Chlorimuron ethyl

Neurotoxicity

Administrative data

Endpoint:

neurotoxicity: acute oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 424 (Neurotoxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

ethyl 2-({[(4-chloro-6-methoxypyrimidin-2-yl)carbamoyl]amino}sulfonyl)benzoate

EC Number:

618-690-2

Cas Number:

90982-32-4

Molecular formula:

C15H15ClN4O6S

IUPAC Name:

ethyl 2-({[(4-chloro-6-methoxypyrimidin-2-yl)carbamoyl]amino}sulfonyl)benzoate

Test material form:

solid

Remarks:

White

Details on test material:

95 to >99% purity

Specific details on test material used for the study:

97.5% purity

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals were housed in pairs in solid bottom caging with bedding mixed with enrichment (Shepherd's™ Cob + PLUS™). Each cage rack contained only animals of one sex.

Animal rooms were maintained at a temperature of 18-26ºC (64-79ºF) and a relative humidity of 30-70%. Animal rooms were artificially illuminated (fluorescent light) on an approximate 12-hour light/dark cycle. The temperature and relative humidity values in the housing rooms were monitored continuously and recorded automatically at regular intervals each day. The temperature and relative humidity records were reviewed by the laboratory veterinarian and/or designee; a copy of the temperature and relative humidity records was retained with the study records. In addition, temperature and relative humidity were recorded during mortality and moribundity checks, and retained with the study records. There were no deviations from the ranges specified above during the study.

Tap water was provided ad libitum to all rats. PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002 was available ad libitum.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Methylcellulose

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sample Preparation: An aliquot of 0.5 ml of each sample was diluted with MeOH to 100 mL. The diluted 0 mg/mL control sample was analyzed by HPLC directly while other concentration samples were further diluted with the diluted control sample before HPLC analysis. The final nominal concentrations for the diluted samples were 0.0125 or 0.025 mg/mL for analysis.

Chromatographic Conditions

Concentrations of the test substance in diluted dose samples were determined by HPLC with UV detection.

The HPLC details are as follows:

Instrument: Agilent 1100 liquid chromatograph
Column: Agilent Zorbax® SB-C 18, 5 µrn, 150 x 2.1 mm
Flow Rate: 0.400 mL/min.
Stoptime: 5.00 min.
Mobile phase: 55% Acetonitrile/45% 3.1 mM phosphoric acid in water
Detection: UV absorbance at 230 nm
Injection Volume: 5.00 µL
Column Temperature: 30.0°C

Calibration and Quantitation: The analytical standard of the test compound (98.8% purity) was used to make a stock solution in MeOH. The stock solution was further diluted with the diluted control sample to make a set of standard solutions that covered the targeted concentrations of the diluted samples. Peak areas from the HPLC analysis of these standard solutions were used to construct a calibration curve by Agilent's ChemStation software. Measured concentrations for the samplles were determined by applying the peak areas from replicate injections of each sample to the calibration curve.

Homogeneity of the test substance in the samples was evaluated by calculating the relative standard deviation (RSD = standard deviation/average x 100%) of the measured concentrations of the top, middle, and bottom samples for each concentration level.

Concentration verification of the test substance in the samples was evaluated by the average results of the top, middle, and bottom sample analyses for each respective dose level.

Stability of the test substance in the samples was evaluated by using the average results of the top, middle, and bottom sample analyses for the respective dose levels as the baseline for comparing the corresponding stability results.

Duration of treatment / exposure:

Single oral gavage dose

Frequency of treatment:

Single oral gavage dose

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw (total dose)

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw (total dose)

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw (total dose)

No. of animals per sex per dose:

12 males and 12 females

Control animals:

yes, concurrent vehicle

Details on study design:

Groups of 12 male and 12 female Crl:CD(SD) rats were administered a single oral dose by gavage of 0, 100, 500, or 2000 mg/kg of the test substance in 0.5% methylcellulose.

