BIOPESTICIDES REGISTRATION ACTION DOCUMENT

PLANT-INCORPORATED PROTECTANTS:

Bacillus thuringiensis Cry1Ab protein and the Genetic Material

Necessary for its Production 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 Production in Event GHB119 Cotton [PC Code 006600, 

OECD Unique Identifier: BCS-GH5-8];

and

TwinLinkTM Cotton (T304-40 and GHB119 Combination PIP product) 

[OECD Unique Identifier: BCS-GHØØ4-7 x BCS-GH5-8]

U.S. Environmental Protection Agency

Office of Pesticide Programs

Biopesticides and Pollution Prevention Division

January 23, 2012
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM Cotton (Cry1Abx2Ae)

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............................................................................................................................10

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 human health effects – Bacillus thuringiensis Cry 1Ab (Event T304-40) 

in cotton plant incorporated protectant.................................................................................................................................26

APPENDIX B: Product characterization and human health effects - Bacillus thuringiensis Cry 2Ae (Event GHB 119) 

and TwinLinkTM in cotton plant incorporated protectant. ..................................................................................................36

APPENDIX C: Ecological and environmental effects – Bacillus thuringiensis Cry1Ab, Cry2Ae and TwinLinkTM cotton 

plant incorporated protectant.................................................................................................................................................59

APPENDIX D: Insect Resistance Management ....................................................................................................................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

Zigfridas Vaituzis, Ph.D.

Shannon Borges, M.S.

Annabel Waggoner, B.S.

Insect Resistance Management

Jeannette Martinez, M.S.

Alan Reynolds, M.S.

Benefit Assessment

Shannon Borges, M.S.

Office of General Counsel

Chris Kaczmarek

Biopesticides Registration Action Document Regulations

Sheryl Reilly, Ph.D. 

Alan Reynolds, 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/BRS1

 

1 Conducted secondary review of DERs for some product characterization data under work-share program according to the 

EPA-BPPD and USDA-BRS memorandum of understanding [09-2000-0052-MU]. 
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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

oC Temperature in Centigrade or Celsius Degrees

DER Data Evaluation Record

DNA Deoxyribonucleic Acid

EPA Environmental Protection Agency (the “Agency”)

ELISA Enzyme-Linked Immunosorbent Assay

EPA Reg. No. EPA Registration Number

FFDCA Federal Food, Drug, and Cosmetic Act

FIFRA Federal Insecticide, Fungicide, and Rodenticide Act

FQPA Food Quality Protection Act

FR Federal Register

g Gram

IU International Units

IRM Insect Resistance Management

kg kilogram

L Liter

MRID No. Master Record Identification Number

mg Milligram

mL Milliliter

μg Microgram

MP Manufacturing-Use Product

mRNA Messenger RNA

NE No Effect

ng nanogram (10-9gram)

NIOSH National Institute for Occupational Safety and Health

OPP Office of Pesticide Programs

OCSPP Office of Chemical Safety and Pollution Prevention

OECD Organisation 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-Transcriptional Gene Silencing

RNA Ribonucleic Acid

rRNA Ribosomal RNA

T-DNA transfer DNA from Agrobacterium

TGAI Technical Grade of the Active Ingredient

USDA United States Department of Agriculture

U.S. EPA United States Environmental Protection Agency

U.S. FDA United States Food and Drug Administration
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EXECUTIVE SUMMARY

The United States Environmental Protection Agency (U.S. EPA, Agency) proposes to register three new 

cotton plant-incorporated protectant (PIP) products as described in this Biopesticide Registration Action 

Document (BRAD).These registration actions represent one new PIP active ingredient and a new event 

of a PIP that has been previously registered in corn. The expression of the PIPs in these products

represent their first food and first outdoor uses in cotton. 

The Agency established a policy in October 1, 2009 to inform and provide the public an opportunity to 

comment on proposed registration decisions before they occur. The draft BRAD and supporting 

documents were available for 30 days in the Agency’s docket (EPA-HQ-OPP-2011-0988) for this 

purpose. No comments were received from the public at the end of the public comment period on 

January 13, 2012.

Bayer CropScience, LP (BCS) developed and sought federal registration of two transgenic cotton 

(Gossypium hirsutum) PIPs, Event T304-40 and Event GHB119. Each expresses an insecticidal protein

(also known as “Cry” proteins) derived from the soil bacterium Bacillus thuringiensis (Bt). These cotton 

events are intended for use as PIPs for conventional breeding to produce a product, TwinLinkTM Cotton 

(referred to in this document as “TwinLinkTM”) that expresses both proteins.

This BRAD presents EPA’s evaluations of the data and information submitted by BCS for the proposed 

registrations of: 

1. EPA File Symbol 264-RNOU: Event T304-40 cotton expressing Bt Cry1Ab 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 transfer 

DNA (TDNA) vector pTDL008. 

2. EPA File Symbol 264-RNOL: Event GHB119 cotton expressing Bt Cry2Ae insecticidal protein. 

It was created by Agrobacterium-mediated transformation using vector pTEM12. It contains the 

cry2ae gene isolated from Bt subspecies dakota.

3. EPA File Symbol 264-RNOA: Genetic material from Events T304-40 and GHB119 are 

combined by conventional breeding to express the combined proteins, Bt Cry1Ab and Bt 

Cry2Ae, in TwinLinkTM Cotton. 

The genes that are responsible for the Bt Cry1Ab and Bt Cry2Ae proteins, cry1Ab and cry2Ae, have

been modified for their expression in plants. These proteins are referred to as Cry1Ab (or Event T304-

40), and Cry2Ae (or Event GHB 119), respectively, in this BRAD. Both events also include the pat gene 

that produces Phosphinothricin Acetyltransferase (PAT) enzyme, 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 

(Cry1Ab) cotton on February 7, 2006 and was extended to expire on December 31, 2011. An EUP for 

GHB119 (Cry2Ae) and the combination PIP product, TwinLinkTM (expressing Cry1Ab 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, Cry1Ab protein (40 CFR § 
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174. 511), and the inert enzyme, PAT protein (40 CFR § 174. 522), and a temporary tolerance 

exemption for the active ingredient, Cry2Ae protein (40 CFR § 174. 530).

BCS applied for pesticide registrations of Cry1Ab and Cry2Ae proteins as expressed in Events T304-40 

and GHB119 cotton, respectively, and a new combination PIP product, TwinLinkTM cotton, created 

through conventional breeding of these events for use as PIPs in 2009, under section 3 of the Federal 

Insecticide, Fungicide, and Rodenticide Act (FIFRA). BCS concurrently filed 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 registrations

proposed by BCS.

I. ACTIVE INGREDIENT OVERVIEW

Active Ingredients

and Office of 

Pesticide Programs 

(OPP) Chemical 

Code and File 

Symbol:

a. Bt Cry1Ab insecticidal protein and the genetic material necessary for its 

production in Event T304-40 cotton (PC Code 006525); OECD Unique 

Identifier: BCS-GHØØ4-7;

EPA File Symbol 264-RNOU.

b. Bt Cry2Ae insecticidal protein and the genetic material necessary for its 

production in Event GHB119 cotton (PC Code 006600); OECD Unique 

identifier: BCS-GH5-8; 

 EPA File Symbol 264-RNOL.

c. Bt Cry1Ab and Bt Cry2Ae insecticidal proteins and the genetic material 

necessary for their production in TwinLinkTM cotton (T304-40 x GHB119) 

combination PIP product; OECD Unique Identifier: BCS-GHØØ4-7 x BCSGH5-8;

EPA File Symbol 264-RNOA.

Applicant/

Manufacturer:

Bayer CropScience – BioScience (BCS)

P.O. Box 12014

2 T.W. Alexander Drive

Research Triangle Park, NC 27709

Type of Pesticide: Plant Incorporated Protectant (PIP)

Use: Insecticide

Target Pest: Cotton bollworm (CBW, Helicoverpa zea), pink bollworm (PBW, Pectinophora 

gossypiella), tobacco budworm (TBW, Heliothis virenscens) larvae, beet 

armyworm (Spodoptera exigua), and fall armyworm (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 Cry1Ab and Cry2Ae proteins in the
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proposed PIPs. There is no mechanistic evidence that Bt proteins pose risks for humans or mammals

during their use as pesticides or as PIPs (OECD, 2007). When Cry proteins are ingested by an insect,

they are dissolved under the alkaline conditions specific to the insect’s gut, and then activated upon 

cleavage by the insect’s midgut enzymes (proteases). These activated Cry proteins must bind 

specifically to glycoprotein or glycolipid receptors on microvillar membrane of the insect’s midgut to 

initiate the pore-forming process that ultimately kills the insect. The fragment of the protein that is toxic 

to insects then interacts with specific high-affinity receptors on the microvilli of the target insect’s 

midgut epithelium (stomach), in particular brush border membrane vesicles (BBMV) (Ferré and Van 

Rie, 2002). Such receptors are not present in vertebrate species. Cry proteins must enter the cell 

membrane and form a pore. 

Although receptor binding appears to be essential for the insecticidal activity of the crystal 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 forming the pore. 

Consequently, the ion gradient across the membrane is compromised, and the columnar cells of the 

midgut swell and lyse osmotically. Lysis leads to disruption of the gut epithelium, resulting in

starvation, and insect death (Ferré and Van Rie, 2002).

II. REGULATORY BACKGROUND 

Applications for use of Cry1Ab, Cry2Ae and TwinLinkTM PIPs

A. Cry 1Ab protein

1. Experimental Use Permit and Temporary Tolerance Exemption 

a. Cry1Ab cotton Permit (EPA Reg. No. 264-EUP-140)

EPA granted an Experimental 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 Cry1Ab protein production in Events T303-3 and 

T304-40. The company intended to research the potential of this cotton PIP, Cry1Ab 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 amended 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 performance, and breeding lines. Also, seed for future plantings of experimental and 

regulatory field trials were produced.

 
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b. Cry1Ab Temporary Tolerance Exemption

A temporary tolerance exemption for Cry1Ab was not required, since a permanent tolerance exemption 

exists in 40 CFR § 174.511 for Cry1Ab protein in all food and animal feed commodities (discussed 

below in IIA.2.b.).

2. Registration Application and Permanent Tolerance Exemption

a. Cry1Ab PIP FIFRA Section 3 Registration 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 Cry1Ab insecticidal protein 

(Event T304-40; EPA File Symbol 264-RNOU), not included in any currently registered pesticide 

products for use in cotton. No public comments were received in response to this publication.

b. Cry1Ab protein - Permanent Tolerance exemption

Residues of Cry1Ab 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 Cry1Ab 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 the 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 Cry1Ab protein is not event-specific and applies to all 

Cry1Ab protein and any other event producing the Cry1Ab protein that might be found in the food 

supply. On April 25, 2007, the tolerance exemption for Cry1Ab protein 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 Cry1Ab protein expressed in this cotton PIP. 

B. Bt Cry2Ae and TwinLinkTM PIPs

1. Experimental Use Permit and Temporary Tolerance Exemption 

a. Cry2Ae and TwinLinkTM Cotton PIPs Experimental Use Permit 

In addition to the EUP for Cry1Ab cotton PIP, EPA granted an EUP to BCS for the Cry2Ae cotton PIP 

and the combined traits of Cry 1Ab x Cry2Ae proteins in TwinLinkTM Cotton (EPA Reg. No. 264-EUP143) 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 lines 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 
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acres were to be planted to Cry2Ae parental cotton and 307 acres to Cry1Ab x Cry2Ae combined trait 

TwinLinkTM Cotton. 

The company was researching the potential for Cry2Ae PIP and combined Cry1Ab and Cry2Ae proteins 

(TwinLinkTM 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 armyworm (Spodoptera frugiperda), and 

Beet armyworm (Spodoptera exigua) All cotton plants evaluated under this EUP contained 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 (cry1Ab) and Event GHB119 or GHB714

(cry2Ae).

b. Cry2Ae Protein: 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 

PIP EUP. Relevant company data/information in support 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 protein in/on cotton food/feed commodities (73 

FR 52591; 9/10/2008). It will expire on December 31, 2012.

2. Registration Application and Permanent Tolerance Exemption

a. Cry2Ae and TwinLinkTM Cotton PIPs - 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 

Cry2Ae protein (EPA File Symbol 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 cotton Events GHB119 and T304-40, thus

expressing the combined traits of the two new active ingredients, Cry1Ab, and Cry2Ae proteins.

b. Cry2Ae PIP – Permanent Tolerance Exemption

Concurrently with the FIFRA section applications, BCS submitted a petition (PP9F7514) establish a 

permanent exemption from the requirement of a tolerance for residues of Bacillus thuringiensis Cry2Ae 

protein in or on all food commodities. 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 enzyme 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 comments were received to any of the Federal Register announcements for Cry2Ae or 

TwinLinkTM Cotton PIPs, or the notice of filing of the pesticide petition for Cry2Ae protein published in 

the Federal Register.
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III. RISK ASSESSMENT SUMMARIES

EPA assessed the data submitted by BCS in 2009 for the proposed PIP cotton registrations and is 

presenting summaries of the product characterization and human health risk assessments in Appendices 

A (Cry1Ab) 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, respectively. In its assessment, the EPA relied upon data and other information 

submitted by the applicant and general knowledge of best available scientific technology. 

The classifications that are found for each data submission are assigned by Agency science reviewers 

and are an indication of the usefulness of the information contained in the documents for risk 

assessment. A rating of “ACCEPTABLE” indicates the study is scientifically sound and is useful for 

risk assessment. A “SUPPLEMENTAL” rating indicates the data provide some information that can be 

useful for risk assessment. The studies may have certain aspects determined not to be scientifically

acceptable (“SUPPLEMENTAL: UPGRADABLE”). If a study is rated as “SUPPLEMENTAL: 

UPGRADABLE,” the Agency always provides an indication of what is lacking or what can be provided 

to change the rating to “ACCEPTABLE.” If there is simply a “SUPPLEMENTAL” rating, the reviewer 

will often state that the study is not required 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 “UNACCEPTABLE” 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 I indicates the highest toxicity and Toxicity Category IV indicates 

the lowest toxicity. 

A. Product Characterization 

All product characterization data requirements for the Bt cotton PIPs containing Cry1Ab, 

Cry2Ae and their combined traits in TwinLinkTM have been satisfied. The two cotton events developed 

by BCS to express these PIPs are:

1. T304-40 cotton: expresses Cry1Ab insecticidal 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 sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) analyses of Cry1Ab 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 Cry1Ab protein standard at 

approximately 66 kilodaltons (kDa).

2. Event GHB119 cotton: expresses Cry2Ae protein, derived from Bt subspecies dakota. It

was created by Agrobacterium-mediated transformation using vector pTEM12, and 

contains the cry2ae gene isolated from Bacillus thuringiensis subspecies dakota. SDSPAGE demonstrates that Cry2Ae protein has a molecular weight of 71 kDa. Its sequence 

is modified for optimal expression in plants.
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These events are combined by conventional breeding in another PIP, TwinLinkTM that expresses Cry1Ab 

and Cry2Ae proteins to provide protection against feeding damage by lepidopteran insect larvae. 

The initial product characterization data submitted by BCS to support the FIFRA Section 3 Registration 

of PIP cotton Events T304-40, and GHB119 and their combination by conventional breeding in 

TwinLinkTM 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 considered insufficient to support the FIFRA section 3 registrations

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 

Cry1Ab and Appendix B for Cry2Ae and TwinLinkTM Cotton. The summaries include discussions of the 

following: 

 Transformation System

 Characterization of the DNA Inserted in the Plant and Inheritance and Stability

 Protein Characterization 

 Protein Equivalence 

 Analytical Method

 Product Characterization

As discussed in the summaries, Cry1Ab and Cry2Ae 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 terms of registration, EPA requires confirmation of the enforcement 

analytical methodology 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 these

actions to register Cry1Ab, Cry2Ae and TwinLinkTM Cotton 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 

subgroups of consumers, including infants and children. 

All toxicology data requirements for the Bt cotton PIPs containing Cry 1Ab, Cry2Ae and their combined 

traits in TwinLinkTM have been satisfied. Acceptable Tier I mammalian toxicology data/information 

support the proposed registrations of these active ingredients. Furthermore, Tier II and Tier III studies 

were not required for either Cry1Ab or Cry2Ae proteins, based on the lack of acute toxicity or 

pathogenicity in the Tier I studies. Refer to Tables A.3 and B.3 (in Appendices A, and B, respectively)

for brief summaries of data evaluated to satisfy toxicity and allergenicity data requirements for these 

PIPs.
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Acute toxicity

Cry1Ab and Cry2Ae proteins

The following summarizes the final determinations regarding acute toxicology data requirements for 

Cry1Ab and 2Ae proteins. See Appendices A and B in this BRAD for more detailed summaries. 

TABLE 1: Tier 1 Mammalian Toxicology Data Requirements and Associated OCSPP Test 

Guidelines

Study Conclusion

Acute Oral Toxicity/Pathogenicity OCSPP 885.3050 – Toxicity Category IV by tests.

Acute Dermal Toxicity OCSPP 885.3100 – Waived.

Acute Pulmonary 

Toxicity/Pathogenicity

OCSPP 885.3150 – Waived.

Acute Injection 

Toxicity/Pathogenicity

OCSPP 885.3200 – superseded by acute oral test for Cry2Ae

protein (See Appendix B).

Hypersensitivity Incidents* OCSPP 885.3400 – none reported yet but required if they occur.

Cell Culture OCSPP 885.3500 – only required for viruses.

Acute Oral Toxicity OCSPP 870.1100 – waived for End-use Product TwinLinkTM.

Acute Dermal Toxicity OCSPP 870.1200 - waived

Acute Inhalation Toxicity OCSPP 870.1300- waived

Acute Eye Irritation OCSPP 870.2400 - waived

Primary Dermal Irritation OCSPP 870.2500 - waived

Hypersensitivity Incidents: Reporting is required when incidents occur. No hypersensitivity incidents, 

including immediate-type or delayed-type reactions in humans or animals, occurred during the 

manufacture and field trials of the Bt Cry1Ab, Cry2Ae and their combination in TwinLinkTM Cotton 

PIPs. Should any future hypersensitivity incidents occur, they must be reported to the Agency.

a. Acute Oral Toxicity

Cry1Ab and Cry2Ae proteins

Acute oral toxicity Tier I studies 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) proteins are non-toxic to laboratory

mice. The acute oral toxicity levels of Cry1Ab and Cry2Ae proteins were assessed in separate tests. The 

mice were given purified, bacterially 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 LD50 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 A.1 and 

B.1 in Appendices A and B). Cotton 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. 
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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 Cry1Ab or Cry 2Ae

proteins at high dose levels in the acute oral toxicity tests, they are not considered to be toxic. Further, 

amino acid sequence comparisons showed no similarities between Cry1Ab or Cry2Ae proteins and 

known toxic proteins in protein databases that would raise a safety concern (U.S.EPA DERs from R. 

Edelstein, 2008 to S. Bacchus; 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 oral toxicity and other 

adverse effects in the Tier I acute oral tests in mice, EPA did not require Tier II and Tier III testing for 

either Cry1Ab or Cry2Ae protein. This conclusion is similar to the Agency’s position regarding toxicity

testing and the requirement of residue data for the registered microbial Bt products containing the 

species from which these PIPs were derived (see 40 CFR §§ 158.2130(d)(1)(i) and 158.2140(d)(7)). 

b. Allergenicity Assessment

Since Cry1Ab and Cry2Ae are proteins, 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 

factors 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 Cry1Ab and Cry2Ae proteins are derived, is not considered to be a source of 

allergenic proteins.

2. Amino acid sequence. Separate comparisons of the amino acid sequence of Cry1Ab and

Cry2Ae proteins 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 analyses of Cry1Ab and Cry2Ae proteins are 

present at relatively low levels. Dietary exposure is extremely limited and expected to be 

correspondingly low. Cry1Ab 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. Cry1Ab protein was digested 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 

glycosylated [Codex Alimentarius Commission (CAC), 2003]. Neither Cry1Ab nor Cry2Ae 

protein expressed in cotton was shown to be glycosylated.

Considering all of the available information, EPA has concluded that the potential for Cry1Ab and 

Cry2Ae to be food allergens is minimal. 

c. Tolerance Exemptions
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Permanent tolerance exemptions have been established for residues of Cry1Ab and PAT, as discussed in 

Chapter II under REGULATORY BACKGROUND, above. 

EPA’s reviews of the toxicology data support granting a permanent exemption from tolerance for 

residues of Cry2Ae protein 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 BCS for cotton food/feed commodities in 

association with an Experimental Use Permit, EPA Reg. No. 264-EUP-143. The toxicological profile of 

Cry2Ae protein was previously described in the Federal Register (73 FR 52591; 9/10/2008: 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 Chapter II, REGULATORY BACKGROUND).

On the basis of evaluations of all relevant data submissions for Cry2Ae, EPA has determined that the 

data support amendment of 40 CFR §174.530 to establish a permanent tolerance exemption for residues 

of Cry2Ae in/on cotton food feed commodities. The exposure analysis and evaluation of additional data 

to establish this permanent exemption from tolerance are based upon the expression of Bacillus 

thuringiensis Cry2Ae protein in cotton. No other uses of this protein as a PIP in other food or animal 

feed commodities exist. As a result, there has been no effort to date to ensure that transformation events 

in plants other than cotton that express Cry2Ae protein have the same safety characteristics as those 

described in this evaluation. Consequently, the final tolerance exemption for Cry2Ae protein residues 

that the Agency is granting varies from what the petitioner sought in as much as it is limited to residues 

of Cry2Ae protein in/on certain cotton food/feed commodities when Cry2Ae protein is used as a PIP in 

cotton. In the future, additional uses in other food or animal feed crops may be proposed, and data or 

other information that demonstrate the same safety 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 “naturally 

occurring estrogen, or other such endocrine effects as the Administrator may designate.” The EDSP 

employs a two-tiered approach to making the statutorily required determinations. Tier 1 consists of a 

battery of 11 screening assays to identify the potential of a chemical substance to interact with the 

estrogen, androgen, or thyroid (E, A, or T) hormonal systems. Chemicals that go through Tier 1 

screening and are found to have the potential to interact with E, A, or T hormonal systems will proceed 

to the next stage of the EDSP where the Agency will determine which, if any, of the Tier 2 tests are 

necessary based on the available data. Tier 2 testing is designed to identify any adverse endocrinerelated effects caused by the substance, and establish a quantitative relationship between the dose and 

the E, A, or T effect.

Between October 2009 and February 2010, the Agency issued test orders/data call-ins for the first group 

of 67 chemicals, which contains 58 pesticide active ingredients and 9 inert ingredients. This list of 

chemicals was selected based on the potential for human exposure through pathways such as food and 

water, residential activity, and certain post-application agricultural scenarios. This list should not be 

construed as a list of known or likely endocrine disruptors.
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Cry1Ab and Cry2Ae proteins are not among the group of 58 pesticide active ingredients on the initial 

list to be screened under the EDSP. Under FFDCA section 408(p), the Agency must screen all pesticide 

chemicals. Accordingly, 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 Quality 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 exemption 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 Cry1Ab or Cry2Ae protein, 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 cotton 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 Cry1Ab and Cry2Ae proteins can get in the water supply the same way that other 

proteins can migrate in crop debris from cotton into ground, and, maybe, drinking water. Such potential 

dietary exposure from cotton or drinking water is expected to be several orders of magnitude lower than 

the amounts of these proteins shown to have no toxicity in mammalian tests. EPA concludes that even 

negligible exposure via food and drinking water would present no harm, based on the lack of 

mammalian toxicity and allergenicity potential, and the rapid digestibility demonstrated for the PIPs. 

Non-occupational dermal and inhalation exposure is not expected, since the PIPs are expressed and 

contained within cotton plant cells. The uses of these PIPs are agricultural, so there would be no 

exposure to infants and children from residential, school or lawn use. The amino acid homology

assessments for Cry1Ab and Cry2Ae proteins included similarity to known aeroallergens. It has been 

demonstrated that there is no evidence of occupationally related respiratory symptoms, 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. 

b. Cumulative Effects from Substances with a Common Mechanism of Toxicity
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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 Cry1Ab nor Cry2Ae protein 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 Cry1Ab and Cry2Ae 

proteins do not have a common mechanism of toxicity with other substances. Thus, EPA concludes that 

there are no cumulative effects associated with Cry1Ab or Cry2Ae protein that need be considered. For 

information regarding EPA’s efforts to determine which chemicals have a common mechanism of 

toxicity and to evaluate the cumulative effects of such chemicals, see EPA’s website at 

http://www.epa.gov/pesticides/cumulative

c. Determination of Safety for U.S. 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 protein. 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 allergenicity 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 specifically 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 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 

safety for infants and children in the case of threshold effects to account for prenatal and postnatal 

toxicity and the completeness of the data base unless EPA determines that a different margin of safety

will be safe for infants and children. 

Based on its review and consideration of all the available information, as discussed in this BRAD, EPA 

concluded that there are no threshold effects of concern and, as a result, that an additional margin of 

safety for infants and children is unnecessary in this instance.

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 residue limits (MRLs) established by the Codex Alimentarius

Commission (Codex), as required by FFDCA section 408(b)(4). The Codex Alimentarius is a joint U.N. 

Food and Agriculture Organization/World Health Organization food standards program, and it is 

recognized as an international food safety standards-setting organization in trade agreements to which 

the United States is a party. EPA may establish a tolerance that is different from a Codex MRL; 

however, FFDCA section 408(b)(4) requires that EPA explain the reasons for departing from the Codex 
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level. No Codex maximum residue level exists for the PIPs, Bacillus thuringiensis Cry1Ab or Cry2Ae 

protein or their combination Cry1Ab x Cry2Ae pyramided protein.

C. Environmental Effects Assessment 

Ecological Effects Data for Event T304-40 (Bt Cry 1Ab) and Event GHB119 (Bt Cry 2Ae)

All ecological and environmental Effects data requirements for the Bt cotton PIPs containing Cry1Ab, 

2Ae proteins and their combined traits in TwinLinkTM 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 these PIPs (Cry1Ab, Cry2Ae, and their combination in 

TwinLinkTM Cotton) 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 PIP registrations to 

meet the 40 CFR Part 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. Limit 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 heavily on beneficial nontarget invertebrates, since they are most closely 

related to organisms susceptible to the insecticidal action of Bt toxins. The Cry1Ab and Cry2Ae proteins 

are meant to target species within the order Lepidoptera (moths and butterflies). 

Bt toxins are known typically to have a limited host range, however, to address any unforeseen change in 

activity spectrum as a result of laboratory protein synthesis 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 Cry1Ab and Cry2Ae proteins 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, and 

honeybee studies. 

Since exposure may also occur to other nontarget organisms, EPA has received data to comply with the 

Agency’s published non-target data requirements on other nontarget organisms. Tests to determine soil 

degradation of Cry1Ab and Cry2Ae proteins have also been submitted. The individual results for 

nontarget organism and soil degradation testing for Cry1Ab and Cry2Ae proteins, and where applicable, 

for their combination Cry1Ab x Cry2Ae in TwinLinkTM Cotton, are summarized in Appendix C. 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, particularly 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 Cry1Ab and Cry2Ae protein, respectively. 

Comparative analyses have been performed to verify the equivalence of the bacterially-produced and 

purified Cry1Ab and Cry2Ae and the Cry1Ab and Cry2Ae proteins produced in T304-40 or GHB119, 

respectively, and TwinLinkTM cotton plants. Product characterization data demonstrate the 
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bioequivalence of the plant-expressed and microbially expressed proteins (Waggoner 2011a, 2011b). 

Consequently, on the basis of the environmental risk assessment and evidence of bioequivalence, EPA 

concludes that Cry1Ab 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 the potential for gene capture and expression of 

Cry1Ab or Cry2Ae protein by wild or weedy relatives of cotton within the U.S. or its territories does not 

pose a significant risk as long as the current sales and distribution restrictions on Bt cotton remain in 

place. Available data indicate that Cry proteins do not have any measurable adverse effect on microbial 

populations in the soil, nor has horizontal transfer of genes from transgenic plants to soil bacteria been 

demonstrated.

Endangered Species

Because of the selectivity of Cry1Ab and Cry2Ae proteins for lepidopteran species, endangered species 

concerns are also expressed for insect species in the order Lepidoptera. The Agency has previously 

determined that any potential concern regarding range overlap of endangered Lepidoptera with cotton 

production was mainly 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 hybridization 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 by competition with such entities.

Cry1Ab protein 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. 

EPA, therefore, makes a “No Effect” determination for direct and indirect effects to listed species of 

these taxa for Cry1Ab protein expressed in Event T304-40 cotton. 

Since Cry2Ae protein 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 direct and indirect effects to 

listed species of these taxa for Cry2Ae protein 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, including Bt cotton. IRM typically 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 
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(should resistance occur), grower compliance with IRM requirements, and grower education. The 

standard risk mitigation strategy 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 susceptible insects to mate 

with any 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., soybean, peanut, tobacco). 

BCS submitted an IRM plan and data with their application for registration of TwinLinkTM cotton 

(MRID#s 47640014 and 47634823). TwinLinkTM is a pyramided Bt cotton PIP expressing Cry2Ae and 

Cry1Ab proteins 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 (MRID#s 

48480010, 48480011, and 484951-01) that were reviewed by 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 TwinLinkTM cotton was acceptable.

BPPD’s major IRM conclusions from its assessment of TwinLinkTM Cotton are detailed below. The 

IRM assessment included data developed for TwinLinkTM Cotton (Cry2Ae x Cry1Ab) as well as its 

single toxin constituent PIPs, GHB119 (Cry2Ae) and T304-40 (Cry1Ab).

