Pioneer Hi-Bred International, Inc. Page 1 of 34

Study Number: PHI-2012-183

TITLE

Petition for an Exemption from the Requirement of a Tolerance for Glycine max Herbicide-Tolerant 

Acetolactate Synthase (GM-HRA) Inert Ingredient as Expressed in Soybean

DATA REQUIREMENT

None

AUTHOR

Nicole C. Jansen

Pioneer Hi-Bred International, Inc.

7100 NW 62nd Avenue

Johnston, IA 50131

USA

STUDY COMPLETION DATE

June 25, 2012

STUDY NUMBER

PHI-2012-183

EPA REGISTRATION NUMBER

29964-x

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Study Number: PHI-2012-183

TABLE OF CONTENTS PAGE

Statement of Data Confidentiality Claim ...........................................................................................2

Transmittal Document ......................................................................................................................3

Section I. Administrative Materials...................................................................................................6

A.1. EPA Form 8570-1....................................................................................................................12

A.2.EPA Form 8570-35...................................................................................................................17

Section II. GM-HRA Tolerance Exemption Petition .......................................................................14

Section A. Name, Chemical Identity, and Composition of Pesticide Chemical ..............................14

Section B. Amount, Frequency, and Time of Application of the Pesticide Chemical......................16

Section C. Safety of the Pesticide Chemical ..................................................................................17

Section D. Amount of Residue Remaining, Including Description of Analytical Method.................23

Section E. Practical Methods for Removing Residues That Exceed Proposed Tolerance ............25

Section F. Proposed Tolerances for the Pesticidal Chemical if Tolerances are Proposed ............26

Section G. Reasonable Grounds in Support of the Petition............................................................27

References .....................................................................................................................................29

Appendix I: Petitioner’s Notice of Filing Summary.........................................................................33

Tables

Table 1. GM-HRA Protein Concentrations in Seed in Events Containing gm-hra ........................23

Table 2. Dietary Risk Calculations for Events Containing GM-HRA Protein .................................24
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Study Number: PHI-2012-183

TRANSMITTAL DOCUMENT

SUBMITTER

Pioneer Hi-Bred International, Inc.

7100 NW 62nd Ave.

Johnston, IA 50131

REGULATORY ACTION IN SUPPORT OF WHICH THIS DOCUMENT IS SUBMITTED

Application for a Tolerance Exemption for an Inert Ingredient of a Plant-Incorporated Protectant

Pursuant to FIFRA §3(c)(5):

Petition for an Exemption from the Requirement of a Tolerance for Glycine max Herbicide-Tolerant

Acetolactate Synthase (GM-HRA) Inert Ingredient as Expressed in Soybean

.

29964-x

TRANSMITTAL DATE

June 25, 2012

STUDY NUMBER

PHI-2012-183
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Study Number: PHI-2012-183

LIST OF ACCOMPANYING VOLUMES

VOL. FIFRA 

10(G) VOLUME/STUDY TITLE MRID

A

Jansen, N.C. (2012). Administrative Volume for Application for a 

Tolerance Exemption for an Inert Ingredient of a Plant-Incorporated 

Protectant

Study Number: PHI-2012-183 June 25, 2012

Pages: 1-34

ADMIN

1 B

Characterization of GM-HRA Protein Derived from a Microbial 

Expression System

Buffington, J. (2006)

Study Number: PHI-2006-016 September 21, 2006

Pages: 1-28 3 copies

2 B

Characterization of GM-HRA Protein Derived From DP-Ø82117-3 

Soybean

Zhang, J. (2012). 

Study Number: PHI-2011-225 May 18, 2012

Pages: 1-24 3 copies

3 B

Description, Derivation, Mode of Action and Familiarity of the GM-HRA 

Enzyme

Locke, M et al. (2012)

Study Number: PHI-2012-020 June 25, 2012

Pages: 1-22 3 copies

4 B

GM-HRA: Acute Oral Toxicity Study in Mice

Finlay, C. (2006)

Study Number: PHI-2006-008 August 9, 2006

Pages: 1-39 3 copies

5 B

Comparison of the Amino Acid Sequence Identity Between the GMHRA Protein and Known Protein Allergens

Krauss, A. (2012)

Study Number: PHI-2006-070/070 May 10, 2012

Pages: 1-23 3 copies

6 B

Evaluation of the Amino Acid Sequence Similarity of the GM-HRA 

Protein to the NCBI Protein Sequence Datasets

Krauss, A. (2012)

Study Number: PHI-2006-071/070 May 11, 2012

Pages: 1-8249 3 copies

7 B

Characterization of the In Vitro Pepsin Resistance of GM-HRA using 

Western Blot Analysis

Schmidt, J. (2007)

Study Number: PHI-2006-199 January 16, 2007

Pages: 1-16 3 copies

8 B

Characterization of the In Vitro Pancreatin Resistance of GM-HRA

Comstock, B. (2007)

