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


BIOPESTICIDES REGISTRATION ACTION DOCUMENT
Bacillus thuringiensis Cry3Bbl Protein and the Genetic Material Necessary for Its Production (Vector
PV-ZMIR13L) in MON 863 Corn (OECD Unique Identifier: MON-00863-5)
PC Code: 006484
Bacillus thuringiensis Cry3Bbl Protein and the Genetic Material Necessary for Its Production (Vector
PV-ZMIR39) in MON 88017 Corn (OECD Unique Identifier: MON-88017-3)
PC Code: 006498
U.S. Environmental Protection Agency
Office of Pesticide Programs
Biopesticides and Pollution Prevention Division

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Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)	September 2010
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. 20031 - Corn Event MON 863	219
B.	2010 Update - Corn Event MON 863 (and MON 863 x MON 8101	222
C.	Initial Registration (December 15. 20051 - 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
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Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)
September 2010
Bacillus thuringiensis Cry3Bbl 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.
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Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)
September 2010
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) Cry3Bbl
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-00863-
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
Cry3Bbl product (MON 88017; EPA Reg. No. 524-551) and several stacked and/or pyramided plant-
incorporated protectants (PIPs), expressing Cry3Bbl along with other proteins, for either commercial or
limited breeding purposes. A complete list of the currently registered products expressing Cry3Bbl—
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 Cry3Bbl:
(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 Cry3Bbl 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,
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Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)
September 2010
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. 20101
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 Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl registrations by extending the current expiration
dates in accordance with the scheme explained in section 111(E) of this Biopesticides Registration Action
Document (BRAD). The basis for this decision can be found in both the risk assessment for the
Cry3Bbl corn products, which is characterized throughout this BRAD, and the Agency's response to
comments document.
All data and findings for the Cry3Bbl 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 Cry3Bbl 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 information21 is also available for public examination:
•	CrylF and Cryl Ab BRAD (Draft - August 2010; Final - September 2010)
•	Cry3Bbl 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.
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Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)
September 2010
•	Cry 1 A. 105 and Cry2Ab2 BRAD (Draft - August 2010; Final - September 2010)
•	Optimum® AcreMax™ 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, Cry3Bbl, and Cry34/35Abl 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 registrant-
produced 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.
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Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)
September 2010
B. Use Profile
1. Corn Event MON 863
Pesticide Name: Bacillus thuringiensis Cry3Bbl protein and the genetic material necessary for
its production (vector PV-ZMIR13L) in MON 863 corn (OECD Unique
Identifier: MON-00863-5)
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.
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Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)
September 2010
2. MON 88017
Pesticide Name:
Trade and Other
Names:
OPP Chemical Code:
Basic Manufacturer:
Type of Pesticide:
Use:
Target Pests:
Products Expressing
This Pesticide:
Bacillus thuringiensis Cry3Bbl protein and the genetic material necessary for
its production (vector PV-ZMIR39) in MON 88017 corn (OECD Unique
Identifier: MON-88017-3)
MON 88017
006498
Monsanto Company
800 North Lindbergh Boulevard
St. Louis, Missouri 63167
Plant-Incorporated Protectant
Field and Sweet Corn
western corn rootworm (Diabrotica virgifera virgifera), northern corn
rootworm (.Diabrotica barberi), and Mexican corn rootworm (.Diabrotica
virgifera zeae)
See complete list in Appendix A.
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Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)
September 2010
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 Cry 1, 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 Resister (FR) 52998s)
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 68681s)
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-EUP-
93 (involving testing of Bacillus thuringiensis Cry3Bb protein and the
genetic material necessary for its production (vector ZMIR13L) in corn
only).
(65 FR 82352s)
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-EUP-
90), 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 4020s)
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Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)
September 2010
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. Arolicant: Monsanto Comranv. 700
Chesterfield Parkwav North. St. Louis. MO 63198. Product Name: Event
MON 863: Corn Rootworm Protected Corn (ZMIR13IA Tvpe of Product:
Plant-pesticide. Active Ingredient: Bacillus thurinsiensis Crv3Bb d rote in
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 15435s)
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 Cry3Bbl 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 Cry3Bbl 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
Cry3Bbl 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)
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Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)
September 2010
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 fox Bacillus thuringiensis Cry3Bbl 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-EUP-
93 (involving testing of Bacillus thuringiensis Cry3Bb protein and the
genetic material necessary for its production (vector ZMIR13L) in corn
only).
(61 FR 7687s)


Notice announcing receipt of the following application for a product
containing a new active ingredient:
March 13, 20022
Federal Register
Publication
(Notice of Receipt)
(1) File Symbol: 524-LEI. Arolicant: Monsanto Comranv. 700
Chesterfield Parkwav North. St. Louis. MO 63198. Product Name: Event
MON 863: Corn Rootworm Protected Corn (ZMIR13L). Tydc of Product:
Plant-pesticide. Active Ingredient: Bacillus thurinsiensis Crv3Bb d rote in
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)
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Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)
September 2010
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.
(61 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).
*ExpirationDate: Mav 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. Arolicant: Monsanto Comoanv. 700
Chesterfield Parkwav North. St. Louis. MO 63198. Product Name:
YieldGard Plus Corn. Tvpe of Product: Plant-incoroo rated protectant.
Active Ingredients: Bacillus thuringiensis Crv3Bbl orotein and the senetic
material necessary for its production (vector ZMIR13L) in corn and
Bacillus thuringiensis Cry lAb protein and the genetic material necessary
for its production in corn.
(68 FR 16036s)
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
Cry3Bbl 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-EUP-
93 (involving testing of Bacillus thuringiensis Cry3Bbl protein and the
genetic material necessary for its production (vector ZMIR13L) in corn
only).
(68 FR 19995s)
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Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)
September 2010
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 Cry3Bbl 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)
*ExpirationDate: Mav 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 Cry3Bbl protein and the genetic material
necessary for its production (vector ZMIR13L) in corn MON 863 and
Bacillus thuringiensis CrylAb 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.
(69FR917)
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 Cry3Bbl protein and the genetic material necessary for its
production (vector ZMIR39) in corn ZMIR39 and Bacillus thuringiensis
CrylAb 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.
(69FR917)
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Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)
September 2010
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-EUP-
96 (involving testing of Bacillus thuringiensis Cry3Bbl protein and the
genetic material necessary for its production (vector ZMIR39) in corn
ZMIR39 and Bacillus thuringiensis CrylAb 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 10040s)
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 Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl 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: Julv 31. 2006
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Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)
September 2010
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. Arolicant: Monsanto Company. 800 North
Lindbersh Boulevard. St. Louis. MO 63167. Product Name: MON 88017.
Tydc of Product: Plant-incorporated protectant. Active Insredient: Bacillus
thuringiensis Cry3Bbl 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
Lindbersh Boulevard. St. Louis. MO 63167. Product Name: MON 88017 x
MON 810. Tvpe of Product: Plant-incoroorated protectant. Active
Insredients: Bacillus thuringiensis Crv3Bbl d rote in and the eenetic
material necessary for its production (vector ZMIR39) in MON 88017 corn
and Bacillus thuringiensis Cry lAb 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 Cry3Bbl protein and the
genetic material necessary for its production (vector ZMIR39) in corn
ZMIR39 and Bacillus thuringiensis CrylAb 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 76732s)
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-EUP-
96 (involving testing of Bacillus thuringiensis Cry3Bbl protein and the
genetic material necessary for its production (vector ZMIR39) in corn
ZMIR39 and Bacillus thuringiensis CrylAb 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 2160s)
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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 Cry3Bbl
protein and the genetic material necessary for its production (vector
ZMIR39) in corn ZMIR39 and Bacillus thuringiensis Cry lAb 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 4,683 acres of field corn; Effective from February 18,
2005 to March 1, 2006.
(10 FR 46510)
October 19, 20052
Federal Register
Publication
(Notice of
Issuance)
Notice announcing conditional approval of two products:
CI) Event MON 863: Corn Rootworm Protected Corn (ZMIR13L)(EPA
Res. No. 524-528) - Resistered under FIFRA section 3(c¥7¥Q and
containing Bacillus thuringiensis Cry3Bbl 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 Res. No. 524-545) - Resistered under
FIFRA section 3(c)(7)(B) and containing Bacillus thuringiensis Cry3Bbl
protein and the genetic material necessary for its production (vector
ZMIR13L) in corn and Bacillus thuringiensis Cry lAb delta-endotoxin and
the genetic material necessary for its production in corn.
*Note: Over 900 comments were received bv the Asencv 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-HQ-
OPP-2004-0182 at www.resulations.sov.
(10 FR 60826s)
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
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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 Cry3Bbl was redesignated from 40 CFR §
180.1214 to 40 CFR § 174.518 and changed to the following:
"Residues of Bacillus thuringiensis Cry3Bbl 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."
C72 FR 2043 T)
August 15, 20073
Federal Register
Publication
(Notice of
Issuance)
Notice announcing conditional approval of two products:
m MON 88017 (EPA Res. No. 524-551) - Resistered under FIFRA
section 3(c)(7)(C) and containing Bacillus thuringiensis Cry3Bbl protein
and the genetic material necessary for its production (vector ZMIR39) in
Event 88017 corn (OECD Unique Identifier: MON-88017-3), an active
ingredient not included in any previously registered product.
(!) MON 88017 x MON 810 (EPA Res. No. 524-552) - Resistered under
FIFRA section 3(c)(7)(C) and containing Bacillus thuringiensis Cry3Bbl
protein and the genetic material necessary for its production (vector
ZMIR39) in Event 88017 corn (OECD Unique Identifier: MON-88017-
3)(an active ingredient not included in any previously registered product)
and Bacillus thuringiensis Cry lAb delta-endotoxin and the genetic material
necessary for its production (vector PV-ZMCT01) in Event MON 810 corn
(OECD Unique Identifier: MON-00810-6).
(72 FR 45807s)
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
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Date
Action Type
Description
June 2008-December
20093
Registration
The Agency registered several combination PIP products expressing
Cry3Bbl 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.
(15 FR 52329s)
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 IIKED of this Biooesticides 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
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September 2010
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 Cry3Bbl-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, Cry3Bbl 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-
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September 2010
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 non-
specific 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 /^-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 Cry3Bbl 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) body weight of test animal. Isolating the amount of purified protein, required to dose
several animals, from /^-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
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Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)
September 2010
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 11(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 Cryl Ab 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 11(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 Cry3Bbl corn. The extraction and detection method as described for Cry3Bbl
protein appears to be adequate for analysis of Cry3Bbl 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 Cry3Bbl, 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 Cry3Bbl in grain and oilseeds has
been completed (USDA 2004); thus, the requirement for EPA laboratory method validation for Cry3Bbl
has also been satisfied.
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September 2010
2. Corn Event MON 863 (Organization for Economic Cooperation and Development
(OECD) Unique Identifier: 00863-5) Expressing Cry3Bbl
a. Data Cited/Submitted for Initial Registration of Corn Event MON 863 (Prior to
February 2003)
Cry3Bbl protein is a delta-endotoxin from Bacillus thuringiensis subspecies kumamotoensis and has
activity against certain beetles. The wild-type cry3Bbl gene was modified to enhance the protein's
activity against the corn rootworm complex. Two Cry3Bbl variants were engineered for expression in
the bacterium, Bacillus thuringiensis strains EG11098 and EG11231. Cry3Bb 1 protein resulting from
these strains differed from wild-type Cry3Bbl protein by 5 and 4 amino acid substitutions (see Tablel).
Corn was genetically modified to express the Cry3Bb 1.11231 protein (resulting in corn line MON 853)
or the Cry3Bbl. 11098 protein (resulting in corn line MON 863). At the 5' end of the c/j3Bbl 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.
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Biopesticides Registration Action Document (BRAD)
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Table 1. Cry3Bbl Protein Variants: Amino Acid Sequence Percent Identities and Position Differences
Cry3Bbl Variant0
% Identity
Wild-Type
Amino Acid Positionsab
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
Ciy3Bbl.11231
99.4
N/A
D
R
L
T
K
Q
Ciy3Bbl.11098
99.2
N/A
G
R
L
T
K
Q
Ciy3Bbl.11098
(Q349R)
98.9
A
G
R
L
T
K
R
Cry 3Bbl.pvzmir39
99.1
A
D
R
L
T
K
R
Plant-Produced Protein/Product
Ciy3Bb 1.11231/
MON 853
99.2
A
D
R
L
T
K
Q
Ciy3Bbl.11098
(Q349R)/MON 863
98.9
A
G
R
L
T
K
R
Cry 3Bb 1 ,pvzmir3 9/
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 Cry3Bbl protein variants contain 652 amino acids. The Escherichia coli- and plant-produced Cry3Bbl
protein variants contain 653 amino acids due to the insertion of an alanine residue at position 2, resulting from the assembly
of the cry3 Bb l gene into the E. coli- or plant-transformation vector.
0 All Cry3Bbl protein variants, except for Cry3Bbl.pvzmir39, are discussed inMRID No. 454240-09, while the wild-type
Cry3Bbl is discussed in Donovan el 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 cry3Bbl 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, Cry3Bb 1.11098 (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 Cry3Bb 1.11098, Cry3Bbl.l 1098 (Q349R), and Cry3Bbl.11231 proteins are variants of the
Cry3Bbl protein. Since these variants do not differ significantly from the Cry3Bbl protein in terms of
biochemical or toxicological characteristics, the Cry3Bbl. 11098, Cry3Bbl.l 1098(Q349R), and
23

