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Administrative data

Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
GLP and guideline study.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information
Short description of key information:
No adverse effects on spermatogenic endpoints (testicular and epididymal sperm numbers, sperm production rate, motility, and the percentage of morphological normal sperm) could be observed in a subchronic 90- day oral toxicity study. Also in females no effect on the duration of the estrous cycle and no histopathology changes in ovary and other reproductive tissues could be observed (see section 7.5.1).

Justification for selection of Effect on fertility via oral route:
In a oral repeated dose toxicity study spermatogenic endpoints were evaluated. Also in females estrous cycle was monitored.

Effects on developmental toxicity

Description of key information
No fetal malformations or developmental variations were attributed to the test item. In addition no test item- related clinical findings or effects on maternal body weight, body weight gains, or food consumption were observed at any dosage level.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 July 2011 to 14 June 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
other: Crl:CD(SD)
Details on test animals and environmental conditions:
- ANIMAL HOUSING
Upon arrival and until pairing, all rats were individually housed in clean, stainless steelwire-mesh cages suspended above cage-board. The cage-board was changed at least 3 times per week. The rats were paired for mating in the home cage of the male. Following positive evidence of mating, the females were returned to individual suspended wire-mesh cages; nesting material was not required as the females were euthanized prior to the date of expected parturition. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). The animal facilities at WIL Research are fully accredited by AAALAC International.

- DIET, DRINKING WATER, AND MAINTENANCE
The basal diet used in this study, PMI Nutrition International, LLC Certified Rodent LabDiet® 5002, was a certified feed with appropriate analyses performed by the manufacturer and provided to WIL Research. Feed lots used during the study were documented in the study records. The feeders were changed and sanitized once per week. Municipal water supplying the facility was regularly sampled for contaminants according to WIL Research’s SOPs. The results of the diet and water analyses are maintained at WIL Research. No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of this study. Reverse osmosis-purified (on-site) drinking water, delivered by an automatic watering system, and the basal diet were provided ad libitum throughout the acclimation period and during the study.

- ENVIRONMENTAL CONDITIONS
All rats were housed throughout the acclimation period and during the study in an environmentally controlled room. The room temperature and humidity controls were set to maintain environmental conditions of 71°F ± 5°F (22°C ± 3°C) and 50% ± 20%, respectively. Room temperature and relative humidity data were monitored continuously and were scheduled for automatic collection on an hourly basis. Actual mean daily temperature ranged from 69.9°F to
70.5°F (21.1°C to 21.4°C) and mean daily relative humidity ranged from 43.0% to 55.3% during the study. Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. The light status (on or off) was recorded once every 15 minutes. The 12-hour light/12-hour dark photoperiod was interrupted as necessary to allow for the performance of scheduled maintenance. Air handling units were set to provide a minimum of 10 fresh air changes per hour.
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
The vehicle and test item formulations were administered orally by gavage, via an appropriately sized flexible, Teflon®-shafted, stainless steel ball-tipped dosing cannula (Natume, Japan), once daily during gestation days 6-19. The dosage volume for all groups was 10 mL/kg. Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose. All animals were dosed at approximately the same time each day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability of the test item (concentration range between 50 and 100 mg/mL) in deionized water was demonstrated to be stable for at least 10 days of refrigerated (approximately 4°C) storage. Prior to the initiation of dose administration, samples for batch homogeneity and resuspension homogeneity determination were collected from the top, middle, and bottom strata of the 25 and 100 mg/mL non-dosing test item batch formulations. Aliquots approximately equivalent in volume to a daily dispensation aliquot were then transferred from the pre-initiation batch into the same type of storage container that was used for the daily aliquots for dosing and stored refrigerated (approximately 4°C) for 10 days. Following the storage period, each aliquot was resuspended for a minimum of 30 minutes using a magnetic stirrer, and samples were collected from the top and bottom strata of each aliquot for assessment of aliquot resuspension homogeneity and stability. Samples for concentration analysis were collected from the middle of each dosing formulation during the in-life phase of the study; however, only the samples collected from the first and last dosing formulations were analyzed. One set of samples from each collection was subjected to the appropriate analyses. The remaining set of samples was stored refrigerated (approximately 4°C) as back-up. All analyses were conducted by the WIL Research Analytical Chemistry Department using a validated gas chromatography method using flame ionization detection.
Details on mating procedure:
At the conclusion of the acclimation period, all available females were weighed and examined in detail for physical abnormalities. At the discretion of the Study Director, each animal judged to be in good health and meeting acceptable body weight requirements was placed in a suspended wire-mesh cage with a resident male from the same strain and source for breeding. Resident males were untreated, sexually mature rats utilized exclusively for breeding. These rats were maintained under similar laboratory conditions as the females. A breeding record containing the male and female identification numbers and the dates of cohabitation was maintained. The selected females were approximately 13 weeks old when paired for breeding.

Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage and verified by a second biologist. Each mating pair was examined daily. The day on which evidence of mating was identified was termed gestation day 0 and the animals were separated.
Duration of treatment / exposure:
Day 6 to day 19 of gestation
Frequency of treatment:
1 per day
Duration of test:
Mating (day 0) to gestation day 20.
Remarks:
Doses / Concentrations:
0 mg/kg bw/day
Basis:
other: gavage
Remarks:
Doses / Concentrations:
250 mg/kg bw/day
Basis:
other: gavage
Remarks:
Doses / Concentrations:
500 mg/kg bw/day
Basis:
other: gavage
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
other: gavage
No. of animals per sex per dose:
25 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosage levels were determined from results of a previous dose range-finding study and that 1000 mg/kg/day is the usual limit dose for developmental toxicity studies (OECD Guideline 414). In the range-finding study, female Crl:CD(SD) rats (6/group) were administered the test item at dosage levels of 500, 750, and 1000 mg/kg/day once daily during gestation days 6 though 19. There was no evidence of maternal or embryo/fetal developmental toxicity at any dosage level. Based on the results of that study and consultation with the Sponsor Representative, dosage levels of 250, 500, and 1000 mg/kg/day were chosen to be used in the current study.

- Rationale for animal assignment: The experimental design consisted of 3 test item-treated groups and 1 control group, composed of 25 rats per group. The bred females were assigned to groups using a WTDMS™ computer program which randomized the animals based on stratification of the gestation day 0 body weights in a block design.
Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: from gestation day 0 to day 20 (prior to dose administration during the treatment period).

BODY WEIGHT and GRAVID UTERINE WEIGHT: Yes
- Time schedule for examinations: Individual maternal body weights were recorded on gestation days 0 and 6-20 (daily). Gravid uterine weight was collected and net body weight (the gestation day 20 body weight exclusive of the weight of the uterus and contents) and net body weight change (the gestation day 0-20 body weight change exclusive of the weight of the uterus and contents) were calculated.

FOOD CONSUMPTION: Yes
- Time schedule: gestation days 0 and 6-20 (daily).
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all viable per litter
- Soft tissue examinations: Yes: all viable per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test item-treated group to the control group. Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means and standard deviations on the summary and individual tables may differ slightly. Therefore, the use of reported individual values to calculate subsequent parameters or means will, in some instances, yield minor variations from those listed in the report data tables. Where applicable, the litter was used as the experimental unit.

