1,3-Benzenedimethanamine, reaction products with polyethylene glycol mono-Bu ether and TDI

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:

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: 71 days.
- Weight at treatment start: Males: minimum 346 g, maximum 392 g,
Females: minimum 226 g, maximum 273 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: 6 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): 21 ± 2°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 body weight.

- 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_GAH0058".
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 14 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).
- Concentrations (verified at first and last treatment week) of the test material formulations were confirmed at each dose level.
- Chemical analysis confirmed that the mean concentrations of WS400517 in prepared formulations were 95.5% to 101.5% of the corresponding
nominal concentration, thus confirming accuracy of formulation.

Duration of treatment / exposure:

- Treatment period, males & toxicity phase females: Daily, for five consecutive weeks, in males commencing 14 days prior to mating
- Treatment period, main phase females (i.e. reproductive subgroup): 44 to 57 days (from 14 days prior to mating 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

Remarks:

Doses / Concentrations:
0 (vehicle control), 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested

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 male survivors 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.
- Functional Observation Battery: During treatment week 5 (before dosing) on all toxicity subgroup animals (5 parental males/group inclusively)*.
- Body weight, all males: Weekly throughout the study.
Body weight, Toxicity Females: Weekly throughout the study.
Body weight, Repro. Females: Weekly for pre-pairing period; on gestation days 0, 6, 13, 20; on lactation days 1 & 7.
- Food consumption, all males: Weekly for pre-pairing period and for the period after mating.
Food cons., Toxicity Females: 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_GAH0059"

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 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 and toxicity subgr. females: Killed in Week 6, after completion of the Treatment Week 5 investigations.
(1 mid dose male was found dead on the day of scheduled termination)
- reproductive subgr. females & offspring: Killed on Day 7 post partum.
(1 low dose female was killed following the death of her litter on lactation day 1)

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

Organs Weights:
- main phase and tox. subgr. adults: Adrenals, brain, epididymides, heart, kidneys, liver, lungs & bronchi, ovaries, pituitary, prostate, seminal
vesicles & coagulation gland, spleen, testes, thymus, thyroid with parathyroids, uterus with cervix & oviducts

Histopathology:
- toxicity subgroups: The following organs were microscopically observed for the control and 1000 mg/kg bw/day groups:
Brain, eyes, Harderian glands, optic nerves, pituitary gland, thyroid with parathyroids, heart, thymus, liver,
spleen, adrenals, kidneys, testes, epididymides, ovaries, lung, trachea, esophagus, 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, skin with mammary glands,
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.
- reproductive subgroups All above organs/tissues from premature deaths (1 low dose female and 1 mid dose male) 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.
Full macroscopic examination of decedent and surviving pups including assessment of the presence of milk in the stomach, where possible.
(Missing or grossly autolysed or cannibalised pups 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_GAH0059"

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 and 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

Description (incidence and severity):

attributable to treatment with the test material

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):

in toxicity phase females and parental males. In parental females only gross lesions were histopathologically examined.

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY
One female of the low dose group (100 mg/kg/day) was killed following the death of her litter on lactation day 1 and one male of the mid dose group (300 mg/kg/day) was found dead on the day of scheduled termination. These deaths were not attributable to treatment with WS400517 and their causes could not be elucidated. In the mammary glands of the affected dam only slight secretory activity was evident.

Toxicologically relevant clinical signs were not evident. Transient chin rubbing occurring more frequently with increasing dose and isolated incidences of salivation in a few animals at all dose levels may have been related to palatability of the dose formulations rather than being indicative of toxicity.

BODYWEIGHT, WEIGHT GAIN AND FOOD CONSUMPTION
Bodyweight and food consumption were unaffected by treatment with WS400517 in treated males and nulliparous, nonpregnant females. In main phase females receiving 300 or 1000 mg/kg bw/day, mean bodyweight gain was slightly higher than concurrent control during gestation days 0-6 and mean food consumption slightly higher during gestation days 0-13. These minor differences from concurrent controls were not considered to represent an adverse effect.

