BIOPESTICIDES REGISTRATION ACTION DOCUMENT

Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 Proteins and the Ge netic Material Necessary for Their Production (PHP17662 T-DNA) in Event DAS-59122-7 Co rn (OECD Unique Identifier: DAS-59122-7)

PC Code: 006490

U.S. Environmental Protection Agency Office of Pesticide Programs Biopesticides and Pollution Prevention DivisionBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

2 

### Table of Contents

I. OVERVIEW

A. Background 4

B. Use Profile 7

C. Regulatory History 8

II. SCIENCE ASSESSMENT

A. Product Characterization 14

B. Human Health Assessment 41

C. Environmental Assessment 66

D. Insect Resistance Management 117

E. Benefits 172

III. REGULATORY POSI TION FOR EVENT DAS-59122-7 CORN

A. Initial Registration (August 31, 2005) 193

B. 2010 Update 195

C. Period of Registration 197

APPENDIX A 199Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

3 Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 Proteins and the Ge netic Material Necessary for Their Production (PHP17662 T-DNA) in Event DAS -59122-7 Corn Regulatory Action Team

Product Characterization and Human Health Rebecca Edelstein, Ph.D. John Kough, Ph.D. Annabel Waggoner Chris Wozniak, Ph.D.

Environmental Fate and Effects Joel Gagliardi, Ph.D. Hillary Hill Tessa Milofsky Robyn Rose Zigfridas Vaituzis, Ph.D. Annabel Waggoner

Insect Resistance Management

Jeannette Martinez Sharlene Matten, Ph.D. Tessa Milofsky Alan Reynolds

Benefits Assessment

Edward Brandt Sharlene Matten, Ph.D. Tessa Milofsky

Registration Support

Mike Mendelsohn Sheryl Reilly, Ph.D.

Ann Sibold

Biopesticides Registration Ac tion Document Team Leaders

Jeannine Kausch

Mike Mendelsohn

Office of General Counsel Angela Huskey, Esq.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

4 I. OVERVIEW

A. Background

On August 31, 2005, the Environmental Protection Ag ency (EPA) issued time-limited, conditional registrations to Mycogen Seeds c/o Dow AgroScie nces LLC (EPA Reg. No. 68467-5) and Pioneer HiBred International, Incorporated (EPA Reg. No. 29964-4) for event DAS-59122-7 corn, a plantincorporated protectant expr essing the active ingredient, Bacillus thuringiensis ( Bt ) Cry34Ab1 and Cry35Ab1 proteins and the genetic material necessary for their pr oduction (PHP17662 T-DNA) in event DAS-59122-7 corn (Organization for Economic Cooperation and Development (OECD) Unique Identifier: DAS-59122-7). Event DAS-59122-7 corn, at the time of 2005 registration, expressed only the second plant-incorporated protectant (PIP) active ingredient to offer protection ag ainst corn rootworm, and expectations were that adaptation of this new technology would result in further reduction of conventional insecticide use by grower s attempting to control the highly destructive corn rootworm and maintain their crop yields. Prior to registration and after extensive review of copious amounts of data/information submitted by the appli cants, the Agency determined that the use of this pesticide was in the public interest and that it would not cause any unreasonable a dverse effects on the environment during the period of time-limited (5 year), conditional registration.

Subsequent to registration of th e single-trait products in 2005, the Agency registered stacked and/or pyramided plant-incorporated protectants (PIPs) , expressing Cry34/35Ab1 along with other proteins, and two seed blends for either commercial or limited breeding purposes. A comple te list of the currently registered products expressing Cry34/35Ab1—including their respective registra tion numbers, product names, registrants, initial dates of registration, pr oteins (or active ingred ients) expressed, and any limitations/special notes—can be found in Appendix A. In conj unction with the 2010 evaluation (explained in the paragraphs that follow), the Agency attempted to better deta il and describe product characterization, human health, enviro nmental effects, and insect resist ance management data that were submitted to support the registrations of the follo wing stacked and/or pyramided products expressing Cry34/35Ab1:

(1) Herculex® XTRA Insect Protection Co rn (EPA Reg. Nos. 68467-6 and 29964-5) (2) 1507 x 59122 x MON 810 (EPA Reg. No. 29964-8) (3) 59122 x MON 810 (EPA Reg. No. 29964-9)

When the first products containing Cry34/35Ab1 were registered by the Agency, Mycogen Seeds c/o Dow AgroSciences LLC and Pioneer Hi-Bred Intern ational, Incorporated were issued time-limited, conditional registrations under sectio n 3(c)(7)(C) of the Fede ral Insecticide, Fungi cide, and Rodenticide Act (FIFRA). Along with several requirements fo r further product charact erization, environmental effects, and insect resistance management data, th e registration notices also clearly established an absolute expiration date of September 30, 2010. Th e follow-on stacks and/or pyramids, specifically mentioned above, all had the same expiration da te with the exception of 1507 x 59122 x MON 810 and 59122 x MON 810, which are set to expire on Oct ober 31, 2010. The registrants for the Cry34/35Ab1Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

5 single-trait and combination PIPs, due to expire on either September 30, 2010 or October 31, 2010, formally requested that the Agency amend their a ssociated products to extend the expiration dates.

On October 1, 2009, EPA announced a policy to provide a more meaningful opportunity for the public to participate on major registration d ecisions before they occur. According to this policy, EPA intends to provide a public comment period prior to making a re gistration decision for, at minimum, the following types of applications: new active ingr edients; first food uses; first outdoo r uses; first residential uses; and other actions for which the Agency anticipates th at there will be signi ficant public interest.

Consistent with the policy of ma king registration actions more transparent, the amendments to the expiring Cry34/35Ab1 corn products were subject to a 30-day comment period because the Agency believed, given past experiences with PIPs in general, these actions would be of significant interest to the public. During this comment period, several comments were received from the following stakeholders: Mycogen Seeds c/o Dow AgroSciences LLC; Pioneer Hi-Bred International, Incorporated; Monsanto Company; National Corn Growers Associ ation; Agricultural Biotechnology Stewardship Technical Committee; Center for Sc ience in the Public Interest; a nd Association of American Seed Control Officials. After reviewing and considering all of the public comments received, the Agency still maintains that, based on all data submitted in suppor t of the Cry34/35Ab1 corn registrations (both for initial registrations and as responses to conditions of registration), it is in the best interest of the public and the environment to amend the currently ex isting Cry34/35Ab1 registra tions by extending their expiration dates in accordance w ith the scheme explained in section III(C) of this Biopesticides Registration Action Document (BR AD). The basis for this decision can be found in both the risk assessment for the Cry34/35Ab1 corn products, which is characterized throughout this BRAD, and the Agency’s response to comments document.

All data and findings for the Cry34/35Ab1 corn pr oducts are presented within the standard BRAD configuration for PIPs (i.e., information is placed into separate and distinct chapters according to scientific discipline or regulatory focus); this should be the most familiar format to outside stakeholders interested in reading further about these actions. In addition to th e Cry34/35Ab1 corn products, there are

other Bt corn PIPs, expressing different proteins effective in controlling corn borers or corn rootworm, that were due to expire in 2010, and for which the asso ciated registrants formally requested an extension to expiration dates. Therefore, within the same docket (EPA-HQ-O PP-2010-0607) as this document, the following information a is also available for public examination:

● Cry1F and Cry1Ab BRAD (Draft - August 2010; Final - September 2010)

● Cry3Bb1 BRAD (Draft - July 2010; Final - September 2010)

● mCry3A BRAD (Draft - July 2010; Final - September 2010)

● Cry1A.105 and Cry2Ab2 BRAD (Draft - August 2010; Final - September 2010)

a Each of the Biopesticides Registration Action Documents in this action are modified from previous versions to account for data/information submitted to fulfill terms and conditions of registration (see draft and final versions) and to respond, in pa rt,
to comments received on the informati on presented in Docket Numb er EPA-HQ-OPP-2010-0607 (see final versions only). All documents presented in the list can be retrieved from the following website: http://www.regulations.gov

.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

6 ● Optimum® AcreMaxTM B

. t . Seed Blends BRAD (Draft - August 2010; Final - September 2010)

● Current Registration Terms and Conditions for Bt Corn Registrations Set to Expire in 2010

● Proposed Registration Terms and Conditions for Bt Corn Registrations Set to Expire in 2010

● Registration Terms and Conditions Established with the Finalized Amendments

● BPPD mCry3A, Cry3Bb1, and Cry34/35Ab1 Corn Rootworm Monitoring Reviews (June 2010)

● Public Comments on EPA Dock et Number EPA-HQ-OPP-2010-0607

● EPA’s Response to Comments

EPA made the decision to amend the regi strations of eighteen (18) expiring Bt corn PIP registrations to extend the expiration dates. We conducted comprehe nsive assessments of each of these registrations, considering all toxicity and envi ronmental effects data, data from insect resistance monitoring, and insect resistance refuge compliance reports, rece ived and obtained since the last comprehensive evaluation of these products in 2001. Based upon our comprehensive assessment, we reached significant conclusions regarding the posit ive environmental impact of Bt corn PIPs, and we took several actions to strengthen the insect resistance management requireme nts to ensure continued success in the prevention of the evolution of resistance in target pests.

Since the commercialization of Bt crops, there have been a significan t number of published field studies that, combined with the post-registration field stud ies required to be submitted to the Agency, have demonstrated that non-target inverteb rates are generally more abundant in Bt cotton and Bt corn fields than in non-transgenic fields mana ged with chemical insecticides. T hus, these published and registrantproduced studies demonstrate that, not only are the Bt crops not causing any unreasonable adverse effects in the environment, but, arthropod prevalence and diversity is greater in Bt crop fields.

To strengthen insect resistance management of these corn PIPs and to address reports that compliance with the mandated refuge requirements has been decreasing, EPA is requiring enhanced compliance assurance programs (CAPs), and a phased requiremen t for seed bag labeling that clearly shows the refuge requirements. Also, given the increasing va riety of PIP products and combinations, and the differing risk of resistance evolution that the various products represent, we are granting registrations for the corn PIP products for different time frames, ba sed on assessments of thei r likelihood of forestalling

the evolution of insect resistance. We are registering di ffering categories of pr oducts for differing time periods to reflect the assessed leve l of risk of resistance posed by the various corn PIP products. The scheme that we are following includes registration periods generally of five, ei ght, and twelve years; with the possibility of a fifteen-y ear registration period for products that are demonstrated to meet specified criteria. We retain, howev er, the discretion to register produc ts for time periods differing from

these defaults where circumstances warrant.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

7 B. Use Profile

Pesticide Name: Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins and the genetic material necessary for their produ ction (PHP17662 T-DNA) in event DAS59122-7 corn (OECD Unique Identifier: DAS-59122-7)

Trade and Other

Names: Event DAS-59122-7 Corn, Herculex® Root worm Insect Protection, Herculex® RW Insect Protection, Herculex® RW Rootworm Protection, Herculex® RW

OPP Chemical Code: 006490

Basic Manufacturers: Mycogen Seeds c/o Dow AgroSciences LLC

9330 Zionsville Road Indianapolis, IN 46268-1054

Pioneer Hi-Bred Intern ational, Incorporated

7100 N.W. 62nd Avenue

P.O. Box 1000 Johnston, IA 50131-1000

Type of Pesticide: Plant-Incorporated Protectant

Use: Field Corn

Target Pests: western corn rootworm ( Diabrotica virgifera virgifera ), northern corn

rootworm ( Diabrotica barberi ), and Mexican corn rootworm ( Diabrotica virgifera zeae )

Products Expressing

This Pesticide: See complete list in Appendix A.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

8 C. Regulatory History

Date Action Type Description

March 7, 2003 Federal Register

Publication

(Notice of Filing) Notice of Filing summarizing information submitted and cited by Mycogen Seeds c/o Dow AgroSciences LLC in support of a request for establishment of a temporary exemption from the requi rement of a tolerance for residues of the plant-incorporated protectant, Bacillus thuringiensis Cry34/35Ab1 insecticidal crystal protein and the genetic material necessary for its production in corn.

( 68 Federal Register (FR) 11100 )

March 13, 2003 Federal Register

Publication

(Notice of Receipt) Notice announcing receipt of appli cations 68467-EUP- 3, 68467-EUP-5, 68467-EUP-T, 68467-EUP-I, 29964-EUP-1, 29964-EUP-3, 29964-EUP-U, and 29964-EUP-L from Mycogen Seeds c/o Dow AgroSciences LLC and Pioneer Hi-Bred International, Incorporated requesting experimental use permits and experimental use permit amendments for the following:

(1) Bacillus thuringiensis Cry34/35Ab1protein and the genetic material necessary for its production (from the insert of plasmid PHP 14352) in corn (2) Bacillus thuringiensis Cry34/35Ab1 protein and the genetic material necessary for its production (from the insert of plasmid PHP 12560) in corn (3) Bacillus thuringiensis Cry34/35Ab1 protein and the genetic material necessary for its production (from the insert of plasmid PHP 17662) in corn (4) Bacillus thuringiensis Cry34/35Ab1 protein and the genetic material necessary for its production (from the insert of plasmid PHP 17658) in

corn
( 68 FR 12073 )Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

9 Date Action Type Description

July 7, 2003 Federal Register

Publication

(Final Rule) The following temporary exemption from the requirement of a tolerance was established under 40 Code of Federal Regulations (CFR) § 180.1242:

“ Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins and the genetic material necessary for their production in corn are temporarily exempted from the requirement of a tolerance when used as plantincorporated protectants in the food and feed commodities of field corn, sweet corn and popcorn. This temporary exemption from the requirement of a tolerance will permit the use of the food commodities in this paragraph when treated in acco rdance with the provisions of the experimental use permits 68467-EUP-3, 68467-EUP-5, 68467-EUP-T(7), 68467-EUP-I(8), 29964-EUP-1, 29964-EUP-3, 29964-EUP-U(4), and 29964-EUP-L(5) which may be issued and amended/extended under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), as amended (7 U.S.C. 136). This temporary exemption from the requirement of a tolerance expires and is revoked April 30, 2006. This temporary exemption from the requirement of a tolerance may be revoked at any time if the experimental use permit is revoked or if any experience with or scientific data on this pesticide indicate that the tolerance

is not safe.”

( 68 FR 40178 )

January 6, 2004 Federal Register

Publication

(Notice of Issuance) Notice announcing issuance of experimental use permits to Mycogen Seeds c/o Dow AgroSciences LLC (68467-EUP-7) and Pioneer Hi-Bred International, Incorporated (29964-EUP -5). Each permit allowed for use of Bacillus thuringiensis Cry34/35Ab1 proteins and the genetic material necessary for their production (from the insert of plasmid PHP17662) in corn on 624 acres of field corn and was effective from June 24, 2003 to May 1, 2004.

( 69 FR 658 )

March 10, 2004 Federal Register

Publication

(Notice of Receipt) Notice announcing receipt of applica tions from Mycoge n Seeds c/o Dow AgroSciences LLC and Pioneer Hi-Bred International, Incorporated requesting amendments/extensions to their experimental use permits (68467-EUP-7 and 29964-EUP-5, respectively).

( 69 FR 11431 )

August 31, 2004 Federal Register

Publication

(Notice of Filing) Notice of Filing summarizing information submitted and cited by Mycogen Seeds c/o Dow AgroSciences LLC in support of a request for the establishment of a permanent exem ption from the requirement of a tolerance for residues of the plant-incorporated protectant, Bacillus thuringiensis Cry 34Ab1 and Cry35Ab1 proteins and the genetic material necessary for their production in corn.

( 69 FR 53060 )Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

10 Date Action Type Description

September 1, 2004 Federal Register

Publication

(Notice of Receipt) Notice announcing receipt of the following applications for products containing new active ingredients:

(1) File Symbol : 29964-U. Applicant : Pioneer Hi-Bred International, A DuPont Company, 7250 N.W. 62nd Ave., P.O. Box 552, Johnston, IA

50131-0552. Product Name : Pioneer Brand B

. t . Cry34/35Ab1 Insect Resistant Corn Seed. Plant-incorpor ated protectant. Active Ingredient : Bacillus thuringiensis Cry34/35Ab1 insecticidal crystal protein and the genetic material for its production (plasmid insert PHP 17662) in event

DAS-59122-7 corn. (2) File Symbol : 68467-L. Applicant : Mycogen Seeds c/o Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268. Product

Name : Mycogen Brand B

. t . Cry34/35Ab1 Construct 17662 Corn. Plantincorporated protectan t. Active Ingredient : Bacillus thuringiensis Cry34/35Ab1 insecticidal crystal protein and the genetic material for its production (plasmid insert PHP 17662) in event DAS-59122-7 corn.

( 69 FR 53434 )

December 22, 2004 Federal Register

Publication

(Notice of Issuance) Notice announcing extension/amendment of experimental use permits previously approved for Mycogen Seeds c/o Dow AgroSciences LLC (68467-EUP-7) and Pioneer Hi-Bred International, Incorporated (29964- EUP-5).

(1) 68467-EUP-7 : For use of 0.94 pounds of Bacillus thuringiensis Cry34/35Ab1 proteins and the genetic material necessary for their production (from the insert of plasmid PHP17662) in corn on 1,177 acres of field corn; Effective from April 29, 2004 to April 30, 2005.

(2) 29964-EUP-5 : For use of 3.97 pounds of Bacillus thuringiensis Cry34/35Ab1 proteins and the genetic material necessary for their production (from the insert of plasmid PHP17662) in corn on 4,690 acres of field corn; Effective from April 29, 2004 to April 30, 2005.

( 69 FR 76732 )Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

11 Date Action Type Description

June 3, 2005 Federal Register

Publication

(Notice of Receipt) Notice announcing receipt of the following applications for products containing new active ingredients:

(1) File Symbol : 29964-L. Applicant : Pioneer Hi-Bred International, A DuPont Company, 7250 N.W. 62nd Ave., P.O. Box 552, Johnston, IA

50131-0552. Product Name : Herculex Xtra Insect Protection. Plantincorporated protectan t. Active Ingredient : Bacillus thuringiensis Cry34/35Ab1 insecticidal crystal protein and the genetic material for its production (plasmid insert PHP 17662) in event DAS-59122-7 corn and Bacillus thuringiensis Cry1F protein and the genetic material for its production (plasmid insert PHI 8999) in corn. (2) File Symbol : 68467-A. Applicant : Mycogen Seeds c/o Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268. Product

Name : Mycogen Seeds Herculex Xtra Insect Protection. Plant-incorporated protectant. Active Ingredient : Bacillus thuringiensis Cry34/35Ab1 insecticidal crystal protein and the genetic material for its production (plasmid insert PHP 17662) in event DAS-59122-7 corn and Bacillus thuringiensis Cry1F protein and the genetic material for its production (plasmid insert PHI 8999) in corn.

( 70 FR 32610 )

August 10, 2005 Federal Register

Publication

(Notice of Issuance) Notice announcing extension/amendment of experimental use permits previously approved for Mycogen Seeds c/o Dow AgroSciences LLC (68467-EUP-7) and Pioneer Hi-Bred International, Incorporated (29964- EUP-5).

(1) 68467-EUP-7 : For use of 2,734.85 grams Cry34Ab1 and 10.88 grams Cry35Ab1 of the insecticides Cry34/35Ab1 proteins and the genetic material necessary for their production (from the insert of plasmid PHP17662) in corn on 3,096 acres of field corn; Effective from January 21, 2005 to April 30, 2006.

(2) 29964-EUP-5 : For use of 1,813.6 grams Cry34Ab1 and 47.2 grams Cry35Ab1 of the insecticides Cry34/35Ab1 proteins and the genetic material necessary for their production (from the insert of plasmid PHP17662) in corn on 5,115 acres of field corn; Effective from January 21, 2005 to April 30, 2006.

( 70 FR 46510 )Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

12 Date Action Type Description

August 31, 2005 Registration The Agency issued time-limited, co nditional registration notices (under FIFRA section 3(c)(7)(C)) for event DAS-59122-7 corn to Mycogen Seeds c/o Dow AgroSciences LLC (EPA Reg. No. 68467-5) and Pioneer Hi-Bred International, Incorporated (EPA Reg. No. 29964-4).

*Expiration Date : September 30, 2010

September 21, 2005 Federal Register

Publication

(Final Rule) The following permanent exemption from the requirement of a tolerance was established under 40 CFR § 174.457:

“ Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins and the genetic material necessary for their production in corn are exempted from the requirement of a tolerance when used as plant-incorporated protectants in the food and feed commodities of corn; corn, field; corn, sweet; and corn, pop.”

( 70 FR 55254 )

October 27, 2005 Registration The Agency issued time-limited, co nditional registration notices (under FIFRA section 3(c)(7)(A)) for Herc ulex® Xtra Insect Protection to Mycogen Seeds c/o Dow AgroSciences LLC (EPA Reg. No. 68467-6) and Pioneer Hi-Bred International, Incorporated (EPA Reg. No. 29964-5).

*Expiration Date : October 15, 2008

April 25, 2007 Federal Register

Publication

(Direct Final Rule) The tolerance exemption for Cry34/35Ab1 proteins was redesignated from 40 CFR § 174.457 to 40 CFR § 174.506 and changed to the following:

“Residues of Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn are exempted from the requirementof a tolerance when used as plantincorporated protectants in the food and feed commodities of corn; corn, field; corn, sweet; and corn, pop.”

( 72 FR 20431 )

August 15, 2007 Federal Register

Publication

(Notice of Issuance) Notice announcing conditional approval of two products (Herculex® Rootworm Insect Protection, EPA Reg. No. 29964-4; Herculex® RW Insect Protection, EPA Reg. No. 68467-5) containing Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins and the genetic materialnecessary for their production (plasmid insert PHP17662) in event DAS59122-7 corn, an active ingredient not included in any previously registered product.

( 72 FR 45807 )Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

13 Date Action Type Description

October 8, 2008 Amendment The Herculex® Xtra Insect Protection registrations (EPA Reg. Nos. 29964- 5 and 68467-6) were amended by the Agency to extend the expiration date set forth in the original registration notices.

*New Expiration Date : September 30, 2010

July 2009–April

2010 Registration The Agency registered several co mbination PIP products expressing Cry34/35Ab1 and another protein(s), as well as two seed blends. See Appendix A for the complete list.

September 2010 Amendment The Herculex® Xtra Insect Protection (EPA Reg. Nos. 29964-5 and 68467-6), Herculex® Rootworm Insect Protection (EPA Reg. No. 29964- 4), and Herculex® RW Insect Protection (EPA Reg. No. 68467-5) registrations were amended by the Agency to extend the expiration date in accordance with the scheme explained in section III(C) of this Biopesticides Registration Action Document (BRAD).

*New Expiration Date : September 30, 2015Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

14 II. SCIENCE ASSESSMENT

The classifications that are found for each data submission are assigned by Environmental Protection Agency (EPA) science reviewers and are an indication of the usefulness of the information contained in the documents for risk assessment. A rating of “A CCEPTABLE” 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 (“SUPPLEMEN TAL: UPGRADABLE”). If a study is rated as “SUPPLEMENTAL: UPGRADABLE,” EP A always provides an indication of what is lacking or what can be provided to change the rating to “ACCE PTABLE.” If there is simply a “SUPPLEMENTAL” rating, the reviewer will often stat e 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 the study is not useful for risk assessment and cannot be upgraded.

A. Product Characterization

1. Event DAS-59122-7 Corn (Organization for Economic Cooperation and Development (OECD) Unique Identifier: DAS-59 122-7) Expressing Cry34Ab1 and Cry35Ab1

a. Data Cited/Submitted for Initial Registra tions of Event DAS-59122-7 Corn (Prior to August 2005)

The Agency’s detailed assessment of the product characterization for Bacillus thuringiensis ( Bt ) Cry34/35Ab1 corn is found in U.S. EPA (2004). Portions of the product characteri zation data were also reviewed in U.S. EPA (2 001a, 2002, 2003a, 2003c, 2003d, and 2003e). Bacillus thuringiensis Cry34/35Ab1 corn was produced by Agrobacterium tumefaciens -mediated transformation of public corn line (Hi-II) with th e transfer deoxyribonucleic acid (T-DNA) from plasmid PHP17662 a , which contains

cry 34Ab1, cry 35Ab1, phosphinothricin acetyltransferase ( pat ), and regulatory sequences necessary for the expression of the genes. The cry 34Ab1 and cry 35Ab1 transgenes were optimized for expression in maize, but the amino acid sequence of the expressed proteins is identical to the native proteins from Bt

. Characterization of the deoxyribonuc leic acid (DNA) isolated from Bt Cry34/35Ab1 corn, using restriction enzyme digests and Southern blot anal ysis (Master Record Identification Numbers (MRID Nos.) 461239-08 and 461239-09; reviewed in U.S. EP A (2004)), indicated that the T-DNA from plasmid PHP17662 inserted as a sing le, intact copy into the corn genome. In addition, DNA analysis indicated stability and inherita nce of the inserted DNA within and across several generations.

Protein characterization data demons trated that the plant-produced pr oteins have characteristics and activities that are equivalent to those of the proteins produced in Pseudomonas fluorescens transformed to produce Cry34Ab1 and Cry35Ab1 (MRID Nos. 461239-05 and 461239-06; reviewed in U.S. EPA

a Early in the development of this product, other plasmids were used and other events tested. Event DAS-59122-7, where plasmid PHP17662 was used, has been chosen for commercialization.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

15 (2004)). The following techniques were used to char acterize and compare the pl ant-produced proteins and the microbially produced proteins: sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal amino acid sequencing, ma trix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF-MS), glycos ylation analysis, enzy me-linked immunosorbent assays (ELISAs), and western blot analysis. Glycosylation analysis indicated that the Cry34Ab1 and Cry35Ab1 proteins are not glycosylated. Biol ogical activity of the plant-produced and P

. fluorescens

produced proteins was also assessed and co mpared; Cry34Ab1 and Cry35Ab1 from both sources displayed similar insecticidal activity to western corn rootworm larvae. These analyses justified the use of microbially produced proteins in toxicity studies.

Studies on the mode of action of Cry34Ab1 a nd Cry35Ab1 indicated that, similar to other Btdeltaendotoxins, Cry34Ab1 and Cry35Ab1 appear to target midgut epithelial cells in susceptible larvae (Master Record Identification Number (MRID No.) 457906-01; reviewed in U.S. EPA (2003c)). Cry34Ab1 appears to cause pore formation in phospho lipid membranes, and addition of Cry35Ab1 resulted in pores remaining open longer and improved membrane permeability (Masson et al . 2004). Ribosomal inhibition activity wa s also investigated (MRID No. 461239-10; reviewed in U.S. EPA (2004)). The results demonstrated that the inse cticidal activity of Cr y34Ab1 and Cry35Ab1 is not

associated with the inhibi tion of protein synthesis.

Submitted studies on heat stability of the Cry34Ab1 and Cry35Ab1 proteins demonstrated that these proteins are inactivated at 90  C and 60  C, respectively (MRID Nos. 453584-01, 455845-01, 458086-01, and 458602-01; reviewed in U.S. EPA (2001a, 2002, and 2003a)).

Data on expression levels of the prot eins in transgenic corn tissues were also provided for both inbred and hybrid lines from several different events (M RID No. 461239-04; reviewed in U.S. EPA (2004)). The protein expression levels were comparable between hybrid and inbred lines. Data on expression levels in a hybrid line are summarized in Table 1.

Table 1. Mean Expression Levels of Cry34Ab1 a nd Cry35Ab1 in Plant Tissues from a Hybrid Line Containing Event DAS-59122-7.

Tissue Cry34Ab1 (ng/mg tissue dry weight + standard deviation)* Cry35Ab1 (ng/mg tissue dry weight + standard deviation)*

Leaf 50 + 8 – 220 + 38 41 + 7 – 85 + 19

Root 37 + 9 – 50 + 20 3 + 2 – 8 + 3

Whole Plant 32 + 16 – 77 + 10 7 + 2 – 14 + 2

Pollen 74 + 7 0.02 + 0.04

Stalk 33 + 4 10 + 2 Forage 53 + 19 12 + 3

Grain 50 + 16 1 + 0.3 * Ranges reflect the range of mean s at different growth stages.

The product characterization studies that were submitted in support of event DAS-59122-7 corn are

summarized in Table 2. Some of the studies were conducted on events other than DAS-59122-7, andBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

16 some of the studies refer to the Cry34Ab1 and Cry35Ab1 proteins as PS149B 1 13.6- or 14-kiloDalton (kDa) and 43.8- or 44-kDa insecticidal crystal protei ns. The term, PS149B1 proteins, was used prior to the proteins receiving their Bt toxin nomenclature de signations (Crickmore et al . 2010). The PS149B1 13.6-kDa or 14-kDa proteins and 43. 8-kDa or 44-kDa proteins are the same as the Cry34Ab1 and Cry35Ab1 proteins, respectively.

Table 2. Product Characterization Data for Ev ent DAS-59122-7 Corn (Reviewed in U.S. EPA (2004) Unless Otherwise Noted).

Study Title Summary MRID No.

Product Characterization

Data for Bacillus thuringiensis PS149B1

13.6 kDa and 43.8 kDa Insecticidal Crystal Proteins Expressed in Transgenic Maize

Plants b Maize immature embryo tissues were transformed using a linearized fragment from plasmid PHP12560 encoding two insecticidal crystal protein (ICP) genes, 8v6 (13.6 kDa) and 7v7 (43.8 kDa), each driven by the maize ubiquitin promoter, and the pat gene, driven by the CaMV 35S promoter. The two ICP genes from Bt PS149B1 are modified in coding sequence to optimize for plant expression. Ami no acid sequences are, however, identical to the bacterial source proteins. Microprojectile bombardment of tissues, followed by selection on glufosinate, produced plants expressing the two insecticidal crystal proteins (ICPs) of PS149B1 and PAT. Maize plants expressing these genes were also produced by Agrobacterium

- mediated transformation of immature embryos. Western corn rootworm (WCRW) bioassays were used to assess the bioactivity of the expressed ICPs. Within 48 hours of feeding on transgenic maize plants, WCRW larvae had midgut symptoms consistent with feeding on delta-endotoxins (e.g., swelling, lysis, blebbing, or vacuolization). WCRW feeding on control plants did not show pathological changes in midgut epithelium. The

two ICPs were also tested for adenos ine diphosphate (ADP)-ribosylating or glucosylating activities as well as other enzymic activities, but none were

detected. Classification: Acceptable 452422-01

Characterization of

Inserted DNA of Event 5639 Transgenic Corn Plants – Interim Report b Based upon the Southern blot data presented, the most likely conclusion is

that there are three insertions of the 8v6

, 7v7 , and pat genes into event 5639, although only one may be fully intact. The complexity of the hybridization patterns does not allow one to say with certainty that three insertions are present at this time. A more detailed analysis of gene insertion will be required as this pro duct develops to fully ascertain the nature of the integration events. Data indicated that event 5639 does not

contain a kan r sequence. Classification: Acceptable 452422-02

b Reviewed in U.S. EPA (2001a)Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

17 Study Title Summary MRID No.

Equivalency of

Microbial- and MaizeExpressed PS149B1 proteins c Biochemical, molecular, and serological data presented indicate that the proteins produced in the P

. fluorescens system are equivalent to the proteins produced in transgenic maize plants. Relative molecular mass and serological reactivity to polyclonal antibodies were the same for the 14- kDa proteins produced in either sy stem. SDS-PAGE and western blots indicated identity between the two production systems. This was also true for the 44-kDa protein and its proteo lytic cleavage prod uct, the 42-kDa protein. The 44-kDa protein predominated in the fresh plant material and in

the P

. fluorescens production system. Storage of leaf material led to the production of the 42-kDa protein as the predominant form due to proteolytic processing. No glycosylation was detected on either protein from the maize system. Tryptic digestion of PS149B1 proteins and subsequent analysis by MALDI-TO F-MS, N-terminal sequencing, and electrospray ionization tandem mass sp ectrometry (ESI MS/MS) spectra all indicated that PS149B1 proteins produced by P

. fluorescens and the transgenic maize are equivalent for toxicological testing to be performed with proteins from either source. In insect bioassays, proteins produced in the transgenic maize system and the microbial system both showed activity against the southern corn rootworm (SCRW) and WCRW larvae. While the transgenic maize assay indicated no reduction in damage from European corn borer (ECB) feeding, the diet-b ased overlay with the microbially produced protein indicated some slight inhibition of growth for ECB larvae. Additionally, the PS149B1 mai ze plants sustained greater leaf damage from ECB, based upon visual estimates, than the control plants. It

is not clear if this difference was sta tistically significant since there was no analysis provided. Classification: Acceptable 452422-03

Microbial PS149B1 Binary Delta-Endotoxin:

Maize-Insect-Pest Susceptibility Study c The relative sensitivity of the test insects in the feeding bioassays suggests that larvae of coleopteran species ar e far more sensitive to the 14-kDa and 44-kDa proteins of PS149B1 than the lepidopteran or homopteran larval insects evaluated. Adult WCRW were not sensitive to the delta-endotoxins, however. Rootworms (northern corn rootworm (NCRW), WCRW, and SCRW) were the most susceptible to the PS149B1 proteins. European corn borer and corn earworm (CEW) demonstrated some inhibition of growth at higher concentrations of the test subs tance than the rootworm larvae. Black cutworm (BCW) was the least sensitive of the lepidopteran species tested. No activity was seen against the corn leaf aphid (CLA ), a homopteran. Relative susceptibilities of the insect species to PS149B1, based upon the more sensitive measure of 50% growth inhibition (GI 50 ), were as follows: (most susceptible) WCRW, NCRW, SCRW > ECB, CEW >> WCRW adult, CLA, BCW (least susceptible). Classification: Acceptable 452422-04

c Reviewed in U.S. EPA (2001a)Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

18 Study Title Summary MRID No.

Quantitative ELISA Analysis of PS149B1 Protein Expression Levels in Hybrid and

Inbred Lines of Maize

Event TC5639 d Leaf, pollen, root, and senescent whole plant tissue samples were taken from field-grown plants cultured in the U.S. corn belt. Both test hybrid and

inbred lines of event 56 39 and control hybrid and inbred lines were compared for expression of the 14-kDa and 44-kDa PS149B1 proteins. Leaf, pollen, root, and senescent whole plant tissues expressed the 14-kDa and 44-kDa proteins at measurable le vels in the modified maize lines. Hybrid and inbred lines had comparab le levels of expression of these proteins when analyzed on a dry we ight basis. On a total extractable protein basis, though, root tissues had the highest expression and pollen had the lowest expression. Control ti ssues were negative for the presence of the 14-kDa or the 44-kDa proteins. Coefficients of variation (CVs) were lacking for data presented and need to be included in order to adequately

assess the results of the ELISAs. The use of samples spiked with known quantities of the two proteins separately is also needed to assess the possible influence of maize tissu e extracts on the enzyme-linked immunosorbent assay (ELISA) values observed. These data and an evaluation of possible cross-reactivity of the primary antibody with other Cry proteins will be needed for Section 3 registration. Classification: Acceptable 452422-13

Lateral Flow Test Kit

for the Detection of the

PS149B1 Protein in

Maize Grain e The double antibody sandwich format of the lateral flow test strip from Strategic Diagnostics was capable of correctly detecting 48 positive aliquots of 24 extracts from 24 single kernel samples of PS149B1 maize. Conversely, 48 aliquots of 24 extracts from 24 kernels of a non-PS149B1 inbred line all tested negative, as expected. Ratios of positive (PS149B1) and negative (control) kernels were mixed and extracted. When 0.5% or greater PS149B1 kernels were present in the mixture, they were correctly detected as containing Cry34Ab1 protein. No ratios of positive to negative below 0.5% were evaluated in this study. Classification: Acceptable 453834-01

d Reviewed in U.S. EPA (2001a)

e Reviewed in U.S. EPA (2003b)Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

19 Study Title Summary MRID No.

Characterization of Cry34Ab1 and Cry35Ab1 from

Recombinant

Pseudomonas fluorescens and Transgenic Maize f Cry34Ab1 and Cry35Ab1 proteins were separated from maize leaf extracts and from microbial production sources on SDS-PAGE gels and then blotted to nitrocellulose for serological detection. Both proteins were detected on the western blots by polyclonal antibody preparations and appeared at the appropriate M

r for both proteins on individual blots (i.e., 14 kDa - Cry34Ab1; 44 kDa - Cry35Ab1). A seco nd protein band reacted with the Cry35Ab1-specific antibody in the microbially produced protein preparation at approximately 40 kDa, which is presumably a degradation product from protease activity. This band or signal was noted in the

western blot of 3 of 6 maize leaf ex tracts representing the 6 events but at

reduced amounts relative to the microbi al preparation. Both Cry proteins from the maize extracts migrated very similarly to the Cry proteins produced in the microbial production system and, by this criterion (i.e., mobility in SDS-PAGE), are cons idered as indistinguishable. Classification: Acceptable 457904-01

f Reviewed in U.S. EPA (2003c)Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

20 Study Title Summary MRID No.

Characterization of

DNA Inserted into Transgenic Corn Events E4497.42.1.34, E4497.45.2.16, E4497.59.1.10, E4497.66.1.27, E4497.71.1.29, and

E4497.71.1.33 g Southern blots were used to discern the number of insertions from the plasmid constructs, PHP17662 and PHP17658, used in the transformation of maize. An analysis of integra tion sites in events E4497.42.1.34,E4497.45.2.16, and E4497.59.1.10 indicated a single integration of the TDNA sequence into the maize genome. Each reactive band present in the

blot resulted from the XhoI cleavage of the incorp orated T-DNA region (containing cry34Ab1

, cry35Ab1 , and pat from PHP17622 in a single fragment) and a second XhoI restriction site in the adjacent maize genome. Hence, the bands represented individual, unique integrations of T-DNA in the maize genome. The size of the r eactive bands cannot be determined a priori since the size of the flanking or border fragment is not known as it is determined by the distance from the single XhoI site within the T-DNA region. Events E4497.42.1.34 and E4 497.45.2.16 each had single reactive hybridization events (bands) on the bl ot, indicating the presence of a single

T-DNA insertion. The comm onality of band sizes (i.e., 9.4 or >22 kilobase (kb)) suggests that the T-DNA insertion is the result of a single insertional event. A 9.4 kb signal in E4497.59.1.10 also indicates the integration of one full T-DNA containing cry34Ab1

, cry35Ab1 , and pat . The presence of a second hybridizing signal (7.4 kb) in the blot of genomic DNA from E4497.59.1.10 probed with cry35Ab1 suggests that a partial construct insertion occurred containing only the cry35Ab1 portion and not cry34Ab1

or pat

. StuI cuts once in PHP17658 and was used to define integration sites in events E4497.66.1.27, E4497.71.1.29, and E4497.71.1.33. Events E4497.71.1.29 and E4497.71.1.33 appear to both contain a single T-DNA insertion based on the reactive band signals being present at 11 and 8.3 kb for all three probes. Additionally, ther e was a reactive band at 14 kb and

2.7 kb for the pat -probed blots of E4497.71.1.29 and E4497.71.1.33, respectively. The second reactive band in the pat - probed blots was a consequence of the position of the cleavage site for the StuI enzyme being

within the pat open reading frame (ORF). Genomic DNA from control plants (non-transgenic) was spiked with restricted plasmid DNA of PHP17661 and PHP17657 at a concentration of one copy per genome equivalent to serve as positive controls. Classification: Acceptable 457904-02

g Reviewed in U.S. EPA (2003c)Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

21 Study Title Summary MRID No.

Product Characterization

Data for Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 Proteins Expressed in Transgenic

Maize Plants (PHP17662) h Genes encoding the PAT, Cry34Ab1, and Cry35Ab1 proteins were transferred into the T-DNA region of the disarmed A

. tumefaciens strain LBA4404. This Ti-plasmid was then referred to as PHP17662 and is approximately 50 kb in size. This strain carrying PHP17662 was used to transform immature maize embryo tissues by co-cultivation for 6 days. Coding sequences for the Cry34Ab1 (14-kDa) and Cry35Ab1 (44-kDa) proteins were synthesized from the native sequence to optimize expression in maize. The PAT protein produced from a synthetic gene is not altered in its amino acid sequence as compared to the native protein. The gene and protein are considered as inert ingr edients in this plant-incorporated protectant. Gut cells of the control ma ize-fed insects and the starved insects appeared normal, although the latter group showed some collapse of the gut lumen. After 48 hours of feedin g on the transgenic maize plants, WCRW larvae had evidence of cell lysis, blebbing, and vacuolization, as would be expected from intoxication with delta-endotoxins in a susceptible insect. Subsequently, the gut tissues showed further collapse and more fragmentation. Enzyme assays were evaluated to determine if either Cry34Ab1 or Cry35Ab1 proteins were capable of activity: glucosylating or ADP-ribosylating of midgut proteins, protease at acid and neutral pH, neuraminidase (sialidase), acid and al kaline phosphatase activity. Protein synthesis inhibition of an in vitro system was also tested. All assays were negative for their corresponding enzyme activities. Amino acid sequences of the three proteins and micrographs of histopathological midgut responses were presented. Classification: Acceptable 457906-01

h Reviewed in U.S. EPA (2003c)Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

22 Study Title Summary MRID No.

Quantitative ELISA Analysis of Cry34Ab1 and Cry35Ab1 Proteins Expressed in Maize

Plants Transformed with

the Vector PHP17662 i ELISA was used to determine the quantity of the two ICPs present in leaf, pollen, root, and seed of transgenic maize from three separate transformation events (E4497.42.1.34 , E4497.45.2.16, and E4497.59.1.10). Direct double antibody sandwich ELISA was used to quantify the Cry34Ab1 and Cry35Ab1 present in plant samples. The primary antibodies raised against Cry34Ab1 and Cry35Ab1 were used to capture the proteins (individually in separate reactions) in the microplate well, and a second specific antibody was used to detect the protein that was bound. This second antibody was conjugated to biotin, which was detected using streptavidin linked to alkaline phosphatase (AP). The colorimetric reaction catalyzed by the AP provides for a m eans of measuring optical density or absorbance and correlating it to the amount of target protein present based upon comparison to a standard curve generated from reference standards. The values obtained from the ELISA readings are converted to amounts of Cry34Ab1 or Cry35Ab1 protein and expressed as nanograms Cry34Ab1 protein per milligram total extractable protein (TEP). Since data for water content exists for the tissue types sampled, the conversion to nanograms Cry34Ab1 protein per milligram dry tissue is possible with conversion factors. Reference standards used to generate the standard curve were run in triplicate within each 96-well microplate. For Cry34Ab1, the magnitude of expression for tissue types was as follows in descending order: leaf, pollen, kernel, and root. All control tissues were negative for the expression of Cry34Ab1. Due to the Mendelian segregation of the transgenes, approximately 50% of the seeds were found to be expressing Cry34Ab1. For Cry35Ab1, the magnitude of expression for tissue types was as follows in descending order: leaf, root, and kernel. All control tissues were negative for the expression of Cry35Ab1. Cry35Ab1 protein was not detected in pollen. Due to the Mendelian segregation of the transgenes, approximately 50% of the seeds were found to be expressing Cry35Ab1. Seeds that were observed to contain no Cry proteins by ELISA were not included in the summary statistics since they represent segregants from the heterozygous populations of the three events. Mean values are presented in tabular format for a ll three events for each tissue type. Classification: Acceptable 458332-01

i Reviewed in U.S. EPA (2003c)Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

23 Study Title Summary MRID No.

Probe MOA Studies to

Assess Potential for Protein Synthesis Inhibition by Bacillus thuringiensis PS149B1 Cry34Ab1/Cry35Ab1

Proteins in a RabbitReticulocyte Assay: ReExamination of Lab

Notebook Data j Two experiments were performed in order to assess the potential for ribosomal inhibition activity by either Cry34Ab1 or Cry35Ab1. The first experiment concluded, based upon a sensitive luciferase (chemiluminescence) assay, that the Cry 34Ab1 and Cry35Ab1 proteins did not show the properties of ribosomal inhibition proteins (RIPs). This was largely based upon comparison to a bovine serum albumin (BSA) control and phosphate buffered saline (PBS) buffer control. A second experiment, with a different treatment regime and altered protocol, concluded that there was some ribosomal inhibition protein (RIP) activity

associated with the PS 149B1 proteins. Further, digesting the 14-kDa protein with trypsin may have also reduced translation a little. The two experiments used different amounts of BSA (i.e., 1x and 2x), and this added variable further confounded the interpretation of results. The lack of proper controls and different treatment regimes in the experiments made any assessment of the potential RIP activity dubious. The experiments would also have benefitted by including a greater number of samples per treatment (at least triplicate) and a ppropriate controls. A positive control (i.e., a protein with known RIP activity) is crucial to the interpretation of results. Note that the source (lot) of the proteins used for the two experiments was different as well. It is also probable that confounding stabilization reagents (e.g., glycerol , azide) were present in the reaction during the second experiment, further complicating any analysis of results. It is not possible, given the present data set and variance in methodologies, to assess the results accurately. The RI P assay has not been considered as a typical part of the submission process for plant-incorporated protectants.

There is no a priori reasoning or similarity between the delta-endotoxins of

Bt

or Bacillus sphaericus insecticidal toxins and proteins with known RIP activity to expect Cry34Ab1 or Cry35Ab1 to exhibit such action. Proteins with this activity also require a means of intracellular vectoring in order to reach the ribosome target. Classification: Supplemental 459428-01

Thermolability ofPS149B1 Binary DeltaEndotoxin k Results indicate that the PS149B1 prot eins, or at least one of them, are inactivated after exposure to 60  C for 30 minutes, based upon activity against the southern corn rootworm. It is possible that shorter times or lower temperatures may have also denatured the protein(s), but these were not tested. With the current protocol design, it is not possible to determine if one or both proteins are inactivated following heat treatment. To address this issue, the applicant should consider repeating the study under a method that would allow this distinction to be made as part of the submission for Section 3 registration. Classification: Acceptable 453584-01

j Reviewed in U.S. EPA (2003d)

k Reviewed in U.S. EPA (2001a)Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

24 Study Title Summary MRID No.

Heat Lability of

Individual Proteins of the PS149B1 Binary

ICP l The percent growth inhibition of larvae, as observed in a southern corn rootworm feeding assay, was used as a measure or indicator that the Cry34Ab1 and Cry35Ab1 proteins were degraded to a point they have lost insecticidal activity. The decrease in growth inhibition activity following heating of the 14-kDa and 44-kDa mixtur e suggests that heat treatment at

60  C for 30 minutes is sufficient to de nature at least one of the necessary components of the ICP. No mortality e ffect was seen with the Cry protein treatments, as expected. The author suggested that the degree of inhibition (30 to 42% larval growth inhibition) seen following heat treatment of the mixture is typical of an inactive protein preparation as observed in previous assays. This difference between the la rval weights observed with the ICPtemperature treatments and the buffer control was a non-specific effect attributable to the presence of proteins in the diet irrespective of the amino acid sequence and conformation of the ICPs. The 44-kDa protein in the ICP was denatured by treatment at 60  C and higher temperatures. The decrease in insect growth inhibition, se en in the 44-kDa spike of the heated ICP as temperature of incubation wa s increased, indicates that the 14-kDa protein was more heat stable than the 44-kDa protein. The concomitant decrease in insect activity (i.e., gr owth inhibition) with temperature increase shows the relative stability of this protein at 60  C and 75  C but its denaturation to background levels (i.e., 36% growth inhibition compared to buffer control) at 90  C. Classification: Supplemental 455845-01

l Reviewed in U.S. EPA (2002)Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

25 Study Title Summary MRID No.

Summary of Heat Lability Studies with Cry34Ab1/Cry35Ab1 m

Slide Presentation Summarizing Cry34Ab1/Cry35Ab1

Heat Inactivation

Studies m Using an insect bioassay as the detection method for PS149B1 proteins (Cry34Ab1, Cry35Ab1) provides for a qualitative assessment of the protein(s) remaining after treatment but does not allow for quantitation. This assay utilized the decrease in growth of larval SCRW, measured as mass, as a means of assessing activity remaining in the Cry protein(s) but did not give a direct correlation with protein concentration. Nonetheless, the assay provided for a determination as to the conformational changes (denaturation) that occur following heat treatment. The two studies previously submitted (MRID Nos. 455845-01 and 453584-01), regarding heat stability of the Cry34Ab1 and Cry35Ab1 proteins, demonstrated that these proteins are inactivated at < 90  C and < 60  C, respectively. The two current submissions served to clarify the lability of the proteins to heat. However, differences in the execution of the two studies complicated the interpretation and comparison of the results. The feeding bioassays, using the southern corn rootworm, indicated that the proteins are no longer active following their respective heat treatments. This is taken as evidence of protein denaturation. The SCRW assay was based upon growth inhibition of the larvae and not mortality. A different diet formulation was used in each study with a greater than 10-fold increase in larval growth occurring

in control insects with the artificial diet in the second study (MRID No. 455845-01). Additionally, the presence of salts and buffer impurities from the purification process used for the synthesis of the test substance (Cry34Ab1 and Cry35Ab1 proteins from a bacterial production system) resulted in experimental variance not expected or attributable to the insecticidal proteins being tested. Bo th Cry proteins evaluated herein are susceptible to heat denaturation under the conditions tested, albeit with different denaturation profiles. Classification: Acceptable 458086-01

458602-01

Independent Laboratory

Validation of Pioneer Hi-Bred International, Incorporated ELISA

Method for the Quantification of Cry34Ab1 Protein from Transgenic Plants This study was conducted to provide independent laboratory validation (ILV) data for the determination of Cry34Ab1 insecticidal crystal protein in corn matrices and to support the stated limit of quantitation (LOQ) of 0.072 nanograms per milligram (ng/mg). The reported data satisfied the requirements of U.S. EPA Guidelin e Office of Chemical Safety and Pollution Prevention (OCSPP) 860.1340(c)(6) and the study protocol. Individual recovery values for each fo rtified control sample were within the range of 70 – 120% and averaged 78% and 98% at the LOQ and 2x the LOQ, respectively. The relative standard deviation (RSD) of replicate recovery measurements did not exceed 20% at or above the LOQ, and

interferences were minor. Classification: Acceptable 461239-01

m

Reviewed in U.S. EPA (2003a)Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

26 Study Title Summary MRID No.

Independent Laboratory

Validation of Dow AgroSciences Method GRM 03.13,

“Determination of Cry35Ab1 Insecticidal

Protein in Maize Tissue by Enzyme-Linked Immunosorbent Assay” This study was conducted to provide ILV data for the determination of Cry35Ab1 insecticidal crystal protein in corn matrices and to support the stated LOQ of 0.06 ng/mg. The reported data satisfy the requirements of U.S. EPA Guideline OCSPP 860.1340(c)(6) and the study protocol. Individual recovery values for each fo rtified control sample were within the range of 70 – 120% and averaged 93 and 101% at the LOQ and 2x the LOQ, respectively. The RSD of repli cate recovery measurements did not exceed 20% at or above the LOQ, and interferences were minor. Classification: Acceptable 461239-02

Cry34/Cry35 Protein

Distribution and Familiarity DNA hybridization and polymerase chai n reaction (PCR) analysis of total genomic DNA from collections of strains of Bt showed that homologs of

cry 34/35 genes were present in strains from North and South America and from Australasia taken from a variety of environments. The overall rate of

occurrence of cry 34/35 genes in Bt strains was 1.2%; this rate of occurrence is comparable to that of the cry 3Aa gene, which encodes a previously registered insect control protein (EPA Registration Number 524-474). Sequence comparisons demons trated that the Cry35 protein has some homology (26 to 29%, respectively) to the 42- and 51-kDa mosquitocidal proteins from B

. sphaericus strain 2362 ( B

. sphaericus H5a5b strain 2362 is a registered biopesticide). Classification: Acceptable 461239-03

Agronomic Characteristics, Quantitative ELISA and Nutrient Composition Analysis of Hybrid Maize Lines Containing

the cry 34Ab1,

cry 35Ab1, and pat
Genes: Chile Locations The data from this study indicated that control and transgenic lines (containing events DAS-45216-6, DAS-59122-7, and DAS-45214-4) are similar with respect to agronomic ch aracteristics and nutrient composition. Expression levels of Cry34Ab1, Cry35Ab1, and PAT proteins were analyzed by ELISA for various plant tis sues at different growth stages for transgenic hybrid and inbred lines and compared with cont rol lines for both herbicide-sprayed plants and non-sprayed plants. On a dry weight basis, the concentrations found for Cry34Ab1 ranged from a low of 31 ng/mg in R1 stalks to a high of 500 ng/mg in R4 leaves; concentrations for Cry35Ab1 ranged from a low of 0.01 ng/mg in pollen to a high of 120 ng/mg in R4 leaves; and concentrations for PAT ranged from 0 ng/mg in pollen to 36 ng/mg in R1 leaves. Transgenic protein levels were independent of spraying status, comparable between hybrid and inbred lines, and the proteins were not detected in any control samples. Classification: Acceptable 461239-04Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

27 Study Title Summary MRID No.

Biological Equivalency of Cry34Ab1/Cry35Ab1 Insecticidal Crystal Protein in Transgenic

Plants and Derived from Transgenic

Pseudomonas fluorescens Three transgenic corn lines (DAS 45214-4, 45216-6, and 59122-7), carrying the cry 34Ab1 and cry 35Ab1 genes that produce a binary insecticidal protein, and control (non-transgenic) corn were field-tested for their resistance to damage from we stern corn rootworm, northern corn rootworm, black cutworm, and European corn borer. The transgenic corn

lines were all shown to have a statisti cally significant effect against western

and northern corn rootworm but were not effective against European corn borer and black cutworm. These results are consistent with the efficacy profiles observed in previous studies on other corn events expressing the same insecticidal protein (DAS Re port 000367) and with the efficacy profiles obtained with purified binary insecticidal protein isolated from P

. fluorescens (DAS Report 000366). Classification: Acceptable 461239-05

Characterization of Cry34Ab1 and Cry35Ab1 Proteins

Derived from Transgenic Maize EventE4497.59.1.22 (DAS59122-7) Selected biochemical properties of Cry34Ab1 and Cry35Ab1 proteins,

isolated from leaf tissue from Bt transgenic corn event E4497.59.1.22 (DAS-59122-7) [59.1.22], were evaluated and compared to biochemical properties of the proteins obtained from a P

. fluorescens bacterial expression system containing the two Bt transgenes. The results were also compared with results from a previous ly submitted study on Cry34Ab1 and Cry35Ab1 isolated from Bt transgenic corn even t 5638. SDS-PAGE and western blot analysis indicated the re lative molecular weights of Cry34Ab1 and Cry35Ab1 from all sources were ~14 kDa and ~44 kDa, respectively. In addition, the Cry35Ab1 protein from both transgenic corn (event 5638)

and P

. fluorescens has been shown previously to be proteolytically degraded to a 40-kDa form (with an intact N–terminus), and the Cry35Ab1 protein isolated from corn event 59.1.22 also show ed a 40-kDa degradation product. Glycosylation analysis indicated that Cry34Ab1 and Cry35Ab1 derived from event 59.1.22 are not glycosylated; this result was consistent with the earlier studies of these two proteins derived from corn event 5638

and from P

. fluorescens . MALDI-TOF-MS and N– terminal sequencing provided additional evidence that Cry34Ab1 and Cry35Ab1 proteins

derived from corn event 59.1.22 are identical to those produced by the P

. fluorescens bacterial expression system and by corn event 5638. Classification: Acceptable 461239-06Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

28 Study Title Summary MRID No.

Characterization of Phosphinothricin Acetyltransferase (PAT)

Derived from Transgenic Maize Event

E4497.59.1.22 SDS-PAGE, western blot analysis, and ELISA were used to compare PAT protein derived from transgenic maize plant event E4497.59.1.22 with PAT protein from a recombinant Escherichia coli expression system. SDSPAGE demonstrated that the E

. coli -produced PAT protein had the expected molecular weight. Leaf extr act samples from event E4497.59.1.22 showed an immunoreactive band in the western blot analysis that corresponded to the exp ected molecular weight for the PAT protein and comigrated with the E

. coli -produced PAT. ELISA results demonstrated similar expression of the PAT protein in all of the transgenic leaf samples tested. The control plants did not co ntain immunoreactive proteins. Results were consistent with earlier studies of PAT protein derived from E

. coli and from transgenic maize event 5638, which used MALDI-TOF-MS peptide mass fingerprinting (Korjagin 2000). The present study further supports the use of microbe-derived PAT protein as an appropriate surrogate for the PAT protein produced in maize plants. Classification: Acceptable 461239-07

Characterization of

DNA Inserted into Transgenic Corn Events

DAS-45216-6 and DAS59122-7 Southern blot analysis was used to characterize the DNA inserted in transgenic corn events DAS-45216-6 and DAS-59122-7. The restriction enzyme fragments observed in the Southern blots matched the predicted fragments, indicating that a single, intact T-DNA from plasmid PHP17662 was integrated into the corn genome at a single locus. The data also

demonstrated the absence of the tetr acycline and spectinomycin resistance genes, the vir G gene, and vector backbone DNA regions immediately outside of the left and the right T-DNA border, indicating that only DNA contained within the T-DNA borders was integrated into the transgenic corn. Identical fragment sizes were obse rved for all samples (two distinct generations, four plants each), indicating stability of inheritance across and within generations. Classification: Acceptable 461239-08

Detailed

Characterization of

DNA Inserted into Transgenic Corn Events

DAS-45216-6 and DAS59122-7 Southern blot analysis was used to analyze the inserted DNA in events DAS-45216-6 and DAS-59122-7 and to develop detailed restriction enzyme maps of the insertions. Frag ments of the expected sizes were observed, indicating that a single, intact T-DNA inserted into the corn genome at one locus without any rea rrangements of the inserted DNA. Classification: Acceptable 461239-09Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

29 Study Title Summary MRID No.

Evaluation of MicrobeDerived Cry34Ab1 and Cry35Ab1 Proteins for Protein Synthesis Inhibition Activity The rabbit reticulocyte assay was used to determine if Cry34Ab1 and Cry35Ab1 proteins inhibit protein synthesis. Ricin, used as a positive control, produced a 50% inhibition in protein synthesis with a concentration of 0.182 nanomolar (nM). In contrast, Cry34Ab1 and Cry35Ab1, as well as BSA used as a negative control, showed no inhibition of protein synthesis at concentrations up to 2,880 nM. The results suggest that the insecticidal activity of Cry34Ab1 and Cry35Ab1 is not associated with the inhibition of protein synthesis. Classification: Acceptable 461239-10

b. Terms and Conditions of the Event DAS -59122-7 Corn Registrations (August 2005 – September 2010)

When event DAS-59122-7 corn was initially re gistered on August 31, 2005, the Agency issued registration notices to both Mycogen Seeds c/o Dow AgroSciences LLC (EPA Reg. No. 68467-5) and Pioneer Hi-Bred International, Incorporated (EPA Reg. No. 29964-4) that contained the following requirement for further product characterization information:

“Provide to the EPA laboratory (Ft. Meade, MD) methodology and/or reagents necessary for validation of a Cry34/35Ab1 analytical method within 6 months of the date of registration.”

More recently, the Agency has decided to allow this requirement to be satisfied by the Grain Inspection, Packers and Stockyards Administra tion (GIPSA) of the United States Department of Agriculture (USDA) performance verification of a qualitative rapid test kit fo r detecting the presence of the biotechnology event in grains a nd oilseeds (USDA 2004). In the case of Cry34/35Ab1, the Agency has confirmed that a test kit has been verified by GIPSA and, therefore, the aforementioned requirement has been satisfied for both registrations.

