Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

1-Generation Rat Diet: Not a reproductive toxin. No guideline specified; Reliability = 1

2-Generation Rat Diet: Not a reproductive toxin. OECD 416; Reliability = 1

Link to relevant study records

Referenceopen allclose all

Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
A one-generation reproduction study was conducted with test substance in which rats were fed a diet that contained the test substance at different targeted concentrations during the premating and gestation periods. During postnatal day (PND) 0 to 42 (F1 adults), which included lactation, daily dosage concentrations were reduced by 40% to achieve targeted concentration intake. Following a 28-day premating period, P1 males and females were cohoused for up to 2 weeks within their respective treatment groups to produce F1 litters. Dams were allowed to deliver and rear their offspring until weaning (postpartum day 21). F1 litters were culled to 4 pups per sex per litter (litter size permitting) on postnatal day 4 and all remaining pups were discarded without further evaluation. At weaning, F1 offspring were randomly selected for continued evaluation including the onset of puberty until postnatal day 60. Clinical observations, body weight, and food consumption were determined at specified intervals throughout the study. Litter examinations (live, dead, or missing pups, individual pup weights, and clinical observations) were determined at birth, on PND 4, and weekly during the lactation period. The age and body weight at either vaginal opening or preputial separation was recorded for the F1 generation. Gross postmortem examinations were performed on selected animals and selected organs were weighed and/or retained for histopathological examination.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
The rat was selected for this study because it is a preferred species for reproductive toxicity testing and recommended by regulatory guidelines. The Crl:CD(SD) strain was chosen because extensive background data are available from the literature, the supplier, and previous studies at test facility. This strain is also considered suitable relative to longevity, hardiness, and incidence of spontaneous disease.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories International, Inc., Kingston, New York
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: Approximately 57 days
- Weight at study initiation: (P1) Males: 260.5 to 291.5 g; Females: 184.6 to 221.6 g
- Housing: All male and female rats were singly housed in solid-bottom caging with bedding containing species-appropriate enrichment. Enrichment was omitted for up to one week before and after expected deliveries. Each cage rack contained only animals of one sex except during cohabitation when the animals were housed as breeding pairs (female in male’s cage). Fourteen days after the first day of cohabitation, females without evidence of copulation were housed singly. During lactation periods, adult females were housed with their litters.
- Diet: PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002 ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-26ºC
- Humidity: 30-70%
- Photoperiod: 12-hour light/dark cycle
Route of administration:
oral: feed
Vehicle:
other: Diet
Details on exposure:
Test substance was added to the diet and thoroughly mixed for a period of time that was adequate to ensure homogeneous distribution in the diet. Control diets were mixed for the same period of time. Diets were prepared biweekly and stored refrigerated until used. Homogeneity of the diets was verified by measuring the concentrations of test substance in diet samples collected from the top, middle, and bottom regions of the diets. The average of homogeneity samples was used to verify concentration at study start. The stability of the samples is done by storing the samples at room temperature for up to 22 days. The measured concentration for the stability samples was compared with the mean measured values of the top, middle, and bottom homogeneity samples. At regular intervals during the study, samples from each dietary level were taken and used to verify concentration.

DIET PREPARATION
- preparation of diet: Test substance was added to the diet and thoroughly mixed for a period of time that was adequate to ensure homogeneous distribution in the diet. Control diets were mixed for the same period of time. Diets were prepared biweekly and stored refrigerated until used.
- Mixing appropriate amounts with: PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002
- Storage temperature of food: frozen in refrigerator
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Until a period of 2 weeks
- Proof of pregnancy: Intravaginal copulation plug or sperm in the vaginal lavage sample referred to as day 0 of pregnancy
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Diet samples containing test substance at concentrations of 400, 1500 and 6000 ppm formulated were submitted for homogeneity, concentration verification, and stability analyses. Diet samples containing test substance at the concentrations of 240, 900, 1500, and 3600 ppm, prepared were submitted for concentration verification analysis. A 0 ppm control diet sample was included with each set of sample for analysis. Stability sample were stored in room temperature. The study samples were frozen when not in use.
Diet samples were extracted with acetonitrile (ACN) or further diluted with diluted control sample. The concentrations of test sample were determined by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection.
The results showed that the test substance was homogeneously mixed at all targeted concentrations, and stable when stored at room temperature for up to 22 days in the diet from 400 to 6000 ppm, and up to 24 days in the diet at 240 ppm.
Test substance was not detected in control samples.
Duration of treatment / exposure:
Dietary administration of the test substance for P1 animals began on test day 0 and continued until the day of sacrifice. Measured dietary administration of the test substance for F1 animals began on PND 21, and continued until the day of sacrifice.
Frequency of treatment:
Daily
Dose / conc.:
400 ppm
Dose / conc.:
1 500 ppm
Dose / conc.:
6 000 ppm
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily Animal Health Observations: at least twice daily, General Clinical Observations: at least once daily between 6:00 am and noon except on days when careful clinical observations were performed.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once before study start and at least once weekly throughout the premating, cohabitation, gestation, and lactation phases for the P1 parental rats.