1. DOSE

a. BPPD concludes that the diet incorporated and overlay assays together (using verification 

method 1) suggest that TwinLinkTM Cotton (Cry2Ae x Cry1Ab proteins) and GHB119 (Cry2Ae

protein) may express a high-dose against CBW but that T304-40 (Cry1Ab protein) may express 

less than high-dose against the target pest.

b. BPPD concludes that diet incorporated and overlay assays (using verification method 1) together 

suggest that Events GHB119 (Cry2Ae protein) and T304-40 (Cry1Ab protein) may express a 

high-dose and TwinLinkTM (Cry2Ae x Cry1Ab proteins) may express an effective high-dose 

against TBW. 

c. BPPD concludes that the single toxin Events GHB119 (Cry2Ae) and T304-40 (Cry1Ab) both 

express less than a high dose against PBW, but that TwinLinkTM (Cry2Ae x Cry1Ab) expresses a 

high dose against the insect.

2. CROSS RESISTANCE

a. Based on submitted data, BPPD concludes that there is low cross resistance potential between 

the Cry2Ae and Cry1Ab toxins that are expressed in TwinLinkTM.

b. BPPD notes that there is a potential for cross resistance between Cry2Ae and Cry2A2 proteins 

(registered in Bollgard II cotton). Should resistance develop to either toxin, the second toxin 

could also be compromised.
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c. Similarly, there is cross resistance potential between Cry1Ab (also registered in VipCot cotton) 

and Cry1Ac (registered in Bollgard and WideStrike cotton) and Cry1F (registered in WideStrike 

cotton). Competitive binding assays have shown that these toxins share binding sites in insect 

midgets which can lead to cross resistance (though the potential is somewhat less for Cry1F).

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 by Dr. Michael Caprio (Mississippi State University). 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 heavily 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 Cry1Ab corn in the model landscape. BCS responded to these deficiencies with 

revised modeling that employed more conservative parameter values. The revised model was run 

for 1,000 simulations; resistance did not evolve to either Cry1Ab or Cry2Ae proteins (within the 

30 year time horizon of the model) in any of the simulations.

c. BCS conducted two modeling analyses for TBW: i) a stochastic, spatially-explicit model, and ii) 

a deterministic, spatially-implicit model. As with CBW, the TBW models were designed and 

conducted by Dr. Michael Caprio. The modeling generally took a conservative approach by 

including “worst case” simulations for certain parameters (dose mortality and dominance) and 

incorporated other Cry1A cotton products in the landscape. Results from the stochastic model 

showed that resistance did not evolve during the 31 year 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 years (both 

toxins) under the benchmark parameters. Deterministic simulations with worst case assumptions 

included resulted in a durability estimate of 80 years (a 2.8 fold difference from the benchmark 

simulations). A separate deterministic simulation was also run with a single trait Bt cotton 

product (Cry1Ac) with a 20% structured refuge that resulted in an estimated durability of 18.8 

years.

d. Based on the modeling results, BPPD concluded that TwinLinkTM cotton deployed with a natural 

refuge should not be expected to pose a greater risk of resistance evolution than other Bt cotton 

products (e.g., Bollgard II) currently registered with a natural refuge strategy.

4. REFUGE STRATEGY

a. BCS provided adequate information to demonstrate that the use of a natural refuge with 

TwinLinkTM cotton should provide high durability for both toxins (Cry1Ab and Cry2Ae). The 

risk of resistance for TwinLinkTM is not expected to be greater than other registered Bt cotton 

products (Bollgard II, WideStrike, VipCot) that employ a natural refuge strategy.
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b. The use of natural refuge has been assessed only for southeastern cotton growing regions. 

Western cotton regions (including west Texas, New Mexico, Arizona, and California) should 

still employ structured refuges (as have been required for other Bt cotton products). The 

structured refuge options are : i) 5% external, unsprayed non-Bt cotton refuge, ii) 20% external, 

sprayed non-Bt cotton refuge, iii) embedded refuge – either 5% or 1 row of non-Bt cotton per 6-

10 rows of Bt cotton.

c. Registrations have been proposed for Events GHB119 (Cry2Ae) and T304-40 (Cry1Ab), both of 

which are single trait products. These products are “breeding” registrations (for the production of 

TwinLinkTM) and are not intended to be commercially sold. BCS has requested that no refuge be 

required for these products. Breeding registrations typically 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 Cry1Ab and Cry2Ae proteins. 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 by 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 

TwinLinkTM 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 by the Arizona Bt Cotton 

Working Group (which has been employed 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 informed within 30 days 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). 
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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 onfarm assessments in addition to anonymous phone (or internet) surveys. BPPD recommends that 

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 TwinLinkTM cotton (i.e., structured 

refuge).

b. It is also recommended that BCS develop a grower guide that at a minimum describes the 

technology (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 TwinLinkTM

should be no greater than for other currently registered Bt cotton pyramids (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 Cry1Ab, Cry2Ae and TwinLinkTM Cotton PIPs are available as Appendix D of this BRAD.

IV. 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 location, cultural practices, or other factors, may have atypical, unusually high exposure to 

these PIP proteins Cry1Ab, Cry2Ae and their combination in TwinLinkTM compared to the general 

population. 

For additional information regarding environmental justice issues, please visit EPA’s web site at 

http://www.epa.gov/compliance/environmentaljustice/index.html.
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document January 23, 2012

Page 23 of 153

V. RISK MANAGEMENT AND PROPOSED REGISTRATION DECISION

Section 3(c)(5) of FIFRA provides for the registration of a new active ingredient if it is determined that 

(A) its composition is such as to warrant the proposed claims for it; (B) its labeling and other materials 

required to be submitted comply with the requirements of FIFRA; (C) it will perform its intended 

function without unreasonable adverse effects on the environment; and (D) when used in accordance 

with widespread and commonly recognized practice, it will not generally cause unreasonable adverse 

effects on the environment. 

The four criteria of the Eligibility Determination for Pesticidal Active Ingredients are satisfied by the 

science assessments supporting the PIPs containing Bacillus thuringiensis Cry1Ab, Cry2Ae and their 

combination, Cry1Ab x Cry2Ae, in TwinLinkTM. These 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 Cry1Ab, Cry2Ae and their combination, 

Cry1Ab x Cry2Ae, in TwinLinkTM 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. Analytical method: OCSPP 860.1340 (40 CFR § 158.2120)

The following validation 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 Cry1Ab 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 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 testing 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)

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 testing 

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 January 23, 2012

Page 24 of 153

3. Term of Registration for TwinLinkTM (T304-40 x GHB119) combination PIP product (EPA File 

Symbol 264-RNOA). Any outstanding data submitted to satisfy the terms of registration for PIP 

cotton Events T304-40 and GHB119 (EPA File Symbol 264-RNOU and 264-RNOL, 

respectively) must be submitted and found acceptable in order to complete the database for 

TwinLinkTM 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 TwinLinkTM

cotton: tobacco budworm, cotton bollworm, and pink bollworm. In addition, baseline 

susceptibility and diagnostic concentration determinations for tobacco budworm, cotton 

bollworm, and pink bollworm to Cry2Ae and Cry1Ab must be submitted. 

2. BCS must submit a final remedial action plan for tobacco budworm and cotton bollworm. The 

remedial action plan must include definitions of “suspected” and “confirmed” resistance and 

steps to take in the event of confirmed resistance.

3. BCS 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., western 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 any grower purchase contract, license agreement, or similar legal document 

and must clearly 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 system which is reasonably likely to assure that 

persons purchasing TwinLinkTM will affirm annually that they are contractually bound to comply 

with the requirements of the IRM program. BCS must submit a copy of the grower 

agreement/stewardship documents and a written description of a system 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. Hypersensitivity Incidents

All incidents of hypersensitivity (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 January 23, 2012

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 January 23, 2012

Page 26 of 153

APPENDIX A: Product characterization and human health effects – Bacillus 

thuringiensis Cry1Ab (Event T304-40) in cotton plant incorporated protectant.

A.1. BACKGROUND

BCS has developed a cotton line from Event T304-40 (EPA File Symbol 264-RNOU and OECD Unique 

Identifier: BCS-GHØØ4-7) that express a Cry1Ab plant-incorporated protectant (PIP) protein derived 

from Bacillus thuringiensis (Bt) subspecies berliner, which is intended to control lepidopteran larvae 

such as bollworm (CBW, Helicoverpa zea), tobacco budworm (TBW, Heliothis virenscens) larvae, and 

fall armyworm (FAW, Spodoptera frugiperda). BCS 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. It was extended on March 8, 2007 to expire on December 31, 2011.

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). BCS later submitted a FIFRA 

section 3 registration application containing new product characterization data and updated toxicological 

data for Cry1Ab protein. These data were reviewed by 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-BRS 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 

numbers (US EPA, 2011). 

Event T304-40 cotton expresses Cry1Ab protein as an active ingredient and phosphinothricin 

acetyltransferase (PAT) enzyme as an inert ingredient. The status of the existing tolerance exemptions 

for Cry1Ab protein (40 CFR 174.511) and PAT (40 CFR 174.522) has been previously discussed in 

Chapter IIA.2.b. of this BRAD. Residues of Bt Cry1Ab protein are exempt from the requirement of a 

tolerance when used as plant-incorporated protectants in all food commodities and the PAT enzyme is 

exempt from the requirement of a tolerance when used as plant-incorporated protectant inert ingredient 

in all food commodities. 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 expressing Cry1Ab protein

a. Transformation System

Event T304-40 was created through Agrobacterium-mediated transformation of cotton variety Coker 

315 with plasmid pTDL008. The cry1Ab gene sequence was modified for expression in plants and 

consists of one nearly complete copy of the T-DNA flanked by an inverted incomplete copy of the 

cry1Ab gene cassette and one additional 3’me1 terminator. The Cry1Ab protein encoded by the gene 

inserted into cotton is expected to have 617 amino acids and a molecular weight of approximately 69 
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document January 23, 2012

Page 27 of 153

kDa. In addition, the deduced amino acid sequence of BCS’s Cry1Ab protein is identical to the amino 

acid sequence of the Cry1Ab protein in a previously registered PIP (Event Bt11 corn, OPP chemical 

code 006444) with the exception of one amino acid at the C- terminus and two amino acids at the Nterminus (MRID No. 467474406; US EPA 2006). Plasmid pTDL008 also contains the bar gene as a 

selectable marker, which is derived from the soil microorganism, Streptomyces hygroscopicus 

(described by Thomson et al. 1987). The bar gene encodes the enzyme phosphinothricin 

acetyltransferase (PAT), which is co-expressed with Cry1Ab protein in Event T304-40 cotton as a PIP 

inert ingredient. The PAT enzyme provides the plant with tolerance to herbicide applications containing 

glufosinate-ammonium by acetylating the herbicide glufosinate-ammonium and making it unable to bind 

glutamine synthase. The PAT enzyme encoded by 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 enzyme digests and 

Southern blot analyses as well as DNA sequencing indicates that the transgenic insert was inserted in the 

cotton genome, consisting of one nearly 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 pTDL0008 were found in T304-40. Inheritance and stability studies of the 

cry1Ab gene in Event T304-40 verified that it is stably integrated into the cotton genome, segregating in 

an expected Mendelian fashion.

c. Protein Characterization

Event T304-40 cotton expresses Cry1Ab protein produced by Bacillus thuringiensis subsp. berliner. The 

Cry1Ab protein after cleavage of the transit peptide has 617 amino acids. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) analyses of Cry1Ab protein, as expressed in Event

T304-40 showed a molecular weight close to the deduced MW for Cry1Ab protein and Western blot 

analyses showed a single immunoreactive band. Cry1Ab protein was also found to be non-glycosylated. 

N-terminal sequencing also confirmed that the transit peptide of the plant form was cleaved at the 

expected residue. The biological activity of Cry1Ab protein was assessed by an insect feeding bioassay

using Heliothis virescens first instar larvae and the LC50 for the plant- produced Cry1Ab protein was 74 

ng/cm2. 

d. Protein Expression

Expression level data were provided for Cry1Ab protein in different plant tissues and at different growth 

stages 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. Grain is the same as delinted seed, also commonly referred to 

as cottonseed. The distinction is made when distinguishing between grain intended for processing into 

cottonseed etc. or seed intended to be planted to grow cotton plants.

Table A.1. Mean Cry1Ab Protein 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
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document January 23, 2012

Page 28 of 153

Tissue Type

A.1. Cry1Ab Protein 

(μg/g dry weight ± SD)*

Leaves 0.45 ± 0.08 – 4.6 ± 0.6

Squares 2.90 ± 1.0

Apex 1.3 ± 0.3

Bolls 0.38 ± 0.2

Whole plants 2.2 ± 1.1

Pollen NAa

Nectar NAa

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 enzyme-linked immunosorbent assay

(ELISA) test kits for the analytical detection of Cry1Ab protein residues in cottonseed and cotton leaf 

tissue. Results showed the test kits are able to detect Cry1Ab 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 analytical method for the detection of Cry1Ab protein in Event T304-40 

cotton is needed. In addition, a validation study conducted by an independent 3rd party laboratory is 

needed to evaluate the performance of the designated residue analytical method for the detection of 

Cry1Ab 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 Cry1Ab protein.

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 Protein Summary MRID No

Characteristics of 

Cry1Ab cotton 

plants derived from 

transformation 

Events number 

T303-3 and T304-

402

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 

molecular analysis, including the protocol used and Southern blot data. 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.

46708801

[This 

study 

supersedes 

MRID No. 

46474401]

Characteristics of 

Cry1Ab cotton 

plants derived from 

transformation 

Events number 

T303-3 and T304-

401

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

 

2

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)

Biopesticides Registration Action Document January 23, 2012

Page 29 of 153

Study Title A.2. Product Characterization - Bt Cry1Ab Protein Summary MRID No

PAT and Cry1Ab 

protein in cotton 

tissues of T303-03

and T304-40 

Events, 

preliminary 

report1

PAT and Cry1Ab 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 Cry1Ab cotton Events T303-.3 and T304-40. 

Average Cry1Ab protein and PAT enzyme concentrations, as determined by 

ELISA in ground cotton seed from Events T303-3 and T304-40, were 16 g/g 

and 2.2 g/g fresh weight, respectively, for Cry1Ab protein and 117 and 129

g, respectively, for PAT. The dry weight equivalents in seeds are: 2 and 16 g 

of Cry1Ab protein per 0.91g dry seed (or 0.0002 and 0.0017% dry weight) and 

117 and 129 g of PAT per 0.91g dry seed (or 0.013 and 0.014% dry weight). 

Cry1Ab protein concentrations in early flowering cotton leaf tissues grown 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 Cry1Ab protein 

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 Cry1Ab protein in leaves was 

estimated to be: 0.2 to 2.3 g of Cry1Ab protein per 0.12 g dry leaf. From 

Event T303-3 and T304-40 cotton grown in the field, Cry1Ab protein was 

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) was not presented in the study report.

CLASSIFICATION: SUPPLEMENTAL- No additional data needed.

46708804 

[This 

study 

supersedes 

MRID No. 

46474404]

Product 

Characterization of 

Cry1Ab Cotton 

Event T304-403

The purpose of this study was to determine if the transfer DNA (T-DNA) 

containing the intended cry1Ab gene from plasmid pTDL008 was inserted into 

the genomic DNA of cotton Event T304-40, and the backbone DNA from 

pTDL008 was not inserted into cotton Event T304-40. Southern blot analyses 

showed that the inserted transgenic sequence in T304-40 consisted of one nearly 

complete copy of cry1Ab flanked 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 was negative using 4 of the 7 

backbone probes. 

However, 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 was stably integrated and segregated according to 

Mendelian laws of inheritance over several successive generations of Event 

T304-40 cotton were not addressed in this study.

CLASSIFICATION: SUPPLEMENTAL- Data gaps addressed in MRID No. 

48480005.

47634801 

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 

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. SDSPAGE analyses of Cry1Ab 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 Cry1Ab protein standard (~66 kDa). 

Western blot analyses showed immunologically reactive bands at the same 

position (~66kDa) for Cry1Ab protein expressed in cotton Event T304-40 and 

TwinLinkTM to the E. coli-derived Cry1Ab protein. However, 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. SDS47634805 

 

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)

Biopesticides Registration Action Document January 23, 2012

Page 30 of 153

Study Title A.2. Product Characterization - Bt Cry1Ab Protein Summary MRID No

hirsutum2 PAGE analyses showed identical electrophoretic mobilities and similar 

molecular 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 bands 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 

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- expressed protein sources 

cannot be confirmed from these data. 

CLASSIFICATION: SUPPLEMENTAL- Data gaps addressed in MRID No. 

48480004.

Analyses of Raw 

Agricultural 

Commodity (Fuzzy 

Seed) of Cry1Ab 

Cotton Event T304-

40 for PAT/bar and 

Cry1Ab and its 

Non-transgenic 

Counterpart for 

PAT/bar and 

Cry1Ab Proteins2

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 nontransgenic cotton line, Coker 312. Fuzzy seed was 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 combined 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 0.000434 ± 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 PAT analyte proteins were expressed in the kernels, 

lint coats and, by inference, in the fuzzy seeds derived from the transgenic T304-

40 cotton plants. 

CLASSIFICATION: ACCEPTABLE

47634804

Protein Expression 

Analysis of Cotton 

Event T304-40, 

Expressing Cry1Ab 

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, flowers and grain or delinted 

seed. 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

47634803
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document January 23, 2012

Page 31 of 153

Study Title A.2. Product Characterization - Bt Cry1Ab Protein Summary MRID No

grain. 

On a dry weight basis, the overall averages for the PAT protein content were 

reported to be 115 and 95.1 μg/g for roots at two growth stages (ca. 4 weeks and 

10 weeks, respectively); 100 and 44.5 (μg/g for stems at two growth stages (ca. 4 

weeks and 10 weeks, respectively); 193, 134 and 57.9 μg/g for leaves at 3 growth 

stages (ca. 4 weeks, 7 weeks, and 10 weeks, respectively); 180 μg/g for squares 

(ca. 7 weeks); 88.9 μg/g for apex (ca. 10 weeks); 39.3 μg/g for bolls (ca. 8 

weeks); 134 μg/g for whole plant (ca. 10 weeks); 0.13 μg/g for pollen fresh 

weight data only due to insufficient availability of material) (ca. 7 weeks); 0.005 

μg/g for nectar fresh weight data only due to insufficient availability of material) 

(ca. 7 weeks); 187 μg/g for flowers (ca. 10 weeks); and 39.6 μg/g for grain (ca. 

22 weeks). 

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 Event T304-40 cotton. 

CLASSIFICATION: ACCEPTABLE

Detailed insert 

characterization of

Gossypium 

hirsutum

transformation 

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 were 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-DNA flanked by an inverted incomplete copy of the cry1Ab gene cassette 

and one additional 3’me1 terminator. 

CLASSIFICATION: ACCEPTABLE

48480003

[This 

study 

supersedes 

MRID No. 

47634801]

Confirmation of 

the absence and 

presence of

vector backbone 

sequences in

Gossypium

hirsutum

transformation 

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 EcoRV and 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 were 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

48480004

[This 

study 

supersedes 

MRID No. 

47634801]

Characterization of

Cry1Ab and 

PAT/bar proteins 

produced in Event 

The purpose of this study was to determine the biochemical and functional 

equivalence of plant-expressed Cry1Ab protein produced in Event T304-40 

cotton plants with bacterial-expressed Cry1Ab protein (Test substances ID No. 

MIN1443 and AW200308) produced from recombinant Escherichia Coli over48480005

[This 

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

Biopesticides Registration Action Document January 23, 2012

Page 32 of 153

Study Title A.2. Product Characterization - Bt Cry1Ab Protein Summary MRID No

T304-40 cotton, 

Gossypium

hirsutum

expression system, for its use as a suitable test substance surrogate in 

toxicological testing. Equivalence studies were also conducted for plantexpressed 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 

included: 1) estimation of molecular weight by observing protein mobility in 

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 for 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. The 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 

between 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 peptide 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 

and AW200308) and PAT (Test substance ID No. NB010905P44P2) proteins 

would 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 

supersedes 

MRIDs 

No. 

47634805 

and 

47640006]

Protein Detection 

Methods. Enzyme 

Linked Immuno 

Sorbent Assay 

(ELISA)

Non-quantitative detection of Cry1Ab protein 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 protein.

CLASSIFICATION: SUPPLEMENTAL pending verification of the specific 

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 Protein

EPA evaluated human health toxicology data for Cry1Ab protein in Event T304-40 as summarized 

below in Table A.3.

Table A.3. Human Health Data for Event T304-40 cotton expressing Bt Cry1Ab protein

(EPA File Symbol264-RNOU)

Study Title A.3. Human health - Summary – Bt Cry1Ab protein MRID No
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document January 23, 2012

Page 33 of 153

Study Title A.3. Human health - Summary – Bt Cry1Ab protein MRID No

Cry1Ab protein -

Overall amino acid 

sequence homology 

search with known 

toxins and allergens4

The amino acid sequence of the Cry1Ab protein encoded by the plant-optimized 

Cry1Ab gene from Bt subspecies berliner 1715 was compared with all protein 

sequences present in the following databases: SwissProt, trEMEL, GeneSeqProt, PIR, PDB, DAD, and GenPept. No similarities between the Cry1Ab 

protein and known allergens or toxins other than Bt toxins were found using the 

criterion of 35% identity over an 80 amino acid segment.

Classification: ACCEPTABLE

46708802 

[This study

supersedes 

MRID No. 

46474402]

Cry1Ab protein -

Epitope homology 

search.

The amino acid sequence of the Cry1Ab protein was compared 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 Homology, 

N-glycosylation and 

Overall Amino 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-glycosylation 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 

Cry1Ab protein 

amino 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 amino 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 two 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 Na2CO3 (pH 9.6) within a 4 

hour time period on the day of treatment. Animals were observed for 15 days. 

At test end, there were no mortalities, and no treatment-related adverse effects 

observed. Therefore, the acute oral LD50 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: ACCEPTABLE

47651103

Cry1Ab Protein: 

Heat stability study

An in vitro heat stability study was 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 SDSPAGE and Western blot analyses showed the Cry1Ab protein had no significant 

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)

Biopesticides Registration Action Document January 23, 2012

Page 34 of 153

Study Title A.3. Human health - Summary – Bt Cry1Ab protein MRID No

changes in protein stability after heat treatment at 60°C for 10, 30 and 60 

minutes 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, 

denaturation was observed after 10 and 30 minutes, and finally at 60 minutes, a 

marked but not complete degradation of the Cry1Ab protein was observed after 

incubation at 90°C. Additional protein bands of lower molecular weight (mostly 

likely representing degradation products) also appeared as the ~69 kDa Cry1Ab 

protein band disappeared. Results showed consistent patterns of decreasing 

structural stability of the Cry1Ab protein in between both analyses tested and 

was 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 simulated human gastric fluid containing pepsin (pH 1.2) to assess 

the susceptibility of Cry1Ab protein to proteolytic 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 simulated human intestinal fluid (SIF) containing pancreatin (pH 7.5) 

to assess the susceptibility of Cry1Ab protein to proteolytic degradation. The 

Cry1Ab protein was incompletely degraded into several stable fragments in 

simulated intestinal fluid containing pancreatin (ph 7.5).

CLASSIFICATION: ACCEPTABLE

47651101

ORF Analysis -

Toxicology (Human 

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 (European Molecular Biology Open Software 

Suite) tools. Two ORFs of interest were found. No significant similarities were 

identified between 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 polypeptide has 

any polymerase activity even if it was expressed. In addition, no records were 

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 Protein– 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)

Biopesticides Registration Action Document January 23, 2012

Page 35 of 153

Vanderbruggen H., van Montagu M., Zabeau M.,Vaeck M. (1986) Structural and functional 

analysis of a cloned delta 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 enzyme. 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. 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 Symbol 264-RNOU), T304-40 cotton expressing Bt CrylAb (EPA File Symbol

264-RNOL); and TwinLinkTM cotton (EPA File Symbol 264-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. 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 ServicesFood and Drug Administration (Docket No. 92N-0139)

http://www.cfsan.fda.gov/~lrd/biotechm.html#reg

Van Rie J. 2004. Comparative analysis of the Cry1Ab protein amino acid sequence. Project Number: 

None; Unpublished study prepared by Bayer CropScience LP. MRID No. 46474406
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document January 23, 2012

Page 36 of 153

APPENDIX B: Product characterization and human health effects - Bacillus 

thuringiensis Cry 2Ae (Event GHB 119) and TwinLinkTM 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 product characterization and mammalian 

toxicology of submitted studies for Bacillus thuringiensis Cry 2Ae and TwinLinkTM (combined Cry1Ab 

x Cry2Ae proteins).

BCS 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 armyworm (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 tolerance of Cry2Ae protein residues on food/feed commodities in cotton. The 

EUP also included testing TwinLinkTM cotton (EPA File Symbol 264-RNOA), a new combination PIP 

product, developed by conventional breeding of the parental Events T304-40 x GHB119. 

The product characterization 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 in this PIP cotton line (40 CFR § 174.530). Event GHB119 cotton 

also expresses phosphinothricin acetyltransferase (PAT) enzyme, 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 registrations for PIP Events GHB119, 

T304-40 and TwinLinkTM cotton, their associated combination PIP product developed by traditional 

breeding methods. Concurrently, BCS filed a petition to establish a permanent tolerance exemption for 

Cry2Ae protein in all food commodities 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. Several product characterization studies and additional data were required 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 registration 

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 PIP are available in this Appendix.
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document January 23, 2012

Page 37 of 153

FFDCA FOOD SAFETY ASSESSMENT

Event GHB119 Cotton (OECD Unique Identifier BCS-GH5-8) Expressing Cry2Ae protein

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 armyworm (FAW, Spodoptera frugiperda).

a. Transformation System

Event GHB119 cotton was produced by 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 thuringiensis 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 region 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 membrane. Plasmid pTEM12 also 

contains the bar gene as a selectable marker, which is derived from the soil microorganism, 

Streptomyces hygroscopicus (described by Thomson et al. 1987). The bar gene encodes the enzyme 

phosphinothricin acetyltransferase (PAT), which is co-expressed with Cry2Ae protein in Event GHB119 

cotton as a PIP inert ingredient. The PAT enzyme provides the plant with tolerance to herbicide 

applications containing glufosinate-ammonium by acetylating the herbicide glufosinate-ammonium and 

making it unable to bind glutamine synthase. The PAT enzyme encoded by 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 enzyme digests and 

Southern blot analyses as well as DNA sequencing indicates that the DNA was inserted in the cotton 

genome at a single locus, and the insert contains 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, 

segregating 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 dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) analyses of Cry2Ae protein as expressed in Event 

GHB119 showed a molecular weight at approximately 71 kDa. Western blot analyses showed a single 

immunoreactive band at ~71 kD. Cry2Ae protein was also found to be non-glycosylated. 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 
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document January 23, 2012

Page 38 of 153

feeding bioassay using Heliothis virescens first instar larvae and the LC50 for the plant- produced 

Cry2Ae protein was 296 ng/cm2. 

d. Protein Expression

Expression level data were provided for Cry2Ae in different plant tissues and at different growth stages 

in Event GHB119 cotton. A summary of the results obtained using an ELISA method is provided in 

Table B.1 below. 

Table B.1. Mean Cry2Ae Protein 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)

Biopesticides Registration Action Document January 23, 2012

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 PIP - Product Characterization MRID No

Characteristics of 

Cry2Ae Cotton 

Plants Derived from

Transformation 

Events GHB119 and 

GHB714: 

Preliminary Report 

and Supplemental 

Information5

This submission includes descriptions of the genetic construct used in 

Events GHB119 and GHB714, the sources of the introduced genes, the 

modes of action of the expressed proteins, and preliminary molecular 

analysis. Restriction enzyme digestion of genomic DNA from leaf 

material from Events GHB119 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 Report1

BCS conducted preliminary protein expression analysis for Cry2Ae 

cotton. Average Cry2Ae protein concentrations, as determined by 

ELISA in ground cotton seed from Events GHB119, GHB714, and 

GHB866 were 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 GHB119, GHB714, and 

GHB866 were 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 flower: 

0.018 to 0.02 1%. The PAT content of cotton tissues (other than seed) 

was 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 

Characterization of 

Cry2Ae Cotton 

Event GHB1196

The purpose of this study was to determine if the transfer DNA (TDNA) containing the intended cry2Ae gene from plasmid pTEM12 was 

inserted into the genomic DNA of cotton Event GHB119 and the 

backbone DNA from pTEM12 was not inserted into cotton Event 

GHB119. Southern blot analyses demonstrated a single copy of the 

intended cry2Ae gene from plasmid pTEM12 was 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

However, 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 laws 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

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)

Biopesticides Registration Action Document January 23, 2012

Page 40 of 153

Study Title B.2. Summary- Cry2Ae PIP - Product Characterization MRID No

successive generations of Event GHB199 cotton were 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 cry2Ae 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 Proteins 

Produced in Bacillus 

thuringiensis (Bt) 

and Escherichia coli

to Cry2Ae and 

PAT/bar Proteins in 

GHB119 Cotton, 

Gossypium 

hirsutum2

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 protein expressed in Bacillus 

thuringiensis (Bt) and the PAT/bar protein expressed in Escherichia 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 protein standard, and TwinLinkTM cotton had identical 

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 PAT/bar E. coli

standard and the TwinLinkTM cotton. However, 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 single-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 relationship 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 source of preparation. 

