Chlorimuron ethyl

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1983

Report date:

1983

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

no

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

Details on test material:

Purity: 90.6-99.6%

Specific details on test material used for the study:

Approximately 1.14 kg of H14697-01 (90.6% purity) and 2.39 kg of H14697-02 (93.2% purity) were mixed to make a single homogeneous sample numbered H14697-03 with a purity of 92.5%. H14697-03 was used in the preparation of test diets through 12/29/82; H14697-04 (99.6% purity) was used for the remainder of the study.

Test animals

Species:

rat

Strain:

other: Crl:CD®

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test animals: Source: Charles River Breeding Laboratories, Kingston, NY;
Age at study initiation: Approximately 6 weeks;
Weight at study initiation: 126.4-148.5 g (males) and 110.3-128.3 g (females);
Housing: The rats were doubly housed, sexes separate, in stainless steel wire-mesh cages;
Diet: Certified Purina® Laboratory Chow #5002;
Water: Tap water ad libitum- Acclimation period: 8 days;
Environmental conditions: Temperature, Humidity, and Photoperiod were not specified.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: Certified Purina® Laboratory Chow #5002

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The diet feed samples were analyzed by HPLC. The results of the homogeneity study are also included.

Ten grams of each diet sample was ultrasonically extracted with either 50 mL or 100 mL of 2% acetic acid in methylene chloride, depending on the level of the test substance expected. An aliquot of the resulting slurry was filtered and injected into a liquid chromatograph. The test substance was then measured using the following chromatographic conditions:

Column: μ Porasil® 30 cm x 4.1 mm i.d.
Mobile phase: 0.16% H2O, 2.0% acetic acid in methylene chloride
Flow rate: 2 mL/min
Detector: Micromeritics® Model 786 variable-wavelength ultraviolet set at 254 nm
Sample volume injected: 10 μL
Quantitation: Peak height measurement
Standard solutions: 0.005, 0.125, 0.50 mg/mL test substance
Retention time of the test substance: Approximately 6 minutes

All results were above 98% of the nominally-prepared levels. Quantitative recoveries were obtained on
spiked control samples. All measurements were made using a reference standard of 93% purity.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 ppm

Remarks:

91-97 ppm analytical

Doses / concentrations 2

Dose / conc.:

2 500 ppm

Remarks:

2250-2540 ppm analytical

Doses / concentrations 3

Dose / conc.:

7 500 ppm

Remarks:

7130-7310 ppm analytical

No. of animals per sex per dose:

16 males and 16 females

Control animals:

yes, plain diet

Details on study design:

Groups of 16 male and 16 female Crl:CD® rats were fed for approximately 90 days with diets that contained 0, 100, 2500, or 7500 ppm of the test substance. Clinical indices were measured in urine and blood at approximately one, two, and three months after study initiation. At the end of the 90-day period, ten rats per sex from each diet group were sacrificed, necropsied, and select tissues examined histologically. The remaining six rats per sex per diet group were mated for the purpose of a one-generation, one-litter reproduction sub-study.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

During the 90-day feeding phase of the study all rats were weighed once weekly and were observed at least twice daily for abnormal behavior or appearance and moribundity. The amount of food consumed by each group was determined weekly during the 90-day feeding phase of the study. Food consumption and body weight data were used to calculate, mean daily food consumption, food efficiency (utilization of food for body growth), and mean daily intake of the test substance.

At approximately one, two, and three months after the study's initiation, hematological, clinical chemical, and urine analytical evaluations were conducted on the ten rats in each treatment group designated for evaluation of subchronic toxicity. Rats were individually housed in metabolism cages to facilitate the collection of urine. Following a 24-hour acclimation period, the rats were deprived of food overnight (approximately 16 hr.) during which time urine was collected. Blood samples were subsequently collected via tail cut from the fasted rats.

The hematological parameters examined at each interval consisted of erythrocyte, leukocyte, differential leukocytes, and platelet counts, hemoglobin contents, and mean corpuscular volumes (MCV). Hematocrits, mean corpuscular hemoglobin contents (MCH), and mean corpuscular hemoglobin concentrations (MCHC) were calculated. Blood smears for reticulocytes were prepared and counted. Clinical chemical evaluation of serum consisted of measures of alkaline phosphatase (AP), alanine-amino transferase (ALT), and aspartate-amino transferase (AST) activities, blood urea nitrogen (BUN), total serum protein, albumin, globulin (calculated), creatinine, cholesterol, glucose, calcium, sodium, and potassium. Urine analysis consisted of quantitative measures of urine volume, pH, and osmolality and semi-quantitative measures of glucose, protein, bilirubin, urobilinogen, ketones, and occult blood. Urine color and appearance were recorded and sediment from the urine samples was examined microscopically.