BODY WEIGHT: Yes
- Time schedule for examinations: once a week.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

Sperm parameters (parental animals):
Seminal vesicles, prostate, tsetes, coagulating glanda (with fluids), testis weight, epididymis weight
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, litter sex ratio
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals survived were sacrificed after siring litters
- Maternal animals: Pregnant females were sacrificed on-day of weaning litters (Day 21 postpartum) and Non pregnant females are sacrificed with pregnant females only.

GROSS NECROPSY
- Gross necropsy consisted of external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 & 2 were prepared for microscopic examination are weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- F1 generation culled pups were sacrificed on day 4 postpartum
- F1 generation weanlings were sacrificed on day of weaning- Day 21 Postpartum. Three of 4 rats/sex/litter (when possible) postnatal day 21
- F1 adults were sacrificed on post natal day 60

GROSS NECROPSY
- Gross necropsy consisted of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.
Statistics:
- For parameters like body weight, body weight gain, food consumption, food efficiency, organ weight, gestation length, implantation site numbers, number of pups per litter, 0-4 Day viability, vaginal patency, preputial separation, mean pup weights, the preliminary test conducted was Levene’s test for homogeneity and Shapiro-Wilk test for normality. The method of statistical analysis if the preliminary test is significant is transformation of the data to achieve normality and variance homogeneity will be used. The order of transforms attempted will be log, square-root, and rank-order. If the log and square-root transforms fail, the rank-order will be used. The method of statistical analysis if the preliminary test is not significant is One-way analysis of variance followed by Dunnett's test.

- For parameters like percent born alive, sex ratio, lactation index, viability index, postimplantation loss, the statistical analysis was done by transforming data as arcsin square-root of percent/100, then one-way analysis of variance followed by Dunnett's test.

- For parameters like mating index, fertility index, gestation index, litter survival, the statistical analysis was done by fisher’s Exact test with Bonferroni-Holm adjustment for the number of comparisons to the control.

- Significance was judged at p <0.05. Separate analyses was performed on the data collected for each sex. For litter parameters, the proportion of affected fetuses per litter or the litter mean was used as the experimental unit for statistical evaluation
Reproductive indices:
The following lists the 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
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- One female from control group was sacrificed on test day 54 due to moderate acute inflammation of the uterus and placenta due to dystocia. one animal in the 6000 ppm female group was sacrificed on test day 60 due to death of litter.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- At 6000 ppm in P1 males, there were test substance-related and adverse reductions in body weight parameters. Mean body weight on test day 28 was 11% lower than the control and mean final body weight on day 67 was 12% lower than the control group mean. At 400 and 1500 ppm test substance related effects were limited to transient reductions in weight gain during the first week of the study. These reductions were not considered adverse because there was no adverse impact on mean body weight at these concentrations. Mean body weights at these concentrations were within 3% of the control group means throughout the study and mean cumulative body weight gains from days 0-28 and days 0-67 were 30 and 26% lower than controls, respectively. Mean body weight on test day 28 was 11% lower than the control and mean final body weight on day 67 was 12% lower than the control group mean. Test substance-related effects on body weight parameters at 400 and 1500 ppm were limited to transient reductions in weight gain during the first week of the study. These reductions were not considered adverse because there was no adverse impact on mean body weight at these concentrations. Mean body weights at these concentrations were within 3% of the control group means throughout the study.