2. Herculex® XTRA Insect Protection Corn (OECD Unique Identifi er: DAS-Ø15Ø7-1 x DAS- 59122-7) Expressing Cry1F, Cry34Ab1, and Cry35Ab1

a. Data Cited/Submitted for Initial Registration s of Herculex® XTRA In sect Protection Corn (Prior to October 2005)

Herculex® XTRA Insect Protecti on Corn was developed through conve ntional breeding of a corn line containing event DAS-01507-1 (also known as event TC1507 and Herculex® I), which expresses the Cry1F (truncated) and PAT proteins, and a corn line containing event DAS-59122-7, which expresses the Cry34Ab1, Cry35Ab1, and PAT proteins. This product is considered a stacked plant-incorporated protectant because it targets both le pidopteran and coleopteran pests a nd contains two separate activeBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

30 ingredients. DAS-01507-1 is brie fly summarized in the paragraph that follows, while additional information on DAS-59122-7, specifi cally related to product char acterization, can be found in section II(A)(1) of this Biopesticides Regist ration Action Document (BRAD).

DAS-01507-1 produces the Bacillus thuringiensis var. aizawai Cry1F protein to selectively control larvae of European corn borer ( Ostrinia nubilalis ) and other lepidopteran pests. In addition, DAS01507-1 also produces the PAT protein from Streptomyces viridochromogenes , which confers tolerance to glufosinate-ammonium herbicides. On May 18, 2001, the Agency issued Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) secti on 3 registrations to Mycogen Seeds c/o Dow AgroSciences LLC (EPA Reg. No. 68467- 2) and Pioneer Hi-Bred Interna tional, Incorporated (EPA Reg. No. 29964-3) for Herculex® I, expressing Bacillus thuringiensis Cry1F protein and the genetic material necessary (plasmid insert PHI8999A ) for its production in corn even t TC1507 (OECD Unique Identifier: DAS-Ø15Ø7-1). The product characterization data s upporting the registration of Herculex® I, including the submitted study titles, conclusions, and th eir MRID Numbers, can be found in the 2001 Bt Crops Reassessment, August 2005 Cry1F BRAD, and th e September 2010 Cry1Ab and Cry1F BRAD (U.S. EPA 2001b, 2005a, and 2010b).

The data submitted for Herculex® XTRA Insect Pr otection Corn, which include DNA analysis of the inserts in and protein expressi on data for the stacked corn product, are summarized in Table 3.

Table 3. Product Characterization Data for Hercu lex® XTRA Insect Protection Corn (Reviewed in U.S. EPA (2005b)).

Study Title Summary MRID No.

Characterization of Stacked Hybrid of Transgenic Corn Lines

DAS-01507-1 and DAS-59122-7 by Comparison to the

Individual Lines Southern blot analysis was conducted on restriction enzyme digests of DNA from the stacked hybrid of transgenic corn lines DAS-01507-1 and

DAS-59122-7 as well as the indi vidual lines. Hybridization of Hind III and

Sac I digests with probes for the cry 1F, cry 34Ab1, cry 35Ab1, and pat genes gave the expected bands on Southern blots, based on restriction enzyme maps of both of the inserts in the individual lines. The results

indicate that the inserts in the 1507 x 59122 stacked hybrid are equivalent

to the individual inserts in the inbred lines.

Classification: Acceptable 462971-02Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

31 Study Title Summary MRID No.

Agronomic Characteristics, Quantitative ELISA,

and Nutrient Composition Analysis of Hybrid Maize Lines Containing the cry 1F,

cry 34Ab1, cry 35Ab1,

and pat Genes: U.S. and

Canada Locations Agronomic characteristics, nutrient composition, and protein expression levels, for transgenic corn line DAS-01507-1 x DAS-59122-7 grown at five locations in the U.S. and Canada, were analyzed and compared with results from a non-transgenic control. The presence of the transgenes for the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins had no notable effect on agronomic parameters, such as early and final population, time to silking and to pollen shed, and plant and ear height. Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins were detected by EL ISA in all plant tissues except that PAT was not detected in pollen. On a tissue dry weight basis, mean expression levels had the following ranges: Cry34Ab1 – a low of 43 ng/mg in grain to a high of 340 ng/mg in leaf tissue at the R4 growth stage; Cry35Ab1 – a low of 0.14 ng/mg in pollen to a high of 104 ng/mg in leaf tissue at the R4 growth stage; Cry1F – a low of 0.14 ng/mg in pollen to a high of 25 ng/mg in leaf tissue at th e R4 growth stage; and PAT – a low of <LOQ in pollen to a high of 13 ng/mg in leaf tissue at the R4 and R1 growth stages and whole plant sa mples from the V9 growth stage. Herbicide spraying did not affect protein expression levels.

Classification: Acceptable 463438-01

b. Terms and Conditions of the Herculex® XTRA Insect Protection Corn Registrations (October 2005 – September 2010)

When Herculex® XTRA Insect Protection Corn wa s initially registered on October 27, 2005, the Agency issued registration notices to both Mycoge n Seeds c/o Dow AgroSciences LLC (EPA Reg. No. 68467-6) and Pioneer Hi-Bred Intern ational, Incorporated (EPA Re g. No. 29964-5) that contained the following requirement:

“Submit all data required to support the in dividual plant-incor porated protectants in event TC1507 (Hercule x I) and event DAS-59122-7 (Herculex Rootworm)

corn....”

All requirements for additional product characteriz ation information for events TC1507 (see U.S. EPA (2010b)) and DAS-59122-7 (see section II(A)(1)(b) of this BRAD) have been satisfied for both registrations.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

32 3. 1507 x 59122 x MON 810 (OECD Unique Iden tifier: DAS-Ø15Ø7-1 x DAS-59122-7 x MON-ØØ81Ø-6) Expressing Cry1F, Cr y34Ab1, Cry35Ab1, and Cry1Ab and 59122 x MON 810 (OECD Unique Identifier: DAS -59122-7 x MON-ØØ81Ø-6) Expressing Cry34Ab1, Cry35Ab1, and Cry1Ab

a. Data Cited/Submitted for Initial Registrations of 1507 x 59122 x MON 810 and 59122 x MON 810 (Prior to February 2010)

The stacked and pyramided product, 1507 x 59122 x MON 810, and the stacked product, 59122 x MON 810, are hybrids created by crossing the indi viduals events, TC1507 (Cry1F), DAS-59122-7 (Cry34/35Ab1), and MON 810 (Cry1Ab), via traditi onal breeding methods. Product characterization background on DAS-59122-7 and TC1507 was disc ussed or referenced in sections II(A)(1) and II(A)(2) of this BRAD, respectively; MON 810 is briefly su mmarized in the paragraph that follows.

MON 810 produces Bacillus thuringiensis subsp. kurstaki strain HD-1 Cry1Ab protein to selectively control larvae of European corn borer ( O

. nubilalis ) and other lepidopteran pests. On December 20, 1996, the Agency issued a FIFRA section 3 registra tion to Monsanto Company for MON 810 (EPA Reg. No. 524-489), expressing Bacillus thuringiensis Cry1Ab protein and the genetic material necessary (PVZMCT01) for its production in corn event MON 810 (OECD Unique Identifier: MON-ØØ81Ø-6). The product characterization data supporting the regi stration of MON 810, including the submitted study titles, conclusions, and their MRID Nu mbers, can be found in both the 2001 Bt Crops Reassessment and the September 2010 Cry1Ab and Cr y1F BRAD (U.S. EPA 2001b, 2010b).

The data submitted for 1507 x 59122 x MON 810 and 59122 x MON 810, which include molecular characterization data, protein expr ession analyses, and a synergism study, are summarized in Table 4.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

33 Table 4. Product Characterization Data for 1507 x 59122 x MON 810 and 59122 x MON 810 (Reviewed in U.S. EPA (2010a)).

Study Title Summary MRID No.

Molecular

Characterization of

Maize Combined Trait

Product DAS-01507-1 x

DAS-59122-7 x MON00810-6 Using Southern Blot Analysis and Event-Specific Polymerase Chain Reaction (Product Characterization) Molecular analyses (restriction enzyme digests and Southern blots) were performed to compare the integrity of the transgenic inserts in events TC1507, DAS-59122-7, and MON 810 corn with the transgenic DNA inserts in the combination plant-incorporated protectant (PIP) 1507 x 59122 x MON 810 corn product, produced by conventional plant breeding. The Southern blot data showed the predicted DNA hybridization patterns

of the cry 1F gene from TC1507 corn, the cry 34Ab1 and cry 35Ab1 genes from DAS-59122-7 corn, and the cry 1Ab gene from MON 810 corn. These data demonstrated equivalence among events because the transgenic inserts were stably integrated and retained wh en the parental lines are crossed to create 1507 x 59122 x MON 810 corn. In addition, event-specific PCR methods detected and confirmed the presence of each expected transgenic

DNA insert in the combination PIP corn event.

Classification: Acceptable 476778-01

Additional Addendum to Expressed Trait

Protein Concentration

of Maize Lines Containing Events TC1507, DAS-59122-7, MON 810, and

Combination PIPs 1507 x MON 810, 59122 x MON 810, and 1507 x

59122 x MON 810 Corn Products (Protein Expression Levels) A field study was conducted using quantitative ELISA methods to statistically compare the level of expression for the Cry1F, Cry34Ab1, Cry35Ab1, Cry1Ab, and PAT proteins expressed in 1507 x 59122 x MON 810, 1507 x MON 810, and 59122 x MON 810 combination PIP corn events to the individual parental events. No statistically significant differences were found for Cry1F, Cry34Ab1, Cry35Ab1, and Cry1Ab protein expression between 1507 x 59122 x MON 810 and parental corn events 1507, 59122, or MON 810. No statistical differences were observed in any plant tissue for Cry1F, PAT, and Cry1Ab protein expression when TC1507 x MON 810 was compared to events 1507 and MON 810 or when event 59122 was compared to event MON 810. Based on the results, the protein concentrations of Cry1F, Cry34Ab1, Cry35Ab1, and Cry1Ab expressed in combination PIP events 1507 x 59122 x MON 810, 1507 x MON 810, and 59122 x MON 810 are comparable to the respective protein concentrations of their respect ive single parental events.

Classification: Acceptable

Note: This study supersedes MRID Nos. 475109-04 and 478654-01. 478800-01Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

34 Study Title Summary MRID No.

Laboratory Characterization of Key Lepidopteran Pest Response to Pyramided Events (Synergism Study) A seven-day laboratory sensitive insect bioassay was conducted to determine if the combination PIP product, 1507 x 59122 x MON 810, has a synergistic effect in comparison to the individual parental events, TC1507 (expressing Cry1F protein) and MON 810 (expressing Cry1Ab protein), on target lepidopteran pests. The pests used in the bioassay were European corn borer (ECB, Ostrinia nubilalis ), southwestern corn borer (SWCB, Diatraea grandiosella ), fall armyworm (FAW, Spodoptera frugiperda ), and corn earworm (CEW, Helicoverpa zea ). The observed and expected larval mortality in the 1507 x 59122 x MON 810 group were similar and mean larval weight of the survivors exposed to 1507 x 59122 x MON 810 leaf tissue was not significantly different from the single parental events or the negative control of non- Bt maize. These results indicate that the Cry1F and Cry1Ab proteins act independently and do not have a synergistic or antagonistic effect on the target pests, other than an additive effect. Quantitative ELISA results also confirmed that the expression of each of the proteins in the combination PIP was not affected by the presence of the other protein.

Classification: Acceptable 476778-02

b. Terms and Conditions of the 1507 x 59122 x MON 810 and 59122 x MON 810 Registrations (February 2010 – September 2010)

When 1507 x 59122 x MON 810 (EPA Reg. No. 29964-8) and 59122 x MON 810 (EPA Reg. No. 29964-9) were initially registered on February 24, 2010, the Agency issued registration notices to Pioneer Hi-Bred International, Incorporated that containe d the following requirements:

 For 1507 x 59122 x MON 810

–

“The data submitted by Pioneer are sufficient to support registration for the combination PIP product...provi ded that the registrant submits/cites any data required to support the PIP registrations of the indivi dual parental events...as

well as the combinat ion PIP product TC1507 (DAS -Ø15Ø7-1) x DAS-59122-7....”

 For 59122 x MON 810

–

“The data submitted by Pioneer are sufficient to support registration for the combination PIP product...provi ded that the registrant submits/cites any data required to support the PI P registrations of the indi vidual parental events....”

All requirements for additional product characteriz ation information for individual events TC1507 (see U.S. EPA (2010b)), MON 810 (see U.S. EPA (2010b)), and DAS-59122-7 (see section II(A)(1)(b) ofBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

35 this BRAD), as well as for combination PIP product TC1507 x 59122-7 (see section II(A)(2)(b) of this BRAD), have been satisfied.

4. References

Crickmore N, Zeigler DR, Schnepf E, Van Rie J, Lereclus D, Baum J, Bravo A, Dean DH. 2010. Bacillus thuringiensis toxin nomenclature (2010). Available from: http://www.lifesci.sussex.ac.uk/Home/Neil_Crickmore/Bt/

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Korjagin VA. 2000. Characterization of Escherichia coli Produced and Transgenic Maize Expressed Phosphinothricin Acetyltransferase (P AT) Protein (DAS Study Number 000369). Unpublished study prepared by Dow AgroSciences LLC.

Masson L, Schwab G, Mazza A, Brousseau R, Potvin L, Schwartz J-L. 2004. A novel Bacillus thuringiensis (PS149B1) containing a Cry34Ab1/Cry35Ab1 binary t oxin specific for the

western corn rootworm Diabrotica virgifera virgifera LeConte forms ion channels in lipid membranes. Biochemistry 43(38):12349–12357.

MRID No. 452422-01. Narva K, Duck N, Kahn T. 2000. Product Characterization Data for Bacillus thuringiensis PS149B1 13.6 kDa and 43.8 kDa Insecticidal Crystal Proteins Expressed in Transgenic Maize Plants. Lab Study ID: GH-C 5117. Unpublished study prepared by Dow AgroSciences LLC, 398 pages.

MRID No. 452422-02. Ernest A. 2000. Character ization of Inserted DNA of Event 5639 Transgenic Corn Plants: Interim Report . Lab Project Number: 000300. Unpublished study prepared by Dow AgroSciences LLC, 55 pages.

MRID No. 452422-03. Herman R, Gilbert J, Patter son V. 2000. Equivalency of Microbial- and Maize Expressed PS149B1 Proteins. Lab Project Number: 000367. Unpublished study prepared by Dow AgroSciences LLC, 85 pages.

MRID No. 452422-04. Herman R. 2000. Microbial PS 149B1 Binary Delta-Endotoxin: Maize-Insect- Pest Susceptibility Study. Lab Project Nu mber: 000366. Unpublished study prepared by Dow AgroSciences LLC, 29 pages.

MRID No. 452422-13. Zeph L, Taggart J, Schmid t J. 2000. Quantitative ELISA Analysis of PS149B1 Protein Expression Levels in Hybrid and Inbred Lines of Maize Event TC5639: Interim Report. Lab Project Number: PHI 99-004. Unpublished study prepared by Pioneer Hi-Bred International, Incorporated, 119 pages.

MRID No. 453584-01. Herman R. 2000. Thermolabil ity of PS149B1 Binary Delta-Endotoxin. Lab Project Number: 001041. Unpublished study prep ared by Dow AgroSciences LLC, 18 pages.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

36 MRID No. 453834-01. Collins R. 2001. Lateral Flow Te st Kit Method Validation for the Detection

of the PS149B1 14 kDa Protein in Mai ze Grain. Lab Projec t Number: GH-C 5212. Unpublished study prepared by Dow AgroSciences LLC, 30 pages.

MRID No. 455845-01. Herman R. 2002. Heat Lability of Individual Proteins of the PS149B1 Binary ICP. Lab Project Number: 010144. Unpublished study prepared by Dow AgroSciences LLC, 15 pages.

MRID No. 457904-01. Schafer B. 2002. Character ization of Cry34Ab1 and Cry35Ab1 from

Recombinant Pseudomonas fluorescens and Transgenic Maize. Lab Project Number: GH-C 5545. Unpublished study prepared by Dow AgroSciences LLC, 17 pages.

MRID No. 457904-02. Locke M, Nirunsuksiri W. 2002. Characterization of DNA Inserted into Transgenic Corn Events (Cry34Ab1 a nd Cry35Ab1). Lab Project Number: GH-C 5550. Unpublished study prepared by Dow AgroSciences LLC, 28 pages.

MRID No. 457906-01. Coats I, Herman R, Narv a K. 2002. Product Characterization Data for Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 Proteins Expr essed in Transgenic Maize Plants (PHP17662). Lab Project Number: PHI-2002-046. Unpublished study prepared by

Pioneer Hi-Bred Internationa l, Incorporated, 283 pages.

MRID No. 458086-01. Herman R. 2002. Summar y of Heat Lability Studies with Cry34Ab1/Cry35Ab1. Lab Project Number: GH-C 5603. Unpublished study prepared by Dow AgroSciences LLC, 21 pages.

MRID No. 458332-01. Essner R. 2002. Quantitative ELISA Analysis of Cry34Ab1 and Cry35Ab1 Proteins Expressed in Maize Plants Transf ormed with the Vector PHP17662. Lab Project Number: PHI-2002-049. Unpublishe d study prepared by Pioneer Hi-Bred International, Incorporated, 110 pages.

MRID No. 458602-01. Hunst P, Herman R. 2002. Slide Presentation Summarizing Cry34Ab1/Cry35Ab1 Heat Inactivation Stud ies. Lab Project Number: DN0006331. Unpublished study prepared by Dow AgroSciences LLC, 18 pages.

MRID No. 459428-01. Evans S, Layton R. 2003. Probe M OA Studies to Assess Potential for Protein Synthesis Inhibition by Bacillus thuringiensis PS149B1 Cry34Ab1/Cry35Ab1 Proteins in a Rabbit Reticulocyte Assay: Re-Examination of Lab Notebook Data. Lab Project Number: GH-C 5662. Unpublished study prepared by Dow AgroSciences LLC, 77 pages.

MRID No. 461239-01. Ojala B. 2003. Independent Laboratory Validation of Pioneer Hi-Bred International, Incorporated ELISA Method for the Quantification of Cr y34Ab1 Protein from Transgenic Plants. Project Number: BS 03/02. Unpublished study pr epared by Biolab Solutions, 46 pages.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

37 MRID No. 461239-02. Ojala B. 2003. Independent La boratory Validation of Dow AgroSciences Method GRM 03.13, “Determination of Cry35Ab1 In secticidal Crystal Protein in Maize Tissue by Enzyme-Linked Immunosorbent A ssay.” Project Number: BS03/01. Unpublished study prepared by Biolab Solutions, 45 pages.

MRID No. 461239-03. Narva K, Schnepf H, Nygaar d L. 2003. Cry 34/35 Prot ein Distribution and Familiarity Project Number: GH/C/5702. Unpub lished study prepared by Dow AgroSciences LLC, 26 pages.

MRID No. 461239-04. Essner R. 2003. Agronomic Characteristics, Quantitative ELISA and Nutrient Composition Analysis of Hybrid Maize Lines Containing the cry 34Ab1, cry 35Ab1

and pat Genes: Chile Locations. Project Number: PHI/2002/050. Unpublished study prepared by Pioneer Hi-Bred Internati onal, Incorporated and EPL Bi o-Analytical Services, 1,971

pages.

MRID No. 461239-05. Herman R. 2003. Biological E quivalency of Cry35Ab1 In secticidal Crystal Protein in Transgenic Plants and Derived from Transgenic Pseudomonas fluorescens . Project Number: GH/C/5696. Unpublished study prepared by Dow AgroSciences LLC, 17 pages.

MRID No. 461239-06. Schafer B. 2003. Characteriz ation of Cry34Ab1 and Cry35Ab1 Proteins Derived from Transgenic Maize Ev ent E4497.59.1.22 (DAS-59122-7). Project Number: 030033. Unpublished study prepared by Dow AgroSciences LLC, 50 pages.

MRID No. 461239-07. Schafer B. 2003. Characterizati on of Phosphinothricin Acetyltransferase (PAT) Derived from Transgenic Ma ize Event E4497.59.1.22. Project Number: 030027. Unpublished study prepared by Dow AgroSciences LLC, 30 pages.

MRID No. 461239-08. Locke M, Igo E. 2003. Characterization of DNA Inserted into Transgenic

Corn Events DAS-45216-6 and DAS-59122-7. Project Number: PHI/2002/038. Unpublished study prepared by E.I. Dupont de Nemours and Company, 81 pages.

MRID No. 461239-09. Locke M, Dietrich N, Webe r N. 2003. Detailed Characterization of DNA Inserted into Transgenic Corn Events DAS-45216-6 and DAS-59122-7. Project Number: PHI/2002/041. Unpublished study prepared by E.I. Dupont de Nemours and Company, 62 pages.

MRID No. 461239-10. Schafer B. 2003. Evaluation of Microbe-Derived Cry34Ab1 and Cry35Ab1 Proteins for Protein Synt hesis Inhibition Activity. Project Number: 031139. Unpublished study prepared by Dow AgroSciences LLC, 37 pages.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

38 MRID No. 462971-02. Weber N, Pfrogner B. 2004. Ch aracterization of a Stacked Hybrid of Transgenic Corn Lines DAS-01507-1 and DAS-59122-7 by Comparison to the Individual Lines. Project Number: PHI/2003/049. Unpublished study prepared by E.I. Dupont de Nemours and Company, 35 pages.

MRID No. 463438-01. Buffington J. 2004. Agronomic Char acteristics, Quantitative ELISA, and Nutrient Composition Analysis of Hybrid Maize Lines Containing cry 1F, cry 34Ab1,

cry 35Ab1, and pat Genes: U.S. and Canada Locations : Amended Final Report. Project Number: PHI/2003/017. Unpublished study prep ared by Dow AgroSciences LLC, EPL Bio-Analytical Services, and Bennett Agri cultural Research and Consulting, 1,114 pages.

MRID No. 475109-04. Werning M. 2008. Expressed Tra it Protein Concentration of Maize Lines Containing Events DAS-01507-1, DAS- 59122-7, MON-00810-6, and DAS-01507-1 x DAS- 59122-7 x MON-00810-6. Project Number s: PHI/2008/006, PHI/2008/006/012, and PHI/2008/006/011. Unpublished study prepared by Pioneer Hi-Bred International, Incorporated, 174 pages.

MRID No. 476778-01. Brink K, Dietrich N. 2009. Mo lecular Characterization of Maize Combined

Trait Product DAS-01507-1 x DAS-59122- 7 x MON-00810-6 x MON-00603-6 Using Southern Blot Analysis and Event-Specific Polymerase Chain Reaction. Project Number: PHI/2008/082. Unpublished study prepared by E.I. Dupont de Nemours and Company, 42 pages.

MRID No. 476778-02. Binning R. 2009. Laboratory Ch aracterization of Key Lepidopteran Pest Response to Pyramided Events. Project Nu mber: PHI/2008/101. Unpublished study prepared by Pioneer Hi-Bred Internati onal, Incorporated, 16 pages.

MRID No. 478654-01. Staley J, Rood T, Johnson T. 2009. Addendum to Expressed Trait Protein

Concentration of Maize Lines Contai ning Events DAS-01507-1, DAS-59122-7, MON- 00810, and Combined Trait Products DAS-01507-1 x MON-00810, DAS-59122-7 x MON-00810, and DAS-01507-1 x DAS-59122-7 x MON-00810: Final Report. Project Number: PHI/2008/006, PHI/2009/182. Unpublishe d study prepared by Pioneer Hi-Bred International, Incorporated, 21 pages.

MRID No. 478800-01. Staley J, Rood T, Johnson T. 2009. Expressed Trait Protein Concentration of Maize Lines Containing Events DAS-01507-1, DAS-59122-7, MON-00810-6, and

Combined Trait Products DAS-01507-1 x MON-00810-6, DAS-59122-7 x MON-00810-6,

and DAS-01507-1 x DAS-59122-7 x MON-00810-6: Additional Addendum. Project Number: PHI/2009/182. Unpublishe d study prepared by Pioneer Hi-Bred International, Incorporated, 110 pages.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

39 U.S. EPA. 2001a. Review of Product Characterizati on, Expression Analysis and Acute Oral Toxicity Studies for PS149B1 Binary Insect Contro l Proteins as Expressed in Maize, a Bacillus thuringiensis -Based Plant-Pesticide, for an Experi mental Use Permit. Memorandum from C.A. Wozniak, Ph.D. and J.L. Kough, P h.D. to M. Mendelsohn dated June 7, 2001.

U.S. EPA. 2001b. Biopesticides Registration Action Document – Bacillus thuringiensis Plant- Incorporated Protectants. Available from: http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm

.

U.S. EPA. 2002. Review of Heat Stability for PS 149B1 Binary Insect Control Proteins, Cry34 and Cry35, as Expressed in Maize, a Bacillus thuringiensis -Based Plant-Incorporated Protectant, for Approval of a Temporary Food Tolerance. Memorandum from C.A. Wozniak, Ph.D. and J.L. Kough, Ph.D. to M. Mendelsohn dated September 10, 2002.

U.S. EPA. 2003a. Summary of Heat Lability Studies for PS149B1 Bina ry Insect Control Proteins, Cry34Ab1 and Cry35Ab1, as Expressed in Maize, a Bacillus thuringiensis -Based Plant- Incorporated Protectant, fo r Approval of a Temporary Food Tolerance. Memorandum from C.A. Wozniak, Ph.D. and J.L. Kough, P h.D. to M. Mendelsohn dated March 18, 2003.

U.S. EPA. 2003b. Review of SDS-PAGE Sensitiv ity and Lateral Flow Detection Method for PS149B1 Binary Insect Control Prot eins as Expressed in Maize, a Bacillus thuringiensis

- Based Plant-Incorporated Pr otectant, for an Experimental Use Permit. Memorandum from C.A. Wozniak, Ph.D. and J.L. Kough, P h.D. to M. Mendelsohn dated April 3, 2003.

U.S. EPA. 2003c. Review of Product Characteriza tion Data for PS149B1 Bina ry Insect Control Proteins, Cry34Ab1 and Cry35Ab1, as Expressed in Maize Following Transformation with Construct PHP17662, a Bacillus thuringiensis -Based Plant-Incorporated Protectant, for an Experimental Use Permit. Memorandum from C.A. Wozniak, Ph.D. and J.L. Kough, Ph.D. to M. Mendelsohn dated May 21, 2003.

U.S. EPA. 2003d. Review of Product Characteriza tion Data for PS149B1 Bina ry Insect Control Proteins, Cry34Ab1 and Cry35Ab1, as Expressed in Maize Following Transformation with Construct PHP17662, a Bacillus thuringiensis -Based Plant-Incorporated Protectant, for an Experimental Use Permit. Memorandum from C.A. Wozniak, Ph.D. and J.L. Kough, Ph.D. to M. Mendelsohn dated June 12, 2003.

U.S. EPA. 2003e. Assessment of Ribosomal Inhibition Protein (RIP ) Activity in PS149B1 Binary Insect Control Proteins, Cry34Ab1 and Cr y35Ab1, as Expressed in Maize Following Transformation with Construct PHP17662, a Bacillus thuringiensis -Based Plant-Incorporated Protectant, for an Experimental Use Perm it. Memorandum from C.A. Wozniak, Ph.D. and J.L. Kough, Ph.D. to M. Mendelsohn dated June 19, 2003.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

40 U.S. EPA. 2004. Review of Product Characterizati on and Expression Analysis for Registration of

B

. t . Cry34/35AB1 Construct PHP17662 Corn. Memora ndum from R.L. Edel stein, Ph.D. and J.L. Kough Ph.D. to M. Mendelsohn dated December 6, 2004.

U.S. EPA. 2005a. Biopesticides Registration Action Document – Bacillus thuringiensis Cry1F Corn. Available from: http://www.epa.gov/oppbppd1/biopesticid es/ingredients/ tech_docs/brad_006481.pdf

.

U.S. EPA. 2005b. Review of Product Characterizati on and Human Health Data for Registration of

HerculexTM Xtra Insect Resist ant Corn. Memorandum from R.L. Edelstein, Ph.D. and J.L. Kough, Ph.D. to M. Mendelsohn dated September 22, 2005.

U.S. EPA. 2010a. Review of Human Health and Product Characterization Data for Registration of Bacillus thuringiensis ( Bt ) Cry1F, Cry34Ab1, Cry35Ab1, and Cr y1Ab Proteins and the Genetic Material Necessary for Their Producti on in the Combination Plant-Incorporated Protectant (PIP) Products: 1507 x MON 810 [EPA Reg. No. 29964-T], 59122 x MON 810 [EPA Reg. No. 29964-O], and 1507 x 59122 x MON 810 [EPA Reg. No. 29964-I]. Memorandum from A. Waggoner and J. L. Kough, Ph.D. to A. Sibold dated February 19, 2010.

U.S. EPA. 2010b. Biopesticides Registration Action Document – Bacillus thuringiensis Cry1Ab and Cry1F Corn (Updated September 2010). Available from: http://www.regulations.gov (see “Supporting & Related Material s” within Docket Number EPA-HQ-OPP-2010-0607).

USDA. 2004. Performance verification of qualitative mycotoxin and biotech rapid test kits (Directive 9181.2). Available from: http://www.gipsa.usda.gov/GIPSA/documents/GIPSA_Documents/9181-2.pdf

.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

41 B. Human Health Assessment

1. Human Health Assessment of Cry34Ab1 and Cry35Ab1

The detailed Agency human health assessment of Cry34Ab1/Cry35Ab1 corn is found in U.S. EPA (2005a and 2005d). Portions of the data used in the human health assessment are reviewed in U.S. EPA (2001a, 2002, 2003, and 2004a). A summary of the key findings is provided below.

a. Mammalian Toxicity and Allergenicity Assessment

Based upon the human health data provided, there is mini mal risk of toxic and/or allergenic effects to humans or animals due to exposure to the Cry34 Ab1 and Cry35Ab1 proteins. Based on review of the data, there is a reasonable certain ty of no harm to humans and animals posed by the aggregate exposure to residues of these proteins.

i. Mammalian Toxicity

Acute oral toxicity data have been submitted dem onstrating the lack of mammalian toxicity at high levels of exposure to the pure Cr y34Ab1 and Cry35Ab1 proteins separately and combined. These data demonstrate the safety of the products at levels we ll above maximum possible exposure levels that are reasonably anticipated in the crops. Basing this conclusion on acute oral toxicity data without requiring further toxicity testing and residue data is simila r to the Agency position regarding toxicity and the requirement of residue data for the microbial Bacillus thuringiensis products from which these plantincorporated protectants we re derived (see 40 CFR §§ 158.2130( d)(1)(i) and 158.2140(d)(7)). For microbial products, further toxicity testing and residue data are trigge red by significant acute effects in studies, such as the mouse oral toxi city study, to verify and quantify the observed effects and clarify the source of these effects (Tiers II and III).

Three acute oral toxicity studies on Cry34Ab1 a nd Cry35Ab1 in mice were submitted, which indicated that these proteins ar e non-toxic to humans.

In an oral toxicity study of Cry34Ab1 alone (M aster Record Identificat ion Number (MRID No.) 452422-07; reviewed in U.S. EP A (2005a)), Cry34Ab1 produced fr om microbial culture was administered to five male mice (5,000 milligrams/kilo gram (mg/kg) body weight) by oral gavage as a 20% mixture in a 0.5% aqueous met hylcellulose vehicle. All animals survived the two-week study. No clinical signs were noted for any animals during the study. An initial weight loss was observed in 3 mice at test days 1 and 2, but they gain ed weight for the remainder of the study. The two other animals gained weight throughout the study. No treatment

- related gross pathologic changes were ob served during the study. Under the conditions of this study, the acute oral median lethal dose (LD 50 ) for the test substance in male CD-1 mice was greater than 5,000 mg/kg. Si nce the test substance contained Cry34Ab1 at 54% purity, the acute oral LD 50 for the pure Cry34Ab1 protei n was greater than 2,700 mg/kg.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

42 In an oral toxicity study of Cry35Ab1 alone (M RID No. 452422-08; reviewed in U.S. EPA (2005a)), Cry35Ab1 produced from microbial culture was administered to five male mice (5,000 mg/kg body weight) by oral gavage as a 20% mixture in a 0.5% aqueous methyl cellulose vehicle. All animals survived the two-week study. No c linical signs were noted for any animal during the study. An initial weight loss was observed in 2 mice at test days 1 and 2, but they gained weight for the remainder of the study. One animal had fluctuating body weight. The other two animals gained weight throughout the study. No treatment

- related gross pathologic changes were observed during the study. Under the conditions of this study, the acute oral LD 50 for the test substance in male CD-1 mice was greater than 5,000 mg/kg. Since the test substance containe d Cry35Ab1 at 37% purity , the acute oral LD 50 for the pure Cry35Ab1 protein was greater than 1,850 mg/kg.

Finally, in an oral toxici ty study of Cry34Ab1 and Cry35Ab1 combined (MRID No. 452422-09; reviewed in U.S. EPA (2001a)), a mixture of the microbially produced Cry34Ab1 and Cry35Ab1 proteins (5,000 milligrams (mg) test material , containing 482 mg pure Cry34Ab1 and 1,520 mg pure Cry35Ab1 (corresponding to an equimolar ratio), per kilogram (kg) body wei ght) was administered by oral gavage to 5 female and 5 male mice as a 20% mixture in 0.5% aqueous methylcellulose. All animals survived the 2-week study. One female mouse exhibited protruding or bulging eyes on days 6 and 7, but this resolved th ereafter. This observation was not attributed to the treatment as it was an isolated observation (i.e., no other animals exhibited this). No other clinical signs were noted for any animals during the study. An initial weight loss was observed in 2 mi ce at test days 1 and 2, but both gained weight for the remainder of the study. All ot her animals gained weight throughout the study. No treatment-related gross pathologic changes were noted. Under the condi tions of the study, the acute oral

LD 50 of the test material in male and female CD-1 mice was greater th an 5,000 mg/kg body weight, corresponding to 2,000 mg/kg of an equimo lar ratio of the pure proteins.

In addition, a study was submitted where the am ino acid sequences of the Cry34Ab1 and Cry35Ab1 proteins were compared with prot ein sequences in publicly available databases (GenPept dataset) to identify any potential similarities with known t oxins (MRID No. 465847-01; reviewed in U.S. EPA (2005d)). No similarities were identifi ed that would raise a safety concern.

When proteins are toxic, they are known to act via acute mechanisms and at very low dose levels (Sjoblad et al . 1992). Therefore, since no effects were show n to be caused by the plant-incorporated protectants, even at relatively high dose levels, the Cry34Ab1 and Cr y35Ab1 proteins are not considered toxic. Further, amino acid sequence comparisons showed no similarity between the Cry34Ab1 and Cry35Ab1 proteins and known toxi c proteins available in public protein databases.

ii. Allergenicity Assessment

Since Cry34Ab1 and Cry35Ab1 are proteins, allergen ic potential was also considered. Currently, no definitive tests for determining the allergenic potential of novel proteins exist. Therefore, EPA uses a weight-of-the-evidence approach where the following factors are considered: source of the trait; amino acid sequence similarity w ith known allergens; prevalence in food; and biochemical properties of the protein, including in vitro digestibility in simulated gastric fluid (SGF) and glycosylation. CurrentBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

43 scientific knowledge sugg ests that common f ood allergens tend to be resistant to degradation by acid and proteases

, may be glycosylated, and can be present at high concentrations in the food. In the past, EPA has also considered heat stability in assess ing allergenicity potential; however, at a March 1–2, 2005 meeting, the Federal Insecticid e, Fungicide, and Rodenticide Ac t (FIFRA) Scientific Advisory Panel (SAP) stated that heat stability based on a bio activity assay is of minimal to no value in predicting the allergenicity potentia l of novel proteins, and EPA agrees. Th erefore, EPA did not consider heat stability of these proteins in its weight-of-the-evidence approach.

Source of the trait

. Bacillus thuringiensis is not considered a sour ce of allergenic proteins.

Amino acid sequence

. A comparison of amino acid sequences of Cry34Ab1 and Cry35Ab1 with known allergens (MRID No. 452422-05; re viewed in U.S. EPA (2001a)) showed no overall sequence similarities or homology at the level of 8 contiguous amino acid residues.

Prevalence in food

. Expression level analysis indicated that th e proteins are presen t at relatively low levels in corn; on a dry weight basis, Cry34Ab1 is present at a concentration of approximately 50 nanograms per milligram (ng/mg) in grain from event 59122-7, and Cry35Ab1 is present at a concentration of approximately 1 ng/mg in gr ain from event 59122-7 (MRID No. 461239-04; reviewed in U.S. EPA (2004b)). Thus, expression of the Cr y34Ab1 and Cry35Ab1 proteins in corn kernels has been shown to be in the parts per million range.

Digestibility

. Two in vitro digestibility studies (MRID No. 452422- 12; reviewed in U.S. EPA (2001a)) were conducted to determine the stability of the Cry34Ab1 and Cry35Ab1 proteins in simulated gastric fluid (i.e., an acid environment cont aining pepsin; SGF). In the first in vitro digestibility study, the proteins were incubated in SGF (pepsin concentra tion: 3.2 milligrams per milliliter (mg/mL); pH 1.2; 37°C) with a pepsin to protein substrate ratio of approximately 20:1, moles to moles (mol/mol) (equivalent to 60:1, weight-to- weight (w/w) for Cry34Ab1 and 17:1, w/w for Cry 35Ab1). Samples taken at 1, 5, 7, 15, 20, 30, and 60 minutes were anal yzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot. Cry35A b1 was no longer visible at the five-minute time point using both SDS-PAGE stained with Cooma ssie Brilliant Blue and western blot detection. Cry34Ab1 was visible on the stained ge l for the 15-minute sample but not in later sample time points. In the western blot analysis, Cry34Ab1 was visible in the 20-minute sample but not in later sample time points. In conclusion, this firs t study showed that Cry34Ab1 was di gested within 30 minutes, and Cry35Ab1 was digested within 5 minutes in SGF under the conditions of the study.

Because Cry34Ab1 appeared to be somewhat resist ant to SGF in the study that used the time-todisappearance endpoint (as described in the preced ing paragraph), Dow AgroSciences LLC submitted a second study on the in vitro digestibility of Cry34Ab1 in SGF us ing a kinetic approach (MRID No. 455845-02; reviewed in U.S. EPA (2002)). The digest ion was performed under the same conditions as the previous study, except that re action mixtures were shaken dur ing incubation and samples were analyzed at 1, 2, 3, 5, 7.5, 10, 15, and 20 minutes. The previous study on pepsin digestibility of Cry34Ab1 and Cry35Ab1, as well as other pepsin digestibility studies used in allergenicity assessments, focused on the time required for the protein to beco me undetectable and, therefore, the results areBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

44 dependent on the detection limit of the analytical method used. In this second study, Dow AgroSciences LLC determined the rate of pe psin digestion of Cry34Ab1 by m easuring the relative amounts of Cry34Ab1 at each of the time points based on SDS-P AGE densitometry estimates. Under the conditions of the study, the rate of decay fit a first-order mo del (with respect to Cry34Ab1 concentration), and Dow AgroSciences LLC estimated the DT 50 (half-life) and DT 90 (time until 90% decay) to be 1.9 minutes and 6.2 minutes, respectively. In this experiment, Cry34A b1 was visible on gels and blots in 15-minute timepoint samples but not in 20-minute time-point samples.

Because the digestibility of Cry34Ab1 was assessed using a differe nt method (i.e., the kinetic approach) rather than the typical end-point method that has been used previ ously, comparison studies, using the kinetic approach to assess the digestibility of known allergens and non-allergens, were submitted to validate the method and allow comparison of the digestibility of Cry34Ab1 with known allergens and non-allergens (Master Record Id entification Numbers (MRID No s.) 461239-20 and 463886-01). In the comparison study where the conditions used were the same as those used in the kinetic study on the digestibility of Cry34Ab1 (MRID No. 463886-01; revi ewed in U.S. EPA (2005a)), two allergens and two non-allergens were shown to digest simila rly to Cry34Ab1. From these studies and published studies, EPA concludes that Cry35Ab1 is rapidly digested, and Cry34Ab1 is digested at a moderate rate in SGF; Cry34Ab1 appears to digest slower than previously registered proteins and ma ny other proteins that are not considered allerg ens but faster than most pr eviously tested allergens.

On March 1–2, 2005, EPA held a FIFRA SAP meeting to address the scientific i ssues that arose during the human health safety assessment of Cry34Ab1 and Cry35Ab1. The SAP report (U.S. EPA 2005b) is summarized below and in U.S. EPA (2005c). EPA as ked the SAP to comment on EPA’s allergenicity assessment of Cry34Ab1. The SAP agreed with EPA’ s preliminary assessment that the allergenicity potential of Cry34Ab1 is low. However, the Panel ba sed its conclusion, in part, on statements made by Dow AgroSciences LLC that Cry34Ab1 and Cry35Ab1 do not aggregate in solution. The Panel was concerned that if the proteins were to aggregate, protease binding site s could be masked, and the rate of digestion could be slower than was observed for the individual proteins. Therefore, EPA asked Dow AgroSciences LLC to submit da ta supporting the claim that Cr y34Ab1 and Cry35Ab1 do not associate

with one another in solution.

To support the digestibility studies on the individual proteins, Dow AgroSciences LLC submitted a study using size exclusion chromatography, which demonstrated that Cry34Ab1 and Cry35Ab1 do not

associate with one another in solution under acid ic conditions (MRID No. 465568-01; reviewed in U.S. EPA (2005d)).

Glycosylation

. Cry34Ab1 and Cry35Ab1 expressed in corn were shown not to be glycosylated (MRID No. 461239-06; reviewed in U.S. EPA (2004b)).

Conclusion . Considering that (1) Cry34Ab1 and Cry35Ab1 or iginate from a non-allergenic source, (2) Cry34Ab1 and Cry35Ab1 have no overall sequence similar ities or homology at the level of 8 contiguous amino acid residues with known allergens, (3) Cry34Ab1 and Cry35Ab1 will onl y be present at low levels in food, (4) Cry35Ab1 is rapidly digested in SG F, (5) Cry34Ab1 is digested at a moderate rate inBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

45 SGF, and (6) Cry34Ab1 and Cry35Ab1 are not glycos ylated when expressed in maize, EPA has concluded that the potential for the Cry34Ab1 and Cry35Ab1 proteins to be food allergens is minimal. The FIFRA SAP that met on March 1–2, 2005 agreed with this conclusion regard ing the allergenicity potential of Cry34Ab1. There were no triggers to raise concern about the allergenicity of Cry35Ab1, so

the SAP was not asked to comment specifically on Cry35Ab1. As noted above, toxic proteins typically

act as acute toxins with low dose levels. Therefore, since no effect s were shown to be caused by the plant-incorporated protectants, ev en at relatively high dose levels , the Cry34Ab1 and Cry35Ab1 proteins

are not considered toxic.

SAP Opinion on the Kinetic Approach

In general, the Panel supported using a kinetic ap proach, rather than the disappearance endpoint, for assessing proteins that are not ra pidly degraded because using severa l time points is “inherently more accurate than the single point assay.” The Panel point ed out, however, that many of the statements and assumptions Dow AgroSciences LLC made about the kinetic model are incorre ct. In particular, Dow AgroSciences LLC has stated that, as long as first-or der conditions are met, first-order rate constants and half-lives are unaffected by changes in substrate and enzyme concentrat ion (when enzyme is saturating) and, therefore, can be used to predict relative digestion efficien cies for proteins even if the concentrations are varied among experiments. The Pa nel stated that it was “impossible to comment” on these statements without knowing the affinity of the substrate for the enzyme and emphasized the need for standardized assay conditions. In additi on, the Panel report stated the following:

“A poor fit of early time points to Michaelis-Menten kinetics indicates a problem with the model. The Panel questioned the application of single

substrate Michaelis-Menten kinetics to th e registrant data. The registrant did not adequately address the problems of applying classical Michaelis-Menten kinetics to a situation where [E 0 ] >> [S 0 ]. They incorrectly applied the term

V

max /K M to this scenario.”

In addition, “the Panel noted that at high enzyme to substrate rati os, the simple quasi-steady-state Michaelis-Menten equation cannot be applied.”

Some Panel members expressed concern about omitti ng early time points to achieve a good fit to firstorder decay but in general “ag reed that modeling of the late first order decay was an adequate/conservative approach for a given simula ted gastric fluid (SGF) de gradation assay...provided that a significant fraction of the di gestion takes place during this slow phase.” The Panel report did not specify what a “significant fracti on” is but pointed out that, in the case of Cry34Ab1, one-half of the substrate had been consumed by the first time point of one minute. The report stat ed that “one does not

know whether or not the reaction foll ows first order kinetics during the initial 50% of the reaction,” but in this case, “this may not be impor tant since one is interested in the time course for total substrate hydrolysis.” However, the Panel also poi nted out that the late phase “may be hard to define in a practical setting and consequently impossible to standardize.” In conclusion, although the Panel pointed out someBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

46 problems with the kinetic approach, it recommended usi ng the approach for protei ns that are not rapidly digested.

SAP Opinion on Assay Conditions

The Panel emphasized the need for standardized assa y conditions throughout the report and gave some useful information on appropriate conditions. The Panel report clearly stated that a fixed amount of enzyme activity units should be used in digestion studi es, and a standardized prot ocol should be used to determine enzyme activity.

In addition, the Panel indicated that the pepsin to substrate ratio and the concentrations of pepsin and substrate have a pronounced effect on the rate of hydrolysis, implying that both the ratio and

concentrations should be standardized.

EPA asked if the concentration of test protein should be fixed on a weight basi s (mg/mL) or a mole basis (mol/L). There was no consensus from the Panel on this issue: “Some Panel members recommended comparison of test proteins on a weight/mL, while others recommended a mole/mL.”

One Panel member stated that a standardized pH should also be used for digestibility assays and indicated that there is no evidence that using more than one pH is necessary for predicting allergenicity. The Panel did not indicate one particular pH as being more appropriate than another.

The Panel report discussed the pros and cons of the different methods that can be used for monitoring digestion reactions (i.e., SDS-PA GE with staining, western blot analysis, and high-performance liquid chromatography (HPLC)). Typically, applicants use SD S-PAGE with staining and western blot analysis. The Panel did not provide any information that would suggest a change to the current methodology.

When asked about the pros and cons of using one digestion reaction and rem oving aliquots at various time points for monitoring or setting up separate reactions for each of the time points, the Panel responded that “there would be no difference...as long as there are no pipetting errors and the reactions are homogeneous.” In addition, several Panel members indicated that using a single digestion reaction gives a “slightly better ability to control extraneous factors that could affect experimental variability.” The report also pointed out that different statistical models would be required, depending on which approach is used.

SAP Opinion on Digestibility Studies in Allergenicity Assessments

When asked what weight in vitro digestibility studies should be given in the overall allergenicity assessment compared with other criteria, the majority of the Panel indicated that digestibility is of some value but is less important than the source of the protein, sequen ce homology, or a validated animal

model. The Panel also stated that “t he use of digestibility data also is only of value in the context of a total weight of evidence approach.”Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

47 When asked about setting acceptable/unacceptable limits fo r digestibility in assessing the safety of a protein, the Panel stated that it is “difficult if not impossible given the lack of consistency between digestibility and allergenicity.” The Panel also noted that the relative digestibility of proteins depends on the assay conditions used, and a da tabase of digestibili ties under standard conditions needs to be

established.

When asked about the significance of the rate of digestion of protein fragments, the Panel recommended determining digestion rates for all fragments of a molecular mass >1500 Daltons (Da) (~10–15 amino acids) since a number of a llergens have been shown to be peptid es. The Panel recommended this size range because smaller peptides are not detectable using SDS-PAGE.

SAP Opinion on Cry34Ab1 Allergenicity Assessment

The Panel agreed with EPA’s assessment that th e weight-of-the-evidence i ndicates that Cry34Ab1 is unlikely to be a food allergen. The Panel reached this decision based on the following: (1) Cry34Ab1 is moderately digested in SGF, a nd it does not self-aggregate or aggr egate with Cry35Ab1 in physiologic solutions; (2) Cry34Ab1 originates from a non-alle rgenic source; (3) Cry34Ab1 has no sequence similarity with known allergens “based on data presen ted on the lack of identity of 8 contiguous amino acids or more than 35% identity over 80 amino acids with known inhalant and ingested allergens”; and (4) Cry34Ab1 is not glycosylated when expressed in maize.

The majority of Panel members indicated that heat st ability is of minimal to no value in predicting the allergenicity potential of Cry34Ab1. Th e Panel also stated that “there is no convincing evidence that B. thuringiensis is an allergen in that it has never conclusively been docum ented to provoke an allergic reaction.” The Panel report also noted that “there are no data indicating th at crystal proteins are allergens.”

b. Aggregate Exposures

In examining aggregate exposure, section 408 of the Federal Food, Drug, and Cosmetic Act (FFDCA)

directs EPA to consider available informa tion 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).

The Agency has considered available information on the aggregate exposure levels of consumers (and major identifiable subgroups of c onsumers) to the pesticide chemi cal residue and to other related

substances. These considerations include dietary exposure under the tolerance exempti on and all other tolerances or exemptions in effect for the plant-in corporated protectants’ ch emical residue, and exposure from non-occupational sources. Exposure via the sk in or inhalation is not likely since the plantincorporated protectants are contained within plant cells, which essentially eliminates these exposure routes or reduces these exposure routes to negligible. Exposure via residential or lawn use to infants and children is also not expected because the use sites for the Cry34Ab1 and Cry35Ab1 proteins are all agricultural for control of insects. Oral exposure, at very low levels, may occur from ingestion ofBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

48 processed corn products and, potentia lly, drinking water. Oral toxic ity testing, however , did not show any adverse effects. Furthermore, the expressi on of the Cry34Ab1 and Cry35Ab1 proteins in corn

kernels has been shown to be in the parts per million range, which ma kes the expected dietary exposure several orders of magnitude lower than the amounts of Cry34Ab1 and Cry35Ab1 proteins shown to have no toxicity. Therefore, even if ne gligible aggregate exposure should occur, the Agency concludes that such exposure would result in no harm due to the lack of mammalian toxicity and low potential for allergenicity demonstrated for the Cry34Ab1 and Cry35Ab1 proteins.

c. Cumulative Effects from Substances with a Common Mechanism of Toxicity

Section 408(b)(2)(D)(v) of FFDCA re quires that, when considering wh ether to establish, modify, or revoke a tolerance, the Agency consider “available information concerning the cumulative effects of [a particular pesticide’s] residues and other substanc es that have a common mechanism of toxicity.”

EPA has considered available information on the cumulative effects of such residues and other

substances that have a common me chanism of toxicity. These considerations included the cumulative

effects on infants and children of such residues and other substances with a common mechanism of toxicity. Because there is no indi cation of mammalian toxicity resul ting from the plant-incorporated protectants, the Agency conclude s that there are no cumulative e ffects for the Cry34Ab1 and Cry35Ab1 proteins. For information regarding EPA’s effort s to determine which ch emicals have a common mechanism of toxicity and to evaluate the cumulativ e 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

i. Toxicity and Allergenicity Conclusions

The data submitted and cited regarding potential h ealth effects for the Cry34Ab1 and Cry35Ab1 proteins include the characterization of the expressed Cry 34Ab1 and Cry35Ab1 proteins in corn, as well as the acute oral toxicity and in vitro digestibility of the proteins. The resu lts of these studies were determined applicable to evaluate human risk, and the validity, completeness, and reliability of the available data

from the studies were considered.

Adequate information was submitted to show that the Cry34Ab1 and Cry35Ab1 protein test material

derived from microbial cultures wa s biochemically and functionally si milar to the protein produced by the plant-incorporated protectant ingredients in corn. Production of microbially produced protein was

chosen in order to obtain su fficient material for testing.

The submitted acute oral toxicity data support the prediction that the Cry 34Ab1 and Cry35Ab1 proteins

would be non-toxic to hu mans. As mentioned in section II(B)(1)(a)(i) of this Biopesticides Registration Action Document (BRAD), wh en proteins are toxic, they are known to act via acute mechanisms and at very low dose levels (Sjoblad et al . 1992). Since no effects were show n to be caused by the Cry34Ab1 and Cry35Ab1 proteins, even at re latively high dose levels, the Cry34Ab1 and Cry35Ab1 proteins areBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

49 not considered toxic. Basing this co nclusion on acute oral toxicity data without requiring further toxicity testing and residue data is similar to the Agency position regarding toxicity and the requirement of

residue data for the microbial Bt products from which these plant-in corporated protectants were derived (see 40 CFR §§ 158.2130(d)(1)(i) and 158.2140(d)(7)). For mi crobial products, further toxicity testing and residue data are triggered by si gnificant acute effects in studies, such as the mouse oral toxicity study, to verify and quantify the observed effects and cl arify the source of these e ffects (Tiers II and III).

Cry34Ab1 and Cry35Ab1 protein residue chemistry data were not required for a human health effects assessment of the subject plant-incorporated protect ant ingredients because of the lack of mammalian toxicity. Notwithstanding that, submitted data de monstrated low levels of the Cry34Ab1 and Cry35Ab1 proteins in corn tissues.

Since Cry34Ab1 and Cry35Ab1 are proteins, their potential allergenicity is also cons idered as part of the toxicity assessment. Consideri ng that (1) Cry34Ab1 and Cry35Ab1 orig inate from a non-allergenic source, (2) Cry34Ab1 and Cry35Ab1 have no overall seque nce similarities or homology at the level of 8 contiguous amino acid residues with known a llergens, (3) Cry34Ab1 and Cry35Ab1 are not glycosylated when expressed in maize, (4) Cry34 Ab1 and Cry35Ab1 will only be present at low levels in food, (5) Cry35Ab1 is rapidly digest ed in SGF, and (6) Cry34Ab1 is di gested at a moderate rate in SGF, EPA has concluded that the potential fo r the Cry34Ab1 and Cry35Ab1 proteins to be food allergens is minimal. The FIFRA SAP that me t on March 1–2, 2005 agreed with this conclusion regarding the allergenicit y potential of Cry34Ab1. There were no tr iggers to raise concern about the allergenicity of Cry35Ab1, so the SAP was not asked to comment specifically on Cry35Ab1.

Neither available information concerning the diet ary consumption patterns of consumers (and major identifiable subgroups of consumers including infants and children) nor safety factors that are generally recognized as appropriate for the use of animal e xperimentation data were evaluated. The lack of mammalian toxicity at high levels of exposure to the Cry34Ab1 and Cry35Ab1 proteins, as well as the minimal potential to be a food allergen, demonstrat e the safety of the produc t at levels well above possible maximum exposure leve ls anticipated in the crop.

The genetic material necessary for the production of the plant-incorpor ated protectant active ingredients are the nucleic acids (deoxyribonuc leic acid (DNA) and ribonucleic aci d (RNA)) that comprise genetic material encoding these proteins and their regulatory regions. Th e genetic material (DNA and RNA) necessary for the production of the Cry34Ab1 and Cry35Ab1 proteins have been exempted under the blanket exemption for all nuc leic acids (40 CFR § 174.507).

ii. Infants and Children Risk Conclusions

FFDCA section 408(b)(2)(C ) provides that EPA shall assess the available information about consumption patterns among infants and children, speci al susceptibility of infants and children to pesticide chemical residues, and the cumulative effect s on infants and children of the residues and other substances with a common mechanism of toxicity.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

50 In addition, FFDCA section 408(b)(2 )(C) also provides that EPA sh all 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 completene ss of the data base unless EPA determines that a different margin of safety will be safe for infants and children.

In this instance, based on all the available informa tion, the Agency concludes th at there is a finding of no toxicity for the Cry34Ab1 and Cry35Ab1 proteins and the genetic material necessary for their production. Thus, there are no threshol d effects of concern and, as a re sult, an additional margin of safety for infants and chil dren is not necessary.

iii. 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 Cry34Ab1 and Cry35Ab1 protei ns and the genetic material necessary for their production. This includes all anticip ated dietary exposures a nd all other exposures for

which there is reliable information.

The Agency has arrived at this conclusion because, as discussed thus far in this chapter, no toxicity to mammals has been observed, nor has there been any i ndication of allergenicity potential for the plantincorporated protectants.

e. Other Considerations

i. Analytical Enforcement Methodology

Validated enzyme-linked immunosor bent assays, for the detection and quantification of Cry34Ab1 and Cry35Ab1 in corn tissue, have been submitted and found acceptable by the Agency (MRID Nos. 461239-01 and 461239-02; reviewed in U.S. EPA (2004b)).

ii. International Residue Limits

In making its tolerance decisions, EPA seeks to harm onize U.S. tolerances with international standards whenever possible, consistent with U.S. food safety standards and agricu ltural practices. In this context,

EPA considers the international maximum residue lim its (MRLs) established by the Codex Alimentarius Commission (Codex), as required by FFDCA section 408(b)(4). The C odex Alimentarius is a joint United Nations Food and Agriculture Organization/Wo rld Health Organization food standards program, and it is recognized as an internatio nal food safety standards-setting or ganization in trade agreements to which the United States is a party. EPA may estab lish a tolerance that is different from a Codex maximum residue level (MRL); however, FFDCA sec tion 408(b)(4) requires that EPA explain the reasons for departing from the Codex level.

The Codex has not established a MRL for the Cry34Ab1 and Cry35Ab1 proteins in corn.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

51 f. Endocrine Disruptors

As required under FFDCA section 408( p), the Agency has developed th e Endocrine Disruptor Screening Program (EDSP) to determine whether certain su bstances (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 effect s 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. Ch emicals that go through Tier 1 screening and are found to have the potential to inte ract with E, A, or T hor monal systems will proceed to the next stage of the EDSP where the Agency w ill determine which, if any, of the Tier 2 tests are necessary based on the available data. Tier 2 testi ng is designed to identify any adverse endocrinerelated effects caused by the substance, and estab lish a quantitative relations hip between the dose and the E, A, or T effect.

Between October 2009 and February 2010, the Agency issu ed test orders/data cal l-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 agri cultural scenarios. Th is list should not be

construed as a list of known or likely endocrine disruptors.

The Cry34Ab1 and Cry35Ab1 proteins are not among the group of 58 pesticide ac tive 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 anticipat es issuing future EDSP orders/data call-ins for all pesticide active ingredients.

For further information on the status of the EDSP, th e policies and procedures, th e list of 67 chemicals, the test guidelines and the Tier 1 scre ening battery, please visit our website: http://www.epa.gov/endo/

.

g. Tolerance Exemption

Certain data submitted and reviewed for experiment al use permits, as well as the FIFRA section 3 registrations of event DAS-59122-7 corn, also supported the petition for a tolerance exemption for

residues of the Bt Cry34Ab1 and Cry35Ab1 proteins. Given the available data and information as summarized in Table 1, the Agency established a permanent exemption from the requirement of a

tolerance for residues of the Bt Cry34Ab1 and Cry35Ab1 proteins and th e genetic material necessary for their production in corn when used as plant-incorporated protectants in the food and feed commodities of corn; corn, field; corn, sw eet; and corn, pop under 40 CFR § 174.457 ( 70 Federal Register (FR)

55254 ; September 21, 2005). Subsequently, administra tive revisions were made to existing plantincorporated protectant tolerance exemptions (e.g., some tolerance exemptions were moved from 40 CFR part 180 to 40 CFR part 174).Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

52 The original Cry34Ab1 and Cry35Ab1 tolerance exemp tion was subject to these revisions as it was redesignated from 40 CFR § 174.457 to 40 CFR § 174.506 and changed to the following ( 72 FR 20431 ; April 25, 2007):

“ § 174.506 Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn; exemption from the requirement of a tolerance.

