## B **IOPESTICIDES REGISTRATION ACTION DOCUMENT** 

**PLANT -**  **INCORPORATED PROTECTANT S**  **:** Bacillus thuringiensis Cry1A c P rotein and the Genetic Material Necessary

for its Prod

uction [ PC Code 00652 7 ] and Bacillus thuringiensis Cry 1F P rotein and the Genetic Material Necessary

for its Prod

uction [ PC Code 006 528 ] as expressed i n Event DAS - 81419 - 2 Soy bean ( PIP product) [ OECD Unique Identifier: DAS - 81419 - 2 ]

**U.S. Environmental Protection Agency**  **Office of Pesticide Programs Biopesticides and Pollution Prevention Division** 

January 9 , 201 4Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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### Bacillus thuringiensis **Cry1A c**  **and Cry**  **1F as**  **DAS -**  **81419 -**  **2 S o**  **ybea n**  **Table of Contents BIOPESTICIDES REGISTRATION ACTION DOCUMENT TEAM**  **................................ ................................**  **................ 3**  **GLOSSARY OF ACRONYMS AND ABBREVIATIONS ................................**  **................................ ................................**  **.... 4**  **EXECUTIVE SUMMARY ................................**  **................................ ................................**  **................................ .......................**  **5 I.**  **ACTIVE IN GREDIENT OVERVIEW**  **................................ ................................**  **................................ ...........................**  **7 II.**  **REGULATORY BACKGROUND ................................**  **................................ ................................**  **................................ ..**  **8 III.**  **RISK ASSESSMENT SUMMARIES ................................**  **................................ ................................**  **.............................. 9**  A. Product Characterization ................................ ................................ ................................ ................................ ................ 9 B. Human Health Assessment ................................ ................................ ................................ ................................ ............ 10 C. Environmental Effects Assessment ................................ ................................ ................................ ................................ .. 16 D. Insect Resistance Management ................................ ................................ ................................ ................................ ....... 19 **IV. ENVIRONMENTAL JUSTICE**  **................................ ................................**  **................................ ................................**  **. 20**  **V. RISK MANAGEMENT AND PROPOSED REGISTRATION DECISION**  **................................ ..........................**  **21 VI.**  **TERMS AND CONDITIONS OF THE REGISTRATION(S) ................................**  **................................ .................**  **21 APPENDIX A: Product characterization and hum**  **an health effects –**  **Bacillus thuringiensis Cry 1A**  **c and**  **Cry1 F p**  **roteins expressed**  **in Event**  **DAS -**  **81419 -**  **2 S**  **o ybea**  **n plant -**  **incorporated protectant. ................................**  **............. 23**  **APPENDIX B**  **: Ecological and environmental effects –**  **Bacillus thuringiensis Cry1A**  **c and**  **Cry 1F**  **proteins expressed in Event**  **DAS -**  **81419 -**  **2 S**  **o ybea**  **n plant -**  **incorporated protectant ................................**  **................................ .....**  **34 APPENDIX**  **C : Insect Resistance Managem**  **ent ................................**  **................................ ................................**  **.................... 62**  **APPENDIX D**  **: References ................................**  **................................ ................................**  **................................ ......................**  **69 APPENDIX E: Bibliography of data submitted for registration of**  **Bacillus thuringiensis Cry1Ac and Cry1F**  **proteins expressed in Event DAS -**  **81419 -**  **2 Soybean plant -**  **incorporated protectant...... ........................**  **...76**Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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### BIOPESTICIDES REGISTRATION ACTION DOCUMENT TEAM Microbial Pesticides Branch Product Characterization and Human Health John Kough, Ph.D. Chris Wozniak, Ph.D. Environmental Fate and Effects Shannon Borges, M.S. Annabel Waggoner, B.S. Insect Resistance Management Alan Rey nolds, M.S. Office of General Counsel Angela Huskey Chris Kaczmarek Biopesticides Registration Action Document Regulations Denise Greenway , B . S . Kimberly Nesci, M.S. Alan Rey nolds, M.S.Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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### GLOSSARY OF ACRONYMS AND ABBREVIATIONS APHIS Animal and Plant Health Inspection Service (USDA) b p Base pairs BPPD Biopesticides and Pollution Prevention Division BRAD Biopesticides Registration Action Document b.w . body weight CAS Chemical Abstracts Service 40 CFR Title 40 of the Code of Federal Regulations cDNA Copied (or Copy) DNA cfu colony forming units dsRNA Double - Stranded RNA cos Cosmid o C Tem perature in Centigrade or Celsius Degrees DER Data Evaluation Record DNA Deoxyribonucleic Acid EPA Environmental Protection Agency (the “Agency”) ELISA Enzyme - Linked Immunosorbent Assay EPA Reg . No. EPA Registration Number FFDCA Federal Food, Drug, and Cosmetic Act FIFRA Federal Insecticide, Fungicide, and Rodenticide Act FQPA Food Quality Protection Act FR Federal Register g Gram IU International Units IRM Insect Resistance Management kg kilogram L Liter MRID No. Master Record Identification Number mg Milligram mL Milliliter μg Microgram MP Manufacturing - Use Product mRNA Messenger RNA NE No Effect ng nanogram (10 - 9 gram) NIOSH National Institute for Occupational Safety and Health OPP Office of Pesticide Programs OCSPP Office of Chemical Safety and Pollution Prevention OECD Organisati on for Economic and Community Development PC Pesticide Chemical PCR Polymerase Chain Reaction PIP Plant - In corporated Protectant pg picogram (10 - 12 gram) ppb parts per trillion ppm parts per million ppt parts per trillion PTGS Post - Transcriptional Gene Silencing RNA Ribonucleic Acid rRNA Ribosomal RNA T - DNA transfer DNA from Agrobacterium TGAI Technical Grade of the Active Ingredient USDA United States Department of Agriculture U . S . EPA (EPA) United States Environmental Protection Agency U . S . FDA United States Food and Drug AdministrationBt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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### EXECUTIVE SUMMARY The United States Environmental Protection Agency (U . S . EPA) proposes to regist er two new Bacillus thuringiensis (

Bt ) s o yb ean plant - incorporated protectant (PIP) active ingredients as described in this Biopesticide Registration Action Document (BRAD) . On July 24, 2012 , Dow AgroSciences LLC (Dow ) submitted an application for a breeding/ seed increase registration of a new transgenic s o ybean ( G lycine m ax) PIP, Event DAS - 81419 - 2 Soy bean , EPA File Symbol 68467 - EN . The proposed PIP is a new transformation event that expresses lepidopteran - active insecticidal protein s (also known as “Cry” proteins) derived from the soil bacterium Bacillus thuringiensis ( Bt) . The proposed new PIP expresses both the Cry 1Ac and Cry 1F proteins in soy bean. Different events of Cry 1Ac have been previously registered in corn, cotton, and so ybean. Similarly, separate events containing Cry 1F have been registered in corn and cotton. However, the expression of Cry 1F and Cry 1Ac proteins in Event DAS - 81419 - 2 S oy bean represent s their first use together in soy bean. Dow concurrentl y filed a petition for a permanent exemption from the requirement of a tolerance for Cry 1F protein residues in or on soy bean food and feed commodities . On October 1, 2009, the EPA announced a new policy to provide a more meaningful opportunity for the public to participate in major registration decisions before they occur. According to this policy , the EPA intends to provide a public comment period prior to making a registration decision for, at minimum, the following types of applications: new active ingredients; first food uses; first outdoor uses; first residential uses; or any other registration actions for which the Agency believes there may

be significant public interest. Consistent wi th the policy of making registration actions more transparent, the proposed Bt Plant - In corporated Protectant (PIP) product, DAS - 81419 - 2 Soy bean (EPA File Sy

mbol 68467 - EN), expressing both the Cry 1Ac and Cry 1F proteins, is subject to a 15 - day comment period as a new active ingredient . The docket identification number associated with these registration actions, and accessed through either http://www.regulations.gov or http://www.epa.gov/pesticides/regulating/registration -status.html , is EPA - HQ - OPP - 2013 - 070 3 . The following documents are available for comment in EPA - HQ - OPP - 2013 - 0 70 3 : (1) This draft Biopesticides Registration Action Document (BRAD); (2) The December 30, 2013, Environmental Risk A ssessment for PIP Event DAS - 81419 - 2 ; (3) The November 7, 2013, Review of Insect Resistance Management (IRM) Considerations for Bt soy bean event DAS - 84149 - 2 (4) The November 22, 2013, Review of Human Health and Product Characterization Data for Registration of B. thuringiensis Cry 1Ac and Cry 1F Proteins and the Genetic Material Necessary for their Production in DA S - 81419 - 2 Soy bean. and ( 5 ) a draft label for the PIP ( EPA File Sy mbol 68467 - EN)Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 6 of 78 While a final decision on registration is contingent upon review and consideration of public comments, EPA believes that, based upon the data and information submitted in support of the proposed Bt s oy

bean Plant - i ncorporated Protectant (PIP) pesticide prod uct, DAS - 81419 - 2 Soy bean , it is appropriate to issue the registration. The basis for this preliminary decision can be found in the risk assessment s for the proposed Bt s oy bean Plant - Incorporated Protectant (PIP) pesticide product, DAS - 81419 - 2 Soy bean , whic h are characterized throughout this BRAD. This BRAD presents EPA’s evaluations of the data and information submitted by Dow for the proposed registration of

: 1. EPA File Sy

mbol 6 8 4 67 - EN: Event DAS - 81419 - 2 S o yb ean express ing Bt Cry 1Ac and Bt Cry 1F insecticidal protein s . The genes that are responsible for the Bt Cry 1A c and Bt Cry 1F proteins, cry1A c and cry 1F , have been modified for their expression in plants . These proteins are referred to as Cry 1A c and Cry 1F , respectivel y, in this BRAD. The e vent also include s the pat gene th at produces Phosphinothricin Acetyl transferase ( PAT ) enzy me, an inert ingredient in the proposed product that confers tolerance of the s o ybean plant to the herbicide, gl ufosinate. DAS - 81419 - 2 was developed by Agrobacterium - m ediated transformation of soy bean using the 2T - DNA plasmid vector pDAB9582 and produces Bacillus thuringiensis protein δ - endotoxins, Cry 1Ac and Cry 1F. A single T - DNA insert containing each of the intact plant transcription units (PTUs) for the cry1Fv3, cry1Ac(synpro), and pat genes was integrated into soy bean to create DAS - 81419 - 2. This protein is intended to provide protection from feeding damage caused by a number of lepidopteran pests. Dow applied for pesticide registration of Cry 1A c and Cry 1F pro teins as co - expressed in Event DAS - 81419 - 2 s o yb ean as a new PIP product, DAS - 81419 - 2 S o ybean , for use as a PIP in 20 14 , under section 3 of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) . Dow concurrently filed a petition for a permanent ex emption from the requirement of a tolerance for Cry 1F protein residues in or on soy bean food and feed commodities . The EP A believes that, based upon its assessment of the data and information submitted by Dow , it is in the interest of the public and the environment to issue the registration proposed b y Dow .Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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### I. **ACTIVE INGREDIENT OVERVIEW Active**  **Ingredients and Office of**  **Pesticide Programs (OPP)**  **Chemical Code and File Symbol:**  Bt Cry 1A c insecticidal protein and the genetic material necessary for i t s production in DAS - 81419 - 2 S o ybean PIP product : 006527 Bt Cry 1F insecticidal protein and the genetic material necessary for i t s production in DAS - 81419 - 2 S o ybean PIP product : 006528 EPA File Sy

mbol 6 8 4 67 - EN. OECD Unique Identifier: DAS - 81419 - 2 **Applicant/ Manufacturer:**  Dow AgroSciences LLC (Dow)

9330 Zionsville Road Indianapolis, In 46268 **Type of Pesticide:** Plant - i ncorporated Protectant (PIP) **Use:** Insecticide **Target Pest s**  **:** Larval stage lepidopteran pests of soy bean , such as soy

bean looper ( Pseudoplusia includens ), velvetbean caterpillar ( Anticarsia gemmatalis ), beet arm ywo rm (Spodoptera exigua ) , c otton bollworm/ corn earworm ( Helicoverpa zea ), green cloverworm (Plathypena scabra ), and cutworms ( Agrotis spp.) **Mode of action:** Insecticides formulated with microbial Bt have been in use worldwide since 1961. These microbial active ingredients are the source of the genes that express the Cry1A c and Cry 1F proteins in the proposed Bt Soy bean PIP. There is no mechanistic evidence that Bt proteins pose risks for humans or mammals during their use as pesticides or as PIPs (OECD, 2007). When Cry proteins are ingested b y an insect , they are dissolved under the alkaline conditions specific to the insect ’s gut , and then activated upon cleavage b y the ins ect’s midgut enzymes (proteases ) . These activated Cry

proteins must bind specifically to glycoprotein or gl ycolipid receptors on microvillar membrane of the insect’s midgut to initiate the pore - forming process that ultimately kills the insect . The fragment of the protein that is toxic to insects then interacts with specific high - affinity

receptors on the microvilli of the target insect’s midgut epithelium (stomach), in particular brush border membrane vesicles (BBMV) (Ferré and Van Rie , 2002). Such receptors are not present in vertebrate species. Cry proteins must enter the cell membrane and form a pore. Although receptor binding appears to be essential for the insecticidal activity of the cry stal proteins, binding by itself may not l ead to toxicity . It appears that the initial protein - receptor interaction or binding is a reversible process, while the irreversible insertion of at least part of the Cry protein (the fragment that is toxic to insects ) into the cell membrane is responsible for form ing the pore. Consequently , the ion gradient across the membrane is compromised , and the columnar cells of the midgut swell and l ys e osmotically . Ly sis lead s to disruption of the gut epithelium, resulting in starvation, and insect death (Ferré and Van Rie, 2002 ).Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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### II. **REGULATORY BACKGROUND Applications for use of Cry1A**  **c and Cry1F expressed in**  **DAS -**  **81419 -**  **2 Soybean PIP**  **A. Cry 1Ac**  **and Cry1A F**  **protein s**  1. Registration Application and Permanent Tolerance Exemption a. Cry 1Ac and Cry 1F PIP FIFRA Section 3 Registration Application On November 22 , 20 1 3 , EPA published a Notice of Receipt in the Federal Register ( 78 FR 70043 ) , announcing that Dow submitted an application to register a new active ingredient, Bt Cry 1A c and Cry 1F insecticidal protein s expressed in soy bean ( DAS - 81419 - 2 Soy

bean ; EPA File Sy mbol 6 8 4 67 - EN ), not included in any currentl y registered pesticide products . No public comments were received in response to this publication .

b. Cry 1Ac protein - Permanent Tol erance E xemption Residues of Cry 1A c protein are exempt from the requirement of a tolerance when used as PIPs in all food commodities ( 40 CFR § 174.510 ) . EPA granted an exemption from tolerance for Cry 1Ac protein in all food and feed commodities on April 11, 1997 (62 FR 17722) . The tolerance exemption wa s published in the Code of Federal Regulations ( 40 CFR § 180.1155 ) . In Octob er 2001, EPA completed a reassessment of this tolerance exemption considering al l of the existing data, public l iterature, and public comments. The reassessment determined that the tolerance exemption met all the required scientific and regulatory standards. This to lerance exemption for the Cry 1Ac protein is not e vent - sp ecific and applies to all Cry 1Ac protein and any other e vent producing the Cry 1Ac protein that might be found in the food supply . On April 25, 2007 , th e tolerance exemption for Cry 1Ac protein was reassigned to 40 CFR § 174.51 0 ( 72 FR 20435) . Based on this existing to lerance exemption , a petition to establish a tolerance or tolerance exemption was not required for the Cry 1Ac protein expressed in this soy bean PIP. c. Cry 1F protein - Permanent Tolerance Exemption Concurrentl y with the FIFRA section application, Dow submitted a petition (PP 2F8066) to establish a permanent exemption from the requirement of a tolerance for residues of Bacillus thuringiensis Cry 1F protein in or on soy bean. EPA published a Notice of Filing of the petition in the Federal Register o n Novemb er 22, 2013 ( 78 FR 70007) and the public was given a 30 - day comment period . No public comments were received in response to this publication . Event DAS - 81419 - 2 Soybean also expresses the PAT enzy me that is exempt from the requirement of a tolerance when used as a PIP inert ingredient in all food commodities ( 40 CFR § 174.522 ) .Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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### I **II .**  **RISK ASSESSMENT SUMMARIES** EPA assessed the data submitted by Dow in 20 12 for the proposed PIP s o ybean registration and is presenting s ummaries of the product characterization and human health risk assessment s in Appendi x A (Cry 1A c and Cry 1F expressed in soy bean DAS - 81419 - 2 Soy

bean) . Summaries of ecological and environmental risk assessments and Insect Resistance Management (IRM) for the PIP product are presented in Appendices B and C , respectivel y. In its assessment, the EPA relied upon data and other information submitted by the applicant and general knowledge of best available scientific technology . The classifications that are found for each data submission are assigned b y Agency science reviewers and are an indication of the usefulness of the information contained in the documents for risk assessment. A rating of “ ACCEPTABLE ” indicates the study is scientifically

sound and is useful for risk assessment. A “SUPPLEMENTAL” rating indicates the data provide some information that can be useful for risk assessment. The studies may have certain aspects determined not to be scientifi cally acceptable (“SUPPLEMENTAL: UPGRADABLE”). If a study is rated as “SUPPLEMENTAL: UPGRADABLE,” the Agency always provides an indication of what is lacking or what can be provided to change the rating to “ACCEPTABLE.” If there is simply

a “SUPPLEMENTAL” rating, the reviewer will often state that the study is not required b y the current 40 Code of Federal Regulations (CFR) part 158. Both “ACCEPTABLE” and “SUPPLEMENTAL” studies may be used in the risk assessment process as appropriate. An “UNACCEPTABLE” rat ing indicates that new data need to be submitted. For the acute toxicity

data requirements, toxicity categories are assigned based on the hazard(s) identified from studies and/or other information submitted to the Agency in support of a pesticide registration. The active ingredient or particular product is classified into Toxicity Category I , II, III, or IV, where Toxicity Category Iindicates the highest toxicity and Toxicity Category IV indicates the lowest toxicity . 

### A. **Product Characterization** All product characterization data requirements for the Bt soybea n PIP containing the Cry 1A c and Cry 1F proteins and their co - expressed traits in DAS - 81419 - 2 Soy bean have been satisfied. The soy bean e vent developed by Dow to express these PIPs is : Event DAS - 81419 - 2 Soybean : co - expresses Cry 1A c and Cry 1F protein s , derived from Bacillus thuringiensis ( Bt ) . It was developed by Agrobacterium - mediated transformation of so ybean using the 2T - DNA plasmid vector pDAB9582 and produces Bacillus thuringiensis protein δ - endotoxins, Cry 1Ac and Cry 1F. A single T DNA insert containing each of the intact plant transcription units (PTUs) for the cry1Fv3, cry1Ac(synpro), and pat genes was integrated into soy bean to create DAS - 81419 - 2. DAS - 81419 - 2 Soy

bean co - expres ses Cry 1A c and Cry 1F proteins to provide protection against feeding dama ge b y lepidopteran insect larvae.Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 10 of 78 For summaries of the data evaluated for product characterization for th ese PIPs, see Appendix A . Th e summaries include discussions of the following:

 Product Characterization  Transformation S ys tem and Genetic Elements  Protein Characterization  Analy tical Detection Method s  Protein Expression As discussed in the summaries, Cry 1A c and C ry 1F proteins are expressed at very low parts per million (ppm) levels in various parts of the transgenic soy bean plant and are shown to be equivalent to the microbially expressed proteins 

### B. **Human Health Assessment 1**  **. Toxicological Profile**  EPA reviewed the available scientific data and other relevant information submitted in support of th is action to register Cry1A c and Cry 1F as the PIP DAS - 81419 - 2 , and considered its validity , completeness and reliability , and the relationship of this information to human risk . EPA has also considered available information concerning the variability of the sensitivities of major identifiable subgroups of consumers, including infants and children . All toxicology

data requirements for the Bt s o yb ean PIP containing Cry 1A c and Cry 1F proteins co - expressed in DAS - 81419 - 2 Soy bean have been satisfied**.** Acceptable Tier I mammalian toxicology

data/information support the proposed registration of the active ingredients . Furthermore, Tier II and Tier III studies were not required for either Cry 1A c or Cry 1F proteins , based on the lack of acute toxicity or pathogenicity

in the Tier I studies. Refer to Table s in Appendi x A for brief summaries of data evaluated to satisfy toxicity and allergenicity data requirements for these PIPs. **Acute toxicity Cry1Ac**  **and Cry 1F**  **proteins** The following summarizes the final determinations regarding acute toxicolog y data requirements for Cry 1A c and Cry 1F proteins . See Appendi x A in this BRAD for more detail ed summaries . **Cry1Ac** Previously

submitted acute oral toxicity data demonstrated a lack of mammalian toxicity at high levels of exposure (ingestion) to the pure Cry 1Ac protein. These data demonstrate the safety of this protein at a level well above maximum possible consumption levels that are reasonabl y anticipated as expressed in the crop. Basing this conclusion on acute oral toxicity data without requiring further toxicity

testing and residue data is similar to the Agency position regarding toxicity testing and the requirement of residue data for the microbial Bacillus thuringiensisBt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 11 of 78 products from which this plant - incorporated protectant was derived (See 40 CFR Sec. 158.740(b)(2)(i)). For microbial products, further toxicity testing (Tiers II & III) and residue data are triggered b y significant adverse acute effects in studies, such as the acute oral toxicity study , to verify the observed adverse effects and clarify the source of these effects. An acute oral toxicity

study in mice (MRID No. 45542313) indicated that Cry 1Ac is n on - toxic to humans and other mammals. Cry 1Ac protein is a δ - endotoxin from B. thuringiensis that has been used extensively

in both microbial and plant - incorporated protectants as a means of insect pest management. An existing exemption from the requirement of a tolerance for Cry 1Ac (CFR 40 Section 174.510; 72 FR 20435, April 25, 1997) in all food and feed commodities precluded the need for a separate tolerance action in conjunction with this review of product characterization and human health assessment. **C ry1F**  An acute oral toxicity

study in mice (MRID No. 45542314) indicated that Cry 1F is non - toxic to humans and other mammals. Cry 1F protein is a δ - endotoxin from B. thuringiensis that has been used extensively

in plant - incorporated protectants as a means of insect pest management. An existing exemption from the requirement of a tolerance for Cry 1F (CFR 40 Section 174.504; 72 FR 20434 , Apr. 25, 2007 ) in cotton and corn (CFR 40 Secti on 174. 520; 72 FR 2043 5 Apr. 25, 2007 ) , precluded the need for a separate tolerance action in conjunction with this review of product characterization and human health assessment . Cry 1F was previously granted an exemption from the requirement of a toleranc e for expression in cotton (CFR 40 Section 174.504) and maize (CFR Section 174.520); a separate petition for an exemption from the requirement of a tolerance for Cry 1F as expressed in soy bean (PP2F8066) is included with the submission from Dow for registration of DAS - 81419 - 2 soy bean. When proteins are toxic, they are known to act via acute mechanisms and at very low dose levels (Sjoblad et al., 1992). Therefore, since no acute effects were shown to be caused by Cry 1Ac and Cry 1F; even at relativel y high dose levels, the Cry1Ac and Cry 1F protein s are not considered toxic to humans and other mammals . Further, amino acid sequence comparisons showed no similarities that would raise a safety concern between the Cry 1Ac and Cry 1F protein s and known toxic proteins in protein databases **Hypersensitivity Incidents** : Reporting is required when incidents occur . No hy persensitivity incidents, including immediate - ty pe or delayed - ty

pe reactions in humans or animals, occurred during the manufacture of Bt Cry 1A c and Cry 1F proteins a s DAS - 81419 - 2 Soy bean PIP. Should any future hy persensitivity

incidents occur, they

must be reported to the Agency . **Allergenicity Assessment** Since Cry 1Ac is a protein, allergenic potential was also considered. Currently , no definitive tests for determining the allergenic potential of novel proteins exist. Therefore, EPA uses a weight- ofevidence approach where the following factors are considered: source of the trait; amino acid sequence comparison with known al lergens; and biochemical properties of the protein, including in vitro digestibility

in simulated gastric fluid (SGF) followed by simulated intestinal fluid (SIF), and gl yc os yl ation. This approach is consistent with the approach outlined in the Annex to th eBt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 12 of 78 Codex Alimentarius “Guideline for the Conduct of Food Safety Assessment of Foods Derived from Recombinant - DNA Plants.” The allergenicity assessment for Cry 1Ac follows: 1. Source of the trait. Bacillus thuringiensis is not considered to be a source of allergenic proteins. 2. Amino acid sequence. A comparison of the amino acid sequence of Cry 1Ac with known allergens showed no significant overall sequence similarity or identity

at the level of eight contiguous amino acid residues. 3. Digestibility . (MRID No. 455 42319) The Cry 1Ac protein was digested rapidly in simulated gastric fluid containing pepsin. Small peptides remaining following gastric simulated digestion were completely degraded to amino acid residues in SIF upon contact (US EPA 2005 , 2010a). 4. Glycos yl ation. Cry 1Ac expressed in soy bean was shown not to be gl ycos yl ated. 5. Conclusion: Considering all of the available information, EPA has concluded that the potential for Cry 1Ac to be a food allergen is minimal. Since Cry 1F is a protein, allergenic potential was also considered. Currently, no definitive tests for determining the allergenic potential of novel proteins exist. Therefore, EPA uses a weight- ofevidence approach where the following factors are considered: source of the trait; amino acid sequence co mparison with known allergens; and biochemical properties of the protein, including in vitro digestibility

in simulated gastric fluid (SGF) followed by simulated intestinal fluid (SIF), and gl yc os yl ation. This approach is consistent with the approach outli ned in the Annex to the Codex Alimentarius “Guideline for the Conduct of Food Safety Assessment of Foods Derived from Recombinant - DNA Plants.” The allergenicity assessment for Cry 1F follows: 1. Source of the trait. Bacillus thuringiensis is not considered t o be a source of allergenic proteins. 2. Amino acid sequence. A comparison of the amino acid sequence of Cry 1F with known allergens showed no significant overall sequence similarity or identity

at the level of eight contiguous amino acid residues. 3. Digestibi lity . (MRID No. 45542318) The Cry 1F protein was digested rapidly (< 5 min) in simulated gastric fluid containing pepsin. Small peptides remaining following gastric simulated digestion were completely degraded to amino acid residues in SIF upon contact (US EPA 2005 , 2010b). 4. Glycos yl ation. Cry 1F expressed in soy bean was shown not to be gl ycos yl ated. 5. Conclusion: Considering all of the available information, EPA has concluded that the potential for Cry 1F to be a food allergen is minimal. Cry 1Ac and Cry 1F were shown not to be gl ycos yl ated in extracts of DAS - 81419 - 2 soy bean ; it is unlikely to be gl ycos yl ated in any other crops because in order for a protein to be gl ycosl yat ed, it needs to contain specific recognition sites for the enzy mes involved in gl yc osylation, and the mechanisms of protein glycosylation are similar in different plants (Lerouge et al., 1998). Considering all of the available information, EPA has concluded that the potential for Cry 1A c and Cry 1F to be food allergen s is minimal. **Tolerance Exemptions**Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 13 of 78 Permanent tolerance exemptions have been established for residues of Cry1 A c and PAT , as discussed in Chapter II under REGULATORY BACKGROUND , above . EPA’s reviews of the toxicology data support granting a permanent exemption from tol erance for residues of Cry 1F protein in/on soy bean food/feed commodities under section 408 of the Federal Food, Drug, and Cosmetic Act ( FFDCA). On the basis of evaluations of all relevant data submissions for Cry 1F , EPA has determined that the data support amendment of 40 CFR §174 to establish a permanent tolerance exemption for residues of Cry 1F in/on soy bean food and feed commodities. **2. Endocrine Disruptors** As required under FFDCA section 408(p), the Agency has developed the Endocrine Disruptor Screening Program (EDSP) to determine whether certain substances (including pesticide active and other ingredients) may have an effect in humans or wildlife similar to an effect produced by a “naturally