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Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD) September 2010
Cry3Bbl. 11231 protein variants are all covered by the Cry3Bbl 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) Un
ess 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), PV-
ZMIR13L (MON 863), or PV-ZMIR14L (MON 853 and MON 860)) via a
particle bombardment mechanism. The m'3Bb land neomycin
phosphotransferase II (nptll) 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 Cry3Bbl
polyclonal antisera confirm the relationship of Cry3Bb 1.11098 and
Cry3Bb 1.11231 to wild-type Cry3Bbl. 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—
Cry3Bb 1.11098 and Cry3Bb 1.11231—have been confirmed as Cry3Bbl
protein variants.
Classification: Acceptable
454240-03
Assessment of the
Physiochemical
Equivalence of
Cry3Bb 1.11098 and
NPTII Proteins in Corn
Event MON 863 to
Microbial Sources
Based upon the data provided, it appears that both the Cry3Bb 1.11098 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
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Bacillus thuringiensis Cry3Bb1 Corn
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September 2010
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 Cry3Bb 1.11098
and Cry3Bbl. 11231 proteins produced inE. coli and cornrootworm
(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
Cry3Bbl Protein
Produced in Corn Event
MON 863
Two genetic variants, designated as m'3Bbl. 11098 and m'3Bbl. 11231.
produce the delta-endotoxin proteins, Cry3Bbl.11098 and Cry3Bbl.11231,
respectively. Cry3Bbl.11098 differs from the wild-type Bacillus
thuringiensis (B.t.) protein by 5 amino acids, while the Cry3Bb 1.11231
protein differs by 4 amino acids. The ciy3 Bb 1.11098 gene was used to
develop maize line MON 863 and variant ay3 Bb 1.11231 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) Ciy3Bbl proteins,
respectively. Cry3Bbl 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 matrix-
assisted 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 N-
terminal amino acids. Data from MALDI-TOF-MS and N-terminal
sequencing indicate that the deduced amino acid sequences of
Cry3Bb 1.11098, as present in MON 863 and in H.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
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Biopesticides Registration Action Document (BRAD)
September 2010
Study Title
Summary
MRID No.
Primary Structural
Protein Characterization
of Corn Event MON 863
Cry3Bb 1.11098 Protein
Using N-Terminal
Sequencing and
MALDI-TOF
Spectrometry
Techniques
Transformation Event MON 863 (maize) produces the 74-kiloDalton (kDa)
Cry3Bb 1.11098 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 Cry3Bb 1.11098
protein for MALDI-TOF-MS and N-terminal sequence analyses. Of the
653 amino acids present in the 74-kDa form of the Cry3Bbl 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 N-
terminus, 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 N-
terminus of the corn-derived Cry3Bb 1.11098 protein.
Classification: Acceptable
(Reviewed in U.S. EPA (2002b))
454240-11
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September 2010
Study Title
Summary
MRID No.
Characterization and
Equivalence of the
Cry3Bbl Protein
Produced by E. coli
Fermentation and Corn
Event MON 863
MALDI-TOF analysis of the microbial and corn Cry3Bbl. 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 N-
terminal sequencing could be detected. In the E. coli Cry3Bbl, the
sequence started at both positions 2 and 32. In the corn Cry3Bbl, three
different starts were detected at positions 19, 25, and 36. The immunoblot
analysis gave similar positive band patterns, indicating the Cry3Bbl
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
Cry3Bbl proteins compared to the corn extracts. The purity for Cry3Bbl
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 Cry3Bbl extracts gave no positive carbohydrate staining regions for
either the microbial or corn samples in the expected regions for Cry3Bbl
protein. The results of the bioassays for the two Cry3Bblextracts 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
27

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Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)
September 2010
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 Cry3Bbl 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 Cry3Bbl protein levels in MON
853 in microgram Cry3Bbl 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 Cry3Bbl protein levels in MON 860 in microgram
Cry3Bb 1 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 Cry3Bbl and
NPTII proteins.
Classification: Acceptable
454240-02
451568-01
B.t. Cry3Bb 1.11098 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 Cry3Bbl 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 Cry3Bbl protein levels in MON863 in microgram Cry3Bbl
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
28

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Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)
September 2010
Study Title
Summary
MRID No.
Validated Method for
Extraction and Direct
ELISA Analysis of
Cry3Bbl in Corn Grain
The extraction and detection method, as described for Cry3Bbl protein,
uses Cry3Bbl-specific antisera for direct sandwich ELISA of Cry3Bbl
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 Cry3Bbl 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 Cry3Bbl analytical method within 6 months
of the date of registration. The extraction and detection method as described for
Cry3Bbl protein appears to be adequate for analysis of Cry3Bbl 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.
29

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Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)	September 2010
Table 3. Product Characterization Data for Corn Event MON 863 (Reviewed in U.S. EPA
	(2006)).	
Study Title
Summary
MRID No.
TraitChek™ Ciy3Bbl
Lateral Flow Test Strip
and SeedChek™
Cry3Bbl ELISA
Performance
Verification for Corn
Seed, Leaf, and
Composite Testing
The TraitChek™ Cry3Bb 1 lateral flow strip test and the SeedChek ™
Cry3Bbl ELISA were evaluated for qualitative detection of Cry3Bbl
protein in corn seed and leaves. Three lots of each test kit were used to
evaluate each of 100 known Cry3Bbl corn seed or corn leaf samples and
100 non-transgenic control samples. In single-seed testing of known
Cry3Bbl kernels, both tests were 100% accurate. In composite-seed testing
(1 known Cry3Bbl kernel in 800 total kernels), the TraitChek™ test had a
false negative rate of 0.3% after a five-minute reading but was 100%
accurate after ten minutes. The SeedChek ™ test was 100% accurate at
both reading times. Both tests showed 100% accuracy in detecting
Cry3Bbl in leaf samples from 13 varieties of Cry3Bbl corn. No cross-
reactivity with other purified recombinant proteins or corn events
expressing other recombinant proteins was observed for either test.
Classification: Acceptable
*Note for 2010: Althoueh the reauirement for an independent laboratory
validation of the Cry3Bbl 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 Cry3Bbl, 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
30

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Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)
September 2010
Study Title
Summary
MRID No.
Cry3Bbl and NPTII
Protein Levels in Corn
Tissues from MON 863
Produced in 2003 U.S.
Field Trials
The levels of proteins Cry3Bbl 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 Cry3Bbl 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 (|ig/g) dwt. Levels of Cry3Bbl 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 Cry3Bbl levels (mean of 55 and 80 jxg/g dwt, respectively), and the
lowest Cry3Bbl levels were in pollen, stover, senescent root, and grain
(mean of 20-35 jxg/g dwt). NPTII protein was only measured in grain,
where it was not detected.
The frozen storage stability of Cry3Bbl 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 Cry3Bbl 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-00863-5 x MON- 00810-6)
Expressing Cry3Bbl and CrylAb
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 Cry3Bbl and NPTII proteins, and Event MON 810, which
expresses the CrylAb 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 11(A)(2) of this BRAD.
31

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Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)
September 2010
MON 810 produces Bacillus thuringiensis subsp. kurstaki strain HD-1 Cryl Ab 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 Cryl Ab protein and the genetic material necessary (PV-
ZMCT01) for its production in corn Event MON 810 (OECD Unique Identifier: MON-00810-6). 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 Cryl Ab and CrylF 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 (cry 1 Ab) and MON 863 (cry3Bb 1). was examined for
the presence of these two genes encoding delta-endotoxins in the resulting
hybrid. Probes for the cryl Ab and cry 3 Bb 1 genes were obtained from
previous studies and corresponded to the first 900 base pairs (cryl Ab) or
the entire length of the gene (cry3Bb 1). The radiolabeled (32P) probe for
cryl Ab 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 cryl Ab in
the MON 810 and MON 810 x MON 863 hybrid plant samples. When
DNA samples were probed with the cry3Bb 1 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 cry 1 Ab and cry3Bb 1 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
32

-------
Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)
September 2010
Study Title
Summary
MRID No.
Cry3Bbl, CrylAb 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.
Cry3Bb 1 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 Cry3Bbl, CrylAb, 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
Cry3Bbl and CrylAb 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 Cry3Bbl or CrylAb were
found to occur in pollen for Cry3Bbl (79.6 |ig/g fresh weight) and in leaf
(17.9 |ig/g fresh weight) for CrylAb.
Classification: Acceptable
457917-02
Table 5. Comparison of CrylAb and Cry3Bbl Protein Levels in Dual-Trait and Single-Trait
Hybrids

Average CrylAb Protein Levels (ju.g/g
fwt) (Range)
Average Cry3Bbl Protein Levels (ju.g/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
33

-------
Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)	September 2010
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 11(A)(2)(b) of this BRAD) have been satisfied for the purposes of
this registration.
4. MON 88017 (OECD Unique Identifier: MON-88017-3) Expressing Cry3Bbl
a. Data Cited/Submitted for Initial Registration of MON 88017 (Prior to December 2005)
The Cry3Bbl protein, as produced in MON 88017, is a variant of the wild-type Cry3Bbl 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 Cry3Bbl.pvzmir39 variant (MON
88017) differs by seven amino acids from the wild-type Cry3Bbl protein and by a single amino acid
from the Cry3Bbl. 11098 (Q349R) protein (MON 863). The Cry3Bbl 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 11(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 Cry3Bbl protein in MON 88017 were found to be similar with
the Cry3Bbl protein in MON 863. Moreover, the protein expression, functional activity, and field
efficacy data of Cry3Bbl 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.
34

-------
Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)
September 2010
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 Sea I restriction fragment. The plasmid vector
contains the cry3Bbl.pvzmir39 and cp4 epsps expression cassettes within
the borders. No additional elements from the transformation vector PV-
ZMIR39 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, Cry3Bbl and CP4 EPSPS, are encoded by
the insert in corn Event MON 88017.
Classification: Acceptable
461817-02
465783-01
Cry3Bbl 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 Cry3Bbl and CP4 EPSPS
protein levels using validated ELISA methods. The mean Cry3Bbl 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 |ig/g dry tissue weight, respectively. The mean Cry3Bbl protein levels
for pollen, forage, silk, and grain tissue were 25, 95, 380, and 15 |ig/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 |ig/g dry tissue weight, respectively. The mean CP4 EPSPS protein
levels for pollen, forage, and grain tissue were 390, 57, and 5.8 |ig/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
Cry3Bbl 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
35

-------
Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)
September 2010
Study Title
Summary
MRID No.
Evaluation of the
Functional Equivalence
of Two Cry3Bbl
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, Cry3Bb 1.11098
(Q349R) produced in MON 863 and Cry3Bbl.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 (|ig/mL) (ranging from 0.79-1.11
Hg/mL) forthe Cry3Bbl.11098 (Q349R) protein and 0.84 |ig/mL (ranging
from 0.64-1.06 |ig/mL) for diets containing the Cry3Bbl.pvzmir39
protein. For WCRW larvae, the mean dietary median lethal concentrations
were 100 |ig/mL (rangingfrom73.2-137 |ig/mL) forthe Cry3Bbl.11098
(Q349R) protein and 139 ng/mL (ranging from 74.6-231 ng/mL) in diets
containing the Cry3Bbl.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
11(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-88017-3 x MON-00810-6)
Expressing Cry3Bbl and CrylAb
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 (Cry3Bbl) and MON 810 (CrylAb), via traditional breeding methods. Product
characterization background on MON 88017 was discussed in section 11(A)(4) of this BRAD; MON 810
was briefly summarized in section 11(A)(3)(a). but more comprehensive information can be found in
either the 2001 Bt Crops Reassessment or the September 2010 CrylAb and CrylF 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.
36

-------
Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)
September 2010
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 Cry3Bbl and CrylAb
proteins, respectively.
Classification: Acceptable
461850-02
Cry3Bbl,CP4 EPSPS,
and CrylAb Protein
Levels in Corn Tissues
Collected from MON
88017 xMON 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 Cry3Bbl, CP4
EPSPS, and CrylAb protein levels using validated ELISA methods. The
levels of Cry3Bbl and CrylAb 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 Cry3Bbl protein levels in MON
88017 x MON 810 corn for root tissue harvested at the V2-V3 stage
ranged from 140-350 |ig/g dwt. The mean Cry3Bbl protein levels for leaf,
pollen, grain, and forage tissues were 670, 27, 9.3, and 100 |ig/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 |ig/g dwt, respectively.
Finally, the mean CrylAb protein levels in MON 88017 x MON 810 corn
for leaf, grain, and forage tissues were 110, 0.39, and 14|ig/g dwt,
respectively. The mean CrylAb protein level for pollen was below the
limit of detection (LOD, 0.090|ig/g fwt). The results demonstrate that the
range and mean Cry3Bbl and CrylAb 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
RegistrationNos. 524-489, 524-528...."
37

-------
Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)
September 2010
"Submit expression level data regarding CrylAb 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 11(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 CrylAb
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
CrylAb 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 |-ig/g fresh weight, and limit for quantification was 0.40 )ag/g fresh
weight. ELISA revealed mean CrylAb protein levels in MON 88017 x
MON 810 corn tissues across all sites were 75 )ag/g dwt in young root and
12 |-ig/g dwt in forage root; similar to the mean CrylAb protein levels in
MON 810 corn, which were 78 )ag/g dwt in young root and 13 )ag/g dwt in
forage root.
Classification: Acceptable
470045-01
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Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)
September 2010
Table 9. Cry3Bbl and CrylAb Protein Levels in MON 88017 x MON 810 and MON 88017 & MON
810 Tissues.
Tissue
Tvoe
Growth
Staae
Crv3Bb1 Protein Levels (ua/a dwt)
CrvlAb Protein Levels (ua/a dwt)


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


Mean
Standard
Deviation
Range
n=9
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
100
12
89-130
Young
Root
V2-V3
350
150
88-560
370
80
240-510
75*
26*
14-130*
78*
21*
51-130*
Pollen
R1
27
5.7
N/A-34
25
4.2
17-32
N/A
N/A
N/A
N/A
N/A
N/A
Forage
R4-R6
(early
dent)
100
23
71-150
95
19
75-130
14
2.1
11-17
14
3.4
8.4-19
Forage
Root
R4-R6
(early
dent)
140
29
89-180
130
29
98-170
12*
4.7*
2.7-22*
13*
5.1*
6.6-21*
Grain
R6
9.3
3.4
3.9-13
15
3.6
10-22
0.39
0.13
0.16-
0.63
0.43
0.091
0.27-
0.54
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
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Bacillus thuringiensis Cry3Bb1 Corn
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September 2010
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, TonnuB, Albee L. 2000. B.t. Cry3Bb 1.11098 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 Cry3Bb 1.11098 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 Cry3Bbl 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.
Cry3Bbl.l 1098 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.
40