Residues of Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins in corn are exempted from the requirement of a tolerance when used as plant-incorporated protectants in the food a nd feed commodities of corn; corn, field; corn, sweet; and corn, pop.”

Table 1. Human Health Assessment Data for Cry34Ab1 and Cry35Ab1.

Study Title Summary MRID No.

Comparison of the Amino Acid Sequence

of the Bacillus thuringiensis strain

PS149B1 13.6 kDa and

43.8 kDa Insecticidal Crystal Proteins to

Known Protein Allergens a Based upon the search requirements th at at least 8 contiguous amino acids are identical to a known allergenic sequence, PS149B1 13.6-kiloDalton (kDa) and 43.8-kDa proteins are unrelated to any known allergens in that they do not share any linear epitopes with known protein allergens. Classification: Acceptable 452422-05

PS149B1 14 kDa and

44 kDa Proteins: Acute Oral Toxicity Study in

CD-1 Mice a All animals survived the two-week study. One female mouse exhibited protruding or bulging eyes on days 6 and 7, but this resolved thereafter.

This observation was not attributed to the treatment as it was an isolated observation (i.e., no other animals exhibited this). No other clinical signs were noted for any animals during the study. An initial weight loss was observed in 2 mice at test days 1 and 2, but both gained weight for the remainder of the study. All other animals gained weight throughout the study. No gross treatment-related observations were recorded during the study as represented by gross pathologic observations. An acute oral LD 50 was calculated for this study based upon a dosage of a 1:4.6 ratio mixture of PS149B1 proteins at greater than 5,000 mg/kg and greater than 2,000 mg/kg for an equimolar mixture (1:3) of the pure proteins. Classification: Acceptable 452422-09

a Reviewed in U.S. EPA (2001a)Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

53 Study Title Summary MRID No.

In vitro Digestibility of

PS149B1 Proteins b Pseudomonas fluorescens strains MR1253 and MR1256 were used to prepare the 14-kDa and 44-kDa PS 149B1 proteins, respectively, as inclusion bodies. A simulated gastric fluid was produced to determine the lability of these proteins in an acid environment containing pepsin. The bovine serum albumin (BSA) positive control was rapidly digested (within 1 minute) and the b-lactoglobulin re mained intact for 60 minutes, as expected, the duration of the experiment. The 14-kDa protein was visible on the SDS-PAGE gel at the 15-minute sample point but not afterwards. It was detected on the western blot, which has greater sensitivity, at the 20- minute time point but not in later sample points. A band was also noted on the western blot at approximately 30 kDa. This band was noted in an earlier study and considered the result of protein aggregation. For the 44- kDa protein western blot, bands were observed at approximately 42 kDa and 14 kDa. A single band was observed on the 44-kDa SDS-PAGE atapproximately 15 to 16 kDa. These bands were only observed at the oneminute time point but not afterwards. It is concluded that both insecticidal crystal proteins (ICPs) are susceptible to degradation in the simulated gastric environment and di ffer in their lability to these conditions (i.e., the 44-kDa protein is more rapidly degraded). Classification: Acceptable 452422-12

In vitro Simulated

Gastric Fluid Digestibility Study of Microbially Derived Cry34Ab1 Protein c Bovine serum albumin, the positive contro l protein, was digested rapidly in the assay and could not be readily detected after 1 minute in the SGF. In contrast, the negative control protein, b-lactoglobulin, was detectable and appeared to change little during the assay time frame. When averaged across the 3 duplicate gels containing Cry34Ab1, the protein appeared to have approached full degradation by 7.5 minutes. Volumes remaining at the 10- and 15-minute time points were excluded from the calculations since they were below background le vels. Using this first-order decay model, the DT 50 and DT 90 for this protein in the SGF were estimated to be 1.9 and 6.2 minutes, respectively. The Cry34Ab1 protein was rapidly degraded in the SGF using this assay and detection methodology. The conditions of the assay were biologically appropriate in temperature, pH, and chemical makeup of the digestive solution. The first-order decay rate kinetics accurately portrayed the digestion of Cry34Ab1. Classification: Acceptable 455845-02

b Reviewed in U.S. EPA (2001a)

c Reviewed in U.S. EPA (2002)Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

54 Study Title Summary MRID No.

SDS-PAGE Sensitivity Analysis for Cry35Ab1 in Support of the

Simulated Gastric Fluid Digestion Study d GelCode Blue staining of the SDS- PAGE gel used to electrophorese Cry35Ab1 samples indicated that the am ount of protein remaining that was visualized by dye binding, 15.6 nanograms (ng), represented 2.6% of the total load (0.61 mg) in that lane. Therefore, 97% of the protein was digested by the simulated gastric digestion assay (pepsin treatment) at this 5-minute time point. The GelCode Staining Reagent does, however, have a sensitivity limitation, which may or may not allow for detection ofCry35Ab1 at levels of 8 ng or less. The sequential sample coelectrophoresed on the gel examined was a 7.8 ng Cry35Ab1 protein/lane, which was not detected by visual examination following staining and destaining. Classification: Supplemental 457904-08

Digestion of Allergenic and Non-Allergenic

Proteins in Simulated

Gastric Fluid e The rate of digestion of allergenic and non-allergenic proteins in SGF was compared. Proteins were incubated in SGF (pH 1.2 and 2.0; pepsin:substrate ratio approximately 3:1, w/w; reaction not shaken), and samples analyzed by SDS-PAGE at various time points. The relative amount of protein rema ining at each time point was estimated by densitometry, and the decay was analyzed using a first-order kinetic model. Under the conditions used in the study, the five allergenic proteins evaluated or a digestion fragment of the allergenic protein had half-lives in SGF of over 16 minutes, while the four non-allergenic proteins or a digestion fragment had half-lives of under 14 minutes (and 3 of those 4 had half-lives of less than 0.5 minutes). Classification: Supplemental 461239-20

Digestion Efficiency ofAllergens and NonAllergens in Simulated

Gastric Fluid f The digestion of seven allergens and eight non-allergens was assessed in SGF using a first-order kinetic model. Half-lives for the proteins tested ranged from <30 seconds to >60 minutes. Allergens tended to be more stable in SGF than non-allergens, but a strong correlation between digestion rate and allergenicity was not observed for this set of proteins. The data fit well to a first-order decay model, except for early time points, and half-lives calculated using initial substrate concentrations that differed by 5-fold were fairly consistent. Classification: Acceptable 463886-01

d Reviewed in U.S. EPA (2003)

e Reviewed in U.S. EPA (2004a)

f Reviewed in U.S. EPA (2005a)Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

55 Study Title Summary MRID No.

PS149B1 14 kDa

Protein: Acute Oral Toxicity Study in CD-1

Mice g PS149B1 14-kDa protein (Cry34Ab1; 54% pure) was evaluated for acute oral toxicity. Five male CD-1 mice r eceived 5,000 mg of test material per kg body weight by oral gavage as a 20% mixture in a 0.5% aqueous methylcellulose vehicle. All animals survived the two-week study. No clinical signs were noted for any animals during the study. An initial weight loss was observed in 3 mice at test days 1 and 2, but they gained weight for the remainder of the study. The two other animals gained weight throughout the study. No treatment-related gross pathologic observations were observed during the study. Under the conditions of this study, the

acute oral LD 50 for the test substance in male CD-1 mice was greater than 5,000 mg/kg; since the test substance contained PS149B1 14-kDa protein (Cry34Ab1) at 54% purity, the acute LD 50 for the pure protein was greater than 2,700 mg/kg. Classification: Acceptable 452422-07

PS149B1 44 kDa

Protein: Acute Oral Toxicity Study in CD-1

Mice g PS149B1 44-kDa protein (Cry35Ab1; 37% pure) was evaluated for acute oral toxicity. Five male CD-1 mice r eceived 5,000 mg of test material per kg body weight by oral gavage as a 20% mixture in a 0.5% aqueous methylcellulose vehicle. All animals survived the two-week study. No clinical signs were noted for any animals during the study. An initial weight loss was observed in 2 mice at test days 1 and 2, but they gained weight for the remainder of the study, and one animal had fluctuating body weight. The other two animals gained weight throughout the study. No treatment-related gross pathologic observations were observed during the study. Under the conditions of this study, the acute oral LD 50 for the test substance in male CD-1 mice was greater than 5,000 mg/kg; since the test substance contained PS149B1 44-kDa protein (Cry35Ab1) at 37% purity,

the acute LD 50 for the pure protein was greater than 1,850 mg/kg. Classification: Acceptable 452422-08

Lack of Cry34Ab1/Cry35Ab1

Co-Association in

Solution h Size exclusion chromatography was used to investigate whether Cry34Ab1 and Cry35Ab1 associate with one another in solution under acidic conditions. Two studies were conducted, one at pH 3.6 and one at pH 2.0. In both studies, Cry34Ab1 and Cry35Ab1 eluted as separate peaks, indicating that the proteins do not as sociate with one another in solution

under these conditions. Classification: Acceptable 465568-01

g Reviewed in U.S. EPA (2005a)

h Reviewed in U.S. EPA (2005d)Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

56 Study Title Summary MRID No.

Evaluation of the Sequence Similarities of the Cry34Ab1, Cry35Ab1, and PAT

Proteins with the Public Protein Sequence

Datasets i The sequences of the Cry34Ab1, Cry35Ab1, and PAT proteins were compared with protein se quences in publicly available databases (GenPept dataset) to identify any potential si milarities with known toxins. The BlastP2.2.6 algorithm was used with a cutoff expectation (E) value of 1.0. The results of the Cry34Ab1 search id entified 10 proteins, 5 of which are Cry proteins from Bt that are closely related or identical to the Cry34Ab1 protein. The other proteins represent putative microbial collagenases and hypothetical proteins from genome-se quencing projects with a low degree of similarity to the Cry34Ab1 protei n. The Cry35Ab1 search identified 22 proteins, 7 of which are similar or identical Cry proteins from Bt . Other significant similarities were with proteins from a related species, Bacillus sphaericus , which has mosquitocidal activity. The PAT search identified 148 accessions. Only 18 of these represent actual GenPept accessions and are either phosphinothricin acetyltran sferases or other acetyltransferases. The remaining 130 proteins are unidentified and/or hypothetical proteins from genome-sequencing data. The Cry34Ab1, Cry35Ab1, and PAT proteins do not appear to have similarities with any proteins that would raise safety concerns. Classification: Acceptable 465847-01

2. Human Health Assessment of PAT (Expre ssed in Event DAS-59122-7 Corn, Herculex® XTRA Insect Protection Corn, 59122 x MON 810, and 1507 x 59122 x MON 810), Cry1F (Expressed in Herculex® XTRA Insect Protection Corn and 1507 x 59122 x MON 810), and Cry1Ab (Expressed in 59122 x MON 810 and 1507 x 59122 x MON 810)

Based on previously completed Agency human health assessments, permanent tolerance exemptions have been established for the PAT, Cry1F, and Cry1Ab proteins:

“ § 174.522 Phosphinothricin Acetyltransferase (PAT); exemption from the re quirement of a tolerance.

Residues of the Phosphinothricin Acetyl transferase (PAT) enzyme are exempt from the requirement of a tolerance wh en used as plant-incorporated protectant inert ingredients in all food commodities.”

**original under 40 CFR § 180.1151 ( 62 FR 17717 ; April 11, 1997)

**revised under 40 CFR § 174.522 ( 72 FR 20431 ; April 25, 2007)

i Reviewed in U.S. EPA (2005d)Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

57 “ § 174.520 Bacillus thuringiensis Cry1F protein in corn; exemption from the requirement of a tolerance.

Residues of Bacillus thuringiensis Cry1F protein in corn are exempt from the requirement of a tolerance when used as plant-incorporated protectants

in the food and feed commodities of co rn; corn, field; corn, sweet; and corn,

pop.

**original under 40 CFR § 180.1217 ( 66 FR 30321 ; June 6, 2001)

**revised under 40 CFR § 174.520 ( 72 FR 20431 ; April 25, 2007)

“ § 174.511 Bacillus thuringiensis Cry1Ab protein in all plants; exemption from the requirement of a tolerance.

Residues of Bacillus thuringiensis Cry1Ab protein in a ll plants are exempt from the requirement of a toleranc e when used as plant-incorporated protectants in all food commodities.”

**original under 40 CFR § 180.1173 ( 61 FR 40340 ; August 2, 1996)

**revised under 40 CFR § 174.511 ( 72 FR 20431 ; April 25, 2007)

The toxicological and allergenicity data supporting the establishment of these tolerance exemptions, as well as the associated regist rations of MON 810 (expressing Cr y1Ab protein) and Herculex® I (expressing Cry1F and PAT proteins ), can be found in the 2001 Bt Crops Reassessment, the August 2005 Cry1F BRAD, and/or the September 2010 Cry1Ab and Cry1F BRAD (U.S. EPA 2001b, 2005e, and 2010b).

The individual data generated for the Cry1Ab, Cr y1F, and PAT proteins (a nd Cry34Ab1 and Cry35Ab1) support their inclusion, as expressed in particular stacked and/or pyramided plant-incorporated protectants (i.e., Herculex® XTRA Insect Protection Corn, 59122 x MON 810, and 1507 x 59122 x MON 810), into the appropriate tolerance exemptions si nce the mode of action fo r the proteins does not suggest a synergistic activity in combination for mammalian species.

Human health assessment data, provided specifica lly in relation to the Herculex® Insect Protection Corn, 59122 x MON 810, and/or 1507 x 59122 x MON 810 registrations, are summarized in Table 2.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

58 Table 2. Human Health Assessment Data for PAT, Cry1F, and Cry1Ab.

Study Title Summary MRID No.

Herculex® XTRA Insect Protection Corn Data

Summary Information in Support of the Section 3 Registration

of Herculex® Xtra

Resistant Corn j Summary information is presented in support of the Section 3 registration of Herculex® Xtra Insect Resistant Corn, which contains Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins. The information focuses on the individual traits (from Herculex® I and Herculex® RW) that comprise Herculex® Xtra and potential synergies of the proteins with respect to

mammalian and environmental risk. Studie s of the individual traits, as well as the stacked traits, indicate expected efficacy to certain lepidopteran and coleopteran pests in field corn and no synergistic effect from the combined proteins. Studies indicate no concerns for safety of humans, non-target organisms, or threatened and endanger ed species. Data waiver requests and rationale are presented in this submission.

Classification: Acceptable 462971-01

Equimolar Ratio of Cry1F (tr) + Cry35Ab1 + Cry34Ab1: Acute Oral Toxicity Study in

CD-1 Mice k The acute oral toxicity of a mixture of microbially produced Cry1F (truncated), Cry34Ab1, and Cry35Ab1 proteins was assessed by administering an equimolar mixture of the proteins to five female mice and five male CD-1 mice. A dose of 525 mg pure Cry1F (truncated)/kg body weight (bw), 355 mg pure Cry35Ab1/kg bw, and 113 mg pure Cry34Ab1/kg bw was given as a suspension in 0.5% aqueous methylcellulose by two fractional gavage doses. All animals survived the two-week study and gained weight, no clinical signs were noted, and there were no gross pathologic changes observed at necropsy. The oral LD 50 was greater than 993 mg/kg for the equimolar mixture of the pure Cry proteins.

Classification: Acceptable 463843-01

59122 x MON 810 & 1507 x 59122 x MON 810 Data

Evaluation of the Amino Acid Sequence Similarity of the Cry1Ab Protein from

MON 810 to the NCBI Protein Sequence Datasets (Toxicity Study) l The 818 amino acid sequence of event MON 810 corn expressing Cry1Ab protein was sequentially searched an d compared for protein sequence homology to known toxins contained in the National Center for Biotechnology Information (NCBI) Protein Sequence datasets. The BLASTP search showed no significant amino acid sequence homologies, using the Cry1Ab protein sequence as a query, with any proteins known to be toxins. Therefore, the Cry1Ab protein does not share any amino acid sequence homology with any proteins known to be toxins.

Classification: Acceptable 477864-06

j Reviewed in U.S. EPA (2005f)

k Reviewed in U.S. EPA (2005f)

l Reviewed in U.S. EPA (2010a)Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

59 Study Title Summary MRID No.

Evaluation of the Amino Acid Sequence Similarity of the Cry1F

Protein from TC1507 to

the NCBI Protein Sequence Datasets (Toxicity Study) m The 605 amino acid sequence of even t TC1507 corn expressing Cry1F protein was sequentially searched for comparisons to known toxins contained in the NCBI Protein Sequ ence datasets. The BLASTP search showed no significant amino acid sequence homologies, using the Cry1F protein sequence as a query, with any proteins known to be toxins. Therefore, the Cry1F protein does not share any amino acid sequence homology with any proteins known to be toxins.

Classification: Acceptable 477864-05

Evaluation of the Amino Acid Sequence Similarity of the PAT

Protein to the NCBI Protein Sequence Datasets (Toxicity Study) m No significant amino acid sequence homologies were identified, using the PAT protein sequence as a query, with any proteins known to be toxins

contained within the NCBI Protein Sequence datasets. Therefore, the updated amino acid homology search verifies no changes in the results and conclusions of the previously submitted toxin homology assessment for the PAT protein (see MRID No. 465847-01).

Classification: Acceptable 477864-07

Evaluation of the Amino Acid Sequence

Similarities of the Cry34Ab1 and Cry35Ab1 Proteins to

the NCBI Protein Sequence Datasets (Toxicity Study) m No significant amino acid sequence homologies were identified, using the Cry34Ab1 and Cry35Ab1 protein sequences as a query, with any proteins known to be toxins contained within the NCBI Protein Sequence datasets. Therefore, the updated amino acid homology search verifies no changes in the results and conclusions of the previously submitted toxin homology assessment for the Cry34Ab1 and Cry35Ab1 proteins expressed in event DAS-59122-7 corn (see MRID No. 465847-01).

Classification: Acceptable 477864-08

Comparison of the Amino Acid Sequence Identity Between the Cry1F Protein and

Known Protein Allergens (Allergenicity Study) m No identity matches of ~35% over ~80 amino acid residues were observed for the Cry1F protein sequence agains t the protein sequences of known allergens. In addition, there were no 8 or greater contiguous identical amino acid matches observed with the Cry1F protein sequence. Therefore, the updated amino acid homology search verifies no changes in the results and conclusions of the previously submitted allergen homology assessment for the Cry1F protein expressed in event TC1507 corn (see MRID No. 449717-01).

Classification: Acceptable 477864-01

m

Reviewed in U.S. EPA (2010a)Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

60 Study Title Summary MRID No.

Comparison of the Amino Acid Sequence Identity Between the Cry34Ab1 and Cry35Ab1 Proteins and

Known Protein Allergens (Allergenicity Study) n No identity matches of ~35% over ~80 amino acid residues were observed for the Cry34Ab1 and Cry35Ab1 protein sequences against the protein sequences of known allergens. In ad dition, there were no 8 or greater contiguous identical amino acid matches observed with the Cry34Ab1 and Cry35Ab1 protein sequences. Therefore, the updated amino acid homology search verifies no changes in the resu lts and conclusions of the previously submitted allergen homology assessment for the Cry34Ab1 and Cry35Ab1 proteins expressed in event DAS-59122-7 corn (see MRID No. 452422- 05).

Classification: Acceptable 477864-02

Comparison of the Amino Acid Sequence Identity Between the

PAT Protein and

Known Protein Allergens (Allergenicity Study) n No identity matches of ~35% over ~80 amino acid residues were observed for PAT protein sequence against the protein sequences of known allergens. In addition, there were no 8 or greater contiguous identical amino acid matches observed with the PAT protein sequence. Therefore, the updated amino acid homology search verifies no changes in the results and conclusions of the previously submitted allergen homology assessment for the PAT protein (see MRID No. 449717-01).

Classification: Acceptable 477864-03

Comparison of the Amino Acid Sequence Identity Between the Cry1Ab Protein from

Event MON 810 and

Known Protein Allergens (Allergenicity Study) n No identity matches of ~35% over ~80 amino acid residues were observed for Cry1Ab protein sequence agains t the protein sequences of known allergens. In addition, there were no 8 or greater contiguous identical amino acid matches observed with the Cry1Ab protein sequence. Therefore, the updated amino acid homology search verifies no changes in the results and conclusions of the previously submitted allergen homology assessment for the Cry1Ab protein expressed in event MON 810 corn (see MRID No. 458789-02).

Classification: Acceptable 477864-04

3. References

MRID No. 449717-01. Meyer T. 1999. Comparison of Amino Acid Sequence Similarity of Cry1F and PAT Proteins to Known Allergen Protei ns: Final Report. Lab Pr oject Number: PHI99- 013. Unpublished study prepared by Trait and Technology Development, 24 pages.

n Reviewed in U.S. EPA (2010a)Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

61 MRID No. 452422-05. Stelman S. 2000. Comparis on of the Amino Acid Sequence of the Bacillus thuringiensis Strain PS149B1 13.6 kDa and 43.8 kDa Insect icidal Crystal Proteins to Known Protein Allergens. Lab Project Number : GH-C 5140. Unpublished study prepared by Dow AgroSciences LLC, 188 pages.

MRID No. 452422-07. Brooks K, DeWildt P. 2000. PS 149B1 14 kDa Protein: Acute Oral Toxicity Study in CD-1 Mice. Lab Project Number : 001130. Unpublished study prepared by Dow AgroSciences LLC, 39 pages.

MRID No. 452422-08. Brooks K, DeWildt P. 2000. PS 149B1 44 kDa Protein: Acute Oral Toxicity Study in CD-1 Mice. Lab Project Number : 001129. Unpublished study prepared by Dow AgroSciences LLC, 39 pages.

MRID No. 452422-09. Brooks K, DeWildt P. 2000. PS149B1 14 kDa and 44 kDa Proteins: Acute Oral Toxicity Study in CD-1 Mice. La b Project Number: 001128. Unpublished study prepared by Dow Chemical Company, 45 pages.

MRID No. 452422-12. Korjagin V. 2000. In vitro Digestibility of PS149B1 Proteins. Lab Project Number: 000302. Unpublished study prepared by Dow AgroSciences LLC, 37 pages.

MRID No. 455845-02. Korjagin V, Herman R, Hunst P. 2002. In vitro Simulated Gastric Fluid Digestibility of Microbi ally Derived Cry34Ab1 Protei n. Lab Project Number: 010111. Unpublished study prepared by Dow AgroSciences LLC, 95 pages.

MRID No. 457904-08. Herman R, Schafer B, Korjagin V. 2002. SDS-Page Sensitivity Analysis for Cry35Ab1 in Support of the Simulated Gastric Fluid Digestion Study. La b Project Number: GH-C 5513. Unpublished study prepared by Dow AgroSciences LLC, 11 pages.

MRID No. 458789-02. Heileman R, Silvanovich A, Rice E. 2002. Bioinformatics Evaluation of the Cry1Ab Protein Produced in Corn Event MON 810 Utilizing an Allergen Database. Lab Project Number: 17452: 01-01- 39-44. Unpublished study prep ared by The Monsanto Company, 47 pages.

MRID No. 461239-01. Ojala B. 2003. Independent Laboratory Validati on Pioneer Hi-Bred International, Incorporated ELISA Method for the Quantification of Cr y34Ab1 Protein from Transgenic Plants. Project Number: BS 03/02. Unpublished study pr epared by Biolab Solutions, 46 pages.

MRID No. 461239-02. Ojala B. 2003. Independent La boratory Validation of Dow AgroSciences Method GRM 03.13, “Determination of Cry35Ab1 In secticidal Crystal Protein in Maize Tissue by Enzyme-Linked Immunosorbent A ssay.” Project Number: BS03/01. Unpublished study prepared by Biolab Solutions, 45 pages.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

62 MRID No. 461239-04. Essner R. 2003. Agronomic Characteristics, Quantitative ELISA and Nutrient Composition Analysis of Hybrid Maize Lines Containing the cry 34Ab1, cry 35Ab1

and pat Genes: Chile Locations. Project Number: PHI/2002/050. Unpublished study prepared by Pioneer Hi-Bred Internati onal, Incorporated and EPL Bi o-Analytical Services, 1,971

pages.

MRID No. 461239-06. Schafer B. 2003. Characteriz ation of Cry34Ab1 and Cry35Ab1 Proteins Derived from Transgenic Maize Ev ent E4497.59.1.22 (DAS-59122-7). Project Number: 030033. Unpublished study prepared by Dow AgroSciences LLC, 50 pages.

MRID No. 461239-20. Herman R. 2003. Digestion of A llergenic and Non-Allergenic Proteins in Simulated Gastric Fluid. Project Number: GH/C/5700. Unpublished study prepared by Dow AgroSciences LLC, 73 pages.

MRID No. 462971-01. Ernest A, Hunst P. 2004. Summar y Information in Support of the Section 3 Registration of Herculex® Xtra Insect Resistant Corn. Proj ect Number: GH/C/5736. Unpublished study prepared by Dow AgroSciences LLC, 55 pages.

MRID No. 463843-01. Wilson D, Brooks K, Dowdney R. 2004. Equimolar Rati on of Cry1F (tr) + Cry35Ab1 + Cry34Ab1: Acute Oral Toxicity St udy in CD-1 Mice. Project Number: 041011. Unpublished study prepared by The Dow Chemical Company, 31 pages.

MRID No. 463886-01. Herman R, Woolhiser M, Ladics G. 2004. Digestion Efficiency of Allergens and Non-Allergens in Simulated Gastric Fluid: Bacillus thuringiensis Cry34/35Ab1 Construct PHP17662. Project Number: GH/C/ 5761. Unpublished study prepared by Dow AgroSciences LLC, Pioneer Hi-Bred Corn Company Production Department, and Dow Chemical Company, 302 pages.

MRID No. 465568-01. Herman R, Evans S, Zhuang M. 2005. Lack of Cry34Ab1/Cry35Ab1 Co- Association in Solution. Project Number: GH/C/5805. Unpublished study prepared by Dow AgroSciences LLC, 36 pages.

MRID No. 465847-01. Cressman R. 2003. Evaluation of the Sequence Similarities of the Cry34Ab1, Cry35Ab1, and PAT Proteins to the Public Pr otein Sequence Datasets. Project Number: PHI/2003/046. Unpublished study prepared by E. I. du Pont de Nemours and Company, Incorporated, 211 pages.

MRID No. 477864-01. Krauss A, Cressman R. 20 09. Comparison of the Amino Acid Sequence Identity Between the Cry1F Protein and K nown Protein Allergens. Project Number: PHI/2008/236. Unpublished study prepared by E. I. du Pont de Nemours and Company, 23

pages.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

63 MRID No. 477864-02. Krauss A, Cressman R. 20 09. Comparison of the Amino Acid Sequence Identity Between the Cry34Ab1 and Cry35Ab1 Pr oteins and Known Protein Allergens. Project Number: PHI/2008/237. Unpublished stu dy prepared by E.I. du Pont de Nemours and Company, 35 pages.

MRID No. 477864-03. Krauss A, Cressman R. 20 09. Comparison of the Amino Acid Sequence Identity Between the (Inert Ingredient) Pr otein and Known Protein Allergens. Project Number: PHI/2008/238. Unpublishe d study prepared by E.I. du Pont de Nemours and Company, 22 pages.

MRID No. 477864-04. Krauss A, Cressman R. 20 09. Comparison of the Amino Acid Sequence Identity Between the Cry1Ab Protein from Event MON 810 and Known Protein Allergens. Project Number: PHI/2008/239. Unpublished stu dy prepared by E.I. du Pont de Nemours and Company, 24 pages.

MRID No. 477864-05. Cressman R, Krauss A. 2009. Evaluation of the Amino Acid Sequence Similarity of the Cry1F Protein to the NCBI Protein Sequence Datasets. Project Number: PHI/2008/240. Unpublished study prepared by E. I. du Pont de Nemours and Company, 291

pages.

MRID No. 477864-06. Krauss A, Cressman R. 20 09. Evaluation of the Amino Acid Sequence Similarity of the Cry1Ab Protein from Event MON 810 to the NCBI Protein Sequence Datasets. Project Number: PHI/2008/241. Unpublis hed study prepared by E.I. du Pont de Nemours and Company, 354 pages.

MRID No. 477864-07. Krauss A, Cressman R. 20 09. Evaluation of the Amino Acid Sequence Similarity of the (Inert Ingredient) Protein to the NCBI Protein Sequence Datasets. Project Number: PHI/2008/242. Unpublishe d study prepared by E.I. du Pont de Nemours and Company, 278 pages.

MRID No. 477864-08. Krauss A, Cressman R. 20 09. Evaluation of the Amino Acid Sequence Similarities of the Cry34Ab1 and Cry35Ab1 Pr oteins to the NCBI Protein Sequence Datasets. Project Number: PHI/2008/243. Unpublis hed study prepared by E.I. du Pont de Nemours and Company, 43 pages.

Sjoblad RD, McClintock JT, Engl er R. 1992. Toxicological consider ations for protein components of biological pesticide products. Regulatory Toxicology and Pharmacology 15(1):3

– 9.

U.S. EPA. 2001a. Review of Product Characterizati on, Expression Analysis and Acute Oral Toxicity Studies for PS149B1 Binary Insect Contro l Proteins as Expressed in Maize, a Bacillus thuringiensis -Based Plant-Pesticide, for an Experi mental Use Permit. Memorandum from C.A. Wozniak, Ph.D. and J.L. Kough, P h.D. to M. Mendelsohn dated June 7, 2001.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

64 U.S. EPA. 2001b. Biopesticides Registration Action Document – Bacillus thuringiensis Plant- Incorporated Protectants. Available from: http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm

.

U.S. EPA. 2002. Review of in vitro Digestibility for PS149B1 Binary Insect Control Proteins, Cry34 and Cry35, as Expressed in Maize, a Bacillus thuringiensis -Based Plant-Incorporated Protectant, for Approval of a Temporary F ood Tolerance. Memorandum from C.A. Wozniak, Ph.D. and J.L. Kough, Ph.D. to M. Mendelsohn dated November 5, 2002.

U.S. EPA. 2003. Review of SDS-PAGE Sensitiv ity and Lateral Flow Detection Method for PS149B1 Binary Insect Control Prot eins as Expressed in Maize, a Bacillus thuringiensis

- Based Plant-Incorporated Pr otectant, for an Experimental Use Permit. Memorandum from C.A. Wozniak, Ph.D. and J.L. Kough, P h.D. to M. Mendelsohn dated April 3, 2003.

U.S. EPA. 2004a. Review of Digestion of Allergen ic and Non-Allergenic Pr oteins in Simulated Gastric Fluid Analyzed by a Kinetic A pproach for Support of Registration of Bt Cry34/35Ab1 Construct PHP17762 Corn. Memora ndum from R.L. Edelstein, Ph.D. and J.L. Kough, Ph.D. to M. Mendelsohn dated August 17, 2004.

U.S. EPA. 2004b. Review of Product Characterizati on and Expression Analysis for Registration of

B

. t . Cry34/35AB1 Construct PHP17662 Corn. Memora ndum from R.L. Edel stein, Ph.D. and J.L. Kough Ph.D. to M. Mendelsohn dated December 6, 2004.

U.S. EPA. 2005a. Review of Human Health Data for Registration of B

. t . Cry 34/35Ab1 Construct

PHP17662 Corn and Risk Assessment. Memorandu m from R.L. Edelstein, Ph.D. and J.L. Kough, Ph.D. to M. Mendelsohn dated February 4, 2005.

U.S. EPA. 2005b. SAP Report No. 2005-02. A Set of Sc ientific Issues Bei ng Considered by the Environmental Protection Agency Regarding: Sc ientific Issues Associated with the Human Health Assessment of the Cry34Ab1 Prot ein. Dated April 5, 2005. Available from: http://www.epa.gov/scipoly/sap/meeti ngs/2005/march/finalm ar2005sapmtg.pdf

.

U.S. EPA. 2005c. Evaluation of FIFRA SAP Meeting Minutes Dated April 5, 2005 from the March 1–2, 2005 Meeting on Scient ific Issues Associated with the Human Health Assessment of the Cry34Ab1 Protein. Memorandum from R.L. Edelstein, Ph.D. and J.L. Kough, Ph.D. to M. Mendelsohn dated May 12, 2005.

U.S. EPA. 2005d. Addendum to Review of Human Health Data for Registration of B

. t . Cry

34/35Ab1 Construct PHP17662 Corn and Risk Assessment. Memorandum from R.L. Edelstein, Ph.D. and J.L. Kough, Ph.D. to M. Mendelsohn dated July 7, 2005.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

65 U.S. EPA. 2005e. Biopesticides Registration Action Document – Bacillus thuringiensis Cry1F Corn. Available from: http://www.epa.gov/oppbppd1/biopesticid es/ingredients/ tech_docs/brad_006481.pdf

.

U.S. EPA. 2005f. Review of Product Characterizati on and Human Health Data for Registration of

HerculexTM Xtra Insect Resist ant Corn. Memorandum from R.L. Edelstein, Ph.D. and J.L. Kough, Ph.D. to M. Mendelsohn dated September 22, 2005.

U.S. EPA. 2010a. Review of Human Health and Product Characterization Data for Registration of Bacillus thuringiensis ( Bt ) Cry1F, Cry34Ab1, Cry35Ab1, and Cr y1Ab Proteins and the Genetic Material Necessary for Their Producti on in the Combination Plant-Incorporated Protectant (PIP) Products: 1507 x MON 810 [EPA Reg. No. 29964-T], 59122 x MON 810 [EPA Reg. No. 29964-O], and 1507 x 59122 x MON 810 [EPA Reg. No. 29964-I]. Memorandum from A. Waggoner and J. L. Kough, Ph.D. to A. Sibold dated February 19, 2010.

U.S. EPA. 2010b. Biopesticides Registration Action Document – Bacillus thuringiensis Cry1Ab and Cry1F Corn (Updated September 2010). Available from: http://www.regulations.gov (see “Supporting & Related Material s” within Docket Number EPA-HQ-OPP-2010-0607).Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

66 C. Environmental Assessment

1. Tiered Hazard and Risk Assessment Process

To minimize data requirements and avoid unnecessary tests, risk assessm ents are structured such that

risk is determined first from estimates of hazar d 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 studi es suggest potentially unacceptable risk, additional studies are designed to assess risk under more rea listic 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 cohesi ve and coherent manner and eliminating unnecessary lines of investigation. Lower tie r, high-dose screening studies also allow tighter control over experimental variables and exposure conditions, resulting in a greater ability to produce statistically reliable results at relatively low cost. a

Tiered tests are designed to firs t represent unrealistic worst-case s cenarios and ONLY progress to realworld field scenarios if the earlier tiered tests fail to indicate adequate certa inty 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 ex treme exposure suggest that population effects are likely to be negligible given rea listic field exposur e scenarios.

The Environmental Protection Agency (EPA) uses a tiered (Tiers I

– IV) testing system to assess the toxicity of a plant-incorp orated protectant (PIP) to representa tive non-target organisms that could be exposed to the toxin in the field e nvironment. Tier I high-dose studies reflect a screening approach to testing designed to maximize any toxic effects of th e test substance on the te st (non-target) organism. The screening tests evaluate single species in a la boratory setting with morta lity as the endpoint. Tiers

II

– IV generally encompass definitive hazard level determinations, longer term greenhouse or field testing, and are implemented when unacceptable eff ects are seen at the Tier I screening level.

Testing methods, which utilize the tiered approach, were last published by EPA as Harmonized Office of Prevention, Pesticides, and Toxi c Substances (OPPTS) Testing Gu idelines (now Harmonized Office

a Non-target invertebrate hazard tests of ten are conducted at exposure concentratio ns 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 tes ting. Lower levels of mortality under these conditions of extre me exposure suggest that population effects ar e 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 appr oach, 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 eff ects of less than 50% indicate that further experiments are not needed to evaluate risk.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

67 of Chemical Safety and Pollution Prevention (OCSPP) Testing Guidelines), Series 850 and 885 (EPA 712-C-96-280, February 1996). b These guidelines apply to microbes a nd microbial toxins when used as pesticides, including those that are naturally occurri ng and those that are strain improved either by natural selection or by deliberate genetic manipulation. Therefore, plant-incorpor ated protectants (PIPs) containing microbial toxins are also covered by thes e testing guidelines.

The Tier I screening maximum hazard dose (MHD) a pproach 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 th e environment, or the Estimated Environmental Concentration (EEC). c 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 assess ment, and such tests can be well standardized. The

Guidelines call for initial screeni ng testing of a single group or severa l groups of test animals at the

maximum hazard dose level. The Guidelines call for testing of one treatment group of at least thirty animals or three groups of ten test animals at the screening test c oncentration. The Guidelines further

state that the duration of all Tier I tests should be approximately 30 days. So me test species, notably non-target insects, may be difficult to culture an d the suggested test duration has been adjusted accordingly. Control and treated insects should be obser ved for at least 30 days, or in cases where an insect species cannot be cultured for 30 days, unt il negative control morta lity rises above 20%.

Failing the Tier I (10x EEC) screeni ng at the MHD does not necessarily indicate the presence of an unacceptable risk in the field, but it tr iggers the need for additional testing. d A less than 50% mortality

effect at the MHD is taken to indicate minimal risk. Greater than 50% mortality, however, 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 haza rds are detected in Tier I testing (i.e., mortality is greater than 50%), additional information at lower test doses is required, which can serve to confirm whether any effect might still be detected at more realistic field (1x EEC) concentrations and routes of

exposure. e

b General OCSPP Harmonized Testin g Guidelines available from: http://www.epa.gov/ocspp/pubs/frs/home/guidelin.htm

. Series 850 Testing Guidelines available from: http://www.epa.gov/ocspp/pubs/frs/public ations/OPPTS_Harmonized/850_Ecologica l_Effects_Test_Guidelines/Drafts/index.

html

. Series 885 Testing Guidelines available from: http://www.epa.gov/ocspp/pubs/frs/public ations/Test_Guidelines/series885.htm

. c 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 orga nism feeding habits or other factors. Hi gh-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.

d Note that the 10x EEC MHD testing approach is not equivalent to what is commonly known as “testing at a 10x safety factor,” where any adverse effect is consid ered significant. Tier I screen testing is not “safety factor te sting.” In a “10x sa fety factor” test, any adverse effect noted is a “level of concern,” whereas in the EPA environmental risk as sessment scenario any adverse effect is viewed as a concern only at 1x the field exposure.

e The 1x EEC test dose is based on plant tissue content and is considered the highest do se in a worst-case scenario (sometimes referred to as the Highest Estimated Environmental Concentration or HEEC). This 1x EEC is still much greaterBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

68 When screening tests indicate a need for additio nal data, the OCSPP Harmoni zed Guidelines call for testing at incrementally lower doses in order to establish a definitive LD 50 (i.e., dose that will kill 50% of the test organisms with in a designated period) and to quantify the hazard. In the definitive testing, the number of doses and test organisms evalua ted must be sufficient to determine an LD 50 value and, when necessary, the Lowest Observed Adverse Effect C oncentration (LOAEC), No Observed Adverse Effect Level (NOAEL), or reproductive and beha vioral 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 co nclusion is made. These tests offe r 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 leve ls of replication, test species numbers, and/or repetition are needed to enhance statistical power in these circumstances.

Data that shows less than 50% mortality at the maximum hazard dosage level (i.e., LC 50 , ED 50 , or LD 50 >10x EEC) is sufficient to evaluate adverse effects, making lower field exposure dose definitive testing unnecessary. It is also notable that the recomme nded >10x EEC maximum hazard dose level is a highly conservative factor. The published EPA Level of Con cern (LOC) is 50% mortal ity at 5x EEC (U.S. EPA 1998). f

Validation : The tiered hazard assessment approach was de veloped for EPA by the American Institute of Biological Sciences (AIBS) and confirmed in 1996 as an acceptable method of environmental hazard assessment by a Federal Insecticide, Fungicide, and Rodenticide Act (F IFRA) 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 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 genetically modified (GM) cr op fields should be conducted. Testing of Bacillus thuringiensis ( Bt ) Cry proteins on species not closely related to th e target insect pest was not recommended, although it is still performed to fulfill the published EPA non-t arget 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 indi cator organisms should be selected on the basis of potential for field exposure to the subj ect protein (U.S. EPA 2000, 2001a, 2002, and 2004b).

Chronic studies : Since delayed adverse effects and/or accu mulation of toxins through the food chain are not expected to result from exposur e to proteins, protein toxins are not routinely tested for chronic effects on non-target organisms. Bu t, the 30-day test duration requirement does amount to subchronic

than any amount which any give n non-target organism may be ingesting in th e field because most no n-target organisms do not ingest plant tissue.

f The established peer and EPA Science Boar d reviewed guidance on screening test leve ls of concern is 50% mortality at 5x environmental concentration for terrestrial and 10x for aquatic species. 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.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

69 testing when performed at field exposure test doses . Proteins do not bioaccu mulate. The biological nature of proteins makes them readily susceptible to metabolic, microbial, and abiotic degradation once they are either ingested or excret ed into the environment. Although there are report s that some proteins (Cry proteins) bind to soil particles, it has also been shown that thes e proteins are degraded rapidly by soil microbial flora upon elution from soil particles.

Conclusion : The tiered approach to test gui delines ensures, to the greatest extent possible, that the Agency requires the minimum amount of data needed to make scientifically sound regulatory decisions.

EPA believes that maximum hazard dos e Tier I screening testing presen ts a reasonable approach for evaluating hazards related to the use of biological pesticides and for id entifying negative results with a high degree of confidence. The Agency expects that Tier I testing for short-term hazard assessment will be sufficient for most studies submitted in support of PIP registrations. If long-range adverse effects must be ascertained, however, then higher tier, longer term field testing will be required. As noted above, the October 2000 SAP and the National Academ y of Sciences (NAS 2000) recommended testing non-target organisms directly in the field. This ap proach, 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 and due to the lack of baseline data on the potential for long-term environmental effect s from the cultivation of PIP- producing plants, the Agency has required long-term field studies on invertebrate populations/communities and Cry protein accumulation

in soils as conditions of past PIP registrations.

Since the commercialization of Bt crops, the number of field studies pu blished 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 invert ebrate community structure in Bt crop fields has since been adequately addressed. Specifically, a meta-analysis g 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 shortand long-term field studies on the effects of invertebrate populations in Bt corn and cotton fields

indicated that no unreasonable adverse effects are taking plac e 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 une xpected 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 reducti ons in the abundance of the pests as prey.

Overall, the Agency is in agreemen t with the conclusions of these st udies and, collectively, these results provide extensive da ta 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

g This research was funded by EPA grant CR-832147-01. The Bt crop non-target effects database can be found on the National Center for Ecological Analys is and Synthesis (NCEAS) Web Site: http://delphi.nceas.ucsb.edu/btcrops/

.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

70 testing of the specialist predator s and parasitoids of target pest s may eventually be considered

unnecessary.

2. Event DAS-59122-7 Corn (Organization for Economic Cooperation and Development (OECD) Unique Identifier: DAS-59 122-7) Expressing Cry34Ab1 and Cry35Ab1

a. Data Cited/Submitted for Initial Registra tions of Event DAS-59122-7 Corn (Prior to August 2005)

i. Background

In 2003, Mycogen Seeds c/o Dow AgroSciences LLC a nd Pioneer Hi-Bred International, Incorporated requested registrations for Bacillus thuringiensis Cry34/35Ab1 binary protein and the genetic material necessary for its production in all corn lines and vari eties. This protein cont rols the corn rootworm (CRW, Diabrotica spp.), a primary pest of corn in the Unite d States. Corn rootworm larvae feed on corn roots, resulting in lodging and a reduction in a plant’ s ability to absorb water a nd nutrients from soil. In areas where the CRW is a pest (e.g., Corn Belt), significant financial loss es are realized from decreased corn yields and increased expenditures on chemi cal pest control agents, including organophosphate, carbamate, and pyrethroid insecticides.

EPA conducted an environmental hazard assessment of the Cry34/35Ab1-producing corn lines. The general topics covered include gene flow to related wild plants, de velopment of weediness, effects on wildlife, and fate of Cry34/35Ab1 protein in the environment. The assessment is based on data submitted to the Agency during the development of the corn lines; additional data submitted for registration; FIFRA SAP recommendations; consultations with scientific expert s; and previous public comments on plant-incorporated protectant regulation.

ii. Non-Target Wildlife Hazard Assessment

Two separate SAP reports (October 2000 and August 2002) recommended that non-target testing should focus on species exposed to the crop being regist ered. Following SAP recommendations, the Agency determined that non-target organisms with the greate st exposure potential to prot ein in transgenic corn fields are beneficial insects, whic h feed on corn pollen and nectar, and soil invertebrates, particularly coleopteran species. Therefore, maximum hazard dose t oxicity testing on represen tative beneficials from several taxa was performed. The toxicity of the Cry34/35Ab1 protein has been evaluated on several species of invertebrates, includ ing larval honey bees, a parasitic hymenoptera, green lacewings, lady beetles, collembola, and earthworms. Reproductive and developmental observations were also made on collembola, honey bee, and lady beetle larva.

The non-target organisms tested are chosen as representative indicato rs of the major groups of wildlife and on the potential for field exposure as deduced from data on Cry34/35Ab1 protein expression in the plant. Although Bt Cry proteins are very specific in their ac tivity to only certain insect species, for Cry34/35Ab1 protein in corn, the Agency has examined the toxicity to birds, fish, honey bees, andBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

71 certain other beneficial insects even though the October 2000 SAP recommende d against testing of nontarget species not related to those susceptible to the specific activity of Bt Cry proteins. In order to comply with the Agency’s published data requirement s for registration of microbial toxins (40 CFR § 158.2150), data submissions or waiver justifications were submitted to address these requirements. Collembola and earthworm studies were also conducted and voluntarily submitted to the Agency by the applicants to ascertain effects of Cry34/35Ab1 on beneficial decom poser species because prolonged exposure to Cry34/35Ab1 protein in soil was a possibility. Honey bee effects on brood were also required as exposure of honey bee larvae to th e Cry34/35Ab1 protein in pollen is expected.

The form of the test substa nces used in the studies for this assessment are plant material such as leaves, roots, pollen, or purified, bacterially produced Cr y34/35Ab1 protein incorporated into the test species diet. The October 2000 SAP provided guidance to the Ag ency that, while actual plant material is the preferred test material, bacterially de rived protein is also a valid test substance, particularly in testing where the test animals do not consume corn plan t tissue and where large amounts of Cry protein are needed for maximum hazard dose testing. Consistent with the OCSPP Harmonized Testing Guidelines, the adult insect studies were generally of 30 days duration or until the negative control mortality reached

20%. Larval studies were carried out th rough pupation and adult emergence.

The results for each study on environmental effe cts for Cry34/35Ab1 are summarized in Table 1. Additionally, the results are presente d in a more descriptive format in subsequent sections of this Environmental Assessment chapter. Complete reviews of each study can be found in the individual Data Evaluation Reports.

Table 1. Environmental Effects Data for Ev ent DAS-59122-7 Corn (Reviewed in U.S. EPA (2005c) Unless Otherwise Noted).

Guideline

Number Study Results MRID

Number

885.4050 Avian Dietary Testing There were no treatment-related deaths or clinical signs observed in broilers fed a diet containing 59% (starter ration) or 63% (grower/finisher ration) Cry34/35 Bt corn for 42 days. Corn containing the test protein supported rapid growth of broiler chickens and was similar in performance to non-transgenic varieties. No

adverse effects on avian wildlife are expected from consumption of Cry34/35Ab1 corn.

Classification: Acceptable 461239-11Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

72 Guideline

Number Study Results MRID

Number

885.4150 Wild Mammal Testing Mammalian wildlife exposure to Cry34/35Ab1 protein is considered likely; however, the Cry34/35Ab1 toxicity data, as described in the Human Health Assessment (see section II(B)(1) of this Biopesticides Registration Action Document (BRAD) ), indicate that there is no significant toxicity to rodents from testing at the maximum hazard dose. Therefore, no hazard to ma mmalian wildlife is anticipated, and data on wild mammal testing are not required. N/A

850.1075 Freshwater Fish Testing h There were no treatment-related deaths or clinical signs observed in rainbow trout fed 100 milligrams (mg) PS149B1 insecticidal crystal (IC) binary protein/kilogram (kg) diet for 8 days. Based on these observations, the 8-day LD 50 for rainbow trout was > 100 mg PS149B1 IC protein. No freshwater fish hazard is expected from commercial cultivation of Cry34/35Ab1 corn.

Classification: Supplemental 457904-03

850.1010 Aquatic

Invertebrate

Acute Toxicity Test

( Daphnia

magna ) h A static-renewal, 48-hour limit test was performed on Daphnia

magna . The test material was purified PS149B1 binary protein at a target concentration of 100 mg protein/Liter (L) water. No adverse

effects were noted for the acute lim it test. No hazards to daphnia are expected from incidental exposure to Cry34/35Ab1-containing corn pollen.

Classification: Supplemental

*Note for 2010 : There is an update to this summary. See section II(C)(2)(b) (“Terms and Conditions of the Event DAS-59122-7 Corn Registrations (August 2005 – September 2010)”) of this BRAD. 457904-04

885.4280 Estuarine and

Marine

Animal Testing The estuarine and marine animal st udies are not required for this product because Cry34/35Ab1 is not intended for direct application into the estuarine or marine environment and there is very low to no potential for exposure to Cry34/35Ab1 protein from field corn. N/A

885.4300 Non-Target

Plant Studies The active ingredient is an insect toxin ( Bt

endotoxin) that is nontoxic to aquatic and terrestrial plants. Consequently, non-target plant studies have been waived for this product. N/A

885.4380 Honey Bee Larva Testing i The median lethal concentration (LC 50 ) for honey bee larvae and adult bee emergence was found to be >5.6 micrograms (μg) Cry34/35Ab1 protein (100x the concentration in pollen). Also, no behavioral or morphologic abnormalities were seen in bees exposed to 2 mg/larva Cry34/35Ab1 corn pollen. Therefore, no hazard to honey bee larvae and adult bee emergence is anticipated.

Classification: Acceptable 453407-01

h Reviewed in U.S. EPA (2005a)

i Reviewed in U.S. EPA (2004a)Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

73 Guideline

Number Study Results MRID

Number

885.4340 Parasitic Hymenoptera Larva Testing The LC 50 for parasitic Hymenoptera was determined to be >280 parts per million (ppm) Cry34/35Ab1 prot ein. No hazard to parasitic Hymenoptera is expected. Testing of a species more common to corn

fields is recommended.

Classification: Acceptable 457904-05

885.4340 Dietary Toxicity to

Green Lacewing

Larvae The LC 50 for green lacewing larvae was determined to be >280 ppm Cry34/35Ab1 protein (10x field exposure). No hazard to green lacewing is expected. It is questionable whether the test species ingests the test material in feeding trails. Consequently, another species (e.g., minute pirate bug or predatory carabid) with greater likelihood of exposure should be tested.

Classification: Acceptable

*Note for 2010 : There is an update to this summary. See section II(C)(2)(b) (“Terms and Conditions of the Event DAS-59122-7 Corn Registrations (August 2005 – September 2010)”) of this BRAD. 457904-07

885.4340 Lady Beetle

Larval Feeding Study ( Coleomegilla

maculata ) Significant growth inhibition (80% growth reduction in 7-day study)

was observed in Coleomegilla maculata larvae feeding on Cry34Ab1/Cry35Ab1 diet, compared to larvae feeding on control diets. However, since toxic effect s were not seen at the field dose

level when Bt corn pollen- or Bt corn plant-fed aphids were used as diet, there is reasonable certainty that Cry34/35Ab1 corn will not adversely affect C

. maculata.

Classification: Acceptable (MRID Nos. 452422-10 and 452422-11) Supplemental (MRID No. 461239-12) because the study was terminated prior to pupation. 452422-10 j

452422-11 j

461239-12

885.4340 Collembola

Chronic Dietary Toxicity Study k The LC 50 for collembola was found to be >12.7 mg PS149B1 active ingredient/kg (10x field exposure). No adverse survival or reproductive effects were noted. No hazard to beneficial soilinhabiting decomposers is expected.

Classification: Acceptable 457904-06

850.6200 Earthworm Toxicity

Study l The LC 50 for earthworm was found to be >76 mg Cry34/35Ab1 protein/kg dry soil (20x field exposure). No hazard to earthworms is expected.

Classification: Supplemental 453602-01

j Reviewed in U.S. EPA (2001b)

k Reviewed in U.S. EPA (2005b)

l Reviewed in U.S. EPA (2004a)Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

74 Guideline

Number Study Results MRID

Number

N/A Insecticidal Activity Spectrum

Study m The relative sensitivity of the test insects in the feeding bioassays suggests that larvae of coleoptera n species are far more sensitive to the 14-kiloDalton (kDa) and 44-kDa proteins of PS149B1 than the lepidopteran or homopteran larval insects evaluated. Adult western corn rootworm (WCRW) were not sensitive to the delta-endotoxins, however. Rootworms (northern corn rootworm (NCRW), WCRW, and southern corn rootworm (SCRW)) were the most susceptible to the PS149B1 proteins. European corn borer (ECB) and corn earworm (CEW) demonstrated some inhibition of growth at higher

concentrations of the test substance than the rootworm larvae. Black cutworm (BCW) was the least sensitiv e of the lepidopteran species tested. No activity was seen agains t the corn leaf aphid (CLA), a homopteran. Relative susceptibilities of the insect species to PS149B1, based upon the more sensitive measure of 50% growth inhibition (GI 50 ), were as follows: (most susceptible) WCRW, NCRW, SCRW > ECB, CEW >> WCRW adult, CLA, BCW (least susceptible).

Classification: Acceptable 452422-04

N/A Non-Target Organism

Field Scale

Risk

Assessment Field monitoring results from two corn-growing seasons confirmed a lack of adverse effects on non-target invertebrates when exposed to Cry34/35Ab1 protein. These studies are supplemental to Tier I maximum hazard dose testing and are of inadequate statistical power for long-term effects determination.

Classification: Supplemental

*Note for 2010 : There is an update to this summary. See section II(C)(2)(b) (“Terms and Conditions of the Event DAS-59122-7 Corn Registrations (August 2005 – September 2010)”) of this BRAD. 461239-14

885.5200 Soil Fate The GI 50 value for SCRW fed a representative soil containingCry34/35Ab1 protein was 3.2 days. This finding suggests that soilincorporated binary insecticidal protein degrades over time.

Classification: Supplemental

*Note for 2010 : There is an update to this summary. See section II(C)(2)(b) (“Terms and Conditions of the Event DAS-59122-7 Corn Registrations (August 2005 – September 2010)”) of this BRAD. 452422-14

m

Reviewed in U.S. EPA (2001c)Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

75 Guideline

Number Study Results MRID

Number

N/A Endangered Species Impact

Assessment Pioneer Hi-Bred International, In corporated conducted a hazard

assessment, exposure assessment, and risk characterization to demonstrate that Cry34/35Ab1 does not pose risk to endangered coleoptera. The Agency’s independ ent assessment is in agreement

with these conclusions.

Classification: Acceptable 461239-17

N/A Non-Target

Invertebrate Ecological

Risk

Assessment Submitted data indicate that Cry34/35Ab1 corn will not pose

unreasonable adverse effects to corn field flora and fauna and that minimal short-term accumulation of Cry34/35Ab1 protein in agricultural soil is expected. Further, no adverse effect to endangered and threatened species listed by the U.S. Fish and Wildlife Service is expected from the proposed Cry34/35Ab1 CRW resistant corn registration.

Classification: Acceptable 461239-13

I. Non-Target Wildlife Study Summaries

a. Mammalian Wildlife

Mammalian wildlife exposure to Cry34/35Ab1 protei n is considered likely; however, the mammalian toxicology information gathered to date on Bt Cry proteins does not show a hazard to wild or domesticated mammals. The Cry34/35A b1 toxicity data, as described in the Human Health Assessment (see section II(B)(1) of this BRAD), indicate th at there is no significant toxi city to rodents from acute oral testing at the maximum hazard dose. Therefore, no hazard to mammalian wildlife is anticipated, and

data on wild mammal tes ting is not required.

b. Avian Species

Published data and studies on file at EPA show that consumption of Bt corn has no measurable deleterious effects on avian species. Nonetheless, the following broiler study was submitted to the Agency in support of the Cr y34/35Ab1 corn registrations.

i. Broiler Study (Master Record Identification Number (MRID No.) 461239-11)

Day-old commercial broiler chickens (Cobb x Cobb) we re fed diets containing ei ther transgenic corn line event TC15344 (Cry34Ab1 or Cry35Ab1), a non-transg enic isoline corn (near isoline of event TC15344), or commercial corn from one of two sources (A or B) for 42 days. A starter ration containing approximately 59% wet weight corn was fed ad libitum during days 1–20 (Phase I), and a grower/finisher ration containing approxima tely 63% wet weight corn was provided ad libitum during days 21–42 (Phase II) of the test period. No biological ly significant differences in live weight, carcassBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

76 weight, or feed intake efficiency were seen among treatment groups. These data suggest that the hybrid maize line containing event TC15344 was nutritionally equivalent to the non-tr ansgenic isoline and commercial maize sources and that long-term exposur e to Cry34/35Ab1 corn is not expected to pose a

hazard to avian wildlife.

c. Aquatic Species

There is no evidence for sensitivity of aquatic (inc luding endangered) species to anti-coleopteran Cry proteins. Aquatic species toxicity studies with coleopteran-active Cry proteins have not revealed hazard for fish or invertebrates exposed to either corn pollen or to bacteria lly expressed Cry protein. Furthermore, aquatic exposure from Bt crops is extremely small.

i. Freshwater Fish (MRID No. 457904-03)

The Harmonized Testing Guidelines requirement for a static-renewal fr eshwater fish toxicity study is usually waived based on low to none xistent exposure to Cry protein produced in corn. Exposure from corn pollen, if it does take place, will be of a very short duration and quantity and is not expected to have any detectable effect on fres hwater fish. Nonetheless, an ei ght-day limit study was performed and submitted for review. This study is scientifically sound but was not performed according to OCSPP microbial testing guidelines.

This limit test was conducted at a target concentration of 100 mg PS149 B1 IC binary protein/kg diet, fed at 10% approximate fish wet body weight per day for 8 days, in a static-renewal system with water and test chambers replaced every 24 hours after feed ing. A control group was fed unamended diet only. A set of 30 fish (3 replicates, n = 10) were tested for each of the control (diet only) and treatment (diet with added PS149B1 IC binary protein) groups. Fish were observed for mort ality and sublethal effects every 24 hours during the 8-day test period. Feed consump tion ranged from 70–90% (as reported) during the 20-minute feeding period. No fish mortality or subletha l effects were reported in either the treatment or control groups. Based on thes e observations, the 8-day LD 50 for rainbow trout was > 100 mg PS149B1 IC protein/kg diet fed at10% approximate fish wet body weight/da y. No statistical test was performed because it was a single dose (limit) test. Biologic al loading exceeded guideline parameters of 0.5 grams (g) fish/L

.

