1-(3-chloropyridin-2-yl)-N-[4-cyano-2-methyl-6-(methylcarbamoyl)phenyl]-3-{[5-(trifluoromethyl)-2H-tetrazol-2-yl]methyl}-1H-pyrazole-5-carboxamide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Two generation reproductive toxicity, oral (OECD 416), rat:

reproductive toxicity: NOAEL >= 12000 ppm (equivalent to 896 and 1032 mg/kg bw/day for males and females of the P0 generation, respectively; equivalent to 1138 and 1218 mg/kg bw/day for males and females of the P1 generation, respectively)

systemic toxicity: NOAEL = 2700 ppm (equivalent to 196 and 224 mg/kg bw/day for males and females of the P0 generation, respectively; equivalent to 253 and 266 mg/kg bw/day for males and females of the P1 generation, respectively)

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Jun 2014 - 03 Jun 2015

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:

adopted in 2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Version / remarks:

adopted in 1998

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No 8147

Version / remarks:

adopted in 2000

GLP compliance:

yes (incl. QA statement)

Remarks:

Department of Health, United Kingdom

Limit test:

yes

Species:

rat

Strain:

other: RCCHanTM; WIST rat.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan (UK) Ltd.
- Age at study initiation: (P) 40 - 46 days; (F1) 28 ± 2 days
- Weight at study initiation: (P) 135 - 197 g (males); 112 - 152 g (females); (F1) Males: 77 - 84 g; Females: 72 - 78 g
- Housing: Pre-pairing, males after pairing to termination and females after weaning to termination: groups up to 4 animals per sex per cage in polycarbonate cages with a stainless steel mesh lid and softwood based bark-free fibre bedding; Pairing: 1 male and 1 female in grid bottomed cages above absorbent paper with a stainless steel mesh lid; Females after mating: individually in polycarbonate cages with a stainless steel mesh lid and softwood based bark-free fibre bedding; Females during littering: one female and litter in polycarbonate cages with a stainless steel mesh lid and softwood based bark-free fibre bedding; an aspen chew block and a plastic shelter were provided to each cage except during pairing and lactation
- Diet: SDS VRF1 powdered diet, ad libitum
- Water: potable water from the public supply, ad libitum
- Acclimation period: 18 days

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

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: weekly
- Mixing appropriate amounts with: SDS VRF1 powdered diet
- Storage temperature of food: ambient (nominally 21°C)

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: up to two weeks
- Proof of pregnancy: ejected copulation plugs in cage tray and sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individual in solid polycarbonate bottom cages with a stainless stell mesh lid with softwood based bark-free fibre bedding and an aspen chew block and plastic shelter as enrichment

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulations prepared at nominal concentrations of 150 and 18000 ppm (168 and 20160 ppm formulated concentrations of material as supplied, respectively) were evaluated for stability and homogeneity using HPLC-UV analysis in a previous study. Stability and homogeneity after storage for up to 22 days at either ambient temperature (nominally 21°C) or frozen (nominally -20°C) were confirmed. For concentration analysis via HPLC-UV analysis during the present study, samples of the following formulations were collected: F0 generation (Week 1), F0 gestation, F0-1 generation (final week in lactation), F1 pairing, F1-F2 generation (final week in lactation), F2 maturation (final study week). The mean concentrations of the test substance in test formulations were between -7.7 and +4.7% of nominal values which is within applied limits +10%/-15% of nominal concentrations, confirming accurate formulation.

Duration of treatment / exposure:

approximately 20 weeks (P and F1 generations; males: ten weeks before pairing until termination; females: ten weeks before pairing, throughout pairing, gestation and lactation)
approximately 8 weeks (F2 generation; from weaning until termination on Day 70 of age)

Frequency of treatment:

continously (via diet)

Details on study schedule:

- Selection of parents from F1 generation when pups were 20 days of age.

Dose / conc.:

300 ppm

Remarks:

during Weeks 1 - 10:
equivalent to 22 and 25 mg/kg bw/day in males and females of the P0 generation, respectively and equivalent to 28 and 30 mg/kg bw/day in males and females of the P1 generation, respectively

Dose / conc.:

600 ppm

Remarks:

during Weeks 1 - 10:
equivalent to 44 and 51 mg/kg bw/day in males and females of the P0 generation, respectively and equivalent to 57 and 63 mg/kg bw/day in males and females of the P1 generation, respectively

Dose / conc.:

2 700 ppm

Remarks:

during Weeks 1 - 10:
equivalent to 196 and 224 mg/kg bw/day in males and females of the P0 generation, respectively and equivalent to 253 and 266 mg/kg bw/day in males and females of the P1 generation, respectively

Dose / conc.:

12 000 ppm

Remarks:

during Weeks 1 - 10:
equivalent to 896 and 1032 mg/kg bw/day in males and females of the P0 generation, respectively and equivalent to 1138 and 1218 mg/kg bw/day in males and females of the P1 generation, respectively

Dose / conc.:

300 ppm

Remarks:

during gestation:
equivalent to 22 and 23 mg/kg bw/day in females of the P0 and the P1 generation, respectively

Dose / conc.:

600 ppm

Remarks:

during gestation:
equivalent to 43 and 47 mg/kg bw/day in females of the P0 and the P1 generation, respectively

Dose / conc.:

2 700 ppm

Remarks:

during gestation:
equivalent to 197 and 211 mg/kg bw/day in females of the P0 and the P1 generation, respectively

