Chlorimuron ethyl

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

supporting 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:

1985

Report date:

1985

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

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: 96-99.7%

Specific details on test material used for the study:

99.7% purity

Test animals

Species:

rat

Strain:

other: Crl:CD®(SD)BR

Sex:

male/female

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on mating procedure:

Each F0 female was then housed for 15 days with a F0 male from the test group fed the same dietary concentration of the test substance. During the 15-day mating period, the females were checked daily for the presence of copulation plugs.

Ten days after the last Fla litter was weaned, the F0 females were mated again, in the same manner. To produce the F1b litters, the F0 males were paired to different females within the same test group.

These F1b rats were fed the diets for their respective groups for approximately ninety days and then mated twice within their respective dietary concentration groups to produce the F2a and F2b litters.

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, 0.05, 0.25 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:

2 generation and 4 litters

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

25 ppm

Doses / concentrations 2

Dose / conc.:

250 ppm

Doses / concentrations 3

Dose / conc.:

2 500 ppm

No. of animals per sex per dose:

20 males and 20 females

Control animals:

yes, plain diet

Positive control:

No

Examinations

Parental animals: Observations and examinations:

No examination was done. These rats, after weaning of the second generation, were returned to the main, 2-year feeding study

Oestrous cyclicity (parental animals):

No estrous examination was done

Sperm parameters (parental animals):

No sperm examinations were done

Litter observations:

The total pup weight for each of the Fla litters was determined approximately 24 hours and four days after birth. Litters with more than ten pups were reduced to this number on day 4 postpartum. An equal number of male and female pups were retained when possible. The pups selected were, by gross appearance, representative of the health status of all pups in the litter. Extra pups were sacrificed by inhalation of 100% carbon dioxide and discarded without pathological evaluation.

Postmortem examinations (parental animals):

No examination was done. These rats, after weaning of the second generation, were returned to the main, 2-year feeding study

Postmortem examinations (offspring):

After weaning of the second litter, ten male pups and ten female pups from each test concentration group were selected for necropsy and pathological evaluation. The following tissues were examined histopathologically: kidneys, liver, trachea, heart, lungs, brain, eyes, urinary bladder, testes, epididymides, ovaries, uterus (horn, corpus, cervix), vagina, bone and bone marrow (sternum), spleen, thymus, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, adrenals, thyroid, parathyroid, pancreas, and any gross lesions. Liver, brain, kidneys, and testes were weighed and mean organ-to-body-weight ratios were calculated. All tissues from the control and high dose groups were evaluated histologically. Only the brain, liver, and gross lesions from the low- and intermediate-dose groups were evaluated histopathologically.

Statistics:

Reproduction and lactation data were analyzed with the Mann-Whitney U test to compare each treatment group with its respective control group. Tests for comparison of means were considered significant at the p < 0.05 level.

Reproductive indices:

Reproduction and lactation indices were calculated according to the following formulas:

Fertility(a) = total number of litters delivered/total number of females mated x 100

Gestation Index (%) = total number of litters with at least one live pup/ total number of litters delivered x 100

Percent Pups(b) Born Alive (per litter) = number of pups born alive/number of pups born x 100

Lactation(c) Index (%) (per litter) = number of pups alive at weaning/ number of pups alive after reduction (reduction occurred at four days postpartum) x 100

Litter Survival (%) = total number of litters at weaning/total /number of litters delivered x 100

a = Excluding female rats that died during the mating phase or before the last litter of that generation's test group was delivered.

b = Indices were calculated on a per litter basis for female rats bearing litters. The percentage was calculated for each litter and the average percentage for each group is reported.

c = Indices were calculated on a per litter basis for female rats bearing litters with at least one live pup. The percentage was calculated for each litter and the average percentage for each group is reported.

