Ferrate(4-), hexakis(cyano-C)-, methylated 4-[(4-aminophenyl)(4-imino-2,5-cyclohexadien-1-ylidene)methyl]benzenamine copper(2+) salts

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Toxicity to Reproduction:

Based on the available results and applying the weight of evidence approach, the test chemical can be considered to non-reprotoxic when exposed to the test chemical orally. The NOAEL value can be considered to be 1000 mg/kg/day.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Weight of evidence approach based on various test chemicals

Justification for type of information:

Weight of evidence approach based on various test chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: Weight of evidence approach based on various test chemicals

Principles of method if other than guideline:

Weight of evidence approach based on various test chemicals

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

other: 2. Wistar; 3. long-Evans

Sex:

male/female

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

not specified

Vehicle:

other: 2. water; 3. diet

Details on exposure:

2.PREPARATION OF DOSING SOLUTIONS: The dosing solutions were prepared at concentrations 0,250, 500 and 1000 mg/kg such that a volume of 10 ml/kg was administered daily.
DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): No data available
- Storage temperature of food: No data available
VEHICLE
- Justification for use and choice of vehicle (if other than water): Water
- Concentration in vehicle: 0, 250, 500 and 1000 mg/kg/day
- Amount of vehicle (if gavage): 10 ml/kg
- Lot/batch no. (if required): No data available
- Purity: No data available
3. no data available

Details on mating procedure:

2. Details on mating procedure
- M/F ratio per cage: 1 : 1 ratio
- Length of cohabitation: Overnight
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pr
egnancy Sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): Individually
- Any other deviations from standard protocol: This procedure was repeated over several consecutive days until at least 30 females had been allocated to each treatment group.
3. The F0 generation rats were mated twice, the F1a litters being necropsied at weaning, and selectedanimals (10 males,20 females) from the F1b litters were used for breeding.Following an 80-day growth period, animals from the F1b generation were mated 3 times and the offspring of the F2a and F2b generations were treated identically to the F1a and F1b generations.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

2. 19 days
3. 80-day ( 2 weeks before the first mating, and dosing continued throughout the gestation, lactation, and post-weaning phases for three successive generations.)

Frequency of treatment:

2. daily
3. daily

Details on study schedule:

2. Dose selection rationale: No data available
- Rationale for animal assignment (if not random): The day of sperm detection was designated day 0 of pregnancy and the mated females were randomly allocated to one of four treatment groups. This procedure was repeated over several consecutive days until at least 30 females had been allocated to each treatment group.
- Rationale for selecting satellite groups: No data available
- Post-exposure recovery period in satellite groups: No data available
3. - F1 parental animals not mated until [...] weeks after selected from the F1 litters.The F0 generation rats were mated twice,
- Selection of parents from F1 generation when pups were [...] days of age.:the F1a
litters being necropsied at weaning, and selected animals (10 males, 20 females) from the F1b
litters were used for breeding.Following an 80-day growth period, animals from the F1b generation were mated 3 times and the offspring of the F2a and F2b generations were treated identically to theF1a and F1b generations.
- Age at mating of the mated animals in the study: [...] weeks:80days
- Section schedule rationale (if not random): No data available

Remarks:

Study 2: 0, 250, 500 and 1000 mg/kg/day

Remarks:

study 3. 0, 10, 100, 300, or 1,000 mg/kg b.w./day

No. of animals per sex per dose:

2. Total: 120
0 mg/kg/day: 30 female
250 mg/kg/day: 30 female
500 mg/kg/day: 30 female
1000 mg/kg/day: 30 female
3. Total:300
F0 generation
0 mg/kg bw/day:10male and 20 females
10mg/kg bw/day:10male and 20 females
100mg/kg bw/day:10male and 20 females
300 mg/kg bw/day:10male and 20 females
1000mg/kg bw/day: 10male and 20 females
F1 generation
0 mg/kg bw/day:10male and 20 females
10mg/kg bw/day:10male and 20 females
100mg/kg bw/day:10male and 20 females
300 mg/kg bw/day:10male and 20 females
1000mg/kg bw/day: 10male and 20 females

Control animals:

yes, concurrent vehicle

Details on study design:

no data available

Positive control:

no data available

Parental animals: Observations and examinations:

2.BODY WEIGHT: Yes / No / No data: yes
- Time schedule for examinations:Each animal was weighed on alternate days, commencing on day 0 of pregnancy.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes / No / No data
- Food consumption for each animal determined and mean daily diet consumption calculated as g foo
d/kg body weight/day: Yes / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

POST-MORTEM EXAMINATIONS: Yes / No / No data: yes
- Sacrifice on gestation day # : On day 20 of pregnancy, 30 females from each group were killed by c
ervical dislocation and a postmortem examination was carried out
- Organs examined: a postmortem examination was carried out, during which the gross appearance of the maternal tissues was noted and the foetuses were removed. The numbers of corpora lutea and implantation sites and the numbers and positions of the sites with dead, live or resorbed foetuses were recorded.
OTHER: At 7 wk of age vaginal smears were prepared and examined for each female. Those females in pro-oestrus (indicated by the presence of mainly cornified and epithelial cells) were caged
individually overnight with a mature male.
3.CAGE SIDE OBSERVATIONS: Yes
- Time schedule: no data available
- Cage side observations checked in table [No.?] were included.
DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule: no data available
BODY WEIGHT: Yes
- Time schedule for examinations:
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations:

Oestrous cyclicity (parental animals):

2. corpora lutea per ovary was examined.
3. corpora lutea per ovary was examined.

