Document ID: EPA-HQ-OPP-2011-0307-0002
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2011-04-20T04:00Z

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 Metabolism
    
    DuPont has investigated the behaviour of 14C-glyphosate (sesquisodium or mono potassium salts) and 14C-N-acetylglyphosate in tobacco, corn, soybean, and canola plants containing various versions of the gat gene.  Plant metabolism investigations were conducted under laboratory (excised tobacco, corn and soybean seedlings) and greenhouse (corn, soybean and canola whole plant) conditions.  
    N-Acetylglyphosate was the principal metabolite formed in gat plants after treatment with [14]C-glyphosate, and is an appropriate residue to be quantified in Optimum[(R)] GLY canola. The other major plant residue observed was glyphosate.   
    
    2. Analytical Methods
    
    An analytical method was developed, and validated, for the determination of glyphosate and degradate residues in transgenic crop and crop fraction matrices.  The analytes examined included glyphosate and N-acetylglyphosate, AMPA (aminomethylphosphonic acid), and N-acetyl AMPA. N-acetylglyphosate, is the principal metabolite associated with transgenic crops containing the glyphosate N-acetyltransferase (gat) enzyme.  The method target limit of quantitation (LOQ) 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 LC/MS/MS system operating with an electrospray interface (ESI) in positive ion mode detection.  The recoveries from samples of various corn and soybean matrices fortified at 0.050 mg/kg (LOQ) and 0.50 mg/kg (10xLOQ) or higher fortification level support the satisfactory performance of this method.  Additionally, validation analyses 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 enzyme and would not contain N-acetylglyphosate as a glyphosate 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 glyphosate and extracting incurred plant residues.  In addition, consistent residue results for glyphosate residues from field trial samples of corn forage, grain, and stover were determined using this method and a current glyphosate enforcement method.
    
    An analytical method was developed, and validated, for the determination of glyphosate, N-acetylglyphosate, AMPA, and N-acetyl AMPA in animal matrices including milk, eggs, muscle, kidney, liver, and fat. The method target limit of quantitation (LOQ) in glyphosate equivalents for each analyte was 0.025 mg/kg in egg, milk, and muscle matrices and 0.050 mg/kg in kidney, liver, and fat matrices. The method was validated at the respective LOQ and 10xLOQ level for each matrix using a LC/MS/MS system operating with an electrospray interface (ESI) in positive or negative ion mode detection. The recoveries 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 of glyphosate, N-acetylglyphosate, AMPA, and N-acetyl AMPA in Optimum[(R)] GLY canola that has been treated with an herbicide containing the active ingredient glyphosate. These trials demonstrate that, when applied to Optimum[(R)] GLY canola according to a registered treatment regime, combined residues of glyphosate plus N-acetylglyphosate, the principal plant metabolite, do not exceed the relevant tolerances established for glyphosate per se. 
    
    B. TOXICOLOGICAL PROFILE
    
    1. Acute Toxicity
    
    Data from an acute oral toxicity study conducted in laboratory animals demonstrates that N-acetylglyphosate 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 termination.  The N-acetylglyphosate oral LD50 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-acetylglyphosate, for its mutagenic potential in the absence and presence of an exogenous metabolic activation system (Aroclor-1254 induced rat liver S9) in Chinese hamster ovary (CHO) cells. To establish a range of doses for the mutagenesis assay, a preliminary cytotoxicity 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/mL for both test conditions (-S9 and +S9). No positive responses, i.e., mutant frequencies >40 mutants per 106 clonable cells, were observed in the mutagenesis assay. No substantial toxicity, i.e., cloning efficiency <50% of the vehicle control, was observed at any dose level. No visible precipitate was observed in the treatment medium at any concentration. Under the conditions of this study N-acetylglyphosate 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-Acetylglyphosate 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-acetylglyphosate for 3 hours at doses ranging from 19.0 to 2,800 μg/mL with or without metabolic activation, and harvested approximately 20 hours after treatment initiation.  An additional set of cultures was treated for 20 hours without metabolic activation.  No significant increase in cells with chromosomal aberrations, polyploidy or endoreduplication was observed in treated cultures.
    
    The test substance, N-acetylglyphosate, was evaluated for its ability to induce micronuclei in bone marrow polychromatic erythrocytes (PCEs) in male and female Crl:CD1(ICR) 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 observed 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 mortality occurred during the study. No statistically significant increases in micronucleated PCE frequency were observed in any evaluated test substance-treated group of male or female animals at either timepoint. There were no statistically significant decreases in PCEs among 1000 erythrocytes. The positive control groups exhibited a response consistent with the micronucleated PCE historical control data. Under the conditions of this study, N-acetylglyphosate did not induce statistically significant or biologically relevant increases in micronucleated polychromatic erythrocytes in animal bone marrow. The test substance was concluded to be negative in this in vivo study.
     
     N-Acetylglyphosate was negative in the bacterial reverse mutation (Ames) assay with and without metabolic activation.  N-Acetylglyphosate 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 system.   N-Acetylglyphosate was negative for reverse mutations in E. coli tester strain WP2uvrA at the tryptophan locus in the presence or absence of mammalian microsomal metabolic activation system.
    
    3. Subchronic Toxicity
    
    A 90-day feeding study was conducted to assess the potential subchronic toxicity of N-acetylglyphosate 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 ppm N-acetylglyphosate for approximately 90 days (95 days in males and 96 days in females). Samples of the diets were analyzed to assess homogeneity, stability, and concentration of the test substance in the diet. Body weights, food consumption, and detailed clinical observations were evaluated weekly. 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, urinalysis) were evaluated at the end of the exposure period. After approximately 90 days 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 ppm 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-observed-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, respectively. The NOAEL is based on a lack of adverse, test substance-related effects in either sex at 18,000 ppm, the highest concentration tested.
    
    4. Animal Metabolism
    
     The absorption, metabolism, and elimination of N-acetylglyphosate were evaluated in male rats following a single oral administration of [[14]C]N-acetylglyphosate.  N-Acetylglyphosate is incompletely absorbed from the gastrointestinal tract and rapidly eliminated in the urine and feces.  Greater than 90% of the administered dose was eliminated by 48 hours after dosing.  Approximately 66% of a dose of [[14]C]N-acetylglyphosate was absorbed following ingestion, and eliminated entirely unchanged in the urine.  Approximately 26% of the [[14]C]N-acetylglyphosate 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 N-acetylglyphosate; glyphosate was the only metabolite present in the feces, comprising less than 1% of the radiolabeled dose administered.  The maximum N-acetylglyphosate concentrations in blood and plasma were reached at 1 and 2 hours post-dose, respectively.  Radioactivity in blood and plasma consisted entirely of unchanged [[14]C] N-acetylglyphosate; no metabolites were present. 
     
     N-Acetylglyphosate 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-acetylglyphosate that is absorbed is eliminated in the urine with a half-life of less than one day, the remainder is eliminated in the feces largely unchanged.  The absorption, metabolism and elimination of N-acetylglyphosate are similar to those reported in the literature for glyphosate.  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 glyphosate to Optimum[(R)] GLY canola, combined residues of glyphosate plus N-acetylglyphosate (expressed as glyphosate per se) do not exceed tolerances currently established at 40 CFR 180.364 for canola seed. Therefore, no additional risk assessments have been conducted since N-acetylglyphosate has been shown to be toxicologically equivalent to glyphosate, and no change in the magnitude of the relevant tolerances is proposed.

    D. INTERNATIONAL TOLERANCES
    
    Numerous countries have established tolerances for glyphosate on canola. No tolerances have been established to date for N-acetylglyphosate in canola.