- At 6000 ppm in P1 females, there were test substance-related and adverse reductions in body weight parameters. During the first week of the study, there was a mean body weight loss of 1.8 grams compared with a gain of 24.3 grams in the control group. Mean body weight gain during the premating period, days 0-28, was 37% lower than controls resulting in a mean body weight that was 9% lower than control on day 28. The effects on body weight persisted during gestation during which the mean cumulative weight gain was 19% lower than for the control resulting in a mean body weight on gestation day 20 that was 17% lower than the control group mean. Mean body weight at the end of lactation was 12% lower than the control group mean. Test substance-related effects on body weight parameters at 400 and 1500 ppm were limited to transient reductions in gain during the first week of the study. These reductions were not considered adverse because there was no adverse impact on mean body weight at these concentrations. Mean body weights at these concentrations were within 4% of the control group means throughout the study.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- At 6000 ppm in both males and females, test substance-related and adverse reductions in mean food consumption were evident beginning during the first week of the study and persisted throughout the 28-day premating period. Mean food consumption from days 0-28 was 18% and 22% was lower than for the control group in males and females respectively. During gestation, mean food consumption was 25% lower than for the control group and during lactation, mean food consumption was 17% lower than control.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
- In P1 males, at 6000 ppm, mean food efficiency was also reduced and was 14% lower than for the control group.

- In P1 females, at 6000 ppm, there is slight increase in mean food efficiency during lactation as a reflection of the smaller than typical loss of body weight during lactation.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- In P1 adults, test substance-related centrilobular hepatocellular hypertrophy was present in all treated male groups and equivocally in females fed 6000 ppm. In males, incidences were 0/10, 4/10, 9/10, and 10/10 in the 0, 400, 1500, and 6000 ppm groups, respectively. In females, incidences were 1/10, 1/10, 1/10, and 3/10, respectively. Hypertrophy was graded as minimal to mild in the 6000 ppm males and minimal in all other affected groups.

- In the liver, minimal vacuolation of periportal hepatocytes was present in 5/10 male rats in the 6000 ppm group but was not observed in other male or female groups.

- Mucification of vaginal epithelium was present at a minimal to mild level in animals from all female groups. Incidences were 5/8, 6/7, 3/8, and 7/8 in the 0, 400, 1500, and 6000 ppm groups, respectively. All groups had changes varying in severity from minimal to mild, with the exception of the 6000 ppm group, in which all animals had mild changes.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
- At 6000 ppm, there were test substance-related and adverse effects on mean number of implantation sites. The mean number of implants was 12.3 compared with a control group mean of 16.1. This reduction in implantation sites resulted in smaller litter sizes at birth. However, the mean litter size was proportionally accurate therefore, there were no test substance-related post-implantation losses.

- Adverse test substance-related effects at 6000 ppm included reduced pup weights at birth. The reduction at birth persisted and increased in magnitude throughout lactation. Mean pup weights were 12, 18, 20, 22, and 27% lower on PND 0, 4, 7, 14, and 21, respectively.
Key result
Dose descriptor:
NOAEL
Effect level:
1 500 ppm
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
food efficiency
other: Reduction in the number of implantation sites in P1 females at 6000 ppm
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- In F1 males, at 6000 ppm, there were adverse test substance-related reductions in body weight parameters. Mean cumulative weight gain from days 0-39 (corresponding to PND 21-60) was 23% lower than for the control resulting in a mean final weight that was 23% lower than the control group mean. There were no test substance-related effects on body weight parameters at 400 or 1500 ppm.

- In F1 females, at 6000 ppm, there were adverse test substance-related reductions in body weight parameters. Mean cumulative weight gain from days 0-39 (corresponding to PND 21-60) was 19% lower than for the control resulting in a mean final weight that was 20% lower than the control group mean. There were no test substance-related effects on body weight parameters at 400 or 1500 ppm.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- At 6000 ppm in F1 males, test substance-related and adverse reductions in mean food consumption were observed. Mean food consumption from test days 0-39 (corresponding to PND 21 to 60) was 22% lower than control.

- At 6000 ppm in F1 females, test substance-related and adverse reductions in mean food consumption were observed. Mean food consumption from test days 0-39 (corresponding to PND 21 to 60) was 23% lower than control.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
- At 6000 ppm in both males and fenales, mean food efficiency was not similarly reduced reflecting that the lower food consumption was consistent with the reduced body weights at this concentration.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- In F1 weanlings (PND 21), Statistically significant organ weight changes were observed in the brain, spleen and thymus in the 6000 ppm male and female F1 weanling groups. Interpretation of these changes is complicated by the marked decrements in nutritional/body weight parameters present in these groups during their critical growth phase (terminal body weights were decreased by 29% and 24% relative to controls in males and females, respectively). As such, the pattern of statistically significant changes in organ weight parameters in these groups included decreases in some absolute organ weights (such as brain) that are generally body weight stable in more mature animals.

