## B **IOPESTICIDES REGISTRATION ACTION DOCUMENT** 

**PLANT -**  **INCORPORATED PROTECTANTS:** Bacillus thuringiensis Cry1Ab protein and the Genetic Material Necessary

for its Prod

uction in event T304 - 40 Cotton [ PC Code 006525, OECD Unique Identifier: BCS - GHØØ4 - 7 ] ; Bacillus thuringiensis Cry2Ae protein and the Genetic Material Necessary

for its Prod

uction in event GHB119 Cotton [ PC Code 006600, OECD Unique Identifier: BCS - GH   5 - 8 ] ;

and TwinL inkTM cotton (T304 - 40 and GHB119 Combination PIP product) [ OECD Unique I dentifier: BCS - GHØØ4 - 7 x BCS - GH   5 - 8 ]

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

December 12, 2011Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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### Bacillus thuringiensis **Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae) Table of Contents**  **BIOPESTICIDES REGISTRATION ACTION DOCUMENT TEAM ................................**  **................................ ................**  **3 GLOSSARY OF ACRONYMS AND ABBREVIATIONS**  **................................ ................................**  **................................ .....**  **4 EXECUTIVE SUMMARY**  **................................ ................................**  **................................ ................................**  **....................... 5**  **I. ACTIVE INGREDIENT OVERVIEW**  **................................ ................................**  **................................ ............................**  **6 II.**  **REGULATORY BACKGROUND ................................**  **................................ ................................**  **................................ ...**  **7 III.**  **RISK ASSESSMENT SUMMARIES ................................**  **................................ ................................**  **.............................. 9**  A. Product Characterization ................................ ................................ ................................ ................................ .............. 10 B. Human Health Assessment ................................ ................................ ................................ ................................ ............. 11 C. Environmental Effects Assessment ................................ ................................ ................................ ................................ .. 17 D. Insect Resistance Management ................................ ................................ ................................ ................................ ....... 18 **IV. ENVIRONMENTAL JUSTICE**  **................................ ................................**  **................................ ................................**  **. 22**  **V. RISK MANAGEMENT AND PROPOSED REGISTRATION DECISION**  **................................ ..........................**  **23 VI. TERMS AND CONDITIONS OF THE REGISTRATION(S)**  **................................ ................................**  **.................... 23**  **APPENDIX A: Product characterization and hum an health e**  **ffects –**  **Bacillus thuringiensis Cry 1Ab (Event T304**  **- 40)**  **in cotton plant incorporated protectant. ................................**  **................................ ................................**  **................................ 26**  **APPENDIX B: Product characterization and hum an health effects**  **- Bacillus thuringiensis**  **Cry 2Ae (Event GHB 119) and TwinLinkTM in cotton plant incorporated prote**  **ctant. ................................**  **................................ ................................**  **.. 36**  **APPENDIX C: Ecological and environmental effects –**  **Bacillus thuringiensis Cry1Ab, Cry2Ae and TwinLinkTM cotton**  **plant incorp orated protectant**  **................................ ................................**  **................................ ................................**  **................. 59**  **APPENDIX D: Insect Resistance Managem ent**  **................................ ................................**  **................................ ....................**  **86 APPENDIX E: References**  **................................ ................................**  **................................ ................................**  **.................... 135** Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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### BIOPESTICIDES REGISTRATION ACTION DOCUMENT TEAM Microbial Pesticides Branch Product Characterization and Human Health John Kough, Ph.D. Annabel Waggoner, B.S. Chris Wozniak, Ph.D. Environmental Fate and Effects Zi gfridas Vaituzis, Ph.D. Shannon Borges, M.S. Annabel Waggoner, B.S. Insect Resistance Management Jeannette Martinez, M.S. Alan Rey nolds, M.S. Benefit Assessment Shannon Borges, M.S. Biopesticides Registration Action Document Regulations Sheryl Reill y, Ph.D. Alan Rey nolds, M.S. Shanaz Bacchus, M.S., MBA Denise Greenway , B . S . Previous U.S. EPA Reviewers Rebbeca Edelstein, Ph.D. Sharlene Matten, Ph.D . USDA/BRS Work share Program Patricia Beetham , Ph.D. USDA/BRS 1

1 Conducted secondary review of DERs for some product characterization data under w ork - share program according to the EPA - BPPD and USDA - BRS mem orandum of understanding [09 - 2000 - 0052 - MU].Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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### GLOSSARY OF ACRONYMS AND ABBREVIATIONS APHIS Animal and Plant Health Inspection Service (USDA) 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 Deox yribonucleic 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 Organisat i on for Economic and Community Development PC Pesticide Chemical PCR Polymerase Chain Reaction PIP Plant - Incorporated Protectant pg picogram (10 - 12 gram) ppb parts per trillion ppm parts per million ppt parts per trillion PTGS Post - T ranscriptional 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 United States Environmental Protection Agency U . S . FDA United States Food and Drug AdministrationBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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### EXECUTIVE SUMMARY The United States Environmental Protection Agency (U . S . EPA, Agency) intends to register three new cotton plant - incorporated protectant (PIP) products as described in this draft Biopesticide Registration Action Document (BRAD) . Th ese proposed registration actions represent two new PIP active ingredients. Their expression s in the proposed product s are first f ood and first outdoor use s of the PIPs. The A genc y established a policy in October 1, 2009 to inform and provide the public an opportunity to comment on proposed registration decisions before they occur . T his draft BRAD and supporting documents are now available for 30 day s in the Agency ’s docket (EPA - HQ - OPP - 20 11 - 09 88 ) for this purpose . All constructive comments relevant to the proposed decisions, in addition to the Agency 's responses , will be included in the docket when the registrations are finalized. Ba ye r CropScience, LP (BCS) developed , and i s seeking federal registration of , two transgenic cotton [ Gossypium hirsutum ] e vent s , T304 - 40 and GHB119 . E ach express es an insecticidal protein (also known as “Cry ” proteins) derived from the soil bacterium Bacillus thuringiensis ( Bt) . These cotton events are intended for use a s PIPs for conventional breeding to produce a product , TwinL inkTM C otton (referred to in this document as “TwinLinkTM ”) that expresses both proteins , This draft BRAD presents EPA’s evaluations of the data and information submitted by BCS for the proposed registrations of : 1. EPA File Sy

mbol 264 - RNOU: Event T304 - 40 cotton express ing Bt Cry 1Ab insecticidal protein . The cry1Ab gene that expresses this protein is derived from Bt subspecies berliner . It was created by Agrobacterium - mediated transformation of Gossypium hirsutum cv. Coker with the TDNA vector pTDL008. 2. EPA File Sy

mbol 264 - RNOL : Event GHB119 cotton express ing Bt Cry2Ae insecticidal protein . It was created b y Agrobacterium - mediated transformation using vector pTEM12. It contains the cry2ae gene isolated from Bt subspecies dakota . 3. EPA File Sy

mbol 264 - RNOA: Genetic material from e vents T304 - 40 and GHB119 are combined by

conventional breeding to express the combined proteins, Bt Cry 1Ab and Bt Cry2Ae, in TwinL inkTM cotton. The genes that are responsible for the Bt Cry 1Ab and Bt Cry2Ae proteins, cry1Ab and cry2Ae , have been modified for their expression in plants . These proteins are referred to as Cry 1Ab ( or event T304 - 40 ) , and Cry2Ae ( or event GHB 119 ) , respectively , in this draft BRAD. Both e vents also include the pat gene th at produces Phosphinothricin Acet yl transferase ( PAT ) enzy me, an inert ingredient in the proposed products that confers tolerance of the cotton plant to the herbicide, glufosinate. An Experimental Use Permit (EUP) was issued to BCS to conduct field trials with event T304 - 40 (Cry 1Ab) cotton on February 7, 2006 and was extended to expire on December 31, 2011. An EUP for GHB119 (Cry2Ae) and the combination PIP product, TwinL inkTM ( expressing Cry 1Ab and Cry2Ae proteins ) was issued on September 1, 2008 , and will expire on December 31, 2011 . These EUPs were associated with permanent tolerance exemptions for the active ingredient, Cry 1Ab ( 40 CFR § 174. 511 ), and the inert enz ym e, PAT protein ( 40 CFR § 174. 522 ) , and a temporary tolerance exemption for the active ingredient, Cry2Ae ( 40 CFR § 174. 530 ).Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document - DRAFT December 12, 2011

Page 6 of 153 BCS applied for pesticide registrations of Cry 1Ab and Cry2Ae proteins as expressed in events T304 - 40 and GHB119 cotton, respectivel y, and a new combination PIP product, TwinL inkTM cotton , c reated through conventional breeding of these events for use as PI Ps in 2009, under section 3 of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) . BCS concurrentl y f iled a petition for a permanent exemption from the requirement of a tolerance for Cry2Ae protein residues in or on food and feed commodities in cotton . The Agency believes that, based upon its assessment of the data and information submitted by

BCS , it is in the interest of the public and the environment to issue the registration s proposed b y BCS . 

### I. **ACTIVE INGREDIENT OVERVIEW** Active Ingredients and Office of Pesticide Programs (OPP) Chemical Code and File Sy mbol: a. Bt Cry 1Ab insecticidal protein and the genetic material necessary for its production in event T304 - 40 cotton (PC Code 006525); OECD Unique Id entifier: BCS - GHØØ4 - 7; EPA File Sy

mbol 264 - RNOU. b. Bt Cry2Ae insecticidal protein and the genetic material necessary f or its production in event GHB119 cotton (PC Code 006600); OECD Unique identifier: BCS - GH   5 - 8; EPA File Sy

mbol 264 - RNOL. c. Bt Cry 1Ab and Bt Cry2Ae insecticidal proteins and the genetic material necessary for their production in TwinL inkTM cotton (T304 - 40 x GHB119) combination PIP product; OECD Unique Identifier: BCS - GHØØ4 - 7 x BCS GH   5 - 8; EPA File Sy

mbol 264 - RNOA. Applicant/ Manufacturer: Ba ye r CropScience

– BioScience (BCS)

P.O. Box 12014 2 T.W. Alex an der Drive Rese arch Tri angle Park, NC 27709 Ty pe of Pesticide: Plant Incorporated Protectant (PIP) Use: Insecticide Target Pest: C otton bollworm (CBW, Helicoverpa zea ), pink bollworm (PBW, Pectinophora

gossypiella ), tobacco budworm (TBW, Heliothis virenscens ) larvae, beet army worm ( Spodoptera exigua ), and fall arm ywo rm ( Spodoptera frugiperda ) . 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 Cry 1Ab and Cry2Ae proteins in the proposed PIPs. There is no mechanistic evidence that Bt proteins pose risks for humans or mammals during th eir use as pesticides or as PIPs (OECD, 2007). When Cry proteins are ingested b y an insect ,Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 7 of 153 they are dissolved under the alkaline conditions specific to the insect ’s gut , and then activated upon cleavage b y the insect’s midgut enzy mes (proteases ) . These ac tivated Cry proteins must bind specificall y to gl ycoprotein or gl ycolipid receptors on microvillar membrane of the insect’s mid gut 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. C ry 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 lead 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). 

### II. **REGULATORY BACKGROUND** Applications for use of Cry 1Ab, Cry2Ae and TwinL inkTM PIPs **A. Cry 1Ab**  1. Experimental Use Permit and Temporary Tolerance Exemption a. Cry 1Ab cotton Permit (EPA Reg. No. 264 - EUP - 140) EPA granted an Experime ntal Use Permit (EUP) for the Cry 1Ab cotton PIP to BCS on February 7, 2006 (71 FR 41020; July

19, 2006; FRL - 8060 - 6) . These experimental trials were conducted with cotton PIPs containing the genetic material necessary for Cry 1Ab protein production in events T303 - 3 and T304 - 40 . The compan y intended to research the potential of this cotton PIP, Cry 1Ab protein, for control of lepidopteran larvae , such as cotton bollworm ( Helicoverpa zea ) and tobacco budworm (Heliothis virescens ), which are common pests of cotton . The EUP was amended on March 8, 2007 (72 FR 34009; June 20, 2007; FRL - 8133 - 5), on August 28, 2008 (73 FR 58949; October 8, 2008; FRL - 8384 - 9), and on November 24, 2008 (74 FR 10571; March 11, 2009; FRL - 8398 - 2) , to permit planting until December 31, 2010. An additional extension was granted to extend the ame nded program to run from January 1, 2011 until December 31, 2011 . The proposed program was conducted in the States of Alabama, Arkansas, Arizona, California, Florida, Georgia, Louisiana, Mississippi, North Carolina, South Carolina, Tennessee, Texas, and in the United States territory of Puerto Rico . The EUP included trials to evaluate insect and herbicide efficacy , agronomic perform ance, and breeding lines . Also, seed for future plantings of experimental and regulatory

field trials were produced .Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 8 of 153 b. Cry 1Ab Temporary

Tolerance Exemption A temporary

tolerance exemption for Cry 1Ab was not required , since a permanent tolerance exemption is established for Cry 1Ab in 40 CFR § 174.51 1 for all food and animal feed commodities ( discussed in IIA.2.b , below ) . 2. Registration Application and Permanent Tolerance Exemption a. Cry 1Ab FIFRA Section 3 R egistration Application On April 8, 2009 , EPA published a Notice of Receipt in the Federal Register ( 74 FR 15978 ) , announcing that BCS submitted an application to register a new active ingredient, Bt Cry 1Ab (event T304 - 40 ; EPA File Sy mbol 264 - RNOU ), not included in any currently registered pesticide products for use in cotton . No public comments were received i n response to this publication .

b. Cry 1Ab - Permanent Tolerance exemption Residues of Cry 1Ab protein are exempt from the requirement of a tolerance when used as PIPs in all food commodities ( 40 CFR § 174.511 ) . EPA granted an exemption from tolerance for Cry 1Ab protein in all food and feed commodities on August 2, 1996. The tolerance exemption is published in the Code of Federal Regulations ( 40 CFR § 180.1173 ) . In September 2001, EPA completed a reassessment of this tolerance exemption considering all of th e existing data, public literature, and public comments. The reassessment determined that the tolerance exemption met all the required scientific and regulatory standards. This tolerance exemption for the Cry 1Ab protein is not event - specific and applies to all Cry 1Ab protein and an y other event producing the Cry 1Ab protein that might be found in the food supply . On April 25, 2007 , the tol erance exemption for Cry 1Ab was reassigned to 40 CFR § 174.511 ( 72 FR 20435) . Based on this existing tolerance exemption , a petition to establish a tolerance or tolerance exemption was not required for the Cry 1Ab expressed in this cotton PIP. **B. Bt**  **Cry2Ae and TwinLink**  **TM** 1. Experimental Use Permit and Temporary Tolerance Exemption a. Cry2Ae and TwinLinkTM Permit (EPA Reg. No. 264 - EUP - 143) In addition to the EUP for Cry 1Ab, EPA granted an EUP to BCS for the Cry2Ae cotton PIP and the combined traits of Cry

1Ab x Cry

2Ae in TwinLinkTM (EPA Reg. No. 264 - EUP - 143) on September 1, 2008 (73 FR 65848; November 5, 2008; FRL - 8388 - 6) , with an expiration date of December 31, 2010. The permit was extended on December 30, 2010 to allow further evaluation of these cotton plant l ines in a wider range of environmental conditions between January 1, 2011 and December 31, 2011. Testing was intended to include insect efficacy trials, agronomic performance evaluations, and herbicide efficacy evaluations, as well as the production of sample material for conducting regulatory studies . Tests were allowed in 12 states: Alabama, Arkansas, Arizona, California, Florida, Georgia, Louisiana, Mississippi, North Carolina, South Carolina, Tennessee and Texas and the United States territory of Puerto Rico. Testing was authorized on a total of 1,919 acres of which 152 acres were to be planted to Cry2Ae and 307 acres to Cry 1Ab x Cry2Ae combined trait cotton (TwinLinkTM Cotton) .Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 9 of 153 The compan y was researching the potential for Cry2Ae and combined Cry1Ab and Cry2Ae proteins (TwinL inkTM Cotton) produced by the inserted genetic material in these cotton PIPs for control of lepidopteran larvae , such as Cotton bollworm ( Helicoverpa zea ), Tobacco budworm ( Heliothis virescens ), Pink bollworm ( Pectinophora gossypiella ), Fall army worm ( Spodoptera frugiperda ), and Beet arm yworm ( Spodoptera exigua ) All cotton plants evaluated under this EUP contain ed the Cry2Ae protein , and were derived from either a transformation event ( GHB119 or GHB714 ) or combinations derived from either transformation event T303 - 3 or T304 - 40 ( cry 1Ab ) and event GHB119 or GHB714 ( cry 2Ae ) . b. Cry2Ae: Temporary Tolerance Exemption A notice of filing of a pesticide petition (PP7F7912) by BCS was published in the Federal Register of August 8, 2007 ( 72 FR 44521 - 44523) (FRL - 8139 - 7) in connection with the above referenced Cry2Ae EUP. Relevant compan y data/information in sup port of the application was posted in the docket, EPA HQ - OPP - 2007 - 0573. Following EPA’s evaluation of these submissions, a temporary exemption from tolerance was approved for residues of Bt Cry2Ae in/on cotton food/feed commodities ( 73 FR 52591 ; 9/10/2008). It is set to expire on December 31, 2012. 2. Registration Application and Permanent Tolerance Exemption

a. Cry2Ae and TwinLinkTM - FIFRA section 3 application On April 8, 2009 , EPA published a Notice of Receipt (NOR) in the Federal Register ( 74 FR 15978 ) , announcing that BCS submitted an application to register a new active ingredient, Bacillus thuringiensis Cry 2Ae (EPA File S ymbol 264 - RNOL ; event GHB 119) as a PIP in cotton . BCS also proposed the registration of another new PIP product, TwinlinkTM Cotton (EPA File Symbol 264 - RNOA), developed by conventional breeding of the aforesaid c otton ev ents GHB119 and T304 - 40 , thus expressing the combined traits of the two new active ingredients, Cry 1Ab , and Cry2Ae.

b. Cry2Ae

– Permanent Tolerance Exemption BCS concurrently

submitted a petition (PP9F7514) to establish an exemption from the requirement of a tolerance for residues of Cry 2Ae and the genetic material necessary for its production with the FIFRA Section 3 registration s for this PIP and TwinlinkTM ( EPA File Sy mbols 264 - RNOl and 264 - RNOA) . EPA published a Notice of Filing of the petition in the Federal Register on April 8, 2009 ( 74 FR 15970 ), and the public was given a 30 - day comment period . Event GHB119 cotton 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 ) . No public comm ents were received to an y of the Federal Register announcements for Cry2Ae or TwinL inkTM , or the notice of filing of the pesticide petition for Cry2Ae published in the Federal Register . 

### I **II .**  **RISK ASSESSMENT SUMMARIES**Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 10 of 153 EPA assessed the data submitted by BCS in 2009 for the proposed PI P cotton registration s and is presenting s ummaries of the product characterization and human health risk assessment s in Appendices A (Cry 1Ab) and B (Cry

2Ae and TwinLinkTM ) . Summaries of ecological and environmental risk assessments and Insect Resistance Management (IRM) for all three PIP products are presented in Appendices C and D, respectivel y. In its assessment, the EPA relied upon data and other information submitted by

the applicant and general knowledge of best ava ilable 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 “ ACC EPTABLE ” indicates the study is scientifically

sound and is useful for risk assessment. A “SUPPL EMENTAL” rating indicates the data provide some information that can be useful for risk assessment. The studies may have certain aspects determined not to be sc ientifically acceptable (“SUPPL EMENTAL : UPGRADABLE”). If a study is rated as “SUPPL EMENTAL: UPGRADABLE,” the Agency alway s provides an indication of what is lacking or what can be provided to change the rating to “ACCEPTABLE.” If there is simply a “SUPPL EM ENTAL” rating, the reviewer will often state that the study is not required by

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 “UNACCEPTAB LE” rating 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 I V indicates the lowest toxicity . 

### A. **Product Characterization** All product characterization data requirements for the Bt cotton PIPs containing Cry 1Ab, 2Ae and their combined traits in TwinL inkTM have been satisfied . The two cotton events developed b y BCS to express these PIPs are:

1. T304 - 40 cotton: expresses Cry 1Ab in secticidal protein derived from Bt subspecies berliner . It was created by Agrobacterium - mediated transformation of Gossypium hirsutum cv. Coker with the TDNA vector pTDL008. Sodium dodecyl sulfate poly acrylamide gel electrophoresis (SDS - PAGE) analy ses of C ry 1Ab protein isolated from event T304 - 40 cotton leaves showed a very faint band estimated to be at a similar molecular weight as the band exhibited for the E. coli - derived Cry 1Ab protein standard at approximately

66 kilodaltons (kDa). 2. Event GHB119 cotto n: expresse s Cry2Ae protein, derived from Bt subspec ies dakota. It was created by Agrobacterium - m ediated transformat ion using vecto r pTEM12 , and contains the cry2ae gene isol ated from Bacillus thuringiensis subspecies dakota . SDS PAGE demonstrates that Cry2Ae has a molecular weight of 71 kDa . Its sequence is modified for optimal expression in plants.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 11 of 153 These events are combined by conventional breeding in another PIP, TwinLinkTM that expresses Cry 1Ab and Cry2Ae proteins to provide protection against feed ing dama ge by lepidopteran insect larvae. Th e initial product characterization data submitted by BCS to support the FIFRA Sec tion 3 Registration of PIP cotton events T304 - 40, and GHB119 and their combination by conventional breeding in TwinL inkTM were reviewed by the Agency

and found Supplemental (U.S. EPA 02/ 04 /11) **.** There were several data deficiencies that the applicant had to address before EPA could continue its review for these events. Consequently , the data were consid ered insufficient to sup port the FIFRA section 3 registration of cotton events GHB119, T304 - 40, and TwinLinkTM its respective combination PIP product (U.S. EPA DER dated February

4, 2011, from A. Waggoner to S. Bacchus and D. Greenway) . Additional data , submitted by

BCS in May 2011 , were evaluated and found acceptable . For summaries of the data evaluated for product characterization for these PIPs, see Appendix A for Cry 1Ab and Appendix B for Cry2Ae and TwinLinkTM . T h e summaries include discussions of the following:

 Transformation S ys tem  Characterization of the DNA Inserted in the Plant and Inheritance and Stability  Protein Characterization  Protein Equivalence  Analy tical Method  Product Characterization As discussed in the summaries, Cry 1Ab and C ry2Ae proteins are expressed at very low parts per million (ppm) levels in various parts of the transgenic cotton plant and are shown to be equivalent to the microbially

expressed proteins. As term s of registration, EPA requires confirmation of the e nforcement analy

tical me thodology as discussed in Chapter VI of this BRAD. 

### B. **Human Health Assessment 1**  **. Toxicological Profile**  EPA reviewed the available scientific data and other relevant information submitted in support of th ese action s to register Cry 1Ab, Cry2Ae and TwinLinkTM PIPs and considered their 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 subgroup s of consumers, including infants and children . All toxicology

data requirements for the Bt cotton PIPs containing Cry 1Ab, 2Ae and their combined traits in TwinL inkTM have been satisfied**.** Acceptable Tier I mammalian toxicology data/information support the proposed registration s of these active ingredients. Furthermore, Tier II and Tier III studies were not required for either Cry 1Ab or Cry2Ae , based on the lack of acute toxicity /pathogenicit y in the Tier I studies. Refer to Table s A. 3 and B.3 ( in Appendices A, and B , respectively) for brief summar ies of data evaluated to satisfy toxicity and allergenicity data requirements for these PIPs.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 12 of 153 Acute toxicity Cry 1Ab and Cry2Ae The following summarizes the final determinations regarding acute toxicolog y data requirements for Cry 1Ab and 2Ae. See Appendices A and B in this BRAD for more detail ed summaries . **TABLE 1**  **: Tier 1 Mamm alian Toxicology Data Requirements**  **and Associated OCSPP Test Guidelines**  Study Conclusion Ac ute Oral Toxicity/Pathogenicity OCSPP 885.3050

– Toxicity Category IV by tests. Ac ute Dermal Toxicity OCSPP 885.3100

– Waived. Ac ute Pulmonary Toxicity/Pathogenicity OCSPP 885.3150

– Waived. Ac ute Injection Toxicity/Pathogenicity OCSPP 885.3200

– superseded by acute oral test for Cry 2Ae (See Appendix B) . Hy persensitivity Incidents ***** OCSPP 885.3400

– none reported yet but required if they occur. Cell Culture OCSPP 885.3500

– only required for viruses. Ac ute Oral Toxicity OCSPP 870.1100

– wa ived for End - use Product TwinLinkTM . Ac ute Dermal Toxicity OCSPP 870.1200 - wa ived Ac ute Inhalation Toxicity OCSPP 870.1300 - wa ived Ac ute Eye Irritation OCSPP 870.2400 - wa ived Primary Dermal Irritation OCSPP 870.2500 - wa ived **Hypersensitivity Incidents** : Reporting is required when incidents occur . No hy persensitivity

incidents, including immediate - ty

pe or delay ed - ty

pe reactions in humans or animals, occurred during the manufacture and field trials of the genetic material for Bt Cry 1Ab, Cry2Ae and their combination in TwinL inkTM . Should any future hy persensitivity

incidents occur, they

must be reported to the Agency . a. Acute Oral Toxicity Cry 1Ab and Cry2Ae A cute oral toxicity

Tier I stud ies in mice indicated that Cry 1Ab ( U.S. EPA DER from A. Waggoner, 2011a to S. Bacchus and D. Greenway) and Cry2Ae ( U.S.EPA DER from R. Edelstein, 2008 to S. Bacchus; A. Waggoner, 2011b to S. Bacchus and D. Greenway) are non - toxic to laboratory mice . The acute oral toxicity levels of Cry 1Ab and Cry2Ae w ere assessed in separate tests . The m ice were given purified, bacteriall y produced protein by oral gavage at a dosage of 2,000 milligrams/kilograms of body weight (mg/kg b.w. ) . All of the treated animals gained weight , and no signs of toxicity or other adverse effects occurred, and no test material - related findings were found at necropsy . The results of the study demonstrated that the acute oral LD 50 of each of these proteins is greater than 2000 mg/kg b.w . The results of these studies demonstrate that no adverse effects are expected at a level that is well above the maximum possible exposure levels that are reasonably anticipated to be present in the crop (i.e. approximately

1 microgram/gram - equivalent to 1mg/kg - dry weight cottonseed

– see Tables B.1 and B.4, Appendix B). C otton itself is not an edible food commodity in the human diet . Dietary exposure to processed cottonseed flour is also expected to be at lower levels than those for which no adverse effects were observed in these studies .Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 13 of 153 When proteins are toxic, they are known to act via acute mechanisms and at very low dose levels ( Sjoblad, Roy

D. et al . (1992) . Since no acute effects were shown to be caused by Cry 1Ab or Cry

2Ae at high dose levels in the acute oral toxicity test s , the y are not considered to be toxic. Further, amino acid sequence comparisons sh owed no similarities between either Cry 1Ab or Cry2Ae and known toxic proteins in protein databases that would raise a safety concern ( U.S.EPA DERs from R. Edelstein, 2008 to S. Ba cchus; from A. Waggoner, 2011a, 2011b to S. Bacchus and D. Greenway) . For microbial products, further Tiers II and III toxicity testing and residue data are only required if adverse effects are observed during Tier I tests. Based on the lack of acute ora l toxicity and other adverse effects in the Tier I acute oral test s in mice , EPA did not require Tier II and Tier III testing for either Cry 1Ab or Cry2Ae. This conclusion is similar to the Agency ’s position regarding toxicity testing and the requirement o f residue data for the registered microbial Bt products containing the species from which th ese PIPs were derived (see 40 CFR §§ 158.2130(d)(1)(i) and 158.2140(d)(7)). b. Allergenicity Assessment Since Cry 1Ab and Cry2Ae are protein s , allergenic sensitivities were considered for each in separate tests for each active ingredient . Currently , no definitive tests exist for determining the allergenic potential of novel proteins. Therefore, EPA uses a weight - of - evidence approach where the following fac tors are considered ( U.S. EPA DERs 2011a and 2011b from Waggoner to Bacchus and Greenway ; Appendices A and B) : 1. **Source of the trait.** Bacillus thuringiensis, the microorganism from which the genetic materials encoding Cry

1Ab and Cry2Ae protein s are derived, is not considered to be a source of allergenic proteins. 2. **Amino acid sequence.** Separate c omparison s of the amino acid sequence of Cry 1Ab and Cry2Ae with known allergens showed no overall sequence similarity using the criterion of 35% identity over an 80 amino acid segment or identity at the level of eight contiguous amino acid residues. 3. **Prevalence in food**

**.** Protein expression level analy ses of Cry 1Ab and Cry2Ae protein s are present at relativel y low levels. Dietary exposure is extremely lim ited and expected to be correspondingl y low . Cry 1Ab and Cry2Ae expression in event GHB119 leaf, root and pollen has been shown to be in the parts per million ranges, and cotton is not a directly consumed food commodity . 4. **Digestibility.** Cry

1Ab was dige sted within 2 minutes, while the Cry2Ae protein was rapidly digested within 30 seconds in simulated mammalian gastric fluid (SGF) containing pepsin at a pH of 1.2 . 5. **Glycosylation.** Current scientific knowledge also suggests that common food allergens may be gl ycos yl ated [ Codex Alimentarius Commission (CAC), 2003 ] . Neither Cry1Ab nor Cry2Ae protein expressed in cotton was shown to be gl yc osylated . Considering all of the available information, EPA has concluded that the potential for Cry 1Ab and Cry2Ae to be food allergen s is minimal.

c. Tolerance ExemptionsBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 14 of 153 Permanent tolerance exemptions have been established for residues of Cry1Ab and PAT , as discussed in Chapter II under REGULATORY BACKGROUND , above . The toxicology database for Cry2Ae supports granting a permanent exemption from tolerance for residues of Cry2Ae in/on cotton food/feed commodities under section 408 of the Federal Food, Drug, and Cosmetic Act ( FFDCA ) . On September 10, 2008 , a temporary exemption from tolerance for residues of Cry2Ae PIP ( 40 CFR §174.530) was granted to B CS for cotton food/feed commodities in association with an Experimental Use Permit, EPA Reg. No. 264 - EUP - 143 . T he toxicological profile of Cry2Ae protein was previously described in the Federal Regis ter (73FR 52591: FRL - 8380 - 1) . Since then, BCS submitted additional data/information , and in 2009 petitioned EPA to establish a permanent exemption from the requirement of a tolerance for residues of Cry2Ae in or on all food commodities (see Regulatory

Back ground). On the basis of evaluations of all relevant data submissions for Cry2Ae, EPA has determined that the database supports amendment of 40 CFR §174.530 to establish a permanent tolerance exemption for residues of Cry2Ae in/on cotton food feed commod ities. EPA only considered th e petition as supporting a permanent exemption from the requirement of a tolerance for the residues of the protein in cotton and its food/feed commodities since the data submitted were associated with the use in cotton and the r e are no other proposed uses in other crops . In the future, additional uses in other food or animal feed crops may be proposed, and data or other information that demonstrates the same safet y characteristics described for its expression in cotton must be submitted along with a petition to amend the tolerance exemption to include those uses. **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 “naturall y occurring estrogen, or other such endocrine effects as the Administrator may designate.” Th e 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) h ormonal 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 which, if any , of the Tier 2 tests are n ecessary based on the available data. Tier 2 testing is designed to identify any adverse endocrine related effects caused b y the substance, and establish a quantitative relationship between the dose and the E, A, or T effect. Between October 2009 and Febr uary 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 pathwa ys such as food and water, residential activit y, and certain post - application agricultural scenarios. This list should not be construed as a list of known or likely endocrine disruptors. Cry 1Ab and Cry2Ae 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 pesticideBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 15 of 153 chemicals. Accordingl y, the Agency anticipates issuing future EDSP orders/data call - ins 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/ . **1. Food Qu**  **ality Protection Act (FQPA) Considerations a.**  **Aggregate Exposures** 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 available 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 e xemption and all other tolerances or exemptions in effect for the PIP residue, and exposure from non - occupational sources. As discussed in Appendices A and B, the oral toxicity studies conducted at a dose of 2000 mg/kg b.w. in separate tests showed no adverse effects for either Cry 1Ab or Cry2Ae, which were also shown to be rapidly digested in vitro . As previously stated, these proteins are expressed at very low ppm levels in the cotton PIPs, which are not directly consumed as food commodities . Although co tton is not a directly consumed food commodity , humans may be exposed to extremely low levels in the diet, potentially from ingestion of processed cotton products (e.g. cottonseed flour and oil). There is also a very remote possibility

that Cry 1Ab and Cry2 Ae can get in the water suppl y the same way that other proteins can migrate in crop debris from cotton into ground, and, may be, drinking water. Such potential dietary exposure from cotton or drinking water is expected to be several orders of magnitude lowe r than the amounts of these proteins shown to have no toxicity in mammalian tests. EPA concludes that even negligible exposure via food and drinking water would present no harm, based on the lack of mammalian toxicity

and allergenicit y potential, and the r apid digestibility demonstrated for the PIP s . Non - occupational dermal and inhalation exposure is not expected, since the PIP s are expressed and contained within cotton plant cells. The uses of th ese PIP s are agricultural, so there would be no exposure t o infants and children from residential, school or lawn use. The amino acid homology assessment s for Cry 1Ab and Cry2Ae included similarity to known aeroallergens. It has been demonstrated that there is no evidence of occupationally related respiratory sy

mp toms, based on a health survey on migrant workers after exposure to Bt pesticides (Bernstein et al . 1999). This observation is also relevant to the low potential for non - occupational inhalation exposure at levels far below those expected in occupationally exposed populations. 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.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 16 of 153 **b. Cumulative Effects from Substances with a**  **Common Mechanism of Toxicity** 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 .” Neither Cry 1Ab nor Cry2Ae is considered toxic . They do not share a common mechanism of toxicity with any other substances, nor do they appear to produce a toxic metabolite produced by other substances. For the purposes of this assessment , therefore, EPA has assumed that Cry 1Ab and Cry2Ae do not have a common mechanism of toxicity with other substances. Thus , EPA concludes that there are no cumulative effects associated with Cry2Ae th at 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 .**  **D etermination of Safety for U.S. Population, 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 Cry2Ae pro tein. This evaluation included the low levels of expression of Cry2Ae proteins in cotton, 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 allergenicit y and the non - toxic source of the protein. Because of this lack of demonstrated mammalian toxicity , no protein residue chemistry data for Cry2Ae 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 et y 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 safet y for infants and children is unnecessary in this instan ce. **2. 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 considers the international maximum residu e 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 recognize d as an international food safet y 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 section 408(b)(4) requires that EPA explain the rea sons for departing from the CodexBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 17 of 153 level. No Codex maximum residue level exists for the PIPs, Bacillus thuringiensis Cry 1Ab or Cry2Ae protein or their combination Cry 1Ab and Cry2Ae . 

### C. **Environmental Effects Assessment Ecological Effects Data for Event**  **T304 -**  **40 (**  **Bt Cry 1Ab)**  **and Event GHB119 (**  **Bt Cry 2Ae)**  All e cological and e nvironmental Effects data requirements for the Bt cotton PIPs containing Cry 1Ab, 2Ae and their combined traits in TwinL inkTM have been satisfied . For a more comprehensive discussion of the Agency ’s assessment of the data and information submitted concerning ecological and environmental risks for the registration of the se PIPs ( Cry 1Ab, Cry2Ae, and their combination in TwinL inkTM ) refer to Appendix C. 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 PI P registrations to meet the 40 CFR P art 158 data requirements for microbial pesticides . These requirements include testing on birds, mammals, nontarget insects, honey bee, plants, and aquatic species, and information has been submitted to address these requirements . Li mit dose testing on representative organisms from several taxa was performed in support of the T304 - 40 and GHB119 Section 3 FIFRA registrations . As stated above, BPPD’s risk assessments focus heavil y on beneficial nontarget invertebrates, since they are most closely related t o organisms susceptible to the insecticidal action of Bt toxins . The Cry 1Ab and Cry2Ae proteins are meant to target species within the order Lepidoptera (moths and butterflies). Bt toxins are known ty

pically to have a limited host range, however, to addr ess an y 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 non - target insect evaluations should include several invertebrate species that are not related to the target pests. Earthworm studies are also recommended . The toxicity of the Cry 1Ab and Cry2Ae has been evaluated on several species of invertebrates including the lady beetle, green lacewing, collembola, Daphnia , honey be e, and earthworm . Reproductive and/or developmental observations were also examined in the lady beetle, Daphnia , collembola, and honeybee studies. Since exposure may

also occur to other nontarget organisms, EPA has received data to compl y with the Agency ’s published non - target data requirements on other nontarget organisms . Tests to determine soil degradation of Cry 1Ab and Cry2Ae have also been submitted . The individual results for nontarget organism and soil degradation testing for Cry 1Ab and Cry2Ae , and where applicable, for the ir combination Cry 1Ab and Cry2Ae in TwinL inkTM , are summarized in Appendix C. F ull reviews of each study for each event can be found in the individual Data Evaluation Records. The October 2000 SAP recommended that while actual plant material is the preferred test material, bacteria - derived protein is also a valid test substance, particularl y in scenarios where test animals do not normally consume cotton plant tissue and where large amounts of Cry protein are needed for maximum h azard dose testing . In support of the T304 - 40 and GHB119 registrations, test substances used in the submitted studies included bacteria - produced purified Cry 1Ab and Cry2Ae protein, respectively . Comparative anal ys es have been performed to verify the equiva lence of the bacteriall y - produced and purified Cry 1Ab and Cry2Ae and the Cry 1Ab and Cry2Ae proteins produced in T304 - 40 or GHB119, respectivel y, and TwinLinkTM cotton plants. Product characterization data demonstrate the bioequivalence of the plant - expresse d and microbially expressed proteins (Waggoner 2011a, 2011b ) .Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 18 of 153 Consequently , on the basis of the environmental risk assessment and evidence of bioequivalence, EPA concludes that Cry 1Ab and Cry2Ae PIPs are not likely to pose a hazard to nontarget organisms. On the basis of the ecological effects assessment, EPA concludes that significant adverse effects are not expected on birds, mammals, nontarget insects, honey bees, freshwater and marine/estuarine fish and invertebrates, and terrestrial and aquatic plants as a result of the use of Event T304 - 40 and Event GHB119 in cotton . EPA has also determined that there is no significant risk of gene capture and expression of Cry 1 Ab or Cry2Ae protein by wild or weed y relatives of cotton within the U.S. or its territorie s as long as the current sales and distribution restrictions on Bt cotton are in place. Available data indicate that Cry

proteins do not have an y measurable adverse effect on microbial populations in the soil, nor has horizontal transfer of genes from tran sgenic plants to soil bacteria been demonstrated. **Endangered Species** Because of the selectivity of Cry 1Ab and Cry2Ae proteins for lepidopteran species, endangered species concerns are also expressed for insect species in the order Lepidoptera . The Agency has previousl y determined that an y potential concern regarding range overlap of endangered Lepidoptera with cotton production was mainl y restricted to the Kern primrose sphinx moth ( Euproserpinus euterpe ). However, the Agency

determined that cotton is not a host plant for this species nor do host - range considerations for this species or other endangered insects place habitat in or near cotton fields (US EPA 2008a) . As previously

noted, there is a possibility for gene transfer in locations where wild or feral cotton relatives exist . As a result, EPA requires stringent sales and distribution restrictions on Bt cotton within these areas to preclude outcrossing or h yb ridization from the crop to sexually compatible relatives . Therefore, EPA does not expect that any threatened or endangered species will be affected by outcrossing to wild relatives or b y competition with such entities. Cry 1Ab has not been shown to have toxic effects on mammals, birds, freshwater invertebrates, terrestrial nontarget inv ertebrates, and plants, and exposure is not anticipated in aquatic environments . EPA, therefore, makes a “No Effect” determination for direct and indirect effects to listed species of these taxa for Cry 1Ab expressed in Event T304 - 40 cotton . Since Cry2Ae has not been shown to have toxic effects on birds, mammals, freshwater invertebrates, terrestrial nontarget invertebrates, and plants, and also because exposure is not anticipated in aquatic environments, the Agency makes a “No Effect” determination for di rect and indirect effects to listed species of these taxa for Cry2Ae expressed in Event GHB119 cotton. 

### D. **Insect Resistance Management** The goal of insect resistance management (IRM) is to mitigate the potential risk of resistance in Bt crops, includin g Bt cotton . IRM t ypicall 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 (in the event of resistance) , grower compliance with IRM requirements, and grower education . The standardBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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for IRM has been the use of structured refuges, which are non - PIP portions of the crop (or other suitable plant hosts) that provide a source of susceptib le insects to mate with an y resistant insects emerging from the PIP field (thus diluting the frequency of resistance genes in the pest population) . Some Bt cotton products have been approved with “natural refuge” instead of structured refuges . Natural refuges include non - cotton plant hosts of the target pests such as weeds, wild hosts, or other cultivated crops (e.g., so ybean, peanut, tobacco). BCS submitted an IRM plan and data with their application for registration of TwinL inkTM cotton (MRID#s 476400 - 14 and 476348 - 23) . TwinLinkTM is a py ramided Bt cotton PIP expressing Cry2Ae and Cry 1Ab and is targeted against three major cotton pests: tobacco budworm (TBW, Heliothis virescens ), cotton bollworm (CBW, Helicoverpa zea ), and pink bollworm ( PBW, Pectinophora gossypiella ). BPPD reviewed this initial submission and identified a number of deficiencies (review dated April 22, 2010) . BCS responded to these deficiencies with several resubmissions (MRI D#s 484800 - 10, 484800 - 11, and 484951 - 01) that we re reviewed b y BPPD (reviews dated September 28, 2010 and June 28, 2010) . BPPD concluded that these resubmissions satisfied the outstanding deficiencies and that the IRM proposal for TwinL inkTM cotton was acceptable. BPPD’s major IRM conclusions from its a ssessment of TwinL inkTM cotton are detailed below . The IRM assessment included data developed for TwinLinkTM (Cry2Ae x Cry 1Ab) as well as its single toxin constituent PIPs, GHB119 (Cry2Ae) and T304 - 40 (Cry 1Ab) . **1. DOSE**  a. BPPD concludes that the diet incorpo rated and overlay assay s together (using verification method 1) suggest that TwinL inkTM (Cry2Ae x Cry 1Ab) and GHB119 (Cry2Ae) may express a high - dose against CBW but that T304 - 40 (Cry 1Ab) may express less than high - dose against the target pest. b. BPPD concludes that diet incorporated and overlay assay s (using verification method 1) together suggest that events GHB119 (Cry2Ae) and T304 - 40 (Cry 1Ab) may express a high - dose and TwinL inkTM (Cry2Ae x Cry 1Ab) may express an effective high - dose against TBW. c. BP PD concludes that the single toxin events GHB119 (Cry2Ae) and T304 - 40 (Cry 1Ab) both express less than a high dose against PBW, but that TwinL inkTM (Cry2Ae x Cry 1Ab) expresses a high dose against the insect. **2. CROSS RESISTANCE**  a. Based on submitted data, BPP D concludes that there is low cross resistance potential between the Cry2Ae and Cry 1Ab toxins that are expressed in TwinL inkTM . b. BPPD notes that there is a potential for cross resistance between Cry2Ae and Cry2A2 (registered in Bollgard II cotton) . Should resistance develop to either toxin, the second toxin could also be compromised. c. Similarly , there is cross resistance potential between Cry 1Ab (also registered in VipCot cotton) and Cry 1Ac (registered in Bollgard and WideStrike cotton) and Cry 1F (registere d in WideStrikeBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 20 of 153 cotton) . Competitive binding assay s have shown that these toxins share binding sites in insect midgets which can lead to cross resistance (though the potential is somewhat less for Cry 1F). **3. SIMULATION MODELING**  a. BCS conducted separate models to evaluate the durability of TwinLinkTM for CBW and TBW . Both models were intended to evaluate durability with “natural refuges” (i.e., non - cultivated plant hosts such as weeds or cultivated crops other than cotton that can serve as sources of susceptible insects). b. Modeling for CBW was conducted b y Dr. Michael Caprio (Mississippi State Universit y) . The model was deterministic with a probabilistic function in which parameters were established using PERT distributions (i.e. a distribution in which the mean value is weighted more heavil y than minimum or maximum values) . BPPD had a number of concerns regarding some of the modeling assumptions including dose mortality , initial resistance allele frequency , and the lack of inclusion of Cry 1Ab corn in the model landscape . BCS responded to these deficiencies with revised modeling that employ ed more conservative parameter values . The revised model was run for 1,000 simulations; resistance did not evolve to either Cry 1Ab or Cry2Ae (within the 30 y ear time horizon of the model) in any of the simulations.

c. BCS conducted two modeling anal ys es for TBW: i) a stochastic, spatiall y - explicit model, and ii) a deterministic, spatially - implicit model . As with CBW, the TBW models were designed and conducted b y Dr. Michael Caprio . The modeling generall y took a conservative approach b y including “worst case” simulations for certain parameters (dose mortality and dominance) and incorporated other Cry 1A cotton products in the landscape . Results from the stochastic model showed that r esistance did not evolve during the 31 y ear model time frame . However, when dominance values (for resistance alleles) and late season survival were increased, the time to resistance decreased . With the deterministic model, the time to resistance was 224 y ears (both toxins) under the benchmark parameters . Deterministic simulations with worst case assumptions included resulted in a durability estimate of 80 ye ars (a 2.8 fold difference from the benchmark simulations) . A separate deterministic simulation was also run with a single trait Bt cotton product (Cry 1Ac) with a 20% structured refuge that resulted in an estimated durability of 18.8 ye ars. d. Based on the modeling results, BPPD concluded that TwinL inkTM cotton deploy ed with a natural refuge should not be e xpected to pose a greater risk of resistance evolution than other Bt cotton products (e.g., Bollgard II) currentl y registered with a natural refuge strategy. **4. REFUGE STRATEGY**  a. BCS provided adequate information to demonstrate that the use of a natural refuge with TwinL inkTM cotton should provide high durability for both toxins (Cry 1Ab and Cry2Ae) . The risk of resistance for TwinL inkTM is not expected to be greater than other registered Bt cotton products (Bollgard II, WideStrike, VipCot) that employ a natural refuge strategy . b. The use of natural refuge has been assessed onl y for southeastern cotton growing regions . Western cotton regions (including west Texas, New Mexico, Arizona, and California) shouldBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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structured refuges (as have been required for other Bt cotton products) . The structured refuge options are : i) 5% external, unspray ed non - Bt cotton refuge, ii) 20% external, spray ed non - Bt cotton refuge, iii) embedded refuge

– either 5% or 1 row of non - Bt cotton per 6 - 10 rows of Bt cott on. c. Registrations have been proposed for events GHB119 (Cry2Ae) and T304 - 40 (Cry 1Ab), both of which are single trait products . These products are “breeding” registrations (for the production of TwinL inkTM ) and are not intended to be commercially sold . BCS has requested that no refuge be required for these products . Breeding registrations ty picall y do not have refuge requirements, however, BPPD recommends the use of acreage/geographic limitations for the single trait products as a means to mitigate potential resistance development. **5. RESISTANCE MONITORING AND REMEDIAL ACTION**  a. BCS has indicated that they are developing baseline susceptibility data for TBW, CBW, and PBW to Cry 1Ab and Cry2Ae . They plan to have the studies completed in 2012 (i.e., after product registration) . Given the importance of baseline data to the resistance monitoring program, BPPD recommends that these data be generated b y the first growing season of commercial use of TwinLinkTM . b. BCS has proposed to monitor for resistance with all three target pests in areas of high TwinL inkTM adoption . BPPD agrees with this proposal but also recommends that monitoring efforts also focus on regions in states with little natural refuge (e.g. Mississippi). c. For PBW, BCS plans to use the remedial action plan developed b y the Arizona Bt Cotton Working Group (which has been emplo yed for other registered Bt cotton products) . For TBW and CBW, the remedial action plan will be refined once baseline susceptibility data are available (i.e., to define “suspected” and “confirmed” resistance) . The base elements of the plan proposed by BCS are similar to other Bt cotton products; however, BPPD recommends that the plan be adopted to include a provision that EPA will be info rmed within 30 day s of a confirmed resistance event.” Additionally , a stipulation should be added that in the event of resistance, BCS will develop a long - term resistance management action plan according to the characteristics of the resistance event(s) and local agronomic practices. d. BPPD recommends that BCS submit final versions of the resistance monitoring and remedial action plans for all pests as a term or condition of registration. **6 .**  **COMPLIANCE** a. A natural refuge strategy for TBW and CBW would eliminate the need for growers to plant a refuge where alternate hosts are abundant. However, there are cotton - growing regions where natural refuge is not applicable and where planting a refuge is still required (i.e., western states) . For those regions it is recommended that BCS develop, implement, and report to EPA on programs to evaluate and promote growers’ compliance with IRM requirements. b. Grower compliance in regions where structured refuge is required should be assessed with on farm assessments i n addition to anony mous phone (or internet) survey s. BPPD recommends thatBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 22 of 153 BCS use the existing grower compliance paradigm that has been established for other Bt cotton registrations. **7 .**  **GROWER EDUCATION** a. It is recommended that BCS develop a grower education program that targets states and counties where a non -Bt corn variety must be planted as the refuge for TwinL inkTM cotton (i.e., structured refuge). b. It is also recommended that BCS develop a grower guide that at a minimum describes the technolog y (including the concept of natural refuge) and includes unexpected damage guidelines . **8. ANNUAL REPORTING**  BPPD recommends that BCS submit annual reports for the following:

i) Sales data; ii) Annual resistance monitoring results; and iii) Compliance results for cotton growing regions where Natural Refuge is not applicable. Overall, BPPD concluded that BCS has submitted adequate resistance management data and information to support commercial use of the product with a natural refuge . The risk of resistance for Tw inLinkTM should be no greater than for other currentl y registered Bt cotton p yramids (including Bollgard II, WideStrike, and VipCot) . There are no data gaps, though it is recommended that BCS submit additional information on resistance monitoring (baseline data and a final monitoring plan), remedial action, compliance, and grower education (i.e., grower guides) as described above . A more detailed discussion of IRM including full summaries of BPPD’s reviews of the supporting data for is Cry 1Ab, Cry2Ae and TwinL inkTM is available as Appendix D of this BRAD. 

### I **V .**  **ENVIRONMENTAL 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 locati on, cultural practices, or other factors, may have at ypical, unusually high exposure to these PIP proteins Cry 1Ab, Cry2Ae and their combination in TwinL inkTM compared to the general population . For additional information regarding environmental justice is sues, please visit EPA’s web site at http://www.epa.gov/compliance/environmentaljustice/index.html .Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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### 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 r equirements 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 generall y 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 PI Ps containing Bacillus thuringiensis Cry 1Ab, Cry2Ae and their combination, Cry 1Ab, Cry2Ae, in TwinL inkTM . Th ese PIPs are not expected to cause unreasonable adverse effects and are likely to provide protection as claimed when used according to label instructions. The Agency

believes, therefore , that Bacillus thuringiensis Cry 1Ab, Cry2Ae and their combination, Cry 1Ab, Cry2Ae, in TwinL inkTM are eligible for registration for the proposed uses. 

