BIOPESTICIDES REGISTRATION ACTION DOCUMENT 

Modified Cry3A Protein and the Genetic Material Necessary for its Production (Via Elements of 

pZM26) in Event MIR604 Corn SYN-IR604-8

 U.S. Environmental Protection Agency 

 Office of Pesticide Programs 

 Biopesticides and Pollution Prevention Division 
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 

Table of Contents

I. Overview .....................................................................................................................4 

 A. Background..................................................................................4 

 B. Executive Summary........................................................................4 

 

 C. Use Profile....................................................................................9 

 

 

II. Science Assessment ...................................................................................................10 

 A. Product Characterization .......................................................................................10 

 

 B. Human Health Assessment ...................................................................................24 

 

 C. Environmental Assessment ...................................................................................34 

 D. Insect Resistance Management .............................................................................72 

 E. Benefits and Public Interest Finding........................................................... .........121 

III. Regulatory Position for Event MIR604 Corn............ ................................136
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Biopesticide Registration Action Document (BRAD) September, 2010 

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Modified Cry3A protein and the Genetic Material Necessary for its Production (Via Elements of 

pZM26) in Event MIR604 corn SYN-IR604-8 Corn Regulatory Action Team

Product Characterization and Human Health

John Kough, Ph.D. 

Annabel Waggoner

Environmental Fate and Effects

Zigfridas Vaituzis, Ph.D. 

Tessa Milofsky, M.S. 

Mika Hunter 

Insect Resistance Management

Sharlene Matten, Ph.D. 

Alan Reynolds, M.S. 

Tessa Milofsky, M.S. 

Mika Hunter 

Benefit Assessment

Tessa Milofsky, M.S. 

Sharlene Matten, Ph.D. 

Registration Support

Mike Mendelsohn

Matt Thompson 

Ann Sibold 

Biopesticides Registration Action Document Team Leader

Mike Mendelsohn 

 

Office of General Counsel

Angela Huskey, Esq. 
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 

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I. Overview 

 

A. Background 

On October 3, 2006, EPA conditionally registered (with an expiration date of September 30, 2010), 

Syngenta Seeds Inc.’s active ingredient, modified Cry3A protein and the genetic material necessary for 

its production (via elements of pZM26) in event MIR604 corn SYN-IR604-8. Additionally, on 

January 24, 2007, the Agency conditionally registered (with an expiration date of October 15, 2008, 

subsequently extended to September 30, 2010) Syngenta Seed Inc.’s stacked product MIR 604 and 

Bt11 with mCry3A and Cry1Ab. The Agency determined that the use of these pesticides was in the 

public interest and that they would not cause any unreasonable adverse effects on the environment 

during the time of conditional registration. 

Results of efficacy trials conducted in 2002, 2003, and 2004 indicated that MIR604 corn provides 

effective control of key rootworm pests of field corn. MIR604 corn has unique biochemical properties 

which may benefit insect resistance management for this and other CRW-protected corn products. 

MIR604 and Bt 11 with mCry3A and Cry1Ab effectively controls key rootworm pests and European 

corn borers. The availability of multiple CRW-protected corn products and CRW/CB-protected corn 

products will increase grower choice and price competition, resulting in lower seed prices for 

consumers and higher adoption rates. Registration of these products is expected to result in further 

reduction of chemical insecticide use by growers. This is of special importance since many pesticides 

registered for CRW-control are highly toxic to humans and the environment, while mCry3Aexpressing corn poses no foreseeable human health or environmental risks. 

In order to reduce the possibility of corn rootworm or European corn borer developing resistance to Bt, 

EPA required Syngenta Seeds, Inc. to ensure that 20 percent of the planted acreage of this product be 

set aside where non-CRW-protected Bt corn will be grown to serve as a “refuge.” These refuge areas 

will support populations of corn rootworm not exposed to the Bt corn. The insect populations in the 

refuges will help prevent resistance development when they cross-breed with insects in the Bt fields. 

This resistance management strategy was developed as a condition of the registration, and EPA will 

require routine monitoring and documentation that these measures are followed. The submitted insect 

resistance management data support a registration until 2010. 

A tolerance exemption (40 CFR Part 174.505) was established for Bacillus thuringiensis modified 

Cry3A protein and the genetic material necessary for its production in corn. 

B. Executive Summary 

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 
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 

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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 mCry3A 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 mCry3A 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 mCry3A registrations 

by extending their expiration dates in accordance with the scheme explained in section III(C) of this 

Biopesticides Registration Action Document (BRAD). The basis for this decision can be found in both 

the risk assessment for the mCry3A corn products, which is characterized throughout this BRAD, and 

the Agency’s response to comments document. 

All data and findings for the mCry3A 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 mCry3A corn products, 

there are other Bt corn PIPs, expressing different proteins effective in controlling corn borers or corn 

rootworm, that were due to expire in 2010, and for which the associated registrants formally requested 

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

document, the following informationa

 is also available for public examination: 

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

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

● Cry34/35Ab1 BRAD (Draft - July 2010; Final - September 2010) 

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

● Optimum® AcreMaxTM B.t. Seed Blends BRAD (Draft - August 2010; Final - September 2010) 

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

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

● Registration Terms and Conditions Established with the Finalized Amendments 

 

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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Biopesticide Registration Action Document (BRAD) September, 2010 

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

● Public Comments on EPA Docket Number EPA-HQ-OPP-2010-0607 

● EPA’s Response to Comments 

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

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

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

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

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

significant conclusions regarding the positive environmental impact of Bt corn PIPs, and we took 

several actions to strengthen the insect resistance management requirements to ensure continued 

success in the prevention of the evolution of resistance in target pests. 

Since the commercialization of Bt crops, there have been a significant number of published field 

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

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

fields than in non-transgenic fields managed with chemical insecticides. Thus, these published and 

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. 

Product Characterization

The modified Cry3A (mCry3A) Bacillus thuringiensis (Bt) insect control protein is produced in 

transgenic corn plants derived from transformation Event MIR604 and has activity against certain 

beetles. A cry3A gene from Bt subsp. tenebrionis was recreated synthetically to optimize for 

expression in corn. Additional changes in this corn-optimized gene were made, such that the encoded 

mCry3A protein has enhanced activity against larvae of the western corn rootworm and northern corn 

rootworm. 
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Biopesticide Registration Action Document (BRAD) September, 2010 

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Protein characterization data demonstrate that the plant-produced protein is of sufficiently similar 

biological activity to that of the two modified Cry3A protein variants produced in the recombinant E. 

coli system (designated as test material MCRY3A-0102) for the purposes of human health and 

ecological effect risk assessments. Although the MCRY3A-0102 test material was not as active 

towards target pests as the plant-produced modified Cry3A protein, the doses in submitted studies were 

much higher than would occur via the modified corn. 

Bt corn Events MIR 604 and Bt11 with modified Cry3A and Cry1Ab is a stacked product that has 

activity against the rootworm varieties previously mentioned and European corn borer. The product 

characterization and protein expression analyses of Bt Cry1Ab and mCry3A insect control proteins and 

the genetic material necessary for its production in maize (corn) plants derived from Event Bt11 x 

MIR604 hybrid cross via traditional plant breeding were found to be similar and functionally 

equivalent to the Cry1Ab protein expressed in Bt11 and to mCry3A protein expressed in MIR604. 

Human Health Assessment

There is a reasonable certainty that no harm will result from aggregate exposure to the U.S. population, 

including infants and children, to the mCry3A protein and the genetic material necessary for its 

production, or MIR 604 and Bt11 with mCry3A and Cry1Ab. 

An acute oral toxicity study was submitted for the mCry3A protein and MIR 604 and Bt11 with 

mCry3A and Cry1Ab. Both studies support the prediction that the products would be non-toxic to 

humans. 

 

Amino acid sequence comparisons showed no similarity between the mCry3A protein or MIR 604 and 

Bt11 with mCry3A and Cry1Ab to known toxic proteins available in public protein databases. 

Amino acid sequence comparisons showed no similarity between the mCry3A protein or MIR 604 and 

Bt11 with mCry3A and Cry1Ab to known allergens available in public allergen databases. 

Environmental Assessment

The Agency is aware of no identified significant adverse effects of mCry3A proteins or MIR 604 and 

Bt11 with mCry3A and Cry1Ab on the abundance of non-target beneficial organisms in any population 

in the field environment, whether they are pest parasites, pest predators, or pollinators. Further, the 

EPA believes that cultivation of these products may have fewer adverse impacts on non-target 

organisms than use of chemical pesticides for corn production, because under normal circumstances, 

these products require substantially fewer applications of chemical pesticides, compared to production 

of non-Bt corn. And fewer chemical insecticide applications generally result in increased populations 

of beneficial organisms that control secondary pests, such as aphids and leafhoppers, in corn fields. 
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 

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In addition, no adverse effect on endangered and threatened species listed by the US Fish and Wildlife 

Service is expected from the proposed MIR604 CRW resistant corn registration. Further, the EPA has 

determined that there is no significant risk of gene capture and expression of mCry3A protein or MIR 

604 and Bt11 with mCry3A and Cry1Ab by wild or weedy relatives of corn in the U.S., its 

possessions, or territories, available data do not indicate that Cry proteins have any measurable adverse 

effect on microbial populations in the soil, nor has horizontal transfer of genes from transgenic plants 

to soil bacteria been demonstrated. 

Insect Resistance Management

 

The proposed IRM strategy and data to support it are “acceptable” except that the in-field strip refuge 

must be at least 4 rows wide based on recent larval movement data. If resistance is recessive, then the 

proposed IRM plan using a 20% structured refuge will be adequate to delay resistance for at least 15 

years given the assumptions of Syngenta’s model. If MIR604 maize is planted in areas with 

observable rotation-resistance in WCRW, then planting transgenic corn only in rotated maize fields is 

a good IRM strategy that will delay the evolution of resistance by at least 15 years regardless of gene 

expression. 

Benefits

Registration of modified Cry3A protein and the genetic material necessary for its production (via 

elements of pZM26) in event MIR604 corn SYN-IR604-8 is in the public interest because: 

1. Results of efficacy trials conducted in 2002, 2003, and 2004 indicate that MIR604 corn 

provides effective control of key rootworm pests of field corn. 

2. MIR604 corn has unique biochemical properties which may benefit insect resistance 

management for this and other CRW-protected corn products. 

3. If MIR604 corn is registered, it will be the third CRW-protected Bt corn product on the market. 

The availability of multiple CRW-protected corn products will increase grower choice and 

price competition, resulting in lower seed prices for consumers and higher adoption rates. 

4. Registration of MIR604 corn is expected to result in further reduction of chemical insecticide 

use by growers. This is of special importance since many pesticides registered for CRW-control 

are highly toxic to humans and the environment, while mCry3A-expressing corn poses no 

foreseeable human health or environmental risks. 
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Biopesticide Registration Action Document (BRAD) September, 2010 

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C. Use Profile 

1. Pesticide Name: Modified Cry3A protein and the genetic material necessary for its production (via 

elements of pZM26) in event MIR604 corn SYN-IR604-8 

 

Trade and Other Names: Agrisure RW Rootworm-Protected Corn; MIR 604 corn 

EPA Registration Number: 67979-5 

 

OPP Chemical Code: 006509 

Basic Manufacturer: Syngenta Seeds, Inc, 

Type of Pesticide: Plant-Incorporated Protectant

Uses: Field Corn

Target Pest(s): Corn Rootworm.

2. Pesticide Names: Modified Cry3A protein and the genetic material necessary for its production 

(via elements of pZM26) in event MIR604 corn SYN-IR604-8 

 and 

Bacillus thuringiensis Cry1Ab delta-endotoxin and the genetic material necessary for its 

production (Plasmid Vector pZ01502) in corn 

Trade and Other Names: Bt11 x MIR 604 Corn Seed; Agrisure CB/LL/RW, Bt11 x SYN-IR6Ø4-5 

corn seed Bt11 x SYN-IR6Ø4-8 corn seed 

EPA Registration Number: 67979-8 

 

OPP Chemical Code: 006509 and 006505 

Basic Manufacturer: Syngenta Seeds, Inc, 

Type of Pesticide: Plant-Incorporated Protectant

Uses: Field Corn

Target Pest(s): European Corn Borer and Corn Rootworm 
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Biopesticide Registration Action Document (BRAD) September, 2010 

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II. Science Assessment 

The classifications that are found for each data submission are assigned by the EPA science reviewer 

and are an indication of the usefulness of the information contained in the documents and if the data 

meet the intent of the test guidelines. A rating of “ACCEPTABLE” indicates the study is scientifically 

valid and has been satisfactorily performed according to accepted EPA guidelines or other justified 

criteria. A “SUPPLEMENTAL” rating indicates the data provide some information that can be useful 

for risk assessment. However, the studies may either have certain aspects not determined to be 

scientifically acceptable (SUPPLEMENTAL, but UPGRADABLE) or that the studies have not been 

done to fulfill a specific EPA guideline requirement. If a study is rated as “SUPPLEMENTAL, but 

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 current EPA guidelines or does not need to be 

reclassified as “ACCEPTABLE.” Both ACCEPTABLE and SUPPLEMENTAL studies may be used 

in the risk assessment process as appropriate. 

II.A. Product Characterization 

1. 2007 Position. 

The Modified Cry3A (mCry3A) Bacillus thuringiensis (Bt) insect control protein is produced in 

transgenic corn plants derived from transformation Event MIR604. The Agency’s detailed assessment 

of the product characterization for Bt mCry3A is found in Fellman (2005a, b, e and 2006a and c). A 

cry3A gene from Bt subsp. tenebrionis was recreated synthetically to optimize for expression in corn. 

Additional changes in this corn-optimized gene were made, such that the encoded mCry3A protein has 

enhanced activity against larvae of the western corn rootworm (WCRM; Diabrotica virgifera 

virgifera) and northern corn rootworm (NCRW; D. longicornis barberi). Introduced via

transformation vector pZM26, the mcry3A gene, consisting of 1797 base pairs (bp), was incorporated 

between an MTL promoter (2556 bp) from the Zea mays metallothionein-like gene and a terminator 

sequence from the nopaline synthase (NOS) gene of Agrobacterium tumefaciens used to provide a 

polyadenylation site. An Escherichia coli manA gene (1176 bp) encoding a phosphomannose 

isomerase (PMI) was incorporated between a promoter region from the Zea mays polyubiquitin gene 

(ZmUbInt (1993 bp)) and the same NOS terminator sequence described above. This PMI gene, which 

was introduced along with the mcry3A gene via the same pZM26 transformation vector, encodes the 

enzyme phosphomannose isomerase (PMI), which is employed as a selectable marker during the 

process of regenerating plant material following transformation. The PMI protein is a common 

enzyme involved in carbohydrate metabolism and allows for selection of transformants in cell culture 

by enabling only transformed corn cells to utilize mannose as a sole carbon source, while corn cells 

lacking the pmi gene fail to grow. 
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Biopesticide Registration Action Document (BRAD) September, 2010 

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The native Cry3A protein of Bt subsp. tenebrionis is a ca. 73 kDa polypeptide of 644 amino acids. By 

comparison, the mCry3A protein expressed in Event MIR64 corn is a ca. 67 kDa polypeptide of 598 

amino acids. The amino acid sequence of the mCry3A protein corresponds to that of the native Cry3A 

protein, except: 1) Its N-terminus corresponds to methionine-48 of the native protein; and 2) A 

cathepsin-G protease recognition site has been introduced, beginning at amino acid residue 155 of the 

native protein. This cathepsin-G recognition site has the sequence alanine-alanine-prolinephenylalanie, and has replaced the amino acids valine-155, serine- 156, and serine-157 in the native 

protein (MRID No. 461556-01, reviewed in Fellman, 2005a). 

The molecular properties of the mcry3A gene and the pmi gene, both present in Event MIR604 corn, 

were evaluated by utilizing Southern blotting analyses, DNA sequencing of the T-DNA insert, and by 

studying the Mendelian inheritance of the transgene insert (MRID No. 461556-02, reviewed in 

Fellman, 2005a). Maize genomic DNA was digested with KpnI restriction enzyme and hybridized to 

probes for mcry3A (1797 bp), pmi (1176 bp) and plasmid backbone (5309 bp; not inserted in the host 

genome). The T-DNA insert (via the pZM26 plasmid) in MIR604 was analyzed via Southern blot 

analyses and single copies of mcry3A and the pmi genes were present and closely linked in MIR604. 

The results also verified that the backbone DNA sequence from the transformation plasmid pZM26 in 

MIR604 was not inserted into MIR604. 

DNA sequencing revealed that there was a 44 and 43 bp truncation at the right and left break points of 

the T-DNA insert, respectively, during the transformation process that resulted in MIR604. Therefore, 

the overall integrity of the insert and the contiguousness of the functional elements were confirmed. In 

addition, T5 generation plants generated from T4 generation plants known to be heterozygous for the 

mcry3A and pmi genes were analyzed by an enzyme-linked immunosorbent assay (ELISA) of mCry3A 

and Polymerase Chain Reaction (PCR) analysis for both the mcry3A and pmi genes. Results showed 

that the two genes were closely linked with the expected Mendelian inheritance ratio. 

Expression levels of the mCry3A protein in transgenic corn tissues were also provided for both inbred 

and hybrid varieties (MRID 461556-04, reviewed in Fellman, 2005a) and were found comparable. 

The data on expression levels in two hybrids lines are summarized in Table 1. 
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TABLE 1. mCry3A Levels on a Dry Weight Basis in Event MIR604-Derived Corn Plants

Tissue Genotype Whorl Anthesis Seed Maturity Senescence

 mean μg mCry3A/g dry weight ± S. D. (range)

Leaves MIR604-B 

Hybrid 

18.64 ± 2.08 

(16.27 - 20.90) 

15.93 ± 3.65 

 (11.40-20.87) 

13.29 ± 2.37 

(9.61 - 15.40) 

5.05 ± 2.37 

(1.69 - 8.24) 

 MIR604-C 

Hybrid 

25.76 ± 4.11 

 (22.23 - 31.86) 

16.83 ± 2.88 

 (13.16-21.18) 

24.49 ± 3.34 

(19.58-28.87) 

6.57 ± 2.97 

 (3.65 - 10.33)

Roots MIR604-B 

Hybrid 

25.50 ± 1.36 

(23.55 - 27.07) 

15.57 ± 4.35 b 

(10.58 - 18.57) 

7.29 ± 1.96 

(4.99 - 9.03) 

10.31± 6.34 

(1.68 - 19.36) 

 MIR604-C 

Hybrid 

19.41 ± 2.17 

(16.29 - 21.18) 

14.46 ± 2.57 

 (11.56 - 18.30) 

12.96 ± 5.36 

(7.10 - 19.46) 

9.87 ± 5.43 

(5.45 - 17.60) 

Kernels MIR604-B 

Hybrid 

N/A N/A 1.09 ± 0.45 

(0.74 - 1.83) 

0.77 ± 0.20 

(0.59 - 0.99) 

 MIR604-C 

Hybrid 

N/A N/A 1.95 ± 0.74 

(1.26 - 3.13) 

0.94 ± 0.47 

(0.43 - 1.59) 

Silk MIR604-B 

Hybrid 

N/A <0.34 

(ND-DNQ) 

0.99 d N/A 

 MIR604-C 

Hybrid 

N/A <0.56 

(DNQ) 

3.04 ± 0.78 a 

(2.31 - 3.92) 

N/A 

Pollen MIR604-B 

Hybrid 

N/A ND e

 N/A N/A 

 MIR604-C 

Hybrid 

N/A ND e N/A N/A 

Whole 

Plant

MIR604-B 

Hybrid 

7.31 ± 3.40 

(1.33 - 9.51) 

11.16 ± 5.48 

(4.23- 16.99) 

16.97 ± 8.24 

(5.88 - 28.88) 

8.96 ± 3.96 

(5.03 - 15.40) 

 MIR604-C 

Hybrid 

9.83 ± 1.83 

(8.56 - 13.01) 

11.22 ± 2.87 

 (9.05 - 14.41) 

23.77 ± 4.91 a 

(16.82 - 28.35) 

11.85 ± 3.51 

(7.44 - 16.78) 

Except where noted otherwise, five samples were used to determine the means and standard deviations. a

 Four samples were used to determine the mean and standard deviation. 

b Three samples were used to determine the mean and standard deviation. c

 Two samples were used to determine the mean and standard deviation. 

d One sample analyzed. 

 e Result of one pooled sample. 

 N/A Not analyzed at this stage. 

 ND Not detectable. 

DNQ. Detectable but not quantifiable 
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Biopesticide Registration Action Document (BRAD) September, 2010 

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Production of bacterial-derived mCry3A protein was chosen in order to obtain sufficient material for 

testing, therefore, its equivalence to the plant-derived protein was determined. The mCry3A protein 

was produced in recombinant E. coli by over-expressing the same modified cry3A gene that was 

introduced into Event MIR604 corn plants. 

The similarity of mCry3A protein expressed in corn event MIR604 and the recombinant E. coli test 

system (designated as test material MCRY3A-0102) were evaluated (MRID 461556-03, reviewed in 

Fellman, 2005a). The mCry3A proteins from both sources had the same approximate molecular 

weight (ca. 67,700 Da.) based on mass spectral analysis. Moreover, Western blot analysis of the test 

material showed a single immunoreactive band corresponding to the predicted molecular weight of ca. 

67,700 Da. SDS-PAGE showed the purity of mCry3A in the test material to be ca. 90.3% by weight. 

The mCry3A protein from both sources was immunologically cross-reactive with the same antimCry3A antibody. Both proteins produced comparable toxicities toward Western Corn Rootworm 

larvae, based on LC50 values. There was also no evidence of post-translational glycosylation of 

mCry3A protein from either source. Therefore, it was concluded that the mCry3A proteins from corn 

event MIR604 and from recombinant E. coli were equivalent.

However, further testing revealed that the test material, MCRY3A-0102, contained two closely related 

components in a ratio of ca. 2:3 via SDS-PAGE and MALDI TOF mass spectrometry (MRID 461556-

06, reviewed in Fellman, 2005a). The lesser of the two components, with the lower molecular weight, 

corresponded to the intended mCry3A protein with 598 amino acids. The other component contained 

the same 598 amino acids as the first component but also contained an additional 16 amino acids at the 

N–terminal end of the protein. To satisfy Agency’ concerns with the long form component of 

mCry3A, Syngenta conducted an in-depth examination of the nucleotide sequence of the cry1Ac 

promoter in the cassette utilized for expression of the mCry3A protein in the MCRY3A-0102 test 

substance. The registrant reported that an “ATG” initiation codon sequence was identified within the 

cry1Ac promoter indicating the presence of an additional putative open reading frame (ORF). This 

demonstrated that the resultant putative ORF would be in-frame with the mcry3A gene and would 

result in an additional 16 amino acids on the N-terminus of the intended mCry3A protein (MRID 

464667-01, reviewed in Fellman, 2005b). 

Visual confirmation of the molecular weights for the long and short forms of mCry3A protein was also 

provided via SDS-PAGE and western blot analysis. These gels indicated single intense bands 

consistent with the predicted molecular weights of ca. 69,500 Da and ca. 67,700 Da for the mCry3ALF (long form) and mCry3A-SF (short form) samples, respectively. Therefore, the identity of the two 

components was conclusively determined by peptide mapping using tandem (MS/MS) mass 

spectrometry and by MALDI TOF mass spectrometry of the intact proteins (MRID 461556-07). The 

molecular weight data showed the two proteins to have masses of 67,519 and 69,138 Da. 

Because both forms of mCry3A were insecticidally active against WCRW and taking into account the 

high degree of structural homology (97.4% amino acid identity), the two forms of mCry3A in test 

material MCRY3A-0102 were considered equivalent. Moreover, the test material was re-analyzed ca. 
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Biopesticide Registration Action Document (BRAD) September, 2010 

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9 months after its initial characterization and found to be substantially stable when stored at -20 °C 

(MRID 461556-05, reviewed in Fellman, 2005a). 

On March 14-15, 2006, EPA held a FIFRA Scientific Advisory Panel (SAP) meeting, 

http://www.epa.gov/scipoly/sap/meetings/2006/index.htm#march to address the scientific issues that 

arose during the risk assessment of mCry3A. The SAP Report (SAP, 2006) is reviewed in Fellman 

(2006c). EPA asked the SAP to comment on the equivalence of the mCry3A proteins from corn event 

MIR604 and from recombinant E. coli - specifically the presence of two forms in the bacterially 

produced mCry3A protein and the differences in bioactivity in the WCRW bioassay. The majority of 

the Panel concluded that the two forms of the mCry3A are of relatively comparable biological activity 

for the purposes of the human health assessments based on the amino acid sequence identity, lack of 

glycosylation, and general stability. 

2. Product Characterization Data. The product characterization data is summarized in Table 2. 

Table 2. Product characterization studies (2010 Update) 

MRID Title Summary

461556-

01 

Review of 

Characterization 

and Safety of 

Modified 

Cry3A protein 

and maize 

(corn) plants 

derived from 

Event MIR604 

with 

comparison to 

native Cry3A 

protein 

The mCry3A protein contains 598 amino acids (ca. 67 kDa), whereas the 

native Cry3A protein is ca. 73 kDa polypeptide of 644 amino acids. The 

amino acid sequence of the mCry3A protein corresponds to that of the 

native Cry3A protein, except: 1) Its N-terminus corresponds to 

methionine-48 of the native protein; and 2) A cathepsin-G protease 

recognition site has been introduced, beginning at amino acid residue 155 

of the native protein. The susceptibility of insect pest species and 

insecticidal properties, such as solubilization, proteolytic processing, 

receptor binding, and membrane pore forming properties, were also 

determined for mCry3A protein and differentiated with native Cry3A 

protein. The mCry3A protein has a similar spectrum of activity to the 

native Cry3A, but with enhanced toxicity to NCRW and WCRW. 

Classification: Acceptable 
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 

 15 

MRID Title Summary

461556-

02 

Molecular 

characterization 

of event 

MIR604 maize 

(corn) 

expressing a 

modified Cry3A 

bacillus 

thuringiensis 

protein 

Corn Event MIR604 does not contain any of the backbone sequences from 

the transforming plasmid pZM26. Three nucleotide changes were 

identified, one in a regulatory region associated with the mcry3A gene and 

two in the pmi coding sequence. The mcry3A and pmi genes are closely 

linked. 

Classification: Acceptable 

461556-

03 

Characterization 

of modified 

Cry3A protein 

produced in 

event MIR604-

derived maize 

(corn) and 

comparison 

with modified 

Cry3A protein 

expressed in 

recombinant E. 

coli

This study evaluated the similarity of modified Cry3A (mCry3A) 

insecticidal protein expressed in corn event MIR604 and mCry3A protein 

expressed in a recombinant E. coli test system. The mCry3A protein 

derived from corn event MIR604 and recombinant E. coli had the same 

approximate molecular weight (ca. 67,700 Da.) based on mass spectral 

analysis (from MRID 461556-06). The mCry3A protein from both sources 

was immunologically cross-reactive with the same anti-mCry3A antibody. 

Both proteins produced comparable toxicities toward Western Corn 

Rootworm larvae, based on LC50 values. There was no evidence of posttranslational glycosylation of mCry3A protein from either source. It was 

concluded that the mCry3A proteins from corn event MIR604 and from 

recombinant E. coli were substantially the same. 

Classification: Acceptable 

461556-

04 

Quantification 

of modified 

Cry3A and PMI 

proteins in 

transgenic 

maize (corn) 

tissues, whole 

plants, and 

silage derived 

from 

transformation 

event MIR604 

The plant extracts (including leaves, roots, kernels, silk, pollen, silage, and 

whole plants) from inbred and hybrid corn varieties derived from MIR604 

field plants were quantitatively analyzed for mCry3A by ELISA. The 

magnitude of expression for tissue types was as follows in descending 

order: leaves, roots, silage, and kernels. All control tissues were negative 

for the expression of mCry3A. The mean extraction efficiency for mCry3A 

over all tissues (except for pollen or silk tissue, where levels were too low 

to be determined) was 76.6%. Low, but quantifiable, levels of PMI protein 

were found in most of the Event MIR604-derived plant tissues analyzed 

including pollen. The mCry3A and PMI proteins were stably expressed in 

four backcross generations in leaf tissue analyzed at anthesis stage. Mean 

levels across all backcross generations were ca. 11.8 - 15.5 μg/g dry weight 

and 1.1 - 1.3 μg/g dry weight for mCry3A and PMI proteins, respectively. 

Classification: Acceptable
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 

 16 

MRID Title Summary

461556-

05 

Characterization 

of modified 

Cry3A test 

substance 

mCry3A-0102 

and certificate 

of analysis 

This study characterized test material MCRY3A-0102, a microbially 

produced protein preparation containing a modified Cry3A (mCry3A) 

protein. The purity of mCry3A in the test material was shown to be ca. 

90.3% by weight using SDS-PAGE analysis. Western blot analysis of the 

test material showed a single immunoreactive band corresponding to the 

predicted molecular weight of ca. 67,700 Da. The test material was 

insecticidally active and had a 144-hour LC50 of 1.4 μg/mL diet (95% 

confidence interval: 0.7 - 2.2 μg/mL) against Western corn rootworm 

(WCRW) larvae. Two forms of mCry3A were found in the test material, 

designated mCry3A-SF and mCry3A-LF, respectively, and were both 

insecticidally active against WCRW. On this basis, and taking into account 

the high degree of structural homology (97.4% amino acid identity), the 

two forms of mCry3A in test material MCRY3A-0102 were considered to 

be equivalent. The test material was re-analyzed ca. 9 months after its 

initial characterization and found to be substantially stable when stored at -

20 °C. 

Classification: Acceptable

461556-

06 

Further 

Characterization 

of Modified 

Cry3A Test 

Substance 

MCRY3A-0102

The test material, MCRY3A-0102, containing mCry3A protein was shown, 

by SDS-PAGE and MALDI TOF mass spectrometry, to contain two 

closely related components in a ratio of ca. 2:3. The lesser of the two 

components, with the lower molecular weight, corresponded to the 

intended mCry3A protein with 598 amino acids. The other component 

contained the same 598 amino acids as the first component but also 

contained an additional 16 amino acids at the N–terminal end of the 

protein. The identity of the two components was conclusively determined 

by peptide mapping using tandem (MS/MS) mass spectrometry and by 

MALDI TOF mass spectrometry of the intact proteins. The molecular 

weight data showed the two proteins to have masses of 67,519 and 69,138 

Da. 

Classification: Acceptable 
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 

 17 

MRID Title Summary

461556-

09 

Analysis for the 

presence of 

modified Cry3A 

protein in wet 

and dry milled 

fractions, corn 

oil and corn 

chips from corn 

(maize) event 

MIR604 

Among the wet-milled fractions, the medium fiber (0.46 μg mCry3A/g), 

fine fiber (0.26 μg mCry3A/g), and gluten meal (0.24 μg mCry3A/g) 

fractions yielded quantifiable amounts of mCry3A. Among the dry-milled 

fractions the highest concentrations were found in the flaking grits (2.12 μg 

mCry3A/g), the corn hulls (1.42 μg mCry3A/g), and the coarse grit (0.92 

μg mCry3A/g) fractions. Levels of mCry3A found in the other dry-milled 

fractions, including fine grits, corn meal, corn cone and corn flour, were 

between 0.32 and 0.69 μg mCry3A/g. Although the concentration of 

mCry3A protein measured in the flour used to prepare the corn chips was 

0.32 μg mCry3A /g, no mCry3A protein was detected in the corn chips. 

Similarly, mCry3A protein was not detectable in oil, whereas the starting 

material, flaking grits, contained 2.12 μg mCry3A/g. 

Classification: Acceptable

465974-

01 

Analytical 

Method for the 

Detection of the 

PlantIncorporated 

Protectant 

Modified 

Cry3A Protein 

in Event 

MIR604 Corn 

Grain and 

Independent, 

Third-Party 

Validation of 

Said Method 

A monoclonal antibody based enzyme-linked immunosorbent assay 

(ELISA) method was developed by Syngenta to detect modified Cry3A 

(mCry3A) protein expressed in MIR604 seed and leaf tissues. An 

independent, third party laboratory (EnviroLogix, Inc.) utilized the assay 

methodology, Event MIR604 seed, negative isoline seed, and three lots of 

mCry3A monoclonal antibody for validation of assay protocol, sensitivity 

and cross-reactivity, according to the USDA GIPSA directive. The assay 

sensitivity was estimated at 1 positive Event MIR604 kernel in 999 nonMIR604 kernels (0.1%), which was based on a minimum of 120 ground 

seed samples (with a LOD of 0.33 ppb for seed material). The average 

quantification was 2.07 ppb (ng/g fresh weight corn). No cross reactivity 

with other commercial, conventional and transgenic corn was detected with 

the exception of products expressing the Cry3Bb1 protein. Moreover, 

extraction efficiency was determined at 59% for ground seed and 75% for 

leaf tissue. Therefore, the monoclonal antibody-based commercial ELISA 

detection assay (tested by EnviroLogix, Inc.) satisfies the EPA Residue 

Chemistry Guidelines OPPTS 860.1340(c)(6) Residue Analytical Methods 

and PR Notice 96-1. EPA’s Analytical Method Laboratory located in Fort 

Meade (Maryland) will have to independently validate Syngenta’s ELISA 

protocol for accuracy, precision, and sensitivity. 

Classification: Acceptable
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 

 18 

MRID Title Summary

67956-

01 

Comparative 

Southern 

Analysis of a 

Bt11 x MIR604 

Maize Hybrid 

with the 

Parental Event 

Bt11 and Event 

MIR604 Maize 

Inbreds 

The purpose of this study was to use Southern blot analysis to confirm the 

presence of the cry1Ab and pat genes from the parental Event Bt11 and 

mcry3A and pmi genes from parental Event MIR604 in the hybrid Bt11 x 

MIR604 in a predictable manner. For the cry1Ab-specific probe, the NdeI 

digest resulted in a single hybridization band of ~4.6 kb in both Event Bt11 

and Bt11 x MIR604. For the pat-specific probe, the NdeI digest resulted in 

a single hybridization band of ~1.9 kb in both Event Bt11 and Bt11 x 

MIR604. For the mcry3A-specific probe, the KpnI digest resulted in a 

single hybridization band of ~5.6 kb in both Event MIR604 and Bt11 x 

MIR604. Likewise, for the pmi-specific probe, the KpnI digest resulted in a 

single hybridization band of ~5.2 kb in both Event MIR604 and Bt11 x 

MIR604. The predicted DNA hybridization patterns were retained and 

stability of the transgenic locus from parent to progeny was demonstrated. 

However, a more detailed explanation with documentation and/or literature 

reference for the unexpected cross hybridization of the Bt11 transformation 

plasmid control lane (observed on Figure 7, lane 8) and the commercial 

DNA ladder is needed to verify the results of the Bt11 x MIR604 Southern 

blot, hybridized with the mcry3A-specific probe. 

CLASSIFICATION: SUPPLEMENTAL but UPGRADEABLEpending submission of a more detailed explanation for the unexpected 

hybridization band detected in the Bt11 transformation plasmid control 

lane on the Bt11 x MIR604 Southern blot, hybridized with the mcry3Aspecific probe. 
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 

 19 

MRID Title Summary

469165-

01 

Comparison of 

Transgenic 

Protein 

Expression in 

Event Bt11, 

Event MIR604 

and Stacked 

Bt11 x MIR604 

Maize (corn) 

Hybrids 

The purpose of this study was to compare expression of the four transgenic 

proteins (Cry1Ab, mCry3A, PAT and PMI) in a Bt11 x MIR604 maize 

(field corn) hybrid with expression in corresponding near-isogenic hybrids 

derived from the individual transformation events to determine whether 

any unexpected differences in protein expression had occurred as a result 

of combining the traits by breeding via ELISA. 

Mean Cry1Ab concentrations in leaves and roots of the Bt11 x MIR604 

hybrid ranged from ca. 19.7 to 27.7 μg/gdw and ca. 5.6 to 10.0 μg/gdw, 

respectively. Mean Cry1Ab concentrations in kernels were ca. 1.7 μg/gdw. 