- In F1 adults (PND 60), Liver weight relative to body weight was increased in the 6000 ppm male and female groups (22% and 20% greater than controls, respectively). Statistically significant organ weight changes were observed in several organs in the 6000 ppm male and female F1 groups. Interpretation of these changes is complicated by the marked decrements in nutritional/body weight parameters present in these groups during their critical growth phase (terminal body weights were decreased by 23% and 20% relative to controls in males and females, respectively). As such, the pattern of statistically significant changes in organ weight parameters in these groups included decreases in some absolute organ weights (such as brain) that are generally body weight stable in more mature animals.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- All gross observations were consistent with incidental findings common to rats of this strain and age and were unrelated to test substance exposure
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
In F1 adults, minimal atrophy of epididymal tubules was present in 4/9 male rats in the 6000 ppm group. This change was most apparent in the cauda of the epididymides and was not associated with changes in epididymal sperm density or with testicular changes. Thus, these changes are likely secondary to the body weight decrements and growth retardation that occurred at this dietary concentration and not indicative of primary target organ effects.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Developmental Landmarks: At 6000 ppm, there was an apparent delay in the time to preputial separation in F1 males. Mean day of achievement of criterion was 49.0 compared with 43.9 in the control group. For perspective, the test facility historical control mean day of achievement is 43.2 and ranges from 39.9 to 48.8. Delays in preputial separation have been shown to occur as a consequence secondary to reductions in body weight. There were no test substance-related changes in the onset of puberty in F1 females.

- Additional adverse test substance-related effects at 6000 ppm included reduced pup weights at birth. The reduction at birth persisted and increased in magnitude throughout lactation. Mean pup weights were 12, 18, 20, 22, and 27% lower on PND 0, 4, 7, 14, and 21, respectively.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 500 ppm
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
food efficiency
other: Delay in the onset of puberty in F1 males
Key result
Reproductive effects observed:
no
Conclusions:
NOAEL (P1): 1500 ppm (Body weight and food consumption parameters)
NOAEL (F1): 1500 ppm (body weight and food consumption parameters and number of implantation sites)
Executive summary:

A one-generation reproduction study was conducted with test substance in which Crl:CD(SD) rats (10/sex/dietary concentration) were fed a diet of PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002 that contained the test substance at targeted concentrations of 0, 400, 1500, or 6000 ppm during the premating and gestation periods. During postnatal day (PND) 0 to 42 (F1 adults), which included lactation, daily dosage concentrations were reduced by 40% to 0, 240, 900, or 3600 ppm, respectively, to achieve targeted concentration intake. The test diets were sampled and analyzed during the course of the study and were confirmed to be at targeted concentrations, homogeneously mixed, and stable under the conditions of the study.

Following a 28-day premating period, P1 males and females were cohoused for up to 2 weeks within their respective treatment groups to produce F1 litters. Dams were allowed to deliver and rear their offspring until weaning (postpartum Day 21). F1litters were culled to 4 pups per sex per litter (litter size permitting) on postnatal Day 4 and all remaining pups were discarded without further evaluation. At weaning, F1 offspring were randomly selected for continued evaluation including the onset of puberty until postnatal Day 60.

Clinical observations, body weight, and food consumption were determined at specified intervals throughout the study. Litter examinations (live, dead, or missing pups, individual pup weights, and clinical observations) were determined at birth, on PND 4, and weekly during the lactation period. The age and body weight at either vaginal opening or preputial separation was recorded for the F1 generation.

Gross postmortem examinations were performed on selected animals and selected organs were weighed and/or retained for histopathological examination. 

There was no test substance-related mortality and there were no test substance-related clinical observations at any dietary concentration tested.

At 6000 ppm, adverse test substance-related effects included significant reductions in body weight and food consumption parameters for P1 males and females. For P1 males, mean final body weight was 12% lower than the control mean. For P1 females, mean final body weight at the end of the premating period was 9% lower than the control mean; mean final weight on day 21 of lactation was 21% lower than for controls. During the 28-day premating period, mean food consumption was 18 and 22% lower than for controls for P1 males and females, respectively.

