BIOPESTICIDES REGISTRATION ACTION DOCUMENT 

Bacillus thuringiensis Cry3Bb1 Protein and the Genetic Material Necessary for Its Production (Vector 

PV-ZMIR13L) in MON 863 Corn (OECD Unique Identifier: MON-ØØ863-5) 

PC Code: 006484 

Bacillus thuringiensis Cry3Bb1 Protein and the Genetic Material Necessary for Its Production (Vector 

PV-ZMIR39) in MON 88017 Corn (OECD Unique Identifier: MON-88Ø17-3) 

PC Code: 006498

U.S. Environmental Protection Agency 

Office of Pesticide Programs 

Biopesticides and Pollution Prevention Division
Bacillus thuringiensis Cry3Bb1 Corn 

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Table of Contents

I. OVERVIEW

A. Background 4 

 

B. Use Profile 7 

C. Regulatory History 9 

 

 

II. SCIENCE ASSESSMENT

A. Product Characterization 19 

 

 B. Human Health Assessment 45

 

C. Environmental Assessment 65

D. Insect Resistance Management 134

E. Benefits 184

 

III. REGULATORY POSITIONS FOR CORN EVENT MON 863 AND MON 88017

A. Initial Registration (February 24, 2003) – Corn Event MON 863 219

B. 2010 Update – Corn Event MON 863 (and MON 863 x MON 810) 222

C. Initial Registration (December 15, 2005) – MON 88017 and MON 88017 x MON 810 223

D. 2010 Update – MON 88017 and MON 88017 x MON 810 225

E. Period of Registration 227

APPENDIX A 229
Bacillus thuringiensis Cry3Bb1 Corn 

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Bacillus thuringiensis Cry3Bb1 Corn Regulatory Action Team 

Product Characterization and Human Health

Joel Gagliardi, Ph.D. 

John Kough, Ph.D. 

Annabel Waggoner 

Michael Watson, Ph.D. 

Chris Wozniak, Ph.D. 

Environmental Fate and Effects

Joel Gagliardi, Ph.D. 

Anna Gross 

John Kough, Ph.D. 

Tessa Milofsky 

Robyn Rose 

Gail Tomimatsu, Ph.D. 

Zigfridas Vaituzis, Ph.D. 

Annabel Waggoner 

Chris Wozniak, Ph.D. 

Insect Resistance Management

Jeannette Martinez 

Sharlene Matten, Ph.D. 

Tessa Milofsky 

Alan Reynolds 

Robyn Rose 

Benefits Assessment

Edward Brandt 

Sharlene Matten, Ph.D. 

Alan Reynolds 

Registration Support

Mike Mendelsohn 

Sheryl Reilly, Ph.D. 

Biopesticides Registration Action Document Team Leaders

Jeannine Kausch 

Mike Mendelsohn 

Matt Thompson 

 

Office of General Counsel

Chris Kaczmarek, Esq. 
Bacillus thuringiensis Cry3Bb1 Corn 

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I. OVERVIEW 

A. Background 

On February 24, 2003, the Environmental Protection Agency (EPA) issued a time-limited, conditional 

registration to Monsanto Company (“Monsanto”) for Corn Event MON 863 (EPA Reg. No. 524-528), a 

plant-incorporated protectant expressing the active ingredient, Bacillus thuringiensis (Bt) Cry3Bb1 

protein and the genetic material necessary for its production (vector PV-ZMIR13L) in MON 863 corn 

(Organization for Economic Cooperation and Development (OECD) Unique Identifier: MON-ØØ863-

5). Corn Event MON 863, at the time of 2003 registration, expressed the first plant-incorporated 

protectant (PIP) active ingredient to offer protection against corn rootworm, and expectations were that 

adaptation of this new technology would result in reduction of conventional insecticide use (e.g., 

organophosphates, carbamates, and synthetic pyrethroids) by growers 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 applicant, the Agency determined that the use 

of this pesticide was in the public interest and that it would not cause any unreasonable adverse effects 

on the environment during the period of time-limited (less than a year), conditional registration. The 

time limitation on this particular registration was extended twice by the Agency: first from May 1, 2004 

to July 31, 2006 and then from July 31, 2006 to September 30, 2010. 

Subsequent to registration of the single-trait product in 2003, the Agency registered another single-trait 

Cry3Bb1 product (MON 88017; EPA Reg. No. 524-551) and several stacked and/or pyramided plantincorporated protectants (PIPs), expressing Cry3Bb1 along with other proteins, for either commercial or 

limited breeding purposes. A complete list of the currently registered products expressing Cry3Bb1—

including their respective registration numbers, product names, registrants, initial dates of registration, 

proteins (or active ingredients) expressed, and any limitations/special notes—can be found in Appendix 

A. In conjunction with the 2010 evaluation (explained in the paragraphs that follow), the Agency 

attempted to better detail and describe product characterization, human health, environmental effects, 

and insect resistance management (IRM) data that were submitted to support the registration of MON 

88017 and the following stacked products expressing Cry3Bb1: 

(1) MON 863 x MON 810 (EPA Reg. No. 524-545) 

(2) MON 88017 x MON 810 (EPA Reg. No. 524-552) 

When the first products containing the two Cry3Bb1 protein variants were registered by the Agency, 

Monsanto was issued time-limited, conditional registrations under section 3(c)(7)(C) of the Federal 

Insecticide, Fungicide, and Rodenticide Act (FIFRA). Along with several requirements for further 

product characterization, environmental effects, and IRM data, the registration notices also clearly 

established absolute expiration dates. Although the registrations—specifically Corn Event MON 863, 

MON 863 x MON 810, MON 88017, and MON 88017 x MON 810—began with varying absolute 

expiration dates, they were set to expire on September 30, 2010. Monsanto formally requested that the 

Agency amend their MON 88017 and MON 88017 x MON 810 registrations to extend the current 

expiration date. Conversely, for the Corn Event MON 863 and MON 863 x MON 810 products, 
Bacillus thuringiensis Cry3Bb1 Corn 

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Monsanto did not seek an extension to the expiration date; therefore, these registrations expired on their 

own terms, and the Agency issued a cancellation order, outlining provisions for existing stocks (75 FR 

52329, August 25, 2010). 

On October 1, 2009, EPA announced a policy to provide a more meaningful opportunity for the public 

to participate on major registration decisions before they occur. According to this policy, EPA intends to 

provide a public comment period prior to making a registration decision for, at minimum, the following 

types of applications: new active ingredients; first food uses; first outdoor uses; first residential uses; and 

other actions for which the Agency anticipates that there will be significant public interest. 

Consistent with the policy of making registration actions more transparent, the amendments to the 

expiring Cry3Bb1 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 Association; Agricultural Biotechnology Stewardship Technical 

Committee; Center for Science in the Public Interest; and 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 support of the Cry3Bb1 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 existing Cry3Bb1 registrations by extending the current expiration 

dates in accordance with the scheme explained in section III(E) of this Biopesticides Registration Action 

Document (BRAD). The basis for this decision can be found in both the risk assessment for the 

Cry3Bb1 corn products, which is characterized throughout this BRAD, and the Agency’s response to 

comments document. 

All data and findings for the Cry3Bb1 corn products 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 the Cry3Bb1 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 associated registrants formally requested an extension 

to expiration dates. Therefore, within the same docket (EPA-HQ-OPP-2010-0607) as this document, the 

following informationa 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) 

 

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 part, 

to comments received on the information presented in Docket Number 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 Cry3Bb1 Corn 

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

● 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 Docket Number EPA-HQ-OPP-2010-0607 

● EPA’s Response to Comments 

EPA made the decision to amend the registrations of eighteen (18) expiring Bt corn PIP registrations to 

extend the expiration dates. We conducted comprehensive assessments of each of these registrations, 

considering all toxicity and environmental effects data, data from insect resistance monitoring, and 

insect resistance refuge compliance reports, received and obtained since the last comprehensive 

evaluation of these products in 2001. Based upon our comprehensive assessment, we reached significant 

conclusions regarding the positive environmental impact of Bt corn PIPs, and we took several actions to 

strengthen the insect resistance management requirements 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 significant number of published field studies 

that, combined with the post-registration field studies required to be submitted to the Agency, have 

demonstrated that non-target invertebrates are generally more abundant in Bt cotton and Bt corn fields 

than in non-transgenic fields managed with chemical insecticides. Thus, 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 requirement for seed bag labeling that clearly shows the 

refuge requirements. Also, given the increasing variety 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, based on assessments of their likelihood of forestalling 

the evolution of insect resistance. We are registering differing categories of products for differing time 

periods to reflect the assessed level of risk of resistance posed by the various corn PIP products. The 

scheme that we are following includes registration periods generally of five, eight, and twelve years; 

with the possibility of a fifteen-year registration period for products that are demonstrated to meet 

specified criteria. We retain, however, the discretion to register products for time periods differing from 

these defaults where circumstances warrant. 
Bacillus thuringiensis Cry3Bb1 Corn 

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B. Use Profile 

1. Corn Event MON 863 

Pesticide Name: Bacillus thuringiensis Cry3Bb1 protein and the genetic material necessary for 

its production (vector PV-ZMIR13L) in MON 863 corn (OECD Unique 

 Identifier: MON-ØØ863-5) 

Trade and Other 

Names: Corn Event MON 863 and YieldGard® Rootworm

 

OPP Chemical Code: 006484

Basic Manufacturer: Monsanto Company 

800 North Lindbergh Boulevard 

St. Louis, Missouri 63167 

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 Cry3Bb1 Corn 

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2. MON 88017 

Pesticide Name: Bacillus thuringiensis Cry3Bb1 protein and the genetic material necessary for 

its production (vector PV-ZMIR39) in MON 88017 corn (OECD Unique 

 Identifier: MON-88Ø17-3) 

Trade and Other 

Names: MON 88017 

 

OPP Chemical Code: 006498

Basic Manufacturer: Monsanto Company 

800 North Lindbergh Boulevard 

St. Louis, Missouri 63167 

Type of Pesticide: Plant-Incorporated Protectant

Use: Field and Sweet 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 Cry3Bb1 Corn 

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C. Regulatory History 

Date Action Type Description 

October 10, 19971 Federal Register 

Publication 

(Notice of Filing) 

Notice of Filing summarizing information submitted and cited by 

Monsanto Company in support of a request for establishment of an 

exemption from the requirement of a tolerance for residues of the plant 

pesticides consisting of Bacillus thuringiensis Cry1, Cry2, and Cry3 

classes of proteins and the genetic material necessary for the production of 

these proteins in or on all raw agricultural commodities. 

(62 Federal Register (FR) 52998) 

December 8, 19992 Federal Register 

Publication 

(Notice of Receipt) 

Notice announcing receipt of applications 524-EUP-ON, 524-EUP-OE, and 

524-EUP-OG from Monsanto Company requesting experimental use 

permits for the following: 

(1) Bacillus thuringiensis Cry3Bb protein and the genetic material 

necessary for its production (vector ZMIR14L) in corn 

(2) Bacillus thuringiensis Cry3Bb protein and the genetic material 

necessary for its production (vector ZMIR12L) in corn 

(3) Bacillus thuringiensis Cry3Bb protein and the genetic material 

necessary for its production (vector ZMIR13L) in corn 

(64 FR 68681) 

December 28, 20002 Federal Register 

Publication 

(Notice of Receipt) 

Notice announcing receipt of an application from Monsanto Company 

requesting amendment/extension to its experimental use permit, 524-EUP93 (involving testing of Bacillus thuringiensis Cry3Bb protein and the 

genetic material necessary for its production (vector ZMIR13L) in corn 

only). 

(65 FR 82352)

January 17, 20012 Federal Register 

Publication 

(Notice of 

Issuance) 

Notice announcing issuance of experimental use permits to Monsanto 

Company (524-EUP-90, 524-EUP-92, and 524-EUP-93). The permits 

allowed for use of (1) Bacillus thuringiensis Cry3Bb protein and the 

genetic material necessary for its production (vector ZMIR14L) in corn, 

(2) Bacillus thuringiensis Cry3Bb protein and the genetic material 

necessary for its production (vector ZMIR12L) in corn, and (3) Bacillus 

thuringiensis Cry3Bb protein and the genetic material necessary for its 

production (vector ZMIR13L) in corn on 1,343 acres of corn (524-EUP90), on 416 acres of corn (524-EUP-92), and on 1,092 acres of corn (524-

EUP-93). All of these experimental use permits were effective from April 

6, 2000 to April 31, 2001. The permits were issued with the limitation that 

all treated crops were to be genetically contained and destroyed or used for 

research purposes only. 

(66 FR 4020) 
Bacillus thuringiensis Cry3Bb1 Corn 

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Date Action Type Description 

March 19, 20012 Federal Register 

Publication 

(Notice of Receipt) 

Notice announcing receipt of the following application for a product 

containing a new active ingredient: 

(1) File Symbol: 524-LEI. Applicant: Monsanto Company, 700 

Chesterfield Parkway North, St. Louis, MO 63198. Product Name: Event 

MON 863: Corn Rootworm Protected Corn (ZMIR13L). Type of Product: 

Plant-pesticide. Active Ingredient: Bacillus thuringiensis Cry3Bb protein 

and the genetic material (vector ZMIR13L) necessary for its production in 

corn. 

*For 1 year, contained, 22,875 acre pre-commercial inbred seed 

propagation and hybrid seed production registration. Plantings are 

proposed for the states of California, Hawaii, Illinois, Iowa, Indiana, 

Kansas, Michigan, Nebraska, South Dakota, Texas, and Wisconsin. 

(66 FR 15435) 

May 11, 20011 Federal Register 

Publication 

(Final Rule) 

The following temporary exemption from the requirement of a tolerance 

was established under 40 Code of Federal Regulations (CFR) § 180.1214: 

“Bacillus thuringiensis Cry3Bb1 protein and the genetic material necessary 

for its production in corn are exempt from the requirement of a tolerance 

when used as plant-pesticides in the food and feed commodities of field 

corn, sweet corn, and popcorn. Genetic material necessary for its 

production means the genetic material which comprise genetic material 

encoding the Cry3Bb1 protein and its regulatory regions. Regulatory 

regions are the genetic material, such as promoters, terminators, and 

enhancers, that control the expression of the genetic material encoding the 

Cry3Bb1 protein. This exemption from the requirement of a tolerance will 

expire on May 1, 2004.” 

(66 FR 24061) 

July 27, 20012 Federal Register 

Publication 

(Notice of 

Issuance) 

Notice announcing extension/amendment of an experimental use permit 

previously approved for Monsanto Company (524-EUP-93): 

For use of 7.4 pounds of Bacillus thuringiensis Cry3Bb protein and the 

genetic material necessary for its production (vector ZMIR13L) in corn on 

4,000 acres of field corn; Effective from April 27, 2001 to April 2002. 

(66 FR 39163) 
Bacillus thuringiensis Cry3Bb1 Corn 

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Date Action Type Description 

September 11, 20012 Federal Register 

Publication 

(Notice of Receipt) 

Notice announcing receipt of an application from Monsanto Company 

requesting amendment to its experimental use permit, 524-EUP-93 

(involving testing of Bacillus thuringiensis Cry3Bb protein and the genetic 

material necessary for its production (vector ZMIR13L) in corn only). 

Amendment requested to allow livestock feeding studies and lift the crop 

destruct provisions, given the temporary tolerance exemption established 

on May 11, 2001 for Bacillus thuringiensis Cry3Bb1 protein and the 

genetic material necessary for its production in corn. 

(66 FR 47218) 

February 20, 20022 

Federal Register 

Publication 

(Notice of Receipt) 

Notice announcing receipt of an application from Monsanto Company 

requesting amendment/extension to its experimental use permit, 524-EUP93 (involving testing of Bacillus thuringiensis Cry3Bb protein and the 

genetic material necessary for its production (vector ZMIR13L) in corn 

only). 

(67 FR 7687) 

March 13, 20022 Federal Register 

Publication 

(Notice of Receipt) 

Notice announcing receipt of the following application for a product 

containing a new active ingredient: 

(1) File Symbol: 524-LEI. Applicant: Monsanto Company, 700 

Chesterfield Parkway North, St. Louis, MO 63198. Product Name: Event 

MON 863: Corn Rootworm Protected Corn (ZMIR13L). Type of Product: 

Plant-pesticide. Active Ingredient: Bacillus thuringiensis Cry3Bb protein 

and the genetic material (vector ZMIR13L) necessary for its production in 

corn. 

*Updated the March 19, 2001 Notice of Receipt in that Monsanto 

Company changed their application for registration from limited use (in 

certain states, for a certain amount of acreage, and for seed increase 

purposes only) to full commercial use. 

(67 FR 11333) 

May 1, 20022 Federal Register 

Publication 

(Notice of 

Extension of 

Comment Period) 

Notice announcing extension of the comment period for the updated Notice 

of Receipt published in the Federal Register on March 13, 2002. 

Originally, the comment period ended on April 12, 2002, but the Agency 

made the decision to extend the comment period to May 31, 2002. 

(67 FR 21669) 
Bacillus thuringiensis Cry3Bb1 Corn 

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Date Action Type Description 

June 26, 20022 Federal Register 

Publication 

(Notice of 

Issuance) 

Notice announcing extension/amendment of an experimental use permit 

previously approved for Monsanto Company (524-EUP-93): 

Removed the crop destruct requirement set forth with the original issuance 

of this experimental use permit; For use of Bacillus thuringiensis Cry3Bb 

protein and the genetic material necessary for its production (vector 

ZMIR13L) in corn on 9,400 acres of field corn; Effective from April 10, 

2002 to February 28, 2003. 

(67 FR 43115) 

February 24, 20032 Registration The Agency issued a time-limited, conditional registration notice (under 

FIFRA section 3(c)(7)(C)) for Corn Event MON 863 (EPA Reg. No. 524-

528). 

*Expiration Date: May 1, 2004 

April 2, 20032 Federal Register 

Publication 

(Notice of Receipt) 

Notice announcing receipt of the following application for a product 

containing an active ingredient involving a changed use pattern: 

(1) File Symbol: 524-LUL. Applicant: Monsanto Company, 700 

Chesterfield Parkway North, St. Louis, MO 63198. Product Name: 

YieldGard Plus Corn. Type of Product: Plant-incorporated protectant. 

Active Ingredients: Bacillus thuringiensis Cry3Bb1 protein and the genetic 

material necessary for its production (vector ZMIR13L) in corn and 

Bacillus thuringiensis Cry1Ab protein and the genetic material necessary 

for its production in corn. 

(68 FR 16036) 

April 2, 20033 Federal Register 

Publication 

(Notice of Receipt) 

Notice announcing receipt of application 524-EUP-OA from Monsanto 

Company requesting an experimental use permit for Bacillus thuringiensis

Cry3Bb1 protein and the genetic material necessary for its production 

(vector ZMIR39) in corn. 

(68 FR 16050) 

April 23, 20032 

Federal Register 

Publication 

(Notice of Receipt) 

Notice announcing receipt of an application from Monsanto Company 

requesting amendment/extension to its experimental use permit, 524-EUP93 (involving testing of Bacillus thuringiensis Cry3Bb1 protein and the 

genetic material necessary for its production (vector ZMIR13L) in corn 

only). 

(68 FR 19995) 
Bacillus thuringiensis Cry3Bb1 Corn 

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Date Action Type Description 

October 22, 20031 Federal Register 

Publication 

(Notice of Filing) 

Notice of Filing summarizing information submitted and cited by 

Monsanto Company in support of a request for establishment of an 

exemption from the requirement of a tolerance for the plant-incorporated 

protectant, Bacillus thuringiensis Cry3Bb1 protein and the genetic material 

necessary for its production in corn in or on field corn, sweet corn, and 

popcorn. 

(68 FR 60371) 

October 31, 20032 Registration The Agency issued a time-limited, conditional registration notice (under 

FIFRA section 3(c)(7)(B)) for YieldGard® Plus Corn (EPA Reg. No. 524-

545) 

*Expiration Date: May 1, 2004 

January 7, 20042 Federal Register 

Publication 

(Notice of 

Issuance) 

Notice announcing extension/amendment of an experimental use permit 

previously approved for Monsanto Company (524-EUP-93): 

For use of Bacillus thuringiensis Cry3Bb1 protein and the genetic material 

necessary for its production (vector ZMIR13L) in corn MON 863 and 

Bacillus thuringiensis Cry1Ab delta-endotoxin and the genetic material 

necessary for its production (vector PV-ZMCT01) in corn MON 810 on 

2,304 acres of field corn; Effective from June 20, 2003 to December 31, 

2003. 

(69 FR 917) 

January 7, 20043

Federal Register 

Publication 

(Notice of 

Issuance) 

Notice announcing issuance of an experimental use permit to Monsanto 

Company (524-EUP-96). The permit allowed for use of Bacillus 

thuringiensis Cry3Bb1 protein and the genetic material necessary for its 

production (vector ZMIR39) in corn ZMIR39 and Bacillus thuringiensis

Cry1Ab delta-endotoxin and the genetic material necessary for its 

production (vector PV-ZMCT01) in corn MON 810 (ZMIR39, MON 810, 

and ZMIR39 x MON 810 hybrids) on 829.7 acres of corn. This 

experimental use permit was effective from July 2, 2003 to December 31, 

2003. 

(69 FR 917) 
Bacillus thuringiensis Cry3Bb1 Corn 

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Date Action Type Description 

March 3, 20043 Federal Register 

Publication 

(Notice of Receipt) 

Notice announcing receipt of an application from Monsanto Company 

requesting amendment/extension to its experimental use permit, 524-EUP96 (involving testing of Bacillus thuringiensis Cry3Bb1 protein and the 

genetic material necessary for its production (vector ZMIR39) in corn 

ZMIR39 and Bacillus thuringiensis Cry1Ab delta-endotoxin and the 

genetic material necessary for its production (vector PV-ZMCT01) in corn 

MON 810 (ZMIR39, MON 810, and ZMIR39 x MON 810 hybrids)). 

(69 FR 10040) 

March 31, 20041 Federal Register 

Publication 

(Final Rule) 

The following permanent exemption from the requirement of a tolerance 

was established under 40 CFR § 180.1214: 

“Bacillus thuringiensis Cry3Bb1 protein and the genetic material necessary 

for its production in corn are exempt from the requirement of a tolerance 

when used as plant-incorporated protectants in the food and feed 

commodities of field corn, sweet corn, and popcorn. Genetic material 

necessary for its production means the genetic material which comprise 

genetic material encoding the Cry3Bb1 protein and its regulatory regions. 

Regulatory regions are the genetic material, such as promoters, terminators, 

and enhancers, that control the expression of the genetic material encoding 

the Cry3Bb1 protein.” 

(69 FR 16809) 

April 23, 20042 Amendment 

The Corn Event MON 863 (EPA Reg. No. 524-528) and YieldGard® Plus 

Corn (EPA Reg. No. 524-545) registrations were amended by the Agency 

to extend the expiration date set forth in the original registration notices. 

*New Expiration Date: July 31, 2006 
Bacillus thuringiensis Cry3Bb1 Corn 

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Date Action Type Description 

December 22, 20043 Federal Register 

Publication 

(Notice of Receipt) 

Notice announcing receipt of the following applications for products 

containing a new active ingredient: 

(1) File Symbol: 524-LLR. Applicant: Monsanto Company, 800 North 

Lindbergh Boulevard, St. Louis, MO 63167. Product Name: MON 88017. 

Type of Product: Plant-incorporated protectant. Active Ingredient: Bacillus 

thuringiensis Cry3Bb1 protein and the genetic material necessary for its 

production (vector ZMIR39) in MON 88017 corn. 

(2) File Symbol: 524-LLE. Applicant: Monsanto Company, 800 North 

Lindbergh Boulevard, St. Louis, MO 63167. Product Name: MON 88017 x 

MON 810. Type of Product: Plant-incorporated protectant. Active 

Ingredients: Bacillus thuringiensis Cry3Bb1 protein and the genetic 

material necessary for its production (vector ZMIR39) in MON 88017 corn 

and Bacillus thuringiensis Cry1Ab and the genetic material necessary for 

its production in corn. 

(69 FR 76716) 

December 22, 20043 Federal Register 

Publication 

(Notice of 

Issuance) 

Notice announcing extension/amendment of an experimental use permit 

previously approved for Monsanto Company (524-EUP-96): 

For use of 2.8 pounds of Bacillus thuringiensis Cry3Bb1 protein and the 

genetic material necessary for its production (vector ZMIR39) in corn 

ZMIR39 and Bacillus thuringiensis Cry1Ab delta-endotoxin and the 

genetic material necessary for its production (vector PV-ZMCT01) in corn 

MON 810 (ZMIR39, MON 810, and ZMIR39 x MON 810 hybrids) on 

2,530 acres of field corn; Effective from April 27, 2004 to February 28, 

2005. 

(69 FR 76732) 

January 12, 20053 Federal Register 

Publication 

(Notice of Receipt) 

Notice announcing receipt of an application from Monsanto Company 

requesting amendment/extension to its experimental use permit, 524-EUP96 (involving testing of Bacillus thuringiensis Cry3Bb1 protein and the 

genetic material necessary for its production (vector ZMIR39) in corn 

ZMIR39 and Bacillus thuringiensis Cry1Ab delta-endotoxin and the 

genetic material necessary for its production (vector PV-ZMCT01) in corn 

MON 810 (ZMIR39, MON 810, and ZMIR39 x MON 810 hybrids)). 

(70 FR 2160) 
Bacillus thuringiensis Cry3Bb1 Corn 

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Date Action Type Description 

August 10, 20053 Federal Register 

Publication 

(Notice of 

Issuance) 

Notice announcing extension/amendment of an experimental use permit 

previously approved for Monsanto Company (524-EUP-96): 

For use of 3.63 pounds of the insecticides, Bacillus thuringiensis Cry3Bb1 

protein and the genetic material necessary for its production (vector 

ZMIR39) in corn ZMIR39 and Bacillus thuringiensis Cry1Ab deltaendotoxin and the genetic material necessary for its production (vector PVZMCT01) in corn MON 810 (ZMIR39, MON 810, and ZMIR39 x MON 

810 hybrids), on 4,683 acres of field corn; Effective from February 18, 

2005 to March 1, 2006. 

(70 FR 46510) 

October 19, 20052 Federal Register 

Publication 

(Notice of 

Issuance) 

Notice announcing conditional approval of two products: 

(1) Event MON 863: Corn Rootworm Protected Corn (ZMIR13L)(EPA 

Reg. No. 524-528) – Registered under FIFRA section 3(c)(7)(C) and 

containing Bacillus thuringiensis Cry3Bb1 protein and the genetic material 

necessary for its production (vector ZMIR13L) in corn, an active ingredient 

not included in any previously registered product. 

(2) YieldGard Plus Corn (EPA Reg. No. 524-545) – Registered under 

FIFRA section 3(c)(7)(B) and containing Bacillus thuringiensis Cry3Bb1 

protein and the genetic material necessary for its production (vector 

ZMIR13L) in corn and Bacillus thuringiensis Cry1Ab delta-endotoxin and 

the genetic material necessary for its production in corn. 

*Note: Over 900 comments were received by the Agency in response to the 

Notices of Receipt for EPA Reg. Nos. 524-528 and 524-545. The Agency’s 

response to these comments can be found in Docket Number EPA-HQOPP-2004-0182 at www.regulations.gov. 

 

(70 FR 60826) 

December 13, 20053 Registration The Agency issued time-limited, conditional registration notices (under 

FIFRA section 3(c)(7)(C)) for MON 88017 (EPA Reg. No. 524-551) and 

MON 88017 x MON 810 (EPA Reg. No. 524-552) 

*MON 88017 Expiration Date: September 30, 2010 

*MON 88017 x MON Expiration Date: October 15, 2008 
Bacillus thuringiensis Cry3Bb1 Corn 

 Biopesticides Registration Action Document (BRAD) September 2010 

17

Date Action Type Description 

July 27, 20062 Amendment Corn Event MON 863 (EPA Reg. No. 524-528) and YieldGard® Plus Corn 

(EPA Reg. No. 524-545) were amended by the Agency to extend the 

expiration date set forth in previous regulatory correspondence. 

*Corn Event MON 863 New Expiration Date: September 30, 2010 

*YieldGard® Plus Corn New Expiration Date: October 15, 2008 

April 25, 20071

Federal Register 

Publication 

(Direct Final Rule) 

The tolerance exemption for Cry3Bb1 was redesignated from 40 CFR § 

180.1214 to 40 CFR § 174.518 and changed to the following: 

“Residues of Bacillus thuringiensis Cry3Bb1 protein in corn are exempt 

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.” 

(72 FR 20431) 

August 15, 20073 Federal Register 

Publication 

(Notice of 

Issuance) 

Notice announcing conditional approval of two products: 

(1) MON 88017 (EPA Reg. No. 524-551) – Registered under FIFRA 

section 3(c)(7)(C) and containing Bacillus thuringiensis Cry3Bb1 protein 

and the genetic material necessary for its production (vector ZMIR39) in 

Event 88017 corn (OECD Unique Identifier: MON-88Ø17-3), an active 

ingredient not included in any previously registered product. 

(2) MON 88017 x MON 810 (EPA Reg. No. 524-552) – Registered under 

FIFRA section 3(c)(7)(C) and containing Bacillus thuringiensis Cry3Bb1 

protein and the genetic material necessary for its production (vector 

ZMIR39) in Event 88017 corn (OECD Unique Identifier: MON-88Ø17-

3)(an active ingredient not included in any previously registered product) 

and Bacillus thuringiensis Cry1Ab delta-endotoxin and the genetic material 

necessary for its production (vector PV-ZMCT01) in Event MON 810 corn 

(OECD Unique Identifier: MON-ØØ81Ø-6). 

(72 FR 45807) 

October 10, 20081 Amendment 

YieldGard® Plus Corn (EPA Reg. No. 524-545) and MON 88017 x MON 

810 (EPA Reg. No. 524-552) were amended by the Agency to extend the 

expiration date set forth in previous regulatory correspondence. 

*New Expiration Date: September 30, 2010 
Bacillus thuringiensis Cry3Bb1 Corn 

 Biopesticides Registration Action Document (BRAD) September 2010 

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Date Action Type Description 

June 2008–December 

20093

Registration The Agency registered several combination PIP products expressing 

Cry3Bb1 and another protein(s). See Appendix A for the complete list. 

August 25, 20102 Federal Register 

Publication 

(Cancellation 

Order) 

Monsanto did not request an extension to their Corn Event MON 863 (EPA 

Reg. No. 524-528) or MON 863 x MON 810 (EPA Reg. No. 524-545) 

registrations; therefore, these registrations expired on their own terms on 

September 30, 2010. The Agency considers the expiration of a conditional, 

time-limited registration to be a cancellation under FIFRA section 3. A 

cancellation order, effective September 30, 2010, and appropriate 

provisions for disposition of existing stocks published in the Federal 

Register on August 25, 2010. 

(75 FR 52329) 

September 20103 Amendment 

The MON 88017 (EPA Reg. No. 524-551) and MON 88017 x MON 810 

(EPA Reg. No. 524-552) registrations were amended by the Agency to 

extend the expiration date in accordance with the scheme explained in 

section III(E) of this Biopesticides Registration Action Document (BRAD). 

*New Expiration Date: September 30, 2015 

1 Applies to both Corn Event MON 863 and MON 88017 

2

 Applies only to Corn Event MON 863 

3

 Applies only to MON 88017
Bacillus thuringiensis Cry3Bb1 Corn 

 Biopesticides Registration Action Document (BRAD) September 2010 

19

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 “ACCEPTABLE” indicates the study is scientifically 

sound and is useful for risk assessment. A “SUPPLEMENTAL” rating indicates the data provide some 

information that can be useful for risk assessment. The studies may have certain aspects determined not 

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

“SUPPLEMENTAL: UPGRADABLE,” EPA always provides an indication of what is lacking or what 

can be provided to change the rating to “ACCEPTABLE.” If there is simply a “SUPPLEMENTAL” 

rating, the reviewer will often state that the study is not required by the current 40 Code of Federal 

Regulations (CFR) Part 158. Both “ACCEPTABLE” and “SUPPLEMENTAL” studies may be used in 

the risk assessment process as appropriate. An “UNACCEPTABLE” rating indicates that the study is 

not useful for risk assessment and cannot be upgraded. 

A. Product Characterization

1. General Background on Cry3Bb1-Expressing Products 

Product characterization is critical to understanding the way in which the product was made and the 

unique characteristics that need to be assessed. The product characterization data provide information on 

the specific transformation systems used for each product, the actual deoxyribonucleic acid (DNA) 

inserted into the plant, the inheritance and stability of these traits in the plant, biochemical 

characteristics of the protein, and protein expression levels for various plant tissues. 

 a. Transformation System

Except for MON 88017, Cry3Bb1 plant-incorporated protectants were transformed into corn tissue via a 

method employing bombardment of particles coated with DNA encoding the intended insert. MON 

88017, on the other hand, was produced by the Agrobacterium-mediated transformation of corn cells 

with plasmid vector PV-ZMIR39. Each plasmid description includes a reference to the strains of 

Bacillus thuringiensis (Bt) used as the source of the DNA sequence for the toxin protein. In addition, the 

sources for marker proteins, promoters, terminators, and enhancers, as well as the fragment size, 

orientation, and any modifications to the original DNA sequence to enhance expression in the plant, are 

given. All the other DNA sequences, improving or restricting expression of the introduced traits, are also 

described. Finally, the plasmid discussion includes a description of any modifications made to the DNA 

(e.g., codon modifications to improve eukaryotic expression). 

b. Characterization of the DNA Inserted in the Plant

Inserted DNA is characterized with Southern blot data of the DNA in the plant genome. The analysis 

usually consists of DNA isolation from the transformed plant, digestion of this DNA with several 

different endonucleases, and hybridization of these restriction endonuclease fragments with labeled-
Bacillus thuringiensis Cry3Bb1 Corn 

 Biopesticides Registration Action Document (BRAD) September 2010 

20

DNA that is complementary to the introduced traits. This analysis includes not only probes specific for 

the entire insert, but also probes recognizing just the coding regions of the traits or DNA elements 

outside the coding region. Polymerase chain reaction (PCR) assays—utilizing various specific and nonspecific primers, genome walking, cosmid libraries, and DNA sequencing—have also been employed 

with sensitive Southern blotting techniques to more completely describe the inserted DNA and 

surrounding regions. The information available from these blots can indicate the presence of all the 

elements of the expected insert, as well as information about possible deletions and other errors 

associated with DNA introduction by transformation. Comparison of Southern blots of genomic DNA, 

digested using a range of restriction endonucleases, can also reveal the copy number of the genes 

introduced and suspected linkage of the traits. Alternatively, the intensity of the radioactive label from 

binding the probe DNA can also estimate the number of insert copies incorporated in the plant genome. 

c. Inheritance and Stability after Transformation

The data generated for this endpoint examine progeny from crosses between selected elite lines with the 

transformed Bt-expressing line, looking for the independent segregation of the introduced traits in the 

progeny. Traditional breeding work done during the development of the plant line by backcrossing can 

reveal the linkage of the introduced traits, as well as changes in trait expression. The inheritance data is 

the ratio of progeny expressing the hemizygous trait based on expected Mendelian inheritance. Stability 

data implies an examination of either the expression of the trait or tracking of the DNA itself over 

several plant generations. One of the main concerns with stability is spontaneous loss of the inserted 

DNA or loss of efficacy due to gene silencing. Neither Corn Event MON 863 nor MON 88017 showed 

independent assortment of the introduced traits with their marker protein genes (neomycin 

phosphotransferase II and CP4 enolpyruvylshikimate-3-phosphate, respectively). This indicates that, in 

both Corn Event MON 863 and MON 88017, the Cry3Bb1 and marker protein traits were on the same 

chromosome and closely linked (crossover events were not detected). 

d. Protein Characterization and Expression

Data has been presented to demonstrate that the protein expressed from the inserted DNA is similar to 

what was produced in the source bacterium and is active as expected against the intended target insect. 

Some protein characterization data demonstrate that microbially produced Bt protein is the equivalent to 

that expressed in the plant. This apparent scientific tautology (where plant-produced protein is the same 

as microbial protein is the same as the plant-produced protein) has been used to justify the use of the 

microbially produced protein as a test substance in toxicity tests. Because the expression level of these 

proteins is so low in plants, and the maximum hazard dose acute oral toxicity test is required as part of 

the human health risk assessment for these proteins, the ability to produce the protein in an industrial 

microbe is essential. The acute oral test requires between 2,000 and 5,000 milligrams (mg) of protein per 

kilogram (kg) bodyweight of test animal. Isolating the amount of purified protein, required to dose 

several animals, from Bt-expressing plants would be a tremendous burden involving harvesting and 

processing large volumes of plant material (environmental effects testing differs and is addressed in the 

Environmental Assessment chapter of this Biopesticides Registration Action Document (BRAD)). 

Proper characterization of the equivalency between these microbial proteins and plant-expressed 
Bacillus thuringiensis Cry3Bb1 Corn 

 Biopesticides Registration Action Document (BRAD) September 2010 

21

proteins provides an alternative to purifying the test material as the plant-produced protein from large 

volumes of tissue. 

Much of the characterization data describes the procedures used to isolate the protein or a highly Bt

protein-enriched fraction of plant extract. The tests done to support the equivalence of microbial and 

plant-produced Bt protein include the following: molecular sizing by sodium dodecyl sulfate 

polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis; immunorecognition using 

enzyme-linked immunosorbent assay (ELISA) and western blot analysis; N-terminal amino acid 

sequencing; matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis of protein 

digests; confirmation of the lack of glycosylation in the plant-produced protein; and bioactivity against a 

range of insects (often pest species including the target pest). Since the issues surrounding non-target 

effects are considered essential for the environmental effects assessment, these non-target pest tests are 

also discussed in the Environmental Assessment chapter of this BRAD. 

The Bt protein expression level in various tissues throughout the growing season have been determined 

for Corn Event MON 863, MON 810 x MON 863, MON 88017, and MON 88017 x MON 810. The data 

for Corn Event MON 863, however, was presented on a fresh weight basis and data in terms of dry 

weight leaf, root, pollen, seed, and whole plant were required as a condition of registration. These data 

(Master Record Identification Number (MRID No.) 464799-02; reviewed in U.S. EPA (2006)) were 

received and determined acceptable (see section II(A)(2)(b) of this BRAD for a brief summary of these 

data). Also, to support MON 88017 x MON 810, expression level data regarding Cry1Ab protein levels 

in MON 810 and MON 88017 x MON 810 young root and forage root were required as a condition of 

registration. These data (MRID No. 470045-01; reviewed in U.S. EPA (2010a)) were received and 

determined acceptable (see section II(A)(5)(b) of this BRAD for a brief summary of these data). 

 e. Residue Analytical Methods

Independent laboratory method validation (Office of Chemical Safety and Pollution Prevention 

(OCSPP) Harmonized Guideline 860.1340) and EPA laboratory method validation were required to 

complete the database for Cry3Bb1 corn. The extraction and detection method as described for Cry3Bb1 

protein appears to be adequate for analysis of Cry3Bb1 protein in corn grain. The independent 

laboratory validation study (MRID No. 463942-01; reviewed in U.S. EPA (2006)), required as a 

condition of registration, was received and found acceptable. Further, in lieu of having the EPA 

laboratory in Fort Meade, Maryland validate the analytical method for Cry3Bb1, the Agency has 

confirmed that the Grain Inspection, Packers and Stockyards Administration (GIPSA) performance 

verification of a qualitative rapid test kit for detecting the presence of Cry3Bb1 in grain and oilseeds has 

been completed (USDA 2004); thus, the requirement for EPA laboratory method validation for Cry3Bb1 

has also been satisfied. 
Bacillus thuringiensis Cry3Bb1 Corn 

 Biopesticides Registration Action Document (BRAD) September 2010 

22

2. Corn Event MON 863 (Organization for Economic Cooperation and Development 

 (OECD) Unique Identifier: ØØ863-5) Expressing Cry3Bb1 

a. Data Cited/Submitted for Initial Registration of Corn Event MON 863 (Prior to 

 February 2003)

Cry3Bb1 protein is a delta-endotoxin from Bacillus thuringiensis subspecies kumamotoensis and has 

activity against certain beetles. The wild-type cry3Bb1 gene was modified to enhance the protein’s 

activity against the corn rootworm complex. Two Cry3Bb1 variants were engineered for expression in 

the bacterium, Bacillus thuringiensis strains EG11098 and EG11231. Cry3Bb1 protein resulting from 

these strains differed from wild-type Cry3Bb1 protein by 5 and 4 amino acid substitutions (see Table1). 

Corn was genetically modified to express the Cry3Bb1.11231 protein (resulting in corn line MON 853) 

or the Cry3Bb1.11098 protein (resulting in corn line MON 863). At the 5' end of the cry3Bb1 gene’s 

reading frame, the vectors used for making MON 853 and MON 863 corn coded for an additional amino 

acid residue due to creation of a restriction enzyme site necessary to construct the vectors. 
Bacillus thuringiensis Cry3Bb1 Corn 

 Biopesticides Registration Action Document (BRAD) September 2010 

23

Table 1. Cry3Bb1 Protein Variants: Amino Acid Sequence Percent Identities and Position Differences 

Amino Acid Positionsab

Cry3Bb1 Variantc % Identity 

Wild-Type 2 165/166 231/232 311/312 313/314 317/318 348/349 

Bacterial-Produced Protein

Wild-Type N/A N/A D H S N E Q 

Cry3Bb1.11231 99.4 N/A D R L T K Q 

Cry3Bb1.11098 99.2 N/A G R L T K Q 

Cry3Bb1.11098 

(Q349R) 98.9 A G R L T K R 

Cry3Bb1.pvzmir39 99.1 A D R L T K R 

Plant-Produced Protein/Product

Cry3Bb1.11231/ 

MON 853 

99.2 A D R L T K Q 

Cry3Bb1.11098 

(Q349R)/MON 863 98.9 A G R L T K R 

Cry3Bb1.pvzmir39/ 

MON 88017 

99.1 A D R L T K R 

a

 A = alanine; G = glycine; D = aspartic acid; R = arginine; H = histidine; L = leucine; S = serine; T = threonine; 

N = asparagine; K = lysine; E = glutamic acid; Q = glutamine; N/A = not applicable 

b

 The B.t.-produced Cry3Bb1 protein variants contain 652 amino acids. The Escherichia coli- and plant-produced Cry3Bb1 

protein variants contain 653 amino acids due to the insertion of an alanine residue at position 2, resulting from the assembly 

of the cry3Bb1 gene into the E. coli- or plant-transformation vector. c All Cry3Bb1 protein variants, except for Cry3Bb1.pvzmir39, are discussed in MRID No. 454240-09, while the wild-type 

Cry3Bb1 is discussed in Donovan et al. (1992). 

Monsanto Company (“Monsanto”) subsequently submitted additional data regarding the MON 863 corn 

line. The vector, used to transform MON 863 corn, coded for an arginine residue at position 349 instead 

of glutamine (as previously thought) within the cry3Bb1 gene’s reading frame. Since the bacterially 

produced protein used in human health safety studies had the glutamine at position 349 and not arginine 

(as produced in MON 863), Monsanto generated another package of characterization and toxicology 

data for this variant, Cry3Bb1.11098 (Q349R). The Agency reviewed the additional data submitted by 

Monsanto in connection with Corn Event MON 863 and concluded the data support the contention that 

the Cry3Bb1.11098, Cry3Bb1.11098 (Q349R), and Cry3Bb1.11231 proteins are variants of the 

Cry3Bb1 protein. Since these variants do not differ significantly from the Cry3Bb1 protein in terms of 

biochemical or toxicological characteristics, the Cry3Bb1.11098, Cry3Bb1.11098(Q349R), and 
Bacillus thuringiensis Cry3Bb1 Corn 

 Biopesticides Registration Action Document (BRAD) September 2010 

24

Cry3Bb1.11231 protein variants are all covered by the Cry3Bb1 tolerance exemption (40 CFR § 

174.518). 

The product characterization studies that were submitted in support of Corn Event MON 863 are 

summarized in Table 2. 

Table 2. Product Characterization Data for Corn Event MON 863 (Reviewed in U.S. EPA 

 (2002a) Unless Otherwise Noted). 

Study Title Summary MRID No.

Data in Support of an 

Application for 

Experimental Use 

Permit for Genetically 

Modified Corn, 

Producing a Protein that 

Provides Control of 

Corn Rootworm 

MON 853, MON 860, MON 862, and MON 863 were produced by the 

incorporation of one of three constructs (PV-ZMIR12L (MON 862), PVZMIR13L (MON 863), or PV-ZMIR14L (MON 853 and MON 860)) via a 

particle bombardment mechanism. The cry3Bb1and neomycin 

phosphotransferase II (nptII) genes were stably introduced into the corn 

genomes, as determined by at least three generations of greenhouse and 

field studies. 

Classification: Acceptable 

(Reviewed in U.S. EPA (2000))

448779-01 

Characterization of B.t. 

Protein 11098 and B.t. 

Protein 11231 Produced 

by Fermentation 

The N-terminal sequence analysis and the immunoreactivity to Cry3Bb1 

polyclonal antisera confirm the relationship of Cry3Bb1.11098 and 

Cry3Bb1.11231 to wild-type Cry3Bb1. Further confirmatory data include 

protein molecular weight analysis and bioactivity. There are some amino 

acid changes (four or five) in the two test proteins compared to wild-type. 

These changes, however, do not appear to significantly affect the 

bioactivity nor the immunoreactivity of the variant proteins. Based upon 

the data submitted, the two proteins produced by fermentation— 

Cry3Bb1.11098 and Cry3Bb1.11231—have been confirmed as Cry3Bb1 

protein variants. 

Classification: Acceptable 

454240-03 

Assessment of the 

Physiochemical 

Equivalence of 

Cry3Bb1.11098 and 

NPTII Proteins in Corn 

Event MON 863 to 

Microbial Sources 

Based upon the data provided, it appears that both the Cry3Bb1.11098 and 

NPTII proteins produced in Event MON 863 have equivalent molecular 

weights and antigenic properties with these same proteins produced in B.t.

and E. coli, respectively. 

Classification: Acceptable 

(MRID No. 451568-03 reviewed in U.S. EPA (2001a))

451568-03 

454240-05 
Bacillus thuringiensis Cry3Bb1 Corn 

 Biopesticides Registration Action Document (BRAD) September 2010 

25

Study Title Summary MRID No.

Assessment of the 

Equivalence of B.t. 

Protein 11098, B.t. 

Protein 11231, and 

NPTII Protein 

Expressed in Corn 

Events MON 853 and 

MON 860 to Microbial 

Sources

This report compares the physical (molecular weight, N-terminal 

sequencing) and functional (bioassay) characteristics of Cry3Bb1.11098 

and Cry3Bb1.11231 proteins produced in E. coli and corn rootworm 

(CRW)-protected corn. The data show that the proteins have equivalent 

molecular weight, immunological reactivities, N-terminal sequences, and 

comparable median lethal concentration (LC50) values. Furthermore, the 

data supports the determination of the equivalence of the bacteria- and 

plant-produced proteins, and the use of the bacterially produced proteins to 

support registration of the CRW corn product. 

Classification: Acceptable

454240-04 

Additional 

Characterization of the 

Cry3Bb1 Protein 

Produced in Corn Event 

MON 863 

Two genetic variants, designated as cry3Bb1.11098 and cry3Bb1.11231, 

produce the delta-endotoxin proteins, Cry3Bb1.11098 and Cry3Bb1.11231, 

respectively. Cry3Bb1.11098 differs from the wild-type Bacillus 

thuringiensis (B.t.) protein by 5 amino acids, while the Cry3Bb1.11231 

protein differs by 4 amino acids. The cry3Bb1.11098 gene was used to 

develop maize line MON 863 and variant cry3Bb1.11231 was used in the 

development of MON 853 for control of the corn rootworm complex. 

Further manipulations, during cloning and insertion into the maize genome, 

brings the total amino acid differences for these two transformants to 7 and 

5 for the 11098 (MON 863) and 11231 (MON 853) Cry3Bb1 proteins, 

respectively. Cry3Bb1 protein was purified from Event MON 863 grain by 

immunoaffinity chromatography and then analyzed by N-terminal 

sequencing and MALDI-TOF. Trypsin fragments, subjected to matrixassisted laser desorption/ionization time-of-flight mass spectrometry 

(MALDI-TOF-MS), provided for identification or verification of 38% of 

the total protein by mass matching when coupled with sequencing of 29 Nterminal amino acids. Data from MALDI-TOF-MS and N–terminal 

sequencing indicate that the deduced amino acid sequences of 

Cry3Bb1.11098, as present in MON 863 and in B.t. strain EG11098, are 

accurate. A comparison of functionality and physicochemical 

characteristics strongly suggests that the two protein variants are nearly 

equivalent. Proteins from the fermentation of B.t. strains EG11098 and 

EG11231 were used for mammalian and ecotoxicology studies, as well as 

in assays relying on immunorecognition of proteins. These proteins are 

considered as biologically suitable for these studies based upon structural 

data indicating only minor changes in the shape of the delta-endotoxin 

proteins. 

Classification: Acceptable 

(Reviewed in U.S. EPA (2002b))

454240-10 
Bacillus thuringiensis Cry3Bb1 Corn 

 Biopesticides Registration Action Document (BRAD) September 2010 

26

Study Title Summary MRID No.

Primary Structural 

Protein Characterization 

of Corn Event MON 863 

Cry3Bb1.11098 Protein 

Using N-Terminal 

Sequencing and 

MALDI-TOF 

Spectrometric 

Techniques 

Transformation Event MON 863 (maize) produces the 74-kiloDalton (kDa) 

Cry3Bb1.11098 protein for control of the corn rootworm complex. 

Modifications to this protein for expression in planta bring the differences 

between the wild-type and MON 863-expressed variant to seven amino 

acids. Grain from Event MON 863 was used as a source of Cry3Bb1.11098 

protein for MALDI-TOF-MS and N-terminal sequence analyses. Of the 

653 amino acids present in the 74-kDa form of the Cry3Bb1 protein, 225 

were identifiable as to position based upon mass matching. Three 

fragments, from the N-terminal region of the protein, were also among 

those matched, representing 43 amino acids. One fragment included the Nterminus, indicating the loss of the terminal methionine and the acetylation 

of the alanine added at position two. This potentially explains the difficulty 

in sequencing the N-terminus of the 66-kDa form of the protein eluted 

from polyvinylidene fluoride (PVDF) blots. Protein samples, obtained from 

elution off of PVDF membranes of both the 74-kDa and 66-kDa proteins, 

were subjected to Edman degradation chemistry, but the larger peptide 

revealed no sequence data, presumably due to blockage of the terminal 

amino acid residue. When the bacterially produced version of this protein 

was subjected to N-terminal sequencing procedures, N-terminal sequence 

data were obtained successfully. The presumed reason for this rests with 

the post-translational modifications that are typical of eukaryotes (e.g., 

plants) and are lacking in prokaryotes (e.g., bacteria). Such modification 

could explain the blockage noted during the attempt to sequence the Nterminus of the corn-derived Cry3Bb1.11098 protein. 

 

Classification: Acceptable 

(Reviewed in U.S. EPA (2002b))

454240-11 
Bacillus thuringiensis Cry3Bb1 Corn 

 Biopesticides Registration Action Document (BRAD) September 2010 

27

Study Title Summary MRID No.

Characterization and 

Equivalence of the 

Cry3Bb1 Protein 

Produced by E. coli

Fermentation and Corn 

Event MON 863 

MALDI-TOF analysis of the microbial and corn Cry3Bb1.11098 (Q349R) 

proteins yielded an agreement of 42 to 50 amino acid fragments predicted 

from the theoretical sequence. The N-terminus of the microbial form 

lacked the terminal methionine, which is commonly cleaved in expressed 

proteins. The corn form was apparently not only lacking the terminal 

methionine, but the N-terminal alanine residue was acetylated as indicated 

by a 42 Dalton greater weight. The N-terminal amino acid sequence 

analyses were flawed in that unequivocal determinations were not possible 

due to the presence of multiple residues in most cycles; however, by 

comparison to the expected sequences, several different start sites for Nterminal sequencing could be detected. In the E. coli Cry3Bb1, the 

sequence started at both positions 2 and 32. In the corn Cry3Bb1, three 

different starts were detected at positions 19, 25, and 36. The immunoblot 

analysis gave similar positive band patterns, indicating the Cry3Bb1 

protein produced in both corn and E. coli had essentially the same 

electrophoretic mobility and immunoreactivity. The positive bands were 

sometimes rather broad (74–66 kDa), but no series of distinct bands could 

be discerned from the photographs provided. The molecular weight and 

purity analyses for the corn and microbial extracts indicate that the 

microbially produced samples were nearly two-fold higher purity in 

Cry3Bb1 proteins compared to the corn extracts. The purity for Cry3Bb1 

was 92.6% and 53.9% for microbial and corn extracts, respectively. Total 

protein concentrations for the two extracts were determined as 0.58 

milligrams per milliliter (mg/mL) and 0.46 mg/mL for microbial and corn 

extracts, respectively, by colormetric assays. The glycosylation analysis for 

the Cry3Bb1 extracts gave no positive carbohydrate staining regions for 

either the microbial or corn samples in the expected regions for Cry3Bb1 

protein. The results of the bioassays for the two Cry3Bb1extracts against 

Colorado potato beetle larvae indicate that there was a dose/response in all 

tests, and the LC50 values were similar and had overlapping 95% 

confidence intervals. 

Classification: Acceptable 

455382-01 
Bacillus thuringiensis Cry3Bb1 Corn 

 Biopesticides Registration Action Document (BRAD) September 2010 

28

Study Title Summary MRID No.

B.t. Protein 11231 and 

NPTII Protein Levels in 

Samples Collected from 

Corn Events MON 853 

and MON 860 in the 

1998 U.S. Field Trials 

The protein titer data provided for MON 860 and MON 853 show the 

ranges of Cry3Bb1 protein in various parts of the plant, as well as 

geographical variation. Overall, based upon the ranges provided, there 

appears to be significant variation between the samples analyzed on 

different days post-planting and at different sites. The registrant mentions a 

potential difference between decreasing titer in MON 853 and mid-season 

increasing titer in MON 860. Such a determination, however, cannot be 

made based upon the data provided in the submission. Even if such a trend 

was supported by additional data for MON 860, the difference in the 

protein titers is much smaller than the variation seen for MON 853 on days 

44, 55, and 100 post-planting. Ranges of Cry3Bb1 protein levels in MON 

853 in microgram Cry3Bb1 protein per gram of fresh weight tissue were 

7.01–68.98 (leaf), 1.66–17.64 (root), and 1.23–29.06 (aboveground whole 

plant). Ranges of Cry3Bb1 protein levels in MON 860 in microgram 

Cry3Bb1 protein per gram of fresh weight tissue were 32.61–91.11 (leaf), 

2.24–10.33 (root), and 0.63–13.95 (aboveground whole plant). 

Classification: Acceptable 

(Reference unknown) 

449043-02 

Agronomic Equivalency 

of Corn Event MON 863 

Hybrids as Determined 

in Year 2000 Field 

Trials 

The data included in this submission appear to support the agronomic 

equivalency of corn Event MON 863 hybrids. Results of the study show 

that there are some differences in the properties of the transgenic plants 

versus the control lines used in the tests. Some of the variation identified 

included differences in corn ear height, plant height, weight, grain 

moisture, and yield, but in each case, the difference was small. Based upon 

the data provided, however, it appears that none of these differences would 

have a significant agronomic impact on the crops and are likely similar to 

typical differences seen in different plant lines and/or those differences 

caused by differing ecological effects. 

 

Classification: Acceptable 

453484-03 

Molecular Analysis of 

Corn Event MON 863 

The data presented in this submission describe the DNA insert for Event 

MON 863. The data provided support the finding that Event MON 863 

contains 1 intact copy of the insert that encodes for both Cry3Bb1 and 

NPTII proteins. 

Classification: Acceptable 

454240-02 

451568-01

B.t. Cry3Bb1.11098 and 

NPTII Protein Levels in 

Tissue Samples 

Collected from Corn 

Event MON 863 Grown 

in 1999 Field Trials 

The protein titer data provided show the ranges of Cry3Bb1 protein in 

various parts of the plant, as well as geographical variation. Overall, based 

upon the ranges provided, there appears to be significant variation between 

the samples analyzed on different days post-planting and at different sites. 

Ranges of Cry3Bb1 protein levels in MON863 in microgram Cry3Bb1 

protein per gram of fresh weight tissue were 30–93 (leaf), 49–86 (grain), 

30–93 (pollen), 3.2–66 (root), and 13–54 (aboveground whole plant). 

Classification: Acceptable 

454240-01 

451568-02 
Bacillus thuringiensis Cry3Bb1 Corn 

 Biopesticides Registration Action Document (BRAD) September 2010 

29

Study Title Summary MRID No.

Validated Method for 

Extraction and Direct 

ELISA Analysis of 

Cry3Bb1 in Corn Grain 

The extraction and detection method, as described for Cry3Bb1 protein, 

uses Cry3Bb1-specific antisera for direct sandwich ELISA of Cry3Bb1 

protein in corn grain. This ELISA method does not involve a commercially 

available test kit (with pre-coated wells) and is not intended to be used as 

an enforcement method. The EPA laboratory (Fort Meade, Maryland) has 

concluded that this work was done early in the development of the biotech 

event and represents research that led to the implementation of the test kits. 

This method is not suitable for method validation as it is not a practical 

method to be used to routinely evaluate field samples. 

(Reviewed in U.S. EPA (2007)) 

453731-01 

b. Terms and Conditions of the Corn Event MON 863 Registration (February 2003 –

 September 2010)

 

When Corn Event MON 863 (EPA Reg. No. 524-528) was initially registered on February 24, 2003, the 

Agency issued a registration notice to Monsanto that contained the following requirements for further 

product characterization information: 

 “Submit independent laboratory method validation (under OPPTS Guidelines 

 860.1340) to complete the database for Cry3Bb1 corn within 12 months of the date 

 of registration. Provide the EPA laboratory (Fort Meade, MD) methodology and/or 

 reagents necessary for validation of a Cry3Bb1 analytical method within 6 months 

 of the date of registration. The extraction and detection method as described for 

 Cry3Bb1 protein appears to be adequate for analysis of Cry3Bb1 protein in corn 

 grain. However, this method must be validated by both an independent laboratory 

 and the EPA Biological and Economic Analysis Division laboratory before it can 

 be considered a valid method....” 

 “Submit expression data in terms of dry weight, as the amount of protein present in 

 the given tissue. Tissues for which expression data must be provided include: leaf, 

 root, pollen, seed, and whole plant. In addition, data for each of these tissues should 

 be provided for young plants in rapid growth, during flowering, and mature plants 

 before harvest when that part of the plant is present. Data obtained for roots should 

 also include typical times when corn rootworm when be feeding. Data are due within 

 24 months of the date of registration....” 

For the Corn Event MON 863 registration, the abovementioned requirements for additional product 

characterization data have been satisfied by submission of appropriate studies and information; 

summaries of this information are presented in Table 3. 
Bacillus thuringiensis Cry3Bb1 Corn 

 Biopesticides Registration Action Document (BRAD) September 2010 

30

Table 3. Product Characterization Data for Corn Event MON 863 (Reviewed in U.S. EPA 

 (2006)).

Study Title Summary MRID No.

TraitChekTM Cry3Bb1 

Lateral Flow Test Strip 

and SeedChekTM 

Cry3Bb1 ELISA 

Performance 

Verification for Corn 

Seed, Leaf, and 

Composite Testing 

The TraitChekTM Cry3Bb1 lateral flow strip test and the SeedChek TM

Cry3Bb1 ELISA were evaluated for qualitative detection of Cry3Bb1 

protein in corn seed and leaves. Three lots of each test kit were used to 

evaluate each of 100 known Cry3Bb1 corn seed or corn leaf samples and 

100 non-transgenic control samples. In single-seed testing of known 

Cry3Bb1 kernels, both tests were 100% accurate. In composite-seed testing 

(1 known Cry3Bb1 kernel in 800 total kernels), the TraitChekTM test had a 

false negative rate of 0.3% after a five-minute reading but was 100% 

accurate after ten minutes. The SeedChek TM test was 100% accurate at 

both reading times. Both tests showed 100% accuracy in detecting 

Cry3Bb1 in leaf samples from 13 varieties of Cry3Bb1 corn. No crossreactivity with other purified recombinant proteins or corn events 

expressing other recombinant proteins was observed for either test. 

Classification: Acceptable 

*Note for 2010: Although the requirement for an independent laboratory 

validation of the Cry3Bb1 analytical method was satisfied by submission 

of this information (MRID No. 463942-01), the reviewer also concluded 

that “EPA’s Analytical Method Laboratory located in Fort Meade 

(Maryland) will have to independently validate Monsanto’s lateral flow 

strip test and ELISA protocol for accuracy, precision, and sensitivity.” 

More recently, the Agency has decided to allow this particular requirement 

to be satisfied by the GIPSA performance verification of a qualitative rapid 

test kit for detecting the presence of the biotechnology event in grains and 

oilseeds (USDA 2004). In the case of Cry3Bb1, the Agency has now 

confirmed that a test kit has been verified by GIPSA and, therefore, the 

science reviewer’s concern has been addressed, and the original condition 

has been satisfied for the Corn Event MON 863 registration. 

463942-01 
Bacillus thuringiensis Cry3Bb1 Corn 

 Biopesticides Registration Action Document (BRAD) September 2010 

31

Study Title Summary MRID No.

Cry3Bb1 and NPTII 

Protein Levels in Corn 

Tissues from MON 863 

Produced in 2003 U.S. 

Field Trials 

The levels of proteins Cry3Bb1 and NPTII were determined in tissues of 

transgenic corn MON 863 generated in five 2003 U.S. field trials. The two 

proteins were quantitated by validated ELISA and presented in dry tissue 

weight (dwt). The highest Cry3Bb1 levels were in leaf, whole plant, and 

root samples, which had, respectively, mean levels of 180–240, 130–340, 

and 140–290 micrograms per gram (μg/g) dwt. Levels of Cry3Bb1 tended 

to be lower at the V10–V12 growth stage in whole plants and roots than for 

the earlier stages (V2–V3, V4–V5, V6–V7). Forage and forage root had 

similar Cry3Bb1 levels (mean of 55 and 80 μg/g dwt, respectively), and the 

lowest Cry3Bb1 levels were in pollen, stover, senescent root, and grain 

(mean of 20–35 μg/g dwt). NPTII protein was only measured in grain, 

where it was not detected. 

 

The frozen storage stability of Cry3Bb1 protein was not established for any 

unprocessed tissues or for processed whole plant, forage, stover, pollen, 

and grain samples. The storage stability of the NPTII protein was unknown 

for processed grain. Storage stability, however, was established for root 

tissue within the time frame it was analyzed, which is the most important 

factor in determining dose/susceptibility for pests. This is because the 

susceptible pest species, corn rootworm larvae, most actively feeds on root 

tissue at this stage of plant development. Thus, it is important that the 

protein expression levels, in particular root tissues, be accurately 

established to demonstrate the efficacy of the plant-incorporated protectant 

(PIP) plant. Moreover, Monsanto states that the levels of Cry3Bb1 from 

MON 863 were consistent with previously submitted tissue expression data 

(from MON 863 corn grown in 1999 field trials). Therefore, although some 

samples were analyzed outside the demonstrated storage stability time 

frame, it is unlikely that these samples were compromised in -80°C 

storage. 

Classification: Acceptable 

464799-02 

3. MON 863 x MON 810 (OECD Unique Identifier: MON-ØØ863-5 x MON- ØØ81Ø-6) 

 Expressing Cry3Bb1 and Cry1Ab

a. Data Cited/Submitted for Initial Registration of MON 863 x MON 810 (Prior to October 

2003)

MON 863 x MON 810 (also known as YieldGard® Plus Corn) was developed by crossing inbred lines 

of Event MON 863, which expresses the Cry3Bb1 and NPTII proteins, and Event MON 810, which 

expresses the Cry1Ab protein. This product is considered a stacked plant-incorporated protectant 

because it targets both lepidopteran and coleopteran pests and contains two separate active ingredients. 

