2-(2,6-diethyl-4-methyl-phenyl)propanediamide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

NOEL systemic effects, male & female rats = 15 mg/kg bw/day

NOEL reproductive toxicity, male & female rats = 150 mg/kg bw/day

NOAEL reproductive toxicity, male & female rats = 1000 mg/kg bw/day

NOAEL offspring toxicity, male & female rats = 1000 mg/kg bw/day

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Remarks:

The two-generation reproductive toxicity study was conducted solely to comply with a non-EU national registration requirement, and has been provided here in accordance with REACH, Article 22(1)e.

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 April 2016 to 06 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF, 12 - Nousan No. 8147

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, CT9 4LT, England.
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: (P) 6-8 wks (males), 5-7 wks (females)
- Weight at study initiation: (P) Males: 181-236 g; Females: 116-158 g
- Fasting period before study: No
- Housing: The P generation animals and selected F1 generation animals were housed in groups of four, by sex, until pairing and for males post-pairing in grid-floor cages suspended over paperlined trays. For pairing, 1 male and 1 female were housed together in grid-floor cages suspended over paper-lined trays. On confirmation of mating, the males were returned to the group cages and the mated females were housed individually and subsequently with their litter, in solid-floor cages with appropriate bedding material.
- Diet: Pelleted rodent diet, VRF1 (manufactured by SDS) supplied by Charles River (UK) Limited, Margate, Kent, CT9 4LT, England ad libitum.
- Water: Mains tap water ad libitum
- Acclimation period: 11 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40-70
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 / 12
IN-LIFE DATES: From: 11 April 2016 To: 06 July 2017

Route of administration:

oral: gavage

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- The test item was formulated within the known stability period, for each group separately, as a suspension in the vehicle. The required amount of test item was added to a container and tared on a balance. The test item was then wetted with a small quantity of vehicle and initially made into a smooth paste. After further addition of vehicle and mixing, the suspension was made up to final weight with vehicle and stirred until visibly homogeneous. Formulations were then placed in an ultrasonic bath for a minimum of 15 minutes (in short bursts, as necessary, to prevent heating of the formulation) to break up any remaining large particulate. Formulations were then divided into daily aliquots for dosing and stored refrigerated (2°C to 8°C). Test item formulations were stirred for at least 15 minutes before the start of dosing and until completion of dosing, to ensure thorough re-suspension and homogeneity.
VEHICLE
- 0.5 % (w/v) carboxymethylcellulose with 0.1 % (v/v) Tween 80.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: Up to 14 days
- Proof of pregnancy: Sperm in vaginal smear, referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: Individually in solid-floor cages with appropriate bedding material

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulations of the test substance in 0.5 % w/v aqueous carboxymethylcellulose with 0.1 % v/v Tween 80 at concentrations between 1 mg/mL and 150 mg/mL, spanning those used in this study (1.5 mg/mL to 100 mg/mL), have been shown to be stable for up to 6 days when stored at room temperature, up to 12 days when stored refrigerated and 1 month when stored frozen (approximately - 20 ºC) so stability was not assessed in this study.
Samples were taken from each test item formulation. Formulations prepared for use on the first day of dosing were analysed to assess their homogeneity and achieved concentrations. Having confirmed satisfactory homogeneity for the first day of dosing, subsequent formulations prepared at approximately 2-monthly intervals were analysed to determine achieved concentration only. Samples from vehicle used to dose Controls on Day 1 and at 2-monthly intervals thereafter were analysed to confirm absence of test item. All samples were analysed for test substance using a validated method.
Results: Achieved concentrations of the test item formulations used to dose animals during Weeks 1 and 8, 16/17, 24/25 and 32/33 of the study were within 9% of nominal, meeting the acceptance criteria (± 10 %). No test substance was detected in the vehicle used to dose Control animals. It is considered therefore that formulations were accurately prepared.

Duration of treatment / exposure:

10 weeks prior to mating of the P generation, during pairing, gestation and lactation until necropsy of the P generation. Four groups of 24 males and 24 females were selected from the weaned parental (P) generation litters to form the filial (F1) generation and were dosed once daily by oral gavage at the same dose levels as the P generation from Day 21 of age for approximately 10 weeks before pairing, during pairing, gestation and lactation and until necropsy.

Frequency of treatment:

All animals were dosed once daily, using a rubber catheter and disposable syringe, for at least 10 weeks before pairing and then until the day before necropsy. Males were dosed before, during and after pairing; females were dosed before and during pairing, during gestation and until Day 20 of lactation. Animals that were mid-parturition at the time of dosing were not dosed on that day. The selected F1 generation were dosed from Day 21 of age. Individual dose volumes were based on the most recently recorded body weight using a dose volume of 10 mL/kg/body weight.

Details on study schedule:

- F1 parental animals not mated until 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.

Dose / conc.:

15 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected in consultation with the Sponsor after examining existing toxicity data. The top dose level of 1000 mg/kg bw/day was selected on the basis of the results of 90 day and reproductive toxicity screening studies with the test substance. In these studies 1000 mg/kg bw/day was well tolerated, with no detrimental effects on body weight or food intake, and no adverse clinical observations indicating excessive toxicity were observed. 1000 mg/kg bw/day was, therefore, considered to be an appropriate top dose level in this study. This is also the limit dose recommended in the OECD guideline for this study type.
- Rationale for animal assignment: Allocation to groups was performed using a stratified randomisation procedure based on individual body weights recorded on arrival.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes (mortality and morbidity)
- Time schedule: Twice daily

CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Male body weights were recorded on the first day of dosing, at weekly intervals throughout the study and then on the day of necropsy. Female body weights were recorded on the first day of dosing and then at weekly intervals until the day of mating. Females were also weighed on Days 0, 7, 14 and 20 of gestation and on Days 0 (where required for dose volume calculations), 1, 4, 7, 14 and 21 of lactation and on the day of necropsy.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: The amount of food consumed by each cage of males was recorded at weekly intervals during their pre-pairing and post-pairing periods. Food intake of the females was recorded weekly during the pre-pairing period and over Days 0 to 4, 4 to 7, 7 to 10, 10 to 14, 14 to 17 and 17 to 20 of gestation and over Days 1 to 4, 4 to 7, 7 to 10, 10 to 14, 14 to 17 and 17 to 21 of lactation. No food intake was recorded for females that had not littered.

SEXUAL DEVELOPMENT OBSERVATIONS:
- All F1 generation females were examined daily from Day 25 of age for vaginal opening. All F1 generation males were examined daily from Day 35 of age for balano-preputial separation. Body weights were recorded on the day that these indicators of sexual maturation were observed.

PARTURITION OBSERVATIONS:
- Females were observed from Day 21 of gestation until the start of the working day when the last pregnant female was on Day 26 after mating. Following completion of parturition, pups were designated as Day 0 of age with the next day classified as Day 1 of age. Dead offspring were not removed from the litter until parturition had been completed.