In view of the lack of demonstrated toxicity to rainbow trout and minimal aquatic exposure, no freshwater fish hazard is expected from commercial cultivation of Cry34/35Ab1 corn.

ii. Freshwater Aquatic In vertebrates (MRID No. 457904-04)

This study was conducted according to procedures sp ecified in Series 72 of EPA’s Registration Guidelines, Pesticide Assessment Guidelines, FI FRA Subdivision E, Hazard Evaluation for acute toxicity testing of pesticidal substances to freshwater aquatic invertebrates.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

77 This static-renewal limit test was performed on Daphnia magna , a freshwater invertebrate. The test material consisted of purified Bacillus thuringiensis PS149B1 crystal binary protein added to water at a target concentration of 100 mg PS149B1 insect icidal crystal protein (ICP)/L. The study was procedurally sound, and no treatment mortality or behavior changes were reported between the dosed and control replicates duri ng the 48-hour exposure period.

The October 2000 and August 2002 SAP reports recomm ended that non-target testing be focused on species exposed to the crop being regi stered (i.e., beneficial insects found in corn fields). Nevertheless, acute aquatic invertebrate species testing was performed, and no substantial aquatic exposure to Cry34/35Ab1 protein contained within corn plant ti ssue is expected. Therefore, no hazard from the registered uses of Cry34/35Ab1-c ontaining corn is anticipated.

*Note for 2010 : There is an update to this summary. See section II(C)(2)(b) (“Terms and Conditions of the Event DAS-59122-7 Corn Registrations (August 2005 – September 2010)”) of this BRAD.

iii. Estuarine and Marine Animals

The estuarine and marine animal study was not requi red for this product because of no or very low potential for exposure to the Cry34/35Ab1 protein in these environments.

d. Terrestrial and Aquatic Plants

Plant toxicity studies were waived for this product because the active ingredient is an insect toxin ( Bt endotoxin), and we are unaware of any demonstrated toxicity to plants.

e. Non-Target Insect Testing

i. Effects on Honey Bee Larvae (MRID No. 453407-01)

An acceptable study was conducted based on OCSPP Ha rmonized Test Guideline 885.4380 (Honey Bee Testing, Tier I). This study was conducted in accor dance with Good Laboratory Practice Standards (40 CFR Part 160) with certain exceptions that di d not affect the integrity of the test.

Testing was conducted with three- to five-day-old honey bee ( Apis mellifera ) larvae. Individual larvae

were dosed with one of the followi ng treatments: 2 mg po llen collected from non-genetically modified maize; 2 mg (0.056 μg PS149B1 ICP) pollen collected from corn plants expressing Bt strain PS149B1 ICP; 5.6 μg bacterially produced PS149B1 binary IC P (100x protein concentration present in 2 mg PS149B1 pollen); 3.2 μg 14-kDa protein; 2.4 μg 44-kDa protein; 20 μg potassium arsenate (positive control); or untreated cell (negative control). La rval survival was evaluated 6 and 12 days after treatment, and adult emergence was evaluated 26 da ys after treatment. Ther e were no statistical differences (p = 0.05) in larval mortality betw een those fed PS149B1 pollen, unmodified pollen, or bacterially produced PS149B1 binary ICP. Based on re sults presented in this study, it can be concluded that honey bee development and survival are not a ffected by exposure to the Cry34/35Ab1 corn pollen.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

78 There was 92.5% mortality among larvae treated with potassium arsenate, the positive control, which indicates that bees were adequa tely exposed to the treatments.

ii. Parasitic Hymenoptera Testing (MRID No. 457904-05)

The study was conducted in accordance with Good La boratory Practice Standards (40 CFR Part 160) with certain exceptions that did no t affect the integrity of the test. This study was conducted based on OCSPP Harmonized Test Guideline 885.4340 (Non- Target Insect Testing, Tier I).

A dietary toxicity study was conducte d to evaluate the effect that Bt strain PS149B1 binary protein (purity equal to 37% 44-kDa pr otein and 54% 14-kDa protein) has on parasitic Hymenoptera ( Nasonia vitripennis ). The wasp test diet consisted of sugar wate r mixed with Cry protein at a rate of 280 ppm (combined total of 120 ppm 44-kD a protein and 160 ppm 14-kDa prot ein) PS149B1 protein, which is approximately equivalent to 10x the maximum protei n concentration present in PS149B1 corn pollen.

Based on this test, the LC 50 for adult parasitic Hymenoptera expos ed to dietary Cry PS149B1 protein was determined to be >280 ppm. It should also be noted that parasitic Hymenopt era do not feed directly on corn plant tissues, including polle n; therefore, minimal exposure of parasitic Hymenoptera to Cry protein is expected. As a result of these findings, no hazard to N

. vitripennis is expected from exposure to Cry34/35Ab1 corn.

The N

. vitripennis study was found acceptable by the Agency. However, the August 27, 2002, SAP concluded that the pa rasitic Hymenoptera ( N

. vitripennis ) is not an appropriate test species for coleopteran-active Bt corn because the dipteran parasitoid is not found in corn fields. A more appropriate parasitoid, that occurs in corn fields (e.g., Tricogramma spp. or Macrocentrus grandii ),

should be considered for future studies.

iii. Green Lacewing Larva Testing (MRID No. 457904-07)

An acceptable study was conducted in accordance w ith Good Laboratory Practice Standards (40 CFR Part 160) with certain exceptions th at did not affect the integrity of the test. This study was conducted

based on OCSPP Harmonized Test Guideline 8 85.4340 (Non-Target Insect Testing, Tier I).

Green lacewing larvae were fed PS149B1 insect icidal crystal protein in a moth egg ( Sitotroga spp.) and water meal diet at a rate of 280 ppm, which is approximately equivalent to 10x the maximum protein concentration in plant tissue. There was 28% mo rtality in the negative control group on day 10. Compared to the negative control, at day 10, ther e was no significant increase in green lacewing larval mortality when fed 10x (280 ppm) the maximum PS149B 1 protein concentration found in plant tissue.

The data show that the LC 50 for green lacewing larvae exposed to PS149B1 in diet was >280 ppm. Based on these results, it is not expected that th e green lacewing will be adversely affected when exposed to Cry34/35Ab1 corn in the field.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

79 The August 27, 2002, SAP concluded that green lacewing ( Chrysoperla carnea ) is not an appropriate test species for several reasons. Green lacewing are di fficult to test in the la boratory because of a high rate of mortality. For example, in the study outlined above (MRID No. 457904-07), the test was terminated after 10 days because th ere was >28% mortality in the ne gative control. In addition, it is questionable whether green lacewings ingest the protein on coated moth eggs, since green lacewing have piercing-sucking mouthparts and do not consume the external surface of eggs. For these reasons, the applicants should conduct a laboratory insect toxicity test on an alternate organism, such as the minute pirate bug ( Orius insidiosus ). This egg predator, which feeds on polle n when prey is scarce, is typically found in corn fields. An appropriate evaluation would involve feeding O

. insidiosus pollen, a natural food source, and purified protein in diet in two separate diet bioassa ys. The purified pr otein assay would be useful in determining toxicity at the maximu m hazard dose, while the pollen assay would provide information on the potential effect of an actual exposure scenario. As noted in Table 2, the Agency has requested that the applicants submit this study as a condition of registration.

*Note for 2010 : There is an update to this summary. See section II(C)(2)(b) (“Terms and Conditions of the Event DAS-59122-7 Corn Registrations (August 2005 – September 2010)”) of this BRAD.

iv. Lady Beetle Testing

The Cry34/35Ab1 protein specifically targets coleopteran (beetle) insects. Consequently, particular attention should be paid to the potential effects that Cry34/35Ab1 protein may have on lady beetles, which are in the family Coccinellidae.

 MRID No. 452422-10 – In a pure Bt binary crystal protein adult lady beetle feeding study, no mortality or abnormal clinical si gns were observed in treatment or control groups on Day 0 at 3/4

and 1 3/4 hours after test initiation. At test termination (Day 11), th ere was 22% (33 of 150) mortality in the negative contro l group. Beetles in th e control group appeared normal, except for five lethargic individuals. The 280 μg active i ngredient/milliliter (mL) treatment group resulted in 13% (20 of 150) mortality at test termination. All beetles in the treatment group appeared normal except for two immobile beetles. There wa s a lower mortality rate and fewer clinical signs of abnormality in the 280μg active ingred ient/mL treatment group then the control group. Mortality and abnormal behavior was not treatment related. The dietary LC 50 value for ladybird beetles exposed to PS149B1 ICP was determined to be greater than 280 μg active ingredient/mL, which was the highest concentration tested.

 MRID No. 452422-11 – A tri-trophic interaction study be tween field corn expressing PS149B1, corn leaf aphids ( Rhopalosiphum maidis ), and lady beetle ( C

. maculata ), in larval and adult stages, was also submitted and reviewed. Field corn plants expressing the PS149B1 protein event TC5638 and non-transgenic isolines were planted in the greenhouse and infested with naturally occurring aphid populations. Aphid colonies were allowed to establish on each plant before being removed and fed to lady beetles. One to three aphids were fed to individual lady beetle

adults and larvae in 7 mL scintillation vial s daily. Mortality and time between molts was monitored daily until day 10. No significant differen ce in adult lady beetle mortality or weightBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

80 was found between lady beetles feeding on PS149B1- intoxicated aphid and aphids that fed on non-transgenic corn. Feeding on PS149B1-intoxicated aphids also did not affect the length of

time between larval molts.

 MRID No. 461239-12

– Two diet assays were conducted: (1) a Tier I assay in which lady beetle larvae ( C

. maculata DeGeer) were fed purified Cry34/35Ab1 pr otein mixed with artificial diet, and (2) a Tier II assay in which larvae were exposed to a diet consisting of 50% inbred Cry34/35Ab1 pollen from events E4497.45.2.16, E4497.59.1.22 (renamed as DAS-59122-7,

which is the event bei ng registered), E4497.45.2.14, and E4497.71.1.33 corn pollen and 50% ground corn earworm eggs. These assays are described in more detail below.

Tier I Assay – Lady beetle larvae ( C

. maculata ) were fed an artificial diet containing purified Cry34/35Ab1 protein at a concen tration equal to approximately 10 times the expected environmental exposure concentration through po llen. Cry34Ab1 protein wa s incorporated at 900 ppm and Cry35Ab1 protein at 1 ppm (ratio based on Cry34 a nd Cry35 expression in event DAS-59122-7 corn pollen). The purified proteins were suspended in deionized water and mixed with commercially available lady beetle diet. Ne onate larvae were placed in individual bioassay wells and allowed to feed for 7 days. Larvae were then assessed for mortality and weight.

There was no significant di fference in mortality among C

. maculata larvae fed for seven days on one of two control diets, wh ich did not contain active protein, and the treatment containing purified Cry34/35Ab1 toxin. However, significant growth inhibition (80% growth reduction) was observed in C

. maculata larvae feeding on the Cry34/35Ab1 diet compared to larvae feeding on the control diets.

Tier II Assay – Corn pollen may comprise up to 50% of a lady beetle larva’s diet during corn anthesis. Consequently, a study was conduc ted to evaluate the effect that event Cry34/35Ab1 corn pollen may have on lady beetle la rvae. Pollen from four different events,

which were tested as four diffe rent treatments, was fed to lady beetle larvae in a diet consisting of 50% Cry34/35Ab1 corn pollen an d 50% ground corn earworm eggs. First neonate larvae were placed in individu al bioassay wells and allowed to feed ad libitum throughout the 14-day larval growth pe riod.

There was no significant difference in mortal ity, development, or adult weight among

C

. maculata larvae fed a control diet, which did not contain active protein, and treatments containing Cry34/35Ab1 pollen. Among th e four events tested, protein expression for Cry34Ab1 proteins ranged from 116 to 175 nanograms per milligram (ng/mg). Among reported events (values were not provided for tw o of the four events because they were not quantifiable or exceeded the uppe r limit of detection), Cry35A b1 protein expression levels ranged from 75.5 to 83.5 ng/mg. For the regist ered event (DAS-59122- 7), Cry34Ab1 protein expression was 117 ng/mg; Cry35Ab1 expression was not determined because one or more of the entries were not quantifiable (a dditional information on Cry34Ab1 and Cry35Ab1 protein expression leve ls may be found in section II(A)(1)(a) of this BRAD).Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

81 Conclusions – Submitted data show that Cry34/35Ab1 protein is toxic to C

. maculata at dose levels that exceed field exposure (Tier I A ssay). However, since t oxic effects were not seen at the field dose level (Tier II Assay) when C

. maculata larvae were fed natural prey and pollen, there is reasonable certainty that Cry34/35Ab1 corn will not adversely affect C

. maculata

.

Since the Cry34/35Ab1 protein is toxic to Coleopteran species, evaluations of other appropriate non-target beetle species should be submitted as a component of this risk assessment. The 2002 SAP suggested that Carabi ds (ground beetles) would be suitable for PIP Tier I testing because b eetles found within this family play important ecological and economic roles within agroecosystems, includ ing corn fields. As not ed in Table 2, the Agency is requesting that the applicants s ubmit this study as a condi tion of registration.

v. Collembola Feeding Study (MRID No. 457904-06)

This study was conducted in accordance with Good Laboratory Practice Standa rds (40 CFR Part 160) with certain exceptions that did no t affect the integrity of the test. This study was conducted based on OCSPP Harmonized Test Guideline 885.4340 (Non- Target Insect Testing, Tier I).

Juvenile collembola ( Folsomia candida ) were fed diets consisting of purified PS149B1 protein (purity = 54% 14-kDa protein and 37% 44-kDa protein) mixed with dry granul ated Brewer’s yeast at a single treatment level of 12.7 mg PS149B1 active ingredient/kg. Fresh diet was provided to test organisms every third day. On days 0 and 28, mortality and observations of sublethal effects on surviving

collembola were recorded.

Results of this study show no adverse effects on su rvival and reproduction of collembola exposed to PS149B1 insecticidal crystal protein at 10x concentrati ons found in transgenic co rn plants. It is noted that the primary route of collembol a exposure to Cry34/35Ab1 protein in the field is from decaying root tissue, which is expressed in corn roots at a range of 3–66 micr ograms per gram (μg/g) and is significantly lower than the treatment level used in this test.

This study adequately addresse s potential concerns for Cry34/ 35Ab1 protein to collembola ( F

. candida ), a representative of beneficial soil insect species. The results of this study demonstrate that Cry34/35Ab1 protein found in transgenic corn poses no hazard to soil-inhabiting collembola species, and by inference, to other beneficial non-coleopteran soil insects. It is noted that the Octobe r 2000 Scientific Advisory Panel stated that it is unnecessary to conduct non-target testing on invertebrate orders that are not known to be affected by the Cry protein in question.

vi. Earthworm Toxicity Testing

Earthworm feeding studies submitted to the Agency demonstrate that Cry proteins are not toxic to

earthworms at the worst-case environmental c oncentration. Although some public comments haveBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

82 questioned whether earthworm test organism s actually ingested the soil incorporated Bt Cry proteins, recently published data show that earthworms do ingest and excret e soil-incorporated Bt Cry proteins.

 MRID No. 453602-01 – This study complied with Good Laboratory Practice Standards (40 CFR Part 160) and OECD Principles of Good Laboratory Practice with certain exceptions that did not affect the integrity of the test. The testi ng was conducted based on OCSPP Harmonized Test Guideline 850.6200 (Earthworm Subchronic Toxici ty Test) and OECD Guideline 207. Thisstudy meets current testing requirements for assessing risks to earthworms from plantincorporated protec tants derived from Bt

.

The 14-day LC 50 for earthworms exposed to PS149B1 ICP in an artificial soil substrate was determined to be greater than 76 mg active ingr edient/kg dry soil (the highest concentration tested), or greater than 20 times the expected field concentration. Ea rthworm mortality and changes in average body weights were not statistically different (p >0.05) among the controls and protein-amended soils. The LC 50 value for earthworms exposed to chloroacetamide, the positive control, was approximately 19.4 mg active ingredient/kg dry soil. This finding was consistent with historical results, and further confirmed the adequacy and consistency of the protocol used in the definitive test. The submitted data suggest that earthworms will not be adversely affected by Cry34/35Ab1 corn plants.

vii. Insecticidal Activity Spectrum Study (MRID No. 452422-04)

The relative sensitivity of the test insects in the feeding bioassays suggested that larvae of coleopteran species are far more sensitive to the 14-kDa and 44- kDa proteins of PS149B1 than the lepidopteran or homopteran larval insects evaluate d. Adult WCRW were not sensitive to the delta-endotoxins, however. Rootworms (NCRW, WCRW and SCRW ) were the most susceptible to the PS149B1 proteins. European corn borer and CEW demonstrated some inhibition of growth at higher concentrations of the test

substance than the rootworm larvae. Black cutworm was the least sensitive of the lepidopteran species tested. No activity was seen against the CLA, a homopteran. Relative susceptibilities of the insect species to PS149B1, based upon the more sensitive measure of GI 50 , were as follows: (most susceptible) WCRW, NCRW, SCRW > ECB, CEW >> WCRW a dult, CLA, BCW (least susceptible).

viii. Field Evaluation of Cry34/35Ab1 Corn Exposure on Non-Target Invertebrates (MRID No. 461239-14)

This Tier IV field experiment was submitted as a supplement to the Tier I maximum hazard dose findings presented above.

Methods: The field study was conducted over two growing seas ons (2001–2002) at two locat ions in the central Corn Belt (York, Nebraska and Johnston, Iowa). The experimental design was a randomized complete block design with two replicates for each of five treatments. Treatments were the following: (1) Cry34/35Ab1 maize hybrid TC5639; (2) Cry34/35Ab1 maize hybrid TC15344; (3) non-transgenicBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

83 maize hybrid with a planting time application of tefluthrin (Force® 3G); (4) non-transgenic maize hybrid sprayed with an application of bifenthrin (Cap ture® 2EC) at the V10 and R2 corn growth stages to control first and second generation CRW adults; and (5) non-transg enic maize hybrid with no corn rootworm control measures taken. Treatment plots covered approximately 2,800 sq uare feet (70 feet x 40 feet) and were separated by 18 feet of non-tran sgenic corn.

Arthropod abundance was evaluated in soil, on the so il surface, and at the crop canopy level. Methods used were the following: (1) visual observations; (2) soil sampling w ith Berlese-Tullgren funnels for invertebrate extraction; (3) soil surface collection using pitfall traps; and (4) canopy level sampling using yellow sticky traps. Sample s were taken at specified interv als throughout the season, and four samples were collected within ea ch plot for each collection met hod at each sampling interval.

Although data were collected on individual inverteb rate families, some families were grouped for statistical analyses. At the comm unity level, groupings were based on a family’s abundance in corn fields, as well as their function within the agri cultural ecosystem. Repres entatives from several functional groups (decomposers, herbivores, predators, parasitoids, and genera list feeders) served as indicator species.

Two analyses were applied. Principal response curves (Van den Brink and Ter Braak 1999) were used to investigate and describe treatment effects at the community level, wh ile analysis of variance (ANOVA) was performed for each key indicator species and ta xa group to detect invertebrate abundance among

treatments.

Results: Neither Cry34/35Ab1 event showed signs of adverse effects on non-target arth ropods at the community level or in key taxa. The two primary effects obser ved were an overall abunda nce decrease at the 50% pollen shed sampling date in the foliar insecticide-tr eated plots compared to the untreated control plots,

and an increase in collembola a bundance at the R2 and R5 sampling stages of the foliar insecticidetreated plots compared to the untreated control pl ots. It is noted that the two Cry34/35Ab1 events presented in this study are not plan ned for commercial release; however , they represent equal or greater Cry34/35Ab1 expression levels when co mpared to commercial event DAS-59122-7.

Conclusions: According to submitted data, Cry34/35Ab1 corn does not adversely impact the abundance of non-target

invertebrates found in corn fields. Th e utility of these data, however, is limited because the plot size (70 feet × 40 feet) was too small, and the experiment incl uded only two replicates at each of two locations. In addition, only four samples, for each collectio n method, were taken from each plot. The August 2002 SAP concluded that field experiments must be appr opriately designed (larger fields, more replicates, more samples) to provide a measure of ecological im pacts and, further, that a two-year field study would not be sufficient to determine if Cry34/35Ab1 corn will have long-term impact on non-target invertebrates. However, since the endpoint for field census studies has not been identified, it is difficult to determine the appropriate field si ze, number of replicat es, and number of samples needed per plot.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

84 The data provided in this repor t suggest that co rn events containing the Cry34/35Ab1 protein did not adversely affect non-target arthropods. Supplemen tal studies, employing larger plot sizes, more replicates, and more samples per plot, are recommended for further verification of a long range no adverse effect finding. In response to these concer ns, as requested by the Agency in Table 2, the applicants are preparing additional field census data that will be submitted to the Agency as a condition of registration.

Although the experimental design of the field assays needs improvement, the submitted field census data

are useful for short-term hazard assessment and as supplementary information that supports the nohazard trend seen in the maximum h azard dose single species laboratory te sting described above. It is an accepted practice in the Office of Pesticide Programs to use the trends seen in several supplemental studies for hazard assessment when a perfect study is not available.

*Note for 2010 : There is an update to this summary. See section II(C)(2)(b) (“Terms and Conditions of the Event DAS-59122-7 Corn Registrations (August 2005 – September 2010)”) of this BRAD.

II. Soil Fate (MRID No. 452422-14)

Soil organisms may be exposed to Cry34/35Ab1 protein through contact with corn plant roots (by direct feeding), corn plant root exudate s, incorporation of aboveground pl ant tissues into soil following harvest, or by soil-deposited pollen. Some evidence s uggests that acidic soils (pH 5.6), and those which are high in clays and humic acids, are more likely to bind Cry protein, and thus decrease the rate of protein degradation by soil microorganisms. It is noted, however, that th e pH factor should not contribute to protein binding in co rn fields, since maize is generall y grown on neutral soils (above pH 5.6). Despite evidence that soils high in clay and hu mic acids may bind Cry proteins, and thus interfere with the microbial degradation processes, the we ight-of-evidence suggests that Cry proteins do not accumulate in soil to arthropod-toxic levels. Nonethele ss, the Agency requires soil fate evaluations for each new insect-protected crop.

This study was conducted in accordance with Good La boratory Practice Standa rds (40 CFR Part 160).

Methods: An insect bioassay was conducted to measure the ra te of purified PS149B1 delta-endotoxin degradation in a representative silt loam field soil . The southern corn rootworm (SCRW, Diabrotica undecimpunctata subsp. howardi ) was selected as the test species for this bioassay due to its susceptibility to PS149B1 insecticidal proteins. An aqueous mixture of the test material, composed of 14-kDa and 44-kDa binary insecticid al proteins, was pipetted onto 1 gram (dry weight) soil at a concentration of 5 mg per protein per gram of soil. The soil-protein mixture was transferred to 50 mL perforated vials (to allo w gas exchange) and incuba ted at approximately ~25  ̊ C for 0, 1, 3, 7, 14, or 28 days. Four vials were prepared for each incubation period. Aliquot s (50 microliters ( μ L)) of these dilutions were applied to the surface of small wells (1.5 square centimeters in diameter) containing 500

μ L of artificial insect diet. Soil-ag ar and agar-only negative controls were also included in bioassay trays. Following treatment application, single neona te rootworm larvae were introduced into each wellBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

85 and housed for six days at ~27  ̊ C. To reach the artificial diet, larv ae had to eat through the soil-protein surface layer and thus consume the insecticidal prot eins. Fourteen to 16 larv ae were tested at each dilution level, for a total of 94–96 larvae per incuba tion interval. Larvae mort ality and weights were

collected for all treatments.

Results: Larval mortality was negligible a nd did not correlate with insecticidal protein content. Consequently, protein degradation at each inc ubation interval was defined as the amount of insecticidal protein required to reduce larval growth by 50% (GI 50 ). GI 50 s were calculated from treatment averages for each soil-protein incubation interval, along with th eir 95% upper and lower confidence limits. Results indicate that larval growth inhi bition was inversely correlated with soil-protein incubation periods, suggesting that protein degradation incr eased with time. Regression of the GI 50 s with time (0 to 7 days) showed a degradation half-life of 3.2 days for the insecticidal protei n. The 14- and 28-day GI 50 s did not fit the first-order decay model and, consequentl y, were excluded from half-life calculations.

Conclusions: Based on these results, it may be c oncluded that purified PS149B1 insec ticidal proteins degrade rapidly in silt loam soil; however, persistence and accumulation at low levels is not addressed. Silt loam soil is just one of many soil classes used for corn produc tion in the United States. A more useful study would evaluate protein degradation/accumu lation in a range of soil types, including those with high clay and humic acid content, due to their known binding affinity for proteins.

In addition, this study utilized field so il spiked with purified insecticidal protein. This appr oach is useful because dose responses can be eas ily quantified. However, the degradation and accumulation of Cry proteins found within decaying plant tissue may behave differently than proteins in artificially spiked soil. Thus, the relevance of these study results is uncle ar other than to show that degradation in soil does take place.

To account for the above concerns, it is recommended that additional studies be conducted to evaluate insecticidal protein degradation, acc umulation, and persistence in a vari ety of soil types, including those high in clay and humic acids into which all non-harves ted corn plant material is incorporated. Sampling should be conducted at the end of three years in a field sown with continuous Cry34/35Ab1 corn. Soil should be monitored for a minimum of one grow ing season after harvest and continued until the Cry34/35Ab1 protein can no longer be de tected. As noted in Table 2, the Agency has requested that the applicants submit these studies as a condition of registration.

*Note for 2010 : There is an update to this summary. See section II(C)(2)(b) (“Terms and Conditions of the Event DAS-59122-7 Corn Registrations (August 2005 – September 2010)”) of this BRAD.

III. Effects on Soil Microorganisms

Numerous published studies indicate that exposure to Cry protein produced in Bt PIP crop plants does not adversely affect so il microorganisms (Sanvido et al . 2007; Oliveira et al . 2008). In addition, Bt toxinBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

86 released from root exudates and biomass of Bt corn has no apparent effect on earthworms, nematodes, protozoa, bacteria, and fungi in soil (Saxena and Stotzky 2001). Other resear ch findings conclude no Bt

related risks have evolved from the decomposition of Bt -corn leaves for the me so- and macrofauna soil community (Hönemann et al

. 2008). Although a minimal transient in crease and shift in microbial populations may result from the presence of transgenic plant tissue in soil, no a dverse effects have been attributed to the Cry protein.

In addition, there are several ongoing U.S. Department of Agriculture and EPA Offi ce of Research and Development funded research projec ts evaluating the effects of Bt crops on soil microbial flora. If

adverse effects are seen from this or any other research, the Agency will take appropriate action to mitigate potential risks.

With regard to the impact of genetically engineered cr ops on soil, it is important to note that agricultural practices themselves cause large changes in soil and soil microbial composition. Furthermore, factors

such as variations in seasons and weather, plant growth stage, and pl ant varieties, inde pendent of being genetically engineered, are also responsible for signi ficant shifts in soil microbial communities. To date, most studies with genetically e ngineered crops have shown minor or no effects on soil microbes beyond the variation caused by the factors listed above.

IV. Horizontal Transfer of Transgen es from Bt Crops to Soil Organisms

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 in, or have their origin in, soil-inhabiting bacteria. Soil is also the habitat of anthrax, tetanus, and botulinum toxin- producing bacteria. Transfer of these genes and/or toxins to other microorganisms or plants has not been detected. Furthermore, several experiments (published in scientific journals), that were conducted to assess the likelihood of HGT, have been unable to detect gene transfer under ty pical environmental conditions. Hori zontal 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, which s uggest that HGT is at most an artif icial event, and th e fact that the

Bt toxins engineered into Cry34/35Ab1 corn are derived from soil-inhabiting bacteria, EPA has concluded that there is a low probability of risk from HGT of transgenes found in Cry34/35Ab1- producing corn.

V. Gene Flow and Weediness Potential

The movement of transgenes from the host plant in to weeds has been a significant concern for the Agency due to the possibility of novel exposures to th e pesticidal substance. The Agency has determined that there is no significant risk of gene capture and expression of Cry34/35Ab1 protein by wild or weedy relatives of corn in the U.S., its possessions, and/or its territories. In addition, the Animal and PlantBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

87 Health Inspection Service (APHIS) of the United Stat es Department of Agricu lture (USDA) has made

this same determination under its statut ory authority under the Plant Pest Act.

Under FIFRA, the Agency has reviewed the pot ential for gene capture and expression of Bt endotoxins by wild or weedy relatives of corn, cotton, and potatoes in the U.S., its possessions, and/or its territories.

Bt plant-incorporated protectants that have been registered to date have been expressed in agronomic plant species that, for the most part , do not have a reasonable possibility of passing their traits to wild native plants. Feral species related to these crops, as found within the United States, cannot be pollinated by these crops (corn, potato, and cotton) due to di fferences in chromosome number, phenology (i.e., periodicity or timing of events within an organism’s life cycle as related to climate, e.g., flowering time) and habitat. The only exception, however, is the possibility of ge ne transfer from Bt cotton to wild or feral cotton relatives in Hawaii, Florida, and the Caribbean.

The Scientific Advisory Panel meeting held on October 18–20, 2000, further discussed the matter of gene flow and offered some issues for consideration in this matter. The panel agreed that the potential for gene transfer between corn (maize) and any recep tive plants within the U. S., its possessions, and/or its territories was of limited proba bility and nearly risk free.

Concern over the potential for species related to maize ( Zea mays ssp. mays ), such as Tripsacum species and the teosintes, as potential recipients of gene flow from genetically modified Zea mays indicated a need for review of what is known related to gene flow potential of Z

. mays . Some Zea species, such as the teosintes, are known to be interfertile with mai ze and are discussed as poten tial recipients of pollendirected gene flow from maize. This issue is of particular concern based u pon the increased planting of genetically modified maize. Theref ore, the Agency conducted a reeval uation in early 2000, the results of which are reported here.

a. Zea mays ssp. mays - Maize - General Biology

Zea mays is a wind-pollinated, monoecious, annual species with imperfect flowers. This means that spatially separate tassels (male flow ers) and silks (female flowers) ar e found on the same plant, a feature that limits inbreeding. A large variet y of types are known to exist (e.g., dent, flint, flour, pop, sweet) and have been selected for specific seed characteristics through standard breeding techniques. Maize

cultivars and landraces are known to be diploid (2n = 20) and interfer tile to a large degree. Nonetheless, some evidence for genetic incompatibility exists within the species (e.g., popcorn x dent crosses; Mexican maize landraces x Chalco teosinte). Zea mays has been domesticated for its current use by selection of key agronomic character s, such as a non-shattering rachis , grain yield, and resistance to pests. The origin of corn is thought to be in Mexi co or Central America, ba sed largely on archaeological evidence of early cob-like maize in indi genous cultures approximately 7,200 years ago.

A recent study has indicated that cross-pollination of commercial maize cultivars at 100 feet downwind from the source of genetically modified maize wa s 1%, and this proportion declined exponentially to

0.1% at 130 feet and further declin ed to 0.03% at 160 feet. At 1,000 feet , the farthest distance measured, no cross-pollination was detect ed (Jemison and Vayda 2000). For production of Foundation Seed, aBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

88 distance of 660 feet has been generally required to mitigate outcrossing between different genotypes. The relatively large size of corn pollen and its short viability peri od under most conditions reduce long distance transfer for purposes of outcrossing (Sch oper, personal communication,1999). Under conditions of high temperature or low humid ity, corn pollen may only survive fo r a matter of minutes. Under more

favorable conditions in the field or with controll ed handling in the laboratory, pollen life may be

extended to several hours.

b. Tripsacum species - Gama Gr ass - General Biology

Close relatives of corn or maize are found in the genus Tripsacum

. Sixteen species of Tripsacum are known worldwide and generally recognized by taxonomists and agrostologists; most of the 16 different Tripsacum species recognized are native to Mexico, Ce ntral America, and South America, but three

occur within the U.S. Hitc hcock (1971) reports the pr esence of three species of Tripsacum in the

continental United States: Tripsacum dactyloides

, Tripsacum floridanum , and Tripsacum lanceolatum

. Of these, T

. dactyloides , Eastern Gama Grass, is the only specie s of widespread occurrence and of any agricultural importance. It is comm only grown as a forage grass and has been the subject of some agronomic improvement (i.e., sele ction and classical breeding). T

. floridanum is known from southern Florida, and T

. lanceolatum is present in the Mule Mountains of Arizona and possibly southern New

Mexico.

For the species occurring in the United States, T

. floridanum has a diploid chromosome number of 2n = 36 and is native to Southern Florida; T

. dactyloides includes 2n = 36 forms, which are native to the central and western U.S., and 2n = 72 forms, whic h extend along the Eastern seaboard and along the

Gulf Coast from Florida to Texas but which have al so been found in Illinois and Kansas; these latter forms may represent tetraploid s (x = 9 or 18)(Lambert, pers onal communication, 1999); and T

. lanceolatum (2n = 72), which occurs in the southwestern U.S. Tripsacum differs from corn in many respects, including chromosome number ( T

. dactyloides n = 18; Z

. mays n = 10). Many species of Tripsacum can cross with Zea , or at least some accessions of each species can cross, but only with difficulty and the resulting hybrids are primarily male and female sterile (Duvick, personal communication, 1999; Galinat 1988; Wilkes 1967). Tripsacum /maize hybrids have not been observed in the field but have been accomplis hed in the laboratory using special techniques under highly controlled

conditions.

Eastern Gama Grass is considered by some to be an ancestor of Z

. mays or cultivated maize (Mangelsdorf 1947), while others dispute this (Galin at 1983; Iltis 1983; Beadle 1980), based largely on the disparity in chromosome number between the two species (m aize n = 10; Gama Grass x = 9 or 18, with diploid, triploid, and tetraploid races existin g; 2n = 36 or 72), as well as radically different phenotypic appearance. Albeit with so me difficulty, hybrids between th e two species have been made (Mangelsdorf and Reeves 1939; DeWald, personal communication,1999). In most cases, these progeny have been sterile or viable only by culturing with in vitro “embryo rescue” techniques.

Even though some Tripsacum species occur in areas where maize is cultivated, gene introgression from maize under natural conditions is highly unlikel y, if not impossible (Beadle 1980). Hybrids ofBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

89 Tripsacum species with Z

. mays are difficult to obtain outside of th e controlled conditions of laboratory and greenhouse. Seed obtained from such crosses are often sterile or prog eny have greatly reduced fertility. Approximately 10–20% of maize- Tripsacum hybrids will set seed when backcrossed to maize,

and none are able to withstand even the mildest wi nters. The only known case of a naturally occurring

Zea

- Tripsacum hybrid is a species native to Guatemala known as Tripsacum andersonii . It is 100% male and nearly 99% female sterile and is thought to have arisen from gene flow to teosinte, but the lineage is uncertain (Doebley, personal communication, 2000). Z

. mays is not known to harbor properties that indicate it has weedy potential and, othe r than occasional volunteer plants in the previous season’s corn field, maize is not c onsidered as a weed in the U.S.

In a telephone conversation with Dr. Chester “Chet” DeWald (Agric ultural Research Service of the USDA; Woodward, Oklahoma), a geneticist working on improvement of grasses, he stated that relatively few accessions of T

. dactyloides will cross with maize, and th e majority of progeny are not

fertile or viable even in those that do. In controlled crosses, if the female parent is maize, there is a greater likelihood of obtaining viable seed. When th ese hybrids have been backcrossed to maize in attempts to introgress Tripsacum genes for quality enhancement or disease resistance, the Tripsacum chromosomes are typically lost in successive gene rations. In many instances where hybridization has been directed between these two species, the resu ltant genome is lacking in most or all of the chromosomal complements of one of the pa rent species in subsequent generations.

Only recently has Dr. DeWald (or anyone else) succeeded in obtaining a true Tripsacum cytoplasm with a maize nuclear background. This was done by using gama grass as the female parent and maize as the male or pollen donor. Numerous accessions were tested and crosses made before this came to fruition.

The Tripsacum -derived mitochondrial chondrome and chloropl ast plastome in these hybrids contribute to the seed qualities of the plants, but the nuclear genome appears to be totally maize in origin (DeWald

et al . 1999).

Dr. DeWald concluded that the possibility of maize contributing genetic material to Eastern Gama Grass through random pollen flow in agricultural or natural situations is extremely remote based upon his experience trying to create hybrids under the best of conditions. He also felt that no other known grass species present in the co ntinental U.S. would inte rbreed with commercial ma ize populations (i.e., be recipients of pollen-directed gene flow). This is in agreement with Holm et al . (1979), who determined that none of the sexually compatible relatives of corn in the U.S. are considered serious, principal, or

common weeds in the U.S.

c. Zea species - Teosintes - Genera l Biology

Teosintes—specifically Zea mays ssp. mexicana (Schrader) Iltis, Zea mays ssp. parviglumis Iltis and Doebley , Zea mays ssp. huehuetenangensis (Iltis and Doebley) Doebley, Zea luxurians (Durieu and Ascherson) Bird , Zea perennis (Hitchc.) Reeves and Mangelsdorf, and Zea diploperennis Iltis, Doebley

and Guzman

— have co-existed and co-evolved in close proximity to maize in the Americas over thousands of years; however, maize and teosinte mainta in distinct genetic constitutions despite sporadic introgression (Doebley 1990).Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

90 The teosintes retain a reduced cob-like fruit/inflores cence that shatters more than cultivated maize but still restricts the movement of seeds as compared to more widely dispersed weedy species. Hence, the dispersal of large numbers of seeds, as is typical of weeds, is not char acteristic of teosintes or maize. In their native habitat, some teosinte s have been observed to be spread by animals feeding on the plants.

Teosintes and teosinte-mai ze hybrids do not survive even mild wi nters and could not propagate in the U.S. Corn Belt. Additionally, some types have strict day length requirements that preclude flowering within a normal season (i.e., they would be induced to flower in November or December) and, hence, seed production under our temperate climate (Bea dle 1980; Iltis, person al communication, 2000; Wilkes, personal communication, 2000; Wilkes 1967).

Since both teosinte and Tripsacum are included in botanical gardens in the U.S., the possibility exists (although unlikely) that exchange of genes could occur between corn and its wild relatives. The Agency is not aware, however, of any such case being repo rted in the United States . Gene exchange between

cultivated corn and transformed co rn would be similar to what natu rally occurs at the present time within cultivated corn hybrids and landraces. Pl ant architecture and reproductive capacity of the intercrossed plants will be similar to normal corn, a nd the chance that a weedy type of corn will result from gene flow with cultivated corn is extremely remote.

Like corn, Z

. mays ssp. mexicana (annual teosinte) and Z

. diploperennis (diploid perennial teosinte) have 10 pairs of chromosomes, are wind pollinated, and tend to outcross but are highly variable species that are often genetically compatible and interfertile w ith corn, especially when maize acts as the female

parent. Z

. perennis (perennial teosinte) has 20 pairs of chromosomes and fo rms less stable hybrids with maize (Edwards et al . 1996; Magoja and Pischedda 1994). Corn a nd compatible species of teosinte are capable of hybridization when in pr oximity to each other. In Mexico and Guatemala, teosintes exist as weeds around the margins of corn fields. The F 1 hybrids have been found to va ry in their fertility and vigor. Those that are fertile are capable of backcrossing to corn. A few isolated populations of annual and perennial teosinte were said to exist in Fl orida and Texas, respectively (USDA APHIS 1997). The Florida populations were presumably an escape from previous use of Z

. mays ssp. mexicana as a forage grass, but local botanists have not documented any natural populati ons of this species for approximately twenty-five years (Bradley, personal communication, 2000; Hall, personal communication, 2000; Wunderlin, personal communication, 2000).

Consultation with botanists and ag ronomists familiar with Texas flora suggested that no teosinte populations exist in the state (Benz, personal communication, 2000; Rea d, personal communication, 2000; Orzell, personal communication, 2000; Wilson, personal communication, 2000). Further, given the day length characteristics of Z

. diploperennis , it is highly unlikely a su staining population would result from introduction of this species. Z

. mays ssp. mexicana

, Z

. mays ssp. parviglumis

, Z

. luxurians

, and Z

. diploperennis may cross with maize to produce fertil e hybrids in many instances (Wilkes 1967). None of these teosinte species ha ve, however, been shown to be aggr essive weeds in their native or introduced habitats (Schoper, pe rsonal communication, 1999). Except for special plantings as noted above, teosinte is not present in th e U.S. or its territories. Its natura l distribution is limited to Mexico, Honduras, Nicaragua, El Salv ador, and Guatemala.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

91 Given the cultural and biological rela tionships of various teosinte spec ies and cultivated maize over the previous two millennia, it would ap pear that significant gene exchange has occurred (based upon morphological characters) between these two grou ps of plants, and that no weedy types have successfully evolved as a result. More recent cytogenetic, biochemical, and molecular analyses have indicated that the degree of gene exchange is fa r less than previously thou ght (Doebley 1984; Doebley et
al . 1987; Kato 1997a; Kato 1997b; Smith et al . 1985). Partial and complete gametophytic incompatibility has been documented among cultivated maize, landraces, and teosinte (Kermicle 1997). The former is demonstrated by differential pollen gr owth and a skewed recove ry of alleles linked to incompatibility genes. Complete inco mpatibility mechanisms serve to is olate a species or subspecies and are evidenced as pollen exclusion or non-functioning of pollen types on certain genotypes. Attempts to cross six collections of Z. mays ssp. mexicana with U.S. maize cultivars (W22, W23) yielded no or few seeds in five of the six gr oups (Kermicle and Allen 1990).

Based on the ability of maize to hybridize with some teosintes, th e suggestion of previous genetic exchange amongst these species over centuries, and their general grow th habits, any in trogression of genes into wild teosinte from Z

. mays is not considered a significant ag ricultural or environmental risk. The growth habits of teosintes are such that the potential for serious weedy propagation and development is not biologically plausible in the United States.

d. Conclusion

The potential for pollen-directed gene flow from maize to Eastern Gama Grass is extremely remote. This is evidenced by the difficulty with which T

. dactyloides x Z

. mays hybrids are produced in structured breeding programs. Additionally, the genus does not represent any spec ies considered as serious or pernicious weeds in the United States or its territories. Any introgression of genes into this species as a result of cross fertilization with genetically modified maize is not expected to result in a species that is weedy or difficult to control. In many instances wh ere hybridization has been directed between these two species, the resultant genome is lacking in most or all of the maize chromosomal complement in subsequent generations.

Many of the Zea species loosely referred to as “teosintes ” will produce viable offspring when crossed

with Z

. mays ssp. mays . None of these plants are known to har bor weedy characteristics and none of the native teosinte species, subspeci es, or races are considered aggr essive weeds in their native or introduced habitats. In fact, many are on the brink of extinction where they are indigenous and will be lost without human intervention (i.e., conservation measures). Further, none of the landraces or

cultivated lines of Z

. mays are considered to have weedy pot ential and are generally considered incapable of survival in the wild as a result of breeding practices (i.e ., selection) during domestication of the crop.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

92 iii. Impacts on Endangered Species (MRID No. 461239-17)

The primary route of exposure to Cry34/35Ab1 protein in corn is through ingesti on of corn tissue. There are no reports of threatened or endangered species f eeding on corn plants; therefore, such species would not be exposed to corn tissue containing the Cry protein. Since Cry34/35Ab1 corn pollen has shown no toxicity at the estimated environmental concentrati on rates to mammals, birds, plants, aquatic species, insects, and other invertebrate sp ecies tested, a “may affect” situa tion for endangered land and aquatic species is not anticipated. In a ddition, EPA does not expect that a ny threatened or endangered plant species will be affected by outcrossing to wild relati ves or by competition with such entities. Hybrid corn does not exist in the wild, nor are there wild plants that can interbreed with corn in the United

States.

Because of the selectivity of Cr y34/35Ab1 protein for coleopteran spec ies, endangered species concerns are mainly restricted to the order Coleoptera. Exam ination of an overlay ma p showing the county-level distribution of the 16 endangered/threat ened coleopteran species (as curren tly listed by the U.S. Fish and Wildlife Service) relative to corn production countie s in the United States cl early indicated that any potential concern regarding range overlap with corn production was mainly restricted to the American burying beetle ( Nicrophorus americanus ). The American burying beetle is the largest carrion beetle in North America and is only found today in limited ar eas in Rhode Island and th e portions of the Great Plains into Arkansas and Georgia. Adults are nocturnal and feed on carrion and sometimes prey on other arthropods. Larvae feed exclusivel y on buried carrion provided by their parents. The American burying

beetle’s habitat is variable and often includes deciduous forest, grassland, and agricultural areas. Considering that both larvae and adult insects fe ed exclusively on carrion with some limited adult predation, even if American burying beetles did occur in proximity to Bt corn fields, there would be little chance of exposure to Bt protein due to their feeding habits. After careful review of the available data, EPA determined that exposure of American bur ying beetle to harmful levels of Cry34/35Ab1 corn tissue is not expected. Likewise, a re view of the preferred habitats of other coleopteran species, listed as endangered by the U.S. Fish and Wildlife Service, i ndicated that no exposure to harmful levels of Cry34/35Ab1 protein would take place. The main reas ons for the lack of expos ure are geographical and habitat limitations. These other speci es are located in non-corn production areas and/or their habitat does not encompass agricultural areas.

Likewise, other endangered/threatened insect specie s in the orders Diptera, Hemiptera, Lepidoptera, Odonata, and Orthoptera are found in dune, meadow/prairie, or open forest habitats and are not closely associated with row crop production often times due to the specificity of the habi tat of their host plants. The reviewed toxicological data show the rela tive insensitivity of a range of insects from nonColeopteran orders to the Cry34/35 Ab1 protein, indicating that th e Cry34/35Ab1 corn hybrids will not likely cause detrimental effects to the non-Coleopteran insects on the endangered/thre atened species list.

Likewise, several insect species li sted are aquatic species and are unl ikely to come in contact with Cry34/35Ab1 corn. Many of the endangered and threaten ed beetles occur in cave or aquatic habitats. Since movement of Cry34/35Ab1 in soil and into water bodies is expected to be negligible, pollen drift was considered the primary source of potential hazar d to end angered aquatic Coleoptera. According toBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

93 estimates based on published studies, if 100% of the po llen grains leaving a corn field were deposited in a 1 hectare pond with 2 meters depth and located ≥ 1 meter from the edge of the corn field, <0.0001 μg Cry34/35Ab1/mL of water would be expected. This is a few orders below the t oxic level to any insect.

Conclusions: The reviewed non-target data confirm the expectati on that Cry34/35Ab1 corn is not likely to jeopardize the continued existence of any endangered and/or threatened species listed by the U.S. Fish and Wildlife Service, including mammals, birds, or terrestrial and aquatic plants a nd invertebrate species. Therefore,

no consultation with the U.S. Fish and Wildlife Serv ice is required under the Endangered Species Act.

iv. Environmental Assessment Summary (MRID No. 461239-13)

The Agency is using a maximum hazard dose tiered system for biopesticide non- target wildlife hazard

assessment. When no adverse effects are observed at the maximum hazard dose, the Agency concludes

that there are no unreasonable adverse effects from the use of the pestic ide. From all of the required and voluntarily developed indicator an d host range species test data on Cry34/35Ab1 corn, the Agency

concludes that the levels of Cr y34/35Ab1 protein in corn will not pos e unreasonable adverse effects to

corn field flora and fauna. Available data also indicate that there should be minimal short-term accumulation of Cry34/35Ab1 protein in agricultural so il. In addition, no adverse effect on endangered and threatened species, as listed by the U.S. Fish a nd Wildlife Service, is expected from the proposed Cry34/35Ab1 CRW resistan t corn registration.

At present, the Agency is aware of no identified significant adverse effect s of Cry34/35Ab1 protein on the abundance of non-target beneficial organisms in any population in the field, whether they are pest parasites, pest predators, or pollinators. Field test ing and field census data submitted to the Agency show minimal to undetectable changes in beneficial insect abundance or diversity. To da te, available field test data show that, compared to crops treated with c onventional chemical pesticid es, the transgenic crops

have no detrimental effect on the abunda nce of non-target insect populations.

The Agency believes that cultivation of Cry34/35Ab1 corn may result in fewer adverse impacts to nontarget organisms than result from the use of chemical pesticides. Under normal circumstances, Cry34/35Ab1 corn requires substantially fewer applicati ons of chemical pesticides. This should result in fewer adverse impacts to non-target organisms b ecause application of non-specific conventional chemical pesticides is known to have an adverse effect on non-target benefi cial organisms found living in the complex environment of an agricultural field. Many of these beneficial organisms are important integrated pest management controls for secondary pests, such as aphids a nd leafhoppers. The overall result of Cry34/35Ab1 corn cultivation is that the number of chemical insect icide applications for nontarget pest control is reduced for management of multiple pest problems.

The movement of transgenes from Cry34/35Ab1 host pl ants into weeds and other crops has also been considered. The Agency has determin ed that there is no significant ri sk of gene capture and expression of Cry34/35Ab1 protein by wild or weedy relatives of corn in the U.S., its possessions, and/ or territories. The fate of Cry34/35Ab1 protein in so ils and indirect effects on soil biot a have also been evaluated. TestBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

94 data show that most of the Cry protein deposited into soil is quickly degraded, although a residual amount may persist in biologically activ e form for a much longer period of time. It is also reported that the same degree of Bt Cry protein persistence takes place in so ils that have been exposed to repeat Bt spray applications when compared to soil exposed to growing Bt crops. Limited data do not indicate that Cry proteins have any measurable effect on microbial populations in the soil. Horizontal transfer of genes from transgenic plants to soil bacteria has not been demonstrated. Published studies of Bt Cry protein in soil show no effect on bact eria, actinomyces, fungi, protozoa, algae, nematodes, springtails, or earthworms. In addition, new plants planted in Bt Cry protein-containing soil do not take up the Bt protein.

Conclusion: At the present time and for the purposes of a time-lim ited registration, this risk assessment finds no

hazard to the environment from cultivation of corn expressing the Cry34/35Ab1 protein.

v. Supplemental Studies Needed for Lo ng-Term Cry34/35Ab1 Non-Target Hazard

Assessment

The Agency has sufficient information to believe th at there is no risk from the proposed uses of Cry34/35Ab1 corn to non-target wildlife, aquatic, and soil organisms. Nonetheless, in response to the August 2002 SAP recommendations, the Agency is requesting supplementary studies that will evaluate more appropriate non-target inverteb rates (e.g., those found in corn fields ) and facilitate identification of potential adverse effects that may result from long-term use of Cry34/35Ab1 corn. The Agency does not believe that these data requirements were r easonably foreseeable by Mycogen Seeds c/o Dow AgroSciences LLC and Pioneer Hi-Bred Internationa l, Incorporated at the time of their respective applications.

Table 2. Supplemental Data Requirements.

Testing Category Type of Data

Non-target insect more appropriate for corn fields Conduct a maximum hazard dose la boratory toxicity test with Orius insidiosus (minute pirate bug).

Non-target insect more appropriate for corn fields Conduct a maximum hazard dose laboratory toxicity test with a carabid (ground beetle).

Ecosystem effects Additional long-range field studies should be conducted based on recommendations from the August 2002 SAP (as presented in summary form in the conclusions

section of the MRID No. 461239-14 review).

Soil fate studies Additional long-range soil degradation field studies should be conducted to include the parameters outlined by the August 2002 SAP (as presented in summary form in the conclusions section of the MRID No. 452422-14 review).

*Note for 2010 : There is an update to this summary. See section II(C)(2)(b) (“Terms and Conditions of the Event DAS-59122-7 Corn Registrations (August 2005 – September 2010)”) of this BRAD.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

95 b. Terms and Conditions of the Event DAS -59122-7 Corn Registrations (August 2005 – September 2010)

When event DAS-59122-7 corn was first registered on August 31, 2005, the Agency issued registration notices to both Mycogen Seeds c/o Dow AgroScie nces LLC (EPA Reg. No. 68467-5) and Pioneer HiBred International, Inco rporated (EPA Reg. No. 29964-4) that, gi ven the conclusions set forth in the initial environmental risk assessment (see section II(C)(2)(a) of this BRAD), contained the following three requirements for further environmental effects data:

“Submit field degradation studies evaluating accumula tion and persistence of Cry34/35Ab1 in several different soils in va rious strata. Representative fields must have been planted with Cry34/35Ab1 corn and include both conventional tillage and no-till samples and be harvested under typical agronomic conditions. Sampling must

continue until the limit of detection is re ached. Studies should include soils with high levels of a variety of clays. Both ELISA and insect bioassays need to be conducted and compared to determine if Cry34/35Ab1 is accumulating or persisting in soil samples... A final report regarding da ta from fields that have had three continuous years of cultivation of event DAS-59122-7 corn is due by January 31, 2010.”

“Submit laboratory toxicity tests with Orius insidiosus (minute pirate bug) [and] carabid (ground beetle) within 24 m onths of the date of registration....”

“Additional 3 year full-scale field or semi-field studies for evaluation of Cry34/35Ab1 event DAS-59122-7 corn exposure on non-ta rget invertebrates must be conducted. Full-scale field experiments must be appr opriately designed to pr ovide a measure of ecological impacts (larger fields, more re plicates, more samples per plot based on recommendations of the August 2002 SAP). The previously submitted two-year field study is not sufficient to determine if Cr y34/35Ab1 corn will have long term impact on non-target invertebrates...A fina l report is due September 30, 2009.”

For both registrations, the abovementioned requirements for additional environmental effects data have been satisfied by submission of appropriate studies; summaries of the studies are presented in Table 3.

Current environmental effects data , to include those conditional data referenced in Table 3, and EPA reviews of Cry34/35Ab1 protein support the Agency’s original determination that adverse effects will not occur to non-target organisms. Due to a demonstr ated lack of toxicity and/or exposure, no effects from Cry34/35Ab1 protein are anticipated for any non-target species, incl uding federally listed threatened and endangered (“listed”) lepidopteran a nd coleopteran species and their designated critical habitats. The Agency is therefor e upholding its determination that the registered uses of Cry34/35Ab1 will have “No Effect,” direct or indirect, on endangere d or threatened terrestrial or aquatic species as listed by the U.S. Fish and Wildlife Service and the National Marine Fisheries Service.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

96 When the docket for the expiring Bt corn registrations was opened for public comment on August 4, 2010 (Docket Number EPA-HQ-OPP-2010-0607), the Ag ency noted its awareness of a recently published laboratory study showing reduced growth in shredding caddis flies exposed to antilepidopteran Cry1A protein corn litter (Rosi-Marshall et al . 2007). Given the findings of this particular study, the Agency proposed requiring additional a quatic invertebrate data for the Cry34Ab1 and Cry35Ab1 proteins—either a 7- to 14-day D

. magna study or a dietary study ev aluating the effects of these proteins on an aquatic invert ebrate that represents the functi onal group of a l eaf shredder in

headwater streams. Since the 2007 Rosi-Marshall et al . publication, numerous researchers have published peer-reviewed studies that identify issues with the scientific merit and relevance of the original caddis fly study (Swan et al . 2009, Jensen et al . 2010, summarized by Beachy et al . 2008, Parrott 2008, and Wolt and Peters on 2010). In response to comments received on the proposed terms and conditions for the extension of the 2010 expiring Bt corn registrations, the Agency conducted a literature review of these recently published studies. Criticisms of the Rosi-Marshall et al . study included several findings: (1) advers e effects were not caused by toxic ity of Cry1A but, rather, by other differences between plant test substances (Jensen et al . 2010); (2) the abundan ce of Trichoptera in streams containing residues of Cry1A was not reduced (Chambers et al . 2007); and (3) while postharvest crop residue was identified as th e most likely route of exposure (Carstens et al . 2010), aquatic exposure to biotech crops has been s hown to be limited temporally and spatially with low to negligible exposure concentrations of Cry proteins in post-harvest crop tissues (Swan et al . 2009, Chambers et al

. 2010, Jensen et al . 2010, Wolt and Peterson 2010, Carstens et al . 2010). In light of these results, the Agency is not requiring additional aquatic inverteb rate studies to assess h azard to aquatic shredder species for existing Cry pr otein PIP registrations.

Table 3. Environmental Effects Data Submitted in Response to Conditions of Registration for

Event DAS-59122-7 Corn

Study Title Summary MRID No.

Three-Year Field Monitoring of Cry34/35Ab1 and Cry1F x Cry34/35Ab1Maize Hybrids for NonTarget Arthropod

Effects n A multi-year field study evaluated the impact of event DAS-59122-7 maize (expressing Cry34Ab1 and Cry35Ab1 proteins) and its respective stacked maize hybrid, DAS-01507-1 x DAS-59122-7 (expressing Cry1F and Cry34/35Ab1 proteins), on non-target arthropods after continuous cropping at three locations in the Corn Belt (Iowa, Nebraska, and Indiana). Community-level analyses and key indi cator species data were collected using visual observations, sticky traps, pitfall traps, and litter bag traps. Results showed similar dynamics on non-target arthropod populations

between non- Bt isogenic maize hybrid fields and the Bt transgenic PIP corn events, DAS-59122-7 and DAS-59122-7 x TC1507. Therefore, there is negligible potential for long-term advers e effects on non-target arthropods on the community level and key taxon abundance after continuous

cultivation of both Herculex® RW and Herculex® XTRA Bt corn events.
Classification: Acceptable 478703-01

n Reviewed in U.S. EPA (2010c)Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

97 Study Title Summary MRID No.

Soil Accumulation of Cry34Ab1 and Cry35Ab1 Proteins after

Three Years ofCropping with DAS59122-7 Corn o After 3 years of continuous cropping with DAS-59122-7 corn, the enzymelinked immunosorbent assay (ELISA) analyses showed no detectable residues of Cry34Ab1 and Cry35Ab1 proteins in soil (bulk and rhizosphere) extracted from 3 different field sites (Illinois, Minnesota, and North Carolina) containing a range of soil properties. In addition, no biological activity was observed in insect bioassays of southern corn rootworm ( Diabrotica undecimpunctata howardi ) neonates after exposure to rhizosphere soil samples. Therefore, there is negligible potential for Cry34Ab1 and Cry35Ab1 proteins to pe rsist or accumulate in agricultural

soils in various soil matrices across cr opping cycles of DAS-59122-7 corn.

Classification: Acceptable 479595-01

Summary Report of

Carabid Beetle Laboratory Toxicity Study Using Cry34Ab1 and Cry35Ab1 p In a laboratory bioassay, ground beetle ( Poecilus cupreus ) larvae were fed blowfly ( Calliphora vomitoria ) pupae containing target concentrations of a combination of 1,000 nanograms (ng) Cry34Ab1 + 333.3 ng Cry35Ab1/mg of pupa. A negative control group of larvae was fed pupae treated with deionized water, and a positive control group was fed pupae treated with teflubenzuron. Survival of the ground beetle larvae was monitored throughout pupation and adult emerge nce. There was no statistically significant difference in pre-imag inal mortality between the negative control and Cry34Ab1 + Cry35Ab1 groups (15% and 8%, respectively), while mortality in the positive control group was 83%. Mean development time and mean adult weight of the negative control and Cry34Ab1 + Cry35Ab1 groups were comparable. This study shows that when carabid beetle larvae are fed diet containing Cry34/35Ab1 protein, no negative adverse effect is observed. Therefore, at field concentra tions, it is unlikely that Cry34/35Ab1 will have negative a dverse effects on carabid beetles.