Sperm parameters (parental animals):

2. no data available

Litter observations:

2.Weight of fetuses, Dead, live or resorbed fetuses were recorded.

Postmortem examinations (parental animals):

2. Gross appearances were observed.
3. SACRIFICE: After third mating, half of the pregnant dams were sacrificed on day 19 of gestation.
GROSS NECROPSY : Gross necropsies were performed on all parent animals and on F1a, F2a, F2c, and F3a offspring at weaning.
HISTOPATHOLOGY / ORGAN WEIGHTS : stomach, ileum, jejunum, colon, liver, spleen, heart, lungs, adrenals,kidneys, urinary bladder, thyroid, ovaries, and uterus or testes was examined histologically.

Postmortem examinations (offspring):

2.
- External examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data: yes
- Soft tissue examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data: yes
- Skeletal examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data: yes
- Head examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data: no data available
3. GROSS NECROPSY:yes
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]: Gross necropsies were performed on all parent animals and on F1a, F2a, F2c, and F3a offspring at weaning.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.: stomach, ileum, jejunum, colon, liver, spleen, heart, lungs, adrenals,kidneys, urinary bladder, thyroid, ovaries, and uterus or testes was examined histologically.

Reproductive indices:

2. Number of corpora lutea, implantation sites, Pre-implantation losses, Early resorptions, Late resorptions and Post-implantation losses was recorded.
3. pregnancy and fertility rates, gestation length

Offspring viability indices:

2. Viability of fetuses were observed.
3. offspring survival,weights and sex, litter survival,resorption rates

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

3. no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

2. No statistically significant differences were observed in treated female rats as compared to control.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

3. No effects attributable to treatment were observed with respect to food consumption.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

2. No effect were observed on number of corpora lutea, implantation sites, Pre-implantation losses, early resorptions, late resorptions and post-implantation losses of treated female rats as compared to control.
3. No effects attributable to treatment were observed with respect to mating performance,pregnancy and fertility rates, gestation length.

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

2. No effect were observed on number of corpora lutea, implantation sites, Pre-implantation losses, early resorptions, late resorptions and post-implantation losses of treated female rats as compared to control.
3. No effects attributable to treatment were observed with respect to mating performance,pregnancy and fertility rates, gestation length.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

gross pathology

reproductive function (oestrous cycle)

reproductive performance

Critical effects observed:

not specified

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

3. No effects attributable to treatment were observed with respect to adult mortality, offspring survival.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

3. No effects attributable to treatment were observed with respect to body weight.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

3. No changes attributable to treatment were observed with respect to food consumption.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

3. No effects attributable to treatment were observed with respect to food consumption, body weight, adult mortality, mating performance,pregnancy and fertility rates, gestation length, offspring
survival, weights and sex, litter survival, resorption rates, or necropsy findings.

Gross pathological findings:

no effects observed

Description (incidence and severity):

3. No effects attributable to treatment were observed with respect to food consumption, body weight, adult mortality, mating performance,pregnancy and fertility rates, gestation length, offspring
survival, weights and sex, litter survival, resorption rates, or necropsy findings.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

3. No effects attributable to treatment were observed with respect to food consumption, body weight, adult mortality, mating performance,pregnancy and fertility rates, gestation length, offspring
survival, weights and sex, litter survival, resorption rates, or necropsy findings.

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

3. No effects attributable to treatment were observed with respect to food consumption, body weight, adult mortality, mating performance,pregnancy and fertility rates, gestation length, offspring
survival, weights and sex, litter survival, resorption rates, or necropsy findings.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

body weight and weight gain

organ weights and organ / body weight ratios

reproductive function (oestrous cycle)

reproductive performance

Critical effects observed:

no

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality / viability:

no mortality observed

Description (incidence and severity):

2. No effect were observed on Live foetuses treated female rats as compared to control
3. No effects attributable to treatment were observed with respect to adult mortality, offspring survival.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

2. No significant effect was observed on fetuses of treated female rats as compared to control.
3. No effects attributable to treatment were observed with respect to body weight

Food consumption and compound intake (if feeding study):

not specified

Description (incidence and severity):

3. No effects attributable to treatment were observed with respect to food consumption.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not specified

Anogenital distance (AGD):

not specified

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

2. Statistically significant increase in fetuses with mucus in tracea were observed in 250, 500 and 1000mg/kg/day treated female rats as compared to control.This findings were observed at all dose levels and the incidence was not dose related.Ossification of proximal phalanges and fourth metacarpals of Skeletal tissue were observed in 500 and 1000 mg/kg/day treated fetuses as compared to control. Despite this association with treatment, the nature of thefinding does not indicate an adverse effect.
3. No effects attributable to treatment were observed with respect to food consumption, body weight, adult mortality, mating performance,pregnancy and fertility rates, gestation length, offspring
survival, weights and sex, litter survival, resorption rates, or necropsy findings.