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Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)
September 2010
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, PylaP, 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 Cry3Bb 1.11098 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 Cry3Bbl
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 Cry3Bbl
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 Cry3Bb 1.11098 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 Cry3Bbl 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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Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)
September 2010
MRID No. 457917-02. Dudin Y, Tonnu B, Lirette R. 2001. Cry3Bbl, CrylAb 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. BhaktaN, Hartmann A, Jennings J. 2003. Cry3Bbl 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 Cry3Bbl 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. BhaktaN, Hartmann A, Jennings J. 2003. Cry3Bbl, CP4 EPSPS, and CrylAb
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. Cry3Bbl 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.
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Bacillus thuringiensis Cry3Bb1 Corn
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September 2010
MRID No. 465783-02. Bhakta N, Hartmann A, Jennings J. 2005. Cry3Bbl 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 Cry3Bbl 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 CrylAb 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 Cry3Bb 1.11098 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/oppbppdl/biopesticides/pips/bt brad, htm.
U.S. EPA. 2002a. EPA Review of the Application for an Exemption from the Requirement of a
Tolerance for Cry3Bbl 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 Cry3Bbl Insect Control Protein as
Expressed in Maize, a Bacillus thuringiensis-B&sed 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 CrylAb / Cry3Bbl 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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Bacillus thuringiensis Cry3Bb1 Corn
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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 Cry3Bbl
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 Cry3Bbl and Cryl Ab 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 Cry3Bbl 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 Cry3Bbl 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 CrylA(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
Cryl Ab and CrylF 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: ic ii'ic. giysa. usda. gov/GIPSA/documents/GIPSA Documents/9181-2, ydf.
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B. Human Health Assessment
September 2010
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 11(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 ol. 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 Cry3Bbl
The detailed Agency human health assessment of Cry3Bbl 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, 200 Id,
200 le, and 2005a). A summary of the key findings is provided below.
a. Mammalian Toxicity and Allergenicitv 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 Cry3Bbl 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 Cry3Bbl 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 §§ 158.213 0(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 Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl protein would be non-toxic to
humans. Male and female mice (10 of each) were dosed with 36, 396, or 3,780 milligrams/kilograms
body weight (mg/kg bwt) of Cry3Bbl protein for one variant. The mice were dosed with 38.7, 419, or
2,980 mg/kg bwt of Cry3Bbl protein for the second variant. The mice were dosed with 300, 900, or
2,700 mg/kg bwt of Cry3Bbl 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 Cry3Bbl proteins are not considered toxic. Further,
amino acid sequence comparisons showed no similarity between the Cry3Bbl proteins and known toxic
proteins available in public protein databases.
ii. Allergenicitv Assessment
Since Cry3Bbl 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 Cry3Bbl proteins are rapidly degraded by gastric
fluid in vitro. In a solution of simulated gastric fluid (pH 1.2), complete degradation of detectable
Cry3Bbl 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 Cry3Bbl,
even at the level of 8 contiguous amino acids residues.
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iii. Conclusion
The potential for the Cry3Bbl proteins to be food allergens is minimal. Regarding toxicity to the
immune system, the acute oral toxicity data submitted support the prediction that the Cry3Bbl 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 ol. 1992). Therefore, since no effects were shown to be caused
by the plant-incorporated protectants, even at relatively high dose levels, the Cry3Bbl 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 plant-
incorporated 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 Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl 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: ic ii'ic. epa. gov/yesticides/cumulative.
d. Determination of Safety for U.S. Population, Infants, and Children
i. Toxicity and Allergenicitv Conclusions
The data submitted and cited regarding potential health effects for the Cry3Bbl proteins include the
characterization of the expressed Cry3Bbl 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 Cry3Bb 1 protein test material derived from
microbial cultures was biochemically and functionally similar to the protein produced by the plant-
incorporated 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 Cry3Bbl proteins would be non-
toxic 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 Cry3Bbl proteins, even at relatively high dose levels (3,780 mg/kg bwt
of Cry3Bbl protein), the Cry3Bbl 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).
Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl 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 plant-
incorporated protectants.
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e. Other Considerations
i. Analytical Enforcement Methodology
Methods for extraction and direct enzyme-linked immunosorbent assay (ELISA) analysis of Cry3Bbl 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 Cry3Bbl 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 endocrine-
related effects caused by the substance, and establish a quantitative relationship between the dose and
the E, A, or T effect.
Between October 2009 and February 2010, the Agency issued test orders/data call-ins for the first group
of 67 chemicals, which contains 58 pesticide active ingredients and 9 inert ingredients. This list of
chemicals was selected based on the potential for human exposure through 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 Cry3Bbl 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.sov/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 Seralini el 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 Cry3Bbl protein produced in MON 88017 is based on
studies conducted with Cry3Bbl protein variants (Cry3Bbl. 11098 and Cry3Bbl. 11231). The
physicochemical characteristics of the Cry3Bbl protein in MON 88017 were found to be similar
with the Cry3Bbl protein in MON 863. Moreover, the protein expression levels, insect bioactivity,
and field efficacy data of Cry3Bbl protein in MON 88017 were compared to MON 863 and found
to be similar and functionally equivalent. Therefore, because the Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl 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. 2007V
"§ 174.518 Bacillus thuringiensis Cry3Bbl protein in corn;
exemption from the requirement of a tolerance.
Residues of Bacillus thuringiensis Cry3Bbl 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 Cry3Bbl (Reviewed in U.S. EPA (2002a) Unless
Otherwise
Voted).
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
Cry3Bbl 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 9803 5M3-007 and 9803 5F3-004 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
Cry3Bb 1.11098 protein exposure related. Based upon the data provided,
the median lethal dose (LD50) for Cry3Bbl. 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 Cry3Bbl.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 Cry3Bb 1.11231 protein
exposure related. Based upon the data provided, the LD50 for
Cry3Bbl.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
Cry3Bbt.11098
(Q349R) Protein
There did not appear to be significant adverse affects to animals resulting
from exposure to Cry3Bb 1.11098 (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 Cry3Bbl.ll098(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 Cry3Bbl proteins are not
stable to digestion in simulated gastric fluid. Incubation of Cry3Bb 1.11098
and Cry3Bbl.11231 in SGF results in the loss of detectable protein by the
30- and 15-second observation points, respectively, as detected by SDS-
PAGE. 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 Cry3Bbl proteins are not
stable to digestion in simulated gastric fluid. Incubation of Cry3Bb 1.11098
and Cry3Bbl.11231 in SGF results in the loss of detectable protein by the
30- and 15-second observation points, respectively, as detected by SDS-
PAGE. 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
Cry3Bbl Protein
Purified from Corn
Event MON 863 and
Cry3Bbl Protein
Purified from E. coli
The tests performed in this study show that the Cry3Bbl proteins are
degraded in simulated gastric fluid. Incubation of corn-produced and
Escherichia coli-produced Cry3Bbl protein in SGF results in the loss of
detectable protein by the 15-second observation point, as detected by SDS-
PAGE.
Classification: Acceptable
455382-03
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Study Title
Summary
MRID No.
Assessment of the in
vitro Digestibility of the
Cry3Bbl.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 Cry3Bb 1.11098 (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: Immuno-
Detectability of
Cry3Bbl.11098 and
Cry3Bbl.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 Cry3Bb 1.11098 found in
MON 863 corn. The Cry3Bbl immunoreactivity was not detectable in both
an immunoblot and ELISA format for MON 863. For MON 853, Cry3Bbl
was not recognizable in an immunoblot and reduced more than 1000-fold
in an ELISA format. Since the rabbit anti-Cry3Bbl 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 Cry3Bb 1.11098 and Cry3Bb 1.11231 to known
protein toxins. The TOXIN4 database was compiled to allow for
comparison of Cry3Bbl.11098 and Cry3Bbl.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 Cry3Bbl. 11098 nor
Cry3Bb 1.11231 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 Cry3Bbl to known protein allergens and
gliadins. The UPDATE2 database was compiled to allow for comparison of
Cry3Bb 1.11098 and Cry3Bb 1.11231 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 Cry3Bbl 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
Cry3Bbl 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 Cry3Bb 1.11098 delta-
endotoxin when compared to the sequence as found in wild type Bt. Bt
strains EG11231 and EG11098 contain variants of the Cry3Bb 1 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
Cry3Bb 1.11098 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
Cry3Bbl 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 Cry3Bbl 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 Cry 3Bb 1 proteins, their lack of homology to known toxins
and allergens, and the safety of the microbial biopesticide Raven®, which
expresses Cry3Bbl proteins, the Cry3Bbl 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 Cry3Bbl
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 Cry3Bbl
protein produced in MON 88017.
All physiochemical characteristics in the Cry3Bbl protein in MON 88017
were found to be similar to the Cry3Bbl protein in MON 863. Moreover,
the protein expression levels, insect bioactivity, and field efficacy data of
Cry3Bbl protein in MON 88017 were compared to MON 863 and found to
be similar and functionally equivalent. Based on substantial similarity of
Cry3Bbl protein in MON 88017 and the Cry3Bbl 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 Cry3Bbl 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
Cry3Bbl protein in transgenic corn MON 88017.
Classification: Acceptable
(Reviewed in U.S. EPA (2005a))
461817-01
3. Human Health Assessment of CrylAb (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 CrylAb, neomycin phosphotransferase II (NPTII), and CP4 5-
enolpyruvylshikimate-3-phosphate (CP4 EPSPS) proteins:
"§ 174.511 Bacillus thuringiensis CrylAb protein in all plants;
exemption from the requirement of a tolerance.
Residues of Bacillus thuringiensis CrylAb 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 Cryl Ab 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 Cryl Ab and CrylF
BRAD (U.S. EPA 200If and 2010).
The individual data generated for the Cryl Ab, NPTII, and CP4 EPSPS proteins (and Cry3Bbl) 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 CrylAb and Cry3Bbl 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.
co//'-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 Cry3Bbl.l 1098 and Cry3Bbl.l 1231 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
Cry3Bbl 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 Cry3Bbl
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 Cry3Bbl.l 1098(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
Cry3Bbl Protein Purified from Corn Event MON 863 and Cry3Bbl 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.
MR TP No. 455770-02. Kileman R, Leach J, Astwood J. 2001. Assessment of the in vitro Digestibility of
the Cry3Bbl.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 Cry3Bbl Protein Produced in MON 88017.
Project Number: MSL/18835. Unpublished study prepared by Monsanto Company, 64 pages.
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Seralini 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.sov/scipolv/sap/meetinss/2000/iune/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. sov/oppbppdl/biopesticides/pips/bt brad. htm.
U.S. EPA. 2002a. EPA Review of the Application for an Exemption from the Requirement of a
Tolerance for Cry3Bbl 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 Cry3Bbl Insect Control Protein as
Expressed in Maize, a Bacillus thuringiensis-B&sed 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 Cry3Bbl and CrylAb 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 Cry3Bbl
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
Cry3Bbl [40 CFRPart 180.1214] and CrylAb [40 CFR 180.1173] Apply to Cry3Bbl Protein
Expressed in Event MON 88017 Corn [EPA Reg. No. 524-LLR] and the Combination of
Cry3Bbl and CrylAb 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
CrylAb and CrylF 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 real-
world field scenarios if the earlier tiered tests fail to indicate adequate certainty of acceptable risk.
Screening (Tier I) non-target organism hazard tests are conducted at exposure concentrations several
times higher than the highest concentrations expected to occur under realistic field exposure scenarios.
This has allowed an endpoint of 50% mortality to be used as a trigger for additional higher tier testing.
Less than 50% mortality under these conditions of extreme exposure suggest that population effects are
likely to be negligible given realistic field exposure scenarios.
The 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 (lOx 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 (lx EEC) concentrations and routes of
exposure.6
b General OCSPP Harmonized Testing Guidelines available from: http://www.epa.sov/ocspp/pubs/frs/home/suidelin.htm.
Series 850 Testing Guidelines available from:
http://www.epa.sov/ocspp/pubs/frs/publications/OPPTS Harmonized/850 Ecolosical Effects Test Guidelines/Drafts/index.
html.
Series 885 Testing Guidelines available from:
http://www.epa.sov/ocspp/pubs/frs/publications/Test Guidelines/series885.htm.
0 The dose margin can be less than lOx 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 lx EEC.
d Note that the lOx EEC MHD testing approach is not equivalent to what is commonly known as "testing at a lOx safety
factor," where any adverse effect is considered significant. Tier I screen testing is not "safety factor testing." In a "lOx safety
factof' 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 lx the field exposure.
e The lx 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 lx 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 lOx 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-analysis8 of the data collected from 42 field studies indicated that non-
target invertebrates are generally more abundant in Bt cotton and Bt maize fields than in non-transgenic
fields managed with insecticides (Marvier et al. 2007). In addition, a comprehensive review of short-
and long-term field studies on the effects of invertebrate populations in Bt corn and cotton fields
indicated that no unreasonable adverse effects are taking place as a result of wide-scale Bt crop
cultivation (Sanvido et al. 2007). Another review of field tests published to date concluded that the
large-scale studies in commercial Bt cotton have not revealed any unexpected non-target effects other
than subtle shifts in the arthropod community caused by the effective control of the target pests (Romeis
et al. 2006). Slight reductions in some invertebrate predator populations are an inevitable result of all
pest management practices, which result in reductions in the abundance of the pests as prey.
Overall, the Agency is in agreement with the conclusions of these studies and, collectively, these results
provide extensive data to support that Bt crops have not caused long-term environmental effects, on a
population level, to organisms not targeted by Bt proteins. Based on these considerations, regulatory
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: httv://delvhi.nceas.ucsb.edu/btcrovs/.
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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-00863-5) Expressing Cry3Bbl
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 Cry3Bbl
protein and the genetic material necessary for its production (vector PV-ZMIR13L) in MON 863 corn
(OECD Unique Identifier: MON-00863-5). 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 Cry3Bbl-producing corn lines. The general
topics covered include gene flow to related wild plants, development of weediness, effects on wildlife,
and fate of Cry3Bbl 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 plant-
incorporated protectant regulation.
ii. Non-Target Wildlife Hazard Assessment
Two separate SAP reports (October 2000 and August 2002) recommended that non-target testing should
focus on species exposed to the crop being registered. In addition to testing species directly exposed to
the CryBbl 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 Cry3Bbl 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 worst-
case soil concentration, soil degradation studies show that Cry3Bbl 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 Cry3Bbl protein expression in the plant.
Although Bt Cry proteins are very specific in their activity to only certain insect species, for Cry3Bbl
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 Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl 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
mrii)
Number
885.4050
Avian Dietary
Testing
The dietary median lethal concentration (LC50) value for Cry3Bbl
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 Cry3Bbl 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 uodate to this summary. See section
11(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 Cry3Bbl protein is considered
likely; however, the Cry3Bbl toxicity data, as described in the
Human Health Assessment (see section 11(B)(2) of this Bioocsticidcs
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% Cry3Bbl 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 Cry3Bbl proteins produced in corn crops.
(Reviewed in U.S. EPA (2003g))
N/A
71