Classification: Acceptable 467141-01

o Reviewed in U.S. EPA (2010c)

p Reviewed in U.S. EPA (2010b)Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

98 Study Title Summary MRID No.

Evaluation of Potential Dietary Effects of Cry34/35Ab1 Protein

on Insidious Flower Bugs, Orius insidiosus q Four separate 10- to 14-day tests we re conducted from December 2005 to July 2007 to evaluate the dietary effects of combined Cry34Ab1 and Cry35Ab1 proteins on survival and development of insidious flower bug ( O

. insidious ) nymphs. Each test included a group given a combination of 750 ng Cry34Ab1 + 85 ng Cry35Ab1/mg of diet (bee pollen), a positive control group given potassium arsenate (8 ng active ingredient/mg of diet volume by weight), and an assay control group given purified reagent grade water mixed with diet. In addition, one test included a group given 750 ng of heat-treated Cr y34Ab1 + 85 ng of heat-treated Cry35Ab1/mg of diet. The control group mortality in each test exceeded the allowable rate specified in the test protocol of less than 20% control mortality. However, the survival of the test species in the control groups and test groups was

similar in each test. It is concluded that, at field concentrations, it is unlikely that Cry34/35Ab1 will have negative adverse effects on the insidious flower bug. Classification: Supplemental 474367-01

3. Herculex® XTRA Insect Protection Corn (OECD Unique Identifi er: DAS-Ø15Ø7-1 x DAS- 59122-7) Expressing Cry1F, Cry34Ab1, and Cry35Ab1

a. Data Cited/Submitted for Initial Registration s of Herculex® XTRA Ins ect Protection Corn (Prior to October 2005)

i. Background

In 2004, Mycogen Seeds c/o Dow AgroSciences LLC a nd Pioneer Hi-Bred International, Incorporated requested full and unrestricted FIFRA section 3 re gistrations for commerci alization of Herculex® XTRA Insect Protection Corn, a st acked product containing the following registered active ingredients: Cry1F (EPA Reg. No. 68467-2 and EPA Reg. No. 29964-3; Herculex® I) and Cry34/35Ab1 (EPA Reg. No. 68467-5 and EPA Reg. No. 29964-4; Event DAS-59122 -7 corn or Herculex® RW). Herculex® XTRA was developed by crossing inbred line s of Cry1F lepidopteran-active and Cry34/35Ab1 coleopteran-active corn to produce hybrid maize line 1507 x 59122. Southern blot analyses

demonstrated that the insertions in the Herculex® XTRA hybrid are equivalent to those of the

Herculex® I and Herculex® RW lines and indicated th at the stacked hybrid was a successful cross of the two inbred lines, 1507 and 59122 (MRID No. 462971-02; reviewed in U. S. EPA (2005f)). In addition to the insecticidal genes, the phosphinothricin acetyltransferase gene, which encodes PAT protein, is also present in the stacked trait corn plants. The inclusion of the pat gene provides tolerance to glufosinate-ammonium herbicides.

The insecticidal proteins expr essed in single-trait events DAS -01507-1 (Herculex® I) and DAS-59122-7 (Herculex® RW) have been previously evaluated fo r adverse impacts on non-target organisms and have

qReviewed in U.S. EPA (2010b)Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

99 been shown to pose negligible risk. Thus, the ev aluation of Herculex® XTRA is simplified, requiring only demonstration of no synergy in the biological activity of the combined traits from Herculex® I and

Herculex® RW.

ii. Non-Target Wildlife Hazard Assessment

An environmental risk assessment was conducted to char acterize the risk of advers e impacts of the traits expressed in Herculex® XTRA on n on-target organisms, particularly invertebrates found in or near agro-ecosystems. Concerns over pos sible effects on endangered and th reatened species, especially butterfly (Lepidoptera) and beetle (Cole optera) species were also addressed.

Expression of the Cry1F, Cry34Ab1, and Cry35Ab1 protei ns in Herculex® XTRA was determined in various tissue types at various stages of devel opment utilizing ELISA kits specific for each protein (MRID No. 463438-01; reviewed in U. S. EPA (2005f)). These data indi cate that protein levels are comparable between the Herculex® I, Herculex® RW, and Herculex® XTRA hybrids. Therefore, the

environmental effects data submitted and reviewed in support of the Herculex® I and Herculex® RW registrations, including studies submitted as conditi ons of registration, may be bridged to support the Herculex® XTRA product registrations . The environmental risk assessments for the insecticidal proteins (Cry1F and Cry34/Cry35Ab1) expresse d in the single-trait events ha ve been evaluated for adverse impacts on non-target organisms and have been s hown to pose negligible risk (see U.S. EPA 2001d, 2005d, and 2010d for Cry1F; see section II(C)(2) of this BRAD for Cry34/35Ab1).

Of unique concern to the environmental risk assessmen t of stacked PIPs, is the potential for synergistic effects that may result from the insertion of mu ltiple active ingredients into a single crop plant. Therefore, the conceptual model for evaluation of the stacked Herculex® XTRA hybrid is focused on the demonstration of no synergy in the biological act ivity of the two traits, Herculex® I and Herculex®

RW. To address this issue and in support of the Herculex® XTRA regi strations, four new studies were submitted: three comparing the effects of the single and stacked proteins in the sensitive target insect pests and two non-target insect spec ies and one evaluating the effects of the stacked proteins on broiler

chickens. These studies were reviewed in U.S. EP A (2005f) and are summarized in the sections that

follow.

I. Non-Target Wildlife Study Summaries

a. Potential Synergistic Interact ion Between the Cry1F and Cry34/35Ab1 Proteins (MRID No. 463438-06)

Laboratory bioassays were conducted to determine if combining the Cry1F and Cry34/35Ab1 proteins increased their activity against southern corn rootworm or European corn borer larvae. No synergism or antagonism between Cry1F and Cry34/35Ab1 was seen in the test s. Therefore, the absence of additive or synergistic effects in sensitive organisms ma kes it extremely unlikely th at the Cry1F x Cry34/35Ab1 proteins produced in Herculex® XTRA will have a ny adverse effect on non-target organisms different

from what was seen in in dividual Cry protein testing.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

100 b. Lady Beetle Study (MRID No. 463438-07)

In a 12-day laboratory feedi ng study, ladybird beetle ( C

. maculata ) larvae were provided a prepared test diet consisting of 50% Cry1F x Cry34/35Ab1 maize pollen and 50% ground corn earworm eggs until adult emergence. Larvae fed the treated diet for 12 days did not show any developmental delays compared to those fed the control diet. There was no statistically significant difference in larval mortality between the treated and control groups (7% and 0%, respec tively). The average weight of adults in the treated and control groups was co mparable. These results demonstrated no additional

adverse effect to C. maculata , at the expected field exposure dosag e, from the mixture in Herculex®

XTRA over that observed with the Cr y1F and Cry34/35Ab1 proteins alone.

Since the Cry34/35Ab1 protein toxicity is specific to coleopteran species, evaluations of other appropriate non-target beetle species should be submitted in support of Cry34/35Ab1-containing corn lines. Studies submitted in support of Herculex® RW may be bridged to support the Herculex® XTRA registrations.

c. Monarch Butterfly Testing (MRID No. 463438-08)

In a 4-day laboratory feeding study, first-instar monarch butterfly ( Danaus plexippus ) larvae were exposed to transgenic (Cry1F x Cry34/35Ab1 stack) or non-transgenic pollen at densities of 50, 100, 200, 400, 800, or 1600 pollen grains/square centimeter on milkweed leaves. After 96 hours, the larvae

were transferred to clean milkweed leaves for an additional 120 hours. Exposure to the transgenic pollen did not significantly reduce larval su rvival, weight gain, developmental life stage, or leaf consumption during the 216-hour observation period. These data indicate that subchron ic effects were not seen when

D

. plexippus was exposed to the Cry1F and Cry34/35Ab1 prot eins at the recommended field dosage for 4 days. Therefore, no hazard to monarch butterflies is expected from incident al exposure to Herculex® XTRA corn pollen.

d. Avian Study (MRID No. 463438-03)

Broiler chickens were fed diet s containing transgenic maize gr ain (containing Cry1F x Cry34/35Ab1 and PAT proteins), a non-transgenic near-isoline maize grain, or non-transgenic commercial reference maize grains. Feeding began on the day of hatch and continued for 42 days. There were no statistically significant differences in broile r mortality, body weight, body weight gain, feed efficiency, or carcass/carcass part yields for any of the diets. These results indicate th at hybrid maize line 1507 x 59122 (Cry1F x Cry34/35Ab1) was nutritionally equiva lent to the near-isoline and commercially available non-transgenic hybrids and, like with the corn events produc ing the individual Cry proteins,

there were no adverse effects noted on the growth and development ofbroiler chickens. Therefore, longterm exposure to Herculex® XTRA is not exp ected to pose a hazard to avian wildlife.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

101 e. Other Non-Targets Organism Risk Assessments

Waiver rationales were also submitted for the additional non-target studies normally required for environmental risk assessments: Avian Oral (O CSPP Harmonized Guideline 885.4050); Avian Injection (now Avian Inhalation) (OCSPP Harmonized Gu ideline 885.4100); Freshwater Fish (OCSPP Harmonized Guideline 885.4200); Fr eshwater Invertebrate (OC SPP Harmonized Guideline 885.4240); Estuarine and Marine Animal (OCSPP Harmoni zed Guideline 885.4280); Non-Target Plant (OCSPP Harmonized Guideline 885.4300); Non-Target In sect (OCSPP Harmonized Guideline 885.4340); and Honey Bee (OCSPP Harmonized Guideline 885.4380) . A poultry broiler study was submitted, so a standard avian oral study is not required. The rationale for the waiver from avian injection and

freshwater fish is that the active i ngredient is not an infectious agent and is expressed at very low levels in plants. The rationale for the waiver from estuar ine and marine animal studies is that the active ingredient is not an infecti ous agent, is expressed at very low levels in plants, and ther e is very low to no potential for exposure to the prot ein from field corn. The rationale for waivers from freshwater invertebrate, non-target insect, and honey bee studies is that appropria te studies were completed for the single-trait products (Cry34/35Ab1 a nd Cry1F). The rationale for the waiver from non-target plant studies is that the active ingr edient is an insect toxin ( Bt endotoxin) that is non-toxic to aquatic and terrestrial plants. Lastly, toxicity data submitted for the Human Health Assessment (see section II(B)(2) of this BRAD) indicate that ther e is no significant toxicity to r odents from testing at the maximum hazard dose and, therefore, no hazard to mammalian wildlife is anticipated.

Additional support for a complete e nvironmental risk assessment is ga ined from an overview of the assessments done for the individual Cry1F (H erculex® I) and Cry34/35Ab1 (Herculex® RW) registrations. A brief summary of th e Cry1F environmental risk assessment is provided in the section that follows, while the comprehensive environmen tal risk assessment for Cry34/35Ab1 can be found in section II(C)(2) of this BRAD.

f. Herculex® I (Lepidopteran-Active Cry1F Protein) Environmental Risk Assessment

EPA has conducted an extensive review of effects of the Cry1F protein presen t in Herculex® I to nontarget organisms in an environmental risk assessm ent as part of the regi stration process (U.S. EPA 2001d, 2005d, and 2010d). EPA reviewed studies conducted on representative non-t arget species with Cry1F protein and performed risk or impact assessm ents on plants, wild mammals, birds, fish, aquatic invertebrates, estuarine and marine animals, earthwo rms, terrestrial non-targe t insects (including honey bee adults and larvae, parasitic wasps, green la cewings, lady beetles, springtails (collembola toxicity/reproduction), and mona rch butterflies), field inverteb rate abundance studies, soil degradation/persistence studies, a nd endangered species with special emphasis on the Karner blue butterfly. In addition, weediness and gene fl ow assessments (via pollen and Cry protein deoxyribonucleic acid (DNA) uptake by plants and soil microorganisms) were also performed.

The Agency concluded that, considering all availa ble information, the weight -of-evidence indicates no unreasonable adverse effects of Cry1F proteins in corn to non-target wildlife, plants, or beneficial

invertebrates.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

102 iii. Endangered Species Considerations

The reviewed non-target data confirm the expectati on that Cry1F x Cry34/35Ab1 corn is not likely to jeopardize the continued existence of any endangered and/or threatened species listed by the U.S. Fish and Wildlife Service, including mammals, birds, or terrestrial and aquatic pl ants and invertebrate species. Herculex® XTRA is not likely to adversely affect Karner blue butterf lies because exposure to harmful levels of Cry1F corn pollen is not expected (U.S. EPA 2001d, 2005d, and 2010d). Likewise, a review of the preferred habitats of other lepidopteran species listed as endangered by the U.S. Fish and Wildlife Service, including the endangered Mitche ll satyr butterfly, indicates that no exposure to harmful levels of Cry1F protein-containing pollen will take place. Furthermore, there is also no overlap of endangered beetle species habitat with corn pr oduction. Therefore, EPA believes that this action will have “No Effect” on listed species. This finding was triggered by the reviewed Cry protein toxicity and exposure data on terrestrial and aquatic species. As a result, no endangered sp ecies labeling is required for the registration of Herculex® XTRA corn.

iv. Environmental Assessment Summary

From all of the required and volunt arily developed indicator and host range species test data on Cry1F, Cry34/35Ab1, and Cry1F x Cry34/35Ab1 corn, the Agency concludes that the levels of Cry1F x Cry34/35Ab1 protein in Herculex® XTRA corn will not pose unreasonable adverse effects to corn field

flora and fauna. Available data also indicate that there should be minimal short-term accumulation of Cry1F x Cry34/35Ab1 protein in agri cultural soil. No evidence of synergy between the lepidopteranactive and coleopteran-active insect icidal proteins expr essed by Herculex® XTRA corn was found in laboratory studies of target and non-target insects evaluated for these registrations. Endangered/threatened species assessments conducted in support of individual trait (Herculex® I and Herculex® RW) registrations were sufficient to demonstrate a “N o Effect” finding for the stacked product. In conclusion, Herculex® XTRA is projected to control certain lepi dopteran and coleopteran pests in field corn without concerns of risk to non-target organisms.

Conclusion: At the present time and for the purposes of a time-lim ited registration, this risk assessment finds no

hazard to the environment from cultivation of corn expressing the Cry1F and Cry34/35Ab1 proteins.

v. Supplemental Studies Needed for Long-Term Cry1F x Cry34/35Ab1 Non-Target Hazard

Assessment

The Agency has sufficient information to believe that there is no evidence of risk from the proposed uses of Herculex® XTRA corn to non-target wild life, aquatic, and soil organisms. However, after consultation with the Scientific Advisory Panel in August 2002, and based upon several public comments, the Agency is requesting more data. Th e supplementary studies would provide additional weight to support the Agency’s conclusions. The Agen cy does not believe that these data requirementsBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

103 were reasonably foreseeable by Mycogen Seeds c/o Dow AgroSciences LLC and Pioneer Hi-Bred International, Incorporated at the tim e of their respectiv e applications.

The following data (Table 4) may be required to as certain any possible adverse environmental effects from long-term use of Herculex® XTRA corn.

Table 4. Supplemental Data Requirements.

Testing Category Type of Data

Toxicity data on non-target insects

more appropriate for corn fields, ecosystem effects, and soil persistence determination Laboratory toxicity testing with Orius insidiosus (minute pirate bug) and a carabid (ground beetle) may be required, as well as long range effects on invertebrate populations in the field and soil persistence studies with Cry1F x Cry34/35Ab1 corn as per the parameters outlined by the August 2002 FIFRA SAP. In the event that Cry34/35Ab1 (Herculex® RW) studies suffici ently demonstrate a lack of long range adverse effects, no additional data w ith Herculex® XTRA will be required.

*Note for 2010 : There is an update to this summary. See section II(C)(3)(b) (“Terms and Conditions of the Herculex® XTRA Insect Protection Corn Regist rations (October 2005 – Se ptember 2010)”) of this

BRAD.

b. Terms and Conditions of the Herculex® XT RA Insect Protection Corn Registrations (October 2005 – September 2010)

When Herculex® XTRA Insect Protection Corn wa s initially registered on October 27, 2005, the Agency issued registration notices to both Mycoge n Seeds c/o Dow AgroSciences LLC (EPA Reg. No. 68467-6) and Pioneer Hi-Bred Intern ational, Incorporated (EPA Re g. No. 29964-5) that contained the following requirement in relation to further environmental effects data:

“Submit all data required to support the in dividual plant-incor porated protectants in event TC1507 (Herculex I) and event DAS-59122-7 (Herculex Rootworm) corn...In the event that the Agen cy concludes Cry34/35Ab1 (Herculex Rootworm) studies do not sufficiently demonstrate a lack of long range adverse effects, additional data with Herculex Xtra must be submitted. This data may include a) laboratory toxicity testing with Orius insidiosus (minute pirate bug), b) laboratory toxicity testing with a carab id (ground beetle), c) long range effects testing on invertebrate populati ons in the field, and d) long range soil persistence testing.”

All requirements for additional environmental effects data for events TC1507 (see U.S. EPA (2010d)) and DAS-59122-7 (see section II(C)(2)(b) of this BRAD), as set fo rth in the October 27, 2005

Herculex® XTRA Insect Protection Co rn registration notices, have been satisfied for both registrations.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

104 Moreover, after evaluating the c onditional data submitted to support the registrations of event DAS59122-7 corn, the Agency has concluded that these da ta do not demonstrate the potential for long-range

adverse effects to the environment as a result of the cultivation of Cry34/35Ab1 corn. It should be noted that Mycogen Seeds c/o Dow AgroSciences LLC a nd Pioneer Hi-Bred Intern ational, Incorporated, although not specifically requested by the Agency, did include Herculex ® XTRA Insect Protection Corn in the three-year non-target arthropod field study (MRID No. 478703-01; re viewed in U.S. EPA 2010(b)).

4. 1507 x 59122 x MON 810 (OECD Unique Iden tifier: DAS-Ø15Ø7-1 x DAS-59122-7 x MON-ØØ81Ø-6) Expressing Cry1F, Cr y34Ab1, Cry35Ab1, and Cry1Ab and 59122 x MON 810 (OECD Unique Identifier: DAS -59122-7 x MON-ØØ81Ø-6) Expressing Cry34Ab1, Cry35Ab1, and Cry1Ab

a. Data Cited/Submitted for Initial Registrations of 1507 x 59122 x MON 810 and 59122 x MON 810 (Prior to February 2010)

The stacked and pyramided product, 1507 x 59122 x MON 810, and the stacked product, 59122 x MON 810, are hybrids created by crossing the indi viduals events, TC1507 (Cry1F), DAS-59122-7 (Cry34/35Ab1), and MON 810 (Cry1Ab), via tradi tional breeding methods. Although background information on individual events DAS-59122-7, TC1507, and MON 810 is provided throughout this BRAD, the most specific information on each of these events is discussed or referenced in the Product Characterization chapter (see sections II(A)(1)–II(A)(3) of this BRAD).

To support their registration applications for 1507 x 59122 x MON 810 and 59122 x MON 810, Pioneer Hi-Bred International, Incorporated submitted mo lecular characterization data, protein expression analyses, and a synergism study. Colle ctively, these data (as summarized in the paragraphs that follow) justified bridging the existing find ings and conclusions from the e nvironmental assessments conducted for the single PIP events (TC1507, DAS-59122-7, a nd MON 810) to the combination PIP products (1507 x 59122 x MON 810 and 59122 x MON 81 0) (MRID Nos. 476778-01, 476778-02, and 478800- 01; reviewed in U.S. EPA (2010a)).

The molecular characterization of Bt Cry1F, Cry34Ab1, Cry35Ab1, and/or Cry1Ab insect control proteins and the genetic material necessary for their production in 59122 x MON 810 and 1507 x 59122 x MON 810 combination PIP corn products demonstrat ed the stability and in tegrity of the intended transgenic DNA inserts of Cry1F protein e xpressed in TC1507, Cry34Ab1 and Cry35Ab1 proteins expressed in DAS-59122-7, and/or Cry1Ab protein expressed in MON 810 corn events, when combined through traditional plant breeding. Protein expression an alyses also demonstrated similar levels of Cry1F, Cry34Ab1, Cry35Ab1, and/or Cry1Ab protein concentrations of each respective parental event when compared to 59122 x MON 810 and 1507 x 59122 x MON 810 combination PIP corn events.

Laboratory assays, using sensitive target pest species, found no antagoni stic or synergistic effects among the Cry proteins expressed in each combination PIP product. This dete rmination demonstrates functional equivalence among the single parental events and th e combination PIP products, and the results andBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

105 conclusions of the non-target organi sm studies (conducted for each single parental event) can be bridged (see U.S. EPA 2001d, 2005d, and 2010d for Cry1F; see section II(C)(2) of this BRAD for Cry34/35Ab1; and see U.S. EPA 2001d and 2010d for Cry1Ab). Ther efore, no unreasonable adverse effects on nontarget organisms and the environment are e xpected from 59122 x MON 810 and 1507 x 59122 x MON

810.

b. Terms and Conditions of the 1507 x 59122 x MON 810 and 59122 x MON 810 Registrations (February 2010 – September 2010)

When 1507 x 59122 x MON 810 (EPA Reg. No. 29964-8) and 59122 x MON 810 (EPA Reg. No. 29964-9) were initially registered on February 24, 2010, the Agency issued registration notices to Pioneer Hi-Bred International, Incorporated that containe d the following requirements:

 For 1507 x 59122 x MON 810

–

“The data submitted by Pioneer are sufficient to support registration for the combination PIP product...provi ded that the registrant submits/cites any data required to support the PI P registrations of the indi vidual parental events...as well as the combination PIP product TC1507 (DAS-Ø15Ø7-1) x DAS-59122-7....”

 For 59122 x MON 810

–

“The data submitted by Pioneer are sufficient to support registration for the combination PIP product...provi ded that the registrant submits/cites any data required to support the PI P registrations of the indi vidual parental events....”

All requirements for additional environmental effects data for individual events TC1507 (see U.S. EPA (2010d)), MON 810 (see U.S. EPA (2010d)), and DAS-59122-7 (see section II(C)(2)(b) of this BRAD), as well as for combination PIP product TC1507 x 59122-7 (see section II(C)(3)(b) of this BRAD), as set forth in the February 24, 2010 1507 x 59122 x M ON 810 and 59122 x MON 810 registration notices,

have been satisfied.

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Rootworm and Northern Corn Rootworm. Memorandum from R. Rose and Z. Vaituzis, Ph.D. to M. Mendelsohn dated May 15, 2001.

U.S. EPA. 2001c. Review of Product Characterizati on, Expression Analysis and Acute Oral Toxicity Studies for PS149B1 Binary Insect Contro l Proteins as Expressed in Maize, a Bacillus thuringiensis -Based Plant-Pesticide, for an Experime ntal Use Permit. Memorandum from C.A. Wozniak, Ph.D. and J.L. Kough, Ph.D. to M. Mendelsohn dated June 7, 2001.

U.S. EPA. 2001d. Biopesticides Registration Action Document – Bacillus thuringiensis Plant- Incorporated Protectants. Available from: http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm

.

U.S. EPA. 2002. SAP Meeting Minutes No. 2002-05. A Set of Scientific Issues Being Considered by the Environmental Protection Agency Regarding: Co rn Rootworm Plant-Incorporated Protectant Non-Target Insect and Insect Resistance Management Issues. Dated November 6, 2002.

Available from: http://www.epa.gov/scipoly/sap/mee tings/2002/august/august2002final.pdf

.

U.S. EPA. 2004a. Reviews of Honeybee Dietar y and Earthworm Acute Toxicity Studies for Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 Proteins and the Genetic Material Necessary for

Their Production in Corn. Memorandum from H. Hill and Z. Vaituzis, Ph.D. to M. Mendelsohn dated March 30, 2004.

U.S. EPA. 2004b. SAP Report No.2004-05. Product Characterization, Human Health Risk, Ecological Risk, and Insect Resistance Management for Bacillus thuringiensis ( Bt ) Cotton Products. Dated August 19, 2004. Available from: http://www.epa.gov/scipoly/sap /meetings/2004/ju ne/final1a.pdf

.

U.S. EPA. 2005a. Review of PS149 B1 Binary Insecticidal Crystal Protein TGAI Non-Target Studies. Memorandum from J. Gagliardi, Ph.D. and Z. Vaituzis, Ph.D. to M. Mendelsohn dated May 5,

2005.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

115 U.S. EPA. 2005b. Assessment of Chr onic Toxicity of Diet Containing Bacillus thuringiensis PS149B1 Insecticidal Crystal Protein to Collembola ( Folsomia candida ). Memorandum from Z. Vaituzis, Ph.D. to T. Milofsky and D. Szuhay dated May 18, 2005.

U.S. EPA. 2005c. Environmental Risk Assessment for Cry34/35Ab1 Bacillus thuringiensis Binary

Protein and the Genetic Materi al Necessary for Its Production in Corn. Memorandum from T. Milofsky and Z. Vaituzis, Ph.D. to M. Mendelsohn dated July 19, 2005.

U.S. EPA. 2005d. Biopesticides Registration Action Document – Bacillus thuringiensis Cry1F Corn. Available from: http://www.epa.gov/oppbppd1/biopesticid es/ingredients/ tech_docs/brad_006481.pdf

.

U.S. EPA. 2005e. Environmental Effects Assess ment of HerculexTM Xtra Stacked Cry34/35Ab1 x Cry1F Proteins and the Genetic Material Necessary for Thei r Production in Corn. Memorandum from T. Milofsky and Z. Vaituzis, Ph.D. to M. Mendelsohn dated September 15, 2005.

U.S. EPA. 2005f. Review of Product Characterizati on and Human Health Data for Registration of

HerculexTM Xtra Insect Resist ant Corn. Memorandum from R.L. Edelstein, Ph.D. and J.L. Kough, Ph.D. to M. Mendelsohn dated September 22, 2005.

U.S. EPA. 2010a. Review of Human Health and Product Characterization Data for Registration of Bacillus thuringiensis ( Bt ) Cry1F, Cry34Ab1, Cry35Ab1, and Cr y1Ab Proteins and the Genetic Material Necessary for Their Producti on in the Combination Plant-Incorporated Protectant (PIP) Products: 1507 x MON 810 [EPA Reg. No. 29964-T], 59122 x MON 810 [EPA Reg. No. 29964-O], and 1507 x 59122 x MON 810 [EPA Reg. No. 29964-I]. Memorandum from A. Waggoner and J. L. Kough, Ph.D. to A. Sibold dated February 19, 2010.

U.S. EPA. 2010b. Review of Carabid (MRID No. 467141-01) and Orius (MRID No. 474367-01) Insect Studies Submitted as a Condition of Registratio n for Mycogen Seeds/Dow AgroSciences and Pioneer Hi-Bred International Cry34Ab1 and Cr y35Ab1 Protein in Corn (HerculexTM EPA Reg. No. 68467-5). Memorandum from Z. Vaituzis, P h.D. to M. Mendelsohn dated July 8, 2010.

U.S. EPA. 2010c. Review of Multi-Year Field St udies on Non-Target Arth ropod Populations and SoilPersistence for PIP Corn Events: TC1507 [Herculex ITM, EPA Reg. 68467-2, 29964-3], DAS59122-7 [Herculex RWTM, EPA Reg. 68467-5, 29964-4] , and Its Associated Stacked DAS59122-7 x TC1507 Hybrid [Herculex XTRA TM, EPA Reg. 68467-6, 29964-5], Expressing Bt Cry1F, Cry34Ab1/Cry35Ab1, and Cry1F x Cr y34Ab1/Cry35Ab1, Respectively. Memorandum from A. Waggoner and Z. Vaituzis, Ph.D . to M. Mendelsohn dated July 12, 2010.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

116 U.S. EPA. 2010d. Biopesticides Registration Action Document – Bacillus thuringiensis Cry1Ab and Cry1F Corn (Updated September 2010). Available from: http://www.regulations.gov (see “Supporting & Related Material s” within Docket Number EPA-HQ-OPP-2010-0607).

USDA APHIS. 1997. USDA/APHIS Petition 97-265-01 for Determination of Nonregulated Status for Bt Cry9C Insect Resistant and Glufosinate To lerant Corn Transformation Event CBH- 351: Environmental Assessment. USDA, APHIS, Riverdale, Maryland.

Van den Brink P, Ter Braak CJF. 1999. Principal Re sponse Curves: Analysis of Time-Dependent Multivariate Responses of Bi ological Community to Stress. Environmental Toxicology and Chemistry 18:138–148.

Wilkes GH. 1967. Teosinte: The closest relative of maize. Bussey Institute, Harvard University, Cambridge, MA.

Wilkes GH. 2000. Personal communication. Professor of Plant Genetics, University of Massachusetts, Amherst, Massachusetts (617-287-6662).

Wilson H. 2000. Personal communication. Professo r of Biology, Texas A&M University, College Station, Texas (409-845-3354).

Wolfenbarger LL, Naranjo SE, Lundgren JG, Bit zer RJ, Watrud LS. 2008. Bt effects on functional guilds of non-target arthropods: a me ta-analysis. PLoS ONE 3(5):e2118. http://dx.doi.org/10.1371/journal.pone.0002118

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Wolt JD, Peterson RK. 2010. Prospective formulation of environmental risk assessments: Probabilistic screening for Cry1A(b) maize risk to aquatic insects. Ecotoxicology and Environmental Safety 73(6):1182–1188.

Wunderlin R. 2000. Personal communication. Professo r of Botany, Institute for Systematic Botany, University of South Florida, Tampa, Florida (813-974-2359).Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

117 D. Insect Resistance Management (IRM)

1. Supporting Information for Development of the Cry34/35 Event DAS-59122-7 Corn Durability Plan (2005 Registration)

a. Pest Biology

A clear understanding of pest biol ogy is essential to the development of a sound IRM plan. Cry34/35 Bacillus thuringiensis ( Bt ) corn was developed to control three target pest species: western corn rootworm ( Diabrotica virgifera virgifera LeConte, WCRW), northern corn rootworm ( Diabrotica

barberi Smith and Lawrence, NCRW) and Mexican corn rootworm ( Diabrotica virgifera zeae Krysan and Smith, MCRW). Key factors be lieved to influence corn rootwo rm (CRW) adaptation to Cry34/35 corn include distribution, univoltinism, adult dispersal among fields, adult dispersa l within fields, larval dispersal across rows, larval mortality due to de nsity-dependent processes, insecticide use, egg mortality, fecundity, and adult and larval population de nsity. Some of these fact ors are discussed below.

Distribution

WCRW is the most prevalent target pest in the United States and throughout most of the Corn Belt 1

. NCRW, also found throughout the Corn Belt, is consider ed the second most preval ent rootworm pest in the United States and is the primary ta rget pest of the north-central region 2 . MCRW, the third target pest, is limited to Texas, is considered a relativel y minor pest in the United States, and is generally excluded from discussion in the data package. Sout hern corn rootworm (SCRW) , also a minor pest of corn in the U.S., is not identified as a target pest of Cr y34/35-protected corn.

Life Cycle

The traditional life cycle of CRW is as follows: adult female rootworms deposit eggs in corn fields during late summer; eggs overwinter and hatch in late-spring, generally between late May and mid-June;

larvae feed on corn roots for three to four weeks, during which time they complete three instar stages (most significant damage to corn plants caused during this period); the third instar transforms into a pupa, which develops for one to two weeks; pupae matu re to become adult beetles, which emerge from the soil in mid-July to feed on corn foliage, pollen, and silks; adults remain active for 10 to 12 weeks, during which time they feed, mate, and deposit eggs.

Recently, deviations from the traditional life-cycle have emerged. A biotype of WCRW is depositing eggs in soybean fields, while a NCRW biotype has broken from the traditional univoltine life cycle by utilizing an extended diapause. In a ffected regions, these adaptations di minish the pest control benefits

1 WCRW is the primary rootworm pest in Colorado, Kansas , Nebraska, Ohio, Indiana, Illinois, Iowa, Missouri, and Michigan.

2 NCRW is the primary rootworm pest in Wisconsin, Minnesota, South Dakota, and northern Iowa.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

118 associated with corn-soybean/corn-alternate crop ro tations, a management practice that has long been recognized as the most effective means of controlling CRW.

Dispersal/Movement

Much remains to be learned about CRW larval and adult dispersal patterns; however, some preliminary research has been completed. In their study of larval movement between corn rows, Hibbard et al

. (2003) reported that 0.75% and 6% of larvae mo ved across rows. Larval movement was largely dependent on the density of plants within a row, food availability, soil porosity, and level of soil compaction.

In their discussion of adult disper sal factors (reference to model disc ussion), the applicants referenced

Nowatzki et al .’s research (Nowatzki et al . 2003), which described a study of male and female WCRW dispersal in a commodity grain field. Results of this research indica te that males m oved at least 184 meters (m) in 4 days, or 45 m per day, while female s took around 14 days to move the same distance, traveling up to 13 m per day.

The applicants also referenced Hi ll and Mayo’s (1980) research on interf ield dispersal, which shows that NCRW was three times more prevalen t than WCRW in first-year cor n, yet that NCRW populations were

half of WCRW in s econd-year corn. Lance et al . (1988) later reported that NC RW adults are more likely than WCRW to disperse from mature corn and, furt her, that NCRW has a te ndency to disperse from corn to feed on flowers and pollen of other plants (Naranjo 1994).

Biopesticides and Pollution Prevention Divisi on (BPPD)’s Analysis of Pest Biology

Sufficient information has been provided on the pest biology of western corn rootworm, northern corn rootworm, and Mexican corn rootworm to develop an IRM strategy. Because there are still information gaps, additional information on WCRW, NCRW , and MCRW biology and ecology could improve understanding of resistance ri sks and lead to improvement s in the IRM strategies. Specific areas to be examined include the following: dispersal behavi or (categorized by sex and developmental stage); reproductive biology, including mating ha bits and frequency, ovipositiona l patterns, role of densitydependent mortality, use of non-corn hosts, and fecundity; level of Cry34/35 expression in corn roots relative to developmental stage of CRW pests; dete rmination of whether IRM strategies designed for WCRW and NCRW are appropriate for MCRW; and the effect that new rotation-resistant WCRW and NCRW biotypes may have on the development of appropriate IRM strategies. Thes e research efforts are

recommended to be continued and bolstered. New info rmation published since the trait durability plan was submitted to the Environmental Protection Agency (EPA) regarding CRW larval dispersal in the

field and use of non-maize host as unstructured ref uge should be considered, as appropriate, in the model (Hibbard et al ., 2003; Hibbard et al ., 2004; Moeser and Hibbard 2005; Oyediran et al . 2004; Clark and Hibbard 2004).Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

119 b. Mode of Action and Cross-Resistance

Cry34Ab1 (14-kiloDalton (kDa)) and Cry35Ab1 (44-kDa) proteins are both required for high activity against WCRW larvae (Ellis et al . 2002). The mode of action app ears to be similar to other Bt proteins. Experimental feeding studies conducted by Moellenbeck et al . (2001) with WCRW neonates and later (2 nd and 3 rd ) instar indicated that these proteins bi nd to midgut receptors, followed by membrane disruption. As a result, high levels of resistance to Cry34/35Ab1 will likely result from loss of binding or loss of activity following bindings, as reported for other Cry proteins (Ferré and Van Rie 2002).

Amino acid sequence comparisons did not show any si gnificant similarity between the 14- and 44-kDa proteins (Cry34Ab1 and Cry35Ab1, respectively) and other registered Bt plant-incorporated protectants, including the Cry3 family (Ellis et al . 2002). These proteins also share no sequence homology with the

Bt vegetative insecticidal protei ns (VIPs). Because of this l ack of sequence homology with known Bt plant-incorporated protectants (PIPs) and VIPs, St orer and Lefko (2003, Master Record Identification Number (MRID No.) 461239-18) assume that high leve ls of cross-resistance development is unlikely with any other rootworm-targeted Bt proteins. Field trials conducte d in areas where methyl-parathion resistance is present and absent showed no a ssociated difference in adult emergence (Storer et al . 2003, MRID No. 461239-15). Similarly, field trials in areas where rotational-adapted populations (behavioral resistance) of NCRW and WCRW are present and ab sent showed no associated differences in adult emergence (Storer et al . 2003, MRID No. 461239-15). Agricultural systems in which Cry34/35 corn is deployed will likely reduce the select ion pressure for resistance to alternative corn rootworm control tactics, including other PIPs. Simulation models ha ve shown that the use of Cry34/35 corn can reverse

selection for rotation resistance in WC RW (Storer 2003b, MRID No. 461239-19).

The 14-kDa protein (Cry34Ab1) alone shows some insec ticidal activity against southern corn rootworm ( Diabrotica undecimpunctata howardi Barber), but was enhanced by the 44-kDa protein (Cry35Ab1), indicating that both proteins are necessary for maximal insecticidal activity (Herman et al . 2002). A recent study by Masson et al . (2004) provided evidence th at Cry34Ab1/Cry35Ab1 causes pore formation in the midgut, which results in membrane permeability. This study provides evidence that the

mode of action is similar to that previously identified for other B

. thuringiensis and Bacillus sphaericus

toxins.

Receptor binding sites for Cry34Ab1 and Cry35Ab1 have not been identified and characterized. Ellis et
al . (2002) indicate that there is little sequence homology between the Cry34Ab1 and Cry35Ab1 proteins and other registered Cry pr oteins and VIPs. Lack of sequence sim ilarity would suggest that high levels

of cross-resistance conferre d by Cry34Ab1 and Cry35Ab1 would be unlikely. There is some evolutionary relatedness between the larger Cry35Ab1 protein ( 44-kDa) and the 42-kDa and 51-kDa dipteran-active toxins from B. sphaericus (Baumann et al . 1988). However, since B

. sphaericus biopesticide products act against dipt erans rather than coleopterans, cr oss-resistance with these toxins should not be an issue. Field trials (Storer et a l . 2003, MRID No. 461239-15) suggest that there is little possibility of cross-resistance between Cry34Ab1/Cry35Ab1 and any ot her rootworm-control tool. The use of Cry34Ab1/Cry35Ab1 corn will likely reduce the se lection pressure for resistance to other corn rootworm control tactics, in cluding other PIPs. Simulation models predict that use ofBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

120 Cry34Ab1/Cry35Ab1 corn can reverse selection for ro tation resistance in WCRW (Storer 2003b, MRID No. 461239-19).

BPPD’s Analysis of Mode of Action and Cross-Resistance

To confirm that high levels of Cry34Ab1 or Cry35Ab1 cross-resistance with other Bt proteins is unlikely, it is recommended that re search efforts should focus on determining the likelihood of crossresistance. Midgut binding receptors for Cry34Ab1 and Cry35Ab1 are recommended to be identified and characterized. Resistant colonies might be used to study the cross-resistan ce potential. Models could

be run with scenarios that include the possibility for cross-resistan ce to see how the selection for resistance is impacted. Resistance may evolve through a detoxification or binding modification mechanism, for example, and/or through a behavior al modification mechanism. Multiple mechanisms

should be considered as one evalua tes the cross-resistance potential. This information would provide further evidence that cross-resistance is unlikely.

There is also a potential “repellent ” behavior that should be further investigated. It is required that additional research efforts focus on characterizing and understanding the implications of this mode of

action on selection for WCRW and NCRW resistance.

c. Genetics and Mechanisms of Resistance

The resistance mechanism of the greatest concern is that which could occur through a single gene mutation (i.e., monogenic) through loss of eff ective receptor binding. In nearly all known Bt resistance cases that have conferred high leve ls of resistance, this is the si ngle most common mechanism (Ferré and Van Rie 2002). Other forms of resistance, such as metabolic resistance or increased midgut protease activity, do not confer high levels of resistance becau se multiple mutations in individual insects would be required to have high fitness on the transgenic plants. The IRM plan is based on the worst-case assumption that resistance will be monogenic, with tw o alleles, one conferring resistance (r-allele) and the other conferring suscep tibility (S-allele). In addition, there is another worst-case assumption that homozygous resistant insects will be comp letely resistant to Cry34Ab1/Cry35Ab1 corn.

Tabashnik et al . (2003) reports that most resi stance (laboratory-selection) to Cry proteins is incomplete meaning that less than 100% of target pests survive on a Bt crop. Incomplete resistance will evolve more slowly than complete resistance since the relative f itness of the r-allele compared with the S-alleles will be lower. The IRM plan for Cry34Ab1/Cry35Ab1 co rn has made the worst-case assumption that resistance will be complete and that homozygous re sistant individuals would have the same fitness on transgenic and non-transgenic plan ts (i.e., complete resistance).

The fitness costs of rootworm resistance to Cry34Ab1/Cry35Ab1 are unknow n. Resistance to other Bt proteins has generally been shown to have high fi tness costs (Ferré and Van Rie 2002). Despite this evidence, the IRM plan assumes the worst-case a ssumption that there will be no loss of fitness

associated with resistance.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

121 Receptor modifications are the most likely cause of resistance. Resistance will be incompletely to completely recessive, and heterozygotes will re tain partially functioning Cry protein receptors. Resistance associated with recept or modifications usually results in low resistance ratios for heterozygotes. IRM plans develope d for lepidopter an-protected Bt corn products have assumed that heterozygous insects will be 25-fold resistant to the associated Cry proteins, an assumption that was deemed sufficiently conservative for PIPs (U.S. EPA 1998 and 2001a). The IRM plan for Cry34Ab1/Cry35Ab1 corn assumes that heterozygous insects will be 25-fold resistant to Cry34Ab1/Cry35Ab1. This assumption en ables the estimation of the fu nctional dominance of the ralleles and, therefore, heterozygot e survival used in the Storer model (Storer 2003b, MRID No. 461239- 19). Based on the dose information, the functional dominance is predicted to be around 0.08 in WCRW

and between 0.15 and 0.37 in NCRW (Storer 2003b, MRID No. 461239-19).

Given the limited availability of information on the genetics of resistance to Cry34/35, the IRM plan utilizes “worst-case” assumptions of resistance po tential in the modeling simulations of target pest adaption to Cry34Ab1/Cry35Ab1 corn (Storer 2003b, MRID No. 461239-19). These assumptions include the following: (1) all re sistance to Cry34Ab1/Cry35Ab1 is mo nogenic; (2) all resistance is complete; (3) resistance does not confer a fitness cos t; and (4) heterozygotes will be 25-fold resistant to Cry34Ab1/Cry35Ab1. BPPD agrees with this approach.

BPPD’s Analysis of Genetics and Mechanism of Resistance

When resistance does occur, little is known about how that resistance would a ffect rootworm fitness. Most studies of target pest resistance to Bt products and Cry proteins ha ve shown that resistance is unstable, most likely due to fitne ss costs associated with resistance genes or with other closely linked loci (Ferré and Van Rie 2002). These findings suggest that the affected midgut receptors may perform alternate functions. Despite insigh ts provided by these preliminary i nvestigations, the trait durability plan acknowledges that “the fitness costs of r ootworm resistance to Cry34/35Ab1 are currently

unknown” and that further research on fitness costs is needed.

Given that functional dominance and/or recessivene ss of resistance to Cry 34/35 is presently unknown

and will remain unknown until field resistance occurs , resistant (homozygous or heterozygous) colonies would be useful to study functional dominance. It is recommended that the applicants establish Cry34/35 resistant CRW laboratory col onies to study possible mechanisms, genetics, and inheritance of

resistance. While useful information is likely from the study of resistan t colonies, it is important to note

that conclusions made from laborator y studies may not necessarily be ex tended to situations in the field. However, even without this confirmatory info rmation, the IRM durability plan is sufficiently

conservative and assumes the worst case that there w ill be no fitness costs associated with resistance.

Laboratory assays submitted by the applicants also s uggest a “repellent” behavi or. While larvae feeding on control plants tended to feed in one location, CRW larvae failed to become established on Cry34/35- protected corn roots. It follows that inconsistent feeding patterns could contribute to poor larval development and decreased fitness. A combinat ion of both behavioral and binding modification

resistance mechanisms would also slow the rate of resistance evolution. It is recommended that theBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

122 applicants further study the “repellent” or behavioral mode of acti on and its implications in the selection

of resistance.

d. Dose

The term “high dose” is not well characterized for the CRW complex, and the previous dose definition developed for lepidopteran pests (“a level of toxin 25 tim es greater than is needed to kill all susceptible insects”) may not apply to CRW speci es. Dose evaluation for CRW-targeted PIPs is also complicated by the fact that (1) CRW larvae and a dults both feed on corn tissues and each life stage must be evaluated separately for dose; (2) CRW larv ae feed underground, making direct observations of mortality difficult; and (3) CRW are notoriously difficu lt to rear in the laboratory.

In terms of adult susceptibility and dose, Dow Ag roSciences LLC/Pioneer Hi-Bred International, Incorporated (Dow/Pionee r) conducted adult feeding studies w ith WCRW and Cry34/35 corn tissue.

These studies indicated that a dult WCRW feeding (solely) on Cry34/35Ab1 aboveground corn tissue does not reduce adult fitness as characterized by longe vity, fecundity, or egg vi ability. Therefore, dose considerations for Cry34/35 corn can be relegated to the larval life stage.

To investigate larval dose, Dow/Pioneer conducted a se ries of single-season fiel d trials that measured adult emergence while correcting for density-dependent mortality to provide an indirect estimate of larval mortality. These trials indicated that WCRW larval mortality ranged between 99.82% and 99.98%, while NCRW larval mortality ranged betw een 92.77% and 99.14%. Laboratory feeding studies were also conducted with neonate, as well as 2 nd and 3 rd instar, WCRW and NC RW. The lab studies documented evidence of repellent or avoidance behavi or but did not provide es timates of mortality or

dose measures.

The mortality estimates obtained from the field indi cate that it is likely that Cry34/35 corn expresses slightly less than a high dose for CR W (perhaps a “borderline” high dose) when evaluated relative to the previously used “25x” high dose definition. These data for dose were directly applied to CRW models to evaluate the durability of the IRM plan.

BPPD’s Analysis of Dose

The studies (both larval and adult dose) are acceptable for th e purposes of registration and the development of an IRM plan for Cry34/35 corn. None theless, there are still significant uncertainties regarding the CRW dose expressed in Cry34/35 corn. At best, the results are an educated estimate of the true effects of Cry34/35 corn on CRW. The exact toxic ity (i.e., mode of action) of the Cry34/35 proteins

to CRW is unclear: the field studie s indicated high levels of mortal ity, while the laboratory studies suggested less toxic mechanisms such as growth inhibition or feeding deterrence. Given these uncertainties, it is recommended that Dow/Pioneer cont inue to research the dos e issue, including the concept of dose for CRW, specific assays to evalua te dose for rootworm-protected corn hybrids, and the role of density dependence in dose determinations.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

123 e. Refuge (General Considerations)

Unstructured refuge

Several non-maize grassy weeds are known to suppor t CRW species. Earlier wo rk characterizing nonmaize hosts of WCRW and NCRW (Branson and Ortm an 1970; Branson and Or tman 1971; Siegfried and Mullin 1990) has been augmented by the work of Oyediran et al . (2004) and Clark and Hibbard (2004) on prairie and forage grasses, and weedy species. The results of these new studies indicate that WCRW can readily develop to ad ulthood by feeding on non-maize hosts and, in the case of pubescent wheatgrass, the number of reared adults was not significantly different from that produced on maize (Oyediran et al . 2004).

When alternate hosts are present in close proximity to Cry34/35 corn fields, it is assumed that some percentage of target pests develop on alternate plant hosts. As a result, alternate hosts may be considered unstructured refuge for target pests. Although much re mains to be learned about the role of alternate hosts as refuge, the Storer CRW model (Store r 2003a; Storer 2003b, MRID No. 461239-19) makes the assumption that 0.5% of larvae attacking Cry34/ 35 maize have developed on non-maize hosts.

Technology adoption is expected to be incomplete (a nd will likely never reach 100%). Therefore, not all fields will be planted to Cry34/35 corn, and these will serve as essent ially unstructured refuge. This will reduce the selection pressure and pr ovide susceptible adults that can mate with any re sistant survivors from Cry34/35 corn fields. It is important to note, however, that the trait durability plan proposed by Dow/Pioneer does not rely on this source of unstructured refuge.

Alternative hosts, such as common gr assy weeds, should not be consider ed as refuge for CRW-protected transgenic corn until more information is available regarding those plants’ ability to produce susceptible

CRW individuals. Potential areas of investigation could include:

• Expanded listing of potential alte rnate hosts, including identification of most promising hosts (e.g., most attractive to target pests, most prev alent near Cry34/35 produ ction regions, etc.);

• CRW movement between maize and non-maize plants at different points in target pest life cycle;

• Impact that movement between Cry34/35 maize and non-maize plants could have on resistance/susceptibility of targ et pests to Cry proteins.

Even though 100% adoption of Cry34/35 corn is unlikel y, unstructured refuge consisting of corn fields not planted in Cry34/35 corn is not suffi ciently reliable in all production areas.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

124 Structured refuge

A structured refuge of non-Cry34/35 co rn will function to delay monogenic 3 resistance evolution by (1) reducing population-wide selection pressure for resistance and (2) pr oviding a source of non-selected ( SS genotype) adults to mate with any partially or fully-resistant (genotypes rr or rS ) adults that may survive in the Cry34/35 corn fields. The higher the dose, the more effective the refuge, and thus a smaller refuge is required for a given durability.

Refuge location is important, especially for higher dos e traits. Susceptible inse cts must be produced in sufficiently close proximity so that any resistant survivors in the Bt fields are likely to mate with susceptible insects rather than amongst themselv es. CRW biology potentially favors this inter-mating. Males emerge 3–5 days before females, and they activel y disperse to seek female s. Females, by contrast, tend to mate while they are teneral and cannot fly and, therefore, are in close proximity to their emergence site. Although CRW long-range disp ersal remains poorly understood, a preliminary investigation of WCRW movement in a commodity grain field showed that male and female adults moved 45 m and 13 m per day, respectively (Nowatzki et al . 2003). Another study, which imposed high target pest infestation rates on e xperimental fields, documented betw een-row CRW larval movement of 0.76 m in susceptible corn and betwee n-plant larval movement equal to “three plants down the row,” or approximately 0.54 centimeter (cm)(Hibbard et al . 2003, 2004). This degree of larv al dispersal is density dependent (increasing movement with increasing density) and is associat ed with significant host plant damage (less food results in grea ter movement). These studies support the assumption that CRW larvae and adults are poorly mobile. Because long-distan ce movement by WCRW and NCRW prior to mating is still poorly understood, refuge areas should be close to or within the Cry34/35 corn fields to be most

effective.

Another important factor in refuge management is the use of insecticides in Cry34/35 fields and accompanying refuge to control CRW and non-CRW pe sts. An effective refuge must produce an abundance of susceptible CRW adults that are available to breed with resistant adults from the Cry34/35 field. To encourage technology adoption and refuge deployment, it is im portant for growers to have the option of treating the refuge (protecting it against root worm larval feeding), but this must be balanced against maintaining the refuge as a source of large numbers of su sceptible Cry34/35 insects. Pest management should be carefully considered. Consequent ly, insecticide applicati ons that function to kill refuge-produced adult CRW must also be applied to the Cry34/35 field to nullify any negative impact on the value of the refuge.

Adulticide treatments can be very effective at reduci ng CRW adult numbers if applied at the right time (i.e., appearance of gravid females) and thus mana gement through adult contro l should be discouraged in the refuge

3 Monogenic resistance is assumed given what is presently known about the genetics of resistance.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

125 Use of seed and granular insectic ide treatments to control CRW larvae could be allowed on refuge acres, even if not applied to the Cry34/ 35 field, since these treatments are shown to be non-high dose controls (Meinke et al . 1998). Data have been collected that suppor t the premise that ba nded insecticides and seed treatments will allow considerable survival of rootworms to adult (Sutter et al . 1991; Cormier and Martel 1997). There is ongoing resear ch as to the impact of clothian idin and Cruiser® seed treatments on rootworm fitness, but preliminary data suggest th at there is a minimal impact of CRW exposed to

these seed treatments.

Crop rotation will have a high impact on larval survival. If a refuge is planted on rotated ground, it will encourage adaptation to Cry34/35 unless the Cry34/35 corn is also planted on rotated ground.

A structured refuge is necessary to reduce the population-wide sele ction pressure for resistance and providing a source of non-selected ( SS genotype) adults to mate with any partially or fully resistant (genotypes rr or rS ) adults that may survive in the Cry34/35 co rn fields. Four basic aspects should be examined: refuge size, refuge deployment, refuge management, and grower f easibility. The higher the dose, the smaller the refuge need ed. Because of the uncertainties associated with CRW long-range dispersal, the refuge should be placed adjacent to or within the same field as Cry34/35 corn. Refuge management should consider the role s of chemical insecticides, crop ro tation, and other practices in the context of an integrated pest management program . For a detailed discussion of structured refuge specific for Cry34/35 corn, see th e “Analysis of Dow/Pioneer’s Proposed Cry34/35 Event 59122-7 Corn Durability Plan (2005 Assessment)” section later in this chapter.

f. Computer Simulation Models

Simulation models were used to predict the impact of various factors on th e rate of adaptation to Cry34/35 (event DAS-59122-7) corn by western corn rootworm and northern corn rootworm. In designing the Cry34/35 durability plan, Dow and Pion eer used simulation models. Simulation modeling, leading to the development of the proposed trait durability plan, is summarized and documented in Storer (2003b). The produc t durability plan based on the simulated modeling is discussed in Storer and Lefko (2003). These materials and published CR W resistance management models (Onstad et al . 2001; Storer 2003a) were al so considered.

Overview of the Storer CRW model

The CRW model used in the submission (Storer 2 003b, MRID No. 461239-19) is described in detail in Storer (2003a). A brie f overview is presented in this sec tion. The model is stochastic. Mortality, dispersal, development, and crop development are probabilistic. The model de scribes an agricultural ecosystem in a spatially explicit manne r, with 25-hectare (ha) square fiel ds arranged in a grid of size 10 x 10 or 12 x 12 fields. The fields can be planted in eith er corn or soybean. Corn fields can be planted to

conventional or rootworm-resistant cor n. Also, corn fields can be subdivide d into blocks or strips so that

a rootworm-resistant field can have an internal refuge of conventional corn. Extern al refuge fields can also be simulated, including adjacen t refuge fields or randomly distri buted refuge fields. The model can

simulate continuous corn cu ltivation, such as practiced in the we stern Corn Belt, or crop rotation withBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

126 soybean, as practiced in much of the central and eastern Corn Belt. The model can examine the rate of adaptation of two types of rootwo rm-protected corn planted simu ltaneously as a mosaic in the environment. The following processes were modeled: crop development; larval development, survival, and movement; adult emergence, development, and survival; mating and oviposition; and adult dispersal.

Input Parameters

Dow has determined that Cry34/35 corn expresses a dose that kills at least 99.75% of susceptible WCRW larvae (using the lower end of the range of field studies to estimate dose; see previous

discussion in section II(D)(1)(d) of this Biopesticides Registra tion Action Document (BRAD)). For NCRW, there was greater uncertainty in the inte nsity of density-depend ent mortality, and two

conservative estimates of dose were derived: 95.0% and 99.0% (midpoint of the range of the lower dose estimate and higher dose estimate, respectively; see previous discussion in section II(D)(1)(d) of this BRAD). The expected mortality of the heterozygotes was calculated by assuming that the dose-response curves were parallel and that he terozygotes are 25 times more tole rant of the proteins than are homozygous susceptibles (Storer 2003a). Although it is not possible to calculate a dose-mortality relationship from a limited set of data points, the relationships for both WCRW and NCRW species areconsistent with a default assumption of a slope of 1.0. Based on the dose estimates, assumptions of doseresponse slopes, and the assumption that heterozygot es are 25x resistant, th e functional dominance of resistance for WCRW is estimated as 0.08 and, for NCRW, between 0.34 and 0.15. Previous research submitted by Dow (Storer and Lang 2003, MRID 461239-16) showed that Cry34/35 corn has no effect on adults that feed on it, so the dose ag ainst adults was 0 for all genotypes.

The default model parameters and se nsitivity analysis of the parameters for the runs with wild-type WCRW are summarized in the Dow modeling submission (Storer 2003b, MRID No. 461239-19). Additional discussion is provide d on the following parameters.

1. Larval movement . A default value of 2.5% of larvae moving one row was used. Hibbard et al

. (2003) showed that between 0.75% and 6% of larvae moved across rows. This represents a relatively high-end estimate of the number of larvae that cross rows. Homozygous susceptible ( SS ) larvae that crossed from transgenic to refuge were assumed to suffer 100% mortality after feeding on transgenic plants, while those crossing in reverse were assu med to suffer 90% mortality. Partially adapted heterozygotes ( rS ) were assumed to suffer 50% of the mort ality after crossing from transgenic to refuge plants, while those crossing in reverse were assume to suffer 30% of the mortality. Adapted homozygous resistant larvae did not suffer any mortalit y. Field data showing the level of survival of

SS and rS larvae when crossing between transgenic Cry34/ 35 and refuge rows when collected can be incorporated into the model.

2. Adult in-field movement

. Dow cited to a study by Nowatzki et al . (2003) that described male and female WCRW intrafield dispersal using rubidium as a marker. Based on this study, females were

shown to move about one third the distances of ma les in irrigated corn fields. By the time females reached the same distance as males, they were assumed to be mated. On average, females matedBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

127 within 2 rows of their emergence row. For block refuge scenarios, the destination of dispersing adults is always the other block (i .e., transgenic to refuge or vice-v ersa). For strip refuge scenarios, the probability that those leaving eac h strip end up in the opposite kind of strip (i.e., transgenic strip to refuge strip or vice-versa) is equal to the proportion of the fiel d that is in the opposite kind of strip.

3. Soybean biotype WCRW biology parameters

. The soybean-biotype WCRW lays eggs in crops other than corn. Only the dispersal parameters di ffer from that of the wild-type WCRW (see Table 1 in Storer (2003b)). Investigations on durability of Cry34/35 corn in areas where soybean-adapted populations are prevalent we re initialized with an allele frequency of 0.5.

4. Wild-type NCRW biology parameters . The parameter values for NCRW are assumed to be the same as for wild-type WCRW, except for adult survival, daily fecundit y, adult between-field dispersal, pre-oviposition period, and density depe ndence. The survival probability decreases from 0.99 at the first day of adult emergence, rather than from 0.999 for WCRW. A value of 16 eggs per female per day is used to ensure the population was sustainable. Th e longevity of NCRW is shorter than WCRW, the fecundity is lower, and the equilibrium population is around 6-fold lower. Between-field dispersal is three times higher for NCRW than for WCRW . The pre-oviposition period of 10 to 14 days was the same for both NCRW and WCRW. Less information is available regarding density-dependent mort ality in NCRW than in WCRW, and the WCRW parameter for density-dependent survival was modified.

Model Output

The model output is the predicted rate of resistance evolution expre ssed relative to a benchmark rate. The same benchmark used in Storer (2003a) was used in the model subm ission (Storer 2003b, MRID No. 461239-19). The benchmark rate is for runs with:

 A product that kills 100% of the susceptible rootworm larvae, 90% of larvae heterozygous for resistance, and 0% of resistant larvae;

 A product planted on 100% of the acreag e with the exception of the refuge;

 20% refuge in external fields whos e location is randomized each year;

 Default values for all model parameters as described in Storer (2003a).