Histopathological findings:

not specified

Other effects:

not specified

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

body weight and weight gain

gross pathology

Critical effects observed:

no

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality / viability:

no mortality observed

Description (incidence and severity):

3. No effects attributable to treatment were observed with respect to food consumption, body weight, adult mortality, mating performance,pregnancy and fertility rates, gestation length, offspring
survival, weights and sex, litter survival, resorption rates, or necropsy findings.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

3. No effects attributable to treatment were observed with respect to adult mortality,offspring survival

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

3. No effects attributable to treatment were observed with respect to food consumption.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not specified

Anogenital distance (AGD):

not specified

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

3. No effects attributable to treatment were observed with respect to food consumption, body weight, adult mortality, mating performance,pregnancy and fertility rates, gestation length, offspring
survival, weights and sex, litter survival, resorption rates, or necropsy findings.

Gross pathological findings:

not specified

Description (incidence and severity):

3. No effects attributable to treatment were observed with respect to food consumption, body weight, adult mortality, mating performance,pregnancy and fertility rates, gestation length, offspring
survival, weights and sex, litter survival, resorption rates, or necropsy findings.

Histopathological findings:

no effects observed

Description (incidence and severity):

3. No effects attributable to treatment were observed with respect to food consumption, body weight, adult mortality, mating performance,pregnancy and fertility rates, gestation length, offspring
survival, weights and sex, litter survival, resorption rates, or necropsy findings.

Other effects:

not specified

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

Dose descriptor:

NOAEL

Generation:

F2a

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

histopathology: non-neoplastic

Dose descriptor:

NOAEL

Generation:

F2b

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

gross pathology

histopathology: non-neoplastic

Critical effects observed:

not specified

System:

other: not specified

Reproductive effects observed:

no

Treatment related:

no

Conclusions:

Based on the available results and applying the weight of evidence approach, the test chemical can be considered to non-reprotoxic when exposed to the test chemical orally. The NOAEL value can be considered to be 1000 mg/kg/day.

Executive summary:

Data available from different studies were reviewed to determine the reproductive toxicity of test chemical. The studies are as mentioned below:

Teratogenicity and embryotoxicity study of the test chemical was performed on rats. Daily oral doses of 0 (control), 250, 500 or 1000 mg/kg body weight were given to groups of 30 pregnant Wistar rats on days 0-19 of pregnancy.The dosing solutions were prepared at concentrations such that a volume of 10ml/kg was administered daily. The controls received water. Each animal was weighed on alternate days, commencing on day 0 of pregnancy. On day 20 of pregnancy, 30 females from each group were killed by cervical dislocation and a postmortem examination was carried out, during which the gross appearance of the maternal tissues was noted and the foetuses were removed. The numbers of corpora lutea and implantation sites and the numbers and positions of the sites with dead, live or resorbed fetuses were recorded. The live foetuses were weighed and examined for gross abnormalities.This treatment did not adversely influence maternal body weight, the numbers of implanations, pre- or post implantation losses or of live foetuses, the sex ratio or the weight of the litters or foetuses. No definite abnormalities were seen and the only finding in the examination of stained skeletons was a slightly more advanced ossification of the forelimbs of the offspring from females given 500 or 1000 mg /kg/day. More foetuses with mucus in the trachea were found in the treated groups than in the controls but this was not considered to be a teratogenic effect. Thus no embryotoxic or teratogenic effects were detected with doses of up to 1000 mg/kg/day throughout pregnancy.