CLASSIFICATION: SUPPLEMENTAL; Data gaps addressed in 

MRID No. 48480003

47641905 

Analyses of Raw 

Agricultural 

Commodity (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 was ground 

into fractions of lint coats and kernels and analyzed separately for 

Cry2Ae and PAT proteins and total extractable protein. To quantitate 

47641904
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document January 23, 2012

Page 41 of 153

Study Title B.2. Summary- Cry2Ae PIP - Product Characterization MRID No

GHB119 for 

PAT/bar and 

Cry2Ae and its Nontransgenic 

Counterpart for 

PAT/bar and 

Cry2Ae Proteins2

the 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 protein content of transgenic fuzzy seed from all 

test sites, without and with herbicide spraying, on a dry weight basis 

was 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 50.7 ± 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 showed 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 nontransgenic cotton line Coker 312. Extracts of plant matrices were 

prepared from the roots, stems, leaves, squares, apex, bolls, whole plant, 

pollen, nectar, flowers and cottonseed. An enzyme-linked 

immunosorbent assay (ELISA) was used for determining the protein 

levels. There was a range of extraction efficiencies for each 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 two growth stages; 5.58 and 5.26 μg/g for stems at two 

growth stages; 37.5, 33.3, and 17.8 μg/g for leaves at three growth 

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 flowers, 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 were found in all plant matrices taken from the 

transgenic cotton Event GHB119 (except nectar, where only PAT 

protein was detected), while these proteins were absent from all of the 

cotton matrices derived from the non-transgenic cotton Coker 312. 

Pollen and nectar contained the least amount of both Cry2Ae and PAT 

proteins.

CLASSIFICATION: ACCEPTABLE

47641903

Protein Detection 

Methods. Enzyme 

Linked Immuno 

Sorbent Assay 

(ELISA)

Semi- and full quantitative detection of Cry2Ae protein 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)

Biopesticides Registration Action Document January 23, 2012

Page 42 of 153

Study Title B.2. Summary- Cry2Ae PIP - Product Characterization MRID No

designated analytical method for the detection of Cry2Ae protein 

residues is needed.

Product 

Characterization –

Nutritional 

Characterization of 

Cry2Ae cotton 

Event GHB1197

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 antinutritional 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. The nutritional and compositional analysis 

of Event GHB119 cotton falls under the regulatory purview of FDA.

47641902

Detailed insert 

characterization of 

Gossypium hirsutum

transformation 

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 

was digested with one of the following restriction enzymes or enzyme 

complexes: DraI, EcoRV, HindIII, NcoI, BamHI, EcoRI, PstI, AvaI, 

NdeI, XhoI, EcoRI/PstI and EcoRI/NdeI). 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-cry2Ae 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 inserted 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 laws of inheritance.

CLASSIFICATION: ACCEPTABLE

48480001

[This study 

supersedes 

MRID No. 

47641901]

Confirmation of the 

absence of vector 

backbone sequences 

in Gossypium 

hirsutum

transformation 

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 cry2Ae 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 EcoRV 

and NotI, using seven overlapping vector backbone probes that 

48480002

[This study 

supersedes 

MRID No. 

47641901]

 

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 GHB119 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 plant varieties, including those developed through biotechnology (FDA, 

1992). The FDA evaluation 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 PIP - 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 be rehybridized with the T-DNA probe, as a positive control. Genomic DNA 

from the Event GHB119 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 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

transformation 

Event GHB119

The purpose of this study was to determine the biochemical and 

functional equivalence of plant-expressed Cry2Ae protein produced in 

Event GHB119cotton plants with bacterial-expressed Cry2Ae proteins 

(Batch Nos. VMLV1041-1 and NB210806P25 produced from 

recombinant Bacillus thuringiensis and Escherichia coli overexpression system, respectively) for their use as suitable test substance 

surrogates in toxicological 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 assess 

the biochemical and functional equivalence, which included: 1) 

estimation of molecular weight by observing protein mobility in SDSPAGE, 2) verification of immunoreactivity using Western blot analysis, 

3) analysis of glycosylation using the reaction 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 bioactivity 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 were found to have a similar molecular 

weight after electrophoresis, similar immunoreactive bands based on 

Western Blot analyses and non-glycosylated. Identity of the plantproduced Cry2Ae protein and the bacterial-produced protein batches 

was 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 VMLV1041-1 and 

75% of bacterial-produced batch NB210806P25 were identified. The Nterminal 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 LC50 for the plant-produced 

Cry2Ae protein was 296 ng/cm2, while the bacterial-produced Cry2Ae 

proteins from Batch Nos. VMLV1041-1 and NB210806P25 were 311 

ng/cm2and 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 NB210806P25) and PAT (Batch No. NB010905P44P2) proteins are 

48480006

[This study 

supersedes 

MRID No. 

47641905 

and 

47641906]
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document January 23, 2012

Page 44 of 153

Study Title B.2. Summary- Cry2Ae PIP - Product Characterization MRID No

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: ACCEPTABLE

B. II. HUMAN HEALTH ASSESSMENT – Cry2Ae PIP

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) only 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 certainty that no harm will result from aggregate 

exposure to the pesticide chemical residue, including all anticipated dietary exposures and all other 

exposures for which there is reliable information.” This includes exposure through drinking water and in 

residential settings, but does not include occupational exposure. Pursuant to section 408(c)(2)(B), in 

establishing or maintaining in effect an exemption from the requirement of a tolerance, EPA must take 

into account the factors set 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 pesticide chemical residue... .” 

Additionally, section 408(b)(2)(D) of the FFDCA requires that the Agency consider “available 

information concerning the cumulative effects of a particular pesticide’s residues” and “other 

substances that have a common mechanism of toxicity.” EPA performs a number of analyses to 

determine the risks from aggregate exposure to pesticide residues. First, EPA determines the toxicity of 

pesticides. Second, EPA examines exposure to the pesticide through food, drinking water, and through 

other exposures that occur as a result of pesticide use in residential settings. 

a. Toxicological Profile

BCS submitted a pesticide tolerance petition (PP 9F7514) under the FFDCA, requesting 40 CFR Part 

174 be amended by 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 exemption for Cry2Ae protein in or on cotton food/feed commodities (40 CFR 

§174.530). The 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 

analyses in cotton. No data were submitted for use of this protein as a PIP in other crops nor do any

currently 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 available 
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document January 23, 2012

Page 45 of 153

information concerning the variability of the sensitivities of major identifiable subgroups of consumers, 

including infants and children.

1) Mammalian Toxicity and Allergenicity Assessment – Cry2Ae PIP

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 protein 

was assessed by orally dosing (via gavage) to five female mice with 2,000 milligrams/kilograms 

bodyweight of purified bacterial-produced Cry2Ae protein test substance (Batch No. NB210806P25, 

>93% purity). All treated animals gained weight and had no test material-related clinical signs and no 

test material-related findings at necropsy. The acute oral LD50 of the Cry2Ae protein is greater than 2000 

mg/kg body weight. The Cry2Ae protein does not appear to cause any significant adverse effects at an 

exposure level of up to 2000 mg/kg bodyweight and supports the finding that the Cry2Ae protein would 

be non-toxic to mammals.

These data demonstrate the safety of the product at a level of 2000 mg/kg b.w. that is well above 

maximum possible ppm exposure levels that are reasonably 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 products 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 by 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 by Cry2Ae protein, even at 

relatively high dose levels, the Cry2Ae protein is not considered toxic. Further, amino acid sequence 

comparisons showed no similarities that would raise a safety concern between the Cry2Ae protein and 

known toxins in protein databases.

Since Cry2Ae is a protein, allergenic sensitivities 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 glycosylation (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 glycosylated; and present at high concentrations 

in the food. 

Summaries of the allergenicity assessment for Cry2Ae protein include: 

1. Source 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 known 
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document January 23, 2012

Page 46 of 153

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 (MRIDs No. 

47641908 and 47641909).

3. Prevalence in food. Protein expression level analyses of Cry2Ae protein confirm its presence 

at relatively low levels. Dietary exposure is extremely limited and expected to be 

correspondingly 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 rapidly 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 glycosylated. 

(MRIDs No. 48471901 and 48480006).

Considering all of the available information, EPA concluded that the potential for Cry2Ae protein 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 or 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 consumers) 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 nonoccupational 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 negligible. The amino acid homology assessment included similarity to known 

aeroallergens. It has been demonstrated that there is no evidence of occupationally related respiratory 

symptoms, based on a health survey on migrant workers after exposure to Bt pesticides (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 directly 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. 

The oral toxicity study conducted at a dose of 2,000 mg/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 TwinLinkTM, Cry2Ae protein is expressed in the ppm levels (see Allergenicity

Assessment above, and Tables B.1 and B.4 in this Appendix). These 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 upon the lack of mammalian toxicity and the 

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

Biopesticides Registration Action Document January 23, 2012

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 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.”

Cry2Ae protein is not considered to be toxic, and does not share a common mechanism of toxicity with 

any other substance. In addition, Cry2Ae protein does not appear to produce a toxic metabolite produced 

by other substances. EPA concludes that there are no cumulative effects associated with Cry2Ae protein 

that need be considered. For information regarding EPA’s efforts to determine which chemicals have a 

common mechanism of toxicity and to evaluate the cumulative effects of such chemicals, see EPA’s 

website at http://www.epa.gov/pesticides/cumulative

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. The 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 fully in Appendix B.II.a., above, the acute oral toxicity data submitted supports the 

prediction that the Cry2Ae protein would be nontoxic to humans. Moreover, Cry2Ae protein has no 

sequence similarity to any known toxin. Because of this lack of demonstrated mammalian toxicity, no 

protein residue chemistry data for Cry2Ae were required for a human health effects assessment. Even 

so, expression level analysis showed Cry2Ae protein is present at relatively low levels. Dietary exposure 

is expected to be correspondingly low. 

Since Cry2Ae is a protein, its allergenic potential was considered as part of the toxicity assessment. Data 

considered as part of the allergenicity 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 allergens;

c) was readily degraded by pepsin; and 

d) was not glycosylated 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 specifically 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 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 

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

Biopesticides Registration Action Document January 23, 2012

Page 48 of 153

Based on all of the available information described in this BRAD,, the Agency concludes that there is a 

reasonable certainty 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 material 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 readily in dietary exposure and is derived 

from a source not known to exert an influence on the endocrine system. 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 enzyme-linked immunosorbent assay (ELISA) analyses for the detection of Cry2Ae protein 

residues in Event GHB119 cotton. Results showed the test kit is able to detect Cry2Ae protein residues 

in cotton with sufficient accuracy, precision, and sensitivity. However, a validation study conducted by

an independent 3rd party laboratory is still needed to evaluate the performance of the ELISA test kit as 

the designated residue analytical method for 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 (MRLs) established by the Codex Alimentarius

Commission (Codex), as required by FFDCA section 408(b)(4). The Codex Alimentarius is a joint U.N. 

Food and Agriculture Organization/World Health Organization food standards program, and it is 

recognized as an international food safety standards-setting organization in trade agreements to which 

the United States is a party. EPA may establish a tolerance that is different from a Codex MRL; 

however, FFDCA 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 Cry2Ae 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 for 

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 any indication of allergenicity potential for the plant-incorporated 

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

Biopesticides Registration Action Document January 23, 2012

Page 49 of 153

The data submitted and reviewed for Cry2Ae protein support the petition for an exemption from 

the requirement of tolerance for Bacillus thuringiensis Cry2Ae protein when used as plant–

incorporated protectant in or on cotton food/feed commodities. 

G. Supporting Data 

The human health studies submitted to support the safety of Cry2Ae protein are summarized in Table 

B.3 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.3. Human Health Data for Event GHB119 cotton expressing Bt Cry2Ae protein

(EPA File Symbol 264-RNOL)

Study Title B.3. Summary – Human health – Cry2Ae PIP MRID No

Description of the 

Amino Acid 

Sequence of the 

Cry2Ae Protein8

The 631 amino acid sequence of the Cry2Ae protein is provided in this 

submission. No information is provided on how the sequence was 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 western blot 

analyses of the plant-produced protein are provided in MRID 46708907, which 

indicate that the protein has the expected molecular weight. 

Classification: SUPPLEMENTAL; Preliminary study used to support EUP

46708902

Cry2Ae (GEM2) 

Protein: Overall 

Amino Acid 

Sequence Homology 

Search with Known 

Toxins and 

Allergens4

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 similarities 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 addressed for Sec. 3 registration in MRID No. 47641910

46708903

Cry2Ae (GEM2) 

Protein: Epitope 

Homology and NGlycosylation 

Searches4

The amino acid sequence of the Cry2Ae protein was compared to known protein 

sequences in the Allergen database to identify any similarities between 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 segment 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 

Fluid4

The digestibility of the trypsin resistant core of microbially produced Cry2Ae 

protein was 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 incubation in simulated 

gastric fluid at pH 1.2. Horseradish peroxidase and ovalbumin were degraded 

rapidly and slowly, respectively, as expected. However, because the protein that 

is produced in cotton is the full-length Cry2Ae protein, digestibility of the fulllength 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)

Biopesticides Registration Action Document January 23, 2012

Page 50 of 153

Study Title B.3. Summary – Human health – Cry2Ae PIP 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 

Mouse4

The acute toxicity of the trypsin resistant core of microbially produced Cry2Ae 

was investigated by intravenous injection in mice. The protein (>95% purity) 

was suspended in PBS buffer (phosphate buffered saline) and administered as a 

single 1 or 5 mg/kg body weight intravenous dose. Melittin protein was used as a 

positive control, and aprotinin protein and buffer alone were used as negative 

controls. Mice were observed for clinical signs daily for the duration of the study 

(15 days), and gross necropsy was 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 were required

46708906

Analysis to 

Determine if the 

Cry2Ae (GEM2) 

Protein from Cotton 

Leaves is 

Glycosylated4

Cry2Ae protein, purified from Bacillus thuringiensis and from cotton leaves from 

Events GHB119, GHB714, and GHB866, was analyzed for glycosylation using a 

commercially available glycosylation test kit. The results indicate that the 

Cry2Ae protein is not glycosylated. 

Classification: ACCEPTABLE 

48471901

[This study 

supersedes 

MRID No. 

46708907]

Cry2Ae (GEM2) 

Protein: Acute 

Toxicity by Oral 

Gavage in Mice4

The acute oral toxicity of Cry2Ae protein 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 were no mortalities, and no 

treatment-related adverse effects observed. Therefore, the acute oral LD50 of the 

Cry2Ae protein is greater than 2000 mg/kg body weight. 

Classification: ACCEPTABLE 

47076902

Cry2Ae (GEM2) 

Protein: In Vitro 

Digestibility Study 

in Simulated Gastric 

Fluid4

The susceptibility of the Cry2Ae protein to proteolytic degradation in simulated 

gastric fluid containing pepsin (SGF) was investigated using Bacillus 

thuringiensis-produced full-length Cry2Ae protein. The band for the Cry2Ae 

protein was no longer visible on the Coomassie blue stained gel after incubation 

in SGF for 0.5 minutes. 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 was 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 protein 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 

band was 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 protein and no significant deterioration after 

incubation at 60oC for 10, 30, or 60 minutes. At incubation of 75oC, the protein 

was 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 consistent 

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 

47641911
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document January 23, 2012

Page 51 of 153

Study Title B.3. Summary – Human health – Cry2Ae PIP MRID No

90°C at 60 minutes.

Classification: ACCEPTABLE

Toxicology (Human 

Health Assessment) 

Summary of 

Analysis of Open 

Reading Frame 

Homology 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 algorithms were used to 

search publically available databases (Uniprot-Swissprot, 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 between 

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 digestibility study was conducted on bacterial-expressed Cry2Ae 

protein in simulated 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 homology 

and N-glycosylation 

searches

No significant amino acid sequence similarities between eight linear contiguous 

amino acid blocks of the Cry2Ae protein with known allergens in the publically 

available AllergenOnline database (release 8.0, 1313 sequences) were found. 

Results suggest the Cry2Ae protein should not cross-react with known allergenic 

epitopes. Eleven potential N-glycosylation sites were identified.

Classification: ACCEPTABLE

47641908

[This study 

supersedes 

MRID No. 

47076904]

Cry2Ae protein with 

transit peptide 

overall amino acid 

sequence homology 

search with known 

toxins and allergens

Bioinformatic searches were conducted to evaluate any significant amino acid 

sequence similarities between 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 similarities 

between 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 

gastric fluid, overall 

amino acid sequence 

homology search 

with known toxins 

and allergens, and 

epitope homology 

and N-glycosylation 

Bioinformatic studies on Cry2Ae protein were repeated in 2008 and showed no 

amino acid sequence similarities between Cry2Ae protein and known toxins and 

allergens (MRID No. 47641909); and no amino acid sequence similarities with 

known allergen epitopes and N-glycosylation sites (MRID No. 47641908), thus 

confirming original results from previously conducted studies 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 protein to pepsin, thus confirming original results from 

the previously conducted in vitro digestibility SGF study in 2005 (MRID No. 

46708905).

47641910

[This study is a 

supplemental 

report to 

MRIDs 

46708905, 

46708903 and 

46708904]
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document January 23, 2012

Page 52 of 153

Study Title B.3. Summary – Human health – Cry2Ae PIP MRID No

searches 

Classification: SUPPLEMENTAL- This study is a supplemental to MRIDs 

46708905, 46708903 and 46708904
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document January 23, 2012

Page 53 of 153

B.III. 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 Symbol

264-RNOA) for use as an alternative insect resistant and herbicide tolerant combination PIP cotton 

product. TwinLinkTM cotton was developed by conventional breeding of the parental Events T304-40 x 

GHB119 to express Cry1Ab, 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 Cry1Ab insecticidal protein, 

which also confers resistance to lepidopteran pest species. The safety of Cry1Ab protein, as expressed in 

the cotton Event T304-40, was previously 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 submitted for its registration were found acceptable

(US EPA 2011c). Considerable safety data have been established for both Cry2Ae protein expressed in 

GHB119 and Cry1Ab protein expressed in T304-40 cotton. The applicant submitted the following 

equivalency studies in order to bridge from these existing acceptable toxicological data for the parental 

Events for registration of TwinLinkTM Cotton.

a. Product Characterization

TwinLinkTM cotton (T304-40 x GHB119 combination PIP product) contains cry1Ab, cry2Ae, and bar

transgenes. Events T304-40 and GHB119 cotton were individually developed by Agrobacteriummediated transformation to insert T-DNA containing the cry1Ab and cry2Ae gene cassettes via plasmid 

vectors pTDL008 and pTEM12, respectively. The resulting cotton lines were then crossed by 

conventional breeding to create TwinLinkTM (GHB119 x T304-40) cotton.

Characterization of DNA isolated from TwinLinkTM (T304-40 x GHB119) cotton using restriction 

enzyme digests and Southern blot analyses were used to confirm the genetic stability and integrity of the 

T304-40 and GHB119 transgenic inserts in TwinLinkTM cotton. The analyses showed the presence of the 

expected hybridization fragments in all tested transgenic DNA samples when hybridized with each of 

the three probes. In addition, results showed the cry1Ab, cry2Ae, and bar DNA inserts are stably 

integrated in TwinLinkTM cotton, based on Southern blot analyses of combination PIP product plants 

obtained after five generations from the original TwinLinkTM cross. Data from the comparative 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 

TwinLinkTM cotton.

b. Analytical Detection Methods

BCS provided validation studies for commercial enzyme-linked immunosorbent assay (ELISA) test kits 

for the analytical detection of Cry2Ae and Cry1Ab protein residues in cottonseed and cotton leaf tissue. 

The studies showed the test kits are able to detect Cry2Ae and Cry1Ab protein in Events GHB119 and 

T304-40 cotton, respectively, with sufficient accuracy, precision, and sensitivity. Verification of the 

specific ELISA test kit method to be used as the proposed residue analytical method for the detection of 

Cry1Ab 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 analytical method for the detection of Cry2Ae and Cry1Ab proteins 
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document January 23, 2012

Page 54 of 153

expressed in cotton Events GHB119 and T304-40, respectively.

c. Protein Expression

Protein expression levels were provided for Cry2Ae, PAT, and Cry1Ab 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 

TwinLinkTM cotton are comparable to those observed in plant tissues from its parental Events GHB119 

and T304-40 cotton. The PAT protein expression levels in TwinLinkTM cotton were higher than those 

expressed in its parental events. These results were expected since TwinLinkTM cotton inherited two 

copies of the bar gene, which were individually inserted in its parental events.

Table B.4. Mean Expression Levels of Cry2Ae, PAT and Cry1Ab Proteins 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 means tested at different growth stages 

d. Conclusion

The DNA characterization study and protein expression analyses field study considered with comparable 

biological activity in between the parental cotton Events GHB119 and T304-40 and no evidence of 

either a synergistic or antagonistic interaction between Cry2Ae and Cry1Ab proteins in tobacco 

budworm (TBW, Heliothis virescens) (see Dean, B. et al. 2010; MRID No. 48480009) reviewed in the 

ecological risk assessment memo for this product (US EPA 2011b) demonstrate that the Cry2Ae and 

Cry1Ab proteins expressed in the parental cotton Events GHB119 and T304-40 are biochemically and 

functionally equivalent to the Cry2Ae and Cry1Ab proteins expressed in TwinLinkTM cotton. Thus, the 

existing toxicological data of parental Events GHB119 and T304-40 cotton can be bridged to support the 

safety of TwinLinkTM Cotton. 

e. Supporting Data

The product characterization studies submitted to support the biochemical equivalence of Cry2Ae and 

Cry1Ab proteins expressed in TwinLinkTM cotton to Cry2Ae and Cry1Ab expressed in its constituent 

parental Events GHB119 and T304-40, respectively, are summarized in Table 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). 
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document January 23, 2012

Page 55 of 153

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

(EPA File Symbol 264-RNOA)

Study Title B.5. Summary - TwinLinkTM - Product Characterization MRID No.

TwinLinkTM 

Product 

Characterization -

Molecular 

Characterization9

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 showed band patterns 

obtained using each probe were 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.

However, no Southern blot analyses were conducted using the pat/bar as a probe on 

the parental events and TwinLinkTM Cotton. In addition, details were missing on the 

test materials, methods used to conduct the study. There were also no plasmid maps 

or schematic 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 Cotton5

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 was 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. 

However, the significant differences may be attributed to the limited number of 

samples, replicates, and trials since 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 

47640003 

 

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

Biopesticides Registration Action Document January 23, 2012

Page 56 of 153

Study Title B.5. Summary - TwinLinkTM - Product Characterization MRID No.

Analyses of Raw 

Agricultural 

Commodity (Fuzzy 

Seed) of

TwinLinkTM Cotton 

for PAT/bar, 

Cry2Ae and 

Cry1Ab and its 

Non-transgenic 

Counterpart for 

PAT/bar, Cry2Ae 

and Cry1Ab 

Proteins

The purpose of this study was to quantify the expression of Cry1Ab, Cry2Ae and 

PAT proteins in the fuzzy seeds of transgenic TwinLinkTM cotton and the nontransgenic Coker 315 cotton line using enzyme-linked immunoabsorbent assay 

(ELISA). Fuzzy seed were separated 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 

was received from the field. 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 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 ± 0.317μg/g, respectively. 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 transgenic fuzzy seed from all test sites, 

without and with herbicide spraying, on a dry weight basis, was 9.35 ± 2.93μg/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 0.00485 ± 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, was 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

47640004

Structural stability 

analysis of 

Gossypium hirsutum

combined 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 combination 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 from 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 was subjected to Southern blot 

analysis using the cry1Ab-bar TDNA probe, the cry1Ab gene probe, and the cry2Aebar 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 TwinLinkTM cotton, based on Southern blot analyses of 

combination PIP product plants obtained after five generations from the original 

TwinLinkTM cross. Therefore, data from the comparative 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 TwinLinkTM Cotton.

CLASSIFICATION: ACCEPTABLE

48480007

[This study 

supersedes 

MRID No. 

47640001]

Protein expression 

analysis for GlyTol 

x TwinLinkTM

(GHB614 x T304-40 

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 combination PIP product: GlyTol x TwinLinkTM (GHB614 x T304-40 x 

GHB119). Cotton Event T304-40 expresses the Cry1Ab and PAT/bar proteins, 

48480008

[This study 
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document January 23, 2012

Page 57 of 153

Study Title B.5. Summary - TwinLinkTM - Product Characterization MRID No.

x GHB119) cotton 

grown in the field, 

USA, 2010

Event GHB119 expresses the Cry2Ae and PAT/bar proteins, and Event GHB6141

expresses the 2mEPSPS (5-enolpyruvyl-shikimate-3-phosphate synthase) protein. 

Combination PIP product cotton tissue samples from leaves, roots, squares, bolls, 

and fuzzy seeds were collected from three different field trial sites at one or more 

developmental stages (T1 though T4). A total of 36 samples of each tissue type 

(matrix) were assayed for protein concentrations. Expression of all proteins (CrylAb, 

Cry2Ae and PAT/bar) was observed in all matrices (leaves (T1, T2, and T3), roots 

(T1, T2, and T3), squares (T3), bolls (T3), and fuzzy seed (T4)). 

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 product were comparable to the protein expression levels 

in its respective parental cotton 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 relative to the PIP parental Events GHB119 

and T304-40, when these parental lines are combined via traditional breeding 

methods.

CLASSIFICATION: ACCEPTABLE 

supersedes 

MRID No. 

47871604]

REFERENCES – Cry2Ae and TwinLinkTM Cotton PIPs

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, Italy. 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). Bayer CropScience Internal Report -

Insect Bioassay from Monheim. MRID No. 48480009 

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) Structural and functional 

analysis 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” Regulatory 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 enzyme. EMBO Journal. 6(9): 2513-2518
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document January 23, 2012

Page 58 of 153

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 Data for an EUP Application 

for Bt. Cry2Ae Insecticidal Protein and Cry2Ae x Cry1Ab as Expressed in Cotton. Memorandum 

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 Analytical Method 

(EPA 712–C–96–174); Document ID: EPA-HQ-OPPT-2009-0155-0007; Docket ID: EPA-HQOPPT-2009-0155 

U.S. EPA. 2011a. Review of Product Characterization Data in support of Sec. 3 Registration of PlantIncorporated Protectant (PIP) cotton Events: GHB119 cotton expressing Bacillus thuringiensis 

Cry2Ae protein (EPA File Symbol 264-RNOU), T304-40 cotton expressing Bt CrylAb (EPA 

File Symbol 264-RNOL); and TwinLinkTM cotton (EPA File Symbol 264-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) Cry1Ab and Bt 

Cry2Ae insect control proteins as expressed in Event T304-40 cotton and GHB119 cotton, 

respectively, and in the combined Event, T304-40 x GHB119 (TwinLinkTM) cotton (EPA File 

Symbols 264-RNOU, -RNOL, -RNOA), submitted by Bayer 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 

Registration of Plant-Incorporated Protectant (PIP) Event T304-40 cotton expressing Bacillus 

thuringiensis Cry1Ab insecticidal protein (EPA File Symbol 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 ServicesFood and Drug Administration (Docket No. 92N-0139)

http://www.cfsan.fda.gov/~lrd/biotechm.html#reg

Van Rie J. 2004. Comparative analysis of the Cry1Ab protein amino acid sequence. Project Number: 

None; Unpublished study prepared by Bayer CropScience LP. MRID No. 46474406.
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APPENDIX C: Ecological and environmental 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:

 Cry1Ab protein and the genetic material necessary for its production in Event T304-40 

transgenic cotton [Gossypium hirsutum] plant line;

 Cry2Ae protein and the genetic material necessary for its production in transgenic Event 

GHB119 cotton plant line; and 

 the associated pyramid in Event T304-40 x GHB119 (referred to hereafter as TwinLinkTM) 

Cotton, which produces both proteins. 

BPPD does not anticipate any adverse effects of the Cry1Ab and Cry2Ae proteins on nontarget 

organisms or on their abundance in any field population. BPPD additionally concludes that the proposed 

Section 3 registrations of Cry1Ab and Cry2Ae proteins as expressed in Event T304-40 x Event 

GHB119, or TwinLinkTM Cotton 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 Cry1Ab and Cry2Ae proteins used in toxicity testing and 

plant-expressed Cry1Ab and Cry2Ae proteins. The ecological risk assessment for the combined Events

expressed in TwinLinkTM cotton is also dependent on equivalent expression levels of Cry1Ab and 

Cry2Ae protein in TwinLinkTM cotton plants compared to the T304-40 and GHB119 parent lines [see 

Product Characterization - Appendix A (Cry1Ab) and B (Cry2Ae)].

BACKGROUND

BCS developed Event T304-40 and Event GHB119 transgenic cotton [Gossypium hirsutum] plant lines, 

which each express a Cry protein insect control gene. Event T304-40 expresses a Cry1Ab 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. Both genes have been modified for their 

expression in plants. Cry1Ab 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 armyworm (Spodoptera exigua), and fall armyworm 

(Spodoptera frugiperda). Events T304-40 and GHB119 also express the PAT gene, which confers 

herbicide tolerance.

Experimental use permits (EUP) 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 currently requesting a FIFRA Sec. 3 registration for Bt insect control protein Cry1Ab as 

expressed in Event T304-40 cotton, Cry2Ae as expressed in Event GHB119 cotton, and the associated 

breeding stack, TwinLinkTM cotton, produced by a traditional breeding cross. This memorandum 

contains the Agency’s environmental risk assessment of the Cry1Ab and Cry2Ae proteins produced in 

T304-40 and GHB119 cotton lines, respectively, as well as in TwinLinkTM Cotton. 
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I. Environmental Risk Assessment for Cry1Ab as Expressed in T304-40 Cotton and Cry2Ae 

as Expressed in GHB119 Cotton PIPs

The paragraphs below describe the process and rationale developed by 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 early in an investigation tend to be 

broad in scope but relatively simple in design, and can be used to demonstrate acceptable risk under 

most conceivable conditions. When screening studies suggest potentially unacceptable risk additional 

studies are designed to assess risk under more realistic field exposure conditions. These later tests are 

more complex than earlier screening studies. Use of this “tiered” testing framework saves valuable time 

and resources by organizing the studies in a cohesive and coherent manner and eliminating unnecessary

lines of investigation. Lower tier, high dose screening studies also allow tighter control over 

experimental variables and exposure conditions, resulting in a greater ability to produce statistically

reliable results at relatively low cost10. 

