Aluminium, 6-hydroxy-5-[(2-methoxy-5-methyl-4-sulfophenyl)azo]-2-naphthalenesulfonic acid complex

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Based on the available results and according to the weight of evidence approach, the NOAEL for the maternal toxicity effects caused by the test chemical can likely to be estimated as 1000mg/kgbw/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. THe study 2,3 are referred as study 1,2

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

other: 1. Osborne-Mendel; 2. Charles River CD

Sex:

male/female

Route of administration:

other: 1. oral: gavage; 2. oral: feed

Vehicle:

other: 1. water; 2. feed

Details on exposure:

1. Test chemical was dissolved in distilled water (w/v). Fresh solutions were prepared daily and administered by gavage in a volume of 1 ml/100 g body weight, at approximately the same time each day. Controls received an equivalent volume of distilled water.
2. PREPARATION OF DOSING SOLUTIONS: test material mixed with food

Details on mating procedure:

1. - M/F ratio per cage: 1:2
- Length of cohabitation: 1 day
- Proof of pregnancy: sperm in vaginal smear was considered day 0 of gestation
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: no data
- Further matings after two unsuccessful attempts: no data
- After successful mating each pregnant female was caged (how): no data
- Any other deviations from standard protocol: no data

2.

Duration of treatment / exposure:

1. Gestation day 0 to day 19

Frequency of treatment:

1. DAILY
2. Pre-Mating Exposure / Males and Females: 60 days and test material adminstered upto three generation

Remarks:

Study 1: 0, 30, 75, 150, 300, 600 or 1000mg/kg/day

Remarks:

Study 2: 0, 5, 50, 150 or 500 mg/kg bw/day

No. of animals per sex per dose:

1. 42-43 female/dose groups
2. 10 males and 20 females/group/generation were used

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

1. The rats were weighed daily and food consumption was measured weekly. Water intake was not measured.
2. Parental animals observation and examinations
CAGE SIDE OBSERVATIONS: yes
Clinical observations were recorded twice daily with at least 5 hours between observations

DETAILED CLINICAL OBSERVATIONS: Yes

Time schedule: Detailed physical examinations and palpation for masses were performed weekly.



BODY WEIGHT: Yes
Time schedule for examinations: weekly for the first fourteen weeks, bi-weekly for the next 12 weeks and every 4 weeks thereafter until the end of the study.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes weekly for the first fourteen weeks, bi-weekly for the next 12 weeks and every 4 weeks thereafter until the end of the study..

Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes ,

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
Time schedule for examinations:

OTHER: Haematology tests, including hemoglobin, hematocrit, erythrocyte and total and differential leukocyte counts, and erythrocyte morphology, were conducted on ten randomly selected animals at months 3, 6, 12, 18 and 24 of the study.

Litter observations:

1. Each live foetus was promptly weighed, sexed and examined for gross external malformations, and the crown-rump length was measured. Any foetus that weighed less than 70% of the average weight of the concurrent male or female controls was considered to be a runt.

Postmortem examinations (parental animals):

1. On day 20 of gestation, starting at 1 pro, the females were examined for gross abnormalities for the last time before being killed by CO, asphyxiation. Caesarean sections were performed. Corpora lutea were counted. The uterus was opened and examined in situ. The uterine positions of all implantation sites were noted and their condition (early or late resorptions, living or dead foetuses) was determined.
2. SACRIFICE
- Male animals: All surviving animals [describe when, e.g. as soon as possible after the last litters in each generation were produced.]
- Maternal animals: All surviving animals [describe when, e.g. after the last litter of each generation was weaned.]

GROSS NECROPSY
Necropsies were conducted on all animals dying prior to study termination, killed in a moribund condition or killed on schedule,Tissues examined included adrenal glands, aorta, blood smear, brain, cecum, colon, duodenum, epididymus or uterus, esophagus, eyes, femur including marrow, tissue masses, gallbladder, heart, ileum, jejunum, duodenum, kidneys, liver, lungs and bronchi, mammary gland, nerves (sciatic), ovaries, lymph nodes, pancreas, parathyroids, pituitary gland, prostrate, rectum, skin, spleen, seminal vesicles, skeletal muscle, testes with ep ididymides, stomach, thymus, thyroid gland including parathyroid, trachea, urinary bladder, uterus


HISTOPATHOLOGY / ORGAN WEIGHTS
Histological examinations were conducted on all animals from both control groups, the highest dose group (2.0 or 5.0%) from each study and also on 10 rats randomly selected from each group for an interim sacrifice at 12 months. Histology was also performed on any animal with gross lesions or masses

Postmortem examinations (offspring):

1. Approximately one-half of the foetuses were fixed in alcohol, stained with Alizarin Red S and examined under a dissecting microscope for all skeletal variations. The remaining half of the foetuses were fixed in Bouin's solution, serially sectioned by razor blade and examined under a dissecting microscope for internal variations of the soft tissues.