This is supported by the results of the 3-generation reproduction study carried out on the test chemical in male and female Long-Evans rats. The test material was mixed with feed and administered at dose levels of 0, 10, 100, 300, or 1,000 mg/kg b.w./day. The first generation parents (10 males, 20 females) were given the appropriate dose of test material in the diet 2 weeks before the first mating, and dosing continued throughout the gestation, lactation, and post-weaning phases for three successive generations. The F0 generation rats were mated twice, the F1a litters being necropsied at weaning, and selected animals (10 males, 20 females) from the F1b litters were used for breeding. Following an 80-day growth period, animals from the F1b generation were mated 3 times and the offspring of the F2a and F2b generations were treated identically to the F1a and F1b generations. Following the third mating, half of the pregnant dams were sacrificed on day 19 of gestation, the uterine contents were examined for total embryos/resorption sites, and the corpora lutea per ovary were recorded. The other half were allowed to deliver normally (F2c) and were sacrificed at weaning. The F2b animals were mated once and allowed to raise their offspring to weaning when both parents and offspring were culled.Gross necropsies were performed on all parent animals and on F1a, F2a, F2c, and F3a offspring at weaning. Selected tissues from 5 animals of each sex/dose from the F1b parents and the F3a generation at weaning were fixed at necropsy, and the following tissues examined histologically from the control and high-dose group: stomach, ileum, jejunum, colon, liver, spleen, heart, lungs, adrenals, kidneys, urinary bladder, thyroid, ovaries, and uterus or testes. No effects attributable to treatment were observed with respect to food consumption, body weight, adult mortality, mating performance, pregnancy and fertility rates, gestation length, offspring survival, weights and sex, litter survival, resorption rates, or necropsy findings. There were no macroscopic or microscopic tissue abnormalities of either F1b- or F3a-generation animals considered to be attributable to treatment. The No observed adverse effect level (NOAEL) for reproductive toxicity was considered to be 1000 mg/kg bw /day,as  there were no adverse effects on food consumption, body weight, adult mortality, mating performance, pregnancy and fertility rates, gestation length, offspring survival, weights and sex, litter survival, resorptions rates, or necropsy findings at dose concentration 1000 mg/kg bw /day.

Based on the available results and applying the weight of evidence approach, the test chemical can be considered to non-reprotoxic when exposed to the test chemical orally. The NOAEL value can be 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

Experimental exposure time per week (hours/week):

168

Species:

rat

Quality of whole database:

Klimisch Rating 2

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Data available from different studies were reviewed to determine the reproductive toxicity of test chemical. The studies are as mentioned below:

Teratogenicity and embryotoxicity study of the test chemical was performed on rats. Daily oral doses of 0 (control), 250, 500 or 1000 mg/kg body weight were given to groups of 30 pregnant Wistar rats on days 0-19 of pregnancy.The dosing solutions were prepared at concentrations such that a volume of 10ml/kg was administered daily. The controls received water. Each animal was weighed on alternate days, commencing on day 0 of pregnancy. On day 20 of pregnancy, 30 females from each group were killed by cervical dislocation and a postmortem examination was carried out, during which the gross appearance of the maternal tissues was noted and the foetuses were removed. The numbers of corpora lutea and implantation sites and the numbers and positions of the sites with dead, live or resorbed fetuses were recorded. The live foetuses were weighed and examined for gross abnormalities.This treatment did not adversely influence maternal body weight, the numbers of implanations, pre- or post implantation losses or of live foetuses, the sex ratio or the weight of the litters or foetuses. No definite abnormalities were seen and the only finding in the examination of stained skeletons was a slightly more advanced ossification of the forelimbs of the offspring from females given 500 or 1000 mg /kg/day. More foetuses with mucus in the trachea were found in the treated groups than in the controls but this was not considered to be a teratogenic effect. Thus no embryotoxic or teratogenic effects were detected with doses of up to 1000 mg/kg/day throughout pregnancy.

This is supported by the results of the 3-generation reproduction study carried out on the test chemical in male and female Long-Evans rats. The test material was mixed with feed and administered at dose levels of 0, 10, 100, 300, or 1,000 mg/kg b.w./day. The first generation parents (10 males, 20 females) were given the appropriate dose of test material in the diet 2 weeks before the first mating, and dosing continued throughout the gestation, lactation, and post-weaning phases for three successive generations. The F0 generation rats were mated twice, the F1a litters being necropsied at weaning, and selected animals (10 males, 20 females) from the F1b litters were used for breeding. Following an 80-day growth period, animals from the F1b generation were mated 3 times and the offspring of the F2a and F2b generations were treated identically to the F1a and F1b generations. Following the third mating, half of the pregnant dams were sacrificed on day 19 of gestation, the uterine contents were examined for total embryos/resorption sites, and the corpora lutea per ovary were recorded. The other half were allowed to deliver normally (F2c) and were sacrificed at weaning. The F2b animals were mated once and allowed to raise their offspring to weaning when both parents and offspring were culled.Gross necropsies were performed on all parent animals and on F1a, F2a, F2c, and F3a offspring at weaning. Selected tissues from 5 animals of each sex/dose from the F1b parents and the F3a generation at weaning were fixed at necropsy, and the following tissues examined histologically from the control and high-dose group: stomach, ileum, jejunum, colon, liver, spleen, heart, lungs, adrenals, kidneys, urinary bladder, thyroid, ovaries, and uterus or testes. No effects attributable to treatment were observed with respect to food consumption, body weight, adult mortality, mating performance, pregnancy and fertility rates, gestation length, offspring survival, weights and sex, litter survival, resorption rates, or necropsy findings. There were no macroscopic or microscopic tissue abnormalities of either F1b- or F3a-generation animals considered to be attributable to treatment. The No observed adverse effect level (NOAEL) for reproductive toxicity was considered to be 1000 mg/kg bw /day,as  there were no adverse effects on food consumption, body weight, adult mortality, mating performance, pregnancy and fertility rates, gestation length, offspring survival, weights and sex, litter survival, resorptions rates, or necropsy findings at dose concentration of 1000 mg/kg bw /day.

