Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented and reliable GLP and guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: OPPTS 870.3550 Reproduction/Developmental toxicity screening test
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid
Details on test material:
Please refer to confidential details on test material.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Crl:WI(Han); Charles River Laboratories, Inc., Raleigh, NC, U.S.A.
- Age at arrival: 69 days
- Weight at study initiation: 258 g to 285 g for males, 177 g to 207 g for females
- Housing: Adult rats were housed 2 or 3 per sex per dose group; During cohabitation, each pair of rats was housed in the male rat's nesting box; female rats were housed individually beginning on Day 0 of presumed gestation; each dam and delivered litter was housed in a common nesting box during the postpartum period.
- Diet: Certified Rodent Diet (ad libitum)
- Water: in individual bottles (ad libitum)
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25°C
- Humidity: 30-70%
- Air changes: 10 changes per hour
- Photoperiod: 12-hours light: 12-hours dark

IN-LIFE DATES: From: 2012-06-25 To: 2012-08-16

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG 300
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared weekly, stored refrigerated (2 to 8°C) and protected from light, and stirred continuously during sample collection and dose administration.

Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days
- Proof of pregnancy: sperm in vaginal smear [referred to as day 0 of pregnancy]
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Sample collection and analysis
Dose formulation samples were collected for analysis, stored refrigerated (2°C to 8°C) and protected from light until shipment for analysis.

Concentration analysis
Duplicate sets of samples (1 mL) were collected from the middle of the control and middle concentrations on the first day of preparation and all concentrations at the midpoint and on the last day of preparation for concentration analysis and stored refrigerated (2°C to 8°C) and protected from light until shipment to the analytical laboratory. Duplicate sets of samples (1 mL) were collected from each specified concentration in the same manner on each occasion and were retained at the Testing Facility as backup samples.

Homogeneity analysis
Duplicate sets of samples (1 mL) were collected from the top, middle, and bottom of the low and high concentrations on the first day of preparation for homogeneity analysis and stored refrigerated (2°C to 8°C) and protected from light until shipment to the analytical laboratory. Duplicate sets of samples (1 mL) were collected from the low and high concentrations in the same manner on the same occasion and were retained at the Testing Facility as backup samples.
Duration of treatment / exposure:
Males: 14 days prior to cohabitation, through cohabitation and continuing through the day before euthanasia.
Female: 14 days prior to cohabitation, during cohabitation, and continuing through the postpartum period to include Day 4 of lactation (DL 4), the day before euthanasia (DL 5).
Frequency of treatment:
once a day
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 250, 700 mg/kg bw/day (20 mg/mL, 50 mg/mL, 140 mg/mL)
Basis:
actual ingested
No. of animals per sex per dose:
10 rats
Control animals:
yes, concurrent vehicle
Details on study design:
Justification of route and dose levels
The oral (gavage) route was selected for use because the exact dose can be accurately administered and to maximize systemic exposure.
Dose levels were selected for this study on the basis of a range-finding maternal toxicity study in Wistar rats.
Positive control:
not applicable

Examinations

Parental animals: Observations and examinations:
VIABILITY CHECK
Each animal was assessed for viability at least twice daily.

CLINICAL OBSERVATIONS
Clinical observations were recorded twice during the acclimation period, daily during the dose period (including DG 0 for females) and on the day of scheduled euthanasia.
Postdose observations were recorded between 1 and 2 hours of administration and at the end of the normal working day.

MATERNAL OBSERVATIONS
Maternal behavior was recorded on DLs 1 and 5. Observed abnormal behavior was recorded daily.

BODY WEIGHTS
Body weights were recorded twice during the acclimation period, daily during the dose period (including DG 0 for females) and on the day of scheduled euthanasia.

FOOD CONSUMPTION
For male rats, food consumption values were recorded twice during the acclimation period, weekly during Day 1 through 36, and twice weekly for the remainder of the dose period. A food left value was recorded on the day of scheduled euthanasia. For female rats, food consumption values were recorded twice during the acclimation period and during the premating dose period, on DGs 0, 7, 10, 14, 18, 21, and 25 (as necessary) and DLs 1 and 5. During cohabitation, when a male and a female rat occupied the same nesting box with 1 food jar, replenishment of the food jar was documented, but individual values were not recorded.

