USDA Petition for No nregulated Status of DAS

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Dow AgroSci ences LLC

Contains No Confidential Business Information

Petition for Determination of Nonregulated Status for Insect

- Resistant DAS

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- 2 Soybean

OEC D Unique Identifier: DAS

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- 2

The undersigned submits 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 not be regulated under 7 CFR 340.

Submitting Company : Dow AgroSciences LLC 9330 Zionsville Road Indianapolis, IN 46268

Submitted by:

_________________________________

Lei Han, Ph.D. Regulatory Manager Regulatory Sciences & Government Affairs Dow AgroSciences LLC 9330 Zionsville Road Indianapolis, IN 46268 Phone: (317) 337

- 3398 Fax: (317) 337

- 4523 Email: lhan@dow.com

September 26 , 2012 Revised November 2 , 2012 Revised December 19, 2012USDA Petition for No nregulated Status of DAS

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Dow AgroSci ences LLC

Contains No Confidential Business Information Release of Information Dow AgroSciences LLC (DAS) is submitting the information in this petition for deregulation to USDA APHIS as p art of the regulatory process. By s ubmitting this information, DAS do es not authorize release of this information to any third party except to the extent the information is requested under the Freedom of Information Act (FOIA), 5 U.S.C., Section 522. In the ev ent that USDA receives a FOIA request covering all or some of the info rmation in this submission, DAS expec t s that, in advance of the release of the document(s), USDA will provide DAS with a copy of the material proposed to be released and the opportunity to object to the release of any information based upon appropriate legal grounds, e.g. , responsiveness, confidentiali ty and/or competitive concerns. DAS expec t s that no information that has been identified as CBI (confidential business information) will be provi ded to any third party. DAS understand s that a CBI

- deleted copy of this information may be made available to the public as par t of the public comment per iod. Except in acc ordance with the foregoing, DAS do es not authorize the release, publication or other distribution of this information (including website posting) without prior notice and consent from DAS

.

© 2012 Dow AgroSciences LLC. All Rights R eserve d. This document is property of Dow AgroSciences LLC and is for use by the regulatory authority to which it has been submitted, and only in support of actions requested by Dow AgroSciences. All data and information herein must not be used, reproduced or di sclosed to third parties for any other purpose without the written consent of Dow AgroSciences.USDA Petition for No nregulated Status of DAS

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Dow AgroSci ences LLC

Contains No Confidential Business Information Certification The undersigned certifies that, to the best knowledge and belief of the undersigned, this petition includes all information and views on which to base a determination, and that it includes all relevant data and information known to the petitioner that are unfavorable to the petition.

Lei Han, Ph.D. Regulatory Manager Dow AgroSciences LLC 9330 Zionsville Road Indianapolis, Indiana 46268 Phone: (317) 337

- 3398 Fax: (317) 337

- 4523 Email: lhan@dow.comUSDA Petition for No nregulated Status of DAS

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Dow AgroSci ences LLC

Contains No Confidential Business Information Summary Dow AgroSciences LLC (herein referred to as “DAS”) is submitting a Petition for Determination of Nonregulated Status for DAS

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- 2 soybean. DAS requests a determination from USDA Animal and Plant Health Inspection Service (APHIS) that s oybean transformation event DAS

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- 2 and any soybean lines derived from crosses between DAS

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- 2 soybean and conventional soybean or biotechnology

- derived soy bean granted non

- regulated status by APHIS no longer be considered regulated articles under 7 CFR Part 340.
DAS

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- 2 Soybean DAS

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- 2 soybean is a transgenic soybean that expresses the insecticidal proteins Cry1Ac and Cry1F originally from the naturally

- occurring soil bacterium, Bacillus thuringiensis. Cry1Ac and Cry1F provide p rotection against several lepidopteran pests of soybean, including soybean looper ( Chrysodeixis includens, formerly Pseudoplusia includens ) , velvetbean caterpillar ( Anticarsia gemmatalis ) , fall armyworm ( Spodoptera frugiperda ) and tobacco budworm ( Heliothis virescens )

. In addition, DAS

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- 2 soybean expresses the phosphinothricin acetyltransferase (PAT) protein from the soil bacterium Streptomyces viridochromogenes. The PAT protein provides tolerance to the herbicide glufosinate and was used as a selectable marker during the developme nt of DAS

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- 2 soybean. The transgenes for Cry1Ac, Cry1F and PAT expression were introduced into soybean via Agrobacterium

- mediated transformation to create DAS

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- 2 soybean.
DAS

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- 2 soybean provides an effective pest management tool for lepidop teran pests that could reduce or replace current insecticide applications in regions where these insect pests cause significant yield loss. In the U.S., the majority of soybeans are grown in the north c entral states and the remaining in the s outheastern a nd Mississippi delta region. Insect pressure is generally greatest in the s outheast, where climate allows the major soybean insect pests to overwinter and facilitates multiple generations per year. Substances that are pesticides, as defined under the Fede ral Insecticide, Fungicide and Rodenticide Act (FIFRA), are subject to regulation by the U.S. Environmental Protection Agency (EPA). Pesticides produced in planta, known as plant

- incorporated protectants (PIPs), are also subject to regulation by the EPA u nder FIFRA. DAS has submitted an application for a FIFRA Section 3 seed increase registration with EPA to support future breeding and seed increase activities in the U.S. Under a Section 3 seed increase registration, commercial sale of DAS

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- 2 soybea n in the U.S. is prohibited. At present, DAS does not plan to commercialize DAS

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- 2 soybean in the U.S. due to the limited number of acres that consistently experience sufficient lepidopteran insect pressure. DAS will apply for a FIFRA Section 3 comm ercial use registration if DAS decides to commercialize DAS

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- 2 soybean in the U.S.
Evaluation of Plant Pest Potential of DAS

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- 2 Soybean The data and information presented in this petition demonstrate that DAS

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- 2 soybean is no more likely to become a plant pest than conventional soybean or to have an adverse environmental impact. This conclusion is based on the outcome of extensive evaluations including i) detailed molecular characterization of the inserted DNA, ii) detailed biochemical chara cterization of the intr oduced proteins, iii) compositional assessment of forage and grain, iv)USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information agronomic and phenotypic characteristics and ecological interaction s assessments, and v) assessment on the potential impact on non

- target organisms and thr eatene d and endangered species.
DAS

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- 2 soybean was genetically modified to express two insecticidal proteins, Cry1Ac and Cry1F, and phosphinothricin acetyltransferase (PAT) protein. Cry1Ac consists of 1156 amino acids from three components: the N

- terminal toxin core from Cry1Ac1 originating from B. thuringiensis subsp. kurstaki, followed by a very small portion of Cry1Ca3 originating from B. thuringiensis subsp. aizawai , and the C

- terminal sequence from Cry1Ab1 originating from B. thuringiensis subsp. Berliner. Cry1F consists of 1148 amino acids from three components: the N

- terminal toxin core from Cry1Fa2 following by a very small portion of Cry1Ca3 originating from B. thuringiensis subsp. aizawai, and the C

- terminal sequence from Cry1Ab1 originating from B. thuringiensis subsp. Berliner. Cry1Ac and Cry1F expressed in DAS

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- 2 soybean are 100% identical in amino acid sequence to Cry1Ac and Cry1F expressed in the deregulated events comprising WideStrike  cotton ; DAS

- 21023

- 5 (also described as 3006

- 2 10

- 23 expressing Cry1Ac) and DAS

- 24236

- 5 (also described as 281

- 24

- 236 expressing Cry1F). The PAT enzyme, originating from Streptomyces viridochromogenes , acetylates the primary amino group of phosphinothricin rendering it inactive. The PAT enzyme express ed in DAS

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- 2 soybean is 100% identical in amino acid sequence to PAT expressed in a number of deregulated events, including LibertyLink  soybean ACS

- GM ØØ 5

- 3 (also described as A2704

- 12 ) ( USDA, 1996 ) , Herculex  I corn DAS

- 01507

- 1 (also described as TC1507) ( USDA, 2001 ) , DAS

- 21023

- 5 (also described as 3006

- 210

- 23) and DAS

- 24236

- 5 (also described as 281

- 24

- 236) comprising WideStrike  cotton ( USDA, 2004 ) , and Herculex  RW DAS

- 59122

- 7 ( USDA, 2005 )

. The transgenes cry1Ac(synpro), cry1Fv3 and pat expressing Cry1Ac, Cry1F and PAT prote ins were introduced into DAS

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- 2 soybean using Agrobacterium

- mediated transformation. Molecular characterization by Southern blot analyses of DAS

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- 2 soybean confirmed that a single, intact DNA insert containing the cry1Ac(synpro), cry1Fv3, and pa t gene expression cassettes was integrated into the soybean genome and the intact DNA insert was stably inherited in the five breeding generations tested. Southern blot analyses confirmed the absence of the plasmid backbone DNA in DAS

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- 2 soybean. An alyses of the segregating generations confirmed that segregation of the DNA insert followed the predicted Mendelian inheritance pattern. These data confirmed the stability of DAS

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- 2 soybean during t raditional breeding procedures. The expression level s of Cry1Ac, Cry1F and PAT proteins in DAS

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- 2 soybean were determined by protein

- specific enzyme linked immunosorbent assays (ELISA). Protein expression was analyzed in leaf, root, whole

- plant and grain tissues collected throughout the growing season from DAS

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- 2 soybean plants grown in the major soybean production regions in the U.S. Mean protein expression levels across sites ran ged from 0.39 ng/mg dry weight (dwt) in root to 25.44 ng/mg dwt in V5 leaf tissue for Cry1Ac, 5.23 ng/mg dwt in root to 56.75 ng/mg dwt in V5 leaf tissue for Cry1F, and 0.63 ng/mg dwt in root to 5.60 ng/mg dwt in V10

- 12 leaf tissue for PAT. Expression of the Cry1Ac and Cry1F proteins peaked during the

 WideStrike and Herculex are registered trademarks of Dow AgroSciences LLC.  LibertyLink is a registered trademark of Bayer.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information vegetative growth stage, providing protection against target lepidopt eran insect pests and potential yield loss as a result of plant damage. The overall low levels of Cry1Ac, Cry1F and PAT proteins in DAS

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- 2 soybean indicate a low exposure to human s and animals. Cry1Ac and Cry1F have a long history of safe use. The proteins originate from the naturally occurring soil bacterium B. thuringiensis. The safety of the proteins has been demonstrated in sprayable Bt formulations for pest control in agriculture for over half a century ( Mendelsohn et al. , 2003 ; EPA, 2011 ; Sanahuja et al. , 2011 )

. Both proteins are expressed in WideStrike  cotton which is authorized for cultivation in the U.S. and Brazil and for food and feed use in Australia, Brazil, Canada, European Union, Japan, Korea, Mexico, New Zealand and U.S. ( www.biotradestatus.com ). Bt corn and Bt cott on expressing variations of Cry1Ac or Cry1F have been cultivated for commercial use in the U.S. and other countries for more than a decade. In 1997, EPA established an exemption from the requirement of a tolerance for the plant

incorporated protectant Cry 1Ac in all plants (40 CFR §174.510). Later , EPA established an exemption from the requirement of a tolerance for the plant

- incorporated protectant Cry1F in cotton (40 CFR §174.504) and in corn (40 CFR §174.520). The exemptions were based on safety assess ments of the proteins including digestibility in simulated gastric fluid, lack of homology to known allergens and protein toxins, and lack of mammalian toxicity as demonstrated by acute oral mouse gavage studies. DAS has filed a petition with EPA for an
e xemption from the requirement of a tolerance for Cry1F a s expressed in soybean in 2012. An extensive set of biochemical evaluations confirmed the identity of the Cry1Ac and Cry1F proteins produced in DAS

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- 2 soybean. Moreover DAS

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- 2 soybean

- deriv ed Cry1Ac and Cry1F were determined to be biochemically equivalent to the corresponding proteins purified from a microbial expression host organism Pseudomonas fluorescens. The Cry1Ac and Cry1F purified from P. fluorescens had been extensively assessed to establish the safety of the proteins. The assessments inclu ded acute oral toxicity in mice and protein digestability in simulated gastric fluid

. The proteins have a very low acute toxicity potential and are rapid ly degraded in simulated gastric fluid. Updated b ioinformatic analyses showed that n either Cry1Ac nor Cry1F share meaningful amino acid sequence similarities with known allergens. No significant homology was identified when either protein sequence was compared with known allergen using the sear ch criteria of either a match of eight or more contiguous identical amino acids, or greater than 35% identity over 80 amino acid residues. Likewise, neither protein share meaningful amino acid sequence similari ties with known protein toxins. The PAT prote in was assessed for any potential adverse effects to human s and animals resulting from the environmental release of crops containing the PAT protein. A step

- wise, weight

- of

evidence approach was used to assess the potential for toxic or allergenic effects from the PAT protein. Updated bioinformatic analyses revealed no meaningful homologies to known or putative allergens or toxins for the PAT amino acid sequence. The PAT protein hydrolyzed rapidly in simulated gastric fluid. There was no evidence of acu te toxicity in mice at a dose of 5000 mg/kg body weight of PAT protein. The low level expression of the PAT protein presents a low exposure risk to human s and animals, and the results of the overall safety assessment of the PAT protein indicate that it is unlikely to cause allergenic or toxic effects in humans or animals. The safety of the PAT protein has been assessed previously and it has been approved fo r use in canola, corn, cotton, rice, soybeans, and sugar beets.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Nutrient c omposition al analyses of forage and grain were conducted to compare the composition of DAS

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- 2 soybean with that of a non

- transgenic control. Compositional analyses were used t o evaluate any changes in the levels of key nutrients and anti

- nutrients in DAS

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- 2 soybean. A total of 88 analytes were evaluated, nine in forage and the remaining 79 in seed including protein, fat, ash, moisture, carbohydrate, mineral, amino acids, fatty acids, vitamins, and bioactives. Seventeen analytes were excluded from statistical analysis because more than 50% of the samples were below the limit of quantitation. Of the remaining 71 analytes, the results indicate that there were no statistical differences between DAS

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- 2 and the non

transgenic control for 61 analytes based on overall treatment effects and pair

- wise comparisons. The statistical differences observed for the remaining ten analytes based on unadjusted P

- values
were non
- existen t after adjustment for multiplicity using the false discovery rate method. The numerical differences in mean values observed for the ten analytes between DAS

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- 2 and
non
- transgenic control were small relative to natural variation. The mean values of DAS

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- 2 soybean were within the literature ranges and/or within the range of the reference varieties included in the study. Nutrient compositional analyses indicate that DAS

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- 2 soybean is substantially equivalent to conventional soybean and will not exhibit unexpected or unintended effects with respect to plant pest risk.
DAS

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- 2 soybean has been field tested in the major soybean growing regions in the U.S. All field tests were conducted under USDA APHIS notifications . Agronomic performance and phenotypic assessments were conducted on DAS

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- 2 soybean in multi

- site field studies to measure characteristics such as emergence, seedling vigor, days to flowering, days to maturity, lodging, plant height, plant morphology, final population, shatt ering, and yield. All field trials were also observed for opportunistic disease or insect stressors as well as normal phenotypic characteristics. Laboratory studies were carried out to evaluate DAS

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- 2 soybean for seed dormancy and germination charac teristics and to compare to that of the non

- transgenic control. Agronomic, phenotypic and ecological evaluations indicate that DAS

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- 2 soybean is substantially equivalent to conventional soybean and does not possess a selective advantage that would re sult in increased pla n t pest risk compared to conventional soybean. A review of potential environmental impact indicates that DAS

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- 2 soybean is unlikely to have adverse effects on non

- target organisms including threatened or endangered species under
n ormal agricultural practices. This conclusion is based on several lines of evidence including Cry protein specificity and long history of safe use, Cry protein content in DAS

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- 2 plant tissues and estimated likelihood for exposure, environmental fate, hazard characterization for representative non

- target organisms including vertebrate and invertebrate species, significant margins of exposure, and non

- target arthropod field trials comparing DAS

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- 2 soybe an with
non
- transgenic soybean.
Because DAS

- 81 419

- 2 soybean is agronomically and compositionally similar to conventiona l soybean, and Cr y1Ac, Cry1F and PAT proteins have a history of safe use , no significant impact is expected on current crop production practices, non

- target or endangered species, cro p rotation, volunteer management, or commodity food and feed soybean products. The availability of DAS

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- 2 soybean is expected to have a beneficial impact on insect pest management by providing another tool t o address insect control needs.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Information collected during field trials and laboratory analyses presented herein demonstrate that DAS

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- 2 soybean exhibits no plant pathogenic properties or weediness characteristics. DAS

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- 2 soybean is no more likely to become a plant pest than convention al soybean, and the Cry1Ac, Cry1F and PAT proteins are unlikely to increase the weediness potential of any other cultivated plant or wild species. DAS hereby requests a determination from APHIS that insect

- resistant DAS

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- 2 soybean and all progeny der ived from crosses between DAS

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- 2 soybean and conventional soybean or biotechnology

- derived soybean granted non

- regulated status by APHIS no longer be considered regulated articles under 7 CFR Part 340.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Table of Contents
Release of Information
................................ ................................ ................................ ................. 2 Certification
................................ ................................ ................................ ................................ ... 3 Summary
................................ ................................ ................................ ................................ ........ 4 Table of Contents
................................ ................................ ................................ .......................... 9 List of Figures
................................ ................................ ................................ .............................. 12 List of Tables
................................ ................................ ................................ ............................... 15 Acronyms and Scientific Terms
................................ ................................ ................................ . 16 1. Rationale for the Development of DAS

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................................ .......................... 18 1.1. Basis for the Request for Nonregulated Status

................................ ..................... 18 1.2. Rationale for the Development of DAS

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- 2 Soybean and Benefits

............. 18 1.3. Submission to Other Regulatory Agen cies

................................ ........................... 20 2. The Biology of Soybean
................................ ................................ ................................ .. 21 2.1. O verview of Soybean Biology

................................ ................................ .............. 21 2.2. Characterization of the Recipient Soybean Cultivar

................................ ............. 21 3. Development of DAS

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................................ ................................ ......... 22 3.1. Description of the Transformation System

................................ ........................... 22 3.2. Selection of Comparators for DAS

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................................ .......... 26 4. Donor Genes and Regulatory Sequences
................................ ................................ ...... 27 4.1. Identity and Source of Genetic Material in pDAB9582

................................ ....... 27 5. Genetic Characterization
................................ ................................ ............................... 31 5.1. Overview of Genetic Characterization

................................ ................................ .. 31 5.2. Analysis of the Insert and Its Genetic Elements

................................ ................... 41 5.2.1. Number of Insertion Sites

................................ ................................ ............... 41 5.2.2. Structure of the Insert and Genetic Elements

................................ ................. 42 5.2.3. Absence of Plasmid Backbone Sequences

................................ ...................... 44 5.2.4. Stability of the Insert Across Generations

................................ ...................... 44 5.2.5. Conclusions

................................ ................................ ................................ ..... 44 5.3. Segregation Analysis

................................ ................................ ............................ 66 5.3.1. Analysis of a Segregating Generation

................................ ............................. 66 5.3.2. Segregation Analysis of Breeding Generations

................................ .............. 66 5.4. Summary of the Genetic Characterization

................................ ............................ 67 6. Characterization of the Introduced Proteins
................................ ............................... 68 6.1. Cry1Ac and Cry1F

................................ ................................ ................................ 68 6.1.1. Cry1 Protein Mode of Action

................................ ................................ ......... 68 6.1.2. Identity of the Cry1Ac Protein

................................ ................................ ........ 68 6.1.3. Biochemical Characterization of the Cry1Ac Protein

................................ .... 69 6.1.4. Expression of the Cry1Ac Protein in Plant Tissues

................................ ........ 70 6.1.5. Identity of the Cry1F Protein

................................ ................................ .......... 71 6.1.6. Biochemical Characterization of the Cry1F Protein

................................ ....... 72 6.1.7. Expression of the Cry1F Protein in Plant Tissues

................................ .......... 73 6.1.8. Food and Feed Safety Assessment of Cry1Ac and Cry1F

.............................. 74 6.1.9. Summary of Cry1Ac and Cry1F Characterization

................................ .......... 76 6.2. PAT

................................ ................................ ................................ ....................... 78 6.2.1. Mode of Action of t he PAT Protein

................................ ................................ 78 6.2.2. Identity of the PAT Protein

................................ ................................ ............. 78USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information 6.2.3. Biochemical Characterization of the PAT Protein

................................ .......... 78 6.2.4. Expression of the PAT Protein in Plant Tissues

................................ ............. 79 6.2.5. Food and Feed Safety Assessment for PAT Protein

................................ ....... 80 6.2.6. Summary of PAT Protein Characterization

................................ .................... 80 7. Grain and Forage Composition
................................ ................................ ..................... 81 7.1. Field Study Design and Data Analysis

................................ ................................ . 81 7.2. Composition Analysis Results

................................ ................................ .............. 83 7.2.1. Proximate, Fiber, and Mineral Analysis of Forage

................................ ......... 83 7.2.2. Proximate and Fiber Analysis of Seed

................................ ............................ 86 7.2.3. Mineral Analysis of Seed

................................ ................................ ................ 89 7.2.4. Amino Acid Analysis of Seed

................................ ................................ ......... 93 7.2.5. Fatty Acid Analysis of Seed

................................ ................................ ........... 99 7.2.6. Vitamin Analysis of Seed

................................ ................................ ............. 104 7.2.7. Bioactive Analysis of Seed

................................ ................................ ........... 109 7.3. Summary of Composition Analysis

................................ ................................ .... 112 8. Agronomic Characteristics
................................ ................................ .......................... 113 8.1. Agronomic Study

................................ ................................ ................................ 113 8.2. Ecological Evaluations

................................ ................................ ........................ 119 8.3. Germination and Dormancy Evaluations

................................ ............................ 121 8.4. Summary of Agronomic Characteristics

................................ ............................. 122 9. Impact on Environment and Agronomic Practices
................................ ................... 123 9.1. Potential Impact on Environment

................................ ................................ ....... 123 9.1.1. Efficacy against Target Pests

................................ ................................ ........ 123 9.1.2. Potential Impact on Non

- Target Organisms

................................ ................. 127 9.1.3. Potential Impact on Threatened and Endanger ed Species

............................ 129 9.1.4. Environmental Fate of Introduced Proteins and Impact on Soil

- dwelling

Organisms

................................ ................................ ................................ ..... 130 9.1.5. Weediness Potential

................................ ................................ ...................... 130 9.1.6. Gene Flow Assessment

................................ ................................ ................. 131 9.2. Current Agronomic Practices for Soybean and Potential Impact

....................... 131 9.2.1. Soybean Production

................................ ................................ ...................... 131 9.2.2. Weeds in Soybean and Weed Management

................................ .................. 133 9.2.3. Crop Rotation and Tillage Practices

................................ ............................. 134 9.2.4. Management of Insect Pests

................................ ................................ .......... 135 9.2.5. Insect Resistance Management

................................ ................................ ..... 138 9.3. Summary of Impact on Environment and Agronomic Practices

........................ 140 10. Adverse Consequences of Introduction
................................ ................................ ...... 141 11. Appendices
................................ ................................ ................................ ..................... 142 Appendix 1. Materials and Methods for Molecular Characterization of DAS

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Soybean

................................ ................................ ................................ ............... 143 Appendix 2. Materials, Methods and Results for Characterization of Cry1Ac Protein

......... 145 Appendix 3. Materials, Methods and Results for Characterization of Cry1F Protein

........... 164 Appendix 4. Materi als, Methods and Results for Characterization of PAT Protein

.............. 183 Appendix 5. Materials and Methods for Cry1Ac, Cry1F and PAT Protei n Expression

Analysis

................................ ................................ ................................ ............... 188 Appendix 6. Analytical Methods for Compositional Analysis

................................ .............. 191USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Appendix 7. Literature Ranges for Compositional Analysis

................................ ................. 198 Appendix 8. Non

- Target Arthropod Field Study

................................ ................................ ... 201 Appendix 9. USDA Notifications for DAS

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................................ .............. 202 Appendix 10. Stewardship of Insect

- Resistant DAS

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................................ 203 Appendix 11. References

................................ ................................ ................................ ....... 204USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information List of Figures Figure 1. Plasmid map of pDAB9582.

................................ ................................ .......................... 23 Figure 2. Diagram of intended T

- DNA insert from plasmid pDAB9582.

................................ .... 23 Figure 3. Event sorting and selection process for DAS

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- 2 soybean.

................................ .. 24 Figure 4. Breeding diagram of DAS

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- 2 soybean.

................................ ............................... 25 Figure 5. Location of the probes on pDAB 9582 used in Southern blot analysis of DAS

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- 2 soybean.

................................ ................................ ................................ ..... 35 Figure 6. Plasmid map of pDAB9582 with selected restriction enzyme sites used for Southern analysis.

................................ ................................ ................................ ............. 40 Figure 7. Actual T

- DNA insert in DAS

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- 2 soybean with restriction enzymes used for DNA dig estion and sizes of the expected hybridization bands.

................................ .. 41 Figure 8. Southern blot analysis of DAS

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- 2 soybean digested with Nco I (A) and Sph I (B); AtUbi10 promoter probe.

................................ ................................ .................. 45 Figure 9. Southern blot analysis of DAS

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- 2 soybean digested with Nco I (A) and Sph I (B); cry1F

- 5 ′ probe

. ................................ ................................ ................................ .. 46 Figure 10. Southern blot analysis of DAS

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- 2 soybean digested with Nco I (A) and Sph I (B); cry1F

- 3 ′

A pr obe

. ................................ ................................ ............................... 47 Figure 11. Southern blot analysis of DAS

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- 2 soybean digested with Nco I (A) and Sph I (B); cry1F

- 3 ′ B probe

. ................................ ................................ .............................. 48 Figure 12. Southern blot analysis of DAS

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- 2 soybean digested with Nco I (A) and Sph I (B); AtuORF23

- A 3′ UTR probe

. ................................ ................................ .............. 49 Figure 13. Southern blot analysis of DAS

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- 2 soybean digested with Nco I (A) and Sph I (B); CsVMV promoter probe.

................................ ................................ .................. 50 Figure 14. Southern blot analysis of DAS

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- 2 soybean digested with Nco I (A) and Sph I (B); cry1Ac

- 5′ probe.

................................ ................................ ................................ 51 Figure 15. Southern blot analysis of DAS

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- 2 soybean digested with Nco I (A) and Sph I (B); cry1Ac

- 3′ A probe.

................................ ................................ ............................. 52 Figure 16. Southern blot analysis of DAS

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- 2 soybean digested with Nco I (A) and Sph I (B); cry1Ac

- 3′ B probe.

................................ ................................ ............................. 53 Figure 17. Southern blot analysis of DAS

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- 2 soybean digested with Nco I (A) and Sph I (B); Atu ORF23

- B 3′ UTR probe.

................................ ................................ .............. 54 Figure 18. Southern blot analysis of DAS

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- 2 soybean digested with Nco I (A) and Sph I (B); pat probe.

................................ ................................ ................................ ........... 55 Figure 19. Southern blot analysis of DAS

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- 2 soybean digested with Nco I (A) and Sph I (B); AtuORF1 3′ UTR probe.

................................ ................................ ................... 56 Figure 20. Southern blot analysis of DAS

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- 2 soybean digested with Not I/ Sph I; (A) AtUbi10 promoter probe, (B) cry1F

- 5 ′ probe.

................................ ................................ .. 57 Figure 21. Southern blot analysis of DAS

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- 2 soybean digested with Not I/ Sph I; (A) cry1F

- 3′ A probe, (B) cry1F

- 3′ B probe.

................................ ................................ ............ 58 Figure 22. Southern blot analysis of DAS

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- 2 soybean digested with Not I/ Sph I; (A) AtuORF23

- A 3′ UTR probe, (B) CsVMV promoter probe.

................................ .............. 59 Figure 23. Southern blot analysis of DAS

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- 2 soybean digested with Not I/ Sph I; (A) cry1Ac

- 5′ probe, (B) AtuORF23

- B 3′ UTR probe.

................................ ............................ 60 Figure 24. Southern blot analysis of DAS

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- 2 soybean digested with Not I/ Sph I;

............... 61 Figure 25. Southern blot analysis of DAS

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- 2 soybean digested with Not I/ Sph I;

............... 62USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 26. Southern blot analysis of DAS

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- 2 soybean digested with (A) Sph I; Ori probe, (B) Nco I; SpecR probe.

................................ ................................ .......................... 63 Figure 27. Southern blot analysis of DAS

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- 2 soybean digested with (A) Nco I; Backbone 1 probe, (B) Sph I; Backbone 2 probe.

................................ .............................. 64 Figure 28. Southern blot analysis of DAS

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- 2 soybean digested with Nco I; Backbone 3 and 4 probes.

................................ ................................ ................................ . 65 Figure 29. Amino acid sequence of Cry1Ac protein.

................................ ................................ ... 69 Figure 30. Amino acid sequence of Cry1F protein.

................................ ................................ ...... 72 Figure 31. Mode of action of the PAT protein.

................................ ................................ ............. 78 Figure 32. Amino acid sequence of the PAT protein.

................................ ................................ ... 78 Figure 33. Proximates, fiber and minerals in control, DAS

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- 2 and reference variety soybean forage.

................................ ................................ ................................ ................. 85 Figure 34. Proximates and fiber in control, DAS

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- 2, and reference variety soybean seed.

................................ ................................ ................................ ................................ .. 88 Figure 35. Minerals in control, DAS

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- 2, and reference variety soybean seed.

................... 91 Figure 36. Amino acids in control, DAS

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- 2, and reference variety soybean seed (% total amino acid).

................................ ................................ ................................ .............. 96 Figure 37. Fatty acids in control, DAS

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- 2, and reference variety soybean seed (% total fatty acid).

................................ ................................ ................................ ............... 103 Figure 38. Vitamins in control, DAS

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- 2, and referen ce variety soybean seed (mg/kg dry weight).

................................ ................................ ................................ ..................... 107 Figure 39. Bioactives in control, DAS

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- 2, and reference variety soybean seed.

.............. 111 Figure 40. Lateral flow strip assay for the Cry1Ac protein expression in Event DAS

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- 2 and Maverick grain extracts.

................................ ................................ ... 149 Figure 41. Western blot analysis of microbe

- and soybean

- derived Cry1Ac proteins.

.............. 150 Figure 42. SDS

- PAGE analysis of immunoaffinity

- purified soybean

- derived Cry1Ac.

............ 1 51 Figure 43. SDS

- PAGE analysis of microbe

- and soybean

- derived Cry1Ac protein stained with GelCode Blue total protein and glycoprotein stains.

................................ .............. 152 Figure 44. Theoretical cleavage of the Cry1Ac protein with trypsin generated in silico using Protein Analysis Worksheet (PAWS) from Proteometrics LLC.

.......................... 153 Figure 45. Theoretical cleavage of the Cry1Ac protein with chymotrypsin generated in silico using Protein Analysis Worksheet (PAWS) from Proteometrics LLC.

................ 154 Figure 46. Tryptic digest sequence coverage map for Cry1Ac (DAS

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- 2) by MALDI

- TOF MS and MALDI TOF/TOF. Cys residues were alkyla ted with iodoacetamide. Sequence coverage was 67.9% with PMF data and 67.9% by tandem MS.

................................ ................................ ................................ ..................... 156 Figure 47. Chymotryptic digest sequenc e coverage map for Cry1Ac (DAS

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- 2) by MALDI

- TOF MS and MALDI TOF/TOF. Cys residues were alkylated with iodoacetamide. Sequence coverage was 53.5% with PMF data and 49.0% by tandem MS.

................................ ................................ ................................ ..................... 158 Figure 48. Overall sequence coverage of trypsin and chymotrypsin digests for Cry1Ac (DAS

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- 2) by MALDI

- TOF MS and MALDI TOF/TOF. Overall sequence coverage w as 86.1% with PMF data and 83.2% by tandem MS.

................................ ... 160 Figure 49. Lateral flow strip assay for the Cry1F protein expression in Event DA S

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- 2 and Maverick grain extracts.

................................ ................................ ... 168 Figure 50. Western blot analysis of microbe

- and soybean

- derived Cry1F proteins.

................. 169USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 51. SDS

- PAGE analysis of immunoaffinity

- purified soybean

- derived Cry1F.

.............. 170 Figure 52. SDS

- PAGE analysis of microbe

- and soybean

- derived Cry1F protein stained with GelCode Blue total protein and glycoprotein stains.

................................ .............. 171 Figure 53. Theoretical cleavage of the Cry1F protein with trypsin generated in silico using Protein Analysis Worksheet (PAWS) from Proteometrics LLC.

.......................... 172 Figure 54. Theoretical cleavage of the Cry1F protein with chymotrypsin generated in silico using Protein Analysis Worksheet (PAWS) from Pr oteometrics LLC.

................ 173 Figure 55. Tryptic digest sequence coverage map for Cry1F (DAS

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- 2) by MALDI

TOF MS and MALDI

- TOF/TOF. Cys residues were alkylated with iodoacetamide. Sequence coverage was 65.1% with PMF data and 65.1% by tandem MS.

................................ ................................ ................................ ..................... 175 Figure 56. Chymotryptic digest sequence coverage map for Cry1F (DAS

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- 2) by MALDI

- TOF MS and MALDI TOF/TOF. Cys residues were alkylated with iodoacetamide. Sequence coverage was 53.8% with PMF data and 52.4% by tandem MS.

................................ ................................ ................................ ..................... 177 Figure 57. Overall sequence coverage of trypsin and chymotryps in digests for Cry1F (DAS

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- 2) by MALDI

- TOF MS and MALDI TOF/TOF. Overall sequence coverage was 81.7% with PMF data and 81.7% by tandem MS.

................................ ... 179 Figure 58. Lateral flow strip assay for PAT protein expression in Event DAS

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- 2 and Maverick leaf extracts.

................................ ................................ ............................. 185 Figure 59. SDS

- PAGE and Western blot analysis of microbe

- derived PAT, transgenic soybean Event DAS

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- 2, and non

- transgenic Maverick extracts.

.......................... 186USDA Petition for No nregulated Status of DAS

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Dow AgroSci ences LLC

Contains No Confidential Business Information List of Tables Table 1. Genetic elements from plasmid pDAB9582.

................................ ................................ .. 29 Table 2. Genetic ele ments in DAS

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- 2 soybean.

................................ ................................ .. 32 Table 3. List of probes and their positions in plasmid pDAB9582.

................................ ............. 34 Table 4. Predicted and observed sizes of hybridizing fragments in Southern blot analyses of DAS

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- 2 soybean.

................................ ................................ ................................ 36 Table 5. Results of F2 individual plants tested with LFS and event

- specific PCR.

..................... 66 Table 6. Results of the individual plants from DAS

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- 2 soybean tested with event

specific PCR within segregating BC1F2 generations.

................................ ...................... 67 Table 7. Expression of Cry1Ac in DAS

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- 2 soybean.

................................ .......................... 71 Table 8. Expressio n of Cry1F in DAS

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- 2 soybean.

................................ ............................ 74 Table 9. Expression of PAT in DAS

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- 2 soybean.

................................ ............................... 79 Table 10. Composition analytes.

................................ ................................ ................................ ... 82 Table 11. Summary of the proximate, fiber, and mineral analysis of soybean for age from all sites and literature range.

................................ ................................ ............................. 84 Table 12. Summary of the proximate and fiber analysis of soybean seed from all sites and literature range.

................................ ................................ ................................ ................. 87 Table 13. Summary of the mineral analysis of soybean seed from all sites and literature range.

................................ ................................ ................................ ................................ . 90 Table 14. Summary of the amino acid analysis of soybean seed from all sites and literature range.

................................ ................................ ................................ ................. 94 Table 15. Summary of the fatty acid analysis of soybean seed from all sites and literature range.

................................ ................................ ................................ ............................... 100 Table 16. Summary of the vitamin analysis of soybean seed from all sites and literature range.

................................ ................................ ................................ ............................... 105 Table 17. Summary of the bioactive analysis of soybean seed from all sites and literature range.

................................ ................................ ................................ ............................... 110 Table 18. Agronomic characteristics.

................................ ................................ ......................... 114 Table 19. Summary of agronomic characteristics for the combined site analysis.

..................... 117 Table 20. Insect and disease present by si te.

................................ ................................ .............. 119 Table 21. Disease and insect incidence.

................................ ................................ ...................... 120 Table 22. Germination percentage for DAS

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- 2 and non

- transgenic control soybean.

...... 122 Table 23. Efficacy of DAS

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- 2 soybean compared to ‘Maverick’.

................................ .... 126 Table 24. Estimated margins of exposure (MOE) to non

- target organisms based on high

end exposure estimates (HEEE) for Cry1Ac and Cry1F proteins produced in DAS

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- 2.

................................ ................................ ................................ ................. 128 Table 25. Soybean

- derived PAT extraction buffer composition.

................................ ............... 185USDA Petition for No nregulated Status of DAS

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Dow AgroSci ences LLC

Contains No Confidential Business Information Acronyms and Scientific Terms
ADF Acid detergent fiber ANOVA Analysis of variance APHIS Animal and Plant Health Inspection Service, USDA AtUbi10 Ubiquitin promoter from Arabidopsis th aliana AtuORF1 Open reading frame 1 of Agrobacterium tumefaciens pTi15955 AtuORF23 Open reading frame 23 of Agrobacterium tumefaciens pTi15955 bp Base pair Bt Bacillus thuringiensis CFR US Code of Federal Regulations Cry1 Insecticidal proteins from Bacillus thuringiensis Cry1Ac Cry1Ac insecticidal protein derived from Bacillus thuringiensis subsp. kurstaki cry1Ac(synpro) Coding sequence for Cry1Ac protein Cry1F Cry1F insecticidal protein derived from Bacillus thuringiensis subsp. aizawai cry1Fv3 Coding sequence for Cry1F protein CsVMV Promoter from cassava vein mosaic virus DAS Dow AgroSciences LLC DAS

- 21023

- 5 OECD identifier for the cotton eve nt expressing the Cry1Ac and PAT protein s . DAS

- 21023

- 5 is also described as 3006

- 210

- 23 DAS

- 24236

- 5 OECD identifier for the cotton eve nt expressing the Cry1F and PAT protein s . DAS

- 24236

- 5 is also described as 281

- 24

- 236 DAS

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- 2 OECD identifier for the soybean eve nt expressing the Cry1Ac, Cry1F, and PAT proteins DNA Deoxyribonucleic acid dwt Dry weight of tissue EEC Estimated environmental concentration ELISA Enzyme

- linked immunosorbent assay EPA US Environmental Protection Agency ESI

- LC/MS Electrospray ionization

- liquid chromatography mass spectrometry FDA US F ood and Drug Administration FDR False Discovery Rate FESTF FIFRA Endangered Species Task Force FFDCA Federal Food, Drug, and Cosmetic Act FIFRA Federal Insecticide, Fungicide and Rodenticide Act HAFT Highest average field trial HEEE High

- end exposure estimate ILSI International Life Sciences Institute IPM Integrated Pest Management IRM Insect Resista n ce Management Kb Kilobase pair kDa Kilodalton L Liter LOD Limit of Detection LOQ Limit of QuantitationUSDA Petition for No nregulated Status of DAS

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Dow AgroSci ences LLC

Contains No Confidential Business Information MALDI

- TOF MS Matrix

- assisted laser desorption/ionization time

- of

- flight mass spectrometry Maverick Publicly available soybean line used in transformat ion to produce DAS

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- 2 soybean MOE Margin of exposure NDF Neutral detergent fiber NOEC No observed effect concentration NTO Non

- target organism OECD Organisation for Economic Co

- operation and Development ORF Open reading frame Ori Origin of replication pat Gene from Streptomyces viridochromogenes which encodes the PAT protein PAT Phosphinothricin N

- acetyl transferase PBN US FDA Pre

- market Biotechnology Notice PCR Polymerase chain reaction pDAB 9582 DNA vector carrying the cry1Ac(synpro), cry1Fv3 and pa t expression cassettes PIP Plant

- incorporated protectant PPT Phosphinothricin PTU Plant transcription unit consisting of promoter, gene, and terminator sequences RCB Randomized complete block SCN Soybean cyst nematode SDS

- PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SE Standard error SGF Simulated gastric fluid Std. De v. Standard deviation STMR Supervised trials mean residue subsp subspecies T

- DNA Transfer DNA USDA United States Department of Agriculture USDA ARS United States Department of Agriculture Agricultural Research Service USDA ERS United States Department of Agriculture Economic Research Service USDA NASS United States Department of Agriculture National Agricultural Statistics Service UTR Untranslated region w/v Weight per volumeUSDA Petition for No nregulated Status of DAS

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Dow AgroSci ences LLC

Contains No Confidential Business Information 1. Rational e for the Development of DAS

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- 2
1.1. Basis for the Request for Nonregulated Status 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) to prevent the introduction or dissemination of plant pests into or within the United States. 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 ri sk and therefore should no longer be regulated. Dow AgroSciences LLC (herein referred to as ” DAS ” ) is submitting data for genetical ly engineered insect

- resistant DAS

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- 2 soybean and request s a determination from APHIS that DAS

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- 2 soybean and cross es of DAS

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- 2 with nonregulated soybean lines no longer be considered regulated articles under 7 CFR 340.
1.2. Rational e for the Development of DAS

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- 2 Soybean and Benefits Large scale soybean production began in the U.S. in the 1920s. Since then, the acres planted to soybeans have expanded rapidly due to ideal planting conditions, increased crop rotation, rising yield, and low er production cost ( USDA ERS, 2012 )

. With the exception of 2007, over the last 15 years soybeans have become the second most planted field crop in the U.S. after corn with
more than 70 million acres ( USDA NASS, 2012 )

. Soyb eans are the dominant oilseed in the U.S. accounting for about 90 percent of U.S. oilseed production. In 2011 soybeans were planted on 75 million acres producing 3.056 billion bushels of soybeans with a farm value of more than $35.7 billion. About 1.615 b illion bushels were crushed to yield soybean meal and soybean oil for domestic use and 1.275 billion bushels were processed for export worldwide. These sales accounted for 37% of the world’s soybean trade valued at $21.5 billion ( USDA ERS, 2012 ; USDA NASS, 2012 )

. In the U.S. the majority of soybeans are grown in the north central states with the remaining primarily grown in the southeaster n and Mississippi delta r egion. Insect injury has not traditionally resulted in substantial economic losses in most North American growing regions. However, in recent years, losses have been severe in some southern regions of the U.S. ( Catchot, 2011 ) . Higher market prices for soybeans have lowered economic thresholds for mitigating damage caused by soybean pests, resulting in expansion of the area and number of insecticide treatments applied to soybeans ( USDA ERS, 2011 ) . Recent increases in pes t insect populations in soybean have been observed; increases are attributed by some to global warming or insect adaptation to the soybean plant ( Way, 1994 ) . Lower economic thresholds coupled with higher insect populations favor more aggressive pest management strategies ( Pedigo, 1996 )

. Insect pressure on soybeans in the southern U.S. states is derived primarily from four lepidopteran insect pests. Of these fou r, v elvetbean caterpillar and soybean looper are the most damag ing defoliating insects in the s outh ( Way, 1994 ; Gianessi, 2009 )

. On average about one

third of the soyb ean acres in Georgia, Louisiana, and North Carolina have been sprayed regularl y with insecticides since 1991

. Insecticides are used on approximately 50% of the soybean acreage in Georgia for lepidopteran pests with velvetbean caterpillar being the most targeted pest ( Gianessi et al. , 2002 )

. Approximately 40% of the soybean acreage in Louisiana is treated withUSDA Petition for No nregulated Status of DAS

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Dow AgroSci ences LLC

Contains No Confidential Business Information insecticides for lepidopteran pests, with soybean looper being the main target ( Gianessi et al. , 2002 )

. Chemical insecticides can have limited efficacy in controlling lepidopteran infestations in soybean. Narrow application windows, the emergence of insecticide resistance, and public pressure for reduced pesticide use limit the desirability of this approach to pest management ( Thomas and Boethel, 1994 )

. Soybean looper has developed extensive insecticide resistance ( Thomas and Boethel, 1994 ) ; resistance to pyrethroids is widespread across the southern U.S. ( Felland et al. , 1990 ; Leonard et al. , 1990 )

. Insecticides remain effective against velvetbean caterpillar. However, infestations can quickly reach damaging levels and cause economic loss if insecticides are not applied promptly. Bt crops expressing one or more insecticidal proteins of Bacillus thuringiensis are effective in targeting specific insect pests

. I n creasing adoption rates of Bt corn and cotton since 1996 have coincided with increased yields ( USDA ERS, 2006 ) . Pesticid e use has also decreased since the introduction of these insect

- resistant Bt crops with one study showing an 8% reduction in inse cticide use for adopters of Bt corn as compared to non
- adopters ( USDA ERS, 2006 )

. Impor tantly, Bt crops have also be en credited with increasing yields, even when economic thresholds have not been reached in either Bt

- or non
- Bt

- fields due to regio nal insect population reductions ( Hutchison et al. , 2010 )

. DAS developed a transgenic soybean that is resistant to target lepidopteran pests. The unique identifier for the transgenic soybean, in accordance with the Organization for Economic Co

operation and Development’s (OECD) “Guidance for the Designation of a Unique Identifier for Transgenic Plants” ( OECD, 2004 ) , is DAS

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- 2.
DAS

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- 2 soybean expresses two insecticidal proteins , Cry1Ac and Cry1F , originally from the naturally

- occurring soil bacterium, Bacillus thuringiensis. Cry1A c and Cry1F provide plant protection against several target lepidopteran pests. In addition, DAS

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- 2 soybean expresses the phosphinothricin acetyltransferase (PAT) protein from the soil bacterium Streptomyces viridochromogenes. The PAT protein provid es tolerance to the herbicide glufosinate and was used as a selectable marker during the development of DAS

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- 2 soybean. The transgenes for Cry1Ac, Cry1F and PAT expression were introduced into soybean via Agrobacterium

- mediated transformation to crea te DAS

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- 2 soybean. Cry1Ac and Cry1F are expressed in the leaf tissues of DAS

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- 2 soybean plants throughout the growing season, providing excellent control of target lepidopteran pests of soybeans. DAS

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- 2 soybean offers several potential ben efits, in particular:

 Efficient and environmentally sound alternative to chemical insecticides. DAS

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- 2 soybean offers effective control of multiple lepidopteran pests of soybean

. The availability of DAS

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- 2 soybean could reduce the need for inse ct scouting, preserve beneficial insect population s , and provide increased convenience and greater performance consistency.

 Greater durability and improved insect resistance management. DAS

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- 2 soybean expresses two insecticidal proteins, Cry1Ac and Cry1F, whose modes of action differ with respect to receptor binding. For example, Cry1Ac and Cry1F have been shown to bind to different receptors in the midgut of the target soybean inse ct pest tobaccoUSDA Petition for No nregulated Status of DAS

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Dow AgroSci ences LLC

Contains No Confidential Business Information budworm ( H. virescens ) ( Jurat

- Fuentes and Adang, 2001 )

. Cry1Ac binds to at least three sets of receptors while Cr y1F binds to at least two, only one of which also binds Cry1Ac. The major receptor for Cry1Ac is not recognized by Cry1F ( Jurat

- Fuentes and Adang, 2006 )

. Such incomplete shared binding is expected to lead to incomplete cross

- resistance when resistance is mediated by receptor changes. Bt gene pyramiding offered by DAS

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- 2 soybean offers greater durability than Bt crops carrying a single Bt trait and provides protection against the development o f insect resistance. Due to narrow commercial applicability in the U.S., DAS submitted an application for a FIFRA Section 3 seed increase registration with EPA to support future breeding and seed increase activities in the U.S

. Under a Section 3 seed inc rease registration, commercial sale of DAS

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- 2 soybean in the U.S. is prohibited. If DAS chooses to market DAS

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- 2 soybean for commercial purposes in the U.S. in the future, DAS would seek a FIFRA Section 3 commercial use registration from EPA. D AS

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- 2 soybean is targeted for commercialization in Brazil and Argentina where insect pressure is heavy, resulting in significant yield loss. DAS expects to make submissions beginning 2012 to the regulatory authorities of trade partners for import clea rance and cultivation approval. Deregulation of DAS

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- 2 soybean by APHIS is a prerequisite in achieving regulatory approvals in many of these countries.
1.3. Submission to Other Regulatory Agencies DAS

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- 2 soybean falls within the scope of the FDA policy statement, published in the Federal Register on May 29, 1992, concerning regulation of products derived from new plant varieties, including those developed vi a biotechnology. DAS intend s to submit a pre

- market biotechnology n otification (PBN) to FD A in 2012

. Substances that are pesticides, as defined under the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) [7 U.S.C. §136(u)], are subject to regulation by the Environmental Protection Agency (EPA). Pesticides produced in planta , referred to as plant

- incorporated protectants (PIPs), are also subject to regulation by the EPA under FIFRA. DAS submitted an application for a FIFRA Section 3 seed increase registration of DAS

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- 2 soybean on July 2 7 th , 2012. Concurrent with the Section 3 app lication, DAS also petitioned the EPA for a permanent exemption from the requirement of a tolerance for Cry1F and the genetic material required for its production in soybean, under Federal Food, Drug and Cosmetic Act (FFDCA) Section 408. An exemption from the requirement of tolerance for Cry1Ac in all crops has already been established by the EPA and is published in the Code of Federal Regulations (40 CFR §174.510). Under seed increase registration, commercial sale of DAS

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- 2 soybean in the U.S. is pr ohibited by law. If DAS chooses to cultivate DAS

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- 2 soybean for commercial purposes in the U.S. in the future, DAS will apply for a FIFRA Section 3 commercial use registration with EPA and will develop a specific IRM program in connection with t he co mmercial use registration.
DAS intend s to s ubmit dossiers beginning in 2012 to the regulatory authorities of trade partners for import clearance and production approval which may include Canada, Japan, Korea, Taiwan, European Union, Australia/New Zealand, South Africa, Brazil, Argentina and Mexico.USDA Petition for No nregulated Status of DAS

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Dow AgroSci ences LLC

Contains No Confidential Business Information 2. The Biology of Soybean
2.1. Overv iew of Soybean Biology Refer to the OECD Consensus Document on the Biology of Glycine max (L.) Merr. (Soybean) ( OECD, 2000 ) , for information related to the following aspects of soybean biology:

 general description, including taxonomy, morphology, and the uses of soybean as a crop plant

 agronomic practices

 centers of origin

 reproductive biology

 cultivated Glycine max as a volunteer weed

 ability to cross inter

- species/genus, introgressions into relatives, and interactions with other organisms

 summary of the ecology of Glycine max The vegetative and reproductive stages of a soybean plant are described using the following nomenclature ( Pedersen, 2004 ; Gaska, 2006 ) : Vegetative Stages Reproductive Stages VE Emergence R1 Beginning bloom VC Unrolled unifoliate leaves R2 Full bloom V1 First

- trifoliate R3 Beginning pod V2 Second

- trifoliate R4 Full pod V3 Third

- trifoliate R5 Beginning seed V(n) n th

- trifoliate R6 Full seed R7 Beginning maturity R8 Full maturity

2.2. Characterization of the Recipient Soybean Cultivar The publicly available cultivar ‘Maverick’ was used as the recipient line f or the generation of DAS

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- 2 soybean. Maverick was originally developed by the Missouri and Illinois Agricultural Experiment Stations at the Universities of Missouri and Illinois, respectively, and released in 1996 ( Sleper et al. , 1998 )

. Maverick was developed because of its resistance to the soybean cyst nematode (SCN) and higher yield compared with SCN

- resistant cultivars of similar maturity. Maverick is classified as a late Group III maturity (relative maturity 3.8). Maverick has purple flowers, grey pubescence, brown pods at maturity, and dull yellow seed with buff hila. Maverick is resistant to phytophthora rot but is susceptible to brown stem rot and sudden death syndrome.USDA Petition for No nregulated Status of DAS

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Dow AgroSciences LLC

Contains No Confidential Business Information 3. Development of DAS

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- 2 Soybean
3.1. Description of the Transformation System Transgenic soybean ( Glycine max ) was generated through Agrobacterium

- mediated transformation of soybean cotyledonary node explants . The disarmed Agrobacterium tumefaciens strain EHA101 ( Hood et al. , 1986 ) , carrying the binary vector, pDAB9582, with the selectable marker (

pat ) and the genes of interest ( cry1Ac(synpro) and cry1Fv3 ) within the T

DNA region, was used to initiate transformation

. Agrobacterium

- mediated transformation was carried out using a modified procedure of Zeng et al

. ( 2004 )

. Briefly, s oybean seeds (cv Maverick) were germinated on basal media and cotyledonary nodes were isolated and infected with Agrobacterium EHA101 carrying pDAB9582

. After infect ion with Agrobacterium , cotyledonary nodes were cultured on the cocultivation medium for 5 days before transferring to shoot initiation medium

. All media, including s hoot initiation, shoot elongation, and rooting media were supplemented with cefotaxime, timentin and v ancomycin to inhibit the growth of Agrobacterium . Glufosinate selection (3

- 8 mg/L) was also employed in those media to inhibit the growth of non

- transformed shoots. Selected shoots were transferred to rooting medium for root development and then transferred to soil mix fo r acclimatization of plantlets.
Terminal leaflets of regenerat ed plantlets were painted with glufosinate (0.05%

- 2% w/v) to screen for putative transformants. Th os e pla ntlets exhibiting tolerance were transferred to the greenhouse, allowed to acclimate and then leaf painted again with glufosinate (0.05%

- 2% w/v) to reconfirm herbicide tolerance. The glufosinate tolerant plants were sampled and analyzed at the molecular level to confirm the presence of the selectable marker gene and/or the genes of interest. Specifically, for T0 plants, PCR analysis was performed to verify the absence of the spectinomycin sequence as well as the presence of the cry1Ac (synpro) and c ry1F v 3 genes. Invader  assay ( Kwiatkowski et al. , 1999 ) was carried out for copy number detection for pat , c ry1Ac (synpro) , and cr

y1F v3 genes. Selected T0 plants were allowed to sel f

- fertilize in the greenhouse to give rise to T1 seed. For T1 plants, Invader assay and Southern blot analyse s were performed to determine copy number, integrat ion number, and PTU integrity. Figure 1 shows a plasmid map of pDAB9582. Figure 2 shows a diagram of the intended T

- DNA insert in plasmid pDAB9582. Figure 3 outlines the development of DAS

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- 2 soybean. Figure 4 shows a breeding diagram for DA S

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- 2 soybean including identification of the generations used in various safety assessment studies.USDA Petition for No nregulated Status of DAS

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Dow AgroSciences LLC

Contains No Confidential Business Information Figure 1 . Plasmid map of pDAB9582.