Based on the available results and applying the weight of evidence approach, the test chemical can be considered to non-reprotoxic when exposed to the test chemical orally. The NOAEL value can be considered to be 1000 mg/kg/day.

Effects on developmental toxicity

Description of key information

Developmental Toxicity:

Based on the available results and applying the weight of evidence approach, the test chemical can be considered to non-teratogenic when exposed to the test chemical orally. The NOAEL value can be considered to be 1000 mg/kg/day.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Weight of evidence approach based on various test chemicals

Justification for type of information:

Weight of evidence approach based on various test chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: Weight of evidence approach based on various test chemicals

Principles of method if other than guideline:

Weight of evidence approach based on various test chemicals

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

other: 2. Wistar; 3. Long-Evans

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

not specified

Vehicle:

other: 2. water; 3.

Details on exposure:

2. PREPARATION OF DOSING SOLUTIONS: The dosing solutions were prepared at concentrations 0, 250, 500 and 1000 mg/kg such that a volume of 10 ml/kg was administered daily.

DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): No data available
- Storage temperature of food: No data available

VEHICLE
- Justification for use and choice of vehicle (if other than water): Water
- Concentration in vehicle: 0, 250, 500 and 1000 mg/kg/day
- Amount of vehicle (if gavage): 10 ml/kg
- Lot/batch no. (if required): No data available
- Purity: No data available
3.

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

2. - M/F ratio per cage: 1 : 1 ratio
- Length of cohabitation: Overnight
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy Sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): Individually
- Any other deviations from standard protocol: This procedure was repeated over several consecutive days until at least 30 females had been allocated to each treatment group.
3. - Impregnation procedure: [artificial insemination / purchased timed pregnant / cohoused]: cohoused
- If cohoused: yes
- M/F ratio per cage: 1:2
- Other: The F0 generation rats were mated twice, the F1a litters being necropsied at weaning, and selected animals (10 males,20 females) from the F1b litters were used for breeding.Following an 80-
day growth period, animals from the F1b generation were mated 3 times and the offspring of the F2a and F2b generations were treated identically to the F1a and F1b generations
- F1 parental animals not mated until [...] weeks after selected from the F1 litters.The F0 generation rats were mated twice,
- Selection of parents from F1 generation when pups were [...] days of age.:the F1a litters being necropsied at weaning, and selected animals (10 males, 20 females) from the F1b litters were used
for breeding.Following an 80-day growth period, animals from the F1b generation were mated 3 times and the offspring of the F2a and F2b generations were treated identically to the F1a and F1b
generations.
- Age at mating of the mated animals in the study: [...] weeks:80 days

Duration of treatment / exposure:

2. 19 days
3. 80-day (2 weeks before the first mating, and dosing continued throughout the gestation, lactation,andpost-weaning phases for three successive generations)

Frequency of treatment:

2. daily
3. daily

Duration of test:

2. 19 days
3. 80-day (2 weeks before the first mating, and dosing continued throughout the gestation, lactation,and post-weaning phases for three successive generations)

Remarks:

Study 2: 0, 250, 500 and 1000 mg/kg bw/day

Remarks:

Study 3: 0, 10, 100, 300, or 1,000 mg/kg b.w./day

No. of animals per sex per dose:

2. Total: 120
0 mg/kg/day: 30 female
250 mg/kg/day: 30 female
500 mg/kg/day: 30 female
1000 mg/kg/day: 30 female
3. Total:300
F0 generation
0 mg/kg bw/day:10male and 20 females
10mg/kg bw/day:10male and 20 females
100mg/kg bw/day:10male and 20 females
300 mg/kg bw/day:10male and 20 females
1000mg/kg bw/day: 10male and 20 females
F1 generation
0 mg/kg bw/day:10male and 20 females
10mg/kg bw/day:10male and 20 females
100mg/kg bw/day:10male and 20 females
300 mg/kg bw/day:10male and 20 females
1000mg/kg bw/day: 10male and 20 females

Control animals:

yes, concurrent vehicle

Maternal examinations:

2. BODY WEIGHT: Yes / No / No data: yes
- Time schedule for examinations:Each animal was weighed on alternate days, commencing on day 0 of pregnancy.

POST-MORTEM EXAMINATIONS: Yes / No / No data: yes
- Sacrifice on gestation day # : On day 20 of pregnancy, 30 females from each group were killed by cervical dislocation and a postmortem examination was carried out
- Organs examined: a postmortem examination was carried out, during which the gross appearance of the maternal tissues was noted and the foetuses were removed. The numbers of corpora lutea and implantation sites and the numbers and positions of the sites with dead, live or resorbed foetuses were recorded.
OTHER: At 7 wk of age vaginal smears were prepared and examined for each female. Those females in pro-oestrus (indicated by the presence of mainly cornified and epithelial cells) were caged
individually overnight with a mature male.