DELIVERY OBSERVATIONS
Female rats were evaluated for:
- Adverse clinical signs observed (during parturition);
- Duration of Gestation (DG 0 to the day the first pup was observed);
- Litter size (defined as all pups delivered);
- Pup viability at birth
Oestrous cyclicity (parental animals):
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Vaginal lavage samples were collected for 14 consecutive days beginning with the first day of dose administration and then until spermatozoa were observed in a smear of the vaginal contents and/or a copulatory plug was observed in situ during the cohabitation period.
Litter observations:
VIABILITY
Litters were observed for dead pups at least twice daily. The pups in each litter were counted once daily.

CLINICAL OBSERVATIONS
Clinical observations were recorded at least once daily.

BODY WEIGHTS
Body weights were recorded on Postnatal days 1 (PND 1; birth) and 5.
Postmortem examinations (parental animals):
METHOD OF EUTHANASIA
P generation rats were euthanized by carbon dioxide asphyxiation.

SCHEDULED EUTHANASIA
MALE RATS
On Day 47 (after completion of the cohabitation period and after natural delivery observations had begun), male rats were euthanized, and a gross necropsy was performed
FEMALE RATS
Female rats were euthanized on DL 5. Female rats without a confirmed mating date that did not deliver a litter were euthanized on an estimated DG 25.

OVARIAN AND UTERINE EXAMINATIONS
The number and distribution of corpora lutea and implantation sites were recorded. Uteri of apparently nonpregnant females were examined while being pressed between glass plates to confirm the absence of implantation sites.

NECROPSY
All rats were subjected to a gross necropsy of the thoracic, abdominal, and pelvic viscera. Special attention was paid to the organs of the reproductive system.

ORGAN WEIGHTS
The organs identified in Table 1 (see "Any other information on material and methods incl. tables") were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals found dead or euthanized in poor condition. Paired organs were weighed together, unless indicated otherwise in Table 1. Organ to body weight ratios (using the terminal body weight obtained at necropsy) were calculated.

HISTOLOGY
With the exception of the tissues required for sperm staging, all tissues identified in Table 1 from all rats were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin.

HISTOPATHOLOGY
Histopathological evaluation was performed by a board-certified veterinary pathologist. Tissues were examined from all rats that survived until scheduled euthanasia, as well as any rats that were euthanized prior to scheduled termination or found dead. All gross lesions were examined microscopically.

SPERM STAGING
Stage-dependent qualitative evaluation of spermatogenesis was conducted. Sections of both testes and epididymides from all terminal necropsy male rats were prepared and stained with Periodic Acid Schiff’s-Hematoxylin (PASH). A detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. Both testes and epididymides from all terminal necropsy male rats were evaluated histologically. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Cell- or stage-specificity of testicular findings were noted.


Postmortem examinations (offspring):
UNSCHEDULED DEATHS
One pup was found dead on Postnatal Day 1 (PND 1) before examination of the litter for pup viability and was evaluated for vital status at birth. The lungs were removed and immersed in water. The lungs of this pup did float, and the pup was identified as liveborn but dying shortly after birth. This pup was examined for gross lesions, and the carcass was discarded.
One pup was found dead on PND 2 before scheduled termination and was examined for gross lesions and the cause of death on the day the observation was made.