Figure 2
. Diagram of intended T

- DNA insert from plasmid pDAB9582.USDA Petition for No nregulated Status of DAS

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Dow AgroSciences LLC

Contains No Confidential Business Information

Figure 3 . Event sorting and selection process for DAS

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- 2 soybean. Assembled Agrobacterium binary vector pDAB9582 carrying the c ry1Ac (synpro) , c ry1F v3 and pat gene cassettes.

Introduced the c ry1Ac (synpro) , c ry1F v3 and pat gene cassettes into the soybean line Maverick via Agrobacterium tumefaciens

- mediated transformation.

Screened transformed plants based on in vitro tolerance to glufosinate herbicide. Selected T0 soybean plants for regeneration.

Screened regenerated T0 plants for the absence of vector backbone, presence of genes of interest, copy number and tolerance to glufosinate herbicide. Allowed self pollination to produce T1.

Continued evaluation of T1 and subsequent generations resulting from self pollina tion. Evaluated transgenic plants via molecular characterization, protein expression, agronomic performance and resistance to lepidopteran pests.

Selected homozygous plants and carried out crossing and backcrossing for further characterization.

Selected DAS

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- 2 as the lead commercial candidate.USDA Petition for No nregulated Status of DAS

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Dow AgroSciences LLC

Contains No Confidential Business Information Analysis Petition Section(s) DAS

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- 2 Soybean Generations Control

Genetic Characterization 5.2 T1, T2, T3, T4, F2 Non

- transgenic soybean Maverick Segregation Analysis 5.3 F2, BC1F2 None Protein Characterization 6.1.3 , 6.1.6 , 6.2.3 T4 , T5 Non

- transgenic soybean Maverick Protein Expression 6.1.4 , 6.1.7 , 6.2.4 T4 Non

- transgenic soybean Maverick Agronomics 8 T4 Non

- transgenic soybean Maverick Germination/Dormancy 8.3 T4 Non

- transgenic soybean Maverick Composition 7 T4 Non

- transgenic soybean Maverick NTO 9.1.2 T4 Non

- transgenic soybean Maverick Efficacy 9.1.1 T4 Non

- transgenic soybean Maverick

Figure 4 . Breeding diagram of DAS

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- 2 soybean.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information 3.2. Select ion of Comparators for DAS

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- 2 Soybean To ensure the accurate assessment of the impact of transgene insertion on various characteristics of DAS

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- 2 soybean, a proper selection of comparator plants is important. The control plants should have a genetic background similar to that of DAS

- 8141 9

- 2 soybean but lack the transgenic insert. In all cases, the non

- transgenic variety Maverick was used as the control. Maverick is the recipient variety that was transformed to generate DAS

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- 2 soybean. The T0 plants and all subsequent self

- pollinated generations derived post

transformation were essentially genetically identical to Maverick soybean with the exception of the transgenic insert DNA.USDA Petition for No nregulated Status of DAS

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Dow AgroSciences LLC

Contains No Confidential Business Information 4. Donor Genes and Regulatory Sequences
4.1. Identity and Source of Genetic Material in pDAB 9582 DAS

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- 2 soyb ean was generated by Agrobacterium

- mediated transform ation using plasmid pDAB9582 ( Figure 1 ). The T

- DNA insert in the plasmid contains two synthetic gene s from Bacill us thuringiensis , cry1Ac(synpro) and cry1Fv3 , as well as a pat gene from Streptomyces viridochromogenes ( Figure 2 ). A summary of the genetic elements in pDAB9582 is provided in Table 1

. Three gene expression casset tes are present in the T

- DNA region of plasmid pDAB9582 for insertion into soybean. The cry1Fv3 expression cassette is de signed to express a synthetic version of the Cry1F protein . The cry1Fv3 gene is comprised of three parts ; at the 5 ′ end , a t oxin
core that was optimized from the native cry1Fa2 gene originally isolated from Bacillus thuringiensis subsp . aizawai strain PS811 ; in the middle , a very small portion of cry1Ca3 which was originally isolated from B. thuringiensis subsp

. aizawai strain PS811 ; and at the 3 ′ end , a tail that was optimized from the native cry1Ab1 tail originally isolated from B. thuringiensis subsp. Berliner 1715. The cry1Fv3 gene encodes the Cry1F prot ei n that is comprised of 1148 amino acids and has a molecular weight of ~130. 2 kDa. The amino acid sequence of the Cry1F protein is identical to that expressed in cotton event DAS

- 24236

- 5 (also described as 281

- 24

- 236, expressing Cry1F) which was deregulated by USDA APHIS in 2004 ( USDA, 2004 )

. Expr ession of cry1Fv3 is controlled by the AtUbi10 promoter from Arabidopsis thaliana and the AtuORF23 3 ′ UTR sequence from Agrobacterium tumefaciens plasmid pTi15955. The AtUbi10 promoter is known to drive constitutive expression of the genes it controls ( Norris et al. , 1993 )

. The function of AtuORF23 (GenBank Accession: CAA25184) in pTi15955 (GenBank Accession: X00493) was not identified ( Barker et al. , 1983 )

. The cry1Ac(synp ro) expression cassette is designed to express a synthetic version of the Cry1 Ac protein . The cry1 Ac(synpro) gene is comprised of three parts ; at the 5′ end , a toxin core that was optimized from the native cry1Ac1 gene originally isolated from B. thuringi ensis subsp

. kurstaki strain HD73; in the middle , a very small portion of cry1Ca3 which was originally isolated from B. thuringiensis subsp

. aizawai strain PS811 ; and at the 3′ end , a tail that was optimized from the native cry1Ab1 tail originally isolated from B. thuringiensis subsp

. Berliner 1715. The cry1 Ac(synpro) gene encodes the Cry1Ac protein that is comprised of 1156 amin o acids and has a molecular weight of ~130.7 kDa. T he cry1Ac(synpro) gene sequence and the correspondin g Cry1Ac amino acid sequence are identical to that expressed in cotton event DAS

- 21023

- 5 (also described as 3006

- 210

- 23, expressing Cry1Ac) which w as deregulated by USDA APHIS in 2004 ( USDA, 2004 )

. Expression of cry1Ac(synp ro) is controlled by the CsVMV promoter from Cassava Vein Mosaic virus and the AtuORF23 3′ UTR sequence from Agrobacterium tumefaciens plasmid pTi15955. The cassava vein mosaic virus is a double stranded DNA virus which infects cassava plants ( Manihot escu lenta Crantz) and has been characterized as a plant pararetrovirus belonging to the caulimovirus subgroup. The CsVMV promoter is known to drive constitutive expression of the genes it controls ( Verdaguer et al. , 1996 ) . The function of AtuORF23 (GenBank Accession: CAA25184) in pTi15955 (GenBank Accession: X00493) was not identified ( Barker et al. , 1983 )

.USDA Petition for No nregulated Status of DAS

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Dow AgroSciences LLC

Contains No Confidential Business Information The pat expression cassett e is designed to express the phosphinothricin N

- acetyl transferase ( PAT ) protein. The pat gene originates from the common soil bacterium Streptomyces viridochromogenes ( Wohlleben et al. , 1988 )

. Expression of PAT protein in soybean plants confers tolerance to glufosinate and was used as a selectable marker during DAS

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- 2 soybean development . The pat gene encodes a protein of 183 amino acids that has a molecular weight of approximately 20.6 kDa. The pat gene has been widely used both a s a selectable marker and herbicide tolerance trait in previously deregulated products ( USDA, 1996 , 2001 , 2004 , 2005 )

. Expression of the pat gen e is controlled by the CsVMV promoter from cassava vein mosaic virus and AtuORF1 3′ UTR sequence from Agrobacterium tumefaciens plasmid pTi15955. The CsVMV promoter driving pat expression is the same as that driving expression of cry1Ac(synpro). The func tion of AtuORF1 (GenBank Accession: CAA25163) in pTi15955 (GenBank Accession: X00493) was not identified ( Barker et al. , 1983 ) , but its translated amino acid sequence has a significant similarity with an indole

- 3

- lactate synthase (GenBank Accession: AAK90967) from Agrobacterium tumefaciens str. C58.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Table 1 . Genetic elements from plasmid pDAB9582.

Feature Name Feature

Start Feature

Stop Feature Length Description

T

- DNA Border B 1 24 24 T

- DNA Border B sequence r equired for transfer of DNA from Agrobacterium tumefaciens into plant cells ( Barker et al. , 1983 ) Intervening sequence 25 295 271 Non

- specific DNA sequences necessary for cloning

AtUbi10 promoter 296 1617 1322 AtUbi10 promoter along with the 5' untranslated region and intron from Arabidopsis thaliana polyubiquitin 10 (UBQ10) gene ( Norris et al. , 1993 ) Intervening sequence 1618 1625 8 Non

- specific DNA sequences necessary for cloning

cry 1F v3 1626 5072 3447 cry 1F v3 (synthetic version of the cry1F gene from Bacillus thuringiensis subsp

. aizawai strain PS811 ) ( Cardineau et al. , 2001 ; Gao et al. , 2006 ) Intervening sequence 5073 5174 102 Non

- specific DNA sequences necessary for cloning

AtuORF23 3' UTR 5175 5631 457 AtuORF23 3' UTR ( 3' untranslated region (UTR) comprising the transcriptional terminator and polyadenylation site of open reading frame 23 (ORF23) of Agrobacterium tumefaciens p Ti15955 ) ( Barker et al. , 1983 ) Intervening sequence 5632 5694 63 Non

- specific DNA sequences necessary for cloning

CsVMV promoter 5695 6211 517 CsVMV p romoter along with the 5' untranslated region derived from Cassava Vein Mosaic virus ( Verdaguer et al. , 1996 ) Intervening sequence 6212 6220 9 Non

- specific DNA sequences necessary for cloning

cry 1Ac(synpro) 6221 9691 3471 cry 1Ac (synpro) (synthetic version of the cry1Ac gene from Bacillus thuringiensis subsp

. kurstaki strain HD73 ) ( Adang et al. , 1985 ; Gilroy and Wilcox, 1992 ; Cardineau et al. , 2001 ) Intervening sequence 969 2 9724 33 Non

- specific DNA sequences necessary for cloning

AtuORF23 3' UTR 9725 10181 457 AtuORF23 3' UTR (3' untranslated region (UTR) comprising the transcriptional terminator and polyadenylation site of open reading frame 23 (ORF23) of Agrobacterium tumefaciens pTi15955) ( Bark er et al. , 1983 ) Intervening sequence 10182 10295 114 Non

- specific DNA sequences necessary for cloning

CsVMV promoter 10296 10812 517 CsVMV p romoter along with the 5' untranslated region derived from Cassava Vein Mosaic virus ( Verdaguer et al. , 1996 ) Intervening sequence 10813 10819 7 Non

- specific DNA sequences necessary for cloningUSDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Feature Name Feature

Start Feature

Stop Feature Length Description

pat 10820 11371 552 pat ( synthetic version of the phosphinothri cin acetyl transferase gene from Streptomyces viridochromogenes ) ( Wohlleben et al. , 1988 ) Intervening sequence 11372 11473 102 Non

- specific DNA sequences necessary for cloning

AtuORF1 3' UTR 11474 12177 704 AtuORF1 3' UTR ( 3' untranslated region (UTR) comprising the transcriptional terminator and polyadenylation site of open reading frame 1 (ORF1) of Agrobacterium tumefaciens pTi15955 ) ( Barker et al. , 1983 ) Intervening sequence 12178 12405 228 Non

- specific DNA sequences necessary for cloning

T

- DNA Border A 12406 12429 24 T

- DNA Border A sequence r equired for transfer of T

DNA insert from Agrobacterium tumefaciens into plant cells ( Barker et al. , 1983 ) Intervening sequence 12430 12448 19 Non

- specific DNA sequences necessary for cloning

T

- DNA Border A 12449 12472 24 T

- DNA Border A sequence r equired for transfer of T

DNA insert from Agrobacterium tumefaciens into plant cells ( Barker et al. , 1983 ) Intervening sequence 12473 12759 287 Non

- specific DNA sequences necessary for cloning

T

- DNA Border A 12760 12783 24 T

- DNA Border A sequence r equired for transfer of T

DNA insert from Agrobacterium tumefaciens into plant cells ( Barker et al. , 1983 ) Intervening sequence 12784 13162 379 Plasmid backbone sequences from gram
- negative bacteria broad host

- range RK2 plasmid ( Stalker et al. , 1981 )

ori 13163 14182 1020 ori ( Replication origin sequences from gram
- negative bacteria broad host

- range RK2 plasmid ) ( Stalker et al. , 1981 ) Intervening sequence 14183 14727 545 Plasmid backbone sequences from gram
- negative bacteria broad host

- range RK2 plasmid ( Stalker et al. , 1981 ) trfA 14728 15876 1149 trfA ( Plasmid replication sequences for Trf A protein from gram
- negative bacteria broad host

- range RK2 plasmid ) ( Stalker et al. , 1981 ) Intervening sequence 15877 17080 1204 Plasmid backbone sequences from gram
- negative bacteria broad host

- range RK2 plasmid ( Stalker et al. , 1981 ) Spec R 17081 17869 789 Spec R (s pectinomycin resistance gene from Escherichia coli Tn7 transposon ) ( Fling et al. , 1985 ) Intervening sequence 17870 18143 274 Plasmid backbone sequences for cloningUSDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information 5. Genetic Characterization
5.1. Overview of Genetic Characterization Molecular characterization of DAS

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- 2 soybean was conducted b y Southern blot analyse s and DNA sequencing

. The r esults demonstrate d that the transgene insert in DAS

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- 2 soybean occurred as a single integration of the T

- DNA insert from plasmid pDAB9582 , including a single, intact copy of each of the plant t ranscription u ni ts (PTUs) for the cry1F v3 , cry1Ac (synpro ) , and pat genes. In addition, a minor (<100 bp) fragment of the cry1Ac(synpro) gene was identified at the 5′ end of the T

- DNA insert in a complementary orientation. The sequence of the insert in DAS

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- 2 soybean was confirmed and the genetic elements identified in DAS

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- 2 soybean are provided in Table 2

. The full insert w a s stably integrated and inherited across breeding generations, and no plasmid backbone sequences are present in DAS

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- 2 soybean. Detailed Southern blot analys es were conducted using probes specific to the gene s , promoters, terminators, and other regul atory elements contained in the transformation plasmid pDAB9582. Locations of each probe on plasmid pDAB9582 are described in Table 3 and Figure 5

. The expected and observed fragment sizes with specific digest and probe combinations, based on the known restriction enzyme sites of plasmid pDAB9582 and the actual T

- DNA insert in DAS

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- 2 soybean are shown in Figure 6 and Figure 7 , respectively. The Southern blot analyses described here made use of two types of restriction fragments: a) internal fragments generated by known restriction enzyme recognition sites contained within the T

- DNA insert of pDAB9582, and b) border fragme nts generated by one known restriction enzyme recognition site located within the T

- DNA insert and a nother site located in the soybean genome flanking the insert. Border fragment sizes vary by event s because they rely on the location of the restriction
en zyme recognition sites in the DNA sequence of flanking genomic region. Since integration sites are unique for each event, border fragments provide a mean s to determine the number of transgene insertions and to specifically identify the event.
Genom ic DNA for Southern blot analyse s was prepared from leaf material of individual DAS

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- 2 soybean plants from five distinct breeding generations. Genomic DNA from leaves of non

- transgenic variety Maverick was used as the control material. Plasmid DNA of pDAB9 582 added to genomic DNA from the non
- transgenic variety Maverick served as the positive c ontrol for Southern blot analyse s. Materials and methods used for Southern analyses are further described in Appendix 1

. The expected restriction fragments of the inserted DNA are shown in Table 4 and Figure 7

. Southern blot analysis results are shown in Figure 8 to Figure 28

. Southern blot analyses showed that DAS

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- 2 soybean contains a single intact copy of each of the PTUs for the cry1Fv3, cry1Ac(synpro), and pat genes from pDAB9582 (Section 5.2.1 and Section 5.2.2 )

. In addition, a minor (<100 bp) fragment of the cry1Ac(synpro) gene was identified at the 5′ end of the T

- DNA insert

. No plasmid backbone sequences were detected in DAS

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- 2 soybean (Section 5.2.3 )

. The hybridization patt erns across five generations of DAS

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- 2 soybean (T1, T2, T3, T4, and F2) were identical, indicating that the insert is stably integrated in the soybean genome (Section 5.2.4 ) . The inheritance of DAS

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- 2 soybean insert in segregating generations was investigated using event

- specific PCR and detection of the PAT protein expression. All results confirmed the predicted inheritance of the transgene in a single locus (Section 5.3 ).USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Table 2 . Genetic elements in DAS

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- 2 soybean.

Feature Name Feature

Start Feature

Stop Feature Length Description

5' Flanking border 1 1297 1297 Soybean genomic DNA flanking the 5' end of the insert in DAS

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- 2 soybean
Re

- arranged sequence 1298 132 1 2 4 Re

- arranged DNA fragment at the 5' end of the insert

cry1Ac(synpro) partial fragment 132 2 1419 9 8 Complementary cry1Ac(synpro ) partial fragment at the 5' end of the inse rt that is 99% identical to 1990

- 208 7 bp of the full

- length cry1Ac(synpro) gene
Re

- arranged sequence 1420 1432 13 Re

- arranged DNA fragment at the 5' end of the insert

Partial T

- DNA Border B 1433 1433 1 Last nucleotide from T

- DNA B order B which is required for transfer of DNA from Agrobacterium tumefaciens into plant cells ( Barker et al. , 1983 ) Intervening sequence 1434 1704 271 Non

- specific DNA sequences necessary for cloning

AtUbi10 promoter 1705 3026 1322 AtUbi10 promoter along with the 5' untranslated region and intron from Arabidopsis thaliana polyubiquitin 10 (UBQ10) gene ( Norris et al. , 1993 ) Intervening sequence 3027 3034 8 Non

- specific DNA sequences necessary for cloning

cry 1F v3 3035 6481 3447 cry 1F v3 (synthetic version of the cry1F gene from Bacillus thuringiensis subsp. aizawai strain PS811 ) ( Cardineau et al. , 2001 ; Gao et al. , 2006 ) Intervening sequence 6482 6583 102 Non

- specific DNA sequences necessary for cloning

AtuORF23 3' UTR 6584 7040 457 AtuORF23 3' UTR ( 3' untranslated region (UTR) comprising the transcriptional terminator and polyadenylation site of open reading frame 23 (ORF23) of Agrobacterium tumefaciens pTi15955 ( Barker et al. , 1983 ) Intervening sequence 7041 7103 63 Non

- specific DNA sequences necessary for cloning

CsVMV promoter 7104 7620 517 CsVMV p romoter along with the 5' untranslated region derived from Cassava Vein Mosaic virus ( Verdaguer et al. , 1996 ) Intervening sequence 7621 7629 9 Non

- specific DNA sequences necessary for cloning

cry 1Ac(synpro ) 7630 11100 3471 cry 1Ac (synpro) (synthetic version of the cry1Ac gene from Bacillus thuringiensis subsp

. kurstaki strain HD73 ) ( Adang et al. , 1985 ; Gilroy and Wilcox, 1992 ; Cardineau et al. , 2001 ) Intervening sequence 11101 11133 33 Non

- specific DNA sequences necessary for cloningUSDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Feature Name Feature

Start Feature

Stop Feature Length Description

AtuORF23 3' UTR 11134 11590 457 AtuORF23 3' UTR (3' untranslated region (UTR) comprising the transcriptional terminator and polyadenylation site of open reading frame 23 (ORF23) of Agrobacterium tumefaciens pTi15955) ( Barker et al. , 1983 ) Intervening sequence 11591 11704 114 Non

- specific DNA sequences necessary for cloning

CsVMV promoter 11705 12221 517 CsVMV p romoter along with the 5' untranslated region derived from Cassava Vein Mosaic virus ( Verdaguer et al. , 1996 ) Intervening sequence 12222 12228 7 Non

- specific DNA sequences necessary for cloning

pat 12229 12780 552 pat (synthetic version of phosphinothricin acetyl transferase gene from Streptomyces viridochromogenes ) ( Wohlleben et al. , 1988 ) Intervening sequence 12781 12882 102 Non

- specific DNA sequences necessary for cloning

AtuORF1 3' UTR 12883 13586 704 AtuORF1 3' UTR ( 3' untranslated region (UTR) comprising the transcriptional terminator and polyadenylation site of open reading frame 1 (ORF1) of Agrobacterium tumefaciens pTi15955 ) ( Barker et al. , 1983 ) Intervening sequence 13587 13784 198 Non

- specific DNA sequences necessary for cloning
Re

- arranged sequence 13785 13793 9 Re

- arranged DNA fragment at the 3' end of the insert 3' Flanking border 13794 15172 1379 Soybean genomic DNA flanking the 3' end of the insert in DAS

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Contains No Confidential Business Information Table 3
. List of probes and their positions in plasmid pDAB9582
.

Probe Name Location in pDAB9582 Size (bp) AtUbi10 Promoter cry1F

- 5 ′ 29

- 1620 1631

- 3376 1592 17 46 cry1F

- 3 ′ A 3354

- 3874 521 cry1F

- 3 ′ B 3851

- 5081 1231 At u ORF23

- A 3′ UTR 5080

- 5630 551 CsVMV Promoter 5664

- 6250 587 cry1Ac

- 5 ′ 6221

- 7940 1720 cry1Ac

- 3 ′ A 7918

- 8352 435 cry1Ac

- 3 ′ B 8329

- 9778 1450 At u ORF23

- B 3′ UTR CsVMV Promoter 9714

- 10180 10292

- 10817 467 526 pat 10820

- 11371 552 AtuORF1 3′ UTR 11342

- 12412 1071 Backbone 3 12389

- 13178 790 Ori 12804

- 14182 1379 Backbone 2 14149

- 15876 1 728 Backbone 1 15846

- 17079 1234 SpecR 1 7081

- 1 8116 1 036 Backbone 4 17426

- 18143 718USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 5
. Location of the probes on pDAB9582 used i n Southern blot analysis of DAS

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- 2 soybean. Note: * Since the CsVMV promoter element in the cry1Ac(synpro) and pat PTUs are 100% identical based on the nucleotide sequences, CsVMV Promoter probe generated from cry1Ac(synpro) PTU was used to hybridize CsVMV promoter in pat PTU.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Table 4 . Predicted and observed sizes of hybridizing fragments in Southern blot analyses of DAS

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- 2 soybean.

Probe Restric tion

E nzyme Sample Lane Expected Fragment S izes (bp) 1 Observed

Fragment S izes (bp) 2 Fig ure

Number

AtUbi10 promoter Nco I pDAB9582 2 3071 ~3100 Figure 8 A Maverick 3 none none DAS

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- 2 4

- 18 >1737 ~7500
Sph I pDAB9582 2 18143 ~18100 Figure 8 B Maverick 3 none none DAS

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- 2 4

- 18 >12346 ~18100

cry1F

- 5′ Nco I pDAB9582 2 14544 ~14500 Figure 9 A Maverick 3 none none DAS

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- 2 4

- 18 >10759 ~14500
Sph I pDAB9582 2 18143 ~18100 Figure 9 B Maverick 3 none none DAS

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- 2 4

- 18 >12346 ~18100

cry1F

- 3′A Nco I pDAB9582 2 14544 ~14500 Figure 10 A Maverick 3 none none DAS

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- 2 4

- 18 >10759 ~14500
Sph I pDAB9582 2 18143 ~18100 Figure 10 B Maverick 3 none none DAS

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- 2 4

- 18 >12346 ~18100

cry1F

- 3′B Nco I pDAB9582 2 14544 ~14500 Figure 11 A Maverick 3 none none DAS

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- 2 4

- 18 >10759 ~14500
Sph I pDAB9582 2 18143 ~18100 Figure 11 B Maverick 3 none none DAS

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- 2 4

- 18 >12346 ~18100

AtuORF23

- A 3′ UTR Nco I pDAB9582 2 14544 ~14500 Figure 12 A Maverick 3 none none DAS

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- 2 4

- 18 >10759 ~14500
Sph I pDAB9582 2 18143 ~18100 Figure 12 B Maverick 3 none none DAS

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- 2 4

- 18 >12346 ~18100USDA Petition for No nregulated Status of DAS

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Dow AgroSciences LLC

Contains No Confidential Business Information Probe Restric tion

E nzyme Sample Lane Expected Fragment S izes (bp) 1 Observed

Fragment S izes (bp) 2 Fig ure

Number

CsVMV Promoter Nco I pDAB9582 2 14544 ~14500 Figure 13 A Maverick 3 none none DAS

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- 2 4

- 18 >10759 ~14500
Sph I pDAB9582 2 18143 ~18100 Figure 13 B Maverick 3 none none DAS

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- 2 4

- 18 >12346 ~18100

cry1Ac

- 5′ Nco I pDAB9582 2 14544 ~14500 Figure 14 A Maverick 3 none none DAS

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- 2 4

- 18 >10759 ~14500
Sph I pDAB9582 2 18143 ~18100 Figure 14 B Maverick 3 none none DAS

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- 2 4

- 18 >12346 ~18100

cry1Ac

- 3′A Nco I pDAB9582 2 14544 ~14500 Figure 15 A Maverick 3 none none DAS

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- 2 4

- 18 >10759, >1737 ~14500, ~7500
Sph I pDAB9582 2 18143 ~18100 Figure 15 B Maverick 3 none none DAS

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- 2 4

- 18 >12346 ~18100

cry1Ac

- 3′B Nco I pDAB9582 2 14544 ~14500 Figure 16 A Maverick 3 none none DAS

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- 2 4

- 18 >10759 ~14500
Sph I pDAB9582 2 18143 ~18100 Figure 16 B Maverick 3 none none DAS

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- 2 4

- 18 >12346 ~18100

AtuORF23

- B 3′ UTR Nco I pDAB9582 2 14544 ~14500 Figure 17 A Maverick 3 none none DAS

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- 2 4

- 18 >10759 ~14500
Sph I pDAB9582 2 18143 ~18100 Figure 17 B Maverick 3 none none DAS

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- 2 4

- 18 >12346 ~18100USDA Petition for No nregulated Status of DAS

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Dow AgroSciences LLC

Contains No Confidential Business Information Probe Restric tion

E nzyme Sample Lane Expected Fragment S izes (bp) 1 Observed

Fragment S izes (bp) 2 Fig ure

Number

pat Nco I pDAB9582 2 14544 ~14500 Figure 18 A Maverick 3 none none
DAS

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- 2 4

- 18 >10759 ~14500

Sph I pDAB9582 2 18143 ~18100 Figure 18 B Maverick 3 none none
DAS

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- 2 4

- 18 >12346 ~18100

AtuORF1 3′ UTR Nco I pDAB9582 2 14544 ~14500 Figure 19 A Maverick 3 none none DAS

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- 2 4

- 18 >10759 ~14500

Sph I pDAB9582 2 18143 ~18100 Figure 19 B Maverick 3 none none DAS

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- 2 4

- 18 >12346, >150 ~18100, ~4100

AtUbi10 promoter

Not I/ Sph I

(Release cry1Fv3 PTU) pDAB9582 2 5393, 4697 ~5400, ~4700 Figure 20 A Maverick 3 none none DAS

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- 2 4

- 18 5393, >353 ~5400, ~6500

cry1F

- 5′ pDAB9582 2 5393 ~5400 Figure 20 B Maverick 3 none none DAS

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- 2 4

- 18 5393 ~5400

cry1F

- 3′A pDAB9582 2 5393, 4550 ~5400, ~4600 Figure 21 A Maverick 3 none none DAS

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- 2 4

- 18 5393, 4550 ~5400, ~4600

cry1F

- 3′B pDAB9582 2 5393, 4550 ~5400, ~4600 Figure 21 B Maverick 3 none none DAS

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- 2 4

- 18 5393, 4550 ~5400, ~4600

AtuORF23

- A 3′ UTR pDAB9582 2 5393, 4550 ~5400, ~4600 Figure 22 A Maverick 3 none none DAS

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- 2 4

- 18 5393, 4550 ~5400, ~4600USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Probe Restric tion

E nzyme Sample Lane Expected Fragment S izes (bp) 1 Observed

Fragment S izes (bp) 2 Fig ure

Number

CsVMV Promoter

Not I/ Sph I

(Release cry1Ac(syn pro) PTU) pDAB9582 2 4550, 1970 ~4600, ~2000 Figure 22 B Maverick 3 none none DAS

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- 2 4

- 18 4550, 1970 ~4600, ~2000

cry1Ac

- 5′ pDAB9582 2 4550 ~4600 Figure 23 A Maverick 3 none none DAS

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- 2 4

- 18 4550 ~4600

cry1Ac

- 3′A pDAB9582 2 5393, 4550 ~5400, ~4600 Figure 24 A Maverick 3 none none DAS

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- 2 4

- 18 >353, 4550, 5393 ~6500, ~4600,

~5400

cry1Ac

- 3′B pDAB9582 2 5393, 4550 ~5400, ~4600 Figure 24 B Maverick 3 none none DAS

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- 2 4

- 18 5393, 4550 ~5400, ~4600

AtuORF23

- B 3′ UTR pDAB9582 2 5393, 4550 ~5400, ~4600 Figure 23 B Maverick 3 none none DAS

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- 2 4

- 18 5393, 4550 ~5400, ~4600

pat Not I/ Sph I

(Release pat PTU) pDAB9582 2 1970 ~2000 Figure 25 A Maverick 3 none none DAS

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- 2 4

- 18 1970 ~2000
AtuORF1 3′ UTR pDAB9582 2 1453, 1970, ~1500, ~2000 Figure 25 B Maverick 3 none none DAS

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- 2 4

- 18 >150, 1970 ~4000, ~2000

Ori Sph I pDAB9582 2 18143 ~18100 Figure 26 A Maverick 3 none none DAS

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- 2 4

- 18 none none
SpecR Nco I pDAB9582 2 3071 ~3100 Figure 26 B Maverick 3 none none DAS

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- 2 4

- 18 none none

Backbone 1 Nco I pDAB9582 2 14544, 3071, 528 ~14500, ~3100,

~500 Figure

27 A Maverick 3 none none DAS

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- 2 4

- 18 none noneUSDA Petition for No nregulated Status of DAS

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Dow AgroSciences LLC

Contains No Confidential Business Information Probe Restric tion

E nzyme Sample Lane Expected Fragment S izes (bp) 1 Observed

Fragment S izes (bp) 2 Fig ure

Number

Backbone 2 Sph I pDAB9582 2 18143 ~18100 Figure 27 B Maverick 3 none none DAS

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- 2 4

- 18 none none
Backbone 3 + 4 Nco I pDAB9582 2 14544, 3071 ~14500, ~3100 Figure 28 Maverick 3 none none DAS

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- 2 4

- 18 none none 1 Expected fragment sizes are based on the plasmid map of pDAB9582 ( Figure 6 ) and the T

- DNA insert in DAS

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- 2 soybean ( Figure 7 ). 2 Observed fragment sizes are considered approximately from these analyses and are based on the indicated sizes of the DIG

- labeled DNA Molecular Weight Marker fragments. Due to the incorporation of DIG molecules for visualization, the Marker fragments typically run approximately 5

- 10% larger than their actual indicated molecular weight.

Figure 6

. Plasmid map of pDAB9582 with selected restriction enzyme sites used for Southern analysis. pDAB9582 18143 bp trfA

pat specR

c ry1Ac( synpro ) c ry1Fv3 AtUbi10 promoter

CsVMV promoter CsVMV promoter Ori Rep

AtuORF23 3' UTR AtuORF1 3' UTR

AtuORF23 3' UTR T

- DNA Border A T

- DNA Border A T

- DNA Border A T

- DNA Border B

Sph I (12234) Nco I (1625) Nco I (16169) Nco I (16697)
Not I (241)

Not I (5634)

Not I (10184) Not I (10224) Not I (10264) Not I (13687)USDA Petition for No nregulated Status of DAS

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Dow AgroSciences LLC

Contains No Confidential Business Information Figure 7
. Actual T

- DNA insert in DAS

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- 2 soybean with restriction enzymes used for DNA digestion and sizes of the expected hybridization bands.

5.2. Analysis of the Insert and Its Genetic Elements
5.2.1. Number of Insertion Sites The restriction enzymes Nco I and Sph I wer e chosen to determine the number of insertions in DAS

- 81419

- 2 soybean ( Figure 7 ). Probes derived from the DNA sequences of AtUbi10 Promoter, cry1Fv3 (split into three overlapping regions cry1F

- 5′, cry1F

- 3′A, and cry1F

- 3′B), AtuORF23 3′ UTR ( probes A and B contain small pieces of different intervening sequences at the 5 ′ and 3′ ends of the AtuORF23 3′ UTR sequence), CsVMV promoter, cry1Ac(synpro) (split into three overlapping regions cry1 Ac

- 5′, cry1 Ac

- 3′A, and cry1 Ac

- 3′B), pat , and AtuORF1 3′UTR were then hybridized to the digested genomic DNA to determine the n umber of insertion sites in DAS

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- 2 soybean. When digested with restriction enzyme Nco I and in dependently hybridized with the cry1F

- 5 ′ , cry1F

- 3 ′A, cry1F

- 3 ′B, AtuORF23

- A 3′ UTR , CsVMV Promoter

, cry1Ac

- 5′, cry1Ac

- 3 ′A, cry1Ac

- 3 ′ B , AtuORF23

- B 3′ UTR , pat , and AtuORF1 3′ UTR probes, a single hybridization band of ~14500 bp was detected in DAS

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- 2 soybean samples, consistent with the predicted size of >10759 bp for the Nco I fragment as shown in Table 4 and Figure 9 A

- Figure 19 A. When Nco I digested samples were hybridized with AtUbi10 Promoter, a single band of ~7500 bp was detected in DAS

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- 2 soybean samples, consistent with the expected result of > 1737 bp ( Figure 8 A). When digested with restriction enzyme Sph I and probed with the AtUbi10 Promoter, cry1F

- 5 ′ , cry1F

- 3 ′A, cry1F

- 3 ′B, AtuORF23

- A 3′ UTR , CsVMV Promoter

, cry1Ac

- 5′, cry1Ac

- 3 ′A,USDA Petition for No nregulated Status of DAS

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Dow AgroSciences LLC

Contains No Confidential Business Information cry1Ac

- 3 ′B , AtuORF23

- B 3′ UTR , and pat probes individually, a single band of ~18100 bp was detected in DAS

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- 2 soybean samples , consistent with the expected result of > 12346 bp ( Table 4 , Figure 8 B

- Figure 18 B ). When Sph I digested samples were hybridized with AtuORF1 3′ UTR probe, two fragments of ~18100 bp and ~4100 bp were detected in DAS

- 81419

- 2 soybean samples , consistent with the expected r esult of >12346 bp and >15 0 bp ( Figure 19 B). Specific hybridization bands were detected in all of the positive samples at the expected sizes, while no specific hybridi zation bands were detected in any of the negative control samples. All these data indicate that there is a single insertion of the T

- DNA containing all the expected elements from pDAB9582 in DAS

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- 2 soybean genome.

5.2.2. Structure of the Insert and Genetic Elements According to the plasmid and T

- DNA restriction maps shown in Figure 6 and Figure 7 , the plant transcription units (PTUs) for cry1F v3 , cry1Ac (synpro) , and pat could be released by restriction digestion with Not I

/ Sph I. These digestions were performed to characterize the structure of the insert in DAS

- 81419

- 2 soybean. When digested with Not I/ Sph I and hybridized with AtUbi10 Promoter probe, two bands of ~5400 bp and ~6500 bp were detected in all DAS

- 81419

- 2 soybean and the positive control samples , consistent with the expected sizes of 5393 bp and > 353 bp for the cry1F v 3 PTU and the border fragment that the AtUbi10 promoter probe extended into, respectively ( Figure 20 A). When digested with Not I/ Sph I and hybridized with cry1F

- 5 ′ probe, a single band of ~5400 bp
was detected in DAS

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- 2 soybean and the positive control samples , consistent with the expected size of 5393 bp for the cry1F v3 PTU ( Figure 20 B).
S ince both cry1Fv3 and cry1Ac(synpro) genes contain synthetic cry 1Ab sequence with 89% identity at nucleotide level, it is expected that the probes derived from the 3 ′ region of the cry1F gene ( cry1F

- 3′A, cry1F

- 3′ B ) would cross

- hybridize with the respective DNA fragments containing the 3 ′ region of the cry1Ac gene. When digested with Not I/ Sph I and hybridized with cry1F

- 3′A and cry1F

- 3′B probes individually , two bands of ~5400 bp and ~4 6 00 bp which are consistent with the expected bands wi th size s of 5393 bp for cry1Fv3 PTU and 4550 bp for cry1Ac(synpro) PTU ( Figure 21 ), were detected in DAS

- 81419

- 2 soybean and the positive control samples . Since the A tuORF23 3 ′ UTR element in the cry1F v3 and cry1Ac (synpro) PTUs are 100% identical based on the nucleotide sequences ( Figure 5 ), it is expected that probes derived from AtuORF23 3 ′ UTR element (AtuORF23

- A 3 ′ UTR and AtuORF23

- B 3 ′ UTR) would cross

- hybridize with the respective DNA fragments containing the AtuORF23 3 ′ UTR. When digested with Not I/ Sph I and hybridized with AtuORF23

- A 3′ UTR probe, two hybridization bands of 5393 bp and 4550 bp are expected for DAS

- 81419

- 2 soybean and positive control samples. As shown in Figure 22 A, two bands of ~5400 bp and ~4600 bp were detected in DAS

- 81419

- 2 soybean and the positive control samples, consistent with the predicted sizes for cry1F v3 and cry1Ac (synpro) PTUs. These data suggest that an intact cry1F v3 PTU is present in all generations tested. Since the CsVMV promoter element in the cry1Ac (synpro ) and pat PTUs are 100% identical based on the nucleotide sequences ( Figure 5 ), it is e xpected that probe derived from CsVMV promoter (CsVMV Promoter probe) would cross

- hybridize with the respective DNA fragments containing the CsVMV promoter element. When digested with Not I/ Sph I and hybridized withUSDA Petition for No nregulated Status of DAS

- 81419

- 2 Soybean Page 43 of 214

Dow AgroSciences LLC

Contains No Confidential Business Information CsVMV Promoter probe, two hybridization ba nds of 4550 bp and 1970 bp are expected for DAS

- 81419

- 2 soybean and the positive control samples. As shown in Figure 22 B, two bands of ~4600 bp and ~2000 bp were detected in DAS

- 81419

- 2 soybean and the positive control samples, consistent with the predicted sizes for cry1Ac (synpro) and pat PTUs. When digested with Not I/ Sph I and hybridized with cry1Ac

- 5′ probe, a single ba nd of ~4600 bp was detected in DAS

- 81419

- 2 soybean and the positive control samples , consistent with the expected size of 4550 bp for the cry1Ac (synpro) PTU ( Figure 23 A). According to the sequence information , a 98

- bp partial cry1Ac (synpro) fragment was also identified at the 5′end of the T

- DNA insert in DAS

- 81419

- 2 soybean ( Figure 7 ). This 98

- bp partial cry1Ac (synpro) fragment is 99% identical to 1990

- 2087 bp region of the cry1Ac (synpro) gene in a complementary orientation. As a consequence, when digested with Not I/ Sph I and hybridized with the cry1Ac

- 3 ′A probe, two hybridization bands, one of ~4600 bp which is consistent with the expected size of 4550 bp for full length cry1Ac (synpro) PTU and the other one of ~ 6500 bp which is consistent with the expected result of >353 bp for the DNA fragment containing the partial cry1Ac (synpro) , were detected in DAS

- 81419

- 2 soybean samples In addition, a very faint band of ~5400 bp was also detected in DAS

- 81419

- 2 soybean and the positive control samples, consistent with the expe cted size of 5393 bp for cry1Fv3 PTU which is expected to be cross

- hybridized by the cry1Ac

- 3 ′A probe ( Figure 24 A ). When digested with Not I/ Sph I and hybridized with cry1Ac

- 3 ′ B probe , two hybridization bands of 5393 bp and 4550 bp are expected for DAS

- 81419

- 2 soybean samples along with positive control sample. As shown in Figure 24 B, two bands of ~5400 bp and ~4600 bp were detected in DAS

- 81419

- 2 soybean and positive control samples, consistent with the predicted sizes for cry1Ac (synpro) and cry1Fv3 PTUs. When digested with Not I/ Sph I and hybridized with AtuORF23

- B 3′ UTR probe, two bands of 5393 bp and 4550 bp are expected for DAS

- 81419

- 2 soybean and positive control samples. As shown in Figure 23 B, two bands of ~5400 bp and ~4600 bp were d etected in DAS

- 81419

- 2 soybean and positive control samples, consistent with the predicted sizes for cry1F v3 and cry1Ac (synpro) PTUs. These data suggest that an intact cry1Ac (synpro) PTU and a small partial cry1Ac(synpro) fragment is pre sent in all generations tested. When digested with Not I/ Sph I and hybridized with pat probe, a single band of ~2000 bp was detected in all DAS

- 81419

- 2 soybean and the positive control samples , consistent with the predicted size of 1970 bp for the pat PTU ( Figure 25 A). When digested with Not I/ Sph I and hybridized with AtuORF1 3′ UTR probe, two bands of ~4000 bp and ~2000 bp were detected in all DAS

- 81419

- 2 soybean and the positive c ontrol samples, consistent with expected bands of >150 bp and 1970 bp ( Figure 25 B). These data suggest that an intact pat PTU is pre sent in all generations tested. Hybridization bands of the expected sizes were detected in all of the positive samples , while no specific hybridization band was detected in the non

- transgenic soybean negative control samples as expected. The hybridization pattern is consistent across a ll generations with all the tested restriction enzyme and probe combinations.USDA Petition for No nregulated Status of DAS

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- 2 Soybean Page 44 of 214

Dow AgroSciences LLC

Contains No Confidential Business Information Taken together, the Southern blot analyses reveal that the single insert in DAS

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- 2 soybean contains an intact PTU for each of the cry1F v3, cry1Ac (synpro) , and pat genes alon g with a partial cry1Ac (synpro) fragment at the 5 ′ end of the insert.

5.2.3. Absence of Plasmid Backbone Sequences To verify that no plasmid vector backbone sequences exist in DAS

- 81419

- 2 soybean, six probes covering the entire region outside of the T

- DNA in pDAB 9582 were hybridized with Nco I or Sph I digested samples ( Table 3 , Figure 5 ). The hybridization results demonstrated that no specific hybridization signals were detected in any DAS

- 81419

- 2 samples or the negative control samples, while hybridization fragments of the expected size were detected in the respective positive controls ( Figure 26

- Figure 28 ). These data indicate that no backbone sequences from pDAB9582 have been integrated into DAS

- 81419

- 2 soybean.

5.2.4. Stability of the Insert Across Generations Southern blot analyses were conducted with samples across five distinct generations (T1, T2, T 3 , T 4, and F2) of DAS

- 81419

- 2 soybean . Prior to ini tiation of Southern blot analyse s, all plants were tested for PAT protein expression usi ng a lateral flow strip assay to identify PAT expression

- po sitive plants. All of the genetic element probes: AtUbi10 Promoter, cry1F

- 5 ′ , cry1F

- 3 ′A, cry1F

- 3 ′B, AtuORF23

- A 3′ UTR , CsVMV Promoter

, cry1Ac

- 5′, cry1Ac

- 3 ′A, cry1Ac

- 3 ′B , AtuORF23

- B 3′ UTR , pat , and AtuORF1 3′ UTR , as well as the probes covering the entire backbone regions of plasmid pDAB9582 , were hybridized with the DAS

- 81419

- 2 soybean samples . Results across all DAS

- 81419

- 2 soybean samples in the five generations were consistent with what were expected ( Table 4 ), indicating stable integration and inheritance of the intact, single copy transgene insert across multiple generations of DAS

- 81419

- 2 soybean.

5.2.5. C onclusions The Southern blot analyses confirm that DAS

- 81419

- 2 s oybean contains a single insertion of the T

- DNA from plasmid pDAB9582 with each of the intact PTUs for cry1Fv3, cry1Ac(synpro), and pat genes

. In addition, a minor (<100 bp) fragment of the cry1Ac(synpro) gene was identified at the 5′ end of the T

- DNA ins ert. Identical hybridization patterns were observed across five distin ct generations of DAS

- 81419

- 2 soybean , indicating stable inheritance of the transgene insert a cross multiple generations. Moreover, the absence of the backbone sequences from plasmid pDAB9582 in DAS

- 81419

- 2 soybean was confirmed using probes covering the entire region flanking the T

- DNA insert.USDA Petition for No nregulated Status of DAS

- 81419

- 2 Soybean Page 45 of 214

Dow AgroSciences LLC

Contains No Confidential Business Information Figure 8
. Southern blot analysis of DAS

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- 2 soybean d igested with Nco I (A) and Sph I (B); AtUbi10 p romoter prob e. Approximately 10  g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 at approximately one transgene copy per soybean genome and 10  g genomic DNA from non

- transgenic soybean Maverick. Fragment sizes in bp of the DIG

- labeled DNA Molecular Weight Marker II and DIG

- labeled DNA Molecular Weight Marker VII are indicated adjacent to image. Note: Bands in lanes 7

- 9 of panel B may not be clear ly visible on printed report.

Lane Sample Lane Sample 1 DIG Molecular Weight Marker II 11 DAS

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- 2

- T 2

- 2 2 Maverick + pDAB9582 12 DAS

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- 2

- T 2

- 3 3 Maverick 13 DAS

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- 2

- T 3

- 1 4 DAS

- 81419

- 2

- F2

- 1 14 DAS

- 81419

- 2

- T 3

- 2 5 DAS

- 81419

- 2

- F2

- 2 15 DAS

- 81419

- 2

- T3

- 3 6 DAS

- 81419

- 2

- F2

- 3 16 DAS

- 81419

- 2

- T 4

- 1 7 DAS

- 81419

- 2

- T 1

- 1 17 DAS

- 81419

- 2

- T 4

- 2 8 DAS

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- 2

- T 1

- 2 18 DAS

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- 2

- T 4

- 3 9 DAS

- 81419

- 2

- T 1

- 3 19 DIG Molecular Weight Marker V II 10 DAS

- 81419

- 2

- T 2

- 1USDA Petition for No nregulated Status of DAS

- 81419

- 2 Soybean Page 46 of 214

Dow AgroSciences LLC

Contains No Confidential Business Information Figure 9
. Southern b lot a nalysis o f DAS

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- 2 s oybean d igested with Nco I (A) and Sph I (B); cry1F

- 5 ′ probe

. Approximately 10  g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 at approximately one transgene copy per soybean genome and 10  g genomic DNA from non

- transgenic soybean Maverick. Fragment sizes in bp of the DIG

- labeled DNA Molecular Weight Marker II and DIG

- labeled DNA Molecular Weight Marker VII are indicated adjacent to image.