Study Number: PHI-2006-074 December 10, 2007

Pages: 1-19 3 copies

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Study Number: PHI-2012-183

SECTION I. ADMINISTRATIVE MATERIALS

A.1. EPA Form 8570-1: Application for Pesticide Registration

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Study Number: PHI-2012-183

A.2. EPA Form 8570-35: Data Matrix for GM-HRA Tolerance Exemption




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SECTION II. GM-HRA TOLERANCE EXEMPTION PETITION

Section A. Name, Chemical Identity, and Composition of Pesticide Chemical

Soybean event DP-Ø82117-3 (hereafter referred to as 82117 soybean) was produced by particle

bombardment with the PHP30987A fragment and expresses the GM-HRA protein. GM-HRA provides 

tolerance against specific classes of herbicides and can be used as a selectable marker for soybean 

transformation. Sufficient concentrations of GM-HRA protein in transgenic events may mitigate crop 

injury from ALS-inhibiting herbicides and potentially enable the use of additional ALS-inhibiting 

herbicides typically not tolerated by soybean. As part of a genetic construct introduced into a soybean 

plant’s genome, GM-HRA itself does not have pesticidal activity and is therefore functionally inert as 

part of a plant-incorporated protectant.

ALS enzymes are widely distributed in nature, as als genes have been isolated from bacteria, fungi, 

algae and plants (Friden et al., 1985; Falco et al., 1985; Reith and Munholland, 1995; Mazur et al., 

1987). ALS is a key enzyme that catalyzes the first common step in the biosynthesis of the essential 

branched-chain amino acids isoleucine, leucine, and valine (LaRossa and Schloss, 1984; LaRossa 

and Falco, 1984; Duggleby and Pang, 2000; Coruzzi and Last, 2000). ALS has been shown to be 

inhibited by five chemical classes of herbicides (including sulfonylureas, imidazolinones, 

triazolopyrimidines, pyrimidinylthio (or oxy)-benzoates, and sulfonylamino-carbonyltriazolinones [Tan 

et al., 2006]) and these compounds inhibit plant growth by inactivating ALS. 

The gene encoding the GM-HRA 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 ALSinhibiting herbicides, allowing its use as a selectable marker during transformation and potentially a 

herbicide-tolerant trait in soybeans.

To obtain the protein amounts sufficient to conduct toxicological and biochemical studies, the GMHRA protein was overexpressed in E. coli strain BL 21 (DE3) RIPL using recombinant DNA 

technology. Various characterization tests were conducted to confirm protein identity, as well as

equivalency of the microbially expressed and in planta produced GM-HRA proteins. A detailed 

description of methods used in these protein characterization studies and the resulting data are 

included in Volumes 1 and 2 (PHI-2006-016 and PHI-2011-225, respectively) of this application. 

Characterization of the physicochemical properties of the plant and microbially expressed GM-HRA 

proteins was accomplished through the use of the following techniques:

1) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) coupled with 

Coomassie-based staining to determine the purity and confirm molecular weight of the 

proteins;

2) Western blot analysis to confirm the molecular weight and immunoreactivity of the proteins;

3) Glycoprotein staining of the proteins to determine the post-translational modification 

(glycosylation);
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4) Mass determination of the tryptic peptides by matrix assisted laser desorption ionization mass 

spectrometry (MALDI-MS) to confirm the identity of the proteins;

5) N-terminal amino acid sequence analysis to further confirm the identity of the proteins.

Utilizing the above characterization methods, the identity of both the microbially expressed and the 

82117 soybean plant-derived GM-HRA proteins was verified, and a comparison of the physiochemical 

properties confirmed that the two proteins were equivalent (Volume 1, PHI-2006-016; Volume 2, PHI2011-225). Based on this confirmation of identity and equivalence, the microbial-expressed GM-HRA 

protein is suitable as a substitute for the plant-expressed GM-HRA protein for in vitro and in vivo

safety assessment studies. 
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Section B. Amount, Frequency, and Time of Application of the Pesticide Chemical

GM-HRA is a selectable marker that is part of a plant-incorporated protectant (PIP) and is not applied 

to crops in the same manner as traditional pesticides.
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Section C. Safety of the Pesticide Chemical

A weight of evidence approach was applied to evaluate the allergenicity and toxicity potential of the 

GM-HRA protein (Codex Alimentarius Commission, 2003; Volume 10, PHI-2012-200). This approach 

takes into account a variety of relevant factors and experimental observations used to derive an 

overall assessment of the allergenic and toxicity potential of a novel protein. The toxic and allergenic

potential of GM-HRA was assessed by: 1) ) review of the gm-hra gene source and history of use or 

exposure (Volume 3, PHI-2012-020); 2) studying the acute oral toxicity of the GM-HRA protein in mice

(Volume 4, PHI-2006-008); 3) a bioinformatic comparison of the amino acid sequence of the GM-HRA 

protein with known protein allergen and toxin sequences (Volume 5, PHI-2006-070/070; Volume 6, 

PHI-2006-071/070); 4) evaluation of the lability of the microbially produced and purified GM-HRA 

protein from E. coli using in vitro gastric and intestinal digestion models, as well as heat treatment

(Volume 7, PHI-2006-199; Volume 8, PHI-2006-074; Volume 9, PHI-2006-135); and 5) determination 

of the protein glycosylation status (Volume 2, PHI-2011-225). The results from this assessment 

demonstrate the lack of allergenicity and toxicity potential of the GM-HRA protein.