SCHEDULED EUTHANASIA
Surviving pups were euthanized on PND 5 and examined for gross lesions. One gross lesion was preserved in neutral buffered 10% formalin for possible future evaluation. Necropsy included a single cross-section of the head at the level of the frontal-parietal suture and examination of the cross-sectioned brain for apparent hydrocephaly.
Statistics:
Clinical observations and other proportional data were analyzed using the Variance Test for Homogeneity of the Binomial Distribution. Continuous data (e.g., body weights, body weight changes, and food consumption) were analyzed using Bartlett’s Test of Homogeneity of Variances and the Analysis of Variance, when appropriate [i.e., Bartlett’s Test was not significant (p>0.001)]. If the Analysis of Variance was significant (p≤0.05), Dunnett’s Test was used to identify the statistical significance of the individual groups. If the Analysis of Variance was not appropriate [i.e., Bartlett’s Test was significant (p≤0.001)], the Kruskal-Wallis Test was used (≤75% ties). In cases where the Kruskal-Wallis Test was statistically significant (p≤0.05), Dunn’s Method of Multiple Comparisons was used to identify the statistical significance of the individual groups. If there were greater than 75% ties, Fisher’s Exact Test was used to analyze the data.
Count data were evaluated using the procedures described above for the Kruskal-Wallis Test.
The following parameters were reported:
Fertility and Gestation Indices
• Fertility Index (percentage of matings that result in pregnancies); reported for each sex
• Mating index
• Gestation Index (percentage of pregnancies that result in birth of live litters)
• Live birth index
• Number and sex of offspring per litter (live and dead pups)
• Number of implantation sites
• Post-implantation loss (on litter basis)
Litter Data
• General condition of dam and litter during the postpartum period
• Litter size and viability (tabulated at birth [day 1] and on PND 5)
• Viability Indices (percentages of pups born that survive to PNDs 1 and 5)
• Percent Survival and Sex Ratio (tabulated at birth [day 1] and on PND 5)
• Pup body weights PNDs 1 and 5
Reproductive indices:
Fertility index, mating index, gestation index, live birth index
Offspring viability indices:
Viability incidices for PND1 and 5
Percent survival on PND1 and 5
Sex Ratio

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

MORTALITY
No deaths were attributed to administration of the test substance at dose levels as high as 700 mg/kg bw/day.
Mortality occurred in 1 male rat in the 250 mg/kg body weight/day dose group that was euthanized due to adverse clinical condition as the result of an intubation accident on Day 20 of study (DS 20). Mortality occurred in 1 female rat in the 250 mg/kg body weight/day dose group that was found dead on Day 4 of study (DS 4). This death was not considered related to administration of the test substance because it did not occur in a dose-dependent manner.

CLINICAL OBSERVATIONS
Clinical signs attributed to administration of the test substance occurred at 100, 250, and 700 mg/kg bw/day. The number of male rats with urine-stained abdominal fur achieved statistical significance (p ≤ 0.01) at 100, 250, and 700 mg/kg bw/day, in comparison with the control group. Urine stained abdominal fur was also observed for females at 100, 250, and 700 mg/kg bw/day; the number of animals affected with the clinical sign achieved statistical significance (p ≤ 0.01) during the lactation period in the 700 mg/kg bw/day group, in comparison with the control group. All other clinical signs were considered unrelated to administration of the test substance.

BODY WEIGHTS AND BODY WEIGHTS CHANGES
Male rats
Mean body weights among the male rats assigned to the 4 dose groups were generally comparable throughout the study and did not significantly differ. Effects on mean body weight change were observed at 700 mg/kg bw/day for male rats. Mean body weight change on Days 1 to 8 was reduced (53% of the control group value) at 700 mg/kg bw/day. This reduction was attributed to administration of the test substance because the effects occurred following the initiation of dose administration. Significant reductions (p ≤ 0.05 or p ≤ 0.01; 78%, 66%, and 72% of the control group value) also occurred on Days 21 to 28 at 100, 250 and 700 mg/kg bw/day, respectively. These reductions did not occur in a dose-dependent manner and were not attributed to administration of the test substance. Mean body weight changes during the overall study period (Days 1 through 47) were 92%, 86%, and 98% of the control group value at 100, 250 and 700 mg/kg bw/day, respectively.