Lane Sample Lane Sample 1 DIG Molecular Weight Marker II 11 DAS

- 81419

- 2

- T 2

- 2 2 Maverick + pDAB9582 12 DAS

- 81419

- 2

- T 2

- 3 3 Maverick 13 DAS

- 81419

- 2

- T 3

- 1 4 DAS

- 81419

- 2

- F2

- 1 14 DAS

- 81419

- 2

- T 3

- 2 5 DAS

- 81419

- 2

- F2

- 2 15 DAS

- 81419

- 2

- T3

- 3 6 DAS

- 81419

- 2

- F2

- 3 16 DAS

- 81419

- 2

- T 4

- 1 7 DAS

- 81419

- 2

- T 1

- 1 17 DAS

- 81419

- 2

- T 4

- 2 8 DAS

- 81419

- 2

- T 1

- 2 18 DAS

- 81419

- 2

- T 4

- 3 9 DAS

- 81419

- 2

- T 1

- 3 19 DIG Molecular Weight Marker V II 10 DAS

- 81419

- 2

- T 2

- 1USDA Petition for No nregulated Status of DAS

- 81419

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Dow AgroSciences LLC

Contains No Confidential Business Information Figure 10
. Southern b lot a nalysis o f DAS

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- 2 s oybean d igested with Nco I (A) and Sph I (B); c ry1F

- 3 ′

A probe

. Approximately 10  g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 at approximately one transgene copy per soybean genome and 10  g genomic DNA from non

- transgenic soybe an Maverick. Fragment sizes in bp of the DIG

- labeled DNA Molecular Weight Marker II and DIG

- labeled DNA Molecular Weight Marker VII are indicated adjacent to image.

Lane Sample Lane Sample 1 DIG Molecular Weight Marker II 11 DAS

- 81419

- 2

- T 2

- 2 2 Maverick + pDAB9582 12 DAS

- 81419

- 2

- T 2

- 3 3 Maverick 13 DAS

- 81419

- 2

- T 3

- 1 4 DAS

- 81419

- 2

- F2

- 1 14 DAS

- 81419

- 2

- T 3

- 2 5 DAS

- 81419

- 2

- F2

- 2 15 DAS

- 81419

- 2

- T3

- 3 6 DAS

- 81419

- 2

- F2

- 3 16 DAS

- 81419

- 2

- T 4

- 1 7 DAS

- 81419

- 2

- T 1

- 1 17 DAS

- 81419

- 2

- T 4

- 2 8 DAS

- 81419

- 2

- T 1

- 2 18 DAS

- 81419

- 2

- T 4

- 3 9 DAS

- 81419

- 2

- T 1

- 3 19 DIG Molecular Weight Marker V II 10 DAS

- 81419

- 2

- T 2

- 1USDA Petition for No nregulated Status of DAS

- 81419

- 2 Soybean Page 48 of 214

Dow AgroSciences LLC

Contains No Confidential Business Information Figure 11
. Southern b lot a nalysis o f DAS

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- 2 s oybean d igested with Nco I (A) and Sph I (B); c ry1F

- 3 ′ B probe

. Approximately 10  g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 at approximately one transgene copy per soybean genome and 10  g genomic DNA from non

- transgenic soybean Maverick. Fragment sizes in bp of the DIG

- labeled DNA Molecu lar Weight Marker II and DIG

- labeled DNA Molecular Weight Marker VII are indicated adjacent to image.

Lane Sample Lane Sample 1 DIG Molecular Weight Marker II 11 DAS

- 81419

- 2

- T 2

- 2 2 Maverick + pDAB9582 12 DAS

- 81419

- 2

- T 2

- 3 3 Maverick 13 DAS

- 81419

- 2

- T 3

- 1 4 DAS

- 81419

- 2

- F2

- 1 14 DAS

- 81419

- 2

- T 3

- 2 5 DAS

- 81419

- 2

- F2

- 2 15 DAS

- 81419

- 2

- T3

- 3 6 DAS

- 81419

- 2

- F2

- 3 16 DAS

- 81419

- 2

- T 4

- 1 7 DAS

- 81419

- 2

- T 1

- 1 17 DAS

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- 2

- T 4

- 2 8 DAS

- 81419

- 2

- T 1

- 2 18 DAS

- 81419

- 2

- T 4

- 3 9 DAS

- 81419

- 2

- T 1

- 3 19 DIG Molecular Weight Marker V II 10 DAS

- 81419

- 2

- T 2

- 1USDA Petition for No nregulated Status of DAS

- 81419

- 2 Soybean Page 49 of 214

Dow AgroSciences LLC

Contains No Confidential Business Information Figure 12
. Southern blot a nalysis o f DAS

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- 2 soybean d igested with Nco I (A) and Sph I (B); AtuORF23

- A 3′ UTR probe

. Approximately 10  g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 at approximately one transgene copy per soybean genome and 10  g genomic DNA from non

- transgenic soybean Maverick. Fragment sizes in bp of the DIG

- labeled DNA Molecu lar Weight Marker II and DIG

- labeled DNA Molecular Weight Marker VII are indicated adjacent to image.

Lane Sample Lane Sample 1 DIG Molecular Weight Marker II 11 DAS

- 81419

- 2

- T 2

- 2 2 Maverick + pDAB9582 12 DAS

- 81419

- 2

- T 2

- 3 3 Maverick 13 DAS

- 81419

- 2

- T 3

- 1 4 DAS

- 81419

- 2

- F2

- 1 14 DAS

- 81419

- 2

- T 3

- 2 5 DAS

- 81419

- 2

- F2

- 2 15 DAS

- 81419

- 2

- T3

- 3 6 DAS

- 81419

- 2

- F2

- 3 16 DAS

- 81419

- 2

- T 4

- 1 7 DAS

- 81419

- 2

- T 1

- 1 17 DAS

- 81419

- 2

- T 4

- 2 8 DAS

- 81419

- 2

- T 1

- 2 18 DAS

- 81419

- 2

- T 4

- 3 9 DAS

- 81419

- 2

- T 1

- 3 19 DIG Molecular Weight Marker V II 10 DAS

- 81419

- 2

- T 2

- 1USDA Petition for No nregulated Status of DAS

- 81419

- 2 Soybean Page 50 of 214

Dow AgroSciences LLC

Contains No Confidential Business Information Figure 13
. Southern b lot a nalysis o f DAS

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- 2 soybean d igested with Nco I (A) and Sph I (B); CsVMV p romoter probe
. Approximately 10  g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 at approximately one transgene copy per soybean genome and 10  g genomic DNA from non

- transgenic soybean Maverick. Fragment sizes in bp of the DIG

- labeled DNA Molecu lar Weight Marker II and DIG

- labeled DNA Molecular Weight Marker VII are indicated adjacent to image.

Lane Sample Lane Sample 1 DIG Molecular Weight Marker II 11 DAS

- 81419

- 2

- T 2

- 2 2 Maverick + pDAB9582 12 DAS

- 81419

- 2

- T 2

- 3 3 Maverick 13 DAS

- 81419

- 2

- T 3

- 1 4 DAS

- 81419

- 2

- F2

- 1 14 DAS

- 81419

- 2

- T 3

- 2 5 DAS

- 81419

- 2

- F2

- 2 15 DAS

- 81419

- 2

- T3

- 3 6 DAS

- 81419

- 2

- F2

- 3 16 DAS

- 81419

- 2

- T 4

- 1 7 DAS

- 81419

- 2

- T 1

- 1 17 DAS

- 81419

- 2

- T 4

- 2 8 DAS

- 81419

- 2

- T 1

- 2 18 DAS

- 81419

- 2

- T 4

- 3 9 DAS

- 81419

- 2

- T 1

- 3 19 DIG Molecular Weight Marker V II 10 DAS

- 81419

- 2

- T 2

- 1USDA Petition for No nregulated Status of DAS

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Dow AgroSciences LLC

Contains No Confidential Business Information Figure 14
. Southern b lot a nalysis o f DAS

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- 2 soybean d igested with Nco I (A) and Sph I (B); cry1Ac

- 5′ probe. Approximately 10  g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 at approximately one transgene copy per soybean genome and 10  g genomic DNA from non

- transgenic soybean Maverick. Fragment sizes in bp of the DIG

- labeled DNA Molecu lar Weight Marker II and DIG

- labeled DNA Molecular Weight Marker VII are indicated adjacent to image.

Lane Sample Lane Sample 1 DIG Molecular Weight Marker II 11 DAS

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- 2

- T 2

- 2 2 Maverick + pDAB9582 12 DAS

- 81419

- 2

- T 2

- 3 3 Maverick 13 DAS

- 81419

- 2

- T 3

- 1 4 DAS

- 81419

- 2

- F2

- 1 14 DAS

- 81419

- 2

- T 3

- 2 5 DAS

- 81419

- 2

- F2

- 2 15 DAS

- 81419

- 2

- T3

- 3 6 DAS

- 81419

- 2

- F2

- 3 16 DAS

- 81419

- 2

- T 4

- 1 7 DAS

- 81419

- 2

- T 1

- 1 17 DAS

- 81419

- 2

- T 4

- 2 8 DAS

- 81419

- 2

- T 1

- 2 18 DAS

- 81419

- 2

- T 4

- 3 9 DAS

- 81419

- 2

- T 1

- 3 19 DIG Molecular Weight Marker V II 10 DAS

- 81419

- 2

- T 2

- 1USDA Petition for No nregulated Status of DAS

- 81419

- 2 Soybean Page 52 of 214

Dow AgroSciences LLC

Contains No Confidential Business Information Figure 15
. Southern blot a nalysis o f DAS

- 81419

- 2 s oybean d igested with Nco I (A) and Sph I (B); cry1Ac

- 3′ A probe. Approximately 10  g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 at approximately one transgene copy per soybean genome and 10  g genomic DNA from non

- transgenic soybean Maverick. Fragment sizes in bp of the DIG

- labeled DNA Molecu lar Weight Marker II and DIG

- labeled DNA Molecular Weight Marker VII are indicated adjacent to image. Note: Fuzzy smear bands at ~18000 bp in lanes 4

- 18 of panel A may be a result of DNA degradation and/or incomplete digestion.

Lane Sample Lane Sample 1 DIG Molecular Weight Marker II 11 DAS

- 81419

- 2

- T 2

- 2 2 Maverick + pDAB9582 12 DAS

- 81419

- 2

- T 2

- 3 3 Maverick 13 DAS

- 81419

- 2

- T 3

- 1 4 DAS

- 81419

- 2

- F2

- 1 14 DAS

- 81419

- 2

- T 3

- 2 5 DAS

- 81419

- 2

- F2

- 2 15 DAS

- 81419

- 2

- T3

- 3 6 DAS

- 81419

- 2

- F2

- 3 16 DAS

- 81419

- 2

- T 4

- 1 7 DAS

- 81419

- 2

- T 1

- 1 17 DAS

- 81419

- 2

- T 4

- 2 8 DAS

- 81419

- 2

- T 1

- 2 18 DAS

- 81419

- 2

- T 4

- 3 9 DAS

- 81419

- 2

- T 1

- 3 19 DIG Molecular Weight Marker V II 10 DAS

- 81419

- 2

- T 2

- 1USDA Petition for No nregulated Status of DAS

- 81419

- 2 Soybean Page 53 of 214

Dow AgroSciences LLC

Contains No Confidential Business Information Figure 16
. Southern b lot a nalysis o f DAS

- 81419

- 2 s oybean d igested with Nco I (A) and Sph I (B); cry1Ac

- 3′ B probe. Approximately 10  g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 at approximately one transgene copy per soybean genome and 10  g genomic DNA from non

- transgenic soybean M averick. Fragment sizes in bp of the DIG

- labeled DNA Molecular Weight Marker II and DIG

- labeled DNA Molecular Weight Marker VII are indicated adjacent to image. Note: Bands in lane 8 of panel A may not be clearly visible on printed report.

Lane Sample Lane Sample 1 DIG Molecular Weight Marker II 11 DAS

- 81419

- 2

- T 2

- 2 2 Maverick + pDAB9582 12 DAS

- 81419

- 2

- T 2

- 3 3 Maverick 13 DAS

- 81419

- 2

- T 3

- 1 4 DAS

- 81419

- 2

- F2

- 1 14 DAS

- 81419

- 2

- T 3

- 2 5 DAS

- 81419

- 2

- F2

- 2 15 DAS

- 81419

- 2

- T3

- 3 6 DAS

- 81419

- 2

- F2

- 3 16 DAS

- 81419

- 2

- T 4

- 1 7 DAS

- 81419

- 2

- T 1

- 1 17 DAS

- 81419

- 2

- T 4

- 2 8 DAS

- 81419

- 2

- T 1

- 2 18 DAS

- 81419

- 2

- T 4

- 3 9 DAS

- 81419

- 2

- T 1

- 3 19 DIG Molecular Weight Marker V II 10 DAS

- 81419

- 2

- T 2

- 1USDA Petition for No nregulated Status of DAS

- 81419

- 2 Soybean Page 54 of 214

Dow AgroSciences LLC

Contains No Confidential Business Information Figure 17
. Southern blot a nalysis o f DAS

- 81419

- 2 soybean d igested with Nco I (A) and Sph I (B); Atu ORF23

- B 3′ UTR probe. Approximately 10  g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 at approximately one transgene copy per soybean genome and 10  g genomic DNA from non

- transgenic soybean Maverick. Fragment sizes in bp of the DIG

- labeled DNA Molecular Weight Marker II and DIG

- labeled DNA Molecular Weight Marker VII are indicated adjacent to image.

Lane Sample Lane Sample 1 DIG Molecular Weight Marker II 11 DAS

- 81419

- 2

- T 2

- 2 2 Maverick + pDAB9582 12 DAS

- 81419

- 2

- T 2

- 3 3 Maverick 13 DAS

- 81419

- 2

- T 3

- 1 4 DAS

- 81419

- 2

- F2

- 1 14 DAS

- 81419

- 2

- T 3

- 2 5 DAS

- 81419

- 2

- F2

- 2 15 DAS

- 81419

- 2

- T3

- 3 6 DAS

- 81419

- 2

- F2

- 3 16 DAS

- 81419

- 2

- T 4

- 1 7 DAS

- 81419

- 2

- T 1

- 1 17 DAS

- 81419

- 2

- T 4

- 2 8 DAS

- 81419

- 2

- T 1

- 2 18 DAS

- 81419

- 2

- T 4

- 3 9 DAS

- 81419

- 2

- T 1

- 3 19 DIG Molecular Weight Marker V II 10 DAS

- 81419

- 2

- T 2

- 1USDA Petition for No nregulated Status of DAS

- 81419

- 2 Soybean Page 55 of 214

Dow AgroSciences LLC

Contains No Confidential Business Information Figure 18
. Southern b lot a nalysis o f DAS

- 81419

- 2 s oybean d igested with Nco I (A) and Sph I (B); pat probe. Approximately 10  g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 at approximately one transgene copy per soybean genome and 10  g genomic DNA from non

- transgenic soybean Maverick. Fragment sizes in bp of the DIG

- labeled DNA Molecular Weight Marker II and DIG

- labeled DNA Molecular Weight Marker VII are indicated adjacent to image. Note: Bands in lane 4 of panel A may not be clearly visible on printed report.

Lane Sample Lane Sample 1 DIG Molecular Weight Marker II 11 DAS

- 81419

- 2

- T 2

- 2 2 Maverick + pDAB9582 12 DAS

- 81419

- 2

- T 2

- 3 3 Maverick 13 DAS

- 81419

- 2

- T 3

- 1 4 DAS

- 81419

- 2

- F2

- 1 14 DAS

- 81419

- 2

- T 3

- 2 5 DAS

- 81419

- 2

- F2

- 2 15 DAS

- 81419

- 2

- T3

- 3 6 DAS

- 81419

- 2

- F2

- 3 16 DAS

- 81419

- 2

- T 4

- 1 7 DAS

- 81419

- 2

- T 1

- 1 17 DAS

- 81419

- 2

- T 4

- 2 8 DAS

- 81419

- 2

- T 1

- 2 18 DAS

- 81419

- 2

- T 4

- 3 9 DAS

- 81419

- 2

- T 1

- 3 19 DIG Molecular Weight Marker V II 10 DAS

- 81419

- 2

- T 2

- 1USDA Petition for No nregulated Status of DAS

- 81419

- 2 Soybean Page 56 of 214

Dow AgroSciences LLC

Contains No Confidential Business Information Figure 19
. Southern b lot a nalysis o f DAS

- 81419

- 2 s oybean d igested with Nco I (A) and Sph I (B); AtuORF1 3′ UTR probe. Approximately 10  g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 at approximately one transgene copy per so ybean genome and 10  g genomic DNA from non

- transgenic soybean Maverick. Fragment sizes in bp of the DIG

- labeled DNA Molecular Weight Marker II and DIG

- labeled DNA Molecular Weight Marker VII are indicated adjacent to image. Note: Bands in lanes 7

- 8 of pan el B may not be clearly visible on printed report.

Lane Sample Lane Sample 1 DIG Molecular Weight Marker II 11 DAS

- 81419

- 2

- T 2

- 2 2 Maverick + pDAB9582 12 DAS

- 81419

- 2

- T 2

- 3 3 Maverick 13 DAS

- 81419

- 2

- T 3

- 1 4 DAS

- 81419

- 2

- F2

- 1 14 DAS

- 81419

- 2

- T 3

- 2 5 DAS

- 81419

- 2

- F2

- 2 15 DAS

- 81419

- 2

- T3

- 3 6 DAS

- 81419

- 2

- F2

- 3 16 DAS

- 81419

- 2

- T 4

- 1 7 DAS

- 81419

- 2

- T 1

- 1 17 DAS

- 81419

- 2

- T 4

- 2 8 DAS

- 81419

- 2

- T 1

- 2 18 DAS

- 81419

- 2

- T 4

- 3 9 DAS

- 81419

- 2

- T 1

- 3 19 DIG Molecular Weight Marker V II 10 DAS

- 81419

- 2

- T 2

- 1USDA Petition for No nregulated Status of DAS

- 81419

- 2 Soybean Page 57 of 214

Dow AgroSciences LLC

Contains No Confidential Business Information Figure 20
. Southern b lot a nalysis o f DAS

- 81419

- 2 s oybean d igested with Not I / Sph I; (A) AtUbi10 p romoter probe , (B) cry1F

- 5 ′ probe
. Approximately 10  g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 at approximately one transgene copy per soybean genome and 10  g genomic DNA from non

- transgenic soybean Maverick. Fragment sizes in bp of the DIG

- labeled DNA Molecu lar Weight Marker II and DIG

- labeled DNA Molecular Weight Marker VII are indicated adjacent to image.

Lane Sample Lane Sample 1 DIG Molecular Weight Marker II 11 DAS

- 81419

- 2

- T 2

- 2 2 Maverick + pDAB9582 12 DAS

- 81419

- 2

- T 2

- 3 3 Maverick 13 DAS

- 81419

- 2

- T 3

- 1 4 DAS

- 81419

- 2

- F2

- 1 14 DAS

- 81419

- 2

- T 3

- 2 5 DAS

- 81419

- 2

- F2

- 2 15 DAS

- 81419

- 2

- T3

- 3 6 DAS

- 81419

- 2

- F2

- 3 16 DAS

- 81419

- 2

- T 4

- 1 7 DAS

- 81419

- 2

- T 1

- 1 17 DAS

- 81419

- 2

- T 4

- 2 8 DAS

- 81419

- 2

- T 1

- 2 18 DAS

- 81419

- 2

- T 4

- 3 9 DAS

- 81419

- 2

- T 1

- 3 19 DIG Molecular Weight Marker V II 10 DAS

- 81419

- 2

- T 2

- 1USDA Petition for No nregulated Status of DAS

- 81419

- 2 Soybean Page 58 of 214

Dow AgroSciences LLC

Contains No Confidential Business Information Figure 21
. Southern b lot a nalysis o f DAS

- 81419

- 2 s oybean d igested w ith Not I / Sph I; (A) cry1F

- 3′ A probe, (B) cry1F

- 3′ B probe
. Approximately 10  g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 at approximately one transgene copy per soybean genome and 10  g genomic DNA from non

- transgenic soybean Maverick. Fragment sizes in bp of the DIG

- labeled DNA Molecu lar Weight Marker II and DIG

- labeled DNA Molecular Weight Marker VII are indicated adjacent to image. Note: Faint bands of ~ 4600 bp in positive control and DAS

- 81419

- 2 soybean samples observed in panel A are the expected cross

- hybridization results of the cry1F

3’A probe binding to the cry1Ac(synpro) PTU.

Lane Sample Lane Sample 1 DIG Molecular Weight Marker II 11 DAS

- 81419

- 2

- T 2

- 2 2 Maverick + pDAB9582 12 DAS

- 81419

- 2

- T 2

- 3 3 Maverick 13 DAS

- 81419

- 2

- T 3

- 1 4 DAS

- 81419

- 2

- F2

- 1 14 DAS

- 81419

- 2

- T 3

- 2 5 DAS

- 81419

- 2

- F2

- 2 15 DAS

- 81419

- 2

- T3

- 3 6 DAS

- 81419

- 2

- F2

- 3 16 DAS

- 81419

- 2

- T 4

- 1 7 DAS

- 81419

- 2

- T 1

- 1 17 DAS

- 81419

- 2

- T 4

- 2 8 DAS

- 81419

- 2

- T 1

- 2 18 DAS

- 81419

- 2

- T 4

- 3 9 DAS

- 81419

- 2

- T 1

- 3 19 DIG Molecular Weight Marker V II 10 DAS

- 81419

- 2

- T 2

- 1USDA Petition for No nregulated Status of DAS

- 81419

- 2 Soybean Page 59 of 214

Dow AgroSciences LLC

Contains No Confidential Business Information Figure 22
. Southern b lot a nalysis o f DAS

- 81419

- 2 s oybean d igested w ith N ot I / Sph I; (A) AtuORF23

- A 3′ UTR probe, (B) CsVMV p romoter probe
. Approximately 10  g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 at approximately one transgene copy per soybean genome and 10  g genomic DNA from non

- transgenic soybean Maverick. Fragment sizes in bp of the DIG

- labeled DNA Molecu lar Weight Marker II and DIG

- labeled DNA Molecular Weight Marker VII are indicated adjacent to image.

Lane Sample Lane Sample 1 DIG Molecular Weight Marker II 11 DAS

- 81419

- 2

- T 2

- 2 2 Maverick + pDAB9582 12 DAS

- 81419

- 2

- T 2

- 3 3 Maverick 13 DAS

- 81419

- 2

- T 3

- 1 4 DAS

- 81419

- 2

- F2

- 1 14 DAS

- 81419

- 2

- T 3

- 2 5 DAS

- 81419

- 2

- F2

- 2 15 DAS

- 81419

- 2

- T3

- 3 6 DAS

- 81419

- 2

- F2

- 3 16 DAS

- 81419

- 2

- T 4

- 1 7 DAS

- 81419

- 2

- T 1

- 1 17 DAS

- 81419

- 2

- T 4

- 2 8 DAS

- 81419

- 2

- T 1

- 2 18 DAS

- 81419

- 2

- T 4

- 3 9 DAS

- 81419

- 2

- T 1

- 3 19 DIG Molecular Weight Marker V II 10 DAS

- 81419

- 2

- T 2

- 1USDA Petition for No nregulated Status of DAS

- 81419

- 2 Soybean Page 60 of 214

Dow AgroSciences LLC

Contains No Confidential Business Information Figure 23
. Southern b lot a nalysis o f DAS

- 81419

- 2 s oybean d igested w ith N ot I / Sph I; (A) cry1Ac

- 5′ probe, (B) AtuORF23

- B 3′ UTR probe
. Approximately 10  g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 at approximately one transgene copy per soybean genome and 10  g genomic DNA from non

- transgenic soybean Maverick. Fragment sizes in bp of the DIG

- labeled DNA Molecu lar Weight Marker II and DIG

- labeled DNA Molecular Weight Marker VII are indicated adjacent to image.

Lane Sample Lane Sample 1 DIG Molecular Weight Marker II 11 DAS

- 81419

- 2

- T 2

- 2 2 Maverick + pDAB9582 12 DAS

- 81419

- 2

- T 2

- 3 3 Maverick 13 DAS

- 81419

- 2

- T 3

- 1 4 DAS

- 81419

- 2

- F2

- 1 14 DAS

- 81419

- 2

- T 3

- 2 5 DAS

- 81419

- 2

- F2

- 2 15 DAS

- 81419

- 2

- T3

- 3 6 DAS

- 81419

- 2

- F2

- 3 16 DAS

- 81419

- 2

- T 4

- 1 7 DAS

- 81419

- 2

- T 1

- 1 17 DAS

- 81419

- 2

- T 4

- 2 8 DAS

- 81419

- 2

- T 1

- 2 18 DAS

- 81419

- 2

- T 4

- 3 9 DAS

- 81419

- 2

- T 1

- 3 19 DIG Molecular Weight Marker V II 10 DAS

- 81419

- 2

- T 2

- 1USDA Petition for No nregulated Status of DAS

- 81419

- 2 Soybean Page 61 of 214

Dow AgroSciences LLC

Contains No Confidential Business Information Figure 24
. Southern b lot a nalysis o f DAS

- 81419

- 2 s oybean d igested w ith N ot I / Sph I; (A) cry1Ac

- 3′ A probe , (B) cry1Ac

- 3′ B probe
. Approximately 10  g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 at approximately one transgene copy per soybean genome and 10  g genomic DNA from non

- transgenic soybean Maverick. Fragment sizes in bp of the DIG

- labeled DNA Molecu lar Weight Marker II and DIG

- labeled DNA Molecular Weight Marker VII are indicated adjacent to image. Note: Faint bands of ~ 5400 bp in positive control and DAS

- 81419

- 2 soybean samples observed in panel A are the expected cross

- hybridization bands of the c ry1Ac

3’A probe binding to the cry1Fv3 PTU. Faint bands at ~9500 bp observed in lanes 2 and 4 in panel A are most likely a result of incomplete digestion.

Lane Sample Lane Sample 1 DIG Molecular Weight Marker II 11 DAS

- 81419

- 2

- T 2

- 2 2 Maverick + pDAB9582 12 DAS

- 81419

- 2

- T 2

- 3 3 Maverick 13 DAS

- 81419

- 2

- T 3

- 1 4 DAS

- 81419

- 2

- F2

- 1 14 DAS

- 81419

- 2

- T 3

- 2 5 DAS

- 81419

- 2

- F2

- 2 15 DAS

- 81419

- 2

- T3

- 3 6 DAS

- 81419

- 2

- F2

- 3 16 DAS

- 81419

- 2

- T 4

- 1 7 DAS

- 81419

- 2

- T 1

- 1 17 DAS

- 81419

- 2

- T 4

- 2 8 DAS

- 81419

- 2

- T 1

- 2 18 DAS

- 81419

- 2

- T 4

- 3 9 DAS

- 81419

- 2

- T 1

- 3 19 DIG Molecular Weight Marker V II 10 DAS

- 81419

- 2

- T 2

- 1USDA Petition for No nregulated Status of DAS

- 81419

- 2 Soybean Page 62 of 214

Dow AgroSciences LLC

Contains No Confidential Business Information Figure 25
. Southern b lot a nalysis o f DAS

- 81419

- 2 s oybean d igested w ith N ot I / Sph I; (A) pat probe , (B) AtuORF1 3′ UTR probe
. Approximately 10  g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 at approximately one transgene copy per soybean genome and 10  g genomic DNA from non

- transgenic soybean Maverick. Fragment sizes in bp of the DIG

- labeled DNA Molecular Weight Marker II and DIG

- labeled DNA Molecular Weight Marker VII are indicated adjacent to image.

Lane Sample Lane Sample 1 DIG Molecular Weight Marker II 11 DAS

- 81419

- 2

- T 2

- 2 2 Maverick + pDAB9582 12 DAS

- 81419

- 2

- T 2

- 3 3 Maverick 13 DAS

- 81419

- 2

- T 3

- 1 4 DAS

- 81419

- 2

- F2

- 1 14 DAS

- 81419

- 2

- T 3

- 2 5 DAS

- 81419

- 2

- F2

- 2 15 DAS

- 81419

- 2

- T3

- 3 6 DAS

- 81419

- 2

- F2

- 3 16 DAS

- 81419

- 2

- T 4

- 1 7 DAS

- 81419

- 2

- T 1

- 1 17 DAS

- 81419

- 2

- T 4

- 2 8 DAS

- 81419

- 2

- T 1

- 2 18 DAS

- 81419

- 2

- T 4

- 3 9 DAS

- 81419

- 2

- T 1

- 3 19 DIG Molecular Weight Marker V II 10 DAS

- 81419

- 2

- T 2

- 1USDA Petition for No nregulated Status of DAS

- 81419

- 2 Soybean Page 63 of 214

Dow AgroSciences LLC

Contains No Confidential Business Information Figure 26
. Southern b lot a nalysis o f DAS

- 81419

- 2 s oybean d igested w ith (A) Sph I; Ori p robe, (B) Nco I; SpecR probe. Approximately 10  g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 at approximately one transgene copy per soybean genome and 10  g genomic DNA from non

- transgenic soybean Maverick. Fragment sizes in bp of the DIG

- labeled DNA Molecu lar Weight Marker II and DIG

- labeled DNA Molecular Weight Marker VII are indicated adjacent to image.

Lane Sample Lane Sample 1 DIG Molecular Weight Marker II 11 DAS

- 81419

- 2

- T 2

- 2 2 Maverick + pDAB9582 12 DAS

- 81419

- 2

- T 2

- 3 3 Maverick 13 DAS

- 81419

- 2

- T 3

- 1 4 DAS

- 81419

- 2

- F2

- 1 14 DAS

- 81419

- 2

- T 3

- 2 5 DAS

- 81419

- 2

- F2

- 2 15 DAS

- 81419

- 2

- T3

- 3 6 DAS

- 81419

- 2

- F2

- 3 16 DAS

- 81419

- 2

- T 4

- 1 7 DAS

- 81419

- 2

- T 1

- 1 17 DAS

- 81419

- 2

- T 4

- 2 8 DAS

- 81419

- 2

- T 1

- 2 18 DAS

- 81419

- 2

- T 4

- 3 9 DAS

- 81419

- 2

- T 1

- 3 19 DIG Molecular Weight Marker V II 10 DAS

- 81419

- 2

- T 2

- 1USDA Petition for No nregulated Status of DAS

- 81419

- 2 Soybean Page 64 of 214

Dow AgroSciences LLC

Contains No Confidential Business Information Figure 27
. Southern b lot a nalysis o f DAS

- 81419

- 2 s oybean d igested w ith (A) Nco I; Backbone 1 p robe, (B) Sph I; Backbone 2 probe. Approximately 10  g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 at approximately one transgene copy per soybean genome and 10  g genomic DNA from non

- transgenic soybean Maverick. Fragment sizes in bp of the DIG

- labeled DNA Molecu lar Weight Marker II and DIG

- labeled DNA Molecular Weight Marker VII are indicated adjacent to image.

Lane Sample Lane Sample 1 DIG Molecular Weight Marker II 11 DAS

- 81419

- 2

- T 2

- 2 2 Maverick + pDAB9582 12 DAS

- 81419

- 2

- T 2

- 3 3 Maverick 13 DAS

- 81419

- 2

- T 3

- 1 4 DAS

- 81419

- 2

- F2

- 1 14 DAS

- 81419

- 2

- T 3

- 2 5 DAS

- 81419

- 2

- F2

- 2 15 DAS

- 81419

- 2

- T3

- 3 6 DAS

- 81419

- 2

- F2

- 3 16 DAS

- 81419

- 2

- T 4

- 1 7 DAS

- 81419

- 2

- T 1

- 1 17 DAS

- 81419

- 2

- T 4

- 2 8 DAS

- 81419

- 2

- T 1

- 2 18 DAS

- 81419

- 2

- T 4

- 3 9 DAS

- 81419

- 2

- T 1

- 3 19 DIG Molecular Weight Marker V II 10 DAS

- 81419

- 2

- T 2

- 1USDA Petition for No nregulated Status of DAS

- 81419

- 2 Soybean Page 65 of 214

Dow AgroSciences LLC

Contains No Confidential Business Information Figure 28
. Southern b lot a nalysis o f DAS

- 81419

- 2 s oybean d igested with Nco I; Backbone 3 and 4 probes. Approximately 10  g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 at approximately one transgene copy per soybean genome and 10  g genomic DNA from non

- transgenic soybean Maverick. Fragment sizes in bp of the DIG

- labeled DNA Molecular Weight Marker II and DIG

- labeled DNA Molecular Weight Marker VII are indicated adjacent to image.

Lane Sample Lane Sample 1 DIG Molecular Weight Marker II 11 DAS

- 81419

- 2

- T 2

- 2 2 Maverick + pDAB9582 12 DAS

- 81419

- 2

- T 2

- 3 3 Maverick 13 DAS

- 81419

- 2

- T 3

- 1 4 DAS

- 81419

- 2

- F2

- 1 14 DAS

- 81419

- 2

- T 3

- 2 5 DAS

- 81419

- 2

- F2

- 2 15 DAS

- 81419

- 2

- T3

- 3 6 DAS

- 81419

- 2

- F2

- 3 16 DAS

- 81419

- 2

- T 4

- 1 7 DAS

- 81419

- 2

- T 1

- 1 17 DAS

- 81419

- 2

- T 4

- 2 8 DAS

- 81419

- 2

- T 1

- 2 18 DAS

- 81419

- 2

- T 4

- 3 9 DAS

- 81419

- 2

- T 1

- 3 19 DIG Molecular Weight Marker V II 10 DAS

- 81419

- 2

- T 2

- 1 23130 9416 6557
4361

2322 2027 8576 7427 6106 4899
3639
2799
1953 1882 1515 1482 1164 992
718/710 bp 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

564 492 359USDA Petition for No nregulated Status of DAS

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Dow AgroSciences LLC

Contains No Confidential Business Information 5.3. Segregation Analysis
5.3.1. Analysis of a Segregating Generation The inheritance pattern of the transgene insert within a segregating generation was demonstrated with a l ateral flow strip assay and event

- specific PCR analys e s of individual plants within a F2 g eneration of DAS

- 81419

- 2 soybean. The F1 generation was generated by crossing homozygous T3 plants of DAS

- 81419

- 2 soybean with a non
- transgenic soybean line. The F1 plants were self polli nated to produce the F2 seeds. A total of 123 plants from the F2 generation of DAS

- 81419

- 2 soybean were tested f or PAT protein expression by a lateral flow strip assay. Of the 123 F2 plants tested, 91 plants were positive and 32 were negative (segregated nulls) for PAT protein expression. One plant died before suffici ent tissue sample was harvested for genomic DNA extraction and thus was not subjected to event

- specific PCR analysis. As a consequence, a total of 122 F2 plants were tested by event

- specific PCR to determine the presence or absence of the DAS

- 81419

- 2 tran sgene insert

. Of the 122 plants tested, 90 plants were positive for the presence of DAS

- 81419

- 2 transgene insert, and the remaining 32 plants wer e negative (segregated null). All plants that tested positi ve for PAT protein expression by a lateral flow st rip assay were also positive for the DAS

- 81419

- 2 transgene insert by event

- specific PCR analysis . Similarly, all plants that tested negative for PAT protein expression were also negative for the presence of the DAS

- 81419

- 2 transgene insert by event

- specif ic PCR ( Table 5 ). This result confirm ed that the phenotypic segregation matched the genotypic makeup of the tested F2 generation. Statistical analysis using
a chi

- sq uare g oodness of fit test indicated the ratio of 91 positive to 32 null segregants for the LFS test, and the ratio of 90 positive to 32 null segregants for the event

- specific PCR analysis, did not significantly differ from the expected Mendelian 3:1 segre gation pattern for a single independent locus

. Table 5 . Results of F2 individual plants tested with LFS and event

- specific PCR.
Tested Method Total plants

tested Positive Negative Expected ratio P

- value a

LFS 123 91 32 3:1 0.7946
Event

Specific PCR 122 90 32 3:1 0.7538

a Based on a chi

- squared g oodness of fit test

5.3.2. Segregation Analysis of Breeding Generations Chi

- square goodness of fit analyse s o f trait inheritance data from three populations of the BC1F2 breeding generation was conducted to determine the Mendelian inheritance of the transgene insert in DAS

- 81419

- 2 soybean . The presence or absence of t he trans gene insert was determined using an event

- specific PCR assay for DAS

- 81419

- 2 soybean. The expected segregation ratio of 3 :1 for plants containing the transgene insert versus plants that do not contain the transgene insert (segregated nulls) was observed ( Table 6 )

.USDA Petition for No nregulated Status of DAS

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Dow AgroSciences LLC

Contains No Confidential Business Information Table 6
. Results of the individual plants from DAS

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- 2 soybean tested with event
specific PCR within segregating BC1F2 generation s.
Generation Total

plants tested Transgene

insert positive (with event

specific PCR) Transgene

insert negative (with event
specific PCR) Expected

ratio P

- value a

BC1F2 48 34 14 3:1 0.5050 BC1F2 30 22 8 3:1 0.8330 BC1F2 294 220 74 3:1 0.9463

a Based on a chi

- square goodness of fit test

5.4. Summary of the Genetic Characterization Molecular characterization of DAS

- 81419

- 2 soybean by Southern blot analyse s confirmed that a single T

- DNA insert containing each of the intact PTUs for the cry1F v3 , cry1Ac (synpro) , and pat genes from plasmid pDAB9582, were integrated into DAS

- 81419

- 2 soybean

. In addition to the full

- length insert, a minor (<100 bp) fragment of the cry1Ac(synpro) gene was identified on the 5′ end of the T

- DNA insert. T he inserted DNA wa s stably inherited across the five generation s (T1, T2, T3, T4, and F2) evaluated. No transf ormation plasmid backbone sequence s were identified in DAS

- 81419

- 2 soybean as demonstrated by Southern blot analyse s using probes covering the entire region of the plasmid flanking the T

- DNA insert . Moreover, the T

- DNA insert displayed the expected Mendel ian inheritance pattern for a single independent insert/locus in segregating generation s (F2 and BC1F2 )

.USDA Petition for No nregulated Status of DAS

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Dow AgroSciences LLC

Contains No Confidential Business Information 6. Characterization of the Introduced Proteins
6.1. Cry1Ac and Cry1F
6.1.1. Cry1 Protein Mode of Action Cry1 insecticidal crystal protein s from Bacillus thuringiensis are a class of structurally related delta endotoxins having three distinct structural domains

. These proteins are generally toxic to a subset of lepidopteran larvae, and t his class of Cry proteins has been investigated most thoroughly with regard to their mode of action. The most widely accepted hypothesis for Cry1 mode of action is the two

- receptor mode l proposed by ( Bravo et al. , 2004 ) . In this model, upon ingestion, the protein is solubilized in the midgut of the insect and is processed to an active core structure by proteases located within the midgut. The processed core then binds to a cadherin protein through the interaction of specific residues on domains II and III of the protein

. Cadherins are a superfamily of proteins made up of a series of 5 to 34 calcium binding repeat elements. The first report of a cadherin in teracting with a Cry protein was in M anduca sexta L. (Lepidoptera: Sphingidae) ( Vadlamudi et al. , 1993 ) . The cadherin was shown to bind Cry1Ac, Cry1Aa and Cry1Ab. Cry1Aa and Cry1Ac competed with Cry1Ab binding, suggesting a common epitope within the cadherin protein. Subsequently, cadherins have been implicated in Cry1 susceptibility in a number of different insect species ( Gahan et al. , 2001 ; Morin et al. , 2003 ; Coates et al. , 2005 ; Yang et al. , 2011 )

. Upon binding to the cadherin receptor, structural changes in the Cry1 active core allow for further processing of the N

- terminus enabling it to oligomerize to a tetramer. In additi on to cadherins, receptors in the aminophosphatase

- N family have been identified in several lepidopteran species wh ile alkaline phosphatases are also believed to be involved. Thus the mode of action of Cry1 protein corresponds to proteolytic processing of the protein and sequential multiple receptor binding interactions leading to pore formation in the insect gut.
6.1.2. Identity of the Cry1Ac Protein The Cry1Ac protein expressed in DAS

- 81419

- 2 soybean is a synthetic version of Cry1Ac1, from B. t huringiensis subs p

. kurstaki strain HD73. In this synthetic version, the first 612 amino acids are comprised of the insect

- active portion of Cry1Ac1; the remaining portion consists of C

- terminal sequences from Cry1Ca3 ( B. thuringiensis subsp

. aizawai PS81I), and Cry1Ab1 ( B. thuringiensis subsp

. Berliner 1715). Together, the portions of Cry1Ca3 and Cry1A b1 that comprise the chimeric C

- terminal domain are approximately those removed by alkaline proteases in the lepidopteran mid

- gut during formation of the active Cry1Ac core toxin. The synthetic Cry1Ac protein is comprised of 1156 amino acids a nd has a molecular weight of ~130.7 kDa ( Figure 29 ). The amino acid sequence of the Cry1Ac prot ein expressed in DAS

- 81419

- 2 soybean is identical to that expressed in WideStrike  cotton event DAS

- 21023

- 5 (also described as 3006

- 210

- 23 expressing Cry1Ac ) ( Dow AgroSciences, 2003a )

.USDA Petition for No nregulated Status of DAS

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Dow AgroSciences LLC

Contains No Confidential Business Information

1 MDNNPNINECIPYNCLSNPEVEVLGGERIE 30 31 TGYTPIDISLSLTQ FLLSEFVPGAGFVLGL 60 61 VDIIWGIFGPSQWDAFLVQIEQLINQRIEE 90 91 FARNQAISRLEGLSNLYQIYAESFREWEAD 120 121 PTNPALREEMRIQFNDMNSALTTAIPLFAV 150 151 QNYQVPLLSVYVQAANLHLSVLRDVSVFGQ 180 181 RWGFDAATINSRYNDLTRLIGNYTDYAVRW 210 211 YNTGLERVWGPDSRDWVRYNQFRRELTLTV 2 40 241 LDIVALFPNYDSRRYPIRTVSQLTREIYTN 270 271 PVLENFDGSFRGSAQGIERSIRSPHLMDIL 300 301 NSITIYTDAHRGYYYWSGHQIMASPVGFSG 330 331 PEFTFPLYGTMGNAAPQQRIVAQLGQGVYR 360 361 TLSSTLYRRPFNIGINNQQLSVLDGTEFAY 390 391 GTSSNLPSAVYRKSGTVDSLDEIPPQNNNV 420 421 PPRQGFSHRLSHVSMFRSGFSNSSVSIIRA 450 451 PMFSWIHRSAEFNNIIASDSITQIPAVKGN 480 481 FLFNGSVISGPGFTGGDLVRLNSSGNNIQN 510 511 RGYIEVPIHFPSTSTRYRVRVRYASVTPIH 540 541 LNVNWGNSSIFSNTVPATATSLDNLQSSDF 570 571 GYFESANAFTSSLGNIVGVRNFSGTAGVII 600 601 DRFEFIPVTAT LEAESDLERAQKAVNALFT 630 631 SSNQIGLKTDVTDYHIDRVSNLVECLSDEF 660 661 CLDEKKELSEKVKHAKRLSDERNLLQDPNF 690 691 RGINRQLDRGWRGSTDITIQGGDDVFKENY 720 721 VTLLGTFDECYPTYLYQKIDESKLKAYTRY 750 751 QLRGYIEDSQDLEIYLIRYNAKHETVNVPG 780 781 TGSLWPLSAPSPIGKCAHHSHHFSLDI DVG 810 811 CTDLNEDLGVWVIFKIKTQDGHARLGNLEF 840 841 LEEKPLVGEALARVKRAEKKWRDKREKLEW 870 871 ETNIVYKEAKESVDALFVNSQYDRLQADTN 900 901 IAMIHAADKRVHSIREAYLPELSVIPGVNA 930 931 AIFEELEGRIFTAFSLYDARNVIKNGDFNN 960 961 GLSCWNVKGHVDVEEQNNHRSVLVVPEWEA 990 991 EVS QEVRVCPGRGYILRVTAYKEGYGEGCV 1020 1021 TIHEIENNTDELKFSNCVEEEVYPNNTVTC 1050 1051 NDYTATQEEYEGTYTSRNRGYDGAYESNSS 1080 1081 VPADYASAYEEKAYTDGRRDNPCESNRGYG 1110 1111 DYTPLPAGYVTKELEYFPETDKVWIEIGET 1140 1141 EGTFIVDSVELLLMEE 1156

Figure 29 . Amino acid sequence of Cry1Ac protein.

6.1.3. Biochemica l Characterization of the Cry1Ac Protein Large quantities of purified Cry1Ac protein are required to perf orm safety assessment studies. As it is technically infeasible to extract and purify sufficient amounts of recombinant protein from transgenic plants ( Evans, 2004 ) , the Cry1Ac protein was produced in Pseudomonas fluorescens

. Characterization studies were performed to confi rm the equivalency of the Cry1Ac protein expressed in DAS

- 81419

- 2 soybean with the P . fluorescens

- derived Cry1Ac protein.USDA Petition for No nregulated Status of DAS

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- 2 Soybean Page 70 of 214

Dow AgroSciences LLC

Contains No Confidential Business Information Sodium dodecyl sulfate polyacrylamide g el electrophoresis (SDS

- PAGE), W estern blot, glycoprotein detection, and protein sequence analysis by matrix assisted laser desorption/ionization time

- of

- flight mass spectrometry (MALDI

- TOF M S) and MALDI

- TOF MS/MS were used to characterize the biochemical properties of the protein s. Using these methods, the Cry1Ac protein from P . fluorescens and DAS

- 81419

- 2 soybean were shown to be biochemically equivalent, thereby supporting the use of the m icrobe

- derived protein in safety assessment studies

. The methods and results of the biochemical characterization of DAS

- 81419

- 2 soybean

- and microbe

- derived Cry1Ac proteins are described in detail in Appendix 2

. Briefly, both the plant and P . fluorescens

- derived Cry1Ac proteins showed the expected molecular weight of ~130 kDa by SDS

- PAGE and were immunoreactive to Cry1Ac protein

- specific polyclonal antibodies by W este rn blot analysis. There was no evidence of any post

- translational modifications (i.e. glycosylation) of the DAS

- 81419

- 2 soybean

- derived Cry1Ac protein. The amino acid sequence was confirmed by enzymatic peptide mass fingerprinting usi ng MALDI

- TOF MS and MALDI

TOF MS/MS and was shown to be as expected and was identical to the protein expressed in P. fluorescens

. The result is consistent with those for the Cry1Ac protein expressed in WideStrike  cotton event DAS

- 21023

- 5 (also described as 3006

- 210

- 23 ) ( Dow AgroSciences, 2003a )

.

6.1.4. Expression of the Cry1Ac Protein in Plant Tissues A field expression study was conducted at ten locations in U.S. during 2011 ( Appendix 5 ) . The test sites represented regions of diverse agronomic practices and environmental conditions for soybean in North America . Plant tissues sampled included leaf, grain, root, and forage. Leaf tissues were collected at V5 and V10

- 12 stages, and root and forage were collected at the R3 stage of development. The grain was collected at the R8 stage of development ( Gaska, 2006 )

. The soluble, extractable Cry1Ac protein was measured using a validated enzyme

- linked im munosorbent assay (ELISA) . Cry1Ac protein levels for all tissue types were calculated on ng/mg dry w eight basis. Methods used for tissue sampling and quantification of protein expression by ELISA are detailed in Appendix 5

. A summary of the Cry1Ac pr otein concentrations (averaged across sites) in the various soybean matrices is shown in Table 7 . Average expression values ranged from 0.39 ng/mg dry weight in R3 stage root to 25.44 ng/mg dry weight in V5 stage leaf tissue. No Cry1Ac protein was detected in the control (Maverick) tis sues across the ten locations.USDA Petition for No nregulated Status of DAS

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Dow AgroSciences LLC

Contains No Confidential Business Information Table 7
. Expression of Cry1Ac in DAS

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- 2 soybean
. Cry1Ac ng/mg Tissue Dry Weight
Matrix Description Overall Mean Std. Dev.