Examinations

Observations and clinical examinations performed and frequency:

Clinical signs of toxicity were assessed daily from test day 0 through the day of sacrifice (except for the days of neurobehavioral evaluation).

Neurobehavioural examinations performed and frequency:

A neurobehavioral test battery, consisting of motor activity and functional observational battery assessments, was conducted on all study rats prior to test substance administration in order to obtain baseline measurements. This neurobehavioral test battery was conducted again on test day 0 approximately 2 hours after administration of the test substance. The neurobehavioral test battery was repeated again on test days 7 and 14.

Sacrifice and (histo)pathology:

On test day 16, six rats per sex per group were anesthetized and underwent whole-body in situ perfusion. Tissues from the control and 2000 mg/kg groups were processed for histopathology and examined.

Other examinations:

The rats were weighed on test days 0, 1, 7, and 14. Food consumption was determined for the intervals of test days 0-1, 0-7, 7-14, and 0-14.

Positive control:

Procedures and data describing the effects of multiple positive control substances are available. These positive control studies are the basis of training certification for the personnel making judgments in the neurobehavioral and neuropathology tests. The data also document that the equipment and procedures are capable of detecting effects that may be seen in neurotoxicity studies of this type.

Statistics:

Parameter: Body Weight, Body Weight Gain, Food Consumption, and Food Efficiency: For the Preliminary Test: Levene’s test for homogeneity and Shapiro-Wilk test for normality.

If the preliminary test is not significant: One-way analysis of variance followed by Dunnett's test.

If preliminary test is significant: Transforms of the data to achieve normality and variance homogeneity were used. The order of transforms attempted was log, square-root, and rank-order. If the log and square-root transforms failed, the rank-order was used.

Parameter: Grip Strength, Foot Splay, Body Temperature, Rearing, and Motor Activity : For the Preliminary Test: Levene’s test for homogeneity and Shapiro-Wilk test for normality.

If the preliminary test is not significant: Repeated measures analysis of variance followed by linear contrasts.

If preliminary test is significant: (1) A normalizing, variance stabilizing transformation followed by repeated measures analysis of variance or (2) sequential application of the Jonckheere-Terpstra trend test .

Parameter: Incidence of FOB Descriptive Parameters: Cochran-Armitage test for trend. If the incidence is not significant, but a significant lack of fit occurs, then Fisher’s Exact test with a Bonferroni-Holm correction is used.

For each parameter analyzed with a trend test, the test was applied to the data sequentially. If a significant dose-response was detected, data from the top dose group was excluded and the test repeated until no significant trend is detected.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related clinical signs of toxicity were observed in males or females. Clinical signs observed were not considered test substance related as they were observed at low incidences and with no dose response.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No adverse, test substance-related effects occurred on body weight or weight gain in males or females. No test substance-related or statistically significant effects occurred on body weights in males or females. Test substance-related effects occurred on mean body weight gain in male and female rats dosed at 2000 mg/kg. Male rats dosed at 2000 mg/kg lost a mean of 0.6 g over the interval of test days 0-1 compared to the control males that gained 4.8 g (statistically significant); and was 9.7% lower than the control value over the interval of test days 0-7. The mean body weight gain of female rats dosed at 2000 mg/kg was decreased 23% compared to control (statistically significant) in the interval days 0-7. However, body weight gain recovered such that the values were similar to control by the end of the 14-day observation period for both males and females. There were no effects on body weight or weight gain in males or females administered 100 or 500 mg/kg. Since the transient decreases in body weight gain did not affect the mean body weights for males or females, they were not considered to be adverse.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No adverse, test substance-related effects occurred on food consumption in males or females. Food consumption was 9.6% lower (not statistically significant) for 2000 mg/kg male rats during the interval of test days 0-1. In females, the mean food consumption of female rats dosed at 2000 mg/kg was 21% lower than control (statistically significant) in the interval days 0-1. These decreases were considered to be test substance related. The mean food consumption of female rats dosed at 100 mg/kg was 12% higher than control (statistically significant). This increase was not considered to be test substance-related because the difference did not exhibit a dose response.