3. CAGE SIDE OBSERVATIONS: Yes / No / No data: yes
- Time schedule: throughout the study
- Cage side observations checked in table [No.?] were included. : adult mortality and offspring mortality was observed for all generations.

DETAILED CLINICAL OBSERVATIONS: Yes / No / No data: no data available
- Time schedule: no data available

BODY WEIGHT: Yes / No / No data: yes
- Time schedule for examinations: throughout the study.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data : yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data: no data available
- Time schedule for examinations: no data available

POST-MORTEM EXAMINATIONS: Yes / No / No data: yes
- Sacrifice on gestation day # : Gross necropsies were performed on all parent animals and on F1a, F2a, F2c, and F3a offspring at weaning.
- Organs examined: Selected tissues from 5 animals of each sex/dose from the F1b parents and the F3a generation at weaning were fixed at necropsy, and the following tissues examined histologically from the control and high-dose group: stomach, ileum, jejunum, colon, liver, spleen, heart, lungs, adrenals, kidneys, urinary bladder, thyroid, ovaries, and uterus or testes

Ovaries and uterine content:

2. The ovaries and uterine content was examined after termination: Yes / No / No data: yes
Examinations included:
- Gravid uterus weight: Yes / No / No data: yes
- Number of corpora lutea: Yes / No / No data: yes
- Number of implantations: Yes / No / No data: yes
- Number of early resorptions: Yes / No / No data: yes
- Number of late resorptions: Yes / No / No data: yes
3. The ovaries and uterine content was examined after termination: Yes / No / No data: yes
Examinations included:
- Number of corpora lutea: Yes / No / No data: yes
- Number of implantations: Yes / No / No data: yes
- Number of early resorptions: Yes / No / No data: yes
- Number of late resorptions: Yes / No / No data: yes

Fetal examinations:

2. - External examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data:yes
- Soft tissue examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data: yes
- Skeletal examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data: yes
- Head examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data: yes
3.- External examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data:yes
- Soft tissue examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data: yes
- Skeletal examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data: yes
- Head examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data: yes

Statistics:

no data available

Indices:

2. Viability of fetuses were observed.
3. pregnancy and fertility rates, gestation length

Historical control data:

no data available

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

3. No effects attributable to treatment were observed with respect to adult mortality

Body weight and weight changes:

no effects observed

Description (incidence and severity):

2. There were no statistically significant differences in body weight between treated and control females
3. No effects attributable to treatment were observed with respect to body weight

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

3. no effects attributable to treatment were observed with respect to food consumption

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

2. At autopsy, all the maternal animals given the test chemical were found to have colouring in the gastro-intestinal tract and the placental tissue also appeared to the slightly green. Apart from this,
no abnormalities were found in the tissues of the maternal animals.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

2. There were no differences between the control and treated groups in the results of the examination of the reproductive system and the foetuses
3. There were no macroscopic or microscopic tissue abnormalities of either F1b- or F3a-generation animals considered to be attributable to treatment

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Description (incidence and severity):

2. No effect were observed on number of corpora lutea, implantation sites, Pre-implantation losses, early resorptions, late resorptions and post-implantation losses of treated female rats as compared to control.

Number of abortions:

no effects observed

Description (incidence and severity):

2. No effect were observed on number of corpora lutea, implantation sites, Pre-implantation losses, early resorptions, late resorptions and post-implantation losses of treated female rats as compared to control

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

2. No effect were observed on number of corpora lutea, implantation sites, Pre-implantation losses, early resorptions, late resorptions and post-implantation losses of treated female rats as compared to control
3.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

2. No effect were observed on number of corpora lutea, implantation sites, Pre-implantation losses, early resorptions, late resorptions and post-implantation losses of treated female rats as compared to control
3. No effects attributable to treatment were observed with respect to resorption rates.

Early or late resorptions:

no effects observed

Description (incidence and severity):

2. No effect were observed on number of corpora lutea, implantation sites, Pre-implantation losses, early resorptions, late resorptions and post-implantation losses of treated female rats as compared to control
3. No effects attributable to treatment were observed with respect to resorption rates.

Dead fetuses:

no effects observed

Description (incidence and severity):

2. No effect were observed on number of corpora lutea, implantation sites, Pre-implantation losses, early resorptions, late resorptions and post-implantation losses of treated female rats as compared to control

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

3. No effects attributable to treatment were observed with respect to gestation length

Changes in number of pregnant:

not specified

Other effects:

no effects observed

Description (incidence and severity):

3. No effects attributable to treatment were observed with respect to food consumption, body weight, adult mortality, mating performance, pregnancy and fertility rates, gestation length, offspring survival, weights and sex, litter survival, resorption rates, or necropsy findings.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

early or late resorptions

effects on pregnancy duration

histopathology: non-neoplastic

maternal abnormalities

necropsy findings

number of abortions

pre and post implantation loss

total litter losses by resorption

Abnormalities:

no effects observed

Fetal body weight changes:

not specified

Reduction in number of live offspring:

not specified

Changes in sex ratio:

not specified

Changes in litter size and weights:

not specified

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

3. No effects attributable to treatment were observed with respect to offspring survival

External malformations:

no effects observed

Description (incidence and severity):