MON 810 is briefly summarized in the paragraph that follows, while additional information on MON 

863, specifically related to product characterization, can be found in section II(A)(2) of this BRAD. 
Bacillus thuringiensis Cry3Bb1 Corn 

 Biopesticides Registration Action Document (BRAD) September 2010 

32

MON 810 produces Bacillus thuringiensis subsp. kurstaki strain HD-1 Cry1Ab protein to selectively 

control larvae of European corn borer (Ostrinia nubilalis) and other lepidopteran pests. On December 

20, 1996, the Agency issued a FIFRA section 3 registration to Monsanto 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 registration of MON 810, including the submitted study 

titles, conclusions, and their MRID Numbers, can be found in both the 2001 Bt Crops Reassessment and 

the September 2010 Cry1Ab and Cry1F BRAD (U.S. EPA 2001b, 2010b). 

The data submitted for MON 863 x MON 810, which include DNA analysis of the inserts in and protein 

expression data for the stacked corn product, are summarized in Tables 4 and 5. 

 

Table 4. Product Characterization Data for MON 863 x MON 810 (Reviewed in U.S. EPA 

 (2003)). 

Study Title Summary MRID No.

Confirmation of the 

Molecular Identity of 

YieldGard and Corn 

Rootworm-Protected 

Combined Trait Corn 

Hybrid MON 810 x 

MON 863 by Southern 

Blot Analysis 

The corn hybrid, MON 810 x MON 863, containing the two transformation 

events, MON 810 (cry1Ab) and MON 863 (cry3Bb1), was examined for 

the presence of these two genes encoding delta-endotoxins in the resulting 

hybrid. Probes for the cry1Ab and cry3Bb1 genes were obtained from 

previous studies and corresponded to the first 900 base pairs (cry1Ab) or 

the entire length of the gene (cry3Bb1). The radiolabeled (32P) probe for 

cry1Ab hybridized to restricted DNA samples on nylon membranes and 

resulted in a lack of any signal detection for the samples from MON 846 

(non-transgenic) and MON 863 but did detect the presence of cry1Ab in 

the MON 810 and MON 810 x MON 863 hybrid plant samples. When 

DNA samples were probed with the cry3Bb1 sequence, hybridization 

confirmed the presence of this gene in MON 863 plants and the MON 810 

x MON 863 hybrid, but again failed to detect the presence of this gene in 

the negative control, MON 846. Plasmid DNA from plasmids containing 

either gene separately did react positively with the appropriate probes when 

the restricted plasmid DNA was co-electrophoresed with MON 846 DNA 

and hybridized with the respective probe. From these results, it is evident 

that MON 810 x MON 863 contains the cry1Ab and cry3Bb1 genes. 

Additionally, the restriction patterns noted on the Southern blot suggest 

that there have been no major alterations or rearrangements in the 

conventional cross of these two events (hybrids) for these two gene inserts. 

Classification: Acceptable 

457917-01 
Bacillus thuringiensis Cry3Bb1 Corn 

 Biopesticides Registration Action Document (BRAD) September 2010 

33

Study Title Summary MRID No.

Cry3Bb1, Cry1Ab and 

NPTII Protein Levels in 

the Dual-Trait Maize 

Hybrid MON 863 x 

MON 810 Produced in 

Argentina Field Trials 

Conducted During the 

1999–2000 Growing 

Season 

ELISA values for field-grown maize samples (grain, forage, root, leaf, and 

pollen) were examined with a double antibody sandwich technique. 

Cry3Bb1 and NPTII protein values were adjusted for method bias to 

optimize the accuracy based upon extraction efficiency, and for variance in 

storage stability of extracts in some instances. The ranges of protein levels 

observed for Cry3Bb1, Cry1Ab, and NPTII were similar across all four 

sites for the MON 863 x MON 810 hybrid and the MON 863 or MON 810 

hybrids as appropriate for the transgene in question. Averages for the 

Cry3Bb1 and Cry1Ab proteins were, however, somewhat higher in the 

MON 863 x MON 810 hybrid versus the single trait hybrids. NPTII protein 

levels and ranges in the MON 863 x MON 810 and MON 863 hybrids were 

similar. The highest levels of expression of Cry3Bb1 or Cry1Ab were 

found to occur in pollen for Cry3Bb1 (79.6 μg/g fresh weight) and in leaf 

(17.9 μg/g fresh weight) for Cry1Ab. 

Classification: Acceptable 

457917-02 

Table 5. Comparison of Cry1Ab and Cry3Bb1 Protein Levels in Dual-Trait and Single-Trait 

 Hybrids 

Average Cry1Ab Protein Levels (μg/g 

fwt) (Range) 

Average Cry3Bb1 Protein Levels (μg/g 

fwt) (Range) 

Tissue Type and Collection 

Time (Days Post-Planting) 

MON 863 x MON 

810 MON 810 MON 863 x MON 810 MON 863 

Young Leaf (18) 17.9 

(14.1–27.5) 

13.0 

(9.8–15.4) 

46.7 

(35.5–53.2) 

30.0 

(21.3–47.2) 

Forage (90) 7.9 

(3.9–11.9) 

5.6 

(3.0–8.2) 

23.6 

(6.7–39.7) 

12.8 

(<0.22–28.8) 

Grain (117) 0.84 

(0.63–1.2) 

0.46 

(0.24–0.77) 

61.1 

(38.5–83.1) 

43.7 

(<0.096–84.1) 

Pollen (60) <0.08 

(<0.08–0.18) 

<0.08 

(<0.08) 

79.6 

(65.1–96.5) 

60.4 

(29.7–90.7) 

Mature Root (90) N/A N/A 19.7 

(6.0–41.7) 

16.2 

(<0.76–49.8) 

Over-Season Root (46) N/A N/A 22.0 (N/A) 20.0 (N/A) 

fwt= fresh weight 

N/A= Not Applicable 

Data from MRID No. 457917-02, pages 10–11 
Bacillus thuringiensis Cry3Bb1 Corn 

 Biopesticides Registration Action Document (BRAD) September 2010 

34

 b. Terms and Conditions of the MON 863 x MON 810 Registration (October 2003 –

September 2010)

When MON 863 x MON 810 (EPA Reg. No. 524-545) was initially registered on October 31, 2003, the 

Agency issued a registration notice to Monsanto that contained the following requirement: 

 “Submit all data required to support the individual plant-incorporated protectants 

 in MON 810 and MON 863 corn, EPA Registration Nos. 524-489 & 524-528.” 

All requirements for additional product characterization information for MON 810 (see U.S. EPA 

(2010b)) and MON 863 (see section II(A)(2)(b) of this BRAD) have been satisfied for the purposes of 

this registration. 

4. MON 88017 (OECD Unique Identifier: MON-88Ø17-3) Expressing Cry3Bb1 

 a. Data Cited/Submitted for Initial Registration of MON 88017 (Prior to December 2005)

The Cry3Bb1 protein, as produced in MON 88017, is a variant of the wild-type Cry3Bb1 protein from 

Bt subsp. kumamotoensis, and it protects the roots of corn plants from feeding damage caused by the 

coleopteran pest, corn rootworm. The amino acid sequence of the Cry3Bb1.pvzmir39 variant (MON 

88017) differs by seven amino acids from the wild-type Cry3Bb1 protein and by a single amino acid 

from the Cry3Bb1.11098 (Q349R) protein (MON 863). The Cry3Bb1 protein variants in MON 88017 

and MON 863 share an amino acid sequence identity of >99.8%, differing from one another by only 1 of 

653 amino acids at position 166 in MON 863, where glycine is present instead of aspartic acid (see 

Table 1 in section II(A)(2)(a) of this BRAD). MON 88017 also expresses the 5-enolpyruvylshikimate-3-

phosphate synthase protein from Agrobacterium spp. strain CP4 (CP4 EPSPS), which confers tolerance 

to glyphosate (Roundup®) herbicides. 

All physicochemical characteristics (including immunoreactivity, amino acid sequence, molecular 

weight, and glycosylation status) in the Cry3Bb1 protein in MON 88017 were found to be similar with 

the Cry3Bb1 protein in MON 863. Moreover, the protein expression, functional activity, and field 

efficacy data of Cry3Bb1 protein in MON 88017 were compared to MON 863 and found to be 

functionally equivalent. 

The product characterization studies that were submitted in support of MON 88017 are summarized in 

Table 6. 
Bacillus thuringiensis Cry3Bb1 Corn 

 Biopesticides Registration Action Document (BRAD) September 2010 

35

Table 6. Product Characterization Data for MON 88017 (Reviewed in U.S. EPA (2005a)). 

Study Title Summary MRID No.

Molecular Analysis of 

YieldGard 

Rootworm/Roundup 

Ready Corn Event 

MON 88017 

Corn Event MON 88017 contains one copy of the transfer 

deoxyribonucleic acid (T-DNA) at a single integration locus on an 

approximately 13 kilobase Sca I restriction fragment. The plasmid vector 

contains the cry3Bb1.pvzmir39 and cp4 epsps expression cassettes within 

the borders. No additional elements from the transformation vector PVZMIR39 or plasmid backbone sequences were detected in the corn genome 

of MON 88017. Insert stability was confirmed over multiple generations. 

PCR and DNA sequence analyses confirmed the organization of the 

elements within the insert and determined the complete DNA sequence of 

the insert in corn Event MON 88017. These data confirm that only the two 

expected full-length proteins, Cry3Bb1 and CP4 EPSPS, are encoded by 

the insert in corn Event MON 88017. 

Classification: Acceptable

461817-02 

465783-01 

Cry3Bb1 and CP4 

EPSPS Protein Levels 

in Corn Tissues 

Collected from MON 

88017 Corn Produced in 

U.S. Field Trials 

Conducted in 2002 

Tissue samples were collected at various times throughout the growing 

season from MON 88017 corn grown in U.S. field trials at three field sites 

in 2002. Tissue samples were analyzed for Cry3Bb1 and CP4 EPSPS 

protein levels using validated ELISA methods. The mean Cry3Bb1 levels 

across three field sites for leaf, whole plant, and root tissues harvested 

throughout the growing season ranged from 260–570, 88–500, and 100–

370 μg/g dry tissue weight, respectively. The mean Cry3Bb1 protein levels 

for pollen, forage, silk, and grain tissue were 25, 95, 380, and 15 μg/g dry 

tissue weight, respectively. The mean CP4 EPSPS protein levels for leaf 

and root tissue throughout the growing season ranged from 150–220 and 

70–150 μg/g dry tissue weight, respectively. The mean CP4 EPSPS protein 

levels for pollen, forage, and grain tissue were 390, 57, and 5.8 μg/g dry 

tissue weight, respectively. CP4 EPSPS protein levels were not assessed in 

whole plant or silk tissue. These data establish the protein levels of 

Cry3Bb1 and CP4 EPSPS proteins on a fresh weight and dry weight basis 

in the various tissues throughout the growing season. 

 

Classification: Acceptable 

461817-03 

465783-02 
Bacillus thuringiensis Cry3Bb1 Corn 

 Biopesticides Registration Action Document (BRAD) September 2010 

36

Study Title Summary MRID No.

Evaluation of the 

Functional Equivalence 

of Two Cry3Bb1 

Protein Variants 

Against Susceptible 

Coleopteran Species 

Both Colorado potato beetle (CPB) and western corn rootworm (WCRW) 

diet bioassays indicate that there are no significant differences in the 

biological activity between the two protein variants, Cry3Bb1.11098 

(Q349R) produced in MON 863 and Cry3Bb1.pvzmir39 produced in MON 

88017. The two protein variants are functionally equivalent in biological 

activity against the susceptible coleopteran insect species, CPB and 

WCRW. The mean dietary median lethal concentrations for CPB larvae 

were 0.95 micrograms per milliliter (μg/mL) (ranging from 0.79–1.11 

μg/mL) for the Cry3Bb1.11098 (Q349R) protein and 0.84 μg/mL (ranging 

from 0.64–1.06 μg/mL) for diets containing the Cry3Bb1.pvzmir39 

protein. For WCRW larvae, the mean dietary median lethal concentrations 

were 100 μg/mL (ranging from 73.2–137 μg/mL) for the Cry3Bb1.11098 

(Q349R) protein and 139 μg/mL (ranging from 74.6–231 μg/mL) in diets 

containing the Cry3Bb1.pvzmir39 protein. 

Classification: Acceptable 

461817-04 

465783-03 

 b. Terms and Conditions of the MON 88017 Registration (December 2005 – September 2010)

When MON 88017 (EPA Reg. No. 524-551) was initially registered on December 13, 2005, the Agency 

issued a registration notice to Monsanto that contained the following requirement: 

 “Submit all data required to support the individual plant-incorporated protectant 

 in Event MON863 (YieldGard Rootworm), 524-528....” 

All requirements for additional product characterization information for MON 863 (see section 

II(A)(2)(b) of this BRAD) have been satisfied for the purposes of this registration. 

5. MON 88017 x MON 810 (OECD Unique Identifier: MON-88Ø17-3 x MON-ØØ81Ø-6) 

 Expressing Cry3Bb1 and Cry1Ab 

 a. Data Cited/Submitted for Initial Registration of MON 88017 x MON 810 (Prior to 

 December 2005)

The stacked product, MON 88017 x MON 810, is a hybrid created by crossing the individuals events, 

MON 88017 (Cry3Bb1) and MON 810 (Cry1Ab), via traditional breeding methods. Product 

characterization background on MON 88017 was discussed in section II(A)(4) of this BRAD; MON 810 

was briefly summarized in section II(A)(3)(a), but more comprehensive information can be found in 

either the 2001 Bt Crops Reassessment or the September 2010 Cry1Ab and Cry1F BRAD (U.S. EPA 

2001b, 2010b). 

The data submitted for MON 88017 x MON 810, which include molecular characterization data and 

protein expression analyses, are summarized in Table 7. 
Bacillus thuringiensis Cry3Bb1 Corn 

 Biopesticides Registration Action Document (BRAD) September 2010 

37

Table 7. Product Characterization Data for MON 88017 x MON 810 (Reviewed in U.S. EPA 

(2005b)). 

Study Title Summary MRID No.

Confirmation of the 

Identity of MON 88017 

x MON 810 Corn by 

Southern Blot Analysis 

Southern blot fingerprints confirmed the presence of the event-specific 

fingerprints for MON 88017 and MON 810 in the Roundup Ready® 

hybrid, MON 88017 x MON 810. The fingerprints of MON 88017 x MON 

810 were consistent with previously reported event-specific fingerprints for 

MON 88017 and MON 810, which produce the Cry3Bb1 and Cry1Ab 

proteins, respectively. 

Classification: Acceptable

461850-02 

Cry3Bb1,CP4 EPSPS, 

and Cry1Ab Protein 

Levels in Corn Tissues 

Collected from MON 

88017 x MON 810 Corn 

Produced in U.S. Field 

Trials Conducted in 

2002 

Tissue samples were collected at various times throughout the growing 

season from MON 88017 x MON 810 corn grown during 2002 at three 

field sites in the U.S. Tissue samples were analyzed for Cry3Bb1, CP4 

EPSPS, and Cry1Ab protein levels using validated ELISA methods. The 

levels of Cry3Bb1 and Cry1Ab protein in tissue samples from the control 

hybrid were below the limit of quantitation (LOQ) or limit of detection 

(LOD) for each tissue assay. The mean Cry3Bb1 protein levels in MON 

88017 x MON 810 corn for root tissue harvested at the V2–V3 stage 

ranged from 140–350 μg/g dwt. The mean Cry3Bb1 protein levels for leaf, 

pollen, grain, and forage tissues were 670, 27, 9.3, and 100 μg/g dwt, 

respectively. The mean CP4 EPSPS protein levels in MON 88017 x MON 

corn for grain and forage tissues were 4.3 and 51 μg/g dwt, respectively. 

Finally, the mean Cry1Ab protein levels in MON 88017 x MON 810 corn 

for leaf, grain, and forage tissues were 110, 0.39, and 14μg/g dwt, 

respectively. The mean Cry1Ab protein level for pollen was below the 

limit of detection (LOD, 0.090μg/g fwt). The results demonstrate that the 

range and mean Cry3Bb1 and Cry1Ab expression levels in each tissue of 

MON 88107 x MON 810 were similar to the corresponding levels in the 

single-trait events, MON 88017 and MON 810, respectively. Finally, the 

levels of the inert ingredient, CP4 EPSPS protein, produced in MON 88017 

x MON 810 were similar to the levels observed for the same CP4 EPSPS 

protein produced in MON 88017. 

Classification: Acceptable 

461850-03 

 b. Terms and Conditions of the MON 88017 x MON 810 Registration (December 2005 –

September 2010)

When MON 88017 x MON 810 (EPA Reg. No. 524-552) was initially registered on December 13, 2005, 

the Agency issued a registration notice to Monsanto that contained the following requirements: 

 “Submit all data required to support the individual plant-incorporated protectants 

 in MON 810 (YieldGard), Event MON863 (YieldGard Rootworm)..., EPA 

 Registration Nos. 524-489, 524-528....” 
Bacillus thuringiensis Cry3Bb1 Corn 

 Biopesticides Registration Action Document (BRAD) September 2010 

38

 “Submit expression level data regarding Cry1Ab protein levels in MON 810 and 

 MON 88017 x MON 810 young root and forage root within 12 months of the date 

 of registration.” 

All requirements for additional product characterization information for MON 810 (see U.S. EPA 

(2010b)) and MON 863 (see section II(A)(2)(b) of this BRAD) have been satisfied for the purposes of 

this registration. For the MON 88017 x MON 810-specific conditional data requirement (i.e., expression 

level data), a study was submitted to the Agency and found acceptable (see Tables 8 and 9). 

Table 8. Product Characterization Data for MON 88017 x MON 810 (Reviewed in U.S. EPA 

 (2010a)).

Study Title Summary MRID No.

Assessment of Cry1Ab 

Protein Levels in Corn 

MON 88017 x MON 

810 Root Tissue 

Produced in U.S. Field 

Trials in 2006 

A traditionally crossed corn hybrid of MON 88017 with MON 810 was 

grown along with conventional seed and MON 810 corn at five locations in 

2006 using a randomized complete block design and sampling scheme. 

Young root tissues were sampled at V2–V3 and forage root tissues at early 

dent or 1/3 milkline. Samples were stored and shipped on dry ice for 

Cry1Ab analysis of trypsinized, extracted tissues. Extraction efficiency was 

92%, spike recovery was 77%, and the trypsinization factor was 2. The 

coefficient of variation was 14% between assays. Limit of detection was 

0.13 g/g fresh weight, and limit for quantification was 0.40 g/g fresh 

weight. ELISA revealed mean Cry1Ab protein levels in MON 88017 x 

MON 810 corn tissues across all sites were 75 g/g dwt in young root and 

12 g/g dwt in forage root; similar to the mean Cry1Ab protein levels in 

MON 810 corn, which were 78 g/g dwt in young root and 13 g/g dwt in 

forage root. 

Classification: Acceptable

470045-01 
Bacillus thuringiensis Cry3Bb1 Corn 

 Biopesticides Registration Action Document (BRAD) September 2010 

39

Table 9. Cry3Bb1 and Cry1Ab Protein Levels in MON 88017 x MON 810 and MON 88017 & MON 

 810 Tissues. 

Tissue 

Type

Growth 

Stage Cry3Bb1 Protein Levels (μg/g dwt) Cry1Ab Protein Le

MON 88107 x MON 810 MON 88017 MON 88017 x MON 810 

 Mean Standard Deviation Range n=9 Mean Standard Deviation Range n=9 Mean Standard Deviation Range n=9 

Young Leaf V2–V3 670 130 550-920 570 170 230-820 110 17 85-140 Young Root V2–V3 350 150 88-560 370 80 240-510 75* 26* 14-130* Pollen R1 27 5.7 N/A-34 25 4.2 17-32 N/A N/A N/A Forage 

R4–R6 (early dent) 100 23 71-150 95 19 75-130 14 2.1 11-17 

Forage Root 

R4–R6 (early dent) 140 29 89-180 130 29 98-170 12* 4.7* 2.7-22* 

Grain R6 9.3 3.4 3.9-13 15 3.6 10-22 0.39 0.13 

0.16-

0.63 

dwt= dry weight N/A= not applicable (as levels were below the level of detection) 

Data from MRID No. 461850-01, page 14 *Data from MRID No. 470045-01 
Bacillus thuringiensis Cry3Bb1 Corn 

 Biopesticides Registration Action Document (BRAD) September 2010 

40

6. References 

Donovan WP, Rupar MJ, Slaney AC, Malvar T, Gawron-Burke MC, Johnson TB. 1992. 

 Characterization of two genes encoding Bacillus thuringiensis insecticidal crystal proteins toxic 

 to Coleoptera species. Applied and Environmental Microbiology 58:3921–3927. 

MRID No. 448779-01. Pilacinski W. 1999. Data in Support of an Application for Experimental Use 

 Permit for Genetically Modified Corn, Producing a Protein that Provides Control of Corn 

 Rootworm (Diabrotica spp.) – Amended. Lab Project Number: 99-533E2. Unpublished study 

 prepared by Monsanto Company, 14 pages. 

MRID No. 449043-02. Cavato T, Dudin Y, Bhakta N. 1999. B.t. Protein 11231 and NPTII Protein 

 Levels in Samples Collected from Corn Events MON 853 and MON 860 in the 1998 U.S. Field 

 Trials. Lab Project Number: 98-01-39-08: 15710. Unpublished study prepared by Monsanto 

 Company, 27 pages. 

MRID No. 451568-01. Cavato T, Rigden E, Mittanck D. 2000. Molecular Analysis of Crop Event MON 

 863. Lab Project Number: 99-01-39-27: MSL-16505. Unpublished study prepared by Monsanto 

 Company, 59 pages. 

MRID No. 451568-02. Dudin Y, Tonnu B, Albee L. 2000. B.t. Cry3Bb1.11098 and NPTII Protein 

Levels in Tissue Samples Collected from Corn Event MON 863 Grown in 1999 Field Trials. Lab 

Project Number: 99-01-39-22: MSL-16559. Unpublished study prepared by Monsanto Company, 

29 pages. 

MRID No. 451568-03. Hollenschak G, Hileman R, Astwood J. 2000. Assessment of the Physiochemical 

 Equivalence of Cry3Bb1.11098 and NPTII Proteins in Corn Event MON 863 to Microbial 

 Sources. Lab Project Number: 99-01-39-31: MSL-16596: GEN-POL-005. Unpublished study 

 prepared by Monsanto Company, 38 pages. 

MRID No. 453484-03. Pilcher C, McFerson J, Ward D. 2001. Agronomic Equivalency of Corn Event 

 MON 863 Hybrids as Determined in Year 2000 Field Trials. Lab Project Number: 99-894E. 

 Unpublished study prepared by Monsanto Company, 11 pages. 

MRID No. 453731-01. Kolwyck D, Tonnu B, Dudin Y. 2001. Validated Method for Extraction and 

Direct ELISA Analysis of Cry3Bb1 in Corn Grain. Lab Project Number: 99-640E-1. 

Unpublished study prepared by Monsanto Company, 19 pages. 

MRID No. 454240-01. Dudin Y, Tonnu B, Albee L. 2001. Amended Report for MSL-16559: B.t. 

 Cry3Bb1.11098 and NPTII Protein Levels in Tissue Samples Collected from Corn Event MON 

 863 Grown in 1999 Field Trials. Lab Project Number: 99-01-39-22: 17181: MSL-17181. 

 Unpublished study prepared by Monsanto Company, 30 pages. 
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MRID No. 454240-02. Cavato T, Rigden E, Mittanck D. 2001. Amended Report for MSL-16505: 

 Molecular Analysis of Corn Event MON 863. Lab Project Number: 99-01-39-27: MSL-17152. 

 Unpublished study prepared by Monsanto Company, 30 pages. 

MRID No. 454240-03. Hileman R, Pyla P, Lee T. 2001. Amended Report for MSL-16505: 

 Characterization of B.t. Protein 11098 and B.t. Protein 11231 Produced by Fermentation. Lab 

 Project Number: 98-01-39-05: MSL-17219: 17219. Unpublished study prepared by Monsanto 

 Company, 36 pages. 

MRID No. 454240-04. Holleschak G, Lee T, Pyla P. 2001. Amended Report for MSL-15835: 

 Assessment of the Equivalence of B.t. Protein 11098, B.t. Protein 11231, and NPTII Protein 

 Expressed in Corn Events MON 853 and MON 860 to Microbial Sources. Lab Project Number: 

 98-01-39-18: MSL-17222: 17222. Unpublished study prepared by Monsanto Company, 53 

 pages. 

MRID No. 454240-05. Holleschak G, Hileman R, Astwood J. Amended Report to MSL-16596: 

 Assessment of the Physiochemical Equivalence of Cry3Bb1.11098 and NPTII Proteins in Corn 

 Event MON 863 to Microbial Sources. Lab Project Number: 99-01-39-31: MSL-17220: 17220. 

 Unpublished study prepared by Monsanto Company, 40 pages. 

MRID No. 454240-09. Hileman R, Astwood J, McKee M. 2001. Safety Assessment of Cry3Bb1 

Variants in Corn Rootworm Protected Corn. Lab Project Number: 17225: MSL-17225. 

Unpublished study prepared by Monsanto Company, 51 pages. 

MRID No. 454240-10. Hileman R, Astwood J. 2001. Additional Characterization of the Cry3Bb1 

 Protein Produced in Corn Event MON 863. Lab Project Number: 17137: MSL-17137. 

 Unpublished study prepared by Monsanto Company, 22 pages. 

MRID No. 454240-11. Thoma R, Holleschak G, Hileman R. 2001. Primary Structural Protein 

 Characterization of Corn Event MON 863 Cry3Bb1.11098 Protein Using N-Terminal 

 Sequencing and MALDI-TOF Spectrometric Techniques. Lab Project Number: 17154: MSL- 

 17154. Unpublished study prepared by Monsanto Company, 21 pages. 

MRID No. 455382-01. Hileman R, Holleschak G, Turner L. 2001. Characterization and Equivalence of 

 the Cry3Bb1 Protein Produced by E. coli Fermentation and Corn Event MON 863. Lab Project 

 Number: 17274: 01-01-39-30. Unpublished study prepared by Monsanto Company, 75 pages. 

MRID No. 457917-01. Borovkov I, Cavato T, Lirette R. 2001. Confirmation of the Molecular Identity 

 of YieldGard and Corn Rootworm-Protected Combined Corn Trait Corn Hybrid MON 810 x 

 MON 863 by Southern Blot Analysis. Lab Project Number: 01-01-39-39: MSL-17466. 

 Unpublished study prepared by Monsanto Company, 27 pages. 
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MRID No. 457917-02. Dudin Y, Tonnu B, Lirette R. 2001. Cry3Bb1, Cry1Ab and NPTII Protein Levels 

 in the Dual-Trait Maize Hybrid MON 863 x MON 810 Produced in Argentina Field Trials 

 Conducted During the 1999–2000 Growing Season. Lab Project Number: 00-01-39-44: MSL- 

 17266. Unpublished study prepared by Monsanto Company, 49 pages. 

MRID No. 461817-02. Beazley K, Anderson H, Wimberley P. 2002. Molecular Analysis of YieldGard 

 Rootworm/Roundup Ready Corn Event MON 88017. Project Number: 01/01/50/11, MSL/17609. 

 Unpublished study prepared by Monsanto Company, 63 pages. 

MRID No. 461817-03. Bhakta N, Hartmann A, Jennings J. 2003. Cry3Bb1 and CP4 EPSPS Protein 

 Levels in Corn Tissues Collected from MON 88017 Corn Produced in U.S. Field Trials 

 Conducted in 2002. Project Number: 02/01/50/17, MSL/18823. Unpublished study prepared by 

 Monsanto Company, 31 pages. 

MRID No. 461817-04. Duan J, Paradise M, Jiang C. 2003. Evaluation of the Functional Equivalence of 

 Two Cry3Bb1 Protein Variants Against Susceptible Coleopteran Species. Project Number: 

 MSL/18799, 03/01/39/11. Unpublished study prepared by Monsanto Company, 24 pages. 

MRID No. 461850-02. Rigden E, Reiser S. 2003. Confirmation of the Identity of MON 88017 x MON 

 810 Corn by Southern Blot Analysis. Project Number: 03/01/50/10, MSL/18797. Unpublished 

 study prepared by Monsanto Company, 24 pages. 

MRID No. 461850-03. Bhakta N, Hartmann A, Jennings J. 2003. Cry3Bb1, CP4 EPSPS, and Cry1Ab 

 Protein Levels in Corn Tissues Collected from MON 88017 x MON 810 Corn Produced in U.S. 

 Field Trials Conducted in 2002. Project Number: 02/01/50/18, MSL/18824. Unpublished study 

 prepared by Monsanto Company, 34 pages. 

MRID No. 463942-01. TraitChek Cry3Bb Lateral Flow Test Strip and SeedChek Cry3Bb ELISA 

Performance Verification for Corn Seed, Leaf, and Composite Testing. Project Number: 

MSL/19581. Unpublished study prepared by Strategic Diagnostics, Incorporated, 16 pages. 

MRID No. 464799-02. Ledesma B, Bookout J, Chinnadurai P. 2005. Cry3Bb1 and NPTII Protein Levels 

 in Corn Tissues from MON 863 Produced in 2003 U.S. Field Trials. Project Number: 

 04/01/39/20, 19597. Unpublished study prepared by Monsanto Company, 30 pages. 

MRID No. 465783-01. Beazley K, Anderson H, Wimberley P. 2005. Molecular Analysis of YieldGard 

 Rootworm/Roundup Ready Corn Event MON 88017: Supplemental Information. Project 

 Number: 03/CR099E/5, BR/EQ/0065/01, BR/EQ/0445/01. Unpublished study prepared by 

 Monsanto Company, 64 pages. 

 
Bacillus thuringiensis Cry3Bb1 Corn 

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MRID No. 465783-02. Bhakta N, Hartmann A, Jennings J. 2005. Cry3Bb1 and CP4 EPSPS Protein 

 Levels in Corn Tissues Collected from MON 88017 Corn Produced in U.S. Field Trials 

 Conducted in 2002: Supplemental Information. Project Number: 03/CR/099E/05, 

 BR/EQ/0923/01, BR/ME/0197/05. Unpublished study prepared by Monsanto Company, 54 

 pages. 

MRID No. 465783-03. Duan J, Paradise M, Jiang C. 2005. Evaluation of Functional Equivalence of 

 Two Cry3Bb1 Protein Variants Against Susceptible Coleopteran Species: Supplemental 

 Information. Project Number: 03/CR/099E/05, BR/ME/0044/02, BR/EQ/0376/01. Unpublished 

 study prepared by Monsanto Company, 74 pages. 

 

MRID No. 470045-01. Niemeyer K, Silvanovich A. 2006. Assessment of Cry1Ab Protein Levels in 

 Corn MON 88017 x MON 810 Root Tissue Produced in U.S. Field Trials in 2006. Project 

 Number: 06/01/50/10, 0020590. Unpublished study prepared by Monsanto Company, 20 pages. 

U.S. EPA. 2000. Data Submitted by Monsanto Company to Support Their Request for Three EUPs for 

 Genetically Modified Corn that Provides Control of Corn Rootworm. Memorandum to M.T. 

 Watson, Ph.D. and J.L. Kough, Ph.D. to M. Mendelsohn dated January 7, 2000. 

U.S. EPA. 2001a. Data Evaluation Report on MRID No. 451568-03 (“Assessment of the 

Physiochemical Equivalence of Cry3Bb1.11098 and NPTII Proteins in Corn Event MON 863 to 

Microbial Sources”). Completed by M.T. Watson, Ph.D. and J.L. Kough, Ph.D. on April 25, 

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. 2002a. EPA Review of the Application for an Exemption from the Requirement of a 

 Tolerance for Cry3Bb1 Protein Expressed in All Food Commodities. Memorandum from C. 

 Wozniak, Ph.D. and J.L. Kough, Ph.D. to M. Mendelsohn dated March 29, 2002. 

U.S. EPA. 2002b. Assessment of the Product Characterization of the Cry3Bb1 Insect Control Protein as 

 Expressed in Maize, a Bacillus thuringiensis-Based Plant-Incorporated Protectant for Control of 

 the Corn Rootworm. Memorandum from C.A. Wozniak, Ph.D. and J.L. Kough, Ph.D. to M. 

 Mendelsohn dated March 29, 2002. 

U.S. EPA. 2003. Assessment of the Product Characterization of the Cry1Ab / Cry3Bb1 Combined Trait, 

 Insect Control Proteins as Expressed in Maize, a Bacillus thuringiensis-Based Plant-Incorporated 

 Protectant for Control of the Corn Borer and Rootworm Complexes, and Consideration of a Data 

 Waiver Request for Further Acute Oral Toxicity Evaluation. Memorandum from C.A. Wozniak, 

 Ph.D. and J.L. Kough, Ph.D. to M. Mendelsohn dated October 17, 2003. 
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U.S. EPA. 2005a. Review of Product Characterization, Expression Analysis, and Human Health Data 

 for Plant-Incorporated Protectant Bacillus thuringiensis subspecies kumamotoensis Cry3Bb1 

 Insect Control Protein and the Genetic Material Necessary for Its Production (Vector ZMIR39) 

 in Maize (Corn) Plants Derived from Event MON 88017 (EPA Reg. No. 524-LLR) in Support 

 for Sec. 3 Registration. Memorandum from A. Fellman and J.L. Kough, Ph.D. to M. Mendelsohn 

 dated July 28, 2005. 

U.S. EPA. 2005b. Review of Product Characterization, Expression Analysis, and Human Health Data 

 for Plant-Incorporated Protectant Bacillus thuringiensis Cry3Bb1 and Cry1Ab Insect Control 

Proteins as Expressed in Corn Event MON 88017 x MON 810 [EPA Reg. No. 524-LLE] for 

Control of the Corn Borer and Rootworm Complexes, in Support for Sec. 3 Registration. 

Memorandum from A. Fellman and J.L. Kough, Ph.D. to M. Mendelsohn dated October 19, 

2005. 

U.S. EPA. 2006. Review of Independent Laboratory Method Validation and Protein Expression Levels 

 of Plant Tissues as Conditions of Registration (No. 5 and 6, Respectively) for Plant-Incorporated 

 Protectant Bacillus thuringiensis subspecies kumamotoensis Cry3Bb1 Insect Control Protein and 

 the Genetic Material Necessary for Its Production (Vector ZMIR13L) in Maize (Corn) Plants 

 Derived from Event MON 863 (EPA Reg. No. 524-528). Memorandum from A. Fellman and 

 J.L. Kough, Ph.D. to M. Mendelsohn dated July 26, 2006. 

U.S. EPA. 2007. Summary of PIP Method Validation Pre-Review for Cry3Bb1 in Corn. Memorandum 

 from M. Rindal and S. Lawrence to S. Reilly, Ph.D. dated April 10, 2007. 

U.S. EPA. 2010a. Expression Level Data for Cry1A(b) from MON 88017 x MON 810 Corn. 

 Memorandum from J.V. Gagliardi, Ph.D. and J.L. Kough, Ph.D. to M. Mendelsohn dated 

 June 24, 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 Materials” 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. 
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B. Human Health Assessment 

1. Background 

The basic premise relied on for the toxicology assessment is the fact that all the Bacillus thuringiensis

(Bt) plant-incorporated protectants are proteins. Proteins are commonly found in the diet and, except for 

a few well described phenomena, present little risk as a mammalian hazard. 

Several types of data are required for the Bt plant-incorporated protectants to provide a reasonable 

certainty that no harm will result from the aggregate exposure to these proteins. The information is 

intended to show that the Bt protein behaves as would be expected of a dietary protein, is not 

structurally related to any known food allergen or protein toxin, and does not display any oral toxicity 

when administered at high doses. These data consist of an in vitro digestion assay, amino acid sequence 

homology comparisons, and an acute oral toxicity test. The acute oral toxicity test is done at a maximum 

hazard dose using purified protein of the plant-incorporated protectant as a test substance. Due to 

limitations of obtaining sufficient quantities of pure protein test substance from the plant itself, an 

alternative production source of the protein, such as the Bt source organism or an industrial fermentation 

microbe, is often used. The justification for employing this alternative source of pure protein is the 

equivalence data discussed section II(A)(2)(a) of this Biopesticides Registration Action Document 

(BRAD). 

In general, the Environmental Protection Agency (EPA) believes that protein instability in digestive 

fluids and the lack of adverse effects using the maximum hazard dose approach eliminate the need for 

longer term testing of Bt protein plant-incorporated protectants. Dosing of animals with the maximum 

hazard dose, along with product characterization data, should identify potential toxins and allergens and 

provide an effective means to determine the safety of these proteins. The adequacy of the current testing 

requirements was discussed at the June 7, 2000 Federal Insecticide, Fungicide, and Rodenticide Act 

(FIFRA) Scientific Advisory Panel (SAP) meeting. In their final report, the SAP agreed in principle 

with the methods used by EPA to assess the toxicity of proteins expressed in plants, especially the 

maximum hazard dose approach (U.S. EPA 2000). 

a. In vitro Digestibility Assay

The intent of this assay is to demonstrate that the Bt protein is degraded into small peptides or amino 

acids in solutions that mimic digestive fluids. Usually, only gastric fluid is tested since Cry protein is 

known to be stable in intestinal fluid. In order to track the breakdown, the proteins were added to a 

solution of the digestive fluids, and a sample was either removed or quenched at given time points 

(usually at time 0, one to several minutes later, and one hour later). The time-point samples were then 

electrophoresed on either a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel 

and further analyzed by western blot or tested in a bioassay against the target pest. 
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As has been stated in several public fora, the in vitro digestibility test is basically a test to confirm the 

biochemical characteristic of instability of the protein in the presence of digestive fluids. The 

digestibility test is not intended to provide information on the toxicity of the protein or imply that 

similar breakdown will happen in all human digestive systems. The in vitro digestibility assay may also 

provide information about the potential of a protein to be a food allergen. The in vitro digestion assays 

confirm that the protein is being broken down in the presence of typical digestive fluids and is not 

unusually persistent in the digestive system. One of the limitations of the test is that it usually only 

tracks protein breakdown to fragments still recognized by the immunological reagents employed. 

 b. Amino Acid Homology

An additional characteristic that is considered as an indication of possible relation to a food allergen is a 

protein’s amino acid sequence when compared to known food allergens. 

c. Acute Oral Toxicity

One of the bases for addressing the toxicity of proteins primarily through the use of acute oral toxicity is 

that, when demonstrated to be toxic, proteins are toxic at low doses (Sjoblad et al. 1992). Therefore, 

when no effects are shown to be caused by the protein plant-incorporated protectants, even at relatively 

high-dose levels in the acute oral exposure test, the proteins are not considered toxic. 

2. Human Health Assessment of Cry3Bb1 

The detailed Agency human health assessment of Cry3Bb1 corn is found in U.S. EPA (2002a). Portions 

of the data used in the human health assessment are reviewed in U.S. EPA (2001a, 2001b, 2001c, 2001d, 

2001e, and 2005a). A summary of the key findings is provided below. 

 a. Mammalian Toxicity and Allergenicity Assessment

Based upon the human health data provided, there is minimal risk of toxic and/or allergenic effects to 

humans or animals due to exposure to the Cry3Bb1 proteins. Based on review of the data, there is a 

reasonable certainty of no harm to humans and animals posed by the aggregate exposure to residues of 

these proteins. 

i. Mammalian Toxicity

Data have been submitted demonstrating the lack of mammalian toxicity at high levels of exposure to 

the pure Cry3Bb1 proteins. These data demonstrate the safety of the products at levels well 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 similar to the 

Agency position regarding toxicity and the requirement of residue data for the microbial Bt products 

from which these plant-incorporated protectants were derived (see 40 CFR §§ 158.2130(d)(1)(i) and 

158.2140(d)(7)). For microbial products, further toxicity testing and residue data are triggered by 
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significant acute effects in studies, such as the mouse oral toxicity study, to verify and quantify the 

observed effects and clarify the source of these effects (Tiers II and III). 

Three acute oral studies were submitted for the Cry3Bb1 proteins (Master Record Identification 

Numbers (MRID Nos.) 449043-05, 449043-06, and 455382-02; reviewed in U.S. EPA (2001a, 2001b, 

and 2002a)). These studies were done with three variants of the Cry3Bb1 protein engineered with either 

four or five internal amino acid sequence changes to enhance activity against the corn rootworm. The 

acute oral toxicity data submitted support the prediction that the Cry3Bb1 protein would be non-toxic to 

humans. Male and female mice (10 of each) were dosed with 36, 396, or 3,780 milligrams/kilograms 

bodyweight (mg/kg bwt) of Cry3Bb1 protein for one variant. The mice were dosed with 38.7, 419, or 

2,980 mg/kg bwt of Cry3Bb1 protein for the second variant. The mice were dosed with 300, 900, or 

2,700 mg/kg bwt of Cry3Bb1 protein for the third variant. In one study, two animals in the high-dose 

group died within a day of dosing. These animals both had signs of trauma, probably due to dose 

administration (i.e., lung perforation or severe discoloration of lung, stomach, brain, and small 

intestine). No clinical signs were observed in the surviving animals and body weight gains were 

recorded throughout the 14-day study for the remaining animals. Gross necropsies indicated no findings 

of toxicity attributed to exposure to the test substance in any of the three studies. No other mortality or 

clinical signs attributed to the test substance were noted during any study. 

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 shown to be caused by the plant-incorporated 

protectants, even at relatively high dose levels, the Cry3Bb1 proteins are not considered toxic. Further, 

amino acid sequence comparisons showed no similarity between the Cry3Bb1 proteins and known toxic 

proteins available in public protein databases. 

 ii. Allergenicity Assessment

Since Cry3Bb1 is a protein, allergenic sensitivities were considered. Current scientific knowledge 

suggests that common food allergens tend to be resistant to degradation by acid and proteases, may be 

glycosylated, and can be present at high concentrations in the food. 

Data have been submitted that demonstrate that the Cry3Bb1 proteins are rapidly degraded by gastric 

fluid in vitro. In a solution of simulated gastric fluid (pH 1.2), complete degradation of detectable 

Cry3Bb1 protein occurred within 30 seconds. Insect bioassay data indicated that the protein lost 

insecticidal activity within 2 minutes of incubation in simulated gastric fluid (SGF). Incubation in 

simulated intestinal fluid resulted in a protein digestion product of ~59 kiloDaltons (kDa). A comparison 

of amino acid sequences of known allergens uncovered no evidence of any homology with Cry3Bb1, 

even at the level of 8 contiguous amino acids residues. 
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iii. Conclusion

The potential for the Cry3Bb1 proteins to be food allergens is minimal. Regarding toxicity to the 

immune system, the acute oral toxicity data submitted support the prediction that the Cry3Bb1 proteins 

would be non-toxic to humans. 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 shown to be caused 

by the plant-incorporated protectants, even at relatively high dose levels, the Cry3Bb1 proteins are not 

considered toxic. 

b. Aggregate Exposures

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

directs EPA to consider available information concerning exposures from the pesticide residue in food 

and all other non-occupational exposures, including drinking water from ground water or surface water 

and exposure through pesticide use in gardens, lawns, or buildings (residential and other indoor uses). 

The Agency has considered available information on the aggregate exposure levels of consumers (and 

major identifiable subgroups of consumers) to the pesticide chemical residue and to other related 

substances. These considerations include dietary exposure under the tolerance exemption and all other 

tolerances or exemptions in effect for the plant-incorporated protectants’ chemical residue, and exposure 

from non-occupational sources. Exposure via the skin 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. Oral exposure, at very low levels, may occur from 

ingestion of processed corn products and, potentially, drinking water. However, a lack of mammalian 

toxicity and the digestibility of the plant-incorporated protectants have been demonstrated. The use sites 

for the Cry3Bb1 proteins are all agricultural for control of insects. Therefore, exposure via residential or 

lawn use to infants and children is not expected. Even if negligible exposure should occur, the Agency 

concludes that such exposure would present no risk due to the lack of mammalian toxicity and low 

potential for allergenicity demonstrated for the Cry3Bb1 proteins. 

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

Section 408(b)(2)(D)(v) of FFDCA requires that, when considering whether to establish, modify, or 

revoke a tolerance, the Agency consider “available information concerning the cumulative effects of [a 

particular pesticide’s] residues and other substances that have a common mechanism of toxicity.” 

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

substances that have a common mechanism 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 indication of mammalian toxicity resulting from the plant-incorporated 

protectants, the Agency concludes that there are no cumulative effects for the Cry3Bb1 proteins. For 

information regarding EPA’s efforts to determine which chemicals have a common mechanism of 
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toxicity and to evaluate the cumulative effects of such chemicals, see EPA’s website at 

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

 d. Determination of Safety for U.S. Population, Infants, and Children

 i. Toxicity and Allergenicity Conclusions

The data submitted and cited regarding potential health effects for the Cry3Bb1 proteins include the 

characterization of the expressed Cry3Bb1 proteins in corn, as well as the acute oral toxicity and in vitro

digestibility of the proteins. The results 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 Cry3Bb1 protein test material derived from 

microbial cultures was biochemically and functionally similar to the protein produced by the plantincorporated protectant ingredients in corn. Production of microbially produced protein was chosen in 

order to obtain sufficient material for testing. 

The submitted acute oral toxicity data support the prediction that the Cry3Bb1 proteins would be nontoxic to humans. As mentioned in section II(B)(2)(a)(i) of this BRAD, when 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 shown to be caused by the Cry3Bb1 proteins, even at relatively high dose levels (3,780 mg/kg bwt 

of Cry3Bb1 protein), the Cry3Bb1 proteins are not considered toxic. Basing this conclusion 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-incorporated protectants were derived (see 40 CFR §§ 158.2130(d)(1)(i) and 

158.2140(d)(7)). For microbial products, further toxicity testing and residue data are triggered by 

significant acute effects in studies, such as the mouse oral toxicity study, to verify and quantify the 

observed effects and clarify the source of these effects (Tiers II and III). 

Cry3Bb1 protein residue chemistry data were not required for a human health effects assessment of the 

subject plant-incorporated protectant ingredients because of the lack of mammalian toxicity. 

Neither available information concerning the dietary 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 experimentation data were evaluated. The lack of 

mammalian toxicity at high levels of exposure to the Cry3Bb1 proteins, as well as the minimal potential 

to be a food allergen, demonstrate the safety of the product at levels well above possible maximum 

exposure levels anticipated in the crop. 

The genetic material necessary for the production of the plant-incorporated protectant active ingredients 

are the nucleic acids (deoxyribonucleic acid (DNA), ribonucleic acid (RNA)) that comprise genetic 
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material encoding these proteins and their regulatory regions. “Regulatory regions” are the genetic 

material—such as promoters, terminators, and enhancers—that control the expression of the genetic 

material encoding the proteins. DNA and RNA are common to all forms of plant and animal life, and the 

Agency knows of no instance where these nucleic acids have been associated with toxic effects related 

to their consumption as a component of food. These ubiquitous nucleic acids, as they appear in the 

subject active ingredients, have been adequately characterized by the applicant. Therefore, no 

mammalian toxicity is anticipated from dietary exposure to the genetic material necessary for the 

production of the subject active plant pesticidal ingredients. Further, the genetic material (DNA, RNA) 

necessary for the production of the Cry3Bb1 proteins have been exempted under the blanket exemption 

for all nucleic 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, special susceptibility of infants and children to 

pesticide chemical residues, and the cumulative effects on infants and children of the residues and other 

substances with a common mechanism of toxicity. 

In addition, FFDCA section 408(b)(2)(C) also provides that EPA shall apply an additional tenfold 

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

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

of safety will be safe for infants and children. 

In this instance, based on all the available information, the Agency concludes that there is a finding of 

no toxicity for the Cry3Bb1 proteins and the genetic material necessary for their production. Thus, there 

are no threshold effects of concern and, as a result, an additional margin of safety for infants and 

children 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 Cry3Bb1 proteins and the genetic material necessary for their 

production. This includes all anticipated dietary exposures and all other exposures for which there is 

reliable information. 

The Agency has arrived at this conclusion because, as discussed thus far in this chapter, no toxicity to 

mammals has been observed, nor has there been any indication of allergenicity potential for the plantincorporated protectants. 
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 e. Other Considerations 

 i. Analytical Enforcement Methodology

Methods for extraction and direct enzyme-linked immunosorbent assay (ELISA) analysis of Cry3Bb1 in 

corn grain have been submitted and found acceptable by the Agency. 

 ii. International Residue Limits

In making its tolerance decisions, EPA seeks to harmonize U.S. tolerances with international standards 

whenever possible, consistent with U.S. food safety standards and agricultural practices. In this context, 

EPA considers the international maximum residue limits (MRLs) established by the Codex Alimentarius 

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

United Nations Food and Agriculture Organization/World Health Organization food standards program, 

and it is recognized as an international food safety standards-setting organization in trade agreements to 

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

maximum residue level (MRL); however, FFDCA section 408(b)(4) requires that EPA explain the 

reasons for departing from the Codex level. 

The Codex has not established a MRL for the Cry3Bb1 proteins in corn. 

 

 f. Endocrine Disruptors 

As required under FFDCA section 408(p), the Agency has developed the Endocrine Disruptor Screening 

Program (EDSP) to determine whether certain substances (including pesticide active and other 

ingredients) may have an effect in humans or wildlife similar to an effect produced by a “naturally 

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

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

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

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

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

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

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

the E, A, or T effect. 

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

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

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

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

construed as a list of known or likely endocrine disruptors. 
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The Cry3Bb1 proteins are not among the group of 58 pesticide active ingredients on the initial list to be 

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

chemicals. Accordingly, the Agency anticipates issuing future EDSP orders/data call-ins for all pesticide 

active ingredients. 

For further information on the status of the EDSP, the policies and procedures, the list of 67 chemicals, 

the test guidelines and the Tier 1 screening battery, please visit our website: http://www.epa.gov/endo/. 

 g. MON 863 90-Day Rat Feeding Study

The EPA and the Food and Drug Administration (FDA) did not request or receive the 90-day dietary 

study nor was there any formal review of the full study by either agency. Both agencies completed the 

safety evaluations for MON 863 corn and did not need the 90-day oral toxicity study to reach a finding 

on human dietary safety. However, other governments often request additional information or studies, 

and summary information on these studies are frequently provided to the U.S. governmental agencies on 

request. A summary of the study report was provided to the EPA. 

The 90-day oral toxicity study (CV-2000-260) was performed by Convance Labs (Madison, Wisconsin) 

with high percentages of MON 863 corn (11 and 33%) used to make diet for the test rodents. The study 

was done according to Organization for Economic Cooperation and Development (OECD) guideline 

protocol 408 for a 90-day oral toxicity study and performed under the good laboratory practices 

guidelines. These testing requirements, routinely used for regulatory purposes, are actually more 

stringent in quality control procedures and record keeping than studies routinely reported in the 

scientific literature. An appropriate isoline corn control (a corn hybrid similar to MON 863 but not 

expressing the beetle control protein), and reference corn varieties (six different non-transgenic, 

commercial corn varieties) were employed to address the possible effects of different corn compositions 

on rat nutrition. The prepared diets were also analyzed to confirm that the proper test corn lines were 

used to make the animal feed. All test animals received diets specially prepared by Purina Mills 

formulated to contain 33% corn (conventional corn, MON 863 corn, or a combination) and provide a 

balanced diet. 

It is important that a balanced diet be provided rather than feeding the test animals 100% corn so that 

any abnormalities that occur can be assigned to the effect of the corn component itself rather than any 

dietary insufficiency. It is also important that more than one level of MON 863 corn in the diet be 

included to establish if a dose-response is present. The dose-response shows that, if an effect is seen at a 

low dose, a more dramatic and similar effect will also occur at a higher dose. While dose-response is a 

key component to a toxicology assessment, it is difficult to establish in a whole food study. This is one 

of the reasons that EPA requests high dose purified protein toxicity studies for plant-incorporated 

protectants and compositional analysis is used for examining whole food. 

The 90-day rat oral study with MON 863 was reviewed by both the Robert Koch Institute (RKI), as the 

German competent authority, and subsequently the European Food Safety Authority (EFSA). As could 

be anticipated by having such a large study with numerous treatments and comparisons, some 
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differences were found between treatments. In understanding the significance of these findings, it is 

important to be aware of the natural variation of biological systems in a population of test organisms and 

the range of responses to a treatment. As discussed in information provided by both RKI and EFSA, the 

decreases in male kidney weight and some changes in hematological parameters (lymphocyte and 

reticulocyte counts) for the highest MON 863-treatment groups were significantly different compared to 

the control isoline corn group. However, when compared to the included reference corn variety 

treatments, the values were found to be within the realm of normal biological variation. Since there were 

questions about possible kidney pathology, independent veterinary pathologists were asked to examine 

tissue samples and render an opinion. No significant findings relevant to an adverse toxicity 

determination were noted. Therefore, both RKI and EFSA found that there were no resultant concerns 

over the safety of MON 863 due to the results of the 90-day toxicity test. 

Since the earlier examination of the study summaries of the 90-day feeding study for MON 863, a more 

recent publication by Séralini et al. (2007) has reanalyzed the data from the study. The paper states that 

there are significant effects due to MON 863 corn being present in the diet of the treated rodents. The 

authors postulate that new analyses of growth curves and blood chemistry parameters suggest significant 

hepatorenal effects. The Agency considers these new findings open to a different interpretation. The 

original studies did not demonstrate any changes in the blood chemistry parameters or body weight 

gains that showed dose-dependent responses. In addition, the organs most likely to be impacted by these 

chemistry changes (liver and kidney) were examined histologically in the original reports by 

independent, competent veterinary pathologists. The reports on these tissues indicated no signs of 

cellular toxicity were found in the hepatic or renal tissues. Without signs of cellular tissue damage in the 

relevant organs, the variations in blood chemistry should not be considered signs of toxicity. 

h. MON 88017 Considerations (Reviewed in U.S. EPA (2005b))

The evaluation of mammalian toxicity of the Cry3Bb1 protein produced in MON 88017 is based on 

studies conducted with Cry3Bb1 protein variants (Cry3Bb1.11098 and Cry3Bb1.11231). The 

physicochemical characteristics of the Cry3Bb1 protein in MON 88017 were found to be similar 

with the Cry3Bb1 protein in MON 863. Moreover, the protein expression levels, insect bioactivity, 

and field efficacy data of Cry3Bb1 protein in MON 88017 were compared to MON 863 and found 

to be similar and functionally equivalent. Therefore, because the Cry3Bb1 proteins produced in 

MON 88017 and MON 863 share an amino acid sequence identity of >99.8% and the 

aforementioned similarities, the data (including the acute mouse gavage studies and in vitro

digestibility studies previously submitted for MON 863) were bridged to support the finding that 

there is a reasonable certainty that no harm will result to from aggregate exposure to the U.S. 

population, including infants and children, to the Cry3Bb1 protein as expressed in MON 88017. 

 i Tolerance Exemption 

Certain data submitted and reviewed for past experimental use permits, as well as the FIFRA section 3 

registration of Corn Event MON 863 (EPA Reg. No. 524-528), also supported the petition for a 
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tolerance exemption for residues of the Bt Cry3Bb1 protein. 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 Cry3Bb1 protein and the genetic material necessary for its production in 

corn when used as plant-incorporated protectants in the food and feed commodities of field corn, sweet 

corn, and pop corn under 40 CFR § 180.1214 (69 Federal Register (FR) 16809; March 31, 2004). 

Subsequently, administrative revisions were made to existing plant-incorporated protectant tolerance 

exemptions (e.g., some tolerance exemptions were moved from 40 CFR part 180 to 40 CFR part 174). 

The original Cry3Bb1 tolerance exemption was subject to these revisions as it was redesignated from 40 

CFR § 180.1214 to 40 CFR § 174.518 and changed to the following (72 FR 20431; April 25, 2007): 

 “§ 174.518 Bacillus thuringiensis Cry3Bb1 protein in corn; 

 exemption from the requirement of a tolerance. 

 Residues of Bacillus thuringiensis Cry3Bb1 protein in corn are exempt 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.” 

Table 1. Human Health Assessment Data for Cry3Bb1 (Reviewed in U.S. EPA (2002a) Unless 

 Otherwise Noted). 

Study Title Summary MRID No.

Acute Oral Toxicity of 

B.t. Protein 11098 in 

Mice 

There did not appear to be significant adverse effects to animals dosed with 

Cry3Bb1 at corrected dose amounts of 38.7, 419, or 2,980 milligrams per 

kilogram (mg/kg) bodyweight. Two animals died during the study—animal 

numbers 98035M3-007 and 98035F3-004 in the 2,980 mg/kg treatment 

group. These deaths appeared to be the result of trauma from dosing rather 

than from the test substance. In addition, although there were some minor 

weight loss and minor abnormal observations at gross necropsy, these 

occurred in both test and control groups and, therefore, do not appear to be 

Cry3Bb1.11098 protein exposure related. Based upon the data provided, 

the median lethal dose (LD50) for Cry3Bb1.11098 is greater than 2,980 

mg/kg bodyweight in mice. 

Classification: Acceptable 

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

449043-06 

Acute Oral Toxicity 

Study of B.t. Protein 

11231 in Mice 

There were no apparent adverse effects identified in mice dosed orally with 

36, 396 and 3,780 mg/kg bodyweight of Cry3Bb1.11231 protein. There 

was some minor weight loss in a few animals and some minor abnormal 

observations via gross necropsy, but these occurred in both the test and 

control groups and, therefore, do not appear to be Cry3Bb1.11231 protein 

exposure related. Based upon the data provided, the LD50 for 

Cry3Bb1.11231 is greater than 3,780 mg/kg bodyweight in mice. 

Classification: Acceptable 

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

449043-05 
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Study Title Summary MRID No.

An Acute Oral Toxicity 

Study in Mice with E. 

coli Produced 

Cry3Bb1.11098 

(Q349R) Protein 

There did not appear to be significant adverse affects to animals resulting 

from exposure to Cry3Bb1.11098 (Q349R) at dose amounts of 300, 900, 

and 2,700 mg/kg body weight. Observations included some minor clinical 

effects and a relatively insignificant lack of weight gain in two animals; 

however, these do not appear to be related to exposure to the test substance 

because these occurred in the various test groups. Based upon the data 

contained in this submission, the LD50 for Cry3Bb1.11098(Q349R) is 

greater than 3,200 mg/kg body weight in mice. 

Classification: Acceptable 

455382-02 

Assessment of the in 

vitro Digestibility of B.t. 

Protein 11098 and B.t. 

Protein 11231 Utilizing 

Mammalian Digestive 

Fate Models 

The tests performed in this study show that the Cry3Bb1 proteins are not 

stable to digestion in simulated gastric fluid. Incubation of Cry3Bb1.11098 

and Cry3Bb1.11231 in SGF results in the loss of detectable protein by the 

30- and 15-second observation points, respectively, as detected by SDSPAGE. Insect bioassay data indicated that the protein lost insecticidal 

activity within 2 minutes of incubation in SGF. Incubation in the simulated 

intestinal fluid (SIF) resulted in a ~59 kDa digestion product that retained 

its insecticidal activity for at least 30 minutes. 

Classification: Acceptable 

(Reviewed in U.S. EPA (2001c)) 

449043-07 

Amended Report for 

MSL-15704: 

Assessment of the in 

vitro Digestibility of B.t. 

Protein 11098 and B.t. 

Protein 11231 Utilizing 

Mammalian Digestive 

Fate Models 

The tests performed in this study show that the Cry3Bb1 proteins are not 

stable to digestion in simulated gastric fluid. Incubation of Cry3Bb1.11098 

and Cry3Bb1.11231 in SGF results in the loss of detectable protein by the 

30- and 15-second observation points, respectively, as detected by SDSPAGE. Insect bioassay data indicated that the protein lost insecticidal 

activity within 2 minutes of incubation in SGF. Incubation in the SIF 

resulted in a ~59 kDa digestion product that retained its insecticidal activity 

after at least 30 minutes incubation. 

Classification: Acceptable 

454240-06 

Assessment of the in 

vitro Digestibility of 

Cry3Bb1 Protein 

Purified from Corn 

Event MON 863 and 

Cry3Bb1 Protein 

Purified from E. coli

The tests performed in this study show that the Cry3Bb1 proteins are 

degraded in simulated gastric fluid. Incubation of corn-produced and 

Escherichia coli-produced Cry3Bb1 protein in SGF results in the loss of 

detectable protein by the 15-second observation point, as detected by SDSPAGE. 

Classification: Acceptable

455382-03 
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Study Title Summary MRID No.

Assessment of the in 

vitro Digestibility of the 

Cry3Bb1.11098 

(Q349R) Protein in 

Simulated Intestinal 

Fluid 

Simulated intestinal fluid activity was verified to be present and at a level 

deemed acceptable by SOP GEN-PRO-058-01. The gels provided indicate 

that the Cry3Bb1.11098 (Q349R) protein is present as a single band at 74 

kDa, which rapidly degraded to two bands of 68 kDa and 57 kDa at the 

first assay time point of 1 minute. The subsequent samples (from 5 minutes 

to 24 hours) all gave a single 57 kDa band that did not appear to decrease 

in intensity. This lack of degradation by intestinal fluids is similar to the 

majority of Cry proteins, which are resistant to the action of trypsin. 

Classification: Acceptable

455770-02 

Amended Report for 

MSL-16597: ImmunoDetectability of 

Cry3Bb1.11098 and 

Cry3Bb1.11231 

Proteins in the Grain of 

Insect Protected Corn 

Events MON 863 and 

MON 853 After Heat 

Treatment 

Heating the corn flour samples at 204C for 30 minutes destroys both the 

immunoreactivity and insect bioactivity of the Cry3Bb1.11098 found in 

MON 863 corn. The Cry3Bb1 immunoreactivity was not detectable in both 

an immunoblot and ELISA format for MON 863. For MON 853, Cry3Bb1 

was not recognizable in an immunoblot and reduced more than 1000-fold 

in an ELISA format. Since the rabbit anti-Cry3Bb1 antibody employed was 

polyclonal immunoglobulin G (IgG), it is also suggestive that epitopes 

were destroyed and not just rendered unrecognized by alteration of the 

three-dimensional configuration. 

Classification: Acceptable 

454240-07 

Bioinformatics Analysis 

of B.t. Protein 11098 

and B.t. Protein 11231 

Sequences Utilizing 

Toxin and Public 

Domain Genetic 

Databases 

Several amino acid database comparison tools were employed to compare 

the amino acid sequence of Cry3Bb1.11098 and Cry3Bb1.11231 to known 

protein toxins. The TOXIN4 database was compiled to allow for 

comparison of Cry3Bb1.11098 and Cry3Bb1.11231 to these known toxin 

proteins. All of the protein similarities identified were to insecticidal 

protein with no similarity to proteins known to be toxic to humans and/or 

animals. Based upon these data, it does not appear that Cry3Bb1.11098 nor 

Cry3Bb1.11231 share significant structural, biological, or immunological 

similarity with known protein toxins other than those affecting insects. 

Classification: Acceptable 

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

449043-08 
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Study Title Summary MRID No.

Bioinformatics Analysis 

of B.t. Protein 11098 

and B.t. Protein 11231 

Sequences Utilizing an 

Allergen Database 

Several amino acid database comparison tools were employed to compare 

the amino acid sequence of Cry3Bb1 to known protein allergens and 

gliadins. The UPDATE2 database was compiled to allow for comparison of 

Cry3Bb1.11098 and Cry3Bb1.11231 to these proteins. The level of 

similarity identified does not indicate significant similarity to any of the 

proteins or gliadins contained in the database. In addition, no contiguous 

stretch of 8 identical amino acids was identified in either the FASTA or 

IDENTITYSEARCH algorithms, suggesting a lack of immunological 

similarity. Based upon these data, it does not appear that Cry3Bb1 shares 

significant structural, biological, or immunological similarity with known 

protein allergens or gliadins. 

Classification: Acceptable 

(MRID No. 449043-09 reviewed in U.S. EPA (2001e)) 

449043-09 

454240-08 
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Study Title Summary MRID No.

Safety Assessment of 

Cry3Bb1 Variants in 

Corn Rootworm 

Protected Corn 

Plants transformed for corn rootworm control (Event MON 863) contained 

a total of seven amino acid changes within the Cry3Bb1.11098 deltaendotoxin when compared to the sequence as found in wild type Bt. Bt

strains EG11231 and EG11098 contain variants of the Cry3Bb1 protein, 

which differ from the wild type delta-endotoxin by 4 and 5 amino acids, 

respectively. Two further alterations in amino acid sequence were made for 

Cry3Bb1.11098 during cloning and insertion into the maize genome. 

