Registration Dossier

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:
guideline study
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 material form:
other: semi-solid, wax-like (at 20°C)
Details on test material:
Name of the test material (as cited in the Study): WS400102

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: 10 - 11 weeks
- Weight at study initiation: Males: 340 g – 395 g, Females: 230 g - 265 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

According to the results of a stability study, the test material was stable in Propylene glycol in concentration range of 20-200 mg/mL for 24 hours at room temperature (RT, 15-30ºC) and for up to three days when stored refrigerated at 2-8ºC (protected from light) and the homogeneity of the formulations was satisfactory.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: for up to 6 days
- 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 WS400102 formulations for concentration and homogeneity was performed using validated HPLC-UV method (CiToxLAB study code 11/350-316AN). The concentration analysis was performed on 3 occasions, during the first, fourth and last weeks of the treatment period. Recovery of WS400102 from propylene glycol was in the range from 97 to 105%.
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: 12-13 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:
Main groups: 12 animals/sex/dose
[Satellite group (20 femal rats): 5 animals/dose]
Offspring were not dosed.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Based on available data and information from previous experimental work, including the results of a preliminary dose range finding study in the rat (Endpoint study report 7.5.1 Dose Range Finding Study 7 days rat_WS400102) , the dose levels selected for this study were 100, 300 and 1000 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
- 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.
Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.
Litter observations:
STANDARDISATION OF LITTERS Not performed. The study ended on Lactation Day 4.

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:
The statistical evaluation of appropriate data (marked † below) was performed 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
Mortality:
no mortality 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:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Findings noted in the liver of males at 1000 mg/kg bw/day (High dose). Slight effects in kidneys of females.
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
see Table on Reproductive Performance attached in attached background material

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There was no mortality during the study.
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. Body weights were comparable to the control in all treated groups.
There was no adverse effect of treatment on food consumption.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There was no effect of treatment on the oestrus cycle or reproductive parameters.There were no differences between the Control and test material-treated groups with regard to reproductive ability or in the mating or gestation indices, or effects considered adverse or toxicologically significant in correlation with WS400102 administration.

The mating indices were 100% in all groups, while fertility indices were 92% in control and Mid groups, 83% in Low group and 75% in High dose animals.
There is no statistical difference to control, but a relationship with treatment cannot be excluded. Any differences are considered to be probably related to maternal toxicity.
WS400102 was considered to have no impact on the duration of the mating period. Successful coitus generally occurred within up to 6 days of pairing.
The mean duration of pregnancy was similar in the control and test material treated groups and varied between 22 and 23 days. The differences were considered as individual animal variation.

The gestation index was 100% in control, Low and Mid groups and 89% at 1000 mg/kg bw/day (High dose). In the High dose group, the slightly lower gestation index value was due to one female, pregnant with two implantation sites but not delivered. However, values are comparable with concurrent control data in Wistar rats. (see Table 1 "Any other information on results incl. tables")
All the parturitions were normal.

Slightly increased pre-implantation, total intrauterine and postnatal mortality values in the High dose group were observed.These small differences were considered to be probably secondary to maternal toxicity.

ORGAN WEIGHTS, GROSS PATHOLOGY, HISTOPATHOLOGY (PARENTAL ANIMALS)
Animals had slightly increased liver weights (by approximately 23-26% in males and 16-19% in females) accompanied by a minimal/mild changes in males in the form of diffuse hepatocellular vacuolation of all lobes (6/9 males) and centrilobular hypertrophy (4/9 males).
Females had histological minimal changes in kidneys in the form of bilateral vacuolation and presence of cytoplasmic eosinophilic granules in the cortical tubules (2/5 females).

No test material-related microscopic changes were noted in the reproductive organs at a dose level of 1000 mg/kg bw. 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.
Randomly distributed changes across control and treated groups were regarded as incidental or common background.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 300 - <= 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:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
other: slight changes in haematology, clinical chemistry, kidney (females only), liver (males only) in high dose animals
Organ:
kidney
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
see Tables on F1 generations attached in attached background material
Body weight and weight changes:
no effects observed
Description (incidence and severity):
see Table on body weight of F1 generation attached in attached background material
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Description (incidence and severity):
No abnormalities were observed at external examination.
Histopathological findings:
not examined
Other effects:
no effects observed

Details on results (F1)

VIABILITY/CLINICAL SIGNS (OFFSPRING)
WS400102 administered to parental generation at up to 1000 mg/kg bw/day did not lead to 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.
The number of viable pups on PND4 was slightly lower at 1000 mg/kg bw/day, when compared to control (p<0.05), which was in accordance with slightly lower number of live-born pups noted on PND0.

The survival indices of pups on PND0 and PND4 were comparable to control values at up to and including 1000 mg/kg bw/day. The incidence of mortality was negligible.
The sex ratios were similar in the Control and treated groups.

BODY WEIGHT (OFFSPRING)
There was no adverse effect of treatment on the offspring body weight or body weight gain.
Mean body weights values evaluated for all pups or on litter basis were similar in all treated groups.
Compared to control, slightly lower mean overall body weight gain values (PND0-4) were noted at 1000 mg/kg. The differences were in the range of 5% (litter basis) and 7% (all pups) and the differences attained statistical significance for all pups basis (p<0.05).

GROSS PATHOLOGY
No abnormalities in structure or behaviour were noted, therefore no necropsy with macroscopic examination was necessary.

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: NOAEL = highest dose tested, administered to the parental animals, offspring were not dosed, 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 WS400102 to Wistar rats at dose levels of 100, 300 and 1000 mg/kg bw/day was associated with week signs of reproduction toxicity in the High dose group and the NOAEL was considered to be 300 mg/kg bw/day. These effects were considered to be secondary to parental toxicity.