Statistics:

1. All data analyses were performed by the Division of Mathematics at the FDA. Data on maternal initial body weights and food consumption were analysed by straight analysis of variance (ANOVA) and two-tailed t-test, and a regression analysis. The number of dams affected was analysed by a Fisher's exact test. Data on maternal weight gain were submitted to an analysis of covariance (ANOCOVA) and a two-tailed t-test. Data on the numbers of implants, corpora lutca, total viable young and viable males and females were analysed by ANOVA followed by a one-tailed t-test. Data on implantation efficiency, early deaths, late deaths and total resorptions (early and late deaths) were transformed by using the Freeman-Tukey arc-sine transformation (Freeman and Tukey, 1950) and then analysed by ANOVA and a one-tailed t-test. Data on litters having one or more or two or more resorptions were analysed by a Fisher's exact test. Similar tests were applied to the number of runts per litter. Data on foetal body weights, crown-!o-rump lengths and foetal ossified vertebrae were analysed by nested ANOVA and a one-tailed t-test. The ANOVA included a correction for unequal sample size (Sokal and Rohlf, 1981). Data on the average number of foetuses per litter with skeletal, sternebral, combined missing plus incomplete plus bipartite sternebrae or soft-tissue variations were transformed by using the Frceman-Tukey arc-sine transformation and then analysed by ANOVA and a one-tailed t-test. Litters with foetuses with at least one, at least two, etc. skeletal, sternebral, combined missing plus incomplete plus bipartite sternebrae, or soft-tissue variations, and specific variations were analysed by Fisher's exact test.
2.

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

2. No treatment-related effects were reported on survival

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

1. Initial body weight at day 0 and maternal body-weight gain during gestation were similar in all groups

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

1. Mean food consumption on days 0-7, 7-14, 14 -20 and 0-20 by the treated animals was similar to that of the control animals

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Description (incidence and severity):

2. No treatment-related effects were reported on haematological finding

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

2. No treatment-related effects were reported on clinical chemistry

Urinalysis findings:

no effects observed

Description (incidence and severity):

2. No treatment-related effects were reported on urinalysis.

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

2. no compound related lesions in any tissue examined histologically, including kidneys and adrenal glands from parental rats.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

2. Histological evaluation revealed a variety of lesions, including neoplasms, present at similar incidences in control and treated animals.The authors considered the lesions to be spontaneous and not related to administration of the test material.

Other effects:

not specified

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

1. The pregnancy rate ranged from 85.71 to 95.35% with no evidence of dose correlation.
The mean numbers of corpora lutea and implants per female were similar in all groups. No litters were totally resorbed. The mean number of viable foetuses per female was similar in all groups. The number of viable male foetuses was increased over the control value in the groups given 30 and 1000mg/kg, but because of the lack of relation to dosage, these increases were considered to be random. The number of viable female foetuses was not affected in any group. The number of early deaths per litter and the number of early plus late deaths per litter were greatest in the 600 mg/kg group, but these appeared to be random occurrences. The percentage of females with at least one resorption was similar in all groups. The percentage of females given 600mg/kg that had at least two resorptions was significantly greater than the percentage in the control females, but there was no dose related effect in the percentage of females with at least two resorptions.

2. There were no compound related effects on fertility, gestation, pup viability or lactation indices, on reproductive organs of females, or on organ weights among parents.