OBSERVATIONS OF LITTERING FEMALES:
- The females were allowed to rear their offspring to weaning on Day 21 of lactation. Abnormalities of nesting or nursing behaviour were recorded.

Oestrous cyclicity (parental animals):

For 21 days before the start of the pairing period, vaginal smears were taken daily by lavage. The smear was examined under light microscopy and the stage of the oestrous cycle was determined by the type of cell present.

Sperm parameters (parental animals):

Sperm motility and concentration were assessed for all males killed at scheduled necropsy using the Hamilton Thorn IVOS Computer Assisted Sperm Analysis (CASA) system. The assessment was performed using fluid from 1 cauda epididymis. The cauda epididymis was weighed separately. A sample of the epididymal fluid was retained in neutral buffered formalin, a smear was prepared for each Control and high dose male and at least 200 sperm per sample were examined for morphological abnormalities. After weighing of the testes, the tunica albuginea of 1 testis was removed and the testis was snap frozen in liquid nitrogen and stored at -20 °C until required. For each Control and high dose male, the testis was thawed, homogenised and the resistant spermatids counted using the CASA system.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS: Yes

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: Approximately 6 weeks after completion of the mating phase (after successful littering).
- Maternal animals: Females with litters were killed on Day 21 of lactation at weaning of their litters. The remaining females, including those apparently non-pregnant and those where all the litter had died before Day 21 of age, were also killed at this time.
- The animals were killed by exposure to carbon dioxide gas in a rising concentration.

GROSS NECROPSY
- The body weight was recorded and the cranial, thoracic and abdominal cavities were opened and the major organs and uterus were examined. The number of implantation scars/sites for each female was recorded. The uterus of any apparently non-pregnant female was given a visual assessment under magnification to confirm pregnancy status. Organs or tissues showing any macroscopic abnormalities were recorded and retained. For each male, one epididymis was processed for sperm evaluation.

ORGAN WEIGHTS: The following organs were weighed after trimming of fat and other contiguous tissue (contralateral organs were weighed together): adrenals, pituitary, brain (including olfactory bulbs), prostate, seminal vesicles (including coagulating gland), epididymides, epididymis cauda, testes, kidneys, spleen, liver, thyroids (including parathyroids), ovaries, uterus (including uterine cervix and oviducts).

HISTOPATHOLOGY
The following tissues, from control, high dose and any premature decedents, were prepared and examined microscopically: adrenal glands, prostate & seminal vesicles (including coagulating gland), epididymides, testes, liver, thyroids (including parathyroids), ovaries, uterus (including uterine cervix and oviducts), vagina, all gross lesions. Subsequently (due to microscopic changes in the high dose animals), the liver, thyroids, adrenals and ovaries were examined microscopically for intermediate and low dose animals in each of the P and F1 generations. Additionally, reproductive organs of males and females that failed to mate, non-pregnant females, males that failed to sire a pregnancy and females that failed to deliver healthy offspring were also examined microscopically.

OVARIAN FOLLICLE EXAMINATION: Quantitative evaluation of follicles was performed for the F1 generation only. For all F1 females, the centre of the left ovary was sectioned using the butterfly technique (5 sections, approximately 100 μm apart) and stained with haematoxylin and eosin for microscopic examination. The following follicles were then counted for Control and high dose females:
• Primordial follicles and naked oocytes (Types 1 and 2 on the Pederson and Peters classification).
• Primary follicles (Type 3a on the Pederson and Peters classification).

Postmortem examinations (offspring):

SACRIFICE
- With the exception of pups culled on Day 4 of lactation, which were not examined further, a necropsy was conducted on all pups killed or found dead during lactation and all unselected pups on Day 21 of age.
-The pups were killed by an intraperitoneal injection of sodium pentobarbitone solution (for those up to the age of 14 days) or by exposure to carbon dioxide gas in a rising concentration (for older pups).

GROSS NECROPSY
- The cranial, thoracic and abdominal cavities were opened and the major organs were examined. For 1 male and 1 female pup per litter, the tissues listed below were retained. All other pups had a gross macroscopic examination and only gross abnormalities were retained: brain, thymus, spleen, all gross lesions'

ORGAN WEIGTHS: For 1 male and 1 female pup from each litter, the following organs were weighed after trimming of fat and other contiguous tissue: brain, spleen, thymus.

HISTOPATHOLOGY: No

Statistics:

All statistical tests were two-sided with minimum significance levels of 5% and 1%. Non-parametric statistics were not routinely conducted. When used, Dunnett’s test was conducted regardless of the outcome of the analysis of variance (ANOVA) or analysis of covariance (ANCOVA). Body weight, food intake, cumulative body weight gain from the start of dosing (throughout gestation and lactation), pup body weights, cumulative pup body weight gain, litter size, gestation length, total litter weight, mean cycle length and mean number of cycles per female during the pre-mating vaginal smears, the day of balanopreputial separation and vaginal opening, sperm velocities (VSL and VAP), right cauda weight, total sperm number in the right cauda, total sperm number in the right testis, the number of sperm per g right cauda and the number of sperm per g right testis, mean anogenital distance and the number of primordial, small and growing follicles were analysed using ANOVA. Parental and pup organ weights were analysed using ANOVA for the absolute weights and by analysis of covariance (ANCOVA) using terminal kill body weight as covariate. Pup survival, pup sex ratios, litter based mean percentages, fertility and copulation indices, sperm motility and sperm abnormalities were analysed using a parametric ANOVA, following a double arcsine transformation. For all of the parameters evaluated initially by ANOVA or ANCOVA, Dunnett’s test was used to compare the control and treated groups, based on the error mean square in the ANOVA or ANCOVA.

Reproductive indices:

Female copulation index, male copulation index, female ferility index, male fertility index, gestation index, post-implantation loss

Offspring viability indices:

Live birth index, viability index (Day 4 pre-cull), viability index Day 7, viability index Day 14, viability index Day 21, Lactation index, Cumulative survival index

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

On Day 22 of gestation, a female (1000 mg/kg/day) was killed due to poor clinical condition, including decreased activity, abnormal gait, slow laboured breathing and twitching. At necropsy, there was a rupture in the oesophagus, which was confirmed by histopathology. The pathology findings indicate that the poor clinical condition of the animal was due to a dosing error. On Day 24 of gestation, a female (1000 mg/kg/day) was killed, due to dystocia. Dystocia is seen occasionally in this strain of rat at these laboratories and as the remaining females in this group littered successfully, this was considered not to be related to the test substance.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

