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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
reproductive toxicity, other
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2001
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Guideline on Detection of Toxicity to Reproduction for Medicinal Products, (ICH Harmonised Tripartite Guideline, S5A) implemented at Step 5
Deviations:
yes
Remarks:
, but the observed deviations were not considered to have compromised the validity or integrity of the study.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: brown cream oily paste
Details on test material:
Batch No. CD01.281
Storage conditions: at +4°C, under nitrogen atmosphere.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
- Test animals are Specific Pathogen Free (S.P.F.).
- Breeder: Charles River Laboratories - IFFA CREDO (L'Arbresle, France).
- Age: on the day of the first treatment (D-14 before mating in the females and on D-28 before mating in the males), males and females were approximately 10 weeks old. On the day of the mating, males were about 14 to 17 weeks of age, females about 12 to 15 weeks.
- Mating: females were mated at the breeder's facilities. The day of confirmed mating (visual assessment: vaginal plug) was designated as day 0 of gestation (D0).
- Sex: females were nulliparous and primiparous, sexually mature at the start of the study. Males were virgin at the start of the study and were sexually mature.
- Acclimation: rats were acclimated for a period of 5 days before the beginning of the treatment period (D6).
- The animal room conditions are set as follows:
. temperature : 19-23°C
. relative humidity : 45-65%
. light/dark cycle : 12h dark/12h light (7:30 a.m. - 7:30 p.m.)
. ventilation : approximately 10 cycles/hour of filtered, non-recycled air.
- The animals were housed in cages of 4 males or females from each treatment group before the mating period, in pairs during the mating period and individually after the mating period in standard size cages..
- The animals had free access to food and water.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Each male rat was given the dosage forms once a day (50, 200, 750 mg/kg b.w./day or vehicle), at approximately the same time, for 28 days before being paired with females, throughout the mating period (21 days maximum) and until terminal sacrifice.
Each female rat was given the dosage forms once a day (50, 200, 750 mg/kg b.w./day or vehicle), at approximately the same time, for 14 days before being paired with males, throughout the mating period (21 days maximum) and until implantation (D6).
The dosage forms were administered by gavage.
The quantity of the dosage form administered to each animal was adjusted according to the most recently recorded body weight.
A constant dosage-volume of 5 mL/kg b.w. was used. Control animals (group 1) received the vehicle alone.
Suspensions to be administered were prepared daily.
Details on mating procedure:
Mating took place at least 28-day dosing of males and 14-day dosing of females. This type of study uses 1:1 (1 male per female) mating. Pairing of each couple was done by randomised assignment, at the start of the study.
Each female was paired with a male from the same group until pregnancy or, should this fail to occur, for up to seven days.
Females were examined twice a day, in the morning and at the end of the working day. This examination included a vaginal smear and seeked the presence of sperm and/or of a vaginal plug. The trays underneath the cages were checked twice a day for the presence of ejected copulation plugs.
Day 0 of pregnancy (D0) was taken to be the day when ejected plugs or sperms are found.
In case of failure to mate within seven days, the male was removed and another from the same group, that has previously mated was substituted. This second pairing continued for up to a further seven days. Vaginal smears were taken and pairs were separated as previously described.
In case of failure to mate, this procedure was repeated one more time. The third pairing continued for up to a further seven days. Vaginal smears were taken and pairs were separated as previously described.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
No concentration assays were performed.
Duration of treatment / exposure:
Each male rat was given the dosage forms once a day, at approximately the same time, for 28 days before being paired with females, throughout the mating period (21 days maximum) and until terminal sacrifice.
Each female rat was given the dosage forms once a day, at approximately the same time, for 14 days before being paired with males, throughout the mating period (21 days maximum) and until implantation (D6).
Frequency of treatment:
Once a day, at approimately the same time.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
50 mg/kg b.w./day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
200 mg/kg b.w./day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
750 mg/kg b.w./day
Basis:
actual ingested
No. of animals per sex per dose:
Group 1 (0 mg/kg b.w./day): 19 males and 20 females
Group 2 (50 mg/kg b.w./day): 19 males and 23 females
Group 3 (200 mg/kg b.w./day): 17 males and 20 females
Group 4 (750 mg/kg b.w./day): 17 males and 18 females
Control animals:
yes, concurrent vehicle
Details on study design:
The number of animals forming each group corresponds to the number of mated animals and not to the number of really pregnant animals at the end of the study.
Positive control:
None.

Examinations

Parental animals: Observations and examinations:
Mortality/morbidity: recorded twice a day.

General clinical examination: at least once a day during the study. During the treatment period, animals were examined at a time when any possible effects were most pronounced ( about 1 hour after dosing). Females showing signs of abortion or of premature delivery were sacrificed on the day of such findings.