Structural data indicate that these alleles of this protein maintained a very 

similar structure to the native form. The initial transformation event used to 

evaluate the rootworm-protected maize was MON 853, which encodes 

Cry3Bb1 variant 11231. Protein produced by fermentation of Bt cells 

expressing variant 11231 was used in toxicology studies for environmental 

and mammalian concerns. Functional and physicochemical equivalence 

between variant 11231 produced in Bt and that produced in MON 853 were 

demonstrated. Maize was also transformed with variant 11098, resulting in 

transformation Event MON 863. These two variants, 11098 and 11231, 

were shown to be physicochemically and functionally equivalent. The 

registrant stated that an examination of toxicity toward catfish, bobwhite 

quail, Daphnia magna, Collembola (Folsomia candida), adult and larval 

honeybees, a ladybird beetle, a green lacewing, a parasitic wasp, and 

earthworms resulted in a NOEC (No Observable Effect Concentration) 

being established that exceeded the concentration of Cry3Bb1 toxins 

expected in the maximum environmental exposure. No Observable Effect 

Concentrations surpassed maximum predicted environmental 

concentrations by 3 to 141 fold, hence, the risk to non-target organisms 

from the culture of MON 863 is indicated to be minimal. However, this 

aspect is the subject of another review and outside the purview of this 

report. Given the lack of known mechanisms of mammalian toxicity from 

Bt delta-endotoxins, their widespread use in agriculture, the rapid 

digestibility of Cry3Bb1 proteins, their lack of homology to known toxins 

and allergens, and the safety of the microbial biopesticide Raven®, which 

expresses Cry3Bb1 proteins, the Cry3Bb1 protein is expected to have a 

reasonable certainty of causing no harm in its aggregate exposure. 

Classification: Acceptable 

(Reviewed in U.S. EPA (2002b)) 

454240-09 
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Study Title Summary MRID No.

Human Health and 

Environmental 

Assessment of the 

Plant-Incorporated 

Protectant Bacillus 

thuringiensis Cry3Bb1 

Protein Produced in 

MON 88017 

This report is a summary of the product characterization studies and the 

human health assessment for the plant-incorporated protectant, Bt Cry3Bb1 

protein produced in MON 88017. 

All physiochemical characteristics in the Cry3Bb1 protein in MON 88017 

were found to be similar to the Cry3Bb1 protein in MON 863. Moreover, 

the protein expression levels, insect bioactivity, and field efficacy data of 

Cry3Bb1 protein in MON 88017 were compared to MON 863 and found to 

be similar and functionally equivalent. Based on substantial similarity of 

Cry3Bb1 protein in MON 88017 and the Cry3Bb1 protein produced in the 

registered MON 863 corn product, data were bridged from the studies 

previously submitted for MON 863. Collectively, these studies 

demonstrated that the Cry3Bb1 protein variants are not toxic to mammals, 

are rapidly and completely digested in simulated gastric fluid, do not share 

any amino acid homology to known toxins or allergens, and are unlikely to 

produce a toxic or allergic response in humans. Therefore, there is a 

reasonable certainty that no harm will result from potential exposure to the 

Cry3Bb1 protein in transgenic corn MON 88017. 

Classification: Acceptable 

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

461817-01 

3. Human Health Assessment of Cry1Ab (Expressed in MON 863 x MON 810 and MON 88017 x 

 MON 810), NPTII (Expressed in Corn Event MON 863 and MON 863 x MON 810), and CP4 

 EPSPS (Expressed in MON 88017 and MON 88017 x MON 810)

Based on previously completed Agency human health assessments, permanent tolerance exemptions 

have been established for the Cry1Ab, neomycin phosphotransferase II (NPTII), and CP4 5-

enolpyruvylshikimate-3-phosphate (CP4 EPSPS) proteins: 

 “§ 174.511 Bacillus thuringiensis Cry1Ab protein in all plants; 

 exemption from the requirement of a tolerance. 

 Residues of Bacillus thuringiensis Cry1Ab protein in all plants are exempt 

 from the requirement of a tolerance 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) 
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 “§ 174.521 Neomycin phosphotransferase II; 

 exemption from the requirement of a tolerance. 

 Residues of the neomycin phosphotransferase II (NPTII) enzyme are 

 exempted from the requirement of a tolerance in all food commodities 

 when used as a plant-incorporated protectant inert ingredient.” 

 **original under 40 CFR § 180.1134 (59 FR 49351; September 28, 1994) 

 **revised under 40 CFR § 174.521 (72 FR 20431; April 25, 2007) 

 

 “§ 174.523 CP4 Enolpyruvylshikimate-3-phosphate (CP4 EPSPS) synthase in all plants; 

 exemption from the requirement of a tolerance. 

 Residues of the CP4 Enolpyruvylshikimate-3-phosphate (CP4 EPSPS) 

 synthase enzyme in all plants are exempt from the requirement of a 

 tolerance when used as plant-incorporated protectant inert ingredients 

 in all food commodities.” 

 

 **original under 40 CFR § 180.1174 (61 FR 40338; August 2, 1996) 

 **revised under 40 CFR § 174.523 (72 FR 20431; April 25, 2007) 

The toxicological and allergenicity data supporting the establishment of the Cry1Ab tolerance 

exemption, as well as the associated registrations of MON 863 x MON 810 and MON 88017 x MON 

810, can be found in the 2001 Bt Crops Reassessment and/or the September 2010 Cry1Ab and Cry1F 

BRAD (U.S. EPA 2001f and 2010). 

The individual data generated for the Cry1Ab, NPTII, and CP4 EPSPS proteins (and Cry3Bb1) support 

their inclusion, as expressed in particular stacked plant-incorporated protectants (i.e., MON 863 x MON 

810 and MON 88017 x MON 810), into the appropriate tolerance exemptions since the mode of action 

for these proteins does not suggest a synergistic activity in combination for mammalian species (U.S. 

EPA 2003, 2005b). This lack of synergism is also suggested by the absence of enhanced responses in 

sensitive target species tested with the combination of Cry1Ab and Cry3Bb1 proteins. 

Human health assessment data, provided specifically in relation to the Corn Event MON 863 

registration, are summarized in Table 2. 
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Table 2. Human Health Assessment Data for NPTII (Reviewed in U.S. EPA (2002a)). 

Study Title Summary MRID No.

Immuno-Detectability 

of NPTII Protein in the 

Grain of Insect 

Protected Corn Event 

MON 863 After Heat 

Treatment 

The immunoblot shows that extraction of the MON863 corn grain spiked 

with NPTII yielded an immunoreactive band that comigrated with the E. 

coli-produced NPTII. The blot also showed that, regardless of the 

extraction buffer used, the heat treatment effectively removed any 

immunoreactive bands from the samples. The results suggest that, even if 

detectable levels of NPTII were present in MON863 corn grain, the heat 

treatment would remove them. Unfortunately, the use of a mouse 

monoclonal antibody limits the ability of this data to be extrapolated. A 

heat treatment, significantly above the 95.8C used for sample preparation 

for SDS-PAGE, destroyed the epitope(s) recognized by the anti-NPTII 

antibody used. 

Classification: Acceptable 

455382-09 

4. References 

MRID No. 449043-05. Bechtel C. 1999. Acute Oral Toxicity Study of B.t. Protein 11231 in Mice. Lab 

 Project Number: 16216: ML-98-142: MSE-N 98036. Unpublished study prepared by Monsanto 

 Company, 212 pages. 

MRID No. 449043-06. Bechtel C. 1999. Acute Oral Toxicity of B.t. Protein 11098 in Mice. Lab Project 

 Number: 16215: ML-98-141: MSE-N 98035. Unpublished study prepared by Monsanto 

 Company, 212 pages. 

MRID No. 449043-07. Leach J, Hileman R, Martin J. 1999. Assessment of the in vitro Digestibility of 

B.t. Protein 11098 and B.t. Protein 11231 Utilizing Mammalian Digestive Fate Models. Lab 

 Project Number: 98-01-39-02: 15704. Unpublished study prepared by Monsanto Company, 40 

 pages. 

MRID No. 449043-08. Astwood J, Hileman R. 1999. Bioinformatics Analysis of B.t. Protein 11098 and 

B.t. Protein 11231 Sequences Utilizing Toxin and Public Domain Genetic Databases. Lab Project 

 Number: 15870. Unpublished study prepared by Monsanto Company, 294 pages. 

MRID No. 449043-09. Astwood J, Hileman R. 1999. Bioinformatics Analysis of B.t. Protein 11098 and 

B.t. Protein 11231 Sequences Utilizing an Allergen Database. Lab Project Number: 15873. 

 Unpublished study prepared by Monsanto Company, 38 pages. 
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MRID No. 454240-06. Leach J, Hileman R, Martin J. 2001. Amended Report for MSL-15704: 

 Assessment of the in vitro Digestibility of B.t. Protein 11098 and B.t. Protein 11231 Utilizing 

 Mammalian Digestive Fate Models. Lab Project Number: 98-01-39-02: 17166: MSL-17166. 

 Unpublished study prepared by Monsanto Company, 41 pages. 

MRID No. 454240-07. Holleschak G, Hileman R, Astwood J. 2001. Amended Report for MSL-16597: 

 Immuno-Detectability of Cry3Bb1.11098 and Cry3Bb1.11231 Proteins in the Grain of Insect 

 Protected Corn Events MON 863 and MON 853 After Heat Treatment. Lab Project Number: 99- 

 01-39-32: MSL-17223: 17223. Unpublished study prepared by Monsanto Company, 36 pages. 

MRID No. 454240-08. Hileman R, Rice E, Goodman R. 2001. Bioinformatics Evaluation of the 

 Cry3Bb1 Protein Produced in Corn Event MON 863 Utilizing Allergen, Toxin and Public 

 Domain Protein Databases. Lab Project Number: 01-01-39-23: 17140: MSL-17140. Unpublished 

 study prepared by Monsanto Company, 102 pages. 

MRID No. 454240-09. Hileman R, Astwood J, McKee M. 2001. Safety Assessment of Cry3Bb1 

 Variants in Corn Rootworm Protected Corn. Lab Project Number: 17225: MSL-17225. 

 Unpublished study prepared by Monsanto Company, 51 pages. 

MRID No. 455382-02. Bonnette K, Pyla P. 2001. An Acute Oral Toxicity Study in Mice with E. coli 

 Produced Cry3Bb1.11098(Q349R) Protein: Amended Final Report. Lab Project Number: 

 3044.856: SB-2001-085: MSL-17382. Unpublished study prepared by Springborn Laboratories, 

 Incorporated, 154 pages. 

MRID No. 455382-03. Leach J, Hileman R, Astwood J. 2001. Assessment of the in vitro Digestibility of 

 Cry3Bb1 Protein Purified from Corn Event MON 863 and Cry3Bb1 Protein Purified from E. 

 coli. Lab Project Number: 17292: 01-01-39-13. Unpublished study prepared by Monsanto 

 Company, 43 pages. 

MRID No. 455382-09. Holleschak G, Hileman R, Astwood J. 2001. Immuno-Detectability of NPTII 

 Protein in the Grain of Insect Protected Corn Event MON 863 After Heat Treatment. Lab Project 

 Number: 01-01-39-36: MSL-17300. Unpublished study prepared by Monsanto Company, 25 

 pages. 

MRID No. 455770-02. Kileman R, Leach J, Astwood J. 2001. Assessment of the in vitro Digestibility of 

 the Cry3Bb1.11098 (Q349R) Protein in Simulated Intestinal Fluid. Lab Project Number: 17530: 

 01-01-62-11. Unpublished study prepared by Monsanto Company, 36 pages.

MRID No. 461817-01. Sidhu R. 2004. Human Health and Environmental Assessment of the Plant- 

 Incorporated Protectant Bacillus thuringiensis Cry3Bb1 Protein Produced in MON 88017. 

 Project Number: MSL/18835. Unpublished study prepared by Monsanto Company, 64 pages. 
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Séralini GE, Cellier D, de Vendomois JS. 2007. New analysis of a rat feeding study with a 

 genetically modified maize reveals signs of hepatorenal toxicity. Archives of Environmental 

 Contamination and Toxicology 52(4):596–602. 

Sjoblad RD, McClintock JT, Engler R. 1992. Toxicological considerations for protein components 

 of biological pesticide products. Regulatory Toxicology and Pharmacology 15(1):3–9. 

U.S. EPA. 2000. SAP Report No. 2000-03B. Sets of Scientific Issues Being Considered by the 

Environmental Protection Agency Regarding: Session II – Mammalian Toxicity Assessment 

Guidelines for Protein Plant Pesticides. Dated September 28, 2000. Available from: 

http://www.epa.gov/scipoly/sap/meetings/2000/june/finbtmamtox.pdf. 

 

U.S. EPA. 2001a. Data Evaluation Report on MRID No. 449043-06 (“Acute Oral Toxicity Study of B.t. 

 Protein 11098 in Mice”). Completed by M.T. Watson, Ph.D. and J.L. Kough, Ph.D. on April 25, 

 2001. 

U.S. EPA. 2001b. Data Evaluation Report on MRID No. 449043-05 (“Acute Oral Toxicity Study of B.t. 

 Protein 11231 in Mice”). Completed by M.T. Watson, Ph.D. and J. L. Kough, Ph.D. on 

 April 25, 2001. 

U.S. EPA. 2001c. Data Evaluation Report on MRID No. 449043-07 (“Assessment of the in vitro 

 Digestibility of B.t. Protein 11098 and B.t. Protein 11231 Utilizing Mammalian Digestive Fate”). 

 Completed by M.T. Watson, Ph.D. and J.L. Kough, Ph.D. on April 25, 2001. 

U.S. EPA. 2001d. Data Evaluation Report on MRID No. 449043-08 (“Bioinformatics Analysis of B.t. 

 Protein 11098 and B.t. Protein 11231 Sequences Utilizing Toxin and Public Domain Genetic 

 Databases”). Completed by M.T. Watson, Ph.D. and J.L. Kough, Ph.D. on April 25, 2001. 

U.S. EPA. 2001e. Data Evaluation Report on MRID No. 449043-09 (“Bioinformatics Analysis of B.t. 

 Protein 11098 and B.t. Protein 11231 Sequences Utilizing an Allergen Database”). Completed by 

 M.T. Watson, Ph.D. and J.L. Kough, Ph.D. on April 25, 2001. 

 

U.S. EPA. 2001f. Biopesticides Registration Action Document – Bacillus thuringiensis Plant- 

 Incorporated Protectants. Available from: 

 http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm. 

U.S. EPA. 2002a. EPA Review of the Application for an Exemption from the Requirement of a 

 Tolerance for Cry3Bb1 Protein Expressed in All Food Commodities. C. Wozniak, Ph.D. and 

 J.L. Kough, Ph.D. to M. Mendelsohn dated March 29, 2002. 
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U.S. EPA. 2002b. Assessment of the Product Characterization of the Cry3Bb1 Insect Control Protein as 

 Expressed in Maize, a Bacillus thuringiensis-Based Plant-Incorporated Protectant for the Control 

 of the Corn Rootworm. C.A. Wozniak, Ph.D. and J.L. Kough, Ph.D. to M. Mendelsohn dated 

 March 29, 2002. 

U.S. EPA. 2003. Applicability of Existing Tolerances for Cry3Bb1 and Cry1Ab Protein to the Stacked 

 Product Expressing Both Proteins. Memorandum from J.L. Kough, Ph.D. to M. Mendelsohn 

 dated June 20, 2003. 

U.S. EPA. 2005a. Review of Product Characterization, Expression Analysis, and Human Health Data 

 for Plant-Incorporated Protectant Bacillus thuringiensis subspecies kumamotoensis Cry3Bb1 

 Insect Control Protein and the Genetic Material Necessary for Its Production (Vector ZMIR39) 

 in Maize (Corn) Plants Derived from Event MON 88017 (EPA Reg. No. 524-LLR) in Support 

 for Sec. 3 Registration. A. Fellman and J.L. Kough, Ph.D. to M. Mendelsohn dated July 28, 

 2005. 

U.S. EPA. 2005b. Determination if the Existing Exemptions from the Requirement of a Tolerance for 

 Cry3Bb1 [40 CFR Part 180.1214] and Cry1Ab [40 CFR 180.1173] Apply to Cry3Bb1 Protein 

 Expressed in Event MON 88017 Corn [EPA Reg. No. 524-LLR] and the Combination of 

 Cry3Bb1 and Cry1Ab Proteins Expressed in Event MON 88017 x MON 810 Corn [EPA Reg. 

 No. 524-LLE]. Memorandum from A. Fellman and J.L. Kough, Ph.D. to M. Mendelsohn dated 

 December 7, 2005. 

U.S. EPA. 2010. Biopesticides Registration Action Document – Bacillus thuringiensis 

Cry1Ab and Cry1F Corn (Updated September 2010). Available from: 

http://www.regulations.gov (see “Supporting & Related Materials” within Docket Number 

EPA-HQ-OPP-2010-0607). 
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C. Environmental Assessment 

1. Tiered Hazard and Risk Assessment Process 

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

risk is determined first from estimates of hazard under “worst-case” exposure conditions. A lack of 

adverse effects under these conditions would provide enough confidence that there is no risk and no 

further data would be needed. Hence, such screening tests conducted early in an investigation tend to be 

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

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

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

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

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

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

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

reliable results at relatively low cost.a

 

Tiered tests are designed to first represent unrealistic worst-case scenarios and ONLY progress to realworld field scenarios if the earlier tiered tests fail to indicate adequate certainty of acceptable risk. 

Screening (Tier I) non-target organism hazard tests are conducted at exposure concentrations several 

times higher than the highest concentrations expected to occur under realistic field exposure scenarios. 

This has allowed an endpoint of 50% mortality to be used as a trigger for additional higher tier testing. 

Less than 50% mortality under these conditions of extreme exposure suggest that population effects are 

likely to be negligible given realistic field exposure scenarios. 

The Environmental Protection Agency (EPA) uses a tiered (Tiers I–IV) testing system to assess the 

toxicity of a plant-incorporated protectant (PIP) to representative non-target organisms that could be 

exposed to the toxin in the field environment. Tier I high-dose studies reflect a screening approach to 

testing designed to maximize any toxic effects of the test substance on the test (non-target) organism. 

The screening tests evaluate single species in a laboratory setting with mortality as the endpoint. Tiers 

II–IV generally encompass definitive hazard level determinations, longer term greenhouse or field 

testing, and are implemented when unacceptable effects are seen at the Tier I screening level. 

Testing methods, which utilize the tiered approach, were last published by EPA as Harmonized Office 

of Prevention, Pesticides, and Toxic Substances (OPPTS) Testing Guidelines (now Harmonized Office 

 

a

 Non-target invertebrate hazard tests often are conducted at exposure concentrations several times higher than the maximum 

concentrations expected to occur under realistic exposure scenarios. This has customarily allowed an endpoint of 50% 

mortality to be used as a trigger for additional higher tier testing. Lower levels of mortality under these conditions of extreme 

exposure suggest that population effects are likely to be negligible given realistic exposure scenarios. Thus, it follows that the 

observed proportion of responding individuals can be compared to a 50% effect to determine if the observed proportion is 

significantly lower than 50%. For example, using a binomial approach, a sample size of 30 individuals is sufficient to allow a 

treatment effect of 30% to be differentiated from a 50% effect with 95% confidence using a one-sided Z test. A one-sided test 

is appropriate because only effects of less than 50% indicate that further experiments are not needed to evaluate risk. 
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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 and microbial toxins when used as 

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

natural selection or by deliberate genetic manipulation. Therefore, plant-incorporated protectants (PIPs) 

containing microbial toxins are also covered by these testing guidelines. 

The Tier I screening maximum hazard dose (MHD) approach to environmental hazard assessment is 

based on some factor (whenever possible >10) times the maximum amount of active ingredient expected 

to be available to terrestrial and aquatic non-target organisms in the environment, 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 assessment, and such tests can be well standardized. The 

Guidelines call for initial screening testing of a single group or several groups of test animals at the 

maximum hazard dose level. The Guidelines call for testing of one treatment group of at least thirty 

animals or three groups of ten test animals at the screening test concentration. The Guidelines further 

state that the duration of all Tier I tests should be approximately 30 days. Some test species, notably 

non-target insects, may be difficult to culture and the suggested test duration has been adjusted 

accordingly. Control and treated insects should be observed for at least 30 days, or in cases where an 

insect species cannot be cultured for 30 days, until negative control mortality rises above 20%. 

Failing the Tier I (10x EEC) screening at the MHD does not necessarily indicate the presence of an 

unacceptable risk in the field, but it triggers 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 hazards are detected in Tier I testing (i.e., mortality 

is greater than 50%), additional information at lower test doses is required, which can serve to confirm 

whether any effect might still be detected at more realistic field (1x EEC) concentrations and routes of 

exposure.e

 

 

b

 General OCSPP Harmonized Testing 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/publications/OPPTS_Harmonized/850_Ecological_Effects_Test_Guidelines/Drafts/index.

html. 

Series 885 Testing Guidelines available from: 

http://www.epa.gov/ocspp/pubs/frs/publications/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 organism feeding habits or other factors. High-dose testing also may not be necessary 

where many species are tested or tests are very sensitive, although the test concentration used must exceed 1x EEC. 

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 considered significant. Tier I screen testing is not “safety factor testing.” In a “10x safety 

factor” test, any adverse effect noted is a “level of concern,” whereas in the EPA environmental risk assessment scenario any 

adverse effect is viewed as a concern only at 1x the field exposure. 

e

 The 1x EEC test dose is based on plant tissue content and is considered the highest dose in a worst-case scenario 

(sometimes referred to as the Highest Estimated Environmental Concentration or HEEC). This 1x EEC is still much greater 

than any amount which any given non-target organism may be ingesting in the field because most non-target organisms do 
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When screening tests indicate a need for additional data, the OCSPP Harmonized Guidelines call for 

testing at incrementally lower doses in order to establish a definitive LD50 (i.e., dose that will kill 50% of 

the test organisms within a designated period) and to quantify the hazard. In the definitive testing, the 

number of doses and test organisms evaluated must be sufficient to determine an LD50 value and, when 

necessary, the Lowest Observed Adverse Effect Concentration (LOAEC), No Observed Adverse Effect 

Level (NOAEL), or reproductive and behavioral effects such as feeding inhibition, weight loss, etc. In 

the final analysis, a risk assessment is made by comparing the LOAEC to the EEC; when the EEC is 

lower than the LOAEC, a no risk conclusion is made. These tests offer greater environmental realism, 

but they may have lower statistical power. Appropriate statistical methods, and appropriate statistical 

power, must be employed to evaluate the data from the definitive tests. Higher levels 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., LC50, ED50, or LD50

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

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

conservative factor. The published EPA Level of Concern (LOC) is 50% mortality at 5x EEC (U.S. EPA 

1998).f

 

Validation: The tiered hazard assessment approach was developed 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 (FIFRA) Scientific Advisory Panel 

(SAP) on microbial pesticides and microbial toxins. The December 9, 1999 SAP agreed that the tiered 

approach was suitable for use with 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) crop fields should be conducted. Testing of Bacillus thuringiensis 

(Bt) Cry proteins on species not closely related to the target insect pest was not recommended, although it 

is still performed to fulfill the published EPA non-target species data requirements. In October 2000, 

another SAP also recommended that field testing should be used to evaluate population-level effects on 

non-target organisms. The August 2002 SAP, and some public comments, generally agreed with this 

approach, with the additional recommendation that indicator organisms should be selected on the basis of 

potential for field exposure to the subject protein (U.S. EPA 2000a, 2001a, 2002f, and 2004a). 

Chronic studies: Since delayed adverse effects and/or accumulation of toxins through the food chain are 

not expected to result from exposure to proteins, protein toxins are not routinely tested for chronic 

effects on non-target organisms. But, the 30-day test duration requirement does amount to subchronic 

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

 not ingest plant tissue. 

f

 The established peer and EPA Science Board reviewed guidance on screening test levels 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. 
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nature of proteins makes them readily susceptible to metabolic, microbial, and abiotic degradation once 

they are ingested or excreted into the environment. Although there are reports that some proteins (Cry 

proteins) bind to soil particles, it has also been shown that these proteins are degraded rapidly by soil 

microbial flora upon elution from soil particles. 

Conclusion: The tiered approach to test guidelines ensures, to the greatest extent possible, that the 

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

EPA believes that maximum hazard dose Tier I screening testing presents a reasonable approach for 

evaluating hazards related to the use of biological pesticides and for identifying negative results with a 

high degree of confidence. The Agency expects that Tier 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 Academy of Sciences (NAS 2000) recommended testing 

non-target organisms directly in the field. This approach, with an emphasis on testing invertebrates 

found in corn fields, was also recommended by the August 2002 SAP and was supported by several 

public comments. Based on these recommendations and due to the lack of baseline data on the potential 

for long-term environmental effects 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 published in scientific literature, in 

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

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

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

addressed. Specifically, a meta-analysisg

 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 place as a result of wide-scale Bt crop 

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

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

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

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

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

Overall, the Agency is in agreement with the conclusions of these studies and, collectively, these results 

provide extensive data to support that Bt crops have not caused long-term environmental effects, on a 

population level, to organisms not targeted by Bt proteins. Based on these considerations, regulatory 

 

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 Analysis and Synthesis (NCEAS) Web Site: http://delphi.nceas.ucsb.edu/btcrops/.
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testing of the specialist predators and parasitoids of target pests may eventually be considered 

unnecessary. 

2. Corn Event MON 863 (Organization for Economic Cooperation and Development 

 (OECD) Unique Identifier: MON-ØØ863-5) Expressing Cry3Bb1 

a. Data Cited/Submitted for Initial Registration of Corn Event MON 863 (Prior to

 February 2003)

 i. Background

In 2000, Monsanto Company (“Monsanto”) requested registration for Bacillus thuringiensis Cry3Bb1 

protein and the genetic material necessary for its production (vector PV-ZMIR13L) in MON 863 corn 

(OECD Unique Identifier: MON-ØØ863-5). This protein controls the corn rootworm (CRW, Diabrotica

spp.), a primary pest of corn in the United States. Corn rootworm larvae feed on corn roots, resulting in 

lodging and a reduction in a plant’s ability to absorb water and nutrients from soil. In areas where the 

CRW is a pest (e.g., Corn Belt), significant financial losses are realized from decreased corn yields and 

increased expenditures on chemical pest control agents, including organophosphate, carbamate, and 

pyrethroid insecticides. 

EPA conducted an environmental hazard assessment of the Cry3Bb1-producing corn lines. The general 

topics covered include gene flow to related wild plants, development of weediness, effects on wildlife, 

and fate of Cry3Bb1 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 experts; and previous public comments on plantincorporated 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 registered. In addition to testing species directly exposed to 

the CryBb1 protein in the field, however, the full battery of non-target wildlife species testing was 

conducted to comply with the published Agency non-target data requirements for microbial toxins. The 

Agency has determined that the non-target organisms most likely to be exposed to the protein in 

transgenic corn fields are beneficial insects feeding on corn pollen and nectar, and soil invertebrates, 

particularly coleopteran species. Initially, in lieu of extensive and difficult laboratory soil coleopteran 

toxicity testing followed by an extrapolation to community risk assessment, direct field census data and 

data on coleopteran insect effects and abundance in the field were requested, received, and evaluated. 

The August 2002 SAP, however, found the field census data unsatisfactory because of low statistical 

power. Therefore, maximum hazard dose toxicity testing on representative beneficials from several taxa 

was performed. The toxicity of the Cry3Bb1 protein has been evaluated on several species of 

invertebrates, including adult and larval honey bees, a parasitic hymenopteran (Nasonia vitripennis), 
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green lacewings, lady beetles, collembola, monarch butterfly, and earthworms. Reproductive and 

developmental observations were also made on collembola, honey bee, and lady beetle larva. The 

August 2002 SAP (as well as several public comments), however, found the green lacewing and 

parasitic wasp studies lacking and recommended testing of alternative species. Lastly, based on worstcase soil concentration, soil degradation studies show that Cry3Bb1 protein in corn tissue is no longer 

detectable in agricultural field soil after 22 to 28 days. The August 2002 SAP (as well as several public 

comments), however, suggested that additional soil degradation testing is desirable in a larger variety of 

soils and climatic conditions. 

The non-target organisms tested are chosen as representative indicators of the major groups of wildlife 

and on the potential for field exposure as deduced from data on Cry3Bb1 protein expression in the plant. 

Although Bt Cry proteins are very specific in their activity to only certain insect species, for Cry3Bb1 

protein in corn, the Agency has examined the toxicity to birds, fish, honey bees, and certain other 

beneficial insects even though the October 2000 SAP recommended against testing of non-target species 

not related to those susceptible to the specific activity of Bt Cry proteins. Nevertheless, in order to 

comply with the Agency’s published data requirements for registration of microbial toxins (40 CFR § 

158.2150), the Agency asked for avian and aquatic invertebrate toxicity data, as well as data on 

collembola (springtail) and earthworm species to ascertain effects on beneficial soil invertebrates 

because prolonged exposure to Cry3Bb1 protein in soil was a possibility. Earthworm studies were also 

conducted and submitted to demonstrate a lack of Corn Event MON 863 effects on beneficial 

decomposers. Honey bee effects on brood, as well as adults, were also required as exposure of honey 

bees to the Cry3Bb1 protein in pollen is expected. 

 

The form of the test substances used in the studies for this assessment are plant material such as leaves, 

roots, pollen, or purified, bacterially produced Cry3Bb1 protein incorporated into the test species diet. 

The October 2000 SAP provided guidance to the Agency that, while actual plant material is the 

preferred test material, bacterially derived protein is also a valid test substance, particularly in testing 

where the test animals do not consume corn plant tissue and where large amounts of Cry protein are 

needed for maximum hazard dose testing. As per 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 through pupation and adult emergence. 

The results for each study on environmental effects for Cry3Bb1 are summarized in Table 1. 

Additionally, the results are presented 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. 
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Table 1. Environmental Effects Data for Corn Event MON 863. 

Guideline 

Number Study Results

MRID 

Number

885.4050 Avian Dietary 

Testing 

The dietary median lethal concentration (LC50) value for Cry3Bb1 

corn grain to juvenile northern bobwhite was greater than 70,000 

parts per million (ppm) (10% of the diet) in a 8-day study (8-day 

observation). No adverse effects on avian wildlife are expected from 

incidental field exposure to Cry3Bb1 corn. A higher corn 

concentration and longer duration broiler study with MON863 corn 

is recommended. 

Classification: Supplemental 

(Reviewed in U.S. EPA (2003a and 2003g) 

*Note for 2010: There is an update to this summary. See section 

II(C)(2)(b) (“Terms and Conditions of the Corn Event MON 863 

Registration (February 2003 – September 2010)”) of this BRAD. 

449043-15 

885.4150 Wild Mammal 

Testing 

Mammalian wildlife exposure to Cry3Bb1 protein is considered 

likely; however, the Cry3Bb1 toxicity data, as described in the 

Human Health Assessment (see section II(B)(2) 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 mammalian wildlife is anticipated, and 

data on wild mammal testing are not required. 

(Reviewed in U.S. EPA (2003g)) 

N/A 

885.4200 Freshwater 

Fish Testing 

In an 8-week subchronic study, no treatment mortality or behavior 

change was observed among channel catfish when fed diets 

containing 35% Cry3Bb1 corn lines MON 853 and MON 859. 

Classification: Acceptable 

(Reviewed in U.S. EPA (2003b and 2003g))

449043-19 

885.4200 Freshwater 

Fish Testing 

The requirement for a freshwater fish (rainbow trout) static-renewal 

toxicity study has been waived based on a lack of any substantial 

exposure of fish to the Cry3Bb1 proteins produced in corn crops. 

(Reviewed in U.S. EPA (2003g)) 

N/A 
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Guideline 

Number Study Results

MRID 

Number

850.1010 Aquatic 

Invertebrate 

Acute 

Toxicity Test 

(Daphnia 

magna) 

The 48-hour LC50 value for Cry3Bb1 corn pollen, when administered 

to neonate daphnids, was >120 milligrams (mg) pollen/liter (L), a 

maximum hazard dose. No other adverse effects were noted. 

Therefore, no hazards to daphnia are expected from incidental 

exposure to Cry3Bb1-containing corn pollen. 

Classification: Acceptable 

(Reviewed in U.S. EPA (2003c and 2003g)) 

*Note for 2010: There is an update to this summary. See section 

II(C)(2)(b) (“Terms and Conditions of the Corn Event MON 863 

Registration (February 2003 – September 2010)”) of this BRAD.

449043-18 

885.4280 Estuarine and 

Marine 

Animal 

Testing 

The estuarine and marine animal studies are waived for this product 

because of very low to no potential for exposure to Cry3Bb1 protein 

from field corn. 

(Reviewed in U.S. EPA (2003g)) 

N/A 

885.4300 Non-Target 

Plant Studies 

Since the active ingredient in this product is an insect toxin (Bt

endotoxin) that has never shown any toxicity to aquatic or terrestrial 

plants, these studies have been waived for this product. Outcrossing 

issues in section II(C)(2)(a)(ii)(V) of this BRAD. 

(Reviewed in U.S. EPA (2003g)) 

N/A 

885.4380 Honey Bee 

Larva Testing 

The LC50 for honey bee larvae and maturation to adult bees was 

determined to be >1,790 ppm Cry3Bb1 protein (100x the 

concentration in pollen) in a maximum hazard dose study. Therefore, 

no hazard to honey bee larvae and adult bee emergence is 

anticipated. 

Classification: Acceptable 

(Reviewed in U.S. EPA (2002a, 2002e, 2003f, and 2003g))

449043-10 

885.4380 Adult Honey 

Bee Testing 

An adult honey bee maximum hazard dose feeding study showed the 

LC50 of the Cry3Bb1 protein to be >360 micrograms per milliliter 

(μg/mL) (20x the concentration found in pollen). Therefore, no 

hazard from the Cry3Bb1 protein to honey bees is expected. 

Classification: Acceptable 

(Reviewed in U.S. EPA (2002a, 2002e, 2003f, and 2003g))

449043-11 
Bacillus thuringiensis Cry3Bb1 Corn 

 Biopesticides Registration Action Document (BRAD) September 2010 

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Guideline 

Number Study Results

MRID 

Number

885.4340 Parasitic 

Hymenoptera 

Larva Testing 

The LC50 for parasitic Hymenoptera was determined to be >8,000 

ppm Cry3Bb1 protein. Parasitic Hymenoptera are not expected to 

feed directly on corn plant tissue. Therefore, minimal exposure and 

no hazard to parasitic Hymenoptera from Cry3Bb1 protein are 

expected. Testing of a species more common to corn fields is 

recommended. 

Classification: Acceptable 

(Reviewed in U.S. EPA (2002a, 2002e, 2003f, and 2003g)) 

*Note for 2010: There is an update to this summary. See section 

II(C)(2)(b) (“Terms and Conditions of the Corn Event MON 863 

Registration (February 2003 – September 2010)”) of this BRAD.

449043-13 

885.4340 Dietary 

Toxicity to 

Green 

Lacewing 

Larvae 

The LC50 for green lacewing larvae was determined to be >8,000 

ppm Cry3Bb1 protein (20x field exposure). Based on these results, it 

can be concluded that green lacewing will not be adversely affected 

when exposed to Cry3Bb1 in the field. Because of questionable 

ingestion of the test material, another species (e.g., minute pirate bug 

or predatory carabid), more likely to be exposed to Cry3Bb1, should 

be tested. 

Classification: Acceptable 

(Reviewed in U.S. EPA (2002a, 2002e, 2003d, and 2003g)) 

*Note for 2010: There is an update to this summary. See section 

II(C)(2)(b) (“Terms and Conditions of the Corn Event MON 863 

Registration (February 2003 – September 2010)”) of this BRAD.

449043-12 

885.4340 Effects of Bt

Protein 11231 

on Adult Lady 

Beetles 

(Hippodamia 

convergens) 

This maximum hazard dose study showed that the LC50 for Cry3Bb1, 

when fed to adult H. convergens is >8,000 micrograms (μg) purified 

Bt protein/milliliter (mL) diet, equivalent to 20x the maximum Bt

protein concentration in plant tissue. A follow-up pollen feeding 

study was requested. 

Classification: Acceptable 

(Reviewed in U.S. EPA (2002e and 2003g))

449043-14 

885.4340 Lady Beetle 

Larval 

Feeding Study 

(Coleomegilla 

maculata) 

The LC50 for Cry3Bb1 expressed in pollen is >93 micrograms per 

gram (μg/g) fresh pollen weight. The larvae were observed through 

pupation to adult emergence. It can be concluded from this study that 

Coleomegilla maculata larvae will not be adversely affected by 

Cry3Bb1 field corn pollen. 

Classification: Acceptable 

(Reviewed in U.S. EPA (2002a, 2002e, and 2003g)) 

455382-04 
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Guideline 

Number Study Results

MRID 

Number

885.4340 Adult Lady 

Beetle Pollen 

Feeding Study 

(Coleomegilla 

maculata) 

No significant adverse effects were noted in a 30-day 50% pollen 

feeding study. Based on these results, no hazard to C. maculata is 

expected when feeding on Cry3Bb1 corn pollen in the field. 

Classification: Acceptable 

(Reviewed in U.S. EPA (2002d, 2002e, and 2003g))

453613-01 

885.4340 Adult Lady 

Beetle Pollen 

Feeding Study 

(Hippodamia 

convergens) 

No significant adverse effects were noted in a 15-day 50% pollen in 

honey water feeding study. Based on these results, no hazard to H.

convergens is expected if feeding on Cry3Bb1corn pollen in the 

field. 

Classification: Acceptable 

(Reviewed in U.S. EPA (2002d, 2002e, and 2003g))

453613-02 

885.4340 Collembola 

Chronic 

Dietary 

Toxicity Study 

The LC50 of the Cry3Bb1 protein for collembola was found to be 

>872.5 μg (50% corn leaf tissue in the diet). No adverse reproductive 

effects were noted. It can be concluded from this test that 

Cry3Bb1protein does not pose a hazard to collembola, a 

representative of a beneficial decomposer soil-inhabiting species. 

Classification: Acceptable 

(Reviewed in U.S. EPA (2002a, 2002e, and 2003g)) 

449043-17 

850.6200 Earthworm 

Toxicity Study 

A maximum hazard dose 14-day LC50 for earthworms exposed to 

Cry3Bb1 protein in an artificial soil substrate was determined to be 

>570 mg Cry3Bb1 protein/kilogram (kg) dry soil, or greater than 10x 

the maximum EEC of the protein. The data show that no adverse 

effects to earthworms are expected from exposure to corn plants 

producing Cry3Bb1 protein. 

Classification: Supplemental 

(Reviewed in U.S. EPA (2002e, 2003e, and 2003g)) 

449043-16 

N/A Earthworm 

Toxicity Study 

There were no earthworm mortalities or other remarkable 

observations during the 14-day study. The LC50 value is greater than 

the highest maximum hazard concentration tested (166.6 mg 

Cry3Bb1 protein variant 11098 (Q349R)/kg dry soil). 

Classification: Acceptable 

(Reviewed in U.S. EPA (2003f and 2003g))

457571-01 
Bacillus thuringiensis Cry3Bb1 Corn 

 Biopesticides Registration Action Document (BRAD) September 2010 

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Guideline 

Number Study Results

MRID 

Number

885.4340 Monarch 

Butterfly 

Larval Pollen 

Feeding Study 

This study demonstrated that corn pollen expressing the Cry3Bb1 

protein will not result in acute toxic or developmental effects to 

monarch larvae. The SAP recommended testing Tetraopes (red 

milkweed) beetles as a more logical choice than the monarch 

butterfly. 

Classification: Supplemental 

(Reviewed in U.S. EPA (2002a, 2002e, 2003d, and 2003g)) 

*Note for 2010: There is an update to this summary. See section 

II(C)(2)(b) (“Terms and Conditions of the Corn Event MON 863 

Registration (February 2003 – September 2010)”) of this BRAD. 

455382-05 

N/A Insecticidal 

Activity 

Spectrum 

Study 

Bioassays of six Families of the Order Coleoptera and two 

Lepidoptera species detected activity only against beetle species of 

the family Chrysomelidae (corn rootworm and Colorado potato 

beetle). 

Classification: Supplemental 

(Reviewed in U.S. EPA (2003g))

455328-07 

N/A Field 

Evaluation of 

Cry3Bb1 Corn 

Exposure on 

Non-Target 

Organisms 

Preliminary results from two-year Tier IV field census studies. These 

studies are supplemental to Tier I maximum hazard dose testing. The 

data do not show any MON 863 corn-related adverse effect on nontarget and beneficial invertebrate abundance in the field. 

Classification: Supplemental 

(Reviewed in U.S. EPA (2002a, 2002e, 2003d, and 2003g)) 

*Note for 2010: There is an update to this summary. See section 

II(C)(2)(b) (“Terms and Conditions of the Corn Event MON 863 

Registration (February 2003 – September 2010)”) of this BRAD. 

455382-06 
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Guideline 

Number Study Results

MRID 

Number

N/A Non-Target 

Organism 

Field Scale 

Risk 

Assessment 

Final report for MRID No. 455382-06 two-year field census study. 

MON863 showed no overall differences in the abundance of nontarget invertebrates and had less impact on certain beneficial insects 

compared to traditional insecticides, especially soil and foliar 

applications. These studies are supplemental to Tier I maximum 

hazard dose testing and are of inadequate statistical power for a longterm effects determination. 

Classification: Supplemental 

(Reviewed in U.S. EPA (2003d and 2003g)) 

*Note for 2010: There is an update to this summary. See section 

II(C)(2)(b) (“Terms and Conditions of the Corn Event MON 863 

Registration (February 2003 – September 2010)”) of this BRAD.

457916-01 

N/A Field and 

Laboratory 

Invertebrate 

Studies 

Summary (without data) of preliminary findings from several oneyear, higher tier field and laboratory studies not triggered by Tier I 

maximum hazard dose testing data. Final report of studies to be 

submitted. 

Classification: Supplemental 

(Reviewed in U.S. EPA (2002b, 2002e, 2003d, and 2003g)) 

*Note for 2010: There is an update to this summary. See section 

II(C)(2)(b) (“Terms and Conditions of the Corn Event MON 863 

Registration (February 2003 – September 2010)”) of this BRAD.

456530-03 

885.5200 Aerobic Soil 

Degradation 

of the 

Cry3Bb1 

Protein 11098 

Finely ground corn leaf tissue in sandy loam field soil degradation 

data at worst-case field concentrations show that the Cry3Bb1 

protein half-life (DT50), based on insect bioassays and enzyme-linked 

immunosorbent assay (ELISA), was 2.37 and 2.76 days, 

respectively. The time until 90% decay (DT90) estimates for the 

insect bioassays and ELISA were 7.87 and 9.16 days, respectively. 

At 28 days, the CryBb1 protein was below the detection level. 

These results verify that the Cry3Bb1 protein degrades rapidly and 

does not accumulate in the soil. Additional testing in different soil 

types is requested. 

Classification: Supplemental 

(Reviewed in U.S. EPA (2002c, 2002e, and 2003g)) 

*Note for 2010: There is an update to this summary. See section 

II(C)(2)(b) (“Terms and Conditions of the Corn Event MON 863 

Registration (February 2003 – September 2010)”) of this BRAD. 

451568-04 
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 Biopesticides Registration Action Document (BRAD) September 2010 

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Guideline 

Number Study Results

MRID 

Number

885.5200 Aerobic Soil 

Degradation 

of Cry3Bb1 

Produced by 

CRWProtected 

Corn Event 

MON 863 

The DT50 values for Cry3Bb1in several dosing regimes and soil 

types ranged from 0.6 days to 2.3 days, and the DT90 values ranged 

from 4.03 days to 50 days. Cry3Bb1 levels in soil sample extracts 

show that concentrations were near or below the ELISA limit of 

quantitation (LOQ) (0.16 μg/g) after 2 months of incubation. 

Additional studies with whole plant tissue are requested. 

Classification: Supplemental 

(Reviewed in U.S. EPA (2002g and 2003g) 

*Note for 2010: There is an update to this summary. See section 

II(C)(2)(b) (“Terms and Conditions of the Corn Event MON 863 

Registration (February 2003 – September 2010)”) of this BRAD.

457571-02 

N/A Endangered 

Species 

Impact 

Assessment 

Monsanto conducted a hazard assessment, exposure assessment, and 

risk characterization to demonstrate that Cry3Bb1 does not pose a 

risk to endangered Coleoptera. The Agency performed an 

independent assessment and has determined that Cry3Bb1 Event 

MON 863 will not result in a “may effect” for endangered and/or 

threatened species listed by the U.S. Fish and Wildlife Service, 

including mammals, birds, terrestrial and aquatic plants, and 

invertebrate species. Therefore, no consultation with the U.S. Fish 

and Wildlife Service is required under the Endangered Species Act. 

Classification: Acceptable 

(Reviewed in U.S. EPA (2002a, 2002e, and 2003g)) 

455770-03 

I. Non-Target Wildlife Study Summaries 

 a. Mammalian Wildlife

Mammalian wildlife exposure to Cry3Bb1 protein 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 Cry3Bb1 toxicity data, as described in the Human Health Assessment (see 

section II(B)(2) of this BRAD), indicate that there is no significant toxicity to rodents from acute oral 

testing at the maximum hazard dose. Therefore, no hazard to mammalian wildlife is anticipated, and 

data on wild mammal testing is not required. 
Bacillus thuringiensis Cry3Bb1 Corn 

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 b. Avian Species

 i. Northern Bobwhite Study (Master Record Identification Number 

 (MRID No.) 449043-15) 

The dietary LC50 value for Cry3Bb1 corn grain (MON 853, MON 854, and MON 855), when fed to 

juvenile northern bobwhite for 5 days, was reported to be greater than 70,000 ppm (10% of the diet), the 

only concentration tested. No adverse effects on bobwhite quail were seen in eight days. These data 

show that there will be no hazard to avian wildlife from incidental field exposure to Cry3Bb1 corn. 

These data, however, are not sufficient to make a hazard assessment from repeated exposure to higher 

doses of Cry3Bb1 corn. The concentration tested (10% corn in the diet) is too low. A six-week broiler 

study with 60%–70% MON 863 corn in the diet is required to assess hazard to non-target birds from 

continuous exposure to high levels of Cry3Bb1 protein. 

*Note for 2010: There is an update to this summary. See section II(C)(2)(b) (“Terms and Conditions of 

the Corn Event MON 863 Registration (February 2003 – September 2010)”) of this BRAD. 

 c. Aquatic Species 

There is no evidence for sensitivity of aquatic (including endangered) species to Cry proteins. Toxicity 

studies with Cry proteins on aquatic organisms show no hazard for fish or invertebrates exposed to 

either corn pollen or to bacterially expressed Cry protein. In addition, aquatic exposure from Bt corn is 

extremely small. When a simple standard pond scenario (1-hectare pond, 2 meters deep draining a 10-

hectare watershed planted with corn) was used to develop a worst case EEC for Cry3Bb1 protein on the 

basis of corn pollen loadings from airborne pollen deposition and agricultural runoff from corn plant 

tissue left in the field at the end of harvest (assuming that no degradation of the protein takes place), 

airborne and agricultural runoff is calculated to be 3.9 nanograms (ng) Cry3Bb1 protein/mL. Thus, total 

water concentration of less than 3.9 ng Cry3Bb1 protein/mL is projected under worst-case conditions. 

 

 i. Freshwater Fish (MRID No. 449043-19) 

The Harmonized Testing Guidelines requirement for a static-renewal freshwater fish toxicity study is 

usually waived based on low to nonexistent 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 freshwater fish. Nevertheless, a subchronic eight-week farmed channel 

catfish feeding study was performed and submitted for review. The study was conducted in compliance 

with EPA FIFRA Good Laboratory Practice Regulations (40 CFR Part 160) with three minor deviations, 

none of which had an impact on the integrity of the study. 

The study is scientifically sound, and no treatment mortality or behavior change was observed among 

channel catfish fed diets containing finely ground corn grain from two insect-protected Cry3Bb1 corn 

lines (MON 853 and MON 859) for eight weeks. The results indicate that corn grain derived from the 

two transgenic lines producing Cry3Bb1 can be used as a feed ingredient in channel catfish diets at 
Bacillus thuringiensis Cry3Bb1 Corn 

 Biopesticides Registration Action Document (BRAD) September 2010 

79 

levels of up to 35% without adverse effect on fish growth, feed conversion efficiency, survival, 

behavior, or body composition. Significant differences were observed only as lower percentage fillet 

moisture among fish fed corn grain of the line MON 859; however, these are relatively unremarkable 

and are unlikely related to the different diets. There were no significant differences noted in feed 

consumption, weight gain, feed conversion ratio, survival, and percentage visceral fat, or percentages 

fat, protein, or ash in fillets of channel catfish fed the different test diets. No abnormal fish behavior was 

observed in the study. 

In view of the lack of demonstrated toxicity to channel catfish and minimal aquatic exposure, no 

freshwater fish hazard is expected from the uses of Cry3Bb1 protein in corn crops. 

 ii. Freshwater Aquatic Invertebrates (MRID No. 449043-18) 

This study was conducted according to procedures specified in Series 72 of EPA’s Registration 

Guidelines, Pesticide Assessment Guidelines, FIFRA Subdivision E, Hazard Evaluation for acute 

toxicity testing of pesticidal substances to freshwater aquatic invertebrates. 

The study was performed on D. magna, a freshwater invertebrate. The test material consisted of corn 

pollen from corn plants (line MON 858). The Cry3Bb1 content was estimated to be 18.8 μg/g fresh 

weight pollen. The study was procedurally sound, and no treatment mortality or behavior change was 

reported between the dosed and control replicates for the 48-hour exposure period. 

The October 2000 and August 2002 SAP reports recommended that non-target testing be focused on 

species exposed to the crop being registered. The Agency has determined that the non-target organisms 

most likely to be exposed to the protein in transgenic corn fields are beneficial insects feeding on corn 

pollen and nectar, and soil invertebrates, particularly coleopteran species. Therefore, testing of aquatic 

invertebrates was performed primarily to satisfy the testing requirements for microbial toxins published 

in 40 CFR Part 158. No substantial aquatic exposure to Cry3Bb1 protein contained within corn plant 

tissue is expected, except for possibly small amounts of pollen. Several public comments have raised 

questions about using corn pollen in aquatic invertebrate testing with D. magna because corn pollen is 

thought to be too large for ingestion by these filter feeders. However, there is some observational 

evidence that daphnids do ingest pollen. As indicated in some study reports reviewed by the Agency, 

daphnids were actually yellow in color, which can be indicative of ingestion of the yellow pollen test 

material. However, there is no clear evidence that D. magna is capable of ingesting particles as large as 

pollen. Therefore, only a statement of no effect from exposure to pollen, and no statement on lack of 

toxicity can be made from this study. Nonetheless, since the Cry3Bb1 protein is confined to corn tissue, 

and the worst case aquatic EEC is calculated to be 3.9 ng Cry3Bb1 protein/mL, there is no substantial 

exposure to aquatic invertebrates; no hazard from the registered used of Cry3Bb1-containing corn is 

anticipated. As a result, no further aquatic invertebrate testing is required at this time. 

*Note for 2010: There is an update to this summary. See section II(C)(2)(b) (“Terms and Conditions of 

the Corn Event MON 863 Registration (February 2003 – September 2010)”) of this BRAD. 
Bacillus thuringiensis Cry3Bb1 Corn 

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iii. Estuarine and Marine Animals

The estuarine and marine animal studies are waived for this product because of very low to no potential 

for exposure to CryBb1 protein from field corn. 

d. Terrestrial and Aquatic Plants

Since the active ingredient in this product is an insect toxin (Bt endotoxin) that has never shown any 

toxicity to plants, these studies have been waived for this product. Outcrossing issues are addressed in 

section II(C)(2)(a)(ii)(V) of this BRAD. 

 

 e. Non-Target Insect Testing

The October 2000 SAP concluded that invertebrates, such as earthworms and springtails (collembola), 

are appropriate indicator species for Cry protein testing because of the specific nature of the Cry protein 

toxicity to select target species. When it initially reviewed the applications for PIP products that were 

registered in 1995, EPA considered requiring studies evaluating effects upon the representative 

beneficial soil invertebrates, collembola and earthworms. The Agency was concerned (1) that such soil 

organisms may be subject to long-term exposure as a result of soil incorporation of crop residues or 

when crop residues are left on the soil surface and (2) that adverse effects on such soil organisms could 

result in an accumulation of plant detritus in fields. Recent reports of exudation of Cry proteins by corn 

roots throughout the growing season add to this concern. The Agency understands, however, that routine 

agronomic practices have included the long-term use of chemical insecticides, which have adverse 

effects on soil organisms, but there has not been an accumulation of significant amounts of plant detritus 

in soils. Thus, Cry3Bb1 corn, which is expected to have less impact on these species than chemical 

pesticides, should not result in any increased build up of plant detritus or Cry proteins at toxic levels. 

Supporting this conclusion are data received by EPA that indicate that such proteins are known to 

degrade rapidly in field soils. Cry proteins that are bound to soil particles have been shown to be rapidly 

degraded by soil microbes upon elution from the soil particles. Therefore, the potential for significant 

soil buildup and adverse effects to non-target soil organisms are not anticipated. It has been confirmed in 

published literature that Bt Cry protein released from root exudates and biomass of Bt corn plants has no 

apparent effect on earthworms, nematodes, protozoa, algae, bacteria, actinomyces, and fungi in soil in 

spite of the fact that enough detectable Cry protein is bound to soil particles to show toxicity to the 

target pest. These results suggest that, despite its presence in soil, the Cry protein released in root 

exudates of Bt corn, or from the degradation of the biomass of Bt corn, is not toxic to a variety of 

organisms in the soil environment. It has also been reported that the same degree of Bt Cry protein 

persistence takes place in soils that have been exposed to repeated Bt microbial spray applications. In 

addition, new plants grown in Bt-containing soil do not take up the Bt protein. Nevertheless, data on 

insects closely related to the target pest, as well as other studies to address the published data 

requirements for registration of microbial toxins (40 CFR Part 158), have been received and reviewed. 
Bacillus thuringiensis Cry3Bb1 Corn 

 Biopesticides Registration Action Document (BRAD) September 2010 

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i. Effects on Honey Bee Larvae (MRID No. 449043-10)

An acceptable study was conducted based on OCSPP Harmonized Test Guideline 885.4380 (Honey Bee 

Testing, Tier I). This study was conducted in accordance with Good Laboratory Practice Standards (40 

CFR Part 160) with certain exceptions that did not affect the integrity of the test. 

Testing was conducted with Bt Cry 3B2.11231 protein (purity 96%; 1.79 mg active protein/mL water; 

current nomenclature refers to this protein as Cry3Bb1) inoculated directly into larval brood cells prior 

to capping. Within 18 days after treatments were administered, all larvae emerged from capped brood 

cells. All larvae (100%) treated with Cry3Bb1 protein survived to pupation or “capping”; whereas, 

97.5% (2.5% mortality) of the honey bee larvae in the control group survived to pupation. There was no 

statistical difference (p = 0.05) in total percent mortality during the larval development or adult 

emergence stages between treated and control groups. Based on the results presented in the study, it can 

be concluded that honey bee development and survival are not affected by exposure to the Cry3Bb1 

protein. There was 88.8% mortality of larvae treated with the reference substance, potassium arsenate, 

which indicated that bees were exposed to the treatments. The LC50 for honey bee larvae was 

determined to be >1,790 ppm Cry3Bb1 protein. 

According to the OCSPP Harmonized Testing Guidelines, non-target insects should be tested at 10–

100x the field dosage. This test was conducted at an acceptable level 100x the concentration in pollen or 

1,790 ppm Cry3Bb1 protein. Since potential exposure of honey bees to Cry3Bb1 will be from pollen, 

this test was conducted at an appropriate maximum hazard dose. Therefore, no hazard to honey bee 

larvae and their development is expected from exposure to the Cry3Bb1 protein in corn pollen. 

ii. Adult Honey Bee Testing (MRID No. 449043-11) 

An acceptable study was conducted based on OCSPP Harmonized Test Guideline 885.4380 (Honey Bee 

Testing, Tier I). This study was conducted in accordance with Good Laboratory Practice Standards (40 

CFR Part 160) with certain exceptions that did not affect the integrity of the test. 

The testing consisted of a control group fed 30% sucrose in deionized water, a reference group fed 

100μg/mL potassium arsenate, a test group fed 360 μg/mL of Cry3Bb1 protein, and a water only group. 

The study concluded that 360 μg/mL Cry3Bb1 protein did not affect survival or behavior of adult honey 

bees. The maximum hazard dose, an LC50 greater than 360 μg/mL, is 20x the concentration found in 

pollen. Therefore, no hazard to adult honey bees is expected from exposure to the Cry3Bb1 protein in 

corn pollen. 

 iii. Parasitic Hymenoptera Testing (MRID No. 449043-13) 

This study was conducted based on OCSPP Harmonized Test Guideline 885.4340 (Non-Target Insect 

Testing, Tier I). This study was conducted in accordance with Good Laboratory Practice Standards (40 

CFR Part 160) with certain exceptions that did not affect the integrity of the test. 
Bacillus thuringiensis Cry3Bb1 Corn 

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A dietary toxicity study with the parasitic Hymenoptera (N. vitripennis) was conducted with Bt Cry 

3B2.11231 (Cry3Bb1) protein (purity 96%; 34.5 mg active protein/mL water). Wasps were tested at 

rates of 400 and 8,000 ppm Cry3Bb1 protein, which is approximately equivalent to 1x and 20x the 

maximum protein concentration in plant tissue. The LC50 for parasitic Hymenoptera was determined to 

be >8,000 ppm Cry3Bb1 protein. When an adjustment for mortality in the control group is considered, 

mortality in the 8,000 ppm treatment group is 45%. Although differences in mortality between the 

control and treatment groups were not significantly different (p>0.05), a treatment effect at 20x EEC 

could not be precluded in this study. At test termination, mortality for the 100 ppm potassium arsenate 

reference group was 33% (24 of 73) and mortality for the 1,000 potassium arsenate reference group was 

100% (70 of 70). 

Based on this test, the LC50 for adult parasitic Hymenoptera exposed to dietary Cry3Bb1 is >8,000 ppm. 

The hazard assessment is based on 4,000 ppm Cry3Bb1 protein, which is 10x the field concentration in 

plants. However, because parasitic Hymenoptera do not feed directly on corn plant tissues, minimal 

exposure of parasitic Hymenoptera to Cry3Bb1 protein is expected. As a result, no hazard to N. 

vitripennis is expected from exposure to MON 863 Cry3Bb1 corn. 

The preliminary review of the N. vitripennis study was initially found acceptable by the Agency (U.S. 

EPA 2002a). However, the August 27, 2002 SAP concluded that the parasitic Hymenoptera (N.

vitripennis) testing was not appropriate. The SAP concluded that “[t]he levels of exposure of...Nasonia

to active protein were not, for example, determined throughout their respective tests. The test 

protein...within a diet broth...could have degraded considerably.” Not only were the procedures in this 

study questioned by the SAP, the appropriateness of testing this organism was questionable. N.

vitripennis is a dipteran parasitoid that does not occur in corn fields. A more appropriate parasitoid that 

occurs in corn fields (e.g., Tricogramma or Macrocentrus grandii) should be considered. Since 

Tricogramma and Macrocentrus are lepidopteran parasitoids, testing another beneficial organism rather 

than a parasitoid is appropriate. Therefore, the Agency is requiring additional maximum hazard dose 

laboratory testing of a beneficial coleopteran, such as a carabid (ground beetle). 

*Note for 2010: There is an update to this summary. See section II(C)(2)(b) (“Terms and Conditions of 

the Corn Event MON 863 Registration (February 2003 – September 2010)”) of this BRAD. 

 iv. Green Lacewing Larva Testing (MRID No. 449043-12) 

This study was conducted based on OCSPP Harmonized Test Guideline 885.4340 (Non-Target Insect 

Testing, Tier I). This study was conducted in accordance with Good Laboratory Practice Standards (40 

CFR Part 160) with certain exceptions that did not affect the integrity of the test. 

Green lacewing larvae were fed the Cry3Bb1 protein in a moth egg (Sitotroga spp.) and water meal diet 

at rates of 400 and 8,000 ppm, which is approximately equivalent to 1x and 20x the maximum protein 

concentration in plant tissue. There was 20% mortality in the negative control group on day 10. 

Compared to the negative control, at day 10, there was no significant increase in green lacewing larval 
Bacillus thuringiensis Cry3Bb1 Corn 

 Biopesticides Registration Action Document (BRAD) September 2010 

83 

mortality when fed 1x (400 ppm) and 20x (8,000 ppm) the maximum Cry3Bb1 protein concentration 

found in plant tissue. At test termination, mortality was 43% (13 of 30) for the 1,000 ppm reference 

group and 100% for the 10,000 ppm reference group (potassium arsenate). The data show that the LC50

for green lacewing larvae exposed to Cry3Bb1 in diet is >8,000 ppm. Based on these results, it is not 

expected that the green lacewing will be adversely affected when exposed to Cry3Bb1 in the field. 

The preliminary review of the green lacewing larva study was initially found acceptable by the Agency. 

However the August 27, 2002 SAP concluded that the green lacewing (Chrysoperla carnea) testing was 

not appropriate. Several public comments also addressed this issue. The SAP concluded that “[t]he 

levels of exposure of Chrysoperla to active protein were not, for example, determined throughout their 

respective tests. The test protein was held for a week within a diet broth in the Chrysoperla test 

chamber, and could have degraded considerably.” 

Additional problems were recognized with the Chrysoperla laboratory study. Green lacewing are 

difficult to test in the laboratory because of a high rate of mortality. In this instance (MRID No. 449043-

12), the test was terminated after 10 days because there was >20% mortality in the negative control. In 

addition, it is questionable whether the green lacewings were ingesting the Cry3Bb1 protein that was 

coated around moth eggs in a diet. Since green lacewing have piercing-sucking mouthparts, they may 

not be exposed to the protein on the external surface of the egg diet. Therefore, Monsanto must conduct 

a laboratory insect toxicity test on an alternate organism. The minute pirate bug (Orius insidiosus) would 

be a more appropriate species to test than the green lacewing. Orius typically occur in corn fields as egg 

predators and they typically feed on pollen. Therefore, a laboratory study, feeding O. insidiosus both 

pollen and purified protein in diet, is required. Feeding O. insidiosus Cry3Bb1 protein in diet will allow 

for a test at the maximum hazard dose; whereas, feeding O. insidiosus pollen expressing the Cry3Bb1 

protein will provide an evaluation of potential effects from actual exposure scenarios. 

*Note for 2010: There is an update to this summary. See section II(C)(2)(b) (“Terms and Conditions of 

the Corn Event MON 863 Registration (February 2003 – September 2010)”) of this BRAD. 

 v. Lady Beetle Testing

Since the Cry3Bb1 protein specifically targets coleopteran (beetle) insects, particular attention is 

warranted regarding potential effects of MON 863 on lady beetles. In addition to a dietary exposure 

study to the purified Cry protein, the Agency requested a test demonstrating the effect on lady beetles 

feeding on corn pollen containing Cry3Bb1. Monsanto conducted three additional laboratory studies on 

two different lady beetle species (C. maculata and H. convergens) in response to this request. 

Adult Lady Beetle Protein Dietary Study (MRID No. 449043-14)

A diet containing purified Cry protein and honey was fed to the adult lady beetle (H. convergens) at 

rates one and 20 times the maximum protein concentration found in corn leaf tissue. When the negative 

control group reached 20% mortality on day 10, the results showed no significant differences in the 
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mortality rate between lady beetles fed 400 and 8,000 μg Cry3Bb1/mL of diet. Results from this study 

showed that the LC50 for Cry3Bb1, when incorporated in diet and fed to H. convergens, is >8,000 μg 

Cry3Bb1 protein/mL diet. Mortality for the 1,000 and 10,000 μg potassium arsenate/mL diet groups 

were 55% and 95%, respectively, at day 10. This demonstrates that toxicity can be measured by mixing 

a test substance in the lady beetle diet. Lady beetles do not feed on corn plant tissue. They do, however, 

feed on corn pollen and prey on pest insects that may feed on corn tissue and contain Cry3Bb1 in their 

gut, thus resulting in exposure to the Bt protein. There is approximately 390 μg Cry3Bb1/gram (g) fresh 

weight corn tissue. Lady beetle exposure is expected to be significantly lower than this since the corn 

tissue would be metabolized, eliminated, or otherwise degraded within the prey species. Since the 

maximum hazard dose LC50 was found to be 8,000 μg Cry3Bb1/mL diet, which is 20 times higher than 

maximum expected exposure levels, no hazard from Cry3Bb1 in corn plants to adult lady beetles is 

anticipated. 