On Day 22 of gestation, a female (1000 mg/kg/day) was killed due to poor clinical condition, including decreased activity, abnormal gait, slow laboured breathing and twitching. At necropsy, there was a rupture in the oesophagus, which was confirmed by histopathology. The pathology findings indicate that the poor clinical condition of the animal was due to a dosing error.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, group mean male body weights and body weight gains were slightly higher over Weeks 0 to 3 (p≤0.05) compared with Controls. Thereafter, body weight gains were similar to Controls. Body weights and body weight gains at doses of 15 or 150 mg/kg/day were similar to Controls throughout dosing. At 150 or 1000 mg/kg/day, group mean female body weight gains were significantly higher than Controls over Weeks 0 to 3 of the pre-pairing period (p≤0.05, p≤0.01, respectively), so that at the end of the pre-pairing period, females in the group given 1000 mg/kg/day, were statistically significantly heavier than the Controls (p≤0.01). This resulted in higher group mean body weights at 1000 mg/kg/day during gestation and lactation (p≤0.05 to p≤0.01), however, gains were unaffected during these periods. There was no effect of the test substance on body weights or body weight gains at 15 mg/kg/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day group mean food intake was increased for females throughout the pre-pairing period (p≤0.05 to p≤0.01) compared with Controls, however, food intake for males at this dose level was only statistically significantly increased on occasion ( p≤0.05). Group mean food intake was similar to Controls for males and females at doses of 15 or 150 mg/kg/day and there was no effect of the test item on food intake during gestation or lactation.

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:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Liver: A dose-related centrilobular hepatocyte hypertrophy (minimal, slight or moderate) was found in animals given 150 or 1000 mg/kg/day but not in animals given 15 mg/kg/day.
Thyroids: Follicular cell hypertrophy (minimal) was found in animals given 150 or 1000 mg/kg/day but not in animals given 15 mg/kg/day. The hypertrophy was associated with colloid alteration / basophilia in the affected animals. The incidence of hypertrophy was dose-related.
Adrenals: There was an increased incidence of cortical vacuolation in males given 150 or 1000 mg/kg/day and in females given 1000 mg/kg/day. In the males, the vacuolation was mainly in the zona fasciculata but females showed vacuolation only in the zona glomerulosa. There was no effect at 15 mg/kg/day.
Ovaries: Hypertrophy and vacuolation of the interstitial gland (dose-related) was found in most of the females given 150 or 1000 mg/kg/day but not in females given 15 mg/kg/day.
A variety of spontaneous microscopic changes was noted in Control and animals given the test substance with no indication of an effect of the test item. The spectrum of these findings is
generally consistent with changes encountered in rats of this age kept under laboratory conditions.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

There was no effect of the test substance on fertility or mating performance at any dose level. All animals given the test item mated, with all but 1 female given 150 mg/kg/day and 3 females given 1000 mg/kg/day pregnant, this was within the historical control data range, indicating no effect on fertility.

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day (nominal)

System:

other: Integumentary, hepatobiliary, urinary, endocrine and female reproductive organ

Organ:

adrenal glands

kidney

liver

ovary

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs related to the test substance were limited to non-adverse dose-related excessive salivation and yellow stained bedding for males and females at all doses.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

On Day 13 of gestation, a female (1000 mg/kg/day) was killed due to poor clinical condition, including decreased activity, abnormal gait, slow breathing, partially closed eyes, hunched posture and piloerection. At necropsy, there was distension of the stomach (with food) and red areas in the non-glandular region. The duodenum was also distended (ingesta). Histopathology revealed ulceration in the stomach and duodenum with associated changes (haemorrhage/inflammation/epithelial hyperplasia). As this was an isolated finding this death was considered not to be related to the test substance.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, males and females began the pre-pairing period slightly lighter than the Controls (p≤0.01); however, group mean male body weight gains were similar to Control throughout the pre-pairing period while females in this group gained slightly more weight than the Controls over Weeks 0 to 3 (p≤0.01). Group mean absolute body weights during gestation and lactation were unaffected despite increased body weight gain over Days 1 to 14 of lactation (p≤0.05; p≤0.01). There was no effect on body weights or body weight gains at 15 or 150 mg/kg/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day group mean female food intake was higher than Controls during Weeks 4 to 6 and 9 to 10 of the pre-pairing period (p≤0.05 to p≤0.01), however, overall food intake was unaffected. Group mean food intake was similar to Controls for males and females at doses of 15 or 150 mg/kg/day and there was no effect on food intake during gestation or lactation.

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:

effects observed, treatment-related

Description (incidence and severity):

Group mean absolute and adjusted liver weights were higher than Controls for both sexes given 150 or 1000 mg/kg/day (p≤0.05 to p≤0.01). These findings correlated to microscopic changes in the liver. Group mean absolute kidney weights were higher than Controls for males given 1000 mg/kg/day (p≤0.01). In addition, the group mean adjusted kidney weights were higher than Controls for the males at 150 or 1000 mg/kg/day (p≤0.01, respectively). The decreases in thymus weights were considered to be test item-related although there was no microscopic correlate for this finding. Group mean absolute and adjusted thyroid weights were higher than Controls for males given 150 or 1000 mg/kg/day (p≤0.05 to p≤0.01). These findings correlated to microscopic changes in the thyroid. The increases in liver, thyroid and kidney weights were considered to be test item-related. With the exception of the mean body weight related liver weights (male and female) and thyroid (male) at 1000 mg/kg/day which were above the historical Control data range, mean body weight related weights were within the historical data range. Other statistically significant inter-group differences were considered to reflect normal biological variation rather than an effect of the test substance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A variety of spontaneous macroscopic changes was noted in Control and animals given the test substance with no indication of an effect of the test item. The spectrum of these findings is generally consistent with changes encountered in rats of this age kept under laboratory conditions.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Liver: Dose-related centrilobular hepatocyte hypertrophy (minimal, slight or moderate) was found in males and females given 1000 mg/kg/day and in males given 150 mg/kg/day. The incidence and severity of this finding were dose-related in males.
Thyroids: Follicular cell hypertrophy (minimal) was found in animals given 150 or 1000 mg/kg/day but not in animals given 15 mg/kg/day. The hypertrophy was associated with colloid alteration / basophilia in the affected animals. The incidence of hypertrophy was dose-related.
Adrenals: There was an increased incidence of cortical vacuolation in some males and females given 150 or 1000 mg/kg/day. In the males, the vacuolation was mainly in the zona fasciculata but females showed vacuolation mainly in the zona glomerulosa.
Ovaries: A dose-related hypertrophy and vacuolation of the interstitial gland was found in most of the females given 150 or 1000 mg/kg/day but not in females given 15 mg/kg/day.
A variety of spontaneous microscopic changes was noted in Control and animals with no indication of an effect of the test item. The spectrum of these findings is generally consistent with changes encountered in rats of this age kept under laboratory conditions.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Sexual development observations: There was no effect on sexual maturation in either sex; the day of attainment of balano-preputial separation or vaginal opening was similar to Controls in males or females, respectively.

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

There was an increased incidence of post-implantation loss at 1000 mg/kg/day (p≤0.01) compared with Controls. These losses were above the historical control data range and, therefore, are considered to be related to the test substance. There was no effect on the mean number of pups born alive, on litter survival indices or on the mean sex ratio of the pups.

