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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
multi-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
no
Qualifier:
according to
Guideline:
other: MAFF Japan Test Guidelines for Agricultural Chemicals 2-1-17 Notification 12-Nousan-8147 (2000)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Purity: 99.4%
Impurities: Not reported

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: Diet
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
P1: From age of 56 days until sacrifice
F1: From age of 21 days until sacrifice
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 ppm
Remarks:
P1: 6.29 to 8.20 mg/kg/d; F1: 6.59 to 7.94 mg/kg/d
Dose / conc.:
500 ppm
Remarks:
P1: 30.69 to 41.79 mg/kg/d; F1: 32.66 to 41.79 mg/kg/d
Dose / conc.:
1 500 ppm
Remarks:
P1: 92.80 to 124.64 mg/kg/d; F1: 95.32 to 121.26 mg/kg/d
Dose / conc.:
3 000 ppm
Remarks:
P1: 182.33 to 256.16 mg/kg/d; F1: 192.78 to 252.91 mg/kg/d
No. of animals per sex per dose:
30
Control animals:
yes, plain diet

Examinations

Reproductive indices:
The following lists of indices of reproductive function that were calculated for the P1 and F1 parental animals.
- Mating index
- Fertility index
- Gestation index
- Post implantation loss
- Pups born alive
- Viability index
- Lactation index
- Litter survival
Offspring viability indices:
The following lists of indices were calculated for offsprings:
- Live born index
- Viability index
- lactation index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- In P1 males, there were statistically significant reductions in mean body weight at 1500 and 3000 ppm that were evident beginning after one and two weeks of exposure at 3000 and 1500 ppm, respectively. Mean body weights at the end of the study (test day 120) were 8 and 10% lower than the control group mean at 1500 and 3000 ppm, respectively.

- In P1 females during premating, there were test substance-related reductions in mean body weight at 3000 ppm. Mean body weight was significantly lower than the respective control group mean after one week of exposure and the reduction persisted until the end of the premating period; mean body weights on test day 71 were 7% lower than the control mean.

- In P1 females during the gestation period, there were test substance-related reductions in mean maternal body weight. Mean maternal weights were lower or significantly lower at 1500 and 3000 ppm beginning on gestation day 0 and persisting until the end of gestation. Mean body weights on gestation day 20 were 6 and 10% lower than the control mean at 1500 and 3000 ppm, respectively.

- In P1 females, during the lactation period, there were test substance-related reductions in mean maternal body weight. Mean maternal weights were lower or significantly lower at 1500 and 3000 ppm beginning on lactation day 0 and persisting until the end of lactation. Mean body weights on lactation day 21 were 5 and 6% lower than the control mean at 1500 and 3000 ppm, respectively.

- In P1 males, cumulative mean body weight gains from test Days 1 to 120 were 13 and 17% lower than the respective control group mean at 1500 and 3000 ppm, respectively.

- In P1 females, during premating period, consistent with the body weight data, there was a significant test substance-related reduction in mean body weight gain in 3000 ppm P1 females during the premating period. Cumulative gain from test days 1 to 71 was 27% lower than for the control. A significant test substance-related reduction in mean body weight gain at 1500 ppm was evident but was not considered adverse. The cumulative gain from test days 1 to 71 was 10% lower than for the control.

- In P1 females, during gestation period, mean body weight gains during gestation were consistent with the resulting mean body weights and were sporadically and generally lower or significantly lower at 1500 and 3000 ppm throughout gestation. Cumulative mean body weight gains from gestation days 0 to 20 were 8 and 10% lower than the control at 1500 and 3000 ppm, respectively.

- In P1 females, during lactation period, mean body weight gains during lactation revealed a tendency toward increased body weight gain at 1500 and 3000 ppm. The mean gains were occasionally statistically significant and the cumulative mean gains from lactation days 0 to 21 were 63 and 102% higher than the control group mean.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- In P1 males, there were test substance-related reductions in mean food consumption at 1500 and 3000 ppm. At 3000 ppm, these reductions were evident during the first week of feeding and persisted until the end of the premating period. At 1500 ppm, these reductions were evident and were lower or significantly lower beginning test days 50 to 57 and persisted until the end of the premating period. Mean cumulative food consumption values from test days 1 to 71 were 6 and 9% lower than the control mean at 1500 and 3000 ppm, respectively.

- In P1 females during the premating period, there were test substance-related effects on mean food consumption at 1500 and 3000 ppm. At 3000 ppm, mean cumulative food consumption from test days 1 to 71 was 15% lower than the control group and at 1500 ppm, mean cumulative food consumption from test days 1 to 71 was 7% lower than the control group.

- In P1 females during gestation, there were test substance-related reductions in mean maternal food consumption at 1500 and 3000 ppm. At 3000 ppm, these reductions were evident throught the gestation period and the cumulative mean food consumption from gestation days 0 to 20 was 12% lower than for the control group.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
- In P1 males, mean cumulative food efficiency values from test days 1 to 71 were 7 and 10% lower than the control mean at 1500 and 3000 ppm, respectively.