Female rats
Mean body weights and body weight gains were unaffected by administration of the test substance at dose levels as high as 250 mg/kg bw/day. Mean body weight changes were slightly decreased throughout gestation at 700 mg/kg bw/day. A significant reduction (p ≤ 0.01; 53% of the control group value) in mean body weight change occurred on Days 7 to 10 of gestation (DGs 7 to 10) at 700 mg/kg bw/day. Consequently, a significant reduction (p ≤ 0.01; 83% of the control group value) in mean body weight change was also observed at 700 mg/kg bw/day during the overall gestation period (DGs 0 through 20). These reductions were attributed to administration of the test substance. Mean body weights among the female rats assigned to the 4 dose groups were generally comparable throughout the study and did not significantly differ.

FOOD CONSUMPTION
Male rats
Mean absolute (g/nesting box/day) food consumption values were unaffected by administration of the test substance at dose levels as high as 250 mg/kg bw/day. A mild reduction (90% of the control group value) in mean absolute food consumption occurred at 700 mg/kg bw/day on Days 1 to 8. This slight reduction correlated with reductions in mean body weight change in this dose group following the initiation of the dose period and was attributed to administration of the test substance. Mean absolute food consumption values at 250 and 700 mg/kg bw/day were significantly increased (p ≤ 0.05 or p ≤ 0.01; 124% and 118% of the control group value, respectively) on Days 42 to 47. These increases did not occur in a dose-dependent manner and were not attributed to administration of the test substance. Mean absolute food consumption values during the overall study period (Days 1 through 47) were 99%, 109%, and 102% of the control group value at 100, 250, and 700 mg/kg bw/day, respectively.

Female rats
Mean absolute (g/nesting box/day or g/day) and/or relative (g/kg bw/day) food consumption values were unaffected by administration of the test substance at dose levels as high as 250 mg/kg bw/day. Mean absolute food consumption values were significantly reduced (p ≤ 0.05) at 700 mg/kg bw/day on Days 1 to 8 (88% of the control group value). This reduction occurred following the initiation of the dose period and was attributed to administration of the test substance. Mild reductions (93% and 92% of the respective control group values) in mean absolute and relative food consumption values occurred at 700 mg/kg bw/day on DGs 0 to 7; the reduction in mean relative food consumption achieved statistical significance (p ≤ 0.05), in comparison with the control group value. Food consumption values were generally comparable among the 4 dose groups during all other intervals and did not significantly differ.

ESTROUS CYCLING; MATING AND FERTILITY
Male rats
Mating and fertility parameters in the male rats were unaffected by administration of the test substance at dose levels as high as 700 mg/kg bw/day.

Female rats
Estrous cycling, mating and fertility parameters in the female rats were unaffected by administration of the test substance at dose levels as high as 700 mg/kg bw/day.

GROSS PATHOLOGY
Male rats
Administration of the test substance at dose levels as high as 700 mg/kg bw/day did not result in any gross lesions that could be attributed to the test substance.
At gross examination, the seminal vesicles and prostates appeared small in 2 of 10 male rats in the 700 mg/kg body weight/day dose group. These gross findings correlated with microscopic findings of mild atrophy and decreased secretions. However, at least 1 of the 2 male rats
successfully mated a female rat, the incidence and severity scores were low, and no histopathological findings were observed in these same organs in a repeat-dose 90-day toxicity study of C-1701 B_C_3 at a daily dose of 1000 mg/kg body weight/day (Protocol 20027338;
BASF Project 50C0473/11X497). Therefore, this finding was considered incidental.

Female rats
There were no gross lesions in the P generation dams. Persistent adverse clinical signs were confirmed at necropsy examination, when possible. These clinical signs included sparse hair coat.

TERMINAL BODY WEIGHTS, ORGAN WEIGHTS, AND RATIOS (%) OF ORGAN WEIGHTS TO TERMINAL BODY WEIGHTS
Male rats
Administration of the test substance at dose levels as high as 700 mg/kg bw/day did not result in any differences in terminal body weights, reproductive organ weights, or ratios of reproductive organ weights to terminal body weights that could be attributed to the test substance. Mean terminal body weights, absolute weights of the left and right epididymides and testes, and the ratios of these reproductive organ weights to terminal body weights were generally comparable among the 4 dose groups and did not significantly differ.