### VI. **TERMS AND CONDITIONS OF THE REGISTRATION(S)** As terms of registration, the applicant must submit or provide the following: A. Analy tical method: OCSPP 860.1340 (40 CFR § 158.2120) The following v alidation studies must be conducted by an independent third party laboratory , as a term of the proposed registrations: 1. Term of Registration for Cry1Ab - event T304 - 40 (EPA File Symbol 264 - RNOU) Verification of the specific ELISA test kit to be used as the proposed analytical method for the detection of Cry 1Ab protein residues expressed in event T304 - 40 cotton is needed. In addition, a validation study

conducted by

an independent third party laboratory

is also needed to evaluate the performance of the designated analytical method for the detection of Cry1Ab protein residues expressed in cotton event T304 - 40. This study shou ld use the prescribed methodology and the reagents necessary for validating the analytical method used in the test kit and should be conducted according to the study parameters in the EPA residue chemistry testing guideline OCSPP 860.1340 Residue Analy

tica l Method and Pesticide Registration (PR) Notice 96 - 1: Tolerance Enforcement Methods - Independent Laboratory Validation by Petitioner (February 7, 1996). (Optional: Consultation with the Agency is highly recommended) 2. Term of Registration for Cry2Ae - event GHB119 (EPA File Symbol. 264 - RNOL) A validation study conducted by an independent third party laboratory

is still needed to evaluate the performance of the ELISA test kit as the designated analytical method for the detection of Cry2Ae protein residues expressed in event GHB119 cotton. This study should use the prescribed methodology

and the reagents necessary for validating the analytical method used in the test kit and should be conducted according to the study parameters in the EPA residue chemistry tes ting guideline OCSPP 860.1340 Residue Analytical Method and Pesticide Registration (PR) Notice 96 - 1: Tolerance Enforcement Methods - Independent Laboratory Validation by Petitioner (February 7, 1996). (Optional: Consultation with the Agency is highly recommended)Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document - DRAFT December 12, 2011

Page 24 of 153 3. Term of Registration for TwinL inkTM (T304 - 40 x GHB119) combination PIP product (EPA File Sy mbol 264 - RNOA). Any

outstanding data submitted to satisfy the terms of registration for PIP cotton events T304 - 40 and GHB119 (EPA File S ymbol 264 - RNOU and 26 4 - RNOL, respectivel y) must be submitted and found acceptable in order to complete the database for TwinL inkTM cotton, as the associated combination PIP product developed by conventional breeding of the parental events T304 - 40 x GHB119 cotton B. Insect Resistance Management 1. BCS must submit a detailed resistance monitoring plan for the major pests of TwinL inkTM cotton: tobacco budworm, cotton bollworm, and pink bollworm . Inaddition, baseline susceptibility

and diagnostic concentration determinations for tobacco budworm, cotton bollworm, and pink bollworm to Cry2Ae and Cry1Ab must be submitted. 2. B CS must submit a final remedial action plan for tobacco budworm and cotton bollworm . The remedial action plan must include definitions of “suspected” and “confir med” resistance and steps to take in the event of confirmed resistance. 3. B CS must submit a compliance assurance program (CAP) for TwinLinkTM that must include a “phased compliance approach” that outlines instances of non - compliance to the IRM requirements and options of responding to non - compliant growers . The CAP must be developed for regions that have structured refuge requirements (i.e., we stern cotton - growing regions); a CAP is not necessary

for regions with natural refuge requirements. 4. Persons purchasing TwinLinkTM cotton must sign a grower agreement . The term “grower agreement” refers to an y grower purchase contract, license agreement, or similar legal document and must clearl y set forth the terms of the current IRM program . By signing the grower agreement, a grower must be contractually bound to comply

with the requirements of the IRM program . In addition, BCS must implement a sy stem whic h is reasonabl y likely to assure that persons purchasing TwinLinkTM will affirm annually that they are contractually

bound to compl y with the requirements of the IRM program . BCS must submit a copy of the grower agreement/stewardship documents and a written description of a s ys tem assuring that growers will sign the grower agreement. C. Adverse Effects Reports of all incidents of adverse effects to the environment must be submitted to the Agency under the provisions stated in FIFRA Section 6(a)(2). D. Hy persens itivity

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). E. Annual Reporting As discussed under IRM in Chapter IV of this BRAD, BPPD recommends that BCS submit annual reports for the following:Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document - DRAFT December 12, 2011

Page 25 of 153 a. Sales data; b. Annual resistance monitoring results; and c. Compliance results for cotton growing regions where Natural Refuge is not applicable

.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document - DRAFT December 12, 2011

Page 26 of 153 

### APPEND **IX A: Product characterization and hum**  **an health effects –**  **Bacillus thuringiensis**  **Cry 1Ab (Event T304**  **- 40) in cotton plant incorporated protectant.**  **A.1. BACKGROUND** B CS has developed a cotton line from event T304 - 40 ( EPA File S ymbol 264 - RNOU and OECD Unique Id entifier: BCS - GHØØ4 - 7 ) that express a Cry 1Ab plant - incorporated protectant (PIP) protein derived from Bacillus thuringiensis (Bt) subsp ecies berliner , which is intended to control lepidopteran larvae such as bollworm (CBW, Helicoverpa zea), tobacco budworm (TBW, Heliothis viren scens) larvae, and fall arm yworm (FAW, Spodoptera frugiperda). B CS previously submitted an EUP application to the Agency for conducting experimental field trials on event T304 - 40 cotton, which was granted on February 7, 2006 and extended on March 8, 2007 . The product characterization and human health data submitted for event T304 - 40 PIP cotton were reviewed and found acceptable for the EUP applications (US EPA, 2006). B CS later submitted a FIFRA section 3 r egistration application containing new product characterization data and updated toxicological data for Cry 1Ab. These data were reviewed b y the Agency and several product characterization studies were found supplemental . BCS submitted additional data to address the data deficiencies identified in those studies . Summaries of the resubmission data for product characterization , as well as the remaining toxicological data submitted to register event T304 - 40 , are provided in this Appendix . A number of studies were reviewed under a work - share program, under an EPA - BPPD and USDA - B RS Memorandum of Understanding (09 - 2000 - 0052 - MU) . For more detailed information, please refer to the individually reviewed studies, summarized in individual Data Evaluation Reports (DERs) , by the assigned MRID number s (US EPA, 2011) . Event T304 - 40 cotton expresses Cry 1Ab as an active ingredient and phosphinothricin acet yl transferase (PAT) enzy

me as an inert ingredient . The status of the existing tolerance exemptions for Cry 1Ab (40 CFR 174.511) and PAT ( 40 CFR 174.522) has been pre viously discussed in Chapter IIA.2.b. of this BRAD . Residues of Bt Cry 1Ab protein are exempt from the requirement of a tolerance when used as plant - incorporated protectants in all food commodities and the PAT enzy me is ex empt from the requirement of a tole rance when used as plant - incorporated protectant inert ingredient in all food commodities (40 CFR 174.522). The data and information discussed below support these existing tolerance exemptions. **A.2. PRODUCT CHARACTERIZATION**  **[ 40 CFR § 158.2120**  **(c) ]**  **1) Event T304**  **- 40**  **Cotton e**  **xpressing Cry1Ab protein**  **a .**  **Transformation System** E vent T304 - 40 was created through Agrobacterium - mediated transformation of cotton variet y Coker 315 with plasmid pTDL 008. The cry1Ab gene sequence was modified for expression in plants and consists of one nearly

complete copy

of the T - DNA flanked b y an inverted incomplete cop y of the cry1Ab gene cassette and one additional 3’me1 terminator. The Cry 1Ab protein encoded b y the gene insert ed into cotton is expected to have 617 amino acids and a molecular weight of approximately 69Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document - DRAFT December 12, 2011

Page 27 of 153 kDa. In addition, the deduced amino acid sequence of BCS’s Cry 1Ab protein is identical to the amino acid sequence of the Cry1Ab protein in a previously registered PI P (event Bt11 corn, OPP chemical code 006444) with the exception of one amino acid at the C - terminus and two amino acids at the N terminus ( MRID No. 467474406; US EPA 2006). Plasmid pTDL 008 also contains the bar gene as a selectable marker, which is de rived from the soil microorganism, Streptomyces hygroscopicus (described b y Thomson et al. 1987). The bar gene encodes the enzy me phosphinothricin acet yl transferase (PAT), which is co - expressed with Cry 1Ab protein in event T304 - 40 cotton as a PIP inert ingredient. The PAT enzy me provides the plant with tolerance to herbicide applications containing glufosinate - ammonium by acet yl ating the herbicide glufosinate - ammonium and making it unable to bind glutamine s ynthase. The PAT enzy me encoded b y the bar gene is a homodimer of 183 amino acids and has an expected molecular weight of approximately 22 kDa. **b .**  **Characterization of the DNA Inserted in the Plant and Inheritance and Stability** Characterization of the DNA isolated from event T304 - 40 cotton using restriction enzy me digests and Southern blot analy ses as well as DNA sequencing indicates that the transgenic insert was inserted in the cotton genome, consisting of one nearl y complete copy of the T - DNA flanked by an inverted incomplete copy of the cry1Ab gene and one additional 3’me1 terminator . No vector backbone sequences from the transformation plasmid pTDL 0008 were found in T304 - 40 . Inheritance and stability studies of the cry1Ab gene in event T304 - 40 verified that it is stably integrate d into the cotton genome, segregating in an expected Mendelian fashion. **c .**  **Protein Characterization** Event T304 - 40 cotton expresses Cry 1Ab protein produced b y Bacillus thuringiensis subsp. berliner . The Cry 1Ab protein after cleavage of the transit peptide has 617 amino acids. Sodium dodecyl sulfate poly acrylamide gel electrophoresis (SDS - PAGE) analy ses of Cry 1Ab protein , as expressed in event T304 - 40 showed a molecular weight close to the deduced MW for Cry 1Ab and Western blot anal ys es showed a sing le immunoreactive band. Cry 1Ab was also found to be non - gly cos yl ated . N - terminal sequencing also confirmed that the transit peptide of the plant form was cleaved at the expected residue. The biological activity

of Cry 1Ab protein was assessed by an insect f

eeding bioassay

using Heliothis virescens first instar larvae and the LC 50 for the plant - produced Cry 1Ab protein was 74 ng/cm2 . **d .**  **Protein Expression** Expression level data were provided for Cry 1Ab in different plant tissues and at different growth stag es in event T304 - 40 cotton and a summary of the results are provided in Table A. 1 below. The data were produced using an ELISA method .

**Table A.**  **1. Mean Cry1Ab Expression levels in Event T304 -**  **40 Cotton Tissue Type**  **A.1. Cry1Ab Protein**  **(μg/g dry weight ± SD)** * Roots 3.8 ± 0.9

– 8.6 ± 2.6 Stems 0.88 ± 0.37

– 6.2 ± 1.1 Leaves 0.45 ± 0.08

– 4.6 ± 0.6 Squares 2.90 ± 1.0 Apex 1.3 ± 0.3 Bolls 0.38 ± 0.2Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 28 of 153 **Tissue Type A.1.**  **Cry1Ab Protein (μg/g dry weight ±**  **SD)*** Whole plants 2.2 ± 1.1 Pollen NA a Nectar NA a Flowers 10.6 ± 1.9 Cottonseed 4.10 ± 1.6 * Range reflects means at different growth stages **a** Not applicable due to the insufficient availability of material. **e .**  **Analytical Method** BCS CropScience has provided validation studies for commercial enzy me - linked immunosorbent assay (ELI SA) test kits for the anal yt ical detection of Cry 1Ab protein residues in cottonseed and cotton leaf tissue . Results showed the test kits are able to detect Cry 1Ab protein residues in cotton with sufficient accuracy , precision, and sensitivity . However, verification of the specific ELISA test kit method to be used as the proposed residue anal yt ical method for the detection of Cry 1Ab protein in event T304 - 40 cotto n is needed. In addition, a validation study conducted by

an independent 3 rd party

laboratory

is needed to evaluate the performance of the designated residue anal yt ical method for the detection of Cry 1Ab protein expressed in cotton event T304 - 40. Table A.2 summarizes the product characterization EPA evaluated and found acceptable for the FIFRA section 3 registration of Cry 1Ab.

**Table A.**  **2 .**  **Product Characterization Data for Event T304 -**  **40 cotton expressing**  **Bt Cry1Ab protein**  **( EPA File Symbol**  **264 -**  **RNOU )**  **Study Title A.2.**  **Product Characterization -**  **Bt Cry1Ab**  **Summary MRID No**  **Characteristics of Cry1Ab cotton**  **plants derived from transform**  **ation events number**  **T303 -**  **3 and T304 -**  **40 2**  This submission replaces MRID 46474401 and includes descriptions of the

genetic constructs used in events T303 - 3 and T304 - 40, the sources of the introduced genes, the modes of action of the expressed proteins, and preliminary mo lecular analysis, including the protocol used and Southern blot data. The mo lecular analysis indicated that in each event, one copy of the cry1Ab gene was inserted into cotton ( Gossypium hirsutum ). **CLASSIFICATION: SUPPLEMENTAL .**  Data gaps addressed in MRID No. 48480003. **46708801 [This**  **study s**  **upersedes MRID No.**  **46474401] Characteristics of**  **Cry1Ab cotton plants**  **derived from transform**  **ation events number**  **T303 -**  **3 and T304 -**  **40 1**  This submission includes descriptions of the genetic constructs used in events

T303 - 3 and T304 - 40, the sources of the introduced genes, the modes of action of the expressed proteins, and preliminary molecular analysis. The study authors state that the molecular analysis indicated that in each event, one copy of the cry1Ab gene was inserted into cotton ( Gossypium hirsutum ). **CLASSIFICATION: SUPPLEMENTAL**

– Data gaps addressed in MRID No. 48480003 . **46474401**

**PAT and Cry1Ab protein in cotton**  **tissues of T303 -**  **03 and T304**  **- 40**  **events, preliminary** PAT and Cry 1 Ab protein in cotton tissues of T303 - 03 and T304 - 40 events,

preliminary report

—BCS conducted preliminary protein expression analysis in support of the EUP request for Cry 1 Ab cotton events T303 - .3 and T304 - 40. Average Cry 1 Ab and PAT concentrations, as determined by ELISA in ground cotton seed from events T303 - 3 and T304 - 40, w ere 16  g/g and 2.2  g/g fresh **46708804 [This**  **study supersedes**  **2** Study submitted with EUP application and reviewed in memorandum fr om R. Edelstein through J. Kough to S. Matten, dated January 27, 2006.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document - DRAFT December 12, 2011

Page 29 of 153 **Study Title A.2.**  **Product Characterization -**  **Bt Cry1Ab**  **Summary MRID No**  **report 1**  weight, respectively, for Cry 1 Ab and 117 and 129  g , respectively, for PAT.

The dry weight equivalents in seeds are: 2 and 16  g of Cry 1 Ab per 0.91g dry seed (or 0.0002 and 0.0017% dry weight) and 117 and 129  g of PAT per 0.91 g dry seed (or 0.013 and 0.014% dry weight) . Cry 1A b concentrations in early flowering cotton leaf tissues grow n in a greenhouse averaged 1.9  g/g fresh weight for T303 - 3 and 0.3  g/g for T304 - 40. In leaf tissue collected six weeks after flowering, the Cry 1 Ab concentrations were 2.1  g/g fresh weight for event T303 - 3 and 0.16  g/g for event T304 - 40. The dry weight content of Cry 1 Ab in leaves was estimated to be: 0.2 to 2.3  g of Cry 1 Ab per 0.12 g dry leaf. From event T303 - 3 and T304 - 40 cotton grown in the field, Cry 1 Ab w as detected in the leaves, squares, flower and boils at concentrations ranging from 0.01 to 0.09 % total soluble protein. The PAT content of cotton tissues (other than seed) w as not presented in the study report. **CLASSIFICATION: SUPPLE ME**  **NTAL** - No additional data needed. **MRID No. 46474404]** 

**Product Characterization of**  **Cry1Ab Cotton Event T304**  **- 40**  **3** The purpose of this study was to determine if the transfer DNA (T - DNA)

containing the intended cry1Ab gene from plasmid pTDL008 w as inserted into the genomic DNA of cotton event T304 - 40, and the backbone DNA from pTDL008 w as not inserted into cotton event T304 - 40 . Southern blot analyses showed that the inserted transgenic sequence in T304 - 40 consisted of one ne arly complete copy of cry1Ab f lanked by an inverted, incomplete copy of the cry1Ab gene cassette, and one additional 3' me1 terminator. Southern blot analysis for the presence of vector backbone in T304 - 40 w as negative using 4 of the 7 backbone probes. H ow ever, there were several data missing from this report and several regions of the vector backbone sequence were not confirmed as absent after the transformation process to create T304 - 40 cotton . Additional analyses such as demonstrating the cry1Ab gene w as stably integrated and segregated according to Mendelian la ws of inheritance over several successive generations of event T304 - 40 cotton we re not addressed in this study. **CLASSIFICATION: SUPPLEMENTAL -**  Data gaps addressed in MRID No. 48480005. **476348 01** 

**Structural and Functional**  **Equivalence of Cry1Ab and**  **PAT/bar Proteins Produced in**  **Escherichia coli to**  **Cry1Ab and PAT/bar Proteins**  **from Event T304**  **- 40 and TwinLinkTM**  **Cotton, Gossypium**  **hirsutum 2**  The purpose of this study was to demonstrate the biochemical and functional

equivalency of the Cry1Ab and PAT/ bar proteins from cotton event T304 - 40 with the Cry1Ab and PAT/ bar proteins expressed in Escherichia coli and TwinLinkTM cotton, based on molecular weight and immunoreactivity. SDS PAGE analyses of Cry1Ab pr otein isolated from event T304 - 40 cotton leaves showed a very faint band estimated to be at a similar molecular weight as the band exhibited for the E. coli - derived Cry1Ab protein standard (~66 kDa). Western blot analyses showed immunologically reactive band s at the same position ( ~66kDa) for Cry1Ab protein expressed in cotton event T304 - 40 and TwinLinkTM to the E. coli - derived Cry1Ab protein. How ever, it is unclear in the study report if the biochemical data presented in the study were derived from the same source of the bacterial - expressed Cry1Ab protein test substance . SDS PAGE analyses showed identical electrophoretic mobilities and similar mo lecular weights (~20 kDa) for the PAT/bar protein isolated from event T304 - 40 cotton leaves, from the PAT/bar E. coli standard and the TwinLinkTM cotton . Western blot analyses showed immunologically reactive band s at the same position ( ~20 kDa) for all three PAT protein sources . Several single - dose sensitive insect bioassays were conducted to confirm bioactivity, but these data **476348 05** 

**3** Study submitted with Sec.3 application and reviewed in memorandum from A. Waggoner through J. Kough to S. Bacchus and D. Greenway, dated February 4, 2011.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 30 of 153 **Study Title A.2.**  **Product Characterization -**  **Bt Cry1Ab**  **Summary MRID No** 

did not provide the quantitative results to allow an adequate comparison of the insecticidal activity for each source of protein. In general, the study report lacked sufficient details on the materials/ methods used, the experimental design, and reported results to properly document the bioequivalence of the bacterial - derived Cry1Ab protein test substance to the T304 - 40 cotton plant - expressed Cry1Ab protein. Therefore, the biochemical and functional equivalence of the plant - and bacterial - expres sed protein sources cannot be confirmed from these data . **CLASSIFICATION: SUPPLEMENTAL -**  Data gaps addressed in MRID No. 48480004. **Analyses of Raw Agricultural**  **Comm odity (Fuzzy**  **Seed) of Cry1Ab Cotton Event T304**  **- 40 for PAT/** **bar and**  **Cry1Ab and its**  **Non -**  **transgenic Counterpart for**  **PAT/ bar**  **and Cry1Ab Proteins**  **2** The purpose of this study was to quantify the expression of Cry1Ab and PAT

proteins in the fuzzy seeds of transgenic cotton event T304 - 40 and in a non transgenic cotton line, Coker 312. Fuzzy seed w as grinded into fractions of lint coats and kernels and analyzed separately for Cry1Ab and PAT proteins and total extractable protein. To quantitate the amount of the protein analytes in the fuzzy seeds, ELISA results of separate assays were c om bined for the kernel and lint coat fractions. The average Cry1Ab protein content of transgenic fuzzy seed from all test sites, without and with herbicide spraying, was 0.955 ±0.317 μg/g and 1.03 ± 0.378 μg/g, respectively. As a percent of total crude protein, Cry1Ab protein comprised an average of 0.0 00434 ± 0.000167% and 0.000465 ± 0.000191% of the total crude protein in unsprayed and sprayed fuzzy seed, respectively. The average PAT protein content of transgenic fuzzy seed from all test sites, without and with herbicide spraying, was 95.4 ± 17.0 μg/g and 100 ± 16.8 μg/g, respectively. As a percent of total crude protein, PAT protein comprised an average of 0.0428 ± 0.00810% and 0.0445 ± 0.00804% of the total crude protein in unsprayed and sprayed fuzzy seed, respectively. Results showed Cry1Ab and P AT analyte proteins were expressed in the kernels, lint coats and, by inference, in the fuzzy seeds de rived from the transgenic T304 - 40 cotton plants . **CLASSIFICATION: ACCEPTABLE 47634804** 

**Protein Expression Analysis of Cotton**  **Event T304 -**  **40, Expressing C**  **ry1Ab and PAT/bar**  **Proteins, USA, 2007**  The purpose of this study was to quantify the expression of Cry1Ab and PAT

proteins in transgenic cotton event T304 - 40, and in a non - transgenic cotton line Coker 315. Extracts of plant matrices were prepared from roots, stems, leaves, squares, apex, bolls, whole plant, pollen, nectar, flow ers and grain. An enzyme linked immunosorbent assay (ELISA) was used for determining the protein levels. On a dry weight basis, the overall averages for the Cry1Ab protein content were 8.6 and 3.8 μg/g for roots at the two growth stages; 6.2 and 0.88 μg/g for stems at the two growth stages; 4.6, 1.4 and 0.45 μg/g for leaves at the three growth stages; 2.9 μg/g for squares; 1.3 μg/g for apex; 0.38 μg/g for bolls; 2.2 μg/g for whole plant; 0.05 μg/g for pollen (fresh weight data only due to insufficient availability of material); 0.002 μg/g for nectar (fresh weight data only due to insufficient availability of material); 10.60 μg/g for flowers and 4.10 μg/g for grain. On a dry weight basis, the overall averages for the PA T protein content were reported to be 115 and 95.1 μg/g for roots at tw o growth stages ( ca . 4 w eeks and 10 w eeks, respectively); 100 and 44.5 (μg/g for stems at two grow th stages ( ca . 4 weeks and 10 w eeks, respectively); 193, 134 and 57.9 μg/g for leaves at 3 growth **47634803**Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 31 of 153 **Study Title A.2.**  **Product Characterization -**  **Bt Cry1Ab**  **Summary MRID No** 

stages ( ca . 4 w eeks, 7 weeks, and 10 w eeks, respectively); 180 μg/g for squares ( ca . 7 w eeks); 88.9 μg/g for apex ( ca . 10 w eeks); 39.3 μg/g for bolls ( ca . 8 weeks); 134 μg/g for whole plant ( ca . 10 w eeks); 0.13 μg/g for pollen fresh wei ght data only due to insufficient availability of material) ( ca . 7 w eeks); 0.005 μg/g for nectar fresh weight data only due to insufficient availability of material) ( ca . 7 w eeks); 187 μg/g for flowers ( ca . 10 w eeks); and 39.6 μg/g for grain ( ca . 22 w eeks) . The recoveries of both proteins from the spiked sample matrices were generally good (means of 59.9 - 121%) thus validating the use of the ELISA used in this study as a suitable test kit for the detection of both Cry1Ab and PAT proteins as expressed in ev ent T304 - 40 cotton. **CLASSIFICATION: ACCEPTABLE Detailed insert**  **characterization of Gossypium**  **hirsutum transform**  **ation event T304**  **- 40**  Southern blot analyses were conducted to determine the organization of the

cry1Ab and bar gene cassette in transformation event T304 - 40 cotton. Genomic DNA extracted from leaves of event T304 - 40 cotton w ere digested with ten different restriction enzymes and the resulting DNA fragments were separated by agarose gel electrophoresis, transferred to a membrane and sequentially hybridized with six different probes (5’e1 - Ps7s7 promoter, cry1Ab gene, 3’me1 terminator, P35S3 promoter, bar gene and 3’nos terminator), each representing a fragment of the transforming gene cassette, and with the complete T - DNA probe. The Southern blot data for the expected and observed hybridization fragments, as well as the hybridization strategy demonstrated that the inserted transgenic sequence in the cotton event T304 - 40 consists of one nearly complete copy of the T - D NA flanked by an inverted incomplete copy of the cry1Ab gene cassette and one additional 3’me1 terminator. **CLASSIFICATION: ACCEPTABLE 484800**  **03 [This**  **study supersedes**  **MRID No. 47634801]** 

**Confirm ation of**  **the absence and presence of**  **vector backbone sequences in**  **Gossypium hirsutum**  **transform ation**  **event T304 -**  **40** The purpose of this study was to determine the presence of any vector backbone

sequences in event T304 - 40 cotton after insertion of the intended cry1Ab gene cassette via the pTDL008 transformation vector to produce event T304 - 40 cotton . Isolated genomic DNA samples from leaves of cotton event T304 - 40 and from the non - transgenic negative control were subjected to Southern blot analysis, after digestion with restriction enzymes Eco RV an d NotI, using seven overlapping vector backbone probes that encompassed the entire backbone of the pTDL008 transformation vector used to produce this transformation event. The vector backbone probes w ere then stripped from the membranes so that they could be re - hybridized with the T - DNA probe, as a positive control. Genomic DNA from the event T304 - 40 cotton showed no hybridization with any of the vector backbone probes , while the positive control showed hybridization as expected . The results confirmed the absence of vector backbone sequences after insertion of the intended cry1Ab gene cassette via the pTDL008 transformation vector to produce event T304 - 40 cotton. **CLASSIFICATION: ACCEPTABLE 484800**  **04 [This**  **study supersedes**  **MRID No. 47634801]** 

**Characterization of Cry1Ab and**  **PAT/ bar**  **proteins produced in Event**  **T304 -**  **40 cotton, Gossypium**  **hirsutum** The purpose of this study was to determine the biochemical and functional

equivalence of plant - expressed Cry1Ab protein produced in event T304 - 40 cotton pl ants with bacterial - expressed Cry1Ab (Test substances ID No. MIN1443 and AW200308) produced from recombinant Escherichia Coli ove r expression system, for its use as a suitable test substance surrogate in toxicological testing . Equivalence studies were al so conducted for plant expressed PAT protein in event T304 - 40 plants with bacterial - expressed PAT protein (Test substance ID No. NB010905P44P2) for use as a test substance surrogate . Six analytical tests were used to assess the equivalence, which include d: 1) estimation of molecular weight by observing protein mobility in **48480005** [ **This study**  **supersedes MRIDs**  **No. 47634805**  **and**Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 32 of 153 **Study Title A.2.**  **Product Characterization -**  **Bt Cry1Ab**  **Summary MRID No** 

SDS - PAGE, 2) verification of immunoreactivity using western blot analysis, 3) analysis of biological activity using enzymatic activity for the PAT/bar protein and insecticidal activity f or the Cry1Ab protein, 4) analysis of glycosylation using the reaction with glycostain, 5) mass spectroscopic peptide mapping using HPLC/Electrospray Mass Spectrometry (LC/MS) of peptides, and 6) analysis of N - terminal sequencing using Edman degradation. T he molecular weights, immunoreactivity and biological activity were shown to be comparable for each respective form of bacterial - and plant - expressed Cry1Ab and PAT proteins. Glycosylation was not detected in either protein from either species. Except for a few amino acids at the N - terminal end, the PAT/ bar protein was shown to be identical in both species in the LC/MS analysis. A high degree of similarity was also shown between the Cry1Ab proteins of both species, with sequences betw een residues 2 and 617 being identical. Slight differences are present in both the N - and C - terminal peptides. Sequencing of the N - terminal end of the Cry1Ab protein using Edman degradation in cotton was unsuccessful, yet consistent with the findings in mass spectroscopic peptid e mapping using LC/MS. The comparison between species in the LC/MS assay on the PAT/ bar protein showed comparable results and confirmed the biochemical and functional equivalence . Thus, both the bacterial - expressed Cry1Ab (Test substances ID No. MIN1443 an d AW200308) and PAT (Test substance ID No. NB010905P44P2) proteins wo uld be suitable test substance surrogates for use in toxicological testing to establish the safety of the plant - expressed Cry1Ab and PAT proteins as expressed in event T304 - 40 cotton. **CLASSIFICATION: ACCEPTABLE 47640006]** 

**Protein Detection Me**  **thods. Enzyme Linked Immuno**  **Sorbent Assay (ELISA)**  Non - quantitative detection of Cry1Ab in extracts of plant material from event

T304 - 40 cotton can accomplished using the EnviroLogix QuantiPlate Kit or the GMO √ Bt Maize Test Kit, each of which uses enzyme

- linked immunosorbent assay (ELISA) to detect the presence of Cry1Ab. **CLASSIFICATION: SUPPLEMENTAL** pending verification of the specif ic ELISA test kit method to be used as the proposed residue analytical method for the detection of Cry1Ab protein in event T304 - 40 cotton in addition to an independent laboratory validation study to evaluate the designated test kit’s performance. **47634826**

**A. III**  **. ACUTE TOXICOLOGY**  **Cry1Ab** EPA evaluated human health toxicology data for Cry 1Ab in event T304 - 40 as summarized below in Table A. 3 . **Table A.**  **3 . Hum**  **an Health Data for Event T304 -**  **40 cotton expressing**  **Bt Cry1Ab protein**  **( EPA File Symbol**  **264 -**  **RNOU )**  **Study Title**  **A.3. Hum**  **an health -**  **Summary –**  **Bt Cry1Ab**  **MRID No Cry1Ab**  **protein -**  **Overall amino acid sequence hom**  **ology** The amino acid sequence of the Cry1Ab protein encoded by the plant - optimized Cry1Ab gene from Bt subspecies berliner 1715 w as com pared with all protein sequences present in the following databases: SwissProt, trEMEL, GeneSeq **46708802 [This study** Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 33 of 153 **Study Title**  **A.3. Hum**  **an health -**  **Summary –**  **Bt Cry1Ab**  **MRID No search**  **with known toxins and allergens**  **4** Prot, PIR, PDB, DAD, and GenPept. No similarities betw een the Cry1Ab protein and known allergens or toxins other than B t toxins were found using the criterion of 35% identity over an 80 amino acid segment. **Classification: ACCEPTABLE supersedes**  **MRID No. 46474402**  **] Cry1Ab**  **protein -**  **Epitope homology search.**  The amino acid sequence of the Cry1Ab protein w as com pared to amino acid sequences of proteins in the Allergen database to identify any short sequence similarities with known allergens. No similarities between the Cry1Ab protein and known allergens were identified based on 100% identity over an eight amino acid segment. **Classification: ACCEPTABLE 46708803**  **[This study supersedes**  **MRID No. 46474403]**  **Cry1Ab Protein: Epitope Hom**  **ology, N**  **- glycosylation and**  **Overall Am ino Acid**  **Sequence Homology Search with Known**  **Toxins and Allergens**  **- Supplemental**  **data** Updated studies (conducted in 2008) on amino acid sequence homology of Cry1Ab protein with known toxins and allergens and for N - glyco sylation sites showed no changes from the previously conducted studies done in 2005 (MRIDs No. 46708802 and 46708803, respectively).

**Classification: SUPPLEMENTAL ; No additional data needed.**  **47634809 [This study is**  **supplemental data to**  **MRIDs No. 46708802 and**  **46708803] Comparative**  **analysis of the Cry**  **1 Ab protein**  **am ino acid sequence**  The amino acid sequence of the Cry1Ab protein encoded by the gene in BCS CropScience’s (BCS) Bt cotton plants was compared with the sequence of the wild type Cry1Ab5 protein and the Cry1Ab protein in a previously registered PIP in corn, Bt11. The deduced ami no acid sequence of BCS’s Cry1Ab protein is identical to the amino acid sequence of the Bt11 Cry1Ab (OPP chemical code 006444) with the exception of one amino acid at the C terminus and two amino acids at the N - terminus. In addition, BCS’s Cry1Ab is identical to a portion of the wild type Cry1Ab5 protein produced in Bt subspecies berliner 1715, except for tw o amino acids at the N - terminus. **Classification: ACCEPTABLE 46708806**  **[This study supersedes**  **MRID No. 46474406]** 

**Acute Toxicity by Oral Gavage in**  **Mice -**  **Cry1Ab Protein**  The acute oral toxicity of Cry1Ab protein was assessed on a group of five female mice by administering bacterial - produced Cry1Ab protein by oral gavage at a total dose of 2000 mg/kg body weight . Due to the limitation of the solubility of the test material, the group of 5 female mice were given two doses of 1000 mg/kg body weight in a solution of 50 mM Na 2 CO 3 (pH 9.6) within a 4 hour time period on the day of treatment. Animals were observed for 15 days. At test end, there w ere no m ort alities, and no treatment - related adverse effects observed. Therefore, the acute oral LD 50 of the Cry1Ab protein is greater than 2000 mg/kg body weight, which places Cry1Ab protein in EPA TOXICITY CATEGORY III for acute oral exposure. **CLASSIFICATION: ACC**  **EPTABLE 47651103** 

**Cry1Ab Protein:**  **Heat stability study** An in vitro heat stability study w as conducted on bacterial - expressed Cry1Ab protein at temperatures of 60, 75 or 90°C for incubation periods of 10, 30 or 60 minutes and examined by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS - PAGE) and by Western blot analysis. Results of the SDS PAGE and Western blot analyses showed the Cry1Ab protein had no significant changes in protein stability after heat treatment at 60°C for 10, 30 and 60 m inutes and at 75°C for 10 and 30 minutes. After incubation for 60 minutes at 75°C, the Cry1Ab protein was slightly decreased. For the 90°C treatment, **47651104**

**4** Study submitted with EUP application and reviewed in memorandum from R. Edelstein through J. Kough to S. Matten, dated January 27, 2006.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 34 of 153 **Study Title**  **A.3. Hum**  **an health -**  **Summary –**  **Bt Cry1Ab**  **MRID No**

denaturation was observed after 10 and 30 minutes, and finally at 60 minutes, a marked but not complete de gradation of the Cry1Ab protein w as observed after incubation at 90°C. Additional protein bands of low er m olecular weight (mostly likely representing degradation products) also appeared as the ~69 kDa Cry1Ab protein band disappeared. Results showed consist ent patterns of decreasing structural stability of the Cry1Ab protein in between both analyses tested and wa s concluded as partially heat - stable at 90°C for 60 minutes. **CLASSIFICATION: ACCEPTABLE Cry1Ab**  **Protein: In**  **vitro digestibility**  **study in human simulated gastric**  **fluid** An in vitro digestibility study was conducted on bacterial - expressed Cry1Ab

protein in simu lated human gastric fluid containing pepsin (pH 1.2) to assess the susceptibility of Cry1Ab protein to proteol ytic degradation . SDS - PAGE analysis of the Cry1Ab protein solution showed the expected molecular weight of ~69 kDa at the initial time point. After incubation in SGF for 2 minutes, the band for the Cry1Ab protein was no longer visible on the Coomassie blue stained gel. This study indicates that the Cry2Ae protein is rapidly digested in SGF containing pepsin at pH 1.2. **CLASSIFICATION: ACCEPTABLE 47651102** 

**Cry1Ab Protein:**  **In vitro**  **digestibility study in simulated**  **intestinal fluid** An in vitro digestibility study was conducted on bacterial - expressed Cry1Ab protein in simu lated human intestinal fluid (SIF) containing pancreatin (pH 7.5) to assess the susceptibility of Cry1Ab protein to proteolytic degradation . The Cry1Ab protein w as incom pletely degraded int o several stable fragments in simulated intestinal fluid containing pancreatin (ph 7.5). **CLASSIFICATION: ACCEPTABLE 47651101** 

**ORF Analysis -**  **Toxicology (Hum an**  **Health Assessment)** The purpose of this study was to evaluate newly created ORFs that span the 5' or 3' junction of the transgenic insert (T - DNA) in cotton event T304 - 40 or the two internal junctions that were created within the T - DNA insert during the transformation process. ORFs were identified using the GetORF search program from the EMBOSS (Euro pean Molecular Biology Open Software Suite) tools. T wo ORFs of interest w ere found. No significant similarities were identified betw een the ORF - 1 putative amino acid sequence and sequences from the databases. The ORF - 2 putative amino acid sequence was identical to a fragment of the polymerase from the rice ragged stunt virus (RRSV), with a 100% match over the 28 putative amino acids generated by this ORF. Since the putative ORF - 2 sequence is only 28 amino acids long, while the RRSV polymerase has 1357 amino acids, it is unlikely that the ORF - 2 poly peptide has any polymerase activity even if it was expressed. In addition, n o records w ere on potential allergenicity or toxicity in animals associated with this virus polymerase protein or with the RRSV organism . Overall, neither of the two putative ORF amino acid sequences of the cotton transformation event T304 - 40 generated any significant amino acid sequence similarities with known toxins and known allergens. **CLASSIFICATION: ACCEPTABLE 47634806** 

**REFERENCES –**  **Cry1Ab –**  **Product Characterization and Human Health** Höfte H., de Greve H., Seurinck J., Jansens S., Mahillon J., Ampe C., Vandekerckhove J.,Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 35 of 153 Vanderbruggen H., van Montagu M., Zabeau M.,Vaeck M. (1986) Structural and functional anal ys is of a cloned delt a endotoxin of Bacillus thuringiensis berliner 1715. Eur. J. Biochem. 161:273 - 280 Thompson C., Van Montagu M. and Leemans J. (1987) Engineering herbicide resistance in plants by expression of a detoxifying enz ym e. EMBO Journal . 6(9): 2513 - 2518 U . S . EPA . 20 06 . The product characterization and human health data su bmitted in support of the EUP. Memorandum from R. Edelstein, through J. Kough, to S. Matten dated January 27, 2006. US Environmental Protection Agency . Washington DC U . S . EPA. 2011 . Review of Product Characterization Data for the Registration of Plant - Incorporated Protectant (PIP) cotton events: GHB119 cotton expressing Bacillus thuringiensis Cry2Ae protein ( EPA File Sy

mbol 264 - RNOU ) , T304 - 40 cotton expressing Bt CrylAb ( EPA File Sy mbol 264 - RNOL ) ; and TwinLinkTM cotton ( EPA File Sy mbol 264 - RNOA ) , its associated combination PIP product developed b y conventional breeding of co tton events GHB119 and T304 - 40. Memorandum from A. Waggoner, through J. Kough, to S. Bacchus and D. Greenway dated February 4, 2011. US EPA. Washington DC U . S . FDA . 1992. Statement of Policy : Foods Derived From New Plant Varieties. Federal Register: Vol. 57 No. 104 p. 22984. Dated: of May 29, 1992. Department Of Health And Human Services Food and Drug Administration ( Docket No. 92N - 0139 ) http://www.cfsan.fda.gov/~lrd/biotechm.html#reg Van Rie J. 2004. Comparative anal ys is of the Cry 1Ab protein amino acid sequence. Project Number: None; Unpublished study prepared b y Ba ye r CropScience LP. MRID No. 467474406Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 36 of 153 

### APPENDIX B: **Product characterization and human health effects -**  **Bacillus thuringiensis**  **Cry 2Ae (Event GHB 119)**  **and TwinLink**  **TM in cotton plant**  **incorporated protectant. Event GHB119**  **Cotton (OECD Unique Identifier BCS**  **- GH**    **5 -**  **8 )**  **Expressing Cry2Ae protein**  Appendix B contains summaries of EPA’s reviews of the p roduct c haracteriz ation and mammalian toxicology

of submitted studies for Bacillus thuringiensis Cry 2Ae and TwinL inkTM (combined Cry 1Ab and 2Ae) . B CS has developed event GHB119 cotton line expressing Cry2Ae insecticidal protein derived from Bacillus thuringiensis (

Bt ), which is intended to control certain lepidopteran pest species such as bollworm (CBW, Helicoverpa zea ) , pink bollworm (PBW, Pectinophora gossypiella ) , tobacco budworm (TBW, Heliothis virescens ) larvae, and fall arm yworm (FAW, Spodoptera frugiperda ) . In 2004, BCS submitted product characterization and human health data for the purposes of conducting time - limited experimental field trials with GHB119 cotton as a new PIP and a petition for a temporary exemption from the requirement of a toleranc e of Cry2Ae protein residues on food/feed commodities in cotton. The EUP also includ ed testing TwinLinkTM cotton ( EPA File Sy mbol 264 - RNOA) , a new combination PIP product, developed b y conventional breeding of the parental events T304 - 40 x GHB119 . The product charact erization and human health data submitted for event GHB119 and TwinLinkTM cotton were reviewed and found acceptable for the EUP application (US EPA, 2008). As part of that regulatory

action, the Agency

also conducted a preliminary food safety

assessment for the Cry2Ae protein expressed in event GHB119 cotton and a temporary tolerance exemption was granted for Cry2Ae protein residues produced i n this PIP cotton line ( 40 CFR § 174.530 ) . Event GHB119 cotton also expresses phosphinothricin acet yl transferase (PAT) enzy me , which is exempt from the requirement of a tolerance in all food commodities when used as a PIP inert ingredient ( 40 CFR § 174.522 ) . In May 2009, BCS submitted applications for FIFRA section 3 r egistration s for PIP events GHB119, T304 - 40 and TwinLinkTM cotton, their associated combination PIP product developed by traditional breeding methods. Concurrentl y, BCS filed a petition to establish a permanent tolerance exemption for Cry2Ae on the basis of the submitted data (see REGULATORY BACKGROUND, Chapter II.B.2.b). These data were reviewed by the Agency and were found supplemental . S everal product characterization studies and addition al data were requ ired by the Agency

to address the data deficiencies identified in those studies (US EPA, 2011a). In May 2011, BCS resubmitted studies to address the data deficiencies. EPA reviewed the additional data submitted for event GHB119 (Cry2Ae) cotton and determined they are acceptable for r egistration of the PIP and for establishing the permanent tolerance exemption for Cry2Ae expressed in event GHB119 (US EPA 2011c). Summaries of EPA’s evaluation of product characterization and the remaining toxicological data for Cry2Ae are available in this Appendix . **FFDCA FOOD SAFETY ASSESSMENT Event GHB119 Cotton (OECD Unique Identifier BCS**  **- GH**    **5 -**  **8) Expressing Cry2Ae protein**Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 37 of 153 **B. I. PRODUCT CHARACTERIZATION**  BCS developed event GHB119 cotton line expressing Cry2Ae insecticidal protein derived from Bacillus thuringiensis (

Bt ), which is intended to control certain lepidopteran pest species such as bollworm (CBW, Helicoverpa zea), pink bollworm (PBW, Pectinophora gossypiella), tobacco budworm (TBW, Heliothis virescens) larvae, and fall arm yworm (FAW, Spodoptera frugiperda). **a . Transformation System**  Event GHB119 cotton was produced b y Agrobacterium tumefaciens - mediated transformation of Gossypium hirsutum L. cultivar Coker 312 using transformation vector pTEM12 carrying T - DNA containing the cry2Ae gene, which encodes for Cry2Ae protein and confers insect resistance to certain lepidopteran pest species. The cry2Ae gene was isolated from Bacillus thuringien sis subspecies dakota and its sequence modified for optimal expression in plants. A sequence encoding a 58 amino acid transit peptide from the small subunit of the rubisco gene from Arabidopsis thaliana (TpssuAt) was added at the 5’ end of the coding regio n of the cry2Ae gene. BCS states that the presence of this transit peptide targets the Cry2Ae protein to the chloroplasts in the transformed cells and that the transit peptide is cleaved off during the transfer of the protein through the choloroplast membr ane. Plasmid pTEM12 also contains the bar gene as a selectable marker, which is derived from the soil microorganism, Streptomyces hygroscopicus (described b y Thomson et al. 1987). The bar gene encodes the enz ym e phosphinothricin acet yl transferase (PAT), wh ich is co - expressed with Cry2Ae protein in event GHB119 cotton as a PIP inert ingredient. The PAT enz ym e provides the plant with tolerance to herbicide applications containing glufosinate - ammonium by acet yl ating the herbicide glufosinate - ammonium and makin g it unable to bind glutamine s ynthase. The PAT enzy me encoded b y the bar gene is a homodimer of 183 amino acids and has an expected molecular weight of approximately 22 kDa. **b . Characterization of the DNA Inserted in the Plant and Inheritance and**  **Stability** Characterization of the DNA isolated from event GHB119 cotton using restriction enzy me digests and Southern blot analy ses as well as DNA sequencing indicates that the DNA was inserted in the cotton genome at a single locus, and the insert contai ns one copy of the cry2Ae gene . No vector backbone sequences from the transformation plasmid pTEM12 were found in GHB119 . Inheritance and stability studies of the cry2Ae gene in GHB119 verified that it is stably integrated into the cotton genome, segregati ng in an expected Mendelian fashion. **c . Protein Characterization**  Event GHB119 cotton expresses Cry2Ae protein produced by Bacillus thuringiensis subsp. dakota 1715. The Cry2Ae protein after cleavage of the transit peptide has 631 amino acids . Sodium dod ecyl sulfate poly acrylamide gel electrophoresis (SDS - PAGE) analy ses of Cry2Ae protein as expressed in event GHB119 showed a molecular weight at approximately 71 kDa. Western blot analy ses showed a single immunoreactive band at ~71 kD. Cry2Ae was also found to be non - gl ycosylated. N - terminal sequencing of the plant expressed form also confirmed that the transit peptide of the plant form was cleaved at the expected residue. The biological activity of Cry2Ae protein was assessed by an insect feeding bioassay u sing Heliothis virescens first instar larvae and the L C 50 for the plant - produced Cry2Ae protein was 296 ng/cm2. **d . Protein Expression** Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 38 of 153 Expression level data were provided for Cry2Ae in different plant tissues and at different growth stages in event GH B119 cotton . A summary of the results obtained using an ELI SA method is provided in Table B. 1 below.

**Table B.**  **1. Mean Cry2Ae Expression levels in Event GHB119 Cotton**

**Tissue Type Cry2Ae Protein**  **(μg/g dry weight ± SD)*** Roots 11.2 ± 2.4

– 16.9 ± 2.8 Stems 5.26 ± 0.87

– 5.58 ± 1.72 Leaves 17.8 ± 7.1

– 37.5 ± 13.6 Squares 8.69 ± 3.72 Apex 8.38 ± 2.29 Bolls 4.13 ± 0.80 Whole plants 30.4 ± 13.2 Pollen ND a Nectar NA b Flowers 5.59± 0.75 Cottonseed 0.99 ± 0.61 *Range reflects means at different growth stages **a** Not detected. **b** Not applicable due to the insufficient availability of material. **e . Supporting Data**  The product characterization studies to support the applicant ’s registration of PIP cotton event GHB119 are summarized in Table 2. A number of studies were reviewed under a work - share program according to the EPA - BPPD and USDA - BRS memorandum of understanding ( 09 - 2000 - 0052 - MU ) . For detailed information of each study and secondary reviewer information, please refer to the individually reviewed studies in the Data Evaluation Reports (DERs) according to its respective assigned MRID number).Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 39 of 153 **Table B.**  **2 .**  **Product Characterization Data for Event GHB119 cotton expressing Bt**  **Cry2Ae protein (**  **EPA File Symbol**  **264 -**  **RNOL )**  **Study Title B.2.**  **Summary -**  **Cry2Ae -**  **Product Characterization MRID No**  **Characteristics of Cry2Ae Cotton**  **Plants Derived from Transformation**  **Events GHB119 and GHB714:**  **Preliminary Report and Supplemental**  **Inform ation**  **5** This submission includes descriptions of the genetic construct used in events GHB119 and GHB714, the sources of the introduced genes, the mo des of action of the expressed proteins, and preliminary molecular analysis. Restriction enzyme digestion of genomic DNA from leaf material from events GHB 119 and GHB714 cotton and hybridization with the complete T - DNA probe gave the expected results, indicating that the events contain a single, intact copy of the expected T - DNA. **CLASSIFICATION: ACCEPTABLE 47125101** 

**PAT and Cry2Ae Protein in Cotton**  **Tissues of Events GHB119 and**  **GHB714. Preliminary Report**  **1** BCS conducted preliminary protein expression analysis for Cry2Ae cotton. Average Cry2Ae concentrations, as determined by ELISA in ground cotton seed from events GHB1 1 9, GHB714, and GHB866 w ere 3.5 ± 0.9  g/g, 2.9 ± 0.8  g/g, and 1.9 ± 0.3  g/g fresh weight, respectively. Average PAT concentrations, as determined by ELISA in ground cotton seed from events GHB1 19, GHB714, and GHB866 w ere 114 ± 25  g/g, 131 ± 23  g/g, and 100 ± 20  g/g, fresh weight, respectively. The percent dry weight equivalents in seeds are: Cry2Ae: 0.0002 - 0.00037% and PAT: 0.01 - 0.0 14%. Leaf, boll, square, and flower samples were also taken at various growth stages and analyzed for Cry2Ae from plants grown both in the field and in a greenhouse. The combined results from all three events gave the following ranges on a percent total soluble protein basis: leaf: 0.026 to 0.068%; boll: 0.007 to 0.036%; square: 0.008 to 0.02 1%; and flow er: 0.018 to 0. 02 1%. The PAT content of cotton tissues (other than seed) w as not presented in the study report. **Classification: SUPPLEMENTAL;** Sufficient to support EUP; Data gaps were addressed for Sec. 3 registration in MRID No. 47641903 **46708908**

**Product Character**  **ization of Cry2Ae Cotton**  **Event GHB119 6**  The purpose of this study was to determine if the transfer DNA (T DNA) containing the intended cry2Ae gene from plasmid pTEM12 w as inserted into the genomic DNA of cotton event GHB119 and the backbone DNA from pTEM12 wa s not inserted into cotton event GHB119. Southern blot analyses demonstrated a single copy of the intended cry2Ae gene from plasmid pTEM12 w as inserted into the genomic DNA of cotton event GHB119 and the configuration of the inserted DNA corresponds to that of the original transformation vector. Results also demonstrated the absence of most of the vector backbone DNA from pTEM12 after transformation event GHB119 How ever, there were several data missing from this report and one region of the vector backbone sequence was not confirmed as absent after the transformation process to create GHB119 cotton . Additional analyses such as demonstrating the cry2Ae gene was stably integrated and segregated according to Mendelian la ws of inheritance over several **47641901**

**5** Study submitted with EUP application and reviewed in memorandum from R. Edelstein through J. Kough to S. Bacchus, dated February 12, 2008. **6** S tudy submitted with Sec.3 application and reviewed in memorandum from A. Waggoner through J. Kough to S. Bacchus and D. Greenway, dated February 4, 2011.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 40 of 153 **Study Title B.2.**  **Summary -**  **Cry2Ae -**  **Product Characterization MRID No** 

s uccessive generations of event GHB199 cotton we re not addressed in this study. Additional data are needed to complete the molecular characterization of event GHB119 . These include: a plasmid map with all the enzymatic cleavage sites; a comparison of the expected DNA base pairs of each restriction enzyme digest to the actual fragment sizes observed , confirmation of the absence of all regions in the vector backbone sequence after transformation; and demonstration of the integration and stability of the cry2

Ae gene over several successive generations of event GHB1199 cotton. **CLASSIFICATION: SUPPLEMENTAL;** Data gaps were addressed for Sec. 3 registration in MRID No. 48480002 and 48480003 **Structural and Functional**  **Equivalence of Cry2Ae and**  **PAT/bar P roteins**  **Produced in Bacillus**  **thuringiensis (Bt)**  **and Escherichia coli**  **to Cry2Ae and PAT/bar Proteins in**  **GHB119 Cotton, Gossypium**  **hirsutum 2**  The purpose of this study was to demonstrate the biochemical and functional equivalence of the Cry2Ae and PAT/bar proteins expressed in cotton event GHB119 with the Cry2Ae expressed in Bacillus thuringiensis (Bt) and the PAT/bar protein expressed in Escher ichia coli (E. coli ), and the Cry2Ae and PAT/bar proteins expressed in TwinLinkTM cotton . The results from SDS - PAGE analyses showed that the Cry2Ae protein isolated from event GHB119 cotton leaves, the Cry2Ae E. coli standard, and TwinLinkTM cotton had ident ical electrophoretic mobilities and similar molecular weights (~66 kDa). SDS - PAGE analyses also showed identical electrophoretic mobilities and similar molecular weights (~20 kDa) for the PAT/bar protein isolated from event GHB119 cotton leaves, from the P AT /bar E. coli standard and the T wi nLinkTM cotton . How ever, it is unclear in the study report if the biochemical data presented in the study were derived from the same source of the bacterial - expressed Cry2Ae protein test substance . The study report lacked sufficient details on the materials/ methods used, the experimental design, and reported results to properly document the bioequivalence of the bacterial - derived Cry2Ae protein test substance to the T304 - 40 cotton plant - expressed Cry2Ae protein. Several si ngle - dose sensitive insect bioassays were also conducted to confirm bioactivity, but these data did not provide the quantitative results to allow

an adequate comparison of the insecticidal activity for each source of protein. Therefore, the biochemical and functional equivalence of the plant - and bacterial - expressed protein sources cannot be confirmed from these data. An additional sensitive insect bioassay study is required to characterize the insecticidal activity and establish the dose - response rel ationship of Cry2Ae protein against the target pest . Additional information is also needed to verify the validity of the results that were reported for Cry2Ae bacterial - derived protein substances to confirm that samples were tested from the same original s ource of preparation. **CLASSIFICATION: SUPPLEMENTAL** ; Data gaps addressed in MRID No. 48480003 **47641905**

**Analyses of Raw Agricultural**  **Comm odity (Fuzzy**  **Seed) of Cry2Ae Cotton Event**  The purpose of this study was to quantify the expression of Cry2Ae and PAT proteins in the fuzzy seeds of transgenic cotton event GHB119 and in a non - transgenic cotton line, Coker 312. Fuzzy seed w as ground into fractions of lint coats and kernels and anal yzed separately for Cry2Ae and PAT proteins and total extractable protein. To quantitate the **47641904**Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 41 of 153 **Study Title B.2.**  **Summary -**  **Cry2Ae -**  **Product Characterization MRID No**  **GHB119 for PAT/** **bar and**  **Cry2Ae and its Non**   ** transgenic** **Counterpart for PAT/** **bar and**  **Cry2Ae Proteins 2**  amount of the protein analytes in the fuzzy seeds, ELISA results of separate assays were combined for the kernel and lint coat fractions. The average Cry2Ae prote in content of transgenic fuzzy seed from all test sites, without and with herbicide spraying, on a dry weight basis wa s 1.55 ± 0.19 μg/g and 1.47 ± 0.07 μg/g, respectively. As a percent of total crude protein, Cry2Ae protein comprised an average of 0.00084 ± 0.00010% and 0.00082 ± 0.00007% of the total crude protein in unsprayed and sprayed fuzzy seed, respectively . The average PAT protein content of transgenic fuzzy seed from all test sites, without and with herbicide spraying, on a dry weight basis, was 5 0.7 ± 4.1 μg/g and 49.9 ± 3.5 μg/g, respectively. As a percent of total crude protein, PAT protein comprised an average of 0.027 ± 0.002% and 0.028 ± 0.003 % of the total crude protein in unsprayed and sprayed fuzzy seed, respectively. Overall results sho wed Cry2Ae and PAT analyte proteins were expressed in the kernels, lint coats and, by inference, in the fuzzy seeds derived from the transgenic GHB119 cotton plants. **CLASSIFICATION: ACCEPTABLE Protein Expression**  **Analysis of Cotton Event**  **GHB119, Expressing Cry2Ae**  **and PAT/ bar**  **Proteins, USA, 2007** The purpose of this study was to quantify the expression of Cry2Ae and PAT proteins in transgenic cotton event GHB119 and in a non transgenic cotton line Coker 312. Extracts of plant matrices were p repared from the roots, stems, leaves, squares, apex, bolls, whole plant, pollen, nectar, flowers and cottonseed. An enzyme - linked immunosorbent assay (ELISA) w as used for determining the protein levels. There w as a range of extraction efficiencies for eac h plant matrix and for each protein (means of 64.2 - 142%) . On a dry weight basis, the overall averages for the Cry2Ae protein content were 16.9 and 11.2 μg/g for roots at tw o growth stages; 5.58 and 5.26 μg/g for stems at two grow th stages; 37.5, 33.3, and 17.8 μg/g for leaves at three grow th stages; 8.69 μg/g for squares; 8.38 μg/g for apex; 4.13 μg/g for bolls; 30.4 μg/g for the whole plant; not analyzed for pollen due to the insufficient availability of material, non - detectable for nectar, 5.59 μg/g for f lowers, and 0.99 μg/g for cottonseed. Generally, the average PAT protein concentrations reported in the plant matrices from GHB119 were ca . an order of magnitude greater than the Cry2Ae protein concentrations . This study showed that quantifiable levels of Cry2Ae and PAT proteins w ere found in all plant matrices taken from the transgenic cotton event GHB119 (except nectar, w here only PAT protein w as detected), w

hile these proteins were absent from all of the cotton matrices derived from the non - transgenic co tton Coker 312. Pollen and nectar contained the least amount of both Cry2Ae and PAT proteins. **CLASSIFICATION: ACCEPTABLE 47641903** 