The relative rate of adaptation (RRA) is calc ulated by the following equation: RRA = (1/ Y ln q γ / q 0 )/ 0.327; where, the q γ equals the r-allele frequency after Y years and 0.327 is the adaptation rate (yr -1 ) for the baseline run (Storer 2003a). The re lative rate of adaptation is calc ulated when the r-allele frequency first exceeds 0.075, when the rootworm egg populati on in the autumn falls below 20,000 per field, or after 10 years, whichever is soonest. The relative rate of adaptation for the benchmark setting is 1.0. A relative rate of 2.0 implies resistance would evolve twice as fast as th e benchmark, while a relative rateBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

128 of 0.5 implies resistance would evolve twice as sl owly. From the equation de scribed above, a relative rate of adaptation of 1 implies that the modeled r-allele frequency would increase 100-fold (e.g., from 0.001 to 0.1) in approximately 14 model years.

Runs or Scenarios Conducted

Several scenarios were modeled to understand how various refuge options may affect the rate at which WCRW and NCRW adapt to Cry34/35 corn. One set of simulations was designe d to investigate the effects of refuge size, refuge placement, and refu ge strip width on WCRW adaptation. Other model runs were designed to look at the durability obtained with Cry34/35 against WCRW compared with a lower

dose event. Another set of simulatio ns was designed to examine the e ffects of incomplete adoption of the technology, incomplete deployment of the ref uge, and a competitive mosaic with an alternate rootworm plant-incorporated (PIP) expressed at a lo wer dose. Simulations also included, adaptation in a crop-rotation system, where 50% of the WCRW popul ation is rotation resist ant (soybean biotype). Adaptation by NCRW was compared with wild-type and ex tended diapause biotype. Each run was conducted at two refuge sizes (10% and 20%) repeated for a total of five runs at each setting to investigate the effect s of stochasticity.

Analysis of Variance

Output from the runs was subjected to analysis of variance for a completely randomized factorial design to identify the parameters that ha d a significant effect on RRA. All fa ctors were treated as fixed. Means separation was conducted using Tukey’s method to identify significant di fferences among refuge placement options.

Sensitivity Analysis

Conservative estimates of the mean values of biologi cal and genetic parameters in the models were used for the simulations, based on empirical data. The un certainty in these values, as they affect the confidence of product durability, was studied using a Monte Carlo analysis (the WCRW model was run 2,000 times). Twenty-one parameters were analyzed a nd a 10% in-field refuge was used. The relative importance of each of the parameters was analyzed through rank correlation between parameter value and adaptation rate (Storer 2003b, MR ID No. 461239-19). The parameters with the largest effects on the relative rate of adaptation were the functional do minance of the r-allele (explaining 58.5% of the variation) and the dose (explaining 33 .5% of the variation). The next most important parameters were the fitness cost of resistance (3.1% of the variatio n), winter survival (2.9%), and fecundity (1.1%). Winter survival and fecundity were correlated. All ot her parameters accounted for less than 1% of the

variation.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

129 Model Results

Western Corn Rootworm

1. How does dose affect durability? An in-field block refuge design wa s used, and the position of the ref uge within the field was not held constant from year to year. Refuge sizes were 10 % and 20%. The simulations predict that higher doses result in longer durability (i.e., lower relative rate of adaptation), and refuge size was most important at higher doses.

2. How does refuge size affect durability of Cry34/35 corn? With a 20% refuge, the mean RRA of the moderate dose (one that kills 90% of susceptibles with afunctional dominance of 0.5) line was 3.17 (standard error = 0.01), while for the Cry34/35 event DAS59122-7 line, the mean RRA was 0.64 (s tandard error = 0.002). These results indicate that the adaptation would take five times longer for the Cry34/35 corn th an for a moderate dose rootworm corn. With a 10% refuge, the mean RRA for Cry34/35 event DAS-59122-7 was 1.24 (standard error = 0.009) or approximately half the durability as predicted with the 20% refuge. This means that a 4% refuge used with Cry34/35 event DAS-59122-7 corn lines would have the same predicted durability as a 20% refuge

used with a moderate dose event corn line.

3. How does refuge placement affect durability? The effect of different refuge designs, using the conservative dose estimate of 99.75% (WCRW) for the Cry34/35 corn line, was simulated. The following depl oyment strategies were compared: in-field single block with an adjacent field and a random pattern of Cry34/35 and refuge fields. A 20% refuge was used

because this allowed the model to place every Cry34/35 field directly adjacent to one refuge field. The model predicts that the RRA was lowe r (approximately half the relative rate of adaptation) for all three scenarios where the refuge was in the same locati on each year (i.e., a fixed refuge) than for refuge locations randomized each year. As discussed in Storer (2003a), th e fixed refuge allows a higher population of susceptible insects fr om year to year. For randomized locations, the RRA was higher the greater the distance between the re fuge and the Cry34/35 fields, but the differences among these three scenarios was relatively small.

4. How does refuge strip width affect durability? The effect of different strip widths for the ref uge on the durability of Cry34/35 was simulated. These simulations assume that the strips are not in the sa me place each year, so that the distribution of hatching eggs across the field is independent of the position of the refuge a nd the Cry34/35 strips. The survival estimates were based on the assumption that the he terozygous individuals the move between the refuge and the Cry34/35 plants have a high er probability of surviving than either the heterozygous individuals that remain on the Cry34/35 plants or homozygous susceptible individual s that move between the refuge and Cry34/35 plants. The overall conclu sion is that strip width is not lik ely to greatly affect predictions of durability with either a 10% or 20% refuge. That is, there is very little differen ce in the relative rate of adaptation between a two-row strip and a ten-row st rip, although there are so me minor benefits. The model predicts that blocks are more durable than strips. This is because there are more interfacesBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

130 between Cry34/35 and refuge strips , allowing greater larval moveme nt and thus greater survival

differential between SS and rS genotypes, and the adults that disp erse from Cry34/35 strips are more likely to end up in other Cry34/35 strips rather than refuge strips because there are more Cry34/35 strips

relative to the number of refuge strips. Based on these simulations, as strip width increases, the durability will increase up to the point of a single bloc k, but the effect will be small due to the practical range of planting wider strips.

5. How does crop rotation affect durability? Farmers may use Cry34/35 corn (or other CRW-protected Bt corn) to help manage rotation-resistant WCRW (soybean biotype) or NCRW (extended diapau se biotype). The model was used to investigate the possible effects crop rotation (i.e ., corn-soybean rotations vs. corn -corn) may have on durability of Cry34/35 corn under two refuge deploy ment structures: in-fie ld block and external fixed block. Both a 10% and 20% refuge were considered. Based on the model predictions, crop rotation reduced the relative rate of adaptation for both in-field and external refuge options . There was a significant interaction between refuge size and rotation in the in-f ield refuge scenarios. For external refuges, there was a main effect of crop rotation, bu t no interaction with refuge size.

6. How does rotation-resistant WCRW affect durability? In certain parts of the eastern range of the WCRW (i .e., Illinois, Indiana, part of Michigan), WCRW has adapted to crop rotation by laying eggs in fields that are not planted in corn, most notably in soybean fields (“soybean biotype”). The soybean biotype lays its eggs in soybean fields and they hatch when it is

rotated to corn. The model was used to examine th e effect of this behavior on durability of Cry34/35 corn. Three refuge scenarios were run: fixed external refuge (c ontinuous corn), with Cry34/35 corn rotated with soybean; in-field refuge, with all fields in corn-soybean ro tation; and in-field refuge, with all fields planted to continuous corn. All scenar ios were run with both 10 % and 20% refuge size.

Rotation resistance is assumed to be controlled by a single gene. Based on the modeling simulations, rotation resistance did not significantly affect the durability of any refuge scheme. That is, the soybean biotype adapted to Cry34/35 at the same rate as wild-type WCRW. Howe ver, there were differences in effects on the rate of relative adaptation caused by different deployment schemes. The external, fixed refuge served as a refuge for both Cry34/35 susceptib ility and rotation suscepti bility and, therefore,

reduced the incidence of rotation resi stance. These results suggest that Cry34/35 corn has the potential to

decrease the incidence of WCRW ro tation resistance if used in rotati on with soybean while an external refuge is planted to continuous non-rootworm protected corn.

7. How do refuge implementation rates and tec hnology adoption rates affect durability? Adoption of Cry34/35 corn will not be 100% in all locations from the time of commercialization and will vary from area to area. The ra te of technology adoption is affect ed by grower attitudes, market acceptance, perceived need for the technology, and e ffectiveness of the technology. The model was used to investigate the impact of Cry34/35 corn adoption and refuge implementation on Cry34/35 corn durability. A grid of 12 x 12 fields planted to conti nuous corn was divided into 16 farms, each consisting of 3 x 3 fields with each farm designated as either an adopter or non-adopter of Cry34/35 corn. Adopting farms were further designated by those either depl oying the required refuge block within each field or not deploying a refuge at all (i.e., 100% Cry34/35 corn). Adoption rates were between 25% and 100%Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

131 and refuge deployments were between 50% and 1 00%. Based on the modeling simulations, rootworm adaptation rate is least when adoption is lowe st (i.e., 25%). When a doption was 25%, refuge implementation had no effect on the overall r-allele frequencies. Refuge size (whether 10% or 20%) had a significant effect on the relative rate of ad aptation only when there was 100% adoption of the technology. At 50% and 75% levels of technology adoption, refuge implementation had virtually no impact on the relative rate of adaptation.

Modeling simulations indicated that th e spatial patterns of population size and r -allele frequencies are

the inverse of one another. The overall r -allele frequency was dominated by populations in the farms with the refuge and non-adopting farm s. Sufficient dispersa l of the adults from these farms to those without a refuge would prevent the r -allele frequencies from becoming locally elevated.

8. How does a mosaic with an alternative root worm PIP affect durability against WCRW? Dow modeled the effect of a spatia l mosaic of two rootworm PIP produc ts on durability. It is expected that multiple rootworm PIP products will be grown as a spatial mosaic across the landscape. Currently, there are two registered rootworm PIPs, YieldGar d® Rootworm Corn (expresses the Cry3Bb1 protein, Event MON 863, EPA Reg. No. 524-528) and YieldGard® Plus Corn (expresses both the Cry3Bb1 and Cry1Ab proteins, EPA Reg. No. 524-545). The 2002 Scient ific Advisory Panel (SAP) concluded that MON 863 expressed a moderate dose to control r ootworm (U.S. EPA 2002). For the simulations, MON 863 was assumed to kill 90% of the insects susceptib le to it, and the functional dominance was assumed

to be 0.5. It was assumed there is no cross- resistance between Cry3Bb1 and Cry34/35 proteins. Simulations were run with 0, 25, 50, 75, and 100% of the rootworm-protected corn expressing Cry34/35, and corresponding 100, 75, 50, 25, and 0% expressing Cry3B b1. All corn fields had an in-field refuge. EPA has required that both YieldGard® Rootworm Corn and YieldGard® Plus Corn have a 20% structured refuge (U.S. EPA 2007a , 2010d). For Cry34/35 corn, the refuge size was assumed to be 10%.

The modeling simulations indicate th at the relative rate of adapta tion for Cry34/35 corn was lower at comparable market share levels than Cry3Bb1 corn. For example, at equal deployment of 50% market share for Cry3Bb1 corn and 50% market share for Cr y34/35 corn, the model predicts that adaptation to Cry34/35 would take four times longer (RRA = 0.33, standard error = 0.005) than at 100% Cry34/45 market share (RRA = 1.25, standard error = 0.016). There is a more dr amatic reduction in the rate of adaptation to Cry34/35 corn as its market share increas es than the rate of adaptation to Cry3Bb1 corn as

its market share increase s. This is because the Cry34/35 fields produce very few Cry3Bb1-susceptible insects. However, these simulations suggest that in a competitive spatial mosaic, both products would benefit in terms of durability from the presence of the other.

Northern Corn Rootworm

1. How is the durability prediction alte red for Northern Corn Rootworm? The model was run to compare differences in pred ictions of durability between NCRW and WCRW. A range of doses and refuge sizes were run. The output was compared with WCRW. Dose and refuge size significantly affected the relative rate of adaptation. The predicted values for NCRW showed a very similar relationship to dose and refuge as for W CRW, although for any given dose and refuge size, theBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

132 relative rate of adaptation for NCRW is lower than that for WCRW . It is thought that a lower estimate for dose of Cry34/35 corn would be 95% and a hi gher estimate for dose of Cry34/35 corn would be

around 99% for control of NCRW. Based on the simu lations, a 20% refuge had lower comparable relative rate of adaptation values than a 10% refuge at both the lowe r and higher estimates of dose. For example, at the lower dose estimate, the 20% ref uge increased durability by about 50% compared with the 10% refuge, while for the higher dose estimate , the 20% refuge increased durability two-fold compared with the 10% refuge.

2. How do crop rotation and rotation-res istant NCRW affect durability? The model was run to examine how crop rotation a nd rotation-resistant NCRW affected durability of Cry34/35 corn. The model predicted th at crop rotation had the biggest advantage when practiced only on the Cry34/35 corn fields and when the refuge fields we re planted in the same location each year. Just as for WCRW, there are two modes of ac tion operating in the rotated Cry34/ 35 corn fields: the direct effect of Cry34/35 and the effect on the progeny of insects laying their eggs in corn fields that could not survive since they hatched in soybean fields.

Rotation-resistant NCRW spend two winters as eggs in the soil; therefore, they act as a temporal refuge from selection for refuge. The model predicted that for in-field refuge options, rotation resistance approximately doubled the durability of Cry34/35 corn when the corn/soybean rotation was maintained and increased the durability 60% to 80% when corn was grown continuously. On the other hand, if the refuge was external in fixed locations, while Cry 34/35 corn was rotated with soybean, the rotation

resistance accelerated resistance e volution to Cry34/35. This is in c ontrast to the re lative rates of adaptation predicted in WCRW. The NCRW rotation -resistant genotype was onl y exposed to Cry34/35 every second year, while the wild type was expos ed every year. Each year, Cry34/35 corn would kill half of the proportion of rotation-resi stant biotype as compared to the wild type and, thus, the frequency

of the rotation-resistant biotype was increased from its initial va lue of 0.5. The rate of increase is affected by how Cry34/35 corn and the refuge were de ployed. When the refuge was planted in the same location each year (continuous corn ) and the Cry34/35 corn was rota ted with soybean, the rate of

increase was lowest.

Storer Modeling Summary

Using the lower end of the range of field studies to estimate dose, Cry34/35 event DAS-59122-7 corn expresses a dose that kills at leas t 99.75% of susceptible WCRW larvae. The model predicts that the durability is likely to be adequate with a 10% refuge, planted in-field or in an adjacent field. Durability

is extended even further when there is market co mpetition with other transgenic and non-transgenic rootworm management options. Cry34/35 corn has th e potential to decrease the incidence of WCRW rotation resistance if used in rotation with soybean wh ile an external refuge is planted to continuous nonrootworm protected corn. Both the Onstad et al . (2001) model and the Storer model (Storer 2003a; Storer 2003b, MRID No. 461239-19) pr edict that a 10% refuge woul d provide acceptable durability against WCRW resistance.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

133 In contrast, Cry34/35 event DAS-59122- 7 expresses a dose (midpoint of the range of the lower dose estimate and higher dose estimate, respectively) that kills at least 95 to 99 % of susceptible NCRW larvae. The model predicts that the durability of a 20% refuge for wild-type NCRW at the higher dose estimate results in comparable durability of a 10% refuge for wild-type WCRW, and less durability at

the lower dose estimate. Rotation resistance through extended diapause results in more than 50% longer durability.

Onstad and Guse WCRW model

Dow also discussed the WCRW simulation mo del of Onstad and his colleagues (Onstad et al . 2001).

The Onstad et al . (2001) model of WCRW adap tation differs from the Storer model in that it is deterministic and non-spatial (the Storer models are st ochastic and spatial). Two refuge structures were

considered: external blocks and in-fie ld strips. It is assumed that the block refuge is fixed (same location annually for maintaining large, non -selected populations) a nd the strip refuge m oves (planted randomly each year). The difference between fixed and moving refuge drives the model. The most important parameter in the Onstad et al . (2001) model affecting durability of a rootworm-protected PIP was the

functional dominance of the resistance alle le (expressed as “allele expression”).

If the allele was fully recessive, re sistance did not occur in this model within 99 years. If the allele was fully dominant, resistance always occurred within 2–5 years for refuge sizes between 5% and 30%. For incompletely recessive alleles, the time to resi stance depended on both dose and refuge size. For the 95%, 90%, and 80% doses, the functional dominance was calculated to be 0.5. Th e model predicted that

the time to resistance in these runs was 3 to 9 mo del years with a 20% refuge, depending on whether the refuge was fixed (block) or moved fr om year to year (row strips). Fo r a dose of 99.9% (i.e., a “practical high dose”), the functional dominance was calculated to be 0.009. In this instance, the time to resistance with the moving 20% refuge (strip s) was 29 model years and with a moving 10% refuge was 22 model years. At the theoretical high dose, 100%, the functional dominance wa s 0, and resistance did not evolve within 99 model years with a 20% moving refuge and evolved in 52 model years with a 5% moving refuge. For a fixed 5% refuge, resistance did not e volve within 99 model years at 99.9% or 100% dose.

Using a low-end estimate, Cry34/35 event DAS-59122- 7 corn expresses a dose of at least 99.75% against WCRW, which is similar to Onstad et al .’s (2001) designation of a “pra ctical high dose.” At this dose, the model predicts that a 5% refuge would give more than four times th e durability of a 80%, 90%, or 95% dose PIP with a 20% refuge. A 10% refuge would give more than 20 model years of durability if the refuge is moved around each year (strips), and greater than 99 model years of durability if the refuge is fixed (blocks).

BPPD’s Analysis of Proposed Computer Models

BPPD agrees with Dow and Pioneer’s assessment of CRW adaptation using the Storer simulation models (Storer 2003a; Storer 2003b, MRID No. 461239-19) and Onstad et al .’s (2001) simulation model. Using the lower end of the range of fiel d studies to estimate dos e, Cry34/35 event DAS-59122-7 corn expresses a dose that kills at least 99.75% of susceptible WCRW larvae. The Storer simulationBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

134 model (Storer 2003b, MRID No. 461239-19) predicts that the durability is likely to be adequate with a 10% refuge, planted in-field or in an adjacent field. Durability is extended even further when there is market competition with other tr ansgenic and non-transgenic rootwo rm management options. Cry34/35 corn has the potential to decrease the incidence of WCRW rotation resistance if used in rotation with soybean while an external refuge is planted to continuous non-rootworm pr otected corn. Both the

Onstad et al . (2001) model and the Storer models (S torer 2003a; Storer 2003b, MRID No. 461239-19) predict that a 10% refuge would provide acc eptable durability agai nst WCRW resistance.

In contrast, Cry34/35 event DAS-59122- 7 expresses a dose (midpoint of the range of the lower dose estimate and higher dose estimate, respectively) that kills at least 95% to 99% of susceptible NCRW larvae. The Storer model (Storer 2003b, MRID No. 461239-19) predicts that th e durability of a 20% refuge for wild-type NCRW at the higher dose estimate results in comparable durability of a 10% refuge for wild-type WCRW, and less dur ability at the lower dose estim ate. Rotation resistance through extended diapause results in more than 50% longer durability.

g. Technology Adoption

Product durability is affected by market penetr ation. Modeling efforts (Storer 2003b, MRID No. 461239-19) make the conservative assu mption of complete adoption (i.e., all corn acres are planted in Cry34Ab1/35Ab1 corn) because the app licants do not wish the durability of the product to depend upon the presence of competing products (Storer a nd Lefko 2003, MRID No. 461239-18). The applicants stated, however, that actual adopt ion is likely to be significantly less than 100% and governed by multiple parameters (availability of alternate contro ls, economic and market factors, etc.)(Storer and Lefko 2003, MRID No. 461239-18). Implementation of a va riety of controls, the most likely scenario, discourages over-reliance on a sing le control method and, consequent ly, helps to delay resistance development to control options (Gray 2001; Ostlie 2001; Carrière et al . 2004).

The assumption of 100% adoption, while not likely, is a reasonable one on which to base a conservative IRM plan to manage the Cr y34Ab1/Cry35Ab1 corn durability.

2. Analysis of Dow/Pioneer’s Proposed Cry34/ 35 Event 59122-7 Corn Durability Plan (2005 Assessment)

Dow and Pioneer proposed a Cry34/35 event 59122-7 corn durability plan that has the following elements: (1) structured ref uge, (2) resistance monitoring, (3) remedial action plan, and (4) compliance

and education. Simulation models were used to assi st in evaluating and comp aring structured refuge options (discussed previously). Us ing the model predictions, Dow and Pioneer were able to propose specific refuge guidelines. Given the uncertainties in understanding the dose, genetics and mechanism(s) of resistance, functional dominance, pest biology and ecology, and level of adoption of the technology, Dow and Pioneer have proposed a co nservative insect resistance ma nagement (durability) plan. The proposed plan is “acceptable,” with one exception—that of the size of the in-field strips (see discussion below). Additional data are needed to support independent treatment of the refuge for other pests, other

than corn rootworm when corn r ootworm may be present, but not targeted and its impact on cornBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

135 rootworm resistance management. Additional research should be conducted to further evaluate the sustainability of the proposed durab ility plan and confirm the assu mptions made in the simulation models. Each aspect of Dow and Pioneer’s prop osed durability plan will be discussed below.

a. Structured Refuge

The guidelines for structured refuge address the following: refuge size, refuge deployment (proximity of the refuge to the Cry34/35 corn fields), and refuge management (acceptable chemical management of target pests in the transgenic fi elds and refuge and agronomic mana gement). In developing the refuge guidelines, Dow and Pioneer stated that a goal was to ensure that the structured refuge would remain productive for the grower, while serving as an eff ective reservoir of susceptible target pests. The applicants’ plan is designed around NCRW for which there is gr eater uncertainty surrounding the estimation of the Cry34/35 dose and, thus, the extent of functional dominance of resistance alleles. Based on the modeling simulations by Storer (2003b), this plan woul d then be more conservative for

WCRW. Since WCRW is re lated to MCRW (Clark et al . 2001), the plan should also be applicable (and conservative) for MCRW. Additionally, the simulati ons by Storer (2003b) i ndicated that rotation resistance in the WCRW soybean biotype did not affect durability of Cry34/35 corn, while rotation resistance in NCRW populations (extended diapause) increased the durability of Cry34/35 corn. These

simulations indicated the trait dura bility plan could reverse the spread of the soybean biotype WCRW.

Several assumptions make this plan conservativ e. The Monte Carlo simulations (see the earlier

simulation models section ) indicate that the choice of default para meter settings lie within the 5-percent worse-case settings. The assumptions of the genetics of resistance are conservative because it is unlikely that there will be incomplete recessiveness of the resi stance allele, rather the more realistic expectation is for complete or near-complete functional recessiveness. Finally, the trait durabil ity plan is based on the unrealistic expectation that 100% of the corn in a given area will be planted to Cry34/35 corn. Storer’s simulations (Storer 2003b, MR ID No. 461239-19) indicate that th e plan would be effective at managing region-wide resistance ev olution if only 75% of farms co rrectly implement the refuge.

Dow and Pioneer’s Proposed Structured Refuge Guidelines

1. Refuge size . The use of Cry34/35 corn from event DAS-59122-7 would require an accompanying 20% refuge. The refuge size is governed by the great er uncertainties in the dose determination of NCRW to Cry34/35. Based on the modeling simula tions for WCRW and NC RW (Storer 2003b, MRID No. 461239-19), the refuge size is considered to be conservative for WCRW (and MCRW).

2. Refuge location

. Given the uncertainties regarding rootwo rm long-range dispersal, the rootworm refuge would be required to be planted within or adjacent (e.g., across the road) to the Cry34/35 corn

field.

3. Refuge management options . The rootworm refuge must be manage d in such a way th at there is little or no yield loss to rootworms, but it is sufficiently productive of susceptible rootworm adults. Dow and Pioneer propose the following refuge management guidelines.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

136  The in-field refuge options may be planted as a single block or as a series of strips measuring at least two crop rows wide. (BPPD does not agre e with this portion of the proposed plan as discussed later; at least four crop rows wide should be required.) M odeling (Storer 2003b, MRID No. 461239-19) showed that the strip width had a very small effect on durability. Single-row strips could be too narrow a nd allow too much larval movement across rows to sufficiently

maintain low functional recessiveness.

 Seed mixtures of Cry34/35 and refuge corn are not permitted. Larval movement within rows would compromise the value of the effective dose of the Cry34/35 corn and allow enhanced heterozygote survival.

 If the refuge is planted on ro tated ground, then Cry34/35 corn mu st also be planted on rotated ground. Refuge on rotated ground is likely to produce few individuals. Therefore, adult production would need to be mirrored in the Cry34/35 corn field to nullify the effect on

resistance evolution.

 If the refuge is planted in continuous corn, the Cry34/35 fi eld may be planted on either continuous or rotated land (option encouraged where WCRW rotation-resistant biotype may be present). The Storer simulations (Storer 2003b, MRID No. 461239-19) predicted that planting the refuge on continuous corn ground and rotating the Cry34/35 corn with a second crop can lead to a reduction in the prevalence of rotation-resistant WCRW.

 Banded application of soil insecticide is permitted in the refuge. As discussed previously, there

are several studies that have shown that this control option permits very high survival of rootworm and, therefore, will not affect refuge performance but will protect the yield in the refuge making this option attract ive to growers. Simulations by Storer (2003a and 2003b) were based on the assumption that the refuge will be managed by soil-insecticides in banded applications when egg popul ations warrant protection.

 Seed treatment is permitted in the refuge, either at a high rate for rootworm protection or at a low rate for controlling secondary soil pests. As di scussed previously, insecticide seed treatments have been shown to have little impact on th e number or timing of adult emergence and, therefore, are acceptable for use in a Cry34/35 corn structured refuge.

 If aerial insecticides are applied to the refuge for control of CRW adults, the same treatment must also be applied to Cry34/35 co rn. It is important that aerial applications be properly timed and that egg population reductions are not reduce d more in the refuge than the Cry34/35 corn fields. Application of adult-ta rgeted aerial insecticides, app lied at the same time to Cry34/35 corn areas and the refuge, will nullify any negative effects on the value of the refuge.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

137  The refuge and Cry34/35 corn may be treated with independent insecticide applications that comply with local integrated pest management guidelines, to control pests other than adult rootworms. Foliar sprays targeted at other pests, other than rootworm adults, will have a much lower effect on the rootworm egg populations. The tim ing of the applications is also important to avoid any significant impact on future adult emer gence. (BPPD does not agree with this portion of the proposed plan at this time. Additional data are needed to address independent treatment of the refuge for other pests (not corn rootworm ) and its impact on corn rootworm resistance management.)

 The rootworm refuge can be planted to any corn hybrid that does not express PIPs for rootworm control (e.g., lepidopteran-protected Bt corn, herbicide-tolerant co rn, or conventional corn).

 The refuge and Cry34/35 corn should be sown on the same date, or with the shortest window possible between planting dates, to ensure that corn root developm ent is similar among varieties.

 Based on simulations by Storer (2003a and 2003b) and Onstad et al . (2001), it may be best for growers to plant the rootworm refuge in the same location each year, as it allows the rootworm population to remain high and the durability of the trait is extended. This refuge can be protected with banded application of soil insecticides or with seed treatments. This option may be preferable to growers who wish to only think of their refuge design once and for growers who grow continuous corn. However, for those grow ers who need to employ crop rotation, a fixed refuge would be impractical.

BPPD’s Analysis of Dow and Pioneer’s Proposed Structured Refuge Guidelines

Given the uncertainties in understanding the dose, ge netics and mechanism(s) of resistance, functional dominance, pest biology and ecol ogy, and level of adoption of the technology, Dow and Pioneer have proposed a conservative insect resistance management (trait durability) plan for Cry34/35 corn. The trait durability plan, while based on the best available sc ientific information regarding rootworm biology and ecology, has also strongly considered grower feas ibility and practicality. If the grower does not implement the structured refuge and manage it correc tly, then the value of the trait durability plan is minimal. The refuge guidelines are designed arou nd NCRW to which there is greater uncertainty surrounding the estimation of the Cry34/35 dose and, t hus, the extent of f unctional dominance of resistance alleles. Based on the modeling simulati ons by Storer (2003a and 2003b) , this plan would be

more conservative for WCRW. Since WCRW is related to MCRW, the plan should also be applicable to and conservative for MCRW. Based on BPPD’s analys is, the proposed plan is acceptable with two modifications: one to the proposed size of the in-field strips and the ot her to the agronomic treatment of the refuge.

1) BPPD disagrees with Dow a nd Pioneer’s recommendation that the in-field strip width be ≥ 2 row strips.

Recent larval movement data, published by Hibbard et al . (2003), showed that between 0.75% and 6%Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

138 of larvae moved across rows. This represents a relati vely high-end estimate of the number of larvae that

could cross rows. This means that much narrower in-fie ld strips should be suffici ent to provide adequate protection from sublethal selecti on caused by CRW larval movement across rows and maintain low functional recessiveness. Any increase in sublethal selection would be o ffset by a greater probability that potentially resistant adults emerging from the Bt corn rows would mate with susceptible adults from the refuge row. Simulations by Storer (2003b) incorporated the Hibbard et al . (2003) larval movement data to compare how strip width can affect the durability. These simulations predicted that narrower in-field strips, between 2 and 10 rows, did not affect trait dur ability. Single-row strips could be too narrow and

allow too much larval movement across rows to su fficiently maintain low functional recessiveness. While the Storer simulations (S torer 2003b, MRID No. 461239-19) indica te that trait durability is virtually unaffected by strip width, in-field strips of ≥ 4 rows would provide some marginal, additional protection (see Figure 4 of MRID No. 461239-19) and also provide the advantage of being more compatible with the current in-field strip width requirement, ≥ 4 row strips ( ≥ 6 row strips preferred) for lepidopteran-protected Bt corn hybrids. In-field strips of ≥ 4 rows would also be pr actical and flexible for the grower, just as a ≥ 2 row strips. Because Cry34/35 will likely be stacked with Cry1F, for example, a

recommendation of ≥ 4 row strips will provide the grower a mo re easily understandable and consistent message regarding the width of in-field strips and reduce confusion associated with in-field row strips for lepidopteran-protected Bt corn hybrids, rootworm-protected Bt corn hybrids, and those Bt corn hybrids that have both traits. Overall, BPPD believes that a requirement of ≥ 4 row in-field strips will simplify refuge deployment and potentially increase compliance with refuge requirements.

Note: Herculex® RW (Cry34/35 corn) was registered with an in-field strip widt h refuge requirement of

at least 4 rows.

2) BPPD disagrees with Dow and Pioneer’s recommendation for independent treatment of the refuge for other pests (not corn rootworm).

Properly timed aerial sprays are very effective at reducing the corn r ootworm egg population in the field, and it is important that the refuge productivity is not reduced more than the Cry34/35Ab1 corn field productivity. Sprays targeted at a dults in the current year’s refuge may significantly reduce the number of adults available for mating with Cry34/35-resistant survivors in Cry34/35 corn fields. Sprays targeted

at adults in the area to be used as refuge the next year may signifi cantly reduce the number of adults emerging from that area the following year, reducing refuge effectiveness. BPPD agrees with Dow and

Pioneer’s conclusion that insecticide applications that function to ki ll refuge-produced adult CRW must also be applied to the Cry34/35 corn field to nullify any negative impact on the value of the refuge.

Foliar sprays targeted at pests othe r than rootworm adults are thought to have a much lower effect on the rootworm egg populations. If the sprays are applied be fore peak adult emergence, then residual activity may be too low to have a significant impact on future adult emergence. Similarly, if the sprays are applied after peak egg-laying, they are likely to have a reduced impact on egg num bers. Therefore, if the treatment windows for other pests do not overlap with the critical period in which corn rootworms would be treated, growers could be permitted to use aeria l sprays on any fields (refuge or not) if they are needed for management of other pests besides adult corn rootworm. Dow and Pioneer proposedBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

139 allowing independent treatment of the refuge and Bt fields if pests other than corn rootworm are targeted. While this would prev ent unnecessary sp raying of the Bt corn fields, additional data are needed

to determine if the treatment windows for othe r insect pests, e.g., European corn borer (2 nd generation), Southwestern corn borer, spider mites and corn root worm overlap and what impact treatment for other pests would have on the effectivenes s of the corn rootworm refuge. At this time, pests other than adult corn rootworms can only be treated with CRW-labele d insecticide on the refuge acres without treating the Cry34/35 acres if treatment occurs when corn rootworms are not present. Pests on the Cry34/35

acres can be treated as needed w ithout having to treat the refuge.

BPPD agrees with Dow and Pioneer that the use of seed and granular insecticide treatments to control CRW larvae should be allowed on refuge acres, even if not applied to the Cry34/35 corn field, since these treatments are shown to be non-high dose controls (Meinke et al . 1998). Data have been collected that support the premise that banded insecticides and seed treatments w ill allow considerable survival of rootworms to adult (Sutter et al . 1991; Cormier and Martel 1997) . There is ongoing research that suggests that clothianidin and Crui ser® seed treatments have a mi nimal impact on CRW fitness.

Crop rotation will have a high impact on larval surviv al, e.g., corn-soybean rotations. If a refuge is planted on rotated ground, it will encourage adaptati on to Cry34/35 unless the Cry34/35 corn is also planted on rotated ground. This is because susceptib le corn rootworm production is lower on rotated ground (i.e., soybean is not a host of corn rootworms) . BPPD agrees with Dow and Pioneer that if the refuge is planted on rotated ground, then Cry34/35 corn must also be planted on rotated ground.

Based on simulation models (Storer 2003a; Onstad et al . 2001), it may be best for growers to plant the rootworm refuge in the same location each year, as it allows the rootworm population to remain high and the durability of the rootworm-protection trait to be extended. This refuge can be protected with banded application of soil insecticides or with seed treatments. For those growers who need to employ crop rotation, a fixed refuge would be impractical.

Based on BPPD’s analysis, a 20% structured non-lepidopteran Bt corn refuge planted adjacent to, or as

≥ 4 row strips within the Cry34/ 35 corn field (rather than ≥ 2 row strips), is sufficiently conservative to mitigate CRW resistance to the Cry34Ab1 and Cry35Ab1 proteins expressed in event DAS-59122-7 corn hybrids. BPPD agrees with all of Dow and Pioneer’s proposed agronomic management recommendations except for one. Additional data are needed to address independent treatment of the refuge for other pests (not corn rootworm) and its impact on corn rootworm resistance management. Additional research on corn rootworm pest biology and ecology, genetics and m echanisms of resistance, functional dominance, fitness costs, cross-resistan ce potential, dose, and mode of action will also be useful to evaluate the proposed durability plan and conf irm the assumptions made in the simulation

models.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

140 Note: Herculex® RW (Cry34/35 corn) was registered w ith the following insecticid e use restrictions to address the concerns regarding re fuge treatment discussed above:

 If aerial insecticides are applied to the refuge for control of CRW adults, the same treatment must also be applied in the same time-frame to Cry34/35 corn.

 Pests other than adult corn rootworms can only be treated with CRW-labeled insecticide on the refuge acres without treating the Cry34/35 acres if treatment occu rs when adult corn rootworms are not present. Pests on the Cry34/ 35 acres can be treated as need ed without having to treat the refuge.

 Application of soil insecticide is permitted in the refuge.

 Seed treatment is permitted in the refuge, either for rootworm protection or for controlling secondary soil pests.

b. Resistance Monitoring

The need for proactive resistance detection and monitoring is critical to the survival of Bt technology. Consequently, the Agency mandates that a resistan ce monitoring plan must be implemented for all registered Bt corn products. Resistance can evolve regiona lly or as a local in crease in resistance ( r -) allele frequency. The resistance monitoring pl an designed for Cry34/35, an adaptation of the Agricultural Biotechnology Stewardship Techni cal Committee (ABSTC) program developed for lepidopteran-protected corn, will atte mpt to detect either local or re gional resistance early enough to

initiate effective remedial acti on (see below). Resistance monitoring will be implemented through a twopronged approach, field reports of unexpected damage and population testing and sampling.

Field Reports of Unexpected Damage

Field monitoring is intended to detect localized resi stance. The applicants are working to accumulate a data set of Cry34/35 field efficacy under a wide range of pest pressures, soil types, and environmental conditions. This information will be used to develop efficacy guidelines that applicants will use to determine whether the level of rootworm damage in th eir fields is considered “normal” or outside the range of normal.

Once field guidelines have been set, reports of une xpected CRW damage will be evaluated to determine if crop damage is due to failure of Cry34/35 or to so me alternative factor. Specifically, the applicant will confirm that plants under question were expr essing Cry34/35Ab1 proteins, that the Cry34Ab1 and Cry35Ab1 proteins were expre ssed at expected levels in corn plan t roots, and that damage was caused by target CRW pests. If, after having completed th e confirmatory procedures described above, the damage report still points to Cry34/35 failure, the cas e will be considered “suspected resistance” and remedial action will be implemented. Eggs or adults will also be collected for the “Population Testing and Sampling” program.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

141 Population Testing and Sampling

Population sampling and testi ng will be used to identif y area-wide increases in r -allele frequency before widespread field failure occurs. R -allele detection programs will employ dose-response and discriminating dose bioassays to detect non-recessive and fully recessive r -alleles in the homozygous state (ABSTC 2003).

CRW species are challenging to rear in the laboratory. Of the identified target pest species, scientists have had the most success rearing WCRW and have had little success with NCRW. Consequently, monitoring through population sampling will focus on WCRW and include NCRW populations when

available.

A bioassay that “accurately and reliably” determin es dose-response relationships for WCRW is being used by a third-party laboratory to establish baseline sensitivity. Efforts to establish baseline sensitivity will continue through the early year s of commercial Cry34/35 corn deployment, before significant selection pressure is placed on rootworm populations . Dow and Pioneer are also working to develop a “high-throughput diagnostic screen,” which uses Cry34/35 seedlings to determine discriminating dose. The applicants state that this screen will be able to identify potential field-resistant insects, may be able to detect heterozygotes for r -alleles, and will provide estimates of phenotype and genotype frequencies.

European and southwestern corn borer are multivoltin e insects that produce one to three generations per year. Monitoring programs developed for these pest species evaluate every s econd or third generation for resistance. In contrast, rootworm species ar e univoltine, meaning that they produce a single generation of insects per year. Sinc e corn borer is evaluated every s econd or third generation (two or three generations produced per year), the applicants argue that a sim ilar level of monitoring intensity would be achieved by testing four or five CRW populations per year.

Rootworm populations will be collected from targeted areas where resistance is most likely to develop. Market penetration of Cry34/35 is co nsidered the most important fact or in determining the risk for resistance. Consequently, county sales data will be used to identify areas with the greatest Cry34/35 adoption rates.

BPPD’s Analysis of the Propos ed Resistance Monitoring Plan

BPPD agrees with Dow and Pioneer’s approach to resistance monitoring. De tecting shifts in the frequency of resistance genes (i .e., susceptibility changes) thr ough resistance monitoring can be an aggressive method for detecting the onset of resistance prio r to widespread crop failure. As such, the

utilization of sensitive and effective resistance mon itoring techniques is critical to the success of an IRM plan.

Dow and Pioneer state that a thir d-party laboratory is using a bioa ssay that “accurately and reliably” determines dose-response relationships for WCRW to establish baseline sensitivity. While BPPD agreesBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

142 that a sensitive and reliable bioassa y should be developed, these effort s are currently under development. Once this work is completed, the applicants need to de velop guidelines as to what level of root damage will be expected under various conditions, and what level of rootworm cont rol is normally achieved. Applicants will investigate grower reports of reduced product performance to determine whether damage is unexpected. However, without guidelines as to what is “acceptable” rootworm damage, then it will not be possible to determin e what the “unexpected” rootworm da mage is. Once these guidelines are established, it will be possible to define what is “suspected resistance” as described under the “Remedial Action Plans” to mitigate the spread of putative resistant populations. Because of the importance of these guidelines, it sh ould be required that the applic ants develop interim rootworm damage guidelines by 2008 and final guidelines by 2010 and submit these to the Agency for review.

Dow and Pioneer also mention that they are working to develop a “high-throughput diagnostic screen,” which uses Cry34/35 seedlings to dete rmine discriminating dose. The premise is that this screen will be able to identify potential field-resistant inse cts and provide estimates of phenotype and genotype frequencies. The applicants state that the screen should also be able to detect heterozygotes for r -alleles. While this type of screen would be highly valuab le, the submission does not provide enough information about this diagnostic test. It is requ ired that the applicants provide B PPD with a detailed explanation and validation (steps for) of the “high-throughput diagnostic screen” if it is to be considered an acceptable addition to present monitoring strategies.

Finally, it is recommended that Cry34/35 corn be given the following resistance monitoring requirements:

1. The applicants should monitor for resistance and/ or trends in increase d tolerance for corn rootworm. Sampling should be focused in those ar eas in which there is the highest risk of resistance development. The applicants should submit to EPA an appropriate sampling protocol as part of its monitoring plan.

2. The applicants should provide EPA a description of its resistance monitoring plan by January 31, 2006. The description would include the following: sampling (number of locations and samples per locations), sampling methodology, bioassa y methodology, standardization procedures (including quality assurance/quality control provis ions), detection technique and sensitivity, and the statistical analysis of the probability of de tecting resistance. A final resistance monitoring plan is required by January 31, 2008.

3. The applicants should develop an appropriate di scriminating or diagnostic dose assay by January 31, 2008.

4. The applicants should follow-up on grower, extens ion specialist, or c onsultant reports of unexpected damage or control failures for corn rootworm.

5. The applicants should provide EPA with an annual resistance monitoring report.

Note: A discussion of the ongoing resistance monito ring program for Cry34/35, including the conditions listed above and results from the annual testing, is contained in the “Conditionally Required Data for Cry34/35 Corn (2010 Update)” section of this chapter.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

143 c. Remedial Action

The remedial action plan is designed as a tier ed approach for mitigating WCRW, NCRW, and MCRW resistance development to the Cry34/35Ab1 proteins . The following program summary describes, in order of events, the steps that will be taken to imple ment a remedial action plan if resistance to target pests is confirmed.

1. Definition of Suspected Resistance : Resistance will be suspected if investigations of unexpected damage reports show that:

a. Implicated corn plant roots were expressing Cry34/35Ab1 proteins at the expected levels;

b. Alternative causes of damage or lodging, such as non-target pe st insect species, weather, physical damage, larval movement from alternat e hosts, planting errors , and other reasonable

causes for the observations , have been ruled out;

c. The level of damage exceeds gui delines for expected damage.

If resistance is “suspected,” the ap plicants will instruct affected growers to use alternate pest control

measures such as adulticide treatme nt, crop rotation the following year, or soil or seed insecticides the following year. These measures are intended to reduce the possibility of potenti ally resistant insects contributing to the following year’s pest population.

2. Confirmation of Resistance : Resistance will be confirmed if a ll of the following criteria are met by progeny from the target pest species sampled from the area of “suspected resistance”:

a. The proportion of larvae that can feed and surv ive on Cry34/35 roots from neonate to adult is significantly higher than th e baseline proportion (curren tly being established);

b. The median lethal concentration (LC 50 ) of the test population exceed s the upper limit of the 95%

confidence interval for the LC 50 of a standard unselected popul ation and/or survival in the diagnostic assay is significantly greater than that of a standard unselected population, as established by the ongoing base line monitoring program;

c. The ability to survive is heritable;

d. Cry34/35 plant assays determine that damage caused by surviving insects would exceed economic thresholds;

e. The identified frequency of field resistance could lead to widespr ead product failure if subsequent collections in the affected field area(s) demonstrated similar bioassay results.

3. Response to Confirmed Resistance : When resistance is “confirmed,” the following steps will be

taken:

a. EPA will receive notification within 30 days of confirming resistance;

b. Affected customers and extension agents will be notified about confirmed resistance;Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

144 c. Affected customers and extension agents will be encouraged to employ alternative CRW control

measures; d. Sale and distribution of Cry34/35 corn in the affected area will cease immediately;

e. A long-term resistance management action plan will be devised according to the characteristics of the resistance event and local agronomic needs.

BPPD’s Analysis of Pro posed Remedial Action Plan

BPPD agrees with the general framework for the Re medial Action Plan; however, because the “baseline sensitivity” has not been calibrated, this plan cannot be implemented. The submission states that mitigation measures will be initiated when unexp ected levels of CRW damage occur. However,

confirmation of insect resistance cannot be done until levels of baseline sensitiv ity are determined (see previous discussion on “Resistance Monitoring”). C onsequently, it should be required that baseline sensitivity be established within two years of pro duct commercialization. Unexpected damage guidelines and confirmation of target pest resistance (using baseli ne sensitivity information) will be used to initiate a remedial action plan when needed.

Note: Baseline susceptibility data through the 2008 growing season are discussed in the “Conditionally Required Data for Cry34/35 Corn (2010 Update)” section of this chapter. Howeve r, a credible diagnostic assay may need to be developed fo r resistance confirmation (e.g., Subl ethal Seedling Assay; Nowatzki

et al . 2008) so that a func tional remedial action plan can be enacted.

d. Compliance

Compliance programs are important in that they enc ourage growers to comply with IRM requirements, while providing mechanisms by which registrants can be held accountable fo r noncompliant growers. The compliance program presented in this s ubmission mirrors those developed for existing Bt corn registrations. Program components include the follo wing: grower and sales force education/training programs that include workshops and educati onal pamphlets/brochures ; contractual technology agreements that require grower adherence to IRM requirements; public relati on activities, including media interviews, press releases, and conferences; a nd an annual affirmation program, which includes use of seed bag tags and tag language.

Grower Education

The applicants state that “grower education is th e single most important element of any strategy for promoting compliance with the IRM requirements.” Th e grower education prog ram described in this submission is similar to that developed for other regi stered insect-protected corn products, so growers are familiar with the proposed concepts.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

145 BPPD’s Analysis of Grower Educat ion and Compliance Assurance Program

Grower education and compliance are critical to the success of the trait durability plan. The applicants’ grower education plans are acceptable. However, the applicants have not presented any additional information as to their compliance assurance progra m analogous to what has previously been required

for all Bt corn PIP products. It is recommended that a compliance assurance program be required and submitted to the Agency that is in concert with existing Bt corn PIP products. Computer simulations by Storer (2003b) have shown that the level of refuge deployment needed fo r the trait durability plan to be fully effective depends on the level of technolog y adoption (see previous discussion under “Computer Simulation Models”). The proposed trait durability pl an is based on 100% adopti on, a very conservative assumption. However, as a worst-case scenario, re fuge implementation by 75% of farmers would be needed for the proposed plan to be fully effective. At lower levels of adopti on such as 75%, only refuge implementation by 50% of farmers would be effectiv e in managing region-wide resistance. To ensure refuge implementation is high, a complianc e assurance program should be required.

Note: A discussion of the ongoing compliance progr am for Cry34/35 corn is contained in the “Conditionally Required Data for Cry34/35 Corn (2010 Update)” section of this chapter.

e. Annual Reports

It is required that the applicants provide BPPD with the follo wing annual reports: resistance monitoring, compliance assurance program, and sales data at the st ate level (county level data to be available if requested). Data from the resist ance monitoring and compliance repo rts are discussed in the next

section.

3. Conditionally Required Data for Cry34/35 Corn (2010 Update)

As part of the terms and conditions of the Herculex® RW (Cry34/35 corn) registrations, the registrants were required to submit programs for resistance m onitoring and refuge compliance. In addition, the registrants were required to submit annual repor ts summarizing the data collected under these requirements. The following elements of resistance monitoring and compliance were required:

 Resistance Monitoring

o Description of the steps to be taken to esta blish corn rootworm baseline sensitivity and damage guidelines (due January 31, 2006);

o Submission of plan . Description of the program incl uding baseline sensitivity, sampling (number of locations and samples per locations), sampling methodology, bioassay methodology, standardization procedures, det ection technique, sensitivity, and the statistical analysis of the probability of detec ting resistance, and an interim description of rootworm damage guidelines (due January 31, 2008);

o Submission of rootworm damage guidelines (f or unexpected pest da mage) (due January 31, 2010);Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

146 o Development of diagnostic dose assay/high throughput screen (due January 31, 2008);

o Annual report of th e insect resistance monitoring program . Results of monitoring and investigations of damage reports (due August 31 st each year, beginning in 2008).

 Compliance

o Written description of Compliance Assurance Program (due January 31, 2006);

o Grower agreements . Proposed system to assure grow ers sign grower agreements and affirmation system to assure annual affirma tion by growers of their IRM obligations (due January 31, 2006);

o Annual report on Compliance Assurance Program (CAP) activities and results . Thirdparty grower survey, on-farm visitation pr ogram, phased-compliance report, tips and complaints, and grower education programs (due January 31 st each year, beginning in 2007);

o Grower education program for Cry34/35 cor n. Subsequent changes to the grower education program must be in cluded in the annual compliance assurance program report (due January 31, 2007).

Dow and Pioneer have submitted all of the required reports as mandate d under the terms of registration. A discussion of the reports and data for the resistance monitoring and compliance programs for Cry34/35 corn follows in the next sections.

a. Resistance Monitoring

Monitoring Plan for Cry34/35 Corn (2008)

A preliminary resistance monitoring program was submitted to EPA in 2006 (reviewed in U.S. EPA 2006a). This submission described work to establis h baseline susceptibility data for Cry34/35, develop a diagnostic bioassay (sublethal s eedling assay), and develop unexpect ed pest damage guidelines.

Dow provided a revised monitoring plan in 2008 (MRID No. 473342-01), which is based on the

framework of the ABSTC document tit led “Updated Monitoring Plan for Bt Corn.” Specifically, Dow addressed the following elements of monitoring: initial baseline sens itivity, sampling plan, bioassay methodologies and analyses, probability of detecting resistance, and an interim description of process for developing rootworm damage guidelines.

Monitoring for CRW resistance to the Bt crystal protein Cry34/35A b1 relies on two approaches: population screening (random sampling, F 1 screen with diet bioassays, and possible Sublethal Seedling Assay) and field observations of pr oduct performance by growers, consul tants, and extension agents. To understand the biological relevance of any future va riation in target pest response to Cry34/35Ab1 and whether changes in susceptibility are due to selection to the prot ein, the population variation in sensitivity needs to be understood.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

147 Sampling for CRW resistance will focus on WCRW (one of three Diabrotica target pests in the US) because this species represents the worst-case scenario (wide geographic distribution, greater abundance, and hence more like ly to evolve resistance to Bt protein first) and is coordinated by the IRM

technical subcommittee of ABSTC. Collection efforts will focus on regions where WCRW pest pressure is greatest, and CRW-protected Bt corn adoption is gr eatest. Such areas of focus are Illinois, Iowa, and Nebraska; however, Indiana, Missouri, Wisconsin, Minnesota, and Ka nsas are also potential candidates for sampling collections. ABSTC’s core sa mpling area is divided into three regions: Region 1

consists of eastern Illinois and Indi ana (rotation-resistant variant); Region 2 consists of western Illinois, Iowa, and Missouri (wild-type variant); and Re gion 3 consists of Nebraska and Kansas (organophosphate-resistant variant). Sampling locatio ns will change on a yearly basis, and sampling intensity will reflect adoption of CRW-protected Bt corn. ABSTC will obtain coordinates of sampling locations so that re-sampling in subsequent years can occur when needed. Fifteen or 16 population subsamples from different counties across the sampli ng region will be collected, and colonies reared and

maintained in the lab. Each collection should consis t of 2,000 adult beetles to ensure a minimum of 5,000 eggs from at least eight of the total samples collected.

The diet bioassay tool is available to monitor for changes in WCRW susceptibil ity to Cry34/35Ab1 and

the Sublethal Seedling Assay (SSA) (Nowatzki et al . 2008) may be available as well. It is Dow’s intention to eventually select for use a single met hod once the advantages and validity of each approach

have been verified. A final descri ption of how the SSA can be used in resistance monitoring should be

available in 2010.

BPPD’s Analysis of Cry34/35 Resistance Monitoring Plan

BPPD has reviewed Dow’s revised monitoring pl an for DAS-59122-7 and has found it adequate for Cry34/35 (see review in U.S.EPA 2009c). BPPD recomm ends that Dow/Pioneer c onsider the use of the Sublethal Seedling Assay (Nowatzki et al . 2008) for detecting shifts in sus ceptibility rather than the diet bioassay approach (EC 50 /LC 50 data). Nowatzki et al . (2008) were able to show that the SSA was more sensitive in detecting shifts in su sceptibility than the bioassay because it used the increased sensitivity of

a sublethal measure.

BPPD also recommends that Dow/Pioneer addres s monitoring for resistance with northern corn rootworm. Baseline susceptibility da ta have been developed in the pa st by the Siegfried lab at the University of Nebraska, which now can serve as a refe rence. Past difficulties w ith rearing of NCRW and arguments of limited geographic distribution should not be the driving factors for failure to monitor for resistance in this corn pest. E fforts to develop reliable mass rear ing and bioassay methods for NCRW

should be continued.

Unexpected Pest Damage Guidelines (2010)

The few follow-ups to unexpected pest damage re ports showed that most crop damage was due to

weather-related causes rather than trait failure. The “ interim unexpected pest damage guidelines ” (submitted in 2008) encompassed the variability seen in registrant-generated fi eld trial data from 2005–Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

148 2007. The Iowa State 0–3 Node Injury Scale (NIS) was used to assess root damage and assign ratings. To create worst-case guidelines, only results from locations where the average damage to the untreated isoline control exceeded a rating of 1.0 were used. Since environmental and ecological factors affect both trait performance and manifesta tion of rootworm damage and can cause outliers from the expected distribution, the interim guidelines for unexpected pest damage call fo r assessment of reports of root damage to be considered on a case-by-case basis.

Final unexpected pest damage guidelines were submitted in 2010 (MRID No. 480455-01). Dow reported that corn rootworm damage on Cry34/35-protected Bt corn was a function of the non-high dose effects of the crystal toxin and environmen tal conditions. Because of the multitude of factors playing a potential role in root-feeding damage, stal k and root lodging, Dow stated th at it was impossible to derive a threshold damage level (i.e., NIS score) that woul d qualify as “unexpected” fo r all possible scenarios. The following are factors that affect the root damage levels:

● Rootworm population pressure ● Soil chemical environment

● Rootworm egg distribution across fields ● Soil fertility

● Field history (previous crop) ● Winter conditions (e.g., temperature,

● Soil conditions in the previous year snow cover, freeze-thaw cycles) (affecting egg distribution) ● Weather events before and during the

● Soil structure and type period of rootworm feeding (storms,

● Use of insecticides drought, etc.)

● Use of fertilizers ● Presence of CRW pathogens and

● Presence of grassy weeds as alternate predators larval plant hosts ● Density-dependent CRW larval

● Presence of off-type plants not expressing competition for feeding sites

the toxin ● Phenotypic variation in expression of

● Presence of volunteer corn the proteins

● Soil moisture at rootworm egg hatch ● Temporal and spatial pattern of

● Timing of rootworm egg hatch Cry34/35 concentration in roots and corn root growth

● Duration of rootworm egg hatch ● Rootworm ability to find suitable host

● Size of root mass at the time of rootworm tissue feeding ● Chronic effects of Cry34/35 on larvae

● Re-growth of corn roots following feeding ● Environmental impacts on corn growth

● Root damage due to other agents and development

other than corn rootworm

● CRW species mix

Both Dow and Pioneer have conducted field trials with their corn rootworm products expressing DAS59122-7 and have found that, under extreme conditions and in the absence of resistance, Cry34/35 may not provide economic prot ection in part due to its non-high dose nature.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

149 Dow and Pioneer have therefore se t up their own protocol for invest igating reports of unexpected CRW damage on DAS-59122-7 corn. A fiel d visit will occur as soon as reasonably possible following a grower complaint. Lodging, stunting, and other symp toms will be investigated through the entire affected area on DAS-59122-7 expressing Bt and non- Bt corn plants, and the cause of damage will be determined (CRW or other pest damage, adverse weathe r, or other factors). If causes other than CRW or planting error do not explain the damage, then plants will be sampled from the affected field and root injury scored and plant samples will be tested to verify the presence of the trait. If the trait was expressed in the injured plants, th en Dow proposed to collect more root samples from the affected field(s) to identify whether inadvertent seed mixi ng occurred. If the trait was expressed in these additional samples and non-expressing plants were not present in significant numbers, then Dow would

escalate the incident for further investigation.

Further investigation involves collecting adult CRW and root tissue for expression analysis. If the expression analysis verified the expected expression level in roots and other f actors did not explain the damage levels observed, then the collected CRW sample s would be submitted to a third-party laboratory for bioassay analysis, which would determine whet her the population sample s are less sensitive to Cry34/35 than normal.

Dow expects this process to last up to a year. However, when damage reports come in too late to collect adults, then the investigation w ould start in the next growing s eason. The field and surrounding area would be assessed and, if damage cannot be explai ned by any other factors, then adults would be collected for rearing and cons equent bioassay purposes.

Dow proposes that resistance would be confirmed if two years of unexplained damage and significantly lower than normal sensitivity to Cry34/35 are observed in offspring of collected adults. Dow’s response to confirmed resistance was outlined in the registra tion letters and triggers the long-term resistance management remedial action plan accor ding to the characteristics of the Bt trait and local agronomic

needs.

BPPD’s Analysis of Unexpected Damage Guidelines

Dow proposed that a damage threshold was no longer f easible to use as an i ndicator for potential CRW

field resistance because other non-toxin related factors could act together in such a manner as to mimic

field resistance. BPPD does not co ncur with Dow’s proposition nor with their arguments to completely abandon the “damage threshold level” as an indica tor for CRW field resistance and proposes a revised approach instead (see review in U.S. EPA 2010c). At the time, the 2002 SAP proposed a Node Injury Score threshold level of 0.5 for Cry3Bb1 as an in dicator of “damage across the economic threshold

level.” BPPD notes that this value cannot be transferred to other single toxins, or even pyramided toxins, and that each report of unexpected CRW damage needs to be dealt with on an individual basis. The “damage threshold level” as expressed by node injury scores is an important tool for alerting the stakeholder community and EPA that some unusual event in Bt corn has occurred; however, this tool

should not be used alone and out of context.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

150 The following points should be considered when follo wing up with a reported incident of unexpected pest damage:

1) The inherent dose of the toxin to cont rol CRW (high dose vs. non-high dose control); 2) Prior use and crop history in the Bt field where excessive damage was observed; 3) Damage on non- Bt plants in the same field or immediately adjacent to the Bt plants; 4) Insect pressure during that corn-growi ng season (low vs. moderate vs. high); and 5) Weather pattern during the corn-g rowing season and possible effects on Bt protein expression and pest population dynamics.

When greater node injuries are observed on the refuge corn than on the Bt corn, then this could be an indication that damage is not due to resistance but some other factors. Likewise, if Bt plants have less damage than refuge plants that were treated with ins ecticides, then the cause of damage is less likely due to resistance. However, if damage levels on Bt plants exceed the level observed on non- Bt plants, then

field resistance could be a concer n. A NIS damage threshold level of 1.0 may be suitable for single toxin, non-high dose CRW products such as DAS- 59122-7. When CRW pressure is exceedingly high, however, then both Bt and refuge corn will incur greater dama ge. Under these circumstances, a threshold level of 1.5 may be appropriate to use by the registrants.