Regarding adverse effects on reproductive performance and offspring at 6000 ppm, test substance-related reductions in mean litter size at birth were observed; mean litter size was 11.9 compared with 15.3 in controls. However, this reduction resulted from significantly fewer implantation sites; mean number of implants was 12.3 compared with 16.1 for controls. Mean pup weights were lower at birth and the reduction in weight persisted and increased in magnitude during lactation. On PND 0, 4, 7, 14, and 21, reduction in respective mean pup weight was 12, 18, 20, 22, and 27% lower than for controls. Otherwise, there were no effects on mating index, fertility index, live born index, 0-4 viability index, or lactation index. There were no effects on pre-coital interval, gestation length, post-implantation losses, or sex ratio. There were no test substance-related clinical observations in pups.

At 6000 ppm, effects in F1 males and females that were selected for further evaluation after weaning included marked reductions in body weight and food consumption parameters. Mean final body weights were 23 and 20% lower than controls for males and females, respectively. Mean food consumption values were 22 and 23% lower than for controls during the post-weaning period, respectively. The time to preputial separation in F1 males was slightly delayed and the mean day of achievement was 49.0 compared with 43.9 in controls. The relevant historical control data mean is 43.2 and ranges from 39.9 to 48.8 days. This apparent delay in pubertal onset is considered secondary to the marked body weight effects at this dietary concentration. 

At 400 and 1500 ppm in P1 males and females, effects during the in-life phase were limited to transient reductions in body weight gain during the first week of the study that were recovered during the second week of the study. These reductions were not considered to be adverse. There were no test substance-related effects on reproductive performance or offspring at either of these levels. 

With regard to organ weights, and gross and microscopic pathologic evaluations, test substance-related findings were observed that were considered to be either secondary to body weight effects and/or non-adverse. Liver weight parameters were increased in all treated male groups and in the 6000 ppm female groups, and were associated with microscopic centrilobular hepatocellular hypertrophy. These liver changes were likely secondary to induction of liver metabolizing enzymes, and were considered nonadverse. Also observed microscopically in the liver of 6000 ppm P1 males was minimal vacuolation of periportal hepatocytes, possibly due to mobilization of fat stores due to reduced food consumption, and also considered nonadverse. Increased thyroid weight parameters in the 6000 ppm male group were not associated with correlative histological changes and were also considered nonadverse. Changes in other organ weight parameters and microscopic observations within the reproductive tract in the 6000 ppm P1 females occurred secondary to decrements in body weights in these groups. In F1 offspring potentially exposed to the test substance in utero and during lactation, and in the diet through PND 60, effects were limited to increases in liver weight parameters in the 6000 ppm male and female groups with weight changes in other organs and microscopic observations in the epididymides occurring secondary to body weight decrements in these groups.

In conclusion, test substance-related effects that were considered to be adverse occurred at 6000 ppm and included reductions in body weight and food consumption parameters in P1 and F1 males and females, and a reduction in the number of implantation sites in P1 females. Therefore, the no-observed-adverse-effect level (NOAEL) was considered to be 1500 ppm. Under the conditions of the current study, the test substance was not demonstrated to be a selective reproductive toxicant.

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
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
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
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
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
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
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: oestrous cycle:
effects observed, non-treatment-related
Reproductive function: sperm measures:
effects observed, non-treatment-related
Reproductive performance:
effects observed, non-treatment-related
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
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.
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
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
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
Reproductive effects observed:
no
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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

A one-generation reproduction study was conducted with test substance in which Crl:CD(SD) rats (10/sex/dietary concentration) were fed a diet that contained the test substance at targeted concentrations of 0, 400, 1500, or 6000 ppm during the premating and gestation periods. During postnatal day (PND) 0 to 42 (F1 adults), which included lactation, daily dosage concentrations were reduced by 40% to 0, 240, 900, or 3600 ppm, respectively, to achieve targeted concentration intake. Test substance-related effects that were considered to be adverse occurred at 6000 ppm and included reductions in body weight and food consumption parameters in P1 and F1 males and females, and a reduction in the number of implantation sites in P1 females. Therefore, the no-observed-adverse-effect level (NOAEL) was considered to be 1500 ppm. Under the conditions of the current study, the test substance was not demonstrated to be a selective reproductive toxicant.