Dose descriptor:

NOAEL

Effect level:

> 500 - <= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

body weight and weight gain

reproductive performance

Critical effects observed:

not specified

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality / viability:

no mortality observed

Description (incidence and severity):

1. no compound related effects on pup viability or lactation indices.
2. no compound related effects on pup viability or lactation indices.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean foetal weights of males and females and crown rump lengths were similar in all groups. The number of litters with runts was similar in all groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

2. Food consumption was similar for control and treated animals at the lower dietary levels, but was slightly higher in the 500 mg/kg bw dose group although not statistically significant.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Description (incidence and severity):

2. No treatment-related effects were reported on Haematological finding

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

2. No treatment-related effects were reported on clinical chemistry

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

Description (incidence and severity):

2. no compound related effects on organ weights

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Aside from haemorrhages present in foetuses in all groups in similar numbers, there were no other external variations in any of the dosed groups. Two foetuses had multiple anomalies: one foetus from the control group had a club foot, a short tail and four digits on the hind legs, and one foetus from the 150-mg/kg group had exencephalus and hydrocephalus. Three foetuses in a single litter of a female given 75 mg/kg were oedematous.
No dose-related increase in sternebral variations was seen among the foetuses with reduced ossification, bipartite, missing, malaligned or fused sternebrae,nor in the numbers of litters containing these foetuses. The incidence of specific skeletal variations was similar in all groups of foetuses, except for a significant increase in the incidence of 14th rib bud in foetuses from the 300 mg/kg group which was considered to be a random occurrence.
2. Necropsies did not reveal any treatment-related gross or microscopic changes.

Histopathological findings:

no effects observed

Description (incidence and severity):

2. There were no compound related lesions in any tissue examined histologically, including kidneys and adrenal glands from offspring.

Other effects:

no effects observed

Description (incidence and severity):

2. There were no compound related effects on fertility, gestation, pup viability or lactation indices, on reproductive organs of females, or on organ weights among offspring.There were no compound related effects on fertility, gestation, pup viability or lactation indices, on reproductive organs of females, or on organ weights among offspring.

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 500 - <= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

gross pathology

Critical effects observed:

not specified

Reproductive effects observed:

no

Treatment related:

not specified

Conclusions:

Based on the available results and according to the weight of evidence approach, the NOAEL for the maternal toxicity effects caused by the test chemical can likely to be estimated as 1000mg/kgbw/day.

Executive summary:

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

Study 1:

The developmental toxicity study of test material was performed on male and female Osborne-Mendel rats. The test material was dissolved in distilled water (w/v). Fresh solutions were prepared daily. The dose concentration of 0, 30, 75, 150, 300, 600 or 1000mg/kg/day was administered by oral gavage route in volume 1ml/100g body weight .On mating days , two females were randomly mated with one male at approximately 4.30pm. The following morning, a vaginal smear was obtained from each female to determine whether copulation had taken place. Sperm positive dams were considered to be at day 0 of gestation. 42-43 female/dose groups were used. All the animals were observed for signs of toxicity. The rats were weighed daily and food consumption was measured weekly. Water intake was not measured. On day 20 of gestation, the females were examined for gross abnormalities for the last time before being killed by CO, asphyxiation. Caesarean sections were performed. Corpora lutea were counted. The uterus was opened and examined in situ. The uterine positions of all implantation sites were noted and their condition (early or late resorptions, living or dead foetuses) was determined. Each live foetus was promptly weighed, sexed and examined for gross external malformations, and the crown-rump length was measured. Any foetus that weighed less than 70% of the average weight of the concurrent male or female controls was considered to be a runt. Approximately one-half of the foetuses were fixed in alcohol, stained with Alizarin Red S and examined under a dissecting microscope for all skeletal variations. The remaining half of the foetuses were fixed in Bouin's solution, serially sectioned by razor blade and examined under a dissecting microscope for internal variations of the soft tissues.

No unusual behaviours were observed in the animals during the study. The external maternal findings were unremarkable. One female in the group given 300mg/kg died at day 12 of gestation as a result of gavage difficulties unrelated to dosage. Initial body weight at day 0 and maternal body-weight gain during gestation were similar in all groups. Mean food consumption on days 0-7, 7-14, 14 -20 and 0-20 by the treated animals was similar to that of the control animals. The pregnancy rate ranged from 85.71 to 95.35% with no evidence of dose correlation.