Offspring viability indices:

0-4 day(c) Viability Index (%) = number of pups alive at four days postpartum/number of pups born alive x 100

1-4 day(c) Viability Index (%) (per litter) = number of pups alive at four days postpartum/ number of pups alive at 24 hours x 100

c = Indices were calculated on a per litter basis for female rats bearing litters with at least one live pup. The percentage was calculated for each litter and the average percentage for each group is reported.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry 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

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

The first mating of F0 rats resulted in lower than expected fertility indices for two test groups. In the control group, the fertility index was 80% (16 of 20 mated dams bore litters). A low fertility index (57.9%) also resulted for the group fed 250 ppm diets; only 11 of 19 mated dams bore litters. Fertility indices for the 25 ppm and 2500 ppm groups were both 95%. In the absence of any dose-response trend, the low index for the 250 ppm group was determined not to be related to intake of the test compound.

Although the fertility index for the 250 ppm group was substantially lower than the fertility index for controls, this same group of dams had a low fertility index for the first mating, and six dams had no litters at either mating. As in the first mating, no dose-response trend existed for reduced fertility, and the low index was not considered to be a compound-related effect. The 2500 ppm group had a gestation index and percent Flb litter survival of 94.4%. Gestation indices for the other test groups were 100%. The lower indices for the high-dose group resulted because one dam (#344429) died after bearing one pup. This pup was dead at birth. All other indices for F1b litters were similar for all test groups, and none of the pup count data for groups fed test compound were significantly different from pup count data for the control group.

In the F2a generation, fertility indices for all groups fed the test compound were lower than the fertility index for controls. The lowest index (68.4%) occurred for the 25 ppm group, in which only thirteen of nineteen dams bore litters. Gestation indices and percent litter survival were 100% for all groups except the 250 ppm group. One dam in this group (#360383) had only one pup, which was born dead. Statistical differences in pup counts occurred when the 2500 ppm group was compared to controls. The mean number of pups born, pups born alive, pups alive at 24 hours, and pups alive at day 4 were all lower for the group fed 2500 ppm diets than for the control group. In addition, the reduced number of pups at day 4, number of pups at day 12, and total number of pups on day 21 were significantly lower for the 25 ppm group than for the controls. None of these lower counts appeared to be related to the intake of test compound.

The F1b rats were again mated to produce the second litter in this generation (F2b). The fertility index was lower than expected for all groups, but was lowest (63.2%) for the 25 ppm group. The gestation index was 100.0% for all groups. All other indices were slightly reduced for the 2500 ppm group, because one dam (#360394) in this group fed 2500 ppm diets had a litter of 15 pups, and none survived to day 21 weaning. Therefore, none of these low pup counts or indices could be attributed to intake of the test substance.

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During the two-generation reproduction study, lower pup weights were seen in the litters produced by rats fed diets that contained 2500 ppm than in litters produced by control group rats. Lower pup weights were evident at weaning in all four sets of litters (two litters/generation). In addition, significantly lower weights were observed for F2b pups at 24 hours and at day 4, prior to litter reduction. Also, the dams in the 2500 ppm diet groups had lower body weights at all four pup weaning times compared to the dams in the control groups. The lower pup weights and dam weights were considered to be compound related.

Although not statistically significant, the mean body weights and total weight gain for the females fed 2500 ppm diets were lower than controls during this period.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Several statistically significant differences in organ weights were observed in weanling rats. Mean absolute liver weight and kidney weight for the male weanlings from the group fed 2500 ppm diets were lower than those organ weights in control group weanlings. However, total body weight for male weanlings in this group was also significantly lower than control weanlings, and relative weights for these organs were not significantly different for controls. Likewise, testes and brain relative weights were higher for the 2500 ppm group than for the control group, but this was likely to have been related to the lower mean body weights. For female rats in the 2500 ppm group, mean absolute kidney weights were lower than controls. Relative liver and brain weights were higher for 2500 ppm female weanlings than for female weanlings in the control group. Mean total body weight for the female weanlings was statistically lower than total body weight for controls, and the differences in organ weights were apparently related to the lower body weights in weanlings from the 2500 ppm group.

Gross pathological findings:

effects observed, non-treatment-related

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Cerebellum effects, exhibited by cellularity differences, were observed on histopathological examination of male and female weanlings produced by rats fed 2500 ppm diets. These histological changes in the cerebellum were considered to be compound related.

