arthropod Species Parasitic Hymenoptera Nasonia vitripennis (Hymenoptera) Family: Pteromalidae Purified protein in honey diet 35 μg/g Cry1F in

pollen c LC 50 >320 p pm

diet >9 Green lacewing Chrysoperla carnea (Neuroptera) Family: Chrysopidae Purified protein in moth egg diet 35 μg/g Cry1F in

pollen c LC 50 >480 ppm

diet >13 Honeybee Apis mellifera (Hymenoptera) Family: Apidae Purified protein in sucrose solution 35 μg/g Cry1F in

pollen c NOEC >640 ng

per larvae e >9 dPioneer Hi - Bred International, Inc. Page 155 of 25 8

4114 Maize **Table 32**  **. 4114 Maize Estimated Margins of Exposure to Non -** **Target Organisms (Cry1F) (continued)**  **Organism: Common Name**  **Species (Order)**  **Cry1F Test**

**Material Expected** 

**Environmental Concentration**  **(EEC) fr om 4114**  **Maize Results**  **Margin of Exposure**  **(MOE) a**  Representative Arthropod Species Water flea Daphnia magna (Cladocera) Family: Daphniidae Purified protein dissolved in water 0.0046 μg/ml Cry1Ff LC 50 and NOEC >100 mg/L >21000

a The margin of exposure is calculated by dividing the NOEC (or LC 50) by the EEC b For 4114 maize, the concentration of Cry1F protein in soil can be calculated based on estimating the amount of protein from senescent tissue in a field. Senescent tissue mean concentration is for R6 whole plant tissue as presented in Section 2 - F . A modified version of the calculation used by Hunst and Rood (2004) was used to calculate the concentration of protein in soil. New assumptions were used for this calculation; plants per acre was increased to 30,000, based on current average maize plant population density (Farnham, 2001; Nielsen and Thomison, 2002) and the weight of the maize plant was now based on dry weight, since protein concentration is based on dry weight (Nguyen and Jehle, 2007 ). Protein in soil: (30,000 plants/acre x 300 g/plant (dry weight) x 4.1 μg/g Cry1F R6 whole plant tissue)/9.08 x 10 5 kg soil/acre (Hunst and Rood, 2004). This number is 0.041 mg/kg soil. c Mean concentration as reported in Section 2 - F . d This number is cal culated based on a number of metrics: the weight of an average pollen grain is 250 ng (Fonseca et al. , 2003); the high pollen deposition rate in a maize field is 1000 pollen grains/cm 2 (Pleasants et al., 2001); the approximate weight of a milkweed leaf (1 7.7 mg/cm 2 ); and the amount of Cry1F per pollen grain based on the mean concentration as presented in Section 2 - F and the weight of a pollen grain (0.0088 ng Cry1F/grain). The EEC = [0.0088 ng Cry1F/pollen grain X 1000 pollen grains/cm 2]/17.7 mg/cm 2

. e The MOE assumes that honey bee larvae will ingest 2 mg of pollen (dividing the final number by a factor of two). Taking this behavioral component into consideration, the tested concentration would be equivalent to an MOE of 9. However, if the pollen cons umption is not factored in, the tested concentration would be equivalent to an MOE of 18. f Estimate based on the protein from 4114 senescent maize tissue moving into a neighboring aquatic environment, using worst-case assumptions modified from the GENE EC model and Raybould and Vlachos (2011 ), as follows: 1) assume 10% of senescent maize tissue from a 10 hectare field will be deposited into a 1 hectare x 2 meter deep pond (20,000,000 L); 2) assume there are 30,000 plants per acre (average plant populati on density, as above), which is equivalent to 74,100 plants per hectare (1 hectare = 2.47 acres); 3) assume the dry weight of a maize plant is 300 g, since protein concentrations are expressed in dry weight, as above. Cry1F concentration in a pond = [4.1 μg/g (dry weight) X 74,100 plants/ha X 300 g/plant]/20,000,000L. Based on these assumptions, the concentration of Cry1F that would enter a pond under worst-case assumptions is 0.0046 μg/ml. The additional contribution of Cry1F protein via aerial deposit ion of maize pollen is negligible (low part per billion).Pioneer Hi - Bred International, Inc. Page 156 of 25 8

4114 Maize **Table 33**  **. 4114 Ma ize Estimated Margins of Exposure to Non** **- Target Organisms (Cry34/35Ab1)**  **Organism: Common Name**  **Species (Order)**  **Cry34/35Ab1**

**Test Material Expected** 

**Environmental Concentration**  **(EEC) from 4114**  **Maize Results**  **Margin of Exposure**  **(MOE) a**  Representative Soil Dwelling Species Springtail Folsomia candida (Collembola) Purified protein in yeast diet 0.56 mg/kg in soil b LC 50 and NOEC >12.7 mg/kg diet >22 Earthworm Eisenia fetida (Haplotaxida) Purified protein in soil 0.56 mg/kg in soil b LC 50 and NOEC >25.3 mg/kg dry soil c >45 Representative Coleopteran Species Ladybird beetle Hippodamia convergens (Coleoptera) Family: Coccinellidae Purified protein in sugar water diet 9.54 μg/g

Cry34/35Ab1 i n pollen d LC 50 and NOEC

>280 μg/mL diet >29 Ladybird beetle Coleomegilla maculata (Coleoptera) Family: Coccinellidae Purified protein in artificial diet 9.54 μg/g

Cry34/35Ab1 in pollen d LC 50> 901 ppm

EC 50 < 901ppm 94 Representative Arthropod Species Pa rasitic Hymenoptera Nasonia vitripennis (Hymenoptera) Family: Pteromalidae Purified protein in sugar water diet 9.54 μg/g

Cry34/35Ab1 in pollen d LC 50 >280 μg/mL

diet >29 Green lacewing Chrysoperla carnea (Neuroptera) Family: Chrysopidae Solution of purified protein in moth egg diet 9.54 μg/g

Cry34/35Ab1 in pollen d LC 50 and NOEC

>280 ppm diet >29 Honeybee Apis mellifera (Hymenoptera) Family: Apidae Purified protein in sucrose solution 9.54 μg/g

Cry34/35Ab1 in pollen d NOEC >5600 ng

per larvae e >294fPioneer Hi - Bred International, Inc. Page 157 of 25 8

4114 Maize **Tab le**  **33 . 4**  **114 Maize Estimated Margins of Exposure to Non -** **Target Organisms (Cry34/35Ab1) (continued)**  **Organism: Common Name**  **Species (Order)**  **Cry34/35Ab1**

**Test Material Expected** 

**Environmental Concentration**  **(EEC) from 4114 Maize**  **Results Margin of**  **Exposur e**  **(MOE) a**  Representative Arthropod Species Water flea Daphnia magna (Cladocera) Family: Daphniidae Purified protein dissolved in water 0.0634 μg/ml

Cry34/35Ab1 f LC 50 and NOEC

>100 mg/L >1500

a The MOE is calculated by dividing the NOEC (or LC50) by th e EEC b For 4114 maize, the concentration of Cry34/35Ab1 protein in soil can be calculated based on estimating the amount of protein from senescent tissue in a field. Senescent tissue concentration is the sum of the mean concentrations of Cry34Ab1 and C ry35Ab1 in R6 whole plant tissue as presented in Section 2 - F . A modified version of the calculation used by Hunst and Rood (2004) was used to calculate the concentration of protein in soil. New assumptions were used for this calculation; plants per acre was increased to 30,000, based on current average maize plant population density (Farnham, 2001; Nielsen and Thomison, 2002) and the weight of the maize plant was now based on dry weight, since protein concentration is based on dry weight (Nguyen and Jehle, 2007 ). Protein in soil: [30,000 plants/acre x 300 g/plant (dry weight) x (36 μg/g Cry34Ab1 + 21 μg/g Cry35Ab1 R6 whole plant tissue)]/9.08 x 10 5 kg soil/acre. This number is 0.56 mg/kg soil. c The final concentration of the experiment was previously reported as 76 mg/kg (EPA, 2010b); however, th e original calculation was based on 76 mg in 3 kg of soil; therefore, the final concentration in the soil was 25.3 mg/kg. d This concentration is the sum of the individual protein mean concentration s as reported in Section 2 - F . Cry34Ab1 expression in pollen is 9.2 μg/g and Cry35Ab1 expression is 0.34 μg/g. e The MOE assumes that honey bee larvae will ingest 2 mg of pollen (dividing the final number by a factor of two). Taking this behavioral component into consideration, the tested concentration would be equivalent to an MOE of 294. However, if the pollen consumption is not factored in, the tested concentration would be equivalent to an MOE of 587. f Estimate based on the protein from 4114 senescent maiz e tissue moving into a neighboring aquatic environment, using worst-case assumptions modified from the GENEEC model and Raybould and Vlachos (2011 ), as follows: 1) assume 10% of senescent maize tissue from a 10 hectare field will be deposited into a 1 hectare x 2 meter deep pond; 2) assume there are 30,000 plants per acre (average plant population density, as above), which is equivalent to 74,100 plants per hectare (1 hectare = 2.47 acres); 3) assume the dry weight of a maize plant is 300 g, since protein concentrations are expressed in dry weight, as above. Cry34/35Ab1 concentration in a pond = [ (36 μg/g Cry34Ab1 + 21 μg/g Cry35Ab1) (dry weight) X 74,100 plants/ha X 300 g/plant]/20,000,000L. Based on these assumptions, the concentration of Cry34/35Ab1 protein that would enter a pond under worstcase assumptions is 0.0634 μg/ml. The additional contribution of Cry34/35Ab1 protein via aerial deposition of maize pollen is negligible (low part per billion).Pioneer Hi - Bred International, Inc. Page 158 of 25 8

4114 Maize **Potential Impact on Non -**  **Target Coleoptera** Toxicity of the Cry1F and Cry34/35Ab1 protein on representative coleopteran species was evaluated for both 1507 and 59122 maize (EPA, 2010a; EPA, 2010b; Hunst and Rood, 2004; Shanahan and Stauffer, 2000; USDA-APHIS, 2001; USDA-APHIS, 2005). Hippodamia convergens was tested with the Cry1F and Cry34/35Ab1 proteins (Tables 32 and 33, respectively). I n addition, Coleomegilla maculata was also tested for Cry34/35Ab1 (Table 33 ). The results from the laboratory assays are given in Tables 32 and 33 . The EEC of the proteins is based on the mean concentrations of the proteins in 4114 maize pollen (Section 2 - F ). N one of the coleopteran species tested showed significant adverse effects from the proteins and the margins of exposure were greater than 13 -fold (Tables 32 and 33, Representative Coleopteran Species ). In addition, the EPA reviewed data from two studies that provided additional evidence for lack of impact of Cry34/35Ab1 on non -target Coleoptera. I n one study, the toxicity of the purified Cry34/35Ab1 protein to carabid beetles ( Poecilus cupreus ) was assessed (EPA, 2010b). N o statistically significant differences were observed in the populations fed Cry34/35Ab1 and the control. This study confirmed that, at field concentrations, there would be no impact of the p roteins on carabid beetles. I n the second study, the possibility of impact of the Cry34/35Ab1 protein in predator- prey feeding interactions was examined (EPA, 2010b). There was no effect on ladybird beetle ( Coleomegilla maculata) larvae that were consumi ng aphids that had been feeding on maize plants expressing the Cry34/35Ab1 protein. Because levels of the Cry34/35Ab1 protein in 4114 maize pollen are comparable to or lower than those of 59122 maize, as described in Section 2- F, these studies and their conclusions are relevant to 4114 maize. Based on these studies, non -target coleopteran species are unlikely to be impacted by the Cry1F and Cry34/35Ab1 proteins at the expected environmental concentrations for 4114 maize. **Potential Impact on Non -**  **Target Le pidoptera**  Due to the established specificity of the Cry1F protein for Lepidoptera, the potential impact of 1507 maize on a representative lepidopteran species of conservation interest, the monarch butterfly ( Danaus plexippus), was studied . The data indicated that 1507 maize would not pose a risk to monarch butterfly larvae (EPA, 2010a; Shanahan and Stauffer, 2000; USDA-APHIS, 2001) . The most likely route of exposure of non -target Lepidoptera to transgenic proteins from 4114 maize would be via pollen deposition on host plants (i.e., milkweed) in maize fields and in field margins. Weed control practices make the likelihood of having host plants growing within the confines of the maize field remote; therefore, the most realistic exposure of monarch butterfl y to maize pollen would be via incidental ingestion of maize pollen on milkweed plants located in field margins. The density of maize pollen deposition within maize fields and field margins hasPioneer Hi - Bred International, Inc. Page 159 of 25 8

4114 Maize been extensively studied (Pleasants et al. , 2001) and these values can be used to estimate the EEC for monarch butterfly to maize pollen. Pollen deposition rates were reported to be 1000 ( high in -field), 171 (in -field average), and 63 (field edge) pollen grains/cm 2 (Pleasants et al. , 2001). In order to calculate t he EEC and margin of exposure, the 1000 grains/cm 2 rate was used (Table 32 ); however, the more realistic case of pollen deposition would be the value at the field edge. The EEC was calculated based on a number of metrics: t he weight of an average pollen grain; a high pollen deposition rate in a maize field ; the approximate weight of a milkweed leaf per cm 2; and the amount of Cry1F per pollen grain based on the concentration as presented in Section 2 - F (Table 32 ). The results of the monarch butterfly stud y are provided in Table 32 . Based on the concentration tested in the study and the EEC, the margin of exposure was calculated to be 20- fold (Table 32 ). Furthermore, a laboratory feeding study was conducted that exposed monarch butterfly larvae to 1507x59 122 maize pollen, containing both the Cry1F and Cry34/35Ab1 proteins, at up to 1600 pollen grains/cm 2 on milkweed leaves (EPA, 2010b). The results of the feeding study showed no reduction in the survival, weight gain, development, and leaf consumption of the larvae and no sub-chronic effects were seen (EPA, 2010b). The pollen deposition was higher than the maximum expected pollen deposition rate described above and the assay also contained the Cry1F and Cry34/35Ab1 proteins; therefore, the results of this study would be applicable to assessment of 4114 maize. Based on these studies, non -target lepidopteran species are unlikely to be impacted by incidental exposure to the Cry1F and Cry34/35Ab1 proteins from 4114 maize pollen at the expected pollen depositi on rates. **Potential Impact on Other Non -** **Target Arthropods** Laboratory assays for four representative arthropods of the orders Hymenoptera ( parasitic hymenoptera, Nasonia vitripennis , and honeybee, Apis mellifera), Neuroptera (green lacewing, Chrysoperla carnea), and Cladocera ( Daphnia magna ) were also conducted using purified Cry1F and Cry34/35Ab1 proteins at doses exceeding expected environmental exposures (Tables 32 and 33 ). These studies were submitted for 1507 and 59122 maize and supported the conclus ion that Cry1F and Cry34/35Ab1 exposure does not have an adverse impact on non target, beneficial arthropods (EPA, 2010a; EPA, 2010b; Hunst and Rood, 2004; Shanahan and Stauffer, 2000; USDA-APHIS, 2001; USDA-APHIS, 2005) . The results from these assays are provided in Table 32 and 33 . For three of the species tested ( parasitic hymenoptera, green lacewing, and honeybee), t he EEC of the proteins wa s based on the concentrations of the proteins in 4114 maize pollen (Section 2 - F ). For Daphnia magna, the potent ial EEC in an aquatic environment was estimated from a modified GENEEC model, which was used to make worstcase estimates for the amount of protein that could move into pond water from a neighboring field (Raybould and Vlachos, 2011 ). None of the other arthropod species tested showed significant adverse effects from the proteins and the margins of exposure were greater than 9- fold (Tables 32 and 33, Representative Arthropod Species ).Pioneer Hi - Bred International, Inc. Page 160 of 25 8

4114 Maize In addition, to further test the impacts of Cry34/35Ab1 on non -target arthropods, EPA reviewed an additional study on the minute pirate bug ( Orius insidiosus) in the order Hymenoptera (EPA, 2010b). E PA concluded that at field concentrations, it is unlikely that Cry34/35Ab1 would have any adverse effects on this insect. These studies have relevance for 4114 maize, expressing the Cry1F and Cry34/Cry35Ab1 proteins. Based on the conclusions of these studies, it is unlikely that 4114 maize would have an adverse impact on non -target, beneficial arthropods. **Field Monitoring for Ef**

**fects of Cry1F and Cry34/35Ab1 on Non -**  **Target Arthropods** In addition to early tier laboratory studies, multiple field studies have be en conducted to investigate the potential effects of cultivating transgenic maize hybrids containing an insectresistant protein on non -target arthropod abundance. One field study reviewed by USDA showed that 1507 maize expressing the Cry1F protein had no effect on beneficial arthropod abundance (Shanahan and Stauffer, 2000; USDA-APHIS, 2001). A second three-year field study showed no impact on abundance of several different non -target organisms, including detritovores, herbivores, and predatory arthropods, by comparing fields of 1507 maize with near- isoline control fields (Higgins et al. , 2009). Similar field studies were also conducted for 59122 maize and were reviewed by different U.S. regulatory agencies; results indicated that 59122 maize expressing the Cry34/35Ab1 proteins had no effect on beneficial arthropod abundance (EPA, 2010b; Hunst and Rood, 2004; USDAAPHIS, 2 005). At hree-year field study was evaluated by the EPA to determine the impact of 59122 maize, expressing the Cry34/Cry35Ab1 proteins, and 1507x59122 maize, expressing both Cry1F and Cry34/35Ab1 proteins, on non -target arthropods after continuous croppin g at three locations in Iowa, Nebraska, and Indiana (EPA, 2010b). S imilarly, the results showed no impact on non -target arthropod populations between fields of the control maize and 59122 and 1507x59122 maize hybrids (EPA, 2010b). The EPA concluded in th eir assessment that there is negligible potential for long-term adverse effects on non -target arthropods after continuous cultivation of both a maize line containing the Cry34/Cry35Ab1 proteins and a stacked line that includes the Cry34/35Ab1 and Cry1F proteins (EPA, 2010b). Results from these field studies confirm laboratory testing and support the hypothesis that 1507 and 59122 maize are unlikely to adversely impact non -target arthropods occurring in maize fields. Because of comparable Cry1F and Cry34/35Ab1 protein expression, 4114 maize is not expected to adversely impact beneficial non -target arthropods in fields.Pioneer Hi - Bred International, Inc. Page 161 of 25 8

4114 Maize **Potential Impact of 4114 Maize on Threatened and Endangered Arthropods** The impact of 4114 maize on threatened and endangered arthropods can also be evaluated by comparing to p revious assessments of 1507 and 59122 maize . These assessments concluded that threatened and endangered species would unlikely be impacted by the cultivation of either maize line (EPA, 2010a; EPA, 2010b; USDA-APHI S, 2001; USDA-APHIS, 2005) . As of July 2011, 60 insect species are listed on the US Fish and Wildlife Service website as threatened or endangered (US FWS, 2011). Twenty-one of these insects are in the order Lepidoptera and 15 are in Coleoptera, with the remaining representing insects of other orders. Almost all of the species of Lepidoptera and Coleoptera currently listed were added prior to the deregulation of 1507 and 59122 maize, except for one coleopteran species, the Salt Creek Tiger Beetle ( Cicindela nevadica lincolniana; Family: Carabidae) (US FWS, 2010; US FWS, 2011). For 1507 maize, USDA considered the impact of the Cry1F protein on two lepidopteran species, the Karner blue butterfly ( Lycaeides melissa samuelis) and Mitchell’s satyr butterfly ( US FWS, 2011; USDA-APHIS, 2001). U SDA concluded that both species would not be expected to be present in or close to maize fields; therefore it is unlikely there would be any impact of 1507 maize cultivation (USDA-APHIS, 2001). For 59122 maize, USDA cons idered the impact on one primary threatened and endangered coleopteran species, the American burying beetle (US FWS, 2011; USDA-APHIS, 2005). F rom review of preferred habitats, this beetle was unlikely to be found in active maize fields and therefore woul d not be exposed significantly to the Cry34/35Ab1 protein from 4114 maize (USDA-APHIS, 2005). For other lepidopteran and corn rootworm resistant maize events, EPA has made similar conclusions for the Karner blue butterfly and the American burying beetle a nd has not identified any new threatened or endangered species that would be impacted by cultivation (EPA, 2009; EPA, 2010b; EPA, 2010c). F urthermore, EPA examined the habitats of the other threatened and endangered insect species in the orders Diptera, H emiptera, Odonata and Orthoptera and found that they primarily occupy dune, meadow or prairie, or open forest habitats and are not closely associated with row crop production, often times due to the specificity of the habitat of their host plants (EPA, 2009; EPA, 2010b; EPA, 2010c). As mentioned earlier, only one coleopteran species, the Salt Creek Tiger Beetle ( Cicindela nevadica lincolniana; Family: Carabidae), was added in 2005 to the threatened and endangered insect species list (US FWS, 2010; US FWS, 2011). This beetle is found in Nebraska (Lancaster and Saunders counties), the third largest state for maize cultivation (US FWS, 2010; USDA- NASS, 2010). However, the beetle’s critical habitats have been characterized as non -vegetated stream banks or ed ges that are in saline or freshwater wetlands and the beetles prefer to be within a few meters of these habitats (US FWS, 2010). Wetland areas, stream edges, or saline wetlands are unlikely to be planted with maize. I n addition, the beetle family is Cara bidae, which is unrelated to those of corn rootworm species (Chrysomelidae). Therefore, it is highly unlikely that this beetle will be exposed to the Cry34/35Ab1 protein from 4114 maize or will be impacted due to the specificity of the Cry34/35Ab1 protein to corn rootworm species.Pioneer Hi - Bred International, Inc. Page 162 of 25 8

4114 Maize Similar to the conclusions for 1507 and 59122 maize, it is unlikely that threatened and endangered species such as the Karner blue and Mitchell’s satyr butterflies and the American burying beetle would be impacted by 4114 maize cultivation. Based on critical habitat and the Cry34/35Ab1 target insect specificity, it is also unlikely that the Salt Creek Tiger Beetle would be adversely affected. F urthermore, the target specificity of the introduced proteins and the lack of habitat overlap with regions of maize cultivation also support the conclusion that 4114 maize would not adversely impact the other listed threatened and endangered insects. **Potential Impact on Non -**  **Target Vertebrate Species** Wildlife in and around maize fields are of interest due to potential exposure to 4114 maize grain and plant residue. A wide variety of toxicology studies conducted over the past 40 years have established the safety of the microbial preparations of B. thuringiensis, includin g their expressed i nsecticidal Cry proteins (Betz et al. , 2000). As discusse d in Section 3, the allergenicity and toxicity of the Cry1F, Cry34Ab1, and Cry35Ab1 proteins has been assessed and all three are unlikely to be potential allergens or toxins to humans or animals. Based on quail and mouse toxicological studies for Cry1F, both EPA and USDA concluded that the protein was unlikely to have an adverse effect on vertebrates, including non -target birds, mammals, and humans (EPA, 2010a; USDA-APHIS, 2001). E PA and USDA made similar conclusions for Cry34Ab1 and Cry35Ab1 on the basis of poultry, mouse, and rainbow trout toxicological studies (EPA, 2010b; USDA-APHIS, 2005). P roviding additional evidence for the lack of toxicity of the Cry1F and Cry34/35Ab1 proteins, recently pu blished rodent feeding studies showed no toxicological effects of diets containing 1507 and 1507x59122 maize grain (Appenzeller et al. , 2009; MacKenzie et al. , 2007). Additional feeding studies for laying hens, swine, beef cattle, and dairy cows with diets containing 1507 and 59122 maize have all shown no differences in nutritional equivalency from diets containing conventional maize (Faust et al. , 2007; Huls et al. , 2008; Jacobs et al. , 2008; Scheideler et al. , 2008; Sindt et al. , 2007; Stein et al. , 2009 ). Results from these studies are relevant for 4114 maize, which expresses the Cry1F, Cry34Ab1, and Cry35Ab1 proteins, and provide evidence that 4114 maize is unlikely to have adverse effects on non -target vertebrate species, including birds, mammals, and humans.Pioneer Hi - Bred International, Inc. Page 163 of 25 8

4114 Maize **Conclusions for the Environmental Impact of the Introduced Proteins in 4114 Maize** The environmental impact of 4114 maize focused on the introduced Cry1F and Cry34/35Ab1 proteins and considered the potential hazard and the level of environmental exposure to non target organisms. D ue to the presence of the same proteins and similar or lower protein expression, the previously conducted environmental safety studies for 1507, 59122, and 1507x59122 maize were relevant to the assessment of 4114 maize. As previously established for 1507 and 59122 maize, the Cry1F and Cry34/Cry35Ab1 proteins have insecticidal activity against lepidopteran and corn rootworm species, and there is low risk of hazard to non -target orders. The low hazard to several represen tative non -target organisms was confirmed in the laboratory at protein concentrations greater than those expected in the environment and the potential environmental exposure to the Cry1F and Cry34/35Ab1 proteins was determined. Based on this analysis, the re is a sufficient margin of exposure for the proteins in the environment and , therefore, the cultivation of 4114 maize is unlikely to adversely impact non target organisms. Likewise, multiple field studies have also confirmed that the abundance of non -ta rget organisms under field conditions is not impacted by maize expressing the Cry1F and Cry34/35Ab1 proteins. Similar to the conclusions for 1507 and 59122 maize, the cultivation of 4114 maize is unlikely to have adverse effects on non -target arthropods, including threatened and endangered arthropods, and non -target vertebrates, including birds, mammal, and humans.Pioneer Hi - Bred International, Inc. Page 164 of 25 8

4114 Maize **6. Overall Conclusions**  In conclusion, based on the data contained herein, Pioneer requests that APHIS grant the request for a determinati on of nonregulated status for 4114 maize and any crosses of this line with other nonregulated Zea mays . 4 114 maize was modified to produce the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins; these proteins and associated genetic elements in 4114 maize are id entical to those in previously approved 1507, 59122, and 1507x59122 maize. I n addition, t he Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins are unlikely to be allergens or toxins and are safe for the food and feed supply . 4114 maize contains all the gene cassettes that express the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins at a single genetic locus. F urthermore, molecular characterization confirmed that 4114 maize contains a single copy, intact insert ion that is stable and segregates according to Mendel ’s laws of genetics. Nutrient composition and agronomic comparative assessments were conducted to determine if 4114 maize would present any new or greater risks relative to maize varieties that have a history of safe use in the environment and as food and feed. These analyses indicated that 4114 maize is as safe as conventional maize varieties and does not pose a greater risk than conventional maize varieties in food, feed, and the environment. It is unlikely that 4114 maize will pose a plant pest risk o r impact non -target organisms in the environment. M aize is not considered a weed, and 4114 maize does not exhibit any characteristics that would indicate it is any more likely than conventional maize to become a weed or plant pest. Because sexually compatible wild relative populations are limited in the U.S. and there is low potential for gene flow, it is unlikely that the inserted DNA in 4114 maize would be introgressed significantly into these wild relative populations. Based on the evidence supporting the environmental safety of the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins, it is unlikely that 4114 maize will pose a risk to non -target organisms including beneficial, threatened and endangered species, as well as non -target vertebrates including birds, mammals, and humans. In support for the safety of 4114 maize, 1507 maize has been commercially available in the U.S. since the 2003 growing season and 59122 and 1507x59122 maize have been available in the U.S. since 2006 with no adverse food, feed, or e nvironmental effects. M aize products containing 1507x59122 maize were grown on more than 14 million acres in 2010.Pioneer Hi - Bred International, Inc. Page 165 of 25 8

4114 Maize **A ppendix 1. 4114 Maize USDA Release Permits and Notifications and Planted Acreage**  Plantings through May 25, 2011 are listed **Year of**  **Planti ng**  **Permit Name Permit**  **Valid Date State**  **Number of Counties Where 4114 Maize** **a was Planted**  **Acreage** 2006 06-019-03R 5/2/2006 PR 2 0.010