Mean maternal body weights (absolute and net), body weight changes (absolute and net), food consumption, gravid uterine weights, numbers of corpora lutea, implantation sites, viable fetuses, and fetal body weights (separately by sex and combined) were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test was used to compare the test item-treated groups to the control group. Mean litter proportions (percent per litter) of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and fetal sex distribution), total fetal malformations and developmental variations (external, visceral, skeletal, and combined) and each particular external, visceral, and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test was used to compare the test item-treated groups to the control group.
Indices:
Group Mean Litter Basis:
Postimplantation Loss/ Litter = #dead fetuses,resorptions(early,late) per group / # gravid females per group

Proportional Litter Basis:
Summation per group (%) = Sum of Postimplantation Loss per Litter (%) / # Litters per Group
Postimplantation Loss per Litter (%) = (#dead fetuses,resorptions(early,late) per litter / # Implantation Sites per Litter) x 100
Historical control data:
Yes, for laparohyserectomy, fetal morphological data and external, visceral and skeletal malformations and variations.
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
- All females in the control, 250, 500, and 1000 mg/kg/day groups survived to the scheduled necropsy on gestation day 20. No test item-related clinical findings were noted at the daily examinations or 1-2 hours following dose administration at any dosage level.
- Maternal body weight, gravid uterine weights and maternal food consumption were not affected by any test item dose.
- At the scheduled necropsy on gestation day 20, no test item-related internal findings were observed at dosage levels of 250, 500, and 1000 mg/kg/day.
- Intrauterine growth and survival were unaffected by test item administration at dosage levels of 250, 500, and 1000 mg/kg/day.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
EXTERNAL MALFORMATIONS AND VARIATIONS:
No test item-related external malformations were noted at any dosage level. External malformations were noted for 4(2), 2(2), 1(1), and 0(0) fetuses (litters) in the control, 250, 500, and 1000 mg/kg/day groups, respectively. Because these findings occurred in single fetuses, in a non-dose-dependent manner, and/or the mean litter proportions were not statistically significantly different from the concurrent control group and within the ranges of the WIL historical control data, they were not considered to be related to test item administration. No external developmental variations were noted at any dosage level.

VISCERAL MALFORMATIONS AND VARIATIONS:
No test item-related visceral malformations were noted at any dosage level. A visceral malformation was noted in 1 fetus in the control group. No test item-related visceral developmental variations were noted at any dosage level. Low incidences of a variety of visceral developmental variations were noted in the control and test item-treated groups. These developmental variations occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related. Additionally the mean litter proportions of these findings were not statistically significantly different from the control group and/or were within the ranges of the historical control data, and were therefore not considered to be test item-related.

SKELETAL MALFORMATIONS AND VARIATIONS:
Skeletal malformations were observed in 1(1), 0(0), 2(2), and 0(0) fetuses (litters) in the control, 250, 500 and 1000 mg/kg/day groups, respectively. These findings occurred similarly in the control group and/or in a manner that was not dose-related and the mean litter proportions were not statistically significantly different from the concurrent control group and were within the ranges of the WIL historical control data, and were therefore not considered to be test substance-related. No test substance-related skeletal developmental variations were noted at any dosage level.
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
Based on the results of this study, a dosage level of 1000 mg/kg/day, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and embryo/fetal development when the test item was administered orally by gavage to bred Crl:CD(SD) rats.
Executive summary:
Objective:

The objective of the study was to determine the potential of the test item 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 study was conducted in compliance with GLP regulations and in accordance with regulatory guideline OECD 414.

Study design:

The test item, in the vehicle (deionized water), was administered orally by gavage to 3 groups of 25 bred female Crl:CD(SD) rats once daily from gestation days 6 through 19. Dosage levels were 250, 500, and 1000 mg/kg/day administered at a dosage volume of 10 mL/kg. Dosages were selected following a range-finding study in which systemic exposure was demonstrated in the pregnant rat. A concurrent control group composed of 25 bred females received the vehicle on a comparable regimen. The females were approximately 14 weeks of age at the initiation of dose administration. 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 20, a laparohysterectomy was performed on each 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. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

Results:

The analyzed dosing formulations were homogeneous, and were stable after 10 days of refrigerated storage. All females survived to the scheduled necropsy on gestation day 20. There were no test item-related clinical observations noted at any dosage level. Additionally, there were no test item-related maternal macroscopic findings noted at the scheduled necropsy. There were no test item-related effects on body weights, body weight gains, net body weights, net body weight gains, or food consumption at any dosage level tested. Based on the parameters evaluated, including postimplantation loss, litter size, mean fetal body weights, and fetal sex ratios, intrauterine growth and survival were unaffected by test item administration at all dosage levels tested. There were no test item-related external, visceral, or skeletal malformations or developmental variations observed at any dosage level tested.