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Several significant differences in weight parameters occurred for the Fla generation. Mean male pup weights, mean female pup weights, and mean dam weights for the group fed 2500 ppm diets were significantly lower at Fla weaning than the mean weights of pups and dams fed control diets. In addition, although not significantly different, the mean weight of Fla pups in the 2500 ppm group at 24 hours and at day 4 were lower than the mean weight of control pups on the same day. These weight differences were considered to be compound related.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Results: F2 generation

General toxicity (F2)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significant differences considered to be related to dietary intake of the test substance occurred in several of the weight parameters measured for the F2a litters. Pup total litter weights at 24 hours and at day 4 before litter reduction were significantly lower for the 2500 ppm group than for the control group. Also, at weaning, the mean male pup weight, mean female pup weight, and mean dam weight for the 2500 ppm group were lower than the day 21 weaning weights for the same groups of controls. The lower pup weights and dam weights were considered to be compound related.

Dam weights and pup weights for the F2a generation indicated possible compound-related effects for the group fed 2500 ppm diets. At weaning, mean weights for female pups in the 2500 ppm group were significantly lower than female pup weights in the control group. In addition, although differences were not statistically significant, the mean pup weights on day 4 and weights of male pups and dams at weaning on day 21 were lower for the 2500 ppm group than for the control group. The lower pup weights and dam weights were considered to be compound related.

Significantly lower weights were observed for F2b pups at 24 hours and at day 4, prior to litter reduction. Also, the dams in the 2500 ppm diet groups had lower body weights at all four pup weaning times compared to the dams in the control groups. The lower pup weights and dam weights were considered to be compound related.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant differences in organ weights were observed for F2b weanlings sacrificed at the end of the reproduction study. All of the organ weights that were significantly different from control organ weights occurred in weanlings produced by rats fed diets that contained 2500 ppm of the test substance. These included lower mean absolute liver and kidney weights and higher relative testes and brain weights in male pups, and lower mean absolute kidney weights and higher relative liver and brain weights in female pups.

Gross pathological findings:

no effects observed

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

Histological examination indicated that there were statistically significant effects in the cerebellum and liver for male and female weanlings from the 2500 ppm group. In the cerebellum, there were areas of decreased cellularity in the internal granular layer coupled with increased cellularity in the external germinal layer. These types of changes in the cerebellum have been noted in studies of under-nutrition and alcohol toxicity. The liver lesion, decreased cytoplasmic vesiculation, was statistically higher at 250 and 2500 ppm when males and females were combined. However, this lesion was determined to be an artifact related to the order of pup sacrifice. The cerebellum effects observed in the 2500 ppm group were determined to be compound related.

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

Dose descriptor:

NOEL

Generation:

F2

Effect level:

250 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Applicant's summary and conclusion

Conclusions:

In a 2-generation, 4 litter reproduction study, groups of male and female rats were fed diets containing 0, 25, 250, or 2500 ppm of the test substance. The notable effects attributed to dietary intake of the test substance were the low pup weights and histologic cerebellum effects in pups produced by rats fed 2500 ppm diets. Therefore, the no-observable-effect level was 250 ppm.

Executive summary:

In the long-term feeding study, male and female rats were fed diets that contained the test substance for 24 months. Four groups of 80 male and four groups of 80 female rats were fed 0, 25, 250, or 2500 ppm diets. The rats were weighed at regular intervals and food consumption was measured throughout the study. Clinical evaluations that included hematology, clinical chemistry, and urology were perfomed 3, 6, 9, 12, 18, and 24 months after study initiation. At twelve months, ten male and ten female rats per group were randomly selected for sacrifice and pathological evaluation. At 24 months all surviving rats were necropsied, and selected tissues were examined microscopically.

A two-generation, four-litter reproduction substudy was initiated approximately three months after the start of the feeding study. All rats in the reproduction substudy were fed diets that contained the same concentrations of the test substance as those used in the 24-month feeding phase. Twenty male and twenty female rats from each group were selected for the first generation matings. Males were mated to females that were fed diets that contained the same concentration of the test compound. After the weaning of the second litter, the mated rats were returned to the long-term feeding study. Twenty male and twenty female weanlings from each dietary concentration group in the second litter were selected to produce the second generation of the substudy. These rats were fed for 90 days with diets that contained 0, 25, 250, or 2500 ppm test substance. Body weights and food consumption were monitored during that period. The rats were then mated to produce two litters in the second generation. At weaning of the second litter, ten male pups and ten female pups from each test concentration group were selected for necropsy and pathological evaluation.