06-019-04R 5/3/2006 HI 1 0.014

2007 06-019-03R 5/2/2006 CA 1 0.009 PR 1 0.039 06-019-04R 5/3/2006 HI 1 0.039

07-040- 101rm 5/3/2007 CA 1 0.009 HI 1 0.146 IA 2 0.072 IL 4 0.092 IN 1 0.030 MN 1 0.030 MO 1 0.020 NE 1 0.020 PR 2 0.110 WI 1 0.020

2008 07-040- 101rm 5/3/2007 HI 1 0.020 PR 1 0.130

08-014- 111n 4/16/2008 CA 1 0.050 IA 2 0.230 IL 4 0.270 IN 2 0.160 MN 1 0.012 MO 1 0.060 NE 2 0.110 PR 2 1.510 WI 1 0.070 08-014- 131n 3/31/2008 HI 1 0.280

08-095 - 105n 5/14/2008 IA 1 0.070 IL 2 0.060 MN 1 0.014 NE 1 1.077 OK 1 0.050 TX 1 0.040

2009 08-014- 111n 4/16/2008 PR 1 0.001

09-013 - 108n 4/1/2009 IA 5 0.750 IL 6 0.370 IN 2 0.280 MO 1 0.114 NE 1 0.186 09-016- 103n 2/15/2009 IL 1 0.032 NE 1 0.023Pioneer Hi - Bred International, Inc. Page 166 of 25 8

4114 Maize **4114 Maize USDA Release Permits and Notifications and Planted Acreage (continued)**  **Year of Planting**  **Permit Name Permit**  **Valid Date State**  **Number of Counties Where 4114 Maize** **a was Planted**  **Acreage**

2009 09-035 - 109n 3/17/2009 AR 1 0.018 CO 1 0.057 DE 1 0.020 GA 1 0.129 HI 1 0.128 KS 1 0.018 MN 1 0 .134 PR 3 2.980 TN 1 0.018 WI 1 0.098 09-264- 102n 10/21/2009 HI 1 0.064 PR 2 2.370

2010 09-264- 102n 10/21/2009 HI 1 0.083 PR 1 0.281

10-015 - 106n 3/1/2010 b IA 3 0.090 IL 3 0.080 IN 1 0.011 MI 1 0.011 MN 1 0.034 N E 1 1.057 10-050- 115n 3/9/2010 b NE 1 0.016

10-052 - 101n 3/19/2010 b AR 1 0.017 CA 1 0.200 CO 2 0.060 GA 1 1.006 HI 1 0.630 IA 9 2.500 IL 6 2.210 IN 4 0.920 KS 1 0.224 MI 1 0.286 MN 2 0.310 MO 3 0.540 NE 1 0 .619 PA 1 0.186 PR 4 1.020 SD 1 0.039 TN 1 0.068 WI 1 0.372 10- 118- 105n 5/6/2010 b IL 1 0.186 10-281 - 101n 10/30/2010 b PR 1 0.069 10-284- 101n 11/16/2010 b PR 2 1.470Pioneer Hi - Bred International, Inc. Page 167 of 25 8

4114 Maize **4114 Maize USDA Release Permits and Notifications and Planted Acre age (continued)**  **Year of Planting**  **Permit Name Permit**  **Valid Date State**  **Number of Counties Where 4114 Maize** **a was Planted**  **Acreage**

2011 11 -040- 123n 3/17/2011 b PR 2 0.404

10-281 - 101n 10/30/2010 b TN 1 0.104 10-284- 101n 11/16/2010 b HI 1 0.030 PR 1 0.684 11 -019- 109n 3/1/2011 b IA 1 0.023 IL 2 0.046 NE 1 0.023 OK 1 0.023 TX 1 0.003

11 -040- 123n 3/17/2011 b CO 1 0.049 GA 1 0.260 HI 1 0.698 IA 7 2.880 IL 6 4.019 IN 5 2.114 KS 1 0.654 MI 1 0.554 MN 2 2.137 MO 3 1.182 NE 1 0.847 SD 1 0.498 TN 1 0.041 WI 2 0.518 11 -040- 124n 3/17/2011 b HI 1 0.033 IA 1 0.002 a 4114 maize is also referred to as DP-004114-3 and EA-2244.04.1.14 in USDA final reports. b Final field permit test reports not yet due to USDA.Pioneer Hi - Bred International, Inc. Page 168 of 25 8

4114 Maize **Appendix 2. Equivalency of the Proteins Produced in 4114 Maize to Those Produced in 1507 and 59122**  **Maize 2.1**  **. Overview and Summary**  As discussed earlier in Section 2, the introduced cry1F, cry34Ab1, cry35Ab1, and pat gene cassettes in 41 14 maize are identical to those cassettes in 1507 and/or 59122 maize. Based on the DNA sequence of the genes, the translated protein sequences are also identical. P rior protein safety studies for 1507 and 59122 maize were conducted on the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins and are described in Section 3. These protein safety studies confirmed that none of the proteins are potential allergens or toxins. I n order to verify the applicability and relevance of these protein safety studies for 4114 m aize, the identity and equivalency of the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins should be established. In order to verify the identity of the introduced proteins in 4114 maize and the equivalency to the proteins in 1507 and/or 59122 maize, western blot analysis of the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins was conducted in sample extracts of 4114 maize and 1507x59122 maize. The western blot analysis demonstrated that the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins derived from 4114 maize have equivalent molecular weights and immunoreactivity to the proteins expressed in 1507x59122 maize. I n all four western blots, the near- isoline control showed no immunoreactive proteins. As described in Section 2, the western blot analysis in conjunction with other analyses confirmed that the proteins in 4114 maize are the same as those of 1507 and/or 59122 maize; therefore, prior protein safety studies conducted for 1507 and/or 59122 maize on Cry1F, Cry34Ab1, Cry35Ab1, and PAT are relevant to the safety assessment of 4114 maize. **2.2 .**  **Western Blot Analysis of the Introduced Proteins Cry1F**  Western blot analysis using anti-Cry1F antibodies (Figure 2.1 ) demonstrated that the Cry1F protein derived from 4114 maize (Lane 3) had the same molecular weight an d relative immunoreactivity as the Cry1F protein derived from 1507x59122 maize (Lane 4). Both protein samples migrated as two bands of approximately 60 kDa and 62 kDa in size. The double banding pattern was expected because plant-derived Cry1F protein ca n be partially degraded by plant proteases to a smaller, more stable truncated protein, therefore appearing as two bands (the larger intact protein and the smaller truncated protein). Relative amounts of the two bands can vary from sample to sample. The Cry1F standard protein included on the blot (Lane 2) consisted of the truncated form of the protein and migrated at the expected approximately 60 kDa.Pioneer Hi - Bred International, Inc. Page 169 of 25 8

4114 Maize **Cry34Ab1** Western blot analysis using anti-Cry34Ab1 antibodies (Figure 2.2 ) demonstrated that the Cry34Ab1 protein derived from 4114 maize (Lane 3) had the same molecular weight and relative immunoreactivity as the Cry34Ab1 protein derived from 1507x59122 maize (Lane 4), with each sample migrating at approximately 14 kDa. S imilarly, the Cry34Ab1 standard protein (Lane 2) migrated at approximately 14 kDa and appeared to be equivalent to the plant-derived proteins. **Cry35Ab1** Western blot analysis using anti-Cry35Ab1 antibodies (Figure 2.3 ) demonstrated that the Cry35Ab1 protein derived from 4114 maize (Lane 3) had the same molecular weight and relative immunoreactivity as the Cry35Ab1 protein derived from 1507x59122 maize (Lane 4). Both protein samples migrated as two bands of approximately 40 kDa and 44 kDa in size. Like the Cry1F protein, the Cry35Ab1 protein can be partially degraded to a more stable truncated protein by plant proteases, and the relative amounts of each protein can vary from sample to sample. The Cry35Ab1 standard protein (Lane 2) consisted of the truncated form of the protein and migrated at the expected approximately 40 kDa. **PAT Protein** Western blot analysis using anti - PAT antibodies (Figure 2.4 ) demonstrated that the PAT protein derived from 4114 maize (Lane 3) had the same molecular weight and relative immunoreactivity as the PAT p rotein derived from 1507x59122 maize (Lane 4), with each sample migrating at approximately 21 kDa. The PAT standard protein (Lane 2) migrated at the same molecular weight as the plant-derived PAT proteins (approximately 21 kDa). **2.3 .**  **Conclusions** The western blot analyses demonstrated that the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins derived from 4114 maize have equivalent molecular weights and immunoreactivity to the proteins expressed in 1507x59122 maize. I n all four western blots, the near- isoli ne control showed no immunoreactive proteins. These data, in addition to the identical DNA sequences encoding the proteins, demonstrates equivalency of the transgenic proteins in 4114 maize to those in 1507 and/or 59122 maize. Therefore, prior safety stu dies conducted on Cry1F, Cry34Ab1, Cry35Ab1, and PAT for 1507 and/or 59122 maize are applicable to 4114 maize.Pioneer Hi - Bred International, Inc. Page 170 of 25 8

4114 Maize

**1 2**  **3 4 5**  **Lane Sample ID**  1 Molecular Weight Marker 2 Cry1F Standard (~4 ng) 3 Protein Extract of R1 Leaf from 4114 Maize (undiluted) 4 Protein Extract of R1 Leaf from 1507x59122

Maize (undiluted) 5 Protein Extract of R1 Leaf from Near- Isoline

Control Maize (undiluted) **Figure 2.1**  **. Western Blot Comparison of Cry1F Protein** Plant-derived Cry1F protein can be partially degraded by plant proteases to a smaller, more stable truncated protein, therefore appearing as two bands (~60 kDa and ~62 kDa). The Cry1F standard protein consisted of the truncated protein only (~60 kDa).

**Cry1F kDa** 

**188**

**62 49**  **38 28**  **18 14** 

**6 3** Pioneer Hi - Bred International, Inc. Page 171 of 25 8

4114 Maize

**1 2 3 4 5**  **Lane Sample ID**  1 Molecular Weight Marker 2 Cry34Ab1 Standard (~10 ng) 3 Protein Extract of R1 Leaf from 4114 Maize (1:2 dilut ion) 4 Protein Extract of R1 Leaf from 1507x59122

Maize (1:3 dilution) 5 Protein Extract of R1 Leaf from Near- Isoline

Control Maize (undiluted) **Figure 2.2.**  **Western Blot Comparison of Cry34Ab1 Protein** Plant-derived and standard Cry34Ab1 proteins migrated at the expected mass of ~14 kDa.

**Cry34Ab1 kDa** 

**188**

**62 49**  **38 28**  **18 14** 

**6 3**  **3**Pioneer Hi - Bred International, Inc. Page 172 of 25 8

4114 Maize

**1 2 3 4 5 Lane**  **Sample ID** 1 Molecular Weight Marker 2 Cry35Ab1 Standard (~10 ng) 3 Protein Extract of R1 Leaf from 41 14 Maize (1:2 dilution) 4 Protein Extract of R1 Leaf from 1507x59122

Maize (1:2 dilution) 5 Protein Extract of R1 Leaf R1 from Near- Isoline

Control Maize (undiluted) **Figure 2.3.**  **Western Blot Comparison of Cry35Ab1 Protein** Plant-derived Cry35Ab1 protein can be partially degraded by plant proteases to a smaller, more stable truncated protein, therefore appearing as two bands (~40 kDa and ~44 kDa). The Cry35Ab1 standard protein consisted of the truncated protein only (~40 kDa).

**Cry35Ab1 kDa** 

**188**

**62 49**  **38 28** 

**18 14**  **6 3**  **3**Pioneer Hi - Bred International, Inc. Page 173 of 25 8

4114 Maize

**1 2 3 4 5 Lane**  **Sample ID** 1 Molecular Weight Marker 2 PAT Standard (~4 ng) 3 Protein Extract of R1 Leaf from 4114 Maize (undiluted) 4 Protein Extract of R1 Leaf from 1507x59122

Maize (1:2 dil ution) 5 Protein Extract of R1 Leaf from Near- Isoline

Control Maize (undiluted) **Figure 2.4.**  **Western Blot Comparison of PAT Protein** Plant-derived and standard PAT proteins showed the expected mass of ~21 kDa. **PAT kDa** 

**188**

**62 49**  **38 28**  **18 14**  **6 3**  **3**Pioneer Hi - Bred International, Inc. Page 174 of 25 8

4114 Maize **2.4. Experimental Methods 2.4.1. Exp** **erimental Design** One maize leaf tissue sample from each of 4114 maize, 1507x59122 maize, and the control maize was extracted and evaluated using western blot analysis for expression of the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins. **2.4.2. Sample Colle ction, Processing, and Storage**  Maize leaf tissue samples were collected at the R1 growth stage (Ritchie et al. , 2005). O ne sample from each of 4114 maize, 1507x59122 maize, and the control maize were collected and processed as desc ribed in Appendix 5. A rep resentative leaf sub-sample from each maize line was weighed at a target weight of 10 mg + 5% into an individual 1.2 ml tube, assigned a unique identification number, and stored in a temperature- monitored freezer at ≤ - 10 °C until prepared for extracti on. **2.4.3. Sample Extraction and Preparation for SDS -**  **PAGE** Two chilled (2 -8 ° C) 5/32” chrome balls and 0.6 ml of chilled (2 -8 ° C) extraction buffer (PBST: phosphate buffered saline, pH 7.4, with 0.05% Tween b

- 20 ) were added to each tube, and the tubes we re covered securely with strip caps. The samples were extracted for 30 seconds at a setting of 1500 strokes per minute on a Geno/Grinderc (SPEX CertiPrep, Inc., Metuchen, NJ ) . The extracts were then centrifuged for 10 minutes at a setting of 4000 rpm at 4 °C and the supernatants were transferred to new tubes. The samples were diluted as needed in PBST to adjust for relative band intensity in the western blots; the 4114 maize extract was not diluted for the PAT and Cry1F western blots but was diluted 1:2 for the Cry34Ab1 and Cry35Ab1 western blots, and the 1507x59122 maize extract was not diluted for the Cry1F western blot but was diluted 1:3 for the Cry34Ab1 western blot and 1:2 for the Cry35Ab1 and PAT western blots. **2.4.4. Preparation of Protein Stand ard Solutions**  The Cry1F and PAT protein standards were each diluted to a concentration of 200 ng/ml in PBST, and the Cry34Ab1 and Cry35Ab1 protein standards were each diluted to a concentration of 500 ng/ml in PBST.

b Registered trademark of ICI Americas c Registered trademark of SPEX CertiPrep, Inc.Pioneer Hi - Bred International, Inc. Page 175 of 25 8

4114 Maize **2.4.5. Sodium Dodecyl Sulfate Polyac rylamide Gel Electrophoresis (SDS** **- PAGE)**  All sample extracts and protein standard solutions were prepared for electrophoresis by adding 25% NuPAGE d 4X LDS (Invitrogen Corporation, Carlsbad, CA) and 10% 10X reducing buffer containing dithiothreitol (DTT) (I nvitrogen), heated for approximately seven minutes at 95  C, and then loaded onto two NuPAGE Novex 4- 12% Bis-Tris 12 -well gels (Invitrogen). Samples were loaded at 20 l/well and SeeBlue d Pre-Stained Standard (Invitrogen) was loaded at 10 l/well. Electrophoresis was conducted with a XCell SureLockd Mini-Cell electrophoresis unit (Invitrogen) with NuPAGE MES SDS running buffer (Invitrogen) at a constant voltage of 200V for approximately 40-44 minutes. **2.4.6. Western Blot Analysis** After electrophoresis, gels were removed from the gel cassette and proteins were electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes (Invitrogen) for approximately seven minutes using an iBlot d module (Invitrogen). Following transfer, the PVDF membrane blots were blocked with a 5% non -fat dry milk (NFDM)/PBST solution for approximately 30 minutes at 20- 25  C, followed by an incubation of approximately 1 hour at 20- 25 °C in a specific monoclonal primary antibody (Table 2.1 ), which was diluted 1:2000 in 1% NFDM/PBST. The blots were then washed with PBST three times, for at least 10 minutes per wash. Following the third wash, the blots were incubated in a secondary antibody (Table 2.1; Promega U.S., Madison, WI ), which was conjugated to the enzyme horserad ish peroxidase (HRP) and diluted 1:10,000 in 1% NFDM/PBST, for approximately 1 hour at 20- 25 °C, after which the blots were washed with PBST three times for at least 10 minutes per wash. The blots were developed using SuperSignal e West Dura Extended Durat ion Substrate detection kit (Pierce Bi otechnology, Inc., Rockford, IL) followed by image capture using the Kodak Image Station 4000R Pro imaging system (Carestream Health, Inc., Rochester, NY). The resulting images were evaluated for similarities between 4114 maize and 1507x59122 maize.

d Registered trademark of Invitrogen Corporation e Registered trademark of Pierce Biotechnology, Inc.Pioneer Hi - Bred International, Inc. Page 176 of 25 8

4114 Maize **Table 2.1**  **. P** **rimary and**  **Secondary Antibody**  **Descriptions Used**  **for Qualitative**  **Western Blot**  **Analysis Target**  **Protein Primary Antibody**  **Secondary Antibody** Cry1F Cry1F Monoclonal Antibody

205A62.1 Anti - Mouse IgG HRP Conjugate (Promega) Cry34Ab1 Cry34Ab1 Monoclonal Antibody 1E1.G6 Anti - Mouse IgG HRP Conjugate (Promega) Cry35Ab1 Cry35Ab1 Monoclonal Antibody

8B5.1A10 Anti - Mouse IgG HRP Conjugate (Promega) PAT PAT Monoclonal Antibody 22G6 Anti - Mouse IgG HRP Conjugate (Promega)Pioneer Hi - Bred International, Inc. Page 177 of 25 8

4114 Maize **Appendix 3**  **. Materials and Methods for Genetic Characterization of 4114 Maize**  **3. 1.**  **Southern Blot Characterization of 4114 Maize**  Southern blot analysis was conducted to characterize the DNA insertion in 4114 maize . Individual plants of the T2, T3, BC3F1 * 3, and BC3F2 * 2 generation s were analyzed by Southern blot to determine the number of each of the genetic elements of the expression cassette that were inserted and to verify that the integrity of the PHP2 7118 T- DN A was maintained upon integrati on. The integration patterns of the insertion in 4114 maize were investigated with Bcl I and Hin d II I restriction enzymes. Southern blot analysis was conducted on individual plants of the four generations to confirm stability of the insertion across gene rations and to verify the absence of backbone sequences from plasmid PHP2 7118 . **3. 2.**  **Test Material** Seeds from the T2, T3, BC3F1 * 3, and BC3F2 * 2 generations of 4114 maize were planted and leaf tissue harvested from individual plants was used for genomic DN A extraction. **3 .3.**  **Control Material** Seeds from the unmodified Pioneer maize proprietary inbreds PHWWE and PH09B were planted and leaf tissue harvested from individual plants was used for genomic DNA extraction. **3 .4.**  **Reference Material** Plasmid DNA fro m PHP2 7118 was prepared from E. coli (Invitrogen, Carlsbad, CA) and was used as a positive control for Southern blot analysis to verify probe hybridization and to verify sizes of fragments internal to the plasmid. The plasmid stock was a copy of the plasm id used for transformation to produce 4114 maize and was digested with restriction enzymes to confirm the plasmid map. The probes used in this study were derived from plasmid PHP2 7118 or from a plasmid containing equivalent genetic elements. The Bcl I re cognition sequence contains a Dam methylase recognition sequence ( 5’ GATC3’ ) (New England Biolabs, 2002). The PHP27118 plasmid used in this analysis was prepared in a dam + strain of E. coli and thus the central adenine residue in all Bcl I sites (recogniti on sequence 5’TGATCA3’ ) was methylated and did not digest as expected (P ioneer data not shown ). Therefore, the Bcl I -digested plasmid served only as a positive control to demonstrate probe hybridization and not to provide any band size data. As Dam methylase is specific to bacteria and not found in maize plants, maize genomic DNA will be digested normally by Bcl I. P lasmid PHP27118 digested with Hin d III was included on some of the Bcl I Southern blots to show that the plasmid used in this analysis was of sufficient quality and cut properly when digested with an enzyme that is not sensitive to methylation.Pioneer Hi - Bred International, Inc. Page 178 of 25 8

4114 Maize DNA molecular weight markers for gel electrophoresis and Southern blot analysis were used to determine approximate molecular weights. For Southern an alysis, DNA Molecular Weight Marker VII, digoxigenin (DIG) labeled (Roche, Indianapolis, IN), was used as a size standard for hybridizing fragments. ΦX174 RF DNA/Hae III Fragments (Invitrogen, Carlsbad, CA) was used as a molecular weight standard to deter mine sufficient migration and separation of the fragments on the gel. **3 .**  **5. Identification of the 4114 Maize Plants Used for Southern Blot Analysis**  Phenotypic analysis of 4114 maize plants and control plants was carried out by the use of lateral flow devices able to detect the Cry1F, Cry34Ab1 and PAT proteins to confirm the absence or presence of the se protein s in material used for Southern blot analysis. Leaf extracts were prepared by grinding leaf punches to homogeneity in 400 μl of EB2 extraction buffer (Envirologix, Inc., Portland, ME). Lateral flow devices (Envirologix) were placed in the homogenate and allowed to develop. After incubation, the results were read from the lateral flow devices. As ingle stripe indicated a negative result and a doubl e stripe indicated the sample was positive for the Cry1F, Cry34Ab1 or PAT proteins. Genotypic analysis of the 4114 maize and control maize plants was carried out by real -time polymerase chain reaction (PCR) using assays specific for the DNA insertion. A leaf sample was taken from each test and control plants for event-specific PCR analysis. D NA was extracted from each leaf sample using the Extract- N -Ampf Plant PCR kit using the described procedure (Sigma -Aldrich, St. Louis, MO). Real -time PCR was perfor med on each DNA sample utilizing an ABI PRISM g 7500 Fast Real -Time PCR System (Applied Biosystems, Inc., Foster City, CA). TaqMan g probe (Applied Biosystems, Inc.) and primer sets (Integrated DNA Technologies, Coralville, IA) were designed to detect target sequences from the insertion in 4114 maize . I n addition, a second TaqMan probe and primer set for a reference maize endogenous gene was used to confirm the presence of amplifiable DNA in each reaction. The assay analysis consisted of real -time PCR dete rmination of qualitative positive/negative calls. The extracted DNA was assayed using TaqMan Universal PCR Master Mix, No AmpEraseg UNG (Applied Biosystems, Inc.). I nitial incubation was at 95  C for 10 minutes followed by 40 cycles as follows: 95  C for 1 5 seconds, 60 C for one minute. Positive or negative determination for each plant was based on comparison of the threshold cycle (CT) of the insertion target PCR to that of the maize endogenous reference target. I f the event-specific and endogenous PCR targets amplified above CT, then the plant was scored as positive for the 4114 event. I f the endogenous target amplified and the event target did not, then the plant was scored as negative.

f Registered t rademark of Sigma -Aldrich g Registered Trademarks of Applied Biosystems, Inc.Pioneer Hi - Bred International, Inc. Page 179 of 25 8

4114 Maize A subset of 4114 maize plants that were identified as containing the inserted DNA and expressing the Cry1F, Cry34Ab1, and PAT proteins from the four generations described previously were selected for Southern blot analysis. **3 .**  **6. Genomic DNA Extraction**  Genomic DNA was extracted from leaf tissue harvested from individu al test and control plants. The tissue was pulverized in tubes containing grinding beads using a Geno/Grinderh (SPEX CertiPrep, Inc., Metuchen, NJ) instrument and the genomic DNA was isolated using a urea based procedure (modification from Chen and Dellaporta, 1994). A pproximately 1 gram of ground tissue per sample was extracted with 5 ml Urea Extraction Buffer (7 M urea, 0.34 M NaCl, 0.05 M Tris- HCl, pH 8.0, 0.02 M EDTA, 1% N - lauroylsarcosine) for 15 - 18 minutes at 37  C, followed by two extractions with p henol/chloroform/isoamyl alcohol (25:24:1) and one extraction with water saturated chloroform. The DNA was precipitated from the aqueous phase by the addition of 1/10 volume of 3 M NaOAc (pH 5.2) and 1 volume of isopropyl alcohol, followed by centrifugati on to pellet the DNA. After washing the pellet twice with 70% ethanol, the DNA was dissolved in 0.5 ml distilled water and treated with 10 g ribonuclease A for 15 minutes at 37  C. The sample was then washed with 70% ethanol. After drying, the DNA was re-dissolved with 0.5 ml distilled water and stored at 4 C. **3 .**  **7. Quantific** **ation of Genomic DNA** Following extraction, the DNA samples were quantified on a spectrofluorometer using PicoGreen i reagent (Molecular Probes, Inc., Eugene, OR) following a standa rd procedure. The DNA was also visualized on a n agarose gel to confirm quantification values from the PicoGreen analysis and to determine DNA quality. **3 .8.**  **Digestion of DNA for Southern Blot Analyses** Genomic DNA samples extracted from selected 4114 ma ize and control maize plants were digested with restriction enzymes following a standard procedure. A pproximately 3 to 6 g of genomic DNA was digested using 50 units of enzyme according to manufacturer’s recommendations. The digestions were carried out at 37  C for approximately three hours, followed by ethanol precipitation with 1/10 volume of 3 M NaOAc (pH 5.2) and 2 volumes of 100% ethanol. After incubation at ≤-5  C and centrifugation, the DNA was allowed to dry and then re-dissolved in TE buffer (10mM Tris, 1 mM EDTA, pH 7.5). The reference plasmid, PHP2 7118, was spiked into a control plant DNA sample in an amount equivalent to approximately one or three gene copies per maize genome and digested with the same enzyme to serve as a positive control for p robe hybridization and to verify sizes of fragments internal to the plasmid on the Southern blot.

h Registered trademark of SPEX CertiPrep, Inc. i Registered trademark of Molecular Probes, Inc.Pioneer Hi - Bred International, Inc. Page 180 of 25 8

4114 Maize **3 .9.**  **Electrophoretic Separation and Southern Transfer** Following restriction enzyme digestion, the resultant DNA fragments were electrophoretically separate d by size through an agarose gel. Am olecular weight standard [ΦX174 RF DNA/Hae III Fragments (Invitrogen)] was used to determine sufficient migration and separation of the fragments on the gel. D IG labeled DNA Molecular Weight Marker VII (Roche), which i s visible after DIG detection as described below, was used to determine hybridizing fragment size on the Southern blots. Agarose gels containing the separated DNA fragments were depurinated, denatured, and neutralized in situ, and transferred to a nylon m embrane in 20x SSC buffer (3M NaCl, 0.3 M sodium citrate) using the method as described for the TURBOBLOTTERj Rapid Downward Transfer System (Whatman, Inc., Piscataway, NJ). The DNA was then bound to the membrane by UV crosslinking (Stratalinker, Stratage ne, La Jolla, CA). **3 .10.**  **DNA Probe Labeling for Southern Blot Hybridization** Probes for the cry1F, cry34Ab1, cry35Ab1 and pat genes were used to detect genes within the T- DNA insertion. P robes for the ubiZM1 promoter, ubiZM1 5’ UTR and intron region, OR F25 terminator, pin II terminator, TA peroxidase promoter, and CaMV 35S promoter and terminator regions were used to detect regulatory regions within the T- DNA insertion. To determine whether PHP27118 backbone was incorporated during T- DNA insertion, the R ight Border (RB) and Left Border (LB) backbone probes were used to analyze the backbone regions directly outside the T- DNA borders, and probes for the spectinomycin resistance (spc), tetracycline resistance ( tet), and virG genes were used to confirm absence of these genes and other portions of the backbone. DNA fragments of the probe elements were generated by PCR from plasmid PHP2 7118 or a plasmid with equivalent elements using specific primers. P CR fragments were electrophoretically separated on an agarose gel, excised and purified using a gel purification kit (Qiagen, Valencia, CA). D NA probes were generated from these fragments by PCR that incorporated a DIG labeled nucleotide, [DIG - 11] -dUTP, into the fragment. P CR labeling of isolated fragments was carried out according to the procedures supplied in the PCR DIG Probe Synthesis Kit (Roche). **3 .11.**  **Probe Hybridization and Visualization** Labeled probes were hybridized to the target DNA on the nylon membranes for detection of the specific fragments usin g the procedures essentially as described for DIG Easy Hyb solution (Roche). After stringent washes, the hybridized DIG - labeled probes and DIG - labeled DNA standards were visualized using CDP-Star Chemiluminescent Nucleic Acid Detection System with DIG Was h and Block Buffer Set (Roche). B lots were exposed to X- ray film for one or more time points to detect hybridizing fragments and to visualize molecular weight standards bound to the nylon membrane. I mages were digitally captured by detection with the Lum inescent j Registered trademark of Whatman, Inc.Pioneer Hi - Bred International, Inc. Page 181 of 25 8