C.1. Source and History of Exposure (Volume 3, PHI-2012-020)

The gm-hra gene, encoding the GM-HRA protein, was obtained from soybeans which have a long 

history of human consumption, with broader U.S. consumption occurring over the last 80 years. 

Soybean is commonly considered as one of the oldest cultivated crops. It’s widely accepted that 

domesticated soybean originated in China, however there’s some controversy as to when this first 

occurred. Early accounts were that the first recording of domesticated soybeans was in a series of 

books known as Pen Ts'ao Kong Mu written by the Chinese emperor, Sheng Nung, in the year 2838 

BC, in which various plants of China are described (Morse, 1950). However, more recent 

investigations reveal evidence indicating that soybean domestication occurred in China sometime 

around 1,100 BC, or about 3,000 years ago (Hymowitz and Shurtleff, 2005; Hymowitz, 2008). 

Soybeans were brought into the United States in 1765 (Hymowitz and Harlan, 1983), with most of the 

early U.S. soybean usage for forage crop rather than harvested for seed. For many years, soybean 

acreage increased very slowly until a World War II disruption of trade routes and the necessity to find 

U.S. based alternatives to imported edible fats and oils, resulted in a rapid expansion of soybean 

acreage in the U.S. (Hansen and Huntrods, 2011). Increased domestic use of soybean for human 

food applications beginning in the 1940’s logically increased the dietary exposure of the U.S. 

population to soybeans through consumption of these products. Currently in the United States, nearly 

all soybeans are crushed to extract the oil from the resulting meal; a comparatively small amount of 

whole soybeans are used for seed, roasted for snacks or on-farm dairy feed, and consumed as 

traditional soy-containing foods such as tofu (USDA-ERS, 2010).

As discussed more fully in Volume 1 (PHI-2012-020), the herbicide-tolerant gm-hra gene, coding for 

the GM-HRA protein, 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; accordingly, GM-HRA is the two amino-acid modified version of the soybean native 

acetolactate synthase I (ALS I) protein and the proteins are >99% homologous. ALS enzymes occur 

in soybean, but they are also widely distributed in nature; als genes have been isolated from bacteria, 

fungi, algae and plants (Friden et al., 1985; Falco et al., 1985; Reith and Munholland, 1995; Mazur et 

al., 1987). The ubiquitous nature of ALS enzymes is also supported by the BLASTP analysis yielding 

12,451 structurally or functionally related protein accessions (Volume 6, PHI-2006-071). Importantly, 

ALS enzymes have been in the human food supply by virtue of their presence in soybean and other 

identified sources without reported adverse health effects. 
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From structural information it can be concluded that GM-HRA can be considered a familiar protein. 

The two amino acid changes in GM-HRA, resulting in a >99% homology to the endogenous soybean 

ALS I enzyme, reflect naturally evolved mutations identified in ALS proteins derived from multiple ALStolerant plant species, supported by the over 12,000 related protein accessions mentioned above 

(Volume 6, PHI-2006-071/070). These two specific mutations are equivalent to the locations of the 

commonly found natural tolerance mutations P197A and W574L in Arabidopsis ALS (Duggleby and 

Pang, 2000). Moreover, there are five commonly occurring mutations in ALS proteins that confer 

tolerance to ALS-inhibiting herbicides; mutations similar or identical to the two in GM-HRA (P183A and 

W560L) are present in several commercially available, non-transgenic, herbicide-tolerant crop 

varieties with a history of safe consumption (see list of commercially available crops in Volume 1, PHI2012-020). 

C.2. Acute Toxicity in Mice (Volume 4, PHI-2006-008)

A single target dose of GM-HRA protein preparation (containing approximately 29% microbially 

expressed, purified GM-HRA protein) was administered by oral gavage to groups of five fasted male 

and five fasted female Crl:CD®

-1(ICR)BR mice at a target dose of 2000 mg/kg. The actual dose of 

purified full-length GM-HRA protein was 436 mg/kg, accounting for 75% of intact GM-HRA protein. 

Two control groups of five fasted male and five fasted female mice were administered bovine serum 

albumin (BSA) at a dose of 2000 mg/kg or water alone at an equivalent dose volume to the GM-HRA 

treated mice.

The mice were observed for mortality, body weight gain, and clinical signs for 14 days post dosing, 

after which they were euthanized and subjected to gross necropsy to detect observable evidence of 

organ or tissue damage or dysfunction. Mortality, clinical signs and body weights were recorded. No 

clinical signs of toxicity, body weight losses, gross lesions, or mortality were observed in male and 

female mice at a dose of 436 mg/kg full-length GM-HRA protein. 