Larval Lady Beetle Pollen Feeding Study (MRID No. 455382-04)

At certain times, corn pollen may comprise up to 50% of lady beetle larva’s diet. Therefore, the effects 

of corn pollen containing event MON 863 Cry3Bb1 protein on lady beetle larvae (C. maculata) was 

evaluated. Pollen was fed to lady beetle larvae in a diet consisting of equal amounts of lyophilized 

tephritid fruit fly eggs and bee pollen. Diets contained 50% pollen (93 μg Cry3Bb1/g fresh pollen 

weight), since this is the potential level of field exposure, and an equal amount of the tephritid fruit fly 

diet. First instar lady beetle larvae were individually placed in test arenas to avoid cannibalism. There 

was not a statistically significant difference between developmental time of larvae to pupae and/or 

adults, nor was there a difference in adult weight survival between larvae fed bee pollen or corn pollen. 

There was also no difference between larvae fed Bt and non-Bt pollen. There was a significant 

difference between the reference group (potassium arsenate) and other test groups since no larvae 

survived in the reference group. The 100% mortality observed in the reference group verified that the 

lady beetles were ingesting the diet. This test was conducted with pollen levels greater than or equal to 

levels lady beetle larvae are expected to be exposed to in the field. Therefore, the LC50 for Cry3Bb1 

expressed in corn pollen is >93 μg/g fresh pollen weight. This study demonstrates that lady beetle larvae 

will not be adversely affected by Cry3Bb1 field corn. 

Adult Lady Beetle Pollen Feeding Studies (MRID Nos. 453613-01 and 453613-02)

C. maculata lady beetle adults were fed diets of transgenic corn pollen mixed with fruit fly eggs to 

determine the potential effects of transgenic pollen to beetles (MRID No. 453613-01). The corn (MON 

863) test pollen (assayed at the time of testing) contained the Cry3Bb1 protein at a concentration of 37.4 

μg/g pollen. After 30 days of diet exposure, 83.3% and 80.0% of adult C. maculata survived in the test 

and control pollen groups, respectively. While these survival rates were significantly less than that in the 

assay control group (bee pollen, which exhibited 100% survival), there were no significant differences 

between the test and control pollen groups. All adults in the positive control (arsenate-treated corn 

pollen) died in less than 8 days. Results indicated that transgenic Bt corn pollen expressing the variant 

Cry3Bb1 protein have no significant negative effects on the survival of C. maculata adults. 
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H. convergens adults were fed diets of transgenic corn pollen in honey to determine the potential effects 

of transgenic pollen to non-target beetles (MRID No. 453613-02). The corn (MON 863) test pollen 

(assayed at the time of testing) contained the Cry3Bb1 protein at a concentration of 37.4 μg/g pollen. 

After 15 days of diet exposure, 84% and 81% of adult H. convergens survived in the test pollen and 

control pollen groups, respectively. There were no significant differences in survival among the test 

pollen, control pollen, and the assay control (honey only) treatment groups. Only 5% of beetles exposed 

to the positive control (arsenate-treated corn pollen) survived. Results demonstrate that transgenic Bt

corn pollen expressing the variant Cry3Bb1 protein had no significant negative effects on the survival of 

H. convergens adults from dietary exposure. 

No adverse effects were detected when C. maculata and H. convergens were fed MON 863 pollen in 

diet in the laboratory. Pollen levels consumed by the lady beetles in this study exceeded concentrations 

that are expected to be encountered in the field. Therefore, it can be concluded MON 863 will not pose a 

hazard to lady beetle adults in the field. 

 vi. Collembola Feeding Study (MRID No. 449043-17) 

This study was conducted based on OCSPP Harmonized Test Guideline 885.4340 (Non-Target Insect 

Testing, Tier I). This study was conducted in accordance with Good Laboratory Practice Standards (40 

CFR Part 160) with certain exceptions that did not affect the integrity of the test. 

Collembola (Folsomia candida) were fed diets consisting of transgenic corn leaf tissue containing 

Cry3Bb1 protein mixed with dry granulated Brewer’s yeast. Diets contained a ratio of 0.50, 5.0 and 50% 

corn leaf tissue in Brewer’s yeast, which was equivalent to 8.73, 87.3 and 872.5 μg Cry protein/g diet, 

respectively. The corn leaf tissue contained 1,745 μg Cry3Bb1 protein/g dried leaf tissue. 

These results show a LC50 >872.5 μg/g diet of Cry3Bb1 protein. The study also noted that a diet 

containing 50% corn leaf tissue expressing the Cry3Bb1 Bt protein (a maximum hazard dose) did not 

adversely affect reproduction of collembola. This test was conducted at concentration levels much 

greater than collembola are expected to be exposed to in the field. The primary route by which 

collembola would be exposed to Cry3Bb1 in the field is through decaying root tissue (and possibly from 

pollen to a much lesser degree). MON 863 is expressed in corn roots in the range of 3–66 μg/g, which is 

significantly lower than the levels used in this test. 

This study adequately addresses potential concerns for Cry3Bb1 protein expressed in transgenic corn to 

collembola (F. candida), a representative of beneficial soil insect species. The results of this study 

demonstrate that Cry3Bb1 protein found in transgenic corn poses no hazard to soil-inhabiting 

collembola species and, by inference, to other beneficial non-coleopteran soil insects. Notably, one of 

the October 2000 SAP’s recommendations was that invertebrates of different orders than those known to 

be affected by the Cry protein in question not be tested. 
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 vii. Earthworm Toxicity Testing 

Earthworm feeding studies submitted to the Agency for all of the registered Cry proteins demonstrate 

that the Cry proteins are not toxic to earthworms at the worst-case environmental concentration. Some 

public comments have voiced concerns as to whether the earthworms actually ingested the Bt Cry 

proteins when these are incorporated into the soil in the test systems used. Recently published data show 

that the earthworms do, however, ingest the Bt Cry proteins with the soil without harmful effects. The 

data also show that there were no significant differences in the percent mortality and weight of 

earthworms after 40 days in soil planted with Bt or non-Bt corn, in fallow fields, or after 45 days in soil 

amended with biomass of Bt or non-Bt corn or not amended. The Bt Cry protein was shown to be present 

in both the casts and guts of the worms.

 MRID No. 449043-16 – 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 testing was conducted based on OCSPP Harmonized Test 

Guideline 850.6200 (Earthworm Subchronic Toxicity Test) and OECD Guideline 207. This 

study meets current testing requirements for assessing risks to earthworms from plantincorporated protectants derived from Bt. 

The 14-day LC 50 for earthworms exposed to Cry3Bb1 protein 11231 in an artificial soil 

substrate was determined to be greater than 570 mg Cry3Bb1 protein/kg dry soil. However, the 

percent mortality reported was 38%. The mortality in the 57.0 milligrams per kilogram (mg/kg) 

group was 8%. It was noted in the study design that the levels of buffer salt in the test groups 

were higher than expected because of a miscalculation. The actual concentration of sodium 

bicarbonate salt in the 57.0 and 570 mg Cry3Bb1 protein/kg treatment groups was 70 and 699 

mg/kg, respectively. The higher concentrations did not appear to have any influence on the 

overall conclusions of the study. Nonetheless, another earthworm study (MRID No. 457571-01) 

was performed. 

 MRID No. 457571-01 – The submitted study is classified as acceptable, is scientifically sound, 

and is consistent with current testing requirements for earthworm hazard assessment. The 14-day 

LC50 for earthworms exposed to purified 11098 Cry3Bb1 (Escherichia coli-produced) protein in 

an artificial soil substrate was determined to be greater than 166.6 mg/kg dry soil (the highest 

concentration tested), or greater than 20 times the worst-case EEC in a corn field. There was no 

apparent effect of the phosphate buffer on the earthworms. There were no earthworm mortalities 

in any of the controls or Cry protein-treated soils during the 14-day study. Changes in average 

body weights were not statistically different (p>0.05) among the controls and protein-amended 

soils. There were no other remarkable observations. At the end of the study, mortality in the 10 

and 20 mg chloroacetamide/kg soil was 2.5% (1 of 40) and 85% (34 of 40), respectively. Percent 

mortality of earthworms in the reference substance (chloroacetamide) groups was consistent with 

historical results and further confirmed the adequacy and consistency of the protocol used in the 

definitive test. 
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The reviewed data show that no adverse effects to earthworms are expected in fields growing Cry3Bb1 

corn plants. 

viii. Monarch Butterfly Larval Pollen Feeding Study (MRID No. 455382-05) 

This study was not required nor requested for Cry3Bb1 because it is a coleopteran-active protein that is 

not expected to affect lepidopterans such as the monarch butterfly. In addition, extensive research 

conducted on the potential affects of monarch feeding on lepidopteran-active Bt corn pollen has shown a 

lack of concern for subchronic toxicity. Due to recent public concern for possible adverse effects of 

Cry3Bb1 corn on monarchs, Monsanto sponsored this study and submitted it to the Agency for review. 

This study has demonstrated that corn pollen expressing the Cry3Bb1 protein will not result in 

subchronic toxic or developmental effects to monarch larvae. Neonate monarch survival was not 

affected after feeding on milkweed dusted with up to 3,200 pollen grains/square centimeter (cm2

) 

expressing Cry3Bb1 for 2, 4, or 10 days of pollen exposure. Larval development, weight gain, and 

milkweed leaf consumption were also not affected by feeding on Bt pollen 96 hours and 10 days after 

exposure. Pollen densities in the field are not expected to be as great as 3,200 grains/cm2. Pollen 

densities in the field average 150 grains/cm2. Levels of 400 and 800 pollen grains/cm2 would probably 

be rare. Therefore, results of this study indicate that young monarch larvae (at the most sensitive stage) 

will not be adversely affected by exposure to corn pollen expressing Cry3Bb1 in the field. Since 

Cry3Bb1 expressed in MON 863 is a coleopteran-active protein, however, the August 27, 2002 SAP 

concluded that the monarch butterfly was not an appropriate indicator organism to be tested. The SAP 

recommended testing Tetraopes (red milkweed beetles) as a more logical choice than the monarch 

butterfly. 

*Note for 2010: There is an update to this summary. See section II(C)(2)(b) (“Terms and Conditions of 

the Corn Event MON 863 Registration (February 2003 – September 2010)”) of this BRAD. 

 ix. Insecticidal Activity Spectrum Study (MRID No. 455328-07)

Insecticidal spectrum of activity bioassays were conducted on six Coleoptera Families and two 

Lepidoptera species. A series of six to eight concentrations of CryBb1 protein (1 ppm–8,000 ppm) in 

standard insect diet preparations were used to conduct seven-day mortality testing. Significant 

insecticidal activity was seen only in the family Chrysomelidae (corn rootworm and Colorado potato 

beetle) of the Order Coleoptera. No activity was seen against the cowpea weevil (Bruchidae), lady bird 

beetle (Coccinellidae), red flour beetle (Tenebrionidae), cotton boll weevil, pepper weevil, and rice 

weevil (Curculionidae). The Lepidoptera corn earworm (Noctuidae) and European corn borer 

(Crambidae) were also not affected. This efficacy study was reviewed for environmental assessment 

purposes to expand the number of insect species examined for possible toxicity of the Cry3Bb1 protein. 

The results confirm the assertion that Bt Cry proteins have a very specific and narrow range of target 

species. 
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 x. Field Evaluation of Cry3Bb1 Corn Exposure on Non-Target Invertebrates 

The Scientific Advisory Panels (October 2000 and August 2002) recommended that non-target testing 

be focused on species exposed to the crop being registered. The Agency has determined that the nontarget organisms most likely to be exposed to the Cry3Bb1 protein in transgenic corn fields were 

beneficial insects feeding on corn pollen and nectar, and soil invertebrates, particularly coleopteran 

species. In addition to extensive and difficult maximum hazard dose single species soil coleopteran 

toxicity testing followed by an extrapolation from the results to a community risk assessment, and the 

fact that all of the species cannot be tested in the laboratory, direct field test and field census data on 

coleopteran insect effects and abundance were requested, received, and evaluated. These studies were 

conducted in several states by Monsanto and several independent university scientists. Results of these 

studies are summarized below. Some of the submissions consist of preliminary results of studies in 

progress. The Agency requested that Monsanto submit the final reports of these studies as they become 

available. 

These preliminary field test and field census data with the study design methodologies have been 

presented to an SAP (August 2002). The SAP commented that the study designs lack appropriate 

statistical power, but that methodology for conducting statistically valid field census studies at the scale 

necessary to determine ecosystem effects is not available. Such methodology is yet to be developed. As 

a result, the Agency is reviewing the available field studies as data supplemental to the maximum hazard 

dose single species laboratory testing but useful for short-range assessment of non-target invertebrate 

abundance in Cry3Bb1 corn test plots. 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. 

a) Preliminary Invertebrate Field Census Data (MRID No. 455382-06)

These two-year Tier IV field studies are intended to supplement the Tier I maximum hazard dose 

findings. Invertebrates in Cry3Bb1 corn field plots were sampled from the soil, soil surface, and foliage. 

Soil-dwelling invertebrates were collected using a “pan trap,” which utilized a modified Burlese 

extraction method. Surface-dwelling invertebrates were sampled in the field with pitfall traps. Foliagedwelling invertebrates were monitored by yellow sticky traps (Pherocon AM) set in the field at 

canopy level and adjusted as the season progressed. Sampling for lady beetles was also done using a 

drop-cloth technique. Preliminary results do not show any MON 863 corn-related adverse effect on nontarget and beneficial invertebrate abundance in the field. The final report (MRID No. 457916-01) is 

discussed below. 

b) Final Invertebrate Field Census Data (MRID No. 457916-01) 

Methods:

During the 2000 and 2001 growing seasons, event MON 863 CRW-protected corn and non-transgenic 

corn (hybrid RX670) were grown in Warren County near Monmouth, Illinois. Corn was planted in both 

fields the previous year and soybeans were planted two years prior to conducting these field trials. All 
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experimental plots were managed according to typical cultural practices of commercially grown corn in 

the region and included the application of the herbicides acetochlor and atrazine after planting and 

before emergence. 

Bt (MON 863) and control hybrids (RX670) were the main plots planted in a split-plot design with four 

replications planted 20 feet (ft) apart. Rows were planted 30 inches apart, seeded at a rate of 

approximately 1.7 seeds/ft, and planted 1.5–1.75 inches deep. Plots (240 ft  60 ft) were divided into 24 

row subplots (60 ft  60 ft) that served as replications receiving one of 4 insecticide regimes. Insecticide 

treatments of the Bt and non-Bt plots included the following: 

(1) No insecticide 

(2) Seed treated with Gaucho® prior to planting 

(3) Granular insecticide, Force 3G®, applied and incorporated in furrows at planting 

(4) Foliar insecticide, Pounce 3.2 EC®, applied at the V10 and R2 corn growth stages to 

 control 1st and 2nd generation CRW adults. 

A four-row buffer of non-transgenic corn was planted around each plot to minimize edge effects from 

adjacent subplots. 

Data were collected on agronomic and phenotypic characteristics, pest (insect and disease) 

susceptibility, soil quality and fertility, microbial populations, and non-target invertebrate abundance. 

Eight-inch deep soil samples were taken to evaluate quality and fertility. Four samples were taken 

during the growing season and two were taken post-harvest in 2000; two samples were taken during and 

after the growing season in 2001 for a total of ten samples. Microbial populations were evaluated from 

test and control plots that received no insecticide regime a total of 14 times during the growing season 

from the top six inches of soil within six inches of the rhizosphere. Samples were taken in 2000 and 

2001 during the V2, V4, V8, R1, and R6 stages, as well as after tillage and the following spring. Soil 

samples from the 2000 growing season were analyzed for bacteria, mold, and yeast by a heterotrophic 

plate count method. Four of the seven soil samples collected during the 2001 growing season were 

analyzed for bacteria, actinomycetes, and fungi by the “viable plate count method.” 

Soil-dwelling invertebrates were collected from root balls, including soil during the V6, V10, and R1 

growth stages during the 2000 and 2001 growing seasons. Three eight-inch root balls were collected 

during the V6, V10, and R1 corn growth stages from all control and test plots and processed through a 

Berlese funnel to extract invertebrates. Earthworms were also collected from soil samples by hand 

sorting. Ground surface-dwelling invertebrates were collected from all test and control plots under all 

insecticide regimes using pitfall traps. Four pitfall traps were placed in each subplot from the V6 to R4 

corn growth stages for three-day periods. Key invertebrates from pitfall traps were counted and 

identified to family level. Flying and foliage-dwelling invertebrate were collected from each subplot 

using yellow sticky traps. Three traps per subplot were placed at canopy level from the V6 to R4 corn 

growth stages. Traps were left in the field for seven days and all key taxa were counted and identified to 
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family or genus level. Data were analyzed using a mixed linear repeated measures model for each 

invertebrate collected by each sample method. 

Results:

Among the three sample methods (soil, pitfall, and sticky trap), there was a total of 156,572 organisms 

from 16 orders and 36 families identified during the 2000 and 2001 growing seasons. Collected 

invertebrates included pests, predators, parasitoids, detritivores, and decomposers. The predominant 

non-target invertebrates collected in each sample method are summarized in Table 2. 

Table 2. Predominant Non-Target Invertebrates Collected in Each Sample Method.

Sample Method Order (Family)

Soil and Root Samples 

(soil-dwelling invertebrate) 

Diplura (Japygidae), Chilopoda, Aranea, Acari, Oligocaeta (earthworms), 

Coleoptera (Carabidae, Staphylinidae, Nitidulidae, Lanthridiidae), 

Hymenoptera (Formicidae) 

Pitfall Trap Samples 

(ground surface-dwelling invertebrate) 

Orthoptera (Gryllidae), Coleoptera (Carabidae, Staphylinidae, Nitidulidae, 

Scarabeidae, Chrysomelidae), Hymenoptera (Formicidae), Araneae, 

Chilopoda 

Yellow Sticky Trap Samples (flying & 

foliage-dwelling invertebrate) 

Coleoptera (Chrysomelidae, Nitidulidae, Coccinellidae), Hymenoptera, 

Homoptera (Aphididae, Cicadellidae), Hemiptera (Anthocoridae), Diptera 

(Syrphidae), Neuroptera (Chrysopidae, Hemerobiidae), Aranea 

 

Overall, there was no statistical difference between MON 863 and non-Bt plots (RX670) in the 

abundance of the predominant invertebrates collected in soil samples. According to the soil sample data, 

the number of Japygidae (diplurans) did not differ between Bt and non-Bt plots; however, there were 

significantly less collected in the insecticide-treated plots. Of the coleopteran insects collected, there 

were generally more carabids (ground beetles) and staphylinids (rove beetles) than nitidulids (sap 

beetles) and lanthridiids (minute brown scavenger beetles). There was no statistical difference in the 

number of coleopteran insects captured between the MON 863 and non-transgenic corn isolines. 

Statistical analysis showed that insecticide treatments significantly reduced the number of carabids in 

2000 and the number of staphylinids on the last sample date in 2001. Invertebrates from the Araneae 

(spider) and Acari (mite) families were also not significantly different between Bt and non-Bt plots; 

however, the number of acarids (mites) was statistically greater in the plots treated with foliar 

insecticides in 2001. The number of chilopods (centipedes), the most abundant non-insect arthropod 

sampled, was not different between Bt and non-Bt isolines but more were collected in plots treated with 

seed, soil, and foliar insecticides in 2001, particularly the MON 863-treated plots. Although the number 

of earthworms collected did not differ between Bt and non-Bt plots, there were significantly less 

earthworms in the plots treated with foliar insecticides than the other insecticide regimes. The handsorting method also showed no differences in the number of earthworms collected in MON 863 and nontransgenic plots, nor was there a difference found between insecticide regimes. 

Overall, there was no statistical difference between MON 863 and non-Bt plots (RX670) in the 

abundance of the predominant invertebrates collected in pitfall trap samples. Of the coleopterans 
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captured in pitfall traps, there were more nitidulids (sap beetles), carabids (ground beetles), and 

staphylinids (rove beetles) captured than chrysomelids and scarabids collected. There was no statistical 

difference between MON 863 and RX670 field plots in the overall number of coleopterans collected in 

pitfall traps. The different insecticide regimes tested resulted in varied and inconsistent effects on the 

abundance of the predominant Coleoptera sampled. The number of gryllids (crickets) and formicids 

(ants) collected in pitfall traps was not statically different between Bt and non-Bt plots or different 

insecticide regimes. Chilopod (centipede) and Araneae (spider) abundance was not different between 

MON 863 and RX670 plots nor was there an effect from insecticide regimes in most cases. Statistical 

analysis showed that there were significantly fewer Araneae (spider) in the plots with soil and foliar 

insecticide treatments in 2000. 

Based on yellow sticky trap counts, there were consistently less corn rootworm (CRW; Diabrotica 

virgifera and Diabrotica barberi) in the MON 863 plots than the non-Bt plots in all insecticide regimes. 

The difference in the number of CRW captured, however, was not statistically different. In both 2000 

and 2001, there were no significant differences between MON 863 and RX670 plots across all 

insecticide regimes in the number of nitidulids (sap beetles), coccinellids (lady beetles), aphidids (grass 

hoppers), cicadellids (potato leaf hoppers), braconids (Macrocentrus grandii - a parasitoid), syrphids 

(syrphid or hover flies), hemerobiids (brown lacewing), chrysopids (green lacewing) and Araneae 

(spiders). There was a reduction in the abundance of C. maculata (lady beetles) and M. grandii and an 

increase in Empoasca fabae (potato leaf hopper) in plots receiving a foliar spray. Syrphid fly abundance 

was reduced in 2000 by soil insecticide treatments. 

Field Census Summary:

Data collected during the 2000 and 2001 growing seasons indicate that MON 863 and RX670 corn are 

agronomically and phenotypically equivalent, and there are not differences in their susceptibility to 

pathogens. Soil quality and fertility were also found to be consistent among MON 863 and RX670 field 

plots. Sampling data collected in 2000 and 2001 also showed that there are no overall differences in the 

abundance of non-target invertebrate collected in MON 863 and RX670 plots. However, corn pests, 

such as D. virgifera, Chaetochnema pulicaria, and Rhopalosiphum maidis, as well as predators (e.g., O.

insidiosus and C. maculata (lady beetle)), parasitoids (e.g., M. grandii), and decomposers (e.g., 

earthworms and diplurans) were significantly impacted by insecticide regimes. Foliar sprays and soil 

treatments resulted in the greatest impact on non-target organisms such as carabids, spiders, O.

insidiosus (a generalist predator), C. maculata (lady beetle), and M. grandii (parasitic wasp). Therefore, 

the report concluded: “MON 863 had less impact on certain beneficial insects compared to traditional 

insecticide control programs, especially soil and foliar applications. Thus, the use of MON 863 for corn 

rootworm control can lead to reduced use of insecticides and increased compatibility with Integrated 

Pest Management programs in corn.” 

Field Census Conclusions:

According to the data submitted to the Agency by Monsanto, MON 863 corn does not adversely impact 

the abundance of non-target invertebrate found in corn fields. Nonetheless, plot size (240 ft  60 ft plots 

divided into 24 row 60 ft  60 ft subplots) was small and only replicated four times. In addition, each 
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plot only included three root and sticky trap samples and four pitfall trap samples. The August 2002 

SAP concluded that field experiments must be appropriately designed to provide a measure of ecological 

impacts. In addition, the SAP opinion was that a two-year field study would not be sufficient to 

determine if MON 863 corn will have long-term impact on non-target invertebrates. Several public 

comments also expressed this concern. Short-term field studies are not adequate to draw conclusions on 

the variations in non-target invertebrate populations. Large field-scale studies, conducted for at least 

three to four years, would be needed to draw a conclusion on non-target impacts. The Panel generally 

concluded that “the state-of-the-science” needed for long-term studies must improve for the research to 

be appropriately conducted to provide meaningful results. The statistical power (avoiding Type II 

experimental error) needed to gain useful results from field studies would require very large fields, more 

replications, and more samples per plot (e.g., 10 soil and pitfall samples) plus the addition of visual plant 

samples (e.g., >50/plot). Since the endpoint for field census studies has not been determined, it is 

difficult to determine how large the fields should be, how many replications are needed, and how many 

samples per plot are needed to achieve appropriate statistical power. Therefore, additional field census 

studies should not be conducted until the endpoints and logistics of the study have been determined. If 

Tier I maximum hazard dose single species laboratory studies show a hazard, intermediate field or semifield studies between laboratory and full-scale field studies should be conducted. Additional full-scale 

field or semi-field studies with appropriate end points and statistical power should also be considered 

based on recommendations of the August 27, 2002 SAP. 

The submitted field census data, demonstrating an abundant presence and diversity of invertebrates in 

the corn CryBb1 corn field, are useful for short-term hazard assessment as supplementary information, 

which shows the same no-hazard trend seen in the maximum hazard dose single species laboratory 

testing. 

c) Year 2001: Field and Laboratory Invertebrate Studies (MRID No. 456530-03)

A summary of preliminary findings from several one-year supplemental higher tier field and laboratory 

studies was submitted (MRID No. 456530-03). These studies were not triggered by Tier I maximum 

hazard dose testing data; however, they appear to have value for assessing possible long-term effects on 

invertebrate populations. These studies are being conducted in Kansas, Nebraska, Illinois, Virginia, 

South Dakota, and New York to evaluate the ecological impact of MON 863 Bt corn, grown under 

different insecticide regimes, on abundance of non-target organisms relative to non-transgenic corn. The 

submitted summary of preliminary findings shows some possible effects of MON 863 on corn field 

insects. Final reports of these studies are required to be submitted to the Agency for review. 

*Note for 2010: There is an update to this summary. See section II(C)(2)(b) (“Terms and Conditions of 

the Corn Event MON 863 Registration (February 2003 – September 2010)”) of this BRAD. 

II. Soil Fate 

Soil organisms may be exposed to Cry3Bb1 protein by exposure to roots, incorporation of aboveground 

plant tissues into soil after harvest, or by pollen deposited on the soil. Root exposure may occur by 
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feeding on living or dead roots or, theoretically, by ingestion or absorption after secretion of the Cry 

protein into the soil. In addition, some evidence suggests that Cry proteins, while bound to some soil 

components (e.g. clays and humic acids), are recalcitrant to degradation by soil microorganisms but 

without eliminating their insect toxicity. Several factors influence either the affinity of binding or the 

rate of degradation. In particular, a neutral pH generally substantially increases degradation. Corn does 

not grow well below ~pH 5.6; therefore, most corn-growing soils are expected to be at a higher pH. 

Under most production conditions, corn would not be grown in soils that would inhibit the rate of 

degradation compared to what is seen at near neutral pH. Nevertheless, these issues are being evaluated 

on a case-by-case basis by environmental fate studies designed to determine the rate of Cry protein 

degradation over sufficiently long periods to assure an accurate assessment of degradation in agricultural 

soils. 

MRID No. 451568-04

This study was conducted in accordance with Good Laboratory Practice Standards (40 CFR Part 160). 

Methods: 

An insect bioassay and an ELISA were conducted to measure the level of functional and non-functional 

Cry3Bb1 protein present in field soil samples. The amount of lyophilized corn tissue added to the field 

soil in this study was based on the amount of plant tissue that could potentially be incorporated into the 

top six inches of soil under field conditions. Since field incorporation of plant tissue usually will not take 

place until the fall, this amount of Cry3Bb1 protein represents the worst-case scenario during the 

growing season, including possible exudation of Cry protein through the roots into soil. Based on this 

calculation, 0.03 g (rounded up from 0.028 g) dry weight plant tissue was added to each gram of dry 

sandy loam field soil (from Fayette County of Lexington, Kentucky); therefore, 3% of the dry weight of 

soil was dry weight of lyophilized plant tissue. An additional test was conducted with 10% of the soil 

containing lyophilized dry weight plant tissue (0.10 g leaf tissue/g soil). Insect bioassays included a 

mixture of test and control substances with an agar-based insect diet added to wells of bioassay trays. 

Colorado potato beetle (CPB; Leptinotarsa decemlineata) larvae were added 1 larva/well, and each 

treatment bioassay was replicated twice for a total of 16 CPB/replicate. CPB are more sensitive to the 

Cry3Bb1 protein then CRW and were, therefore, expected to result in a more measurable response than 

CRW, the target species. In addition to an insect bioassay, an ELISA was conducted to measure the level 

of Cry3Bb1 protein present in samples. The ELISA test will only show extractable protein and does not 

distinguish between functional and non-functional proteins. 

Results:

Results from this study show the DT50 and DT90 (degradation time) for Cry3Bb1 in leaf tissues in sandy 

loam soil based on the ELISA test to be 2.76 and 9.16 days, respectively. The 21-day ELISA sample 

was the last to show traces of Cry protein. At 28 days, the Cry3Bb1 protein was below the detection 

level. The value of these results, however, needs to be considered with regard to biological activity 

because it is unknown if the extractable protein in the ELISA test was functional or non-functional. 

Therefore, the insect bioassays were performed with CPB and determined the DT50 and DT90 to be 2.37 

and 7.87 days, respectively. The no-detection level was in the range of the results obtained by ELISA. 
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Conclusions:

Based on these results, Cry3Bb1 protein does degrade rapidly and does not accumulate in sandy loam 

soil; however, corn is not necessarily grown in sandy loam soil in all regions. Corn is grown in other soil 

types, such as clay loam and silt loam soils in various regions of the U.S. Testing clay soils would be 

considered a “worst-case scenario.” In addition, this test does not account for all plant tissue, such as 

roots, or root exudation of the Cry protein in the field. It is possible that root tissue is degrading slower 

then leaf tissue in the soil, which may result in a longer duration of degradation time of the Cry3Bb1 

protein. Therefore, it was recommended that field testing should be continued in a variety of soil types, 

including clay and humic acid soils, over a three years to determine the long-term degradation rate and 

accumulation/persistence of Cry3Bb1 protein in soil. 

Since the submission and review of this study (MRID No. 451568-04), the Agency convened an SAP on 

August 27, 2002 to address corn rootworm PIP non-target issues, including the degradation of Bt protein 

in soil. The aerobic soil degradation study (MRID No. 457571-02) below was designed to address the 

deficiencies identified in MRID No. 451568-04 and comments made by both the August 27, 2002 SAP 

and the public. 

MRID No. 457571-02

This study is intended to address the issues raised in MRID No. 451568-04: 

“Supplemental to conducting the study in the field with all plant tissue 

 incorporated into the soil in fields that have had MON 863 corn grown for 

 one to three consecutive years. Studies should also be conducted in a variety 

 of soil types particularly soil high in clay and humic acids.” 

In addition, the previously submitted aerobic soil degradation study (MRID No. 451568-04) utilized 

plant tissue from Cry3Bb1 transformation event MON 859 rather than MON 863. The study described in 

the paragraphs that follow involved use of MON 863 corn root and shoot tissue. 

Methods:

The test substance consisted of finely ground and lyophilized root and shoot tissue of MON 863 

containing the Cry3Bb1 insecticidal protein. The concentration of Cry3Bb1 protein in the lyophilized 

corn tissue, determined by ELISA, was 487 μg/g in the root and 468 μg/g in the shoot. A purified 

Cry3Bb1 protein, obtained from a genetically modified E. coli strain containing a sequence identical to 

MON 863 corn, was also included in the study. MON 863 corn shoot and root tissues were collected 

from field grown plants in Richland, Iowa. The test matrix consisted of three soils collected from the top 

six inches, Horizon A, of corn fields in Carlyle, Illinois; Monmouth, Illinois; and Richland, Iowa. Soil 

properties for these three soils (Table 3) and a microbial analysis were characterized. Soil viability was 

confirmed at test initiation and at approximately four and eight weeks of incubation. Soils were shown 

to remain active and viable throughout the study (144–228 microbial biomass carbon/50 g soil). The 

highest concentration of Cry3Bb1 protein in roots 35 days after planting is 66 μg/g. On this basis, the 

maximum field loading of Cry3Bb1 in corn shoots is 3.93 μg/g soil (equivalent to 8 mg shoots/g soil); 
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for corn root tissue, the loading is 2.79 μg/g soil (equivalent to 6 mg of roots/g soil). For additional 

conservatism, the maximum values were exaggerated 3x for dosing of soils. Therefore, 24 mg of shoot 

tissue and 18 mg of root tissue was added to soils. Also, the purified Cry3Bb1 control dosing 

concentration was exaggerated ~25x to model the unlikely scenario that large amounts of protein would 

be exuded by roots into the soil throughout the growing season. Approximately 48 μg of purified protein 

was added to 0.5 g of soil. Soils from the three test locations were air dried and dosed with the Cry3Bb1 

test substance at these rates. All vials were mixed thoroughly and soil moisture adjusted with deionized 

water to obtain soil moisture of 75% field capacity at 0.33 bar. 

 

Table 3. Physicochemical Characteristics of Soils Collected from Horizon A (Top 6 Inches). 

Soil Source

Parameter Carlyle, IL Monmouth, IL Richland, IA 

USDA 

Textural Class Silt Loam Silt Loam Silt Loam 

Particle Size 

Distribution 

(%) 

Sand 

Silt 

Clay 

21 

58 

21 

18 

56 

26 

13 

62 

25 

Bulk Density 

(g/cm3) 1.00 1.03 1.07 

% Organic 

Matter 2.5 4.6 4.0 

Cation 

Exchange 

Capacity 

(meq/100 g) 

16.6 23.9 20.7 

Field Moisture 

Capacity 

@ 1/3 Bar 

@ 15 Bar 

28.2 

15.7 

30.7 

17.9 

30.3 

17.2 

*Table taken from page 29 of MRID No. 457571-02. 

Results:

Cry3Bb1 protein levels in soil sample extracts determined by ELISA show that Cry3Bb1 protein 

concentrations were near or below the ELISA detection limit (LOQ) of 0.16 μg/g after 2 months of 

incubation. After two months of soil incubation, Cry3Bb1 concentrations were at least an order of 

magnitude below the initial concentration of Cry3Bb1 in the dosed samples. The DT50 values for all 

dosing regimes and soil types ranged from 0.6 days to 2.3 days, and the DT90 values ranged from 4.03 

days to 50 days (Table 4). Purified Cry3Bb1 protein degraded faster than when corn shoot or root tissue 

was applied. Visual observation verified that root tissues are slower to degrade in soil than shoot tissue. 
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These results also indicate, as expected, that the longer DT50 and DT90 values for corn tissues are due to 

the time required for the tissue to decay and for the Cry3Bb1 protein to move from tissue to soil. In 

addition, the rapid degradation (DT90 of 4.0 to 5.2 days) of the purified Cry3Bb1 protein suggests that 

any Cry3Bb1 protein reaching the soil by root exudation or release from slowly decaying plant tissue 

would be >90% degraded in less than 6 days. 

Table 4. DT50 and DT90 Estimates for the Dissipation of Cry3Bb1 Protein in Soils. 

Protein Source Soil Source DT50a

 (Days) DT90

b (Days) 

Carlyle, IL 0.63 (0.49, 0.78)c 4.03 (3.67, 4.42) 

Monmouth, IL 0.64 (0.50, 0.80) 4.18 (3.77, 4.62) Purified 

Protein 

Richland, IA 0.73 (0.50, 1.00) 5.23 (4.48, 6.07 

Carlyle, IL 1.74 (0.78, 3.20) 27.29 (14.52, 50.58) 

Monmouth, IL 1.19 (0.86, 1.57) 12.48 (9.90, 15.67) 

MON 863 

Corn Root 

Tissue 

Richland, IA 2.27 (1.68, 2.98) 50.02 (34.57, 72.18) 

Carlyle, IL 1.45 (0.82, 2.30) 18.68 (12.61, 27.46) 

Monmouth, IL 0.90 (0.61, 1.24) 7.43 (6.01, 9.12) 

MON 863 

Corn Shoot 

Tissue 

Richland, IA 1.77 (1.40, 2.21) 28.59 (23.16, 35.23) 

a DT50 = Time to 50% dissipation of original protein concentration 

b DT90 = Time to 90% dissipation of original protein concentration c Lower and upper 95% confidence interval on the DT value 

*Table taken from page 36 of MRID No. 457571-02. 

Conclusions:

It is difficult to determine a DT50 or DT90 for Cry3Bb1 expressed in corn tissue in the field from this 

study because corn shoot and root tissue were analyzed separately and not all plant material was 

included. Therefore, methods utilized in this study do not represent actual field conditions. It is unknown 

whether these laboratory results can be adequately correlated to the field. Additional field studies should 

be conducted that include the incorporation of all non-harvested plant tissue in a variety of soil types, 

particularly areas high in clay (>26% tested here) and humic acids. These studies should be conducted 

for at least one growing season after harvest and continue until no Cry3Bb1 protein is detected. In 

addition, the persistence of the Cry3Bb1 protein under less than optimum conditions (e.g., high or low 

temperatures; high or low soil moisture content) should be examined. Additional studies, conducted to 

address the degradation of Cry3Bb1 protein in the soil, should include an insect bioassay utilizing a 

known sensitive species (e.g., Colorado potato beetle). 

These conclusions are based in part on the August 27, 2002 SAP and several public comments. The 

Panel concluded that several different soils should be examined and monitored for a minimum of one 

growing season after harvest and continued until the Cry3Bb1 protein can no longer be detected. The 
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Panel also recommended that an additional sample or two should be examined to verify that an 

analytical error was not the cause for the lack of detection. According to the Panel, at least two 

additional soil types should be evaluated for Cry3Bb1 persistence. Soils that are high in organic matter 

and clay should be the focus, since there is the highest potential of persistence in these soil types. Other 

soils, however, should still be considered. The Panel also recommended that the soil degradation studies 

be conducted under less than optimum conditions, such as high or low temperatures or high or low 

moisture content. Since corn roots grow deep into the soil to areas with reduced microbial activity, 

degradation rates may be reduced. Therefore, degradation of Cry3Bb1 from deep sites should also be 

examined. The Panel also addressed the protein source that is appropriate for the soil degradation 

studies. Future studies should utilize plant material that is representative of actual field conditions. For 

example, whole plant tissue should be incorporated. Plant tissue should not be ground prior to 

incorporation because it artificially increases the surface area exposed to microorganisms, which may 

then lead to an increase in the rate of degradation of the protein. Since more protein may be present than 

is detected by an ELISA, an insect bioassay, using a sensitive species such as the Colorado potato beetle, 

should be conducted. The SAP concluded that “[r]eal life or true persistence is likely to be equal to or 

less than that measured with ELISA.” If an ELISA is conducted, the results should be compared to 

results from a beetle bioassay. 

This study, although rated as supplemental, provides further evidence that the Cry3Bb1 protein in MON 

863 produces no short-term risk of unreasonable adverse effects for the environment. 

*Note for 2010: There is an update to this summary. See section II(C)(2)(b) (“Terms and Conditions of 

the Corn Event MON 863 Registration (February 2003 – 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 soil microorganisms (Sanvido et al. 2007; Oliveira et al. 2008). In addition, Bt toxin 

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 research findings conclude no Btrelated risks have evolved from the decomposition of Bt-corn leaves for the meso- and macrofauna soil 

community (Hönemann et al. 2008). Although a minimal transient increase and shift in microbial 

populations may result from the presence of transgenic plant tissue in soil, no adverse effects have been 

attributed to the Cry protein. 

In addition, there are several ongoing U.S. Department of Agriculture and EPA Office of Research and 

Development funded research projects 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 crops on soil, it is important to note that agricultural 

practices themselves cause large changes in soil and soil microbial composition. Furthermore, factors 
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such as variations in seasons and weather, plant growth stage, and plant varieties, independent of being 

genetically engineered, are also responsible for significant shifts in soil microbial communities. To date, 

most studies with genetically engineered crops have shown minor or no effects on soil microbes beyond 

the variation caused by the factors listed above. 

IV. Horizontal Transfer of Transgenes 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 typical environmental conditions. Horizontal gene transfer to soil 

organisms has only been detected with very promiscuous microbes under laboratory conditions designed 

to favor transfer. 

As a result of these findings, which suggest that HGT is at most an artificial event, and the fact that the 

Bt toxins engineered into Cry3Bb1 corn are derived from soil-inhabiting bacteria, EPA has concluded 

that there is a low probability of risk from HGT of transgenes found in Cry3Bb1-producing corn. 

V. Gene Flow and Weediness Potential

The movement of transgenes from the host plant into weeds has been a significant concern for the 

Agency due to the possibility of novel exposures to the pesticidal substance. The Agency has determined 

that there is no significant risk of gene capture and expression of Cry3Bb1 protein by wild or weedy 

relatives of corn in the U.S., its possessions, and/or its territories. In addition, the Animal and Plant 

Health Inspection Service (APHIS) of the United States Department of Agriculture (USDA) has made 

this same determination under its statutory authority under the Plant Pest Act. 

Under FIFRA, the Agency has reviewed the potential 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 differences 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 gene 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 
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for gene transfer between corn (maize) and any receptive plants within the U.S., its possessions, and/or 

its territories was of limited probability 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 maize and are discussed as potential recipients of pollendirected gene flow from maize. This issue is of particular concern based upon the increased planting of 

genetically modified maize. Therefore, the Agency conducted a reevaluation 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 flowers) and silks (female flowers) are found on the same plant, a feature 

that limits inbreeding. A large variety 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 interfertile to a large degree. However, 

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 characters, such as a non-shattering rachis, grain yield, and resistance to 

pests. The origin of corn is thought to be in Mexico or Central America, based largely on archaeological 

evidence of early cob-like maize in indigenous 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 was 1%, and this proportion declined exponentially to 

0.1% at 130 feet and further declined to 0.03% at 160 feet. At 1,000 feet, the farthest distance measured, 

no cross-pollination was detected (Jemison and Vayda 2000). For production of Foundation Seed, a 

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 period under most conditions reduce long 

distance transfer for purposes of outcrossing (Schoper, personal communication,1999). Under conditions 

of high temperature or low humidity, corn pollen may only survive for a matter of minutes. Under more 

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

extended to several hours. 

 b. Tripsacum species - Gama Grass - 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, Central America, and South America, but three 

occur within the U.S. Hitchcock (1971) reports the presence of three species of Tripsacum in the 

continental United States: Tripsacum dactyloides, Tripsacum floridanum, and Tripsacum lanceolatum. 
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Of these, T. dactyloides, Eastern Gama Grass, is the only species of widespread occurrence and of any 

agricultural importance. It is commonly grown as a forage grass and has been the subject of some 

agronomic improvement (i.e., selection 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, which extend along the Eastern seaboard and along the 

Gulf Coast from Florida to Texas but which have also been found in Illinois and Kansas; these latter 

forms may represent tetraploids (x = 9 or 18)(Lambert, personal 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 accomplished 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 (Galinat 1983; Iltis 1983; Beadle 1980), based largely on 

the disparity in chromosome number between the two species (maize n = 10; Gama Grass x = 9 or 18, 

with diploid, triploid, and tetraploid races existing; 2n = 36 or 72), as well as radically different 

phenotypic appearance. Albeit with some difficulty, hybrids between the 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 unlikely, if not impossible (Beadle 1980). Hybrids of 

Tripsacum species with Z. mays are difficult to obtain outside of the controlled conditions of laboratory 

and greenhouse. Seed obtained from such crosses are often sterile or progeny 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 winters. 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, other than occasional volunteer plants in the previous 

season’s corn field, maize is not considered as a weed in the U.S. 

In a telephone conversation with Dr. Chester “Chet” DeWald (Agricultural 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 the 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 
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greater likelihood of obtaining viable seed. When these 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 generations. In many instances where hybridization has 

been directed between these two species, the resultant genome is lacking in most or all of the 

chromosomal complements of one of the parent 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 chloroplast 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 continental U.S. would interbreed with commercial maize 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 - General 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 maintain distinct genetic constitutions despite sporadic 

introgression (Doebley 1990). 

The teosintes retain a reduced cob-like fruit/inflorescence 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 characteristic of teosintes or maize. In 

their native habitat, some teosintes have been observed to be spread by animals feeding on the plants. 

Teosintes and teosinte-maize hybrids do not survive even mild winters 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 (Beadle 1980; Iltis, personal 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 
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is not aware, however, of any such case being reported in the United States. Gene exchange between 

cultivated corn and transformed corn would be similar to what naturally occurs at the present time 

within cultivated corn hybrids and landraces. Plant architecture and reproductive capacity of the 

intercrossed plants will be similar to normal corn, and 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 with corn, especially when maize acts as the female 

parent. Z. perennis (perennial teosinte) has 20 pairs of chromosomes and forms less stable hybrids with 

maize (Edwards et al. 1996; Magoja and Pischedda 1994). Corn and compatible species of teosinte are 

capable of hybridization when in proximity to each other. In Mexico and Guatemala, teosintes exist as 

weeds around the margins of corn fields. The F1 hybrids have been found to vary 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 Florida 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 populations of this species for approximately 

twenty-five years (Bradley, personal communication, 2000; Hall, personal communication, 2000; 

Wunderlin, personal communication, 2000). 

Consultation with botanists and agronomists familiar with Texas flora suggested that no teosinte 

populations exist in the state (Benz, personal communication, 2000; Read, 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 sustaining 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 fertile hybrids in many instances (Wilkes 1967). 

None of these teosinte species have, however, been shown to be aggressive weeds in their native or 

introduced habitats (Schoper, personal communication, 1999). Except for special plantings as noted 

above, teosinte is not present in the U.S. or its territories. Its natural distribution is limited to Mexico, 

Honduras, Nicaragua, El Salvador, and Guatemala. 

Given the cultural and biological relationships of various teosinte species and cultivated maize over the 

previous two millennia, it would appear that significant gene exchange has occurred (based upon 

morphological characters) between these two groups 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 far less than previously thought (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 growth and a skewed recovery of alleles linked to 

incompatibility genes. Complete incompatibility mechanisms serve to isolate 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 groups (Kermicle and Allen 1990). 
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Based on the ability of maize to hybridize with some teosintes, the suggestion of previous genetic 

exchange amongst these species over centuries, and their general growth habits, any introgression of 

genes into wild teosinte from Z. mays is not considered a significant agricultural 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 species 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 where 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 harbor weedy characteristics and none of the 

native teosinte species, subspecies, or races are considered aggressive 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 potential and are generally considered 

incapable of survival in the wild as a result of breeding practices (i.e., selection) during domestication of 

the crop. 

 iii. Endangered Species Considerations

Cry3 proteins, including Cry3Bb1, are known to be highly specific against coleopteran insects and are 

not hazardous to vertebrate animals. It has been generally demonstrated that Cry3 proteins do not pose a 

hazard to non-target animals or invertebrates. The Cry3Bb1 protein appears to be specifically toxic to 

Chrysomelid beetles, including corn rootworm (Diabrotica spp.) and Colorado potato beetle 

(Leptinotarsa decemlineata) (MRID No. 455328-07). Currently, there are no Chrysomelid species listed 

on the endangered species list, and no other species are known to be sensitive to Cry3Bb1. Therefore, no 

adverse effects from Cry3Bb1 (Event MON 863) are expected against endangered species. Nevertheless, 

all endangered/threatened beetle species habitats, found in the counties where corn is grown, were 

examined to determine possible exposure to corn pollen. Their habitat (and breeding grounds) was found 

not to overlap with corn fields. Endangered beetles will not be exposed to potentially harmful levels of 

corn tissue or pollen containing Cry3Bb1 protein. 
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Terrestrial and aquatic exposure were considered in this assessment since non-target coleopterans may 

be exposed to the Cry3Bb1 protein within corn fields or in surrounding areas from plant tissue (e.g., 

pollen) movement offsite. The distance pollen moves outside of the corn field must be considered. 

Published data show that less than 25 grains of pollen per square centimeter are expected 4–5 meters 

from the corn field edge. A relative comparison of surface ratio of milkweed to other substrates (e.g., 

other host plants, arthropod prey, and animal carrion) can be used as a basis for estimating the amount of 

pollen that may leave the field. The maximum concentration of Cry3Bb1 protein has been determined to 

be 93 μg/g fresh weight pollen. Based on this concentration, <0.03 μg Cry3Bb1 protein/g of diet would 

be expected to be deposited 4–5 meters from the field edge. The potential for aquatic organisms to be 

exposed to the Cry3Bb1 protein is minimal. Such exposure would occur from runoff of the protein 

(either free or sequestered in plant debris) into adjacent water bodies or pollen drift. Since movement of 

Cry3Bb1 in soil into water bodies is expected to be negligible, pollen drift was considered the primary 

source of potential hazard to endangered aquatic Coleoptera. According to estimates based on published 

studies, if 100% of the pollen grains leaving the field were deposited in a 1 hectare pond with 2 meters 

depth and located 1 mile from the edge of the corn field, <0.0001 μg Cry3Bb1/mL of water would be 

expected. This is a few orders below the toxic level to any insect. 

Many of the endangered and threatened beetles occur in cave or aquatic habitats. None of these 

endangered beetles are expected to occur in or near corn fields. The American burying beetle may occur 

in old fields or cropland hedge rows. Based upon the feeding habits of the American burying beetle, 

however, it is not expected to occur within corn fields or be exposed to Cry3Bb1 protein. Adult 

American burying beetles are classified as opportunistic scavengers that feed on anything dead and bury 

vertebrate carcasses on which their larvae feed. Carrion regurgitated by adults is fed to larvae until they 

are able to feed directly on a carcass. 

In addition, Monsanto conducted a hazard assessment, exposure assessment, and risk characterization to 

demonstrate that Cry3Bb1 does not pose a risk to endangered Coleoptera (MRID No. 455770-03). This 

endangered species assessment was based on the Hazard Evaluation Division, Standard Evaluation 

Procedure – Ecological Risk Assessment (U.S. EPA 1986). The Agency reviewed this assessment and 

found it acceptable. 

An examination of the endangered bird and bat species shows that their breeding habitats are mostly 

non-agricultural. Insectivorous bats do not prey on larvae; they rely on flying insects. Taking these, and 

other pertinent issues into consideration, it becomes apparent that reduction in the target pests of corn 

would not have an effect on the food source of endangered birds and bats. Of those that do encroach on 

agricultural fields and in the rare instances where these species may feed on the target pests, the 

reduction in the pest species will merely cause them to rely on other plentiful insects as a source of food. 

Submitted and published field data show that a wide variety of insects remain abundant in Cry3Bb1 corn 

fields, as opposed to non-Bt fields when conventional insect pest control practices are used. Therefore, 

the data show that Bt crops should actually be beneficial to bird and bat populations. 

The reviewed non-target data confirm the expectation that Cry3Bb1 corn will have no effects on 

endangered and/or threatened species listed by the U.S. Fish and Wildlife Service, including mammals, 
Bacillus thuringiensis Cry3Bb1 Corn 

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birds, or terrestrial and aquatic plants and invertebrate species. Therefore, no consultation with the U.S. 

Fish and Wildlife Service is required under the Endangered Species Act. 

 iv. Environmental Assessment Summary 

The Agency is using a MHD tiered system for biopesticide non-target wildlife hazard assessment. When 

no adverse effects at the maximum hazard dose are observed, the Agency concludes that there are no 

unreasonable adverse effects from the use of the pesticide. From all of the required and voluntarily 

developed indicator and host range species test data on Cry3Bb1 corn, including the supplementary twoyear field data, the Agency concludes that the levels of Cry3Bb1 protein in 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 Cry3Bb1 protein in agricultural soil. In addition, no effects on 

listed endangered and threatened species are expected from the proposed Cry3Bb1 CRW-resistant corn 

registration. 

At present, the Agency is aware of no identified significant adverse effects of Cry3Bb1 proteins on the 

abundance of non-target beneficial organisms in any population in the field, whether they are pest 

parasites, pest predators, or pollinators. Field testing and field census data submitted to the Agency show 

minimal to undetectable changes in the beneficial insect abundance or diversity. In corn fields, densities 

of predatory and non-target insects are generally higher on Cry3Bb1 corn than non-Bt corn, primarily 

because the Cry3Bb1 corn is not subjected to the same number of applications of nonspecific pesticides. 

Two-year invertebrate abundance studies do not show a shift in the biodiversity in Cry3Bb1 corn, except 

in cases where the predators are dependent on the pest insect as prey. In contrast, treatment with 

chemical pesticides, when studied, had significant effects on the total numbers of insects and on the 

numbers within the specific groups. To date, the available field test data show that, compared to crops 

treated with conventional chemical pesticides, the transgenic crops have no detrimental effect on the 

abundance of non-target insect populations. Despite these conclusions, annual insect monitoring of 

representative commercial fields will continue for long-term biodiversity effects assessment. 

The Agency believes that cultivation of Cry3Bb1 corn may result in fewer adverse impacts to non-target 

organisms than result from the use of chemical pesticides. Under normal circumstances, Cry3Bb1 corn 

requires substantially fewer applications of chemical pesticides. This should result in fewer adverse 

impacts to non-target organisms because application of nonspecific conventional chemical pesticides is 

known to have an adverse effect on non-target beneficial 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 and leafhoppers. The overall result of 

cultivation of corn expressing Cry3Bb1 proteins is that the number of chemical insecticide applications 

for non-target pest control is reduced for management of multiple pest problems. 

The movement of transgenes from Cry3Bb1 host plant into weeds and other crops has also been 

considered. The Agency has determined that there is no significant risk of gene capture and expression 

of Cry3Bb1 protein by wild or weedy relatives of corn in the U.S., its possessions, and/or territories. The 
Bacillus thuringiensis Cry3Bb1 Corn 

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fate of Cry3Bb1 protein in soils and indirect effects on soil biota has also been evaluated. Test data show 

that most of the Cry protein deposited into soil is quickly degraded, although a residual amount may 

persist in biologically active form for a much longer period of time. It is also reported that the same 

degree of Bt Cry protein persistence takes place in soils that have been exposed to repeat Bt spray 

applications when compared to soil exposed to growing Bt crop. Limited data do not indicate that Cry 

proteins have any measurable effect on microbial populations in the soil. Horizontal transfer from 

transgenic plants to soil bacteria has not been demonstrated. Published studies of Bt Cry protein in soil 

show no effect on bacteria, 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. 

This assessment finds no hazard to the environment, at the present time, from cultivation of corn 

expressing Cry3Bb1 protein. 

 v. Supplemental Studies Needed for Long-Term Cry3Bb1 Non-Target Hazard

 Assessment

The Agency has sufficient information to believe that there is no risk from the proposed uses of 

Cry3Bb1 corn to non-target wildlife, aquatic, and soil organisms. Nonetheless, after consultation with 

the FIFRA Scientific Advisory Panel in August, 2002 and in response to several public comments, the 

Agency is requesting additional data, which could provide more weight to support the Agency's 

conclusions. Specifically, the Agency is requesting the data, as set forth in Table 5, to ascertain any 

possible adverse environmental effects from long-term use of Cry3Bb1 and to test on more appropriate 

non-target invertebrates found in corn fields. The Agency does not believe that these data requirements 

were reasonably foreseeable by Monsanto at the time of its application. 
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Table 5. Supplemental Data Requirements.

Testing Category Type of Data

Avian chronic exposure 

testing 

The submitted avian dietary toxicity data are not sufficient to make a final avian hazard 

assessment from repeated exposure(s) to higher doses of Cry3Bb1 corn. A six-week 

broiler dietary study with 60%–70% MON 863 corn in the diet is needed to assess 

hazard to wild and domesticated fowl from chronic exposure to high levels of Cry3Bb1 

protein. 

Non-target insect more 

appropriate for corn fields 

Conduct a maximum hazard dose laboratory 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). 

Non-target insect more 

appropriate for corn fields 

Maximum hazard testing with Tetraopes (red milkweed beetle) should be performed 

because they are a more logical choice than the monarch butterfly. 

Field community effects Submit final results to field studies previously summarized in MRID No.456530-03. The 

carabid and nematode data are of particular interest. 

Ecosystem effects Additional long-range field studies should also be conducted based on recommendations 

of the August, 2002 SAP. 

Soil fate studies Additional long-range soil degradation field studies should also be conducted, including 

the parameters outlined by the August, 2002 SAP. 

*Note for 2010: There is an update to this summary. See section II(C)(2)(b) (“Terms and Conditions of 

the Corn Event MON 863 Registration (February 2003 – September 2010)”) of this BRAD. 

b. Terms and Conditions of the Corn Event MON 863 Registration (February 2003 –

 September 2010)

When Corn Event MON 863 was first registered on February 24, 2003, the Agency issued a registration 

notice to Monsanto that, given the conclusions set forth in the initial environmental risk assessment (see 

section II(C)(2)(a) of this BRAD), contained the following four requirements for further environmental 

effects data: 

 “Submit field degradation studies evaluating accumulation and persistence of Cry3Bb1 

 in several different soils in various strata. Representative fields must have been planted 

 with MON 863 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 reached. 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 Cry3Bb1 is accumulating or persisting in soil samples...a final report is 

 due two years from the date of registration.” 
Bacillus thuringiensis Cry3Bb1 Corn 

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 “Submit laboratory toxicity tests with Orius insidiosus (minute pirate bug), carabid 

 (ground beetle), and Tetraopes (red milkweed beetle) within 24 months of the date of 

 registration....” 

 “Full-scale field or semi-field studies with appropriate end points and statistical power 

 must be conducted. Submit intermediate and multi-year non-target organism field 

 studies with statistical power. You must submit final results to field studies previously 

 summarized in MRID No. 456530-03....” 

 “Submit a six week broiler dietary study with 60%–70% MON 863 corn in the diet that 

 is of appropriate duration to represent the start and growing periods of the test species. 

 Balanced diets should be formulated according to the National Research Council 

 Guidelines (“Nutrient Requirements of Poultry,” Ninth Revised Edition, 1994) with the 

 energy requirements of the test species being met by the inclusion of corn in the diet 

 to assess hazards from chronic exposure of wild and domesticated fowl....” 

For the Corn Event MON 863 registration, the abovementioned requirements for additional 

environmental effects data have been satisfied by submission of appropriate studies and a request for a 

waiver from conducting the laboratory toxicity test with the Tetraopes; summaries of this information 

are presented in Table 6. 

Current ecological effects data, to include those conditional data referenced in Table 6, and EPA reviews 

of Cry3Bb1 protein support the Agency’s original determination that adverse effects will not occur to 

non-target organisms. Due to a demonstrated lack of toxicity and/or exposure, no effects from Cry3Bb1 

protein are anticipated for any non-target species, including federally listed threatened and endangered 

(“listed”) lepidopteran and coleopteran species and their designated critical habitats. The Agency is 

therefore upholding its determination that the registered uses of Cry3Bb1 will have “No Effect,” direct 

or indirect, on endangered or threatened terrestrial or aquatic species as listed by the U.S. Fish and 

Wildlife Service and the National Marine Fisheries Service. 

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 Agency 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 aquatic invertebrate data for the Cry34Ab1 and 

Cry35Ab1 proteins—either a 7- to 14-day D. magna study or a dietary study evaluating the effects of 

these proteins on an aquatic invertebrate that represents the functional group of a leaf 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 Peterson 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 
Bacillus thuringiensis Cry3Bb1 Corn 

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109 

literature review of these recently published studies. Criticisms of the Rosi-Marshall et al. study 

included several findings: (1) adverse effects were not caused by toxicity of Cry1A but, rather, by other 

differences between plant test substances (Jensen et al. 2010); (2) the abundance of Trichoptera in 

streams containing residues of Cry1A was not reduced (Chambers et al. 2007); and (3) while postharvest crop residue was identified as the most likely route of exposure (Carstens et al. 2010), aquatic 

exposure to biotech crops has been shown to be limited temporally and spatially with low to negligible 

exposure concentrations of Cry proteins in post-harvest crop tissues (Swan et al. 2009, Chamber 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 invertebrate studies to assess hazard to aquatic shredder 

species for existing Cry protein PIP registrations. 

Table 6. Environmental Effects Data/Information Submitted in Response to Conditions of 

 Registration for Corn Event MON 863. 

Study Title Summary MRID No.

Waiver Request from 

Conducting a 

Laboratory Toxicity 

Test with Tetraopes

(Red Milkweed Beetle) 

The Agency notes that further investigation of the biology and life cycle of 

the red milkweed beetle demonstrates that there will be little or no 

exposure of larvae under natural conditions. Although adults may be 

exposed to Bt corn pollen while feeding on milkweed leaves, Bt is typically 

less toxic to adults than larvae. Further, there is no protocol for rearing red 

milkweed beetles in the laboratory, and the development of such a 

laboratory assay would be difficult due to the red milkweed beetle’s long 

development time. Therefore, on October 1, 2003, the Agency granted 

Monsanto’s request to waive the requirement for conducting a red 

milkweed beetle study as set forth in the notice of registration (February 

24, 2003). 

(Unknown reference) 

N/A 

Comparison of Broiler 

Performance When Fed 

Diets Containing Events 

MON 863, Parental 

Line or Commercial 

Corn 

Day-old commercial broiler chickens (Ross x Ross) were fed diets 

containing either transgenic corn line MON 863 (containing Bacillus 

thuringiensis CRY3BB protein), a non-transgenic parental corn line, or a 

non-transgenic commercial corn reference line for 42 days. There were no 

treatment-related, biologically significant differences among groups for 

mortality; live weight; feed intake or efficiency; carcass weight; fat pad, 

breast meat, thigh, drum, or wing weights; or breast and thigh moisture, fat, 

and protein content. 

Classification: Acceptable 

(Reviewed in U.S. EPA (2010b)) 

459415-01 
Bacillus thuringiensis Cry3Bb1 Corn 

 Biopesticides Registration Action Document (BRAD) September 2010 

110

Study Title Summary MRID No.

Assessment of the 

Environmental Fate of 

the Cry3Bb1 Protein in 

Corn Fields Planted 

with MON 863 (Interim 

Report) 

This interim report summarizes study progress through 2003 and includes 

information concerning site selection, soil characterization assays, soil 

specimen collection, and agronomic activities that occurred in 2003. 

Analysis of soil specimens for the presence of Cry3Bb1 protein has not yet 

been performed. 

Classification: Acceptable 

(Reviewed in U.S. EPA (2006a)) 

462001-01 

Evaluation of Dietary 

Effects of Cry3Bb1 

Protein on the Ground 

Beetle, Poecilus 

chalcites (Coleoptera: 

Carabidae) 

First instar larvae of the ground beetle (Poecilus chalcites) were fed 

artificial diet containing Cry3Bb1 protein (930 μg/g), a 10 millimolar 

(mM) sodium carbonate/bicarbonate buffer control (0.147 milliliters per 

gram (mL/g)), a potassium arsenate reference material (200 μg/mL), or a 

diet-only negative control for 28 days. There were no statistically 

significant differences in survival, growth, or development among larvae in 

the Cry3Bb1, buffer control, or negative control groups. 

Classification: Acceptable 

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

464799-04 

Evaluation of Dietary 

Effects of a Cry3Bb1 

Protein Variant on 

Minute Pirate Bugs 

(Orius insidiosus) 

Orius insidiosus nymphs were exposed for 14 days to a Cry3Bb1 protein 

variant (930 μg active ingredient/g) diet, a potassium arsenate (8.9 μg 

active ingredient/g) positive control diet, an E64 protease inhibitor (53 

μg/g) positive control diet, a sodium carbonate/bicarbonate buffer control 

diet, or a negative control diet. There was no statistically significant 

difference in survival among the Cry3Bb1, negative control, or buffer 

control diet groups, while survival in the positive control diet groups was 

0%. The percent of nymphs developing to adults and the mean number of 

days to develop to adults were comparable among the Cry3Bb1, negative 

control, and buffer control diet groups. 

Classification: Acceptable 

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

464799-05 

Research on the Effects 

of Corn Rootworm 

Protected Transgenic 

Corn on Nontarget 

Organisms: Publications 

and Manuscripts 

Several studies were conducted between 2000 and 2002 to evaluate the 

effects of the transgenic corn MON 863, which contains Cry3Bb1 protein, 

on non-target organisms. These studies included broad field surveys of 

surface-dwelling and soil-dwelling arthropods and other invertebrates, field 

studies of the abundance and community structure of Collembola and 

carabids, and laboratory and field studies of soil mites. In general, no 

consistent adverse effects on non-target taxa were found, although most of 

the studies used small test plot sizes. 

Classification: Supplemental 

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

462627-02 
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 Biopesticides Registration Action Document (BRAD) September 2010 

111 

Study Title Summary MRID No.

Statistical Power 

Analysis of a Two-Year 

Field Study Evaluating 

the Ecological Effect of 

Corn Event MON 863 

The statistical power of a previous two-year field study (MRID No. 

457916-01) to evaluate the effects of MON 863 corn on terrestrial 

invertebrate populations was determined. For the analysis, a biologically 

significant effect was defined as a 50% difference in the mean abundance 

of individual invertebrate taxa between MON 863 and control corn plots, 

and a statistical power of ≥80% to detect the biological effect was desired. 