occurring estrogen, or other such endocrine effects a s the Administrator may designate.” The EDSP employ s a two - tiered approach to making the statutorily required determinations. Tier 1 consists of a battery of 11 screening assay s to identify the potential of a chemical substance to interact with the estrogen, androgen, or thyroid (E, A, or T) hormonal sy stems. Chemicals that go through Tier 1 screening and are found to have the potential to interact with E, A, or T hormonal sy stems will proceed to the next stage of the EDSP where the Agency will determine wh ich, if any, of the Tier 2 tests are necessary based on the available data. Tier 2 testing is designed to identify any adverse endocrine - related effects caused by the substance, and establish a quantitative relationship between the dose and the E, A, or T effect. Between October 2009 and February 2010, the Agency

issued test orders/data call - ins for the first group of 67 chemicals, which contains 58 pesticide active ingredients and 9 inert ingredients. This list of chemicals was selected based on the potential for human exposure through pathway s such as food and water, residential activity , and certain post - application agricultural scenarios. This list should not be construed as a list of known or likely endocrine disruptors. Cry 1A c and Cry 1F proteins are not among the group of 58 pesticide active ingredients on the initial list to be screened under the EDSP. Under FFDCA section 408(p), the Agency must screen all pesticide chemicals. Accordingl y, the Agency anticipates issuing future EDSP orders/data call - i ns for all pesticide active ingredients. For further information on the status of the EDSP, the policies and procedures, the list of 67 chemicals, the test guidelines and the Tier 1 screening battery , please visit our website: http://www.epa.gov/endo/ . **3 .**  **Food Quality Protection Act (FQPA) Considerations a.**  **Aggregate Exposures**Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 14 of 78 In examining aggregate exposure, section 408 of FFDCA directs EPA to consider available information concerning exposures from the pesticide residue in food and all other non occupational exposures, including drinking water from ground water or surface water and exposure through pesticide use in gardens, lawns, or buildings (residential and other indoor uses). EPA considered availabl e information on the aggregate exposure levels of consumers (including major identifiable subgroups of consumers) to the PIP residue and to other related substances. These considerations include dietary exposure under the tolerance exemption and all other tolerances or exemptions in effect for the PIP residue, and exposure from non - occupational sources. As previously

discussed, data indicated that Cry 1F protein is non - toxic to humans and other mammals, and Cry 1F protein was shown to be rapidly digested in vitro. Exemptions from the requirement of a tolerance already have been established for Bacillus thuringiensis Cry 1F protein in cotton (40 CFR § 174.504) and corn (40 CFR § 174.520). The EP A has considered dietary exposure under the tolerance exemption and all other exemptions in effect for the PIP residue and exposure from non - occupational sources. When Cry 1F protein is used as a PIP in soy bean, it i s expressed at very

low levels in the plant. Humans may be exposed to extremel y low levels in the diet. There is also a very remote possibility

that Cry 1F protein can get in the water suppl y the same way that other proteins in crop debris can migrate into the ground, and, possibly, drinking water . Because such potential dietary exposure from soy

bean or drinking water is expected to be several orders of magnitude lower than the amounts of these proteins shown to have no toxicity in mammalian tests, EPA concludes that negligible exposure via food an d drinking water would present no harm, based on the lack of mammalian toxicity and allergenicity

potential, and the rapid digestibility

demonstrated in SGF for the PIP. Non - occupational dermal and inhalation exposure is not expected, since the PIP is expressed and contained within soy

bean plant cells, which essentially eliminates these exposure routes or reduces these exposure routes to negligible. The use sites of this PIP are all agricultural, for insect control, so there would be no exposure to inf ants and children from residential, school or lawn use. The amino acid sequence homology of known aeroallergens was included in the amino acid comparison of Cry 1F protein with known food allergens, and the results indicated that no respiratory allergeni city would be expected if Cry1F protein were inhaled. The amino acid sequence results are discussed in Section II.B., above. Additionally , soy bean flowers in a self

pollinating fashion wherein the anthers generall y release pollen within a closed or covered fl oral tube. Pollen movement from soy bean is minimal and highl y localized when it happens (i.e., the pollen does not move readily on wind currents). Taking all these data and information into consideration, EPA concludes that even if negligible aggregate exposure should occur it would present no harm to the U.S. human population due to the lack of mammalian toxicity and the rapid digestibility demonstrated for the Cry 1F protein. **b. Cumulative Effects from Substances with a Common Mechanism of Toxicity** Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 15 of 78 Section 408(b)(2)(D)(v) of FFDCA requires that, when considering whether to establish, modify , or revoke a tolerance, the Agency consider “available information” concerning the cumulative effects of a particular pesticide’s residues and “other substances that have a common mechanism of toxicity .” Cry 1F protein is not considered toxic . Itdoes not share a common mechanism of toxicity with any other substances, nor do es it appear to produce a toxic metabolite produced b y other substances. For the purposes of this assessment , therefore, EPA has assumed that Cry 1 F protein does not have a common mechanism of toxicity with other substances. Thus , EPA concludes that there are no cumulative effects associated with Cry 1 F protein that need be considered . For information regarding EPA’s efforts to determine which chemicals have a common mechanism of toxicity

and to evaluate the cumulative effects of such chemicals, see EPA’s website at http://www.epa.gov/pesticides/cumulative **c .**  **Determination of Safety for U.S. Pop ulation, Infants and Children**  To evaluate human risk, EPA considered the validity , completeness, and reliability of the available data from the studies cited in Unit III regarding potential health effects for Cry 1F protein . This evaluation included the l ow levels of expression of Cry 1F protein in soy bean, as well as the lack of acute oral toxicity at high dose levels, heat stability , and in vitro digestibility of this protein. EPA also considered the minimal potential for allergenicity and the non - toxic s ource of the protein. Because of this lack of demonstrated mammalian toxicity , no protein residue chemistry

data for Cry

1F were required for a human health effects assessment. Finally , and specificall y with regards to infants and children, FFDCA section 408(b)(2)(C) provides that EPA shall assess the available information about consumption patterns among infants and children, special susceptibility of infants and children to pes ticide chemical residues, and the cumulative effects on infants and children of the residues and other substances with a common mechanism of toxicity . In addition, FFDCA section 408(b)(2)(C) provides that EPA shall apply

an additional tenfold margin of saf ety for infants and children in the case of threshold effects to account for prenatal and postnatal toxicity and the completeness of the data base unless EPA determines that a different margin of safety will be safe for infants and children. Based on its review and consideration of all the available information, as discussed in this BRAD , EPA concluded that there are no threshold effects of concern and, as a result, that an additional margin of safety for infants and children is unnecessary in this instan ce. **4. International Residue Limits**  In making its tolerance decisions, EPA seeks to harmonize U.S. tolerances with international standards whenever possible, consistent with U.S. food safety standards and agricultural practices. In this context, EPA con siders the international maximum residue limits (MRL s) established by

the Codex Alimentarius Commission (Codex), as required b y FFDCA section 408(b)(4). The Codex Alimentarius is a joint U.N. Food and Agriculture Organization/World Health Organization food standards program, and it is recognized as an international food safety standards - setting organization in trade agreements to which the United States is a party. EPA may establish a tolerance that is different from a Codex MRL ; however, FFDCA sectionBt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 16 of 78 408(b)(4) requires that EPA explain the reasons for departing from the Codex level. No Codex maximum residue level exists for Bacillus thuringiensis Cry 1A c and Cry 1F protein s as expressed in Event DAS - 81419 - 2 S oy bean . 

### C. **Environmental Effects Assessment Ecological Effects Data for**  **Cry1F and Cry1Ac Insecticidal Proteins Expressed in Event**  **DAS -**  **81419 -**  **2 Soybean**  All e cological and environmental e ffects data requirements for the Bt soy bean PIPs containing Cry 1A c and Cry 1F proteins expressed in DAS - 81419 - 2 Soy bean have been satisfied. The submission of results of a confirmatory soil degradation study , however, is needed to evaluate the degradation rate of the Cry 1Ac and Cry 1F proteins sampled specifically from representative soy bean soil sy stems. The completed soil degradation study must be received by

the EPA within 18 months of the DAS - 81419 - 2 Soy bean Notice of Pesticide Registration date. For a more comprehensive discussion of the Agency ’s assessment of the data and information submitted concer ning ecological and environmental risks for the registration of the se PIPs ( Cry 1A c , Cry 1F , and their combination in DAS - 81419 - 2 Soy bean) refer to Appendix B . The tiered approach EPA uses to evaluate ecological and environmental effects , and summaries of the data evaluated , are included. In the absence of PIP - specific risk assessment guidance, EPA requires applicants for PIP registrations to meet the 40 CFR Part 158 data requirements for microbial toxins. These requirements include testing on birds, mammals, nontarget insects, honey bee, plants, and aquatic species, and information has been submitted to address these requirements. Limit dose testing on representative organisms from several taxa was performed in support of Section 3 FIFRA registration of ev ent DAS - 81419 - 2 expressing Bt- derived Cry 1F and Cry 1Ac insecticidal proteins. As stated above, BPPD’s risk assessments focus more greatl y on beneficial nontarget invertebrates, since they

are most closel y related to organisms susceptible to the insecticidal action of Bt toxins. The Cry 1F and Cry 1Ac proteins are meant to target species within the order Lepidoptera (e.g. moths). Bt toxins are known ty pically to have a limited host range, however, to address any unforeseen change in activity spectrum as a result of laboratory protein sy

nthesis and to fulfill the published registration data requirements EPA requires that test species used for nontarget insect evaluations should include several invertebrate species that are not related to the target pests. Earthw orm studies are also recommended. The applicant has requested to bridge to the Agency database of previousl y reviewed toxicity studies of select nontarget organisms (NTOs) that supported the currentl y registered WideStrike  PIP cotton product ( EPA Reg. No. 68467 - 3). The WideStrike  PIP cotton line was produced b y conventional cross breeding of cotton lines DAS - 21023 - 5 (also described as 3006 - 210 - 23) expressing Cry 1F protein and DAS - 24236 - 5 (also described as 281 - 24 - 236) expressing Cry 1Ac protein . The environmental risk assessment for the insecticidal proteins (Cry 1Ac and Cry 1F) expressed in WideStrike  cotton was previously evaluated for adverse impacts on nontarget organisms and have been shown to pose negligible risk (US EPA 2005). Since exposure may

also occur to other nontarget organisms, EPA has received a number ofBt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 17 of 78 studies and data waiver rationales to comply with the Agency ’s published data requirements on other nontarget organisms. The toxicity

of Cry 1F and Cry 1Ac proteins has been previousl y ev aluated on several species of invertebrates including: lady beetle (Hippodamia convergens ), green lacewing ( Chrysoperla rufilabris ), honey

bee ( Apis mellifera ), collembola ( Folsomia candida ), parasitic wasp ( Nasonia vitripennis ) and earthworm ( Eisenia fetida ). Two new dietary toxicity studies were submitted in support for event DAS - 81419 - 2 soy bean, which includes Northern Bobwhite ( Colinus viginianus ) and Rainbow Trout, ( Oncorhynchus Mykiss ) . The individual results for the cited and newly submitted nontarge t organism studies in support for Cry 1F and Cry1Ac are summarized in Table 1. The studies are described in more detail below, and full reviews of each study can be found in the individual Data Evaluation Records. To support data bridging from the previou sl y conducted environmental risk assessment for Cry 1F and Cry 1Ac insecticidal proteins co - expressed in WideStrike  PIP cotton line, an environmental risk assessment was conducted using a “weight - of - evidence” approach based on the following lines of evidence to support the registration of event DAS - 81419 - 2 soy bean: 1) Confirmation of biological equivalency of Cry1F and Cry 1Ac proteins expressed in event DAS - 81419 - 2 soy

bean plant and previously characterized microbial - derived Cry 1F and Cry 1Ac protein test substances; 2) No significant increase in exposure to NTOs based on calculated margins of exposure for protein concentrations in event DAS - 81419 - 2 plant tissue matrices relative to the dose concentrations used in the NTO laboratory toxicity tests co nducted on Cry 1Ac and Cry 1F ; and 3) No adverse effects on nontarget arthropods on a population and community level observed from a field monitoring study and other supporting supplemental information (e.g. meta - anal ys is, target feeding studies, and biolog ical specificity to the target pest). Exposure estimates were compared with endpoints reported from Tier I toxicity studies previously

conducted on various representative nontarget test species to determine a Margin of Exposure (MOE), the ratio of the endpoint value to the EEC. The MOEs for representative NTOs were recalculated based on high - end exposure estimates (HEEEs) (US EPA, 2009) derived from Cry 1Ac and Cry 1F protein expressi on levels quantified from various plant tissues of event DAS 81419 - 2 soy

bean in a field study . A MOE of 50% mortality at 10X the estimated environmental exposure is regarded as sufficient to demonstrate negligible risk and was also the threshold for consid eration of uncertainties in the risk assessment (US EPA 1998; US EPA 2007). If subsequent expression characterization in the commercial event leads to a safety margin of <10X, then the consequences are considered in terms of uncertainties in the ecological risk assessment below (US EPA 2007). **Endangered Species** Because of the selectivity of Cry 1F and Cry 1Ac insecticidal cry stal proteins for lepidopteran species and lack of evidence of effects on other nontarget species, the Agency has investigatedBt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 18 of 78 concer ns for Federall y listed threatened and endangered insect species in the order Lepidoptera. Because so yb ean pollen is not expected to move beyond the planted soy bean field and its immediate margins, as discussed above, any exposure to lepidopterans would be expected to occur within those areas. Exposure could occur via direct consumption of PIP event DAS - 81419 - 2 soy bean plants or consumption of DAS - 81419 - 2 pollen that falls on non - soy bean plants within the soy bean field and its immediate margins. However, or al exposure to significant amounts of Cry 1F and Cry 1Ac via pollen consumption is not likely . Little pollen is expected to be released from soy bean flowers, since soy

bean plants are predominantly self - pollinated and anthers usually dehisce and release polle n before flowers open. Airborne pollen concentrations have been measured at very

low levels within soy

bean fields (mean of 0.18 grains/cm2 /day) (Yoshimura et al. 2006). Based on this analy sis, BPPD concludes that exposure resulting from pollen falling on p otential non - soy

bean food plants in the field and immediate margins is not sufficient to cause effects in listed lepidopterans. Therefore, any significant exposure would have to occur through consumption of the DAS - 81419 - 2 soy bean plants on the field or vi a tritrophic effects, in which predatory beneficial arthropod species consume soy bean pest species feeding on PIP event DAS 81419 - 2 soy

bean plant tissues. A search of EPA’s LOCATES database indicates that three species of listed lepidopterans are present in U.S. counties in which soy beans are grown. These are the Karner blue butterfl y ( Lycaeides melissa samuelis ), St. Francis’s Satyr Butterfl y ( Neonympha mitchellii fransisci), and Mitchell’s Saty

r Butterfly

( Neonympha mitchellii mitchellii). The potential effects of Bt PIPs in corn on the Karner blue butterfly was extensively anal yz ed in BPPD’s Bt Crops Reassessment (USEPA 2001b, 2004b and 2010a and b) and in the endangered species assessment section in the BRAD for event MON87701 soy bean e xpressing Cry 1Ac protein (see US EPA 2010c). As previously

discussed, soybean pollen is not expected to be deposited on plants in or around soy bean fields in amounts sufficient to cause effects in sensitive lepidopterans. Ecology and life history informati on demonstrated that habitat requirements for larvae and adults of two of the identified species (Mitchell's Satyr butterfly and Saint Francis' Satyr butterfly) do not overlap with commercial so ybean acreage, and are therefore not expected to be impacted b y P IP event DAS - 81419 - 2 cultivation (Bartel and Sexton 2009; Barton and Bach 2005; US FWS 1997). There is a possible overlap between the geographic range for Karner blue butterfly and soy bean use sites. EPA has previously reviewed information on the proxim ity of Karner blue habitats, and none are known to exist immediately adjacent to agricultural fields (“adjacent to” was defined as 0 - 3 m for corn fields, which is reasonabl y applied to soybean fields) (US EPA 2001b). Karner blue larvae feed only

on lupines (

Lupinus spp.) and soy

bean is neither identified as a larval food source, nor is so ybean open - pollinated. Thus, pollen from event DAS - 81419 - 2 soy bean is highly unlikely to be deposited on lupine leaves (NatureServe 2013). Furthermore, soy bean is not identified as a nectaring source for Karner blue adults. Therefore, the risk posed to the Karner blue butterfl y was considered to be negligible. Based on the above anal ysis, EPA determines that there will be no direct effect to listed lepidopteran species as a result of the cultivation of DAS - 81419 - 2 soy beans as proposed. Obligate relationships between insectivorous listed species with lepidopterans that are expected to be found in soy

bean fields, especiall y pest species that feed on DAS - 81419 - 2 plants, are no t currentl y known. Since the Cry1F and Cry1Ac in DAS - 81419 - 2 soy beans targets onl y lepidopteran insects, loss of the pest insects as a result of DAS - 81419 - 2 are expected to be offsetBt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 19 of 78 by the presence of other insects that could act as food sources for liste d species, including beneficial insects that are known not to be affected by Cry 1F and Cry 1Ac. Effects on species other than insects have also been determined to be very unlikely because of the specificity of Cry 1F and Cry 1Ac. Lastl y, since the Agency

pre viously

determined Cry 1F and Cry 1Ac proteins are not expected to have adverse effects on mammals, birds, plants, freshwater and estuarine/marine fish and invertebrates, nontarget insects and other invertebrate species at the EEC, a “No Effect” determinatio n is made for direct and indirect effects to Federally listed threatened and endangered species and their designated Critical Habitats . In addition, EPA does not expect that any threatened or endangered plant species will be affected by outcrossing to wild relatives or b y competition with such entities. Hy brid soy bean does not exist in the wild in the United States, nor do any wild plants that can interbreed with soybean in the United States. 

### D. **Insect Resistance Management** The goal of insect resistanc e management (IRM) is to mitigate the potential risk of resistance in Bt crops, including Bt soy bean . IRM typicall y involves two phases: 1) data submitted to assess the risk of resistance for a certain Bt toxin and crop including information and data on pest biology , dose expression of the toxins in the PIP, simulation modeling, cross resistance potential and baseline susceptibility of the target pests to the toxin(s); and 2) a post - registration stewardship program to implement the resistance mitigation measures which includes resistance monitoring, remedial action ( should resistance occur), grower compliance with IRM requirements, and grower education . Dow submitted IRM considerations with their application for registration of DAS - 81419 - 2 Soy bean (MRID No. 4 8828415 and its replacement, MRID No. 4 9024110 ) . BPPD concluded that Dow’s IRM submissions satisfied the IRM requirements for DAS - 81419 - 2 Soy bean when used for breeding/seed increase . BPPD’s major IRM conclusions from its assessment of DAS - 81419 - 2 Soy bean are detailed below . **Conclusions and Re quirement**  **s** 1) Because DAS - 81419 - 2 S oy bean is intended for breeding/seed increase, Dow did not propose a species - specific IRM plan for the product. Rather, the company proposed to mitigate resistance risk by acreage limitations on a county (1,000, 10,000, or 25,000 acres) and national (250, 000 acres) scale. BPPD agrees that given the low acreage projected b y Dow, it is unlikely that there will be a significant risk of resistance to the main soy bean lepidopteran pests in the United States. These insects include soy bean looper, velvetbean cat erpillar, beet army worm, cotton bollworm, green cloverworm, tobacco budworm, and cutworms ( Agrotis spp.). This conclusion is further supported b y the biology of the target insects, which are highly poly phagous (feeding on a number of wild hosts and cultiv ated crops) and (for some species) have limited overwintering in non - tropical areas.Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 20 of 78 2) Should a full commercial registration or expanded acreage be sought in the future, D ow must submit a complete, species - specific IRM assessment for DAS - 81419 - 2 Soy bean . Data needed to support such a review should include additional information on pest biology , dose, simulation modeling, cross resistance, resistance monitoring, and potential impacts on the natural refuge strategy for Bt cotton. 3) The use of DAS - 81419 - 2 Soy bean on a limited basis for seed increase purposes should not significantl y impact the natural refuge strategy in place for Bt cotton PIPs. Soybean is one of the non - cotton crops that have been considered as part of natural refuge for tobacco budworm a nd cotton bollworm. While it can be expected that plantings of DAS - 81419 - 2 soy bean will remove some of the currentl y- available natural refuge for Bt cotton, this potential reduction will likely be small relative to the total amount of natural refuge present in southern cotton regions. Dow has proposed to further mitigate the impact on natural refuge b y limiting DAS - 81419 - 2 to 10,000 acres in counties with > 25,000 total acres of so ybean and 1,000 acres in counties with < 25,000 total acres of so yb ean. 4 ) As a term of registration, Dow must submit a resistance monitoring and remedial action plan for DAS - 81419 - 2 S oy

bean. A resistance monitoring plan limited to investigations of unexpected pest damage is appropriate for a capped acreage registration. BPPD n otes that monitoring of two potential target insects, tobacco budworm and cotton bollworm, is already being conducted as part of the WideStrike Bt cotton monitoring program. Dow must also compile annual sales and acreage data for each state and provide a D AS - 81419 - 2 Soy bean - specific report to EPA if requested. Since DAS - 81419 - 2 Soy bean will be deployed without a structured refuge requirement, a compliance assurance plan and grower education program is not necessary . Overall, BPPD concluded that Dow has submitted adequate resistance management data and information to support breeding/seed increase use of the product . There are no data gaps, though Dow must submit additional information , described above , to support any future request for either expande d acreage or commercial registration . A full discussion of BPPD’s IRM review of the supporting data for the Cry 1Ac and Cry 1F proteins expresse d as DAS - 81419 - 2 Soy bean PIP, including technical references, is available as Appendix C of this BRAD. 

### I **V .**  **ENV IRONMENTAL JUSTICE**  EPA seeks to achieve environmental justice, the fair treatment and meaningful involvement of all people, regardless of race, color, national origin, or income, in the development, implementation, and enforcement of environmental laws, regulations, and policies . To help address potential environmental justice issues, the Agency seeks information on any groups or segments of the population who, as a result of their location, cultural practices, or other factors, may have aty pical, unusual ly

high exposure to the Bt proteins Cry 1A c and Cry 1F a s expressed in PIP Event DAS - 81419 - 2 Soybean compared to the general population . For additional information regarding environmental justice issues, please visit EPA’s web site at http://www.epa.gov/compliance/environmentaljustice/index.html .Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 21 of 78 

### V. **RISK MANAGEMENT AND PROPOSED REGISTRATION DECISION**  Section 3(c)(5) of FIFRA provides for the registration of a new active ingredient if it is determined that (A) its composition is such as to warrant the proposed claims for it; (B) its labeling and other materials required to be submitted comply with the requirements of FIFRA; (C) it will perform its intended function without unreasonable adverse effects on the environment; and (D) when used in accordance with widespread and commonly recognized practice, it will not generally cause unreasonable adverse effe cts on the environment. The four criteria of the Eligibility Determination for Pesticidal Active Ingredients are satisfied b y the science assessments supporting the PIPs containing Bacillus thuringiensis proteins Cry 1A c and Cry 1F a s expressed in Event DAS - 81419 - 2 Soy bean . Th ese PIPs are not expected to cause unreasonable adverse effects and are likel y to provide protection as claimed when used according to label instructions. The EP A believes, therefore, that DAS - 81419 - 2 Soy bean is eligible for registration for the proposed uses. 