Mean body weights of male and female rats fed diets that contained 2500 test substance for 24 months were lower than the mean body weights of their respective control groups. These lower weights were statistically significant for males during the first three months and the last twelve months of the study, and for females throughout the entire 24 month feeding period. By the end of the study, the mean body weight for male rats fed 2500 ppm diets was 8% less than the mean body weight for male controls; females fed the 2500 ppm diets, had a mean body weight that was 24% less than controls. In addition, food consumption and food efficiency were lower for the female group fed 2500 ppm diets than for the female control group. The mean daily intake values for male rats fed 25, 250, and 2500 ppm diets were 1.0, 10, and 110 mg test substance/kg body wt/day, respectively. The intake values for females fed the same respective diet concentrations were 1.4, 13, and 140 mg test substance/kg body wt/day.

No effects from the dietary intake of the test substance were seen in clinical observations in this study nor in the incidences of unscheduled mortalities. In addition, the clinical pathology evaluations indicated that, although several clinical parameters for compound-treated groups were significantly different from controls, none of the observed effects occurred consistently for any test group, and none of the differences were considered to be related to intake of the test compound.

Several statistical differences in mean organ weights were observed at the 12-month and 24-month sacrifices for the male and female groups fed diets that contained test substance when compared to mean organ weights of their respective control groups. At the 12-month sacrifice, the significant differences for the 2500 ppm male group included lower mean absolute and relative spleen weights. Females fed this dietary concentration had a higher mean relative liver weight. The 250 ppm male group had an elevated mean absolute brain weight. At the two-year sacrifice, statistically significant organ weights were again present for the 2500 ppm male and female groups. These included lower mean absolute heart weight and higher mean relative testes weight in males and lower mean absolute heart and kidney weights, and higher mean relative brain and liver weights in females. No gross or histopathologic changes attributable to dietary intake of the test substance were observed in rats that died or were sacrificed in extremis during the study or in rats sacrificed by design at 12 or 24 months. The differences observed in organ weights appeared to be related to the lower total body weights for rats in the 2,500 ppm groups since no unusual histological effects were observed in these organs.

During the two-generation reproduction study, lower pup weights were seen in the litters produced by rats fed diets that contained 2500 ppm test substance than in litters produced by control group rats. Lower pup weights were evident at weaning in all four sets of litters (two litters/generation). In addition, significantly lower weights were observed for F2b pups at 24 hours and at day 4, prior to litter reduction. Also, the dams in the 2500 ppm diet groups had lower body weights at all four pup weaning times compared to the dams in the control groups. The lower pup weights and dam weights were considered to be related to the dietary intake of the test substance.

No notable effects upon food consumption, food efficiency, clinical observations, or mortalities were seen in the F1b rats fed the test substance in the diet for 90 days. Although not statistically significant, the mean body weights and total weight gain for the females fed 2,500 ppm diets were lower than controls during this period.

Statistically significant differences in organ weights were observed for F2b weanlings sacrificed at the end of the reproduction study. All of the organ weights that were significantly different from control organ weights occurred in weanlings produced by rats fed diets that contained 2500 ppm test substance. These included lower mean absolute liver and kidney weights and higher relative testes and brain weights in male pups, and lower mean absolute kidney weights and higher relative liver and brain weights in female pups. No compound-related effects were observed in the weanlings at gross pathological examinations; however, cerebellum effects, exhibited by cellularity differences, were observed on histopathological examination of male and female weanlings produced by rats fed 2500 ppm diets. These histological changes in the cerebellum were considered to be compound related.

In this study the major effects that could be attributed to the dietary intake of the test substance included the lower mean body weights of rats fed 2,500 ppm diets for 24 months, the lower mean pup weights for all litters produced by rats fed 2500 ppm diets, and the histopathological cerebellum changes observed in the 2500 ppm group of F2b weanlings from the reproduction substudy. The no-observable effect level for rats fed diets that contained test substance for two years was 250 ppm, and the no-observable-effect level for the reproduction substudy was also 250 ppm.

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