Tiered tests are designed to first represent unrealistic worst case scenarios and ONLY progress to real 

world field scenarios if the earlier tiered tests fail to indicate adequate certainty of acceptable risk. 

Screening (Tier I) 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 system to assess the toxicity of a PIP to representative nontarget organisms that could be exposed to the toxin in the field environment. Tier I high dose studies 

reflect a screening approach to testing designed to maximize any toxic effects of the test substance on 

the test (non-target) organism. The screening tests evaluate single species in a laboratory setting with 

mortality as the end point. Tiers II – IV generally 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 by 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 allowed an endpoint of 50% 

mortality 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 scenarios. 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 30 individuals is 

sufficient to allow a treatment effect of 30% to be differentiated from a 50% effect with 95% confidence using a one-sided Z 

test. A one-sided test is 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 website: 

http://www.epa.gov/opptsfrs/publications/OPPTS_Harmonized/885Microbial_Pesticide_Test_Guidelines/Series
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guidelines, as defined in 40 CFR 152.20, apply to microbes and microbial toxins when used as 

pesticides, including those that are naturally occurring, and those that are strain-improved, either by

natural selection or by deliberate genetic manipulation. Therefore, PIPs containing microbial toxins are 

also covered by 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 expected 

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 hypotheses. Elevated test doses, therefore, add certainty to the 

assessment, and such tests can be well standardized. The Guidelines call for initial screening testing of a 

single group or several groups of test animals at the maximum hazard dose level. The Guidelines call for 

testing of one treatment group of at least 30 animals or three groups of 10 test animals at the screening 

test concentration. The Guidelines further state that the duration of all Tier I tests should be 

approximately 30 days. Some test species, notably non-target insects, may be difficult to culture and the 

suggested test duration has been adjusted accordingly. Control and treated insects should be observed 

for at least 30 days, or, in cases where an insect species cannot be cultured, for 30 days, 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 necessarily indicate the existence of unacceptable risk in the field, but it does trigger the need to 

collect additional dose-response information and a refinement of the exposure estimation before 

deciding if the risk is acceptable or unacceptable. Where potential hazards are detected in Tier I testing 

(i.e. mortality is greater than 50%), additional information at lower test doses is required which can 

serve to confirm whether any effect might still be detected at more realistic field [1X EEC] 

concentrations and routes of exposure14. 

When screening tests indicate a need for additional data, the OPPTS Harmonized Guidelines call for 

testing at incrementally lower doses in order to establish a definitive LD50 and to quantify the hazard. In

the definitive testing, the number of doses and test organisms evaluated must be sufficient to determine 

an LD50 value and, when necessary, the Lowest Observed Effect Concentration (LOEC), No Observed 

Adverse Effect Level (NOAEL) , or reproductive and behavioral effects such as feeding inhibition, 

weight loss, etc. In the final analysis, 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 employed 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 many 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 non-target organism may be ingesting in the 

field because most non-target organisms do not ingest plant tissue.
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levels of replication, the number of test species, and/or repetition are needed to enhance statistical power 

in these circumstances. 

Data that show less than 50 % mortality at the maximum hazard dosage level – (i.e. LC50, ED50, or LD50

>10 X EEC) is sufficient to evaluate adverse effects, making lower field exposure dose definitive testing 

unnecessary. It is also notable that the recommended >10X EEC maximum hazard dose level is a highly

conservative factor. The published 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 Institute 

of Biological Sciences (AIBS) and confirmed in 1996 as an acceptable method of environmental hazard 

assessment by a FIFRA Scientific Advisory Panel (SAP) on microbial pesticides and microbial toxins. 

The December 9, 1999 SAP agreed that the Tiered approach was suitable for use with Plant-Incorporated 

Protectants (PIPs); however, this panel recommended that, for PIPs with insecticidal properties, additional 

testing of beneficial invertebrates closely 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 performed 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 delayed 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 requirement does amount to 

subchronic testing when performed at field exposure test doses. Proteins also do not bioaccumulate. The 

biological nature of proteins makes them readily susceptible to metabolic, microbial, and abiotic 

degradation once they are ingested 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 rapidly by 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 I screening testing 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 sufficient for most studies submitted in support of PIP registrations. However, if long range adverse 

effects must be ascertained, then higher-tier longer-term field testing will be required. As noted above, 

the October 2000 SAP and the National Academy of Sciences (NAS 2000) recommended testing nontarget organisms directly in the field. This approach, with an emphasis on testing invertebrates found in 

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 The established peer and EPA Science Board reviewed guidance on screening test levels of concern is 50% mortality at 5X 

environmental concentration. 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 may be tested at the 

maximum hazard dose.
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due to the lack of baseline data on the potential for long-term environmental effects from the cultivation 

of PIP-producing plants.

Since the commercialization of Bt crops, the number of field studies published in scientific literature in 

combination with the post-registration field studies submitted to the Agency has accumulated to a level 

where empirical conclusions can be made. As a result, the issue of long range effects of cultivation of 

these Cry proteins on the invertebrate community structure in Bt crop fields has since been adequately

addressed. Specifically, a meta-analysis16 of the data collected from 42 field studies indicated that nontarget invertebrates are generally more abundant in Bt cotton and Bt maize fields than in non-transgenic 

fields managed with insecticides (Marvier et al., 2007). In addition, a comprehensive review of short 

and long term field studies on the effects of invertebrate populations in Bt corn and cotton fields 

indicated that no unreasonable adverse effects are taking place as a result of wide scale Bt crop 

cultivation (Sanvido et al. 2007). Another review of field tests published to date concluded that the 

large-scale studies in commercial Bt cotton have not revealed any unexpected non-target effects other 

than subtle shifts in the arthropod community caused by the effective control of the target pests (Romeis 

et al., 2006). Slight reductions in some invertebrate predator populations are an inevitable result of all 

pest management practices, which result in reductions in the abundance of the pests as prey. 

Overall, the Agency is in agreement with the conclusions of these studies and collectively, 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 by 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 determined that non-target organisms with the 

greatest exposure potential to Cry protein in transgenic cotton fields are beneficial insects that feed on 

cotton pollen and nectar, particularly lepidopteran insects, and soil invertebrates. While EPA’s risk 

assessments of Bt cotton have focused primarily on these taxa, BPPD recognizes that exposure to other 

nontarget organisms can occur and has required testing on representative species.

The EPA risk assessment is centered on adverse effects at the field exposure rates, which are typically

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 system is low or where high concentrations of test material are not 

possible to achieve due to test organism feeding habits. BPPD may also allow for testing at lower doses 

in cases where many species are tested or tests are very sensitive, although the concentration used must 

exceed the EEC. For the purposes of the 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 

Cry1Ab or Cry2Ae protein expressed in the tissue(s) with the highest concentration based on protein 

expression studies (MRIDs 47634803, 47641903). 

 

16 This research was 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/).
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B. Ecological Effects Data for Event T304-40 and Event GHB119

In the absence of PIP-specific risk assessment guidance, EPA requires applicants for PIP registrations to 

meet the 40 CFR Part 158 data requirements for microbial toxins. These requirements include testing on 

birds, mammals, nontarget insects, honey bee, plants, and aquatic species, and information has been 

submitted to address these requirements. Limit dose testing on representative organisms from several taxa 

was performed in support of the T304-40 and GHB119 Section 3 FIFRA registrations. As stated above, 

BPPD’s risk assessments focus heavily on beneficial nontarget invertebrates, since they are most closely 

related to organisms susceptible to the insecticidal action of Bt toxins. The Cry1Ab and Cry2Ae proteins 

are meant to target species within the order Lepidoptera (moths and butterflies). Bt toxins are known 

typically to have a limited host range. However, to address any unforeseen change in activity spectrum as 

a result of laboratory protein synthesis 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 

Cry1Ab and Cry2Ae proteins 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, and honeybee 

studies. 

Since exposure may also occur to other nontarget organisms, EPA has received data to comply with the 

Agency’s published non-target data requirements on other nontarget organisms. Tests to determine soil 

degradation of Cry1Ab and Cry2Ae proteins have also been submitted. The individual results for 

nontarget organism and soil degradation testing for Cry1Ab and Cry2Ae proteins are summarized in 

Tables 1 and 2, respectively. 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, particularly 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 Cry1Ab and Cry2Ae protein, respectively. 

Comparative analyses have been performed to verify the equivalence of the bacterially-produced and 

purified Cry1Ab and Cry2Ae and the PIP Cry1Ab and Cry2Ae proteins produced in T304-40 or 

GHB119, respectively, and TwinLinkTM cotton plants. Product characterization data (see Appendices A 

and B) demonstrate functional equivalence between the bacteria-derived Cry1Ab and Cry2Ae proteins 

used in toxicity testing and plant-expressed Cry1Ab and Cry2Ae proteins. The ecological risk 

assessment for the combined events expressed in TwinLinkTM cotton is also dependent on equivalent 

expression levels of Cry1Ab and Cry2Ae protein in TwinLinkTM cotton plants compared to the T304-40 

and GHB119 parent lines. This bioequivalence has been demonstrated [see Product Characterization -

Appendices A (Cry1Ab) and B (Cry2Ae and TwinLinkTM Cotton)].

Table C.1. Summary of environmental effects studies and waiver justifications for Cry1Ab as expressed in 

T304-40 cotton submitted to comply with data requirements published in 40 CFR § 158.2150(d).

Data 

Requirement 

OPPTS

Guideline

Test Substance C.1. Cry1Ab PIP – Ecological Effects -

Results Summary and Classification

MRID 

No. 

Avian dietary 

testing, 

885.4050 TwinLinkTM

(T304-40 x 

A 42-day dietary study showed no adverse 

affects to broiler chickens when fed a diet 

47640011
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Data 

Requirement 

OPPTS

Guideline

Test Substance C.1. Cry1Ab PIP – Ecological Effects -

Results Summary and Classification

MRID 

No. 

broiler chicken, 

Gallus 

domesticus 

GHB119) cotton 

seed meal 

composed of 10% TwinLinkTM cottonseed meal 

containing both Cry1Ab x Cry2Ae proteins. 

However, the presence of the proteins in the test 

material was 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 waiver rationale was submitted to justify 

that testing is not necessary on 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 Microbiallyproduced 

Cry1Ab

No adverse effects on survival or reproduction 

of Daphnia magna exposed to 48 μg Cry1Ab

protein/L in a 10-day static renewal test. The 48-

hr and 10-day EC50 values were determined to 

be > 48 μg Cry1Ab protein/L; the NOAEC is ≥ 

48 μg Cry1Ab protein/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 Microbiallyproduced 

Cry1Ab protein

No adverse effects on survival, time to pupation, 

and mean adult weight for Coleomegilla 

maculata larvae exposed to 19 μg Cry1Ab 

protein/g diet over 21 days. The LC50 was >19 

μg Cry1Ab protein/g diet and the NOAEC is ≥ 

19 μg Cry1Ab protein/g diet. 

Classification: Acceptable

47634815

Non-target 

insect testing, 

green lacewing, 

Chyrsoperla 

rufilabris

885.4340 Microbiallyproduced 

Cry1Ab protein

No adverse effects on survival or pupation were 

observed in Chrysoperla rufilabris exposed to 

20 μg Cry1Ab protein/g diet (nominal) in a 10-

day laboratory bioassay. The nominal LC50 is 

>20 μg Cry1Ab protein/g diet and the NOAEC 

is ≥20 μg Cry1Ab protein/g diet. 

Classification: Acceptable

47692701

Non-target 

insect testing, 

collembola, 

Folsomia 

candida

885.4340 Microbiallyproduced 

Cry1Ab protein

No adverse effects on survival or reproduction 

of collembola (Folsomia candida) exposed to 20 

μg of Cry1Ab protein /g diet in a 28-day

laboratory bioassay. The nominal LC50 was 

determined to be > 20 μg Cry1Ab protein/g diet 

and the nominal NOAEC is ≥20 μg. 

Classification: Acceptable

47651106

Honeybee 

testing, 

Honeybee 

885.4380 Microbiallyproduced 

Cry1Ab protein

No adverse effects on survival were observed in 

honey bee larvae treated in natal cells with 20 

μg/L solution of Cry1Ab protein. The LC50 was 

47651105
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Data 

Requirement 

OPPTS

Guideline

Test Substance C.1. Cry1Ab PIP – Ecological Effects -

Results Summary and Classification

MRID 

No. 

larvae,

Apis mellifera

determined to be > 20 μg Cry1Ab protein/mL.

Classification: Acceptable

Earthworm 

toxicity, 

Eisenia foetida

850.6200 N/A Acceptable Waiver Rationale 47634818

Soil fate and 

degradation

885.5200 Microbiallyproduced 

Cry1Ab protein

Based on the measured bioactivity in tobacco 

budworm (Heliothis virescens), the average 

DT50 for Cry1Ab protein in three different soils 

was 3.6 days. 

Classification: Acceptable

47634820

Table C.2. Summary of environmental effects studies and waiver justifications for GHB119 submitted to comply with 

data requirements published in 40 CFR § 158.2150 (d).

Data 

Requirement 

OPPTS

Guideline

Test 

Substance

C.2. Cry2Ae PIP - 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 

Cry1Ab and Cry2Ae proteins. However, the 

presence of the proteins in the test material was 

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 waiver rationale was submitted to justify that 

testing is not necessary on the basis of lack of 

exposure.

Classification: Supplemental

A 5-day feeding study with Northern bobwhite 

showed no adverse effects at 10% raw GHB119 

cotton seed meal in the diet. 

Classification: Acceptable

47903301

48177501

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 Microbiallyproduced 

Cry2Ae

protein

Daphnia magna were exposed to a nominal 

concentration of 330 μg Cry2Ae protein/L both in 

water and in food for 10 days. No adverse effects 

on immobilization/survival or reproduction were 

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, 

885.4340 Microbiallyproduced No statistically significant effects on mortality, 

body weight, mean number of days to pupation, and 

47641915
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Data 

Requirement 

OPPTS

Guideline

Test 

Substance

C.2. Cry2Ae PIP - Ecological Effects –

Results Summary and Classification

MRID No. 

pink-spotted 

lady beetle, 

Coleomegilla 

maculata

Cry2Ae

protein

mean number of days to adulthood were observed 

in Coleomegilla maculata exposed to 64 μg Cry2Ae 

protein/g of diet for 21 days. The nominal LC50 is > 

64 μg Cry2Ae protein/g diet and the NOAEC is ≥ 

64 μg Cry2Ae protein/g diet.

Classification: Acceptable

Non-target 

insect testing, 

green lacewing, 

Chrysoperla 

rufilabris

885.4340 Microbiallyproduced 

Cry2Ae

protein

No statistically significant differences in mortality 

or % pupation were observed in green lacewing 

exposed to a nominal concentration of 10 or 110

μg Cry2Ae protein/g of diet for 10 days. The 

nominal LC50 is >110 μg Cry2Ae protein/g diet 

and the nominal NOAEC is ≥ 110 μg Cry2Ae

protein/g diet.

Classification: Acceptable

47692501

Non-target 

insect testing, 

collembolan, 

Folsomia 

candida

885.4340 Microbiallyproduced 

Cry2Ae

protein

Limit test: The number of offspring per adult 

exposed to Cry2Ae protein was significantly 

reduced in Collembola exposed to 66 μg Cry2Ae

protein/g diet for 28 days. 

Classification: Supplemental

Definitive test: No statistically significant 

differences in survival or reproduction among the 

test material and negative control groups were 

observed in Collembola exposed to 9.9, 20, 40, 80, 

or 160 μg of Cry2Ae protein/g diet. The nominal 

LC50 is >160 μg Cry2Ae protein/g diet and the 

nominal NOAEC is ≥160 μg Cry2Ae protein/g 

diet.

Classification: Acceptable

47641917a

47641917b

Earthworm 

toxicity, 

Eisenia foetida

850.6200 Microbiallyproduced

Cry2Ae

protein

No adverse effects were observed on burrowing 

time, mortality, and weight change for earthworms 

(Eisenia fetida) were exposed to a nominal 

concentration of 100 mg Cry2Ae protein/kg of 

artificial soil for 14 days. The nominal 7- and 14-

day LC50 was determined 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 Microbiallyproduced 

Cry2Ae

protein

Larval survival did not differ significantly between 

the Cry2Ae protein treated group and the controls 

at capping and at emergence for honey bee (Apis 

mellifera) larvae exposed to 50 μg Cry2Ae

protein/mL. Therefore, the LC50 is >50 μg Cry2Ae

protein/mL. 

Classification: Acceptable

47641914

Soil fate and 

degradation

885.5200 Microbiallyproduced 

Cry2Ae

protein

Based on the measured bioactivity in tobacco 

budworm (Heliothis virescens), the average DT50

for Cry2Ae protein in three different soils was 3.4 

days. 

Classification: Acceptable

47641920
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1. Non-target Organism Study Summaries for T304-40 expressing Cry1Ab Protein

 a. Avian Wildlife

Avian Dietary Toxicity

In a 42-day study (MRID 47640011), newly-hatched Ross #308 broiler chickens were fed formulated 

diet containing 10% TwinLinkTM 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 statistically significant treatment-related effects on survival, body weight, body weight gain, 

feed consumption, feed conversion ratio, or weight of chilled carcass, leg, thigh, wing, or breast among 

any of the test groups. DNA analysis of the prepared diets confirmed the presence of transgenic genetic 

material in the TwinLinkTM diet, however the presence of the Cry1Ab and Cry2Ae proteins were not 

confirmed. The study 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 Cry1Ab or 

Cry2Ae proteins 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 Cry1Ab protein 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 Cry1Ab 

protein expressed in Event T304-40 cotton (MRID 47903201). The justification rested primarily on the 

expected minimal exposure of birds to Cry1Ab protein 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 Inhalation Toxicity

Avian inhalation toxicity testing is typically 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 with 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 effects on body weight were observed on mice dosed at 

2000 mg/kg body 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
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Data waiver rationale (MRID 47871403) was submitted to fulfill the data requirements for freshwater 

fish for Cry1Ab protein 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 heavy to be 

dispersed by wind. As a result, little pollen is expected to move off field and enter aquatic systems. 

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 systems is expected to be low, and testing of freshwater fish 

is determined to be unnecessary. 

Because of concerns raised in a study with the leaf shredding (caddis fly) trichopteran, Lepidostoma liba

(Rosi-Marshall, et al. 2007), EPA is requiring applicants of Bt corn and cotton to submit 7-10 day

freshwater invertebrate toxicity studies to reduce uncertainty 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 Cry1Ab protein in T304-40 cotton. Daphnia were exposed to a nominal concentration of 48 μg 

Cry1Ab protein/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, with exposure in 

both water and in food treated with Cry1Ab protein, no mortalities and no significant differences in the 

number of offspring produced were observed. Therefore, the EC50 was determined to be > 48 μg 

Cry1Ab protein/L, and the NOAEC was determined to be ≥ 48 μg Cry1Ab protein/L. 

Estuarine and marine animals

BPPD typically 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 previous conclusions about exposure to Cry proteins in aquatic environments. 

 d. Terrestrial and aquatic plant species

BPPD typically 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 47871403) that was consistent with BPPD’s position on nontarget plant

risks to Bt -endotoxin. 

 e. Invertebrate species 

In a study with the ladybird beetle (Coleomegilla maculata, MRID 47634815), larvae were exposed to a 

nominal concentration of 19 μg Cry1Ab protein/g of artificial diet for 21 days. This amount is meant to 

represent 10X the mean expression level of Cry1Ab protein in the cotton plants. Larval and adult 

mortality, time to pupation, time to adulthood, and adult weight did not differ significantly from the 

negative control group. Larval mortality was 7.5% and 12.5% in the treated and control groups, 

respectively; adult mortality was 0% and 2.9% in the treated and control groups, respectively. The 

nominal LC50 was determined to be >19 μg Cry1Ab protein/g of diet and the NOAEC was determined to 

be ≥ 19 μg Cry1Ab protein/g of diet.
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A study with green lacewing (Chrysoperla rufilabris, MRID 47692701) larvae exposed 25 individuals to 

a nominal concentration of 20 μg Cry1Ab protein/g of artificial diet for 10 days. The amount to which 

the larvae were exposed throughout the test was meant to represent 10X the mean fresh weight 

expression level of Cry1Ab protein in T304-40 cotton plants. Mortality and pupation measured in the 

test group (94% and 76%, respectively) did not differ significantly from the negative control (96% and 

76%, respectively). Based on the results, the LC50 for Cry1Ab protein in C. rufilabris is >20 μg a.i./g 

and the NOAEC was determined to be ≥ 20 μg Cry1Ab protein /g of diet.

Collembola (Folsomia candida) are soil dwelling insects that consume detritus. In a 28-day study

(MRID 47651106), 40 collembola were exposed to 20 μg Cry1Ab protein/g diet and observed for 

mortality and number of offspring produced per adult. Mortality of the collembola was 25% for the 

groups exposed to Cry1Ab protein and 12.5% for the control groups. The number of offspring produced 

per adult was 46.9 and 43.9 for the Cry1Ab treated and control groups, respectively. Neither of these 

endpoints differed significantly between treated and control groups. Based on the results, the, the LC50

based on the nominal concentration is > 20 μg Cry1Ab protein/g diet and the NOAEC was established to 

be ≥ 20 μg Cry1Ab protein/g diet. 

Data waiver rationale (MRID 47634818) was presented to justify that toxicity testing for Cry1Ab protein

as expressed in T304-40 cotton is not necessary with the earthworm. The 14-day LC50 for earthworms 

exposed to Cry1Ab protein derived from E. coli was previously determined to be > 200 mg/kg of 

artificial soil (U.S. EPA 2010, Mendelsohn et al. 2003). Since there is no evidence of toxicity of Cry1Ab 

protein 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 μL droplet of solution containing 20 μg/mL Cry1Ab protein, which was intended to 

represent >10X the mean fresh weight expression concentration of Cry1Ab protein in T304-40 cotton 

leaves, and > 400 times the exposure to the amount of Cry1Ab protein expressed in T304-40 pollen. 

Mortality at capping and at emergence was observed, and neither of these differed significantly from 

controls. Emergence was considered to be complete on Day 16. Of the larvae treated with Cry1Ab

protein, 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 LC50 was determined to 

be > 20 μg Cry1Ab protein/mL.

2. Non-target Organism Study Summaries for GHB119 expressing Cry2Ae protein

 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 protein expressed in GHB119 cotton. Since there is 

no assurance that the test animals were exposed to Cry1Ab or Cry2Ae protein in the diet throughout the 

study, it cannot be used for avian risk assessment for Cry2Ae protein. A request to bridge to data from 

this study to the assessment of Cry2Ae protein expressed in GHB119 cotton (MRID 47871504) was not 

reviewed because the study was determined to be unacceptable.
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Avian Oral Toxicity 

Rationale was presented to justify waiving the avian oral toxicity data requirement for the Cry2Ae 

protein expressed in Event GHB119 cotton (MRID 47903301). The justification rested primarily on the 

expected minimal exposure of birds to Cry2Ae protein in GHB119 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 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 cotton 

seed meal (containing Cry2Ae protein) at 10% of their daily diet for five days (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. This 

study does not sufficiently test hazard at higher dietary concentrations. EPA has previously determined 

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 exposure 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 protein is not likely to cause adverse effects in 

birds. However, a study in which birds are exposed to higher doses of Cry2Ae protein would reduce 

uncertainties related to actual field exposure. 

Avian Inhalation Toxicity

Avian inhalation toxicity testing is typically not required for Bt cotton event registrations, since 

exposure to Cry proteins via inhalation is 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 registration, and in this study no mortalities or treatmentrelated 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 

mammals, 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 protein 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 Cry1Ab protein was presented for Cry2Ae

protein, and this is consistent with BPPD’s previous conclusions that little aquatic exposure is expected 
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from the use of cotton expressing Cry proteins. Since BPPD has concluded that exposure in aquatic 

systems 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 Cry1Ab protein in T304-40 cotton. Daphnia were exposed to a 

nominal concentration of 330 μg Cry2Ae protein/L contained in water alone and water 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 produced were observed. Therefore, the EC50 was determined to be > 330 μg 

Cry2Ae protein/L, and the NOAEC was determined to be ≥ 330 μg Cry2Ae protein/L. 

Estuarine and marine animals

BPPD typically 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 the same as that discussed above for exposure of 

freshwater fish to Cry1Ab protein and was determined to be sufficient based on BPPD’s conclusions 

about exposure in aquatic environments. 

d. Terrestrial and aquatic plant species

BPPD typically 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 position on nontarget plant 

risks to Bt -endotoxin. 

 e. Invertebrate species 

A study with ladybird beetle (Coleomegilla maculata, MRID 47641915) larvae exposed 40 individuals 

to a nominal concentration of 64 μg Cry1Ab protein/g of artificial diet for 21 days. This amount was 

approximately 7X the fresh weight expression level of Cry2Ae protein 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 significantly from the negative control group. Larval mortality was 5.0% and 

12.8% in the treated and corresponding control group, respectively; adult mortality was 0% and 3.3% in 

the treated and control groups, respectively. The nominal LC50 was determined to be >64 μg Cry1Ab

protein/g diet and the NOAEC based on development and body weight is ≥ 64 μg Cry2Ae protein/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 protein/g of artificial diet for 10 days. These 

concentrations were meant to test effects at 1X and 10X the fresh weight expression level of Cry2Ae 

protein 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 protein/g diet test groups and the 

buffer controls was 100%, 96%, and 100%, respectively. Pupation in these groups was 84%, 72%, and 
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88%, respectively. The results establish a nominal LC50 of >100 μg Cry2Ae protein/g diet and a nominal 

NOAEC of ≥ 100 μg Cry2Ae protein/g diet. 

Collembola (Folsomia candida) are soil dwelling insects that consume detritus. In a 28-day single-dose 

study (MRID 47641917a), collembola were exposed to 66 μg Cry2Ae protein/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 protein and 22.5% for the control groups. The number of offspring produced 

per adult was 10.3 and 44.5 for the Cry2Ae protein 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 protein/g diet 

(MRID 47641917b). Negative and positive controls were also included, and a bioassay on a sensitive 

species (Heliothis virescens) confirmed bioactivity. Mortality 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 

LC50 was determined to be >160 μg Cry2Ae protein/g diet and the NOAEC was determined to be ≥160 

μg Cry2Ae protein/g diet. 

A 14-day earthworm toxicity study (MRID 47641918) was submitted, wherein earthworms were exposed 

to 100 mg Cry2Ae protein/gram of artificial soil. This concentration was intended to be approximately

10X the fresh weight concentration of Cry2Ae protein 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 by 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 LC50 was determined to be >100 mg Cry2Ae

protein/kg, and the NOAEC was ≥100 mg Cry2Ae protein/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 μL droplet of solution containing 50 μg/mL Cry1Ab protein, which was intended to 

represent >50X the exposure to the amount of Cry2Ae protein expressed in GHB119 cotton pollen 

(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 the larvae treated with Cry2Ae protein, 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 LC50 is >50 μg Cry2Ae protein/mL.

3. Soil Fate Studies for Cry1Ab and Cry2Ae PIPs

Soil organisms may be exposed to Cry1Ab 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 by soil-deposited pollen. Some evidence suggests that soils which are high in clays 

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 on 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 any 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 soilinhabiting bacteria, Bacillus thuringiensis, and found in commercial microbial insecticides (De Maagd et 

al., 2003 and Graser and Song, 2006), the degradation of Cry1Ab and Cry2Ae from Event T304-40 and 
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Event GHB119 cotton is expected to be similar to that of these proteins that are produced naturally from 

soil bacteria. Nonetheless, BPPD required the following 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 determine the DT50 (time for 50% of the 

initial concentration of bioactive material to dissipate) for Cry1Ab 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 Cry1Ab protein, and then sampled 

periodically over 45 days. These samples were used in sensitive insect bioassays. 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 

Cry1Ab protein in the soils was conducted to generate a dose-response curve to determine the 

degradation of Cry1Ab protein over time based on the bioactivity response. The DT50 was determined 

from the Cry1Ab protein concentration first-order exponential regression curve. The average DT50 for 

the three soils was 3.6 days. The soils were also analyzed for Cry1Ab protein content by enzyme linked 

immunosorbent assay (ELISA), which showed that Cry1Ab protein steadily degraded in soil during the 

study. 

A similar study was conducted to determine the DT50 for Cry2Ae protein in soil. Soils were collected 

from the same locations as above for the study with Cry1Ab protein. The soils were treated once with 50 

μg/g Cry2Ae protein and then sampled periodically over 45 days. A sensitive insect bioassay using the 

same species (Heliothis virescens) was conducted using identical methods as with Cry1Ab protein,

except that the concurrent six-day dose response study exposed H. virescens larvae to 0.056 to 3.4 μg/g 

Cry2Ae protein to generate a standard curve for determination of Cry2Ae protein concentration based on 

bioactivity response. Based on the bioassays, the average DT50 was determined to be 3.4 days. The soils 

were also analyzed for Cry2Ae protein content by ELISA, which showed that Cry2Ae protein steadily

degraded in soil during the study.