4114 Maize Image Analyzer LAS-3000 (Fujifilm Medical Systems, Stamford, CT). D igital images were compared to original X- ray film exposures as verification for use in this submission. The sizes of detected bands were documented for each digest and each probe. **3 .12.**  **Stripping of Probes and Subsequent Hybridizations** Following hybridization and detection, membranes were stripped of DIG - labeled probe to prepare the blot for subsequent re- hybridization to additional probes. Membranes were rinsed briefly in di stilled, de- ionized water and then stripped in a solution of 0.2 M NaOH and 0.1% SDS at 37°C with constant shaking. The membranes were then rinsed in 2x SSC and used directly for subsequent hybridizations. The alkali - based stripping procedure effectively removes probes labeled with the alkali - labile DIG.Pioneer Hi - Bred International, Inc. Page 182 of 25 8

4114 Maize **Appendix 4. Materials and Methods for Segregation Analysis of Five Generations of 4114 Maize**  Five generations of 4114 maize were evaluated using polymerase chain reaction (PCR) analyses and herbicide tolerance testing to confirm Mendelian inheritance of the genotype and phenotype. **4.1. Greenhouse Experimental Design**  Five separate generations (F1 *1, BC2F1 *1, BC3F1 *1, BC2F1 *2, and BC3F1 *2 ) of 4114 maize were planted and grown in a greenhouse under sta ndard maize production environmental conditions. Leaf punch samples were collected from each generation and analyzed using PCR analyses specific for the event DP-ØØ4114

-3, cry1F gene, cry34Ab1 gene, cry35Ab1 gene, and pat gene. After sample collection, a ll plants were treated with a broadcast application of glufosinate and then visually evaluated for herbicide resistance. **4.2. Planting and Leaf Sample Collection** Maize seeds, approximately 126 for each generation, were planted in separate cell -divided flats and grown in a greenhouse using typical greenhouse procedures. Ten days after planting, each generation was thinned to a final population of approximately 100 plants. When plants were at approximately the V2 growth stage (the growth stage when the collar of the second leaf is visible ) and p rior to herbicide application, leaf samples were collected from each plant. The samples each consisted of one leaf punch distributed into an individual bullet tube and placed on dry ice until they were transferred to a freezer ( ≤ - 10°C) for storage. Individual plants and corresponding leaf punch samples were uniquely labeled to allow a given sample to be traced back to the originating plant. After the data from the original entries were analyzed, it was dete rmined that additional plants for the BC3F1 *2 generation were needed to verify the original result and increase the statistical power. A n additional 126 seeds from same BC3F1 *2 seed source and 91 seeds from a second BC3F1 *2 generation seed source were pla nted and sampled following the previously outlined procedures. No thinning was performed on the plants from the second seed source before leaf punch samples were collected. **4.3 .**  **Genotypic Analysis** Leaf punch samples were analyzed using event-specific PCR analysis to confirm the presence or absence of event DP-ØØ4114

-3 as well as gene-specific PCR analysis to confirm the presence or absence of the cry1F, cry34Ab1, cry35Ab1, and pat genes.Pioneer Hi - Bred International, Inc. Page 183 of 25 8

4114 Maize **4.4. Phenotypic Analysis** After sample collection, all p lants were treated with a tank mixture containing Ignite k 280 SL herbicide ( Bayer CropScience AG, Monheim am Rhein, Germany) and ammonium sulfate. The Ignite 280 SL herbicide contained 24.5% glufosinate (by weight) in the form of glufosinate-ammonium, equ ivalent to 2.34 pounds glufosinate active ingredient per gallon (0.28 kilograms glufosinate active ingredient per liter). The tank mix was applied at an approximate rate of 22 fluid ounces per acre (1.6 liters per hectare) and the spray volume was 21.6 ga llons per acre (202 liters per hectare). Each plant was visually evaluated 7 days after herbicide application for the presence or absence of herbicide injury, and was identified as presenting either an herbicide-tolerant phenotype (plant exhibited no he rbicidal injury) or an herbicide-susceptible phenotype (plant exhibited severe herbicide injury). For the additional BC3F1 *2 generation plants Ignite 280 SL herbicide was applied with a spray volume of 19.7 gallons per acre (184 L/ha) without the additi on of ammonium sulfate and plants were visually evaluated for herbicide injury 4 days after herbicide application. **4.5 . Statistical Analysis**  A chi -square analysis (95%) was performed on the segregation results of each 4114 maize generation to compare th e observed segregation ratio to the expected segregation ratio (1:1).

k Registered trademark of Bayer CropSciencePioneer Hi - Bred International, Inc. Page 184 of 25 8

4114 Maize **Appendix 5. Materials and Methods for Determination of Cry1F, Cry34Ab1, Cry35Ab1, and PAT Protein Concentrations**  Concentrations of the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins were determined in various tissues of 4114 maize using enzyme- linked immunosorbent assays (ELISA). Plant tissues were collected from 4114 maize plants from a 2010 field trial and analyzed as described further below. **5.1. F** **ield Trial Experimental Design** 4114 maize plants of the F1 *5 generation were grown at five sites in 2010 located in commercial maize growing regions of North America, with four sites in the United States and one site in Canada [ Bagley, IA; Atlantic, IA; Wyoming, IL; York, NE; and Branchton, ON ] . Each field site utilized a randomized complete block design with 4114 maize, 1507 maize, 59122 maize, 1507x59122 maize, and the control maize planted in two- row plots within four replicate blocks. A pproximately 30 seeds were planted in each 25 ft (7.6 m) row resulting in a seed spacing of approximately 10 inches (25 cm). Row spacing was approximately 30 inches (76 cm) and every two- row plot was bordered on either side by one row of commercial maize of similar maturity. I n addition, blocks were separated by an alley distance of at least 36 inches (0.9 m) and each site was surrounded by a minimum of four external border rows. I n order to ensure grain purity, ear shoots of test and control plants were covered prior to silk emergence and the prima ry ear was self- pollinated by hand. To control experimental bias in this study, the following procedures were utilized: non -systematic selection of trial and plot areas within each site, randomization of maize entries within each block, and uniform mainte nance across blocks in each field site. **5.2. Plant Material Collection, Shipping, Processing, and Storage 5.2.1.**  **Tissue Collection** Leaf, root, whole plant, pollen, forage, and grain samples for protein concentration analysis were collected from impa rtially selected, healthy, representative plants from each entry. E ach sample was placed on dry ice after collection before being transferred to a freezer ( ≤ - 10 ° C) for storage until shipment. The following tissues were collected with the plant growt h stages indicated in parenthese s: leaf (V6, V9, R1, R4, and R6), root (V6, V9, R1, R4, and R6), whole plant (V9, R1, and R6), pollen (R1), forage (R4), and grain (R6) . Plant growth stages are explained in Table 5.1 ( Ritchie et al., 2005 ) .Pioneer Hi - Bred International, Inc. Page 185 of 25 8

4114 Maize **Table 5.1.**  **Maize Growth Stage Descriptions Growth**  **Stage a**  **Description a**  V6 The collar of the sixth leaf becomes visible V9 The collar of the ninth leaf becomes visible R1 The silks become visible R4 The material within the kernel produces a doughy consistency R6 The typical harvest maturity for grain; regarded as physiological maturity a Growth stages are as described in Ritchie et al. , 2005

Leaf Four leaf samples (one sample per plant) were each collected from 4114 maize, 1507 maize, 59122 maize, and 1507x5 9122 maize and one sample was collected from the control maize at each site . Samples were obtained from the youngest healthy leaf that had emerged at least 8 inches (20 cm) from the whorl. Root Four root samples (one sample per plant) were each collected from 4114 maize, 1507 maize, 59122 maize, and 1507x59122 maize and one sample was collected from the control maize at each site . Samples were obtained by cutting a circle 10- 15 inches (25 -38 cm) in diameter around the base of the plant to a depth of 7-9 inches (18- 23 cm). The root ball was removed from the soil, shaken to remove excess soil, and thoroughly cleaned with water. A representative sub-sample of root tissue was collected. Whole Plant Following the collection of all other samples for a respective growth stage, whole plant samples were collected from the remaining aerial portion of the plants. Four whole plant samples (one sample equals one plant) were each collected from 4114 maize, 1507 maize, 59122 maize, and 1507x59122 maize and one sample was collected from the control maize at each site . Samples were obtained by cutting the plants from the root system approximately 1 inch (2.5 cm) above the soil surface line. The plants were chopped into sections less than 3 inches (7.5 cm) in length.Pioneer Hi - Bred International, Inc. Page 186 of 25 8

4114 Maize Pollen Four pollen samples (one sample per plant) were each collected from 4114 maize, 1507 maize, 59122 maize, and 1507x59122 maize and one sample was collected from the control maize at each site . The tassel selected for sampling had half to thre e-quarters of the main spikes shedding and was bagged no more than one day prior to collection. Samples were obtained by shaking or tapping the bagged tassel to dislodge the pollen. Forage Following the collection of R4 leaf and root samples, forage sam ples were collected from the remaining aerial portion of the plants. Four forage samples (one sample equals one plant) were each collected from 4114 maize, 1507 maize, 59122 maize, and 1507x59122 maize and one sample was collected from the control maize at each site . A ll plants used for sampling contained self- pollinated primary ears. A ny secondary or tertiary ears with exposed silks were removed prior to sampling. Samples were obtained by cutting the plants from the root system approximately 1 inch (2. 5 cm) above the soil surface line. The plants were chopped into sections less than 3 inches (7.5 cm) in length. Grain Four grain samples (one sample equals the grain from one self pollinated primary ear per plant) were each collected from 4114 maize, 1507 maize, 59122 maize, and 1507x59122 maize and one sample was collected from the control maize at each site . The grain samples were obtained by husking and shelling each ear. Arepresentative sub-sample of 15 kernels from each ear was collected. H usks and cobs were retained for inclusion with the R6 whole plant samples. **5.2.2. Sample Shipping, Processing, and Storage**  All tissue samples were shipped frozen and stored at ≤ -5 °C. W hole plant and forage samples were coarsely homogenized. Leaf, root, w hole plant, pollen, forage, and grain samples were lyophilized under vacuum until dry. Following lyophilization, leaf, root, whole plant, forage, and grain samples were finely homogenized and stored frozen until analysis. **5.3. Protein Concentration Dete rmination**  Concentrations of the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins were determined using specific quantitative ELISA methods. **5.3.1 . Protein Extraction**  Analytical samples were weighed into 1.2- ml tubes at target weights (±5%) of 10 mg for l eaf; 20 mg for root and grain; 30 mg for forage, and whole plant; and 5 mg for pollen. Each sample analyzed for protein concentrations was extracted with 0.6 ml of chilled PBST buffer (PhosphatePioneer Hi - Bred International, Inc. Page 187 of 25 8

4114 Maize Buffered Saline, pH 7.45 and 0.05% Tween l

- 20). Following ce ntrifugation, supernatants were removed, diluted, and analyzed. **5.3. 2. Cry1F ELISA Procedure**  The Cry1F ELISA method utilized a sequential ELISA format to determine the concentration of the Cry1F protein in sample extracts. The Cry1F ELISA kit employed was obtained from Envirologix, Inc. (Portland, Maine). Standards (analyzed in triplicate wells) and diluted sample extracts (analyzed in duplicate wells) were incubated in a plate pre-coated with a Cry1F-specific antibody. Following incubation, unbound s ubstances were washed from the plate. Ad ifferent Cry1F-specific antibody conjugated to the enzyme horseradish peroxidase (HRP) was added to the plate and incubated. The unbound substances were washed from the plate. D etection of the bound Cry1F-antibod y complex was accomplished by the addition of substrate, which generated a colored product in the presence of HRP. The reaction was stopped with an acid solution and the optical density (OD) of each well was determined using a plate reader. An average of the results from duplicate wells was used to determine the concentration of the Cry1F protein in ng/mg sample weight. **5.3. 3. Cry34Ab1 ELISA Procedure**  A similar procedure was used as for the Cry1F ELISA, except a Cry34Ab1-specific antibody was used on th e pre-coated plate and a second Cry34Ab1-specific antibody conjugated to HRP was used. The Cry34Ab1 ELISA kit employed was obtained from Envirologix, Inc. **5.3. 4. Cry35Ab1 ELISA Procedure**  A similar procedure was used as for the Cry1F ELISA, except a Cry35Ab1-specific antibody was used on the pre-coated plate and a second Cry35Ab1-specific antibody conjugated to HRP was used. The Cry35Ab1 ELISA kit employed was obtained from Acadia BioScience, LLC (Portland, Maine) . **5.3. 5. PAT ELISA Procedure**  A simila r procedure was used as for the Cry1F ELISA, except a PAT-specific antibody was used on the pre-coated plate and a second PAT-specific antibody conjugated to HRP was used. The PAT ELISA kit employed was obtained from Envirologix, Inc. **5.3.6 . Calculation**  **s for Determining Protein Concentrations** SoftMaxm Pro GxP (Molecular Devices, Sunnyvale, CA) software was used to perform the calculations required to convert OD values obtained by the microtiter plate reader to protein concentration values.

l Registered trademark of ICI Americas, Inc.Pioneer Hi - Bred International, Inc. Page 188 of 25 8

4114 Maize **Standard Curv e**  A standard curve was included on each ELISA plate. The equation for the standard curve was generated by the software, which used a quadratic fit to relate the mean OD values obtained for the standards to the respective standard concentration (ng/ml). The quadratic regression equation was applied as follows: y = Cx2 + Bx + A Where x = known standard concentration and y = respective mean absorbance value (OD) **Sample Concentration** Interpolation of the sample concentration (ng/ml) was accomplished by solving for x in the above equation using values for A, B, and C determined by the standard curve. Sample concentration (ng/ml) = 

###   C 2 4C B B OD

sample - A - 2   The sample concentration values were adjusted for a dilution factor expressed as 1:N by multiplying the interpolated co ncentration by N. Adjusted sample concentration values obtained from SoftMax Pro GxP software were converted from ng/ml to ng/mg sample weight as follows: ng/mg Sample Weight = ng/ml x Extraction Volume (ml)/Sample Weight (mg) **Lower Limit of Quantifica tion (LLOQ)**  The LLOQ, in ng/mg sample weight, was calculated as follows: LLOQ = Reportable Assay LLOQ x Extraction Volume Sample Target Weight **5.4 . Statistical Analysis**  Statistical analyses were conducted using SAS n software, Version 9. 2 (SAS Inst itute, Inc. , Cary, NC) to estimate mean concentrations of Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins derived from 4114 maize, 1507 maize, 59122 maize, and 1507x59122 maize.

m Registered trademark of Molecular Devices Corporation n Registered tra demark of SAS Institute, Inc.Pioneer Hi - Bred International, Inc. Page 189 of 25 8

4114 Maize **5.4.1. Processing of Data**  **Transformation** Protein concentrations were first subjected to a natural logarithm transformation “ln(y)” before statistical analyses. Residuals were examined for validation of the normality and homogeneous variance assumptions. W hen these assumptions were questionable, other types of transforms or non e were considered. The following proteins and tissue types did not undergo “ln(y)” transformation because the original data achieved model validation assumptions.

 Cry1F in leaf (V9), root (R4, V6, and V9), and whole plant (V9)  Cry34Ab1 in leaf (R1 and R4), root (R1, R4, and V6), and whole plant (V9)  Cry35Ab1 in leaf (R4 and R6), root (R4 and R6), and grain (R6)  PAT in leaf (R1, R4 and V9), forage (R4), and root (V9) If the protein concentration underwent “ln(y)” transformation, the statistical analyses were conducted based on the transformed data. The estimated mean values and the confidence limits were then back-transformed to the original data scale for reporting purposes. **Partial LLOQ Sample Values** For a given protein in a given tissue, the num ber of samples below the LLOQ determined whether statistical analysis was conducted. The following rules were implemented: If < 80% of samples for each entry were below the LLOQ, then the statistical model considering LLOQ was utilized to conduct statistical analysis . If ≥ 80% of samples for a single entry within the study were below the LLOQ, then statistical analysis was not conducted. **5.4.2. Statistical Models**  Default Model For a given protein concentration in a given tissue, data were analyzed using the following linear mixed model:Pioneer Hi - Bred International, Inc. Page 190 of 25 8

4114 Maize yijk = μ i + l

j + r k(j) + (μl)ij + εijk Model 1 l j ~ iid N(0, σ

2Site), r k(j) ~ iid N(0,σ

2Rep), (μl) ij ~ iid N(0, σ 2Ent×Site), and εijk ~ iid N(0, σ 2Error ) Where μ i denotes the mean of the i th entry (fixed effect), l j denotes the effect of the j th site (random effect), r

k(j) denotes the effect of the kth block within the j th site (random effect), (μl) ij denotes the interaction between the entries and sites (random effect) and εijk denotes the effect of the plot assigned the i th entry in the kth block of the j th site (random effect or residual). Notation ~iid N(0, σ 2a) indicates random variables that are identically independently distributed ( iid) as normal with zero mean and variance σ 2a . The residual maximum li kelihood (REML) estimation procedure was utilized to generate estimates of variance components and entry means. The estimated means are known as least squares means (LS- Means). SAS PROC MIXED was utilized to fit Model 1 and to generate LS- Means, 95% co nfidence intervals . Model Considering LLOQ For Cry1F in leaf (R6), PAT in root (R4 and R6), and whole plant (R6), <80% of sample values were detected below the assay LLOQ. Sample results below the LLOQ were treated as leftcensored observations at the respective assay LLOQ value. Data were analyzed using the following linear mixed model: yijk = μ i + l

j + r k(j) + (μl)ij + εijk Model 2 l j ~ iid N(0, σ

2Site), r k(j) ~ iid N(0,σ

2Rep), (μl) ij ~ iid N(0, σ 2Entry×Site), εijk ~ iid N(0, σ 2Error ) Where μ i denotes the mean of the i th entry (fixed effect), l j denotes the effect of the j th site (random effect), r

k(j) denotes the effect of the kth block within the j th site (random effect), (μl) ij denotes the interaction between the entries and sites (rando m effect) and εijk denotes the residual for the observation obtained from the plot assigned to the i th entry in the kth block of the j th site. Model 2 can also be written as: yijk = μ ijk + εijk = μ i + l j + r k(j) + (μl) ij + εijk Where μ ijk denotes the mean of yijk. The conditional likelihood for each observation, given the random effects, was formulated according to the status of the observation (i.e. observed or leftcensored) (Thiébaut and Jacqmin -Gadda, 2004 ):Pioneer Hi - Bred International, Inc. Page 191 of 25 8

4114 Maize 

     



        censored left c c y P observed

y y y P y f ijk ijk ijk ijk ijk ijk ijk )

( ) ( )

( ) ( ) | ( * *

    

Where  denotes the vector of a ll random effects,  denotes the standard normal density function,  denotes the standard normal cumulative distribution function, y *ijk denotes the observed sample value of yijk, and c denotes the assay LLOQ value. The conditional likelihood function was a product of all individual conditional likelihoods, and the marginal likelihood function was formed when the conditional likelihood function was integrated over all random effects. The maximum likelihood (ML) procedure was then used to generate esti mates of variance components and entry means (i.e. LS- Means). SAS PROC NLMIXED was utilized to fit Model 2 and generate LS- Means and 95% confidence intervals. **5.4. 3**  **. Reported Statistics** For each expressed trait protein in each tissue that was stati stically analyzed, entry LS- Mean (back-transformed, if needed), range, and 95% confidence interval (back-transformed, if needed) (labeled as Mean, Range, and CI, respectively) are provided in Section 2; Tables 10- 13 . In these tables, descriptive statistics (arithmetic means and ranges) are reported for proteins and tissue s that were not statistically analyzed using mixed model analyses due to insufficient above- LLOQ samples. The Cry1F, Cry34Ab1, and Cry35Ab1 protein mean concentrations in 4114 maize in ea ch tissue were divided by the respective protein mean concentrations in 1507, 59122, and/or 1507x59122 maize to provide an “expression ratio” (Section 2; Table 14) . When the expression ratio was close to one, this indicated that 4114 maize had comparable expression to 1507, 59122, and/or 1507x59122 maize. I f the expression ratio was less than one, this indicated that 4114 maize had lower expression. For values greater than one, expression was determined to be higher in 4114 maize.Pioneer Hi - Bred International, Inc. Page 192 of 25 8

4114 Maize **Appendix 6. Materi als and Methods for Nutrient Composition**  Nutrient composition was determined in 4114 maize using plants from a 2010 field trial and analyzed as described further below. **6.1. Field Trial and Experimental Design** 4114 maize plants of the F1 *5 generation were grown at six sites in 2010 in commercial maize growing regions of North America, with four sites in the U.S. and two sites in Canada [Richland, IA; Wyoming, IL; Geneva, MN; York, NE; Branchton, ON Canada; and Thorndale, ON Canada]. Each field site uti lized a randomized complete block design with 4114 maize and the control maize planted in two- row plots within four replicate blocks. Approximately 30 seeds were planted in each 25 ft (7.6 m) row resulting in a seed spacing of approximately 10 inches (25 cm). Row spacing was approximately 30 inches (76 cm) and every two- row plot was bordered on either side by one row of commercial maize of similar maturity. I n addition, blocks were separated by an alley distance of at least 36 inches (0.9 m) and each site was surrounded by a minimum of four external border rows. Maintenance fertilizer, herbicides, and pesticides were applied uniformly to 4114 maize and the control maize at each site, as needed. In order to ensure grain purity for compositional analyses, ear shoots of test and control plants were covered prior to silk emergence and the primary ear was self- pollinated by hand. To control experimental bias in this study, the following procedures were utilized: non -systematic selection of trial and plot are as within each site, randomization of maize entries within each block, and uniform maintenance across blocks in each field site. **6.2. Plant Material**  **Collection, Shipping, Processing, and Storage 6.2.1. Tissue Collection**  All samples were collected fro m impartially selected, healthy individual plants. A ll control maize samples were collected prior to collection of 4114 maize samples. A ll forage and grain samples were assigned unique sample identification numbers that described the sample by site, entry, block, sample number, and tissue type. Samples were placed on dry ice within thirty minutes of collection and were maintained in coolers on wet or dry ice and/or in the freezers until shipment. Forage One forage sample ( composite of 3 whole plants) was collected from 4114 maize and the control maize from each of the four blocks. A ny secondary or tertiary ears with exposed silks were removed prior to sampling. Forage samples were obtained by cutting the aerial portion of the plants from the root system approximately 1 inch (2.5 cm) above the soil surface line. The plants (including ears) were then cut into sections approximately 3 inches (7.5 cm) or less, and approximately 1/3 of the total sample was collected in a pre- labeled plastic- lined cloth b ag.Pioneer Hi - Bred International, Inc. Page 193 of 25 8

4114 Maize Grain One grain sample (five pooled ears equaled one sample) was collected from 4114 maize and the control maize from each of the four blocks at typical harvest maturity. Each ear was husked and shelled, and the grain was collected into a pre- label ed plastic- lined cloth bag. After collection, forage and grain samples were shipped from each field site to EPL BioAnalytical Services (EPL- BAS) for processing and nutrient composition analysis. E ach sample was labeled by site, entry, block, sample nu mber, tissue type, and growth stage. Au nique sample identification number was also included on each label. **6.2.2. Sample Shipping, Processing, and Storage** After collection, forage and grain samples were shipped and stored frozen at - 20 °C. Samples were lyophilized, ground, and homogenized before nutrient composition analysis. **6.3. Nutrient Composition Analyses** Nutrient composition analyses of maize forage and grain were conducted and included the analysis methods described in Table 6.1.

**Tabl e 6.1.**  **Methods for Compositional Analysis of 4114 Maize Nutritional Analyte**  **Method** Moisture in forage and grain The analytical procedure for moisture determination was based on a method published by the Association of Official Analytical Chemists (AOAC) . Samples were assayed to determine the percentage of moisture by gravimetric measurement of weight loss after drying in a forced air oven (forage) or vacuum oven (grain ) . Ash in forage and grain The analytical procedure for ash determination was based on a method published by the AOAC. Samples were analyzed to determine the percentage of ash by gravimetric measurement of the weight loss after ignition in a muffle furnace. Crude protein in forage and grain The analytical procedure for crude protein determination utilized an automated Kjeldahl technique based on a method provided by the manufacturer of the titrator unit (Foss-Tecator). Ground samples were digested in the presence of a catalyst. The digestate was then distilled and titrated with a Foss-Tecator Kjeltec titrator unit.Pioneer Hi - Bred International, Inc. Page 194 of 25 8

4114 Maize **Table 6.1. Methods for Compositional Analysis of 4114 Maize (continued)**  **Nutritional Analyte Method**  Crude fat in forage and grain The analytical procedure for crude fat determination was based on methods provided by the manufacturer of the hydrolysis and extraction apparatus (Ankom Technology). Samples were hydrolyzed with 3N hydrochloric acid at 90 °C for 60 minutes. The hydrolysates were extracted with a petroleum ether/ethyl ether/ethyl alcohol solution at 90 °C for 60 minutes. The ether extracts were evaporated and the fat residue remaining determined gravimetrically. Carbohydrate in forage and grain The carbohydrate content in maize grain on a dry weight basis was calculated using a formula obtained from the U nited States Department of Agriculture “Energy Value of Foods, ” in which the percent dry weight of crude protein, crude fat, and ash was subtracted from 100%. Crude fiber in forage and grain The analytical procedure for crude fiber determination was based on methods provided by the manufacturer of the extraction apparatus (Ankom Technology). Samples were analyzed to determine the percentage of crude fiber by digestion and solubilization of other materials present. Neutral detergent fiber in forage and grain The analytical procedure for neutral detergent fiber (NDF) determination was based on a method provided by the manufacturer of the extraction apparatus (Ankom Technology). Samples were analyzed to determine the percentage of NDF by digesting with a ne utral detergent solution, sodium sulfite, and alpha amylase. The remaining residue was dried and weighed to determine the NDF content. Acid detergent fiber in forage and grain The analytical procedure for acid detergent fiber (ADF) determination was base d on a method provided by the manufacturer of the extraction apparatus (Ankom Technology). Samples were analyzed to determine the percentage of ADF by digesting with an acid detergent solution and washing with reverse osmosis water. The remaining was dri ed and weighed to determine the ADF content. Minerals in forage and grain The analytical procedure for the determination of minerals is based on methods published by the AOAC and CEM Corporation. The maize forage minerals determined were calcium and phos phorus. The maize grain minerals determined were calcium, copper, iron, magnesium, manganese, phosphorus, potassium sodium, and zinc. The samples were digested in a microwave based digestion system and the digestate was diluted using deionized water. Sa mples were analyzed by inductively coupled plasma optical emission spectroscopy (ICP-OES). Tryptophan in grain The analytical procedure for tryptophan determination was based on an established lithium hydroxide hydrolysis procedure with reverse phase ultra performance liquid chromatography (UPLC) with ultraviolet (UV) detection published by the Journal of Micronutrient Analysis .Pioneer Hi - Bred International, Inc. Page 195 of 25 8

4114 Maize **Table 6.1. Methods for Compositional Analysis of 4114 Maize (continued)**  **Nutritional Analyte Method**  Cystine and methioni ne in grain The analytical procedure for cystine and methionine determination was based on methods obtained from Waters Corporation, AOAC, and Journal of Chromatography A . The procedure converts cystine to cysteic acid and methionine to methionine sulfone, after acid oxidation and hydrolysis, to the 6-aminoquinolyl - N - hydroxysuccinimidyl carbamate derivatives which are then analyzed by reverse phase UPLC with UV detection. Additional amino acids in grain Along with tryptophan, cystine, and methionine, 15 additional amino acids were determined. The analytical procedure for analysis of these amino acids was based on methods obtained from Waters Corporation and the Journal of Chromatography A . The procedure converts the free acids, after acid hydrolysis, to the 6-aminoquinolyl - N - hydroxysuccinimidyl carbamate derivatives, which are analyzed by reverse phase UPLC with UV detection. Fatty acids in grain The analytical procedure for determination of fatty acids was based on methods published by the AOAC and American Oil Chemist Society (AOCS). The procedure converts the free acids, after ether extraction and base hydrolysis, to the fatty acid methyl ester (FAME) derivatives, which are analyzed by gas chromatography with flame ionization detection (GC/FID). Results are reported as percent total fatty acids but presented in the raw data as %DB. Thiamine (vitamin B1) and Riboflavin (vitamin B2) in grain The analytical procedure for the determination of Thiamine (Vitamin B1) and Riboflavin (Vitamin B2) was based on a method published by the American Association of Cereal Chemists (AACC). The samples were extracted with 10% acetic acid/4.3% trichloroacetic acid solution. A 50 fold dilution was performed then the samples were analyzed by reverse phase HPLC tand em mass spectrometry (MS/MS).