Sacrifice and pathology:

On days 95 and 96 of the study, the surviving male and female rats, respectively, from each treatment group designated to be evaluated for subchronic toxicity were sacrificed and necropsied. The order of sacrifice was random among all rats within a sex. All rats that died or were sacrificed before the end of the study were also necropsied. Brain, heart, liver, spleen, kidneys, lungs, thymus, adrenals, pituitary and testes of all necropsied rats except those found dead were weighed and, for rats in the terminal sacrifice, organ weight/final body weight ratios were calculated. The following tissues taken from rats designated to be evaluated for subchronic toxicity that died (tissue integrity permitting) or were sacrificed prior to the end of the 90-day feeding study, and from rats in the high-dose and control groups, were examined microscopically: thymus, spleen, femoral bone marrow, lymph nodes, heart, aorta-thoracic, trachea, lungs, salivary glands, esophagus, stomach, small intestine (duodentnn, jejuntnn and
ileum), large intestine (colon and cecum), liver, pancreas, kidneys, bladder, pituitary, thyroid - parathyroid, adrenals, testes, epididymides, prostate, ovaries, corpus and cervix uteri, vagina, brain, eye, muscle (thigh), bone (femur), and all gross lesions. All gross lesions, heart, liver, kidney, and target organs from rats in the low- and intermediate-dose groups were also examined histopathologically. The other tissues listed above from rats in the low- and intermediate-dose groups were collected, processed to the block stage of preparation, but were not evaluated histologically.

Statistics:

Body weights and organ weights were subjected to analyses of variance. The least significant difference [LSD] from control values and Dunnett's statistics were calculated whenever the ratio of variances (F-ratio) indicated significant differences existed among the test groups.
Significance was judged at the p <0.05 probability level.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Rat No. 333368 (male 7500 ppm group) - alopecia - leg
Rat No. 333369 (male 7500 ppm group) - alopecia - leg and neck; skin sore – leg
Rat No. 333444 (female 7500 ppm group)- alopecia - back
Rat No. 333456 (female 7500 ppm group)- alopecia - legs and shoulder

No adverse clinical signs were observed in the control or other treatment groups.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Rat No. 333445 (female 7500 ppm group), was found dead on Day 68 of the study. The probable cause of death was not determined.

Rat No. 333432 (female 2500 ppm group) and Rat No. 333443 (female 7500 ppm group) were interchanged and fed the incorrect diets following the one-month clinical examination. These rats were sacrificed on Day 29 of the study. Tissues from these rats were held in block stage and excluded from histologic evaluation.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weight and overall weight gains of male rats in the 7500 ppm group were significantly lower than the control group from weeks 3-13 of the study.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Food efficiency (g Weight Gain/g Diet Consumed) was significantly lower in the male 7500 ppm group compared to control group.

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The one-month evaluation revealed a statistically-significant decrease in mean corpuscular hemoglobins (MCH), mean corpuscular hemoglobin concentrations (MCHC), platelets, and serum AST/GOT activities, and a significant increase in serum concentrations of calcium, cholesterol, creatinine, total protein, albumin, and globulin and decreased urine volume in the male 7500 ppm group. Increased serum calcium concentrations and reticulocyte counts were observed in the male 2500 ppm group.

At the one-month evaluation, female rats in the 2500 and 7500 ppm groups had statistically-significant decreased erythrocyte counts and hematocrits; increased MCH, MCHC, and serum potassium levels relative to controls. Decreased serum alkaline phosphatase activities and increased serum cholesterol concentrations were also observed in the female 7500 ppm group.

At the two-month evaluation, the male 2500 and 7500 ppm groups had decreased hemoglobins, MCHC, and serum AST/GOT activities, and increased serum calcium concentrations. Also observed were decreased MCH and decreased absolute numbers of lymphocytes in the male 7500 ppm group; increased serum globulin levels in the male 2500 ppm group; and decreased numbers of reticulocytes in the male 100 and 7500 ppm groups.

At the two-month evaluation, female rats in the 7500 ppm group had increased absolute numbers of monocytes and decreased serum albumin concentrations while increased absolute numbers of eosinophils occurred in the female 2500 ppm group.

At the three-month evaluation, the male 2500 and 7500 groups had decreased erythrocyte counts and hematocrits, and increased MCH and MCHC, while increased absolute numbers of eosinophils were observed only in the male 2500 ppm group. Decreased serum AST/GOT activities were observed in all male treated groups.

At the three-month evaluation, female rats had increased MCH, MCHC, and platelet counts in the 2500 ppm group, and increased serum globulin concentrations in the 7500 ppm group.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The one-month evaluation revealed a statistically-significant decrease in mean corpuscular hemoglobins (MCH), mean corpuscular hemoglobin concentrations (MCHC), platelets, and serum AST/GOT activities, and a significant increase in serum concentrations of calcium, cholesterol, creatinine, total protein, albumin, and globulin and decreased urine volume in the male 7500 ppm group. Increased serum calcium concentrations and reticulocyte counts were observed in the male 2500 ppm group. Female rats in the 2500 and 7500 ppm groups had statistically-significant decreased erythrocyte counts and hematocrits; increased MCH, MCHC, and serum potassium levels; and excreted less urine relative to controls. Decreased serum alkaline phosphatase activities and increased serum cholesterol concentrations were also observed in the female 7500 ppm group.