(n=10) Min/Max

Range STMR a HAFT b

V5 Leaf DAS

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- 2 25.44 6.61 12.10

- 40.20 26.90 35.25

V10

- 12 Leaf DAS

- 81419

- 2 23.16 6.17 10.70

- 37.45 22.15 34.61
Forage DAS

- 81419

- 2 5.54 2.54 1.38

- 11.83 5.44 10.28

Root DAS

- 81419

- 2 0.39 0.24 [0.12] *

- 1.12 0.32 0.97

Grain DAS

- 81419

- 2 1.04 0.10 0.79

- 1.40 1.04 1.20

a Supervised Trials Mean Residue is calculated as the median of all the individual results across different sites. b Highest Average Field Trial is the maximum value of all the means across different sites. * Expression level below LOQ

The Limit of Detection (LOD) and Limit of Quantitation (LOQ) of the Cry1Ac ELISA in the tissue matrices were as follows: Cry1Ac (ng/mg sample dry weight) Matrix LOD LOQ Leaf V5 0. 1 0. 2 Leaf V10

- 12 0. 1 0. 2 Root 0. 1 0. 2 Forage 0. 1 0. 2 Grain 0. 1 0. 2

6.1.5. Identity of the Cry1F Protein The Cry1F protein expressed in DAS

- 81419

- 2 soybean is a synthetic version of Cry1F from Bacillus thuringiensis subsp

. aizawai strain PS81I. In this synthetic version, the first 604 amino acids are comprised of the insect

- active portion of Cry1F; the remaining portion consists of C

- terminal sequences from Cry1Ca3 ( B. thuringiensis subsp

. aizawai PS81I), and Cry1Ab1 ( B. thuringiensis subsp

. Berliner 1715). Together, the portions of Cr y1Ca3 and Cry1A b1 that comprise the chimeric C

- terminal domain are approximately those removed by alkaline proteases in the lepidopteran mid

- gut during the formation of the active Cry1F core toxin. The synthetic Cry1F protein is comprised of 1148 amino ac ids a nd has a molecular weight of ~130.2 kDa ( Figure 30 ). The amino acid sequence of the Cry1F protein expressed in DAS

- 81419

- 2 soybean is identical to that expressed in WideStrike  cotton event DAS

- 24236

- 5 (also described as 281

- 24

- 236) ( Dow AgroSciences, 2003b )

.USDA Petition for No nregulated Status of DAS

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Dow AgroSciences LLC

Contains No Confidential Business Information

1 MENNIQNQCVPYNCLNNPEVEILNEERSTG 30 31 RLPLDISLSLTRFLLSEFVPGVGVAFGLFD 60 61 LIWGFITPSDWSLFLLQIEQLIEQRIETLE 90 91 RNRAITTLRGLADSYEIYIEALREWEANPN 120 121 NAQLREDVRIRFANTDDALITAINNFTLTS 150 151 FEIPLLSVYVQAANLHLSLLRDAVSFGQGW 180 181 GLDIATVNNHYNRLINLIHRYTKHCLDTYN 210 211 QGLENLRGTNTRQWARFNQFRRDLTLTVLD 240 241 IVALFPNYDVRTYPIQTSSQLTREIYTSSV 270 271 IEDSPVSANIP NGFNRAEFGVRPPHLMDFM 300 301 NSLFVTAETVRSQTVWGGHLVSSRNTAGNR 330 331 INFPSYGVFNPGGAIWIADEDPRPFYRTLS 360 361 DPVFVRGGFGNPHYVLGLRGVAFQQTGTNH 390 391 TRTFRNSGTIDSLDEIPPQDNSGAPWNDYS 420 421 HVLNHVTFVRWPGEISGSDSWRAPMFSWTH 450 451 RSATPTNTIDPERITQIPLVKAHTLQS GTT 480 481 VVRGPGFTGGDILRRTSGGPFAYTIVNING 510 511 QLPQRYRARIRYASTTNLRIYVTVAGERIF 540 541 AGQFNKTMDTGDPLTFQSFSYATINTAFTF 570 571 PMSQSSFTVGADTFSSGNEVYIDRFELIPV 600 601 TATLEAESDLERAQKAVNALFTSSNQIGLK 630 631 TDVTDYHIDRVSNLVECLSDEFCLDEKKEL 660 661 SEK VKHAKRLSDERNLLQDPNFRGINRQLD 690 691 RGWRGSTDITIQGGDDVFKENYVTLLGTFD 720 721 ECYPTYLYQKIDESKLKAYTRYQLRGYIED 750 751 SQDLEIYLIRYNAKHETVNVPGTGSLWPLS 780 781 APSPIGKCAHHSHHFSLDIDVGCTDLNEDL 810 811 GVWVIFKIKTQDGHARLGNLEFLEEKPLVG 840 841 EALARVKRAEKKWRDKREKLEWETNIVYKE 870 871 AKESVDALFVNSQYDRLQADTNIAMIHAAD 900 901 KRVHSIREAYLPELSVIPGVNAAIFEELEG 930 931 RIFTAFSLYDARNVIKNGDFNNGLSCWNVK 960 961 GHVDVEEQNNHRSVLVVPEWEAEVSQEVRV 990 991 CPGRGYILRVTAYKEGYGEGCVTIHEIENN 1020 1021 TDELKFSNCV EEEVYPNNTVTCNDYTATQE 1050 1051 EYEGTYTSRNRGYDGAYESNSSVPADYASA 1080 1081 YEEKAYTDGRRDNPCESNRGYGDYTPLPAG 1110 1111 YVTKELEYFPETDKVWIEIGETEGTFIVDS 1140 1141 VELLLMEE Figure 30 . Amino acid sequence of Cry1F protein.
6.1.6. Biochemical Characterization of the Cry1F Protein Large quantities of purified Cry1F protein are required to perf orm safety assessment studies. As it is technically infeasible to extract and purify sufficient amounts of recombinant protein from transgenic plants ( Evans, 2004 ) , the Cry1F protein was pr oduced in Pseudomonas fluorescens

. Characterization studies were performed to confi rm the equivalency of the Cry1F protein expressed in DAS

- 81419

- 2 soybean with the P . fluorescens

- derived Cry1F protein. Sodium dodecyl sulfate polyacrylamide g el electroph oresis (SDS

- PAGE), W estern blot, glycoprotein detection, and protein sequence analysis by matrix assisted laser desorption/ionization time

- of

-USDA Petition for No nregulated Status of DAS

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- 2 Soybean Page 73 of 214

Dow AgroSciences LLC

Contains No Confidential Business Information flight mass spectrometry (MALDI

- TOF MS) and MALDI

- TOF MS/MS were used to characterize the biochemical properties o f the protein s ( Gao et al. , 2006 )

. Using these methods, the Cry1F protein from P . fluorescens and DAS

- 81419

- 2 soybean were shown to be biochemically equivalent, thereby supporting the use of the microbe

- derived protein in safety assessment studies

. The methods and results of the biochemical characterization of DAS

- 81419

- 2 soybean

- and microbe

- derived Cry1F proteins are described in detail in Appendix 3 . Briefly, both the plant and P. fluorescens

- derived Cry1F proteins showed the expected molecular weight of ~130 kDa by SDS

- PAGE and were immunoreactive to Cry1F protein

- spe cific polyclonal antibodies by W estern blot analysis. There was no evidence of any post

- translational modifications (i.e. glycosylation) of the DAS

- 81419

- 2 soybean

- derived Cry1F protein. The amino acid sequence was confirmed by enzymatic peptide mass fingerprinting using MALDI

- TOF MS and MALDI

TOF MS/MS and was shown to be as exp ected and was identical to the protein expressed in P. fluorescens . The result is consistent with those for the Cry1F protein expressed in WideStrike  cotton event DAS

- 24236

- 5 (also described as 281

- 24

- 236 ) ( Dow AgroSciences, 2000 ; Gao et al. , 2006 )

. 6.1.7. Expression of the Cry1F Protein in Plant Tissues A field expression study was conducted at ten locations in U.S. during 2011 ( Appendix 5 ). The test sites represented regions of diverse agronomic practices and environmental conditions for soybean in North America . Plant tissues sampled included leaf, grain, root, and forage. Leaf tissues were collected at V5 and V10

- 12 stages, and root and forage were collected at the R3 sta ge of development. The grain was collected at the R8 stage of development ( Gaska, 2006 )

. The soluble, extractable Cry1F protein was measured using a validated enzyme

- linked immunoso rbent assay (ELISA) . Cry1 F protein levels for all tissue types were calculated on ng/mg dry weight basis. Methods used for tissue sampling and quantif ication of protein expression by ELISA are detailed in Appendix 5

. A summary of the Cry1F protein concentrations (averaged across sites) in the various soybean matrices is shown in Table 8 . Average expression values ranged from 5.23 ng/mg dry weight in R3 stage root to 56.75 ng/mg dry weight in V 5 stage leaf tissue. No Cry1F protein was detected in the control (Maverick) tissues across the ten locations.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Table 8 . Expression of Cry1F in DAS

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- 2 soybean.
Cry1F ng/mg Tissue Dry Weight
Matrix Description Overall Mean Std. Dev.

(n=10) Min/Max

Range STMR a HAFT b

V5 Leaf DAS

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- 2 56.75 15.03 24.60

- 99.50 56.30 76.05

V10

- 12 Leaf DAS

- 81419

- 2 39.07 16.60 12.75

- 76.71 38.70 59.98
Forage DAS

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- 2 20.28 11.29 5.34

- 44.62 20.64 40.23

Root DAS

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- 2 5.23 3.74 1.09

- 16.08 4.12 14.21

Grain DAS

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- 2 13.80 1.24 10.41

- 16.95 13.71 16.21

a Supervised Trials Mean Residue is calculated as the median of all the individual results across different sites. b Highest Average Field Trial is the maximum value of all the means across different sites.

The Limit of Detection (LOD) and Limit of Quantitation (LOQ) of the Cry1F ELISA in the tissue matrices were as follows: Cry1F (ng/mg sample dry weight) Matrix LOD LOQ Leaf V5 0.25 0.5 Leaf V10

- 12 0.25 0.5 Root 0.15 0.3 Forage 0.05 0.1 Grain 0.2 0.4

6.1.8. Food and Feed Safety Assessment of Cry1Ac and Cry1F The food and feed safety assessment of the Cry1Ac and Cry1F proteins expressed in DAS

- 81419

- 2 soybean considers several factors including safety of the donor organism, history of safe use, allergenic potential, toxicity potential, and dietary risk assessment based on
consum ption patterns. Results of the overall safety assessments of Cry1Ac and Cry1F indicate that each protein is unlikely to cause an allergenic reaction in humans or be a toxin to humans or animals. The donor organism, Bacillus thuringiensis, is a naturally occurring microorganism commonly found in soil. B. thuringiensis was first registered as a pesticide in the U.S. in 1961. Today there are more than 100 registered pesticide products containing B. thuringiensis as an active ingredient ( EPA, 2011 )

. An exemption from the requirement of tolerance for residues of B. thuringiensis was first established by FDA in 1960 and was republished and then amen ded after EP A was established.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Cry1Ac and Cry1F have a long history of safe use. The safety of the proteins has been demonstrated in sprayable Bt formulations for pest control in agriculture for over half a century ( Mendelsohn et al. , 2003 ; EPA, 2011 ; Sanahuja et al. , 2011 )

. Both proteins are expressed in events comprising Do w AgroSciences’ WideStrike  cotton authorized for cultivation in the U.S. and Brazil and for food and feed use in Australia, Brazil, Canada, European Union, Japan, Korea, Mexico, N ew Zealand and U.S. ( www.biotrad estatus.com ). Bt corn and Bt cotton expressing variations of either Cry1Ac or Cry1F have been cultivated for commercial use in the U.S. and other countries for more than a decade. In 1997, EPA established an exemption from the requirement of a tolerance for the plant

- incorporated protectant Cry1Ac in all plants (40 CFR §174.510). Later EPA established an exemption from the requirement of a tolerance for the plant

- incorporated protectant Cry1F in cotton (40 CFR §174.504) and in corn (40 CFR §174.520). Th e exemptions were based on safety assessments of the proteins including digestibility in simulated gastric fluid, lack of homology to known allergens and protein toxins, and lack of mammalian toxicity as demonstrated by acute oral mouse gavage studies. DA S has filed a petition with EPA for an exemption of a tolerance for Cry1F as expressed in soybean in 2012. Allergenicity assessments of Cry1Ac and Cry1F found no evidence of allergenic potential for either protein

. Both Cry1Ac and Cry1F were rapidly digested in simulated gastric fluids in less than one minute indicating that the proteins are unlikely to elicit allergenic reactions when consumed. Glycosylation analysis revealed no detectable covalently linked carbohy drates in the Cry1Ac and Cry1F proteins expressed in DAS

- 81419

- 2 soybean. Bioinformatics analysis of Cry1Ac and Cry1F amino acid sequences using an updated allergen database (Food Allergy Research and Resource Program (FARRP), www.allergenonline.org , Allergen Database Version 12, Released February, 2012) demonstrated that the proteins do not share any amino acid sequence similarities with known allergens. No significant homology was identified when either protei n sequence was compared with known allergen using the search criteria of either a match of eight or more contiguous identical amino acids, or greater than 35% identity over 80 amino acid residues. Toxicity assessments identified no potential for toxic effe cts in humans or animals for Cry1Ac or Cry1F. Mouse acute oral toxicity studies demonstrated that neither protein was acutely toxic or ca used any adverse effects with doses up to 700 mg/kg body weight and 600 mg/kg body weight for Cry1Ac and Cry1F, respec tively

. BLASTp search of Cry1Ac and Cry1F amino acid sequences against an up

- to

- date GenBank non

- redundant protein sequences (nr) database ( www.ncbi.nlm.nih.gov/genbank ; updated to May 18, 2012 ) demonstrat ed that neither Cry1Ac nor Cry1F shared any amino acid sequence similari ties with known protein toxins. In 2005, EPA concluded, based upon the human health assessment of Cry1Ac and Cry1 F expressed in WideStrike  cotton events DAS

- 21023

- 5 (also described as 3006

- 210

- 23 expressing Cry1Ac) and DAS

- 24236

- 5 (also described as 281

- 24

- 236 expressing Cry1F) , that “there is a reasonable certainty that no harm will result from exposure to Cry1F and Cry1Ac” and “data provided by D ow AgroSciences support issuance of a tolerance exemption for the Bacillus thuringiensis Cry1F protein and the genetic material necessary for its productin in or on cotton” ( EPA, 2005 )

. Cry1Ac and Cry1F expressed in DAS

- 81419

- 2 soybean are identical in amino acid sequence to Cry1Ac and Cry1F expressed in WideStrike  cotton events DAS

- 21023

- 5 and DAS

- 24236

- 5 (Sections 6.1.2 and 6.1.5 )

. The Cry proteins expressed in DAS

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- 2USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information soybean are biochemically equivalent to Cry1Ac and Cry1F (Sections 6.1.3 and 6.1.6 ) that had been previously assessed in terms of allergenic and toxicity potential. Potential dietary risk to humans and livestock from the consumption of the Cry1Ac and Cry1F proteins in food and feed derived from DAS

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- 2 soybean were evaluated by determining margins of exposure (MOE). MOE is the ratio of the No Observable Effect Level (NOEL) of Cry1Ac or Cry1F determined by the mouse acute oral toxicity study (700 mg/kg body weight for Cr y1Ac, 600 mg/kg body weight for Cry1F) to estimated dietary intake of the respective Cry protein. To evaluate dietary risk for human food consumption, MOEs were determined for the overall U.S. population and for several subpopulations based on mean protein expression levels in the seed . The dietary risk assessments revealed that the MOEs for the overall U.S. population were greater than 1067073 and 68918 for Cry1Ac and Cry1F, respectively. The MOEs for infants (an age group with highest soybean intake) we re greater than 268817 and 17366 for Cry1Ac and Cry1F, respectively. It is a common regulatory practice that MOE values greater than 100 are considered protective when using animal data to protect human health. Large MOE values for the overall U.S. popula tion and for each subpopulation indicate there is no concern for Cry1Ac and Cry1F from acute dietary exposure through DAS

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- 2 soybean. The U.S. livestock dietary assessments considered several soybean commodity forms as potential animal feeds includin g seed, meal, hulls, aspirated grain fractions and optional forage. For the typical diet without soybean forage, the highest exposed animal in the U.S. is beef cow. For the worst

case diet with soybean forage, the highest exposed animal in the U.S. is da iry cow. Using the worst

- case diet for assessments the MOEs were greater than 2941 and 596 for Cry1Ac and Cry1F, respectively, indicating an adequate margin of safety for livestock.

6.1.9. Summary of Cry1Ac and Cry1F Characterization The Cry1Ac protein expressed in DAS

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- 2 soybean is a synthetic version of Cry1Ac1, from B. thuringiensis subsp

. kurstaki strain HD73. The Cry1Ac protein is comprised of 1156 amino acids a nd has a molecular weight of ~130 .7 kDa

. Detailed biochemica l characterization of the Cry1A c protein d erived from DAS

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- 2 soybean confirmed the identity of the protein

. Moreover, biochemical characterization demonstrated that the DAS

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- 2 soybean

- derived and Pseudomonas

- derived Cry1Ac proteins are equivalent thereby supporting the use of Pseudomonas

- derive d Cry1Ac protein for safety assessment. Additionally, characterization of Cry1Ac p rotein expression in DAS

- 81419

- 2 soybean over the growing season was determined by analyzing leaf, root, whole plant, and grain tissues. The Cry1F protein expressed in DAS

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- 2 soybean is a synthetic version of Cry1F from B. thuringiensis subsp

. aizawai strain PS81I. The Cry1F protein is comprised of 1148 amino acids
a nd has a molecular weight of ~130 .2 kDa

. Detailed biochemica l charac terization of the Cry1F protein d erived from DAS

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- 2 soybean confirmed the identity of the protein. Moreover, biochemical characterization demonstrated that the DAS

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- 2 soybean

- derived and Pseudomonas

- derived Cry1F proteins are equivalent, thereby supporting the use of Pseudomonas

- derived Cry1F protein for safety assessment

. Additionally, characterization of Cry1F p rotein expression in DAS

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- 2 soybean over the growing season was determined by analyzing leaf, root, whole plant, and grain tissues

. A step

- wise, weight

- of

- evidence approach was used to assess the potential for toxic or allergenic effects from the Cry1Ac and Cry1F protein s

. Bioinformatic analyses revealed no meaningfulUSDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information homologies to known or putative allergens or toxins for the Cry1A c and Cry1F amino acid sequence. Both Cry1Ac and Cry1F hydrolyze d rapi dly in simulated gastric fluid and glycosylation analysis revealed no detectable covalently linked carbohydrates in Cry1Ac and Cry1F

. Additionaly, n either protein caused adverse effects in mouse acute oral toxicity studies

. Therefore , the low level Cry1Ac and Cry1F content in DAS

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- 2 soybean relative to total plant proteins presents a low exposure risk to human s and animals, and the results of th e overall safety assessment of the Cry1Ac and Cry1F protein s indicate that the proteins are unlikely to cause allergenic or toxi c effects in humans or animals.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information 6.2. PAT
6.2.1. Mode of Action of the PAT Protein The phosphinothricin acetyltransferase (PAT) has been ex pressed in a variety of crops to provide tolerance to the herbicide glufosinate, which contains the active ingredient phosphinothricin (PPT). The L

- isomer of PPT is a potent inhibitor of glutamine synthetase (GS) in plants and is used as a non

- selective he rbicide ( OECD, 1999 )

. Inhibition of GS by PPT causes rapid accumulation of intracellular ammonia which leads to cessation of photorespiration and results in the death of the plant cell ( Duan et al. , 2009 )

. The pat gene which encodes phosphinothricin acetyltransferase (PAT) acetylates the free NH 2 group of PPT ( in the presence of acetyl coenzyme A) and thereby prevents autotoxicity in the producin g organism ( Figure 31 , ( Duke, 1996 ) )

.

Figure 31 . Mode of action of the PAT protein.

6.2.2. Identity of the PAT Protein The phosphinothricin acetyltransferase (PAT) protein was d erived from Streptomyces viridochromogenes , a gram
- positive soil bacterium ( Strauch et al. , 1988 ; OECD, 1999 )

. The pat transgene in DAS

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- 2 encodes a protein sequence that is identical to the native PAT protein ( UniProt Accession N umber : Q57146 )

. PAT is comprised of 183 amino acids a nd has a molecular weight of ~20.6 kDa ( Figure 32 )

.

1 MSPERRPVEIRPATAADMAAVCDIVNHYIE 30 31 TSTVNFRTEPQTPQEWIDDLERLQDRYPWL 60 61 VAEVEGVVAGIAYAGPWKARNAYDWTVEST 90 91 VYVSHRHQRLGLGSTLYTHLLKSMEAQGFK 120 121 SVVAVIGLPNDPSVRLHEALGYTARGTLRA 150 151 AGYKHGGWHDVGFWQRDFELPAPPRPVRPV 180 181 TQI Figure 32
. Amino acid sequence of the PAT protein
.

6.2.3. Biochemical Characterization of the PAT Protein Characterization of the biochemical properties of the DAS

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- 2 soybean

- derived PAT protein was accomplished through the use of sodium dodecyl sulfate polyacrylamide g elUSDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information electrophoresis (SDS

- PAGE) , W estern blot analysis , protein sequence alignment, and a lateral flow strip assay

. The methods and results are described in Appendix 4

. Using these methods , the PAT protein produced in DAS

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- 2 soybean was shown to be substantially equivalent to that produced in Escherichia coli

. Furthermore, the sequence of the PAT protein is identical to the PAT protein expressed in other deregulated transgenic crops ( USDA, 1996 , 2001 , 2004 , 2005 )

. 6.2.4. Expression of the PAT Protein in Plant Tissues A field expression study was conducted at ten locations in U.S. during 2 011 ( Appendix 5 ). The test sites represented regions of diverse agronomic practices and environmental conditions for soybean in North America . Plant tissues sampled included leaf, grain, root, and forage. Leaf tissues were collected at V5 and V10

- 12 stages, and root and forage were collected at the R3 stage of development. The grain was collected at the R8 stage of development ( Gaska, 2006 )

. The soluble, extractable PAT protein was measured using a validated enzyme

- li nked immunosorbent assay (ELISA) method. PAT protein levels for all tissue types were calculated on ng/mg dry weight basis. Methods used for tissue sampling and quantification of protein expression by ELISA are detailed in Appendix 5

. A summary of the PAT protein concentrations (averaged across sites) in the various soybean matrices is shown in Table 9 . Average expression values ranged from 0.63 ng/mg dry weight in R3 stage root to 5.60 ng/mg dry weight in V 10

- 12 stage leaf tissue. No PAT protein was detected in the control (Maverick) tissues acros s the ten locations.
Table 9 . Expression of PAT in DAS

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- 2 soybean. PAT ng/mg Tissue Dry Weight
Matrix Description Overall Mean Std. Dev. Min/Max Range STMR a HAFT b

V5 Leaf DAS

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- 2 5.23 0.88 3.25

- 7.35 5.30 6.93

V10

- 12 Leaf DAS

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- 2 5.60 1.14 2.55

- 7.56 5.76 7.32
Forage DAS

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- 2 4.06 1.30 1.24

- 6.12 4.02 5.69

Root DAS

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- 2 0.63 0.12 0.44

- 1.05 0.63 0.85

Grain DAS

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- 2 0.86 0.13 0.63

- 1.12 0.83 1.06

a Supervised Trials Mean Residue is calculated as the median of all the individual results across different sites. b Highest Average Field Trial is the maximum value of all the means across different sites. The Limit of Detection (LOD) and Limit of Quantitation (LOQ) of the PAT ELISA in the tissue matrices were as follows: PAT (ng/mg sample dry weight) Matrix LOD LOQ Leaf V5 0.06 0.12 Leaf V10

- 12 0.06 0.12USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information PAT (ng/mg sample dry weight) Matrix LOD LOQ Root 0.06 0.12 Forage 0.06 0.12 Grain 0.06 0.12
6.2.5. Food and Feed Safety Assessment for PAT Protein The PAT protein expressed in DAS

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- 2 soybean originates from a common soil bacterium Streptomyces viridochromogenes ( Wohlleben et al. , 1988 )

. The protein is 100% identical in amino acid sequence to PAT expressed in other tr ansgenic crops that have been previously deregulated by USDA ( USDA, 1996 , 2001 , 2004 , 2005 )

. The food and feed safety of PAT was assessed in th ese products and in published findings ( OECD, 1999 ; Herouet et al. , 2005 ) and shown to present no signif icant food or feed safety risk. The PAT protein is hydrolyzed rapidly in simulated gastric fluid and there wa s no evidence of acute toxicity in mice at a dose of 5000 mg/kg body wei ght of PAT protein ( OECD, 1999 )

. Additionally, updated b ioinformatic analyses revealed no meaningful homologies to known or putative allergens or toxi ns for the PAT amino acid sequenc e

. Additionally, the US EPA has concluded, after reviewing data on the acute toxicity and digestibility of the PAT protein, that there is a reasonable certainty that no harm will result from aggregate exposure to the U.S. p opulation, including infants and children, to the PAT protein and the genetic material necessary for its production ( EPA, 1997 )

. The US EPA has consequently established an exemption from tolerance requirements pursuant to FFDCA section 408(j)(3) for PAT and the genetic material nece ssary for its production in all plants. The dietary risk assessments showed large margins of exposure for the overall U.S. populations as well as for infants (age group with the highest soybean consumption). The analyses also revealed large margins of exposure for poultry and livestock. Large margins of exposure indicate lack of meaningful risks to humans and animals from dietary exposure.

6.2.6. Summary of PAT Protein Characterization The phosphinothricin acetyltra nsferase (PAT) protein was derived from Streptomyces viridochromogenes , a gram
- positive soil bacterium. PAT is comprised of 183 amino acids a nd has a molecular weight of ~20.6 kDa . T he PAT protein , as expressed in DAS

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- 2 , has the
same amino acid seq uence as the PAT expressed in several other transgenic event s

. Western blot analysis and lateral flow strip assays demonstrated that the PAT protein expressed in DAS

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- 2 soybean had the expected molecular weight and immunorea ctivity. Characterization of PAT protein expression in DAS

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- 2 soybean was determined by analyzing leaf, root, whole plant, and grain tissues over the growing season. The PAT protein has a long history of safe use and the food and feed safety of PAT has previo usly been assessed in other products and in published findings. T he low level expression of the PAT protein in DAS

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- 2 soybean presents a low exposure risk to humans and animals, and the results of the overall safety assessment of the PAT protein indi cate that it is unlikely to cause allergenic or toxi c effects in humans or animals.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information 7. Grain and Forage Composition Field trials with DAS

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- 2 soybean, the non

- transgenic control (Maverick), and reference lines were conducted in 2011 at ten sites located in the U.S. This study used the same plots that were used for protein expression ( Section 6 ) and agronomic characterization ( Section 8 ) studies. No biologically meaningful unintended compositional differences were observed between the
non
- transgenic near

- isogenic control (Maverick) and DAS

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- 2 soybean. Results from t his study demonstrate compositional equivalence between event DAS

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- 2 soybean and non

transgenic soybean.
7.1. Field Study Design and Data Analysis A crop composition study with DAS

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- 2 soybean, a near

- isogenic non

- transgenic control (Maverick) and six non
- transgenic reference lines (IL 3503, Porter 75148, DSR 75213

- 72, Pioneer 93M62, HiSOY 38C60, Williams 82) was conducted in 2011 at ten sites located in Richland, Iowa; Atlantic, Iowa; Carlyle, Illinois; Wyoming, Illinois; Frankfort, Indiana; Fisk, Mis souri; La Plata, Missouri; York, Nebraska; Brunswick, Nebraska; and Germansville, Pennsylvania.
Each trial site included event DAS

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- 2, the non

- transgenic near

- isogenic control (Maverick), and three non

- transgenic reference lines. At each of the ten sites, all entries were arranged in a randomized complete block design with four blocks. Across all sites, e ach control (Maverick) and DAS

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- 2 entry was represented by a total of 40 plots (10 sites, 4 replicate plots per entry at e ach site). Three of the six reference lines were included at each site by randomizing across sites in a balanced incomplete

- block design. Each of the six reference lines was assigned to five sites; therefore, each reference line was represented by a tota l of 20 plots across sites (5 sites per reference line, 4 replicate plots per entry at each site). At each site, four replicate plots of each entry were established, with each plot consisting of four 25 ft (7.62 m) rows. Soybean seeds were planted at a s eeding rate of approximately 125 seeds per row with seed spacing within each row of approximately 2.4 inches (6 cm). Each soybean plot was bordered by two rows of a non

- transgenic soybean cultivar of similar maturity. The entire trial site was surrounded by a minimum of four rows (10 ft or 3.0 m) of a non

- transgenic soybean cultivar of similar maturity. Appropriate insect, weed, and disease control practices were applied to produce an agronomically acceptable crop. Soybean forage samples used for compos ition analysis were composed of the above

- ground portion of three whole plants that collected from the center two rows of each four row plot at the R3 growth stage. Soybean seed samples used for composition analysis were composed of a representative 500 g sample of the soybean seed harvested from the center two rows of each four row plot. Composition Analysis Samples of soybean forage and seed were analyzed for nutrient and antinutrient content ( Appendix 6 ). The analytes examined are presented in Table 10

.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Table 10 . Composition analytes.

Matrix Category Analyte Matrix Category Analyte Forage Proximate Protein Grain Amino Acid Valine Forage Proximate Fat Grain Fatty Acid 8:0 Caprylic Forage Proximate Ash Grain Fatty Acid 10:0 Capric Forage Proximate Moisture Grain Fatty Acid 12:0 Lauric Forage Proximate Carbohydrates Grain Fatty Acid 14:0 Myristic Forage Fiber ADF Grain Fatty Acid 14:1 Myristoleic Forage Fiber NDF Grain Fatty Acid 15:0 Pentadecanoic Forage Mineral Calcium Grain Fatty Acid 15:1 Pentadec e noic Forage Mineral Phosphorus Grain Fatty Acid 16:0 Palmitic Grain Proximate Protein Grain Fatty Acid 16:1 Palmitoleic Grain Proximate Fat Grain Fatty Acid 17:0 Heptadecanoic Grain Proximate Ash Grain Fatty Acid 17:1 Heptadecenoic Grain Proximate Moisture Grain Fatty Acid 18:0 Stearic Grain Proximate Carbohydrates Grain Fatty Acid 18:1 Oleic Grain Fiber ADF Grain Fatty Acid 18:2 Linoleic Grain Fiber NDF Grain Fatty Acid 18:3 Linole n ic Grain Fiber Total Dietary Fiber Grain Fatty Acid 18:3 γ

- Linole n ic Grain Mineral Calcium Grain Fatty Acid 20:0 Arachidic Grain Mineral Copper Grain Fatty Acid 20:1 Eicosenoic Grain Mineral Iron Grain Fatty Acid 20:2 Eicosadienoic Grain Mineral Magnesium Grain Fatty Acid 20:3 Eicosatrienoic Grain Mineral Manganese Grain Fatty Acid 20:4 Arachidonic Grain Mineral Phosphorus Grain Fatty Acid 22:0 Behenic Grain Mineral Potassium Grain Vitamin Vitamin A (Beta Carotene) Grain Mineral Selenium Grain Vitamin Vitamin B1 (Thiamine HCl) Grain Mineral Sodium Grain Vitamin Vitamin B2 (Riboflavin) Grain Mineral Zinc Grain Vitamin Vitamin B3 (Niacin) Grain Amino Acid Alanine Grain Vitamin Vitamin B5 (Pantothenic Acid) Grain Amino Acid Arginine Grain Vitamin Vitamin B6 (Pyridoxine HCl) Grain Amino Acid Aspartic Acid Grain Vitamin Vitamin B9 (Folic Acid) Grain Amino Acid Cystine Grain Vitamin Vitamin C (Ascorbic Acid) Grain Amino Acid Glutamic Acid Grain Vitamin α

- t ocopherol (Vitamin E) Grain Amino Acid Glycine Grain Vitamin β

- tocopherol Grain Amino Acid Histidine Grain Vitamin γ

- tocopherol Grain Amino Acid Isoleucine Grain Vitamin δ

- tocopherol Grain Amino Acid Leucine Grain Vitamin Total tocopherol Grain Amino Acid Lysine Grain Bioactive Lectin Grain Amino Acid Methionine Grain Bioactive Phytic Acid Grain Amino Acid Phenylalanine Grain Bioactive Raffinose Grain Amino Acid Proline Grain Bioactive Stachyose Grain Amino Acid Serine Grain Bioactive Trypsin Inhibitor Grain Amino Acid Threonine Grain Bioactive Total Daidzein Equivalent Grain Amino Acid Tryptophan Grain Bioactive Total Genistein Equivalent Grain Amino Acid Tyrosine Grain Bioactive Total Glycitei n Equivalent

The results of the compositional analysis for soybean forage and seed were compared with values reported in the literature ( Kakade et al. , 1972 ; Iskander, 1987 ; Hartwig and Kilen, 1991 ; Padgette et al. , 1996 ; Taylor et al. , 1999 ; OECD, 2001 ; McCann et al. , 2005 ; Harrigan et al. , 2007 ; Bilyeu et al. , 2008 ; Lundry et al. , 2008 ; Berman et al. , 2009 , 2010 ; ILSI, 2010 ; Berman et al. , 2011 ; Zhou et al. , 2011 )

. A summary of the compositional data from the literature can be found in Appendix 7

.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Statistical Analysis Analysis of variance was conducted across field sites (combined

- site a nalysis) for composition data using a mixed model ( SAS Institute Inc., 2009 )

. Entry was considered a fixed effect, and location, block within location, and location

- by

- entry, were designated as random effec ts. Significant differences were declared at the 95% confidence level. Data were not rounded off for statistical analysis. The significance of an overall treatment e ffect was estimated using an F

- test. Comparisons were made between DAS

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- 2 and the control entry using t

- tests. Due to the large number of comparisons made in this study, multiplicity was an issue. Multiplicity is an issue when a large number of comparisons are made in a single study to look for unexpected effects. Under these conditions, the probability of falsely declaring differences based on compa rison

- wise P

- values is very high (1

- 0.95 number of comparisons ). In th is study there
w ere 71 analytes analyzed for composition ; therefore, 71 comparisons were made in the combined

- site composition analysis. As a result , the probability of declaring one or more false differences ba sed on unadjusted P

- values was 97.38 % (1

- 0.95 71 )

. One method to account for multiplicity is to adjust P

- val u es to control the experiment

- wise error rate, but when many comparisons are made in a study, the power for detecting speci fic effects can be reduced significantly. An alternative with much greater power is to adjust P

- values to control the probability that each declared difference is significant ( Curran

- Everett, 2000 )

. This can be accomplished using a False Discovery Rate (FDR) control procedure ( Benjamini and Hochberg, 1995 ) ; FDR methods are commonly applied in studies examining transgenic crop s ( Herman et al. , 2007 ; Coll et al. , 2008 ; Huls et al. , 2008 ; Jacobs et al. , 2008 ; Stein et al. , 2009 ; Herman et al. , 2010 )

. Therefore, the P

- values from the composition contra sts were each adjusted using the FDR method to improve discrimination of true differences among treatments from random effects (false positives). Differences were considered significant if the FDR

- adjusted P

value was less than 0.05.
7.2. Composition Analysis Results An across

- site summary and statistical analysis of composition data from the non

- transgenic
near
- isogenic control (Maverick) and DAS

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- 2 soybean is found in Table 11 to Tab le 17 and Figure 33 to Figure 39 . For each analyte and entry , the least

- square mean, standard error, and minimum and maximum sample value are reported. Also for comparison, the minimum and maximum values for the six reference lines and literature range are reported (values are for individual plot results except where noted). Arithmetic means from each field site are plotted in figures and literature ranges are shaded (literature ranges reported as not detected or <LO D are plotted as zeros).

7.2.1. Proximate, Fiber, and Mineral Analysis of Forage S oybean forage samples from the non
- transgenic control (Maverick) , DAS

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- 2, and reference variety entries were analyzed for proximates, fiber, and minerals (nine analytes) . A
su mmary of the r esults across all locations is presented in Table 11 and Figure 33

. No statistical differences were observed in the combined

- site analysis between the control and DAS

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- 2 entries for all proximate, fiber, and mineral analytes tested. Additionally, a ll mean values were within literature ranges (when available) a nd within ranges for reference varieties included in the study.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Table 11 . Summary of the proximate, fiber, and mineral analysis of soybean forage from all sites and literature range.

Analytical Component (Units) a Overall Treatment Effect (Pr > F) b Control

(Maverick) DAS

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- 2 Reference Variety Range Combined

Literature Range d
Mean ± SE Mean ± SE Min

- Max Min

- Max Min

- Max Min

- Max

(P

- value, Adj.P) c Proximate Protein 0.786 20.4 ± 0.8 20.6 ± 0.8 13.9

- 29.8 15.8

- 25.8 14.0

- 35.5 11.2

- 24.71 (% DW) (0.786, 0.828) Fat 0.274 2.49 ± 0.19 2.70 ± 0.20 0.898

- 3.89 0.857

- 4.32 0.685

- 5.32 1.01

- 9.87 (% DW) (0.274, 0.589) Ash 0.926 8.96 ± 0.31 8.99 ± 0.32 7.01

- 16.9 7.51

- 10.9 6.80

- 15.2 4.68

- 10.782 (% DW) (0.926, 0.931) Moisture 0.578 79.7 ± 0.8 79.4 ± 0.8 75.8

- 84.3 75.5

- 83.6 75.3

- 86.6 32.05

- 84.60 (% FW) (0.578, 0.719) Carbohydrates e 0.510 68.2 ± 1.1 67.7 ± 1.1 57.2

- 76.4 60.3

- 75.7 50.1

- 75.6 59.8

- 80.18 (% DW) (0.510, 0.696) Fiber Acid Detergent 0.310 34.7 ± 2.1 33.3 ± 2.1 Fiber (ADF) 22.4

- 56.7 22.2

- 45.4 19.4

- 64.1 22.72

- 59.03 (% DW) (0.310, 0.595) Neutral Detergent 0.300 41.6 ± 2.6 39.9 ± 2.6 Fiber (NDF) 27.8

- 70.9 27.2

- 59.3 25.2

- 82.0 19.61

- 73.05 (% DW) (0.300, 0.592) Mineral Calcium 0.333 1378 ± 64 1401 ± 64 940

- 1840 908

- 1740 874

- 2000 NR (mg/100g dry wt.) (0.333, 0.615) Phosphorus 0.793 266 ± 7 268 ± 7 206

- 327 201

- 342 187

- 381 NR (mg/100g dry wt.) (0.793, 0.828) Abbreviations: NR = Not Reported.

a Unit of measure was not converted prior to analysis, except for calcium and phosphorus. Unit of measure for calcium and phosphorus was converted from % DW to mg/100g dry wt. prior to analysis.
b Overall treatment effect estimated using an F

- test.

c P

- value = Comparison to the control using t

- tests; Adj. P = P

- val ues adjusted using False Discovery Rate (FDR) procedure; P

- values < 0.05 were considered significant.

d Combined range from Appendix 7

. e % Carbohydrates = 100 %

- (% Protein + % Fat + % Ash + % Moisture)USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 33 . Proximates, fiber and minerals in control, DAS

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- 2 and reference variety soybean forage. moisture = % fresh weight, calcium and pho sphorous = mg/100g dry wt., all others = % dry weight Symbols for each location shown: open circle = IA1,  = IA2, + = IL1, open triangle = IL2, open square = IN, open diamond = MO1, filled circle = MO2, filled triangle = NE1, filled square = NE2, filled d iamond = PA. Literature range is shaded for each analyte.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 33 (Cont)

. Proximates, fiber and minerals in control, DAS

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- 2 and reference variety soybean forage. moisture = % fresh weight, calcium and phosphorous = mg/100g dry wt., all others = % dry weight Symbols for each location shown: open circle = IA1,  = IA2, + = IL1, open triangle = IL2, open square = IN, open diamond = MO1, filled circle = MO2, filled triangle = NE1, filled square = NE2, filled diamond = PA. Literature range (when available) is shaded for each analyte.

7.2.2. Proximate and Fiber Analysis of Seed Soybean seed samples from the control (Maverick) , DAS

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- 2, and reference variety entries were analyzed for proximate s and fiber (eight analytes). A summary of the results across all locations is presented in Table 12 and Figure 34 . All mean result s were within literature ranges (when available) and within ranges for reference varieties included in the study. Statistically significant overall treatment effects were found for fat, ash, and moisture , and pair

- wise comparisons between the control and DAS

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- 2 were also significant for those analytes based upon unadjusted P

- values (but not after adjustment for multiplicity using FDR methods). However, these differences were small relative to natural variation and not biologically meaningful, as all results were within literature ranges and within the ranges of the reference varieties included in this study.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Table 12 . Summary of the proximate and fiber analysis of soybean seed from all sites and literature range.

Analytical Component (Units) a Overall Treatment Effect (Pr > F) b Control

(Maverick) DAS

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- 2 Reference Variety Range Combined

Literature Range d
Mean ± SE Mean ± SE Min

- Max Min

- Max Min

- Max Min

- Max

(P

- value, Adj.P) c Proximate Protein 0.442 37.9 ± 0.6 38.1 ± 0.6 34.3

- 41.9 32.9

- 42.8 34.4

- 46.0 32

- 48.4 (% DW) (0.442, 0.662) Fat 0.008 18.2 ± 0.5 17.7 ± 0.5 15.4

- 21.5 14.2

- 21.0 14.1

- 22.7 8.104

- 24.7 (% DW) ( 0.008 , 0.102) Ash 0.026 5.06 ± 0.07 5.18 ± 0.07 4.62

- 5.68 4.57

- 6.05 3.79

- 6.79 3.885

- 6.994 (% DW) ( 0.026 , 0.233) Moisture 0.019 12.3 ± 0.9 11.7 ± 0.9 8.29

- 19.2 7.56

- 17.9 7.91

- 22.7 4.7

- 34.4 (% FW) ( 0.019 , 0.196) Carbohydrates e 0.372 38.8 ± 0.7 39.0 ± 0.7 33.6

- 41.2 33.3

- 43.3 29.9

- 40.8 29.3

- 50.2 (% DW) (0.372, 0.643) Fiber Acid Detergent 0.813 15.3 ± 0.7 15.2 ± 0.7 Fiber (ADF) 10.6

- 22.8 10.5

- 22.5 10.2

- 21.0 7.81

- 26.26 (% DW) (0.813, 0.837) Neutral Detergent 0.576 17.5 ± 0.8 17.7 ± 0.8 Fiber (NDF) 11.7

- 24.2 11.6

- 25.5 10.6

- 22.6 8.53

- 23.90 (% DW) (0.576, 0.719) Total Dietary 0.689 23.8 ± 0.9 24 .0 ± 0.9 Fiber 17.6

- 29.1 17.4

- 31.3 16.1

- 29.5 NR (% DW) (0.689, 0.765) Abbreviations: NR = Not Reported.

a Unit of measure was not converted prior to analysis.
b Overall treatment effect estimated using an F

- test.

c P

- value = Comparison to the control using t

- tests; Adj. P = P

- values adjusted using False Discovery Rate (FDR) procedure; P

- values < 0.05 were considered significant.

d Combined range from Appendix 7

. e % Carbohydrates = 100 %

- (% Protein + % Fat + % Ash + % Moisture)USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 34 . Proximates and fiber in control, DAS

- 81419

- 2, and reference variety soybean seed. moisture = % fresh weight, all others = % dry weight Symbols for each location shown: open circle = IA1,  = IA2, + = IL1, open triangle = IL2, open square = IN, open diamond = MO1, filled circle = MO2, filled triangle = NE1, filled square = NE2, filled di amond = PA. Literature range is shaded for each analyte.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 34 (Cont) . Proximates and fiber in control, DAS

- 81419

- 2, and reference variety soybean seed. mois ture = % fresh weight, all others = % dry weight Symbols for each location shown: open circle = IA1,  = IA2, + = IL1, open triangle = IL2, open square = IN, open diamond = MO1, filled circle = MO2, filled triangle = NE1, filled square = NE2, filled diamon d = PA. Literature range (when available) is shaded for each analyte.

7.2.3. Mineral Analysis of Seed Soybean seed samples from the control (Maverick) , DAS

- 81419

- 2, and reference variety entries were analyzed for mineral content (10 analytes). A summary of t he results across all locations is presented in Table 13 and Figure 35 . For sodium, statistical analysis was not performed because more than 50% of the samples were found to be below the LOQ. All mean results were within literature ranges (when available) and/or within ranges for reference varieties included in the study. Addit ionally, no statistical differences were observed in the combined

- site analysis between the control and DAS

- 81419

- 2 entries for all mineral analytes.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Table 13 . Summary of the mineral analysis of soybean seed from all sites and literature range.

Analytical Component (Units) a Overall Treatment Effect (Pr > F) b Control

(Maverick) DAS

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- 2 Reference Variety Range Combined

Literature Range d
Mean ± SE Mean ± SE Min

- Max Min

- Max Min

- Max Min

- Max

(P

- value, Adj.P) c Calcium 0.137 270 ± 8 267 ± 8 233

- 335 205

- 328 181

- 339 116.55

- 510 (mg/100 g DW) (0.137, 0.456) Copper 0.655 1.32 ± 0.06 1.33 ± 0.06 0.922

- 1.64 0.894

- 1.72 0.693

- 1.86 0.632

- 1.092 (mg/100 g DW) (0.655, 0.747) Iron 0.500 9.56 ± 1.40 10.26 ± 1.40 6.61

- 27.3 6.21

- 42.9 6.33

- 151 3.734

- 10.954 (mg/100 g DW) (0.500, 0.696) Magnesium 0.663 233 ± 4 232 ± 4 204

- 256 197

- 257 205

- 278 219.40

- 312.84 (mg/100 g DW) (0.663, 0.747) Manganese 0.548 2.64 ± 0.09 2.67 ± 0.09 2.13

- 3.09 2.01

- 3.96 2.22

- 7.18 2.52

- 3.876 (mg/100 g DW) (0.548, 0.719) Phosphorus 0.118 607 ± 14 619 ± 14 536

- 704 494

- 708 471

- 759 506.74

- 935.24 (mg/100 g DW) (0.118, 0.456) Potassium 0.168 1799 ± 21 1819 ± 21 1660

- 1940 1490

- 1980 1650

- 2050 1868.01

- 2510 (mg/100 g DW) (0.168, 0.459) Selenium 0.401 468 ± 188 507 ± 187 <LOQ

- 2370 <LOQ

- 2560 <LOQ

- 3060 NR (ppb DW) (0.401, 0.647) Sodium NA NA NA <LOQ <LOQ <LOQ

- 22.2 4.05

- 30 (mg/100 g DW) Zinc 0.066 4.53 ± 0.15 4.63 ± 0.15 3.66

- 5.73 3.66

- 5.83 3.12

- 6.33 4.98

- 7.578 (mg/100 g DW) (0.066, 0.381) Abbreviations: NA (Not Available) = analysis not performed, majority of data was < LOQ (Limit of Quantitation); NR = Not Reported

a Unit of measure was not converted prior to analysis.
b Overall treatment effect estimated using an F

- test.

c P

- value = Comparison to the control using t

- tests; Adj. P = P

- values adjusted using False Discovery Rate (FDR) procedure; P

- values < 0.05 were considered significant.

d Combined range f

rom Appendix 7

.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 35 . Minerals in control, DAS

- 81419

- 2, and reference variety soybean seed. selenium = ppb dry weight, all others = mg/100 g dry weight Symbols for each location shown: open circle = IA1,  = IA2, + = IL1, open triangle = IL2, open square = IN, open diamond = MO1, filled circle = MO2, filled triangle = NE1, filled square = NE2, filled dia mond = PA. Literature range is shaded for each analyte.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 35 (Cont)

. Minerals in control, DAS

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- 2, and reference variety soybean seed. selenium = ppb dry weight, all others = mg/100 g dry weight Symbols for each location shown: open circle = IA1,  = IA2, + = IL1, open triangle = IL2, open square = IN, open diamond = MO1, filled circle = MO2, filled triangle = NE1, filled square = NE2, filled diamond = PA. Literature range (when available) is shaded for each analyte.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information

7.2.4. Amino Acid Analys is of Seed Soybean seed samples from the control (Maverick) , DAS

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- 2, and reference variety entries were analyzed for the content of 18 amino acids. A summary of the results across all locations is presented in Table 14 and Figure 36

. All mean results were within literature ranges (when available) and /or within ranges for reference varieties included in the study. With the exception of phenylalanine, no statistical differences were observed in the combined

- site analysis between the control and DAS

- 81419

- 2 entries for amino acid content. The overall treatment ef fect and pair

- wise comparison between the control and DAS

- 81419

- 2 were significant for phenylalanine based on unadjusted P

- values (but not after adjustment for multiplicity using FDR methods). However, these differences were small relative to natural vari ation and not biologically meaningful, as all results were within literature ranges and within the range of the reference varieties included in this study.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Table 14 . Summary of the amino acid analysis of soybean seed from all site s and literature range.

Analytical Component (Units) a Overall Treatment Effect (Pr > F) b Control

(Maverick) DAS

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- 2 Reference Variety Range Combined

Literature Range d
Mean ± SE Mean ± SE Min

- Max Min

- Max Min

- Max Min

- Max

(P

- value, Adj.P) c Alanine 0.212 4.57 ± 0.03 4.59 ± 0.03 4.31

- 4.76 4.30

- 4.82 4.28

- 4.75 4.16

- 4.74 (% total amino acid) (0.212, 0.520) Arginine 0.111 7.49 ± 0.05 7.44 ± 0.05 7.04

- 7.79 7.05

- 7.74 7.22

- 8.20 6.41

- 8.41 (% total amino acid) (0.111, 0.456) Aspartic Acid 0.099 11.51 ± 0.01 11.48 ± 0.01 11.29

- 11.64 11.30

- 11.76 9.99

- 11.74 11.37

- 12.68 (% total amino acid) (0.099, 0.440) Cystine 0.283 1.610 ± 0.028 1.629 ± 0.028 1.432

- 1.946 1.448

- 1.861 1.240

- 1.792 1.02

- 1.87 (% total amino acid) (0.283, 0.591) Glutamic Acid 0.161 17.32 ± 0.11 17.23 ± 0.11 16.29

- 18.01 16.28

- 17.98 17.04

- 18.56 17.71

- 20.48 (% total amino acid) (0.161, 0.456) Glycine 0.745 4.50 ± 0.02 4.50 ± 0.02 4.34

- 4.67 4.30

- 4.66 4.14

- 4.54 4.19

- 4.62 (% total amino acid) (0.745, 0.802) Histidine 0.567 2.714 ± 0.016 2.703 ± 0.016 2.583

- 2.818 2.165

- 2.836 2.434

- 2.776 2.49

- 2.89 (% total amino acid) (0.567, 0.719) Isoleucine 0.931 4.80 ± 0.02 4.80 ± 0.02 4.65

- 4.96 4.52

- 4.99 4.61

- 4.99 4.13

- 5.11 (% total amino acid) (0.931, 0.931) Leucine 0.605 7.65 ± 0.01 7.64 ± 0.01 7.51

- 7.83 7.49

- 7.75 7.45

- 7.98 7.46

- 8.29 (% total amino acid) (0.605, 0.728)

a Unit of measure was converted from % DW to % total amino acid prior to analysis.
b Overall treatment effect estimated using an F

- test.

c P

- value = Comparison to the control using t

- tests; Adj. P = P

- values adjusted using False Discovery Rate (FDR) procedure; P

- values < 0.05 were considered significant. d Combined range from Appendix 7

.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Table 14 (Cont)

. Summary of the amino acid analysis of soybean seed from all sites and literature range.

Analytical Component (Units) a Overall Treatment Effect (Pr > F) b Control

(Maverick) DAS

- 81419

- 2 Reference Variety Range Combined

Literature Range d
Mean ± SE Mean ± SE Min

- Max Min

- Max Min

- Max Min

- Max

(P

- value, Adj.P) c Lysine 0.151 6.32 ± 0.08 6.44 ± 0.08 5.90

- 7.51 5.92

- 7.41 5.61

- 7.29 6.23

- 7.38 (% total amino acid) (0.151, 0.456) Methionine 0.211 1.419 ± 0.011 1.435 ± 0.011 1.305

- 1.608 1.325

- 1.607 1.223

- 1.618 1.18

- 1.71 (% total amino acid) (0.211, 0.520) Phenylalanine 0.009 5.15 ± 0.01 5.11 ± 0.01 4.99

- 5.25 4.92

- 5.22 4.88

- 5.37 4.91

- 5.44 (% total amino acid) ( 0.009 , 0.102) Proline 0.240 5.14 ± 0.04 5.20 ± 0.04 4.86

- 5.97 4.91

- 5.73 4.80

- 6.02 4.75

- 5.62 (% total amino acid) (0.240, 0.550) Serine 0.466 5.13 ± 0.02 5.16 ± 0.02 4.74

- 5.37 4.85

- 5.38 4.81

- 5.53 3.25

- 6.04 (% total amino acid) (0.466, 0.674) Threonine 0.381 4.19 ± 0.03 4.20 ± 0.03 3.99

- 4.38 3.96

- 4.44 3.86

- 4.25 3.15

- 4.24 (% total amino acid) (0.381, 0.643) Tryptophan 0.650 1.519 ± 0.016 1.524 ± 0.016 1.423

- 1.640 1.401

- 1.653 1.271

- 1.686 0.95

- 1.49 (% total amino acid) (0.650, 0.747) Tyrosine 0.418 3.97 ± 0.01 3.96 ± 0.01 3.88

- 4.10 3.83

- 4.06 3.82

- 4.16 2.62

- 3.72 (% total amino acid) (0.418, 0.659) Valine 0.532 4.98 ± 0.02 4.96 ± 0.02 4.74

- 5.22 4.70

- 5.32 4.59

- 5.18 4.28

- 5.57 (% total amino acid) (0.532, 0.712)

a Unit of measure was converted from % DW to % total amino acid prior to analysis.
b Overall treatment effect estimated using an F

- test.

c P

- value = Comparison to the control using t

- tests; Adj. P = P

- values adjusted using False Discovery Rate (FDR) procedure; P

- values < 0.05 were considered significant. d Combined range from Appendix 7

.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 36
. Amino acids in control, DAS

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- 2, and reference variety soybean seed (% total amino acid). Symbols for each location shown: open circle = IA1, × = IA2, + = IL1, open triangle = IL2, open square = IN, open diamond = M O1, filled circle = MO2, filled tr iangle = NE1, filled square = NE2, filled diamond = PA. Literature range is shaded for each analyte.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 36 (Cont)

. Amino acids in control, DAS

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- 2, and reference variety soybean seed (% total amino acid). Symbols for each location shown: open circle = IA1, × = IA2, + = IL1, open triangle = IL2, open square = IN, open diamond = M O1, filled circle = MO2, filled triangle = NE1, filled square = NE2, fil led diamond = PA. Literature range is shaded for each analyte.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 36 (Cont)

. Amino acids in control, DAS

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- 2, and reference variety soybean seed (% total amino acid). Symbols for each location shown: open circle = IA1, × = IA2, + = IL1, open triangle = IL2, open square = IN, open diamond = MO1, filled circle = MO2, filled triangle = NE1, filled square = NE2, filled diamond = PA. Literature range is shaded for each analyte.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information

7.2.5. Fatty Acid Analysis of Seed Soybean seed samples from the control (M averick) , DAS

- 81419

- 2, and reference variety entries were analyzed for the content of 22 fatty acids. A summary of the results across all locations is presented in Table 15 and Figure 37

. S tatistical analysis was not performed on the following fatty acids because greater than 50% of the samples were found to be below the LOQ of 0 .02% fresh weight : 8:0 c aprylic, 10:0 c apric, 12:0 l auric, 14:0 m yristic, 14:1 m yristoleic, 15:0 p entadecanoic, 15:1 p entadecenoic, 16:1 p almitoleic, 17:0 h eptadecanoic, 17:1 h eptadecenoic, 18:3 γ

- linolenic , 20:2 e icosadienoic, 20:3 e icosatrienoic, 20:4 a rachidonic

. The mean values of the remaining fatty acids were within literature ranges (when available) and /or within ranges for reference varieties included in the study. Overall treatment effect s and pair

- wise comparisons between the control and DAS

- 81 419

- 2 were significant for 16:0 palmitic, 18:3 linolenic, and 20:1 eicosenoic. However, these differences were small relative to natural variation and not biologically meaningful, as all results were within literature ranges and/or within the range of the reference varieties included in this study.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Table 15 . Summary of the fatty acid analysis of soybean seed from all sites and literature range.

