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Effects on fertility

Link to relevant study records
Reference
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
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
of 1996
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Sprague Dawley rats, strain: Crl:CD(SD) with appropriate range of bodyweight at study start.
- Source: Charles River (UK) Ltd.
- Age at treatment start: ca. 72 days.
- Weight at treatment start: Males: minimum 325 g, maximum 387 g,
Females: minimum 229 g, maximum 280 g.
- Housing Inside a barriered rodent facility:
all animals pre-pairing + toxicity subgroups: In groups up to 5 by sex in solid floor polycarbonate cages.
during pairing (1 male+1 female/cage): In RB3 modified polypropylene cages with stainless steel grid-floor over absorbent paper-lined trays.
males after pairing: In groups up to 5 in solid floor polycarbonate cages.
females during gestation and lactation: Females housed individually (+litter) in solid floor polycarbonate cages.
- Bedding material (in solid floor cages): Wood based bedding, sterilised by autoclaving before use.
- Cage enrichment: Aspen chew block + plastic shelter (except during pairing or post Gestation Day 20).
- Diet (ad libitum): Standard rodent diet (SDS VRF1 Certified) without antibiotic, chemotherapeutic or prophylactic agent.
- Fasting (diet withheld): Main phase males and Toxicity phase females overnight before blood sampling for clinical pathology.
- Water (ad libitum): Potable drinking water from the public supply.
- Acclimation period: 7 days before treatment start, under laboratory conditions.

Routine analysis of the batch of diet used and water, chew blocks and bedding material did not provide evidence of contamination that might have prejudiced the study.

IN-LIFE DATES:
- Duration of test, males & toxicity phase females: Five weeks
Duration of test, main phase females (i.e. reproductive subgroup): From 14 days prior to pairing to day 7 of lactation.
Duration of test, offspring: From birth to day 7 of lactation.

ENVIRONMENTAL CONDITIONS

Air conditioned room kept at positve pressure without re-circulation of the filtered fresh air supplied to the room.
Controlled environment, environmental conditions were set at:
- Temperature (°C): 22 ± 3°C
- Relative Humidity (%): 40 to 70%
- Photoperiod (artificial lighting): 12 hrs day / 12 hrs night
- Rate of air exchange: At least 15 changes/h
Deviations from the target ranges for temperature and relative humidity were not evident.

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
Treatment of parental animals by oral gavage administration. Test substance was not directly administered to F1 animals.

- Concentration in vehicle: The concentration of the test material in vehicle varied between dose groups thus allowing constant dosage volume in terms of mL/kg bw/day.
- Amount (dose volume by gavage): 5 mL/kg bw/day.
Actual dose volumes were calculated at about weekly or shorter intervals accounting for the latest bodyweight.

- For concentrations of test material in vehicle at different dose levels, see Table 1 in "Any other information on materials and methods incl. tables"

- Justification for choice of vehicle:
The suitability of propylene glycol as a vehicle was established during the 7-day range-finding study:
Endpoint study record "7.5.1 Repeated dose toxicity: oral - 7d_range-finding_gavage_HLS_GAH0116".
In addition, in the present main study, concentrations of dose formulations were chemically analysed.


Details on mating procedure:
- Male/female ratio per cage: 1/1
- Length of cohabitation: At the most 12 days, until proof of pregnancy was confirmed. 
- Proof of successful mating: Formation of at least one copulation plug and a sperm positive vaginal smear.
The day this was found was referred to as day 0 of gestation.
(During cohabitation, females were checked every morning for pregnancy).