The Cry1Ab concentrations in pollen from all three locations ranged from 

below the LOQ to 0.06 μg/gdw. Mean mCry3A concentrations in leaves 

and roots of Bt11 x Event MIR604 hybrid ranged from ca. 33.4 to 46.3 

μg/gdw and ca. 18.9 to 23.9 μg/gdw, respectively. Mean mCry3A 

concentrations in kernels were ca. 0.7 μg/gdw. The mCry3A levels in the 

pollen collected at anthesis from all three locations range from below the 

LOQ to ca. <0.03 μg/gdw. Mean PAT concentrations in leaves and roots 

of the Bt11 x MIR604 hybrid ranged from ca. <0.05 to 0.17 μg/gdw and 

ca. 0.08 to 0.19 μg/gdw, respectively. Mean PAT concentrations in the 

kernels were both <LOQ to <0.04 μg/gdw. The PAT levels in the pollen 

collected at anthesis from all three locations range from below the LOQ to 

ca. <0.034 μg/gdw. Mean MIR604 PMI concentrations in leaves and roots 

of the Bt11 x MIR604 hybrid ranged from ca. 5.7 to 10.4 μg/gdw and ca. 

2.3 to 6.0 μg/gdw, respectively. Mean MIR604 PMI concentrations in 

kernels were ca. 1.9 μg/gdw. The PMI levels in the pollen collected at 

anthesis from all three locations range from 39.3 to 56.9 μg/gdw. 

Overall, concentrations of Cry1Ab, mCry3A, PAT, and PMI protein levels 

were found comparable and all control tissues were negative for the 

expression of Cry1Ab, PAT, mCry3A and PMI. Therefore, transgenic 

protein expression in the Bt11 x MIR604 hybrid are not substantially 

different from that of the hybrids derived from the individual Bt11 and 

MIR604 transformation events. 

Classification: Acceptable
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 

 20 

MRID Title Summary

470062-

01 

Response to 

EPA questions 

regarding 

mcry3A 

Southern blot in 

the Bt11 x 

MIR604 

Comparative 

Southern 

Analysis Report 

(MRID No. 

467956-01, 

Figure 7) 

On November 22, 2006, EPA and representatives from Syngenta Seeds Inc., held a conference call 

to discuss the deficiencies noted in MRID 467965-01. EPA required clarification for the Southern 

blot (Figure 7) where an unexpected hybridization band (as 6.2 kb) was observed in the Bt11 

transformation plasmid (the NotI-digested pZO1502) control lane. 

In response, Syngenta submitted a report that explained that the unexpected hybridization band was 

due to a small amount of pUC plasmid present in the KB ladder (used as the molecular weight 

marker). Syngenta ascertained this after discussion with a tech service representative of Stratagene 

(the KB ladder supplier), who indicated trace amounts of pUC plasmid may remain as contaminants 

in the KB ladder stock (as a result of the manufacturing process). Due to the pUC plasmid presence 

in the KB ladder, Syngenta subsequently hybridized the membrane with KB ladder probe, mcry3Aspecific probe, and pUC plasmid probe. 

Syngenta provided an additional rationale based on the generated Southern data for another project 

(Bt11 x MIR604 x GA21) in which they utilized a different ladder (Analytical Marker DNA, Wide 

Range, Promega Cat. No. DB1931). There was no unexpected hybridization band in the pZO1502 

lane on the blot probed with the mcry3A-specific probe. This further confirmed that the unexpected 

hybridization band (Figure 7, lane 8) was due to the radiolabeled pUC plasmid, present in the KB 

ladder, hybridizing to COLE1in pZO1502. 

EPA also questioned Syngenta’s method of radiolabeling the ladder DNA. Syngenta stated a 

preference to hybridize blots with the ladder and element-specific probes simultaneously so that the 

ladder can be visualized directly on the blot. The registrant utilizes this technique to eliminate the 

potential for introducing errors when manually transferring the position of stained ladder bands from 

the gel to the membrane. 

Syngenta also stated that they are using a different molecular weight marker preparation (consisting 

of a mixture of restriction enzyme digests of lambda DNA and Phi X174 DNA) to avoid any future 

potential non-specific sequence binding of the labeled ladder probe. Since this ladder is made from 

phage DNA, no unexpected hybridization bands in the plasmid control lanes is either expected nor 

observed in other Southern analyses (when probing with a combination of the radiolabeled 

Analytical Marker DNA, Wide Range ladder and a radiolabeled element-specific probe).

Syngenta also noted that choosing different enzymes would not eliminate the presence of any of the 

plasmid backbone sequences in the plasmid control lanes, because the plasmid controls on the 

Southern blots contain all the fragments of the digested plasmids. 

EPA Reviewer’s Comment: The registrant has submitted a sufficient explanation for the nonspecific binding in the Southern blot (Figure 7, lane 8). However, visual verification is needed to 

confirm the lack of any unexpected hybridization band in the pZO1502 lane on the blot probed with 

the mcry3A-specific probe. Therefore, Syngenta should submit a Southern blot containing genomic 

DNA from Bt11, MIR604, Bt11 x MIR604 and a negative control as well as the plasmid controls 

pZO1502 and pZM26 hybridized with a combination of the mcry3A-specific and the newly 

employed Analytical Marker DNA, Wide Range ladder, which does not contain the COLE1 origin 

of replication vector sequence. 

Classification: Supplemental, But Upgradeable- pending submission of an additional Bt11 x 

MIR604 Southern blot probed with the mcry3A-specific probe to confirm the lack of non-specific 

sequence binding in the Bt11 transformation pZO1502 plasmid control lane as confirmatory data to 

complete the Bt11 x MIR604 database. 
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 

 21 

MRID Title Summary

472648-

01 

Supplement to 

Report Titled: 

Comparative 

Southern 

Analysis of a 

Bt11 x MIR604 

Maize Hybrid 

with the 

Parental Event 

Bt11 and Event 

MIR604 Maize 

Inbreds 

The original submission of Southern blot data for the registration of this 

combination PIP product expressing both Cry1Ab and mCry3A proteins 

did not have clear results for the reactivity of the molecular weight 

standards with labeled plasmid components. Syngenta was required as a 

condition of the registration to submit new Southern blot data to clarify the 

cross reactivity and other issues. 

The study presents Southern blot data with appropriate plasmid maps to 

explain the expected fragment banding patterns. The combination of 

several endonuclease digestions and probes gives the necessary 

background to judge the results from the single parental lines as well as the 

hybrid cross Bt11 x MIR604. There was no aberrant probe binding to the 

molecular weight markers. The control samples in each gel were free from 

probe reactivity except in the case of positive controls. 

These data demonstrate stability and presence of both the Cry1Ab and 

mCry3A proteins and PAT and PMI marker genes in the parental line and 

the hybrid cross. 

Classification: Acceptable

none Provide to the 

EPA Laboratory 

(Ft. Meade, 

MD) 

methodology 

and/or reagents 

necessary for 

validation of 

mCry3A 

analytical 

method. 

More recently, the Agency decided to allow this requirement to be satisfied 

when the Grain Inspection, Packers and Stockyards Administration 

(GIPSA) of the United States Department of Agriculture (USDA) has 

verified the performance of a qualitative rapid test kit for detecting the 

presence of the biotechnology event in grains and oilseeds. In the case of 

mCry3A, the Agency has confirmed that a test kit has been verified by 

GIPSA and, therefore, the aforementioned requirement has been satisfied. 

Classification: Acceptable

3. 2010 Update: Terms and Conditions of the Event MIR 604 expressing modified Cry3A 

1. Validation of analytical method for mCry3A. When Event MIR 604 expressing modified Cry3A 

Corn (EPA Reg. No. 67979-5) was initially registered on October 03, 2006, the Agency issued 

registration notices to Syngenta Seeds, Inc. that contained the following requirement for further 

product characterization information: 

“Provide to the EPA laboratory (Ft. Meade, MD) methodology and/or reagents 

necessary for validation of a mCry3A analytical method within 6 months from the 

date that the Agency requests them.” 
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 

 22 

More recently, the Agency decided to allow this requirement to be satisfied when the Grain Inspection, 

Packers and Stockyards Administration (GIPSA) of the United States Department of Agriculture 

(USDA) has verified the performance of a qualitative rapid test kit for detecting the presence of the 

biotechnology event in grains and oilseeds. In the case of mCry3A, the Agency has confirmed that a 

test kit has been verified by GIPSA and, therefore, the aforementioned requirement has been satisfied. 

2. Product Characterization for Events MIR 604 x Bt11, expressing the proteins mCry3A and Cry1Ab 

The product Agrisure CB/LL/RW contains Bt corn events MIR 604 x Bt11 expressing the proteins 

mCry3A and Cry1Ab. This stacked product targets both lepidopteran corn pests that are susceptible to 

Cry1Ab as well as corn rootworm that is susceptible to Cry3A. Both Bt11 and MIR 604 have been 

previously registered as single gene products. Specific product characterization information on this 

stacked product is presented below. 

The purpose of the Transgenic Protein Expression Study was to compare expression of the four 

transgenic proteins (Cry1Ab, mCry3A, PAT and PMI) in a Bt11 x MIR604 maize (field corn) hybrid 

with expression in corresponding near-isogenic hybrids derived from the individual transformation 

events to determine whether any unexpected differences in protein expression had occurred as a result 

of combining the traits by breeding via ELISA. 

Overall, concentrations of Cry1Ab, mCry3A, PAT, and PMI protein levels were found comparable and 

all control tissues were negative for the expression of Cry1Ab, PAT, mCry3A, and PMI. Therefore, 

transgenic protein expression in the Bt11 x MIR604 hybrid are not substantially different from that of 

the hybrids derived from the individual Bt11 and MIR604 transformation events. 

The Southern blot analysis is used to confirm the presence of the cry1Ab and pat genes from the 

parental Event Bt11 and mcry3A and pmi genes from parental Event MIR604 in the hybrid Bt11 x 

MIR604 in a predictable manner. The predicted DNA hybridization patterns were retained and 

stability of the transgenic locus from parent to progeny was demonstrated. However, a more detailed 

explanation with documentation and/or literature reference for the unexpected cross hybridization of 

the Bt11 transformation plasmid control lane and the commercial DNA ladder was needed to verify the 

results of the Bt11 x MIR604 Southern blot, hybridized with the mcry3A-specific probe. 

On November 22, 2006, the EPA and representatives from Syngenta Seeds Inc., held a conference call 

to discuss the deficiencies noted in the Southern Blot data (MRID 467965-01). The Agency wanted 

further clarification for the Southern blot where an unexpected hybridization band (as 6.2 kb) was 

observed in the Bt11 transformation plasmid (the NotI-digested pZO1502) control lane. 

In response, Syngenta submitted a report that explained that the unexpected hybridization band was 

due to a small amount of pUC plasmid present in the KB ladder (used as the molecular weight marker). 

Syngenta ascertained this after discussion with a tech service representative of Stratagene (the KB 
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 

 23 

ladder supplier), who indicated trace amounts of pUC plasmid may remain as contaminants in the KB 

ladder stock (as a result of the manufacturing process). Due to the pUC plasmid presence in the KB 

ladder, Syngenta subsequently hybridized the membrane with KB ladder probe, mcry3A-specific 

probe, and pUC plasmid probe. 

Syngenta provided an additional rationale based on the generated Southern data for another project 

(Bt11 x MIR604 x GA21) in which they utilized a different ladder (Analytical Marker DNA, Wide 

Range, Promega Cat. No. DB1931). There was no unexpected hybridization band in the pZO1502 lane 

on the blot probed with the mcry3A-specific probe. This further confirmed that the unexpected 

hybridization band was due to the radiolabeled pUC plasmid, present in the KB ladder, hybridizing to 

COLE1in pZO1502. 

EPA also questioned Syngenta’s method of radiolabeling the ladder DNA. Syngenta stated a 

preference to hybridize blots with the ladder and element-specific probes simultaneously so that the 

ladder can be visualized directly on the blot. The registrant utilizes this technique to eliminate the 

potential for introducing errors when manually transferring the position of stained ladder bands from 

the gel to the membrane. 

Syngenta also stated that they are using a different molecular weight marker preparation (consisting of 

a mixture of restriction enzyme digests of lambda DNA and Phi X174 DNA) to avoid any future 

potential non-specific sequence binding of the labeled ladder probe. Since this ladder is made from 

phage DNA, no unexpected hybridization bands in the plasmid control lanes is either expected nor 

observed in other Southern analyses (when probing with a combination of the radiolabeled Analytical 

Marker DNA, Wide Range ladder and a radiolabeled element-specific probe).

Syngenta also noted that choosing different enzymes would not eliminate the presence of any of the 

plasmid backbone sequences in the plasmid control lanes, because the plasmid controls on the 

Southern blots contain all the fragments of the digested plasmids. 

EPA found that the registrant submitted a sufficient explanation for the non-specific binding in the 

Southern blot. Visual verification was needed, however, to confirm the lack of any unexpected 

hybridization band in the pZO1502 lane on the blot probed with the mcry3A-specific probe. The 

registrant should submit a Southern blot containing genomic DNA from Bt11, MIR604, Bt11 x 

MIR604 and a negative control as well as the plasmid controls pZO1502 and pZM26 hybridized with a 

combination of the mcry3A-specific and the newly employed Analytical Marker DNA, Wide Range 

ladder, which does not contain the COLE1 origin of replication vector sequence. 

Syngenta submitted a supplemental study which showed Southern blot data with appropriate plasmid 

maps to explain the expected fragment banding patterns. The combination of several endonuclease 

digestions and probes gave the necessary background to judge the results from the single parental lines 

as well as the hybrid cross Bt11 x MIR604. There was no aberrant probe binding to the molecular 

weight markers. The control samples in each gel were free from probe reactivity except in the case of 
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 

 24 

positive controls. These data demonstrated stability and presence of both the Cry1Ab and mCry3A 

proteins and PAT and PMI marker genes in the parental line and the hybrid cross. 

II. B. Human Health Assessment 

1. Mammalian Toxicity and Allergenicity Assessment 

Consistent with section 408(b) (2) (D) of the FFDCA, EPA has reviewed the available scientific data 

and other relevant information in support of this action and considered its validity, completeness and 

reliability and the relationship of this information to human risk. EPA has also considered available 

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

consumers, including infants and children. 

Data have been submitted demonstrating the lack of mammalian toxicity at high levels of exposure to 

the pure mCry3A protein. These data demonstrate the safety of the products at levels well above 

maximum possible exposure levels that are reasonably anticipated in the crops. This is similar to the 

Agency position regarding toxicity and the requirement of residue data for the microbial Bacillus 

thuringiensis products from which this plant-incorporated protectant was derived (See 40 CFR Sec. 

158.2130 and 158.2140).158.740(b)(2)(i)). 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 

the observed effects and clarify the source of these effects (Tiers II and III). 

An acute oral toxicity study was submitted for the mCry3A protein (MRID 461556-10, reviewed in 

Fellman, 2005a). The acute oral toxicity data submitted support the prediction that the mCry3A protein 

would be non-toxic to humans. Male and female mice (5 of each) were dosed with 2,377 

milligrams/kilograms bodyweight (mg/kg bwt) of mCry3A protein. With the exception of one female 

in the test group that was euthanized on day 2 (due to adverse clinical signs consistent with a dosing 

injury), all other mice survived the study, gained weight, had no test material-related clinical signs, and 

had no test material-related findings at necropsy. 

 

When proteins are toxic, they are known to act via acute mechanisms and at very low dose levels 

(Sjoblad, Roy D., et al. 1992). Therefore, since no effects were shown to be caused by the plantincorporated protectants, even at relatively high dose levels, the mCry3A protein is not considered 

toxic. Amino acid sequence comparisons showed no similarity between the mCry3A protein and 

known toxic proteins available in public protein data bases (MRID 461556-11, reviewed in Fellman, 

2005a). According to the Codex Alimentarius guidelines, the assessment of potential toxicity also 

includes stability to heat (FAO/WHO Standards Programme, 2001). Further data demonstrate that 

mCry3A is inactivated against WCRW, when heated to 95 C for 30 minutes (MRID 461556-08) 

Since mCry3A is a protein, allergenic sensitivities were considered. Currently, no definitive tests for 

determining the allergenic potential of novel proteins exist. Therefore, EPA uses a weight of the 
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 

 25 

evidence approach where the following factors are considered: source of the trait; amino acid sequence 

similarity with known allergens; prevalence in food; and biochemical properties of the protein, 

including in vitro digestibility in simulated gastric fluid (SGF), and glycosylation. Current scientific 

knowledge suggests that common food allergens tend to be resistant to degradation acid and proteases; 

may be glycosylated; and are present at high concentrations in the food. 

Data have been submitted that demonstrate that the mCry3A protein is rapidly degraded by gastric 

fluid in vitro (MRID 461556-07, review in Fellman, 2005a). In a solution of simulated gastric fluid 1 

mg/mL mCry3A test protein mixed with simulated gastric fluid (pH 1.2, containing 2 mg/mL NaCl, 14 

μL 6 N HC1, and 2.7 mg/mL pepsin) resulting in 10 pepsin activity units/ μg protein (complies with 

2000 US Pharmacopoeia recommendations), complete degradation of detectable mCry3A protein 

occurred within 2 minutes. A comparison of amino acid sequences of known allergens uncovered no 

evidence of any homology with mCry3A, even at the level of 8 contiguous amino acids residues 

(MRID 461556-12). Further data demonstrate that mCry3A is not glycosylated and is present in low 

levels in corn tissue. 

EPA also asked the March 14-15, 2006 FIFRA SAP to comment on EPA’s conclusions regarding the 

lack of mammalian toxicity and allergenicity of the mCry3A protein- specifically the impact of the less 

potent mCry3A form on the results of the acute oral toxicity tests and the usefulness of in vitro 

digestibility studies and amino acid sequence homology analysis as part of the risk assessment (SAP, 

2006 and reviewed in Fellman, 2006c). Overall, the Panel was more concerned with the quality of 

data, i.e. inadequately described methods and poor reproduction of data images. The Panel specifically 

noted that the amino acid sequence analysis to known toxins were missing the following data: a full, 

technical description of each specific toxin sequence compared to the mCry3A amino acid sequence in 

the NCBI database; specification of which version of NCBI database was utilized; descriptions of 

parameters utilized; and dates accessed for the BLAST search. EPA recognizes that these are 

important parameters to include in a description of an amino acid analysis, however, the parameters 

used and dates of the BLAST search were provided in the reference list and in a footnote, respectively, 

in the toxin homology report by the registrant. EPA is requiring submission of a detailed description 

of each amino acid sequence of known toxins compared to mCry3A by Syngenta Seeds, Inc. However, 

the Agency maintains that the conclusions of the amino acid sequence analysis are still valid for the 

purpose of the risk assessment. EPA reached this decision based on the following: 1) lack of 

mammalian toxicity of mCry3A protein as shown by the acute oral mouse study; 2) mCry3A protein is 

rapidly digested in SGF; 3) mCry3A protein originates from a non-allergenic source; 4) lack of 

sequence identity of mCry3A protein with 8 contiguous amino acids or more than 35% identity over 80 

amino acids with known allergens; and 5) mCry3A protein is not glycosylated when expressed in corn. 

Therefore, the potential for the mCry3A protein to be a food allergen is minimal. As noted above, toxic 

proteins typically act as acute toxins with low dose levels. Therefore, since no effects were shown to 

be caused by the plant-incorporated protectant, even at relatively high dose levels, the mCry3A protein 

is not considered toxic. 
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Biopesticide Registration Action Document (BRAD) September, 2010 

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A summary of the toxicity and allergenicity data is presented in Table 3. 

Table 3. Mammalian Toxicity and Allergenicity studies (2010 Update)

MRID Title Summary 

461556-

10 

Acute oral 

toxicity study of 

modified Cry3A 

protein 

(MCRY3A-0102) 

in the mouse 

MCRY3A-0102 was not acutely toxic to mice. There was no 

evidence of toxicity at 2,632 mg MCRY3A-0102/kg body weight, 

representing ca. 2,377 mg mCry3A protein/kg body weight. The 

estimated LD50 value for pure mCry3A protein in male and female 

mice was > 2,377 mg/kg body weight, the single dose used. 

Classification: Acceptable

461556-

07 

In vitro 

digestibility of 

modified Cry3A 

protein 

(MCRY3A-0102 

and IAPMIR604-

0103) under 

simulated 

mammalian 

gastric conditions 

The susceptibility of mCry3A protein to proteolytic degradation was 

evaluated in simulated gastric fluid (SGF) containing pepsin. 

Modified Cry3A protein from transgenic corn and recombinant E. 

coli (test material M MCRY3A -0102) was readily degraded in SGF. 

The data support a conclusion that mCry3A protein expressed in 

transgenic plants will be readily digested as conventional dietary 

protein under typical mammalian gastric conditions. 

Classification: Acceptable 

461556-

08 

Effect of 

temperature on 

the stability of 

modified Cry3A 

protein 

(MCRY3A-0102) 

At 95°C mCry3A protein was completely inactivated. At 4°C, 25°C, 

and 37° C there was little or no effect on mCry3A bioactivity, while 

at 65°C there was some reduction in the bioactivity. 

Classification: Acceptable

461556-

11 

Modified Cry3A 

protein as 

expressed in 

transgenic maize 

event MIR604: 

assessment of 

amino acid 

homology with 

known toxins 

The National Center for Biotechnology Information (NBI) GenBank 

Database (NBI, 2003) containing all publicly available protein 

sequences was queried for proteins with amino acid sequences 

having significant homology to mCry3A protein and that were 

toxins. The query found no significant amino acid homology 

between any protein toxin and the mCry3A protein. 

Classification: Acceptable 
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Biopesticide Registration Action Document (BRAD) September, 2010 

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MRID Title Summary 

461556-

12 

Modified Cry3A 

protein as 

expressed in 

transgenic maize 

event MIR604: 

assessment of 

amino acid 

homology with 

known allergens 

No significant similarity was found between any of the mCry3A 80-

amino acid peptides and any entries in the SBI Allergen Database. 

Also, there were no alignments of eight or more contiguous amino 

acids between the mCry3A protein and any of the proteins in the 

allergen database. Overall, the mCry3A protein showed no 

significant amino acid homology to any known or putative 

allergenic protein. 

Classification: Acceptable

461556-

13 

Phosphomannose 

Isomerase as 

expressed in 

transgenic maize 

event MIR604: 

assessment of 

amino acid 

homology with 

known toxins 

Two nucleotide changes were discovered in the pmi gene sequence 

inserted in corn Event MIR604. This resulted in two changes in the 

PMI protein; valine-61 was replaced by alanine, and glutamine-210 

was replaced by histidine. These substitutions have not resulted in 

any apparent functional change in the PMI protein. The NCBI 

GenBank Database containing all publicly available protein 

sequences was queried for proteins with amino acid sequences 

having significant homology to this modified PMI protein that were 

toxins. The query found no significant amino acid homology 

between any protein toxin and the PMI protein expressed in corn 

Event MIR604. 

Classification: Acceptable

464252-

01 

Phosphomannose 

Isomerase as 

expressed in 

transgenic maize 

event MIR604: 

assessment of 

amino acid 

homology with 

known allergens 

No significant similarity was found between any of the PMI 80-

amino acid peptides and any entries in the SBI Allergen Database. 

However, in the eight or more contiguous amino acids homology 

search, there was an alignment between the PMI protein and a 

recently identified allergen, α-parvalbumin from Rana species 

CH2001 (a frog of Indonesian origin). However, a serum 

screening concluded that there is no cross-reactivity between PMI 

and serum IgE (obtained from an allergic individual who displayed 

food-induce anaphylaxis from α-parvalbumin). Bovine serum 

albumin was also tested as an internal check. The observed low 

degree of sequence identity between MIR604 PMI and αparvalbumin is not biologically relevant. 

Classification: Acceptable
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Biopesticide Registration Action Document (BRAD) September, 2010 

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MRID Title Summary 

none Information 

provided in a 

letter dated July 1, 

2010, to 

supplement 

MRIDs 46155612 

and 46155611. 

In a review dated July 22, 2010, the Agency found that the letter 

provided the details of the NCBI database versions accessed, the 

parameters of the FASTA and BLASTP searches and the date of the 

searches themselves. This information supplements the risk 

assessments done for the mCry3A protein and fulfills the 

recommended additional information as reflected in the minutes of 

the FIFRA-Scientific Advisory Panel on the mCry3A corn PIP 

product. 

2. 2010 Update: Terms and Conditions of the Event MIR 604 expressing mCry3A and 

Events MIR 604 x Bt11 expressing mCry3A and Cry1Ab proteins. 

When products containing Event MIR 604 expressing mCry3A protein (EPA Reg. No. 67979-5), and 

events MIR 604 x Bt11 expressing mCry3A + Cry1Ab proteins (EPA Reg. No. 67979-8) were initially 

registered on October 03, 2006 and January 25, 2007 respectively, the Agency issued registration 

notices to Syngenta Seeds, Inc that contained the following requirement: 

“Submit the following data to augment the mCry3A amino acid sequence 

analysis to known toxins and allergens within six months of the date of 

registration: specification of which version of NCBI database was utilized; 

descriptions of parameters utilized; and dates accessed for the BLAST 

search.“ 

 

Syngenta responded to the request for additional information in a letter dated July 1, 2010. In a review 

dated July 22, 2010, the Agency found that this letter provided the details of the NCBI database 

versions accessed the parameters of the FASTA and BLASTP searches and the date of the searches 

themselves. This information supplemented the risk assessments done for the mCry3A protein and 

fulfilled the recommended additional information requested. 

3. Aggregate Exposures 

In examining aggregate exposure, section 408 of the 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 
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Biopesticide Registration Action Document (BRAD) September, 2010 

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tolerances or exemptions in effect for the plant-incorporated protectant chemical residue, and exposure 

from non-occupational sources. Exposure via the skin or inhalation is not likely since the plantincorporated protectant is contained within plant cells, which essentially eliminates these exposure 

routes or reduces these exposure routes to negligible. Exposure via residential or lawn use to infants 

and children is also not expected because the use sites for the mCry3A protein are all agricultural for 

control of insects. Oral exposure, at very low levels may occur from ingestion of processed corn 

products and, potentially, drinking water. 

However, oral toxicity testing done at a dose in excess of 2 gm/kg showed no adverse effects. 

Furthermore, the expression of the modified Cry3A protein in corn kernels has been shown to be in the 

parts per million ranges, which makes the expected dietary exposure several orders of magnitude lower 

than the amounts of mCry3A protein shown to have no toxicity. Therefore, even if negligible 

aggregate exposure should occur, the Agency concludes that such exposure would present no harm due 

to the lack of mammalian toxicity and the rapid digestibility demonstrated for the mCry3A protein. 

4. Cumulative Effects 

Pursuant to FFDCA section 408(b)(2)(D)(v), 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 protectant, we conclude that there are no cumulative 

effects for the mCry3A protein. 

5. Determination of Safety for U.S. Population, Infants and Children 

 a) Toxicity and Allergenicity Conclusions 

The data submitted and cited regarding potential health effects for the mCry3A protein include the 

characterization of the expressed mCry3A protein in corn, as well as the acute oral toxicity, heat 

stability, 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 mCry3A protein test material derived from 

microbial cultures was biochemically and functionally similar to the protein produced by the plantincorporated protectant ingredients in corn. Microbially produced protein was chosen in order to 

obtain sufficient material for testing. 

The acute oral toxicity data submitted supports the prediction that the mCry3A protein would be nontoxic to humans. As mentioned above, when proteins are toxic, they are known to act via acute 

mechanisms and at very low dose levels (Sjoblad, Roy D., et al. 1992). Since no effects were shown to 

be caused by mCry3A protein, even at relatively high dose levels (2,377 mg mCry3A/kg bwt), the 
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Biopesticide Registration Action Document (BRAD) September, 2010 

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mCry3A protein is not considered toxic. This is similar to the Agency position regarding toxicity and 

the requirement of residue data for the microbial Bacillus thuringiensis products from which this plantincorporated protectant was derived. (See 40 CFR 158.740(b)(2)(i)). Moreover, mCry3A showed no 

sequence similarity to any known toxin and was not efficacious against WCRW when inactivated by 

heat.

Protein residue chemistry data for mCry3A were not required for a human health effects assessment of 

the subject plant-incorporated protectant ingredients because of the lack of mammalian toxicity. 

However, data submitted demonstrated low levels of mCry3A in corn tissues with less than 2 

micrograms mCry3A protein/gram dry weight in kernels and less than 30 micrograms mCry3A 

protein/gram dry weight of whole corn plant. 

Since modified Cry3A is a protein, its potential allergenicity is also considered as part of the toxicity 

assessment. Data considered as part of the allergenicity assessment include that the modified Cry3A 

protein came from Bacillus thuringiensis which is not a known allergenic source, showed no sequence 

similarity to known allergens, was readily degraded by pepsin, and was not glycosylated when 

expressed in the plant. Therefore, there is a reasonable certainty that modified Cry3A protein will not 

be an allergen. 

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 mCry3A protein, 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 (DNA, RNA) which comprise genetic material encoding these 

proteins and their regulatory regions. The genetic material (DNA, RNA) necessary for the production 

of mCry3A protein has been exempted under the blanket exemption for all nucleic acids (40 CFR 

174.475). 

b) 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(bfl2)(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 
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Biopesticide Registration Action Document (BRAD) September, 2010 

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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 mCry3A protein and the genetic material necessary for their production. Thus, there 

are no threshold effects of concern and, as a result, the provision requiring an additional margin of 

safety does not apply. Further, the provisions of consumption patterns, special susceptibility, and 

cumulative effects do not apply. 

c) 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 mCry3A protein and the genetic material necessary for its 

production. This includes all anticipated dietary exposures and all other exposures for which there is 

reliable information. The Agency has arrived at this conclusion because, as previously discussed, no 

toxicity to mammals has been observed, nor any indication of allergenicity potential for the plantincorporated protectant. 

6. Other Considerations 

a) Endocrine Disruptors 

2010 Updated Position. As required under FFDCA section 408(p), EPA 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 EPA 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, EPA 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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Biopesticide Registration Action Document (BRAD) September, 2010 

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Modified Cry3A is not among the group of 58 pesticide active ingredients on the initial list to be 

screened under the EDSP. Under FFDCA § 408(p) the Agency must screen all pesticide chemicals. 

Accordingly, EPA anticipates issuing future EDSP orders/data call-ins for all Registration Review 

cases, including those for which EPA has already opened a Registration Review docket for a pesticide 

active ingredient. 

For further information on the status of the EDSP, the policies and procedures, the list of 67 chemicals, 

the test guidelines and the Tier 1 screening battery, please visit our website: 

http://www.epa.gov/endo/. 

b) Analytical Method(s) 

A method for extraction and ELISA analysis of mCry3A protein in corn was submitted and found 

acceptable by the Agency. 

2010 Updated Position. More recently, the Agency decided to allow this requirement to be satisfied 

when the Grain Inspection, Packers and Stockyards Administration (GIPSA) of the United States 

Department of Agriculture (USDA) has verified the performance of a qualitative rapid test kit for 

detecting the presence of the biotechnology event in grains and oilseeds. In the case of mCry3A, the 

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

requirement has been satisfied. 

c) Codex Maximum Residue Level 

No Codex maximum residue levels exist for the plant-incorporated protectant Bacillus thuringiensis 

mCry3A protein and the genetic material necessary for its production in corn. 

7. Tolerance Exemptions 

The data submitted and reviewed for Modified Cry3A support the petition for an exemption from the 

requirement of tolerance for Bacillus thuringiensis mCry3A protein and the genetic material necessary 

for its production in corn. 

An exemption from tolerance was established for residues of mCry3Aprotein when used as plantincorporated protectant in the food and feed commodities corn, field corn, sweet corn and popcorn. (40 

CFR 174.505) 
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Biopesticide Registration Action Document (BRAD) September, 2010 

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References 

Fellman, A., 2005a. EPA Review of Product Characterization and Expression Analysis for Modified 

Cry3A (mCry3A) Bacillus thuringiensis insect control protein and maize (corn) plants Derived 

from Event MIR604 in support for a temporary exemption from tolerances and an 

Experimental Use Permit (EUP) Application. Memorandum dated February 11, 2005. 

Fellman, A. 2005b. EPA Addendum to Product Characterization and Expression Analysis for Modified 

Cry3A (mCry3A) B.t. insect control protein and maize (corn) plants Derived from Event 

MIR604. Memorandum dated February 11, 2005. 

Fellman, A., 2005c. EPA Addendum No. 2 to Product Characterization and Expression Analysis for 

Modified Cry3A (mCry3A) B.t. insect control protein and maize (corn) plants derived from 

Event MIR604. Memorandum dated February 23, 2005. 

Fellman, A., 2005d. EPA Evaluation of Phosphomannose isomerase protein (PMI) as expressed in 

Transgenic maize Event MIR604 with E. coli-derived PMI protein in support for a temporary 

tolerance exemption for Modified Cry3A B.t. insecticidal protein and the genetic material 

necessary for its production in corn plants derived from Event MIR604. Memorandum dated 

March 3, 2005. 

Fellman, A., 2005e. EPA Review of Product Characterization and Expression Analysis for Modified 

Cry3A B.t. insect control protein and maize plants derived from Event MIR604 in support for 

an exemption from the requirement of tolerance and Sec. 3 Registration. Memorandum dated 

May 31, 2005. 

Fellman, A., 2006a. EPA Review of Product Characterization and Human Health Data for Modified 

Cry3A B.t. insect control protein and maize plants derived from Event MIR504 in support for a 

an exemption from the requirement of tolerance and Sec. 3 Registration. Memorandum dated 

February 15, 2006. 

Fellman, A., 2006b. EPA Review of Application to Extend/Amend the Experimental Use Permit and 

existing temporary exemption from the requirement of tolerance for Modified Cry3A B.t. insect 

control protein as expressed in Event MIR604 corn plants. Memorandum dated February 24, 

2006. 

Fellman, A., 2006c. EPA Evaluation of FIFRA SAP meeting minutes dated June 1, 2006 from March 

14 – 15, 2006 meeting on scientific issues associated with the Product Characterization and 

Human Health Assessment for Modified Cry3A B.t. insect control Protein and maize plants 

derived from Event MIR604. Memorandum dated August 31, 2004. 

 
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Biopesticide Registration Action Document (BRAD) September, 2010 

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Food and Agriculture Organization of the United Nations and World Health Organization, 2003. Foods 

Derived from Biotechnology. Codex Alimentarius. Sec. 4, No.38, pg.16. 

Kough, J., 2004. EPA Review of Characterization and Safety of Phosphomannose Isomerase (PMI), a 

selectable marker expressed in Event 3243M-derived maize plants. Memorandum dated 

January 30, 2004. 

Kough, J., no date. EPA Review of Southern Blot data for Bt11 X MIR604 Corn from Syngenta 

Seeds, Inc. Memorandum to M. Mendelsohn. 

Kough, J., EPA Review of Specifics of the Bioinformatics Analyses for the mCry3A PIP Protein 

Expressed in Corn from Syngenta Seeds, Inc. Memorandum to M. Mendelsohn dated July 22, 

2010. 

Scientific Advisory Panel (SAP), 2006. Minutes of the FIFRA Scientific Advisory Panel Meeting 

Held March 14 – 15, 2006 on Event MIR604 Modified Cry3A Protein B.t. corn- Plant - 

Incorporated Protectant, Report dated June 1, 2006 (SAP Report No. 2006-02). 

Sjoblad, R.D., J.T. McClintock and R. Engler, 1992. Toxicological considerations for protein 

components of biological pesticide products. Regulatory Toxicol. Pharmacol. 15: 3-9. 

USDA. 2004. Performance verification of qualitative mycotoxin and biotech rapid test kits (Directive 

9181.2). Available from: 

http://www.gipsa.usda.gov/GIPSA/documents/GIPSA_Documents/9181-2.pdf. 

Waggoner, A., and J. Kough, 2007. Review of Product Characterization, Expression Analysis and 

Human Health Data for Plant-Incorporated Protectant Bacillus thuringiensis Cty1Ab and 

mCry3A insect control proteins and the genetic material necessary for their production in 

Maize (Corn) Plants as expressed in Event Bt11 x MIR 604 (EPA Reg. No. 67979-I) in support 

for Sec. 3 Registration, submitted by Syngenta Seeds, Inc. – Field Crops – NAFTA. 

Memorandum to M. Mendelsohn, dated Jan 10, 2007. 

II.C. Environmental Assessment 

Background 

Syngenta Ltd. has requested a registration for Bacillus thuringiensis mCry3A protein and the genetic 

material, which includes the PMI inert marker gene, necessary for its production in all corn lines and 

varieties. This protein is intended to control 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 
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Biopesticide Registration Action Document (BRAD) September, 2010 

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(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. 

The EPA has conducted an environmental risk assessment of mCry3A when expressed in corn. 

General topics covered in this assessment include effects on wildlife, gene flow to related wild plants 

and its potential effects, and fate of mCry3A protein in the environment. This assessment is based on 

data submitted to EPA during the development of Event MIR604 corn lines, additional data submitted 

for registration, Federal Insecticide Fungicide and Rodenticide Act (FIFRA) Scientific Advisory Panel 

(SAP) recommendations, consultations with scientific experts, and public comments on PlantIncorporated Protectant (PIP) regulation. 