### VI. **TERMS AND CONDITIONS OF THE REGISTRATION**  **( S**  **)** As a term of registration, the applicant must submit or provide the following: Expression in a Terrestrial Environment: OCSPP 885.5200 (40 CFR § 158.2150) Although the registrant submitted a sufficient rationale of rapid degradation of Cry 1F and Cry 1Ac proteins in other crop soil sy stems, a soil degradation study has not been evaluated in soy bean for this specific PIP event. A previous EPA FIFRA SAP noted that in soi l degradation and field persistence studies, toxin degradation rates may vary with the crop, the toxin produced by the transformation event, the microbial community in the recipient soil microcosm, the statistical characterization of toxin persistence, and /or the initial dose of toxin in the experiment (US EPA 2001a). These differences in Cry toxin concentration may

be due to the fact that different genetic constructs were used and/or inserted at different locations in the plant genome (Yu et al . 2012). Plant genoty pe and parental backgrounds have also been recognized as important factors influencing protein expression (Kranthi et al. 2005; Adamczy k and Sumerford 2001; and Torres et al. 2006). Other important factors that affect degradation of Cry toxins inc lude environmental conditions such as temperature, light, drought, and soil properties (e.g. pH, texture, clay

content) (Adamczy

k et al. 2004; Chen et al. 2005; Jiang et al. 2006; Rochester 2006; Dong et al. 2008; Hallikeri et al. 2009; Addison and Rogers 2010; Chen et al. 2011; Ranjithkumar et al. 2011). To reduce this uncertainty , a soil fate study to determine the degradation rate (DT 50 ) of Cry 1F and Cry 1Ac proteins sampled from representative soils of soy bean growing regions i s t o be conducted as confirmatory data. The completed soil degradation study must be received b y the EPA within 18 months of the DAS - 81419 - 2 Soy bean Notice of Pesticide Registration date. Insect Resistance Management Dow must submit a resistance monitoring and remedial action plan for DAS - 81419 - 2 S oy bean. Although a full resistance monitoring plan with pest sampling and detection bioassay s is notBt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 22 of 78 warranted for this seed increase registration, an approach based on inve stigations of unexp ected pest damage reports is appropriate. Monitoring for the subject proteins is conducted as part of the WideStrike cotton (EPA Registration Number 68467 - 3) monitoring program, and a remedial action plan (should resistance be documented) aligned with the plans in place for Bt corn or cotton (e.g., a “ stop sale” if resistance develops) can be adapted for DAS - 81419 - 2 Soy bean . Additionally , annual sales and total acreage data reports for each state must be compiled by Dow for submission to the EP A (upon request). The subject registration is limited to seed increase and to a total of 250,000 acres per ye ar with no more than 20,000 acres per county (in non - cotton growing regions); 10,000 acres per county (in cotton - growing counties with at least 25,000 acres of so yb ean); or 1,000 acres (in cotton growing counties with less than 25,000 acres of soy bean) per year A. Adverse Effects Reports of all incid ents of adverse effects to the environment must be submitted to the EPA under the provisions stated in FIFRA Section 6(a)(2). B. Hy persensitivity

Incidents All incidents of hy

persensitivity

(including both suspected and confirmed incidents) must be reported to the Agency

under the provisions of 40 CFR §158.2140(d).Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 23 of 78 

### APPEND **IX A: Product characterization and human health effects**  **– Bacillus thuringiensis**  **Cry 1A**  **c and Cry1F Proteins**  **expressed in**  **Event DAS**  **- 81419**  **- 2 S**  **o ybea**  **n plant -**  **incorporated protectant. A.1.**  **BACKGROUND** Event DAS - 81419 - 2 expresses the δ - endotoxins Cry 1Ac and Cry 1F derived from Bacillus thuringiensis . Product characterization and human health data submitted for seed increase registration of DAS - 81419 - 2 are summarized below. **A.2. PRODUCT CHARACTERIZATION**  Event DAS - 81419 - 2 was developed b y Agrobacterium - mediated transformation of soy bean using the 2T - DNA plasmid vector pDAB9582 and produces Bacillus thuringiensis protein δ endotoxins, Cry 1Ac and Cry 1F. A single T - DNA insert contai ning each of the intact plant transcription units (PTUs) for the cry1Fv3, cry1Ac(synpro), and pat genes was integrated into soy bean to create DAS - 81419 - 2. This protein is intended to provide protection from feeding damage caused b y a number of lepidopteran pests. **a .**  **Transformation System and Genetic Elements**  Molecular characterization of DAS - 81419 - 2 soy bean by Southern blot analy sis confirmed that a single T - DNA insert containing each of the intact plant transcription units (PTUs) for the cry1Fv3, cry1Ac(synpro), and pat genes from plasmid pDAB9582, was integrated into DAS 81419 - 2 soy

bean. In addition, a minor (<100 bp) fragment of the cry1Ac(synpro) gene was identified on the 5 ′ end of the T - DNA insert. Data were generated to evaluate the integrati on and integrity of the cry1Fv3, cry1Ac(synpro) , and pat genes inserted into the soybean genome. Characterization of the integration of non - coding regions (designed to regulate the coding regions), such as promoters and terminators was also performed. The inserted DNA was stably inherited across the five generations (T1, T2, T3, T4, and F2) evaluated. No transformation plasmid backbone sequence was found in DAS - 81419 - 2 soy bean as demonstrated by

Southern blot analy sis using probes covering the entire regio n of the plasmid flanking the T - DNA insert. **b .**  **Protein Characterization** Protein characterization data demonstrate that the plant - produced Cry 1Ac and Cry 1F have biochemical and functional activities that are similar to those of the Pseudomonas fluorescen s produced proteins that were used in several toxicity studies. The following techniques were used to characterize and compare the plant - produced and the Pseudomonas fluorescens - produced proteins: sodium dodecyl sulfate polyacrylamide gel electrophoresis ( SDS - PAGE), western blot anal ys is, densitometry , matrix - assisted laser desorption/ionization time - of - flight (MALDI - TOF) /mass spectrometry , gl yc osylation anal ys is, N - terminal amino acid sequencing, and insecticidal activity bioassay s. Gl yc oslyation anal ys i s indicated that the protein is not gl ycosl yated. These anal ys es demonstrated the structural and functional similarity between the plant - produced andBt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 24 of 78 the Pseudomonas fluorescens - produced Cry 1Ac and Cry 1F proteins and justified the use of Pseudomonas fluorescens - produced protein in toxicity studies. **c. Analytical Detection Methods**  An exemption from the requirement of a food tolerance is already in existence (since 1997) for Cry 1Ac protein for all food commodities (40 CFR 174.510) and an appropriate anal ytical detection method was previously reviewed for Cry 1Ac. An exemption from the requirement of a food tolerance exists for Cry 1F, however, the exemption is limited to expression in maize (40 CFR 174.520) and cotton (40 CFR 174.504) . A petition for an exemption from the requirement of a food tolerance for Cry1F in soy bean was included with the application from D ow . Dow AgroSciences LLC anal ytical method 110674, "Method Validation for the Determination Cry 1Ac Protein in Soy

bean Tiss ues by

Enzy me - Li nked Immunosorbent Assay (ELISA)", was demonstrated to be suitable for its intended purpose. The method was validated over the concentration range of 0.1 to 2 ng Cry 1Ac/mg dry weight (DW) and has a validated limit of quantitation (L OQ) in s oy bean tissues of 0.2 ng/mg DW and a limit of detection (LOD) of 0.1 ng/mg DW. The Cry 1Ac protein was recovered at acceptable levels from soy bean tissues. The Cry 1Ac protein was efficiently extracted from all tested soy bean tissues. The assay

was shown to have acceptable accuracy, precision and ruggedness, and no false positive or false negative results were seen below the target LOD. It is concluded that this Cry 1Ac ELISA method has been demonstrated to be suitable for quantitative measurements of the Cry1 Ac protein in soy bean tissue. Dow AgroSciences LLC anal ytical method 110675, "Determination of Cry1F Protein in Soy bean Tissues Using an Enz ym e - Linked Immunosorbent Assay (ELISA)", was demonstrated to be suitable for its intended purpose. The method was validated over the concentration range of from 0.05 ng/mg to 1 ng/mg DW for root R3, from 0.15 ng/mg to 1.5 ng/mg DW for forage R3, from 0.2 ng/mg to 2 ng/mg DW for grain, and from 0.25 ng/mg DW to 2.5 ng/mg DW for leaf V5 and leaf V10 - 12. The method has a validated limit of quantitation (L OQ) of 0.1 ng/mg DW for root R3, 0.3 ng/mg DW for forage R3, 0.4 ng/mg DW for grain, and 0.5 ng/mg DW for leaf V5 and leaf V10 - 12, and a limit of detection (LOD) of 0.05 ng/mg DW for root R3, 0.15 ng/mg DW for forage R3, 0.20 ng/mg DW for grain, and 0.25 ng/mg DW for leaf V5 and leaf V10 - 12. Cry 1F protein was recovered at acceptable levels from soy bean tissues. This Cry 1F ELISA method has been demonstrated to be suitable for quantitative measurements of the Cry1F protein in soy bean tissue. **d .**  **Protein Expression** Expression level data were provided for Cry 1Ac in different plant tissues and at different growth stages. Cry 1Ac protein is expressed at relativel y low levels in event DAS - 81419 - 2 soy bean. The data were produced using ELISA methods for this protein. Summary results are provided below in Table A. 1. Table A. 2 . provides summaries of the product characterization studies and data provided. **Table A.1. Protein Expression in DAS**  **- 81419**  **- 2 s**  **oybean**Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 25 of 78 **Summary of Cry1Ac Protein Expression**

Test Cry1Ac ng/mg Tissue Dry Weight Matrix Entry Description Overall Mean Std. Dev.

(n=10) Min/Max

Range STMRa HAFT b V5 Leaf 7 DAS - 81419 - 2 25.44 6.61 12.10 - 40.20 26.90 35.25 V10 - 12 Leaf 7 DAS - 81419 - 2 23.16 6.17 10.70 - 37.45 22.15 34.61 Forage 7 DAS - 81419 - 2 5.54 2.54 1.38 - 11.83 5.44 10.28 Root 7 DAS - 81419 - 2 0.39 0.24 [0.12] - 1.12 0.32 0.97 Grain 7 DAS - 81419 - 2 1.04 0.10 0.79 - 1.40 1.04 1.20

a Supervised Trials Mean Residue b Highest Average Field Trial **Summary of Cry1F Protein Expression**

Test Cry1F ng/mg Tissue Dry Weight Matrix Entry Description Overall Mean Std. Dev.

(n=10) Min/Max

Range STMRa HAFT b V5 Leaf 7 DAS - 81419 - 2 56.75 15.03 24.60 - 99.50 56.30 76.05 V10 - 12 Leaf 7 DAS - 81419 - 2 39.07 16.60 12.75 - 76.71 38.70 59.98 Forage 7 DAS - 81419 - 2 20.28 11.29 5.34 - 44.62 20.64 40.23 Root 7 DAS - 81419 - 2 5.23 3.74 1.09 - 16.08 4.12 14.21 Grain 7 DAS - 81419 - 2 13.80 1.24 10.41 - 16.95 13.71 16.21

a Supervised Trials Mean Residue b Highest Average Field Trial **Summary of PAT Protein Expression**

Test PAT ng/mg Tissue Dry Weight Matrix Entry Description Overall Mean Std. Dev.

(n=10) Min/Max

Range STMRa HAFT b V5 Leaf 7 DAS - 81419 - 2 5.23 0.88 3.25 - 7.35 5.30 6.93 V10 - 12 Leaf 7 DAS - 81419 - 2 5.60 1.14 2.55 - 7.56 5.76 7.32 Forage 7 DAS - 81419 - 2 4.06 1.30 1.24 - 6.12 4.02 5.69 Root 7 DAS - 81419 - 2 0.63 0.12 0.44 - 1.05 0.63 0.85 Grain 7 DAS - 81419 - 2 0.86 0.13 0.63 - 1.12 0.83 1.06

a Supervised Trials Mean Residue b Highest Average Field TrialBt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 26 of 78 **Table A.**  **2 .**  **Summary of Product Characterization**  **Data for PIP E**  **vent DAS -**  **81419 -**  **2 soybean**  **(EPA Reg. No. 68467**  **- EN)**  **Study Type/Title Summary**  **MRID No.**  Molecular Characterization of DAS - 81419 - 2 Soybean Data from multiple Southern blots probed with various fragments of

the cry and pat genes, as well as promoter and 3’ UTR sequences, indicate that a single insertion of T - DNA is present in DAS - 81419 - 2 S oybean. Samples from five generations of DAS - 81419 - 2 soybean yielded identical patterns of hybridization indicating that the insertion is stably introduced into the plant genome. Further, the absence of pDAB9582 backbone sequences was demonstrated with probing of Southern blots using 4 different sequences (i.e., Spec r , Backbone, 1, 2 and 3) outside of the T - DNA region of the plasmid. All blots included genomic DNA from a non - transgenic soybean, Maverick, and a Maverick DNA sample spiked with pDAB9582 DNA as negative and positive controls, respectively. **Classification: ACCEPTABLE** 48828401

Protein Expression of a Transformed Soybean Cultivar Containing Cry1Ac, Cry1F, and Phosphinothricin Acetyltransferase (PAT) - Event DAS 81419 - 2 Measures of protein accumulation at different growth stages and in different organs of DAS - 81419 - 2 S oybean plants were obtained using appropriate methodology. The authors of this study quantified Cry1Ac, Cry1F and PAT proteins in leaf, forage (aerial portions of plant withou

t pods), root and seed of soybean cultivated in 10 different localities typical for this crop. The ELISA -based methods were typical for this technique and the findings were statistically acceptable (i.e., variance is within acceptable limits). Sample handl ing and processing were also performed appropriately. **Classification: ACCEPTABLE** 48828402

Biological Equivalency of (DAS - 81419 - 2) and microbe -produced Cry1Ac and Cry1F Proteins With respect to biological activity of the plant -produced and microbe -produced Cry1Ac and Cry1F proteins, LC 50 data for the target pest Chrysodeixis includens (soybean looper) indicate that the plant - and microbe - derived Cry proteins retain similar activity against this insect. LC 50 values vary by less than 15%, whic h is an acceptable level of variance for such a biological system. **Classification: ACCEPTABLE** 48828403

Method Validation for the Determination of Cry1Ac Protein in Soybean Tissues by Enzyme - Linked Immunosorbent Assay (ELISA) Analytical method 110674 was validated to perform robustly in the quantitation of Cry1Ac protein in soybean organs in the range of 0.2 ng/mg DW to 2.9 ng/mg DW using an ELISA - based technique with two monoclonal antibodies raised against Cry1Ac. The method performed with acceptable level of accuracy in the quantitation of Cry1Ac in soybean organ samples, including leaf, root, forage (aerial portions of plant without pods), and seed. Given some matrix effects, a 2X dilution of samples is recommended for soybe an samples to more accurately quantify Cry1Ac. Variability in results was within 48828404Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 27 of 78 acceptable limits as the Coefficient of Variation (CV) values were all less than 20%. No cross reactivity with a variety of nontarget proteins was detected and extraction effi ciencies from soybean samples were at acceptable levels. **Classification: ACCEPTABLE** Method Validation for the Determination of Cry1F Protein in Soybean Tissues Using an Enzyme Linked Immunosorbent Assay (ELISA) The ELISA - based double antibody sandwich method employed by the study authors accurately quantified target protein Cry1F in soybean organs sampled (root, forage, leaves, seed). The analytical method 110675 was validated over a range of protein concentration values appropriate for the le vels as found in cultivated soybean spiked with known quantities of Cry1F protein. The range is, however, on the lower end of most transgenic soy samples as evidenced in other volumes of this submission as some leaf samples contained concentrations of Cry proteins in the 25 to 56 ng/mg DW range. Matrix effects were adequately resolved and indicate that in practice, dilution of root, leaf, seed and forage samples will need to be performed at 1X, 5X, 4X, and 3X respectively to minimize these effects on quantification of Cry1F in soybean. The variance recorded among samples and between different analysts was less than 20%, an acceptable amount when working with complex plant organ extracts, with the exception of root samples spiked to yield quantities of Cry 1F protein at or near the LOD (0.1 ng/mg DW). Higher variability at or near an LOD is not unexpected. The ELISA -based method 110675 was not confounded by false positives or negatives and only minimal cross reaction with Cry1Ac (0.114 %) was observed while other nontarget proteins were similarly non - cross reactive. The method appears robust enough for practical use in the detection of Cry1F in soybean. **Classification: ACCEPTABLE** 48828405

Characterization of the Cry1Ac Protein Derived from Transgenic Soybean Event DAS - 81419 - 2 The study authors employed several techniques to adequately demonstrate that the Cry1Ac protein from soybean event DAS 81419 - 2 is biochemically equivalent to the Cry1Ac protein as expressed in Pseudomonas fluorescens . Lateral test flow strips (an ELISA -based method), immunoprecipitation, detection of glycosyl residues, Western blot, MALDI - TOF / MS, MALDI - TOF/TOF, MS/MS, and SDS - PAGE analyses all support the contention that the two proteins are biochemically equivalent. Samples were processed, stored and handled in an appropriate manner to maintain integrity of these protein samples. **Classification: ACCEPTABLE** 48828416

Characterization of the Cry1F Protein Derived from Transgenic Soybean Event DAS - 81419 - 2 The study authors employe d several techniques to adequately demonstrate that the Cry1F protein from soybean event DAS - 81419 - 2 is biochemically equivalent to the Cry1F protein as expressed in Pseudomonas fluorescens . Lateral test flow strips (an ELISA -based method), immunoprecipita tion, detection of glycosyl residues, 4 8828417Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 28 of 78 Western blot, MALDI - TOF / MS, MALDI - TOF/TOF, MS/MS, and SDS - PAGE analyses all support the contention that the two proteins are biochemically equivalent. Samples were processed, stored and handled in an appropriate mann er to maintain integrity of these protein samples. **Classification: ACCEPTABLE** Amended Response to EPA Comment on Characterization of the Cry1F Protein Derived from Transgenic Soybean Event DAS - 81419 - 2 Study

– Address amino acid sequence similarity between Dow’s Cry1F microbial protein and soybean produced Cry1F: Related to MRID 488284 - 17 The synthetic Cry1F protein expressed in DAS - 81419 - 02 S oybean

and the Cry1F protein expressed in Pseudomonas fluorescens strain MR872 differ at 4 amino acid positions in the full length proteins. The variations or alterations in amino acid residues for these 4 positions (residues 604, 608, 624, 629) do not affect the biochemical or biological properties evaluated in this ri sk assessment. For the purposes of testing, as performed, the proteins are considered equivalent.

**Classification: ACCEPTABLE** 49081501

**A. 3**  **. HUMAN HEALTH ASSESSMENT**  Section 408(c)(2)(A)(i) of the Federal Food, Drug, and Cosmetic Act (FFDCA) allows EPA to establish an exemption from the requirement for a tolerance (the legal limit for a pesticide chemical residue in or on a food) onl y if EPA determines that the exemption is “safe.” Section 408(c)(2)(A)(ii) of the FFDCA defines “safe” to mean that “th ere is a reasonable certainty that no harm will result from aggregate exposure to the pesticide chemical residue, including all anticipated dietary

exposures and all other exposures for which there is reliable information.” This includes exposure through drinking water and in residential settings, but does not include occupational exposure. Pursuant to section 408(c)(2)(B), in establishing or maintaining in effect an exemption from the requirement of a tolerance, EPA must take into account the factors set f orth in section 408(b)(2)(C), which require EPA to give special consideration to exposure of infants and children to the pesticide chemical residue in establishing a tolerance and to “ensure that there is a reasonable certainty that no harm will result to infants and children from aggregate exposure to the pesticide chemical residue... .” Additionally , section 408(b)(2)(D) of the FFDCA requires that the Agency consider “available information concerning the cumulative effects of a particular pesticide’s re sidues” and “other substances that have a common mechanism of toxicity .” EPA performs a number of analy ses to determine the risks from aggregate exposure to pesticide residues. First, EPA determines the toxicity of pesticides. Second, EPA examines exposure to the pesticide through food, drinking water, and through other exposures that occur as a result of pesticide use in residential settings.Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 29 of 78 Dow submitted a pe sticide tolerance petition (PP2 F 8 066 ) under the FFDCA, requesting 40 CFR Part 174 be amended b y establishing an exemption from the requirement of a tolerance for residues of Bacillus thuringiensis ( Bt) Cry 1F insect control protein and the genetic material necessary for its production in foo d commodities of soy bean . Consistent with section 408(b)(2)(D) of the FFDCA, EPA has reviewed the available scientific data and other relevant information in support of this action and considered its validity, completeness and reliability , and the relatio nship of this information to human risk. EPA has also considered available information concerning the variability of the sensitivities of major identifiable subgroups of consumers, including infants and children.

**a. Toxicological Profile**  EPA has reviewed the available scientific data and other relevant information in support of this action and considered its validity , completeness and reliability and the relationship of this information to human health risk. No indication of toxicity toward mammals was noted in the review of the two Cry

proteins expressed in soy bean. It should be noted that the allergenicity of Cry 1Ac protein was evaluated previously (Wozniak, 2010) as evidenced in MRID No . 478417 09 (US EPA, 2010). Based upon sequence comparisons to known allergens and digestibility assay s (MRID No. 47841708), Cry 1Ac was considered to be of little potential to function as an allergen. An exemption from the requirement of a food tolerance was established previously for phosphinothricin ac etyl transferase (PAT; 40 CFR 174.522) as expressed as part of a plant incorporated protectant, so no further toxicological review of this protein is contained herein. The human health studies submitted for Cry 1Ac and Cry 1F are summarized in Table A. 3 . be low. **Table A.3. Summary of**  **Human Health Data**  **conducted for PIP E vent DAS**  **- 81419**  **- 2**  **soybean (EPA Reg. No.**  **68467 -**  **EN) Study Type/Title**  **Summary MRID**  **No.** Sequence Similarity Assessment of Cry1F Protein to Known Allergens by Bioinformatics Analysi s The study authors have utilized appropriate search methods in comparing the sequence of their Cry1F protein with the ‘allergenonline’ database of known allergens. The default parameters applied to the search are sufficient to identify sequences with > 35% homo logy over any 80 amino acid sequence and to detect any individual 8 amino acid sequences which may represent epitopes of allergens. Given the exhaustive analysis of Cry proteins, including Cry1F and closely related δ - endotoxins, over many years for the pre sence of sequences representing potential allergenic epitopes and the general lack of demonstrated IgE - mediated immune responses to those exposed to preparations of biopesticidal Bacillus thuringiensis , 48828406Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 30 of 78 the findings are not surprising or unexpected. Its hould be noted that the allergenicity of Cry1F protein was evaluated previously (Wozniak, 2002; Matten 2005) as evidenced in MRID No. 44971701 and discussed in a Biopesticide Registration Action Document (US EPA, 2005; ibid 2010). Based upon sequence comparisons to known allergens and digestibility assays (MRID No. 45542312), Cry1F was considered to be of little potential to function as an allergen. **Classification: ACCEPTABLE** Sequence Similarity Assessment of Cry1F to Known Toxins by Bioinformatics Analysis The study authors queried appropriate databases containing sequences of proteins, including those with potential toxicity to vertebrates. Parameters chosen for the search were selected to detect proteins with minimal homology in seq uence such that even those with insufficient similarity to trigger a toxic reaction may be uncovered. Of the 672 proteins or hypothetical protein sequences returned from the search E - value < 1.0, including 646 with E -value <0.01, 618 were clearly related t o δ - endotoxins from Bacillus thuringiensis and two were related to proteins from other Bacillus species (e.g, B. cereus ). Of the remaining 28 protein hits from the databases, some of the sequences were from parasporal proteins of related species (e.g., L ysinibacillus sphaericus , Paenibacillus lentimorbus ) and also from hypothetical proteins (of no known function) from eubacteria, slime molds / Mycetozoa and a fungal species (Fusarium oxysporum ). These proteins and sequences of hypothetical proteins are no t known to represent toxic moieties with respect to vertebrates. As is typical with previously characterized Cry proteins of B. thuringiensis , no toxicity to vertebrates, including man, is expected from consumption or contact with these proteins. **Classi fication: ACCEPTABLE**  48828407

Sequence Similarity Assessment of Cry1Ac Protein to Known Allergens by Bioinformatics Analysi s The study authors have utilized appropriate search methods in comparing the sequence of their Cry1Ac protein with the ‘ allergenonline’ database of known allergens. The default parameters applied to the search are sufficient to identify sequences with > 35% homology over any 80 amino acid sequence and to detect any individual 8 amino acid sequences which may represent epitopes of allergens. Given the exhaustive analysis of Cry proteins, including Cry1Ac and closely related δ - endotoxins, over many years for the presence of sequences representing potential allergenic epitopes and the general lack of demonstrated IgE - mediated i mmune responses to those exposed to preparations of biopesticidal Bacillus thuringiensis , the findings are not surprising or unexpected. Itshould be noted that the allergenicity of Cry1Ac protein was evaluated previously as evidenced in MRID No. 4554231 9 (US EPA, 2005). Based upon sequence comparison s to known allergens and 48828408Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 31 of 78 digestibility assays (MRID No. 47841708), Cry1Ac was considered to be of little potential to function as an allergen. **REFERENCES:** US Environmental Protection Agency (2010) BIOPESTICIDE REGISTRATION ACT IO N DOCUMENT, Bacillus thuringiensis Cry1Ac Protein and the Genetic Material (Vector PV - GMIR9) Necessary for Its Production in MON 87701 (OECD Unique Identifier: MON 877Ø1 - 2) Soybean [P C Code 006532] http://www.epa.gov/pesticides/biopesticides/pips/bt - cry1ac protien.pdf **Classification: ACCEPTABLE** Similarity Assessment of Cry1Ac Protein to Known Toxins by Bioinformatics Analysi s The study authors queried appropriate databases containing sequences of proteins, including those with potential toxicity to vertebrates. Parameters chosen for the search were selected to detect proteins with minimal homology in sequence such that even those with insufficient similarity to trigger a to xic reaction may be uncovered. Of the 684 proteins or hypothetical protein sequences returned from the search, 662 hits were clearly related (E - value <0.01) to δ - endotoxins from Bacillus thuringiensis and two were related to proteins from other Bacillus species. Of the remaining 52 protein hits from the databases, some of the sequences were from parasporal proteins of related species (e.g., Bacillus cereus , Paenibacillus lentimorbus ) and also from hypothetical proteins (of no known function) from eubac teria, slime molds / Amoebozoa and fungal species. These proteins and sequences of hypothetical proteins are not known to represent toxic moieties with respect to vertebrates. As is typical with previously characterized Cry proteins of B. thuringiensis , no toxicity to vertebrates, including man, is expected from consumption or contact with these proteins. **Classification: ACCEPTABLE** 48828409

**b. Mammalian Toxicity and Allergenicity Assessment**  Previously

submitted acute oral toxicity data demonstrated a lack of mammalian toxicity at high levels of exposure (ingestion) to the pure Cry 1Ac protein. These data demonstrate the safety of this protein at a level well above maximum possible consumptio n levels that are reasonabl y anticipated as expressed in the crop. Basing this conclusion on acute oral toxicity data without requiring further toxicity

testing and residue data is similar to the Agency position regarding toxicity testing and the requirem ent of residue data for the microbial Bacillus thuringiensis products from which this plant - incorporated protectant was derived (See 40 CFR Sec. 158.740(b)(2)(i)). For microbial products, further toxicity testing (Tiers II & III) and residue dataBt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 32 of 78 are trig gered b y significant adverse acute effects in studies, such as the acute oral toxicity study , to verify the observed adverse effects and clarify the source of these effects. An acute oral toxicity

study in mice (MRID No. 45542313) indicated that Cry 1Ac i s non - toxic to humans and other mammals. Cry 1Ac protein is a δ - endotoxin from B. thuringiensis that has been used extensively

in both microbial and plant - incorporated protectants as a means of insect pest management. An existing exemption from the requirement of a tolerance for Cry 1Ac (CFR 40 Section 174.510; 72 FR 20435, April 25, 1997) in all food commodities precluded the need for a separate tolerance action in conjunction with this review of product characterization and human health assessment. When proteins are toxic, they are known to act via acute mechanisms and at very low dose levels (Sjoblad et al., 1992). Therefore, since no acute effects were shown to be caused by Cry 1Ac, even at relativel y high dose levels, the Cry1Ac protein is not cons idered toxic. Further, amino acid sequence comparisons showed no similarities that would raise a safety concern between the Cry 1Ac protein and known toxic proteins in protein databases. Since Cry 1Ac is a protein, allergenic potential was also considered. Currently , no definitive tests for determining the allergenic potential of novel proteins exist. Therefore, EPA uses a weight- ofevidence approach where the following factors are considered: source of the trait; amino acid sequence comparison with kno wn allergens; and biochemical properties of the protein, including in vitro digestibility

in simulated gastric fluid (SGF) followed by simulated intestinal fluid (SIF), and gl yc os yl ation. This approach is consistent with the approach outlined in the Annex to the Codex Alimentarius “Guideline for the Conduct of Food Safety Assessment of Foods Derived from Recombinant - DNA Plants.” The allergenicity assessment for Cry 1Ac follows: 1. Source of the trait. Bacillus thuringiensis is not considered to be a source of allergenic proteins. 2. Amino acid sequence. A comparison of the amino acid sequence of Cry 1Ac with known allergens showed no significant overall sequence similarity or identity

at the level of eight contiguous amino acid residues. 3. Digestibility . (MRID No. 455423 19 ) The Cry 1Ac protein was digested rapidly in simulated gastric fluid containing pepsin. Small peptides remaining following gastric simulated digestion were completely degraded to amino acid residues in SIF upon contact (US EPA 2005; 2010a). 4. Gl ycos yl ation. Cry 1Ac expressed in soy bean was shown not to be gl ycos yl ated. 5. Conclusion: Considering all of the available information, EPA has concluded that the potential for Cry 1Ac to be a food allergen is minimal. An acute oral toxicity

study in mice ( MRID No. 45542314) indicated that Cry 1F is non - toxic to humans and other mammals. Cry 1F protein is a δ - endotoxin from B. thuringiensis that has been used extensively

in plant - incorporated protectants as a means of insect pest management. E xisting exemption s from the requirement of a tolerance for Cry 1F in cotton (CFR 40 Section 174.504; 72 FR 20434 , April 25, 2007 ) and corn (CFR 40 Section 174. 520; 72 FR 2043 5, April 25, 2007 ) precluded the need for a separate tolerance action in conjunction with this review of product characterization and human health assessment. Cry 1F was previously granted an exemption from the requirement of a tolerance for expression in cotton (CFR 40 Sec tionBt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 33 of 78 174.504) and maize (CFR Section 174.520); a separate petition ( PP 2F8066) for an exemption from the requirement of a tolerance for Cry 1F as expressed in soy bean is included with the submission from Dow for registration of DAS - 81419 - 2 S oy bean. Since C ry 1F is a protein, allergenic potential was also considered. Currently, no definitive tests for determining the allergenic potential of novel proteins exist. Therefore, EPA uses a weight- ofevidence approach where the following factors are considered: source of the trait; amino acid sequence comparison with known allergens; and biochemical properties of the protein, including in vitro digestibility

in simulated gastric fluid (SGF) followed by simulated intestinal fluid (SIF), and gl yc os yl ation. This approac h is consistent with the approach outlined in the Annex to the Codex Alimentarius “Guideline for the Conduct of Food Safety Assessment of Foods Derived from Recombinant - DNA Plants.” The allergenicity assessment for Cry 1F follows: 1. Source of the trait. Bac illus thuringiensis is not considered to be a source of allergenic proteins. 2. Amino acid sequence. A comparison of the amino acid sequence of Cry 1F with known allergens showed no significant overall sequence similarity or identity

at the level of eight contiguous amino acid residues. 3. Digestibility . (MRID No. 455423 18 ) The Cry 1F protein was digested rapidly (< 5 min) in simulated gastric fluid containing pepsin. Small peptides remaining following gastric simulated digestion were completely degraded to amino acid residues in SIF upon contact (US EPA 2005; 2010b). 4. Glycos yl ation. Cry 1F expressed in soy bean was shown not to be gl ycos yl ated. 5. Conclusion: Considering all of the available information, EPA has concluded that the potential for Cry 1F to be a food allergen is minimal. Cry 1Ac and Cry 1F were shown not to be gl ycos yl ated in extracts of DAS - 81419 - 2 soy bean ; it is unlikely to be gl ycos yl ated in any other crops because in order for a protein to be gl ycosl yat ed, it needs to contain specific recognition s ites for the enzy mes involved in gl yc osylation, and the mechanisms of protein glycosylation are similar in different plants (Lerouge et al., 1998). **c. Overall Safety Conclusions**  The continued use of Cry1Ac and Cry 1F proteins in plant expression sy stems (plant - incorporated protectants) and in microbial biopesticides is fully supported by the information presented in this registration submission relative to human and animal health c oncerns. The lack of mammalian toxicity and allergenicity effects following thorough examination of pertinent information, as well as the efficacy

noted in the insect bioassay s, indicates that the specificity of the δ endotoxins Cry 1Ac and Cry 1F as an inse ct management mechanism is safe as proposed for use in soy bean intended for cultivation, and human and animal consumption.Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 34 of 78 

