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EPA BIOPESTICIDES AND POLLUTION PREVENTION DIVISION COMPANY 

NOTICE OF FILING FOR PESTICIDE PETITIONS PUBLISHED IN THE 

FEDERAL REGISTER 

EPA Biopesticides and Pollution Prevention Division contact: hollis.linda@epa.gov; 

Syngenta Seeds, Inc.

[Insert petition number]

EPA has received a pesticide petition ([insert petition number]) from Syngenta 

Seeds, Inc., P.O. Box 12257, 3054 E. Cornwallis Road, Research Triangle Park, NC 

27709-2257 proposing, pursuant to section 408(d) of the Federal Food, Drug, and 

Cosmetic Act (FFDCA), 21 U.S.C. 346a(d), to amend 40 CFR part 174.532

1. NA-Remove

2. NA-Remove

3. to establish an amendment/expansion of an existing tolerance exemption for 

the

1. NA-Remove

2. NA-Remove

3. plant-pesticide Bacillus thuringiensis eCry3.1Ab insect control protein in or 

on corn.

Pursuant to section 408(d)(2)(A)(i) of the FFDCA, as amended, Syngenta Seeds, 

Inc. has submitted the following summary of information, data, and arguments in support 

of their pesticide petition. This summary was prepared by Syngenta Seeds, Inc. and EPA 

has not fully evaluated the merits of the pesticide petition. The summary may have been 

edited by EPA if the terminology used was unclear, the summary contained extraneous 

material, or the summary unintentionally made the reader conclude that the findings 

reflected EPA’s position and not the position of the petitioner.

I. Syngenta Seeds, Inc. Petition Summary

[Insert petition number]

A. Product Name and Proposed Use Practices
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This notice of filing summarizes the information submitted and cited by Syngenta 

Seeds in support of a request for a temporary exemption from the requirement of a 

tolerance for the Bacillus thuringiensis eCry3.1Ab protein and the genetic material 

necessary for its production in corn. The eCry3.1Ab protein is highly specific to the 

targeted coleopteran species, and is nontoxic to mammalian species. 

Upon commercial introduction as a plant-incorporated protectant, eCry3.1Ab corn 

will offer growers a new option for corn rootworm pest control and integrated pest 

management. eCry3.1Ab corn will be an attractive and more effective alternative to 

conventional insecticides. eCry3.1Ab corn also offers environmental and worker safety 

benefits over conventional insecticide control options for control of coleopteran pests.

B. Product Identity/Chemistry

1. Identity of the pesticide and corresponding residues. The Bacillus 

thuringiensis eCry3.1Ab protein has been incorporated into corn plants to provide control 

of some coleopteran insect pests, for example Western corn rootworm. It is a chimeric 

protein of Bacillus thuringiensis modified Cry3A and Cry1Ab proteins. The ecry3.1Ab

transformed plants are derived from transformation event 5307 that contain the 

eCry3.1Ab protein via Agrobacterium mediated transformation.

2. Magnitude of residues at the time of harvest and method used to determine the

residue. A determination of the magnitude of residues at harvest is not required for 

residues that are temporarily exempt from tolerances.

3. A statement of why an analytical method of detecting and measuring the levels 

of the pesticide residue are not needed. This petition is a request for a temporary 

exemption from the requirement of a tolerance without numerical limitation, thus an 

analytical detection method should not be required. However, in response to an Agency 

request the Petitioner has submitted an immunoassay method for determination of 

eCry3.1Ab in corn tissues.

C. Mammalian Toxicological Profile

The eCry3.1Ab protein is derived from Bacillus thuringiensis. It is a chimeric 

protein of Bacillus thuringiensis modified Cry3A and Cry1Ab proteins. Bacillus 

thuringiensis proteins have been used as safe and effective pest control agents in 

microbial formulations for many years. The numerous toxicology studies conducted with 

these microbial products show no significant adverse effects, and demonstrate that the 

products are practically nontoxic to mammals.

It has been demonstrated that eCry3.1Ab protein display insect-specific toxicity 

following ingestion by sensitive coleopteran species. In the insect digestive tract, 

partially digested eCry3.1Ab protein can bind to specific receptors which are then 

inserted into the gut membrane to form ion-specific pores. The formation of membrane 
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pores results in electrolyte imbalances that cause death of the insect. This mode of action 

is not active in humans and other mammals as demonstrated in an acute mouse toxicology 

study where results confirmed that eCry3.1Ab is nontoxic to mice. 

The mammalian safety of eCry3.1Ab has been confirmed in studies summarized 

herein, that demonstrate the lack of toxicity of the eCry3.1Ab protein following high-dose 

acute oral exposures to mice, rapid degradation of eCry3.1Ab upon exposure to simulated 

mammalian gastric fluid, and the lack of amino acid sequence similarity of the eCry3.1Ab

protein to proteins known to be mammalian toxins or human allergens. These studies all 

support that eCry3.1Ab will be nontoxic to humans. 