Dose / conc.:

12 000 ppm

Remarks:

during gestation:
equivalent to 875 and 1000 mg/kg bw/day in females of the P0 and the P1 generation, respectively

Dose / conc.:

150 ppm

Remarks:

during lactation:
equivalent to 23 and 23 mg/kg bw/day in females of the P0 and the P1 generation, respectively

Dose / conc.:

300 ppm

Remarks:

during lactation:
equivalent to 47 and 47 mg/kg bw/day in females of the P0 and the P1 generation, respectively

Dose / conc.:

1 350 ppm

Remarks:

during lactation:
equivalent to 211 and 215 mg/kg bw/day in females of the P0 and the P1 generation, respectively

Dose / conc.:

6 000 ppm

Remarks:

during lactation:
equivalent to 890 and 947 mg/kg bw/day in females of the P0 and the P1 generation, respectively

No. of animals per sex per dose:

24

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The highest dose (12000 ppm) was selected based on a preliminary study of reproduction performance in the rat where treatment-related findings at 15000 ppm consisted of a slightly low body weight gain during late gestation in the F0 parent females compared with the controls together with a slight loss in body weight during late lactation. In the offspring at the high dose level, body weight gain of both male and females was reduced. For animals selected to form the P1 generation, body weight gain continued to be statistically significantly reduced associated with a delay in sexual maturation. The high dose level of 12000 ppm on the two generation reproduction study was expected to approximate to a limit dose of 1000 mg/kg/day during the F0 pre-mating phase of the study. A high intermediate dose level of 2700 ppm provided an approximate 4.5 fold factor to the highest dose level. The selected low intermediate dose of 600 ppm was anticipated to be a NO(A)EL for both parents and offspring. In the event of toxicity observed at 600 ppm, a dose level of 300 ppm was expected to provide a clear NO(A)EL. During the lactation phase the dietary concentration for females was lowered by 50% to 6000, 1350, 300 and 150 ppm so that the achieved dose for the P0/P1 females in the high dose group does not increase markedly above the limit dose of 1000 mg/kg/day as the maternal food consumption increased.
- Rationale for animal assignment:
P0 generation: Animals were allocated to the P0 generation of the study by sex. Animals showing signs of ill health were excluded. Animals at the extreme of the weight range or litters showing large variation in individual weights were not selected if alternatives were available. The method of allocation of animals to the P0 generation ensured that no more than one offspring of each sex from each litter was present in each group.
P1/F2 generation: The offspring with the lowest within-litter identification per sex from each selected litter will be selected to form the P1/F2 generations, after exclusion of grossly atypical animals. Where possible, either two males and two females or one male and one female were selected from each selected litter. If more were required, offspring were taken from randomly selected litters from each group.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations included: mortality, ill-health, reaction to treatment

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly and on Days 0, 5, 12, 18 and 20 after mating and on Days 1, 7, 14 and 21 of lactation for P0 and P1 animals

BODY WEIGHT: Yes
- Time schedule for examinations: P0 males were weighed on the day of treatment, weekly thereafter and at necropsy. P0 females were weighed on the day of treatment, weekly thereafter until mating, on Days 0, 7, 14 and 20 after mating and on Days 1, 4, 7, 14, 21, 25 and 28 post-partum. P1, F1 and F2 animals were weighed at the same frequency P0 animals following selection (nominally 4 weeks of age).

FOOD CONSUMPTION AND COMPOUND INTAKE:
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded weekly until paired for mating or termination for P0 males and females. From these records the mean weekly consumption per animal (g/animal/week) was calculated for each cage. The different weights were also recorded for P0 females on Days 0-6, 7-13 and 14-19 after mating and Days 1-3, 4-6, 7-13 and 14-20 of lactation. From these records the mean daily consumption (g/animal/day) was calculated for each animal. For P1/F1/F2 animals the weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded at the same frequency as P0 animals following selection, at approximately 4 weeks of age.

WATER CONSUMPTION: No

Oestrous cyclicity (parental animals):

Smears were taken daily for 22 days before pairing, using cotton swabs moistened with saline. The smears were subsequently examined to establish the duration and regularity of the oestrous cycle. After pairing with the male, daily smearing was continued using pipette lavage, until evidence of mating was observed. For 4 days before scheduled termination, Days 25 to 28 post-partum for P0 and P1 females and Days 67 to 70 of age for F1 and F2 females, daily vaginal smears were taken and used to determine the stage of the oestrous cycle at termination.

Sperm parameters (parental animals):

Parameters examined in P/F1 male parental generations:
testis weight, epididymis weight, sperm motility, sperm morphology, sperm count in epididymides, other: homogenisation-resistant spermatids count

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 8 pups/litter (4/sex/litter wherever possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
number and sex of pups, anogenital distance (AGD), postnatal mortality, other: clinical signs, sex ratio, individual offspring body weights

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead; an assessment for the presence of milk in the stomach was included

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: all surviving animals when the majority of litters had weaned after 18 weeks of treatment for the P0 males or 18 weeks of the F1 generation for the P1 males
- Maternal animals: all surviving animals on Day 28 post-partum

GROSS NECROPSY
- Gross necropsy consisted of a complete macroscopic examination. For all females of the parental generations the number of implantation sites was recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of a complete macroscopic examination.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table 2 were prepared for microscopic examination and weighed, respectively.