Food efficiency:

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Motor activity

Transient test substance-related decreases in motor activity were observed in 500 mg/kg and above males and females on test day 0. The mean duration of movement was statistically significantly lower in the 500 and 2000 mg/kg males during the 2nd, 3rd, and 4th 10-minute intervals. Total duration of movement was 40% and 50% lower than control values for 500 and 2000 mg/kg males, respectively (statistically significant only for the 2000 mg/kg males). Similarly, on test day 0, mean number of movements was statistically significantly lower in 500 and 2000 mg/kg males during the 2nd and 3rd 10-minute intervals and total number of movements were 21% lower compared to control. These decreases in duration and number of movements on test day 0 in 500 mg/kg and above males were considered adverse and test substance related. On test day 0, 100 mg/kg males had significantly lower duration of movement during the 3rd 10-minute interval and significantly lower number of movements during the 2nd 10-minute interval. However, these statistical differences were minor and did not affect total duration or number of movements, and therefore were not considered to be adverse.

On test day 0, 2000 mg/kg males had significantly higher number of movements during the 9th 10-minute interval, however this statistical difference reflects the normal variability observed during this phase of the evaluation and was not considered test substance related. On test day 14, 2000 mg/kg males had a significantly lower number of movements during the 9th 10-minute interval. However, since total number of movements were similar to control, this statistical difference was not considered to be test substance related.

On test day 0, mean duration of movement was statistically significantly lower in the 500 mg/kg females during the 1st, 3rd, and 4th 10-minute intervals. Mean duration of movement was statistically significantly lower in 2000 mg/kg females during the 1st, 2nd, 3rd, and 4th 10-minute intervals. Total duration of movement was decreased 40% and 64% in 500 and 2000 mg/kg females, respectively, and is considered to be test substance related and adverse. On test day 0, mean number of movements was statistically significantly lower in 2000 mg/kg females during the 1st, 2nd, and 3rd 10-minutes intervals, and total number of movements was 41% lower than control. These decreases were considered adverse and test substance related.

Males administered 2000 mg/kg had transient, statistically significant increases in the incidence of curled up posture in the home cage on test day 0 (4/12 compared to 0/12 for controls). The increased incidence of curled up posture in the home cage for 2000 mg/kg males correlated with the reduced motor activity in this group and was considered test substance related and adverse. The incidence of low arousal in the open field was also significantly increased in 500 and 2000 mg/kg males on test day 0 (3/12 and 3/12, compared to 0/12 in the control group). However, a dose-response relationship was not present on test day 0, the incidence was low, and similar incidences were also observed in 100 mg/kg males on test day 7 (3/12), and in 500 mg/kg males on test day 14 (4/12) compared to an incidence of 1/12 in the 2000 mg/kg males on both days. Therefore, the significantly higher incidence of low arousal in 500 and 2000 mg/kg males on test day 0 was not considered test substance related or adverse.

The incidence of curled up posture in the home cage in 2000 mg/kg females on test day 14 (incidence of 2/12) and sleeping in home cage in 2000 mg/kg females on test day 0 (incidence of 2/12) and test day 14 (incidence of 2/12) were significantly increased compared to the control incidences of 0/12 for each of those days. These increased incidences were not considered to be test substance related since they occurred at a low incidence and were observed with a similar lower frequency during the baseline evaluation.

Rearing

On test day 0, female rats dosed at 2000 mg/kg had a statistically significantly decreased mean number of rearing movements compared to the control value. This decrease was considered to be test substance-related and adverse. The number of rearing movements for the 2000 mg/kg male and female groups were similar to control on test days 7 and 15.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

2 000 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No neurotoxicity effects at the highest dose tested

2

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Systemic toxicity including associated effects on motor activity, rearing, and/or low arousal on day of dosing at 500 mg/kg

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, the NOAEL in males and females was considered to be 100 mg/kg, based on evidence of systemic toxicity accompanied by associated effects on motor activity, rearing, and/or low arousal parameters on the day of dosing at 500 mg/kg. The NOAEL for neurotoxicity was 2000 mg/kg based on the absence of effects on neuropathological and several neurobehavioral parameters. When neurobehavioral parameters were affected (e.g., on test day 0), these effects were transient rather than prolonged and were considered related to system toxicity observed on the day of dosing.

