Petition to Amend Tolerance Expression: COMMODITIES

Glyphosate and N - Acetylglyphosate Canola Seed I . E.I. du Pont de Nemours and Company PP No . 1F7840 EPA has received a pesticide petition (PP No. 1F7840 ) from E.I. du Pont de Nemours & Company, 1007 Market Street, Wilmington, DE 19898 , proposing pursuant to section 408 (d) of the Federal Food, Drug, and Cosmetic Act, 21 U.S.C. 346a (d), to amend 40 CFR Part 180.364 (a) by moving canola seed from paragraph (1) to paragraph (2). EPA has determined that the petition contains data or information regarding the elements set forth in section 408 (d) (2) of the FFDCA; however, EPA has not fully evaluated the sufficiency of the submitted data at this time or whether the data supports granting of the petition. Additional data may

be needed before EPA rules on the petition. A. RESIDUE CHEMISTRY 1. Plant Metabo lism DuPont has investigated the behaviour of 14 C - gl yphosate (sesquisodium or mono potassium salts) and 14 C -N

- acetylg ly phosate in tobacco, corn, soy bean , and canola plants containing various versions of the gat gene. Plant metabolism investigations were conducted under laborat ory

(excised tobacco, corn and soy bean seedlings) and greenhouse (corn , soy bean and canola whole plant) conditions. N - A cet yl gl yphosate was the principal metabolite formed in gat plants a fter treatment with 14 C - gl yphosate, and is an appropriate residue to be quantified in O ptimum ® GLY canola . The other major plant residue observed was gl yphosate. 2. Analytical Methods An anal yt ical method was developed, and validated, for the determination of gl yphosate and degradate residues in transgenic crop and crop fraction matrices. The analy tes examined included gl yphosate and N - acetylglyphosate, AMPA ( aminomethylphosphonic acid) , and N - acetyl AMPA. N - acet yl gl yphosate , is the principal metabolite associated with transgenic crops containing the gl yphosate N - acetyltransferase (

gat) enzy me . The method target limit of quantitation (L OQ) in each matrix examined was 0.050 mg/kg (ppm). The method was validated at 0.050 mg/kg and 0.50 mg/kg or higher fortification level using a L C/MS/MS sy stem operating with an electrospray interface (ESI) in positive ion mode detection. The recoveries from samples of various corn and so yb ean matrices fortified at 0.050 mg/kg (LOQ) and 0.50 mg/kg (10×LOQ) or higher fortification level support the satisfactory performance of this method. Additionally , validation analy ses were conducted for plum and lime matrices to support method performance in watery and acid crop matrices, even though these crop types do not contain gat enzy me and would not contain N - acetyl gl yphosate as a gl yphosate herbicide residue. The extraction procedure used in this method is consistent with the method developed for corn RACs in a pilot plant metabolism study using radiolabeled gl yphosate and extracting incurred plant residues. In addition, consistentPetition to Amend Tolerance Expression: COMMODITIES

Glyphosate and N - Acetylglyphosate Canola Seed residue results for gly

phosate residues from field trial samples of corn forage, grain, and stover were determined using this method and a current gl yphosate enforcement method. An anal yt ical method was developed, and validated, for the determination of gl yphosate, Nacet yl gl yphosate, AMPA, and N - acetyl AMPA in animal matrices including milk, eggs, muscle, kidney , liver, and fat. The method target limit of quantitation (L OQ) in gl yphosate equivalents for each analy te was 0.025 mg/kg in egg, milk, and muscle matri ces and 0.050 mg/kg in kidney , liver, and fat matrices. The method was validated at the respective LOQ and 10×LOQ level for each matrix using a L C/MS/MS system operating with an electrospray

interface (ESI) in positive or negative ion mode detection. The r ecoveries from matrix samples fortified at the respective LOQ and higher levels support the satisfactory performance of this method. 3. Magnitude of Residue in Canola DuPont has conducted a full set of at - harvest residue studies to determine residues o f gl yphosate , N