Statistics:

For body weight, food consumption, litter size and survival indices, sexual maturation and organ weight data, a parametric analysis was performed if Bartlett's test for variance homogeneity was not significant (1% level). The F1 approximate test was applied. If the F1 approximate test for monotonicity of dose-response was not significant (1% level), Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, Dunnett's test was performed instead. A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. The H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied. If the H1 approximate test for monotonicity of dose-response was not significant at the 1% level, Shirley's test for a monotonic trend was applied. If the H1 approximate test was significant, Steel's test was performed instead. For litter size and survival indices and sexual maturation, if 75% of the data (across all groups) were the same value, for example c, Fisher’s Exact tests were performed (pairwise comparison). Sex ratio were analysed by generalised mixed linear model with binomial errors, a logit link function and litter as a random effect. Each treated group was compared to control using a Wald chi-square test. For sex ratio, the numerator was number of males, the denominator was number of live fetuses. For gestation length an exact two-tailed Linear-by-linear test, with equally spaced scores, was applied to all groups. If the test was statistically significant (p<0.05), the highest dose group was excluded and the test re-applied. This ‘step-down’ process was repeated until the test was no longer statistically significant (p≥0.05). If the exact version of the Linear-by-linear test could not be calculated (due to the size of the table containing the data), the asymptotic version was used instead.

Reproductive indices:

Percentage mating = (number animals mating / animals paired) x 100
Conception rate (%) = (number animals achieving pregnancy / animals mated) x 100
Fertility index (%) = (number animals achieving pregnancy / animals pairing) x 100
Gestation index (%) = (number live litters born / number pregnant) x 100
Post-implantation survival index (%) = (total number of offspring born / total number of uterine implantation sites) x 100
Percentage males = (number of males in litter / total number of offspring in litter) x 100

Offspring viability indices:

Live birth index (%) = (number of live offspring on Day 1 after littering / total number of offspring born) x 100
Viability index (%) = (number of live offspring on Day 4 before culling / number of live offspring on Day 1 after littering) x 100
Lactation index (%) = (number of live offspring on Day 21 after littering / number of live on Day 4 after culling) x 100

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

FEMALES
12000 ppm: decreased mean body weight gain when compared to controls during the first and second week of gestation (Days 0 - 7 and 7 - 14); increased body weight gain when compared to controls from Day 1 - 4 of lactation (both adverse during Days 0 - 7)
2700, 600 and 300 ppm: decreased mean body weight gain when compared to controls during the second week of gestation (Days 7 - 14)

(see table 3)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

MALES
12000, 2700 and 600 ppm: decreased food consumption when compared with controls during Week 1 (adverse at the highest dose)
12000 and 2700 ppm: decreased food consumption when compared with controls during Week 2 (adverse at the highest dose)
12000, 2700, 600 and 300 ppm: decreased food consumption when compared with controls during Weeks 5,7 and 8 (adverse at the highest dose)

FEMALES
12000, 2700, 600 and 300 ppm: decreased food consumption when compared with controls during Week 1; decreased food consumption when compared with controls during the second week of gestation (adverse at the highest dose)

(see table 4)

Food efficiency:

no effects observed

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:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

FEMALES
12000 ppm: significant shift towards shorter gestation periods with the percentage exhibiting a gestation length of 22 days exceeding the historical control data range (non-adverse)
(see table 5)

Pre-coital interval, mating performance, fertility and gestation index were unaffected by treatment

For details on results including tables see the attached document.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

2 700 ppm

Based on:

test mat.

Remarks:

equivalent to 196 and 224 mg/kg bw/day in males and females, respectively

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

>= 12 000 ppm

Based on:

test mat.

Remarks:

equivalent to 896 and 1032 mg/kg bw/day for males and females, respectively

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

MALES
12000, 2700, 600 and 300 ppm: decreased mean body weight at the beginning of the P1 generation (Week 0) and during Weeks 1, 2, 3, 4, 5 and 6 (adverse at the highest dose)

FEMALES
12000, 2700, 600 and 300 ppm: decreased mean body weight at the beginning of the P1 generation (Week 0) and during all weeks before pairing; decreased weight gain between Weeks 0 - 10 before pairing, decreased mean body weights during gestation and lactation, increased weight gain during Days 1 - 4 of lactation (all effects adverse at the highest dose)

(see table 6)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

MALES
12000 and 2700 ppm: decreased food consumption during Week 1 (adverse at the highest dose)
600 ppm: increased food consumption during Week 2
12000, 2700, 600 and 300 ppm: decreased food consumption during Weeks 3 and 4 (adverse at the highest dose)

FEMALES
12000 and 2700 ppm: decreased food consumption during Week 1 (adverse at the highest dose)
600 ppm: increased food consumption during Week 2
12000, 2700, 600 and 300 ppm: decreased food consumption during Week 4 (adverse at the highest dose)

(see table 7)

Food efficiency:

no effects observed

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:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

For details on results including tables see the attached document.

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Pre-coital interval, mating performance and fertility were unaffected by treatment. Assessment of vaginal cytology prior to termination did not reveal any differences across the groups. Assessment of ovarian follicle counts of control females and females at the highest dose (12000 ppm and 6000 ppm during lactation phase) did not reveal any difference.
There was a slight shift in gestation length with a higher percentage of females at 12000 ppm showing a shorter gestation length compared with controls, although, the gestation length for all females at 12000 ppm was within the normal range of 22 to 23.5 days. The shift did not attain statistical significance, however the percentage of females showing a 22-day gestation length exceeded the historical control data range, whilst the percentage of females showing a 22.5 and 23-day gestation length was below the historical control data range. It should be noted however, that the concurrent control data was also outside the historical control data range.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

2 700 ppm

Based on:

test mat.