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Chemical analysis of test material formulations by high performance liquid chromatography coupled with a mass spectrometer (HPLC-MS/MS).
- Mean concentrations (verified for first and last treatment week) of the test material formulations were confirmed at each dose level.
Chemical analysis confirmed that the mean concentrations of WS400123 in prepared formulations were 97.0% to 112.9% of the corresponding
nominal concentration, thus confirming acceptable accuracy of formulation for dosing of the animals.
- Homogeneity and stability of test material formulations at 2 and 200 mg/L and at storage and handling conditions similar to those adopted for dosing of the animals were confirmed.
Duration of treatment / exposure:
- Treatment period, males & toxicity phase females: Daily, for five consecutive weeks, in males commencing 14 days prior to pairing
- Treatment period, main phase females (i.e. reproductive subgroup): 43 to 47 days (from 14 days prior to pairing to day 6 of lactation)
- Offspring were not dosed
Frequency of treatment:
Daily, 7 days/week (during parturition, dosing omitted as appropriate)
Details on study schedule:
- Age at mating of the mated animals in the study: 12 to 14 weeks
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:
Toxicity phase animals: */ 5 females
Main phase animals (i.e. reproductive subgroups): 10 males / 10 females
*Explanatory note by the notifier:
Examinations assigned to the toxicity phase females to meet the requirements of a 28-day repeat dose oral toxicity study were also assigned to 5 (for some examinations to 10) main phase males per dose group. Therefore, these 5 main phase males per dose group are called also "toxicity subgroup" in the present robust study summary for clarification. After pairing with main phase females, all males were killed at the same time (Week 6).
Control animals:
yes, concurrent vehicle
Details on study design:
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 (toxicity phase) and reproductive subgroup animals (main phase), whereby 5 of the 10 F0 males (used for pairing) per dose group formed the toxicity male subgroups.

Dose selection was based on the results of a 7-day preliminary oral toxicity study in the rat in which dose levels of 100, 300 or 1000 mg/kg/day did not have any overt treatment-related effects on young adult animals (females nulliparous and non-pregnant).
Positive control:
Not included in the study.
Parental animals: Observations and examinations:
Clinical observations performed and frequency:
- Clinical signs : At least twice a day (before and after administration)
- Detailed physical examination
and arena observations: Before treatment start and at least once a treatment week (except 1 or 2 females taking 5 to 12 days for pairing).
- Functional Observation Battery: During treatment week 5 (before dosing) on all toxicity subgroup animals (5 parental males/group inclusively)*.
- Body weight, all males: About weekly throughout the study.
Body weight, Toxicity Females: About weekly throughout the study.
Body weight, Repro. Females: Weekly for pre-pairing period; on gestation days 0, 6, 13, 20; on lactation days 1, 4 & 7.
- Food consumption, all males: Weekly for pre-pairing period and for the period after mating.
Food cons., Toxicity Females: About weekly throughout the study.
Food cons., Repro. Females: Weekly for pre-pairing period, during gestation for days 0-6, 6-13, 13-20, during lactation for days 1-4 & 4-7.
- Hematology: During treatment week 5 after functional observation battery*
- Blood (plasma) chemistry: During treatment week 5 after functional observation battery*

* Examinations confined to toxicity subgroup animals are marked above with an asterisk*
and are detailed in the separate endpoint study record "7.5.1 Repeated dose toxicity: oral - Repeat dose tox combined_gavage_rat_HLS_GAH0117"

Explanatory note
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 (toxicity phase) and reproductive subgroup animals (main phase), whereby 5 of the 10 F0 males (used for pairing) per dose group formed the toxicity male subgroups.
Oestrous cyclicity (parental animals):
Frequency of vaginal oestrus was determined by examination of vaginal smears taken daily from all main phase (i.e. reproductive subgroup) females from the beginning of the treatment period to the day of confirmed copulation.
- Regular: All observed cycles of 4 or 5 days
- Irregular: At least one cycle of 2, 3 or 6 to 10 days
- Acyclic: At least 10 days without oestrus
Sperm parameters (parental animals):
Parameters examined in male parental animals:
- testis weight,
- epididymis weight
- 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 7.

LITTER PARAMETERS EXAMINED
- From Day 20 post copulation 3 times a day checks for evidence of parturition, any difficulties and numbers of live and dead offspring.
- Total litter size on day 1 of age and mortality/live litter size on each day until 7 days after littering.
- Sex ratio expressed as percentage males and calculated for total offspring on Day 1 and for live offspring on Days 1 & 7
(No. of male pups in litter/No. of offspring in litter) x 100
- Gestation index (No. of live litters born on day 0/No. of living pregnant females) x 100
- Clinical signs, recorded daily
- Body weight of live pups (on days 1, 4 and 7 after littering) and weight change from Days 1-4, 4-7 and 1-7.



Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes, see below
WEIGHING OF ORGANS: Yes, see below
HISTOPATHOLOGY: Yes, see below

Terminal sacrifice
- all males (F0) and toxicity subgr. females: Killed in Week 6, after completion of the Treatment Week 5 investigations.
- reproductive subgr. females & offspring: Killed on Day 7 post partum.
(1 control & 1 mid dose female were killed on the respective Day 25 after mating, as they had failed to litter)
(1 mid dose female was found dead on Treatment Day 8 due to an intubation error)
(1 high dose female was found dead on Gestation Day 6 due to an intubation error)

Gross pathology:
- adult/parental animals: Full macroscopic examination.

Organs Weights:
- toxicity subgroups: Adrenals, brain, epididymides, heart, kidneys, liver, ovaries, prostate, seminal vesicles with coagulation
gland, spleen, testes, thymus, uterus with cervix & oviducts.
- dams: Ovaries

Histopathology:
- toxicity subgroups*: The following organs were microscopically observed for the control and 1000 mg/kg bw/day groups:
Brain, eyes, pituitary gland, thyroid with parathyroids, heart, thymus, liver, spleen, adrenals, kidneys, testes,
epididymides, ovaries, lung, trachea, oesophagus, stomach, duodenum, jejunum, ileum, caecum, rectum, colon, Peyer's patch, lymph node (axillary, mesenteric), urinary bladder, uterus (with cervix & oviducts), vagina,
spinal cord, sciatic nerve, skeletal muscle, sternum with marrow, seminal vesicle & coagulation gland, prostate.
In addition, any gross lesions for all adult animals from all dose groups were examined by light microscopy.

* Histopathology examination only on control & high dose groups, 5 males and 5 females per group

- reproductive subgroups All above organs/tissues from premature deaths (1 mid dose & 1 high dose females) and any gross lesions from all adult animals from all dose groups were examined by light microscopy.
Postmortem examinations (offspring):
Pup survivors were killed on Day 7 post partum and had a careful external macroscopic examination for gross abnormalities.

Externally abnormal offspring and premature deaths had an internal macroscopic examination including assessment of the presence of milk in the stomach, where possible. (Missing or grossly autolysed or cannibalised offspring could not be examined).
Statistics:
As detailed in Endpoint study record "7.5.1 Repeated dose toxicity: oral - Repeat dose tox combined_gavage_rat_HLS_GAH0117"
Reproductive indices:
- Pre-coital interval (pairing days until detection of mating)
- No. of animals mating (evidence of successful copulation, i.e. at least one copulation plug or a sperm positive vaginal smear)
- No. of animals achieving pregnancy
- Percentage mating (No. of animals mating/No. of animals paired) x 100
- Fertility index (No. of animals achieving pregnancy/ No. or animals paired) x 100
- Conception rate (No. of animals achieving pregnancy/No. of animals mated) x 100
- Gestation length (time elapsing between detection of mating and commencement of parturition)
- No. of living pregnant females
- For further reproductive parameters, see also the above section "Litter observations" and section "Offspring viability indices" below.
Offspring viability indices:
- Post-implantation survival index  (Total no. of pups born/Total no. of uterine implantation sites) x 100
- Live birth index (No. of live pups on Day 1 after littering/Total no. of pups born) x 100
- Viability index (No. of live pups on Day 4 after littering /No. of live pups on Day 1 after littering) x 100
- Lactation index (No. of live pups on Day 7 after littering /No. of live pups on Day 1 after littering) x 100
- For further parameters indicative of the viability of the offspring, see also the above section "Litter observations"
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One mid dose female and one high dose female of the reproductive subgroups were found dead following intubation errors.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY
There were no premature deaths, clinical signs or adverse effects on neurobehaviour attributable to treatment with the test material. One mid dose female and one high dose female of the reproductive subgroups were found dead following intubation errors. One control and one mid dose females were killed on the respective Day 25 after evidence of mating, because they had failed to litter.

BODYWEIGHT, WEIGHT GAIN AND FOOD CONSUMPTION
Bodyweight and food consumption were unaffected by treatment with WS400123 in treated males and nulliparous, nonpregnant females. In dams receiving 1000 mg/kg bw/day, mean bodyweight gain was slightly lower than in concurrent controls during Lactation Days 4-7, but did not differ statistically significantly from controls overall during Lactation Days 1-7 and therefore was considered to represent normal biological variation.

