Pioneer Hi - Bred International, Inc. Page 1 of 34 Study Number: PHI - 2012 - 183



### T ITLE

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



### D ATA 

### R EQUIREMENT None



### A UTHOR Nicole C. Jansen Pioneer Hi - Bred Intern ational, Inc. 7100 NW 62 nd Avenue

Johnston, IA 50131 USA



### S TUDY 

### C OMPLETION 

### D ATE June 25 , 2012 

### S TUDY 

### N UMBER

PHI - 2012 - 183



### EPA R EGISTRATION 

### N UMBER 29964 - xPioneer Hi - Bred International, Inc. Page 3 of 34 Study Number: PHI - 2012 - 183



### T ABLE OF 

### C ONTENTS 

### P AGE 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. Am ount, 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 Resid ues 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 Summ ary ................................ ................................ ......... 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

................................ . 24Pioneer Hi - Bred International, Inc. Page 4 of 34 Study Number: PHI - 2012 - 183



### T RANSMITTAL 

### D OCUMENT



### S UBMITTER Pione er Hi - Bred International, Inc. 7100 NW 62 nd Ave. Johnston, IA 50131



### R EGULATORY 

### A CTION IN 

### S UPPORT OF 

### W HICH 

### T HIS 

### D OCUMENT IS 

### S UBMITTED 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



### T RANSMITTAL 

### D ATE June 25 , 2012



### S TUDY 

### N UMBER

PHI - 2012 - 183Pioneer Hi - Bred International, Inc. Page 5 of 34 Study Number: PHI - 2012 - 183 

### L IST OF 

### A CCOMPANYING 

### V OLUMES

V OL . FIFRA 10(G) V OLUME /S TUDY 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 Jun e 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). S tudy 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 , 201 2 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 GM HRA Protein and Known Protein Al lergens 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 copiesPioneer Hi - Bred International, Inc. Page 7 of 34 Study Number: PHI - 2012 - 183 

### S ECTION 

### I. A DMINISTRATIVE 

### M ATERIALS A. 1 . EPA Form 8570 - 1 : Application for Pesticide RegistrationPioneer Hi - Bred International, Inc. Page 9 of 34 Study Num ber: PHI - 2012 - 183 A.2 . EPA Form 8570 - 35 : Data Matrix for GM - HRA Tolerance ExemptionPioneer Hi - Bred International, Inc. Page 14 of 34 Study Num ber: PHI - 2012 - 183 

### S ECTION 

### II. GM 

### - HRA TOLERANCE EXEMPT ION PETITION 

### Section A. Name, Chemical Identi ty, 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 class es 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 - in hibiting herbicides typically not tolerated by soybean. A s 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 c hemical 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 G lycine m ax and is a n h erbicide - r esistant a llele. The herbicide - tolerant gm - hra gene was made by isolating the herbicide - sensitive soybean gm - als I gene and introducing chang es 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 fo und 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 transforma tion and potentially a herbicide - tolerant trait in soybean s . To obtain the protein amounts sufficient to conduct toxicological and biochemical studies, the GM HRA 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 us e 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 w eight and immunoreactivity of the proteins; 3) Glycoprotein staining of the proteins to determine the post - translational modification (glycosylation);Pioneer Hi - Bred International, Inc. Page 15 of 34 Study Num ber: PHI - 2012 - 183 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 w as verified , and a comparison of the physiochemical properties confirmed that the two proteins were equivalent (Volume 1, PHI - 2006 - 016; Volume 2, PHI 2011 - 225)

. Based on this confirmation of identity and equivalence, the mic robial - 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.Pioneer Hi - Bred International, Inc. Page 16 of 34 Study Num ber: PHI - 2012 - 183 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.Pioneer Hi - Bred International, Inc. Page 17 of 34 Study Num ber: PHI - 2012 - 183 Section C. Safety of the Pesticide Chemical A weight of evidence approach was applied to evalua te 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 assess ment 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 toxic ity 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 ( V olume 5 , PHI - 2006 - 070/070 ; Volume 6 , PHI - 2006 - 071 / 070 ) ; 4 ) evaluati on 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 ) deter mination 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