Remarks:

equivalent to 253 and 266 mg/kg bw/day for males and females, respectively

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

>= 12 000 ppm

Based on:

test mat.

Remarks:

equivalent to 1138 and 1218 mg/kg bw/day for males and females, respectively

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

MALES
12000/6000 ppm (parental animal before lactation phase/during lactation phase): decreased offspring body weight gain when compared with controls from Day 7 of age up to weaning on day 21 of age
12000, 2700, 600 and 300 ppm (treatment following weaning at Days 21 - 25 in selected animals): decreased offspring body weight gain when compared to controls

FEMALES
12000/6000 ppm (parental animal before lactation phase/during lactation phase): decreased offspring body weight gain when compared with controls from Day 7 of age up to weaning on day 21 of age
2700/1350, 600/300 and 300/150 ppm (parental animal before lactation phase/during lactation phase): decreased offspring body weight gain when compared with controls from Day 7 to 14 of age

(see table 8)

Body weight and weight changes during the 10 week pre-mating period of the F1 generation becoming P1 generation are shown in section "Results: P1 generation".

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption during the 10 week pre-mating period of the F1 generation becoming P1 generation is shown in section "Results: P1 generation".

Food efficiency:

no effects observed

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:

effects observed, non-treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

For details on results including tables see the attached document.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Generation:

F1

Effect level:

2 700 ppm

Based on:

test mat.

Remarks:

equivalent to 253 and 266 mg/kg bw/day in males and females, respectively

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Generation:

F1

Effect level:

>= 12 000 ppm

Based on:

test mat.

Remarks:

equivalent to 1138 and 1218 mg/kg bw/day in males and females, respectively

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

MALES
12000/6000 ppm (parental animal before lactation phase/during lactation phase): decreased offspring weight gain when compared with controls from Day 14 to 21 of age; decreased absolute weights from Day 14 to 25
12000 ppm: decreased absolute body weights when compared with controls on Weeks 0, 1, 2, 4 and 5 (adverse)

FEMALES
12000/6000 ppm (parental animal before lactation phase/during lactation phase): decreased offspring weight gain when compared with controls from Day 14 to 21 of age; decreased absolute weights from Day 21 to 25
12000 ppm: decreased absolute body weights when compared with controls on Weeks 1, 4 and 5 (adverse)

(see tables 12 and 13)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

MALES
12000 and 2700 ppm: decreased food consumption during Week 3

(see table 14)

Food efficiency:

no effects observed

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:

effects observed, non-treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

MALES
12000/6000 ppm (parental animal before lactation phase/during lactation phase): increased brain weight relative to body weight (see table 16)
12000 ppm (selected animals terminated on Day 70 of age): decreased absolute and body weight relative ovarian weights (see table 17)

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

For details on results including tables see the attached document.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Generation:

F2

Effect level:

2 700 ppm

Based on:

test mat.

Remarks:

equivalent to 307 and 312 mg/kg bw/day for males and females, respectively

Sex:

male/female

Basis for effect level:

sexual maturation

body weight and weight gain

food consumption and compound intake

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Generation:

F2

Effect level:

>= 12 000 ppm

Based on:

test mat.

Remarks:

equivalent to 1361 and 1392 mg/kg bw/day for males and females, respectively

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

For details on results including tables see the attached document.

 

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

896 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.7, of Regulation (EC) No 1907/2006.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A reliable study regarding toxicity to reproduction is available for the test substance.

Toxicity to reproduction in the rat

The influence of the test substance on reproductive performance was assessed in a two generation reproductive toxicity study in RCCHanTM;WIST rats performed according to OECD Guideline 416 and in compliance with GLP (M-569724-02-1). For the P0 generation, four groups of 24 male rats received the test substance orally, via the diet, at concentrations of 300, 600, 2700 or 12000 ppm for ten weeks before pairing until termination. For the P0 generation, four groups of 24 female rats received the test substance orally, via the diet, at concentrations of 300, 600, 2700 or 12000 ppm for ten weeks before pairing, throughout pairing and gestation and at concentrations of 150, 300, 1350 or 6000 ppm during lactation. A similarly constituted control group received untreated basal diet for the same duration.

P0 generation

The mean achieved dose during Weeks 1 to 10 of treatment at 300, 600, 2700 and 12000 ppm was 22, 44, 196 and 896 mg/kg/day for males and 25, 51, 224 and 1032 mg/kg/day for females, respectively. During gestation and lactation the achieved doses were 22, 43, 197 and 875 mg/kg bw/day for females at 300, 600, 2700 and 12000 ppm and 23, 47, 211 and 890 mg/kg bw/day for females at 150, 300, 1350 and 6000 ppm, respectively.

P1 and F1 generation

The mean achieved dose during Weeks 1 to 10 of the P1 generation at 300, 600, 2700 and 12000 ppm was 28, 57, 253 and 1138 mg/kg/day for males and 30, 63, 266 and 1218 mg/kg bw/day for females, respectively. During gestation and lactation the achieved doses were 23, 47, 211 and 1000 mg/kg bw/ day for females at 300, 600, 2700 and 12000 ppm and 23, 47, 215 and 947 mg/kg bw/day for females at 150, 300, 1350 and 6000 ppm, respectively.