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Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)
September 2010
Guideline
Number
Study
Results
mrii)
Number
850.1010
Aquatic
Invertebrate
Acute
Toxicity Test
(Daphnia
magna)
The 48-hour LC50 value for Cry3Bbl 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 Cry3Bbl-containing corn pollen.
Classification: Acceptable
(Reviewed in U.S. EPA (2003c and 2003g))
*Note for 2010: There is an uodate to this summary. See section
11(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 Cry3Bbl 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(CV2VaVii)(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 Cry3Bbl protein (lOOx 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 Cry3Bbl protein to be >360 micrograms per milliliter
(Hg/mL) (20x the concentration found in pollen). Therefore, no
hazard from the Cry3Bbl protein to honey bees is expected.
Classification: Acceptable
(Reviewed in U.S. EPA (2002a, 2002e, 2003f, and 2003g))
449043-11
72

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Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)
September 2010
Guideline
Number
Study
Results
mrii)
Number
885.4340
Parasitic
Hymenoptera
Larva Testing
The LC50 for parasitic Hymenoptera was determined to be >8,000
ppm Cry3Bbl protein. Parasitic Hymenoptera are not expected to
feed directly on corn plant tissue. Therefore, minimal exposure and
no hazard to parasitic Hymenoptera from Cry3Bbl 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 uodate to this summary. See section
11(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 Cry3Bbl protein (20x field exposure). Based on these results, it
can be concluded that green lacewing will not be adversely affected
when exposed to Cry3Bbl 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 Cry3Bbl, should
be tested.
Classification: Acceptable
(Reviewed in U.S. EPA (2002a, 2002e, 2003d, and 2003g))
*Note for 2010: There is an uodate to this summary. See section
11(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
convergent)
This maximum hazard dose study showed that the LC50 for Cry3Bbl,
when fed to adult H. convergens is >8,000 micrograms (|ig) 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 Cry3Bbl expressed in pollen is >93 micrograms per
gram (|ig/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
Cry3Bbl field corn pollen.
Classification: Acceptable
(Reviewed in U.S. EPA (2002a, 2002e, and 2003g))
455382-04
73

-------
Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)
September 2010
Guideline
Number
Study
Results
mrii)
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 Cry3Bbl 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
convergent)
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 Cry3Bblcorn 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 Cry3Bbl protein for collembola was found to be
>872.5 |ig (50% corn leaf tissue in the diet). No adverse reproductive
effects were noted. It can be concluded from this test that
Cry3Bblprotein 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
Cry3Bbl protein in an artificial soil substrate was determined to be
>570 mg Cry3Bbl protein/kilogram (kg) dry soil, or greater than lOx
the maximum EEC of the protein. The data show that no adverse
effects to earthworms are expected from exposure to corn plants
producing Cry3Bbl 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
Cry3Bbl protein variant 11098 (Q349R)/kg dry soil).
Classification: Acceptable
(Reviewed in U.S. EPA (2003f and 2003g))
457571-01
74

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Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)
September 2010
Guideline
Number
Study
Results
mrii)
Number
885.4340
Monarch
Butterfly
Larval Pollen
Feeding Study
This study demonstrated that corn pollen expressing the Cry3Bbl
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 uodate to this summary. See section
11(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
Cry3Bbl 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 non-
target 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 uodate to this summary. See section
11(C)(2)(b) ("Terms and Conditions of the Corn Event MON 863
Registration (February 2003 - September 2010)") of this BRAD.
455382-06
75

-------
Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)
September 2010
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 non-
target 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 long-
term effects determination.
Classification: Supplemental
(Reviewed in U.S. EPA (2003d and 2003g))
*Note for 2010: There is an uodate to this summary. See section
11(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 one-
year, 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 uodate to this summary. See section
11(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
Cry3Bbl
Protein 11098
Finely ground corn leaf tissue in sandy loam field soil degradation
data at worst-case field concentrations show that the Cry3Bbl
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 CryBbl protein was below the detection level.
These results verily that the Cry3Bbl 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 uodate to this summary. See section
11(C)(2)(b) ("Terms and Conditions of the Corn Event MON 863
Registration (February 2003 - September 2010)") of this BRAD.
451568-04
76

-------
Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)
September 2010
Guideline
Number
Study
Results
mrii)
Number
885.5200
Aerobic Soil
Degradation
of Cry3Bbl
Produced by
CRW-
Protected
Corn Event
MON 863
The DT50 values for Cry3Bblin 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. Cry3Bbl levels in soil sample extracts
show that concentrations were near or below the ELIS A limit of
quantitation (LOQ) (0.16 |ig/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 uodate to this summary. See section
11(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 Cry3Bbl does not pose a
risk to endangered Coleoptera. The Agency performed an
independent assessment and has determined that Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl toxicity data, as described in the Human Health Assessment (see
section 11(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.
77

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Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)
September 2010
b. Avian Species
i. Northern Bobwhite Study (Master Record Identification Number
(MRID No.) 449043-15)
The dietary LC50 value for Cry3Bbl 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 Cry3Bb 1 corn.
These data, however, are not sufficient to make a hazard assessment from repeated exposure to higher
doses of Cry3Bbl 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 Cry3Bbl protein.
*Note for 2010: There is an update to this summary. See section 11(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 Cry3Bbl 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) Cry3Bbl protein/mL. Thus, total
water concentration of less than 3.9 ng Cry3Bbl 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 Cry3Bbl corn
lines (MON 853 and MON 859) for eight weeks. The results indicate that corn grain derived from the
two transgenic lines producing Cry3Bbl can be used as a feed ingredient in channel catfish diets at
78

-------
Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)
September 2010
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 Cry3Bbl 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 Cry3Bbl content was estimated to be 18.8 |ig/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 Cry3Bbl 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/), 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 Cry3Bbl protein is confined to corn tissue,
and the worst case aquatic EEC is calculated to be 3.9 ng Cry3Bbl protein/mL, there is no substantial
exposure to aquatic invertebrates; no hazard from the registered used of Cry3Bbl-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 11(C)(2)(b) ("Terms and Conditions of
the Corn Event MON 863 Registration (February 2003 - September 2010)") of this BRAD.
79

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Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)
September 2010
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 CryBbl 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, Cry3Bbl 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 /^-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.
80

-------
Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)
September 2010
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 Cry3Bbl) 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 Cry3Bbl protein survived to pupation or "capping"; whereas,
97.5%) (2.5%o 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 Cry3Bbl
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 Cry3Bbl protein.
According to the OCSPP Harmonized Testing Guidelines, non-target insects should be tested at 10-
lOOx the field dosage. This test was conducted at an acceptable level lOOx the concentration in pollen or
1,790 ppm Cry3Bbl protein. Since potential exposure of honey bees to Cry3Bbl 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 Cry3Bbl 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
100|ig/mL potassium arsenate, a test group fed 360 |ag/mL of Cry3Bbl protein, and a water only group.
The study concluded that 360 |ag/m L Cry3Bbl protein did not affect survival or behavior of adult honey
bees. The maximum hazard dose, an LC50 greater than 360 |ig/mL, is 20x the concentration found in
pollen. Therefore, no hazard to adult honey bees is expected from exposure to the Cry3Bbl 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.
81

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Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)
September 2010
A dietary toxicity study with the parasitic Hymenoptera (N. vitripennis) was conducted with Bt Cry
3B2.11231 (Cry3Bbl) protein (purity 96%; 34.5 mg active protein/mL water). Wasps were tested at
rates of 400 and 8,000 ppm Cry3Bbl protein, which is approximately equivalent to lx and 20x the
maximum protein concentration in plant tissue. The LC50 for parasitic Hymenoptera was determined to
be >8,000 ppm Cry3Bbl 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 Cry3Bbl is >8,000 ppm.
The hazard assessment is based on 4,000 ppm Cry3Bbl protein, which is lOx the field concentration in
plants. However, because parasitic Hymenoptera do not feed directly on corn plant tissues, minimal
exposure of parasitic Hymenoptera to Cry3Bbl protein is expected. As a result, no hazard to N.
vitripennis is expected from exposure to MON 863 Cry3Bbl 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 11(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 Cry3Bbl protein in a moth egg (Sitotroga spp.) and water meal diet
at rates of 400 and 8,000 ppm, which is approximately equivalent to lx 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
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mortality when fed lx (400 ppm) and 20x (8,000 ppm) the maximum Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl 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 earned) 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 Cry3Bbl 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 0. insidiosus both
pollen and purified protein in diet, is required. Feeding O. insidiosus Cry3Bbl protein in diet will allow
for a test at the maximum hazard dose; whereas, feeding O. insidiosus pollen expressing the Cry3Bbl
protein will provide an evaluation of potential effects from actual exposure scenarios.
*Note for 2010: There is an update to this summary. See section 11(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 Cry3Bbl 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 Cry3Bbl. 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 |ig Cry3Bbl/mL of diet. Results from this study
showed that the LC50 for Cry3Bbl, when incorporated in diet and fed to H. converge/is^ is >8,000 |ig
Cry3Bbl protein/mL diet. Mortality for the 1,000 and 10,000 |ig 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 Cry3Bbl in their
gut, thus resulting in exposure to the Bt protein. There is approximately 390 |ig Cry3Bbl/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 |ig Cry3Bbl/mL diet, which is 20 times higher than
maximum expected exposure levels, no hazard from Cry3Bbl 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 Cry3Bbl 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 |ig Cry3Bbl/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 Cry3Bbl
expressed in corn pollen is >93 |ig/g fresh pollen weight. This study demonstrates that lady beetle larvae
will not be adversely affected by Cry3Bbl 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 Cry3Bbl protein at a concentration of 37.4
|ig/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
Cry3Bbl 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 Cry3Bbl protein at a concentration of 37.4 |ig/g pollen.
After 15 days of diet exposure, 84% and 81% of adult//, 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 Cry3Bbl 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
Cry3Bbl 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 |ig Cry protein/g diet,
respectively. The corn leaf tissue contained 1,745 |ig Cry3Bbl protein/g dried leaf tissue.
These results show a LC50 >872.5 |ig/g diet of Cry3Bbl protein. The study also noted that a diet
containing 50% corn leaf tissue expressing the Cry3Bbl 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 Cry3Bbl 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 jug/g, which is
significantly lower than the levels used in this test.
This study adequately addresses potential concerns for Cry3Bbl protein expressed in transgenic corn to
collembola (F. Candida), a representative of beneficial soil insect species. The results of this study
demonstrate that Cry3Bbl 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 plant-
incorporated protectants derived from Bt.
The 14-day LC 50 for earthworms exposed to Cry3Bbl protein 11231 in an artificial soil
substrate was determined to be greater than 570 mg Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl (Escherichia co/z'-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 Cry3Bbl
corn plants.
viii. Monarch Butterfly Larval Pollen Feeding Study (MRID No. 455382-05)
This study was not required nor requested for Cry3Bbl 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
Cry3Bbl corn on monarchs, Monsanto sponsored this study and submitted it to the Agency for review.
This study has demonstrated that corn pollen expressing the Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl in the field. Since
Cry3Bbl 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 11(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 CryBbl 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 Cry3Bbl 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 Cry3Bbl 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 non-
target organisms most likely to be exposed to the Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl 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. Foliage-
dwelling 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 non-
target 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 x 60 ft) were divided into 24
row subplots (60 ft x 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 VI0 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, Rl, 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, VI0, and Rl
growth stages during the 2000 and 2001 growing seasons. Three eight-inch root balls were collected
during the V6, VI0, and Rl 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 hand-
sorting method also showed no differences in the number of earthworms collected in MON 863 and non-
transgenic 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) andM 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, Chaetochnemapulicaria, and Rhopalosiphum maidis, as well as predators (e.g., 0.
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), andM 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 x 60 ft plots
divided into 24 row 60 ft x 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 semi-
field 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 CryBbl 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 11(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 Cry3Bbl 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.
MRU) 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
Cry3Bbl 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 Cry3Bbl 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
Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl 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, Cry3Bbl 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 Cry3Bbl
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 Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl insecticidal protein. The concentration of Cry3Bbl protein in the lyophilized
corn tissue, determined by ELISA, was 487 |ig/g in the root and 468 |ig/g in the shoot. A purified
Cry3Bb 1 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 Cry3Bbl protein in roots 35 days after planting is 66 jug/g. On this basis, the
maximum field loading of Cry3Bbl in corn shoots is 3.93 |ig/g soil (equivalent to 8 mg shoots/g soil);
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for corn root tissue, the loading is 2.79 |ig/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 Cry3Bbl 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 |ig of purified protein
was added to 0.5 g of soil. Soils from the three test locations were air dried and dosed with the Cry3Bbl
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).
Parameter
Soil Source
Carlyle, IL
Silt Loam
Monmouth, IL
Silt Loam
Richland, IA
Silt Loam
USD A
Textural Class