A. Environmental Hazard Assessment 

I. The Hazard Assessment Process 

1. Tiered Testing Hazard Assessment 

To minimize data requirements and avoid unnecessary testing, 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, 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. 

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 (NTO) 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 highertier testing. Less than 50% mortality under these conditions of extreme exposure suggest that 

population effects are likely to be negligible given realistic field exposure scenarios. 

The EPA uses a tiered (Tiers I-IV) testing system to assess the toxicity of a Cry protein (Bt endotoxin) 

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 
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Biopesticide Registration Action Document (BRAD) September, 2010 

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test substance on the test (non-target) organism. The screening tests evaluate single species in a 

laboratory setting with mortality as the end point. Tiers II – IV generally encompass definitive hazard 

level determinations, longer term greenhouse or field testing, and are implemented when unacceptable 

effects are seen at the Tier I screening level. 

Testing methods which utilize the tiered approach were last published by the EPA as Harmonized 

OPPTS Testing Guidelines, Series 850 and 885 (EPA 712-C-96-280, February 1996). These 

guidelines, as defined in 40 CFR 152.20, apply to microbes and microbial toxins when used as 

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

natural selection or by deliberate genetic manipulation. Therefore Bt Cry protein in corn, being a 

microbial toxin, is also covered by these testing guidelines. 

The Tier I screening maximum hazard dose approach to environmental hazard assessment is based on 

some factor (whenever possible >10) times the maximum amount of active ingredient expected to be 

available to terrestrial and aquatic non-target organisms in the environment (EEC). 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 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 (MHD). The Guidelines call for 

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

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

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

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

observed for at least 30 days after dosing, or in cases where an insect species cannot be cultured for 30 

days, until negative control mortality rises above 20 percent. 

Failing Tier I screening does not necessarily indicate the presence of an unacceptable risk in the field 

but it triggers the need for additional testing.a

 A less than 50% mortality effect at the MHD is taken to 

indicate minimal risk. However, greater than 50% mortality does not necessarily indicate the existence 

of unacceptable risk in the field, but it does trigger the need to collect additional dose-response 

information and a refinement of the exposure estimation before deciding if the risk is acceptable or 

unacceptable. Where potential hazards are detected in Tier I testing (i.e. mortality is greater than 

50%), additional information at lower test doses is required which can serve to confirm whether any 

effect might still be detected at more realistic field (1X EEC)concentrations and routes of exposure. 

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

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

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

 

a

 It is notable that that the 10 X EEC MHD testing approach is not equivalent to what is commonly known as “testing at a 

10X SAFETY FACTOR” where any adverse effect at 10X must be considered significant. 
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Biopesticide Registration Action Document (BRAD) September, 2010 

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

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

inhibition, weight loss, etc. In the final analysis, a risk assessment is made by comparing the LOAEC 

to the EEC; when the EEC is lower than the LOAEC, a no risk conclusion is made. Appropriate 

statistical methods, and appropriate statistical power, must be employed to evaluate the data from the 

definitive tests. These tests offer greater environmental realism, but they may have lower statistical 

power. Higher levels of replication, test species numbers 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

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

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

is a highly conservative factor. The published EPA Level of Concern (LOC) is 50% mortality at 5X 

EEC (see footnotes 5 and 6). 

The tiered hazard assessment approach was developed for the EPA by the American Institute of 

Biological Sciences and confirmed, in 1996, as an acceptable method of environmental hazard 

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

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

Incorporated Protectants (PIP); however, this panel recommended that, for PIPs with insecticidal 

properties, additional testing of beneficial invertebrates closely related to target species and/or likely to 

be present in GM crop fields should be conducted. Testing of Bt Cry proteins on species not closely 

related to the target insect pest was not recommended, although it is still performed to fulfill the 

published EPA non-target species data requirements [40 CFR § 158.740 (d)]. In October 2000, another 

SAP also recommended that field testing should be used to evaluate population-level effects on nontarget 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.b

 

 

b

 EPA-SAP. February 4, 2000. Characterization and non-target organism data requirements for protein plantpesticides. SAP report No. 99-06A for FIFRA Scientific Advisory Panel Meeting held December 8, 1999, held at the 

Sheraton Crystal City Hotel, Arlington, VA. 

EPA-SAP. March 12, 2001. Bt plant-pesticides risk benefit assessments. SAP report No. 2000-07 for FIFRA 

Scientific Advisory Panel Meeting held October 18-20 at the Marriott Crystal City Hotel, Arlington, VA. 

EPA-SAP. November 6, 2002. Corn rootworm plant-incorporated protectant insect resistance management and 

non-target insect issues. Transmittal of meeting minutes of the FIFRA Scientific Advisory Panel Meeting held August 27-

29 at the Marriott Crystal City Hotel, Arlington, VA. 

EPA-SAP. August 19, 2004. Product characterization, human health risk, ecological risk, and insect resistance 

management for Bacillus thuringiensis (Bt) cotton products. Transmittal of meeting minutes of the FIFRA Scientific 

Advisory Panel Meeting held June 8-10 at the Holiday Inn Ballston, Arlington, VA. 

 
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 

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The tiered approach to test guidelines ensures, to the greatest extent possible, that the Agency requires 

the minimum amount of data needed to make scientifically sound regulatory decisions. The EPA 

believes that maximum hazard dose Tier I testing presents a reasonable approach for evaluating 

hazards related to the use of biological pesticides and for identifying negative results with a high 

degree of confidence. The Agency expects that Tier 1 testing for short-term hazard assessment will be 

sufficient for most studies submitted in support of plant-incorporated Bt Cry protein registrations. 

However, if long range adverse effects must be ascertained, then higher-tier longer-term field testing 

will be required. As noted above, the October 2000 SAP and the National Academy of Sciences (NAS 

2000) recommended direct field testing to ascertain long range environmental effects. 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. 

Since delayed adverse effects and/or accumulation of toxins through the food chain are not expected to 

result from exposure to Bt Cry proteins, these protein toxins are not routinely tested for chronic effects 

on non-target organisms. The 30 day test duration requirement does, however, amount to subchronic 

testing when performed at field exposure test doses. Bacillus thuringiensis Cry endotoxins are proteins 

and proteins do not bioaccumulate. The biological nature of protein makes Bt Cry toxins readily 

susceptible to metabolic, microbial, and abiotic degradation once they are ingested or excreted into the 

environment. Although there are reports that Cry proteins bind to soil particles, it has also been shown 

that these proteins are degraded rapidly by microbes upon elution from soil. The same sources also 

report that Bt proteins present in soil collected from Bt corn fields have no detectable adverse effect on 

soil invertebrates or culturable microbial flora. 

2. MIR604 Scientific Advisory Panel Comments 

The FIFRA Scientific Advisory Panel (SAP) Meeting Held March 14 - 15, 2006 on Event MIR604 

Modified Cry3A Protein Bt Corn had several panel members specifically assigned to evaluate the 

Agency’s Environmental Risk Assessment. (These) “Panel members expressed a diversity of opinions 

concerning the adequacy of the Agency’s analysis of the ecological studies submitted by the 

Registrant. These opinions ranged from nearly unqualified acceptance to qualified rejection.” (p. 9, 

SAP Minutes). 

The panel members who understood the Agency’s concept of maximum hazard dose screening (limit) 

testing (outlined in Section A.I.1 above) concluded that ... “If one takes the NTO (Non-Target 

Organism) toxicity assays of this Tier I hazard assessment at face value, none of them reported high 

acute toxicity. Rather, in most instances mortality was low or did not occur at the ETCs (Estimated 

Test Concentrations) achieved. Although the ETCs frequently did not meet the standard for maximum 

hazard dose testing, these results do support the conclusion of the absence of a strong, toxic response 

in the NTOs.” (p. 19, SAP Minutes). 
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Biopesticide Registration Action Document (BRAD) September, 2010 

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In reality, the absence of a strong (50%) mortality response at 5X the field concentration is the Agency 

standard (endpoint) for maximum hazard dose Tier I screening testsc (see Section A.I.1 above). The 

results of the testing performed for MIR604 were far below this published Agency standard (Level of 

Concern) for maximum hazard dose testing. There is an absence of 50% mortality in all of the nontarget tests performed, and no mortality at all attributable to the test substance was noted. 

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

field exposure scenarios if the earlier tiered tests fail to indicate an adequate certainty of acceptable 

risk (i.e. show greater than 50% mortality of the test species). Many of the current Panel comments 

were made as if field exposure scenario testing was being reviewed, when in fact the worst case 

(screening) scenarios did not trigger a need for field exposure rate (sublethal/definitive) testing. 

Therefore many of the Panel’s comments are superfluous to the established EPA hazard assessment 

process which is based on the results of screening tests (when these show less than 50% mortality of 

the test species). 

The EPA risk assessment is centered only on adverse effects at the field exposure rates (1X EEC), and 

not on adverse effects at greater concentrations. “The Panel was split on the importance of the 

Registrant not adhering to the 10X (EEC test dose) standard. Several Panel members considered this a 

serious deficiency but a deficiency that easily could have been avoided. Other Panel members 

considered the test concentrations/doses adequate to support the Agency’s finding of no likely adverse 

ecological effects.” (p. 16 SAP Minutes)d

 The dose margin can be less than 10x where uncertainty in 

the system is low or where high concentrations of test material are not possible to achieve due to test 

organism feeding habits. High dose testing also may not be necessary where many species are tested 

or tests are very sensitive, although the concentration used must exceed 1X EEC. 

The purpose of convening the SAP was to comment on the Agency’s conclusion that the MIR604 corn 

would not cause unreasonable and/or irreversible adverse effects to the environment as determined by 

 

c

 See footnotes 5& 6 

d

 It is notable those panel members who perceived any non-adherence to the 10X standard as a serious deficiency did so as 

a result of an impression that all testing must be done at a 10X “safety” factor. In reality the 10 X EEC screen testing 

approach is not equivalent to what is commonly known as “testing at a 10X SAFETY FACTOR” where any adverse effect 

at 10X must be considered significant. In addition, those panel members who perceived non-adherence to the 10X standard 

did so as a result of an impression that the purified, bacterially produced Cry protein test substance had half of the activity

of the plant produced Cry protein. This impression may not be justified because the intrinsically variable insect bioassay 

results which appear to show a two fold difference in activity were in fact within the range of variability for insect 

bioassays. In addition, in the event that the test concentration was at half of the plant Cry protein activity level, the test

concentrations are still at or above the EPA Level of Concern (5X EEC) and in all cases above the 1X field exposure rate 

(EEC) at which the assessment of adverse ecological effects is made. The 1X EEC (based on plant tissue content of Cry 

protein) is in turn still much greater than any amount which any given non-target organism may be actually ingesting in the 

field. 
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 

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existing EPA risk assessment SOPs. As noted above, some panel members responded positively to 

this charge, whereas others chose to comment on the EPA testing and risk assessment SOPs. The 

MIR604 SAP was not asked to revise the EPA pesticide program's assessment system which has been 

developed through many years of evaluation both within the government and through outside experts. 

The methods have already undergone several layers of SAP, public, peer and EPA Science Advisory 

Board review and have been found to be adequate.e

 (See also Section A.I.1 above and footnote 2) 

In addition to not commenting on the adequacy of the Agency’s risk conclusions at field exposure rates 

(i.e. 1X EEC), some panel members were not prepared to accept the concept of screening (limit) 

testing. Some viewed the high dose screen tests designed to detect LD50’s as if these were definitive 

determinations where any effect (at a limit test dose) is also a level of concern at field exposure 

concentrations. Tier I screening tests were also referred to as 10X “safety factor” tests by some Panel 

members (p.9, SAP Minutes). Tier I screen testing is not ‘safety factor testing’. In a “10X safety 

factor” test any adverse effect noted is a “level of concern”f

, whereas in the EPA risk assessment 

scenario any adverse effect is viewed as a concern only at 1X the field exposure. As a result the panel 

comments on the testing procedures were not addressing screening methods but the definitive (1X) 

testing procedures that are triggered only when the screening results show greater than 50% 

mortality. This level of toxicity was not seen in any screening test performed for mCry3A protein. 

Therefore definitive testing (at 1X EEC) was not necessary and any comments made by the panel 

applicable only to the definitive (1X) testing methods are superfluous.g In addition, the EPA 

Guidelines require three replicates of 10 test animals, or one replicate of 30 for the tier I screening 

tests, therefore the SAP comments on insufficient replication and numbers of test species are also 

superfluous. 

However, in principle the Agency is in agreement with these SAP comments and the OPPTS Testing 

Guidelines are already implementing them as much as they apply to definitive testing procedures (See 

Section A.I.1 above). The SAP methodological comments do not, however, apply to Tier I screening 

tests which in this case did not trigger additional, definitive testing for MIR604 mCry1A proteins 

There was no effort by some panel members to distinguish 'screening testing' (which is performed to 

 

e

 Environmental Protection Agency (USEPA) (1998). “Guidelines for Ecological Risk 

Assessment.” EPA 630/R-95-002F. Washington, DC, USA. (Federal Register, May 14, 1998. 63(93): 26846-26924.] 

f

 The established (see footnote 5 above), peer and USEPA Science Advisory Board reviewed guidance on screening test 

levels of concern is 50% mortality at 5X environmental concentration. The appropriate endpoints in high dose 

limit/screening testing are based on mortality of the treated, as compared to the untreated (control) non-target organisms. A 

single group of 30 test animals may be tested at the maximum hazard dose. 

 

g

 The 1X EEC test dose is based on plant tissue content and is considered a high worst case dose of the Cry protein. This 

1X EEC is in turn still much greater than any amount which any given non-target organism may be ingesting in the field. 

Therefore the actual exposure scenario would have to be further refined in the event that adverse effects are noted at the 

(1X) EEC based on plant tissue analysis. 
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 

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determine intrinsic toxicity of an insecticidal protein) from ‘definitive testing’ triggered by positive 

screen test results and which is much more complicated and uses more realistic environmental 

exposure rates. 

A tiered risk assessment as performed by the Agency is recognized as being the most appropriate and 

rigorous approach to assess non-target affects from both scientific and regulatory standpoints. This 

process is designed to optimize the use of resources and to identify and define sources of potential risk. 

Where no unreasonable hazard is detected at Tier I limit (screen/high dose) testing, costly and 

unnecessary testing at field exposure rates is prevented from taking place. Both hazard and exposure 

can be evaluated within different levels or “tiers” that progress from worst-case hazard and exposure to 

more realistic scenarios. Lower tier tests serve to identify potential hazards, and they are conducted in 

the laboratory at high dose levels which increase the statistical power to test the hypotheses and 

therefore do not require high replication or large numbers of test species. Where potential hazards are 

detected in Tier I testing (i.e. test species mortality is greater than 50%), additional information at 

lower test doses is required which can serve to confirm whether any effect might still be detected at 

more realistic field (1X EEC) concentrations and routes of exposure. The confirmatory tests offer 

greater environmental realism, but they may have lower statistical power. Higher levels of replication 

or repetition are needed to enhance statistical power in these circumstances. Tier I high dose/screening 

tests do not require multiple replication and repetition as was recommended by some panel members. 

In addition, screening tests without multiple replications are of publication quality, and numerous 

screening rests are published in peer reviewed literature. 

Some panel members also listed deficiencies in the testing procedures of non-target wildlife other than 

insects. The precision of some assay methods was also criticized. As discussed above, these comments 

are valid, but they do not apply to screening testing, and additionally, testing of wildlife other than 

insects related to the target pest is not essential for an environmental risk assessment of mCry3A 

proteins. This fact was recognized by some members of the SAP who state that: “Cry3A is a protein 

endotoxin produced naturally by the tenebrionis strain of Bacillus thuringiensis subsp. morrisoni (H 

8a8b) that upon ingestion and proteolytic activation exhibits a high level of insecticidal activity to 

certain species of coleopteran insects, but otherwise is apparently non-toxic to other types of insects, as 

well as vertebrates” (p.29, SAP minutes). Therefore any deficiencies as noted by the panel on testing 

of non-target organisms other than insects would not have had an adverse effect on the environmental 

risk assessment of mCry3A protein in corn because they were (a) superfluous tests for Bt Cry proteins 

and (b) none of the screening tests showed unacceptable effects according to the EPA risk assessment 

guidelines. 

In summary, some panelists agreed with the Agency’s risk assessment. Other panelists recommended a 

variety of refinements to field exposure rate testing protocols which do not apply to the high dose 

screening tests performed for MIR604 corn and therefore do not change the “no apparent 

environmental risk” assessment for MIR604 corn. The Panel’s critical comments are valid for field 

exposure dose definitive testing which was not triggered by the screening rests performed with 
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 

 42 

mCry3A protein. However, the Panel’s comments and recommendations are being reviewed for 

incorporation into the Agency’s definitive testing protocols as may be necessary. 

The following comments address additional observations found in the SAP Minutes. 

A no “may effect” finding was made by the Agency to the endangered Hungerford’s crawling water 

beetle due to low aquatic exposure from corn, a terrestrial crop. In addition, the submitted studies with 

mCry3A protein did not show any adverse effects at terrestrial field use rates to non-target insect 

species most closely related to the endangered aquatic beetle. The EPA Environmental Risk 

Assessment for MIR604 states: “There is no evidence for sensitivity of aquatic (including endangered) 

species to anti-coleopteran Bt Cry proteins. Furthermore, aquatic exposure to mCry3A is extremely 

small or non-existent since mCry3A is not expressed in MIR604 pollen.” and: “... Since mCry3A 

protein has not been shown to have toxicity effects on ... aquatic species, insects and other invertebrate 

species at the EEC, a ‘may affect’ situation for endangered land and aquatic species is not 

anticipated.”; and “...Further, several of the federally listed insect species are aquatic and 

consequently, are unlikely to come in contact with MIR604 maize plant material.” Therefore it is the 

Agency’s position that the MIR604 Risk Assessment has adequately addressed the endangered aquatic 

beetle species. 

Contrary to the perceptions of some panel members, the testing performed did meet, and in many cases 

actually exceeded the Agency’s published Tier I testing standards. Where the 10X standard was not 

met it was due to insect biology (feeding habit) factors that did not permit exposure to 10X EEC 

concentrations. 

The large variability in re-isolation of Cry protein in test soils or test diets is inherent in such testing, 

especially when insect bioassays are used. It is not realistic to expect precision or accuracy in such 

assays. For that reason the Agency relies on these tests primarily as a measure that the test material 

was actually added to, and is present in the test systems. 

The determination of a slope (as was done in the soil degradation study) from experimental data points 

is a standard procedure in OPP and is not considered inadequate. 

The maturation rates (weight gain) of male and female birds are naturally different in some avian 

species and therefore the Agency does not consider the different weight gain rates to be an effect of 

Cry protein ingestion. 

The death of one test animal (fish or bird) in a screening high test dose study (where 50% mortality is 

the end point) is not considered by the Agency to be an indication of hazard at real world field 

exposure rates. 

Criticism of the use of agar pour plates is not considered a critical issue by the Agency. Agar pour 

plates have been used for more than 50 years to enumerate microbial and viral particles, and the 50o

C 
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 

 43 

temperature used to prepare these plates does not affect the function of (protein) enzymes or viability 

of bacteria or virus particles. Therefore there is no reason to expect that the use of the pour plate 

method to prepare insect diets will have adverse effects on the functionality of Cry proteins. 

3. 2010 Update: Terms and Conditions of the Event MIR 604 expressing modified Cry3A 

and Events MIR 604 x Bt11 expressing the proteins modified Cry3A and Cry1Ab. 

When Event MIR 604 expressing mCry3A Corn (EPA Reg. No. 67979-5) and events MIR 604 x Bt11 

expressing mCry3A and Cry1Ab (EPA Reg. No. 67979-8) were initially registered on October 03, 

2006 and January 25, 2007 respectively, the Agency issued registration notices to Syngenta Seeds, Inc 

that contained the following requirement for further Environmental Assessment information: 

“Submit field degradation studies evaluating accumulation and persistence of 

mCry3A in several different soils in various strata. Representative fields must have 

been planted with mCry3A corn and include both conventional tillage and no-till 

samples and be harvested under typical agronomic conditions. Sampling must 

continue until the limit of detection is 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 mCry3A is accumulating or persisting in 

soil samples. A protocol is due within 90 days of the date of registration. Should the 

registration expiration date be extended, a final report regarding data from fields 

that have had three continuous years of cultivation of Event MIR604 corn is due by 

January 31, 2011.” 

In response to this requirement, Syngenta Seeds, Inc. – Field Crops – NAFTA submitted a request to 

waive the conditionally-required soil persistence field study for the plant-incorporated protectant (PIP) 

Bacillus thuringiensis (Bt.) mCry3A insecticidal protein and the genetic material necessary for its 

production in Event MIR604 corn [EPA Reg. No. 67979-5] in support of the Section 3 registration. 

Soil microorganism exposure to Bt proteins can occur through contact with corn plant roots (by direct 

feeding), corn plant exudates, incorporation of above-ground plant tissues into soil following harvest, 

or by soil-deposited pollen. Scientists have generated data on Bt proteins in crop residues and their 

persistence in soil for long periods of time following exposure to a wide range of environmental field 

conditions. 

The results of these studies, summarized in Table 4, show that there is no detectable Cry protein 

accumulation in agricultural soils during commercial planting of currently registered Cry proteinproducing crops (Icoz and Stotzky, 2007; Sanvido, et. al, 2007). Likewise, no unexpected 

accumulation of Cry proteins has been seen in numerous studies submitted directly to EPA for 

currently registered Cry proteins. There are various published studies that measured Bt protein 

degradation in a laboratory setting relating the dissipation time of the protein to bioactivity (DT50); 

data predict that Cry proteins in the field degrade rapidly and do not persist or accumulate in the soil to 
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 

 44 

any extent that would affect nontarget organisms (NTOs). The DT50 (based on a simple first-order 

kinetic model) for mCry3A in an artificial soil environment was 7.6 days (MRID No. 462656-14). The 

results of this study demonstrate a similar rate of degradation to other Cry proteins in artificial soil 

systems (U.S. EPA, 2001) and, along with the literature cited below, indicate that mCry3A is likely to 

have a similar field degradation profile to other Bt proteins. In addition, data already exist to support 

the lack of effects of the mCry3A protein on NTOs (U.S. EPA, 2006; Raybould et al., 2007), which is 

the primary focus when considering soil fate data. 

 II. Non-Target Wildlife Hazard Assessment

Two separate SAP reports (October 2000 and August 2002) recommended that non-target testing of Bt

Cry proteins should focus on invertebrate species exposed to the crop being registered. Following SAP 

recommendations, the EPA determined that non-target organisms with the greatest exposure potential 

to Cry protein in transgenic corn fields are beneficial insects, which feed on corn pollen and nectar, 

and soil invertebrates, particularly Coleoptera species. Therefore, maximum hazard dose toxicity 

testing on representative beneficial organisms from several taxa was performed in support of this 

Section 3 FIFRA registration. The toxicity of the mCry3A protein has been evaluated on several 

species of invertebrates including the lady beetle, Carabid beetle, rove beetle, flower bug, honey bee, 

and earthworm. Reproductive and developmental observations were also made in the lady beetle, rove 

beetle and honeybee studies. 

Although the mCry3A protein is known to be very host specific, conferring toxic effects on corn 

rootworm, Colorado potato beetle, and closely related species, and despite the October 2000 and 

August 2002 SAP’s recommendations against testing of non-target species not related to susceptible 

target pests, EPA has done a risk assessment on a range of non-target wildlife to comply with the 

Agency’s published non-target data requirements (in the absence of PIP-specific risk assessment 

guidance, EPA requires applicants for PIP registrations to meet the 40 CFR Part 158 data requirements 

for microbial toxins). These requirements include birds, mammals, plants and aquatic species. In 

addition, an earthworm study was voluntarily submitted to the Agency to ascertain the potential effects 

of mCry3A on beneficial decomposer species. 

Test substances (i.e. source of mCry3A protein) used for studies submitted in support of the mCry3A 

registration included bacterially-produced purified mCry3A protein (referred to as mCry3A-0102) and 

corn grain. The October 2000 SAP recommended that while actual plant material is the preferred test 

material, bacterially-derived protein is also a valid test substance, particularly in scenarios where test 

animals do not normally consume corn plant tissue and where large amounts of Cry protein (Cry 

protein concentrations that exceed levels present in plant tissue) are needed for maximum hazard dose 

testing. An insect feeding study, which compared the relative potency of plant produced mCry3A 

protein to microbe produced mCry3A-0102, indicated that plant produced protein was twice as toxic as 

microbe produced protein (see Section A.II.1.d.vii). However, since exposure to mCry3A-0102 was at 

least several times the EEC for all submitted non-target studies, EPA determined that exposure to 

mCry3A protein was adequate despite the lower potency of microbe produced mCry3A-0102. In 
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 

 45 

accordance with OPPTS Harmonized Testing Guidelines, adult insect studies were generally 

conducted for 30 days or until mortality in the negative control reached, or exceeded, 20% and larval 

studies were carried out through pupation and adult emergence. 

The results of ecological effects studies submitted in support of the MIR604 Section 3 FIFRA 

registration are summarized in Table 4 and presented in a more descriptive format in subsequent 

sections of this risk assessment document. Full reviews of each study can be found in the individual 

Data Evaluation Reports (DERs) and accompanying memos. 

Table 4. Summary of environmental effects studies and waiver justifications submitted to 

comply with data requirements published in 40 CFR § 158.740 (d). (Update 2010).

Guideline Study Results MRID

885.4150 Wild 

Mammal 

Testing, Tier 

I 

Mammalian wildlife exposure to mCry3A protein is 

considered likely; however, mCry3A-0102 toxicity data 

indicate that, when tested at the maximum hazard dose 

level, there was no significant toxicity to rodents. 

Therefore no hazard to mammalian wildlife is anticipated 

and data on wild mammal testing is not required. 

N/A 

850.2100 Avian Acute 

Oral Toxicity 

Test, Tier 1 

(Northern 

Bobwhite 

quail, Colinus 

virginianus) 

Young adult northern bobwhite quail were administered a 

single nominal oral dose of 722 mg mCry3A-0102/kg 

body wt and observed for 14 days. There were no 

treatment-related adverse clinical signs or mortality. 

Body weight and feed consumption of the test birds were 

comparable to those of the controls. Classification: 

Acceptable 

461556-16 

885.4050 Avian Oral, 

Tier 1 

(Broiler, 

Gallus 

domesticus) 

In a 49-day feeding study, commercial broiler chickens 

were fed formulated diets containing one of the following 

ingredients: MIR604 corn (contained mCry3A protein); 

an MIR604 isoline (no mCry3A protein); or a nontransgenic commercial corn hybrid (no mCry3A protein). 

No adverse clinical signs were noted, and carcass yield 

and mortality were not significantly different among 

treatment groups. Classification: Acceptable 

462656-15 
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Biopesticide Registration Action Document (BRAD) September, 2010 

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Guideline Study Results MRID

885.4200 

Freshwater 

Fish Testing, 

Tier 1 

(Rainbow 

trout, 

Onchorhynch

us mykiss) 

In a 28-day toxicity study, granular fish feed containing 

50% by weight Event MIR604 corn grain did not produce 

statistically significant mortality or sublethal effects 

when fed twice daily to juvenile rainbow trout. 

Classification: Acceptable 

461556-17 

462656-02 

885.4280 

Estuarine and 

Marine 

Animal 

Testing, Tier 

I 

Estuarine and marine animal studies are not required for 

this product, because mCry3A is not intended for direct 

application to estuarine or marine environments and there 

is very low potential that these ecosystems will be 

exposed to mCry3A protein in field corn. 

N/A 

885.4300 

Nontarget 

Plant Studies, 

Tier I 

The active ingredient is an insect toxin (Bt endotoxin) 

that is non-toxic to aquatic and terrestrial plants. 

Consequently, non-target plant studies have been waived 

for this product. 

N/A 

885.4340 

Nontarget 

Insect 

Testing, Tier 

1 (Lady 

beetle, 

Coccinella 

septempuncta

ta) 

Lady beetle larvae were fed live pea aphids that were 

dipped in a solution containing: 50μg mCry3A-0102/mL 

Agral 90 solution (a non-ionic surfactant); Agral 90 

solution only (negative control); or 0.5 mL of Nemolt 

(teflubenzuron)/L Agral 90 solution (positive control). 

The rate of pupal development was not significantly 

different between the negative control and mCry3A-0102 

treatments. However, the number of days to adult 

emergence was significantly lower in the mCry3A-0102 

treatment. Classification: Acceptable 

462656-03 

462656-04 

885.4340 

Nontarget 

Insect 

Testing, Tier 

1 (Carabid 

beetle, 

Poecilus 

cupreus) 

Twenty-four to 48 hour-old Carabid beetle larvae were 

fed daily until pupation with blowfly pupae that had been 

injected with one of three treatments. Results showed no 

significant difference, in the percent of pre-imaginal 

mortality or mean weight of emerged adults, between the 

mCry3A treatment and the negative control group. 

Classification: Acceptable 

462656-05 

462656-06 
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Biopesticide Registration Action Document (BRAD) September, 2010 

 47 

Guideline Study Results MRID

885.4340 Nontarget 

Insect 

Testing, Tier 

1 (Rove 

beetle, 

Aleochara 

bilineata) 

Rove beetles were provided approximately 0.2 g of 

minced beef treated with: 50 μg mCry3A protein/g meat; 

10 mL deionized water/90 g meat (negative control); 

teflubenzuron at a rate of 0.01 mg a.i./g meat (positive 

control). Beetle mortality and reproductive capacity were 

not adversely affected by feeding on a test diet composed 

of 45.85 μg mCry3A/g diet for 35 days. Classification: 

Acceptable 

462656-07 

462656-08 

885.4340 Nontarget 

Insect 

Testing, Tier 

1 (Insidious 

flower bug, 

Orius. 

insidiosis) 

Nymphal flower bugs were fed, on a daily basis, diet with 

one of three treatments: 50 μg mCry3A-0102 /g of diet; 

20 mL deionized water/per 180 g diet (negative control); 

or teflubenzuron at a rate of 0.01 mg a.i./g diet. Mortality 

in the mCry3A-0102, negative control, and positive 

control treatments were 18, 23, and 98%, respectively. 

Average development time for all treatments was not 

significantly different. Classification: Acceptable 

462656-09 

462656-10 

885.4380 

Honey Bee 

Testing, Tier 

1 (Apis 

mellifera)

Honeybees were exposed to sucrose solution containing 

50 μg mCry3A protein/g sucrose solution, or a positive or 

negative control. Results suggest that incidental ingestion 

of mCry3A proteins would not adversely affect the hive 

condition, survival of larvae in brood cells, or exposed 

adult worker bees. Classification: Acceptable 

461556-18 

885.4240 Aquatic 

Invertebrate 

Acute 

Toxicity Test, 

Tier 1 

The only plausible potential route of exposure of 

freshwater invertebrates to insecticidal proteins produced 

by transgenic corn plants is corn pollen drift into aquatic 

habitats. However, since the pollen of Event MIR604 corn 

plants has no detectable mCry3A protein, exposure of 

freshwater aquatic invertebrates to mCry3A protein will 

be negligible. Classification: Acceptable 

Waiver 

justification 

850.620 Earthworm 

Subchronic 

Toxicity 

Study 

(Eisenia 

fetida)

Earthworms were exposed to soil containing mCry3A0102 at a nominal concentration of 370 μg/g dry soil for 

14 days, or one of two control treatments. At test end, a 

mortality rate of 5% and a mean weight loss of 5.8% 

were recorded for mCry3A-0102 treated worms. For the 

negative control, mortality was 0%, and mean weight loss 

was 11.4%. Mortality was 100% for the positive control. 

Classification: Acceptable 

462656-11 

462656-12 
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 

 48 

Guideline Study Results MRID

N/A Insecticidal 

Activity 

Spectrum 

Study 

The mCry3A protein has a similar spectrum of activity to 

native Cry3A, but with enhanced toxicity to NCRW and 

WCRW. Modified Cry3A produced in E. coli and maize 

were found to be active against WCRM with 144 hour 

LC50 values of 0.43 μg mCry3A-0102/mL diet and 0.20 

μg mCry3A/mL diet surface, respectively.. 

Classification: Acceptable

461556-01 

461556-03 

885.5200 

Expression in 

a Terrestrial 

Environment 

(Soil Fate) 

A simple first-order kinetic model, based on CPB larvae 

feeding data, determined that the DT50 for mCry3A in 

this silty clay loam soil was 7.6 days. This finding 

suggests that soil incorporated mCry3A protein degrades 

over time. Classification: Acceptable 

462656-14 

NA Environment

al Fate 

Assessment 

MIR604 corn plants have been shown to express 

mCry3A protein in leaves, kernels, roots, and silks, but 

the protein was not detected in corn pollen. Due to corn’s 

lack of invasive characteristics and the low probability 

that the mCry3A gene from Event MIR604 would transfer 

to a wild relative of corn, it is unlikely that mCry3A will 

spread beyond cultivated sites and persist in weedy 

populations. It is also unlikely that genes present in 

MIR604 corn would be subject to horizontal gene 

transfer at a frequency that exceeds the rate of transfer in 

other plants. Classification: Acceptable 

462656-13 

NA Endangered 

Species 

Assessment 

The primary route of exposure to mCry3A protein in corn 

is through ingestion of corn tissue. There are no reports 

of threatened or endangered species feeding on corn 

plants, therefore such species would not be exposed to 

corn tissue containing Cry protein. Since mCry3A protein 

has not been shown to have toxicity effects on mammals, 

birds, plants, aquatic species, insects and other 

invertebrate species at the EEC, a "may affect" situation 

for endangered land and aquatic species is not 

anticipated. In addition, EPA does not expect that any 

threatened or endangered plant species will be affected 

by outcrossing to wild relatives or by competition with 

such entities. Hybrid corn does not exist in the wild, nor 

are there wild plants that can interbreed with corn in the 

United States. 

Because of the selectivity of mCry3A protein for 

462656-01 
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 

 49 

Guideline Study Results MRID

coleopteran species, endangered species concerns are 

mainly restricted to the order Coleoptera. Examination of 

an overlay map showing the county level distribution of 

the 16 endangered/threatened coleopteran species 

(currently listed by the U.S. Fish and Wildlife Service) 

relative to corn production counties in the United States 

clearly indicated that any potential concern regarding 

range overlap with corn production was mainly restricted 

to the American burying beetle (Nicrophorus 

americanus). The American burying beetle is the largest 

carrion beetle in North America and is only found in 

limited areas of Rhode Island and portions of the Great 

Plains, including Arkansas and Georgia. Adults are 

nocturnal and feed on carrion and sometimes prey on 

other arthropods. Larvae feed exclusively on buried 

carrion provided by their parents. The American burying 

beetle’s habitat is variable and often includes deciduous 

forest, grassland and agricultural areas. Considering that 

both larvae and adult insects feed exclusively on carrion, 

it appears that even if American burying beetles did 

occur in proximity to Bt corn fields, there would be little 

chance of exposure to Bt protein due to their feeding 

habits. After careful review of available data, the EPA 

determined that exposure of American burying beetle to 

harmful levels of MIR604 corn tissue is not expected. 

Likewise, a review of the preferred habitats of other 

coleopteran species listed as endangered by the U.S. Fish 

and Wildlife Service indicated that exposure to harmful 

levels of mCry3A protein would not take place. The main 

reasons for the lack of exposure are geographical and 

habitat limitations. These species are located in non-corn 

production areas and/or their habitat does not encompass 

agricultural areas. 

Likewise, other insect species in the orders Diptera, 

Hemiptera, Lepidoptera, Odonata and Orthoptera that are 

listed as endangered/threatened species are found in 

dune, meadow/prairie or open forest habitats and are not 

closely associated with row crop production, often times 

due to the specificity of the habitat of their host plants. 

The reviewed toxicological data shows the relative 
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 

 50 

Guideline Study Results MRID

insensitivity of a range of insects in non-Coleopteran 

orders to the mCry3A proteins, indicating that MIR604 

maize hybrids are not likely to have detrimental effects 

on non-Coleopteran insects included on the 

endangered/threatened species list. 