 

In the two-generation rat reproduction toxicity study, the test substance was administered in the diet to male and female rats at concentrations of 0, 100, 500, 1500, and 3000 ppm. The NOAEL for parental toxicity was 500 ppm based on reductions in body weight and nutritional parameters in P1 and F1 males and females at 1500 ppm and above. There were no test substance-related deaths or clinical observations, and no adverse effects on gross observations, organ weights, or microscopic alterations in P1 or F1 parental rats. The NOAEL for reproductive and fertility effects was 3000 ppm based on a lack of adverse, test substance-related effects in either the P1 or F1 generation at 3000 ppm, the highest dose tested. The data for mating, fertility, precoital interval length, gestation length, and implantation site counts were comparable across all groups for both generations. Additionally, there were no adverse, treatment-related effects noted on pup survival indices, estrous parameters, or sperm parameters at any concentration for either generation. The NOAEL for effects on pup growth and development was 1500 ppm based on a reduction in the body weight of F1 and F2 pups from birth and throughout lactation at 3000 ppm. A slight delay in preputial separation observed at 3000 ppm was considered secondary to the effects on body weight.

Effects on developmental toxicity

Description of key information

Rat Developmental Diet: Not a unique developmental toxin. OECD 414; Reliability = 1

Rabbit Developmental Diet: Not a unique developmental toxin. OECD 414; Reliability = 1

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
other: MAFF Japan Test Guidelines for Agricultural Chemicals 12-Nousan-8147 (2000)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Route of administration:
oral: gavage
Vehicle:
other: 0.5% methylcellulose with 0.1% Tween 80
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
GD 6-20
Frequency of treatment:
Once daily
Duration of test:
15 days
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
No. of animals per sex per dose:
20 females/dose
Control animals:
yes, concurrent vehicle
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):
Mean maternal body weights were statistically significantly reduced (4-5%) at 200 mg/kg/day from gestation days 18 through 21, compared to the control group. Overall body weight gains were 15% lower (statistically significant) than control group means and corresponded to reduced weight gains observed during the treatment period. These reductions were most pronounced during the first two days of dosing when the reduction was 77% lower than control group. At this same level, adjusted (minus products of conception) final body weight and overall body weight gains were 3% (not statistically significant) and 27% lower (statistically significant) than control group, respectively.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 200 mg/kg/day, mean maternal food consumption was lower than control group during treatment resulting in an 11% overall (days 6-21G) reduction (statistically significant) in food consumption compared to the control group.
Gross pathological findings:
effects observed, non-treatment-related
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
External malformations:
effects observed, non-treatment-related
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
There was a statistically significant increased incidence of short (primarily 13th) ribs at 100 and 200 mg/kg/day. The increased incidence of short ribs was not considered adverse since there was no correlative vertebral findings (i.e vertebral ossification delays) observed at this level and delays in ossification of the 13th rib are expected to resolve postnatally.
Visceral malformations:
effects observed, non-treatment-related
Key result
Dose descriptor:
NOAEL
Remarks:
Developmental toxicity
Effect level:
> 200 mg/kg bw/day (nominal)
Sex:
male/female
Remarks on result:
other: Highest concentration tested
Key result
Developmental effects observed:
no
Conclusions:
NOAEL (Maternal toxicity): 100 mg/kg bw/d (Body weight and food consumption)
NOAEL (Developmental toxicity): >200 mg/kg bw/d (Highest concentration tested)
Executive summary:

The study was conducted according to guidelines, U.S. EPA OPPTS 870.3700 and OECD 414 to evaluate the potential maternal and developmental toxicity of the test substance in pregnant rats. Formulations of the test substance in 0.5% methylcellulose with Tween 80 were administered to presumed pregnant rats once daily by gavage on gestation days (GD) 6-20. The day of mating was defined as gestation day 0.

The dose levels used in the current study were 0, 25, 50, 100, and 200 mg/kg/day; control group animals were administered the vehicle. The dose volume was 10 mL/kg for all groups. Samples of the dosing formulations were collected and analyzed near the beginning and end of the dosing period. The results of these analyses confirmed that the formulations were at targeted concentrations, uniformly mixed, and stable under the experimental conditions used during the study.