REPRODUCTIVE ENDPOINTS
Oestrous cycles, pre-coital interval, mating performance, fertility, gestation length, gestation index and litter size were not affected by treatment.

GROSS PATHOLOGY
Macroscopic findings attributable to treatment with the test material were not evident. The numbers of uterine implantation sites in main phase females recorded on Lactation Day 7 were unaffected by treatment with WS400123.

ORGAN WEIGHTS
Toxicologically significant effects on organ weights were not evident in males and nulliparous females following 5 treatment weeks, and ovary weights were unaffected by treatment with the test material in dams on Lactation Day 7. In addition, there were no histopathological correlates to any minor deviations in organ weights from concurrent controls.

HISTOPATHOLOGY
Microscopic pathology findings attributable to treatment with the test material were not evident in main phase males and toxicity subgroup females (nulliparous and non-pregnant) after five weeks of treatment. In addition, evaluation of the seminiferous testicular tubules regarding their stage in the spermatogenic cycle and regarding the integrity of the various cell types present within the different stages did not reveal any cell or stage abnormalities. Main phase females (reproductive subgroup) killed on Lactation Day 7 were not histopathologically examined. The two premature deaths (1 mid dose & 1 high dose females macroscopically and microscopically examined) were attributed to trauma during dose administration (intubation error), rather than to an adverse effect of the test material itself.
Key result
Dose descriptor:
NOEL
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'
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
on bodyweight or bodyweight gain to Day 7, the day of scheduled sacrifice of the pups.
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
disclosed by careful external macroscopic examination of all pups surviving to Day 7 and by necropsy of externally abnormal pups and decedents
Histopathological findings:
not examined
Other effects:
no effects observed
There was no indication of any effect of parental treatment with WS400123 on litter size and survival, clinical condition, sex ratio, bodyweight, bodyweight gain or development of the offspring until Lactation Day 7.
Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: NOEL = highest dose tested. Offspring development up to Day 7 of age.
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In the subchronic oral toxicity study in rats, no effects were observed on reproductive organs and tissues. Estrous cycles were NOT AFFECTED after 12 and 13 weeks of dosing up to and including the highest dose level of 1000 mg/kg/day. At the end of this subchronic study also no effects were observed on sperm parameters (sperm motility, testicular spermatid numbers and sperm morphology).

Effects on developmental toxicity

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Oct 2018 - Jul 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Ltd
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approx. 75 days
- Weight at study initiation: females 225-270 g
- Fasting period before study: no
- Housing: 4 females per cage during acclimatisation, 1 male and 1 female per cage during pairing ,1 female per cage during gestation
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 9 days

DETAILS OF FOOD AND WATER QUALITY: SDS VRF1 certified pelleted diet; potable water from public supply

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 40-70%
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 h / 12 h
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amounts of test material were weighed and approx. 50% of the final volume of vehicle was added and mixed with a magnetic stirrer until homogeneous. Then the final volume was adjusted and stirred until homogeneous.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 20, 60, 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg bw
- Lot/batch no. (if required):
- Purity:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations of dose formulations were analysed by HPLC/MS. The mean concentrations of WS400123 analysed in test formulations were within 5% of the nominal concentrations.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1 : 1
- Length of cohabitation: until evidence of mating
- Verification of same strain and source of both sexes: yes, a colony of stud males was maintained
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
from day 6 till day 19 of gestation
Frequency of treatment:
daily
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:
20 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: In an OECD422 study in Sprague Dawley rats, oral administration of WS400123 at dose levels of 100, 300 and 1000 mg/kg/day was well tolerated without adverse effects.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations : visual inspection for ill-health or reaction to treatment

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: days 0, 5, 12, 18, and 20

BODY WEIGHT: Yes
- Time schedule for examinations: days 0, 3, and daily from 6-20