Female rats
Administration of the test substance at dose levels as high as 700 mg/kg bw/day did not result in any differences in terminal body weights, reproductive organ weights, or ratios of reproductive organ weights to terminal body weights that could be attributed to the test substance. Mean terminal body weights, absolute paired ovary weights, and the ratios of the paired ovary weights to terminal body weights were generally comparable among the 4 dose groups and did not significantly differ.

MICROSCOPIC EVALUATION
Male rats
Administration of the test substance at dose levels as high as 250 mg/kg bw/day to male rats did not result in any microscopic lesions that could be attributed to the test substance. There were no alterations in the testes or epididymides related to administration of the test substance. Normal progression of the spermatogenic cycle, with the expected cell associations and cell proportions in the various stages of spermatogenesis, was present. At gross examination, the seminal vesicles and prostates appeared small in 2 male rats at 700 mg/kg bw/day. These gross findings correlated with microscopic findings of mild atrophy and decreased secretions. This finding only occurred in 2 male rats with no apparent effects on fertility, and was therefore not attributed to administration of the test substance. The other gross and microscopic findings noted were considered incidental findings for male rats of this strain and age, and were not attributed to administration of the test substance.

Female rats
Administration of the test substance at dose levels as high as 700 mg/kg bw/day did not result in any microscopic lesions that could be attributed to the test substance. There were no alterations in the ovaries related to administration of the test substance. Ovaries of female rats administered to the test substance were unremarkable histologically, contained the expected numbers of follicles and corpora lutea, and were similar histologically to the respective tissues of the control females. The other gross and microscopic findings noted were considered incidental findings for female rats of this strain and age, and were not attributed to administration of the test substance.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Decreased body weight; food consumption
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
700 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no test substance related adverse effets observed

Results: F1 generation

Details on results (F1)

NATURAL DELIVERY AND LITTER OBSERVATIONS
Natural delivery and litter observation parameters were unaffected by administration of the test substance at dose levels as high as 250 mg/kg bw/day. Mean pup weights per litter on DLs 1 and 5 were significantly reduced (p ≤ 0.05; 91% and 86% of the respective control group values) at 700 mg/kg bw/day. These decreased pup weights were concurrent with slightly higher mean litter sizes (11.2 versus 10.4 in the control group) and decreased mean body weight gains in the dams during gestation (approximately 83% of the control group values). It is known from literature (Fleeman et al., 2005) that reductions in fetal body weights frequently occur concurrent with reduced maternal food consumption and maternal body weights, similar to the current study. Values for the duration of gestation (DG 0 to the day the first pup is observed), implantation sites per delivered litter, percentage of postimplantation loss, dams with stillborn pups, dams with no liveborn pups, gestation index (number of rats with live offspring/number of pregnant rats), dams with all pups dying, pups delivered, liveborn and stillborn pups, pups found dead or presumed cannibalized, viability index, surviving pups per litter, percent male pups per number of pups sexed, and live litter sizes at weighing were generally comparable among the 4 dose groups and did not significantly differ.

Reference:
Fleeman TL, Cappon GD, Chapin RE, Hurtt ME (2005). The effects of feed restriction during organsogenesis on embryo-fetal development in the rat. Birth Defects Res B Repro Toxicol 74 (5):442-449.

CLINICAL OBSERVATIONS
None of the clinical signs observed in the F1 generation pups were attributed to administration of the test substance to the P generation dams.

NECROPSY OBSERVATIONS
There were no test substance related gross lesions in the F1 generation pups.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reduced pups weights

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Dose Formulation Analysis

Analysis of the dose formulation samples revealed that all actual concentrations were within the acceptance criteria of ± 15% of the respective theoretical concentrations. All dose formulation samples met acceptance criteria for homogeneity (the relative standard deviation [RSD] of concentrations was < 5 % for each). Control stubstance samples contained no detectable concentrations of the test substance.

Applicant's summary and conclusion