2-Butenedioic acid (Z)-, ester with 1,2-propanediol, compd. with 2-(dibutylamino)ethanol

Endpoint summary

Key value for chemical safety assessment

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:

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.

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)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system 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

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

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

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

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: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

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.

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

Key result

Critical effects observed:

no

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

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.

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.

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The reproduction toxicity screening test is adequate for an initial assessment of effects on fertility; it is of high quality and reliability. The NOAEL was derived at the highest dose tested, i.e. 300 mg/kg/day.

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 a sub-chronic oral toxicity study in rats at the end of the study period no effects were seen on estrous cycles as well as on sperm parameters up to the highest dose level.

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:

Jan. 2017 - Sep. 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BYK Chemie LS-15-109A, LS-15-109B, LS-15-109C
- Expiration date of the lot/batch: 1-Nov-2018
- Purity test date: UVCB

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient temperature in the dark
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: stable

Species:

rat

Strain:

other: RccHan; WIST rat

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS Ltd
- Age at study initiation: 77 - 84 days (at Day 0 of gestation)
- Weight at study initiation: 180-218 g (at Day 0 of gestation)
- Fasting period before study: no
- Housing: 1 animal per cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days before commencement of pairing

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

IN-LIFE DATES: From: To:

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amounts of test material were weighed. Approx. 50% of the final volume of vehicle was added and magnetically stirred until the test item was uniformly mixed. The remaining vehicle was added to achieve the required volume and the formulation was mixed using a magnetic stirrer until homogeneous.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 0, 16, 40, 100 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 GC with flame ionisation detector. Mean concentrations were within an acceptable range 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.:

80 mg/kg bw/day (actual dose received)

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Dose / conc.:

500 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:
The dose levels selected for investigation in this study (0, 80, 200 and 500 mg/kg/day) were chosen in conjunction with the Sponsor based on the results of a preliminary rat embryo-fetal study conducted at these laboratories (Envigo study No. PW46CS), in which dose levels of 300, 500 or 700 mg/kg/day were employed. At 700 mg/kg/day there were marked effects on maternal weight gain, mean food intake was low throughout the treatment period and a spectrum of post-dosing signs were apparent including tremors, piloerection, reduced body temperature (cold to touch), underactive behaviour, partially closed eyes, abnormal/unsteady gait, repetitive movements, pallor and hunched posture. In addition, one female experienced a short-lasting convulsion at approximately 10-15 minutes after dosing on Day 11 of gestation and although this event did not impact upon subsequent maternal body weight gain, food intake or litter data (compared to the other females in the group), placental and fetal weights in this litter were very low. Based on all of these effects, the dose level of 700 mg/kg/day was considered unsuitable for further investigation in pregnant rats.

At 500 mg/kg/day, although maternal body weight gain and food intake were lower than Control and some post dosing observations were apparent, the magnitude, nature and incidence of the effects were lower than observed at 700 mg/kg/day, and since the OECD414 guideline states that “the highest dose level should be chosen with the aim to induce some developmental and/or maternal toxicity”, 500 mg/kg/day was considered suitable for selection as the high dose level in this current study. The low and intermediate dose levels of 80 and 200 mg/kg/day were chosen to achieve a dose response and/or aid in the determination of a No-Observed-Adverse-Effect-Level (NOAEL).

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
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: 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 external: 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 7 studies performed during 2016 with the same rat strain are provided in the report.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no signs observed at routine physical examination that were related to treatment with BYK-LP X 20954.
Test item-related signs in relation to dose administration were primarily restricted to animals given 500 mg/kg/day and comprised a low incidence of underactive behaviour, reduced body temperature (cold to touch), piloerection, body tremors, reduced body tone, unsteady gait and flat or hunched posture. A very low incidence of piloerection was also observed at 200 mg/kg/day.

At 500 mg/kg/day there was also a low incidence of salivation, post-salivation coat staining and chin rubbing; a single incidence of salivation was recorded in the 200 mg/kg/day group. These signs are commonly observed with oral gavage administration and are considered to represent distaste of the formulations and not an effect of treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There was one premature death during the course of the study which was unrelated to treatment with BYK-LP X 20954. Female No. 38 in the 80 mg/kg/day group was killed for reasons of animal welfare on Day 19 of gestation due to signs of piloerection, a firm area on the ventral abdomen and a prominent vagina. Macroscopic examination revealed the uterus and vagina were distended with a large amount of yellow fluid; this female was pregnant with 14 live and grossly normal fetuses.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During Days 6-8 of gestation, mean body weight stasis or slight mean body weight loss (-2 grams) was apparent among females given 200 or 500 mg/kg/day respectively when compared to the performance of Controls (+3 grams); these differences from Control attained statistical significance. In the 500 mg/kg/day group, mean body weight gain was 26% lower than Control during Days 8-15 of gestation, with statistical significance attained, but was similar to Control thereafter. Overall mean weight gain from Day 6-20 of gestation in the 500 mg/kg/day group was statistically significantly lower than Control (-13%), but was unaffected by treatment at 80 or 200 mg/kg/day.
The mean gravid uterine weight on Day 20 of gestation was similar in all groups. When overall mean body weight gain was adjusted for the contribution of the gravid uterus, net mean body weight gain was 45% lower than Control at 500 mg/kg/day, with statistical significance attained, but was similar to Control at 80 or 200 mg/kg/day.

see "overall remarks, attachments" : Table Body weight and body weight change

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption was statistically significantly lower than Control in a dosedependent manner (ranging from 12% to 29%) for females given 200 or 500 mg/kg/day during Days 6-9 of gestation. At 500 mg/kg/day, mean food consumption remained 21% lower than Control during Days 10-13 of gestation. From Day 18 of gestation, mean food consumption was marginally lower than Control at 200 or 500 mg/kg/day, although in the absence of a dose response relationship.
see "overall remarks, attachments" : Table Food consumption

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:

not specified

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

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

>= 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks on result:

other: NOAEL = highest dose tested

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
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined

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:

effects observed, non-treatment-related

Description (incidence and severity):

Incidental fetal findings were unrelated to maternal treatment.
see "overall remarks, attachments" : Table Fetal examinations

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Incidental fetal findings were unrelated to maternal treatment.
see Table Fetal examinations

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Incidental fetal findings were unrelated to maternal treatment.
see "overall remarks, attachments" : Table Fetal examinations

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

>= 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: NOAEL = highest dose tested

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

At the highest dose level tested, 500 mg/kg bw/day, maternal toxicity was observed. No developmental toxicity was observed at the highest dose level.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

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

Following the results of the available studies classification of WS400402 regarding reproductive or developmental toxicity is not warranted according to REGULATION (EC) 1272/2008.