. I t’s wide ly accepted that domesticated soybean originated in China, however there’s some controversy as to when this first occurred . Early account s were that t he first recording of domesticated soybeans was in a series of books known as Pen Ts'ao Kong Mu written b y 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 year s, 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 soy beans 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 dis cussed more fully in Volume 1 (PHI - 2012 - 020), t he 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 ami no acid changes in the protein ; accordingly, G M - 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 distribut ed 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 yiel ding 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 e ffects.Pioneer Hi - Bred International, Inc. Page 18 of 34 Study Num ber: PHI - 2012 - 183 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 ident ified in ALS proteins derived from multiple ALS tolerant plant species , supported by the over 1 2 ,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, t here are five commonly occurring mutations in ALS proteins that confer tolerance to ALS - inhibiting herbicides; mutations similar or identical to the two i n GM - HRA (P183A and W560L) are present in several commercially available, non - transgenic, herbicide - tolerant crop varieties with a history of safe consumptio n (see list of commercially available crops in Volume 1 , PHI 2012 - 020). **C.2. Acute Toxicity in Mic**  **e** (Volume 4 , PHI - 2006 - 0 0 8 ) 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 a lbumin (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 wer e recorded. No clinical signs of toxicity, body weight losses, gross lesions, or mortality were observed in male and f emale 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 allergenici ty 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 sequ ence 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 alle rgenic 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 Nebra ska, 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 ami no acid sequence and those of proteins in the allergen database were evaluated using the FASTA35 sequence alignment program (Pearson and Lipman,Pioneer Hi - Bred International, Inc. Page 19 of 34 Study Num ber: 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 Assessmen t (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 tra nslation 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 seque nces (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 ac etolactate 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 identifie d 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 charac terized 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.Pioneer Hi - Bred International, Inc. Page 20 of 34 Study Num ber: 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 parame ters 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, 1 0, 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 r apidly and persisted in pepsin, verifying that the assay performe d 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 durin g 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 pe ptide 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 SDS PAGE 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 stainin g 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. T hermal Lability ( Volume 10 , PHI - 2006 - 135) The objective of this study w as 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 a ssay 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.Pioneer Hi - Bred International, Inc. Page 21 of 34 Study Num ber: 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 pro tein, 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 oth er transgenic soybean events, DP - 3Ø5423 - 1 high oleic s oybean and DP - 356Ø43 - 5 glyphosate - t oleran t 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 FD A 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 co mmonly 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

. , 200 7). 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 differe nt 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 deregul ated 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 ac etolactate 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 pr otein, 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 proper ties.Pioneer Hi - Bred International, Inc. Page 22 of 34 Study Num ber: PHI - 2012 - 183 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; U SDA - 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 GM HRA protein in all events is expect ed 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 e vents 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 (linole ic and linolenic) and to a lesser extent, palmitic acid. In DP 3Ø5423 - 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 tolera nce to the ALS inhibiting 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) Range 1 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 e t al., 2006; Pavely et al., 2007; US - FDA, 2007a; US - FDA,Pioneer Hi - Bred International, Inc. Page 23 of 34 Study Num ber: PHI - 2012 - 183 2009a). In these submissions, the r isk 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 (full le ngth and non - full length) that was safely consumed in the GM - HRA mouse acute oral toxicology study , PHI - 2006 - 0 08 (Volume 4) (582 mg/kg bw)

. In order to assess the dietary impact of the full length 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 - HR A 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 consum e enough raw soybean seed to expose them to the amount of the full - length GM - HRA protein safely consumed in the toxic ity testing study ( 436 mg/kg bw) . Accordingly, the margin of safety estimated by these calculations, using the expression values from thes e 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 expres sed at concentrations comparable to the ranges of DP - 3Ø5423 - 1 and DP - 356Ø43 - 5 (0 - 4.9 ng/mg dry tissu e weight). Subsequent dietary exposure calculations using values in this expression range would be comparable to those in Table 2