Single-year comparisons did not achieve the desired power level of ≥80% 

for detecting a 50% difference in population size for 38 of 68 (56%) 

comparisons made. An analysis of joint data for both years, however, 

found ≥80% power was obtained for 30 of 32 comparisons, and the 

remaining two reached 74% and 77%. The findings indicate that the 

experimental design used in the two-year field study was adequate to detect 

a 50% difference in abundance of invertebrates between plots of MON 863 

and conventional corn. 

Classification: Supplemental 

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

462627-03 

Supplemental Statistical 

Analysis of Data from a 

Two-Year Field Census 

Study with Corn Event 

MON 863 

The statistical power of a previously conducted two-year field study to 

evaluate the effects of MON 863 corn on terrestrial invertebrate 

populations was determined. For the analysis, a biologically significant 

effect was defined as a 50% difference in the mean abundance of 

individual invertebrate taxa between MON 863 and control corn plots, and 

a statistical power of ≥80% to detect that biologically significant effect was 

desired. A previous statistical power analysis (MRID No. 462627-03) 

showed that analyzing data for each of the two years separately failed to 

achieve the 80% level of power in more than half of the statistical 

comparisons between MON 863 and RX 670 plots. The present study 

repeated the statistical analysis with the data for both years pooled and 

analyzed jointly. Results showed that the joint analysis achieved a ≥80% 

power level for 30 of 32 group comparisons, sufficient to determine that 

the original field study design was adequate for its intended purpose. 

Classification: Supplemental 

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

463942-02 
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 Biopesticides Registration Action Document (BRAD) September 2010 

112

Study Title Summary MRID No.

Environmental Fate of 

Cry3Bb1 Protein in 

Corn Fields Planted 

with MON 863

Soil samples were collected from six field sites, representing seven 

different soil types, in six different U.S. Corn-Belt states. Prior to study 

initiation, none of the plots had ever been planted in MON 863. Sampling 

occurred at planting, 30, 60, and 90 days after planting, six weeks after 

harvest, and prior to the following year’s planting. Field treatments were 

the following: MON 863 corn with tillage; no-till MON 863 corn; RX670 

corn with tillage (negative control); or no-till RX670 corn (negative 

control). Soil samples that were collected from the treated plots before, 

during, and after corn production were analyzed for persistence and 

accumulation of Cry3Bb1 protein using ELISA (LOQ 0.1 μg/g soil) and a 

Colorado potato beetle (CPB) mortality bioassay (limit of detection (LOD) 

20 μg Cry3Bb1/g soil). ELISA did not detect Cry3Bb1 in any soil sample, 

and the CPB bioassay showed now statistically significant differences, 

between MON 863 and RX670 negative plots, that were attributable to the 

presence of Cry3Bb1. 

These results suggest that Cry3Bb1 protein did not persist or accumulate in 

soil to levels that could be detected by ELISA and/or affect the mortality of 

the Colorado potato beetle. If the field-based, three-year soil degradation 

study that is to be submitted in support of the MON 810 x MON 863 stack 

suggests that there is persistence and/or accumulation of Cry3Bb1 protein 

in soil samples, the MON 863 field-based soil degradation study should be 

repeated using soil samples collected from fields on which MON 863 has 

been grown for three consecutive years. 

Classification: Acceptable 

(Reviewed in U.S. EPA (2006b)) 

465103-01 
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 Biopesticides Registration Action Document (BRAD) September 2010 

113 

Study Title Summary MRID No.

Field Studies Assessing 

Arthropod Nontarget 

Effects in Bt Transgenic 

Crops 

This submission includes complete references and additional information 

that has become available on field data regarding possible impacts of Bt

corn on non-target invertebrates. Most papers focused on large-scale field 

studies using transgenic corn, including discussions of study design and 

non-target invertebrate populations. EPA has reviewed the submitted 

information and a discussion summary of each paper, relevant to MON 

863, is provided below. Overall, the published literature did not report any 

consistent adverse impacts on non-target invertebrates as a result of multiyear commercial Bt corn cultivation. Slight reductions in some invertebrate 

predator populations are seen; however, these are an inevitable result of all 

pest management practices, which tend to result in reductions in the 

abundance of the pests as prey. The continually expanding body of 

literature provides EPA, academia, and the public with a better 

understanding of the impact of transgenic crops on non-target organisms 

and provides useful information and considerations for those conducting 

large-scale field studies. 

(1) Bhatti et al. (2005a) compared ground-dwelling invertebrate abundance 

between Bt (MON863, Cry3Bb1) and non-Bt corn. Invertebrates were 

collected over a three year period using pan and pitfall traps. Of the 14-taxa 

collected, only two resulted in significant Bt treatment effects. However, 

the observed effects were not consistent and varied within and among 

years. 

(2) In a companion study to Bhatti et al. (2005a), Bhatti et al. (2005b) 

looked at the abundance of foliage-dwelling arthropods in Bt (MON863, 

Cry3Bb1) corn and non-Bt corn. Arthropods were collected over a threeyear period using sticky traps. No consistent adverse impacts of Bt corn on 

the relative abundance of seven orders and 17 families of arthropods were 

observed. 

(3) Bitzer et al. (2005) was conducted over two years and evaluated the 

diversity and abundance of surface-active and subsurface springtails 

(Collembola) in Iowa and Illinois. Springtails were collected using pitfall 

traps and coil cores in plots planted with Bt (Cry3Bb1) and non-Bt corn. 

Using several measurements of diversity, the authors found no significant 

differences in the abundance of individual species between Bt and non-Bt

plots. 

(Reviewed in U.S. EPA (2010d)) 

467129-01 
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 Biopesticides Registration Action Document (BRAD) September 2010 

114

3. MON 863 x MON 810 (OECD Unique Identifier: MON-ØØ863-5 x MON- ØØ81Ø-6) 

Expressing Cry3Bb1 and Cry1Ab 

 a. Data Cited/Submitted for Initial Registration of MON 863 x MON 810 

 (Prior to October 2003) (U.S. EPA 2003h)

MON 863 x MON 810 was produced by conventional breeding of single PIP trait corn lines MON 810 

(YieldGard® Corn Borer) and MON 863 (YieldGard® Rootworm). 

Non-target beneficial insect data were waived because the susceptibility of target pests to MON 863 x 

MON 810 is comparable to their susceptibility to the single trait Cry1Ab and Cry3Bb1 corn. Therefore, 

the non-target data and the environmental risk assessment for the single PIP trait corn lines are 

applicable to the MON 863 x MON 810 corn line (Cry3Bb1 – see section II(C)(2) of this BRAD; 

Cry1Ab – see U.S. EPA (2001b and 2010e)). Since there is no change in susceptibility among 

susceptible insects, then it is unlikely that there will be a difference in effects of the stacked versus 

single-trait hybrids on non-target insects. 

 i. Protein Interaction (MRID No. 460697-01)

Studies were conducted and submitted to test the hypothesis that the Cry1Ab and Cry3Bb1 proteins do 

not interact when combined in MON 863 x MON 810. It was concluded from leaf disk, whole plant, and 

in vitro studies with purified Bt protein that there are no interactive effects on susceptible insect pests 

when the Cry1Ab and Cry3Bb1 proteins are combined in MON 863 x MON 810. Since combining these 

proteins in MON 863 x MON 810 does not change the level of susceptibility of susceptible pests 

compared to the single-traits (MON 810 and MON 863 corn), it can be concluded that there will not be a 

difference for non-target insects not susceptible to the Cry1Ab or Cry3Bb1 proteins. As was required for 

the single PIP trait products, however, EPA is requiring non-target invertebrate field studies and Cry 

protein field degradation studies on MON 863 x MON 810. 

*Note for 2010: There is an update to this summary. See section II(C)(3)(b) (“Terms and Conditions of 

the MON 863 x MON 810 Registration (October 2003 – September 2010)”) of this BRAD. 

b. Terms and Conditions of the MON 863 x MON 810 Registration (October 2003 –

September 2010)

When MON 863 x MON 810 was first registered on October 31, 2003, the Agency issued a registration 

notice to Monsanto that, given the conclusions set forth in the initial environmental risk assessment (see 

section II(C)(3)(a) of this BRAD), contained the following two requirements for further environmental 

effects data: 

 “Submit small and large-scale field studies...conducted with YieldGard® 

 Plus Corn with appropriate end points and statistical power to verify 

 there are no adverse ecological effects to non-target invertebrate populations....” 
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 “Submit field degradation studies evaluating accumulation and persistence of 

 Cry1Ab and Cry3Bb1 from YieldGard® Plus Corn in several different soils 

 in various strata. Representative fields must have been planted with YieldGard® 

 Plus Corn for at least three consecutive years and include both conventional 

 tillage and no-till samples and be harvested under typical agronomic conditions. 

 Sampling must begin after three consecutive years of YieldGard® Plus Corn 

 planting and continue until the limit of detection is reached. 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 Cry1Ab and Cry3Bb1 are 

 accumulating or persisting in soil samples...a final report is due November 15, 2007.” 

For the MON 863 x MON 810 registration, the abovementioned requirements for additional 

environmental effects data have been satisfied by submission of appropriate studies; summaries of the 

studies are presented in Table 7. 
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Table 7. Environmental Effects Data Submitted in Response to Conditions of Registration for 

 MON 863 x MON 810 (Reviewed in U.S. EPA (2009)). 

Study Title Summary MRID No.

Two-Year Field 

Assessment of the 

Effect of Combined 

Trait Bt Corn MON 863 

x MON 810 on 

Nontarget Organisms 

Field trials were conducted in 2001 and 2002 at different sites each year in 

Warren County near Monmouth, Illinois. A split-plot design was used with 

four block replications. The main plots were MON 863 x MON 810, MON 

863, MON 810, and RX670, and the subplots contained four insect 

regimes: no insecticide, seed insecticide applied before planting, soil 

insecticide applied at planting, and foliar insecticide. Data were collected 

using pan traps (for sampling soil-dwelling invertebrates), pitfall traps (for 

sampling surface-dwelling invertebrates), and sticky traps (for sampling 

foliage-dwelling invertebrates). In addition, earthworm sampling was 

conducted by digging soil cores and sorting through soil samples; there 

were no significant effects of corn lines or insecticide regimes on the 

abundance of earthworms in both 2001 and 2002. The results of the twoyear field study consistently showed that the combined-trait Bt corn MON 

863 x MON 810 did not have any significant adverse effects on the 

abundance of diverse groups of non-target taxa including predators, 

parasitoids, and decomposers or detritivores in comparison to non-Bt corn 

(RX670), as well as the single-trait Bt corn controls (MON 863 and MON 

810). In contrast, insecticides applied under varying regimes had 

significant impacts not only on target and non-target corn pests, but also on 

diverse groups of predators, parasitoids, and decomposers. Although 

statistically significant interactions between corn lines, insecticide regimes, 

and/or dates were detected for a few non-target taxa, these interactions 

were not consistent between years and are not considered biologically 

relevant. The combined-trait Bt corn (MON 863 x MON 810), as well as 

the single-trait Bt corn lines, consistently exhibited little impact on the 

abundance and diversity of beneficial insects captured during the two-year 

field study. 

Classification: Acceptable 

472829-01 

Environmental Fate of 

the Cry3Bb1 and 

Cry1Ab Proteins in 

Corn Fields Planted 

with MON 863 x MON 

810 for Three 

Consecutive Years 

Field studies at six sites (AR, CO, IA, IL, MN, and NE) with a range of 

soil properties and two sampling depths (0–6 and 6–12 inches) had no 

detectable Cry3Bb1 or Cry1Ab protein in the soil after 3 years of 

continuous cropping with YieldGard Plus® Corn (Mon 863 x MON 810) 

as determined by ELISA analysis or soil dietary bioassay with CPB or 

European corn borer (ECB) neonates. This 3-year field study is consistent 

with earlier studies indicating no accumulation or persistence of the 

Cry3Bb1 and Cry1Ab proteins in soils. There is negligible potential for 

plant-produced Cry3Bb1 and Cry1Ab proteins to persist or accumulate in 

agricultural soils across cropping cycles with YieldGard Plus® Corn. 

Classification: Acceptable 

472829-02 
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4. MON 88017 (OECD Unique Identifier: MON-88Ø17-3) Expressing Cry3Bb1 and MON 88017 

x MON 810 (OECD Unique Identifier: MON-88Ø17-3 x MON- ØØ81Ø-6) Expressing 

Cry3Bb1 and Cry1Ab 

 a. Data Cited/Submitted for Initial Registrations of MON 88017 and MON 88017 x MON 810 

(Prior to December 2005)(U.S. EPA 2005a and 2005b)

i. Background

The Cry3Bb1 protein expressed in MON 88017 is a variant of the wild-type Cry3Bb1 protein from Bt

subsp. kumamotoensis that protects the roots of corn plants from feeding damage caused by the 

coleopteran pest, corn rootworm (Diabrotica spp.). Previously described product characterization 

studies (see section II(A)(4) of this BRAD) demonstrated the functional and physicochemical 

equivalence of the Cry3Bb1 protein expressed in MON 88017 with the Cry3Bb1 protein expressed in 

Corn Event MON 863. That is, the Cry3Bb1 proteins expressed in both Corn Event MON 863 and MON 

88017 share an amino acid sequence identity of >99.8%, differ from one another by only one of 653 

amino acids, express similar protein levels in plant tissues, show similar biological activity against 

larvae of the Colorado potato beetle and western corn rootworm, and exhibit comparable field efficacy 

against corn rootworm. Given the similarities between the two proteins, all environmental effects data 

submitted to support Corn Event MON 863 were bridged to MON 88017. 

ii. MON 88017 (Coleopteran-Active Cry3Bb1 Protein) Risk Assessment

For registration of MON 863 (also applicable to MON 88017), EPA reviewed studies conducted on 

representative non-target species with several Cry3Bb1 protein variants and performed risk assessments 

on plants, wild mammals, birds, fish, aquatic invertebrates, estuarine and marine animals, earthworms, 

terrestrial non-target insects (including honey bee adults and larvae, parasitic wasps, green lacewings, 

several lady beetle species, springtails (collembola toxicity/reproduction), and monarch butterflies), field 

evaluations of the effects of Cry3Bb1 exposure on non-target invertebrates, soil degradation/persistence, 

and endangered species (see section II(C)(2) of this BRAD). In addition, gene flow and weediness 

assessments, via pollen and Cry protein DNA uptake by plants and soil microorganisms, were also 

performed. The Agency has sufficient information to believe that there is no risk from the proposed uses 

of Cry3Bb1 corn to non-target wildlife, aquatic, and soil organisms. The Agency has requested 

additional data in hopes that it will provide weight to the some of the initial conclusions made regarding 

use of Cry3Bb1 corn. These additional data consist of long-range soil fate studies, long-range field 

effects on invertebrates studies, and toxicity studies on additional Coleoptera, specifically the ground 

beetle and the minute pirate bug. Whether any additional non-target or long-range field studies are 

required on MON 88017 will be determined by the results of these reviews. In the event that these 

studies sufficiently demonstrate a lack of long-range adverse effects, no additional data will be required. 

The evaluation of the submitted Cry3Bb1 long-range field studies will be based on recommendations 

from the August 27, 2002 FIFRA SAP. 
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*Note for 2010: There is an update to this summary. See section II(C)(4)(b) (“Terms and Conditions of 

the MON 88017 and MON 88017 x MON 810 Registrations (December 2005 – September 2010)”) of 

this BRAD. 

iii. MON 810 (Lepidopteran-Active Cry1Ab Protein) Risk Assessment

Likewise, the EPA has conducted an extensive review of effects of the Cry1Ab protein present in MON 

810 to non-target organisms in an ecological risk assessment as part of the reassessment of Bt plantincorporated-protectants (U.S. EPA 2001b). EPA reviewed studies conducted on representative nontarget species with Cry1Ab protein and performed risk assessments on plants, wild mammals, birds, fish, 

aquatic invertebrates, estuarine and marine animals, earthworms, terrestrial non-target insects (including 

honey bee adults and larvae, parasitic wasps, green lacewings, lady beetles, springtails (collembola 

toxicity/reproduction), monarch butterflies, and black swallow tail butterflies), field invertebrate 

abundance, soil degradation/persistence, and endangered species with special emphasis on the Karner 

blue butterfly. In addition, weediness and gene flow assessments, via pollen and Cry protein DNA 

uptake by plants and soil microorganisms, were also performed. The Agency concluded, considering all 

available information, the weight-of-evidence indicates no unreasonable adverse effects of Bt Cry 

proteins in plants to non-target wildlife, plants, or beneficial invertebrates (U.S. EPA 2001b). 

 iv. MON 88017 x MON 810 Corn (Cry3Bb1 x Cry1Ab Coleopteran/Lepidopteran-Active 

 Protein) Risk Assessment

The YieldGard® Plus Corn (Cry protein content equivalent to MON 88017 x MON810 corn) did not 

enhance or diminish European corn borer (ECB), corn earworm (CEW), fall armyworm (FAW), or 

southwestern corn borer (SWCB) leaf feeding damage compared to single-trait MON 810 corn 

containing the Cry1Ab protein in five in planta assays. YieldGard® Plus Corn also did not enhance or 

diminish western corn rootworm (WCRW) and southern corn rootworm (SCRW) larval feeding on roots 

compared to single-trait MON 863 corn containing the Cry3Bb1 protein. Leaf disk assays resulted in no 

difference in insecticidal activity against FAW between YieldGard® Plus Corn and single-trait MON 

810 corn. The presence of Cry3Bb1 in YieldGard® Plus Corn did not affect FAW nor did the presence 

of Cry1Ab affect Colorado potato beetle (CPB) in leaf disk assays. Insect bioassays conducted with 

purified protein verified that Cry3Bb1 will not affect ECB survival, and Cry1Ab will not affect CPB 

survival. LC50 values for ECB and CPB were similar for the single-trait hybrids (MON 810 and MON 

863) and dual-trait hybrid (Cry3Bb1 x Cry1Ab), and dose response curves did not differ. 

Collectively, these data provide evidence that the Cry1Ab and Cry3Bb1 proteins do not interact in an 

antagonistic, additive, or synergistic manner. Results of these assays verify that no interactive affects 

occur (which was expected), since different physiological conditions are needed for the Cry1Ab and 

Cry3Bb1 proteins to function. Protection against lepidopteran and coleopteran target pests were 

equivalent for the single-trait and stacked hybrids. Based on the lack of interactive effects on susceptible 

pests, it is extremely unlikely that the Cry3Bb1 and Cry1Ab proteins contained in a single plant will 

impart any safety concerns for non-target organisms exposed to these hybrids in the environment. 
Bacillus thuringiensis Cry3Bb1 Corn 

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119 

It can be concluded from the leaf disk, whole plant, and in vitro studies with purified Bt protein that 

there are no interactive effects on susceptible insect pests when the Cry1Ab and Cry3Bb1 proteins are 

combined in YieldGard® Plus Corn. Since combining these proteins in YieldGard® Plus Corn does not 

change the level of susceptibility of susceptible pests compared to single-trait MON 810 and MON 863 

corn, it can be concluded that there will not be a difference for non-target insects not susceptible to the 

Cry3Bb1 or Cry1Ab proteins. Therefore, development of a new set of non-target organism effects data 

were waived for MON 88017 x MON 810 corn. 

The Agency has sufficient information to believe that there is no risk from the proposed uses of MON 

88017 x MON 810 corn to non-target wildlife, aquatic, and soil organisms. The Agency has requested 

additional data on Cry3Bb1 in corn (Corn Event MON 863); these supplementary studies should provide 

weight to the Agency’s initial conclusions regarding the use of Cry3Bb1 corn. The additional data 

consist of long-range soil fate studies, long-range field effects on invertebrates studies, and toxicity 

studies on additional Coleoptera, specifically the ground beetle and the minute pirate bug. In the event 

that these studies sufficiently demonstrate a lack of long-range adverse effects, no additional data with 

MON 88017 x MON 810 will be required. Cry1Ab protein levels in MON 810 and MON 88017 x MON 

810 young root and forage root are required regardless of the outcome of the MON 863 studies being 

conducted to fulfill conditions of registration. 

*Note for 2010: There is an update to this summary. See section II(C)(4)(b) (“Terms and Conditions of 

the MON 88017 and MON 88017 x MON 810 Registrations (December 2005 – September 2010)”) of 

this BRAD. 

 b. Terms and Conditions of the MON 88017 and MON 88017 x MON 810 Registrations 

 (December 2005 – September 2010)

When MON 88017 and MON 88017 x MON 810 were initially registered on December 13, 2005, the 

Agency issued registration notices to Monsanto that contained the following requirements in relation to 

further environmental effects data: 

 For MON 88017 – 

 “Submit all data required to support the individual plant-incorporated protectant in 

 Event MON 863 (YieldGard Rootworm), 524-528. In the event that the Agency 

 concludes MON 863 (YieldGard Rootworm) studies do not sufficiently demonstrate 

 a lack of significant adverse effects, additional data with MON 88017 corn must 

 be submitted. These data may include a) laboratory toxicity testing with Orius 

 insidiosus (minute pirate bug), b) laboratory toxicity testing with a carabid (ground 

 beetle), c) long range effects testing on invertebrate populations in the field, and 

 d) long range soil persistence testing.” 
Bacillus thuringiensis Cry3Bb1 Corn 

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 For MON 88017 x MON 810 – 

 “Submit all data required to support the individual plant-incorporated protectant in 

 MON 810 (YieldGard), Event MON 863 (YieldGard Rootworm), MON 88017 corn 

 EPA Registration Nos. 524-489, 524-528. In the event that the Agency 

 concludes MON 863 (YieldGard Rootworm) studies do not sufficiently demonstrate 

 a lack of significant adverse effects, additional data with MON 88017 x MON 810 

 corn must be submitted. These data may include a) laboratory toxicity testing with Orius 

 insidiosus (minute pirate bug), b) laboratory toxicity testing with a carabid (ground 

 beetle), c) long range effects testing on invertebrate populations in the field, and 

 d) long range soil persistence testing.” 

 “Submit expression level data regarding Cry1Ab protein levels in MON 810 

 and MON 88017 x MON 810 young root and forage root within 12 months of the 

 date of registration.” 

All requirements for additional environmental effects data for MON 810 (see U.S. EPA (2010e)) and 

Corn Event MON 863 (see section II(C)(2)(b) of this BRAD), as set forth in the December 13, 2005 

registration notices, have been satisfied for both registrations (see Table 8). Moreover, after evaluating 

the conditional data submitted to support the registration of Corn Event MON 863, the Agency has 

concluded that these data do not demonstrate the potential for long-range adverse effects to the 

environment as a result of the cultivation of Cry3Bb1 corn. 

 

Table 8. Environmental Effects Data Submitted in Response to Conditions of Registration for 

 MON 88017 x MON 810 (Reviewed in U.S. EPA (2010a)). 

Study Title Summary MRID No.

Assessment of Cry1Ab 

Protein Levels in Corn 

MON 88017 x MON 

810 Root Tissue 

Produced in U.S. Field 

Trials in 2006 

A traditionally crossed corn hybrid of MON 88017 with MON 810 was 

grown along with conventional seed and MON 810 corn at five locations in 

2006 using a randomized complete block design and sampling scheme. 

Young root tissues were sampled at V2–V3 and forage root tissues at early 

dent or 1/3 milkline. Samples were stored and shipped on dry ice for 

Cry1Ab analysis of trypsinized, extracted tissues. Extraction efficiency was 

92%, spike recovery was 77%, and the trypsinization factor was 2. The 

coefficient of variation was 14% between assays. Limit of detection was 

0.13 g/g fresh weight, and limit for quantification was 0.40 g/g fresh 

weight. ELISA revealed mean Cry1Ab protein levels in MON 88017 x 

MON 810 corn tissues across all sites were 75 g/g dry tissue weight (dwt) 

in young root and 12 g/g dwt in forage root; similar to the mean Cry1Ab 

protein levels in MON 810 corn, which were 78 g/g dwt in young root 

and 13 g/g dwt in forage root. 

Classification: Acceptable 

470045-01 
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Number: WL-98-299: 16166. Unpublished study prepared by Monsanto Company and Wildlife 

International Limited, 41 pages. 

 

MRID No. 449043-15. Gallagher S, Grimes J, Beavers J. 1999. Bacillus thuringiensis Protein 11231 in 

Corn Grain: A Dietary Toxicity Study with the Northern Bobwhite. Lab Project Number: WL99-014: 16161: 139-44. Unpublished study prepared by Monsanto Company and Wildlife 

International Limited, 43 pages. 

MRID No. 449043-16. Hoxter K, Palmer S, Krueger H. 1999. Bacillus thuringiensis Protein 11231: An 

Acute Toxicity Study with the Earthworm in an Artificial Soil Substrate. Lab Project Number: 

WL-99-013: 16162: 139-443A. Unpublished study prepared by Monsanto Company and 

Wildlife International Limited, 45 pages. 

MRID No. 449043-17. Teixeira D. 1999. Assessment of Chronic Toxicity of Corn Tissue Containing the 

Bacillus thuringiensis Protein 11098 to Collembola (Folsomia candida). Lab Project Number: 

SB-98-296: 15988: 252.6149. Unpublished study prepared by Monsanto Company and 

Springborn Laboratories, 55 pages. 

MRID No. 449043-18. Drottar K, Krueger H. 1999. Bacillus thuringiensis Protein 11098 in Corn 

Pollen: A 48-Hour Static-Renewal Acute Toxicity Test with the Cladoceran (Daphnia magna). 

Lab Project Number: WL-98-295: 16163: 139A-236. Unpublished study prepared by Monsanto 

Company and Wildlife International Limited, 31 pages. 

MRID No. 449043-19. Meng L, Robinson E. 1999. Evaluation of Insect Protected Corn Lines MON 853 

and MON 859 as a Feed Ingredient for Catfish. Lab Project Number: XX-98-297: 16164. 

Unpublished study prepared by Monsanto Company and Mississippi State University, 32 pages. 
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MRID No. 451568-04. Martin J, McKee M, Dubelman S. 2000. Aerobic Soil Degradation of the B.t. 

Protein 11098 as a Component of Insect Protected Corn. Lab Project Number: XX-99-015: 

MSL-16440: 1281. Unpublished study prepared by PTRL East, Incorporated and Monsanto 

Company, 70 pages. 

MRID No. 453613-01. Duan J, Head G, McKee M. 2001. Dietary Effects of Transgenic Bacillus 

thuringiensis (Bt) Corn Pollen Expressing a Variant of Cry3Bb1 Protein on Adults of the 

Ladybird Beetle, Coleomegilla maculata. Lab Project Number: MSL-16936: 00-01-39-37. 

Unpublished study prepared by Monsanto Company, 35 pages. 

MRID No. 453613-02. Bryan R, Porch J, Krueger H. 2001. Dietary Effects of Transgenic Bt Corn 

Pollen Expressing a Variant of Cry3Bb1 Protein on Adults of the Ladybird Beetle, Hippodamia 

convergens – Final Report. Lab Project Number: WL-2000-158: MSL-17171: 139-453. 

Unpublished study prepared by Wildlife International Limited, 36 pages. 

MRID No. 455382-04. Duan J, McKee M, Nickson T. 2001. Dietary Effects of Transgenic Bacillus

thuringiensis (Bt) Corn Pollen Expressing a Variant of Cry3Bb1 Protein on Larvae of the 

Ladybird Beetle, Coleomegilla maculata. Lab Project Number: MSL-16907: 00-01-39-26. 

Unpublished study prepared by Monsanto Company, 39 pages. 

MRID No. 455382-05. Sears M, Mattila H. 2001. Determination of the Toxicity of Corn Pollen 

Expressing a Cry3Bb1 Variant Protein to First Instar Monarch Butterfly Larvae (Danaus 

plexippus) via Laboratory Bioassay. Lab Project Number: MSL-17235: 01-01-39-26. 

Unpublished study prepared by University of Guelph, 33 pages. 

MRID No. 455382-06. Bhatti M, Pilcher C, McKee M. 2001. Field Evaluation for the Ecological Impact 

of Corn Rootworm Insect-Protected Corn on Non-Target Organisms. Lab Project Number: 00-

01-39-16: MSL:17179. Unpublished study prepared by Monsanto Agronomy Center and 

Monsanto Company, 42 pages. 

MRID No. 455382-07. Head G, Pleau M, Sivausupramanian S. 2001. Insecticidal Spectrum of Activity 

for Cry3Bb Protein in vitro. Lab Project Number: C3NTO. Unpublished study prepared by 

Monsanto Company, 12 pages. 

MRID No. 455770-03. Duan J, McKee M, Head G. 2002. Endangered Species Impact Assessment for 

Cry3Bb1 Protein in Transgenic Corn Event MON 863. Lab Project Number: MSL-17614. 

Unpublished study prepared by Monsanto Company, 17 pages. 

MRID No. 456530-03. Head G. 2002. Research on the Effects of Corn Rootworm Protected Transgenic 

Corn Events on Nontarget Organisms: Preliminary Results. Lab Project Number: 00-CR-032E-7. 

Unpublished study prepared by Monsanto Company, 70 pages. 
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MRID No. 457571-01. Sindermann A, Porch J, Krueger H. 2002. Evaluation of a Cry3Bb1 Protein 

Variant in an Acute Toxicity Study with the Earthworm in an Artificial Soil Substrate. Lab 

Project Number: MSL-18137: 139-457: WL-2001-129. Unpublished study prepared by Wildlife 

International Limited and Monsanto Company, 42 pages. 

MRID No. 457571-02. Dubelman S, Ayden B, Mueth M. 2002. Aerobic Soil Degradation of the 

Bacillus thuringiensis Cry3Bb1 Variant Protein Produced by Corn Rootworm Protected Corn 

Event MON 863. Lab Project Number: 01-01-39-01: MSL-17102. Unpublished study prepared 

by Monsanto Company and Agvise Laboratories, 80 pages. 

MRID No. 457916-01. Bhatti M, Duan J, Pilcher C. 2002. Ecological Assessment for Non-Target 

Organisms in the Plots of Corn Rootworm Insect Protected Corn Hybrid Containing MON 863 

Event: 2000–2001 Field Trials. Lab Project Number: 00-01-39-16: MSL-17531. Unpublished 

study prepared by Monsanto Agronomy Center, AGVISE Laboratories, Monsanto Company, and 

Key Agricultural Services, Incorporated, 143 pages. 

MRID No. 459415-01. George B. 2001. Comparison of Broiler Performance When Fed Diets 

Containing Events MON 863, Parental Line or Commercial Corn. Lab Project Number: MSL17243: MSL-18126: MN-00-1. Unpublished study prepared by Colorado Quality Research, 

Incorporated, 172 pages. 

MRID No. 460697-01. Vaughn T, Brown C, McKee M. 2003. An Investigation into the Potential for 

Interactions Between the Cry1Ab and Cry3Bb1 Proteins Produced in YieldGard Plus Corn. 

Project Number: MSL/18726, 18726. Unpublished study prepared by Monsanto Company, 17 

pages. 

MRID No. 462001-01. Dubelman S, Bhatti M, Ayden B. 2004. Interim Report: Assessment of the 

Environmental Fate of the Cry3Bb1 Protein in Corn Fields Planted with MON 863. Project 

Number: 03/01/39/14, MSL/18931. Unpublished study prepared by Monsanto Agricultural 

Company, Agvise, Incorporated, and North Dakota State University, 70 pages. 

MRID No. 462627-02. Head G. 2004. Research on the Effects of Corn Rootworm Protected Transgenic 

Corn on Nontarget Organisms: Publications and Manuscripts. Project Number: 03/CR/097/13. 

Unpublished study prepared by Monsanto Company, 148 pages. 

MRID No. 462627-03. Duan J, Jiang C, McKee M. 2004. Statistical Power Analysis of a Two-Year 

Field Study Evaluating the Ecological Effect of Corn Event MON 863. Project Number: 

MSL/19246. Unpublished study prepared by Monsanto Company, 30 pages. 

MRID No. 463942-02. Duan J, Jiang C, Brown C. 2004. Supplemental Statistical Analysis of Data from 

a Two-Year Field Census Study with Corn Event MON 863. Project Number: MSL/19329, 

MSL/17531, 00/01/39/16. Unpublished study prepared by Monsanto Company, 43 pages. 
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MRID No. 464799-04. Duan J, Paradise M. 2005. Evaluation of Dietary Effects of Cry3Bb1 Protein on 

the Ground Beetle, Poecilus chacites (Coleoptera: Carabidae). Project Number: MSL/19631. 

Unpublished study prepared by Monsanto Company, 71 pages. 

MRID No. 464799-05. Teixeira D. 2005. Evaluation of Dietary Effects of a Cry3Bb1 Protein Variant on 

Minute Pirate Bugs (Orius insidiosus). Project Number: 525/6194, 04/01/39/23, MSL/19697. 

Unpublished study prepared by Springborn Smithers Laboratories, 69 pages. 

MRID No. 465103-01. Dubelman S, Bhatti M, Ayden B. 2005. Environmental Fate of the Cry3Bb1 

Protein in Corn Fields Planted with MON 863. Project Number: 03/01/39/14, MSL/19285. 

Unpublished study prepared by Monsanto Company, Agvise, Incorporated, and North Dakota 

State University, 230 pages. 

MRID No. 467129-01. Head G, Reding H. 2005. Field Studies Assessing Arthropod Nontarget Effects 

in Bt Transgenic Crops. Project Number: 95/152E. Unpublished study prepared by Monsanto 

Company, 202 pages. 

MRID No. 470045-01. Niemeyer K, Silvanovich A. 2006. Assessment of Cry1Ab Protein Levels in 

 Corn MON 88017 x MON 810 Root Tissue Produced in U.S. Field Trials in 2006. Project 

 Number: 06/01/50/10, 0020590. Unpublished study prepared by Monsanto Company, 20 pages. 

MRID No. 472829-01. Duan J, Bhatti M, Brown C. 2007. Two-Year Field Assessment of the Effect of 

Combined Trait Bt Corn MON 863 x MON 810 on Nontarget Organisms. Project Number: 

01/01/39/34, MSL 19696. Unpublished study prepared by Monsanto Research Farm, Key 

Agricultural Services, Incorporated, and Monsanto Company, 176 pages. 

MRID No. 472829-02. Dubelman S, Ayden B, Colyer J. 2007. Environmental Fate of the Cry3Bb1 and 

Cry1Ab Proteins in Corn Fields Planted with MON 863 x MON 810 for Three Consecutive 

Years. Project Number: 04/01/39/07, MSL0020589. Unpublished study prepared by Monsanto 

Agricultural Company, 432 pages. 

Muir WM, Howard RD. 2001. Fitness components and ecological risk of transgenic release: a model 

using Japanese medaka (Oryzias latipes). The American Naturalist 158:1–16. 

National Academy of Sciences (NAS). 2000. Environmental Effects of Transgenic Plants: The Scope 

and Adequacy of Regulation is available from the National Academies Press, 500 Fifth Street 

NW, Lockbox 285, Washington, D.C. 20055; (888) 624-8373 or (202) 334-3313 (in the 

Washington D.C. metropolitan area); http://www.nap.edu/. 

Oliveira AP, Pampulha ME, Bennett JP. 2008. A two-year field study with transgenic Bacillus 

 thuringiensis maize: Effects on soil microorganisms. Science of the Total Environment 405:351–

357. 
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Orzell S. 2000. Personal communication. Botanist/Ecologist, United States Air Force, Avon Park Air 

Force Range, Florida, 2000. 

Parrott W. 2008. Study of Bt impact on caddisflies overstates its conclusions: response to Rosi-Marshall 

et al. Proceedings of the National Academy of Sciences of the United States of America, pp. E10. 

Available from: http://www.ask-force.org/web/Bt/Parrott-Rosi-Marshall-2008.pdf. 

Read J. 2000. Personal communication. Professor, Texas Agricultural Experiment Station, Dallas, Texas 

(972-231-5362). 

Romeis J, Meissle M, Bigler F. 2006. Transgenic crops expressing Bacillus thuringiensis toxins and 

 biological control. Nature Biotechnology 24:63–71. 

Rosi-Marshall EJ, Tank JL, Royer TV, Whiles MR, Evans-White M, Chambers C, Griffiths NA, 

 Pokelsek J, Stephen ML. 2007. Toxins in transgenic crop byproducts may affect headwater 

 stream ecosystems. Proceedings of the National Academy of Sciences of the United States of 

 America 104(41):16204–16208. 

Rosi-Marshall EJ, Tank JL, Royer TV, Whiles MR. 2008. Reply to Beachy et al. and Parrott: study 

 indicates Bt corn may affect caddisflies. Proceedings of the National Academy of Sciences of the 

 United States of America, 105, 7, pp. E11–E11. Available from: 

http://www.ask-force.org/web/Bt/Rosi-Marschall-Bt-Aquatic-reply-2008.pdf. 

Sanvido O, Romeis J, Bigler F. 2007. Ecological impacts of genetically modified crops: ten years of 

field research and commercial cultivation. Advances in Biochemical Engineering and 

Biotechnology 107:235–278. 

Saxena D, Stotzky G. 2001. Bacillus thuringiensis (Bt) toxin released from root exudates and biomass of 

Bt corn has no apparent effect on earthworms, nematodes, protozoa, bacteria, and fungi in soil. 

Soil Biology and Biochemistry 33:1225–1230. 

Schoper J. 1999. Personal communication. Geneticist, Pioneer Hi-Bred International, Johnston, IA. 

 (515-270-3544). 

Smith JSC, Goodman MM, Stuber CW. 1985. Relationships between maize and teosinte of Mexico and 

Guatemala: Numerical analysis of allozyme data. Economic Botany 39:12–24. 

Swan CM, Jensen PD, Dively GP, Lamp WO. 2009. Processing of transgenic crop residues in stream 

ecosystems. Journal of Applied Ecology 46:1304–1313. 
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U.S. EPA. 1996. Hazard Evaluation Division, Standard Evaluation Procedure – Ecological Risk 

Assessment. Office of Pesticide Programs. 

U.S. EPA. 1998. “Guidelines for Ecological Risk Assessment.” EPA 630/R-95-002F. Washington, 

D.C., USA. [Federal Register, May 14, 1998. 63(93): 26846–26924.] 

U.S. EPA. 2000a. SAP Report No. 99-06. Sets of Scientific Issues Being Considered by the 

Environmental Protection Agency Regarding: Section I – Characterization and Non-Target 

Organism Data Requirements for Protein Plant-Pesticides. Dated February 4, 2000. Available 

from: http://www.epa.gov/scipoly/sap/meetings/1999/december/report.pdf. 

U.S. EPA. 2000b. Review of Ecological Non-Target Lady Beetle Effects for Monsanto’s EUP Request 

 to Allow Testing and Further Development of Their Bacillus thuringiensis Cry3Bb1 Field Corn. 

 Memorandum from R. Rose and Z. Vaituzis, Ph.D. to M. Mendelsohn dated March 10, 2000. 

U.S. EPA. 2001a. SAP Report No. 2000-07. Sets of Scientific Issues Being Considered by the 

Environmental Protection Agency Regarding: Bt Plant-Pesticides Risk and Benefit Assessments. 

Dated March 12, 2001. Available from: 

http://www.epa.gov/scipoly/sap/meetings/2000/october/octoberfinal.pdf. 

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. 2002a. Review of Ecological Non-Target Insect Studies for Bacillus thuringiensis Cry3Bb1 

Protein. Memorandum from R. Rose and Z. Vaituzis, Ph.D. to M. Mendelsohn dated May 20, 

2002. 

U.S. EPA. 2002b. Data Evaluation Report on MRID No. 456530-03 (“Research on the Effects of Corn 

 Rootworm Protected Transgenic Corn Events on Nontarget Organisms: Preliminary Results”). 

 Completed by R. Rose on July 9, 2002. 

U.S. EPA. 2002c. Review of Aerobic Soil Degradation Study Submitted by Monsanto Co. in Support of 

Registering Bacillus thuringiensis Cry3Bb1 Protein Expressed in Field Corn. Memorandum from 

R. Rose and C. Wozniak, Ph.D. to M. Mendelsohn dated July 10, 2002. 

U.S. EPA. 2002d. Review of Laboratory Pollen Feeding Studies on Two Species of Lady Beetles 

(Coleomegilla maculata and Hippodamia convergens) in Support of Monsanto’s Request to 

Register Bacillus thuringiensis (Bt) Cry3Bb1 Protein (Event MON 863) Expressed in Field Corn 

Pollen. Memorandum from R.I. Rose to M. Mendelsohn dated July 11, 2002. 
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U.S. EPA. 2002e. Preliminary Risk Assessment for Soil, Soil Surface, and Foliar Invertebrates for 

Bacillus thuringiensis Cry3Bb Protein. Memorandum from Z. Vaituzis, Ph.D. and R. Rose to M. 

Mendelsohn dated July 23, 2002. 

U.S. EPA. 2002f. SAP Meeting Minutes No. 2002-05. A Set of Scientific Issues Being Considered by 

the Environmental Protection Agency Regarding: Corn Rootworm Plant-Incorporated Protectant 

Non-Target Insect and Insect Resistance Management Issues. Dated November 6, 2002. 

Available from: http://www.epa.gov/scipoly/sap/meetings/2002/august/august2002final.pdf. 

U.S. EPA. 2002g. Review of Aerobic Soil Degradation Study Submitted by Monsanto Co. in Support of 

Registering Bacillus thuringiensis Cry3Bb1 Protein Expressed in Field Corn. Memorandum from 

R. Rose to M. Mendelsohn dated December 23, 2002. 

U.S. EPA. 2003a. Data Evaluation Report on MRID No. 449043-15 (“Bacillus thuringiensis Protein 

 11231 in Corn Grain: A Dietary Toxicity Study with the Northern Bobwhite”). Completed by G. 

 Tomimatsu, Ph.D. and Z. Vaituzis, Ph.D. on January 14, 2003. 

U.S. EPA. 2003b. Data Evaluation Report on MRID No. 449043-19 (“Evaluation of Insect Protected 

Corn Lines MON 853 and MON 859 as a Feed Ingredient for Catfish”). Completed by G. 

Tomimatsu, Ph.D. and Z. Vaituzis, Ph.D. on January 14, 2003. 

U.S. EPA. 2003c. Data Evaluation Report on MRID No. 449043-18 (“Bacillus thuringiensis Protein 

11098 in Corn Pollen: A 48-Hour Static-Renewal Acute Toxicity Test with the Cladoceran 

(Daphnia magna”). Completed by G. Tomimatsu, Ph.D. and Z. Vaituzis, Ph.D. on January 14, 

2003. 

U.S. EPA. 2003d. Supplemental Review of Ecological Assessment of Non-Target Organisms for 

Bacillus thuringiensis Cry3Bb1 Field Corn. Memorandum from R. Rose and Z. Vaituzis, Ph.D. 

to M. Mendelsohn dated January 14, 2003. 

U.S. EPA. 2003e. Data Evaluation Report on MRID No. 449043-16 (“Bacillus thuringiensis Protein 

11231: An Acute Toxicity Study with the Earthworm in an Artificial Soil Substrate”). Completed 

 by G. Tomimatsu, Ph.D. and Z. Vaituzis, Ph.D. on January 14, 2003. 

U.S. EPA. 2003f. Data Evaluation Report on MRID No. 457571-01 (“Evaluation of a Cry3Bb1 Protein 

Variant in an Acute Toxicity Study with the Earthworm in an Artificial Soil Substrate”). 

Completed by G. Tomimatsu, Ph.D. and Z. Vaituzis, Ph.D. on January 14, 2003. 

U.S. EPA. 2003g. Ecological Hazard Assessment for Bacillus thuringiensis Cry3Bb1 Protein. 

Memorandum from Z. Vaituzis, Ph.D. and R. Rose to M. Mendelsohn dated February 12, 2003. 
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U.S. EPA. 2003h. Review of Monsanto’s Application for a Section 3 Registration of Bacillus 

thuringiensis Cry1Ab and Cry3Bb1 Proteins and the Genetic Material Necessary for Their 

Production in YieldGard® Plus Corn. Memorandum from R. Rose and Z. Vaituzis, Ph.D. to M. 

Mendelsohn dated October 2, 2003. 

U.S. EPA. 2004a. 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/june/final1a.pdf. 

U.S. EPA. 2005a. Review of Product Characterization, Expression Analysis, and Human Health Data 

 for Plant-Incorporated Protectant Bacillus thuringiensis subspecies kumamotoensis Cry3Bb1 

 Insect Control Protein and the Genetic Material Necessary for Its Production (Vector ZMIR39) 

 in Maize (Corn) Plants Derived from Event MON 88017 (EPA Reg. No. 524-LLR) in Support 

 for Sec. 3 Registration. A. Fellman and J.L. Kough, Ph.D. to M. Mendelsohn dated July 28, 

 2005. 

U.S. EPA. 2005b. Environmental Effects Assessment of MON 88017 and the Stacked MON 88017 x 

MON 810 Bt Corn. Memorandum from T. Milofsky and Z. Vaituzis, Ph.D. to M. Mendelsohn 

dated October 25, 2005. 

U.S. EPA. 2006a. Review of the Interim Report Submitted in Support of Monsanto’s Cry3Bb1 (EPA 

Reg. No. 524-528) Soil Fate Study. Memorandum from T. Milofsky and Z. Vaituzis, Ph.D. to M. 

Mendelsohn dated March 28, 2006. 

U.S. EPA. 2006b. Review of the Soil Fate Study (MRID No. 465103-01) That Was Required as a 

Condition of Registration for Monsanto’s Cry3Bb1 (EPA Reg. No. 524-528) Corn. 

Memorandum from T. Milofsky and Z. Vaituzis, Ph.D. to M. Mendelsohn dated November 15, 

2006.. 

U.S. EPA. 2009. Review of the Non-Target Organism Field Study and 3-Year Soil Fate Study (MRID 

Numbers 472829-01 and 472829-02) That Were Required as a Condition of Registration for 

Monsanto’s YieldGard Plus Corn (MON 863 x MON 810). Memorandum from A. Gross and Z. 

Vaituzis, Ph.D. to M. Mendelsohn dated July 23, 2009. 

U.S. EPA. 2010a. Expression Level Data for Cry1A(b) from MON 88017 x MON 810 Corn. 

 Memorandum from J.V. Gagliardi, Ph.D. and J.L. Kough, Ph.D. to M. Mendelsohn dated June 

 24, 2010. 

U.S. EPA. 2010b. Data Evaluation Report on MRID No. 459415-01 (“Comparison of Broiler 

 Performance When Fed Diets Containing Events MON 863, Parental Line or Commercial 

 Corn”). Completed by G. Tomimatsu, Ph.D. on June 30, 2010. 
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U.S. EPA. 2010c. Review of Orius (MRID No. 464799-05) and Carabid (MRID No. 464799-04) Insect 

 Studies Submitted as a Condition of Registration for Monsanto Maize Event MON863 Cry3Bb1 

 (EPA Reg. No. 524-528) and Supporting Published Literature on the Effects of Corn Rootworm 

 Protected Transgenic Corn on Nontarget Organisms (Including Statistical Power Analyses) 

 (MRID Numbers: 463942-02, 462627-03, 462627-02). Memorandum from Z. Vaituzis, Ph.D. to 

 M. Mendelsohn dated July 7, 2010. 

U.S. EPA. 2010d. Review of ABSTC Submission, “Field Surveys of Non-Target Invertebrate 

 Populations in Bt Corn.” Memorandum from Z. Vaituzis, Ph.D. to M. Mendelsohn dated July 7, 

 2010. 

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 Materials” 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 Tolerant Corn Transformation Event CBH- 351: 

Environmental Assessment. USDA, APHIS, Riverdale, Maryland. 

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. Professor of Biology, Texas A&M University, College 

Station, Texas (409-845-3354). 

Wolfenbarger LL, Naranjo SE, Lundgren JG, Bitzer RJ, Watrud LS. 2008. Bt effects on functional 

 guilds of non-target arthropods: a meta-analysis. PLoS ONE 3(5):e2118. 

http://dx.doi.org/10.1371/journal.pone.0002118. 

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. Professor of Botany, Institute for Systematic Botany, 

University of South Florida, Tampa, Florida (813-974-2359). 
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D. Insect Resistance Management (IRM) 

1. Background

Corn expressing the Cry3Bb1 protein provides protection against certain species of the corn rootworm 

(CRW), including western corn rootworm (WCRW, Diabrotica virgifera virgifera), northern corn 

rootworm (NCRW, Diabrotica barberi), and Mexican corn rootworm (MCRW, Diabrotica virgifera 

zea). In order to delay the onset of insect resistance to Cry3Bb1 corn, an acceptable IRM plan is 

necessary. The IRM plan consists of the following: (1) a 20% structured refuge placed adjacent to or 

within the YieldGard® Rootworm corn (MON 863) field, in-field strips must be at least four rows wide; 

(2) a resistance monitoring program; (3) a remedial action plan; and (4) an IRM grower compliance and 

education program. The Environmental Protection Agency (EPA) also required that Monsanto Company 

(“Monsanto”) provide to the Agency annual resistance monitoring, compliance (and education), and 

sales reports. 

 

2. MON 863 (YieldGard® Rootworm; EPA Reg. No. 524-528) 

 a. Regulatory Background 

MON 863 was registered for commercial use for the 2003 growing season and is targeted against corn 

rootworm (CRW, Diabrotica spp.). Prior to registration, Monsanto submitted several documents in 

support of an interim Cry3Bb1 IRM plan. An IRM plan for MON 863 corn dated June 20, 2000 was 

submitted to the Agency (Master Record Identification Number (MRID No.) 451568-05). This 

submission included information on dose, CRW biology, simulation models of resistance development, 

and grower surveys. Research reports and results of grower surveys were also included in the appendices 

of the June 2000 submission. An amended IRM plan dated January 8, 2002 was submitted to the Agency 

for review (MRID No. 455770-01). The amended plan titled “An Interim Insect Resistance Management 

Plan for Corn Event MON 863: A Transgenic Corn Rootworm Control Product” was intended to 

supersede MRID No. 451568-05. Therefore, MRID No. 451568-05 was used for additional information 

and as reference material but was not formally reviewed (see review of interim IRM plan in U.S. EPA 

(2002a)). An additional preliminary research report dated February 20, 2001 was submitted to the 

Agency by Monsanto (MRID No. 453484-01). 

A Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) Scientific Advisory Panel (SAP) was 

convened in August 2002. The August 2002 SAP comments regarding Monsanto’s interim IRM plan 

were documented in a memorandum from Paul Lewis to Marcia Mulkey dated November 6, 2002 (U.S. 

EPA 2002b). In response to the SAP, Monsanto submitted additional information to EPA in a document 

from Dennis Ward to Janet Andersen dated December 13, 2002 (Ward 2002). This additional 

information, along with additional clarifications provided to the Agency by Dr. Michael Caprio on 

December 20, 2002, Dr. David Andow on December 23, 2002, and Dr. Fred Gould on February 12, 

2003, was incorporated into the final review (U.S. EPA 2003a). 
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As terms and conditions of registration in 2003, Monsanto was required to submit resistance allele 

frequency data and additional IRM research data to increase the understanding of corn rootworm 

biology and other factors related to resistance management. The following areas of research were to be 

addressed: 

 Research regarding adult and larval movement and dispersal, mating habits, ovipositional 

patterns, number of times a female can mate, and fecundity;

 Research to determine if IRM strategies designed for WCRW and NCRW are appropriate for 

MCRW;

 Research regarding the mechanism of potential resistance of CRW to MON 863 is necessary to 

develop an appropriate long-term IRM strategy. Monsanto must attempt to develop resistant 

CRW colonies to aid in determining selection intensity;

 Research regarding the effect of WCRW ovipositing in soybean prior to overwintering and 

extended diapause in NCRW on an IRM strategy needs further investigation;

 Detailed summaries of the four data-sets identified in Monsanto’s December 13, 2002 letter 

should be submitted to the Agency to support their conclusion that the initial resistance allele 

frequency is ≤ 0.01;

 Baseline susceptibility studies currently underway should be continued for WCRW and initiated 

for NCRW and monitoring techniques, such as discriminating dose concentration assays, need to 

be thoroughly investigated for their feasibility as resistance monitoring tools.

Monsanto was to provide protocols for the proposed research (within 90 days of the date of registration) 

and a progress report by January 31, 2004. A final report was to be submitted by January 31, 2006. 

Monsanto submitted 12 protocols for review (submitted May 23, 2003; no MRID No.) and a progress 

report (submitted January 30, 2004; MRID No. 461865-01) covering the first four research areas; both 

the protocols and the 2004 progress report were found to be acceptable (U.S. EPA 2004c). 

In April 2005, Monsanto submitted a second progress report, providing the Agency with an update of 

the ongoing insect resistance management research. This second progress report (MRID No. 466066-01) 

was found to be acceptable (U.S. EPA 2006b). The final report (summarized below) was submitted 

January 24, 2006 and was reviewed in U.S. EPA (2006c). 

Monsanto submitted a full report on four data sets regarding the initial Cry3Bb1 resistance allele 

frequency in CRW. Although this report satisfied the condition of registration, there is still significant 

uncertainty regarding the mode of action of Cry3Bb1 corn, the nature of potential CRW resistance, and 

the frequency of resistance allele(s) in pest populations (U.S. EPA 2004d). 
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Summary of Final IRM Research Submissions 

Monsanto submitted fourteen studies to provide additional information on the biology and ecology of 

the corn rootworm pest complex. They are divided into the following subject areas: CRW larval 

movement (Hibbard et al. 2003; Hibbard et al. 2004; Hibbard et al. 2005), CRW adult movement 

(Spencer et al. 2003; Kim and Sappington 2005), CRW hosts (Clark and Hibbard 2004; Oyediran et al. 

2004; Wilson and Hibbard 2004), rotation-resistant WCRW (Rondon and Gray 2004; Crowder et al. 

2005; Onstad et al. 2003), extended diapause NCRW (Mitchell and Onstad 2005), and baseline 

susceptibility of CRW to Cry3Bb1 (Siegfried et al. 2005). Data from the CRW larval movement studies 

have been used to make changes to the in-field refuge strip width requirement for YieldGard® 

Rootworm, YieldGard® Plus, MON 88017, and MON 88017 X MON 810 ( i.e., in-field strip width was 

changed from at least 6 to 12 rows wide to at least 4 rows wide). A list of the literature citations for the 

studies is provided in the “References” section of this IRM chapter. Findings from these studies are 

incorporated into the next section of this document, “Corn Rootworm Biology and Factors Related to 

Resistance Management.” 

 b. Corn Rootworm Biology and Factors Related to Resistance Management

 i. Pest Biology (Information Considered in the Original Risk Assessment) 

This section contains data and information reviewed as part of the original IRM risk assessment for 

MON 863 corn. Pest biology data that were required as a condition of registration are discussed in the 

subsequent section of this document. 

In order to develop an appropriate IRM strategy for MON 863 corn, as well as all insect-protected 

transgenic crops, it is important to consider the biology of the target pest. Knowledge of pest biology is 

imperative in determining optimal size and placement of refuges that will encourage random mating 

between pests in Bt and non-Bt corn fields. Based on the movement of CRW adults, a non-Bt corn 

refuge should be planted adjacent to or within MON 863 fields. 

Characteristics of pest biology that are relevant to IRM (e.g., movement, feeding habits, and 

ovipositional habits) differ for WCRW and NCRW. WCRW and NCRW adults will feed on corn silks, 

pollen, and young kernels in the ear tip; however, only WCRW feed on leaves. Since NCRW adults do 

not feed on corn leaves, they leave the field after pollination to find a field with pollen available 

(Branson and Krysan 1981). Since adult and larval CRW feed on various parts of the corn plant, both 

life stages may be exposed to the Bt protein and extended selection pressure may result (Meinke et al. 

2001). Severe root damage from larval feeding will lead to plant lodging (where damaged corn stocks 

fall over making mechanical harvesting impossible) and yield losses. 

WCRW and NCRW are univoltine in most of the Corn Belt (Branson and Krysan 1981; Meinke et al. 

2001). CRW typically oviposit where the adults are feeding, which is almost exclusively in corn fields 

(Branson and Krysan 1981; Levine and Oloumi-Sadeghi 1991). In general, CRW adult emergence varies 

based on species, geography, weather, management practices (such as insecticide use), population 
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density, and sex. For instance, males typically emerge before females, and emergence, as well as 

fecundity, longevity, and egg viability, are reduced in corn planted later in the season (Boetel and Fuller 

1997; Levine and Oloumi-Sadeghi 1991; Meinke et al. 2001). It is unknown what effect corn rootwormprotected transgenic corn will have on phenology, sex ratio, and adult emergence patterns. 

Asynchronous adult emergence for Bt corn fields and non-Bt refuges may lead to nonrandom or 

assortative mating, which may lead to an increase in the rate of resistance evolution. Nonrandom or 

assortative mating may also occur if Bt corn disrupts the synchrony of male and female CRW adult 

emergence (Meinke et al. 2001). Mating typically occurs within 24 to 48 hours of female adult 

emergence within the corn fields they emerged from or nearby (Meinke et al. 2001). 

CRW larval movement is limited, particularly in areas with low population densities (Meinke et al. 

2001). Published and unpublished articles have reported varying distances that CRW larvae move. 

WCRW larvae may move from 12 to 16 inches and have been found in corn rows planted up to 40 

inches apart (Suttle et al. 1967; Short and Luedtke 1970; Gray 1999). These studies suggest that CRW 

larvae hatching from eggs between rows are capable of finding and injuring corn roots regardless of row 

spacing. Since field corn is typically planted approximately 24 to 30 inches apart, CRW may move up to 

two rows according to current research. Additional information is needed, however, to verify the 

distance CRW larvae move within and between rows. In general, young CRW larvae (e.g., 1st, 2nd, and 

sometimes 3rd instars) tend to move toward actively growing corn roots. Larval movement toward 

respiring, growing corn roots is probably because of their ability to detect and move toward carbon 

dioxide sources (Strnad and Bergman 1987; Gray 1999). Young larvae will feed on the distal portion of 

corn roots and move through the soil to feed on new, short roots as they develop into later instars (Strnad 

and Bergman 1987; Gray 1999). It is possible, therefore, that a RS heterozygous larva with a partially 

recessive resistance trait will begin feeding on transgenic corn roots and finish its development on 

adjacent non-transgenic roots, which would result in a non-lethal dose of MON 863 and potential 

survival of that larva. 

NCRW- and WCRW-mated adults may be very mobile and have potentially high dispersal capabilities 

(Meinke et al. 2001). Nonetheless, local dispersal is more common and involves movement within or 

among adjacent fields; migratory dispersal over long distances occurs in a small portion of individuals 

and usually involves females (Meinke et al. 2001). Dispersal capabilities of the WCRW are greater than 

the NCRW. The WCRW is also a greater competitor and displaced the NCRW in Nebraska by 1980 

(Hill and Mayo 1980). WCRW post-mating dispersal may be local or migratory. Published data suggest 

that some WCRW females may leave the field after mating to oviposit elsewhere (Coates et al. 1986). 

While sustained flights by mated female CRW are possible, movement by unmated females is limited. 

Knowledge of the maximum and average distance an adult CRW moves is limited. Additional research, 

regarding adult and larval WCRW and NCRW dispersal potential, is needed to determine placement of 

non-Bt corn refuges. 

Additional information was required on various aspects of CRW pest biology as it relates to a long-term 

IRM strategy. Characteristics of pest biology that are relevant to IRM (e.g., movement, feeding habits, 

and ovipositional habits) differ for WCRW and NCRW; therefore, additional information on the biology 
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of the WCRW and NCRW was needed. According to the August 2002 SAP, the WCRW and MCRW 

are subspecies and much of the data collected on biology will relate to both species. The Panel 

concluded, however, that data on adult mating behavior, male and female migration, and reproductive 

biology and fecundity of females would be needed to determine if the IRM plan is suitable for MCRW. 

Although the SAP concluded that the same IRM strategy may be appropriate for the WCRW and 

NCRW, the Panel recommended additional research on the NCRW and suggested collecting data from 

several geographic locations of the WCRW. There are behavioral differences in WCRW populations 

from the western and eastern regions of their distribution. Thus, studies on aspects of pest biology (e.g., 

movement) should be conducted in several areas. Since the biology of the southern corn rootworm 

(SCRW, Diabrotica undecimpunctata howardi) is very different from the other Diabrotica spp. and it is 

not adequately controlled by MON 863, SCRW should not be considered. 

The August 2002 SAP identified several areas of additional research needed to fully understand CRW 

biology as it relates to an IRM strategy. The SAP concluded that male and female adult movement, as 

well as fitness in MON 863 and non-transgenic corn, should be evaluated in large-scale field studies. 

Data needed on movement include, but are not limited to, the distance males and females will move over 

time and the rate adults leave the natal field. Research may also be needed on the movement of NCRW 

male and female adults since little is currently known. The SAP also recommended an evaluation of “the 

impact of adult density on migration patterns of adults, whether a delay in male emergence from MON 

863 affects male fitness and lowers the chances of mating, and whether there are sublethal effects of 

MON 863 on female fecundity, offspring quality and other fitness parameters.” 

The NCR-46 (a technical committee consisting of research and extension CRW specialists and other 

cooperators) submitted a letter dated May 29, 2001 to the EPA that outlines additional CRW biology 

research. The August 2002 SAP recommended that the EPA consider the recommendations made by the 

NCR-46. 

Based on reviews of available data and the SAP and NCR-46 recommendations, the Agency concluded 

that more information on CRW movement, host utilization, mating habits, ovipositional patterns, the 

number of times a female can mate, and fecundity would be useful for CRW IRM. In addition, further 

investigation of the IRM impacts of behavioral resistance adaptations, including the effects of WCRW 

oviposition in soybean prior to overwintering and extended diapause in NCRW, was recommended. In 

previous submissions (Master Record Identification Numbers (MRID Nos.) 453484-01 and 455770-01), 

Monsanto listed and summarized studies underway at the time of the MON 863 registration application 

that related to the biology of CRW. Additional CRW biology, ecology, and genetics data were required 

as conditions of the MON 863 (YieldGard® and YieldGard® Plus) registrations. Results of these 

studies, as well as additional research, were submitted to EPA after registration and are summarized in 

the next section. 
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 ii. Pest Biology (Conditionally Required Studies)

As a condition of registration, Monsanto submitted fourteen studies to provide additional information on 

the biology and ecology of the corn rootworm pest complex. These data address the following subject 

areas: (1) CRW larval movement; (2) CRW adult movement; (3) CRW hosts; (4) rotation-resistant 

WCRW; (5) extended diapause NCRW; and (6) baseline susceptibility of CRW to Cry3Bb1 (discussed 

in the resistance monitoring section). Further information on some of the research objectives (i.e., 

applicability of the IRM plan to MCRW and development of resistant colonies) was provided in 

progress reports. The Agency has reviewed these data (and other published studies) and summarized the 

findings by discipline in this section. 

CRW Larval Movement 

Larval movement data published by Hibbard et al. (2003) show that between 0.75% and 6% of larvae 

moved across corn rows. This represents a relatively high-end estimate of the number of larvae that 

cross rows. This means that much narrower in-field strips should be sufficient to provide adequate 

protection from sublethal selection caused by CRW larval movement across rows and maintain low 

functional recessiveness. Any increase in sublethal selection would be offset by a greater probability that 

potentially resistant adults emerging from the Bt corn rows would be mated by susceptible adults from 

the refuge row. Single-row strips would likely be too narrow and allow too much larval movement 

across rows to sufficiently maintain low functional recessiveness. Therefore, in-field strips of ≥4 row 

strips should provide sufficient CRW resistance management within the field based both on the 

consideration of the current understanding of larval movement and selection, as well as grower 

feasibility, practicality, consistency, and compliance. 

CRW larval movement is density dependent at high infestation levels (Onstad et al. 2006). According to 

Hibbard et al. (2004), however, density-dependent dispersal for neonates did not occur during a threeyear field study, even under high-density pressure (with artificial infestation). Subsequent investigation 

by Hibbard et al. (2005) found that late-instar movement occurred from sufficiently damaged non-Bt

plants to surrounding transgenic plants. Work by Dr. Hibbard indicates that it is more realistic to assume 

high larval movement from late instars and little to no movement for neonates. On the other hand, 

statistically significant neonate movement from Bt plants to non-Bt plants was observed in one year of 

their field study (Hibbard et al. 2005). 

Adult CRW Movement

Spencer et al. (2003) developed a new technique that used ingested transgenic corn tissue as a marker 

for measuring movement of CRW adults between corn and soybean fields. This method used lateral 

flow strips to detect the Cry3Bb1 protein in the gut of insects that had ingested YieldGard® Rootworm. 