2. a lack of teratogenic effect was indicated by the absence of any gross malformations of severe defects in the examination of the skeletal or soft parts.
3. There were no macroscopic or microscopic tissue abnormalities of either F1b- or F3a-generation animals considered to be attributable to treatment

Skeletal malformations:

no effects observed

Description (incidence and severity):

2. a lack of teratogenic effect was indicated by the absence of any gross malformations of severe defects in the examination of the skeletal or soft parts.
3. There were no macroscopic or microscopic tissue abnormalities of either F1b- or F3a-generation animals considered to be attributable to treatment

Visceral malformations:

no effects observed

Description (incidence and severity):

2. a lack of teratogenic effect was indicated by the absence of any gross malformations of severe defects in the examination of the skeletal or soft parts.
3.There were no macroscopic or microscopic tissue abnormalities of either F1b- or F3a-generation animals considered to be attributable to treatment

Other effects:

no effects observed

Description (incidence and severity):

2. Ossification of proximal phalanges and fourth metacarpals of Skeletal tissue were observed in 500 and 1000 mg/kg/day treated fetoses as compared to contorl. Despite this association with treatment, the nature of the finding does not indicate an adverse effect.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

external malformations

skeletal malformations

visceral malformations

Abnormalities:

no effects observed

Developmental effects observed:

not specified

Conclusions:

Based on the available results and applying the weight of evidence approach, the test chemical can be considered to non-teratogenic when exposed to the test chemical orally. The NOAEL value can be considered to be 1000 mg/kg/day.

Executive summary:

Data available from different studies were reviewed to determine the teratogenic toxicity potential of test chemical. The studies are as mentioned below:

Teratogenicity and embryotoxicity study of the test chemical was performed on rats. Daily oral doses of 0 (control), 250, 500 or 1000 mg/kg body weight were given to groups of 30 pregnant Wistar rats on days 0-19 of pregnancy.The dosing solutions were prepared at concentrations such that a volume of 10ml/kg was administered daily. The controls received water. Each animal was weighed on alternate days, commencing on day 0 of pregnancy. On day 20 of pregnancy, 30 females from each group were killed by cervical dislocation and a postmortem examination was carried out, during which the gross appearance of the maternal tissues was noted and the foetuses were removed. The numbers of corpora lutea and implantation sites and the numbers and positions of the sites with dead, live or resorbed foetuses were recorded. The live foetuses were weighed and examined for gross abnormalities.This treatment did not adversely influence maternal body weight, the numbers of implanations, pre- or post-implantation losses or of live foetuses, the sex ratio or the weight of the litters or foetuses. No definite abnormalities were seen and the only finding in the examination of stained skeletons was a slightly more advanced ossification of the forelimbs of the offspring from females given 500 or 1000 mg /kg/day. More foetuses with mucus in the trachea were found in the treated groups than in the controls but this was not considered to be a teratogenic effect. Thus no embryotoxic or teratogenic effects were detected with doses of up to I000 mg/kg/day throughout pregnancy

This is supported by the results of the 3-generation reproduction study carried out on the test chemical in male and female Long-Evans rats. The test material mixed with feed and administered at dose levels of 0, 10, 100, 300, or 1,000 mg/kg b.w./day. The first generation parents (10 males, 20 females) were given the appropriate dose of test material in the diet 2 weeks before the first mating, and dosing continued throughout the gestation, lactation, and post-weaning phases for three successive generations. The F0 generation rats were mated twice, the F1a litters being necropsied at weaning, and selected animals (10 males, 20 females) from the F1b litters were used for breeding. Following an 80-day growth period, animals from the F1b generation were mated 3 times and the offspring of the F2a and F2b generations were treated identically to the F1a and F1b generations. Following the third mating, half of the pregnant dams were sacrificed on day 19 of gestation, the uterine contents were examined for total embryos/resorption sites, and the corpora lutea per ovary were recorded. The other half were allowed to deliver normally (F2c) and were sacrificed at weaning. The F2b animals were mated once and allowed to raise their offspring to weaning when both parents and offspring were culled.Gross necropsies were performed on all parent animals and on F1a, F2a, F2c, and F3a offspring at weaning. Selected tissues from 5 animals of each sex/dose from the F1b parents and the F3a generation at weaning were fixed at necropsy, and the following tissues examined histologically from the control and high-dose group: stomach, ileum, jejunum, colon, liver, spleen, heart, lungs, adrenals, kidneys, urinary bladder, thyroid, ovaries, and uterus or testes. No effects attributable to treatment were observed with respect to food consumption, body weight, adult mortality, mating performance, pregnancy and fertility rates, gestation length, offspring survival, weights and sex, litter survival, resorption rates, or necropsy findings. There were no macroscopic or microscopic tissue abnormalities of either F1b- or F3a-generation animals considered to be attributable to treatment. The No observed adverse effect level (NOAEL) for reproductive and developmental toxicity was considered to be 1000 mg/kg bw /day,as  there were no adverse effects on food consumption, body weight, adult mortality, mating performance, pregnancy and fertility rates, gestation length, offspring survival, weights and sex, litter survival, resorptions rates, or necropsy findings were observed in rat at dose concentration 1000 mg/kg bw /day.