Executive summary:

The purpose of this study was to assess the potential neurotoxicity of the test substance to rats after acute exposure. Young adult male and female Crl:CD(SD) rats (12 rats/dose/sex) were administered a single oral dose by gavage of 0, 100, 500, or 2000 mg/kg of the test substance in 0.5% methylcellulose. Samples of the dosing solution were analyzed to verify the concentration of the test substance. The rats were weighed on test days 0, 1, 7, and 14. Food consumption was determined for the intervals of test days 0-1, 0-7, 7-14, and 0-14. Clinical signs of toxicity were assessed daily from test day 0 through the day of sacrifice [except for the days of neurobehavioral evaluation] (OECD 424).

 

A neurobehavioral test battery, consisting of motor activity and functional observational battery assessments, was conducted on all study rats prior to test substance administration in order to obtain baseline measurements. This neurobehavioral test battery was conducted again on test day 0 approximately 2 hours after administration of the test substance. The neurobehavioral test battery was repeated again on test days 7 and 14. On test day 16, six rats per sex per group were anesthetized and underwent whole-body in situ perfusion. Tissues from the control and 2000 mg/kg groups were processed for histopathology and examined.

 

No unscheduled deaths occurred during the study. No test substance-related clinical signs of toxicity were observed in males or females.

 

No adverse test substance-related effects occurred on body weight, weight gain, food consumption, or food efficiency in males or females. Test substance-related effects occurred on mean body weight gain in male and female rats dosed at 2000 mg/kg; however, since there were no significant effects on mean body weights these were not considered as adverse.

There was a transient, but non-statistically significant reduction in food consumption in 2000 mg/kg males for interval days 0-1. There were no test substance-related or statistically significant effects on food consumption in males for the remainder of the study. Test substance-related and statistically significant reduction in food consumption was observed during this same interval in female rats dosed at 2000 mg/kg. The mean food efficiency of male rats dosed at 2000 mg/kg was statistically significantly decreased compared to control in the interval days 0-1 and corresponded to the decreased body weight gain in that group. No test substance-related or statistically significant effects occurred on food efficiency in females. However, since there were no significant effects on body weight, these transient changes in food consumption and/or efficiency were not considered to be adverse.

 

No test substance-related or statistically significant effects occurred on forelimb or hindlimb grip strength or hindlimb footsplay for either males or females administered any dose of the test substance. Decreased body temperature on test day 0 in 2000 mg/kg males and in 500 and 2000 mg/kg females was considered to be test substance related but not adverse. Transient but statistically significant decreases in motor activity were observed on test day 0 in 500 and 2000 mg/kg males and females. These changes were primarily observed during the first few 10-minute intervals (intervals 1 through 4, but not 5 through 9). There was correlation of these motor activity changes with reduced rearing and increased incidence of low arousal in males anddecreased rearing incidence in females. However, in the absence of test substance-related gross or microscopic anatomic pathology effects or other neurological parameters in either males or females administered any dose of the test substance, these changes are considered to reflect systemic toxicity.

Under the conditions of the study, the no-observed-adverse effect level (NOAEL) in males and females was considered to be 100 mg/kg, based on evidence of systemic toxicity including associated effects on motor activity, rearing, and/or low arousal on the day of dosing at 500 mg/kg. The NOAEL for acute neurotoxicity was 2000 mg/kg based on the absence of effects on neuropathological and several neurobehavioral parameters. When neurobehavioral parameters were affected (e.g., on test day 0), these effects were transient rather than prolonged and were considered related to systemic toxicity observed on the day of dosing.