- acetylglyphosate, AMPA, and N - acetyl AMPA i n O ptimum ® GLY canola that has been treated with an herbicide containing the active ingredient gl yphosate. These trials demonstrate that, when applied to O ptimum ® GLY canola according to a registered treatment regime, combined residues of gl yphosate plus N - acetyl gl yphosate , the principal plant metabolite, do not exceed the relevant tolerances established for gly phosate per se . B. TOXICOLOGICAL PROFILE 1. Acute Toxicity Data from an acute oral toxicity study conducted in laboratory

animals demonstrates that Nacet yl gl yphosate is of low toxicity to mammals following acute exposure. Five male and five female rats received a limit dose of 5,000 mg/kg by gavage. One female died on the day of dosing, and one male and one female were found dead the day after dosing. All clinical signs of toxicity

were resolved in the remaining seven rats by day three. All surviving animals gained weight from initiation of dosing to study ter mination. The N

acet yl gl yphosate oral LD 50 for both male and female rats is greater than 5,000 mg/kg body weight. 2. Genotoxicity The CHO/HGPRT test was used to evaluate the test substance, N - acetylg ly phosate , for its mutagenic potential in the absence and presence of an exogenous metabolic activation sy stem (Aroclor - 1254 induced rat liver S9) in Chinese hamster ovary (CHO) cells. To establish a range of doses for the mutagenesis assay , a preliminary cy totoxic ity assay was conducted. Based on the findings in the preliminary study , the concentrations chosen for the mutagenesis assay

were 250, 500, 1000, 1500, and 2091 μg/mLfor both test conditions ( - S9 and +S9). No positive responses, i.e., mutant frequencies > 40 mutants per 10 6 clonable cells, were observed in the mutagenesis assay . No substantial toxicity , i.e., cloningPetition to Amend Tolerance Expression: COMMODITIES

Glyphosate and N - Acetylglyphosate Canola Seed efficiency <50% of the vehicle control, was observed at any dose level. No visible precipitate was observed in the treatment medium at any con centration. Under the conditions of this study N - acet yl gl yphosate was not found to induce forward mutations at the HGPRT gene locus of CHO cells in either the nonactivated or S9 - activated test conditions. The test substance was concluded to be negative in this in vitro test. N - Acet yl gl yphosate did not cause structural chromosomal aberrations in Chinese hamster ovary (CHO) cells in an in vitro chromosome aberration assay either with or without exogenous metabolic activation. Replicate cultures of CHO cells were treated with N acet yl gl yphosate for 3 hours at doses ranging from 19.0 to 2,800 μg/mLwith or without metabolic activation, and harvested approximately 20 hours after treatment initiation. An additional set of cultures was treated for 20 hours witho ut metabolic activation. No significant increase in cells with chromosomal aberrations, poly ploidy or endoreduplication was observed in treated cultures. The test substance, N

- acetylg ly phosate, was evaluated for its ability to induce micronuclei in bone marrow pol ychromatic ery throcy tes (PCEs) in male and female Crl:CD1(I CR) mice. Based on range finding results, doses of 0, 500, 1000, or 2000 mg/kg of the test substance were selected for the main study . No clinical signs of toxicity or mortality

were obse rved in the rangefinder experiment. All animals were given a single dose by oral intubation. In the main study , there were no significant changes in body weight or body weight gain in either male or female animals administered the test substance, or in the vehicle or positive control groups. No clinical signs of toxicity were observed at any

dose level in test substance - treated male and female mice exposed to the test substance. No abnormalities were detected in the vehicle or positive control groups. No mo rtality occurred during the study. No statistically significant increases in micronucleated PCE frequency were observed in an y evaluated test substance - treated group of male or female animals at either timepoint. There were no statisticall y significant dec reases in PCEs among 1000 erythrocy tes. The positive control groups exhibited a response consistent with the micronucleated PCE historical control data. Under the conditions of this study , Nacet yl gl yphosate did not induce statistically significant or biol ogically relevant increases in micronucleated pol yc hromatic ery throcy tes in animal bone marrow. The test substance was concluded to be negative in this in vivo study . N - Acet yl gl yphosate was negative in the bacterial reverse mutation (Ames) assay with and without metabolic activation. N - Acet yl gl yphosate was negative for reverse mutations in Salmonella typhimurium tester strainsTA98, TA100, TA1535, and TA1537 at the histidine locus in the presence or absence of mammalian microsomal metabolic activation sy st em. N - Acet yl gl yphosate was negative for reverse mutations in E. coli tester strain WP2 uvr A at the try ptophan locus in the presence or absence of mammalian microsomal metabolic activation sy stem. 3. Subchronic ToxicityPetition to Amend Tolerance Expression: COMMODITIES