C.3. Bioinformatics Analysis to Known Protein Allergens and Toxins

Allergenicity Assessment (Volume 5, PHI-2006-070/070)

Bioinformatic analyses were conducted to evaluate the potential allergenicity of the GM-HRA protein.

The original criteria established by FAO/WHO (Food and Agricultural Organization of the United 

Nations/World Health Organization) in 2001 for determining identity were 1) 35% or greater FASTA 

identity with an allergen protein sequence over a region of 80 amino acids in length or greater, and 2) 

contiguous matches of 6 amino acid residues or greater (FAO/WHO, 2001; Codex, 2003). The use of 

a 6 contiguous amino acid match for identifying potentially cross-reactive allergenic proteins is not 

scientifically supported; matches occur too commonly between unrelated proteins and therefore, do 

not reliably predict allergenic potential (Hileman, 2002; Stadler, 2003; Ladics, 2006; Silvanovich, 

2006). The searches performed in this study use an 8 contiguous amino acid match as originally 

suggested (Metcalfe et al., 1996). 

The amino acid sequence of the GM-HRA protein was compared to a database of allergens derived 

from the Food Allergy Research and Resource Program (FARRP), University of Nebraska, Allergen 

Database (Release 12 - February 2012), which contains 1603 entries representing both putative and 

proven food, environmental, and contact allergens as well as proteins implicated in celiac disease. 

Potential identities between the GM-HRA amino acid sequence and those of proteins in the allergen 

database were evaluated using the FASTA35 sequence alignment program (Pearson and Lipman, 
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Study Number: PHI-2012-183

1988) set to the default parameters. Alignments were reviewed for identities greater than or equal to 

35% over 80 or greater residues. 

Results of the evaluation showed no eight or greater contiguous identical amino acid stretches in 

common between the GM-HRA protein sequence and any of the protein sequences in the allergen 

dataset. None of the FASTA35 alignments between the GM-HRA protein sequence and the 

sequences in the allergen dataset exceeded the 35% threshold over 80 or greater amino acids. These 

data indicate that the GM-HRA protein does not show sequence identity with known allergens.

Toxicity Assessment (Volume 6, PHI-2006-071)

A global sequence similarity search of the GM-HRA amino acid sequence against the National Center 

for Biotechnology Information (NCBI) Protein dataset was conducted using the BLASTP algorithm. A 

sequence file comprising the translation of the gm-hra gene was queried using the BLASTP 2.2.25 

algorithm against Release 188.0 (02/15/12) of the NCBI Entrez Protein dataset, which incorporates 

non-redundant entries from all GenBank and RefSeq nucleotide translations along with protein 

sequences from SWISS-PROT (http://www.expasy.org/sprot/), PIR (http://pir.georgetown.edu/), PRF 

(http://www.prf.or.jp/index-e.html), and PDB (http://www.wwpdb.org/). 

One of the most important parameters to monitor when performing similarity searches is the 

expectation, or E score. This score represents the probability that a particular alignment is due to 

random chance and can be used to evaluate the significance of an alignment. The calculated E score 

depends on the overall length of the aligned sequences (including inserted gaps), the number of 

identical and conserved residues within the alignment, and the size of the database (Pearson and 

Lipman, 1988; Baxevanis and Ouellette, 2005). When examining an alignment between two protein 

sequences, a very small E score is more likely to reflect a true similarity, while a large E score is more 

likely to be produced by chance and therefore less biologically relevant.

A cutoff expectation (E) value of 1.0 was used to generate biologically meaningful similarity between 

the protein of interest (GM-HRA) and proteins in the NCBI datasets. Although a statistically significant 

sequence similarity generally requires a match with an expectation value less than 0.01, a cutoff of E 

≤ 1.0 insures that proteins with even limited similarity will not be overlooked in the search (Pearson, 

2000). The scoring matrix used was the default (BLOSUM62), low complexity filtering was turned off, 

and the number of alignments returned was set to 20,000 using additional BLAST options.

The GM-HRA similarity search identified 12,451 protein accessions with an E score of less than 1.0, 

demonstrating the widespread presence of similar proteins in nature. The highest E score returned 

was 0.99, confirming that sequences with limited similarity were not overlooked in the search. Two 

hundred and sixty-one of the accessions returned by the searches displayed complete significance (E 

= 0) and represent very closely related acetohydroxyacid synthase (AHAS) or acetolactate synthase 

(ALS) proteins from various plant species. Twenty-seven of these accessions are labeled as 

hypothetical, predicted, or unnamed proteins, and are classified separately from the ALS/AHAS 

proteins. A total of 5,044 sequences were identified as either AHAS or ALS proteins from various 

bacterial, archaebacterial, and eukaryotic species. The remaining 7,407 hits represented a variety of 

proteins, the great majority of which were functionally related by the possession of one or more well 

characterized conserved thiamine pyrophosphate (TPP) binding domains. 