. Therefore , t he overall dietary risk of GM - HRA protein expressing events that are controlled by the gm - hra gene would be similarly low.Pioneer Hi - Bred International, Inc. Page 24 of 34 Study Num ber: PHI - 2012 - 183 Section E. Practical Methods for Removing Residues That Exceed Proposed Tolerance An exemption from the requirement of a tolerance is being reques ted; therefore removal methods are not necessary.Pioneer Hi - Bred International, Inc. Page 25 of 34 Study Num ber: 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 soyb ean 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.”Pioneer Hi - Bred International, Inc. Page 26 of 34 Study Num ber: 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 allerge nic 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 differen t 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 pe r 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 protei n 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 am ino 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 th an 1 minute as demonstrated by W estern 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 pla nt - 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 bo th 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; US FDA, 2009b) . Additionally, a safety conclusion regar ding 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 DP 3 56Ø43 - 5), expression ranges and dietary exposure of GM - HRA from 8211 7 soybean are expected to be similarly low.Pioneer Hi - Bred International, Inc. Page 27 of 34 Study Num ber: PHI - 2012 - 183 References Bannon, G.A., Goodman, R.E., Leach, J.N., Rice, E., Fuchs, R.L., and Astwood, J.D. 2002, Digestive stability in the context of assess ing the potential allergenicity of food proteins. Comments on Toxicol., 8:271 - 285. Baxevanis, A.D. and Ouellette, B.F.F. (2005) Bioinformatics: A practical guide to the analysis of genes and proteins. 3rd Edition, John Wiley & Sons, Inc., New York. Creaso n, G.L. and Chaleff, R.S. (1988) A second mutation enhances resistance of a tobacco mutant to sulfonylurea herbicides. Theoretical and Applied Genetics 76: 177 - 182. Coruzzi, G. and Last, R. (2000) Amino acids. In: Buchanan, B., Gruissem, W., and Jones, R., eds, Biochemistry and Molecular Biology of Plants, Chapter 8, American Society of Plant Physiologists, Rockville, MD, pp 358 - 410. Duggleby, R.G. and Pang, S.S. (2000) Acetohydoxyacid synthase. Journal of Biochemistry and Molecular Biology 33: 1 - 36. Falco, S.C., Dumas, K.D., and Livak, K.J. (1985) Nucleotide sequence of the yeast ILV2 gene which encodes acetolactate synthase. Nucleic Acids Research 13: 4011 - 4027. Friden, P., Donegan, J., Mullen, J., Tsui, P., Freundlich, M., Eoyang, L., Weber, R., and Silve rman, P.M. (1985) The ilvB locus of Escherichia coli K - 12 is an operon encoding both subunits of acetohydroxyacid synthase I. Nucleic Acids Research 13: 3979 - 3993. Food and Agriculture Organization of the United Nations (FAO/WHO) (2001). Report of a Joint FAO/WHO Expert Consultation on Allergenicity of Foods Derived from Biotechnology 22

– 25 January 2001. http://www.who.int/foodsafety/publications/biotech/en/ec_jan2001.pdf. Hansen R, Huntrods, D. (2011) Soyfood Profile. Agricultural Marketing Resource Ce nter, 