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:
Cry3Bbl protein levels in soil sample extracts determined by ELISA show that Cry3Bbl protein
concentrations were near or below the ELISA detection limit (LOQ) of 0.16 |ig/g after 2 months of
incubation. After two months of soil incubation, Cry3Bbl concentrations were at least an order of
magnitude below the initial concentration of Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl protein to move from tissue to soil. In
addition, the rapid degradation (DT90 of 4.0 to 5.2 days) of the purified Cry3Bbl protein suggests that
any Cry3Bbl 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 Dr
"90 Estimates for the Dissipation of Cry3Bbl Protein in Soils.
Protein Source
Soil Source
DTsoa (Days)
DT90b(Days)
Purified
Protein
Carlyle, IL
0.63 (0.49, 0.78)°
4.03 (3.67,4.42)
Monmouth, IL
0.64 (0.50, 0.80)
4.18 (3.77,4.62)
Richland, IA
0.73 (0.50, 1.00)
5.23 (4.48, 6.07
MON 863
Corn Root
Tissue
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)
Richland, IA
2.27(1.68,2.98)
50.02 (34.57, 72.18)
MON 863
Corn Shoot
Tissue
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)
Richland, IA
1.77(1.40,2.21)
28.59 (23.16,35.23)
aDT50= Time to 50% dissipation of original protein concentration
bDT90= Time to 90% dissipation of original protein concentration
0 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 Cry3Bbl 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 Cry3Bbl protein is detected. In
addition, the persistence of the Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl 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 11(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 eial. 2008). In addition, Bi 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 Bt-
related risks have evolved from the decomposition of Bl-com leaves for the meso- and macrofauna soil
community (Honemann 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 Cry3Bbl corn are derived from soil-inhabiting bacteria, EPA has concluded
that there is a low probability of risk from HGT of transgenes found in Cry3Bbl-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 Cry3Bbl 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 (USD A) 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 pollen-
directed 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; litis 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 maizz-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
USD A; 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
etal. 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) litis, Zea mays ssp. parviglumis litis and
Doebley, Zea mays ssp. huehuetenangensis (litis and Doebley) Doebley, Zea luxurians (Durieu and
Ascherson) Bird, Zeaperennis (Hitchc.) Reeves and Mangelsdorf, and Zea diploperennis litis, 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; litis, 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 etal. 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 Fi 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. parviglamis, 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 Cry3Bbl, 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 Cry3Bbl 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 Cry3Bbl. Therefore, no
adverse effects from Cry3Bbl (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 Cry3Bbl protein.
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Terrestrial and aquatic exposure were considered in this assessment since non-target coleopterans may
be exposed to the Cry3Bbl 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 Cry3Bbl protein has been determined to
be 93 |ig/g fresh weight pollen. Based on this concentration, <0.03 |ig Cry3Bbl 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 Cry3Bbl 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
Cry3Bbl 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 |ig Cry3Bbl/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 Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl corn will have no effects on
endangered and/or threatened species listed by the U.S. Fish and Wildlife Service, including mammals,
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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 Cry3Bbl corn, including the supplementary two-
year field data, the Agency concludes that the levels of Cry3Bbl 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 Cry3Bbl protein in agricultural soil. In addition, no effects on
listed endangered and threatened species are expected from the proposed Cry3Bbl CRW-resistant corn
registration.
At present, the Agency is aware of no identified significant adverse effects of Cry3Bbl 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 Cry3Bbl corn than non-Bt corn, primarily
because the Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl corn may result in fewer adverse impacts to non-target
organisms than result from the use of chemical pesticides. Under normal circumstances, Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl protein by wild or weedy relatives of corn in the U.S., its possessions, and/or territories. The
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fate of Cry3Bbl 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 Cry3Bbl protein.
v. Supplemental Studies Needed for Long-Term Cry3Bbl Non-Target Hazard
Assessment
The Agency has sufficient information to believe that there is no risk from the proposed uses of
Cry3Bbl 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 Cry3Bbl 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
)ata 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 Cry3Bbl 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 Cry3Bbl
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 11(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 11(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 Cry3Bbl
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 Cry3Bbl is accumulating or persisting in soil samples.. .a final report is
due two years from the date of registration."
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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 Cry3Bbl 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 Cry3Bbl
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 Cry3Bbl 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 anti-
lepidopteran Cryl A 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 Cry34Abl and
Cry35Abl 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
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literature review of these recently published studies. Criticisms of the Rosi-Marshall etal. study
included several findings: (1) adverse effects were not caused by toxicity of Cryl A but, rather, by other
differences between plant test substances (Jensen etal. 2010); (2) the abundance of Trichoptera in
streams containing residues of Cryl A was not reduced (Chambers et al. 2007); and (3) while post-
harvest 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
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Study Title
Summary
MRID No.
Assessment of the
Environmental Fate of
the Cry3Bbl 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 Cry3Bbl protein has not yet
been performed.
Classification: Acceptable
(Reviewed in U.S. EPA (2006a))
462001-01
Evaluation of Dietary
Effects of Cry3Bbl
Protein on the Ground
Beetle, Poecilus
chalcites (Coleoptera:
Carabidae)
First instar larvae of the ground beetle (Poecilus chalcites) were fed
artificial diet containing Cry3Bbl protein (930 |ig/g). a 10 millimolar
(mM) sodium carbonate/bicarbonate buffer control (0.147 milliliters per
gram (mL/g)), a potassium arsenate reference material (200 |ig/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 Cry3Bbl, buffer control, or negative control groups.
Classification: Acceptable
(Reviewed in U.S. EPA (2010c))
464799-04
Evaluation of Dietary
Effects of a Cry3Bbl
Protein Variant on
Minute Pirate Bugs
(iOrius insidiosus)
Orius insidiosus nymphs were exposed for 14 days to a Cry3Bbl protein
variant (930 |ig active ingredient/g) diet, a potassium arsenate (8.9 |ig
active ingredient/g) positive control diet, anE64 protease inhibitor (53
|ig/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 Cry3Bbl, 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 Cry3Bbl, 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 Cry3Bbl 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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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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Study Title
Summary
MRID No.
Environmental Fate of
Cry3Bbl 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 Cry3Bbl protein using ELISA (LOQ 0.1 |ig/g soil) and a
Colorado potato beetle (CPB) mortality bioassay (limit of detection (LOD)
20 |ig Cry3Bbl/g soil). ELISA did not detect Cry3Bbl 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 Cry3Bbl.
These results suggest that Cry3Bbl 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 Cry3Bbl 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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September 2010
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 multi-
year 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 el al. (2005a) compared ground-dwelling invertebrate abundance
between Bt (MON863, Cry3Bbl) and non-5/ 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,
Cry3Bbl) corn and non-5/ corn. Arthropods were collected over a three-
year 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 (Cry3Bbl) and non-5/ corn.
Using several measurements of diversity, the authors found no significant
differences in the abundance of individual species between Bt and non-5/
plots.
(Reviewed in U.S. EPA (2010d))
467129-01
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3. MON 863 x MON 810 (OECD Unique Identifier: MON-00863-5 x MON- 00810-6)
Expressing Cry3Bbl and CrylAb
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 CrylAb and Cry3Bbl 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 (Cry3Bbl - see section 11(C)(2) of this BRAD;
CrylAb - 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 (MRU) No. 460697-01)
Studies were conducted and submitted to test the hypothesis that the CrylAb and Cry3Bbl 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 CrylAb and Cry3Bbl 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 CrylAb or Cry3Bbl 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 11(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 11(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
CrylAb and Cry3Bbl 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 CrylAb and Cry3Bbl 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
xMON 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 two-
year 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-5/ 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 Cry3Bbl and
CrylAb 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 Cry3Bbl or CrylAb 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
Cry3Bbl and CrylAb proteins in soils. There is negligible potential for
plant-produced Cry3Bbl and CrylAb 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-88017-3) Expressing Cry3Bbl and MON 88017
x MON 810 (OECD Unique Identifier: MON-88017-3 x MON- 00810-6) Expressing
Cry3Bbl and CrylAb
a. Data Cited/Submitted for Initial Registrations of MON 88017 and MON 88017 x MON 810
(Prior to December 2005HU.S. EPA 2005a and 2005b)
i.	Background
The Cry3Bbl protein expressed in MON 88017 is a variant of the wild-type Cry3Bbl 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 11(A)(4) of this BRAD) demonstrated the functional and physicochemical
equivalence of the Cry3Bbl protein expressed in MON 88017 with the Cry3Bbl protein expressed in
Corn Event MON 863. That is, the Cry3Bbl 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 Cry3Bbl Protein) Risk Assessment
For registration of MON 863 (also applicable to MON 88017), EPA reviewed studies conducted on
representative non-target species with several Cry3Bbl 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 Cry3Bbl exposure on non-target invertebrates, soil degradation/persistence,
and endangered species (see section 11(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 Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl 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 11(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 CrylAb Protein) Risk Assessment
Likewise, the EPA has conducted an extensive review of effects of the CrylAb protein present in MON
810 to non-target organisms in an ecological risk assessment as part of the reassessment of Bt plant-
incorporated-protectants (U.S. EPA 2001b). EPA reviewed studies conducted on representative non-
target species with CrylAb 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 (Cry3Bbl x CrylAb 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 CrylAb protein in five inplanta 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 Cry3Bbl 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 Cry3Bbl in YieldGard® Plus Corn did not affect FAW nor did the presence
of CrylAb affect Colorado potato beetle (CPB) in leaf disk assays. Insect bioassays conducted with
purified protein verified that Cry3Bbl will not affect ECB survival, and CrylAb 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 (Cry3Bbl x CrylAb), and dose response curves did not differ.
Collectively, these data provide evidence that the CrylAb and Cry3Bbl 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 CrylAb and
Cry3Bbl 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 Cry3Bbl and CrylAb proteins contained in a single plant will
impart any safety concerns for non-target organisms exposed to these hybrids in the environment.
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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 CrylAb and Cry3Bbl 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
Cry3Bbl or CrylAb 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 Cry3Bbl in corn (Corn Event MON 863); these supplementary studies should provide
weight to the Agency's initial conclusions regarding the use of Cry3Bbl 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. CrylAb 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 11(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."
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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 CrylAb 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 11(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 Cry3Bbl 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 CrylAb
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
CrylAb 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 |-ig/g fresh weight, and limit for quantification was 0.40 )ag/g fresh
weight. ELISA revealed mean CrylAb protein levels in MON 88017 x
MON 810 corn tissues across all sites were 75 )ag/g dry tissue weight (dwt)
in young root and 12 )ag/g dwt in forage root; similar to the mean CrylAb
protein levels in MON 810 corn, which were 78 )ag/g dwt in young root
and 13 )ag/g dwt in forage root.
Classification: Acceptable
470045-01
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Bacillus thuringiensis Protein 11098 to Collembola (Folsomia Candida). Lab Project Number:
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thuringiensis (Bt) Corn Pollen Expressing a Variant of Cry3Bbl Protein on Larvae of the
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MRID No. 455382-05. Sears M, Mattila H. 2001. Determination of the Toxicity of Corn Pollen
Expressing a Cry3Bbl Variant Protein to First Instar Monarch Butterfly Larvae (Danaus
plexippus) via Laboratory Bioassay. Lab Project Number: MSL-17235: 01-01-39-26.
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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
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for Cry3Bb Protein in vitro. Lab Project Number: C3NTO. Unpublished study prepared by
Monsanto Company, 12 pages.
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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 Cry3Bbl 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 Cry3Bbl 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
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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: MSL-
17243: MSL-18126: MN-00-1. Unpublished study prepared by Colorado Quality Research,
Incorporated, 172 pages.
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Interactions Between the CrylAb and Cry3Bbl Proteins Produced in YieldGard Plus Corn.
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pages.
MR TP No. 462001-01. Dubelman S, Bhatti M, Ayden B. 2004. Interim Report: Assessment of the
Environmental Fate of the Cry3Bbl 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 onNontarget Organisms: Publications and Manuscripts. Project Number: 03/CR/097/13.
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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.
127