Niacin (vitamin B3) in grain The analytical procedure for the determination of Niacin (vitamin B3) was based on a method published by the AACC. Niacin (vitamin B3) was extracted from the sample by adding deionized (DI) water and autoclaving. A tube array was prepared using three different dilutions of the samples. This tube array was inoculated with Lactobacillus plantarum and allowed to incubate for approximately 18 to 22 hours. After incubation, the bacterial growth wa s determined using a spectrophotometer at an absorbance of 660 nm. The absorbance readings were compared to a standard curve generated using known concentrations of nicotinic acid.Pioneer Hi - Bred International, Inc. Page 196 of 25 8

4114 Maize **Table 6.1. Methods for Compositional Analysis of 4114 Maize (continu**  **ed) Nutritional Analyte**  **Method**

Pantothenic acid (vitamin B5) in grain The analytical procedure for the determination of pantothenic acid (Vitamin B5) was based on a method from the AOAC. Pantothenic acid (Vitamin B5) content was determined using a micro biological assay. Pantothenic acid (Vitamin B5) was extracted from the sample by an acetic acid buffer solution, consisting of acetic acid adjusted to a pH of 5.65 with sodium hydroxide, and autoclaving the samples. A tube array was prepared using three different dilutions of the samples. This tube array was inoculated with Lactobacillus plantarum and allowed to incubate for approximately 18-22 hours. After incubation, the bacterial growth was determined using a spectrophotometer at an absorbance of 660 nm. The absorbance readings were compared to a standard curve generated using known concentrations of D- pantothenic acid hemicalcium salt.

Pyridoxine (vitamin B6) in grain The analytical procedure for the determination of pyridoxine (Vitamin B6) was based on a method from the AACC. Pyridoxine (Vitamin B6) was determined using a microbiological assay. Pyridoxine (Vitamin B6) was extracted from the sample by adding sulfuric acid and autoclaving. The pH was adjusted and a tube array was prepared using fo ur different dilutions of the samples. This tube array was inoculated with Saccharomyces cerevisiae and allowed to incubate for approximately 18-22 hours. After incubation, the microbial growth was determined using a spectrophotometer at an absorbance of 600 nm. The absorbance readings were compared to a standard curve generated using known concentrations of pyridoxine hydrochloride.

Total folate as folic acid (vitamin B9) in grain The analytical procedure for determination of total folate as folic acid was based on a microbiological assay published by the AACC. Samples were hydrolyzed and digested by protease and amylase enzymes to release the folate from the grain. A conjugase enzyme was used to convert the naturally occurring folypolyglutamates. An aliquot of the extracted folates was mixed with a folate and folic acid free microbiological growth medium. The mixture was inoculated with Lactobacillus casei. The total folate content was determined by measuring the turbidity of the Lactobacillus casei growth response in the sample and comparing it to the turbidity of the growth response with folic acid standards. Tocopherols (vitamin E) in grain The analytical procedure for determination of tocopherols (Vitamin E) was based on methods from the Journal of the American Oil Chemists’ Society and Analytical Sciences . Alpha, beta, delta, and gamma -tocopherols were extracted with hot hexane and the extracts were analyzed by normal phase HPLC with fluorescence detection. Beta -carotene in grain The analyti cal procedure for determination of beta -carotene was based on a method published by the AOAC. Fat-soluble pigments from the ground maize grain were extracted and determined spectrophotometrically and expressed as carotene.Pioneer Hi - Bred International, Inc. Page 197 of 25 8

4114 Maize **Table 6.1. Methods for Compo** **sitional Analysis of 4114 Maize (continued) Nutritional Analyte**  **Method** Trypsin inhibitor in grain The analytical procedure for the determination of trypsin inhibitor was based on a method published by the AOCS. Trypsin inhibitor was extracted with sodiu m hydroxide. Trypsin was added and reacted with trypsin inhibitor. The amount of trypsin present in the sample was measured using a spectrophotometer, and the amount of inhibitor was calculated based on how much trypsin remained. Furfural in grain The a nalytical procedure for the determination of furfural was based on methods published in the Journal of Agricultural and Food Chemistry. G round maize grain was analyzed for furfural content by reverse phase HPLC with UV detection. p-Coumaric and ferulic acid in grain The analytical procedure for the determination of p-coumaric and ferulic acids was developed based on methods published in Journal of Agricultural and Food Chemistry and The Journal of Chemical Ecology. Ground maize grain was analyzed to determine the amounts of p-coumaric acid and ferulic acid by separating the total content of phenolic acids using reverse phase HPLC and UV detection. Phytic acid in grain The analytical procedure for the determination of phytic acid was based on a method p ublished by the AOAC. The samples were analyzed to determine the amount of phytic acid by extracting the phytic acid with dilute hydrochloric acid (HCl) and isolating it using an aminopropyl silica solid phase extraction column. Once isolated and eluted, the phytic acid was analyzed for elemental phosphorus by ICP-OES.Pioneer Hi - Bred International, Inc. Page 198 of 25 8

4114 Maize **6.4. S** **tatistical Analyses** Statistical analyses were conducted using SASo software, Version 9.2 (SAS Institute, Inc. , Cary, NC) to evaluate and compare the nutrient composition of forage and grain derived from 4114 maize and the control maize. **6.4.1. Processing of Data Fatty Acids**  For some fatty acid analytes, absolute sample values were detected below the assay lower limit of quantification (LLOQ). W hen sample values for each fatty acid analyte were converted from an absolute value to a relative proportion (percentage of total fatty acids), sample results below the LLOQ were reported as zero to reflect a negligible proportion. However, these zeros were not “true” zeros, but we re some unknown small positive values. Therefore, these “zero” sample values were treated as missing values during subsequent statistical analysis. **Data Transformation** A natural logarithmic “ln(y)” transformation was performed for the raw data of all analytes before statistical analyses. For each analyte, residuals were examined for validation of the normality and homogeneous variance assumptions. Residual distributions skewed to the left for the following analytes after “ln(y)” transformation and therefore, either no transformation, a cubic “(y) 3” transformation, a square “(y) 2” transformation, or a square root “√y” transformaon was performed to the raw data instead:

 Vitamin B9 - No transformation was performed.  Crude protein in both forage an d grain, and tryptophan and valine in grain - A cubic “(y) 3” transformation was performed.  NDF, alanine, aspartic acid, glutamic acid, isoleucine, leucine, phenylalanine, proline, and threonine - A square “(y) 2” transformation was performed.  Vitamin B6 – A square root “√y” transformaon was performed. The model assumptions were reasonably satisfied for these analytes after the moreappropriate transformation or non -transformation. For a given analyte, the same type of transformation or non -transformation was used fo r all statistical analyses and comparisons performed. The statistical results were then backtransformed to the original data scale for reporting purposes.

o Registered trademark of SAS Institute, Inc.Pioneer Hi - Bred International, Inc. Page 199 of 25 8

4114 Maize **6.4.2. Statistical Analyses** For a given analyte, the number of samples below the assay LLOQ value determined whether a statistical analysis was conducted. The following rules were implemented:

 If, for each entry, <80% of samples were below the LLOQ, the n the analysis was conducted.  If, for a single entry, ≥80% of samples were below the LLOQ, then the analys i s was not conducted. If, for a given analyte, a statistical analysis was not conducted due to insufficient data, the logistic regression likelihood - ratio test was used to test if the proportion of assay values below the LLOQ was different between 4114 maize and the control maize. Default Model For a given analyte, data were analyzed using the following linear mixed model: yijk = μ i + l

j + r k(j) + (μl)ij + εijk Model 1 l j ~ iid N(0, σ

2Site ), r k(j) ~ iid N(0,σ

2Rep ), (μl) ij ~ iid N(0, σ 2Ent×Site ), and εijk ~ iid N(0, σ 2Error ) Where μ i denotes the mean of the i th entry (fixed effect), l j denotes the effect of the j th site (random effect), r k(j) d enotes the effect of the kth block within the j th site (random effect), (μl) ij denotes the interaction between the entries and sites (random effect) and εijk denotes the effect of the plot assigned the i th entry in the kth block of the j th site (random effect or residual). Notation ~iid N(0, σ 2a) indicates random variables that are identically independently distributed ( iid) as normal with zero mean and variance σ 2a . The residual maximum likelihood (REML) estimation procedure was utilized to generate esti mates of variance components and entry means. The estimated means are known as least squares means (LS- Means). The statistical comparison was conducted by testing for differences in LS- Means between 4114 maize and the control maize . The approximated deg rees of freedom for the statistical test were derived by the Kenward - Roger (KR) method ( Kenward and Roger, 1997 ). SAS PROC MIXED was utilized to fit Model 1 and to generate LS- Means, 95% confidence intervals, and statistical comparisons (P-values). By default, the variance components in Model 1 are all constrained to be non - negative. W hen the estimated value of σ 2Ent×Site is zero, the KR method pools degrees of freedom for the interaction term with the degrees of freedom for residuals. Consequently, the degrees of freedom for the statistical test could be larger than what was expected under the original experimental design. I n order to stabilize the degrees of freedom across all analytes, effect (μl)ij in Model 1 was combined with εijk and the compound symmetry (CS) structure was used to model the corresponding residual variance structure. This approach allows σ 2Ent×Site to take negative values without affecting the degrees of freedom ( Littell et al., 2006 ).Pioneer Hi - Bred International, Inc. Page 200 of 25 8

4114 Maize Model Considering LLOQ For the analytes sodium, vitamin B1, vitamin B5, and raffinose, <80% of sample values were detected below the assay LLOQ. Sample results below the LLOQ were treated as left-censored observations at the respective assay LLOQ value. Data were analyzed using the following li near mixed model: yijk = μ i + l

j + r k(j) + (μl)ij + εijk Model 2 l j ~ iid N(0, σ

2Site ), r k(j) ~ iid N(0,σ

2Rep ), (μl) ij ~ iid N(0, σ 2Entry×Site ), εijk ~ iid N(0, σ 2Error ) Where μi denotes the mean of the ith entry (fixed effect), l j denotes the effect of the j th site (random effect), r k(j) denotes the effect of the k th block within the j th site (random effect), (μl) ij denotes the interaction between the entries and sites (random effect) and εijk denotes the residual for the observation obtained from the plot assigned to the ith entry in the k th block of the j th site. N otation ~iid N(0, σ 2a ) indicates random variables that are identically independently distributed (iid ) as normal with zero mean and variance σ 2a. Model 2 can also be written as: yijk = μijk + εijk = μi + l j + r k(j) + (μl) ij + εijk Where μijk denotes the mean of yijk , The conditional likelihood for each observation, given the random effects, was formulated according to the status of the observation (observed or left-cen sored) (Thiébaut and Jacqmin Gadda, 2004):       



        censored left c c y P observed

y y y P y f ijk ijk ijk ijk ijk ijk ijk )

( ) ( )

( ) ( ) | ( * *

    

Where  denotes the vector of all random effects,  denotes the standard normal density function,  denotes the standard normal cumulative distribution function, y*ijk denotes the observed sample value of yijk, and c denotes the assay LLOQ value. The conditional likelihood function was a product of all individual conditional likelihoods, and the marginal likelihood function was formed when the conditional likelihood function was integrated over all random effects. The maximum likelihood procedure was then used to generate estimates of variance components and entry means (LS- Means). The statistical comparison was conducted by testing for difference in LS- Means between 4114 maize and the control maize . The approximated degrees of freedom for the statistical test were derived by the KR method. SAS PROC NLMIXED was utilized to fit Model 2 and generate LS- Means, 95% confidence intervals, and statistical comparisons (P-values).Pioneer Hi - Bred International, Inc. Page 201 of 25 8

4114 Maize **FDR Adjustment** The FDR method of Benjamini and Hoch berg ( Benjamini and Hochberg, 1995; Westfall et al., 1999 ) was applied as a post- hoc procedure to account for multiple comparisons due to multiple compositional analytes, and P-values were adjusted accordingly. The FDR adjustment was conducted for eac h set of comparisons. As ignificant difference was identified if an adjusted P-value <0.05. SAS PROC MULTTEST was utilized to provide adjusted P-values. **6.4.3. Interpretations of Statistical Results** Where a statistically significant difference (adjusted P-value <0.05) was identified for a given analyte in the analysis between 4114 maize and the control maize, the respective range of individual values from 4114 maize was compared to a tolerance interval. Tolerance intervals containing 99% of the values for corresponding analytes of the conventional maize population with 95% confidence level ( Graybill, 1976 ) were derived from data collected under previous studies. In each of these studies, four non - modified commercially available maize lines were grown at six sites in North America, and were harvested, processed, and analyzed using methods similar to those employed in this nutrient composition study for 4114 maize. The selected maize varieties represent the non - modified maize population with a history of safe use, and the selected environments (site and year combinations) represent maize growth under a range of environmental conditions (i.e., soil type, temperature, precipitation, and irrigation) and maize maturity group zones similar to the sites used in the 4114 maize agronomic study. Ranges containing individual values outside the tolerance interval for a given analyte were then compared to the respective literature range obtained from publi shed literature ( Codex, 1996; Codex, 2005; ILSI, 2006; OECD, 2002; Watson, 1982; Watson, 1987 ). **6.4.4. Reported Statistics** For each analyte that was statistically analyzed using mixed model analysis, entry LS- Mean (back-transformed, if needed), range, and 95% confidence interval (back-transformed, if needed) (la beled as Mean, Range, and CI, respectively) are provided in Sectio n 4; Tables 15- 22 . Both the non -adjusted P-values and FDR-adjusted P-values (labeled as P-Value and Adjusted PValue, res pectively) are provided for comparisons between 4114 maize and the control maize . For each analyte, a tolerance interval ( Section 4; Tables 15- 22 ) and a literature range ( Section 4; Tables 15 -22 ), if available, are provided. In Section 4, Tables 15 -22, descri ptive statistics (arithmetic means and ranges) are reported fo r analytes that were not statistically analyzed using mixed model analysis. Note: for fatty acid analytes, means and ranges were calculated based on assay values above the LLOQ. W hen all fatty acid samples values were below the LLOQ for a given entry, m eans and ranges were 0 and 0-0, respectively, and were not reported .Pioneer Hi - Bred International, Inc. Page 202 of 25 8

4114 Maize **Appendix 7. Material and Methods for Analysis of Agronomic Characteristics and Germination Evaluation**  **7.1. Germination and Dormancy Evaluation**  4114 maize and two commercial Pionee r® hybrid maize reference lines ( 32D78 and 34P88 ) were evaluated for germination rates under warm, cold, and diurnal growing conditions. **7.1.2. Experimental Design** Three separate germination tests (warm, cold, and diurnal) were conducted. For a given germination test, approximately 400 seed from each of 4114 maize, the control maize, 32D78 maize, and 34P88 maize were evaluated. The seed from each maize line were arranged into eight individual replicates, with approximately 50 seed per replicate. N o broken or damaged seed were included in any of the germination tests. To control experimental bias in this study, the following procedures were utilized: randomization of each sample replication within the growth chamber and uniform maintenance of environm ental conditions across all entries and replicates within the growth chamber. **7.1.3. Germination Tests** The warm germination test simulated optimal maize growth conditions. E ach replicate was placed between sheets of moist germination toweling and rolle d up. The rolls were then placed in a dark growth chamber at a continuous setting of 25 °C and 90% relative humidity for 10 days. After 10 days, the number of germinated seed in each replicate was counted. The cold germination test simulated early sprin g planting conditions in the midwestern United States. Each replicate was placed between sheets of moist germination toweling and rolled up. The rolls were then placed in a dark growth chamber at a continuous setting of 10 °C and 90% relative humidity fo r 10 days, followed by three days at a continuous setting of 25 °C and 90% relative humidity. After 13 days, the number of germinated seed in each replicate was counted. The diurnal germination test simulated daily weather conditions in the midwestern Un ited States. Each replicate was placed between sheets of moist germination toweling and rolled up. The rolls were then placed in a growth chamber at a cyclical setting of 10 °C and 90% relative humidity for 16 hours and then 25 °C and 90% relative humidity for 8 hours, repeated daily for 10 days. After 10 days, the number of germinated seed in each replicate was counted. **7.1.4. Seed Evaluation Classification of Germinated and Non** **- Germinated Seed**  At the end of each germination test, each seed was defi ned as either germinated or non germinated. Ag iven seed was classified as germinated if any of the essential structuresPioneer Hi - Bred International, Inc. Page 203 of 25 8

4114 Maize necessary to produce a normal plant under favorable conditions had emerged, or non -germinated if these structures had not emerged. N on -germinated seed were further defined into three categories: h ard (i.e., did not absorb water), imbibed (i.e., absorbed water but did not show signs of growth during the germination test), or dead (i.e., absorbed water, did not show signs of growth duri ng the germination test, and displayed distinct signs of decay such as an extremely soft interior that did not hold shape under gentle pressure). Descriptions of all germination classifications are summarized in Table 7. 1.

**Table 7.**  **1. Description of Germination Test Classifications**  **Germination Classification**  **Description** Germinated Seed Visible emergence of any structures necessary to produce a healthy plant.

Non Germinated Seed Hard No emergence of structures necessary to produce a healthy

plant . Seed did not absorb water during the germination test. Imbibed No emergence of structures necessary to produce a healthy

plant. Seed did absorb water during the germination test but showed no signs of growth. Dead No emergence of structures necessa ry to produce a healthy

plant. Seed did absorb water during the germination test but showed no signs of growth. Seed displayed distinct signs of decay (i.e. , extremely soft interior that did not hold shape under gentle pressure).Pioneer Hi - Bred International, Inc. Page 204 of 25 8

4114 Maize **Evaluation of Non -** **Ger minated Seed Viability**  Germinated seed were considered viable. N on -germinated seed classified as dead were considered non -viable and no further assessments of viability were conducted. Non -germinated seed classified as hard or imbibed were further evalu ated for viability using a tetrazolium chloride (TZ) test (AOSA, 2005 ). In non -germinated hard seed, potential dormancy can be indicated by seed viability; however, seed dormancy is not commonly observed in maize. The TZ test was conducted as follows: seeds were bisected longitudinally and placed in a separate 50 ml vial for each entry, then stained with 1% TZ solution (prepared by dissolving 10 g of 2,3,5 -triphenyltetrazolium chloride into one liter of water) for approximately two hours at 25 °C. A ny l iving cells were stained a reddish - pink color by the TZ solution, allowing identification of viable tissues. Seed with staining patterns indicative of 100% viable tissue in the essential seed structures (i.e., radical, embryo axis, plumule, and coleoptile s), were considered viable. Seed with staining patterns (or non -staining) indicative of less than 100% viable tissue in the essential seed structures were considered to be non -viable. **7.1.5. Statistical Analysis** Statistical analyses of germination data were conducted to evaluate the germination rate of seed derived from 4114 maize compared to the germination rate of seed derived from the control maize. Statistical analyses were conducted separately for each of the three germination tests (warm, cold, and diurnal) using SAS p software, Version 9.2 (SAS Institute, Inc. , Cary, NC). For a given germination test, when the minimum value of the expected count of total non -germinated seed derived from either 4114 maize or the control maize was less than five, a Fisher’s exact test was conducted to compare total germination frequencies. As ignificant difference was identified if the P-value < 0.05. SAS PROC FREQ was utilized to conduct Fisher’s exact tests. For a given germination test, if the expected count of total non -germinated seed derived from both 4114 maize and the control maize was five or more, then a Generalized Linear Mixed Model (GLMM) assuming binomial distribution with the “logit” link function was utilized to analyze the data. M aximum likeliho od method with Laplace approximation (SAS Institute Inc., 2008; Vonesh, 1996 ) was utilized to estimate and compare mean germination rates between 4114 maize and the control maize. As ignificant difference was identified if the P-value < 0.05. SAS PROC GL IMMIX was utilized to implement this type of analysis. Let yij represent the number of germinated seed in the j th replicate (each replicate contained a total of 50 seed) of the i th entry, j = 1, 2, ..., 8. yij ~ Binomal (n ij,  ij), where nij denotes the total

p Registered trademark of SAS Institute, Inc.Pioneer Hi - Bred International, Inc. Page 205 of 25 8

4114 Maize number of seed in the j th replicate of the i th entry, and  ij denotes the probability of a seed being germinated in the j th replicate of the i th entry. The “logit” link function was expressed as: logit ( ) log 1 ij ij ij ij

   

     

 

   Which transformed the parameter  ij on the unit scale into a parameter on the linear predictor scale, denoted as  ij. Logit link is default fo r binomial data. The GLMM on the linear predictor scale was:  ij =  i + r j(i) Where μ

i denotes the mean response for the i th entry (fixed effect) and r j(i) den otes the effect of the j th replicate within the i th entry (random effect nested within fixed effect). For this model, it was assumed that random effects r j(i) ~ N(0, 2R

) were independently and identically distributed of each other. Reported statistics for each germination test included descriptive statistics (total germination frequency, mean, and range of individual replicate germination rates) for 4114 maize and the control maize, and P-values for statistical comparisons in Section 4; Tables 24- 26 . When GLMM was utilized, estimated mean germination rate and standard error of the estimate were also provided (labeled as LS- Mean and SEM, respectively) for 4114 maize and the control maize Section 4; Tables 24- 26 . For each germination test, the range of germination rates for individual replicates across reference maize lines (labeled as Reference Range) is provided Section 4; Tables 24- 26 . **7.2. Agronomic Characteristics Evaluation** Agronomic characteristics were evaluated in 4114 maize using plants fro m two experiments in 2010 and analyzed as described below.Pioneer Hi - Bred International, Inc. Page 206 of 25 8

4114 Maize **7.2.1. Field Trial and Experimental Design** 4114 maize plants of the F1 *5 generation were grown in two experiments at a total of 17 sites in 2010, with 15 sites in the U.S. and two sites in Cana da. For those sites in the same location ( e.g. , 3 and 12), the field trials were conducted independently of each other. **Site Country**  **Location Experiment A: 2010 Field Trials**  1

U.S. Sheridan, IN 2 Deerfield, MI 3 Richland, IA 4 Rochelle, IL 5 Bagley, IA 6 Seymour, IL 7 York, NE 8 Carlyle, IL 9 Wyoming, IL 10 Atlantic, IA 11 Geneva, MN **Experiment B: 2010 Field Trials** 12 U.S. Richland, IA 13 Wyoming, IL 14 Geneva, MN 15 York, NE 16 Canada Branchton, ON 17 Thorndale, ON Ea ch field site utilized a randomized complete block design with 4114 maize and the control maize planted in two- row plots within four replicate blocks. Approximately 30 seeds were planted in each 25 ft (7.6 m) row. Row spacing was approximately 30 inches (76 cm) and every two- row plot was bordered on either side by one row of commercial maize of similar maturity. In addition, blocks were separated by an alley distance of at least 36 inches (0.9 m) and each site was surrounded by a minimum of four external border rows. Maintenance fertilizer, herbicides, and pesticides were applied uniformly to 4114 maize and the control maize at each site, as needed. In plots where grain yield was collected, plants were allowed to open - pollinate . To control experimental bias in this study, the following procedures were utilized: non -systematic selection of trial and plot areas within each site, randomization of maize entries within each block, and uniform maintenance across blocks in each field site.Pioneer Hi - Bred International, Inc. Page 207 of 25 8

4114 Maize **7.2.2. Agronomic Data Collection**  Agronomic characteristics were recorded for each maize line within all blocks at each site. Descriptions of maize growth stages are provided in Table 7.2 .