The mean numbers of corpora lutea and implants per female were similar in all groups. No litters were totally resorbed. The mean number of viable foetuses per female was similar in all groups. The number of viable male foetuses was increased over the control value in the groups given 30 and 1000mg/kg, but because of the lack of relation to dosage, these increases were considered to be random. The number of viable female foetuses was not affected in any group. The number of early deaths per litter and the number of early plus late deaths per litter were greatest in the 600 mg/kg group, but these appeared to be random occurrences. The percentage of females with at least one resorption was similar in all groups. The percentage of females given 600mg/kg that had at least two resorptions was significantly greater than the percentage in the control females, but there was no dose related effect in the percentage of females with at least two resorptions. Mean foetal weights of males and females and crown rump lengths were similar in all groups. The number of litters with runts was similar in all groups. Aside from haemorrhages present in foetuses in all groups in similar numbers, there were no other external variations in any of the dosed groups. Two foetuses had multiple anomalies: one foetus from the control group had a club foot, a short tail and four digits on the hind legs, and one foetus from the 150-mg/kg group had exencephalus and hydrocephalus. Three foetuses in a single litter of a female given 75 mg/kg were oedematous. No dose-related increase in sternebral variations was seen among the foetuses with reduced ossification, bipartite, missing, malaligned or fused sternebrae, nor in the numbers of litters containing these foetuses. The incidence of specific skeletal variations was similar in all groups of foetuses, except for a significant increase in the incidence of 14th rib bud in foetuses from the 300 mg/kg group which was considered to be a random occurrence.

Hence the NOAEL was considered to be 1000mg/kg bw as no effects on development of fetus was observed, when Osborne-Mendel male and female rats were treated with test material orally in 20 days.

Study 2

Three generation reproductive study of test material was performed on male and female Charles River CD rats. Animals were housed individually and fed the test diet ad libitum.

The test material mixed with food in dose concentration 0, 5, 50, 150 or 500 mg/kg bw/day and 10 males and 20 females/group/generation were used .Pre-Mating Exposure / Males and Females were 60 days. Clinical observations were recorded twice daily with at least 5 hours between observations. Detailed physical examinations and palpation for masses were performed weekly. Body weights and food consumption were determined weekly for the first fourteen weeks, bi-weekly for the next 12 weeks and every 4 weeks thereafter until the end of the study. The intake of the test substance was determined from body weight, food consumption and dietary concentration. Haematology tests, including haemoglobin, haematocrit, erythrocyte and total and differential leukocyte counts, and erythrocyte morphology, were conducted on ten randomly selected animals at months 3, 6, 12, 18 and 24 of the study. Necropsies were conducted on all animals dying prior to study termination, killed in a moribund condition or killed on schedule. Histological examinations were conducted on all animals from both control groups, the highest dose group (2.0 or 5.0%) from each study and also on 10 rats randomly selected from each group for an interim sacrifice at 12 months. Histology was also performed on any animal with gross lesions or masses. Also tissues examined included adrenal glands, aorta, blood smear, brain, cecum, colon, duodenum, epididymus or uterus, esophagus, eyes, femur including marrow, tissue masses, gallbladder, heart, ileum, jejunum, duodenum, kidneys, liver, lungs and bronchi, mammary gland, nerves (sciatic), ovaries, lymph nodes, pancreas, parathyroids, pituitary gland, prostrate, rectum, skin, spleen, seminal vesicles, skeletal muscle, testes with epididymides, stomach, thymus, thyroid gland including parathyroid, trachea, urinary bladder, uterus.

At the end of study, no treatment-related effects were reported on survival. No treatment-related changes were reported at gross necropsy. Histological evaluation revealed a variety of lesions, including neoplasms, present at similar incidences in control and treated animals. The authors considered the lesions to be spontaneous and not related to administration of the test material. There were no compound related effects on fertility, gestation, pup viability or lactation indices, on reproductive organs of females, or on organ weights among parents and offspring. There were no compound related lesions in any tissue examined histologically, including kidneys and adrenal glands from parental rats or from offspring. There was no toxicity to either the F1 or F2 generation. Food consumption was similar for control and treated animals at the lower dietary levels, but was slightly higher in the high-dose study, although not statistically significant. Haematological, clinical chemistry and urinalysis parameters did not differ significantly from the controls. Necropsies at one year did not reveal any treatment-related gross or microscopic changes.

Hence NOAEL was considered to be 500mg/kg bw/day for F0, F1 and F2 generation when male and female Charles River CD rats were treated with test material orally.