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day (nominal)

System:

other: Hepatobiliary, Endocrine and female reproductive system

Organ:

adrenal glands

liver

ovary

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day absolute pup body weights were lower than Controls from Day 14 of age (p≤0.01) resulting in an overall decrease in pup body weight gain (p≤0.01). At 15 or 150 mg/kg/day there was no effect on absolute pup body weight or pup body weight gain.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Group mean absolute and adjusted thymus weights were lower than Controls for males given 1000 mg/kg/day (p≤0.01 and p≤0.05, respectively). Only the absolute thymus weight was
lower for females at this dose (p≤0.05). The male mean body weight related thymus weights were below the historical Control data range, however the female weight was within. The decreases in thymus weights were considered to be test item-related although there was no microscopic correlate for this finding. Other statistically significant inter-group differences were considered to reflect normal biological variation rather than any effect of the test substance.

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

not specified

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

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

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Group mean absolute and adjusted thymus weights were lower than Controls for both sexes given 1000 mg/kg/day (p≤0.01, p≤0.05, respectively). The mean body weight related thymus weights were below the historical Control data range. The decreases in thymus weights were considered to be test item-related although there was no microscopic correlate for this finding. Other statistically significant inter-group differences were considered to reflect normal biological variation rather than any effect of the test item.

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

Administration of the test substance, once daily, by oral gavage at 15, 150 or 1000 mg/kg/day to male and female Crl:WI(Han) rats over two consecutive generations, was well tolerated, but elicited test item-related liver, thyroid, kidney, adrenal and ovarian changes at 150 and 1000 mg/kg/day. There was also an increased incidence of post-implantation loss in F1 animals dosed at 1000 mg/kg/day.

The No Observed Effect Level (NOEL) for both sexes for systemic toxicity was considered to be 15 mg/kg/day.
The No Observed Effect Level (NOEL) for both sexes for reproductive toxicity was considered to be 150 mg/kg/day.
The No Observed Adverse Effect Level (NOAEL) for both sexes for the offspring was considered to be 1000 mg/kg/day.

Executive summary:

Four groups of 24 male and 24 female rats of the Crl:WI(Han) strain were dosed by oral gavage at dose levels of 0 (0.5% carboxymethylcellulose with 0.1 % Tween 80), 15, 150 or

1000 mg/kg/day test substance for 10 weeks before pairing for mating, during pairing, gestation and lactation until necropsy. Four groups of 24 males and 24 females were selected from the weaned parental (P) generation litters to form the filial (F1) generation; these animals were dosed once daily by oral gavage at the same dose levels as the P generation from Day 21 of age for approximately 10 weeks before pairing, during pairing, gestation and lactation and until necropsy. Animals were dosed using a dose volume of 10 mL/kg. All animals were examined for effects on general condition, body weight and food intake. The stage of the oestrous cycle was recorded for 21 days before pairing for P and F1 parental females, and during the pairing period vaginal smears were taken daily until sperm were found in the smear. The females were allowed to litter and rear their offspring to weaning. The day of sexual development was recorded for all selected F1 animals. The P and F1 parental males were subjected to macroscopic necropsy once successful littering was completed. The testes and epididymides were removed and weighed and sperm evaluation was conducted. The P and F1 parental females were killed and subjected to necropsy on Day 21 of lactation. A macroscopic necropsy was performed and the number of implantation scars was recorded. For the P and F1 parental males and females, a selection of organs were weighed, fixed and examined microscopically. Unselected F1a animals and all F2a animals were killed on Day 21 of age. A gross macroscopic necropsy was performed on all pups and, for one male and one female pup per litter from the P and F1 generations, the brain, spleen and thymus were weighed. Homogenisation resistant testicular spermatids were counted for the P and F1 parental males and ovarian follicle evaluation was performed for the F1 generation parental females.

There were no test item-related deaths. Clinical signs related to the test substance were limited to non-adverse, dose-related excessive salivation and yellow stained bedding for adult animals at all doses in the P and F1 generations. There were no test item-related clinical signs in the litters. During the P generation, the females given 150 mg/kg/day and the males and females given 1000 mg/kg/day, gained more weight than Controls over the first 3 weeks of the pre-pairing period, while for the F1 generation, only females given 1000 mg/kg/day gained more weight than the Controls during the first weeks of the pre-pairing period. There was no effect of the test substance on body weight gain during the gestation or lactation periods and no effect on body weight of the F1 generation pups. There was a decreased body weight gain of the P generation pups at 1000 mg/kg/day.

Throughout the pre-pairing period there was an increase in mean food intake for P generation females given 1000 mg/kg/day, however, food intake for P generation males and F1

generation females at this dose level was only sporadically increased. Mean food intake for both generations was unaffected for males and females given 15 or 150 mg/kg/day.

There was no effect of the test item on oestrous cycling, fertility and mating performance or on gestation length for either generation at any dose level. At 1000 mg/kg/day there was an increased incidence of post-implantation loss for F1 generation females but pregnant females gave birth to a similar number of pups, and there was no effect of the test item on sex ratio or on the postnatal survival of the P or F1 generation litters to Day 21 of age. There was no effect of the test substance on sexual maturation of the F1 generation, or on sperm parameters for males from either generation, or the primordial follicles of the F1 generation females. For both generations, group mean absolute and adjusted liver weights were higher than Controls for both sexes given 150 or 1000 mg/kg/day. For the P generation, at mean absolute and adjusted adrenal weights higher for females. For the F1 generation, group mean thyroid weights were higher than Controls for males given 150 or 1000 mg/kg/day. There were no test item-related macroscopic findings. In the P and F1 generation animals given 150 or 1000 mg/kg/day, test item-related changes were found in the liver (centrilobular hepatic hypertrophy), thyroids (follicular cell hypertrophy) and adrenals (vacuolation in the zona fasciculate/glomerulosa) of males and females and in the ovaries (vacuolation/hypertrophy of interstitial gland) of females.

Administration of the test substance, once daily, by oral gavage at 15, 150 or 1000 mg/kg/day to male and female Crl:WI(Han) rats over two consecutive generations, was well tolerated, but elicited test item-related liver, thyroid, kidney, adrenal and ovarian changes at 150 and 1000 mg/kg/day. There was also an increased incidence of post-implantation loss in F1 animals dosed at 1000 mg/kg/day.

The No Observed Effect Level (NOEL) for both sexes for systemic toxicity was considered to be 15 mg/kg/day.

The No Observed Effect Level (NOEL) for both sexes for reproductive toxicity was considered to be 150 mg/kg/day.