Analytical Component (Units) a Overall Treatment Effect (Pr > F) b Control

(Maverick) DAS

- 81419

- 2 Reference Variety Range Combined

Literature Range d
Mean ± SE Mean ± SE Min

- Max Min

- Max Min

- Max Min

- Max

(P

- value, Adj.P) c 8:0 Caprylic NA NA NA <LOQ <LOQ <LOQ <LOQ

- 0.148 (% total fatty acid) 10:0 Capric NA NA NA <LOQ <LOQ <LOQ <LOQ

- 0.27 (% total fatty acid) 12:0 Lauric NA NA NA <LOQ <LOQ <LOQ <LOQ

- 0.132 (% total fatty acid) 14:0 Myristic NA NA NA <LOQ <LOQ <LOQ <LOQ

- 0.238 (% total fatty acid) 14:1 Myristoleic NA NA NA <LOQ <LOQ <LOQ <LOQ

- 0.125 (% total fatty acid) 15:0 Pentadecanoic NA NA NA <LOQ <LOQ <LOQ <LOQ

- <LOQ (% total fatty acid) 15:1 Pentadecenoic NA NA NA <LOQ <LOQ <LOQ <LOQ

- <LOQ (% total fatty acid) 16:0 Palmitic <0.001 11.12 ± 0.08 11.56 ± 0.08 10.04

- 11.59 10.88

- 12.27 9.12

- 11.53 1.40

- 15.77 (% total fatty acid) ( <0.001, <0.001 ) Abbreviations: NA (Not Available) = analysis not performed, majority of data was < LOQ (Limit of Quantitation); NR = Not Reported.

a Unit of measure was converted from % dry wt. to % total fatty acid prior to analysis.
b Overall treatment effect estimated using an F

- test.

c P

- value = Comparison to the control using t

- tests; Adj. P = P

- values adjusted using False Discovery Rate (FDR) procedure; P

- values < 0.05 were considered significant.

d Combined range from Appendix 7

.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Table 15 (Cont)

. Summary of the fatty acid analysis of soybean seed from all sites and literature range.

Analytical Component (Units) a Overall Treatment Effect (Pr > F) b Control

(Maverick) DAS

- 81419

- 2 Reference Variety Range Combined

Literature Range d
Mean ± SE Mean ± SE Min

- Max Min

- Max Min

- Max Min

- Max

(P

- value, Adj.P) c 16:1 Palmitoleic NA NA NA <LOQ <LOQ

- 0.236 <LOQ <LOQ

- 0.194 (% total fatty acid) 17:0 Heptadecanoic NA NA NA <LOQ

- 0.128 <LOQ

- 0.132 <LOQ

- 0.133 <LOQ

- 0.146 (% total fatty acid) 17:1 Heptadecenoic NA NA NA <LOQ <LOQ <LOQ <LOQ

- 0.087 (% total fatty acid) 18:0 Stearic 0.18 4.40 ± 0.12 4.46 ± 0.12 3.67

- 5.27 3.62

- 5.22 3.19

- 5.07 0.50

- 5.88 (% total fatty acid) (0.180, 0.474) 18:1 Oleic 0.08 21.6 ± 0.4 21.2 ± 0.4 19.6

- 25.2 19.8

- 25.5 18.8

- 24.6 2.60

- 45.68 (% total fatty acid) (0.080, 0.404) 18:2 Linoleic 0.124 54.1 ± 0.4 53.8 ± 0.4 51.2

- 55.8 50.4

- 55.7 53.6

- 57.5 7.58

- 58.8 (% total fatty acid) (0.124, 0.456) 18:3 Linolenic 0.04 7.97 ± 0.15 8.17 ± 0.15 6.91

- 8.76 7.32

- 8.94 6.58

- 9.88 1.27

- 12.52 (% total fatty acid) ( 0.040 , 0.314) Abbreviations: NA (Not Available) = analysis not performed, majority of d ata was < LOQ (Limit of Quantitation);

a Unit of measure was converted from % dry wt. to % total fatty acid prior to analysis.
b Overall treatment effect estimated using an F

- test.

c P

- value = Comparison to the control using t

- tests; Adj. P = P

- values adjusted using False Discovery Rate (FDR) procedure; P

- values < 0.05 were considered significant.

d Combined range f

rom Appendix 7

.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Table 15 (Cont) . Summary of the fatty acid analysis of soybean seed from all sites and literature range.

Analytical Component (Units) a Overall Treatment Effect (Pr > F) b Control

(Maverick) DAS

- 81419

- 2 Reference Variety Range Combined

Literature Range d
Mean ± SE Mean ± SE Min

- Max Min

- Max Min

- Max Min

- Max

(P

- value, Adj.P) c 18:3 γ

- Linolenic NA NA NA <LOQ <LOQ <LOQ <LOQ

- <LOQ (% total fatty acid) 20:0 Arachidic 0.068 0.319 ± 0.007 0.325 ± 0.007 0.276

- 0.370 0.282

- 0.372 0.254

- 0.383 0.038

- 0.57 (% total fatty acid) (0.068, 0.381) 20:1 Eicosenoic 0.048 0.157 ± 0.004 0.153 ± 0.004 <LOQ

- 0.189 <LOQ

- 0.171 <LOQ

- 0.196 0.024

- 0.35 (% total fatty acid) ( 0.048 , 0.339) 20:2 Eicosadienoic NA NA NA <LOQ <LOQ <LOQ <LOQ

- 0.245 (% total fatty acid) 20:3 Eicosatrienoic NA NA NA <LOQ <LOQ <LOQ <LOQ

- <LOQ (% total fatty acid) 20:4 Arachidonic NA NA NA <LOQ <LOQ <LOQ <LOQ

- <LOQ (% total fatty acid) 22:0 Behenic 0.159 0.317 ± 0.003 0.321 ± 0.003 0.283

- 0.347 0.263

- 0.345 0.277

- 0.390 0.043

- 0.65 (% total fatty acid) (0.159, 0.456) Abbreviations: NA (Not Available) = analysis not performed, majority of data was < LOQ (Limit of Quantitation); NR = Not Reported.

a Unit of measure was converted from % dry wt. to % total fatty acid prior to analysis.
b Overall treatment effect estimated using an F

- test.

c P

- value = Comparison to the control using t

- tests; Adj. P = P

- values adjusted using False Discovery Rate (FDR) procedure; P

- values < 0.05 were considered significant.

d Combined range from Appendix 7

.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 37
. Fatty acids in control, DAS

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- 2, and reference variety soybean seed (% total fatty acid). Symbols for each location shown: open circle = IA1, × = IA2, + = IL1, open triangle = IL2, open square = IN, open diamond = M O1, filled circle = MO2, filled tria ngle = NE1, filled square = NE2, filled diamond = PA. Literature range is shaded for each analyte.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 37 (Cont). Fatty acids in control, DAS

- 81419

- 2, and refe rence variety soybean seed (% total fatty acid). Symbols for each location shown: open circle = IA1, × = IA2, + = IL1, open triangle = IL2, open square = IN, open diamond = M O1, filled circle = MO2, filled triangle = NE1, filled square = NE2, filled diamon d = PA. Literature range is shaded for each analyte.

7.2.6. Vitamin Analysis of Seed Soybean seed samples from the control (Maverick) , DAS

- 81419

- 2, and reference variety entries were analyzed for the content of 13 vitamins. A summary of the results across all locations is presented in Table 16 and Figure 38

. S tatistical analysis was not performed on Vitamin A because greater than 50% of the samples were found to be below the LOQ

. All mean results were within literature ranges (when available) and /or within ranges for reference varieties included in the study. With the exception of γ

- tocopherol and Vitamin B5, no statistical differences were observed in the combined

- site analysis between the control and DAS

- 81419

- 2 entries for vitamin content. The overall treatment effect and pair

- wise comparison between the control and DAS

- 81419

- 2 were significant for γ

- tocopherol and Vitamin B5 based on unadjusted P

- values (but not after adjustment for multiplicity using FDR methods). However, these differen ces were small relative to natural variation and not biologically meaningful, as all results were within the range of the reference varieties included in this study.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Table 16 . Summary of the vitamin analysis of soybean seed from a ll sites and literature range.

Analytical Component (Units) a Overall Treatment Effect (Pr > F) b Control

(Maverick) DAS

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- 2 Reference Variety Range Combined

Literature Range d
Mean ± SE Mean ± SE Min

- Max Min

- Max Min

- Max Min

- Max

(P

- value, Adj.P) c Vitamin A NA NA NA (β

- Carotene) <LOQ <LOQ <LOQ

- 0.244 NR (mg/kg DW) Vitamin B1 0.447 3.51 ± 0.24 3.43 ± 0.24 (Thiamine) 2.35

- 5.44 2.20

- 5.16 1.82

- 4.92 1.01

- 2.54 (mg/kg DW) (0.447, 0.662) Vitamin B2 0.146 3.40 ± 0.08 3.51 ± 0.08 (Riboflavin) 2.63

- 4.65 2.58

- 4.64 2.42

- 5.00 1.90

- 3.21 (mg/kg DW) (0.146, 0.456) Vitamin B3 0.07 25.0 ± 0.7 25.6 ± 0.7 (Niacin) 20.2

- 30.5 20.3

- 32.1 20.5

- 29.0 NR (mg/kg DW) (0.070, 0.381) Vitamin B5 0.004 14.8 ± 0.5 14 ± 0.5 (Pantothenic Acid) 12.3

- 19.5 11.8

- 16.8 8.97

- 18.0 NR (mg/kg DW) ( 0.004 , 0.074) Vitamin B6 0.721 5.23 ± 0.11 5.18 ± 0.11 (Pyridoxine) 4.42

- 6.43 4.53

- 6 3.01

- 6.36 NR (mg/kg DW) (0.721, 0.787) Vitamin B9 0.599 4.21 ± 0.20 4.15 ± 0.20 (Folic Acid) 3.05

- 5.62 2.75

- 5.51 2.94

- 5.59 2.386

- 4.709 (mg/kg DW) (0.599, 0.728) Vitamin C 0.135 141 ± 13 133 ± 13 (Ascorbic Acid) 75.2

- 231 74

- 230 49.2

- 210 NR (mg/kg DW) (0.135, 0.456) Abbreviations: NA (Not Available) = analysis not performed, majority of data was < LOQ (Limit of Quantitation); NR = Not Reported.

a Unit of measure was not converted prior to analysis.
b Overall treatment effect estimated using an F

- test.

c P

- value = Comparison to the control using t

- tests; Adj. P = P

- values adjusted using False Discovery Rate (FDR) procedure; P

- values < 0.05 were considered significant.

d Combined range fro m Appendix 7

.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Table 16 (Cont). Summary of the vitamin analysis of soybean seed from all sites and literature range.

Analytical Component (Units) a Overall Treatment Effect (Pr > F) b Control

(Maverick) DAS

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- 2 Reference Variety Range Combined

Literature Range d
Mean ± SE Mean ± SE Min

- Max Min

- Max Min

- Max Min

- Max

(P

- value, Adj.P) c

α

- Tocopherol 0.267 14.3 ± 1.3 13.6 ± 1.3 8.56

- 29.3 9.62

- 26.6 6.51

- 25.0 1.934

- 84.9 (mg/kg DW) (0.267, 0.589) β

- Tocopherol NA NA NA <LOQ <LOQ <LOQ NR (mg/kg DW)
γ

- Tocopherol 0.004 69.4 ± 2.8 74.7 ± 2.8 41.3

- 83.1 45.4

- 90.8 49.7

- 104.0 NR (mg/kg DW) ( 0.004 , 0.074) δ

- Tocopherol 0.338 168 ± 8 172 ± 8 97

- 220 118

- 219 77.5

- 240 NR (mg/kg DW) (0.338, 0.615) Total Tocopherol e 0.096 237.5 ± 6.9 247.1 ± 6.9 159.5

- 280.5 194.7

- 282.3 150.5

- 299.9 NR (mg/kg DW) (0.096, 0.440) Abbreviations: NA (Not Available) = analysis not performed, majority of data was < LOQ (Limit of Quantitation); NR = Not Reported.

a Unit of measure was not converted prior to analysis.
b Overall treatment effect estimated using an F

- test.

c P

- value = Comparison to the control using t

- tests; Adj. P = P

- values adjusted using False Discovery Rate (FDR) procedure; P

- values < 0.05 were considered significant.

d Combined range from Appendix 7

.
e Total Tocopherol = α

- Tocopherol + β

- Tocopherol + γ

- Tocopherol + δ

- TocopherolUSDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 38
. Vitamins in control, DAS

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- 2, and reference variety soybean seed (mg/kg dry weight). Symbols for each location shown: open circle = IA1, × = IA2, + = IL1, open triangle = IL2, open square = IN, open diamond = M O1, filled circle = MO2, filled triangle = NE1, filled square = NE2, filled diamond = PA. Literature range (when available) is shaded for each analyte.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 38 (Cont)

. Vitamins in control, DAS

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- 2, and reference variety soybean seed (mg/kg dry weight). Symbols for each location shown: open circle = IA1, × = IA2, + = IL1, open triangle = IL2, open square = IN, open diamond = M O1, filled circle = MO2, filled triangle = NE1, filled square = NE2, fil led diamond = PA. Literature range (when available) is shaded for each analyte.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information

7.2.7. Bioactive Analysis of Seed Soybean seed samples from the control (Maverick) , DAS

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- 2, and reference variety entries were analyzed for the content of eight bioactives. A summary of the results across all locations is presented in Tab le 17 and Figure 39

. All mean results were within literature ranges and /or within ranges for reference varieties included in this study. With the exception of total glycitein equivalent, no statistical differences were observed in the combined

- site analysis between the control and DAS

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- 2 entries for bioactive content. The overall treatment effect and pair

wise comparison between the control and DAS

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- 2 were significant for total glycitein equivalent based on unadjusted P

- values (but not after adjustment fo r multiplicity using FDR methods). However, these differences were small relative to natural variation and not biologically meaningful, as all results were within the literature range and the range of the reference varieties included in this study.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Tab le 17 . Summary of the bioactive analysis of soybean seed from all sites and literature range.

Analytical Component (Units) a Overall Treatment Effect (Pr > F) b Control

(Maverick) DAS

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- 2 Reference Variety Range Combined

Literature Range d
Mean ± SE Mean ± SE Min

- Max Min

- Max Min

- Max Min

- Max

(P

- value, Adj.P) c Lectin 0.401 30.8 ± 2.1 32.2 ± 2.1 37

- 323 (H.U./mg protein 13.9

- 50.1 12.4

- 52.6 7.89

- 45.2 DW) (0.401, 0.647) Phytic Acid 0.447 1.22 ± 0.06 1.24 ± 0.06 0.41

- 2.68 0.857

- 2.02 0.911

- 1.52 0.678

- 1.71 (% DW) (0.447, 0.662) Raffinose 0.475 0.750 ± 0.038 0.766 ± 0.038 0.212

- 1.85 0.505

- 1.02 0.475

- 0.977 0.570

- 1.16 (% DW) (0.475, 0.674) Stachyose 0.648 3.68 ± 0.08 3.69 ± 0.08 1.21

- 6.65 3.19

- 4.14 3.15

- 4.29 3.01

- 5.28 (% DW) (0.648, 0.747) Trypsin Inhibitor 0.226 29.1 ± 1.2 30.2 ± 1.2 18.14

- 118.68 22.2

- 46.5 21.3

- 49.9 19.5

- 53.8 (TIU/mg) (0.226, 0.536) Total Daidzein 0.299 950 ± 48 932 ± 48 Equivalent 462

- 1200 504

- 1190 585

- 1460 25

- 2453.5 (mcg/g DW) (0.299, 0.592) Total Genistein 0.362 1296 ± 63 1276 ± 63 Equivalent 808

- 1680 922

- 1620 753

- 1950 28

- 2837.2 (mcg/g DW) (0.362, 0.643) Total Glycitein 0.002 197 ± 6 180 ± 6 Equivalent 156

- 266 140

- 237 40.3

- 259 15.3

- 349.19 (mcg/g DW) ( 0.002 , 0.074)

a Unit of measure was not converted prior to analysis.
b Overall treatment effect estimated using an F

- test.

c P

- value = Comparison to the control using t

- tests; Adj. P = P

- values adjusted using False Discovery Rate (FDR) procedure; P

- values < 0.05 were considered significant.

d Combined range from Appendix 7

.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 39 . Bioactives in control, DAS

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- 2, and reference variety soybean seed. Lectin = H.U./mg protein dry w eight, Trypsin inhibitor = TIU/mg, total daidzein equivalent, total genistein equivalent, and total glycitein equivalent = mcg/gram dry weight, all others = % dry weight Symbols for each location shown: open circle = IA1, × = IA2, + = IL1, open triangle = IL2, open square = IN, open diamond = MO1, filled circle = MO2, filled triangle = NE1, filled square = NE2, filled diamond = PA. Literature range is shaded for each analyte.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 39 (Cont)

. Bioactives in control, DAS

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- 2, and reference variety soybean seed. Lectin = H.U./mg protein dry weight, Trypsin inhibitor = TIU/mg, total daidzein equivalent, total genistein e quivalent, and total glycitein equivalent = mcg/gram dry weight, all others = % dry weight Symbols for each location shown: open circle = IA1, × = IA2, + = IL1, open triangle = IL2, open square = IN, open diamond = M O1, filled circle = MO2, filled triangle = NE1, filled square = NE2, filled diamond = PA. Literature range is shaded for each analyte. 7.3. Summary of Composition Analysis All overall mean values for the control (Maverick) and DAS

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- 2 were within literature ranges (when available) for soybean a nd/or within ranges for non

- transgenic soybean reference varieties included in the study. A limited number of statistically significant differences were observed between DAS

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- 2 and the control ; however, the se differences were not biologically meaning ful because the differences were small relat ive to natural variation and the results were within ranges found for non

- transgenic soybean. In conclusion, forage and grain derived from DAS

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- 2 soybean are compositionally equivalent to those of non
- transgenic soybean.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information 8. Agronomic Characteristics Field trials with DAS

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- 2 soybean , a non
- transgen ic control (Maver ick), and reference varieties were conducted in 2011 at ten sites located in Iowa (2 sites), Illino is (2 sites), Indiana, Missouri (2 sites), Nebraska (2 sites) , and Pennsylvania

. This study used the same plots that were used for protein expression (Secti on 6 ) and nutrient composition studies (Section 7 ). No biologically relevant differences , other than resistance to target insect pests , were identified between DAS

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- 2 soybean and non

- transgenic soybean. A gronomic characteristics of DAS

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- 2 were statistically indistinguishable from the non

- transgenic control Maverick or were within the reference variety ranges for non

- transgenic soybean. Ecological evaluations indicated that there were no statistically significant differences between DAS

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- 2 soybean and the non

- transgenic control Maverick in susceptibility to and interactions with diseases and
non
- target insects

. The g ermination and dormancy evaluation indicated that seed dormancy characteristi cs have not been altered in DAS

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- 2 soybean. Taken together, r esults from the field and laboratory studies demonstrate agronomic equivalence be tween DAS

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- 2 soybean and non

- transgenic soybean.
8.1. Agronomic Study An agronomic study with DAS

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- 2 soybean, a non

- transgenic near

- isogenic control (Maverick) and six non

- transgenic reference lines (IL 3503, Porter 75148, DSR 75213

- 72, Pioneer 93M62, HiSOY 38C60, Williams 82) was conducted in 2011 at ten sites located in Richland, Iowa; Atlantic, Iowa; Carlyle, Illinois; Wyoming, Illinois; Fr ankfort, Indiana; Fisk, Missouri; La Plata, Missouri; York, Nebraska; Brunswick, Nebraska; and Germansville, Pennsylvania.
Each trial site included event DAS

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- 2, the non

- transgenic near

- isogenic control (Maverick), and three non

- transgenic reference lines. At each of the ten sites, all entries were arranged in a randomized complete block design with four blocks. Across all sites, e ach control (Maverick) and DAS

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- 2 was represented by a total of 40 plots (10 sites, 4 replicate plots per entry at each site). Three of the six reference lines were included at each site by randomizing across sites in a balanced incomplete

- block design. Each of the six reference lines was assigned to five sites; therefore, each reference line was represented by a tot al of 20 plots across sites (5 sites per reference line, 4 replicate plots per entry at each site). At each site, four replicate plots of each entry were established, with each plot consisting of four 25 ft (7.62 m) rows. O bservations of agronomic charac teristics were conducted on the center two rows of each plot; row number one and four were included as additional border rows. Soybean seeds were planted at a seeding rate of approximately 125 seeds per row with seed spacing within each row of approximatel y 2.4 inches (6 cm). Each soybean plot was bordered by two rows of a non

- transgenic soybean cultivar of similar maturity. The entire trial site was surrounded by a minimum of four rows (10 ft or 3.0 m) of a non

- transgenic soybean cultivar of similar matu rity. Appropriate insect, weed, and disease control practices were applied to produce an agronomically acceptable crop. Agronomic Data Collection The following agronomic characteristics were measured and recorded for all test entries at each location on a per plot basis ( Table 18 ).USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Table 18 . Agronomic characteristics.
Trait Evaluation

Timing Description Scale

Early Population (Stand Count)  V2 Number of plants in a representative 1 meter section of one row per plot Number of emerged plants in 1 meter

Seedling Vigor  V2 Visual estimate of average vigor of plants in each plot 0

- 100% r ating s cale (e.g., 0 = all plants are short with small, thin leaves; 100 = all plants are tall with robust leaves). N ot base d on g rowth of the control entries; Did not consider germination/ emergence (stand count)
Days to 50% Flowering  R1/R2 Date at which  50% of plants are flowering Record ed the date when  50% of the plants in each plot we re flowering . Days since planting calculated.

Disease Incidence  R6 Visual estimate of disease incidence 0

- 100%; Estimate d % plant tissue/leaf area diseased over all plants in plot; did not record % of plants in plot that had detectable disease; 100% = all plant tissues in plot were diseased;
r ecord ed type of disease
Insect Damage  R6 Visual estimate of insect damage 0

- 100%; Estimate d % plant tissue/leaf area damaged over all plants in plot; Did not reco rd % of plants in plot that had detectable damag e; 100% = all plant tissues had feeding damage; Record ed type of damage, e.g. chewing, stippling, distortion; Record ed t ype of insect(s) if present
Days to Maturity  R8 Date at which  95% of plant s had reached physiological maturity/dry down color Record ed the date when  95% of the plants in each plot reached physiological maturity/dry down color ; Days since planting cal culated
Lodging  R8 Visual estimate of incidence of lodging severity 0

- 100%; Estimate d % of plants lodged in plot; 100% = all plants in plot we re lodged
Plant Height  R8 Average plant height: from soil surface to growing tip (at senescence / after leaf shed) Record ed the average height of all plants in plot (stand) in centimeters (cm); On e value for each plot; If plot wa s lodged, a representative group of plants was held up to obtain a measurement
Final Population (Stand Count)  R8 Number of plants in a representative 1 meter section of one row per plot Number of plants in 1 met er; Did not sample a section where plants were removed during previous sampling

Plant Morphology (number of seeds and pods)  R8 (prior to

harvest) Number of pods and seeds

from 5 plants collected from each plot Record ed the number of pods and seeds present on 5 representative plants collected from each plotUSDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Trait Evaluation

Timing Description Scale

Shattering  R8 (prior to

harvest) Visual estimate of pod

shattering 0

- 100%; Estimated % of shattered p ods for each plot; 100% = all pods shattered

Yield  R8 Weight of grain harvested from each plot Record ed the weight in grams of grain harvested from each plot
100 seed weight  R8 Weight of 100 representative seeds from bulk yield sample in grams Record ed the weight in grams for 100 representative seeds taken from the bulk yield sample

Analysis of variance was conducted across field sites (combined

- site analysis) for agronomic data using a mixed model ( SAS Institute Inc., 2009 )

. Entr y was considered a fixed effect and location, b lock within locati on, and location

- by

- entry were designated as random effec ts. Significant differences were declare d at the 95% confidence level. The significance of an overall treatment effect was estimated using an F

- test. Comparisons were made betwee n DAS

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- 2 soybean and the control (Maverick) using t

- tests. Due to the large number of comparisons made in this stu dy, multiplicity was an issue. Multiplicity is an issue when a large number of comparisons are made in a single study t o look for unexpec ted effects. Under these conditions, the probability of falsely declaring differences based on comparison

- wise P

- values is very high (1

- 0.95 number of comparisons ). In this study , there was one comparison per endpoint (14 analyzed observation types for agronomics), resulting in 14 comparisons made in the combined

- site agronomic analysis. Therefore, the probability of declaring one or more false differences base d on unadjusted P

- values was >51 % for agronomics (1

- 0.95 14 )

. One method to account for multiplicity is to adjust P

- val u es to control the experiment

- wise error rate, but when many comparisons are made in a study, the power for detecting specific effects can be reduced significantly. An alternative with much greater power is to adjust P

- values to control the probability that each decla red difference is significant. This can be accomplished using False Discovery Rate (FDR) control procedures ( Benjamini and Hochberg, 1995 ) ; FDR methods are commonly applied in studies examining transgenic crops ( Herman et al. , 2007 ; Coll et al. , 2008 ; Huls et al. , 2008 ; Jacobs et al. , 2008 ; Stein et al. , 2009 ; Herman et al. , 2010 )

. Therefore, the P

- values from the agronomics evaluations were each adjusted using the FDR method to improve discrimination of true differences among treatments from ran dom effects (false positives)

. Differences were considered significant if the FDR

- adjusted P

- value was less than 0.05. Agronomic Results
A statistical analysis of the agronomic data collected from the control (Maverick) and DAS

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- 2 entries was conducted. For each agronomic charac teristic and entry, the least squares mean, standard error, and minimum and maximum sample values are reported.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Additionally, the minimum and maximum values from reference varieties across all sites (reference variety ranges) for each agronomic character i stic are provided for comparison . Each minimum and maximum value is an individual data point reported for a single test plot.
For the combined

- site analysis ( Table 19 ) , no statistically significant differences were observed following False Discovery Rate (FDR) adjustment of P

- values for all of the agronomic characteristics evaluated: early population, seedling vigor, days to flowering, disease incidence, insect damage, days to maturity, lodging, plant height, final population, number of pods per five plants, number of seeds per five plants (plant morphology) , shattering, yield, and 100 seed weight. Una djusted P

- values were significant at the 0.05 level for paired t

- tests for days to flowering and number of pods per five plants (plant morphology) . For each significant unadjusted P

- value, differences in mean values between DAS

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- 2 and control (Maveri ck) entries were negligible and the mean values of DAS

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- 2 were within the range observed for reference varieties included in the study. Results from this study demonstrate agronomic equivalence between event DAS

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- 2 and non

- transgenic soybean.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Table 19 . Summary of agronomic characteristics for the combined site analysis.

Overall Control

(Maverick) DAS

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- 2 Reference Variety Agronomic Component (Units) a Tr eatment Mean ± SE Mean ± SE Range Effect Min

- Max Min

- Max Min

- Max (Pr > F) b (P

- value, Adj.P) c Early Population

- V2 0.267 17 ± 0 16 ± 0 (number of plants in a 13

- 21 11

- 21 7

- 22 1 m section of row) (0.267, 0.386) Seedling Vigor

- V2
0.074 86 ± 5 82 ± 5 ( 0

- 100% scale, 0% = all plants are 60

- 100 50

- 100 60

- 100 short with small thin leaves; 100% = all plants are tall with robust leaves) (0.074, 0.137) Days to Flowering

- R1/R2 0.008 42 ± 2 43 ± 2 (days since planting) 32

- 58 33

- 58 32

- 58 ( 0.008 , 0.053) Disease Incidence

- R6 0.343 4 ± 3 5 ± 3 (0

- 100% scale, 0% = no disease 0

- 30 0

- 30 0

- 50 100% = all plants diseased) (0.343, 0.446) Insect Damage

- R6 0.504 7 ± 3 6 ± 3 (0

- 100% scale, 0% = no damage 0

- 30 0

- 30 0

- 30 100% = all plants damaged) (0.504, 0.596) Days to Maturity

- R8 0.957 114 ± 3 114 ± 3 (days since planting) 91

- 130 93

- 133 96

- 133 (0.957, 0.957) Lodging

- R8 0.053 19 ± 6 7 ± 6 (0

- 100% scale, 0% = no lodging 0

- 90 0

- 30 0

- 35 100% = all plants lodged) (0.053, 0.137) Plant Height

- R8 0.066 108 ± 4 103 ± 4 (Representative plant height in cm) 80

- 135 71

- 126 63

- 129 (0.066, 0.137) Final Population

- R8 0.742 14 ± 1 14 ± 1 (number of plants in a 7

- 22 8

- 22 7

- 17 1 m section of row) (0.742, 0.804) Number of Pods

- R8 0.006 331 ± 10 304 ± 10 (number of pods on 5 plants) 203

- 476 172

- 408 153

- 808 ( 0.006 , 0.053) Number of Seeds

- R8 0.059 783 ± 38 728 ± 38 (number of seeds from 5 plants) 491

- 1154 318

- 1031 313

- 1244 (0.059, 0.137)

a Unit of measure was not converted prior to analysis.
b Overall treatment effect estimated using an F

- test.

c P

- value = Comparison to the control using t

- tests; Adj. P = P

- values adjusted using False Discovery Rate (FDR) procedure; P

- values < 0.05 were considered significant.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Table 19 (Cont) . Summary of agronomic characteristics for the combined site analysis
.

Overall Control

(Maverick) DAS

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- 2 Reference Variety
Agronomic Component (Units) a Tr eatment Mean ± SE Mean ± SE Range Effect Min

- Max Min

- Max Min

- Max (Pr > F) b (P

- value, Adj.P) c Shattering

- R8 NA NA NA (0

- 100% scale, 0% = no shattering 0

- 0 0

- 0 0

- 0 100% = all pods shattered) Yield

- R8 0.092 37 ± 4 35 ± 4 (bushels per acre) 5

- 55 6

- 54 7

- 62 (0.092, 0.149) 100 Seed Weight

- R8 0.051 14 ± 1 13 ± 1 (grams) 9.76

- 17.61 10.83

- 16.65 11.69

- 25.82 (0.051, 0.137) Abbreviations: NA (Not Available) = analysis not performed, insufficent variation in response variable to enable ANOVA.

a Unit of measure was not converted prior to analysis except for yield. Unit of measure for yield was converted from grams per plot to bushels per acre prior to analysis using the formula: (X g/125 ft 2 ) × (43560 ft 2 /A) × (1 kg/1000 g) × (1 bu./27.2 155 kg) , where X is the individual sample value.
b Overall treatment effect estimated using an F

- test.

c P

- value = Comparison to the control using t

- tests; Adj. P = P

- values adjusted using False Discovery Rate (FDR) procedure; P

- values < 0.05 were considered significant.

Conclusions
A gronomic characteristics of DAS

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- 2 soybean were e valuated in field trials in 2011

. DAS

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- 2 agronomic characteristics were statistically indistinguishable from the control or were within the reference variety ranges for non

- transgenic soybean. N o unintended agronomic effects were observed for DAS

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- 2 soybean. Results from this study demonstrate agronomic equival ence between event DAS

- 81419

- 2 and non

- transgenic soybean.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information 8.2. Ecological Evaluations The DAS

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- 2 soybean field trials were monitored and observed by personnel familiar with soybean cultivation practices (breeders, field station managers, field agronomists, field associates). The personnel conducting the field tests visuall y monitored the incidence of plant disease and pests on DAS

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- 2 soybeans compared with the conventional soybean varieties, including Maverick and six reference lines, in the same trials. Disease and insect damage was rated on a numerical scale of 0

- 10 0%, with 0% representing no damage due to disease incidence or insect pests. The insect and disease pressure observed in these trials was typical of the growing locations. Table 20 summarizes the insects and diseases that caused the insect damage and disease incidence at each field site. In all cases, the insects and diseases observed were found on both test and control plots. Table 21 summarizes the results obtained from the field trials conducted in 2011 at ten sites

. There were no statist ically significant differences between DAS

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- 2 soybean and the non

- transgenic control Maverick in susceptibility to and interactions with diseases and insects. It should be noted that e fficacy evaluation of DAS

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- 2 soybean was not the objective of this study (refer to Section 9.1.1 for efficacy evaluation of DAS

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- 2 soybean). All plots were uniformly treated with pest control measures at each location based on pressure in the non

- transgenic plots such that low incidence of t hese pests was expected. This design mimics commercial practice and allows comparison to typical cultivation conditions for non

- transgenic soybean. The results do not indicate an ecological impact of DAS

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- 2 soybean on non
- target insects or disease.
Table 20 . Insect and disease present by site.
Site Insects Present Diseases Present

110000IA1 Not Recor d ed a None Present

110000IA2 Grasshoppers Bacterial Pustule

110000IL1 Green Cloverworms, Bean Leaf Beetles,

Grasshoppers None Present

110000IL2 Not Recor d ed a Not Recor d ed a

110000IN Bean Leaf Beetles None Present

110000MO1 None Present Not Recor d ed a

110000MO2 None Present None Present

110000NE1 Wooly Bear Caterpillars Not Recor d ed a

110000NE2 Caterpillars Mold, Mildew, Blight

110000PA Loopers, Skippers None Present

a Insects or Diseases were not recorded due to their presence on less than 30% of the site.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Table 21 . Disease and insect incidence.

Overall Control DAS

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- 2 Reference Variety
Agronomic Component
(Units) a Tr eatment Mean ± SE Mean ± SE Range

Effect Min

- Max Min

- Max Min

- Max
(Pr > F) b (P

- value, Adj.P) c

Disease Incidence *

- R6

0.343 4 ± 3 5 ± 3

0

- 30 0

- 30 0

- 50

(0.343, 0.446)
Insect Damage **

- R6

0.504 7 ± 3 6 ± 3

0

- 30 0

- 30 0

- 30

(0.504, 0.596)

a Unit of measure was not converted prior to analysis.
b Overall treatment effect estimated using an F

- test.

c P

- value = Comparison to the control using t

- tests; Adj. P = P

- values adjusted usi ng False Discovery Rate (FDR) procedure; P

- values < 0.05 were considered significant. *Disease incidence 0

– 100% scale. 0% = no disease; 100% = all plants diseased. **Insect damage 0

– 100% scale. 0% = no damage; 100% = all plants damaged.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information 8.3. Germination and Dormancy Evaluations The germination of DAS

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- 2 soybean seed compared with the non

- transgenic control (Maverick) under warm and cold conditions was evaluated to determine any impact on seed dormancy characteristics. The cold and warm germination tests consisted of four replications of each entry with 100 seeds per replication in a completely randomized design.
Cold Germination Methods: The seeds were covered with approximately 1/8 inch of a soil mixture and pl aced in an incubator set at 10° C for 7 days; the tempe rature was then raised to 25° C for an additional 7 days. After the seven

- day incubation period at 25° C, the number of seedlings germinated was determined in accordance with the AOSA Seedling Evaluation guidelines ( AOSA, 2010b ) and documented. This method is adapted from the AOSA Seed Vigor Testing Handbook ( AOSA, 2009 )

. Warm Germination Methods: Twenty

- five soybean seeds per petri dish were saturated with water and positioned with four dishes placed closely together in a square to make one replication

. The dishes were pl aced in an incubator set at 25° C and o n day five, the number of seeds germinated in each replication was r ecorded. On day 8, all remaining seeds were scored as germinated (normal seedlings), abnormal, or hard seeds. This method is adapted from the AOSA Rules for Testing Seeds ( AOSA, 2010a ) . Seedlings were evaluated in accordance wit h the AOSA Seedling Evaluation G uidelines ( AOSA, 2010b )

. Data were transformed using the arcsine of the square root of the decimal fraction of seeds germinated per replicate and then subjected to analysis of variance based on a completely randomized design ( SAS Institute Inc., 2009 )

. Germination data and P

- values for the significance of the effect of entry on ger mination are provided in Table 22 . Germination of DAS

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- 2 soybean did not differ significantly (α = 0.05 ) from that of the non

- transgenic, near

- isogenic control Maverick under cold ( P = 0.5372) and warm ( P = 0.6129) conditions. The results indicate that s eed dormancy characteristics have not been changed in DAS

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Contains No Confidential Business Information Table 22 . Germination percentage for DAS

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- 2 and non

- transgenic control soybean. Soybean Germination (%) Temperature Entry Rep 1 Rep 2 Rep 3 Rep 4 Mean (±SE) P
Cold DAS

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- 2 88 93 90 91 90.5 (±1.0) 0.5372 Cold Control 90 91 90 88 89.8 (±0.6)
Warm DAS

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- 2 99 98 * 100 99 99.0 (±0.4) 0.6129 Warm Control 98 * 100 100 99 99.3 (±0.5)

* One hard seed was found in these replicates.

8.4. Summary of Agronomic Characteristics The agronomic study evaluating plant growth characteristics throughout the growing season demonstrated the equivalence of DAS

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- 2 soybean with conventional non

- transgenic soybean. Agronomic and phenotypic characteristics, including response to ecological stressors as indicated by susceptibility to disease and insect pressure , were not different between DAS

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- 2 soybean and conventional soybean grown across diverse environments. In addition, no differences were detected in germination and dormancy of seed from DAS

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- 2 compared with control soybean. The data support th e conclusion that agronomic, disease, and pest characteristics of DAS

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- 2 soybean are not significantly different from that of conventional soybean, and there is no indication that DAS

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- 2 soybean will pose an increased plant pest risk.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information 9. Impact on Environment and Agronomic Practices This section describes the potential impact of DAS

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- 2 soybean on non

- target organisms including threatened and endangered species and an assessment on the impact of introducing DAS

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- 2 soybean on the current ag ronomic practices for soybean in the U.S. and insect resistance management. This sectio n also provides an evaluation of weediness potential and gene flow.
9.1. Potential Impact on Environment
9.1.1. Efficacy against Target Pests The efficacy of DAS

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- 2 soybean against lepidopteran pests was evaluated under field conditions and through laboratory bioassay of field collected foliage at the Dow AgroSciences Southern U.S. Research Center located near Greenville , Mississippi

. DAS

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- 2 soybean was
c ompared directly with the non
- transgenic soybean control Maverick . T4 seed (four generations post

- transformation) bearing DAS

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- 2 were planted for field evaluation. Bioassay of excised leaves with soybean looper ( Chrysodeixis includens, formerly Pseudoplusi a includens ) , velvetbean caterpillar (

Anticarsia gemmatalis ) , fall armyworm ( Spodoptera frugiperda ) , and tobacco budword ( Heliothis virescens ) was used to measure the efficacy . In addition, the number of natural C. includens larvae and associated levels of leaf damage were observed in the DAS

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- 2 and ‘Maverick’ plots. Excellent e fficacy against soybean looper, velvetbean caterpillar , fall armyworm , and tobacco budworm was demonstrated. Trial Design: Plots were planted in r andomized complete block with 4 replicates of each entry replicated one time per block. Each replicate plot consisted of 4 rows, 20 ft ( 6.1 m ) in length, spaced 40 in ches ( 1

. 02 m ) apart. There were 160 seeds planted per row, spaced 1.5 in ches ( 3.8
cm ) apart within the row. Two a dditional rows of ‘Maverick’ were planted between the experimental plots and four rows of ‘Maverick’ were planted adjacent to the outermost experimental plots to attract natural insect pest populations

. Dates of Efficacy Evalua tions: Leaves for the first bioassay were collected 15 July 201 1, when t he soybean plants were in the R2 growth stage . Leaves for the second bioassay were collected from the same plants on 12 August 2011, when t he soybean plants were in the R 5 growth stag e

. Field populations of C. includens were sampled on 8, 15, 22, and 29 August 2011. Bioassay of Excised Leaves at Growth Stage R2: Fully

- expanded, mainstem trifoliate leaves, showing no signs of damage or discoloration and located four nodes below the meristem , were excised for the feeding bioassay. A single trifoliate leaf was excised from each of 15 plants per replicate; 4 trifoliates per replicate were used for the bioassay of C. includens (formerly P. includens ) , 4 trifoliates per replicate were us ed for the bioassay of S. frugiperda , 4 trifoliates per replicate were used for the bioassay of H. virescens , and 3 trifoliates per replicate were used for the bioassay of A. gemmatalis

. T he two side leaflets of each trifoliate were excised and placed in s eparate, labeled petri plates containing a thin layer of agar. T wo second instar s of C. includens , A. gemmatalis , S. frugiperda
or H. virescens were placed on each leaflet. For C. includens , S. frugiperda , and H. virescens , 16 larvae per replicate (64 la rvae in total) were exposed to leaf tissue from event DAS

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- 2 and ‘Maverick’. For A. gemmatalis , 12 larvae per replicate (48 larvae in total) were exposed to leafUSDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information tissue from event DAS

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- 2 and ‘Maverick’

. All insect larvae were provided by the Dow AgroSciences Insectary in Indianapolis, I ndiana . The petri plates holding the infested leaflets were held at 25  C and 40% RH. After 2 days, larvae were determined to be dead (no movement when stimulated with a sharp probe), moribund (larva responds to stimulus but unable to right itself if placed on side), stunted (smaller in size than larvae held on ‘Maverick’ leaves), or alive (normal in size and response to stimulus). Also, t he percentage of each leaflet consumed was estimated visually. If no fe eding damage was evident, 0% consumption was recorded. If only

“ pinholes ” in the leaf tissue were evident, 0.5% consumption was recorded. For greater amounts of consumption, a photographic guide was used to aid in estimating the percentage consumed ( Rice, 2002 )

. Larval condition and leaf consumption were observed again 4 days after larvae had been placed on leaflets. Bioassay of Excised Leaves at Growth Stage R5: The feeding bioassa y procedure at R5 was the same as the procedure used at R2 with the exception that for S

. frugiperda , and H

. virescens , all three leaflets were excised from each trifoliate and a single, second instar was placed on each leaflet. This resulted in 1 2 larvae per replicate (48 larvae in total) of S. frugiperda and H. virescens being exposed to leaflets from DAS

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- 2 and ‘Maverick’

. Field Sampling of Natural Chrysodeixis includens Infestations: Insect counts were initiated when the natural C. includens (formerly P. includens ) population began to cause damage in ‘Maverick’ plots, and continued weekly while C. includens larvae were present

. The center two rows of each plot were sampled. A 3 ft × 3 ft ( 91 cm × 91 cm ) white cloth was placed at a randomly
s elected location between the center two rows. The plants in the section of row adjacent to one edge of the sheet were bent over the cloth and shaken 15 times to dislodge any insects present. This process was repeated for the row on the opposite edge of t he cloth. Larvae were counted by species and size: larvae < 0.25 in ( < 6 mm ) in length were counted as small larvae and larvae ≥ 0.25 in ( ≥ 6 mm ) in length were counted as large larvae. All insects were removed from the cloth before taking the next measurement. The cloth was moved to a second randomly selected location between the two center rows and the sampling process was repeated, resulting in two sub samples per plot at each sampling date. On the last sample date, p ercent defoliation for 20 plants per replicate was estimated visually on the trifoliate at the fifth mainstem node below the meristem with the aid of a photographic guide ( Rice, 2002 )

. Data Analysis: Given the binomial nature of the insect mortality data (alive and healthy vs. dead, moribund or stunted), the data were arranged as a 2×2 contingency table (as shown below) and subjected to Pearson’s chi

- square test. The calculated chi

- square values and significance levels ( P values) of these chi

- square values are presented in Table 23

.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information DAS

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- 2 ‘Maverick’
Total number of live, healthy larvae a b Row 1 total

Total number of dead, moribund, or stunted larvae c d Row 2 total

Column 1 total Column 2

total Grand total

All other results were subjected to analysis of variance to determine if significant treatment or replicate effects existed. There were no significant replication effects for any variable measured. Bartlett’s test was used to test for the equality of tre atment variances. Where variances were unequal, the ln (

x +1) transformation was applied. If the ln ( x +1) transformation did not equalize treatment variances, the square root( x +0.5) transformation was applied. If the square root( x +0.5) transformation did n ot equalize treatment variances, the arcsine square root transformation was applied. The calculated F values and significance levels ( P values) of these F values are presented in Table 23

. Results Bioassays of field

- grown foliage of s oybean event DAS

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- 2 demonstrated excellent control of C. includens , A. gemmatalis , S. frugiperda , and H. virescens at both the R2 and R5 plant development stages ( Table 23 ). Likewise, field evaluations indicated excellent control of C. includens

.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Table 23 . Efficacy of DA S

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- 2 soybean compared to ‘ Maverick’
.

C. includens (formerly P. includens).
In conclusion, l aboratory bioassay of field grown event DAS

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- 2 soybean foliage, and evaluation of field infestations, demonstrate the efficacy of this event against four lepidopteran pests of soybean.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information

9.1.2. Potential Impact on Non

- Target Organisms A risk assessment was conducted to characterize the potential for adverse effects of cultivating DAS

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- 2 soybean, expressing Cry1Ac and Cry1F proteins, on non

- target organisms (NTOs). The assessment focused on non

- target arthropods associated with soybean agroecosystems with special emphasis on beneficial taxa and threatened or endangered species. Assessment factors included: established safety data for Cry proteins including data supporting previous deregulations for corn and cotton products expressing the Cry1Ac and Cry1F proteins, the expression levels of the Cry1Ac and Cry1F proteins in DAS

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- 2 soybean (Section 6 ) , the environmental fate of the Cry1Ac and Cry1F proteins (Section 9.1.4 ), feeding tests of Cry1Ac and Cry1F proteins to representative NTOs, results from a field survey of non

- target arthropods associated with DAS

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- 2, and information from key databases for threatened and endangered species. Results from the assessment indicate that DAS

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- 2 is unlikely to have
a dverse effects on non

- target organisms , including threatened or endangered species , under normal agricultural practices. Regulatory guidelines for risk assessment of genetically modified crops expressing plant

incorporated protectants (PIPs) were developed by the U.S. EPA ( EPA, 1998 ) , and followed by Scientific Advisory Panel (SAP) meetings ( EPA, 2001c , 2001b , 2004 ) , and publications that provide refinements applicable to Bt PIPs ( EPA, 2007 ; Wolt et al. , 2010 ; Carstens et al. , 2011 )

. The assessment for DAS

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- 2 focused on non

- target arthropods associated with s oybean agroecosystems using a step

- wise process that included problem formulation, hazard identification and characterization, exposure assessment, and risk characterization. Problem formulation accounted for the familiarity of mode of action for Cry prot eins ( Aronson and Shai, 2001 ; Rang et al. , 2004 ; Lee et al. , 2006 ; Gouffon et al. , 2011 ; Liu et al. , 2011 ) , the narrow spectrum of activity for Cry proteins ( McClintock et al. , 1995 ; Romeis et al. , 2006 ; Mar vier et al. , 2007 ; Duan et al. , 2008 ; Wolfenbarger et al. , 2008 ; Duan et al. , 2009 ) , the demonstrated history of safe use for Bt cops ( Naranjo, 2005 ; Sanvido et al. , 2007 ) and the agronomic equivalence of DAS

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- 2 soybean to conventional soybean (Section 8 )

. The hazard characterization was informed by existing safety data for the Cry1Ac and Cry1F proteins , spectrum of activity data that were produced to support the deregulation and registration of Cry1Ac

- and Cry1F

- expressing WideStrike ® cotton events DAS

- 21023

- 5 (also described as 3006

- 210

- 23) and DAS

- 24236

- 5 (also described as 281

- 24

- 236) ( EPA, 2005 ) , and lack of synergy between these two proteins ( EPA, 2005 )

. The exposure characterization was based on the expression analyses for Cry1Ac and Cry1F in DAS

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- 2 soybean (Section 6 ) and environmental fate of Cry proteins (Section 9.1.4 ). Special consideration for threatened o r endangered species is addressed in Section 9.1.3

. L aboratory studies used Cry1Ac and Cry1F proteins that have been demonstrated to be equivalent with the Cry1Ac a nd Cry1F proteins produced by DAS

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- 2 (Section 6 )

. Laboratory d ose levels were based on Cry1Ac and Cry1F protein expression levels in WideStrike  cotton events DAS

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- 5 and DAS

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- 5, and therefore for the present assessment, the margins of exposure (MOE) for representative NTOs were recalculated based on high

- end exposure estimates (HEEEs) ( EPA, 2009 ) derived from Cry1Ac and Cry1F protein expression in DAS

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- 2 . Based on the level of concern (LOC) of 50% mortality at 10× the estimated environmental concentration (EEC) ( EPA, 2007 ) , and HEEEs for Cry1Ac and Cry1F in DAS

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- 2 cropping systems, the resulting MOE values support the conclusion ofUSDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information negligible risk to NTOs representing diverse taxonomic groups and exposure scenarios ( Table 24 ).
Table 24 . Estimated margins of exposure (MOE) to non

- target organisms based on hi gh

end exposure estimates (HEEE) for Cry1Ac and Cry1F proteins produced in DAS

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- 2. Organism Protein 1 HEEE 2 NOEC 3 MOE 4 Mouse Cry1Ac 28.33 ng/mg > 700 mg/kg > 100 (CD

- 1) Cry1F 63.32 ng/mg > 600 mg/kg > 100 Earthworm 5 Cry1Ac 0.0910 mg/kg soil 107 mg/kg soil > 100 ( Eisenia fetida ) Cry1F 0.345 mg/kg soil 247 mg/kg soil > 100 Collembola 5 Cry1Ac 0.0910 mg/kg soil > 22.6 mg/kg diet > 100 ( Folsomia candida ) Cry1F 0.345 mg/kg soil > 702 mg/kg diet > 100 Lady beetle Cry1Ac 4.76 ng/mg fw > 20.5 ng/mg fw > 4 ( Hippodamia convergens ) Cry1F 10.6 ng/mg fw > 273 ng/mg fw > 25

1 Microbe

- derived Cry1Ac and Cry1F proteins used in laboratory studies have been demonstrated to be equivalent with the Cry1Ac and Cry1F proteins expressed in DAS

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- 2 ( Section 6 ).