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 was unaffected by treatment with WS400517.

ORGAN WEIGHTS
Toxicologically significant effects on organ weights were not evident in males and females following 5 treatment weeks or in females on lactation Day 7. In addition, there were no histopathological correlates to any minor deviations in organweights 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.

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:

Remarks on result:

not determinable due to absence of adverse toxic effects

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 examined

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Several pups of one female of the low dose group (100 mg/kg/day) were found dead from commencement of parturition and after its completion leading to total litter loss by lactation day 1. There was no clear pathology to suggest why the litter had died, but in the mammary glands of the affected dam only slight secretory activity was evident. As the affected dam did not exhibit any clinical signs or adverse effect of treatment on bodyweight, and necropsy and subsequent examinations failed to identify any cause of death/litter loss, these deaths were not attributed to treatment.

There was no indication of adverse effects of parental treatment with WS400517 on offspring survival, clinical signs, sex ratio, bodyweight, development or macropathology findings.

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

>= 1 000 other: mg/kg bw/day; dosing refers to the dose of the lactating dam.

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Reproductive effects observed:

not specified

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

Short description of key information:
WS400517 was tested in the rat in a Combined Repeated Dose Oral (Gavage) Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD 422) at dose levels of 0 (vehicle control), 100, 300 and 1000 mg/kg bw/day. Signs of toxicity in parental animals or effects on reproductive and developmental endpoints were not evident. Therefore, the NOEL derived for all endpoints was the highest dose level applied, i.e. 1000 mg/kg bodyweight/day.

In a sub-chronic oral (gavage) toxicity study with WS400517 in rats the highest dose of 600 mg/kg bw/day was derived as NOAEL. No effects were observed on reproduction organs/tissues, i.e. oestrous cycles as well as sperm motility and morphology were unaffected after 11 and 13 weeks of dosing, respectively.

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:

Sep. 2016 - 2017

Reliability:

1 (reliable without restriction)

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 HJ-15-097
- Expiration date of the lot/batch: 1-Dec-2016
- 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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: grinding

Species:

rat

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK)
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: 71-72 days
- Weight at study initiation: females 233-277 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: 5 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:

other: propylene glycol / water (1 : 1)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amounts of test material were weighed. Small amounts of the vehicle were added and mixed with the test material in a mortar using a pestle. Any agglomerates were broken down to produce a smooth paste. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was poured into a measuring cylinder which had been wetted with the vehicle. The mortar was also rinsed with the vehicle and added to the cylinder and the suspension was made up to the required volume with vehicle. The suspension was transferred into a mixing container and mixed, using a Silverson mixer, carefully until a slight bitty suspension was achieved (not a paste) and then stirred magnetically for at least 30 minutes.
A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test item.


VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 10, 30, 100 mg/ml
- Amount of vehicle (if gavage): 10 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 with evaporative light scattering detector. Mean concentrations were within 10% 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:
The dose levels selected for investigation in this study of embryo-fetal development (0, 100, 300 and 1000 mg/kg/day) were selected based on the results of a combined repeat dose toxicity study and reproductive/developmental toxicity screening study in the rat. In that study the same dose levels were investigated and there were no treatment-related effects observed; it was concluded that, in terms of general toxicity, reproductive function and fertility, the high dose of 1000 mg/kg/day was the No-Observed-Effect-Level (NOEL).

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: 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 11 studies performed during 2016 with the same rat strain are provided in the report.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

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:

>= 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

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:

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:

not examined

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

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 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

In the OECD 422 Study, it was demonstrated that treatment with WS400517 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.

In the developmental toxicity / teratogenicity study no effects were observed including the highest dose of 1000 mg/kg bw/day.

There were no findings or adverse effects warranting the classification of WS400517 regarding reproductive or developmental toxicity according to European classification rules [REGULATION (EC) 1272/2008].