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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)
Deviations:
no
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: Males: 350 g – 382 g, Females: 183 g - 218 g
- Fasting period before study: overnight prior to treatment
- Housing: 5 animals of the same sex and group/cage with the exception of the mating and gestation/delivery period, when they
were paired or individually housed, respectively. (cage: Type II and/or III polycarbonate)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20,1-25
- Humidity (%): 36-70
- Air changes (per hr):15-20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
VEHICLE
- Concentration in vehicle: 20, 60, 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg bw

The test material and the vehicle was warmed up on a water bath to 50 C for approximately 10 minutes separately and mixed up on a hot stirrer plate. Once a suitable formulation was obtained, the container was removed from the plate. Pending administration to the animals, the dose formulations were stirred on a magnetic stirrer at room temperature and were protected from light.
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 WS400104 formulations for concentration and homogeneity was performed using validated HPLC method (CiToxLAB study code 11/352-316AN). The concentration analysis was performed on 3 occasions, during the first, fourth and last weeks of the treatment period. Recovery of WS400104 from propylene glycol ranged between 92% and 106%.
Duration of treatment / exposure:
Main males: 35 days (14 days pre-mating, 14 days mating/post-mating period followed by an additional week)
Main females: ca. 47 days (14 days pre-mating, for up to 7 days mating period, through gestation til PPD 4)
Satellite females (nulliparous and nonpregnant): 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.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12 animals/sex/dose
(Satellite female group: 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 WS400104 (administered via oral gavage to Wistar rats for 7 consecutive days at dose levels of 100, 300 and 1000 mg/kg bw) showed no overt adverse effects related to the test material (7.5.1 Dose Range Finding Study 7 days_WS400104).
Based on these results, the dose levels selected for the main study were 0, 100, 300 and 1000 mg/kg bw/day.

This study (OECD Guideline 422) was conducted to examine both repeated dose toxicity and reproductive/developmental toxicity as an OECD screening combined study . 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.
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
Description (incidence and severity):
Variant findings were not considered toxicologically significant.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Variant findings were not considered toxicologically significant.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Observations in high dose groups (1000 mg/kg bw/day).

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)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There was no mortality during the study and there were no clinical signs related to treatment.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
There was no adverse effect of test material on body weight or body weight gain.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Compared to control, differences attaining statistical significance (p<0.05) were noted for males and females at 300 mg/kg (Mid dose) during mating period (lower values), and for males at 300 mg/kg (Mid dose) following the mating period on Weeks 3 and 4 (higher values). The individual values remained within the normal ranges and the finding was not considered toxicologically significant or to reflect an adverse effect of WS400104.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There was no effect of treatment on the oestrus cycle or reproductive parameters.
There was no effect of treatment noted during gestation, parturition or the post-partal period.
The mean duration of pregnancy was similar in the control and test item treated groups and varied from 22.8 days (control), 22.7 days (100 mg/kg, Low dose), 22.6 days (300 mg/kg, Mid dose), to 22.8 days (1000 mg/kg, High dose group). All the parturitions were normal.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Compared to controls, the absolute and body weight relative weights of testes were slightly increased at 100 mg/kg bw/day (Low dose). The differences were in the range of 7% and were statistically significant for absolute values (p<0.05). The values were within physiological range, and were not considered to reflect an adverse effect.
For main study females, slightly higher weights of uterus were recorded at 1000 mg/kg bw/day (High dose), when compared to controls. The differences were no more than 10% for absolute and for brain related mean values and were not statistically significant.
In the satellite group of females, no significant differences in organ weights were recorded.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Test material related microscopic findings were found at 1000 mg/kg bw/day (High dose) in stomach, in the form of hyper/paraceratosis of the nonglandular gastric mucosa. This minimal to mild multifocal change occurred in 4 of 5 males and 2 of 5 females.

There was no evidence of test item-related histological findings 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 revealed normal histological pictures. 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:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

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) / CLINICAL SIGNS (OFFSPRING)
There was/were no mortality or any adverse effects considered related to treatment or toxicologically significant in the F1 generation. No abnormal behaviour of the pups was noted. No external abnormalities ascribed to treatment were detected at the clinical or external macroscopic examinations of the pups. In single pups at 100, 300 and 1000 mg/kg haemorrhage was observed on PND0.
The sex ratios were similar in the Control and treated groups.

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:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: 1000 mg/kg bw/day was the highest dose tested. Offspring development up to Day 4 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:
For the reproduction toxicity endpoints the NOAEL was considered to be 1000 mg/kg day. Dose levels of 1000 mg/kg body weight to Wistar rats for 35 consecutive days caused a minimal/mild hyper/paraceratosis of the nonglandular gastric mucosa in stomach in 4 of 5 males and 2 of 5 females. This finding is considered to be indicative of local irritation in a structure of the stomach that does not exist in man. Systemic effects were not observed.