The No Observed Adverse Effect Level (NOAEL) for both sexes for the offspring was considered to be 1000 mg/kg/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP study according to OECD TG 416, fully reliable with no restrictions

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Information from the two-generation reproductive toxicity study (OECD TG 416)

The potential adverse effects of the substance on fertility and reproduction were investigated under GLP in a two-generation reproductive toxicity study according to OECD TG 416. The experiments were conducted in rats of the Crl:WI(Han) strain. The test substance was administered daily by oral gavage in 0.5% carboxymethylcellulose with 0.1% Tween 80 to four groups of 24 female and 24 male rats at 0 (vehicle only), 15, 150 or 1000 mg/kg bw/day using a dose volume of 10 mL/kg bw. Animals were dose for 10 weeks before pairing, during pairing, gestation and lactation until necropsy. Four groups of 24 males and 24 females were selected from the weaned parental (P) generation litters to form the filial (F1) generation; these animals were dosed once daily by oral gavage at the same dose levels as the P generation from Day 21 of age for approximately 10 weeks before pairing, during pairing, gestation and lactation and until necropsy.

There were no test item-related deaths. Clinical signs related to the test substance were limited to non-adverse, dose-related excessive salivation and yellow stained bedding for adult animals at all doses in the P and F1 generations. There were no test item-related clinical signs in the litters. During the P generation, the females given 150 mg/kg/day and the males and females given 1000 mg/kg/day gained more weight than Controls over the first 3 weeks of the pre-pairing period, while for the F1 generation, only females given 1000 mg/kg/day gained more weight than the Controls during the first weeks of the pre-pairing period. There was no effect of the test substance on body weight gain during the gestation or lactation periods and no effect on body weight of the F1 generation pups. There was a decreased body weight gain of the P generation pups at 1000 mg/kg/day.

Throughout the pre-pairing period there was an increase in mean food intake for P generation females given 1000 mg/kg/day, however, food intake for P generation males and F1 generation females at this dose level was only sporadically increased. Mean food intake for both generations was unaffected for males and females given 15 or 150 mg/kg/day.

There was no effect of the test item on oestrous cycling, fertility and mating performance or on gestation length for either generation at any dose level. At 1000 mg/kg/day there was an increased incidence of post-implantation loss for F1 generation females, but pregnant females gave birth to a similar number of pups, and there was no effect of the test item on sex ratio or on the postnatal survival of the P or F1 generation litters to Day 21 of age. There was no effect of the test substance on sexual maturation of the F1 generation, or on sperm parameters for males from either generation, or the primordial follicles of the F1 generation females.

For both generations, group mean absolute and adjusted liver weights were higher than Controls for both sexes given 150 or 1000 mg/kg/day. For the P generation, at mean absolute and adjusted adrenal weights were higher for females. For the F1 generation, group mean thyroid weights were higher than Controls for males given 150 or 1000 mg/kg/day. There were no test item-related macroscopic findings. In the P and F1 generation animals given 150 or 1000 mg/kg/day, test item-related changes were found in the liver (centrilobular hepatic hypertrophy), thyroids (follicular cell hypertrophy) and adrenals (vacuolation in the zona fasciculate/glomerulosa) of males and females and in the ovaries (vacuolation/hypertrophy of interstitial gland) of females.

Administration of the test substance, once daily, by oral gavage at 15, 150 or 1000 mg/kg/day to male and female Crl:WI(Han) rats over two consecutive generations, was well tolerated, but elicited test item-related liver, thyroid, kidney, adrenal and ovarian changes at 150 and 1000 mg/kg/day. There was no adverse effect on female and male reproductive organs, oestrous cycle, sperm motility, concentration and morphology, fertility, mating performance and gestation length. An increased incidence of post-implantation loss in F1 animals dosed at 1000 mg/kg/day was observed, but no associated adverse effect on the mean number of pups born alive, on litter survival indices or on the mean sex ratio of the pups.

Effects on developmental toxicity

Description of key information

NOAEL embryo-foetal development = 15 mg/kg bw/day

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Remarks:

The prenatal developmental toxicity study was conducted solely to comply with a non-EU national registration requirement, and has been provided here in accordance with REACH, Article 22(1)e.

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 August 2016 to 05 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF, 12 - Nousan No. 8147

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI (Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, CT9 4LT, UK
- Age at study initiation: 8 to 10 weeks old
- Weight at study initiation: 177 to 246 g
- Fasting period before study: No
- Housing: Animals were housed individually in solid-floor cages with appropriate bedding provided.
- Diet (e.g. ad libitum): VRF1 manufactured by SDS available ad libitum
- Water (e.g. ad libitum): Mains tap water available ad libitum
- Acclimation period: 2 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 40 - 70
- Photoperiod (hrs dark / hrs light): 12 h dark / 12 h light cycle

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% (w/v) aqueous CMC with 0.1% (v/v) Tween 80

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:The required amount of test item was added to a container and tared on a balance. The test item was then wetted with a small quantity of vehicle and initially made into a smooth paste. After further addition of vehicle and mixing, the suspension was made up to final weight with vehicle and stirred until visibly homogeneous. Formulations were then placed in an ultrasonic bath for a minimum of 15 minutes (in short bursts, as necessary, to prevent heating of the formulation) to break up any remaining large particulate. Formulations were then divided into daily aliquots for dosing and stored refrigerated (2 °C to 8 °C). Test item formulations were stirred for at least 15 minutes before the start of dosing and until completion of dosing, to ensure thorough re-suspension and homogeneity.

- Concentration in vehicle: 0, 1.5, 15 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were taken from each test item formulation prepared for the first day of dosing and were analysed for test item using a validated method to confirm homogeneity and also achieved concentrations. Having confirmed satisfactory homogeneity for the first day of dosing, triplicate samples were taken from all test item formulations prepared towards the end of the dosing period and analysed to determine achieved concentrations only. Triplicate samples were taken from formulations prepared for the first day of dosing and towards the end of the dosing period from the vehicle used to dose Controls and were analysed to confirm absence of test item.
All remaining samples were retained and were discarded once the final formulation analysis
results were accepted.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant from Charles River (UK) Limited.
Each female had been mated with a sexually mature male of the same strain. The day on which mating was detected was designated Day 0 of gestation.

Duration of treatment / exposure:

Animals were dosed from Day 6 to Day 19 of gestation.

Frequency of treatment:

Animals were dosed once daily.

Duration of test:

Dosing started on Day 6 of gestation and animals were killed on Day 20 of gestation.

Dose / conc.:

15 mg/kg bw/day

Dose / conc.:

150 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected in consultation with the Sponsor after examining existing toxicity data. The top dose level of 1000 mg/kg/day was selected on the basis of the results from a 90-day study and a reproduction and developmental toxicity screening study. In these studies, 1000 mg/kg/day was well tolerated with no detrimental effects on body weight or food intake and no adverse clinical observations. Therefore, 1000 mg/kg/day was considered to be an appropriate high dose level for use in this study. This is also the limit dose recommended in the OECD guideline for this study type.