None of the protein sequences returned by the BLASTP search identified safety concerns that might 

arise from the expression of GM-HRA in genetically modified plants.
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Study Number: PHI-2012-183

C.4. Digestibility of the GM-HRA Protein

Simulated Gastric Fluid (Volume 7, PHI-2006-199)

Simulated gastric fluid (SGF) was used to assess the susceptibility of the GM-HRA protein to 

proteolytic digestion by pepsin in vitro. The SGF formulation, time course, and experimental 

parameters followed in the evaluation of GM-HRA were similar to conditions employed in the ILSI 

multi-laboratory evaluation (Thomas et al., 2004).

The GM-HRA protein was incubated in SGF containing pepsin at pH 1.2 for specific time intervals (0, 

0.5, 1, 2, 5, 10, 20, 30 and 60 minutes) and analyzed by sodium dodecyl sulfate polyacrylamide gel 

electrophoresis (SDS-PAGE) and Western blot. Coomassie-based stain was used to detect protein 

bands. Bovine serum albumin (BSA) and β-lactoglobulin were employed as positive and negative 

control substances in the assay, and respectively disappeared rapidly and persisted in pepsin, 

verifying that the assay performed as expected. 

The GM-HRA protein was rapidly (< 30 seconds) digested in SGF containing pepsin at pH 1.2 as 

demonstrated by both Western blot and SDS-PAGE analysis. Peptide fragments that migrate with the 

loading dye front at the bottom of the gel during SDS-PAGE have been confirmed to have masses <3 

kDa. There are theoretical limits to the peptide size required to cross-link two IgE antibodies on the 

surface of mast cells or basophils and induce degranulation. Data from the literature indicate that 

peptide fragments between 1.5 and 3.5 kDa in size are unlikely to elicit mast cell degranulation in 

sensitized individuals (Kane et al., 1988; FAO/WHO, 2001; Bannon et al., 2002; Lack et al., 2002; 

Thomas et al., 2004).

Simulated Intestinal Fluid (Volume 9, PHI-2006-074)

Simulated intestinal fluid (SIF) was used to assess the susceptibility of the GM-HRA protein to 

proteolytic digestion by pancreatin in vitro. The GM-HRA protein was incubated in SIF containing 

porcine pancreatin prepared as described in the United States Pharmacopoeia (USP-NF, 2000) for 

specific time intervals (0, 0.5, 1, 2, 5, 10, 20, 30 and 60 minutes), analyzed by SDS-PAGE and stained 

with Coomassie-based stain. β-lactoglobulin and BSA were used as controls. In addition to the SDSPAGE gel, western blot analysis was conducted to ensure that the multiple bands in the pancreatin 

digestion mixture did not interfere with the ability to detect GM-HRA-derived fragments.

The GM-HRA protein was detected following SDS-PAGE/Coomassie-based staining and western blot 

analyses at 30 seconds and one minute, respectively. The GM-HRA protein was rapidly (< 1 minute) 

and completely digested in SIF containing pancreatin at pH 7.5. 

Thermal Lability (Volume 10, PHI-2006-135)

The objective of this study was to characterize the thermal stability of the GM-HRA enzyme. Purified 

GM-HRA protein was incubated at 2 °C incremental temperatures ranging from 36-60 °C, and was 

then subjected to the acetolactate synthase/acetohydroxyacid synthase (ALS/AHAS) activity assay for 

GM-HRA protein in the presence and absence of an herbicide containing the active ingredient 

chlorsulfuron.

The results of the study showed that the GM-HRA enzyme, either in the presence or absence of, 

chlorsulfuron, was inactivated when incubated for 15 minutes at 50 °C.
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Study Number: PHI-2012-183

C.5. Glycosylation Analysis (Volume 2, PHI-2011-225)

To examine whether the GM-HRA protein isolated from 82117 soybean tissue was glycosylated, the 

protein was electrophoresed (SDS-PAGE) together with a glycoprotein-positive control (horseradish 

peroxidase) and glycoprotein-negative control (soybean trypsin inhibitor). Potential glycoproteins were 

detected with a GelCode Glycoprotein Staining Kit from Pierce according to the manufacturer’s 

instructions. Results showed that GM-HRA protein, isolated and purified from whole plant tissue from 

82117 soybean plants was not glycosylated.

C.6. Additional Safety Reviews

The GM-HRA safety data presented above has also been previously evaluated by both FDA and 

USDA as part of submissions for two other transgenic soybean events, DP-3Ø5423-1 high oleic

soybean and DP-356Ø43-5 glyphosate-tolerant soybean(US-FDA, 2007a; US-FDA, 2009a; Rood et 

al., 2006; Pavely et al., 2007). 

In addition to reviewing safety data related to each event, both USDA and FDA reviewed nutrient 

composition information. Notably, in its submissions to the Agencies for both the high-oleic event and 

GAT tolerant events, Pioneer Hi-Bred International, Inc. (DuPont Pioneer) shared that the 

concentrations of two minor fatty acids commonly found in soybean oil, heptadecanoic acid (C17:0)

and heptadecenoic acid (C17:1), were slightly increased due to the mutation in the GM-HRA protein

(US-FDA, 2007a; US-FDA, 2009a; Rood et al., 2006; Pavely et al., 2007). DuPont Pioneer evaluated 

the potential for health risks from dietary consumption of C17:0 and C17:1 fatty acids and concluded 

no safety or nutritional issues were expected as a result of a slight increase in exposure (US-FDA, 

2007a; US-FDA, 2009a; Rood et al., 2006; Pavely et al., 2007). 