### http://www.agmrc.org/commodities__products/grains__oilseeds/soy/soyfood_profile.cfm Hileman, R.E., Silvanovich, A., Goodman, R.E., Rice, E.A., Hollesc hak, G., Astwood, J.D., and Hefle, S.L. (2002). Bioinformatic methods for allergenicity assessment using a comprehensive allergen database. Int. Arch. Allergy Immunol. 128: 280 - 291. Hymowitz, T. (2008) The History of the Soybean In Johnson, L.A., White, P. J., Galloway, R., Soybeans - Chemistry, Production Processing, and Utilization, Volume 2 (pp. 1 - 31). AOCS Press. Hymowitz, T. and J. R. Harlan (1983). Introduction of soybeans to North America by Samuel Bowen in 1765. Econ. Bot. 37: 371 - 379. 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. Lack, G., Chapman, M., Kalshekert, N., King, V., Robinson, C., and Venables, K. (2002) Report on the potential allergenicity of genetically modified organisms and their products. Clin. Exp. Allergy 32:1131 - 1143.Pioneer Hi - Bred International, Inc. Page 28 of 34 Study Num ber: PHI - 2012 - 183 Ladics G.S., Bardina L., Cressman R.F., Mattsson J.L., and Sampson H.A. (2006). Lack of cross reactivity between the Bacillus thuringiensis derived protein Cry1F in maize grain and dust mite Der p7 protein with human sera positive fo r 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 herbi cide 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., Dunsmu ir, 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 Determina tion 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 sequen ce 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) Complet e 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.Pioneer Hi - Bred International, Inc. Page 29 of 34 Study Num ber: PHI - 2012 - 183 Tan, S., Evans, R., and Singh, B. (2006) Herbicidal inhibitors of amino ac id 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 Safe ty 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

. USDA - APHIS (2010) Determination of Nonregulated Status for Genetically Engineered High - oleic Soybeans. Federal Register 75: 32356 - 32357. USDA - ERS (2010) Soybeans and Oil Crops: Background. United States Department of Agriculture, Economic Research Servic e, http://www.ers.usda.gov/Briefing/SoybeansOilcrops/background.htm

. US - FDA (2007a) Biotechnology Consultation Note to the File BNF No. 000108. United States Food and Drug Ad ministration, http://www.fda.gov/Food/Biotechnology/Submissions/ucm155604.htm

. US - FDA (2007b) Biotechnology Consultation Agency Response Letter BNF No. 000108. United States F ood and Drug Administration, http://www.fda.gov/Food/Biotechnology/Submissions/ucm155575.htm

. US - FDA (2009a) Biotechnology Consultation Note to the File BNF No. 000110. United States Food and Drug Administration, http://www.fda.gov/Food/Biotechnology/Submissions/ucm155595.htm

. US - FDA (2009b) Biotechnology Consultation Agency Response Letter BNF No. 0 00110. United States Food and Drug Administration, http://www.fda.gov/Food/Biotechnology/Submissions/ucm155567.htm

.Pioneer Hi - Bred International, Inc. Page 30 of 34 Study Num ber: PHI - 2012 - 183 

### Appendix I i EPA B IOPESTICIDES 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 62 nd 

### 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 (Option s (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. m icrobial 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 argumen ts 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 thePioneer Hi - Bred International, Inc. Page 31 of 34 Study Num ber: 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 Pra ctices

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 event s. 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 a nd corresponding residues. 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

- a ls 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 add itionally 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 sele ctable 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 GM

HRA 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 measurin g 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). ALSPioneer Hi - Bred International, Inc. Page 32 of 34 Study Num ber: 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; R eith 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 identi ty 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 seq uence (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 G M

- 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 (Volum e 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). Neith er agency identified safety concerns with the protein (USDA

- APHIS, 2008; USDA

APHIS, 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

- 1Pioneer Hi - Bred International, Inc. Page 33 of 34 Study Num ber: 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 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 expr ession 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 demonstrate d. 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 glyc osylated. GM

- HRA was also highly digestible in vitro , as well as thermally inactivated at 50  ̊C. Further, t he dietary risk for other transgenic soybean events expressing GM

- HRA ( DP

- 3Ø5423

- 1 and DP

- 356Ø43

- 5) using genetic elements identical to those conta ined 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 c ause 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 d erived 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 notPioneer Hi - Bred International, Inc. Page 34 of 34 Study Num ber: PHI - 2012 - 183 

### glycosylated. GM

- HRA was also highly digestible in vitro , as well as thermally inactivated at 50  ̊C. Further, t he 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. 821 17 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 ag e 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 fo r 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 cert ain 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 r equirement 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 profil es 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 revi ew 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.