2 High

- End Exposure Estimate (HEEE) represents the 90% upper bound of the reported expression; HEEE = mean expression concentration + (t 0.1, upper tail, n

- 1 × std. dev.) / n 1/2 ; HEEE values based on Cry1Ac and Cry1F expression level determined for DAS

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- 2 plant tissue relevant to the potential exposure route for the organism of interest; values for earthworm and Collembola are expressed as estimated environ mental concentrations for soil (soil EECs) based on the HEEEs for forage tissue at the R3 growth stage; values for mice and lady beetles were based on leaf expression at the V5 growth stage.

3 No Observed Effect Concentration (NOEC).

4 Margin of Exposure ( MOE) was calculated based on the ratio of the NOEC to HEEE.

5 The MOE values for earthworm and Collembola were calculated using the following parameter assumptions: 175,000 soybean plants/acre, soybean plant dry weight of 71.2 g/plant, soil bulk density of 1500 kg/cubic meter, soil volume in a one

- acre area with a depth of 0.15 m, and Cry1Ac and Cry1F soil EEC values of 6.65 and 25.22 ng/mg dw, respectively, for forage tissue at the R3 growth stage. The estimated MOEs ranged from approximately 4 to greater than 100. Where the MOE for lady beetles ( > 4×) in the Cry1Ac test was not demonstrated to be greater than 10× NOEC , the potential risk was examined in the context of the known spectrum of activity of Cry1Ac being restricted to the order Lepidoptera, an ad ditional high

- dose laboratory study of the Cry1Ac protein against lady beetles ( Li et al. , 2011 ) as well as data for coccinellids monitored in DAS

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- 2 field trials that demonstrated no adverse effects on lady beetle performance and abundance, respectively. Given no detrimental effec ts were observed in independent laboratory and field studies, risk to lady beetles was concluded to be negligible. A lack of adverse effects following exposure to Cry1Ac and/or Cry1F proteins has also been shown for collembolans ( Folsomia candida and Xenyl la grisea ) ( Sims and Martin, 1997 ) , the minute pirate bug ( Orius albidipennis ) ( González

- Zamora et al. , 2007 ) , green lacewing ( Chrysopa carnea ) ( Rodrigo

- Simón et al. , 2006 ) , hymenopteran parasitoids ( Nasonia vitripennis ) , honeybees (

Apis mellifer

a ), and bobwhite quail . Agreement between laboratory and field observations for transgenic Bt proteins ( Duan et al. , 2009 ) has been supported throughUSDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information meta

- analyses of NTO data at the order

- ( Naranjo, 2009 ) and species

- level ( Duan et al. , 2008 )

. The meta

- analysis for honeybees conducted by Duan et al. (2008) included six studies incorporating Cry1Ac or Cry1F (truncated or full lengt h protein), suggesting that negative effects on pollinators are unlikely given exposure to the Cry1Ac and Cry1F proteins expressed in DAS

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- 2

. To confirm previous laboratory findings and to address any uncertainties in the risk assessment focused on ta xonomic groups that were most likely to be exposed to the Cry proteins produced by DAS

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- 2 soybean in field settings , the potential for adverse effects of DAS

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- 2 on
non
- target arthropod populations was assessed in a US field study. Field study de tails are provided in Appen dix 8

. Assessment of population abundance and composition was conducted for several taxonomic groups, including: Oribatida (soil mites), A raneae (foraging and web spiders), Collembola (springtails), Hemiptera (e.g. Orius spp.), Coleoptera (e.g. ground beetles , lady beetles ), Neuroptera (e.g. Chrysopa spp.), Lepidoptera, Diptera, and Hymenoptera (e.g. parasitoid wasps). Consistent across stu dy site locations and sampling methods, arthropod populations associated with DAS

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- 2 were similar to those of non

- transgenic soybean. Field monitoring confirmed the results of laboratory studies examining direct exposure to Cry1Ac and Cry1F proteins, and demonstrated no adverse effects of DAS

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- 2 cultivation on
non
- target arthropod communities. Field trial results for DAS

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- 2 contribute to a series of studies demonstrating no adverse effects of Bt crops on NTOs across a diversity of receiving environments ( Pilcher et al. , 1997 ; Lozzia et al. , 1998 ; Bhatti et al. , 2005 ; Bitzer et al. , 2005 ; Daly and Buntin, 2005 ; Dively, 2005 ; Head et al. , 2005 ; Lopez et al. , 2005 ; Naranjo, 2005 ; Pilcher et al. , 2005 ; Torres and Ruberson, 2005 ; Whitehouse et al. , 2005 )

. The Cry1Ac and Cry1F proteins expressed in DAS

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- 2 have been previously demonstrated to have no adverse effects on fish (rainbow trout) or aquatic invertebrates (e.g. Daphnia magna ). Risk posed to aquatic invertebrates (including the aquatic Lepidoptera) through DAS

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- 2 cultivation was concluded to be negligible due to a low likelihood of exposure ( Carstens et al. , 2011 ) and no verified instances of sensitive species. Th erefore, the existing studies in Daphnia and rainbow trout were considered to be adequate in supporting the conclusion that DAS

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- 2 deployment poses little risk to aquatic organisms.

9.1.3. Potential I mpact on Threatened and Endangered Species Given the narrow spectrum of activity observed for Cry proteins ( McClintock et al. , 1995 ; Romeis et al. , 2006 ; Marvier et al. , 2007 ; Wolfenbarger et al. , 2008 ; Duan et al. , 2009 ) consideration for threatened and endangered (T/E) species in the assessment for DAS

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- 2 focused on taxa within the order Lepidoptera. Proximity relationships between potenti al growing sites where DAS

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- 2 could be planted and locations of T/E lepidopteran species were determined from the information contained in the FIFRA Endangered Species Task Force (FESTF) databases. Ecology and life history information for species ide ntified as being present in soybean

- growing counties was accessed from the US Fish and Wildlife Service and NatureServe Explorer database s ( http://www.natureserve.org/explorer/ ). Four lepidopteran butterfl y species (Karner blue, Lycaeides melissa samuelis ; Mitchell's satyr, Neonympha mitchellii mitchellii ; Saint Francis' satyr, Neonympha mitchellii francisci ; andUSDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Uncompahgre fritillary, Boloria acrocnema ) were indicated to co

- occur with soybean use sites in 66 counties located in 13 states. Ecology and life history information demonstrated that habitat requirements for larvae and adults of three of the identified species (Mitchell's satyr, Saint Francis' satyr, and Uncompahgre fritillary) do not overlap wit h commercial soybean acreage , and are therefore not expected to be impacted by DAS

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- 2 cultivation . There is a possible overlap between the geographic range for Karner blue butterfly and soybean use sites; however, the potential for direct exposure to the Cry proteins produced by DAS

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- 2 soybean is negligible. Karner blue larvae feed only on lupines ( Lupinus spp.) and soybean is n either identified as a larval food source, nor is soybean open

- pollinated so DAS

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- 2 soybean pollen is highly unlik ely to be deposited on lupine leaves. Furthermore, soybean is not identified
as a nectaring source for Karner blue adults . Therefore, the risk posed to the Karner blue butterfly was considered to be negligible . In agreement with previous T/E species ass essments for Cry1

- containing corn ( Herculex ® ) and cotton (WideStrike ® ) products , the assessment for DAS

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- 2 supports a conclusion of negligible risk to T/E species.

9.1.4. Environmental Fate of Introduced Proteins and Impact on Soil

- dwelling Organisms Proteins are not known to persist in the environment due to the ubiquitous nature of proteases in microbes. The Cry1Ac protein has been used in sprayable Bt formulations for over half a century a nd is present in both WideStrike  cotton event DAS

- 21023

- 5 ( also described as 3006

- 210

- 23) and Bollgard  cotton ( event MON 531, as well as a discontinued corn event: DBT418Cry1Ac corn ) ( Mendelsohn et al. , 2003 ; Sanahuja et al. , 2011 ) . The Cry1F protein has also been used in sprayable Bt formulati ons and is present in WideStrik e  cotton event DAS

- 24236

- 5 (also described as 281

- 24

- 236) , as well as, in Herculex  and SmartStax  corn (event TC1507) ( Mendelsohn et al. , 2003 ; Sanahuja et al. , 2011 ) . Laboratory experiments have shown that both Cry1Ac and Cry1 F are quickly inactivated, and field studies have shown no accumulation of the proteins as a result of continuous planting of crops containing these proteins ( Head et al. , 2002 ; Herman et al. , 2002 ; Shan et al. , 2008 )

. It has further been suggested that the Cry proteins in general are unlikely to represent a significant risk in the soil environment ( Icoz and Stotzky, 2008 )

.

9.1.5. Weediness Potential Commercial soybea n varieties in the U.S. are not considered w eeds and are not effective at in vading established ecosystems. Cultivated soybean seed r arely displays any dormancy characteristics and only under certain environmental conditions grows as a volunteer in the year following cultivation. If this should occur, volunteers do not compete well with the succeeding crop, and can easily be controlled mechanically or chemically. The soybean pl ant is not weedy in character. In North America, Glycine max is not found outside of cultivation. In managed ecosystems, soybean does not effectively compete with other cultivated plants o r primary colonizers ( OECD, 2000 )

.

 Bollgard and SmartStax are registered trademarks of Monsanto Technology LLC.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information The introduction of insecticidal proteins Cry1Ac a nd Cry1F into soybean does not alter the weedine ss characteristics of soybean. Agr onomic properties of DAS

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- 2 soybean related to weediness, such as emergence ( Section 8.1 ) , seedling vigor ( Section 8.1 ) , response to environmental stressors ( Section 8.2 ) , and germination (Section 8.3 ), have been shown to be equivalent to conventional soybean. These findings support the conclusion that DAS

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- 2 soybean does not have increased weediness potential compared to conventional soybean.

9.1.6. Gene Flow Assessment
Vertical Gene Flow Soybean is considered to be a self

- pollinated species, although natural crossing can occur at low rates ( OECD, 2000 ) . The level of cross pollination can vary as a function of growing conditions, spatial arrangement of plants, and genotype. Cross pollin ation levels ranging from 0.09% up to 3.6% in adjacent rows have been reported ( Beard and Knowles, 1971 ; Ahrent and Caviness, 1994 ) , but outcrossing frequency rapidl y declines with distance. Cross pollination rates have been reported to be <0.1% at distances of >5 m ( Caviness, 1966 ; Ray et al. , 2003 ) . Cultivated soybeans can cross only with members of its subgenus Soja . Wild soybean species of the subgenus Soja are native to Korea, Japan, Taiwan, northeastern China, and areas of the former USSR, but do not exist naturally in the United States ( OECD, 2000 ) . Therefore, there is no potential for gene flow from DAS

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- 2 soybeans to wild soybean relatives in the United States.
Horizontal Gene Flow There is no known me chanism for, or definitive demonstration of, DNA transfer from plants to microbes ( Conner et al. , 2003 ) . Even if such a transfer were to take place, transfer of the cry1Ac ( synpro ) , cry1Fv3 and pat genes from DAS

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- 2 soybean would not present a human health or plant pest risk, based on the safety data presented in this petition.

9.2. Current Agronomic Practices for Soybean and Potential Impact
9.2.1. Soybean Production Large scale soybean production in the U.S. began in the early 1920s ( USDA ERS, 2012 )

. According to USDA NASS ( 2012 ) , approximately 1.5 million acres were planted to soybean in 1924. Over the following two decades soy bean acreage expanded rapidly reach ing 15 million
acres. Since 1997 more than 70 million acres were planted to soybean every year with an exception in 2007 when an increase in corn acreage limited soybean cropland

. This i ncrease in soybean acreage is largely due to ideal planting conditions, increased crop rota tion, rising yield, and low er production cost s ( USDA E RS, 2012 )

. Average soybean yield per acre varied from 33.9 bushels to 44 bushels between 1997 and 2011 with the lowest yield recorded in 2003 and the highest in 2009. Total annual soybean production during the same time period ranged from approximatel y 2.45 billion bushels in 2003 to 3.36 billion bushels in 2009. Farm value of U.S. soybean production ranged from $12.21 billion in 1999 to $37.55 billion in 2010 ( USDA NASS, 2012 )

. An average of 1.6 billion bushels of soybean were crushed annually in the last ten years to produce soybean meal for l ivestock and soybean oil for consumption and industrial products ( Soy Stats, 2012 )

.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information In the U.S. the majority of soybean s are grown in the north central states with the remaining crop primarily grown in the southeastern and Mississippi

- delta regions. About 80% of the planted soybean acres and 80% of the harvested soybean acres are concentrated in 11 states, including Iowa, Illinois, Minnesota, Indiana, Missouri, Nebraska, Ohio, South Dakota, North Dakota, Kansas and Arkansas, where growers achieve higher yields at lower per

- acre production cost ( USDA ERS, 2012 )

. Soybean yield in the north central region in 2011 ranged from 27 bushels per acre in Kansas to 53.5 bushels per acre in Nebraska ( USDA NASS, 2012 )

. Soybean yield is largely dependent on variety selection, environmental conditions and agronomic decisions. Variety selection is critical in achieving high yield while environmental conditions and agronomic decisions such as weeds and disease management an d fertilizer application have a significant impact on genetic yield potential. In 2011, seed, chemicals and fertilizers contributed to 70% of the operating cost ( USDA ERS, 2012 )

. In the southern U.S. states higher fertilizer and chemical insecticide applications per acre soybean planted contributed to hi gher operating cost ( Soy Stats, 2012 )

. U.S. is the world’s leading soybean exporter. In 2011, U.S. exported 1.275 bill ion bushels of soybean which accounted for 42% of the U.S. production and 37% of the world’s soybean trade ( Soy Stats, 2012 ; USDA ERS, 2012 )

. U.S. soybean and soybean product exports exceeded $21.5 billion in 2011 with soybean accoun ting for 82%, soybean meal 13% and soybean oil 6% ( Soy Stats, 2012 )

. China was the largest export market for U.S. soyb ean with purchases exceeding $10.4 billion. Mexico was the second largest market for U.S. soybean with purchases of nearly $1.6 billion. Other significant export markets for U.S. soybean are Japan, Indonesia and Taiwan. Canada was the largest export mar ket for U.S. soybean meal in 2011 with purchases reaching $375 million, Mexico was second with $369 million followed by Venezuela at $260 million. T he t op three export markets for U.S. soybean oil were Morocco, Mexico and China with combined purch ases exc eeding $659 million ( Soy Stats, 2012 )

. Soybean seed for commercial planting is produced throughout most of the U.S. soybean growing regions. Soybean varieties are developed and adapted to certain geographical zones and are separated into ten maturity groups ( OECD, 2000 ) . Seed production for these maturity groups is grown in respective geographical zone for each maturity group.
Seed quality , including genetic purity, vigor, and presence of weed seed, seed

- borne diseases, and inert materials , is a major factor affecting crop yield. Genetic purity in commercial seed production is generally regulated through a system of seed certificati on which is intended to ensure that the desired traits in the seed are maintained throug hout all stages in cultivation.
The U.S

. Federal Seed Act , enacted in 1939 , recognizes seed certification and official certifying agencies

. Implementing regulations fu rther recognize land history, field isolation, and varietal purity standards for seed

. States have developed laws to regulate the quality of seed available to farmers ( Bradford, 2006 )

. Most of the laws are similar in nature and have general guidelines for providi ng information on the label for the following: 1) Commonly accepted n ame of agricultural seed, 2) Approximate total percentage by weight of purity, 3) Approximate total per centage of weight of weed seeds, 4) Name and approximate number per pound of each ki nd of noxious weed seeds, 5) Approximate percen tage of germination of the seed, and 6) Month and year the seed was tested.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information The Association of Official Seed Certifying Agencies ( AOSCA, 2012 ) defines the classes of seed as follows: 1) breeder seed, 2) foundation seed, 3) registered seed, and 4) certified seed. Breeder seed is seed directly controlled by the originating or sponsoring pla nt breeding organization. Foundation seed is the progeny of B reeder seed or Foundation seed that is handled to maintain specific genetic identity and purity. Registered seed is the progeny of Breeder or Foundation seed that is so handled as to maintain s atisfactory genetic identity and purity. Certified s eed is the progeny of Breeder, Foundation, or Registered seed that is handled to maintain satisfactory genetic identity and purity. Production of all classes of certified seed requires that 1) each cert ifying agency shall determine that genetic purity and identity are maintained at all stages of certification including seeding, harvesting, processing, and labeling of the seed; 2) the unit of certification shall be a clearly defined field or fields; 3) on e or more field inspection shall be made prior to harvest and when genetic purity and identity can best be determined; and 4) a certification sample shall be drawn in a manner approved by the certifying agency from each cleaned lot of seed eligible for
cer tification (7 CFR §201.72). Federal regulation 7 CFR §201.76 specifies minimum land, isolation, field, and seed standards required for soybean Foundation, Registered and Certified seed. To produce all classes of certified seed, the land requirement is th at the crop shall not be grown on land where soybeans were grown the previous year unless the preceding soybean crop was planted with a class of certified seed of the same variety or unless the preceding soybean crop and the variety being planted have an i dentifiable character difference. Isolation requirement varies somewhat among states and seed producers. Some states require an isolation distance of five feet while other states and seed producers require 10 feet of isolation distance. To qualify as Cer tified seed, only one plant of another variety in 200 soybean plants and 0.5% of seed of other varieties or off

- types are permitted (7 CFR §201.76). To qualify as Registered seed, only one plant of another variety in 500 soybean plants and 0.2% of seed of other variety or off

- types are permitted. To be certified as Foundation seed, only one plant of another variety in 1,000 soybean plants and 0.1% of seed of other variety or off

- types are permitted.
DAS

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- 2 does not differ from conventional soybean in agronomic characteristics (Section 8 ) and thus is not expected to impact U.S. soybean grain or seed production.

9.2.2. Weeds in Soybean and Weed Management Weeds in soybean compete with the crop for light, nutrients and soil moisture. When weeds are uncontrolled for the entire season, yield losses can exceed 68 to 75% ( Krausz et al. , 2001 ; Dalley et al. , 2002 )

. Hartzler and Pringnitz ( 2005 ) define t he critical period of competition in soybeans as the point of time when weeds that emerge with th e crop begin to impact yields. Most studies have found that soybean yields are protected if weeds are controlled before they reach a height of 6 to 8 inches

. T he critical perio d varies widely, depending upon weed species and densities, environmental conditions, and cultural practices. To maximize soybean yields, weeds must be removed or controlled when they are no more than 6 inches tall ( Loux et al. , 2006 )

. Field trials conducted in Ohio in 2001 found that applying glyphosate when weeds are already 9 to 12 inches tall resulted in a 6

- 10% yield loss due to weed competition prior to control of the weeds ( Loux et al. , 2006 )

. Dalley et al. ( 2002 ) studied the eff ect of row width and weed competition on soybean yield in 1998

- 2001. They found that weedUSDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information competition could reduce soybean yields by the time weeds reached 6, 6, and >12 inches in height in 7.5, 15, and 30 inch rows, respectively. The most common weed pro blems in soybeans are the annual grass and broadleaf weeds

. Some fields are also infested with perennial weeds (quackgrass, Johnsongrass, field bindweed, Canada thistle, others) which are more difficult to control since they can reproduce from seed or und erground root buds or rhizomes.
In 200 6 , USDA NASS surveyed 1 9 states ( Arkansas, Illinois, Indiana, Iowa, Kansas, Kentucky, Louisiana, Michigan, Minnesota, Mississippi, Missouri, Nebraska, North Carolina, North Dakota, Ohio, South Dakota, Tennessee, Virgin ia, and Wisconsin ; about 9 6 % of the total U.S. soybean acreage) and found that 98% of the planted soybean acreage was treated with herbicides, an indication of the intensive weed management that is used i n U.S. soybeans ( USDA NASS, 2007 )

. In 2006, 97.5 % of the planted soybea n acres received an average of 2.2 herbicide applications to contro l weeds ( USDA ERS ARMS, 2006 )

. Many herbicides are registered for pre
- plant, pre

- emergent and/or post

- emergent application to selectively control most weed speci es commonly found in soybeans. In general, soybeans sometimes receive a soil applied or burndown herbicide prior to planting or at plant, but alm ost always receive a post

- em ergence herbicide application. In 200 6 , the most widely used herbicide in soybeans was glyphosate, driven by the high adoption o f glyphosate tolerant soybeans ( USDA NASS, 2007 )

. S oybeans received an average of 1. 7 applications of glyphosate on 9 6 % of the acres planted in 200 6. Herbicides such as 2,4

- D ( 10 % of acres), chlorimuron (4% of acres), and trifluralin (2% of acres) were applied on significantly fewer acres. All other herbicides applied in soybeans that year comprised 3% or less of the to tal acres planted. Integrated Weed M anagement (IWM) programs advocate the use of a combination of preventive, cultural, mechanical and chemical tools to keep weed pressure below threshold levels that reduce yield and profits ( Knezevic, 2010 )

. DAS

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- 2 soybean expresses the phosphinothricin acetyltransferase (PAT) protein and therefore it is tolerant to the herbicide glufosinate. With the exception of glufosinate tolerance, no impact i s expected from the deregulation of DAS

- 81419

- 2 soybean on current weed management practices.
DAS

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- 2 soybean expresses the phosphinothricin acetyltransferase (PAT) protein and therefore it is tolerant to the herbicide glufosinate. As glufosinate tole rance in soybeans is currently available today to U.S. growers, no impact is expected from the deregulation of DAS

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- 2 soybean on current weed management practices.

9.2.3. Crop Rotation and Tillage Practices In 2006, 71% of U.S. soybean acres were grown in ro tation after corn, 13% were grown after soybeans, and 16% were grown after small grains, cotton, fallow or other crops ( USDA ERS ARMS )

. Crop rotation is a widespread management practice that has been recognized and exploited for centuries to increase crop yields ( Lauer, 2007 )

. In the Midwestern U.S., a corn

soybean rotation produces at least 10% greater yields in both crops, and sometimes as much as 19% higher corn yields. T he exact mechanism for the rotation effect is unknown, but may be influenced by increases in organic matter and soil fertility, as well as management of diseases, insects, and weeds.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information The introduction of numerous herbicides in the decades following the lau nch of 2,4

- D in the mid

- 1940s allowed reduced and conservation tillage systems to become more feasible and popular ( Givens et al. , 2009 )

. The introduction of glyphosate

- resistant (GR) crops in 1996 brought a technology that enabled many producers to adopt reduced

- tillage production systems. By 2002, only 17% of U.S. soybeans were produced under conventional tillage (<15% residue) systems ( USDA ERS ARMS )

. Some form of conservation tillage was being used on the other 83% of U.S. soybean acres, as follows: 18% reduced tillage ( 15

- 30% residue), 32% mulch till, 1

- 2% ridge till, and 31% no

- till. Increased yields coupled with decreases in erosion and water runoff and lower fuel use and fuel cost strongly support the premise that soybean produced with conservation tillage in any pro duction system is more economically and environmentally sustainable ( CAST, 2009 )

. Given the data presented in this petition demonstrating no difference between DAS

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- 2 and conventional soybean in agronomic characteristics (Section 8 ), no impact is expected from the deregulation of DAS

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- 2 soybean on current crop rotation and tillage practices.

9.2.4. Management of Insect Pests Agricultural prod uction of soybean is a relatively recent development; soybean did not become a major export crop in North America until the 1970s ( Smith, 1994 ) . Insec t injury has not traditionally resulted in economic losses in most North American growing regions. However, in recent years, losses have been severe in some southern regions of the United States ( Catchot, 2011 ) . Economic thres holds , which are the level of insect pressure where the benefits of treatment cover the cost of that treatment, have traditionally been based on $4

- 6 per bushel soybeans. However, recent prices of $12

- 15 per bushel have significantly lowered economic thre sholds associated with many soybean pests, expanding the area and number of insecticide treatments applied to soybeans ( USDA ERS, 2011 ) . Increases in pest insect populations in soybean have also been observed; increases are attributed by some to glob al warming or insect adaptation to the soybean plant ( Way, 1994 ) . Lower economic thresholds coupled with higher insect populations favor more aggressive p est management strategies ( Pedigo, 1996 )

. Insect injury can negatively impact yield, plant maturity, and seed quality. Insect injury is most often defined by the plant parts that are injured: roots, stems, foliage, or pods. Root

- and stem

feeding insect groups typically remain undetected until damage has occurred. Although leaf

feeding insects are the most diverse group, pod

- feeding insects are generally considered to be proportionately more important, because high

- value soybean reproductive parts are affected. Many soybean foliar feeders are caterpillars (Lepidopt era larvae) in the family Noctuidae. These include the soybean looper, Chrysodeixis includens formerly Pseudoplusia includens Walker, velvetbean caterpillar, Anticarsia gemmatalis Hübner and green cloverworm, Hypena scabra (Fab.)

. These species commonly occur sympatrically and procedures are being developed for making management decisions that acknowledge combined effects of these pests. A major effect of foliar feeding is reduced light intercep tion by the soybean canopy; which ultimately decreases yield ( Higley, 1994 )

. Soybean plants can withstand up to 35% leaf loss pre

bloom without lowering yield ( de Freitas Bueno et al. , 2011 ) . However, defoliation during the flowering and pod

- filling stages poses a greater threat to yield because the plant has less time to compensate for injury. From pod

- set to maturity, the plant can tolerate only a 20% defoliationUSDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information level before yield is affected ( de Freitas Bueno et al. , 2011 ) . The tolerance of soybeans for defoliation varies somewhat depending on cultivar ( de Freitas Bueno et al. , 2011 )

. In addition to the plant tissue attacked, soybean responses to injury are influenced by the mode of feeding. For example, yield loss incurred from pod

- feeding stink bugs, such as the southern green stinkbug Nezara viridula (L.) (Hemiptera: Pentatomidae) , is linear in re lation to the amount of plant tissue damage caused by feeding. In contrast, for s eedcorn maggot, Delia platura (Meigen) (Diptera: Anthomyiidae), larval feeding on roots reduces yield only when injury levels are relatively high ( Higley, 1994 )

. Environmental factors also impact the response of soybean to insect injury; yield reductions from insect injury are more severe under conditions associ ated with water stress.
Althou gh soybean is attacked by many pests, only a few pose an economic threat. Damage is concentrated in certain production regions ( Way, 1994 )

. Eight insect species are responsible for most insect damage in U.S. soybean production: velvetbean caterpillar, soybean looper, green cloverworm, Mexican bean beetle, Epilachna varivestis Mulsant (Coleoptera: Coccinellidae), southern green stinkbug, bean l eaf beetle, Cerotoma trifurcata Forster (Coleoptera: Coccinellidae), southern green podworm/corn earworm, Helicoverpa zea (Boddie) (formerly Heliothis zea ) (Lepidoptera: Noctuidae) , and green stink bug (Higley and Boethel, 1994). Soybean insect pest popul ations and extent of soybean damage varies annually and regionally due to differences in climatic and weather conditions, species distributions and environmental tolerances and production practices ( Way, 1994 )

. Generally, soybean insect pest problems are less severe in the Midwest states of the U.S. than in other soybean producing areas ( Way, 1994 )

. Green cloverworm is the only known lepidopteran pest that occurs frequently in the Midwest; cutworms, Agrostis ipsilo n (Hufnagel) and Peridroma saucia (Hübner ) (Noctuidae ) and Painted Lady , Vanessa cardui L. ( Nymphalidae ) have also been reported but are less commonly economically important. Economic insect problems increased in the 1990s ( Hammond, 2012 ) with greater increases in the north

- central U.S. around 2000 ( McWilliams et al. , 1999 ) . Generally, in sect challenges still remain below economic threshold levels for the U.S. Midwest. Several insect pests have been reported in the northeastern United States and Southern Canada in numbers high enough to cause economic losses in soybean ( Penn State Extension, 2012 )

. Defoliators include: green cloverworm, Japanese beetle, Popillia japonica Newman (Coleoptera: Scarabaeidae), Mexican bean beetle, bean leaf beetle, and grasshoppers, Melanoplus spp. (Orthoptera: Acrididae) . Sa p
- sucking insects include: soybean aphid, Aphis glycines Matsumura ( Hemiptera: Aphididae) , potato leafhopper, Empoasca fabae (Harris) (Hemiptera: Cicadellidae) and stink bugs. Stink bugs in particular will feed on both foliage and pods. Seedcorn maggots feed on seed s

. Insect pressure is generally greatest in the southern U.S. states bordering the Gulf of Mexico and the Atlantic Ocean. Four of the major soybean insect pests in the Southeast region are lepidopteran insect pests. Velvetbean caterpillar and soybean looper infestations are greatest in the southeastern states because of their close proximity to the tropics. The warm climate allows these pests to overwinter and facilitates multiple generations per year.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Stink bugs ( Nezara viridula, Acrosternum hilare, Euschistus servus ) are responsible for most infestations and greatest economic losses in the southeastern United States ( McPherson et al. , 1997 ; Guillebeau et al. , 2008 ) . Lepidopteran insects, primarily soybean loope r, velvetbean caterpillar, corn earworm (soybean podworm), and lesser cornstalk borer, Elasmopalpus lignosellus (Zeller) (Lepidoptera: Pyralidae) are responsible for most of the remaining damage ( McPherson et al. , 1997 ; Guillebe au et al. , 2008 )

. Velvetbean caterpillar and soybean looper are considered the most damaging defoliating insects in the South ( Way, 1994 ; Hammond, 2012 )

. Insecticides are used on approximately 50% of the soybean acreage in Georgia for lepidopteran pests with velvetbean caterpillar being the most targeted pest ( Gianessi et al. , 2002 )

. Approximately 40% of the soybean acreage in Louisiana is treated with insecticides fo r lepidopteran pests, with soybean looper being the main target ( Gianessi et al. , 2002 )

. Although insecticides provide effective and economical control or suppression of soybean insect pests that reach e conomic thresholds, Integrated P est M anagement (IPM) programs that incorporate preventive pest management with insecticidal control have proven to be highly effecti ve in managing pest problems ( Pedigo, 1996 ; Kogan, 1998 ) . Effective management of insects in soybean requires consideration of agronomic, economic and biological factors. For example, preventive pest management practices are most important where pest problems are anticipated. Agric ultural practices can be adjusted to adversely affect pest species or aid beneficial species. Similarly, variety selection, crop rotation, tillage, planting dates, and adjacent crops all impact the potential for pest outbreaks and the success of natural e nemies in suppressing pest populations. As it is not possible to eliminate all economic losses due to insects, IPM programs are implemented to minimize these losses. IPM practice includes monitoring fields for insect growth stage, insect development and p opulation density, and occasionally natural enemy development and population density. Management decisions for insect populations in individual fields are based on economic injury level, which is defined as the lowest population density of each insect lik ely to cause economic damage. The economic injury level usually changes during the growing season, with later stages of development warranting action at a lower level of defoliation. IPM programs integrate chemical control and biological control, cultura l control and plant resistance to minimize insecticide resistance and reduce dependence on insecticides ( Pedigo, 1996 ) . Effective IPM requires intense insect scouting and is justified in high value crops or where insect pressure is common. However, the majority of soybean fields are not scouted because damage is infrequent. Failure to scout risks allowing damage that greatly exceeds the economic threshold to occur unnoticed; discovery may occur too late for subsequent insecticide applications to be effe ctive. The growing problem of soybean insect pest pressure is evidenced by increasing insecticide treatments in recent years ( USDA NASS, 2007 ) . According to USDA

- NASS statistics, about 16% of the U.S. soybean acreage in 2006 received an insecticide treatment, with chlorpyrif os (6%), esfenvalerate (5%), and lambda

- cyhalothrin (3%); these accounted for nearly all treated
acres ( USDA NASS, 2007 ) . Each of these insecticides controls a similar broad spectrum of insect pests including lepidopteran and non

- lepidopteran pests. USDA

- NASS (2007) also
re ported that approximately 15% of the soybean acreage in the Midwest region and approximately 25% of the soybean acreage in the Southeast and Eastern Coastal regions of theUSDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information U.S. received an insecticide treatment in 2006 with some states in the Southeast req uiring treatments on up to 75% of their acreage ( USDA NASS, 2007 ) Chemical insecticides can have limited efficacy in controlling lepidopteran infestations in soybean. Narrow application windows, the emergence of insecticide resistance, and public pressure for reduced pestic ide use limit the desirability of this approach to pest management ( Thomas and Boethel, 1994 )

. S oybean looper has developed extensive insecticide resistance ( Thomas and Boethel, 1994 ) ; resistan ce to pyrethroids is widespread across the southern U.S. ( Felland et al. , 1990 ; Leonard et al. , 1990 )

. Insecticides remain effective against velvetbean caterpillar. However, infestations can quickly reach damaging levels and cause economic loss if insecticides are not applied promptly.

Bt crops (expressing at least one of several insecticidal proteins isolated from Bacillus thuringiensis ) are effective in targeting s pecific insect pests, easy to adopt, and cost effective; I ncreasing adoption rates of Bt corn and cotton since 1996 have coincided with increased yields ( USDA ERS, 2006 ) . Pesticide use has also decreased since the introduction of these insect

resistant crops, with one study showing an 8% reduction in inse cticide use for adopters of Bt corn than non

- adopters ( USDA ERS, 2006 ) . Importantly, Bt crops have also be credited with increasing yields, even when economic thresholds have not been reac hed in either Bt

- or non
- Bt

fields due to regional insect population reductions ( Hutchison et al. , 2010 )

. Like Bt corn and cotton, DAS

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- 2 soybean offers an efficient and e nvironmentally sound alternative to chemical insecticides for controlling lepidopteran soybean pests. Genetically modified crops are now an important component of many IPM programs, providing effective management of lepidopteran pests with reduced insecticide use ( Perlak et al. , 2001 ; Pilcher and Rice, 2003 ) . Additional benefits from DAS

- 81419

- 2 soybean include a reduced need for scouting, preservation of beneficial ins ect populations, increased convenience and greater performance consistency.

9.2.5. Insect Resistance Management DAS

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- 2 soybean expresses two insecticidal proteins , Cry1Ac and Cry1F , originally from the naturally occurring soil bacterium Bacillus thuringiensi s. Data presented in this petition demonstrate that Cry1Ac and Cry1F are expressed in the tissues of the field

- grown DAS

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- 2 soybean throughout the growing season

. DA S

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- 2 soybean provide s an effective pest management tool while reducing or repla cing current insecticide applications to control lepidopteran pests in regions where these insect pests cause significa nt plant damage and yield loss. Substances that are pesticides, as defined under the Federal Insecticide, Fungicide and Rodenticide Act ( FIFRA), are subject to regulation by the U.S. Environmental Protection Agency (EPA). Pesticides produced in planta, known as plant

- incorporated protectants (PIPs), are also subject to regulation by the EPA under FIFRA. To prevent or delay the onset of ins ect resistance to Bt proteins expressed in transgenic crops, EPA has mandated specific insect resistance management (IRM) programs to protect the benefits of Bt crops and sustainable long

term use of Bt crops ( EPA, 2001a )

. EPA requires that an IRM plan is based on the characteristics of the crop, the biology and sensitivity of the target insect pests, and the cropping systems in which the product will be deployed. A core element of an IRM program is the use ofUSDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information refuges, whic h are non

- Bt host plants of the target insects that support susceptible target insects and reduce selection pressure for resistance. Susceptible insects produced from the refuge may mate with any resistant insects that survive in the Bt crop producing het erozygote insects that may be susceptible to the Bt crop ( EPA, 2001a )

. Given that the registration DAS is currently seeking from EPA for planting of DAS

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- 2 soybean in the U.S. will be limited in acreage for breeding and seed increase purp oses to support product development, DAS has propose d that planting of a refuge is not necessary and an IRM program for DAS

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- 2 soybean is not needed. The proposal is based on previous registration decisions, impact on the natural refuge for key target pests of Bt cotton, durability of DAS

- 81419

- 2 soybean, and target pest biology. A summary of the analyses is presented below. Detailed analyses will be provided to EPA in connection with the FIFRA Section 3 seed inc rease registration application. In prev ious registration decisions, EPA indicated that cultivation of crops containing PIPs for seed production on up to 20,000 acres per county and 250,000 acres total does not require planting of a refuge (for example, Herculex  I corn, EPA registration number 68467

- 2 as amended on September 29 th , 2010). Similarly, on July 27 th 2012, EPA issued registrations under FIFRA Section 3 that permit cultivation of each Bt cotton, Cry1Ac Insect Protected cotton seed (EPA registration number 68467

- 17) or Cry1F Insect Pro tected cotton seed (EPA registration number 68467

- 18), on up to a total of 20,000 acres per county and up to a combined total of 30,000 acres per year without planting of a refuge for breeding, agronomic testing and seed production purposes. In the U.S., the majority of the soybeans are grown in the north central states and the remaining in the southeastern and Mississippi delta regions. In the southern and southeastern U.S., soybean is a component of the Helicoverpa zea (bollworm) natural refuge for Bt c otton ( Jackson et al. , 2008 ) and DAS

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- 2 soybean is expected to cause reduced survival of H. z ea larvae feeding on it relative to non

- Bt soybean. EPA has oversight of insect resistance management for Bt crops in the U.S. Through the terms of registration for DAS

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- 2 soybean, EPA will impose resistance management requirements to promote the durability of Bt traits in cotton, corn, and soybean

. DAS

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- 2 soybean expresses two insecticidal proteins whose modes of action differ with respect to receptor binding. For example Cry1Ac and C ry1F have been shown to bind to different receptors in the midgut of the target soybean insect pest tobacco budworm H. virescens ( Jur at

- Fuentes and Adang, 2001 )

. Cry1Ac binds to at least three sets of receptors while Cry1F binds to at least two, only one of which also binds Cry1Ac. The major receptor for Cry1Ac is not recognized by Cry1F ( Jurat

- Fuentes and Adang, 2006 )

. Such incomplete shared binding is expected to lead to incomplete cross

- resistance when resistance is mediated by receptor changes. Bt gene pyramiding offered by DAS

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- 2 soybean provides additional protec tion against the development of insect resistance and offers greater durability than Bt crops carrying a single Bt trait.
In addition to H. zea , other lepidopteran pests of soybean that are sensitive to the Cry1F and/or Cry1Ac Bt proteins, such as s oybean l ooper ( Chrysodeixis includens, formerly Pseudoplusia includens ) , v elvetbean c aterpillar ( Anticarsia gemmatalis ) , and f all a rmyworm ( Spodoptera frugiperda ) , are polyphagous, feeding on numerous wild and cultivated plants ( Sparks, 1979 ; Waters and Barfield, 1989 ; Jost and Pitre, 2002 )

. With other Bt crops grown for breeding andUSDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information seed increase purposes, plantings of up to 20,000 acres per county are permitted by EPA without a refuge because with these limits the selection pressure for resistance in sensitive insect species is negligible . The natural refuge of non

- Bt crops and wild hosts for these polyphagous species that are pests of soybean further reduces sel ection pressure for resistance. The information presented here indicates that planting of DAS

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- 2 soybean under limited
acre age for breeding and seed increase purposes in the U.S. will have very little effect on resistance development in H. zea to Bt cotton. Furthermore, the level of selection pressure on other lepidopteran pests of soybean will be negligible under the propose d acreage limits. We therefore anticipate that EPA will not require a structured refuge for DAS

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- 2 soybean under the conditions of use. If DAS chooses to cultivate DAS

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- 2 soybean for commercial purposes in the U.S. in the future, DAS will apply for a FIFRA Section 3 commercial use registration with EPA and will develop a specific IRM program in connection with t he commercial use registration. Under the terms of the EPA’s registrations for commercial cultivation of Bt cotton and Bt corn, DAS is r equired to monitor for resistance in several lepidopteran pests of cotton that are also pests of soybean. This monitoring will continue after the introduction of DAS

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- 2 soybean.
9.3. Summary of Impact on Environment and Agronomic Practices The efficacy study demonstrat e s that Cry1Ac and Cry1F expressed in DAS

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- 2 soybean provide protection against target lepidopteran pests . There are no new phenotypic characte ristics in DAS

- 81419

- 2 soybean to indicate it is any different from conventional soybean in weediness potential, and like conventional soybean, the ri sk of gene flow from DAS

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- 2 soybean to wild relat ives in the U.S. is negligible. No significant impact is expected on curre nt crop management practices, non

- target or endangered species, or crop rotation from the introduction of DAS

- 81419

- 2 s oybean. DAS

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- 2 offers an efficient and environmentally sound alternative to chemical insecticides. In addition, DAS

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- 2 offers g reater durability and therefore improved insect resistance management. The availability of DAS

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- 2 is expected to reduce the need for scouting, preserve beneficial insect population, and provide increased convenience and g reater performance consistenc y.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information 10. Adverse Consequences of Introduction Field and laboratory testing of DAS

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- 2 soybean demonstrated that the transgenic soybean is substantially equivalent to non

- transgenic conventional soybean apart from the intended change of insect resistance. D AS knows of no study results or other observations associated with DAS

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- 2 soybean that indicate there would be anticipated to be adverse consequences from introduction.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information 11. Appendices Appendix 1 . Materials and Methods for Molecular C haracterization of DA S

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- 2 S oybean Appendix 2

. Materials, Methods and Result s for Characterization of Cry1Ac Protein Appendix 3

. Materials, Methods and Results for Characterization of Cry1F Protein Appendix 4

. Materials, Methods and Result s for Characterization of PAT Protein Appendix 5

. Materials and Methods for Cry1Ac, Cry1F and PAT Protein Expression Analysis Appendix 6

. Analytical Methods for Compositional Analysis Appendix 7

. Literature Ranges for Compositional Analysis Appen dix 8

. Non

- Target Arthropod Field Study Appendix 9

. USDA Notifications for DAS

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. St ewardship of Insect

- Resistant DAS

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Contains No Confidential Business Information Appendix 1 . Materials and Methods for Molecular C haracterization of DA S

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DAS

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- 2 Soybean Material Transgenic soybean seeds from five distinct generations of soybean containing event DAS

81419

- 2 were planted in the greenhouse. After at least one week of growth for emerged seedlings , leaf punches were taken from each plant and were tested for PAT protein expression using a l ateral flow s t rip assay according to the manufacturer’s instructions (Envirologix Inc.)

. Each plant was given a “+” or “

- ” for the presence or absence of the PAT protein. Control Soybean Material
Seeds from the non
- transgenic soybean line Maverick were planted in the greenhouse. The Maverick seeds had a genetic background representative of the transgenic seeds but did not contain the cry1Ac(synpro), cry1Fv3, or pat gene s

. Reference Materials DNA of the plasmid pDAB9582 was added to samples of the non
- transgenic control genomic DNA at a ratio approximately equivalent to 1 copy of the transgene per soybean genome with a soybean genome size of ~1.1 × 10 9 bp ( Arumuganathan and Earle, 1991 ) and used as the positive control for the Southern hybridization

. DIG

- labeled DNA Molecular Weight Marker II a nd DIG

- labeled DNA Molecular Weight Marker VII (Roche Diagnostics), each containing a mixture of DNA fragments with different sizes, served as size standards for agarose gel electrophoresis and Southern blot analysis. DNA Probe Preparation DNA probes wer e generated by a PCR

- based inco rporation of a digoxigenin ( DIG) labeled nucleotide, [DIG ]

- dUTP, into fragments generated by primers specific to gene tic elements and backbone regions from plasmid pDAB9582 . Generation of DNA probes by PCR synthesis was
carr ied out using a PCR DIG Probe Synthesis Kit (Roche Diagnostics)

. Labeled probes were purified from agarose gels and were quantified by PicoGreen reagent (Invitrogen). Sample Collection and DNA Extraction

L eaf samples were collected from greenhouse

- grown p lants for genomic DNA extrac tion

. Genomic DNA was extracted following a modified CTAB method. Briefly, leaf samples were individually ground in liquid nitrogen followed by addition of CTAB extraction buffer (~5:1 ratio milliliter CTAB extraction buffer: gram leaf tissu e) and RNase

- A (>10 μL) (Qiagen). After approximately 1 hour of incubation at ~65 °C with gentle shaking, samples were spun down and the supernatants were extracted with equal volume s of chloroform:octanol = 24:1 (Sigma). DNA was precipitat ed by mixing the supernatants with equal volume s of CTAB preci pitation buffer

. The precipitated DNA was diss olved in high salt TE buffer followed by preci pitation with isopropyl alcohol. The precipitated DNA was rinsed with 70% ethanol, air

- dried, and di ssol ved in appropriate volume of 1 × TE buffer (pH 8.0). The DNA was quantified with PicoGreen reagent (Invitrogen), and was visualized on an agarose gel to check for genomic DNA quality.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information DNA Digestion and Electrophoretic Separation of the DNA Fragments
G enomic DNA extracted from the soybean leaf tissue was digested with restriction enzy mes by combining approximately 10  g of ge nomic DNA with approximately 5

- 10 units of the selected restriction enzyme per  g of DNA in the corresponding reaction buffer. Ea ch sample was incubated at 37 o C overnight for digestion . The digested DNA samples were precipitated with Quick

- Precip (Edge BioSystems) and re

- suspended to achieve the desired volume for gel loading. The DNA samples and molecular size markers were then e lectrophoresed through 0.8% agarose gels with 1  TBE buffer at 3 5

- 65 V for 18

- 22 h r to achieve fragment separation. The gels were stained with ethidium bromide and the DNA was visualized under UV light. A photographic record was made for each stained gel

. Southern Transfer DNA fragments in t he agarose gels were depurinated, denatured, neutralized in situ , and transferred to nylon membrane s in 10  SSC buffer using a wicking system. After transfer to the membrane, the DNA was fixed to the membrane by cross linking through UV treatment. Hybridization Labeled probes were hybridized to the target DNA on the nylon membranes using the DIG Easy Hyb Solution according to manufacturer’s instructions (Roche Diagnostics). DIG

- labeled DNA molecular weight marker II and VII were used to determine the hybridizing fragment size on the Southern blots.
Detection
DIG

- labeled probes bound to the nylon m embranes after stringent wash were incubated with Alkaline Phosphatase (AP)

- conjugated anti

- Digoxigenin antibody for ~1 h r at room temperature. The anti

- DIG antibody specifically bound to the probes was then visualized using CDP

- Star Chemiluminescent Nucleic Acid Detection System (Roche Diagnostics). Blots were exposed to chemiluminescent film to detect the hybridizing frag ments and to visualize the molecular weight markers . The images were then scanned and stored. The number and size of all the detected band were documented for each digest and probe combination

. Once the data were recorded, membranes were rinsed with mill i

- Q water and then stripped of the bound probe in a solution containing 0.2 M NaOH and 1.0% SDS. The alkali

- based stripping procedure successfully removes the labeled probes from the membranes, allowing them to be re

hybridized with a different DNA probe.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Appendix 2
. Materials, Methods and Result s for Characterization of Cry1Ac Protein
Materials and Methods Test Substance/Test System The test substance was the Cry1Ac protein expressed in and extracted from grain of transgenic soybean event DAS

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- 2 : Source ID: YX10MX500029.0001 (T4 generation) . The seeds were planted, grown, and harvested under DAS protocol number 110000 . After harvest, the grain was frozen, lyophilized, ground, and stored at

- 20  C. The presence of Cry1Ac protein in the soybean grain was confirmed by a commercially available lateral flow assay kit from EnviroLogix Inc

. , as described below.
Control Substances The control substance used in this study was a non

- transgenic soybean grain grown from seed ( Glycine max cv Maverick )

. Grain of the Maverick soybean line ( Source ID: YX10MX030001.0002

– T4 generation) were planted, grown, harvested, and processed under the same conditions as the transgenic plants described above. The absence of Cry1Ac protein in the non

- transgenic soybean tissue was confirmed by a commercially available lateral flow assay kit as described below

. Recombinant Cry1Ac microbial protein, ( TSN102591, Lot #: 1757

- 66 ), has a molecular weight of ~ 130 kDa and a concentration of 144  g/mg

. The microbial preparation was produced and purified from recombinant Pseudomonas fluorescens at Dow AgroSciences and a n aliquot of the purified sample was sent to the Test Substance Coordinator located in Indianapolis , IN for dispensation. Reference Su bstances The commercially available (non

- GLP) reference substances used in this study are listed in the following table: Reference Substance Product Name Lot Number Assay Reference Mass Spectrometry Mass Standards Kit Mass Standards Kit for Calibration of AB SCIEX TOF/TOF Instruments A1068 Protein sequence analysis AB SCIEX

Soybean Trypsin Inhibitor (STI) A component of the GelCode glycoprotein staining kit MH161385 Glycosylation assay ThermoFisher

Horseradish Peroxidase (HRP) A component of the GelCode glycoprotein staining kit ND171686 Glycosylation assay ThermoFisher

Bovine Serum Albumin (BSA) Pre

- diluted BSA

protein assay standard set NA165380 & NE170914 SDS

- PAGE &

glycosylation assay ThermoFisher

Unstained Molecular Weight Markers Novex Sharp

unstained protein standards 1143231 SDS

- PAGE Invitrogen: Molecular Weight Markers of 260, 160, 110, 80, 60, 50, 40, 30, 20, 10, and 3.5 kDaUSDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Reference Substance Product Name Lot Number Assay Reference Prestained Molecular Weight Markers Novex Sharp

prestained protein standards 1095889, 1141762, & 1022458 SDS

- PAGE,

western blot & glycosylation assay Invitrogen: Molecular

Weight Markers of 260, 160, 110, 80, 60, 50, 40, 30, 20, 10, and 3.5 kDa
Lateral Flow Strip Assay The soybean grain tissues of the transgenic event DAS

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- 2 and non

- transgenic Maverick were planted, grown, and harvested under protocol 110000 in 2011. After harvest, the samples were shipped frozen, ground, and stored at

– 20  C until use. To confirm the presence/absence of the Cry1Ac protein in the ground grain, approximately 250

- mg samples of the grain were weighed out in 2

- mL microfuge tubes and tested by the lateral flow strip assay as described by Envirologix Inc. Briefly, t he soluble proteins we re extracted by adding 1.5 mL of EB2 extraction buffer and grinding i n a Geno

- Grinder for 3 minutes at 500 strokes per minute. The test strips were added to the tubes and incubated at room temperature in the samples for 10 minutes to develop. After the a ssay was complete, the strips were removed and allowed to air dry and the results were recorded.
SDS

- PAGE and Western Blot SDS

- PAGE and western blot analysis of the crude protein extracts prepared from DAS

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- 2 soybean grain and non

- transgenic Maverick soybean grain were performed with Bio

- Rad Criterion gels fitted in a Criterion Gel chamber with XT MES running buffer (Bio

- Rad). Extracts were prepared by Geno

- Grinding ~15 mg of the ground soybean grain in 1.5 mL PBS/Triton/ascorbic acid buffer for 3 min utes in a 2 mL micro

- centrifuge tube. The supernatants were clarified by briefly centrifuging the samples at >20,000×g (4°C), and 250  L of each extract was mixed with 250  L of Laemmli sample buffer (Bio

- Rad) containing freshly added 

- mercaptoethanol (Bio

- Rad) and heated for 5 minutes at ~95  C. After a brief centrifugation (2 min at 20,000 ×g), 40 μL of each supernatant was loaded directly on the gel. The reference standard, microbe

- derived Cry1Ac (TSN102591), and control standard, BSA (ThermoScienti fic), were diluted to an appropriate concentration and combined with Laemmli sample buffer containing 

- mercaptoethanol. The electrophoresis was conducted at a constant voltage of 150 V for ~60 minutes. After separation, the gel was cut in half and one h alf was stained with ThermoScientific GelCode Blue protein stain and scanned with a densitometer (GE Healthcare) to obtain a permanent record of the gel. The remaining half of the gel was electro

- blotted to a nitrocellulose membrane (Bio

- Rad) with a Crite rion transfer cell (Bio

- Rad) for 60 minutes under a constant voltage of 100V. The transfer buffer contained 20% methanol and Tris/glycine buffer from Bio

- Rad. After transfer, the membrane was probed with a Cry1Ac specific polyclonal rabbit antibody (α

- Cr y1Ac PAb, NB1434

- 63, 1.46 mg/mL) for 60 minutes (1:2500 dilution) at room temperature

. A 1:5000 dilution of conjugated goat anti

- rabbit IgG (H+L) with horseradish peroxidase (ThermoScientific) was used as the secondary antibody. GE Healthcare ECL chemilu minescent substrate was used for development and visualization of the immunoreactive protein bands. The membranes were exposed to detection film (ThermoScientific) for various time points and subsequently developed with an All

- Pro 100 Plus film developer. Purification of Soybean

- Derived Cry1Ac from Event DAS

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- 2 The Cry1Ac protein was extracted from the ground soybean grain with a modified RIPA buffer (phosphate buffered saline containing an additional 0.1 M NaCl, 12.5 mM EDTA, 1% Tween

- 20,USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information 0.1% SDS, a nd 0.5% sodium deoxycholate) by weighing ~25 grams of ground grain into a chilled Waring blender cup and adding ~175 mL of buffer and 150 μL of protease inhibitor cocktail mix. The tissue was blended on high speed four times in 20 second pulses and the
so lution was filtered through 2 layers of pre

- wetted miracloth (Calbiochem) and clarified by centrifugation at 38,000×g for 30 minutes. The supernatant was removed and filtered through 2 layers of miracloth and another 150 μL of protease inhibitor cocktail was added to the extract and held on ice. The Cry1Ac protein was purified from the supernatant by immuno

- precipitation using monoclonal antibodies (cell line #158E6, lot #: 200.687

- 3

- 5 (6 mg/mL) or cell line #158E7 (5.8 mg/ml)) cross

- linked to Thermo Scien tific’s Protein A/G Agarose resin at 1.0 μg of antibody per mL of resin. For each 15 mL of clarified supernatant, 100 μL (100  g of antibody) of coupled resin was added and allowed to incubate on a rotating mixer for 30 minutes at 4°C. The resin
was reco vered by centrifugation at 500×g for 5 minutes at 4°C and then resuspended in an additional 15 mL of extract for a total of 30 mL of extract applied to each resin preparation. The resin was then washed with 15 mL of extraction buffer for 15 minutes at 4o C, followed by two washes with 15 mL of wash buffer (PBS, 0.7 M NaCl, 25 mM EDTA, 25 mM galactose, 1% Tween 20). After the final wash, spin, and decant, the resin was transferred to a 1.5 mL Eppendorf tube with 1 mL of wash buffer and centrifuged for 2 mi nutes at 500×g at 4°C to pellet the resin. The supernatant was carefully aspirated and the pellet was washed an additional two times with 1 mL of PBS, 5 mM EDTA. The pelleted resin was resuspended in 1 mL of PBS, 5 mM EDTA and 500 μL of the slurry was re moved and centrifuged at 21000×g for 1 minute to pellet the resin. After the supernatant was removed, the resin was stored at

- 80°C for later use. From the remaining 500 μL of slurry, the resin was pelleted as described above and the bound proteins were eluted by incubating the resin for 5 minutes in 80 μL of 2% SDS at room temperature. The resin was pelleted with a brief centrifugation and 75 μL of supernatant was removed and the elution procedure was repeated for a total of 150 μL of eluted protein. T he extracted resin, eluted protein, and other fractions were all stored at

- 80°C for future analysis. Detection of Post

- Translational Glycosylation The immunoaffinity

- purified, soybean

- derived Cry1Ac protein was mixed with Laemmli sample buffer (37.5 μL + 12.5 μL of 4x LSB) and heated at 95°C for 10 minutes. The microbe

- derived Cry1Ac, soybean trypsin inhibitor, bovine serum albumin, and horseradish peroxidase were diluted with 2x LSB to the approximate concentration of the purified soybean

- derived Cry1Ac protein. After mixing the proteins with Laemmli sample buffer, the proteins were heated at ~95  C for 10 minutes and centrifuged at 20,000×g for 2 minutes to obtain a clarified supernatant. The resulting supernatants were applied directly to a Bio

- Rad Min i

- Protean TGX gel and electrophoresed at 100V for ~90 minutes. After electrophoresis, the gel was cut in half and one half was stained with GelCode Blue stain for total protein according to the manufacturers’ protocol. After the staining was complete, th e gel was scanned with a densitometer to obtain a permanent visual record of the gel. The remaining half of the gel was stained with a GelCode Glycoprotein Staining Kit (ThermoScientific) according to the manufacturers’ protocol to visualize the glycoprot eins. The procedure for glycoprotein staining is briefly described as follows: After electrophoresis, the gel was fixed in 50% methanol for 30 minutes and rinsed with 3% acetic acid. This was followed by an incubation period with the oxidation solution from the staining kit for 15 minutes. The gel was once again rinsed with 3% acetic acid and incubated with GelCode glycoprotein staining reagent for 15 minutes. Finally, the gel was immersed in theUSDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information reduction solution for 5 minutes, and rinsed with 3% ace tic acid. The glycoproteins (with a detection limit as low as 0.625 ng per band) were visualized as magenta bands on a light pink background. After the glycoprotein staining was complete, the gel was scanned with a GE Healthcare densitometer to obtain a permanent visual record of the gel.