F2 generation

The mean achieved dose during Weeks 1 to 5 of the F2 generation at 300, 600, 2700 and 12000 ppm was 34, 69, 307 and 1361 mg/kg bw/day for males and 34, 68, 312 and 1392 mg/kg bw/day for females,respectively.

Conclusion

Dietary administration to Han Wistar rats of the test substance at concentrations of 300, 600, 2700 and 12000 ppm (adjusted to 150, 300, 1350 and 6000 ppm for females during lactation) was generally well tolerated. There were no signs of any overt systemic toxicity among the P0, P1, F1 or F2 adult male and female animals and their general condition, mating performance, fertility and reproductive organs were unaffected by treatment. Differences in body weight and food consumption at the mid and low dose compared to control were marginal, with no dose response and no effect on food conversion efficiency; in isolation, these findings were considered to be non-adverse at these dose levels. At the high dose, the difference in body weight and food consumption was slightly more pronounced andwas observed in all generations. In light of the slightly more pronounced effects on bodyweight and food consumption at the high dose, which were associated with the observed delay in vaginal opening (F1) and preputial separation (F2), these findings were considered to be adverse at this dose level. Thus, under the conditions of this study, it is considered that 2700 ppm (equivalent to the mean achieved dose before pairing: 196 mg/kg/day for P0 males; 224 mg/kg/day for P0 females; 253 mg/kg/day for F1 and P1 males; 266 mg/kg/day for F1 and P1 females; 307 mg/kg/day for F2 males; 312 mg/kg/day for F2 females) represents the NOAEL in this study for both the P0, P1, F1 and F2 adult animals and the F1 and F2 offspring. In terms of reproductive effects, no treatment-related findings were observed at any dose level tested; therefore, the high dose of 12000 ppm (equivalent to the mean achieved dose before pairing: 896 mg/kg/day for P0 males; 1032 mg/kg/day for P0 females; 1138 mg/kg/day for F1 and P1 males; 1218 mg/kg/day for F1 and P1 females; 1361 mg/kg/day for F2 males; 1392 mg/kg/day for F2 females) is considered to be the NOAEL for reproductivetoxicity.

Effects on developmental toxicity

Description of key information

Teratogenicity study, oral (OECD 414), rat: NOAEL >= 1000 mg/kg bw/day, NOEL = 250 mg/kg bw/day

Teratogenicity study, oral (OECD 414), rabbit: NOEL >= 1000 mg/kg bw/day

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Aug 2013 - 18 Sep 2014

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:

adopted in 2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted in 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted in 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: M.A.F.F. in Japan, notification 12 Nousan N°8147 guideline

Version / remarks:

adopted in 2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD (SD) Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Sulzfeld, Germany
- Age at study initiation: 11 - 13 weeks
- Weight at study initiation: 239 - 328 g (at mating)
- Housing: individual in suspended polycarbonate wire mesh cages
- Diet: A04C-10 pelleted rodent diet (Scientific Animal Food and Engineering, Augy, France), ad libitum
- Water: filtered and softened tap water from the municipal water supply, ad libitum
- Acclimation period: at least 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 0.5% aqueous methylcellulose 400

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Two dosing solutions were prepared during the study by dissolving appropriate amounts of the test material in an aqueous solution of methylcellulose 400 at 0.5% yielding final concentrations of 6.25, 25 and 100 g/L. A complementary formulation at the highest dose level for the last two days of the study was prepared for the treatment of one animal.

VEHICLE
- Concentration in vehicle: 6.25, 25 and 100 g/L
- Amount of vehicle (if gavage): 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The first formulation (F1) prepared for the study was evaluated for homogeneity and concentration using HPLC-UV analysis. Triplicate samples were collected of the lowest and the highest dietary concentrations from the top, the middle and the bottom of the formulation. In addition triplicate samples of the intermediate dose level (F1) and all dose levels of the second formulation (F2) were collected from the surface, middle and the bottom of the formulation for concentration analysis. One of the three collected samples was analyzed and the others were stored for possible verification analysis. Data indicate homogenous mixture of the test material in the vehicle. The measured values for the individual homogeneity samples ranged between 97 and 101% of nominal. Additionally, samples were at the targeted concentration (98 – 100% of nominal). The stability of test item in 0.5% aqueous methylcellulose was demonstrated in a previous study at concentrations of 0.3 and 250 g/L for up to 28 days under similar conditions of usage and storage to those of the current study (2012).

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: overnight
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear or sperm plug in situ referred to as day 0 of pregnancy

Duration of treatment / exposure:

day 6 - 20 of gestation

Frequency of treatment:

daily, 7 days/week

Duration of test:

day 21 of gestation

Dose / conc.:

62.5 mg/kg bw/day

Dose / conc.:

