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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented and reported study fully adequate for assessment. The study was conducted according to internationally accepted technical guidelines and in compliance with GLP in a recognized contract research organization.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS: Wistar Crl:WI rats
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld
- Age at study initiation: 11 weeks
- Weight at study initiation: Males: 352 g – 402 g, Females: 210 g - 246 g
- Housing:up to 5 animals of the same sex and dose group/cage (Type II and/or III polycarbonate), with the exception of the mating and gestation/delivery period, when they were paired or individually housed, respectively.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.9 – 23.9°C
- Humidity (%): 36 - 70%
- Air changes (per hr): 15-20

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test material and vehicle were mixed and homogenized using stirrer for approximately 10-15 minutes at room temperature. Pending administration to the animals, the dose formulation(s) were stirred on a magnetic stirrer at room temperature and were protected from light. Formulations were prepared fresh prior administrations, according to stability assessment results (stable in Propylene glycol in concentration range of 5- 250 mg/mL for 4 hours at room temperature (RT, 15-30ºC) and for up to two days when stored refrigerated at 2-8ºC).

VEHICLE
- Justification for use and choice of vehicle (if other than water): appropriate for formulation and its dilution; compatible to the test system
- Concentration in vehicle: 0, 6, 20, 60 mg/mL
- Amount of vehicle (if gavage): 5 ml/kg b.w.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: until copulation occurred, for up to 7 days.
- Proof of pregnancy: Vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy.
- After successful mating each pregnant female was caged individually.
- Mating of siblings was avoided.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of WS400402 formulations for concentration and homogeneity was performed using validated GC method (CiToxLAB study code 11/351-316AN). Recovery of WS400402 from propylene glycol, tested 3 times - during the first and last weeks and approximately midway during the treatment, ranged from 94 to 105% for all dose groups.
Duration of treatment / exposure:
Main males: 35 days (premating 14 days, mating and postmating 14, plus an additional week)
Main females: ca. 47-48 days (premating 14 days, mating 7 days, gestation ca. 22-23 days, lactation period 4 days)
Satellite females (not mated): 35 days
Frequency of treatment:
daily, 7 days/week
Details on study schedule:
- Age at mating of the mated animals in the study: 13 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12 animals/sex/dose
(Satellite female group (not mated): 20 femal rats: 5 animals/dose - for Repeated Dose Toxicity Testing according to OECD 407)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose finding toxicity study with WS400402 (administered via oral gavage to Wistar rats for 7 consecutive days at dose levels of 50, 300 and 800 mg/kg bw) showed a decrease in body weight, lower body weight gain and decreased food consumption in animals treated at 800 mg/kg. In addition, minor changes in clinical pathology parameters were observed at 800 mg/kg.(CiToxLAB study code 11/351-220PE) - 7.5.1 Dose Range Finding Study 7 days_WS400402.
Based on these results, the dose levels selected for the main study were 0, 30, 100 and 300 mg/kg bw/day.

This study was conducted to examine both repeated dose toxicity and reproductive/developmental toxicity as an OECD screening combined study (OECD 422 test guideline). Therefore, animals initially entering the study were divided into toxicity subgroup animals (Satellite females) and reproductive subgroup animals (Main females and males), whereby 5 of the 12 Main males (used for pairing) per dose group formed the toxicity male Subgroup A.
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
All animals were monitored for pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality.
Delivery process was observed as carefully as possible. Dams were observed to record whether they form a nest from the bedding material and cover their new-borns or not.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least weekly (observations in a standard arena)

BODY WEIGHT: Yes
- Time schedule: Parent females were weighed on gestation Days GD 0, 7, 14 and 20 and on postpartal Days PPD0 (within 24 hours after parturition), and PPD5 (before termination).
Parent males were weighed on Day 0 and at least weekly.




Sperm parameters (parental animals):
Parameters examined in male parental generations, all males (12/dose):
testes, epididymides (total and cauda), prostate, seminal vesicles with coagulating glands.
Detailed qualitative histopathology examination of the testes taking into account the tubular stages of the spermatogenic cycle. This was 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 in the lumen. Any cell- or stage-specificity of testicular findings was noted.
Litter observations:
STANDARDISATION OF LITTERS Not performed. The study ended on Lactation Day 4.

PARAMETERS EXAMINED
The following parameters were examined in F1- offspring:
number and sex of pups, stillbirths, live births; postnatal mortality; presence of gross anomalies, weight gain (PPD0-PPD4), physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes; for external abnormalities, any pups showing abnormalities in structure or behaviour were subjected to necropsy with macroscopic examination.

Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals on Day 35.
- Maternal animals: All surviving animals on Day 5 of lactation (PND5).

GROSS NECROPSY
- Gross necropsy consisted of external examinations including the cervical, thoracic, and abdominal viscera.
- Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea were recorded in the Main females as applicable.

ORGAN WEIGHTS
Weight of the following organs of all adult animals were determined:
- With a precision of 0.01 g: uterus (with and without cervix), vagina, testes, epididymides (total and cauda), prostate, seminal vesicles with coagulating glands, brain
- With a precision of 0.001 g: ovaries, pituitary

HISTOPATHOLOGY :
Detailed histological examinations was performed in all main adults of control and high dose groups.
Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.
Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring on Day 4:
Pups were carefully examined at least externally for gross abnormalities. Any pups showing abnormalities in structure or behaviour were subjected to necropsy with macroscopic examination. The probable cause of death of dead pups were recorded if it can be identified, e.g. cannabilism.

Macroscopic examination included assessment of the presence of milk in the stomach, where possible.
Statistics:
Performance with the statistical program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test,the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed as feasible.
Reproductive indices:
Formulas for Calculation of Mating and Fertility Indices
Male Mating Index: Number of males with confirmed mating : Total Number of males cohabited x 100
Female Mating Index: Number of sperm-positive females : Total Number of females cohabited x 100
Male Fertility Index: Number of males impregnating a female : Total Number of males cohabited x 100
Female Fertility Index: Number of pregnant females : Number of sperm-positive females x 100
Gestation Index: Number of females with live born pups : Number of pregnant females x 100
Offspring viability indices:
Formulas for Calculation of Pups’ Mortality and Sex Ratio Indices
Survival Index: Number of live pups (at designated time) : Number of pups born x 100
Pre-implantation mortality: (Number of Corpora lutea − Number of Implantations) : Number of Corpora lutea x 100
Intrauterine mortality: (Number of implantations - Number of liveborns) : Number of implantations x 100
Total mortality: (Number of implantations - Number of viable pups (d4)) : Number of implantations x 100
Post-natal mortality: (Number of viable pups (d0) - Number of viable pups (d4)) : Number of viable pups (d0) x 100
Sex ratio: (Number of pups exa min ed − Number of males) : Number of pups examined x 100

Results and discussion

Results: P0 (first parental generation)

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
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Observed effects were regarded as common background.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Test material administration was considered to have no impact on the duration of the mating period. Successful coitus generally occurred within up to 7 days of pairing (cohabitation).The mating and fertility indices were 100% in all groups. The gestation index was also 100%.
The mean duration of pregnancy was similar in the control and test material treated groups and varied from 22.8 days (control), 22.8 days (30 mg/kg, Low dose), 22.5 days (100 mg/kg, Mid dose), to 22.7 days (300 mg/kg, High dose group). All the parturitions were normal.
There was no effect of treatment on the oestrus cycle or reproductive parameters. There were no differences or effects that could be ascribed to treatment on the pre/post-implantation, post-natal or total mortality values (%) at up to and including 300 mg/kg bw/day.
The number of corpora lutea and implantation sites were comparable in the treated groups up to and including 300 mg/kg bw/day with the mean value recorded in the Control group.

ORGAN WEIGHTS (PARENTAL ANIMALS)
The values were within physiological range, and were not considered to reflect an adverse effect.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There was no evidence of test material-related histological findings in the High dose animals or macroscopic observations from all groups in the reproductive organs.
Histopathological evaluation of the male gonads as well as testicular interstitial cell structure, the spermatogenic cells representing different phases of the development and differentiation of the spermatozoons were similar in Control and High Dose males. The follicular, luteal and interstitial compartments of the ovary as well as epithelial capsule and stroma were similar histological structure in both Control and High Dose females.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: NOAEL = highest dose tested

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

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:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
Few pups were cannibalised and the incidence was similar across all experimental groups including control.
The number of viable pups on PND4 as well as pups survival indices on PND0 and PND4 were comparable to control values at up to and including 300 mg/kg bw/day.
The sex ratios were similar in the Control and treated groups.

CLINICAL SIGNS (OFFSPRING)
No external abnormalities ascribed to treatment were detected at the clinical or external macroscopic examinations of the pups.

BODY WEIGHT (OFFSPRING)
There was no effect of treatment on the offspring body weight or body weight gain.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: NOAEL = highest dose tested, F1 observations up to 4 days of age.

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
In conclusion, in this study - OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test) - daily oral gavage administration of WS400402 to Wistar rats at dose levels of 30, 100 and 300 mg/kg day was not associated with signs of reproduction toxicity and the NOAEL was considered to be 300 mg/kg day.