Fatty acids, C18, unsatd., dimers, reaction products with N,N-dimethyl-1,3-propanediamine and 1,3-propanediamine

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

WS400130 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. The NOEL regarding fertility was set at 1000 mg/kg/day, i.e. the highest dose level applied.

In the sub-chronic oral toxicity study no effects on reproduction organs and tissues were observed up to and including the highest dose of 1000 mg/kg/d.

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 an internationally accepted technical guideline 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)

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: 70 days.
- Weight at treatment start: Males: minimum 357 g, maximum 403 g,
Females: minimum 225 g, maximum 267 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 of 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_GAH0091".
In addition, in the present main study, concentrations of dose formulations were determined by chemical analysis.

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 WS400130 in prepared formulations were 93.5% to 99.5% 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): 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

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 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) necessitating any reduction of target dose levels in the present OECD 422 combined repeat dose toxicity and reprotoxic/develpmental toxicity screening study.

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: 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_GAH0092"

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 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 low dose female was killed on treatment day 6 in poor health condition resulting from an intubation error)
(1 mid dose female without viable litter was killed on lactation day 1)
(1 high dose female was killed on gestation day 19 in poor health condition resulting from an intubation error)

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

Organ 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, colon, rectum, 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 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_GAH0092"

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

Description (incidence and severity):

attributable to treatment with the test material

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY
There were no deaths attributable to treatment with the test substance. One female of the low dose group (100 mg/kg/day) was killed on treatment day 6 and one female of the high dose group (1000 mg/kg/day) was killed on gestation day 19. Both animals were in poor health condition resulting from an intubation error. One female of the mid dose group (300 mg/kg/day) and its litter were killed on lactation day 1 for reasons of animal welfare. Its litter comprised of a single female offspring that had a bruised head and no evidence of milk in stomach and therefore low survival prognosis.

Toxicologically relevant clinical signs were not evident. A low incidence of chin rubbing occurring in females at 0 (vehicle control), 100 and 300 mg/kg/day, and a low incidence of salivation in females at 100, 300 and 1000 mg/kg/day may have been related to palatability of the formulations and therefore were considered to be of no toxicological importance.

BODYWEIGHT, WEIGHT GAIN AND FOOD CONSUMPTION
There were no adverse effects on bodyweight, weight gain and food consumption.

REPRODUCTIVE ENDPOINTS
Oestrous cycles, pre-coital interval mating performance, fertility, gestation length, gestation index and litter size were unaffected by treatment.

GROSS PATHOLOGY
Macroscopic findings attributable to treatment with the test material were not evident. The mean numbers of uterine implantation sites in main phase females recorded on lactation day 7 were unaffected by treatment with WS400130.

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 dams of the high dose group, ovary and spleen weights adjusted to terminal bodyweight were statistically significantly higher than concurrent controls. In the absence of consistency of these findings within or between sexes and of macroscopic or microscopic pathology correlates, the changes in organ weight were toxicologically not significant.

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 spermatogenic 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: no adverse effects observed

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

attributable to parental treatment with the test material

Mortality / viability:

no mortality observed

Description (incidence and severity):

attributable to parental treatment with the test material

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):

attributable to parental treatment with the test material

Histopathological findings:

not examined

There was no indication of adverse effects of parental treatment with WS400130 on offspring survival up to Day 7 of age, clinical signs, sex ratio, bodyweight, development or macropathology findings.

One dam of the mid dose group (300 mg/kg/day) and its litter were killed on lactation day 1 for reasons of animal welfare. Its litter comprised of a single female offspring that had a bruised head and no evidence of milk in stomach and therefore low survival prognosis. This isolated litter mortality was not considered to be attributable to treatment with the test material.

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: no adverse effects observed. 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

Effects on developmental toxicity

Description of key information

WS400130 was tested in the rat in a developmental toxicity Study (OECD 414) at dose levels of 0, 100, 300 and 1000 mg/kg bw/day. The NOAEL regarding maternal and developmental toxicity was set at 1000 mg/kg/day, i.e. the highest dose level applied. 

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

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

Species:

rat

Strain:

Wistar

Remarks:

Hannover Wistar rats (CRL:WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Sulzfeld, Germany
- Age at study initiation: at least 13 weeks old at mating
- Weight at study initiation: 203 - 257 g
- Fasting period before study: no
- Housing: individual housing
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: at least 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.3 - 24.8°C
- Humidity (%): 33 - 65%
- Air changes (per hr): 15 - 20 per hour
- 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:

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 mg/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test substance was dispersed in propyleneglycol. This dispersion was diluted with tetrahydrofuran (1:2) and directly measured by near infrared (NIR) spectroscopy between 908 and 1676 nm (transmission).