During the in-life portion of the study, body weights, food consumption, and clinical observations before and after dosing were collected on a daily basis. All dams were euthanized on GD 21 and the gross necropsy included an examination and description of uterine contents including counts of corpora lutea, implantation sites, resorptions, and live and dead fetuses. All live fetuses were examined externally and euthanized; following euthanasia, fresh visceral and head examinations were performed on selected fetuses. The fetal carcasses were then processed and examined for skeletal alterations.

Under the conditions of this study, maternal toxicity was observed at 200 mg/kg/day as evidenced by treatment-related adverse effects on body weight and food consumption parameters when compared with current control group values. There was no early mortality or adverse treatment-related clinical signs of toxicity at any level tested. Additionally, there were no gross pathological findings associated with treatment on this study. Intrauterine growth and survival were unaffected by maternal treatment. There were no treatment-related increases in external, visceral, or skeletal malformations. Non-adverse, treatment-related fetal findings were limited to an increased incidence of short (primarily 13th) ribs at 100 and 200 mg/kg/day. This fetal anomaly was classified as a developmental variation and was considered non-adverse, as ossification was expected to be completed shortly after birth and was, therefore, not a reflection of developmental toxicity.

Based on the results of this study, the no-observed-adverse-effect level (NOAEL) for maternal toxicity was 100 mg/kg/day based on effects on body weight and food consumption parameters at 200 mg/kg/day. The NOAEL for developmental toxicity was considered to be greater than 200 mg/kg/day.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
other: JMAFF 12 Nousan 8147 (2000)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
other: Hra:(NZW)SPF
Route of administration:
oral: gavage
Vehicle:
other: 0.5% methylcellulose (4000 centipoise [cps]) with 0.1% Tween® 80
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
gestation days 7 through 28
Frequency of treatment:
once daily
Duration of test:
Approximately one month
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
22 females/dose
Control animals:
yes, concurrent vehicle
Historical control data:
Test facility has historical control data on the background incidence of fetal malformations and developmental variations in the New Zealand White rabbit. This animal model has been proven to be susceptible to the effects of developmental toxicants.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
A test substance-related increase in the incidence of decreased defecation was noted in the 500 mg/kg/day group compared to the control group at the daily examinations beginning as early as gestation day 8.
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A significant lower mean body weight gains were noted in the 500 mg/kg/day group generally throughout the treatment period. Mean body weights in this group were not affected by these lower mean body weight gains.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean food consumption in the 500 mg/kg/day group was generally lower than the control group throughout the treatment period (gestation days 7-29). The differences were occasionally significant (p <0.05 or p <0.01). The lower mean food consumption in this group generally corresponded to decreased defecation and mean body weight losses and lower mean body weight gains.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related effects on hematology in the 500 mg/kg/day group consisted of lower mean red blood cell and eosinophil counts, hematocrit and hemoglobin levels and higher MCV and MCH levels and reticulocyte counts. The differences from the control
group were of small magnitude but generally significant (p <0.05 or p <0.01). Slightly lower mean hematocrit and hemoglobin levels in the 500 mg/kg/day group also corresponded to the lower mean red blood cell count and the differences from the control group were not statistically significant.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related higher mean spleen weights (absolute and relative to brain weight), correlating with the minimal effect on red blood cell parameters, were noted in the 500 mg/kg/day group and the differences from the control group were significant (p <0.05 or p <0.01).
Gross pathological findings:
effects observed, non-treatment-related
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
haematology
organ weights and organ / body weight ratios
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, non-treatment-related
Changes in sex ratio:
effects observed, non-treatment-related
Changes in litter size and weights:
effects observed, non-treatment-related
External malformations:
effects observed, non-treatment-related
Skeletal malformations:
effects observed, non-treatment-related
Visceral malformations:
effects observed, non-treatment-related
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Based on the lack of effects on embryo-fetal development at any dosage level
Remarks on result:
other: Highest concentration tested
Key result
Developmental effects observed:
no
Conclusions:
NOAEL (Maternal toxicity): 250 mg/kg/day (Based on clinical signs, body weight gains, food consuption, Haematology, organ weights)
NOAEL (Embryo-fetal toxicity): 500 mg/kg/day (Highest dosage level tested)
Executive summary:

The study was conducted according to guidelines, OECD 414 and U.S. EPA OPPTS 870.3700 to determine the potential of test substance to induce developmental toxicity after maternal exposure from implantation to 1 day prior to expected parturition, to characterize maternal toxicity at the exposure levels tested, and to determine a no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity.