Further, several of the federally listed insect species are 

aquatic and consequently, are unlikely to come in contact 

with MIR604 maize plant material. Many of the 

endangered and threatened beetles occur in cave or 

aquatic habitats. Since movement into water bodies of 

soil containing mCry3A 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 a corn field were 

deposited in a 1 ha pond with 2 m depth and located ≥1 

m from the edge of a corn field, <0.0001 μg mCry3A/mL 

of water would be expected. This is a few orders of 

magnitude below the toxic level to any insect. 
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 

 51 

Guideline Study Results MRID

NA Long-term 

field 

degradation 

study on the 

accumulation 

and 

persistence of 

active 

ingredient 

Soil microorganism exposure to Bt proteins can occur 

through contact with corn plant roots (by direct feeding), 

corn plant exudates, incorporation of above-ground plant 

tissues into soil following harvest, or by soil-deposited 

pollen. Scientists have generated data on Bt proteins in 

crop residues and their persistence in soil for long periods 

of time following exposure to a wide range of 

environmental field conditions. 

The results of these studies show that there is no 

detectable Cry protein accumulation in agricultural soils 

during commercial planting of currently registered Cry 

protein-producing crops (Icoz and Stotzky, 2007; 

Sanvido, et. al, 2007). Likewise, no unexpected 

accumulation of Cry proteins has been seen in numerous 

studies submitted directly to EPA for currently registered 

Cry proteins. There are various published studies that 

measured Bt protein degradation in a laboratory setting 

relating the dissipation time of the protein to bioactivity 

(DT50); data predict that Cry proteins in the field degrade 

rapidly and do not persist or accumulate in the soil to any 

extent that would affect nontarget organisms (NTOs). 

The DT50 (based on a simple first-order kinetic model) 

for mCry3A in an artificial soil environment was 7.6 days 

(MRID No. 462656-14). The results of this study 

demonstrate a similar rate of degradation to other Cry 

proteins in artificial soil systems (U.S. EPA, 2001) and, 

along with the literature cited below, indicate that 

mCry3A is likely to have a similar field degradation 

profile to other Bt proteins. In addition, data already 

exist to support the lack of effects of the mCry3A protein 

on NTOs (U.S. EPA, 2006; Raybould et al., 2007), which 

is the primary focus when considering soil fate data. 

RECOMMENDATION: The data and rationales in 

support of the request to waive the need for a soil 

persistence field study are ACCEPTABLE for mCry3A 

protein, as expressed in MIR604 corn. 

478477-01 
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 

 52 

1. Non-target Wildlife Testing and Hazard Assessment 

 a. Mammalian Wildlife 

Mammalian wildlife exposure to mCry3A protein is considered likely; however, mammalian 

toxicology information gathered to date on Bt Cry proteins does not show a hazard to wild mammals. 

And an acute oral toxicity test, submitted to EPA in support of the MIR604 registration (see Human 

Health Risk Assessment), indicated that no significant toxicity was seen when rodents were exposed to 

mCry3A at the maximum hazard dose level. Therefore, no hazard to mammalian wildlife is anticipated 

and data on wild mammal testing is not required for this registration. 

 b. Avian hazard assessment

Published data and studies on file at EPA show that consumption of Bt corn has no measurable 

deleterious effects on avian species. However, to comply with published data requirements, the 

following studies were submitted to EPA in support of the MIR604 product registration. These studies 

were GLP compliant and, when considered together, meet EPA data requirements for avian species. 

i. Northern Bobwhite Quail 

This study meets current EPA Guideline requirements for acute toxicity testing of incidental exposures 

of plant incorporated Cry proteins to non-target birds in the wild. 

Young adult (25 week old) northern bobwhite quail (Colinus virginianus) were administered a single 

nominal oral dose of 722 mg mCry3A-0102/kg body wt and observed for 14 days. There were no 

adverse treatment-related clinical signs or mortality. Body weight and feed consumption of the test 

birds were comparable to those of the negative control. The acute oral LD50 of mCry3A-0102 was 

shown to be greater than a nominal concentration of 722 mg mCry3A-0102/kg body wt (approximately 

652 mg mCry3A protein/kg body wt). These data show that there will be no adverse effects on avian 

wildlife from incidental field exposure to mCry3A corn. 

 ii. Broiler study 

The submitted study was not EPA GLP compliant, but was conducted according to accepted scientific 

methods. 

In a 49-day avian feeding study, one day-old commercial broiler chickens (Gallus domesticus, Ross 

344 males and feather-sexable Ross 308 females) were fed formulated diets containing one of the 

following ingredients: MIR604 corn (contained mCry3A protein); an MIR604 isoline (no mCry3A 

protein), or a non-transgenic commercial corn hybrid (no mCry3A protein). Starter, grower (days 16-

31), and finisher diets (days 31-49) contained 57.5, 63.0 and 67.5% corn, respectively. The 

concentration of plant produced mCry3A in the transgenic diets was reported to be 0.04, 0.06, and 0.08 

μg/g dry weight of the starter, grower, and finisher diets, respectively. Chicks were separated by sex 

and placed into single sex pens containing 25 birds each. Each treatment group contained 6 cages each 
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 

 53 

of males and females (12 cages x 25 birds/cage = 300 birds). Pen weights (25 birds/pen) were recorded 

at days 1 (hatch), 16, 31, and 49. On the later three dates, feed conversion ratios were determined. 

Feeding was terminated approximately16 hours before slaughter on day 51. Body weight, feed 

conversion, and survival data were recorded. Results indicate that sex had a significant effect on body 

weight and survival, with males weighing more and having higher mortality (which is normal for this 

species). No adverse clinical signs were noted, and carcass yield and mortality were not significantly 

different among treatment groups. 

Table 5. Mean body weight of broiler chickens fed mCry3A positive or mCry3A negative corn 

grain.

Treatment Body Weight (g) 

Day 1 (hatch) Day 16 Day 31 Day 49 

MIR604 (mCry3A positive) 44.60 ± 0.19 

a* 

547.8 ± 9.6 

a 

1684.1 ± 50.6 

a 

3468.4 ± 129.5 

a 

MIR604 (mCry3A negative) 44.66 ± 0.18 a 563.0 ± 5.4 

a 

1690.5 ± 40.2 

a 

3469.4 ±113.1 

a 

Commercial hybrid (mCry3A 

negative) 

44.70 ± 0.19 a 551.1 ± 9.6 

a 

1634.0 ± 44.3 

b 

3365.2 ± 117.6 

b 

* Within sampling days, means followed by different letters differ significantly (p≤0.05). 
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 

 54 

Table 6. Carcass and parts yield at day 51 for broiler chickens fed mCry3A positive or mCry3A 

negative corn grain.

Males

Dressed 

carcass* 

Thighs Pectoralis 

major 

Pectoralis 

minor 

Treatment 

----------------------------------------- g -------------------------------------

MIR604 (mCry3A positive) 2947.2 ± 57.44 

a** 

526.3 ± 14.05 

a 

638.8 ± 19.18 

a 

150.8 ± 4.63 a

MIR604 (mCry3A negative) 2936.7 ± 56.88 a 484.3 ± 15.10 

ab 

609.6 ± 16.73 

a 

145.7 ± 4.57 a

Commercial hybrid (mCry3A 

negative) 

2812.9 ± 61.49 a 454.6 ± 20.30 

b 

605.1 ± 23.09 

a 

151.3 ± 4.97 a

Females

MIR604 (mCry3A positive) 2248.3 ± 45.53 a 380.8 ± 7.6 a 515.4 ± 18.9 a 126.2 ± 3.3 a 

MIR604 (mCry3A negative) 2291.0 ± 41.4 a 361.9 ± 9.2 a 511.8 ± 13.8 a 128.0 ± 3.5 a 

Commercial hybrid (mCry3A 

negative) 

2204.2 ± 54.8 a 366.2 ± 11.3 a 487.1 ± 22.0 a 125.9 ± 3.4 a 

* Fresh carcass without head, neck, feet, feathers, viscera, and blood 

** Within carcass parts categories, means followed by different letters differ significantly (p≤0.05) 

 

 c. Aquatic species testing

There is no evidence for sensitivity of aquatic (including endangered) species to anti-coleopteran Bt

Cry proteins. Furthermore, aquatic exposure to mCry3A is extremely small or non-existent since 

mCry3A was not detectable in MIR604 pollen. 

 i. Freshwater Fish 

The Harmonized Testing Guidelines requirement for a static renewal freshwater fish toxicity study is 

usually waived for Bt corn PIPs due to the low potential for exposure to Cry protein produced in this 

crop. Nonetheless, a 28 day flow-through study was performed and submitted for review. This study is 

scientifically sound. 

In this 28-day toxicity study, juvenile rainbow trout (Onchorhynchus mykiss) were fed fish feed 

containing 50% w/w Event MIR604 (0.09 μg mCry3A/g test diet) or non-transgenic (negative control) 

corn grain. Prior to test initiation, 40 fish were placed in each of two test vessels, the exposure tank and 

the control tank. Mortality and symptoms of toxicity were assessed on a daily basis and detailed 
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 

 55 

observations of symptoms and feeding responses were made on days 4, 7, 10, 15, and 22. No 

significant differences were detected in the weight of the control or test fish at 0, 14, or 28 days. No 

significant difference in length was seen at 14 or 28 days. In the MIR604 test group, transient 

discoloration, sounding, and surfacing were seen in one to three fish after day 15, and one fish (2.5% 

of test group) was found dead on day 21. No mortality was seen in the control group. Due to the lack 

of demonstrated toxicity of the mCry3A protein to juvenile rainbow trout and the low probability that 

aquatic systems will be exposed to the protein, no fresh water fish hazard is expected from commercial 

cultivation of Event MIR604 corn. 

 ii. Aquatic invertebrates 

Update (July, 2010). In light of recently published laboratory studies showing reduced growth in 

shredding caddis flies exposed to anti-lepidopteran Cry1A protein corn litter (Rosi-Marshall, et al. 

2007), additional aquatic invertebrate data are required. A 7 to 14 day Daphnia magna study as per the 

885 series OPPTS Guidelines needs to be performed. The study may be submitted as a condition of 

registration. Alternatively, a dietary study of the effects on an aquatic invertebrate, representing the 

functional group of a leaf shredder in headwater streams, can be performed and submitted in lieu of the 

Daphnia study. 

Update (September 2010). 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, EPA conducted a literature 

review of these recently published studies. Criticisms of the Rosi-Marshall et al. study included 

several findings: (1) adverse effects were not caused by toxicity of Cry1A but, rather, by other 

differences between plant test substances (Jensen et al. 2010); (2) the abundance of Trichoptera in 

streams containing residues of Cry1A was not reduced (Chambers et al. 2007); and (3) while postharvest crop residue was identified as the most likely route of exposure (Carstens et al. 2010), aquatic 

exposure to biotech crops has been shown to be limited temporally and spatially with low to negligible 

exposure concentrations of Cry proteins in post-harvest crop tissues (Swan et al. 2009, Chambers et al. 

2009, Jensen et al. 2010, Wolt and Peterson 2010, Carstens et al. 2010). In light of these results, EPA 

is not requiring additional aquatic invertebrate studies to assess hazard to aquatic shredder species for 

existing Cry protein PIP registrations. 

iii. Estuarine and Marine Animals

Estuarine and marine animal studies were not required for this product, because of the low probability 

that aquatic systems will be exposed to the mCry3A protein produced in MIR604 corn plant tissues. 

iv. Terrestrial and Aquatic Plants

Plant toxicity studies were not required for this product because the active ingredient is an insect toxin 

(Bt endotoxin) that has never shown any toxicity to plants. 
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 

 56 

 

d. Non-Target Insect Testing 

The mCry3A protein specifically targets corn rootworm species, which are within the order Coleoptera 

(beetles). Since Bt toxins are known to have a limited host range, EPA requires that test species used 

for non-target insect evaluations should include several species that are related to the target pests 

(coleopteran species), since it is expected that these species will be most susceptible to the Bt toxin. 

i. Ladybird Beetle 

This study complies with the testing requirements outlined in OPPTS Series 885.4340 (Nontarget 

Insect Testing, Tier 1), the Organization for Economic Development (OECD) Principles of Good 

Laboratory Practice, and the UK Good Laboratory Practice Regulations. 

Four–day old lady beetle larvae (Coccinella septempunctata) were fed live pea aphids that were dipped 

in one of three solutions for a period of 14 days. For each treatment, aphids were immersed for 30 

seconds in a solution containing: mCry3A dissolved in a solution of Agral 90 (a non-ionic surfactant) 

at a concentration of 50 μg mCry3A protein/mL solution; Agral 90 solution only (negative control); or 

0.5 mL of Nemolt (teflubenzuron)/L Agral 90 solution (positive control). Freshly-treated live aphids 

were provided to lady beetle larvae daily until pupation, and the number of pea aphids provided at each 

feeding time increased with larval age. Beetle larvae were assessed daily for developmental stage and 

mortality. Following adult emergence from pupae, beetles were fed 50 treated aphids 3 times a week 

for 14 days. Adults were assessed for mortality at each feeding. 

There was no significant difference in the rate of pupal development between the negative control and 

mCry3A-0102 treatment. However, the mean number of days to adult emergence was significantly 

lower in the mCry3A treatment. There was no significant difference in pre-imaginal or adult survival 

among the negative control and mCry3A treatments. All larvae in the positive control died in the preimaginal stage. Since lady beetles are known to feed on corn pollen or insect prey, rather than corn 

leaves, and since mCry3A concentration in pollen (undetected) and prey is much lower than in corn 

leaves, it is not expected that lady beetles will be adversely affected by MIR604 corn in a field 

environment. 

 
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 

 57 

Table 7. Mean developmental time for lady beetles exposed to mCry3A-0102 or a control 

treatment. 

Treatment Concentration Larvae to Pupae Larvae to Adult 

Mean Standard 

Deviation 

Mean Standard 

Deviation 

Negative 

control 

Agral 90 5.48 0.50 9.8 0.68 

mCry3A0102 50 μg 

mCry3A/mL 

Agral solution 

5.33 0.47 9.48* 0.59 

*Significantly different from control (p<0.05) 

Table 8. Mortality assessment for lady beetles exposed to mCry3A-0102 or a control treatment. 

Pre-Imaginal Mortality Adult Mortality 

Mortality Corrected 

Mortality 

Mortality Corrected 

Mortality 

Treatment Concentration 

---------------------------------- % ---------------------------------- 

Negative 

control 

Agral 90 0 - 7.5 - 

mCry3A0102 50 μg 

mCry3A/mL 

Agral solution 

0 0 15.0 8.1 

Nemolt 0.5 mL/L Agral 

solution 

100* 100 - - 

* Treatment differed significantly from the negative control (p<0.001) 

It is noted that the actual amount of mCry3A protein consumed by larvae and adult ladybird beetles 

was 9 μg mCry3A/g aphid, which is below the targeted concentration of 50μg/g aphid (10 x mCry3A 

concentration in corn leaves). However, since lady beetles are known to feed on corn pollen or insect 

prey, rather than corn leaves, and since mCry3A concentration in pollen (undetected) and prey is much 

lower than in corn leaves, it is not expected that lady beetles will be adversely affected by MIR604 

corn in a field environment. 

 

 ii. Carabid Beetle 

This study complies with the testing requirements outlined in OPPTS Series 885.4340 (Nontarget 

Insect Testing, Tier 1), the Organization for Economic Development (OECD) Principles of Good 

Laboratory Practice, and the UK Good Laboratory Practice Regulations. 

Twenty-four to 48 hour-old Carabid beetle larvae (Poecilus cupreus) were fed daily with blowfly 

pupae until pupation. For each treatment, blow fly pupae were injected with: mCry3A-0102 at a rate of 
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 

 58 

50μg mCry3A/g pupa; deionized water only (negative control); or teflubenzuron at a rate of 0.664 ng 

a.i./g fly pupae (positive control). Fly pupae were replaced daily with freshly defrosted pupae until 

beetle larvae entered pupation. Beetle larvae were assessed three times per week for the first two 

weeks, and two times per week thereafter until day 32 of the study, at which time test containers were 

checked daily for emerging adult beetles. Adults were sexed, and overall weight and mortality were 

statistically analyzed. Results showed no significant difference, in the percent of pre-imaginal 

mortality or mean weight of emerged adults, between the mCry3A treatment and the negative control 

group. The positive treatment resulted in 100% pre-imaginal mortality (Table 9). 

Table 9. Larval mortality and adult weight of Carabid beetles fed blowfly pupae treated with or 

without mCry3A-0102 protein. 

Treatment Percent Larval 

Mortality 

Percent Corrected 

Larval Mortality 

Mean Adult 

Weight 

mCry3A-0102 10 0 82.9 

Negative control 20 - 81.5 

Positive control 100 100 - 

Analysis of the test diet showed that the actual concentration of mCry3A in blowfly pupae was 12 μg/g 

pupae. This amount is less than 10X (50 μg/g) the expressed concentration in maize leaves. However, 

the most likely route of carabid exposure to mCry3A protein is through consumption of prey that has 

eaten MIR604 plant tissue and studies indicate that the concentration of mCry3A in prey is at least 

1.4X lower than the protein concentration in plant tissue. 

 iii. Rove Beetle 

This study complies with the testing requirements outlined in OPPTS Series 885.4340 (Nontarget 

Insect Testing, Tier 1), the Organization for Economic Development (OECD) Principles of Good 

Laboratory Practice, and the UK Good Laboratory Practice Regulations.

Rove beetles (Aleochara bilineata) were obtained from parasitized onion fly (Delia antique) pupae and 

adults were four days old (physiologically) at study initiation. During this 35 day feeding trial, beetles 

were provided one of three treatments of cooked minced beef. For each treatment, beetles were given 

approximately 0.2 g of minced beef treated with: 50 μg mCry3A protein/g meat; 10 mL deionized 

water/90 g meat (negative control); or teflubenzuron at a rate of 0.01 mg a.i./g meat (positive control). 

For the first 7 days, beetles (10 female and 10 male) were kept in round plastic pots. From days 7 to 

35, test arenas were comprised of polystyrene boxes filled with at least 4 cm of quartz sand. On days 1, 

7 and 35 living, moribund, dead, and missing beetles were noted. To assess beetle fecundity, 

approximately 500 onion fly pupae were incorporated beneath the sand surface in each test box on 

days 14, 21, and 28. After approximately seven days, fly pupae were removed from the sand and 

placed in plastic pots. F1 beetles emerging from onion fly pupae were recorded every 1 to 4 days 

through day 76. The study was concluded when the mean number of beetles emerging per replicate 

declined to less than two per day in the control treatment. The mean number of F1 progeny was 647 for 
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 

 59 

the negative control and 663 beetles for the mCry3A treatment and these results were not significantly 

different. At day 35, mortality in the mCry3A, negative treatment, and positive treatments were 31, 34, 

and 35%, respectively and did not differ significantly (Table 10). Results also indicate that the 

reproductive capacity of beetles feeding on the mCry3A test diet was not adversely affected. 

Table 10. Mortality and number of progeny of rove beetles supplied mCry3A-0102 or a control 

treatment for 35 days.

Treatment % Mortality at 35 

days*

% Corrected 

Mortality

Mean no. of 

F1 Progeny

% Effect on 

Reproduction

mCry3A-0102 

(50 μg mCry3A/g diet) 

31 0 663 ± 219 -2.5 

Negative control 34 - 647 ± 169 - 

Teflubenzuron 

(10 μg/g diet) 

35 2 3 ± 5** 99.5 

* Treatments did not differ significantly from the negative control (p>0.05) 

** Treatment differed significantly from the negative control (p0.001) 

 iv. Flower Bug 

This study complies with the testing requirements outlined in OPPTS Series 885.4340 (Nontarget 

Insect Testing, Tier 1), the Organization for Economic Development (OECD) Principles of Good 

Laboratory Practice and the UK Good Laboratory Practice Regulations. 

Nymphal Orius insidiosis were fed, on a daily basis, diet consisting of cooked beef, liver, yeast, honey, 

egg, sugar, water, and Nipagin (chemical preservative) for 21 days. The diet was treated with one of 

three treatments: 50 μg mCry3A/g diet; deionized water (negative control); or teflubenzuron at a rate 

of 0.01 mg a.i./g diet. For each treatment, approximately 0.2 g of diet was placed into a small plastic 

cup covered with parafilm; insects pierced the parafilm to reach the diet. Nymphs were assessed for 

mortality and vitality daily until adulthood, or until 21 days after test initiation. Bugs that were 

missing, squashed, or injured during the study were excluded from data analysis. Mortality in the 

mCry3A, negative control, and positive control treatments were 18, 23, 98%, respectively (Table 11). 

Average development time for all treatments was not significantly different (p>0.5). Analysis of the 

test diet showed that protein expression was 47.8 μg mCry3A/g diet, or 95.6% of the nominal 

concentration (50 μg/g diet). In addition, a bioassay in which Colorado potato beetle, CPB 

(Leptinotarsa decemlineata) larvae were fed a diet containing 10 or 20% mCry3A-treated diet resulted 

in mortality of 83 and 90%, respectively. Although control mortality was 23% (test guidelines state 

that negative control mortality should not exceed 20%), it is unlikely that study conclusions were 

affected by this high rate of mortality. 
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 

 60 

Table 11. Mortality of flower bugs supplied with diet containing mCry3A-0102 or a control diet.

Treatment % Pre-imaginal Mortality % Corrected 

Mortality

mCry3A-0102 18 0 

Negative control 23 - 

Teflubenzuron 

(10 μg/g diet) 

98* 97 

* Significantly different from negative control (p0.001) 

 v. Honey Bee Larvae 

An acceptable study was conducted based on OPPTS Series 885.4380 (Honey Bee Testing, Tier I), in 

accordance with Good Laboratory Practice Standards as published by the U.S. Environmental 

Protection Agency in 40 CFR Part 160. 

Honeybees (Apis mellifera) were exposed via in-hive feeders to sucrose solution containing 50 μg/g 

sucrose solution, a negative control of 50% w/v sucrose solution, or a positive control of 480 g/L 

diflubenzuron insect growth regulator (Dimilin Flo ) in sucrose solution. Fresh treatment solutions 

were provided to each hive daily for five days. Egg cell mortality in the negative control, test, and 

positive control groups was 28.5, 27.3, and 100%, respectively. Larval cell mortality was 6.0, 6.8, and 

100%, respectively. There was no significant difference in mortality between the test and negative 

control groups for cells with eggs or larvae. There was also no significant difference in pre- and posttest hive condition between the test and negative control treatments. Results for the positive control 

treatment were significantly different from the other treatments for both mortality and hive condition 

(Table 12). Adult bees were not affected by any of the treatments. These study results suggest that 

incidental ingestion of mCry3A proteins would not adversely affect the hive condition, survival of 

larvae in brood cells, or exposed adult worker bees. 
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Table 12. Honey bee egg and larval cell development and brood count per frame when exposed to 

mCry3A-0102 or a control treatment. 

Brood Development Assessments Mean Brood per Frame 

Egg Cells Larvae Cells 

Mortality Corrected

Mortality 

Mortality Corrected 

Mortality 

Before 

Treatment 

After 

Treatment

Treatment 

---------------------------------------------------%------------------------------------ 

Negative 

control 

28.5 - 6.0 - 36.4 49.6 

mCry3A0102 27.3 0 6.8 0.9 34.2 40.7 

Dimilin Flo 100.0* 100.0 100.0 100.0* 34.5 27.7* 

* Significantly different from negative control (p<0.01) 

 vi. Freshwater Aquatic Invertebrate (waiver justification) 

The only plausible potential route of exposure of freshwater invertebrates to insecticidal proteins 

produced by transgenic corn plants is corn pollen drift into aquatic habitats. However, since the pollen 

of Event MIR604 corn plants has no detectable mCry3A protein, exposure of freshwater aquatic 

invertebrates to mCry3A protein will be negligible. 

 vii. Earthworm Toxicity Testing 

This study complies with the testing requirements outlined in OPPTS Series 850.6200 (Earthworm 

Subchronic Toxicity Study), Good Laboratory Practice Standards as published by the EPA in 40 CFR 

Parts 160 and 792, and the Organization for Economic Development (OECD) Principles of Good 

Laboratory Practice. 

In a laboratory test, earthworms (Eisenia fetida) were exposed to test substance mCry3A-0102, 

incorporated into artificial soil, at a nominal concentration of 370 μg/g dry soil (334 μg mCry3A 

protein/g dry soil) for 14 days. A negative control of deionized water and artificial soil and a positive 

control of 10, 20, 30 40, or 50 mg 2-chloroacetamide/kg dry soil were also used in the test. At test end, 

a mortality rate of 5% and a mean weight loss of 5.8% were recorded for mCry3A treated worms 

(Table 13). Worms included in the negative control had a mortality rate of 0% and a mean weight loss 

of 11.4%. Positive control worms exposed to 30 mg 2-chloroacetamide/kg of dry soil had a mortality 

rate of 100% and the LC50 value for earthworms exposed to 2-chloroacetamide was approximately 18 

mg active ingredient/kg dry soil. The 14-day LC 50 for earthworms exposed to mCry3A-0102 in an 

artificial soil substrate was determined to be greater than 370 μg test substance/g dry soil (equivalent to 

334 μg/g mCry3A active ingredient/g dry soil, the highest concentration tested), or 67 times greater 

than the expected field concentration of 5.5 μg/g soil (based on mCry3A concentration in senescent 

MIR604 plant roots). These study findings, which are consistent with historical Bt cry protein feeding 

results, indicate that earthworms should not be adversely affected by MIR604 corn plants. 
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Although some public comments have questioned whether earthworm test organisms actually ingested 

the soil incorporated Bt Cry proteins, recently published data show that earthworms do ingest and 

excrete soil incorporated Bt Cry proteins. 

Table 13. Mortality and weight loss of earthworms exposed to mCry3A-0102 or a control 

treatment.

Treatment Cumulative mortality (%)

Day 7 Day 14

14-Day Weight loss 

(%)

mCry3A-0102 2.5 5.0 5.8 

Negative control 0 0 11.4 

viii. Insecticidal Activity Spectrum Study 

Insect susceptibility studies showed that native Cry3A is primarily active against Colorado potato 

beetle and has minimal activity against northern corn rootworm (NCRW); both species are members of 

the Chrysomelidae family of beetles. The mCry3A protein has a similar spectrum of activity to native 

Cry3A, but with enhanced toxicity to NCRW and western corn rootworm (WCRW).

The bioactivity of the recombinant E. coli and maize event MIR604 mCry3A proteins were compared 

using a diet incorporation bioassay with first instar WCRW. Modified Cry3A from both E. coli and 

maize were found to be active against WCRW with 144 hour LC50 values of 0.43 μg mCry3A/mL diet 

and 0.20 μg mCry3A/mL diet surface, respectively (Table 14). 

Table 14. Toxicity of mCry3A protein derived from recombinant E. coli (mCry3A-0102) or from 

corn event MIR604 on WCRW larvae. 

Sample LC50 

μg mCry3A protein/mL 

diet

95% Confidence 

Interval

LPMIR604-0103 (corn-derived 

protein) 

0.20 (0.09 - 0.41) 

mCry3A-0102 (bacterial-derived 

protein) 

0.43 (0.14 - 0.94) 

2. Soil Fate 

Soil organisms may be exposed to mCry3A protein through contact with corn plant roots (by direct 

feeding), corn plant root exudates, incorporation of above-ground plant tissues into soil following 

harvest, or by soil-deposited pollen. Some evidence suggests that acidic soils (pH 5.6), and those 

which are high in clays and humic acids, are more likely to bind Cry protein, and thus decrease the rate 

of protein degradation by soil microorganisms. It is noted, however, that the pH factor should not 

contribute to protein binding in corn fields, since maize is generally grown on neutral soils (above pH 
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5.6). And despite evidence that soils high in clay and humic acids may bind cry proteins, and thus 

interfere with the microbial degradation processes, the weight of evidence suggests that Cry proteins 

do not accumulate in soil to arthropod-toxic levels. Nonetheless, the Agency requires soil fate 

evaluations for each new insect protected crop. 

This study was conducted in accordance with Good Laboratory Practice Standards as published by the 

U.S. Environmental Protection Agency, Office of Pesticide Programs in 40 CFR Part 160. 

The test soil was a silty clay loam collected from a corn-growing region of Iowa. Treatments were the 

following: mCry3A test mixture (Bt cry protein and water) mixed with soil at a nominal dose of 230 

μg mCry3A/g d/w soil, and sampled after 0, 1, 3, 7, 12, and 30 days of incubation; a negative control 

consisting of soil dosed with deionized water; and a positive control consisting of mCry3A mixed with 

test diet (no soil), at a concentration of 230 μg mCry3A/g diet (equivalent test dose level at Day 0). 

For the CPB bioassay, mCry3A-treated soil collected at each incubation time point was incorporated 

into a stock diet at a concentration of 10% w/w, and the resulting suspension was poured into Petri 

dishes. Negative and positive controls were prepared in the same manner. Ten freshly-hatched CPB 

larvae were placed in each Petri dish, which were then covered and maintained under ambient 

laboratory conditions. Test material and negative control treatments were replicated 12 times (120 

larvae/treatment) and the positive control was replicated four times (40 larvae). Larval mortality was 

assessed at 72 hours. 

Mean CPB larval mortality in mCry3A- treated soil ranged from 48-54% during the first week, then 

declined rapidly to 9% on Day 30 (Table 15). The DT50 value (time for 50% of initial bioactivity to 

dissipate) for degradation of mCry3A in this soil was estimated to be 7.6 days. Biomass determinations 

of soil at study start and end showed that microbial activity was maintained during the study. 

Table 15. mCry3A protein bioactivity in treated soil as measured 

by CPB larvae mortality.

Treatment % CPB Mortality 

Negative control 18 

Positive control 53 

Test Treatment 

Day 0 53 

Day 1 54 

Day 3 48 

Day 7 51 

Day 12 11 

Day 30 9 
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Based on these results, it may be concluded that purified mCry3A-0102 insecticidal proteins degrade 

rapidly in silty clay loam soil. However, silty clay loam soil is just one of many soil classes used for 

corn production in the United States. A more useful study would evaluate protein degradation, 

accumulation, and/or persistence in a range of soil types, including those with high clay and humic 

acid content, due to their known binding affinity for proteins. 

In addition, this study utilized field soil spiked with purified insecticidal protein. This approach is 

useful because dose responses can be easily quantified. However, the degradation and accumulation of 

Cry proteins found within decaying plant tissue may behave differently than proteins in artificially 

spiked soil. Thus, the relevance of these study results is unclear other than to show that degradation in 

soil does take place. 

To account for the above concerns, it is recommended that additional studies should be conducted to 

evaluate insecticidal protein degradation, accumulation, and persistence in a variety of soil types, 

including those high in clay and humic acids, into which all non-harvested corn plant material is 

incorporated. Sampling should be conducted each year for three years in a field sown with continuous 

MIR604 corn. Soil should be monitored for a minimum of one growing season after harvest and 

monitoring should continue until mCry3A protein can no longer be detected. As noted in Table 16 

below, the Agency has requested that the applicant submit this study as a condition of registration. 

These recommendations are based in part on the August 27, 2002 SAP and public comments provided 

to EPA at that time. This Panel concluded that several different soils should be examined and 

monitored for a minimum of one growing season after corn harvest and that monitoring should 

continue until the Cry protein can no longer be detected. According to the Panel, at least three soil 

types should be evaluated for Cry protein persistence and these soils should be high in organic matter 

and clay, since the potential of persistence is highest in these soil types. The Panel also recommended 

that soil degradation studies may be conducted under less than optimum environmental conditions, 

such as high or low temperatures, since protein persistence may be affected by varying environmental 

conditions. The Panel further suggested that since corn roots grow into deep soil with reduced 

microbial activity, degradation rates in these zones should also be examined. With regards to protein 

source, the Panel recommended that studies should utilize plant material that is representative of actual 

field conditions. For example, soil samples should be collected from field sites where whole plant 

tissue has been incorporated into the field. And further, plant tissue should not be ground prior to 

incorporation, because grinding artificially increases the plant tissue surface area exposed to 

microorganisms, and increased surface area may lead to an increase in the rate of protein degradation. 

Finally, the SAP stated that “(r)eal life or true persistence (or Cry protein) is likely to be equal to or 

less than that measured with ELISA.” So if an ELISA is conducted, results should be compared to a 

bioassay that uses a sensitive species such as the Colorado potato beetle. 

 

 
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3. Effects on Soil Microorganisms

2010 Update: Numerous published studies indicate that exposure to Cry protein produced in Bt PIP 

crop plants does not adversely affect soil microorganisms (Sanvido et al. 2007; Oliveira et al. 2008). In 

addition, Bt toxin released from root exudates and biomass of Bt corn has no apparent effect on 

earthworms, nematodes, protozoa, bacteria, and fungi in soil (Saxena and Stotzky 2001). Other 

research findings conclude no Bt-related risks have evolved from the decomposition of Bt-corn leaves 

for the meso- and macrofauna soil community (Hönemann et al. 2008). Although a minimal transient 

increase and shift in microbial populations may result from the presence of transgenic plant tissue in 

soil, no adverse effects have been attributed to the Cry protein. 

In addition, there are several ongoing U.S. Department of Agriculture and EPA Office of Research and 

Development funded research projects evaluating the effects of Bt crops on soil microbial flora. If 

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

mitigate potential risks. 

With regard to the impact of genetically engineered crops on soil, it is important to note that 

agricultural practices themselves cause large changes in soil and soil microbial composition. 

Furthermore, factors 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. 

 

4. Horizontal Transfer of Transgenes from Bt Crops to Soil Organisms

2010 Update: 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 mCry3A corn are derived from soil-inhabiting bacteria, EPA has concluded 

that there is a low probability of risk from HGT of transgenes found in mCry3A-producing corn. 
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5. Gene Flow and Weediness Potential 

Conclusions gathered from this review process are as follows: 

 The potential for pollen-directed gene flow from corn to Eastern gamagrass is extremely 

remote (DeWald et al. 1999). This is evidenced by the difficulty with which Tripsacum 

dactyloides x Z. mays hybrids are produced in structured breeding programs. Additionally, the 

genus Zea does not represent any species considered as serious or pernicious weeds in the U.S., 

its possessions, and/or its territories (Holm et al. 1979). Any introgression of genes into this 

species as a result of cross fertilization with genetically modified corn 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 

corn chromosomal complement in subsequent generations (DeWald, personal communication, 

1999). 

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

crossed with Zea mays ssp. mays. However, none of these plants are known to harbor weedy 

characteristics and none of the native teosinte species, subspecies, or races are considered to be 

aggressive weeds in their native or introduced habitats (Schoper, personal communication, 

1999). 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 

to be incapable of survival in the wild as a result of breeding practices (i.e., selection) during 

domestication of the crop. 

The October 2000 Scientific Advisory Panel agreed that the potential for gene transfer between corn 

and any receptive plants within the U.S., its possessions, and/or its territories was of limited probability 

and nearly risk free. Based on these findings, the EPA has determined that there is no significant risk 

of gene capture and expression of the mCry3A protein by wild or weedy relatives of corn in the U.S., 

its possessions, or its territories. 

2010 Update: Movement of transgenes from crop plants into weeds is a significant concern, due to 

uncertainty regarding the effect that a new pest resistance gene may have on plant populations in the 

wild. Under FIFRA, EPA 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. To date, Bt plant-incorporated protectants have been registered for use in agronomic plant 

species that do not have a reasonable possibility of passing their traits to wild native plants. However, 

due to concern over the possibility that species related to corn (Zea mays ssp. mays), such as 

Tripsacum species and the teosintes, could be recipients of gene flow from genetically modified Z.

mays, EPA conducted a thorough review of the scientific literature on what is known about the gene 

flow potential of Z. mays (U.S. EPA 2001). 
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6. Impacts on Endangered Species 

The primary route of exposure to mCry3A protein in corn is through ingestion of corn tissue. There are 

no reports of threatened or endangered species feeding on corn plants, therefore such species would not 

be exposed to corn tissue containing Cry protein. Since mCry3A protein has not been shown to have 

toxicity effects on mammals, birds, plants, aquatic species, insects and other invertebrate species at the 

EEC, a "may affect" situation for endangered land and aquatic species is not anticipated. In addition, 

EPA does not expect that any threatened or endangered plant species will be affected by outcrossing to 

wild relatives or by competition with such entities. Hybrid corn does not exist in the wild, nor are there 

wild plants that can interbreed with corn in the United States. 

Because of the selectivity of mCry3A protein for coleopteran species, endangered species concerns are 

mainly restricted to the order Coleoptera. Examination of an overlay map showing the county level 

distribution of the 16 endangered/threatened coleopteran species (currently listed by the U.S. Fish and 

Wildlife Service) relative to corn production counties in the United States clearly indicated that any 

potential concern regarding range overlap with corn production was mainly restricted to the American 

burying beetle (Nicrophorus americanus). The American burying beetle is the largest carrion beetle in 

North America and is only found in limited areas of Rhode Island and portions of the Great Plains, 

including Arkansas and Georgia. Adults are nocturnal and feed on carrion and sometimes prey on other 

arthropods. Larvae feed exclusively on buried carrion provided by their parents. The American burying 

beetle’s habitat is variable and often includes deciduous forest, grassland and agricultural areas. 

