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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
15/07/1999-15/03/2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented report of a guideline study conducted according to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): OS#132086
- Molecular formula (if other than submission substance): C7H16N204S
- Molecular weight (if other than submission substance): 224.28
- Smiles notation (if other than submission substance): [NH4+].O=S(=O)(O)CC(C)(C)/N=C(\[O-])C=C
- InChl (if other than submission substance): InChI=1/C7H13NO4S.H3N/c1-4-6(9)8-7(2,3)5-13(10,11)12;/h4H,1,5H2,2-3H3,(H,8,9)(H,10,11,12);1H3
- Substance type: organic
- Physical state: liquid
- Analytical purity: 50% (in aqueous solution
- Impurities (identity and concentrations): water (50%)
- Composition of test material, percentage of components: 100%
- Purity test date: not specified
- Lot/batch No.: not specified
- Expiration date of the lot/batch: not specified
- Stability under test conditions: stable
- Storage condition of test material: not specified

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Kingston, New York, USA
- Age at study initiation: approximately 10 weeks
- Weight at reception: Males: 238 to 263 grams; Females: 201 to 244 grams
- Fasting period before study: no
- Housing: individually (except during cohabitation for mating)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.3 to 26.1 (65 to 79°F)
- Humidity (%): 30 to 70
- Air changes (per hr): 10 to 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 31/08/1999 To: 23/10/1999

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For each test article dose group, a specified amount of the test article was weighed into a beaker. A sufficient quantity of RO-Di water was added to the beaker to achieve the desired concentration and the mixture was stirred for 15 minutes. Each test article solution was prepared fresh weekly and dispensed into daily aliquots which were stored refrigerated in amber glass containers. The vehicle, RO-Di water, was also dispensed into daily aliquots for administration to control animals and stored refrigerated in amber glass containers. The physical state of the control and each dosing solution was recorded during each preparation. The vehicle and test article dosing mixtures were clear colorless solutions. Daily aliquots of the dosing solutions were allowed to equilibrate to room temperature prior to dispensing. The dosing solutions were stirred prior to dispensation and then continuously until dosing was complete.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: day of confirmed copulation or for 14 days if no copulation observed
- After successful mating each pregnant female was caged: single
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity of the test article in the vehicle was evaluated by analysing duplicate samples from the top, middle and bottom of the 10 and 100 mg/mL concentrations. Stability of the test article in the vehicle was assessed by analysing duplicate samples obtained from the 10 and
100 mg/mL concentrations following 24 hours of room temperature storage, and following 3 and 10 days of refrigerated storage. In addition, duplicate samples were obtained from the vehicle and each test article dosing mixture prepared for weeks 1 , 3, 5 and 7 for concentration analysis.
Duration of treatment / exposure:
Dosing preparations were administered orally, by gavage, as a single dose daily to F0 males and females beginning two weeks prior to mating.
The F0 males were dosed for approximately seven weeks, including two weeks prior to mating, during mating and post-mating
The F0 females were dosed throughout the study, including two weeks prior to mating, during
mating, during gestation, and following parturition. Individual doses were adjusted based on the most recent body weight data.
Both F0 males and females were dosed up to and including the day prior to scheduled euthanasia.
Frequency of treatment:
7 days/week
Details on study schedule:
Dosing preparations were administered orally, by gavage, as a single dose daily to F0 males and females beginning two weeks prior to mating. The F0 males were dosed for approximately seven weeks, including two weeks prior to mating, during mating and post-mating. The F0 females were dosed throughout the study, including two weeks prior to mating, during mating, during gestation, and following parturition. Individual doses were adjusted based on the most recent body weight data. Both F0 males and females were dosed up to and including the day prior to scheduled euthanasia.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 500 or 1000 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dosage levels were selected by the Sponsor based on the results of previous studies with the test article, which indicated that the limit level of 1000 mg/kg/day could be utilized as the high-dose for this study. The mid- and low-dose levels were selected as lower multiples of the high-dose level.
- Rationale for animal assignment: random
Positive control:
No

Examinations

Parental animals: Observations and examinations:
F0 Clinical Observations
Mortality/general health checks were performed twice daily for the F0 parental animals, once in the morning and once in the afternoon. Detailed clinical observations were performed weekly for the F0 males and females. In addition, cage-side observations were performed daily, within approximately one-half hour to two hours following dosing. During gestation and lactation, detailed clinical observations were performed daily for F0 females. Detailed clinical observations were also performed on all F0 males on the day of scheduled euthanasia.