**Table 7.2 . Maize Growth Stage Descriptions**  **Growth Stage** **a Description**  **a** V2 The stage when the collar of the second leaf becomes

visible. V4 The stage when the collar of the fourth leaf becomes

visible. V6 The stage when the collar of the sixth leaf becomes visible. V7 The stage when the collar of the seventh leaf becomes

visib le. V9 The stage when the collar of the ninth leaf becomes

visible. R1 The stage when silks become visible. R4 The stage when the material within the kernel produces a

doughy consistency. R6 Typical grain harvest would occur. This stage is regarded as

physiological maturity. a Growth stages as described in Ritchie et al. , 2005

The following characteristics were recorded: **Early Population** The total number of plants emerged per plot was determined between V2 and V4 growth stages. **Seedling Vigor** Seedling vigor was visually estimated per plot when plants were between V2 and V4 growth stages using a 1 -9 scale, with 1 corresponding to poor vigor (short plants with small, thin leaves) and 9 corresponding to good vigor (tall plants with large, robust leaves).Pioneer Hi - Bred International, Inc. Page 208 of 25 8

4114 Maize **Time to Silking** The number of accumulated heat units q was calculated per plot from the planting date to the date when approximately 50% of plants had produced silks, except for two sites, which did not record 50% silking date for 4114 maize in Blocks 2, 3, and 4; and the control maize in Block 4. **Time to Pollen Shed** The number of accumulated heat units was calculated per plot from the planting date to the date when approximately 50% of plants had shed pollen , except for one site which did n ot record the dates to 50% pollen shed . **Pollen Viability** Pollen viability was only assessed at six of the 17 sites, with four sites in the U.S. and two sites in Canada. P ollen viability has been correlated to pollen shape and color ( Luna et al. , 2001 ) . Pollen viability per plot was assessed indirectly by evaluating pollen shape and color when approximately 50% of the plants had shed pollen. The percentage of pollen grains with collapsed walls r and intense yellow colors were recorded at four time poin ts (0 minutes, 30 minutes, 60 minutes, and 120 minutes). **Plant Height** Plant height was measured in centimeters from the soil surface to the tip of the tassel for ten plants per plot at the R4 growth stage, except for two field sites which measured plant height at the R4 or R5 growth stages. **Ear Height** Ear height was measured in centimeters from the soil surface to the base of the primary ear for ten plants per plot at the R4 growth stage, except for two field sites which measured ear height at the R4 or R5 growth stages. **Stalk Lodging** Stalk lodging severity was visually estimated per plot as the percentage of plants broken below the primary ear at the R6 growth stage.

q Heat units = ((maximum temperature + minimum temperature)/2) – 50 °F If the maximum temperature was greater than 86 °F then 86 °F was used. If the minimum temperature was less than 50 °F, 50 °F was used. Heat units were calculated for each growing day and summed to give a total accumulated heat unit value. If a daily heat unit was negative, 0 (zero) was assigned. r Percentage of collapsed pollen compared to spherical pollen. s Percentage of pollen with intense yellow color compared to pollen white in color.Pioneer Hi - Bred International, Inc. Page 209 of 25 8

4114 Maize **Root Lodging** Root lodging severity was visually estimated per plot as the pe rcentage of plants leaning approximately 30 degrees or more in the first 2 feet (0.6 m) above the soil surface at the R6 growth stage. **Final Population** The total number of remaining plants per plot was recorded at the R6 growth stage. P reviously sampl ed plants were included in the final population total. **Stay Green** Overall plant health was visually estimated per plot at the R6 growth stage using a 1 to 9 scale, with 1 corresponding to no visible green tissue remaining, 5 corresponding to approximately 50% green tissue remaining, and 9 corresponding to approximately ≥90% green tissue remaining. **Disease Incidence** Foliar disease incidence was visually estimated per plot at the R6 growth stage using a 1 -9 scale, with 1 corresponding to poor disease resistance (high infection) and 9 corresponding to excellent disease resistance (low infection). **Insect Damage**  Insect damage was visually estimated per plot at the R6 growth stage using a 1-9 scale, with 1 corresponding to poor insect resistance (high da mage) and 9 corresponding to excellent insect resistance (low damage). **Yield** Yield was only assessed at 11 of the 17 sites in the U.S. during the 2010 growing season. Maize grain was collected from each plot at typical harvest maturity (R6 growth stage). Yield was determined based on the weight (kg) and moisture content (%) of the grain. The following formulas were used to determine yield: Grain weights from each plot were adjusted to 0% moisture content. Grain (dry weight) (kg) = Grain (fresh weight) (kg) – (Grain (fresh weight) (kg) * % actual moisture) Example : Grain (fresh weight) = 9.66 kg, Actual Moisture = 21.5% Grain (dry weight) = 9.66 kg – (9.66 kg * 21.5%) = 7.58 kgPioneer Hi - Bred International, Inc. Page 210 of 25 8

4114 Maize The grain dry weights were then adjusted to 15.5% moisture content. Grain (@ 15.5% moisture) (kg) = Grain (dry weight) (kg) / (1 – 15.5% moisture) Example : Grain (dry weight) = 7.58 kg Grain (@ 15.5% moisture) = 7.58 kg / (1 - 15.5%) = 8.98 kg The grain weights at 15.5% moisture were then converted to bu/A.

Yield (bu/A) = Grain (@ 15.5% moisture) (kg) * 43560 ft 2/A

Plot Area (ft 2) * 25.4 kg/bu Example : Grain (@ 15.5% moisture) = 8.98 kg, Plot Area = 125 ft 2

Yield (bu/A) = 8.98 kg * 43560 ft 2/A = 123 bu/A

125 ft 2 * 25.4 kg/bu **7.2.3. Statistical Methods** Statistical analyses were conducted separately for two studies to evaluate and compare agronomic characteristics of 4114 maize and the control maize using SAS software, Version 9.2 (SAS Institute, Inc. Cary, NC). **7.2.4. Processing of Data Plant and Ear Height**  Pla nt height and ear height were measured on ten plants per plot. Plot average (average of ten plants) was treated as the response. **Transformation** Pollen viability (color and shape) at 0, 30, and 60 minutes, were percentage data. They were converted to proportion data. E arly population and final population were binomial count data. They were also converted to proportion data according to: y= number of stand count / max (target seedling rate, which is 60. M aximum stand count across plots which could b e more or less than 60)Pioneer Hi - Bred International, Inc. Page 211 of 25 8

4114 Maize Proportion data was subjected to the arcsine square- root transformation, arcsin( ) z y  where y denotes proportion value, and z denotes transformed value. The transformed data met statistical assumptions of mixed model analyses, name ly normality and constant variance assumptions. The statistical results were then transformed back to the original scale. **7.2.5. Statistical Analyses** In one of the two experiments, greater than 80% of data values for root lodging and stalk lodging we re at a uniform value (i.e., 0% lodging); therefore, the analysis was not conducted for these two characteristics. Mixed Model Analysis For a given agronomic characteristic, data were analyzed using the following linear mixed model: 

### yijk = 

### μi + 

### l

j + 

### r k(j) + 

### (μl) ij + 

### εijk Model 1 

### l j ~ iid N(0, σ

2Site ), 

### r k(j) ~ iid N(0,σ

2Rep ), 

### (μl) ij ~ iid N(0, σ 2Ent×Site ), and 

### εijk ~ iid N(0, σ 2Error ) where μ

i denotes the mean of the i th entry (fixed effect), l j denotes the effect of the j th site (random effect), r

k(j) denotes the effect of the kth block within the j th site (random effect), (μl)ij denotes the interaction between the entries and sites (random effect) and ε ijk denotes the effect of the plot assigned the i th entry in the kth block of the j th site (random effect or residual). Notation ~ iid N(0, σ 2a ) indicates random variables that are identically independently distributed ( iid) as normal with zero mean and variance σ 2a. The residual maximum likelihood (REML) estimation p rocedure was utilized to generate estimates of variance components and entry means. The estimated means are known as least squares means (LS- means). The statistical comparison was conducted by testing for difference in LS- means for contrasts of interest. The approximated degrees of freedom for statistical test were derived by Kenward - Roger (KR) meth od ( Kenward and Roger, 1997 ). SAS PROC MIXED was utilized to fit Model 1 and generate LS- means and 95% confidence intervals. The same procedure also provi ded statistical comparisons (P-values). By default, the variance components in Model 1 are all constrained to be non - negative. W hen the estimated value of σ

2 Ent×Site is zero, the KR method pools degrees of freedom for the interaction term with the degrees of freedom for residuals. Consequently, the degrees of freedom for the statistical test could be drastically larger than what was expected under the original experimental design.Pioneer Hi - Bred International, Inc. Page 212 of 25 8

4114 Maize In order to make the degrees of freedom remain stable across all agronomi c characteristics, effect 

### (μl)ij in Model 1 was combined with 

### ε ijk and the compound symmetry (CS) structure was used to model the corresponding residual variance structure. This approach allows σ 2 Ent×Site to take negative values without affecting the degrees of freedo m ( Littell et al. , 2006 ). However, expanding the parameter space sometimes can cause the model to fail to converge. I n one of the two studies, for time to silking, the model with CS structure failed to converge and parameters were estimated using the default setting. Generalized Cochran - Mantel - Haenszel (CMH) Test In one agronomic study the characteristics insect damage, stalk lodging, and root lodging had very discrete values. I n the other agronomic study, the characteristics disease incidence, insect damage, seedling vigor, and pollen viability (color and shape) at 120 minutes, had very discrete values. This violated the normality assumption in Model 1; hence, it was not feasible to apply the mixed model approach on these characteristics. I nstead, comparisons of entries were based on the generalized CMH test. The generalized CMH test was developed specifically for stratified nominal - by-ordinal contingency tables (Agresti, 2002; Koch et al. , 1990 ). It compares entries (a nominal variable) based on their values (recorded on an ordinal scale) while controlling for location (the stratifying variable). The test’s P-values can be directly interpreted as testing for the difference between the arithmetic means for each entry, because the data values were used as the scores in the generalized CMH test. Note that when applying the generalized CMH test, the block- nested -within - location effect and the location - by-entry effect in the design were ignored. This does not present any issues because for these agronomic characteristics, the block- nested -within - location effect and the location - by-entry effect are often negligible compared with the location effect. SAS PROC FREQ was used to perform the generalized CMH test. **7.2.6. FDR (False Discovery Rate) Adjustment** The F DR method of Benjamini and Hochberg ( Benjamini and Hochberg, 1995; Westfall et al. , 1999 ) was applied as a post- hoc procedure to account for multiple comparisons due to multiple agronomic characteristics and P-values were adjusted accordingly. The FDR adj ustment was conducted for results. A significant difference was identified if an adjusted P-value <0.05. SAS PROC MULTTEST was utilized to provide adjusted P-values. **7.2.7. Interpretation of Statistical Results** Where a statistically sign ificant difference (adjusted P-value <0.05) was identified for a given agronomic characteristic, the respective range of individual values was compared to a tolerancePioneer Hi - Bred International, Inc. Page 213 of 25 8

4114 Maize interval. Tolerance intervals containing 99% of the values for corresponding characteristics of the co nventional maize population with 95% confidence level ( Graybill, 1976 ) were derived from data collected under multiple historical studies. In those studies, a total of eight non modified commercial maize lines were grown in a total of twelve sites in North America and a total of six non - modified commercial maize lines were grown in a total of four sites in Chile under normal agronomic practices. The selected maize lines represent a non - modified maize population with a history of safe use, and the selected environments (site and year combinations) represent maize growth under a wide range of environmental conditions (i.e., soil type, temperature, precipitation, and irrigation) and maize maturity group zones similar to the sites used in these 4114 maize agron omic studies. **7.2.8. Reported Statistics** For each agronomic characteristic that was statistically analyzed using mixed model analysis, entry LS- mean (back-transformed, if needed), range, and 95% confidence interval of the mean (labeled as Mean, Range, a nd CI, respectively) are provided in Section 4; Tables 30 and 31 . Both the non -adjusted P-values and the FDR-adjusted P-values (labeled as P-Value and Adjusted P-Value, respectively) are provided for statistical comparisons. For each agronomic characteri stic, a tolerance interval, if available, is provided for the results in Section 4; Tables 30 and 31 . In the tables (Section 4; Tables 30 and 31 ), d escriptive statistics (arithmetic means and ranges) are reported for characteristics that were not statisti cally analyzed using mixed model analysis.Pioneer Hi - Bred International, Inc. Page 214 of 25 8

4114 Maize **Appendix 8. Field Insect and Disease Observations**  4114 maize has been field tested in the U.S. and Puerto Rico since 2006, as authorized by USDAAPHIS permits and notifications (Appendix 1). For each trial, a survey of the naturally occurring insects and diseases and any unexpected differences in the response of 4114 maize as compared to the control line (near- isoline and/or conventional maize lines) were recorded by experienced plant breeders and field staff at least every four weeks. The plant breeders and field staff were familiar with plant pathology and entomology and also recorded the severity of any insect or disease in the field . These observations provide a means to determine if 4114 maize will res pond differently from conventional maize lines to insects or diseases in the environment. A summary of the naturally-occurring insects present in the fields and any unexpected differences seen between 4114 maize and control lines is presented in Table 8 .1. A summary of diseases present in the fields is presented in Table 8.2. The following scale was used to evaluate 4114 maize and control lines (Tables 8.1 and 8.2; “Range of Severity in 4114 Maize”):

 Mild – very little disease or insect injury (<10% ) visible  Moderate – noticeable plant tissue damage (10% to 30%)  Severe – significant plant tissue damage (>30%) In every case, the 4114 maize did not exhibit any unexpected responses to naturally-occurring insects or diseases as compared to control line .Pioneer Hi - Bred International, Inc. Page 215 of 25 8

4114 Maize **Table 8. 1.**  **Observations of Insects Present and Comparison B** **etween 4114 Maize and Control Year of**  **Planting Permit Number**  **State County**  **Insect Overall Severity in**  **Field Unexpected** 

**Difference in**  **Comparison ?** 

2006 06-019-03R PR Guayama Fall armyworm (Spodoptera frugiperda) mild to moderate no Corn earworm ( Helicoverpa zea) mild no 06-019-04R HI Kauai Corn earworm ( Helicoverpa zea) mild to severe no Rose beetle (Adoretus sinicus) mild no Leafhopper (Cicadellidae) mild to moderate no Thrips ( Frankliniella spp.) mild no Spider mites (Tetranychidae) mild no 06-019-03R PR Salinas Corn earworm ( Helicoverpa zea) mild no Fall armyworm (Spodoptera frugiperda) mild, moderate no

Aphids (Aphididae) mild no

2007 07-040- 101rm CA Yolo Painted ladies ( Vanessa spp.) mild no HI Kauai Corn earworm ( Helicoverpa zea) mild no Aphids (Aphididae) mild no Leafhopper (Cicadellidae) mild no Rose beetle (Adoretus sinicus) mild no Fall armyworm (Spodoptera frugiperda) mild no

IA Linn Corn rootworm ( Diabrotica spp.) mild to moderate no Japanese beetle ( Popillia japonica) mild no European corn borer ( Ostrinia nubilalis) moderate no Polk Corn rootworm ( Diabrotica spp.) mild no

European corn borer ( Ostrinia nubilalis) mild no Corn earworm ( Helicoverpa zea) mild no Bean leaf beetles ( Cerotoma trifurcata) mild no IL Bureau European corn borer (Ostrinia nubilalis) mild no Corn rootworm ( Diabrotica spp.) mild no Champaign Corn rootworm ( Diabrotica spp.) mild no

Grasshoppers (Orthoptera) mild no McDonough Corn rootworm ( Diabrotica spp.) mild to moderate noPioneer Hi - Bred International, Inc. Page 216 of 25 8

4114 Maize **Table 8. 1.**  **Observations of Insects Present and**  **Comparison B etween 4114 Maize and Control (continued)**  **Year of Planting**  **Permit**

**Number State**  **County Insect**  **Overall Severity in Field**  **Unexpected**

**Difference in Comparison?** 

2007 07-040- 101rm IL McDonough European corn borer ( Ostrinia nubilalis) mild to moderate no Ogle Corn rootworm ( Diabrotica spp.) mild no

Japanese beetle ( Popillia japonica) mild no IN Tipton Corn rootworm ( Diabrotica spp.) mild no MN Blue Earth Aphids (Aphididae) mild no Corn rootworm ( Diabrotica spp.) mild no PR Juana Diaz Corn earworm ( Helicoverpa zea) mild no

Fall armyworm (Spodoptera frugiperda) mild to moderate no Salinas Corn earworm ( Helicoverpa zea) mild no

Fall armyworm (Spodoptera frugiperda) mild to moderate no WI Rock Corn rootworm ( Diabrotica spp.) moderate no Aphids (Aphididae) mild no

NE York European corn borer ( Ostrinia nubilalis) mild no Lady beetles (Coccinellidae) mild no Aphids (Aphididae) mild to moderate no Corn rootworm ( Diabrotica spp.) mild to moderate no Spider mites (Tetranychidae) mild no Lacewings (Neuroptera) mild no

2008 07-040- 101rm PR Salinas Corn earworm ( Helicoverpa zea) mild no Fall armyworm (Spodoptera frugiperda) mild to moderate no Corn planthoppers (Delphacidae) mild no HI Kauai Leafhopper (Cicadellidae) mild no Spider mites (Tetranychidae) mild to moderate no Corn earworm ( Helicoverpa zea) moderate no 08-014- 111n CA Yolo Painted ladies ( Vanessa spp.) mild no

IN Gibson Japanese beetle ( Popillia japonica) mild no Tipton Corn rootworm ( Diabrotica spp.) mild no

European corn borer ( Ostrinia nubilalis) mild noPioneer Hi - Bred International, Inc. Page 217 of 25 8

4114 Maize **Table 8. 1.**  **Observations of Insects Present and Comparison B** **etween 4114 Maize and Control (continued) Year of**  **Planting Permit** 

**Number State**  **County Insect**  **Overall Severity in Field**  **Unexpected**

**Difference in Comparison?** 

2008 08-014- 111n IA Polk European corn borer (Ostrinia nubilalis) mild no Japanese beetle ( Popillia japonica) mild no Linn Japanese beetle ( Popillia japonica) mild to moderate no

European corn borer ( Ostrinia nubilalis) mild no

NE Nance Euro pean corn borer ( Ostrinia nubilalis) mild no Lady beetles (Coccinellidae) mild no Corn rootworm ( Diabrotica spp.) mild no Aphids (Aphididae) mild no Grasshoppers (Orthoptera) mild no Stink bug (Pentatomidae) mild no

York Europe an corn borer ( Ostrinia nubilalis) mild to moderate no

Lady beetles (Coccinellidae) mild to moderate no Stink bug (Pentatomidae) mild no Beetles (Coleoptera) mild to moderate no Grasshoppers (Orthoptera) mild to moderate no Aphids (Aphididae) mild to moderate no Corn rootworm ( Diabrotica spp.) mild to moderate no Lacewings (Neuroptera) mild no MN Blue Earth European corn borer (Ostrinia nubilalis) mild to moderate no

Corn rootworm ( Diabrotica spp.) moderate no

P R Juana Diaz Fall armyworm (Spodoptera frugiperda) mild to moderate no

Corn earworm ( Helicoverpa zea) mild to moderate no Aphids (Aphididae) severe no Salinas Fall armyworm (Spodoptera frugiperda) mild to severe no

Corn earworm ( Helicoverpa zea) mild to severe no Cornsilk fly ( Euxesta stigmatias) mild no Aphids (Aphididae) mild no Grasshoppers (Orthoptera) mild to moderate noPioneer Hi - Bred International, Inc. Page 218 of 25 8

4114 Maize **Table 8. 1.**  **Observations of Insects Present and Comparison B** **etween 4114 Maize and Control (continu ed)**  **Year of Planting**  **Permit**

**Number State**  **County Insect**  **Overall Severity in Field**  **Unexpected**

**Difference in Comparison?** 

2008 08-014- 111n IL Bureau Corn rootworm ( Diabrotica spp.) mild to moderate no Champaign Corn rootworm ( Diabrotica spp.) mild no McDonough Japanese beetle ( Popillia japonica) mild no

Corn rootworm ( Diabrotica spp.) mild no Ogle Japanese beetle ( Popillia japonica) severe no

Corn rootworm ( Diabrotica spp.) severe no WI Rock Japanese beetle ( Popillia japonica) mild no Corn rootworm ( Diabrotica spp.) mild no Aphids (Aphididae) mild no Grasshoppers (Orthoptera) mild no

08-014- 131n HI Kauai Leafhopper (Cicadellidae) mild no Corn earworm ( Helicoverpa zea) mild to moderate no Spider mites (Tetranychidae) mild to moderate no Rose beetle (Adoretus sinicus) mild no Thrips ( Frankliniella spp.) mild no Leafhopper (Cicadellidae) mild no

08-095 - 105n OK Caddo Fall armyworm (Spodoptera frugiperda) mild to moderate no Green June beetles ( Cotinus nitida) mild no Corn earworm ( Helicoverpa zea) moderate no

IL Clinton Corn earworm ( Helicoverpa zea) mild no

Fall armyworm (Spodoptera frugiperda) mild no Japanese beetle ( Popillia japonica) mild no Stark Corn rootworm ( Diabrotica spp.) mild no

Aphids (Aphididae) mild no European corn borer ( Ostrinia nubilalis) mild no IA Jefferson European corn borer ( Ostrinia nubilalis) mild no Corn rootworm ( Diabrotica spp.) mild no Corn earworm ( Helicoverpa zea) mil d noPioneer Hi - Bred International, Inc. Page 219 of 25 8

4114 Maize **Table 8. 1.**  **Observations of Insects Present and Comparison B** **etween 4114 Maize and Control (continued) Year of**  **Planting Permit** 

**Number State**  **County Insect**  **Overall Severity in Field**  **Unexpected**

**Difference in Comparison?** 

2008 08-095 - 105n MN Freeborn Aph ids (Aphididae) mild no European corn borer ( Ostrinia nubilalis) mild no Corn rootworm ( Diabrotica spp.) mild no NE York Corn rootworm ( Diabrotica spp.) mild no Aphids (Aphididae) mild no Grasshoppers (Orthoptera) mild no Corn e arworm ( Helicoverpa zea) mild to moderate no TX Wharton Corn earworm ( Helicoverpa zea) mild no Corn stalk borers (Pyralidae) mild no Fall armyworm (Spodoptera frugiperda) mild no

2009 08-014- 111n PR Salinas Fleahoppers (Miridae) moderate to severe no Corn earworm ( Helicoverpa zea) mild to severe no Corn sap beetle ( Carpophilus spp.) severe no Fall armyworm (Spodoptera frugiperda) moderate no

09-013 - 108n IL Bureau Corn rootworm ( Diabrotica spp.) mild no Japanese beetle ( Popillia japonica) mild no Fulton Corn rootworm ( Diabrotica spp.) mild no Aphids (Aphididae) mild no Grasshoppers (Orthoptera) mild no McDonough European corn borer ( Ostrinia nubilalis) mild no

Corn rootworm ( Diabrotica spp.) mild no IN Gibson Japanese beetle ( Popillia japonica) mild no Tipton Japanese beetle ( Popillia japonica) mild no

Corn rootworm ( Diabrotica spp.) mild no European corn borer (Ostrinia nubilalis) mild no IA Dallas Bean leaf beetles ( Cerotoma trifurcata) mild no

European corn borer ( Ostrinia nubilalis) mild no Corn rootworm ( Diabrotica spp.) mild noPioneer Hi - Bred International, Inc. Page 220 of 25 8

4114 Maize **Table 8. 1.**  **Observations of Insects Present and Comparison B** **etween 4114 Maize and Control (continued) Year of**  **Planting Permit** 

**Number State**  **C ounty**  **Insect Overall Severity in**  **Field Unexpected** 

**Difference in Comparison?** 

2009 09-013 - 108n IA Dallas Aphids (Aphididae) mild no Lady beetles (Coccinellidae) mild no Stink bug (Pentatomidae) mild no Kossuth Corn rootworm ( Diabrotica spp.) m ild no Aphids (Aphididae) mild no Linn Corn earworm ( Helicoverpa zea) mild no

Japanese beetle ( Popillia japonica) mild no Corn earworm ( Helicoverpa zea) mild no Polk Beetles (Coleoptera) mild no

Aphids (Aphididae) mild no

NE York European corn borer ( Ostrinia nubilalis) mild no

Corn blotch leafminers (Agromyza parvicornis) mild no Western bean cutworm ( Richia albicosta) mild no Corn earworm ( Helicoverpa zea) mild no Corn rootworm ( Diabrotica spp.) mild no Aphids (Aphididae) mild no

09-035 - 109n AR Crittenden Corn earworm ( Helicoverpa zea) mild to severe no European corn borer ( Ostrinia nubilalis) severe no Fall armyworm (Spodoptera frugiperda) severe no CO Weld Spider mites (Tetranychidae) mild no GA Grady Fall armyworm (Spodoptera frugiperda) mild to moderate no Aphids (Aphididae) moderate no Spider mites (Tetranychidae) moderate no HI Kauai Leafhopper (Cicadellidae) mild no Spider mites (Tetranychidae) mild no Rose beetle (Adoretus sinicus) mild no Aphids (Aphididae) mild no Cornsilk fly ( Euxesta stigmatias) mild noPioneer Hi - Bred International, Inc. Page 221 of 25 8

4114 Maize **Table 8. 1.**  **Observations of Insects Present and Comparison B** **etween 4114 Maize and Control (continued) Year of**  **Planting Permit** 

**Number State**  **County Insect**  **Overall Severity in Field**  **Unexpected**

**Difference in Comparison?** 

2009 09-035 - 109n HI Kauai Corn sap beetle ( Carpophilus spp.) mild no Beet armyworm (Spodoptera exigua) mild no Rose beetle (Adoretus sinicus) mild no Corn earworm ( Helicoverpa zea) mild no MN Blue Earth European corn borer (Ostrinia nubilalis) mild no

Corn rootworm ( Diabrotica spp.) mild no Aphids (Aphididae) mild no

PR Guayama Fall armyworm (Spodoptera frugiperda) mild to severe no Corn earworm ( Helicoverpa zea) mild to moderate no Aphids (Aphididae) moderate to severe no Corn sap beetle ( Carpophilus spp.) mild no

Juana Diaz Fall armyworm (Spodoptera frugiperda) mild to severe no

Corn earworm ( Helicoverpa zea) mild to moderate no Corn sap beetle ( Carpophilus spp.) mild to severe no Cornsilk fly ( Euxesta stigmatias) mild to severe no Aphids (Aphididae) mild to severe no Spider mites (Tetranychidae) moderate no Corn planthoppers (Delphacidae) mild no Leafhopper (Cicadellidae) mild no Fleahoppers (Miridae) moderate no Whiteflies (Aleyrodidae) moderate no Salinas Corn earworm ( Helicoverpa zea) mild to severe no

Fall armyworm (Spodoptera frugiperda) mild to severe no Fleahoppers (Miridae) mild to severe no Cornsilk fly ( Euxesta stigmatias) moderate no TN Obion European corn borer (Ostrinia nubilalis) mild no

Southwestern corn borer ( Diatraea grandiosella) mild no

Grasshoppers (Orthoptera) mild noPioneer Hi - Bred International, Inc. Page 222 of 25 8

4114 Maize **Table 8. 1.**  **Obs ervations of Insects Present and**  **Comparison B etween 4114 Maize and Control (continued)**  **Year of Planting**  **Permit**

**Number State**  **County Insect**  **Overall Severity in Field**  **Unexpected**

**Difference in Comparison?** 

2009 09-035 - 109n TN Obion Fall armyworm (Spodoptera f rugiperda) mild no Corn earworm ( Helicoverpa zea) mild no WI Rock Aphids (Aphididae) mild no Japanese beetle ( Popillia japonica) mild no Corn rootworm ( Diabrotica spp.) mild to moderate no

09-264- 102n HI Kauai Corn earworm ( Helicoverpa zea) mild no Beetles (Coleoptera) mild no Corn flea beetle ( Chaetocnema pulicaria) mild no Whiteflies (Aleyrodidae) mild no Leafhopper (Cicadellidae) mild no Lacewings (Neuroptera) mild no Wasps (Hymenoptera) mild no Spid er mites (Tetranychidae) mild no Aphids (Aphididae) mild no Lady beetles (Coccinellidae) mild no

PR Juana Diaz Fall armyworm (Spodoptera frugiperda) mild to moderate no

Aphids (Aphididae) mild to moderate no Spider mites (Tetranychid ae) mild no Corn earworm ( Helicoverpa zea) mild no Corn sap beetle ( Carpophilus spp.) mild no Cornsilk fly ( Euxesta stigmatias) mild no

Salinas Fall armyworm (Spodoptera frugiperda) mild to severe no

Grasshoppers (Orthoptera) mild n o Corn planthoppers (Delphacidae) mild to moderate no Corn earworm ( Helicoverpa zea) mild to moderate no Corn sap beetle ( Carpophilus spp.) mild to severe no Cornsilk fly ( Euxesta stigmatias) mild to moderate no Spider mites (Tetra nychidae) mild to severe noPioneer Hi - Bred International, Inc. Page 223 of 25 8

4114 Maize **Table 8.1. Observations of Insects Present and**  **Comparison B etween 4114 Maize and Control (continued)**  **Year of Planting**  **Permit**

**Number State**  **County Insect**  **Overall Severity in Field**  **Unexpected**

**Difference in Comparison?**  2009 09-2 64- 102n PR Salinas Aphids (Aphididae) mild to severe no

09-016- 103n IL Stark Aphids (Aphididae) mild no European corn borer ( Ostrinia nubilalis) mild no NE York Black cutworms (Agrotis ipsilon ) mild no European corn borer ( Ostrinia nubilalis) mild no