Considering that both larvae and adult insects feed exclusively on carrion, it appears that even if 

American burying beetles did occur in proximity to Bt corn fields, there would be little chance of 

exposure to Bt protein due to their feeding habits. After careful review of available data, the EPA 

determined that exposure of American burying beetle to harmful levels of MIR604 corn tissue is not 

expected. Likewise, a review of the preferred habitats of other coleopteran species listed as endangered 

by the U.S. Fish and Wildlife Service indicated that exposure to harmful levels of mCry3A protein 

would not take place. The main reasons for the lack of exposure are geographical and habitat 

limitations. These species are located in non-corn production areas and/or their habitat does not 

encompass agricultural areas. 

Likewise, other insect species in the orders Diptera, Hemiptera, Lepidoptera, Odonata and Orthoptera 

that are listed as endangered/threatened species are found in dune, meadow/prairie or open forest 

habitats and are not closely associated with row crop production, often times due to the specificity of 

the habitat of their host plants. The reviewed toxicological data shows the relative insensitivity of a 

range of insects in non-Coleopteran orders to the mCry3A proteins, indicating that MIR604 maize 

hybrids are not likely to have detrimental effects on non-Coleopteran insects included on the 

endangered/threatened species list. 

Further, several of the federally listed insect species are aquatic and consequently, are unlikely to come 

in contact with MIR604 maize plant material. Many of the endangered and threatened beetles occur in 

cave or aquatic habitats. Since movement into water bodies of soil containing mCry3A is expected to 
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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 a 

corn field were deposited in a 1 ha pond with 2 m depth and located ≥1 m from the edge of a corn 

field, <0.0001 μg mCry3A/mL of water would be expected. This is a few orders of magnitude below 

the toxic level to any insect. 

2010 Update: Current ecological effects data and EPA reviews of mCry3A protein, as expressed in 

MIR 604 and in the stacked event MIR 604 x Bt11, expressing mCry3A x Cry1Ab, support the 

Agency’s determination that adverse effects will not occur to nontarget organisms. Due to a 

demonstrated lack of toxicity and/or exposure, no effects from mCry3A protein, as expressed in MIR 

604 and in the stacked events expressing Bt11 x mCry3A x Cry1Ab, are anticipated for any nontarget 

species, including federally-listed threatened and endangered (“listed”) lepidopteran and coleopteran 

species and their designated critical habitats. EPA has also determined that there are no indirect effects 

on endangered and threatened plant species, such as impacts on lepidopteran pollinators that are 

important and/or essential to an endangered or threatened plant. The Agency is therefore upholding its 

determination that the registered uses of mCry3A protein as expressed in MIR 604 and in the stacked 

events expressing Bt11 x mCry3A x Cry1Ab 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 (USFWS) and 

the National Marine Fisheries Services (NMFS). 

B. Environmental Risk Assessment

The EPA uses a Maximum Hazard Dose 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. 

At present, the Agency is aware of no identified significant adverse effects of mCry3A proteins on the 

abundance of non-target beneficial organisms in any population in the field environment, whether they 

are pest parasites, pest predators, or pollinators. Further, the EPA believes that cultivation of mCry3A 

corn may have fewer adverse impacts on non-target organisms than use of chemical pesticides for corn 

production, because under normal circumstances, mCry3A corn requires substantially fewer 

applications of chemical pesticides, compared to production of non-Bt corn. And fewer chemical 

insecticide applications generally result in increased populations of beneficial organisms that control 

secondary pests, such as aphids and leafhoppers, in corn fields. In addition, no adverse effect on 

endangered and threatened species listed by the US Fish and Wildlife Service is expected from the 

proposed MIR604 CRW resistant corn registration (see Section A.II.6 above). Further, the EPA has 

determined that there is no significant risk of gene capture and expression of mCry3A protein by wild 

or weedy relatives of corn in the U.S., its possessions, or territories (see Section A.II.5 above), 

available data do not indicate that Cry proteins have any measurable adverse effect on microbial 

populations in the soil (see Section A.II.3 above), nor has horizontal transfer of genes from transgenic 

plants to soil bacteria been demonstrated (see Section A.II.4 above). In conclusion, this risk assessment 
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finds no hazard to the environment at the present time from cultivation of mCry3A protein expressing 

MIR604 corn for a time-limited registration. 

C. Supplemental Studies Needed for Long Term MCry3A Non-Target Hazard Assessment

The Agency has sufficient information to believe that there is no risk from the proposed uses of 

mCry3A corn to non-target wildlife, aquatic, and soil organisms. However, in response to the August 

2002 SAP recommendations, the Agency is requesting supplementary studies that will evaluate the 

persistence of mCry3A in the soil and the long range effects of cultivation of mCry3A on the 

invertebrate community structure in corn fields. This will facilitate identification of potential adverse 

effects which may result from long-term use of this product. 

Table 16. Supplemental data requirements for Event MIR604 corn.

Testing Category Type of Data

Ecosystem effects Long range field studies should be conducted based on 

recommendations of the August, 2002 SAP. 

Soil fate studies Long range soil degradation field studies should be conducted. 

Studies should follow guidelines outlined by the August 2002 SAP, 

which are presented in summary form in the conclusion section of 

the soil fate review (see Section A.II.2 above). 

Update September 2010. The soil fate studies have been waived, but the nontarget long range field 

studies data requirement is still outstanding. 

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II. D. Insect Resistance Management 

1. BACKGROUND 

Syngenta submitted an amended insect resistance management strategy in support of their FIFRA 

Section 3 Registration for Event MIR604-derived transgenic maize that expresses modified Cry3A 

(mCry3A) protein (MRID# 465296-01, McCaffery et al., 2005). Modified Cry3A in MIR604 is 

expressed at a non-high dose against infestation by the Western corn rootworm (WCRW; Diabrotica 

virgifera virgifera Le Conte) and the Northern corn rootworm (NCRW; D. longicornis barberi Smith 

and Lawrence). A behavioral avoidance mechanism is also present. BPPD reviewed Syngenta’s 

previously submitted IRM strategy and noted several deficiencies in the simulation modeling and 

evaluation of cross-resistance potential (BPPD, 2005). These deficiencies were addressed in the 

amended IRM strategy submission (McCaffery et al., 2005, MRID# 465296-01) and are discussed in 

this chapter. 

2. SUMMARY OF SYNGENTA’S SUBMISSION (2004 - 2005)

Syngenta’s submission is divided into three main parts: 1) Sustainable deployment of Event MIR604 

maize through effective insect resistance management (IRM), 2) IRM plan for MIR 604, and 3) 

Justification for the proposed IRM plan for MIR604. The first two sections of the revised Syngenta’s 

submission were originally submitted by Syngenta (McCaffery and Stein, 2004) and reviewed by 

BPPD (BPPD, 2005). There were significant deficiencies identified in the IRM modeling and 

evaluation of cross-resistance potential. Syngenta has addressed these deficiencies in the section 

entitled, “Justification for the proposed IRM plan for MIR604.” 

Syngenta’s IRM plan for MIR604 maize contains the following elements: 

1. 20% structured refuge and its deployment and management; 

2. IRM as a component of an integrated pest management program; and 

3. Product stewardship program. 

The product stewardship program consists of the following aspects: 

1. Grower education plans; 

2. Grower obligations; 

3. Grower compliance; 

4. Annual resistance monitoring; 

5. Action plan in the event of unexpected levels of CRW damage; 

6. Remedial action plans; and 

7. Annual IRM plan review. 
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Syngenta has provided the following areas of support for its proposed IRM plan for MIR604 maize: 

1. Dose of mCry3A expressed in MIR604 (root expression assays, laboratory feeding assays 

using excised roots, field efficacy, and adult emergence studies); 

2. Simulation models for development of resistance; 

3. Impact on MIR604 maize on biology of CRW; 

4. Cross-resistance potential. 

3. BPPD REVIEW (BPPD, 2005; BPPD, 2006a) 

a. Nature and Derivation of Syngenta’s MIR604 CRW-Control Maize 

A cry3A gene from Bacillus thuringiensis subsp. tenebrionis (B.t.t.) (Sekar et al., 1987) was 

synthesized by Syngenta scientists for optimal expression in maize. The gene was then modified 

(hereinafter referred to as “mCry3A” or “modified Cry3A”) to enhance activity against the Diabrotica 

virgifera virgifera (Western corn rootworm, WCRW) and Diabrotica longicornis barberi Smith and 

Lawrence (Northern corn rootworm, NCRW). The introduction of a particular serine protease 

recognition site (both chymotrypsin and cathepsin G are serine proteases) into the native Cry3A results 

in a much more rapid and complete processing of the 67 kDa protein to 55 kDa and thus increased 

activity against WCRW and NCRW. After binding to the receptor, the mCry3A protein inserts rapidly 

and irreversibly into the plasma membrane of the cell to form ion permeable pores in a similar fashion 

as other Cry toxins (Van Rie et al., 1989; Garcia-Alonso and Vlachos, 2003). These ion channels 

cause a loss in membrane potential and the formation of these lesions leads to death resulting from 

septicaemia and/or starvation (Schnepf et al., 1998). 

b. Corn Rootworm Species Controlled 

Native Cry3A is primarily active against Colorado potato beetle (Leptinotarsa decemlineata) and has 

minimal activity against NCRW, both members of the Chrysomelidae family of beetles. The mCry3A 

protein has a similar spectrum of activity to the native Cry3A, but with enhanced toxicity to NCRW 

and WCRW, both major coleopteran pests of maize in the USA. Modified Cry3A has some activity 

against Diabrotica balteata (Banded cucumber beetle). Trials were conducted in 2005 (data not 

available for this review) to determine the level of control of Diabrotica virgifera zea (Mexican corn 

rootworm; MCRW) by mCry3A expressing maize plants (Event MIR604). Modified Cry3A has no 

activity against non-coleopteran pests including various lepidopteran pests of maize. MIR604 maize is 

most effective against larvae, especially the first instar. Syngenta’s IRM strategy focuses entirely on 

rootworms. 

c. Pest Biology 

A clear understanding of pest biology and ecology is essential to the development of a sound IRM 

plan. MIR604 Bt corn was developed to control two primary corn rootworm species: western corn 
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rootworm (Diabrotica virgifera virgifera LeConte, WCRW) and northern corn rootworm (D. Barberi

Smith & Lawrence, NCRW). WCRW is the most prevalent target pest in the United States and 

throughout most of the Corn Belt1

. NCRW, also found throughout the Corn Belt, is considered the 

second most prevalent rootworm pest in the United States, and is the primary target pest of the northcentral region2

. 

Key factors believed to influence CRW adaptation to MIR604 corn include distribution, univoltinism, 

adult dispersal among fields, adult dispersal within fields, larval dispersal across rows, larval mortality 

due to density-dependent processes, insecticide use, egg mortality, fecundity, and adult and larval 

population density. Syngenta has provided a sufficient summary of the biology and ecology of the 

corn rootworm target pest(s) both in the current IRM strategy submission (McCaffery et al., 2005, 

MRID# 465296-01) and the original IRM strategy submission (McCaffery and Stein, 2004, MRID# 

462656-17). See BPPD (2005) for the review of Syngenta’s original IRM strategy submission and pest 

biology information. 

d. IRM Plan for MIR604

Syngenta’s IRM plan for MIR604 maize contains three major components: 1) a 20% structured 

refuge; 2) IRM is part of an integrated pest management program; and 3) product stewardship. Each of 

these components will be discussed below. 

1) 20% structured refuge and its deployment and management

There are two ways a grower can implement the refuge requirement: a non-Bt corn refuge can be 

planted as a continuous block adjacent to the MIR604 maize fields or as non-transgenic strips planted 

within transgenic field. Considering the limited movement of CRW larvae, planting refuges close to 

transgenic fields in large blocks is preferred to narrow strips (Gray 1999, Meinke et al. 2001). 

Syngenta will require growers to plant a minimum structured refuge of non-corn rootworm-control 

(CRW) maize on at least 20% of their maize acres. Refuge fields must not be planted with other 

transgenic corn used to control rootworm because neither acts as a refuge for the other. Use of a 20% 

refuge with MIR604 maize complements other technologies and provides a degree of uniformity for 

CRW control as well as with products with both CRW and lepidopteran control. This uniformity 

provides growers with a straightforward and understandable message about refuge requirements and 

promotes compliance. Justification of the 20% refuge requirement is discussed later in this review. 

 

1

 WCRW is the primary rootworm pest in Colorado, Kansas, Nebraska, Ohio, Indiana, Illinois, Iowa, 

Missouri, and Michigan. 

2

 NCRW is the primary rootworm pest in Wisconsin, Minnesota, South Dakota and northern Iowa. 
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Syngenta will require growers to plant the structured refuge as blocks, strips, perimeter borders or 

pivot corners. Encouragement will be given to growers to plant these non-CRW control corn acres 

within their CRW-control acres. Syngenta has proposed that in-field strips be at least 6 to 12 nonrootworm protected Bt corn rows, although this row width size may be larger than necessary based on 

the current understanding of rootworm biology. BPPD has reviewed larval movement data published 

by Hibbard et al. (2003). This study indicated that between 0.75% and 6% of larvae moved across 

corn rows. These results represent 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 sub-lethal 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, seed mixes 

would not be a good refuge strategy for rootworms. In-field strips of ≥4 rows would provide the 

advantage of being more compatible with the current in-field strip width requirement for lepidopteranprotected Bt corn hybrids (≥4 row strips, with ≥6 row strips preferred). In-field strips of ≥4 rows 

would also be more practical and flexible for the grower because of the compatibility with split-planter 

designs. Because mCry3A will be stacked with Cry1Ab (submission pending), a recommendation of 

≥4 row strips will provide the grower a more easily understandable and consistent message regarding 

the width of in-field strips. Overall, BPPD’s recommendation is that Syngenta require growers to 

plant in-field strip refuges with widths of ≥4 rows (≥6 rows preferred) for rootworm-protected Bt corn 

hybrids. This will simplify refuge deployment and potentially increase grower compliance with refuge 

requirements. 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. 

Management of the structured refuge must conform to the rotational and management practices to 

achieve synchronous CRW development in both refuge and MIR604 maize fields. Growers will have 

the option of treating the refuge with conventional insecticides (seed treatments, soil applications and 

foliar applications) to control severe damage by CRW and to control secondary pests. These 

treatments will impact the overall number of beetles that emerge from the refuge fields even though 

their efficacy is less than 100%. Syngenta’s modeling study examines one scenario in which a 

nominal 50% of refuge insects are removed by insecticide control that impacted the potential 

development of resistance (see modeling section below). Syngenta indicates that there is no evidence 

for any cross-resistance, synergy, or antagonism between the mCry3A in MIR604 maize and Cry1Ab 

and other Bt toxins in Lepidoptera-control maize varieties. There are also no known interactions 

between mCry3A and insecticides. 

Syngenta notes that the use of continuous corn acres for refuge fields is always to be encouraged. It is 

also permissible for growers to plant the refuge acres on first-year corn acres, but only where the 

CRW-control corn is also planted on first-year corn acres. This distinction is made for areas in which 

WCRW has adapted to crop rotation and sizable populations of these insects will emerge from first-
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year corn. There is also a variant of NCRW in which there is extended diapause. Both rotationresistant CRW variants impact the efficacy of crop rotation. 

2) IRM is Part of an Integrated Program 

Syngenta stressed that the IRM strategy for MIR604 maize is part of an overall package of integrated 

crop management techniques. These tactics include: 

1. Crop rotation. Growers will be encouraged to maintain crop rotation as a vital part of CRW 

management. The presence of rotation-resistant phenotypes, however, may render such an 

approach ineffective in some areas. 

2. Refuge quality. Growers will be educated on management practices to maximize the 

effectiveness of their refuges. 

3. Insecticide use. When moderate to high CRW population pressures in the refuge, growers will 

be encouraged to treat the refuge with a soil insecticide or use seed treatments in a manner to 

maintain overall survival of CRW to function effectively as refuge mating partners. 

3) Product Stewardship

Growers are crucial to the success of any IRM plan because they are responsible its implementation on 

the practical level. Syngenta has developed a product stewardship program that stresses the 

importance of implementation of the IRM plan for MIR604 maize. Syngenta’s product stewardship 

program consists of the following aspects: 

1. Grower education plans (e.g., Syngenta guide); 

2. Grower obligations (grower agreements); 

3. Grower compliance; 

4. Annual resistance monitoring; 

5. Action plan in the event of unexpected levels of CRW damage; 

6. Remedial action plans; and 

7. Annual IRM plan review. 

Compliance programs are important in that they encourage growers to comply with IRM requirements, 

while providing mechanisms by which registrants can be held accountable for noncompliant growers. 

The compliance program presented in this submission mirrors those developed for existing 

lepidopteran-protected Bt maize registrations. The specific compliance assurance program 

components are: 1) Annual IRM Survey, 2) Mechanism for Handling Tips and Complaints, 3) OnFarm Visits, and 4) Phased Compliance Approach. Syngenta will respond to instances of noncompliance through the Phased Compliance Approach and will also use grower agreements and an 

annual affirmation scheme to reinforce grower understanding and compliance. Grower education 

programs give growers a clear understanding of the importance of IRM and its implementation. The 

IRM program will be communicated to growers by Syngenta through its authorized sales agents using 

both print and other media including workshops, educational pamphlets/brochures, and an assortment 
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of public relation activities. BPPD finds Syngenta’s compliance assurance program to be 

“acceptable” with the following caveats. The EPA-approved changes (granted on June 16, 2006) to 

the annual IRM reporting requirements and the Compliance Assurance Program for the lepidopteranprotected Bt maize registrations should be incorporated into Syngenta’s IRM Compliance Assurance 

Program for MIR604 Corn Products. 

The need for proactive resistance detection and monitoring is critical to the survival of Bt technology. 

Consequently, the Agency mandates that a resistance monitoring plan must be implemented for all 

registered Bt maize products. Resistance can evolve regionally or as a local increase in resistance (r-) 

allele frequency. The resistance monitoring plan designed for MIR604 maize is an adaptation of the 

ABSTC program developed for lepidopteran-protected Bt maize. This program will attempt to detect 

either local or regional resistance early enough to initiate effective remedial action. As for all other Bt

maize products, Syngenta’s resistance monitoring program will be implemented through a twopronged approach, including field reports of unexpected damage and population testing and sampling. 

The initial emphasis of their monitoring plan will be on establishing the baseline susceptibility for 

WCRW. Growers will be asked to report to Syngenta any unexpected field damage. Diagnostic 

bioassays of larvae from eggs laid by field-collected WCRW adults will be used to survey 

susceptibility of the insects to mCry3A. The monitoring efforts will focus on regions of the Corn Belt 

where WCRW populations are known to regularly reach high numbers and where the highest MIR604 

adoption is expected. Detecting shifts in the frequency of resistance genes (i.e., susceptibility changes) 

through resistance monitoring can be an aggressive method for detecting the onset of resistance prior 

to widespread crop failure. As such, the utilization of sensitive and effective resistance monitoring 

techniques is critical to the success of an IRM plan. BPPD finds that Syngenta’s basic resistance 

monitoring plan and remedial action plan for the mCry3A protein expressed in MIR604 maize are 

“acceptable,” although certain issues need to be addressed. These are discussed below. 

1. The monitoring plan only focuses on WCRW. CRW species are challenging to rear in the 

laboratory. Of the identified target pest species, scientists have had the most success rearing 

WCRW, and have had little success with NCRW. Consequently, BPPD agrees with Syngenta 

that initial monitoring through population sampling will focus on WCRW, but should include 

NCRW populations when available. No information exists on MCRW. These data should be 

provided to BPPD when they become available. 

2. A diagnostic method cannot be developed until the dose-response relationships for WCRW to 

establish the baseline sensitivity to mCry3A. While BPPD agrees that a sensitive and reliable 

bioassay should be developed, these efforts are currently under development. Once this work is 

completed, the registrants must develop guidelines as to what level of root damage will be 

expected under various conditions, and what level of rootworm control is normally achieved. 

Growers will have to report any “unexpected damage.” Without such guidelines, i.e., what is 

“acceptable” rootworm damage, then it will not be possible to determine what the “unexpected” 

rootworm damage is. Once these guidelines are established it will be possible to define what is 

“suspected resistance” as described under the “Remedial Action Plans” to mitigate the spread 

of putative resistant populations. Because of the importance of these guidelines, it should be 
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required that the registrant develops an interim rootworm damage guidelines by 2008 and final 

guidelines by 2010 and submits these to the Agency for review. 

 

It is recommended that MIR604 maize be given the following resistance monitoring requirements: 

1. The registrants should monitor for resistance and/or trends in increased tolerance for corn 

rootworm. Sampling should be focused in those areas in which there is the highest risk of 

resistance development. The registrants should submit to EPA an appropriate sampling 

protocol as part of its monitoring plan. 

2. The registrants should provide EPA a description of its resistance monitoring plan by January 

31, 2007. The description would include: sampling (number of locations and samples per 

locations), sampling methodology, bioassay methodology, standardization procedures 

(including QA/AC provisions), detection technique and sensitivity, and the statistical analysis 

of the probability of detecting resistance. A final resistance monitoring plan is required by 

January 31, 2008. 

3. The registrants should develop an appropriate discriminating or diagnostic dose assay by 

January 31, 2008. 

4. The registrants should follow-up on grower, extension specialist or consultant reports of 

unexpected damage or control failures for corn rootworm. 

5. The registrants should provide EPA with an annual resistance monitoring report. 

The remedial action plan is designed as a tiered approach for mitigating potential WCRW, NCRW, and 

MCRW resistance development to the Cry34/35Ab1 protein. BPPD agrees with the general framework 

for the Remedial Action Plan; however, because the “baseline sensitivity” has not been calibrated, this 

plan cannot be implemented. The submission states that mitigation measures will be initiated when 

unexpected levels of CRW damage occur. But, levels of “expected” damage cannot be identified until 

baseline sensitivity is determined (see discussion above “Resistance monitoring”). Consequently, it 

should be required that baseline sensitivity be established within two years of product 

commercialization, so that expected levels of crop damage and target pest resistance can be 

established, and a remedial action plan initiated when needed. 

The following program summary describes, in order of events, the steps that should be taken to 

implement a remedial action plan if resistance to target pests is confirmed (this general process has 

been implemented for other lepidopteran and CRW Bt corn products). 
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1. Definition of Suspected Resistance: Resistance will be suspected if investigations of unexpected 

damage reports show that: 

a. Implicated maize plant roots were expressing the mCry3A protein at the expected level; 

b. Alternative causes of damage or lodging, such as non-target pest insect species, weather, 

physical damage, larval movement from alternate hosts, planting errors, and other 

reasonable causes for the observations, have been ruled out; 

c. The level of damage exceeds guidelines for expected damage. 

If resistance is “suspected”, the registrants will instruct affected growers to use alternate pest control 

measures such as adulticide treatment, crop rotation the following year, or use of soil or seed 

insecticides the following year. These measures are intended to reduce the possibility of potentially 

resistant insects contributing to the following year’s pest population. 

2. Confirmation of Resistance: Resistance will be confirmed if all of the following criteria are met 

by progeny from the target pest species sampled from the area of “suspected resistance”: 

a. The proportion of larvae that can feed and survive on mCry3A roots from neonate to adult 

is significantly higher than the baseline proportion (currently being established); 

b. The LC50 of the test population exceeds the upper limit of the 95% confidence interval for 

the LC50 of a standard unselected population and/or survival in the diagnostic assay is 

significantly greater than that of a standard unselected population, as established by the 

ongoing baseline monitoring program; 

c. The ability to survive is heritable; 

d. mCry3A plant assays determine that damage caused by surviving insects would exceed 

economic thresholds; 

e. The identified frequency of field resistance could lead to widespread product failure if 

subsequent collections in the affected field area(s) demonstrated similar bioassay results. 

3. Response to Confirmed Resistance: When resistance is “confirmed”, the following steps will be 

taken: 

a. EPA will receive notification within 30 days of resistance confirmation; 

b. Affected customers and extension agents will be notified about confirmed resistance; 

c. Affected customers and extension agents will be encouraged to employ alternative 

CRW control measures; 

d. Sale and distribution of mCry3A maize in the affected area will cease immediately; 

e. A long-term resistance management action plan will be devised according to the 

characteristics of the resistance event and local agronomic needs. [The details of such a 

plan should be approved by EPA and all appropriate stakeholders. This process may 

take longer than 30 days following confirmation of resistance.] 
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e. Support for Syngenta’s IRM Plan for MIR604 Maize 

1) 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 MIR604 maize. Syngenta conducted a series 

of field efficacy trials across the Corn Belt in 2003 and 2004 to assess dose. This information was 

used in the modeling study detailed later in this review. Additional data were obtained from root 

expression assays and in vitro feeding assays in the laboratory. Based on the review of Syngenta’s 

data, it can be concluded that MIR604 maize does not provide a high dose for CRW control. 

a) Root expression assays

Levels of expression in leaves, roots, and whole plants were determined at two developmental stages 

for two MIR604 hybrids. Levels of expression were numerically higher in leaves than in roots at both 

the whorl stage and at anthesis and somewhat higher in roots at the whorl stage than at anthesis, but 

there were few, if any statistically significant differences at p<0.05. Root expression was found to be 

uniform throughout the root architecture implying that there are no microhabitats. Therefore, larvae 

feeding on MIR604 roots should be uniformly exposed to the mCry3A toxin. Average values of 

mCry3A in roots range from 1.8 to 3.0 μg mCry3A/g fresh weight. 

b) WCRW laboratory feeding assays

In the first laboratory feeding study, nine replicate groups of ten larvae were placed in plastic dishes 

containing 3 cm root sections excised from MIR604 hybrids. Five of nine replicates exhibited only 

minimal root damage (≤ 1 feeding hole) with an average of 22% mortality; while, three of nine 

replicates exhibited two to three feeding holes with an average of 13.3% mortality. Only one replicate 

had more than four or more feeding holes. 

In the second laboratory feeding study, nine replicate groups of ten larvae were placed in plastic dishes 

were given a choice of two 3 cm root sections excised from MIR604 and non-transgenic (control) 

hybrids. In this second experiment, larvae preferred the non-transgenic hybrid to the MIR604 hybrid. 

Approximately 75% of non-transgenic hybrid roots were heavily damaged with more than four feeding 

holes per root section. As in the first experiment, the mortality levels on transgenic root sections were 

low, only 17%. A third experiment, using a similar choice protocol, produced similar results to the 

second experiment, but there was considerably more variation. 

Following investigations by Syngenta and its co-operators, there is evidence for both a direct mortality 

mechanism of first instar CRW larvae as well as evidence that MIR604 hybrids have properties that 

might deter feeding. Thus, in terms of root protection, MIR604 plants exhibit a much higher dose than 

would otherwise be assumed from the consideration of the mortality data alone. The behavioral 

deterrence mechanism needs to be studied further by Syngenta. 
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c) Field efficacy

In 2003 and 2004 Syngenta conducted field-based studies of MIR604’s efficacy against WCRW and 

NCRW and study results were used to develop models of MIR604. Trials included naturally and 

artificially infested sites and study endpoints were survival until adult stage, timing of adult 

emergence, sex ratio, and adult weight. Both artificial and natural infestation trials were conducted. A 

brief summary of the results is provided below. 

Efficacy trials compared root damage among MIR604 and non-MIR604-derived corn hybrids. 

Experimental plots were artificially infested (400 to 1450 eggs/trial), located in fields with naturally 

occurring populations of corn rootworm, or both approaches to infestation were combined. When 

artificially infestation was used, at the V2 to V3 leaf stage rootworm eggs were mechanically placed 3 

inches deep in soil, within 2 to 3 inches of the corn stalk base. 

i. Artificial Infestation with WCRW:

The percentage of artificially infested CRW eggs known to survive to the larval stage is low, and even 

lower for adult survival. Consequently, at each trial location the ratio of viable eggs to larvae was 

assessed, and comparative estimates of mortality made. 

Results indicate that MIR604 provided better control of WCRW than untreated non-transgenic 

controls. Emergence data from 2003 show that MIR604 controlled an average of 89.9% of CRW, with 

a low of 74% at Walcott, IA and a high of 97% at Bloomington, IL (Table 1). On an emergence per 

hectare basis, non-transgenic control plots averaged 740,410 beetles per hectare, while MIR604 plots 

averaged 72,019 beetles. 
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Table 1. Summary of artificial infestation trials that evaluate control of WCRW by MIR604 

corn. Product efficacy is estimated by the reduction in adult emergence from MIR604 plants 

relative to untreated, non-transgenic control plants. 

Location 

2003 

Walcott 

IA 

2003 

Stanton 

MN 

2003 

Bloomington 

IL 

2003 

Stanton 

MN 

Artificial 

infestation 

totals 

Artificial 

infestation 

averages 

Plants/ha 61,507 61,507 61,507 61,507 61,507 

Viable eggs/plant 350 428 405 224 352 

Initial density 

eggs/ha 21.6 M 26.4 M 25.0 M 13.8 M 21.7 M 

Emergence in 

MIR604 total 

(F+M) 

38 48 14 18 118 30 

Emergence in 

negative control 

total (F+M) 

145 314 406 299 1164 291 

Area evaluated 

per entry (ha) 0.00047904 0.00038323 0.00038323 0.00038323 0.0016287 0.00040718 

MIR604 total 

(F+M)/ha 79,325 125,250 36,531 46,969 288,075 72,019 

Negative cont. 

total (F+M)/ha 302,688 819,344 1,059,407 780,203 2,961,642 740,410 

% control 73.8% 84.7% 96.6% 94.0% 90.3% 90.3% 

ii. Natural Infestation with WCRW:

Field trials using natural infestation were implemented at six locations in 2004. Three of these 

locations (Clay Center, NE, Ames, IA, and Higginsville, MO) were lightly infested – characterized by 

few beetles and minimal plant root damage. The remaining three locations (Bloomington, IL, Urbana, 

IL, and Mead, NE) had a high level of infestation – characterized by high beetle population densities 

and a high level of plant root damage. 

Percent control was determined by comparing emergence in MIR604 plots against the non-transgenic 

control. This method assumed that the natural infestation of WCRW was evenly distributed across 

treatments (an unlikely scenario), although the procedure may provide a rough estimate of the efficacy 

of MIR604. At the lightly infested locations, MIR604 provided greater control of WCRW than nontransgenic controls, with a mean of 92.2% beetle control compared to the controls (Table 2). At 

heavily infested locations MIR604 provided an average of 11.1% beetle control, with a low of -13.5% 

and a high of 44.5%, compared to non-transgenic control treatments (Table 3). 
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Table 2. Summary of light pressure natural infestation trials that evaluate control of WCRW 

by MIR604 corn. Product efficacy is estimated by the reduction in adult emergence from 

MIR604 plants relative to untreated, non-transgenic control plants.

Location 

2004 

Clay Center 

NE 

2004 

Ames 

IA 

2004 

Higginsville 

MO 

Light 

pressure 

natural 

infestation 

totals 

Light pressure 

natural 

infestation 

averages 

Plants/ha 61,507 61,507 61,507 61,507 

Emergence in 

MIR604 

total (F+M) 

23 35 16 74 25 

Emergence in 

negative 

control total (F+M) 

263 276 412 951 317 

Area evaluated 

per entry 

(ha) 

0.00038323 0.00038323 0.00038323 0.0011497 0.00038323 

MIR604 total 

(F+M)/ha 60,016 91,328 41,750 193,094 64,365 

Negative cont. total 

(F+M)/ha 686,266 720,188 1,075,063 2,481,517 827,172 

% control 91.3% 87.3% 96.1% 92.2% 92.2% 
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Table 3. Summary of heavy pressure natural infestation trials that evaluate control of WCRW 

by MIR604 corn. Product efficacy is estimated by the reduction in adult emergence from 

MIR604 plants relative to untreated, non-transgenic control plants. 

Location 

2004 

Bloomington 

IL 

2004 

Urbana 

IL 

2004 

Mead 

NE 

Heavy pressure 

natural 

infestation 

totals 

Heavy pressure 

natural 

infestation 

averages 

Plants/ha 61,507 61,507 61,507 61,507 

Emergence in MIR604 

Total (F+M) 126 386 345 857 286 

Emergence in negative 

control 

total (F+M) 

227 340 397 964 321 

Area evaluated per entry 

(ha) 0.00038323 0.00038323 0.00038323 0.0011497 0.00038323 

MIR604 total (F+M)/ha 328,781 1,007,219 900,235 2,236,235 745,412 

Negative cont. total 

(F+M)/ha 592,328 887,188 1,035,922 2,515,438 838,479 

% control 44.5% -13.5% 13.1% 11.1%

iii. Natural Infestation with NCRW:

High natural infestations of NCRW were seen at one location in 2003 and six locations in 2004. 

Infestations were light at Stanton, MN, Cavour, SD, Bloomington, IL, and Mead, NE, while pressure 

was heavier in Walcott, IA, Ames, IA, and Higginsville, MO. Percent control was determined by 

comparing emergence in MIR604 plots against the non-transgenic control. This method assumed that 

the natural infestation of NCRW was evenly distributed across treatments (an unlikely scenario), 

although the procedure may provide a rough estimate of the efficacy of MIR604. At the Ames and 

Higginsville locations NCRW emergence reached one third of total CRW adult emergence. For these 

locations, control of NCRW ranged from 45 to 95%. Control ranged from 34.7 to 100% in areas with 

lower NCRW pressure (Table 4). 
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Table 4. Summary of natural infestation trials that evaluate control of NCRW by MIR604 corn. 

Product efficacy is estimated by the reduction in adult emergence from MIR604 plants relative 

to untreated, non-transgenic control plants.

Location 

2003 

Walcott 

IA 

2004 

Stanton 

MN 

2004 

Cavour 

SD 

2004 

Bloomington 

IL 

2004 

Ames 

IA 

2004 

Higginsville 

MO 

2004 

Mead 

NE 

Plants/ha 61,507 61,507 61,507 61,507 61,507 61,507 127,859 

Emergence in MIR604 

Total (F+M) 28 2 0 5 63 10 32 

Emergence in negative 

control total (F+M) 73 22 53 12 115 218 49 

Area evaluated per 

entry (ha) 0.00047904 0.00038323 0.00038323 0.00038323 .00038323 0.00038323 0.00038323 

MIR604 total 

(F+M)/ha 58,450 5,219 0 13,047 164,391 26,094 83,500 

Negative cont. total 

(F+M)/ha 152,388 57,406 138,297 31,313 300,078 568,844 127,859 

% control 61.6% 90.9% 100.0% 58.3% 45.2% 95.4% 34.7% 

d) Adult emergence

To make realistic judgments regarding the likelihood that a resistant CRW beetle will pass on 

resistance alleles to the next generation, adult fitness and timing of emergence must be understood. 

Both the fitness of the surviving beetles and the timing of their emergence from the crops have a major 

impact on their ability to mate with other WCRW adults that will have an impact on their ability to 

contribute genetically to the subsequent generation. Based on all MIR604 replicate treatments, there 

was a delay in the emergence of adults from the crop when compared to the untreated negative isoline 

control treatments. 

Over all locations, a mean delay of 7.7 days was seen for adult emergence in all MIR604 treatments 

when compared to non-transgenic controls. No discernable differences were seen between treatments 

with light or heavy pressure and between male and female beetles. 

i) Artificial infestation with WCRW:

In trials that used artificial infestation, delays of 6 to 10 days compared to non-transgenic controls were 

commonly seen among male and female beetles (Table 5). 
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Table 5. Emergence delay times (days) for adult male and female WCRW in emerging from 

artificially infested MIR604 corn fields in 2003 and 2004. 

Location 2003 

Walcott IA 

2003 

Stanton MN 

2003 

Bloomington 

IL 

2004 

Stanton 

MN 

Artificial 

infestation 

averages 

Emergence parameter Emergence (days) 

5% male emergence 6.1 4.2 0.1 18.4 7.2 

50% male emergence 4.6 9.8 0.9 11.0 6.6 

80% male emergence 2.6 14.3 1.6 10.2 7.1 

5% female emergence 14.8 3.5 1.3 20.4 10.0 

50% female emergence 6.9 8.4 -0.3 9.0 6.0 

80% female emergence 5.0 8.1 6.6 8.5 7.0 

Mean emergence delay for M+F 6.7 8.0 1.7 12.9 7.3 

ii) Natural infestation with WCRW:

Emergence delays of over 10 days were seen among female CRW beetles in locations with light 

natural infestation (Table 6). Few males were identified in these plots, so emergence time could not be 

reliably measured. Emergence delays in locations with heavy natural infestation were shorter than 

delays seen in sites with light emergence (Table 7). This finding was heavily influenced by the Urbana 

IL results, which were atypical and for efficacy purposes, considered an outlier. Removing the Urbana 

results from the heavily infested data set brings the mean value for emergence to 6.5 days, which is 

more similar to results seen in the artificial infestation trial. 