F0 Body Weights
The F0 parental animals were weighed on a weekly basis following the commencement of dosing. The specific weighing schedules for F0 males and females were as follows:
a. Males: recorded once per week and on the day of scheduled euthanasia.
b. Females: recorded once per week prior to confirmation of mating.
Females with positive mating signs and females that delivered were weighed as follows:
Gestation: days 0, 7, 14 and 20.
Lactation: days 1 and 4.
Females without evidence of mating were weighed on a weekly basis.

F0 Food Consumption
Individual food weights were collected on the same day as the body weights, except during cohabitation when food consumption was not measured.

F0 Breeding
Following 14 days of treatment, each F0 female was cohabited with a single F0 male randomly selected from the same treatment group. Each mating pair was observed daily for evidence of copulation. Evidence of mating was determined by the presence of a copulatory plug in the vagina or a sperm positive vaginal smear. The day evidence of copulation was confirmed was designated as day 0 of gestation and the female was returned to its cage. If no evidence of copulation was observed after 14 days of mating, the female was separated from the male and the mating phase was concluded.

F0 Parturition and Lactation
On gestation day 18, F0 females with confirmed copulation were transferred to individual plastic cages containing nesting material. Each F0 female was observed for signs of parturition a minimum of twice daily. The time parturition was first detected and the time parturition was completed were recorded, when possible. Signs of difficult or prolonged delivery were recorded, if observed. The day on which parturition was judged complete was designated as lactation day 0. The F0 females and their offspring remained together until in-life conclusion on lactation day 4. Abnormal nursing and nesting behaviours were recorded, if observed, during the abbreviated lactation period. The offspring were designated as the F1 generation. F0 females with no evidence of mating were examined for parturition beginning 19 days following initiation of the mating phase.
Sperm parameters (parental animals):
F0 Breeding
Following 14 days of treatment, each F0 female was cohabited with a single F0 male randomly selected from the same treatment group. Each mating pair was observed daily for evidence of copulation. Evidence of mating was determined by the presence of a copulatory plug in the vagina or a sperm positive vaginal smear. The day evidence of copulation was confirmed was
designated as day 0 of gestation and the female was returned to its cage. If no evidence of copulation was observed after 14 days of mating, the female was separated from the male and the mating phase was concluded.
Litter observations:
F1 Lactation Observations, Pup Identification and Offspring Parameters
On lactation day 0, pups in each litterwere individually identified using a toe marking (tattooing) system. The following parameters were recorded for each pup during lactation:
a. Viability: daily from days 0 to 4.
b. External examinations: days 0 and 4.
c. Sex Determinations: days 0 and 4.
d. Body Weights: days 1 and 4.

F1 Unscheduled Deaths
All intact (noncannibalized) F1 pups which were found dead during lactation were necropsied, with emphasis on developmental morphology. Non-viable cannibalized F1 pups with no apparent deformities were discarded without necropsy.
Postmortem examinations (parental animals):
F0 Euthanasia and Necropsy
All F0 males and females were euthanised by carbon dioxide inhalation and subjected to a complete gross necropsy examination. The F0 males were euthanised after approximately seven weeks of treatment (days 49 and 50). F0 females that delivered were euthanised on lactation day 4. F0 females that failed to deliver were euthanised 25 days after evidence of mating was first detected (post-breeding day 25). F0 females with no evidence of mating were euthanised 25 days after completion of the mating period (post-breeding period day 25). The necropsy examination included evaluation of the external surfaces of the body, all orifices, and the cranial, thoracic, abdominal and pelvic cavities and their contents. All gross abnormalities were recorded. Uterine contents were examined and the number of implantation sites and number of corpora lutea (per ovary) were recorded. Uteri with no macroscopic evidence of implantation were opened and placed in 10% aqueous ammonium sulfide solution for detection of early embryolethality as described by Salewski. The testes of all F0 males were preserved in Bouin’s fixative for 48 to 96 hours, rinsed and then retained in 70% isopropanol. The following tissues/organs from all F0 parental animals were preserved in 10% neutral buffered formalin for possible future histopathological examination:
Gross lesions
Ovaries
Prostate
Epididymides
Seminal vesicles (with coagulating glands)
Uterus (with oviducts and cervix)
Vagina

F0 Organ Weights
The testes and epididymides of all F0 males were weighed fresh at scheduled necropsy (paired organ weights).