Details on mating procedure:

- If cohoused:
- M/F ratio per cage: 1 : 1
- Length of cohabitation: females with appropriate oestrous cycle were paired in the morning for approx. 2 hours
- Proof of pregnancy: vaginal plug or sperm in vaginal smear was referred to as day 0 of pregnancy

Duration of treatment / exposure:

from gestation day 6 till gestation day 19

Frequency of treatment:

once 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:

24

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at the first day of treatment and then weekly

BODY WEIGHT: Yes
- Time schedule for examinations: on GD 0, 3, 6, 8, 10, 12, 14, 16, 18, 20

FOOD CONSUMPTION
- Food consumption for each animal determined on GD 0, 3, 6, 8, 10, 12, 14, 16, 18, 20
- mean daily diet consumption calculated as g food/kg body weight/day: Yes, for intervals of days on which body weight and food consumption were determined

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: the dams' viscera were examined macroscopically

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

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Statistics:

The statistical evaluation of data was performed with the program package SAS v9.2 in case of Provantis v.9, or SPSS PC+4.0 (SPSS Hungary Kft, Budapest) in case of data tabulated in Excel, by an appropriate statistical method.

In case of the SAS v9.2 software package (within the validated Provantis system) the following decision tree was applied automatically for statistical evaluation of numeric data. The normality and heterogeneity of variance between groups were checked by Shapiro-Wilk and Levene tests using the most appropriate data format (logtransformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunnett’s (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate.

In case of the SPSS PC+4.0 program package, the heterogeneity of variance between groups was checked by Bartlett's test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, then Duncan's Multiple Range test was used to assess the significance of inter-group differences.

Indices:

- Number of corpora lutea: mean ± S.D.
- Number of implantations: mean ± S.D.
- Number and percentage of live foetuses: mean ± S.D.
- Number and percentage of intrauterine mortality: mean ± S.D.
Classified according to time of death: preimplantation loss, postimplantation loss, early and late embryonic loss, as well as foetal death

Foetal Data:
- Sex distribution: %, group mean
- Foetal body weight : mean ± S.D.
- External abnormalities/litter: %, group mean
- Visceral abnormalities/litter: %, group mean
- Skeletal abnormalities/litter: %, group mean

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

A minor, but statistically significant (p<0.01) difference was noted in the body weight gain of the High dose group in GD 8-10 period when compared to the control, in the period GD 6-8 a similar low weight gain was observed, but was not statistically significant. From GD 10 the weight gain for this group was comparable with controls. This was a minor, transient effect with little or no consequences on the overall body weight values. It is not unusual to have such transient effects in the first few days of treatment, with full recovery after animals acclimatise to the test item. There was no effect on weight gain for the Mid or Low dose groups. The difference at the high dose was so small and transient, with no consequences on the overall body weight, and no statistical difference on the overall weight gain, that the small change is not considered as a clear adverse effect.

see Table 2 in the attachments

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Reduced daily food consumption was recorded for the High dose group at the beginning of the treatment (by 9% in the GD 6-8 period and by 13% in the GD 8-10 period. This transient difference was statistically significant at p<0.01) when compared to the control values (smaller changes without statistical significance were also recorded in the Mid dose group). The minor, transient difference at the high dose corresponds to the body weight gain data. The overall mean daily food consumption values in the treatment period or entire study did not differ more the 6% from the control values in any test item treated groups and there was no dose response in any case. Similar trends were seen in the total food consumption and food utilization parameters. Overall, no test item related effect on the food consumption was observed in the Mid or Low dose groups; a minor, transient reduced food intake in the High dose group was seen. The effect at the High dose was so small and transient with no significant on the overall study food intake that the difference is not considered to be adverse.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

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 Table 4 in the attachments

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

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:

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: no adverse effects observed

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

see Table 5 in the attachments
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): see Table 5 in the attachments

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

see Table 5 in the attachments

Changes in sex ratio:

no effects observed

Description (incidence and severity):

see Table 5 in the attachments

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

see Table 5 in the attachments

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

see Table 6 in the attachments

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

see Table 10 in the attachments

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

see Table 8 in the attachments

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: no adverse effects observed

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

In this developmental toxicity study the test substance did not exhibit any adverse toxic effects to maternal animals and to foetuses up to and including the highest dose level of 1000 mg/kg/day.

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

There were no findings or adverse effects in the Combined Repeated Dose Oral (Gavage) Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD 422) regarding reproductive or developmental toxicity.

In a 90-day oral toxicity study in rats (OECD 408) the NOAEL was derived at 1000 mg/kg/d; no effects were observed in reproduction organs and tissues.

In a prenatal development toxicity study (OECD 414) no effects on foetuses was observed up to and including the highest dose level of 1000 mg(/kg/d.

Based on absence of adverse effects on reproduction and development WS400130 does not need to be classified according to European classification rules [REGULATION (EC) 1272/2008].