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MRID No. 464799-04. Duan J, Paradise M. 2005. Evaluation of Dietary Effects of Cry3Bbl 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 Cry3Bbl 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.
MR TP No. 465103-01. Dubelman S, Bhatti M, Ayden B. 2005. Environmental Fate of the Cry3Bbl
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Unpublished study prepared by Monsanto Company, Agvise, Incorporated, and North Dakota
State University, 230 pages.
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Corn MON 88017 x MON 810 Root Tissue Produced in U.S. Field Trials in 2006. Project
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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
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Agricultural Company, 432 pages.
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357.
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U.S. EPA. 1996. Hazard Evaluation Division, Standard Evaluation Procedure - Ecological Risk
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U.S. EPA. 1998. "Guidelines for Ecological Risk Assessment." EPA 630/R-95-002F. Washington,
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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.sov/scwolv/sap/meetinss/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 Cry3Bbl 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:
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U.S. EPA. 2001b. Biopesticides Registration Action Document - Bacillus thuringiensis Plant-
Incorporated Protectants. Available from:
http://www. epa. sov/oppbppdl/biopesticides/pips/bt brad, htm.
U.S. EPA. 2002a. Review of Ecological Non-Target Insect Studies for Bacillus thuringiensis Cry3Bbl
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 Cry3Bbl 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 corner gens) in Support of Monsanto's Request to
Register Bacillus thuringiensis (Bt) Cry3Bbl 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/scipolv/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 Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl 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 CrylAb and Cry3Bbl 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.sov/scipolv/sap/meetinss/2004/iune/finalla.pdf.
U.S. EPA. 2005a. Review of Product Characterization, Expression Analysis, and Human Health Data
for Plant-Incorporated Protectant Bacillus thuringiensis subspecies kumamotoensis Cry3Bbl
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 Cry3Bbl (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 Cry3Bbl (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 CrylA(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 Carahid {MK\D No. 464799-04) Insect
Studies Submitted as a Condition of Registration for Monsanto Maize Event MON863 Cry3Bbl
(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
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EPA-HQ-OPP-2010-0607).
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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.
htty://dx. doi. org/10.13 71/journal, pone. 0002118.
Wolt JD, Peterson RK. 2010. Prospective formulation of environmental risk assessments: Probabilistic
screening for Cry 1 A(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 Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl resistance allele
frequency in CRW. Although this report satisfied the condition of registration, there is still significant
uncertainty regarding the mode of action of Cry3Bbl 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 Cry3Bbl (Siegfried el 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 rootworm-
protected 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 Cry3Bbl (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 three-
year 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 Cry3Bbl 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 Cry3Bbl-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 etal. (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.
CRWHosts
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 etal. 2004; Wilson and Hibbard 2004) and,
therefore, may serve as additional refuge to the structured non-Cry3Bbl corn refuges. This additional
source of refuge could reduce the risk of Cry3Bbl 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 etal. (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, fence-
row/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 Cry 3Bbl -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 2n 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 Cry3Bbl-
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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 Fi 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 ol. (2008) were able to select for
Cry3Bbl 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 Cry3Bbl. 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, Cry3Bbl 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 Cry3Bbl (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 el 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 non-
transgenic 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 Cry3Bbl. 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
Cryl Ac and Cryl Ab. 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 etal. 2003; Ferre 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 Cry3Bbl 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%o 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 (MRU) 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 Cry3Bbl endotoxin, as compared to isoline corn
without Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl 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 (|ig/cm2) vs. 13.00 |ig/cm2). In all four studies, predicted results, based on the Hardy-
Weinberg 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 Cry3Bbl 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 (Cry3Bbl) 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
Cry3Bbl 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 etal. 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 etal. (2003) that showed between 0.15% 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 lepidopteran-
protected 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 Cry3Bbl 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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Cry3Bbl Monitoring Strategy
As a condition of registration, Monsanto was required to develop a resistance monitoring program for
Cry3Bbl 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 Cry3Bbl registration).
• Rootworm damage guidelines for unexpected pest damage (required for MON 88017, a
subsequent Cry3Bbl registration).
Monsanto submitted a resistance monitoring strategy for MON 863 and Cry3Bbl 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
Cry3Bbl) in 2005. The Cry3Bbl 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 Cry3Bbl 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 etal. (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 Cry3Bbl should be
established using baseline susceptibility and annual monitoring data, as well as other historical
information for WCRW and Cry3Bbl. 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 Cry3Bbl-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 Cry3Bbl-expressing plants have >1 root nodes destroyed by suspected resistant
populations under controlled lab conditions.
Unexpected Pest Damage
Since Cry3Bbl, 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 plant-
incorporated protectant (PIP) that expresses Cry3Bbl) 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 V2 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 /^-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 Cry3Bbl
As required by the terms and conditions of the MON 863 registration, baseline susceptibility levels of
western corn rootworm populations to Cry3Bbl have been measured (Siegfried etal. 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 Cry3Bbl
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 Cry3Bbl toxin (expressed in MON 863).
Cry3Bbl 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 Cry3Bbl of 170.8 |ig/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 Cry3Bbl.
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Table 2. Percent Mortality and Mass of Surviving Larvae at the Putative Diagnostic
Concentration of 170 ng/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 Cry3Bbl 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 el 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).
Cry3Bbl Resistance Monitoring Data
Resistance monitoring data for Cry3Bbl 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
Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl protein for bioassay. Neonate larvae were used for
diet overlay bioassays and exposed to different Cry3Bbl concentrations (10.7 |ig/cm2, 21.4 |ig/cm2, 42.7
|ig/cm2, 85.4 |ig/cm2, and 170.8 |ig/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 |ig/cm2, representing a 4-fold
difference in susceptibility, and were comparable to the EC50 range obtained in 2007 (14.2-33.4
|ig/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 Cry3Bbl 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 |ig/cm2 and (2) LC90 range: 218.2-43,931.0 |ig/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 Cry3Bbl for Three Counties in the Corn Belt as Measured by
EC50 and LC50 Data 1
2005-2008).
Population
Mean EC50
Mean LC50
Uig/cnr)
(HS/cnr)
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 Cry3Bbl protein.
Table 4. CRW Susceptibility to Cry3Bbl - Data Ranges for All Populations Sampled in the Corn
Belt (2005-2C
108).
Year
ECso ((.ig/cnr)
LCso (|-ig/cm2)
LC9o (i-ig/cnr)
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 Cry3Bbl 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 Cry3Bbl 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 long-
term 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 (Cry3Bbl 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 Cry3Bbl (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 Cry3Bbl, 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 Cry3Bbl-containing
diet. If heritability is confirmed, survivors will be placed on Cry3Bbl 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 Cry3Bbl-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
Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl
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 Cry3Bbl 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
(Cry3Bbl, mCry3A, and Cry34/35Abl). 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
Adhcrcnec to Refuse3
Size
93
89
80
74
Adherence to Distance
Requirements3
87
82
79
63
Awareness of I RIM
Requirements
97
93
97
96
Unaided Recall of Refuse
Size
51
57
63
72
Unaided Recall of Refuse
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
Adhcrenee to Refuse Size1
78
70
724
Adherence to Distance Requirements1
92
66
663
Awareness of IRM Requirements
95
96
97
Unaided Recall of Refuse Size
59
62 and 552
81
Unaided Recall of Refuse 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 rootworm-
protected 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 Cry3Bbl 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 rootworm-
protected PIPs, including varieties expressing the Cry3Bbl, mCry3A, and Cry34/35Abl 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

2006
2007
2008
Growers Assessed
395
247
134
Refuge Distanee Deviations2
13
N/A
N/A
Refuge Size Deviations
7
N/A
N/A
Signifieant Deviations
11 (2.8%)
16 (6.5%)
12 (9.0)
Insignifieant 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%)
'Tabic 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

2006
2007
2008
Growers Assessed
600
1069
1799
Refuge Distanee Deviations
512
N/A
N/A
Refuge Size Deviations
8
N/A
N/A
Signifieant Deviations
45 (7.5%)
77 (7.2%)
863
Insignifieant 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 V2 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
• Invoice monitoring
• Warning letter
• Technical assistance

• Compliance assistance contact
• Grower IRM training

prior to planting
• Reaffirmation of IRM

• Compliance assessment
obligations

contact for the following
• Deny access to the Bt corn

growing season
product for other deviations that

• Deny access to the Bt corn
are repeated over a period of

product for any significant
years

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

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 "On-
Farm 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
Loss of Access to

Deviations3
Technology
2006
62
0
l4
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 Cry3Bbl (MON 863) and Cryl Ab (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 CrylAb and Cry3Bbl
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 Cry3Bbl protein. Corn that only contains the CrylAb 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 V2 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
Cry3Bbl/CrylAb PIPs are summarized in Table 12.
Resistance Monitoring
Monsanto is required to monitor for pest resistance to the CrylAb and Cry3Bbl proteins in YieldGard®
Plus Corn. Monsanto has folded YieldGard® Plus Corn into the existing resistance monitoring plans for
lepidoptera (CrylAb) and CRW (Cry3Bbl). A complete discussion of the resistance monitoring
program for CrylAb and lepidopteran pests is included in the U.S. EPA (2010b). Resistance monitoring
for Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl from each hybrid
to determine LC50 values for the test insects. The results were similar for both Cry3Bbl variants: (1) for
CPB, the MON 88017 variant (Cry3Bbl.pvzmir39) had an LC50 of 0.84 microgram per milliliter
(|ig/mL), while the MON 863 variant (Cry3Bb 1.11098) had anLCso of0.95 |ig/mL; (2) for WCRW, the
LC50 was 139 |ig/mL for the MON 88017 variant and 100 |ag/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 Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl 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 (Cry3Bbl) IRM plan is found in section 11(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 Cry3Bbl and CrylAb 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 Cry3Bbl toxins produced in both MON 88017 and MON 863. The Cry3Bbl
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 CrylAb 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 Cry3Bbl and CrylAb 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 Cry3Bbl expression, which
was found to be almost identical. Using ELISA techniques, young leaf, young root (Cry3Bbl only),
pollen (Cry3Bbl only), forage (leaf), forage root (Cry3Bbl only), and grain tissues were analyzed for
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the amount of Cry3Bbl and CrylAb protein both in dry weight and fresh weight tissues. The results
showed that the Cry3Bbl 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 CrylAb, 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
<'/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.
Same corn rotation as YGRW
(e.g., first-year corn or corn
followed by corn).
Same corn rotation as YG
Plus (e.g., first-year corn or
corn followed by corn).