At the two-month evaluation, the male 2500 and 7500 ppm groups had decreased hemoglobins, MCHC, and serum AST/GOT activities, and increased serum calcium concentrations. Also observed were decreased MCH and decreased absolute numbers of lymphocytes in the male 7500 ppm group; increased serum globulin levels in the male 2500 ppm group; and decreased numbers of reticulocytes in the male 100 and 7500 ppm groups. Female rats in the 7500 ppm group had increased absolute numbers of monocytes and decreased serum albumin concentrations while increased absolute numbers of eosinophils occurred in the female 2500 ppm group.

At the three-month evaluation, the male 2500 and 7500 groups had decreased erythrocyte counts and hematocrits, and increased MCH and MCHC, while increased absolute numbers of eosinophils were observed only in the male 2500 ppm group. Decreased serum AST/GOT activities were observed in all male treated groups. In female rats, increased MCH, MCHC, and platelet counts were demonstrated in the 2500 ppm group, and increased serum globulin concentrations in the 7500 ppm group.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

At the one-month evaluation, decreased urine volume was seen in the male 7500 ppm group. Female rats in the 2500 and 7500 ppm groups excreted less urine relative to controls.

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Relative liver weight in males (grams)

Group Relative liver weight

Control 3.652
100 ppm 3.572
2500 ppm 3.696
7500 ppm 4.414*

* Statistically-significant (p ≤ 0.05) differences

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Compound-related histopathological effects were present in the livers of the male 2500 and 7500 ppm groups and the female 7500 ppm group. Margination of hepatocyte cytoplasmic contents in the centrilobular areas occurred in the male 2500 and 7500 ppm groups and the female 7500 ppm group. These changes were interpreted to represent a non-degenerative, adaptive response by the liver.

Histopathological findings: neoplastic:

not examined

Effect levels

Key result

Dose descriptor:

NOEL

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

haematology

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The no-observable-effect level (NOEL) for the administration of the test substance for the 90-day feeding study was 100 ppm for both male and female rats.

Executive summary:

Groups of 16 male and 16 female Crl:CD® rats were fed for approximately 90 days with diets that contained 0, 100, 2500, or 7500 ppm of the test substance. Clinical indices were measured in urine and blood at approximately one, two, and three months after study initiation. At the end of the 90-day period, ten rats per sex from each diet group were sacrificed, necropsied, and select tissues examined histologically. The remaining six rats per sex per diet group were mated for the purpose of a one-generation, one-litter reproduction sub-study (equivalent to OECD 408).

The administration of the test substance in the feed for a 90-day period resulted in significantly decreased body weights in the male 7500 ppm group and the female 2500 and 7500 ppm groups. Since there were no meaningful differences in food consumption among these groups compared to their respective control group, the decreased body weights were interpreted to represent a compound-related toxic effect or possibly an indirect effect of the test substance on nutrient absorption.

 

Rats in this study did not demonstrate any adverse clinical signs with the exception of alopecia which was observed in two males and two females in the 7500 ppm groups. Since alopecia is a relatively common clinical observation among CD® rats, it is not clear that the low incidence of alopecia observed in this study was related to the administration of the test substance.

 

Numerous, statistically-significant changes in hematological and clinical chemical parameters were reported for both male and female rats at each of the evaluation periods during the study. Decreased erythrocyte counts and hematocrits and decreased or increased MCH and MCHC are believed to have resulted from non-compound-related in vitro hemolysis. Decreased serum AST activity which occurred in the male 2500 and 7500 ppm groups after two and three months on test appeared to be compound-related but is not believed to be biologically significant. Other hematological and clinical chemical parameters which were affected were within the expected range of biological variation and are not believed to be compound-related.

 

Numerous, statistically-significant differences in absolute and relative organ weights were observed between rats in the control and treatment groups. However, none of the organs affected demonstrated any compound-related histopathological effects. Although the biological significance of these changes in organ weights remains unclear, these changes may simply reflect the decreased body weights observed throughout the study. The liver was the only organ affected histologically. Margination of hepatocyte cytoplasmic contents in the centrilobular areas occurred in the male 2500 and 7500 ppm groups and the female 7500 ppm group. These changes were interpreted to represent a non-degenerative, adaptive response by the liver.

 

The no-observable-effect level (NOEL) for the administration of the test substance for the 90-day feeding study was 100 ppm for both male and female rats.