Insects feeding on YieldGard® Rootworm could be detected for at least 16 hours after feeding but not 

32 hours. This technique allows the impact of factors, such as temperature, precipitation, and wind 

speed, on short-term adult movement to be studied. Spencer et al. (2003) found that 85.3% of males and 
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females moved ≤4.6–9.1 meters per day (m/d) through R2-R3 stage corn. For Cry3Bb1-positive adults 

that moved out of corn fields into an adjacent soybean field, 86.4% of males and 93.1% of females 

moved ≤4.6–9.1 m/d through soybean. Data suggest that plant-to-plant movement was motivated by a 

search for food and was density dependent because plant damage was density dependent. This technique 

is a tool to better estimate rates of beetle movement away from transgenic corn than currently employed 

techniques, such as fluorescent powder mark-recapture methods, and offers an opportunity to better 

study dispersal of CRW and other insect herbivores. 

Separately, Nowatzki et al. (2003a and 2003b) conducted a field study (mark-recapture) with WCRW 

using a rubidium labeling technique to quantify short-range, in-field movement of adult beetles from the 

time of their emergence. The data (from trials in Nebraska) showed that males moved an average of 13.9 

m/d prior to female emergence and 15.2 m/d during peak female emergence (data were pooled across 

two years). Females moved on average 1.9 m/d during peak female emergence and 13.1 m/d during 

post-female emergence (data pooled across years). 

Kim and Sappington (2005) studied the population genetic structure of 10 western corn rootworm 

populations (595 individuals sampled) from nine U.S. states (western Texas and Kansas to New York 

and Delaware) based on microsatellite loci analysis. These researchers found that all populations 

exhibited high levels of genetic diversity, with the mean allelic diversity ranging from 7.3 to 8.6 and 

mean expected heterozygosity ranging from 0.600 to 0.670. Little genetic differentiation, as a whole, 

was observed across the geographic range sampled, with a global fixation index (Fst) of 0.006. Pairwise 

Fst estimates also indicated little genetic differentiation. The researchers concluded that the western corn 

rootworm population had not had sufficient time for substantial genetic structuring since its recent 

eastward range expansion from the Great Plains approximately 50 years ago. 

CRW Hosts 

A variety of grass species were studied to determine their suitability as a refuge host for corn rootworms. 

Several prairie grasses and forage grasses were shown to support larval growth of western corn 

rootworm larvae (Clark and Hibbard 2004; Oyediran et al. 2004; Wilson and Hibbard 2004) and, 

therefore, may serve as additional refuge to the structured non-Cry3Bb1 corn refuges. This additional 

source of refuge could reduce the risk of Cry3Bb1 resistance evolving. 

Clark and Hibbard (2004) examined larval survivorship and growth parameters of western corn 

rootworm on the roots of 29 plant species comprised of maize, maize-field weeds, native prairie grasses, 

forage grasses, and small grain crops. Adults were recovered from five plants species in addition to 

maize, and larvae survived at least 6 days after infestation on 27 species and 24 days on 23 plant species. 

Oyediran et al. (2004) evaluated 21 prairie grass species as larval hosts of western corn rootworm. 

Maize and sorghum were included as positive and negative controls, respectively. Overall, adults were 

produced from 14 of 23 species evaluated. 
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Wilson and Hibbard (2004) monitored larval development and survivorship of western corn rootworm 

on 22 plant species, including maize, maize-field weeds, and selected native prairie grasses, fencerow/forage grasses, and small grain crops planted in greenhouse trials. Adults were recovered from 10 

species. Larvae survived at least 14 days on 21 species and 26 days on 18 species. 

The potential for rootworm larvae to move between weeds within or adjacent to a maize field could be 

an important factor in resistance management of transgenic-rootworm maize. The long-term implication 

of such movement for a low-dose transgenic event has not yet been worked out. 

Rotation-Resistance in WCRW 

Traditionally, farmers rotate the planting of corn and soybeans in a field as a means to manage corn 

rootworm. In recent years, however, the effectiveness of this practice has been diminished because of a 

soybean-variant of the corn rootworm that also deposits eggs in soybean fields. Rondon and Gray (2004) 

studied the oviposition patterns of the soybean-variant and found that, although corn can be the preferred 

oviposition site among crops, similar egg densities can be found in soybean and oat stubble. These 

results indicate that producers who choose to rotate corn with soybean or other crops, such as alfalfa, 

may be at risk to economic larval injury to corn roots. 

Crowder et al. (2005) modeled pest management strategies from both a biological and an economic 

perspective. Based on these modeling efforts, greater doses were the most effective at preventing 

resistance to transgenic corn with the standard management strategies. This was especially true in areas 

without rotation-resistant phenotypes. Returns with the dynamic adoption strategies were always similar 

when compared with the standard strategy with a medium or greater dose. If the pest management 

industry can achieve a high dose of toxin, farmers can plant 80% of their corn fields to a transgenic 

cultivar with confidence that this strategy will be beneficial biologically and economically. Results 

indicate that, in areas without rotation-resistance, planting 80% transgenic corn (required refuge is 20%) 

in the continuous corn field each year generated the greatest economic returns with a medium toxin dose 

or greater. Where rotation-resistance was a problem, planting transgenic corn in the rotated corn field 

was the most effective strategy. These results support the current IRM strategy for MON 863. 

Onstad et al. (2003) also modeled management strategies for rotation-resistance over a 15-year 

timeframe and concluded that using corn rootworm-resistant corn was an economically valuable 

approach using a 2-year or 3-year rotation strategy. 

Overall, these studies indicate that YieldGard® Rootworm corn can be a useful tool for managing 

rotation-resistance. Furthermore, the existing IRM plan, using a 20% refuge for YieldGard® Rootworm, 

should be appropriate whether or not rotation-resistant corn rootworms are present. 
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Extended Diapause in NCRW 

Some northern corn rootworm populations have developed an extended diapause period, resulting in 

synchronization of egg hatch with the planting of corn and thus circumventing crop rotation 

management strategies. Mitchell and Onstad (2005) developed a population genetics model to study the 

impact of northern corn rootworm populations with extended diapause on current IRM strategies. The 

model produced mixed results depending on various other factors, such as insecticide use and farmer 

practices, but overall showed that extended diapause tended to reduce the rate of resistance evolution. 

No changes to the existing IRM plan for YieldGard® Rootworm corn were indicated by these modeling 

efforts. 

Applicability of the IRM Strategy to Mexican Corn Rootworm 

Although this research requirement was not addressed in the final report, information was previously 

submitted in the progress reports (described in MRID No. 466066-01; reviewed in U.S. EPA 2006b). 

Monsanto submitted the results of a MCRW field efficacy evaluation designed to (1) evaluate and 

compare performance of MON 863 vs. conventional soil insecticides in protecting corn roots from 

injury by MCRW; and (2) evaluate performance of MON 863 hybrids vs. conventional soil insecticides 

and experimental seed treatments in protecting corn roots from MCRW. 

Of the 12 efficacy trials conducted, four were (unintentionally) planted in areas with insignificant 

MCRW pressure for the 2003 growing season (e.g., 0.25 Node Injury Scale (NIS) in untreated check); 

thus, only eight trials yielded useful data. MON 863 provided corn root damage control of MCRW 

greater than or equal to that provided with furrow treatment by the commercial insecticides. MON 863 

provided better MCRW control than non-Bt isoline seed treatment with clothianidin at 1.25 milligrams 

(mg) active ingredient/kernel. The average NIS for MON 863 was 0.10, while MON 863 plus 

clothianidin was 0.07. Comparatively, the NIS for the isoline seed alone was 1.2 and the isoline plus 

clothianidin was 0.32. Since the summary of these results was not presented with accompanying 

statistical analyses, it was not possible to determine whether significant differences occurred between 

MON 863 alone and the highest non-Bt isoline seed treatment tested. 

Mechanism of Potential Resistance of CRW/Development of a Cry3Bb1-Resistant Colony 

EPA did not receive a final report from Monsanto detailing efforts to investigate potential resistance and 

develop a resistant colony. Information on this research objective, however, was provided in a 

previously submitted progress report (described in MRID No. 466066-01; reviewed in U.S. EPA 

2006b). 

The first study (conducted by P. Clark and J. Foster, Department of Entomology, University of 

Nebraska) was intended to identify differences in 1st, 2nd, and 3rd instar larval feeding on transgenic 

maize compared with its non-transgenic isoline on the basis of larval feeding behavior, neonate feeding 

location, and larval survivorship. The research design incorporated the novel feature of a transparent 

corn plant growth medium that permitted direct visual observation of WCRW larval feeding in situ
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without disturbing the feeding larvae or growing roots. Qualitative observations (feeding location, larval 

movement in and around the transgenic and non-transgenic plants, etc.) were recorded, and quantitative 

measurements were made on root wet and dry weight, larval wet and dry weight, head capsule width, 

and larval mortality. Observational results showed that 1st instar larvae aggregate and initiate feeding at 

the root tip meristem on both transgenic and non-transgenic corn. Following this initial aggregation 

phase, larval behavior differs, depending on whether the plant is transgenic or not. On non-transgenic 

corn, larvae feed into the root interior, leaving an outer “shell” of 1–2 layers of epidermal root tissue; 

continued feeding results in larval movement into older and elongated root tissues over time. On MON 

863 corn, 1st and 2nd instar larvae began feeding on meristematic tissue but terminated feeding before 

entering the root interior; further, larvae on MON 863 fed less frequently, did not become established at 

feeding sites, and moved more frequently than same-stage instars on conventional corn. Survival of 1st

and 2nd instar larvae to successful molt on MON 863 was approximately 1%. Larvae that fed and molted 

to 2nd instar on transgenic roots exhibited the same growth rate as did larvae in non-transgenic isoline; 

further, there were no significant differences for the parameters of head capsule width and larval wet and 

dry weight. These results seem consistent with a “repellent factor” in roots or root exudate that may 

contribute to the overall efficacy of MON 863. 

In the second study (conducted by T. Clark, B. Hibbard, and I. Oyediran, Department of Entomology, 

University of Missouri), the objective was to determine selection intensity and associated fitness 

parameters of WCRW on transgenic, rootworm-resistant corn in a Missouri environment. During 2004, 

the study was expanded to include sites in Nebraska, Iowa, and South Dakota. Replicate plots of 

glyphosate-resistant and MON 863 and non-MON 863 isoline corn were established at the field sites and 

infested at V2 with two densities of WCRW eggs. At pupation, emergence cage monitoring of each plot 

was performed daily and continued for 3 weeks until emergence ceased. Adults were counted and sexed, 

and results were analyzed for effects of environment (weather, soil information, and agronomics), 

infestation level, and corn type. In the experiment, selection intensity was defined as beetle emergence 

from a known egg load. When comparing low- and high-level infestations separately, beetle emergence 

ranged from 1.2% to slightly over 7.2% for isoline corn, while MON 863 beetle emergence ranged from 

0.006% to 0.145%. All categories had a female bias with MON 863 showing the greatest bias for beetle 

emergence. General conclusions were that the factors of treatment, environment, and treatment plus 

environment played significant roles in the selection intensity. It is recognized that interpreting data 

from selection studies from single locations or narrow geographies can be misleading; thus, adult 

emergence data may need to be collected on a greater scale from various geographies. Further types of 

similar analysis may need to be conducted to better understand the factors that may influence selection 

intensity. 

A third study was conducted to initiate development of a MON 863-resistant colony (conducted by T. 

Clark, B. Hibbard, and I. Oyediran, Department of Entomology, University of Missouri). As of the 2004 

progress report, four nondiapausing colonies, stemming from one wild-type x nondiapausing colony, 

were being reared and fed optimally as adults but differing in larval diet: 0) non-Bt (isoline) only, 1) 

exposed to Bt corn as neonates but reared on isoline, 2) Bt corn only from second instar to pupation and 

3) reared solely on Bt corn. Numbering started at 0 to reflect the amount of exposure to Cry3Bb1-
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expressing corn. Virgin female wild-type insects were crossed with nondiapausing males and virgin 

female nondiapausing beetles were crossed with wild-type males. From the resulting eggs (combined 

reciprocal crosses), a total of 4,262 F1 beetles emerged that produced 241,000 eggs. From these eggs, 

colonies 0 through 3 were initiated and reared in the greenhouse. Results (as of 2004) showed that 

significant numbers of adults emerged with colonies 0 and 3 producing the most adults. Time from egg 

hatch to first adult was 27 days for colonies 0, 1, and 2 and 31 days for colony 3. The goal was to rear at 

least four to five generations of each colony per year to reach 10 generations. Generations 3, 6, and 10 

were to be broadly tested for mechanisms of survival. The nondiapausing trait appeared to be completely 

dominant in the wild-type x nondiapausing colony that was created. 

 

In separate research (not part of the MON 863 report), Miehls et al. (2008) were able to select for 

Cry3Bb1 resistance within three generations in greenhouse experiments. This work also suggested that 

the resistance trait could have non-recessive inheritance and could lead to rapid response to selection 

without adequate refuge. 

 iii. Dose

Identifying the level of dose, as related to selection intensity, is crucial when determining size and 

structure of a refuge needed to delay CRW resistance to MON 863 corn. CRW feeding behavior and 

survival and root expression data can be used to identify the dose of MON 863. From data currently 

available, it can be concluded that MON 863 corn does not provide a high dose for CRW control. The 

August 2002 SAP suggested that it is not necessary to determine the difference between a low and 

moderate dose. It is adequate to differentiate between high dose and non-high dose products when 

determining effective refuge size. Therefore, MON 863 should be characterized as a non-high dose 

product. 

According to the August 2002 SAP, comparing measures of fitness levels of susceptible homozygotes 

on MON 863 and non-Bt corn would provide a good approximation of selection intensity. The SAP 

suggested that the first step in approximating selection intensity would be to measure efficacy of MON 

863 corn against CRW larvae. The Panel pointed out, however, that selection intensity based on larval 

efficacy may be underestimated if sublethal effects or fitness costs occur. Selection intensity based on 

larval survival may also be underestimated if density-dependent mortality is occurring. Resistant 

colonies of CRW should be developed to aid in determining selection intensity. 

The SAP based their determination that MON 863 is a non-high dose product on the SS (homozygous 

susceptible) survival rate. The Panel also concluded that Monsanto’s artificial diet assays had 

deficiencies but were adequate to determine the median lethal concentration (LC50) for first instar larvae, 

level of larval resistance, and dose. 

 iv. Simulation Models of Resistance

In Monsanto’s three-year interim IRM plan, they recommended planting a 20% non-Bt corn refuge to 

delay the potential of CRW resistance to Cry3Bb1. Monsanto’s conclusion that a 20% refuge would be 
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adequate to delay resistance to MON 863 corn was based on CRW biology, Cry3Bb1 effective dose, 

preliminary modeling results, and agronomic considerations. The Agency concluded that a 20% non-Bt

corn refuge planted within or adjacent to MON 863 corn fields is expected to adequately delay the risk 

of CRW developing resistance to Cry3Bb1 (U.S. EPA 2002a). Monsanto’s IRM interim plan and EPA’s 

review of Monsanto’s plan were addressed by the August 2002 SAP (U.S. EPA 2002b). 

According to the SAP, the current models (Monsanto’s modified Caprio model; Onstad et al. 2001; 

Andow and Alstad 2002) show that the time to resistance does not substantially differ when the refuge 

size ranges from 10–25%. While the SAP agreed that resistance would not occur during an initial 3 

years regardless of the size of the refuge, the majority of the Panel recommended a 50% refuge would be 

a desirable conservative approach since resistance would be delayed substantially longer. The SAP also 

stated that the amount of gene frequency increases during an interim period is of greater importance than 

years to resistance because of the potential future impact on IRM. Since MON 863 is a non-high dose 

product, the Panel suggested that the potential for heritable quantitative variation and rapid evolution of 

resistance should be considered. In addition, the models only consider monogenic (single locus) 

resistance, but the SAP suggested that the models consider the potential for polygenic resistance in a 

non-high dose product. 

Additional comments were made by the Panel regarding initial resistance allele frequency. Each of the 

models (Monsanto’s modified Caprio model; Onstad et al. 2001; Andow and Alstad 2002) submitted in 

support of Monsanto’s IRM plan designated the initial resistance allele frequency as 0.001. The Panel 

suggested that the initial resistance allele frequency may be as low as 0.1 in a non-high dose product. 

Therefore, the Panel recommended that studies be conducted to determine if the initial resistance allele 

frequency is less than 0.01, and models should be run that investigate the full range of dominance 

values. 

In a letter dated December 13, 2002, Monsanto responded to the August 2002 SAP (Ward 2002). Within 

the letter, Monsanto summarized results from four data sets from research they sponsored on the 

efficacy of MON 863. The first and third data sets consisted of field data collected from 1999 to 2002 by 

22 scientists from 15 universities located in 15 states. The second data set included data collected by Dr. 

Bruce Hibbard (University of Missouri), and the fourth data set was from research conducted by Dr. 

Blair Siegfried (University of Nebraska). According to Monsanto, results of these four data sets 

demonstrate that the initial allele frequency is ≤0.01. Detailed summaries of these four data sets will be 

submitted to the Agency for confirmation. 

The first data set looked at 7,500 corn plants artificially infested with ≥1,200 CRW eggs/plant from 

naturally occurring populations. If the initial resistance allele frequency is 0.01 and Hardy-Weinberg is 

assumed, then 24 CRW/plant (24 = 1,200(1-(1-0.01)2

) would be resistant, and the damage rating on the 

Iowa scale would be 3.1. Weiss et al. (1985) showed that <20 CRW = 3.1 on the Iowa scale. Since the 

average damage recorded in the first data set was 1.6, it can be concluded that the initial resistance allele 

frequency is ≤0.01. 
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The second data set summarized by Monsanto evaluated larval survival. In this study, ≥30 larvae were 

recovered per non-Bt corn plant at a wide range of egg infestation rates. If the initial resistance allele 

frequency is 0.01 and Hardy-Weinberg is assumed, then 0.6 resistant larvae (0.6 = 30(1-(1-0.01)2) would 

occur per MON 863 corn plant. Since an average of 0.7 larvae were recovered (but not feeding 

normally), a ≤0.01 initial resistance allele frequency can be assumed. 

The third data set evaluated the number of surviving adult CRW. This data set included several studies 

that infest corn plants with over 1,200 eggs. Of the 1,200 eggs, an average of 30 adults survived on nontransgenic corn. If the initial resistance allele frequency is 0.01 and Hardy-Weinberg is assumed, then 

0.6 resistant adults (0.6 = 30(1-(1-0.01)2) would occur per MON 863 plant, and the damage rating on the 

Iowa scale would equal 3. Since damage averages 1.6 on the Iowa scale, a ≤0.01 initial resistance allele 

frequency can be assumed. 

The final data set (#4) examined 11 field-collected adult female CRW populations reared in the lab. 

Between 134 and 489 larvae per population were examined for susceptibility to Cry3Bb1. These larvae 

demonstrated less than 6-fold difference between the most and least susceptible populations, which is 

similar to or less than populations of European corn borer and corn earworm in their susceptibilities to 

Cry1Ac and Cry1Ab. If the initial resistance allele frequency is 0.01 and Hardy-Weinberg is assumed, 

then 2% (>20) of the larvae assayed would be resistant. Monsanto asserted that no putatively resistant 

large larvae were recovered at high doses, suggesting no larvae survived and there was low variation 

(lower than with lepidopterans); therefore, a ≤0.01 initial resistance allele frequency can be assumed. 

Products with a resistance allele frequency ≥0.01 would not have enough efficacy to justify 

commercialization (Bourguet et al. 2003; Ferré and Van Rie 2002). If the initial resistance allele 

frequency were 0.1, then the efficacy of MON 863 corn would be so poor that it would not be a 

marketable product. At initial resistance allele frequencies of 0.1 and 0.01, damage would be greater 

than 4.6 and equal to 3 on the Iowa scale, respectively. The economic threshold in corn is a 3 on the 

Iowa scale. Monsanto has demonstrated that the average damage rating is 1.6. Since MON 863 

consistently provides enough protection to result in much less than a 3 root rating, it can be concluded 

that the initial resistance allele frequency is ≤0.01 based upon product performance. 

Monsanto modified Caprio’s model to include an initial resistance allele frequency of 0.01 and 

submitted these results to EPA (Ward 2002). Results of running this model showed that a 20% refuge 

would delay resistance for approximately 7–16 years (Figure 2 on page 13 and Figure 3 on page 14 in 

Ward (2002)). For this model, SS survival was set at 0.5, and RS survival was set at 0.8, which is partial 

dominance. Based on data collected by Monsanto and its cooperators, MON 863 has been shown to 

control an average of 50% of the homozygous susceptible (SS) CRW. Therefore, the SS survival was 

designated 0.5 in the modified Caprio model. 

According to Monsanto, RS survival (dominance) probably equals 0.7. Therefore, basing dominance on 

≥0.8 would be considered a very conservative approach. Monsanto modeled RS survival to range from 

0.5 to 1. If a RS survival of 1 (absolute worst case) were to occur and the initial resistance allele 

frequency is assumed to be 0.01, then resistance would be delayed for approximately 13 years with a 
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20% refuge (Figure 1 on page 8 in Ward (2002)). If RS survival is designated 0.8, then resistance will 

occur in approximately 16 years. According to Dr. David Andow (University of Minnesota), RS survival 

ranges between 0.3 and 0.8 (Andow, personal communication, 2002). Therefore, a likely case 

assessment would be to designate RS as 0.8, which suggests that 80% of the heterozygotes survive. 

Monsanto also provided the Agency with additional runs of the modified Caprio model that included 

conservative parameters representing a worst-case scenario. These additional models included initial 

resistance allele frequencies of 0.01 and 0.001, RS dominance values of 0.7 and 0.8, and SS survival 

ranging from 0.1 to 0.8. Results of the model, incorporating these conservative input parameters (e.g., 

initial allele frequency = 0.01; RS dominance value = 0.8; SS survival = 0.1), suggested that CRW 

resistance to Cry3Bb1 will not occur for at least 7 years assuming 100% MON 863 market penetration 

and 100% IRM compliance (Table 1). 

 

Table 1. Predictions for MON 863 Durability with a 20% Refuge. 

SS Survival RS Dominance Allele Frequency = 0.01 Allele Frequency = 0.001 

0.1 0.7 7 years 9 years 

0.1 0.8 7 years 9 years 

0.3 0.7 11 years 15 years 

0.3 0.8 10 years 13 years 

0.5 0.7 20 years 30 years 

0.5 0.8 16 years 23 years 

0.8 0.7 >100 years >100 years 

0.8 0.8 >50 years >50 years 

Monsanto also commented on the SAP’s recommendation to consider polygenic resistance in the 

simulation models. According to Monsanto, results of the model will not differ if polygenic resistance is 

considered rather than monogenic resistance. Dr. Mike Caprio (Mississippi State University) agreed 

with Monsanto’s conclusion. According to Dr. Caprio, applying monogenic or polygenic resistance to 

the models does not affect the outcome in the absence of refuge (Caprio 1998; Caprio, personal 

communication, 2002). Groeters and Tabashnik (2000) concluded “that the intensity of selection, rather 

than the number of loci conferring resistance, is central in determining rates of resistance evolution and 

effectiveness of refuges.” This new information, provided to the Agency by Monsanto after the August 

2002 SAP, suggests that assuming CRW resistance to MON 863 is polygenic rather than monogenic will 

not affect the results of the models. 

Based on the additional information submitted by Monsanto after the August 2002 SAP and results of 

running Caprio’s modified model with a 0.01 initial resistance allele frequency, it can be concluded that 

a 20% refuge will delay resistance for approximately 7 to 16 years and probably longer since the model 

also assumes 100% adoption. However, Monsanto assumes that 50% of the susceptible homozygotes 
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(SS) will be controlled. Efficacy data submitted thus far shows 17% to 62% larval survival on MON 863 

corn. If the SS input parameter were changed to a lower level of efficacy (e.g., 0.3), then the years to 

resistance may decrease. 

Based on the results presented in Monsanto’s submission and recommendations from national experts, 

including the NCR-46, a 20% refuge should be adequate to delay resistance for 7 to 16 years. In 

addition, because growers are familiar with the 20% refuge required for currently registered Bt corn 

products, better compliance can be expected based on grower familiarity, feasibility, and presenting a 

consistent message to growers; a 20% refuge should be planted adjacent to or within fields. 

 v. Resistance Allele Frequency Data (MRID No. 459438-01)

Greenhouse and field efficacy studies, adult emergence trials, and laboratory feeding studies have 

generated data for estimating initial resistance allele frequency in corn rootworm populations feeding on 

transgenic MON 863 corn containing the genes for Cry3Bb1 endotoxin, as compared to isoline corn 

without Cry3Bb1 expression. These four studies (root damage ratings in the greenhouse and field; larval 

establishment in the field; adult survival in the field; and larval susceptibility to Cry3Bb1 in a laboratory 

bioassay) were conducted between 2000 and 2002. Monsanto and university trials, the work of Hibbard 

et al., and data from Siegfried and Spencer were summarized in this report. Artificial infestations (eggs) 

of WCRW were used to challenge both greenhouse and field populations of MON 863. Bioassay of 

artificial diet, top-loaded with five concentrations of purified Cry3Bb1 protein, and control were tested 

with larvae reared from eggs laid by adult female WCRW collected from 11 distinct field populations in 

six Midwestern states. Bioassay results for the MON 863 low-to-moderate dose product exhibited a 6-

fold regional variation in larval susceptibility on the basis of LC50 values (2.22 micrograms per square 

centimeter (μg/cm2

) vs. 13.00 μg/cm2). In all four studies, predicted results, based on the HardyWeinberg law and the assumption of an r-allele frequency of either 0.1 or 0.01 (for parameters of 

damage to MON 863 plants, number of larvae on each MON 863 plant, and number of resistant larvae), 

were compared with measured observations. 

The evidence presented in the four data sets is largely circumstantial—r-allele frequency was estimated 

using three field efficacy studies and the results from Cry3Bb1 baseline susceptibility work. Major 

assumptions were made regarding density-dependent mortality, the genetics of potential resistance 

(assumed to be dominant), and the mode of action of MON 863 corn. While these indirect approaches 

provided some support to Monsanto’s hypothesis that the r-allele frequency for CRW is <0.01, there was 

too much uncertainty to definitively prove that this was the case. In addition, there is still significant 

uncertainty regarding the mode of action of MON 863 (Cry3Bb1) corn and the nature of potential CRW 

resistance (U.S. EPA 2004d). 

Monsanto has been required to continue to pursue the r-allele issue through the development of resistant 

CRW colonies (required as a condition of registration) and other research efforts. A better understanding 

of the mode of action (i.e., toxic or repellent effects) would also aid in the understanding of potential 

CRW resistance. In addition, research on dose, fitness, behavior, and possible polygenic inheritance 

could also be useful to further the understanding of CRW resistance. 
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 vi. Refuge

A 20% non-Bt corn refuge is necessary to produce an adequate number of CRW susceptible to the 

Cry3Bb1 protein. There are two ways a grower can implement the refuge requirement. Non-Bt corn 

refuge can be planted as a continuous block adjacent to the MON 863 field or as non-transgenic strips 

planted within the MON 863 field. Considering the limited movement of CRW larvae, planting refuges 

close to transgenic fields in large blocks is preferred to narrow strips (Gray 1999; Meinke et al. 2001). If 

the 20% refuge is planted as row strips within a corn field, the non-Bt corn strips must be at least four or 

more consecutive rows wide. Use of an in-field strip refuge is not intended for fields planted to increase 

inbred seed since these fields need to be isolated from external corn pollen sources. An in-field or 

adjacent non-Bt corn refuge would be inconsistent with inbred seed production practices. 

MON 863 corn was originally registered with a row width requirement of at least six rows for in-field 

strip refuges. Monsanto amended the terms of registration in 2005 to modify this requirement to at least 

four or more consecutive rows. This amendment was supported by larval movement data published by 

Hibbard et al. (2003) that showed between 0.75% and 6% of larvae moved across rows. This likely 

represents a relatively high-end estimate of the number of larvae that cross rows. Given this finding, 

narrower in-field strips should be sufficient to provide adequate protection from sublethal selection 

caused by CRW larval movement across rows. In addition, in-field strips of four or more rows for CRW 

provide the advantage of compatibility with the in-field strip width requirement for lepidopteranprotected Bt corn plant-incorporated protectants (PIPs) (also ≥4 rows), possibly increasing refuge 

compliance (U.S. EPA 2005d). 

Insecticides, applied to the soil to control CRW larvae, are acceptable on refuge acres. The ability to 

treat refuges with larval insecticides is necessary to avoid the potential for severe damage and economic 

impact. It is not acceptable, however, to treat refuges for adult CRW control since these treatments may 

diminish the effectiveness of the refuge. If growers spray their corn fields with insecticides to control 

pests other than CRW, then all acres (Bt and non-Bt) should be treated identically. 

Bt fields and non-Bt refuge acres should be treated with identical agronomic practices, such as irrigating 

all corn (Bt and non-Bt) at the same time. To ensure the production of similar numbers of CRW, Bt and 

non-Bt corn should be planted in fields with similar backgrounds. For example, if MON 863 hybrids are 

planted on continuous corn fields, then the non-Bt refuge should be planted on continuous corn fields or 

both should be planted on first-year corn acres. Likewise, non-Bt refuges should be planted on first-year 

corn fields if the MON 863 hybrids are planted on first-year corn fields. 

vii. Monitoring for Resistance

A resistance monitoring strategy for Bt corn is needed to test the effectiveness of the resistance 

management programs. Detecting shifts in the frequency of resistance genes (i.e., susceptibility changes) 

through resistance monitoring can be an aggressive method to detect the onset of resistance before 
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widespread crop failure occurs. As such, the utilization of sensitive and effective resistance monitoring 

techniques is critical to the success of an IRM plan. Monitoring techniques, such as discriminating dose 

concentration assays, need to be thoroughly investigated for Diabrotica spp. for their feasibility as 

resistance monitoring tools. 

Grower participation (e.g., reports of unexpected damage) is an important step in resistance monitoring. 

Resistance monitoring is also important because it provides validation of biological parameters used in 

models. Resistance detection/monitoring, however, is a difficult and imprecise task. It requires both high 

sensitivity and accuracy. Good resistance monitoring should have well-established baseline 

susceptibility data so changes in pest susceptibility over time can be monitored. 

The August 2002 SAP recommended a two-tiered approach to monitoring for CRW resistance to MON 

863. The Panel recommended tier 1 monitoring methods should identify locations that would merit tier 2 

laboratory bioassays. Early detection monitoring should be directed to areas with the highest rate of 

MON 863 adoption since these areas represent the highest risk of resistance occurring. 

The SAP suggested that current methods used for early detection of resistance probably do not have the 

necessary level of sensitivity. Therefore, the Panel recommended potential alternatives to the insect 

bioassay using artificial diet. For instance, susceptibility of neonate larvae to corn lines expressing 

varying levels of the Cry3Bb1 protein (e.g., Events MON 863, MON 862, MON 853, and MON 854) 

could be explored. Measuring larval mortality and growth data with various corn lines rather than 

artificial diet would be easier and may eliminate some of the problems associated with the feeding 

bioassay, such as mold growth on the artificial diet. Susceptibility data should also be collected for the 

NCRW and MCRW. 

The SAP also suggested that data on root damage may be used as a monitoring tool; however, a method 

of using root damage ratings to monitor for resistance has not been developed or validated at this time. It 

also may be possible to use data on emergence patterns in the MON 863 and non-Bt corn refuges. More 

females than males from susceptible populations tend to emerge from MON 863. It may be possible to 

evaluate the percentage of males emerging and be correlated with resistance. 

Monitoring will become more important after the accrual of multiple growing seasons of exposure and 

grower adoption increases. In addition to baseline susceptibility data, information is needed to determine 

how many individuals need to be sampled and in how many locations. The chance of finding a resistant 

larva in a Bt crop depends on the level of pest pressure, the frequency of resistant individuals, the 

location and number of samples that are collected, and the sensitivity of the detection technique. 

Therefore, as the frequency of resistant individuals or the number of collected samples increases, the 

likelihood of locating a resistant individual increases (Roush and Miller 1986). If the phenotypic 

frequency of resistance is 1 in 1,000, then more than 3,000 individuals must be sampled to have a 95% 

probability of one resistant individual (Roush and Miller 1986). 
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Cry3Bb1 Monitoring Strategy 

As a condition of registration, Monsanto was required to develop a resistance monitoring program for 

Cry3Bb1 in MON 863 corn. The following elements were required for the plan: 

 CRW sampling should be focused in those areas in which there is the highest risk of resistance 

development. 

 The registrant must follow up on grower, extension specialist, or consultant reports of less than 

expected results or control failures for the corn rootworm. 

 Baseline susceptibility studies for WCRW and NCRW and the investigation of techniques, such 

as discriminating dose concentration assays, for feasibility as resistance monitoring tools. 

 Develop and validate an appropriate discriminating or diagnostic dose assay (required for MON 

88017, a subsequent Cry3Bb1 registration). 

 Rootworm damage guidelines for unexpected pest damage (required for MON 88017, a 

subsequent Cry3Bb1 registration). 

Monsanto submitted a resistance monitoring strategy for MON 863 and Cry3Bb1 that was largely based 

on the established paradigm for lepidopteran Bt corn insects (reviewed in U.S. EPA 2004b). This 

monitoring plan was subsequently revised with the registration of MON 88017 (which also expresses 

Cry3Bb1) in 2005. The Cry3Bb1 monitoring plan (as developed for MON 88017; MRID No. 473547-

01) is described below (reviewed in U.S. EPA (2009b and 2010)) and is applicable to MON 863 corn. 

Monitoring for CRW resistance consists of two main parts: (1) monitoring for unexpected field damage 

by growers, extension agents, consultants, and company agronomists, and (2) monitoring for resistance 

through targeted population sampling and testing. Monitoring for unexpected damage will reveal the 

occurrence of localized resistance (or hot spots) before resistance will have spread. Resistance 

monitoring through targeted field sampling should reveal changes in susceptibility of geographically 

representative populations. 

Population sampling will focus on the WCRW species, which will serve as a worst-case surrogate for 

northern corn rootworm and Mexican corn rootworm. Because of their widespread distribution and 

abundance, but similarity in life cycles compared to NCRW and MCRW, it is more likely that resistance 

due to exposure to Cry3Bb1 will evolve in WCRW first. 
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Monsanto proposed to focus its geographic sampling in areas where MON 863 and MON 88017 

adoption has been highest and selection pressure is greatest. These areas are as follows: 

Region 1, includes rotation-resistant variant 

 Eastern Illinois 

 Western Indiana 

Region 2, wild-type variant 

 Western Illinois 

 Iowa 

 Missouri 

Region 3, organophosphate-resistant variant 

 Nebraska 

 Kansas 

The breakdown into these three regions has been determined based on the three WCRW biotypes found 

in the U.S.: soybean/corn rotation-resistant, wild-type, and organophosphate-resistant. Monsanto 

proposed to target between 4–6 populations, but no less than 3, in these areas with different biotypes. 

Also, not all states specifically listed above may be represented by the sample collection. Actual sample 

sites are decided by DM Crop Research Group based on beetle abundance and environmental 

conditions. A periodic review of sales information may warrant modification of sample areas, though the 

Agency will be informed of such changes. 

Population susceptibility will be assessed using a diet-based bioassays approach as described by 

Siegfried et al. (2005). The dose-response curve will be determined for each population and compared to 

historical data from populations in the same regions. A diagnostic concentration for Cry3Bb1 should be 

established using baseline susceptibility and annual monitoring data, as well as other historical 

information for WCRW and Cry3Bb1. Once a diagnostic concentration has been established, 

approximately 400 neonates (4 replicates total) will be tested at the assumed discriminating 

concentration. 

Unexpected survivors at the discriminating dose will be reared to adults and mated amongst themselves 

or single-pair mated with individuals from a susceptible lab colony if numbers are low. The resulting 

progeny will once more be exposed to the diagnostic concentration bioassays to determine heritability of 

survival on a Cry3Bb1-containing diet. If heritability is confirmed, survivors will be placed on MON 

88017 corn plants to assess whether level of resistance is enough to cause severe root damage. Whether 

an increase in susceptibility has occurred will be assessed with a discriminating concentration bioassay 

when such dose has been established. Until then, either of the following criteria below will serve to 

confirm resistance; however, Monsanto stated that their working definition of resistance will be refined 

based upon continued research and experience: 
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 The LC50 of the standard bioassay exceeds the 95% confidence interval of the mean historical 

LC50 for susceptible pests according to the baseline measurements. 

 Over 50% of Cry3Bb1-expressing plants have ≥1 root nodes destroyed by suspected resistant 

populations under controlled lab conditions. 

Unexpected Pest Damage 

Since Cry3Bb1, as expressed in MON 88017, is not high dose, it will not control CRW at the same level 

as registered Lepidoptera protected Bt corn products. Hence, this requires a different approach to discern 

between unexpected pest damage and damage caused by CRW due to non-high dose control. Monsanto 

reported (MRID No. 473547-01) that the 2002 SAP Advisory Panel suggested consideration of the 

following factors specifically related to CRW resistance monitoring: 

 CRW survival and some degree of root damage are expected in fields planted with MON 88017. 

 A single corn root system supports numerous rootworm larvae. Therefore, the effect of resistant 

individuals on the overall root structure will not be easy to detect unless the resistant individuals 

represent a significant proportion of the population on that root system. 

 Root damage caused by corn rootworm larvae feeding is not readily visible; plants must be dug 

up, and roots washed to assess damage. 

 Aboveground symptoms of root damage, such as lodging, often have causes other than larval 

feeding (e.g., high winds in combination with high soil moisture content). 

 Environmental factors can be significant determinants of the amount of damage caused by 

rootworm larvae. 

Monsanto proposed final root damage guidelines of unexpected damage reports in a letter to EPA (dated 

January 20, 2010). The guidelines were submitted for MON 88017 (a subsequently registered plantincorporated protectant (PIP) that expresses Cry3Bb1) but are also applicable to MON 863. The letter 

defined unexpected damage as: “[t]he level of root damage in MON 88017 field must be equal to or 

greater than that in the refuge field, assuming the MON 88017 and refuge fields are comparable with 

respect to management practices and the damage in the refuge is above 1.5 on the 0–3 nodal injury scale 

(NIS). In circumstances when a comparable refuge field is not available (e.g., refuge flooded, etc.), then 

guidelines for establishing suspect resistance are as follows: 

1) Average root damage in the MON 88017 or MON 88017 x MON 810 field is >1.5 on the 0–3 

NIS; and 

2) The frequency of MON 88017 or MON 88017 x MON810 corn plants with >1.5 nodes destroyed 

exceeds 50% of the sampled plants.” 

Monsanto stated that these guidelines reflected the empirical experience of four years of CRW 

monitoring, which have allowed Monsanto to quantify the maximum amount of damage that could be 

expected under heavy infestation and favorable environmental conditions for rootworm feeding. This 

cumulative experience has led to an increase in the threshold NIS from the originally proposed 1.0 to 1.5 
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on the nodal injury scale. With the new threshold, Monsanto has found that a few false positive events 

still could be expected, making these guidelines a conservative choice. 

Monsanto had previously indicated (MRID No. 473547-01) that if the above conditions were met and 

complaints of unexpected pest damage were received from the same growers in two consecutive years, 

they would attempt to collect CRW populations from the fields the following year. Collections would be 

undertaken when CRW flight is at its peak and approximately 1⁄2 mile away from the nearest MON 

88017 fields. 

EPA’s review of Monsanto’s unexpected damage strategy identified a number of concerns with the 

proposed approach (U.S. EPA 2010a). A damage threshold level of 1.0 may be more appropriate and 

conservative for moderate insect pressure and single, non-high dose CRW products, such as MON 

88017 and MON 863. The review recommended that Monsanto keep the threshold level for unexpected 

pest damage at 1.0 when insect pressure is low to moderate. When CRW pressure is exceedingly high 

during a corn-growing season, however, then Bt-protected and refuge corn will likely incur greater 

damage. Under these circumstances, a threshold level of 1.5 may be more appropriate as proposed by 

Monsanto. The review also recommended consideration of the following factors for unexpected pest 

damage: 

1) The inherent dose of the toxin to control 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-growing season (moderate vs. high); and 

5) Weather pattern during the corn-growing season and possible effects on Bt protein expression 

and pest population dynamics. 

Further, concerns were identified with Monsanto’s approach to respond to unexpected damage only after 

reports have been received in two consecutive years by the same grower. This approach may be too 

protracted (two years before sample collection occurs and four to five years total before resistance is 

confirmed) and could lead to the undetected spread of resistance in that region. It was recommended that 

Monsanto respond to reports of unexpected pest damage during the same growing season when the 

report has been filed, or no later than July of the following growing season (U.S. EPA 2009b and 2010). 

Baseline Susceptibility of Corn Rootworm to Cry3Bb1 

As required by the terms and conditions of the MON 863 registration, baseline susceptibility levels of 

western corn rootworm populations to Cry3Bb1 have been measured (Siegfried et al. 2005). These data 

serve as the baseline for measuring any annual shifts in the susceptibility of these populations. Results 

indicated that the representative WCRW populations collected in 2004 were susceptible to the Cry3Bb1 

toxin and that slight differences in susceptibility among the populations were due to natural variation in 

responses. In addition to the baseline susceptibility studies conducted on western corn rootworm, 

populations of the northern corn rootworm and Mexican corn rootworm were being collected as part of 

the required monitoring program and to determine baseline susceptibility levels for these species. 
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Improved methods for collecting Mexican corn rootworm adults have been published (Spurgeon et al. 

2004) and may help in this on-going effort to monitor rootworm populations. 

EPA reviewed (U.S. EPA 2006a) the baseline monitoring data provided by Monsanto from 2001–2004 

(no data available for 2003) and concluded that all representative WCRW populations were susceptible 

to the Cry3Bb1 toxin (expressed in MON 863). 

Cry3Bb1 Discriminating (Diagnostic) Concentration for CRW 

As a condition of registration, Monsanto was required to investigate the feasibility of employing a 

discriminating (or diagnostic) concentration assay for CRW monitoring. Diagnostic concentration assays 

function by identifying potential resistant individuals that can survive high levels of the controlling 

toxin. For lepidopteran target pests of other Bt corn PIPs, these assays have been based on the LC99 of 

susceptible populations. 

Monsanto developed a diagnostic concentration for Cry3Bb1 of 170.8 μg/cm2 against WCRW (MRID 

No. 478846-01). Low and variable survival was still apparent at this level; however, surviving larvae 

were severely stunted as observed in field-collected populations of 2007 and 2008. Table 2 summarizes 

the percent mortality and mean mass of survivors of field populations collected during the two years and 

compares the results to those obtained from the susceptible lab reference strain. The weight of larval 

survivors from field populations ranged from 0.01–0.04 mg compared to the mean mass of control 

larvae (0.22 mg). Monsanto concluded that this concentration appeared to fit the needs for an effective 

diagnostic concentration where rootworm response is measured as larval mass at the end of the assay. 

Should field-derived survivors show significant development, Monsanto would further investigate this 

population for potential resistance to Cry3Bb1. 
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Table 2. Percent Mortality and Mass of Surviving Larvae at the Putative Diagnostic 

 Concentration of 170 μg/cm2. 

Population Average % mortality 

across replicates 

Average mass of survivors 

(mg) across replicates 

Maximum mass (mg) across 

replicates 

Montgomery Co., IN 77.8 0.02 0.04 

McLean Co., IL 85.4 0.01 0.02 

Dewitt Co., IL 88.9 0.01 0.02 

Scott Co., IA 86.1 0.01 0.02 

York Co., NE 86.1 0.01 0.02 

Polk Co., NE 73.6 0.02 0.03 

Seward Co., NE 47.2 0.03 0.05 

Piatt Co., IN 75.0 0.02 0.03 

Henry Co., IL 44.4 0.03 0.05 

Palo Alto Co., IA 54.2 0.04 0.05 

Logan Co, NE 48.6 0.03 0.04 

Clinton Co., IA 76.4 0.02 0.03 

Monsanto Lab 62.5 0.04 0.07 

Population Range 44.4–88.9 0.01–0.04 0.02–0.07 

Field population control 

– No Cry3Bb1 exposure 

Average % mortality 

across replicates 

Average mass of survivors 

(mg) across replicates 

Maximum mass (mg) across 

replicates 

All sites 13.3 0.22 0.41 

*Table data extracted from MRID No. 478846-01. 

EPA has also recommended that Monsanto investigate the sublethal seedling assay (SSA) developed by 

Nowatzki et al. (2008), in addition to their effective diagnostic bioassays, to determine which approach 

is more sensitive to detect shifts in CRW susceptibility. Nowatzki et al. (2008) tested the sensitivity of 

SSA side-by-side with a diet bioassay. They found that the SSA, measuring survival and age structure of 

larval populations in three potential instar-groups, was able to detect shifts in susceptibility of CRW at a 

much smaller scale than the diet bioassay, which measured mortality and growth inhibition responses. 

Diet bioassay endpoints (LC50 and EC50) were relatively insensitive to detecting shifts in susceptibility 

(treatments were 0%, 5%, 25%, and 50% selected individuals mixed into susceptible population samples 

of CRW), while the SSA was most sensitive to changes in susceptibility when selected individuals were 

present at <25%. Nowatzki et al. (2008) stated that the SSA may be a more sensitive tool to measure 

shifts in susceptibility than the bioassay because it uses the increased sensitivity of a sublethal measure 

(developmental shifts of larvae into three instar stages/cohorts). 

Cry3Bb1 Resistance Monitoring Data 

Resistance monitoring data for Cry3Bb1 and WCRW have been tabulated from 2005 through the 2008 

growing season. For 2008 (MRID No. 478846-01), DM Crop Research Group (an independent party 

that collects corn pest samples) made thirteen field collections from areas of high CRW pressure and 

Cry3Bb1 adoption to represent populations with the highest potential risk of resistance evolution. 

Custom Bio-Products in Maxwell (IA), an independent laboratory that has conducted bioassays for 

Monsanto since 2006, maintained the insect collections and conducted the bioassays. Populations were 
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maintained using standard protocol for this species; in addition, a nondiapausing lab population was 

supplied by Monsanto as a reference. The form of Cry3Bb1 used was a solution of 4.1 milligrams per 

milliliter (mg/mL) supplied by Monsanto Company. This solution was diluted with 0.1% Titron-X 100 

to obtain a series of concentrations of the Cry3Bb1 protein for bioassay. Neonate larvae were used for 

diet overlay bioassays and exposed to different Cry3Bb1 concentrations (10.7 μg/cm2, 21.4 μg/cm2, 42.7 

μg/cm2, 85.4 μg/cm2, and 170.8 μg/cm2). Thirteen micrograms of each dilution was applied to 12 

individual wells and allowed to dry prior to larval introduction. Larvae were non-systematically selected 

and placed into wells of the tissue culture tray. After three to four days, mortality and survival was 

recorded. Six replicates were conducted for each field and the Monsanto lab population. A replication 

was considered valid if control mortality did not exceed 25%. 

Observed EC50 values from the 2008 populations ranged from 7.3–30.4 μg/cm2

, representing a 4-fold 

difference in susceptibility, and were comparable to the EC50 range obtained in 2007 (14.2–33.4 

μg/cm2). The mean EC50 of the lab reference strain was comparable to those of most field populations as 

measured by the 95% confidence interval overlap. Based on the data, Monsanto concluded that the 

variation observed in EC values was due to the nature of the Cry3Bb1 protein standard rather than 

changes in susceptibility of field populations. LC50 values were more variable than EC values: (1) LC50

range: 24.5–335.0 μg/cm2

 and (2) LC90 range: 218.2–43,931.0 μg/cm2. Higher LC values may be an 

artifact of the assay system because larvae are able to survive without feeding. 

Several portions of the Corn Belt have been sampled consistently since 2005 (Scott Co., IA; Seward Co., 

NE; and Henry Co., IL). A comparison of the CRW susceptibility data in these geographic areas 

(summarized in Table 3 below) showed that susceptibility (as measured by EC50 and LC50) appeared to 

decrease during the 2005–2008 sampling period. The susceptibility of the field population, however, has 

been comparable to the lab reference strain. 
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Table 3. CRW Susceptibility to Cry3Bb1 for Three Counties in the Corn Belt as Measured by 

 EC50 and LC50 Data (2005–2008). 

Population Mean EC50 

(μg/cm2)

Mean LC50 

(μg/cm2)

Scott County, IA (2005) 0.3 0.5 

Scott County, IA (2006) 1.7 6.6 

Scott County, IA (2007) 15.5 63.8 

Scott County, IA (2008) 25.8 40.0 

Henry County, IL (2005) 1.9 3.2 

Henry County, IL (2006) 1.9 5.6 

Henry County, IL (2007) 16.2 50.2 

Henry County, IL (2008) 14.8 300.9 

Seward County, NE (2005) 2.6 3.3 

Seward County, NE (2006) 1.3 9.3 

Seward County, NE (2007) 14.2 64.2 

Seward County, NE (2008) 9.4 335.0 

Monsanto Reference Strain (2007) 12.9 22.3 

Monsanto Reference Strain (2008) 21.8 87.9 

*Table generated from data submitted by Monsanto. 

Susceptibility data (EC50, LC50, and LC90) from population samples collected across the Corn Belt 

during 2005–2008 are summarized in Table 4 below. These data show that there has been no apparent 

increase in susceptibility in CRW across the Corn Belt and support Monsanto’s conclusion that WCRW 

remain susceptible to the Cry3Bb1 protein. 

Table 4. CRW Susceptibility to Cry3Bb1 - Data Ranges for All Populations Sampled in the Corn 

 Belt (2005–2008). 

Year EC50 (μg/cm2

) LC50 (μg/cm2) LC90 (μg/cm2)

2005 0.25–1.28 0.42–1.36

2006 0.64–1.88 1.43–22.22

2007 12.91–33.46 22.29–289.25 97.03–1E+6 

2008 7.3–30.4 24.5–335 218.2–43,931 

*Table generated from data submitted by Monsanto. 
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 viii. Remedial Action

Background and Recommendations for Remedial Action 

The initial observation of unexpected CRW damage or suspected resistance will likely occur by the 

grower. Unexpected damage will probably be observed as lodged corn plants in the fields. Growers 

should be required to report any unexpected CRW damage, such as lodged plants, to the registrant. The 

August 2002 SAP identified the following four steps a registrant should take to determine if further 

testing is needed to confirm resistance is occurring: 

 “request the grower check planting records” 

 “rule out damage from nontarget insects, weather, or other environmental factors” 

 “conduct tests to verify MON 863 was planted and that the correct percentage of plants are 

expressing” 

 “if plants are MON 863 and damage approaching a 0.5 (Node Injury Scale) is found on any 

expressing plant, evaluate roots from the corresponding refuge” 

Resistance should be confirmed by a standard diet bioassay or evaluation of root node injury. An insect 

diet bioassay with the Cry3Bb1 protein that results in a LC50 that exceeds the upper limit of the 95% 

confidence interval of the LC50 established from baseline measurements of susceptible populations could 

be used to confirm resistance. Alternatively, resistance may be confirmed when one or more root nodes 

of at least 50% of Cry3Bb1 plants grown in the laboratory are destroyed. A discriminating concentration 

bioassay may also be used to confirm resistance; however, this method may take a long time to develop. 

The August 2002 SAP also recommended investigating the potential of using samples of populations 

surviving on Bt corn or an evaluation of larval root tunneling to confirm resistance. 

Confirmed resistance should be reported to EPA as soon as possible but no later than 30 days. Once 

resistance has been confirmed, alternative control measures to reduce or control the local target pest 

population should be recommended to customers, extension agents, consultants, university cooperators, 

seed distributors, processors, state regulatory authorities, EPA regional and national authorities, and any 

other pertinent personnel of the incidence(s) of resistance in the affected area. Where appropriate, 

customers and extension agents in the affected area should apply insecticides and/or crop rotation 

practices to control any potentially resistant individuals. 

As soon as possible following confirmation of resistance, but within 90 days, Monsanto should notify 

the Agency of the immediate mitigation measures that were implemented and submit a proposed longterm resistance management action plan for the affected area. Monsanto should work closely with the 

Agency in assuring that an appropriate long-term remedial action plan for the affected area is 

implemented. A remedial action plan that is approved by EPA should be implemented that consists of 

some or all the following elements, as warranted: (1) Inform customers and extension agents in the 

affected area of pest resistance; (2) Increase monitoring in the affected area, ensuring that local target 

pest populations are sampled on an annual basis; (3) Recommend alternative measures to reduce or 
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control target pest populations in the affected area; (4) Implement intensified local IRM measures in the 

affected area based on the latest research results (implementation of such measures will be coordinated 

by the Agency with other registrants); and (5) Monsanto should cease sales of all MON 863 Bt corn 

hybrids in the affected area until resistance has abated. During the sales suspension period, Monsanto 

may sell and distribute in these counties only after obtaining EPA approval to study resistance 

management in those counties. The implementation of such a strategy should be coordinated with the 

Agency.

For the growing season(s) following a confirmed resistance incident(s), Monsanto should maintain the 

sales and distribution suspension of all MON 863 hybrids potentially affected by the resistant pest 

populations and/or areas in which resistance is considered to be serious. This should be done within the 

affected region or if undetermined, the affected county(ies) and proximate surrounding counties. This 

sales suspension should remain in place until resistance has been determined to have subsided (within 

5% to 10% or one standard deviation of baseline levels). In addition, Monsanto should develop, 

recommend, and implement alternative resistance management strategies for controlling the resistant 

pest(s) on corn with all necessary personnel (e.g., growers, extension agents, consultants, seed 

distributors, processors, university cooperators, and state/federal officials) in the affected 

region/county(ies) and surrounding counties of the resistance situation. All necessary personnel (e.g., 

growers, consultants, extension agents, seed distributors, processors, university cooperators, and 

state/federal authorities) in the affected region/county(ies) and surrounding counties of the resistance 

situation should be informed. Monitoring and surveillance in the affected area(s) for resistance and 

defining the boundaries of the affected region should be intensified, and studies on the rate of decline of 

resistance in the field should be conducted. Monsanto should continue to work with the Agency, states, 

grower groups, extension agents, consultants, university cooperators, or other expert personnel and 

stakeholders to ensure the implementation and development of appropriate mitigation measures for 

resistance in the affected areas. 

Remedial Action Plan for MON 863 (Cry3Bb1 Corn) 

As a term of registration, Monsanto was required to develop a remedial action plan for MON 863 corn 

to address many of the issues discussed above. Details of the process used to confirm resistance in 

suspected CRW populations are described in the resistance monitoring plan for Cry3Bb1 (submitted for 

MON 88017, MRID No. 473547-01). A stepwise remedial action plan was submitted separately by 

Monsanto to fulfill the terms of registration (submission dated January 22, 2004, No MRID No.). 

As described in the resistance monitoring plan for Cry3Bb1, unexpected survivors in discriminating 

dose assays will be reared to adults and mated amongst themselves or single-pair mated with individuals 

from a susceptible lab colony if numbers are low. The resulting progeny will once more be exposed to 

the diagnostic concentration bioassays to determine heritability of survival on a Cry3Bb1-containing 

diet. If heritability is confirmed, survivors will be placed on Cry3Bb1 corn plants to assess whether level 

of resistance is enough to cause severe root damage. Whether an increase in susceptibility has occurred 

will be assessed with a discriminating concentration bioassay when such dose has been established. 
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Until then, either of the following criteria will serve to confirm resistance; however, Monsanto stated 

that their working definition of resistance will be refined based upon continued research and experience: 

 The LC50 of the standard bioassay exceeds the 95% confidence interval of the mean historical 

LC50 for susceptible pests according to the baseline measurements. 

 Over 50% of Cry3Bb1-expressing plants have ≥1 root nodes destroyed by suspected resistant 

populations under controlled lab conditions. 

Monsanto proposed a 2.5-year timeframe from the moment of initial detection of CRW resistance to 

Cry3Bb1 to the actual implementation of an appropriate remediation plan. This is the same timeframe 

that has been proposed for lepidopteran resistance in Bt corn. Because PIPs containing Cry3Bb1 are not 

expressed at high dose levels, resistance is likely to be additive and potentially dominant. ABSTC 

(2003) demonstrated in their lepidopteran monitoring plan that if resistance is additive and dominant, 

detection needs to occur at resistance allele frequency levels ≤0.03 and 0.002, respectively, to allow 

detection 2.5 years before the population has become resistant. The sample size needed is 1,000 insects 

in order to detect a particular allele frequency with 80% or 95% confidence when resistance is 

incomplete or dominant. Failure to detect resistance with 1,000 genomes suggests that the resistance 

allele frequency is less than 0.001. The upper 80% and 95% confidence limits of this estimate (0.001) 

are 0.0016 and 0.003, respectively, which are compatible with the allele frequency detection thresholds 

(0.03 and 0.002) needed for a 2.5-year remedial action timeframe before resistance occurs. 

Monsanto’s proposed remedial action plan and steps are more suitable to situations where field 

resistance is detected through product performance monitoring and subsequently confirmed in the lab. 

These steps may include the following: 

 Confirm that resistance is heritable; 

 Confirm field resistance; 

 Use crosses to determine the nature of resistance; 

 Estimate the r-allele frequency in the original population; 

 Determine whether the r-allele frequency is increasing by analyzing field collections; 

 Sample from the site in subsequent years where the resistant allele(s) was originally collected 

and determine if resistance is still detectable; 

 Determine the geographic distribution of the r-allele by analyzing field collections in subsequent 

years from sites surrounding the site where the resistant allele was originally collected; 

 If the r-allele frequency is determined to be increasing or spreading, design an appropriate 

remedial action plan based on the knowledge of the genetics and level of resistance it confers in 

the field. 
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Monsanto’s remedial action plan for MON 863 (as described in the January 22, 2004 submission) is as 

follows: 

1. In cases of suspected resistance, the registrant will instruct growers to do one or more of the 

following: 

a. During the present season, use conventional insecticides to control the adult stage of the 

suspected pest; 

b. During the following season, use an alternative pest control method to deter 

establishment of potentially resistant insects. 

2. If resistance is confirmed, Monsanto will: 

a. Increase resistance monitoring in the affected area. 

b. Notify affected growers, consultants, extension agents, seed distributors, university 

cooperators, and state/federal authorities of the resistance event. 

c. Instruct affected growers and extension agents to use alternative CRW control measures. 

d. Report the incident to EPA within 30 days of confirming pest resistance. 

e. Within 90 days of confirmed pest resistance: 

i. Notify the Agency of any mitigation measures that have been implemented; 

ii. Work with the Agency to develop an appropriate (in agreement with current 

research on IRM) resistance management action plan for the affected region; 

iii. Submit a proposed action plan to the Agency. 

3. If resistance mitigation efforts (described above) fail, Monsanto will stop the sale and 

distribution of YieldGard® CRW in the remedial action zone (may be less than a single county, a 

single county, or multiple counties), until an effective mitigation plan has been approved by 

EPA. 

4. The EPA-approved management plan for the affected area may consist of some or all of the 

following elements: 

a. Annual IRM education for relevant parties (e.g., growers, consultants, extension agents, 

seed distributors, university cooperators, and state/federal authorities). 

b. Annual resistance monitoring. 

c. Use of alternative pest control measures to reduce or control target pest populations. 

d. Development of alternative resistance management strategies. 

e. Suspension of YieldGard® CRW sales until an EPA-approved resistance management 

plan is in place. 

EPA’s review of the remedial action plan (U.S. EPA 2004b) found the framework of the plan to be 

acceptable. 

 ix. Compliance

Grower compliance with refuge and IRM requirements is a critical element for resistance management. 

Significant non-compliance with IRM among growers may increase the risk of resistance for Cry3Bb1 

corn. 
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MON 863 Compliance Assurance Program 

As a term of the MON 863 registration, Monsanto was required to develop and submit to EPA a 

compliance assurance program (CAP) to ensure grower adherence to IRM requirements. The terms of 

registration mandated a number of components for the compliance program including the following: 

 Grower Agreements: Contractual arrangement between the registrant and grower to obligate 

adherence to IRM requirements. 

 Annual IRM Survey: A survey (conducted anonymously by an independent research firm) 

intended to provide a statistically representative sample of growers 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 registrant is required to develop an on-site assessment program in 

which trained personnel from each company 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 registrant must establish a means for the reporting and investigation 

of incidences of refuge non-compliance. 

 Phased Compliance Approach (PCA): A consistent set of procedures (for all Bt corn 

registrants) to be employed to address non-compliance among growers and seed dealers. 

 

The Agricultural Biotechnology Stewardship Technical Committee (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 (see discussion in the U.S. EPA (2010b)). MON 863 (and 

other Cry3Bb1 registrations) 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 (described below) can be found in U.S. 

EPA (2004a, 2007, 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 grower survey functions to 

measure compliance adherence to refuge size and distance requirements at a regional level and to 

identify educational opportunities to increase grower compliance with IRM requirements. More than 

500 growers from 4 separate regions are anonymously surveyed annually. The methodology for 

conducting the grower survey has remained virtually unchanged since it was first conducted by 

Marketing Horizons, Incorporated 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 

standardize the grower survey across insect-protected traits, Marketing Horizons, Incorporated utilized 

an internet-based survey approach. 
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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 5 

below. Results from the stacked (lepidopteran + rootworm) Bt corn surveys are tabulated separately 

(Table 6) and also include all registered rootworm PIP traits. 

Table 5. Summary of Telephone (2005–2006) and Online (2007–2008) Survey Results for 

 Rootworm-Protected Bt Corn Growers. 

Survey Question 

20051

% Respondents 

20062

% Respondents 

2007 

% Respondents 

2008 

% Respondents 

Adherence to Refuge3

Size 93 89 80 74 

Adherence to Distance 

Requirements3 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 surveyed
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Table 6. 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 Size1 78 70 724

Adherence to Distance Requirements1 92 66 663

Awareness of IRM Requirements 95 96 97 

Unaided Recall of Refuge Size 59 62 and 552

 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 all four regions surveyed 2 First number listed is for ECB and the second 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 requirements for both single-trait 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 and stacked rootworm PIPs. Compliance with refuge proximity was 

lower; in 2008, ≤66% of rootworm PIP growers deployed refuges 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 below 40% in 2008. Refuge distance requirements for 

rootworm-protected Bt corn products may be more challenging for growers because the refuge must be 

deployed either within or immediately adjacent to the Bt field. Stacked products present additional 

challenges due to the need to plan either two refuges (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 the CAP that identifies individual 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 enhance 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 (for lepidopteran registrations) and has typically 

encompassed more than 2,000 growers per season (for all types of Bt corn). On-farm assessments for 

rootworm-protected PIPs (including Cry3Bb1 products) began in 2006. 

Data from the on-farm assessments (2006 through 2008) of rootworm-protected Bt corn PIPs are 
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summarized in Table 7 below. These on-farm assessments encompass all growers planting rootwormprotected PIPs, including varieties expressing the Cry3Bb1, mCry3A, and Cry34/35Ab1 toxins. Results 

for the on-farm assessments of stacked (lepidopteran + rootworm) PIPs are detailed in Table 8. The 

assessments do not have the statistical power associated with the consistently stratified and randomized 

telephone/on-line surveys and are not used to measure representative rates of non-compliance. In 2007 

and 2008, no information was provided regarding specific non-compliance with refuge size and 

distance. This information should be provided in future reports to be consistent with previously collected 

data and to illustrate how growers are out of compliance (U.S. EPA 2009a). 

Table 7. Cumulative Results for the On-Farm Assessments of Coleopteran-Protected Bt Corn 

 Growers (2006–2008)1

. 

2006 2007 2008 

Growers Assessed 395 247 134 

Refuge Distance Deviations2 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 other than refuge size or distance; thus, the total of refuge distance and size 

deviations does not equal the number of non-compliant growers. 

Table 8. Cumulative Results of the On-Farm Assessments of Stacked Bt Corn 

 (Rootworm + Corn Borer) Growers (2006–2008)1. 

2006 2007 2008 

Growers Assessed 600 1069 1799 

Refuge Distance Deviations 512 N/A N/A

Refuge Size Deviations 8 N/A N/A

Significant Deviations 45 (7.5%) 77 (7.2%) 863

Insignificant Deviations 16 33 (3.1%) 363

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 other than refuge size or distance; thus, the total of refuge distance and size 

deviations 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. 
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Tips and Complaints 

As required by the terms of registration, Bt corn registrants must have a “tips and complaints” system as 

a mechanism for individuals (e.g., growers, sales representatives, etc.) to report alleged instances of IRM 

non-compliance. The number of tips and complaints (summarized for all Bt corn registrations, including 

lepidopteran and rootworm varieties) received through 2008 is summarized in Table 9 below. Each of 

these growers identified through the tips and complaints mechanism were 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 9. Anonymous Tips and Complaints about Non-Compliance with IRM Requirements 

 (Data from ABSTC Annual Reports 2003 through 2008). 