Insecticides can be used in
refuge to control ECB &
southwestern corn borer
when above economic
thresholds.
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.
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.
(Not applicable)
Microbial Bt insecticides are
not allowed.
Refuge Corn
Types
Conventional (Non-5/)
Conventional (Non-5/)
YGCB (a corn borer refuge
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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 Cry3Bbl 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 Cry3Bbl 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 etal. 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 Cry3Bbl 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.
8. References
ABSTC. 2003. Updated Monitoring Plan for Bt Corn. Unpublished report submitted to the Agency by
the Agricultural Biotechnology Stewardship Technical Committee on January 31, 2003.
Andow DA. 2002. University of Minnesota. Personal communication with Robyn Rose (U.S. EPA) on
December 23, 2002.
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Andow DA, Alstad DN. 2002. Preliminary draft: efficacy of between field refuges for resistance
management of transgenic corn rootworm insecticidal corn: model results.
MRIDNo. 455770-01.
Boetel MA, Fuller BW. 1997. Seasonal emergence-time effects on adult longevity, fecundity, and egg
viability of northern and western corn rootworms (Coleoptera: Chrysomelidae). Environ.
Entomol. 26:1208-1212.
Bourguet D, Chaufaux J, Seguin M, Buisson C, Hinton JL, Stodola TJ, Porter P, Cronholm G,
Buschman LL, Andow DA. 2003. Frequency of alleles conferring resistance to Bt maize in
French and U.S. corn belt populations of the European corn borer, Ostrinia nubilalis. Theor.
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Branson TF, Krysan JL. 1981. Feeding and oviposition behavior and life cycle of Diabrotica: an
evolutionary view with implications for pest management. Environ. Entomol. 10(6):826-831.
Caprio M. 1998. Evaluating resistance management strategies for multiple toxins in the presence of
external refuges. J. Econ. Entomol. 91:1021-1031.
Caprio M. 2002. Mississippi State University. Personal communication with Robyn Rose (U.S. EPA) on
December 20, 2002.
Clark TL, Hibbard BE. 2004. Comparison of nonmaize hosts to support western corn rootworm
(Coleoptera: Chrysomelidae) larval biology. Environ. Entomol. 33:681-689.
Coates SA, Tollefson JJ, Mutchmor JA. 1986. Study of migratory flight in the western corn rootworm
(Coleoptera: Chrysomelidae). Environ. Entomol. 15:1-6.
Crowder DW, Onstad DW, Gray ME, Mitchell PD, Spencer JL, Brazee RJ. 2005. Economic analysis of
dynamic management strategies utilizing transgenic corn for control of western corn rootworm
(Coleoptera: Chrysomelidae). J. Econ. Entomol. 98:961-75.
Ferre J, Van Rie J. 2002. Biochemistry and genetics of insect resistance to Bacillus thuringiensis.
Annu. Rev. Entomol. 47:501-533.
Gray ME. 1999. Transgenic insecticidal cultivars for corn rootworms: resistance management
considerations. In: Proceedings of the Crop Protection Technology Conference. University of
Illinois, Urbana-Champaign. pp 50-55.
Groeters FR, Tabashnik BE. 2000. Roles of selection intensity, major genes, and minor genes in
evolution of insecticide resistance. J. Econ. Entomol. 93(6): 1580-1587.
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Hibbard BE, Durran PN, Ellersieck MR, Ellsbury MM. 2003. Post-establishment movement of western
corn rootworm larvae (Coleoptera: Chrysomelidae) in central Missouri corn. J. Econ. Entomol.
96(3):599-608.
Hibbard BE, Higdon ML, Duran DP, Schweikert YM, Ellersieck MR. 2004. Role of egg density on
establishment and plant-to-plant movement by western corn rootworm larvae (Coleoptera:
Chrysomelidae). J. Econ. Entomol. 97:871-882.
Hibbard BE, Vaughn TT, Oyediran IO, Clark TL, Ellersieck MR. 2005. Effect of Cry3Bbl expressing
transgenic corn on plant-to-plant movement by western corn rootworm larvae (Coleoptera:
Chrysomelidae). J. Econ. Entomol. 98:1126-1138.
Hill RE, Mayo ZB. 1980. Distribution and abundance of corn rootworm species as influenced by
topography and crop rotation in eastern Nebraska. Environ. Entomol. 9:122-127.
Kim KS, Sappington TW. 2005. Genetic structuring of western corn rootworm
(Coleoptera: Chrysomelidae) populations in the United States based on microsatellite loci
analysis. Environ. Entomol. 34:494-503.
Levine E, Oloumi-Sadeghi H. 1991. Management of Diabroticite rootworms in corn. Annu. Rev.
Entomol. 36:229-255.
Meinke L etal. 2001. Letter from the NCR-46 to Sharlene Matten, Ph.D. dated May 29, 2001.
Meihls L, Hidgon M, Siegfried B, Miller N, Sappington T, Ellersieck M, Spencer T, Hibbard B. 2008.
Increased survival of western corn rootworm on transgenic corn within three generations of
on-plant greenhouse selection. Proc. Nat. Acad. Sci. 105(49): 19177-19182.
Mitchell PD, Onstad DW. 2005. Effect of extended diapause on evolution of resistance to transgenic
Bacillus thuringiensis corn by northern corn rootworm (Coleoptera: Chrysomelidae). J. Econ.
Entomol. 98:2220-2234.
MRID No. 451568-05. Davis P, Head G, McFerson J. 2000. Insect Resistance Management for a
Transgenic Corn Rootworm Control Product. Unpublished study prepared by Monsanto
Company, 236 pages.
MRID No. 453484-01. Vaughn T. 2001. Preliminary Results of Research on Insect Resistance
Management for a Transgenic Corn Rootworm Control Product. Lab Project Number: 99-894E.
Unpublished study prepared by Monsanto Company, 116 pages.
177
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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. 459438-01. Vaughn T. 2003. Estimating Cry3Bbl Resistance Allele Frequencies in Corn
Rootworm Larvae Feeding on Corn Event MON 863. Lab Project Number: 03-CR-097E-4.
Unpublished study prepared by Monsanto Company, 58 pages.
MRID No. 461817-01. Sidhu R. 2004. Human Health and Environmental Assessment of the
Plant-Incorporated Protectant Bacillus thuringiensis Cry3Bbl Protein Produced in MON 88017.
Project Number: MSL/18835. Unpublished study prepared by Monsanto Company, 64 pages.
MRID No. 461850-01. Sidhu R. 2004. Human Health and Environmental Assessment of the
Plant-Incorporated Protectant Bacillus thuringiensis Cry3Bbl and CrylAb Proteins Produced in
MON 88017 x MON 810. Project Number: MSL/18955. Unpublished study prepared by
Monsanto Company, 24 pages.
MRID No. 461865-01. Vaughn T. 2004. Progress Report on Insect Resistance Management Research
for Corn Event MON 863. Project Number: 03/CR/097E/9. Unpublished study prepared by
Monsanto Company, Texas A&M University, Geochemical and Environmental Research, and
University of Missouri, 100 pages.
MRID No. 466066-01. Vaughn T. 2005. Second Progress Report on Insect Resistance Management
Research for Corn Event MON 863. Project Number: 03/CR/097/22. Unpublished study
prepared by Monsanto, 161 pages.
MRID No. 470444-01. Reding H. 2007. 2006 Insect Resistance Management Compliance Assurance
Program Report for Corn Borer-Protected BT Corn, Corn Rootworm-Protected BT Corn, and
Corn Borer/Corn Rootworm-Protected Stacked BT Corn. Project Number: CAP/2006.
Unpublished study prepared by Agricultural Biotechnology Stewardship Technical Committee,
14 pages.
MRID No. 473396-01. Bailey L. 2008. 2007 Insect Resistance Management Compliance Assurance
Program Report for Corn Borer-Protected Bt Corn, Corn Rootworm-Protected Bt Corn, and Corn
Borer/Corn Rootworm-Protected Stacked Bt Corn. Project Number: CAP/2007. Unpublished
study prepared by Agricultural Biotechnology Stewardship Technical Committee, 17 pages.
MRID No. 473547-01. Head G. 2008. Corn Rootworm Resistance Monitoring Plan for MON 88017 and
MON 88017 xMON 810. Project Number: 07/CR/181E/7. Unpublished study prepared by
Monsanto Company, 12 pages.
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MRID No. 476633-01. Bailey L. 2009. 2008 Insect Resistance Management Compliance Assurance
Program Report for Corn-Borer Protected Bt Corn, Corn Rootworm-Protected Bt Corn, and Corn
Borer/Corn Rootworm-Protected Stacked Bt Corn. Project Number: CAP/2008. Unpublished
study prepared by Agricultural Biotechnology Stewardship Technical Committee, 15 pages.
MRID No. 478846-01. Lang B. 2009. 2008 Season Monitoring for the Susceptibility of Neonate
Western Corn Rootworm Larvae to the Bacillus thuringiensis Cry3Bbl Protein. Project Number:
07/CR/181E/11. Unpublished study prepared by Custom Bio-Products, 12 pages.
Nowatzki TJ, Bradley N, Warren KK, Putnam S, Meinke LJ, Gosselin DC, Harvey FE, Hunt TE,
Siegfried B. 2003a. In-field labeling of western corn rootworm adults (Coleoptera:
Chrysomelidae) with rubidium. J. Econ. Entomol. 96(6): 1750-1759.
Nowatzki TJ, Meinke LJ, Siegfried B. 2003b. Poster presented at the North Central Branch of the
Entomological Society of America Meeting, Cincinnati, Ohio. March, 2003.
Nowatzki T, Lefko SA, Binning RR, Thompson SD, Spencer TA, Siegfried BD. 2008. Validation of a
novel resistance monitoring technique for corn rootworm (Coleoptera: Chrysomelidae) and event
DAS-59122-7 maize. J. Appl. Entomol. 132:177-188.
Onstad DW, Guse CA, Spencer JL, Levine E, Gray ME. 2001. Modeling the dynamics of adaptation to
transgenic corn by western corn rootworm (Coleoptera: Chrysomelidae). J. Econ. Entomol.
94(2): 529-540.
Onstad DW, Crowder DW, Mitchell PD, Guse CA, Spencer JL, Levine E, Gray ME. 2003. Economics
versus alleles: balancing integrated pest management and insect resistance management for
rotation-resistant western corn rootworm (Coleoptera: Chrysomelidae). J. Econ. Entomol.
96:1872-1885.
Onstad DW, Hibbard BE, Clark TL, Crowder DW, Carter KG. 2006. Analysis of density-dependent
survival of Diabrotica (Coleoptera: Chrysomelidae) in cornfields. J. Econ. Entomol.
35(5): 1272-1278.
Oyediran IO, Hibbard BE, Clark TL. 2004. Prairie grasses as alternate hosts of the western corn
rootworm (Coleoptera: Chrysomelidae). Environ. Entomol. 33:740-747.
Rondon SI, Gray ME. 2004. Ovarian development and ovipositional preference of the western corn
rootworm (Coleoptera: Chrysomelidae) variant in east central Illinois. J. Econ. Entomol.
97:390-396.
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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 Cry3Bbl 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.sov/scipolv/sap/meetinss/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.sov/scipolv/sap/meetinss/2002/ausust/ausust2002final,pdf.
U.S. EPA. 2003a. Insect Resistance Management Assessment for the Registration of Monsanto
Company's Cry3Bbl 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 CrylAb and Cry3Bbl 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 Cry3Bbl 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
Cryl Ab and CrylF Corn (Updated September 2010). Available from:
http: icii'ic.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 cry 3Bb 1 gene that produces
the insecticidal crystal protein, Cry3Bbl. MON 863 also has a nptllmarker 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. Cry3Bbl 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 LeContej, 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 reregi strati on.
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 Oloumi-
Sadeghi 1991; Levine etal. 1992a) and aNCRW extended diapause (2 year extended) variant, primarily
in parts of Minnesota, Iowa, and South Dakota (Krysan el al. 1986; Levine el 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 Species (Reprinted from
Pages 18-21 o
FMRID No. 456530-01 and
Verified by
EPA).
Product
Active
Ingredients
rp Si
Type
Use Rateb
Use
Classification0
Ambush® Insecticide -
Syngenta
permethrin -
25.6%
SP
0.2 lb/acre
Adult
control
WARNING. Restricted Use; extremely toxic to fish and
aquatic invertebrates, highly toxic to bees
Asanci-XL Insecticide
0.66 Emulsifiable
Concentrate - DuPont
esfenvalerate -
8.4%
SP
0.05 lb/acre
Adult
control
WARNING. Restricted Use; extremely toxic to fish and
aquatic invertebrates, highly toxic to bees
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 to fish and wildlife
Baythroid~2 Emulsifiable
PyrethroidInsecticide -
Bayer
cyfluthrin - 25%
SP
0.04 lb/acre
Adult
control
DANGER. Restricted Use; extremely toxic to fish and
aquatic invertebrates, highly toxic to bees, may cause allergic
skin reactions
Capture~2EC
Insecticide/Miticide -
FMC
bifenthrin -
25.1%
SP
0.3 lb/acre
Larval
control
WARNING. Restricted Use; extremely toxic to fish and
aquatic invertebrates, highly toxic to bees
Chlorfos~15G Insecticide
Granular - Griffin LLC
chlorpyrifos -
15%
OP
2.02 lb/acre
Larval
control
CAUTION. Toxic to birds and wildlife, extremely toxic to
fish and aquatic organisms
Chlorfos~4E Insecticide -
Griffin LLC
chlorpyrifos -
42%
OP
2.52 lb/acre
Adult &
Larval
control
WARNING. Toxic to birds and wildlife, extremely toxic to
fish and aquatic organisms
Countet~CR Systemic
Insecticide-Nematicide -
American Cyanamid
Company
terbufos - 20%
OP
1.30 lb/acre
Larval
control
DANGER. Restricted Use; fatal if swallowed, inhaled, or
absorbed through skin, extremely toxic to fish and wildlife
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Product
Active
Ingredients
rp Si
Type
Use Rateb
Use
Classification0
D-z-n-Dicizinon AG 500
Insecticide - Syngenta
diazinon - 48%
OP
0.48 lb/acre
Adult
control
CAUTION. Restricted Use; highly toxic to birds, fish, and
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 toxic to birds, fish, and
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 swallowed, inhaled, or
absorbed through skin, highly toxic to aquatic invertebrates and
wildlife, highly toxic to bees
Diazinon 500-AG
Organophosphate
Insecticide - UAP
diazinon - 48%
OP
0.48 lb/acre
Adult
control
CAUTION. Restricted Use; highly toxic to birds, fish, and
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 aquatic invertebrates,
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 aquatic invertebrates,
highly toxic to bees
5 lb Dimethoate Systemic
Insecticide - Helena
dimethoate -
57%
OP
0.46 lb/acre
Adult
control
DANGER. Toxic to wildlife and aquatic invertebrates, highly
toxic to bees
Force-3G Insecticide -
Syngenta
tefluthrin - 3%
SP
0.17 lb/acre
Larval
control
CAUTION. Restricted Use; very highly toxic to freshwater
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 wild mammals, birds,
fish, and aquatic invertebrates
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September 2010
Product
Active
Ingredients
rp Si
Type
Use Rateb
Use
Classification0
FortressSG Granular
Insecticide - DuPont
chlorethoxyfos -
5%
OP
0.16 lb/acre
Larval
control
DANGER. Restricted Use; toxic to wild mammals, birds,
fish, and aquatic invertebrates
Furadan-4F Insecticide/
Nematicide - FMC
carbofuran -
44%
C
0.88 lb/acre
Adult &
larval
control
DANGER. Restricted Use; poisonous if swallowed or
inhaled, toxic to fish, birds, and other wildlife, highly toxic to
bees, can seep or leach through soil and can contaminate
groundwater
Lannate-LVInsecticide -
DuPont
methomyl - 29%
C
0.65 lb/acre
Adult
control
DANGER. Restricted Use; fatal if swallowed, toxic to fish,
aquatic invertebrates, and mammals, highly toxic to bees,
known to leach through soil into groundwater
LannateSP Insecticide -
DuPont
methomyl - 90%
C
0.45 lb/acre
Adult
control
DANGER. Restricted Use; fatal if swallowed, may cause
blindness, toxic to fish, aquatic invertebrates, and mammals,
highly toxic to bees, known to leach through soil into
groundwater
Lorsban-15G Granular
Insecticide - Dow
AgroSciences
chlorpyrifos -
15%
OP
2.03 lb/acre
Larval
control
CAUTION. Toxic to birds and wildlife, extremely toxic to
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 wildlife, extremely toxic to
fish and aquatic organisms
Mocap K)% Granular
Nematicide Insecticide -
Aventis CropScience
ethoprop - 10%
OP
3.53 lb/acre
Larval
control
WARNING. Toxic to aquatic organisms and wildlife
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Product
Active
Ingredients
rp Si
Type
Use Rateb
Use
Classification0
Mocap -EC Nematicide-
Insecticide - Aventis
Crop Science
ethoprop -
69.6%
OP
3.34 lb/acre
Larval
control
DANGER. Restricted Use; toxic to aquatic organisms and
extremely toxic to birds
Penncap-
M-Microencapsulated
Insecticide - Elf
Atochem
methyl parathion
- 22%
OP
0.44 lb/acre
Adult
control
WARNING. Restricted Use; highly toxic to aquatic
invertebrates and wildlife
Phorate 20 G
Organophosphate
Insecticide - UAP
phorate - 20%
OP
1.3 lb/acre
Adult &
larval
control
DANGER. Restricted Use; extremely toxic to fish and
wildlife
Pounce® WSB Insecticide
- FMC Corporation
permethrin -
24.7%
SP
0.2 lb/acre
Adult
control
WARNING. Restricted Use; extremely toxic to fish and
aquatic invertebrates, highly toxic to bees
Pounce® 3.2 EC
Insecticide - FMC
Corporation
permethrin -
38.4%
SP
0.2 lb/acre
Adult
control
CAUTION. Restricted Use; extremely toxic to fish and
aquatic invertebrates, highly toxic to bees
Pounce® 25 WP
Insecticide - FMC
Corporation
permethrin -
25%
SP
0.2 lb/acre
Adult
control
WARNING. Restricted Use; extremely toxic to fish and
aquatic invertebrates, highly toxic to bees
Regent~4 SC Insecticide -
Aventis CropScience
fipronil - 39.4%
PP
0.13 lb/acre
Larval
control
WARNING. Restricted Use; toxic to birds, fish and aquatic
invertebrates
Sevin~Brand 80S
Carbaryl Insecticide -
Aventis CropScience
carbaryl - 80%
C
2.0 lb/acre
Adult
control
WARNING. Extremely toxic to aquatic and estuarine
invertebrates, highly toxic to bees
Sevin-BrandXLR PL US
Carbaryl Insecticide -
Aventis CropScience
carbaryl - 44.1%
C
1.76 lb/acre
Adult
control
CAUTION. Extremely toxic to aquatic and estuarine
invertebrates, highly toxic to bees
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September 2010
Product
Active
Ingredients
rp Si
Type
Use Rateb
Use
Classification0
Thimet~20-G Soil and
Systemic Insecticide -
American Cyanamid
phorate - 20%
OP
1.3 lb/acre
Larval
control
DANGER. Restricted Use; extremely toxic to fish and
wildlife
Thimet~20-G Soil and
Systemic Insecticide -
American Cyanamid
phorate - 20%
OP
1.3 lb/acre
Larval
control
DANGER. Restricted Use; extremely toxic to fish and
wildlife
Warrior-Insecticide with
Zeon Technology -
Syngenta
lambda-
cyhalothrin -
11.4%
SP
0.03 lb/acre
Adult
control
WARNING. Restricted Use; extremely toxic to fish and
aquatic organisms and toxic to wildlife, highly toxic to bees
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
MRU) No. 456530-01).
Parameter
Continuous
Corn
First-Year
Corn
All Corn
Acres planted (xl,000)
22,269
57,310
79,579
Total insecticide-treated acres (xl,000)
11,590
12,518
24,108
Total insecticide active ingredient applied
(lb xl,000)
6,332
6,011
12,343
CRW-targeted acres (xl,000)
8,271
5,926
14,197
Active ingredient applied to CRW-targeted acres
(lb xl,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 (xl,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 CRW-
targeted 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 seed-
applied 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 >
\o. 456530-01