BPPD concluded that Dow/Pioneer’s response strategy for dealing with unexpected damage reports in DAS-59122-7 corn is protracted and could be improved (U.S. EPA 2010c). For example, Dow/Pioneer stated that they would first survey the area with unexpectedly high CRW damage, then collect root tissues and conduct tissue tests to verify appropriate Bt expression. If the Bt toxin was expressed, Dow/Pioneer proposed to collect more tissue samples to rule out planting erro r and then, if unable to rule out CRW resistance, begin co llecting adults. With this sequenc e of steps, BPPD noted that

Dow/Pioneer could lose valuable time to obtain adult CRW collections. BPPD recommended that

Dow/Pioneer collect adult CRW imme diately after first bein g alerted to unusually high root damage to

DAS-59122-7 corn. These adults could be held until all the other proposed causative agents have been

ruled out. The benefit to BPPD’s a pproach could be that if field resi stance is the cause of product failure and the unexpected damage reports, then Dow/Pi oneer would already have a population sample available and could start the rearing process, bioassa ys, and have results to report possibly within one year rather than Dow/Pioneer’s worst-case scenario of two years. Subsequent to BPPD’s review, Dow/Pioneer provided clarification that most of the sequential steps outlined to test for resistance do in fact occur during the initial vis it (Dow and Pioneer 2010). This incl udes testing for the presence of Cry34/35 and starting insect collections (except when reports are received too late in the season and CRW are not available to sample). If this is the case , the registrants will have addressed BPPD’s primary concern regarding delayed insect collection.

When CRW adults are collected in response to an unexpected dam age report, BPPD recommends that samples originate from the Bt field of concern and the adjacent refuge corn. However, studies by Naranjo (1990) and Coats et al . (1986) suggest that a significant fraction of the female WCRW population engages in longer distance and pre-ovipositional dispersal. Hence, collections of putativeBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

151 resistant individuals should also be obtained past the 1⁄2 mile radius from the affected field should

resistance be detected.

Resistance Monitoring Results (Through 2008)

In 2008 (report detailed in MRID No. 479008-01), nine populations of western corn rootworm were
collected across the Corn Belt wh ere the species was most abundant and where all three biotypes occurred (see below). At each collection site, 2,000 individuals were collected in one day and maintained for oviposition purposes by Custom Bio-Products in Maxwell, Iowa—an independent laboratory conducting bioassays for Dow since 2006.

ABSTC Region 1, rotation -resistant variant

 McLean County, Illinois

 Piatt County, Indiana

 Henry County, Illinois

ABSTC Region 2, wild-type variant

 Scott County, Iowa

 Palo Alto County, Iowa

 Clinton County, Iowa

ABSTC Region 3, organophosphate-resistant variant

 York County, Nebraska

 Polk County, Nebraska

 Seward County, Nebraska

Concentration response bioassays were conducted usi ng neonates from each of the nine collected 2008 populations; the same protocol was followed as for the baseline susceptibility studies described in Dow’s report to the Agency (MRID No. 473342-01). Th e protein used in the assays consisted of microbially produced, partially purif ied powders mixed in a ratio of 3:1 by active ingredient weight (Cry34 to Cry35). Concentrations tested included 0, 0.56, 1.67, 5.0, 15.0, and 45.0 micrograms per square centimeter (μg/cm 2 ). Growth inhibition (EC 50 ) and mortality (LC 50 ) were measured from neonates of the collected insects and compared to the established baseline suscep tibility. In addition, the variation in sensitivity was measured usi ng a ratio of the greatest to lowest LC 50 and ratio of greatest to

lowest EC 50 and compared to prior results.

Data from 2008 were reported in MRID No. 479008-01 (reviewed in U.S. EPA 2010c). Estimated EC 50 and LC 50 values for the nine geographically distin ct 2008 WCRW populations ranged from 2.0 μg/cm 2
to 7.9 μg/cm 2 and 6.2 μg/cm 2 to 15.4 μg/cm 2 , respectively. For EC 99 and LC 90 measures, the estimates ranged from 83.8 μg/cm 2 to 596.6 μg/cm 2 (EC 99 ) and 58.4 μg/cm 2 to 613.6 μg/cm 2 (LC 90 ). The 2008 results are summarized in Table 1 below, along with da ta from susceptibility assays in previous seasonsBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

152 (2004–2008). Dow concluded that the se nsitivity of WCRW, as measured by the ratio of highest to

lowest LC 50 and EC 50 , was similar to those in previous years (see Figure 1).

Table 1. Cumulative WCRW Cry34/35 Susceptibility Data (2004–2008).

Year EC 50 (μg/cm 2 ) EC 99 (μg/cm 2 ) LC 50 (μg/cm 2 ) LC 90 (μg/cm 2 )

2004–2005 0.8

– 1.1 2.0

– 2.4

2006 0.8

– 2.3 21.0

– 56.6 1.2

– 7.3 9.2

– 78.1

2007 1.0

– 3.7 51.9

– 213.6 3.0

– 11.5 23.9

– 463.5

2008 2.0

– 7.9 83.8

– 596.6 6.2

– 15.4 58.4

– 613.6

BPPD generated table from data submitted by Dow.

Figure 1. Variation in response to Cry34/35 (a s measured by the ratio of high:low LC 50 and EC 50 ) among WCRW field populations co llected between 2004 a nd 2008 (Dow figure extracted from MRID No. 479008-01).

Overall, there has been a trend of decreasing susc eptibility in WCRW to Cry34/35 when the data are evaluated on a qualitative basis. Al l measured and extrapolated mean EC and LC values were higher in 2008 than in previous years (see ranges for EC 99 and LC 90 values in Table 1). From 2006 to 2008, the range of mean EC 99 and LC 90 values obtained in bioassay tests or from extrapolati ons increased from 21.0–56.6 μg/cm 2 to 83.8–596.6 μg/cm 2 and 9.2–78.1 μg/cm 2 to 58.4–613.6 μg/cm 2 , respectively. Dow and Pioneer have indicated, however, that the year-to-year differences in EC 99 and LC 90 are not statistically relevant (D ow and Pioneer 2010). The EC 99 and LC 90 values are extrapolated beyond the concentrations used in the bioassays and have overlapping confidence intervals. Further, the registrantsBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

153 recommended focusing on the LC 50 ratio of the least sensitive CRW population to the most sensitive population for comparison between seasons. An increase in this ratio could re sult if a popul ation shows reduced susceptibility to the toxin. Using this measure, CRW susceptibility to Cry34/35 has not significantly decreased since monitori ng was initiated in 2004 (Figure 1).

As an additional point of analysis, two counties that were previously sampled in 2006 and 2007 were also sampled in 2008, and a more direct data compar ison could be made for these two locations (see Table 2). U.S. EPA (2010c) focused on EC data, since these showed le ss variation than LC data. When

the mean EC 50 and EC 99 values were qualitatively compared across three consecutive years in Henry County (Illinois) and Scott County (Iowa), it was apparent that the extrapolated EC 99 values had

increased. The mean EC 99 values for Henry and Scott County ranged from 21.0 μg/cm 2 to 388.3 μg/cm 2
and 48.7 μg/cm 2 to 95.8 μg/cm 2 , respectively. BPPD concluded that susceptibility to DAS-59122-7 may

have decreased in these areas (Henry County, Illinois and Scott County, Iowa) and recommended that additional sampling occur in these counties during future corn-growi ng seasons to assess whether these susceptibility trends continue. Dow and Pioneer noted th at the apparent decrease in susceptibility may in fact be due to a change in test methodology. The timing of the exposure period in the bioassays was reduced from 4–7 days to 4 days, which could have resulted in lower mortality and growth inhibition (Dow and Pioneer 2010).

Table 2. Cry34/35 Susceptibility Data for WCRW po pulations in Henry County (IL) and Scott County (IA) (2006–2008) (BPPD generated table from data submitted by Dow/Pioneer).

Location/Year EC 50 (μg/cm 2 ) EC 99 (μg/cm 2 )

Henry Co (2006) 1.3 21.0

Henry Co (2007) 2.1 213.6

Henry Co (2008) 4.0 388.3

Scott Co (2006) 0.8 48.7

Scott Co (2007) 1.8 63.2

Scott Co (2008) 2.9 95.8

b. Compliance

Dow/Pioneer’s compliance reports, including grower agreements, grow er affirmation procedures, and CAP, were reviewed in U.S. EPA (2006b). The co mpliance program for DAS-59122-7 (Cry34/35 corn) is aligned with that developed by ABSTC (in consultation with the EPA) for corn-borer protected Bt corn products. Specific elements of the program and additional reports with annual survey data are

discussed below.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

154 DAS-59122-7 Compliance Assurance Program

As a term of the Herculex RW (DAS-59122-7; Cry34/35Ab1) registration, Dow and Pioneer were required to develop and submit to EPA a CAP to en sure grower adherence to IRM requirements. The terms of registration mandated a number of components for the compliance program including:

 Grower Agreements: Contractual arrangement be tween the registrant a nd grower to obligate adherence to IRM requirements.

 Annual IRM Survey: A survey (conducted anony mously by an independent research firm) intended to provide a statistically representative sample of gr owers from various corn-growing regions in the U.S. Results from the survey should assess levels of grower compliance with refuges, as well as grower motivations, attitudes, and reasons for non-compliance.

 On-Farm Assessments: The registra nt is required to develop an on-site assessment program in which trained personnel from each comp any make visits to farms growing Bt corn. During these visits, compliance with refuge requirements is assessed and growers not in compliance are

identified for corrective action under the Phased Compliance Approach.

 Tips and Complaints: The registra nt must establish a means for th e reporting and investigation of incidences of refuge non-compliance.

 Phased Compliance Approach (PCA): A c onsistent set of procedures (for all Bt corn registrants) to be employed to address non-complianc e among growers and seed dealers.

ABSTC, a consortium of Bt corn registrants, previously developed and submitted a CAP for lepidopteran Bt corn PIPs in 2002. Subsequently, ABSTC submitted revised versions of the CAP in 2004 and 2005 in response to EPA reviews of annual growing season reports (s ee discussion in U.S. EPA 2010e). Herculex® RW (and other registered produc ts with Cry34/35) have been included as part of this existing program, though data are tabulated separately for lepidopteran, rootworm, and stacked (lepidopteran + rootworm) Bt corn PIPs. EPA reviews of compliance data for rootworm-protected PIPs (some of which predates the re gistration of Herculex® RW) can be found in U.S. EPA (2004, 2007b, and 2009a).

Annual Grower Surveys

As a condition of each individual Bt corn registration, the registrant must perform an annual third-party survey of a statistically representative sample of Bt corn growers. The grow er survey functions to measure compliance adherence to refuge size and distance requirements at a regional level and to identify educational opportunities to increase grow er compliance with IRM requirements. More than 500 growers from four separate regions are a nonymously surveyed annually. The methodology for conducting the grower survey has remained virt ually unchanged since it was first conducted by Marketing Horizons, Incorporat ed in 2000 for the lepidopteran Bt corn registrations. Starting in 2007, however, due to an increasing complexity of growers’ Bt corn planting practices and a need to

standard ize the grower survey across insect-protected traits, Marketi ng Horizons, Incorporated utilized an internet-based survey approach.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

155 Surveys for the corn rootworm PIPs encompass all growers planting rootworm-protected traits (Cry3Bb1, mCry3A, and Cry34/35Ab1). Cumulative results of the surveys are summarized in Table 3

below. Results from the stack ed (lepidopteran + rootworm) Bt corn surveys are tabulated separately (Table 4) and also include all registered rootworm PIP traits.

Table 3. Summary of Telephone (2005–2006) and Online (2007–2008) Survey Results for

Rootworm-Protected Bt Corn Growers.

Survey Question 2005 1

% Respondents 2006 2

% Respondents 2007

% Respondents 2008

% Respondents

Adherence to Refuge 3 Size 93 89 80 74

Adherence to Distance Requirements 3 87 82 79 63

Awareness of IRM Requirements 97 93 97 96

Unaided Recall of Refuge Size 51 57 63 72

Unaided Recall of Refuge

Distance 58 55 33 34

1 Includes YieldGard® RW and YieldGard® Plus corn growers

2 Includes YieldGard® RW, YieldGard® Plus, Herculex® RW, and Herculex® XTRA corn growers

3 Weighted averages across all four regions surveyedBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

156 Table 4. Summary of Telephone and Online Survey Results for Stacked Bt Corn Growers (2006–2008).

Survey Question 2006

% Respondents 2007

% Respondents 2008 % Respondents

Adherence to Refuge Size 1 78 70 72 4

Adherence to Distance Requirements 1 92 66 66 3

Awareness of IRM Requirements 95 96 97

Unaided Recall of Refuge Size 59 62 and 55 2 81

Unaided Recall of Refuge Distance

CRW 48 39 36

Unaided Recall of Refuge Distance European Corn Borer (ECB) 32 77 86

1 Weighted average across a ll four regions surveyed

2 First number listed is for ECB and the s econd number for CRW refuge compliance.

3 On a per field basis, adherence was 76%.

4 On a per field basis, adherence was 73%.

Overall compliance (per grower) with refuge requir ements for both single-tra it and stacked rootwormprotected PIPs has declined from 2005 to 2008. Grower adherence to the necessary refuge size fell to below 75% in 2008 for single-trait an d stacked rootworm PIPs. Compliance with refuge proximity was lower; in 2008, ≤ 66% of rootworm PIP growers deployed ref uges within the required distance to the Bt field. The percent of growers who were able to recall the correct refuge distance requirements (unaided) for rootworm PIPs drastically declined to belo w 40% in 2008. Refuge distance requirements for rootworm-protected Bt corn products may be more challenging fo r growers because the refuge must be deployed either within or immediately adjacent to the Bt field. Stacked produc ts present additional challenges due to the need to plan either two re fuges (for lepidoptera and rootworm) or a combined refuge for both pest complexes. Grower awareness of the distance requirements has been poor and likely explains much of the reported non-compliance.

On-Farm Assessments

The on-farm assessment program is the portion of th e CAP that identifies i ndividual non-compliant growers (regardless of farm size) for remedial IRM education, follow-up reassessments, and other activities as part of the PCA. It can also serve as a tool to enhan ce the registrant’s understanding of the obstacles growers face in implementing IRM requirements. The mandatory on-farm assessment program was fully implemented for the first time in 2003 (f or lepidopteran registra tions) and has typically encompassed more than 2,000 growers per season (for all types of Bt corn). On-farm assessments for rootworm-protected PIPs (incl uding Cry34/35 products) began in 2006.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

157 Data from the on-farm assessments ( 2006 through 2008) of rootworm-protected Bt corn PIPs are

summarized in Table 5 below. These on-farm asse ssments encompass all growers planting rootwormprotected PIPs, including varie ties expressing the Cr y3Bb1, mCry3A, and Cry34/ 35Ab1 toxins. Results for the on-farm assessments of stacked (lepidopter an + rootworm) PIPs are detailed in Table 6. The

assessments do not have the statisti cal power associated with the consistently stratifie d and randomized telephone/on-line surveys and are not used to meas ure representative rates of non-compliance. In 2007 and 2008, no information was provided regarding sp ecific non-compliance with refuge size and

distance. This information should be provided in future reports to be c onsistent with previously collected

data and to illustrate how growers are out of compliance (U.S. EPA 2009a).

Table 5. Cumulative Results for the On-F arm Assessments of Coleopteran-Protected Bt Corn Growers (2006–2008) 1

. 2006 2007 2008

Growers Assessed 395 247 134

Refuge Distance

Deviations 2 13 N/A N/A

Refuge Size Deviations 7 N/A N/A

Significant Deviations 11 (2.8%) 16 (6.5%) 12 (9.0%)

Insignificant Deviations 10 (4.0%) 8 (3.2%) 7 (5.2%)

Compliant Growers 374 (94.7%) 223 (90.3%) 115 (85.8%)

Non-Compliant

Growers 21 (5.3%) 24 (9.7%) 19 (14.2%)

1 Table adapted from page 12 of MRID No. 470444-01.

2 Some growers had compliance deviations othe r than refuge size or distance; thus, the total of refuge distan ce and size deviati ons does not equal the number of non-compliant growers.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

158 Table 6. Cumulative Results of th e On-Farm Assessments of Stacked Bt Corn (Rootworm + Corn Borer) Growers (2006–2008) 1

. 2006 2007 2008

Growers Assessed 600 1,069 1,799

Refuge Distance

Deviations 51 2 N/A N/A

Refuge Size Deviations 8 N/A N/A

Significant Deviations 45 (7.5%) 77 (7.2%) 86 3

Insignificant Deviations 16 33 (3.1%) 36 3

Compliant Growers 539 (89.8%) 959 (89.7%) 1546 (85.9%)

Non-Compliant

Growers 61 (10.2%) 110 (10.3%) 253 (14.1%)

1 Table adapted from page 12 of MRID No. 470444-01.

2 Some growers had compliance deviations othe r than refuge size or distance; thus, the total of refuge distan ce and size deviati ons does not equal the number of non-compliant growers.

3 The numbers of deviations do not add up to the 253 non-compliant cases reported.

Tips and Complaints

As required by the terms of registration, Bt corn registrants must have a “t ips and complaints” system as a mechanism for individuals (e.g., growers, sales repres entatives, etc.) to report alleged instances of IRM non-compliance. The number of tips a nd complaints (summarized for all Bt corn registrations, including lepidopteran and rootworm varietie s) received through 2008 is summarize d in Table 7 below. Each of these growers identified thr ough the tips and complaints mechanism we re visited as part of the on-farm assessment program. However, it is not possible to determine whether any of the non-compliant growers, identified via the tips and complaints route, were subject to the Phased Compliance Approach.

Table 7. Anonymous Tips and Complaints ab out Non-Compliance with IRM Requirements (Data from ABSTC An nual Reports 2003 through 2008).

Year Number of Tips and Complaints

2003 0

2004 0

2005 5

2006 3

2007 14

2008 5Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

159 Phased Compliance Approach

ABSTC’s CAP for lepidopteran and r ootworm-protected PIPs includes a standard set of procedures (shown in Table 8), known as the Phased Complia nce Approach (PCA), which is to be used by registrants when responding to in stances of grower non-compliance with the IRM requirements. The PCA also established a tiered approach for non-comp liance with “significant” deviations and “other” deviations. For a 20% CRW refuge re quirement (Corn Belt), a significant size deviation is defined as a

Bt grower planting less than 15% non- Bt corn refuge. This definition is also applicable to “combined” refuges planted for both lepi doptera and CRW for stacked Bt corn PIPs. On the other hand, a significant deviation based on refuge proximity has not been clearly defined for CRW refuges and, as of the 2008 CAP report, it is unclear what standards are being used by ABSTC. For lepidopteran Bt corn, a significant deviation is triggere d if fewer than 2/3 of the Bt corn fields are planted within 1⁄2 mile of a

non- Bt corn refuge. However, this de finition is not compatible with CRW refuge because the distance requirement mandates that refuges be placed adjacent to or within the Bt corn field. A reasonable extension of the lepidopteran definition fo r CRW could be “less than 2/3 of the non- Bt refuge is deployed adjacent to or within the Bt field” and “fewer than two-thirds (2/3) of the in-field strips are at least four rows wide” (U.S. EPA 2006b). This formula would also be applicable to combined refuges for

stacked PIPs.

Table 8. Phased Compliance Approach (PCA) – Standards for Bt Corn Refuge Non-Compliance (Submitted with the ABSTC 2002 CAP).
Mandatory Responses Additional Responses Significant Deviations  IRM education

 Warning letter

 Compliance assistance contact prior to planting

 Compliance assessment contact for the following growing season

 Deny access to the Bt corn product for any significant deviation two years in a row

Other Deviations  IRM education

 Letter and/or compliance assistance contact prior to planting

 Compliance assessment contact in the following growing season  Invoice monitoring

 Technical assistance

 Grower IRM training

 Reaffirmation of IRM obligations

 Deny access to the Bt corn product for other deviations that are repeated over a period of

yearsBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

160 Under the PCA, sales are to be suspended to in dividual growers for one y ear after two years of significant deviations. Following the one-year suspension, growers will need to be requalified to purchase seeds. Grower identified as non-compliant (s ignificant or other devi ations) are required to receive a “compliance assessment contact” the following year under th e PCA. Non-compliant growers are typically identified through th e on-farm assessment program (see previous discussion in the “OnFarm Assessments” section ). Table 9 summarizes the numbers of non-compliant growers reassessed under the PCA, and the growers still found to be out of compliance. As of the 2008 growing season, one grower was denied access to Bt corn technology due to a refusal to be reassessed in the following season after significant non-complia nce. Compliance data, including results of on-farm assessments and PCA activities, are detailed in U.S. EPA (2007b and 2009a).

Table 9. Reassessment of Rootworm-Protected and Stacked Bt Corn Growers Under the Phased Compliance Approach (Taken from ABSTC Annual CAP Reports) 1

.

Year Reassessments 2 Significant Deviations 3 Loss of Access to Technology

2006 62 0 1 4

2007 82 0 0

2008 134 0 0

1 The data in this table includes both growers planting singl e-trait rootworm PIPs and stacked (lepidopteran + rootworm) PIPs. The data in the table has been summed for both groups.

2 Reassessments of growers identified with deviations (significant and other) to refuge requirements the previous growing

season.

3 Significant deviations recorded the follo wing season. Two successive years of signif icant deviations results in loss of access

to Bt corn technology.

4 One grower refused to be reassessed in 2006 and was denied access to Bt corn

4. Stacked Bt Corn PIPs Containing Cry34/35

Several “stacked” Bt corn PIPs have been re gistered that contain Cry34/ 35. Stacked products express two or more toxins targeted at se parate pest complexes. Cry34/35 (ro otworm control) has been paired with lepidopteran-active proteins for control of European corn bor er and other pests. The stacked Cry34/35 products (registered as of 2010) are described below. A separa te product has been registered with a “pyramid” (two or more toxins targeted at the same pest complex) of Cry34/35 and Cry3Bb1 for rootworm control. This product (SmartStaxTM) is di scussed in a separate Fact Sheet (U.S. EPA 2009b). Another product containing a seed blend mix of Cry34/35 corn (Optimum® AcreMaxTM 1) has also been registered and is discussed in a separate Biopesticides Regi stration Action Document (U.S. EPA 2010b and 2010f).

a. Herculex® Xtra (DAS 59122-7 x TC1507)

Herculex Xtra (EPA Reg. Nos. 68467-6 and 29964-5) is a stacked product that is the result of a conventional breeding cross between Herculex® I (Event DAS 01507-1; EP A Reg. Nos. 68467-2 andBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

161 29964-3) and Herculex® Rootworm (EventDAS-59122-7; EPA Re g. Nos. 68467-5, 29964-4). Herculex® I (first registered in 2001) expresses the Cry1F Bt toxin and is targeted against stalk-boring lepidopteran pests, while Herc ulex® Rootworm corn (register ed August 31, 2005) expresses the Cry34Ab1/35Ab1 binary toxin for contro l of CRW. The complete list of ta rgeted pests is as follows: European corn borer (ECB, Ostrinia nubilalis ), southwestern corn borer (SWCB, Diatraea grandiosella ), corn earworm (CEW, Helicoverpa zea ), fall armyworm (FAW, Spodoptera frugiperda ), black cutworm (BCW, Agrotis ipsilon ), western bean cutworm (WBCW, Richia albicosta ), western corn rootworm (WCRW, Diabrotica virgifera virgifera ), northern corn rootworm (NCRW, Diabrotica

barberi ), and Mexican corn rootworm (MCRW, Diabrotica virgifera zeae ).

When evaluating a stacked PIP created from previously registered single-trait PIPs, the dose profiles for

each of the toxins in the stack will have been previously established for the isolines. Therefore, the dose

of the stacked PIP can be verified relatively easily through confirmatory studies. Two sets of data are typically analyzed: (1) plant expression data to verify that the toxin expression in the stacked product is at least as high as in the single-ge ne isolines; and (2) efficacy data to confirm that the level of control against the target pests in the stacked product is at least as high as the isolines. Dow/Pioneer submitted efficacy studies (Master Record Identificati on Numbers (MRID Nos.) 463438-04 and 463438-05) that were designed largely to confirm efficacy in the newly created stacked product, Herculex® Xtra. The data revealed that Herculex® Xtra offered compar able levels of protectio n against ECB, FAW, and WCRW as the single-gene hybrids (H erculex® I and Herculex® Rootworm) used to create the stacked product. Other labeled pests (e.g., BCW, SWCB, and NCRW) were not evaluated in these studies. However, since previously submitted data have demons trated efficacy against all the labeled pests in the single-gene hybrids (Herculex® I an d Herculex® Rootworm), it is exp ected that Herculex® Xtra will offer comparable protection. The results are also supported by toxin expr ession data (MRID No. 463438-04), which showed that the stacked hybrid e xpresses both Cry1F and Cry34/35 at comparable (or higher levels) than the singl e-gene hybrids (U.S. EPA 2005c).

IRM must be addressed for lepidopteran and corn rootworm by deploying either separate 20% non- Bt corn refuges (for each pest complex) or a combined 20% refuge that addresses both pest complexes. A combination refuge must address the proximity rest rictions for CRW (i.e., the refuge must be placed adjacent to or within the Bt field and in-field strips must be at least 4 rows wide).

Other aspects of the IRM program for Herculex® Xtra (resistance monito ring, remedial action, and compliance) were incorporated in to the existing programs for the single-toxin PIPs (Cry34/35 and Cry1F). The IRM program for Cry34/35 is discussed in sections II(D)(2) and II(D)(3) of this BRAD; Cry1F is discussed in the August 2005 Cry1F BRAD and the September 2010 Cry1Ab and Cry1F BRAD (U.S. EPA 2005b and 2010d).

b. 59122 x MON 810 and 1507 x 59122 x MON 810

Both 59122 x MON 810 (EPA Reg. No. 29964-9) and 1507 x 59122 x MON 810 (EPA Reg. No. 29964- 8) are stacked products registered by Pioneer for control of rootworm and corn borer pests. The first, 59122 x MON 810, expresses Cry34/35 and Cry1Ab, wh ile 1507 x 59122 x MON 810 expresses Cry1F,Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

162 Cry34/35, and Cry1Ab. Like other stacked Bt corn PIPs, these two product s were supported by efficacy and expression data to confirm the dose profile against the major target pests.

Protein expression data were generated for each of the stacked products (59122 x MON 810 and 1507 x 59122 x MON 810) and the single-toxin constituen t PIPs (MRID No. 475109-04). Studies were conducted in greenhouses, and plant tissues were an alyzed by enzyme-linked immunosorbent assay (ELISA) for protein expression. Pioneer sampled leaf, stalk, root, whole plant, and grain tissues for the analyses (pollen tissue was also coll ected but was often below the limit of detection). Corn tissues were sampled during the R1 corn growth stage, except for grain which was taken from the R6 stage. The results from the ELISA assays showed that Cry1F, Cry1Ab, Cry34Ab1, and Cry35Ab1 protein were expressed at comparable levels in the stacked products to the single-toxin constituent products. Expression was consistent throughout all tissues—only small differences in protein levels, which were not statistically sign ificant, were noted between the stacked and singl e-toxin products.

Pioneer conducted field studies to investigate th e efficacy of their stacked products against ECB, SWCB, FAW, and CRW. The company asserted that testing was not necessary for other labeled pests (i.e., secondary lepidopteran corn pests) given that the consti tuent PIPs (1507, 59122, and MON 810) have well established efficacy profiles. These st udies were described in MRID No. 476778-03. The submitted field efficacy data were su fficient to demonstrate that the Bt toxins in the three proposed stacked products have comparable dose expression profiles as thei r single-trait cons tituent products (U.S. EPA 2010a). Both MON 810 (Cry1Ab) and 1507 (C ry1F) have been considered “high dose” for ECB and likely SWCB but not for CEW. With CRW, 59122 (Cry34/35) is known not to have high dose expression. Given the confirmatory protein expression and efficacy studies, the stacked products can also be considered high dose for ECB a nd SWCB and non-high dose for CEW and CRW.

Similar to other stacked products, refuge must be addressed by planting se parate (corn borer and rootworm) 20% refuges or a combin ation 20% refuge for both pests. Other aspects of the IRM program (resistance monitoring, remedial acti on, and compliance) are largely the same as the existing programs for the single-toxin PIPs (Cr y34/35, Cry1Ab, and Cry1F). The IRM pr ogram for Cry34/35 is discussed

in sections II(D)(2) and II(D)(3) of this BRAD; Cry1Ab and Cry1F are discussed in the 2001 Bt Crops Reassessment, August 2005 Cry1F BRAD, and/or the September 2010 Cry1Ab and Cry1F BRAD (U.S. EPA 2001b, 2005b, and 2010e).

c. Refuges for Stacked Bt Corn Products

Table 10 below describes the re fuge requirements for stacked Bt corn PIPs targeting lepidopteran (e.g., ECB) and rootworm pests. Growers must either plant two separate re fuges for each pest complex or a combined refuge that addr esses both insect groups.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

163 Table 10. Comparison of Lepidopteran (e .g., TC 1507, MON 810) and Rootworm (e.g., DAS-59122-7) IRM Requirements for Stacked Bt Corn PIPs (e.g., Herculex® Xtra)

Requirements (Separate Lepidopteran Refuge) (Separate Rootworm Refuge) (Combined Refuge)

Refuge Size  20%  20%  20%

Refuge

Placement  1⁄2 mile Adjacent or within field Adjacent or within field

Refuge Configuration Block, in-field strips (  4 rows), or edges Block or strips (  4 rows) Block or strips (  4 rows)

Refuge Management Any corn rotation meeting placement and configuration requirements

Insecticides can be used in refuge to control

ECB/SWCB when

above economic

thresholds.

Microbial Bt insecticides

are not allowed. Same corn rotation as the rootworm-protected Bt fields (e.g., first year corn or corn followed by corn)

Conventional insecticides

or seed treatments can be used in refuge to control

CRW larvae and other soil pests. If the refuge is

treated with a foliar

insecticide labeled for

CRW control when CRW adults are present, then the

Bt fields also must be

treated.

(Not applicable) Same corn rotation as the

Bt fields (e.g., first year corn or corn followed by corn)

Conventional insecticides

or seed treatments can be used in refuge to control

CRW larvae and other soil pests. If the refuge is

treated with a foliar

insecticide labeled for

CRW control when CRW adults are present, then the

Bt fields also must be

treated.

Microbial Bt insecticides

are not allowed.

Refuge Corn Types Conventional (Non- Bt ) Conventional (Non- Bt )

Corn borer-protected Bt corn (a corn borer refuge planted  1⁄2 mile also will be required)

Herbicide-tolerant corn (Non- Bt ) Conventional (Non- Bt )

Corn borer-protected Bt corn (an additional refuge

for corn borer will be required)

Herbicide-tolerant corn (Non- Bt )Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

164 5. IRM Terms and Conditions of Registration

The terms and conditions for each of the Cry34/35Ab1 registrations contain a complete description of the IRM requirements. Details are provided on the requirements for refuge (size and structure), resistance monitoring, remedial action, complian ce assurance, grower education, and annual IRM reports. For more information, please refer to th e document, in Docket Number EPA-HQ-OPP-2010- 0607, presenting the registration terms and conditions establis hed with the 2010 amendments.

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.

U.S. EPA. 2004. Technical Review of Monsanto Co.’s 2003 Grower Education Program, Including Proposed Changes for 2004, and IRM CAP for MON 863 Bt Corn (EPA Reg. No. 524-528). Memorandum from T. Milofsky to M. Mendelsohn dated June 23, 2004.

U.S. EPA. 2005a. EPA Review of Dose, Adult Feed ing Effects, and Insect Resistance Management (Trait Durability) Simulations and Plans for BtCry34/35Ab1 Construct PHP17662 (Event DAS59122-7) Corn. Memorandum from S. Matten, P h.D. to M. Mendelsohn dated July 13, 2005.

U.S. EPA. 2005b. Biopesticides Registration Action Document – Bacillus thuringiensis Cry1F Corn. Available from: http://www.epa.gov/oppbppd1/biopesticid es/ingredients/ tech_docs/brad_006481.pdf

.

U.S. EPA. 2005c. Review of Effi cacy Data and Insect Resistance Management (IRM) Plan Submitted

for Herculex Xtra Corn. Memorandum from A. Reynolds to M. Mendelsohn dated September 20, 2005.

U.S. EPA. 2006a. Review of the Preliminary Resist ance Monitoring Plan for Herculex Rootworm Corn Submitted by Dow AgroSciences LLC. Memora ndum from A. Reynolds to M. Mendelsohn dated July 24, 2006.

U.S. EPA. 2006b. Technical Review of Dow AgroSciences’ Grower Ag reements, Grower Affirmation Procedures, and Compliance Assurance Program (C AP) as Required as Part of the Terms and Conditions of the Herculex Rootworm (EPA Re g. No. 68467-5) and Herculex Xtra (EPA Reg. No. 68467-6) Registrations. Memorandum from S. Matten, Ph.D. to M. Mendelsohn dated November 27, 2006.

U.S. EPA. 2007a. Biopesticides Registration Action Document – Bacillus thuringiensis Cry3Bb1 Corn.

Available from: http://www.epa.gov/oppbppd1/biopesticid es/ingredients/t ech_docs/brad_006484.htm

.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

170 U.S. EPA. 2007b. Review of IRM Compliance A ssurance Program (CAP) Components Covered in

ABSTC’s Submission on Behalf of Corn Bo rer-Protected (EPA Reg. Nos. 524-489, 68467-2, 67979-1, and 29964-3), Corn Rootworm-Protected (EPA Reg. Nos. 524-528, 68467-5, and 29964-4), and Stacked (Corn-Borer/Corn Root worm-Protected) (EPA Reg. Nos. 524-545, 68467-6, and 29964-5) Bt Corn Registrations. Memora ndum from T. Milofsky and S. Matten, Ph.D. to M. Mendelsohn dated August 30, 2007.

U.S. EPA. 2009a. EPA Review of ABSTC’s 2007 and 2008 Corn In sect Resistance Management Compliance Assurance Program (EPA Regi stration Nos. 524-489, 68467-2, 67979-1, 29964-3, 524-528, 524-551, 68467-5, 67979-5, 29964-4, 524-545, 524-552, 68467-6, 67979-8, and 29964-5; MRIDs 473396-01 and 476633-01). Memora ndum from J. Martinez to M. Mendelsohn dated April 15, 2009.

U.S. EPA. 2009b. Fact Sheet – SmartStaxTM (MON 89034 x TC1507 x MON 88017 x DAS-59122-7).

Available from: http://www.epa.gov/oppbppd1/biopesticides /pips/smartstax-factsheet.pdf

.

U.S. EPA. 2009c. Review of Dow’s 2007 Corn Root worm Monitoring Report a nd Revised Resistance Monitoring Plan for DAS-59122-7. Memorandum from J. Martinez to M. Mendelsohn dated October 14, 2009.

U.S. EPA. 2010a. Review of Insect Resist ance Management (IRM) Considerations for Bt Corn Events 1507 x MON 810, 59122 x MON 810, 1507 x 59122 x MON 810. Memorandum from A. Reynolds to A. Sibold dated January 7, 2010.

U.S. EPA. 2010b. Biopesticides Registra tion Action Document – Optimum® AcreMaxTM B

. t . Corn

Seed Blends. Available from: http://www.epa.gov/pesticides/biopestic ides/ingredients/ tech_docs/brad_006490_oam.pdf

.

U.S. EPA. 2010c. Review of Dow’s 2008 Corn Root worm Monitoring Report a nd Revised Resistance Monitoring Plan for DAS-59122-7. Memorandum from J. Martinez to M. Mendelsohn dated June 30, 2010.

U.S. EPA. 2010d. Biopesticides Registration Action Document – Bacillus thuringiensis Cry3Bb1 Corn (Updated September 2010). Available from: http://www.regulations.gov (see “Supporting & Related Materials” within Docket Number EPA-HQ-OPP-2010-0607).

U.S. EPA. 2010e. Biopesticides Registration Action Document – Bacillus thuringiensis Cry1Ab and Cry1F Corn (Updated September 2010). Available from: http://www.regulations.gov (see “Supporting & Related Material s” within Docket Number EPA-HQ-OPP-2010-0607).Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

171 U.S. EPA. 2010f. Biopesticides Registra tion Action Document – Optimum® AcreMaxTM B

. t . Corn Seed Blends (Updated September 2010). Available from: http://www.regulations.gov (see “Supporting & Related Materials” within Docket Number EPA-HQ-OPP-2010-0607).Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

172 E. Benefits and Public Interest Finding for Initial Registrations of Event DAS-59122-7 Corn

1. Overview

The Environmental Protection Agency (EPA) has revi ewed the public interest document for event DAS59122-7 corn (Master Record Identificati on Numbers (MRID Nos.) 461227-01 and 461239-21; reviewed in U.S. EPA (2005)) and concludes that, under section 3(c)(7 )(C) of the Federa l Insecticide, Fungicide, and Rodenticide Act (FIF RA), conditional registration of ev ent DAS-59122-7 corn is in the public interest. Cry34/35Ab1 corn rootworm (CRW)-protected corn hybrids (event DAS-59122-7 corn)

will extend the benefits of insecticide use reduction that have been established for the Cry3Bb1 (event MON 863) CRW-protected corn products. Cry34/35A b1 CRW-protected corn is comparatively less risky to human health and the environment than curren tly registered chemical pe sticides. It also provides

a different mode of action for CRW control, comparable or better effi cacy and yield compared to other CRW-control products, expanded product choices for growers, and indirect benefits (e.g., energy savings resulting from reduced chemical insecticide use).

2. Public Interest Finding

The criteria for determining whether registration of a pesticide chemical is in the public interest are set forth in a Federal Register Notice dated March 5, 1986 (51 Federal Register (FR) 7628). There is a presumption that registration of a pe sticide chemical is in the public interest if one of the following criteria is met: (1) the use is for a minor crop; (2) the use is a replacement for anot her pesticide that is of continuing concern to the Agency; (3) the use is one for which an emergency exemption under FIFRA section 18 has been granted (i.e., the basis for the exemption was lack of a registered alternative product); or (4) the use is against a pest of public health significance. Further, EPA may determine that such a registration is in the public interest on the basis of the following criteria: (1) there is a need for the new chemical that is not being met by currentl y registered pesticides; (2) the new pesticide is comparatively less risky to health or the environmen t than currently registered pesticides; or (3) the benefits (including economic benefi ts) from the use of the new activ e ingredient exceed those of alternative registered pesticides and other available non-chemical techniques.

Mycogen Seeds c/o Dow AgroSciences LLC and Pioneer Hi-Bred International, Incorporated (hereafter referred to as Dow and Pioneer, respectively) ha ve provided data to s upport their claim that Cry34/35Ab1 CRW-protected corn is in the public interest. EPA’s analysis supports the following

conclusions:

1. Cry34/35Ab1-protected corn provides effective control of key rootwo rm pests of field corn and may prove more efficacious than chemical insectic ides presently registered for this purpose.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

173 2. Economic models suggest that, under conditi ons of high rootworm pressure, use of Cry34/35Ab1-protected corn will pr ovide greater net returns to fa rmers. Cost benefits include reduced expenditures on insecticides, applica tion equipment, and personnel, complemented by greater potential corn yields. U nder high rootworm pressure, thes e benefits are expected to outweigh the higher cost of seed.

3. Registration of Cry34/35Ab1-protected corn is expected to result in further reduction of chemical insecticide use by growers. This is of special importance since many pesticides registered for CRW-control are highly toxic to humans and the environment, while Cry34/35Ab1-expressing corn poses no foreseeable human health or environmental risks.

4. If Cry34/35Ab1 corn is registered, it will be the second CRW-protected Bacillus thuringiensis ( Bt ) corn product on the market (the first was Cry3Bb1). The availabil ity of multiple CRWprotected corn products will incr ease grower choice a nd price competition, likely resulting in lower seed prices for consumers and higher adoption rates.

5. The Cry34/35Ab1 CRW-protected corn will provide a different mode of action and extend the durability of other CRW control measures, including other Bt CRW-protected corn hybrids.

a. Background

Corn is the most widely cultivated U.S. crop, in terms of acreage planted and net value. In 2004, U.S. corn acreage totaled 80.9 million, yielding 11.8 billion bushels. Corn rootworm (CRW, Diabrotica spp.), one of the most damaging pests of field corn, can cause yield losses in the range of 8 to 16 percent if left

uncontrolled.

Prior to the advent of in sect-protected field cor n, CRW was controlled through the use of crop rotation and insecticides. Although crop rota tion is regarded as an effec tive CRW-control tool (Levine and Oloumi-Sadeghi 1991), behavior cha nges in northern corn rootworm (extended diapause) and western corn rootworm (soybean rotation) have reduced the effectiveness of this management option in some corn-growing regions. Insecticidal control, a pest management a lternative to crop rotation, employs chemicals that are highly toxic to fish, birds, and other wildlife speci es. In addition, resistance to some CRW insecticides, such as methyl parathion and carbaryl (Meinke et al . 1998; Scharf et al . 1999; Zhu et
al . 2001), may result in increased chemical use.

Since 2003, transgenic CRW-protected Bt corn, a third method of CRW control, has been available to

farmers. The first Bt field corn product registered for CR W control was Monsanto’s event MON 863 (expresses the Cry3Bb1 protein). This revi ew concerns the se cond CRW-protected Bt corn product submitted for registration, Dow’s and Pioneer ’s DAS-59122-7, which pr oduces the Cry34Ab1 and Cry35Ab1 insecticidal proteins (originally isolated from Bt strain PS149B1). Similar to event MON 863 corn hybrids, event DAS-59122-7 Bt corn is targeted against th e western corn rootworm (WCRW, Diabrotica virgifera virgifera LeConte ), northern corn rootworm (NCRW, Diabrotica barberi Smith and Lawrence), and Mexican corn rootworm (MCRW, Diabrotica virgifera zeae Krysan and Smith).Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

174 b. Efficacy

i. Summary of Submitted Information

In 2003, Dow and Pioneer conducted field trials at mu ltiple locations to evaluate the performance of Cry34/35Ab1 CRW-protected corn against coleopteran insect pests. Several efficacy trials were designed to compare Cry34/ 35Ab1 hybrids to their non- Bt isolines across multiple genetic backgrounds following exposure to natural and/or artificial CRW infe stations. Results sugge st that DAS-59122-7

offers excellent control of WCRW , NCRW, and MCRW. Further details on studies of individual pest species are provided below.

WCRW : Field trials were conducted to evaluate the efficacy of WCRW control by Cry34/35Ab1 rootworm-protected corn, compared to non- Bt isolines and chemical control measures. Each applicant conducted separate efficacy trials, and Pioneer comp leted two independent evaluations (Dow completed one evaluation). All trials were re plicated in four locat ions, using a randomized complete block design with three blocks per locati on. Fields used in these tria ls had been planted to co rn in previous years and

contained natural infestations of western and north ern corn rootworm. In addition, corn plants were artificially infested with WCRW eggs (1,000 eggs per plant) at plant growth stage V3. Roots were harvested and scored when larvae reached pupation, wh ich was at approximately plant growth stage R1. Event DAS-59122-7 corn roots consistently showed less WCRW damage compared to non- Bt isolines with no chemical control (Tables 1, 2, and 3). Howe ver, results suggest th at efficacy of Cry34/35Ab1 corn is similar, or slightly better, than that of non- Bt isolines treated with chemical insecticides.

Table 1. Efficacy of Cry34/35Ab1 Rootworm-P rotected Corn for Control of WCRW in 2003
(1 st Pioneer Study).

Pioneer Experiment #1

Corn Line York, NE Johnston, IA Janesville, WI Windfall, IN Average

Non- Bt Hybrid 2.46 1 a 2 1.06 a 0.59 a 0.12 a 1.22 a

Force 3G 0.57 b 0.11 b 0.16 b 0.05 a 0.31 b

DAS-59122-7 0.06 b 0.10 b 0.06 b 0.03 a 0.12 c

1 Iowa State University Node Injury Root Rating Scale (Oleson 1998): 0.00 = no feeding damage, 1.00 = one node eaten

back to within two inches of stalk, 2.00 = tw o nodes eaten, and 3 = three or more nodes eaten.

2 Within each column, means that are followed by the same letter are not significantly different.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

175 Table 2. Efficacy of Cry34/35Ab1 Rootworm-P rotected Corn for Control of WCRW in 2003
(2 nd Pioneer Study).

Pioneer Experiment #2

Corn Line York, NE Johnston, IA Janesville, WI Rochelle, IL Average

Non- Bt Hybrid 0.99 1 a 2 0.79 ab 1.96 a 0.78 a 1.03 a

Force 3G 0.32 bc 0.45 bc 1.20 b 0.11 b 0.44 b

Counter 15G 0.25 bc 0.04 c 0.52 c 0.13 b 0.19 c

DAS-59122-7 (hybrid 1) 0.02 c 0.04 c 0.44 c 0.03 b 0.11 c

DAS-59122-7 (hybrid 2) 0.07 c 0.04 c 0.06 c 0.02 b 0.04 c

DAS-59122-7 (hybrid 3) 0.02 c 0.17 c 0.30 c 0.04 b 0.11 c

DAS-59122-7 (hybrid 4) 0.01 c 0.05 c 0.24 c 0.02 b 0.07 c

1 Iowa State University Node Injury Root Rating Scale (Oleson 1998): 0.00 = no feeding damage, 1.00 = one node eaten back to within two inches of stalk, 2.00 = tw o nodes eaten, and 3 = three or more nodes eaten.

2 Within each column, means that are followed by the same letter are not significantly different.

Table 3. Efficacy of Cry34/35Ab1 Rootworm-Pro tected Corn for Control of WCRW in 2003
(Dow Study).

Dow Experiment #1

Corn Line Huxley, IA York, NE Arlington, WI Fowler, IN Average

Non- Bt Hybrid 1.94 a 2 0.93 a 1.03 a 2.43 a 1.58 a

Force 3G 0.15 d 0.11 b 0.19 c 0.57 b 0.25 b

Counter 15G 0.02 d 0.10 b 0.09 c 0.27 b 0.12 b

DAS-59122-7 0.03 d 0.04 b 0.05 c 0.09 b 0.05 b

1 Iowa State University Node Injury Root Rating Scale (Oleson 1998): 0.00 = no feeding damage, 1.00 = one node eaten

back to within two inches of stalk, 2.00 = tw o nodes eaten, and 3 = three or more nodes eaten.

2 Within each column, means that are followed by the same letter are not significantly different.

NCRW : In 2003, each applicant completed a NCRW tria l that compared the efficacy of Cry34/35Ab1 rootworm-protected corn to a non- Bt isoline. Each trial was conduc ted in a single location, using a randomized complete block design with three replicates.

Pioneer’s evaluation of NCRW (nat ural infestations) was conducted in conjunction with their WCRW study (artificial infestations) at Jane sville, WI. Beetle emergence traps sh owed that the ratio of naturally emerging NCRW and WCRW was 1:1, but quantification of natural population pressure was difficult

due to the artificial infestation of WCRW (1,000 WCRW/plant). Root rati ngs (Tables 1 and 2) show thatBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

176 Event DAS-59122-7 is more effi cacious against CRW than non- Bt hybrids with no insecticide treatment and comparable to non- Bt hybrids treated with c onventional insecticides.

Dow’s NCRW efficacy trial, conducted at Lamberton, MN, utilized a randomized complete block design with three replicates. Fields used in this trial had been planted to corn in pr evious years and contained

natural infestations of WCRW and NCRW; however, the rati o of WCRW and NCRW was not determined. Thus, results indicate product efficacy against an undetermined co mbination of NCRW and

WCRW. Roots were harvested and sc ored when larvae reached pupation, at approximately growth stage R1. Results suggest that event DAS-59122-7 corn had less CRW damage compared to the non- Bt hybrids (Table 4).

Table 4. Efficacy of Cry34/35Ab1 Rootworm-Pro tected Corn for Control of NCRW and WCRW
at Lamberton, MN in 2003 (Dow Study).

Corn Line Average Root Rating (1–6 scale) 1

Non- Bt Hybrid 4.38 a 2

DAS-59122-7 2.54 b

1 Modified root rating scale: 1 = no visible damage, 2 = slight feeding damage on one or more roots, 3 = 1–5 roots pruned to

within 1.5 inches of stalk, 4 = one whorl of roots pruned, 5 = two whorls of roots pruned, and 6 = three whorls of roots pruned.

2 Means that are followed by the same le tter are not significantly different.

MCRW : Pioneer conducted a field trial to evaluate the efficacy of MCRW control by event DAS59122-7 corn, compared to a non- Bt isoline and a non- Bt isoline treated with a chemical control. The trial was conducted in Sugarek, TX using a randomi zed complete block design with four replicates. Fields used in this trial had been planted to corn in previous years and containe d natural infestations of MCRW. Results show that event DAS-59122-7 corn had less MCRW damage compared to non- Bt corn with and without chemical treatment (Table 5).

Table 5. Efficacy of Cry34/35Ab1 Rootworm-Pro tected Corn for Control of Mexican Corn
Rootworm at Sugarek, TX in 2003 (Pioneer Study).

Corn Line Average Root Rating

Non-Bt Hybrid 0.80 1 a 2

Non-Bt Hybrid + Aztec 0.21 a

DAS-59122-7 0.05 b

1 Iowa State University Node Injury Root Rating Scale (Oleson 1998).

2 Within each column, means that are followed by the same letter are not significantly different.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

177 ii. Biopesticides and Pollution Prevention Division (BPPD) Review

Results indicated that event DAS-59122-7 corn had less CRW damage when compared to non- Bt corn and comparable efficacy to non- Bt corn treated with c onventional insecticides. Field plots used for WCRW and NCRW efficacy studie s contained natural infestati ons of both pest species and discrimination between the species was not determined at all sites. Despite th e presence of WCRW and NCRW species, efficacy against WCRW is likely to have been adequately measured because WCRW densities were boosted through artifi cial infestations (1,000 eggs per plant). However, efficacy results

for NCRW evaluations could not be clearly determined. At the Janesville, WI location, NCRW populations were dwarfed by artifi cial WCRW infestations and, at the Lamberton, MN location, the

ratio of NCRW to WCRW was not determined. Consequently, one may conclude that low root ratings associated with CRW-protected corn suggests that NCRW was controlled at so me undetermined level.

Mexican corn rootworm efficacy was measured at one location, Sugarek, TX, under natural infestation pressure. Results indicate that event DAS-59122-7 corn was more efficacious against MCRW than nonBt hybrids with and without chemical insecticide tr eatment. Additional efficacy data, collected from multiple locations and years, would be necessary to determine whether lower root ratings were due to high product efficacy, low initial pest populations, or a combination of both.

c. Yield and Agronomic Performance

i. Summary of Submitted Information

Several yield and agronomic performance studies were completed by the applicants. Each evaluation is

described below.

In 2003, Pioneer conducted a yield trial (in conjuncti on with the first WCRW efficacy trial described above) to evaluate the benefit of event DAS-59122-7 hybrids in terms of yield performance, when compared to a non- Bt isoline hybrid treated with and without a chemical insecticide (Force 3G, applied at 5 ounces/1000 row feet). The following agronomic tr aits were evaluated: yi eld, grain density, percent moisture at harvest, accumulated growing degree-d ays to 50% silk, accumulated growing degree-days to 50% shed, percent stalk lodging, root lodging, plant heig ht, ear height, and staygr een; these traits were

evaluated to determine the overall he alth and harvesta bility of a hybrid. Trials were conducted in four locations (see WCRW efficacy trial description above), using a randomized complete block design with three replicates per location, twelve treatments per replicate, and f our rows per treatment. At plant growth stage V2, plants we re artificially infested w ith WCRW eggs at a rate of 1,000 eggs per plant.

Results show that the DAS-59122-7 corn hybrid provi ded similar agronomic performance against CRW when compared to the isoline hybrid with and without applied insecticid e. Yields were not statistically different among treatments, despit e relatively high rootworm pr essure (1,000 eggs per plant).

Dow and Pioneer conducted additional agronomic performance trials in 2003. Performance of Cry34/35Ab1 CRW-protected hybrids (two expr essing the single event DAS-59122-7 and twoBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

178 expressing DAS-59122-7 x event TC1507 (Cry1F)) were compared to that of near-isogenic hybrids containing event TC1507 and near-isogenic hybrids co ntaining no transgenes. An elite hybrid was also included in the trials. The Pioneer trials were plante d at 7 locations, and the Do w evaluations at 12 and 13 locations (Dow completed 2 diffe rent experiments). Trials took pl ace throughout the U.S. Corn Belt and fields were managed according to standard agronomic practices. Trials utilized randomized complete block design, with three rep licates per location and two rows per plot. It is not clear if fields had been planted to corn prior to experiment initiation and/or whether rootworm pressure was severe in

field trial locations.

Results indicate that hybrids w ith and without event DAS-59122-7 performed similarly for most agronomic traits; however, expression of th e Cry34/35Ab1 proteins in event DAS-59122-7 CRWprotected corn was shown to confer advantages in yield and root lodging in some locations with high rootworm pressure. Agronomic performance may have been affected by base genetics of different hybrids and genetic variability in Cry34/35Ab1 corn hybrid lines.

ii. BPPD Review

Agronomic performance of Cry34/35Ab1 CRW-prot ected hybrids, two containing event DAS-59122-7 stacked with event TC1507 expressing Cry1F (Her culex® I) and two cont aining only event DAS59122-7, was compared to that of near-isogenic hybrids containing event TC1507 and near-isogenic hybrids containing no transgenes. An el ite hybrid was also included in th e trials. Based on the review of all of the agronomic trials conducted in 2003, the following conclusions can be made. With low CRW pressure, event DAS-59122-7 (Cry34/35Ab1 CRW-protect ed corn) performed sim ilarly to conventional corn hybrids treated with insecticides. There was no yield drag associated with event DAS-59122-7. Under high CRW pressure, however, some event DAS -59122-7 corn hybrids were shown to confer advantages, in some locations, in both yield and r oot lodging over that of non- transgenic corn treated with granular insecticides. Ther e were no significant differences between stacked Cry34/35Ab1/Cry1F corn hybrids and Cry34/35Ab1 corn hybrids. The overall range of values for the measured agronomic parameters are all within the range of values obtain ed for traditional corn h ybrids and do not indicate

increased weediness.

d. Economic Benefits

i. Summary of Submitted Information

Economic benefit analyses were co nducted to evaluate the impact of Cry34/35Ab1 CRW-protected corn hybrids (event DAS-59122-7) on grower profitabilit y, compared to various insect management alternatives. The following paragraphs describe th e considerations/assumptions used to develop the benefits analyses.

Several key assumptions were developed for economi c modeling based on data collected by universities on insecticide performance under various corn rootworm densities. Published information was used to predict relationships between root damage in untreat ed corn and expected root damage in insecticide-Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

179 treated corn for granular , liquid, and seed treatments. Separate equations were developed using the 0–3 nodal injury scale (25 locations, 1999–2002) and the 1–6 Hills & Peters scale (45 locations, 1997–2001). The relationship between event DAS- 59122-7 and control root ratings wa s determined from 12 Dow and

Pioneer trials conducted in 2003. Base d on slopes of the regression equati ons, the most effective to least effective product classes in reduc ing root damage are as follows: (1) event DAS-59122-7 hybrids, (2) granular insecticides, (3) liquid insecticides, and (4) seed treatments.

The relationship between root ratings and grain yield has been extensiv ely studied under both artificial infestations and natural populations. The economic thresholds for insecticide use typically are exceeded at a root rating of 3 or higher (1–6 scale) but can be higher or lower depending upon environmental

conditions. There is also data that conventional breeding programs and planting rates influence the yield response to corn rootworm damage. To predict the relationship between root damage and yield, data

from 10 corn rootworm trials conduct ed by Iowa State University (Oleson et al . 2000, 2001, 2002, and 2003) were evaluated. The predicted difference in yield between insect icide-treated and untreated plots increases linearly with level of da mage in untreated check plots over a range from 0 to 37 bushels per acre. Regression analysis from 37 university yi eld trials from Nebraska, Minnesota, Indiana, Pennsylvania, Illinois, and Iowa provide a simila r equation from the 1–6 rati ng scale with predicted yield losses ranging from 0.6 to 39.5 bushels per acre.

Another key concern of growers is that rootworm feeding increas es the risk of lodging. Lodged cornfields yield less both due to direct physiologi cal losses and to increased harvest losses. Two additional harvest costs associated with lodging are labor costs due to reduced harvest speed and increased fuel usage if the field must be harveste d in one direction. For this economics assessment, the relationship between level of root damage and lodgi ng was determined using insecticide treatments and untreated check plots (1–6 scale: 13 locations, Figure 6; 0–3 scale, 18 University trials and 49 Pioneer strip trials). Although several other factors (e.g., soil moisture level, wind speed, and root re-growth) influence the severity of lodging, root ratings were fa irly good at predicting leve l of root lodging with R 2

values of 0.3 and 0.6.

Cropping Scenarios Three different farming operations were evaluated, using partial budge t analysis, to illustrate the potential value that Cry34/35Ab1 CRW-protected corn hybrids may bring to the grower. Three farming situations were analyzed: (1) m oderate pressure rootworm in c ontinuous corn, (2) high pressure rootworm in rotated corn, and (3) moderate pressure rootworm in rotated corn. Variable costs for corn following soybeans and continuous corn include fertili zers, herbicides, insecticide, crop insurance, tillage, planting, harvest, and drying. These variable costs were based on values reported by Duffy and Smith (2003) for 150 bushels per acre and a corn price of $2.10 was used in all situations. Note: Tables 6–8 were revised by the applicant, per the Agency’s re quest, to more accurately reflect variable costs.

Example 1: Moderate corn rootwo rm pressure, corn following corn Traditionally, corn planted in fiel ds where corn was planted in prio r years is considered to be at moderate risk for rootworm infestations througho ut the central Corn Belt, including Nebraska and parts of Iowa, Illinois, a nd Kansas. A high proportion of corn growers treat their continuousBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

180 corn acres with a planting-time application of gra nular or liquid insecticid es applied at the full, labeled rate. Adult beetle management, whic h involves scouting and typically two aerial applications of insecticide, is also implemen ted in parts of Nebraska, Colorado, Kansas, and South Dakota (Meinke 1996). Recently, high rate s eed treatment options have become available

and are included in this analysis.

If left untreated, moderate rootworm pressure is expected to reduce harvestable yield 15 bushes per acre. Based on modeling assumptions, neither a two-application adu lt control program nor use of high rate seed treatments increased vari able returns over the control despite improved standability and yield (Table 6) . In contrast, use of event DAS- 59122-7 or full, labeled rates of granular insecticides are expected to return from $10 to $14 per ac re more than untreated fields. Use of the full, labeled rate of liquid insecticides returned sl ightly less than event DAS-59122-7 or granular insecticides ($9–$12 per acre over untreated fields).