250 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

23 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The range of doses has been selected in agreement with the Sponsor Representative and based on results obtained in a range-finding study where pregnant rats received the test substance at 0, 100, 400 or 800 mg/kg/day from GD 6 through 20 (2010). In this study, reduced mean maternal body weight gain between GD 6 and 8 when compared to controls was observed at 800 mg/kg bw/day. Mean maternal corrected body weight change (body weight gain between GD 6 and 21 independent of the gravid uterus weight recorded at cesarean section) was decreased by 60% in comparison to controls (not statistically significant). Decreased mean maternal corrected body weight change was observed at the mid and low dose (not statistically significant). Therefore, 0, 62.5, 250 and 1000 mg/kg/day were selected as the dose levels for this study. A high dose of 1000 mg/kg/day is a limit dose, which based on the range-finding study was not expected to exceed a maximal tolerated dose (MTD) in the dams. A mid dose of 250 mg/kg/day provided a 4 fold factor between the dose levels. A low dose of 62.5 mg/kg/day was expected to be a No Observed (Adverse) Effect Level for both maternal and fetal toxicity, based on the range-finding data.
- Other: If possible, those females having been paired with the same male were not allocated to the same group. Body weight means were checked after the mating period to ensure similar means among all groups.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (once daily at weekends and public holiday)
- Cage side observations included: mortality, moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily, day 0 - 21 of gestation

BODY WEIGHT: Yes
- Time schedule for examinations: day 0, 6, 8, 10, 12, 14, 16, 18 and 21 of gestation

FOOD CONSUMPTION: Yes
Full feeder weights were measured on GD: 1, 6, 8, 10, 12, 14, 16 and 18. Empty feeder weights were measured on GD: 6, 8, 10, 12, 14, 16, 18 and 21. From these records the mean daily consumption was calculated.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: visceral organs (macroscopic examination), liver (weighed), reproductive tract (weighed and dissected; gravid uterine weight), uterine horn(s) without visible implantations (immersed in a 10% solution of ammonium sulfide to visualize any sites which were not apparent)

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: number of live and dead fetuses, sex of live fetuses, individual weights of live fetuses

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data

Statistics:

For most maternal and litter based endpoints Bartlett test was performed to compare the homogeneity of group variances. If the Bartlett test was not significant (p>0.05), means were compared using the analysis of variance (ANOVA). If the ANOVA was not significant (p>0.05), the group means were considered to be homogeneous and no further analysis was performed. If the ANOVA was significant (p≤0.05), means of the exposed groups were compared to the mean of the control group using the Dunnett test (2-sided). If the Bartlett test was significant (p≤0.05), group means were compared using the non- parametric Kruskal-Wallis test. If the Kruskal-Wallis test was not significant (p>0.05), the group means were considered to be homogeneous and no further analysis was performed. If the Kruskal-Wallis test was significant (p≤0.05), means of the exposed groups were compared to the mean of the control group using the Dunn test (2-sided). If the Bartlett test was significant for average food consumption data were transformed using the log transformation the Bartlett test was applied to the transformed data. If the Bartlett test was significant (p≤0.05) even after log transformation, statistical analysis was proceeded as mentioned above (the ANOVA test was also performed with transformed data). For fetal body weight the Levene test was used followed by the ANOVA test in case of non-significance. In case of significance (p≤0.05) data were transformed using the log transformation before the Levene test was applied to the transformed data. If the Levene test was significant (p≤0.05) even after log transformation, statistical analysis was proceeded wth the ANOVA test or the Kruskal-Wallis test as mentioned above (the ANOVA test was also performed with transformed data). If one or more group variance(s) equaled 0, means were compared using non-parametric procedures.

Indices:

- Corrected body weight change (CBWC): CBWC = (BW on day 21 of gestation – BW on day 0 of gestation) – (gravid uterine weight)
- Carcass weight: BW on day 21 of gestation – uterus weight
- Total resorptions = early + late resorptions
- Pre-implantation loss %: (number of corpora lutea – number of implantations / number of corpora lutea) x 100
- Post-implantation loss %: (number of implantations – number of live fetuses / number of implantations) x 100
- Percentage of dead fetuse/ fetal death: number of dead fetuses / total number of fetuses x 100
- Percentage of male fetuses: number of live male fetuses / total number of live male and female fetuses x 100
- Mean fetal body weight: sum of individual weights of live fetuses / number of weighed live fetuses
- Mean fetal body weight per sex (example male fetuses): sum of individual weights of live male fetuses / number of weighed live male fetuses
- Fetal death per litter (%): number of litters with dead fetuses / total number of litters x 100
- For external, visceral and skeletal fetal findings, the percentage of fetuses affected per group for a given parameter: sum of live fetuses affected / number of live fetuses examined x 100
- For external, visceral and skeletal fetal findings, the percentage of litters affected per group for a given parameter: sum of litters with live fetuses affected / number of litter with live fetuses examined x 100

Historical control data:

Historical control data from studies conducted in-house were referred to in order to allow comparison with concurrent controls. The data were generated from sixteen rat studies between 2003 and 2012 and are presented in the report.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

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:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

For details on results including tables see the attached document.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, non-treatment-related

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

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

Details on maternal toxic effects:

For details on results including tables see the attached document.

Key result

Dose descriptor:

NOEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: highest dose tested

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: decreased mean fetal body weight (combined and per sex) compared to control (4%, non-adverse)
(see table 3)
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Skeletal malformations:

effects observed, non-treatment-related

Visceral malformations:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

SKELETAL FETAL VARIATIONS
1000 mg/kg bw/day: increased incidence of of 2 spontaneous variations; “5th and 6th sternebrae: incomplete ossification” at the fetal level and “5th and/or 6th sternebrae: unossified” at the fetal and litter levels (non-adverse)

Details on embryotoxic / teratogenic effects:

For details on results including tables see the attached document.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Dose descriptor:

NOEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: skeletal fetal variations

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

For details on results including tables see the attached document.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Apr - 12 Nov 2014

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:

adopted in 2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted in 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted in 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: M.A.F.F. in Japan, notification 12 Nousan N°8147 guideline