These studies utilized field soil spiked with purified insecticidal protein produced by bacteria. This 

approach is useful because dose responses can be easily quantified. However, the degradation and 

accumulation of Bt Cry proteins found within decaying plant tissue may behave differently 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 

Cry1Ab and Cry2Ae proteins 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 year soil fate studies for the currently registered Cry protein producing crops grown in a 

variety of soils and environmental conditions, as a condition of registration. More recently, a 

comprehensive review of all available scientific data on ecological effects of commercially grown GM 

crops over ten years was completed (Sanvido, et al. 2007). The review concluded “none of the 

laboratory or field studies suggest accumulation of Bt-toxins in soil over several years 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. Collectively, 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 
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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 Cry1Ab and Cry2Ae proteins in soil as 

demonstrated in the insect bioassays 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 proteins (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. 

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 unlikely to consume cotton plant material, 

particularly cotton seeds, due to high concentrations of gossypol, they could be exposed to Cry1Ab 

and/or Cry2Ae proteins 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 Cry1Ab and 

Cry2Ae proteins are not expected to cause adverse effects in birds. There is some uncertainty related to 

the exposure level in the testing with Cry2Ae protein compared to field exposures, but EPA previously

determined that the exposure level in the study was sufficient for avian testing. Testing at higher doses 

would reduce this uncertainty. 

 b. Wild mammals

Wild mammals may be exposed to Cry1Ab protein in Event T304-40 cotton or Cry2Ae protein 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 Cry1Ab or Cry2Ae proteins at doses of 2000 mg/kg 

bodyweight 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 majority of the cotton pollen that is produced is not expected to travel very far off-field. 
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BPPD has previously 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 Cry1Ab or 

Cry2Ae protein 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 Cry1Ab and Cry2Ae protein resulting from Event T304-40 or GHB119 cotton 

use, respectively, are not expected to reach marine/estuarine areas. BPPD concludes that adverse effects 

to fish and invertebrates in these environments are not expected. 

 e. Terrestrial and aquatic plant species

BPPD typically 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. 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 they may also consume pest insects that feed on cotton plant tissue or rarely 

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 decomposing plant tissue and plant 

exudates in soil. Studies submitted to the Agency with Cry1Ab and Cry2Ae proteins in lady bird beetle, 

green lacewing, collembola, and earthworm showed no adverse effects at concentrations well above 

those that would be encountered by 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 Cry1Ab and Cry2Ae proteins above those expected to be encountered in the 

environment. Thus, 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 Cry1Ab 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 adversely affect soil microorganisms (Sanvido et al., 2007). Although a minimal transient increase 

and shift in microbial populations may result from the presence of transgenic plant tissue in soil, no 

adverse effects have been attributed to the Cry protein. In addition, the soil degradation studies above 

show that the Cry1Ab and Cry2Ae proteins degrade quickly 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. 
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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If adverse effects are seen from this or any other research, the Agency will take appropriate action to 

mitigate potential risks (U.S. EPA 2010).

With regard to the impact of genetically 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 

genetically engineered, are also responsible for significant shifts in soil microbial communities. Most 

studies with genetically engineered crops to date have shown minor or no effects on soil microbes 

beyond the variation caused by the factors listed above. 

3. Horizontal Transfer of Transgenes from Bt Crops to Soil Organisms 

The EPA has evaluated the potential for horizontal gene transfer (HGT) from Bt crops to soil organisms 

and has considered possible risk implications if such a transfer were to occur. Genes that have been 

engineered into Bt crops are mostly found, or have their origin, in soil-inhabiting bacteria. Soil is also 

the habitat of other 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 

conducted to assess the likelihood of HGT, have been unable to detect gene transfer under typical 

environmental conditions. Horizontal gene transfer to soil organisms has only been detected with very

promiscuous microbes under laboratory conditions designed to favor transfer. 

As a result of these findings and the fact that the Bt toxins engineered into T304-40 and GHB119 were 

derived from soil-inhabiting bacteria, the EPA has concluded that the risk of HGT of transgenes found 

in Cry1Ab 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 weedy 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 hybridization from the crop to sexually compatible relatives. There are only four areas in 

the United States and its territories wherein cultivated cotton has the opportunity to outcross to wild or 

feral species, which are genetically 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 progeny when crossed with the tetraploids G. hirsutum or G. barbadense. In the 

very south of Florida, feral G. hirsutum exists in apparently self-sustaining populations. Since these 

would readily cross with cultivated cotton, sale of Bt-Cotton is restricted south of Interstate 60. There is 

currently no commercial cotton 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 places; hence, outcrossing is not 

currently 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 weedy relatives of cotton in the U.S., its 
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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possessions or territories, see the EPA Biopesticides Registration Action Document (BRAD) for the 

Bacillus thuringiensis (Bt) Plant-Incorporated 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 Cry1Ab 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 any 

measurable adverse effect on microbial populations in the soil, nor has horizontal transfer of genes from 

transgenic plants to soil bacteria been demonstrated. 

6. Impacts on Endangered Species 

Because of the selectivity of Cry1Ab and Cry2Ae proteins for lepidopteran species, endangered species 

concerns are also expressed for insect species in the order Lepidoptera. The Agency has previously 

determined that any potential concern regarding range overlap of endangered Lepidoptera with cotton 

production was mainly 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 hybridization 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 by competition with such entities.

Since Cry1Ab protein 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 Cry1Ab protein expressed in Event T304-40 cotton. 

Since Cry2Ae protein 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 direct and indirect effects to 

listed species of these taxa for Cry2Ae protein 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 wildlife, terrestrial 

invertebrates, fish and aquatic invertebrates, and plants and soil organisms. As described above, 

uncertainty related to testing with Cry2Ae protein in birds would be reduced by additional testing at 

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

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II. Environmental Risk Assessment for Cry1Ab and Cry2Ae proteins expressed in T304-40 X 

GHB119 (TwinLinkTM) Cotton Hybrid 

A. Background 

BCS developed T304-40 x GHB119 (TwinLinkTM) cotton by conventional breeding of transgenic Event

T304-40 cotton and transgenic Event GHB119 cotton, which express the Cry1Ab and Cry2Ae 

insecticidal proteins, respectively, for control of certain lepidopteran pests. Event T304-40 expresses a 

cry1Ab 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 (TwinLinkTM) cotton hybrid

Environmental effects data are available for studies conducted with Cry1Ab and Cry2Ae proteins. These 

can be used in an assessment of risk for TwinLinkTM cotton, provided there is no evidence of a 

synergistic association between Cry1Ab and Cry2Ae protein and protein expression levels of Cry1Ab 

and Cry2Ae are equivalent between the T304-40 and GHB119 parent lines and the TwinLinkTM hybrid. 

Data have been submitted with which to determine the potential for synergy and protein expression 

levels. Additional toxicity data have been submitted in which the combination of these proteins has been 

tested (TwinLinkTM plant material was used as the test substance) with some nontarget species, 

including honeybee, collembola, and broiler chicken. Additionally, 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 TwinLinkTM cotton are summarized in Table 

C.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 synergism between the 

Cry1Ab and Cry2Ae proteins are necessary for bridging, as well as data showing equivalent expression 

of the proteins within the TwinLinkTM plants and the individual parent lines. A synergism study was 

submitted and is discussed below. Studies showing equivalence were reviewed and found acceptable in 

the product characterization review for TwinLinkTM (BRAD Appendices A and B). BPPD concluded 

that the data from the expression of the proteins in the parent lines can be bridged to those expressed in 

the combined event.

Table C.3. Summary of environmental effects studies and waiver justifications for T304-40 x GHB119 

(TwinLinkTM) 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 

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 Cry1Ab and Cry2Ae

protein. 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

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 

47903101
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Data 

Requirement 

OPPTS

Guideline

Test Substance C.3. TwinLinkTM - Ecological Effects - Results 

Summary and Classification

MRID 

No. 

Colinus 

virginianus

Cry2Ae protein was performed)

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 near 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 survival or reproduction of Collembola.

Classification: Acceptable

47640010

Honeybee testing, 

Honeybee larvae,

Apis mellifera

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 

pollen and observed for survival up to the time of 

emergence. 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. 

Classification: Acceptable

47640009

Synergism N/A Sensitive insect dose-response bioassays with T304-40, 

GHB119, and TwinLinkTM plant material showed that 

Cry1Ab and Cry2Ae proteins expressed in TwinLinkTM 

plants are not synergistic. 

Classification: Acceptable

48480009

1. Nontarget Organism Study Summaries for TwinLinkTM

 a. Avian Wildlife

Avian Dietary Toxicity

The broiler chicken feeding study (MRID 47640011) discussed above in the assessment for Events 

T304-40 and GHB119 was also submitted to provide data on toxicity for TwinLinkTM. As stated above, 

since there is no assurance that the test animals were exposed to Cry1Ab or Cry2Ae proteins in the diet 

throughout the study, the study cannot be used for avian risk assessment.

Avian Oral Toxicity 

Rationale was presented to justify waiving the avian oral toxicity data requirement for the Cry1Ab 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 Cry1Ab and Cry2Ae proteins

in TwinLinkTM 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 currently registered Cry proteins, including Cry1Ab, do not pose hazards to birds (Mendelsohn et al. 
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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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 synergism and protein 

expression equivalence that supports acceptability of bridging these data to TwinLinkTM. 

Avian Inhalation Toxicity

Avian inhalation toxicity testing is typically not required for Bt cotton 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 is acceptable. An avian inhalation test is not required.

 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 Cry1Ab protein in T304-40 and 

Cry2Ae protein 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 synergism and protein expression equivalence below. Protein 

expression is shown to be equivalent between TwinLinkTM cotton and the T304-40 and GHB119 parent 

lines. Consequently, bridging these data to TwinLinkTM is acceptable. 

c. Freshwater animals

Data waiver rationale (MRID 47871603) was submitted to fulfill the data requirements for freshwater 

fish for Cry1Ab and Cry2Ae proteins in TwinLinkTM. 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 consistent with BPPD’s conclusion. 

Toxicity data with Daphnia are available for both Cry1Ab and Cry2Ae proteins for use in the 

TwinLinkTM risk assessment. Results of these studies are presented above. 

d. Estuarine and marine animals

The Agency typically waives 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 the 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 typically 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 47871603) that was consistent with the Agency’s position on 

nontarget plant risks to Bt -endotoxin. 
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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 f. Invertebrate species 

A 28-day study with collembola (Folsomia candida) was conducted to determine the effects of 

TwinLinkTM plant tissue on this soil invertebrate (MRID 47640010). Collembola (Folsomia candida) 

were provided diet containing nominal concentrations of 5.0, 20, or 50% transgenic lyophilized 

TwinLinkTM cotton leaf. The test also included a negative control group of collembola fed diet without 

lyophilized TwinLinkTM cotton leaf, a control substance group fed diet containing 50% lyophilized 

nontransgenic Coker 315 (near isoline) cotton leaf, and a positive control group fed diet containing 

thiodicarb. Mortality in the 5%, 20%, and 50% TwinLinkTM test groups was 12.5%, 10.0%, and 7.5%, 

respectively, while cumulative number of offspring per female were 42.2, 41.7, and 33.4, respectively. 

Mortality in the negative control and Coker 315 groups was 2.5% and 5.0%, respectively, and 

cumulative number of offspring per female was 26.9 and 38.6, respectively. Mortality 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 TwinLinkTM pollen when 

administered to 2-3 day old honey bee larvae (Apis mellifera, MRID 47640009). Honey bee larval cells 

were treated with 10 μL of 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 typically 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 TwinLinkTM; 97.5% for the Coker 315; and 0% for the 

reference substance). Based on statistical analysis, there were no significant differences in survival 

between the TwinLinkTM treated larvae and those treated with Coker 315 pollen.

Additionally, bridging criteria are satisfied. Consequently, data developed for the Cry1Ab and Cry2Ae 

proteins expressed individually in Event T304-40 and Event GHB119 cotton plants, respectively, can 

also be used for the ecological risk assessment for TwinLinkTM.

g. Supplemental Information

BCS submitted a summary of nontarget organism testing with Cry1Ab and Cry2Ae proteins (MRIDs 

47640008), which summarized the toxicity information and data waiver rationale for these proteins and 

TwinLinkTM 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 Cry1Ab and Cry2Ae proteins expressed in TwinLinkTM cotton plants exhibit 

synergistic toxicity. Leaf material from the single event cotton plants expressing either Cry1Ab or 

Cry2Ae protein and the combined event (TwinLinkTM) cotton plants were fed to the larvae at multiple 

concentrations determine the LD50s for the proteins within each plant. Expression of each protein was 

determined to be equivalent between the TwinLinkTM plants and the individual parent lines. Based on 
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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the predicted and actual LD50s for the proteins in each type of plant, a conclusion of no synergism was 

made. The results of toxicity tests with each individual protein are assumed to adequately represent the 

toxicity of these proteins when expressed in TwinLinkTM cotton, based on the equivalent protein 

expression. 

3. Protein Expression Comparability

Data have been submitted to the Agency showing the proteins expression of Cry1Ab in Event T304-40 

cotton, Cry2Ae in Event GHB119 cotton, and both proteins in TwinLinkTM cotton. Additional data have 

been submitted to determine whether the relative potency of plant-produced Cry1Ab and Cry2Ae

proteins is similar to the purified bacterially-produced proteins used in the toxicity testing with each 

individual protein. These data show that each event produced comparable protein expression levels of 

Cry1Ab and Cry2Ae in each respective parent line as well as the T304-40 x GHB119 hybrid, and that 

the relative potency of plant-produced and bacteria-produced Cry1Ab and Cry2Ae proteins are 

equivalent (see Appendices A and B). Consequently, bridging from tests for the individual events is 

appropriate for support of the T304-40 x GHB119 (TwinLinkTM) registration. 

C. CONCLUSION

As long as BCS shows that Cry1Ab 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 TwinLinkTM cotton. This 

conclusion is based on the environmental risk assessments for Cry1Ab and Cry2Ae proteins expressed 

individually in Event T304-40 and GHB119 cotton, as well as additional data developed with 

TwinLinkTM cotton plant material. 

Both Cry1Ab and Cry2Ae proteins have not been shown to have toxic effects on birds, mammals, 

freshwater invertebrates, terrestrial nontarget invertebrates, and plants, and because exposure is not 

anticipated in aquatic environments. Bioequivalence of protein expression has been discussed in 

Appendices A and B. Thus, the Agency makes a “No Effect” determination for direct and indirect 

effects to listed species of these taxa for these proteins as expressed in TwinLinkTM 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., McCreedy, C., Regetz, J. & Kareiva, P. 2007. A meta-analysis of effects of Bt

cotton and maize on nontarget invertebrates. Science 316: 1475–1477.

National Academy 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 metropolitan area); http://www.nap.edu.

Romeis, J., M. Meissle, and F. Bigler. 2006. Transgenic Crops expressing Bacillus 
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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thuringiensis toxins and biological control. Nature Biotechnology 24: 63-71. 

Rosi-Marshall E. J., J. L. Tank, T. V. Royer, M. R. Whiles, M. Evans-White, C. Chambers, N. A. 

Griffiths, J. Pokelsek, and M. L. Stephen. (2007). Toxins in transgenic crop byproducts may

affect headwater stream ecosystems. PNAS: 104(41): 16204–16208.

Sanvido,O., Romeis, J., Bigler, F. 2007. Ecological Impacts of Genetically 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. 

U.S. EPA. 1998. “Guidelines for Ecological Risk Assessment.” EPA 630/R-95-002F. 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/scipoly/sap/1999/index.htm#december

U.S. EPA. 2001a. SAP Report No. 2000-07. Sets of scientific issues being considered by the 

Environmental Protection Agency regarding: Bt plant-pesticides risk and benefit assessments. 

Dated March 12, 2001. Web site: http://www.epa.gov/scipoly/sap/2000/october/octoberfinal.pdf

U.S. EPA. 2001b. Biopesticides Registration Action Document: Bacillus thuringiensis (Bt) 

Incorporated 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 by the 

Environmental Protection Agency regarding: Corn rootworm plant-incorporated protectant 

nontarget 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/meetings/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 Cry1F (synpro) and Cry1Ac (synpro) 

Construct 281/3006 Insecticidal Crystal Proteins as Expressed in Cotton. 

http://www.epa.gov/oppbppd1/biopesticides/ingredients/tech_docs/brad_006512.pdf
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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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 Document – Bacillus thuringiensis modified 

Cry1Ab (SYN-IR67B-1) and Vip3Aa19 (SYN-IR102-7) 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.pdf

U.S. EPA, 2010. Biopesticides Registration Action Document – Cry1Ab and Cry1F Bacillus 

thuringiensis (Bt) Corn Plant-Incorporated Protectants, 

http://www.epa.gov/oppbppd1/biopesticides/pips/cry1f-cry1ab-brad.pdf.

U.S. EPA. 2010. Review of Product Characterization Data in support of Sec. 3 Registration of PlantIncorporated Protectant (PIP) cotton Events: GHB119 cotton expressing Bacillus thuringiensis 

Cry2Ae protein (EPA File Symbol 264-RNOU), T304-40 cotton expressing Bt CrylAb (264-

RNOL); and TwinLinkTM cotton (EPA File Symbol EPA File Symbol 264-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. U.S. EPA. Washington DC.

U.S. EPA. 2011. Review of Product Characterization and Human Health Data in support of Sec. 3 

Registration of Plant-Incorporated Protectant (PIP) Event T304-40 cotton expressing Bacillus 

thuringiensis Cry1Ab insecticidal protein (EPA File Symbol 264-RNOU; OECD Unique 

Identifier: 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.
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APPENDIX D: Insect Resistance Management

Cry1Ab, Cry2Ae proteins and their combination (Cry1Ab x Cry2Ae) in TwinLinkTM

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 BPPD17 review of BCS’ Insect Resistance Management (IRM) plan and supporting data for 

the application to register TwinLinkTM cotton PIP as well as supporting documentation for a Natural 

Refuge request (U.S. EPA memorandum 04/22/10; EPA File Symbol 264-RNOU, MRIDs 4760014 and 

47634823). Deficiencies identified in this application for full commercial registration TwinLinkTM

cotton 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 assays together (using verification 

method 1) suggest that TwinLinkTM 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 assays (using verification method 1) together 

suggest that Events GHB119 and T304-40 may express a high-dose and TwinLinkTM 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 by 

Ellsworth and Li (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 by BCS. 

 

17 The use of BPPD in this review refers to the BPPD IRM team consisting of Jeannette Martinez and Alan Reynolds.
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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 NonHigh 

Dose

Possible 

HighDose

Possible 

Effective 

High-Dose

Inconcl.

*

Inconcl.* Inconcl.* -- -- --

TBW Possible 

Highdose

HighDoseEffective 

High-Dose -- -- -- -- -- --

PBW

-- -- --

Inconcl.

*

Inconcl.* Inconcl.* --- Inconcl.* Inconcl.*

* Insufficient information was submitted to the Agency to make a dose determination 

** Cry1Ab protein could not be tested in this experiment because the lab strain was resistant to Cry1Ab. 

2. PROTEIN EXPRESSION

a. BPPD has reviewed BCS’ 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, TwinLinkTM. Event T304-40 expressed higher levels of toxin in leaves of single event 

plants 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 differences observed. 

b. Expression of GHB119 in leaves from early to late stage appeared somewhat variable or 

unstable. Expression levels were measured in leaves in 2004 and 2005 at three developmental 

stages (early 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 early, 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 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 by 

BCS for T304-40 and GHB119 in different plant tissues. It appears that the same tissue types 

were lumped together for plants expressing single and pyramided events. Since there appears to 

be unequal expression of the proteins in plants expressing either the single or pyramided 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 separated by tissue type for each individual

Event, GHB119, T304-40, and TwinLinkTM, rather than lumped together for the same protein.

d. BPPD recommends that BCS provide more specific and explanatory information together with 

their protein expression results in future data submissions for PIP registrations. 
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3. CROSS-RESISTANCE

a. BPPD concludes that based on BCS’s data there is little concern for cross-resistance between 

Cry1Ab and Cry2Ae proteins as expressed in TwinLinkTM.

b. BPPD notes that should resistance evolve in CBW and TBW to Cry2Ae protein (as expressed in 

TwinLinkTM cotton) that Cry2Ab2 protein (as in Bollgard II cotton) may be compromised as well 

because, as BCS stated in their report, there is a great potential for cross-resistance between the 

two toxins. 

c. BPPD notes that the cross-resistance potential is great between Cry1Ab, Cry1Ac, and Cry1F

(although somewhat less for Cry1F) proteins based on the competition assays; the three toxins 

share high affinity binding sites. Should CBW evolve resistance to Cry1Ab protein, other PIP 

products expressing Cry1Ac and Cry1F proteins 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 analysis, the lack of including Cry1Ab corn 

in the modeling landscape, the lack of establishing 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 commercially and serve only for producing the combination PIP 

TwinLinkTM. For any other combinations, BCS may need to apply 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 previously granted on the basis that the toxins were expressed at a high-dose against TBW. 

A second dose TwinLinkTM-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 TwinLinkTM 

cotton. 

e. If a Natural Refuge strategy 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 in 
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the following Texas counties: Brewster, Crane, Crockett, Culberson, El Paso, Hudspeth, Jeff 

Davis, Loving, Pecos, Presidio, Reeves, Terrell, Val Verde, Ward, and Winkler. 

f. For those States and regions requiring a non-Bt refuge, the following refuge planting 

requirements should apply:

i. 5% External, Unsprayed Refuge

ii. 20% External Sprayed 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 TwinLinkTM is commercialized before 2012. Baseline susceptibility

data should be available by the first growing season of commercial use of the PIP product. BPPD 

recommends that BCS accelerate the development of these data. 

b. The applicant proposed to monitor for resistance evolution in all three target pests (CBW, TBW, 

and PBW) in areas of high TwinLinkTM adoption. BPPD agrees with this proposal but also 

recommends that monitoring efforts additionally focus on regions in states with little natural 

refuge.

c. BCS 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 

TwinLinkTM. 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 seriously and followed up immediately to conclusively 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 TwinLinkTM cotton. 

b. It is recommended that BCS develop a grower guide that at a minimum describes the technology

and includes unexpected damage guidelines.
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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 

anonymous 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 were 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:

i) Baseline and discriminating dose work irrespective of their status;

ii) Sales 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 by BPPD until BCS clarifies how 

mortalities were calculated for the experiments using high-dose verification methods (3) and (4).

Deficiency 2: In the modeling analysis provided by BCS, only CBW was modeled but not 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 highdose 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 highdose 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 analysis be conducted using somewhat lower mortality assumptions based on the study results 

reported by BCS. The bioassay experiments showed that Cry1Ab protein 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 
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is no evidence that the Cry1Ab PIP expresses high-dose against CBW. BCS concluded, too, that 

TwinLinkTM Cotton expressed less than high-dose against CBW due to the target pest being less 

susceptible to Cry1Ab and lower level of expression of Cry1Ab toxin in the parental Event for 

TwinLinkTM Cotton. 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 Cry1Ab (0.0002). Although no field resistance to Cry1Ab has been documented yet 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 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 expected. BPPD recommends that BCS consider modeling a most likely resistance 

allele frequency of 0.001 for Cry1Ab in CBW.

Deficiency 6: BCS needs to include Cry1Ab corn and Cry1Ab cotton in the modeling landscape when 

assessing the risk of resistance evolution in the target pests because Syngenta has registered Bt cotton 

and Bt corn and Monsanto has registered Bt corn products expressing Cry1Ab. These corn and cotton 

PIPs are grown in the Cotton Belt and need to be included in a simulation analysis for TwinLinkTM.

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 

TwinLinkTM. 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. 
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I. BACKGROUND – Insect Resistance Management

TwinLinkTM cotton is a pyramided transgenic cotton trait that expresses Cry1Ab (Event T304-40), 

Cry2Ae (Event GHB119), and a glufosinate ammonium (herbicide) tolerant active ingredient based on 

LibertyLink technology. TwinLinkTM 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 

evaluations with artificially and naturally 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 minimal damage to TwinLinkTM Cotton 

(expressing Cry2Ae and Cry1Ab proteins) during the growing seasons of 2004 and 2005.

Cry1 and Cry2 are effective lepidopteran toxins and share less than 20% amino acid homology. 

Crickmore et al. (1998) constructed a phylogenetic tree using amino acid sequence identity as homology

criteria for all presently known Bt crystal proteins. This tree indicates that Cry1Ab and Cry2Ae proteins 

are genetically 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 Cry1F, Cry1Ab, Cry1Ac, Cry2Ab2, and Vip3Aa19. Pyramids of Cry1F and Cry1Ac, Cry1Ac 

and Cry2Ab2, and Cry1Ab and Vip3A19 are expressed in WideStrike cotton (product of Dow 

AgroSciences), Bollgard II cotton (product of Monsanto), and VipCot (product of Syngenta), 

respectively. All pyramided cotton PIP are effective against 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 

armyworm (Spodoptera frugiperda, FAW) and Beet armyworm (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 study results (MRID 47640014, Vol. 15), which are reported below.

1. Cotton Bollworm (Taken from MRID 47640014)

CBW is highly mobile and can move long distances. The general pattern of CBW migration 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 by Gould et al. (2002): they used 

13C/12C stable isotope assessment of alternative host used by CBW and showed indirectly that late 

season CBW moths captured in Louisiana and Texas could be migrants from the northern Corn Belt. 

Typically, greater than 50% of the moths captured in August through October 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 common wild C4 plants. Cotton, along with wheat, rice, barley and other ancient plants are 

C3 plants, easily differentiated from C4 plants.
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Directly 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 greatly across years; in non-Bt plots densities ranged from 0-67 pupae/ha for 

CBW. Lower densities of CBW pupae (0-214 pupae/ha) were found over the seven-year study in Bt 

cotton fields. Only partial control of CBW was possible with these first generation single gene Bt cotton 

plants expressing only the Cry1Ac protein.

Local temporal and spatial dispersal in CBW (Han and Caprio, 2002) were studied by using 

polymorphic allozyme 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 highly 

polyphagous 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 was 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 

typically captured by 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 4th and 5th 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 significantly reduce larval numbers, but damaged bolls and adults were 

significantly 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 significantly lower levels, only 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 producer of CBW larvae in 

the early 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 originally expected. Beginning in midAugust, soybean and conventional cotton became the major crop sources for CBW production. Pupae 

and adult production followed the same trends. 
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Kurtz et al. (2004) used these estimates (Jackson et al., 2003) and unpublished data by Jackson in their 

computer simulation model. For the first two CBW generations, 90% of the larvae developed on corn, 

5% on cotton and 5% on soybean. 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 soybean. Storer et al. (2001) confirmed these results, finding that CBW adults 

emerging from ear-stage cornfields infest cotton, soybean and other crops In North Carolina. The level 

of adult CBW production was greatly decreased (~80%) 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: approximately 32,000 

larvae/acre in 2002 and approximately 12,000 larvae/acre in 2003; field corn: approximately 20,000 

larvae/acre in 2002 and approximately10,000 larvae/acre in 2003. Larval densities on soybean and on 

non-transgenic and Bt cotton were much lower (<3000 larvae/acre). Larval populations in corn, 

sorghum, maturity group 6 soybean, cotton and Bt cotton peaked on 25 June, 12-14 July, 2-4 August, 2-

4 August, and 11-16 August, respectively. The only crop that exhibited similar temporal synchrony with 

bollworm larvae on Bt cotton was soybean maturity group 6, indicating the possibility of including it in 

an IRM strategy. A more extensive study over two years and over five states demonstrated again that 

non-cotton crops, such as corn, grain sorghum, peanut and soybean, 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 

frequently 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 rarely varied among interfaces, except for highly 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 in sorghum trap crop than in cotton trap crop for two years.

Larval development studies in the lab of CBW on sorghum, corn, soybean and cotton showed a survival 

of 73, 55, 26, and 13%, respectively and a mean development time to pupation of 15.6, 12.4, 18.4, and 

25 days, 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 year period in 

Mississippi and Mexico, garbanzo bean, an important 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. Low densities of adult CBW moths (0-275 

ha/yr) emerged from plots and were not significantly different between the three crops. Cover crops, 

such as legume mixture, crimson and rye seem to be more preferred by heliothines than the cotton crop. 

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). 

Natural mortality of CBW was studied from 1991-95 in short season cotton in Texas with one 

generation per year (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 by Trichogramma egg parasitation, larval parasitation rarely exceeded 5%, but numerically

the most import mortality factor was egg consumption of around 77% by Orius insidiosus (minute pirate 

bug). 
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2. Tobacco Budworm (Taken from MRID 47640014)

TBW is a highly mobile insect that becomes less mobile in the mid to late summer when it prefers 

cotton as a host plant. Directly related to movement is the overwintering of the insect. Recently, 

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 across years: in non-Bt plots 

densities ranged from 0-303 pupae/ha TBW. No TBW pupae were found over the seven-year 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 typically 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 polymorphic 

allozyme and RAPID-PCR makers. TBW populations overall showed little special differentiation, 

indicating a highly effective gene flow rate over the spatial 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, cotton 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 determine the extent of movement 

reduction.

In the Texas High Plains TBW adult populations were monitored weekly from March to November 

between 1982 and 1995 using pheromone traps at Lubbock and Halfway Texas, prior to the introduction 

of Bt cotton (Parajulee et al., 2004). The bollworm/budworm complex consisted of 98% CBW and 2% 

TBW. For weekly moth abundance, a positive correlation was shown with weekly temperature, degreedays and weekly average precipitation, and a negative correlation was shown with the weekly wind 

velocity. Data from this study indicate that the late-season moth catches could be indicative of the 

dynamics of the early-season catches the following year. 

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 TBW were 

typically captured by April 10th. From 1986-1996, the mean number of TBW moths captured per trap 

and per night was 19.5 with a maximum of 500. Since 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 Southeastern US, the first TBW generation develops almost exclusively on weeds; 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 consistently on C3 plants (examples of C3 plants are cotton, soybean, clover, and peanut). 