The test substance in the vehicle was administered orally by gavage to 4 groups of 22 time-mated female New Zealand White rabbits once daily from gestation days 7 through 28. Dosage levels were 50, 100, 250, and 500 mg/kg/day administered at a dosage volume of 10 mL/kg. A concurrent control group of 22 time-mated females received the vehicle on a comparable regimen.

All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. On gestation day 29, blood was collected for haematology evaluation and a laparohysterectomy was performed on each surviving female. The uteri, placentae, and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. Selected organs/tissues from all animals were collected, weighed, and retained for possible future microscopic examination. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

One female in the 500 mg/kg/day group was euthanized in extremis on gestation day 26 due to body weight loss and markedly low food consumption; decreased defecation was also noted for this female. All other females survived to the scheduled necropsy. Lower mean body weight gains were noted in the 500 mg/kg/day group generally throughout the treatment period; mean body weights in this group were not affected by these lower mean body weight gains. Corresponding lower mean food consumption and decreased defecation were also noted in the 500 mg/kg/day group; therefore, the lower mean body weight gains were considered adverse in this group. Mean body weights, body weight gains, and food consumption in the 50, 100, and 250 mg/kg/day groups were not affected by test substance administration. Mean net body weights, body weight changes, and gravid uterine weights in the 50, 100, 250, and 500 mg/kg/day groups were similar to the control group.

Test substance-related effects on haematology in the 500 mg/kg/day group consisted of lower mean red blood cell and eosinophil counts, haematocrit and haemoglobin levels and higher MCV and MCH levels and reticulocyte counts. No test substance-related effects on haematology were noted at 50, 100, and 250 mg/kg/day.

There were no test substance-related macroscopic findings observed at any dosage level. Higher mean spleen weights (absolute and relative to brain weight) were noted in the 500 mg/kg/day group. No test substance-related effects on mean organ weights were noted at 50, 100, and 250 mg/kg/day.

Intrauterine fetal growth and survival were unaffected by test substance administration at 50, 100, 250, and 500 mg/kg/day. There were no test substance-related fetal malformations or developmental variations noted at any dosage level.

No test substance-related effects were noted at 250 mg/kg/day. Therefore, a dosage level of 250 mg/kg/day was considered the no-observed adverse- effect level (NOAEL) for maternal toxicity. Based on the lack of effects on embryo-fetal development at any dosage level, the NOAEL for embryo-fetal toxicity was considered to be 500 mg/kg/day, the highest dosage level tested.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Developmental toxicity studies were conducted with the test substance in rats and rabbits. The test substance was not teratogenic and was not uniquely toxic to the rat or rabbit conceptus.

 

In the developmental toxicity study in rats, the NOAEL for maternal toxicity was 100 mg/kg bw/day based on reduced body weight and food consumption parameters at 200 mg/kg bw/day. No effects on survival, clinical observations, or gross pathology were observed at any dose. The NOAEL for foetal toxicity was 200 mg/kg bw/day based on a lack of adverse effects at the highest dose tested. An increase in incidence of the variation short ribs was observed at 100 and 200 mg/kg bw/day, but was considered non-adverse since it was not associated with correlative vertebral findings, and would be expected to resolve post-natally. There were no test substance-related effects on reproductive outcome or quantitative litter data. The mean number of implantation sites, resorptions, live foetuses, mean foetal weight, and sex ratio were comparable across all groups tested.

 

In the developmental toxicity study in rabbits, the NOAEL for maternal toxicity was 250 mg/kg bw/day based on reduced body weight and food consumption parameters, reduced defecation, and minimal haematology changes with associated increased spleen weights, at 500 mg/kg bw/day. No effects on survival or gross pathology were observed. The NOAEL for foetal toxicity was 500 mg/kg bw/day, the highest dose tested. No adverse effects were observed on intrauterine growth and survival, post-implantation loss, live litter size, mean foetal body weights, or foetal sex ratios. No test substance-related differences in the incidence of foetal variations or malformations were observed.

Justification for classification or non-classification

There is no evidence to suggest that the test substance causes adverse effects on reproduction or development of the offspring. Therefore, the test substance is not classified for reproductive or developmental toxicity according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.