**Protein Detection Me**  **thods. Enzyme Linked Immuno**  **Sorbent Assay (ELISA)**  Semi - and full quantitative detection of Cry2Ae in extracts of plant

material from event GHB119 cotton can be accomplished using the EnviroLogix QuantiPlate Kit for Cry2A, which uses enzyme - linked immunosorbent assay (ELISA) analyses to detect the presence of Cry2A protein residues. **CLASSIFICATION: SUPPLEMENTAL** - An independent laboratory validation study to evaluate the ELISA test kit’s performance as the **47641926**Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 42 of 153 **Study Title B.2.**  **Summary -**  **Cry2Ae -**  **Product Characterization MRID No** 

designated analytical method for the detection of Cry2Ae protein residues is needed. **Product Characterization**  **– Nutritional**  **Characterization of Cry2Ae cotton event**  **GHB119 7**  The purpose of this study was to perform a compositional analysis of

nutritional factors, including protein, fat, carbohydrate, minerals, vitamins, amino acids and fatty acids (cottonseed oil), and anti nutritional factors, including gossypol, phytic acid and cyclic fatty acids present in transgenic cotton event GHB119 (without and with herbicide treatment), and to compare these nutritional values to those found in a non - transgenic cotton line (Coker 315) and to nutritional values generally found in cottonseed reported from other sources. **CLASSIFICATION: SUPPLEMENTAL** ; Data not required for EPA Sec.3 Registration of PIPs . Th e nutritional and compositional analysis of eve nt GHB119 cotton falls under the regulatory purvie w of FDA. **47641902**

**Detailed insert characterization of**  **Gossypium hirsutum transform**  **ation event GHB119 by**  **Southern blot analysis**  Southern blot analyses were conducted to determine the organization of

the cry2Ae and bar gene cassette in transformation event GHB119 cotton. Genomic DNA extracted from leaves of event GHB119 cotton wa s digested with one of the following restriction enzymes or enzyme complexes: Dra I, Eco RV, Hin dIII, Nco I, Bam HI, Eco RI, PstI, Ava I, Nde I, Xho I, Eco RI/

PstI and EcoRI/

Nde I). The resulting DNA fragments were separated by agarose gel electrophoresis, transferred to a membrane and sequentially hybridized with six different probes cassette (3'nos, bar, Pcsvmv, P35S2 - 5'cab22L, TPssuAT - cry2 Ae and 3'35S - RB), each representing a fragment of the transforming gene cassette, and with the complete T - DNA probe. The Southern blot data for the expected and observed hybridization fragments, as well as the hybridization strategy demonstrated that the i nserted transgenic sequence in the cotton event GHB119 is integrated as a single copy in event GHB119 cotton . Based on plant breeding activities, analyses from breeding and backcrossing activities with GHB119 generations, the Chi - square analyses of the ratio of expected vs. observed F1 and F2 progeny demonstrated that the cry2Ae gene insertion was stably integrated and segregated according to Mendelian la ws of inheritance. **CLASSIFICATION: ACCEPTABLE 48480001**  **[This study supersedes**  **MRID No. 476419**  **01]**

**Confirm ation of the**  **absence of vector backbone sequences**  **in Gossypium**  **hirsutum transform**  **ation event GHB119**  The purpose of this study was to determine the presence of any vector

backbone sequences in event GHB119 cotton after insertion of the intended cry2

Ae gene cassette via the pTEM12 transformation vector . Isolated genomic DNA samples from leaves of cotton event GHB119 and from the non - transgenic negative control were subjected to Southern blot analysis, after digestion with restriction enzymes Eco RV an d NotI, using seven overlapping vector backbone probes that **48480002 [This study**  **supersedes MRID No.**  **476419 01]** 

**7** This study is not reviewed in this report because EPA regulates the use, sale, and distribution of pesticides under FIFRA, which includes “plant - incorporated protectants” (in this case - Cry2Ae protein and the genetic material necessary for its production in GHB119 cotton), but not the plant itself. The nutritional and compositional analysis of event GHB 119 cotton as a food/feed commodity falls under the regulatory purview of FDA within the scope of the 1992 FDA policy statement concerning regulation of products derived from new p lant varieties, including those developed through biotechnology (FDA, 1992). The FDA e valuation for this study can be found in the Biotechnology Notification File BNF No. 000118 - Biotechnology Consultation - Note to the File . Subject: Cotton Events T304 - 40 and GHB119; Dated: August 12, 2011; Signed by: Richard E. Bonnette .Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 43 of 153 **Study Title B.2.**  **Summary -**  **Cry2Ae -**  **Product Characterization MRID No** 

encompassed the entire backbone of the pTEM12 transformation vector used to produce this transformation event. The vector backbone probes were then stripped from the membranes so that they could b e re hybridized with the T - DNA probe, as a positive control. Genomic DNA from the event GHB119 cotton show ed no hybridization with any of the vector backbone probes, while the positive control showed hybridization as expected. The results confirmed the abs ence of vector backbone sequences after insertion of the intended cry2Ae gene cassette via the pTEM12 transformation vector to produce event GHB119 cotton. **CLASSIFICATION: ACCEPTABLE Characterization of**  **Cry2Ae and PAT/bar proteins**  **expressed in Gossypium hirsutum**  **transform ation**  **event GHB119** The purpose of this study was to determine the biochemical and functional equivalence of plant - expressed Cry2Ae protein produced in event GHB119cott on plants with bacterial - expressed Cry2Ae proteins (Batch Nos. VMLV1041 - 1 and NB210806P25 produced from recombinant Bacillus thuringiensis and Escherichia coli over expression system, respectively) for their use as suitable test substance surrogates in t oxicological testing . Equivalence studies were also conducted for plant - expressed PAT protein in event GHB119 plants with bacterial - expressed PAT protein (Batch Nos. NB010905P44P2) for use as a test substance surrogate . Six analytical tests were used to a ssess the biochemical and functional equivalence, which included: 1) estimation of molecular w eight by observing protein mobility in SDS PAGE, 2) verification of immunoreactivity using Western blot analysis, 3) analysis of glycosylation using the reactio n with glycostain, 4) mass spectroscopic peptide mapping using HPLC/Electrospray Mass Spectrometry (LC/MS) of peptides, 5) analysis of N - terminal sequencing using Edman degradation, and , 3) analysis of biological activity using a sensitive insect bioactiv ity feeding assay to determine the insecticidal activity for the Cry2Ae proteins and enzymatic activity for the PAT/ bar protein, The Cry2Ae protein purified from GHB119 cotton plants and the bacterial - produced batches w ere found to have a similar molecular weight after electrophoresis, similar immunoreactive bands based on Western Blot analyses and non - glycosylated. Identity of the plant produced Cry2Ae protein and the bacterial - produced protein batches wa s confirmed by peptide mapping using electrospray mass spectrometry. After CNBr treatment and tryptic digest, 66% of the plant Cry2Ae protein, 73% of bacterial - produced batch VMLV10 41 - 1 and 75% of bacterial - produced batch NB210806P25 w ere identified. The N terminal sequence of the Cry2Ae protein batches was confirmed by Edman degradation. N - terminal sequencing also confirmed that the transit peptide of the plant form was cleaved at the expected residue. Biological activity was assessed by an insect bioassay using Heliothis virescens first instar larvae and the obtained LC 50 for the plant - produced Cry2Ae protein was 296 ng/cm2 , while the bacterial - produced Cry2Ae proteins from Batch Nos. VMLV1041 - 1 and NB210806P25 w ere 311 ng/cm2 and 346 ng/cm2 , respectively, demonstrating that the plant - and bacterial - produced proteins have comparable biological activity. Thus, both the bacterial - expressed Cry2Ae (Batch No. VMLV1041 - 1 and NB210806P 25) and PAT (Batch No. NB010905P44P2) proteins **48480006 [This study**  **supersedes MRID No.**  **47641905 and**  **47641906 ]** Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 44 of 153 **Study Title B.2.**  **Summary -**  **Cry2Ae -**  **Product Characterization MRID No** 

are suitable test substance surrogates for use in toxicological testing for establishing the safety of the plant - produced Cry2Ae and PAT proteins as expressed in event GHB119 cotton. **CLASSIFICATION: ACCEPT ABLE**  **B. II.**  **HUMAN HEALTH ASSESSMENT –**  **Cry2Ae** 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 “there is a reasonable certaint y that no harm will result f rom 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 forth 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 pest icide 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 residues” and “other substances that have a common mechani sm 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 thro ugh other exposures that occur as a result of pesticide use in residential settings. **a . Toxicological Profile**  BCS submitted a pesticide tolerance petition (PP 9F7514) 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 ) Cry2Ae insect control protein and the genetic material necessary for its production in or on all food commodities. The toxicological profile of the protein was previously described in the Federal Register of September 10, 2008 (73FR 52591: FRL - 8380 - 1) establishing a temporary

tolerance exempt ion for Cry2Ae in or on cotton food/feed commodities ( 40 CFR §174.530 ) . T he data reviewed to support the petition for establishing a permanent tolerance exemption are based upon the biochemical and functional characteristics of Bt Cry2Ae protein and its expression analy ses in cotton. No data were submitted for use of this protein as a PIP in other crops nor do any currentl y approved PIP events express Cry2Ae protein. Since the submitted data are specific to Cry2Ae protein expressed in cotton, the exemption from the requirement of a tolerance of Cry2Ae protein residues may only be supported in or on cotton food/feed commodities. 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 relationship of this information to human risk. EPA has also considered availableBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 45 of 153 information concerning the variability of the sensitivities of major identifiable s ubgroups of consumers, including infants and children. **1) Mammalian Toxicity** and **Allergenicity Assessment –**  **Cry2Ae** BCS submitted acute oral toxicity data to demonstrate the lack of mammalian toxicity at high levels of exposure to the pure Cry2Ae protein (MRID No. 47076902). The acute oral toxicity of Cry2Ae was assessed b y orally dosing (via gavage) to five female mice with 2,000 milligrams/kilograms bodyw eight of purified bacterial - produced Cry2Ae protein test substance (Batch No. NB210806P25, >93% puri ty). All treated animals gained weight and had no test material - related clinical signs and no test material related findings at necropsy . The acute oral LD 50 of the Cry2Ae protein is greater than 2000 mg/kg body weight. T he Cry2Ae protein does not appear t o cause an y significant adverse effects at an exposure level of up to 2000 mg/kg body weight and supports the finding that the Cry2Ae protein would be non - toxic to mammals. These data demonstrate the safet y of the product at a level of 2000 mg/kg b.w. that is well above maximum possible ppm exposure levels that are reasonabl y anticipated in the crop (see Table B.1. above) . 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 thuringiensis produ cts from which this plant - incorporated protectant was derived (see 40 CFR §§ 158.2130(d)(1)(i) and 158.2140(d)(7)). For microbial products, further toxicity testing 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 (Tiers II & III). Since there were no adverse effects in the Tier I acute oral study, further Tiers II and III toxicity testing and residue data were not required. **2) Allergenicity Assessment** When proteins are toxic, they are known to act via acute mechanisms and at very low dose levels (Sjoblad et al . 1992). Since no acute effects were shown to be caused b y C ry2Ae, even at relativel y high dose levels , the Cry2Ae protein is not considered toxic. Further, amino acid sequence comparisons showed no similarities that would raise a safet y concern between the Cry2Ae protein and known toxins in protein databases. Since Cry2Ae is a protein, allergenic sensiti vities were considered. Currently , no definitive tests exist for determining the allergenic potential of novel proteins. Therefore, EPA uses a weight - of - evidence approach where the following factors are considered: source of the trait; amino acid sequence similarity with known allergens; prevalence in food; and biochemical properties of the protein, including in vitro digestibility

in simulated gastric fluid (SGF) and glycos yl ation (as recommended by Codex Alimentarius Commission, 2003). Current scientific knowledge suggests that common food allergens tend to be resistant to degradation by acid and proteases; may be gl yc os yl ated; and present at high concentrations in the food. Summaries of the allergenicity assessment for Cry2Ae protein include: 1. **Sour ce of the trait.**  Bacillus thuringiensis, the microorganism from which Cry2Ae protein is derived , is not considered to be a source of allergenic proteins (MRID No. 47125101). 2. **Amino acid sequence.** A comparison of the amino acid sequence of Cry2Ae with kn ownBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 46 of 153 allergens showed no overall sequence similarit y using the criterion of 35% identity over an 80 amino acid segment or identity at the level of eight contiguous amino acid residues (MRIDs No. 47641908 and 47641909). 3. **Prevalence in food**

**.** Protein expression level analy ses of Cry2Ae protein confirm its presence at relativel y low levels. Dietary exposure is extremely limited and expected to be correspondingl y low . Expression in event GHB119 ranged from 0.99 to 1.55 μg/g or ppm for cottonseed. Thus, the expression has been shown to be in the parts per million range (MRID No. 47641903). 4. **Digestibility.** The Cry2Ae protein was rapidl y digested within 30 seconds in simulated mammalian gastric fluid containing pepsin at a pH of 1.2 (MRID No. 47125102). 5. **Glycosylation.** The Cry2Ae protein expressed in cotton was shown not to be gl yc os yl ated. (MRIDs No. 48471901 and 48480006). Considering all of the available information, EPA concluded that the potential for Cry2Ae to be a food allergen is minimal. **b . Aggregate Exposures**  Pursuant to FFDCA section 408(b)(2)(D)(vi), EPA considers available information concerning aggregate exposures from the pesticide residue in food and all other non - occupational exposures, including drinking water from ground water o r surface water and exposure through pesticide use in gardens, lawns, or buildings (residential and other indoor uses). The Agency

considered available information on the aggregate exposure levels of consumers (and major identifiable subgroups of consume rs) to the pesticide chemical 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 plant - incorporated protectants chemical residue, and exposure from non occupational sources. Exposure via the skin or inhalation is not likely since the plant - incorporated protectant is contained within plant cells, which essentially eliminates these exposure routes or reduces these exposure routes to negl ig ible. The amino acid homology assessment included similarity to known aeroallergens . It has been demonstrated that there is no evidence of occupationally related respiratory sy mptoms, based on a health survey on migrant workers after exposure to Bt pesti cides ( Bernstein et al . 1999) . Exposure via residential or lawn use to infants and children is also not expected because the use sites for the Cry2Ae protein are all agricultural for control of specific insect pest species. Although cotton is not a directl y consumed food commodity, humans may be potentially

exposed to extremely

low levels in the diet, from ingestion of processed cotton products (e.g. cottonseed flour and oil) and, potentially , drinking water. T he oral toxicity

study conducted at a dose of 2 ,000 m g/kg b.w. showed no adverse effects. Cottonseed may be processed to edible food commodities, cottonseed flour and cottonseed oil . In cottonseed from the parental strain and TwinL inkTM, Cry2Ae is expressed in the ppm levels (see Allergenicit y Assessment above, and Tables B.1 and B.4 in this Appendix). T hese levels are several orders of magnitude lower than the amounts of Cry2Ae protein shown to have no toxicity . Even if low levels of dietary exposure should occur, no harm is expected, based up on the lack of mammalian toxicity and the rapid digestibility

demonstrated for the Cry2Ae protein.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 47 of 153 **c . Cumulative Effects from Substances with a Common Mechanism of Toxicity**  Section 408(b)(2)(D)(v) of FFDCA requires that, when considering whether to e stablish, 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 .” Cry2Ae is not considered to be toxic, and Cry2Ae does not share a common mechanism of toxicity with any other substance . In addition , Cry 2Ae does not appear to produce a toxic metabolite produced by other substances . EPA concludes that there are no cumulative effects associated with Cry2Ae 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 **d . Determination of Safety for U.S. Population, Infants and Children**  The data submitted and cited regarding potential health effects for the Cry2Ae protein include the characterization of the expressed Cry2Ae protein in cotton, as well as the acute oral toxicity , heat stability , and in vitro digestibility of the protein. Th e results of these studies were used to evaluate human risk, and the validity , completeness, and reliability of the available data from the studies were also considered. As discussed more full y i n Appendix B.II.a . , above, the acute oral toxicity data subm itted supports the prediction that the Cry2Ae protein would be nontoxic to humans . Moreover, Cry2Ae has no sequence similarity to any known toxin . Because of this lack of demonstrated mammalian toxicity , no protein residue chemistry

data for Cry2Ae were re quired for a human health effects assessment . Even so, expression level anal ys is showed Cry2Ae protein is present at relativel y low levels. Dietary exposure is expected to be correspondingl y low. Since Cry2Ae is a protein, its allergenic potential was considered as part of the toxicity assessment. Data considered as part of the allergenicit y assessment include that the Cry2Ae protein : a) came from Bacillus thuringiensis , which is not a known allergenic source ; b) showed no sequence similarity to known aller gens ; c) was readily

degraded by

pepsin ; and d) was not gl yc osylated when expressed in the plant. On this basis, EPA concluded there is a reasonable certainty that Cry2Ae protein will not be a food allergen. Considered together, the lack of mammalian toxicity at high levels of exposure to the Cry2Ae protein and the minimal potential for that protein to be a food allergen demonstrate the safety of the product at levels well above possible maximum exposure levels anticipated in the crop. Finally , and specifical ly 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 pesticide chemical residue s, 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 safet y for infants and chi ldren 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.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 48 of 153 Based on all of the available infor mation described in this draft BRAD, , the Agency concludes that there is a reasonable certaint y that no harm will result to the U.S. population, including infants and children, from aggregate exposure to residues of the Cry2Ae protein and the genetic mater ial necessary for its production in cotton. This includes all anticipated dietary exposures and all other exposures for which there is reliable information . The Agency also 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 instance. **e . Other Considerations**  **1) Endocrine Disruptors** The pesticidal active ingredient is a protein that breaks down readil y in dietary exposure and is derived from a source not known to exert an influence on the endocrine s ys tem. Therefore, the Agency is not requiring information on the endocrine effects of the plant - incorporated protectant at this time. **2) Analytical Method**  BCS provided a validation study for EnviroLogix’s QuantiPlateTM Kit for Cry2A (Cat. No. AP005), which uses enzy

me - linked immunosorbent assay (ELISA) anal ys es for the detection of Cry2Ae protein residues in event GHB119 cotton. Results showed the test kit is able to detect Cry2Ae protein resid ues in cotton with sufficient accuracy , precision, and sensitivity . However, a validation study conducted by an independent 3 rd part y l aboratory is still needed to evaluate the performance of the ELISA test kit as the designated residue analy tical method f

or the detection of Cry2Ae protein expressed in event GHB119 cotton. **3) 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 considers 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 safet y 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 section 408(b)(4) requires that EPA explain the reasons for departing from the Codex level. No Codex maximum residue level exists for the plant - incorporated protectant Bacillus thuringiensis Cry

2Ae protein. **f. Overall Safety Conclusion** There is a reasonable certainty that no harm will result from aggregate exposure to the U.S. population, including infants and children, to the residues of Cry2Ae protein and the genetic material necessary fo r its production. This includes all anticipated dietary exposures and all other exposures for which there is reliable information. The Agency has arrived at this conclusion because, as discussed above, no toxicity to mammals has been observed, nor an y indi cation of allergenicity potential for the plant - incorporated protectant.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 49 of 153 The data submitted and reviewed for Cry2Ae support the petition for an exemption from the requirement of tolerance for Bacillus thuringiensis Cry2Ae protein when used as plant

– incor porated protectant in or on cotton food/feed commodities. **G. Supporting Data** The human health studies submitted to support the safet y of Cry2 Ae protein are summarized in Table B. 3 below. For detailed information of each stud y, please refer to the indivi dually reviewed studies in the Data Evaluation Reports (DERs) according to its respective assigned MRID number).

**Table B.**  **3 .**  **Hum an Health Data**  **for Event GHB119 cotton expressing Bt**  **Cry2Ae protein (**  **EPA File Symbol**  **264 -**  **RNOL )**  **Study Title B.3.**  **Summary –**  **Hum an health**  **– Cry2Ae**  **MRID No Description of the**  **Am ino Acid**  **Sequence of the Cry2Ae Protein**  **8** The 631 amino acid sequence of the Cry2Ae protein is provided in this submission. No information is provided on how the sequence w as determined, though it was presumably deduced from the inserted sequence of the gene, and no information is provided on whether the sequence was verified by mass spectrometry or N - terminal amino acid sequencing. SDS - PAGE and w estern blot analyses of the plant - produced protein are provided in MRID 46708907, which indicate that the protein has the expected molecular weight. **Classifi cation: SUPPLEMENTAL;**  Preliminary study used to support EUP **46708902**

**Cry2Ae (GEM2) Protein: Overall**  **Am ino**  **Acid Sequence Homology**  **Search with Known**  **Toxins and**  **Allergens 4**  The amino acid sequence of the Cry2Ae protein was compared to sequences contained in publicly available databases (SwissProt, trEMBL, GeneSeq - Prot, PIR, PDB, DAD, and GenPept.) using the BLASTP 2.2.2 algorithm and the BLOSUM62 scoring matrix. No similariti es between the Cry2Ae protein and known allergens or toxins, other than Cry toxins, were found using the criterion of 35% identity over an 80 amino acid segment. **Classification: SUPPLEMENTAL;** Sufficient to support EUP and tolerance exemption; Data gaps a ddressed for Sec. 3 registration in MRID No. 47641910 **46708903**

**Cry2Ae (GEM2) Protein: Epitope**  **Hom ology and N**  **** **Glycosylation Searches**  **4** The amino acid sequence of the Cry2Ae protein was compared to known protein sequences in the Allergen database to identify any similarities betw een short segments of amino acids. Searches were also performed to identify any possible N - glycosylation motifs. No significant similarities between Cry2Ae protein and known allergens based on a 100% identity over an 8 amino acid segm ent were found. Eleven potential N - glycosylation sites were found in the Cry2Ae protein sequence. **Classification: ACCEPTABLE 46708904** 

**Cry2Ae (GEM2) Protein in vitro**  **Digestibility Study in Simulated Gastric**  **Fluid 4**  The digestibility of the trypsin resistant core of microbially produced Cry2Ae wa s investigated in simulated gastric fluid containing pepsin. Horseradish peroxidase and ovalbumin were included in the study as controls. The trypsinized Cry2Ae protein was degraded within two minutes of incu bation in simulated gastric fluid at pH 1.2. Horseradish peroxidase and ovalbumin were degraded rapidly and slowly, respectively, as expected. How ever, because the protein that is produced in cotton is the full - length Cry2Ae protein, digestibility of the f ull length protein is more relevant for the risk assessment. **46708905**

**8** Study submitted with EUP application and reviewed in memorandum from R. Edelstein through J. Kough to S. Bacchus, dated February 12, 2008.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 50 of 153 **Study Title B.3.**  **Summary –**  **Hum an health**  **– Cry2Ae**  **MRID No**

**Classification: SUPPLEMENTAL -**  Data gaps were addressed for EUP and Sec. 3 registration in MRID No. 47125102 **Cry2Ae (GEM2) Protein**  **- Acute**  **Toxicity by Intravenous**  **Injection in the Mouse**  **4** The acute toxicity of the trypsin resistant core of microbially produced Cry2Ae wa s investigated by intravenous injection in mice. The protein (>95% purity) wa s suspended in PBS buffer (phosphate buffered saline) and administered as a single 1 or 5 mg/kg body weight intravenous dose. Melittin protein w as used as a positive control, and aprotinin protein and buffer alone were used as negative controls. Mice w ere observed for clinical signs daily for the duration of the study (15 days), and gross necro psy w as performed at the end of the study. Four out of five mice treated with melittin at a dose of 5 mg/kg died on day 1. No other deaths occurred during the study. No treatment - related adverse effects were observed in the mice administered Cry2Ae protein at either dose. **Classification: SUPPLEMENTAL;** Data gaps were addressed for EUP in MRID No. 47076902 and no additional data w ere required **46708906**

**Analysis to Determ**  **ine if the Cry2Ae (GEM2)**  **Protein from Cotton**  **Leaves is Glycosylated**  **4** Cry2Ae purified from Bacillus thuringiensis and from cotton leaves from events GHB1 19, GHB714, and GHB866) w as analyzed for glycosylation using a commercially available glycosylation test kit. The results indicate that the Cry2Ae protein is not glycosylated. **Classificati on: ACCEPTABLE**  **48471901 [This study**  **supersedes MRID No.**  **46708907] Cry2Ae (GEM2)**  **Protein: Acute Toxicity by Oral**  **Gavage in Mice 4**  The acute oral toxicity of Cry2Ae was assessed by administering groups of five female mice Bacillus thuringiensis - produced Cry2Ae protein by oral gavage at a dose of 2000 mg/kg body weight. There w ere no m ortalities, and no treatment related adverse effects observed. Therefore, the acute oral LD 50 of the Cry2Ae protein is greater than 2000 mg/kg body weight. **Classification: A CCEPTABLE**  **47076902**

**Cry2Ae (GEM2) Protein: In Vitro**  **Digestibility Study in Simulated Gastric**  **Fluid 4**  The susceptibility of the Cry2Ae protein to proteolytic degradation in simulated gastric fluid containing pepsin (SGF) w as investigated using Bacillus thuringiensis - produced full - length Cry2Ae. The band for the Cry2Ae protein was no longer visible on the Coomassie blue stained gel after incubation in SGF for 0.5 mi nutes. In addition, smaller degradation fragments were not observed. Horseradish peroxidase and ovalbumin were degraded rapidly and slowly, respectively, as expected. This study indicates that the Cry2Ae protein is rapidly digested in SGF at pH 1.2. **Classification: ACCEPTABLE 47125102**  **[This study supersedes**  **MRID No. 46708905 and**  **confirms MRID No. 47125102]**  **Cry2Ae Protein: Heat stability study**  An in vitro heat stability study w as conducted on bacterial - expressed Cry2Ae protein at temperatures of 60, 75 and 90°C for incubation periods of 0, 10, 30 and 60 minutes and examined by SDS - PAGE and by Western blot analysis. Results of the SDS - PAGE showed a ~59.3 kDa band for Cry2Ae and no significant deterioration after incubation at 60oC at all time points, but partial degradation at 75oC for 10 through 60 minutes. After 30 minutes at 90oC, the ban d w as visible, but markedly decreased and by 60 minutes was no longer present. Results of the Western blot analyses showed a ~59.3 kDa immunoreactive band for Cry2Ae and no signifi cant deterioration after incu bation at 60oC for 10, 30, or 60 minutes . At incubation of 75oC, the protein w as partially stable for 10 minutes and further decreased in stain intensity through 60 minutes. At incubation of 90oC, the band was visible at 10 minutes, but no longer present after 30 through 60 minutes. Results showed co nsistent patterns of decreasing structural stability of the Cry2Ae protein in between both analyses tested. This study indicates that the Cry2Ae protein is heat labile at 90°C at 60 **47641911**Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 51 of 153 **Study Title B.3.**  **Summary –**  **Hum an health**  **– Cry2Ae**  **MRID No**

minutes. **Classification: ACCEPTABLE Toxicology (Hum**  **an Health**  **Assessment) Summary of**  **Analysis of Open Reading Fram**  **e Hom**  **ology Searches** The purpose of this study was to evaluate newly created ORFs that span the 5' or 3' junction of the transgenic insert (T - DNA) in cotton event GHB119 and determine if any newly created chimeric ORFs could give rise to amino acid sequences with toxic or allergenic properties. The ORF search was performed by means of the GetORF search program from the EMBOSS (European Molecular Biology Open Software Suite). BLASTP or FindPatterns algorithm s were used to search publically available databases (Uniprot - Sw issprot, Uniprot - TrEMB, DAD, PDB, and GenPept). Three ORFs were found, one spanning the 5' junction and two spanning the 3' junction. No significant similarities were identified betw een any of the putative amino acid sequences generated by these ORFs and sequences found in databases, including known allergens and toxins. **Classification: ACCEPTABLE 47641906** 

**Cry2Ae Protein:**  **In vitro**  **digestibility study in simulated**  **intestinal fluid** An in vitro d igestibility study was conducted on bacterial - expressed Cry2Ae protein in simu lated human intestinal fluid (SIF) containing pancreatin (pH 7.5) to assess the susceptibility of Cry2Ae protein to proteolytic degradation . The Cry2Ae protein was degraded into several stable fragments in the presence of pancreatin, at pH 7.5 during the 60 minute digestion. **Classification: ACCEPTABLE 47641907** 

**Cry2Ae protein with**  **transit peptide epitope hom**  **ology and N**  **- glycosylation**  **searches** No significant amino acid sequence similarities between eight linear contiguous amino acid blocks of the Cry2Ae protein with known aller gens in the publically available AllergenOnline database (release 8.0, 1313 sequences) w ere found. Results suggest the Cry2Ae protein should not cross - rea ct with known allergenic epitopes. Eleven potential N - gly cosylation sites were identified. **Classification: ACCEPTABLE 47641908**  **[This study supersedes**  **MRID No. 47076904]**  **Cry2Ae protein with**  **transit peptide overall am**  **ino acid sequence hom**  **ology search with**  **known toxins and allergens**  Bioinformatic searches were conducted to evaluate any significant amino acid sequence similarities betw een the Cry2Ae protein and any known toxins in public available databases (Uniprot_SwissProt, Uniprot_TrEMBL, PDB, DAD, and GenPept.) using the FASTA algorithm and the BLOSUM62 scoring matrix; and any known allergens in the AllergenOnline database (release 8.0) using the FASTA algorithm and BLOSUM50 scoring matrix. Comparisons were conducted on the complete amino acid sequence of the test protein with matches of >35% identity over an 80 amino acid sequence segment; and then sequentially compared using sub - divided sequences of the test protein into 80 amino acid segments with all known allergens present in the database. No simil arities betw een the Cry2Ae protein and known allergens or toxins, other than Cry toxins, were found. **Classification: ACCEPTABLE 47641909**  **[This study supersedes**  **MRID No. 47076903]** 

**Update on Cry2Ae protein:**  **In vitro digestibility study in**  **human simulated ga**  **stric fluid, overall am**  **ino acid sequence hom**  **ology search with known toxins**  **and allergens, and epitope hom**  **ology and N**  **- glycosylation**  Bioinformatic studies on Cry2Ae protein were repeated in 2008 and show ed no amino acid sequence similarities in between Cry2Ae protein and known toxins and allergens (MRID No. 47641909); and no amino acid sequence similarities with known allergen epitopes and N - glyco sylation sites (MRID No. 47641908), thus confirming original results from previously conducted studi es done in 2005 (MRIDs No. 46708903 and MRID 46708904, respectively). An in vitro digestibility study of Cry2Ae protein in human simulated gastric fluid containing pepsin was repeated in 2007 (MRID No. 47125102) and showed rapid degradation of Cry2Ae prote in to pepsin, thus confirming original results from the previously conducted in vitro digestibility SGF study in 2005 (MRID No . 46708905). **47641910 [This s**  **tudy is a supplemental**  **report to MRIDs**  **46708905, 46708903 and**  **46708904]**Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 52 of 153 **Study Title B.3.**  **Summary –**  **Hum an health**  **– Cry2Ae**  **MRID No searches**  **Classification: SUPPLEMENTAL -**  This study is a supplemental to MRIDs 46708905, 46708903 and 46708904Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 53 of 153 **B.II I**  **. Product characterization and human health effects for TwinLinkTM (T304**  **- 40 x GHB119)**  **Combination PIP Cotton Product (OECD Unique Identifier: BCS -**  **GH**   **4 -**  **7 x BCS -**  **GH**   **5 -**  **8 )**  **Expressing Cry1Ab, PAT, and Cry2Ae Proteins** BCS submitted data in support of FIFRA Sec. 3 registration of TwinLinkTM cotton ( EPA File Sy mbol 264 - RNOA ) for use as an alternative insect resistant and herbicide tolerant combination PIP cotton product . TwinL i nkTM cotton was developed by conventional breeding of the parental events T304 - 40 x GHB119 to express Cry 1Ab, Cry2Ae and PAT proteins. Event T304 - 40 cotton encodes the cry1Ab gene derived from Bt subsp. berliner (described in Höfte et al. 1986) to express C ry 1Ab insecticidal protein, which also confers resistance to lepidopteran pest species. The safet y of Cry1Ab protein , as expressed in the cotton event T304 - 40 , was previousl y reviewed by the Agency and the data were found acceptable to support registration of event T304 - 40 cotton (US EPA 2006, 2008, and 2011a and c). A tolerance exemption exists for Cry1Ab protein residues when used as plant - incorporated protectant in all food commodities ( 40 CFR § 174.511 ) and all final data submitt ed for its registration were found acceptable (US EPA 2011c). C onsiderable safet y data have been established for both Cry2Ae expressed in GHB119 and Cry 1Ab expressed in T304 - 40 cotton . T he applicant submitted the following equivalency studies in order to b ridge from these existing acceptable toxicological data for the parental events for registration of TwinLinkTM cotton . **a . Product Characterization**  TwinL inkTM cotton (T304 - 40 x GHB119 combination PIP product) contains cry1Ab, cry2Ae, and bar transgenes. Events T304 - 40 and GHB119 cotton were individuall y developed by Agrobacterium mediated transformation to insert T - DNA containing the cry1Ab and cry2Ae gene cassettes via plasmid vectors pTDL008 and pTEM12, respectivel y. The resulting cotton li nes were then crossed by conventional breeding to create TwinLinkTM (GHB119 x T304 - 40) cotton. Characterization of DNA isolated from TwinL inkTM ( T304 - 40 x GHB119) cotton using restriction enzy me digests and Southern blot anal ys es were used to confirm the ge netic stability and integrit y of the T304 - 40 and GHB119 transgenic inserts in TwinLinkTM cotton. The anal yses showed the presence of the expected hy

bridization fragments in all tested transgenic DNA samples when hy bridized with each of the three probes. In addition, results showed the cry1Ab, cry2Ae , and bar DNA inserts are stabl y integrated in TwinLinkTM cotton, based on Southern blot anal ys es of combination PIP product plants obtained after five generations from the original TwinL inkTM cross. D ata from the c omparative Southern blot analy ses confirmed the integrity and genetic stability

of the transgenic cry1Ab, cry2Ae, and bar inserts from the individual parental events as preserved during conventional breeding to produce TwinL inkTM cotton. **b . Analytical**  **Detection Methods** BCS provided validation studies for commercial enzy me - linked immunosorbent assay (E LISA) test kits for the analy

tical detection of Cry2Ae and Cry 1Ab protein residues in cottonseed and cotton leaf tissue. The studies showed the test kits are able to detect Cry2Ae and Cry 1Ab protein in events GHB119 and T304 - 40 cotton, respectively , with sufficient accuracy , precision, and sensitivity . V erification of the specific ELISA test kit method to be used as the proposed residue anal yt ical method fo r the detection of Cry 1Ab protein in event T304 - 40 cotton is needed. In addition, individual validation studies conducted by an independent third party laboratory

are still needed to evaluate the performance of each ELISA test kit as the designated residue anal yt ical method for the detection of Cry2Ae and Cry 1Ab proteinsBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 54 of 153 expressed in cotton events GHB119 and T304 - 40, respectivel y. **c . Protein Expression**  Protein expression levels were provided for Cry2Ae, PAT, and Cry 1Ab in different plant tissues from T304 - 40 x GHB119 cotton tested at key developmental growth stages at 4 field sites. The average protein expression levels are shown below in Table 4. The Cry2Ae and Cry1Ab protein levels in TwinL inkTM cotton are comparable to those observed in plant tissues fro m its parental events GHB119 and T304 - 40 cotton . The PAT protein expression levels in TwinL inkTM cotton were higher than those expressed in its parental events . These results were expected since TwinL inkTM cotton inherited two copies of the bar gene, which w ere individuall y inserted in its parental events.

**Table B.**  **4. Mean Expression Levels of Cry2Ae, PAT and Cry1Ab from TwinLinkTM cotton**  **Tissue Type Cry2Ae** 

(μg/g dry weight + standard deviation) **PAT** (μg/g dry weight + standard deviation) **Cry1Ab**

(μg/g dry weight + standard deviation) Leaves (T1 - T3)* 18 ± 6.4

– 70 ± 35 250 ±120

– 1200 ± 650 3.0 ± 2.1

– 8.8 ± 3.8 Squares (T3) 12 ± 4.7 370 ± 160 8.2 ± 3.7 Bolls (T3) 4.7 ±1.7 240 ± 100 6.5 ± 2.9 Cottonseed (T4) 6.7 ± 3.3 280 ± 130 13 ± 6.5 Roots (T1 - T3)* 3.9 ± 1.9

– 11 ± 5.3 170 ± 80

– 290 ± 130 2.5 ± 1.4

– 3.8 ± 1.9 ***Ranges reflect m eans tested at different growth stages**  **d . Conclusion**  The DNA characterization study and protein expression analy ses field study considered with comparable biological activity

in between the parental cotton events GHB119 and T304 - 40 and no evidence of either a sy nergistic or antagonistic interaction between Cry2Ae and Cry 1Ab proteins in tobacco budworm (TBW, Heliothis virescens) (see Dean, B. et al. 2010; MRID No. 484800 - 09) reviewed in the ecological risk assessment memo for this product (US EPA 2011b) demonstrate that the Cry2Ae and Cry 1Ab proteins expressed in the parental cotton events GHB119 and T304 - 40 are biochemically and functionally

equiva lent to the Cry2Ae and Cry 1Ab proteins expressed in TwinL inkTM cotton. Thus, the existing toxicological data of parental events GHB119 and T304 - 40 cotton can be bridged to support the safet y of TwinLinkTM cotton. **e . Supporting Data**  The product characterization studies submitted to support the biochemical equivalence of Cry2Ae and Cry 1Ab proteins expressed in TwinL inkTM cotton to Cry2Ae and Cry 1Ab expressed in its constituent parental events GHB119 and T304 - 40, respectively , are summarized in Tabl e 5 below. For detailed information of each study, please refer to the individually reviewed studies in the Data Evaluation Reports (DERs) according to its respective assigned MRID number).

**Table B.**  **5 .**  **Product Characterization Data for Combination PIP TwinLinkTM** 

**( EPA**  **File Symbol 264**  **- RNOA** **)**Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 55 of 153 **Study Title B.5.**  **Summary -**  **TwinLink TM**  **- Product Characterization**  **MRID No .**  **TwinLinkTM Product**  **Characterization -**  **Molecular Characterization**  **9** Southern blot analyses were used to characterize the genomic DNA to confirm the

integrity of the GHB119 and T304 - 40 inserts in the combination PIP product T304 - 40 x GHB119. Samples from T304 - 40 x GHB119 cotton show ed band patterns obtained using each probe we re repeatable as those observed for the individual events, indicating that no DNA rearrangements occurred during the production of TwinLinkTM developed by conventional breeding of the parental events T304 - 40 x GHB119. How ever, no Southern blot analyses were conducted using the pat / bar as a probe on the parental events and T wi nLinkTM cotton . In addition, details were missing on the test materials, methods used to conduct the study. There w ere also no plasmid maps or schem atic depictions included to show the location of the cleavage sites for each restriction enzyme, the probe positions, and the predicted sizes of each DNA fragments after digestion. Lastly, the lanes for the negative control and the positive control appear to be mislabeled, since the negative and positive control results were opposite to those expected in both gel figures. **CLASSIFICATION: SUPPLEMENTAL -**  Data gaps were addressed in MRID No. 47640007 **47640001**

**Protein Expression Analysis for T304**  **- 40, GHB119 and**  **TwinLinkTM Transgenic Cotton**  **5** This study was conducted to characterize and compare the levels of protein

expression for Cry1Ab, Cry2Ae, and PAT expressed in events T304 - 40, GHB119, and the combination PIP TwinLinkTM cotton, developed by conventional breeding of the parental events T304 - 40 x GHB119. The trial contained 50 plants for each cotton line grown under typical greenhouse conditions including the application of insecticides and fertilizer. Samples of leaves, squares and grain were collected from five randomly selected cotton plants from each genotype . The Cry1Ab, Cry2Ae and PAT proteins were extracted from the plant tissue matrices to measure the amount of protein expressed in the cotton lines, using commercially available enzyme - linked immunosorbent assay (ELISA) test kits. There w as considerable variation in the levels of protein expression reported for each PIP protein expressed in the parental events compared to the levels of protein expression in TwinLinkTM cotton; as well as significant differences reported within the ranges of protein expression levels for certain tissue types, although not statistically analyzed. How ever, the significant differences may be attributed to the limited number of samples, replicates, and trials s ince this study was conducted as a single trial in a greenhouse . Therefore, the dataset used to characterize the protein expression levels may not be representative of the protein levels expressed in the field . In addition , the study does not account for variability in the levels of protein expressed under field conditions, where different environmental factors may also have an effect. **CLASSIFICATION: SUPPLEMENTAL -**  Data gaps were addressed in MRID No. 47640008 **4764000 3** 

**Analyses of Raw Agricultural**  **Comm odity (Fuzzy**  **Seed) of TwinLinkTM Cotton**  **for PAT/ bar** **, Cry2Ae and**  **Cry1Ab and its** The purpose of this study was to quantify the expression of Cry1Ab, Cry2Ae and

PAT proteins in the fuzzy seeds of transgenic T winLinkTM cotton and the non transgenic Coker 315 cotton line using enzyme - linked immunoabsorbent assay (ELISA) . Fuzzy seed were se parated into lint coats and kernels and these fractions were analyzed separately for Cry1Ab, Cry2Ae and PAT proteins and for total extractable protein. The respective values for the kernel and lint coat fractions were combined on a weight basis to calculate the respective values for the fuzzy seed that wa s received from the field. T he limit of detection (LOD) an d limits of quantitation **47640004**

**9** Study submitted with Sec.3 application and reviewed in memorandum from A. Waggoner through J. Kough to S. Bacchus and D. Greenway, dated February 4, 2011Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 56 of 153 **Study Title B.5.**  **Summary -**  **TwinLink TM**  **- Product Characterization**  **MRID No .**  **Non -**  **transgenic Counterpart for**  **PAT/ bar,**  **Cry2Ae and Cry1Ab**  **Proteins** (LOQ), the extract dilution factors, extraction efficiencies, standard curves (ng/mL),

and the calculations for the protein extractions from each tissue samples were reported to establish the validity (sufficient sensitivity and extraction efficiencies) of the ELISA method used in this study. The average Cry1Ab protein content of transgenic fuzzy seed from all test sites, without and with herbicide spraying, on a dry weight basis, was 1.32 ± 0.517 μg/g and 1.57 ± 0.317 μg/g, respec tively. As a percent of total crude protein, Cry1Ab protein comprised an average of 0.000577 ± 0.000204% and 0.000707 ± 0.000155% of the total crude protein in unsprayed and sprayed fuzzy seed, respectively. The average Cry2Ae protein content of transgen ic fuzzy seed from all test sites, without and with herbicide spraying, on a dry weight basis, was 9.35 ± 2.93 μg/g and 10.7 ± 4.17 μg/g, respectively. As a percent of total crude protein, Cry2Ae protein comprised an average of 0.00420 ± 0.00129 % and 0.004 85 ± 0.00204 % of the total crude protein in unsprayed and sprayed fuzzy seed, respectively. The average PAT protein content of transgenic fuzzy seed from all test sites, without and with herbicide spraying, on a dry weight basis, w as 162 ± 32.5 μg/g and 163 ± 27.4 μg/g, respectively. As a percent of total crude protein, PAT protein comprised an average of 0.0726 ± 0.0121% and 0.0737 ± 0.0151% of the total crude protein in unsprayed and sprayed fuzzy seed, respectively. **CLASSIFICATION: ACCEPTABLE Structural stability**  **analysis of Gossypium hirsutum**  **com bined event**  **TwinLinkTM x**  **GlyTol (GHB614)** The purpose of this study was to confirm the integrity and genetic stability of the

intended transgenic inserts contained in com bination PIP TwinLinkTM cotton plants developed by conventional breeding of the parental cotton events T304 - 40 (expressing Cry1Ab and PAT proteins) and GHB119 (expressing Cry2Ae and PAT proteins). TwinLinkTM cotton contains the cry1Ab and bar (cry1Ab - bar) genes on one DNA insert f

rom T304 - 40 cotton and the cry2Ae and bar (cry2Ae - bar) genes on a second DNA insert from GHB119 cotton. Genomic DNA was prepared from 24 individual combination PIP product cotton plants and w as subjected to Southern blot analysis using the cry1Ab - bar TDNA probe, the cry1Ab gene probe, and the cry2Ae bar TDNA probe. The analyses showed the presence of the expected hybridization fragments in all tested transgenic DNA samples when tested with each of the three probes . In addition, results showed the cry1Ab, cry2Ae , and bar DNA inserts are stably integrated in T wi nLinkTM cotton, based on Southern blot analyses of combination PIP product plants obtained after five generations from the original TwinLinkTM cross. Therefore, data from the com parative Southern blot analyses confirmed the integrity and genetic stability of the transgenic cry1Ab, cry2Ae, and bar inserts from the individual parental events as preserved during conventional breeding to produce Tw inLinkTM cotton. **CLASSIFICATION: ACCEPTABLE 48480007**  **[This study**  **supersedes MRID No.**  **47640001]**

**Protein expression analysis for GlyTol**  **x TwinLinkTM**  **(GHB614 x T304 -**  **40 x GHB119) cotton**  **grown in the field, USA, 2010**  The purpose of this study was to use enzyme - linked immunosorbent assay (ELISA)

methods to determine the expression of CrylAb, Cry2Ae, and PAT/ bar proteins in the com bination PIP product: GlyTol x TwinLinkTM (GHB614 x T304 - 40 x GHB119). Cotton event T304 - 40 expresses the Cry1Ab and PAT/ bar proteins, event GHB119 expresses the Cry2Ae and PAT/ bar proteins, and event GHB614 1 expresses the 2mEPSPS ( 5 - enolpyruvyl - shikimate - 3 - phosphate synthase) protein. Com bination PIP product cotton tissue samples from leaves, roots, squares, bolls, and fuzzy seeds were collected from three different field trial sit es at one or more developmental stages (T1 though T4). A total of 36 samples of each tissue type (m atrix) were assayed for protein concentrations. Expression of all proteins (CrylAb, Cry2Ae and PAT/ bar) w as observed in all matrices (leaves (T1, T2, and T3) , roots (T1, T2, and T3), squares (T3), bolls (T3), and fuzzy seed (T4)). **48480008 [This study**  **supersedes MRID No.**  **47871604]**Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 57 of 153 **Study Title B.5.**  **Summary -**  **TwinLink TM**  **- Product Characterization**  **MRID No .** 

Detailed information was provided on the limit of detection (LOD) and limits of quantitation (LOQ), the extract dilution factors, extraction efficiencies, standard curves (ng/mL), and the calculations for the protein extractions from each tissue samples for the purposes of validating the ELISA methods used in this study. Overall, the levels of protein expression for Cry2Ae, Cry1Ab, and PAT proteins in the combination PIP maize produ ct w ere comparable to the protein expression levels in its respective parental cott on events GHB119 and T304 - 40. These results can also be used to establish the biochemical equivalence of the Cry2Ae, Cry1Ab and PAT proteins expressed in combination PIP rel ative to the PIP parental events GHB119 and T304 - 40, when these parental lines are combined via traditional breeding methods. **CLASSIFICATION: ACCEPTABLE REFERENCES**  **– Cry2Ae and TwinLinkTM**  Bernstein, I. L., Bernstein, J.A., Miller, M., Tierzieva, S., Bernstein, D.I., Lummus, Z., Selgrade, M.K., Doerfler DL, Seligy

VL. (1999) Immune responses in farm workers after exposure to Bacillus thuringiensis pesticides. Environ Health Perspect . 107(7):575 - 82 CAC (2003) Alinorm 03/34: Joint FAO/WHO Food Standard Programme. Codex Alimentarius Commission , Twenty - Fifth Session, 30 July 2003. Rome, Ital y. Appendix III: Guideline for Conduct of Food Safety

Assessments of Foods Derived from Recombinant - DNA Plants; Appendix IV: Annex on Assessment of Possible Allergenicity. CAC , 47

– 60 Dean, B., Jansen., S., Becker, A. (2010) Insect bioassay of plant material expressing cotton events T304 - 40 GHB119 and TwinLinkTM (T304 - 40 x GHB119). Bay er CropScience Internal Report - Insect Bioassay from Monheim. MRID No. 484800 - 09 Höfte H., de Greve H., Seurinck J., Jansens S., Mahillon J., Ampe C., Vandekerckhove J., Vanderbruggen H., van Montagu M., Zabeau M.,Vaeck M. (1986) S tructural and functional anal ys is of a cloned delta endotoxin of Bacillus thuringiensis berliner 1715. Eur. J. Biochem. 161:273 - 280 Sjoblad, Roy

D. et al . (1992) Toxicological Considerations for Protein Components of Biological Pesticide Products” Regulato ry Toxicology and Pharmacology 15: 3 - 9 Thompson C., Van Montagu M. and Leemans J. (1987) Engineering herbicide resistance in plants by expression of a detoxifying enz ym e. EMBO Journal . 6(9): 2513 - 2518 U . S . EPA . 2006 . “The product characterization and human health data submitted in support of the EUP.” Memorandum from R. Edelstein, through J. Kough, to S. Matten; dated January 27, 2006. US Environmental Protection Agency . Washington DC U . S . EPA . 2008 . Review of Product Characterization and Human health Dat a for an EUP Application for B t. Cry2Ae Insecticidal Protein and Cry2Ae x Cry 1Ab as Expressed in Cotton. MemorandumBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 58 of 153 from R. Edelstein, through J. Kough, to S. Bacchus ; dated February 12, 2008. US EPA. Washington DC U . S . EPA . 2009 . Residue Chemistry Test Guidelines: OPPTS 860.1340 Residue Anal yt ical Method ( EPA 712

– C

– 96

– 174 ) ; Document ID: EPA - HQ - OPPT - 2009 - 0155 - 0007; Docket ID: EPA - HQ OPPT - 2009 - 0155 U.S. EPA. 2011a. Review of Product Characterization Data in support of Sec. 3 Registration of Plant In corporated Protectant (PIP) cotton events: GHB119 cotton expressing Bacillus thuringiensis Cry2Ae protein ( EPA File Sy mbol 264 - RNOU ) , T304 - 40 cotton expressing Bt CrylAb ( EPA File Sy mbol 264 - RNOL ) ; and TwinL inkTM cotton ( EPA File Sy mbol 26 4 - RNOA ) , its associated combination PIP product developed by conventional breeding of cotton events GHB119 and T304 - 40.” Memorandum from A. Waggoner, through J. Kough, to S. Bacchus and D. Greenway ; dated February 4, 2011. US EPA. Washington DC U . S . EPA. 2011b . Environmental Risk Assessment for Bacillus thuringiensis ( Bt) Cry 1Ab and Bt Cry2Ae insect control proteins as expressed in Event T304 - 40 cotton and GHB119 cotton, respectivel y, and in the combined Event, T304 - 40 x GHB119 (TwinL inkTM ) cotton ( EPA File Sy mbols 264 - RNOU, - RNOL, - RNOA ) , submitted by Bay er CropScience LP. Memorandum from S. Borges to S. Bacchus; dated October 13, 2011. US EPA. Washington DC US EPA. 2011c. Review of Product Characterization and Human Health Data in support of Sec. 3 Registr ation of Plant - In corporated Protectant (PIP) event T304 - 40 cotton expressing Bacillus thuringiensis Cry 1Ab insecticidal protein ( EPA File Sy mbol 264 - RNOU ) . Memorandum from A. Waggoner, through J. Kough, to D. Greenway ; dated November 30, 2011. US EPA. Washington DC U . S . FDA. 1992. Statement of Policy : Foods Derived From New Plant Varieties. Federal Register: Vol. 57 No. 104 p. 22984. Dated: of May 29, 1992. Department Of Health And Human Services Food and Drug Administration ( Docket No. 92N - 0139 ) http://www.cfsan.fda.gov/~lrd/biotechm.html#reg Van Rie J. 2004. Comparative anal ys is of the Cry 1Ab protein amino acid sequence. Project Number: None; Unpublished study prepared b y Ba ye r CropSc ience LP. MRID No. 467474406 .Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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### APPENDIX **C :**  **Ecological and e**  **n vironmental effects**  **– Bacillus thuringiensis**  **Cry1Ab, Cry2Ae and TwinLinkTM cotton plant incorporated protectant** BCS applied for a FIFRA Section 3 registration for two plant incorporated protectants (PIPs) . These include :  Cry 1Ab and the genetic material necessary for its production in Event T304 - 40 transgenic cotton [ Gossypium hirsutum ] plant line ;  Cry2Ae and the genetic material necessary for its production in transgenic Event GHB119 cotton plant line ; and  the associated stack in Event T304 - 40 x GHB119 (referred to hereafter as TwinL inkTM ) cotton, which produces both proteins. BPPD does not anticipate an y adverse effects of the Cry 1Ab and Cry2Ae proteins on nontarget organisms or on their abundance in an y field population . BPPD additionally concludes that the proposed Section 3 registrations of Cry 1Ab and Cry2Ae as expressed in Event T304 - 40, Event GHB119, or TwinL inkTM co tton will not result in direct or indirect effects to nontarget organisms, including federally - listed threatened or endangered species. This assessment references data that were reviewed and found acceptable to demonstrate functional equivalence between the bacteria - derived Cry 1Ab and Cry2Ae proteins used in toxicity testing and plant - expressed Cry 1Ab and Cry2Ae proteins . The ecological risk assessment for the combined event expressed in TwinL inkTM cotton is also dependent on equivalent expression levels of Cry 1Ab and Cry2Ae protein in TwinLinkTM cotton plants compared to the T304 - 40 and GHB119 parent lines [see Product C haracterization - Appendix A (Cry 1Ab) and B (Cry2Ae)] . **BACKGROUND** BCS developed Event T304 - 40 and Event GHB119 transgenic cotton [ Go ssypium hirsutum ] plant lines , which each express a Cry protein insect control gene . Event T304 - 40 expresses a Cry 1Ab gene, which is derived from the soil bacterium, Bacillus thuringiensis ( Bt) subsp. berliner , and Event GHB119 expresses a Cry2Ae gene deri ved from Bt subsp. dakota . Both genes have been modified for their expression in plants . Cry 1Ab and Cry2Ae proteins are intended to control several lepidopteran pests of cotton including cotton bollworm ( Helicoverpa zea ), pink bollworm ( Pectinophora gossypiella ), tobacco budworm (