Year Number of Tips and 

Complaints 

2003 0 

2004 0 

2005 5 

2006 3 

2007 14 

2008 5 

Phased Compliance Approach 

ABSTC’s CAP for lepidopteran- and rootworm-protected PIPs includes a standard set of procedures 

(shown in Table 10), known as the Phased Compliance Approach (PCA), which is to be used by 

registrants when responding to instances of grower non-compliance with the IRM requirements. The 

PCA also established a tiered approach for non-compliance with “significant” deviations and “other” 

deviations. For a 20% CRW refuge requirement (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 lepidoptera 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 triggered if fewer than 2/3 of the Bt corn fields are planted within 1⁄2 mile of a 

non-Bt corn refuge. However, this definition 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 for CRW could be “less than 2/3 of the non-Bt refuge is 

deployed adjacent 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 2007). This formula would also be applicable to combined refuges for 

stacked PIPs. 
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Table 10. 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 

years 

 

Under the PCA, sales are to be suspended to individual growers for one year after two years of 

significant deviations. Following the one-year suspension, growers will need to be requalified to 

purchase seeds. Growers identified as non-compliant (significant or other deviations) are required to 

receive a “compliance assessment contact” the following year under the PCA. Non-compliant growers 

are typically identified through the on-farm assessment program (see previous discussion in the “OnFarm Assessments” section). Table 11 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-compliance. Compliance data, including results of on-farm assessments and PCA 

activities, are detailed in U.S. EPA (2007 and 2009a). 
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Table 11. Reassessment of Rootworm-Protected and Stacked Bt Corn Growers Under the Phased 

 Compliance Approach (Taken from ABSTC Annual CAP Reports)1

. 

Year Reassessments2

 Significant 

Deviations3 

Loss of Access to 

Technology 

2006 62 0 14 

2007 82 0 0 

2008 134 0 0 1

 The data in this table includes both growers planting single-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 following season. Two successive years of significant 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. 

 

 x. Grower Education 

Growers are perhaps the most essential element for the implementation and success of any IRM plan as 

they will ultimately be responsible for ensuring that refuges are planted according to guidelines and that 

Bt fields are monitored for unexpected pest damage. Therefore, a program that educates growers as to 

the necessity of IRM and provides guidance as to how to deploy IRM should be an integral part of any 

resistance management strategy. The 2000 SAP also suggested that a comprehensive education program 

may help increase IRM compliance (U.S. EPA 2001). Ideally, the educational messages presented to 

growers should be consistent (among different registrants, if applicable for CRW) and reflect the most 

current resistance management guidelines. Specific examples of education tools for growers can include 

grower guides, technical bulletins, sales materials, training sessions, internet sites, toll-free numbers for 

questions or further information, and educational publications. 

Monsanto’s grower education program for MON 863 was reviewed in U.S. EPA (2004a) and found to 

be acceptable. Components of the educational program include a Technology Use Guide (for growers), a 

Seed Resource Guide (for seed dealers), an interactive CD-ROM, on-line training module (developed in 

cooperation with the National Corn Growers Association (NCGA)), and educational/promotional 

meetings where IRM issues will be discussed. 

3. MON 863 x MON 810 (YieldGard® Plus; EPA Reg. No. 524-545)

Monsanto has developed an IRM plan for YieldGard® Plus Corn. YieldGard® Plus is a stacked corn 

product that expresses Cry3Bb1 (MON 863) and Cry1Ab (MON 810) for control of corn rootworm and 

corn borers. An amendment to Monsanto’s IRM plan in Volume 1 was submitted to EPA on April 1, 

2003 (reviewed in U.S. EPA (2003b)). Since YieldGard® Plus expresses the Cry1Ab and Cry3Bb1 

proteins, an IRM plan needs to address both European corn borer and corn rootworm. Monsanto’s IRM 
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plan for YieldGard® Plus considers European corn borer and corn rootworm resistance management and 

takes a conservative approach when strategies differ between the target pests. 

Refuge Requirements 

Based upon growers’ agronomic practices and pesticide use, growers may plant one refuge for European 

corn borers and corn rootworms or separate refuges for each pest. A grower that adopts the common 

refuge option would be required to plant a minimum of a 20% non-Bt structured refuge adjacent to or 

within YieldGard® Plus Corn fields. Refuges acres should be planted as continuous blocks adjacent to 

or within fields, perimeter strips, or strips within YieldGard® Plus Corn fields. Monsanto is 

recommending that in-field strips should be at least four row and preferably six rows wide. Agronomic 

practices should be comparable for YieldGard® Plus Corn and refuge acres. For example, if 

YieldGard® Plus Corn acres are planted continuously or as first-year corn, then the non-Bt refuge acres 

should also be planted continuously or as first-year corn, respectively. Non-Bt insecticides may be 

applied to refuge acres to control corn root larvae but may only be applied to refuge acres when corn 

rootworm adults are present if YieldGard® Plus Corn acres are also treated. 

Growers that choose the separate refuge option must plant a distinct refuge for corn rootworm and 

European corn borer. A 20% non-corn rootworm-protected corn refuge must be planted to delay corn 

rootworm resistance to YieldGard® Plus Corn. An additional 20% non-Bt corn must also be planted to 

delay European corn borer resistance. The corn rootworm refuge must be planted with corn that does not 

contain the Cry3Bb1 protein. Corn that only contains the Cry1Ab protein, however, may be planted if a 

separate non-Bt corn refuge is planted to delay European corn borer resistance. The corn rootworm 

refuge should be planted as continuous blocks adjacent to or within fields, perimeter strips, or strips 

within YieldGard® Plus Corn (at least 4 rows), and utilize comparable agronomic practices as the 

YieldGard® Plus Corn acres. European corn borer refuges may be planted within fields as blocks or 

strips, adjacent to fields, or up to 1⁄2 mile from YieldGard® Plus Corn acres. Non-Bt insecticides may be 

applied to refuge acres to control corn rootworm larvae but may only be applied to refuge acres when 

corn rootworm adults are present if YieldGard® Plus Corn acres are also treated. Non-Bt insecticides 

may be applied to refuges to control the European corn borer, corn earworm, or southwestern corn borer 

if economic injury levels occur. The refuge requirements for YieldGard® Plus and stacked 

Cry3Bb1/Cry1Ab PIPs are summarized in Table 12. 

Resistance Monitoring 

Monsanto is required to monitor for pest resistance to the Cry1Ab and Cry3Bb1 proteins in YieldGard® 

Plus Corn. Monsanto has folded YieldGard® Plus Corn into the existing resistance monitoring plans for 

lepidoptera (Cry1Ab) and CRW (Cry3Bb1). A complete discussion of the resistance monitoring 

program for Cry1Ab and lepidopteran pests is included in the U.S. EPA (2010b). Resistance monitoring 

for Cry3Bb1 and CRW is described in section II(D)(2)(b)(vii) of this Biopesticides Registration Action 

Document (BRAD). 
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Remedial Action 

A Remedial Action Plan is required for YieldGard® Plus Corn and must be implemented if resistance is 

detected. This plan is the same as the remedial action plans developed for European corn borer (detailed 

in U.S. EPA (2010b)) and corn rootworm (described in section II(D)(2)(b)(viii) of this BRAD). 

Grower Agreements 

Growers are required to sign an agreement similar to the agreements growers currently sign to plant 

MON 810 or MON 863 corn. This signed agreement contractually obligates growers to comply with 

refuge requirements. 

Compliance Assurance Plan 

Monsanto is required to implement a CAP that will evaluate and promote grower compliance. 

YieldGard® Plus Corn is included with the ABSTC compliance program for Bt corn (described in 

section II(D)(2)(b)(ix) of this BRAD). 

 

Grower Education 

Grower education programs are required for YieldGard® Plus Corn. The elements of this program are 

the same as for MON 863 and MON 810 corn but must describe how to deploy refuges for both 

lepidopteran and rootworm pests. 

4. MON 88017 (YieldGard® VT RW; EPA Reg. No. 524-551) 

The Cry3Bb1 protein expressed in MON 88017 corn is functionally and physiologically similar to that 

expressed in MON 863. The proteins differ by only 1 amino acid of 653 (99.8% homology) and are 

expressed at comparable levels in the plant. To test for functional equivalence, Monsanto conducted 

susceptibility assays with Colorado potato beetle (CPB) and WCRW, as well as field efficacy tests 

against CRW larvae. The susceptibility assays involved diet incorporation of Cry3Bb1 from each hybrid 

to determine LC50 values for the test insects. The results were similar for both Cry3Bb1 variants: (1) for 

CPB, the MON 88017 variant (Cry3Bb1.pvzmir39) had an LC50 of 0.84 microgram per milliliter 

(μg/mL), while the MON 863 variant (Cry3Bb1.11098) had an LC50 of 0.95 μg/mL; (2) for WCRW, the 

LC50 was 139 μg/mL for the MON 88017 variant and 100 μg/mL for the MON 863 variant. Field 

efficacy trials were conducted with MON 88017, MON 863, and non-expressing control plants using 

artificial infestations of WCRW eggs. Efficacy was measured as protection against feeding damage 

using a root rating scale. Seven weeks after infestation, the root damage ratings (RDR = 0.12) were 

identical for MON 88017 and MON 863, both of which were significantly lower than the level of 

damage on the control plants (RDR = 1.47). 
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In addition to structural and functional analysis of the Cry3Bb1 toxin, Monsanto also determined protein 

expression levels in MON 88017 relative to those for MON 863. Using enzyme-linked immunosorbent 

assay (ELISA) techniques, leaf, root, pollen, silk, grain, and stover tissues were analyzed for the amount 

of Cry3Bb1 protein both in dry weight and fresh weight tissues. The results showed that the protein 

expression in MON 88017 was comparable to MON 863: expression was slightly higher in young leaf, 

stover, and silk tissues; slightly lower in pollen; and the same in forage, forage root, and grain tissues. 

Only the expression in silk was significantly different. When tracked through the growing season, the 

amount of Cry3Bb1 protein declined in MON 88017 leaf, whole plant, and root tissue in a manner 

similar to that observed for MON 863. 

The IRM plan developed for MON 863 corn is compatible with MON 88017 corn (MRID No. 461817-

01; reviewed in U.S. EPA (2005b)), and all aspects of the MON 863 IRM plan are to be followed for 

MON 88017. A complete description of the MON 863 (Cry3Bb1) IRM plan is found in section II(D)(2)

of this BRAD.

5. MON 88017 x MON 810 (YieldGard® VT Plus; EPA Reg. No. 524-552) 

Monsanto’s submission indicates that the Cry3Bb1 and Cry1Ab toxins expressed in MON 88017 x 

MON 810 are “physiologically and functionally” equivalent to that expressed in MON 863, MON 

88017, and MON 810. To demonstrate the physiological equivalence, Monsanto investigated the amino 

acid sequences of the Cry3Bb1 toxins produced in both MON 88017 and MON 863. The Cry3Bb1 

proteins produced in MON 88017 and MON 863 share an amino acid sequence identity of >99.8%, 

differing from one another by only 1 of 653 amino acids. Since the Cry1Ab toxin was introduced using 

conventional breeding with MON 810, the toxins in MON 88017 x MON 810 and MON 810 should be 

identical. To test for functional equivalence, field efficacy tests were conducted against CRW and ECB 

larvae. Four treatments were used: MON 88017 x MON 810, MON 88017, MON 810 (crossed with a 

glyphosate-tolerant hybrid), and a non-expressing control (a glyphosate-tolerant hybrid). For ECB, 

evaluations of natural infestations were used, which were supplemented by artificial infestations at the 

whorl stage. Damage (efficacy) was determined by assessing leaf damage (LDR) using the Modified 

Guthrie Scale (0 = no damage; 9 = high damage). CRW efficacy was also evaluated with artificial 

infestations of WCRW, which was done at the second leaf stage (V2). Damage was assessed using a 

RDR scale (Oelson Node Injury Scale). The results for ECB efficacy (tabulated after 21 days) showed 

that both MON 88017 x MON 810 and MON 810 alone had low amounts of leaf damage (LDR = 0.8 

and 0.9, respectively), while the MON 88017 alone and non-expressing control had significantly higher 

levels of damage (LDR = 2.7 for both). For WCRW (determined after 6–7 weeks), both MON 88017 x 

MON 810 and MON 88017 alone had significantly greater root protection (RDR = 0.1 for both) than 

MON 810 alone or the non-expressing control (RDR = 1.24 and 1.35, respectively). 

In addition to the structural and functional analysis of the Cry3Bb1 and Cry1Ab toxins, Monsanto also 

determined protein expression levels in MON 88017 x MON 810 relative to those for MON 88017 and 

MON 810. MON 88017 had been previously compared with MON 863 for Cry3Bb1 expression, which 

was found to be almost identical. Using ELISA techniques, young leaf, young root (Cry3Bb1 only), 

pollen (Cry3Bb1 only), forage (leaf), forage root (Cry3Bb1 only), and grain tissues were analyzed for 
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the amount of Cry3Bb1 and Cry1Ab protein both in dry weight and fresh weight tissues. The results 

showed that the Cry3Bb1 protein expression in MON 88017 x MON 810 was comparable to MON 

88017 in all tissues. Expression in MON 88017 x MON 810 was slightly lower in young root and grain 

tissues and was higher in all other tested tissues, though none of the differences were statistically 

significant. For Cry1Ab, expression in MON 88017 x MON 810 was also comparable to MON 810, 

with only slight insignificant differences in young leaf, forage leaf, and grain tissues. 

The IRM plan developed for YieldGard® Plus (MON 863 x MON 810), consisting of CRW (MON 863) 

and Lepidoptera (MON 810) components, is compatible with MON 88017 x MON 810 corn (MRID No. 

461850-01; reviewed in U.S. EPA (2005c)), and all aspects of the YieldGard® Plus IRM plan are to be 

applied to MON 88017 x MON 810. The refuge requirements for MON 88017 x MON 810 corn are 

summarized in Table 12. 
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Table 12. Comparison of Event MON 810 (Lepidopteran) and MON 863/88017 (Rootworm) IRM 

 Requirements for Monsanto’s Stacked Bt Corn PIPs (YieldGard® Plus and 

 YieldGard® VT Plus Corn). 

Requirements MON 810 

 (Lepidopteran Refuge)

MON 863/88017 

(Rootworm Refuge)

YieldGard® Plus/VT Plus 

Corn 

(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 & configuration 

requirements. 

Insecticides can be used in 

refuge to control ECB & 

southwestern corn borer 

when above economic 

thresholds. 

Microbial Bt insecticides are 

not allowed. 

Same corn rotation as YGRW

(e.g., first-year corn or corn 

followed by corn). 

Conventional insecticides or 

seed treatments can be used 

in the refuge to control CRW 

larvae & other soil pests. If 

the refuge is treated with a 

foliar insecticide labeled for 

CRW control when CRW 

adults are present, then 

YGRW also must be treated. 

(Not applicable) 

Same corn rotation as YG 

Plus (e.g., first-year corn or 

corn followed by corn). 

Conventional insecticides or 

seed treatments can be used 

in the refuge to control CRW 

larvae & other soil pests. If 

the refuge is treated with a 

foliar insecticide labeled for 

CRW control when CRW 

adults are present, then YG 

Plus also must be treated. 

Microbial Bt insecticides are 

not allowed. 

Refuge Corn 

Types

Conventional (Non-Bt) Conventional (Non-Bt) 

YGCB (a corn borer refuge 

planted 1⁄2 mile also will be 

required) 

Roundup Ready corn (NonBt)

Conventional (Non-Bt) 

YGCB (an additional refuge 

for corn borers will be 

required) 

Roundup Ready corn (NonBt)

YGCB = YieldGard® Corn Borer; Bt corn with lepidopteran corn borer protection only (i.e., MON 810).

YGRW = YieldGard® Rootworm; Bt corn with corn rootworm protection only (i.e., MON 863).

6. Conclusions

A 20% non-Bt corn refuge for Cry3Bb1 corn has been employed since the registration of MON 863 in 

2003. As a condition of the MON 863 registration, Monsanto was required to submit data on corn 

rootworm biology, ecology, susceptibility to Cry3Bb1, and genetics of potential resistance. These data, 

as well as other published literature, support the adequacy of the current IRM plan for MON 863, MON 
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863 x MON 810, MON 88017, and MON 88017 x MON 810. Since there are still uncertainties 

associated with potential rootworm resistance to the Cry3Bb1 toxins, however, the adequacy of the IRM 

strategy should be periodically reevaluated. 

The non-Bt corn refuge should be planted as continuous blocks adjacent to the MON 863 fields, as 

perimeter strips, or as non-transgenic strips planted within the transgenic field. A 20% non-Bt corn 

refuge is necessary to produce an adequate number of CRW susceptible to the Cry3Bb1 protein. 

Considering the limited movement of CRW larvae, planting refuges close to transgenic fields in large 

blocks is preferred to narrow strips (Gray 1999; Meinke et al. 2001). If a 20% refuge is planted as row 

strips within a corn field, then it should be planted as at least four or more consecutive rows (Hibbard et 

al. 2003). 

Seed and granular insecticide treatments to control CRW larvae are acceptable on refuge acres. It is not 

acceptable, however, to treat refuges for adult CRW control as these treatments may diminish the 

effectiveness of the refuge. If growers spray their corn fields with insecticides to control pests other than 

CRW, then all acres (Bt and non-Bt) should be treated identically. Bt fields and the non-Bt refuge acres 

should be treated with identical agronomic practices, such as irrigating all corn (Bt and non-Bt) at the 

same time. To ensure the production of similar numbers of CRW, Bt and non-Bt corn should be planted 

in fields with similar backgrounds. For example, if MON 863 hybrids are planted on continuous corn 

fields, then the non-Bt refuge should be planted on continuous corn fields or both should be planted on 

first-year corn acres. Likewise, non-Bt refuges should be planted on first-year corn fields if the MON 

863 hybrids are planted on first-year corn fields. 

7. IRM Terms and Conditions of Registration 

The terms and conditions for each of the Cry3Bb1 registrations contain a complete description of the 

IRM requirements. Details are provided on the requirements for refuge (size and structure), resistance 

monitoring, remedial action, compliance assurance, grower education, and annual IRM reports. For 

more information, please refer to the document, in Docket Number EPA-HQ-OPP-2010-0607, 

presenting the registration terms and conditions established with the 2010 amendments. 

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Roush RT, Miller GL. 1986. Considerations for design of insecticide resistance monitoring programs. 

J. Econ. Entomol. 79:293–298. 

Short DE, Luedtke RJ. 1970. Larval migration of the western corn rootworm. J. Econ. Entomol. 

 63:325–326. 

Siegfried BD, Vaughn TT, Spencer T. 2005. Baseline susceptibility of western corn rootworm 

 (Coleoptera: Chrysomelidae) to Cry3Bb1 Bacillus thuringiensis toxin. J. Econ. Entomol. 

 98:1320–1324. 

Spencer JL, Mabry TR, Vaughn TT. 2003. Use of transgenic plants to measure insect herbivore 

 movement. J. Econ. Entomol. 96:1738–1749. 

Spurgeon DW, Esquivel JF, Suh CP. 2004. Population patterns of Mexican corn rootworm (Coleoptera: 

 Chrysomelidae) adults indicated by different sampling methods. J. Econ. Entomol. 97:687–694. 

Strnad SP, Bergman MK. 1987. Movement of first-instar western corn rootworms (Coleoptera: 

 Chrysomelidae) larvae in corn roots. Environ. Entomol. 16:1193–1198. 

Suttle PJ, Musick GJ, Fairchild ML. 1967. Study of larval migration of the western corn rootworm. 

J. Econ. Entomol. 60:1226–1228. 

U.S. EPA. 2001. SAP Report No. 2000-07. Sets of Scientific Issues Being Considered by the 

Environmental Protection Agency Regarding: Bt Plant-Pesticides Risk and Benefit Assessments. 

Dated March 12, 2001. Available from: 

http://www.epa.gov/scipoly/sap/meetings/2000/october/octoberfinal.pdf. 

U.S. EPA. 2002a. Preliminary Review of Monsanto’s Interim Insect Resistance Management Plan for 

Bacillus thuringiensis Event MON 863 Corn Rootworm Protected Field Corn. Memorandum 

from R. Rose to M. Mendelsohn dated July 23, 2002. 

U.S. EPA. 2002b. SAP Meeting Minutes No. 2002-05. A Set of Scientific Issues Being Considered by 

the Environmental Protection Agency Regarding: Corn Rootworm Plant-Incorporated Protectant 

Non-Target Insect and Insect Resistance Management Issues. Dated November 6, 2002. 

Available from: http://www.epa.gov/scipoly/sap/meetings/2002/august/august2002final.pdf. 

U.S. EPA. 2003a. Insect Resistance Management Assessment for the Registration of Monsanto 

Company’s Cry3Bb1 Proteins Expressed in MON 863 Field Corn. Memorandum from R. Rose 

to M. Mendelsohn dated February 21, 2003. 
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U.S. EPA. 2003b. Review of Monsanto’s Application for a Section 3 Registration of Bacillus 

thuringiensis Cry1Ab and Cry3Bb1 Proteins and the Genetic Material Necessary for Their 

Production in YieldGard Plus Corn. Memorandum from R. Rose to M. Mendelsohn dated 

October 2, 2003. 

U.S. EPA. 2004a. Technical Review of Monsanto Company’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. 2004b. Technical Review of Monsanto’s YieldGard CRW (EPA Reg. No. 524-528) 

Remedial Action Plan (Submitted January 22, 2004). Memorandum from T. Milofsky to M. 

Mendelsohn dated August, 2004. 

U.S. EPA. 2004c. Review of Corn Rootworm Research Protocols and Progress Report for MON 863 

 Field Corn. Memorandum from A. Reynolds to M. Mendelsohn dated December 9, 2004. 

U.S. EPA. 2004d. Agency Review of Resistance Allele Frequency Data Submitted for MON 863 Field 

 Corn. Memorandum from A. Reynolds to M. Mendelsohn dated December 9, 2004. 

U.S. EPA. 2005a. Proposed Changes to the Refuge Requirements for YieldGard® Rootworm Corn 

 (524-528) and YieldGard® Plus Corn (524-545) Based on New Scientific Information. 

 Memorandum from S. Matten, Ph.D. to M. Mendelsohn dated July 13, 2005. 

U.S. EPA. 2005b. Review of Proposed Insect Resistance Management Plan and Benefits Information 

 Submitted by Monsanto for MON 88017 Bt Corn. Memorandum from A. Reynolds to 

 M. Mendelsohn dated October, 2005. 

U.S. EPA. 2005c. Review of Proposed Insect Resistance Management Plan and Benefits Information 

 Submitted by Monsanto for MON 88017 x MON 810 Bt corn. Memorandum from A. Reynolds 

 to M. Mendelsohn dated October, 2005. 

U.S. EPA. 2005d. Agency Review of Monsanto’s Amendment (December 1, 2005) to Change the In- 

 Field Strip Width Refuge Requirement for YieldGard® Rootworm Corn (524-528) and 

 YieldGard® Plus Corn (524-545) Based on New Scientific Information. Memorandum from 

 S. Matten, Ph.D. and T. Milofsky to M. Mendelsohn dated December 5, 2005. 

U.S. EPA. 2006a. Review of Monsanto’s YieldGard CRW (EPA Reg. No. 524-528) Remedial Action 

 and Resistance Monitoring Plans. Memorandum from T. Milofsky to M. Mendelsohn dated 

 June 1, 2006. 
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U.S. EPA. 2006b. Agency Review of Monsanto’s Interim Corn Rootworm IRM Research Progress 

 Report for MON 863, MRID No. 466066-01. Memorandum from S. Matten, Ph.D. to M. 

 Mendelsohn dated June 7, 2006. 

U.S. EPA. 2006c. Agency Review of Monsanto’s Final Corn Rootworm IRM Research Report for MON 

 863. Memorandum from S. Matten, Ph.D. to M. Mendelsohn dated June 16, 2006. 

U.S. EPA. 2007. Review of IRM Compliance Assurance Program (CAP) Components Covered in 

 ABSTC’s Submission on Behalf of Corn Borer-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 Rootworm-Protected) (EPA Reg. Nos. 524-545, 

 68467-6, and 29964-5) Bt Corn Registrations. Memorandum 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 Insect Resistance Management 

 Compliance Assurance Program [EPA Registration 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; MRID Nos. 473396-01 and 476633-01]. Memorandum from J. Martinez to M. 

 Mendelsohn dated April 15, 2009. 

U.S. EPA. 2009b. Review of Monsanto’s Corn Rootworm Monitoring Reports from 2005–2007 for 

Susceptibility to Cry3Bb1 and Revised Corn Rootworm Resistance Monitoring Plan for MON 

88017, MON 88017 x MON 810, and MON 89034 x MON 88017. Memorandum from 

J. Martinez to M. Mendelsohn dated August 24, 2009. 

U.S. EPA. 2010a. Review of Monsanto’s 2008 Corn Rootworm Monitoring Report and Revised Corn 

Rootworm Resistance Monitoring Plan for MON 88017, MON 88017 x MON 810, MON 863, 

MON 863 x MON810, MON 89034 x TC1507 x MON 88017 x DAS-59122-7, and MON 89034 

x MON 88017. Memorandum from J. Martinez to M. Mendelsohn dated June 30, 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 Materials” within Docket Number 

EPA-HQ-OPP-2010-0607). 

Ward DP. 2002. Response Dated December 13, 2002 to FIFRA Scientific Advisory Panel Report 

 Dated November 6, 2002. 

Weiss MJ, Seevers KP, Mayo ZB. 1985. Influence of western corn rootworm larval densities and 

 damage on corn rootworm survival, developmental time, size and sex ratio (Coleoptera: 

 Chrysomelidae). J. Kansas Entomol. Soc. 58(3):397–402. 
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Wilson TA, Hibbard BE. 2004. Host suitability of nonmaize agroecosystem grasses for the western corn 

 rootworm (Coleoptera: Chrysomelidae). Environ. Entomol. 33:1102–1108. 
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E. Benefits and Public Interest Findings for Initial Registrations of Corn Event MON 863 and 

 MON 88017 

1. Background

Corn is the largest cultivated crop grown in the U.S. in terms of acreage planted and net value. The crop 

was planted on 79.5 million acres in the year 2000, yielding 10 billion bushels with a net value of $18.4 

billion. There are nearly 410 million acres of agricultural land being used to grow crops nationwide, 

including Conservation Reserve Program lands. Corn rootworm (CRW, Diabrotica spp.) is one of a 

spectrum of insect pests that a farmer may choose to control. Other insect pests include cutworms, 

wireworms, white grubs, flea beetles, seedcorn maggots, black cutworms, and corn borer species. CRW, 

however, is one of the most damaging insect pests of corn and is responsible for economic damages 

(costs associated with insecticides and crop losses) totaling nearly $1 billion (Gray 2000). The three 

CRW control methods have been (1) use of crop rotation, (2) soil-applied insecticides, and (3) limited 

use of rescue-treatments for CRW adult beetles. 

MON 863 and MON 88017 corn rootworm-protected corn both contain the cry3Bb1 gene that produces 

the insecticidal crystal protein, Cry3Bb1. MON 863 also has a nptII marker gene that encodes neomycin 

phosphotransferase II, while MON 88017 has a cp4 epsps gene that encodes CP4 5-

enolpyruvylshikimate-3-phosphate synthase, conferring resistance to glyphosate. Cry3Bb1 corn is 

targeted against the CRW complex, comprised primarily of the northern corn rootworm (NCRW, 

Diabrotica barberi Smith and Lawrence), western corn rootworm (WCRW, Diabrotica virgifera 

virgifera LeConte), and Mexican corn rootworm (MCRW, Diabrotica virgifera zeae Krysan and Smith). 

One additional Diabrotica species, the southern corn rootworm (SCRW, Diabrotica undecimpunctata 

howardi Barber) is considered a relatively minor pest of corn that inhabits the southeastern coastal 

regions of the U.S. CRW accounts for more chemical pesticide usage on corn than does any other pest; 

approximately 28 million acres of corn are infested with CRW. In the year 2000, approximately 8 

million pounds of insecticidal active ingredient, costing $172 million, were applied to 14 million acres 

of corn to reduce CRW damage. There were approximately 24 million acres of corn treated with 

insecticides for CRW and other pests (e.g., grubs, maggots, cutworms, wireworms). The National 

Agricultural Statistics Service’s figures from 2001 indicate that 9.8 million pounds of insecticide active 

ingredients, specifically registered for CRW control, were applied on more than 31% of the planted 

acres. Left untreated, CRW can cause severe yield loss, typically in the range from 8% to 16%, although 

reductions in yield may be as high as 28%. 

2. 2003 Corn Event MON 863 Public Interest Finding (Reviewed in U.S. EPA (2003a and 2003b)) 

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 pesticide 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 another pesticide that is of 

continuing concern to the Agency; (3) the use is one for which an emergency exemption under section 

18 of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) has been granted (i.e., the basis 
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for the exemption was lack of a registered alternative product); or (4) the use is against a pest of public 

health significance. Further, the Environmental Protection Agency (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 currently registered pesticides; (2) the new pesticide is comparatively 

less risky to health or the environment than currently registered pesticides; or (3) the benefits (including 

economic benefits) from the use of the new active ingredient exceed those of alternative registered 

pesticides and other available non-chemical techniques. 

EPA determines that conditional registration of MON 863 is in the public interest given conventional 

chemicals, currently used for CRW control, that are of continuing concern to the Agency as indicated by 

the following factors: 

1. Special review. Certain pesticides that are used for CRW control have been reviewed under 

EPA Special Review (40 Code of Federal Regulations (CFR) Part 154) because the use of these 

pesticides may result in unreasonable adverse effects to humans or the environment. The 

pesticides in this category are dimethoate, phorate, and terbufos; terbufos and phorate are being 

evaluated for reregistration. 

2. Acute avian risk from granular pesticides. In 1992, EPA issued an analysis that indentified 

14 granular pesticides believed to pose potentially higher risk of killing birds due to their acute 

toxicity and availability in the environment (U.S. EPA 1992). Among these granular pesticides 

are several that are still used for CRW control: carbofuran (no longer used as a granular but still 

a pesticide of Agency concern), phorate, terbufos, and chlorpyrifos. The results of the Agency’s 

initiative to reduce exposure to these highly toxic granular pesticides are presented in the 1994 

EPA report, “Avian Granular Risk Reduction Initiative.” 

3. Restricted use. Many of the granular pesticides used for CRW control have been classified as 

restricted use (due to adverse environmental effects under use practices), limiting the use of these 

chemicals to certified pesticide applicators. Pesticides classified as restricted use include 

chloroethoxyfos, phorate, terbufos, tefluthrin, and the commercial combination of tebupirimfos 

and cyfluthrin (i.e., Aztec®). 

4. Food Quality Protection Act of 1996. EPA must reassess all existing tolerances to be sure 

that they meet the standard of “reasonable certainty of no harm.” The EPA is required to first 

consider those pesticides that pose the highest risk to humans. EPA is reviewing the 

organophosphate and carbamate pesticides because of their known risk of acute and chronic 

toxicity to humans and wildlife. The organophosphates and carbamates share the same mode of 

action. The organophosphate insecticides used for CRW control include chlorpyrifos, terbufos, 

phorate, chloroethoxyfos, dimethoate, and tebupirimphos, while a carbamate insecticide used for 

CRW control is carbofuran. 
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In addition, EPA also determines, in accordance with the criteria set forth in the Federal Register Notice 

dated March 5, 1986, that MON 863 qualifies for a positive public interest finding. To qualify for a 

positive public interest finding, the product must demonstrate advantages in terms of the need for the 

chemical and its comparative benefits, risks, and costs. Monsanto Company (“Monsanto”) has submitted 

two public interest documents and other supporting documents that present the potential benefits of 

MON 863 (Master Record Identification Numbers (MRID Nos.) 450297-01, 456530-01, 456530-02, 

453613-03, 455382-08, 456923-01, 455770-01). EPA has reviewed the submitted documents, public 

comments, syndicated marketing research studies, and published information. The potential benefits 

have been identified and evaluated 

The major proposed benefits of MON 863 corn for CRW control are as follows: 

 Safer for handlers, applicators, growers, and the public than current chemical alternatives 

 Safer for the environment than use of available chemical pesticides 

 Easier and less time consuming for farmers to use than current control options 

 Comparable or improved efficacy relative to the current chemical alternatives 

 Yield benefits 

 Reduced use of current higher risk chemical alternatives 

 Economic benefits to farmers from increased yields and decreased cost of rootworm control as 

 compared with conventional control 

The use of MON 863 CRW-protected corn is presumed to be in the public interest because it will 

replace or reduce the use of a number of higher risk pesticides for CRW control that are of Agency 

concern as discussed previously (e.g., terbufos, chlorpyrifos, and phorate). Additionally, MON 863 also 

has clearly identified benefits. Therefore, EPA concludes that the use of MON 863 CRW-protected corn 

is in the public interest and supports the conditional registration of MON 863 under FIFRA section 

3(c)(7)(C). 

a. Characterization and Use of Chemical Insecticides to Control Corn Rootworm

Three CRW control methods have been used for decades: (1) crop rotation (typically with soybeans), (2) 

soil-applied insecticides to control larvae (approximately 90% of the total CRW-treated acres), and (3) 

use of adulticides to control CRW adult beetles (approximately 10% of the total CRW-treated 

acres)(Levine and Oloumi-Sadeghi 1991). Greater than 90% of the growers use soil-applied insecticides, 

applied at planting to control larvae, due to greater efficacy and ease of application. Historically, crop 

rotation has been the primary method of controlling CRW (Levine and Oloumi-Sadeghi 1991). Crop 

rotation, however, is now far less effective because of the existence of a WCRW soybean rotational 

variant, primarily in Eastern Illinois and Western Indiana, that colonizes soybeans (Levine and OloumiSadeghi 1991; Levine et al. 1992a) and a NCRW extended diapause (2 year extended) variant, primarily 

in parts of Minnesota, Iowa, and South Dakota (Krysan et al. 1986; Levine et al. 1992b). In addition, 

CRW has developed resistance to methyl parathion and carbaryl, both adulticides used in rescue 

treatments (Meinke et al. 1998). Therefore, growers have become increasingly dependent on chemical 

pesticides to limit CRW losses. EPA has registered 36 insecticide products for control of CRW (see 
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Table 1). The insecticides used to control corn rootworm in conventionally grown (non-Bt) corn consist 

mainly of organophosphates (9), carbamates (3), synthetic pyrethroids (6), and phenyl pyrazole (1) 

classes of chemistry. Twenty-five products are classified as “restricted use.” All 36 insecticides are toxic 

to extremely toxic to fish, aquatic invertebrates, bees, and/or wildlife. Three products have been 

involved in the Agency’s Special Review process: dimethoate, phorate, and terbufos. Twenty-six of 

these products contain active ingredients either from the organophosphate or carbamate classes, both of 

which are considered to be top priorities under the Food Quality Protection Act (FQPA) and tolerance 

reassessment. Terbufos, phorate, chlorpyrifos, diazinon, ethoprop, carbofuran, and methomyl have 

presented varying levels of concern regarding avian, aquatic, and mammalian risk. EPA has specified 

specific risk mitigation measures for all of these chemicals and, in many cases, certain uses have been 

eliminated because of either human or environmental risk concerns. 
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Table 1. Insecticide End-Use Products Registered by EPA for Use on Corn for Control of Corn Rootworm S

 Pages 18-21 of MRID No. 456530-01 and Verified by EPA).

Product 

Active 

Ingredients Typea Use Rateb Use Classification

Ambush® Insecticide – Syngenta 

permethrin – 

25.6% SP 0.2 lb/acre Adult control WARNING. Restricted Use; extrem

aquatic invertebrates, highly toxic to b

AsanaXL Insecticide 0.66 Emulsifiable Concentrate – DuPont 

esfenvalerate – 

8.4% SP 0.05 lb/acre Adult control WARNING. Restricted Use; extrem

aquatic invertebrates, highly toxic to b

Aztec2.1% Granular Insecticide – Bayer 

tebupirimfos – 

2.0% 

cyfluthrin – 0.1% 

OP 

SP 

0.15 lb/acre 

0.01 lb/acre 

Larval 

control WARNING. Restricted Use; toxic t

Baythroid2 Emulsifiable Pyrethroid Insecticide – Bayer 

cyfluthrin – 25% SP 0.04 lb/acre Adult control DANGER. Restricted Use; extreme

aquatic invertebrates, highly toxic to bskin reactions 

Capture2EC Insecticide/Miticide – FMC 

bifenthrin – 

25.1% SP 0.3 lb/acre Larval control WARNING. Restricted Use; extrem

aquatic invertebrates, highly toxic to b

Chlorfos15G Insecticide Granular – Griffin LLC 

chlorpyrifos – 

15% OP 2.02 lb/acre Larval control CAUTION. Toxic to birds and wildl

fish and aquatic organisms 

Chlorfos4E Insecticide – Griffin LLC 

chlorpyrifos – 

42% OP 2.52 lb/acre 

Adult & 

Larval 

control 

WARNING. Toxic to birds and wildfish and aquatic organisms 

CounterCR Systemic Insecticide-Nematicide – American Cyanamid Company 

terbufos – 20% OP 1.30 lb/acre Larval control DANGER. Restricted Use; fatal if s

absorbed through skin, extremely toxi
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Product 

Active 

Ingredients Typea Use Rateb Use Classification

D-z-nDiazinon AG500 Insecticide – Syngenta diazinon – 48% OP 0.48 lb/acre Adult control CAUTION. Restricted Use; highly t

other wildlife, highly toxic to bees 

D-z-nDiazinon AG600 WBC Insecticide – Syngenta 

diazinon – 56% OP 0.45 lb/acre Adult control CAUTION. Restricted Use; highly t

other wildlife, highly toxic to bees 

DeclareEmulsifiable Insecticide Concentrate – Griffin LLC 

methyl parathion 

– 45.11% OP 0.22 lb/acre Adult control DANGER. Restricted Use; fatal if s

absorbed through skin, highly toxic to wildlife, highly toxic to bees

Diazinon 500-AG Organophosphate Insecticide – UAP

diazinon – 48% OP 0.48 lb/acre Adult control CAUTION. Restricted Use; highly t

other wildlife, highly toxic to bees

Dimethoate 4 EC Systemic Insecticide – Helena 

dimethoate – 

44.8% OP 0.45 lb/acre Adult control WARNING. Toxic to wildlife and aq

highly toxic to bees 

Dimethoate 400 Systemic Insecticide-Miticide – UAP 

dimethoate – 

43.5% OP 0.44 lb/acre Adult control WARNING. Toxic to wildlife and aq

highly toxic to bees 

5 lb Dimethoate Systemic Insecticide – Helena 

dimethoate – 

57% OP 0.46 lb/acre Adult control DANGER. Toxic to wildlife and aqu

toxic to bees 

Force3G Insecticide – Syngenta tefluthrin – 3% SP 0.17 lb/acre Larval control CAUTION. Restricted Use; very hi

and estuarine fish and invertebrates 

Fortress2.5G Granular Insecticide – DuPont 

chlorethoxyfos – 

2.5% OP 0.16 lb/acre Larval control DANGER. Restricted Use; toxic to 

fish, and aquatic invertebrates 
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Product 

Active 

Ingredients Typea Use Rateb Use Classification

Fortress5G Granular Insecticide – DuPont 

chlorethoxyfos – 

5% OP 0.16 lb/acre Larval control DANGER. Restricted Use; toxic to 

fish, and aquatic invertebrates 

Furadan4F Insecticide/ Nematicide – FMC 

carbofuran – 

44% C 0.88 lb/acre 

Adult & 

larval 

control 

DANGER. Restricted Use; poisonouinhaled, toxic to fish, birds, and other bees, can seep or leach through soil angroundwater 

LannateLV Insecticide – DuPont 

methomyl – 29% C 0.65 lb/acre Adult control DANGER. Restricted Use; fatal if s

aquatic invertebrates, and mammals, hknown to leach through soil into grou

LannateSP Insecticide – DuPont methomyl – 90% C 0.45 lb/acre Adult control 

DANGER. Restricted Use; fatal if sblindness, toxic to fish, aquatic inverthighly toxic to bees, known to leach thgroundwater 

Lorsban15G Granular Insecticide – Dow AgroSciences 

chlorpyrifos – 

15% OP 2.03 lb/acre Larval control CAUTION. Toxic to birds and wildl

fish and aquatic organisms 

Lorsban-4E Insecticide – Dow AgroSciences 

chlorpyrifos – 

44.9% OP 2.69 lb/acre 

Adult & 

larval 

control 

WARNING. Toxic to birds and wildfish and aquatic organisms 

Mocap10% Granular Nematicide Insecticide – Aventis CropScience 

ethoprop – 10% OP 3.53 lb/acre Larval control WARNING. Toxic to aquatic organi
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Product 

Active 

Ingredients Typea Use Rateb Use Classification

MocapEC NematicideInsecticide – Aventis CropScience 

ethoprop – 

69.6% OP 3.34 lb/acre Larval control DANGER. Restricted Use; toxic to 

extremely toxic to birds 

PenncapMMicroencapsulated Insecticide – Elf Atochem 

methyl parathion 

– 22% OP 0.44 lb/acre Adult control WARNING. Restricted Use; highly 

invertebrates and wildlife 

Phorate 20 G Organophosphate Insecticide – UAP 

phorate – 20% OP 1.3 lb/acre 

Adult & 

larval 

control 

DANGER. Restricted Use; extremewildlife

Pounce® WSB Insecticide– FMC Corporation 

permethrin – 

24.7% SP 0.2 lb/acre Adult control WARNING. Restricted Use; extrem

aquatic invertebrates, highly toxic to b

Pounce® 3.2 EC Insecticide – FMC Corporation 

permethrin – 

38.4% SP 0.2 lb/acre Adult control CAUTION. Restricted Use; extreme

aquatic invertebrates, highly toxic to b

Pounce® 25 WP Insecticide – FMC Corporation 

permethrin – 

25% SP 0.2 lb/acre Adult control WARNING. Restricted Use; extrem

aquatic invertebrates, highly toxic to b

Regent4 SC Insecticide – Aventis CropScience fipronil – 39.4% PP 0.13 lb/acre Larval control WARNING. Restricted Use; toxic t

invertebrates 

SevinBrand 80S Carbaryl Insecticide – Aventis CropScience 

carbaryl – 80% C 2.0 lb/acre Adult control WARNING. Extremely toxic to aqua

invertebrates, highly toxic to bees 

SevinBrand XLR PLUS Carbaryl Insecticide – Aventis CropScience 

carbaryl – 44.1% C 1.76 lb/acre Adult control CAUTION. Extremely toxic to aqua

invertebrates, highly toxic to bees 
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Product 

Active 

Ingredients Typea Use Rateb Use Classification

Thimet20-G Soil and Systemic Insecticide – American Cyanamid 

phorate – 20% OP 1.3 lb/acre Larval control DANGER. Restricted Use; extreme

wildlife 

Thimet20-G Soil and Systemic Insecticide – American Cyanamid 

phorate – 20% OP 1.3 lb/acre Larval control DANGER. Restricted Use; extreme

wildlife 

Warrior-Insecticide with Zeon Technology – Syngenta 

lambdacyhalothrin – 

11.4% 

SP 0.03 lb/acre Adult control WARNING. Restricted Use; extrem

aquatic organisms and toxic to wildlif

a – OP: organophosphate; SP: synthetic pyrethroid; C: carbamate; PP: phenyl pyrazole b – maximum labeled use rate expressed in pounds of active ingredient per acre (assume that 1 liquid pt = 1 pound) c – precautionary language as stated on label
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In the year 2000, approximately 8 million pounds of insecticidal active ingredient, costing $172 million, 

were applied to 14 million acres of corn to reduce CRW damage (see Table 2 below). These data were 

independently verified by EPA. There were approximately 24 million acres of corn treated with 

insecticides; growers indicated that CRW was the sole target pest on approximately 7 million acres and 

one of a number of pests on the remaining treated acres. CRW-targeted acres (14 million) that received 

an insecticide treatment represented 18% of the total acres of corn planted in the continental U.S. and 

59% of the total acres receiving any insecticide treatment in 2000. Continuous corn and first-year corn 

acres (rotated acres) received 58% and 42% of the CRW-targeted insecticide applications, respectively. 

Continuous corn use areas include western Iowa, Nebraska, eastern Colorado, eastern South Dakota, the 

panhandle areas of Texas and Oklahoma, northeastern New Mexico, and southern Minnesota. Rotational 

acres are located predominantly in eastern Iowa, most of Minnesota, Wisconsin, Missouri, Illinois, 

Indiana, Michigan and other areas of the Eastern Corn Belt. 

The CRW treatments on 14 million acres of corn do not include acreage where the expected level of 

infestation is below the economic threshold (i.e., where the expected loss is less than the $15.00 cost of 

treatment). National estimates of infested acreage are not published (unlike the Cotton Council, for 

example, that publishes estimates of infested acreage). Market analyses estimate all infested acreage 

with CRW to be around 28 million acres. This estimate is reasonable with what could be expected on the 

basis of yield losses without treatment and prices in year 2000. At depressed corn prices of $2 per bushel 

and yields at 175 bushels/acre, a maximum yield loss of 8.5% would result in a $30 loss. Half of the 

infested acres would be below the $15.00 cost of treatment. If corn prices rise or the cost of control 

decreases, the percent of infested acres that is treated would likely increase. 

The infested acreage is expected to grow by 2.6% per year, implying that the 28 million infested acres in 

year 2000 will grow to 39 million infested acres by the year 2013. These factors need to be considered 

when projecting the amount of chemical acre treatments and adopted acres of MON 863. 

Table 2. Insecticide Usage on Corn in Year 2000 (Reprinted from Page 22 of 

 MRID No. 456530-01).

Parameter 

Continuous 

Corn 

First-Year 

Corn All Corn 

Acres planted (x1,000) 22,269 57,310 79,579 

Total insecticide-treated acres (x1,000) 11,590 12,518 24,108 

Total insecticide active ingredient applied 

(lb x1,000) 6,332 6,011 12,343 

CRW-targeted acres (x1,000) 8,271 5,926 14,197 

Active ingredient applied to CRW-targeted acres 

(lb x1,000) 4,699 3,137 7,836 

Average active ingredient rate applied (lb/ac) 0.568 0.529 0.552 

Average cost per acre $11.95 $12.27 $12.08 

Total cost of CRW insecticide purchased (x1,000) $98,811 $72,699 $171,510 
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Only a few of the 36 products listed in Table 1, registered for control of CRW, dominate the market. 

Table 3 below shows that five active ingredients (tefluthrin, chlorpyrifos, terbufos, fipronil, and 

cyfluthrin/tebupirimfos) are applied to 86% of the acres treated. Three of these active ingredients are 

organophosphates and three are classified as restricted use. In terms of pounds of insecticide applied, 

chlorpyrifos and terbufos (both organophosphates), account for 77% of the total insecticide products 

(almost 6 million pounds) applied to CRW-targeted acres. These 11 active ingredients (in Table 3) 

accounted for 98.3% of the total quantity of insecticide applied to CRW-targeted acres in 2000. 

Table 3. Insecticide Active Ingredients Applied to Corn Rootworm-Targeted Acres in Year 2000 

 (Reprinted from page 24 of MRID No. 456530-01). 

Active Ingredient Acres Treated 

(x1,000) Pounds Applied (Formulated Product)

Carbofuran 342 242,379 

Chlorethoxyfos 361 55,485 

Chlorpyrifos 3,557 3,765,310 

Cyfluthrin/Tebupirimfos 1,326 179,527 

Fipronil 1,498 158,141 

Lambda-cyhalothrin 179 3,846 

Methyl parathion 367 142,011 

Permethrin 246 24,344 

Phorate 508 588,380 

Tefluthrin 3,570 400,339 

Terbufos 2,044 2,146,761 

Total 13,998 7,706,523 

Table 4 provides a listing of the 13 major end-use products that are applied to CRW-targeted areas 

(Table 1 lists end-use products that are labeled for CRW control). The use of five products—Aztec 2.1% 

Granular Insecticide, Counter CR Systemic Insecticide-Nematicide, Force 3G Insecticide, Lorsban 15G 

Granular Insecticide, and Regent 4 SC Insecticide—accounted for applications to 83% of the CRWtargeted areas. These products are all for larvicide rather than adulticide use. All of these products are 

restricted use, except Lorsban 15G Granular Insecticide. A small number of seed-applied insecticides 
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have been recently approved for use: Gaucho seed-applied insecticide (Gustafson, LLC), Prescribe seedapplied insecticide (Gustafson, LLC), and Force ST seed-applied insecticide (Syngenta). Imidacloprid is 

the active ingredient in Gaucho and Prescribe, and tefluthrin is the active ingredient in Force ST. MRID 

No. 456530-01 describes that the performance of these seed-applied insecticides is inconsistent and 

weak under conditions of high CRW pressure and, further, that these products do not perform as well as 

most soil-applied insecticides. 

Table 4. Insecticide End-Use Products Used for Control of Corn Rootworm in Year 2000 

 (Reprinted from page 25 of MRID No. 456530-01) 

Producta Average Cost 

($/A) 

Adult (A) 

Control 

Larval (L) 

Control 

EPA 

Classification 

Acres Treated 

(x1,000) 

Aztec 2.1% Granular Insecticide

(tebupirimfos/cyfluthrin)

$13.05 L Restricted 1,327 

Counter CR Systemic 

Insecticide-Nematicide (terbufos) 

$13.10–$13.50 L Restricted 2,044 

Force 3G Insecticide (tefluthrin) $14.48 L Restricted 3,570 

Fortress 5G Granular Insecticide

(chlorethoxyfor) 

$14.65 L Restricted 361 

Furadan 4F Insecticide/

Nematicide (carbofuran) 

$11.74 L Restricted 342 

Lorsban 15G Granular Insecticide

(chlorpyrifos) 

$11.79 L Unrestricted 3,165 

Lorsban 4E Insecticide

(chlorpyrifos) 

$10.52 A Unrestricted 374 

Penncap-M Microencapsulated 

Insecticide (methyl parathion) 

$6.79 A Restricted 330 

Pounce 3.2 EC Insecticide

(permethrin) 

$4.42 A Restricted 224 
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Producta Average Cost 

($/A) 

Adult (A) 

Control 

Larval (L) 

Control 

EPA 

Classification 

Acres Treated 

(x1,000) 

Regent 4 SC Insecticide (fipronil) $14.65 L Restricted 1,392 

Regent 80 WG Insecticide (fipronil) $8.57 L Restricted 106 

Thimet 20-G Soil and Systemic 

Insecticide (phorate) 

$10.90–$12.74 L Restricted 508 

Warrior Insecticide with Zeon 

Technology (lambda-cyhalothrin) 

$7.37 A Restricted 173 

Total 

Adult Control $4.42–$10.52 8% 

Larval Control $8.57–$14.65 92% 

Restricted Use 73% 

Unrestricted 27% 

 a – active ingredient stated in parentheses 

 b. Comparative Toxicity to Humans ( MRID Nos. 456530-01 and 450297-01)

MON 863 CRW-protected corn is safer for handlers, applicators, farmers, and the public than chemical 

pesticides in current use. Adoption of MON 863 corn hybrids will reduce the occupational, farmer, and 

public risks associated with the manufacture, transportation, storage, handling, application, and disposal 

of conventional insecticides. Many comments were received concerned with the potential contact to 

growers, their families, and communities with the application, drift, and on-farm storage of toxic 

materials. At product maturity, MON 863 hybrids have the potential to reduce insecticide applications 

by millions of pounds. This reduction of insecticide use will lead to both reduced human and 

environmental risks. The potential insecticide use reduction caused by adoption of MON 863 corn 

hybrids is discussed in section II(E)(2)(g) of this Biopesticides Registration Action Document (BRAD). 
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Virtually all of the registered conventional insecticides used to control CRW are of special concern to 

EPA because of risks to humans (see Table 1 and previous discussion in section II(E)(2) of this BRAD). 

Of the 36 insecticides registered for CRW control and listed in Table 1, 25 are classified as “Restricted 

Use” and 12 have the “Danger” label classification. These include products formulated with the 

following active ingredients: chloroethoxyfos, phorate, terbufos, tefluthrin, and the commercial 

combination of tebupirimfos and cyfluthrin (i.e., Aztec®). Each year there are confirmed reports of 

human illness associated with the registered chemical insecticide alternatives. Several of the current 

CRW insecticides are in Agency Special Review (i.e., dimethoate, phorate, and terbufos). Twenty-six of 

the 36 products contain either organophosphate or carbamate active ingredients, which are listed as top 

priorities for tolerance reassessment under FQPA because of their high risk to humans and the 

environment. Because of EPA’s concern with the conventional insecticide alternatives for CRW control, 

special precautions are required during all stages of their life cycle, including manufacture, 

transportation, storage, use, and disposal. 

By contrast, MON 863 corn presents minimal or no risks to humans during any stage of its life cycle, 

from production to ingestion to disposal. Unlike the conventional insecticide alternatives that require a 

tolerance (maximum allowable level of pesticide residue in food), Cry3Bb1 protein and the genetic 

material necessary for its production in corn has been exempted from the requirement of a tolerance 

given the conclusions set forth in the human health risk assessment (see section II(B) of this BRAD for 

additional details). Lastly, use of this new pesticide could potentially reduce use of CRW chemical 

pesticides by millions of pounds per year. 

 c. Comparative Toxicity and Potential for Adverse Environmental Effects 

 (MRID Nos. 456530-01 and 450297-01)

All of the major chemicals used for CRW control can cause major adverse environmental effects under 

conditions of normal use (see Table 1 and previous discussion in section II(E)(2) of this BRAD). These 

products are formulated with the following active ingredients: chlorethoxyfos, phorate, terbufos, 

tefluthrin, methyl parathion, carbofuran, fipronil, bifenthrin, cyfluthrin, esfenvalerate, permethrin, 

diazinon, chlorpyrifos, dimethoate, methomyl, ethoprop, carbaryl, lambda-cyhalothrin, and the 

commercial combination of tebupirimfos and cyfluthrin (i.e., Aztec®). Fifteen products are labeled as 

“toxic,” 6 as “highly toxic,” 1 as “very highly toxic,” and 14 as “extremely toxic” to birds, fish, and 

other wildlife. Each year there are confirmed reports of fish and bird poisonings associated with the 

registered chemical insecticide alternatives. Environmental effects from CRW chemical pesticides 

include toxicity and mortality in fish, birds, terrestrial mammals, aquatic invertebrates, and non-target 

insects. These chemicals can also spread via spray drift and runoff, thus contaminating both land and 

water bodies and impacting non-target organisms. Of the 36 insecticides registered for CRW control 

listed in Table 1, 25 are classified as “Restricted Use” and 12 have the “Danger” label classification. 

Table 5 compared the ecological risk for selected endpoints for the top three CRW insecticides: terbufos, 

chlorpyrifos, and tefluthrin. Together these three insecticides account for 63% of the acres treated (see 

Table 4). Tefluthrin poses lower risk than either chlorpyrifos or terbufos (see Table 5). 
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Table 5. Comparison of Ecological Risks Associated with Terbufos, Chlorpyrifos, and 

 Tefluthrin. 

Endpoint Terbufosa Chlorpyrifosa Tefluthrina

Mammalian Acute RQ 50 1 0.008 

Avian Acute RQ 0.27 0.55 0.0001 

Fish acute RQ 11 2 0.77 

Freshwater invertebrates RQ 50 20 0.77 

Marine/Estuarine Invertebrates RQ 53 162 0.87 

 a Risk is defined as the risk quotient (RQ) >level of concern (LOC). 

 RQ = Toxicity/Exposure. LOC = 1 

Potential adverse effects on non-target organisms, resulting from the exposure to Cry3Bb1 protein, have 

been evaluated in a series of studies with representative avian, aquatic, and terrestrial beneficial 

invertebrate species as discussed in section II(C) of this BRAD. 
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Table 6. Summary of Results from Ecological Effects Tests with the Cry3Bb1 Proteins (Reprinted from pag MRID No. 456530-01). 

Test Organism Test 

Substance 

Resultsa Conclusionsb 

Cladoceran 

(Daphnia magna) 

Pollen NOEC 2.26 μg/L NOEC 141x surface water MEEC 

Collembola 

(Folsomia candida) 

Leaf NOEC 872.5 μg/g NOEC 66x soil MEEC 

Channel Catfish 

(Ictalurus punctatus ) 

Grain No effect on growth or survival at 35% of diet No significant risk 

Bobwhite Quail 

(Colinus virginianus) 

Grain No effect on growth or survival at 10% of diet No significant risk 

Adult Honey Bee 

(Apis mellifera) Purified protein NOEC 360 μg/mL NOEC 3.8x maximum pollen level 

Larval Honey Bee 

(Apis mellifera) 

Purified protein NOEC 1,790 μg/mL as a single dose NOEC 19x maximum pollen level 

Adult Ladybird Beetle 

(Hippodamia convergens) 

Purified protein NOEC 8,000 μg/g NOEC 86x maximum pollen level 
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Test Organism Test 

Substance 

Resultsa Conclusionsb 

Adult Ladybird Beetle 

(Hippodamia convergens) Pollen No effect on growth or behavior at 50% of diet No significant risk 

Larval Ladybird Beetle 

(Coleomegilla maculata) Pollen No effect on growth or survival at 50% of diet No significant risk 

Adult Ladybird Beetle 

(Coleomegilla maculata) Pollen No effect on survival at 50% of diet No significant risk 

Monarch Butterfly Larvae 

(Danaus plexippus) Pollen No effect on growth or survival No significant risk 

Green Lacewing 

Larvae (Chrysoperla carnea) Purified protein NOEC 8000 μg/g NOEC 86x maximum pollen level 

Parasitic Hymenoptera 

(Nasonia vitripennis) Purified protein NOEC = 400 μg/mL NOEC 4.3x maximum pollen level 

Earthworm 

(Eisenia fetifa) Purified protein NOEC = 57 mg/kg NOEC 4.3x MEEC in soil 

a NOEC – No Observable Effect Concentration b MEEC – Maximum Expected Environmental Concentration 
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Results of the environmental fate studies indicate that Cry3Bb1 protein does not accumulate in the 

environment (e.g., air, soil, or water) or in animal tissues. Therefore, non-target soil organisms will be 

minimally exposed to the Cry3Bb1 protein based on its rapid degradation in the soil. 

In summary, Cry3Bb1 poses less risk to the environment than tefluthrin, terbufos, chlorpyrifos, fipronil, 

or any other conventional insecticide labeled for CRW control. MON 863 corn poses minimal risk to 

non-target organisms. The Cry3Bb1 protein is expressed by the corn plant, thus reducing the exposure to 

non-target organisms. In addition, Cry3Bb1 has a narrow target range. Monsanto has performed dietary 

bioassays to determine the insecticidal spectrum of the Cry3Bb1 protein. The protein is effective at 

killing only beetles of the family Chrysomelidae, specifically CRW and Colorado potato beetle 

(Leptinotarsa decemlineata (Say)). There have been no functional receptors for Cry proteins found on 

intestinal cells of fish, birds, or mammals. Finally, Cry3Bb1 is degraded rapidly in the soil (reducing 

non-target exposure). Use of this new pesticide could potentially reduce the use of CRW chemical 

pesticides by millions of pounds per year and substantially reduce non-target organism risk. 

d. Practical, Easier, and Safer for Growers to Use Than Current Alternatives 

 (MRID Nos. 456530-01 and 450297-01)

MON 863 offers many more practical advantages to corn growers than the current alternatives. It can be 

planted early for a longer growing season and potentially higher yield, while ensuring adequate CRW 

protection throughout the growing season. Planting corn early is desirable to boost yield, but it can also 

reduce insecticide performance because of chemical dissipation prior to larval hatch. With MON 863 

corn, the grower can plant early and not have to worry about timing or chemical dissipation. In addition, 

growers should be able to plant their crop quicker because they won’t have to continually stop and refill 

the insecticide boxes. MON 863 seeds can also have seed treatments that will allow even greater control 

of other associated pests such as wireworm, grub, maggots, and cutworms. Thus, growers will have 

multi-pest protection while carrying out insect control in essentially a single step at planting. All of these 

advantages to planting MON 863 corn are practical, easier, and safer for the grower. Planting MON 863 

corn will save the grower money in application, insecticide, labor, fuel, equipment, storage, and disposal 

costs (since there will be no insecticide containers needed for CRW control). Plus, it will provide the 

grower and other occupational workers greater safety, protect water bodies from run-off, and mitigate 

spray drift and non-target effects. Grower interest in MON 863 is high, approximately 70% of growers 

surveyed were either “very interested” or “somewhat interested” in the new CRW trait hybrids (see page 

48 of MRID No. 455770-01). In the first few years, however, there will be a limited amount of seeds 

available, the trait will not be in all corn varieties, and many growers will try out the new technology 

rather than planting the maximum 80% of corn with MON 863. 

Prior to the development of MON 863, the three CRW control methods have been (1) crop rotation, (2) 

soil-applied insecticides, and (3) limited use of rescue-treatments for CRW adult beetles. Historically, 

crop rotation has been the primary method used for controlling CRW (Levine and Oloumi-Sadeghi 

1991). Crop rotation, however, is now far less effective because of the existence of a WCRW soybean 
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rotational variant, primarily in Eastern Illinois and Western Indiana, that oviposits in soybean fields 

(Levine and Oloumi-Sadeghi 1991) and a NCRW extended diapause (2 year extended) variant, primarily 

in parts of Minnesota, Iowa, and South Dakota (Krysan et al. 1986). In addition, CRW has developed 

resistance to methyl parathion and carbaryl, both adulticides used in rescue treatments (Meinke et al. 

1998). Therefore, growers have become increasingly dependent on chemical pesticides to limit CRW 

losses. MON 863 CRW-protected corn offers a way to potentially control CRW behavioral variants and 

insecticide-resistant populations more effectively than through the use of chemical pesticides for CRW 

and still utilize effective corn-soybean (or other crop) rotations. MON 863 corn will likely reduce or 

eliminate the use of certain CRW insecticides (see discussion below). 

MON 863 gives growers equal or higher yields than use of chemical pesticides, while requiring less 

input of time and other resources. Preliminary results put this yield benefit at 1.5–4.5% (see MRID No. 

456430-02). For a reasonable range of prices and yields, the value of this yield benefit to growers is $4–

$12/acre relative to the use of soil-applied insecticides and depending on the CRW pressure. 

Farmers were surveyed (see MRID No. 456923-01) to determine major factors that would be important 

to them in deciding whether to plant transgenic corn with CRW resistance, such as MON 863, in place 

of their current corn. In addition to economic considerations, the farmers indicated the following nonmonetary benefits would also be important: 

 Safety of not handling a toxic insecticide 

 Easy to use and handle 

 All-in-one-product insect control 

 Saving time and labor 

 Better pest control 

Farmers were especially interested in minimizing health and environmental effects of the pesticides they 

use and, if cost and performance are comparable, prefer a general-use product over a restricted-use 

product. Again, the survey (see MRID No. 456923-01) indicates that farmers will favor the pesticide 

that minimizes adverse effects on the environment. 

e. Efficacy of Event MON 863 (MRID Nos. 453613-03 and 455382-08)

Based on the review of the submitted field efficacy studies, MON 863 corn is as effective or more 

effective than chemical insecticides in protecting corn roots from larval CRW feeding damage. 

Chemical pesticides for CRW are usually applied to the soil at the time of planting; however, the 

pesticide may dissipate and no longer be effective by the time the larvae hatch. Timing is not a problem 

with MON 863 corn because the pesticide is incorporated within the corn roots and is produced at a 

relatively constant rate in growing corn. Weather is unlikely to effect the efficacy of MON 863 corn as 

much as it might decrease the effectiveness of the chemical insecticides. Based on the results discussed 

below, the extent of root damage sustained by MON 863 was less than that seen in the control corn, and 
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less than or equal to the damage in corn treated with any of the other chemical pesticides used in the 

comparative analysis. 

 i. Comparing the Efficacy of MON 853 and MON 863 to Three Corn Rootworm Species, 

 Northern Corn Rootworm (D. barberi), Southern Corn Rootworm (D. undecimpunctata 

 howardi), and Western Corn Rootworm (D. virgifera virgifera) (MRID No. 455382-08) 

In this experiment, Monsanto compared the relative efficacy of two transformed corn hybrids, 

expressing the Cry3Bb1 protein (MON 853 and MON 863), in preventing damage from three species of 

corn rootworm larvae. This was accomplished by artificially infesting potted corn plants (treatments 

consisting of the two transformed hybrids and a non-transformed control hybrid) with eggs from each of 

three rootworm species. Each plant (in the V2 stage) was infested with approximately 800 eggs (6–8 

plants per treatment were used). Root damage was scored using the Iowa Root Damage Rating (RDR) 

index (1 = no damage, 6 = extensive damage) after 3–4 weeks of larval feeding. 