2010 09-264- 102n HI Kauai Thrips ( Frankliniella spp.) mild no Leafhopper (Cicadellidae) mild no Fall armyworm (Spodoptera frugiperda) mild no Lacewings (Neuroptera) mild no Lady beetles (Coccinellidae) mild no Corn earwo rm ( Helicoverpa zea) mild no Spider mites (Tetranychidae) mild no Aphids (Aphididae) mild no Wasps (Hymenoptera) mild no

PR Salinas Fall armyworm (Spodoptera frugiperda) mild to moderate no Spider mites (Tetranychidae) mild to moderate no Leafhopper (Cicadellidae) mild to moderate no Corn sap beetle ( Carpophilus spp.) mild to moderate no Cornsilk fly ( Euxesta stigmatias) moderate no Corn earworm ( Helicoverpa zea) mild to moderate no Aphids (Aphididae) mild to moderate no Corn planthoppers (Delphacidae) moderate no 10-050- 101n NE Keith Corn rootworm ( Diabrotica spp.) mild no Grasshoppers (Orthoptera) mild no Corn earworm ( Helicoverpa zea) mild no 10-052 - 101n AR Crittenden Fall armyworm (Spodoptera frugiperda) moderate to severe no Corn earworm ( Helicoverpa zea) moderate to severe no Southwestern corn borer ( Diatraea grandiosella) moderate to severe noPioneer Hi - Bred International, Inc. Page 224 of 25 8

4114 Maize **Table 8. 1.**  **Observations of Insects Present and Comparison B** **etween 4114 Maize and C ontrol (continued)**  **Year of Planting**  **Permit**

**Number State**  **County Insect**  **Overall Severity in Field**  **Unexpected**

**Difference in Comparison?** 

2010 10-052 - 101n AR Crittenden European corn borer ( Ostrinia nubilalis) moderate to severe no CA Yolo Spider mites (Tetranychidae) mild to moderate no CO Philips Grasshoppers (Orthoptera) mild no Corn rootworm ( Diabrotica spp.) mild no Corn earworm ( Helicoverpa zea) mild no Weld Spider mites (Tetranychidae) mild no GA Grady Aphids (Aphididae) mild no

Fall armyworm (Spodoptera frugiperda) mild no Spider mites (Tetranychidae) mild no Aphids (Aphididae) mild no

HI Kauai Aphids (Aphididae) mild no Corn earworm ( Helicoverpa zea) mild no Spider mites (Tetranychidae) mild no Fa ll armyworm (Spodoptera frugiperda) mild no Beet armyworm (Spodoptera exigua) mild no Lacewings (Neuroptera) mild no Thrips ( Frankliniella spp.) mild no Leafhopper (Cicadellidae) mild no Lady beetles (Coccinellidae) mild no

IL B ureau Japanese beetle ( Popillia japonica) mild no Champaign Corn rootworm ( Diabrotica spp.) mild no

European corn borer (Ostrinia nubilalis) mild no Fulton Grasshoppers (Orthoptera) mild no

Lady beetles (Coccinellidae) mild no Corn rootworm ( Diabrotica spp.) mild no Stink bugs (Pentatomidae) mild no Aphids (Aphididae) mild noPioneer Hi - Bred International, Inc. Page 225 of 25 8

4114 Maize **Table 8. 1.**  **Observations of Insects Present and Comparison B** **etween 4114 Maize and Control (continued) Year of**  **Planting Permit** 

**Number State**  **County In**  **sect Overall Severity in**  **Field Unexpected** 

**Difference in Comparison?** 

2010 10-052 - 101n IL La Salle Japanese beetle ( Popillia japonica) mild no McDonough Lady beetles (Coccinellidae) mild no

Grasshoppers (Orthoptera) mild no Corn rootworm ( Diabrotica spp.) mild no Ogle Corn rootworm ( Diabrotica spp.) mild to severe no

Japanese beetle ( Popillia japonica) severe no IN Gibson Japanese beetle ( Popillia japonica) mild no Corn rootworm ( Diabrotica spp.) mild no Tipton Corn rootworm ( Diabrotica spp.) mild no

IA Bremer Corn earworm ( Helicoverpa zea) mild no Corn earworm ( Helicoverpa zea) mild no Dallas Bean leaf beetles ( Cerotoma trifurcata) mild no

Corn rootworm ( Diabrotica spp.) mild no European corn borer (Ostrinia nubilalis) mild no Corn rootworm ( Diabrotica spp.) mild no Lady beetles (Coccinellidae) mild no Fall armyworm (Spodoptera frugiperda) mild no Delaware Corn earworm ( Helicoverpa zea) mild no

Japanese beetle ( Popillia japonica) mild no Kossuth Lady beetles (Coccinellidae) mild no

Corn rootworm ( Diabrotica spp.) mild no Lady beetles (Coccinellidae) mild no Linn Corn earworm ( Helicoverpa zea) mild no

Japanese beetle ( Popillia japonica) mild no Madison Be an leaf beetles ( Cerotoma trifurcata) mild no

Corn rootworm ( Diabrotica spp.) mild no European corn borer (Ostrinia nubilalis) mild no Corn rootworm ( Diabrotica spp.) mild noPioneer Hi - Bred International, Inc. Page 226 of 25 8

4114 Maize **Table 8. 1.**  **Observations of Insects Present and Comparison B** **etwee n 4114 Maize and Control (continued)**  **Year of Planting**  **Permit**

**Number State**  **County Insect**  **Overall Severity in Field**  **Unexpected**

**Difference in Comparison?** 

2010 10-052 - 101n IA Madison Lady beetles (Coccinellidae) mild no Fall armyworm (Spodoptera frugiperda) mild no Polk Corn earworm ( Helicoverpa zea) mild no

Japanese beetle ( Popillia japonica) mild no Beetles (Coleoptera) mild no Corn earworm ( Helicoverpa zea) mild no Van Buren Corn rootworm ( Diabrotica spp.) mild no KS Finney F lea beetle (Galerucinae) mild no Cucumber beetle (Chrysomelidae) mild no MI Gratiot Western bean cutworm ( Richia albicosta) mild no Corn rootworm ( Diabrotica spp.) mild N o Lady beetles (Coccinellidae) mild N o MN Blue Earth European cor n borer (Ostrinia nubilalis) mild N o Corn rootworm ( Diabrotica spp.) mild N o MO Scott European corn borer (Ostrinia nubilalis) mild N o Fall armyworm (Spodoptera frugiperda) mild N o Grasshoppers (Orthoptera) mild N o NE York Corn rootworm ( Diabrotica spp.) mild N o European corn borer (Ostrinia nubilalis) mild N o Western bean cutworm ( Loxagrotis albicosta) mild N o PA Lebanon European corn borer (Ostrinia nubilalis) mild N o Fall armyworm (Spodoptera frugiperda) mild N o

PR Guayama Corn sap beetle ( Carpophilus spp.) mild to severe N o Cornsilk fly ( Euxesta stigmatias) mild to severe N o Fall armyworm (Spodoptera frugiperda) mild to severe N o Grasshoppers (Orthoptera) mild N o Corn planthoppers (Delphacidae ) mild to moderate N o Corn earworm ( Helicoverpa zea) mild to severe N oPioneer Hi - Bred International, Inc. Page 227 of 25 8

4114 Maize **Table 8.1. Observations of Insects Present and**  **Comparison B etween 4114 Maize and Control (continued)**  **Year of Planting**  **Permit**

**Number State**  **County Insect**  **Overall Severity in Fiel**  **d Unexpected** 

**Difference in Comparison?** 

2010 10-052 - 101n PR Guayama Aphids (Aphididae) mild to severe no Fall armyworm (Spodoptera frugiperda) mild to severe no Corn sap beetle ( Carpophilus spp.) mild to severe no Spider mites (Tetranychidae ) mild to severe no Corn flea beetle ( Chaetocnema pulicaria) mild no

Juana Diaz Cornsilk fly ( Euxesta stigmatias) mild to severe no Aphids (Aphididae) mild to severe no Spider mites ( Tetranychidae) mild to moderate no Corn planthoppers (Delphacidae) mild to moderate no Spider mites ( Tetranychidae) moderate to severe no Fall armyworm (Spodoptera frugiperda) mild to moderate no Grasshoppers (Orthoptera) mild to moderate no Corn earworm ( Helicoverpa zea) mild to mode rate no Corn sap beetle ( Carpophilus spp.) mild to severe no Thrips ( Frankliniella spp.) mild to moderate no Salinas Fall armyworm (Spodoptera frugiperda) moderate no Aphids (Aphididae) mild to moderate no Corn planthoppers (Delphac idae) moderate no Cornsilk fly ( Euxesta stigmatias) mild to moderate no Corn sap beetle ( Carpophilus spp.) mild to moderate no Santa Isabel Fall armyworm (Spodoptera frugiperda) mild no

Corn planthoppers (Delphacidae) mild no Corn sap beetle ( Carpophilus spp.) moderate no Spider mites (Tetranychidae) moderate no TN Obion Fall armyworm (Spodoptera frugiperda) mild no European corn borer (Ostrinia nubilalis) mild no Southwestern corn borer ( Diatraea grandiosella) mild no Corn earworm ( Helicoverpa zea) mild noPioneer Hi - Bred International, Inc. Page 228 of 25 8

4114 Maize **Table 8. 1.**  **Observations of Insects Present and Comparison B** **etween 4114 Maize and Control (continued) Year of**  **Planting Permit** 

**Number State**  **County Insect**  **Overall Severity in Field**  **Unexpected**

**Difference in C** **omparison?**

2010 10-052 - 101n TN Obion Grasshoppers (Orthoptera) mild no WI Rock Corn rootworm ( Diabrotica spp.) mild no Grasshoppers (Orthoptera) mild no Japanese beetle ( Popillia japonica) mild no 10- 118- 105n IL Gallatin Japanese beetle ( Popillia japonica) mild no

10-281 - 101n HI Kauai Leafhopper (Cicadellidae) mild no Spider mites (Tetranychidae) mild no Corn earworm ( Helicoverpa zea) mild no

PR Salinas Fall armyworm (Spodoptera frugiperda) mild to moderate no Corn planth oppers (Delphacidae) mild to moderate no Grasshoppers (Orthoptera) mild no Cornsilk fly ( Euxesta stigmatias) mild to moderate no Corn sap beetle ( Carpophilus spp.) mild to moderate no Aphids (Aphididae) mild to moderate no Corn earworm ( Helicoverpa zea) mild to moderate no Spider mites (Tetranychidae) mild to moderate no

10-284- 101n HI Kauai Corn earworm ( Helicoverpa zea) mild no Spider mites (Tetranychidae) mild no Leafhopper (Cicadellidae) mild no Lacewings ( Neuroptera) mild no

PR Salinas Corn sap beetle ( Carpophilus spp.) mild to severe no Corn earworm ( Helicoverpa zea) mild to severe no Cornsilk fly ( Euxesta stigmatias) mild to severe no Spider mites ( Tetranychidae) mild to severe no Corn planthoppers (Delphacidae) mild to severe no Chinch bugs (Lygaeidae) moderate no Thrips ( Frankliniella spp.) mild to severe no Lady beetles (Coccinellidae) mild noPioneer Hi - Bred International, Inc. Page 229 of 25 8

4114 Maize **Table 8. 1.**  **Observations of Insects Present and Comparison B** **etween 4114 Maize and Control (continued)**  **Year of Planting**  **Permit**

**Number State**  **County Insect**  **Overall Severity in Field**  **Unexpected**

**Difference in Comparison?** 

2010 10-284- 101n PR Salinas Leafhopper (Cicadellidae) mild no Lacewings (Neuroptera) mild no Fall a rmyworm (Spodoptera frugiperda) mild to severe no

Aphids (Aphididae) mild to severe no Grasshoppers (Orthoptera) mild to moderate no

Guayama Corn planthoppers (Delphacidae) mild to moderate no

Spider mites (Tetranychidae) moderate no Corn earworm ( Helicoverpa zea) moderate no Fall armyworm (Spodoptera frugiperda) moderate to severe no Corn sap beetle ( Carpophilus spp.) moderate no Thrips ( Frankliniella spp.) moderate no Cornsilk fly ( Euxesta stigmatias) moderate n o Fall armyworm (Spodoptera frugiperda) mild to moderate no

Aphids (Aphididae) mild to moderate no Spider mites (Tetranychidae) moderate no Grasshoppers (Orthoptera) moderate no

10-015 - 106n IL Champaign Fall armyworm (Spodoptera frugi perda) mild no Corn earworm ( Helicoverpa zea) mild no Clinton Corn earworm ( Helicoverpa zea) mild no

Western bean cutworm ( Richia albicosta) mild no Grasshoppers (Orthoptera) mild no Japanese beetle ( Popillia japonica) mild no Stark Corn rootworm ( Diabrotica spp.) mild no

Aphids (Aphididae) mild no European corn borer ( Ostrinia nubilalis) mild no IA Guthrie Grasshoppers (Orthoptera) mild no

European corn borer ( Ostrinia nubilalis) mild no Western bean cutwo rm ( Richia albicosta) mild noPioneer Hi - Bred International, Inc. Page 230 of 25 8

4114 Maize **Table 8. 1.**  **Observations of Insects Present and Comparison B** **etween 4114 Maize and Control (continued) Year of**  **Planting Permit** 

**Number State**  **County Insect**  **Overall Severity in Field**  **Unexpected**

**Difference in Comparison?** 

2010 10-015 - 106n IA Jefferson Corn rootworm ( Diabrotica spp.) mild no European corn borer ( Ostrinia nubilalis) mild no Corn earworm ( Helicoverpa zea) mild no Shelby European corn borer ( Ostrinia nubilalis) mild no

Corn earworm ( Helicoverpa zea) moderate no Aphids (Aphididae) mild to moderate no Grasshoppers (Orthoptera) mild no MI Lenawee Corn earworm ( Helicoverpa zea) mild no Aphids (Aphididae) mild no MN Freeborn Aphids (Aphididae) mild no

European corn borer ( Ostrinia nubilalis) mild no

NE York Corn earworm ( Helicoverpa zea) mild no

European corn borer ( Ostrinia nubilalis) mild no Grasshoppers (Orthoptera) mild no Corn rootworm ( Diabrotica spp.) mild no Beetles (Coleoptera) mild no Picnic beetles (Nitidulidae) mild no Aphids (Aphididae) mild no

2011 10-284- 101n PR Guayama Fall armyworm (Spodoptera frugiperda) mild to severe no Aphids (Aphididae) mild to severe no Spider mites (Tetranychidae) mild to severe no Corn planth oppers (Delphacidae) mild to moderate no Cornsilk fly ( Euxesta stigmatias) mild to severe no Thrips ( Frankliniella spp.) mild to severe no Corn earworm ( Helicoverpa zea) mild to severe no Corn sap beetle ( Carpophilus spp.) moderate no 11 -040- 123n PR Guayama Corn planthoppers (Delphacidae) mild to moderate no Thrips ( Frankliniella spp.) mild to moderate noPioneer Hi - Bred International, Inc. Page 231 of 25 8

4114 Maize **Table 8.2. Observations of Diseases Present and**  **Comparison B etween 4114 Maize and Control**  **Year of Planting**  **Permit**

**Number St** **ate County**  **Disease Overall Severity in**  **Field Unexpected** 

**Difference in Comparison?**  2006 06-019-04R HI Kauai Northern corn leaf blight ( Exserohilum turcicum ) mild no Common corn rust ( Puccinia sorghi) mild no

2007 07-040-

101rm CA Yolo Common smut ( Ustilago zeae) mild no

IA Linn Gray leaf spot ( Cercospora zeae-maydis) mild no

Polk Stewart's wilt ( Pantoea stewartii) mild to moderate no

Northern corn leaf blight ( Exserohilum turcicum ) mild to moderate no Common corn rust ( Puccinia sorghi) mild to moderate no Common smut ( Ustilago zeae) mild to moderate no Gray leaf spot ( Cercospora zeae-maydis) mild to moderate no Southern corn rust ( Puccinia polysora) mild to moderate no Anthracnose ( Colletotrichum graminicola) mild no

IL Bureau Common corn rust ( Puccinia sorghi) mild no Champaign Northern corn leaf blight ( Exserohilum turcicum ) mild no

Common corn rust ( Puccinia sorghi) mild no McDonough Gray leaf spot ( Cercospora zeae-maydis) mild to moderate no

N orthern corn leaf blight ( Exserohilum turcicum ) moderate no Ogle Common corn rust ( Puccinia sorghi) mild no

Common smut ( Ustilago zeae) mild no Gray leaf spot ( Cercospora zeae-maydis) mild no IN Tipton Common corn rust ( Puccinia sorghi) mi ld no Gray leaf spot ( Cercospora zeae-maydis) mild to severe no Northern corn leaf blight ( Exserohilum turcicum ) mild to moderate no MO Saline Gray leaf spot ( Cercospora zeae-maydis) mild to moderate no Southern corn rust ( Puccinia polysora) mild no WI Rock Gray leaf spot ( Cercospora zeae-maydis) mild no Common corn rust ( Puccinia sorghi) mild no NE York Common corn rust ( Puccinia sorghi) mild no Gray leaf spot ( Cercospora zeae-maydis) mild to moderate noPioneer Hi - Bred International, Inc. Page 232 of 25 8

4114 Maize **Table 8.2 .**  **Obse rvations of Diseases Present and**  **Comparison B etween 4114 Maize and Control (continued)**  **Year of Planting**  **Permit**

**Number State**  **County Stressor**  **Overall Severity in Field**  **Unexpected**

**Difference in Comparison?** 

2008 08-014- 111n IN Gibson Northern corn leaf blight ( Exserohilum turcicum ) mild no Tipton Common corn rust ( Puccinia sorghi) mild no

Gray leaf spot ( Cercospora zeae-maydis) mild no Northern corn leaf blight ( Exserohilum turcicum ) mild no

IA Polk Gray leaf spot ( Cercospora zeae-maydis) mil d no Common corn rust ( Puccinia sorghi) mild no Northern corn leaf blight ( Exserohilum turcicum ) mild no Gray leaf spot ( Cercospora zeae-maydis) mild no Southern corn rust ( Puccinia polysora) mild no Crazy top (Sclerophthora macrospora) mild no Linn Common corn rust ( Puccinia sorghi) mild no

Northern corn leaf blight (Exserohilum turcicum ) mild no

NE Nance Common corn rust ( Puccinia sorghi) mild to moderate no Gray leaf spot ( Cercospora zeae-maydis) mild to moderate no Common smut ( Ustilago zeae) mild to moderate no Crazy top (Sclerophthora macrospora) moderate no York Common corn rust ( Puccinia sorghi) mild to moderate no

Gray leaf spot ( Cercospora zeae-maydis) mild to moderate no MO Saline Gray leaf spot ( Cercospora zeae-maydis) mild to moderate no

IL Bureau Common corn rust ( Puccinia sorghi) mild no

Northern corn leaf blight ( Exserohilum turcicum ) mild no Anthracnose ( Colletotrichum graminicola) mild no Gray leaf spot ( Cercospora zeae-maydis) mild no Champaign Gray leaf spot ( Cercospora zeae-maydis) mild no

Northern corn leaf blight ( Exserohilum turcicum ) mild no Common corn rust ( Puccinia sorghi) mild no McDonough Common corn rust ( Puccinia sorghi) mild no

Gray leaf spot ( Cercospora zeae-maydis) mild noPioneer Hi - Bred International, Inc. Page 233 of 25 8

4114 Maize **Table 8.2 .**  **Observations of Diseases Present and Comparison B** **etween 4114 Maize and Control (continued) Year of**  **Planting Permit** 

**Number State**  **County Disease**  **Overall Severity in Field**  **Unexpected**

**Differenc e in**  **Comparison?**

2008 08-014- 111n WI Rock Common corn rust ( Puccinia sorghi) mild to moderate no Gray leaf spot ( Cercospora zeae-maydis) moderate no Northern corn leaf blight ( Exserohilum turcicum ) mild no Gibberella stalk rot ( Gibberella zeae) mild no

08-095- 105n OK Caddo Common smut ( Ustilago zeae) mild no

IL Clinton Gray leaf spot ( Cercospora zeae-maydis) mild no

Stark Anthracnose ( Colletotrichum graminicola) mild no

Gray leaf spot ( Cercospora zeae-maydis) mild no Commo n corn rust ( Puccinia sorghi) mild no IA Jefferson Northern corn leaf blight ( Exserohilum turcicum ) mild no Common corn rust ( Puccinia sorghi) mild no NE York Common corn rust ( Puccinia sorghi) mild to moderate no Gray leaf spot ( Cercospora zeae-maydis) mild no Corn eyespot (Aureobasidium zeae) mild no

2009 09-013- 108n IL Bureau Northern corn leaf blight ( Exserohilum turcicum ) mild no Gray leaf spot ( Cercospora zeae-maydis) mild no Common corn rust ( Puccinia sorghi) mild no Douglas Gray leaf spot ( Cercospora zeae-maydis) mild to severe no

Northern corn leaf blight ( Exserohilum turcicum ) mild to severe no Common corn rust ( Puccinia sorghi) mild no Southern corn rust ( Puccinia polysora) mild no Anthracnose ( Colletotrichum graminicola) mild no Fulton Gray leaf spot ( Cercospora zeae-maydis) mild to moderate no

Common corn rust ( Puccinia sorghi) mild no Northern corn leaf blight ( Exserohilum turcicum ) mild to moderate no McDonough Gray leaf spot ( Cercospora zeae-maydis) mild to moderate no

Northern corn leaf blight ( Exserohilum turcicum ) mild to moderate no IN Gibson Gray leaf spot ( Cercospora zeae-maydis) mild to moderate noPioneer Hi - Bred International, Inc. Page 234 of 25 8

4114 Maize **Table 8.2 .**  **Observations of Diseases Present and Comparis** **on B etween 4114 Maize and Control (continued)**  **Year of Planting**  **Permit**

**Number State**  **County Disease**  **Overall Severity in Field**  **Unexpected**

**Difference in Comparison?** 

2009 09-013- 108n IN Gibson Northern corn leaf blight ( Exserohilum turcicum ) mild to moderate no Southern corn rust ( Puccinia polysora) mild to moderate no Tipton Gray leaf spot ( Cercospora zeae-maydis) mild no

Common corn rust ( Puccinia sorghi) mild no Northern corn leaf blight ( Exserohilum turcicum ) mild no

IA Dallas Norther n corn leaf blight ( Exserohilum turcicum ) mild no Gray leaf spot ( Cercospora zeae-maydis) mild no Common corn rust ( Puccinia sorghi) mild no Southern corn rust ( Puccinia polysora) mild no Kossuth Gray leaf spot ( Cercospora zeae-maydis) m ild to moderate no

Corn eyespot (Aureobasidium zeae) mild no Common corn rust ( Puccinia sorghi) mild to moderate no Northern corn leaf blight ( Exserohilum turcicum ) mild to moderate no Common smut ( Ustilago zeae) mild to moderate no Linn Common corn rust ( Puccinia sorghi) mild no

Common smut ( Ustilago zeae) mild no Polk Northern corn leaf blight ( Exserohilum turcicum ) mild no

Gray leaf spot ( Cercospora zeae-maydis) mild no Common smut ( Ustilago zeae) mild no Common corn rust ( Puccinia sorghi) mild no Corn eyespot (Aureobasidium zeae) mild no NE York Gray leaf spot ( Cercospora zeae-maydis) mild to moderate no Common corn rust ( Puccinia sorghi) mild to moderate no Common smut ( Ustilago zeae) mil d to moderate no Northern corn leaf blight ( Exserohilum turcicum ) mild to moderate no MO Saline Gray leaf spot ( Cercospora zeae-maydis) mild no Common corn rust ( Puccinia sorghi) mild no 09-035- 109n AR Crittenden Common corn rust ( Puccinia sorghi) mild to moderate noPioneer Hi - Bred International, Inc. Page 235 of 25 8

4114 Maize **Table 8.2 .**  **Observations of Diseases Present and Comparison B** **etween 4114 Maize and Control (continued) Year of**  **Planting Permit** 

**Number State**  **County Disease**  **Overall Severity in Field**  **Unexpected**

**Difference in Comparison?** 

2009 09-035- 109n AR Crittenden Southern corn rust ( Puccinia polysora) mild to moderate no Northern corn leaf blight ( Exserohilum turcicum ) moderate no Gray leaf spot ( Cercospora zeae-maydis) mild to severe no GA Grady Northern corn leaf blight ( Exserohilum turcicum ) mild no Southern corn leaf blight ( Bipolaris maydis) mild no Common corn rust ( Puccinia sorghi) mild no HI Kauai Maize chlorotic mottle machlomovirus moderate no

Aspergillus ear and kernel rot (Aspergillus spp.) mild no

Fusarium (Fusarium spp.) mild no MN Blue Earth Northern corn leaf blight ( Exserohilum turcicum ) mild no TN Obion Northern corn leaf blight ( Exserohilum turcicum ) mild no Gray leaf spot ( Cercospora zeae-maydis) mild no Common corn rust ( Puccinia sorghi) mild no Southern corn rust ( Puccinia polysora) mild no Brown Spot ( Physoderma maydis) mild no WI Rock Anthracnose ( Colletotrichum graminicola) mild no Common corn rust ( Puccinia sorghi) mild no Common smut ( Ustilago zeae) mild no Northern corn leaf blight ( Exserohilum turcicum ) mild no Anthracnose ( Colletotrichum graminicola) mild no 09-264- 102n HI Kauai Common corn rust ( Puccinia sorghi) mild no Common smut ( Ustilago zeae) mild no Maize chloroti c mottle machlomovirus mild no 09-016- 103n IL Stark Common corn rust ( Puccinia sorghi) mild no Gray leaf spot ( Cercospora zeae-maydis) mild no 2010 09-264- 102n HI Kauai Maize chlorotic mottle machlomovirus mild no Common smut ( Ustilago zeae) m ild no Maize mosaic rhabdovirus mild noPioneer Hi - Bred International, Inc. Page 236 of 25 8

4114 Maize **Table 8.2 .**  **Observations of Diseases Present and Comparison B** **etween 4114 Maize and Control (continued) Year of**  **Planting Permit** 

**Number State**  **County Disease**  **Overall Severity in Field**  **Unexpected**

**Difference in Co**  **mparison?**

2010 09-264- 102n HI Kauai Maize dwarf mosaic potyvirus mild no Common corn rust ( Puccinia sorghi) mild no

10-050- 101n NE Keith Common corn rust ( Puccinia sorghi) mild no

AR Crittenden Diplodia ear rot (Stenocarpella maydis) mild to mod erate no Fusarium ear mold (Fusarium spp.) mild to moderate no Gray leaf spot ( Cercospora zeae-maydis) moderate no Southern corn rust ( Puccinia polysora) moderate no Southern corn leaf blight ( Bipolaris maydis) moderate no GA Grady N orthern corn leaf blight ( Exserohilum turcicum ) mild to moderate no Common corn rust ( Puccinia sorghi) mild no Southern corn leaf blight ( Bipolaris maydis) mild to moderate no Southern corn rust ( Puccinia polysora) mild to moderate no Common smut ( Ustilago zeae) moderate no HI Kauai Common corn rust ( Puccinia sorghi) mild no Common smut ( Ustilago zeae) mild no Northe r n corn leaf spot ( Bipolaris zeicola) mild no

IL Bureau Gray leaf spot ( Cercospora zeae-maydis) mild no Common corn rust ( Puccinia sorghi) mild no Common corn rust ( Puccinia sorghi) mild no Champaign Gray leaf spot ( Cercospora zeae-maydis) mild no

Northern corn leaf blight ( Exserohilum turcicum ) mild no Southern corn rust ( Puccinia polysora) mild no Common corn rust ( Puccinia sorghi) mild no Fulton Gray leaf spot ( Cercospora zeae-maydis) mild no