- Randomisation: Animals were allocated to groups using a stratified body weight randomisation procedure based on individual body weights recorded at the supplier on Day 0 of gestation (making sure that females mated with the same male were spread across the groups). The cages were positioned in the battery using a randomised cage allocation procedure, starting at the top left-hand corner of the rack and then working from left to right, top to bottom. All groups were allocated to each rack. Each animal was uniquely identified by a subcutaneously implanted micro-identification device.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Day 0 of gestation by the supplier then daily from Day 5 to 20 of gestation, inclusive

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: Days 6 to 9, 9 to 12, 12 to 15, 15 to 18, and 18 to 20 of gestation.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: the thoracic and abdominal cavities were opened by a ventral mid-line incision and the major organs were examined. Organs or tissues showing any macroscopic abnormalities were removed and retained in fixative. The uterus of any apparently non-pregnant female was stained with ammonium sulphide to confirm pregnancy status. The uterus was then retained.

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]
- Decaptiation: Yes [half per litter]
- Soft tissue examinations: Yes: [all - intact foetuses and the bodies of the decapitated foetuses using a combined sectioning/dissection technique ]
- Skeletal examinations: Yes: [all - intact foetuses and the bodies of the decapitated foetuses]. Carcasses were then cleared in potassium hydroxide, stained with Alizarin red S and Alcian blue to visualise the ossified skeleton and cartilage and examined.
- Head examinations: Yes: [ all - half per litter serial section of Bouins fixed heads, remaining half per litter heads of intact foetuses - coronal section made through the head along the frontal parietal suture and brain examined.

Statistics:

Test were two-sided with significance levels of 5% and 1%. The litter, was considered as the experimental unit. When used, Dunnett’s test was conducted regardless of the outcome of the analysis of variance (ANOVA) or analysis of covariance (ANCOVA).
For Quantitative Data: Body weight, cumulative body weight gain from the start of dosing, food intake, terminal body weight, numbers of corpora lutea, implants, live foetuses, dead foetuses, early intrauterine deaths, late intrauterine deaths, gravid uterus weight, total litter weight, placental weight and mean foetal weight (sexes separately and combined) were analysed using a parametric ANOVA.
For Percentages: Pre-implantation loss, post-implantation loss, sex ratios (% male foetuses) and litter based mean percentages were analysed using a parametric ANOVA, following a double arcsine transformation (Freeman and Tukey).
Maternal Performance: (e.g. the proportion of females with live foetuses at termination, abortions, total resorptions) were analysed by a two-tailed Fisher’s Exact Text comparing each treated group to the control group.
Foetal Morphology Data: The incidence of foetal malformations and developmental variations (external, visceral and skeletal) were summarised as the proportion of foetuses affected, the proportion of litters affected and the proportion of foetuses affected within each litter. The proportions of litters affected were analysed by the exact version of the Cochran-Armitage Test. The percentages of foetuses affected within each litter was analysed by the exact version of the Jonckheere Trend Test. In both cases the tests were performed in a step-wise manner where, when a test was significant at the 5 % level, the test was repeated after removing the then top dose, until only the Control group was left. Tests were one-sided looking for increase in treated groups versus the Control group.

Indices:

Pre-implantation loss (%) = (no. of corpora lutea – no. of implantation sites) / no. of corpora lutea (x 100)
Post-implantation loss (%) = (no. of implantation sites – no. of live foetuses) / no. of implantation sites (x 100)
Mean pre- and post-implantation losses were calculated on a proportional litter basis.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Yellow stained bedding was observed in the cages of females given 1000 mg/kg/day. This was considered to be test item-related but non-adverse.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Females in the group given 1000 mg/kg/day ate statistically significantly less (p<0.01) than the Controls from Day 6 to Day 15 of gestation so that their overall mean food intake for the entire dosing period was 13 % lower than the Controls.
At 15 or 150 mg/kg/day mean food intake was similar to Control values.

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

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, the mean incidence of post-implantation loss was slightly higher than that of the Controls; however, this was due to one female with total resorption, this female had only one implantation that was an early intrauterine death. Given the absence of an effect of the test substance on post-implantation loss in the litters with live foetuses at 1000 mg/kg/day, the single occurrence of total resorption with only a single implantation is considered to be an incidental event unrelated to administration of the test item. For this reason, the dam and litter have been excluded from calculation of the group mean data.

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, the mean incidence of post-implantation loss was slightly higher than that of the Controls; however, this was due to one female with total resorption, this female had only one implantation that was an early intrauterine death. Given the absence of an effect of the test substance on post-implantation loss in the litters with live foetuses at 1000 mg/kg/day, the single occurrence of total resorption with only a single implantation is considered to be an incidental event unrelated to administration of the test item. For this reason, the dam and litter have been excluded from calculation of the group mean data.

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

food consumption and compound intake

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day group mean foetal weight (3.35 g) was slightly, but statistically significantly lower than Control (-9 %, p<0.01) and below the historical Control ranges for this laboratory (3.50 g to 3.91 g). There was no effect on foetal weight at 15 mg/kg/day or 150 mg/kg/day.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, the overall incidence of minor foetal abnormalities was significantly higher than the Controls (p<0.001). This included increases in the number of foetuses showing skeletal and cartilage abnormalities of the cervical vertebrae and ribs together with the presence of cervical ribs; the majority of these incidences were outside the historical Control range.
In addition, at 1000 mg/kg/day, there were significantly higher (p<0.001) incidences of related variant findings of the cervical vertebrae and non - or incomplete ossification of the 5th and 6th sternebrae and 5th metacarpal (p<0.05, p<0.01, p<0.001), the majority of these findings were outside the historical Control range.
The minor defect of partially fused jugal and maxilla was noted in 5 foetuses from 3 litters in the group given 1000 mg/kg/day (p<0.05). Although the incidence was slightly higher than the historical Control range, this abnormality is seen in this strain of rats in this laboratory and as such the incidence was considered likely to be spontaneous and not an adverse effect of test substance administration.
At 1000 mg/kg/day, there were significantly higher incidences of the minor defects, irregular ridging of the palate (p<0.01) and incomplete ossification of the nasal bones (p<0.001), compared with Control, both of which were outside the historical Control range.
At 150 mg/kg/day, there were marginal increases in the incidences of minor abnormalities of the cervical vertebrae and incomplete ossification of the nasal bones compared with the Controls which correlated with increases seen also at 1000 mg/kg/day. These incidences were also above the historical Control range.
The findings at 150 or 1000 mg/kg/day related to ossification are considered to be transient in nature and/or of no developmental consequence. The increased incidences of other skeletal and cartilage minor foetal abnormalities and variants were clearly test item-related and considered to represent an adverse effect of the test item on foetal development.