Results from the study showed that both MON 853 and MON 863 experienced significantly less root 

damage from all three rootworm species than the non-transformed control hybrid. In terms of western 

and northern corn rootworm damage, MON 863 had significantly less root damage (<2 RDR) than MON 

853 (~2.3 RDR). For southern corn rootworm, there was no significant difference between MON 853 

and MON 863 (RDR ~3.5–3.8). Southern corn rootworm damage was greater than western or northern 

corn rootworm damage for all treatments. It is noted that Monsanto is only commercializing Event MON 

863. 

 ii. Efficacy of MON 863 Against Corn Rootworm and Comparison to Insecticide Treatments 

 – Results of Year 2000 Field Trials (MRID No. 453613-03)

In this experiment, Monsanto evaluated the relative effectiveness of MON 863 and conventional 

pesticide treatments at preventing damage from corn rootworm feeding in field efficacy trials. The 

pesticides tested (all soil insecticides) included Force 3G (tefluthrin), Counter CR (terbufos), and 

Lorsban 15G (chlorpyrifos). 

The study consisted of three separate field experiments, all of which utilized similar growth stage MON 

863 and non-transgenic control hybrids (negative MON 863 isoline). In each of the experiments, 

treatments were deployed using a randomized block design and were scored for root damage in late July. 

Root damage was assessed using the Iowa Root Damage Rating index (1 = no damage, 6 = extensive 

damage, >3 = economic threshold). For the first experiment (conducted at seven different locations), 

treatments (MON 863, control, Force 3G, Counter CR, and Lorsban 15G) were deployed as four-row 

strips (4 replicates per treatment). Each plot was artificially infested with 800 rootworm eggs/foot 

(species not specified). In the second experiment (conducted at eight different locations), MON 863 was 

evaluated against Force 3G treatment and an untreated control. Treatments were deployed as single rows 

and were artificially infested with 1,600 rootworm eggs/foot (species not specified). In the third 

experiment (conducted at nine test sites), treatments (MON 863, control, Force 3G, Counter CR, and 
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Lorsban 15G) were planted in four-row strips in continuous corn acres or a corn/pumpkin trap crop (no 

artificial rootworm infestation was used). For all tests, RDR damage was analyzed via analysis of 

variance and t-tests to determine significant differences between treatments. Also, a “consistency rating” 

was calculated for each experiment by determining the percentage of root damage in a treatment that is 

below the economic threshold (RDR = 3) when the corresponding control treatment root damage is 

above the threshold. 

The results of the first experiment showed that when summed across all test locations, MON 863 (RDR 

= 2.02), Force 3G (RDR = 2.40), Counter CR (RDR = 2.26), and Lorsban 15G (RDR = 2.40) 

experienced significantly less root damage than the untreated control (RDR = 3.91), although there was 

no significant difference between MON 863 and the insecticide treatments. At three of the seven 

locations, MON 863 had significantly less root damage than all of the other insecticide treatments. For 

the second experiment, when summed across all eight test sites, MON 863 (RDR = 1.41) and Force 3G 

treatment (RDR = 1.91) showed significantly less root damage than the untreated control (RDR = 3.27). 

There was no significant difference between MON 863 and Force 3G, although root damage for MON 

863 was significantly less than that for Force 3G at five of the test sites. In the third experiment, MON 

863 experienced significantly less root damage (RDR = 1.72, summed over all nine locations) than any 

of the insecticide treatments or the control (all insecticide treatments had significantly less damage than 

the control). For all three experiments, the “consistency rating” for MON 863 was close to 100%, 

meaning that damage in the MON 863 hybrids was almost always kept below the economic threshold 

when the control treatment showed damage exceeding the threshold. 

Taken together, the results show that MON 863 prevented root damage from rootworm feeding as well 

or better than rootworm soil insecticides. Root damage ratings for MON 863 were typically between 1.2 

and 2.0, a high level of control relative to untreated control hybrids. In addition, the results were 

generally consistent from location-to-location (test sites included plots in six separate corn-growing 

states). 

 f. Yield Benefits (MRID No. 456530-02)

The field efficacy data discussed above were used to estimate the yield benefit of the MON 863 corn 

hybrids relative to non-transgenic corn hybrids without corn rootworm control and with a soil insecticide 

for corn rootworm control (see MRID No. 456530-02). Field data were collected to estimate the 

proportional yield loss as a function of the root rating difference (1–6 root rating scale of Hills and 

Peters). Three years of data (1994–1996) in 2 locations in Illinois (near Urbana and DeKalb) were used 

for the analysis. Data from efficacy experiments conducted in 1999 and 2000 in several locations were 

used to estimate the impact of Event MON 863 on the root rating. Preliminary estimates, using a 

composed error model for insect damage functions, indicate that the MON 863 corn hybrids have a yield 

benefit of 1.5 to 4.5% relative to control with a soil insecticide and 9 to 28% relative to no control. The 

value of these benefits is estimated to be $4–$12/acre relative to control with a soil insecticide, 

depending on the corn rootworm pressure, and $25–$75/acre relative to no control. Because there is a 

low correlation between root rating difference and yield loss, there is uncertainty in the realized yield 
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benefit. This uncertainty is not due to MON 863 per se but to the numerous environmental and 

agronomic factors determining a corn plant’s yield and yield response to corn rootworm larval damage. 

 g. Grower Benefits (MRID Nos. 456923-01, 456530-01-03, 450297-01, and 455770-01)

Monsanto submitted a study entitled “An Ex Ante Analysis of the Benefits from the Adoption of 

Monsanto’s Corn Rootworm Resistant Varietal Technology - YieldGard® Rootworm.” This study 

examined the potential economic impacts in the U.S. of the commercial adoption of MON 863 corn 

(YieldGard® Rootworm technology). The model estimates the economic impacts if MON 863 corn had 

been available and was priced such that the technology fee per acre would be the same as for a 

representative conventional (non-Bt) CRW control technology. The study used data from the year 2000 

and made certain assumptions where necessary. For the year 2000, almost 8 million pounds of CRW 

insecticide, costing $172 million, were applied to 14 million acres (i.e., 17% of total corn acres planted). 

For a reasonable range of prices and yields, benefit to growers was estimated at $4 to $12/acre, 

depending on corn rootworm pressure. The authors of the study estimated one-year total benefits (in the 

year 2000), with 100 percent adoption of MON 863 corn in year 2000, to be $460 million. This benefit 

includes $171 million to Monsanto and other seed companies, $231 million to farmers from yield gains, 

$58 million to farmers from reduced risk and time savings, and other benefits associated with the 

reduced use of insecticides. 

 i. EPA Projections of Grower Benefits and Environmental Benefits

Grower benefits are a theoretical construct that cannot be directly measured or monitored. They are 

defined as the premium a grower would pay for MON 863, or the difference between the value of MON 

863 and its costs. Grower benefits can be depicted in a graph as the area above the technology fee and 

below the demand curve. This is where product value, as measured by willingness to pay, exceeds the 

technology fee. Grower benefit projections are best confirmed by comparing projected adoption rates 

with actual adoption rates given technology fees. 

The factors that will influence grower demand are the following: CRW-infested acres, comparative 

yields and costs of competing technologies for CRW insect control, U.S. and global market acceptance 

and approval, and other regulatory constraints (e.g., refuge requirements). About 30% of the corn 

acreage (24 million acres) was treated with 12 million pounds of insecticides to control pests over the 

last several years. For the year 2000, almost 8 million pounds of CRW insecticide, costing $172 million, 

were applied to 14 million acres ($12.29 per acre). The EPA estimates and projections use the submitted 

comparative performance studies and yield enhancements, which indicate an increased yield of 1.5 to 

4.5% for use of MON 863 over chemical pesticides when infestation levels are high. 

I. Methodology and Parameter Estimates 

The Agency predicted mature market adoption rates based on a demand simulation model and pricing 

behaviors based on revenue maximization (marginal cost = 0). The demand curve measures adoption at 
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alternative technology fees for MON 863 corn. A discussion of the simulation model is found in section

II(E) EPA’s 2001 Bt Crops Reassessment (U.S. EPA 2001). Briefly, the distribution of MON 863 

perceived grower value and costs are assumed to be uniformly distributed across all infested acreage. 

Single parameter estimates are required for the maximum product value and cost. The maximum value 

(willingness to pay) is derived from estimates of (1) improvements to yield, (2) reductions in chemical 

costs, and (3) the perceived value of a less toxic product. The model also requires an estimate of the 

negative costs associated with marketability discounts/risks, refuge requirements, or any other costs 

associated with this technology. 

The maximum value reflects the acreage with the highest values due to greatest pest pressures and cost 

of rootworm control. An estimate of $15.75 is used for the model and is based on a 4.5% yield 

improvement on $350/acre gross income for corn, including government payments, which is 

characteristic of expectations for 2001. The cost savings from insecticides includes the out-of-pocket 

costs of $12.50 per acre plus a maximum of $2.50 per acre due to perceived value as a general use 

product with less toxic effects to the local environment. The perceived value of a less toxic product is 

not an out-of-pocket cost and is probably of lesser importance to growers. The high market share for 

chlorpyrifos (25 % of the CRW market) may be due, in part, to the fact that it is only major alternative 

registered for general use (i.e., no restricted uses). 

Value due to product performance (yield) $15.75 

Value from lower chemical costs $12.50 

Value from easier and safer use $ 2.50

Max value of Bt corn rootworm $30.75 

It is unlikely, based on past experience with biotechnology products, that European Union approval will 

occur in less than three years after launch (see MRID No. 455770-01). This international regulatory 

constraint imposes an additional cost on adoption of MON 863 corn. Just as was done for Roundup 

Ready® corn, until full European (or global) regulatory approval occurs, Monsanto plans to continue its 

channeling program with growers, dealers, and grain handlers to help ensure that MON 863 corn is 

directed into appropriate global markets (markets with regulatory approval). The simulation model can 

be used to assess the impact of access to global markets. The negative costs associated with limited 

marketability are reflected by the percent of growers who would not use MON 863 even if there was no 

technology fee. That is, adoption would not be 100% even if MON 863 is given away. Based on the 

results of a survey on grower attitudes toward genetically modified organisms (conducted by the 

American Corn Growers Association), sixteen percent of respondents stated they would not be willing to 

grow more non-genetically engineered corn varieties (see MRID No. 456923-01). The 16% of growers 

is consistent with a maximum cost of $10 per acre as compared to a maximum cost of $5 per acre, the 

9% (91% adoption) at $0/acre technology fee. A 16% removal from the target market would reduce 

adoption rates from 43% to 35%, which would translate to lower grower benefits and less use reduction. 

The demand curve and derived marginal revenue curve provide a basis for predicting a technology fee. 

The estimate of $15 per acre technology fee is based on revenue maximization behaviors that are 
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equivalent to profit maximization if marginal cost is zero. The actual technology fee would vary from 

this estimate based on licensed seed companies’ perceptions of the demand curves for specific hybrids 

and marginal costs associated with marketing and sales. 

EPA’s economic assessment and eleven (11)-year projection of aggregate grower benefits is based on 

current chemical prices of alternatives. It neither anticipates nor includes any price changes from 

competing technologies as MON 863 corn is introduced or the effects of new active ingredients 

registered for corn rootworm. The economic assessment is limited to grower benefits and does not 

estimate the reduced cost passed through to final consumers (though in the long run, economic theory 

suggests that the 2% improvement in returns on gross revenue would be passed through to consumers). 

No assessments are made of impacts to foreign trade or agricultural practices. MON 863 corn may lower 

the costs of rootworm control and, therefore, have some effect on acres grown to continuous corn, which 

would increase pest pressures and reduce the environmental benefits of MON 863 (UCS 2002). 

II. Corn Rootworm-Infested Acres

Scouting for the range and level of infestation is done by measuring the density of adult beetles and 

larvae. Gray (2000) noted that CRW-infested acres are increasing due to the geographical expansion of 

the WCRW soybean variant. Based on both the likely geographic spread of the WCRW soybean variant 

and the NCRW extended diapause variant, it is likely the total infested acres will move from 

approximately 28 million acres to closer to 39 million acres in 13 years. This assessment projects the 

range of infestation to increase uniformly by 2.6% per year, and the density to remain such that only 1⁄2 

of the acreage infested is above the economic threshold. To the extent that market forces reduce the 

economic threshold (increased corn prices, increased yields, or reduced cost of CRW control), acreage 

adoption and conventional insecticide use would be higher than currently forecast, and the 

environmental and grower benefits of MON 863 would also be higher than projected in this review. 

EPA has considered the rate of increase of CRW-infested aces as input into its simulation model that 

was used to predict the technology fee, adoption rates, and grower benefits of MON 863 corn. 

III. MON 863 Corn Hybrid Supply 

Adoption of MON 863 corn (acres) is dependent on supply and demand. The supply is constrained by 

corn seed hybrid availability, both for a single hybrid and the total number of hybrids available. The 

commercial hybrid development process requires sequential development that will take several years 

after commercial launch. 

In the first two to three years after commercial launch, adoption of MON 863 corn is predicted to be 

relatively slow because there will only be a limited number of MON 863 corn seed hybrids available. 

Monsanto has projected that MON 863 corn adoption will be similar to Roundup Ready® corn in that it 

will not be available in Pioneer or Syngenta brands nor have European Union approval at the time of 

launch (see MRID No. 455770-01). Roundup Ready® corn had approximately 1% (790,000 acres), 
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2.5% (2 million acres), and 5% (4 million acres) acreage penetration in percent of total corn acres in 

years 1, 2, and 3 from commercial release, respectively. These data were used by EPA in its economic 

analysis. 

IV. Estimating the Demand Curve for MON 863 

The demand curve for MON 863 for year 2013 is shown in Table 7. It is based on a simulation of 

adoption at alternative technology fees for MON 863 as described in section II(E)(2)(g)(i) of this 

BRAD. Marginal revenue is computed using a technology fee of $15 based on revenue maximization. It 

is based on pricing behavior where the marginal costs of increasing seed production are negligible 

(assumes that all seed hybrids are in place). It is the last point where marginal revenue is positive (see 

bolded row in Table 7 below). The total revenue with a $15 technology fee is $252 million for 16.9 

million acres of MON 863 corn planted in the year 2013. Actual technology fees are certain to vary from 

this estimate. At a $15 technology fee, MON 863 adoption is predicted to be 43% of infested acreage. If 

only 50% of infested acreage is treated, then only 7% of infested acreage will still be treated by 

conventional chemical controls (not including any refuge acres required as part of an insect resistance 

management plan). 

Table 7. Simulated Demand Curve for the Year 2013.

Tech Fee 

Schedule 

($) 

Percent Adoption 

(Model Calculation) 

Acres 

Adopted 

(X 106) 

Total Revenue 

(X 106) 

Marginal Revenue 

(X 106) 

Marginal Revenue 

Per Acre 

($) 

27 5% 1.95 52.65 

24 14% 5.46 131.04 78.39 22.33 

21 23% 8.97 188.37 57.33 16.33 

18 34% 13.26 238.68 50.31 11.73 

15 43% 16.77 251.55 12.87 3.67 

12 53% 20.67 248.04 (3.51) (0.90) 

9 63% 24.57 221.13 (26.91) (6.90) 

 6 72% 28.08 168.48 (52.65) (15.00) 

3 83% 32.37 97.11 (71.37) (16.64) 

0 91% 35.49 0.00 (97.11) (31.12) 

 V. Projecting Grower Benefits

Grower benefits are calculated as the sum of the difference between what the grower is willing to pay 

and the actual technology fee. The EPA simulation model computes the average gross benefits for 

adopters and Bt-related costs due to MON 863 corn adoption. The estimated net benefits per acre are 

$6.56, based on a $15 technology fee (see Table 7 above). The estimated gross benefits (primarily, yield 

and insecticide cost reduction) for adopters are $23.94 per acre, and estimated Bt-related costs for 

adopters are $2.38 per acre. 
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The annual change in infested acres, adoption, conventional chemical use, and associated grower 

benefits are projected for each year from 2003 to 2013 (Table 8). Projected acres infested and 

conventional chemical treatments are based on growth rates for infested acreage with a fixed treatment 

percentage of 50% of the total projected CRW-infested acres. 

Table 8. Projected Acreage Infested, MON 863 Adoption, and Conventional Treatments 

 (2003 to 2013). 

Year 

Acres 

Infested

(X 106)

Acres 

Treated 

(X 106)

MON 863 

Acres 

(X 106)

Conventional 

Treatments

(X 106)

2000 28.0 14.0 0.0 14.0 

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% -14.36% 

Annual grower benefits (see Table 9) are based on a constant $6.56 per acre, and the growth in total 

annual benefits are due to the availability of hybrid seed containing Cry3Bb1 over greater areas of CRW 

infestation. Actual grower benefits may be higher in the early years, if supply is first available in those 

areas with highest CRW pest pressure. 

The cumulative sum of the grower benefits for the first three years (2003 to 2005) is $49.2 million and 

for eleven years (2003 to 2013) is $642.7 million (Table 9). The discounted aggregate benefits for year 

2003 to 2013 are $385.31 million, assuming a discount rate of 7%. The 7% discount rate represents the 

Office of Management and Budget rate (see Circular Number A-94, Transmittal Memo Number 64, 

October 29, 1992), a relatively risk-free rate of return similar to that assumed for a long-term U.S. 

Treasury bond. The discount rate is defined as the interest rate used in calculating the present value 

expected yearly for benefits and costs. 
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 Table 9. Annual Grower Benefits of MON 863.

Year 

Target 

Acres 

(X 106) 

Adoption 

Acres 

(X 106) 

 Grower 

Benefits 

($ X 106) 

2003 2.3 1.0 6.6 

2004 5.8 2.5 16.4 

2005 9.3 4.0 26.2 

2006 14.0 6.0 39.4 

2007 16.7 7.2 47.2 

2008 20.1 8.6 56.7 

2009 24.1 10.4 68.0 

2010 27.7 11.9 78.2 

2011 31.9 13.7 90.0 

2012 36.7 15.8 103.5 

2013 39.0 16.8 110.5 

Cumulative 98.0 $ 642.7 

 VI. Projecting Chemical Use with MON 863 (MRID Nos. 456530-01 and 450297-01) 

The insecticides used to control corn rootworm in conventionally grown (non-Bt) corn consist mainly of 

organophosphates (9), carbamates (3), synthetic pyrethroids (6), and phenyl pyrazole (1) classes of 

chemistry (see Table 1). Table 10 shows that there have been significant shifts in the use of insecticides. 

Synthetic pyrethroids have increased at the expense of organophosphates and carbamates. The clear shift 

is away from organophosphate insecticides and toward synthetic pyrethroids, especially the effective, 

relatively new product tefluthrin that is now the market leader. Fipronil was introduced in 1998 and 

accounts for the other category. Table 11 projects future acre treatments, by chemical class, using past 

trends and the projected conventional treatments shown in Table 8. 

 Table 10. Historical Market Shares for Corn Rootworm (Percent of Total Acre Treatments).

Chemical Class 1995 2001

Carbamate 4.5% 2.1%

Synthetic Pyrethroid 21.9% 41.0%

Organophosphate 73.4% 45.8%

Other 0.1% 11.0%

 Total 100.0% 100.0%
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Table 11. Projected Treatments for Corn Rootworm (Millions of Acre Treatments for Corn 

 Rootworm). 

Chemical Class 2002 2003 2004 2005 2006 2007 

Carbamate 0.3 0.3 0.2 0.2 0.2 0.1 

Synthetic 

Pyrethroid 6.2 6.2 5.9 5.6 5.0 4.8 

Organophosphate 6.5 6.0 5.3 4.6 3.8 3.3 

Other 1.7 1.7 1.6 1.5 1.4 1.3 

MON 863 0.0 1.0 2.5 4.0 6.0 7.0 

Total CRWTreated 14.7 15.1 15.5 15.9 16.3 16.5 

Table 12. Projected Use Reduction Associated with MON 863 (Millions of Acre Treatments for 

 Corn Rootworm)

Chemical class 2002 2003 2004 2005 2006 2007 

Carbamate 0.0 0.0 0.0 0.1 0.1 0.1 

Pyrethroid 0.0 0.4 1.1 1.9 2.9 3.5 

Organophosphate 0.0 0.4 1.0 1.5 2.2 2.5 

Other 0.0 0.1 0.3 0.5 0.8 0.9 

total reduction 0.0 1.0 2.5 4.0 6.0 7.0 

These pesticides have varying application rates, many are toxic to humans and non-target wildlife, and 

many have restricted use or specific, mandatory mitigation measures to minimize exposure. The average 

active ingredient rate applied (pounds per acre) has been steadily decreasing, reflecting the shift from 

organophosphates and carbamates to synthetic pyrethroids and fipronil. Rates have gone from an 

average of 0.7 pounds per acre in 1995 to 0.4 pounds per acre in 2001. The application rates shown in 

Table 1 for terbufos, chlorpyrifos, carbofuran, tebupirimfos, and phorate are closer to 1 pound per acre. 

Synthetic pyrethroids and fipronil, for example, are newer chemistries that are used at 0.1 (or less) 

pound per acre. The use reductions shown in Table 12 indicate that, as MON 863 CRW-protected corn 

adoption increases in the next five years, acre treatments will be reduced for all currently registered 

CRW insecticides. The greatest use reductions are seen in both the organophosphate and synthetic 

pyrethroid classes. In 2005, approximately 1.5 million acre treatments of organophosphate insecticides, 

1.9 million acre treatments of synthetic pyrethroid insecticides, 0.1 million acre treatments of carbamate 

insecticides, and 0.5 million acre treatments of other chemical insecticides, including members of the 

phenyl pyrazole class (e.g., fipronil), will be reduced based on 2003 figures. In 2007, the extent of 

insecticide use reduction will be even greater, approximately 2.5 million acre treatments of 

organophosphate insecticides, 3.5 million acre treatments of synthetic pyrethroid insecticides, 0.1 
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million acre treatments of carbamate insecticides, and 0.9 million acre treatments of other chemical 

insecticides are expected to be reduced. 

 ii. Comparing Estimates of Grower Benefits from Other Studies 

Reported estimates may differ with respect to the entities included and target year. Some include the 

grower and chemical producer and reflect the total societal value of MON 863. EPA’s assessment is 

limited to grower benefits. Estimates made in MRID No. 456923-01 are based on an ex ante assessment, 

assuming that MON 863 was available in the year 2000. It is necessary to adjust estimates to the extent 

possible to create valid comparisons. 

The authors of the study (MRID No. 456923-01) estimated one-year total benefits (in the year 2000), 

with 100 percent adoption of MON 863 corn in year 2000, to be $460 million. This benefit includes 

$171 million to Monsanto and other seed companies, $231 million to farmers from yield gains, $58 

million to farmers from reduced risk and time savings, and other benefits associated with the reduced 

use of insecticides. The EPA assessment for year 2013 is based on a higher level of infested acreage. 

Adjusting the analysis (from MRID No. 456923-01) of the ex ante total benefits for the year 2000 of 

$460 million to the year 2013 by an additional 39% gives $640 million in total benefits. 

The EPA estimate of total benefits can be calculated assuming a zero technology fee. This is essentially 

the total area under the demand curve. Total benefits would be $507 million in 2013. This compares 

with the adjusted estimate of $640 million total benefits mentioned in the previous paragraph. 

In a separate analysis, Gray (2000) states that if farmers invested $400 million in this technology 

(technology fees are assumed to $15 per acre), these resources would prevent an economic loss of 

approximately $600 million, for a net gain of $200 million to farmers. Adjusting this estimate to 

coincide with infestation levels in 2013 (a 39% increase) provides total benefits of $834 million. 

EPA’s estimate of total benefits in 2013 ($507 million) is 20% lower than projections from MRID No. 

456923-01 and 40% lower than Gray (2000)’s projections. 

 h. Suggested Measures to Monitor Environmental and Grower Benefits 

The amount of chemical use reduction attributed to MON 863 cannot be directly observed. For example, 

if the economic threshold for CRW treatment is reduced by the increased competition created by MON 

863, then total infested acres treated would increase. The effect of MON 863 on chemical use reduction 

would be less then 1 acre per adopted acre of MON 863. A survey of growers who adopted MON 863 

would be helpful to directly estimate chemical use reduction. 

Translating the environmental benefits of chemical use reduction is a topic that is given increased 

attention for the purpose of strategic planning and measuring results. Measures most likely affected by 

the reduction in insecticide use are reported incidents from workers, accidental spills, and mortality to 

non-target wildlife. 
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EPA’s Environmental Fate and Effects Division in the Office of Pesticide Programs has identified the 

most toxic active ingredients to birds, based on risk assessments of all available information. The list 

contains 10 insecticides currently being used on agricultural crops: aldicarb, methyl parathion, 

dicrotophos, carbofuran, phorate, oxamyl, diazinon, disulfoton, methamidophos, and ethoprophos. 

Methyl parathion, carbofuran, phorate, diazinon, and ethoprophos are active ingredients registered as 

formulated products for the control of corn rootworm (i.e., alternatives to MON 863). The use of methyl 

parathion, carbofuran, and phorate for corn rootworm control accounts for 1.2 million acres—6.6% of 

the total use of the ten insecticides on all agricultural crops. Thus, MON 863 alone can have a significant 

impact on reducing the use of insecticides posing the highest risk to birds. Data are not currently 

available to estimate impact exactly, but additional data may be collected during the time of the MON 

863 conditional registration that would be useful in determining the impact of Bt corn on birds and bird 

populations (e.g., percent of invasive species, native species of management concern, changes in types 

of and/or abundance of bird species, etc.). 

3. 2005 MON 88017 and MON 88017 x MON 810 Public Interest Finding 

 (Reviewed in U.S. EPA 2005) 

MON 88017 (plasmid vector ZMIR39) expresses the Cry3Bb1 Bt toxin and is targeted against corn 

rootworm larvae. The toxin is the same as expressed by MON 863 corn (EPA Reg. No. 525-528), 

registered by Monsanto for the 2003 growing season. The Cry3Bb1 protein produced in MON 88017 

and MON 863 is a variant of the wild-type Cry3Bb1 protein from Bt subsp. kumamotoensis. When 

compared by amino acid sequencing, the Cry3Bb1 protein expressed in MON 88017 has been reported 

to be 99.8% similar to the Cry3Bb1 protein expressed in MON 863. The primary difference between the 

two hybrids is that MON 88017 also expresses a gene for resistance to glyphosate-based herbicides. 

Given the similarities between MON 863 and MON 88017, Monsanto proposed to bridge the public 

interest finding that was made for MON 863 (see section II(E)(2) of this BRAD) to the MON 88017 and 

MON 88017 x MON 810 registrations. The MON 863 benefits assessment concluded that the 

registration of MON 863 would be in the public interest. 

Monsanto identified the following benefits of the MON 863 registration that should also be applicable to 

the MON 88017 and MON 88017 x MON 810 registrations: 

 Replacement of higher risk pesticides 

 Practical benefits for growers (reduced input costs, time and labor savings, etc.) 

 Efficacy (equivalent to conventional insecticides) 

 Human health benefits (reduced toxicity relative to conventional insecticides) 

 Environmental benefits (reduced risks to non-target organisms relative to conventional 

 insecticides) 

 Yield benefits (greater yields than conventional corn) 
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 Grower economic benefits (total benefits up to $6.56 per acre) 

Monsanto also identified several benefits that MON 88017 offers in addition to the MON 863 benefits 

listed above: 

 Herbicide tolerance – MON 88017 (and MON 88017 x MON 810) have been engineered to 

 tolerate glyphosate herbicide applications. This trait will provide economic benefits to growers. 

 Enhanced breeding efficiency – MON 88017 can be bred faster (and selected through the 

 herbicide tolerance trait), which should allow for a greater supply of the product to growers. 

The Biopesticides and Pollution Prevention Division (BPPD) agrees with Monsanto’s rationale to bridge 

the public interest finding for MON 863 to the MON 88017 and MON 88017 x MON 810 registrations. 

The benefits identified for MON 863 (as summarized above and discussed more comprehensively in 

section II(E)(2) of this BRAD) are all applicable to the MON 88017 products. In addition, MON 88017 

and MON 88017 x MON 810 offer further benefits with the addition of herbicide (glyphosate) tolerance. 

These factors address the criteria established in the Federal Register Notice dated March 5, 1986 (51 FR 

7628) in terms of need, comparative risk issues, and comparative benefits. As such, the registration of 

both MON 88017 and MON 88017 x MON 810 can be expected to be in the public interest. 

4. References 

Gray ME. 2000. Prescriptive Use of Transgenic Hybrids for Corn Rootworms: An Ominous Cloud on 

 the Horizon? Proceedings of the January 5–6, 2000 Illinois Crop Protection Technology 

 Conference. University of Illinois at Urbana-Champaign, College of Agricultural, Consumer, and 

 Environmental Sciences, University of Illinois Extension. pp. 97–103. Available from: 

 http://ipm.illinois.edu/education/proceedings/icptcp2000.pdf. 

Krysan JL, Foster DE, Branson TF, Ostlie KR, Cranshaw WS. 1986. Two years before the hatch: 

 rootworms adapt to crop rotation. Bull. Entomol. Soc. Amer. 32:250–253. 

Levine E, Oloumi-Sadeghi H. 1991. Management of Diabroticite rootworms in corn. Annu. Rev. 

Entomol. 36:229–255. 

Levine E, Oloumi-Sadeghi H, Ellis CR. 1992a. Thermal requirements, hatching patterns, and prolonged 

 diapause in western corn rootworm (Coleoptera: Chrysomelidae) eggs. J. Econ. Entomol. 85: 

 2425–2432. 

Levine E, Oloumi-Sadeghi H, Fisher JR. 1992b. Discovery of multiyear diapause in Illinois and South 

 Dakota northern corn rootworm (Coleoptera: Chrysomelidae) eggs and incidence of the 

 prolonged diapause trait in Illinois. J. Econ. Entomol. 85:262–267. 
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Meinke LJ, Siegfried BD, Wright RJ, Chandler LD. 1998. Adult susceptibility of Nebraska western 

 corn rootworm (Coleoptera: Chrysomelidae) populations to selected insecticides. J. Econ. 

 Entomol. 91:594–600. 

MRID No. 449043-10. Maggi V. 1999. Evaluation of the Dietary Effects of Purified Bacillus 

thuringiensis Protein 11231 on Honey Bee Larvae. Lab Project Number: CA-98-169: CAR 158- 

98. Unpublished study prepared by Monsanto Company and California Agricultural Research, 

Incorporated, 46 pages. 

MRID No. 449043-11. Maggi V. 1999. Evaluation of the Dietary Effects of Purified Bacillus 

thuringiensis Protein 11231 on Adult Honey Bees (Apis mellifera L.). Lab Project Number: CA98-171: CAR 160-98: 16169. Unpublished study prepared by Monsanto Company and California 

Agricultural Research, Incorporated, 58 pages. 

MRID No. 449043-12. Palmer S, Krueger H. 1999. Bacillus thuringiensis Protein 11231: A Dietary 

Toxicity Study with Green Lacewing Larvae (Chrysoperla carnea). Lab Project Number: WL98-298: 16165. Unpublished study prepared by Monsanto Company and Wildlife International 

Limited, 40 pages. 

MRID No. 449043-13. Palmer S, Krueger H. 1999. Bacillus thuringiensis Protein 11231: A Dietary 

Toxicity Study with the Parasitic Hymenoptera (Nasonia vitripennis). Lab Project Number: WL98-300: 16167. Unpublished study prepared by Monsanto Company and Wildlife International 

Limited, 50 pages. 

MRID No. 449043-14. Palmer S, Krueger H. 1999. Bacillus thuringiensis Protein 11231 in Corn Grain: 

A Dietary Toxicity Study with the Ladybird Beetle (Hippodamia convergens). Lab Project 

Number: WL-98-299: 16166. Unpublished study prepared by Monsanto Company and Wildlife 

International Limited, 41 pages. 

 

MRID No. 449043-15. Gallagher S, Grimes J, Beavers J. 1999. Bacillus thuringiensis Protein 11231 in 

Corn Grain: A Dietary Toxicity Study with the Northern Bobwhite. Lab Project Number: WL99-014: 16161: 139-44. Unpublished study prepared by Monsanto Company and Wildlife 

International Limited, 43 pages. 

MRID No. 449043-16. Hoxter K, Palmer S, Krueger H. 1999. Bacillus thuringiensis Protein 11231: An 

Acute Toxicity Study with the Earthworm in an Artificial Soil Substrate. Lab Project Number: 

WL-99-013: 16162: 139-443A. Unpublished study prepared by Monsanto Company and 

Wildlife International Limited, 45 pages. 
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MRID No. 449043-17. Teixeira D. 1999. Assessment of Chronic Toxicity of Corn Tissue Containing the 

Bacillus thuringiensis Protein 11098 to Collembola (Folsomia candida). Lab Project Number: 

SB-98-296: 15988: 252.6149. Unpublished study prepared by Monsanto Company and 

Springborn Laboratories, 55 pages. 

MRID No. 449043-18. Drottar K, Krueger H. 1999. Bacillus thuringiensis Protein 11098 in Corn 

Pollen: A 48-Hour Static-Renewal Acute Toxicity Test with the Cladoceran (Daphnia magna). 

Lab Project Number: WL-98-295: 16163: 139A-236. Unpublished study prepared by Monsanto 

Company and Wildlife International Limited, 31 pages. 

MRID No. 449043-19. Meng L, Robinson E. 1999. Evaluation of Insect Protected Corn Lines MON 853 

and MON 859 as a Feed Ingredient for Catfish. Lab Project Number: XX-98-297: 16164. 

Unpublished study prepared by Monsanto Company and Mississippi State University, 32 pages. 

MRID No. 450297-01. Miller D. 2000. Public Interest Document Supporting the Registration and 

 Exemption from the Requirement of a Tolerance for the Plant-Incorporated Protectant, Bacillus 

 thuringiensis Cry3Bb Protein and the Genetic Material Necessary for Its Production in Corn 

 (Vectors ZMIR12L, ZMIR13L, and ZMIR14L). Lab Project Number: 99-781E. Unpublished 

 study prepared by Monsanto Company, 40 pages. 

MRID No. 453613-01. Duan J, Head G, McKee M. 2001. Dietary Effects of Transgenic Bacillus 

thuringiensis (Bt) Corn Pollen Expressing a Variant of Cry3Bb1 Protein on Adults of the 

Ladybird Beetle, Coleomegilla maculata. Lab Project Number: MSL-16936: 00-01-39-37. 

Unpublished study prepared by Monsanto Company, 35 pages. 

MRID No. 453613-02. Bryan R, Porch J, Krueger H. 2001. Dietary Effects of Transgenic Bt Corn 

Pollen Expressing a Variant of Cry3Bb1 Protein on Adults of the Ladybird Beetle, Hippodamia 

convergens – Final Report. Lab Project Number: WL-2000-158: MSL-17171: 139-453. 

Unpublished study prepared by Wildlife International Limited, 36 pages. 

MRID No. 453613-03. Pilcher C. 2001. Efficacy of MON 863 Against Corn Rootworm and Comparison 

 to Insecticide Treatments – Results of Year 2000 Field Trials. Lab Project Number: 00-CR- 

 032E-3. Unpublished study prepared by Monsanto Company, 10 pages. 

MRID No. 455382-04. Duan J, McKee M, Nickson T. 2001. Dietary Effects of Transgenic Bacillus

thuringiensis (Bt) Corn Pollen Expressing a Variant of Cry3Bb1 Protein on Larvae of the 

Ladybird Beetle, Coleomegilla maculata. Lab Project Number: MSL-16907: 00-01-39-26. 

Unpublished study prepared by Monsanto Company, 39 pages. 

MRID No. 455382-05. Sears M, Mattila H. 2001. Determination of the Toxicity of Corn Pollen 

Expressing a Cry3Bb1 Variant Protein to First Instar Monarch Butterfly Larvae (Danaus 

plexippus) via Laboratory Bioassay. Lab Project Number: MSL-17235: 01-01-39-26. 

Unpublished study prepared by University of Guelph, 33 pages. 
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MRID No. 455382-08. Vaughn T, Pleau M, Knutson R. 2001. Comparing the Efficacy of MON 853 and 

 MON 863 to Three Corn Rootworm Species, Northern Corn Rootworm (Diabrotica barberi), 

 Southern Corn Rootworm (D. undecimpunctata howardi), and Western Corn Rootworm (D. 

virgifera virgifera). Lab Project Number: MTC RPT 4. Unpublished study prepared by 

 Monsanto Company, 5 pages. 

MRID No. 455770-01. Vaughn T, Ward D, Pershing J. 2001. An Interim Insect Resistance Management 

 Plan for Corn Event MON 863: A Transgenic Corn Rootworm Control Product. Lab Project 

 Number: MSL-17556: 01-CR-070E. Unpublished study prepared by Monsanto Company, 147 

 pages. 

MRID No. 456530-01. Ward D. 2002. Public Interest Document for Bacillus thuringiensis Cry3Bb1 

 Protein and the Genetic Material (Vector ZMIR13L) Necessary for Its Production in Corn Event 

 MON 863. Lab Project Number: MSL-17766. Unpublished study prepared by Monsanto 

 Company, 53 pages. 

MRID No. 456530-02. Mitchell P. 2002. Yield Benefit of Corn Event MON 863. Lab Project Number: 

 MSL-17782. Unpublished study prepared by Texas A&M University, 65 pages. 

MRID No. 456923-01. Laston J, Hyde J, Marra M. 2002. An Ex Ante Analysis of the Benefits from the 

 Adoption of Monsanto’s Corn Rootworm Resistant Varietal Technology – YieldGard 

 Rootworm, Lab Project Number: MSL-17993. Unpublished study prepared by University of 

 California, 70 pages. 

Union of Concerned Scientists (USC). 2002. Public comments submitted to OPP Docket-30509B. 

 Available from : http://www.regulations.gov (see “YieldGard Plus and MON 863 Gene Stacking 

 Response to Comments Document...” within Docket Number OPP-30509B). 

U.S. EPA. 1992. Comparative Analysis of Acute Avian Risk from Granular Pesticides. Office of 

 Pesticide Programs, March, 1992. 

U.S. EPA. 2001. Biopesticides Registration Action Document – Bacillus thuringiensis Plant- 

 Incorporated Protectants. Available from: 

 http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm. 

U.S. EPA. 2003a. EPA Public Interest Finding and Review of Benefits for Monsanto Company’s Corn 

 Rootworm-Protected Field Corn Product (Event MON 863) for the Section 3(c)(7)(C) Full 

 Commercial Registration. Memorandum from E.B. Brandt, S.R. Matten, Ph.D., and A.H. 

 Reynolds to M. Mendelsohn dated February 13, 2003. 
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U.S. EPA. 2003b. Amended EPA Public Interest Finding and Review of Benefits for Monsanto 

 Company’s Corn Rootworm-Protected Field Corn Product (Event MON 863) for the Section 

 3(c)(7)(C) Full Commercial Registration. Memorandum from E.B. Brandt, S.R. Matten, Ph.D., 

 and A.H. Reynolds to M. Mendelsohn dated February 24, 2003. 

U.S. EPA. 2005. Review of Bridging Rationale for a Public Interest Finding for MON 88017 and MON 

 88017 x MON 810 Bt Corn. Memorandum from A. Reynolds and S. Matten, Ph.D. to M. 

 Mendelsohn dated November 9, 2005. 
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III. REGULATORY POSITIONS FOR CORN EVENT MON 863 AND MON 88017 

A. Initial Registration (February 24, 2003) – Corn Event MON 863

 

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 imposition 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 conditional registration will not cause any unreasonable 

adverse effect on the environment, and (2) the registration 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 imposition of the requirements for the following data: 

1. Independent laboratory analytical method validation. 

 2. Cry3Bb1 protein expression data in terms of dry weight, as the amount of protein present in the 

given tissue. 

 3. Field degradation studies evaluating accumulation and persistence of Cry3Bb1 protein in 

 several different soils in various strata. 

 4. Laboratory toxicity test with Orius insidiosus (minute pirate bug). 

 5. Laboratory toxicity test with carabid (ground beetle). 

 6. Laboratory toxicity test with Tetraopes (red milkweed beetle). 

 7. Intermediate and multi-year non-target organism field studies with statistical power. 

 8. A six-week broiler dietary study. 

 9. Research regarding corn rootworm adult and larval movement and dispersal, mating habits, 

 ovipositional patterns, number of times a female can mate, and fecundity. 

 10. Research to determine if insect resistance management (IRM) strategies designed for western 

 corn rootworm (WCRW) and northern corn rootworm (NCRW) are appropriate for Mexican 

 corn rootworm (MCRW). 

 11. Research regarding the mechanism of potential resistance of corn rootworm (CRW) to MON 

 863. Monsanto must attempt to develop resistant CRW colonies to aid in determining selection 

 intensity. 

 12. Research regarding the effect of WCRW ovipositing in soybean prior to overwintering and 

 extended diapause in NCRW on an IRM strategy. 

 13. Detailed summaries of the four data-sets identified in Monsanto’s December 13, 2002 letter 

 should be submitted to the Agency to support their conclusion that the initial resistance allele 

 frequency is 0.01. 

 14. Continuation of baseline susceptibility studies, currently underway for WCRW, and initiation 

 for NCRW and monitoring techniques, such as discriminating dose concentration assays, as 

 well as investigation of their feasibility as resistance monitoring tools. 
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The registration applicant has submitted or cited data sufficient for EPA to determine that a conditional 

registration under FIFRA 3(c)(7)(C), for the period ending May 1, 2004, will not result in unreasonable 

adverse effects on the environment. The human health effects data and non-target organism effects data 

are considered sufficient for the period of the conditional registration. These data demonstrate that no 

foreseeable human health hazards or ecological effects are likely to arise from the use of the product and 

that the risk of resistance developing to Bacillus thuringiensis Cry3Bb1 protein during the conditional 

registration is not expected to be significant. The data also demonstrate that there is virtually no 

possibility of any risk associated with weediness or outcrossing to wild relatives. 

Registration of Bacillus thuringiensis Cry3Bb1protein and the genetic material necessary for its 

production (vector PV-ZMIR13L) in event MON 863 corn is in the public interest because MON 863 

(YieldGard® Rootworm) corn is less risky to human health and/or the environment than currently 

registered pesticides and the improved season-long protection and practical benefits of ease of MON 863 

use exceed those of the currently registered alternatives, most of which are restricted-use products. 

Specifically, MON 863 is in the public interest for the following reasons: 

(1) For the first 3 years, MON 863 is projected to reduce conventional pesticide use by 12.5 million 

pounds of active ingredient over 7.5 million corn acres (0.1 carbamates, 3.5 pyrethroids, 3.0 

organophosphates, and 0.9 for other chemical classes). This totals to 7.5 million acres of use reduction. 

To the extent that MON 863 is used on acreage that would be uneconomical to otherwise treat, the total 

use reduction would be less than the MON 863 acres adoption. 

(2) It has a predicted yield benefit of 1.5–4.5% per acre greater than conventionally treated corn. 

(3) Grower benefits are estimated at $6.56 per acre vs. conventionally treated corn. 

(4) The total first 3 year economic benefits are estimated at $49.2 million. 

(EPA’s public interest analysis considers three years of MON 863 use because EPA has been informed 

that Monsanto will request that the current tolerance exemption for Cry 3bB1, which expires on May 1, 

2004, be amended to remove the expiration date. If (1) Monsanto requests such an amendment to the 

Cry3Bb1 tolerance exemption, (2) EPA grants such amendment request, and (3) Monsanto subsequently 

requests that the MON 863 registration be amended to expire at a later date, EPA currently believes that 

the data reviewed so far will likely support an extension of the conditional registration for an additional 

two years.) 

In view of these minimal risks and the clear benefits related to YieldGard® Rootworm, EPA believes 

that the use of the product during the limited period of the conditional registration will not cause any 

unreasonable adverse effects. 
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Although the data with respect to this particular new active ingredient are satisfactory, they are not 

sufficient to support an unconditional registration under FIFRA 3(c)(5). Additional data are necessary to 

evaluate the risk posed by the continued use of this product. Consequently, EPA is imposing the data 

requirements specified earlier in this chapter. 

EPA has determined, as explained in section II(E) of this Biopesticides Registration Action Document 

(BRAD), that the third criterion for a FIFRA 3(c)(7)(C) conditional registration has been fulfilled 

because the use of Cry3Bb1 corn under this registration would be in the public interest. 

A tolerance exemption has been granted, pursuant to section 408(d) of the Federal Food, Drug, and 

Cosmetic Act (FFDCA), for Bacillus thuringiensis Cry3Bb1 protein and the genetic material necessary 

for its production in corn. This tolerance exemption expires in May 2004 and, therefore, this registration 

would also expire in May 2004 until such time as the tolerance exemption is amended to extend its 

duration. If the tolerance exemption is amended to extend its duration, EPA believes that current data 

and information reviewed would support an amendment of the conditional registration to expire in three 

years from the date of the original registration. 

Conclusion

The submitted data in support of this registration under section 3(c)(7)(C) of FIFRA have been reviewed 

and determined to be adequate. Studies mentioned previously are included in the terms, conditions, and 

limitations of this registration. EPA determines that, for the period of conditional registration, this 

registration will not cause unreasonable adverse effects to man or the environment and is in the public 

interest. 

Based on the data submitted and cited by the applicant and reviewed by Biopesticides and Pollution 

Prevention Division, EPA has concluded that Monsanto Company’s Cry3Bb1 corn product, containing 

the new active ingredient Bacillus thuringiensis Cry3Bb1 protein and the genetic material necessary for 

its production (vector PV-ZMIR13L) in Event MON863 corn, be REGISTERED under FIFRA section 

3(c)(7)(C), with appropriate limitations. 
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B. 2010 Update – Corn Event MON 863 (and MON 863 x MON 810)

Monsanto did not request an extension to their Corn Event MON 863 (EPA Reg. No. 524-528) or MON 

863 x MON 810 (EPA Reg. No. 524-545) registrations; therefore, these registrations expired on their 

own terms on September 30, 2010. The Agency considers the expiration of a conditional, time-limited 

registration to be a cancellation under Section 3 of the Federal Insecticide, Fungicide, and Rodenticide 

Act (FIFRA). A cancellation order, effective September 30, 2010, and appropriate provisions for 

disposition of existing stocks published in the Federal Register on August 25, 2010 (75 FR 52329). 
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C. Initial Registration (December 15, 2005) – MON 88017 and MON 88017 x MON 810 

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 imposition 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 conditional registration will not cause any unreasonable 

adverse effect on the environment, and (2) the registration and use of the pesticide during the conditional 

registration is in the public interest. EPA determines that all of these criteria have been fulfilled. 

EPA determined that it is appropriate to conditionally register the MON 88017 products under FIFRA 

section 3(c)(7)(C) for a period of time (specified below) reasonably sufficient for the generation and 

submission of certain data that are lacking because insufficient time has elapsed since the imposition of 

those data requirements: 

(1) For MON 88017, all data that was previously required as a condition of registration to support 

 the individual plant-incorporated protectant in Event MON863 (YieldGard® Rootworm), 524- 

528, are necessary. In the event that the Agency concludes that the MON 863 (YieldGard® Rootworm) 

studies required in connection with the MON 863 conditional registration do not sufficiently 

demonstrate a lack of significant adverse effects, additional data with MON 88017 corn must be 

submitted. These data may include the following: (a) laboratory toxicity testing with Orius insidiosus

(minute pirate bug), (b) laboratory toxicity testing with a carabid (ground beetle), (c) long-range effects 

testing on invertebrate populations in the field, and (d) long-range soil persistence testing. 

(2) For MON 88017 x MON 810, all data required to support the individual plant-incorporated 

protectants in MON 810 (YieldGard®), Event MON 863 (YieldGard® Rootworm), MON 88017 corn; 

EPA Registration Nos. 524-489, 524-528, are necessary. In the event that the Agency concludes MON 

863 (YieldGard® Rootworm) studies do not sufficiently demonstrate a lack of significant adverse 

effects, additional data with MON 88017 x MON 810 corn must be submitted. These data may include 

(a) laboratory toxicity testing with Orius insidiosus (minute pirate bug), (b) laboratory toxicity testing 

with a carabid (ground beetle), (c) long-range effects testing on invertebrate populations in the field, and 

(d) long-range soil persistence testing. Additionally, expression level data regarding Cry1Ab protein 

levels in MON 810 and MON 88017 x MON 810 young root and forage root are required within 12 

months of the date of registration. 

At this time, the applicant has submitted or cited data sufficient for EPA to determine that conditional 

registration of Bacillus thuringiensis Cry3Bb1 protein and the genetic material necessary for its 

production (vector PV-ZMIR39) in MON 88017 corn (OECD Unique Identifier: MON-88Ø17-3) under 

FIFRA 3(c)(7)(C) will not result in unreasonable adverse effects to the environment during the period of 

conditional registration, as discussed previously. Monsanto submitted and/or cited satisfactory data 

pertaining to the proposed use. The human health effects data and non-target organism effects data are 
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considered sufficient for the period of the conditional registration. These data demonstrate that no 

foreseeable human health hazards or ecological effects are likely to arise from the use of the products 

during the period of conditional registration and that the risk of resistance developing to Cry3Bb1 

proteins during the conditional registrations are not expected to be significant. 

Registration of MON 88017 and MON 88017 x MON 810 is in the public interest for the following 

reasons: 

 Replacement of higher risk pesticides. 

 Practical benefits for growers (reduced input costs, time and labor savings, etc.). 

 Efficacy (equivalent to conventional insecticides). 

 Human health benefits (reduced toxicity relative to conventional insecticides). 

 Environmental benefits (reduced risks to non-target organisms relative to conventional 

insecticides). 

 Yield benefits (greater yields than conventional corn). 

 Grower economic benefits (total benefits up to $6.56 per acre). 

 Herbicide tolerance: MON 88017 (and MON 88017 x MON 810) have been engineered to 

tolerate glyphosate herbicide applications. This trait will provide economic benefits to growers. 

 Enhanced breeding efficiency: MON 88017 can be bred faster (and selected through the 

herbicide tolerance trait), which should allow for a greater supply of the product to growers. 

In view of these minimal risks and the clear benefits related to MON 88017 (in both the MON 88017 

and MON 88017 x MON 810 products), EPA believes that the use of the products, during the limited 

period of the conditional registration, will not cause any unreasonable adverse effects. 

Although the data with respect to this particular new active ingredient are satisfactory, they are not 

sufficient to support an unconditional registration under FIFRA 3(c)(5). As noted above, additional data 

are necessary to evaluate the risk posed by the continued use of these products. Consequently, the 

Agency has imposed the data requirements specified earlier in this chapter. 

Permanent tolerance exemptions exist and are applicable to the MON 88017 products, MON 88017 and 

MON 88017 x MON 810. 

Based on the data submitted and cited by the applicant and reviewed by EPA, the EPA registered 

Monsanto’s Cry3Bb1 MON 88017 corn products, containing the new active ingredient Bacillus 

thuringiensis Cry3Bb1 protein and the genetic material necessary for its production (vector PVZMIR39) in MON 88017 corn (OECD Unique Identifier: MON-88Ø17-3), be REGISTERED under 

FIFRA section 3(c)(7)(C), with appropriate limitations. 

The expiration date of the registration has been set to September 30, 2010 for MON 88017 and October 

15, 2008 for MON 88017 x MON 810 (due to the MON 810 2008 expiration date). 
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D. 2010 Update – MON 88017 and MON 88017 x MON 810 

Section 3(c)(7)(A) of FIFRA provides for the registration or amendment of a pesticide when the 

pesticide and proposed use “...are identical or substantially similar to any currently registered 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 the 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 the use of any pesticide...” Thus, pursuant to section 

3(c)(7)(A), EPA may conditionally register a pesticide if (1) the pesticide and its proposed use are 

identical or substantially similar to a currently registered pesticide; or (2) the pesticide and its proposed 

use differ only in ways that would not significantly increase the risk of unreasonable adverse effects; and 

(3) approving the registration would not significantly increase the risk of any unreasonable adverse 

effect. 

The Agency concludes that the following Cry3Bb1 corn product registrations, set to expire on 

September 30, 2010 and described in-depth throughout this BRAD, meet both criteria (1) and (2): 

 (1) MON 88017 (EPA Reg. No. 524-551) 

 (2) MON 88017 x MON 810 (EPA Reg. No. 524-552) 

These Cry3Bb1 corn products are identical in both composition and use (corn) to plant-incorporated 

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 cultivation of Cry3Bb1-containing 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 sections II(C)(2)(b), II(C)(3)(b), 

and II(C)(4)(b) of this BRAD, strengthen the Agency’s initial position and also confirm that long-term 

effects on non-target organisms are not anticipated. Lastly, the continued 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 

2003 registration of Corn Event MON 863 and the 2005 registrations of MON 88017 and MON 88017 x 

MON 810 (e.g., reduction in use of conventional insecticides that are highly toxic to both humans and 

the environment). 
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In conclusion, as the expiring Cry3Bb1 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 datesa as 

follows: 

Product Name (EPA Reg. No.) Expiration Date 

MON 88017 (524-551) September 30, 2015 

MON 88017 x MON 810 (524-552) September 30, 2015 

Although data provided were satisfactory to make the determinations required by section 3(c)(7)(A) of 

FIFRA, they were not sufficient to support an unconditional 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 remain as terms or conditions for the purposes of the 

amendments. 

 

a

 See section III(E) of this BRAD for an explanation describing how the proposed expiration dates were determined. 
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E. 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 date of October 15, 

2008. All of the products being assessed at that time were efficacious against lepidopteran pests. EPA 

based this action on the finding that use of Cry1Ab or Cry1F expressed 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 later amended to extend the period of 

registration to an expiration date of September 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 registration twice, to 

February 24, 2008, and September 30, 2010. 

As set forth elsewhere in this document, EPA’s primary concern for the Bt protected transgenic corn 

products is the possibility that target pests will develop 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 stringent requirements intended to countermand the 

potential development of resistance. Registrants similarly have been busily developing various products, 

product mixes (i.e., so-called “pyramids” and “stacks”), and resistance strategies, 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 that the limited market penetration of these early 

crops would be so low as to guarantee that adequate natural refuges 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 management (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 treated 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 certainty and consistency to the market 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 combinations of “pyramided” products 

(i.e., products containing two or more toxins efficacious against the same pest) and “stacked” products 

(i.e., products combining toxins efficacious against different pests), the refuge requirements have begun 

to vary. For example, certain products require a 20% external refuge; some products permit a 5% 
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external refuge; one product incorporates a 10% seed blend refuge; we have applications in process for 

products that propose to incorporate a 5% seed blend refuge; and other permutations are possible. 

Given the profusion of various toxin combinations and refuge options, we can no longer proceed on the 

basis that, as concerns insect resistance management, all products are equal. It was a relatively simple 

proposition when the default requirement of a 20% sprayed refuge applied to almost all of the Bt corn 

crops in the market. Under those circumstances, the relative durability of products against the 

development of resistance was functionally equivalent, and, as a consequence, imposing functionally 

equivalent registration periods was appropriate. That is now no longer the case. 

As part of our continually evolving regulatory approach to the continually evolving product mix 

wrought by developers, we think it appropriate to revise our regulatory 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 particular product, or category of products, likely will pose 

less risk of insect resistance developing to a particular PIP protein, we think it appropriate to grant that 

particular product, or category of products, a registration for a period greater than that granted a 

corresponding product that poses a greater risk of insect resistance developing. This approach is 

reflective of complementary principles: first, to ensure that we apply our limited resources to the 

products that pose greater risk of adverse effects to the environment; and, second, to conserve the 

resources that registrants and applicants must expend in amending the registrations of products that pose 

less risk of adverse effects to the environment. 

The scheme that we are following includes 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 blend or external refuge, qualifies for a twelve-year 

registration; (iv) a product with pyramided PIP toxins (i.e., two or more 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 baseline 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 resistance 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. 
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APPENDIX A

GLOSSARY OF ACRONYMS AND ABBREVIATIONS 

ABSTC Agricultural Biotechnology Stewardship Technical Committee 

AIBS American Institute of Biological Sciences 

APHIS Animal and Plant Health Inspection Service (of the United States Department of Agriculture) 

AR Arkansas 

ARS Agricultural Research Service (of the United States Department of Agriculture) 

BPPD Biopesticides and Pollution Prevention Division 

BRAD Biopesticides Registration Action Document 

Bt/B.t. Bacillus thuringiensis

°C Celsius (degrees) 

C carbamate 

CAP/CAPs Compliance Assurance Program/Compliance Assurance Programs 

CEW corn earworm 

cm

2

 square centimeter/square centimeters 

CP4 EPSPS CP4 5-enolpyruvylshikimate-3-phosphate synthase 

CPB Colorado potato beetle 

CFR Code of Federal Regulations 

Co County 

CO Colorado 

Codex Codex Alimentarius Commission 

CRW corn rootworm 

DNA deoxyribonucleic acid 

DT50 half-life 

DT90 time until 90% decay 

dwt dry tissue weight 

EC50 growth inhibition 

ECB European corn borer 

ED50 median effective dose (produces desired effect in 50% of population) 

EDSP Endocrine Disruptor Screening Program 

EEC Estimated Environmental Concentration 

EFSA European Food Safety Authority 

ELISA enzyme-linked immunosorbent assay 

EPA Environmental Protection Agency (the Agency) 

EPA Reg. No. Environmental Protection Agency Registration Number 

EPA Reg. Nos. Environmental Protection Agency Registration Numbers 

EUPs experimental use permits 

Fst fixation index 

FAW fall armyworm 

FDA Food and Drug Administration 

FFDCA Federal Food, Drug, and Cosmetic Act 

FIFRA Federal Insecticide, Fungicide, and Rodenticide Act 

FQPA Food Quality Protection Act 

FR Federal Register 

ft foot/feet 
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GLOSSARY OF ACRONYMS AND ABBREVIATIONS, CONTINUED 

fwt fresh weight 

g gram/grams 

g/cm3

 grams per cubic centimeter 

GIPSA Grain Inspection, Packers and Stockyards Administration (of the United States Department of 

 Agriculture) 

GM genetically modified 

HEEC Highest Estimated Environmental Concentration 

HGT horizontal gene transfer 

IA Iowa 

IgG immunoglobulin G 

IL Illinois 

ILSI-CERA International Life Sciences Institute Research Foundation – Center for Environmental Risk 

Assessment 

IN Indiana 

IRM insect resistance management 

ISBN International Standard Book Number 

kDa kiloDalton 

kg kilogram/kilograms 

L liter/liters 

lb pound/pounds 

lb/ac pounds per acre 

LC50 median lethal concentration. A statistically 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). 

LD50 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). 

LDR leaf damage 

LLC limited liability company 

LOAEC Lowest Observed Adverse Effect Concentration 

LOC Level of Concern 

LOD limit of detection 

LOQ limit of quantitation 

MALDI-TOF matrix-assisted laser desorption/ionization time-of-flight 

MALDI-TOF-MS matrix-assisted laser desorption/ionization time-of-flight mass spectrometry 

MCRW Mexican corn rootworm 

m/d meters per day 

MD Maryland 

MEEC Maximum Expected Environmental Concentration 

meq milliequivalent 

μg microgram/micrograms 

μg/cm2

 micrograms per square centimeter 

μg/g micrograms per gram 

μg/L micrograms per liter 
Bacillus thuringiensis Cry3Bb1 Corn 

 Biopesticides Registration Action Document (BRAD) September 2010 

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GLOSSARY OF ACRONYMS AND ABBREVIATIONS, CONTINUED 

μg/mL micrograms per milliliter 

mg milligram/milligrams 

mg/kg milligrams per kilogram 

mg/kg bwt milligrams per kilogram bodyweight 

mg/mL milligrams per milliliter 

MHD maximum hazard dose 

mL milliliter/milliliters 

mL/g milliliters per gram 

mM millimolar 

MN Minnesota 

MRID No./MRID Nos. Master Record Identification Number/Master Record Identification Numbers 

MRL/MRLs maximum residue level/maximum residue levels 

N/A not applicable 

NAS National Academy of Sciences 

NCEAS National Center for Ecological Analysis and Synthesis 

NCGA National Corn Growers Association 

NCR-46 Technical committee consisting of research and extension CRW specialists and other 

 cooperators 

NCRW northern corn rootworm 

NE Nebraska 

ng nanograms/nanograms 

NIS Node/Nodal Injury Scale 

NOAEL No Observed Adverse Effect Level 

NOEC No Observable Effect Concentration 

NPTII neomycin phosphotransferase II 

OCSPP Office of Chemical Safety and Pollution Prevention 

OECD Organization for Economic Cooperation and Development 

OP organophosphate 

OPP Office of Pesticide Programs 

OPPTS Office of Prevention, Pesticides, and Toxic Substances 

PC Code Pesticide Chemical Code 

PCA Phased Compliance Approach 

PCR polymerase chain reaction 

PIP/PIPs plant-incorporated protectant/plant-incorporated protectants 

PP phenyl pyrazoles 

ppm parts per million 

PVDF polyvinylidene fluoride 

RDR root damage rating 

RKI Robert Koch Institute 

RNA ribonucleic acid 

RQ risk quotient 

SAP Scientific Advisory Panel (the Panel) 

SCRW southern corn rootworm 

SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis 

SGF simulated gastric fluid 

SIF simulated intestinal fluid 
Bacillus thuringiensis Cry3Bb1 Corn 

 Biopesticides Registration Action Document (BRAD) September 2010 

232

GLOSSARY OF ACRONYMS AND ABBREVIATIONS, CONTINUED 

SP synthetic pyrethroid 

SSA sublethal seedling assay 

SWCB southwestern corn borer 

T-DNA transfer deoxyribonucleic acid 

UCS Union of Concerned Scientists 

U.S. United States 

USDA United States Department of Agriculture 

WCRW western corn rootworm 

YGCB YieldGard® Corn Borer (i.e., MON 810)

YGRW YieldGard® Rootworm (i.e., MON 863)
Bacillus thuringiensis Cry3Bb1 Corn 

 Biopesticides Registration Action Document (BRAD) September 2010 

233 

Table 1. Currently Registered PIPs Expressing Cry3Bb1 Protein. 

EPA 

Registration 

Number 

Registration Name Company and Address Active 

Ingredient(s) 

Initial Date of Registration 

524-528* Corn Event MON 863 Monsanto Company 

800 North Lindbergh Boulevard 

St. Louis, MO 63167 

 Cry3Bb1 February 24, 2003 

524-545* MON 863 x MON 810 Monsanto Company 

800 North Lindbergh Boulevard 

St. Louis, MO 63167 

 Cry3Bb1 

 Cry1Ab 

October 31, 2003 

524-551 MON 88017 Monsanto Company 

800 North Lindbergh Boulevard 

St. Louis, MO 63167 

 Cry3Bb1 December 13, 2005 

524-552 MON 88017 x MON 810 Monsanto Company 

800 North Lindbergh Boulevard 

St. Louis, MO 63167 

 Cry3Bb1 

 Cry1Ab 

December 13, 2005 

524-576 MON 89034 x MON 88017 Monsanto Company 

800 North Lindbergh Boulevard 

St. Louis, MO 63167 

 Cry1A.105 

 Cry2Ab2 

 Cry3Bb1 

June 10, 2008 

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 

524-583 TC1507 x MON 88017 

*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 

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 

524-587 MON 89034 x TC1507 x MON 88017 

*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 

 Cry3Bb1 

October 15, 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 
Bacillus thuringiensis Cry3Bb1 Corn 

 Biopesticides Registration Action Document (BRAD) September 2010 

234

EPA 

Registration 

Number 

Registration Name Company and Address Active 

Ingredient(s) 

Initial Date of Registration 

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 

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 

68467-8 MON 89034 x TC1507 x MON 88017 

*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 

 Cry3Bb1 

October 15, 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 

68467-13 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 

68467-14 TC1507 x MON 88017 

*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 

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 expired on its own terms on September 30, 2010.