Heliothis virenscens ) larvae, beet arm yworm ( Spodoptera exigua ), and fall army worm ( Spodoptera frugiperda ) . Events T304 - 40 and GHB119 also express the PAT gene, which confers herbicide resistance. Experimental use permits (E UP) were previously granted by

EPA to conduct field tests on Event T304 - 40, Event GHB119, and T304 - 40 x GHB119 cotton (EPA Reg. Nos. 264 - EUP - 140 and 264 - EUP - 143) . BCS is currentl y requesting a FIFRA Sec. 3 registration for Bt insect control protein Cry 1Ab as expressed in Event T304 - 40 cotton, Cry2Ae as expressed in Event GHB119 cotton, and the associated breeding stack, TwinL inkTM cotton, produced b y a traditional breeding cross . This memorandum contains the Agency ’s environmental risk assessment of the Cry 1 Ab and Cry2Ae proteins produced in T304 - 40 and GHB119 cotton lines, respectivel y, as well as in TwinL inkTM cotton.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 60 of 153 **I. Environmental Risk Assessment for Cry1Ab as Expressed in T304**  **- 40 Cotton and Cry2Ae**  **as Expressed in GHB119 Cotton** The paragraphs below d escribe the process and rationale developed b y BPPD 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 earl y 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 scree ning studies suggest potentiall y 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 save s valuable time and resources b y organizing the studies in a cohesive and coherent manner and eliminating unnecessary lines of investigation . Lo wer tier, high dose screening studies also allow tighter control over experimental variables and exposure condit ions, resulting in a greater ability to produce statistically reliable results at relatively low cost 10

. Tiered tests are designed to first represent unrealistic worst case scenarios and ONLY progress to real world field scenarios if the earlier tiered t ests fail to indicate adequate certainty of acceptable risk . Screening (Tier I) non - target 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 additional higher - tier testing . Less than 50% mortality

under these conditions of extreme exposure suggest that population effects are likely 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 non target 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 test (non - target) organism . The screening tests evaluate single species in a laboratory setting with mortality as the end point . Tiers II

– IV generall 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. Testing methods which utilize the tiered approach were last published b y the EPA as Harmonized OPPTS Testing Guidelines, Series 850 and 885 (EPA 712 - C - 96 - 280, February 1996) 11 . These 10 Non - target 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 al low ed an endpoint of 50% mo rtality to be used as a trigger for additional higher - tier testing. Lower levels of mortality under these conditions of extreme exposure suggest that population effects are likely to be negligible given realistic exposure scenar ios. Thus, it follows 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 3 0 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 appropriate because only effects of less than 50% indicate that further experiments are not needed to evaluate risk. 11 OPPTS Testing Guidelines, Series 850 and 885 we bsite: http://www.epa.gov/opptsfrs/publications/OPPTS_Harmonized/ 885Microbial_Pesticide_Test_Guidelines/SeriesBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 61 of 153 guidelines, as defined in 40 CFR 152.20, apply to microbes and microbial toxins when used as pesticides, including those that are naturall y occurring, and those that are strain - improved, either by natural selection or by

deliberate genetic manipulation . Therefore, PIPs containing microbial toxins are also covered b y these testing guidelines. The Tier I screening maximum hazard dose (MHD) approach to environmental hazard assessment is based on some factor (whenever possible >10) times the maximum amount of active ingredient expect ed to be available to terrestrial and aquatic non - target organisms in the environment (EEC) 12 . 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 pothes es . Elevated test doses, therefore, add certaint y 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 Guidel ines 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 specie s, notabl y non - target 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 d ay 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. 13 A less than 50% mortality effect at the MHD is taken to indicate minimal risk . However, greater than 50% mortality does not necessaril y indicate the existence of unacceptable risk in the field, but it does trigger the need to collect additional dose - response inf ormation 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 requ ired which can serve to confirm whether any effect might still be detected at more realistic field [1X EEC] concentrations and routes of exposure 14

. When screening tests indicate a need for additional data, the OPPTS Harmonized Guidelines call for testing at incrementall y 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 Obse rved Effect Concentration (L OEC), 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 risk assessment is made by 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 yed to evaluate the data from the definitive tests. Higher

12 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 ma ny species are tested or tests are very sensitive, although the test concentration used must exceed 1X EEC . 13 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 factor 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. 14 The 1X EEC test dose is based 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 no n - target organism may be ingesting in the field because most non - target organisms do not ingest plant tissue.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 62 of 153 levels of 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 de finitive testing unnecessary . Itis also notable that the recommended >10X EEC maximum hazard dose level is a highl y conservative factor . The publish ed EPA Level of Concern [LOC] is 50% mortality at 5X EEC 15 ( U.S. EPA 1998). Validation: The tiered hazard assessment approach was developed for the EPA by the American In stitute of Biological Sciences (AIBS) and confirmed in 1996 as an acceptable method of environmental hazard assessment by

a FIFRA Scientific Advisory Panel (SAP) on microbi al pesticides and microbial toxins. The December 9, 1999 SAP agreed that the Tiered approach was suitable for use with Plant - Incorporated 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 closely related to the target insect pest was not recommended, although it is still per formed to fulfill the published EPA non - target species data requirements . In October 2000, another SAP also recommended that field testing should be used to evaluate population - level effects on non - target organisms. The August 2002 SAP, and some public comments, generally

agreed with this approach, with the additional recommendation that indicator organisms should be selected on the basis of potential for field exposure to the subject protein (U.S. EPA 2000, 2001a, 2002, and 2004). Chronic studies: Since delay ed adverse effects and/or accumulation of toxins through the food chain are not expected to result from exposure to proteins, protein toxins are not routinely tested for chronic effects on non - target organisms . However, the 30 day test duration requir ement does amount to subchronic testing when performed at field exposure test doses. Proteins also do not bioaccumulate. The biological nature of proteins makes them readil y s usceptible to metabolic, microbial, and abiotic degradation once they

are ingeste d or excreted into the environment . Although there are reports that some proteins (Cry

proteins) bind to soil particles, it has also been shown that these proteins are degraded rapidl y b y soil microbial flora upon elution from soil particles. Conclusion : The tiered 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 that maximum hazard dose Tier Iscreening test ing presents a reasonable approach for evaluating hazards related to the use of biological pesticides and for identifying negative results with a high degree of confidence. The Agency expects that Tier 1 testing for short - term hazard assessment will be suf ficient for most studies submitted in support of PIP registrations. However, if long range adverse effects must be ascertained, then higher - tier longer - term fie ld testing will be required. As noted above, the October 2000 SAP and the National Academ y of Sc iences (NAS 2000) recommended testing non target organisms directly

in the field. This approach, with an emphasis on testing invertebrates found in corn fields, was also recommended by the August 2002 SAP and was supported by several public comments. Based on these recommendations, the Agency has required field studies on long term invertebrate population/community and Cry protein accumulation in soils as a condition of registration 15 Th e established peer and EPA Science Board review ed guidance on screening test levels of concern is 50% m ortality at 5X environmental concentrat ion . The appropriate endpoints in high dose limit/screening testing are based on mortality of the treated, as compared to the untreated (control) non - target organisms. A single group of 30 test animals ma y be tested at the maximum hazard dose .Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 63 of 153 due to the lack of baseline data on the potential for long - term environment al 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 accumulate d to a level where empirical conclusions can be made . As a result, the issue of long range effects of cultivation of these Cry proteins on the invertebrate community structure in Bt crop fields has since been adequately addressed . Specificall y, a meta - anal ys is 16 of the data collected from 42 field studies indicated that non target invertebrates are generall y more abundant in Bt cotton and Bt maize fields than in non - transgenic fields managed with insecticides (Marvier et al ., 2007) . In addition, a comprehens ive 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 cultivation (Sanvido et al. 2007) . Ano ther review of field tests published to date concluded that the large - scale studies in commercial Bt cotton have not revealed an y unexpected non - target effects other than subtle shifts in the arthropod community caused by

the effective control of the targe t 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 . Overall, the Agency

is in agreement w ith the conclusions of these studies and collectivel y, these results provide extensive data to support that Bt crops have not caused long term environmental effects on a population level to organisms not targeted b y Bt proteins. Based on these considerations, regulatory testing of the specialist predators and parasitoids of target pests may eventually

be considered unnecessary . **A .**  **Environmental Exposure Assessment** Two separate SAP reports (October 2000 and August 2002) recommended that non - target 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 determ ined that non - target organisms with the greatest exposure potential to Cry prot ein in transgenic cotton fields are beneficial insects that feed on cotton pollen and nectar, particularl y lepidopteran insects, and soil invertebrates . While EPA’s risk assessm ents of Bt cotton have focused primaril y 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 pically based on protein expression levels within the plant for PIPs . Although it is recommended that non - target 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 sy stem 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 man y species are tested or tests are very sensitive, although the concentration used must exceed the EEC . For the purposes of the non - target organism studies submitted in support of Event T304 - 40 and Event GHB119, the test material dose levels were based on an estimated concentration of Cry 1Ab or Cry2Ae protein exp ressed in the tissue(s) with the highest concentration based on protein expression studies (MRIDs 47634803, 47641903). 16 This research w as funded by Environmental Protection Agency grant CR - 832147 - 01. The Bt crop non - target effects database can be found on the National Center for Ecological Analysis and Synthesis (NCEAS) . Website. ( http://delphi.nceas.ucsb.edu/btcrops/).Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 64 of 153 **B .**  **Ecological Effects Data for Event T304 -**  **40 and Event GHB119** In the absence of PIP - specific risk assessment guidance, EPA requi res 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 . Li mit dose testing on representative organisms from several taxa was performed in support of the T304 - 40 and GHB119 Section 3 FIFRA registrations . As stated above, BPPD’s risk assessments focus heavil y on beneficial nontarget invertebrates, since they are most closely related to organisms susceptible to the insecticidal action of Bt toxins . The Cry 1Ab and Cry2Ae proteins are meant to target species w ithin the order Lepidoptera (moths and butterflies). Bt toxins are known ty picall y to have a limited host range, however, to address an y unforeseen change in activity spectrum as a result of laboratory

protein sy

nthesis and to fulfill the published registr ation data requirements EPA requires that test species used for non - target insect evaluations should include several invertebrate species that are not related to the target pests. Earthworm studies are also recommended . The toxicity of the Cry 1Ab and Cry2A e has been evaluated on several species of invertebrates including the lady beetle, green lacewing, collembola, Daphnia , honey bee, and earthworm . Reproductive and/or developmental observations were also examined in the lady beetle, Daphnia , collembola, an d honey bee studies. Since exposure may

also occur to other nontarget organisms, EPA has received data to compl y with the Agency ’s published non - target data requirements on other nontarget organisms . Tests to determine soil degradation of Cry 1Ab and Cry2A e have also been submitted . The individual results for nontarget organism and soil degradation testing for Cry 1Ab and Cry2Ae are summarized in Tables 1 and 2, respectivel y . The studies are described in more detail below, and full reviews of each study for each event can be found in the individual Data Evaluation Records. The October 2000 SAP recommended that while actual plant material is the preferred test material, bacteria - derived protein is also a valid test substance, particularl y in scenarios where test animals do not normally consume cotton plant tissue and where large amounts of Cry protein are needed for maximum hazard dose testing . In support of the T304 - 40 and GHB119 registrations, test substances used in the submitted studies included bacteria - produced purified Cry 1Ab and Cry2Ae protein, respectively . Comparative anal ys es have been performed to verify the equivalence of the bacteriall y - produced and purified Cry 1Ab and Cry2Ae and the Cry 1Ab and Cry2Ae proteins produced in T304 - 40 or GHB119, respe ctivel y, and TwinLinkTM cotton plants . Product characterization data (see Appendix A and B) demonstrate functional equivalence between the bacteria - derived Cry 1Ab and Cry2Ae proteins used in toxicity testing and plant - expressed Cry 1Ab and Cry2Ae proteins . T he ecological risk assessment for the combined event expressed in TwinL inkTM cotton is also dependent on equivalent expression levels of Cry 1Ab and Cry2Ae protein in TwinL inkTM cotton plants compared to the T304 - 40 and GHB119 parent lines . This has also been demonstrated [see Product C haracterization - Appendix A (Cry 1Ab) and B (Cry2Ae and TwinL inkTM)]. **Table C.**  **1 .**  **Summary of environmental effects studies and waiver justifications for Cry1Ab as expressed in**  **T304 -**  **40 cotton submitted to**  **comply with data requiremen ts published in 40 CFR § 158.2150**  **(d). Data**  **Requirement OPPTS** 

**Guideline Test Substance**  **C.1. Cry1Ab –**  **Ecological Effects -**  **Results Summary and Classification MRID**  **No.** Avian dietary testing, broiler chicken, Gallus 885.4050 TwinLinkTM (T304 - 40 x GHB119) cotton seed meal A 42 - day dietary study showed no adverse affects to broiler chickens when fed a diet composed of 10% TwinLinkTM cottonseed meal containing both Cry1Ab and Cry2Ae . However, 47640011Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 65 of 153 **Data Requirement**  **OPPTS**

**Guideline Test Substance**  **C.1. Cry1Ab –**  **Ecological Effects -**  **Results Summary and Classification MRID**  **No.** domesticus the presence of the proteins in the test material wa s not confirmed. **Classification: Unacceptable** Avian inhalation testing 885.4100 N/A **Acceptable waiver rationale** 47871403 Avian oral testing, Northern bobwhite Colinus virginianus 850.2100 N/A Data w aiver rationale was submitted to justify that testing is not necessary o n the basis of lack of exposure and information on the toxicity of registered Cry proteins to birds. **Classification: Acceptable**  47903201

Wild mammal testing 885.4150 N/A **Acceptable bridging rationale to acute oral**  **toxicity test on laboratory mice** 47871403 Freshwater fish testing, 885.4200 N/A **Acceptable waiver rationale** 47871403 Freshwater aquatic invertebrate testing, water flea, Daphnia magna 885.4240 Microbially produced Cry1Ab No adverse effects on survival or reproduction

of Daphnia magna exposed to 48 μg Cry1Ab/L in a 10 - day

static renewal test . The 48 - hr and 10 - day EC 50 values were determined to be > 48 μg Cry1Ab/L; the NOAEC is ≥ 48 μg Cry1Ab/L. **Classification: Acceptable** 47634819

Estuarine and marine animal testing 885.4280 N/A **Acceptable waiver rationale** 47871403 Non - target plant testing 885.4300 N/A **Acceptable waiver rationale** 47871403 Non - target insect testing, pink - spotted lady beetle, Coleomegilla maculata 885.4340 Microbially produced Cry1Ab N o adverse effects on survival, time to pupation,

and mean adult weight for Coleomegilla maculata larvae exposed to 19 μg Cry1Ab protein/g die t over 21 days . The LC 50 wa s >19 μg Cry1Ab/g diet and the NOAEC is ≥ 19 μg Cry1Ab/g diet . **Classification: Acceptable** 47634815

Non - target insect testing, green lacewing, Chyrsoperla rufilabris 885.4340 Microbially produced Cry1Ab No adverse effects on survival or pupation w ere

observed in Chrysoperla rufilabris exposed to 20 μg Cry1Ab protein/g diet (nominal) in a 10 - day laboratory bioassay . The nominal LC 50 is >20 μg Cry1Ab/g diet and the NOAEC is ≥20 μg Cry1Ab/g diet. **Classification: Acceptable** 47692701

Non - target insect testing, collembola, Folsomia candida 885.4340 Microbially produced Cry1Ab No adverse effects on survival or reproduction

of collembola (Folsomia candida ) exposed to 20 μg of Cry1Ab /g diet i n a 28 - day laboratory bioassay . The nominal LC 50 was determined to be > 20 μg Cry1Ab/g diet and the nominal NOAEC is ≥20 μg. **Classification: Acceptable** 47651106

Honeybee testing, Honeybee larvae, Apis mellifera 885.4380 Microbially produced Cry1Ab No adverse effects on survival w ere observed in

honey bee larvae treated in natal cells with 20 μg/L solution of Cry1Ab protein . The LC 50 was determined to be > 20 μg Cry1Ab/mL. **Classification: Acceptable** 47651105Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 66 of 153 **Data Requirement**  **OPPTS**

**Guideline Test Substance**  **C.1. Cry1Ab –**  **Ecological Effects -**  **Results Summary and Classification MRID**  **No.** Earthworm toxicity, Eisenia foetida 850.6200 N/A **Acceptable Waiver Rationale** 47634818 Soil fate and degradation 885.5200 Microbially produced Cry1Ab Based on the measured bioactivity in tobacco

budworm (Heliothis virescens ), the average DT 50 for Cry1Ab in three different soils was 3.6 days. **Classification: Acceptable** 47634820

**Table C.**  **2 .**  **Summary of environm ental effects studies and waiver justifications for GHB119 submitted to com**  **ply with data requirements published in 40 CFR § 158.2150**  **(d). Data**  **Requirement OPPTS** 

**Guideline Test** 

**Substance C.2. Cry2Ae**  **- Ecological Effects**  **–**

**Results Summary and Classification MRID No.**  Avian dietary testing, broiler chicken, Gallus domesticus 885.4050 TwinLinkTM (T304 - 40 x GHB119) cotton seed meal A 42 - day dietary study showed no adverse affects to broiler chickens when fed a diet composed of 10% TwinLinkTM cottonseed meal containing both Cry1A b and Cry2Ae . However, the presence of the proteins in the test material w as not confirmed. **Classification: Unacceptable**  47640011 (same as above) Avian inhalation testing 885.4100 N/A **Acceptable waiver rationale** 47871503 Avian oral testing, Northern bobwhite Colinus virginianus 850.2100 N/A GHB119 cotton seed meal (raw) Data w aiver rationale was submitted to justify that

testing is not necessary on the basis of lack of exposure. **Classification: Supple mental**  A 5 - day feeding study with Northern bobw hite showed no adverse effects at 10% raw GHB119 cotton seed meal in the diet. **Classification: Acceptable** 4790 3301 481 77501 Wild mammal testing 885.4150 N/A **Acceptable bridging rationale to acute oral toxicity test on laboratory mice**  47871503 Freshwater fish testing 885.4200 N/A **Acceptable waiver rationale** 47871503 Freshwater aquatic invertebrate, water flea neonate, Daphnia magna 885.4240 Microbially produced Cry2Ae Daphnia magna we re exposed to a nominal

concentration of 330 μg Cry2Ae/L both in water and in food for 10 days . No adverse effects on immobilization/ survival or reproduction w ere observed. The nominal 48 - hr and 10 - day EC50 values were > 330 μg a.i./L and the nominal NOAEC was ≥ 330 μg a.i./L . **Classification: Acceptable** 47641919

Estuarine and marine animal testing 885.4280 N/A **Acceptable waiver rationale**  47871503 Non - target plant testing 885.4300 N/A **Acceptable waiver rationale** 47871503 Non - target insect testing, pink - spotted lady beetle, Coleomegilla maculata 885.4340 Microbially produced Cry2Ae No st atistically significant effects on mortality,

body weight, mean number of days to pupation, and mean number of days to adulthood w ere observed in Coleomegilla maculata exposed to 64 μg Cry2Ae protein/g of diet for 21 days . The nominal LC 50 is > 64 μg Cry2A e/g diet and the NOAEC is ≥ 64 μg 47641915Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 67 of 153 **Data Requirement**  **OPPTS**

**Guideline Test** 

**Substance C.2. Cry2Ae**  **- Ecological Effects**  **–**

**Results Summary and Classification MRID No.**  Cry2Ae/g diet. **Classification: Acceptable** Non - target insect testing, green lacewing, Chrysoperla rufilabris 885.4340 Microbially produced Cry2Ae N o statistically significant differences in mortality

or % pupation w ere observed in green lacewing exposed to a nominal concentration of 10 or 110 μg Cry2Ae protein/g of diet for 10 days . The nominal LC 50 is >110 μg Cry2Ae/g diet and the nominal NOAEC is ≥ 110 μg Cry2Ae/g diet. **Classification: Acceptable** 47692501

Non - target insect testing, collembolan, Folsomia candida 885.4340 Microbially produced Cry2Ae Limit test : The number of offspring per adult

exposed to Cry2Ae was significantly reduced in Collem bola exposed to 66 μg Cry2Ae/g diet for 28 days. **Classification: Supple**  **mental** Definitive test: N o statistically significant differences in survival or reproduction among the test material and negative control groups w ere observed in Collembola exposed to 9.9, 20, 40, 80, or 160 μg of Cry2Ae/g diet . The nominal LC 50 is >160 μg Cry2Ae/g diet and the nominal NOAEC is ≥160 μg Cry2Ae/g diet. **Classification: Acceptable** 47641917 a 47641917b

Earthworm toxicity, Eisenia foetida 850.6200 Microbially produced Cry2Ae No adverse effects were observed on burrowing

time, mortality, and w eight change for earthworms (Eisenia fetida ) w ere exposed to a nominal concentration of 100 mg Cry2Ae protein/kg of artificial soil for 14 days. The nominal 7 - and 14 - day LC 50 was d etermined to be >100 mg a.i./kg, and the NOAEC was ≥100 mg a.i./kg. **Classification: Acceptable** 47641918

Honeybee testing, Apis mellifera 885.4380 Microbially produced Cry2Ae Larval survival did not differ significantly between

the Cry2Ae treated group and the controls at capping and at emergence for honey bee ( Apis mellifera ) larvae exposed to 50 μg Cry2Ae/mL. Therefore, the LC 50 is >50 μg Cry2Ae/mL. **Classification: Acceptable** 47641914

Soil fate and degradation 885.5200 Microbially produced Cry2A e Based on the measured bioactivity in tobacco

budworm (Heliothis virescens ), the average DT 50 for Cry2Ae in three different soils w as 3.4 days . **Classification: Acceptable** 47641920

**1. Non -**  **target Organism Study Summaries for T304 -**  **40 expressing Cry1Ab a**  **. Avian Wildlife**  Avian Dietary Toxicity In a 42 - day

study (MRID 47640011), newl y - hatched Ross #308 broiler chickens were fed formulated diet containing 10% TwinL inkTM cottonseed meal Control groups were fed formulated diet containing either 10% near isogenic cottonseed meal or 10% non - transgenic commercial cottonseed meal. There were no statisticall y significant treatment - related effects on survival, body weight, bod y weight gain, feed consumption, feed conversion ratio, or weight of chilled carcass, leg, thigh, wing, or breast amongBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 68 of 153 any of the test groups. DNA anal ys is of the prepared diets confirmed the presence of transgenic genetic material in the TwinLinkTM diet, ho wever the presence of the Cry 1Ab and Cry2Ae proteins were not confirmed . The stud y report stated that the cottonseed meal was toasted, and this heating may have degraded the proteins . Thus, there is no assurance that the test animals were exposed to Cry 1Ab or Cry2Ae in the diet throughout the study , and the study cannot be used for avian risk assessment. A request to bridge to data from this study to the assessment of Cry 1Ab expressed in T304 - 40 cotton (MRID 47871404) was not reviewed because the study was determined to be unacceptable. Avian Oral Toxicity Rationale was presented to justify waiving the avian oral toxicity data requirement for the Cry 1Ab protein expressed in Event T304 - 40 cotton (MRID 47903201) . The justification rested primarily on the expected minimal exposure of birds to Cry 1Ab in T304 - 40 cotton as a result of their avoidance of cotton seed consumption, as well as the lack of observed effects of other Bt proteins that have been observed on birds (Mendelsohn et al. 2003).

Avian Inha lation Toxicity Avian inhalation toxicity

testing is ty

pically not required for Bt cotton event registrations, since exposure to Cry

proteins via inhalation is not expected . The applicant submitted data waiver rationale (MRID 47871403) that is consistent with BPPD’s position on inhalation exposure for birds in Bt cotton, and this rationale is acceptable . An avian inhalation test is not required.

**b .**  **Wild mammals** BCS submitted acceptable data waiver rationale (MRID 47871403) to justify that testing wi th wild mammals is not necessary . An acute oral toxicity study with mice (MRID 47651103) has been submitted to EPA in support of the T304 - 40 registration, and in this study no mortalities, clinical signs of treatment - related effects, or treatment - related e ffects on body weight were observed on mice dosed at 2000 mg/kg bod y weight . This study has been determined to be acceptable . There is no indication that laboratory

species would not be representative of wild mammals in this case; therefore, this information is suitable for use in assessment of risk to wild mammals, and no additional testing with wild species is necessary .

**c .**  **Aquatic species** Freshwater animals Data waiver rationale (MRID 47871403) was submitted to fulfill the data requirements for freshwater fish for Cry 1Ab in T304 - 40 cotton . Justification rested primarily on the lack of expected exposure of aquatic environments to Cry proteins produced in cotton, and this rationale was determined to be sufficient . Cotton pollen is widely known to be “sticky” (i.e., to form clumps) and too heav y to be dispersed b y wind . As a result, little pollen is expected to move o ff field and enter aquatic s ys tems . BPPD has also concluded that little Bt cotton plant matter is expected to move off field (e.g., see U.S. EPA 2008a) . Therefore, exposure in aquatic sy stems is expected to be low, and testing of freshwater fish is determi ned to be unnecessary .Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 69 of 153 Because of concerns raised in a study with the leaf shredding (caddis fl y) trichopteran, Lepidostoma liba (Rosi - Marshall, et al. 2007), EPA is requiring applicant s of Bt corn and cotton to submit 7 - 10 day freshwater invertebrate toxicity studies to reduce uncertaint y regarding this issue . A 10 - day study with Daphnia is available with which to determine the risk to freshwater invertebrates as a result of exposure to Cry 1Ab in T304 - 40 cotton. Daphnia were exposed to a nominal concentration of 48 μg Cry1Ab/L, which is based on the mean expression level of protein present in one kilogram of pollen, dispersed in one liter of water (see MRID 47634803) . At this concentration, wi th exposure in both water and in food treated with Cry 1Ab, no mortalities and no significant differences in the number of offspring produced were observed . Therefore, the EC 50 was determined to be > 48 μg Cry 1Ab/L , and the NOAEC was determined to be ≥ 48 μ g Cry 1Ab/L.

Estuarine and marine animals BPPD ty pically waives toxicity

studies with estuarine/marine fish and invertebrates based on an expected minimum exposure in these environments; however, the applicant submitted data waiver rationale (MRID 47871403) to fulfill data requirements for estuarine and marine animals . The waiver rationale presented was the same as that discussed above for freshwater fish and was determined to be sufficient based on BPPD’s prev ious conclusions about exposure to Cry proteins in aquatic environments. **d. Terrestrial and aquatic plant species**  BPPD ty pically waives nontarget plant testing for Bt Cry proteins, since the active ingredient is an insect toxin ( Bt  - endotoxin) that has never shown an y t oxicity to plants . The applicant submitted acceptable data waiver rationale (MRID 47871403) that was consistent with BPPD’s position on nontarget plant risks to Bt  - endotoxin.

**e .**  **Invertebrate species** In a study with the lad ybird beetle ( Coleomegilla maculata , MRID 47634815), larvae were exposed to a nominal concentration of 19 μg Cry 1Ab/g of artificial diet for 21 day s . This amount is meant to represent 10X the mean expression level of Cry 1Ab in the cotton plants . Larval and adult mortality , time to pupation, time to adulthood, and adult weight did not differ significantl y from the negative control group . Larval mortality

was 7.5% and 12.5% in the treated and control groups, respectivel y; adult mortality was 0% and 2. 9% in the treated and control groups, respectively . The nominal L C 50 was determined to be >19 μgCry 1Ab/g of diet and the NOAEC was determined to be ≥ 19 μg Cry 1Ab/g of diet. A study with green lacewing ( Chrysoperla rufilabris , MRID 47692701) larvae expos ed 25 individuals to a nominal concentration of 20 μg Cry 1Ab/g of artificial diet for 10 day s . The amount to which the larvae were exposed throughout the test was meant to represent 10X the mean fresh weight expression level of Cry 1Ab in T304 - 40 cotton plants . Mortality and pupation measured in the test group (94% and 76%, respectivel y) did not differ significantl y from the negative control (96% and 76%, respectively). Based on the results, the L C 50 for Cry 1Ab in C. rufilabris is >20 μg a.i./g and the NOAEC was determined to be ≥ 20 μg Cry 1Ab/g of diet. Collembola (

Folsomia candida ) are soil dwelling insects that consume detritus . Ina 28 - day study (MRID 47651106), 40 collembola were exposed to 20 μg Cry 1Ab/g diet and observed for mortality and number of o ffspring produced per adult . Mortality of the collembola was 25% for the groups exposed toBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 70 of 153 Cry 1Ab and 12.5% for the control groups . The number of offspring produced per adult was 46.9 and 43.9 for the Cry 1Ab treated and control groups, respectively . Neither of these endpoints differed significantl y between treated and control groups . Based on the results, the, the L C 50 based on the nominal concentration is > 20 μg Cry 1Ab/g diet and the NOAEC was established to be ≥ 20 μg Cry 1Ab/g diet. Data waiver r ationale (MRID 47634818) was presented to justify that toxicity

testing for Cry 1Ab as expressed in T304 - 40 cotton is not necessary with the earthworm . The 14 - day L C 50 for earthworms exposed to Cry 1Ab derived from E. coli was previously determined to be > 2 00 mg/kg of artificial soil ( U.S. EPA 2010, Mendelsohn et al. 2003) . Since there is no evidence of toxicity of Cry 1Ab to earthworms, BPPD determined that additional testing with earthworms is not needed. A study with honey

bee ( Apis mellifera ) was also performed . In this study , 2 - 3 day old honey

bee larvae were exposed to a 10 μLdroplet of solution containing 20 μg/mL Cry 1Ab, which was intended to represent >10X the mean fresh weight expression concentration of Cry 1Ab in T304 - 40 cotton leaves, and > 400 times the exposure to the amount of Cry 1Ab expressed in T304 - 40 pollen . Mortality at capping and at emergence was observed, and neither of these differed significantl y from controls . Emergence was considered to be complete on Day 16 . Of the larvae treated with Cry 1Ab, 85.25% survived to capping, and all of these survived to emergence; 82.5% of the controls survived to capping and all of these also survived to emergence . Therefore, the LC 50 was determined to be > 20 μg Cry 1Ab/mL. **2 .**  **Non -**  **target Organism Stu dy Summaries for GHB119 expressing Cry2Ae**  **a .**  **Avian Wildlife** Avian Dietary Toxicity The broiler chicken dietary study described in the summaries above for T304 - 40 (MRID 47640011) was also submitted for this data requirement for Cry2Ae expressed in GHB119 cotton . Since there is no assurance that the test animals were exposed to Cry 1Ab or Cry2Ae in the diet throughout the stud y, it cannot be used for avian risk assessment for Cry2Ae. A request to bridge to data from this study to the assessment of Cry2Ae expressed in GHB119 cotton (MRID 47871504) was not reviewed because the study was determined to be unacceptable. Avian Oral Toxicity Rationale was presented to justify waiving the avian oral toxicity data requirement for the Cry2Ae protein express ed in Event GHB119 cotton (MRID 47903301) . The justification rested primarily on the expected minimal exposure of birds to Cry2Ae in GHB119 cotton as a result of their avoidance of cotton seed consumption, as well as the lack of observed effects of other B t proteins that have been observed on birds . However, this rationale does not account for other routes of oral exposure in birds (e.g., consumption of insects) . BCS submitted a feeding study in which Northern bobwhite were exposed to raw Event GHB119 cot ton seed meal (containing Cry2Ae protein) at 10% of their daily diet for five day s (MRID 48177501) . No mortality or behavioral abnormalities were observed, and significant differences in body weight gain or food consumption were not detected . This study is sufficient to show that hazard to birds exposed to the dietary concentration tested (up to 0.241 ppm diet or 10% of GHB119 cottonseed) is not expected . ThisBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 71 of 153 study does not sufficiently

test hazard at higher dietary concentrations . EPA has previousl y deter mined that an exposure level of 10% raw cottonseed meal containing Cry protein is a sufficient exposure level for determining the effects of Cry proteins expressed in cotton in birds (e.g., U.S. EPA 2005a, U.S. EPA 2005b, U.S. EPA 2008) . Based on these exp osure assumptions, the study was determined to be acceptable; however, there is uncertainty about the potential effects to birds if field exposure occurs at higher concentrations . It is noted that no avian toxicity data submitted to the Agency to date has shown detrimental effects of Cry proteins on birds, including data on the Cry2Ab2 protein ( U.S. EPA 2005a) . Therefore, BPPD concludes that exposure to Cry2Ae is not likely to cause adverse effects in birds . However, a stud y in which birds are exposed to hi gher doses of Cry2Ae would reduce uncertainties related to actual field exposure.

Avian Inhalation Toxicity Avian inhalation toxicity

testing is ty

pically not required for Bt cotton event registrations, since exposure to Cry

proteins via inhalation i s not expected . The applicant submitted data waiver rationale (MRID 47871503) that is consistent with BPPD’s position on inhalation exposure for birds in Bt cotton, and this rationale is acceptable . An avian inhalation test is not required.

**b. Wild mammals**  The applicant submitted acceptable data waiver rationale (MRID 47871503) to justify that testing with wild mammals is not necessary . An acute oral toxicity study with mice (MRID 47076902) has been submitted to EPA in support of the GHB119 registr ation, and in this study no mortalities or treatment related signs of adverse effects were observed on mice dosed at 2000 mg/kg body weight . This study was determined to be acceptable. This information is suitable for use in assessment of risk to wild mamm als, and no additional testing with wild species is necessary at this time.

**c. Aquatic animals**  Data waiver rationale (MRID 47871503) was submitted to fulfill the data requirements for freshwater fish for Cry2Ae in GHB119 cotton . Justification rested primarily on the lack of expected exposure of aquatic environments to Cry proteins produced in cotton, and this rationale was determined to be sufficient . The same rationale as presented above for Cry 1Ab was presented for Cry2Ae, and this is consistent with BPPD’s previous conclusions that little aquatic exposure is expected from the use of cotton expressing Cry

proteins . Since BPPD has concluded that exposure in aquatic s ys tems is expected to be very low, testing of freshwater fish is unnecessary . A 10 - day study with Daphnia magna is available with which to determine the risk to freshwater invertebrates as a result of exposure to Cry 1Ab in T304 - 40 cotton . Daphnia were exposed to a nominal concentration of 330 μg Cry2Ae/L contained in water alone and wate r and food . This exposure level is based on the fresh weight expression level of protein present in one kilogram of pollen (see MRID 47641933), dispersed in one liter of water . No mortalities and no significant differences in the number of offspring produc ed were observed . Therefore, the EC 50 was determined to be > 330 μg Cry2Ae/L, and the NOAEC was determined to be ≥ 330 μg Cry2Ae/L .

Estuarine and marine animalsBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 72 of 153 BPPD ty pically waives toxicity

studies with estuarine/marine fish and invertebrates; however, the applicant submitted data waiver rationale (MRID 47871503) to fulfill data requirements for estuarine and marine animals . The waiver rationale presented was th e same as that discussed above for exposure of freshwater fish to Cry 1Ab and was determined to be sufficient based on BPPD’s conclusions about exposure in aquatic environments.

**d. Terrestrial and aquatic plant species**  BPPD ty pically waives nontarget plant testing for Bt Cry proteins, since the active ingredient is an insect toxin ( Bt  - endotoxin) that has never shown any toxicity to plants . The applicant submitted acceptable data waiver rationale (MRID 47871503) that was consistent with BPPD’s positio n on nontarget plant risks to Bt  - endotoxin .

**e .**  **Invertebrate species** A study with lady bird beetle ( Coleomegilla maculata , MRID 47641915) larvae exposed 40 individuals to a nominal concentration of 64 μg Cry 1Ab/g of artificial diet for 21 day s . This amount was approximately

7X the fresh weight expression level of Cry2Ae in the leaves of GHB119 cotton plants (see MRID 47641903) . Larval and adult mortality , time to pupation, time to adulthood, and adult body weight did not differ significantl y from the negative control group . Larval mortality was 5.0% and 12.8% in the treated and corresponding control group, respectivel y; adult mortality was 0% and 3.3% in the treated and control groups, respectivel y . The nominal L C 50 was determined to be >64 μgCry 1Ab/g diet and the NOAEC based on development and body weight is ≥ 64 μg Cry2Ae/g diet. A study with green lacewing ( Chrysoperla rufilabris , MRID 47692501) larvae exposed 25 individuals to a nominal concentration of 10 μg and 100 μg Cry2Ae/g of arti ficial diet for 10 day s . These concentrations were meant to test effects at 1X and 10X the fresh weight expression level of Cry2Ae as measured in the leaves of GHB119 cotton plants (see MRID 47641903) . Mortality and percent pupation were measured and compared to negative and buffer controls, and no significant differences were observed . Survival of the 10 μg and 100 μg Cry2Ae/g diet test groups and the buffer controls was 100%, 96%, and 100%, respectively . Pupation in these groups was 84%, 72%, and 88%, res pectively . The results establish a nominal L C 50 of >100 μg Cry2Ae/g diet and a nominal NOAEC of ≥ 100 μg Cry2Ae/g diet. Collembola (

Folsomia candida ) are soil dwelling insects that consume detritus . Ina 28 - day single - dose study (MRID 47641917a), collemb ola were exposed to 66 μg Cry2Ae/g diet and observed for mortality and number of offspring produced per adult . Mortality of the collembola was 27.5% for the groups exposed to Cry2Ae and 22.5% for the control groups . The number of offspring produced per adu lt was 10.3 and 44.5 for the Cry2Ae treated and control groups, respectively, and the difference was statistically

significant . Because of this outcome, a definitive test was performed on collembolan exposed to nominal concentrations of 9.9, 20, 40, 80, or 160 μg Cry2Ae/g diet (MRID 47641917b) . Negative and positive controls were also included, and a bioassay on a sensitive species (

Heliothis virescens ) confirmed bioactivity . Mortalit y of the test groups from lowest to highest concentration was 2.5%, 0%, 0% , 13%, and 0%, and mortality in the negative controls was 7.5% . Mean number of offspring did not differ significantly among any

of these groups . With this test, the nominal L C50 was determined to be >160 μg Cry2Ae/g diet and the NOAEC was determined to be ≥160 μg Cry2Ae/g diet .Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 73 of 153 A 14 - day earthworm toxicity

study (MRID 47641918) was submitted, wherein earthworms were exposed to 100 mg Cry2Ae/gram of artificial soil . This concentration was intended to be approximately 10X the fresh weight concentration of C ry2Ae expressed in the leaves of GHB119 cotton plants (see MRID 47641903) . The average burrowing time was equal for both the treated and control groups, and no mortalities were observed in either group . Mean weight decreased b y 22% in the control group and by 20% in the test material group, which was expected since the artificial soil did not contain nutritive material . Based on the results the nominal LC 50 was determined to be >100 mg Cry2Ae/kg, and the NOAEC was ≥100 mg Cry2Ae/kg. A study with honey

bee ( Apis mellifera ) was also performed . In this study , 2 - 3 day old honey

bee larvae were exposed to a 10 μLdroplet of solution containing 50 μg/mL Cry 1Ab, which was intended to represent >50X the exposure to the amount of Cry2Ae expressed in GHB119 cotton p ollen (assuming the test solution is approximately the same weight, see MRID 47641913) . Mortality at capping and at emergence were observed, and neither of these differed significantly from controls . Emergence was considered to be complete on Day 16 . Of th e larvae treated with Cry2Ae, 93.75% survived to capping, and all of these survived to emergence; 95% of the controls survived to capping and all of these also survived to emergence . Therefore, the LC 50 is >50 μg Cry2Ae/mL. **3 .**  **Soil Fate Studies for Cry1Ab and Cry2Ae**  Soil organisms may

be exposed to Cry 1Ab and Cry2Ae protein through contact with cotton plant roots (by direct feeding), cotton plant root exudates, incorporation of above - ground plant tissues into soil following harvest, or b y soil - deposited pollen. Some evidence suggests that soils which are high in clay s and humic acids are more likely to bind Cry

protein . However, neutral pH soils tend to have high microbial activity

and microbes contribute to Cry protein degradation . In addition, a study o n the release of Cry proteins in the root exudates of Bt cotton has shown that no Cry proteins were detected immunologically

or by larvicidal assay

in an y soil or hydroponic solution in which Bt cotton had been grown (Saxena and Stotzky , 2001) . The weight of evidence indicates that Cry proteins do not accumulate in soil to arthropod - toxic levels. Because Cry proteins are toxins derived from soil inhabiting bacteria, Bacillus thuringiensis , and found in commercial microbial insecticides (De Maagd et al. , 2003 and Graser and Song, 2006), the degradation of Cry 1Ab and Cry2Ae from Event T304 - 40 and Event GHB119 cotton is expected to be similar to that of these proteins that are produced naturall y from soil bacteria . Nonetheless, BPPD required the foll owing soil fate evaluations to support the Event T304 - 40 and GHB119 Bt cotton registrations . These studies are summarized here, and detailed evaluations are provided in their associated DERs. A laboratory

aerobic soil degradation study was conducted to de termine the DT 50 (time for 50% of the initial concentration of bioactive material to dissipate) for Cry 1Ab protein . Soil used in the study was collected from three locations throughout the region where transgenic cotton is grown, and each was of a different texture (Proctor, AR: clay loam; Senatobia, MS: silt loam; East Bernard, TX: sandy clay loam) . Each soil was treated once with 15 μg/g bacterially - produced Cry 1Ab, and then sampled periodically over 45 day s . These samples were used in sensitive insect bi oassay s . The bioassay used tobacco budworm (

Heliothis virescens ) larvae exposed to the soil samples incorporated into lepidopteran diet. A concurrent six - day

dose response study

with H. virescens larvae exposed to 0.004 to 0.28 μg/g Cry 1Ab in the soils was conducted to generate a dose - response curve to determine the degradation of Cry 1Ab over time based on the bioactivity response. The DT 50 was determined from the Cry 1Ab concentration first - order exponential regression curve. The average DT 50 for the three soils was 3.6 day s . The soilsBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 74 of 153 were also anal yz ed for Cry 1Ab content by enz ym e linked immunosorbent assay , which showed that Cry 1Ab steadily

degraded in soil during the study . A similar study

was conducted to determine the DT 50 for Cry 2Ae in soil . Soils were collected from the same locations as above for the study with Cry 1Ab . The soils were treated once with 50 μg/g Cry2Ae and then sampled periodically over 45 day s . A sensitive insect bioassay using the same species ( Heliothis virescens ) was conducted us ing identical methods as with Cry 1Ab, except that the concurrent six - day dose response study

exposed H. virescens larvae to 0.056 to 3.4 μg/g Cry2Ae to generate a standard curve for determination of Cry2Ae concentration based on bioactivity response . Based on the bioassay s, the average DT 50 was determined to be 3.4 days . The soils were also anal yz ed for Cry2Ae content by

enzy me linked immunosorbent assay , which showed that Cry2Ae steadil y degraded in soil during the stud y. These studies utilized field soil spiked with purified insecticidal protein produced b y bacteria. This approach is useful because dose responses can be easily quantified. However, the degradation and accumulation of Bt Cry

proteins found within decayingplant tissue may behave differentl y than proteins in artificially

spiked soil . Thus, the presence of low levels of Bt Cry proteins in the soil (at or below the level of detection) is anticipated until all plant tissue is degraded . However, the reviewed data show that Cry 1Ab and Cry2Ae will be quickly degraded upon release from decaying plant tissue . Based on FIFRA Scientific Advisory Panel recommendations and public comments, the Agency has required three ye ar soil fate studies for the currently registered Cry

protein producing crops grown in a variet y of soils and environmental conditions, as a condition of registration . More recentl y, a comprehensive review of all available scientific data on ecological effects of commercially grown GM crops over ten years was completed (Sanvido, et al . 2 007) . The review concluded “none of the laboratory

or field studies suggest accumulation of Bt- toxins in soil over several ye ars of cultivation” and “experience from commercial cultivation indicates that Bt- toxin will not persist for long periods under natural conditions.” The Agency agrees with these conclusions. Collectivel y, the long - term field studies for Bt crops also confirm the previous SAP conclusion that “bioaccumulation is not expected to occur with transgenic proteins because biodegradation mechanisms for proteins are ubiquitous ” (US EPA, 2000) . More importantly , the numerous laboratory studies that demonstrated rapid protein degradation in soil of Bt proteins produced in Bt crops (when performed under realistic environmental conditions) can be considered predictive that Bt protein in soil is not likely to persist or accumulate in soil after continuous cultivation . In light of these published findings and the rapid degradation of Cry 1Ab and Cry2Ae proteins in soil as demonstrated in the insect bioassay s described above, there is no indication that the proteins expressed in Event T304 - 40 and Event GHB119 cotton are likely to persist in soil after continuous cultivation. Therefore, no additional long - term field studies are required for these PIP products. **4 .**  **Supplemental Information** BCS submitted summaries of nontarget organism testing with Cry1Ab and Cry2Ae (MRIDs 47634813 and 47641913, respectively), which summarized the information above, and included an assessment of ecological risk and some discussion of environmental fate . These summaries were determined to be supplemental, but provided little additional information for use in the risk assessment .Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 75 of 153 **C .**  **Environmental Risk Assessment for Event T304**  **- 40 and Event GHB119**  **1 .**  **Effects to Nontarget Wildlife, Invertebrates, and Plants a**  **. Avian Wildlife**  Birds are known to forage in cotton fields . While birds are unlikel y to consume cotton plant material, particularl y cotton seeds, du e to high concentrations of goss ypol, they could be exposed to Cry 1Ab and/or Cry2Ae through consumption of invertebrates that feed on Bt cotton plant tissues . Exposure is expected to be low, and data and rationale were sufficient to conclude that Cry 1Ab an d Cry2Ae are not expected to cause adverse effects in birds . There is some uncertaint y related to the exposure level in the testing with Cry2Ae compared to field exposures, but EPA previously determined that the exposure level in the study

was sufficient f

or avian testing . Testing at higher doses would reduce this uncertainty . **b. Wild mammals** Wild mammals may

be exposed to Cry 1Ab in Event T304 - 40 cotton or Cry2Ae in Event GHB119 cotton by consuming plant parts and/or invertebrates that feed on the cotton plants . Toxicity testing with laboratory

mice showed no adverse of Cry 1Ab or Cry2Ae at doses of 2000 mg/kg body weight each . This amount is much higher than concentrations that wild mammals are expected to encounter in the field . Therefore, based on this information, adverse effects to wild mammals are not expected as a result of the use of Event T304 - 40 and Event GHB119 cotton .

**c. Freshwater animals** BPPD has concluded that exposure in the aquatic environment to the Cry proteins in Bt cotton is expected to be low . Cotton plants are not wind - pollinated, and cotton pollen is widely understood to be spiny and “sticky ,” leading to the formation of clumps of pollen that are not easily dispersed by

wind . As a result, the majorit y of the cott on pollen that is produced is not expected to travel very far off - field . BPPD has previousl y determined that exposure in aquatic environments resulting from deposition of other cotton plant tissues is minimal (e.g., see U.S. EPA 2008a) . Therefore, exposure of Cry 1Ab or Cry2Ae from use of Event T304 - 40 or Event GHB119 cotton, respectively , to freshwater fish and invertebrates is expected to be minimal, and adverse effects to these species are not expected to occur.

**d. Estuarine and marine animals** Significant amounts of Cry 1Ab and Cry2Ae resulting from Event T304 - 40 or GHB119 cotton use, respectivel y, are not expected to reach marine/estuarine areas . Therefore, BPPD concludes that adverse effects to fish and invertebrates in these environments is no t expected .

**e. Terrestrial and aquatic plant species** Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 76 of 153 BPPD ty pically waives nontarget plant testing for Bt Cry proteins, since the active ingredient is an insect toxin ( Bt  - endotoxin) that has never shown any toxicity to plants . Therefore, BPPD has concluded that adverse effects to terrestrial and aquatic plants are not anticipated.