Single high dose exposures to eCry3.1Ab in mice produced no measurable 

adverse effects. The eCry3.1Ab protein, produced in a heterologous fermentation system,

was administered orally to male and female mice; a no observable effect level of 2000 mg 

eCry3.1Ab/kg body weight was indicated and the LD50 can be estimated as >2000 mg 

eCry3.1Ab/kg body weight. No mortality or clinical signs attributable to the test 

substance were observed during the study. Plant and microbially-produced eCry3.1Ab 

proteins have been shown to be equivalent in terms of biological activity, molecular 

weight, immunoreactivity, and lack of post-translational modifications. 

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

low doses (Sjoblad, R. D., J. T. McClintock, and R. Engler, 1992). Therefore, when a 

protein displays no acute oral toxicity in high-dose testing using a standard laboratory 

mammalian test species, a determination can be made that the protein will be nontoxic to 

humans and other mammals, and will not present a hazard under any realistic exposure 

scenario, including long-term exposures. 

The amino acid sequence of eCry3.1Ab is not homologous to any non-B.t. 

proteins identified as, or known to be, toxins described in public databases. The amino 

acid sequence of eCry3.1Ab is not homologous to that of any known or putative allergens 

described in public databases. In addition, the eCry3.1Ab protein is not derived from a 

known source of allergens and does not display characteristics commonly associated with 

allergens, including post-translational glycosylation. Additionally, eCry3.1Ab is readily 

digested in simulated gastric fluid in the presence of pepsin at pH 1.2.

D. Aggregate Exposure

1. Dietary exposure. Information is available to estimate aggregate exposure 

levels for consumers and major identifiable subgroups of consumers to the plantincorporated protectant and related substances. Possible sources of exposure include 

dietary exposure under the tolerance exemption and all other tolerances or exemptions in 

effect for eCry3.1Ab protein residues, and exposure from other nonoccupational sources.

i. Food. Oral exposure, at very low levels may occur from ingestion of corn and

processed corn products; however, a lack of mammalian toxicity and the digestibility of 
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eCry3.1Ab protein have been demonstrated. The use sites for eCry3.1Ab proteins are all 

agricultural for the control of insects.

ii. Drinking Water Exposure. Oral exposure, at very low levels, may occur 

through drinking water; however, a lack of mammalian toxicity and the digestibility of 

eCry3.1Ab protein have been demonstrated. The use sites for eCry3.1Ab proteins are all 

agricultural for the control of insects.

2. Non-dietary exposure. Exposure via the skin or inhalation is not likely since 

eCry3.1Ab is contained within plant cells, which essentially eliminates these exposure 

routes or reduces them to negligible levels.

E. Cumulative Effects

There is no indication of mammalian toxicity potential for eCry3.1Ab protein. 

The Cry class of proteins class display toxicity towards a narrow spectrum of insect 

species. It is reasonable to conclude that there will be no cumulative effects for this 

protein.

F. Safety Determination

1. U.S. population. Based on available data for eCry3.1Ab proteins there are no 

indications of mammalian toxicological effects and no threshold effects of concern. 

Due to its digestibility, lack of toxicity and unlikely potential for allergenicity, eCry3.1Ab

dietary exposure is not anticipated to pose any harm to the U.S. population. There is no 

need for application of an additional safety factor for establishing an adequate margin of 

exposure. Furthermore, special provisions pertaining to unique consumption patterns, 

special susceptibility, and cumulative effects do not apply.

2. Infants and children. Based on available data for eCry3.1Ab protein there are 

no indications of mammalian toxicological effects and no threshold effects of concern. 

Due to its digestibility, lack of toxicity, and unlikely potential for allergenicity, 

eCry3.1Ab dietary exposure is not anticipated to pose any harm to infants and children. 

There is no need for application of an additional safety factor for establishing an adequate 

margin of exposure. Furthermore, special provisions pertaining to unique consumption 

patterns, special susceptibility, and cumulative effects do not apply.

G. Effects on the Immune and Endocrine Systems

The available safety data for eCry3.1Ab protein show no adverse effects in 

mammals at high levels of exposure; thus, no effects on mammalian immune or 

endocrine systems are predicted. Furthermore, the eCry3.1Ab protein is derived from 

Bacillus thuringiensis and Bacillus thuringiensis is known not to exert an influence on 

the endocrine system.
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H. Existing Tolerances

There are no existing tolerances or exemptions from the requirement of a 

tolerance for the eCry3.1Ab protein granted to Syngenta Seeds.

I. International Tolerances

No Codex maximum residue levels exist for the eCry3.1Ab protein and the 

genetic material necessary for their production in plants.