Version / remarks:

adopted in 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Groupe Interministeriel Des Produits Chimiques, Ivry-sur-Seine, France

Limit test:

no

Species:

rabbit

Strain:

other: New Zealand White Crl:KBL (NZW)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Châtillon-sur-Chalaronne, France
- Age at study initiation: 18 ± 1 week
- Weight at study initiation: 2.95 - 3.87 kg
- Housing: individual in suspended stainless steel wire mesh cages
- Diet: 110 C-10 pelleted (Scientific Animal Food and Engineering, Augy, France), ad libitum
- Water: filtered and softened tap water from the municipal water supply, ad libitum
- Acclimation period: at least 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 21
- Humidity (%): 40 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 8/16

Route of administration:

oral: gavage

Vehicle:

other: 0.5% aqueous methylcellulose 400

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Six dosing solutions were prepared during the study by dissolving appropriate amounts of the test material in an aqueous solution of methylcellulose 400 at 0.5% yielding final concentrations of 15.625, 62.5 and 250 g/L.

VEHICLE
- Concentration in vehicle: 15.625, 62.5 and 250 g/L
- Amount of vehicle (if gavage): 4 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The first formulation (F1) prepared for the study was evaluated for homogeneity and concentration using HPLC-UV analysis. Triplicate samples were collected of the lowest and the highest dietary concentrations from the top, the middle and the bottom of the formulation. In addition triplicate samples of the intermediate dose level (F1) and all dose levels of the other formulations (F2, F3, F4, F5, F6) were collected from the surface, middle and the bottom of the formulation for concentration analysis. One of the three collected samples was analyzed and the others were stored for possible verification analysis. Data indicate homogenous mixture of the test material in the vehicle. The measured values for the individual homogeneity samples ranged between 95 and 104% of nominal. Additionally, samples were at the targeted concentration (93 – 104% of nominal). The stability of test item in 0.5% aqueous methylcellulose was demonstrated in a previous study at concentrations of 0.3 and 250 g/L for up to 28 days under similar conditions of usage and storage to those of the current study (2012).

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant
- Verification of same strain and source of both sexes: yes

Duration of treatment / exposure:

day 6 - 28 of gestation

Frequency of treatment:

daily, 7 days/week

Duration of test:

day 29 of gestation

Dose / conc.:

62.5 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

23

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The range of dosages has been selected in agreement with the Sponsor Representative and based on results obtained in a previous range-finding study where pregnant rabbits received the test substance at 0, 100, 300 or 900 mg/kg/day from GD 6 through 28 (2012). In this study no treatment-related effects on maternal or litter parameters or at the external fetal observations were recorded up to the dose of 900 mg/kg/day. Therefore, 0, 62.5, 250 and 1000 mg/kg/day were selected as the dose levels for this study. A high dose of 1000 mg/kg/day was a limit dose, which based on the range-finding study was not expected to exceed a maximal tolerated dose (MTD) in the dams. A mid dose of 250 mg/kg/day provided a 4 fold factor between the dose levels. A low dose of 62.5 mg/kg/day was expected to be a No Observed (Adverse) Effect Level for both maternal and fetal toxicity, based on the range-finding data.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (once daily at weekends and public holidays)
- Cage side observations included: mortality, moribundity, signs of abortion

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily (clinical signs)

BODY WEIGHT: Yes
- Time schedule for examinations: days 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 26 and 29 of gestation

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Full feeder weights were measured on days 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 28 of gestation. Empty feeder weights were measured on days 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 29 of gesation. From these records the mean daily consumption was calculated.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: visceral organs (macroscopic examination), liver (weighed), reproductive tract (weighed and dissected; gravid uterine weight), uterine horn(s) without visible implantations (immersed in a 10% solution of ammonium sulfide to visualize any sites which were not apparent)

OTHER:
- Bioanalytical examination: On the day of sacrifice, a blood sample was collected from the ear of at least five pregnant rabbits from each group just prior to necropsy and plasma was prepared. The plasma samples were analysed for concentrations of the test substance and the major metabolite using HPLC Tandem Mass Spectrometry (HPLC-MS/MS).

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: number of live and dead fetuses, individual weights of live fetuses

Fetal examinations:

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

Statistics:

For most maternal and litter based endpoints Bartlett test was performed to compare the homogeneity of group variances. If the Bartlett test was not significant (p>0.05), means were compared using the analysis of variance (ANOVA). If the ANOVA was not significant (p>0.05), the group means were considered to be homogeneous and no further analysis was performed. If the ANOVA was significant (p≤0.05), means of the exposed groups were compared to the mean of the control group using the Dunnett test (2-sided). If the Bartlett test was significant (p≤0.05), group means were compared using the non- parametric Kruskal-Wallis test. If the Kruskal-Wallis test was not significant (p>0.05), the group means were considered to be homogeneous and no further analysis was performed. If the Kruskal-Wallis test was significant (p≤0.05), means of the exposed groups were compared to the mean of the control group using the Dunn test (2-sided). If the Bartlett test was significant for average food consumption data were transformed using the log transformation and the Bartlett test was applied to the transformed data. If the Bartlett test was significant (p≤0.05) even after log transformation, statistical analysis was proceeded as mentioned above (the ANOVA test was also performed with transformed data). For fetal body weight the Levene test was used followed by the ANOVA test in case of non-significance. In case of significance (p≤0.05) data were transformed using the log transformation before the Levene test was applied to the transformed data. If the Levene test was significant (p≤0.05) even after log transformation, statistical analysis was proceeded wth the ANOVA test or the Kruskal-Wallis test as mentioned above (the ANOVA test was also performed with transformed data). If one or more group variance(s) equaled 0, means were compared using non-parametric procedures.