Example 2: High corn rootworm pressure, corn following soybeans

In east central Illinois and north western Indiana, fields of corn following soybeans are at high risk for root damage due to an eastern variant of the WCRW. If fields in this region have not been scouted, growers are advised to apply a planti ng-time insecticide at full, labeled rate. It is

not unusual to have untreated 1 st year cornfields average two or more nodes of roots destroyed, resulting in high levels of lodgi ng. In a situation where first-year corn is infested with high populations of corn rootworms, harvestable yiel d is predicted to be reduced by 37 bushes per acre if no treatment is applied. Liquid insecticides and high rate seed treatments are not as effective in reducing losses and lodging as gr anular insecticides. Hi gh, consistent efficacy against even high rootworm pressure give s event DAS-59122-7 a significant economic advantage over granular ($6–$13/ acre), liquid ($16–$23/acre), and seed treatment options ($37– $40/acre)(Table 7).

Example 3: Moderate corn rootworm pressure, corn following soybeans

The eastern variant of the WCRW is considered a moderate risk to corn in northeastern Indiana

and northern Illinois. This moderate risk area is expanding into southern Wisconsin, southwestern Michigan, and western Ohio. In ad dition, extended diapause by the northern corn rootworm is placing first-year corn in southe rn Minnesota, northern Iowa, and southwestern South Dakota at significant risk to damage. Both granular and liquid planting-time applications are being used in fields, particul arly in fields with a history of root lodgi ng. Frequently, growers

elect to use a reduced rate of insecticide if rootwo rm pressure in the first-year corn is moderate.

If first-year corn is infested with moderate populations of corn rootworm, use of either hybrids containing event DAS-59122-7, a reduced rate of gra nular insecticides, or reduced rates of liquid insecticides is expected to incr ease variable returns $10 to $17 per acre over the untreated field (Table 8). Use of reduced rate of granular insecticide is slightly more profitable ($2 to $7) than

either event DAS-59122-7 or a reduced rate of liquid insecticides.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

181 Table 6. Economic Analysis of Moderate P ressure Corn Rootworm in Corn Following Corn.

Untreated DAS59122-7 Granular

Insecticide Liquid

Insecticide High rate

IST Adult Control

CRW Damage

Root rating (0-3) 1 1.0 0.08 0.20 0.36 0.67 0.20 % Lodged 2 23% 2% 5% 8% 15% 5%

Variable Cost

----------------------------- $/ acre

----------------------------- Seed @ 30,000/acre 3 $37.50 $37.50 $37.50 $37.50 $37.50 $37.50 CRW premium 0 $13.13–

16.88 0 0 0 0

Low rate IST $4.50 $4.50 0 0 0 $4.50 Scouting 0 0 0 0 0 $7.00

Insecticide 0 0 $12.00–15.00 $12.00–

15.00 $17.25 $12.80–16.00

Insecticide equipment & application 0 0 $2.00 $2.00 0 $6.00

Other variable costs 4 $155.00 $155.00 $155.00 $155.00 $155.00 $155.00 Fuel adjustment 5 0 0 0 0 0 0 Labor adjustment 6 $0.55 0 0 0 $0.55 0 Harvest adjustment 7 -$2.44 -$0.15 -$0.40 -$0.70 -$1.80 -$0.70

SUBTOTAL $195.11 $209.98–

213.73 $206.10–

209.10 $205.80–

208.80 $208.50 $222.10–225.30

Yield

----------------------------- bushels/a cre

----------------------------- Maximum yield 150 150 150 150 150 150

Harvest loss from lodging 8 -3 0 0 0 -3 0

CRW loss 9 -12 -1 -3 -4 -8 -4

SUBTOTAL 135 149 147 146 139 146

----------------------------- $/ acre

-----------------------------
Gross Returns @
$2.10/bushel $283.50 $312.90 $308.70 $306.60 $291.90 $306.60

Returns over Variable

Cost $88.39 $99.17–

102.92 $99.60–102.60 $97.80–

100.80 $83.40 $81.30–84.50

1 Moderate pressure defined as root damage rating (RDR) of 1 on 0–3 rating scale in untreated field. Expected RDR in DAS-59122-7. Expected

RDR in insecticide & IST treatments.

2 % lodging = 0.4175 + 22.083*RDR.

3 Planting rate is 30,000 kernels per acre; seed cost for 80,000 kernels/unit is $100.00.

4 Includes costs for fertilizer, lime, herbicide, drying, storage, mach inery, harvest, insurance, a nd interest (Duffy and Smith 2 003) for 150 bushels/acre production.

5 Fuel adjustment: 50% of normal combine cost @ $7.87/acre (Duffy and Smith 2003) if lodging is >40%.

6 Labor adjustment for harvesting lodged corn: $9.00/hour; normal combine speed 4.5 mph; for lodging > 10% but <40%, combine speed reduced by 25%; for lodging > 40%, combine speed reduced by 50%.

7 Variable costs for harvest (hauling, drying, and handli ng) reduced $0.16/bushel for yields below 150 bushels/acre.

8 Assumes 2% loss for lodging > 10% but <40% and 5% loss for lodging > 40%.

9 CRW loss: Change in yield = 12.254*RDR .Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

182 Table 7. Economic Analysis of High Pressure Corn Rootworm in Corn Following Soybean.

Untreated DAS59122-7 Granular

Insecticide Liquid

Insecticide High rate IST

CRW Damage

Root rating (0-3) 1 2.5 0.08 0.20 0.36 1.70 % Lodged 2 56% 5% 12% 20% 38%

Variable Cost

----------------------------- $/ acre ----------------------------- Seed @ 30,000/acre 3 $37.50 $37.50 $37.50 $37.50 $37.50 CRW premium 0 $13.13–

16.88 0 0 0

Low rate IST $4.50 $4.50 0 0 0

Insecticide 0 0 $12.00–15.00 $12.00–15.00 0 Insecticide equipment & application 0 0 $2.00 $2.00 0

Other variable costs 4 $139.00 $139.00 $139.00 $139.00 $139.00 Fuel adjustment 5 $3.93 0 0 0 0 Labor adjustment 6 $1.65 0 $0.55 $0.55 $0.55 Harvest adjustment 7 -$6.10 -$0.37 -$1.48 -$2.24 -$3.77

SUBTOTAL $180.48 $193.76–

197.51 $189.57–192.57 $188.81–191.81 $190.53

Yield

----------------------------- bushels/a cre

----------------------------- Maximum yield 150 150 150 150 150 Harvest loss from lodging 8 -7 0 -3 -3 -3

CRW loss 9 -31 -2 -6 -11 -21

SUBTOTAL 112 148 141 136 126

----------------------------- $/ acre

-----------------------------
Gross Returns @
$2.10/bushel $234.90 $310.17 $295.54 $285.62 $265.49

Returns over Variable Cost $54.42 $112.66–

115.66 $102.97–105.97 $93.81–96.81 $74.96

1 High pressure defined as RDR of 2.5 on 0–3 rating scale in untreat ed field. Expected RDR in DAS- 59122-7. Expected RDR in insect icides &

IST treatments.

2 % lodging = 0.4175 + 22.083*RDR.

3 Planting rate is 30,000 kernels per acre; seed cost for 80,000 kernels/unit is $100.00.

4 Includes costs for fertilizer, lime, herbicide, drying, storage, mach inery, harvest, insurance, a nd interest (Duffy and Smith 2 003) for 150 bushels/acre production.

5 Fuel adjustment: 50% of normal combine cost @ $7.87/acre (Duffy and Smith 2003) if lodging is >40%.

6 Labor adjustment for harvesting lodged corn: $9.00/hour; normal combine speed 4.5 mph; for lodging > 10% but <40%, combine speed reduced by 25%; for lodging > 40%, combine speed reduced by 50%.

7 Variable costs for harvest (hauling, drying, and handli ng) reduced $0.16/bushel for yields below 150 bushels/acre.

8 Assumes 2% loss for lodging > 10% but <40% and 5% loss for lodging > 40%.

9 CRW loss: Change in yield = 12.254*RDR .Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

183 Table 8. Economic Analysis of Moderate Pre ssure Corn Rootworm in Corn Following Soybean.

Untreated DAS59122-7 Granular

Insecticide Liquid

Insecticide High rate IST

CRW Damage

Root rating (0-3) 1 1.0 0.08 0.2 0.4 0.7 % Lodged 2 23 2 5 8 15

Variable Cost

----------------------------- $/ acre

----------------------------- Seed @ 30,000/acre 3 $37.50 $37.50 $37.50 $37.50 $37.50 CRW premium 0 $13.13–

16.88 0 0 0

Low rate IST $4.50 $4.50 0 0 0

Insecticide 0 0 $9.00–11.25 $9.00–11.25 $17.25 Insecticide equipment & application 0 0 $2.00 $2.00 0

Other variable costs 4 $139.00 $139.00 $139.00 $139.00 $139.00 Fuel adjustment 5 0 0 0 0 0 Labor adjustment 6 $0.55 0 0 0 $0.55 Harvest adjustment 7 -$2.44 -$0.15 -$0.40 -$0.70 -$1.80

SUBTOTAL $174.61 $193.98–

197.73 $187.10–

189.35 $186.80–

189.05 $192.50

Yield

----------------------------- bushels/a cre

----------------------------- Maximum yield 150 150 150 150 150 Harvest loss from lodging 8 -3 0 0 0 -3

CRW loss 9 -12 1 3 4 8

SUBTOTAL 135 149 147 146 139

----------------------------- $/ acre

-----------------------------
Gross Returns @
$2.10/bushel $282.97 $313.07 $309.74 $305.77 $291.41

Returns over Variable Cost $108.36 $115.34–

119.09 $120.39–

122.64 $116.72–

118.97 $98.91

1 Moderate pressure defined as RDR of 1 on 0–3 rating scale in unt reated field. Expected RDR in DAS-59122-7. Expected RDR in inse cticide &

IST treatments .

2 % lodging = 0.4175 + 22.083*RDR.

3 Planting rate is 30,000 kernels per acre; seed cost for 80,000 kernels/unit is $100.00.

4 Includes costs for fertilizer, lime, herbicide, drying, storage, mach inery, harvest, insurance, a nd interest (Duffy and Smith 2 003) for 150 bushels/acre production.

5 Fuel adjustment: 50% of normal combine cost @ $7.87/acre (Duffy and Smith 2003) if lodging is >40%.

6 Labor adjustment for harvesting lodged corn: $9.00/hour; normal combine speed 4.5 mph; for lodging > 10% but <40%, combine speed reduced by 25%; for lodging > 40%, combine speed reduced by 50%.

7 Variable costs for harvest (hauling, drying, and handli ng) reduced $0.16/bushel for yields below 150 bushels/acre.

8 Harvest loss: assumes 2% loss for lodging > 10% but <40% and 5% loss for lodging > 40%.

9 CRW loss: Change in yield = 12.254*RDR .Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

184 ii. BPPD Review

Dow and Pioneer analyzed three different farming ope rations to illustrate th e economic benefits that event DAS-59122-7 (Cry34/35Ab1) corn hybrids may provi de to growers. Per the Agency’s request, Dow and Pioneer revised the original tables (T ables 19–21; Master Record Identification Number (MRID No.) 461239-21) to include va riable cost adjustments, base d on predicted yield, for hauling, drying, and handling. By using a static value for variable costs (origina l tables), economic models were slightly biased in favor of higher yielding treat ments (Cry34/35Ab1 corn hybr ids) and against lower yielding treatments because static va lues do not consider the effect that yield has on operating costs. The

revised tables showed increased re turns over variable costs for the un treated corn hybrids and high-rate insecticide treatments but had minimal imp act on other treatments (Tables 6–8).

BPPD agrees with Dow and Pioneer’s analysis of the economic benefits that event DAS-59122-7 corn hybrids may provide to growers. Potential benefits include higher corn yields, reduced expenditures on insecticides and insecticide appli cation, and the potential for lower ha rvest costs due to reduced lodging.

Additional costs associated with event DAS-59122-7 include higher seed prices and use of a refuge. Net benefits depend on the severity of the corn root worm problem and on the price of event DAS-59122-7 seed. Models suggest that, in continuous corn fiel ds with moderate CRW pressure, hybrids containing event DAS-59122-7 and insecticide-treated non-transg enic varieties should pr ovide similar returns (Table 6). In contrast, slightly hi gher returns are predicted in corn-s oybean rotations with high rootworm pressure (Table 7). Additional bene fits, which are not quantified in th ese models, include the potential for more predictable yields and reduced exposure to in secticides. Future models should also consider the

cost-benefit of resistance management programs.

e. Practical Benefits

i. Summary of Submitted Information

Cry34/35Ab1 (event DAS-59122-7) corn offers many practical advantages to corn growers over other

CRW control measures

. First, longer season varieties can be us ed, because later planting dates, which have traditionally helped to mitigate CRW dama ge, are unnecessary. Second, compared to use of chemical insecticides, growers should be able to plant more quickly becau se they won’t have to stop and refill the insecticide boxes. Third, insecticidal seed treatments may be applied to Cry34/35Ab1 seeds to permit control of associated pests such as wire worm, grub, maggots, and cutw orms, providing protection against multiple pests. Fourth, planting Cry34/35Ab1 co rn is expected to save the grower money in application, insecticide, labor, fuel , equipment, storage, and packaging disposal costs (no pesticide containers). Fifth, Cry34/35Ab1 corn is labeled for general use and t hus, provides an alternative to restricted use products. Sixth, Cr y34/35Ab1 corn is expected to provide the grower and other occupational workers greater safety, as well as fewer adverse environmental impacts, compared to use of

chemical insecticides.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

185 ii. BPPD Review

BPPD agrees that Cry34/35Ab1 co rn offers many practical adva ntages to corn growers over conventional insecticides, as stated above.

f. Market Adoption and Pesticide Use Reduction

i. Summary of Submitted Information

It is expected that growers w ho plant Cry34/35Ab1 CRW-protected corn will significantly reduce use of chemical insecticides for rootworm control. The addition of commercially available Cry34/35Ab1 corn will benefit growers for the following reasons: (1 ) corn varieties containing the Cry34/35Ab1 CRWprotected corn will compete with Cry3Bb1 corn va rieties, creating price co mpetition among transgenic CRW control technologies; and (2) corn varieties containing the Cr y34/35Ab1 offer alternatives to current Cry3Bb1 CRW-protected corn varieties in terms of plant ge netics and performance attributes.

ii. BPPD Review

Rootworm damage may cost U.S. corn growers $1 billion annually in control costs and crop losses (Gianessi et al . 2002). In the year 2000, almost 8 million pounds of CRW insecticide, costing $172 million, were applied to 14 million acres or 17% of U.S. corn acreage ($12.29 per acre). CRW-infested acreage is projected to increase from approximate ly 31.8 million acres in 2005 to 39 million acres by 2013 (Table 9).

Grower demand for CRW control technologies is infl uenced by the level of CR W infestation (acreage and degree of infestation), comparative cost-benefit of competing CRW control technologies, U.S. and global market acceptance and approval of a tech nology, and other regulatory constraints (e.g., refuge requirements). BPPD anticipates, through evaluation of National Agricultural Stat istics Service (NASS) (of the United States Department of Agriculture) a nd Doane® Agricultural Services databases on CRW damage and control costs, that the market for tran sgenic in-plant CRW prot ection will increase by 2.6% per year, reaching 18 to 19 million acres by the year 2013 (Table 9).

Anticipated economic and pesticide use reduction benefits of Cry34/35Ab1 corn largely depend on market penetration. Introduction of commercially available Cry34/35A b1 corn hybrids is expected to create price competition among commercially availa ble transgenic CRW-protected corn products and will offer a greater variety of plant genetics and performance attributes to growers.

Increased adoption of transgenic CRW-protected corn products is expected to result in reduced use of many of the chemical insecticides registered for CR W control, which are highly toxic to humans and the environment, are in Special Review, and have restrict ed use labels. The chemical insecticides (larvicides and adulticides) subject to the greatest use reduction following adop tion of transgenic CRW-protected hybrids are the following: organophosphates (chlorpyr ifos, tebupirimphos, terbuf os, methyl parathion, and chlorethoxyfos), pyrethroids (tefluthrin, cyfluthri n, bifenthrin, and lambda cyhalothrin), carbamatesBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

186 (carbofuran), and pyrazoles (fipronil) (Table 10). Adoption of transgen ic CRW-protected corn products is not expected to result in redu ced use of seed treatments (nico tinoids); however, seed treatment products are generally less toxic than at-plant and post-plant products.

Table 9. Projected Acreage with Corn Rootwo rm Infestation and Breakdown of Associated

CRW Control Practices for the Years 2000 to 2013 (Note: Informat ion Presented for

2000 and 2002 Reflect Actual Infest ation and Insect Control Tactics).

Acres Infested Acres Treated CRW-Protected

Bt Corn Acreage Conventional

Treatments Year

------------------------------------ -----acreage x 10 6

--------------- ------------------------- 2000 28.0 14.0 0.0 14.0

2001 1

- - - - 2002 29.5 14.7 0.0 14.7

2003 30.2 15.1 1.0 14.1

2004 31.0 15.5 2.5 13.0

2005 31.8 15.9 4.0 11.9

2006 32.6 16.3 6.0 10.3

2007 33.5 16.7 7.2 9.5

2008 34.3 17.2 8.6 8.5

2009 35.2 17.6 10.4 7.2

2010 36.1 18.1 11.9 6.1

2011 37.1 18.5 13.7 4.8

2012 38.0 19.0 15.8 3.2

2013 39.0 19.5 16.8 2.7

Annual Growth

Rate 2.58% 2.58% 16.8% -14.36%

1 The Agency does not have data for the 2001 growing season.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

187 Table 10. Use of Chlorpyrifos, Phorate, Tefluthr in, and Terbufos Insecticides for Rootworm

Control in Corn.

Chlorpyrifos Phorate Tefluthrin Terbufos

Source of data 1 G U-97 U-03 G U-97 U-03 G U-97 U-03 G U-97 U-03

State . ------------- ---------------1,000 pounds act ive ingredient -----

------------- ------------

---------------

Colorado 7 - 2 12 1 - 218 125

Illinois 810 2,105 747 120 – 68 57 205 466 1,117 –

Indiana 299 267 621 62 36 31 93 558 – 473

Iowa 1,044 1,248 366 114 – 71 35 – 366 922 –

Kansas 91 – 19 – 226

Maryland 45 5 11

Michigan 135 153 146 47 15 – 155 – –

Minnesota 265 - 214 129 – 21 – – 206 – –

Missouri 232 315 106 25 – 1 – – 23

Nebraska 294 382 - 134 68 270 76 879 748 246

New York 216 80 147 11 24 –

North Dakota 48 1 107 –

Ohio 363 429 67 36 – 46 – – – –

Oklahoma 1 2 14

Pennsylvania 132 151 15 – 15 21 31 –

South Dakota 480 – – 226 10 7 – 502 –

Texas 50 44 – 5 512 228

Wisconsin 286 254 96 70 – 32 32 43 385 – –

Total - sum of rows 4,750 5,153 2,638 1,037 – – 558 432 454 4,681 2,787 1,072

Total - as reported in USDA 5,341 3,024 522 523 3,200 1,660

1 G = Gianessi et al . (2002), data from 1997 U-97 = USDA/NASS (1998), data for 1997 U-03 = USDA/NASS (2004), data for 2003

2 A - indicates that usage data not published for this active ingredientBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

188 g. Direct and Indirect Economic Benefits

i. Summary of Submitted Information

Shifting from conventional CRW cont rol technologies to Cry34/35Ab1 CR W-protected corn is expected to provide direct economic benefits to farmers, by lessening the co sts associated with pest control; transgenic corn seed is generally less expensive than chemical pesticide control programs. Indirect benefits are also expected to result from adoptio n of Cry34/35Ab1 CRW-protec ted corn. Benefits may include reduced energy consumpti on, resulting from a decline in the manufacture, transport, and application of chemical insecticides. Waste products , arising from pesticide manufacturing and residual chemical stocks, may also be reduced pending a dec line in chemical pesticide manufacture and sales.

Dow and Pioneer calculated indirect economic bene fits based on reduced chemical insecticide use following adoption of Cry34/35Ab1 technology. Chemi cal insecticides consume roughly 2.25 x 10 8
British thermal units (BTU) per ton for manufacture and distri bution, and approximately 94,000 BTU per acre are used for a single pe sticide applicat ion. Estimated energy savings are approximately 660,000 barrels of crude oil (3.83 x 10 12 BTU), assuming a targeted adoption of 23.4 x 10 6 acres Cry34/35Ab1 Bt corn by 2014. These calculations are based on 2.25 x 10 6 BTU/ton of [active ingredient] pesticide for manufacturing and distribution, a nd 94,000 BTU/acre for application (one application per acre). Using

Gianessi et al . (2002)’s 14.5 million pound (7,250 tons) estimate of applied CRW insecticide (annually), estimated annual energy savings would be: [(7,250 tons)(2.25 x 10 8 BTU/ton) + (23.4 x 10 6
acres)(94,000 BTU/acre) = 3.83 x 10 12 BTU, at 5.8 x 10 6 BTU/barrel of oil = 660,000 barrels of oil per

year.

ii. BPPD Review

BPPD agrees with Dow and Pioneer’s conclusion that indirect economic benefits will be obtained from adoption of Cry34/35Ab1 CRW-protected corn. However, the applicants ’ estimates of energy savings may be inflated. There are four assumptions to ch eck: (1) energy for pesticide manufacturing and distribution; (2) amount of pesticid e applied; (3) energy fo r application; and (4) number of acres. BPPD agrees with the applicants’ conclusions on assump tions 1, 2, and 4. However, assumption 3 (“energy for application”) may be over estimated a . A value within the range of 15,730 to 25,220 BTU/acre may be more accurate than the appli cants’ estimate of 94,000 BTU/acre.

a From Bhat et al . (1994), energy to manufacture insectic ides is estimated to be 210.7 x 10 6 BTU/ton and for formulation, packaging, and transportation ranges from 11.2 to 33.5 x 10 6 BTU/ton of active ingredient. An estimate of 2.25 x 10 8 BTU/ton of [active ingredient] pesticide for manufacturing and distribution is valid. From a number of crop budgets, a pplication on corn is typically assumed to be done using a 50-foot boom sprayer pulled by a 60-horsepower (hp) trac tor that covers 25.6 acres/hour. A 60-hp tractor consumes 2.64 gallons of fuel/hour based on the American Society of Agricultural Engineer s (ASAE 2001) standards for calcul ating fuel consumption (0.044 gallons of diesel/horsepower-hour). The American Agricultural Economics Association (2000) me thodology for estimating costs of farming incorporates the ASAE standards and also a ssumes that powered equipment, such as tractors, operate 10% longer than is needed fo r the actual field operation. Thus, the 60-hp tractor would consume 2.9 04 gallons of diesel/machine hour in the field (402,760 BTU/ma chine hour based on 138,690 BTU/gallon of diesel). To calculate the area covered by the spra yer, assume it travels 6.5 miles per hour (mph) with a field efficiency of 0. 65 (i.e., it does productive work 65% of the tim e) (ASAE 2001) [6.5 mph * 5280 ft/mi * 50 ft/(43,560 ft 2 /acre) = 25.6 acres/hour]. Based on 25.6 acres/hour, th is is equivalent to 15,730 BTU/acre.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

189 h. Human Health Benefits

i. Summary of Submitted Information

Cry34/35Ab1 CRW-protected corn is e xpected to be safer for handlers, applicators, farmers, and the public than chemical pesticides in current use. Twen ty-five of the 39 registered conventional insecticides

used to control CRW are classified as “Restricted Use,” 12 have the “Danger” label classification, and several are in Agency Special Review (e.g., dimethoate , phorate, and terbufos). Further, each year there are confirmed reports of human illness associated with these registered chemical insecticides.

Adoption of Cry34/35Ab1 corn hybrids has the potent ial to reduce occupationa l, farmer, and public

health risks associated with the manufacture, transportation, storage, handling, application, and disposal

of conventional insecticides , by providing a safer alternative for CRW control.

ii. BPPD Review

BPPD has concluded that the Cry34Ab1 and Cry35Ab1 pr oteins are unlikely to be allergens and that there is reasonable certainty of no harm from aggregate e xposure to the proteins as expressed in corn. (see the Human Health Assessment and Product Characterization chap ters of this Biopesticides Registration Action Document (BR AD) for more information on potential human health effects.)

i. Environmental Benefits

i. Summary of Submitted Information

All of the major chemicals used for CRW control can cause major a dverse environmental effects under conditions of normal use. Further, these chemicals can spread via spray drift and runoff, contaminating both land and water bodies and impacting non-target orga nisms. Fifteen of these products are labeled as “toxic,” 6 as “highly toxic,” 1 as “very highly toxic,” and 14 as “extrem ely toxic” to birds, fish, and

other wildlife. Several of the s ynthetic insecticides, in particul ar organophosphates and synthetic pyrethroids, exhibit moderate to high toxicity to terrestrial non-target species. Of special concern are methyl parathion and carbofuran, both of which ar e implicated in bird kills. The three top CRW insecticides (Table 11)—terbufos, chlorpyrifos, and tefluthrin—accoun t for the majority of the acres treated (63%) for CRW control. These three CRW in secticides pose greater ecological risk than do Cry34/35Ab1 CRW-protected corn hybrids.

In contrast, the Cry34/35Ab1 protei n is essentially non-toxic to non-ta rget species, including endangered and beneficial species. The Cry34Ab1 and Cry35Ab1 prot eins rapidly degrade soil, off-target exposure to non-target species through pollen or soil residues will be insignificant, and there is a minimum potential for runoff; exposure to Cry34Ab1 and Cr y35Ab1 is minimal or non-existent. Reduced runoff reduces environmental pollution. The ecological risk assessment and characte rization of the Cry34Ab1Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

190 and Cry35Ab1 proteins, as expressed in corn, suggests that these protei ns pose no significant ecological risk to non-target species, including endangered and be neficial species.

ii. BPPD Review

See BPPD’s Cry34/35Ab1 CRW-protected corn hybrid Environmental Assessm ent chapter in this BRAD for more information on the pot ential for ecological effects.

Table 11. Comparison of Ecological Risks Asso ciated with Terbufos, Chlorpyrifos, and

Tefluthrin.

Endpoint Terbufos Chlorpyrifos Tefluthrin

Mammalian Acute RQ 50 1 0.008

Avian Acute RQ 0.27 0.55 0.0001

Fish acute RQ 11 2 0.77

Freshwater invertebrate RQ 50 20 0.77

Marine/Estuarine Invertebrate RQ 53 162 0.87

a Risk is defined as the risk quotient (RQ) >level of concern (LOC). RQ = Toxicity/Exposure. LOC = 1

j. Insect Resistance Management Benefits

i. Summary of Submitted Information

There is concern about the ability of target pest s to evolve resistance to CRW control mechanisms, including crop rotation, chemical insecticides, and CRW-protected Bt -corn products. Currently, the favored management practice for CRW control is crop rotation, specifically corn-soybean rotations, complimented by application of chemical insectic ides (e.g., pyrethroids, organophosphates, carbamates, and pyrazoles) and, more recently, neonicotinoids as s eed treatments. Due to continuous use of a small number of active ingredients, CR W has developed resistance to a number of these chemical control products. The recent development of the NCRW ex tended diapause and WCRW soybean resistant variants have further reduced the efficacy of cr op rotation and chemical control options. Since Cry34/35Ab1 CRW-protected corn presents a new m ode of action and has good efficacy against CRW, introduction of this corn product is expected to extend the durabi lity of existing rootworm control measures, including other commercially available CRW-protected Bt -corn products (e.g., MON 863). To ensure the long-term efficacy of Cry34/35Ab1 CRW-pr otected corn hybrids, an insect resistance management plan should be implemented.

ii. BPPD Review

See BPPD’s Cry34/35Ab1 Insect Resistance Management chapter of this BRAD for the analysis of the dose, mode of action, durability modeling, and the proposed insect resistance management plan for Cry34/35Ab1 CRW-protected corn hybrids.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

191 3. References

AAEA.2000. Commodity Costs and Returns Estimati on Handbook, American Agricultural Economics Association, Ames, Iowa.

ASAE. 2001. ASAE Standards, 48 th Edition, American Society of Agri cultural Engineers, St. Joseph, Michigan.

Bhat MG, English BC, Turhollow AF, Nyangito HO. 1994. Energy in Synthetic Fertilizers and

Pesticides: Revisited. Available from: http://www.osti.gov/bridge/purl.cover.js p;jsessionid=B5046E886528DE6ADC405B64E9E49731 ?purl=/10120269-p6yhLc/webviewable/

.

Duffy M, Smith D. 2003. Estimated costs of crop production in Iowa - 2003. FM 1712. Iowa State University, University Extension, Ames, IA.

Gianessi LP, Silvers CS, Sankula S, Carpenter JE . 2002. Plant Biotechnology: Current and Potential Impact for Improving Pest Management in U.S. Agriculture: An Analysis of 40 Case Studies.

Available from: http://www.ncfap.org/40casestudies.html

.

Levine E, Oloumi-Sadeghi H. 1991. Management of Diabroticite rootworms in corn. Annual Review of Entomology 36:229–255.

Meinke LJ. 1996. Adult corn rootworm management. University of Nebraska -Lincoln publication no.

MP63-C.

Meinke LJ, Siegfried BD, Wright RJ, Chandler LD. 1998. Adult susceptib ility of Nebraska western corn rootworm (Coleoptera: Chrysomelidae) populations to selected insecticides. Journal of Economic Entomology 91(3):594–600.

MRID No. 461227-01. Bing J, Coats I, Davis P. 2003. Public Interest Docume nt for Pioneer Brand Bt Cry34/35Ab1 Corn Construct PHP 17662. Proj ect Number: PHI/2003/059. Unpublished study prepared by Pioneer Hi-Bred Intern ational, Incorpor ated, 607 pages.

MRID No. 461239-21. Bing J, Coats I, Ernest A. 2003. Public Interest Document for Mycogen Brand

B

. t . Cry34/35Ab1 Construct PHP17662 Insecticidal Crystal Protein as Expressed in Corn. Project Number: GH/C/5704, 5,704 pages.

Oleson JD. 1998. Joint Meeting, Entomological Society of America and American Phytopathological Society, Display Presentation, D 428 Linear Scale for Evaluating Corn Rootworm Larval Injury. November 8–12, Las Vegas, Nevada. Available from: http://www.ent.iastate.edu/pest/r ootworm/nodeinjury/nodeinjury.html

.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

192 Oleson JD, Nowatzki TM, Wilson TA, Tollefson JJ. 2000. Corn rootworm larval control, 1999. Arthropod Mgmt. Test, v. 25, rep F50.

Oleson JD, Nowatzki TM, Wilson TA, Park Y, Tolle fson JJ. 2001. Corn rootworm larval control, 2000. Arthropod Mgmt. Test, v. 26, rep F33.

Oleson JD, Nowatzki TM, Wilson TA, Park Y, Tollefs on JJ. 2002. Evaluation of soil insecticides for control of corn rootworm larvae, 2001. Arthropod Mgmt. Test, v. 27, rep F27.

Oleson JD, Wilson TA, Park Y, Tollefson JJ. 2003. Evalua tion of soil insecticides for control of corn rootworm larvae, 2002. Arthropod Mgmt. Test, v. 28, rep F32.

Scharf ME, Meinke LJ, Siegfried BD, Wright RJ, Chandler LD. 1999. Carbaryl susceptibility, diagnostic concentration determination, and s ynergism for U.S. populations of western corn rootworm (Coleoptera: Chrysomelidae). Journal of Economic Entomology 92(1):33–39.

USDA/NASS. 1998. Agricultural Chemical Usage: 1997 Field Crops Summary. Available from: http://usda.mannlib.cornell.edu/usda/nass/ AgriChemUsFC/ /1990s/1998/AgriChemUsFC-05-20- 1998.pdf 1998

.

USDA/NASS. 2004. Agricultural Chemical Usage: 2003 Field Crops Summary. Available from: http://usda.mannlib.cornell.edu/usda/nass/ AgriChemUsFC/ /2000s/2004/AgriChemUsFC-05-20- 2004.pdf

.

U.S. EPA. 2005. EPA Public Intere st Finding and Review of the Bene fits Associated with Cry34/35

Rootworm-Protected Field Corn Products for the Section 3(c)(7)(C) Full Commercial Registration. Memorandum from T. Milofsky, S.R. Matten, Ph.D., and E.B. Brandt to M. Mendelsohn dated July 5, 2005.

Zhu KY, Wilde GE, Higgins RA, Sloderbeck PE, Busc hman LL, Shufran RA, Whitworth RJ, Starkey SR, He F. 2001. Evidence of evolving carbaryl resi stance in western corn r ootworm (Coleoptera: Chrysomelidae) in areawide-managed cornfields in north central Kansas. Journal of Economic Entomology 94(4):929–934.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

193 III. REGULATORY POSITION FOR EVENT DAS- 59122-7 CORN

A. Initial Registration (August 31, 2005)

Pursuant to section 3(c)(7)(C) of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), the Environmental Protection Agency (EPA) may conditionally register a new pesticide active ingredient for a period of time reasonably sufficient for the generation and submission of required data that are lacking because insufficient time has elapsed since the impositi on of the data requirement for those data to be developed. EPA may grant such conditional registration only if EPA determines that (1) the use of the pesticide product during the period of the conditiona l registration will not cause any unreasonable adverse effect on the environment, and (2) the regist ration and use of the pesticide during the conditional registration is in the public interest. EPA determines that all of these criteria have been fulfilled.

The first criterion under FIFRA section 3(c)(7)(C ), as mentioned above, has been met because insufficient time has elapsed since the impositi on of the requirements for the following data:

1. Independent laboratory analytical method validation. 2. Field degradation studies evaluating accu mulation and persistence of the Cry34Ab1 and Cry35Ab1 proteins in several di fferent soils in various strata. 3. Laboratory toxicity test with Orius insidiosus (minute pirate bug). 4. Laboratory toxicity test with carabid (ground beetle). 5. Multi-year non-target organism field studies.

The applicants submitted and/or cited data sufficien t for EPA to determine that conditional registration

of the Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins and the genetic material necessary for their production (PHP17662 T-DNA) in event DAS -59122-7 corn (Organization for Economic Cooperation and Development (OECD) Unique Identifier: DAS-59122-7) under FIFRA section 3(c)(7)(C) will not result in unreason able adverse effects to the environment, as discussed above. The applicants submitted and/or cited satisfactory data pertaining to the proposed use. The human health effects and non-target organism eff ects data are considered sufficien t for the period of the conditional registration. These data demonstrate that no foreseeab le human health hazards or environmental effects are likely to arise from the use of the products and, furthermore, that the risk of resistance developing to the Cry34Ab1 and Cry35Ab1 proteins during the conditi onal registrations is not expected to be significant.

Registration of the Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins and the genetic material necessary for their production (PHP17662 T-DNA) in event DAS-59122-7 corn (OECD Unique Identifier: DAS-59122-7) is in the public interest for the following reasons:

1. Cry34/35Ab1-protected corn provides effective control of key rootwo rm pests of field corn and may prove more efficacious than chemical insectic ides presently registered for this purpose.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

194 2. Economic models suggest that, under conditi ons of high rootworm pressure, use of Cry34/35Ab1-protected corn will pr ovide greater net returns to fa rmers. Cost benefits include reduced expenditures on insecticides, applica tion equipment, and personnel, complimented by greater potential corn yields. U nder high rootworm pressure, thes e benefits are expected to outweigh the higher cost of seed.

3. Registration of Cry34/35Ab1-protec ted corn is expected to re sult in further reduction of chemical insecticide use by growers. This is of special importance since many pesticides registered for corn rootworm (CRW) control ar e highly toxic to humans and the environment, while Cry34/35Ab1-expressing corn poses no fores eeable human health or environmental risks.

4. Cry34/35Ab1 corn will be the second CRW-protect ed corn trait on the ma rket (the first was Cry3Bb1). The availability of multiple CRW-protected corn products will increase grower choice and price competition, resulting in lower seed prices for consumers and higher adoption

rates.

5. The Cry34/35Ab1 CRW-protected corn will provide a different mode of action and extend the durability of other CRW control measures, including other Bacillus thuringiensis CRWprotected corn hybrids.

In view of these minimal risks and the clear benefits related to the Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins and the genetic material necessary for their pr oduction (PHP17662 T-DNA) in event DAS-59122-7 corn (OECD Unique Identifier: DAS-59122- 7), EPA believes that the use of the products during the limited period of the c onditional registrations will not cause any unreasonable adverse effects.

Although the data with respect to th is particular new active ingredient are satisfactory, they are not sufficient to support an unconditi onal registration under FIFRA secti on 3(c)(5). Additional data are necessary to evaluate the risk posed by the cont inued use of these products. Consequently, EPA is imposing the data requirements speci fied earlier in this chapter.

EPA has determined, as explained in section II(E) of this Biopesticides Re gistration Action Document (BRAD), that the third criterion for a FIFRA section 3(c)(7)(C) condi tional registration has been

fulfilled because use of the Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 proteins and the genetic material necessary for their production (PHP17662 T-DNA) in event DAS-59122-7 corn (OECD Unique Identifier: DAS-59122-7) under these registra tions is in the p ublic interest.

The submitted data, in support of these registrati ons under FIFRA section 3(c)(7)(C), have been reviewed and determined to be adequate. Studies me ntioned above are included in the terms, conditions, and limitations of these registrations. These registrati ons will not cause unreas onable adverse effects to

man or the environment and ar e in the public interest.

The expiration date of the registratio ns has been set to September 30, 2010.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

195 B. 2010 Update

Section 3(c)(7)(A) of FIFRA provides for the regi stration or amendment of a pesticide when the pesticide and proposed use “...are iden tical or substantially similar to any currently regi stered pesticide and use thereof, or differ only in ways that would not significantly increase the risk of unreasonable adverse effects on the environment, and (ii) approving the registration or amendment in the manner proposed by the applicant would not significantly increase the risk of any unreasonable adverse effect on

the environment.” Unreasonable adverse effects on th e environment are defined under section 2(bb) of FIFRA as “... any unreasonable risk to man or the environment, taking into account the economic, social, and environmental costs and benefits of th e use of any pesticide...” Thus, pursuant to section 3(c)(7)(A), EPA may conditionally re gister a pesticide if (1) the pesticide and its proposed use are identical or substantially similar to a currently regist ered pesticide; or (2) the pesticide and its proposed use differ only in ways that would not significantly incr ease the risk of unreasonable adverse effects; and (3) approving the registration woul d not significantly incr ease the risk of any unreasonable adverse

effect.

The Agency concludes that the following Cry34/35A b1 corn product registrations, set to expire in September 2010 and described in-depth throughout this BRAD, meet both criteria (1) and (2):

(1) Event DAS-59122-7 Corn (EPA Reg. Nos. 68467-5 and 29964-4) (2) Herculex® XTRA Insect Protecti on Corn (EPA Reg. Nos. 68467-6 and 29964-5)

All of these Cry34/35Ab1 corn produ cts are identical in both composition and use (corn) to plantincorporated protectants that are currently registered. Thus, criterion (1) has been fulfilled.

With regard to criterion (2), the Agency maintains, as was previously determined for the original registration of these particular products, that cu ltivation of Cry34/35Ab1-cont aining corn will not cause

unreasonable adverse effects on the environment. The conditional environmental effects data, submitted in response to terms and conditions of registration and summarized in section II(C)(2)(b) of this BRAD, strengthen the Agency’s initial po sition and also confirm that long-te rm effects on non-target organisms are not anticipated. Lastly, the c ontinued use of these products will likely still provide many of the

benefits as were evaluated in section II(E) of this BRAD to support the 2005 registration of Event DAS59122-7 corn (e.g., reduction in use of conventional insecticides that are highly toxic to both humans and the environment).Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

196 In conclusion, as the expiring Cry34/35Ab1 products have met the required criteria under section 3(c)(7)(A) of FIFRA, the Agency is amending these registrations to extend their respective expiration

dates a as follows:

Product Name (EPA Reg. No.) Expiration Date

Herculex® RW Insect Protection (68467-5) September 30, 2015

Herculex® Rootworm Insect Protection (29964-4) September 30, 2015

Herculex® XTRA Insect Protection (68467-6) September 30, 2015

Herculex® XTRA Insect Protection (29964-5) September 30, 2015

Although data provided were satisfac tory to make the determinations required by section 3(c)(7)(A) of FIFRA, they were not sufficient to support an unc onditional registration under FIFRA section 3(c)(5). Additional data, specifically in relation to insect resistance management, are necessary for a finding of registrability under FIFRA section 3(c)(5) and will rema in as terms or conditions for the purposes of the

amendments.

a See section III(C) of this BRAD for an explanation describing how the expiration dates were determined.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

197 C. Period of Registration

In the 2001 Bt Corn reassessment, EPA determined that it was appropriate to amend the then-existing registrations to extend the period of registration of those products to an expiration da te of October 15, 2008. All of the products being assess ed at that time were efficacious against lepidopteran pests. EPA based this action on the finding that use of Cry1Ab or Cry1F expresse d in corn will not significantly

increase the risk of unreasonable adverse effects on the environment “for the limited time period of 7 additional years (to October 15, 2008) .” These registrations were late r amended to extend the period of registration to an expiration date of Septem ber 30, 2010. EPA subsequently granted time-limited registrations to products efficacious against coleopteran corn rootworm pests. For example, EPA registered Cry3Bb1 on February 24, 2003, to May 1, 2004, and extended that re gistration twice, to February 24, 2008, and September 30, 2010.

As set forth elsewhere in this docum ent, EPA’s primary concern for the Bt protected transgenic corn products is the possibility that ta rget pests will devel op resistance to one or more of the plantincorporated protectant (PIP) toxins . Development of resistance to a Bt toxin would be a grave adverse effect, and, for over 15 years, EPA has imposed st ringent requirements in tended to countermand the potential development of resistance. Registrants similarly have been busily developing various products, product mixes (i.e., so-called “pyramids” and “stack s”), and resistance st rategies, to maximize agronomic benefits and address resistance management issues. The result has been a vast array of product combinations and, occurring over the past couple of years, a re-emergence of varying refuge requirements for different products.

As discussed in the 2001 Bt PIP BRAD (at IID13), the earliest Bt corn registrations did not include mandatory refuge requirements. There was a lack of scientific consensus as to what the appropriate refuge requirement should be, and, it was assumed th at the limited market penetration of these early crops would be so low as to gua rantee that adequate natural re fuges would be available from neighboring non- Bt corn fields. From 1995 to 1997, Bt corn registrations included voluntary refuge requirements of 0% to 20% in the Corn Belt. In 1999, the Agricultural Biotechnology Stewardship Technical Committee (ABSTC), in conjunction with the National Corn Growers Association, proposed

uniform insect resistance mana gement (IRM) requirements for Bt corn registrations. With some modifications, this proposal, put in place for the 2000 growing season, formed the baseline IRM requirements for almost all Bt corn registrations for the better part of a decade: farmers were required to plant a 20% refuge that could be tr eated for insects, or a 50% treated refuge in cotton-growing areas; all refuges to be planted within one-half mile of the Bt corn field.

These uniform requirements brought cer tainty and consistency to the ma rket after the initial period where many Bt corn products had different refuge requirements. Recently, however, as product developers have begun to conceive of products with different com binations of “pyramided” products (i.e., products containing two or more toxins efficacious against the sa me pest) and “stacked” products (i.e., products combining toxins efficacious against different pests), the ref uge requirements have begun to vary. For example, certain products require a 20% external refuge; some products permit a 5%Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

198 external refuge; one product incorpor ates a 10% seed blend refuge; we have applications in process for products that propose to incorporat e a 5% seed blend refuge; and ot her permutations are possible.

Given the profusion of various toxi n combinations and refuge options , we can no longer proceed on the basis that, as concerns insect resi stance management, all products are eq ual. It was a relatively simple proposition when the default requir ement of a 20% sprayed refuge applied to almost all of the Bt corn crops in the market. Under thos e circumstances, the relative dura bility of products against the development of resistance was functionally equiva lent, and, as a consequence, imposing functionally equivalent registration periods was appr opriate. That is now no longer the case.

As part of our continually e volving regulatory approach to th e continually evolving product mix wrought by developers, we think it a ppropriate to revise our regulator y requirements in scientifically defensible ways to reflect the comparative level of risks posed by the products that we regulate. Here, for example, where we’ve determined that a partic ular product, or category of products, likely will pose less risk of insect resistance developing to a particul ar PIP protein, we think it appropriate to grant that particular product, or category of products, a regi stration for a period greater than that granted a corresponding product that poses a gr eater risk of insect resistan ce developing. This approach is reflective of complementary principles: first, to en sure that we apply our limited resources to the products that pose greater risk of adverse effect s to the environment; and, second, to conserve the resources that registrants and applicants must expe nd in amending the registrations of products that pose

less risk of adverse effects to the environment.

The scheme that we are following in cludes registration periods of five, eight, and twelve years; a fifteenyear registration period will also be available, if adequately supported by our science assessment. In this scheme, (i) a product with a single PIP toxin, and a 20% external refuge, qualifies for a five-year registration; (ii) a product with pyramided PIP toxins (i.e., two or more toxins with distinct, non-cross reacting modes of action), that are non-high dose (the definition for a high dose product remains unchanged), with either a seed blend or external refuge, qualifies for an eight-year registration; (iii) a product with pyramided PIP toxins (i.e., two or more toxins with distinct, non-cross reacting modes of action), that are high-dose , with either a seed ble nd or external refuge, qu alifies for a twelve-year registration; (iv) a product with py ramided PIP toxins (i.e., two or mo re toxins with distinct non-cross reacting modes of actions), with either a seed blend or external refuge, that has been determined by

EPA’s science assessment to be 150% as durable as the baseline single toxin product with a 20% external refuge, would qualify for a fifteen-year registration. Products determined by EPA’s science

assessment to be less than 100% as durable as the ba seline single toxin product with a 20% external refuge would not qualify for a five-year registration and the registration period for such products will be determined on a case-by-case basis consistent with the level of risk they pose. Similarly, instances where other risk issues may arise, or where novel resi stance concerns may be present, would also be determined on a case-by-case basis, as will novel refuge configurations that may present unique durability profiles.Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

199 APPENDIX A

GLOSSARY OF ACRONYMS AND ABBREVIATIONS

AAEA American Agricultural Economics Association ABSTC Agricultural Biotechnology Stewardship Technical Committee ADP adenosine diphosphate AIBS American Institute of Biological Sciences ANOVA analysis of variance AP alkaline phosphatase APHIS Animal and Plant Health Inspection Service (of the United States Department of Agriculture) ARS Agricultural Research Service (of th e United States Department of Agriculture) ASAE American Society of Agricultural Engineers

BCW black cutworm BLAST Basic Local Alignment Search Tool BLASTP BLAST search that compares an amino acid query sequence with others stored in protein sequence databases BPPD Biopesticides and Pollution Prevention Division BRAD/BRADs Biopesticides Registration Action Document/Biopesticides Registration Action Documents

BSA bovine serum albumin

Bt

/ B

. t

. Bacillus thuringiensis

BTU British thermal units bw body weight °C Celsius (degrees) CAP/CAPs Compliance Assurance Program/Compliance Assurance Programs

CEW corn earworm CLA corn leaf aphid CFR Code of Federal Regulations

cm centimeter/centimeters

Codex Codex Alimentarius Commission

CRW corn rootworm

CVs coefficients of variation

Da Dalton/Daltons DNA deoxyribonucleic acid

DT 50 half-life

DT 90 time until 90% decay

EC 50 growth inhibition ECB European corn borer

ED 50 median effective dose (produces desired effect in 50% of population) EDSP Endocrine Disruptor Screening Program

EEC Estimated Environmental Concentration ELISA/ELISAs enzyme-linked immunosorbent assay/enzyme-linked immunosorbent assays EPA Environmental Protection Agency (the Agency) EPA Reg. No. Environmental Protection Agency Registration Number EPA Reg. Nos. Environmental Protection Agency Registration Numbers ESI MS/MS electrospray ioni zation tandem mass spectrometry FAW fall armyworm FFDCA Federal Food, Drug, and Cosmetic ActBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

200 GLOSSARY OF ACRONYMS AND ABBREVIATIONS, CONTINUED

FIFRA Federal Insecticide, Fungicide, and Rodenticide Act FR Federal Register

ft foot/feet ft/mi feet per mile

ft 2 /acre square feet per acre g gram/grams

GI 50 50% growth inhibition GIPSA Grain Inspection, Packers an d Stockyards Administration (of the United States Department of Agriculture) GM genetically modified

ha hectare/hectares HEEC Highest Estimated Environmental Concentration HGT horizontal gene transfer hp horsepower HPLC high-performance liquid chromatography

IA Iowa IC insecticidal crystal ICP/ICPs insecticidal crystal protein/insecticidal crystal proteins

IL Illinois

ILSI-CERA International Life Sciences Institute Research Foundation – Center for Environmental Risk

Assessment ILV independent laboratory validation

IN Indiana IRM insect resistance management

ISBN International Standard Book Number

K M Michaelis-Menten constant

kb kilobase

kDa kiloDalton kg kilogram/kilograms

L liter/liters

LC 50 median lethal concentration. A st atistically derived concentration of a substance that can be expected to cause death in 50% of test animals. It is usually expressed as the weight of substance per weight or volume of water, air, or feed (e.g., mg/L, mg/kg, or ppm).

LD 50 median lethal dose. A statistically derived single dose that can be expected to cause

death in 50% of the test animals when administered by the route indicated (oral, dermal, inhalation). It is expressed as a weight of substance per unit weight of animal (e.g., mg/kg). LLC limited liability company

LOAEC Lowest Observed Adverse Effect Concentration

LOC Level of Concern LOQ limit of quantitation

m meter/meters MALDI-TOF-MS matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

MCRW Mexican corn rootworm μg microgram/micrograms μg/cm 2 micrograms per square centimeterBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

201 GLOSSARY OF ACRONYMS AND ABBREVIATIONS, CONTINUED

μg/g micrograms per gram μL microliter/microliters mg milligram/milligrams mg/kg milligrams per kilogram mg/mL milligrams per milliliter

MHD maximum hazard dose

mL milliliter/milliliters

MN Minnesota

MO Missouri

MOA mode of action mol/L moles per liter

mol/mol moles to moles mph miles per hour

MRID No./MRID Nos. Master Reco rd Identification Number/Maste r Record Identification Numbers

MRL/MRLs maximum residue level/maximum residue levels N/A not applicable NAS National Academy of Sciences NASS National Agricultural Statistics Service (o f the United States Department of Agriculture) NCBI National Center for Biotechnology Information NCEAS National Center for Ecological Analysis and Synthesis

NCRW northern corn rootworm

NE Nebraska ng nanograms/nanograms ng/mg nanograms per milligram NIS Node Injury Scale

nM nanomolar

NOAEL No Observed Adverse Effect Level OCSPP Office of Chemical Safety and Pollution Prevention OECD Organization for Economic Cooperation and Development OPP Office of Pesticide Programs OPPTS Office of Prevention, Pe sticides, and Toxic Substances ORF open reading frame

PAT/ pat phosphinothricin acetyltransferase PBS phosphate buffered saline

PC Code Pesticide Chemical Code PCA Phased Compliance Approach PCR polymerase chain reaction PIP/PIPs plant-incorporated protectant/plant-incorporated protectants ppm parts per million RDR root damage rating RIP/RIPs ribosomal inhibition protein/ribosomal inhibition proteins

RNA ribonucleic acid RQ risk quotient RRA relative rate of adaptation

RSD relative standard deviation SAP Scientific Advisory Panel (the Panel)

SCRW southern corn rootwormBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

202 GLOSSARY OF ACRONYMS AND ABBREVIATIONS, CONTINUED

SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis SGF simulated gastric fluid SSA Sublethal Seedling Assay

SWCB southwestern corn borer T-DNA transfer deoxyribonucleic acid TEP total extractable protein TGAI technical grade of the active ingredient

TX Texas

tr truncated

U.S. United States USDA United States Department of Agriculture

V

max maximum velocity of the reaction VIPs vegetative insecticidal proteins

WBCW western bean cutworm

WCRW western corn rootworm

WI Wisconsin w/w weight-to-weightBacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

203 Table 1. Currently Registered PIPs Expressing Cry34Ab1 and Cry35Ab1 Proteins.

EPA Registration

Number Registration Name Comp any and Address Active Ingredient(s) Initial Date of Registration

524-581 MON 89034 x TC1507 x MON 88017 x DAS-59122-7 (or SmartStaxTM) Monsanto Company 800 North Lindbergh Boulevard St. Louis, MO 63167  Cry1A.105

 Cry2Ab2

 Cry1F

 Cry3Bb1

 Cry34Ab1

 Cry35Ab1 July 20, 2009

524-584 MON 89034 x DAS-59122-7

Note : This registration is for breeding purposes, agronomic testing, increasing inbred seed stocks, and producing hybrid seed only. Monsanto Company 800 North Lindbergh Boulevard St. Louis, MO 63167  Cry1A.105

 Cry2Ab2

 Cry34Ab1

 Cry35Ab1 December 14, 2009

524-586 MON 88017 x DAS-59122-7

Note : This registration is for breeding purposes, agronomic testing, increasing inbred seed stocks, and producing hybrid seed only. Monsanto Company 800 North Lindbergh Boulevard St. Louis, MO 63167  Cry3Bb1

 Cry34Ab1

 Cry35Ab1 December 14, 2009

524-588 MON 89034 x TC1507 x DAS-59122-7

Note : This registration is for breeding purposes, agronomic testing, increasing inbred seed stocks, and producing hybrid seed only. Monsanto Company 800 North Lindbergh Boulevard St. Louis, MO 63167  Cry1A.105

 Cry2Ab2

 Cry1F

 Cry34Ab1

 Cry35Ab1 December 14, 2009

524-589 MON 89034 x MON 88017 x DAS59122-7

Note : This registration is for breeding purposes, agronomic testing, increasing inbred seed stocks, and producing hybrid seed only. Monsanto Company 800 North Lindbergh Boulevard St. Louis, MO 63167  Cry1A.105

 Cry2Ab2

 Cry3Bb1

 Cry34Ab1

 Cry35Ab1 December 14, 2009

524-590 TC1507 x MON 88017 x DAS-59122-7

Note : This registration is for breeding purposes, agronomic testing, increasing inbred seed stocks, and producing hybrid seed only. Monsanto Company 800 North Lindbergh Boulevard St. Louis, MO 63167  Cry1F

 Cry3Bb1

 Cry34Ab1

 Cry35Ab1 December 14, 2009

29964-4 Herculex® Rootworm Insect Protec tion Pioneer Hi-Bred International, Incorporated

7100 N.W. 62 nd Avenue

P.O. Box 1000 Johnston, IA 50131-1000  Cry34Ab1

 Cry35Ab1 August 31, 2005

29964-5 Herculex® XTRA Insect Protecti on Pioneer Hi-Bred International, Incorporated

7100 N.W. 62 nd Avenue

P.O. Box 1000 Johnston, IA 50131-1000  Cry1F

 Cry34Ab1

 Cry35Ab1 October 27, 2005Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

204 EPA Registration

Number Registration Name Comp any and Address Active Ingredient(s) Initial Date of Registration

29964-6 Optimum® AcreMaxTM 1

Note : This registration is a seed blend. Pioneer Hi-Bred International, Incorporated

7100 N.W. 62 nd Avenue

P.O. Box 1000 Johnston, IA 50131-1000  Cry1F

 Cry34Ab1

 Cry35Ab1 April 30, 2010

29964-8* 1507 x 59122 x MON 810 Pioneer Hi-Bred International, Incorporated

7100 N.W. 62 nd Avenue

P.O. Box 1000 Johnston, IA 50131-1000  Cry1F

 Cry34Ab1

 Cry35Ab1

 Cry1Ab February 24, 2010

29964-9* 59122 x MON 810 Pioneer Hi-Bred International, Incorporated

7100 N.W. 62 nd Avenue

P.O. Box 1000 Johnston, IA 50131-1000  Cry34Ab1

 Cry35Ab1

 Cry1Ab February 24, 2010

29964-10 Optimum® AcreMaxTM RW

Note : This registration is a seed blend. Pioneer Hi-Bred International, Incorporated

7100 N.W. 62 nd Avenue

P.O. Box 1000 Johnston, IA 50131-1000  Cry34Ab1

 Cry35Ab1 April 30, 2010

68467-5 Herculex® RW Insect Protection Mycogen Seeds c/o Dow AgroSciences LLC

9330 Zionsville Road Indianapolis, IN 46268-1054  Cry34Ab1

 Cry35Ab1 August 31, 2005

68467-6 Herculex® XTRA Insect Protection Mycogen Seeds c/o Dow AgroSciences LLC

9330 Zionsville Road Indianapolis, IN 46268-1054  Cry1F

 Cry34Ab1

 Cry35Ab1 October 27, 2005

68467-7 MON 89034 x TC1507 x MON 88017 x DAS-59122-7 (or SmartStaxTM) Mycogen Seeds c/o Dow AgroSciences LLC

9330 Zionsville Road Indianapolis, IN 46268-1054  Cry1A.105

 Cry2Ab2

 Cry1F

 Cry3Bb1

 Cry34Ab1

 Cry35Ab1 July 20, 2009

68467-9 MON 89034 x TC1507 x DAS-59122-7

Note : This registration is for breeding purposes, agronomic testing, increasing inbred seed stocks, and producing hybrid seed only. Mycogen Seeds c/o Dow AgroSciences LLC

9330 Zionsville Road Indianapolis, IN 46268-1054  Cry1A.105

 Cry2Ab2

 Cry1F

 Cry34Ab1

 Cry35Ab1 December 14, 2009

68467-10 MON 89034 x MON 88017 x DAS59122-7

Note : This registration is for breeding purposes, agronomic testing, increasing inbred seed stocks, and producing hybrid seed only. Mycogen Seeds c/o Dow AgroSciences LLC

9330 Zionsville Road Indianapolis, IN 46268-1054  Cry1A.105

 Cry2Ab2

 Cry3Bb1

 Cry34Ab1

 Cry35Ab1 December 14, 2009

68467-11 MON 89034 x DAS-59122-7

Note : This registration is for breeding purposes, agronomic testing, increasing inbred seed stocks, and producing hybrid seed only. Mycogen Seeds c/o Dow AgroSciences LLC

9330 Zionsville Road Indianapolis, IN 46268-1054  Cry1A.105

 Cry2Ab2

 Cry34Ab1

 Cry35Ab1 December 14, 2009Bacillus thuringiensis Cry34/35Ab1 Corn Biopesticides Registration Action Document (BRAD) September 2010

205 EPA Registration

Number Registration Name Comp any and Address Active Ingredient(s) Initial Date of Registration

68467-13 MON 88017 x DAS-59122-7

Note : This registration is for breeding purposes, agronomic testing, increasing inbred seed stocks, and producing hybrid seed only. Mycogen Seeds c/o Dow AgroSciences LLC

9330 Zionsville Road Indianapolis, IN 46268-1054  Cry3Bb1

 Cry34Ab1

 Cry35Ab1 December 14, 2009

68467-15 TC1507 x MON 88017 x DAS-59122-7

Note : This registration is for breeding purposes, agronomic testing, increasing inbred seed stocks, and producing hybrid seed only. Mycogen Seeds c/o Dow AgroSciences LLC

9330 Zionsville Road Indianapolis, IN 46268-1054  Cry1F

 Cry3Bb1

 Cry34Ab1

 Cry35Ab1 December 14, 2009

*Registration expires October 31, 2010.