Anthracnose ( Colletotrichum graminicola) mild no Northe r n corn leaf spot ( Bipolaris zeicola) mild no La Salle Gray leaf spot ( Cercospora zeae-maydis) mild no

Common corn rust ( Puccinia sorghi) mild noPioneer Hi - Bred International, Inc. Page 237 of 25 8

4114 Maize **Table 8.2 .**  **Observations of Diseases Present and Comparison B** **etween 4114 Maize and Control (continued) Year of**  **Planting Permit** 

**Number State**  **County Disease**  **Overall Severi ty in**  **Field Unexpected** 

**Difference in Comparison?** 

2010 10-052- 101n IL La Salle Common corn rust ( Puccinia sorghi) mild no McDonough Gray leaf spot ( Cercospora zeae-maydis) mild no Common smut ( Ustilago zeae) mild no Northern corn leaf blight ( Exserohilum turcicum ) mild no Ogle Common corn rust ( Puccinia sorghi) mild to moderate no

Gray leaf spot ( Cercospora zeae-maydis) mild to moderate no

IN Gibson Gray leaf spot ( Cercospora zeae-maydis) mild no Gray leaf spot ( Cercospora zeae-maydis) mild no Shelby Common corn rust ( Puccinia sorghi) mild no

Northern corn leaf blight ( Exserohilum turcicum ) mild no Gray leaf spot ( Cercospora zeae-maydis) mild no Tipton Northern corn leaf blight ( Exserohilum turcicum ) mild no White Common corn rust ( Puccinia sorghi) mild no

IA Bremer Common corn rust ( Puccinia sorghi) mild no

Dallas Northern corn leaf blight ( Exserohilum turcicum ) mild no

Gray leaf spot ( Cercospora zeae-maydis) mild no Common corn rust ( Puccinia sorghi) mild no Delaware Gray leaf spot ( Cercospora zeae-maydis) mild no Kossuth Common corn rust ( Puccinia sorghi) mild no

Common smut ( Ustilago zeae) mild no Northern corn leaf blight ( Exserohilum turcicum ) mild no Common corn rust ( Puccinia sorghi) mild no Linn Common smut ( Ustilago zeae) mild no

Common corn rust ( Puccinia sorghi) mild no Madison Northern corn leaf blight ( Exserohilum turcicum ) mild no

Gray leaf spot ( Cercospora zeae-maydis) mild no Gray leaf spot ( Cercospora zeae-maydis) moderate no Polk Northern corn leaf blight ( Exserohilum turcicum ) mild to moderate noPioneer Hi - Bred International, Inc. Page 238 of 25 8

4114 Maize **Table 8.2 .**  **Observations of Diseases Present and Comparison B** **etween 4114 Maize and Control (continued) Year of**  **Planting Permit** 

**Number State**  **County Disease**  **Overall Severity in Field**  **Unexpected**

**Difference in Comparison?** 

2010 10-052- 101n IA Polk Common corn rust ( Puccinia sorghi) mild to moderate no Gray leaf spot ( Cercospora zeae-maydis) mild to moderate no Common smut ( Ustilago zeae) moderate no Van Buren Common smut ( Ustilago zeae) mild no KS Finney Common corn rust ( Puccinia sorghi) mild no MI Gratiot Common smut ( Ustilago zeae) mild no Stewart's wilt ( Pantoea stewartii) mild no Common corn rust ( Puccinia sorghi) mild no MN Blue Earth Northern corn leaf blight ( Exserohilum turcicum ) mild no Common smut ( Ustilago zeae) mild no Kandiyohi Common corn rust ( Puccinia sorghi) mild no

Northern corn leaf blight ( Exserohilum turcicum ) mild no Gray leaf spot ( Cercospora zeae-maydis) mild no MO Scott Northern corn leaf blight ( Exserohilum turcicum ) mild no Common corn rust ( Puccinia sorghi) mild no Gray leaf spot ( Cercospora zeae-maydis) mild no NE York Common smut ( Ustilago zeae) mild no Common corn rust ( Puccinia sorghi) mild no Goss's wilt ( Clavibacter michiganensis) mild no Common smut ( Ustilago zeae) mild no Northern corn leaf blight ( Exserohilum turcicum ) mild no PA Lebanon Gray leaf spot ( Cercospora zeae-maydis) mild no Juana Diaz Northern corn leaf blight ( Exserohilum turcicum ) mild no

Common smut ( Ustilago zeae) mild no TN Obion Gray leaf spot ( Cercospora zeae-maydis) mild no Common corn rust ( Puccinia sorghi) mild no Southe rn corn rust ( Puccinia polysora) mild no Brown spot ( Physoderma maydis) mild noPioneer Hi - Bred International, Inc. Page 239 of 25 8

4114 Maize **Table 8.2 .**  **Observations of Diseases Present and Comparison B** **etween 4114 Maize and Control (continued) Year of**  **Planting Permit** 

**Number State**  **County Disease**  **Overall Sever ity in**  **Field Unexpected** 

**Difference in Comparison?** 

2010 10-052- 101n TN Obion Common corn rust ( Puccinia sorghi) mild no WI Rock Northern corn leaf blight ( Exserohilum turcicum ) mild no Common smut ( Ustilago zeae) mild no Gray leaf spot ( Cercospora zeae-maydis) mild no Corn eyespot (Aureobasidium zeae) mild no Common corn rust ( Puccinia sorghi) mild no 10- 118- 105n IL Gallatin Northern corn leaf blight ( Exserohilum turcicum ) mild no Gray leaf spot ( Cercospora zeae-maydis) mild to moderate no 10-281- 101n HI Kauai Common smut ( Ustilago zeae) mild no Common corn rust ( Puccinia sorghi) mild no PR Salinas Maize stripe tenuivirus mild no Corn leaf blight (Pleosporaceae) mild no Common corn rust ( Puccinia sorghi) mild no

10-284- 101n HI Kauai Common smut ( Ustilago zeae) mild no Maize stripe tenuivirus mild no PR Salinas Common corn rust ( Puccinia sorghi) mild no Maize stripe tenuivirus mild no Corn leaf blight (Pleosporaceae ) mild no Guayama Mai ze stripe tenuivirus mild no

Southern corn leaf blight ( Bipolaris maydis) mild no

10-015- 106n IN Clinton Anthracnose ( Colletotrichum graminicola) mild no Common corn rust ( Puccinia sorghi) mild no Gray leaf spot ( Cercospora zeae-maydis) mi ld no Northern corn leaf blight ( Exserohilum turcicum ) mild no IL Champaign Diplodia ear rot (Stenocarpella maydis) mild no Clinton Gray leaf spot ( Cercospora zeae-maydis) mild no

Northern corn leaf blight ( Exserohilum turcicum ) mild no Stark Gray leaf spot ( Cercospora zeae-maydis) mild noPioneer Hi - Bred International, Inc. Page 240 of 25 8

4114 Maize **Table 8.2 .**  **Observations of Diseases Present and Comparison B** **etween 4114 Maize and Control (continued) Year of**  **Planting Permit** 

**Number State**  **County Disease**  **Overall Severity in Field**  **Unexpected**

**Diffe rence in**  **Comparison?**

2010 10-015- 106n IL Stark Anthracnose ( Colletotrichum graminicola) mild no Common corn rust ( Puccinia sorghi) mild no

IA Guthrie Eye spot of corn (Aureobasidium zeae) mild no Gibberella stalk rot ( Gibberella zeae) mild no Common smut ( Ustilago zeae) mild no Jefferson Northern corn leaf blight ( Exserohilum turcicum ) mild no

Common corn rust ( Puccinia sorghi) mild no Southern corn leaf blight ( Bipolaris maydis) mild no Eye spot of corn (Aureobasidium zeae) mild no Shelby Gray leaf spot ( Cercospora zeae-maydis) mild no

Common corn rust ( Puccinia sorghi) mild no Common smut ( Ustilago zeae) mild no MI Lenawee Common corn rust ( Puccinia sorghi) mild no MN Freeborn Northern corn leaf blight ( Exserohilum turcicum ) mild no Common corn rust ( Puccinia sorghi) mild no NE York Common corn rust ( Puccinia sorghi) mild no Gray leaf spot ( Cercospora zeae-maydis) mild noPioneer Hi - Bred International, Inc. Page 241 of 25 8

4114 Maize **References Section 1**  Brookes G, Barfoot P (2009) Global Impact of Biotech Crops: I ncome and Production Effects, 1996- 2007. AgBioForum 12: 184- 208 Brookes G, Barfoot P (2010) GM crops: global socio-economic and environmental impacts 1996- 2008. PG Economics Ltd, Dorchester, UK CCUR (2009) Corn Uses Poster. Iowa State University, Center for Crops Utilization Research, http://www.ccur.iastate.edu/research/cornposter.pdf CERA (2002) Glufosinate Fact Sheet. International Life Sciences Institute, Center for Environmental Risk Assessment, http://www.cera -gmc.org/static/htmfiles/glufosinate.htm EPA (2010a ) Biopesticides Registration Action Document : Cry1Ab and Cry1F Bacillus thuringiensis ( Bt) Corn Plant- Incorpo rated Protectants. United States E nvironmental Protection Agency, www.epa.gov/opp00001/biopesticides/pips/cry1f-cry1ab- brad.pdf EPA (2010 b ) Biopesticides Reg istration Action Document : Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 Proteins and the Genetic Material Necessary for Their Production (PHP17662 T- DNA) in Event DAS-59122 -7 Corn (OECD Unique Identifier: DAS-59122 -7). United States E nvironmental Protection Agency, www.epa.gov/pesticides/biopesticides/pips/cry3435ab1 - brad.pdf EPA (2011a) Current and Previously Registered Section 3 PIP Registrations. United States Environmental Protection Agency, http://www.ep a.gov/oppbppd1/biopesticides/pips/pip_list.htm EPA (2011b) Glufosinate Ammonium Case. United States Environmental Protection Agency, http://www.epa.gov/oppsrrd1/registration_review/glufosinate_ammonium/ FDA (2001 ) Biotechnology Consultation Agency Respon se Letter BNF No. 000073. United States Food and Drug Administration, http://www.fda.gov/Food/Biotechnology/Submissions/ucm155765.htm FDA (2004) Biotechnology Consultation Agency Response Letter BNF No. 000081. United States Food and Drug Administratio n, www.fda.gov/Food/Biotechnology/Submissions/ucm155591.htm GfK Kynetec (2010 ) CropData Table: Doanes HXI, HXX, Smartstax Acres 2000-2010, Market Information. GfK Kynetec, http://www.gfk- kynetec.com/Pioneer Hi - Bred International, Inc. Page 242 of 25 8

4114 Maize Gray ME, Sappington TW, Miller NJ, Moeser J, Bohn MO (2009) Adaptation and Invasiveness Of Western Corn Rootworm: Intensifying Research on a Worsening Pest. A nnual Review of Entomology 54: 303-321 NCGA (2011) 2011 World of Corn. N ational Corn Growers Association, http://www.ncga.com/files/pdf/WOC2011.pdf OECD (2002) Consensus Document on Compositional Considerations for New Varieties of Maize (Zea Mays): Key Food and Feed Nutrients, Anti- Nutrients and Secondary Plant Metabolites. Organisation for Economic Co-operation and Developmen t, ENV/JM/MONO(2002) 25, http://www.olis.oecd.org/olis/2002doc.nsf/LinkTo/NT00002F66/$FILE/JT00130429.PDF Ostlie KR, Hutchison WD, Hellmich RL (eds) (2002) Bt Corn & European Corn Borer: Long-Term Success Through Resistance Management. U niversity of Minnesota Extension Publi cation WW07055, http://www.extension.umn.edu/distribution/cropsystems/dc7055.html USDA-APHIS (2001) Approval of Mycogen Seeds c/o Dow AgroSciences LLC and Pioneer Hi - Bred International, Inc., Request (00- 136-01p) Seeking a Determination of Non - regulated Status for Bt Cry1F Insect Resistant, Glufosinate Tolerant Corn Line 1507: E nvironmental Assessment and Finding of No Significant Impact. United States Department of Agriculture, Animal and Plant Health Inspection Service USDA-APHIS (2005) Mycogen Seeds/ Dow AgroSciences LLC and Pioneer Hi - Bred International Inc.; Availability of Determination of Nonregulated Status for Genetically Engineered Corn: Environmental Assessment and Finding of No Significant Impact. United States Department of Agriculture: Anima l and Plant Health Inspection Service USDA- ERS (2009) Corn: Background. United States Department of Agriculture, Economic Research Service, http://www.ers.usda.gov/Briefing/Corn/background.htm USDA- ERS (2010) Bioenergy: Background. United States Departme nt of Agriculture, Economic Research Service, http://www.ers.usda.gov/Briefing/bioenergy/background.htm USDA- NASS (2007) Ethanol Co- Products Used for Livestock Feed. United States Department of Agriculture, National Agricultural Statistics Service, http://usda.mannlib.cornell.edu/usda/current/EthFeed/EthFeed -06-29-2007_revision.pdf USDA- NASS (2010) Acreage. United States Department of Agriculture, National Agricultural Statistics Service, http://usda.mannlib.cornell.edu/usda/current/Acre/Acre-06-30- 2010. pd f USDA- NASS (2011) Crop Production 2010 Summary. United States Department of Agriculture, National Agricultural Statistics Service, http://usda.mannlib.cornell.edu/usda/current/CropProdSu/CropProdSu -01- 12 - 2011_new_format.pdfPioneer Hi - Bred International, Inc. Page 243 of 25 8

4114 Maize **Section 2** An G, Mitra A, C hoi HK, Costa MA, An K, Thornburg RW, Ryan CA (1989) Functional Analysis of the 3' Control Region of the Potato Wound - Inducible Proteinase Inhibitor II Gene. Plant Cell 1: 115 - 122 Barker RF, Idler KB, Thompson DV, Kemp JD (1983) Nucleotide sequence of the T- DNA region from the Agrobacterium tumefaciens octopine Ti plasmid pTi15955. Plant Molecular Biology 2: 335 -350 Bravo A, Gill SS, Soberón M (2007) Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxico n 49 **:** 423 -435 Chambers JA, Jelen A, Gilbert MP, Jany CS, Johnson TB, Gawron - Burke C (1991) Isolation and characterization of a novel insecticidal crystal protein gene from Bacillus thuringiensis subsp. aizawai. Journal of Bacteriology 173 **:** 3966-3976 Chri stensen AH, Sharrock RA, Quail PH (1992) Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Plant Molecular Biology 18: 675 -689 de M aagd RA, Bravo A, Berry C, Crickmore N, Schnepf HE (2003) Structure, diversity, and evolution of protein toxins from spore-forming enteromopathogenic bacteria. Annual Review of Genetics 37 **:** 409-433 Ellis RT, Stockhoff BA, Stamp L, Schnepf HE, Schwab GE, K nuth M, Russell J, Cardineau GA, Narva KE (2002) Novel Bacillus thuringiensis Binary Insecticidal Crystal Proteins Active on Western Corn Rootworm, Diabrotica virgifera virgifera LeConte. Applied and Environmental Microbiology 68: 1137- 1145 Franck A, Guil ley H, Jonard G, Richards K, Hirth L (1980) Nucleotide sequence of cauliflower mosaic virus DNA. Cell 21: 285 -294 Herman RA, Wolt JD, Halliday WR (2002) Rapid Degradation of the Cry1F Insecticidal Crystal Protein in Soil. Journal of Agricultural and Food Chemistry 50: 7076-7078 Hertig C, Rebmann G, Bull J, Mauch F, Dudler R (1991) Sequence and tissue-specific expression of a putative peroxidase gene from wheat (Triticum aestivum L.). Plant Molecular Biology 16: 171- 174 Hua G, Masson L, Jurat- Fuentes JL, Schwab G, Adang MJ (2001) Binding Analyses of Bacillus thuringiensis Cry δ- Endotoxins Using Brush Border Membrane Vesicles of Ostrinia nubilalis . Applied and Environmental Microbiology 67 **:** 872-879Pioneer Hi - Bred International, Inc. Page 244 of 25 8

4114 Maize Keil M, Sanchez-Serrano J, Schell J, Willmitzer L (1986) P rimary structure of a proteinase inhibitor II gene from potato (Solanum tuberosum). Nucleic Acids Research 14: 5641 -5650 Moellenbeck DJ, Peters ML, J. Bing JW, Rouse JR, Higgins LS, Sims L, Nevshemal T, Marshall L, Ellis RT, Bystrak PG, Lang BA, Stewart J L, Kouba K, Sondag V, Gustafson V, Nour K, Xu D, Swenson J, Zhang J, Czapla T, Schwab G, Jayne S, Stockhoff BA, Narva K, Schnepf HE, Stelman SJ, Poutre C, Koziel M, Duck N (2001) Insecticidal proteins from Bacillus thuringiensis protect corn from corn rootworms. Nature Biotechnology 19: 668-672 New England Biolabs (2002) Dam and Dcm Methylases of E. coli. New England Biolabs 2002 - 2003 Catalog and Technical Reference, pp 253 Odell JT, Nagy F, Chua N - H (1985) Identification of DNA sequences required for act ivity of the cauliflower mosaic virus 35S promoter. Nature 313: 810-812 OECD (2003) Consensus Document on the Biology of Zea Mays Subsp. Mays (Maize). Organisation for Economic Co-operation and Development, ENV/JM/MONO(2003)11, pp 1-49, http://www.olis.oe cd.org/olis/2003doc.nsf/LinkTo/NT0000426E/$FILE/JT00147699.PDF Pietrzak M, Shillito RD, Hohn T, Potrykus I (1986) Expression in plants of two bacterial antibiotic resistance genes after protoplast transformation with a new plant expression vector. Nucleic Acids Research 14: 5857-5868 Ritchie SW, Hanway JJ, Benson GO (2005) How a corn plant develops. Cooperative Extension Service Special Report 48, Iowa State University, Ames, IA Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler DR, Dean DH (1998) Bacillus thuringiensis and Its Pesticidal Crystal Proteins. Microbiology and Molecular Biology Reviews 62 **:** 775-806 Siegel JP (2001) The Mammalian Safety of Bacillus thuringiensis- Based Insecticides. Journal of Invertebrate Pathology 77 **:** 13 - 21 Zhao Z-y, Gu W, Cai T, Tagliani L, Hondred D, Bond D, Schroeder S, Rudert M, Pierce D (2001) High throughput genetic transformation mediated by Agrobacterium tumefaciens in maize. Molecular Breeding 8: 323 -333 **Section 3** Bartsch K, Tebbe CC (1989) Initial Steps in the Degradation of Phosphinothricin (Glufosinate) by Soil Bacteria. Applied and Environmental Microbiology 55 **:** 711-716Pioneer Hi - Bred International, Inc. Page 245 of 25 8

4114 Maize Baumann L, Broadwell AH, Baumann P (1988) Sequence analysis of the mosquitocidal toxin genes encoding 51.4- and 41.9- kilodalton proteins from Bacillus sphaericus 2362 and 2297. Journal of Bacteriology 170 **:** 2045 - 2050 Baumann P, Clark MA, Baumann L, Broadwell AH (1991) Bacillus sphaericus as a Mosquito Pathogen: Properties of the Organism and Its Toxins. Microbiology and Molecular Biology Reviews 55 **:** 425-436 Baxevanis AD (2005) Assessing Pairwise Sequence Similarity: BLAST and FASTA. In AD Baxevanis, BFF Ouellette, eds, Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Ed 3. John Wiley and Sons, New York, pp 295 -324 Berne S, Križaj I, Pohleven F, Turk T, Maček P, Sepčić K (2002) Pleurotus and Agrocybe hemolysins, new proteins hypothetically involved in fungal fruiting. Biochimica et Biophysica Acta 1570 **:** 153 - 159 Berne S, Lah L, Sepčić K (2009) Aegerolysins: Structure, function, and putative biological role. Protein Science 18 **:** 694-706 Berne S, Sepč ić K, Anderluh G , Tu rk T, Maček P, Poklar Ulrih N (2005) Effect of pH on the Pore Forming Activity and Conformational Stability of Ostreolysin, a Lipid Raft- Binding Prote in from the Edible Mushroom Pleurotus ostreatus . Biochemistry 44 **:** 11137- 11147 Bravo A, Gill SS, Soberón M (2007) Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxicon 49 **:** 423 -435 Chambers JA, Jelen A, Gilbert MP, Jany CS, Johnson TB, Gawron - Burke C (1991) Isolation and characterization of a novel insecticidal crystal protein gene from Bacillus thuringiensis subsp. aizawai. Journal of Bacteriology 173 **:** 3966-3976 Codex Alimentarius Commission (2003) Al inorm 03/34: Report of the Third Session of the Codex Ad Hoc Intergovernmental Task Force on Food Derived from Biotechnology. Food and Agriculture Organization of the United Nations, World Health Organization, Rome, pp 47-60 Codex Alimentarius Commission (2009) Foods derived from modern biotechnology. Food and Agriculture Organization of the United Nations, World Health Organization, Rome Dröge- Laser W, Siemeling U, Pühler A, Broer I (1994) The Metabolites of the Herbicide LPhosphinothricin (Glufosinate) : Identification, Stability, and Mobility in Transgenic, HerbicideResistant, and Untransformed Plants. Plant Physiology 105 **:** 159- 166 Ebina K, Sakagami H, Yokota K, Kondo H (1994) Cloning and nucleotide sequence of cDNA encoding Asp- hemolysin from Aspergillus fumigatus . Biochimica et Biophysica Acta 1219 **:** 148- 150Pioneer Hi - Bred International, Inc. Page 246 of 25 8

4114 Maize EPA (1998) Reregistration Eligibility Decision (RED): Bacillus thuringiensis . U nited States Environmental Protection Agency, EPA738- R-98-004, http://www.epa.gov/oppsrrd1/REDs/0247.pdf EPA (20 10a ) Biopesticides Registration Action Document: Cry1Ab and Cry1F Bacillus thuringiensis ( Bt) Corn Plant- Incorporated Protectants. United States Environmental Protection Agency, www.epa.gov/opp00001/biopesticides/pips/cry1f-cry1ab- brad.pdf EPA (2010 b ) Bio pesticides Registration Action Document: Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 Proteins and the Genetic Material Necessary for Their Production (PHP17662 T- DNA) in Event DAS-59122 -7 Corn (OECD Unique Identifier: DAS-59122 -7). United States Environme ntal Protection Agency, www.epa.gov/pesticides/biopesticides/pips/cry3435ab1 - brad.pdf FAO/WHO (2001) Evaluation of allergenicity of genetically modified foods: Report of a Joint FAO/WHO Expert Consultation on Allergenicity of Foods Derived from Biotechnol ogy, 22 - 25 January 2001. Food and Agriculture Organization of the United Nations, Rome Fasoli E, Pastorello EA, Farioli L, Scibilia J, Aldini G, Carini M, Marocco A, Boschetti E, Righetti PG (2009) Searching for allergens in maize kernels via proteomic tools. Journal of Proteomics 72 **:** 501-510 FARRP (2011) Food Allergy Research and Resource Program. University of Nebaska - Lincoln, http://www.allergenonline.com Fasoli E, Pastorello EA, Farioli L, Scibilia J, Aldini G, Carini M, Marocco A, Boschetti E, Righ etti PG (2009) Searching for allergens in maize kernels via proteomic tools. Journal of Proteomics 72 **:** 501-510 Federici BA, Park H -W, Bideshi DK, Wirth MC, Johnson JJ (2003) Recombinant bacteria for mosquito control. The Journal of Experimental Biology 206 **:** 3877-3885 Fernandez Espinar M -T, Labarère J (1997) Cloning and sequencing of the Aa-Pri1 gene specifically expressed during fruiting initiation in the edible mushroom Agrocybe aegerita, and analysis of the predicted amino-acid sequence. Current Genetics 32 **:** 420-424 GfK Kynetec (2010 ) CropData Table: Doanes HXI, HXX, Smartstax Acres 2000-2010, Market Information. GfK Kynetec, http://www.gfk- kynetec.com/ Hefle SL, Nordlee JA, Taylor SL (1996) Allergenic foods. Critical Reviews in Food Science and Nutrition 36 **:** 69-89Pioneer Hi - Bred International, Inc. Page 247 of 25 8

4114 Maize Hernandez E, Ramisse F, Cruel T, le Vagueresse R, Cavallo J - D (1999) Bacillus thuringiensis serotype H34 isolated from human and insecticidal strains serotypes 3a3b and H14 can lead to death of immunocompetent mice after pulmonary infecti on. FEMS Immunology and Medical Microbiology 24 **:** 43 -47 Hérouet C, Esdaile DJ, Mallyon BA, Debruyne E, Schulz A, Currier T, Hendrickx K, van der Klis R-J, Rouan D (2005) Safety evaluation of the phosphinothricin acetyltransferase proteins encoded by the pat and bar sequences that confer tolerance to glufosinate-ammonium herbicide in transgenic plants. Regulatory Toxicology and Pharmacology 41 **:** 134- 149 Hoffmann -Sommergruber K (2002) Pathogenesis- related (PR) - proteins identified as allergens. Biochemical Soc iety Transactions 30 **:** 930-935 Juberg DR, Herman RA, Thomas J, Brooks KJ, Delaney B (2009) Acute and repeated dose (28 day) mouse oral toxicology studies with Cry34Ab1 and Cry35Ab1 Bt proteins used in coleopteran resistant DAS-59122 -7 corn. Regulatory Toxi cology and Pharmacology 54 **:** 154- 163 Kutzner HJ (1981) The Family Streptomycetaceae. In MP Starr, H Stolp, eds, The Prokaryotes: A Handbook on Habitats, Isolation, and Identification of Bacteria, Vol 2. Springer-Verlag, pp 2028- 2090 Ladics GS, Bardina L, Cressman RF, Mattsson JL, Sampson HA (2006) Lack of cross- reactivity between the Bacillus thuringiensis derived protein Cry1F in maize grain and dust mite Der p7 protein with human sera positive for Der p7- IgE. Regulatory Toxicology and Pharmacology 44 **:** 13 6- 143 McClintock JT, Schaffer CR, Sjoblad RD (1995) A Comparative Review of the Mammalian Toxicity of Bacillus thuringiensis- Based Pesticides. Pesticide Science 45 **:** 95 - 105 Moneret-Vautrin DA, Kanny G, Beaudouin E (1998) Does food allergy to maize exist? Allergie et Immunologie 30 **:** 230 NCBI (2009) RefSeq Definitions. National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/RefSeq/key.html#accessions NCBI (2011) Aegerolysin [Streptomyces cf. griseus XylebKG - 1]. National Center for Biotec hnology Information, http://www.ncbi.nlm.nih.gov/protein/ZP_08234223 Pasini G, Simonato B, Curioni A, Vincenzi S, Cristaudo A, Santucci B, Peruffo ADB, Giannattasio M (2002) IgE- mediated allergy to corn: a 50 kDa protein, belonging to the Reduced Soluble Proteins, is a major allergen. Allergy 57 **:** 98- 106Pioneer Hi - Bred International, Inc. Page 248 of 25 8