Indices:

- Corrected body weight change (CBWC): CBWC = (BW on gestation day 29 – BW on gestation day 3) – (gravid uterine weight)
- Carcass weight: BW on gestation day 29 – uterus weight
- Total resorptions = early + late resorptions
- Pre-implantation loss %: (number of corpora lutea – number of implantations / number of corpora lutea) x 100
- Post-implantation loss %: (number of implantations – number of live fetuses / number of implantations) x 100
- Percentage of dead fetus/ fetal death: number of dead fetuses / total number of fetuses x 100
- Percentage of male fetuses: number of live male fetuses / total number of live male and female fetuses x 100
- Mean fetal body weight: sum of individual weights of live fetuses / number of weighed live fetuses
- Mean fetal body weight per sex (example male fetuses): sum of individual weights of live male fetuses / number of weighed live male fetuses
- Fetal death per litter (%): number of litters with dead fetuses / total number of litters x 100
- Mean fetal body weight: sum of mean weights of live fetuses per litter / number of litter with live fetuses
- Mean fetal body weight per sex (example male fetuses): sum of mean weights of live male fetuses per litter / number of litter with live male fetuses
- For external, visceral and skeletal fetal findings, the percentage of fetuses affected per group for a given parameter: sum of live fetuses affected / number of live fetuses examined x 100
- For external, visceral and skeletal fetal findings, the percentage of litters affected per group for a given parameter: sum of litters with live fetuses affected / number of litter with live fetuses examined x 100

Historical control data:

Historical control data from studies conducted in-house were referred to in order to allow comparison with concurrent controls. The data were generated from sixteen rabbit studies between 2000 and 2011 and are presented in the report.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

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, non-treatment-related

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

For details on results including tables see the attached document.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

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): effects observed, non-treatment-related

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

For details on results including tables see the attached document.

Key result

Dose descriptor:

NOEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: highest dose tested

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

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

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

effects observed, non-treatment-related

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Skeletal malformations:

effects observed, non-treatment-related

Visceral malformations:

effects observed, non-treatment-related

Other effects:

effects observed, non-treatment-related

Details on embryotoxic / teratogenic effects:

For details on results including tables see the attached document.

Key result

Dose descriptor:

NOEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

For details on results including tables see the attached document.

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

Quality of whole database:

The available information comprises adequate, reliable (Klimisch score 1) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.7, of Regulation (EC) No 1907/2006.

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

Reliable studies regarding developmental toxicity / teratogenicity are available for the test substance.

Developmental toxicity study in the rat

The teratogenic potential of the test substance was assessed in a developmental toxicity study in Crl:CD (SD) Sprague-Dawley rats performed according to OECD Guideline 414 and in compliance with GLP (M-503758-01-1). Three groups of 23 sperm-positive female rats received the test substance by oral gavage from gestation day (GD) 6 to 20 at doses of 62.5, 250 or 1000 mg/kg bw/day in suspension   in aqueous solution of 0.5% methylcellulose 400. A control group of 23 females received the vehicle. Pregnancy rate was unaffected and there were no treatment-related effects on any parameter assessed in dams up to the highest concentration of 1000 mg/kg bw/day.Litter parameters were unaffected by treatment. There were no treatment-related malformations at the external, visceral and skeletal fetal examination. Additionally, at the skeletal fetal examination, treatment-related changes consisted of an increased incidence of 2 spontaneous variations; “5th and 6th sternebrae: incomplete ossification” at the fetal level and “5th and/or 6th sternebrae: unossified” at the fetal and litter levels. As these findings were limited to a delay in ossification in the 5th and 6th sternebrae, which are commonly seen spontaneous variations that correlated with slightly lower mean fetal body weights, they were considered to represent only a slight and transient delay in fetal development with no adverse long term consequences. Thus, under the conditions of this study, the NOAEL of the test substance in terms of fetal toxicity and the NOEL in terms of maternal toxicity following administration of the test substance to pregnant rats via gavage is 1000 mg/kg bw/day. The NOEL in terms of fetal toxicity is 250mg/kg/day.

Developmental toxicity study in the rabbit

The teratogenic potential of the test substance was assessed in a developmental toxicity study in New Zealand White Crl:KBL (NZW) rabbits performed according to OECD Guideline 414 and incompliance with GLP (M-510211-02-1). Three groups of 23 time-mated pregnant female rabbits received the test substance by oral gavage from gestation day (GD) 6 to 28 at doses of 62.5, 250 or 1000 mg/kg body weight/day in suspension in aqueous solution of 0.5 % methylcellulose 400. A control group of 23 females received the vehicle. Pregnancy rate was unaffected and there were no treatment-related effects on any parameter assessed in dams up to the highest concentration of 1000 mg/kg bw/day. Litter parameters were unaffected by treatment.At the external, visceral and skeletal fetal examinations, there were no treatment-related malformations or variations. Thus, under the conditions of this study, the NOEL of the test substance in terms of maternal and fetal toxicity following administration of the test substance to pregnant rats via gavage is 1000 mg/kgbw/day.

Justification for classification or non-classification

The available data on toxicity to reproduction do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.