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: full macroscopic examination of tissues
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: number of fetuses (live and dead)
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes : all per litter
Statistics:
The following sequence of statistical tests was used for body weight, gravid uterus weight, food consumption, corpora lutea, implantations, live young, fetal, placental and litter weight data:
A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. For pretreatment data, analysis of variance was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using t-tests, with the error mean square from the one-way analysis of variance, were made. For all other comparisons t/The F1 approximate test was applied. This test is designed to detect significant departure from monotonicity of means when the main test for the comparison of the means is a parametric monotonic trend test, such as Williams’ test (Williams 1971, 1972). The test statistic compares the mean square, NMS, for the deviations of the observed means from the maximum likelihood means, calculated under a constraint of monotonicity with the usual error mean square, EMS. The null hypothesis is that the true means are monotonically ordered. The test statistic is F1 = NMS/EMS which can be compared with standard tables of the F-distribution with 1 and error degrees of freedom. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test (Dunnett 1955, 1964) was performed instead.

A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. For pretreatment data, Kruskal-Wallis’ test (Kruskal and Wallis 1952, 1953) was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using Wilcoxon rank sum tests (Wilcoxon 1945) were made.
Indices:
Prenatal losses were separated into pre- and post-implantation phases. Pre-implantation loss was considered to reflect losses due to non-fertilization of ova and failure to implant. It was calculated from the formula:
Pre-implantation loss (%) = (Number of corpora lutea – Number of implantations) x 100 / Number of corpora lutea

Where the number of implantations exceeded the number of corpora lutea observed, pre implantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to have occurred).
Post-implantation loss was calculated from the formula:
Post-implantation loss (%) = (Number of implantations – Number of live fetuses) x 100 / Number of implantations

All group values and SD (as appropriate) were calculated from the individual litter values
Historical control data:
Historical control data from 10 studies performed from April 2018 to February 2019 with the same rat strain are provided in the report.
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
one animal of the control group died due to mal-dosing during gavage administration.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
see "overall remarks, attachments" : Figure Body weight of dams
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
see "overall remarks, attachments" : Table Litter data
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
see "overall remarks, attachments" : Table Litter data
Early or late resorptions:
no effects observed
Description (incidence and severity):
see "overall remarks, attachments" : Table Litter data
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
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:
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
see "overall remarks, attachments" : Table Litter and fetal weights
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
see "overall remarks, attachments" : Table Litter data
Changes in sex ratio:
no effects observed
Description (incidence and severity):
see "overall remarks, attachments" : Table Litter data
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
see "overall remarks, attachments" : Table Litter and fetal weights
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
see "overall remarks, attachments" : Table Fetal major abnormality findings
Skeletal malformations:
no effects observed
Description (incidence and severity):
see Table Fetal minor skeletal abnormality findings
Visceral malformations:
no effects observed
Description (incidence and severity):
see "overall remarks, attachments" : Table Fetal minor visceral abnormality findings
Other effects:
no effects observed
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:
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Executive summary:

Treatment of pregnant rats with WS400123 was well tolerated and there were no test-item related deaths.  Clinical condition, body weight performance, gravid uterine weight, adjusted body weight gain and food consumption were not adversely affected by WS400123 at any dose level investigated.  There were no macroscopic abnormalities detected at scheduled termination.  

There was no effect of maternal treatment with WS400123 on the mean numbers of corpora lutea, implantations, resorptions, live young, pre- and post- implantation losses, sex ratio or on placental, litter and fetal weights at any dose level investigated.

Evaluation of the fetuses did not reveal any adverse major, minor, skeletal or visceral abnormalities which were considered to be related to maternal treatment with WS400123.  

Based on the results of this embryo-fetal toxicity study, it was concluded that the dose level of 1000 mg/kg/day represented the No Observed Adverse Effect Level (NOAEL) for maternal toxicity and for embryo-fetal survival, growth and development.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

In the OECD 422 Study, it was demonstrated that treatment with WS400123 at doses up to and including 1000 mg/kg bw/day for 35 to 57 consecutive days did not induce any adverse effects on reproductive and developmental endpoints. After subchronic oral exposure of rats no effects on estrous cycles and sperm parameters were observed up to and including the highest dose level of 1000 mg/kg/day.

In the developmental toxicity study in rats no effects were observed in dams and embryos up to and including the highest dose level of 1000 mg/kg/day.

Based on these results no classification for reproductive toxicIty is warranted according to European classification rules [REGULATION (EC) 1272/2008].

Additional information