**f. Invertebrate species** Nontarget insects are expected to receive exposure primarily through consumption of Bt cotton pollen and/or nectar, though the y may also consume pest insects that feed on cotton plant tissue or rarel y consume other cotton plant tissues themselves . The principal route of soil - dwelling invertebrates, such as collembola, earthworms, and rove beetles, is assumed to be from decomposin g plant tissue and plant exudates in soil . Studies submitted to the Agency with Cry 1Ab and Cry2Ae in lady bird beetle, green lacewing, collembola, and earthworm showed no adverse effects at concentrations well above those that would be encountered b y nontarget insects in the environment . Therefore, BPPD does not anticipate the occurrence of adverse effects to nontarget invertebrates as a result of the use of T304 - 40 or GHB119 cotton . Additionally , studies with honey bee larvae also showed no adverse effects to honey bees at exposure levels to Cry 1Ab and Cry2Ae above those expected to be encountered in the environment . Therefore risk to honey

bees as a result of the proposed registrations is also expected to be minimal . It should be noted that effects to invertebrates that are recognized as cotton pests (i.e., insects that feed detrimentally

on cotton plants) that are not targeted by the Cry 1Ab and Cry2Ae proteins (but are controlled by

other means) are not included in BPPD’s assessment of risk to nontarget insects . **2 .**  **Effects on Soil Microorganisms** Numerous published studies indicate that exposure to Cry protein produced in Bt PIP crop plants does not adversel y 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 . Inaddition, the soil degradation studies above show that the Cry 1Ab and Cry2Ae proteins degrade qu ickly in soil . In addition, there are several ongoing U.S. Department of Agriculture and EPA Office of Research and Development funded research projects evaluating the effects of Cry protein crops on soil microbial flora. If adverse effects are seen from this or an y other research, the Agency will take appropriate action to mitigate potential risks ( U.S. EPA 2010). With regard to the impact of geneticall y engineered crops on soil, it is important to note that agricultural practices themselves cause large changes in soil and soil microbial composition. Furthermore, factors such variations in seasons and weather, plant growth stage, and plant varieties, independent of being geneticall y engineered, are also responsible for significant shifts in soil microbia l communities. Most studies with genetically

engineered crops to date have shown minor or no effects on soil microbes bey ond the variation caused by the factors listed above. **3 .**  **Horizontal Transfer of Transgenes from Bt**  **Crops to Soil Organisms** The EP A 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 beenBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 77 of 153 engineered into Bt crops are mostly found, or have their o rigin, in soil - inhabiting bacteria. Soil is also the habitat of other toxin - producing bacteria, and transfer of these genes and/or toxins to other microorganisms or plants has not been detected. Furthermore, several published experiments, that were conduct ed to assess the likelihood of HGT, have been unable to detect gene transfer under t ypical 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 T304 - 40 and GHB119 were derived from soil - inhabiting bacteria, the EPA has concluded that the risk of HGT of transgenes found in Cry 1Ab or Cry2Ae producing cotton is low. **4 .**  **Gene Flow and Weediness Potential** Movement of transgenes from crop plants into weeds is a significant concern, due to uncertainty regarding the effect that a new pest resistance gene may have on plant populations in the wild. Under FIFRA, the Agency

has reviewed the potential for gene capture and expression of Cry proteins in commercial Bt cotton by

wild or weed y relatives of cotton in the United States, its possessions or territories. There is a possibility

for gene transfer in locations where wild or feral cotton relatives exist . Therefore, EPA has required stringent sales and distribution restrictions on Bt cotton within these areas to prevent outcrossing or h yb ridization from the crop to sexually compatible relatives . There are onl y fo ur areas in the United States and its territories wherein cultivated cotton has the opportunity to outcross to wild or feral species, which are geneticall y compatible: (1) southern Arizona, (2) Hawaiian islands, (3) southern Florida and 4) Puerto Rico. G. thurberi (Arizona Wild Cotton) is present in the elevated regions of Arizona and does not grow in areas of commercial cotton production. G. thurberi is a diploid and produces sterile, triploid progen y when crossed with the tetraploids G. hirsutum or G. barbadense . In the very south of Florida, feral G. hirsutum exists in apparentl y self - sustaining populations. Since these would readil y cross with cultivated cotton, sale of Bt - Cotton is restricted south of Interstate 60. There is currentl y no commercial c otton production in the southern part of Florida. Evidence from germplasm collections indicates that feral G. barbadense and possibly G. hirsutum exist in the U.S. Virgin Islands . There is presently

no production of commercial cotton in either of these pla ces; hence, outcrossing is not currentl y an issue in these areas . For a detailed review of the Agency ’s assessment of the potential for gene capture and expression of Bt endotoxins by wild or weed y relatives of cotton in the U.S., its possessions or territ ories, see the EPA Biopesticides Registration Action Document (BRAD) for the Bacillus thuringiensis (

Bt ) Plant - In corporated Protectants, dated October 15, 2001. **5 .**  **Conclusions** BPPD concludes that significant adverse effects on birds, mammals, nontarget insects, honey bees, freshwater and marine/estuarine fish and invertebrates, and terrestrial and aquatic plants are not expected as a result of the use of Event T304 - 40 and Event GHB119 in cotton . EPA has also determined that there is no significant risk of gene capture and expression of Cry 1Ab or Cry2Ae protein by wild or weedy relatives of cotton within the U.S. or its territories as long as the current sales and distribution restrictions on Bt cotton are in place. Available data do not indicate that Cry proteins have an y measurable adverse effect on microbial populations in the soil, nor has horizontal transfer of genes from transgenic plants to soil bacteria been demonstrated .Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 78 of 153 **6 .**  **Impacts on Endangered Species** Because of the selectivity of Cry 1Ab and Cry2Ae proteins for lepidopteran species, endangered species concerns are also expressed for insect species in the order Lepidoptera . The Agency has previousl y determined that an y potential concern regarding range overlap of endangered Lepidoptera with cotton production was mainl y restricted to the Kern primrose sphinx moth ( Euproserpinus euterpe ). However, the Agency

determined that cotton is not a host plant for this species nor do host - range considerations for this species or other endangered insects place habitat in or near cotton fields (US EPA 2008a) . As previously

noted, there is a possibility for gene transfer in locations where wild or feral cotton relatives exist . As a result, EPA requires stringent sales and distribution restrictions on Bt cotton within these areas to preclude outcrossing or h yb ridization from the crop to sexually compatible relatives . Therefore, EPA does not expect that any threatened or endangered species will be affected by outcrossing to wild relatives or b y competiti on with such entities. Since Cry 1Ab has not been shown to have toxic effects on mammals, birds, freshwater invertebrates, terrestrial nontarget invertebrates, and plants, and exposure is not anticipated in aquatic environments . Therefore, the Agency

makes a “No Effect” determination for direct and indirect effects to listed species of these taxa for Cry 1Ab expressed in Event T304 - 40 cotton . Since Cry2Ae has not been shown to have toxic effects on birds, mammals, freshwater invertebrates, terrestrial nont arget invertebrates, and plants, and also because exposure is not anticipated in aquatic environments, the Agency makes a “No Effect” determination for direct and indirect effects to listed species of these taxa for Cry2Ae expressed in Event GHB119 cotton. **D .**  **Data Needed to Confirm T304 -**  **40 and GHB119 Non -**  **Target Hazard Assessment** BPPD has sufficient information to believe that there is no significant risk from the proposed uses of Event T304 - 40 and Event GHB119 cotton to non - target avian and mammalian wil dlife, terrestrial invertebrates, fish and aquatic invertebrates, and plants and soil organisms . As described above, uncertaint y related to testing with Cry2Ae in birds would be reduced b y additional testing at higher doses . **II** . **Environmental Risk Asses sm**  **ent for Cry1Ab and Cry2Ae expressed in T304 -**  **40 X GHB119**  **(TwinLinkTM) Cotton Hybrid A**  **. Background**  BCS developed T304 - 40 x GHB119 (TwinL inkTM ) cotton by conventional breeding of transgenic event T304 - 40 cotton and transgenic event GHB119 cotton, which express the Cry 1Ab and Cry2Ae insecticidal proteins, respectivel y, for control of certain lepidopteran pests. Event T304 - 40 expresses a Cry 1Ab gene, which is derived from the soil bacterium, Bacillus thuringiensis ( Bt) subsp. berliner , and Event GHB119 expresses a Cry2Ae gene derived from Bt subsp. dakota . **B .**  **Ecological effects data for T304 -**  **40 x GHB119 (TwinLink TM** **) cotton hybrid** Environmental effects data are available with Cry1Ab and Cry2Ae . These can be used in an assessment of r isk for TwinLinkTM cotton, provided there is no evidence of a s ynergistic association betweenBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 79 of 153 Cry 1Ab and Cry2Ae and protein expression levels of Cry 1Ab and Cry2Ae are equivalent between the T304 - 40 and GHB119 parent lines and the TwinLinkTM hy brid . Data have been submitted with which to determine the potential for sy nergy and protein expression levels . Additional toxicity data have been submitted in which the combination of these proteins has been tested ( TwinL inkTM plant material was used as the test substanc e) with some nontarget species, including honey bee, collembola, and broiler chicken . Additionall y, data waiver rationales were presented for freshwater fish, marine/estuarine fish and invertebrates, nontarget plants, avian oral and inhalation toxicity , and wild mammals . Data available on TwinL inkTM cotton is summarized in Table 3, and descriptions of these studies and waiver rationales are provided below . Additional review of these studies is provided in their associated DERs. For most studies required, BCS is seeking to bridge from data generated on the proteins expressed individually

in the parent lines to the combined event . Data showing a lack of sy nergism between the Cry 1Ab and Cry2Ae proteins is necessary for bridging, as well as data showing equivalent expression of the proteins within the TwinL inkTM plants and the individual parent lines . A sy nergism study

was submitted and is discussed below . Studies showing equivalence are currently under review, and the acceptability

of the data on each protein expr essed individually for bridging is dependent on a finding of equivalence . The equivalence data will be evaluated in the product characterization review for TwinL inkTM ( U.S. EPA 2011). **Table C.**  **3 .**  **Summary of environmental effects studies and w aiver**  **justifications for T304 -**  **40 x GHB119 (TwinLink** 

**TM )**  **submitted to comply with data requirements published in 40 CFR § 158.2150**  **(d). Data**  **Requirement OPPTS** 

**Guideline Test Substance**  **C.3. TwinLinkTM -**  **Ecological Effects -**  **Results Summary and Classification**  **MRID N**  **o.** Avian dietary testing, broiler chicken, Gallus domesticus 885.4050 TwinLinkTM (T304 - 40 x GHB119) cotton seed meal A 42 - day dietary study showed no adverse affects to broiler chickens when fed a diet composed of 10% TwinLinkTM cottonseed meal containing both Cry1Ab and Cry2Ae . However, the presence of the proteins in the test material was not confirmed. **Classification: Unacceptable** 47640011

Avian inhalation testing 885.4100 N/A **Acceptable waiver rationale** 47871603 Avian oral testing, Northern bobwhite Colinus virginianus 850.2100 N/A Data waiver rationale was submitted to justify that testing is not necessary on the basis of lack of exposure. **Classification : Supplemental**  (subsequent testing with Cry2Ae was performed) 47903101

Freshwater fish testing, 885.4200 N/A **Acceptable waiver rationale** 47871603 Estuarine and marine animal testing 885.4280 N/A **Acceptable waiver rationale** 47871603 Non - target plant testing 885.4300 N/A **Acceptable waiver rationale** 47871603 Non - target insect testing, collembola, Folsomia candida 885.4340 Lyophilized TwinLinkTM cotton leaves Collembola ( Folsomia candida ) were fed diet containing nominal concentrations of 0, 5, 20, or 50% lyophilized TwinLinkTM cotton leaf, or 50% lyophilized ne ar isoline cotton leaf for 28 days. No statistically significant differences in survival or reproduction among the test material and control groups were observed, indicating that exposure to TwinLinkTM plant tissue at the levels tested has no effect on surv ival or reproduction of Collembola. **Classification: Acceptable** 47640010

Honeybee testing, Honeybee larvae, 885.4380 TwinLinkTM pollen Honey bee larval cells were treated with 10 μL of a solution containing 2 mg of TwinLinkTM pollen or near isoline 47640009Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 80 of 153 **Data Requirement**  **OPPTS**

**Guideline Test Substance**  **C.3. TwinLinkTM -**  **Ecological Effects -**  **Results Summary and Classification**  **MRID N**  **o.** Apis mellifera pollen and observed for survival up to the time of em ergence . A significant difference in survival between these two groups was not observed, indicating a lack of adverse effects of TwinLinkTM pollen at the tested level on honey bee larvae . **Classif ication: Acceptable**  Synergism N/A Sensitive insect dose - response bioassays with T304 - 40, GHB119, and Twin LinkTM plant material showed that Cry1Ab and Cry2Ae expressed in TwinLinkTM plants are not synergistic . **Classification: Acceptable** 48480009

**1 .**  **Nontarget Organism Study Summaries for TwinLink TM**  **a .**  **Avian Wildlife** Avian Dietary Toxicity The broiler chicken feeding stud y (MRID 47640011) discussed above in the assessment for Events T304 - 40 and GHB119 was also submitted to provide data on toxicity for TwinL inkTM . As stated above, since there is no assurance that the test animals were exposed to Cry 1Ab or Cry2Ae in the diet throughout the stud y, the study cannot be used for avian risk assessment. Avian Oral Toxicity Rationale was pres ented to justify waiving the avian oral toxicity data requirement for the Cry 1Ab and Cry2Ae proteins expressed in Event T304 - 40 x Event GHB119 cotton (MRID 47903101) . The justification rested primarily on the expected minimal exposure of birds to Cry 1Ab an d Cry2Ae in TwinL inkTM cotton as a result of their avoidance of cotton seed consumption, as well as the lack of observed effects of other Bt proteins that have been observed on birds . However, this rationale did not account for other routes of oral exposure in birds (e.g., consumption of insects) . BPPD has concluded that currentl y registered Cry proteins, including Cry1Ab, do not pose hazards to birds (Mendelsohn et al. 2003; U.S. EPA 2005a, 2005b, 2008a, 2008b, 2010) . BPPD also subsequently received additional data to show that Cry2Ae protein is not likely to cause adverse effects when raw Event GHB119 cottonseed meal is included at 10% in the diet (MRID 48177501) . See the discussion of sy nergism and protein expression equivalence below; if protein expression is shown to be equivalent between TwinLinkTM cotton and the T304 - 40 and GHB119 parent lines, then bridging these data to TwinL inkTM is acceptable.

Avian Inhalation Toxicity Avian inhalation toxicity

testing is ty

pically not required for Bt cott on event registrations, since exposure to Cry

proteins via inhalation is not expected . The applicant submitted data waiver rationale (MRID 47871603) that is consistent with BPPD’s position on inhalation exposure for birds in Bt cotton, and this rationale i s acceptable . An avian inhalation test is not required.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 81 of 153 **b. Wild mammals** BCS submitted acceptable data waiver rationale (MRID 47871603) to justify that testing with wild mammals is not necessary . As described above in the risk assessment of Cry 1Ab in T304 - 40 and Cry2Ae in GHB119 cotton, acute oral toxicity studies with mice are available, and the Agency has no evidence to suggest that laboratory mammals are insufficient surrogates for determining toxicity to wild mammals . See the discussion of sy ner gism and protein expression equivalence below; if protein expression is shown to be equivalent between TwinL inkTM cotton and the T304 - 40 and GHB119 parent lines, then bridging these data to TwinL inkTM is acceptable . **c. Freshwater animals** Data waiver rationale (MRID 47871603) was submitted to fulfill the data requirements for freshwater fish for Cry 1Ab and Cry2Ae in TwinL inkTM . Justification rested primarily on the lack of expected exposure of aquatic environments to Cry proteins produced in cotton, and this rationale was determined to be sufficient . BPPD previously determined that exposure to Cry proteins in aquatic environments as a result of registered uses of transgenic cotton is very low (e.g., see US EPA 2008a), and the data waiver rationale was co nsistent with BPPD’s conclusion. Toxicity data with Daphnia are available for both Cry 1Ab and Cry2Ae for use in the TwinL inkTM risk assessment . Results of these studies are presented above . **d. Estuarine and marine animals** The Agency

t ypically waive s toxicity studies with estuarine/marine fish and invertebrates for Bt Cry proteins; however, the applicant submitted data waiver rationale (MRID 47871603) to fulfill data requirements for estuarine and marine animals . The waiver rationale presented was th e same as that discussed above for freshwater fish and was determined to be sufficient based on the Agency ’s conclusions about exposure in aquatic environments . **e. Terrestrial and aquatic plant species**  The Agency

t ypically waives nontarget plant tes ting for Bt Cry proteins, since the active ingredient is an insect toxin (

Bt  - endotoxin) that has never shown any toxicity to plants . The applicant submitted acceptable data waiver rationale (MRID 47871603) that was consistent with the Agency ’s position o n nontarget plant risks to Bt  - endotoxin . **f. Invertebrate species** A 28 - day study with collembola (

Folsomia candida ) was conducted to determine the effects of TwinL inkTM plant tissue on this soil invertebrate (MRID 47640010) . Collembola ( Folsomia candida ) were provided diet containing nominal concentrations of 5.0, 20, or 50% transgenic l yophilized TwinL inkTM cotton leaf

. The test also included a negative control group of collembola fed diet without lyophilized TwinL inkTM cotton leaf, a control substance group fed diet containing 50% l yophilized nontransgenic Coker 315 (near isoline) cotton leaf, and a positive control group fed diet containing thiodicarb . Mortality

in the 5%, 20%, and 50% TwinL inkTM test groups was 12.5%, 10.0%, and 7.5%, res pectivel y, while cumulative number of offspring per female were 42.2, 41.7, and 33.4, respectively .Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 82 of 153 Mortality in the negative control and Coker 315 groups was 2.5% and 5.0%, respectivel y, and cumulative number of offspring per female was 26.9 and 38.6, res pectively . Mortalit y and number of offspring were not statistically significant different among any groups tested . Response in the positive control group was as expected. The results of the study show that exposure to transgenic TwinLinkTM cotton leaves at 5.0, 20, and 50% concentration in the diet has no significant effect on survival or reproduction of Collembola . A study was also conducted to determine the potential dietary effects of TwinL inkTM pollen when administered to 2 - 3 day

old honey

bee larvae (

A pis mellifera , MRID 47640009). Honey bee larval cells were treated with 10 μLof a 30% solution containing 2 mg of TwinLinkTM pollen . Additional treatment groups included: 1) a negative control (pollen from Coker 315) in combination with 30% sucrose; and 2) a positive control (potassium arsenate in 30% sucrose at 2,000 μg/mL) . The amount of pollen administered to the larvae was approximately the maximum amount that is ty picall y fed to honey

bee larvae (Babendreier et al. 2004) . Emergence was complete on Day 15 of the study . Larval survival and adult emergence was determined (96.25% for TwinL inkTM ; 97.5% for the Coker 315; and 0% for the reference substance). Based on statistical anal ys is, there were no significant differences in survival between the TwinL inkTM treated larvae and those treated with Coker 315 pollen. Additionally , if bridging criteria are satisfied, then data developed for the Cry 1Ab and Cry2Ae proteins expressed individually

in Event T304 - 40 and Event GHB119 cotton plants, respectivel y, can also be used for the ecological risk assessment for TwinLinkTM . **g .**  **Supplemental Information** BCS submitted a summary of nontarget organism testing with Cry 1Ab and Cry2Ae (MRIDs 47640008), which summarized the toxicity information and data waiver rationale for these proteins and TwinL inkTM and included an assessment of ecological . This summary was determined to be supplemental, but provided little additional information for use in the risk assessment . **2 .**  **Synergism of Cry1Ab and Cry2Ae proteins** A sensitive - insect dose - response bioassay with Heliothis virescens neonate larvae was performed to determine whether Cry 1Ab and Cry2Ae expressed in TwinL inkTM cotton plants exhibit sy nergistic toxicity . Leaf material from the single event cotton plants expressing eithe r Cry 1Ab or Cry2Ae and the combined event (TwinL inkTM ) cotton plants were fed to the larvae at multiple concentrations determine the LD 50 s for the proteins within each plant. Expression of each protein was determined to be equivalent between the TwinL inkTM p lants and the individual parent lines . Based on the predicted and actual LD 50 s for the proteins in each ty pe of plant, a conclusion of no sy nergism was made . Therefore, the results of toxicity tests with each individual protein are assumed to adequatel y re present the toxicity of these proteins when expressed in TwinL inkTM cotton, assuming equivalent protein expression. **3 .**  **Protein Expression Comparability** Data have been submitted to the Agency showing the proteins expression of Cry 1Ab in event T304 - 40 cott on, Cry2Ae in event GHB119 cotton, and both proteins in TwinL inkTM cotton . Additional data have been submitted to determine whether the relative potency of plant - produced Cry 1Ab and Cry2Ae is similar to the purified bacteriall y - produced proteins used in the toxicity testing with each individual protein . These data are currentl y in review . If these data show that each event produced comparableBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 83 of 153 protein expression levels of Cry 1Ab and Cry2Ae in each respective parent line as well as the T304 - 40 x GHB119 h ybrid, and that the relative potency of plant - produced and bacteria - produced Cry 1Ab and Cry2Ae are equivalent, then bridging from tests for the individual events is appropriate for support of the T304 - 40 x GHB119 (TwinL inkTM ) registration . **C .**  **CONCLUSION** As lo ng as BCS shows that Cry 1Ab and Cry2Ae protein expression is equivalent between TwinLinkTM cotton and the individual T304 - 40 and GHB119 parent lines, then no unreasonable adverse effects to nontarget birds, mammals, aquatic animals, terrestrial and aquatic plants, and nontarget invertebrates will occur as a result of exposure to these proteins from registered use of TwinL inkTM cotton . This conclusion is based on the environmental risk assessments for Cry 1Ab and Cry2Ae proteins expressed individually

in Event T304 - 40 and GHB119 cotton, as well as additional data developed with TwinL inkTM cotton plant material . Both Cry 1Ab and Cry2Ae have not been shown to have toxic effects on birds, mammals, freshwater invertebrates, terrestrial nontarget invertebrates, and p lants, and because exposure is not anticipated in aquatic environments . If protein expression is shown to be equivalent, then the Agency makes a “No Effect” determination for direct and indirect effects to listed species of these taxa for these proteins as expressed in TwinL inkTM cotton . **E. References**  Babendreier, D., N. Kalberer, J. Romeis, and P. Fluri . 2004. Pollen consumption in honey bee larvae: a step forward in the risk assessment of transgenic plants . Apidologie 35: 293 - 300. Marvier, M., McCreed y, C., Regetz, J. & Kareiva, P . 2007 . A meta - anal ys is of effects of Bt cotton and maize on nontarget invertebrates. Science 316: 1475

– 1477. National Academ y of Science (NAS) . 2000 . Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation is available from the National Academy Press, 2101 Constitution Avenue, N.W., Lockbox 285, Washington, DC 20055; (800) 624 - 6242 or (202) 334 - 3313 (in the Washington metropolit an area); http://www.nap.edu . Romeis, J., M. Meissle, and F. Bigler . 2006 . Transgenic Crops expressing Bacillus thuringiensis toxins and biological control. Nature Biotechnology 24: 63 - 71. Rosi - Marshall E. J., J. L . Tank, T. V. Roy er, M. R. Whiles, M. Evans - White, C. Chambers, N. A. Griffiths, J. Pokelsek, and M. L . Stephen. (2007). Toxins in transgenic crop by products may affect headwater stream ecos ys tems. PNAS : 104(41): 16204

– 16208. Sanv ido,O., Romeis, J., Bigler, F. 2007. Ecological Impacts of Geneticall y Modified Crops: Ten Years of Field Research and Commercial Cultivation. Adv Biochem Engin/Biotechnol 107: 235

–278. Saxena, D. and Stotzky , G. 2001 . Bacillus thuringiensis ( Bt) toxin released from root exudates and biomass of Bt corn has no apparent effect on earthworms, nematodes, protozoa, bacteria, and fungi in soil. Soil Biol. Biochem . 33: 1225

– 1230.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 84 of 153 U.S. EPA . 1998. “Guidelines for Ecological Risk Assessment.” EPA 630/R - 9 5 - 002F. Washington, DC, USA. Federal Register, May 14, 1998. 63(93): 26846 - 26924. U.S. EPA . 2000. SAP report No 99 - 06. Sets of scientific issues being considered by 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 website: http://www.epa.gov/scipol y/ sap/1999/index.htm#december U.S. EPA . 2001a. SAP Report No. 2000 - 07. Sets of scientific issues being considered by th e Environmental Protection Agency regarding: Bt plant - pesticides risk and benefit assessments. Dated March 12, 2001. Web site: http://www.epa.gov/scipol y/ sap/2000/october/octoberfinal.pdf U.S. EPA . 2001b . Biopesticides Registration Action Document: Bacillus thuringiensis ( Bt) In corporated Plant Protectants . Washington, DC . http://www.epa.gov/opp00001/biopesticides/pips/bt_brad.htm U.S. 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 n ontarget insect and insect resistance management issues. Dated November 6, 2002 . http://www.epa.gov/scipoly /sap/2002/august/august2002final.pdf U.S. EPA . 2004. SAP Report No.2004 - 05 . Product characterization, human health risk, ecological risk, and insect resistance management for Bt cotton products. Dated August 19, 2004 . http://www.epa.gov/scipoly /sap/m eetings/2004/june/final1a.pdf U.S. EPA . 2005a . Biopesticides Registration Action Document: Bacillus thuringiensis Cry2Ab2 Protein and its Genetic Material Necessary for its Production in Cotton . Washington, DC . http://www.epa.gov/pesticides/biopesticides/ingredients/tech_docs/brad_006487.pdf U.S. EPA . 2005b. Biopesticides Registration Action Document: Bt Cry 1F (sy npro) and Cry 1Ac (s ynpro) Construct 281/3006 In secticidal Cry stal Proteins as Expressed in Cotton. http://www.epa.gov/oppbppd1/biopesticides/ingredients/tech_docs/brad_006512.pdf U . S . EPA . 2006. The product characterization and human health data submitted in support of the EUP . Memorandum from R. Edelstein, through J. Kough, to S. Matten dated January 27, 2006. U.S. EPA. Washington DC U.S. EPA , 2008a. Biopesticides Registration Action Docum ent

– Bacillus thuringiensis modified Cry 1Ab (SYN - IR67B - 1) and Vip3Aa19 (SYN - IR 102 - 7) insecticidal proteins and the genetic material necessary

for their production in COT102 X COT67B cotton, http://www.epa.gov/pesticides/biopesticides/ingredients/tech_docs/brad_006529.pdf U.S. EPA , 2008b. Biopesticides Registration Action Document

– Bacillus thuringiensis Cry2Ab2 protein and its genetic material necessary for its production in cotton . Amended . http://www.epa.gov/oppbppd1/biopesticides/ingredients/tech_docs/brad_006487.pdfBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 85 of 153 U.S. EPA , 2010. Biopesticides Registration Ac tion Document

– Cry 1Ab and Cry 1F Bacillus thuringiensis (

Bt) Corn Plant - Incorporated Protectants, http://www.epa.gov/oppbppd1/biopesticides/pips/cry 1f - cry 1ab - brad.pdf

. U.S. EPA. 2010 . Review of Product Characterization Data in support of Sec. 3 Registration of Plant In corporated Protectant (PIP) cotton events: GHB119 cotton expressing Bacillus thuringiensis Cry2Ae pr otein ( EPA File Sy mbol 264 - RNOU ) , T304 - 40 cotton expressing Bt CrylAb ( 264 - RNOL ) ; and TwinLinkTM cotton ( EPA File Sy mbol EPA File Sy

mbol 264 - RNOA ) , its associated combination PIP product developed by conventional breeding of cotton events GHB119 and T304 - 40. Memorandum from A. Waggoner, thro ugh J. Kough, to S. Bacchus and D. Greenway dated February

4, 2011. U . S . EPA. Washington DC . U . S . EPA . 201 1 . Review of Product Characterization and Human Health Data in s upport of Sec. 3 Registration of Plant - In corporated Protectant (PIP ) event T304 - 40 co tton expressing Bacillus thuringiensis Cry 1Ab insecticidal protein ( EPA File Sy mbol 264 - RNOU; OECD Unique Id entifier: BCS - GHØØ4 - 7 ) ; Memorandum from A. Waggoner, through J. Kough, to S. Bacchus and D. Greenway

dated November 30 , 2011. U.S. EPA. Washington DC.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 86 of 153 

### APPENDIX **D :**  **Insect Resistance Management Cry1Ab**  **, Cry 2Ae**  **and their combination (Cry1Ab and Cry2Ae) in T**  **winLink TM**  Discussions of EPA’s risk assessments for Insect Resistance Management (IRM) are presented in this Appendix . Where deficiencies were identified in the earlier reviews, BCS provided data to satisfy EPA’s requirements. Immediately below is the risk assessment completed by EPA’s J. Martinez on March 22, 2010. Following this risk assessment are those completed on September 28, 2010 and June 2011. **I. Insect Resistance Management**  **Review** Below is the BPPD 17 review of BCS’ Insect Resistance Management (IRM) plan and supporting data for the application to register TwinL inkTM cotton PI P as well as supporting documentation for a Natural Refuge request ( U.S. EPA memorandum 04/22/10; EPA File Sy mbol 264 - RNOU, MRIDs 4760014 and 476348 - 23 ) . Deficiencies identified in this application for full commercial r egistration TwinL inkTM c otton were satisfied in subsequent submissions in 2010 and 2011. Those risk assessments follow this initial review. **CONCLUSION AND RECOMMENDATIONS 1.**  **DOSE** a. BPPD concludes that the diet incorporated and overlay assay s together (using verification method 1) sug gest that TwinL inkTM and GHB119 may express a high - dose against CBW but that T304 - 40 may

express less than high - dose against the target pest. b. BPPD concludes that diet incorporated and overlay assay s (using verification method 1) together suggest that events GHB119 and T304 - 40 may express a high - dose and TwinL inkTM may express an effective high - dose against TBW. c. BPPD cannot make a conclusion about the dose profile for TwinLinkTM and its events and PBW (using verification methods 3 and 4) because it is unclear how the mortality results reported b y Ellsworth and L i (2008) were obtained. When BPPD divided the number of entry holes reported (Tables 9 - 12) by

the number of live larvae and calculated average mortality, the obtained results did not match mortalities reported b y BCS.

17 The use of BPPD in this review refers to the BPPD IRM team consisting of Jeannette Martinez and Alan Reynolds.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 87 of 153 BPPD’s conclusion regarding dose determination (see also Table D.15) **Pest Method #1**  **Method #4 Method #3**  **T304 -**  **40 GHB119**  **TL T304**  **- 40**  **GHB119 TL**  **T304 -**  **40 GHB119**  **TL** CBW Non High Dose Possible

High Dose Possible

Effective High - Dose Inconcl.

* Inconcl.* Inconcl.* -- -- -- TBW Possible High dose High Dose Effective

High - Dose -- -- -- -- -- -- PBW

-- -- -- Inconcl. * Inconcl.* Inconcl.* --- Inconcl.* Inconcl.* * Insufficient information was submitted to the Agency to make a dose determination ** Cry1Ab could not be tested in this experiment because the lab strain was resistant to Cry1Ab. **2. PROTEIN EXPRESSION**  a. BPPD has reviewed B CS’ protein expression results reported in their IRM submission and concludes that the single event T304 - 40 may not express the same levels of toxin as in the dual gene PIP, TwinL inkTM . Event T304 - 40 expressed higher levels of toxin in leaves of single event p lants than leaves collected from TwinLinkTM plants. For flowers and bolls the pattern was reversed. It is unclear whether these differences in expression results were statistically significant . Because BCS did not provide further information on the differen ces observed. b. Expression of GHB119 in leaves from earl y to late stage appeared somewhat variable or unstable. Expression levels were measured in leaves in 2004 and 2005 at three developmental stages (earl y leaf, mid leaf, and late leaf). At developmental stage 1, expression was higher than at developmental stage 2, and at developmental stage 3, expression increased again but to higher levels than observed at developmental stage 1. For earl y, mid, and late boll, Expression level for GHB119 were also assess ed in three different developmental stages of bolls (earl y, mid, and late). Levels of GHB119 were higher in TwinLinkTM than in the single event plant during developmental stage 2 and 3. It appears that the difference was approximately two - fold (see Figure 1 ). c. Table D. 1 in this review summarizes the protein expression level (μg/g - dry weight) submitted b y BCS for T304 - 40 and GHB119 in different plant tissues. It appears that the same tissue ty pes were lumped together for plants expressing single and py ramided events. Since there appears to be unequal expression of the proteins in plants expressing either the single or py ramided events over the course of the growing season, this approach is not providing the m ost information. It would be more useful to have the expression data separated b y tissue type for each individual event, GHB119, T304 - 40, and TwinL inkTM , rather than lumped together for the same protein. d. BPPD recommends that BCS provide more specific and e xplanatory information together with their protein expression results in future data submissions for PIP registrations.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 88 of 153 **3. CROSS**  **- RESISTANCE**  a. BPPD concludes that based on BCS’s data there is little concern for cross - resistance between Cry 1Ab and Cry2Ae as expressed in TwinL inkTM . b. BPPD notes that should resistance evolve in CBW and TBW to Cry2Ae (as expressed in TwinL inkTM cotton) that Cry2Ab2 (as in Bollgard II cotton) may be compromised as well because, as BCS stated in their report, there is a great po tential for cross - resistance between the two toxins. c. BPPD notes that the cross - resistance potential is great between Cry 1Ab, Cry 1Ac, and Cry 1F (although somewhat less for Cry 1F) based on the competition assay s; the three toxins share high affinity bindin g sites. Should CBW evolve resistance to Cry 1Ab, other PIP products expressing Cry 1Ac and Cry 1F will likely be compromised as well. **4. MODELING**  BPPD has reviewed the modeling assessment for CBW and accepted the probabilistic approach presented therein. However, BPPD is concerned about parameter assumptions for initial resistance allele and dose mortality that went into the anal ys is, the lack of including Cry 1Ab corn in the modeling landscape, the lack of est ablishing a high dose for TBW (requested for natural refuge strategy

request) and the oversight of not modeling PIP durability for the TBW target pest. These concerns are all addressed in the Deficiencies 2 - 6 listed below. **5. REFUGE STRATEGY**  a. An IRM plan waiver request for single trait events (T304 - 40 and GHB119) is acceptable if seeds from those events are not sold commerciall y and serve onl y for producing the combination PIP TwinL inkTM . For an y other combinations, BCS may need to appl y for an experimental use permit or product registration. BPPD recommends the use of acreage/geographic limitations for the single trait events as a means to mitigate potential resistance development. BCS should provide information on proposed acreage for these single trait events. b. BPPD verified that BCS cited all the relevant natural refuge studies originally developed by Monsanto Company

and has obtained a data citation permission letter from Monsanto. c. BPPD notes that a second dose verification method is lacking for TBW. Natural Refuge requests were previousl y granted on the basis that the toxins were expressed at a high - dose against TBW. A second dose TwinL inkTM - verification method will need to be submitted to support BCS’ Natural Refuge request, or BCS can design a model with their specific dose data for TBW. d. When BCS has satisfactorily addressed the deficiencies related to the modeling of resistance evolution in CBW and TBW, BPPD will re - assess the natural refuge request for TwinL inkTM cotton. e. If a Natural Refuge strate gy is approved by

BPPD for TwinLinkTM , the requirements for a non -Bt cotton refuge in conjunction with the planting of any acreage of TwinLinkTM cotton should remain in place for the following states and regions: Arizona, California, and New Mexico and inBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 8 9 of 153 th e following Texas counties: Brewster, Crane, Crockett, Culberson, El Paso, Hudspeth, Jeff Davis, L oving, Pecos, Presidio, Reeves, Terrell, Val Verde, Ward, and Winkler. f. For those States and regions requiring a non - Bt refuge, the following refuge plantin g requirements should apply: i. 5% External, Unspray ed Refuge ii. 20% External Spray ed Refuge iii. 5% Embedded Refuge **6. RESISTANCE MONITORING**  a. BCS’ approach for developing baseline data for TBW, CBW, and PBW by 2012 may be too protracted in the case that TwinL inkTM is commercialized before 2012. Baseline susceptibility data should be available by the first growing season of commercial use of the PI P product. BPPD recommends that BCS accelerate the development of these data. b. The applicant proposed to monit or for resistance evolution in all three target pests (CBW, TBW, and PBW) in areas of high TwinL inkTM adoption. BPPD agrees with this proposal but also recommends that monitoring efforts additionally focus on regions in states with little natural refuge. c. B CS should follow the monitoring paradigm established for other Bt cotton registrations; this point is addressed in Deficiency 8. **7. REMEDIAL ACTION PLAN**  a. BCS needs to have an established remedial action plan in place for each target pest of TwinL inkTM . This is addressed in Deficiency 8 below. b. BPPD notes that it will not be sufficient if BCS “attempts” to confirm and characterize resistance upon receiving notification of unexpected damage. Rather, unexpected pest damage reports should be taken seriousl y and followed up immediately to conclusivel y determine whether the cause of crop damage is due to resistance or other reasons. BPPD is aware that it can take months before a determination can be reached. **8. GROWER EDUCATION**  a. It is recommended that BCS develop a grower education program that targets states and counties where a non - Bt corn variety must be planted as the refuge for TwinL inkTM cotton. b. It is recommended that BCS develop a grower guide that at a minimum describes t he technology and includes unexpected damage guidelines.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 90 of 153 **9. GROWER COMPLIANCE**  a. BPPD notes that BCS should have a well developed grower - education program (including grower guide) for all regions with clear indications for regional requirements. b. A natural refuge strategy for TBW and CBW would eliminate the need for growers to plant a refuge where alternate hosts are abundant. However, there are cotton - growing regions where natural refuge is not applicable and where planting a refuge is still required. For those regions it is recommended that BCS develop, implement, and report to EPA on programs to evaluate and promote growers’ compliance with IRM requirements. c. Grower compliance in these regions should be assessed with on - farm assessments in addition to an ony mous phone surveys. BPPD recommends that BCS use the existing grower compliance paradigm for other Bt cotton registrations. **10. REPORTING REQUIREMENTS**  BCS stated that “once the baseline susceptibility data was collected for the three primary target pe sts and a discriminating dose level was established, annual monitoring would be conducted and reported to the agency”. BPPD recommends that BCS submit annual reports for the following: i) Baseline and discriminating dose work irrespective of their status; ii) Sal es data; iii) Annual resistance monitoring; and iv) CAP results for cotton growing regions where Natural Refuge is not applicable. **11. DEFICIENCIES**  **Deficiency 1** : The dose profile reported for PBW cannot be verified b y BPPD until BCS clarifies how mortalities were calculated for the experiments using high - dose verification methods (3) and (4). **Deficiency 2** : In the modeling anal ys is provided by BCS, onl y CBW was modeled but not TBW. For a new refuge strategy

such as Natural Refuge, each main target pest needs to be evaluated separatel y via a simulation model that incorporates biology , genetics, toxicity , and ecology of the target pest. BCS needs to provide simulation modeling for TBW in order to support their natural refuge request. **Deficiency 3** : The natural refug e paradigm was approved for Bollgard II and WideStrike with a high dose profile for TBW. If BCS wishes to pursue the natural refuge strategy for TwinLinkTM , then a second high - dose verification study will need to be submitted to support that the PIP express es a high dose against TBW, or BCS can design a model with their specific dose data for TBW. **Deficiency 4** : BPPD is concerned with the dose assumptions for CBW and recommends that an additional anal ys is be conducted using somewhat lower mortality assumptio ns based on the stud y results reported b y BCS . The bioassay experiments showed that Cry 1Ab caused mortality ranging 88.5 - 100% (see this review). Estimated mortalities from the artificial infestation experiments (2007 - 2008) ranged from 99.5 - 99.7% (see this review). However, BPPD cannot verify the results of the artificial infestation experiments due to lack of information provided (i.e. % egg viability). At the moment, there is noBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 91 of 153 evidence that Cry 1Ab expresses high - dose against CBW. BCS concluded , too , that TwinL inkTM expressed less than high - dose against CBW due to the target pest being less susceptible to Cry 1Ab and lower level of expression of Cry 1Ab in the parental event for TwinL inkTM . Hence BPPD recommends exploring dose mortalities ranging from 90 - 99% i n the simulation model. **Deficiency 5** : BPPD is concerned about the most likely value chosen for initial resistance allele frequency for Cry 1Ab (0.0002). Although no field resistance to Cry 1Ab has been documented y et for corn earworm (aka CBW), the toxin has been in use for over a decade (in B t corn) and selection may likely have increased the frequency of potential resistance alleles. However, because corn earworm does not overwinter in the Corn Belt, BPPD agrees that the most likely value will be somewhat lower than could otherwise be expecte d. BPPD recommends that BCS consider modeling a most likel y resistance allele frequency

of 0.001 for Cry 1Ab in CBW. **Deficiency 6** : BCS needs to include Cry 1Ab corn and Cry 1Ab cotton in the modeling landscape when assessing the risk of resistance evolution in the target pests because Sy ngenta has registered Bt cotton and Bt corn and Monsanto has registered Bt corn products expressing Cry 1Ab. These corn and cotton PIPs are grown in the Cotton Belt and need to be included in a simulation analy sis for TwinL inkTM . **Deficiency 7** : BCS needs to have a well - developed monitoring plan in place before TwinLinkTM is approved for commercialization (i.e. sampling regions, sampling scheme, etc). BPPD recommends that BCS follow the monitoring paradigm established for other Bt cotton registrations. **Deficiency 8** : BCS needs to have an established remedial action plan in place for each target pest of TwinL inkTM . It is not sufficient to provide an outline or general statement about different kinds of approaches. BPPD recommends that BCS follow the existing remedial action paradigm for Bt cotton PIPs.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 92 of 153 **I. BACKGROUND**  **– Insect Resistance Management**  TwinL inkTM cotton is a py ramided transgenic cotton trait that expresses Cry 1Ab (Event T304 - 40), Cry2Ae (Event GHB119), and a glufosinate amm onium (herbicide) tolerant active ingredient based on LibertyLink technology . TwinL inkTM was developed by BCS using conventional breeding techniques. Cry2Ae is a new PIP for Bt cotton that allows for the control of Lepidopteran pests. Small field trial ev aluations with artificially and naturall y infested plots using the three major target pests Cotton bollworm (

Helicoverpa zea , CBW), Tobacco budworm ( Heliothis virescens , TBW), and Pink Bollworm ( Pectinophora gossypiella , PBW) demonstrated that there was mi nimal damage to TwinL inkTM cotton (expressing Cry2Ae and Cry 1Ab) during the growing seasons of 2004 and 2005. Cry 1 and Cry2 are effective lepidopteran toxins and share less than 20% amino acid homology . Crickmore et al. (1998) constructed a ph yl ogenetic tree using amino acid sequence identity as homology criteria for all presently

known Bt cry stal proteins. This tree indicates that Cry 1Ab and Cry2Ae are geneticall y dissimilar entities and share little sequence homology. Currently there are five other Cry

toxins registered in Bt cotton for the control of Lepidopteran pests; those are Cry 1F, Cry 1Ab, Cry 1Ac, Cry2Ab2, and Vip3Aa19. Py ramids of Cry 1F and Cry 1Ac, Cry 1Ac and Cry2Ab2, and Cry 1Ab and Vip3A19 are expressed in WideStrike cotton (product of Dow AgroSciences), Bollgard II cotton (product of Monsanto), and VipCot (product of Sy ngenta), respectivel y . All py

ramided cotton PIP are effective again st the three main target pests CBW, TBW, and PBW. **II. PEST BIOLOGY AND ECOLOGY**  TBW, CBW, and PBW are considered the three major target pests for TwinLinkTM Cotton. Fall army worm ( Spodoptera frugiperda , FAW) and Beet ar my worm ( Spodoptera exigua , BAW) are considered secondary

target pests. A summary

of the biology

and ecology

for major Bt cotton target pest, such as TBW, CBW, and PBW, can be viewed in the IRM section of the Agency ’s 2001 Bt crop reassessment document at http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm . Since the publication of the Agency document, additional biological and ecological information has become available. BCS summarized new stud y results (MRID 476400 - 14, Vol. 15), which are reported below. 1. Cotton Bollworm (Taken from MRID 476400 14) CBW is highly

mobile and can move long distances. The general pattern of CBW migrat ion is a northward movement (U.S. EPA, 2001). At the 2001 Scientific Advisory Panel (SAP) the general consensus was that southward movement to overwinter was not proven but that there was good circumstantial evidence. Further research on south migration was done b y Gould et al. (2002): they used 13C/12C stable isotope assessment of alternative host used by CBW and showed indirectl y that late season CBW moths captured in Louisiana and Texas could be migrants from the northern Corn Belt. Ty pically , greater than 50% of the moths captured in August through Oc tober have isotope ratios indicative of larval feeding on C4 plants (i.e. tropical and prairie grassland plants), even though local C4 crops were harvested at least one month earlier and the authors presume at the Texas trial site that there are also no co mmon wild C4 plants. Cotton, along with wheat, rice, barley and other ancient plants are C3 plants, easily

differentiated from C4 plants.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 93 of 153 Directl y related to migration is the overwintering behavior of the insect. Schneider (2003) studied the overwintering of CBW in cotton fields in the northeast portion of Mississippi. He found that densities of overwintering pupae varied greatl y across ye ars; in non - Bt plots densities ranged from 0 - 67 pupae/ha for CBW. L ower densities of CBW pupae (0 - 214 pupae/ha) were fo und over the seven - ye ar stud y in Bt cotton fields. Only

partial control of CBW was possible with these first generation single gene Bt cotton plants expressing only

the Cry 1Ac protein. Local temporal and spatial dispersal in CBW (Han and Caprio, 2002) wer e studied by using poly morphic allozy

me and RAPID - PCR makers. For CBW, Han and Caprio (2002) found that population differentiation at local and regional areas was very low, not unexpected for a highl y poly phagous and migratory

insect. Contrary

to TBW, the genetic variance was not significantly different between the four seasonal generations for CBW. Similarities amongst pheromone trap catches of CBW in eastern North Carolina suggested a high level of CBW moth movement into and out of localized areas and w as much more significant than anticipated (Jackson et al., 2003). Cotton production in localized areas has little impact on the number of CBW moths in the local environment. Gore et al. (2002) monitored larval CBW movement on cotton and compared movement on Bt cotton (Bollgard) and non - transgenic cotton. On pre - flowering plants at 1, 3, 6, and 24 hours after infestation 47.8%, 39.4%, 20.9%, and 13.0% of the larvae, respectively , remained in the terminals of non - Bt cotton plants. For Bt cotton, this was 28 .7%, 11.4%, 6.3%, and 1.3%, respectively . A day after artificial infestation, larvae were found 2.4 main stem nodes below the terminals on flowering non - Bt plants, for Bt cotton this was 5.7 main stem nodes below the terminals. In the Mississippi Delta, moth populations were monitored from 1986 to 2005 (Adamczyk and Hubbard, 2006) with pheromone traps. During the non - crop months, moderate numbers of moths of CBW were ty picall y captured b y 20 March. From 1986 - 1996, the mean number of CBW moths captured per trap and per night was 15.7 with a maximum of 310. Since 1997, CBW populations have declined, with a mean number per trap and per night of 6.2, and a maximum of 70. The decline may due to wide - scale planting of Bt cotton. Jackson et al. (2004) estimated the number of CBW that developed on conventional and Bt cotton in 2000 - 2002 for North Carolina. Across 7 locations they found 60,283 4 th and 5 th instar living larvae/ha in conventional cotton, which produced 47,078 adults/ha and damaged 367,835 bolls/ha. The Bt cotton (Bollgard event) did not significantl y reduce larval numbers, but damaged bolls and adults were significantl y less. The numbers of larvae were also estimated in fields utilizing the conventional chemical insecticide regime in 2001 - 2002; non - treated fields produced 75,510 larvae/ha, while sprayed fields had significantl y lower levels, onl y 28,973 larvae/ha. Alternative cultivated or wild hosts, which can be used as non - crop or natural refuges, have been extensively

studied since 2001. Jackson et al. (2003) studied the larvae, pupae and adult populations on different hosts in eastern North Carolina and found that corn was the major pro ducer of CBW larvae in the earl y season and stayed a major producer until the last week of August. These results indicated that field corn may

serve as refuge for CBW much longer than originall y expected. Beginning in mid August, so ybean and conventional c otton became the major crop sources for CBW production. Pupae and adult production followed the same trends.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 94 of 153 Kurtz et al. (2004) used these estimates (Jackson et al., 2003) and unpublished data b y J ackson in their computer simulation model. For the first two CBW generations, 90% of the larvae developed on corn, 5% on cotton and 5% on soy bean. For the third CBW generation in eastern North Carolina the estimates dropped to only

17.39% production on corn, with increases to 36.34% production on cotton, 0.05% on peanuts and 40.99% on soy bean. Storer et al. (2001) confirmed these results, finding that CBW adults emerging from ear - stage cornfields infest cotton, soy bean and other crops In North Carolina. The level of adult CBW production was greatly decreased (~8 0%) in Bt corn, while non - Bt corn produced 20 - 30 adults per 100 ears. In Louisiana, Peters et al. (2004) monitored larval CBW populations in several field crops during the 2002 and 2003 growing seasons. Larval populations were highest in sorghum: approxim ately 32,000 larvae/acre in 2002 and approximately 12,000 larvae/acre in 2003; field corn: approximately 20,000 larvae/acre in 2002 and approximately 10, 000 larvae/acre in 2003. Larval densities on soy bean and on non - transgenic and Bt cotton were much lower (<3000 larvae/acre) . Larval populations in corn, sorghum, maturit y group 6 soy bean, cotton and Bt cotton peaked on 25 June, 12 - 14 July , 2 - 4 August, 2 - 4 August, and 11 - 16 August, respectivel y. The only crop that exhibited similar temporal s ynchron y with bo llworm larvae on Bt cotton was soy bean maturity

group 6, indicating the possibility of including it in an IRM strategy . A more extensive study over two y ears and over five states demonstrated again that non - cotton crops, such as corn, grain sorghum, peanut and soy bean, support much larger larval CBW populations than cotton throughout the season. In June and portions of July the larval CBW populations were almost entirely

restricted to corn and in July and August were observed in non - cotton crops more freque ntl y and in larger numbers than in cotton. Again it was shown that CBW moths move extensively from their natal host origins. The number of CBW adults captured in pheromone traps at interfaces of fields of Bt cotton and various crop hosts rarel y varied amon g interfaces, except for highl y attractive corn (Jackson et al., 2008). Tillman and Mullinix (2004) showed that CBW females preferred to oviposit in grain sorghum over cotton; the number of eggs per plant and percentage of plants with CBW eggs was higher i n sorghum trap crop than in cotton trap crop for two y ears. Larval development studies in the lab of CBW on sorghum, corn, soy bean and cotton showed a survival of 73, 55, 26, and 13%, respectivel y and a mean development time to pupation of 15.6, 12.4, 18. 4, and 25 day s, respectively

(Gore et al., 2003). Cotton was the least suitable host, which was also reflected in the significantly

lower pupal weight. Blanco et al. (2007) showed that over a three y ear period in Mississippi and Mexico, garbanzo bean, an i mportant legume crop in Mexico, had an annual CBW level of 96,000 larvae/ha on garbanzo bean, and approximately 5 times less larvae on cotton and approximately

6 times less larvae on velvetleaf, respectively . L ow densities of adult CBW moths (0 - 275 ha/y r) emerged from plots and were not significantly different between the three crops. Cover crops, such as legume mixture, crimson and ry e seem to be more preferred by heliothines than the cotton crop. In sweeps no heliothine could be caught on the cotton plant s; in contrast, around 0.5 heliothine larva/sweep were found on the cover crops (Tillman et al., 2004). Natural mortalit y of CBW was studied from 1991 - 95 in short season cotton in Texas with one generation per ye ar (Sansone and Smith, 2001). Mortality of 71 - 95% could be consistently expected in the egg and first - instar stages. The more detailed studies in 1991 - 1993 partitioned the mortality ; 3.4% was caused b y Trichogramma egg parasitation, larval parasitation rarel y exceeded 5%, but numerically the most import mortality factor was egg consumption of around 77% b y Orius insidiosus (minute pirate bug).Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 95 of 153 2. Tobacco Budworm (Taken from MRID 476400 14) TBW is a highl y mobile insect that becomes less mobile in the mid to late summer when it prefers cotton as a ho st plant. Directl y related to movement is the overwintering of the insect. Recentl y, Schneider (2003) studied the overwintering of TBW in cotton fields in the northeast portion of Mississippi. He found that densities of overwintering pupae varied greatly a cross ye ars: in non - Bt plots densities ranged from 0 - 303 pupae/ha TBW. No TBW pupae were found over the seven - ye ar study in Bt cotton fields. Using pheromone traps to estimate the total area - wide TBW population overwintering, Schneider (2003) found that in the pre - transgenic cotton era (<1996) cotton fields ty pically accounted for up to 10% of the overwintering TBW population. After introduction of Bt cotton, but prior to expanded no - till cotton planting in the 1996 - 2001 era, the estimate was less than 2%. Local temporal and spatial dispersal of TBW (Han and Caprio, 2004) was studied by using poly

morphic allozy me and RAPID - PCR makers. TBW populations overall showed little special differentiation, indicating a highl y effective gene flow rate over the spatia l scale under investigation (Han and Caprio, 2004). Allelic differentiation arose more frequently in the second generation than in the other three generations, suggesting that TBW moth dispersal was more limited during the midsummer. During midsummer, cott on is the main host available for TBW in the Mississippi Delta. Once cotton flowers, moth movement declines along with the availability of a ready nectar source. Because the populations never reached genetic equilibrium, Han and Caprio (2004) could not det ermine the extent of movement reduction. In the Texas High Plains TBW adult populations were monitored weekl y from March to November between 1982 and 1995 using pheromone traps at Lubbock and Halfway Texas, prior to the introduction of Bt cotton (Parajule e et al., 2004). The bollworm/budworm complex consisted of 98% CBW and 2% TBW. For weekl y moth abundance, a positive correlation was shown with weekl y temperature, degree day s and weekl y average precipitation, and a negative correlation was shown with the weekl y wind velocity . Data from this study indicate that the late - season moth catches could be indicative of the dy namics of the earl y - season catches the following y ear. In the Mississippi Delta, moth populations were monitored from 1986 to 2005 (Adamczy k and Hubbard, 2006) with pheromone traps. During the non - crop months, moderate numbers of moths of TBW were ty picall y captured b y April 10 th . From 1986 - 1996, the mean number of TBW moths captured per trap and per night was 19.5 with a maximum of 500. Sinc e 1997, populations have declined for TBW with a mean number per trap and per night of 2.9, and a maximum of 25. The decline may be due to wide scale planting of Bt cotton. In the Southeast ern US, the first TBW generation develops almost exclusively on we eds; throughout the whole growing season cotton is a less important host, because of the abundance of other host plants (Abney et al., 2004). Abney

et al. (2005) also showed, using 13C/12C stable isotope assessment, that TBW fed consistentl y on C3 plants ( examples of C3 plants are cotton, so ybean, clover, and peanut). Unfortunatel y this technique could not be used to determine the natal origin of the TBW. Blanco et al., (2007) showed that over a three year period in Mississippi and Mexico, garbanzo bean, an important legume crop in Mexico, had an annual TBW larval densit y of 287,000 larvae/ha, approximatel y 4 times higher than in cotton, and approximately 5 times higher than in velvetleaf. Also the TBW moth densities emerging from garbanzo bean (20,180 adul ts/ha) were approximately 13 times higher than from cotton and approximately 35 times higher than from velvetleaf.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 96 of 153 Cover crops, such as legume mixtures, crimson , and ry e , seem to be more preferable to heliothines than cotton. In sweeps, no heliothine could be caught on the cotton plants; in contrast, around 0.5 heliothine larva/sweep were found on the cover crops (Tillman et al., 2004). 3. Pink Bollworm ( Excerpted from MRID 476400 - 14, modified by BPPD) Flight - mill experiments by Wu et al. (2006) showed that over a 72h flight the mean accumulated flight distance and flight duration of PBW were 41.25 km and 23.87 h for females and 23.46 km and 14.12 h for males. For unmated moths, the flight activity reac hed a peak after 3 - 5 day s after eclosion , and then reduced gradually . Optimum temperature for flight was from 24 to 28°C and the optimum relative humidity ranged from 75 to 90%. While PBW can be good fliers, local adult movement is limited; in most cases dispersal is limited to <1 km (Carrière et al., 2001a). Besides adult movement, oviposition preference is also important. L iu et al. (2002) found that in greenhouse experiments females did not lay fewer eggs on Bt cotton bolls than on non - Bt cotton bolls. They also revealed that the number of eggs laid per boll was negativel y associated with boll age and positively associated with boll diameter; more eggs per boll were laid on plants with more bolls. The distribution of eggs was clumped, which is an indicat ion that boll quality rather than avoidance of other eggs was the primary factor in oviposition preference. Liu et al. (2002) also studied mining behavior of neonate larvae. The number of entrance holes per boll did not differ between Bt cotton and non - Bt cotton. As with female moths, neonate preferred younger bolls and larger bolls. Carrière et al. (2001c) studied the prediction of spring moth emergence and its use for cultural control of the insect. Accumulation of heat units from January 1st accuratel y predicted the rate of PBW emergence in different regions in Arizona. Emergence was significantly higher in higher altitude regions. Most moths emerge from diapause too early to reproduce on cotton, a phenomenon known as ‘suicidal emergence’. Manipulation o f the planting date could be a good cultural control, but shortening the growing season can reduce yi eld. Most larval stages of PBW are relativel y protected from natural enemies because they feed on seeds inside the cotton boll; eggs laid externall y on ve getative structures, however, are vulnerable to predation and parasitism. Predation rates on sentinel PBW eggs were compared on Bt and non - Bt cotton plants (Sisterson et al., 2004). Many eggs were attacked by

predators with pierce and/or sucking mouthparts and by lacewing larvae. Between Bt and non - Bt plots, no significant differences were found for PBW egg predators; approximately 1.5 - 3 predators per plant were recovered, and PBW egg predation rates were approximately

40 - 50%. In Naranjo (2005), sentinel eggs of PBW also experienced the same rate of predation in Bt and non - Bt cotton. Egg predation rate varied from around 53% in 2001 to approximately 35% in 2003. No parasitism was detected in eggs that survived predation. On average, chewing and sucking pre dators were each responsible for approximately 50% of the total mortality . Naranjo (2005) also found no significant difference for rates of pupal mortality between Bt and non -Bt cotton. Pupal mortalities of around 40% in 2002 and 64% in 2003 were observed. No parasitism was observed in intact pupae. **III. DOSE** Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 97 of 153 TwinL inkTM cotton is a combined trait product made through traditional breeding of events T304 - 40 and GHB119 which express Cry 1Ab and Cry2Ae, respectivel y. BCS submitted 1) protein expression data for different cotton tissues; 2) L C 50 data for Cry 1Ab and Cry2Ae and their target pests; and 3) dose information for Event T304 - 40 (Cry 1Ab), Event GHB119 (Cry2Ae), and TwinL inkTM using methods #1 and #4 as described by the SAP ( U.S. EPA , 2001). a) Protein Expression BCS submitted protein expression results for cotton tissues, which were measured using ELISA based assay s in 2004 and 2005. Bay er determined that protein expression in Cry 1Ab and Cry2Ae cotton as well as TwinL inkTM cotton plants depended on the ty pe as well as maturity of tissues assay ed (see Figure 1). The lowest expression was found in nectar, pollen, and seeds; the highest expression was observed in leaves, roots, squares (flower buds containing male and female reproductive parts), apex, stems, and bolls (fertilized ovum followed by enlargement, filling, and maturation of structure) (see Table 1). Cry 1Ab , as expressed in TwinLinkTM , had consistent expression in leaves and squares with higher expression in bolls and flowers. Cry2Ae , as expressed in TwinL inkTM had relativel y high expression levels in leaves across the growing season and in late bolls (data from 2005). Expression was ty picall y higher in Cry2Ae than Cry1Ab (up to 10X). Cotton plants expressing the single events were anal yz ed in 2004 and showed similar protein expression levels as the plants expressing both events. BCS concluded that there was no suppression or enhancement of protein expression when Cry 1Ab and Cry2Ae were expressed in the same plant.