Based on the information provided in the submissions for each event, including safety information 

related to the GM-HRA protein and C:17 fatty acid content, FDA concluded that the safety profiles of 

both new soybean events were not materially different from that of other marketed soybean varieties, 

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

(US-FDA, 2007b; US-FDA, 2009b). Likewise, subsequent to its review of similar information, USDA 

deregulated both events (USDA-APHIS, 2008; USDA-APHIS, 2010).

A conclusion regarding the dietary safety of GM-HRA has also been published in the scientific 

literature. Mathesius et al. (2009), in the peer-reviewed publication, “Safety assessment of a modified 

acetolactate synthase protein (GM-HRA) used as a selectable marker in genetically modified 

soybeans”, concluded that the GM-HRA protein from genetically engineered soybean is safe and that 

no adverse health effects are expected from exposure to the GM-HRA protein, when used consistent 

with agricultural biotechnology practices. 

C.7. Conclusion

The results from this weight of evidence approach and previous assessments by USDA and FDA 

support the conclusion that the GM-HRA protein lacks allergenic or toxic properties.
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Section D. Amount of Residue Remaining, Including Description of Analytical Method

The gm-hra genetic cassette used by DuPont Pioneer during development of transgenic soybean 

event DP-Ø82117-3 (82117) is the same genetic cassette employed by DuPont Pioneer in two 

additional transgenic soybean events, DP-3Ø5423-1 (high oleic soybean) and DP-356Ø43-5 

(herbicide-tolerant soybean). Both of these events were the subject of review and subsequent 

regulatory clearance by both USDA and FDA (Rood et al., 2006; Pavely et al., 2007; US-FDA, 2007a; 

US-FDA, 2009a; USDA-APHIS, 2008; USDA-APHIS, 2010; US-FDA, 2007b; US-FDA, 2009b). Based 

on the identical nature of the genetic cassette used in all of these events, the expression of the GMHRA protein in all events is expected to be comparable, resulting in a similarly low dietary risk. 

D.1. The gm-hra Gene in Transgenic Soybean Events

The gm-hra genetic cassette used by DuPont Pioneer during development of its transgenic soybean 

events consists of identical genetic elements: a soybean S-adenosyl-L-methionine synthetase (SAMS) 

promoter and control elements conferring constitutive expression of the transgene in soybean tissues; 

the gm-hra gene; and the gm-als terminator. Details relating to the aforementioned GM-HRA 

expressing events appear below. 

Event DP-3Ø5423-1 High Oleic Soybean

Event DP-3Ø5423-1 is a transgenic soybean product that provides soybean seeds with 

increased levels of monounsaturated (oleic) fatty acid and decreased levels of 

polyunsaturated fatty acids (linoleic and linolenic) and to a lesser extent, palmitic acid. In DP3Ø5423-1 soybean, the gm-hra gene is solely used as a selectable marker. 

Event DP-356Ø43-5 Herbicide-Tolerant Soybean

DP-356Ø43-5 soybean has been genetically modified to express the GAT4601 (glyphosate 

acetyltransferase) and GM-HRA proteins. The GAT4601 protein, encoded by the gat4601

gene, confers tolerance to glyphosate-containing herbicides by acetylating glyphosate and 

thereby rendering it non-phytotoxic. The GM-HRA protein confers tolerance to the ALSinhibiting class of herbicides and in this case confers tolerance at commercially viable levels. 

D.2. GM-HRA Expression Ranges and Dietary Risk

The ranges of GM-HRA expression in seed from events DP-3Ø5423-1 and DP-356Ø43-5 (Rood et 

al., 2006; Pavely et al., 2007; US-FDA, 2007a; US-FDA, 2009a) appear below in Table 1. 

Table 1: GM-HRA Protein Concentrations in Seed in Events Containing gm-hra

Event

ng/mg Tissue Dry Weight in Seed

Mean (Standard 

Deviation) Range1

DP-3Ø5423-1 2.5 (1.1) 0 – 4.9

DP-356Ø43-5 0.91(0.17) 0.64-1.2 1

Range denotes the lowest and highest individual value across locations.

Because identical elements are used to control the expression of the GM-HRA protein within these 

events, the concentration of the GM-HRA protein in event 82117 seed is expected to fall within a 

range similar to these two other gm-hra-containing events (Table 1). 

A risk characterization for each protein was also submitted to both USDA and FDA in DuPont 

Pioneer’s petitions for these events (Rood et al., 2006; Pavely et al., 2007; US-FDA, 2007a; US-FDA, 
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Study Number: PHI-2012-183

2009a). In these submissions, the risk for these events was characterized by calculating the amount 

of soybean seed an individual must consume in one day to match the dose of GM-HRA protein (fulllength and non-full length) that was safely consumed in the GM-HRA mouse acute oral toxicology 

study, PHI-2006-008 (Volume 4) (582 mg/kg bw). In order to assess the dietary impact of the fulllength GM-HRA protein, using the same approach previously used in DuPont Pioneer’s USDA and 

FDA petitions, updated dietary risk calculations were conducted using the full-length GM-HRA dose 

(436 mg/kg bw), and those values are presented below in Table 2. 