### APPENDIX **B :**  **Ecological and e**  **n vironmental effects**  **– Bacillus thuringiensis**  **Cry1A c**  **and Cry**  **1F proteins expressed in Event**  **DAS -**  **81419 -**  **2 S o**  **ybea n plant** **** **incorporated protectant** D ow submitted data in support for Sec. 3 Seed - In crease Registration of event DAS - 81419 - 2 soy bean line [EPA Reg. No. 68467 - EN; OECD Unique I dentifier: DAS - 81419 - 2] for use as a Plant - In corporated Protectant (PIP). The proposed PIP is a new transformation event that express es lepidopteran - active Cry 1F and Cry 1Ac protein s in soy bean . The insecticidal proteins Cry 1F and Cry 1Ac (encoded by the cry1F gene and cry1Ac genes, respectively) are co - expressed in WideStrike  cotton (OECD Unique Identifier: DAS - 21Ø23 - 5 x DAS - 24236 - 5), which is currentl y registered for use as a PIP cotton product (US EPA 2005). The cotton line WideStrike  was produced by crossing cotton lines 281 - 24 - 236 (OECD Unique I dentifier: DAS - 24236 - 5) with 3006 - 210 - 23 (OECD Unique I dentifier: DAS - 21Ø23 - 5) via conventional breeding methods. Since these proteins are identical to those expressed in th e proposed PIP product , a “weight - of

- evidence” approach was used to bridge data from WideStrike  cotton in support for the environmental risk assessment of event DAS - 81419 - 2 soy bean . Thus, the findings of the environmental risk assessment for event DAS - 81419 - 2 soy bean include the extrapolation of previously submitted nontarget organism (NTO ) toxicity studies and results from field studies currently found in the Agency

database for the registration of W ideStrike® expressing Cry 1F and Cry 1Ac proteins for use as a PIP in cotton . The biological equivalency of Cry 1F and Cry 1Ac proteins expressed in event DAS - 81419 - 2 soy bean plant was confirmed with the previously

characterized microbial - derived Cry 1F and Cry 1Ac protein test substances. No significant increase in exposure to NTOs was demonstrated based on calculated margins of exposure for protein concentrations in ev ent DAS - 81419 - 2 plant tissue matrices relative to the dose concentrations used in the NTO laboratory toxicity tests conducted on Cry 1Ac and Cry 1F . Lastl y, a field monitoring study showing no differences on nontarget arthropods on a population and community level and other supporting supplemental information (e.g. meta - anal ys is, target feeding studies, and biological specificity to the target pest) provided additional certainty to support the “weight - of

- evidence” that there is negligible risk to NTOs represe nting diverse taxonomic groups and exposure scenarios from the cultivation of DAS - 81419 - 2 soy bean. Therefore, the submitted data supports the applicant’s request and the findings from the environmental risk assessment conducted for WideStrike® cotton can be bridged to support the Sec. 3 Registration of PIP event DAS - 81419 - 2 (US EPA 2005). Based on e xtrapolating from previous studies on Bt proteins, which tend to be very species specific in their activity , the Agency concludes that the cultivation of event DAS - 81419 - 2 soy bean will not result in adverse effects to nontarget organisms, are not likely to persist in the soil, and pose no risk of gene flow and development of weediness in wild relatives, and additionally will have no effect, direct or indirect, on Federally

listed threatened and endangered species and their designated Critical Habitats. **BACKGROUND** Dow has developed event DAS - 81419 - 2 soy bean line [EPA Reg. No. 68467 - EN; OECD Unique Id entifier: DAS - 81419 - 2] for use as a plant - incorporated protectant (PIP). Event DAS - 81419 - 2Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 35 of 78 soy bean was produced by Agrobacterium - mediated transformation with plasmid pDAB958 2. The inserted T - DNA from this plasmid contains three transgenes: 1) s ynthetic truncated cry1F gene from Bacillus thuringiensis ( Bt) var. aizawai ; 2) cry1Ac gene isolated from Bt var. kurstaki ; and 3) a version of the phosphinothricin acetyl transferase ( pat) gene from Streptomyces viridochromogenes that has been optimized for expression in soy bean. The Cry 1F and Cry 1Ac proteins, (encoded b y the cry1F and cry1Ac genes, respectivel y) confer resistance to certain lepidopteran pests. The PAT enzy me encoded b y the pat gene provides the plant with tolerance to herbicide applications containing glufosinate - ammonium by acetyl ating the herbicide glufosinate - ammonium and making it unable to bind glutamine s ynthase. The proposed PIP is intended to provide protection against several lepidopteran pests of so yb ean, including so ybean looper ( Chrysodeixis includens, formerl y Pseudoplusia includens ), velvetbean caterpillar ( Anticarsia gemmatalis ), fall arm yworm (Spodoptera frugiperda ) and tobacco budworm ( Heliothis virescens ). The Cry 1F and Cry 1Ac proteins are co - expressed in the currently registered PIP event WideStrike® cotton (EPA Reg. No. 68467 - 3 ). Dow has requested to bridge several environmental fate and effects studies previously reviewed for the registration of WideStrike® cotton (US EPA 2005) to support the registration of event DAS - 81419 - 2 soy bean PIP product. A “weight - of

- evidence” approach has been used to determine if the supporting data submitted by Dow is acceptable for bridging to the Agency ’s databa se on the Cry 1F and Cry 1Ac proteins expressed in WideStrike® cotton and whether extrapolation of those studies and other supplemental information support the environmental risk assessment of event DAS - 81419 - 2 soy bean. **I. Environmental Risk Assessment Proc**  **ess and Rationale for PIPs under EPA** The paragraphs below describe the process and rationale developed b y the U.S. EPA’s Biopesticides and Pollution Prevention Division (BPPD) within the Office of Pesticide Programs for evaluating hazard of PIPs to nontarget organisms. This process is described in several of BPPD’s documents and is presented again here as background information. To minimize data requirements and avoid unnecessary tests, risk assessments are structured such that risk is determined first from estimates of hazard under “worst - case” exposure conditions. A lack of adverse effects under these conditions would provide enough confidence that there is no risk and no further data would be needed. Hence, such screening tests conducted early in an investigation tend to be broad in scope but relatively simple in design, and can be used to demonstrate acceptable risk under most conceivable conditions. When screening studies suggest potentially

unacceptable risk additional studies are designed to assess risk under more realistic field exposure conditions. These later tests are more complex than earlier screening studies. Use of this “tiered” testing framework saves valuable time and resources b y organizing the studies in a cohesive and coherent manner an d eliminating unnecessary lines of investigation. Lower tier, high dose screening studies also allow tighter control over experimental variables and exposure conditions, resulting in a greater ability to produce statistically

reliable results at relatively low cost 1

. **1** Nontarget invertebrate hazard tests often are conducted at exposure concentrations several times higher than the maximum concentrations expected to occur under realistic exposure scenarios. This has customarily allowed anBt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 36 of 78 Tiered tests are designed to first represent unrealistic worst case scenarios and ONLY progress to real world field scenarios if the earlier tiered tests fail to indicate adequate certainty of acceptable risk. Screening (Tier I) nontarget organism hazard tests are conducted at exposure concentrations several times higher than the highest concentrations expected to occur under realistic field exposure scenarios. This has allowed an endpoint of 50% mortality to be used as a trigger for addition al higher - tier testing. Less than 50% mortality under these conditions of extreme exposure suggest that population effects are likel y to be negligible given realistic field exposure scenarios. The EPA uses a tiered (Tiers I - IV ) testing sy stem to assess the toxicity of a PIP to representative nontarget organisms that could be exposed to the toxin in the field environment. Tier I high dose studies reflect a screening approach to testing designed to maximize any toxic effects of the test substance on the tes t (nontarget) organism. The screening tests evaluate single species in a laboratory

setting with mortality

as the end point. Tiers II

– IVgenerall y encompass definitive hazard level determinations, longer term greenhouse or field testing, and are implemented when unacceptable effects are seen at the Tier I screening level. EPA’s Office of Chemical Safety and Pollution Prevention (OCSPP) has published a series of test guidelines that contain testing methods and standards for conducting, evaluating, and reporting of data needed to support the registration of a pesticide under FIFRA. The testing methods, which utilize the tiered approach, are published in the OCSPP Harmonized Test Guidelines, Series 850 and 885. 2 These guidelines, as defined in 40 CFR § 1 58.70(c), appl y to microbes and microbial toxins when used as biological pesticides, including those that are naturally occurring, and those that are strain - improved, either by natural selection or by deliberate genetic manipulation. Therefore, PIPs contai ning microbial toxins are also covered by these testing guidelines. Further, most registered PIP products express insecticidal proteins derived from Bacillus thuringiensis ( Bt), a common soil bacterium that is also found in several "reduced risk" microbial pesticides and currentl y available commercially to prevent the damage caused b y insect pests. Microbial pesticides containing Bt proteins have a long history of safe use and undergone extensive toxicity testing showing no adverse effects to human or animal health (US EPA 2000 and 2001a; McClintock et al. 1995; Mendelsohn et al. 2003). The Tier I screening maximum hazard dose (MHD) approach to environmental hazard assessment is based on some factor (whenever possible >10) times the Estimated Environmental endpoint of 50% mortality to be used as a trigger for additional higher - tier testing. Lower levels of mortality under these conditions of extreme exposur e suggest that population effects are likely to be negligible given realistic exposure scenarios. Thus, it follow s that the observed proportion of responding individuals can be compared to a 50% effect to determine if the observed proportion is significantly lower than 50%. For example, using a binomial approach, a sample size of 30 individuals is sufficient to allow a treatment effect of 30% to be differentiated from a 50% effect with 95% confidence using a one - sided Z test. A one - sided test is appropri ate because only effects of less than 50% indicate that further experiments are not needed to evaluate risk. **2** O CSPP Harmonized Test Guidelines, Series 850 - Ecological Effects . Available at the EPA website: http://www.epa.gov/ocspp/pubs/frs/publications/Test_Guidelines/series850.htm O CSPP Harmonized Test Guidelines, Series 885 - Microbial Pesticide . Available at the EPA website: http://www.epa.gov/ocspp/pubs/frs/publications/Test_Guidelines/series885.htmBt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 37 of 78 Concentration 3 (EEC), which is the maximum amount of active ingredient expected to be available to terrestrial and aquatic nontarget organisms in the environment. Tier I tests serve to identify potential hazards and are conducted in the laboratory at high dose levels which increase the statistical power to test the hy potheses. Elevated test doses, therefore, add certainty to the assessment, and such tests can be well standardized. The Guidelines call for initial screening testing of a single group or several groups of test animals at the maximum hazard dose level. The Guidelines call for testing of one treatment group of at least 30 animals or three groups of 10 test animals at the screening test concentration. The Guidelines further state that the duration of all Tier I tests should be approximately 30 day s. Some test species, notabl y nontarget insects, may be difficult to culture and the suggested test duration has been adjusted accordingl y. Control and treated insects should be observed for at least 30 day s or in cases where an insect species cannot be cultured for 30 day s, until negative control mortality rises above 20 percent. Failing the Tier I (10 X EEC) screening at the MHD dose does not necessarily indicate the presence of an unacceptable risk in the field but it triggers the need for additional testing. 4 A less than 50% mortality

effect at the MHD is taken to indicate minimal risk. However, greater than 50% mortality

does not necessarily

indicate the existence of unacceptable risk in the field, but it does trigger the need to collect additional dose - response information and a refinement of the exposure estimation before deciding if the risk is acceptable or unacceptable. Where potential hazards are detected in Tier I testing (i.e. mortality is greater than 50%), additional information at lower test doses is required which can serve to confirm whether any effect might still be detected at more realistic field [1X EEC] concentrations and routes of exposure. 5 When screening tests indicate a need for additional data, the OCSPP Harmonized Guidelines call for testing at incrementally lower doses in order to establish a definitive LD 50 and to quantify the hazard. In the definitive testing, the number of doses and test organisms evaluated must be sufficient to determine an LD 50 value and, when necessary , the Lowest Observed Effect Concentration (LOEC), No Observed Adverse Effect Level (NOAEL) , or reproductive and behavioral effects such as feeding inhibition, weight loss, etc. In the final anal ys is, a ri sk assessment is made b y comparing the LOAEC to the EEC; when the EEC is lower than the LOAEC, a no risk conclusion is made. These tests offer greater environmental realism, but they may have lower statistical power. Appropriate statistical methods, and appropriate statistical power, must be emplo ye d to evaluate the data from the definitive tests. Higher levels of

**3** The dose margin can be less than 10x where uncertainty in the system is low or where high concentrations of test material are not possible to achieve due to test organism feeding habits or other factors. High dose testing also may not be necessary where many species are tested or tests are very sensitive, although the test concentration u sed must exceed 1X EEC. **4** It is notable that that the 10 X EEC MHD testing approach is not equivalent to what is commonly known as “testing at a 10X SAFETY FACTOR” where any adverse effect is considered significant. Tier I screen testing is not ‘safety fac tor testing’. In a “10X safety factor” test any adverse effect noted is a “level of concern”, whereas in the EPA environmental risk assessment scenario any adverse effect is viewed as a concern only at 1X the field exposure. **5** The 1X EEC test dose is b ased on plant tissue content and is considered a high worst case dose (sometimes referred to as HEEC). This 1X EEC is still much greater than any amount which any given nontarget organism may be ingesting in the field because most nontarget organisms do no t ingest plant tissue.Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 38 of 78 replication, the number of test species, and/or repetition are needed to enhance statistical power in these circumstances. Data that shows less than 50 % mortality at the maximum hazard dosage level

– (i.e. L C 50 , ED 50 , or LD 50 >10 X EEC) is sufficient to evaluate adverse effects, making lower field exposure dose definitive testing unnecessary . It is also notable that the recommended >10X EEC maxi mum hazard dose level is a highly conservative factor. The published EPA Level of Concern [LOC] is 50% mortality

at 5X EEC 6 (US EPA 1998). Validation: The tiered testing approach was developed for the EPA b y the American Institute of Biological Scienc es (AIBS) and confirmed in 1996 as an acceptable method of environmental hazard assessment b y a FIFRA Scientific Advisory Panel (SAP) on microbial pesticides and microbial toxins. The December 9, 1999 SAP agreed that the Tiered approach was suitable for us e with Plant - In corporated Protectants (PIPs); however, this panel recommended that, for PIPs with insecticidal properties, additional testing of beneficial invertebrates closel y related to target species and/or likely

to be present in GM crop fields should be conducted. Testing of Bt Cry proteins on species not closel y related to the target insect pest was not recommended, although it is still performed to fulfill the published EPA nontarget species data requirements. In October 2000, another SAP also recommended that field testing should be used to evaluate population - level effects on nontarget organisms. The August 2002 SAP, and some public comments, generall y agreed with this approach, with the additional recommendation that test surrogate organisms shoul d be selected on the basis of potential for field exposure to the subject protein (US EPA 2000, 2001a, 2002 and 2004a). A White Paper developed by the U.S. States Department of Agriculture (USDA) and EPA discussed the tiered testing approach and provided additional guidance for conducting nontarget invertebrate ecological risk assessments specifically for crops expressing insecticidal proteins (US EPA 2007). Chronic studies: Additional higher - tier (e.g. Tier II, III, and IV) testing for chronic effects on nontarget organisms are only conducted when they

are triggered (i.e. toxicity studies that show 50% mortality

when tested at least 5X EEC). Since delayed adverse effects and/or accumulation of toxins through the food chain are not expected to result from exposure to Bt PIP proteins, these protein toxins are not routinely tested for chronic effects on nontarget organisms. However, the 30 day

test duration requirement does amount to subchronic testing when performed at field exposure test doses. Conclusion : The tiered testing approach to test guidelines ensures, to the greatest extent possible, that the Agency

requires the minimum amount of data needed to make scientifically sound regulatory

decisions. The EPA believes the Tier I screening maximum hazard dose (MHD) approach presents a reasonable approach for evaluating environmental hazards related to the use of biological pesticides and for identifying negative results with a high degree of confidence (US EPA 2007). The Agency

expects that Tier I testing for short - term hazard assessment will be sufficient for most studies submitted in support of PIP registrations (US EPA 2007). However, if **6** The established peer and EPA Science Board reviewed guidance on screening test levels of concern is 50% mo rtality at 5X the environmental concentration. The appropriate endpoints in high dose limit/screening testing are based on mo rtality of the treated, as compared to the untreated (control) nontarget organisms. A single group of 30 test animals may be tested at the maximum hazard dose.Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 39 of 78 long range adverse effects must be ascertained, then higher- tier longer - term field testing will be required. As noted above, the October 2000 SAP and the National Academy of Sciences (NAS 2000) recommended testing nontarget organisms directly in the field. This approach, with an emphasis on testing invertebrates found in Bt cotton fields, was also recommend ed b y the August 2002 SAP and was supported by several public comments. Based on these recommendations, the Agency has required large - scale, long term field studies as a condition of registration on: 1) monitoring nontarget invertebrate populations (e.g. community level effects); and 2) determining the bioavailability

/persistence of Cry

proteins remaining in soils representative of different growing regions of Bt crops. These studies were conducted as a precautionary measure due to the lack of baseline data on the potential for long - term environmental effects from the cultivation of PIP - producing plants. Since the commercialization of Bt crops, the number of field studies published in scientific literature in combination with the post - registration field studies submitted to the Agency has accumulated to a level where empirical conclusions can be made. As a result, the issue of long term effects of cultivation of these Cry proteins on the invertebrate community structure in Bt crop fields has since been ad equatel y addressed. Specificall y, a meta - analy sis 7 of the data collected from 42 field studies indicated that nontarget invertebrates are generall y more abundant in fields of Bt crops than in non - transgenic fields managed with insecticides (Marvier et al . 2007). In addition, a comprehensive review of short and long term field studies on the effects of invertebrate populations in Bt corn and cotton fields indicated that no unreasonable adverse effects are taking place as a result of wide scale Bt crop cultiv ation (Sanvido et al. 2007). Another review of field tests published to date concluded that the large - scale studies in commercial Bt crops have not revealed any unexpected nontarget effects other than subtle shifts in the arthropod community caused b y the effective control of the target pests (Romeis et al . 2006). Slight reductions in some invertebrate predator populations are an inevitable result of all pest management practices, which result in reductions in the abundance of the pests as prey . Proteins are not known to persist in the environment due to the ubiquitous nature of proteases in microbes. The biological nature of proteins makes them readil y susceptible to metabolic, microbial, and abiotic degradation once they are ingested or excreted into th e environment. Sprayable microbial pesticide formulations containing Bt have a long history of safe use. Several laboratory

studies have demonstrated rapid degradation of Cry proteins in various soils sampled from Bt crop growing regions. L ong term field s tudies have been conducted showing no accumulation (i.e. persistence) of Cry proteins as a result of continuous planting of Bt crops (Head et al. 2002; Herman et al. 2002; Shan et al. 2008). Thus, the issue of long term effects of Cry proteins has also been addressed b y s everal field studies showing a lack of persistence in various soil ty

pes sampled from representative Bt crop growing region. While there are reports that some proteins (Cry

proteins) binding to soil particles, it has also been shown that these proteins are degraded rapidly by soil microbial flora upon elution from soil particles (Icoz and Stozky 2008).

**7** This research was funded by Environmental Protection Agency grant CR - 832147 - 01. The Bt crop n ontarget effects database can be found on the National Center for Ecological Analysis and Synthesis (NCEAS). Website. ( http://delphi.nceas.ucsb.edu/btcrops/).Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 40 of 78 As a result, the Agency

has determined no additional long - term field studie s are required for monitoring nontarget invertebrate populations or soil protein persistence for Bt crops that contain similar Cry

proteins expressed in previousl y registered PIP events with the exception of Tier I laboratory

studies that “trigger” additi onal testing or if the proposed registration is a novel PIP. Overall, the Agency

is in agreement with the conclusions of these studies and collectivel y, these results provide extensive data to support that Bt crops registered to date have not caused l ong term adverse effects on a population level to organisms not targeted b y Bt proteins. Moreover, these field studies further substantiate the “tiered testing approach” b y confirming that the absence of hazard in the Tier I toxicity studies is predictive of an absence of ecological harm in the field. These results are also consistent with earlier studies of Bt strains used as microbial insecticides, which demonstrated no significant detrimental impacts on populations of the nontarget organisms that were st udied. Based on these considerations, the “tiered testing approach” has been demonstrated as an appropriate method for assessing environmental risk of Bt crops while ensuring, to the greatest extent possible, that the Agency requires the minimum amount of data needed to make scientificall y sound regulatory decisions. **A .**  **Environmental Exposure Assessment** Two separate FIFRA Scientific Advisory Panel reports (US EPA SAP 2000 and 2002) recommended that nontarget testing of Bt Cry proteins should focus on invertebrate species exposed to the crop in which the protein(s) will be expressed. Following SAP recommendations, the EPA determined that nontarget organisms with the greatest exposure potential to Cry protein in transgenic soy

bean fields are beneficial insects that feed on so ybean leaf foliage and pods, particularl y lepidopteran insects, and soil invertebrates. While EPA’s risk assessments of Bt soy bean have focused primarily on these taxa, BPPD recognizes that exposure to other nontarget organisms can occur and has required testing on representative species. The EPA risk assessment is centered on adverse effects at the field exposure rates, which are ty picall y based on protein expression levels within the plant for PIPs. Although it is recommended that nontarget testing be conducted at a test dose 10X the EEC whenever possible, the test dose margin can be less than 10X where uncertainty in the s ys tem is low or where high concentrations of test material are not possible to achieve due to test organism feeding habits. BPPD may

also allow for testing at lower doses in cases where many species are tested or tests are very sensitive, although the concentration used must exceed the EEC. For the purposes of the nontarget organis m studies submitted in support of Cry 1F and Cry 1Ac expressed in event DAS - 81419 - 2 soy

bean, the test material dose levels were based on an estimated concentration of Cry 1F and Cry 1Ac proteins expressed in the tissue(s) with the highest concentration calculated from a field study quantifying protein expression levels in several soybean plant tissue parts (MRID No. 48828402). **B .**  **Ecological Effects Data for Cry1F and Cry1Ac Insecticidal Proteins Expressed in**  **Event DAS**  **- 81419**  **- 2**  **Soybean** In the absence o f PIP - specific risk assessment guidance, EPA requires applicants for PIP registrations to meet the 40 CFR Part 158 data requirements for microbial toxins. TheseBt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 41 of 78 requirements include testing on birds, mammals, nontarget insects, honey bee, plants, and aquatic species, and information has been submitted to address these requirements. Limit dose testing on representative organisms from several taxa was performed in support of Section 3 FIFRA registration of event DAS - 81419 - 2 expressing Bt- derived Cry 1F and Cry 1Ac insecticidal proteins. As stated above, BPPD’s risk assessments focus more greatl y on beneficial nontarget invertebrates, since they

are most closel y related to organisms susceptible to the insecticidal action of Bt toxins. The Cry 1F and Cry 1Ac protein s are meant to target species within the order Lepidoptera (e.g. moths). Bt toxins are known ty pically to have a limited host range, however, to address any unforeseen change in activity spectrum as a result of laboratory protein s ynthesis and to fulfill the published registration data requirements EPA requires that test species used for nontarget insect evaluations should include several invertebrate species that are not related to the target pests. Earthworm studies are also recommended. The applicant h as requested to bridge to the Agency database of previousl y reviewed toxicity studies of select nontarget organisms (NTOs) that supported the currentl y registered WideStrike  PIP cotton product ( EPA Reg. No. 68467 - 3). The WideStrike  PIP cotton line was produced b y conventional cross breeding of cotton lines DAS - 21023 - 5 (also described as 3006 - 210 - 23) expressing Cry 1F protein and DAS - 24236 - 5 (also described as 281 - 24 - 236) expressing Cry 1Ac protein . The environmental risk assessment for the insecticidal proteins (Cry 1Ac and Cry 1F) expressed in WideStrike  cotton was previously evaluated for adverse impacts on nontarget organisms and have been shown to pose negligible risk (US EPA 2005). Since exposure may

also occur to other nontarget organisms, EPA has rec eived a number of studies and data waiver rationales to comply with the Agency ’s published data requirements on other nontarget organisms. The toxicity

of Cry 1F and Cry 1Ac proteins has been previousl y evaluated on several species of invertebrates including: lady beetle (Hippodamia convergens ), green lacewing ( Chrysoperla rufilabris ), honey

bee ( Apis mellifera ), collembola ( Folsomia candida ), parasitic wasp ( Nasonia vitripennis ) and earthworm ( Eisenia fetida ). Two new dietary toxicity studies were submitte d in support for event DAS - 81419 - 2 soy bean, which includes Northern Bobwhite ( Colinus viginianus ) and Rainbow Trout, ( Oncorhynchus Mykiss ) . The individual results for the cited and newly submitted nontarget organism studies in support for Cry 1F and Cry1Ac are summarized in Table 1. The studies are described in more detail below, and full reviews of each study can be found in the individual Data Evaluation Records. To support data bridging from the previousl y conducted environmental risk assessment for Cry 1F and Cry 1Ac insecticidal proteins co - expressed in WideStrike  PIP cotton line, an environmental risk assessment was conducted using a “weight - of - evidence” approach based on the following lines of evidence to support the registration of event DAS - 81419 - 2 soy bean: 1) Confirmation of biological equivalency of Cry1F and Cry 1Ac proteins expressed in event DAS - 81419 - 2 soy

bean plant and previously characterized microbial - derived Cry 1F and Cry 1Ac protein test substances; 2) No significant increase in exposure to NTOs based on calculated margins of exposure for protein concentrations in event DAS - 81419 - 2 plant tissue matrices relative to theBt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 42 of 78 dose concentrations used in the NTO laboratory toxicity tests conducted on Cry 1Ac and Cry 1F ; and 3) No adverse effects on nontarget arthropods on a population and community level observed from a field monitoring study and other supporting supplemental information (e.g. meta - anal ys is, target feeding studies, and biological specificity to the target pest). Exposure estimates were compared with endpoints reported from Tier I toxicity studies previously

conducted on various representative nontarget test species to determine a Margin of Exposure (MOE), the ratio of the endpoint value to the EEC. The MOEs for representative NTOs were recalculated based on high - end exposure estimates (HEEEs) (US EPA, 2009) derived from Cry 1Ac and Cry 1F protein expression levels quantified from various plant tissues of event DAS 81419 - 2 soy

bean in a field study . A MOE of 50% mortal ity at 10X the estimated environmental exposure is regarded as sufficient to demonstrate negligible risk and was also the threshold for consideration of uncertainties in the risk assessment (US EPA 1998; US EPA 2007). If subsequent expression characterization in the commercial event leads to a safety margin of <10X, then the consequences are considered in terms of uncertainties in the ecological risk assessment below (US EPA 2007). **Table B**  **. 1**  **. Summary of Environmental Effects Studies conducted for PIP E**  **ven t DAS**  **- 81419**  **- 2 soybean**  **(EPA Reg. No. 68467**  **- EN) and Scientific Rationales in support for bridging**  **Cry1F and Cry1Ac data from the Agency database on WideStrike® PIP cotton ( EP** **A Reg. No.**  **68467 -**  **3) OCSPP**  **Guideline Study Type**  **SUMMARY OF RESULTS AND CLASSIFICATION**  **MRID No. WideStrike® PIP cotton**  **data (EPA Reg. No. 68467**  **- 3)**  885.4150 Wild Mammal