**Figure D.**  **1 . Expression of Cry1Ab and Cry2Ae as % Bt protein/total extractable protein**  **- TL denotes**  **TwinLinkTM** (extracted from MRID 476400 - 14) **Table D.**  **1 . Expression of Cry1Ab and Cry2Ae in plant tissues** Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 98 of 153 **PLANT TISSUE AVE**  **EXPRESSION LEVEL (μg/g -**  **dry weight) Cry1Ab**  **Cry2Ae** Roots 2.9 - 11.2 8.8 - 19.7 Stems 0.5 - 7.3 4.4 - 7.3 Leaves 0.4 - 5.2 10.7 - 63.3 Squares 0.8 - 3.9 3.4 - 12.4 Apex 1.0 - 1.6 6.1 - 10.7 Bolls 0.2 - 0.6 3.3 - 4.9 Flowers 8.7 - 12.5 4.8 - 6.3 Seed 0.1 - 0.7 0.02 - 1.6 Pollen 0.03 - 0.07 0.06 - 0.6 Nectar 0.00001 - 0.005 0 ( E xtracted from MRID 476400 - 14) b) LC 50 Data for Cry 1Ab and Cry2Ae in CBW and TBW BCS provided results from insect bioassay s using purified individual Bt proteins in a surface lay er diet for the target pests of TwinL inkTM cotton. Table 2 shows the mean LC 50 values and ranges for six target pests. BCS described the insect susceptibility for Cry 1Ab as follows: TBW > PBW > Old World BW (OWBW, Helicoverpa amigera) >> CBW. The susceptibility for Cry2Ae was described as : TBW > PBW >> CBW > BAW > FAW > Old World BW. **Table D.**  **2 . Mean LC**  **50 values and ranges for Cry1Ab and Cry2Ae**  **INSECTS Cry1Ab**  **(ng/cm 2**  **) Cry2Ae**  **(ng/cm 2**  **)** Heliothis virescens , TBW 7.2 (4.9 - 10.5) 4.1 (2.9 - 5.5) Helicoverpa zea , CBW 1357 (462 - 2844) 238.9 (107.7 - 473.1) Helicoverpa armigera , Old World BW 78.5 (41.1 - 149.9) 3556 (2824 - 4709) Pectinophora gossypiella , PBW 41.2 (6.9 - 152.0) 11.6 (4.9 - 22.2) Spodoptera frugiperda , FAW >>2000 1154.3 (682.0 - 1680.1) Spodoptera exigua , BAW >>1350 486.4 (336.5 - 685.5) ( E xtracted from MRID 476400 - 14 ) c) Dose Determination The 1998 SAP Subpanel discussed way s to define and measure "high dose" in plants. It was agreed that the definition of high dose as "25 times the toxin concentration needed to kill susceptible larvae" was reasonable based on current empirical data. However, the Subpanel recognized that it was conceivable that a heteroz ygote might develop with higher than 25 - fold resistance. The major problem identified by the Subpanel was in determining if this 25 - fold level was achieved in a specified cultivar. After much discussion, it was concluded that there were at least five way s to assess this 25 - fold level, but that some approaches were more appropriate for specific crop pests. The Subpanel concluded that a cultivar couldBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 99 of 153 be considered to provide a high dose if two of the five approaches described here indicated presence of a high dose. The five approaches generally used for L epidopteran s ys tems are: (1) Serial dilution bioassay with artificial diet conta ining l yophilized tissues of Bt plants (tissue from non -Bt plants serving as controls); (2) Bioassay s using plant lines with expression levels approximately 25 - fold lower than the commercial cultivar (determined b y quantitative ELI SA or some more reliable technique); (3) Survey

large numbers of commercial plants on sentinel plots in the field (e.g., sentinel sweet corn method) to make sure that the cultivar is at the LD 99.99 or higher to assure that 95% of heterozygotes would probabl y be killed. With this approach Bt sweet corn h yb rids are used to attract high densities of ECB and cotton bollworm ( Helicoverpa zea )(Boddie)) (CBW/CEW) moths, sampling can be limited to sweet corn ears in the Bt plot (ca. 1/4 - 1/2 acre block), and a frequency of resistant phen oty

pes can be estimated as the ratio of density of larvae/plant in Bt sweet corn to densit y of larvae/plant in an adjacent planting of non -Bt sweet corn (Andow and Hutchison, 1998; Hutchison, unpublished data); (4) Similar to (3) above, but would use controlled infestation with a laboratory strain of the pest that had an LD 50 value similar to field strains; and (5) Determine if an older instar of the targeted pest could be found with an LD 50 that was about 25 - fold higher than that of the neonate larvae. If so, that stage could be tested on the crop plants to determine if 95% or more of the older stage larvae were killed. BCS applied three methods to test for high - dose of TwinL inkTM and its single events.

 Method (1) utilized lyophilized plant tissue from events T304 - 40, GHB119, and TwinL inkTM mixed with artificial diet to produce 10X and 25X concentrations. The bioassay s were conducted as diet incorporated assays and diet overlay assays with TBW and CBW.  Method (4) involved controlled infestations of Twin Li nkTM cotton plots with susceptible lab strain of CBW and susceptible lab strain of PBW to assess the level of mortality .  Method (3) utilized a homozygous resistant lab strain (Cry 1Ac) of PBW with which y oung cotton bolls were infested. High - Dose Verific ation Method (1) : 1. Cotton Bollworm Lyo philized plant material from greenhouse grown cotton was mixed into the bioassay diets at different concentrations (4% and 10% of Cry - protein). For the negative control, 0, 0.8, 4, and 10% of non - Bt cotton (Coker312) were used. Twent y - four neonate larva e were exposed at each treatment level (see Table D. 3 ); two replicates per treatment were carried out. Mortality was scored at day s 4, 7, 12, 17, 19, 21, and 24, and average mortality was calculated from the two replicates. For ov erlay

assay s, the same leaf material was used and the l yophilized powder was mixed in a 0.2% agar solution. The concentrations tested were 4% and 6.9% of Cry - protein. L iquid diet was poured into each well and allowed to dry . Different concentrations were p repared (see Table D. 4 ) and overlaid onto the solidified diet. Mortality was scored at day

3, 7, 11, 15, 18, and 21.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 100 of 153 The applicant reported that control mortality was difficult to manage using cotton leaf tissue due to the presence of goss ypol. Diet incorporated assays had higher control mortality (17.5 - 29.5% on day 17) than overlay assay s (16.8 - 19% on day 15); hence overlay

assay s w ere best for verifying TwinLinkTM cotton’s high - dose level. BCS concluded that TwinLinkTM expressed less than high - dose against CBW due to the target pest being less susceptible to Cry 1Ab and lower level of expression of Cry 1Ab in the parental event for Twin Link. **Table D.**  **3 . CBW**  **mo rtality to Cry1Ab, Cry2Ae, and**  **TwinLinkTM -**  **diet incorporated assays Treatment**  **% Treatment**

**Concentration % Mortality** 

**Day 4 Day 7**  **Day 12 Day 17**  **Day 19 Day 21**  **Day 24** Coker312 10 0 10.5 17 29.5 All pupae or pre - pupae

4 4 10.8 15 23 All pupae or pre - pupae 0.8 0 8.5 15.5 17.5 All pupae or pre - pupae T304 - 40, Cry1Ab 10 21 85.5 100

4 6 21 41 55 57.5 81.5 89 GHB119, Cry2Ae 10 29 72 100

4 19 31 52 73.5 73.5 84 95 TwinLink TM 10 44 85 100

4 14.8 55.5 70 94 97 100 Modified from MRID 476400 - 14 **Table D.**  **4 . CB**  **W m ortality to Cry1Ab, Cry2Ae, and**  **TwinLinkTM -**  **overlay assay m ethod**  **Treatment % Treatment** 

**Concentration % Mortality** 

**Day 3 Day 7**  **Day 11 Day 15**  **Day 18 Day 21**  Coker312 6.9 0 10.5 16.8 16.8 All pupae or pre pupae 4 0 8 17 19 All pupae or pre pupae T304 - 40, Cry1Ab 6.9 0 35.5 54 88.5 88.5 88.5

4 0 23 47.5 76 84 89.5 GHB119, Cry2Ae 6.9 23 79 100

4 49.5 83 97.5 100 TwinLink TM 6.9 27.5 75 100

4 29 80 97.5 100 Modified from MRID 476400 - 14 2. Tobacco Budworm As for TBW, two ty

pes of assay s were carried out, the diet bioassay and overlay

bioassay using all three toxins. The same methodology was used for TBW as was used for CBW (see above Section III c) 1.). However, concentrations for the negative control of diet bioassay s differed and were 0, 0.8, and 4% of Coker312. Furthermore, mortality was scored at day

4, 7, 12, and 17 and averaged across the two replicates. For overlay

assay s, mortality

was scored at day

3, 7, 11, and 15.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 101 of 153 As for CBW bioassay s, control mortality for TBW was high and difficult to control. The applicant attributed this to the presence of goss ypol in the leaf diet. BCS reported that control mortality was lower in overlay assay s and that therefore those results were best for verifying TwinL inkTM cotton’s dose profile. BCS concludes from the bioassay results using method (1) that there was a high dose expression for TBW in TwinLinkTM cotton and its individual traits. **Table D.**  **5 . TBW m**  **ortality to Cry1Ab, Cry2Ae, and TwinLinkTM**  **- diet incorporated assays**  **Treatment % Treatment** 

**Concentration % Mortality** 

**Day 4 Day 7**  **Day 12 Day 17**  Food* 0 0 8.3 25 25 Coker312 4 0 14.8 23.8 23.8 0.8 2 17 28 28 T304 - 40, Cry1Ab 4 29 73 95.5 100 GHB119, Cry2Ae 4 100 TwinLink TM 4 100 Modified from MRID 476400 - 14 * Unclear what source of food constituted negative treatment. **Table D.**  **6 . TBW m**  **ortality to Cry1Ab, Cry2Ae, and TwinLinkTM**  **- overlay assay m**  **ethod Treatment**  **% Treatment**

**Concentration % Mortality** 

**Day 3 Day 7**  **Day 11 Day**  **15** Coker312 4 0 14.8 17 17 T304 - 40, Cry1Ab 4 41 88.5 95.5 100 GHB119, Cry2Ae 4 76 100 TwinLink TM 4 71 100 Modified from MRID 476400 - 14 High - Dose Verification Method (4) : 1. Cotton Bollworm Coker312, TwinL inkTM , and its two single events were evaluated for efficacy against CBW and PBW. The experiment was carried out at 14 different locations across the US with two replicates each during the 2007 and 2008 cotton growing season. At two locations only , BCS conducte d a larval survival experiment which provided the data for the reported results of high - dose verification method (4). In two locations (in 2007) and three locations (in 2008) of the Cotton Belt (actual locations were not reported b y the applicant ), natural infestation was supplemented with artificial infestations of the target pest; repeated artificial infestations with CBW were conducted at one location in 2007 and three locations in 2008. A total of 7,200 eggs were applied in each row of 60 plants and 50 plants in 2007 and were anal yz ed for presence of larvae. In 2008, a total of 6,600 eggs/plot were applied and each boll was dissected subsequentl y to determine presence of larvae. Percent mortalit y was calculated for 2007 ( T able D. 7 ). The average number of boll examined in each plot was recorded and percent mortality calculated in 2008 ( Table D. 8 ). BCS reported that mortality for all three events (single and dual) was very high and greater than 99.2% in all but one instance. In 2008, one TwinL inkTM plot had slightly less CBW mortality

(98.5%).Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 102 of 153 **Table D.**  **7 . CBW m**  **ortality on artificially infested plots d uring the 2007 growing season**  **Event Total eggs/plot** * **Total live larvae % Mortality**  T304 - 40, Cry1Ab 6000 16 99.73 GHB119, Cry2Ae 6000 1 99.98 TwinLink TM 6000 2 99.97 * 50 plants/60 - plant row were scouted = 83.3% Modified from MRID 476400 - 14 **Table D.**  **8 .**  **CBW m ortality on artificially infested plots during the 2008 growing season**  **Event Total eggs/** 

**3 locations*** **Total fruit**

**(average) Total live** 

**larvae*** **% live**

**larvae Est. live** 

**larvae  ** %**

**Mortality** T304 - 40, Cry1Ab 19800 30422 8 0.84 322.97 99.52 GHB119, Cry2Ae 19800 24996 3 0.27 86.40 99.68 TwinLink TM 19800 32279 7 0.89 309.01 98.26 * 6,600 eggs were applied per plot; 1 plot = 1 location Modified from MRID 476400 - 14 2. Pink Bollworm A similar artificial infestation study as for CBW was carried out for PBW by Drs. Ellsworth and Li at the University

of Arizona. Four plots were planted as a random complete block design (8 replicates) with TwinL inkTM , each single event, and Coker312 as control. Each plot was six rows long (30 - ft) and separated from other blo cks by one empt y row. Young bolls were infested with 100 eggs each from a susceptible PBW strain maintained by USDA - ARS (Maricopa, AZ). Six to eight day s later tagged bolls were brought into the lab for dissection. Attack rates on bolls were evaluated by c ounting the number of entry holes on the exterior of the boll carpel. PBW survival was estimated 6 - 15 day s later which provided enough time for larvae to develop to 3 rd instar. Survival was determined based on the number of exit holes, 3 rd , live 4 th , and p upae. Mortality (s ynonymousl y used with efficacy) was estimated b y dividing the number of surviving PBW b y the number of entry holes and by correcting for control mortality . Mortality

in the Coker line was determined by limiting examinations to mainly thos e bolls with < 20 entry

holes in order to avoid densit y - dependent mortality in control treatments. BCS concluded that all three infestations with Cry 1Ab and TwinLinkTM demonstrated high efficacy (corrected mortality

> 99.5%) against PBW. Two infestations with Cry2Ae demonstrated high efficacy (corrected mortality

>99.1%), while results from one infestation were somewhat lower (corrected mortality 98.7%). BCS hypothesized that the small sample size from the first infestation in addition to the high control mortality may have contributed to this lower mortality estimate. Since control mortalities were high ( Table D. 9 ), corrected mortality rates based on each infestation showed somewhat lower levels of efficacy

for each line. Cry2Ae was somewhat less effective in all infestations. When all data were pooled for each cotton line, the mortality rates were again very

high ( Table D. 10 ).Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 103 of 153 **Table D.**  **9 .**  **Mortality results of susceptib le PBW following artificial infestation of cotton plants**  **Infestation/ Data**  **collection Treatment**  **Entry Holes**  **Live**

**Large Larvae**  **Average**

**Mortality (%)**  **Low er** 

**95% Mean**  **Upper**

**95% Mean Corrected** 

**Mortality (%)**  Ju ly Coker

Cry1Ab Cry2Ae TwinLink TM 1646 2756 1901 331 726

8 11 2 50.26

99.70 99.23 99.96 46.37

99.46 98.65 99.87 54.16

99.94 99.82 100 -

99.34 98.69 99.86 August Coker

Cry1Ab Cry2Ae TwinLink TM 1262 2725 2485 1897 393

0 5 0 59.75

100 99.84 100 54.39

100 99.70 100 65.10

100 99.98 100 -

100 99.35 100 September Coker

Cry1Ab Cry2Ae TwinLink TM 598 2399 1551 1452 182

3 4 0 63.29

99.75 99.65 100 56.63

99.32 99.19 100 69.95

100 100 100 -

99.59 99.15 100 Modified from MRID 476400 - 14 **Table D.**  **10 . Pooled m**  **ortality results of susceptible PBW following artificial infestation of cotton plants**  **Treatment Total** 

**Entries N** 

**Bolls Live** 

**Large Larvae**  **Apparent**

**Mortality Low**  **er**

**95% mean Upper** 

**95% Mean Corrected** 

**Mortality (%)**  Coker 3506 259 1301 56.3 53.4 59.2 - - Cry1Ab 7880 345 11 99.8 99.6 99.9 99.6 Cry2Ae 5937 252 20 99.6 99.4 99.8 99.1 TwinLink TM 6680 302 2 99.9 99.9 100 99.9 Modified from MRID 476400 - 14 High - Dose Verification Method #3 : 1. Pink Bollworm In 2007 and 2008, an artificial infestation study was carried out by

Drs. Ellsworth and Li at the University

of Arizona with a homozygous resistant strain of PBW. A Cry 1Ac - resistant PBW strain (AZP - R) was obtained from Dr. Bruce Tabashnik at the Universit y of Arizona. That strain was resistant to Cry 1Ac with 53% survival in response to 10μg of Cry 1Ac powder/g of die t and demonstrated cross resistance to Cry 1Aa and Cry 1Ab. The artificial infestation experiment was run in the lab with field collected plant tissue of Cockerline (control), TwinL inkTM , Cry 1Ab, and Cry2Ae plant tissue. Young cotton bolls were harvested (1 5 - 19 day s old) after tagging. Each boll was maintained in a water pick bioassay

s ys tem. To measure the efficacy of the transgenic cotton, bolls were infested with an estimated 15 homozygous PBW neonates. The same procedures were followed for attack rate es timates, boll dissection, and PBW survival and mortality were the same as described for the PBW artificial infestation study

using verification method #4. A total of 348 cotton bolls were assay ed. The mean entry holes ranged from 6.2 - 28.5. Attack rates w ere much higher in some cases. Maximum number of entry holes on a boll were 31 (Coker), 48 (Cry 1Ab), 48 (Cry2Ae), and 54 (TwinL inkTM ). The mean percentage of PBW recovered ranged from 80.1 to 94.5%, which was similar to assay results with susceptible PBW. T he criterion for ‘survival’ at the timeBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 104 of 153 of boll dissection was a ‘living > 3 rd instar larva’. Mortality was calculated by

dividing the number of surviving PBW b y the number of entry holes as well as by correcting for control mortality . JMP IN Statistical Di scovery Software, Version 5.1, SAS Institute 2005 was used for the data anal ys es. In two out of three infestations, Cry2Ae demonstrated 100% efficacy ; TwinL inkTM demonstrated 100% efficacy in all three infestations. Cry 1Ab treatments were less effective in the first and second infestation and demonstrated slightly better efficacy in the third infestation. However, corrected mortalities for Cry 1Ab were similar and just below 50%. When all data were pooled for each treatment (or cotton line), the mortality

rates were high. PBW mortalities in Cry2Ae and TwinLinkTM were not significantl y different from 100%. The reported cross - resistance of AZP - R to Cry 1Ab toxin was consistent with the findings in this study . No reported cross - resistance to Cry2Ae was apparent. T he authors concluded that Cry2Ae and TwinLinkTM expressed a high - dose against PBW. The authors stated that PBW survival on Bt plants ( TwinL inkTM and Cry2Ae) could have been higher because 1 st instar larvae might have been exposed to hemizygous rather than p urely homozygous seed tissue. Experimental Bt plot sizes where small, and Coker null lines were in close proximity , which could have facilitated low rates of out - crossing. Also, other factors like environment or altered production of protein due to warting (after bollworm attack) may have led to lower Bt expression in bolls than expected and hence, led to higher survival of PBW on TwinL inkTM and Cry2Ae plants. **Table D.**  **11 . M**  **ortality results for Cry1Ac**  **resistant PBW following artificial infestation of cotton plants**  **Infestation/ Data**  **collection Treatment**  **Entry Holes**  **Live**

**Large Larvae**  **Apparent**

**Mortality (%)**  **Low er** 

**95% Mean**  **Upper**

**95% Mean Corrected** 

**Mortality (%)**  Ju ly Coker

Cry1Ab Cry2Ae TwinLink TM 210 466 448 569 86

99 1 0 51.9

76.8 99.8 100 36.0

71.3 99.4 100 67.7

82.3 100 100 -

48.1 99.5 100 August Coker

Cry1Ab Cry2Ae TwinLink TM 279 431 467 468 89

71 0 0 67.1

79.8 100 100 60.5

73.1 100 100 73.7

86.5 100 100 -

48.4 100 100 September Coker

Cry1Ab Cry2Ae TwinLink TM 111 71 141 327 30

9 0 0 67.5

90.3 100 100 52.6

78.6 100 100 82.4

100 100 100 -

53.1 100 100 Modified from MRID 476400 - 14 **Table D.**  **1 . Pooled m**  **ortality results for Cry1Ac resistant PBW following artificial infestation of cotton plants**  **Treatment Total** 

**Entries N** 

**Bolls Live** 

**Large Larvae**  **Apparent**

**Mortality Low**  **er**

**95% mean Upper** 

**95% Mean Corrected** 

**Mortality (%)**  Coker 600 56 205 64.5 58.3 70.7 - Cry1Ab 968 74 179 80.3 76.0 85.6 45.9 Cry2Ae 1056 85 1 99.95 99.85 100 99.7 TwinLink TM 1364 112 0 100 100 100 100 Modified from MRID 476400 - 14Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 105 of 153 **IV. CROSS**  **- RESISTANCE POTENTIAL**  (MRID 476400 - 14) BCS referred to a series of binding studies (Karim et al., 2000; Luo et al., 2007; Hernandez - Rodriguez et al., 2008) to support that Cry 1 and Cry2 proteins bind to different target sites in the larval midgut of Lepidoptera pests. In addition, work by Hernande z - Rodriguez et al. (in press) showed that Cry2Aa, Cry2Ab, and Cry2Ae share a common binding site in CBW and the Old World bollworm. BCS commissioned Dr. Jurat - Fuentes at the University of Tennessee to conduct competitive binding experiments with radio la beled Cry 1Ab and Cry2Ae proteins using BBMV of TBW and CBW to confirm that these proteins bind to the target pests’ midgut with specificity. Furthermore, Dr. Jurat Fuentes conducted a study with Cry 1F, Cry 1Ab, Cry 1Ac, Cry2Ab, and Cry2Ae to fully characteri ze competitive binding aspects between the two newly proposed Bt toxins for cotton and those toxins alread y found in alread y registered Bt cotton products (see figures and results below). BCS did not conduct binding studies for the third main target pest, PBW; rather the applicant cited publicly available literature. In experiments by Karim et al. (2000), Cry 1Ab and Cry 1Ac bound specificall y and with high affinity to BBMV of PBW, while Cry 1Aa and Cry2Aa bound with less specificit y and lower affinity yet sh owed high binding site concentration. BCS stated that these results confirmed that Cry 1A and Cry2A proteins were unlikely to be for cross - resistant, as was seen in other insect studies. 1. Binding Experiments with Cry1Ab and Cry2Ae using CBW Brush Border Mem brane Vesicles The BBMV experimental results showed that labeled Cry 1Ab (see Figure 2) and Cry2Ae (not shown here) bound with specificity to the midgut of CBW.

**Figure D.**  **2 . BBMV binding assays with increasing CBW BBMV concentrations and fixed radio**  **- labeled Cry1Ab**  (from MRID476400 - 14). Unlabeled and labeled toxin (of constant amount) tend to bind to specific sites first; once the specific receptors are occupied, toxins bind to unspecific sites. Non - specific binding continues to increase linearly because the concentration of non - specific sites increases with increasing BBMV.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 106 of 153 2. Binding Experiments with Cry1Ab and Cry2Ae using TBW Brush Border Membrane Vesicles The BBMV experimental results showed that labeled Cry 1Ab (see Figure 3) bound with specificity to the midgut of TBW. The results for Cry2Ae are not shown here.

**Figure D.**  **3 .**  **BBM V binding assay with increasing TBW BBMV concentrations and fixed radio**  **- labeled Cry1Ab**  (from MRID476400 - 14) 3. Competition Experiments between Cry1Ab, Cry1Ac, Cry1F, Cry2Ab, and Cry2Ae using CBW Brush Border Membrane Vesicle A competitive binding experi ment with Cry 1Ab, Cry 1Ac, Cry 1F, Cry2Ab2, and Cry2Ae showed that Cry 1Ab shared high affinity binding sites with Cry 1Ac and Cry 1F in CBW. However, Cry 1Ab did not compete with Cry2Ab and Cry2Ae suggesting that these proteins do not share binding sites in CBW (See Figure 4).

**Figure D.**  **4 .**  **Com petition binding of Cry1Ab and com**  **petitors to BBMV of CBW** (from MRID 476400 - 14); if two different Bt toxins share binding sites, there will be less binding by the labeled toxin (with constant concentration) as the amount of competitor increases. When there is no competition, a flat line across can be expected for the competitor concentration. **H. zea** Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 107 of 153 4. Competition Experiments between Cry1Ab and Cry2Ae using TBW Brush Border Membrane Vesicles Results of a binding competition experiment between Cry 1Ab and Cry2Ae using TBW BBMV showed that the two toxins did not share binding sites (Figure 5).

**Figure D.**  **5 . Com** **petition assay with Cry1Ab and Cry2Ae using B BM**  **V of TBW** In conclusion, BCS stated that the relative potential for cross resistance was low between variants of Cry 1 and Cry2 toxins as expressed in Bt cotton. Yet, the potential for cross - resistance was high within the group of Cry 1 variants (e.g. Cry 1Ab, Cry 1Ac, and Cry 1F) as well as within the group of Cry2 variants (e.g. Cry2Ab and Cry2Ae) (see Table below). **Table D.**  **2 .**  **BCS’ estim ated potential for cross**  **- resistance between various Bt proteins in TBW, CBW, and PBW** 

**Toxin Cry1Ab**  **Cry1Ac Cry1F**  **Cry2Ab Cry2Ae**  **Cry1Ab N/A**  **+++ ++**  **- -**  **Cry1Ac N/A**  **+ -**  **- Cry1F**  **N/A -**  **- Cry2Ab**  **N/A +++**  Modified from MRID 476400 - 14 +++, ++ = high potential; + = low potential; - = little to no potential **V. MODELING**  BCS commissioned Dr. Mike Caprio (Mississippi State University) to conduct a risk assessment for TwinL inkTM cotton on CBW using a model by Caprio et al. (2009). Dr. Caprio conducted a probabilistic risk assessment using a deterministic model and using PERT distributions (ty pe of Beta distribution with mean was weighted, for example, 4x more than minimum and maximum values) for parameters of interest (with the exception of two cases: dominance associated with fitness costs). One thousand simulations were run with reasonable parameter values chosen at random from distributions derived with expert feedback ( Table D. 14 ). The model included 43 different habitats from early season wild hosts, non - Bt corn, Bt cotton, so yb eans, sorghum, and late season wild hosts. A field of cotton progressed through four different habitats in a growing season: fallow field (unsuitable habitat), pre - bloom cotton, flowering cotton, peak bloom **H. vi** **res cens** Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 108 of 153 cotton, and back to fallow field. Two Bt traits were modeled, Cry 1Ab was assumed to have complete cross - resistance to Cry 1Ac, and Cry2Ae was assumed to have complete cross - resistance to Cry2Ab2. The size of each field was assumed to be 100 acres, and the modeling landscape consisted of 1024 fields. Corn, cotton, and soy b eans accounted for 260 fields each, sorghum for 99 fields, and wild hosts for 145 fields. 100% of cotton was simulated as TwinL inkTM cotton. No Cry 1Ab corn was modeled in the landscape because BCS did not have access to sales information. The rate of resis tance evolution was estimated in two way s: (1) The final R - allele frequency after 31 ye ars was divided by

the initial frequency

(was not constant due to re - sampling) and the result was log transformed. In man y cases, the resistance allele frequency exceede d 90% and, once there, could no longer increase. All cases that approach the frequency of 1.0 received the same score no matter how many y ears it took to approach that value. (2) The rate of change in the resistance allele frequency was measured after thre e ye ars to estimate the rate of resistance evolution. **Table D.**  **3 .**  **Param eters varied and used to define probability function, and function used for random** **draws Parameter**  **Max Min**  **Most Likely Distribution**  Initial R - allele frequency, Cry2Ae 0.01 0.00001 **0.0002** Pert - Log Initial R - allele frequency, Cry1Ab 0.05 0.00001 **0.002** Pert - Log R - allele fitness cost (independently drawn for both alleles) 0.5 0.0 **0.1** Pert R - allele fitness cost dominance (independently drawn for both alleles) 0.2 0.0 **0.05** Mpert (2.0) Mortality from Cry2Ae 0.9999 0.999 **0.99** Pert - Log Mortality from Cry1Ab 0.99999 0.9999 **0.99** Pert - Log Dominance of resistance (independently draw n for both alleles) 0.3 0.00 **0.05** Pert Modified from MRID 476400 - 14 In the model, resistance did not evolve to either toxin in TwinL inkTM cotton in 1000 simulations of 31 ye ars of use; hence, under the conditions modeled there appeared little risk of product failure in a 30 - ye ar time horizon. In none of the simulations did the resistance allele frequency increase more than three - fold (see Figures 6 and 7); in most cases the R - allele frequency decreased. The fitness costs simulated were low (recessive in nature), however, the effects of fitness costs exceeded the effects of s election. The report was concluded with “These results support the contention that even small fitness costs can have a much larger impact on the evolution of resistance to stacked toxins than the case of plants expressing a single toxin”.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 109 of 153 **Figure D.**  **6 . Rate of increase in Cry1Ab resistance allele frequency after three years. Decreases in the resistance allele**  **frequency occur when fitness costs exceeded increases due to selection** (taken out of MRID 476400 - 14)

**Figure D.**  **7 . Rate of increase in Cry2Ae resistance allele frequency after 3 years** Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 110 of 153 **VI. REFUGE STRATEGY**  BCS stated that since 1) both Cry 1Ab and Cry2Ae express high - dose for the major target pests, CBW and TBW, and 2) the two toxins bind to diff erent target sites in the midgut of the three insect pests, that TwinL inkTM cotton should qualify for Natural Refuge. Their request was accompanied by a letter from Monsanto Company

which gave BCS permission to cite relevant studies in support of a natural refuge request. A waiver request for an IRM plan was submitted to the Agency because BCS does not intend to commerciall y sell seeds from the single events Cry1Ab and Cry2Ae. Very small acreages may

be grown for seed production only

in order to test combi nations with other insect control traits that could potentially

be candidate commercial products. BCS submitted a structured refuge request for the target pest pink bollworm. **VII. RESISTANCE MONITORING PROGRAM**  BCS proposed to monitor for resistance evolution in all three target pests in areas of high TwinL inkTM adoption. The applicant proposed to have baseline susceptibility established for Cry 1Ab and Cry2Ae proteins by 2012 (for CBW, TBW, and PBW) prior to commercialization. Sample collection for TBW and CBW would occur in the following manner: 10 - 20 populations for each pest would be collected in the primary cotton growing regions of the USA. For PBW, 4 - 6 populations would be collected from Arizona, New Mexico, West Texas, and California. This preliminary su sceptibility

data will provide valuable baseline information to compare with future annual insect susceptibility monitoring data. BCS will also use this baseline information to develop a discriminating dose for each toxin and each pest. Once TwinLinkTM has been commercialized, BCS has committed to monitoring of the three target pests on an annual basis. **VIII. REMEDIAL ACTION PLAN**  BCS stated that if insect resistance is suspected (monitoring or unexpected crop damage reports), it will attempt to confirm and characterize the resistance. First, the damaged cotton plants would be tested to confirm that the Bt proteins was produced and that a target pest of TwinL inkTM cotton was responsible for the observed damage. If the plants were confirmed to express TwinL inkTM , the insect damage was from a primary

target pest, and all other reasonable causes for the field damage had been ruled out (i.e. weather, cultural reasons), then insect populations would be collected from the region. Protein and plant bioassay s would be con ducted to determine the level and extent of resistance on the sampled insect populations and a colon y established to determine if the putative resistance was heritable. Data from standardized bioassay s would be compared to the original baseline data. Communication with customers and extension agents in the region, the EPA, and other registrants and applicants , whose products could be impacted b y the findings, is another aspect of BCS’ proposed remedial program. Confirmed resistance would define the remedia l action area(s) as well as the approach. The main focus would be to limit the spread of resistant alleles b y eliminating resistant population(s) as quickl y as possible. This might be accomplished through chemical insecticide treatments of the Bt cotton fi eldBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 111 of 153 effective against the resistant insects and/or using crop destruction methods that could limit the growth and reproduction of the resistant population(s). Susceptibility monitoring would be enhanced, especially in the region where resistance was identi fied. Refuge acreage may be modified and/or suspension of product sales could occur in the affected areas, not only TwinLinkTM , but for all products expressing similar Bt proteins that could be potentially cross - resistant. **IX. GROWER EDUCATION**  BCS stated that it will use grower meetings to introduce the TwinLinkTM technology and guide the growers on best practices to capture the highest production yields. Growers have the responsibility to monitor for unexpected crop damage in TwinLinkTM and to re port product failure to BCS. A toll free telephone number will be provided in brochures and other communication tools for efficient tracking and follow - up of unexpected insect damage reports. BCS will ensure that IRM plans are full y communicated to BCS sa les staff and distribution channels and initially , will limit TwinLinkTM seed sales to its own seed company , under FiberMax and Stoneville varieties. This will provide BCS with oversight on communication and control with educational efforts of sales staff

. The transition to a natural refuge for combined trait Bt cotton products will reduce the education/communication needs related to refuge deploy ment and eliminate the need to monitor grower compliance with IRM requirements aside from the required field moni toring for unexpected insect damage. **X. GROWER COMPLIANCE**  Grower compliance with refuge and IRM requirements is a critical element for resistance management. Significant non - compliance with IRM among growers may increase the risk of resistance for Bt cro ps. To minimize the effects of non - compliance, it is necessary to develop a broad compliance program as part of the IRM strategy . Such a program has to include 1) an understanding of the effect of non compliance on IRM; 2) identification of compliance mech anisms to maximize adoption of IRM requirements; 3) measurement of the level of compliance; and 4) establishment of an enforcement structure to ensure compliance and penalize non - compliance. **XI. REPORTING REQUIREMENTS**  Once the baseline susceptibility data i s collected for the three primary target pests and a discriminating dose level has been established, annual monitoring will be conducted and reported to the agency . **XII. BPPD REVIEW**  **A. PROTEIN EXPRESSION**  BPPD reviewed B CS’ protein expression results reported in their IRM submission and concludes that single event T304 - 40 may not express the same levels of toxin as in the dual gene PIP, TwinL inkTM . It appears that T304 - 40 was expressed at higher levels in leaves of single eve nt plants compared to leaves collected from TwinL inkTM plants. For flowers and bolls the pattern was reversed, and TwinL inkTM plants appeared to express T304 - 40 at higher levels in these tissues. It is unclear whether theseBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 112 of 153 differences in expression results were statistically significant because BCS did not provide further information on the differences observed. Expression of GHB119 in leaves from earl y to late stage appeared somewhat variable or unstable. Expression levels were measured in leaves in 2004 and 2005 at three developmental stages (earl y leaf, mid leaf, and late leaf). At developmental stage 1, expression was higher than at developmental stage 2, and at developmental stage 3, expression increased again but to higher levels than observed at deve lopmental stage 1. For earl y, mid, and late boll, Expression level for GHB119 were also assessed in three different developmental stages of bolls (early , mid, and late). Levels of GHB119 were higher in TwinL inkTM than in the single event plant during develo pmental stage 2 and 3. It appears that the difference was approximately two - fold (see Figure D. 1 ). Table D. 1 in this review summarizes the protein expression level (μg/g - dry weight) submitted b y BCS for T304 - 40 and GHB119 in different plant tissues. It appears that the same tissue ty pes were lumped together for plants expressing single and p yr amided events. Since there appears to be unequal expression of the proteins in plants expressing either the single or p yramided events over the course of the growing season, this approach is not providing the most information. It would be more useful to have the expression data broken down by tissue for each individual event, GHB119, T304 - 40, and TwinLinkTM , rather than lumped together for the same protein. BPPD recommends that BCS provide more specific and explanatory information together with their protein expression results in future data submissions for PIP registrations. **B. DOSE**  The determination of dose, or the amount of toxin expressed by the transgenic crop relative to the susceptibility

of the target pests, is a critical component of IRM . Models have shown that a high - dose of toxin, coupled with a non - transgenic refuge to provide a supply of susceptible insec ts, is the most effective strategy

for delaying resistance in Bt crops . The high - dose/refuge strategy assumes that resistance to Bt is recessive and is conferred by a single locus with two alleles resulting in three genot yp es: susceptible homozygotes (SS), heterozygotes (RS), and resistant homozygotes (RR) . The high - dose/refuge strategy also assumes that there will be a low initial resistance allele frequency and extensive random mating between resistant and susceptible adults . In practice, a high - dose PIP should express sufficient quantities of toxin to kill all susceptible insects (SS) as well as heteroz ygous insects with one resistance allele (RS) . Lo wer dose PIPs might allow for survival of insects with at least one susceptibility

allele (SS or RS), alth ough effective IRM may still be possible with a suitable refuge strategy . To be able to demonstrate high - dose, it is recommended that applicant s generate data b y at least two of the five laboratory and field approaches as outlined by the SAP (1998) and des cribed by the Agency in the 1998 Bt Plant - Pesticides and Resistance Management document (US EPA, 1998) and 2001 Biopesticide Registration Action document (US EPA, 2001). The high - dose definition and verification techniques were developed in 1998 when all of the registered Bt crops were single toxin products targeted against L epidopteran pests . In recent ye ars, PIPs in Bt cotton have been approved that contain two genes targeted at the same insect pest . These “p yr amided” products can be beneficial for IRM s ince target pests must overcome two toxins to develop field resistance to the PIP . The benefits are greatest for two toxins with unrelated modes of action (i.e.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 113 of 153 binding to different Bt receptor sites in the midgut) that are expressed at high - doses in the p lant (Roush 1998) . For p yr amided products, the dose of each toxin should be evaluated separately . In cases where the py ramided product is created through conventional breeding and the dose of the single toxin products has alread y been established, the co mbined dose in the py ramided PIP can be determined with comparative efficacy

studies and protein expression data. However, this is not the case for TwinLinkTM cotton. BPPD reviewed B CS’ dose information for TBW, CBW, and PBW and concludes the following: 1. Tobacco Bollworm BCS submitted one dose verification method for TBW rather than two. Since all verification methods may be imperfect at assessing dose against a target pest when used alone, the Scientific Advisory Panel (1998) recommended that at least tw o methods (of five methods described by the panel) be used when demonstrating dose expression in target pests. BP PD notes that a second dose verification method is lacking for TBW. In the past Natural Refuge requests were granted on the basis that the toxi ns expressed high - dose against TBW. A second dose - verification method will need to be submitted if BCS wishes to maintain that GHB119, T304 - 40, and TwinLinkTM express a high - dose against TBW and to support their Natural Refuge request. **High -**  **dose verification method (1):**  From mortality

results obtained with dose verification method (1), BPPD concludes that events GHB119, T304 - 40, and TwinL inkTM may express a high - dose and effective high - dose, respectivel y, against TBW. After day 4 of the diet - inco rporated bioassay , mortality was reported as 100% for TwinLinkTM and Cry2Ae treatments, while control mortality ranged from 0% - 2%. For the overlay assay , mortality

was 100% for TwinLinkTM and Cry2Ae by day 7, while control mortality was 15%. For Cry 1Ab treatments, mortality reported from diet bioassay s did not reach 100% until after day 15. In addition, control mortality was relativel y high (ranging from 23% - 28%). For overlay assay s, mortality for TBW when exposed to Cry 1Ab was 100% by day 15; control mortal ity

at that time had reached 17%. Cry 1Ab may

express a high - dose against TBW. 2. Cotton Bollworm It appears that TwinL inkTM may express a possible effective high - dose against CBW. However, a second dose verification method turned out to be inconclusive during BPPD’s review and therefore, a high - dose cannot be verified. Should BCS wish to maintain that TwinL inkTM expresses a high - dose against CBW, additional information needs to be provided to clarify uncertainties in the experiment using high - dose verificati on method 4. **High -**  **dose verification method (1):** BPPD concludes that the diet - incorporated and overlay assay s together suggest that TwinLinkTM and GHB119 may

express a high - dose, but T304 - 40 may express less than high - dose against CBW.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 114 of 153 In the control treatments, larvae had developed into pupae b y da y 17. BCS did not report whether surviving larvae of the exposure treatments, Cry 1Ab, Cry2Ae, and TL, developed into pupae and consequentl y adults or whether survivors remained in the larval s tage. BPPD notes that for future submission, it is recommended that BCS report whether surviving larvae were developmentall y ‘dead’ (as opposed to ph ys iologically), or whether they continued to mature into pupae and adults. **High -**  **dose verification method ( 4):**  BPPD identified several uncertainties in the methodology section that preclude making a dose determination based on study results obtained with dose verification method (4). BCS stated in the IRM submission that mortality results were obtained from an artificial and natural infestation experiment done in conjunction with the efficacy study described in chapter 13 of their submission (MRID 476400 - 12). W hen BPPD consulted chapte r 13 for raw data or data tables, h owever, mortality data from the infestation experiments were lumped for all lepidopteran pests, and no pest specific mortalit y data were reported. Itis unclear how the data that BCS reported in chapter 15 of their submis sion (Tables 7 and 8) were obtained, and no further details about the study were submitted in an appendix accompan yi ng their submission (MRID 476400 - 14) . Furthermore, BCS stated in study volume 13 that some plots were treated with conventional pesticides, while others were not. It is unclear whether the mortality results reported in Table 7 and Table 8 come from plots that have been spray ed, not spray ed, or both, and what effect spraying might have had on d ose determinations. Coker cotton was planted in the control plot treatments for this natural and artificial infestation experiment; however, no information on control mortality was provided in the submitted report . It cannot be determined whether the fin al CBW mortality results from plots expressing Cry 1Ab, Cry2Ae, and TwinL inkTM have been corrected for control mortality . The applicant may

have conducted an egg hatchability study along with the infestation experiment to estimate egg viability

and larval attack rate. BPPD has not been provided with egg hatchability data or other information that would allow estimating viability and attac k rate. Without such information it is unclear how man y larvae hatched, attacked the Bt cotton plants, and ultimately survived Bt exposure. The applicant did, however, provide the infestation rate per plant. Furthermore, BCS provided BPPD with CBW average mortality across two locations in 2007 , and three locations in 2008, but no results were provided for individual locations. Inaddition to providing average mortality results, BPPD requests that individual treatment mortalities be reported for future data submissions so that minimum mortality per toxin is known. Should BCS wish to pursue the high - dose claim for TwinL inkTM and CBW, BPPD recommends providing clarification and further data for the study using high - dose verification method (4). BPPD further recommends that the applicant provide more in - depth descriptions of results and methodology

for future studies and data submissions. 3. Pink Bollworm **High -**  **dose verification methods (3) and (4):**Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 115 of 153 BPPD cannot make a conclusion about the dose profile for TwinL inkTM and its events and PBW (using verification methods 3 and 4) because it is unclear how the mortality results reported b y Ellsworth and Li (2008) were obtained. When BPPD divided the number of entry holes reported (Tables 9 - 12) b y the number of live lar

vae and calculated average mortality , the obtained results did not match mortalities reported b y BCS. **Deficiency 1** : The dose profile reported for PBW cannot be verified b y BPPD until BCS clarifies how mortalities were calculated for the experiments using dose verification methods (3) and (4). **Table D.**  **4 . BPPD Conclusion Regarding Dose Profile for**  **Events T304**  **- 40, GHB119, and TwinLink**  **TM Pest**  **Method #1 Method #4**  **Method #3 T304**  **- 40**  **GHB119 TL**  **T304 -**  **40 GHB119**  **TL T304**  **- 40**  **GHB119 TL**  CBW Non High Dose Possible

High Dose Possible

Effective High Dose Inconcl .* Inconcl.* I nconcl.* -- -- -- TBW Possible

High dose High Dose Effective

High Dose -- -- -- -- -- -- PBW

-- -- -- Inconcl .* Inconcl.* I nconcl.* --- Inconcl .* Inconcl.* * Insufficient information was submitted to the Agency to make a dose determination ** Cry1Ab could not be tested in this experiment because the lab strain was resistant to Cry1Ab. **C. CROSS**  **- RESISTANCE**  Analy ses of resistance to Bt Cry proteins indicate that cross - resistance occurs most often with proteins that are similar in structure (Tabashnik, 1994; Gould et al., 1995). BCS cited the work b y Crickmore et al. (1998), which supports that Cry 1 and Cry2 Bt proteins do not have more than 20% amino acid sequence homology

in common. Furthermore, the 3 - dimensional cry stal structures of Cry2Aa (very closely related to Cry2Ae as expressed in TwinLinkTM ) and Cry 1Aa (very closely related to Cry 1Ab as expressed in TwinL inkTM proteins have been resolved by Morse et al. (2001) and demonstrate that the proteins have a distinct tertiary structure. This significant difference in structure suggests that these two classes of proteins may

also have unique biochemical mechanisms, specifically with respec t to binding sites. The similarity

in the global architecture of Cry2Aa and Cry 1Aa, however, suggests that the two toxins may

have similar general steps for the mode of action. BPPD concludes that the evidence provided b y BCS supports that cross - resistanc e between Cry 1Ab and Cry2Ae, as expressed in TwinL inkTM cotton, is unlikely to develop in the main target pests CBW, TBW, and PBW. To summarize, the amino acid sequence homology between the two Bt toxins is less than 20%; the two protein structures differ s ubstantially at the third terminal suggesting that the two proteins may have unique mechanisms with respect to binding sites (and other biochemical pathway s); the binding assay

studies by

Jurat - Fuentes showed that Cry 1Ab and Cry2Ae were not competing for the same binding sites in CBW as well as TBW; and experiments by Karim et al. (2000) demonstrated that Cry 1Ab binding to gut membrane of PBW was specific while Cry2A binding was less specific y et with high binding site concentrations (suggesting that toxins were not competing for same binding sites).Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 116 of 153 However, BPPD notes that should resistance evolve in CBW and TBW to Cry2Ae as expressed in TwinL inkTM cotton that Cry2Ab2 (as in Bollgard II) may be compromised as well because, as BCS stated in their report, th ere is a great potential for cross - resistance between the two toxins. BPPD notes that the cross - resistance potential is great between Cry 1Ab, Cry 1Ac, and Cry 1F (although somewhat less for Cry 1F) based on the competition assay s; the three toxins share hi gh affinity binding sites. Should CBW evolve resistance to Cry 1Ab, other PIP products expressing Cry 1Ac and Cry 1F will likely be compromised as well. **D. MODELING**  EPA has used predictive models to compare IRM strategies for Bt crops. Because models cannot be validated without actual field resistance, models have limitations and the information gained from the use of models is only

a part of the weight of evidence used b y EPA in assessing the risks of resistance development. It was the consensus of the SAP (2000) Subpanel that models were an important tool in determining appropriate Bt crop IRM strategies. They agreed that models were “the only scientifically rigorous way

to integrate all of the biological information available, and that without these models, the Agency would have little scientific basis for choosing among alternative resistance management options.” They

also recommended that models must have an agreed upon time frame for resistance protection. For example, conventional growers may desire a maximum planning horizon of five y ears, while organic growers may desire an indefinite planning horizon. The Subpanel recommended that model design should be peer reviewed and parameters validated. Models should also include suc h factors as level of Bt crop adoption, level of compliance, economics, fitness costs of resistance, alternate hosts, spatial components, stochasticity , and pest population dy namics. BPPD reviewed the modeling assessment and accepted the probabilistic app roach presented therein. Based on the choice for parameter values, natural refuge assumptions, and their anal ys is, BCS stated that there is little risk of resistance evolution in CBW to TwinL inkTM within 30 y ears. BPPD needs further clarifications on severa l issues and has some concerns regarding assumptions made for certain important parameters. These are summarized below. **Deficiency 2** : In the modeling anal ys is provided by BCS, onl y CBW was modeled but not TBW. For a new refuge strategy , such as Natural Re fuge, each main target pest needs to be evaluated separatel y via a simulation model that incorporates biology , genetics, toxicity , and ecology of the target pest. BCS needs to provide simulation modeling for TBW in order to support their natural refuge req uest. **Deficiency 3** : The natural refuge paradigm was approved for Bollgard II and WideStrike with a high dose profile for TBW. If BCS wishes to pursue the natural refuge strategy for TwinLinkTM , then a second high - dose verification study will need to be sub mitted to support that the PIP expresses a high dose against TBW, or BCS can design a model with their specific dose data for TBW. **Deficiency 4** : CBW is the pest of most concern since its populations show greater genetic variability to toxins expressed in Bt cotton (Cry 1Ac, Cry 1F, and Cry2Ab2) than other pest (i.e. TBW). BCS’s LC 50 data show that CBW have a wide range of susceptibility against Cry 1Ab (462 - 2844 ng/cm 2 ) as expressed in cotton when compared to TBW (4.9 - 10.5 ng/cm 2 ). BCS concluded in their rep ort that TwinL inkTM expressed less than high - dose against CBW due to the target pest being less susceptible to Cry 1Ab and lower level of expression of Cry 1Ab in the parental event for TwinL inkTM . In addition, bioassay results for Cry 1Ab in corn (BPPD 2009) s howed greater variability as well for Corn earworm,Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 117 of 153 CEW, (aka CBW) than for other pests, such as ECB. In fact, there is up to a three - fold difference in magnitude of reported LC 50 s of CEW compared to ECB. And previous attempts to create a diagnostic concentration for CEW were frustrated b y the fact that the insect was less susceptible to Cry 1Ab than other Lepidopteran pests. Given this information, BPPD is concerned with the dose assumptions for CBW and recommends that an additional anal ys is be conducted us ing somewhat lower mortality assumptions based on the study results reported b y BCS . The bioassay experiments showed that Cry1Ab caused mortalit y ranging 88.5 - 100% (see this review). Estimated mortalities from the artificial infestation experiments (2007 - 2 008) ranged from 99.5 - 99.7% (see this review). However, BPPD cannot verify the results of the artificial infestation experiments due to lack of information provided (i.e. % egg viability). At the moment, there is no evidence that Cry 1Ab expresses high - dose against CBW; hence BPPD recommends exploring dose mortalities ranging from 90 - 99% in the simulation model. **Deficiency 5** : BPPD is concerned about the most likely value chosen for initial resistance allele frequency for Cry 1Ab (0.0002). Although no field resistance to Cry 1Ab has been documented y et for corn earworm (CEW), the toxin has been in use for over a decade (in Bt corn) and selection may likely have increased the frequency of potential resistance alleles. However, because corn earworm does not over winter in the Corn Belt, BPPD agrees that the most likely value will be somewhat lower than could otherwise be expected. BPPD recommends that BCS consider modeling a most likely resistance allele frequency of 0.001 for Cry 1Ab in CBW. **Deficiency 6** : BCS nee ds to include Cry 1Ab corn and Cry 1Ab cotton in the modeling landscape when assessing the risk of resistance evolution in the target pests because Sy ngenta has registered Bt cotton and Bt corn and Monsanto has registered Bt corn products expressing Cry 1Ab. These PIPs are represented in the Cotton Belt and need to be included in a simulation analysis for TwinL inkTM . **E. REFUGE STRATEGY**  An IRM plan waiver request for single trait events (Cry 1Ab and Cry2Ae) is acceptable if seeds from those events are not sold commerciall y and serve only for producing the combination PIP TwinLinkTM . For an y other combinations, BCS may need to apply

for an experimental use permit or section 3 registration. BPPD recommends the use of acreage/geographic limitations for the single tr ait events as a means to mitigate potential resistance development. BCS should provide information on proposed acreage for these single trait events. BPPD notes that a second dose verification method is lacking for TBW. In the past Natural Refuge requests were granted on the basis that the toxins expressed high - dose against TBW. A second dose verification method will need to be submitted to support BCS’ Natural Refuge request, or BCS can design a model with their specific dose data for TBW. BPPD verified that BCS cited all the relevant natural refuge studies originally developed by