Unfortunately 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 density of 287,000 larvae/ha, 

approximately 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 adults/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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Cover crops, such as legume mixtures, crimson, and rye, 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 47640014, 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 reached a peak after 3-5 days 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. Liu 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 negatively 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 indication 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 accurately 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 of the planting date could be a good cultural control, but shortening the 

growing season can reduce yield.

Most larval stages of PBW are relatively protected from natural enemies because they feed on seeds 

inside the cotton boll; eggs laid externally on vegetative 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 predators 

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

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III. DOSE

TwinLinkTM cotton is a combined trait product made through traditional breeding of Events T304-40 

and GHB119 which express Cry1Ab and Cry2Ae proteins, respectively. BCS submitted 1) protein 

expression data for different cotton tissues; 2) LC50 data for Cry1Ab and Cry2Ae proteins and their 

target pests; and 3) dose information for Event T304-40 (Cry1Ab), Event GHB119 (Cry2Ae), and 

TwinLinkTM Cotton 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 

assays in 2004 and 2005. Bayer determined that protein expression in Cry1Ab and Cry2Ae cotton as 

well as TwinLinkTM cotton plants depended on the type as well as maturity of tissues assayed (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). Cry1Ab protein, as expressed in TwinLinkTM, had consistent expression in leaves and squares 

with higher expression in bolls and flowers. Cry2Ae protein, as expressed in TwinLinkTM had relatively

high expression levels in leaves across the growing season and in late bolls (data from 2005). Expression 

was typically higher in Cry2Ae than Cry1Ab PIP (up to 10X). Cotton plants expressing the single events 

were analyzed 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 

Cry1Ab 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 47640014)
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 98 of 153

Table D.1. Expression of Cry1Ab and Cry2Ae proteins in plant tissues

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

(Extracted from MRID 47640014)

b) LC50 Data for Cry1Ab and Cry2Ae proteins in CBW and TBW

BCS provided results from insect bioassays using purified individual Bt proteins in a surface layer diet 

for the target pests of TwinLinkTM cotton. Table 2 shows the mean LC50 values and ranges for six target 

pests. BCS described the insect susceptibility for Cry1Ab protein as follows: TBW > PBW > Old World 

BW (OWBW, Helicoverpa amigera) >> CBW. The susceptibility for Cry2Ae protein was described as: 

TBW > PBW >> CBW > BAW > FAW > Old World BW.

Table D. 2. Mean LC50 values and ranges for Cry1Ab and Cry2Ae proteins

INSECTS Cry1Ab 

(ng/cm2)

Cry2Ae 

(ng/cm2)

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)

(Extracted from MRID 47640014)

c) Dose Determination

The 1998 SAP Subpanel discussed ways 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 heterozygote might develop with higher than 25-fold resistance. The major problem identified by
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 99 of 153

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 ways to assess this 25-fold level, but that some 

approaches were more appropriate for specific crop pests. The Subpanel concluded that a cultivar could 

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 Lepidopteran systems are:

(1) Serial dilution bioassay with artificial diet containing lyophilized tissues of Bt plants (tissue from 

non-Bt plants serving as controls);

(2) Bioassays using plant lines with expression levels approximately 25-fold lower than the commercial 

cultivar (determined by quantitative ELISA 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 LD99.99 or higher to assure that 95% of heterozygotes 

would probably be killed. With this approach Bt sweet corn hybrids 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 phenotypes can be 

estimated as the ratio of density of larvae/plant in Bt sweet corn to density 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 LD50 value similar to field strains; and

(5) Determine if an older instar of the targeted pest could be found with an LD50 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 TwinLinkTM and its single events. 

 Method (1) utilized lyophilized plant tissue from Events T304-40, GHB119, and TwinLinkTM 

mixed with artificial diet to produce 10X and 25X concentrations. The bioassays were conducted 

as diet incorporated assays and diet overlay assays with TBW and CBW. 

 Method (4) involved controlled infestations of TwinLinkTM 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 (Cry1Ac) of PBW with which young 

cotton bolls were infested.

High-Dose Verification Method (1):

1. Cotton Bollworm

Lyophilized 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. Twenty-four neonate larvae were exposed at each treatment level (see 

Table D.3); two replicates per treatment were carried out. Mortality was scored at days 4, 7, 12, 17, 19, 

21, and 24, and average mortality was calculated from the two replicates.
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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For overlay assays, the same leaf material was used and the lyophilized powder was mixed in a 0.2% 

agar solution. The concentrations tested were 4% and 6.9% of Cry-protein. Liquid diet was poured into 

each well and allowed to dry. Different concentrations were prepared (see Table D.4) and overlaid onto 

the solidified diet. Mortality was scored at day 3, 7, 11, 15, 18, and 21. 

The applicant reported that control mortality was difficult to manage using cotton leaf tissue due to the 

presence of gossypol. Diet incorporated assays had higher control mortality (17.5-29.5% on day 17) than 

overlay assays (16.8-19% on day 15); hence overlay assays were 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, and lower level of expression of, Cry1Ab protein in the parental 

event for TwinLink.

Table D.3. CBW mortality to Cry1Ab, Cry2Ae, and TwinLinkTM Cotton - 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

TwinLinkTM

10 44 85 100

4 14.8 55.5 70 94 97 100

Modified from MRID 47640014

Table D.4. CBW mortality to Cry1Ab, Cry2Ae, and TwinLinkTM - overlay assay method

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 prepupae

4 0 8 17 19 All pupae or prepupae

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

TwinLinkTM

6.9 27.5 75 100

4 29 80 97.5 100

Modified from MRID 47640014

2. Tobacco Budworm

As for TBW, two types of assays 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.). 
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 101 of 153

However, concentrations for the negative control of diet bioassays 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 assays, mortality was scored at day 3, 7, 11, and 15.

As for CBW bioassays, control mortality for TBW was high and difficult to control. The applicant

attributed this to the presence of gossypol in the leaf diet. BCS reported that control mortality was lower 

in overlay assays and that therefore those results were best for verifying TwinLinkTM 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 mortality 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

TwinLinkTM 4 100

Modified from MRID 47640014

* Unclear what source of food constituted negative treatment.

Table D.6. TBW mortality to Cry1Ab, Cry2Ae, and TwinLinkTM - overlay assay method 

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

TwinLinkTM 4 71 100

Modified from MRID 47640014

High-Dose Verification Method (4):

1. Cotton Bollworm

Coker312, TwinLinkTM, 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 conducted 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 by 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 analyzed for presence of larvae. In 2008, a total of 6,600 eggs/plot were applied and each boll was 

dissected subsequently to determine presence of larvae. Percent mortality was calculated for 2007 (Table 

D.7). The average number of boll examined in each plot was recorded and percent mortality calculated 

in 2008 (Table D.8).
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Page 102 of 153

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 TwinLinkTM plot had slightly less CBW mortality (98.5%).

Table D.7. CBW mortality on artificially infested plots during 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

TwinLinkTM 6000 2 99.97

* 50 plants/60-plant row were scouted = 83.3%

Modified from MRID 47640014

Table D.8. CBW mortality 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

TwinLinkTM 19800 32279 7 0.89 309.01 98.26

* 6,600 eggs were applied per plot; 1 plot = 1 location

Modified from MRID 47640014

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 TwinLinkTM, each single event, and Coker312 as control. Each plot was six rows long (30-ft) and 

separated from other blocks by one empty row. Young bolls were infested with 100 eggs each from a 

susceptible PBW strain maintained by USDA-ARS (Maricopa, AZ). Six to eight days later tagged bolls 

were brought into the lab for dissection. Attack rates on bolls were evaluated by counting the number of 

entry holes on the exterior of the boll carpel. PBW survival was estimated 6-15 days later which 

provided enough time for larvae to develop to 3rd instar. Survival was determined based on the number 

of exit holes, 3rd, live 4th, and pupae. Mortality (synonymously used with efficacy) was estimated by 

dividing the number of surviving PBW by the number of entry holes and by correcting for control 

mortality. Mortality in the Coker line was determined by limiting examinations to mainly those bolls 

with < 20 entry holes in order to avoid density-dependent mortality in control treatments. 

BCS concluded that all three infestations with Cry1Ab and TwinLinkTM Cotton 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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Table D.9. Mortality results of susceptible PBW following artificial infestation of cotton plants

Infestation/ 

Data 

collection

Treatment Entry 

Holes

Live 

Large 

Larvae

Average 

Mortality 

(%)

Lower 

95%

Mean

Upper 

95% Mean

Corrected 

Mortality 

(%)

July

Coker

Cry1Ab

Cry2Ae

TwinLinkTM

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

TwinLinkTM

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

TwinLinkTM

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 47640014

Table D.10. Pooled mortality results of susceptible PBW following artificial infestation of cotton plants

Treatment Total 

Entries

N 

Bolls

Live 

Large 

Larvae

Apparent 

Mortality

Lower 

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

TwinLinkTM 6680 302 2 99.9 99.9 100 99.9

Modified from MRID 47640014

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 Cry1Ac-resistant PBW strain 

(AZP-R) was obtained from Dr. Bruce Tabashnik at the University of Arizona. That strain was resistant 

to Cry1Ac with 53% survival in response to 10μg of Cry1Ac powder/g of diet and demonstrated crossresistance to Cry1Aa and Cry1Ab. The artificial infestation experiment was run in the lab with field 

collected plant tissue of Cockerline (control), TwinLinkTM, Cry1Ab, and Cry2Ae plant tissue.

Young cotton bolls were harvested (15-19 days old) after tagging. Each boll was maintained in a water 

pick bioassay system. 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 estimates, 

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 assayed. The mean entry holes ranged from 6.2-28.5. Attack rates were 

much higher in some cases. Maximum number of entry holes on a boll were 31 (Coker), 48 (Cry1Ab), 

48 (Cry2Ae), and 54 (TwinLinkTM Cotton). The mean percentage of PBW recovered ranged from 80.1 

to 94.5%, which was similar to assay results with susceptible PBW. The criterion for ‘survival’ at the 
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document January 23, 2012

Page 104 of 153

time of boll dissection was a ‘living >3

rd instar larva’. Mortality was calculated by dividing the number 

of surviving PBW by the number of entry holes as well as by correcting for control mortality. JMP IN 

Statistical Discovery Software, Version 5.1, SAS Institute 2005 was used for the data analyses.

In two out of three infestations, Cry2Ae demonstrated 100% efficacy; TwinLinkTM Cotton demonstrated 

100% efficacy in all three infestations. Cry1Ab treatments were less effective in the first and second 

infestation and demonstrated slightly better efficacy in the third infestation. However, corrected 

mortalities for Cry1Ab 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 

significantly different from 100%. The reported cross-resistance of AZP-R to Cry1Ab toxin was 

consistent with the findings in this study. No reported cross-resistance to Cry2Ae was apparent. The 

authors concluded that Cry2Ae and TwinLinkTM Cotton expressed a high-dose against PBW.

The authors stated that PBW survival on Bt plants (TwinLinkTM and Cry2Ae Cotton) could have been 

higher because 1st instar larvae might have been exposed to hemizygous rather than purely 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 TwinLinkTM and Cry2Ae plants.

Table D.11. Mortality results for Cry1Ac resistant PBW following artificial infestation of cotton plants

Infestation/ 

Data 

collection

Treatment Entry 

Holes

Live 

Large 

Larvae

Apparent

Mortality 

(%)

Lower 

95%

Mean

Upper 

95% Mean

Corrected 

Mortality 

(%)

July

Coker

Cry1Ab

Cry2Ae

TwinLinkTM

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

TwinLinkTM

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

TwinLinkTM

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 47640014

Table D.1. Pooled mortality results for Cry1Ac resistant PBW following artificial infestation of cotton plants

Treatment Total 

Entries

N 

Bolls

Live 

Large 

Larvae

Apparent 

Mortality

Lower 

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

TwinLinkTM 1364 112 0 100 100 100 100

Modified from MRID 47640014
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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IV. CROSS-RESISTANCE POTENTIAL (MRID 47640014)

BCS referred to a series of binding studies (Karim et al., 2000; Luo et al., 2007; Hernandez-Rodriguez et 

al., 2008) to support that Cry1 and Cry2 proteins bind to different target sites in the larval midgut of 

Lepidoptera pests. In addition, work by Hernandez-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 labeled Cry1Ab and Cry2Ae proteins using BBMV of TBW and CBW to 

confirm that these proteins bind to the target pests’ midgut with specificity. Furthermore, Dr. JuratFuentes conducted a study with Cry1F, Cry1Ab, Cry1Ac, Cry2Ab, and Cry2Ae to fully characterize 

competitive binding aspects between the two newly proposed Bt toxins for cotton and those toxins 

already found in already 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), Cry1Ab and Cry1Ac bound 

specifically and with high affinity to BBMV of PBW, while Cry1Aa and Cry2Aa bound with less 

specificity and lower affinity yet showed high binding site concentration. BCS stated that these results 

confirmed that Cry1A 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 Membrane Vesicles

The BBMV experimental results showed that labeled Cry1Ab (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 

MRID47640014). 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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2. Binding Experiments with Cry1Ab and Cry2Ae using TBW Brush Border Membrane Vesicles

The BBMV experimental results showed that labeled Cry1Ab (see Figure 3) bound with specificity to 

the midgut of TBW. The results for Cry2Ae are not shown here.

Figure D.3. BBMV binding assay with increasing TBW BBMV concentrations and fixed radio-labeled Cry1Ab (from 

MRID47640014)

3. Competition Experiments between Cry1Ab, Cry1Ac, Cry1F, Cry2Ab, and Cry2Ae using CBW 

Brush Border Membrane Vesicle

A competitive binding experiment with Cry1Ab, Cry1Ac, Cry1F, Cry2Ab2, and Cry2Ae showed that 

Cry1Ab shared high affinity binding sites with Cry1Ac and Cry1F in CBW. However, Cry1Ab did not 

compete with Cry2Ab and Cry2Ae suggesting that these proteins do not share binding sites in CBW 

(See Figure 4).

Figure D.4. Competition binding of Cry1Ab and competitors to BBMV of CBW (from MRID 47640014); 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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4. Competition Experiments between Cry1Ab and Cry2Ae using TBW Brush Border Membrane 

Vesicles

Results of a binding competition experiment between Cry1Ab and Cry2Ae using TBW BBMV showed 

that the two toxins did not share binding sites (Figure 5).

Figure D.5. Competition assay with Cry1Ab and Cry2Ae using BBMV of TBW

In conclusion, BCS stated that the relative potential for cross resistance was low between variants of 

Cry1 and Cry2 toxins as expressed in Bt cotton. Yet, the potential for cross-resistance was high within 

the group of Cry1 variants (e.g. Cry1Ab, Cry1Ac, and Cry1F) as well as within the group of Cry2 

variants (e.g. Cry2Ab and Cry2Ae) (see Table below).

Table D.2. BCS’ estimated 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 47640014 

+++, ++ = high potential; + = low potential; - = little to no potential

V. MODELING 

BCS commissioned Dr. Mike Caprio (Mississippi State University) to conduct a risk assessment for 

TwinLinkTM 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 (type 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, soybeans, 

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

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cotton, and back to fallow field. Two Bt traits were modeled, Cry1Ab was assumed to have complete 

cross-resistance to Cry1Ac, 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 soybeans accounted for 260 fields each, sorghum for 99 fields, and wild hosts 

for 145 fields. 100% of cotton was simulated as TwinLinkTM cotton. No Cry1Ab corn was modeled in 

the landscape because BCS did not have access to sales information.

The rate of resistance evolution was estimated in two ways: (1) The final R-allele frequency after 31 

years was divided by the initial frequency (was not constant due to re-sampling) and the result was log 

transformed. In many cases, the resistance allele frequency exceeded 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 years it took to approach that value. (2) The rate of change in the resistance allele frequency was 

measured after three years to estimate the rate of resistance evolution. 

Table D.3. Parameters 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 

drawn for both 

alleles)

0.3 0.00 0.05 Pert

Modified from MRID 47640014

In the model, resistance did not evolve to either toxin in TwinLinkTM cotton in 1000 simulations of 31 

years of use; hence, under the conditions modeled there appeared little risk of product failure in a 30-

year 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 

selection. 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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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 47640014)

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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VI. REFUGE STRATEGY

BCS stated that since 1) both Cry1Ab and Cry2Ae express high-dose for the major target pests, CBW 

and TBW, and 2) the two toxins bind to different target sites in the midgut of the three insect pests, that 

TwinLinkTM 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 

commercially sell seeds from the single Events Cry1Ab and Cry2Ae. Very small acreages may be grown 

for seed production only in order to test combinations 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 TwinLinkTM 

adoption.

The applicant proposed to have baseline susceptibility established for Cry1Ab 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 susceptibility 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 TwinLinkTM cotton was responsible 

for the observed damage. If the plants were confirmed to express TwinLinkTM, 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 

bioassays would be conducted to determine the level and extent of resistance on the sampled insect 

populations and a colony established to determine if the putative resistance was heritable. Data from 

standardized bioassays 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 by the findings, is another aspect of BCS’ proposed remedial program.

Confirmed resistance would define the remedial action area(s) as well as the approach. The main focus 

would be to limit the spread of resistant alleles by eliminating resistant population(s) as quickly as 

possible. This might be accomplished through chemical insecticide treatments of the Bt cotton field 
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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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 identified. 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 report 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 fully communicated to BCS sales 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 deployment and eliminate the need to monitor grower 

compliance with IRM requirements aside from the required field monitoring 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 crops. 

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 noncompliance on IRM; 2) identification of compliance mechanisms 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 are 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 BCS’ 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, TwinLinkTM. It 

appears that T304-40 was expressed at higher levels in leaves of single event plants compared to leaves 

collected from TwinLinkTM plants. For flowers and bolls the pattern was reversed, and TwinLinkTM 

plants appeared to express T304-40 at higher levels in these tissues. It is unclear whether these 

differences in expression results were statistically significant because BCS did not provide further 

information on the differences observed. 
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Expression of GHB119 in leaves from early to late stage appeared somewhat variable or unstable. 

Expression levels were measured in leaves in 2004 and 2005 at three developmental stages (early 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 early, 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 

TwinLinkTM than in the single event plant during developmental 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 by BCS 

for T304-40 and GHB119 in different plant tissues. It appears that the same tissue types were lumped 

together for plants expressing single and pyramided events. Since there appears to be unequal expression 

of the proteins in plants expressing either the single or pyramided 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 insects, 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 

genotypes: 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 heterozygous insects 

with one resistance allele (RS). Lower dose PIPs might allow for survival of insects with at least one 

susceptibility allele (SS or RS), although effective IRM may still be possible with a suitable refuge 

strategy. To be able to demonstrate high-dose, it is recommended that applicants generate data by at 

least two of the five laboratory and field approaches as outlined by the SAP (1998) and described 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 Lepidopteran pests. In recent years, PIPs in Bt

cotton have been approved that contain two genes targeted at the same insect pest. These “pyramided” 

products can be beneficial for IRM since 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. 

binding to different Bt receptor sites in the midgut) that are expressed at high-doses in the plant (Roush 

1998). 
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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For pyramided products, the dose of each toxin should be evaluated separately. In cases where the 

pyramided product is created through conventional breeding and the dose of the single toxin products 

has already been established, the combined dose in the pyramided PIP can be determined with 

comparative efficacy studies and protein expression data. However, this is not the case for TwinLinkTM 

cotton.

BPPD reviewed BCS’ 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 two methods (of five methods described by the panel) be used when 

demonstrating dose expression in target pests. 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 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 TwinLinkTM may express a high-dose and effective high-dose, respectively,

against TBW. 

After day 4 of the diet-incorporated 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 Cry1Ab treatments, mortality reported from diet bioassays did not reach 100% until after day 15. In 

addition, control mortality was relatively high (ranging from 23%-28%). For overlay assays, mortality

for TBW when exposed to Cry1Ab was 100% by day 15; control mortality at that time had reached 

17%. Cry1Ab may express a high-dose against TBW. 

2. Cotton Bollworm 

It appears that TwinLinkTM 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 TwinLinkTM expresses a high-dose 

against CBW, additional information needs to be provided to clarify uncertainties in the experiment 

using high-dose verification method 4. 

High-dose verification method (1): 

BPPD concludes that the diet-incorporated and overlay assays together suggest that TwinLinkTM and 

GHB119 may express a high-dose, but T304-40 may express less than high-dose against CBW.

In the control treatments, larvae had developed into pupae by day 17. BCS did not report whether 

surviving larvae of the exposure treatments, Cry1Ab, Cry2Ae, and TL, developed into pupae and 
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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consequently adults or whether survivors remained in the larval stage. BPPD notes that for future 

submission, it is recommended that BCS report whether surviving larvae were developmentally ‘dead’ 

(as opposed to physiologically), 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 47640012). When BPPD consulted chapter 13 for raw data or data tables, however, 

mortality data from the infestation experiments were lumped for all lepidopteran pests, and no pest 

specific mortality data were reported. It is unclear how the data that BCS reported in chapter 15 of their 

submission (Tables 7 and 8) were obtained, and no further details about the study were submitted in an 

appendix accompanying their submission (MRID 47640014).

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 sprayed, not sprayed, or both, and what effect spraying might have had on 

dose 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 final CBW mortality results from plots expressing Cry1Ab, Cry2Ae, 

and TwinLinkTM 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 attack rate. Without such information it is 

unclear how many 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. In addition 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 TwinLinkTM 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):

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 by Ellsworth and 
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Li (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 by BCS. 

Deficiency 1: The dose profile reported for PBW cannot be verified by 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 TwinLinkTM

Pest Method #1 Method #4 Method #3

T304-40 GHB119 TL T304-40 GHB119 TL T304-40 GHB119 TL

CBW NonHigh 

Dose

Possible 

HighDose

Possible 

Effective 

HighDose

Inconcl.* Inconcl.* Inconcl.* -- -- --

TBW Possible 

Highdose

HighDose

Effective 

HighDose-- -- -- -- -- --

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. 

C. CROSS-RESISTANCE

Analyses 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 by Crickmore et 

al. (1998), which supports that Cry1 and Cry2 Bt proteins do not have more than 20% amino acid 

sequence homology in common. Furthermore, the 3-dimensional crystal structures of Cry2Aa (very

closely related to Cry2Ae as expressed in TwinLinkTM) and Cry1Aa (very closely related to Cry1Ab as 

expressed in TwinLinkTM 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 respect to binding 

sites. The similarity in the global architecture of Cry2Aa and Cry1Aa, however, suggests that the two 

toxins may have similar general steps for the mode of action.

BPPD concludes that the evidence provided by BCS supports that cross-resistance between Cry1Ab and 

Cry2Ae, as expressed in TwinLinkTM 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 substantially at the third terminal suggesting that the two proteins 

may have unique mechanisms with respect to binding sites (and other biochemical pathways); the 

binding assay studies by Jurat-Fuentes showed that Cry1Ab 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 

Cry1Ab binding to gut membrane of PBW was specific while Cry2A binding was less specific yet with 

high binding site concentrations (suggesting that toxins were not competing for same binding sites).
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However, BPPD notes that should resistance evolve in CBW and TBW to Cry2Ae as expressed in 

TwinLinkTM cotton that Cry2Ab2 (as in Bollgard II) may be compromised as well because, as BCS 

stated in their report, there is a great potential for cross-resistance between the two toxins. 

BPPD notes that the cross-resistance potential is great between Cry1Ab, Cry1Ac, and Cry1F (although 

somewhat less for Cry1F) based on the competition assays; the three toxins share high affinity binding 

sites. Should CBW evolve resistance to Cry1Ab, other PIP products expressing Cry1Ac and Cry1F 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 by 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 years, 

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 such 

factors as level of Bt crop adoption, level of compliance, economics, fitness costs of resistance, alternate 

hosts, spatial components, stochasticity, and pest population dynamics.

BPPD reviewed the modeling assessment and accepted the probabilistic approach presented therein. 

Based on the choice for parameter values, natural refuge assumptions, and their analysis, BCS stated that 

there is little risk of resistance evolution in CBW to TwinLinkTM within 30 years. BPPD needs further 

clarifications on several issues and has some concerns regarding assumptions made for certain important 

parameters. These are summarized below.

Deficiency 2: In the modeling analysis provided by BCS, only CBW was modeled but not 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 highdose 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 highdose 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 (Cry1Ac, Cry1F, and Cry2Ab2) than other pest (i.e. TBW). BCS’s LC50

data show that CBW have a wide range of susceptibility against Cry1Ab (462 -2844 ng/cm2) as 

expressed in cotton when compared to TBW (4.9-10.5 ng/cm2). BCS concluded in their report that 

TwinLinkTM expressed less than high-dose against CBW due to the target pest being less susceptible to 

Cry1Ab and lower level of expression of Cry1Ab in the parental event for TwinLinkTM. In addition, 

bioassay results for Cry1Ab in corn (BPPD 2009) showed greater variability as well for Corn earworm, 
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CEW, (aka CBW) than for other pests, such as ECB. In fact, there is up to a three-fold difference in 

magnitude of reported LC50s of CEW compared to ECB. And previous attempts to create a diagnostic 

concentration for CEW were frustrated by the fact that the insect was less susceptible to Cry1Ab than 

other Lepidopteran pests. Given this information, BPPD is concerned with the dose assumptions for 

CBW and recommends that an additional analysis be conducted using somewhat lower mortality

assumptions based on the study results reported by BCS. The bioassay experiments showed that Cry1Ab 

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 no evidence that Cry1Ab 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 Cry1Ab (0.0002). Although no field resistance to Cry1Ab has been documented yet 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 

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 likely resistance allele 

frequency of 0.001 for Cry1Ab in CBW.

Deficiency 6: BCS needs to include Cry1Ab corn and Cry1Ab cotton in the modeling landscape when 

assessing the risk of resistance evolution in the target pests because Syngenta has registered Bt cotton 

and Bt corn and Monsanto has registered Bt corn products expressing Cry1Ab. These PIPs are 

represented in the Cotton Belt and need to be included in a simulation analysis for TwinLinkTM.

E. REFUGE STRATEGY

An IRM plan waiver request for single trait Events (Cry1Ab and Cry2Ae) is acceptable if seeds from 

those events are not sold commercially and serve only for producing the combination PIP TwinLinkTM. 

For any 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 trait 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 doseverification 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 TwinLinkTM cotton. 

If a Natural Refuge strategy for TwinLinkTM 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 in the 
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following Texas counties: Brewster, Crane, Crockett, Culberson, El Paso, Hudspeth, Jeff Davis, 

Loving, Pecos, Presidio, 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 preferably 300 feet wide. This refuge 

may not be treated with any insecticide labeled for the control of tobacco budworm, cotton 

bollworm, or pink bollworm. At the pre-squaring cotton stage only, 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 (preferably adjacent to or within 1/4 mile or 

closer) from the Bt cotton fields. 

2) 20% External Sprayed 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 foliar 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 (preferably 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 applicants monitor for insect resistance (measurement of resistanceconferring alleles) to the Bt toxins as an important early 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 difficult 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 samples increases, the 

likelihood of locating a resistant individual increases (Roush & Miller 1986). If the phenotypic 

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 individual (Roush & Miller 1986). Current sampling strategies have a 

target of 100 to 200 individuals per location. Previous experience with conventional insecticides has 

shown than once resistant phenotypes are detected at a frequency >10%, control or crop failures are 
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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 TwinLinkTM is commercialized before 2012. Baseline susceptibility data should be 

available by 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 

TwinLinkTM 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 remedial action plan should clearly 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 fully mitigate resistance, a critical element of 

any remedial action plan should be that once pest resistance is confirmed, sales of all Bt cotton hybrids 

that express a similar protein or a protein in which 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 

TwinLinkTM. 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 seriously and followed up immediately to eventually conclusively 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 variety 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, Loving, Pecos, Presidio, Reeves, Terrell, Val Verde, Ward, and Winkler.
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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 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. 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 

anonymous 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 were 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.
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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 Agency, MRID 47634823

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 crystal proteins. 

Microbiology and Molecular Biology Reviews, Vol. 62 (3): 807-813.

Gould, F., Anderson, A., Reynolds, A., Bumgarner, L., and Moar, W. 1995. Selection and genetic 

analysis of a Heliothis virescens (Lepidoptera: Noctuidae) strain with high levels of resistance to 

Bacillus thuringiensis toxins. J. Econ. Entomol. 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-endotoxins to cotton bollworm (Heliocoverpa zea) and pink bollworm 

(Pectinophora gossypiella) 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 

Cry1Ac-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: 

TwinLinkTM Cotton. Unpublished report submitted by Bayer CropScience to the Agency, MRID 

47640014.

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 pyramiding 

succeed where pesticides mixtures have not? Phil. Trans. Royal Soc. Lond. B (1998): 1777- 1786

Tabashnik, B.E. 1994. Evolution of resistance to Bacillus thuringiensis. Annu. Rev. Entomol. 39:47-79. 
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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U.S. EPA, 1998. FIFRA Scientific Advisory Panel Subpanel on Bacillus thuringiensis (Bt) PlantPesticides 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 

Incorporated 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 47413901). Memorandum from S. Borges to M. Mendelsohn dated April 8th, 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, MRIDs 

4760014 and 47634823). On July 29th, 2010, BCS submitted their response to BPPD’s eight listed IRM 

deficiencies for TwinLinkTM (BPPD 2010). BPPD18 reviewed BCS’ response and additional information 

and concludes that five of the eight deficiencies were satisfactorily 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 days” 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 reported 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 satisfactorily addressed and resolved. BPPD concludes that the single Events 

Cry1Ab and Cry2Ae in cotton both express a less than high-dose against PBW and that TwinLinkTM 

expresses a high-dose against PBW.

B) Discussion regarding Deficiencies 2-3:

Deficiency 2: In the modeling analysis provided by BCS, only 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.

