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Study Number: PHI-2012-183

Appendix I

i

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: [insert name and 

telephone number with area code]

SUBMISSION: Email the completed template to: hollis.linda@epa.gov.

TEMPLATE:

Pioneer Hi-Bred International, Inc.

[Insert petition number]

 EPA has received a pesticide petition ([insert petition number]) from Pioneer Hi-Bred 

International, Inc.(DuPont Pioneer), 7100 NW 62nd Avenue, P.O. Box 1000, Johnston, Iowa, 

50131 requesting, 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 180

(Options (pick one)

1. by establishing a tolerance for residues of-NA-Remove

2. to establish an exemption from the requirement of a tolerance for- GM-HRA

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

NA-Remove

(Options (pick one)

1. microbial pesticide NA-Remove

2. biochemical pesticide NA-Remove

3. plant-pesticide inert GM-HRA in or on soybean.

Pursuant to section 408(d)(2)(A)(i) of FFDCA, as amended, DuPont Pioneer has submitted 

the following summary of information, data, and arguments in support of their pesticide 

petition. This summary was prepared by DuPont Pioneer and EPA has not fully evaluated the 

merits of the pesticide petition. The summary may have been edited by EPA if the
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Study Number: PHI-2012-183

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. DuPont Pioneer Petition Summary

[Insert petition number]

A. Product Name and Proposed Use Practices

GM-HRA is a protein obtained from a genetically modified endogenous soybean gene, als I, 

that provides tolerance against specific classes of herbicides allowing it to be used as a 

selectable herbicide-tolerant marker in soybean transformation events. As part of a genetic 

construct introduced into a plant’s genome, GM-HRA itself does not have insectical activity 

and is therefore functionally inert as part of a plant-incorporated protectant.

B. Product Identity/Chemistry

1. Identity of the pesticide and corresponding residues. The gene encoding the GMHRA protein is designated gm-hra because it is derived from Glycine max and is an 

herbicide-resistant allele. The herbicide-tolerant gm-hra gene was made by isolating the 

herbicide-sensitive soybean gm-als I gene and introducing changes in the DNA sequence that 

result in two specific amino acid changes in the protein. These amino acid substitutions are 

analogous to those in herbicide-tolerant tobacco (Creason and Chaleff, 1988; Lee et al., 

1988), and additionally represent commonly found natural tolerance mutations. Ultimately, 

this two-amino acid modification of the endogenous GM-ALS I protein to GM-HRA protein 

yields an enzyme that is insensitive to ALS-inhibiting herbicides, allowing its use as a 

selectable marker during transformation and potentially a herbicide-tolerant trait in soybeans.

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

residue. Because an exemption from the requirement of a tolerance is requested for GMHRA protein as expressed in soybean, this section of the petition is not applicable. However, 

ranges of GM-HRA protein expression in events containing the gm-hra gene are quite low.

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

the pesticide residue are not needed. Because an exemption from the requirement of a 

tolerance without numerical limitation is requested for GM-HRA protein as expressed in 

soybean, this section of the petition is not applicable.

C. Mammalian Toxicological Profile

1) The donor organism is soybean (Glycine max.), which has a long history of human 

use. The GM-HRA protein is only two amino acids different from the endogenous 

soybean ALS I protein from which it was derived (Volume 3, PHI-2012-020). ALS 
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Study Number: PHI-2012-183

enzymes have also been part of the human diet by virtue of their presence in 

soybeans. 

2) Bioinformatic analyses revealed GM-HRA to be similar to other ALS proteins found 

in bacteria, fungi, algae and plants (Friden et al., 1985; Falco et al., 1985; Reith and 

Munholland, 1995; Mazur et al., 1987).

3) There was no evidence of acute toxicity in mice at a target dose of 2000 mg purified 

protein preparation per kg of body weight (equivalent to approximately 436 mg of 

full-length GM-HRA protein per kg of body weight) (Volume 4, PHI-2006-008). 

4) Bioinformatic analyses revealed no biologically relevant amino acid sequence 

identities between known or putative protein allergens and the GM-HRA protein 

sequence. These data indicate the lack of both amino acid identity and 

immunologically relevant similarities between the GM-HRA protein and known 

protein allergens (Volume 5, PHI-2006-070/070).

5) No biologically relevant amino acid sequence identities were observed between 

known protein toxins and the GM-HRA protein sequence (Volume 6, PHI-2006-

071/070).

6) The GM-HRA protein is rapidly digested in both simulated gastric and intestinal 

fluids (less than 30 seconds in simulated gastric fluid containing pepsin at pH 1.2 as 

demonstrated by SDS-PAGE analysis and Western blot, and less than 30 seconds in 

simulated intestinal fluid containing pancreatin at pH 7.5 as demonstrated by SDSPAGE/Coomassie analysis or less than 1 minute as demonstrated by Western blot 

analysis) (Volume 7, PHI-2006-199; Volume 8, PHI-2006-074). 

7) The GM-HRA protein is heat labile (i.e., heating at 50 °C for 15 min completely 

inhibits the enzyme activity of the GM-HRA protein) (Volume 9, PHI-2006-135).

8) The plant-expressed GM-HRA protein is not glycosylated as demonstrated by 

glycoprotein staining (Volume 2, PHI-2011-225).