4114 Maize Pastorello EA, Farioli L, Pravettoni V, Ispano M, Scibola E, Trambaioli C, Giuffrida MG, Ansaloni R, Godovac-Zimmermann J, Conti A, Fortunato D, Ortolani C (2000) The maize major allergen, which is respon sible for food - induced allergic reactions, is a lipid transfer protein. Journal of Allergy and Clinical Immunology 106 **:** 744-751 Pastorello EA, Pompei C, Pravettoni V, Farioli L, Calamari AM, Scibilia J, Robino AM, Conti A, Iametti S, Fortunato D, Bonomi S, Ortolani C (2003) Lipid -transfer protein is the major maize allergen maintaining IgE- binding activity after cooking at 100°C, as demonstrated in anaphylactic patients and patients with positive double- blind, placebo-controlled food challenge results. Jou rnal of Allergy and Clinical Immunology 112 **:** 775-783 Pearson WR (2000) Flexible sequence similarity searching with the FASTA3 program package. Methods in Molecular Biology 132 **:** 185- 219 Pearson WR, Lipman DJ (1988) Improved tools for biological sequence comparison. Proceedings of the National Academy of Sciences of the United States of America 85 **:** 2444-2448 PIR (2011) Protein Information Resource. Georgetown University, http://pir.georgetown.edu/ PRF (2011) Protein Research Foundation, http://www.prf.or.jp/index-e.html Sakurai N, Kaneko J, Kamio Y, Tomita T (2004) Cloning, expression, and pore-forming properties of mature and precursor forms of pleurotolysin, a sphingomyelin -specific two-component cytolysin from the edible mushroom Pleurotus ostreatus . B iochimica et Biophysica Acta 1679 **:** 65 -73 Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler DR, Dean DH (1998) Bacillus thuringiensis and Its Pesticidal Crystal Proteins. Microbiology and Molecular Biology Reviews 62 **:** 775-806 SWI SS- PROT (2011) ExPASy Bioinformatics Resource Portal. Swiss Institute of Bioinformatics, http://www.expasy.org/sprot/ Thomas K, Aalbers M, Bannon GA, Bartels M, Dearman RJ, Esdaile DJ, Fu TJ, Glatt CM, Hadfield N, Hatzos C, Hefle SL, Heylings JR, Goodman RE, Henry B, Herouet C, Holsapple M, Ladics GS, Landry TD, MacIntosh SC, Rice EA, Privalle LS, Steiner HY, Teshima R, van Ree R, Woolhiser M, Zawodny J (2004) A multi - laboratory evaluation of a common in vitro pepsin digestion assay protocol used in assess ing the safety of novel proteins. Regulatory Toxicology and Pharmacology 39: 87-98 wwPDB (2011) Worldwide Protein Data Bank, http://www.wwpdb.org/Pioneer Hi - Bred International, Inc. Page 249 of 25 8

4114 Maize **Section 4** AOSA (2005) Tetrazolium Testing Handbook, Contribution No. 29 to the Handbook on Seed Testing. Association of Official Seed Analysts, Lincoln, NE AOSA (2007) Rules for testing seeds. Association of Official Seed Analysts, Lincoln, NE Benjamini Y, Hochberg Y (1995) Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. Journal of the Royal Statistical Society B 57: 289 -300 Codex (1996) Corn Oil (Unhydrogenated). In Food Chemical s Codex, Ed 4, National Academy Press, Washington, D.C., pp 110- 111 Codex (2005) Codex Standard for Named Vegetable Oils. Codex alime ntarius, STAN - 210- 1999 ILSI (2006) ILSI Crop Composition Database Version 3.0 . Intern ational Life Sciences Institute, http://www.cropcomposition.org/ Luna V S, Figueroa M J, Baltazar M B, Gomez L R, Townsend R, Schoper JB (2001) Maize Pollen Longevity an d Distance Isolation Requirements for Effective Pollen Control. Crop Science 41 **:** 1551- 1557 NCGA (2011) 2011 World of Corn. N ational Corn Growers Association, http://www.ncga.com/files/pdf/WOC2011.pdf OECD (2002) Consensus Document on Compositional Consi derations for New Varieties of Maize (Zea Mays): Key Food and Feed Nutrients, Anti- Nutrients and Secondary Plant Metabolites. Organisation for Economic Co-operation and Development, ENV/JM/MONO(2002) 25, pp 1-41, http://www.olis.oecd.org/olis/2002doc.nsf/ LinkTo/NT00002F66/$FILE/JT00130429.PDF Pawitan Y, Michiels S, Koscielny S, Gusnato A, Ploner, A (2005) False discovery rate, sensitivity and sample size for microarray studies. Bioinformatics 21: 3017-3024 Ritchie SW, Hanway JJ, Benson GO (2005) How a co rn plant develops. Cooperative Extension Service Special Report 48, Iowa State University, Ames, IA Spelman RJ, Bovenhuis H (1998) Moving from QTL experimental results to the utilization of QTL in breeding p rogrammes. Animal Genetics 29 : 77-84 Watson SA (1982) Corn: Amazing Maize. General Properties. In IA Wolff, ed, CRC Handbook of Processing and Utilization in Agriculture, Vol 2. CRC Press, Boca Raton, pp 3 - 29 Watson SA (1987) Structure and Composition. In SA Watson, PE Ramstad, eds, Corn: chemistry an d technology. American Association of Cereal Chemists, pp 53 -82Pioneer Hi - Bred International, Inc. Page 250 of 25 8

4114 Maize Westfall PH, Tobias RD, Rom D, Wolfinger RD, Hochberg Y (1999) Concepts and Basic Methods for Multiple Comparisons and Tests. In PH Westfall, RD Tobias, D Rom, RD Wolfinger, Y Hochberg, eds, Multiple Comparisons and Multiple Tests Using SAS, SAS Institute Inc, Cary, pp 13 -40 **Section 5** Appenzeller LM, Malley L, MacKenzie SA, Hoban D, Delaney B (2009) Subchronic feeding study with genetically modified stacked trait lepidopteran and coleoptera n resistant (DAS-Ø15Ø7

- 1xDAS-59122 -7) maize grain in Sprague- Dawley rats. Food and Chemical Toxicology 47 **:** 1512 - 1520 Aylor DE (2004) Survival of maize (Zea mays) pollen exposed in the atmosphere. Agricultural and Forest Meteorology 123 **:** 125 - 133 Baker HG (1974) The Evolution of Weeds. Annual Review of Ecology and Systematics 5 **:** 1- 24 Betz FS, Hammond BG, Fuchs RL (2000) Safety and Advantages of Bacillus thuringiensisProtected Plants to Control Insect Pests. Regulatory Toxicology and Pharmacology 32 **:** 156- 1 73 Byrne PF, Fromherz S (2003) Can GM and non -GM crops coexist? Setting a precedent in Boulder County, Colorado, USA. Food, Agriculture and Environment 1 **:** 258- 261 CERA (2011) A Review of the Environmental Safety of the PAT Protein. International Life Sci ences Institute, Center for Environmental Risk Assessment, http://cera gmc.org/docs/cera_publications/pub_05_2011.pdf Clark BW, Phillips TA, Coats JR (2005) Environmental Fate and Effects of Bacillus thuringiensis (Bt) Proteins from Transgenic Crops: a Review. Journal of Agricultural and Food Chemistry 53 **:** 4643-4653 EPA (2009) Biopesticides Reg istration Action Document : Bacillus thuringiensis Vip3Aa20 Insecticidal Protein and the Genetic Material Necessary for Its Production (via Elements of Vector pNOV13 00) in Event MIR162 Maize (OECD Unique Identifier: SYN - IR162 -4) . United States Environmental Protection Agency, http://www.epa.gov/oppbppd1/biopesticides/ingredients/tech_docs/brad_006599.pdf EPA (2010a) Biopesticides Reg istration Action Document : Cry1Ab and Cry1F Bacillus thuringiensis ( Bt) Corn Plant- Incorpo rated Protectants. United States Environmental Protection Agency, www.epa.gov/opp00001/biopesticides/pips/cry1f-cry1ab- brad.pdfPioneer Hi - Bred International, Inc. Page 251 of 25 8

4114 Maize EPA (2010b) Biopesticides Regi stration Action Document: Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 Proteins and the Genetic Material Necessary for Their Production (PHP17662 T- DNA) in Event DAS-59122 -7 Corn (OECD Unique Identifier: DAS-59122 -7). United States Environmental Protection Agency, www.epa.gov/pesticides/biopesti cides/pips/cry3435ab1- brad.pdf EPA (2010c) Biopesticides Registration Action Document: Bacillus thuringiensis Cry3Bb1 Protein and the Genetic Material Necessary for Its Production (Vector PV-ZMIR13L) in MON 863 Corn (OECD Unique Identifier: MON -ØØ863

-5) a nd Bacillus thuringiensis Cry3Bb1 Protein and the Genetic Material Necessary for Its Production (Vector PV-ZMIR39) in MON 88017 Corn (OECD Unique Identifier: MON -88Ø17

-3). United States Environmental Protection Agency, http://www.epa.gov/oppbppd1/biopestic ides/pips/cry3bb1 - brad.pdf EPA (2011) Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins and the genetic material necessary for their production (plasmid insert PHP 17662) in Event DAS-59122 -7 corn and Bacillus thuringiensis Cry1F protein and the genet ic material necessary for its production (plasmid insert PHI8999) in Event TC1507 corn (006490, 006481) Fact sheet. United States Environmental Protection Agency, http://www.epa.gov/oppbppd1/biopesticides/ingredients/factsheets/factsheet_006481 - 006490.htm Farnham D (2001) Corn Planting Guide. I owa State University Extension, http://www.extension.iastate.edu/publications/pm1885.pdf Faust M, Smith B, Rice D, Owens F, Hinds M, Dana G, Hunst P (2007) Performance of Lactating Dairy Cows Fed Silage and Grain from a Maize Hybrid with the cry1F Trait Versus its Nonbiotech Counterpart. Journal of Dairy Science 90 **:** 5706-5713 Fonseca AE, Westgate ME, Grass L, Dornbos DL (2003) Tassel Morphology as an Indicator of Potential Pollen Production in Maize. Crop Manageme nt Herman RA, Evans SL, Shanahan DM, Mihaliak CA, Bormett GA, Young DL, Buehrer J (2001) Rapid Degradation of Cry1F Delta - Endotoxin in Soil. Environmental Entomology 30 **:** 642 -644 Hérouet C, Esdaile DJ, Mallyon BA, Debruyne E, Schulz A, Currier T, Hendrickx K, van der Klis R-J, Rouan D (2005) Safety evaluation of the phosphinothricin acetyltransferase proteins encoded by the pat and bar sequences that confer tolerance to glufosinate-ammonium herbicide in transgenic plants. Regulatory Toxicology and Pharmaco logy 41 **:** 134- 149 Higgins LS, Babcock J, Neese P, Layton RJ, Moellenbeck DJ, Storer N (2009) Three-Year Field Monitoring of Cry1F, Event DAS-Ø15Ø7

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4114 Maize Huls TJ, Erickson GE, Klopfenstein TJ, Luebbe MK, Vander Pol KJ, Rice DW, Smith B, Hinds M, Owens F, Liebergesell M (2008) Effect of Feeding DAS-59122-7 Corn Grain and Nontransgenic Corn Grain to Individually Fed Finishing Steers. The Professional Animal Scientist 24: 572 -577 Hunst P, Rood T (2004) Application for the Determination of Nonregulated Status for B.t. Cry34/35Ab1 Insect- Resistant, Glufosinate-Tolerant Corn: Corn Line 59122. Dow AgroSciences and Pioneer Hi - Bred International, http://www.aphis.usda.gov/brs/aphisdocs/03_35301p.pdf Icoz I, Stotzky G (2008) Fate and effects of insect- resistant Bt crops in soil ecosystems. Soil Biology and Biochemistry 40 **:** 559-586 Iltis HH (2011) Zea L. (Section 26.29). Utah State University, Intermountain Herbarium, http://herbarium. usu.edu/webmanual/info2.asp?name=Zea&type=treatment Jacobs CM, Utterback PL, Parsons CM, Rice D, Smith B, Hinds M, Liebergesell M, Sauber T (2008) Performance of Laying Hens Fed Diets Containing DAS-59122 -7 Maize Grain Compared with Diets Containing Nontransgenic Maize Grain. Poultry Science 87: 475 -479 Jarosz N, Loubet B, Durand B, McCartney A, Foueillassar X, Huber L (2003) Field measurements of airborne concentration and deposition rate of maize pollen. Agricultural and Forest Meteorology 119 **:** 37-51 Keeler KH (1989) Can genetically engineered crops become weeds? Bio/Technology 7 **:** 1134- 1139 Li Y, Romeis J (2010) Bt maize expressing Cry3Bb1 does not harm the spider mite, Tetranychus urticae, or its ladybird beetle predator, Stethorus punctillum . Biologi cal Control 53 **:** 337-344 Lundgren JG, Wiedenmann RN (2005) Tritrophic Interactions Among Bt (Cry3Bb1) Corn, Aphid Prey, and the Predator Coleomegilla maculata (Coleoptera: Coccinellidae). Environmental Entomology 34 **:** 1621- 1625 MacKenzie SA, Lamb I, Schmid t J, Deege L, Morrisey MJ, Harper M, Layton RJ, Prochaska LM, Sanders C, Locke M, Mattsson JL, Fuentes A, Delaney B (2007) Thirteen week feeding study with transgenic maize grain containing event DAS-Ø15Ø7

- 1 in Sprague- Dawley rats. Food and Chemical Toxicology 45 **:** 551-562 Matsuo K, Amano K, Shibaike H, Yoshimura Y, Kawashima S, Uesugi S, Misawa T, Miura Y, Ban Y, Oka M (2004) Pollen dispersal and outcrossing in Zea mays populations: A simple identification of hybri ds detected by xenia using conventional corn in simulation of transgene dispersion of GM corn. Proceedings of the 8th International Symposium on the Biosafety of Genetically Modified Organisms, Montpellier, Poster 31Pioneer Hi - Bred International, Inc. Page 253 of 25 8

4114 Maize Meissle M, Romeis J (2009) The web- bu ilding spider Theridion impressum (Araneae: Theridiidae) is not adversely affected by Bt maize resistant to corn rootworms. Plant Biotechnology Journal 7 **:** 645 -656 Nguyen HT, Jehle JA (2007) Quantitative analysis of the seasonal and tissue-specific express ion of Cry1Ab in transgenic maize MON810. Journal of Plant Diseases and Protection 114: 82 -87 Nielse n RK, Thomison PR (2002) Late- Planted Corn and Seedling Rates. P urdue University Department of Agronomy, http://www.agry.purdue.edu/ext/corn/news/article s.02/Delayed_Planting_Populations0510.html Obrist LB, Klein H, Dutton A, Bigler F (2005) Effects of Bt maize on Frankliniella tenuicornis and exposure of thrips predators to prey- mediated Bt toxin. Entomologia Experimentalis et Applicata 115 **:** 409-416 OE CD (1993) Safety Considerations for Biotechnology: Scale- up of crop plants. Organisation for Economic Co-operation and Development, http://www.oecd.org/dataoecd/26/26/1958527.pdf OECD (1999) Consensus document on general information concerning the genes a nd their enzymes that confer tolerance to phosphinothricin herbicide. Organisation for Economic Cooperation and Development, ENV/JM/MONO(99)13, pp 1 - 26, http://www.oecd.org/dataoecd/16/52/46815628.pdf OECD (2002) Consensus Document on Compositional Cons iderations for New Varieties of Maize (Zea Mays): Key Food and Feed Nutrients, Anti- Nutrients and Secondary Plant Metabolites. Organisation for Economic Co-operation and Development, ENV/JM/MONO(2002) 25, pp 1-41, http://www.olis.oecd.org/olis/2002doc.nsf /LinkTo/NT00002F66/$FILE/JT00130429.PDF OECD (2003) Consensus Document on the Biology of Zea Mays Subsp. Mays (Maize). Organisation for Economic Co-operation and Development, ENV/JM/MONO(2003)11, pp 1-49, http://www.olis.oecd.org/olis/2003doc.nsf/LinkTo/N T0000426E/$FILE/JT00147699.PDF Pleasants JM, Hellmich RL, Dively GP, Sears MK, Stanley- Horn DE, Mattila HR, Foster JE, Clark P, Jones GD (2001) Corn pollen deposition on milkweeds in and near cornfields. Proceedings of the National Academy of Sciences 98 **:** 11919- 11924 Raybould A, Vlachos D (2011) Non -target organism effects tests on Vip3A and their application to the ecological risk assessment for cultivation of MIR162 maize. Transgenic Research 20 **:** 599- 611Pioneer Hi - Bred International, Inc. Page 254 of 25 8

4114 Maize Romeis J, Bartsch D, Bigler F, Candolfi MP, Gie lkens MMC, Hartley SE, Hellmich RL, Huesing JE, Jepson PC, Layton R, Quemada H, Raybould A, Rose RI, Schiemann J, Sears MK, Shelton AM, Sweet J, Vaituzis Z, Wolt JD (2008) Assessment of risk of insect- resistant transgenic crops to nontarget arthropods. Nat ure Biotechnology 26 **:** 203 -208 Romeis J, Hellmich R, Candolfi M, Carstens K, De Schrijver A, Gatehouse A, Herman R, Huesing J, McLean M, Raybould A, Shelton A, Waggoner A (2011) Recommendations for the design of laboratory studies on non -target arthropods for risk assessment of genetically engineered plants. Transgenic Research 20 **:** 1- 22 Romeis J, Meissle M (2011) Non -target risk assessment of Bt crops – Cry protein uptake by aphids. Journal of Applied Entomology 135 **:** 1-6 Rose RI (2007) White paper on tier- based testing for the effects of proteinaceous insecticidal plant- incorporated protectants on non -target invertebrates for regulatory risk assessment. USDA-APHIS and US Environmental Protection Agency, Washington DC, http://www.epa.gov/pesticides/biopesti cides/pips/non -target-arthropods.pdf Scheideler SE, Rice D, Smith B, Dana G, Sauber T (2008) Evaluation of Nutritional Equivalency of Corn Grain from DAS- 15Ø7

- 1 (Herculex* I) in the Diets of Laying Hens. The Journal of Applied Poultry Research 17: 383-389 Sears MK, Stanley- Horn D (2000) Impact of Bt corn pollen on monarch butterfly populations. In C Fairbairn, G Scoles, A McHughen, eds, Proceedings of the 6th International Symposium on the Biosafety of GMOs, University Extension Press, University of Saskatchewan, Saskatoon, pp 120- 130 Shan G, Embrey SK, Herman RA, McCormick R (2008) Cry1F Protein Not Detected in Soil After Three Years of Transgenic Bt Corn (1507 Corn) Use. Environmental Entomology 37 **:** 255 -262 Shanahan D, Stauffer C (2000) Petition for D etermination of Non - Regulated Status of B.t. Cry1F Insect- Resistant, Glufosinate-Tolerant Maize Line 1507. Mycogen Seeds c/o Dow AgroSciences LLC and Pioneer Hi - Bred International, Inc, http://www.aphis.usda.gov/brs/aphisdocs/00_13601p.pdf Sindt J, Droui llard J, Loe E, Kessen T, Sulpizio M, Montgomery S, Rice D, Hinds M, Smith B, Owens F, Dana G, Hunst P (2007) Effect of Corn Containing the Cry1F Protein on Performance of Beef Heifers Fed a Finishing Diet Based on Steam - Flaked Corn. The Professional Anima l Scientist 23 **:** 632-636 Spacie A, McCarty LS, Rand GM (1995) Bioaccumulation and Bioavailability in Multiphase Systems. In GM Rand, ed, Fundamentals of Aquatic Toxicology: Effects, Environmental Fate, and Risk Assessment, Ed 2. Taylor and Francis, New Yor k, pp 493 -521Pioneer Hi - Bred International, Inc. Page 255 of 25 8

4114 Maize Stein HH, Rice DW, Smith BL, Hinds MA, Sauber TE, Pedersen C, Wulf DM, Peters DN (2009) Evaluation of corn grain with the genetically modified input trait DAS-59122 -7 fed to growingfinishing pigs. Journal of Animal Science 87 **:** 1254- 1260 US FWS (2010) Endangered and Threatened Wildlife and Plants; Designation of Critical Habitat for the Salt Creek Tiger Beetle. United States Fish and Wildlife Service, Federal Register 75: 17466- 17509 US FWS (2011) Species Report. United States Fish and Wild life Service, http://ecos.fws.gov/tess_public/SpeciesReport.do?groups=I&listingType=L&mapstatus=1 USDA (2011) The PLANTS Database. U nited States Department of Agriculture, National Resources Conservation Service, http://plants.usda.gov USDA-APHIS (2001) Approval of Mycogen Seeds c/o Dow AgroSciences LLC and Pioneer Hi - Bred International, Inc., Request (00- 136-01p) Seeking a Determination of Non - regulated Status for Bt Cry1F Insect Resistant, Glufosinate Tolerant Corn Line 1507: E nvironmental Assessment and Finding of No Significant Impact. United States Department of Agriculture, Animal and Plant Health Inspection Service USDA-APHIS (2005) Mycogen Seeds/Dow AgroSciences LLC and Pioneer Hi - Bred International Inc.; Availability of Determination of Nonregu lated Status for Genetically Engineered Corn: Environmental Assessment and Finding of No Significant Impact. United States Department of Agriculture: Animal and Plant Health Inspection Service USDA- ERS (2009) Corn: Background. United States Department of Agriculture: Economic Research Service, http://www.ers.usda.gov/Briefing/Corn/background.htm USDA- NASS (2009) 2007 Census of Agriculture. United States Department of Agriculture: National Agricultural Statistics Service, http://www.agcensus.usda.gov/Publi cations/2007/Full_Report/usv1.pdf USDA- NASS (2010) Acreage. United States Department of Agriculture: National Agricultural Statistics Service, http://usda.mannlib.cornell.edu/usda/current/Acre/Acre -06-30- 2010.pdf USDA- NASS (2011) Corn for All Purposes 20 10: Planted Acres by County for Selected States. United States Department of Agriculture: National Agricultural Statistics Service, http://www.nass.usda.gov/Charts_and_Maps/Crops_County/pdf/CR- PL10- RGBChor.pdf USDA- NRCS (2011) Invasive and Noxious Weeds: Federal and State Noxious Weeds. United States Department of Agriculture, Natural Resources Conservation Service, http://plants.usda.gov/java/noxCompositePioneer Hi - Bred International, Inc. Page 256 of 25 8

4114 Maize USGS (2010) Bioaccumulation. United States Geological Survey Toxic Substances Program, http://toxics .usgs.gov/definitions/bioaccumulation.html **Appendix 1**  No references cited in this section. **Appendix 2**  Ritchie SW, Hanway JJ, Benson GO (2005) How a corn plant develops. Cooperative Extension Service Special Report 48, Iowa State University, Ames, IA **Appendix 3**  Chen J, Dellaporta S (1994) Urea - based plant DNA miniprep. In M Freeling, V Walbot, eds, The Maize Handbook. Springer-Verlag, New York, pp 526-527 New England Biolabs (2002) Dam and Dcm Methylases of E. coli. New England Biolabs 2002- 2003 Catalog and Technical Reference, pp 253 **Appendix 4** No references cited in this section. **Appendix 5** Ritchie SW, Hanway JJ, Benson GO (2005) How a corn plant develops. Cooperative Extension Service Special Report 48, Iowa State University, Ames, IA Thié baut R, Jacqmin -Gadda H (2004) Mixed models for longitudinal left-censored repeated measures. Computer Methods and Programs in Biomedicine 74 **:** 255- 260 **Appendix 6** Benjamini Y, Hochberg Y (1995) Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. Journal of the Royal Statistical Society B 57: 289 -300Pioneer Hi - Bred International, Inc. Page 257 of 25 8

4114 Maize Codex (1996) Corn Oil (Unhydrogenated). In Food Chemicals Codex, Ed 4, National Academy Press, Washington, D.C., pp 110- 111 Codex (2005) Codex Standard for Named Vegetable Oils. Codex alimentarius, STAN - 210- 1999 Graybill FA (1976) Applications of the general linear model. I n Theory and Application of the Linear Model, Duxbury Press, Boston, pp 270- 275 ILSI (2006) ILSI Crop Composition Database Version 3.0. Internati onal Life Sciences Institute, http://www.cropcomposition.org/ Kenward MG, Roger JH (1997) Small sample inference for fixed effects from restricted maximum likelihood. Biometrics 53 **:** 983-997 Littell RC, Milliken GA, Stroup WW, Wolfinger RD, Schabenberger O (2006) Example: Variance Component Estimates Equal to Zero. In SAS for Mixed Models, Ed 2, SAS Institute, Inc, Cary, pp 148- 154 OECD (2002) Consensus Document on Compositional Considerations for New Varieties of Maize (Zea Mays): Key Food and Feed Nutri ents, Anti- Nutrients and Secondary Plant Metabolites. Organisation for Economic Co-operation and Development, ENV/JM/MONO(2002) 25, http://www.olis.oecd.org/olis/2002doc.nsf/LinkTo/NT00002F66/$FILE/JT00130429.PDF Thiébaut R, Jacqmin -Gadda H (2004) Mixed models for longitudinal left-censored repeated measures. Computer Methods and Programs in Biomedicine 74 **:** 255- 260 Watson SA (1982) Corn: Amazing Maize. General Properties. In IA Wolff, ed, CRC Handbook of Processing and Utilization in Agriculture, Vol 2. CRC Press, Boca Raton, pp 3 - 29 Watson SA (1987) Structure and Composition. In SA Watson, PE Ramstad, eds, Corn: Chemistry and Technology, American Association of Cereal Chemists, pp 53 -82 Westfall PH, Tobias RD, Rom D, Wolfinger RD, Hochberg Y (1999) Con cepts and Basic Methods for Multiple Comparisons and Tests. In PH Westfall, RD Tobias, D Rom, RD Wolfinger, Y Hochberg, eds, Multiple Comparisons and Multiple Tests Using SAS, SAS Institute Inc, Cary, pp 13 -40 **Appendix 7.** Agresti A (2002) Categorical da ta analysis, Ed 2. John Wiley and Sons, pp 250- 253 AOSA (2005) Tetrazolium Testing Handbook, Contribution No. 29 to the Handbook on Seed Testing. Association of Official Seed Analysts, Lincoln, NEPioneer Hi - Bred International, Inc. Page 258 of 25 8

4114 Maize Benjamini Y, Hochberg Y (1995) Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. Journal of the Royal Statistical Society B 57 **:** 289-300 Graybill FA (1976) Applications of the general linear model. I n Theory and Application of the Linear Model, Duxbury Press, Boston, p p 270- 275 Kenward MG, Roger JH (1997) Small sample inference for fixed effects from restricted maximum likelihood. Biometrics 53 **:** 983-997 Koch GG, Tangen C, Tudor G, Stokes ME (1990) Strategies and Issues for the Analysis of Ordered Categorical Data From Multifactor Studies in Industry. Technometrics 32 **:** 137- 149 Littell RC, Milliken GA, Stroup WW, Wolfinger RD, Schabenberger O (2006) Example: Variance Component Estimates Equal to Zero. In SAS for Mixed Models, Ed 2, SAS Institute, Inc, Cary, pp 148- 154 Luna VS, Figueroa MJ, Baltazar MB, Gomez L R, Townsend R, Schoper JB (2001) Maize Pollen Longevity and Distance Isolation Requirements for Effective Pollen Control. Crop Science 41 **:** 1551- 1557 Ritchie SW, Hanway JJ, Benson GO (2005) How a corn plant develop s. Cooperative Extension Service Special Report 48, Iowa State University, Ames, IA SAS Institute Inc. (2008) The GLIMMIX Procedure. In SAS/STAT® 9.2 User's Guide, SAS Institute Inc., Cary, pp 2222 - 2225 Vonesh EF (1996) A note on the use of Laplace's app roximation for nonlinear mixed -effects models. Biometrika 83 **:** 447-452 Westfall PH, Tobias RD, Rom D, Wolfinger RD, Hochberg Y (1999) Concepts and Basic Methods for Multiple Comparisons and Tests. In PH Westfall, RD Tobias, D Rom, RD Wolfinger, Y Hochberg, eds, Multiple Comparisons and Multiple Tests Using SAS, SAS Institute Inc, Cary, pp 13 -40 **Appendix 8** No references cited in this section.