Monsanto Company and has obtained a data citation permission letter from Monsanto. When BCS has satisfactorily addressed the deficiencies related to the modeling of resistance evolution in CBW and TBW, BPPD will re - assess the natural refuge request for TwinL inkTM cotton. If a Natural Refuge strategy for TwinLinkTM is approved by BPPD for TwinL inkTM , the requirements for a non -Bt cotton refuge in conjunction with the planting of a ny acreage of TwinL inkTM cotton should remain in place for the following states and regions: Arizona, California, and New Mexico and in theBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 118 of 153 following Texas counties: Brewster, Crane, Crockett, Culberson, El Paso, Hudspeth, Jeff Davis, Loving, Pecos, Presid io, Reeves, Terrell, Val Verde, Ward, and Winkler. For those States and regions requiring a non - Bt refuge, the following refuge planting requirements are recommended: 1) 5% External, Unsprayed Refuge At least 5 acres of non -Bt cotton (refuge cotton) must be planted for every 95 acres of Bt cotton. The size of the refuge must be at least 150 feet wide, but preferabl y 300 feet wide . This refuge may not be treated with any

insecticide labeled for the control of tobacco budworm, cotton bollworm, or pink bo llworm . At the pre - squaring cotton stage onl y, the refuge may be treated with any lepidopteran insecticide to control foliage feeding caterpillars. A non -Bt cotton refuge has to be maintained within at least 1⁄2 linear mile (preferabl y adjacent to or within 1/4 mile or closer) from the Bt cotton fields. 2) 20% External Spray ed Refuge At least 20 acres of non - Bt cotton are planted as a refuge for every 80 acres of Bt cotton (total of 100A). The non -Bt cotton may be treated with insecticides (excluding fol iar Btk products) for control of the tobacco budworm, cotton bollworm, or pink bollworm. A non -Bt cotton refuge has to be maintained within at least 1 linear mile (preferabl y within 1⁄2 mile or closer) from the Bt cotton fields. 3) 5% Embedded Refuge The refuge cotton must be planted as at least one single non -Bt cotton row for every six to ten rows of Bt cotton . The refuge may be treated with any

insecticide (excluding foliar Btk products) for the control of pink bollworm whenever the entire field is treated . The in - field refuge rows may not be treated independently of the surrounding Bt cotton field in which it is embedded. **F. RESISTANCE MONITORING**  The need for proactive resistance detection and monitoring is critical to the survival of Bt technology . The Agency

mandates that applicant s monitor for insect resistance (measurement of resistance conferring alleles) to the Bt toxins as an important earl y warning sign to developing resistance in the field and whether IRM strategies are working. Grower participation (e.g., reports of unexpected damage) is also important for monitoring. Resistance monitoring is also important because it provides validation of biological parameters used in models. However, resistance detection/monitoring is a diffic ult and imprecise task. It requires both high sensitivity and accuracy . Good resistance monitoring should have well - established baseline susceptibility data prior to introduction of Bt crops. The chances of finding a resistant larva in a Bt crop depend on the level of pest pressure, the frequency of resistant individuals, the location and number of samples that are collected, and the sensitivity of the detection technique. Therefore, as the frequency of resistant individuals or the number of collected sampl es increases, the likelihood of locating a resistant individual increases (Roush & Miller 1986). If the phenoty pic frequency of resistance is one in 1,000, then more than 3,000 individuals must be sampled to have a 95% probability

of one resistant individu al (Roush & Miller 1986). Current sampling strategies have a target of 100 to 200 individuals per location. Previous experience with conventional insecticides hasBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 119 of 1 53 shown than once resistant phenoty pes are detected at a frequency >10%, control or crop failur es are common (Roush & Miller 1986). Because of sampling limitations and monitoring technique sensitivity , resistance could develop to Bt toxins prior to it being easily detected in the field. ( http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm ) BCS’ approach for developing baseline data for TBW, CBW, and PBW by 2012 may be too protracted in the event that TwinL inkTM is commercialized before 2012. Baseline susceptibility data s hould be available b y the first growing season of commercial use of the PIP product . BPPD recommends that BCS accelerate the development of these data. The applicant proposed to monitor for resistance evolution in all three target pests in areas of high TwinL inkTM adoption. BPPD agrees with this proposal but also recommends that monitoring efforts additionally

focus on regions in states with little natural refuge. **Deficiency 7** : BCS needs to have a well - developed monitoring plan in place before TwinLinkTM is approved for commercialization (i.e. sampling regions, sampling scheme, etc). BPPD recommends that BCS follow the monitoring paradigm established for the other Bt cotton registrations. **G. REMEDIAL ACTION PLAN**  Remedial action plans are a potential response measure should resistance develop to Bt crops. Since resistance may

develop in “localized” pest populations, it may be possible to contain the resistance outbreak before it becomes widespread. A specific reme dial action plan should clearl y indicate what actions the applicant will take in cases of “suspected” resistance (i.e., unexpected damage) and “confirmed” resistance. The remedial action plan can also include appropriate adaptations for regional variation and the inclusion of appropriate stakeholders. To full y mitigate resistance, a critical element of any remedial action plan should be that once pest resistance is confirmed, sales of all Bt cotton hy brids that express a similar protein or a protein in whic h cross - resistance potential has been demonstrated would be ceased in the affected region ( see http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm ) **Deficiency 8** : BCS needs to have an established remedial action plan in place for each target pest of TwinL inkTM . It is not sufficient to provide an outline or general statement about different kinds of approaches. BPPD recommends that BCS follow the existing remedial action paradigm for Bt cotton PIPs. BPPD notes that it will not be sufficient if BCS only attempts to confirm and characterize resistance upon receiving notification of unexpected damage. Rather, unexpected pest damage reports should be taken seriousl y and followed up i mmediatel y to eventually conclusivel y determine whether the cause of crop damage is due to resistance or other reasons. BPPD understands that it can take several months before a determination can be reached. **H. GROWER EDUCATION**  It is recommended that BCS develop a grower education program that targets states and counties where a non - Bt corn variet y must be planted as the refuge for TwinLinkTM cotton. Those regions are Arizona, California, and New Mexico and the following Texas counties: Brewster, Crane, Crockett, Culberson, El Paso, Hudspeth, Jeff Davis, L oving, Pecos, Presidio, Reeves, Terrell, Val Verde, Ward, and Winkler.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 120 of 153 It is recommended that BCS develop a grower guide that at a minimum describes the technology and includes unexpected damage guidelines. **I. GROWER COMPLIANCE**  BPPD notes that BCS should have a well developed grower- education program (including grower guide) for all regions with clear indications for regional requirements. A natural refuge strategy

for TBW and CB W would eliminate the need for growers to plant a refuge where alternate hosts are abundant. However, there are cotton - growing regions where natural refuge is not applicable and where planting a refuge is still required. For those regions it is recommended that BCS develop, implement, and report to EPA on programs to evaluate and promote growers’ compliance with IRM requirements. These states and regions are Arizona, California, and New Mexico and in the following Texas counties: Brewster, Crane, Crockett, Culberson, El Paso, Hudspeth, Jeff Davis, Loving, Pecos, Presidio, Reeves, Terrell, Val Verde, Ward, and Winkler. Grower compliance in these regions should be assessed with on - farm assessments in addition to anony mous phone surveys. BPPD recommends that BCS use the existing grower compliance paradigm for other Bt cotton registrations. **J. REPORTING REQUIREMENTS**  BCS stated that “once the baseline susceptibility data was collected for the three primary target pests and a discriminating dose level was established, annual monitoring would be conducted and reported to the agency . BPPD recommends that BCS submit annual reports for the following:

 Baseline and discriminating dose work irrespective of their status;  Sales data;  Annual resistance monitoring; and  CAP results for cotton growing regions where Natural Refuge is not applicable.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 121 of 153 **REFERENCES** Bushey , D. 2008. Request for a waiver from the requirement to develop an insect resistance management plan. Unpublished report submitted by BCS to the Agen cy , M RID 476348 23 Caprio, M.A., Parker, C.D. and J.C. Schneider. Est. 2009. Future fitness of female insect pests in temporally

stable and unstable habitats and its impact on habitat utility as refuges for insect resistance management. J. Insect Sci. in press Crickmore, N., Zeigler, D.R., Feitelson, J., Schnepf, E, Van Rie, D., Lereclus, D., Baum, J., and Dean, D.H. 1998. Revisions of the Nomenclature for the Bacillus thuringiensis pesticidal cry stal proteins. Microbiology

and Molecular Biology

Reviews, Vol. 6 2 (3): 807 - 813. Gould, F., Anderson, A., Rey nolds, A., Bumgarner, L ., and Moar, W. 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. Ento mol. 88:1545 - 1559. Hernández - Rodriguez, C.S., Van Vliet, A., Bautsoens, N., Van Rie, J. and J. Ferré (est. 2008) Specific binding of Bacillus thuringiensis Cry2A insecticidal proteins to a common site in the midgut of Helicoverpa sp. Appl. Environ. Microbiol. In press. Karim, S., Riazuddin, S., Gould, F., and D.H. Dean. 2000. Determination of receptor binding properties of Bacillus thuringiensis δ - endotoxin s to cotton bollworm (Heliocoverpa zea) and pink bollworm (Pectinophora goss ypiella) midgut brush border membrane vesicles. Pest. Biochem. Physiol. 67:198 - 216. Luo S., Wu K., Tian Y., Liang G., Feng X., Zhang J., and Y. Guo. 2007. Cross - resistance studies of Cry 1Ac - resistant strains of Helicoverpa armigera (Lepidoptera: Noctuidae) to Cry2Ab. J. Econ. Entomol. 100:909 - 915. MacIntosh, S., Holloway, J., Jansens, S., and Van Rie, J. 2008. Insect resistance management plan: TwinL inkTM Cotton. Unpublished report submitted by Bay er CropScience to the Agency , MRID 476400 - 14. Roush, R. T., and G. L . Miller. 1986. Considerations for design of insecticide resistance monitoring programs. Journal of Economic Entomology , Vol. 79: 293 - 298. Roush, R. 1996. Two - toxin strategies for management of insecticidal transgenic crops: can p yramiding succeed where pesticides mixtures have not? Phil. Trans. Roy al Soc. Lond. B (1998): 1777 - 1786 Tabashnik, B.E. 1994. Evolution of resistance to Bacillus thuringiensis. Annu. Rev. Entom ol. 39:47 - 79.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 122 of 153 U . S . EPA, 1998. FIFRA Scientific Advisory Panel Subpanel on Bacillus thuringiensis (Bt) Plant Pesticides and Resistance Management, February 9 and 10, 1998 U . S . EPA, 2000. FIFRA Scientific Advisory Panel Subpanel on Bt Plant - Pesticid es Risk and Benefit Assessments: Insect Resistance Management, October 18 - 20, 2000 U . S . EPA, 2001. Biopesticides Registration Action Document

– Bacillus thuringiensis Plant In corporated Protectants, http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm BPPD. 2009. Review of 2007 monitoring data submitted by ABSTC for lepidopteran Bt corn registrants (MRID 474139 - 01). Memorandum from S. Borges to M. Mendelsohn dated April 8 th , 2009. **Appendix D. II -**  **EPA’s Review of BCS’ Response to Deficiencies** Below is EPA’s review of BCS’ response to the eight IRM deficiencies noted in BPPD’s risk assessment for TwinLinkTM dated 4/22/2010 ( EPA Reg. Nos. 264 - RNOU, 264 - RNOL, and 264 - RNOA, MRID s 4760014 and 476348 23 ). On July 29 th , 2010, BCS submitted their response to BPPD’s eight listed IRM deficiencies for TwinLinkTM (BPPD 2010). BPPD 18 reviewed B CS’ response and additional information and concludes that five of the eight deficiencies were sati sfactoril y addressed and resolved (U.S. EPA 09/28/10) . In BCS’ response to deficiency 8, BPPD recommends including the following: “reporting resistance to Agency

within 30 day s” and “a long - term IRM action plan” as outlined below. The applicant stated that the remaining deficiencies (#2 and #3) would be addressed during the next cotton growing season. BPPD recommends that these two items be included as terms of registration. **A) Discussion regarding Deficiency 1:** Deficiency

1 : The dose profile r eported for Pink bollworm (PBW) cannot be verified by BPPD until BCS clarifies how mortalities were calculated for the experiments using high - dose verification methods (3) and (4). BCS’ response : In high - dose verification experiments (method 3 and 4), BCS reported that the mortality was calculated as follows: Mortality = 1 - number of large larvae/ number of entry holes. BPPD response: BPPD has verified the calculations and confirmed the reported mortality results. This deficiency

has been satisfactoril y addressed and resolved. BPPD concludes that the single events Cry 1Ab and Cry2Ae in cotton both express a less than high - dose against PBW and that TwinL inkTM expresses a high - dose against PBW. **B) Discussion regarding Deficiencies 2 -**  **3:** Deficiency

2 : In the modeling anal ys is provided by BCS, onl y cotton bollworm (CBW) was modeled but not Tobacco budworm (TBW). For a new refuge strategy , such as Natural Refuge, each main target pest needs to be evaluated separately via a simulation model that incorporates bio logy , genetics, toxicity , and ecology

of the target pest. BCS needs to provide simulation modeling for TBW in order to support their natural refuge request.

18 The use of BPPD in this review refers to the BPPD IRM team consisting of Jeannette Martinez and Alan Reynolds.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 123 of 153 Deficiency

3 : The natural refuge paradigm was approved for Bollgard II and WideStrike with a high dose profile for TBW. If BCS wishes to pursue the Natural Refuge strategy for TwinLinkTM , then a second high - dose verification study will need to be submitted to support that the PIP expresses a high dose against TBW, or BCS can design a model with their sp ecific dose data for TBW. BCS’ response: BCS informed BPPD that an additional high - dose study for TBW is scheduled for the next cotton growing season when field infestations are present. Modeling resistance evolution in TBW to TwinL inkTM cotton will be con ducted afterwards incorporating dose profile and Natural Refuge assumptions. BPPD response: BPPD will review the dose data and modeling for TBW in support of a Natural Refuge request when the new data become available. In the meantime, in order to pursue registration with a structured refuge for TwinL inkTM , as proposed by BCS during the June 21 teleconference, this additional information is not required. **C) Discussion regarding Deficiencies 4 -**  **6:** Deficiency

4 : BPPD is concerned with the dose assumptions for CBW and recommends that an additional anal ys is be conducted using somewhat lower mortality assumptions based on the study results reported b y BCS . The bioassay experiments showed that Cry 1Ab caused mortality ranging 88.5 - 100% (see this review). Estimated mortalities from the artificial infestation experiments (2007 - 2008) ranged from 99.5 - 99.7% (see this review). BPPD cannot verify the results of the artificial infestation experiments , however, due to lack of information provided (i.e. % egg viability ). At the moment, there is no evidence that Cry 1Ab expresses high - dose against CBW. BCS also concluded that TwinL inkTM expressed less than high - dose against CBW due to the target pest being less susceptible to Cry 1 Ab and lower level of expression of Cry 1Ab in the parental event for TwinL inkTM . Hence BPPD recommends exploring dose mortalities ranging from 90 - 99% in the simulation model. Deficiency

5 : BPPD is concerned about the most likely value chosen for initial re sistance allele frequency for Cry 1Ab (0.0002). Although no field resistance to Cry 1Ab has been documented y et for corn earworm (aka CBW), the toxin has been in use for over a decade (in Bt corn) and selection may likely have increased the frequency of pote ntial resistance alleles. However, because corn earworm does not overwinter in the Corn Belt, BPPD agrees that the most likely value will be somewhat lower than could otherwise be expected. BPPD recommends that BCS consider modeling a most likel y resistanc e allele frequency

of 0.001 for Cry 1Ab in CBW. Deficiency

6 : BCS needs to include Cry 1Ab corn and Cry 1Ab cotton in the modeling landscape when assessing the risk of resistance evolution in the target pests because Sy ngenta has registered Bt cotton and Bt corn and Monsanto has registered Bt corn products expressing Cry 1Ab. These corn and cotton PIPs are grown in the Cotton Belt and need to be included in a simulation analy sis for TwinL inkTM . BCS’ response: The most likely R - allele value for Cry 1Ab was actua lly set at 0.002 in the original submission; however, the initial allele frequency was set at 0.00001. In order to address BPPD’s concerns, BCS increased the initial R - allele frequency of Cry 1Ab to 0.0001. With respect to incorporating Cry 1Ab corn in the m odeling landscape, all corn crops were converted to 80% Bt (dual gene) corn and 20% non - Bt corn. Mortality rates were based on H. zea data on TwinL inkTM . Results after 2000 simulations (with revised parameters) showed that resistance did not evolve to eithe r toxin inBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 124 of 153 any of the simulations. The rate of increase over the first five y ears for each locus reflected the presence of fitness costs. In man y cases, particularl y with the Cry1Ab resistance locus, the resistance allele frequency actually dropped. This w as likely due to the recessive nature of the fitness costs simulated. The most likely

initial resistance allele frequency was 10 - fold greater for this locus than for the Cry2Ae locus resulting at least initially in greater expression of fitness costs. The recessive nature of fitness costs may explain why

the final resistance allele frequencies at both loci were similar despite the fact that the most likely

value for the initial Cry 1Ab locus frequency was 10 - fold greater. Informall y, several runs were made w ithout the Cry2Ae toxin to compare the rate of increase of the Cry 1Ab resistance allele in a single gene versus a two gene construct, and the mean rate of increase was greater in the single gene case. These results support those of Gould et al. (2006) that not only do dual gene plants increase the expected longevity

of toxins but they

also increase the delay

due to fitness costs. Based on these results , there may be some criticism about the assumption of recessive inheritance of fitness costs given the lac k of extensive genetic data. This assumption is, however, conservative. If fitness costs are additive or even dominant, those fitness costs would be expressed at even lower resistance allele frequencies. The current assumption allowed resistance allele fre quencies to increase to higher levels before fitness costs were expressed. The fact that resistance did not evolve in these simulations despite the conservative assumption provides stronger evidence that the risk of resistance evolving over a 30 ye ar time horizon is small. BPPD response: The additional modeling information has been reviewed and BPPD concludes that the deficiency

has been adequately

addressed and resolved. BCS was able to demonstrate that with more conservative (greater) resistance allele frequencies and the inclusion of Cry1Ab corn, TwinLinkTM corn had a durabilit y of more than 30 years. **D) Discussion regarding Deficiency 7:** Deficiency

7 : BCS needs to have a well - developed monitoring plan in place before TwinLinkTM is approved for commercialization (i.e. sampling regions, sampling scheme, etc). BPPD recommends that BCS follow the monitoring paradigm established for other Bt cotton registrations. BSC response: For TBW and CBW, 10 - 20 population will be collected from the Cotton Belt; for PBW, 4 - 6 populations will be collected from the western USA (Arizona, New Mexico, West Texas, and California) during 2011. These samples in conjunction with data collected prior to commercialization will help establish the baseline susceptibility for all target pests. Resistance monitoring will focus on areas of highest adoption and highest risk for TBW and CBW and states where PBW are an economic pest. Where the risk is highest for resistance evolution in the target pests, BCS will collect at least 20 populations. Monitoring reports will include sampling methodology , bioassay methodology , standardized procedures, detection technique and sensitivity , statistical analy sis of probability to detect resistance. Discriminating dose assay s for TBW, CBW, and P BW will be provided to the Agency on a ye arl y basis. BPPD response: BPPD concludes that B CS’ monitoring plan reflects the paradigm established for other Bt cotton registrations and the deficiency has been satisfied. However BPPD recommends that in the Ter ms and Conditions of the registration, BCS be required to submit baseline susceptibility and a diagnostic assay

data.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 125 of 153 **E) Discussion regarding Deficiency 8:** Deficiency

8 : BCS needs to have an established remedial action plan in place for each target pest of TwinL inkTM . It is not sufficient to provide an outline or general statement about different kinds of approaches. BPPD recommends that BCS follow the existing remedial action paradigm for Bt cotton PIPs. BSC response: For PBW, BSC will use the remedial action plan developed by the Arizona Bt Cotton Working Group. For TBW and CBW, baseline susceptibility data from 2011 and 2012 will define aspects of “suspected” and “confirmed” resistance and field response for each pest. In general, the plan will incorpo rate the following measures to identify or confirm suspected resistance:

 Monitoring (as outlined in MRID 476400 - 14);  Education of customer with respect to monitoring of unexpected levels of damage, reporting suspected resistance, and applying established remedial insecticide treatments;  Confirm resistance when unexpected damage or survival exceed established upper limit of normal baseline levels, LC 50 /L C 99 exceed established upper limit of baseline levels, and resistance is heritable;  Alternate methods to control pests as outlined under “Containment” below;  Sales restrictions/suspension until data provided and reviewed b y EPA support that target pests have regained susceptibility to Bt proteins;  Containment through chemical insecticide spraying, accelerated harvest, crop destruction methods, crop rotation, tillage, use of sterile insects, use of products with different modes of action;  Monitoring will be increased in affected and surrounding areas; change in ty pe and size of refuge should be considered. BPP D response: BPPD has reviewed BCS’s additional remedial action information and notes that the above is short summary

of the applicant ’s actual response. BPPD noted that the remedial action plans needs to explicitly

state that “the Agency

is informed within 30 day s of confirmed resistance”. In addition, BCS should incorporate that “a long - term resistance management action plan will be de signed according to characteristics of resistance events and local agronomic practices”. If BCS makes these changes, then Deficiency 8 and the Agency ’s request for more information will have been satisfied.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 126 of 153 **Appendix D. III –**  **Risk Assessment of data/infor mat**  **ion to respond to remaining IRM deficiencies.**  Below is U.S. EPA ’s review (June 22, 2011) of BCS’ resubmissions for FIFRA sec tion 3 application for TwinL inkTM Cotton expressing a p yr amid with Events T3 04 - 40 and GHB 119 (MRIDs 48480011, 484800 1 0 , and 484951 01), EPA File Sy mbols 264 - RNOA and 264 - RNOL : PC Codes: 006525 (Cry 1Ab), 006600 (Cry

2Ae ) . In the original IRM risk assessment dated April 22, 2010, EPA identified eight deficiencies, five of which were addressed on 09/28/10. BCS provided additi onal data/information to satisfy the remaining deficiencies. BPPD 19 review ed BCS’ submission to address deficiencies noted in the original review of TwinLinkTM Cotton, a two - gene p yramided PIP targeting Heliothine pests (BPPD 2010). BPPD’s conclusions and recommendations are contained in this memo. **CONCLUSIONS AND RECOMMENDATIONS** DOSE VERIFICAT ION FOR TBW (Deficiency 3) BCS provided enough data to resolve concerns with the dose for TwinLinkTM and its single parental events against TBW, and Deficiency 3 has been addressed in a satisfactory manner. Specificall y, based on the field experiment conducted with young cotton plants, BPPD concludes that Cry 1Ab and Cry2Ae express a high dose against Heliothis virescens (TBW) during the earl y cotton growing season (also late season for Cry2Ae) . No larvae on Cry2Ae and Cry 1Ab survived or developed to 3 rd instar during the 11 - day test duration, while 2.8% and 4.5% larvae in control treatments reached or surpassed the 3 rd i nstar developmental stage. The lab experiment conducted with older cotton plants, however, resulted in 2.17% survival ( > 3 rd instar) on Cry 1Ab plants expressing the parental event only suggesting that expression of Cry 1Ab was decreased in older plants comp ared to younger plants. Based on the limited data available at this point, BPPD cautions on the side of error and concludes that Event T304 - 40 does not express a high - dose against TBW during the late cotton - growing season (based on larval mortality on olde r cotton plants in the lab). TwinLinkTM cotton, however, expresses an effective high dose against TBW, and no larval survival was observed in the lab experiment. MODELING FOR TBW AND TWI NLINKTM COTTON WITH NATURALREFUGE (Deficiency 2 ) BCS provided additional modeling to resolve concerns for their “Natural Refuge” request for TwinL inkTM Cotton, and Deficiency 2 has been addressed in a satisfactory manner. BPPD concludes that TwinL inkTM Cotton should not pose a greater risk of resistance evolution in TBW than, for example, Bollgard II Cotton based on the modeling simulations provided in BCS submission. Specificall y, BCS conducted two modeling anal yses (stochastic, spatiall y explicit and deterministic, spatially implicit) and generall y took a conservative approach to modeling the durability of TwinL inkTM targeting TBW. Mainl y, the model landscape accounted for other Cry1A products and model explored several scenarios with increased dominance and lower survival based on the lower protein expressi on for Cry 1Ab cotton based on lab observations. The natural refuge assumptions were based on available natural host information of Mississippi

– a worst - case candidate state for this IRM strategy . The deterministic simulations estimated that durability of TwinL inkTM Cotton would be as low as 80 y ears when all worst - case assumptions were incorporated or as high as 224 ye ars using benchmark

19 The use of BPPD in this review refers to the BPPD IRM Team consisting of Jeannette Martinez and Alan ReynoldsBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 127 of 153 assumptions. This represents a 2.8 fold difference between estimated durabilities. The spatially explicit, stochastic simulations estimated that the durability for TwinLinkTM Cotton would be on average between 20 y ears (possibl y an unrealistic worst - case scenario based on survival percentages used) and 23 y ears (possibly a reasonable worst - case scenario). Assuming benchmark p arameter values for mortality (as used for other Cry 1A products in the past), the mean time to estimated product failure (resistance) was 26 y ears. Whether results differed significantly between any

scenarios modeled was unclear since no such anal ys is and/ or explanation accompanied the report. Additionally , comparative anal ys is between Bollgard II (Monsanto released data used for the natural refuge SAP) and TwinL inkTM Cotton showed that estimated mean durabilities were comparable. Although the likelihood fo r cross - resistance between Cry 1Ab and Cry2Ae has been shown to be small, BPPD notes that it would have been more conservative to consider and incorporate a small degree into the modeling anal ys is of TwinL inkTM Cotton. Other forms of epistasis (expression of a gene is suppressed b y a gene at another locus) were not considered: rather the applicant assumed that survival of genot yp es was multiplicative for both loci (the least conservative assumption). The SAP (2011) suggested that other forms of epistasis shou ld be explored for py ramided PIPs to address the risk of resistance evolution (see conclusion section for discussion). Resistance evolved almost simultaneously to both toxins (under worst case assumptions) when lower protein expression for Cry 1Ab was inco rporated for older plants and raised some concerns with BPPD. Further anal ys is should be conducted to assess whether this outcome was an artifact of the modeling design taken b y the modeler or a function of real temporal as ynchron y from the growing oviposi tion period between generations and delay ed oviposition of adults dispersing from senescing wild hosts to other acceptable hosts. BPPD notes though that the simulations with a lower dominance value for Cry 1Ab did not result in simultaneous resistance evolution of the two toxins during the ten replications. It will be important to determine whether this outcome can be replicated for the durability of other py ramided cotton PIPs that express Cry 1A Bt toxins which have lower expression levels during the later cotton growing season (resulting in greater dominance values for the target pest). BPPD concludes that TwinL inkTM Cotton deploy ed with a natural refuge strategy should not be expected to pose a greater risk of resistance evolution in TBW than for example Bollgard II Cotton based on the modeling simulations provided in BCS ’ submission. **I. BACKGROUND**  TwinL inkTM cotton is a py ramided transgenic cotton trait that expresses Cry 1Ab (Event T304 - 40), Cry2Ae (Event GHB119), and a glufosinate ammonium (herbicide) tole rant trait based on L ibert yLi nk technology . TwinL inkTM was developed b y BCS , using conventional breeding techniques. The Cry 1 and Cry2 Bt toxin group are effective against lepidoptera and share less than 20% amino acid homology . Crickmore et al. (1998) cons tructed a ph yl ogenetic tree using amino acid sequence identity as homology criteria for all presently

known Bt cry stal proteins. This tree indicates that Cry 1Ab and Cry2Ae are geneticall y dissimilar entities and share little sequence homology. On April 2 2 nd , 2010, the BPPD IRM team completed its initial review of BCS’ submission for a FIFRA product registration for TwinL inkTM cotton. During its review, BPPD identified eight IRM deficiencies for which BCS needed to provide clarifications and/or data.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 128 of 153 On Ju ly 29 th , 2010, BCS submitted their response to BPPD’s eight listed IRM deficiencies for TwinL inkTM (BPPD 2010a). BPPD reviewed B CS’ response and additional information and concluded that five of the eight deficiencies were satisfactorily addressed and resol ved (BPPD 2010b). The applicant stated that the remaining deficiencies (simulation modeling and second high - dose verification study , both for Tobacco budworm) would be addressed during the next cotton growing season. The third deficiency

(#8) related to th e remedial action plan and highlighted that BCS needed to explicitly state that “the Agency

is informed within 30 day s of confirmed resistance” . This BPPD review assesses deficiencies 2 and 3 listed below: Deficiency

2 : In the modeling anal ys is provided b y BCS, onl y cotton bollworm (CBW) was modeled but not Tobacco budworm (TBW). For a new refuge strategy , such as Natural Refuge, each main target pest needs to be evaluated separately via a simulation model that incorporates biology , genetics, toxicity , and ecology of the target pest. BCS needs to provide simulation modeling for TBW in order to support their natural refuge request. Deficiency

3 : The natural refuge paradigm was approved for Bollgard II and WideStrike with a high - dose profile for TBW. I f BCS wishes to pursue the Natural Refuge strategy for TwinL inkTM , then either a second high - dose verification study will need to be submitted to support that the PIP expresses a high - dose against TBW, or BCS can design a model with their specific dose data for T BW. **II. BAYER CROPSCIENCE RESUBMISSION MATERIALS**  1. Field Dose Stud y (MRID 484800 - 10) In 2011, a study

was conducted in Mississippi cotton fields (MRID 484800 - 10) to estimate mortality of Heliothis virescens (Tobacco budworm, TBW) on caged plants expressing parental Events Cry 1Ab and Cry2Ae as well as the pyramided PIP. Materials and Methods : Four treatments were tested: 1) Cry 1Ab cotton (T304 - 40), 2) Cry2Ae cotton (GHB119), 3) TwinLinkTM cotton (T304 - 40 x GHB119), and 4) Coker 312 (negative control). A treatment plot consisted of two 38 inch rows of 40 feet in length (0.0029 acres) and each contained approximately 240 plants. Fine mesh netting cages were set up (8 ft x 20 ft) in each plot which covered appr oximately 120 plants. Artificial infestations were conducted with individuals from a laboratory strain from USDA - ARS, Stonesville (MS). Fift y males and 50 females were released into each cage on two dates separated b y one month (two replicates). Mated fema les oviposited onto cotton plants, and first data collections began four day s after introducing the adults into the cages to obtain lower limits of eggs deposited . Ten randoml y selected plants in each cage were inspected and scored using binomial decision rule. If a plant contained at least 30 eggs, it was scored with a ‘y es’. Larval survival was estimated on 20 randomly selected plants within each cage, and survival and developmental stage were recorded. Larvae that survived 11 day s of exposure on the plant and had developed to 3 rd instar were scored as survivors. The lower limit of the proportion of surviving individuals was then computed by assuming that each plant was infested with 30 eggs (actual infestation was higher

– data not shown here) yielding an initial egg infestation cohort of 600 eggs (20 plants x 30 eggs) per replicate of Bt plant ty pe.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 129 of 153 Results : One larva developed to 1 st instar on one Bt plant type (Cry 1Ab), while 17 and 37 developed to ≥3 rd instar in the negative control plots (first and second replicate, respectively). Since no larvae survived on the three Bt treatments, there was no need to compute corrected mortalit y. **Table D**  **.III.1 .**  **TBW larval survival after 11 days of exposure to TwinLinkTM**  **and its parental events Treatm**  **ent Type Numbe**  **r of Eggs (initial cohort**  **– mi**  **ni mum)**  **Total Number of Survivors**  **Number of Survivors**

**( ≥ 3**  **rd instar)**  **Proportion of Survivors**  **– mi**  **ni mum value**  **( ≥ 3**  **rd instar)**  Coker 312 (Control) Replicate 1 600 31 17 0.0283 Replicate 2 600 28 27 0.0450 Cry1Ab (single event) Replicate 1 600 1 0 0 Replicate 2 600 0 0 0 Cry2Ae (single event) Replicate 1 600 0 0 0 Replicate 2 600 0 0 0 Cry1Ab x Cry2Ae (TL) Replicate 1 600 0 0 0 Replicate 2 600 0 0 0 This Table has been modified from the Table in the original submission (p.12, MRID 484800 - 10) Applicant Conclusions : “This study

provided additional evidence for the high susceptibility of TBW to TwinL inkTM as well as its parental (single) events. No survivors ( ≥ 3 rd instar) were found on the parental event, therefore, TwinL inkTM should provide IRM benefits comparable to or exceeding those of other currently registered py ramided Bt cotton products approved for the natural refuge strategy .” 2. Greenhouse Dose Stud y (MRID 484800 - 11) In 2011, BCS conducted a greenhouse trial to provide further evidence that TwinL inkTM Cotton and its parental events express high dose against Heliothis virescens . Materials and Methods : Cotton plants (144 - 184 day s old) were placed into four isolated screened enclosures to hold groups of different Bt phenot ypes. The treatments were 1) Cry1Ab cotton (T304 - 40), 2) Cry2Ae cotton (GHB119), 3) TwinLinkTM cotton (T304 - 40 x GHB119), and 4) non - cotton plants (negative control). Each enclosure contained 12 plants of each treatment (three replicates total). Bt and non - Bt plants were artificially

infested with 50 and 15 (24 - hr old) TBW neonates, respectively. L arval counts were made at day 7 and 14 after infestation. Survival was computed as the proportion of individuals surviving 14 day s and completing development to at least 3 rd instar. Corrected mortality was calculated b y dividing ‘proportional survival on Bt’ b y ‘proportional survival on non - Bt cotton’. Results : Larval survival on non - Bt and Cry 1Ab p lants was 69 and five 3 rd instar larvae out of a total infestation with 540 (on control) and 1800 (on Bt); after correcting for control mortality, survival on Cry1Ab plantsBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 130 of 153 was estimated at 0.0217 (see Table 2). On Cry2Ae and TwinL inkTM cotton plants, no survivors were found (infestation with 1800 larvae). **Table D.**  **III. 5**  **. TBW larval survival on parental events and pyramided PIP TwinLinkTM**  **during the late cotton growing season**  **Treatm ent**  **Total Plants**

**infested Total Number** 

**of Neonates Total Number** 

**of Survivors Me**  **an**

**Proportion Survivors**  **Std.**

**Deviation Proportion**  **Corrected Mean**

**Proportion Survivors**  Non - Bt 36 540 69 0.128 0.0484 Cry1Ab 36 1800 5 0.00278 0.00192 0.0217 Cry2Ae 36 1800 0 0 0 0 TwinLinkTM 36 1800 0 0 0 0 This Table has been modified from the Table in the original submission (p. 11, MRID 4848000 - 11 Conclusions : The level of mortalit y observed in these trials and levels of survival on single - toxin plants was comparable to or exceeded that of the currentl y registered Bt c otton products. L ack of survivors on Cry2Ae plants underscores the highl y effective dose of this protein in the TwinL inkTM py ramid. The low level of survival has not been observed under field conditions but could be due to reduced protein expression levels in older plants tested (late - season decline has also been reported for Cry 1Ac in currentl y registered Bt cotton products). This study shows that plants, even late in the production cycle, continue to cause very

high mortalit y to TBW. This should provide ad ditional support for the use of natural refuge strategy

for T304 - 40 x GHB119. 3. Simulation Modeling for TwinL inkTM Cotton and Tobacco Budworm (MRID 484951 - 01) BCS contracted the simulation modeling to Dr. Mike Caprio from Mississippi State University . Two different models, stochastic and deterministic, were employ ed to estimate the durability of TwinLinkTM Cotton in an environment such as the state of Mississippi (worst - case state for natural refuge option). Materials and Methods : Spatially explicit, stochastic model : This model was based on a previous model b y Caprio et al. (2009) for Helicoverpa zea (cotton bollworm, CBW) and was modified to address TBW spec ifically and to conduct simulations for TwinL inkTM cotton. Onl y the modifications to the model were described in BCS’ modeling resubmission. A total of six different habitats were modeled (prevalence determined from 2002 data): Bollgard cotton, non - Bt cotto n, wild hosts (clover, geranium), and TwinL inkTM (post - cutout and very late season). Alternate hosts were modeled as separate patches, which in turn forced adult TBW to disperse between patches as wild hosts declined with progressing time. This landscape si mulated a situation in the field where alternate hosts would be spatially segregated. All patches were randomly assigned the nature of plant ty pe at the initiation of the simulation. All larvae and pupae were accounted for and placed as pupae in geranium p atches at the beginning of each ye ar. Because of complete mixing during the overwintering routine, the habitats were not redistributed at the beginning of each ye ar. This allowed detecting the effects of habitat distribution between simulations. The dose of Cry 1Ab was modeled at 99.9% for the first three generations and subsequently reduced to address the greater survival seen on cotton plants in the lab expressing parental Cry 1Ab. Four differentBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 131 of 153 levels of decline in protein expression were investigated le ading to 0.1% survival (0% decline), 2% survival (level observed in lab), 5% (see Table 3 for genoty pic survival), and 10% (extreme case). These levels of decline continued for two more generations until the overwintering subroutine began. Two values of do minance were used: 0.1 (high value) and 0.001 (lower value). Multiplicative toxin interactions were assumed. To overcome extinction of relativel y small TBW populations, the fecundity was raised from 50 to 150 eggs/day - female in the model. **Table D**  **.III .**  **3 . T**  **BW Survival on TwinLinkTM**  **Cotton with 5% and 0.1% survival to Cry1Ab and Cry2Ae Toxin**  **Cry1Ab Cry1Ae**  **Genotypes** SS RS RR

Pre - Cutout SS 0.000001 0.0001009 0.001 RS 0.0001009 0.01018081 0.1009 RR 0.001 0.1009 1.0 Post - Cutout SS RS RR SS 0.00005 0.000145 0.001 RS 0.00504 0.01463 0.1009 RR 0.05 0.145 1.0 Table modified from original Table (p.11, MRID 484951 - 01) Deterministic model : The Caprio model (based on a construct previously employ ed b y EPA - ORD) was used to model TBW resistance to TL cotton and modified to include refuges of variable size as well as Cry 1Ab protein expression decline in older plants . Mortality from Cry2Ae was constant at 99.9% for each generation. For Cry 1Ab, mortalit y was 99.9% for the first three generations (surviva l of the susceptible genot ype was set at 0.0001) and declined thereafter to 98%. The natural refuge percentage varied for each generation and was 20%, 45%, 7%, 1%, 3%, and 3% for each consecutive generation (worst case simulations based on refuge data in w orst case county of MS). Fitness for homozygous resistant individuals was 1.0 in all habitats (no fitness cost). Activity of the two toxins was multiplicative (no epistasis). Survivorship of the susceptible genot yp e was 0.0001% (0.01% x 0.01%). Dominance f or TBW was set at 0.001 to compare modeling runs with other models for validation purposes. For further validation, the model was run for a single gene product (Cry 1Ac such as for Bollgard), 20% refuge, and 50% reduction in adult emergence due to spraying. The estimated durability was 18.8 ye ars. Given that Bollgard was used for almost 15 y ears, this would suggest that a dominance value of 0.001 may not be unreasonable and on the high side of realistic. Additionally , comparative modeling was conducted for B ollgard II (Monsanto released data for natural refuge SAP) and TwinLinkTM to assess the relative durability of TwinLinkTM to the alread y registered py ramided PIP. Results : Stochastic model: When dominance was set at 0.001, no resistance evolved to TwinL inkTM within 31 ye ars (including worst - case scenarios with 10% survival after cutout). When dominance was increased to 0.1, results were more complex (see Table 3). With 5% survival in the later season, mean time to resistance was 23.9 ye ars . Greater survival later in the season resulted in faster resistance evolution. Additionally , in almost every simulation resistance to both toxins (Cry 1A and Cry2Ae) occurred almost simultaneously . This was unexpected since Cry 1Ab moved towards a lower dose later in the grow ing season, and the expectation was that resistance would evolve to Cry 1Ab first. Analy sis of data revealed that resistant individuals in two fields became temporaril y isolated from the remaining refuge populationBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 132 of 153 earl y in the season. Toxin expression was high at that time and hence, onl y individuals resistant to both toxins survived. Their homozygous resistant offspring emerged before susceptible individuals dispersed into those fields. This resistance was not caused by isolation as described by Peck et al . (1999) but took place in two separate fields (far enough apart so no dispersal occurred between them) after complete mixing had occurred during the overwintering season (an assumption made by the modeler as described in the methodology

section). This app arent temporal as ynchron y occurred although no developmental delay s were incorporated into the model and was a function of 1) a growing oviposition period between generations (adults emerge over an interval; late emerging cohorts will alway s be late until redistribution occurs during overwintering season) and 2) adults from senescing wild hosts had to disperse first to a new habitat before oviposition could take place (evidence for this phenomena from shorter “tail” of pupal emergence distribution from wild hosts compared to distributions from TLcotton). The data suggest that resistance evolved in “the tails of the generational distributions”. Resistance observed under these model parameters was not specific to TwinLinkTM Cotton but could be generated b y a h ypothetical two - gene PIP consisting of two high dose toxins (99.9% mortality). The modeler contended that the result might be an artifact based on model design aimed at addressing TBW biology . Further research, independent of an y particular PIP, may be war ranted. The model was run using Monsanto’s parameter values and dominance values of 0.001 and 0.1 . With lower dominance, resistance did not evolve in an y of the 10 replicates; with lower dominance values, resistance evolved as early as in 10.1 ye ars and mean time to resistance was 27.3 y ears (comparable to TwinL inkTM results). **Table D**  **.III .**  **6 . Mean tim**  **e to resistance for TBW to TwinLinkTM**  **Cotton Survival to Cry1Ab**  **Post -**  **Cutout Me**  **an Time to**

**Resistance (yrs) Number of**  **Simulations** 0.1% 1 26.0 10 2% 2 23.5 10 5% 23.9 10 10% 20.6 10 Survival to Cry2Ae was held constant during the different simulations 1 default survival value used by registrants based on field data from Cry1A plants prior to cutout . 2 Survival v alue observed by BCS in 2011 greenhouse studies of plants after cutout . Table modified from original Table (p.12, MRID 484951 - 01) Deterministic Model : The deterministic runs of the model resulted in an estimated 224 y ears before resistance evolved in TBW to TwinL inkTM (both toxin s) under benchmark assumptions. When the dominance value was increased to 0.01 (for later generations of TBW in response to lower expression levels in older cotton plants), it took 80 y ears before resistance evolved to TwinL inkTM (see Table D.III. 5 below). Unlike other previous models for TBW and currentl y registered Bt cotton PIPs expressing Cry 1A, BCS incorporated a seasonal decline in Cry 1Ab expression, which somewhat elevated the risk of resistance evolution. **Table D**  **.III .**  **5 . Durability of**  **TwinLinkTM Cotto**  **n based on the deterministic model simulations Dom**  **inance Value for Cry1Ab Time to Resistance (yrs)** 

0.001 224 0.01 1 80 TL simulations run with parameter values as close to Monsanto’s values for Bollgard II 1 Dominance is 10x that of Monsanto’s valueBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 133 of 153 Conclusions : “Based on both stochastic and deterministic models, using worst - case scenarios for refuge size and contrasts of other key

model parameters, TwinLinkTM would not be expected to change the risk of resistance over existing dual - Bt products. Risk of resistance to TwinLinkTM and all other Cry 1A - based dual - Bt products registered in the US may be somewhat higher than indicated by previous simulations, owing to possible late - season decline in Cry 1Ac expression.” **III. BPPD REVIEW AND CONCLUSIONS**  DOSE ( Defic iency 3 ): Based on the field experiment conducted with young cotton plants, BPPD concludes that Cry1Ab and Cry2Ae (individual parental events) express a high dose against Heliothis virescens (TBW) during the early cotton growing season (also late season fo r Cry2Ae) . No larvae on Cry2Ae and Cry 1Ab survived or developed to 3 rd instar during the 11 - day test duration, while 2.8% and 4.5% larvae in control treatments reached or surpassed the 3 rd instar developmental stage. The lab experiment conducted with older cotton plants, however, resulted in 2.17% survival ( > 3 rd instar) on Cry 1Ab plants expressing the parental event onl y suggesting that expression of Cry 1Ab was decreased in older plants compared to y ounger plants. Based on the limited data available at thi s point, BPPD cautions on the side of error and concludes that Event T304 - 40 does not express a high - dose against TBW during the late cotton - growing season (based on larval mortality on older cotton plants in the lab). TwinL inkTM cotton, however, expresses an effective high dose against TBW, and no larval survival was observed in the lab experiment. MODELING (

Deficiency 2 ): BCS conducted two modeling analy ses (stochastic spatially explicit and deterministic, spatially

implicit) and generally

took a conservative approach to modeling the durability of TwinL inkTM Cotton targeting TBW. Dr. Mike Caprio from Mississippi State University incorporated other Cry 1A products into the landscape and explored several scenarios with increased dominance and lower survival ba sed on the lower protein expression observed in the lab for Cry 1Ab cotton. The natural refuge assumptions in the model were based on available host data of Mississippi - a worst - case candidate state for this IRM strategy . The deterministic simulations esti mated that durability of TwinL inkTM Cotton would be as low as 80 ye ars when all worst - case assumptions were incorporated or as high as 224 ye ars using benchmark assumptions. This was a 2.8 fold difference between estimated durabilities. The spatially explic it, stochastic simulations estimated that the durability for TwinL inkTM Cotton was on average between 20 ye ars (probably an unrealistic worst - case scenario based on survival percentages used) and 23 y ears (probabl y reasonable worst - case scenario). Assuming benchmark parameter values for mortality (as used for other Cry 1A products in the past), the mean time to estimated product failure (resistance) was 26 ye ars. Whether results differed significantly between an y scenarios modeled was unclear since no such an al ys is and/or explanation accompanied the report. Additionally , comparative anal ys is between Bollgard II (Monsanto released data presented to SAP for Natural Refuge assessment) and TwinLinkTM Cotton showed that estimated mean durabilities were comparable b etween the two py

ramided PIPs. Although the likelihood for cross - resistance between Cry 1Ab and Cry2Ae has been shown to be small, BPPD notes that it would have been more conservative to consider and incorporate a small degree of epistasis (expression of a gene is suppressed b y a gene at another locus) into the modeling anal ys is of Cotton. Other forms of epistasis were not considered: rather the applicant assumed that survival of genot yp es was multiplicative for both loci (the least conservative assumption) . The SAP (2011) suggested that additional forms of epistasis should be explored for p yr amided PIPs such as, for example,Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 134 of 153 developmentall y restricted expression of low levels of Cry - protease and where resistance at the a locus could be determined b y the mos t rapidly evolving locus, which could drag other resistance loci along and, thereb y, increase the rate of resistance evolution beyond what would be estimated b y a purel y multiplicative approach. The discussion as to wh y resistance evolved almost simultane ously to both toxins (with assumption of increased dominance) raised some concerns with BPPD. Further anal ys is should be conducted to assess whether this outcome was an artifact of the modeling design taken b y the modeler or a function of real temporal as y

nchron y from the growing oviposition period between generations and delay ed oviposition of adults dispersing from senescing wild hosts to other acceptable hosts. BPPD notes though that the simulations with a lower dominance value for Cry1Ab did not result in simultaneous resistance evolution of the two toxins during the ten replications. It will be important to determine whether this outcome can be replicated for the durability of other py

ramided cotton PIPs that express Cry 1A Bt toxins with lower expressio n levels during the later cotton growing season (resulting in greater dominance values for the target pest). BPPD concludes that TwinL inkTM Cotton deploy ed with a natural refuge strategy should not be expected to pose a greater risk of resistance evolutio n in TBW than , for example , Bollgard II Cotton based on the modeling simulations provided in BCS submission.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 135 of 153 

### APPENDIX **E :**  **R eferences**  **Insect resistance Manageme n**  **t** 1. BPPD, 2010a. EPA review of B CS’ insect resistance management plan for a section(3) full commercial registration request for TwinLinkTM Cotton ( EPA File Sy mbol 264 - RNOU, MRIDs 47600 - 14 and 476348 - 23 ) . Memo randum from JC Martinez to S. Bacchus and D. Greenway on April 4. 2. BPPD, 2010b. EPA Review of Bay er’s Response to IRM Deficiencies noted in BPPD’s risk assessment for TwinLinkTM dated 4/22/2010 ( EPA File Sy mbol 264 - RNOU, MRIDs 47600 14 and 476348 - 23 ) . Memo randum from JC Martinez to S. Bacchus and D. Greenway on July 29. 3. Caprio MA, CD Parke r, and JC Schneider. 2009. Future fitness of female insect pests in temporall y stable and unstable habitats and its impact on habitat utility as refuges for insect resistance management. Journal of Insect Science , Vol. 9(44): 1 - 10 4. MA Caprio. 2011. A risk assessment of TwinLinkTM Cotton and Tobacco Budworm (Heliothis virescens) in the Midsouth. Bay er CropScience unpublished report submitted to the Agency , MRID 484951 - 01. 5. Crickmore N, DR Zeigler, J Feitelson, E Schnepf, D Van Rie, D Lereclus, J Baum , and DH Dean. 1998. Revisions of the Nomenclature for the Bacillus thuringiensis pesticidal cry stal proteins. Microbiology and Molecular Biology Reviews , Vol. 62 (3): 807 - 813. 6. EPA Scientific Advisory Panel (SAP). 2011. Transmittal Meetin g Minutes of the FIFRA Scientific Advisory Panel Meeting Held December 8 - 9, 2010 to Address Scientific Issues Associated with Insect Resistance Management for SmartStaxTM Refuge - in - the - Bag, a Plant - Incorporated Protectant (PIP) Corn Seed Blend. Memorandum f rom Dr. Sharlene Matten (DFO) to Dr. Steven Bradbury (Director of OPP) on March 3, 2011. 7. Navacerrad JJ, J Pierson, ICoats, and PLHunst. 2011. High dose study : estimating the mortality of Heliothis virescens in a greenhouse on plants on T304 - 40 x GHB119 (TwinLinkTM ) Cotton (BCS GH004 - 7 x BCS - GH005 - 8) and parental event plants T304 - 40 (Cry 1Ab) and GHB119 (Cry2Ae). Ba ye r CropScience unpublished report submi tted to the Agency , MRID 484800 11 8. W Mullins. 2011. High dose study : estimating the mortality of Heliothis virescens in a Mississippi cotton field on caged plants on T304 - 40 x GHB119 (TwinL inkTM ) Cotton (BCS - GH004 - 7 x BCS GH005 - 8) and parental event plants T304 - 40 (Cry1Ab) and GHB119 (Cry2Ae). Bay er Crop Science unpublished report submi tted to the Agency , MR ID 484800 10 9. Peck SL , F Gould, and SP Ellner. 1999. Spread of resistance in spatiall y - extended regions of transgenic cotton: implications for management of Heliothis virescens (Lepidoptera: Noctuidea). Journal of Economic Entomology , Vol. 92: 1 - 1Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 136 of 153 **REFERENC ES**  Other References : U . S . EPA . 1998. FIFRA Scientific Advisory Panel Subpanel on Bacillus thuringiensis (Bt) Plant Pesticides and Resistance Management, February 9 and 10, 1998 U . S . EPA . 2000. FIFRA Scientific Advisory Panel Subpanel on Bt Plant - Pesticides Risk and Benefit Assessments: Insect Resistance Management, October 18 - 20, 2000 U . S . EPA . 2001. Biopesticides Registration Action Document

– Bacillus thuringiensis Plant In corporated P rotectants, http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm U . S . EPA . BPPD. 2009. Review of 2007 monitoring data submitted by ABSTC for lepidopteran B t corn registrants (M RI D 474139 01). Memorandum from S. Borges to M. Mendelsohn dated April 8 th , 2009. **U.S. EPA REVIEWS –**  **I nsect Resistance Managem**  **ent** U.S. EPA . 2010a. Memorandum J . Martinez and A . Rey nolds. B CS’ Insect Resistance Management Plan for a Section (3) Full Commercial Registration Request of TwinL ink Cotton ( EPA Reg. No. 264 - RNOU, MRIDs 4760014 and 476348 23 ). April 22. U.S. EPA . 2010b. M emorandum. J . Martinez and A . Rey nolds. September 28, 2010. EPA Review of Ba ye r’s Response to IRM Deficiencies noted in BPPD’s risk assessment for TwinLink dated 4/22/2010 ( EPA File Sy

mbols 264 - RNOU, 264 - RNOL, and 264 - RNOA , MRIDs 4760014 and 476348 23). September 28. U.S. EPA . 2011. M emorandum. J . Martinez and A . Rey nolds. EPA review of Bay er CropScience’s resubmissions for TwinLinkTM Cotton sec(3) application expressing a p yr amid with Events T3 04 - 40 and GHB 119 (MRIDs 48480011, 48480010, and 484951 01), EPA File Sy mbol 264 - RNOA, - RNOL: PC Codes: 006 525 (Cry 1Ab), 006 600 (Cry 2Ae). June 28.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 137 of 153 **Appendix E**  **.1 .**  **Bibliography of data submitted for registration of**  **Bt Cry1Ab (event T304**  **- 40)**  **Cry1Ab and TwinLinkTM**  **, its combination with Bt**  **Cry 2Ae ( event GHB119**  **).** Most of the submissions below refer to Cry2Ae , and some of them also refer to TwinL inkTM . There may be repeats of those made for Cry2Ae in the Bibliography cited for Cry 1Ab because they were submitted for TwinLinkTM, which contains both proteins **. I.**  **Cry 1Ab -**  **Data Submissions MRID**  **Citation Reference ( Bt**  **Cry 1Ab PI**  **P )**  47076900 Ba ye r Cropscience LP. (2007) Submission of Toxicity Data in Support of the Experimental Use of Bacillus thuringiensis Cry 1Ab and Bacillus thuringiensis Cry2Ae for Use on Cotton. Transmittal of 2 Studies. 47076902 Rouque, D. (2006) Cry2Ae (GEM2) Protein: Acute Toxicity by Oral Gavage in Mice. Project Number: SA/06235, BBS06/010. Unpublished study prepared by Ba ye r Cropscience. 62 p. 47171700 Ba ye r Cropscience (2007) Submission of Residue Data in Support of the

Experimental Use of Bacillus thuring iensis Cry 1Ab Insecticidal Protein on Cotton Plants. Transmittal of 1 Study . 47171701 Hay es, R. (2007) Final Field Report for 2006 Season of Bacillus thuringiensis

Cry 1Ab Insecticidal Protein as Expressed in Cotton Plants. Project Number: 264/EUP/140. Unp ublished study prepared by

Bayer Cropscience. 6 p. 47634800 Ba ye r CropScience (2008) Submission of Efficacy Data in Support of the Application for Registration of BCS Cry 1Ab Cotton Event T304 - 40. Transmittal of 20 Studies. 47634801 Ferullo, J. (2008) Pro duct Characterization of Cry1Ab Cotton Event T304 - 40.