Weighing:
- Males were weighed on the day of allocation to dosing groups then twice a week until the day of successful mating and/or the day when dosing is stopped if it has never taken place, on the day of successful mating (D0) and on the day of sacrifice (not fasted).
- Females were weighed on the day of allocation to dosing groups then twice a week until the day of successful mating and/or the day when dosing is stopped if it has never taken place, then in case of successful mating on D0 (1st day of pregnancy), then daily until the day of sacrifice (not fasted-D15).

Food and water consumption:
- During the pre-mating period, measured weekly.
- During the mating period and during pregnancy, it was not recorded.
Oestrous cyclicity (parental animals):
During the pre-mating period: daily for 14 days prior to pairing, a vaginal plug was prepared from each female and examined to determine the stage of the oestrus cycle (A- pro-oestrus, B-oestrus, C-met-oestrus, D-di-oestrus).
During the mating period, males and females were monitored twice a day. A vaginal smear was obtained from females and the presence of sperm and/or of a vaginal plug sought. Particular attention was paid during this period to the general behaviour of the pair. The stage of the oestrus cycle was recorded as indicated above.
Sperm parameters (parental animals):
Spermatozoa motility was assessed on epididymal sperm suspension.
Spermatozoa morphology (head, midpiece and tail) was judged on staining smears.
Sperm count was performed on testis homogenate.
Litter observations:
On the day of scheduled sacrifice of mothers (D15), the number of embryos/foetus was assessed.
Embryos/foetus were fixed for possible subsequent examination.
Postmortem examinations (parental animals):
Males were sacrificed by exsanguination after sodium pentobarbital anesthesia by intraperitoneal route. Main organs were weighed and examined (liver, spleen, kidneys, stomach, intestines, gonads/reproductive tract, lungs and heart), including urogenital system (testis, epididymis, prostate, seminal vesicle, coagulating gland).

Females were sacrificed by exsanguination after sodium pentobarbital anesthesia by intraperitoneal route. Main organs were weighed and examined (liver, spleen, kidneys, stomach, intestines, gonads/reproductive tract, lungs and heart), including urogenital system (ovaries, uterus, vagina).
Ceasarian procedure on each pregnant females was as follows:
- count of corpora lutea on ovaries,
- number and uterin location of implantation sites,
- number and uterine location of embryos,
- number and uterine location of resorptions.
Statistics:
Statistical analysis of results were performed.
Reproductive indices:
The following calculations were made:
Copulation index = Number of females with successful copulation*100/Number of females paired
Fertility index = Number of gravid females*100/Number of females with successful copulation
Pre-implantation loss (%) = (Number of corpora lutea - Number of implantation sites)*100/Number of corpora lutea
Post-implantation loss (%) = (Number of implantation sites - Number of live embryos)*100/Number of implantation sites

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
related to dosing incidence, with mainly decrease in locomotor activity, piloerection, salivation, dry blood around the nose and dyspnea.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
lower in females treated at 750 mg/kg b.w./day
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
lower in females treated at 750 mg/kg b.w./day
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
only ovaries weight of females treated with 750 mg/kg b.w./day was significantly lower
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
lower fertility indice observed with females treated at 750 m/kg b.w./day

Details on results (P0)

Mortality:
Five deaths occured, but were related to dosing incident.

Clinical findings:
There were related to dosing incident with mainly decrease in locomotor activity, piloerection, salivation, dry blood around the nose and dyspnea.

Body weight change:
No statistically significant difference was noted between treated and control animals. Only significant decrease was observed in females at 750 mg/kg b.w./day during pregnancy period, but it was due to three females that were not pregnant and did not gain weight after mating.

Food and water consumption:
- For males, food consumption in treated groups was similar to those of control males; main greater water consumption was observed in males treated at 750 mg/kg b.w./day.
- For females, difference on food consumption was observed but was slight and not dose-dependent; main greater water consumption was observed in females treated at 750 mg/kg b.w./day.

Mating results:
No effects on copulation index, lenght of the mating period or oestrus cycle.

Macroscopic examination:
No findings was dose-related.

Organ weights:
For males, no statistically significant difference was noted between treated and control males.
For females, absolute and relative weight of the ovaries of females treated at 750 mg/kg b.w./day were significantly lower than that of control females. This was related to the lower number of corpora lutea reported in those females.

Reproductive performance:
Fertility indices were similar to that of control group, except for female treated at 750 mg/kg b.w./day with a lower index (83.3% vs. 100%).

Sperm evaluation: no statiscally significant difference on motility, sperm count in testis homogenate and sperm morphology.

