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 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 

14C-glyphosate, and is an appropriate residue to be quantified in Optimum® 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 (10×LOQ) 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 
 Petition to Amend Tolerance Expression: COMMODITIES

Glyphosate and N-Acetylglyphosate Canola Seed

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, Nacetylglyphosate, 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 10×LOQ 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® GLY canola that 

has been treated with an herbicide containing the active ingredient glyphosate. These trials 

demonstrate that, when applied to Optimum® 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 Nacetylglyphosate 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 Nacetylglyphosate 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 
 Petition 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 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 Nacetylglyphosate 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, Nacetylglyphosate 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
 Petition 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 Nacetylglyphosate 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 Nacetylglyphosate 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 [14C]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 [14C]N-acetylglyphosate was absorbed following 

ingestion, and eliminated entirely unchanged in the urine. Approximately 26% of the 

[

14C]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 Nacetylglyphosate; 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 [14C] Nacetylglyphosate; no metabolites were present. 
 Petition to Amend Tolerance Expression: COMMODITIES

Glyphosate and N-Acetylglyphosate Canola Seed

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® GLY canola, combined residues of glyphosate plus Nacetylglyphosate (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.