Overall, statistical analysis showed no significant difference in rates of emergence among sexes, 

locations or treatments. The most reliable data were collected from three trials in 2004 that recorded 

over 100 adults in emergence cages. For these sites, a mean delay of 5.8 days was calculated for males 

and females. Consequently, the modeling study (described in modeling section) uses a standard delay 

of 6 days for males and females emerging from MIR604 corn fields. 
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Table 6. Emergence delay times (days) for adult male and female WCRW in emerging from 

light pressure naturally infested MIR604 corn fields in 2003 and 2004. 

Location 2004 

Clay Center NE 

2004 Ames 

IA 

2004 

Higginsville MO 

Light pressure 

natural 

infestation averages 

Emergence parameter Emergence (days) 

5% male emergence N/A* N/A* N/A* N/A* 

50% male emergence N/A* N/A* N/A* N/A* 

80% male emergence N/A* N/A* N/A* N/A* 

5% female emergence 8.6 12.3 11.5 10.8 

50% female emergence 15.4 12.1 7.0 11.5 

80% female emergence 19.4 12.5 5.8 12.6 

Mean emergence delay for 

M+F 14.4 12.3 8.1 11.6 

N/A* - < / = 3 beetles emerged from this entry at this location, and therefore data are excluded from delay calculations 

Table 7. Emergence delay times (days) for adult male and female WCRW in emerging from 

heavy pressure naturally infested MIR604 corn fields in 2003 and 2004. 

Location 2004 

Bloomington IL 

2004 

Urbana IL 

2004 

Mead NE 

Heavy pressure natural 

infestation averages 

Emergence parameter Emergence (days) 

5% male emergence 1.1 6.9 9.5 5.8 

50% male emergence 3.4 4.8 5.0 4.4 

80% male emergence 11.2 -3.2 5.9 4.6 

5% female emergence 2.4 4.5 10.6 5.8 

50% female emergence 7.0 -4.9 9.4 3.8 

80% female emergence 8.7 -6.5 4.1 2.1 

Mean emergence delay for M+F 5.6 0.3 7.4 4.4 

iii) Artificial infestation with NCRW:

In 2003 and 2004 data on NCRW emergence was collected from four locations. Although there was 

some variability among locations, emergence delays ranged from 5.5 to 11 days. As noted with 

WCRW emergence data, beetle sex was not shown to affect rate of emergence. 
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Table 8. Emergence delay times (days) for adult male and female NCRW in emerging from 

naturally infested MIR604 corn fields in 2003 and 2004. 

Location 2003 

Walcott IA 

2004 

Ames IA 

2004 

Higginsville 

MO 

2004 

Mead NE 

NCRW natural 

infestation 

averages 

Emergence parameter Emergence (days) 

5% male emergence 7.5 0.0 N/A* 12.9 6.8 

50% male emergence 2.9 6.1 N/A* 9.5 6.2 

80% male emergence 1.4 3.9 N/A* 19.6 8.3 

5% female emergence 18.4 6.4 13.1 14.4 13.1 

50% female emergence 9.7 10.5 5.6 6.3 8.0 

80% female emergence 6.3 6.1 -0.5 3.2 3.8 

Mean emergence delay 

for M+F 7.7 5.5 6.1 11.0 7.6 

N/A* - < / = 3 beetles emerged from this entry at this location, and therefore data are excluded from delay calculations 

iv) Adult body weight

The body weights of adult WCRW beetles were recorded at the artificially infested trial in Stanton, 

MN. Results show that the mean body weight of adult beetles emerging from MIR604 corn fields was 

significantly greater than the weight of those emerging from non-transgenic plots. 

 

Table 9. Mean dry weight of adult WCRW emerging from artificial infestation field trial at 

Stanton, MN in 2003. 

Treatment % Control w.r.t. untreated 

control 

Mean adult weight 

(g x 10-5) 

MIR604-3-11 87.0 206.5 bc 

MIR604-3-12 90.3 183.4 bc 

Untreated, non-transgenic, 

control 0.0 155.4 c 

Non-transgenic + FORCE 3G 85.9 222.8 ab 
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2) Simulation Models for Development of Resistance 

Simulation modeling was used by Syngenta to predict the evolution of WCRW resistance to the 

mCry3A toxin expressed in MIR604 maize. Models have been useful tools in the development of IRM 

strategies. Previously, Monsanto modified a Caprio model for cotton bollworm (Helicoverpa zea) to 

predict the risk of CRW developing resistance to MON 863 corn (U.S. EPA, 2002). Andow and 

Alstad (2002), Onstad et al. (2001), and Onstad et al. (2003) used deterministic models to predict the 

evolution of WCRW resistance using a variety of management strategies. The Andow and Alstad 

model predicted that, under certain conditions, a 20% refuge would probably delay resistance for 

greater than 15 generations when the dose was low, but would be ineffective for resistance 

management of a high dose (U.S. EPA, 2003). In Onstad et al. (2001), the model showed that when 

resistance was dominant, the resistance allele frequency exceeded 3% within 2-5 years as refuge size 

ranged from 5 to 30% for all doses of toxin, but the resistance allele frequency never exceeded 3% 

when resistance was recessive. This model was further modified to examine the impact of landscape 

on WCRW resistance evolution (Onstad et al., 2003). Both of the Onstad models were further 

expanded to evaluate the risk of resistance by WCRW to both transgenic crops and crop rotation in 

areas with or without rotation-resistant phenotypes (Crowder et al., 2005). Storer (2003) developed a 

stochastic, spatially explicit model to simulate adaptation (resistance) of WCRW in much of the US 

Corn Belt. In this model, the relative rate of adaptation was affected by the refuge size and the manner 

in which the non-transgenic refuge maize was deployed. Specifically, the adaptation rate was lowest if 

the non-transgenic refuge maize was planted in the same fields each year (Storer, 2003). This model 

was further modified to predict the evolution of resistance to Cry34Ab1/Cry35Ab1 and considered the 

influence of rotation-resistant phenotypes (U.S. EPA, 2005). 

3) Syngenta’s Specific Modeling for MIR604 Maize 

a) Methods

Syngenta worked with Dr. David Onstad of the University of Illinois to customize the Crowder et al.

(2005) model using the value for efficacy (i.e., dose) generated by Syngenta (see section on “Dose” 

above). The model includes patches of crops without explicit spatial structure. Each patch had a basic 

spatial unit of a 100 ha farm and was in a homogeneous region consisting of similar farms. The typical 

model landscape had four crops and MIR604 transgenic maize planted to both continuous and rotated 

cornfields. The crops include continuous corn, rotated corn, rotated soybean, and extra vegetation. Six 

scenarios were studied. Scenario I consisted only of 100% continuous maize fields in the landscape 

with no rotation-resistant WCFW in this area. Scenarios II-V had landscapes with 5% extra 

vegetation, 10% continuous corn, 42.5% soybean, and 42.5% rotated corn based on the standard set in 

the work of Crowder et al. (2005). In these four scenarios the area was inhabited by rotation-resistant 

WCRW. Scenario VI was similar to scenario I, but in this case a soil insecticide was used in the 

refuges, giving 50% survival of refuge insects. In scenarios IV-V, transgenic corn was planted only in 

rotated maize fields. Crowder et al. (2005) demonstrated that planting transgenic maize only in 

continuous maize fields in these kinds of landscapes was an inferior strategy so this scenario was not 

evaluated. In areas with rotation-resistant WCRW (scenarios II-V), there were two kinds of landscapes 
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evaluated: one with an initial resistance allele frequency of 0.0001 of the rotation-resistance allele (Y) 

in the rootworm population (scenarios II and IV) and the second had an initial Y-allele frequency of 

0.1 (scenarios III and V). In both cases, the expression of the rotation resistance allele was dominant. 

Previously, Crowder et al. (2005) and Crowder and Onstad (2005) demonstrated that the simulation of 

realistic evolution of rotation resistance required either additive or dominant expression. 

Three refuge levels were evaluated: 10%, 20%, and 30%. The refuge fields and MIR604 fields were 

assumed to be planted together in either continuous maize or in rotated maize. The refuge was 

assumed to be planted in the MIR604 field or adjacent to it. If the refuge was continuous maize, the 

refuge had to remain in the same place every year; otherwise, the refuge in rotation maize fields could 

change location from year to year. 

The time horizon used was 15 years after introduction of MIR604 transgenic maize. This model had a 

daily time step during July-September to simulate adult activity. 

Resistance to MIR604 transgenic maize and rotation was modeled as dominant, recessive, or additive. 

Precise additive expression for resistance to transgenic maize was used. Resistance to crop rotation 

was modeled as dominant (Y allele). 

The initial adult density was assumed to be 50,000/ha of maize for all types. The sex ratio of adults 

was 50:50. The initial allele frequency for resistance to MIR604 transgenic maize was 0.0001. The 

initial allele frequency for resistance to rotation was 0, 0.0001, or 0.1. 

A seed insecticide treatment was not included in most scenarios except for Scenario VI, which 

included soil insecticide treatment in refuge in continuous corn. Larval survival was 50% before 

density-dependent survival. 

The model used a range of MIR604 transgenic maize mCry3A doses derived from the adult emergence 

data provided by Syngenta (see discussion above) and calculated using two larval survival functions 

described by Onstad et al. (2001, 2003). These calculations are not shown in this review, but can be 

found in McCaffery et al. (2005, MRID# 465296-01). Homozygous resistant individuals (RR) were 

assumed to always have 100% survival to MIR604 maize. Heterozygous individuals with R dominant 

(sR) were assumed to always have a 100% survival to the transgenic crop. The survival of 

homozygous susceptible individuals (SS) and heterozygote individuals with R recessive (Sr) were 

assumed to have a survival of 0, 0.001, 0.05, and 0.20 to represent a theoretical high, practical high, 

medium, or low toxin dose, respectively. 

The standard density-dependent survival of larvae per state is 0.21 X exp (-0.058 EGG), where EGG is 

the density of eggs in millions per ha. The maximum larval survival based on this function was 21%. 

It was assumed that density-dependent mortality occurred after mortality due to overwintering and 

toxin exposure. In a second approach, it was assumed that mortality is density-independent based on 

the field data collected by Hibbard et al. (2004); thus, 5% of larvae survived after overwintering and 
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toxin exposure. The Urbana, IL field data collected in 2004 was omitted from the analyses because it 

was considered an outlier. The data from 2003 and 2004 were combined. Assuming that densitydependent survival occurs, the overall mean proportion of survival by susceptibles to the mCry3A 

toxin in the nine trials was 0.093. For the five trials with natural infestations, the mean survival was 

0.14. For the four trials with artificial infestations, the mean survival was 0.038. For densitydependent survival, a range of 0.038, 0.093, to 0.14 for the survival rate of susceptible neonates 

encountering MIR604 transgenic maize was simulated. 

The mean larval survival based on the assumption of density-independent mortality after surviving 

toxin was calculated based on proportional differences between emergence of adults in control cages 

and treated cages. The overall mean survival by susceptibles to the toxin in the nine trials was 0.24. 

For the five trials with natural infestations, the mean survival was 0.34. For the five trials with natural 

infestations, the mean survival was 0.34. For the four trials with artificial infestations, the mean 

survival was 0.125. Larval WCRW survival on MIR604 maize was simulated as 0.125, 0.24, and 0.34. 

Delays in the emergence of adults were determined from data supplied by Syngenta (see discussion on 

adult emergence above). In the modeling done by Crowder et al. (2005), there was no significant 

effect of early mortality and delays of 10 and 14 days. For all cases in 2003-2004 in which there were 

at least 38 adults emerging in the treatment cages, there observed delays ranged from no delay to a 

delay of almost 15 days. A mean delay of 5.8 days was calculated for both males and females for three 

situations in which over 100 adults emerged from the treatment cages (most reliable data). The model 

used a standard delay of 6 days for both susceptible males and females emerging in MIR604 transgenic 

maize fields. Susceptible geneotypes are SS when R is dominant and SS and rS when R is recessive. 

For the additive case, simulations were run with only SS having early mortality, and then other 

simulations were run with both SS and RS having early mortality. 

Sensitivity analyses were conducted. In all sensitivity analyses a refuge size of 20% was simulated. 

In the sensitivity analysis all variables were set to standard conditions (unless otherwise noted) except 

for the function being tested. The effect of initial population size, effect of initial R allele frequencies 

(resistance to transgenic corn) and the effect of lower fecundity by susceptible adults in transgenic 

maize were also included in the sensitivity analyses. The sensitivity analysis also used a 12-day delay 

(in addition to the 6-day delay) to determine how the results were affected by a longer delay. 
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Table 10. Landscape Scenarios Modeled. 

I. No rotation resistance (Y allele frequency = 0); only continuous maize 

II. Rotated maize with Y allele frequency = 0.0001 

5% extra vegetation, 10% continuous maize, 42.5% soybean, 42.5% rotated maize 

Transgenic MIR604 maize in both rotated and continuous maize fields 

III. Rotated maize with Y allele frequency = 0.1 

5% extra vegetation, 10% continuous maize, 42.5% soybean, 42.5% rotated maize 

Transgenic MIR604 maize in both rotated and continuous maize fields 

IV. Rotated maize with Y allele frequency = 0.0001 

5% extra vegetation, 10% continuous maize, 42.5% soybean, 42.5% rotated maize 

Transgenic MIR604 maize only in rotated maize fields 

V. Rotated maize with Y allele frequency = 0.1 

5% extra vegetation, 10% continuous maize, 42.5% soybean, 42.5% rotated maize 

Transgenic MIR604 maize only in rotated maize fields 

VI. No rotation resistance (Y allele frequency = 0); only continuous maize 

Annual soil insecticide use in refuge with larval survival of 50% 

b) Results from Syngenta’s Specific Modeling for MIR604 Maize

Syngenta’s modeling results are found in Appendix 1 of their submission (McCaffery et al., 2005, 

MRID# 465296-01). 

1. Recessive resistance allele. If resistance to transgenic MIR604 maize is recessive, then 

WCRW never became resistant within 15 years in all simulations for all scenarios. 

2. Dominant resistance allele. For scenarios I-III, and VI, either when larval survival was densitydependent or density-independent, resistance to transgenic MIR604 maize evolved in less than 

13 years with a 20% refuge (all three toxin scenarios). For scenarios IV and V, either when 

larval survival was density-dependent or density-independent, resistance to transgenic MIR604 

maize did not evolve within the 15 years of the simulation. Resistance evolution using the 

10% refuge was worse for scenarios I-III, and VI than using the 20% refuge. The 30% refuge 

delayed evolution of resistance several years beyond 15 in Scenario III, IV, V when larval 

survival was density-dependent and density-independent, but resistance still evolved in less 

than 15 years in Scenarios I, II, and VI. 

3. Additive resistance expression. Two simulations representing two possible effects of additive 

expression on early mortality were run for each combination of toxin survival (3 combinations) 

and refuge level (3 levels) for a total of 18 simulations. Like the results for dominant resistance 
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expression, the results for scenarios I-II were much different from those for scenarios IV-V 

when expression was additive. 

Density-Dependent. For Scenarios I and II, with density-dependent survival, resistance evolved in less 

than 15 years using both the 10% and 20% refuge options for all but one simulation that for additive 

expression case #1 in which the toxin survival was 0.14 using a 20% refuge. No resistance was 

predicted if a 30% refuge (with density-dependent survival) was used in any of the six landscape 

scenarios with additive expression (both case #1 and #2) during the 15 years of the simulation. No 

resistance evolved during the 15 years of the model simulations using scenarios IV and V (MIR604 

maize was planted only in rotated maize fields and rotation-resistance existed). 

Density –Independent. Resistance did not evolve during the 15 years of the simulation under scenarios 

I-V using a 20% and 30% refuge if there was density-independent survival except in additive 

expression case #2 when toxin survival was 0.125 for larvae in scenarios I-III. In these three 

simulations, resistance evolved in 13 years. A 10% refuge delayed resistance for at least 10 years in 

scenarios I-III. Resistance did evolve in less than 15 years using all refuge sizes and all toxin 

combinations with scenario VI (continuous maize treated with soil insecticide). In this case, the 20% 

refuge with an annual soil insecticide causing 50% mortality will effectively become a 10% refuge. 

No resistance evolved during the 15 years of the model simulations using scenarios IV and V. 

c) Conclusions from Syngenta’s Specific Modeling for MIR604 Maize

The sensitivity analysis indicated that changes in the initial allele frequency had the greatest effect on 

results. Results were insensitive to recessive expression in MIR604 maize. Simulations using scenario 

V were insensitive to changes in initial adult density, R-allele frequency, and emergence delay. For 

scenario I and dominant expression of resistance to transgenic MIR604 maize, the results were 

generally not sensitive to increases in initial pest density, lengthening of the early-mortality period, no 

reduction in fecundity of survivors in transgenic MIR604 maize fields. For scenario I and additive 

expression or dominant expression, results were sensitive to changes in initial allele frequency. 

If rootworm resistance to the mCry3A toxin as expressed in MIR604 maize is recessive (this is thought 

to be the case), then the modeling study suggests that an IRM plan with a 20% refuge, as proposed by 

Syngenta, will be adequate for delaying the evolution of resistance for at least 15 years. If MIR604 

maize is planted in areas with observable rotation-resistance in the WCRW population, then the 

simulations indicate that planting the transgenic maize only in rotated maize fields is a good IRM plan 

that will delay resistance evolution to the mCry3A toxin expressed in MIR604 maize by at least 15 

years regardless of gene expression. This strategy will also counteract the rotation resistance in 

WCRW (see Crowder et al., 2005). 

The most complicated cases are for areas without rotation-resistance or for framers who want to plant 

continuous corn. The simulations show that resistance to MIR604 maize may occur in 9-12 years with 

a 20% refuge, a toxin survival of 0.093 (the overall mean) and with dominant expression of resistance. 
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If expression is additive, the evolution of resistance is delayed a bit more than for dominant expression 

by 1-2 years. If the initial resistance allele frequency is higher than assumed then resistance can occur 

a few years earlier. If soil insecticides are used on an annual basis in the refuge, then a 20% refuge 

effectively becomes a 10% refuge when resistance expression is dominant or additive and resistance 

evolves more quickly. 

 

4) Impact of MIR604 on Biology of CRW 

In order to develop an effective IRM plan and appropriate deployment strategies for MIR604, 

Syngenta submitted information describing CRW biology, ecology, behavior and toxicology related to 

MIR604. This information is especially important for this product because some individuals survive 

after feeding on the event. Not only is it important to understand the relative fitness of adults that 

emerge after feeding on MIR604, it is also important to understand the behavior of surviving larvae. 

For the most part Syngenta submitted detailed information to address these issues. Syngenta considers 

this research ongoing, however, and BPPD anticipates additional information when it comes available. 

Evidence and observations made by Syngenta show that corn rootworm males normally start to emerge 

before females and this emergence period generally continues for over a month (Hein at al., 1998; 

Elliot and Hein, 1991; Meinke, 1995). Experiments conducted by Syngenta in 2003 and 2004 indicate 

that WCRW adults emerging from MIR604 emerge an average of 6-7 days later than WCRW adults 

emerging from non-transgenic isoline corn. A similar delay in emergence was observed with NCRW. 

Syngenta found no evidence that there is a difference in the delay in emergence between males and 

females (although there was some variability and the emergence profiles did vary). This last 

observation is especially important because it implies that normal mating patterns are not likely to be 

disrupted by any shift in the sex ratios normally found in the field. Given that the emergence period of 

WCRW is over a month, the observed delays of 6-7 days for WCRW adults emerging from MIR604 

plots should not impact their availability to mate with adults from refuge plots. Syngenta also 

investigated the impact of density-dependent and density-independent mortality with respect to delays 

in emergence. These factors were evaluated for their impact on the evolution of resistance in 

Syngenta’s model (discussed above). 

It is clear from Syngenta’s submission that the development of surviving larvae is significantly 

delayed, which is reflected in the delay of emerging adults. Therefore, it is also necessary to establish 

that adults emerging from MIR604 are not physiologically compromised in a way that would prevent 

them from mating with refuge adults. Syngenta is currently working with collaborators to fully 

understand the relative fitness of adults that emerge from MIR604. Currently little is known about 

sub-lethal effects of toxicants on specific Diabrotica species. WCRW females mate soon after they 

emerge and need to feed for about two weeks before they can lay eggs (Hein and Tollefson, 1985; 

Hein et al., 1988). Observations have shown early emerging adults survived longer and were more 

fecund than later emerging adults, conferring reduced fitness to the later emerging results (Boetel and 

Fuller, 1997). In most cases, the fitness of insects that are exposed to sub-lethal doses of an insecticide 

is decreased and the number of offspring is reduced (Haynes, 1988). The reduced relative fitness of 
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insecticide-resistant genotypes is also common among insects (Crow, 1957), which has been 

documented for strains of insects resistant to synthetic insecticides (e.g., Ferrari and Geoghiou, 1981; 

Roush and Plapp, 1982) and strains resistant to Bacillus thuringiensis (Groeters et al., 1994; Alyokhin 

and Ferro, 1999). Syngenta’s continued research regarding the biological impact of MIR604 on CRW 

adults is important. 

It is evident from submitted information that the prevention of damage to MIR604 corn roots is not 

necessarily accompanied by high levels of larval mortality. Syngenta and collaborators conducted 

studies to better understand the interactions between CRW larvae and MIR604 roots and the means by 

which roots are protected. To investigate these interactions, both MIR604 and negative isoline plants 

were from seed and infested with newly hatched WCRW. At several intervals (1, 2, 3, 7 and 14 days) 

after infestation estimates of root weight, larva number, larval wet and dry weights and larval feeding 

activity were recorded. Results showed that the weight of MIR604 roots increased significantly after 

day 3 when compared with the control roots, showing that MIR604 roots were protected from WCRW 

damage. With regard to larval numbers, the numbers of WCRW larvae on both negative isoline and 

MIR604 roots declined after infestation, but on MIR604 this decline occurred more quickly and by day 

7 no larvae were alive. This finding contrasts with the negative isoline roots where significant survival 

was observed. Larval feeding was recorded on each assessment day and significantly fewer larvae 

were observed on the MIR604 roots compared to the control. For example, on days 1 and 2 less than 

10% of the larvae were present on MIR604 roots and none were feeding on day 3, whereas about 50% 

of larvae on the control roots were observed feeding at any one time. As expected, this difference in 

feeding resulted in significant differences in larval weight between the two groups. Both wet and dry 

weights of larvae feeding on MIR604 roots were significantly lower than those feeding on negative 

isoline roots. Possibly the most importing information from Syngenta’s research were the behavioral 

findings. It was observed that the larvae feeding on MIR604 roots became sick and either died within 

the first day or survived until day 2 but did not continue to feed on root tissue. Larvae on the MIR604 

roots appeared to have one of two behaviors: 1) feeding without inhibition followed by death a short 

time later, or 2) movement through the root zone, not feeding on the root tissue, but sampling root 

hairs and continuing to search for food. Applying these finding to a field setting, it is possible that 

larvae can survive to the 2nd instar by feeding on root hairs of MIR604 plants, taking in small amounts 

of root tissue, and supplementing their diet with surrounding grassy weeds and plant roots (Wilson and 

Hibbard, 2004; Clark and Hibbard, 2004). By surviving to the 2nd instar stage, which is less 

susceptible to mCry3A than the 1st instar stage, WCRW could re-establish on MIR604. Although a reestablishment of the pest is possible, it is documented that natural loss of WRCW between root 

penetration and adult emergence is high (Elliot et al., 1989). 

5) Cross-Resistance 

It is important to consider the impact of cross-resistance on the evolution of corn rootworm resistance 

to MIR604 maize. Cross-resistance occur when a pest becomes resistant to one Bt toxin, for example, 

and by virtue of this resistance, it confers resistance to another distinct Bt toxin. The degree to which 

this cross-resistance might occur depends largely on the mechanism of resistance characterizing the 
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original resistance and the degree to which the two (or more) toxins are independently compromised 

by those mechanisms. For example, Cry1Ac and Cry1F, two Bt Cry toxins that target Lepidoptera, 

have a least one midgut receptor in common and if target site modification is responsible for resistance 

then a degree of cross-resistance is probably (Granero et al., 1996; Ballester et al., 1999). 

Syngenta discussed the potential for cross-resistance involving the mCry3A toxin in MIR604 maize in 

the IRM submission. To date, no receptor for Cry3 toxins has been definitely isolated or characterized. 

Recently, a cadherin-link protein has been identified in the midgut of WCRW (Siegfried et al., 2005). 

Syngenta’s event MIR604 CRW-control maize will be deployed into a landscape consisting of 

conventional corn and other CRW-control varieties. At present there are two commercially available 

CRW-control varieties that must be included in the evaluation of cross-resistance: Monsanto’s 

YieldGard® Rootworm (MON863) maize that expresses the Cry3Bb1 toxin and Dow AgroSciences 

Herculex® RW maize that expresses the Cry34Ab1/Cry35Ab1 toxins. The IRM plan for MIR604 

must therefore consider the likelihood and possible consequences of the evolution of resistance in 

CRW that confers cross-resistance to multiple transgenic varieties. 

The mode of action of mCry3A expressed in MIR604 CRW-control maize is similar to that of all 

known Cry toxins (see discussion earlier in this review). The degree of sequence homology may 

influence the potential for cross-resistance, but this depends on the specificity of the mechanism of 

resistance that might arise. For Bt Cry toxins, only two modes of resistance have been observed: 1) 

altered detoxification by protease enzymes in the midgut lumen that cleave the toxin and 2) 

modification of the target site receptor on the brush border membrane of the midgut epithelium so that 

binding of the toxin is prevented or hindered (Ferré and Van Rie, 2002). The latter mechanism was 

observed in diamondback moth (Plutella xylostella) that were selected with formulated microbial Bt 

products that resulted in field resistance as described in Tabashnik et al. (1997). Other non-target site 

mechanisms of Bt resistance have also been described: reduced or impaired gut proteolytic activity in 

Plodia interpunctella (Indian meal moth) (Oppert et al., 1994, 1997; Herrero et al., 2001) and 

alternation of toxin processing or some other metabolic step in laboratory-selected strains of Heliothis 

virescens (tobacco budworm) (Jurat-Fuentes et al., 2003). A non-target site mechanism of resistance 

would be detrimental to IRM strategies based around reduced selection resulting from stacked Cry 

toxin genes or mosaic plantings of varieties expressing different toxins. The degree to which such 

non-target site mechanisms of resistance might impact resistance to Cry toxins in CRW is currently 

unknown. 

As described in Ballester et al. (1999), modifications of the target site binding protein resulting from 

selection are likely to confer a very specific spectrum of resistances to closely related Cry proteins that 

also have some affinity with the binding site in question. Between mCry3A and native Cry3Bb1 there 

is a 61.7% identity and 69.1% similarity in amino acid sequences. Taking into consideration the size 

difference between the native and modified Cry3A proteins (mCry3A starts at residue #48), the 

identity is 74.3% and similarity is 66.5%. This level of amino acid homology is not considered to be 

particularly high and such differences indicate that there may be a differential recognition of these two 
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proteins in the insect midgut. Galitsky et al. (2001) note that there are differences between Cry3A and 

Cry3Bb1 in certain oligomeric structures in domain II and domain III that affect pore formation in the 

midgut membrane, regulation of channel function, and specificity towards target pests. These 

differences may affect the behavior of each of these proteins and reduce the likelihood of target-site 

cross-resistance, although this is unknown. 

The Cry34Ab1 and Cry35Ab1 toxins expressed in the Dow AgroScience’s CRW product, Herculex® 

RW represent a new family of insecticidal crystal proteins (Ellis et al., 2002). Although the target site 

for these CRW-active proteins is completely unknown, it can be argued that cross-resistance at the 

target-site between them and the Cry3 toxins is extremely unlikely give the marked divergence in 

structure of these proteins from the Cry3 group. Therefore, cross-resistance between MIR604 and 

Herculex RW is considered to be unlikely and need not be considered further. 

By in large, the mechanisms of resistance that might evolve to Bt toxins in Coleoptera like the CRW 

are largely unknown. Based on the review of the literature, the only genuine cases of resistance to Bt 

toxins suggest that target site resistance is the most common, and probably is responsible for the 

majority of resistance cases. Whether this would actually be the case for CRW is unknown. Work by 

Siegfried et al. (2005), however, suggested that there is similarity of the target sites in Coleoptera and 

Lepidoptera so that if resistance to Cry3 toxins evolves in CRW, it might involve a target site 

modification. For a brief period when there was a commercially available Cry3A-expressing potato in 

the US, potential resistance mechanisms were studied in the laboratory. Based on these experiments, 

Colorado potato beetle was shown to survive for a short period of time on transgenic plants (Rahardja 

and Whalon, 1997). No further characterization of this resistance was ever undertaken as NatureMark 

stopped marketing these Bt potatoes in 2001. 

More recent studies by Rausell et al. (2004) compared the toxin-binding capacities of proteolyticallyprocessed Cry3A, Cry3B and Cry3C toxins to midgut brush border membranes of Colorado potato 

beetle. In vitro heterologous competition binding experiments showed that the three proteoloyticallyactivated Cry3 toxins all shared a common binding site, but Cry3Aa and Cry3Ca have an extra binding 

site that is not shared with the Cry3Ba toxin. This means that there could be some differences in the 

binding of mCry3A in MIR604 and that of Cry3Bb1 in YieldGard RW. The mCry3A has been 

modified to promote processing so that the availability of active toxin for interaction with the binding 

site may be different for different Cry3 toxins in the natural situation in CRW midguts. 

Receptors for Cry3 toxins have never been isolated and characterized. To date, cadherin and 

aminopeptidase-N are most frequently associated with Cry toxin binding in Lepidoptera (e.g., Gahan 

et al., 2001; Luo et al., 1996), although actin (McNall and Adang, 2003), alkaline phosphatase (McNall 

and Adang, 2003), and glycolipids (Griffith et al., 2005) have also been identified more recently in 

binding. Only insect cadherins have been proven to mutate to give resistance to Cry toxins in 

Lepidoptera (Gahan et al., 2001; Morin et al., 2003). Siegfried et al. (2005) used an expressed 

sequence tag to identify the first Coleopteran cadherin gene in CRW. Cadherin could be a receptor for 

Bt proteins in CRW, but further studies are necessary to confirm this possibility. Genetic and 
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biochemical studies with different insect species have shown that resistance mechanisms based on 

target-site genes such as cadherin are inherited as recessive or incompletely recessive traits (Ferré and 

Van Rie, 2002). On the other hand, non-target site mechanisms, such as altered metabolism or 

processing, are more likely to be inherited as incompletely dominant traits. One would therefore 

predict that a recessively-inherited target site mechanism is the most likely to evolve if resistance 

occurs to mCry3A in CRW. The modeling studies (discussed above) include varying degrees of 

dominance. 

Rausell et al. (2004) found that CryBb1 might confer some cross-resistance to Cry3A (mCry3A) 

through modification of the shared receptor. The reverse, however, is not necessarily true. That is, 

resistance to mCry3A could occur through modification of a unique binding site with which Cry3Bb1 

does not interact. Thus, CRW developing resistance to mCry3A expressed in MIR604 maize may not 

confer complete resistance to Cry3Bb1 maize. While Rausell et al. (2004) did demonstrate specific 

binding of processed toxin they did not demonstrate functional receptor binding. Because there is a 

lack of real information on the nature of resistance, especially in CRW, it is best to assume complete 

cross-resistance between Cry3Bb1 and mCry3A. This “worst-case” was assumed in the modeling 

studies discussed above. It is more likely, however, that if CRW resistance does occur to mCry3A, 

only partial cross-resistance to Cry3Bb1 is expected. 

To study these issues further, it is recommended that Syngenta conduct cross-resistance studies using 

CRW colonies resistant to mCry3A. Experiments should be conducted to investigate the nature, 

inheritance, and fitness costs of specific mechanisms of resistance to the mCry3A protein expressed in 

MIR604 maize. 

4. CONDITIONALLY REQUIRED DATA FOR MIR604 CORN (2010 UPDATE) 

As part of the terms and conditions of the MIR604 (mCryA) registration, the registrant was required to 

submit programs for resistance monitoring and refuge compliance. Annual reports summarizing the 

data collected under these requirements must also be submitted to EPA. Further, data were required to 

address aspects of potential resistance to mCry3A. The following terms and conditions were required 

for resistance monitoring, compliance, and additional research: 

 Resistance Monitoring 

o Description of the steps to be taken to establish corn rootworm baseline sensitivity and 

damage guidelines (due within 90 days of registration -- January 31, 2007); 

o Submission of a detailed resistance monitoring plan. Description of the program 

including baseline sensitivity, sampling (number of locations and samples per 

locations), sampling methodology, bioassay methodology, standardization procedures, 

detection technique, sensitivity, and the statistical analysis of the probability of 

detecting resistance, and an interim description of rootworm damage guidelines (due 

January 31, 2008); 
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o Submission of rootworm damage guidelines (for unexpected pest damage) (due January 

31, 2010); 

o Development and validation of an appropriate discriminating or diagnostic dose assay 

(due January 31, 2010); 

o Annual report of the insect resistance monitoring program. Results of monitoring and 

investigations of damage reports (due August 31st each year, beginning in 2008). 

 Compliance 

o Written description of Compliance Assurance Program (due within 90 days of 

registration -- January 31, 2007); 

o Grower Agreements: proposed system to assure growers sign grower agreements and 

affirmation system to assure annual affirmation by growers of their IRM obligations 

(due within 90 days of registration -- January 31, 2007); 

o Annual report on Compliance Assurance Program (CAP) activities and results: thirdparty grower survey, on-farm visitation program, phased-compliance report, tips and 

complaints, and grower education programs (due January 31st each year, beginning in 

2008); 

o Grower education program for MIR604 corn. Subsequent changes to the grower 

education program must be included in the annual compliance assurance program report 

(due January 31, 2008). 

 Additional IRM Data 

o Initiate establishment of CRW strains that are resistant to mCry3A and investigate the 

nature, inheritance, and fitness costs of specific mechanisms of resistance to the 

mCry3A protein expressed in MIR604 maize (due January 31, 2010); 

o Study the behavioral deterrence (avoidance) mechanism further and submit appropriate 

results (due January 31, 2010); 

o Continue studies on the biological impact of adults surviving on MIR604 maize and 

submit these results (due January 31, 2010). 

Syngenta has submitted all of the required reports as mandated under the terms of registration. A 

discussion of the reports and data for the resistance monitoring and compliance programs for MIR604 

corn follows in the next sections. 

a) Resistance Monitoring 

Monitoring Plan for mCry3A Corn (2008) 

A resistance monitoring program for MIR604 (mCry3A) was submitted to EPA in 2008 (MRID 

473401-01; reviewed in BPPD, 2009a). This submission described the monitoring protocols for 

mCry3A corn as well as progress to establish baseline susceptibility data for mCry3A, develop a 

diagnostic bioassay, create a response protocol for unexpected pest damage, and develop a mCry3Aresistant colony. 
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Like other Bt corn registrations, the monitoring plan for MIR604 is based on the framework of the 

Agricultural Biotechnology Stewardship Technical Committee’s (ABSTC) document titled “Updated 

Monitoring Plan for Bt Corn.” Specifically, Syngenta considered the following elements of 

monitoring: spatial patterns of resistance, geographic and species focus, baseline susceptibility data, 

and an annual sampling plan. 

The monitoring program for MIR604 relies on two main components: reports of unexpected pest 

damage and bioassays of field collected Western corn rootworm (Diabrotica virgifera virgifera, 

WCRW). With respect to bioassays and insect collections, WCRW are considered the only reliable 

species for annual monitoring although the Bt toxin is also active against other CRW pests. The 

regions from which Syngenta plans to collect WCRW are listed below. Modifications to the sampling 

program may occur based on MIR604 corn adoption and changes in pest importance. 

 Region 1: Illinois – soybean variant of WCR 

 Region 2: Nebraska – organophosphate resistant WCR 

 Region 3: Iowa – wild type WCR 

After baseline susceptibility has been established for mCry3A, diet bioassays will continue to be 

conducted for the three regions described above. Any unusually low sensitivity to mCry3A will be 

investigated as soon as practical and tied to field relevance. ‘Unusually low’ sensitivity would be 

investigated by considering the following questions: 

 Is low sensitivity heritable? 