Testing, Tier I Not required; Mammalian wildlife exposure to Cry1F and Cry1Ac

proteins is considered likely; however, the results f rom two acute oral toxicity studies indicated no adverse effects on mice from microbial derived Cry1F and Cry1Ac proteins administered via oral gavage at the maximum hazard dose. The NOAEL exceeded 600 mg/kg body weight for Cry1F (MRID No. 45542312) and 70 0 mg/kg bw for Cry1Ac (MRID No. 45542313 ). These data are sufficiently representative of toxicity that wo uld be expected in wild mammals, and based on the results of this study, risk to wild mammals resulting from exposure to Cry1F and Cry1Ac proteins is not expected. **Classification: ACCEPTABLE for data bridging**  45542312 and 4554231 3

850.1010 Aquatic

Invertebrate Ac ute Toxicity Test, Daphnia magna In a 48 - hour static test with freshwater daphnids ( Daphnia magna ) there were no observed adverse effects with Cry1F and Cry1Ac in combination at respective concentrations of 510 and 2,500 μ g/L. No immobility or other adverse effects were seen during the study. T he 24 - hour and 48hour EC50s for daphnia exposed to the Cry 1F + Cry1Ac mixture were >510 μ g Cry1F/L and >2500 μ g Cry1Ac/L (represents a worst - case exposure of one kg of transgenic cotton pollen per liter of pond water). This rate of fortification represents 298X and >23,000X the anticipated EEC for Cry1F and Cry1A c protein in surface water. Therefore, no hazard to aquatic invertebrates is expected from incidental exposure to WideStrike cotton pollen. 45808412Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 43 of 78 **OCSPP Guideline**  **Study Type SUMMARY OF RESULTS AND**  **CLASSIFICATION MRID No.**  **Classification: ACCEPTABLE for WideStrike Cotton;**  **SUPPLEMENTAL for DAS -**  **81419 -**  **2 soybean.** 885.4280 Estuarine and

Marine Animal testing, Tier I Not required. Exposure in marine and/or estuarine environments is not

expected to be significant. N/A 885.4300 Nontarget Plant

Studies, Tier I Not required . Bt and its proteins are not known plant pathogens or toxins,

and adverse effects in plants are not anticipated. Exposure to nontarget plants is expected to be minimal. N/A 885.4380 Honey Bee

Larva Testing, Tier I ( Apis mellifera ) At 1.98 μg Cry1F + 11.94 μg Cry1Ac per mL sugar water no effect on

survival of larvae to adult emergence was seen. The LC 50 is >4X pollen expression. Therefore no hazard to honey bee larvae and adult bee emergence is anticipated. **Classification: ACCEPTABLE for data bridging**  45 542316

885.4340 Nontarget

Insect Testing, Tier I ( Nasonia vitripennis ) At 5.2 μg Cry1F + 46.8 μg Cry1Ac per mL sugar water at 10 days, no

effect of limit dose with LC 50 > 13X pollen expression was seen on parasitic hymenoptera larva. Minimal exposure and no hazard to parasitic Hymenoptera from Cry1F and Cry1Ac proteins are expected. Testing of a species more common to cotton fields is recommended. **Classification: ACCEPT**  **ABLE for data bridging** 45808411

885.4340 Nontarget

Insect Testing, Tier I ( Chryosperla camea ) A d ietary toxicity study on green lacewing ( Chrysoperla rufilabris ) larvae

wa s conducted by presenting the green lacewings with a moth egg ( Sitotroga sp.) diet incorporated with Bt Cry1F and Cry1Ac proteins. T he dietary LC 50 wa s >48.2 μg/g diet for Cry1Ac and >5.2 μg/g diet for Cry1F , which represented at least at 14 X th e concentration found in pollen . No effect was noted at the Cry1F and Cry1Ac protein levels expressed in pollen that would be encountered by green lacewings in the field. Because of questionable ingestion of the test material another species (e.g, minute pirate bug), w hich is more likely to be exposed , shou ld be tested. **Classification: ACCEPTABLE for data bridging**  45808410

885.4340 Adult Lady

Beetle Testing, Tier I (Hippodamia convergens ) At 300 μg Cry1F + 22.5 μg Cry1Ac per mL sugar water, no effect of limit

dose with LC 50 > 780X Cry1F pollen expression and > 8X Cry1Ac pollen expression on adult lady beetles. Based on these results, no hazard to H. convergens is expected when feeding on WideStrike® cotton pollen in the field. **Classification: ACCEPTABLE for data bridging**  45 542315

885.4340 Collembola

Chronic Dietary Toxicity Study, Tier I (Folsomia candida ) The combination of 709 μg Cry1F + 22.6 μg Cry1Ac per g diet and cotton

leaf tissue showed no effect on adult survival and reproduction at up to 10X the anticipated field level of expression. Therefore, no hazard to decomposers represented by collembola is expected from exposure to WideStrike  cotton in the field. **Classification: ACCEPTABLE for data bridging**  45808409Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 44 of 78 **OCSPP Guideline**  **Study Type SUMMARY OF RESULTS AND**  **CLASSIFICATION MRID No.**  OECD Guideline 207 Earthworm

Toxicity Study, (Eisenia foetida ) A 14 - day study on earthworms exposed to soils treated with microbial produced Cry1Ac and Cry1F, individually and in combination, was performed. The 14 - day

LC50s were >247 mg a.i./kg for Cry1F; >107 mg a.i./kg for Cry1Ac, and > 247 mg a.i./kg Cry1F + >107 mg a.i./kg Cry1Ac in the test with the two proteins combined. There were no overt signs of toxicity to earthworm s exposed to soils containing nominal concentrations of Cry1F and Cry1Ac at 50X the expected worst case EEC [this represents concentrations which are 792X and 5479X higher than the expected EEC for incorporation of defoliated cotton pl ants into the top 15 cm of soil ] . **Classification: SUPPLEMENTAL**  45580701

885.5200 Expression in a

terrestrial Environment Tier II The soil half - life of the plant expressing Cry1F and Cry1Ac was estimated as 1.3 days in a laboratory study with a representative soil from a cotton growing region. The Cry proteins were not detectable after 14 days. These results verify that the Cry1F and Cry1Ac proteins degrade rapidly in cotton soil. **Classification: ACCEPTABLE for WideStrike Cotton;**  **SUPPLEMENTAL for DAS -**  **81419 -**  **2 S**  **oybean.** 45 556801

**DAS -**  **81419 -**  **2 S**  **oybean data**  **(EPA Reg. No. 68467 -**  **EN)** 885.4050 Avian Dietary

Toxicity Study , Tier I ( Colinus viginianus ) In a 14 - day acute dietary toxicity study, Northern bobwhites ( Colinus

virginianus ) we re exposed to avian diet fortified with a 7.9:1.0 mixture of Cry1F and Cry1Ac proteins with a test substance concentration of 128 mg /kg body weight (24.52 mg a.i./kg body weight based on percent a.i.) by oral gavage. Results showed no apparent effects on mortality, s ublethal observations, body weight, and food consumption at this treatment level. The 14 day acute oral LD 50 for the test substance was > 128 mg /kg bw (24.52 mg a.i./kg body weight based on percent a.i.) and the 14 day NOEL for the test substance wa s > 12 8 mg /kg bw (24.52 mg a.i./kg body weight based on percent a.i.) . M onogastric animals like birds cannot safely consume raw soybean seed since it contains endogenous antinutrients (Friedman et al ., 1991). The estimated feed consumption per bird was 33 gram s/day. Therefore, in relation to the daily diet, Cry1F was dosed at 0.660 mg AI/gm (660 ppm) and Cry1Ac was dosed at 0.083 mg AI/gm (83 ppm). The HEEE concentration of Cry1F in raw DAS - 81419 - 2 soy bean seed is 14.34 ng/mg dw (ppm) and 1.08 ng/mg dw (ppm) fo r Cry1Ac (MRID No. 48828413). The MOE s was calculated for Cry1F and Cry1Ac in the quail study relative to raw soy bean seed consumed as 100% of the diet was 46X (660 ppm/14.34 ppm) and 77X (83 ppm/1.08 ppm), respectively, which provides a large safety margi n even if bird could safely ingest raw

soy bean as their sole source of food . Thus, no adverse effects on avian wildlife are expected from incidental field exposure to DAS - 81419 - 2 soybean expressing Bt Cry1F and Cry1Ac insecticidal proteins. **Classification: ACCEPTABLE**  48828410

885.4200 Freshwater Fish

Testing , Tier I ( Onchorynchus mykiss ) An acute oral dietary toxicity study on rainbow trout ( Onchorynchus

mykiss ) was conducted to assess the effects of a basal fish diet fortified with a mixture of microbial - derived Cry1F and Cry1Ac proteins for eight days. No fish mortality and no sublethal effects were observed in any of the control or test treatment organisms during this study. Results show the 8 - day LD 50 value with rainbow trout was > 100 mg a.i./kg diet wet weight 48828411Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

Biopesticides Registration Action Document January 2014

Page 45 of 78 **OCSPP Guideline**  **Study Type SUMMARY OF RESULTS AND**  **CLASSIFICATION MRID No.**  (>200 mg a.i./kg diet dry weight). The NOEC was reported as > 100 mg a.i./kg diet weight (>200 mg a.i./kg diet dry weight) based on the lack of mo rtalities, sublethal effects, and apparent lack of effects on standard length and body weight. In addition, exposure to freshwater fish in aquatic environments is also considered to be limited . In vie w of the lack of toxicity and minimal aquatic exposure , no adverse effects to freshwater fish as a result of the cultivation of DAS - 81419 - 2 soybeans are expected. **Classification: ACCEPTABLE**  Non guideline Nont arget

Arthropod Census Field Study A field study was conducted to mo nitor nontarget arthropods in fields of

DAS - 81419 - 2 soybean in comparison to non - transgenic fields for any statistically significant differences in the types of taxa collected and the ir abundance for determining any adverse effects on a community level to nontarget arthropods. DAS - 81419 - 2 soybean, a near - isogenic non transgenic control (Maverick) and four non - transgenic reference lines (DSR 3590; IL 3505; Porter 75148; Williams 82) were monitored in replicated field trials at two locations in the USA, Richland, Iowa and York, Nebraska. Each trial site included the six soybean lines arranged in a randomized complete block design with four replicate blocks. Three sampling methods were used to monitor arthropod abundance: 1) pitfall trapping, 2) sticky card trapping and 3) vertical beat sheet sampling. A total of 74 taxonomic groupings were detected and assessed across field sites. To determine biological relevance of any statistical differences, results were interpreted in the context of the magnitude of differences, observations in reference plots, and trend consis tency across sampling methods, sampling periods, and locations. Analysis of data found no statistically significant differences of biological relevance in the nontarget arthropod populations present in fields planted with DAS - 81419 - 2 soy bean and the non - tr ansgenic control soybean lines. **Classification: SUPPLEMENTAL**  48828412

Non guideline Environmental

Risk Assessment Summary on Nontarget Arthropods An environment al risk assessment was separately prepared by the

registrant to summarize the potential for adverse effects of cultivating PIP event DAS - 81419 - 2 soybean on nontarget arthropods associated with soy bean agroecosystems with a special emphasis on beneficial taxa and threatened or endangered species. Dow requested data bridging to the existing ecotox icological studies on the effects of Bt Cry1F and Cry1Ac proteins expressed in WideStrike® PIP cotton on representative nontarget insect species, including beneficial s, pollinators, detritivores, and incidentals. Exposure estimates for Cry1F and Cry1Ac pro tein concentrations in PIP event DAS - 81419 - 2 soybean were compared with endpoints reported from toxicity studies conducted for WideStrike  cotton on select test species to determine the MOE, the ratio of the endpoint value to the EEC. Most MOEs exceeded 10 X the estimated environmental concentrations. None of the estimated exposure values exceeded NOECs for any of the tested species, indicating that the most sensitive individual in a population is not likely to be adversely affected. O ther supplemental in formation (i.e. meta - analyses, biological specificity to the pest species, targeted toxicity feeding studies) , the biology of the crop and lack of exposure (due to feeding habits/food preferences) support the conclusion of no adverse effects to NTO species from PIP event DAS 81419 - 2 soybean. Other supporting data includes environmental fate data 4882841 3Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

Biopesticides Registration Action Document January 2014

Page 46 of 78 **OCSPP Guideline**  **Study Type SUMMARY OF RESULTS AND**  **CLASSIFICATION MRID No.**  of the Cry1Ac and Cry1F proteins, results from a field survey showing no effects on the populations of nontarget arthropods associated with PIP event DAS - 81419 - 2, and supplemental information from the FIFRA Endangered Species Task Force databases for identifying threatened and endangered species that may be exposed to PIP event DAS - 81419 - 2 soy bean. **Classification: SUPPLEMENTAL**  A soil fate study is n ee ded to demons trate rapid protein degradation of Cry1F and Cry1Ac in a variety of soil types that are common in soybean cultivation as confirmatory data . Non guideline Supporting

document A r esponse to EPA Comment s on Cry1F(synpro) ICP and

Cry1Ac(synpro) insecticidal crystal protein was submitted for the acute oral toxicity study with the northern bobwhite ( MRID No. 488284 - 10 ). Protein identity and analytical assays confirmed the equivalence of and their respective certificates of analysis were submitte d to demonstrate the equivalence of Dow’s test material #1 01811 to plant -produced protein and p rovide a justification of the test dosage . **Classification: SUPPLEMENTAL**  to MRID No. 4882841 0 49044807

Non guideline Supporting

document A r esponse to EPA Comment s on Cry1F(synpro) and Cry1Ac(synpro)

insecticidal crystal proteins was submitted to demonstrate the equivalence of Dow’s test material #10 1811 to the plant -produced protein used in the 8 - day dietary study with the rainbow trout ( Oncorhynchus mykiss ) WALBAUM (MRID No. 4 88284 - 11 ). **Classification: SUPPLEMENTAL**  to MRID No. 4882841 1 49044808

Non guideline Supporting

document A response to EPA Comments on Cry1F(synpro) ICP and

Cry1Ac(synpro) insecticidal crystal protein was submitted to address several data gaps in the registrant’s environmental risk summary (MRID No. 48828413) for PIP event DAS - 81419 - 2 soybean. Additional data was needed to complete the risk assessment on the environmental fate of Cry1F and Cry1Ac proteins expressed in PIP event DAS - 81419 - 2 soy bean. Supporting literature and data waiver rationales were presented for evaluating soil fate, gene flow and the potential for weediness . **Classification: SUPPLEMENTAL**  to MRID No. 48828413 4904480 9

Additional support for a complete environmental risk assessment is gained from an overview of the assessment conducted to support the WideStrike® cotton EPA Sec. 3 registration (EPA Reg. No. 68467 - 3) . A brief summary of the EP A’s environmental risk assessment is provided in the follo wing section: **C. WideStrike**   **cotton (lepidopteran active) Environmental Risk Assessment**  **Summary (** **EPA Reg. No. 68467**  **- 3)**  Potential adverse effects to nontarget organisms by Cry 1F and Cry 1Ac proteins have been reviewed in Bt Cry 1F/Cry 1Ac WideStrike  cotton BRAD (US EPA 2005). The following is aBt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

Biopesticides Registration Action Document January 2014

Page 47 of 78 summary of the environmental risk assessment for Cry 1F and Cry 1Ac. EPA performed risk assessments on plants, wild mammals, birds, fish, aquatic invertebrates, earthworms, terrestrial nontarget insects (includi ng honey bee, parasitic wasps, green lacewings, lady bird beetle, springtails [Collembola toxicity /reproduction], and monarch butterflies), as well as field evaluations of the effects of Cry 1F and Cry 1Ac exposure on nontarget insects in cotton fields, soil degradation/persistence studies, and an endangered species impact assessment, particularl y for Lepidoptera. In addition, gene flow and weediness assessments via pollen and Cry protein DNA uptake b y plants were also performed. Cry 1F and Cry 1Ac protein in so il has been shown to degrade rapidl y to very low levels. EPA concluded that there is sufficient information to believe that there is no risk from the uses of Cry 1F and Cry 1Ac cotton to nontarget wildlife, aquatic, and soil organisms. These studies were individually reviewed b y the Agency

and reported in Data Evaluation Reports (DERs). For detailed information of each study , refer to the DER according to its respective assigned MRID number in the associated environmental effects data summary

tables for WideS trike  cotton (reviewed in US EPA 2005) . At present, the Agency

is aware of no identified significant adverse effects of Cry 1F and Cry 1Ac proteins on the abundance of nontarget organisms in any population in the aquatic or terrestrial field environment. F ield testing and field census data submitted to the Agency show minimal to undetectable changes in the beneficial insect abundance or diversity . To date the available field test data show that compared to crops treated with conventional chemical pesticides , the transgenic crops have no detrimental effect on the abundance of nontarget invertebrate populations. In addition, no direct or indirect effects on Federally listed endangered and threatened species or effects on their critical habitat are expected. Th e EPA has reviewed the potential for gene capture and expression of Cry1F and Cry 1Ac proteins by wild or weedy relatives of cotton in the United States, its possessions or territories and has found that there is no significant risk in the United States, it s possessions or territories (US EPA 2005). In conclusion, the risk assessment found no unreasonable adverse effects on the environment from cultivation of WideStrike  cotton expressing Cry 1F and Cry 1Ac proteins for use as a PIP. **D. Protein Equivalence**  The October 2000 SAP recommended that while actual plant material is the preferred test material, microbial - derived protein is also a valid test substance, particularly in scenarios where test animals do not normally consume cotton plant tissue and where l arge amounts of Cry protein are needed for maximum hazard dose testing. In support of event DAS - 81419 - 2 soy bean registration, several of the nontarget toxicological studies used microbial - produced, purified Cry 1F and Cry 1Ac protein test substances that were originall y developed for the Widestrike  PIP cotton registration; in addition to plant tissue (ly pholized leaf tissue) from event DAS 81419 - 2 soy

bean. In support of bridging data from previousl y reviewed NTO toxicological studies conducted for WideStrike  cotton (US EPA 2005), the biochemical properties the microbial - derived Cry 1F and Cry 1Ac protein test substances were compared with plant - produced Cry1F and Cry 1Ac proteins expressed in event DAS - 81419 - 2 soy bean to determine that the proteins from the two host sources were biologically equivalent. The proteins were characterized and equivalence wasBt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

Biopesticides Registration Action Document January 2014

Page 48 of 78 evaluated based on a panel of anal ytical tests and assay s. The results of these evaluations provide a detailed characterization of the Cry 1F and Cry 1A c proteins isolated from event DAS - 81419 - 2 soy bean and confirmed their equivalence to the microbial - derived Cry 1F and Cry 1Ac proteins (MRID N o s. 48828403 and 49044803; reviewed in US EPA 2013). Therefore, the results of the NTO toxicity

studies that utiliz ed the microbial - derived Cry 1F and Cry 1Ac test protein substances can be bridged to support the environmental risk assessment of event DAS - 81419 - 2 soy bean. Furthermore, development of additional nontarget organism toxicity testing can be waived for PIP event DAS - 81419 - 2 soy bean and the results and conclusions from the environmental risk assessment conducted for the WideStrike  cotton PIP line can be bridged to support event DAS - 81419 - 2 soy bean (US EPA 2005). **E .**  **Nontarget Organism Effects Cry1F and Cry1Ac**  **insecticidal proteins expressed in**  **Event DAS -**  **81419 -**  **2 Soybean (EPA Reg. No. 68467**  **- EN)**  **1. Avian W** **ildlife** The primary

routes of exposure of birds to Cry 1F and Cry 1Ac insecticidal proteins expressed in PIP event DAS - 81419 - 2 soy bean plants expected to occur through consumption of event DAS 81419 - 2 plant material (e.g., leaf foliage and pods) and invertebrates that feed on event DAS 81419 - 2 soy

bean plant material. Exposure via consumption of soy bean seeds may

be limited, however, b y compounds that interfere wi th digestion and nutrient uptake, such as try psin inhibitors. Other routes of exposure (e.g., inhalation) are not expected to be significant, since Cry 1F and Cry 1Ac proteins are contained primarily within the plant. In a 14 - day

acute dietary

toxicity study, Northern bobwhites ( Colinus virginianus ) were exposed to avian diet fortified with a 7.9:1.0 mixture of Cry 1F and Cry 1Ac proteins by oral gavage (MRID No. 48828410) . Results showed no apparent effects on mortality , sublethal observations, body weight, and food consumption at this treatment level. The 14 day acute oral LD 50 for the test substance was > 128 mg/kg bw (24.52 mg a.i./kg body weight based on percent a.i.) and the 14 day NOEL for the test substance was > 128 mg /kg bw test substance (24.52 mg a .i./kg body weight based on percent a.i.). BPPD estimates the exposure to birds in the field for soy beans is minimal. M onogastric animals like birds cannot safely

consume raw soy

bean seed since it contains endogenous antinutrients (Friedman et al., 1991) . The estimated feed consumption per bird was 33 grams/day . Therefore, in relation to the daily

diet, Cry 1F was dosed at 0.660 mg a.i. /gm (660 ppm) and Cry 1Ac was dosed at 0.083 mg a.i. /gm (83 ppm). The HEEE concentration of Cry 1F in raw DAS - 81419 - 2 soy bean seed is 14.34 ng/mg dw (ppm) and 1.08 ng/mg dw (ppm) for Cry 1Ac (MRID No. 48828413). The MOE was calculated for Cry 1F and Cry 1Ac in the quail study relative to raw soy bean seed consumed as 100% of the diet was 46X (660 ppm/14.34 ppm) and 77X (83 ppm/1.08 ppm), respectively , which provides a large safety margin even if bird s could safel y ingest raw so ybean as their sole source of food. Furthermore, the Cry 1F and Cry 1Ac proteins have been registered as PIPs in several crops with no identified concerns for birds, and there is a history of use with no reported incidents involving birds in the field, including use in a crop that is typically ingested b y birds (corn). Therefore, based on this history , current toxicity data, and the BPPD’s assumptions of the la ck of avianBt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

Biopesticides Registration Action Document January 2014

Page 49 of 78 exposure, no adverse effects to birds are anticipated as a result of the registration of DAS - 81419 - 2 S oy bean. **2 .**  **Wild M**  **ammals** Wild mammals could be exposed to Cry 1F and Cry 1Ac proteins produced by event DAS - 81419 - 2 soy bean plant s by

consumption of plant material and invertebrates that feed on event DAS 81419 - 2 soy

bean plant material. As with birds, these are expected to be the primary sources for exposure. Data are available with which to determine the risk of Cry 1F and Cry 1Ac proteins from DAS 81419 - 2 soy

beans to wild mammals. The results from the acute oral toxicity studies showed no significant toxicity

to mice from acute oral testing with the Cry 1F and Cry1Ac proteins, with a NOAEL that exceeds 600 mg/kg body weight for Cry1 F (MRID No. 45542312) and 700 mg/kg for Cry 1Ac (MRID No. 45542313). Based on the estimated expression level in leaf (V5) tissue (63.3 ng/mg dw) converted to dose (0.00153 mg/kg bw), adverse effects would not be expected at concentrations up to a MOE of >3. 9 x 10 5 X for Cry 1F. Based on the estimated expression level in leaf (V5) tissue (28.3 ng/mg dw) converted to dose (0.000683 mg/kg bw), adverse effects would not be expected at concentrations up to a MOE of >1.0 x 10 6X for Cry 1Ac. Thus, the MOEs greatl y exc eed the required 10X EEC showing no adverse effects when tested at the maximum hazard dose. These data are sufficientl y representative of toxicity that would be expected in wild mammals, and based on the results of this study risk to wild mammals resultin g from exposure to Cry 1F and Cry 1Ac proteins expressed in DAS - 81419 - 2 S oy bean is not expected.