Product3
Average Cost
($/A)
Adult (A)
Control
Larval (L)
Control
EPA
Classification
Acres Treated
(xl,000)
Aztec 2.1% Granular Insecticide
(tebupirimfos/cyflutMn)
$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)
$4.42
A
Restricted
224
195
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Biopesticides Registration Action Document (BRAD)
September 2010
Product3
Average Cost
($/A)
Adult (A)
Control
Larval (L)
Control
EPA
Classification
Acres Treated
(xl,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 11(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 11(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), Cry3Bbl 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 11(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
(MRU) 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 11(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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September 2010
Table 5. Comparison of Ecological Risks Associated with Terbufos, Chlorpyrifos, and
Tefluthrin.
Endpoint
Terbufos3
Chlorpyrifos3
Tefluthrin3
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 Cry3Bbl protein, have
been evaluated in a series of studies with representative avian, aquatic, and terrestrial beneficial
invertebrate species as discussed in section 11(C) of this BRAD.
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September 2010
Table 6. Summary of Results from Ecological Effects Tests with the Cry3Bbl Proteins (Reprinted from page 35 of
MRU) No. 456530-01).
Test Organism
Test
Substance
Results3
Conclusions'3
Reference
Cladoceran
(Daphnia magna)
Pollen
NOEC >2.26 (ig/L
NOEC >14lx surface water MEEC
MRID No. 449043-18
Collembola
(Folsomia Candida)
Leaf
NOEC >872.5 ng/g
NOEC >66x soil MEEC
MRID No. 449043-17
Channel Catfish
(Ictalurus punctatus)
Grain
No effect on growth or
survival at 35% of diet
No significant risk
MRID No. 449043-19
Bobwhite Quail
(Colinus virginianus)
Grain
No effect on growth or
survival at 10% of diet
No significant risk
MRID No. 449043-15
Adult Honey Bee
(Apis mellifera)
Purified protein
NOEC >360 ng/mL
NOEC >3.8x maximum pollen level
MRID No. 449043-11
Larval Honey Bee
(Apis mellifera)
Purified protein
NOEC >1,790 iig/mL as a
single dose
NOEC >19x maximum pollen level
MRID No. 449043-10
Adult Ladybird Beetle
(Hippodamia convergens)
Purified protein
NOEC >8,000 (ig/g
NOEC >86x maximum pollen level
MRID No. 449043-14
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Bacillus thuringiensis Cry3Bb1 Corn
Biopesticides Registration Action Document (BRAD)
September 2010
Test Organism
Test
Substance
Results3
Conclusions'3
Reference
Adult Ladybird Beetle
(Hippodamia convergent)
Pollen
No effect on growth or
behavior at 50% of diet
No significant risk
MRIDNo. 453613-02
Larval Ladybird Beetle
(Coleomegilla maculata)
Pollen
No effect on growth or
survival at 50% of diet
No significant risk
MRID No. 455382-04
Adult Ladybird Beetle
(Coleomegilla maculata)
Pollen
No effect on survival at
50% of diet
No significant risk
MRIDNo. 453613-01
Monarch Butterfly Larvae
(Danaus plexippus)
Pollen
No effect on growth or
survival
No significant risk
MRID No. 455382-05
Green Lacewing
Larvae (Chrysoperla earned)
Purified protein
NOEC >8000 (ig/g
NOEC >86x maximum pollen level
MRID No. 449043-12
Parasitic Hymenoptera
(Nasonia vitripennis)
Purified protein
NOEC = 400 ng/mL
NOEC >4.3x maximum pollen level
MRID No. 449043-13
Earthworm
(Eisenia fetifa)
Purified protein
NOEC = 57 mg/kg
NOEC >4.3x MEEC in soil
MRID No. 449043-16
aNOEC - No Observable Effect Concentration
bMEEC - Maximum Expected Environmental Concentration
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September 2010
Results of the environmental fate studies indicate that Cry3Bbl 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 Cry3Bbl protein based on its rapid degradation in the soil.
In summary, Cry3Bbl 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 Cry3Bbl protein is expressed by the corn plant, thus reducing the exposure to
non-target organisms. In addition, Cry3Bbl has a narrow target range. Monsanto has performed dietary
bioassays to determine the insecticidal spectrum of the Cry3Bbl 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, Cry3Bbl 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
(MRU) 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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September 2010
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 etal. 1986). In addition, CRW has developed
resistance to methyl parathion and carbaryl, both adulticides used in rescue treatments (Meinke el 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 non-
monetary 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. undecimyunctata
howardiX and Western Corn Rootworm (D. vireifera virgifera) (MRU) No. 455382-08)
In this experiment, Monsanto compared the relative efficacy of two transformed corn hybrids,
expressing the Cry3Bbl 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 (MRU) 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 (MRU) 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 (MRU) 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
11(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).
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% (9P/o 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
Value due to product performance (yield)
$15.75
$12.50
$ 2.50
Value from lower chemical costs
Value from easier and safer use
Max value of Bt corn rootworm
$30.75
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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 (1 l)-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 V2
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 /^/-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 ifr-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).

Acres
Acres
MON 863
Conventional
Year
Infested
Treated
Acres
Treatments
(X 106)
(X 106)
(X 106)
(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 Cry3Bbl 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 $385.31 million, assuming a discount rate of 7%. The 7% discount rate represents the
Office of Management and Budget rate (see Circular Number A-94, Transmittal Memo Number 64,
October 29, 1992), a relatively risk-free rate of return similar to that assumed for a long-term U.S.
Treasury bond. The discount rate is defined as the interest rate used in calculating the present value
expected yearly for benefits and costs.
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Table 9. Annual Grower
Senefits of IV
ON 863.
Year
Target
Acres
(X 106)
Adoption
Acres
(X 106)
Grower
Benefits
($ 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 (MR11) 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
iootworm).
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 CRW-
Treated
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 Cry3Bbl 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 Cry3Bbl protein produced in MON 88017
and MON 863 is a variant of the wild-type Cry3Bbl protein from Bt subsp. kumamotoensis. When
compared by amino acid sequencing, the Cry3Bbl protein expressed in MON 88017 has been reported
to be 99.8% similar to the Cry3Bbl 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 11(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 11(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://wm.illinois.edu/education/proceedmss/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: CA-
98-171: 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: WL-
98-298: 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: WL-
98-300: 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: WL-
99-014: 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 Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl 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 (I), undecimpunctata howardi), and Western Corn Rootworm (I),
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 Cry3Bbl
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 : htty://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: ic ii'ic. eya. gov/oyybyydl/bioyesticides/yiys/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. Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl protein 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 3bBl, which expires on May 1,
2004, be amended to remove the expiration date. If (1) Monsanto requests such an amendment to the
Cry3Bbl 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 11(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 Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl corn product, containing
the new active ingredient Bacillus thuringiensis Cry3Bbl 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 Cryl Ab 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 Cry3Bbl protein and the genetic material necessary for its
production (vector PV-ZMIR39) in MON 88017 corn (OECD Unique Identifier: MON-88017-3) 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 Cry3Bbl
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 Cry3Bbl MON 88017 corn products, containing the new active ingredient Bacillus
thuringiensis Cry3Bbl protein and the genetic material necessary for its production (vector PV-
ZMIR39) in MON 88017 corn (OECD Unique Identifier: MON-88017-3), 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 Cry3Bbl 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 Cry3Bbl 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 Cry3Bbl-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 11(C)(2)(b). 11(C)(3)(b).
and 11(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 11(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 Cry3Bbl 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 111(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 Cryl Ab or CrylF 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 Cry3Bbl 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 plant-
incorporated 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 fifteen-
year 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
AIBS
APHIS
AR
ARS
BPPD
BRAD
Bt/B.t.
°C
C
CAP/CAPs
CEW
2
cm
CP4 EPSPS
CPB
CFR
Co
CO
Codex
CRW
DNA
DT50
dt90
dwt
EC50
ECB
ED50
EDSP
EEC
EFSA
ELISA
EPA
EPA Reg. No.
EPA Reg. Nos.
EUPs
F»t
FAW
FDA
FFDCA
FIFRA
FQPA
FR
ft
Agricultural Biotechnology Stewardship Technical Committee
American Institute of Biological Sciences
Animal and Plant Health Inspection Service (of the United States Department of Agriculture)
Arkansas
Agricultural Research Service (of the United States Department of Agriculture)
Biopesticides and Pollution Prevention Division
Biopesticides Registration Action Document
Bacillus thuringiensis
Celsius (degrees)
carbamate
Compliance Assurance Program/Compliance Assurance Programs
corn earworm
square centimeter/square centimeters
CP4 5-enolpyruvylshikimate-3-phosphate synthase
Colorado potato beetle
Code of Federal Regulations
County
Colorado
Codex Alimentarius Commission
corn rootworm
deoxyribonucleic acid
half-life
time until 90% decay
dry tissue weight
growth inhibition
European corn borer
median effective dose (produces desired effect in 50% of population)
Endocrine Disruptor Screening Program
Estimated Environmental Concentration
European Food Safety Authority
enzyme-linked immunosorbent assay
Environmental Protection Agency (the Agency)
Environmental Protection Agency Registration Number
Environmental Protection Agency Registration Numbers
experimental use permits
fixation index
fall armyworm
Food and Drug Administration
Federal Food, Drug, and Cosmetic Act
Federal Insecticide, Fungicide, and Rodenticide Act
Food Quality Protection Act
Federal Register
foot/feet
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GLOSSARY OF ACRONYMS AND ABBREVIATIONS, CONTINUED
fwt
g
g/cm
GIPSA
GM
HEEC
HGT
IA
IgG
IL
ILSI-CERA
IN
IRM
ISBN
kDa
kg
L
lb
lb/ac
LC50
LD
'50
LDR
LLC
LOAEC
LOC
LOD
LOQ
MALDI-TOF
MALDI-TOF-MS
MCRW
m/d
MD
MEEC
meq
ng 2
Hg/cm
Hg/g
Hg/L
fresh weight
gram/grams
grams per cubic centimeter
Grain Inspection, Packers and Stockyards Administration (of the United States Department of
Agriculture)
genetically modified
Highest Estimated Environmental Concentration
horizontal gene transfer
Iowa
immunoglobulin G
Illinois
International Life Sciences Institute Research Foundation - Center for Environmental Risk
Assessment
Indiana
insect resistance management
International Standard Book Number
kiloDalton
kilogram/kilograms
liter/liters
pound/pounds
pounds per acre
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).
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).
leaf damage
limited liability company
Lowest Observed Adverse Effect Concentration
Level of Concern
limit of detection
limit of quantitation
matrix-assisted laser desorption/ionization time-of-flight
matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
Mexican corn rootworm
meters per day
Maryland
Maximum Expected Environmental Concentration
milliequivalent
microgram/micrograms
micrograms per square centimeter
micrograms per gram
micrograms per liter
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GLOSSARY OF ACRONYMS AND ABBREVIATIONS, CONTINUED
Hg/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
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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)
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Table 1. Currently Registered PIPs Expressing Cry3Bbl 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
• Cry3Bbl
February 24, 2003
524-545*
MON 863 x MON 810
Monsanto Company
800 North Lindbergh Boulevard
St. Louis, MO 63167
• Cry3Bbl
• CrylAb
October 31, 2003
524-551
MON 88017
Monsanto Company
800 North Lindbergh Boulevard
St. Louis, MO 63167
• Cry3Bbl
December 13, 2005
524-552
MON 88017 x MON 810
Monsanto Company
800 North Lindbergh Boulevard
St. Louis, MO 63167
• Cry3Bbl
• CrylAb
December 13, 2005
524-576
MON 89034 x MON 88017
Monsanto Company
800 North Lindbergh Boulevard
St. Louis, MO 63167
• Cry 1A.105
• Cry2Ab2
• Cry3Bbl
June 10, 2008
524-581
MON 89034 x TCI507 x MON 88017 x
DAS-59122-7 (or SmartStax™)
Monsanto Company
800 North Lindbergh Boulevard
St. Louis, MO 63167
• Cry 1A.105
• Cry2Ab2
• Cry IF
• Cry3Bbl
• Cry34Abl
• Cry35Abl
July 20, 2009
524-583
TC1507x 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
• Cry IF
• Cry3Bbl
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
• Cry3Bbl
• Cry34Abl
• Cry35Abl
December 14, 2009
524-587
MON 89034 x TCI507 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
• Cry 1A.105
• Cry2Ab2
• Cry IF
• Cry3Bbl
October 15, 2009
524-589
MON 89034 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
• Cry 1A.105
• Cry2Ab2
• Cry3Bbl
• Cry34Abl
• Cry35Abl
December 14, 2009
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EPA
Registration
Number
Registration Name
Company and Address
Active
Ingredient(s)
Initial Date of Registration
524-590
TCI507 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
• Cry IF
• Cry3Bbl
• Cry34Abl
• Cry35Abl
December 14, 2009
68467-7
MON 89034 x TCI507 x MON 88017 x
DAS-59122-7 (or SmartStax™)
Mycogen Seeds
c/o Dow AgroSciences LLC
9330 Zionsville Road
Indianapolis, IN 46268-1054
• Cry 1A.105
• Cry2Ab2
• Cry IF
• Cry3Bbl
• Cry34Abl
• Cry35Abl
July 20, 2009
68467-8
MON 89034 x TCI507 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
• Cry 1A.105
• Cry2Ab2
• Cry IF
• Cry3Bbl
October 15, 2009
68467-10
MON 89034 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
• Cry 1A.105
• Cry2Ab2
• Cry3Bbl
• Cry34Abl
• Cry35Abl
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
• Cry3Bbl
• Cry34Abl
• Cry35Abl
December 14, 2009
68467-14
TC1507x 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
• Cry IF
• Cry3Bbl
December 14, 2009
68467-15
TCI507 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
• Cry IF
• Cry3Bbl
• Cry34Abl
• Cry35Abl
December 14, 2009
*Registration expired on its own terms on September 30, 2010.
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