MALDI

- TOF MS and LC/MS Peptide Mass Fingerprinting and Sequence Analysis of Soybean

and Microbe

- Derived Cry1Ac Sample preparation and deposition Tryptic (in

- gel digest), and Chymotryptic (in

- gel digest) peptides were p urified using Millipore ZipTip C18 as per manufacturer’s procedure. Purified peptides were eluted sequentially with aqueous 10%, 25%, 50%, and 100% ACN (supplemented with 0.1% TFA). The ZipTip C18 fractions were mixed with 4 μL of CHCA matrix (10 mg/mL CHC A in 50% ACN supplemented with 0.1% TFA), and 1 μL of the sample

- matrix mixture was deposited on the MALDI target and allowed to air dry.
MALDI

- TOF MS The sample preparations were analyzed directly by MALDI

- TOF mass spectrometry. All mass spectra were acquired on an AB Sciex 4800 MALDI

- TOF/TOF mass spectrometer (S/N AK011030605H). Mass calibration was performed with a mass standards kit for calibration of AB SCIEX TOF/TOF instruments. The plate wide calibration model was used for MS calibration.
MALDI

- T OF MS, E lectrospray Ionization

- Liquid Chromatography Mass Spectrometry ( ESI

LC/MS), glycoprotein analysis, and N

- terminal and internal protein sequence analysis of the microbe

- derived Cry1Ac (TSN102591) was conducted previously at Dow AgroSciences

. Results Lateral Flow Strip Assay The presence of the Cry1Ac protein in the ground grain of DAS

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- 2 soybean was confirmed using commercially prepared lateral flow strips from EnviroLogix Inc. The strips, capable of detecting one transgenic seed in 400, easily discriminated between transgenic and non

- transgenic tissue. The extracts prepared from DAS

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- 2 soybean tested positive for Cry1Ac protein and the non

- transgenic extracts of Maverick did not contain detectable immunoreactive protein ( Figure 40 ). This result was also confirmed by the western blot analysis using polyclonal antibodies specific to the Cry1Ac protein ( Figure 41 ).USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 40 . Lateral flow strip assay for the Cry1Ac protein expression in Event DAS

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- 2 and Maverick grain extracts. Western Blot Analysis of Transgenic Grain Extracts The microbe

- derived Cry1Ac protein showed a positive signal of the expected size (the full

length Cry1Ac protein is ~130 kDa) by polyclonal antibody western blot analysis ( Figure 41 ). This was also observed in the DAS

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- 2 transgenic soybean grain extract whereas the non

transgenic Maverick extract did not contain any immunoreactive proteins. In the Cry1Ac western blot anal ysis, some immunoreactive proteins of truncated Cry1Ac were observed in the microbe

- derived standard; however no alternate size Cry1Ac proteins (aggregates or degradation products) were observed in the transgenic samples ( Figure 41 ). These results add to the evidence that the protein expressed in soybean is not glycosylated or post

- translationally modified which would add to or subtract from the overall protein molecular weight.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 41 . Western blot analysis of microbe

- and soybean

- derived Cry1Ac proteins.

Lane Sample Amount Loaded M Novex unstained MW m arkers 10  L 1 (gel) Bovine serum albumin 1.3 μ g 1 (blot) Bovine s erum a lbumin 2.5 ng 2 (gel) Cry1Ac protein standard (TSN102591 ) 0.72  g 2 (blot) Cry1Ac protein standard (TSN102591 ) 1.9 ng 3 Maverick crude (non

- transgenic) grain extract 40  L 4 DAS

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- 2 crude (transgenic) grain extract 40  L

Purification of the Cry1Ac Protein from Transgenic Event DAS

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- 2 Grain Extracts Immunoaffinity precipitation was conducted on an aqueous extract of ~25 grams of ground DAS

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- 2 soybean grain. The protein that bound to the mono clonal antibody columns was eluted from the column and examined by SDS

- PAGE which demonstrated that the final concentrated fractions contained the intact Cry1Ac protein at an approximate molecular weight of ~130 k Da and fragments that contain the active core toxin ( Figure 42 ). Once isolated, the soybean

- derived Cry1Ac was compared with the microbe

- derived protein.
SDS

- PAGE Analysis of the Immuno

- purif ied Cry1Ac In the toxicology

- lot preparation of P. fluorescens

- produced Cry1Ac (TSN102591), the major protein bands, as visualized on Coomassie stained SDS

- PAGE gels, were approximately 130, 110, and 90 kDa. As expected, the corresponding soybean

- derived Cry1Ac protein was identical in size to the full

- length microbe

- expressed protein ( Figure 42 ). Predictably, the plant purified fractions contained a minor amount of impurities in addition to the full

- length and truncated Cry1Ac core protein. The co

- eluted proteins were likely retained on the column by weakUSDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information interactions with the column matrix or antibody leaching off of the column under the elution
con ditions. Other researchers have also reported the non

- specific adsorption of proteins, peptides, and amino acids on activated agarose immuno

- adsorbents ( Holroyde et al. , 1976 ; Kennedy and Barnes, 1983 ; Williams et al. , 2006 ) as well as antibody leaching from the column ( Goldberg et al. , 1991 )

.

Figure 42 . SDS

- PAGE analysis of immunoaffinity

- purified soybean

- derived Cry1Ac.

Lane Sample Amount Loaded M Novex prestained MW m arkers 10  L 1 Bovine serum albumin ~1 μ g 2 DAS

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- 2 crude grain extract 3  L 3 Cry1Ac protein standard (TSN102591 ) ~1  g 4 Soybean

- derived Cry1Ac (DAS

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- 2) 20  L 5 Soybean

- derived Cry1Ac (DAS

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- 2) 20  L 6 Soybean

- derived Cry1Ac (DAS

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- 2) 20  L 7 Soybean

- derived Cry1Ac (DAS

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- 2) 20  L

Detection of Glycosylation Potential protein glycosylation of soybean

- derived Cry1Ac was assessed with a GelCode Glycoprotein Staining Kit from ThermoScientific. The immunoaffinity

- purified Cry1Ac protein was electrophoresed simultaneously with a set of control and reference protein standards. A glycoprotein, horseradish peroxidas e, was loaded as a positive indicator for glycosylation, and
non
- glycoproteins, microbe

- derived Cry1Ac, soybean trypsin inhibitor, and bovine serum albumin, were employed as negative controls. The results showed that both the soybean

- and microbe

- derived Cry1Ac proteins had no detectable covalently linked carbohydrates ( Figure 43 ).USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 43 . SDS

- PAGE analysis of microbe

- and soybean

- derived Cry1Ac protein stained with GelCode Blue total protein and glycoprotein stains.

Lane Sample Amount Loaded M Novex prestained MW m arkers 10  L 1 Cry1Ac protein standard (TSN102591 ) ~1 μ g 2 Soybean

- derived Cry1Ac (DAS

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- 2) 20  L 3 Bovine serum albumin (

- control) 0.5  g 4 Horseradish peroxidase (+ control ) 0.5  g 5 Soybean trypsin inhibitor (

- control) 0.5  g

MALDI

- TOF MS and MS/MS Peptide Mass Fingerprinting and Sequence Analysis The Cry1Ac p rotein derived from DAS

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- 2 soybean tissue was separated by SDS

- PAGE ( Figure 42 ) and the respective Cry1Ac bands were excised and subjected to in

- gel digestion by trypsin and chymotrypsin . The resulting peptide mixture was analyzed by MALDI

- TOF MS and sequence verified by MS/MS to determine the peptide sequences . The masses of the detected peptides were compared with expected masses based on trypsin or chymotrypsin cleavage sites within the sequence of the soybean

- derived Cry1Ac protein. Figure 4 4 and Figure 45 illustrates the theoretical cleavage which was gene rated in silico using Protein Analysis Worksheet (PAWS) freeware from Proteometrics LLC. The Cry1Ac protein, once denatured, is readily digested by endoproteases to yield numerous peptide s that are able to be detected using mass spectrometry.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 4 4 . Theoretical cleavage of the Cry1Ac protein with trypsin generated in silico using Protein Analysis Worksheet (PAWS) from Proteometrics LLC.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 45
. Theoretical cleavage of the Cry1Ac protein with chymotrypsin generated in silico using Protein Analysis Worksheet (PAWS) from Proteometrics LLC.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information In the endoprotease digest s of the transgenic

- soybean

- derived Cry1Ac protein, the peptide sequence coverage from peptide mass fingerprint (PMF) data was extensive at 86.1 %. Of the 86.1% sequence coverage from PMF data, 83.2% was confirmed by tandem mass spectrometry sequencing. The detected peptide fragments covered nearly the entire protein sequence with only a few peptide fragment s undetected ( Figure 46

- Figure 48 ). This analysis confirmed the soybean

- derived protein amino acid sequence matched that of the microbe

- derived Cry1Ac protein near the N

- and C

- terminus as well as a major portion of the internal sequence. In the MS spectra, there were unid entified peptides detected in the enzyme digest preparations ( data not shown ). Many factors contribute to the formation of these unidentified peptides, such as over digestion (which results in non

- specific cleavage), self

- digestion products of trypsin and chymotrypsin, as well as random breakage of peptides during ionization. Unidentified peptides do not indicate the protein is different from the predicted amino acid sequence. The results of these analyses indicate that the amino acid sequence of the soybean

- derived Cry1Ac protein was equivalent to the P. fluorescens

- expressed protein previously characterized

.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 46 . Tryptic digest sequence coverage map for Cry1Ac (DAS

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- 2) by MALDI

TOF MS and MALDI TOF/TOF. Cys resi dues were alkylated with iodoacetamide. Sequence coverage was 67.9% with PMF data and 67.9% by tandem MS.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 46 (Cont )
. Tryptic digest sequence coverage map for Cry1Ac (DAS

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- 2) by MALDI

- TOF MS and MALD I TOF/TOF.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 47
. Chymotryptic digest sequence coverage map for Cry1Ac (DAS

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- 2) by MALDI

- TOF MS and MALDI TOF/TOF. Cys residues were alkylated with iodoacetamide. Sequence coverage was 53.5% with PMF data and 49.0% by tandem MS.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 47 (Cont )
. Chymotryptic digest sequence coverage map for Cry1Ac (DAS

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- 2) by MALDI

- TOF MS and MALDI TOF/TOF.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 48
. Overall sequence coverage of trypsin and chymotrypsin digests for Cry1Ac (DAS

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- 2) by MALDI

- TOF MS and MALDI TOF/TOF. Overall sequence coverage was 86.1% with PMF data and 83.2% by tandem MS.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 48 (Cont )
. Overall sequence coverage of trypsin and chymotrypsin digests for Cry1Ac (DAS

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- 2) by MALDI

- TOF MS and MALDI TOF/TOF.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 48 (Cont)
. Overall sequence coverage of trypsin and chymotrypsin digests for Cr y1Ac (DAS

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- 2) by MALDI

- TOF MS and MALDI TOF/TOF.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Conclusions It was demonstrated that the biochemical identity of P. fluorescens

- produced Cry1Ac protein was equivalent to the protein purified from grain of soybean event DAS

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- 2. Both the soybean

- and microbe

- derived Cry1Ac proteins had an apparent molecular weight of ~130 kDa and were immunoreactive to Cry1Ac protein

- specific antibodies in both lateral flow strip and W estern blot assays. The amino acid sequences were confirmed by enzymatic peptide mass fingerprinting by MALDI

- TOF MS and verified by MS/MS. In addition, the lack of glycosylation of the soybean

- derived Cry1Ac protein provided additional evidence that the Cry1Ac protein produced by P. fluorescens and transgenic soybean were essentially equivalent molecules.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Appendix 3
. Materials, Methods and Results for Characterization of Cry1F Protein
Materials and Methods
Test Substance/Test System The test substance was the Cry1F protein expressed and extracted from grain of transgenic soybean event DAS

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- 2 : Source ID: YX10MX500029.0001 (T4 generation). The seeds were planted, grown, and harvested under DAS protocol number 110000 . Afte r harvest, the grain was frozen, lyophilized, ground, and stored at

- 20  C. The presence of Cry1F protein in the soybean grain was confirmed by a commercially available lateral flow assay kit from EnviroLogix Inc

. , as described below.
Control Substances Th e control substance used in this study was a non

- transgenic soybean grain grown from seed ( Glycine max cv Maverick )

. Grain of the Maverick soybean line (Source ID: YX10MX030001.0002) were planted, grown, harvested, and processed under the same conditions as the transgenic plants described above. The absence of Cry1F protein in the non

transgenic soybean tissue was confirmed by a commercially available lateral flow assay kit as describ ed below.
Recombinant Cry1F microbial protein, ( TSN103748, Lot #: 020404 ), has a molecular weight of

~ 130 kDa and a concentration of 233  g/mg

. The microbial preparation was produced and purified from recombinant Pseudomonas fluorescens at Dow AgroSciences and the purified Cry1F protein was sent to the Test Substance Coordinator located in Indianapolis

. Reference Substances The commercially available (non

- GLP) reference substances used in this study are listed in the following table: Reference S ubstance Product Name Lot Number Assay Reference Mass Spectrometry Mass Standards Kit Mass Standards Kit for Calibration of AB SCIEX TOF/TOF Instruments A1068 Protein sequence analysis AB SCIEX

Soybean Trypsin Inhibitor (STI) A component of the GelCode glycoprotein staining kit MH161385 Glycosylation assay ThermoFisher

Horseradish Peroxidase (HRP) A component of the GelCode glycoprotein staining kit ND171686 Glycosylation assay ThermoFisher

Bovine Serum Albumin (BSA) Pre

- diluted BSA protein assay stand ard set NA165380 and NE170914 SDS

- PAGE

& glycosylation assay ThermoFisher

Unstained Molecular Weight Markers Novex Sharp unstained protein standards 1143231 SDS

- PAGE Invitrogen: Molecular Weight Markers of 260, 160, 110, 80, 60, 50, 40, 30, 20, 10, and 3.5 kDaUSDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Reference S ubstance Product Name Lot Number Assay Reference Prestained Molecular Weight Markers Novex Sharp prestained protein standards 1095889 & 1022458 SDS

- PAGE,

western blot & glycosylation assay Invitrogen: Molecular

Weight Markers of 260, 160, 110, 80, 60, 50, 40, 30, 20, 10, and 3.5 kDa

Lateral Flow Test Strip Assay The soybean grain tissues of the transgenic event and non

- transgenic Maverick were planted, grown and harvested under protocol 110000 in 2011. The samples after harvest were shipped frozen, ground, and stored at

- 20  C until use. To confirm the presence/absence of the Cry1F protein in the ground grain, approximately 250

- mg samples of the grain were weighed out in 2

- mL micro

- centrifuge tubes and tested by the lateral flow test strip assay as described by Envirologix In c. Briefly, t he soluble proteins were extracted by adding 1.5 mL of EB2 extraction buffer and grinding i n a Geno

- Grinder for 3 minutes at 500 strokes per minute at room temperature. The test strips were added to the tubes and developed for 10 minutes. A fter the assay was complete, the strips were removed and allowed to air dry and the results were recorded.
SDS

- PAGE and Western Blot SDS

- PAGE and western blot analysis of the crude protein extracts prepared from the transgenic DAS

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- 2 soybean grain and non
- transgenic Maverick soybean grain were performed with Bio

- Rad Criterion gels fitted in a Criterion Gel chamber with XT MES running buffer (Bio

- Rad). Extracts were prepared by geno

- grinding ~15 mg of the ground soybean grain in 1.5 mL PBS/Triton/ascor bic acid buffer for 3 minutes in a 2 mL micro

- centrifuge tube. The supernatants were clarified by briefly centrifuging the samples at >20,000×g (4°C), and 250  L of each extract was mixed with 250  L of Laemmli sample buffer (Bio

- Rad) containing freshly a dded 

- mercaptoethanol (Bio

- Rad) and heated for 5 minutes at ~95  C. After a brief centrifugation (2 min at 20,000 ×g), 40 μL of each supernatant was loaded directly on the gel. The reference standard, microbe

- derived Cry1F (TSN103748), and control standa rd, BSA (ThermoScientific), were diluted to an appropriate concentration and combined with Laemmli sample buffer containing 

- mercaptoethanol. The electrophoresis was conducted at a constant voltage of 150 V for ~60 minutes. After separation, the gel was cut in half and one half was stained with ThermoScientific GelCode Blue protein stain and scanned with a densitometer (GE Healthcare) to obtain a permanent record of the gel. The remaining half of the gel was electro

- blotted to a nitrocellulose membrane (Bio

- Rad) with a Criterion transfer cell (Bio

- Rad) for 60 minutes under a constant voltage of 100 volts. The transfer buffer contained 20% methanol and Tris/glycine buffer from Bio

- Rad. After transfer, the membrane was probed with a Cry1F specific polycl onal rabbit antibody (α

- Cry1F PAb, Lot#: 200.310

- 4

- 63, 6.48 mg/mL) for 60 minutes (1:5000 dilution) at room temperature

. A 1:5000 dilution of conjugated goat anti

- rabbit IgG (H+L) with horseradish peroxidase (ThermoScientific) was used as the secondary an tibody. GE Healthcare ECL chemiluminescent substrate was used for development and visualization of the immunoreactive protein bands. The membranes were exposed to detection film (ThermoScientific) for various time points and subsequently developed with a n All

- Pro 100 Plus film developer.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Purification of Soybean

- Derived Cry1F from Event DAS

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- 2 The Cry1F protein was extracted from the ground soybean grain with a modified RIPA buffer [phosphate buffered saline containing an additional 0.1 M NaCl, 12.5 mM EDTA, 1% Tween

- 20, 0.1% SDS, and 0.5% sodium deoxycholate] by weighing ~25 grams of ground grain into a chilled Waring blender cup and adding ~175 mL of buffer and 150 μL of a broad spectrum protease inhibitor cocktail mix. The tissue was blended on hi gh speed four times in 20 second pulses and the solution was filtered through 2 layers of pre

- wetted miracloth (Calbiochem) and clarified by centrifugation at 38,000×g for 30 minutes. The supernatant was removed and filtered through 2 layers of miracloth and another 150 μL of protease inhibitor cocktail was added to the extract and held on ice. The Cry1F protein was purified from the supernatant by immuno

- precipitation using monoclonal antibodies (cell lines #96A22.2 and 96A19) cross

- linked to Thermo Scien tific’s Protein A/G Agarose resin at 1.0 μg of antibody per mL of resin. For each 15 mL of clarified supernatant, 100 μL (100  g of antibody) of coupled resin was added and allowed to incubate on a rotating mixer for 30 minutes at 4°C. The resin was reco vered by centrifugation at 500×g for 5 minutes at 4°C and then resuspended in an additional 15 mL of extract for a total of 30 mL of extract applied to each resin preparation. The resin was then washed with 15 mL of extraction buffer for 15 minutes at 4°C , followed by two washes with 15 mL of wash buffer (PBS, 0.7 M NaCl, 25 mM EDTA, 25 mM galactose, 1% Tween 20). After the final wash, spin, and decant, the resin was transferred to a 1.5 mL Eppendorf tube with 1 mL of wash buffer and centrifuged for 2 min utes at 500×g at 4°C to pellet the resin. The supernatant was carefully aspirated and the pellet was washed an additional two times with 1 mL of PBS, 5 mM EDTA. The pelleted resin was resuspended in 1 mL of PBS, 5 mM EDTA and 500 μL of the slurry was rem oved and subjected to centrifugation at 21,000×g for 1 minute to pellet the resin. After the supernatant was removed the resin was stored at

- 80°C. From the remaining 500 μL of slurry, the resin was pelleted as described above and the bound proteins were eluted by incubating the resin for 5 minutes in 80 μL of 2% SDS at room temperature. The resin was pelleted with a brief centrifugation and 75 μL of supernatant was removed and the elution procedure was repeated for a total of 150 μL of eluted protein. The extracted resin, eluted protein and other fractions were all stored at

- 80°C. Detection of Post

- Translational Glycosylation The immunoaffinity

- purified, soybean

- derived Cry1F protein was mixed with Laemmli sample buffer (37.5 μL + 12.5 μL of 4x LSB) an d heated at 95 °C for 10 minutes. The microbe

- derived Cry1F, soybean trypsin inhibitor, bovine serum albumin, and horseradish peroxidase were diluted with 2x LSB to the approximate concentration of the purified soybean

- derived Cry1F protein. After mixing the proteins with Laemmli sample buffer, the proteins were heated at ~95

 C for 10 minutes and centrifuged at 20000×g for 2 minutes to obtain a clarified supernatant. The resulting supernatants were applied directly to a Bio

- Rad Mini

- Protean TGX gel and electrophoresed at 100V for ~90 minutes. After electrophoresis, the gel was cut in half and one half was stained with GelCode Blue stain for total protein according to the manufacturers’ protocol. After the staining was complete, the gel was scanned with a densitometer to obtain a permanent visual record of the gel. The remaining half of the gel was stained with a GelCode Glycoprotein Staining Kit (ThermoScientific) according to the manufacturers’ protocol to visualize the glycoproteins. The procedure f or glycoprotein staining is briefly described as follows: After electrophoresis, the gel was fixed in 50% methanol for 30 minutes and rinsedUSDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information with 3% acetic acid. This was followed by an incubation period with the oxidation solution from the staining kit for 15 minutes. The gel was once again rinsed with 3% acetic acid and incubated with GelCode glycoprotein staining reagent for 15 minutes. Finally, the gel was immersed in the reduction solution for 5 minutes, and rinsed with 3% acetic acid. The glycopr oteins (with a detection limit as low as 0.625 ng per band) were visualized as magenta bands on a light pink background. After the glycoprotein staining was complete, the gel was scanned with a GE Healthcare densitometer to obtain a permanent visual recor d of the gel.

MALDI

- TOF MS and LC/MS Peptide Mass Fingerprinting and Sequence Analysis of Soybean

and Microbe

- Derived Cry1F
Sa mple preparation and deposition Tryptic (in

- gel digest), and Chymotryptic (in

- gel digest) peptides were purified using Millipore ZipTip C18 as per manufacturer’s procedure. Purified peptides were eluted sequentially with aqueous 10%, 25%, 50%, and 100% ACN (supplemented with 0.1% TFA). The ZipTip C18 fractions were mixed with 4 μL of CHCA matrix (10 mg/mL CHCA in 50% ACN supplemente d with 0.1% TFA), and 1 μL of the sample

- matrix mixture was deposited on the MALDI target and allowed to air dry.
MALDI

- TOF MS The sample preparations were analyzed directly by MALDI

- TOF mass spectrometry. All mass spectra were acquired on an AB Sciex 480 0 MALDI

- TOF/TOF mass spectrometer (S/N AK011030605H). Mass calibration was performed with a mass standards kit for calibration of AB SCIEX TOF/TOF instruments. The plate wide calibration model was used for MS calibration. MALDI

- TOF MS, E lectrospray Ionizat ion

- Liquid Chromatography Mass Spectrometry ( ESI

LC/MS), glycoprotein analysis, and N

- terminal and internal protein sequence analysis of the microbe

- derived Cry1F (TSN103748) was conducted previously by ( Gao et al. , 2006 )

. Results
Lateral Flow Test Stri

p Assay The presence of the Cry1F protein in the ground grain of event DAS

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- 2 was confirmed using commercially prepared lateral flow test strips from EnviroLogix Inc. The strips, capable of detecting one transgenic seed in 400, easily discriminated b etween transgenic and non

- transgenic tissue. The transgenic extracts were positive for Cry1F protein and the non

- transgenic extracts of Maverick did not contain detectable immunoreactive protein ( Figure 49 ). This result was also confirmed by the western blot analysis using polyclonal antibodies specific to the Cry1F protein ( Figure 50 ).USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 49 . Lateral flow strip assay for the Cry1F protein expression in Event DAS

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- 2 and Maverick grain extracts. Western Blot Analysis of Transgenic Grain Extracts The microbe

- derived Cry1F protein showed a positive signal of the expected size (the full

- length Cry1F protein is ~130 kDa) by polyclonal antibody western blot analysis ( Figure 50 ). This was also observed in the DAS

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- 2 transgenic soybean grain extract whereas the non

- transgenic Maverick extracts did not contain any immunoreactive proteins. In the Cry1F western blot analysis, some immunoreactive proteins of truncated Cry1F were observed in the microbe

der ived standard; however no alternate size Cry1F proteins (aggregates or degradation products) were observed in the transgenic samples ( Figure 50 ). These results add to the evidence that the protein expressed in soybean is not glycosylated or post

- translationally modified which would add to or subtract from the overall protein molecular weight.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 50 . Western blot analysis of microbe

- and soybean

- derived Cry1F proteins.

Lane Sample Amount Loaded M Novex unstained MW m arkers 10  L 1 (gel) Bovine serum albumin 1.3 μ g 1 (blot) Bovine serum albumin 2.5 ng 2 (gel) Cry1F protein standard ( TSN103748 ) 0.47  g 2 (blot) Cry1F protein standard ( TSN103748 ) 1.2 ng 3 Maverick crude (non

- transgenic) grain extract 40  L 4 DAS

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- 2 crude (transgenic) grain extract 40  L

Purification of the Cry1F Protein from Transgenic Event DAS

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- 2 Grain Extracts Immunoaffinity precipitation was conducted on an aqueous extract of ~25 grams of ground DAS

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- 2 transgenic grain. The protein that bound to the monoclonal antibody columns was examined by SDS

- PAGE which demonstrated that the final concentrated fractio ns contained the intact Cry1F protein at an approximate molecular weight of ~130 k Da and fragments that contain the active core toxin ( Figure 51 ). Once isolated, the soybean

- derived Cry1F was then compared with the microbe

- derived protein.
SDS

- PAGE Analysis of the Immuno

- purified Cry1F In the toxicology

- lot preparation of P. fluorescens

- produced Cry1F (TSN103748), the major protein bands, as visualized on Coomassie st ained SDS

- PAGE gels, were approximately 130, 110, and 90 kDa. As expected, the corresponding soybean

- derived Cry1F protein was identical in size to the full

- length microbe

- expressed protein s ( Figure 51 ). Predictably, the plant purified fractions contained a minor amount of impurities in addition to the full

- length and truncated Cry1F core protein. The co

- eluted proteins were likely retained on the column by weakUSDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information int eractions with the column matrix or antibody leaching off of the column under the elution conditions. Other researchers have also reported the non

- specific adsorption of proteins, peptides, and amino acids on activated agarose immunoadsorbents ( Holroyde et al. , 1976 ; Kennedy and Barnes, 1983 ; Williams et al. , 2006 ) as well as antibody leaching from the column ( Goldberg et al. , 1991 )

.

Figure 51 . SDS

- PAGE analysis of immunoaffinity

- purified soybean

- derived Cry1F.

Lane Sample Amount Loaded M Novex prestained MW m arkers 10  L 1 Bovine serum albumin ~1 μ g 2 DAS

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- 2 crude grain extract 3  L 3 Cry1F protein standard ( TSN103748 ) 0.25  g 4 Soybean

- derived Cry1F (DAS

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- 2) 20  L 5 Soybean

- derived Cry1F (DAS

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- 2) 20  L 6 Soybean

- derived Cry1F (DAS

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- 2) 20  L 7 Soybean

- derived Cry1F (DAS

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- 2) 20  L

Detection of Glycosylation Potential protein glycosylation of soybean

- derived Cry1F was assessed with a GelCode Glycoprotein Staining Kit from ThermoScientific. The immunoaffinity

- purified Cry1F protein was electrophoresed simultaneously with a set of control and reference protein standards. A glycoprotein, horseradish peroxidase, was loaded as a positive indicator for glycosylation, and
non
- glycoproteins, microbe

- derived Cry1F, soybean trypsin inhibitor, and bovine serum albumin, were employed as n egative controls. The results showed that both the soybean

- and microbe

- derived Cry1F proteins had no detectable covalently linked carbohydrates ( Figu re 52 ).USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figu re 52 . SDS

- PAGE analysis of microbe

- and soybean

- derived Cry1F protein stained with GelCode Blue total protein and glycoprotein stains.

Lane Sample Amount Loaded M Novex prestained MW m arkers 10  L 1 Cry1F protein standard (TSN103748 ) ~1 μ g 2 Soybean

- derived Cry1F (DAS

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- 2) 20  L 3 Bovine serum albumin (

- control) 0.5  g 4 Horseradish peroxidase (+ control ) 0.5  g 5 Soybean trypsin inhibitor (

- control) 0.5  g

MALDI

- TOF MS and MS/MS Peptide Mass Fingerprinting and Sequence Analysis The Cry1F protein derived from DAS

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- 2 soybean t issue was separated by SDS

- PAGE ( Figure 51 ) and the respective bands were excised and subjected to in

- gel digestion by trypsin and chymotrypsin . The resulting peptide mixture was analyzed by MALDI

- TOF MS and sequence verified by MS/M S to determine the peptide sequences . The masses of the detected peptides were compared with the expected masses based on trypsin or chymotrypsin cleavage sites in the sequence of the soybean

- derived Cry1F protein. Figure 53 and Figure 54 illustrate the theoretical cleavage of the Cry1F protein which was generated in silico usin g Protein Analysis Worksheet (PAWS) freeware from Proteometrics LLC. The Cry1F protein, once denatured, is readily digested by endoproteases to yield numerous peptide s that are able to be detected using mass spectrometry

.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 53 . Theoretical cleavage of the Cry1F protein with trypsin generated in silico using Protein Analysis Worksheet (PAWS) from Proteometrics LLC.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 54
. Theoretical cleavage of the Cry1F protein with chymotrypsin generated in silico using Protein Analysis Worksheet (PAWS) from Proteometrics LLC.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information In the endoprotease digest s of the transgenic

- soybean

- derived Cry1F protein, the peptide sequence coverage fro m peptide mass fingerprint (PMF) data was extensive at 81.7%. Of the 81.7% sequence coverage from PMF data, all peptide sequences were confirmed by tandem mass spectrometry sequencing. The detected peptide fragments covered nearly the entire protein sequ ence with only a few peptide fragment s undetected ( Figure 55 , Figure 56 and Figure 57 ). This analysis confirmed the soybean

- derived protein amino acid sequence matched that of the microbe

- derived Cry1F protein near the N

- and C

- terminus as w ell as a major portion of the internal sequence ( Gao et al. , 2006 ) . In the MS spectra, there were unidentified peptides detected in the enzyme digest preparations ( data not shown ). Many factors contribute to the formation of these unidentified peptides, such as over digestion (which results in non

- specific cleavage), self

- digestion products of trypsin and chymotrypsin, as well as random breakage of peptides during ionization. Unid entified peptides do not indicate the protein is different from the predicted amino acid sequence. The results of these analyses indicate that the amino acid sequence of the soybean

- derived Cry1F protein was equivalent to the P. fluorescens

- expressed prot ein previously characterized ( Gao et al. , 2006 )

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Contains No Confidential Business Information Figure 55 . Tryptic digest sequence coverage map for Cry1F (DAS

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- 2) by MALDI

TOF MS and MALDI

- TOF/ TOF. Cys residues were alkylated with iodoacetamide. Sequence coverage was 65.1% with PMF data and 65.1% by tandem MS.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 55 ( Cont )
. Tryptic digest sequence coverage map for Cry1F (DAS

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- 2) by MALDI

- TOF MS and MALDI

- TOF/ TOF.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 56 . Chymotryptic digest sequence coverage map for Cry1F (DAS

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- 2) by MALDI

- TOF MS and MALDI TOF/TOF. Cys residues were alkylated with iodoacetamide. Sequence coverage was 53.8% with PMF data and 52.4% by tandem MS.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 56 ( Cont )
. Chymotryptic digest sequence coverage map for Cry1F (DAS

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- 2) by MALDI

- TOF MS and MALDI TOF/TOF.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 57
. Overall sequence coverage of trypsin and chymotrypsin digests for Cry1F (DAS

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- 2) by MALDI

- TOF MS and MALDI TOF/TOF. Overall sequence coverage was 81.7% with PMF data and 81.7% by tandem MS.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 57 ( Cont )
. Overall sequence coverage of trypsin and chymotrypsin digests for Cry1F (DAS

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- 2) by MALDI

- TOF MS and MALDI TOF/TOF.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Figure 57 ( Cont )
. Overall sequence coverage of trypsin and chymotrypsin digests for Cry1F (DAS

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- 2) by MALDI

- TOF MS and MALDI TOF/TOF.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Conclusions
It was demonstrated that the biochemical identit y of P. fluorescens

- produced Cry1F protein was equivalent to the protein purified from grain of soybean event DAS

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- 2. Both the soybean

and microbe

- derived Cry1F proteins had an apparent molecular weight of ~130 kDa and were immunoreactive to Cry1F protein

- specific antibodies in both lateral flow strip and western blot assays. The amino acid sequences were confirmed by enzymatic peptide mass fingerprinting by MALDI

- TOF MS and verified by MS/MS. In addition, the lack of glycosylation of the soybean

derived Cry1F protein provided additional evidence that the Cry1F protein produced by P. fluorescens and transgenic soybean were essentially equivalent.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Appendix 4
. Materials, Methods and Result s for Characterization of PAT Protein
Materials and Methods Test Substance/Test System The test substance was the PAT protein expressed and extracted from tissues grown from the T5 seeds of transgenic soybean event DAS

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- 2 : Source ID: YTR11ETR760037 (T SN303074)

. The seeds were planted in the greenhouise and 3 to 7 week old leaf tissue was harvested, frozen, lyophilized, ground, stored at

- 20°C to vent the dry ice, and stored at

- 80  C. The presence of PAT protein in the soybean tissue was confirmed by a commercially available lateral flow assay kit from EnviroLogix Inc

. , as described below.
Control Substances The control substance used in this study was a non

- transgenic soybean plant extract ( Glycine
max cv Maverick )

. Seeds of the Maverick soybean line (Source ID: YX11AX090001) were planted, grown, harvested and processed under the same conditions as the transgenic plants described above. The absence of PAT protein in the non

- transgenic soybean tissue was confirmed b y a commercially available lateral flow assay kit as described below. Recombinant PAT microbial protein, ( TSN031116

- 0001, Lot #: 55238

- 1A) , has a molecular weight of ~21 kDa and a concentration of 810  g/m L

. An enzymatically active microbial preparation was produced in recombinant E. coli and b ased on confirmation of the gene sequence in the E. coli expression system ( Madduri and Sno dderley, 2007 ) , t he E

. coli

- derived PAT protein sequence is identical to the proteins expressed in deregulated transgenic corn, cotton, soybean, canola, event DAS

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- 2, and the native organism S. viridochromogenes ( Wohlleben et al. , 1988 ; AgrEvo, 1994 ; Northrup King, 1995 ; AgrEvo, 1996 , 1997 ; Dow AgroSciences, 2003c , 2003b , 2003a ) . The DAS

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- 2 and E. coli

- derived PAT protein sequences are also identical to the PAT protein sequence described in the OECD consensus do cument ( OECD, 1999 ) and in other publications ( Herouet et al. , 2005 )

. Reference Substances The commercially available (non

- GLP) reference substances used in this study ar e listed in the following table: Reference Substance Product Name Lot Number Assay Reference Prestained Molecular Weight Markers Novex Sharp prestained protein standards 1095889 SDS

- PAGE and Western blot Invitrogen: Molecular

Weight Markers of 260, 160, 110, 80, 60, 50, 40, 30, 20, 15, 10 and 3.5 kDa Unstained Molecular Weight Markers Novex Sharp protein standards 1030454 SDS

- PAGE Invitrogen: Molecular Weight Markers of 260, 160, 110, 80, 60, 50, 40, 30, 20, 15, 10 and 3.5 kDaUSDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Lateral Flow Strip Assay The soybean leaf tissues of the transgenic and non

- transgenic events were harvested fresh as described above and were frozen, lyophilized, ground, and stored at approximately

– 80  C until use. To confirm the presence/absence of the PAT protein in the pooled tissues, approximately 16 mg of the lyophilized tissues (Event DAS

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- 2 and Maverick) were weighed into 2.0

- mL microfuge tubes and tested as described by EnviroLogix Inc. Briefly, the soluble proteins were extracted by adding 2 metal beads to t he extraction buffer (0.25 mL EB2 buffer supplied by the manufacturer) and grinding in a Spex Geno

- Grinder for 3 minutes at 500 strokes per minute. The samples were subjected to centrifugation for 5 minutes at 20,000×g and the resulting supernatants were transferred to a fresh tube and the strips were developed according to the manufacturer’s instructions.

SDS

- PAGE and Western Blot SDS

- PAGE analysis of the transgenic ( DAS

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- 2 ) and non

- transgenic Maverick soybean extracts was performed with Bio

- Rad Crit erion gels fitted in a Criterion Cell gel module with MES running buffer. Extracts were prepared by grinding ~43 mg of tissue for 3 minutes in a Geno

- Grinder with steel ball bearings in ~ 1 mL of PBST based buffer ( Table 25 ). The supernatants were clarified by centrifuging for 5 minutes at 20,000×g, and then 120  L of each extract was mixed with 30  L of 5x Laemmli sample buffer (LSB, 2% SDS, 50 mM Tris pH 6.8, 0.2 mg/mL bromophenol blue, 50% (w/w) glycerol containing 10% freshly added 2

- mercaptoethanol). Samples were heated for 5 minutes at ~95  C, and after a brief centrifugation, 20 μL of the supernatant was loaded directly on the gel. The reference standard, mic robe

derived PAT (TSN031116

- 0001) was diluted with Bio

- Rad 2x LSB containing 5% β

mercaptoethanol and processed as described earlier. The electrophoresis was conducted at a constant voltage of 150 V for ~60 minutes using MES running buffer from Bio

- Rad. After separation, the gel was cut in half and one half was stained with Thermo Scientific GelCode Blue protein stain and scanned with a densitometer to obtain a permanent record of the image. The remaining half of the gel was electro

- blotted to a nitrocel lulose membrane from Bio

- Rad with a Criterion trans

- blot electrophoretic transfer cell for ~60 minutes under a constant voltage of 100 volts. The transfer buffer contained 20% methanol and Tris/glycine buffer from Bio

- Rad. After transfer, the membrane wa s probed with a PAT specific polyclonal rabbit antibody (Lot #: D2976

- 27, 1.1 mg/mL). A conjugate of goat anti

- rabbit IgG (H+L) and horseradish peroxidase from Pierce was used as the secondary (detection) antibody. Pierce ECL chemiluminescent substrate w as used for development and visualization of the immunoreactive protein bands. The membranes were exposed to Thermo Scientific CL

- XPosure detection film for various time points and subsequently developed with a Radiation Services film developer.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information

Table 25 . Soybean

- d erived PAT e xtraction b uffer c omposition
. Ingredient Amount Phosphate Buffered Saline with 0.5% Tween a 20, pH 7.4 5.924 mL

0.5 M EDTA 60  L Protease inhibitor cocktail 6.0  L 

- mercaptoethanol 10  L Note : the extraction buffer was prepared immediately before use.
Results Lateral Flow Strip Assay The presence of the PAT protein in the pooled T5 leaf tissue of DAS

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- 2 was confirmed using a commercially prepared lateral flow test strip from EnviroLogix Inc. T he assay easily discriminated between transgenic and non

- transgenic plants as the non

- transgenic extracts of Maverick did not contain detectable amounts of immunoreactive protein ( Figure 58 ).

Figure 58
. Lateral flow strip assay for PAT protein expression in Event DAS

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- 2 and Maverick leaf extracts. * Note: Event DAS

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- 2 is positive for the PAT protein
SDS

- PAGE and Western Blot Analysis In the toxicology

- lot preparation of E. coli

- produced PAT protein (TSN031116

- 0001), the major protein band, as visualized on Coomassie stained SDS

- PAGE gels, was approximately 21 kDa ( Figure 59 ). As expected, the corresponding soybean

- derived PAT protein was visualized by immunospecific polyclonal antibodies at an identical size to the microbe

- expressed proteins

. In the PAT Western blot analysis, no immunoreactive proteins, consistent with the PAT protein,USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information were observed in the control Maverick extract; however a non

- specific band was detected at ~40 kDa and was detected in both the transgenic and non

- transgenic extracts. In additio n the polyclonal antibody did detect a small amount of a protein dimer in the microbe

- derived PAT protein preparation. These results add to the evidence that the PAT protein expressed in soybean is not post

- translationally modified (the PAT enzyme it does not contain N

- glycosylation sites ( Herouet et al. , 2005 ) or processed which would have added to or subtracted from the overall protein molecular weight.

Figure 59
. SDS

- PAGE an d Western blot analysis of microbe

- derived PAT, transgenic soybean Event DAS

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- 2, and non

- transgenic Maverick extracts.