Unpublished study

prepared by

Bay er BioScience N.V. 22 p. 47634802 Nennstiel, D. (2008) Product Characterization: Nutritional Characterization of

Cry 1Ab Cotton Event T304 - 40. Unpublished study prepared by

Bay er CropScience LP. 8 p. 47634803 Currier, T.; Massengill, J. (2008) Protein Expression Analy sis of Cotton Event T304 -

40, Expressing Cry 1Ab and PAT/bar Proteins. Project Number: CP07B005. Unpublished study

prepared by

Bay er CropScience LP. 158 p. 4 7634804 Martone, A. (2008) Analyses of Raw Agricultural Commodity (Fuzzy Seed) of Cry 1Ab Cotton Event T304 - 40 for PAT/bar and Cry 1Ab and its Non - Transgenic Counterpart for PAT/bar and Cry 1Ab Proteins. Project Number: CP07B011. Unpublished study

prepared by Bay er CropScience LP. 154 p. 47634805 Martone, A. (2008) Structural and Functional Equivalence of Cry 1Ab and PAT/bar

Proteins Produced in Escherichia coli to Cry 1Ab and PAT/bar Proteins from Event T304 - 40 and TwinLinkTM Cotton, Gossypium hirsutum . Project Number: CP08B003. Unpublished study

prepared by

Bay er CropScience LP. 52 p.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 138 of 153 **MRID Citation Reference (**  **Bt Cry 1Ab**  **PI P**  **)** 47634806 Nennstiel, D. (2008) ORF Anal ys is - Toxicology (Human Health Assessment). Unpublished study

prepared by

Bay er Cropscience. 10 p. 47634809 Stephenson, I . (2008) Cry 1Ab P rotein: Epitope Homology , N - gl ycos yl ation and

Overall Amino Acid Sequence Homology Search with Known Toxins and Allergens. Supplement to MRID 46708802 and MRID 46708803. Unpublished study prepared by Bay er BioScience N.V. 6 p. 47634812 Nennstiel, D. (2008) Toxicology (Human Health Assessment): History of Safe Use. Unpublished study

prepared by

Bay er BioScience N.V. 49 p. 47634813 Chalmers, A. (2008) Summary of Non - Target Organism Testing and Assessment of Risk of Gossypium hirsutum : Transformation Ev ent T304 - 40 Expressing Cry 1Ab Protein. Project Number: ECORA1. Unpublished study prepared by

Bay er CropScience. 67 p. 47634815 Patnaude, M. (2008) Laboratory Study to Determine the Effects of Cry 1Ab Protein on the Predatory

Beetle Coleomegilla maculata. P roject Number: EB99L008, 13798/6217. Unpublished study prepared by

Springborn Smithers Laboratories. 60 p. 47634816 Scott, A. (2008) Cry 1Ab Protein: Toxicity to Green L acewing (Chry soperla rufilabris). Project Number: EB99L009. Unpublished study prepared by

Bay er CropScience LP. 4 p. 47634818 Bushey , D. (2008) Request for Waivers from the Requirement to Conduct a Non Target Soil Invertebrate Study : Earthworm. Unpublished study prepared by

Bay er CropScience LP. 6 p. 47634819 Say ers, L. (2008) Cry 1Ab Prote in: Ten Day Toxicity Test to Water Fleas (Daphnia magna) Under Static - Renewal Conditions. Project Number: 13798/6232, EB99L010. Unpublished study

prepared by

Springborn Smithers Laboratories. 45 p. 47634820 Martone, A. (2008) The Use of an Insect Heliothis virescens Bioassay to Determine the DT50 of the Cry 1Ab Protein Produced from Escherichia coli After Aerobic Soil Degradation. Project Number: CP07B010. Unpublished study prepared b y Ba ye r CropScience LP. 152 p. 47634821 Jesudason, P. (2008) Cry1 Ab: Efficacy Assessment (Field and Laboratory). Unpublished study

prepared by

Bay er CropScience LP. 5 p. 47634822 Bushey , D. (2008) Request for a Waiver from the Requirement to Prepare a Public

Interest Document. Unpublished study prepared by

Bay er CropSc ience LP. 6 p. 47634823 Bushey , D. (2008) Request for a Waiver from the Requirement to Develop an Insect

Resistance Management Plan. Unpublished study prepared by

Bay er CropScience LP. 6 p. 47634824 Nennstiel, D. (2008) Detection Methods: rtPCR. Unpublis hed study prepared by Ba ye r BioScience N.V. 6 p. 47634825 Nennstiel, D. (2008) Protein Detection Methods: Lateral Flow Strip (LFS).

Unpublished study

prepared by

EnviroLogix, Inc. 8 p.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document - DRAFT December 12, 2011

Page 139 of 153 **MRID Citation Reference (**  **Bt Cry 1Ab**  **PI P**  **)** 47634826 Nennstiel, D. (2008) Protein Detection Methods: Enzy me Linked Immuno Sorbent Assay (ELISA). Unpublished study prepared by

Strategic Diagnostics, Inc. and EnviroL ogix, Inc. 18 p. 47640000 Ba ye r Cropscience LP (2008) Submission of Product Chemistry , Efficacy , Toxicity ,

Exposure and Risk and Public Interest Data in Support of the Application for Registration of BCS TwinL inkTM Cotton Event T304 - 40 x GHB 119 Cotton. Transmittal of 14 Studies. 47640001 Ferullo J. (2008) TwinL inkTM Product Characterization - Molecular Characterization.

Unpublished study

prepared by Bay er BioScience N.V. 6 p. 47640002 Jesudason, P. (2008) Product Characterization - Nutritional Characterization of

Twinlink. Unpublished study prepared by

Bay er Cropscience LP. 8 p. 47640003 Mackie, S. (2008) Protein Expression Analy sis for T304 - 40, GHB 119 and

TwinL inkTM Transgenic Cotton. Project Number: CP08B002, BBS07/013, BB06/010. Unpublished study prepared by

Bay er Cropscience LP. 155 p. 47640004 Martone, A. (2008) Analyses of Raw Agricultural Commodity (Fuzzy Seed) of TwinL inkTM Cotton for PAT/bar, Cry2Ae and Cry 1Ab and its Non - Transgenic Counterpart for PAT/bar, Cry2Ae and Cry 1Ab Proteins. Project Number: CY07B008, BTS/0066/06, BBS07/006. Unpublished study prepared by

Bay er Cropscience LP. 216 p. 47640005 Martone, A. (2008) Structural and Functional Equivalence of Cry 1Ab, Cry2Ae and

PAT/bar Proteins Produce in Escherichia coli and Bacillus thuringiensis to the Cry 1Ab, Cry2Ae and PAT/bar Proteins from Events T304 - 40, GHB119 and TwinL inkTM Cotton Seed, Gossypi um hirsutum . USA, 2008. Project Number: CP08B011, BTS/0077/08, BBS07/006. Unpublished study prepared by

Bay er Cropscience LP. 70 p. 47640006 Martone, A. (2008) Structural and Functional Equivalence of Cry 1Ab, Cry2Ae and

PAT/bar Proteins Produced in Bacillus thuringiensis ( Bt) and Escherichia coli to Cry 1Ab, Cry2Ae and PAT/bar in TwinL inkTM Cotton, Gossypium hirsutum , USA, 2007. Project Number: CY07B006, BBS07/013, BIOX/001/PROTSUPP/07. Unpublished study

prepared by

Bay er Cropscience LP. 402 p. 47640007 Bus hey , D. (2008) Toxicology : Summary Human Health Assessment. Unpublished study prepared by

Bay er BioScience N.V. 8 p. 47640008 Chalmers, A. (2008) Summary of Non - Target Organism Testing and Assessment of Risk of TwinL inkTM Cotton Expressing Cry 1Ab and Cry2A e Proteins. Project Number: ECORA3. Unpublished study prepared by

Bay er Cropscience LP. 17 p. 47640009 Richards, K. (2008) Evaluation of the Dietary Effect(s) of Pollen from TwinL inkTM on Honey Bee Larvae (

Apis mellifera L. ) Development. Project Number: EB 99X037, CAR/204/08, BTS/0081/08. Unpublished study prepared b y C alifornia Agricultural Research, Inc. and Bay er CropScience Ecotoxicology Department. 48 p. 47640010 Patnaude, M. (2009) Chronic Toxicity to Collembola (Folsomia candida) UsingBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document - DRAFT December 12, 2011

Page 140 of 153 **MRID Citation Reference (**  **Bt Cry 1Ab**  **PI P**  **)** Transgenic Lyophilized TwinL inkTM Cotton Leaf. Project Number: EB99X036, 13798/6223. Unpublished study prepared by

Springborn Smithers Laboratories. 40 p. 47640011 Stafford, J. (2008) Broiler Chicken Nutritional Equivalency Study with TwinL inkTM Cotton. Project Number: 13798/4118, TX99X106, M/310302/01/1. Unpublished study prepared by

Springborn Smithers L aboratories. 223 p. 47640012 Rinehardt, M.; Holloway, J. ; Bus hey , D. (2008) Agronomic Performance and

Efficacy of TwinLinkTM Cotton Obtained by Conventional Breeding of Cry 1Ab Cotton Event T304 - 40 and Cry2Ae Cotton Event GHB119: 2007 - 2008 USA Production Seasons. Project Number: 07/044/102N, IR/7D/GH/MR, 07/059104N. Unpublished study

prepared by

Bay er Cropscience LP. 68 p. 47640013 Maclntosh, S.; Bushey , D.; Christian, M.; et al. (2008) Public Interest Document for

Bacillus thuringiensis Cry1Ab and Cry2Ae Insecticidal Proteins as Expressed in Combined Trait Cotton ( Tw inL inkTM Cotton) Plants. Unpublished study prepared by Ba ye r CropScience LP. 36 p. 47640014 Maclntosh, S.; Holloway, J.; Jansens, S.; et al. (2008) Insect Resistance Management

Plan: TwinL inkTM Cotton. Unpublished study prepared by

Bay er CropScience LP. 88 p. 47651100 Ba ye r Cropscience LP - Bioscience (2008) Submission of Fate and Toxicity Data in

Support of the Application for Registration of BCS Cry 1Ab Cotton Event T304 - 40. Transmittal of 6 Studies. 47651101 Rouquie, D. (2007) Cry 1Ab Protein: In Vitro Di gestibility Study in Simulated Intestinal Fluid. Project Number: M/295264/01/1, SA/07111, LYNX/PSI/N/TX99L035. Unpublished study prepared by

Bay er Cropscience. 54 p. 47651102 Rouquie, D. (2007) Cry 1Ab Protein: In Vitro Digestibility Study in Human Simulated Gastric Fluid. Project Number: M/295272/01/1, SA/07110, LYNX/PSI

N/TX51X006. Unpublished study prepared by

Bay er Cropscience. 56 p. 47651103 Rouquie, D. (2007) Cry 1Ab Protein: Acute Toxicity by Oral Gavage in Mice. Project Number: M/295507/01/1, SA/07109, LYNX/PSI/NTX51X007. Unpublished study prepared b y Ba ye r Cropscience. 58 p. 47651104 Rouquie, D. (2007) Cry 1Ab Protein: Heat Stability Study . Project Number: M/295690/01/1, SA/07112, LYNX/PSI/N/TX99L034. Unpublished study prepared by Bay er Cropsc ience. 51 p. 47651105 Richards, K. (2008) Evaluation of the Dietary Effect(s) of a Cry 1Ab Protein on Honey Bee Larvae (Apis mellifera L.). Project Number: EB99X030, CAR/177/07, BBS07/006. Unpublished study prepared by

California Agricultural Research, Inc . 111 p. 47651106 Patnaude, M. (2008) Chronic Toxicity to Collembola (Folsomia candida) Using Cry 1Ab Protein. Project Number: 13798/6218, EB99X028, BBS07/006. Unpublished study prepared by

Springborn Smithers L aboratories. 61 p.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document - DRAFT December 12, 2011

Page 141 of 153 **MRID Citation Reference (**  **Bt Cry 1Ab**  **PI P**  **)** 47692700 Ba ye r CropScienc e LP (2009) Submission of Toxicity Data in Support of the Application for Registration of BCS Cry 1Ab Cotton Event T304 - 40. Transmittal of 1 Study . 47692701 Patnaude, M. (2009) Cry1 Ab Protein - Toxicity to Green Lacewing (Chry soperla rufilabris) Following OPPTS Guideline 885.4340. Project Number: 13798/6230, EB99L009, 082808. Unpublished study prepared by

Springborn Smithers Laboratories. 48 p. 47850300 Ba ye r CropScience, LP (2009) Submission of Environmental Fate Data in Support of

the Experimental Use of TwinL inkTM Cotton on Cotton Plants. Transmittal of 1 Study . 47850301 Coats, I . (2009) TwinLinkTM Cotton (BCS - GH004 - 7 x BCS - GH005 - 8): Investigation

of Po llen Flow in Puerto Rico. Unpublished study prepared by

Bay er CropScience, LP. 24 p. 47871400 Ba ye r CropSciences (2009) Submission of Product Chemistry , Environmental Fate

and Toxicity

Data in Support of the Application for Registration of BCS Cry 1Ab Cott on Event T304 - 40. Transmittal of 4 Studies. 47871401 Coats, I . (2009) Request for Waivers from the Requirement to Conduct Certain

Product Anal ys is Studies as Outlined in 40 CFR 158.2120 - Cry 1Ab. Unpublished study prepared by

Bay er CropScience LP. 7 p. 47 871402 Coats, I . (2009) Request for Waivers from the Requirement to Conduct Certain

Toxicology

Studies to Test for Health Effects as Outlined in 40 CFR 158.2140 - Cry 1Ab. Unpublished study prepared by

Bay er CropScience LP. 9 p. 47871403 Coats, I . (2009) Request for Waivers from the Requirement to Conduct Certain

Nontarget and Environmental Fate Studies to Test for Ecological Effects as Outlined in 40CFR 158.21 - Cry 1Ab. Unpublished study prepared by

Bay er CropScience LP. 7 p. 47871404 Hunst, P. (2009) Brid ging Data from TwinL inkTM Cotton Avian Nutritional Study to Parental Events T304 - 40 and GHB119. Unpublished study prepared by

Bayer CropScience LP. 7 p. 47871600 Ba ye r CropScience, LP (2009) Submission of Product Chemistry and Toxicity

Data in Support of the Application for Registration of BCS TwinL inkTM Cotton Event T304 - 40xGHB 119 Cotton. Transmittal of 6 Studies. 47871601 Coats, I . (2009) Request for Waivers from the Requirement to Conduct Certain

Product Anal ys is Studies. Unpublished study prepared by Ba ye r CropScience, LP. 7 p. 47871602 Coats, I . (2009) Request for Waivers from the Requirement to Conduct Certain

Toxicology

Studies to Test for Health Effects as Outlined in TwinL inkTM . Unpublished study

prepared by

Bay er CropScience. 9 p. 47871603 Coats , I . (2009) Request for Waivers from the Requirement to Conduct CertainBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document - DRAFT December 12, 2011

Page 142 of 153 **MRID Citation Reference (**  **Bt Cry 1Ab**  **PI P**  **)** Nontarget and Environmental Fate Studies to Test for Ecological Effects - TwinL inkTM . Unpublished studies prepared by Bay er CropScience. 7 p. 47871604 Currier, T. (2009) Protein Expression Analy sis for Pat/bar, Cry 1Ab and Cry2Ae

Proteins in Whole TwinLinkTM Plants Grown in the Field. Project Number: CP09Q005. Unpublished study prepared by

Bay er CropScience, LP. 28 p. 47871605 Hunst, P. (2009) Investigati on of Sy nergistic Effects of the Cry 1Ab and Cry2Ae Proteins expressed in TwinL inkTM Cotton (BCS - GH004 - 7 x BCS - GH005 - 8) on Target Insect Pests Using L eaf Bioassay . Unpublished study prepared by

Bay er CropScience, LP. 8 p. 47871606 Coats, I . (2009) Supplement to MRID # 47640014: Insect Resistance Management

Plan: TwinL inkTM Cotton. Unpublished study prepared by

Bay er CropScience, LP. 7 p. 47903100 Ba ye r CropScience LP (2009) Submission of Toxicity Data in Support of the Application for Registration of BCS TwinL inkTM Cotton Event T304 - 40 x GHB 119 Cotton. Transmittal of 1 Study . 47903101 Hunst, P. (2009) Request for Waiver from the Requirement to Conduct an Avian

Oral Toxicity

Test: TwinLinkTM . Unpublished study prepared by

Bay er CropScience LP. 16 p. 479032 00 Ba ye r CropScience LP (2009) Submission of Toxicity Data in Support of the Application for Registration of BCS CRY1AB Cotton Event T304 - 40. Transmittal of 1 Study . 47903201 Hunst, P. (2009) Request for Waiver from the Requirement to Conduct an Avian

Oral Toxicity

Test: TwinLink. Unpublished study prepared by

Bay er CropScience LP. 16 p. 47928800 Ba ye r CropScience, LP - BioScience. (2009) Submission of Efficacy Data in Support of the Registration of BSC TwinL inkTM Cotton Event T304 - 40 X GHB 119 Cotton. Tr ansmittal of 1 Study . 47928801 Coats, I . (2009) Addendum to Product Chemistry for TwinL ink: Structural and Functional Equivalence of Cry 1Ab and Cry2Ae and PAT/bar Proteins Produced in Bacillus thuringiensis (Bt) and Escherichia coli to Cry 1Ab, Cry2Aa and PAT/Bar in TwinL inkTM Cotton, Gossypium hirsutum , USA, 2007: Final Report. Project Number: CY07B006. Unpublished study prepared by

Bay er CropScience, LP. 7 p. 48166300 Ba ye r CropScience LP - BioScience (2010) Submission of Efficacy Data in Support of the Exp erimental Use of Bacillus thuringiensis Cry2Ae Insecticidal Protein and Bacillus thuringiensis Cry1Ab and Cry2Ae Insecticidal Proteins on Cottons. Transmittal of 1 Study . 48166301 Bushey , D.; Acker, T.; Price, K.; et al. (2010) TwinL inkTM Cotton (BCS - GH004 - 7 x

BCS - GH005 - 8): Investigation of Pollen Flow in Puerto Rico: Year 2: 2009 - 2010. Project Number: M/386577/01/1. Unpublished study prepared by

Bay er CropScience. 21 p.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document - DRAFT December 12, 2011

Page 143 of 153 **MRID Citation Reference (**  **Bt Cry 1Ab**  **PI P**  **)** 48427900 Ba ye r CropScience (2011) Submission of Efficacy Data in Support of the Experi mental Use of T304 - 40 x GHB119 x COT102 x COT67B in Cotton. Transmittal of 1 Study . 48427901 Robinson, T. (2011) Cry1Ab, Cry2Ae, PAT/bar, FL Cry 1Ab, Vip3Aa, APH4 Levels

in Selected Tissues of the Combined Trait Product T304 - 40 x GHB119 x COT102 x COT67B (BCS - GHOO4 - 7 x BCS - GH005 - 8 x SYN - IR1O2 - 7 x SYN - IR O67 - 1). Project Number: 10/BM45L 001. Unpublished study prepared by

Bay er CropScience, LP. 22 p. 48471900 Ba ye r CropScience (2011) Submission of Product Chemistry Data in Support of the Experimental Use of Cry2Ae (GEM2) in Cotton. Transmittal of 1 Study . 48471901 Currier, T. (2011) Anal ysis to Determine if the Cry2Ae (GEM2) Protein from Cotton

Leaves is Gl ycos yl ated: Final Report. Project Number: BK05B003. Unpublished study prepared by

Bay er CropScience. 32 p. 48480000 Ba ye r CropScience LP (2011) Submission of Product Chemistry and Efficacy

Data in Support of the Applications for Registration of BCS Cry 1Ab Cotton Event T304 - 40, BCS Cry2Ae Cotton Event GHB119 and BCS TwinL inkTM Cotton Event T304 - 40 x GHB 119 Cotton, and the Petition for Tolerance of Cry2Ae. Transmittal of 11 Studies. 48480001 Habex, V.; Verhaeghe, S. (2008) Detailed Insert Characterization of Gossypium

hirsutum Transformation Event GHB119 b y Southern Blot Analy sis. Project Number: BBS07/009, M/308404/02/1. Unpublished study prepared by

Bay er BioScience N.V. 43 p. 48480002 Moens, S. (2008) Confirmation of the Absence of Vector Backbone Sequences in

Gossypium hirsutum Transformation Event GHB119. Project Number: BBS06/007, M/311127/02/1. Unpubl ished study prepared by

Bay er BioScience N.V. 40 p. 48480003 Moens, S.; Criel, I . (2008) Detailed Insert Characterization of Gossypium hirsutum

Transformation Event T304 - 40. Project Number: BBS07/014, M/311245/03/1. Unpublished study

prepared by

Bay er BioScience N.V. 62 p. 48480004 Habex, V.; Van Herck, H. (2007) Confirmation of the Absence/Presence of Vector

Backbone Sequences in Gossypium hirs utum Transformation Event T304 - 40: Final Report. Project Number: BBS05/003, M/308400/02/1. Unpublished study prepared by Bay er BioScience N.V. 41 p. 48480005 Currier, T. (2011) Characterization of Cry 1Ab and (Inert Ingredient) Proteins

Produced in Event T 304 - 40 Cotton, Gossypium hirsutum USA, 2011. Project Number: 10/BM99L508, M/406631/01/1. Unpublished study prepared by

Bay er CropScience LP. 31 p. 48480006 Moens, S. (2011) Characterization of Cry2Ae and (Inert Ingredient) Proteins

Expressed in Gossypium hirsutum Transformation Event GHB119. Project Number: BIO2/005/PROTEQUL/512, M/406615/02/1. Unpublished study prepared by

Bay er BioScience N.V. 35 p.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document - DRAFT December 12, 2011

Page 144 of 153 **MRID Citation Reference (**  **Bt Cry 1Ab**  **PI P**  **)** 48480007 Currier, T. (2011) Structural Stability Anal ys is of Gossypium hirsutum Combined Event TwinL inkTM X GlyTol (GHB614): Final Report. Project Number: CP08Q012/2. Unpublished study prepared by

Bayer CropScience LP. 34 p. 48480008 Currier, T.; Harbin, A. (2011) Protein Expression Analy sis for Gl yTol x TwinL inkTM

(GHB614 x T304 - 40 x GHB119) Cotton Grown in the Field, USA 2010. Project Number: 10BM99L440B, M/406245/01/1. Unpublished study prepared by

Bay er CropScience LP. 125 p. 48480009 Bushey , D.; Jansens, S.; Becker, A. (2010) Insect Bioassay of Plant Material Expressing Cotton Events T304 - 40, GHB119, and TwinL inkTM (T304 - 40xGHB119). Project Number: BIO2/005, INSECT/BIOASSAY/FROM/MONHEIM. Unpublished study prepared by

Bay er CropScience. 22 p. 48480010 Mullins, W. (2011) High Dose Study : Estimating the Mortality of Heliothis virescens in a Mississippi Cotton Field on Caged Plants of T304 - 40 x GHB119 (TwinLinkTM ) Cotton (BCS - GHOO4 - 7 x BCS - GHOO5 - 8) and Parental Events T304 - 40 (Cry 1Ab) and GHB119 (Cry2Ae). Unpublished study prepared by

Bay er CropScience LP. 16 p. 48480011 Denneh y, T. (2011) High Dose Study : Estimating the Mortality of Heliothis virescens in a Greenhouse on Plants of T304 - 40 x GHB119 (TwinL inkTM ) Cotton (BCS - GHOO4 - 7 x BCS - GHOO5 - 8) and Parental Event Plants T304 - 40 (Cry 1Ab) and GHB119 (Cry2Ae). Project Number: BA44Y001. Unpublished study prepare d by Bay er CropScience LP. 16 p. 48495100 Ba ye r CropScience (2011) Submission of Efficacy Data in Support of the Application for Registration of the TwinL inkTM . Transmittal of 1 Study . 48495101 Denneh y, T. (2011) A Risk Assessment of TwinL inkTM Cotton and Tobacco

Budworm (Heliothis virescens) in the Midsouth. Unpublished study prepared by Mississippi State University . 15 p.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document - DRAFT December 12, 2011

Page 145 of 153 

### Appendix E **.2. Bibliography of data submitted for**  **Bacillus thuringiensis Bt**  **Cry 2Ae ( event**  **GHB119 )**  **and TwinLink TM, its combination with**  **Bt Cry1Ab (event T304**  **- 40)**  Most of the submissions below refer to Cry2Ae , and some of them also refer to TwinL inkTM . There may be repeats of those made for Cry2Ae in the Bibliography cited for Cry 1Ab because they were submitted for TwinLinkTM, which contains both proteins **. MRID**  **Citation Reference (**  **Bt Cry 2Ae**  **PI P**  **)** 46708900 Ba ye r Cropscience LP (2005) Submission of Product Chemistry , Toxicity and Efficacy Data in Support of the Experimental Use of Cry2Ae for Use on Cotton. Transmittal of 9 Studies. 46708901 Jansens, S.; Rouan, D. (2005) Characteristics of Cry2Ae Cotton Plants Derived From

Transformation Events GHB119 and GHB714. Preliminary Report. Unpublished study prepared by

Aventis Cropscience N.V. 24 p. 46708902 Habex, V. (2005) Description of the Amino Acid Sequence of the Cry2Ae Protein.

Project Number: GEM2/AAS/ 01. Unpublished study prepared by

Aventis Cropscience N.V. 5 p. 46708903 Rouquie, D. (2005) Cry2Ae (GEM2) Protein: Overall Amino Acid Sequence

Homology

Search with Known Toxins and Allergens. Project Number: SA/05133, TX99S005, B004973. Unpublished study prepared by Bay er Cropscience. 120 p. 46708904 Rouquie, D. (2005) Cry2Ae (GEM2) Protein: Epitome Homology and N Gly cos yl ation Searches. Project Number: SA/05134, TX99S006, B004974. Unpublished study

prepared by

Bay er Cropscience. 192 p. 46708905 Rouquie, D. (2005) Cry2Ae (GEM2) Protein: In Vitro Digestibility Study in Simulated Gastric Fluid. Project Number: SA/05032. Unpublished study prepared b y Ba ye r Cropscience. 44 p. 46708906 Rouquie, D. (2005) Cry2Ae (GEM2) Protein: Acute Toxicity by Intravenous In jection in the Mouse. Project Number: SA/05033, B004972. Unpublished study prepared b y Ba ye r Cropscience. 80 p. 46708907 Currier, T. (2005) Anal ysis to Determine if the Cry2Ae (GEM2) Protein from Cotton

Leaves in Gl ycos yl ated. Project Number: BK05B003, B0 04964. Unpublished study prepared b y Ba ye r Cropscience LP. 28 p. 46708908 Saey , B.; Jansens, S. (2005) PAT Cry2Ae Protein in Cotton Tissues of Events

GHB119 and GHB714: Preliminary Report. Unpublished study prepared by

Aventis Cropscience N.V. 9 p. 46708909 Williams, D.; Jansens, S. (2005) Preliminary Efficacy Report of Cry2Ae Cotton Plants Derived from Transformation Events GHB119 and GHB714. Unpublished study prepared by

Aventis Cropscience N.V. 6 p. 47076900 Ba ye r Cropscience LP. (2007) Submissio n of Toxicity Data in Support of the Experimental Use of Bacillus thuringiensis Cry 1Ab and Bacillus thuringiensis Cry2Ae for Use on Cotton. Transmittal of 2 Studies.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document - DRAFT December 12, 2011

Page 146 of 153 **MRID Citation Reference**  **( Bt**  **Cry 2Ae PI**  **P )**  47076902 Rouque, D. (2006) Cry2Ae (GEM2) Protein: Acute Toxicity by Oral Gavage in Mice. Project Number: SA/06235, BBS06/010. Unpublished study prepared by Ba ye r Cropscience. 62 p. 47125100 Ba ye r CropScience LP. (2007) Submission of Product Chemistry and Fate Data in Support of the Experimental Use of Bacillus thuringiensis Cry2Ae Insecticid e Protein in Cotton. Transmittal of 2 Studies. 47125101 Jansens, S.; Rouan, D.; Habex, V. (2006) Characteristics of Cry2Ae Cotton Plants

Derived from Transformation Events GHB119 and GHB714: Preliminary Report and Supplemental Information. Unpublished stu dy prepared by

Aventis Cropscience N.V. 29 p. 47125102 Rouquie, D. (2007) Cry2Ae (GEM2) Protein: In Vitro Digestibility Study in Simulated Gastric Fluid. Project Number: SA/07101, TX99L 005, M/287164/01/1. Unpublished study

prepared by

Bay er Cropscience. 4 2 p. 47640000 Ba ye r Cropscience LP (2008) Submission of Product Chemistry , Efficacy , Toxicity ,

Exposure and Risk and Public Interest Data in Support of the Application for Registration of BCS TwinL inkTM Cotton Event T304 - 40 x GHB 119 Cotton. Transmittal of 14 Studies. 47640001 Ferullo J. (2008) TwinL inkTM Product Characterization - Molecular Characterization.

Unpublished study

prepared by

Bay er BioScience N.V. 6 p. 47640002 Jesudason, P. (2008) Product Characterization - Nutritional Characterization of

Twinlink. Unpublished study prepared by

Bay er Cropscience LP. 8 p. 47640003 Mackie, S. (2008) Protein Expression Analy sis for T304 - 40, GHB119 and

TwinL inkTM Transgenic Cotton. Project Number: CP08B002, BBS07/013, BB06/010. Unpublished study prepared by

Bay er Cropscience LP. 155 p. 47640004 Martone, A. (2008) Analyses of Raw Agricultural Commodity (Fuzzy Seed) of TwinL inkTM Cotton for PAT/bar, Cry2Ae and Cry 1Ab and its Non - Transgenic Counterpart for PAT/bar, Cry2Ae and Cry 1Ab Proteins. Project Number: CY07B 008, BTS/0066/06, BBS07/006. Unpublished study prepared by

Bay er Cropscience LP. 216 p. 47640005 Martone, A. (2008) Structural and Functional Equivalence of Cry 1Ab, Cry2Ae and

PAT/bar Proteins Produce in Escherichia coli and Bacillus thuringiensis to the Cry 1Ab, Cry2Ae and PAT/bar Proteins from Events T304 - 40, GHB119 and TwinL inkTM Cotton Seed, Gossypium hirsutum . USA, 2008. Project Number: CP08B011, BTS/0077/08, BBS07/006. Unpublished study prepared by

Bay er Cropscience LP. 70 p. 47640006 Martone, A. (2008) Structural and Functional Equivalence of Cry 1Ab, Cry2Ae and

PAT/bar Proteins Produced in Bacillus thuringiensis ( Bt) and Escherichia coli to Cry 1Ab, Cry2Ae and PAT/bar in TwinL inkTM Cotton, Gossypium hirsutum , USA, 2007. Project Number: C Y07B006, BBS07/013, BIOX/001/PROTSUPP/07. Unpublished study

prepared by

Bay er Cropscience LP. 402 p.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document - DRAFT December 12, 2011

Page 147 of 153 **MRID Citation Reference**  **( Bt**  **Cry 2Ae PI**  **P )**  47640007 Bushey , D. (2008) Toxicology : Summary Human Health Assessment. Unpublished study prepared by

Bay er BioScience N.V. 8 p. 47640008 Chalmers, A. (2 008) Summary of Non - Target Organism Testing and Assessment of Risk of TwinL inkTM Cotton Expressing Cry 1Ab and Cry2Ae Proteins. Project Number: ECORA3. Unpublished study prepared by

Bay er Cropscience LP. 17 p. 47640009 Richards, K. (2008) Evaluation of the Dietary Effect(s) of Pollen from TwinL inkTM on Honey Bee Larvae (Apis mellifera L.) Development. Project Number: EB99X037, CAR/204/08, BTS/0081/08. Unpublished study prepared b y C alifornia Agricultural Research, Inc. and Bay er CropScience Ecotoxicology Department. 48 p. 47640010 Patnaude, M. (2009) Chronic Toxicity to Collembola (Folsomia candida) Using Transgenic Lyophilized TwinL inkTM Cotton Leaf. Project Number: EB99X036, 13798/6223. Unpublished study prepared by

Springborn Smithers Laboratories. 40 p. 4 7640011 Stafford, J. (2008) Broiler Chicken Nutritional Equivalency Study with TwinL inkTM Cotton. Project Number: 13798/4118, TX99X106, M/310302/01/1. Unpublished study prepared by

Springborn Smithers L aboratories. 223 p. 47640012 Rinehardt, M.; Holloway, J. ; Bushey , D. (2008) Agronomic Performance and

Efficacy of TwinLinkTM Cotton Obtained by Conventional Breeding of Cry 1Ab Cotton Event T304 - 40 and Cry2Ae Cotton Event GHB119: 2007 - 2008 USA Production Seasons. Project Number: 07/044 /102N, IR/7D/GH/MR, 07/059104N. Unpublished study

prepared by

Bay er Cropscience LP. 68 p. 47640013 Maclntosh, S.; Bushey , D.; Christian, M.; et al. (2008) Public Interest Document for

Bacillus thuringiensis Cry1Ab and Cry2Ae Insecticidal Proteins as Expre ssed in Combined Trait Cotton ( TwinL inkTM Cotton) Plants. Unpublished study prepared by Ba ye r CropScience LP. 36 p. 47640014 Maclntosh, S.; Holloway, J.; Jansens, S.; et al. (2008) Insect Resistance Management

Plan: TwinL inkTM Cotton. Unpublished study prep ared b y Ba ye r CropScience LP. 88 p. 47641900 Ba ye r CropScience (2009) Submission of Product Chemistry and Toxicity

Data in Support of the Application for Registration of BCS Cry2Ae Cotton Event GHB119 Cotton and the Petition for Tolerance of Bacillus thur ingiensis Cry2Ae. Transmittal of 26 Studies. 47641901 Ferullo, J. (2008) Product Characterization of Cry2Ae Cotton Event GHB119.

Unpublished study

prepared by

Bay er BioScience N.V. 24 p. 47641902 Jesudason, P. (2008) Product Characterization - Nutritiona l Characterization of

Cry2Ae Protein. Unpublished study prepared by

Bay er CropScience. 6 p. 47641903 Currier, T. (2008) Protein Expression Analy sis of Cotton Event GHB119, Expressing

Cry2Ae and PAT/bar Proteins. Project Number: CY07B005. Unpublished study prepared b y Ba ye r CropScience LP. 161 p. 47641904 Martone, A. (2008) Analyses of Raw Agricultural Commodity (Fuzzy Seed) ofBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document - DRAFT December 12, 2011

Page 148 of 153 **MRID Citation Reference**  **( Bt**  **Cry 2Ae PI**  **P )**  Cry2Ae Cotton Event GHB119 for PAT/bar and Cry2Ae and its Non - Transgenic Counterpart for PAT/bar and Cry2Ae Proteins. Project Numb er: CY07B001. Unpublished study

prepared by

Bay er CropScience. 123 p. 47641905 Martone, A. (2008) Structural and Functional Equivalence of Cry2Ae and PAT/bar

Proteins Produced in Bacillus thuringiensis (Bt) and Escherichia coli to Cry2Ae and PAT/bar Prote ins in GHB119 Cotton, Gossypium hirsutum . Project Number CY07B002. Unpublished study prepared by

Bay er CropScience. 57 p. 47641906 Capt, A.; Nennstiel, D. (2008) Toxicology (Human Health Assessment): Summary of Analy sis of Open Reading Frame Homology Sear ches. Unpublished stud y prepared by Bay er Cropscience. 10 p. 47641907 Rouquie, D. (2008) Cry2Ae Protein: In Vitro Digestibility Study in Simulated Intestinal Fluid. Project Number: SA08127. Unpublished study prepared by

Bay er Cropscience. 52 p. 47641908 Rouquie, D. (2008) Cry2Ae Protein with Transit Peptide Epitope Homology and N Gly cos yl ation Searches. Project Number: SA08209, TX99L 003. Unpublished study prepared b y Ba ye r Cropscience. 30 p. 47641909 Rouquie, D. (2008) Cry2Ae Protein with Transit Peptide Overall Amino Acid

Sequence Homology

Search with Known Toxins and Allergens. Project Number: SA08208, TX99L 004. Unpublished study prepared by

Bay er Cropscience. 444 p. 47641910 Stephenson, I . (2008) Update on Cry2Ae Protein: In Vitro Digestibility Study in Human Simulated Gastric Fluid, Overall Amino Acid Sequence Homology Search with Known Toxins and Allergens, and Epitope Homology and N - Gl ycos yl ation Searches. Unpublished study prepared by

Bay er BioScience N.V. 6 p. 47641911 Rouquie, D. (2008) Cry2Ae P rotein: Heat Stability Study . Project Number: SA08128, TX99L 036. Unpublished study prepared by

Bay er Cropscience. 49 p. 47641912 Nennstiel, D. (2008) Cry2Ae Protein: History of Safe Use. Unpublished study prepared b y Ba ye r BioScience N.V. 7 p. 47641913 C halmers, A. (2008) Summary of Non - Target Organism Testing and Assessment of Risk of Gossypium hirsutum : Transformation Even GHB119 Expressing Cry2Ae Protein. Project Number: ECORA2. Unpublished study prepared by

Bay er CropScience LP. 50 p. 47641914 Richar ds, K. (2008) Evaluation of the Dietary Effect(s) of a Cry2Ae Protein on Honey Bee Larvae. Project Number: EB99 L001, CAR178/07. Unpublished study prepared b y California Agricultural Research, Inc. 109 p. 47641915 Patnaude, M. (2007) Laboratory Study to Determine the Effects of Cry2Ae Protein on the Predatory

Beetle Coleomegilla maculata. Project Number: EB99X006, 13798/6203. Unpublished study prepared by

Springborn Smithers Laboratories. 64 p. 47641916 Scott, A. (2008) Cry2Ae Protein - Toxicity to Green Lacewing (Chry soperla rufilabris). Unpublished study prepared by

Bay er CropScience LP. 4 p.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document - DRAFT December 12, 2011

Page 149 of 153 **MRID Citation Reference**  **( Bt**  **Cry 2Ae PI**  **P )**  47641917 Patnaude, M. (2008) Chronic Toxicity to Collembola (Folsomia candida) using GEM2 Proteins. Project Number: EB99X0081, 13798/6204. Unpublished study prepared by

Springborn Smithers L aboratories. 133 p. 47641918 Patnaude, M. (2008) Cry2Ae Protein - Acute Toxicity to Earthworms (Eisenia fetida). Project Number: 13798/6222, EB99X032. Unpublished study prepared by Springborn Smithers Laboratories. 37 p. 47641 919 Say ers, L. (2008) Cry2Ae Protein - Ten Day Toxicity Test to Water Fleas (Daphnia magna) Under Static - Renewal Conditions. Project Number: 13798/6233, EB99X033. Unpublished study

prepared by

Springborn Smithers Laboratories. 45 p. 47641920 Martone, A. ( 2008) The Use of an Insect Heliothis virescens Bioassay to Determine the DT50 of the C ry2Ae P r otein Produced from Bacillus thuringiensis after Aerobic Soil Degradation. Project Number: CY07B007. Unpublished study prepared by Ba ye r CropScience LP. 152 p. 47641921 Jesudason, P. (2008) Cry2Ae: Efficacy Assessment (Field and Laboratory). Unpublished study

prepared by

Bay er CropScience LP. 5 p. 47641922 Bushey , D. (2008) Request for a Waiver from the Requirement to Prepare a Public

Interest Document (Cry2Ae). Unpublished study prepared by

Bay er CropScience LP. 6 p. 47641923 Bushey , D. (2008) Request for a Waiver from the Requirement to Develop an Insect

Resistance Management Plan (Cry2Ae). Unpublished study prepared by

Bay er CropScience LP. 6 p. 47641924 Nennstiel, D. (2008) Detection Methods: rtPCR (Cry2Ae). Unpublished study

prepared b y Ba ye r BioScience N.V. 7 p. 47641925 Nennstiel, D. (2008) Protein Detection Methods: Lateral Flow Strip (Cry2Ae).

Unpublished study

prepared by

EnviroLogix Inc. 8 p. 476 41926 Nennstiel, D. (2008) Protein Detection Methods: Enzy me Linked Immuno Sorbent Assay (ELISA) (Cry2Ae). Unpublished study prepared by

EnviroLogix, Inc. 10 p. 47692500 Ba ye r CropScience (2009) Submission of Toxicity Data in Support of the Application for Registration of BCS Cry2Ae Cotton Event GHB119 Cotton. Transmittal of 1 Study . 47692501 Patnaude, M. (2009) Cry2Ae Protein - Toxicity to Green Lacewing (Chry soperla rufilabris) Following OPPTS Guideline 885.4340. Project Number: 13798/6229, EB99X025, BBS07/007. Unpublished study prepared by

Springborn Smithers Laboratories. 65 p. 47871400 Ba ye r CropSciences (2009) Submission of Product Chemistry , Environmental Fate

and Toxicity

Data in Support of the Application for Registration of BCS Cry 1Ab Cotton E vent T304 - 40. Transmittal of 4 Studies. 47871403 Coats, I . (2009) Request for Waivers from the Requirement to Conduct Certain

Nontarget and Environmental Fate Studies to Test for Ecological Effects as OutlinedBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document - DRAFT December 12, 2011

Page 150 of 153 **MRID Citation Reference**  **( Bt**  **Cry 2Ae PI**  **P )**  in 40CFR 158.21 - Cry 1Ab. Unpublished study pr epared b y Ba ye r CropScience LP. 7 p. 47871404 Hunst, P. (2009) Bridging Data from TwinL inkTM Cotton Avian Nutritional Study to Parental Events T304 - 40 and GHB119. Unpublished study prepared by

Bayer CropScience LP. 7 p. 47871500 Ba ye r CropScience, LP (200 9) Submission of Product Chemistry and Toxicity

Data in Support of the Application for Registration of Cry2Ae. Transmittal of 4 Studies. 47871501 Coats, I . (2009) Request for Waivers from the Requirement to Conduct Certain

Product Anal ys is Studies as Outlined in 40CFR 158.2120 - Cry2Ae. Unpublished study prepared by

Bay er CropScience, LP. 7 p. 47871502 Coats, I . (2009) Request for Waivers from the Requirement to Conduct Certain

Toxicology

Studies to Test for Health Effects as Outlined in 40CFR 158.2140 - Cry2Ae. Unpublished study prepared by

Bay er CropScience, LP. 9 p. 47871503 Coats, I . (2009) Request for Waivers from the Requirement to Conduct Certain

Nontarget and Environmental Fate Studies to Test for Ecological Effects as Outlined in 40CFR 158.3150 - C ry2Ae. Unpublished study prepared by

Bay er CropScience, LP. 7 p. 47871504 Hunst, P. (2009) Bridging Data from TwinL inkTM Cotton Avian Nutritional Study to Parental Events T304 - 40 and GHB119. Unpublished study prepared by

Bayer CropScience, LP. 7 p. 478716 00 Ba ye r CropScience, LP (2009) Submission of Product Chemistry and Toxicity

Data in Support of the Application for Registration of BCS TwinL inkTM Cotton Event T304 - 40xGHB 119 Cotton. Transmittal of 6 Studies. 47871601 Coats, I . (2009) Request for Waivers from the Requirement to Conduct Certain

Product Anal ys is Studies. Unpublished study prepared by

Bay er CropScience, LP. 7 p. 47871602 Coats, I . (2009) Request for Waivers from the Requirement to Conduct Certain

Toxicology

Studies to Test for Health Effects as Outlined in TwinL ink. Unpublished study prepared by

Bay er CropScience. 9 p. 47871603 Coats, I . (2009) Request for Waivers from the Requirement to Conduct Certain

Nontarget and Environmental Fate Studies to Test for Ecological Effects - TwinL ink. Unpublished studies prepared b y Ba ye r CropScience. 7 p. 47871604 Currier, T. (2009) Protein Expression Analy sis for Pat/bar, Cry 1Ab and Cry2Ae

Proteins in Whole TwinLinkTM Plants Grown in the Field. Project Number: CP09Q005. Unpublished study pre pared by

Bay er CropScience, LP. 28 p. 47871605 Hunst, P. (2009) Investigation of Sy nergistic Effects of the Cry 1Ab and Cry2Ae Proteins expressed in TwinL inkTM Cotton (BCS - GH004 - 7 x BCS - GH005 - 8) on Target Insect Pests Using L eaf Bioassay . Unpublished study prepared by Bay er CropScience, LP. 8 p.Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document - DRAFT December 12, 2011

Page 151 of 153 **MRID Citation Reference**  **( Bt**  **Cry 2Ae PI**  **P )**  47871606 Coats, I . (2009) Supplement to MRID # 47640014: Insect Resistance Management

Plan: TwinL inkTM Cotton. Unpublished study prepared by

Bay er CropScience, LP. 7 p. 47903100 Ba ye r CropScience LP (2009) Submissio n of Toxicity Data in Support of the Application for Registration of BCS TwinL inkTM Cotton Event T304 - 40 x GHB 119 Cotton. Transmittal of 1 Study . 47903101 Hunst, P. (2009) Request for Waiver from the Requirement to Conduct an Avian

Oral Toxicity

Test: TwinLink. Unpublished study prepared by

Bay er CropScience LP. 16 p. 47903300 Ba ye r CropScience LP (2009) Submission of Toxicity Data in Support of the Application for Registration of BCS CRY2AE Cotton Event GHB119. Transmittal of 1 Study . 47903301 Hunst, P. (2009) Request for Waiver from the Requirement to Conduct an Avian

Oral Toxicity

Test: TwinLink. Unpublished study prepared by

Bay er CropScience LP. 16 p. 47928800 Ba ye r CropScience, LP - BioScience. (2009) Submission of Efficacy Data in Support of the Registration of BSC TwinL inkTM Cotton Event T304 - 40 X GHB 119 Cotton. Transmittal of 1 Study . 47928801 Coats, I . (2009) Addendum to Product Chemistry for TwinL ink: Structural and Functional Equivalence of Cry 1Ab and Cry2Ae and PAT/bar Proteins Produced in Bacillus thuringiensis (Bt) and Escherichia coli to Cry 1Ab, Cry2Aa and PAT/Bar in TwinL inkTM Cotton, Gossypium hirsutum , USA, 2007: Final Report. Project Number: CY07B006. Unpublished study prepared by

Bay er CropScience, LP. 7 p. 48177500 Ba ye r CropScience LP (2010) Submission of Toxicity Data in Support of the Applications for Registration of BCS TwinL inkTM Cotton Event T304 - 40 X GHB 119 Cotton, BCS Cry

1Ab Cotton Event T304 - 40, and BCS Cry2Ae Cotton Event GHB119 and the Petition for Tolerance of Cry2Ae. Tr ansmittal of 1 Study . 48177501 Stafford, J. (2010) Northern Bobwhite ( Colinus virginianus ) Dietary Toxicity Test (L C50) with GHB119 (Cry2AE) Cottonseed Meal. Project Number: 13798/4130, TX44L 001. Unpublished study prepared by

Springborn Smithers Laboratories. 60 p. 48427900 Ba ye r CropScience (2011) Submission of Efficacy Data in Support of the Experimental Use of T304 - 40 x GHB119 x COT102 x COT67B in Cotton. Transmittal of 1 Study . 48427901 Robinson, T. (2011) Cry1Ab, Cry2Ae, PAT/bar, FL Cry 1Ab, Vip3Aa, APH4 Levels

in Selected Tissues of the Combined Trait Product T304 - 40 x GHB119 x COT102 x COT67B (BCS - GHOO4 - 7 x BCS - GH005 - 8 x SYN - IR1O2 - 7 x SYN - IR O67 - 1). Project Number: 10/BM45L 001. Unpublished study prepared by

Bay er CropScience, LP. 22 p. 4847 1900 Ba ye r CropScience (2011) Submission of Product Chemistry Data in Support of the Experimental Use of Cry2Ae (GEM2) in Cotton. Transmittal of 1 Study .Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document - DRAFT December 12, 2011

Page 152 of 153 **MRID Citation Reference**  **( Bt**  **Cry 2Ae PI**  **P )**  48471901 Currier, T. (2011) Anal ysis to Determine if the Cry2Ae (GEM2) Protein from Cotton

Leaves is Gl ycos yl ated: Final Report. Project Number: BK05B003. Unpublished study prepared by

Bay er CropScience. 32 p. 48480000 Ba ye r CropScience LP (2011) Submission of Product Chemistry and Efficacy

Data in Support of the Applications for Registration of BCS Cry 1 Ab Cotton Event T304 - 40, BCS Cry2Ae Cotton Event GHB119 and BCS TwinL inkTM Cotton Event T304 - 40 x GHB 119 Cotton, and the Petition for Tolerance of Cry2Ae. Transmittal of 11 Studies. 48480001 Habex, V.; Verhaeghe, S. (2008) Detailed Insert Characterization of Gossypium

hirsutum Transformation Event GHB119 b y Southern Blot Analy sis. Project Number: BBS07/009, M/308404/02/1. Unpublished study prepared by

Bay er BioScience N.V. 43 p. 48480002 Moen s, S. (2008) Confirmation of the Absence of Vector Backbone Sequences in

Gossypium hirsutum Transformation Event GHB119. Project Number: BBS06/007, M/311127/02/1. Unpublished study prepared by

Bay er BioScience N.V. 40 p. 48480003 Moens, S.; Criel, I . (200 8) Detailed Insert Characterization of Gossypium hirsutum

Transformation Event T304 - 40. Project Number: BBS07/014, M/311245/03/1. Unpublished study

prepared by

Bay er BioScience N.V. 62 p. 48480004 Habex, V.; Van Herck, H. (2007) Confirmation of the Absenc e/Presence of Vector

Backbone Sequences in Gossypium hirsutum Transformation Event T304 - 40: Final Report. Project Number: BBS05/003, M/308400/02/1. Unpublished study prepared by Bay er BioScience N.V. 41 p. 48480005 Currier, T. (2011) Characterization of C ry 1Ab and (Inert Ingredient) Proteins

Produced in Event T304 - 40 Cotton, Gossypium hirsutum USA, 2011. Project Number: 10/BM99L508, M/406631/01/1. Unpublished study prepared by

Bay er CropScience LP. 31 p. 48480006 Moens, S. (2011) Characterization of Cry2A e and (Inert Ingredient) Proteins

Expressed in Gossypium hirsutum Transformation Event GHB119. Project Number: BIO2/005/PROTEQUL/512, M/406615/02/1. Unpublished study prepared by

Bay er BioScience N.V. 35 p. 48480007 Currier, T. (2011) Structural Stability Anal ys is of Gossy

pium hirsutum Combined Event TwinL inkTM X GlyTol (GHB614): Final Report. Project Number: CP08Q012/2. Unpublished study prepared by

Bayer CropScience LP. 34 p. 48480008 Currier, T.; Harbin, A. (2011) Protein Expression Analy sis for Gl yTol x TwinL inkTM

(GHB614 x T304 - 40 x GHB119) Cotton Grown in the Field, USA 2010. Project Number: 10BM99L440B, M/406245/01/1. Unpublished study prepared by

Bay er CropScience LP. 125 p. 48480 009 Bushey , D.; Jansens, S.; Becker, A. (2010) Insect Bioassay of Plant Material Expressing Cotton Events T304 - 40, GHB119, and TwinL inkTM (T304 - 40xGHB119). Project Number: BIO2/005, INSECT/BIOASSAY/FROM/MONHEIM. UnpublishedBacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document - DRAFT December 12, 2011

Page 153 of 153 **MRID Citation Reference**  **( Bt**  **Cry 2Ae PI**  **P )**  study prepared by

Bay er CropScien ce. 22 p. 48480010 Mullins, W. (2011) High Dose Study : Estimating the Mortality of Heliothis virescens in a Mississippi Cotton Field on Caged Plants of T304 - 40 x GHB119 (TwinLink) Cotton (BCS - GHOO4 - 7 x BCS - GHOO5 - 8) and Parental Events T304 - 40 (Cry 1Ab) and GHB119 (Cry2Ae). Unpublished study prepared by

Bay er CropScience LP. 16 p. 48480011 Denneh y, T. (2011) High Dose Study : Estimating the Mortality of Heliothis virescens in a Greenhouse on Plants of T304 - 40 x GHB119 (TwinL ink) Cotton (BCS - GHOO4 - 7 x BCS - GHO O5 - 8) and Parental Event Plants T304 - 40 (Cry 1Ab) and GHB119 (Cry2Ae). Project Number: BA44Y001. Unpublished study prepared by Bay er CropScience LP. 16 p. 48495100 Ba ye r CropScience (2011) Submission of Efficacy Data in Support of the Application for Regis tration of the TwinL ink. Transmittal of 1 Study . 48495101 Denneh y, T. (2011) A Risk Assessment of TwinL inkTM Cotton and Tobacco

Budworm (Heliothis virescens) in the Midsouth. Unpublished study prepared by Mississippi State University . 15 p. Other References **Safe use –**  **mode of operation** Ferré, J. and Van Rie, J. 2002. Biochemistry and genetics of insect resistance to Bacillus thuringiensis. Ann. Rev. Entomol. 47:501 - 533. Organization for Economic Co - operation and Development (OECD). 2007. Consensus document on safet y information on transgenic plants expressing Bacillus thuringiensis - derived insect control protein. 109 pages.