Visceral malformations:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: skull

skeletal: forelimb

skeletal: sternum

skeletal: rib

skeletal: supernumerary rib

skeletal: vertebra

Description (incidence and severity):

At 1000 mg/kg/day, the overall incidence of minor foetal abnormalities was significantly higher than the Controls (p<0.001). This included increases in the number of foetuses showing skeletal and cartilage abnormalities of the cervical vertebrae and ribs together with the presence of cervical ribs; the majority of these incidences were outside the historical Control range.
In addition, at 1000 mg/kg/day, there were significantly higher (p<0.001) incidences of related variant findings of the cervical vertebrae and non - or incomplete ossification of the 5th and 6th sternebrae and 5th metacarpal (p<0.05, p<0.01, p<0.001), the majority of these findings were outside the historical Control range.
The minor defect of partially fused jugal and maxilla was noted in 5 foetuses from 3 litters in the group given 1000 mg/kg/day (p<0.05). Although the incidence was slightly higher than the historical Control range, this abnormality is seen in this strain of rats in this laboratory and as such the incidence was considered likely to be spontaneous and not an adverse effect of test substance administration.
At 1000 mg/kg/day, there were significantly higher incidences of the minor defects, irregular ridging of the palate (p<0.01) and incomplete ossification of the nasal bones (p<0.001), compared with Control, both of which were outside the historical Control range.
At 150 mg/kg/day, there were marginal increases in the incidences of minor abnormalities of the cervical vertebrae and incomplete ossification of the nasal bones compared with the Controls which correlated with increases seen also at 1000 mg/kg/day. These incidences were also above the historical Control range.
The findings at 150 or 1000 mg/kg/day related to ossification are considered to be transient in nature and/or of no developmental consequence. The increased incidences of other skeletal and cartilage minor foetal abnormalities and variants were clearly test item-related and considered to represent an adverse effect of the test item on foetal development.

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Table 1: Foetal findings with statistical significance

 

		Dose level (mg/kg/day)				Background data range
Finding		0	15	150	1000
Oral cavity – palate: irregular ridging	No Foetuses No Litters	5 (4.8) 5	5 (5.1) 4	7 (6.0) 6	20 (20.2)** 13***	1.7-8.5 2-5
Skull- nasal- uni- or bilateral: incomplete ossification	No Foetuses No Litters	3 (2.5) 2	5 (5.4) 3	7 (7.1) 4	73 (71.8)*** 19***	2.1-6.3 1-6
Skull- jugal and maxilla- uni or bilateral: partial fusion	No Foetuses No Litters	0 (0.0) 0	0 (0.0) 0	0 (0.0) 0	5 (5.1)* 3*	0.6-3.1 1-2
Cervical vertebra – one or more neural arch: misshapen	No Foetuses No Litters	2 (1.0) 2	7 (3.5) 5	12 (6.5)* 7	63 (31.8)*** 18***	3.6-3.6 4-4
Cervical vertebra – cartilaginous ventral plate - uni- or bilateral: fused	No Foetuses No Litters	1 (0.4) 0	0 (0.0) 0	3 (1.5) 3	10 (4.8)*** 2	0.3-1.0 1-2
Cervical vertebra – additional cartilaginous ventral plate – uni- or bilateral: fused	No Foetuses No Litters	1 (0.4) 0	0 (0.0) 0	3 (1.5) 3	10 (4.6)*** 8***	0.7-0.7 1-1
Rib – uni – or bilateral: cervical	No Foetuses No Litters	6 (3.0) 5	11 (7.8) 6	12 (6.4) 7	71 (33.4)*** 16***	1.2-12.6 2-9
Rib – one or more: costal cartilage fused severe	No Foetuses No Litters	0 (0.0) 0	1 (0.5) 1	0 (0.0) 0	13 (6.0)*** 6***	0.9-0.9 1-1
Rib – cervical rib – left: costal cartilage fused severe	No Foetuses No Litters	0 (0.0) 0	0 (0.0) 0	0 (0.0) 0	8 (3.9)*** 6***	0.0-0.0 0
Rib – cervical rib – uni- or bilateral: with costal cartilage	No Foetuses No Litters	2 (0.9) 1	2 (1.1) 1	1 (0.6) 1	26 (11.9)*** 9***	0.8-3.4 1-3
Sternum – 2nd sternebra: incomplete ossification	No Foetuses No Litters	2 (0.7) 2	3 (1.4) 3	2 (0.9) 1	8 (3.7) 6*	0.8-8.9 1-7
Cervical vertebra – cartilaginous ventral plate – uni- or bilateral: incomplete	No Foetuses No Litters	1 (0.4) 1	0 (0.0) 0	2 (0.9) 2	11 (5.5)*** 9***	0.8-1.7 1-3
Cervical vertebra – cartilaginous ventral plate – uni- or bilateral: on 5th cervical vertebra	No Foetuses No Litters	1 (0.4) 1	5 (2.5) 4	10 (5.7) 7*	57 (29.1)*** 16***	0.9-5.9 2-5
Cervical vertebra – cartilaginous ventral plate – uni- or bilateral: misshapen	No Foetuses No Litters	0 (0.0) 0	2 (1.0) 2	2 (1.2) 1	12 (6.5)*** 8***	0.9-0.9 1-1
Cervical vertebra – additional cartilaginous ventral plate – uni- or bilateral: on 5th cervical vertebra	No Foetuses No Litters	1 (0.4) 1	0 (0.0) 0	3 (1.5) 3	19 (9.2)*** 14***	0.3-2.7 1-2
Sternum – 5th sternebra: not ossified	No Foetuses No Litters	1 (0.4) 1	8 (6.5)* 7	12 (6.5)* 7	97 (45.8)*** 17***	1.1-10.7 1-7
Sternum – 5th sternebra: incomplete ossification	No Foetuses No Litters	6 (2.9) 5	6 (3.7) 4	19 (9.7) 8	24 (12.7)*** 14***	1.7-15.3 2-11
Sternum – 6th sternebra: not ossified	No Foetuses No Litters	1 (0.4) 1	5 (2.6) 1	1 (0.5) 1	10 (4.6)** 7**	0.3-4.7 1-4
Sternum – 6th sternebra: incomplete ossification	No Foetuses No Litters	5 (2.4) 3	8 (4.9) 4	2 (1.2) 2	18 (7.8) 8*	2.1-21.5 2-12
Forelimb – 5th metacarpal – uni- or bilateral: not ossified	No Foetuses No Litters	33 (15.9) 12	34 (21.5) 12	21 (10.5) 12	80(39.1)** 17*	2.5-36.0 3-16

Figures in brackets are group mean percent values

*= p<0.05; **= p<0.01; ***= p<0.001

Conclusions:

Administration of the test substance to the pregnant Crl:WI(Han) rat at 15, 150 or 1000 mg/kg/day once daily by oral gavage from Days 6 to 19 of gestation inclusive, elicited slight maternal toxicity at 1000 mg/kg/day in terms of reduced food intake. A slightly lower mean foetal weight was observed at a dose level that induced maternal toxicity. There were no test item related major foetal abnormalities, at any dose level, however, an increased incidence of minor foetal abnormalities and variants was noted at both 150 and 1000 mg/kg/day above both the concurrent and historical Control ranges. Based on the above findings, the No-Observed-Adverse-Effect-Level (NOAEL) for maternal toxicity was considered to be 1000 mg/kg/day and NOAEL for embryo-foetal development was considered to be 15 mg/kg/day.