- In P1 females during the premating period, cumulative mean food efficiency values from test days 1 to 71 were slightly or significantly lower than the control group value at 1500 and 3000 ppm; mean values from test days 1 to 71 were 3 and 14% lower than for the control group. The most marked reductions in food efficiency were evident during the first week of feeding at these levels.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Histopathological findings: neoplastic:
effects observed, non-treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Reproductive function: sperm measures:
effects observed, non-treatment-related
Reproductive performance:
effects observed, non-treatment-related

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
Systemic toxicity
Effect level:
500 ppm
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
food efficiency
Key result
Dose descriptor:
NOAEL
Remarks:
Reproductive toxicity
Effect level:
3 000 ppm
Sex:
male/female
Remarks on result:
other: Highest concentration tested

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Mortality / viability:
mortality observed, non-treatment-related
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- In F1 males, there were statistically significant reductions in mean body weight at 1500 and 3000 ppm. Mean body weight at 1500 ppm and 3000 ppm on test day 106 was 6% and 11% lower than the control respectively. Cumulative mean body weight gains from test days 1 to 106 were 7 and 11% lower than the control mean at 1500 and 3000 ppm, respectively.

- In F1 females during premating, there were test substance-related reductions in mean body weight at 3000 ppm and mean body weights on test day 71 were 8% lower than the control mean and the Cumulative gain from test days 1 to 71 was 8% lower than for the control.

- In F1 females, during the gestation period, there were test substance-related reductions in mean maternal body weight at 3000 ppm. Mean maternal weights were significantly lower beginning on gestation day 0 and persisting until the end of gestation. Mean body weight on gestation day 20 was 8% lower than the control mean at 3000 ppm.

- During the lactation period, mean body weights were slightly but significantly lower than for the controls at 3000 ppm on lactation days 0 and 7. These reductions were not considered advers. At the end of lactation, mean body weights were within 2% of the control mean at all concentrations tested.

- In F1 offspring, there were lower or significantly lower pup weights throughout lactation at 3000 ppm. Beginning at birth, mean pup weights were 4% lower for the control group. For the remainder of lactation, mean pup weights ranged from 7 to 12% lower than ther respective controls.

- In F1 males, cumulative mean body weight gains from test days 1 to 106 were 7 and 11% lower than the control mean at 1500 and 3000 ppm, respectively.

- In F1 females, during premating, consistent with the body weight data, there was a significant test substance-related reduction in mean body weight gain in 3000 ppm F1 females. Cumulative gain from test days 1 to 71 was 8% lower than for the control.

- In F1 females, during lactation, mean body weight gains during lactation were significantly increased at 1500 and 3000 ppm. The cumulative gain during lactation was significantly higher; mean cumulative gains were 160 and 215% of the control mean at 1500 and 3000 ppm, respectively.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- In F1 males, there were test substance-related reductions in mean food consumption at 1500 and 3000 ppm. Mean cumulative food consumption values from test days 1 to 71 were 4 and 13% lower than the control mean at 1500 and 3000 ppm, respectively.

- In F1 females during the premating period, there were test substance-related effects on mean food consumption at 3000 ppm. At 3000 ppm, mean food consumption was lower or significantly lower beginning with the first week of feeding and persisted until the end of the premating period. Mean cumulative food consumption from test days 1 to 71 was 8% lower than for the control group.

- In F1 females during gestation, there were slight test substance-related reductions in mean maternal food consumption at 3000 ppm throughout gestation which were occasionally statistically significant. Cumulative mean food consumption from gestation days 0 to 20 was 7% lower (not statistically significant) than for the control group.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
- Test substance-related increases or significant increases in mean food efficiency were evident at 1500 and 3000 ppm during lactation.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- Test substance-related organ weight effects were observed in F1 adult females and consisted of increased liver weights (both at 3000 ppm) and increased kidney weights (3000 and ≥1500 ppm, respectively). Both the liver and kidney weight increases were interpreted to be the result of the non-adverse induction of metabolic enzymes.

- A test substance-related decrease in spleen weights was observed in the F1 male (3000 ppm for both) and female (3000 and ≥1500 ppm, respectively) weanlings with no microscopic correlate.
Gross pathological findings:
effects observed, non-treatment-related
Histopathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Sperm parameters (F1 males): No effects observed.
Estrous Cycle Parameters (F1 females): No effects observed.
Reproductive Indices and Precoital Interval (F1 animals): No effects observed.
Developmental Landmarks (F1 animals): In F1 males, there was a significant increase in the mean time to preputial separation at 3000 ppm and there were no test substance-related effects on vaginal patency at any dietary concentration tested in F1 females.