Table 2: Dietary Risk Calculations for Events Containing GM-HRA Protein

Event

Protein 

Concentration

(ng/mg tissue 

dry weight-seed)

Toxicity Test 

Dose (mg/kg bw)

Soybean Consumption Amount 

(kg/day) to Match Toxicity Testing 

Dose

Child (10 kg body 

weight)

Adult (60 kg body 

weight)

DP-3Ø5423-1 2.5 436 1,744 10,464

DP-356Ø43-5 0.91 436 4,791 28,747

The calculated necessary consumption levels in Table 2 are in the thousands and tens of thousands 

of kilograms per day (1,744-28,747 kg/day). There are no plausible circumstances where a child or 

an adult would consume enough raw soybean seed to expose them to the amount of the full-length 

GM-HRA protein safely consumed in the toxicity testing study (436 mg/kg bw). Accordingly, the 

margin of safety estimated by these calculations, using the expression values from these gm-hra

containing events (2.5 and 0.91 ng/mg tissue dry weight, respectively) is clear. Factoring in soybean 

dilution in commodity seed, as well as exposure to heat for food processing (GM-HRA was shown to 

be thermally inactivated at 50  ̊C), the margin of safety for the GM-HRA protein is likely much larger. 

Importantly, in transgenic soybean events (including 82117 soybean) where GM-HRA expression is 

controlled by identical genetic elements, it would be expected that the GM-HRA protein would be 

expressed at concentrations comparable to the ranges of DP-3Ø5423-1 and DP-356Ø43-5 (0-4.9 

ng/mg dry tissue weight). Subsequent dietary exposure calculations using values in this expression 

range would be comparable to those in Table 2. Therefore, the overall dietary risk of GM-HRA protein 

expressing events that are controlled by the gm-hra gene would be similarly low.
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Study Number: PHI-2012-183

Section E. Practical Methods for Removing Residues That Exceed Proposed Tolerance

An exemption from the requirement of a tolerance is being requested; therefore removal methods are 

not necessary.
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Study Number: PHI-2012-183

Section F. Proposed Tolerances for the Pesticidal Chemical if Tolerances are Proposed

An exemption from the requirement of a tolerance is being requested for GM-HRA protein in soybean 

and soybean commodities. 

DuPont Pioneer’s suggested wording for 40 CFR 174.XXX is as follows:

“Residues of Glycine max herbicide resistant acetolactate synthase (GM-HRA) 

are exempt from the requirement of a tolerance, when used as an inert 

ingredient as part of a plant-incorporated protectant, in soybean commodities.”
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Study Number: PHI-2012-183

Section G. Reasonable Grounds in Support of the Petition

In summary, the requested exemption of the GM-HRA protein from the requirement for a tolerance is 

supported by the lack of allergenic or toxic potential of the GM-HRA protein when using a weight of 

evidence approach, enumerated in the points below:

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 enzymes have also been 

part of the human diet by virtue of their presence in soybeans. 

2) Bioinformatic analyses revealed GM-HRA to also 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 SDS-PAGE/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 GMHRA 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; USDA-APHIS, 2010; US-FDA, 2007b; USFDA, 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 and DP356Ø43-5), expression ranges and dietary exposure of GM-HRA from 82117 soybean are 

expected to be similarly low.
Pioneer Hi-Bred International, Inc. Page 27 of 34

Study Number: PHI-2012-183

References

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Baxevanis, A.D. and Ouellette, B.F.F. (2005) Bioinformatics: A practical guide to the analysis of 

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Duggleby, R.G. and Pang, S.S. (2000) Acetohydoxyacid synthase. Journal of Biochemistry and 

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Falco, S.C., Dumas, K.D., and Livak, K.J. (1985) Nucleotide sequence of the yeast ILV2 gene which 

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P.M. (1985) The ilvB locus of Escherichia coli K-12 is an operon encoding both subunits of 

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S.L. (2002). Bioinformatic methods for allergenicity assessment using a comprehensive 

allergen database. Int. Arch. Allergy Immunol. 128: 280-291.

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Hymowitz, T. and J. R. Harlan (1983). Introduction of soybeans to North America by Samuel Bowen 

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Hymowitz, T. and Shurtleff, W.R. (2005). Debunking Soybean Myths and Legends in the Historical 

and Popular Literature. Crop Sci. 45:473-476.

Kane, P.M., Holowka, D., and Baird, B., (1988) Cross-linking of IgE –receptor complexes by rigid 

bivalent antigens >200 A in length triggers cellular degranulation. J. Cell Biol. 107:969-980.