Executive summary:

The purpose of this study was to investigate the effects of the test item on the pregnant rat and developing organism when administered by oral gavage daily, from Day 6 to Day 19 of gestation, inclusive.

Four groups of 22 sexually mature, timed-mated female Crl:WI(Han) rats were dosed with 0 (vehicle), 15, 150 or 1000 mg/kg/day test substance daily, by oral gavage, from Day 6 to Day 19 of gestation, inclusive, at a dose volume of 10 mL/kg body weight. Body weights, food intake and clinical observations were recorded. All animals were killed on Day 20 of gestation, a necropsy was performed and the internal organs examined for gross abnormalities. The progress and outcome of pregnancy were assessed and maternal dead body weight, gravid uterus and placenta weights were recorded. The foetuses were removed from the uterus, weighed, sexed and examined for external, visceral, skeletal and cartilage abnormalities.

There were no deaths or adverse clinical observations during the study. Yellow stained bedding was observed in the cages of females given 1000 mg/kg/day. Group mean body weights and body weight gains were similar to Controls at all dose levels. There was no adverse effect of the test substance on terminal body weight when adjusted for the weight of the gravid uterus. Females in the group given 1000 mg/kg/day ate less than the Controls from Day 6 to 15 of gestation (the difference was statistically significant) so that their overall mean food intake for the entire dosing period was 13% lower than the Controls. At 15 or 150 mg/kg/day mean food intake was similar to Control values. There were no test item-related maternal necropsy findings.

There were 21, 19, 21 and 20 females in the groups given 0, 15, 150 or 1000 mg/kg/day, respectively, with live foetuses on Day 20 of gestation. The mean number of live foetuses per female was similar in all groups. Uterine/implantation parameters at 15 or 150 mg/kg/day were comparable with Controls. At 1000 mg/kg/day group mean foetal weight was slightly, but statistically significantly, lower than Control values (-9 %). There was no effect on foetal weight at 15 or 150 mg/kg/day. Mean placental weight and sex ratio were similar in all groups. There were no major foetal abnormalities considered to be test item-related. At 150 and 1000 mg/kg/day, there were higher incidences of minor and variant foetal abnormalities compared with Control that affected the cervical vertebrae, ribs, sternebrae, skull and palate. These significantly higher incidences of several skeletal minor abnormalities and/or variants (affecting skull, cervical vertebrae, ribs and sternebrae) were outside of the historical Control range seen within this laboratory and were considered to be related to the test item. Increases in the minor or variant foetal abnormalities associated with skeletal ossification were considered to be transient in nature and/or of no developmental consequence.

Administration of the test substance to the pregnant Crl:WI(Han) rat at 15, 150 or 1000 mg/kg/day once daily by oral gavage from Days 6 to 19 of gestation inclusive, elicited slight maternal toxicity at 1000 mg/kg/day in terms of reduced food intake. A slightly lower mean foetal weight was observed at a dose level that induced maternal toxicity. There were no test item related major foetal abnormalities at any dose level, however, an increased incidence of minor foetal abnormalities and variants was noted at both 150 and 1000 mg/kg/day above both the concurrent and historical Control ranges.

Based on the above findings, the No-Observed-Adverse-Effect-Level (NOAEL) for maternal toxicity was considered to be 1000 mg/kg/day and NOAEL for embryo-foetal development was considered to be 15 mg/kg/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

15 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP study according to OECD TG 414, fully reliable with no restrictions

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Information from the developmental toxicity study (OECD TG 414)

One developmental toxicity study in the rat, conducted to OECD TG 414 (2001 revision), is available (King, 2018). Groups of 22 female time-mated Crl:WI(Han) rats were dosed with 0 (vehicle), 15, 150 or 1000 mg/kg bw/day daily, by oral gavage, from Day 6 to Day 19 of gestation, inclusive, at a dose volume of 10 mL/kg body weight.

Females in the group given 1000 mg/kg/day exhibited an overall mean food intake for the entire dosing period 13% lower than the Controls. Group mean body weights and body weight gains were similar to Controls at all dose levels. There was no adverse effect of CA3250 on terminal body weight when adjusted for the weight of the gravid uterus.

There were no test item-related maternal necropsy findings. There were 21, 19, 21 and 20 females with live foetuses on Day 20 of gestation in the groups given 0, 15, 150 or 1000 mg/kg/day, respectively. The mean number of live foetuses per female was similar in all groups. Uterine/implantation parameters at 15 or 150 mg/kg/day were comparable with Controls. At 1000 mg/kg/day group mean foetal weight was slightly, but statistically significantly, lower than Control values (-9 %). There was no effect on foetal weight at 15 or 150 mg/kg/day. Mean placental weight and sex ratio were similar in all groups.

At 150 and 1000 mg/kg/day, there were higher incidences of minor and variant foetal abnormalities compared with Control that affected the cervical vertebrae, ribs, sternebrae, skull and palate.

These significantly higher incidences of several skeletal minor abnormalities and/or variants (affecting skull, cervical vertebrae, ribs and sternebrae) were outside of the historical Control range seen within this laboratory and were considered to be related to the test item. In addition, increases in minor or variant foetal abnormalities associated with skeletal ossification were observed, but were considered to be transient in nature and/or of no developmental consequence. There were no major foetal abnormalities considered to be test item-related.

Justification for classification or non-classification

Reproductive toxicity

No adverse effects on oestrous cycle, sperm motility, concentration and morphology, fertility, reproductive performance or gestation length were seen at any dose level in the available reliable two-generation reproductive toxicity study performed in the rat under GLP and to OECD TG 416. Sub-chronic oral exposure to the substance lead to hypertrophy and vacuolation of the interstitial gland in the ovaries of female rats, but in the absence of a negative impact on fertility and reproduction the adverse nature of these observed effects remains equivocal. Based on the available information, the substance does not meet the criteria for classification for reproductive effects according to the CLP Regulation (EC) No. 1272/2008.

Developmental toxicity

In a developmental toxicity study in the rat performed under GLP and in accordance with OECD TG 414 higher incidences of minor and variant foetal abnormalities compared with the Control that affected the cervical vertebrae, ribs, sternebrae, skull and palate were observed at the mid and high dose. Evaluation of the evidence for effects of the substance on the embryo and foetal development indicates that the available evidence meets the criteria required for classification as suspected human reproductive toxicant, category 2, but does not meet the criteria for classification as a presumed human reproductive toxicant, category 1. The substance should be classified as a suspected human reproductive toxicant, category 2 (H361d: Suspected of damaging the unborn child) in accordance with the criteria set out in Regulation (EC) No. 1272/2008 on the basis of a pattern of minor and variant skeletal abnormalities.