Lane Sample Amount Loaded M Novex unstained MW Markers 10  L 1 PAT Protein Standard (TSN031116

- 0001) ~1 μ g 2 Transgenic Soybean

- Event DAS

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- 2 20  L 3 Non

- transgenic Soybean

– Maverick 20  L 4 PAT Protein Standard (TSN031116

- 0001) ~5 ng 5 Transgenic Soybean

- Event DAS

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- 2 20  L 6 Non

- transgenic Soybean

– Maverick 20  L M Novex Presta ined MW Markers* 10  L *Note: The molecular weight markers were manually transferred to the film after development.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Conclusion The results of this study demonstrated that both the transgenic soybean

- plant extract and the microbe

- derived PAT toxicological lot contained the intact, full

- length PAT protein. This was confirmed by SDS

- PAGE molecular

- weight approximation, Western blot analysis, and immunoreactivity using a commercially available lateral flow strip assay. Together, these biochemical tests indicate that the plant

- and microbe

- derived PAT proteins are substantially equivalent and consistent w ith the published data on PAT.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Appendix 5
. Materials and Methods for Cry1Ac, Cry1F and PAT Protein Expression Analysis Experimental Design This study used the same plots that were used for agronomic performance and nutrient composition studies. The exper im ental design included ten field sites located in Iowa (2 sites), Illinois (2 sites), Indiana, Missouri (2 sites), Nebraska (2 sites), and Pennsylvania. Each trial site included DAS

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- 2 soybean , the non

- transgenic near

- isogenic control (Maverick), and three
non
- transgenic reference lines. At each of the ten sites, all entries were arranged in a randomized complete block design with four blocks. Across all sites, e ach control and DAS

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- 2 was represented by a total of 40 plots (10 sites, 4 replicate plots per entry at each site). Three of the six reference lines were included at each site by randomizing across sites in a balanced incomplete

- block design. Each of the six reference lines was assigned to five sites; therefore, each reference line was represented by a total of 20 plots across sites (5 sites per reference line, 4 replicate plots per entry at each site). At each site, four replicate plots of each entry were established, with each plot consisting of four 25 ft (7.62 m) rows. Soybean seeds were planted at a seeding rate of approximately 125 seeds per row with seed spacing within each row of approximately 2.4 inches (6 cm). Each soybean plot was bordered by two rows of a non

transgenic soybean cultivar of similar maturity. The entire trial site was surrounded by a minimum of four rows (10 ft or 3.0 m) of a non

- transgenic soybean cultivar of similar maturity. Appropriate insect, weed, and disease control practices were applied to produce an agronomically acceptable crop. Sample Collect ion Plant tissue samples were collected as described below and shipped to Dow AgroSciences Regulatory Science s and Government Affairs laboratories where they were maintained frozen until use. Samples of soybean tissues were prepared for expression analysi s by coarse grinding, lyophilizing and/or fine

- grinding with a Geno/Grinder (Certiprep, Metuchen, New Jersey ). Leaf (V5 and V10

- 12) One leaf sample per plot, each sample containing 8 trifoliate set s of leaves collected from separate plants, was collected f or each test and control entry. Each leaf sample was the youngest set of fully expanded trifoliate leaves. Root (R3) One root sample (representing 3 plants) per plot was collected for each test and control entry at the R3 stage by cutting a circle around the base of the plant. The root ball was removed and cleaned. Forage (R3) One forage sample (representing 3 plants) per plot each consisting of the aerial portion ( no roots) of 3 whole plants was collected fro m each test and control entry. Grain (R8

– Ma turity) One individual sample was collected from each plot of each test and control entry. Each sample contained approximately 500

- gram s of grain.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Determination of Cry1Ac Protein Concentration Soybean tissue samples were analyzed using Dow A groSciences me thod 110674

. In this method, the soluble, extractable Cry1Ac protein was quantified using an enzyme

- linked immunosorbent assay (ELISA) kit from Romer Labs, Inc. The Cry1Ac protein was extracted from lyophilized, ground soybean tissues with a phosphate buf fered saline solution containing 0.05% Triton X

- 100 with 1 mg/mL sodium ascorbate (PBS/Triton/AA). The extract was centrifuged, and then the aqueous supernatant was collected and subjected to trypsin digestion to convert full length Cry1Ac to the truncate d Cry1Ac core toxin. After truncation by trypsin, the reaction was stopped by the protease inhibitor phenylmethanesulfonyl fluoride (PMSF). The digested extract was diluted and assayed using a specific Cry1Ac ELISA kit. A sequential sandwich ELISA forma t was applied in this assay. An aliquot of the diluted sample was incubated in the wells of a plate pre

- coated with an immobilized monoclonal anti

- Cry1Ac antibody, and then the unbound samples are removed from the plate by washing with phosphate buffered saline solution containing 0.05% Tween 20 (PBST). An excess amount of enzyme

- conjugated monoclonal antibody specific to the Cry1Ac protein was then added to the wells for incubation. These antibodies bind with Cry1Ac protein in the wells and form a “sand wich” with Cry1Ac protein bound between the soluble and immobilized antibodies. At the end of an incubation period, the unbound reagents are removed from the plate by washing with PBST. The presence of Cry1Ac was detected by incubating the antibody

- bound enzyme conjugate with an enzyme substrate, generating a colored product. Since the Cry1Ac is bound in the antibody sandwich, the level of color development is proportional to the concentration of Cry1Ac in the sample (i.e., lower protein concentrations
r esult in lower color development). The absorbance at 450 nm was measured either using a Grifols Triturus Automated Immunoassay Analyzer with a 620 nm background subtraction or using a microplate reader with a 650 nm background subtraction. A calibration curve was estimated from 7 standard concentrations using a quadratic regression equation with a coefficient of determination > 0.990. Determination of Cry1F Protein in Soybean Tissue Samples Soybean tissue samples were analyzed using Dow AgroSciences method 110675

. In this method, the soluble, extractable Cry1F protein was quantified using an enzyme

- linked immunosorbent assay (ELISA) kit from Romer Labs, Inc. The Cry1F protein was extracted from lyophilized, ground soybean tissues with a phosphate buf fered saline solution containing 0.05% Triton X

- 100 with 1 mg/mL sodium ascorbate (PBS/Triton/AA). The extract was centrifuged; the aqueous supernatant was collected, diluted, and assayed using a specific Cry1F ELISA kit. A sandwich ELISA format was appl ied in this assay. An aliquot of the diluted sample was incubated in the wells of a plate pre

- coated with an immobilized monoclonal anti

- Cry1F antibody coated plate along with an enzyme

- conjugated monoclonal antibody specific to the Cry1F protein. These antibodies bind with Cry1F protein in the wells and form a “sandwich” with Cry1F protein bound between the soluble and immobilized antibodies. At the end of an incubation period, the unbound reagents are removed from the plate by washing with PBST. The p resence of Cry1F was detected by incubating the antibody

- bound enzyme conjugate with an enzyme substrate, generating a colored product. Since the Cry1F isUSDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information bound in the antibody sandwich, the level of color development is proportional to the concentration of Cry1F in the sample (i.e., lower protein concentrations result in lower color devel opment). The absorbance at 450 nm was measured either using a Grifols Triturus Automated Immunoassay Analyzer with a 620 nm background subtraction or using a microplate reader with a 650 nm background subtraction. A calibration curve was estimated from 7 standard concentrations using a quadratic regression equation. Determination of PAT Protein in Soybean Tissue Samples Soybean tissue samples were analyzed using Dow Agro Sciences method GRM 08.05

. In this method, the soluble, extractable PAT protein was quantified using an enzyme

- linked immunosorbent assay (ELISA) kit from EnviroLogix, Inc. The PAT protein was extracted from lyophilized, ground soybean samples with a phos phate buffered saline solution containing 0.05% Tween 20 and 1% polyvinylpyrrolidone (PBST/PVP). The extract was centrifuged; the aqueous supernatant was collected, diluted and assayed using a specific PAT ELISA kit. An aliquot of the diluted sample was incubated with enzyme

conjugated anti

- PAT protein monoclonal antibody in the wells of an anti

- PAT polyclonal antibody coated plate in a sandwich ELISA format. Both antibodies in the sandwich pair capture the PAT protein in the sample. At the end of the i ncubation period, the unbound reagents were removed from the plate by washing with PBST. The presence of PAT was detected by incubating the antibody

- bound enzyme conjugate with an enzyme substrate, generating a colored product. Since the PAT is bound in the antibody sandwich, the level of color development is proportional to the concentration of PAT in the sample (i.e., lower protein concentrations result in lower color devel opment). The absorbance at 450 nm was measured either using a Grifols Triturus A utomated Immunoassay Analyzer with a 620 nm background subtraction or using a microplate reader with a 650 nm background subtraction. A calibration curve was estimated from the 7 standard concentrations using a quadratic regression equation.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Appendix 6
. Analytical Methods for Compositional Analysis Acid Detergent Fiber (ADFA) The ANKOM2000 Fiber Analyzer automated the process of removal of proteins, carbohydrates, and ash. Fats and pigments were removed with an acetone wash prior to analysis. The fibrous residue that was primarily cellulose and lignin and insoluble protein complexes remained in the Ankom filter bag, and was determined gravimetrically. Amino Acid Composition (TALC/TPLC) Total aspartic acid (including asparagine) Total threonine Total serine Total glutamic acid (including glutamine) Total proline Total glycine Total alanine Total valine Total isoleucine Total leucine Total tyrosine Total phenylalanine Total histidine Total lysine Total arginine Total tr yptophan Sulfur

- containing amino acids: Total methionine Total cystine (including cysteine) The samples were hydrolyzed in 6N hydrochloric acid for approximately 24 hours at approximately 106

- 118oC. Phenol was added to the 6N hydrochloric acid to preve nt halogenation of tyrosine. Cystine and cysteine are converted to S

- 2

- carboxyethylthiocysteine by the addition of dithiodipropionic acid. Tryptophan was hydrolyzed from proteins by heating at approximately 110oC in 4.2N sodium hydroxide for approximately 20 hours. The samples were analyzed by HPLC after pre

- injection derivatization. The primary amino acids were derivatized with o

- phthalaldehyde (OPA) and the secondary amino acids are derivatized with fluorenylmethyl chloroformate (FMOC) before injection.

Reference Standards: Component Manufacture Lot No. Purity (%) L

- Alanine Sigma

- Aldrich 1440397 99.9 L

- Arginine Monohydrochloride Sigma

- Aldrich 1361811 100.0 L

- Aspartic Acid Sigma

- Aldrich BCBB9274 100.6 L

- Cystine Sigma

- Aldrich 1418036 1451329 99.9

100 L

- Glutamic Acid Sigma

- Aldrich 1423805 100.2 Glycine Sigma

- Aldrich 1119375 100.0 L

- Histidine Monohydrochloride Monohydrate Sigma

- Aldrich BCBB1348 99.9 L

- Isoleucine Sigma

- Aldrich 1423806 100.0USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Component Manufacture Lot No. Purity (%) L

- Leucine Sigma

- Aldrich BCBB1733 98.6 L

- Lysine Monohydrochloride Sigma

- Aldrich 1362380 100.2 L

- Methionine Sigma

- Aldrich 1423807 99.9 L

- Phenylalanine Sigma

- Aldrich BCBB9200 100 L

- Profile Sigma

- Aldrich 1414414 99.7 L

- Serine Sigma

- Aldrich 1336081 99.9 L

- Threonine Sigma

- Aldrich 1402329 100.0 L

- Tryptophan Sigma

- Aldrich BCBB1284 99.8 L

- Tyrosine Sigma

- Aldrich BCBB5393 99.5 L

- Valine Sigma

- Aldrich 1352709 100.0
Ash (ASHM) All organic matter was driven off when the samples were ignited at approximately 550oC in a muffle furnace for at least 5 hours. The remaining inorganic material was determined gravimetrically and referred to as ash.
Beta Carotene (BCLC) The samples were saponified and extracted with hexane. The samples were then injected on a reverse phase high

- performance liquid chromatogra phy system with ultraviolet light detection. Quantitation was achieved with a linear regression analysis. Reference Standard: Sigma

- Aldrich, Beta Carotene, 99.0%, Lot No. 021M1304V Carbohydrate (CHO) The total carbohydrate level was calculated by differenc e using the fresh weight

- derived data and the following equation: % carbohydrates = 100 %

- (% protein + % fat + % moisture + % ash) Fat by Acid Hydrolysis (FAAH) The samples were hydrolyzed with hydrochloric acid. The fat was extracted using ether and hex ane. The extracts were dried down and filtered through a sodium sulfate column. The remaining extracts were then evaporated, dried, and weighed. Fat by Soxhlet Extraction (FSOX) The samples were weighed into a cellulose thimble containing sodium sulfate and dried to remove excess moisture. Pentane was dripped through the samples to remove the fat. The extract was then evaporated, dried, and weighed. Fatty Acids (FAPM) The lipid was extracted and saponified with 0.5N sodium hydroxide in methanol. The sapon ification mixture was methylated with 14% boron trifluoride in methanol. The resulting methyl esters were extracted with heptane containing an internal standard. The methyl esters of the fatty acids were analyzed by gas chromatography using external standa rds for quantitation.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Reference Standards: Manufacturer Lot No. Component Weight (%) Purity

(%)

Nu

- Chek Prep GLC Reference Standard Covance 1 Covance 2 S27

- V

S28

- V S27

- V S28

- V

Methyl Octanoate 3.0 1.25 99.7 Methyl Decanoate 3.25 1.25 99.6 Methyl Laurate 3.25 1.25 99.8 Methyl Myristate 3.25 1.25 99.8 Methyl Myristoleate 1.0 1.25 99.5 Methyl Pentadecanoate 1.0 1.25 99.6 Methyl Pentadecenoate 1.0 1.25 99.4 Methyl Palmitate 10.0 15.0 99.8 Methyl Palmitoleate 3.0 1.25 99.7 Methyl Heptadecanoate 1.0 1.25 99.6 Methyl 10

- Heptadecenoate 1.0 1.25 99.5 Methyl Stearate 7.0 11.0 99.8 Methyl Oleate 10.0 15.0 99.8 Methyl Linoleate 10.0 15.0 99.8 Methyl Gamma Linolenate 1.0 1.25 99.4 Methyl Linolenate 3.0 1.25 99.5 Methyl Arachidate 2.0 1.25 99.8 Methyl 11

- Eicosenoate 2.0 1.25 99.6 Methyl 11

- 14 Eicosadienoate 1.0 1.25 99.5 Methyl 11

- 14

- 17 Eicosatrienoate 1.0 1.25 99.5 Methyl Arachidonate 1.0 1.25 99.4 Methyl Behenate 1.0 1.5 99.8

Folic acid (FOAN) The samples were hydrolyzed in a potassium phosphate buffer with the addition of ascorbic acid to protect the folic acid during autoclaving. Following hydrolysis by autoclaving, the samples were treated with a chicken

- pancreas enzyme and incubated approximately 18 hours to liberate the bound folic acid. The amount of folic acid was determined by comparing the growth response of the samples, using the bacteria Lactobacillus casei , with the growth response of a folic acid standard. This resp onse was measured turbidimetrically.
Reference Standard: USP, Folic acid, 98.9%, Lot No. Q0G151 ICP Emission Spectrometry (ICPS) The sample was dried, precharred, and ashed overnight in a muffle set to maintain 500  C. The ashed sample was re

- ashed with nitric acid, treated with hydrochloric acid, taken to dryness, and put into a solution of 5% hydrochloric acid. The amount of each element was determined at appropriate wavelengths by comparing the emission of the unknown sample, measured on the inductive ly coupled plasma spectrometer, with the emission of the standard solutions. Inorganic Ventures Reference Standards and Limits of Quantitation:USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information

Mineral Lot N o. Concentration (μg/mL) Calcium E2

- MEB393070MCA, E2

- MEB393072 200 , 1000 Copper E2

- MEB393070MCA, E2

- MEB393071MCA 2.00, 10.00 Iron E2

- MEB393070MCA, E2

- MEB393073 10.0, 50.0 Magnesium E2

- MEB393070MCA, E2

- MEB393071MCA 50.0 , 250 Manganese E2

- MEB393070MCA, E2

- MEB393071MCA 2.0 , 10. 0 Phosphorus E2

- MEB393070MCA, E2

- MEB393072 200, 1000 Potassium E2

- MEB393070MCA, E2

- MEB393072 200 , 1000 Sodium E2

- MEB393070MCA, E2

- MEB393072 200, 1000 Zinc E2

- MEB393070MCA, E2

- MEB393071MCA 10.0, 50. 0

Isoflavones (ASOF) The samples were extracted at approximately 65°C with a 80/20 methanol:water solution and the extract was saponified with dilute NaOH solution. The extract was then acidified, filtered, and diluted. The samples were analyzed on a high

- performance liquid chromatography system with ultraviolet spectrophotometric detection and was co mpared against an external standard curve.

Reference Standards: Component Manufacturer Lot No. Purity (%) Daidzein LC Labs DA

- 121 99.7 Glycitein LC Labs ARH

- 114 99.8 Genistein LC Labs CH

- 148 99.7 Daidzin LC Labs ARF

- 114 99.7 Glycitin LC Labs ARG

- 113 99.6 Genistin LC Labs ARE

- 109 99.4
Lectins (LCTN) The determination of lectins was based on the ability of lectin (a hemagglutinin) to bind to specific sugars present on the surface of red blood cells (RBCs) of different animal species resulting in the agglutination of RBCs. Samples were defatted and extracted with a saline solution. Agglutination of trypsinized rabbit RBCs was measured with a spectrophotometer at a wavelength of 620 nm.
Moisture (M100) The samples were dried in a vacuum oven at approximately 100 ° C. The moisture weight loss was determined and converted to percent moisture. Neutral Detergent Fiber (NDFA) The ANKOM2000 Fiber Analyzer automated the process of the removal of protein, carbohydrate, and ash. Fats and pigments were remo ved with an acetone wash prior to analysis. Hemicellulose, cellulose, lignin and insoluble protein fraction were left in the filter bag and d etermined gravimetrically.
Niacin (NIAP) The samples were hydrolyzed with sulfuric acid and the pH was adjusted to remove interferences. The amount of niacin was determined by comparing the growth response of theUSDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information samples, using the bacteria Lactobacillus plantarum, with the growth response of a niacin standard. This response was measured turbidimetrically.
Reference St andard: USP, Niacin, 99.8%, Lot No. I0E295
Pantothenic Acid (PANN) The samples were diluted with water or treated with an enzyme mixture to liberate the pantothenic acid from coenzyme A and the pH was adjusted to remove interferences. The amount of pantoth enic acid was determined by comparing the growth response of the samples, using the bacteria Lactobacillus plantarum , with the growth response of a calcium pantothenate standard. This growth response was measured turbidimetrically.
Reference Standard: USP, Calcium Pantothenate, 99.0%, Lot No. O1H081 Phytic Acid (PHYT) The samples were extracted using hydrochloric acid and sonication, purified using a silica based anion exchange column, concentrated and injected onto a high

- performance liquid chromatography (HPLC) system with a refractive index detector.
Reference Standard: Sigma

- Aldrich, Phytic Acid Sodium Salt Hydrate, 93.5%, Lot No. BCBF5728V
Protein (PGEN) The BUCHI Automated Digester was used to automate the following process. The protein and other organic nitrogen in the samples were converted to ammonia by digesting the samples with sulfuric acid containing a catalyst mixture. The acid digest was made alkaline. The ammonia was distilled and then titrated with a previously standardized acid. The per cent nitrogen was alculated and converted to equivalent protein using the factor 6.25. Selenium by Inductively Coupled Plasma

- Mass Spectrometry (SEMS) The samples were closed

- vessel microwave digested with nitric acid (HNO 3 ) and water. After digestion, the solution was brought to a final volume with water. To normalize the organic contribution between samples and standards, a dilution was prepared for analysis that contained methanol. The selenium concentration was determined with Se 78 using an inductively coupled plasma

- mass spectrometer (ICP

- MS) with a dynamic reaction cell (DRC) by comparing the counts generated by standard solutions.
Reference Standard: SPEX, Selenium, 1000 mg/L, Lot No. 16

- 177SE Sugar Profile (SUGT) Sugars in the samples were extracted with a 50:50 water:methanol solution. Aliquots were taken, dried under inert gas, and then reconstituted with a hydroxylamine hydrochloride solution in pyridine containing phenyl

- β

- D

- glucopyranoside as the internal standard. The resulting oximes
were conve rted to silyl derivatives by treatment with hexamethyldisilazane and trifluoracetic acid treatment, and then analyzed by gas chromatography using a flame ionization detector.USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information Reference Standards: Component Manufacturer Lot No. Purity (%) D

- (+)

- Raffinose pentahydrate Sigma

- Aldrich 019K1156 99.6 Stachyose hydrate Sigma

- Aldrich 049K3800 98 Total Tocopherols (TTLC) The samples were saponified to break down any fat and release vitamin E. The saponified mixtures were extracted with an organic solvent, dried down and brought to a suitable volume in hexane. The samples were then quantitated by high

- performance liquid chromatography using a silica column.

Reference Standards: Component Manufacturer Lot No. Purity (%) Alpha Tocopherol USP N0F068 98.9
rac
- beta

- Tocopherol Matreya LLC 23260 >99 Gamma Tocopherol ACROS A0083534 99.3 (+)

- δ

- Tocopherol (Delta) Sigma

- Aldrich 090M1916V 92 Total Dietary Fiber (TDF) Duplicate samples were gelatinized with α

- amylase and digested with enzymes to break down starch and protein. Ethanol was added to each of the samples to precipitate the soluble fiber. The samples were filtered, and the residue was rinsed with ethanol and acetone to remove starch and protein degr adation products and moisture. Protein content was determined for one of the duplicates; ash content was determined for the other. The total dietary fiber in the samples were calculated using protein and ash values and the weighed residue fractions. Trypsi n Inhibitor (TRIP) The samples were ground and defatted with petroleum ether. A sample of matrix was extracted with 0.01N sodium hydroxide. Varying aliquots of the sample suspensions were exposed to a known amount of trypsin and benzoy1

- DL

- arginine~p~nitro anilide hydrochloride. The samples were allowed to react for 10 minutes at 37°C. After 10 minutes, the reaction was halted by the addition of acetic acid. The absorbance was determined at 410 nm against a sample blank. Trypsin inhibitor activity was determ ined by photometrically measuring the inhibition of trypsin’s reaction with benzoyl

- DLarginine~p~nitroanilide hydrochloride. Thiamine Hydrochloride (BIDE) The samples were autoclaved under weak acid conditions to extract the thiamine. The resulting solutio ns were incubated with a buffered enzyme solution to release any bound thiamine. The solutions were purified on a cation

- exchange column. Aliquots were reacted with potassium ferricyanide to convert thiamine to thiochrome. The thiochrome was extracted into isobutyl alcohol, measured on a fluorometer, and quantitated by comparison to a known standard.
Reference Standard: USP, Thiamine Hydrochloride, 99.8%, Lot No. O1F236
Vitamin B 2 (Riboflavin) (B2FV)USDA Petition for No nregulated Status of DAS

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Contains No Confidential Business Information The samples were hydrolyzed with dilute hydrochloric acid and the pH was adjusted to remove interferences. The amount of riboflavin was determined by comparing the growth response of the samples, using the bacteria Lactobacillus rhamnosus , with the growth response of multipoint riboflavin standards. The growth response was measured turbidimetrically.
Reference Standard: USP, Riboflavin, 99.7%, Lot No. N1J079 Pyridoxine Hydrochloride The samples were hydrolyzed with dilute sulfuric acid in the autoclave and the pH was adjusted to remove interferences. The amount of pyridoxine was determined by comparing the growth response of the samples, using the yeast Saccharomyces cerevisiae, with the growth response of a pyridoxine standard. The response was measured tur bidimetrically. Results were reported as pyridoxine hydrochloride.
Reference Standard: USP, Pyridoxine hydrochloride, 99.8%, Lot No. Q0G409
Vitamin C (VCF) The vitamin C in the samples were extracted, oxidized, and mixed with o

- phenylenediamine to produce a fluorophor having an activation maximum at approximately 350 nm and a fluorescence maximum at 430 nm. Fluorescence was proportional to concentration. Development of the fluorescence compound with the vitamin was prevented by forming a boric acid

dehydro ascorbic acid complex prior to addition of the o

- phenylenediamine solution. Any remaining fluorescence was due to extraneous material and served as the blank.
Reference Standard: USP, Ascorbic Acid, 99.9%, Lot No. Q1G135USDA Petition for N onregulated Status of DAS

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Contains No Confidential Business Information Appendix 7
. Literature Ranges for Compositional Analysis Matrix Category Analyte Units OECD 1 ILSI 2 Literature Literature Citations Min Max Min Max Min Max Min Max For age Fiber ADF % DW 32 38 NR NR 22.72 59.03 Harrigan et al. 2007 Berman et al. 2010 Forage Fiber NDF % DW 34 40 NR NR 19.61 73.05 Lundry et al. 2008 Berman et al. 2010 Forage P roximate Ash % DW 8.8 10.5 6.718 10.782 4.68 9.24 Harrigan et al. 2007 Lundry et al. 2008 Forage Proximate Carbohydrates % DW NR NR 59.8 74.7 60.61 80.18 Berman et al. 2009 Berman et al. 2010 Forage P roximate Moisture % FW 74 79 73.5 81.6 32.05 84.60 Berman et al. 2009 Berman et al. 2010 Forage Proximate Crude Protein % DW 11.2 17.3 14.38 24.71 11.77 24.29 Berman et al. 2010 Lundry et al. 2008 Forage P roximate Total Fat % DW NR NR 1.302 5.132 1.01 9.87 Berman et al. 2010 Lundry et al. 2008 Forage Mineral Calcium mg/100g dry wt. NR NR NR NR NR NR NR NR Forage M ineral Phosphorus mg/100g dry wt. NR NR NR NR NR NR NR NR Grain Fiber ADF % DW 9 .0 11.1 7.81 18.61 9.22 26.26 Lundry et al. 2008 Lundry et al. 2008 Grain Fi ber NDF % DW 10 .0 14.9 8.53 21.25 10.79 23.90 Lundry et al. 2008 Lundry et al. 2008 Grain Proximate Ash % DW 4.5 6.4 3.885 6.994 4.29 6.44 Padgette et al. 1996 Harrigan et al. 2007 Grain Pr oximate Carbohydrates % DW 31.7 31.8 29.6 50.2 29.3 44.35 Padgette et al. 1996 Harrigan et al. 2007 Grain Proximate Moisture % FW NR NR 4.7 34.4 4.71 14.30 Harrigan et al. 2007 Taylor et al. 1999 Grain Pr oximate Crude Protein % DW 32 43.6 33.19 45.48 32.29 48.4 Berman et al. 2011 Hartwig and Kilen 1991 Grain Proximate Total Fat % DW 15.5 24.7 8.104 23.562 14.10 23.67 Padgette et al. 1996 Berman et al. 2010 Grain Mi neral Calcium mg/100g dry wt. NR NR 116.55 307.10 258 510 Iskander 1987 Bilyeu et al. 2008 Grain Mineral Copper mg/100g dry wt. NR NR NR NR 0.632 1.092 Bilyeu et al. 2008 Bilyeu et al. 2008 Grain Mi neral Iron mg/100g dry wt. NR NR 5.536 10.954 3.734 6.624 Bilyeu et al. 2008 Bilyeu et al. 2008 Grain Mineral Magnesium mg/100g dry wt. NR NR 219.40 312.84 261 280 Iskander 1987 Bilyeu et al. 2008 Grain Mi neral Manganese mg/100g dry wt. NR NR NR NR 2.52 3.876 Iskander 1987 Bilyeu et al. 2008 Grain Mineral Phosphorus mg/100g dry wt. NR NR 506.74 935.24 770 790 Bilyeu et al. 2008 Bilyeu et al. 2008 Grain Mi neral Potassium mg/100g dry wt. NR NR 1868.01 2316.14 1910 2510 Iskander 1987 Bilyeu et al. 2008 Grain Mineral Selenium ppb DW NR NR NR NR NR NR NR NR Grain Mi neral Sodium mg/100g dry wt. NR NR NR NR 4.05 30 Iskander 1987 Bilyeu et al. 2008 Grain Mineral Zinc mg/100g dry wt. NR NR NR NR 4.98 7.578 Iskander 1987 Bilyeu et al. 2008 Grain Am ino Acid Alanine % total amino acid NR NR 4.23 4.74 4.16 4.54 Berman et al. 2011 Harrigan et al. 2007 Grain Amino Acid Arginine % total amino acid NR NR 6.70 8.41 6.41 7.60 Berman et al. 2010 Lundry et al. 2008 Grain Am ino Acid Aspartic Acid % total amino acid NR NR 11.39 12.04 11.37 12.68 Taylor et al. 1999 Berman et al. 2009 Grain Amino Acid Cystine % total amino acid NR NR 1.02 1.87 1.40 1.75 Berman et al. 2010 Berman et al. 2011 Grain Am ino Acid Glutamic Acid % total amino acid NR NR 17.71 19.24 18.25 20.48 Harrigan et al. 2007 Berman et al. 2010 Grain Amino Acid Glycine % total amino acid NR NR 4.19 4.61 4.24 4.62 Berman et al. 2010 Berman et al. 2011 Grain Am ino Acid Histidine % total amino acid NR NR 2.49 2.85 2.65 2.89 Lundry et al. 2008 Berman et al. 2011 Grain Amino Acid Isoleucine % total amino acid NR NR 4.13 5.11 4.51 4.62 Berman et al. 2009 Taylor et al. 1999 Grain Am ino Acid Leucine % total amino acid NR NR 7.62 8.29 7.46 7.88 Berman et al. 2010 Lundry et al. 2008USDA Petition for N onregulated Status of DAS

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Contains No Confidential Business Information Matrix Category Analyte Units OECD 1 ILSI 2 Literature Literature Citations Min Max Min Max Min Max Min Max Gra in Amino Acid Lysine % total amino acid NR NR 6.29 7.16 6.23 7.38 Berman et al. 2011 Berman et al. 2010 Grain Amino Acid Methionine % total amino acid NR NR 1.18 1.71 1.31 1.47 Lundry et al. 2008 Lundry et al. 2008 Grain Am ino Acid Phenylalanine % total amino acid NR NR 4.91 5.44 4.95 5.44 Berman et al. 2010 Berman et al. 2011 Grain Amino Acid Proline % total amino acid NR NR 4.75 5.62 4.96 5.23 Berman et al. 2010 Taylor et al. 1999 Grain Am ino Acid Serine % total amino acid NR NR 3.25 6.04 5.03 5.47 Berman et al. 2010 Lundry et al. 2008 Grain Amino Acid Threonine % total amino acid NR NR 3.15 4.24 3.92 4.08 Berman et al. 2009 Berman et al. 2011 Grain Am ino Acid Tryptophan % total amino acid NR NR 0.95 1.29 0.97 1.49 Lundry et al. 2008 Padgette et al. 1996 Grain Am ino Acid Tyrosine % total amino acid NR NR 2.83 3.72 2.62 3.67 Berman et al. 2010 Padgette et al. 1996 Grain Am ino Acid Valine % total amino acid NR NR 4.28 5.57 4.69 4.93 Padgette et al. 1996 Berman et al. 2011 Grain Fatty Acid 8:0 Caprylic % total fatty acid NR NR 0.148 0.148 ND ND Harrigan et al. 2007 Harrigan et al. 2007 Grain Fa tty Acid 10:0 Capric % total fatty acid NR NR ND ND ND 0.27 Harrigan et al. 2007 Berman et al. 2009 Grain Fatty Acid 12:0 Lauric % total fatty acid NR NR 0.082 0.132 ND ND Harrigan et al. 2007 Harrigan et al. 2007 Grain Fa tty Acid 14:0 Myristic % total fatty acid NR NR 0.071 0.238 ND 0.11 Harrigan et al. 2007 Berman et al. 2009 Grain Fatty Acid 14:1 Myristoleic % total fatty acid NR NR 0.121 0.125 ND ND Harrigan et al. 2007 Harrigan et al. 2007 Grain Fa tty Acid 15:0 Pentadecanoic % total fatty acid NR NR ND ND ND ND Harrigan et al. 2007 Harrigan et al. 2007 Grain Fatty Acid 15:1 Pentadecenoic % total fatty acid NR NR ND ND ND ND Harrigan et al. 2007 Harrigan et al. 2007 Grain Fa tty Acid 16:0 Palmitic % total fatty acid NR NR 9.55 15.77 1.40 12.63 Harrigan et al. 2007 Berman et al. 2009 Grain Fatty Acid 16:1 Palmitoleic % total fatty acid NR NR 0.086 0.194 ND 0.14 Harrigan et al. 2007 Berman et al. 2009 Grain Fa tty Acid 17:0 Heptadecanoic % total fatty acid NR NR 0.085 0.146 ND 0.13 Harrigan et al. 2007 Berman et al. 2009 Grain Fatty Acid 17:1 Heptadecenoic % total fatty acid NR NR 0.073 0.087 ND 0.064 Harrigan et al. 2007 Berman et al. 2009 Grain Fa tty Acid 18:0 Stearic % total fatty acid NR NR 2.70 5.88 0.50 5.63 Harrigan et al. 2007 Berman et al. 2009 Grain Fatty Acid 18:1 Oleic % total fatty acid NR NR 14.3 32.2 2.60 45.68 Harrigan et al. 2007 Berman et al. 2010 Grain Fa tty Acid 18:2 Linoleic % total fatty acid NR NR 42.3 58.8 7.58 57.72 Harrigan et al. 2007 Berman et al. 2009 Grain Fatty Acid 18:3 Linolenic % total fatty acid NR NR 3.00 12.52 1.27 9.90 Harrigan et al. 2007 Berman et al. 2009 Grain Fa tty Acid 18:3 gamma Linolenic % total fatty acid NR NR ND ND ND ND Harrigan et al. 2007 Harrigan et al. 2007 Grain Fatty Acid 20:0 Arachidic % total fatty acid NR NR 0.163 0.482 0.038 0.57 Harrigan et al. 2007 Berman et al. 2009 Grain Fa tty Acid 20:1 Eicosenoic % total fatty acid NR NR 0.140 0.350 0.024 0.35 Harrigan et al. 2007 Berman et al. 2010 Grain Fatty Acid 20:2 Eicosadienoic % total fatty acid NR NR 0.077 0.245 ND 0.065 Harrigan et al. 2007 Berman et al. 2010 Grain Fa tty Acid 20:3 Eicosatrienoic % total fatty acid NR NR ND ND ND ND Harrigan et al. 2007 Harrigan et al. 2007 Grain Fatty Acid 20:4 Arachidonic % total fatty acid NR NR ND ND ND ND Harrigan et al. 2007 Harrigan et al. 2007 Grain Fa tty Acid 22:0 Behenic % total fatty acid NR NR 0.277 0.595 0.043 0.65 Harrigan et al. 2007 Berman et al. 2009 Grain Vitamin Alpha Tocopherol (Vitamin E) mg/kg DW NR NR 1.934 61.693 10

. 8 84.9 Berman et al. 2010 Zhou et al. 2011 Grain Vi tamin Beta Tocopherol mg/kg DW NR NR NR NR NR NR NR NR Grain Vi tamin Delta Tocopherol mg/kg DW NR NR NR NR NR NR NR NR Grain Vi tamin Gamma Tocopherol mg/kg DW NR NR NR NR NR NR NR NR Grain Vitamin Total Tocopherol mg/kg DW NR NR NR NR NR NR NR NRUSDA Petition for N onregulated Status of DAS

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Contains No Confidential Business Information Matri x Category Analyte Units OECD 1 ILSI 2 Literature Literature Citations Min Max Min Max Min Max Min Max Grain Vitamin Vitamin A (Beta Carotene) mg/kg DW NR NR NR NR NR NR NR NR Grain Vitamin Vitamin B1 (Thiamine HCl) mg/kg DW NR NR 1.01 2.54 NR NR NR NR Grain Vitamin Vitamin B2 (Riboflavin) mg/kg DW NR NR 1.90 3.21 NR NR NR NR Grain Vitamin Vitamin B3 (Niacin) mg/kg DW NR NR NR NR NR NR NR NR Grain Vitamin Vitamin B5 (Pantothenic Acid) mg/kg DW NR NR NR NR NR NR NR NR Grain Vitamin Vitamin B6 (Pyridoxine HCl) mg/kg DW NR NR NR NR NR NR NR NR Grain Vitamin Vitamin B9 (Folic Acid) mg/kg DW NR NR 2.386 4.709 NR NR NR NR Grain Vitamin Vitamin C (Ascorbic acid) mg/kg DW NR NR NR NR NR NR NR NR Grain Bioactive Total Daidzein Equivalent mcg/g DW NR NR 60.0 2453.5 25 2100.00 McCann et al. 2005 Berman et al. 2011 Grain Bioactive Total Genistein Equivalent mcg/g DW NR NR 144.3 2837.2 28 2600.70 McCann et al. 2005 Harrigan et al. 2007 Grain Bioactive Total Glycitein Equivalent mcg/g DW NR NR 15.3 310. 0 32 349.19 Berman et al. 2011 Harrigan et al. 2007 Grain Bioactive Lectin HU/mg Protein DW 37 323 NR NR 37 323 Kakade et al. 1972 Kakade et al. 1972 Grain Bioactive Phytic Acid % DW NR NR 0.634 1.960 0.41 2.68 Lundry et al. 2008 Berman et al. 2010 Grain Bioactive Raffinose % DW NR NR 0.212 0.661 0.22 1.85 Harrigan et al. 2007 Berman et al. 2011 Grain Bioactive Stachyose % DW NR NR 1.21 3.50 1.52 6.65 Harrigan et al. 2007 Berman et al. 2011 Grain Bioactive Trypsin Inhibitor TIU/mg DW NR NR 19.59 118.68 18.14 75.5 Berman et al. 2009 McCann et al. 2005 ND = Not Detected, NR = Not Reported 1 ( OECD, 2001 ) Consensus document on compositional considerations for new varieties of soybean: Key food and feed nutrients and anti

- nutrients. ENV/JM/MONO(2001)15. 30. www.oecd.org/dataoecd/15/60/46815135.pdf

. 2 International Life Sciences Institute, Crop Composition Database v4. 2 (accessed January 10 and May 16, 2012)USDA Petition for N onregulated Status of DAS

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Contains No Confidential Business Information Appen dix 8
. Non

- Target Arthropod Field Study
A non

- target arthropod field study with DAS

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- 2 soybean, a near

- isogenic non

- transgenic control (Maverick) and four non

- transgenic reference lines (DSR 3590; IL 3505; Porter 75148; Williams 82) was conducted in 2011 at two sites in the U.S. located in Richland, Iowa and York, Nebraska. Each trial site included six entries, including one entry of DAS

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- 2, one control entry (Maverick), and four entries of non

- transgenic reference varieties. At each site, entries were arranged in a randomized complete block design with four blocks. Across sites, each control, reference, and DAS

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- 2 entry was represented by a total of eight plots (2 sites, 4 replicate plots per entry at each si te). Each study plot (observational unit) measured approximately 335 m 2 (3600 ft 2 ; 24 rows wide, 60 ft length). Soybean seeds were planted at a seeding rate of approximately 300 seeds per row with seed spacing within each row of approximately 2.4 inches (6 cm; 5 seeds per row foot). Study plots were separated by four rows of a non

- transgenic soybean cultivar of similar maturity. The entire trial site was surrounded by a minimum of four rows (3.0 m; 10 ft) of a non

- transgenic soybean cultivar of similar maturity. Therefore, the entire trial area formed a continuous field of soybean. Appropriate weed and disease control practices were applied to produce an agronomically acceptable crop. Arthropod pest management, including sprayed insecticides and mitic ides, was excluded from study. Arthropod abundance was monitored using three sampling methods: pitfall trapping, sticky card trapping, and vertical beat sheet sampling. Sample timings included vegetative and reproductive soybean stages. Arthropods collec ted during the study included representatives from multiple orders and diverse ecological roles, including phytophagous, predatory, parasitic, and saprophagous modes of feeding. Assessment was conducted for several taxonomic groups, including : Oribatida ( soil mites), Araneae (foraging and web spiders), Collembola (springtails), Hemiptera (e.g. Orius spp.), Coleoptera (e.g. ground beetles, lady beetles), Neuroptera (e.g. Chrysopa spp.), Lepidoptera , Diptera, and Hymenoptera (e.g. parasitoid wasps). A step

- wise approach to statistical analysis was used, where community

- level data were first analyzed using redundancy analysis (RDA) and Monte Carlo simulations ( Dively, 2005 ) t o test for differences between arthropod populations associated with DAS

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- 2 and the near

- isogenic control. Redundancy analysis results were used to construct principal response curves (PRCs) to visualize population patterns. When defined criteria were met, a second step assessment included univariate analysis of variance (ANOVA) and application of the false discovery rate (FDR) procedure ( Benjamini and Hochberg, 1995 ; Curran

- Everett, 2000 ) . Results of both the PRC and ANOVA analyses confir med no unintended effects of DAS

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- 2 soybean on any of the arthropod groups monitored. Consistent across locations and sampling methods, arthropod populations associated with DAS

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- 2 soybean were similar to those of non

- transgenic soybean. Field monitoring confirmed the results of established laboratory studies examining the Cry1Ac and Cry1F proteins, and demonstrated no adverse effects of DAS

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- 2 cultivation on non
- target arthr opod communities

.USDA Petition for N onregulated Status of DAS

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Contains No Confidential Business Information Appendix 9
. USDA Notifications for DAS

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- 2 Soybean

USDA Notification Number Notification

Authorization Date Notification

Expiration Date States Number of Trials Trial

Status 1

12

- 088

- 113n 4/23/2012 4/23/2013 AL, AR, GA, IA, IL, IN, MD, MO, NE 24 pending

12

- 072

- 105n 4/2/2012 4/2/2013 IA, IL, MO, NE, PA, IN 8 pending

12

- 060

- 107n 4/1/2012 4/1/2013 IA, IL, MO, NE, PA, IN 12 pending

11

- 305

- 101n 11/30/2011 11/30/2012 PR 1 pending

11

- 216

- 106n 9/9/2011 9/9/2012 PR 8 pending

11

- 088

- 112n 5/6/2011 5/6/2012 IA, IN, MO 4 pending

11

- 088

- 101n 5/9/2011 5/9/2012 IA, NE 2 pending

11

- 087

- 115n 4/21/2011 4/21/2012 AR, GA, IA, IL, IN, MD, MI, MO, MS, NE, PR 26 pending

11

- 087

- 113n 4/15/2011 4/15/2012 IA, IL, IN, MO, NE, PA 10 pending

10

- 243

- 105n 9/30/2010 9/30/2011 PR 3 submitted

10

- 077

- 106n 4/14/2010 4/14/2011 IN 1 submitted

09

- 259

- 107n 9/30/2009 9/30/2010 PR 1 submitted

1 Pending reports as of July 28, 2012 to be submitted within six months of the notification expiration date.USDA Petition for N onregulated Status of DAS

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Contains No Confidential Business Information Appendix 10
. St ewardship of Insect

- Resistant DAS

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- 2 Soybean Dow AgroSciences is committed to promoting the responsible use and stewardship of its products throughout the product life cycle. DAS has a long

- standing commitment to product stewardship and is a founding member of Excellence through Stewardship . In addition, DAS has voluntarily establi shed a Biotechnology Quality Management System (BQMS) which has been verified by third

- party audits as conforming to the USDA APHIS BQMS Program Audit Standard. Consistent with its existing stewardship programs and quality management systems , DAS will impl ement comprehensive stewardship measures for i nsect

- r esistant DAS

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- 2 s oybean. These programs and practices include rigorous field compliance , quality assurance, quality control, and auditing

. DAS has submit ted an application for a FIFRA Section 3 seed increase registration with EPA to support future breeding and seed increase activities in the United States. Under a Section 3 seed increase registration, commercial sale of DAS

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- 2 s oybean in the U.S. is proh ibited. If DAS chooses to cultivate DAS

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- 2 s oybean for commercial purposes in the U.S. in the future, DAS will apply for a FIFRA Section 3 commercial use registration with EPA. As with all products, DAS is committed to the rigorous product stewardshi p of DAS

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- 2
s oybean. This includes having processes in place to restrict and steward the use of DAS

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- 2
s oybean based on the registered uses in the U.S

. All breeding or seed multiplication work will be done directly by DAS or its licensees under a contract. As long as DAS

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- 2 soybean is cultivated in the U.S. under the Section 3 seed increase registration, t he limitations on the EPA registration, combined with contractual production of the seed, will serve to effectively limit use of DAS

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- 2 s oybean and product stewardship requirements will be implemented accordingly. In the future, should DAS choose to register DAS

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- 2 s oybean for commercial production in the U.S., product stewardship measures would be expanded to reflect commercia l grower use and insect resistance management requirements

. Before commercially launching DAS

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- 2 s oybean in any country, DAS will obtain regulatory approval from key soybean import countries with functioning regulatory systems to assure regulatory com pliance, promote product launch stewardship, and support the flow of international trade. DAS is in the process of making submissions to the regulatory authorities of trade partners for import approval and /or cultivation approval. DAS also commits to indu stry practices to maintain plant product integrity and seed purity of DAS

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- 2 s oybean. A detection method will be made available to soybean producers, processors and buyers before commercializing DAS

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- 2 s oybean in any country.USDA Petition for N onregulated Status of DAS

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Contains No Confidential Business Information Appendix 11 . References Adang, M.J., Staver, M.J., Rocheleau, T.A., Leighton, J., Barker, R.F., Thompson, D.V., 1985. Characterized full

- length and truncated plasmid clones of the crystal protein of Bacillus thuringiensis subsp. kurstaki HD

- 73 and their toxicity to Manduca sexta . Gene 36, 289

- 300. AgrEvo, 1994. Petition for determination of nonregulated status: Glufosinate resistant corn transformation events T14 and T25. http://www.aphis.usda.gov/brs/aphisdocs/94_35701p.pdf AgrEvo, 1996. Petition for determination of nonregulated status: Glufosinate resistant soybean transformation events. http://www.aphis.usda.gov/brs/aphisdocs/96_06801p.pdf AgrEvo, 1997. Petition for determination of nonregulated status: Glufosinate tolerant canola transformation event T45. http://www.a phis.usda.gov/brs/aphisdocs/97_20501p.pdf

. Ahrent, D.K., Caviness, C.E., 1994. Natural cross

- pollination of twelve soybean cultivars in Arkansas. Crop Science 34, 376

- 378. AOSA, 2009. Seed vigor testing handbook contribution no. 32. In: Baalbaki, R.J., Mar cos
- Filho, J., Elias, S.G., McDonald, M. (Eds.). AOSA, 2010a. AOSA rules for testing seeds volume 1. Principles and procedures. Association of Official Seed Analysts Inc. AOSA, 2010b. AOSA rules for testing seeds volume 4. Seeding evaluation. Association o f Official Seed Analysts Inc. AOSCA, 2012. Seed certification. http://www.aosca.org/seed%20certification.htm

. Aronson, A.I., Shai, Y., 2001. Why Bacillus thuringiensis insecticidal toxins are so e ffective: Unique features of their mode of action. FEMS Microbiology Letters 195, 1

- 8. Arumuganathan, K., Earle, E., 1991. Nuclear DNA content of some important plant species. Plant Molecular Biology Reporter 9, 208

- 218. Barker, R.F., Idler, K.B., Thompson , D.V., Kemp, J.D., 1983. Nucleotide

- sequence of the T

DNA region from the Agrobacterium tumefaciens octopine Ti plasmid pTi15955. Plant Molecular Biology 2, 335

- 350. Beard, B.H., Knowles, P.F., 1971. Frequency of cross

- pollination of soybeans after seed i rradiation. Crop Science 11, 489

- 492. Benjamini, Y., Hochberg, Y., 1995. Controlling the false discovery rate: A practical and powerful approach to multiple testing. Journal of the Royal Statistical Society. Series B (Methodological) 57, 289

- 300. Berman, K .H., Harrigan, G.G., Nemeth, M.A., Oliveira, W.S., Berger, G.U., Tagliaferro, F.S., 2011. Compositional equivalence of insect

- protected glyphosate

- tolerant soybean MON 87701 × MON 89788 to conventional soybean extends across different world regions and mul tiple growing seasons. Journal of Agricultural and Food Chemistry 59, 11643

- 11651. Berman, K.H., Harrigan, G.G., Riordan, S.G., Nemeth, M.A., Hanson, C., Smith, M., Sorbet, R., Zhu, E., Ridley, W.P., 2009. Compositions of seed, forage, and processed fracti ons from insect

- protected soybean MON 87701 are equivalent to those of conventional soybean. Journal of Agricultural and Food Chemistry 57, 11360

- 11369. Berman, K.H., Harrigan, G.G., Riordan, S.G., Nemeth, M.A., Hanson, C., Smith, M., Sorbet, R., Zhu, E., Ridley, W.P., 2010. Compositions of forage and seed from second

- generation glyphosate

- tolerant soybean MON 89788 and insect

- protected soybean MON 87701 from brazil are equivalent to those of conventional soybean ( Glycine max ). Journal of Agricultural and F ood Chemistry 58, 6270

- 6276.USDA Petition for N onregulated Status of DAS

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Contains No Confidential Business Information Bhatti, M.A., Duan, J.J., Head, G.P., Jiang, C., McKee, M.J., Nickson, T.E., Pilcher, C.L., Pilcher, C.D., 2005. Field evaluation of the impact of corn rootworm (coleoptera: Chrysomelidae)

–

protected Bt corn on foliage

- dwelling arthropods. Environmental Entomology 34, 1336

- 1345. Bilyeu, K.D., Zeng, P., Coello, P., Zhang, Z.J., Krishnan, H.B., Bailey, A., Beuselinck, P.R., Polacco, J.C., 2008. Quantitative conversion of phytate to inorganic phosphorus in soybean seeds expressing a bacterial phytase. Plant Physiology 146, 468

- 477. Bitzer, R.J., Rice, M.E., Pilcher, C.D., Pilcher, C.L., Lam, W.

- k.f., 2005. Biodiversity and community structure of epedaphic and euedaphic springtails (collembola) in transgenic rootworm Bt corn. Environmental Entomology 34, 1346

- 1376. Bradford, K.J., 2006. Methods to maintain genetic purity of seed stocks. ANR Publication 8189, 5. Bravo, A., Gomez, I., Conde, J., Munoz

- Garay, C., Sanchez, J., Miranda, R., Zhuang, M., Gill, S.S., Soberon, M., 2004. Oligomerization triggers binding of a Bacillus thuringiensis Cry1Ab pore

- forming toxin to aminopeptidase N receptor leading to insertion into membrane microdomains. Biochimica et Biophysica Acta 1667, 38

- 46. Cardineau, G.A., Stelman, S.J., Narva, K.E., 2001. Plant

- optimized genes encoding pesticidal toxins. 6,218,188 B1. Carstens, K., Anderson, J., Bachman, P., De Schrijver, A., Dively, G., Federici, B., Hamer, M., Gielkens, M., Jensen, P., Lamp, W., Rauschen, S., Ridley, G., Romeis, J., Waggoner, A., 2011. Genetically modified crops and aquatic ecosystems: Considerations for environmental risk assessment and non

- target organism testing. Tra nsgenic Research, 1

- 30. CAST, 2009. Sustainability of U.S. Soybean production: Conventional, transgenic, and organic production systems. http://www.cast

- science.org/publications/

. Catchot, A., 2011. S oybean insect pest problems in the mid

- south, what to watch for in 2011. Caviness, C.E., 1966. Estimates of natural cross

- pollination in Jackson Soybeans in Arkansas. Crop Science 6, 211

- 212. Coates, B.S., Sumerford, D.V., Hellmich, R.L., Lewis, L.C., 200 5. Sequence variation in the cadherin gene of Ostrinia nubilalis : A tool for field monitoring. Insect Biochemistry and Molecular Biology 35, 129

- 139. Coll, A., Nadal, A., Palaudelmàs, M., Messeguer, J., Melé, E., Puigdomènech, P., Pla, M., 2008. Lack of re peatable differential expression patterns between MON810 and comparable commercial varieties of maize. Plant Molecular Biology 68, 105

- 117. Conner, A.J., Glare, T.R., Nap, J.P., 2003. The release of genetically modified crops into the environment. Part II. Overview of ecological risk assessment. The Plant Journal 33, 19

- 46. Curran

- Everett, D., 2000. Multiple comparisons: Philosophies and illustrations. American Journal of Physiology

- Regulatory, Integrative and Comparative Physiology 279, R1

- R8. Dalley, C. D., Renner, K.A., Kells, J.J., 2002. Weed competition in Roundup Ready soybeans and corn. http://msue.anr.msu.edu/

. Daly, T., Buntin, G.D., 2005. Effect of Bacillus thuringiensis transgenic corn for lepidopteran
con trol on nontarget arthropods. Environmental Entomology 34, 1292

- 1301. de Freitas Bueno, R.C.O., de Freitas Bueno, A., Moscardi, F., Postali Parra, J.R., Hoffmann

Campo, C.B., 2011. Lepidopteran larva consumption of soybean foliage: Basis forUSDA Petition for N onregulated Status of DAS

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Contains No Confidential Business Information developingmu ltiple

- species economic thresholds for pestmanagement decisions. Pest Management Science 67, 170

- 174. Dively, G.P., 2005. Impact of transgenic vip3a × cry1ab lepidopteran

- resistant field corn on the nontarget arthropod community. Environmental Entomology 34, 1267

- 1291. Dow AgroSciences, 2000. Petition for determination of non

- regulated status B.t. Cry1F insect

resistant glufosinate

- tolerant maize line 1507. http://www.aphis.usda.gov/brs/a phisdocs/00_13601p.pdf Dow AgroSciences, 2003a. Petition for determination of non

- regulated status B.t. Cry1Ac insect

resistant cotton event 3006

- 210

- 23. http://www.aphis.usda.gov/brs/aph isdocs/03_03602p.pdf

. Dow AgroSciences, 2003b. Petition for determination of non

- regulated status B.t. Cry1F insect

resistant cotton event 281

- 24

- 236. http://www.aphis.usda.gov/brs/aphisdo cs/03_03601p.pdf

. Dow AgroSciences, 2003c. Petition for the determination of nonregulated status Bt Cry34/35Ab1 insect

- resistant, herbicide

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