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potential allergenicity of genetically modified organisms and their products. Clin. Exp. Allergy 

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Ladics G.S., Bardina L., Cressman R.F., Mattsson J.L., and Sampson H.A. (2006). Lack of crossreactivity between the Bacillus thuringiensis derived protein Cry1F in maize grain and dust 

mite Der p7 protein with human sera positive for Der p7-IgE. Regul. Toxicol. Pharmacol. 44 

(2): 136-43.

LaRossa, R.A. and Falco, S.C. (1984) Amino acid biosynthetic enzymes as targets of herbicide action. 

Trends in Biotechnology 2: 158-161. 

LaRossa, R.A. and Schloss, J.V. (1984) The sulfonylurea herbicide sulfometuron methyl is an 

extremely potent and selective inhibitor of acetolactate synthase in Salmonella typhimurium. 

Journal of Biological Chemistry. 259(4): 8753-8757.

Lee, K.Y., Townsend, J., Tepperman, J., Black, M., Chiu, C.F., Mazur, B., Dunsmuir, P., and 

Bedbrook, J. (1988) The molecular basis of sulfonylurea herbicide resistance in tobacco. 

EMBO Journal 7(5): 1241-1248.

Mathesius, C.A., Barnett, J.F. Jr, Cressman, R.F., Ding, J., Carpenter, C., Ladics, G.S., Schmidt, J., 

Layton, R.J., Zhang, J.X., Appenzeller, L.M., Carlson, G., Ballou, S., Delaney, B. (2009) 

Safety assessment of a modified acetolactate synthase protein (GM-HRA) used as a 

selectable marker in genetically modified soybeans. Regul. Toxicol. Pharmacol. 55(3): 309-

20.

Mazur, B., Chiu, C.-F., and Smith, J.E. (1987) Isolation and characterization of plant genes coding for 

acetolactate synthase, the target enzyme for two classes of herbicides. Plant Physiology 85: 

1110-1117.

Metcalfe, D.D., Astwood, J.D., Townsend, R., Sampson, H.A., Taylor, S.L., and Fuchs, R.L.

(1996). Assessment of the allergenic potential of foods from genetically engineered crop

plants. Critical Reviews in Food Science and Nutrition, 36(S): S165-186.

Pavely C, Fedorova M, Weber N (2007) Petition for the Determination of Nonregulated Status for High 

Oleic 305423 Soybean. Pioneer Hi-Bred International, Inc., 

http://www.aphis.usda.gov/brs/aphisdocs/06_35401p.pdf

Pearson, W.R. (2000) Flexible sequence similarity searching with the FASTA3 program package. 

Methods Mol Biol 132:185-219.

Pearson, W.R. and Lipman, D.J. (1988). Improved tools for biological comparison. Proc. Natl. Acad. 

Sci. USA. 85, 2440-2488.

Reith, M. and Munholland, J. (1995) Complete nucleotide sequence of the Porphyra purpurea 

chloroplast genome. Plant Molecular Biology Reporter 7: 333-335.

Rood T (2006) Petition for the Determination of Nonregulated Status for Herbicide Tolerant 356043 

Soybean. Pioneer Hi-Bred International, Inc., 

http://www.aphis.usda.gov/brs/aphisdocs/06_27101p.pdf

Silvanovich A., Nemeth M.A., Song P., Heman R., Tagliani L., and Bannon G.A. (2006). The value of 

short amino acid sequence matches for prediction of protein allergenicity. Toxicol. Sci. 90 (1): 

252-8.

Stadler, M.B. and Stadler, B.M. (2003). Allergenicity prediction by protein sequence. FASEB J. 17: 

1141- 1143.
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Tan, S., Evans, R., and Singh, B. (2006) Herbicidal inhibitors of amino acid biosynthesis and 

herbicide-tolerant crops. Amino Acids 30: 195-204.

Thomas, K., Aalbers, M., Bannon, G.A.; Bartels, M.; Dearman, R.J.; Esdaile, D.J.; Fu, T.J.; Glatt, 

C.M.; Hadfield, N.; Hefle, S.L.; Heylings, J.R.; Goodman, R.E.; Henry, B.; Herouet, C., Ladics, 

G.S.; Landry, T.D.; MacIntosh, S.C.; Rice, E.A.; Privalle, L.S.; Steiner, H.Y., Teshima; van 

Ree, R.; Woolhiser, M., Zawodny, J., 2004, A Multi-Laboratory Evaluation of a Common In 

vitro Pepsin Digestion Assay Protocol Used in Assessing the Safety of Novel Proteins. Reg. 

Toxicol. Pharmacol. 39:87-98

USDA-APHIS (2008) Determination of Nonregulated Status for Soybean Genetically Engineered for 

Tolerance to Glyphosate and Acetolactate Synthase-Inhibiting Herbicides. Federal Register 

73: 43203-43205.

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Soybeans. Federal Register 75: 32356-32357.

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States Food and Drug Administration, 

http://www.fda.gov/Food/Biotechnology/Submissions/ucm155567.htm.
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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
Pioneer Hi-Bred International, Inc. Page 31 of 34

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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Study Number: PHI-2012-183

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.