 Is the proportion of field collected individuals with unusually low sensitivity that can feed and 

survive on MIR604 corn significantly lower than the baseline proportion? 

 Did plant assays with mCry3A determine that damage exceeded economic threshold? 

Based on 2007 baseline susceptibility data (MRID 480470-02), Syngenta proposed that only 

population samples with LC50 values >8,500 ng/cm2

 would be considered for further investigation. 

This value would be 10-times higher than the mean value observed in past assays while still below the 

maximum concentration used to test field collections. EPA would be informed by Syngenta within one 

month when bioassay results exceeding 8500 ng/cm2

 would be obtained. 

BPPD Analysis of mCry3A Resistance Monitoring Plan

BPPD’s review of the mCry3A monitoring plan (BPPD, 2009a and 2010) concurred with Syngenta 

that it is appropriate to focus the current monitoring efforts on WCRW only. Because of WCRW’s 

widespread distribution and abundance, but similarity in life cycles compared to NCRW and MCRW, 

it may be more likely that they evolve resistance first to mCry3A. BPPD, however, also has concerns 

for NCRW and recommends that Syngenta begin to develop baseline susceptibility data for this target 
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pest. Past difficulties with rearing of NCRW and arguments of limited geographic distribution should 

not be the driving factors for neglecting to monitor for resistance in this important corn pest. 

Syngenta proposed that it would focus its annual monitoring for resistance on 1) using dose response 

curves, 2) field reports of unexpected pest damage, and the 3) development of resistant WCRW 

colonies in the lab. For CRW monitoring and dose-response curves, Syngenta proposed that only 

population samples with LC50 values >8,500 ng/cm2

 would be considered for further investigation. 

BPPD notes that although this value may be 10-times higher than the mean value observed over the 

years, it still appears somewhat arbitrarily chosen. It is not clear how sensitive and reliable this 

particular value will be; LC values may be misleading because larvae can survive on Bt diets for some 

time without feeding. BPPD recommends that Syngenta pursue a sublethal measure to monitor for 

shifts in susceptibility to mCry3A and explore the use of the Sublethal Seedling Assay (SSA) 

developed by Nowatzki et al. (2008). 

Unexpected Pest Damage Guidelines (2010)

Syngenta submitted finalized protocols for monitoring unexpected pest damage in 2010 (MRID 

480470-02). The following strategy was proposed for MIR604 corn: 

In cases of unexpected root damage on MIR604 corn, Syngenta proposed a threshold level of > 2.0 on 

the Node Injury Scale (NIS; Oleson et al., 2005) to trigger investigation. Syngenta set this level based 

on their experience with MIR604 corn. When growers and extension specialists, or consultants report 

to seed dealers that there was unusually high CRW damage (>2.0), then: 

 Syngenta representatives will test for presence of mCry3A in root tissue of damaged plants and 

confirm hybrid and lot number. If tests confirm that mCry3A was present, then additional roots 

will be dug and rate the root damage. At this point it will be determined if other causes were 

responsible for excessive root damage. If damage is again >2.0 and due to CRW, then 

Syngenta will be notified. If confirmed, the representative will dig additional plants and 

determine if the root damage was caused by other corn pests not susceptible to the toxin, 

weather conditions, hybrid genetics, or planting error. If the root node injury in the field is >2 

due to CRW feeding, then Syngenta will be notified. 

 Syngenta will perform more flow strip assays to assess the % of plants expressing mCry3A. If 

more than 95% of the plants tested express the toxin, then Syngenta will collect WCRW for 

analysis. 

BPPD Analysis of Unexpected Pest Damage Guidelines 

BPPD did not concur with Syngenta that the threshold level for unexpected damage for MIR604 

should be >2.0 (see review in BPPD, 2010). BPPD believes that a damage threshold level of 1.0 (NIS) 

may be more appropriate and conservative under moderate insect pressure for single, non-high dose 

CRW products such as are currently registered by Syngenta (MIR604). When insect pressure is 
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exceedingly high, then both Bt protected and refuge corn will incur greater damage. Under these 

circumstances, a threshold level of 1.5 may be more suitable. In addition, for any mCry3A pyramided 

products (i.e., with two toxins expressing a functional high dose), the damage threshold should be 

lower than 1.0 and based on the efficacy against the target pest. 

BPPD recommended that the following be considered when Syngenta follows up with incidents of 

unexpected damage reports (BPPD, 2010): 

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 (low vs. moderate vs. high); and 

5) Weather pattern during the corn growing season and possible effects on Bt protein expression 

and pest population dynamics. 

When greater node injuries are observed on refuge corn than on Bt corn, then this could be an 

indication that damage is not due to resistance but some other factors. Likewise, if Bt plants have less 

damage than refuge plants that were treated with insecticides, then the cause of damage is not likely 

due to resistance. But, if damage levels on Bt plants exceed the level observed on non-Bt plants, then 

field resistance should be suspected. 

Syngenta’s response to unexpected damage reports is protracted but could be made more efficient by 

collecting CRW immediately after unexpected damage reports are received (if adults are still flying). 

After sampling, Syngenta representatives should still be able to proceed with flow strip assays and root 

damage analysis, as well as putative causes for damage. Furthermore, BPPD recommended that 

monitoring samples for potentially resistant insects should be taken from within and adjacent fields 

where the pest damage has occurred. Studies by Naranjo (1990) and Coats et al. (1986) suggest that a 

significant fraction of the female WCRW population engages in longer distance and pre-ovipositional 

dispersal. Hence, BPPD recommended that collections of putative resistant individuals should also 

extend passed a 1⁄2 mile radius from the Bt field with unexpected damage (BPPD, 2010). 

Baseline Susceptibility and Diagnostic Assays for mCry3A (Monitoring Results 2006-2008) 

Syngenta collected baseline susceptibility data for mCry3A from WCRW populations sampled in 2006 

through 2008. DM Crop Research Group, Inc. (a separate company working with Bt corn registrants 

to conduct CRW sampling) collected adult samples of WCRW populations from the three sampling 

regions (IA, IL, and NE) during the 2006 to 2008 growing seasons. Between 11 and 15 populations 

were sampled each year. 

Custom BioProducts, Inc. (IA) (another independent company that conducts the susceptibility testing) 

maintained the WCRW colonies and resulting eggs and performed the subsequent bioassays on the 

populations using neonate larvae (< 24 hours old). Neonates were transferred into 96-well plates 
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containing artificial diet developed for CRW covered with surface applied Bt dilutions containing 

mCry3A at eight different concentrations: 0 (0.1% Triton-X 100 control), 25, 100, 500, 1000, 2500, 

5000, and 10,000 ng mCry3A/cm2

. The protein used in the tests consisted of mCry3A in a lyophilized 

powder at a concentration of 900 μg/mg (supplied by Syngenta). Larvae were allowed to feed for five 

to seven days depending on when fungal contamination appeared. Mortality was recorded when larvae 

did not respond or move when probed. Six replicates of dose response bioassays were conducted for 

every population sample. A replication was considered valid if control mortality was ≤ 25%. Results 

from the assays were used to determine LC50, LC90, and LC99 values for the collected populations. 

Data from the mCry3A susceptibility bioassays are summarized (range of mean susceptibility values 

for the sampled populations) in Table 11 below. Overall, the baseline susceptibility data for mCry3A 

were variable as was evident by the LC50 and LC90 results, although the LC50 ranges were more 

consistent year-to-year than the LC90 values. One population (from McLean Co., IL) collected during 

2008 showed much higher tolerance to mCry3A (LC50: 4822.6 ng/cm2

; LC90: 37,820,880.0 ng/cm2) 

than the other sampled populations (LC50: 191.9 - 1518.4 ng/cm2; LC90: 1430.0 - 225,779.9 ng/cm2). 

Based on the susceptibility testing, Syngenta proposed that only population samples with LC50 values 

> 8,500 ng/cm2 would be considered for further investigation. BPPD noted that although this value 

may be 10-times higher than the mean value observed in the testing through 2008, it still appears to be 

somewhat arbitrarily chosen. It is not clear how sensitive and reliable this particular value will be; LC 

values can be misleading because larvae can survive on Bt diets for some time without feeding (see 

review in BPPD, 2010). 

LC99 values and ranges were submitted for each year but are not shown in Table 11 because the 

extrapolated values were exceedingly high for most populations. Chisquare testing resulted in high 

probabilities values that indicated that the extrapolated mean LC99 values were not a reliable estimate 

for the actual LC99. This suggests that the use of ‘lethal concentrations’ will not be useful to monitor 

for changes in CRW susceptibility to mCry3A and an effective diagnostic concentration (LC99) can not 

be realistically achieved for this toxin. Given these difficulties, Syngenta may need to consider 

sublethal measures (i.e., molting inhibition) to estimate the mCry3A activity against CRW. BPPD 

(2010) recommended that Syngenta use a sublethal measure for detecting shifts in susceptibility to 

mCry3A and/or consider the use of the Sublethal Seedling Assay (SSA) developed by Nowatzki et al. 

(2008) to monitor for shifts in CRW susceptibility. Nowatzki et al. (2008) found that the SSA, which 

measured 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. 
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Table 11. Cumulative Results of mCry3A Susceptibility Testing for WCRW (2006 - 2008; Table 

created from data submitted by Syngenta). 

Year LC50 range (ng/cm2

) LC90 range (ng/cm2) MRID Citation 

2006 357.9 - 1923.6 3788.7 - 28,766.6 473401-01 

2007 209.8 - 1172.3 2470.5 - 52,515.0 480470-02 

2008 191.9 - 4822.6 1430.0 - 37,820,880.0 1 480470-02 1

 One population in 2008 exhibited high tolerance to mCry3A. The other sampled populations had susceptibility ranges of 

191.9 - 1518.4 (LC50) and 1430.0 - 225,779.9 (LC90). 

b) Compliance 

The compliance program for MIR604 (mCry3A corn) is aligned with that developed by ABSTC (in 

consultation with the EPA) for corn-borer protected Bt corn products. Specific elements of the 

program and additional reports with annual survey data are discussed below. 

MIR604 Compliance Assurance Program 

As a term of the MIR604 (mCry3A) registration, Syngenta 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: 

 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 
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reviews of annual growing season reports (see discussion in the Cry1Ab/Cry1F Bt corn Biopesticide 

Registration Action Document). MIR604 (and other registered products with mCry3A) have been 

included as part of this existing program, though data are tabulated separately for lepidopteran, 

rootworm, and stacked (lepidopteran + rootworm) Bt corn PIPs (and not by toxin). EPA reviews of 

compliance data for rootworm-protected PIPs (some of which predates the registration of MIR604) can 

be found in BPPD (2004), BPPD (2007), and BPPD (2009b). 

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 four 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, Inc in 2000 for the lepidopteran Bt corn registrations. But, starting in 2007 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 utilized an internet-based survey approach. 

Surveys for the corn rootworm PIPs encompasses all growers planting rootworm-protected traits 

(Cry3Bb1, mCry3A, and Cry34/35Ab1). Cumulative results of the surveys are summarized in Table 

12 below. Results from the stacked (lepidopteran + rootworm) Bt corn surveys are tabulated 

separately (Table 13) and also include all registered rootworm PIP traits. 
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Table 12. Summary of Telephone (2005-2006) and Online (2007-2008) Survey Results for 

Rootworm-Protected Bt Corn Growers 

Survey Question 20051

% Respondents 

20062

% Respondents 

2007 

% Respondents 

2008 

% Respondents 

Adherence to Refuge3

Size 93 89 80 74 

Adherence to Distance 

Requirements3 87 82 79 63 

Awareness of IRM 

Requirements 97 93 97 96 

Unaided recall of refuge 

size 51 57 63 72 

Unaided recall of refuge 

distance 58 55 33 34 

1 Includes only YieldGard RW and YieldGard Plus corn growers

2 Includes only YieldGard RW, YieldGard Plus, Herculex RW, and Herculex XTRA corn growers 3 Weighted averages across all four regions surveyed

Table 13. Summary of Telephone and Online Survey Results for Stacked (Rootworm + Corn 

Borer) Bt Corn Growers (2006-2008) 

Survey Question 2006 

% Respondents 

2007 

% Respondents 

2008 

% Respondents 

Adherence to Refuge Size1 78 70 723

Adherence to Distance Requirements1 92 66 664

Awareness of IRM Requirements 95 96 97 

Unaided recall of refuge size 59 62 and 552

 81 

Unaided recall of refuge distance 

CRW 48 39 36 

Unaided recall of refuge distance 

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 73% 

4

 On a per field basis, adherence was 76%

Overall compliance (per grower) with refuge requirements for both single trait and stacked rootwormprotected PIPs has declined from 2005 to 2008. Grower adherence to the necessary refuge size fell to 
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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 the 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. Nevertheless, 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 phased compliance approach (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 seasons (for all 

types of Bt corn). On-farm assessments for rootworm-protected PIPs (including mCry3A products) 

began in 2006. 

Data from the on-farm assessments (2006 through 2008) of rootworm-protected Bt corn PIPs are 

summarized in Table 14 below. These on-farm assessments encompass all growers planting 

rootworm-protected PIPs including varieties expressing the Cry3Bb1, mCry3A, and Cry34/35Ab1 

toxins. Results for the on-farm assessments of stacked (lepidopteran + rootworm) PIPs are detailed in 

Table 15. 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 noncompliance. 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 (see EPA review in 

BPPD, 2009b). 
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Table 14. Cumulative Results for the On-Farm Assessments of Coleopteran-Protected Bt Corn 

Growers (2006-2008)1 

2006 2007 2008 

Growers assessed 395 247 134 

Refuge distance 

deviations2 13 N/A N/A 

Refuge size deviations 7 N/A N/A 

Significant deviations 11 (2.8%) 16 (6.5%) 12 (9.0) 

Insignificant deviations 10 (4.0%) 8 (3.2%) 7 (5.2%) 

Compliant growers 374 (94.7%) 223 (90.3%) 115 (85.8%) 

Non-compliant growers 21 (5.3%) 24 (9.7%) 19 (14.2%) 

1

Table adapted from page 12 of MRID 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 15. Cumulative Results of the On-Farm Assessments of Stacked Bt Corn (Rootworm + 

Corn Borer) Growers (2006-2008)1 

2006 2007 2008 

Growers assessed 600 1069 1799 

Refuge distance deviations 512 N/A N/A 

Refuge size deviations 8 N/A N/A 

Significant deviations 45 (7.5%) 77 (7.2%) 86 3

Insignificant deviations 16 33 (3.1%) 36 3

Compliant growers 539 (89.8%) 959 (89.7%) 1546 (85.9%) 

Non-compliant growers 61 (10.2%) 110 (10.3%) 253 (14.1%) 

1 Table adapted from page 12 of MRID 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. 

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 noncompliance. The number of tips and complaints (summarized for all Bt corn registrations 

including lepidopteran and rootworm varieties) received through 2008 is summarized in Table 16 
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below. Each of these growers identified through the tips and complaints mechanism were visited as 

part of the on-farm assessment program. It is not possible, however, to determine whether any of the 

non-compliant growers identified via the tips and complaints route were subject to the Phased 

Compliance Approach. 

Table 16. 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 17), known as the Phased Compliance Approach (PCA), which is to be used by 

registrants when responding to instances of grower noncompliance with the IRM requirements. The 

PCA also established a tiered approach for non-compliance with “significant” deviations and “other” 

deviations. For a 20% CRW refuge requirement (Corn Belt), a significant size deviation is defined as 

a Bt grower planting less than 15% non-Bt corn refuge. This definition is also applicable to 

“combined” refuges planted for both lepidoptera and CRW for stacked Bt corn PIPs. On the other 

hand, a significant deviation based on refuge proximity has not been clearly defined for CRW refuges 

and as of the 2008 CAP report it is unclear what standards are being used by ABSTC. For 

lepidopteran Bt corn, a significant deviation is triggered if fewer than 2/3 of the Bt corn fields are 

planted within 1⁄2 mile of a non-Bt corn refuge. 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 infield strips are at least four rows wide” (see discussion in BPPD, 2006b). This formula would also be 

applicable to combined refuges for stacked PIPs. 
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Table 17. Phased Compliance Approach (PCA) – Standards for Bt Corn Refuge NonCompliance (submitted with the ABSTC 2002 CAP) 

Mandatory Responses Additional Responses

Significant Deviations  IRM education. 

 Warning letter. 

 Compliance assistance 

contact prior to planting. 

 Compliance assessment 

contact for the following 

growing season. 

 Deny access to the Bt 

corn product for any 

significant deviation two 

years in a row. 

Other Deviations  IRM education. 

 Letter and/or compliance 

assistance contact prior to 

planting. 

 Compliance assessment 

contact in the following 

growing season. 

 Invoice monitoring. 

 Technical assistance. 

 Grower IRM training. 

 Reaffirmation of IRM 

obligations. 

 Deny access to the Bt 

corn product for other 

deviations that are 

repeated over a period of 

years. 

 

Under the PCA, sales are to be suspended to individual growers for one year after two years of 

significant deviations. Following the one-year suspension, growers will need to be requalified to 

purchase seeds. Growers identified as non-compliant (significant or other deviations) are required to 

receive a “compliance assessment contact” the following year under the PCA. Non-compliant growers 

are typically identified through the on-farm assessment program (see discussion in the on-farm 

assessment section above). Table 18 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 BPPD (2007) and BPPD (2009b). 
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Table 18. Reassessment of Rootworm-Protected and Stacked Bt Corn Growers Under the 

Phased Compliance Approach (taken from ABSTC annual CAP reports) 1

Year Reassessments 2 Significant 

Deviations 3 

Loss of Access to 

Technology 

2006 62 0 1 4 

2007 82 0 0 

2008 134 0 0 

1

 The data in this table includes both growers planting 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 

c) Additional IRM Data 

As a condition of registration, Syngenta was required to address three areas of research related to the 

nature of potential resistance to mCry3A in CRW. Syngenta addressed these requirements in a 

submission to EPA (MRID# 480470-01; reviewed in BPPD, 2010). The specific requirements and 

responses are described below. 

i) “Initiate establishment of CRW strains that are resistant to mCry3A and investigate the nature, 

inheritance, and fitness costs of specific mechanisms of resistance to the mCry3A protein expressed in 

MIR604 maize.” 

Since 2006, Syngenta has worked with Dr. Bruce Hibbard (USDA-ARS, Columbia, MO) to establish a 

mCry3A-resistant (non-diapausing) WCRW strain. Nine generations of selection have occurred since 

the beginning of the bioassay experiment. A control strain was reared on non-Bt maize as a 

comparison (see Table 19). 

After three generations of selection, the mean LC50 of the treatment colony was 0.259 μg/cm2

 (95% 

C.I.: 0.0906 - 0.5380 μg/cm2) and the LC50 of the unselected control colony was 0.552 (95% C.I.: 

0.318 - 0.917 μg/cm2

). Since the 95% C.I, did overlap, the results did not differ significantly. 

After six generations of selection, the mean LC50 of the treatment colony was 5.541 μg/cm2 (95% C.I.: 

2.361 - 26.429 μg/cm2) and the LC50 of the unselected control colony was 1.301 (95% C.I.: 0.871 - 

2.003 μg/cm2

). The 95% C.I, did not overlap, hence the results differed significantly. 
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After nine generations of selection, the mean LC50 of the treatment colony was 11.25 μg/cm2

 (95% 

C.I.: 5.70 - 32.69 μg/cm2) and the LC50 of the unselected control colony was 0.73 (95% C.I.: 0.51 - 

1.05 μg/cm2). The 95% C.I, did not overlap, hence the results differed significantly. 

Table 19. LC50 Values of a mCry3A-Selected Colony and Lab Reference Strain (Table created 

from data submitted in MRID# 480470-01). 

Generations of Selection Mean LC50 Value with 

95% C.I. (μg/cm2)

Significance between Selected 

and Lab Strain based on 

overlapping 95% C.I. 

Three generations on Bt (treatment 

colony) 

0.259 

(0.0906-0.5380) 

Three generations on non-Bt (lab 

reference strain) 

0.552 

(0.318-0.917) 

not significant 

Six generations on Bt (treatment 

colony) 

5.541 

(2.361-26.429) 

Six generations on non-Bt (lab 

reference strain) 

1.301 

(0.871-2.003) 

significant 

Nine generations on Bt (treatment 

colony) 

11.25 

(5.70-32.69) 

Nine generations on non-Bt (lab 

reference strain) 

0.73 

(0.51-1.05) 

significant 

To determine the field relevance of differences in LC50s, field evaluations were performed and damage 

to MIR604 and non-Bt corn was assessed. 

Despite the differences in the LC50s between selected and non-selected colonies, no statistically 

significant differences in damage, beetle emergence, or larval weight were observed when larvae were 

exposed to MIR604. When the selected colony was placed on Bt and non-Bt plants, no difference in 

emergence, weight, and root damage were observed. At this point, it is unclear whether similar 

performance is related to increasing fitness of the selected colony on MIR604 plants, decreased fitness 

of the selected colony on isoline plants, or a combination of both. 

Additional steps to be performed include maintenance of the colony and further selection in upcoming 

years to investigate the nature, inheritance, and fitness costs of specific mechanisms of resistance to 

mCry3A. 

ii) “Study the behavioral deterrence (avoidance) mechanism further and submit appropriate studies.” 

Syngenta worked with Dr. Hibbard (USDA-ARS, Columbia, MO) to further study any potential 

avoidance mechanisms in CRW larvae to mCry3A expressing corn. The methods by Strnad and Dunn 

(1990) were employed to evaluate host recognition of WCRW and MIR604, non-Bt corn, oat roots, 

and filter paper (negative control). 
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Neonates exposed to MIR604 or isoline diet had moved significantly lower distances and exhibited 

lower velocities than larvae exposed to the negative control or oats. Larvae exposed to isoline 

treatments had significantly higher angular velocities and turn angle as well as lower distances moved 

than neonates exposed to MIR604. These results were consistent with observations of local search 

behavior described for larvae feeding on host roots. 

These results suggest that CRW larvae recognize MIR604 corn as a host and that larvae engage in 

short range searching behavior when exposed to mCry3A in corn diet. Hence, it is unlikely that there 

is an avoidance mechanism unique to mCry3A and WCRW. Syngenta suggested that any avoidance 

behavior may be due to the intoxication from exposure to Bt. 

iii) “Continue studies on the biological impact of adults surviving on MIR604 maize and submit 

results.” 

Syngenta worked with Dr. Lance Meinke (University of Nebraska) to measure potential effects of 

adult WCRW sublethal exposure to MIR604 (field and greenhouse experiments). 

The study design was set up as a 2 x 2 factorial experiment of all possible larva/adult diet 

combinations. The impacts of larval and adult diet on fecundity and longevity were measured. 

Treatments: 

 Larvae reared on MIR604 – adults fed on MIR 604 tissue 

 Larvae reared on MIR604 – adults fed on non-Bt tissue 

 Larvae reared on non-Bt corn – adults fed on MIR604 tissue 

 Larvae reared on non-Bt corn – adults fed on non-Bt tissue 

Results of the study showed no difference in longevity and fecundity between adults of the different 

treatments. Since adult CRW do not seem to detect the difference between Bt and non-Bt corn (no 

repellency to adults), Syngenta concludes that diet switching should not negatively impact the 

effectiveness of any IRM refuge strategy in place. 

Syngenta’s report on these three studies adequately addresses the condition of registration. The 

selection experiment is still ongoing, and BPPD recognizes that it may take significant additional time 

to fully address that condition of registration. 

5. CONCLUSIONS 

The original proposed IRM strategy (submitted in 2005) and data to support it were found to be 

“acceptable” except that the in-field strip refuge must be at least 4 rows wide based on recent larval 

movement data (see EPA reviews in BPPD, 2005 and 2006a). If resistance is recessive, then the 

proposed IRM plan using a 20% structured refuge should be adequate to delay resistance for at least 15 

years given the assumptions of Syngenta’s model. If MIR604 maize is planted in areas with known 
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rotation-resistant WCRW, then planting transgenic corn only in rotated maize fields should be a sound 

IRM strategy that will delay the evolution of resistance by at least 15 years regardless of gene 

expression. 

In accordance with the terms and conditions of registration, Syngenta has submitted additional data 

regarding resistance monitoring, the nature of potential resistance to mCry3A, and refuge compliance. 

These data (as discussed in section 4 above), support the original IRM determination including the use 

of 20% non-Bt corn refuge to mitigate resistance development by CRW to mCry3A. 

6. IRM TERMS AND CONDITION OF REGISTRATION 

The terms and conditions for each of the mCry3A registrations contain a complete description of the 

IRM requirements for the product. Details are provided on the requirements for refuge (size and 

structure), resistance monitoring, remedial action, compliance assurance, grower education, and annual 

IRM reports. For additional information, please refer to the following document: 

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Plant-incorporated Protectant Non-target Insect and Insect Resistance Management Issues. 

U.S. Environmental Protection Agency. 2003. Event MON863 Bacillus thuringiensis Cry3Bbl Corn. 

Biopesticides Registration Action Document. 

U.S. Environmental Protection Agency. 2005. Bacillus thuringiensis Cry34Ab1/Cry34Ab1 Corn. 

Biopesticides Registration Action Document. 
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 

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Van Rie, J., Jansens, S., Hofte, H., Degheele, D. and Van Mellaert, H. 1989. Specificity of Bacillus 

thuringiensis δ-endotoxin: importance of specific receptors on the brush border membrane of the 

midgut of target insects. Eur. J. Biochem. 186: 239-247. 

Wilson, TA and Hibbard, B.E. 2004. Host suitability of nonmaize agroecosystem grasses for the 

Western corn rootworm (Coleoptera: Chrysomelidae). Environ. Entomol. 33: 1102-1108. 

EPA Reviews 

BPPD, 2005. Insect Resistance Management Strategy Preliminary Review and Deficiencies for 

Syngenta’s Event MIR 604 Maize (modified Cry3A). USEPA/OPP/BPPD Memorandum from S. 

Matten to M. Mendelsohn. Dated February 3, 2005. 

BPPD, 2006a. Review of Syngenta’s Revised Insect Resistance Management Strategy for Event 

MIR604 Maize (modified Cry3A). USEPA/OPP/BPPD Memorandum from S. Matten, T. Milofsky, 

and M. Hunter to M. Mendelsohn. Dated September 9, 2006. 

BPPD, 2006b. Technical Review of Dow AgroSciences’ Grower Agreements, Grower Affirmation 

Procedures, and Compliance Assurance Program (CAP) as Required as Part of the Terms and 

Conditions of the Herculex Rootworm (EPA Reg. No. 68467-5) and Herculex Xtra (EPA Reg. No. 

68467-6) Registrations. USEPA/OPP/BPPD Memorandum from S. Matten to M. Mendelsohn dated 

November 27, 2006. 

BPPD, 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. USEPA/OPP/BPPD Memorandum from T. Milofsky and S. Matten to M. Mendelsohn 

dated August 30, 2007. 

BPPD, 2009a. Review of Syngenta’s Corn Rootworm Monitoring Plan and 2006 Baseline 

Susceptibility Data for MIR604 Corn (EPA Reg. No. 67979-5 and 67979-8). USEPA/OPP/BPPD 

Memorandum from J. Martinez to M. Mendelsohn. Dated September 17, 2009. 

BPPD, 2009b. 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; MRIDs 

473396-01 and 476633-01]. USEPA/OPP/BPPD Memorandum from J. Martinez to M. Mendelsohn 

dated April 15, 2009. 
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BPPD, 2010. Review of Syngenta’s Corn Rootworm Monitoring Plan and 2007 and 2008 Baseline 

Susceptibility Data for MIR604 Corn (EPA Reg. No. 67979-5 and 67979-8). USEPA/OPP/BPPD 

Memorandum from J. Martinez to M. Mendelsohn. Dated June 30, 2010. 

Studies Cited by MRID Number 

462656-17 McCaffery, A.; Stein, J. (2004) Insect Resistance Management for Syngenta Event 

MIR604 Maize (Corn). Project Number: MIR604. Unpublished study prepared by 

Syngenta Jealotts Hill International. 47 p. 

465296-01 McCaffery, A.; Stein, J.; Onstad, D. (2005) Amended Insect Resistance Management of 

Syngenta Event MIR604 Maize (Corn). Unpublished study prepared by Syngenta 

Jealotts Hill International, SYNGENTA SEEDS, INC. - FIELD CROPS - NAFTA and 

University of Illinois. 83 p. 

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 p. 

473401-01 Kurtz, R. (2008) Steps Taken to Determine the Baseline Susceptibility of Diabroctica 

virifera virgifera to the mCry3A Protein and the Interim Damage Guidelines for 

MIR604 corn: 2006 Summary. Unpublished study prepared by Syngenta 

Biotechnology, Inc. 18 p. 

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 p. 

480470-01 Kurz, R. (2010) A Summary of Studies Addressing the Conditions of Registration for 

MIR604 and Bt11 x MIR604 Corn Traits. Project Number: SSB/109/10. Unpublished 

study prepared by Syngenta Biotechnology, Inc. 21 p. 

480470-02 Kurtz, R.; Crespo, A. (2010) Steps Taken to Determine the Baseline Susceptibility of 

Diabrotica virgifera virgifera to the MCry3A Protein and the Final Damage Guidelines 

for MIR604 Corn. Project Number: SSB/114/10. Unpublished study prepared by 

Syngenta Biotechnology, Inc. 29 p. 
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II. E. Benefits and EPA Public Interest Finding 

 

1. PUBLIC INTEREST FINDING 

The criteria for determining whether registration of a pesticide chemical is in the public interest are set 

forth in a Federal Register Notice dated March 5, 1986 volume 51, No. 43 (OPP-32500; FRL-2977-2) 

titled Conditional Registration of New Pesticides. There is a presumption that registration of a 

pesticide chemical is in the public interest if one of the following criteria is met: i) the use is for a 

minor crop; (ii) the use is a replacement for another pesticide that is of continuing concern to the 

Agency; (iii) the use is one for which an emergency exemption under FIFRA Section 18 has been 

granted for lack of an alternative pest control method, or (iv) the use is against a pest of public health 

significance. Further, EPA may determine that such a registration is in the public interest on the basis 

of the following criteria: i) there is a need for the new chemical that is not being met by currently 

registered pesticides; ii) the new pesticide is comparatively less risky to health or the environment 

than currently registered pesticides; or iii) the benefits (including economic benefits) from the use of 

the new active ingredient exceed those of alternative registered pesticides and other available nonchemical techniques. 

Syngenta has provided data to support its claim that MIR604 CRW-protected corn is in the public 

interest. EPA’s analysis supports the following conclusions: 

1. Results of efficacy trials conducted in 2002, 2003, and 2004 indicate that MIR604 corn 

provides effective control of key rootworm pests of field corn. 

2. MIR604 corn has unique biochemical properties which may benefit insect resistance 

management for this and other CRW-protected corn products. 

3. If MIR604 corn is registered, it will be the third CRW-protected Bt corn product on the market. 

The availability of multiple CRW-protected corn products will increase grower choice and 

price competition, resulting in lower seed prices for consumers and higher adoption rates. 

4. Registration of MIR604 corn is expected to result in further reduction of chemical insecticide 

use by growers. This is of special importance since many pesticides registered for CRW-control 

are highly toxic to humans and the environment, while mCry3A-expressing corn poses no 

foreseeable human health or environmental risks. 

2. BACKGROUND 

Corn is the most widely cultivated U.S. crop, in terms of acreage planted and net value. In 2004, U.S. 

corn acreage totaled 80.9 million, yielding 11.8 billion bushels. Corn rootworm (CRW, Diabrotica

spp.), one of the most damaging pests of field corn, can cause yield losses in the range of 8 to 16% if 

left uncontrolled. 
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Prior to the advent of insect protected field corn, CRW was controlled through the use of crop rotation 

and insecticides. Although crop rotation is regarded as an effective CRW control tool (Levine and 

Sadeghi, 1991), behavior changes in northern corn rootworm (NCRW, Diabrotica barberi) (extended 

diapause) and western corn rootworm (WCRW, D. virgifera virgifera) (soybean rotation) have reduced 

the effectiveness of this management option in some corn growing regions. Insecticidal control, a pest 

management alternative to crop rotation, employs chemicals that are highly toxic to fish, birds, and 

other wildlife species. In addition, resistance to some CRW insecticides, such as methyl parathion and 

carbaryl, (Meinke et al., 1998; Scharf et al., 1999; Zhu et al., 2001), may result in increased chemical 

use. 

Since 2003, a third method of CRW control, CRW-protected Bt corn, has been available to farmers. 

The first and second Bt field corn products registered for CRW control were Monsanto’s event 

MON863 (expressing the Cry3Bb1 protein) and Dow/Pioneer’s DAS 59122-7 (expressing the 

Cry34Ab1 and Cry35Ab1 proteins) field corn products, respectively. This review concerns the third 

CRW-protected Bt corn product submitted for registration, Syngenta’s MIR604, which produces the 

mCry3A insecticidal protein. Similar to event MON863 and DAS 59122-7 corn hybrids, event 

MIR604 Bt corn is targeted against the WCRW, NCRW, and Mexican corn rootworm (MCRW, D. 

virgifera zeae). 

3. EFFICACY AND YIELD TRIALS 

Early and late maturity MIR604-derived non-commercial, experimental hybrids were evaluated in 

northern, Midwest, and southern U.S. corn-belt locations in 2002, 2003, and 2004. Small plot trials 

utilizing either randomized complete block or split plot designs were conducted. Most trials included 

application of chemical insecticides to assess synergies between MIR604 corn and chemical 

insecticides. Chemical insecticide treatments included one of two seed treatments, ProShield ST®

(tefluthrin) and Cruiser® (thiamethoxam), plus one of three in-furrow soil-applied granular treatments, 

Force 3G® (tefluthrin), Aztec 2.1G® (Tebupirimiphos + cyfluthrin) and Lorsban 15G® (chlorpyrifos). 

a. Efficacy 

Efficacy trials compared root damage among MIR604 and non-MIR604-derived corn hybrids. 

Experimental plots were artificially infested (400 to 1450 eggs/trial), located in fields with naturally 

occurring populations of corn rootworm, or both approaches to infestation were combined. When 

artificially infestation was used, at the V2 to V3 leaf stage rootworm eggs were mechanically placed 3 

inches deep in soil, within 2 to 3 inches of the corn stalk base. At the VT to R1 stage, corn plants were 

manually dug from the ground, soil was washed from the roots and roots were examined and rated for 

corn rootworm damage according to the Iowa State 0-3 node-injury rating scale. 

1) 2002 Field Season 

In 2002, results from trials located in Minnesota and Illinois showed that MIR604 derived hybrids had 

significantly less root damage compared to non-transgenic controls, with and without insecticide 
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applications. In all but one case, MIR604 seed treated with Cruiser or ProShield ST (ProShield is an 

older seed treatment that has been replaced by Cruiser), or plants treated with Force 3G soil-applied 

insecticide, had significantly reduced root damage ratings relative to non-transgenic control hybrids 

with the same chemical treatment. The exception was in Stanton, MN, trial SYN137 which included a 

Force 3G insecticide application, where similar reductions were seen but the difference was not 

statistically significant. 

2) 2003 Field Season 

Eight event MIR604 hybrids were assessed for efficacy at over a dozen locations in Illinois, Iowa, 

Kansas, Minnesota, Missouri, Nebraska, South Dakota, Texas, and Wisconsin in 2003. At all locations, 

MIR604 corn had significantly lower mean root damage, compared to non-transgenic controls with the 

same chemical treatment. And for most trials, untreated Event MIR604 hybrids demonstrated 

significantly greater efficacy compared to negative isoline controls treated with Cruiser at low or high 

rates. No significant difference could be detected between rates of Cruiser applied to MIR604 hybrids; 

however, efficacy of Cruiser seed treatment on negative control plants did appear to increase with 

increasing application rates. Overall, Event MIR604 hybrids seem to provide similar protection against 

CRW, compared to nontransgenic controls treated with Lorsban 15G, Force 3G, or Aztec 2.1 granular 

soil-applied insecticides. In general, MIR604 hybrids delivered equivalent or better efficacy than 

chemical controls currently employed to control root damage from corn rootworm. 

Due to difficulties in rearing NCRW, a limited number of efficacy trials were conducted with this 

rootworm species. In greenhouse trials that employed artificial infestation of NCRW, mean root 

damage ratings show significantly greater efficacy among MIR604 hybrids compared to negative 

controls. In these trials, no significant difference was seen among MIR604 hybrids. The study author 

stated that MIR604 hybrids were exposed to natural populations of NCRW at the majority of locations. 

However, the Willmar, MN location was populated primarily with NCRW and results from this trial 

indicate that MIR604 hybrids provided greater CRW control, compared to negative hybrids with and 

without chemical treatment. 