 

18 The use of BPPD in this review refers to the BPPD IRM team consisting of Jeannette Martinez and Alan Reynolds.
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Deficiency 3: The natural refuge paradigm was approved for Bollgard II and WideStrike with a highdose 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 highdose against TBW, or BCS can design a model with their specific 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 TwinLinkTM cotton will be conducted 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 TwinLinkTM, 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 analysis be conducted using somewhat lower mortality assumptions based on the study results 

reported by BCS. The bioassay experiments showed that Cry1Ab 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 Cry1Ab expresses high-dose against CBW. BCS also concluded that TwinLinkTM 

expressed less than high-dose against CBW due to the target pest being less susceptible to Cry1Ab and 

lower level of expression of Cry1Ab in the parental event for TwinLinkTM Cotton. 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 Cry1Ab (0.0002). Although no field resistance to Cry1Ab has been documented yet 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 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 expected. BPPD recommends that BCS consider modeling a most likely resistance 

allele frequency of 0.001 for Cry1Ab in CBW.

Deficiency 6: BCS needs to include Cry1Ab corn and Cry1Ab cotton in the modeling landscape when 

assessing the risk of resistance evolution in the target pests because Syngenta has registered Bt cotton 

and Bt corn and Monsanto has registered Bt corn products expressing Cry1Ab. These corn and cotton 

PIPs are grown in the Cotton Belt and need to be included in a simulation analysis for TwinLinkTM.

BCS’ response: The most likely R-allele value for Cry1Ab was actually 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 Cry1Ab to 0.0001. With respect to 

incorporating Cry1Ab corn in the modeling 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 TwinLinkTM. Results 

after 2000 simulations (with revised parameters) showed that resistance did not evolve to either toxin in 
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any of the simulations. The rate of increase over the first five years for each locus reflected the presence 

of fitness costs. In many cases, particularly with the Cry1Ab resistance locus, the resistance allele 

frequency actually dropped. This was 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 Cry1Ab locus frequency was 10-fold greater. Informally, several runs 

were made without the Cry2Ae toxin to compare the rate of increase of the Cry1Ab 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 lack 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 frequencies 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 year 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 durability 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 analysis of probability to detect 

resistance. Discriminating dose assays for TBW, CBW, and PBW will be provided to the Agency on a 

yearly basis.

BPPD response: BPPD concludes that BCS’ monitoring plan reflects the paradigm established for other 

Bt cotton registrations and the deficiency has been satisfied. However BPPD recommends that in the 

Terms and Conditions of the registration, BCS be required to submit baseline susceptibility and a 

diagnostic assay data.
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E) Discussion regarding Deficiency 8:

Deficiency 8: BCS needs to have an established remedial action plan in place for each target pest of 

TwinLinkTM. 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 incorporate the following measures to identify or confirm suspected resistance:

 Monitoring (as outlined in MRID 47640014);

 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, LC50/LC99 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 by 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 type and size of refuge 

should be considered.

BPPD 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 days of confirmed resistance”. In 

addition, BCS should incorporate that “a long-term resistance management action plan will be designed 

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. 
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Appendix D. III – Risk Assessment of data/information to respond to remaining IRM deficiencies. 

Below is U.S. EPA’s review (June 22, 2011) of BCS’ resubmissions for FIFRA section 3 application for 

TwinLinkTM Cotton expressing a pyramid with Events T304-40 and GHB 119 (MRIDs 48480011, 

48480010, and 48495101), EPA File Symbols 264-RNOA and 264-RNOL: PC Codes: 006525 

(Cry1Ab), 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 additional data/information 

to satisfy the remaining deficiencies.

BPPD19 reviewed BCS’ submission to address deficiencies noted in the original review of TwinLinkTM 

Cotton, a two-gene pyramided PIP targeting Heliothine pests (BPPD 2010). BPPD’s conclusions and 

recommendations are contained in this memo.

CONCLUSIONS AND RECOMMENDATIONS

DOSE VERIFICATION FOR TBW (Deficiency 3)

BCS provided enough data to resolve concerns with the dose for TwinLinkTM Cotton and its single 

parental events against TBW, and Deficiency 3 has been addressed in a satisfactory manner. 

Specifically, based on the field experiment conducted with young cotton plants, BPPD concludes that 

Cry1Ab and Cry2Ae PIPs express a high dose against Heliothis virescens (TBW) during the early cotton 

growing season (also late season for Cry2Ae). No larvae on Cry2Ae and Cry1Ab cotton survived or 

developed to 3rd instar during the 11-day test duration, while 2.8% and 4.5% larvae in control treatments 

reached or surpassed the 3rd instar developmental stage. The lab experiment conducted with older cotton 

plants, however, resulted in 2.17% survival (> 3

rd instar) on Cry1Ab plants expressing the parental event 

only suggesting that expression of Cry1Ab was decreased in older plants compared 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 older 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 TWINLINKTM COTTON WITH NATURAL REFUGE (Deficiency 2)

BCS provided additional modeling to resolve concerns for their “Natural Refuge” request for 

TwinLinkTM Cotton, and Deficiency 2 has been addressed in a satisfactory manner. BPPD concludes 

that TwinLinkTM 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. 

Specifically, BCS conducted two modeling analyses (stochastic, spatially explicit and deterministic, 

spatially implicit) and generally took a conservative approach to modeling the durability of TwinLinkTM 

targeting TBW. Mainly, the model landscape accounted for other Cry1A products and model explored 

several scenarios with increased dominance and lower survival based on the lower protein expression for 

Cry1Ab 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 TwinLinkTM Cotton would be as low as 80 years 

when all worst-case assumptions were incorporated or as high as 224 years using benchmark 

 

19 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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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 years (possibly an unrealistic worst-case scenario based on survival percentages used) and 23 years 

(possibly a reasonable worst-case scenario). Assuming benchmark parameter values for mortality (as 

used for other Cry1A products in the past), the mean time to estimated product failure (resistance) was 

26 years. Whether results differed significantly between any scenarios modeled was unclear since no 

such analysis and/or explanation accompanied the report. Additionally, comparative analysis between 

Bollgard II (Monsanto released data used for the natural refuge SAP) and TwinLinkTM Cotton showed 

that estimated mean durabilities were comparable.

Although the likelihood for cross-resistance between Cry1Ab and Cry2Ae cotton 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 analysis of TwinLinkTM Cotton. Other forms of epistasis (expression of a gene is 

suppressed by a gene at another locus) were not considered: rather the applicant assumed that survival of 

genotypes was multiplicative for both loci (the least conservative assumption). The SAP (2011) 

suggested that other forms of epistasis should be explored for pyramided 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 Cry1Ab was incorporated for older plants and raised some concerns with BPPD. 

Further analysis should be conducted to assess whether this outcome was an artifact of the modeling 

design taken by the modeler or a function of real temporal asynchrony from the growing oviposition 

period between generations and delayed 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 

pyramided cotton PIPs that express Cry1A 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 TwinLinkTM Cotton deployed 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

TwinLinkTM cotton is a pyramided transgenic cotton trait that expresses Cry1Ab (Event T304-40), and 

Cry2Ae proteins (Event GHB119), and a glufosinate ammonium (herbicide) tolerant trait based on 

LibertyLink technology. TwinLinkTM was developed by BCS, using conventional breeding techniques. 

The Cry1 and Cry2 Bt toxin group are effective against lepidoptera and share less than 20% amino acid 

homology. Crickmore et al. (1998) constructed a phylogenetic tree using amino acid sequence identity

as homology criteria for all presently known Bt crystal proteins. This tree indicates that Cry1Ab and 

Cry2Ae are genetically dissimilar entities and share little sequence homology. 

On April 22nd, 2010, the BPPD IRM team completed its initial review of BCS’ submission for a FIFRA 

product registration for TwinLinkTM 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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On July 29th, 2010, BCS submitted their response to BPPD’s eight listed IRM deficiencies for 

TwinLinkTM (BPPD 2010a). BPPD reviewed BCS’ response and additional information and concluded 

that five of the eight deficiencies were satisfactorily addressed and resolved (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 the remedial action plan and highlighted that BCS needed to explicitly state 

that “the Agency is informed within 30 days of confirmed resistance”. This BPPD review assesses 

deficiencies 2 and 3 listed below:

Deficiency 2: In the modeling analysis provided by BCS, only 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. If BCS wishes to pursue the Natural Refuge strategy for 

TwinLinkTM, 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 TBW.

II. BAYER CROPSCIENCE RESUBMISSION MATERIALS

1. Field Dose Study (MRID 48480010)

In 2011, a study was conducted in Mississippi cotton fields (MRID 48480010) to estimate mortality of 

Heliothis virescens (Tobacco budworm, TBW) on caged plants expressing parental Events Cry1Ab and 

Cry2Ae as well as the pyramided PIP. 

Materials and Methods:

Four treatments were tested: 1) Cry1Ab 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 approximately 120 plants. Artificial 

infestations were conducted with individuals from a laboratory strain from USDA-ARS, Stonesville 

(MS). Fifty males and 50 females were released into each cage on two dates separated by one month 

(two replicates). Mated females oviposited onto cotton plants, and first data collections began four days 

after introducing the adults into the cages to obtain lower limits of eggs deposited. Ten randomly 

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 ‘yes’. Larval survival was estimated on 20 randomly selected plants 

within each cage, and survival and developmental stage were recorded. Larvae that survived 11 days of 

exposure on the plant and had developed to 3rd 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 type. 
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Results:

One larva developed to 1st instar on one Bt plant type (Cry1Ab), while 17 and 37 developed to ≥3rd

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 mortality.

Table D.III.1. TBW larval survival after 11 days of exposure to TwinLinkTM and its parental Events

Treatment Type

Number of Eggs 

(initial cohort –

minimum)

Total Number of 

Survivors

Number of Survivors

(≥ 3rd instar)

Proportion of 

Survivors –

minimum value

(≥ 3rd 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 48480010)

Applicant Conclusions:

“This study provided additional evidence for the high susceptibility of TBW to TwinLinkTM as well as 

its parental (single) events. No survivors (≥ 3rd instar) were found on the parental event, therefore, 

TwinLinkTM should provide IRM benefits comparable to or exceeding those of other currently registered 

pyramided Bt cotton products approved for the natural refuge strategy.”

2. Greenhouse Dose Study (MRID 48480011)

In 2011, BCS conducted a greenhouse trial to provide further evidence that TwinLinkTM Cotton and its 

parental events express high dose against Heliothis virescens. 

Materials and Methods:

Cotton plants (144-184 days old) were placed into four isolated screened enclosures to hold groups of 

different Bt phenotypes. 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. Larval counts were made at 

day 7 and 14 after infestation. Survival was computed as the proportion of individuals surviving 14 days 

and completing development to at least 3rd instar. Corrected mortality was calculated by dividing 

‘proportional survival on Bt’ by ‘proportional survival on non-Bt cotton’.

Results:

Larval survival on non-Bt and Cry1Ab plants was 69 and five 3rd instar larvae out of a total infestation 

with 540 (on control) and 1800 (on Bt); after correcting for control mortality, survival on Cry1Ab plants 
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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was estimated at 0.0217 (see Table 2). On Cry2Ae and TwinLinkTM 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

Treatment Total Plants 

infested

Total Number 

of Neonates

Total Number 

of Survivors

Mean 

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 484800011

Conclusions:

The level of mortality observed in these trials and levels of survival on single-toxin plants was 

comparable to or exceeded that of the currently registered Bt cotton products. Lack of survivors on 

Cry2Ae plants underscores the highly effective dose of this protein in the TwinLinkTM pyramid. 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 Cry1Ac in 

currently registered Bt cotton products). This study shows that plants, even late in the production cycle, 

continue to cause very high mortality to TBW. This should provide additional support for the use of 

natural refuge strategy for T304-40 x GHB119.

3. Simulation Modeling for TwinLinkTM Cotton and Tobacco Budworm (MRID 48495101)

BCS contracted the simulation modeling to Dr. Mike Caprio from Mississippi State University. Two 

different models, stochastic and deterministic, were employed 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 by Caprio et al. (2009) for Helicoverpa zea (cotton 

bollworm, CBW) and was modified to address TBW specifically and to conduct simulations for 

TwinLinkTM cotton. Only 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 cotton, wild hosts (clover, geranium), and TwinLinkTM (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 simulated a 

situation in the field where alternate hosts would be spatially segregated. All patches were randomly

assigned the nature of plant type at the initiation of the simulation. All larvae and pupae were accounted 

for and placed as pupae in geranium patches at the beginning of each year. Because of complete mixing 

during the overwintering routine, the habitats were not redistributed at the beginning of each year. This 

allowed detecting the effects of habitat distribution between simulations.

The dose of Cry1Ab 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 Cry1Ab. Four different 
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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levels of decline in protein expression were investigated leading to 0.1% survival (0% decline), 2% 

survival (level observed in lab), 5% (see Table 3 for genotypic survival), and 10% (extreme case). These 

levels of decline continued for two more generations until the overwintering subroutine began. Two 

values of dominance were used: 0.1 (high value) and 0.001 (lower value). Multiplicative toxin 

interactions were assumed. To overcome extinction of relatively small TBW populations, the fecundity 

was raised from 50 to 150 eggs/day-female in the model.

Table D.III.3. TBW 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 48495101)

Deterministic model: 

The Caprio model (based on a construct previously employed by EPA-ORD) was used to model TBW 

resistance to TL cotton and modified to include refuges of variable size as well as Cry1Ab protein 

expression decline in older plants. Mortality from Cry2Ae was constant at 99.9% for each generation. 

For Cry1Ab, mortality was 99.9% for the first three generations (survival of the susceptible genotype 

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 worst 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 genotype was 0.0001% (0.01% x 0.01%). Dominance for 

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 (Cry1Ac such as for Bollgard), 20% refuge, and 

50% reduction in adult emergence due to spraying. The estimated durability was 18.8 years. Given that 

Bollgard was used for almost 15 years, 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 

Bollgard II (Monsanto released data for natural refuge SAP) and TwinLinkTM to assess the relative 

durability of TwinLinkTM to the already registered pyramided PIP.

Results:

Stochastic model: When dominance was set at 0.001, no resistance evolved to TwinLinkTM within 31 

years (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 years. Greater survival later in the season resulted in faster resistance evolution. 

Additionally, in almost every simulation resistance to both toxins (Cry1A and Cry2Ae) occurred almost 

simultaneously. This was unexpected since Cry1Ab moved towards a lower dose later in the growing 

season, and the expectation was that resistance would evolve to Cry1Ab first. Analysis of data revealed 

that resistant individuals in two fields became temporarily isolated from the remaining refuge population 
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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early in the season. Toxin expression was high at that time and hence, only 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 apparent temporal asynchrony occurred although no developmental 

delays 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 always 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 TL cotton). 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 by a hypothetical 

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 any particular PIP, may be warranted. 

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 any of the 10 replicates; with lower dominance values, 

resistance evolved as early as in 10.1 years and mean time to resistance was 27.3 years (comparable to 

TwinLinkTM results).

Table D.III.6. Mean time to resistance for TBW to TwinLinkTM Cotton 

Survival to Cry1Ab 

Post-Cutout

Mean Time to 

Resistance (yrs)

Number of 

Simulations

0.1%126.0 10

2%223.5 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 value observed by BCS in 2011 greenhouse studies of plants after cutout.

Table modified from original Table (p.12, MRID 48495101)

Deterministic Model: The deterministic runs of the model resulted in an estimated 224 years before 

resistance evolved in TBW to TwinLinkTM (both toxins) 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 years before resistance evolved to TwinLinkTM (see Table 

D.III.5 below). Unlike other previous models for TBW and currently registered Bt cotton PIPs 

expressing Cry1A protein, BCS incorporated a seasonal decline in Cry1Ab protein expression, which 

somewhat elevated the risk of resistance evolution.

Table D.III.5. Durability of TwinLinkTM Cotton based on the deterministic model simulations

Dominance Value for Cry1Ab Time to Resistance (yrs)

0.001 224

0.01180

TL simulations run with parameter values as close to Monsanto’s values for Bollgard II

1 Dominance is 10x that of Monsanto’s value
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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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 Cry1A-based 

dual-Bt products registered in the US may be somewhat higher than indicated by previous simulations, 

owing to possible late-season decline in Cry1Ac expression.”

III. BPPD REVIEW AND CONCLUSIONS

DOSE (Deficiency 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 for Cry2Ae). No larvae on 

Cry2Ae and Cry1Ab survived or developed to 3rd instar during the 11-day test duration, while 2.8% and 

4.5% larvae in control treatments reached or surpassed the 3rd instar developmental stage. The lab 

experiment conducted with older cotton plants, however, resulted in 2.17% survival (> 3

rd instar) on 

Cry1Ab plants expressing the parental event only suggesting that expression of Cry1Ab protein was 

decreased in older plants compared 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 older 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 (Deficiency 2): BCS conducted two modeling analyses (stochastic spatially explicit and 

deterministic, spatially implicit) and generally took a conservative approach to modeling the durability

of TwinLinkTM Cotton targeting TBW. Dr. Mike Caprio from Mississippi State University incorporated 

other Cry1A products into the landscape and explored several scenarios with increased dominance and 

lower survival based on the lower protein expression observed in the lab for Cry1Ab 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 estimated that durability of 

TwinLinkTM Cotton would be as low as 80 years when all worst-case assumptions were incorporated or 

as high as 224 years using benchmark assumptions. This was a 2.8 fold difference between estimated 

durabilities. The spatially explicit, stochastic simulations estimated that the durability for TwinLinkTM 

Cotton was on average between 20 years (probably an unrealistic worst-case scenario based on survival 

percentages used) and 23 years (probably reasonable worst-case scenario). Assuming benchmark 

parameter values for mortality (as used for other Cry1A products in the past), the mean time to estimated 

product failure (resistance) was 26 years. Whether results differed significantly between any scenarios 

modeled was unclear since no such analysis and/or explanation accompanied the report. Additionally, 

comparative analysis between Bollgard II (Monsanto released data presented to SAP for Natural Refuge 

assessment) and TwinLinkTM Cotton showed that estimated mean durabilities were comparable between 

the two pyramided PIPs.

Although the likelihood for cross-resistance between Cry1Ab and Cry2Ae cotton 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 by a gene at another locus) into the modeling analysis of 

Cotton. Other forms of epistasis were not considered: rather the applicant assumed that survival of 

genotypes was multiplicative for both loci (the least conservative assumption). The SAP (2011) 

suggested that additional forms of epistasis should be explored for pyramided PIPs such as, for example, 
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document January 23, 2012

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developmentally restricted expression of low levels of Cry-protease and where resistance at the a locus 

could be determined by the most rapidly evolving locus, which could drag other resistance loci along 

and, thereby, increase the rate of resistance evolution beyond what would be estimated by a purely 

multiplicative approach.

The discussion as to why resistance evolved almost simultaneously to both toxins (with assumption of 

increased dominance) raised some concerns with BPPD. Further analysis should be conducted to assess 

whether this outcome was an artifact of the modeling design taken by the modeler or a function of real 

temporal asynchrony from the growing oviposition period between generations and delayed 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 pyramided cotton PIPs that express Cry1A Bt 

toxins with lower expression levels during the later cotton growing season (resulting in greater 

dominance values for the target pest). 

BPPD concludes that TwinLinkTM Cotton deployed 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.
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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APPENDIX E: References

Insect Resistance Management

1. BPPD, 2010a. EPA review of BCS’ insect resistance management plan for a section(3) full 

commercial registration request for TwinLinkTM Cotton (EPA File Symbol 264-RNOU, MRIDs 

4760014 and 47634823). Memorandum from JC Martinez to S. Bacchus and D. Greenway on April 

4.

2. BPPD, 2010b. EPA Review of Bayer’s Response to IRM Deficiencies noted in BPPD’s risk 

assessment for TwinLinkTM dated 4/22/2010 (EPA File Symbol 264-RNOU, MRIDs 4760014 and 

47634823). Memorandum from JC Martinez to S. Bacchus and D. Greenway on July 29.

3. Caprio MA, CD Parker, and JC Schneider. 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. 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. Bayer CropScience unpublished report submitted to the Agency, MRID 

48495101.

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 crystal proteins. 

Microbiology and Molecular Biology Reviews, Vol. 62 (3): 807-813.

6. EPA Scientific Advisory Panel (SAP). 2011. Transmittal Meeting 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 from Dr. Sharlene Matten (DFO) to Dr. Steven Bradbury

(Director of OPP) on March 3, 2011.

7. Navacerrad JJ, J Pierson, I Coats, and PL Hunst. 2011. High dose study: estimating the mortality of 

Heliothis virescens in a greenhouse on plants on T304-40 x GHB119 (TwinLinkTM) Cotton (BCSGH004-7 x BCS-GH005-8) and parental Event plants T304-40 (Cry1Ab) and GHB119 (Cry2Ae). 

Bayer CropScience unpublished report submitted to the Agency, MRID 48480011

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 (TwinLinkTM) Cotton (BCS-GH004-7 x BCSGH005-8) and parental Event plants T304-40 (Cry1Ab) and GHB119 (Cry2Ae). Bayer Crop Science 

unpublished report submitted to the Agency, MRID 48480010

9. Peck SL, F Gould, and SP Ellner. 1999. Spread of resistance in spatially-extended regions of 

transgenic cotton: implications for management of Heliothis virescens (Lepidoptera: Noctuidea). 

Journal of Economic Entomology, Vol. 92: 1-1
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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REFERENCES

Other References:

U.S. EPA. 1998. FIFRA Scientific Advisory Panel Subpanel on Bacillus thuringiensis (Bt) PlantPesticides 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 

Incorporated Protectants, 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 Bt corn 

registrants (MRID 47413901). Memorandum from S. Borges to M. Mendelsohn dated April 8th, 2009.

U.S. EPA REVIEWS – Insect Resistance Management

U.S. EPA. 2010a. Memorandum J. Martinez and A. Reynolds. BCS’ Insect Resistance Management 

Plan for a Section (3) Full Commercial Registration Request of TwinLink Cotton (EPA Reg. No. 264-

RNOU, MRIDs 4760014 and 47634823). April 22.

U.S. EPA. 2010b. Memorandum. J. Martinez and A. Reynolds. September 28, 2010. EPA Review of 

Bayer’s Response to IRM Deficiencies noted in BPPD’s risk assessment for TwinLink dated 4/22/2010 

(EPA File Symbols 264-RNOU, 264-RNOL, and 264-RNOA, MRIDs 4760014 and 47634823). 

September 28.

U.S. EPA. 2011. Memorandum. J. Martinez and A. Reynolds. EPA review of Bayer CropScience’s 

resubmissions for TwinLinkTM Cotton sec(3) application expressing a pyramid with Events T304-40 and 

GHB 119 (MRIDs 48480011, 48480010, and 48495101), EPA File Symbol 264-RNOA, -RNOL: PC 

Codes: 006525 (Cry1Ab), 006600 (Cry 2Ae). June 28.
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

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Appendix E.1. Bibliography of data submitted for registration of Bt Cry1Ab (Event T304-40) and 

TwinLinkTM Cotton, its combination with Bt Cry2Ae (Event GHB119).

Most of the submissions below refer to Cry2Ae, and some of them also refer to TwinLinkTM. There may

be repeats of those made for Cry2Ae in the Bibliography cited for Cry1Ab because they were submitted 

for TwinLinkTM, which contains both proteins.

I. Cry 1Ab and TwinLinkTM Cotton - Data Submissions 

MRID Citation Reference (Bt Cry 1Ab PIP)

47076900 Bayer Cropscience LP. (2007) Submission of Toxicity Data in Support of the 

Experimental Use of Bacillus thuringiensis Cry1Ab 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

Bayer Cropscience. 62 p.

47171700 Bayer Cropscience (2007) Submission of Residue Data in Support of the 

Experimental Use of Bacillus thuringiensis Cry1Ab Insecticidal Protein on Cotton 

Plants. Transmittal of 1 Study.

47171701 Hayes, R. (2007) Final Field Report for 2006 Season of Bacillus thuringiensis

Cry1Ab Insecticidal Protein as Expressed in Cotton Plants. Project Number: 

264/EUP/140. Unpublished study prepared by Bayer Cropscience. 6 p.

47634800 Bayer CropScience (2008) Submission of Efficacy Data in Support of the 

Application for Registration of BCS Cry1Ab Cotton Event T304-40. Transmittal of 

20 Studies.

47634801 Ferullo, J. (2008) Product Characterization of Cry1Ab Cotton Event T304-40. 

Unpublished study prepared by Bayer BioScience N.V. 22 p.

47634802 Nennstiel, D. (2008) Product Characterization: Nutritional Characterization of 

Cry1Ab Cotton Event T304-40. Unpublished study prepared by Bayer CropScience 

LP. 8 p.

47634803 Currier, T.; Massengill, J. (2008) Protein Expression Analysis of Cotton Event T304-

40, Expressing Cry1Ab and PAT/bar Proteins. Project Number: CP07B005. 

Unpublished study prepared by Bayer CropScience LP. 158 p.

47634804 Martone, A. (2008) Analyses of Raw Agricultural Commodity (Fuzzy Seed) of 

Cry1Ab Cotton Event T304-40 for PAT/bar and Cry1Ab and its Non-Transgenic 

Counterpart for PAT/bar and Cry1Ab Proteins. Project Number: CP07B011. 

Unpublished study prepared by Bayer CropScience LP. 154 p.

47634805 Martone, A. (2008) 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. Project Number: CP08B003. 

Unpublished study prepared by Bayer CropScience LP. 52 p.
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document January 23, 2012

Page 138 of 153

MRID Citation Reference (Bt Cry 1Ab PIP)

47634806 Nennstiel, D. (2008) ORF Analysis - Toxicology (Human Health Assessment). 

Unpublished study prepared by Bayer Cropscience. 10 p.

47634809 Stephenson, I. (2008) Cry1Ab Protein: Epitope Homology, N-glycosylation and 

Overall Amino Acid Sequence Homology Search with Known Toxins and Allergens. 

Supplement to MRID 46708802 and MRID 46708803. Unpublished study prepared 

by Bayer BioScience N.V. 6 p.

47634812 Nennstiel, D. (2008) Toxicology (Human Health Assessment): History of Safe Use. 

Unpublished study prepared by Bayer BioScience N.V. 49 p.

47634813 Chalmers, A. (2008) Summary of Non-Target Organism Testing and Assessment of 

Risk of Gossypium hirsutum: Transformation Event T304-40 Expressing Cry1Ab 

Protein. Project Number: ECORA1. Unpublished study prepared by Bayer 

CropScience. 67 p.

47634815 Patnaude, M. (2008) Laboratory Study to Determine the Effects of Cry1Ab Protein 

on the Predatory Beetle Coleomegilla maculata. Project Number: EB99L008, 

13798/6217. Unpublished study prepared by Springborn Smithers Laboratories. 60 p.

47634816 Scott, A. (2008) Cry1Ab Protein: Toxicity to Green Lacewing (Chrysoperla 

rufilabris). Project Number: EB99L009. Unpublished study prepared by Bayer 

CropScience LP. 4 p.

47634818 Bushey, D. (2008) Request for Waivers from the Requirement to Conduct a NonTarget Soil Invertebrate Study: Earthworm. Unpublished study prepared by Bayer 

CropScience LP. 6 p.

47634819 Sayers, L. (2008) Cry1Ab Protein: 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 Cry1Ab Protein Produced from Escherichia coli After Aerobic Soil 

Degradation. Project Number: CP07B010. Unpublished study prepared by Bayer 

CropScience LP. 152 p.

47634821 Jesudason, P. (2008) Cry1Ab: Efficacy Assessment (Field and Laboratory). 

Unpublished study prepared by Bayer 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 Bayer CropScience 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 Bayer CropScience 

LP. 6 p.

47634824 Nennstiel, D. (2008) Detection Methods: rtPCR. Unpublished study prepared by

Bayer 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 January 23, 2012

Page 139 of 153

MRID Citation Reference (Bt Cry 1Ab PIP)

47634826 Nennstiel, D. (2008) Protein Detection Methods: Enzyme Linked Immuno Sorbent 

Assay (ELISA). Unpublished study prepared by Strategic Diagnostics, Inc. and 

EnviroLogix, Inc. 18 p.

47640000 Bayer 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 TwinLinkTM Cotton Event T304-40 x GHB 119 Cotton. 

Transmittal of 14 Studies.

47640001 Ferullo J. (2008) TwinLinkTM Product Characterization - Molecular Characterization. 

Unpublished study prepared by Bayer BioScience N.V. 6 p.

47640002 Jesudason, P. (2008) Product Characterization - Nutritional Characterization of 

Twinlink. Unpublished study prepared by Bayer Cropscience LP. 8 p.

47640003 Mackie, S. (2008) Protein Expression Analysis for T304-40, GHB119 and 

TwinLinkTM Transgenic Cotton. Project Number: CP08B002, BBS07/013, 

BB06/010. Unpublished study prepared by Bayer Cropscience LP. 155 p.

47640004 Martone, A. (2008) Analyses of Raw Agricultural Commodity (Fuzzy Seed) of 

TwinLinkTM Cotton for PAT/bar, Cry2Ae and Cry 1Ab and its Non-Transgenic 

Counterpart for PAT/bar, Cry2Ae and Cry1Ab Proteins. Project Number: 

CY07B008, BTS/0066/06, BBS07/006. Unpublished study prepared by Bayer 

Cropscience LP. 216 p.

47640005 Martone, A. (2008) Structural and Functional Equivalence of Cry1Ab, Cry2Ae and 

PAT/bar Proteins Produce in Escherichia coli and Bacillus thuringiensis to the 

Cry1Ab, Cry2Ae and PAT/bar Proteins from Events T304-40, GHB119 and 

TwinLinkTM Cotton Seed, Gossypium hirsutum. USA, 2008. Project Number: 

CP08B011, BTS/0077/08, BBS07/006. Unpublished study prepared by Bayer 

Cropscience LP. 70 p.