9) As part of two other transgenic soybean events (DP-3Ø5423-1 and DP-356Ø43-5), 

the GM-HRA protein has been the subject of safety reviews by both USDA and FDA 

(Rood et al., 2006; Pavely et al., 2007; US-FDA, 2007a; US-FDA, 2009a). Neither 

agency identified safety concerns with the protein (USDA-APHIS, 2008; USDAAPHIS, 2010; US-FDA, 2007b; US-FDA, 2009b). Additionally, a safety conclusion 

regarding GM-HRA exists in the scientific literature published by Mathesius et al

(2009).

10) Based on the identical nature of the gm-hra cassette used in 82117 soybean and the 

two other transgenic soybean events expressing the GM-HRA protein (DP-3Ø5423-1 
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and DP-356Ø43-5), expression ranges and dietary exposure of GM-HRA from 82117 

soybean are expected to be similarly low.

D. Aggregate Exposure

1. Dietary exposure. Expression ranges and dietary exposure calculations for GMHRA as expressed in DP-3Ø5423-1 and DP-356Ø43-5 are very low. Based on the identical 

genetic elements used in 82117 soybean, GM-HRA expression and subsequent exposure are 

expected to be similarly quite low.

i. Food. Expression ranges and dietary exposure calculations for GM-HRA as 

expressed in DP-3Ø5423-1 and DP-356Ø43-5 are very low. Based on the identical genetic 

elements used in 82117 soybean, GM-HRA expression and subsequent exposure are expected 

to be similarly quite low.

ii. Drinking water. While not likely, oral exposure at very low levels may occur 

through drinking water; however, a lack of mammalian toxicity has been demonstrated.

2. Non-dietary exposure. Exposure to GM-HRA via inhalation or dermal contact is 

not likely as GM-HRA is contained within plant cells.

E. Cumulative Effects

There is no indication of mammalian toxicity potential for GM-HRA. It is therefore 

reasonable to conclude there will be no cumulative toxic effects for this protein.

F. Safety Determination

1. U.S. population. Based on the available data for the GM-HRA protein, there are no 

indications of mammalian toxicological effects. GM-HRA was derived from a common 

dietary source (soybean), was not acutely toxic in mice, and the amino acid sequences of the 

GM-HRA protein exhibited no significant homology to the amino acid sequences of proteins 

known to be food allergens or toxins; and the protein is not glycosylated. GM-HRA was also 

highly digestible in vitro, as well as thermally inactivated at 50  ̊C. Further, the dietary risk 

for other transgenic soybean events expressing GM-HRA (DP-3Ø5423-1 and DP-356Ø43-5)

using genetic elements identical to those contained in 82117 soybean to drive GM-HRA 

expression, have been calculated and are quite low. 82117 soybean is expected to express 

GM-HRA at ranges comparable to other GM-HRA expressing events, to have a similarly low 

dietary risk, and is not anticipated to cause any harm to the U.S. population, including any 

sensitive sub-populations or vulnerable age groups.

2. Infants and children. Based on the available data for the GM-HRA protein, there 

are no indications of mammalian toxicological effects. GM-HRA was derived from a 

common dietary source (soybean), was not acutely toxic in mice, and the amino acid 

sequences of the GM-HRA protein exhibited no significant homology to the amino acid 

sequences of proteins known to be food allergens or toxins; and the protein is not 
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Study Number: PHI-2012-183

glycosylated. GM-HRA was also highly digestible in vitro, as well as thermally inactivated 

at 50  ̊C. Further, the dietary risk for other transgenic soybean events expressing GM-HRA 

(DP-3Ø5423-1 and DP-356Ø43-5) using genetic elements identical to those contained in 

82117 soybean to drive GM-HRA expression, have been calculated and are quite low. 82117 

soybean is expected to express GM-HRA at ranges comparable to other GM-HRA expressing 

events, to have a similarly low dietary risk, and is not anticipated to cause any harm to the 

U.S. population, including any sensitive sub-populations or vulnerable age groups.

G. Effects on the Immune and Endocrine Systems

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.” 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.

The GM-HRA protein is not among the group of 58 pesticide active ingredients on the initial 

list to be screened under the EDSP.

H. Existing Tolerances

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

GM-HRA protein and the genetic material necessary for its production. However, GM-HRA 

as encoded by the gm-hra gene has been previously evaluated for its safety by the U.S. Food 

and Drug Administration (FDA) and the U.S. Department of Agriculture (USDA) in two 

transgenic soybean events, one designed to offer high oleic soybean oil and one that offered 

glyphosate and ALS-inhibiting herbicide tolerance. Based on the information provided, FDA 

concluded that the safety profiles of the new soybean events including the GM-HRA protein 

were not materially different from that of other marketed soybean varieties, and FDA 

required no further premarket review or approval activities related to the events or the 

proteins. Upon its review of information regarding these events and the GM-HRA protein, 

USDA deregulated both transgenic soybean events.

I. International Tolerances

International tolerances do not exist for this protein, however safety information related to the 

GM-HRA protein has been reviewed by international food safety authorities as part of the 

high oleic and herbicide-tolerant event submissions.