F0 Histopathology
The testes, epididymides and ovaries of F0 control and high-dose males and females were processed histologically and examined microscopically. The tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. The histological processing was performed by Histo Techniques, Powell, Ohio. The slides were examined microscopically by Dr. William H. Baker, a board-certified veterinary pathologist.
Postmortem examinations (offspring):
F1 Scheduled Euthanasia
All surviving F1 pups were euthanized on lactation day 4 by carbon dioxide inhalation or an abdominal cavity injection of sodium pentobarbital and examined macroscopically for structural abnormalities or other pathological changes. Special attention was directed to organs of the reproductive system. All gross lesions were preserved in 10% neutral buffered formalin for possible future histopathological examination.
Statistics:
Body weights, body weight gain, food consumption, organ weights, gestation length, precoital intervals, implantation scar counts, corpora lutea, pup body weights, and mean live litter size were analysed by One-way Analysis of Variance (ANOVA). If significance was observed with ANOVA, control to treatment group comparisons were performed using Dunnett's test. Count data were analyzed using Chi-square test for copulation and fertility indices, pup sex ratios, the number of live and dead pups per group (on lactation day 0) and pup survival (after lactation day 0). The Mann-Whitney U test was used to compare pre-implantation loss and post-implantation loss. Statistical analyses were performed by a Digital MicroVax 3100 computer. All analyses were two-tailed with a minimum significance level of 5% ( p<0.5 )
Reproductive indices:
Precoital interval, gestation length, number with successful copulation, fertility
Offspring viability indices:
Number of litters in each group with live offspring, mean live litter size or pup sex ratios

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
F0 Survival and Clinical Observations:
All animals survived to scheduled euthanasia and no clinical signs of toxicity were noted during the study. Clinical signs which were observed consisted of a low incidence of relatively minor and sporadic findings such as hair loss, malalignment of incisors, scab formation, and dark material around the eyes or nose. Two females in the 500 mg/kg/day group which were euthanized on presumed gestation day 25 (i.e., at 25 days following positive evidence of insemination) were found to be nongravid at necropsy (ammonium sulfide negative). Two other females, one each in the 100 and 500 mg/kg/day groups, were euthanized on post breeding day 25 (i.e., at 25 days following completion of the breeding period, during which no evidence of mating was detected). These females were also found to be nongravid at necropsy (ammonium sulfide negative).

F0 Body Weights and Weight Gain: There were no toxicologically meaningful differences in male or female body weights or weight gains during the study. The only statistical difference consisted of a lower mean body weight gain in the 500 mg/kg/day females during the week prior to mating (week 2 to 3). This difference was not considered toxicologically significant since a similar change was not observed at the 1000 mg/kg/day level. In addition, mean body weights and weight gains were comparable between the control and all female treatment groups (100, 500 and 1000 mg/kg/day levels) throughout gestation and the abbreviated lactation period (i.e.., lactation days 1 and 4). Similarly, male body weights and weight gains were comparable between the control and test article-treatment groups throughout the study.