47640006 Martone, A. (2008) Structural and Functional Equivalence of Cry1Ab, Cry2Ae and 

PAT/bar Proteins Produced in Bacillus thuringiensis (Bt) and Escherichia coli to 

Cry1Ab, Cry2Ae and PAT/bar in TwinLinkTM Cotton, Gossypium hirsutum, USA, 

2007. Project Number: CY07B006, BBS07/013, BIOX/001/PROTSUPP/07. 

Unpublished study prepared by Bayer Cropscience LP. 402 p.

47640007 Bushey, D. (2008) Toxicology: Summary Human Health Assessment. Unpublished 

study prepared by Bayer BioScience N.V. 8 p.

47640008 Chalmers, A. (2008) Summary of Non-Target Organism Testing and Assessment of 

Risk of TwinLinkTM Cotton Expressing Cry1Ab and Cry2Ae Proteins. Project 

Number: ECORA3. Unpublished study prepared by Bayer Cropscience LP. 17 p.

47640009 Richards, K. (2008) Evaluation of the Dietary Effect(s) of Pollen from TwinLinkTM 

on Honey Bee Larvae (Apis mellifera L.) Development. Project Number: EB99X037, 

CAR/204/08, BTS/0081/08. Unpublished study prepared by California Agricultural 

Research, Inc. and Bayer CropScience Ecotoxicology Department. 48 p.

47640010 Patnaude, M. (2009) Chronic Toxicity to Collembola (Folsomia candida) Using 
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document January 23, 2012

Page 140 of 153

MRID Citation Reference (Bt Cry 1Ab PIP)

Transgenic Lyophilized TwinLinkTM 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 TwinLinkTM 

Cotton. Project Number: 13798/4118, TX99X106, M/310302/01/1. Unpublished 

study prepared by Springborn Smithers Laboratories. 223 p.

47640012 Rinehardt, M.; Holloway, J. ; Bushey, D. (2008) Agronomic Performance and 

Efficacy of TwinLinkTM Cotton Obtained by Conventional Breeding of Cry1Ab 

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 Bayer 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 (TwinLinkTM Cotton) Plants. Unpublished study prepared by

Bayer CropScience LP. 36 p.

47640014 Maclntosh, S.; Holloway, J.; Jansens, S.; et al. (2008) Insect Resistance Management 

Plan: TwinLinkTM Cotton. Unpublished study prepared by Bayer CropScience LP. 88 

p.

47651100 Bayer Cropscience LP - Bioscience (2008) Submission of Fate and Toxicity Data in 

Support of the Application for Registration of BCS Cry1Ab Cotton Event T304-40. 

Transmittal of 6 Studies.

47651101 Rouquie, D. (2007) Cry1Ab Protein: In Vitro Digestibility Study in Simulated 

Intestinal Fluid. Project Number: M/295264/01/1, SA/07111, 

LYNX/PSI/N/TX99L035. Unpublished study prepared by Bayer Cropscience. 54 p.

47651102 Rouquie, D. (2007) Cry1Ab Protein: In Vitro Digestibility Study in Human 

Simulated Gastric Fluid. Project Number: M/295272/01/1, SA/07110, 

LYNX/PSIN/TX51X006. Unpublished study prepared by Bayer Cropscience. 56 p.

47651103 Rouquie, D. (2007) Cry1Ab Protein: Acute Toxicity by Oral Gavage in Mice. Project 

Number: M/295507/01/1, SA/07109, LYNX/PSI/NTX51X007. Unpublished study

prepared by Bayer Cropscience. 58 p.

47651104 Rouquie, D. (2007) Cry1Ab Protein: Heat Stability Study. Project Number: 

M/295690/01/1, SA/07112, LYNX/PSI/N/TX99L034. Unpublished study prepared 

by Bayer Cropscience. 51 p.

47651105 Richards, K. (2008) Evaluation of the Dietary Effect(s) of a Cry1Ab 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 

Cry1Ab Protein. Project Number: 13798/6218, EB99X028, BBS07/006. Unpublished 

study prepared by Springborn Smithers Laboratories. 61 p.
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document January 23, 2012

Page 141 of 153

MRID Citation Reference (Bt Cry 1Ab PIP)

47692700 Bayer 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.

47692701 Patnaude, M. (2009) Cry1Ab Protein - Toxicity to Green Lacewing (Chrysoperla 

rufilabris) Following OPPTS Guideline 885.4340. Project Number: 13798/6230, 

EB99L009, 082808. Unpublished study prepared by Springborn Smithers 

Laboratories. 48 p.

47850300 Bayer CropScience, LP (2009) Submission of Environmental Fate Data in Support of 

the Experimental Use of TwinLinkTM Cotton on Cotton Plants. Transmittal of 1 

Study.

47850301 Coats, I. (2009) TwinLinkTM Cotton (BCS-GH004-7 x BCS-GH005-8): Investigation 

of Pollen Flow in Puerto Rico. Unpublished study prepared by Bayer CropScience, 

LP. 24 p.

47871400 Bayer CropSciences (2009) Submission of Product Chemistry, Environmental Fate 

and Toxicity Data in Support of the Application for Registration of BCS Cry1Ab 

Cotton Event T304-40. Transmittal of 4 Studies. 

47871401 Coats, I. (2009) Request for Waivers from the Requirement to Conduct Certain 

Product Analysis Studies as Outlined in 40 CFR 158.2120-Cry1Ab. Unpublished 

study prepared by Bayer CropScience LP. 7 p.

47871402 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-

Cry1Ab. Unpublished study prepared by Bayer 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-Cry1Ab. Unpublished study prepared by Bayer CropScience LP. 7 

p.

47871404 Hunst, P. (2009) Bridging Data from TwinLinkTM Cotton Avian Nutritional Study to 

Parental Events T304-40 and GHB119. Unpublished study prepared by Bayer 

CropScience LP. 7 p.

47871600 Bayer CropScience, LP (2009) Submission of Product Chemistry and Toxicity Data 

in Support of the Application for Registration of BCS TwinLinkTM Cotton Event 

T304-40xGHB 119 Cotton. Transmittal of 6 Studies. 

47871601 Coats, I. (2009) Request for Waivers from the Requirement to Conduct Certain 

Product Analysis Studies. Unpublished study prepared by Bayer 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 TwinLinkTM. 

Unpublished study prepared by Bayer CropScience. 9 p.

47871603 Coats, I. (2009) Request for Waivers from the Requirement to Conduct Certain 
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document January 23, 2012

Page 142 of 153

MRID Citation Reference (Bt Cry 1Ab PIP)

Nontarget and Environmental Fate Studies to Test for Ecological Effects -

TwinLinkTM. Unpublished studies prepared by Bayer CropScience. 7 p.

47871604 Currier, T. (2009) Protein Expression Analysis for Pat/bar, Cry1Ab and Cry2Ae 

Proteins in Whole TwinLinkTM Plants Grown in the Field. Project Number: 

CP09Q005. Unpublished study prepared by Bayer CropScience, LP. 28 p.

47871605 Hunst, P. (2009) Investigation of Synergistic Effects of the Cry1Ab and Cry2Ae 

Proteins expressed in TwinLinkTM Cotton (BCS-GH004-7 x BCS-GH005-8) on 

Target Insect Pests Using Leaf Bioassay. Unpublished study prepared by Bayer 

CropScience, LP. 8 p.

47871606 Coats, I. (2009) Supplement to MRID # 47640014: Insect Resistance Management 

Plan: TwinLinkTM Cotton. Unpublished study prepared by Bayer CropScience, LP. 7 

p.

47903100 Bayer CropScience LP (2009) Submission of Toxicity Data in Support of the 

Application for Registration of BCS TwinLinkTM 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 Bayer CropScience 

LP. 16 p.

47903200 Bayer 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 Bayer CropScience 

LP. 16 p.

47928800 Bayer CropScience, LP-BioScience. (2009) Submission of Efficacy Data in Support 

of the Registration of BSC TwinLinkTM Cotton Event T304-40 X GHB 119 Cotton. 

Transmittal of 1 Study.

47928801 Coats, I. (2009) Addendum to Product Chemistry for TwinLink: Structural and 

Functional Equivalence of Cry1Ab and Cry2Ae and PAT/bar Proteins Produced in 

Bacillus thuringiensis (Bt) and Escherichia coli to Cry1Ab, Cry2Aa and PAT/Bar in 

TwinLinkTM Cotton, Gossypium hirsutum, USA, 2007: Final Report. Project 

Number: CY07B006. Unpublished study prepared by Bayer CropScience, LP. 7 p.

48166300 Bayer CropScience LP-BioScience (2010) Submission of Efficacy Data in Support of 

the Experimental 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) TwinLinkTM 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 Bayer 

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

Biopesticides Registration Action Document January 23, 2012

Page 143 of 153

MRID Citation Reference (Bt Cry 1Ab PIP)

48427900 Bayer 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, FLCry1Ab, 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-IRO67-1). 

Project Number: 10/BM45L001. Unpublished study prepared by Bayer CropScience, 

LP. 22 p.

48471900 Bayer 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) Analysis to Determine if the Cry2Ae (GEM2) Protein from Cotton 

Leaves is Glycosylated: Final Report. Project Number: BK05B003. Unpublished 

study prepared by Bayer CropScience. 32 p.

48480000 Bayer CropScience LP (2011) Submission of Product Chemistry and Efficacy Data 

in Support of the Applications for Registration of BCS Cry1Ab Cotton Event T304-

40, BCS Cry2Ae Cotton Event GHB119 and BCS TwinLinkTM 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 by Southern Blot Analysis. Project 

Number: BBS07/009, M/308404/02/1. Unpublished study prepared by Bayer 

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. Unpublished study prepared by Bayer 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 Bayer BioScience N.V. 62 p.

48480004 Habex, V.; Van Herck, H. (2007) Confirmation of the Absence/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 Bayer BioScience N.V. 41 p.

48480005 Currier, T. (2011) Characterization of Cry1Ab 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 Bayer 

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 Bayer 

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

Biopesticides Registration Action Document January 23, 2012

Page 144 of 153

MRID Citation Reference (Bt Cry 1Ab PIP)

48480007 Currier, T. (2011) Structural Stability Analysis of Gossypium hirsutum Combined 

Event TwinLinkTM 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 Analysis for GlyTol x TwinLinkTM 

(GHB614 x T304-40 x GHB119) Cotton Grown in the Field, USA 2010. Project 

Number: 10BM99L440B, M/406245/01/1. Unpublished study prepared by Bayer 

CropScience LP. 125 p.

48480009 Bushey, D.; Jansens, S.; Becker, A. (2010) Insect Bioassay of Plant Material 

Expressing Cotton Events T304-40, GHB119, and TwinLinkTM (T304-40xGHB119). 

Project Number: BIO2/005, INSECT/BIOASSAY/FROM/MONHEIM. Unpublished 

study prepared by Bayer 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 (Cry1Ab) 

and GHB119 (Cry2Ae). Unpublished study prepared by Bayer CropScience LP. 16 

p.

48480011 Dennehy, T. (2011) High Dose Study: Estimating the Mortality of Heliothis 

virescens in a Greenhouse on Plants of T304-40 x GHB119 (TwinLinkTM) Cotton 

(BCS-GHOO4-7 x BCS-GHOO5-8) and Parental Event Plants T304-40 (Cry1Ab) 

and GHB119 (Cry2Ae). Project Number: BA44Y001. Unpublished study prepared 

by Bayer CropScience LP. 16 p.

48495100 Bayer CropScience (2011) Submission of Efficacy Data in Support of the 

Application for Registration of the TwinLinkTM. Transmittal of 1 Study.

48495101 Dennehy, T. (2011) A Risk Assessment of TwinLinkTM 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 January 23, 2012

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 TwinLinkTM Cotton. 

There may be repeats of those made for Cry2Ae in the Bibliography cited for Cry1Ab because they were 

submitted for TwinLinkTM Cotton, which contains both proteins.

MRID Citation Reference (Bt Cry 2Ae PIP)

46708900 Bayer 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 Bayer Cropscience. 120 p.

46708904 Rouquie, D. (2005) Cry2Ae (GEM2) Protein: Epitome Homology and NGlycosylation Searches. Project Number: SA/05134, TX99S006, B004974. 

Unpublished study prepared by Bayer 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 by 

Bayer Cropscience. 44 p.

46708906 Rouquie, D. (2005) Cry2Ae (GEM2) Protein: Acute Toxicity by Intravenous 

Injection in the Mouse. Project Number: SA/05033, B004972. Unpublished study

prepared by Bayer Cropscience. 80 p.

46708907 Currier, T. (2005) Analysis to Determine if the Cry2Ae (GEM2) Protein from Cotton 

Leaves in Glycosylated. Project Number: BK05B003, B004964. Unpublished study

prepared by Bayer 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 Bayer Cropscience LP. (2007) Submission of Toxicity Data in Support of the 

Experimental Use of Bacillus thuringiensis Cry1Ab and Bacillus thuringiensis

Cry2Ae for Use on Cotton. Transmittal of 2 Studies. 
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document January 23, 2012

Page 146 of 153

MRID Citation Reference (Bt Cry 2Ae PIP)

47076902 Rouque, D. (2006) Cry2Ae (GEM2) Protein: Acute Toxicity by Oral Gavage in 

Mice. Project Number: SA/06235, BBS06/010. Unpublished study prepared by

Bayer Cropscience. 62 p.

47125100 Bayer CropScience LP. (2007) Submission of Product Chemistry and Fate Data in 

Support of the Experimental Use of Bacillus thuringiensis Cry2Ae Insecticide 

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 study 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, TX99L005, M/287164/01/1. 

Unpublished study prepared by Bayer Cropscience. 42 p.

47640000 Bayer 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 TwinLinkTM Cotton Event T304-40 x GHB 119 Cotton. 

Transmittal of 14 Studies.

47640001 Ferullo J. (2008) TwinLinkTM Product Characterization - Molecular Characterization. 

Unpublished study prepared by Bayer BioScience N.V. 6 p.

47640002 Jesudason, P. (2008) Product Characterization - Nutritional Characterization of 

Twinlink. Unpublished study prepared by Bayer Cropscience LP. 8 p.

47640003 Mackie, S. (2008) Protein Expression Analysis for T304-40, GHB119 and 

TwinLinkTM Transgenic Cotton. Project Number: CP08B002, BBS07/013, 

BB06/010. Unpublished study prepared by Bayer Cropscience LP. 155 p.

47640004 Martone, A. (2008) Analyses of Raw Agricultural Commodity (Fuzzy Seed) of 

TwinLinkTM Cotton for PAT/bar, Cry2Ae and Cry 1Ab and its Non-Transgenic 

Counterpart for PAT/bar, Cry2Ae and Cry1Ab Proteins. Project Number: 

CY07B008, BTS/0066/06, BBS07/006. Unpublished study prepared by Bayer 

Cropscience LP. 216 p.

47640005 Martone, A. (2008) Structural and Functional Equivalence of Cry1Ab, Cry2Ae and 

PAT/bar Proteins Produce in Escherichia coli and Bacillus thuringiensis to the 

Cry1Ab, Cry2Ae and PAT/bar Proteins from Events T304-40, GHB119 and 

TwinLinkTM Cotton Seed, Gossypium hirsutum. USA, 2008. Project Number: 

CP08B011, BTS/0077/08, BBS07/006. Unpublished study prepared by Bayer 

Cropscience LP. 70 p.

47640006 Martone, A. (2008) Structural and Functional Equivalence of Cry1Ab, Cry2Ae and 

PAT/bar Proteins Produced in Bacillus thuringiensis (Bt) and Escherichia coli to 

Cry1Ab, Cry2Ae and PAT/bar in TwinLinkTM Cotton, Gossypium hirsutum, USA, 

2007. Project Number: CY07B006, BBS07/013, BIOX/001/PROTSUPP/07. 

Unpublished study prepared by Bayer Cropscience LP. 402 p.
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document January 23, 2012

Page 147 of 153

MRID Citation Reference (Bt Cry 2Ae PIP)

47640007 Bushey, D. (2008) Toxicology: Summary Human Health Assessment. Unpublished 

study prepared by Bayer BioScience N.V. 8 p.

47640008 Chalmers, A. (2008) Summary of Non-Target Organism Testing and Assessment of 

Risk of TwinLinkTM Cotton Expressing Cry1Ab and Cry2Ae Proteins. Project 

Number: ECORA3. Unpublished study prepared by Bayer Cropscience LP. 17 p.

47640009 Richards, K. (2008) Evaluation of the Dietary Effect(s) of Pollen from TwinLinkTM 

on Honey Bee Larvae (Apis mellifera L.) Development. Project Number: EB99X037, 

CAR/204/08, BTS/0081/08. Unpublished study prepared by California Agricultural 

Research, Inc. and Bayer CropScience Ecotoxicology Department. 48 p.

47640010 Patnaude, M. (2009) Chronic Toxicity to Collembola (Folsomia candida) Using 

Transgenic Lyophilized TwinLinkTM 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 TwinLinkTM 

Cotton. Project Number: 13798/4118, TX99X106, M/310302/01/1. Unpublished 

study prepared by Springborn Smithers Laboratories. 223 p.

47640012 Rinehardt, M.; Holloway, J.; Bushey, D. (2008) Agronomic Performance and 

Efficacy of TwinLinkTM Cotton Obtained by Conventional Breeding of Cry1Ab 

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 Bayer 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 (TwinLinkTM Cotton) Plants. Unpublished study prepared by

Bayer CropScience LP. 36 p.

47640014 Maclntosh, S.; Holloway, J.; Jansens, S.; et al. (2008) Insect Resistance Management 

Plan: TwinLinkTM Cotton. Unpublished study prepared by Bayer CropScience LP. 88 

p.

47641900 Bayer 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 thuringiensis Cry2Ae. Transmittal 

of 26 Studies.

47641901 Ferullo, J. (2008) Product Characterization of Cry2Ae Cotton Event GHB119. 

Unpublished study prepared by Bayer BioScience N.V. 24 p.

47641902 Jesudason, P. (2008) Product Characterization - Nutritional Characterization of 

Cry2Ae Protein. Unpublished study prepared by Bayer CropScience. 6 p.

47641903 Currier, T. (2008) Protein Expression Analysis of Cotton Event GHB119, Expressing 

Cry2Ae and PAT/bar Proteins. Project Number: CY07B005. Unpublished study

prepared by Bayer CropScience LP. 161 p.

47641904 Martone, A. (2008) Analyses of Raw Agricultural Commodity (Fuzzy Seed) of 
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document January 23, 2012

Page 148 of 153

MRID Citation Reference (Bt Cry 2Ae PIP)

Cry2Ae Cotton Event GHB119 for PAT/bar and Cry2Ae and its Non-Transgenic 

Counterpart for PAT/bar and Cry2Ae Proteins. Project Number: CY07B001. 

Unpublished study prepared by Bayer 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 Proteins in GHB119 Cotton, Gossypium hirsutum. Project Number 

CY07B002. Unpublished study prepared by Bayer CropScience. 57 p.

47641906 Capt, A.; Nennstiel, D. (2008) Toxicology (Human Health Assessment): Summary of 

Analysis of Open Reading Frame Homology Searches. Unpublished study prepared 

by Bayer Cropscience. 10 p.

47641907 Rouquie, D. (2008) Cry2Ae Protein: In Vitro Digestibility Study in Simulated 

Intestinal Fluid. Project Number: SA08127. Unpublished study prepared by Bayer 

Cropscience. 52 p.

47641908 Rouquie, D. (2008) Cry2Ae Protein with Transit Peptide Epitope Homology and NGlycosylation Searches. Project Number: SA08209, TX99L003. Unpublished study

prepared by Bayer 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, TX99L004. Unpublished study prepared by Bayer 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-Glycosylation 

Searches. Unpublished study prepared by Bayer BioScience N.V. 6 p.

47641911 Rouquie, D. (2008) Cry2Ae Protein: Heat Stability Study. Project Number: 

SA08128, TX99L036. Unpublished study prepared by Bayer Cropscience. 49 p.

47641912 Nennstiel, D. (2008) Cry2Ae Protein: History of Safe Use. Unpublished study

prepared by Bayer BioScience N.V. 7 p.

47641913 Chalmers, 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 Bayer 

CropScience LP. 50 p.

47641914 Richards, K. (2008) Evaluation of the Dietary Effect(s) of a Cry2Ae Protein on 

Honey Bee Larvae. Project Number: EB99L001, CAR178/07. Unpublished study

prepared by 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 (Chrysoperla 

rufilabris). Unpublished study prepared by Bayer CropScience LP. 4 p.
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document January 23, 2012

Page 149 of 153

MRID Citation Reference (Bt Cry 2Ae PIP)

47641917 Patnaude, M. (2008) Chronic Toxicity to Collembola (Folsomia candida) using 

GEM2 Proteins. Project Number: EB99X0081, 13798/6204. Unpublished study

prepared by Springborn Smithers Laboratories. 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.

47641919 Sayers, 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 Cry2Ae Protein Produced from Bacillus thuringiensis after Aerobic 

Soil Degradation. Project Number: CY07B007. Unpublished study prepared by

Bayer CropScience LP. 152 p.

47641921 Jesudason, P. (2008) Cry2Ae: Efficacy Assessment (Field and Laboratory). 

Unpublished study prepared by Bayer 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 Bayer 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 Bayer 

CropScience LP. 6 p.

47641924 Nennstiel, D. (2008) Detection Methods: rtPCR (Cry2Ae). Unpublished study

prepared by Bayer BioScience N.V. 7 p.

47641925 Nennstiel, D. (2008) Protein Detection Methods: Lateral Flow Strip (Cry2Ae). 

Unpublished study prepared by EnviroLogix Inc. 8 p.

47641926 Nennstiel, D. (2008) Protein Detection Methods: Enzyme Linked Immuno Sorbent 

Assay (ELISA) (Cry2Ae). Unpublished study prepared by EnviroLogix, Inc. 10 p.

47692500 Bayer 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 (Chrysoperla 

rufilabris) Following OPPTS Guideline 885.4340. Project Number: 13798/6229, 

EB99X025, BBS07/007. Unpublished study prepared by Springborn Smithers 

Laboratories. 65 p.

47871400 Bayer CropSciences (2009) Submission of Product Chemistry, Environmental Fate 

and Toxicity Data in Support of the Application for Registration of BCS Cry1Ab 

Cotton Event 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 Outlined 
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document January 23, 2012

Page 150 of 153

MRID Citation Reference (Bt Cry 2Ae PIP)

in 40CFR 158.21-Cry1Ab. Unpublished study prepared by Bayer CropScience LP. 7 

p.

47871404 Hunst, P. (2009) Bridging Data from TwinLinkTM Cotton Avian Nutritional Study to 

Parental Events T304-40 and GHB119. Unpublished study prepared by Bayer 

CropScience LP. 7 p.

47871500 Bayer CropScience, LP (2009) 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 Analysis Studies as Outlined in 40CFR 158.2120-Cry2Ae. Unpublished 

study prepared by Bayer 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 Bayer 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-Cry2Ae. Unpublished study prepared by Bayer CropScience, 

LP. 7 p.

47871504 Hunst, P. (2009) Bridging Data from TwinLinkTM Cotton Avian Nutritional Study to 

Parental Events T304-40 and GHB119. Unpublished study prepared by Bayer 

CropScience, LP. 7 p.

47871600 Bayer CropScience, LP (2009) Submission of Product Chemistry and Toxicity Data 

in Support of the Application for Registration of BCS TwinLinkTM Cotton Event 

T304-40xGHB 119 Cotton. Transmittal of 6 Studies. 

47871601 Coats, I. (2009) Request for Waivers from the Requirement to Conduct Certain 

Product Analysis Studies. Unpublished study prepared by Bayer 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 TwinLink. Unpublished 

study prepared by Bayer 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 - TwinLink. 

Unpublished studies prepared by Bayer CropScience. 7 p.

47871604 Currier, T. (2009) Protein Expression Analysis for Pat/bar, Cry1Ab and Cry2Ae 

Proteins in Whole TwinLinkTM Plants Grown in the Field. Project Number: 

CP09Q005. Unpublished study prepared by Bayer CropScience, LP. 28 p.

47871605 Hunst, P. (2009) Investigation of Synergistic Effects of the Cry1Ab and Cry2Ae 

Proteins expressed in TwinLinkTM Cotton (BCS-GH004-7 x BCS-GH005-8) on 

Target Insect Pests Using Leaf Bioassay. Unpublished study prepared by Bayer 

CropScience, LP. 8 p.
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document January 23, 2012

Page 151 of 153

MRID Citation Reference (Bt Cry 2Ae PIP)

47871606 Coats, I. (2009) Supplement to MRID # 47640014: Insect Resistance Management 

Plan: TwinLinkTM Cotton. Unpublished study prepared by Bayer CropScience, LP. 7 

p.

47903100 Bayer CropScience LP (2009) Submission of Toxicity Data in Support of the 

Application for Registration of BCS TwinLinkTM 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 Bayer CropScience 

LP. 16 p.

47903300 Bayer 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 Bayer CropScience 

LP. 16 p.

47928800 Bayer CropScience, LP-BioScience. (2009) Submission of Efficacy Data in Support 

of the Registration of BSC TwinLinkTM Cotton Event T304-40 X GHB 119 Cotton. 

Transmittal of 1 Study.

47928801 Coats, I. (2009) Addendum to Product Chemistry for TwinLink: Structural and 

Functional Equivalence of Cry1Ab and Cry2Ae and PAT/bar Proteins Produced in 

Bacillus thuringiensis (Bt) and Escherichia coli to Cry1Ab, Cry2Aa and PAT/Bar in 

TwinLinkTM Cotton, Gossypium hirsutum, USA, 2007: Final Report. Project 

Number: CY07B006. Unpublished study prepared by Bayer CropScience, LP. 7 p.

48177500 Bayer CropScience LP (2010) Submission of Toxicity Data in Support of the 

Applications for Registration of BCS TwinLinkTM 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. Transmittal of 1 Study.

48177501 Stafford, J. (2010) Northern Bobwhite (Colinus virginianus) Dietary Toxicity Test 

(LC50) with GHB119 (Cry2AE) Cottonseed Meal. Project Number: 13798/4130, 

TX44L001. Unpublished study prepared by Springborn Smithers Laboratories. 60 p.

48427900 Bayer 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, FLCry1Ab, 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-IRO67-1). 

Project Number: 10/BM45L001. Unpublished study prepared by Bayer CropScience, 

LP. 22 p.

48471900 Bayer 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 January 23, 2012

Page 152 of 153

MRID Citation Reference (Bt Cry 2Ae PIP)

48471901 Currier, T. (2011) Analysis to Determine if the Cry2Ae (GEM2) Protein from Cotton 

Leaves is Glycosylated: Final Report. Project Number: BK05B003. Unpublished 

study prepared by Bayer CropScience. 32 p.

48480000 Bayer CropScience LP (2011) Submission of Product Chemistry and Efficacy Data 

in Support of the Applications for Registration of BCS Cry1Ab Cotton Event T304-

40, BCS Cry2Ae Cotton Event GHB119 and BCS TwinLinkTM 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 by Southern Blot Analysis. Project 

Number: BBS07/009, M/308404/02/1. Unpublished study prepared by Bayer 

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. Unpublished study prepared by Bayer 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 Bayer BioScience N.V. 62 p.

48480004 Habex, V.; Van Herck, H. (2007) Confirmation of the Absence/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 Bayer BioScience N.V. 41 p.

48480005 Currier, T. (2011) Characterization of Cry1Ab 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 Bayer 

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 Bayer 

BioScience N.V. 35 p.

48480007 Currier, T. (2011) Structural Stability Analysis of Gossypium hirsutum Combined 

Event TwinLinkTM 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 Analysis for GlyTol x TwinLinkTM 

(GHB614 x T304-40 x GHB119) Cotton Grown in the Field, USA 2010. Project 

Number: 10BM99L440B, M/406245/01/1. Unpublished study prepared by Bayer 

CropScience LP. 125 p.

48480009 Bushey, D.; Jansens, S.; Becker, A. (2010) Insect Bioassay of Plant Material 

Expressing Cotton Events T304-40, GHB119, and TwinLinkTM (T304-40xGHB119). 

Project Number: BIO2/005, INSECT/BIOASSAY/FROM/MONHEIM. Unpublished 
Bacillus thuringiensis Cry1Ab, Cry2Ae, TwinLinkTM (Cry1Ab, 2Ae)

Biopesticides Registration Action Document January 23, 2012

Page 153 of 153

MRID Citation Reference (Bt Cry 2Ae PIP)

study prepared by Bayer 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 (TwinLink) 

Cotton (BCS-GHOO4-7 x BCS-GHOO5-8) and Parental Events T304-40 (Cry1Ab) 

and GHB119 (Cry2Ae). Unpublished study prepared by Bayer CropScience LP. 16 

p.

48480011 Dennehy, T. (2011) High Dose Study: Estimating the Mortality of Heliothis 

virescens in a Greenhouse on Plants of T304-40 x GHB119 (TwinLink) Cotton 

(BCS-GHOO4-7 x BCS-GHOO5-8) and Parental Event Plants T304-40 (Cry1Ab) 

and GHB119 (Cry2Ae). Project Number: BA44Y001. Unpublished study prepared 

by Bayer CropScience LP. 16 p.

48495100 Bayer CropScience (2011) Submission of Efficacy Data in Support of the 

Application for Registration of the TwinLink. Transmittal of 1 Study.

48495101 Dennehy, T. (2011) A Risk Assessment of TwinLinkTM 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 safety information on transgenic plants expressing Bacillus thuringiensis-derived

insect control protein. 109 pages.