Conclusion:

There were no test item-related clinical findings or effects on maternal body weight, body weight gains, or food consumption observed at any dosage level. In addition, there were no test item-related effects on embryo/fetal development at any dosage level. Based on the results of this study, a dosage level of 1000 mg/kg/day, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and embryo/fetal development when the test item was administered orally by gavage to bred Crl:CD(SD) rats.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Species:
rat
Quality of whole database:
GLP and guideline study
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
A screening study was conducted according to OECD TG 414 developmental toxicity screening test. The test item was administered to 6 female rats of strainCrl:CD(SD) per dose by oral gavage at dose level of 0, 500; 750 or 1000 mg/kg bw/day administered at a dosage volume of 10 mL/kg. A concurrent control group composed of 6 bred females received the vehicle (water) on a comparable regimen. On gestation day 20, a laparohysterectomy was performed on each 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. There were no maternal or embryo/fetal toxicity at dosage levels up to 1000 mg/kg bw/day, which is the limit dosage within the OECD 414 guideline. Serum data provided evidence of dose dependent test item absorption, rapid metabolism to two main metabolites and efficient elimination with no expectation of bioaccumulation with time.The dosage of 1000 mg/kg bw/day was selected as the highest dosage for a definitive prenatal developmental toxicity study administered orally by gavage. This study is regarded as acceptable range-finding study and satisfies the guideline requirements. A study was conducted according to OECD TG 414. The objective of the study was to determine the potential of the test item 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 item, in the vehicle (deionized water), was administered orally by gavage to 3 groups of 25 bred female Crl:CD(SD) rats once daily from gestation days 6 through 19. Dosage levels were 250, 500, and 1000 mg/kg/day administered at a dosage volume of 10 mL/kg. Dosages were selected following a range-finding study in which systemic exposure was demonstrated in the pregnant rat. A concurrent control group composed of 25 bred females received the vehicle on a comparable regimen. The females were approximately 14 weeks of age at the initiation of dose administration. 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 20, a laparohysterectomy was performed on each 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. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations. The analyzed dosing formulations were homogeneous, and were stable after 10 days of refrigerated storage. All females survived to the scheduled necropsy on gestation day 20. There were no test item-related clinical observations noted at any dosage level. Additionally, there were no test item-related maternal macroscopic findings noted at the scheduled necropsy. There were no test item-related effects on body weights, body weight gains, net body weights, net body weight gains, or food consumption at any dosage level tested. Based on the parameters evaluated, including postimplantation loss, litter size, mean fetal body weights, and fetal sex ratios, intrauterine growth and survival were unaffected by test item administration at all dosage levels tested. There were no test item-related external, visceral, or skeletal malformations or developmental variations observed at any dosage level tested. There were no test item-related clinical findings or effects on maternal body weight, body weight gains, or food consumption observed at any dosage level. In addition, there were no test item-related effects on embryo/fetal development at any dosage level. Based on the results of this study, a dosage level of 1000 mg/kg/day, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and embryo/fetal development when the test item was administered orally by gavage to bred Crl:CD(SD) rats.

Justification for selection of Effect on developmental toxicity: via oral route:
Only one study available.

Justification for classification or non-classification

Based on the results of the developmental toxicity/ teratogenicity study and the 90-days- oral repeated dose toxicity study (NOAEL = 1000 mg/kg bw/ d) the test item is not classified as reproductive toxicant according to EU Directive 67/548/EEC or EU Regulation (EC) No 1272/2008.