F0 Food Consumption: There were no statistically significant or toxicologically meaningful differences in food consumption of males or females during the study.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Reproductive Indices, Precoital Intervals and Gestation Lengths: There were no statistically significant or toxicologically meaningful differences in copulation or fertility indices among the groups. The copulation index was 100% in the control and the 1000 mg/kg/day groups, and 91.7% in the 100 and 500 groups mg/kg/day. The fertility index was 100% in the control, 100 and 1000 mg/kg/day groups, and 81.8 % in the 500 mg/kg/day. No statistically significant differences were observed in group mean precoital intervals or gestation lengths. Mean precoital intervals in the control, 100, 500 and 1000 mg/kg/day groups were 1.5, 2.8, 3.5 and 2.6 days, respectively. Mean gestation lengths in the control, 100, 500 and 1000 mg/kg/day groups were 22.1, 22.0, 21.8 and 22.1days, respectively.
One 1000 mg/kg/day female (#8646) was found to have delivered pups in her suspended stainless steel cage (prior to transfer to a nesting box) on presumed gestation day 15. Three pups were found in the cage and three pups were found on the litter board. The pups appeared to be full-term (based on body weights), indicating that the presumed gestation day 0 was not correct. Because of these events, gestation body weight and food consumption data for this female, as well as all pup data, were considered invalid. Therefore, these data were excluded from group mean calculations and individual data tables, as appropriate.
One other 1000 mg/kg/day female (#8639) began delivering pups in her suspended stainless steel cage on presumed gestation day 17. Six pups were found in the cage at the a.m. mortality check. The pups appeared to be full-term based on body weights. The female and pups were immediately transferred to a nesting box. Ultimately, the female completed parturition on the same day, resulting in a litter count of 16 pups. At necropsy, this female had 16 implantation scars, indicating that all pups were accounted for. Because of these events, gestation body weight and food consumption data for this female were eliminated from group mean calculations. However, since pup viability and maternal care did not appear to be significantly compromised, pup data for this litter were not excluded from group mean calculations and individual data tables.

ORGAN WEIGHTS (PARENTAL ANIMALS):
There were no statistically significant or toxicologically meaningful differences in absolute or relative testes and epididymes weights between the control and test material-created groups

HISTOPATHOLOGY (PARENTAL ANIMALS):
Histopathological of the testes, ovaries and epididymides from control and high-dose rats did not reveal any test material-related microscopic changes.

OTHER FINDINGS (PARENTAL ANIMALS):
There were no statistically significant or toxicologically meaningful differences between control and test material-treated groups with respect to corpora lutea counts, implantation scar counts, mean number of live pups, or pre- or post-implantation loss.

Effect levels (P0)

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

F1 Pup Viability: There were no toxicologically meaningful differences with respect to F1 pup viability, number of litters in each group with live offspring, mean live litter size or pup sex ratios. The incidence of dead pups on lactation day 0 was slightly but significantly higher in the 500 mg/kg/day group. This difference was not considered toxicologically significant since the incidence of dead pups was comparable between the control and 1000 mg/kg/day groups. Additionally, pup survival after lactation day 0 was comparable between the control, 100, 500 and 1000 mg/kg/day groups.

F1 Pup Observations during Lactation: F1 pup observations during lactation were generally unremarkable. Individual findings tended to be randomly distributed among the groups, with no apparent dose-response pattern(s) emerging.

F1 Pup Body Weights during Lactation: There were no statistically significant or toxicologically meaningful differences in F1 pup body weights during lactation.

F1 Pup Gross Necropsy Observations: For F1 pups found dead or euthanized as scheduled on lactation day 4, gross necropsy did not reveal any findings which would indicate a relationship to treatment with the test article. The most notable gross observations in pups found dead on lactation day 0 consisted of atelectasis and absence of milk in the stomach, indicating that these pups were most likely stillborn. Other necropsy findings tended to be of low incidence and randomly distributed among the groups. There were no indications of treatment-related developmental effects.

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Oral administration of OS#132086 at dosage levels of 100, 500 and 1000 mg/kg/day had no effect on F0 survival, growth, mating behavior, copulation, fertility, precoital intervals, gestation lengths, corpora lutea counts, implantation counts, mean live litter size, prelpost-implantation loss, gross necropsy findings or organ weights (testes and epididymides). Histopathological examination of the testes, ovaries and epididymides from control and high-dose rats did not reveal any test article-related microscopic changes. No test article-related effects were observed in the F1 offspring with respect to survival, clinical observations, body weights or gross necropsy findings. In addition, there were no indications of test article-related developmental effects in the F1 pups at any dosage level tested. Based on the results, a dosage level of 1000 mg/kg/day was considered a no-observed-effect level (NOEL) for this reproduction and developmental screening study in rats.