**3. Freshwater Animals** The registrant submitted an acute oral dietary toxicity study on rainbow trout ( Onchorynchus mykiss ) to assess the effects of consuming a basal fish diet fortified with a mixture of microbial derived Cry 1F and Cry 1Ac proteins for eight days (MRID No. 48828411). No fish mortality and no sublethal effects were observed in any of the control or test treatment organisms during this s tudy . Results show the 8 - day LD 50 value with rainbow trout was > 100 mg a.i./kg diet wet weight (>200 mg a.i./kg diet dry weight). The NOEC was reported as > 100 mg a.i./kg diet weight (>200 mg a.i./kg diet dry weight) based on the lack of mortalities, sub lethal effects, and apparent lack of effects on standard length and body weight. In addition, exposure to freshwater fish in aquatic environments is also considered to be minimal. For freshwater invertebrates, a 48 - hour static toxicity study on daphnids (

Daphnia magna ) showed no adverse effects after exposure to WideStrike® cotton pollen expressing Cry 1F and Cry 1Ac in combination at respective concentrations of 510 and 2,500 μg/L (MRID No. 45808412). Based on a worst - case exposure of one kg of transgenic cotton pollen per liter of pond water, the rate of fortification at these concentration are 298X and >23,000X the anticipated EEC for Cry 1F and Cry 1Ac protein in surface water. However, this study was determined of minimal value for evaluating the potenti al effects to nontartget freshwater invertebrates because the length of the study was not conducted at the Agency’s recommendation of at least 7 - 14 day s, since the mode of action for Bt would likely take effectBt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

Biopesticides Registration Action Document January 2014

Page 50 of 78 after several day s of exposure. In addition, EPA determined that exposure to Cry proteins in the freshwater environment were expected to be low, based on the assumption that exposure in aquatic environments primarily resulted from pollen deposition (see US EPA 2010a) and that post - harvest crop resi due is the most likely route of exposure to aquatic organisms (Carstens et al. 2011). However, in light of published studies showing reduced growth in caddis flies exposed to anti - lepidopteran Cry 1A protein (Rosi - Marshall et al. 2007), concerns regarding the potential for exposure to shredder invertebrate species via consumption of Bt crop plant litter in aquatic environments. EPA determined exposure to shredder invertebrate species from Bt crops is likely to be low, since the concentrations of Cry proteins in post - harvest crop tissues are limited temporally

and spatially

(Swan et al . 2009; Jensen et al. 2010; Wolt and Peterson 2010; Carstens et al . 2011). Nevertheless, EPA has required aquatic invertebrate testing lasting 7 - 10 day s with Daphnia spp. to redu ce uncertainty in past PIP registrations of Bt crops. A study with shredder species is even more appropriate since it would directl y test the invertebrate species expected to be affected b y Bt plant litter that may enter streams. Specificall y for so yb ean, the Agency has determined that the exposure of aquatic sy stems to Cry 1F and Cry 1Ac proteins produced in PIP event DAS - 81419 - 2 soy bean are negligible. Movement of Cry

proteins from off

- field pollen or post - harvest crop residues to aquatic habitats is not e xpected given that soy beans are primaril y s elf

- pollinated and cultivated soy bean plants release pollen prior to flower opening (Abud et al . 2007; Caviness 1966). The Agency previousl y concluded that plant litter from both Bt corn and Bt cotton crops are no t deposited in amounts high enough to result in adverse effects (US EPA 2001b). Since soy bean produces less above ground residue under cultivation (Green and Blackmer 1995), this conclusion is also made for the cultivation of DAS - 81419 - 2 S oy bean. Based on the acute oral toxicity study on rainbow trout, adverse effects to freshwater fish are not anticipated as a result of the registration of Cry 1F and Cry 1Ac as expressed in event DAS 81419 - 2 soy

bean. Risk posed to aquatic invertebrates (including the aquati c Lepidoptera) through DAS - 81419 - 2 cultivation was concluded to be much reduced due to a low likelihood of exposure (Carstens et al. 2011) and no verified instances of sensitive species. In view of the lack of toxicity

and minimal aquatic exposure, the rainbow trout study and the data waiver rationale for freshwater invertebrates are considered to be adequate to support the conclusion that deploy ment of event DAS - 81419 - 2 for use as a PIP in soy bean poses little risk to aquatic organisms. **4. Estuarine and Marine A**  **nimals** BPPD ty pically waives the requirement of studies with estuarine/marine animals for Bt Cry proteins because of an expected lack of exposure in these environments. Therefore, data were not required for Cry 1F and Cry 1Ac for th e registration of DAS - 81419 - 2 S oy bean, and based on expected lack of exposure; adverse effects to these species are not anticipated.

**5. Terrestrial and A**  **quatic Plant S**  **pecies** BPPD ty pically waives nontarget plant testing for Bt Cry proteins, since the active ingredients are insect toxins (

Bt δ - endotoxin) that have never shown any toxicity to plants. Therefore, BPPDBt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 51 of 78 has concluded that adverse effects of Cry 1F and Cry 1Ac insecticidal proteins expressed in DAS 81419 - 2 S oy

bean to terrestrial and aquatic plants are not anticipated. The active ingredients Cry 1Ac and Cry 1F are part of a larger group of insect toxins produced a naturally- occurring Bacillus thuringiensis ( Bt  - endotoxins) that have not shown toxicity to plants. Effects to nontarget plants in the terrestrial and aquatic environments are not expected, and BPPD concludes that adverse effects to terrestrial and aquatic plants as a result of the registration and use of Cry1F and Cry 1Ac insecticidal proteins expressed in event DAS - 81419 - 2 S oy bean are not anticipated.

**6 . Invertebrate S**  **pecies** BPPD assumes that nontarget insects receive exposure primaril y through consumption of DAS 81419 - 2 S oy

bean leaf foliage, pods, and occasionally other so ybean plant tissues as well as indirect exposure through consumpti on of insects that feed on so ybean plant tissue. The principal route of exposure to soil - dwelling invertebrates, such as collembola and earthworms, is assumed to be consumption of decomposing plant tissue, and also possibly plant exudates, in soil during f eeding. The registrant requested data bridging to the existing ecotoxicological studies on the effects of Bt Cry 1F and Cry 1Ac proteins expressed in WideStrike® PIP cotton on representative nontarget species, including nontarget beneficial insects, pollinators, detritivores, and incidentals (MRID No. 48828413). As previously discussed, the Cry 1F and Cry 1Ac proteins produced in DAS 81419 - 2 soy

beans were determined biologicall y equivalent with the microbial - derived Cry 1F and Cry 1Ac protein test substances us ed for producing toxicological data to support WideStrike® cotton (MRID No. 48828403 and 49044803; reviewed in US EPA 2013 ). The high - end exposure estimates (HEEEs) were derived from protein expression levels of Cry 1F and Cry 1Ac via Enzy

me - linked immuno sorbent assay (ELI SA) anal ys es on various soy bean plant tissue parts of PIP event DAS - 81419 - 2. Exposure estimates for Cry 1F and Cry 1Ac protein concentrations in PIP event DAS - 81419 - 2 soy bean were compared with endpoints reported from toxicity studies previ ously

conducted for WideStrike® cotton on various representative nontarget test species to determine a Margin of Exposure (MOE). The results of the MOE calculations are presented in Tables B . 2 . and B . 3. A MOE of 50% mortality at 10X the estimated enviro nmental exposure is regarded as sufficient to demonstrate negligible risk and was also the threshold for consideration of uncertainties in the risk assessment (US EPA 1998; US EPA 2007). If subsequent expression characterization in the commercial event leads to a safety margin of <10X, then the consequences are considered in terms of uncertainties in the ecological risk assessment below (US EPA 2007). The ratio of the endpoint value to the EEC and are presented in Tables B . 2 . and B . 3 . for Cry 1F and Cry 1Ac, respectivel y. The published EPA level of concern is 50% mortality at 5× MEEC (US EPA 1998). It should also be noted that the dose margin can be less than 10× where uncertainty in the test sy stem is low. High dose testing also may not be necessary

where many species are tested or tests are very

sensitive, although the test concentration used must exceed 1× MEEC (US EPA 1998; US EPA 2007). The registrant note that the concentrations tested were much higher than those encountered under field conditions. Since m ost MOEs exceeded 10X the estimatedBt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 52 of 78 environmental concentrations (Tables B . 2 . and B . 3 . ), the results of the MOE calculations indicates the most sensitive individual in a population is not likely to be adversel y affected. **a. Beneficial Arthropods and**  **P ollinators**  Data from a dietary

toxicity study on lady bird beetle previously conducted for WideStrike  cotton showed that adverse effects did not occur to the test insects at >300 μg/mLdiet for Cry 1F and >22.5 μg/mL diet for Cry 1Ac assuming a maximum expected environmental concentration of 10.6 ng/mg fresh weight for Cry 1F and 4.76 ng/mg fresh weight for Cry 1Ac (see MRID No. 45542315). Dow did not anal yz e protein expression levels for pollen or so ybean floral nectar, as Cry 1 protein expression in this se cretion in four cotton lines is negligible (Wolt 2002; MRID No. 45808420). Therefore, the MOE calculation for lady beetle relies on the highest level of protein expression found in the field expression study (V5 leaf). Based on the estimated expression lev el in leaf (V5) tissue, adverse effects would not be expected at concentrations up to 25.6X for Cry 1F and 4.3X Cry 1Ac. The registrant requested data bridging to the dietary toxicity study on green lacewing ( Chrysoperla rufilabris ) larvae, which was conduc ted by presenting the green lacewings with a moth egg (Sitotroga sp.) diet incorporated with Bt Cry 1F and Cry 1Ac proteins (MRID No. 45808410). Results showed that adverse effects did not occur to the test insects at >48.2 μg/g diet for Cry 1Ac and >5.2 μg/g diet for Cry 1F. However, this study was considered supplemental. A previous EPA FIFRA Scientific Advisory Panel (SAP) determined that it is inappropriate to test the activity

of insecticidal proteins by incorporating the Bt protein into a moth egg diet, s ince Bt protein may

bind to the surface of the moth eggs, resulting in limited exposure to lacewings that feed with piercing sucking mouthparts (US EPA - SAP 2002). Furthermore, green lacewings do not consume much pollen in the field (US EPA - SAP 2002) and are not exposed to Bt proteins via consumption of aphids (Head et al. 2001). In the Agency ’s environmental risk assessment of WideStrike  cotton, this study was classified as supplemental and the Agency recommended other generalist predators (e.g. minute pirate bug) as a representative surrogate test organism for Tier I testing the effects of Cry 1F and Cry 1Ac proteins (US EPA 2005). Thus, the green lacewing study on the effects of Cry1Ac and Cry1F proteins expresse d in DAS - 81419 - 2 soy bean can be bridged , but only

offer supplemental information towards the environmental risk assessment of DAS - 81419 - 2 soy bean. Other information on the effects of Cry 1Ac and Cry 1F exist on green lacewing larvae concludes Cry 1Ac and Cry 1F toxins do not have a detrimental effect on the species when they are ingested either directly or through prey

(Rodrigo - Simón 2006 ; T ian et al. 2013). A dietary toxicity study that tested the effects of Cry 1F and Cry 1Ac expressed in DAS - 81419 - 2 soy bean in honey

bee ( Apis mellifera ) was also cit ed for data bridging. Results showed no effect on survival of larvae to adult emergence at >1.98 μg/mLsugar water diet for Cry 1F and >11.9 μg/mLsugar water diet for Cry 1Ac (MRID No. 45542316). Pollen expression data for DAS 81419 - 2 soy

bean was not availa

ble, however, the MOE previously calculated for WideStrike® cotton showed the L C 50 was >4X the levels of pollen expressed. These data can be inferred to represent the MOE for event DAS - 81419 - 2 soy bean, since the same genetic construct that was used to transform WideStrike® cotton was used for event DAS - 81419 - 2 soy bean and the constitutive expression in pollen should be similar. In addition, other information from a meta anal ys is (Duan et al. 2008) of several laboratory acute oral toxicity

studies on hone yb ee isBt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 53 of 78 available, which consistently demonstrated no biological activity in Hy menoptera order of insects from Cry

proteins that target lepidopteran pests. Six Cry 1F and Cry1Ac studies were included in the meta - analy sis, suggesting that regardless of expre ssion level in event DAS 81419 - 2 soy

bean pollen the MOE will be acceptable. An acute toxicity

study on parasitic wasp species (Nasonia vitripennis ) was cited for data bridging. Results showed adverse effects did not occur to the test insects at >5.2 μg/gdiet for Cry 1F, respectivel y, and >46.8 μg/g diet for Cry1Ac, which represented at least at 14X the concentration found in WideStrike® cotton pollen (see MRID No. 45808411). Similar to the green lacewing and a honey bee study , the MOE was not determine d for the parasitic wasp species due to the lack of pollen and nectar expression data, and some uncertainty remains. . However, the HEEE of Cry 1F and Cry 1Ac from V5 leaf sample (as opposed to pollen) relative to the dose concentrations used on honey bees an d nontarget arthropods (likely to be exposed via pollen ingestion) in toxicity studies is considered highl y conservative and the margins of exposure (see Tables B. 2 . and B. 3 . ) are well above dose levels of Cry 1Ac and Cry 1F protein toxicity testing showing no adverse effects. The registrant note d that the concentrations tested were much higher than those encountered under field conditions. Since m ost MOEs exceeded 10X the estimated environmental concentrations (Tables B. 2 . and B. 3 . ), the results of the MOE calculations indicates the most sensitive individual in a population is not likely to be adversel y affected. Further, in regards to exposure of Cry 1F and Cry 1Ac proteins expressed in event DAS - 81419 - 2 soy bean pollen, pollinators and other beneficial ar thropods are anticipated to be less exposed based upon the cleistogamous nature of its flowers (in most cultivars). Soybean is predominantl y self - pollinated and its pollen is essentially contained in the flower, since anthers usually dehisce and release po llen before flowers open (Caviness 1966; OECD 2000). Ty pical outcrossing rates are less than 1% (CPC 2004), so this suggests that contact of honey bees and other beneficial and predatory invertebrates to soy

bean pollen is minimal. So although the MOE to hon ey bees, green lacewings, and lady beetle were calculated from the HEEEs from Cry 1F and Cry 1Ac proteins in leaf tissue (V5) stage instead of pollen, the likelihood of actual field exposure to many nontarget invertebrates via pollen consumption or indirectly through consumption of pollen - feeding invertebrates is expected to be minimal. Since no pollen is expected to travel outside the field borders, off

- field exposure t o many nontarget invertebrates is further reduced. In any case, no adverse effects were found on previously conducted toxicity

testing of Cry 1F and Cry 1Ac proteins on representative nontarget beneficial arthropods and pollinators species (e.g. honey bees, lady

beetles, green lacewings) based on the majority of the MOEs exceeded 10X the estimated environmental concentrations (Tables B. 2 . and B. 3 . ). The results of the MOE calculations indicate the most sensitive individual in a population is not likely to be adversel y affected. Risk to honey

bees and the nontarget arthropods is further reduced due to minimal potential for exposure via pollen. Further, published meta - anal ys is of laboratory and field studies showed no significant differences were noted on populations of nontarget invertebrates on a community

level for Bt crops expressing Cry 1F and/or Cry 1Ac vs. non - transgenic crops (Marvier, et al 2007; Duan et al. 2008). Lastl y, an independent field census study showed no differences in the population of nontarget arthropods (MRID No. 48828412) in field sites cont aining event DAS - 81419 - 2 soy bean vs. non - transgenic conventional soy bean lines. AmongBt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

Biopesticides Registration Action Document January 2014

Page 54 of 78 the taxa showing no significant population impacts were two that were also tested in the laboratory : Coccinellidae (lady beetles) and Parasitica (parasitoid wasps). In v iew of the lack of toxicity and limited exposure via pollen consumption, no adverse effects are anticipated on nontarget beneficial arthropods and pollinators from the cultivation of PIP event DAS - 81419 - 2 S oy bean . **b. Soil Dwelling I**  **nvertebrates** An earthworm study

conducted for WideStrike® cotton was also cited for data bridging. Earthworms (

Eisenia foetida ) were exposed to artificial soils treated with 247 mg/kg and 107 mg/kg of microbial - produced Cry 1Ac and Cry 1F proteins, respectively , for 14 d ay s (MRID No. 45580701). The study showed no effects on mortality or burrowing time resulting from exposure to Cry 1F and Cry 1Ac present in artificial soil at approximately 716X for Cry 1F and 1176X for Cry 1Ac at the maximum EEC. The maximum EEC of the soil was 0.345 mg/kg for Cry 1F and 0.091 mg/kg for Cry 1Ac based on so ybean plant residue in the top 15cm of soil . Data from a toxicity

study on Collembolas (

Folsomia candida ) previousl y conducted for WideStrike  cotton showed that adverse effects did not occur to the test insects at >702 mg/kg soil for Cry 1F and >22.6 mg/kg soil for Cry 1Ac (MRID No. 45808409). The MOE for Cry 1Ac was calculated as >2035X and >248X for Cry 1F, assuming a maximum EEC of 0.345 mg/kg for Cry 1F and 0.091 mg/kg for Cry 1Ac based on so ybean plant residue in the top 15cm of soil. A lack of adverse effects following exposure to Cry 1Ac protein has also been shown for collembolans (

Folsomia candida ) (Sims and Martin, 1997). Based on the studies submitted for DAS - 81419 - 2, as well as other information for Cry 1F and Cry 1Ac, risk to nontarget soil invertebrates resulting from the cultivation of PIP event DAS 81419 - 2 S oy

bean is not expected.Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 55 of 78 **Table B**  **. 2. Margins of Exposure calculated from**  **Cry1F Protein Concentrations in PIP**  **Event DAS**  **- 81419**  **- 2**  **soybean plant tissues relative to Dose Concentrations**  **used in the Nontarget Arthropod Ecotoxicological**  **Studies previously conducted on WideStrike® PIP cot**  **ton plan t**  **- and microbial**  **- derived**  **Cry1F protein a**  **MRID No. Study Type**  **Species Tissue**  **sample HEEE**  **b Tox.**  **Endpoint MOE**  **Comment**

45542316 Honey Bee

Larva Testing, Tier I (885.4380) Honeybee

( Apis mellifera ) Pollen ND >1.98 μg/mL diet NOEC ND No effects found

for honey bee larvae and adults in published meta analysis with Cry1F and/or Cry1Ac expressing Bt crops (Duan et al. 2008)

45808410 Nontarget

Insect Testing, Tier I (885.4340) Green

lacewing ( Chryosperla camea ) Nectar,

pollen ND >5.2 μg/g diet acute oral LC50 ND No effects found

in Cry 1Ac and Cry1F protein lab study (Rodrigo Simón et al. 2006) and tritrophic feeding study on green lacewings exposed to a diet of Cry1F or Cry1Ac resistant prey (Tian et al. 2013) 45542315 Nontarget

Insect Testing, Tier I (885.4340) Lady beetle

(Hippodamia convergens ) Pollen

(used V5 Leaf) 10.6

ng/mg fw >300 μg/mL

diet acute oral LC50 >25.6 Pollen expression

data is not available; Highest expression in leaf converted to fw for MOE calculation. 45808411 Nontarget

Insect Testing, Tier I (885.4340) Parasitic wasp

( Nasonia vitripennis ) Nectar ND >5.2 μg/mL diet acute oral LC50 ND No feeding in

narrow soybean flowers

45808409 Nontarget

Insect Testing, Tier I (885.4340) Collembola

(Folsomia candida ) R3

Forage soil residue 0.345

mg/kg soil >702 mg/kg

soil NOEC >2035 Plant residue in top 15 cm soil

45580701 Earthworm

Toxicity Study Earthworm

(Eisenia foetida ) R3

Forage soil residue 0.345

mg/kg soil 247 mg/kg

soil NOEC 716 Plant residue in top 15 cm soil

**a** Microbe - derived Cry1F protein used in laboratory studies was demonstrated to be equivalent with the Cry1F protein expressed in DAS - 81419 - 2 **b** HEEE = mean expression level + (t 0.1, upper tail, n - 1 x std. dev.) / n 1/2 . HEEE values based on Cry1F expression level determined for DAS - 81419 - 2 plant tissue relevant to the potential exposure route for the organism of interest. ND = Not Determined (data not available) . Table B.2 reproduced from Table 3, page 26 from MRID No. 48828413Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

Biopesticides Registration Action Document January 2014

Page 56 of 78 **Table B**  **. 3. Margins of Exposure calculated f**  **rom Cry1Ac protein concentrations in**  **DAS -**  **81419 -**  **2 Soybean plant tissues relative to dose concentrations used**  **in the Nontarget Arthropod Ecotoxicological Studies**  **previously conducted on WideStrike® PIP cotton plant -**  **and microbial -**  **derived Cry1Ac protein a**  **MRID No.**  **Study Type**

**and OCSPP Guideline**  **No. Species**  **Tissue sample**  **HEEE b**  **Tox.**

**Endpoint MOE**  **Comment** 45580701 Earthworm

Toxicity Study Earthworm

(Eisenia foetida ) R3

Forage soil residue 0.091

mg/kg soil 107 mg/kg

soil NOEC 1176 Plant residue in top 15 cm soil 45808409 Nontarget

Insect Testing, Tier I (885.4340) Collembola

(Folsomia candida ) R3

Forage soil residue 0.091

mg/kg soil >22.6 mg/kg

soil NOEC >248 Plant residue in top 15 cm soil 45542316 Honey Bee

Larva Testing, Tier I (885.4380) Honeybee

( Apis mellifera ) Pollen ND >11.9μg/mL diet NOEC ND No effects found for honey bee larvae and adults in published meta analysis with Cry1F and/or Cry1Ac expressing Bt crops (Duan et al. 2008) 45808410 Nontarget

Insect Testing, Tier I (885.4340) Green

lacewing ( Chryosperla camea ) Nectar,

pollen ND >46.8 μg/g

diet acute oral LC50 ND No effects found

in Cry protein lab study (Rodrigo Simón et al. 2006) and tritrophic feeding studies on green lacewings exposed to a diet of Cry1F or Cry1Ac resistant prey ( Tian et al. 2013) 45542315 Nontarget

Insect Testing, Tier I (885.4340) Lady beetle

(Hippodamia convergens ) Pollen

(used V5 Leaf) 4.76

ng/mg fw >22.5 μg/mL diet acute oral LC50 >4.3 Highest expression

in leaf converted to fw . No effect found in Cry1Ac feeding study in Coleomegilla maculata (Li et al . 2011) 45808411 Nontarget

Insect Testing, Tier I (885.4340) Parasitic wasp

( Nasonia vitripennis ) Nectar ND >46.8 μg/mL diet acute oral LC50 ND No feeding in

narrow soybean flowers

**a** Microbe - derived Cry1Ac protein used in laboratory studies have been demonstrated to be equivalent with the Cry1Ac protein expressed in DAS - 81419 - 2 **b** HEEE = mean expression level + (t 0.1, upper tail, n - 1 x std. dev.) / n 1/2 . HEEE values based on Cry1Ac expression level determined for DAS - 81419 - 2 plant tissue relevant to the potential exposure route for the organism of interest. ND = Not Determined (data not available) . Table B.3 reproduced from Table 3, page 26 from MRID No. 488284 - 13Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

Biopesticides Registration Action Document January 2014

Page 57 of 78 **7 .**  **Supple mental Information**  Where a lack of expression data prevented calculation of high - end exposure values, other supplemental information (i.e. meta - anal ys es, biological specificity to the pest species, targeted toxicity studies, or food preferences) was used as a supporting line of evidence to support the conclusion of no adverse effects on NTOs from Cry1F and Cry 1Ac proteins expressed in DAS 81419 - 2 soy

bean. To address any

uncertainties in the risk assessment focused on taxonomic groups that were most likely to be exposed to Cry1F and Cry 1Ac proteins expressed in PIP event DAS - 81419 - 2 soy

bean in field settings, the potential for adverse effects of DAS - 81419 - 2 on nontarget arthropod populations was assessed in a US field study (MRID No. 48828412). A nontarget arthropod field study with PIP event DAS - 81419 - 2 soy bean, 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 field sites in the U.S. located in Richland, I owa and York, Nebraska. Each trial site included the six soy bean lines arranged in a randomized complete block design with four replicate blocks. Arthropod abundance was sampled during the vegetative and reproductive soy bean stages and monit ored using three sampling methods: pitfall trapping, sticky card trapping, and vertical beat sheet sampling. Arthropods collected during the study included representatives from multiple orders and diverse ecological roles, including phytophagous, predatory , parasitic, and saprophagous modes of feeding. The anal ys es encompassed 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 Hy menoptera (e.g. parasitoid wasps). Consistent across locations and sampling methods, results showed arthropod populations associated with DAS - 81419 - 2 soy

bean were similar to those of non - transgenic so yb ean. Therefore, th e lack of differences in between populations of nontarget arthropod taxa and their abundance from the field monitoring suggests no adverse effects on nontarget arthropod communities from deploy ment of event DAS - 81419 - 2 soy bean cultivation. **F. Soil Fate**  Since proteins are not known to persist in the environment due to the ubiquitous nature of proteases in microbes, the registrant submitted a rationale for demonstrating rapid degradation of Cry 1F and Cry 1Ac proteins in various crop soils. Data from a prev iousl y s ubmitted laboratory study showed that Cry 1F and Cry 1Ac expressed in cotton plants degrades quickly and does not accumulate in soil for WideStrike® PIP cotton (MRID No. 45556801) . The soil half - life of the plant expressing Cry 1F and Cry 1Ac was estim ated as 1.3 day s and the Cry proteins were not detectable after 14 day s. These results verify that the Cry 1F and Cry 1Ac proteins degrade rapidl y in soils sampled from cotton growing regions. The registrant also provided a data waiver rationale to support rapid degradation of Bt Cry 1F and Cry 1Ac proteins in soil based on the history of safe use in sprayable Bt formulations. Cry 1Ac 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: DBT418Cry 1Ac corn) (Mendelsohn et al. 2003; Sanahuja et al. 2011). Cry 1F is also present in WideStrike® cottonBt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

Biopesticides Registration Action Document January 2014

Page 58 of 78 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). Other supporting information included previously conducted field studies showing no accumulation (i.e. persistence) of the proteins as a result of continuous planting of other Bt crops contain ing these proteins (Head et al. 2002; Herman et al. 2002; Shan et al. 2008). As a result, the Agency has determined that no additional long - term field studies are required for PIP event DAS - 81419 - 2 soy beans because currently

registered PIPs have not been s hown to persist in soil. Although the registrant submitted a sufficient rationale of rapid degradation of Cry 1F and Cry 1Ac proteins in other crop soil sy stems, a soil degradation study has not been evaluated in soy bean for this specific PIP event. A previ ous EPA FIFRA SAP noted that in soil degradation and field persistence studies, toxin degradation rates may vary with the crop, the toxin produced by the transformation event, the microbial community in the recipient soil microcosm, the statistical charact erization of toxin persistence, and/or the initial dose of toxin in the experiment (US EPA 2001a). These differences in Cry toxin concentration may

be due to the fact that different genetic constructs were used and/or inserted at different locations in the plant genome (Yu et al . 2012). Plant genoty pe and parental backgrounds have also been recognized as important factors influencing protein expression (Kranthi et al. 2005; Adamczy k and Sumerford 2001; and Torres et al. 2006). Other important factors that a ffect degradation of Cry toxins include environmental conditions such as temperature, light, drought, and soil properties (e.g. pH, texture, clay

content) (Adamczy

k et al. 2004; Chen et al. 2005; Jiang et al. 2006; Rochester 2006; Dong et al. 2008; Hallikeri et al. 2009; Addison and Rogers 2010; Chen et al. 2011; Ranjithkumar et al. 2011). To reduce this uncertainty , a soil fate study to determine the degradation rate (DT 50 ) of Cry 1F and Cry 1Ac proteins sampled from representative soils of soy bean growing regions is to be conducted as confirmatory data. **G. Effects on Soil Microorganisms**  Numerous published studies indicate that exposure to Cry protein produced in Bt PIP crop plants does not adversely

affect soil microorganisms (Sanvido et al . 2007). Although a minimal transient increase and shift in microbial populations may result from the presence of transgenic plant tissue in soil, no adverse effects have been attributed to the Cry protein. In addition, the soil degradation studies discussed in the WideStrike  cotton BRAD show Cry 1F and Cry 1Ac proteins degrade quickl y in soil (US EPA 2005). With regard to the impact of geneticall y engineered crops on soil, EPA has previously noted (US EPA 2010a) that agricultural practices themselves cause large changes in soil and soil microbial composition. Furthermore, factors such as variations in seasons and weather, plant growth stage, and plant varieties, independent of being genetically engineered, are also responsible for significant shifts in soil microb ial communities. Most studies with genetically engineered crops to date have shown minor or no effects on soil microbes beyond the variation caused by the factors listed above. If reports of adverse effects became available, the Agency will take appropriat e action to mitigate potential risks. **H. Horizontal Transfer of Transgenes from**  **Bt Crops to Soil Organisms** Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

