Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Remarks:
from OECD 422 study with reproduction / developmental toxicity screening
Type of information:
experimental study
Adequacy of study:
supporting 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:
other: 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
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
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%.
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.
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
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.

Examinations

Maternal 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: Maternal animals 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).

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Day 5 of lactation (PND5)
- Organs examined: uterus, vagina, ovaries

HISTOPATHOLOGY :
Detailed histological examinations was performed in all maternal animals of control and high dose groups of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes (with and wtihout cervix)
- Number of corpora lutea: Yes
- Number of implantations: Yes

Post implantation survival was determined.

In addition, gestation length (time elapsing between detection of mating and commencement of parturition) was recorded.
Fetal examinations:
- External examinations: from PPD0 to PPD4, all pubs per litter
parameters observed: number and sex of pups, stillbirths, live births; postnatal mortality; presence of gross anomalies, weight gain (PPD0-PPD4), physical or behavioural abnormalities
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.
Indices:
Female Mating Index: Number of sperm-positive females : Total Number of females 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

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: maternal animals

General toxicity (maternal animals)

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
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Behaviour (functional findings):
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
in high dose females of the OECD 422 study microscopic findings were observed in kidney

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
see Tables on F1 generation attached in attached background material
Total litter losses by resorption:
no effects observed
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
see Tables on F1 generation attached in attached background material
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
see Tables on F1 generation attached in attached background material
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
see Tables on F1 generation attached in attached background material
Other effects:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: In High dose group: changes in haematology, in some parameters of clinical chemistry, slightly increased liver weight, histological minimal changes in kidneys

Details on maternal toxic effects:
In the High dose group (1000 mg): Changes in haematology (decrease of 8-12% in erythrocyte count, hemoglobin concentration and hematocrit value) and clinical chemistry parameters (increase in albumin concentration and albumin to globulin ration, higher calcium, sodium and chloride concentrations). Animals had slightly increased liver weights (16-19%) changes. 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).
Slightly increased pre-implantation, total intrauterine and postnatal mortality values and slightly lower number of born pups as well as slightly lower body weight gain of pups were observed. These small differences were considered to be probably secondary to maternal toxicity.

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
>= 300 - <= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
see Tables on F1 generation attached in attached background material
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
see Tables on F1 generation attached in attached background material
Changes in sex ratio:
no effects observed
Description (incidence and severity):
see Tables on F1 generation attached in attached background material
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
see Tables on F1 generation attached in attached background material
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
see Tables on F1 generation attached in attached background material
External malformations:
no effects observed
Description (incidence and severity):
see Tables on F1 generation attached in attached background material
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No external abnormalities ascribed to treatment were detected at the clinical or external macroscopic examinations of the pups. At 300 mg/kg in one male pup aplasia of the tail was detected without any internal abnormality. The incidence of this finding was low, within the physiological range expected in the population of Wistar rats and considered without toxicological significance, or to reflect a test item or adverse effect.

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 and was 1 of 160 in control, 1 of 156 in Low dose, 6 of 155 in Mid dose and 3 of 89 in High dose. Three of 6 dead pups in the Mid dose and 3 of 3 dead pups in the High dose originated from one litter. All of these pups were lost on Day PND1, except one pup of Mid dose (PND4) and control (PND2). These pups were probably cannibalized.The sex ratios were similar in the Control and treated groups.

There was no adverse effect of treatment on the offspring body weight or body weight gain. Slightly higher mean body weights values in the Low dose group and slightly lower mean overall body weight gain values (PND0-4) in the High dose group were regarded as individual, biological variability, without
toxicological significance.

Effect levels (fetuses)

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

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
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.