Glyphosate and N - Acetylglyphosate Canola Seed A 90 - day feeding study

was conducted to assess the potential subchronic toxicity of Nacet yl gl yphosate in rats. Five groups of young adult male and female Crl:CD(SD) rats (10/sex/group) were administered diets that contained 0, 180, 900, 4500, or 18,000 pp m Nacet yl gl yphosate for approximately 90 day s (95 day s in males and 96 day s in females). Samples of the diets were anal yz ed to assess homogeneity , stabilit y, and concentration of the test substance in the diet. Body weights, food consumption, and detailed clinical observations were evaluated weekl y. Ophthalmological and neurobehavioral (abbreviated functional observational battery , grip strength, motor activity) evaluations were performed on all rats prior to the start of dietary exposure and near the end of the exposure period. Clinical pathology

endpoints (hematology , coagulation, clinical chemistry , urinaly sis) were evaluated at the end of the exposure period. After approximately 90 day s of dietary exposure, the surviving rats were sacrificed and given a gross and microscopic pathological examination. No adverse test substance - related effects on any body weight or nutritional parameters were observed. Statistically

significantly

lower overall mean body weight gain (86% of control) was observed in 18,000 p pm males but was not considered adverse as it was not associated with a statistically

significant difference in mean final body weight, or in overall mean food consumption or food efficiency . No test substance - related deaths occurred, and no clinical, ophthalmological, or neurobehavioral observations were attributed to exposure to the test substance. There were no adverse effects on clinical pathology parameters, organ weights, gross pathology , or microscopic pathology

in male or female rats. The no - observe d - adverse - effect level (NOAEL) for male and female rats was 18,000 ppm, equivalent to 1157 and 1461 mg/kg/day in males and females, respectivel y. The NOAEL is based on a lack of adverse, test substance - related effects in either sex at 18,000 ppm, the highe st concentration tested. 4. Animal Metabolism The absorption, metabolism, and elimination of N - acetyl gl yphosate were evaluated in male rats following a single oral administration of [ 14 C] N - acetyl gl yphosate. N

- Acet yl gl yphosate is incompletely

absorbed from the gastrointestinal tract and rapidly eliminated in the urine and feces. Greater than 90% of the administered dose was eliminated b y 48 hours after dosing. Approximately

66% of a dose of [ 14 C] N - acetyl gl yphosate was absorbed following ingestion, and e liminated entirely unchanged in the urine. Approximately 26% of the [ 14 C] N - acetylg ly phosate administered was eliminated in the feces, almost entirely as the parent compound. More than 99% of the radioactivity in the feces was in the form of Nacet yl gl yph osate; gl yphosate was the onl y metabolite present in the feces, comprising less than 1% of the radiolabeled dose administered. The maximum N - acetylg ly phosate concentrations in blood and plasma were reached at 1 and 2 hours post - dose, respectivel y. Radioa ctivity in blood and plasma consisted entirely of unchanged [ 14 C] N

acet yl gl yphosate; no metabolites were present.Petition to Amend Tolerance Expression: COMMODITIES

Glyphosate and N - Acetylglyphosate Canola Seed N - Acet yl gl yphosate ingested from glyphosate - tolerant crops is of low toxicity and will likely be rapidly

eliminated from the body unchanged in the urine and feces. The portion of N - acet yl gl yphosate that is absorbed is eliminated in the urine with a half - li fe of less than one day , the remainder is eliminated in the feces largel y unchanged. The absorption, metabolism and elimination of N - acetyl gl yphosate are similar to those reported in the literature for gly

phosate. Both compounds are incompletely absorbed and rapidly eliminated in the urine and feces. C. AGGREGATE EXPOSURE, CUMULATIVE EFFECTS, AND SAFETY DETERMINATION The residue trials previously described demonstrate that following the application of gl yphosate to O ptimum ® GLY canola , combined residue s of gl yphosate plus Nacet yl gl yphosate (expressed as glyphosate per se ) do not exceed tolerances currentl y established at 40 CFR 180.364 for canola seed . Therefore, no additional risk assessments have been conducted since N - acetyl gl yphosate has been shown to be toxicologicall y equivalent to gl yphosate, and no change in the magnitude of the relevant tolerances is proposed. D. INTERNATIONAL TOLERANCES Numerous countries have established tolerances for gl yphosate on canola . No tolerances have been establish ed to date for N - acetyl gl yphosate in canola .