At two locations in Texas, Event MIR604 corn hybrids showed efficacy toward MCRW, but not to the 

same extent as WCRW and NCRW. At both locations, application of Cruiser seed treatment to Event 

MIR604 hybrids enhanced the efficacy in a rate dependent manner. 

3) 2004 Field Season 

In 2004, root damage ratings were significantly different among untreated MIR604 hybrids and 

negative controls. More specifically, at all locations, except Champaign, IL, MIR604 hybrids treated 

with Cruiser® seed treatment had significantly less root damage than untreated controls. Further, at all 

locations, except Urbana, IL, root damage ratings were not significantly different between untreated 

MIR604 hybrids and MIR604 hybrids treated with Cruiser® insecticide at 0.125 or 0.75 mg AI/seed. 

These findings suggest that negative interactions between mCry3A protein and Cruiser® insecticide did 

not result from use of the seed treatment on MIR604 hybrids. The efficacy of MIR604 corn for 

rootworm protection was also compared to Force 3G®, a granular soil insecticide that is used to control 
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CRW. Results of these trials showed that untreated MIR604 corn, MIR604 corn treated with Cruiser®

insecticide, and a negative control hybrid treated with Force 3G® insecticide provided statistically 

equivalent root protection. These results suggest that the efficacy of MIR604 corn, with or without 

Cruiser® insecticide, is similar to that of Force 3G®, a commercially available corn rootworm 

insecticide. 

Significant natural populations of northern corn rootworm (NCRW, Diabrotica barberi) were 

identified at three trial locations in 2004. At the Redwood Falls, MN location, adults seen in the field 

before and after root evaluations were said to be primarily NCRW. At university trials in Ames, IA and 

Higginsville, MO, adult emergence counts indicated that the NCRW population in these sites was 35% 

and 32%, respectively, of the total rootworm population. Results from the three locations identified 

above were similar to results seen for the overall experiment. Mean root damage ratings for MIR604 

hybrids were found to be significantly lower than damage ratings for the untreated negative control 

hybrid and damage ratings for MIR604 hybrids with or without Cruiser® insecticide applications were 

statistically equivalent to the negative control hybrid treated with Force 3G® insecticide. 

An MIR604 hybrid, identified as “late hybrid 25”, was also shown to be effective at the Texas site, 

which contained large populations of MCRW. As seen in the trials described above, rootworm damage 

on the untreated MIR604 corn hybrid was statistically equivalent to the negative control hybrid treated 

with Force 3G® insecticide. In addition, root damage ratings were not significantly different between 

the MIR604 hybrid treated with and without Cruiser® insecticide. Based on limited data collected 

during the 2003 and 2004 cropping seasons, the registrant concluded that MIR604 corn hybrids 

provide moderately high levels of root damage protection against MCRW and that, on average, greater 

control was provided against WCRW than MCRW. Additional data will be collected in 2005 to 

confirm these findings. 

b. Yield 

Yield trials compared grain production among MIR604 and non-MIR604-derived corn hybrids. 

Experimental plots were located on sites with or without natural CRW pressure and chemical 

insecticides were applied to some sites, but not to all. 

1) 2002 Field Season 

In 2002, Event MIR604 derived hybrids were evaluated for yield (bu/acre) at several field trials in 

Minnesota and Illinois. Growing conditions were normal at the Minnesota locations, while some 

Illinois sites experienced drought and significant CRW feeding pressure. At the Minnesota locations, 

the average yield of MIR604-derived hybrids was statistically equivalent to the negative isogenic 

controls (with and without insecticide). However, at the Illinois locations, MIR604-derived hybrids 

produced greater yields than negative controls with and without chemical insecticide applications. 

2) 2003 Field Season 

MIR604 yield trails were repeated in 2003 at field sites in Illinois, Indiana, Iowa, Kentucky, 

Minnesota, Nebraska, South Dakota and Wisconsin. In contrast to 2002, growing conditions were good 
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at most trial sites. Mean yields from untreated (no chemical insecticides) MIR604 hybrids were, on 

average, statistically equivalent to control hybrids, regardless of maturity group and insecticide 

treatment. This data suggests that where CRW pressure exists, MIR604 has a positive effect on yield, 

and this effect may be magnified under adverse environmental conditions; however, where CRW 

pressure does not exist, yield among MIR604 and non-transgenic plots will be similar. 

3) 2004 Field Season 

Grain yield for MIR604 plots that were untreated or treated with Cruiser® insecticide produced an 

average of 27.5 bushels per acre more than the untreated negative control hybrid. In addition, results at 

Bloomington-2, IL, which experienced extreme rootworm pressure and stressful growing conditions, 

suggest that MIR604 corn is most beneficial where rootworm pressure is greatest. The submission 

states that although some of these trials lacked the precision to show statistical differences among 

treatments, the numerical trends in grain yield were positive for MIR604 corn. 

A multiple location yield trial conducted in the absence of CRW feeding pressure (granular insecticide 

applied to all plots within trial) showed no significant difference in yield between MIR604 hybrids and 

the negative isogenic control at all trials and locations except SYNAM445, where MIR604 hybrid 22 

yielded 10.5 bushels more per acre than the isogenic negative control hybrid. These results suggest 

that, under low stress conditions, MIR604 hybrids perform as well as negative isogenic hybrids. 

c. Conclusions 

Results from the 2002, 2003, and 2004 efficacy trials suggest that MIR604 hybrids provide greater 

protection against CRW than negative control hybrids. Further, the efficacy of MIR604 hybrids 

appears to be similar to that of negative control hybrids treated with Force 3G® insecticide and the 

mCry3A protein that is present in MIR604 hybrids does not seem to interact with Cruiser® seed 

treatment. Conclusions regarding the relative yield from 2002 and 2003 field trials data were obscured 

by poor experimental design and extreme weather conditions. Data from 2004 yield trials indicated 

that under high stress conditions, MIR604 hybrids (±Cruiser® seed treatment have greater yields than 

untreated negative controls, although there was no statistical difference among treatments. Under low 

stress conditions, there was no statistical difference in yield between MIR604 hybrids (±Cruiser® seed 

treatment) and the negative controls (± insecticide). Yield data suggest that MIR604 hybrids perform 

about the same as the isoline hybrids.

4. BENEFITS 

EPA has completed an independent analysis of the potential grower benefits associated with Bt CRWprotected corn hybrid technologies. Grower demand for CRW control technologies is influenced by the 

level of CRW infestation (acreage and degree of infestation), comparative cost-benefit of competing 

CRW control technologies, U.S. and global market acceptance and approval of a technology, and other 

regulatory constraints (e.g. refuge requirements). In the year 2000, almost 8 million pounds of CRW 

insecticide, costing $172 million ($12.29/acre), were applied to 14 million acres, or 17% of U.S. corn 

acreage. And between the years 2005 to 2013, CRW infested acreage is projected to increase from 
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approximately 31.8 million acres to 39 million acres (Table 36). BPPD anticipates, through evaluation 

of USDA/NASS and DOANE databases on CRW damage and control costs, that the market for 

transgenic in-plant CRW protection will increase by 2.6% per year, reaching 18 to 19 million acres by 

the year 2013. 

Data on product efficacy suggests that Event MIR604 corn provides greater protection against CRW 

damage than does unmodified corn (see Efficacy section above). However, submitted yield data was 

inconclusive and should be supported by a third year of field trials (see Yield section above). 

Table 36. Projected acreage with corn rootworm infestation and breakdown of associated CRW 

control practices for the years 2000 to 2013. Information presented for 2000 and 2002 reflect 

actual infestation and insect control tactics.

Acres Infested Acres Treated CRW-Protected 

Bt Corn Acreage 

Conventional 

Treatments 

Year 

-----------------------------------------acreage x 106----------------------------------------

2000 28.0 14.0 0.0 14.0 

20011 - - - - 

2002 29.5 14.7 0.0 14.7 

2003 30.2 15.1 1.0 14.1 

2004 31.0 15.5 2.5 13.0 

2005 31.8 15.9 4.0 11.9 

2006 32.6 16.3 6.0 10.3 

2007 33.5 16.7 7.2 9.5 

2008 34.3 17.2 8.6 8.5 

2009 35.2 17.6 10.4 7.2 

2010 36.1 18.1 11.9 6.1 

2011 37.1 18.5 13.7 4.8 

2012 38.0 19.0 15.8 3.2 

2013 39.0 19.5 16.8 2.7 

Annual Growth 

Rate 

2.58% 2.58% 16.8% -14.36% 

1

 EPA does not have data for the 2001 growing season. 

a. Human Health Benefits 

Event MIR604 CRW-protected corn is expected to be safer for handlers, applicators, farmers, and the 

public than chemical pesticides in current use. Twenty-five of the 39 registered conventional 

insecticides used to control CRW are classified as “Restricted Use”, 12 have the “Danger” label 

classification, and several are in Agency Special Review (e.g., dimethoate, phorate, and terbufos). 

Further, each year there are confirmed reports of human illness associated with these registered 
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chemical insecticides (See EPA’s Incident Data Base, http://www.opp.gov/pesticides). Adoption of 

mCry3A corn hybrids has the potential to reduce occupational, farmer, and public health risks 

associated with the manufacture, transport, storage, handling, application, and disposal of conventional 

insecticides, by providing a safer alternative for CRW control. 

Increased adoption of transgenic CRW-protected corn products is of special importance to EPA, 

because many of the chemical insecticides registered for CRW control are highly toxic to humans and 

the environment, are in Special Review, and have restricted use labels. The chemical insecticides 

(larvicides and adulticides) subject to the greatest use reduction following adoption of transgenic 

CRW-protected hybrids are: organophosphates (chlorpyrifos, tebupirimphos, tebufos, methyl 

parathion, and chlorethoxyfos), pyrethroids (tefluthrin, cyfluthrin, bifenthrin, lambda cyhalothrin), 

carbamates (carbofuran), and pyrazoles (fipronil) (Appendix A). Adoption of transgenic CRWprotected corn products is not expected to result in reduced use of seed treatments (nicotinoids); 

however seed treatment products are generally less toxic than at-plant and post-plant products. 

In contrast to the registered chemical insecticide alternatives, EPA has concluded that there is 

reasonable certainty of no harm from aggregate exposure to the mCry3A protein as expressed in corn. 

See mCry3A Human Health and Product Characterization reviews for more information on potential 

human health effects [Fellman, 2005(a,b); 2006(c)]. 

b. Environmental Benefits 

Chemicals commonly used for CRW control can cause adverse environmental effects when used 

according to label instructions. Of the most commonly used CRW control products, 15 are labeled as 

“toxic,” six as “highly toxic,” one as “very highly toxic,” and 14 as “extremely toxic” to birds, fish, 

and other wildlife. Several of the synthetic insecticides (e.g. organophosphates, carbamates, and 

synthetic pyrethroids), are moderately to highly toxic to terrestrial non-target species. Of special 

concern are methyl parathion and carbofuran, both of which are implicated in bird kills (Appendix A, 

Table 1A). In contrast, the ecological risk assessment and characterization of the mCry3A protein, as 

expressed in corn, suggests that these proteins pose no significant ecological risk to non-target species, 

including endangered and beneficial species (Milofsky, 2006). 

c. Insect Resistance Management Benefits 

There is concern about the ability of target pests to evolve resistance to CRW control mechanisms, 

including crop rotation, chemical insecticides, and CRW-protected Bt corn products. Currently, the 

favored management practice for CRW control is crop rotation, specifically corn-soybean rotations, 

complimented by application of chemical insecticides – pyrethroids, organophosphates, carbamates, 

pyrazoles, and more recently, neonicotinoids as seed treatments. CRW has developed resistance to a 

number of chemical control products used as adulticides. The recent development of the NCRW 

extended diapause and WCRW soybean resistant variants have further reduced the efficacy of crop 

rotation and chemical control options. 
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Since mCry3A CRW-protected corn appears to have good efficacy against CRW, introduction of this 

product is expected to extend the durability of other commercially available CRW-protected Bt corn 

products (e.g., Cry3Bb1 PIPs and Cry34/35Ab1 PIPs). To ensure the long-term efficacy of mCry3A 

CRW-protected corn hybrids, an insect resistance management plan should be implemented (Matten et 

al. 2006). 

5. REFERENCES 

AgroTrak U.S. Crop Chemical Usage Study. 2003 Doane Marketing Research, Inc. 

Chen, E. and Stacy, C. 2003. Modified Cry3A toxins and nucleic acid sequences coding. Patent 

Application WO Patent No. 03/018810 

Federal Register. 2004. Phosphomannose Isomerase and the Genetic Material Necessary for Its 

Production in all Plants; Exemption from the Requirement of a Tolerance. Vol. 69(94); Friday, 

May 14, 2004; pp. 26770-26775; 40 CFR Part 180.1252 

Fellman, A. 2005 (a). Addendum to product characterization and expression analysis for modified 

Cry3A (mCry3A) Bacillus thuringiensis insecticidal protein and the genetic material necessary 

for their production (via pZM26) in transgenic maize plants derived from Syngenta Seeds’ 

transformation Event MIR604. U.S. EPA/BPPD Memorandum from A. Fellman to M. 

Mendelsohn. Dated Feb 11, 2005. 

Fellman, A. 2005 (b). Review of product characterization and expression analysis for modified Cry3A 

(mCry3A) Bacillus thuringiensis insect control protein and maize plants derived from Event 

MIR604 in support for a permanent exemption from tolerances and Section 3 registration, 

submitted by Syngenta Seeds, Inc. – Field Crops- NAFTA. U.S. EPA/BPPD Memorandum 

from A. Fellman to M. Mendelsohn. Dated May 31, 2005. 

Fellman, A. 2006 (c). Review of product characterization and human health data for modified Cry3A 

(mCry3A) Bacillus thuringiensis insect control protein and maize plants derived from Event 

MIR604 in support for a permanent exemption from tolerances and Section 3 registration, 

submitted by Syngenta Seeds, Inc.-field crops-NAFTA. U.S. EPA/BPPD Memorandum from 

A. Fellman to M. Mendelsohn. Dated Feb. 15, 2006. 

Gallagher, S.P., Mitchell, L.R. and Beavers, J.B. 2003. An acute oral toxicity study of modified 

Cry3A protein (MCRY3A-0102) in the northern bobwhite. Syngenta, MRID No. 46155616. 

Garcia-Alonso, M. and Vlachos, D. 2003. Characterization and safety of modified Cry3A Bacillus 

thuringiensis protein and maize (corn) plants derived from event MIR604. EUP submission for 

MIR604. Syngenta, MRID No. 46155601 
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Johnson, I. 2003. Acute oral toxicity study of modified Cry3A protein (MCRY3A-0102) in the mouse. 

Syngenta, MRID No. 46155610. 

Matten, S.R., Milofsky, T., and M. Hunter. 2006. Insect resistance management assessment for 

modified Cry3A (mCry3A) corn rootworm-protected corn (Event MIR 604). U.S. EPA/BPPD 

Memorandum from S. Matten to M. Mendelsohn. Dated Sept. 06, 2006. 

Milofsky, T. 2006. Environmental Risk Assessment for modified Cry3A (mCry3A) Bacillus 

thuringiensis protein and the genetic material necessary for its production in Event MIR604 

corn. U.S. EPA/BPPD. Memorandum from T. Milofsky to M. Mendelsohn, Dated Feb. 9, 

2006. 

NCBI. 2003. National Center for Biotechnology Information 

Rabe, S. 2003. Molecular characterization of event MIR604 maize (corn) expressing a modified Cry3A 

Bacillus thuringiensis protein. Syngenta, MRID No. 46155602 

Raybould, A. 2004. Environmental safety assessment of modified Cry3A protein and event MIR604 

corn to non-target organisms. Syngenta, MRID No. 46265601 

University of Illinois Agricultural Extension Service Bulletins #22 and #25. 2003. 

U.S. Department of Agriculture, Economic Research Service (USDA-ERS). 2001. Commodity costs 

and returns. 

U.S. Department of Agriculture, National Agricultural Statistics Service (USDA-NASS). 2001. 

Agricultural Statistics 2001. 

U.S. Department of Agriculture, National Agricultural Statistics Service (USDA-NASS). 2002. 

Agricultural Statistics 2002. 

U.S. Environmental Protection Agency. 2001. Biopesticides Registration Action Document, Bacillus 

thuringiensis Plant Incorporated Protectants. 

U.S. Environmental Protection Agency. 2003. Biopesticides Registration Action Document, Event 

MON863 Bt Cry3Bb1 

Vlachos, D. and Joseph, R. 2003. Characterization and safety of phosphomannose isomerase (PMI), a 

selectable marker expressed in event MIR604-derived maize (corn) plants. Syngenta, MRID 

No. 45934402 
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 130 Appendix A Table 1A. Insecticide end-use products registered by EPA for use on corn for control of corn rootworm speci

Product

Active 

Ingredients Type a

Use Rate 

b Use Classification 

Ambush® Insecticide – Syngenta 

Permethrin – 25.6% SP 0.2 lb/ac 

Adult 

control 

WARNING. Restricted Use; extrand aquatic invertebrates, highly to

Asana®XL Insecticide 0.66 Emulsifiable Concentrate - DuPont 

Esfenvalerate – 8.4% SP 0.05 lb/ac

Adult 

control 

WARNING. Restricted Use; extrand aquatic invertebrates, highly to

Aztec 2.1% Granular Insecticide – Bayer Corp. 

Tebupirimfos – 2.0% cyfluthrin – 0.1% 

OP 

SP 

0.15 lb/ac0.01 lb/ac

Larval 

control 

WARNING. Restricted Use; toxiwildlife 

Baythroid 2 

Emulsifiable Pyrethroid Insecticide - Bayer 

Cyfluthrin – 25%  SP 0.04 lb/ac

Adult 

control 

DANGER. Restricted Use; extremand aquatic invertebrates, highly tocause allergic skin reactions 

Capture 2EC Insecticide/Miticide – FMC Corp. 

Bifenthrin – 25.1% SP 0.3 lb/ac 

Larval 

control 

WARNING. Restricted Use; extand aquatic invertebrates, highly to

Chlorfos 15G 

Insecticide Granular – Griffin LLC 

Chlorpyrifos – 15% OP 2.02 lb/ac

Larval 

control 

CAUTION. Toxic to birds and witoxic to fish and aquatic organisms 
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 

Product

Active 

Ingredients Type a

Use Rate 

b Use Classification 

Chlorfos 4E 

Insecticide – Griffin LLC 

Chlorpyrifos – 42% OP 2.52 lb/ac

Adult & 

Larval 

control 

WARNING. Toxic to birds and wtoxic to fish and aquatic organisms 

Counter CR Systemic Insecticide-Nematicide– American Cyanamid Company Terbufos – 20% OP 1.30 lb/ac

Larval 

control 

DANGER. Restricted Use; fatal iinhaled or absorbed through skin, efish and wildlife 

D-z-n diazinon AG500 Insecticide - Syngenta Diazinon – 48% OP 0.48 lb/ac

Adult 

control 

CAUTION. Restricted Use; highand other wildlife, highly toxic to b

D-z-n diazinon AG600 WBC Insecticide - Syngenta Diazinon - 56% OP 0.45 lb/ac

Adult 

control 

CAUTION. Restricted Use; highand other wildlife, highly toxic to b

Declare Emulsifiable Insecticide Concentrate

– Griffin LLC 

Methyl parathion – 45.11% OP 0.22 lb/ac

Adult 

control 

DANGER. Restricted Use: fatal 

inhaled or absorbed through skin, h

aquatic invertebrates and wildlife, h

Diazinon 500-AG Organophosphate Insecticide – UAP Diazinon – 48% OP 0.48 lb/ac

Adult 

control 

CAUTION. Restricted Use; highand other wildlife, highly toxic to b

Dimethoate 4 EC Systemic Insecticide - Helena 

Dimethoate – 44.8% OP 0.45 lb/ac

Adult 

control 

WARNING. Toxic to wildlife and invertebrates, highly toxic to bees 
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 132 

Product

Active 

Ingredients Type a

Use Rate 

b Use Classification 

Dimethoate 400 Systemic InsecticideMiticide - UAP Dimethoate – 43.5% OP 0.44 lb/ac Adult control WARNING. Toxic to wildlife and 

invertebrates, highly toxic to bees 

5 lb. Dimethoate Systemic Insecticide - Helena 

Dimethoate – 57% OP 0.46 lb/ac 

Adult 

control 

DANGER. Toxic to wildlife and a

highly toxic to bees 

Force 3G Insecticide - Syngenta Tefluthrin – 3% SP 0.17 lb/ac

Larval 

control 

CAUTION. Restricted Use; very freshwater and estuarine fish and in

Fortress 2.5G granular insecticide - 

DuPont 

Chlorethoxyfos – 2.5% OP 0.16 lb/ac

Larval 

control 

DANGER. Restricted Use; toxic 

birds, fish and aquatic invertebrates 

Fortress 5G granular insecticide – DuPont 

Chlorethoxyfos – 5% OP 0.16 lb/ac

Larval 

control 

DANGER. Restricted Use; toxic 

birds, fish and aquatic invertebrates 

Furadan 4F insecticide/ nematicide– FMC Corp. 

Carbofuran – 44% C 0.88 lb/ac

Adult & 

larval 

control 

DANGER. Restricted Use; poisoninhaled, toxic to fish, birds and othetoxic to bees, can seep or leach throcontaminate groundwater 

Lannate LV insecticide

– DuPont 

Methomyl – 29% C 0.65 lb/ac

Adult 

control 

DANGER. Restricted Use; fatal ito fish, aquatic invertebrates and m

toxic to bees, known to leach througroundwater 
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 

Product

Active 

Ingredients Type a

Use Rate 

b Use Classification 

Lannate SP insecticide- DuPont 

Methomyl – 90% C 0.45 lb/ac

Adult 

control 

DANGER. Restricted Use; fatal icause blindness, toxic to fish, aquatmammals, highly toxic to bees, knosoil into groundwater 

Lorsban 15G Granular Insecticide – Dow Agrosciences 

Chlorpyrifos – 15% OP 2.03 lb/ac

Larval 

control 

CAUTION. Toxic to birds and witoxic to fish and aquatic organisms 

Lorsban -4E Insecticide– Dow Agrosciences 

Chlorpyrifos – 44.9% OP 2.69 lb/ac

Adult & 

larval 

control 

WARNING. Toxic to birds and wtoxic to fish and aquatic organisms 

Mocap 10% Granular Nematicide Insecticide – Aventis CropScience Ethoprop – 10% OP 3.53 lb/ac

Larval 

control WARNING. Toxic to aquatic orga

Mocap EC Nematicide-Insecticide– Aventis CropScience 

Ethoprop – 69.6% OP 3.34 lb/ac

Larval 

control 

DANGER. Restricted Use; toxic and extremely toxic to birds 

Penncap-M

Microencapsulated Insecticide – Elf Atochem 

Methyl Parathion – 22% 

OP 

 0.44 lb/ac

Adult 

control 

WARNING. Restricted Use; highinvertebrates and wildlife 

Phorate 20 G Organophosphate Insecticide - UAP Phorate – 20% OP 1.3 lb/ac 

Adult & 

larval 

control 

DANGER. Restricted Use; extrem

and wildlife 
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 134 

Product

Active 

Ingredients Type a

Use Rate 

b Use Classification 

Pounce® WSB Insecticide – FMC Corporation 

Permethrin - 24.7% SP 0.2 lb/ac 

Adult 

control 

WARNING. Restricted Use; extrand aquatic invertebrates, highly to

Pounce® 3.2 EC Insecticide – FMC Corporation 

Permethrin – 38.4% SP 0.2 lb/ac 

Adult 

control 

CAUTION. Restricted Use; extre

and aquatic invertebrates, highly to

Pounce® 25 WP Insecticide – FMC Corporation 

Permethrin – 25% SP 0.2 lb/ac 

Adult 

control 

WARNING. Restricted Use; extr

and aquatic invertebrates, highly to

Regent 4 SC Insecticide – Aventis CropScience Fipronil – 39.4% PP 0.13 lb/ac

Larval 

control 

WARNING. Restricted Use; toxi

aquatic invertebrates 

Sevin brand 80S Carbaryl Insecticide – Aventis CropScience Carbaryl – 80% C 2.0 lb/ac 

Adult 

control 

WARNING. Extremely toxic to aqinvertebrates, highly toxic to bees 

Sevin brand XLR PLUS Carbaryl Insecticide – Aventis CropScience 

Carbaryl – 44.1% C 1.76 lb/ac

Adult 

control 

CAUTION. Extremely toxic to aqinvertebrates, highly toxic to bees 

Thimet 20-G Soil and Systemic Insecticide – American Cyanamid Phorate – 20% OP 1.3 lb/ac 

Larval 

control 

DANGER. Restricted Use; extremand wildlife 
Modified Cry3A Corn 

Biopesticide Registration Action Document (BRAD) September, 2010 

Product

Active 

Ingredients Type a

Use Rate 

b Use Classification 

Thimet 20-G Soil and Systemic Insecticide – American Cyanamid Phorate – 20% OP 1.3 lb/ac 

Larval 

control 

DANGER. Restricted Use; extremand wildlife 

Warrior® Insecticide with Zeon Technology - Syngenta 

Lambdacyhalothrin – 11.4% SP 0.03 lb/ac

Adult 

control 

WARNING. Restricted Use; extrand aquatic organisms and toxic to toxic to bees 

Cruiser® 5FS - Syngenta Thiamethoxam N 

0.1 lb/ac 

(seeds) 

Larval 

control CAUTION. toxic to aquatic organi

Poncho® 1250 -Gustafson LLC Clothianidin N 

0.1 lb/ac 

(seeds) 

Larval 

control 

CAUTION. This product is toxic tinvertebrates 

Prescribe® - 

Gustafson LLC Imidacloprid N 

0.11 lb/ac

(seeds) 

Larval 

control 

CAUTION. Highly toxic to birds ainvertebrates 

Table reproduced from MON 863 registration and updated with data provided by DAS in MRID # 46123921. a - OP: organophosphate; SP: synthetic pyrethroid; C: carbamate; PP: phenyl pyrazole; N: nicotinoid b – maximum labeled use rate expressed in pounds of active ingredient per acre (assume that 1 liq pt  1 lb) c – precautionary language as stated on label. 
136

III. Regulatory Position for Modified Cry3A Protein and the Genetic Material Necessary for its 

Production (Via Elements of pZM26) in Event MIR604 Corn SYN-IR604-8 

A. Initial Regulatory Position

In 2006 EPA conditionally registered Syngenta Seeds Inc.'s new active ingredient, modified Cry3A 

protein and the genetic material necessary for its production (via elements of pZM26) in event MIR604 

corn SYN-IR604-8. 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 time of 

conditional registration. 

Results of efficacy trials conducted in 2002, 2003, and 2004 indicate that MIR604 corn provides 

effective control of key rootworm pests of field corn. MIR604 corn has unique biochemical properties 

that may benefit insect resistance management for this and other CRW-protected corn products. 

MIR604 contains the third CRW-active Bt corn protein on the market. The availability of multiple 

CRW-protected corn products will increase grower choice and price competition, resulting in lower 

seed prices for growers and higher adoption rates. Registration of MIR604 corn is expected to result in 

further reduction of chemical insecticide use by growers. This is of special importance since many 

pesticides registered for CRW-control are highly toxic to humans and the environment, while 

mCry3A-expressing corn poses no foreseeable human health or environmental risks. 

The new corn plant-incorporated protectant, Event MIR604 corn, produces its own insecticide within 

the corn plant. This protectant, mCry3A protein, is derived from Bacillus thuringiensis (Bt), a naturally 

occurring soil bacterium. The mCry3A protein used in this product controls corn rootworm, a highly 

destructive pest responsible for the single largest use of conventional insecticides in the United States. 

In order to reduce the possibility of corn rootworm developing resistance to Bt, EPA required Syngenta 

Seeds, Inc. to ensure that 20 percent of the planted acreage of this product be set aside in which nonCRW-protected corn will be grown to serve as a "refuge." These refuge areas will support populations 

of corn rootworm not exposed to the CRW-protected corn. The insect populations in the refuges will 

help prevent resistance development when they cross-breed with insects in the CRW-protected fields. 

This resistance management strategy was developed as a condition of the registration, and EPA will 

require routine monitoring and documentation that these measures are followed. The submitted insect 

resistance management data support a registration until 2010. 

A tolerance exemption at 40 CFR Part 174.456 was established for residues of Bacillus thuringiensis

modified Cry3A protein and the genetic material necessary for its production in corn. 

Pursuant to FIFRA section 3(c)(7)(C), 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. 
137

The first criterion under FIFRA Section 3(c)(7)(C) mentioned above has been met because insufficient 

time has elapsed since the imposition of the data requirements for: 

1) Field degradation studies evaluating accumulation and persistence of mCry3A in several different 

soils in various strata. 

2) Three (3) year full-scale field or semi-field studies for evaluation of mCry3A Event MIR604 corn 

exposure on non-target invertebrates. 

3) Data to augment the mCry3A amino acid sequence analysis to known toxins and allergens within six 

months of the date of registration: specification of which version of NCBI database was utilized; 

descriptions of parameters utilized; and dates accessed for the BLAST search. 

4) Insect resistant management data: a) Specific cross-resistance studies. Establish strains of CRW 

that are resistant to mCry3A and investigate the nature, inheritance, and fitness costs of specific 

mechanisms of resistance to the mCry3A protein expressed in MIR604 maize; b) Study the behavioral 

deterrence (avoidance) mechanism further and submit appropriate results; c) Continue studies on the 

biological impact of adults surviving on MIR604 maize and submit these results. 

The applicants submitted or cited data sufficient for EPA to determine that conditional registration of 

modified Cry3A protein and the genetic material necessary for its production (via elements of pZM26) 

in event MIR604 corn SYN-IR604-8 under FIFRA 3(c)(7)(C) will not result in unreasonable adverse 

effects to the environment, as discussed above. The applicants submitted and/or cited satisfactory data 

pertaining to the proposed use. The human health effects 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 risks of resistance developing to mCry3A protein during the conditional registrations are 

not expected to be significant. 

Registration of modified Cry3A protein and the genetic material necessary for its production (via 

elements of pZM26) in event MIR604 corn SYN-IR604-8 is in the public interest because: 

1. Results of efficacy trials conducted in 2002, 2003, and 2004 indicate that MIR604 corn 

provides effective control of key rootworm pests of field corn. 

2. MIR604 corn has unique biochemical properties which may benefit insect resistance 

management for this and other CRW-protected corn products. 

3. If MIR604 corn is registered, it will be the third CRW-protected Bt corn product on the market. 

The availability of multiple CRW-protected corn products will increase grower choice and 

price competition, resulting in lower seed prices for consumers and higher adoption rates. 
138

4. Registration of MIR604 corn is expected to result in further reduction of chemical insecticide 

use by growers. This is of special importance since many pesticides registered for CRW-control 

are highly toxic to humans and the environment, while mCry3A-expressing corn poses no 

foreseeable human health or environmental risks. 

In view of these minimal risks and the clear benefits related to modified Cry3A protein and the genetic 

material necessary for its production (via elements of pZM26) in event MIR604 corn SYN-IR604-8, 

EPA believes that the use of the product 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). 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 Section III. 

EPA has determined, as explained in section II.E., that the third criterion for a FIFRA 3(c)(7)(C) 

conditional registration has been fulfilled because the use of modified Cry3A protein and the genetic 

material necessary for its production (via elements of pZM26) in event MIR604 corn SYN-IR604-8 

under this registration is in the public interest. 

The submitted data in support of this registration under section 3(c)(7)(C) of the Federal Insecticide, 

Fungicide, and Rodenticide Act (FIFRA) have been reviewed and determined to be adequate. Studies 

mentioned above are included in the terms, conditions, and limitations of these registrations. This 

registration will not cause unreasonable adverse effects to man or the environment and is in the public 

interest. 

The expiration date of the registrations has been set to September 30, 2010. 

B. 2010 Update: Regulatory Position

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. 
139

The Agency concludes that the following mCry3A corn product registrations, set to expire in 

September 2010 and described in-depth throughout this BRAD, meet both criteria (1) and (2): 

 (1) Agrisure RW Rootworm-Protected Corn (EPA Reg. No. 67979-5) 

 (2) Agrisure CB/LL/RW (EPA Reg. No.67979-8) 

All of these mCry3A corn products are identical in both composition and use (corn) to plantincorporated protectants that are currently registered. Thus, criterion (1) has been fulfilled. 

With regard to criterion (2), the Agency maintains, as was previously determined for the original 

registration of these particular products, that cultivation of mCry3A-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, strengthen the Agency’s initial position 

and also confirm that long-term effects on non-target organisms are not anticipated. Lastly, the 

continued use of these products will likely still provide many of the benefits as were evaluated in 

section II(E) of this BRAD to support the 2006 registration of Event MIR604 corn (e.g., reduction in 

use of conventional insecticides that are highly toxic to both humans and the environment). 

In conclusion, as the expiring mCry3A products (i.e., Agrisure RW Rootworm-Protected Corn, 

(MIR604 Corn, EPA Reg. No. 67979-5) and Agrisure CB/LL/RW (Bt11 x MIR604 Corn, EPA Reg. 

No.67979-8)) have met the required criteria under section 3(c)(7)(A) of FIFRA, the Agency is 

amending these registrations to extend their respective expiration dates as follows: 

Product Name (EPA Reg. No.) Expiration Date 

Agrisure RW Rootworm-Protected Corn (EPA 

Reg. No. 67979-5)

September 30, 2015 

Agrisure CB/LL/RW (EPA Reg. No. 67979-8) 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, specific in relation to long term nontarget testing and insect resistance management, 

are necessary for a finding of registrability under FIFRA section 3(c)(5) and remain as terms or 

conditions for the purposes of the amendments. 

C. Period of Registration 

In the 2001 Bt Corn reassessment, EPA determined that it was appropriate to amend the then-existing 

registrations to extend the period of registration of those products to an expiration date of October 15, 

2008. All of the products being assessed at that time were efficacious against lepidopteran pests. EPA 

based this action on the finding that use of Cry1Ab or Cry1F expressed in corn will not significantly 

increase the risk of unreasonable adverse effects on the environment “for the limited time period of 7 
140

additional years (to October 15, 2008).” These registrations were later amended to extend the period of 

registration to an expiration date of September 30, 2010. EPA subsequently granted time-limited 

registrations to products efficacious against coleopteran corn rootworm pests. For example, EPA 

registered Cry3Bb1 on February 24, 2003, to May 1, 2004, and extended that registration twice, to 

February 24, 2008, and September 30, 2010. 

As set forth elsewhere in this document, EPA’s primary concern for the Bt protected transgenic corn 

products is the possibility that target pests will develop resistance to one or more of the plantincorporated protectant (PIP) toxins. Development of resistance to a Bt toxin would be a grave adverse 

effect, and, for over 15 years, EPA has imposed stringent requirements intended to countermand the 

potential development of resistance. Registrants similarly have been busily developing various 

products, product mixes (i.e., so-called “pyramids” and “stacks”), and resistance strategies, to 

maximize agronomic benefits and address resistance management issues. The result has been a vast 

array of product combinations and, occurring over the past couple of years, a re-emergence of varying 

refuge requirements for different products. 

As discussed in the 2001 Bt PIP BRAD (at IID13), the earliest Bt corn registrations did not include 

mandatory refuge requirements. There was a lack of scientific consensus as to what the appropriate 

refuge requirement should be, and, it was assumed that the limited market penetration of these early 

crops would be so low as to guarantee that adequate natural refuges would be available from 

neighboring non-Bt corn fields. From 1995 to 1997, Bt corn registrations included voluntary refuge 

requirements of 0% to 20% in the Corn Belt. In 1999, the Agricultural Biotechnology Stewardship 

Technical Committee (ABSTC), in conjunction with the National Corn Growers Association, proposed 

uniform insect resistance management (IRM) requirements for Bt corn registrations. With some 

modifications, this proposal, put in place for the 2000 growing season, formed the baseline IRM 

requirements for almost all Bt corn registrations for the better part of a decade: farmers were required 

to plant a 20% refuge that could be treated for insects, or a 50% treated refuge in cotton-growing areas; 

all refuges to be planted within one-half mile of the Bt corn field. 

These uniform requirements brought certainty and consistency to the market after the initial period 

where many Bt corn products had different refuge requirements. Recently, however, as product 

developers have begun to conceive of products with different combinations of “pyramided” products 

(i.e., products containing two or more toxins efficacious against the same pest) and “stacked” products 

(i.e., products combining toxins efficacious against different pests), the refuge requirements have 

begun to vary. For example, certain products require a 20% external refuge; some products permit a 

5% 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 
141

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.