Biopesticides Registration Action Document January 2014

Page 59 of 78 The EPA has evaluated the potential for horizontal gene transfer (HGT) from Bt crops to soil organisms and has considered possible risk implications if such a transfer were to occur. Genes that have been engineered into Bt crops are mostly found, or have their origin, in soil - inhabiting bacteria. Soil is also the habitat of other tox in - producing bacteria, and transfer of these genes and/or toxins to other microorganisms or plants has not been detected. There is no known mechanism for, or definitive demonstration of, DNA transfer from plants to microbes (Conner et al. 2003). Furthermore, several published experiments, that were conducted to assess the likelihood of HGT, have been unable to detect gene transfer under typical environmental conditions. Horizontal gene transfer to soil organisms has only been detected with very promiscuous microbes under laboratory conditions designed to favor transfer. As a result of these findings and the fact that the Bt toxins engineered into event DAS - 81419 - 2 are derived from soil inhabiting bacteria, the EPA has concluded that the risk of HGT of transg enes found in event DAS - 81419 - 2 soy

bean expressing Cry 1F and Cry1Ac proteins are low. **I. Gene Flow and Weediness Potential**  The movement of transgenes from the host plant into weeds has been a significant concern for EPA due to the possibility of novel exposures to the pesticidal substance. This concern has been considered for each of the B t plant - incorporated protectants currentl y registered, and was extensively

reexamined in 2010 (see US EPA 2010a), and EPA believes that these concerns have been satisf

actorily addressed. The FIFRA EPA Scientific Advisory Panel meeting held on October 18 - 20, 2000 discussed the matter of gene flow and offered some issues for consideration in this matter. The panel agreed that the potential for gene transfer between Gossy pium hirsutum (cotton) and any receptive plants within the U.S., its possessions and territories, was of limited probability and nearly

risks free (US EPA 2000). Since so ybean is also predominantly a self - pollinating species, the dispersal of pollen is lim ited because the anthers mature in the bud and directly pollinate the stigma of the same flower (OECD 2000). Thus, the risk of potential gene transfer between soy bean and any receptive plants is further negligible. Lastl y, there are no known wild or weedy relatives exist in the U.S. with which Glycine max (L.) Merr. can form viable hy brids in nature, and soy

bean is not weedy in character or invasive (US EPA 2007). Therefore, BPPD determined that there is no significant risk of gene capture and expression of any Bt endotoxin by wild or weedy relatives of soy bean in the U.S., its possessions or territories. **J. Endangered Species Considerations**  Because of the selectivity of Cry 1F and Cry 1Ac insecticidal cry stal proteins for lepidopteran species and lack of evidence of effects on other nontarget species, the Agency has investigated concerns for Federall y listed threatened and endangered insect spe cies in the order Lepidoptera. Because so yb ean pollen is not expected to move beyond the planted soy bean field and its immediate margins, as discussed above, any exposure to lepidopterans would be expected to occur within those areas. Exposure could occur via direct consumption of PIP event DAS - 81419 - 2 soy bean plants or consumption of DAS - 81419 - 2 pollen that falls on non - soy bean plants withinBt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 60 of 78 the soy bean field and its immediate margins. However, oral exposure to significant amounts of Cry 1F and Cry 1Ac via p ollen consumption is not likely . Little pollen is expected to be released from soy bean flowers, since soy

bean plants are predominantly self - pollinated and anthers usually dehisce and release pollen before flowers open. Airborne pollen concentrations have b een measured at very

low levels within soy

bean fields (mean of 0.18 grains/cm2 /day) (Yoshimura et al. 2006). Based on this analy sis, BPPD concludes that exposure resulting from pollen falling on potential non - soy

bean food plants in the field and immediate margins is not sufficient to cause effects in listed lepidopterans. Therefore, any significant exposure would have to occur through consumption of the DAS - 81419 - 2 soy bean plants on the field or via tritrophic effects, in which predatory beneficial arthropo d species consume soybean pest species feeding on PIP event DAS 81419 - 2 soy

bean plant tissues. A search of EPA’s LOCATES database indicates that three species of listed lepidopterans are present in U.S. counties in which soy beans are grown. These are the Karner blue butterfl y ( Lycaeides melissa samuelis ), St. Francis’s Satyr Butterfl y ( Neonympha mitchellii fransisci), and Mitchell’s Saty

r Butterfly

( Neonympha mitchellii mitchellii). The potential effects of Bt PIPs in corn on the Karner blue butterfly was extensively anal yz ed in BPPD’s Bt Crops Reassessment (USEPA 2001b, 2004b and 2010a and b) and in the endangered species assessment section in the BRAD for event MON87701 soy bean expressing Cry 1Ac protein (see US EPA 2010c). As previously

discussed, soybean pollen is not expected to be deposited on plants in or around soy bean fields in amounts sufficient to cause effects in sensitive lepidopterans. Ecology and life history information demonstrated that habitat requirements for larvae and adults of two of th e identified species (Mitchell's Satyr butterfly and Saint Francis' Satyr butterfly) do not overlap with commercial so ybean acreage, and are therefore not expected to be impacted b y P IP event DAS - 81419 - 2 cultivation (Bartel and Sexton 2009; Barton and Bach 2005; US FWS 1997). There is a possible overlap between the geographic range for Karner blue butterfly and soy bean use sites. EPA has previously reviewed information on the proximity of Karner blue habitats, and none are known to exist immediately adjacent to agricultural fields (“adjacent to” was defined as 0 - 3 m for corn fields, which is reasonabl y applied to soybean fields) (US EPA 2001b). Karner blue larvae feed only

on lupines (

Lupinus spp.) and soy

bean is neither identified as a larval food source, n or is soy

bean open - pollinated. Thus, pollen from event DAS - 81419 - 2 soy bean is highly unlikely to be deposited on lupine leaves (NatureServe 2013). Furthermore, soy bean is not identified as a nectaring source for Karner blue adults. Therefore, the risk pose d to the Karner blue butterfl y was considered to be negligible. Based on the above anal ysis, the EP A determines that there will be no direct effect to listed lepidopteran species as a result of the cultivation of DAS - 81419 - 2 soy beans as proposed. Obligat e relationships between insectivorous listed species with lepidopterans that are expected to be found in soy

bean fields, especiall y pest species that feed on DAS - 81419 - 2 plants, are not currentl y known. Since the Cry1F and Cry1Ac in DAS - 81419 - 2 soy beans targets onl y lepidopteran insects, loss of the pest insects as a result of DAS - 81419 - 2 are expected to be offset by the presence of other insects that could act as food sources for listed species, including beneficial insects that are known not to be affecte d by Cry 1F and Cry 1Ac. Effects on species other than insects have also been determined to be very unlikely because of the specificity of Cry 1F and Cry 1Ac.Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 61 of 78 Lastl y, since the Agency

previously

determined Cry 1F and Cry 1Ac proteins are not expected to have adverse effects on mammals, birds, plants, freshwater and estuarine/marine fish and invertebrates, nontarget insects and other invertebrate species at the EEC, a “No Effect” determination is made for direct and indirect ef fects to Federally

listed threatened and endangered species and their designated Critical Habitats . In addition, EPA does not expect that any threatened or endangered plant species will be affected by outcrossing to wild relatives or by competition with su ch entities. Hy brid soy bean does not exist in the wild in the United States, nor do any wild plants that can interbreed with soybean in the United States. **K. Data Needed to Confirm**  **DAS -**  **81419 -**  **2 Soybean Environmental Fate Assessment**  Although the registrant submitted a sufficient rationale of rapid degradation of Cry 1F and Cry 1Ac proteins in other crop soil sy stems, the degradation rate (DT 50 ) of Cry 1F and Cry 1Ac proteins sampled from representative soils of so yb ean growing regions has not been evaluated and must be conducted as confirmatory data. The soil degradation study is to be submitted to the EPA within 18 months of the issuance date of the DAS - 81419 - 2 Soy bean Notice of Pesticide Registration. **II .**  **Conclusion** The biological equivalency of Cry 1F and Cry 1Ac proteins expressed in event DAS - 81419 - 2 soy bean plant was confirmed with the previously characterized microbial - derived Cry 1F and Cry 1Ac protein test substances. No significant increase in exposure to NTOs was demonstrated based on calculated m argins of exposure for protein concentrations in event DAS - 81419 - 2 plant tissue matrices relative to the dose concentrations used in the NTO laboratory toxicity tests conducted on Cry 1Ac and Cry 1F . Lastl y, a field monitoring study showing no differences on nontarget arthropods on a population and community level and other supporting supplemental information (e.g. meta - analy sis, target feeding studies, and biological specificity to the target pest) provided additional certainty to support the “weight - of - evid ence” that there is negligible risk to NTOs representing diverse taxonomic groups and exposure scenarios from the cultivation of DAS - 81419 - 2 soy

bean. Therefore, the submitted data supports the applicant’s request and the findings from the environmental risk assessment conducted for WideStrike® cotton can be bridged to support a breeding/seed increase r egistration for PIP event DAS - 81419 - 2 (US EPA 2005). Based on e xtrapolating from previous studies on Bt proteins, which tend to be very species specific in their activity , the Agency

concludes that the cultivation of event DAS - 81419 - 2 soy bean will not result in adverse effects to nontarget organisms, are no t likely to persist in the soil, and pose no risk of gene flow and development of weediness in wild relatives, and additionally will have no effect, direct or indirect, on Federall y listed threatened and endangered species and their designated Critical Ha

bitats.Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 62 of 78 

### APPENDIX **C :**  **Insect Resistance Management Cry1A**  **c and**  **Cry 1F**  **proteins a**  **s DAS**  **- 81419**  **- 2 Soybean**  Discussions of EPA’s risk assessments for Insect Resistance Management (IRM) are presented in this Appendix . Immediately below is the risk assessment completed by EPA’s A . Rey

nolds on November 7 , 201 3 . **I. Insect Resistance Management**  **Review** Below is the BPPD review of Dow ’ s Insect Resistance Management (IRM) plan and supporting data for the application to register DAS - 81419 - 2 Soy bean PIP for seed production and research purposes, as well as supporting documentation concerning n atural refuge for Bt cotton (U.S. EPA memorandum 11/7/ 1 3 ; EPA File Sy mbol 6 8467 - EN, MRID 4 9 0 2 4 110 replaced MRID 48 828415 ) . **CONCLUSION AND R** **EQUIREMENTS** 1) Because DAS - 81419 - 2 soy bean is intended for breeding/seed increase, Dow did not propose a species - specific IRM plan for the product. Rather, the company proposed to mitigate resistance risk by acreage limitations on a county (1,000, 10,000, or 25,000 acres) and national (250,000 acres) scale. BPPD agrees that given the low acreage projected b y Dow, it is unlikely that there will be a significant risk of resistance to the main soy bean lepidopteran pests in the United States. These insects include soy bean looper, velvetbean caterpillar, beet army worm, cotton bollworm, green cloverworm, tobacco budworm, and cutworms ( Agrotis spp.). This conclusion is further supported b y the biology of the target insects, which are highly poly phagous (feeding on a number of wild hosts and cultivated crops) and (for some species) have limited overwintering in non - tropical areas. 2) Should a f

ull commercial registration or expanded acreage be sought in the future, a complete, species - specific IRM assessment for DAS - 81419 - 2 will be required . Data needed to support such a review should include additional information on pest biology , dose, simulation modeling, cross resistance, resistance monitoring, and potential impacts on the natural refuge strategy for Bt cotton. 3) The use of DAS - 81419 - 2 on a limited basis for seed increase purposes should not significantl y impact the natural refuge strategy in place for Bt cotton PIPs. Soy bean is one of the non - cotton crops that have been considered as part of natural refuge for tobacco budworm and cotton bollworm. While it can be expected that plantings of DAS - 81419 - 2 soy bean will remove some of the currentl y- available natural refuge for Bt cotton, this potential reduction will likely be small relative to the total amount of natural refuge present in southern cotton regions. Dow has proposed to further mitigate the impact on natural refuge b y limiting DAS - 814 19 - 2 to 10,000 acres in counties with > 25,000 total acres of so ybean and 1,000 acres in counties with < 25,000 total acres of so yb ean.Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 63 of 78 4) Dow must institute a stewardship program for DAS - 81419 - 2 including resistance monitoring and remedial action (in the event of resistance). A resistance monitoring plan limited to investigations of unexpected pest damage is appropriate for a capped acreage registration. BPPD notes that monitoring of two potential target insects, tobacco budworm and cotton bollworm, is already being conducted as part of the WideStrike Bt cotton monitoring program. Dow must also compile annual sales and acreage data for each state and provide a report to EPA if requested. Since DAS - 81419 - 2 will be deplo yed without a structured refuge requirement, a compliance assurance plan and grower education program should not be necessary . **IRM Rationale and Proposed Plan for DAS -**  **81419 -**  **2 Bt**  **Soybean (MRID No.**  **49024110)** Dow’s proposed DAS - 81419 - 2 soy bean is intended for breeding, seed production, and research in the United States. Therefore, acreage of the product grown in the United States will be limited to a total of 250,000 per year. Individual counties would also be annuall y limited to either 20,000 (in non - cotton growing regions), 10,000 (in cotton growing counties with at least 25,000 acres of soy bean), or 1,000 acres (in cotton - growing counties with < 25,000 soy bean acres). Dow’s IRM rationale for DAS - 81419 - 2 is based on the limited commercialization of the product in the United States. The co mpany reasons that selection pressure should be limited by the acreage caps and notes that EPA has not mandated refuges for similarly- capped Bt corn breeding registrations. Further, the product provides two modes of action (Cry 1F and Cry 1Ac) for control of lepidopteran pests. Soy bean is one of the lepidopteran crop hosts that have been considered as natural refuge for Bt cotton. Dow’s acreage proposal for DAS - 81419 - 2 could therefore reduce the amount of soy bean available for cotton refuge in areas where bo th crops are planted. To estimate the potential effects of DAS - 81419 - 2 on natural refuge, Dow calculated the refuge contribution of soy bean relative to the amount of cotton as such: [(acres of soy bean

– proposed DAS - 81419 - 2 acres)/(acres of so yb ean + cott on) * 100]. DAS acreage was set at either 10,000 or 1,000 per county as described above. Acreage data for county cotton and soy

bean were obtained from USDA -NASS (http://quickstats.nass.usda.gov/) for the 2010 season. Calculations were conducted for all counties with plantings of both soy bean and cotton in the states of Alabama, Arkansas, Georgia, Kansas, L ouisiana, Mississippi, Missouri, North Carolina, Oklahoma, South Carolina, Tennessee, and Georgia. A total of 183 counties were surveyed b y Dow (data contained in Tables 1 - 7 in MRID No. 49024110). Of these, 84 had at least 25,000 acres of soy bean planted during the 2010 season. In these counties up to 10,000 acres of DAS - 81419 - 2 has been proposed. Even with this potential reduction in refuge, the amount of available so ybean refuge averaged 62% among the 84 counties. Dow noted that this level of refuge is well above the 20% structured refuge that was required for Bt cotton prior to approval of natural refuge. In addition, commercial so ybean fields are rarel y treated for cotton bollworm which should further enhance production of susceptible insects.Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 64 of 78 The remaining 99 counties anal yz ed b y Dow, with less than 25,000 total soybean acres, would have a limit of 1,000 DAS - 81419 - 2 acres. Some of these counties grow l ittle soy bean (< 2000 acres) such that the relative contribution to natural refuge from the crop is likely low, regardless of the adoption of DAS - 81419 - 2. Other counties grow larger amounts of soy bean; however, in these counties full adoption of DAS - 81419 - 2 (1,000 acres) would still have a small impact on natural refuge. Dow indicated a number of other factors add to the conservatism of the IRM rationale:  Seed production for so yb ean (the primary intended purpose for DAS - 81419 - 2 in the U.S.) occurs in areas where the crop is favored

– i.e., in counties with large amounts of soy bean (>25,000 acres).  Counties with not as much soy bean (< 25,000 acres) are less likely to be used for seed production. Nonetheless, a “regional” natural refuge may provide susceptible insects from other counties since H. zea is known to be highl y mobile (Jackson et al. 2008).  Soy bean for seed production (non - co mmercial production) is frequentl y treated with insecticides for pod - feeding insects. Therefore, seed production soy bean is likely

to be limited in value as natural refuge regardless of the adoption of DAS - 81419 - 2.  In addition to soy

bean, H. zea is known t o utilize numerous other crop and plant hosts including corn, peanuts, sorghum, and weed species. Accordingl y, natural refuge for the insect is not limited solely to so ybean acres and the county refuge estimates provided b y Dow will be underestimates of th e actual amounts. **BPPD Review** Dow’s proposal to limit the overall acreage and distribution of DAS - 81419 - 2 in the U.S. should be sufficient to mitigate potential resistance development for a seed increase registration. It should be noted, however, that BPPD does not have sufficient information in the submitted materials to completel y assess the risk of resistance for a full commercial registration of DAS 81419 - 2. Target Pests Dow’s submission did not indicate the primary insect pests targeted by

DAS - 8 1419 - 2 soy

bean. BPPD presumes this is because the product is intended for commercialization in South America; U.S. plantings will be solely for seed production. Nonetheless, DAS - 81419 - 2 can be expected to confer protection against susceptible soy bean - feed ing lepidoptera while grown for seed production in the U.S. Soy beans can be affected by

a number of lepidopteran insects, including (among others) soy bean looper ( Pseudoplusia includens ), velvetbean caterpillar ( Anticarsia gemmatalis ), beet arm yworm (Spod optera exigua ), cotton bollworm/corn earworm ( Helicoverpa zea ), green cloverworm ( Plathypena scabra ), and cutworms ( Agrotis spp.). Though not common on soy bean, tobacco budworm (

Heliothis virescens ) has also been known to infest the crop (Sheck and Gould 1 993). Other occasional lepidopteran pests include fall army worm (Spodoptera frugiperda ) and saltmarsh caterpillar (

Estigmene acrea ).Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 65 of 78 Soy bean looper occurs throughout the U.S. where soy beans are grown, but overwinters onl y in tropical regions migrating annually from Mexico, Central America, and the Caribbean (Pedigo 1999). The insect is multivoltine with two (northern regions) to seven (southern states) generations per season. Soy bean looper is known to feed on a wide range of vegetables, field crops (including corn and cotton), and weeds in addition to soy bean (Capinera 2005). Velvetbean caterpillar, similar to soy bean looper, is a tropical species that migrates annuall y northward into the U.S. It can range as far north as New England, but overwinters only in the southern end of the Florida peninsula (Pedigo 1999; Barbara 2008). Velvetbean caterpillar is poly phagous and feeds on a number of field crops (soy bean, alfalfa, cotton, peanut, other legumes) and weeds (Barbara 2008). Green cloverworm also follows a southern overwintering/migration pattern from Gulf Coast areas. The insect feeds on a variety of legumes, including so ybean, and has up to four generations per season (http://ipm.ncsu.edu/AG271/soy beans/green_cloverworm.html ). Cotton bollworm and tobacco budworm are poly phagous Noctuid species that are prim ary target pests of Bt cotton registrations (see descriptions in BPPD 2001 and 2006). Fall army worm and cutworms are secondary

pests targeted by

some Bt corn registrations (see BPPD 2010). IRM Risk Considerations No data were provided in the submission t o evaluate the efficacy of DAS - 81419 - 2 against any of the potential U.S. target pests. Published data for another Bt soy bean cultivar (TIC107 with a cry1A gene) showed high activity against velvetbean caterpillar and soy bean looper (McRae et al., 2005). Cry1Ac and Cry1F are also known to have toxicity to cotton bollworm and tobacco budworm (BPPD 2005), while fall arm yworm is affected b y Cry1F (BPPD 2010). However, without specific data for DAS - 81419 - 2, it is not possible to evaluate dose considerations for the product or determine the resistance risk for targeted insect pests. Rather than formulate an insect - specific IRM approach for DAS - 81419 - 2, Dow has proposed acreage limitations to lower selection pressure for resistance development. A number of other f actors should further reduce the risk of resistance such that a formalized refuge strategy is unnecessary . These considerations include biological aspects of soy bean lepidopteran pests, the abundance of alternative hosts (natural refuge) for these insects, the p yr amiding of two toxins in DAS - 81419 - 2, and anticipated limited plantings of the product for seed increase purposes. Most of the lepidopteran insects that infest soy bean are pol yphagous and feed on numerous other wild and cultivated hosts which should provide a source of natural refuge to reduce selection pressure for Cry 1Ac resistance. Further, so yb ean looper, velvetbean caterpillar, and fall army worm overwinter only

in tropical areas (e.g., Florida in the U. S.); population migrate annually from the Caribbean, Mexico and Central America northward through the U.S. Other key pests, cotton bollworm and green cloverworm, ov erwinter in southern Gulf Coast states. Because of this, it is less likely that any resistant individuals that evolve during the growing season would be able to successfull y overwinter and propagate the resistant trait to future generations.Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 66 of 78 As a p yramid , DAS - 81419 - 2 expresses two toxins (Cry 1Ac and Cry 1F) for lepidopteran control. Py ramided PIPs are thought to increase the respective durability of each toxin relative to mosaics of single toxin PIPs (Roush 1998, Caprio 1998, Zhao et al. 2003, others). BP PD notes that the potential for some cross resistance exists between Cry 1Ac and Cry 1F in cotton bollworm and tobacco budworm (Gould et al. 1995; BPPD 2005, 2010), which could limit the effectiveness of the py ramid for those insects. Nonetheless, py ramiding two toxins in DAS - 81419 - 2 should further decrease the likelihood of resistance development. Acreage limitations have been emplo yed for seed/breeding registrations issued for Bt corn PIPs as a means to lower overall selection pressure for resistance. Thes e restrictions have been implemented on county

(up to 20,000 acres) and national level (250,000 acres total). Dow’s proposed acreage for DAS - 81419 - 2 generally follows this standard, with up to 25,000 acres per county (250,000 nationally) except in counties where so ybean is currently planted (see discussion below). Based on the proposed acreage - limited (breeding) registration and in consideration of the biological aspects of the soy bean pests discussed above, BPPD concludes the risk of resistance to Cry 1Ac and Cry 1F in DAS - 81419 - 2 should be low. Therefore, a refuge strategy is not recommended at this time for DAS - 81419 - 2. However, should any request be made to expand the scope of DAS - 81419 - 2 to a commercial registration (with large or unlimited acreage), BPP D recommends a formal IRM assessment to better evaluate the resistance risk and develop appropriate mitigation measures. Such a review should include appropriate data for dose expression, simulation modeling, and cross resistance. Considerations for Bt Co tton Natural Refuge In addition to the primary target pests of soy

bean, BPPD is also concerned about other insect pests that are targeted b y Bt cotton but may also be exposed to Bt soy bean. These insects, tobacco budworm (TBW, Heliothis virescens ) and co tton bollworm (CBW, Helicoverpa zea ), are highl y polyphagous and are known to exploit numerous crop and wild plant hosts. Cry 1Ac and Cry 1F are expressed in both DAS - 81419 - 2 and WideStrike Bt cotton (Bollgard II also expresses Cry 1Ac). The established resi stance management plan for Bt cotton involves the use of “natural refuge” in which non - PIP hosts (such as weeds and other non - cotton cultivated crops) provide sources of susceptible insects to dilute any potential resistance genes arising from transgenic c otton. One of the non - cotton crops included in calculations of natural refuge is so ybean. Should a significant portion of the soy

bean crop be devoted to a Bt variety , the potential amount of natural refuge available to TBW and CBW could be reduced. To evaluate potential impacts to cotton natural refuge, Dow conducted an analy sis of counties in southern states growing soy bean. The company

tabulated soy

bean acreage in 2010 (USDA NASS data) and projected the impact DAS - 81419 - 2 would have on the natural refuge contribution of soy

bean. In the surveyed counties with large soy bean acreage (>25,000), full adoption of DAS - 81419 - 2 would still leave a sizable (average 62%) contribution to naturalBt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 67 of 78 refuge from non - Bt so yb ean. As could be expected, the potential reductio n in natural refuge was greater in counties with less than 25,000, particularly those with less than 5,000 acres. Soy bean contribution in 19 counties (of 99 total) would be reduced to less than 10% with the full addition of DAS - 81419 - 2. In counties with le ss than 1,000 soy bean acres (3 total), refuge contribution from soy bean could potentially

be eliminated. Generall y, so ybean refuge was lower in Georgia and Texas than the other surveyed states. This was largel y because these states each have a number of co unties with low overall soy bean acreage but relativel y high (> 20,000) cotton planting. Dow’s anal ys is was limited by considering onl y s oy

bean as a contributor to cotton natural refuge. BPPD agrees with the company that numerous other crops (e.g., tobacco , peanut, corn, sorghum, alfalfa) and wild or weedy hosts that support TBW and CBW are present in cotton growing regions. As such, Dow’s assessment of the impact of DAS - 81419 - 2 on natural refuge was likely

highl y conservative and underestimated the amount of refuge relative to cotton. Nonetheless, it can be expected that plantings of DAS - 81419 - 2 soy bean will remove some of the currentl y- available natural refuge for Bt cotton. But this potential reduction will likely be small relative to the total amount of natural refuge available in southern cotton regions. As part of the natural refuge assessment in 2006 - 2007, BPPD reviewed extensive host utilization data developed b y Monsanto (see BPPD 2006, 2007; Jackson et al. 2008). TBW and CBW were sampled for host p reference by testing insect bodies for the presence of bound goss ypol (TBW) or insect wings for the ratio of C3 to C4 plant isotopes (CBW). These data showed that TBW and CBW in cotton - growing areas fed much more frequently on non - cotton hosts than on cott on. In many of the sampled states, 50% or more of sampled TBW developed on crops other than cotton. While soy

bean is a component of these non - cotton hosts, it is unlikely that it is the sole (or even a majority) source of natural refuge, given the relativ ely low county

acreages (< 25,000 soy

bean acres in most counties). This is the case in Georgia, where low so ybean acreage (relative to cotton) in Dow’s analy sis appeared to indicate a small natural refuge contribution from soy bean (< 10% in many counties). However, Monsanto’s host utilization data clearly showed a high proportion of natural refuge in the state, as very few sampled TBW (0 to 10%) developed on cotton (MRID No. 46717201, reviewed in BPPD 2006, 2007). BPPD’s assessment of the impact of DAS - 814 19 - 2 on Bt cotton natural refuge is contingent upon the acreage projections provided by Dow (as discussed in the previous section). A full commercial registration of DAS - 81419 - 2 without acreage limitations would require a more detailed assessment potential ly including revised natural refuge calculations and simulation modeling. Stewardship Activities BPPD ’s review indicated that other aspects of IRM including resistance monitoring and remedial action plan are needed for DAS - 81419 - 2. A full resistance monitoring plan with pest sampling and detection bioassay s should not be warranted for a seed increase registration; rather an approach based on investigations of unexpected pest damage reports should be appropriate. In addition, both TBW and CBW are monitored for Cry 1Ac and Cry 1F susceptibility as part of the WideStrike cotton monitoring program (see BPPD 2005). A remedial action plan, in the event ofBt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 68 of 78 documented resistance, could mimic those in place for Bt corn or cotto n or could rel y on a “stop sale” approach if resistance develops. It wa s also not ed that Dow will need to compile DAS 81419 - 2

– specific annual sales and total acreage data for each state and provide a report to EPA if requested. Since DAS - 81419 - 2 will be d eployed without a structured refuge requirement, a compliance assurance plan and grower education program should not be necessary .Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 69 of 78 

### APPENDIX **D :**  **R eferences**  **Product Characterization and Human Health**  Lerouge, P. Cabanes - Macheteau, M., Rayon, C., Fichette - Lainé, A - C., Gomord, V., and Faye, L., (1998) N - Gl yc oprotein biosy nthesis in plants: recent developments and future trends. Plant Molecular Biology 38: 31 - 48. Sjoblad, Roy