Based on the available results and applying the weight of evidence approach, the test chemical can be considered to non-teratogenic when exposed to the test chemical orally. The NOAEL value can be considered to be 1000 mg/kg/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Experimental exposure time per week (hours/week):

168

Species:

rat

Quality of whole database:

Klimisch rating 2

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

Data available from different studies were reviewed to determine the teratogenic toxicity potential of test chemical. The studies are as mentioned below:

Teratogenicity and embryotoxicity study of the test chemical was performed on rats. Daily oral doses of 0 (control), 250, 500 or 1000 mg/kg body weight were given to groups of 30 pregnant Wistar rats on days 0-19 of pregnancy.The dosing solutions were prepared at concentrations such that a volume of 10ml/kg was administered daily. The controls received water. Each animal was weighed on alternate days, commencing on day 0 of pregnancy. On day 20 of pregnancy, 30 females from each group were killed by cervical dislocation and a postmortem examination was carried out, during which the gross appearance of the maternal tissues was noted and the foetuses were removed. The numbers of corpora lutea and implantation sites and the numbers and positions of the sites with dead, live or resorbed foetuses were recorded. The live foetuses were weighed and examined for gross abnormalities.This treatment did not adversely influence maternal body weight, the numbers of implanations, pre- or post-implantation losses or of live foetuses, the sex ratio or the weight of the litters or foetuses. No definite abnormalities were seen and the only finding in the examination of stained skeletons was a slightly more advanced ossification of the forelimbs of the offspring from females given 500 or 1000 mg /kg/day. More foetuses with mucus in the trachea were found in the treated groups than in the controls but this was not considered to be a teratogenic effect. Thus no embryotoxic or teratogenic effects were detected with doses of up to 1000 mg/kg/day throughout pregnancy

This is supported by the results of the 3-generation reproduction study carried out on the test chemical in male and female Long-Evans rats. The test material wasmixed with feed and administered at dose levels of 0, 10, 100, 300, or 1,000 mg/kg b.w./day. The first generation parents (10 males, 20 females) were given the appropriate dose of test material in the diet 2 weeks before the first mating, and dosing continued throughout the gestation, lactation, and post-weaning phases for three successive generations. The F0 generation rats were mated twice, the F1a litters being necropsied at weaning, and selected animals (10 males, 20 females) from the F1b litters were used for breeding. Following an 80-day growth period, animals from the F1b generation were mated 3 times and the offspring of the F2a and F2b generations were treated identically to the F1a and F1b generations. Following the third mating, half of the pregnant dams were sacrificed on day 19 of gestation, the uterine contents were examined for total embryos/resorption sites, and the corpora lutea per ovary were recorded. The other half were allowed to deliver normally (F2c) and were sacrificed at weaning. The F2b animals were mated once and allowed to raise their offspring to weaning when both parents and offspring were culled.Gross necropsies were performed on all parent animals and on F1a, F2a, F2c, and F3a offspring at weaning. Selected tissues from 5 animals of each sex/dose from the F1b parents and the F3a generation at weaning were fixed at necropsy, and the following tissues examined histologically from the control and high-dose group: stomach, ileum, jejunum, colon, liver, spleen, heart, lungs, adrenals, kidneys, urinary bladder, thyroid, ovaries, and uterus or testes. No effects attributable to treatment were observed with respect to food consumption, body weight, adult mortality, mating performance, pregnancy and fertility rates, gestation length, offspring survival, weights and sex, litter survival, resorption rates, or necropsy findings. There were no macroscopic or microscopic tissue abnormalities of either F1b- or F3a-generation animals considered to be attributable to treatment.

The No observed adverse effect level (NOAEL) for reproductive and developmental toxicity was considered to be 1000 mg/kg bw /day,as  there were no adverse effects on food consumption, body weight, adult mortality, mating performance, pregnancy and fertility rates, gestation length, offspring survival, weights and sex, litter survival, resorptions rates, or necropsy findings were observed in rat at dose concentration 1000 mg/kg bw /day.

Based on the available results and applying the weight of evidence approach, the test chemical can be considered to non-teratogenic when exposed to the test chemical orally. The NOAEL value can be considered to be 1000 mg/kg/day.

Justification for classification or non-classification

Based on the available results and applying the weight of evidence approach, the test chemical can be considered to non-reprotoxic and non-teratogenic when exposed to the test chemical orally. Hence, the test chemical can be classified under the category "Not Classified" as per CLP Regulation.