Litter data:
- Corpora lutea: no statistically difference, however the number was lower in females treated at 750 mg/kg b.w./day.
- Number of implantation sites/pre-implantation loss: the number was lower in females treated at 750 mg/kg b.w./day and was related to a higher preimplantation loss in the treated females.
- Number of embryos/post-implantation loss: the number was significantly lower in females treated at 750 mg/kg b.w./day. This was related to the lower number of implantation sites and to a higher post-implantation loss in the treated females.

Effect levels (P0)

open allclose all
Dose descriptor:
dose level: = 750 mg/kg b.w./day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No effect on male rat fertilty.
Remarks on result:
not measured/tested
Remarks:
Effect level not specified (migrated information)
Dose descriptor:
dose level: = 200 mg/kg b.w./day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No evidence of any adverse effects on the oestrus cycle was observed at any doses, but some interference with the process of implantation and/or very early survival of embryos reported at 750 mg/kg b.w./day.
Remarks on result:
not measured/tested
Remarks:
Effect level not specified (migrated information)

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In males treated with PIAS at any dose levels, there was no evidence of any adverse effects on the sperm maturation, the libido and the mating. There was no influence on male rat fertility.
In female rats treated with PIAS at any dose levels, there was no evidence of any adverse effects on the oestrus cycle. At the high dose of 750 mg/kg b.w./day, there appeared to be some interference with the process of implantation and/or very early survival of embryos (as evidence by a reduction of the number of pregnant females and a slight increase in pre-implantation loss). At the dose levels of 50 mg/kg b.w./day and 200 mg/kg b.w./day, no adverse effects were recorded.
Executive summary:

The study involved 4 group including a control group:

- Group 1 (0 mg/kg b.w./day): 19 males and 20 females

- Group 2 (50 mg/kg b.w./day): 19 males and 23 females

- Group 3 (200 mg/kg b.w./day): 17 males and 20 females

- Group 4 (750 mg/kg b.w./day): 17 males and 18 females

Mortality

All the deaths occuring during the study were related to dosing incident. The physical characteristics of the test substance (oily paste) made it difficult to administer, and this may have been a contributing factor in the number of misgavages, which occured in this study.

Clinical findings

Clinical findings reported in few animals from the control group and from the treated groups were related to dosing incident.

Body weight change

From D13 to D15, mean body weight in females treated at the dose level of 750 mg/kg b.w./day was significantly lower than that of control females at the threshold of 5%. This difference was reported since D10 but was not statistically significant.

This difference was related to the three females that were not pregnant and did not gain weight after mating as demonstrate after excluding all those not pregnant females from the statistical analysis.

Food and water consumption

Despite some slight differences, during the pre-mating period, food consumption in treated males was similar to those of control males. On week 1 and week 2 (pre mating), food consumption in females treated at the dose levels of 50, 200 and 750 mg/kg b.w./day was significantly lower than that of control females at the threshold of 5% or 1%. On week 1 the difference was slight and dose dependent, on week 2 the difference was very slight and was not dose dependent.

Statistical differences were noted for water consumption in treated animals compared with control animals (in both males and females). The main striking difference was reported in animals treated at the dose level of 750 mg/kg b.w./day; this was a greater water consumption than that of control animals.

Mating results

- Number of mating: all the females that were mated had successful copulation

- Length of the mating period: for the first pairing, the length of the mating period was lower than the three days in all groups. Surprisingly the groups with the longer period of pairing was the control group. There was no treatment related changes in the duration of the mating period.

- Oestrus cycle: the number of oestrus cycle during the pre-mating period was about 3 to 4 in all control and treated females. All the stages were noted. There was no predominance of any stage at any time.

Macroscopic examination

The main findings reported at necropsy of few animals were related to dosing incidence. Other findings were commonplace and were those reported usually in young laboratory rats. Non were treatment related.

Organ weights

- Testis/epididymis/prostate: no statistically difference was noted between treated and control males.

- Ovary: absolute and relative weight (% body weight) of the ovaries of females treated at the dose level of 750 mg/kg b.w./day were significantly lower thant that of control females at the threshod of 5% or 1%. This was related to the number of corpora lutea reported in those females.

Reproductive performance

- Fertility indices: one females from the control group out of 19 and 3 females from the high dose group were not pregnant at terminal necropsy despite successful copulation. Indice in females dosed at 750 mg/kg b.w./day was lower than that of control females.

- Litter data: in females dosed at 750 mg/kg b.w./day, the number of corpora lutea, implantation sites and embryos was lower than that of control females. This was related to the higher pre-implantation loss and post-implantation loss in treated females. These differences were reported at a lesser extend in females treated at the dose of 200 mg/kg b.w./day.

- Sperm evaluation: no statistically difference or abnormality observed on motility, sperm count or sperm morphology.