D., et al., (1992) Toxicological Considerations for Protein Components of B iological Pesticide Products. Regulatory Toxicology and Pharmacology 15:3 - 9. U.S. Environmental Protection Agency (2005) BIOPESTI CIDES REGISTRATION ACTION DOCUMENT (Bacillus thuringiensis Cry 1F (s ynpro and Cry 1Ac (s ynpro) Construct 281/3006 Insecticidal Crys tal Proteins as expressed in cotton) (Chemical PC Codes 006512 and 006513, respectivel y) May 2005 Office of Pesticide Programs http://www.epa.gov/oppbppd 1/biopesticides/ingredients_keep/tech_docs/brad_006512.pdf US Environmental Protection Agency (2010a) BIOPESTI CIDE REGI STRATION ACTION DOCUMENT, Bacillus thuringiensis Cry 1Ac Protein and the Genetic Material (Vector PV GMIR9) Necessary for Its Production in MON 87701 (OECD Unique Identifier: MON 877Ø1 - 2) Soy

bean [PC Code 006532] http://www.epa.gov/pesticides/biopesticides/pips/bt cry1ac - protien.pdf US Environmental Protection Agency (2010b) BIOPESTI CIDES REGISTRATION ACTION DOCUMENT Cry 1Ab and Cry 1F Bacillus thuringiensis ( Bt) Corn Plant - Incorporated Protectants http://www.epa.gov/pesticides/biopesticides/pips/cry1f - cry 1ab - brad.pdf **Ecological and Environmental Effects** Abud, S., de Souza, P.I .M., Vianna, G.R., Leonardecz, E., Moreira, C.T., Faleiro, F.G., Junior, J.N., Monteiro, P.M.F.O., Rech, E.L. and F.J.L . Aragao. (2007) Gene flow from transgenic to nontransgenic so ybean plants in the Cerrado region of Brazil. Genetics and Molecular Research 6: 445 - 452. Adamczy k, J.J. Jr, and D.V. Sumerford. (2001) Potential factors impacting season long expression of Cry 1Ac in 13 commercial varieties of Bollgard cotton . J Insect Sci 1: 1

– 6. Adamczy k, J.J., Jr, William, R. and J. Meredith. (2004) Genetic bases for viability of Cry 1Ac expression among commercial transgenic Bacillus thuringensis ( Bt) cotton cultivars in the United States. J Insect Sci 8: 17

–23. Addison, S.J. and D.J. Rogers. (2010) Potential impact of differential production of the Cry2Ab and Cry 1Ac proteins in transgenic cotton in response to cold stress. J Econ Entomol 103: 1206

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Page 70 of 78 Carstens, K.L., Anderson, J.A ., Bachman, P., DeShrijver, A., Divel y, G. Federici, B. Hamer, M., Gielkens, M., Jensen, P., Lamp, W., Raushen, S., Ridley , G., Romeis, J., and A. Waggoner. (2011) Geneticall y modified crops and aquatic ecosystems: considerations for environmental risk ass essment and non - target organism testing. Transgenic Res. 21(4): 813 - 42. Caviness, C.E. (1966) Estimates of natural cross - pollination in Jackson soybeans in Arkansas. Crop Sci . 6: 211. Chen, D.H., Ye, G.Y., Yang, C.Q., Chen, Y. and Y.K. Wu. (2005) The effec t of high temperature on the insecticidal properties of Bt cotton. Environ Exp Bot 53: 333

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–702. Duan, J.J., Marvier, M., Huesing, J., Dively, G., and Z.Y. Huang. (2008) A Meta - Anal ys is of Effects of Bt Crops on Honey Bees (Hy

menoptera: Apidae). PLoS ONE 3(1): e1415. Friedman, M., Brandon, D.L ., Bates, A.H., and T. Hy mowitz. (1991) Comparison of a commercial so ybean cultivar and an isoline lacking the Kunitz trypsin inhibitor: Composition, nutritional value and effects of heating. Journal of Agricultural and Food Chemistry 39: 327 - 335. Green, C.J. and A.M. Blackmer. (1995) Residue decomposition effects on nitrogen availability to corn following corn or soy bean. Soil Science Society of America Journal 59: 1065 - 1070. Hallikeri, S.S., Halemani, H.L ., Katageri, I.S., Patil, B.C., Patil, V.C. and Y.B. Palled. (2009) In fluence of sowing time and moisture regimes on Cry protein concentration and related parameters of Bt- cotton. Karnataka J Agric Sci 22: 995

– 1000. Head, G., Surber, J.B., Watson, J.A., Martin, J.W. , and J.J. Duan. (2002). No detection of Cry 1Ac protein in soil after multiple years of transgenic Bt cotton (Bollgard) use. Environmental Entomology 31: 30 - 36. Richard L. Hellmich, R.L., Siegfried, B.D., Sears, M.K., Stanley- Horn, D.E., Daniels, M.J., M attila, H.R., Spencer, T., Bidne, K.G. and L.C. Lewis (2001) Monarch larvae sensitivity to Bacillus thuringiensis - purified proteins and pollen. Proc Natl Acad Sc 98(21) 11925 - 11930 Herman, R.A., Scherer, P.N., Young, D.L., Mihaliak, C.A., Meade, T., Woods worth, A.T., Stockhoff, B.A., and K.E. Narva. (2002) Binary insecticidal cry stal protein from Bacillus thuringiensis , strain PS149B1: Effects of individual protein components and mixtures in laboratory

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– 320. Kranthi, K.R., Naidu, S., Dhawad, C.S., Tatwawadi, A., Mate, K. Patil E, et al . (2005) Temporal and intra - plant variability of Cry 1Ac expression in Bt cotton and its influence on the survival of the cotton bollworm, Helicoverpa armigera (Hübner) (Noctuidae: Lepidoptera ). Curr Sci 89: 291

–298. Marvier, M., McCreedy, C., Regetz, J. an d P. Kareiva. (2007) A meta - analy sis of effects of Bt corn and cotton on nontarget invertebrates. Science 316: 1475

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of Bacillus thuringiensis - based pesticides. Pesticide Science 45: 95 - 105. Mendelsohn, M., Kough, J., Vaituzis, Z., and K. Matthews. (2003) Are Bt crops safe? Nature Biotechnology 21: 1003 - 1009. National Academ y of Science (2000) Environmental Effects of Transgenic Plants: The Scop e and Adequacy of Regulation is available from the National Academy Press, 2101 Constitution Avenue, N.W., L ockbox 285, Washington, DC 20055; (800) 624 - 6242 or (202) 334 - 3313 (in the Washington metropolitan area); http:// www.nap.edu . NatureServe ( 2013) NatureServe Explorer: An online encyclopedia of life [web application]. Version 7.1. NatureServe, Arlington, Virginia. Available http://www.natureserve.org/explorer [Accessed: November 6, 2013]. OECD (2000). Consensus document on the biology of Glycine max (L.) Merr. (Soy bean). Organisation for Economic Co - operation and Development. Series on Harmonization of Regulatory

Oversight in Biotechnology No. 15. ENV/JM/MONO (2000)9 http://www.oecd.org/dataoecd/16/56/46815668.pdf. Ranjithkumar, L ., Patil, B.V. and N.G. Vijay kumar (2011) Impact of irrigation and fertilizer levels on Cry 1Ac protein content in Bt cotton. Res J Agric Sci 2: 33

– 35. Ray bould, A. (2006) Problem formu lation and hypothesis testing for environmental risk assessments of genetically modified crops. Environmental Biosafety Research 5: 119 - 125. Rochester, I.J. (2006) Effect of genotype, edaphic, environmental conditions and agronomic practices on Cry 1A prote in expression in transgenic cotton. J Cotton Sci 10: 252

–262. Rodrigo - Simón, A., de Maagd, R.A., Avilla, C., Bakker, P.L., Molthoff, J., González - Zamora, J.E., and J. Ferré, (2006) Lack of Detrimental Effects of Bacillus thuringiensis Cry Toxins on the In sect Predator Chrysoperla carnea: a Toxicological, Histopathological, and Biochemical Anal ys is. Environ Microbiol 72: 1595 - 1603.Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 72 of 78 Rosi - Marshall E. J., Tank, J. L ., Royer, T. V., Whiles, M. R., Evans - White, M., Chambers, C., Griffiths, N. A., Pokelsek, J. and M.L . Stephen. (2007) Toxins in transgenic crop by products in Bacillus thuringiensis Cry 1Ac may affect headwater stream ecos ys tems. Proc Natl Acad Sc 104: 16204

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–278. Shan, G., Embrey , S.K., Herman, R.A., and R. McCormick. (2008) Cry 1F protein not detected in soil after three years of transgenic Bt corn (1507 corn) use. Environmental Entomology 37: 255 - 262. Sims, S.R. and J.W. Martin. (1997) E ffect of the Bacillus thuringiensis insecticidal proteins Cry 1A(b), Cry 1A(c), cryiia, and cryiiia on folsomia candida and xenylla grisea (insecta: Collembola). Pedobiologia 41: 412 - 416. Swan, C.M., Jensen, P.D., Dively , G.P., and W.O. Lamp, W.O. (2009) Processing of transgenic crop residues in stream ecosy stems . J. App. Ecol. 46: 1304 - 1313. Tian, J.C., Wang, X.P., Long, L.P., Romeis, J., Naranjo, S.E. et al. (2013) Bt Crops Producing Cry 1Ac, Cry2Ab and Cry 1F Do Not Harm the Green Lacewing, Chrysoperla ru filabris. PLoS ONE 8(3): e60125. Torres, J.B., Ruberson, J.R. and M.J. Adang. (2006) Expression of Bacillus thuringiensis Cry 1Ac protein in cotton plants, acquisition by pests and predators: a tritrophic anal ys is. Agric For Entomol 8: 191

–202. United States Environmental Protection Agency (US EPA) (1998). “Guidelines for Ecological Risk Assessment.” EPA 630/R - 95 - 002F. Washington, DC, USA. [Federal Register, May 14, 1998. 63(93): 26846 - 26924.] US EPA (2000) Scientific Advisory Panel (SAP) Report No. 99 - 06. Sets of scientific issues being considered b y the Environmental Protection Agency regarding: Section I - Characterization and Nontarget Organism Data Requirements for Protein Plant Pesticides. Dated February

4, 2000. Available at the EPA websit e: http://www.epa.gov/scipoly /sap/1999/index.htm#december US EPA (2001a) SAP Report No. 2000 - 07. Sets of scientific issues being considered by the Environmental Protection Agency regarding: Bt plant - pesticides risk and benefit assessments. Dated March 12, 2001. US EPA (2001b) Biopesticides Registration Action Document for Bacillus thuringiensis ( Bt) Plant - In corporated Protectants, dated October 21, 2001. US Environmental Protectio n Agency , Washington, D.C. US EPA (2002) SAP Report No. 2002 - 05. A set of scientific issues being considered b y the Environmental Protection Agency regarding: Corn rootworm plant - incorporated protectant nontarget insect and insect resistance management iss ues. Dated November 6, 2002.Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 73 of 78 US EPA (2004a) SAP Report No.2004 - 05. Product characterization, human health risk, ecological risk, and insect resistance management for Bt cotton products. Dated August 19, 2004. US EPA (2004b) Biopesticides Registration Action Document for e vent TC1507 (Trade name: Herculex TM I) and DAS - 06275 - 8 Maize expressing Bacillus thuringiensis Cry 1F . (Chemical PC Codes 006481 and 006491, respectively); US Environmental Protection Agency , Washington, D.C. US EPA (2005) Biopesticid es Registration Action Document for WideStrike  cotton expressing Bacillus thuringiensis Cry1F (s ynpro) and Cry1Ac (s ynpro) Construct 281/3006 Insecticidal Cry stal Proteins as expressed in cotton (Trade name: WideStrike  cotton; OECD Unique ID No: DAS - 21Ø2 3 - 5 x DAS - 24236 - 5 (Chemical PC Codes 006512 and 006513, respectivel y); US Environmental Protection Agency, Washington, D.C. US EPA (2007) White Paper on Tier- Based Testing for the Effects of Proteinaceous Insecticidal Plant - In corporated Protectants on Non - Target Arthropods for Regulatory Risk Assessments. April 6, 2007. US Environmental Protection Agency , Washington, D.C. US EPA (2010a) Biopesticides Registration Action Document for Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 Proteins and the Genetic Material Necessary for Their Production (PHP17662 T - DNA) in Event DAS - 59122 - 7 Maize (OECD Unique Identifier: DAS - 59122 - 7). US Environmental Protection Agency , Washington, D.C. US EPA (2010b) Biopesticides Registration Action Document

– Modified Cry3A Protein and the Genetic Material Necessary for its Production (Via Elements of PZM26) in Event MIR604 Maize SYN - IR 604 - 8. US Environmental Protection Agency , Washington, D.C. US EPA (2010c) Biopesticides Registration Action Document

– Bacillus thuringiensis Cry 1Ac P rotein and the Genetic Material Necessary for its Production in Event MON8771 (OECD Unique Identifier: MON 877Ø1 - 2) Soy bean. US Environmental Protection Agency , Washington, D.C. US EPA (2013) Review of Product Characterization and Human Health data in sup port for Sec. 3 Registration of PIP event DAS - 81419 - 2 soy bean expressing Cry 1F and Cry 1Ac insecticidal proteins; U.S. EPA Memorandum from C. Wozniak, through J. Kough, to D. Greenway . US Environmental Protection Agency , Washington, D.C. US FWS (1997) Recovery Plan for Mitchell’s Satyr Butterfly ( Neonympha mitchellii mitchellii French), Ft. Snelling, MN. United States Fish and Wildlife Service. US Fish and Wildlife Services (2009) Mitchell’s Saty r Butterfly

Spotlight Species Action Plan. Ft. Snelling, MN. http://ecos.fws.gov/docs/action_plans/doc3038.pdf Wolt, J.D. and R.K.D. Peterson (2010) Prospective formulation of environmental risk assessments: Probabilistic screening for Cry 1A(b) maize risk to aquatic insects. Ecotoxicol. Environ. Safety 73: 1182 - 1188. Yoshimura, Y., K. Matsuo, and K. Yasuda. (2006) Gene flow from GM gl yphosate - tolerant to conventional soy

beans under field conditions in Japan. Environmental Biosafety Research 5: 169 - 173.Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 74 of 78 Yu, H., Li, Y., Xiangju, L., Romeis, J. and K. Wu. (2013) Expression of Cry 1Ac in transgenic Bt soy bean lines and their efficiency in controlling lepidopteran pests. Pest Manag Sci doi: 10.1002/ps.3508. **Insect R esistance Manageme**  **n t**  Barbara, K.A. , 2008. University of Florida, Department of Entomology and Nematology informational web page for velvetbean caterpillar ( Anticarsia gemmatalis ): http://entnemdept.ufl.edu/creatures /field/velvetbean.htm . Publication Number: EENY - 151. BPPD, 2001. Bt plant - incorporated protectants October 15, 2001 Biopesticides Registration Action Document. Available at http://www.epa.gov/pesticides/biopesticides. BPPD, 2005. Biopesticides Registration Action Document: Bacillus thuringiensis var. aizawai Cry 1F and the genetic material (from the insert of plasmid pgma281) necessary for its production in cotton and Bacillus thuringiensis var. kurstaki Cry 1Ac and the genetic material (from the insert of plasmid pmyc3006) necessary for its production in cotton) (Chemical PC Codes 006512 and 006513, respectively). September, 2005. Available at http://www.epa.gov/opp00001/chem_search/reg_actions/registration/decision_PC 006512_1 - Sep - 05.pdf

. BPPD, 2006. Review of data and support materials submitted by Monsanto to amend the insect resistance management requirements for Bollgard II Bt cotton. A. Rey nolds and S. Matten memorandum to L . Cole, May 16, 2006. BPPD, 2007. Scientific review of a proposal b y Monsanto to replace structured refuge with natural refuge for Bo llgard II Bt cotton (resubmitted amendment request to address the concerns of the FIFRA SAP). A. Rey nolds and S. Matten memorandum to M. Mendelsohn, May 3, 2007. BPPD, 2010. Biopesticide Registration Action Document: Cry 1Ab and Cry 1F Bacillus thuringiens is (Bt) Corn Plant - Incorporated Protectants. Updated September, 2010. Available at http://www.epa.gov/pesticides/biopesticides/pips/cry 1f - cry1 ab - brad.pdf

. Capinera, J. L. , 2005. University of Florida, Department of Entomology and Nematology informational web page for soy bean looper (

Pseudoplusia includens ): http://entnemdept.ufl.edu/creatur es/veg/leaf/cabbage_looper.htm . Publication Number: EENY - 116. Caprio, M., 1998. Evaluating resistance management strategies for multiple toxins in the presence of external refuges. J. Econ. Entomol. 91: 1021 - 1031. Gould, F., A. Anderson, A. Rey nolds, L. Bumgarner, and W. Moar, 1995. Selection and genetic anal ys is of a Heliothis virescens (Lepidoptera: Noctuidae) strain with high levels of resistance to Bacillus thuringiensis toxins. J. Econ. Entomol. 88: 1545 - 59. Jackson, R. E., J. R. Bradley , J. W. Van Duy n, B. R. L eonard, R. Luttrell, J. Ruberson, J. J. Adamczy k, J. Gore, D. Hardee, R. Voth, S. Sivasupramaniam, J. W. Mullins and G. Head, 2008. Regional assessment of Helicoverpa zea (Lepidoptera: Noctuidae) populations on cotton and non - cotton crop host s. Entomol. Exp. Appl. 126 (2): 89 - 106.Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 75 of 78 MacRae T.C., M.E. Baur, D.J. Boethel, B.J. Fitzpatrick, A.G. Gao, J.C. Gamundi, L .A. Harrison, V.T. Kabuye, R.M. McPherson, J.A. Miklos, M.S. Paradise, A.S. Toedebusch, A. Viegas, 2005. L aboratory

and field evaluations of transgenic soy bean exhibiting high - dose expression of a sy

nthetic Bacillus thuringiensis cry1A gene for control of Lepidoptera. J Econ Entomol. 98 (2): 577 - 87. Pedigo, L.P., 1999. Entomology and Pest Management (Third Edition). Prentice Hall: New Jersey . 691 pp. Roush, R.T., 1998. Two toxin strategies for management of insecticidal transgenic crops: will py ramiding succeed where pesticide mixtures have not? Phil. Trans. R. Soc. Lond. 353: 1777 - 1786. Sheck, A. L. and F. Gould, 1993. The genetic basis of host range in Heliothis virescens : larval survival and growth. Entomol. Exp. Appl. 69: 157

– 172. Zhao, J., J. Cao, Y. Li, H. Collins, R. Roush, E. Earle, and A. Shelton, 2003. Transgenic plants expressing two Bacillus thuringiensis toxins delay insect resistance evolution. Nature Biotechnology. 21: 1493 - 1497. **Other** Ferré , J and J . Van Rie, 2002 . Biochemistry and genetics of insect resistance to Bacillus thuringiensis , J Annu Rev Entomol , 47:501 - 33 .Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

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Page 76 of 78 

### Appendix **E .**  **Bibliography of data submitted for registration of**  **B acillus**  **t huringiensis**  **Cry1A c and Cry1F Proteins**  **expressed in Event DAS**  **- 81419**  **- 2**  **Soybea n**  **. Table E. 1**  **. D**  **ata Sub mitted for**  **Cry 1A c**  **and Cry1F**  **proteins expressed in DAS**  **- 81419**  **- 2** 

**Soybean MRID**  **No. Citation**  **Receipt Date**  49024100 Dow

AgroSciences LLC (2012) Submission of Pesticide Use, Product Chemistry and Toxicity Data in Support of the Application for Registration of DAS - 81419 - 2 Soybean. Transmittal of 10 Studies. 18 - Dec 2012 49024101 Unknown (2012) Supplement to MRID 488284 - 01: Molecular Characterization of

DAS/81419 - 2 Soybean. Unpublished study prepared by Dow AgroSciences. 5p . 18 - Dec 2012 49024102 Em

brey , S. (2012) Characterization for Soy Leaf Tissue Samples (TSN303074, TSN303075). Project Number: 120416. Unpublished study prepared by Dow AgroSciences LLC. 2p. 18 - Dec 2012 49024103 Unknown (2012) Supplement to MRID 48828409: Similarity Assessment of Cry1Ac Protein

to Known Toxins by Bioinformatics Analysis: (DAS - 81419 - 2 Soy bean). Project Number: 120416. Unpublished study prepared by Dow AgroSciences LLC. 1p. 18 - Dec 2012 49024104 Unknown (2012) Supplement to MRID 48848216: Ch aracterization of the Cry1Ac Protein

Derived from Transgenic Soybean Event DAS - 81419 - 2. Project Number: 110840. Unpublished study prepared by Dow AgroSciences LLC. 3p. 18 - Dec 2012 49024105 Unknown (2012) Supplement to MRID 48828417: Characterization of th e Cry 1F Protein

Derived from Transgenic Soybean Event DAS - 81419 - 2. Unpublished study prepared by Dow AgroSciences LLC. 3p. 18 - Dec 2012 49024106 Unknown (2002) Certificate of Analysis for Test/Reference/Control Substances

(TSN103748): (DAS - 81419 - 2 Soy bean) . Project Number: BIOT023159. Unpublished study prepared by Dow

AgroSciences LLC. 7p. 18 - Dec 2012 49024107 Unknown (2012) Supplement to MRID 488284 - 10: An Acute Oral Toxicity Study with the

Northern Bobw

hite: (DAS - 81419 - 2 Soybean). Unpublished study prepared by Dow AgroSciences LLC. 1p. 18 - Dec 2012 49024108 Unknown (2002) Certificate of Analysis for Test/Reference/Control Substances

(TSN103748): (DAS - 81419 - 2 Soy bean). Project Number: BIOT023159. Unpublished study prepared by Dow

AgroSciences LLC. 7p. 18 - Dec 2012 49024109 Unknown (2012) Supplement to MRID 48828413: Potential Impact of Cry1F and Cry1Ac in

DAS - 81419 - 2 Soy

bean on Non - Target Organisms and Threatened and Endangered Species. Unpublished study prepared by Dow AgroSciences LLC. 2p. 18 - Dec 2012 49024110 Storer, N. (2012) Insect Resistance Management Implications of Seed - Production and

Research Registration for DAS - 81419 - 2 Soybean - Revised. Project Number: IRM/2012/08. Unpublished study prepared by Dow AgroSciences LLC. 19p. 18 - Dec 2012 490448 00 Dow

AgroSciences, LLC (2013) Submission of Product Chemistry, Efficacy, Toxicity and Exposure/Risk Data in Support of the Application for Registration of DAS - 81419 - 2 Soy bean. Transmittal of 9 Studies. 25 - Jan 2013Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

Biopesticides Registration Action Document January 2014

Page 77 of 78 49044801 Storer, N. (2012) Insect Resis tance Management Implications of Seed - Production and

Research Registration for DAS - 81419 - 2 Soybean - Revised. Project Number: IRM/2012/08. Unpublished study prepared by Dow AgroSciences, LLC. 19p. 25 - Jan 2013

49044802 Guttikonda, S. (2013) Response to EPA Comment on Molecular Characterization of DAS 81419 - 2 Soybean Study - Provide Information Concerning the Source of the Promoters and Terminators; Provide the Actual construction of the Cry Proteins; Related to MRID 488284 - 01. Project Number: IRSOY/2013/01. Unpublished study prepared by Dow AgroSciences, LLC. 8p. 25 - Jan 2013

49044803 Han, L. (2013) Response to EPA Comment on Biological Equivalency of (DAS - 81419 - 2)

and Microbe - Produced Cry1Ac and Cry1F Proteins Study - Provide Explanation to Establish the Equivalence of Dow''s Cry1Ac and Cry1F Proteins to those Expressed in the Soybean; Identify the Leaf Powder Diluent Used in the Insect Bioassays: Related to MRID 488284 - 03. Project Number: IRSOY/2013/02. Unpublished study prepared by Dow AgroSciences, LLC. 8 p. 25 - Jan 2013

49044804 Han, L. (2013) Response to EPA Comment on Similarity Assessment of Cry1Ac Protein to

Known Toxins Bioinformatics Analysis Study - Identify the Protein Toxin Source; Explain the Error on Page 11 of 16: Related to MRID 488284 - 09. Pro ject Number: IRSOY/2013/03. Unpublished study prepared by Dow AgroSciences, LLC. 5p. 25 - Jan 2013

49044805 Han, L. (2013) Response to EPA Comment on Characterization of the Cry1Ac Protein

Derived from Transgenic Soybean Event DAS - 81419 - 2 Study - Address Amino Acid Sequence Similarity between Dow''s Cry1Ac Microbial Protein and Soybean - Produced Cry1Ac; Provide a Replacement for the Illegible Figure on Page 55: Related to MRID 488284 - 16. Project Number: IRSOY/2013/04. Unpublished study prepared by Dow AgroSci ences, LLC. 8p. 25 - Jan 2013

49044806 Han, L. (2013) Response to EPA Comment on Characterization of the Cry1F Protein

Derived from Transgenic Soybean Event DAS - 81419 - 2 Study - Address Amino Acid Sequence Similarity between Dow''s Cry1F Microbial Protein and Soy bean - Produced Cry1F: Related to MRID 488284 - 14. Project Number: IRSOY/2013/05. Unpublished study prepared by Dow AgroSciences, LLC. 7p. 25 - Jan 2013

49044807 Herman, R. (2013) Response to EPA Comment on Cry1F(Synpro) ICP and Cry1Ac(Synpro )

ICP: An Acute Oral Toxicity Study with the Northern Bobwhite - Provide Data Demonstrating the Equivalence of Dow''''s Test Material #101811 to Plant -produced Protein; Provide a Justification of the Test Dosage: Related to MRID 488284 - 10. Project Number: IRSOY/2013/06. Unpublished study prepared by Dow AgroSciences, LLC. 14p. 25 - Jan 2013

49044808 Han, L. (2013) Response to EPA Comment on Cry1F(Synpro) and Cry1Ac(Synpro)

Insecticidal Crystal Proteins: An 8 - Day Dietary Study with the Rainbow Trout Oncorhync hus mykiss, WALBAUM - Provide Data Demonstrating the Equivalence of Dow''s Test Material #101811 to Plant -produced Protein: Related to MRID 488284 - 11. Project Number: IRSOY/2013/07. Unpublished study prepared by Dow AgroSciences, LLC. 13p. 25 - Jan 2013

490 44809 Han, L. (2013) Response to EPA Comment on Potential Impact of Cry1F and Cry1Ac in

DAS - 81419 - 2 Soy

bean on Non - target Organisms and Threatened and Endangered Species Study - Provide Gene Flow and Weediness Information to Address Environmental Fate Conc erns; Address Soil Degradation: Related to MRID 488284 - 13. Project Number: IRSOY/2013/08. Unpublished study prepared by Dow AgroSciences, LLC. 6p. 25 - Jan 2013 49081500 Dow

AgroSciences, LLC (2013) Submission of Pesticide Use Data in Support of the Application for Registration of DAS - 81419 - 2 Soybean. Transmittal of 1 Study. 14 - Mar 2013 49081501 Han, L. (2013) Amended Response to the EPA Comment on Characterization of the Cry1F 14 - Mar -Bt Cry1Ac and Cry1F proteins expressed in DAS - 81419 - 2 Soybean

Biopesticides Registration Action Document January 2014

Page 78 of 78 Protein Derived from Transgenic Soybean Event DAS - 81419 - 2 Study - Address Amino Acid Sequence Similarity Between Dow's Cry1F Microbial Protein and Soybean - Produced Cry1F. Project Number: IRSOY/2013/05. Unpublished study prepared by Dow AgroSciences, LLC. 7p. 2013