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
Systemic toxicity
Generation:
F1
Effect level:
500 ppm
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
food efficiency
Key result
Dose descriptor:
NOAEL
Remarks:
offspring
Generation:
F1
Effect level:
1 500 ppm
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Remarks:
Reproduction
Generation:
F1
Effect level:
3 000 ppm
Sex:
male/female
Remarks on result:
other: Highest dose tested

Results: F2 generation

General toxicity (F2)

Clinical signs:
effects observed, non-treatment-related
Mortality / viability:
mortality observed, non-treatment-related
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In F2 offspring, there were lower or significantly lower pup weights during lactation at 3000 ppm. Beginning at birth, mean pup weights were 5% lower for the control group. For the remainder of lactation, mean pup weights ranged from 10 to 19% lower than the respective controls. At 1500 ppm, mean pup weights were 7% lower than for the respective control on lactation day 21. This reduction was statistically significant. Based on the fact that pups are consuming test diet at this point, this is considered to reflect systemic toxicity based on direct exposure to the test substance.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- A test substance-related decrease in spleen weights was observed in the F2 male (3000 ppm for both) and female (3000 and ≥1500 ppm, respectively) weanlings with no microscopic correlate.
Gross pathological findings:
effects observed, non-treatment-related
Histopathological findings:
effects observed, non-treatment-related

Effect levels (F2)

Key result
Dose descriptor:
NOAEL
Remarks:
offspring
Generation:
F2
Effect level:
1 500 ppm
Sex:
male/female
Basis for effect level:
body weight and weight gain

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
NOAEL for systemic, offspring, and reproductive toxicity were 500, 1500, and 3000 ppm, respectively.
Executive summary:

The study was conducted according to guidelines, OECD Guideline 416, U.S. EPA OPPTS 870.3800 to evaluate the effect of the test substance on the gonadal function, conception, parturition, and growth and development of male and female Crl:CD(SD) rats over two generations involving the production of one set of litters in each generation. The test substance was administered orally because it is a potential route for human exposure and is a route recommended by regulatory agencies for this type of study. In the current study, groups of Crl:CD(SD) rats (30/sex/concentration) were exposed to the test substance via the diet at concentrations of 0, 100, 500, 1500, or 3000 ppm. During lactation and for the first three weeks post-weaning for the F1 generation adults, the dietary concentrations were reduced to 0, 60, 300, 900, or 1800 ppm. The dietary concentrations were reduced in an attempt to maintain relatively constant mean daily intake levels during lactation when the maternal food consumption is increased and during late lactation and early post-weaning which is a time of rapid offspring growth consistent with increased food consumption in these young animals. Samples of the test diets were analyzed and confirmed to be at targeted concentrations, homogeneously mixed, and stable under the conditions of use for the study.

Mean daily intake values (mg/kg/day) for the various phases of the study are in range of 6.29 to 8.20, 30.69 to 41.79, 92.80 to 124.64 and 182.33 to 256.16 mg/kg bw/d at 100, 500, 1500 and 3000 ppm respectively.

Following at least 10 weeks of exposure to the test substance (premating), the P1 and F1 generation males and females were co-housed within their respective treatment groups to produce F1 and F2 litters, respectively. Dams were allowed to deliver and rear their offspring until weaning on postnatal day 21 (PND 21). F1 and F2 litters were culled to 4 pups/sex/litter (litter size permitting) on PND 4. All remaining pups were discarded without further evaluation. At weaning, selected F1 offspring (one rat per sex per litter when possible) were randomly selected to serve as parents for the F2 generation. F2 litters were terminated at weaning.

Clinical observations, body weight, and food consumption were determined weekly throughout the study. Litter examinations (live, dead, or missing pups, individual pup weights, clinical observations) were determined at birth, on PND 4, and weekly during the 21-day lactation period. Estrous cycle parameters were evaluated daily for 3 weeks prior to cohabitation and up to the day of presumed mating in P1 and F1 adult rats. The age at either vaginal opening or preputial separation was recorded for the F1 generation. Sperm motility, morphology, concentration in the cauda epididymis, and spermatid concentration in the testis were determined for P1 and F1 adult rats at the terminal sacrifice.

Gross postmortem examinations were performed on selected animals, and selected organs were weighed and/or retained for histopathological examination. A quantitative evaluation of ovarian follicles was conducted on 10 lactating F1 females from the control and high-dose (3000 ppm) groups.

Adverse test substance-related systemic toxicity was limited to reductions in body weight and nutritional parameters at 1500 and 3000 ppm in adult P1 and F1 male and female rats. There were no adverse effects on body weight or nutritional parameters in P1 or F1 adult rats at 500 ppm or lower. There was no test substance-related mortality nor were there any test substance-related clinical observations. There were no adverse effects on gross observations, organ weights, or microscopic alterations in P1 or F1 parental rats or F1 or F2 offspring at any exposure concentration.

Adverse test substance-related reductions in offspring weight occurred at 3000 ppm. A slight delay in preputial separation in F1 males at 3000 ppm was considered secondary to the effects on body weight.

There were no adverse test substance-related effects on reproductive outcomes or on offspring at any exposure concentration tested in either the P1 or F1 generations. The data for mating, fertility, precoital interval length, gestation length, and implantation site counts were comparable across all groups tested for each respective generation. Additionally, there were no adverse, treatment-related effects noted on pup survival indices, estrous parameters, or sperm parameters at any concentration for either generation.

Therefore, the no-observed-adverse-effect-levels (NOAELs) for systemic, offspring, and reproductive toxicity were 500, 1500, and 3000 ppm, respectively.