Based on the available results and according to the weight of evidence approach, the NOAEL for the maternal toxicity effects caused by the test chemical can likely to be estimated as 1000mg/kgbw/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch Rating - 2

Additional information

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

Study 1:

The developmental toxicity study of test material was performed on male and female Osborne-Mendel rats. The test material was dissolved in distilled water (w/v). Fresh solutions were prepared daily. The dose concentration of 0, 30, 75, 150, 300, 600 or 1000mg/kg/day was administered by oral gavage route in volume 1ml/100g body weight .On mating days , two females were randomly mated with one male at approximately 4.30pm. The following morning, a vaginal smear was obtained from each female to determine whether copulation had taken place. Sperm positive dams were considered to be at day 0 of gestation. 42-43 female/dose groups were used. All the animals were observed for signs of toxicity. The rats were weighed daily and food consumption was measured weekly. Water intake was not measured. On day 20 of gestation, the females were examined for gross abnormalities for the last time before being killed by CO, asphyxiation. Caesarean sections were performed. Corpora lutea were counted. The uterus was opened and examined in situ. The uterine positions of all implantation sites were noted and their condition (early or late resorptions, living or dead foetuses) was determined. Each live foetus was promptly weighed, sexed and examined for gross external malformations, and the crown-rump length was measured. Any foetus that weighed less than 70% of the average weight of the concurrent male or female controls was considered to be a runt. Approximately one-half of the foetuses were fixed in alcohol, stained with Alizarin Red S and examined under a dissecting microscope for all skeletal variations. The remaining half of the foetuses were fixed in Bouin's solution, serially sectioned by razor blade and examined under a dissecting microscope for internal variations of the soft tissues.

No unusual behaviours were observed in the animals during the study. The external maternal findings were unremarkable. One female in the group given 300mg/kg died at day 12 of gestation as a result of gavage difficulties unrelated to dosage. Initial body weight at day 0 and maternal body-weight gain during gestation were similar in all groups. Mean food consumption on days 0-7, 7-14, 14 -20 and 0-20 by the treated animals was similar to that of the control animals. The pregnancy rate ranged from 85.71 to 95.35% with no evidence of dose correlation.

The mean numbers of corpora lutea and implants per female were similar in all groups. No litters were totally resorbed. The mean number of viable foetuses per female was similar in all groups. The number of viable male foetuses was increased over the control value in the groups given 30 and 1000mg/kg, but because of the lack of relation to dosage, these increases were considered to be random. The number of viable female foetuses was not affected in any group. The number of early deaths per litter and the number of early plus late deaths per litter were greatest in the 600 mg/kg group, but these appeared to be random occurrences. The percentage of females with at least one resorption was similar in all groups. The percentage of females given 600mg/kg that had at least two resorptions was significantly greater than the percentage in the control females, but there was no dose related effect in the percentage of females with at least two resorptions. Mean foetal weights of males and females and crown rump lengths were similar in all groups. The number of litters with runts was similar in all groups. Aside from haemorrhages present in foetuses in all groups in similar numbers, there were no other external variations in any of the dosed groups. Two foetuses had multiple anomalies: one foetus from the control group had a club foot, a short tail and four digits on the hind legs, and one foetus from the 150-mg/kg group had exencephalus and hydrocephalus. Three foetuses in a single litter of a female given 75 mg/kg were oedematous. No dose-related increase in sternebral variations was seen among the foetuses with reduced ossification, bipartite, missing, malaligned or fused sternebrae, nor in the numbers of litters containing these foetuses. The incidence of specific skeletal variations was similar in all groups of foetuses, except for a significant increase in the incidence of 14th rib bud in foetuses from the 300 mg/kg group which was considered to be a random occurrence.

Hence the NOAEL was considered to be 1000mg/kg bw as no effects on development of fetus was observed, when Osborne-Mendel male and female rats were treated with test material orally in 20 days.

Study 2

Three generation reproductive study of test material was performed on male and female Charles River CD rats. Animals were housed individually and fed the test diet ad libitum.

The test material mixed with food in dose concentration 0, 5, 50, 150 or 500 mg/kg bw/day and 10 males and 20 females/group/generation were used .Pre-Mating Exposure / Males and Females were 60 days. Clinical observations were recorded twice daily with at least 5 hours between observations. Detailed physical examinations and palpation for masses were performed weekly. Body weights and food consumption were determined weekly for the first fourteen weeks, bi-weekly for the next 12 weeks and every 4 weeks thereafter until the end of the study. The intake of the test substance was determined from body weight, food consumption and dietary concentration. Haematology tests, including haemoglobin, haematocrit, erythrocyte and total and differential leukocyte counts, and erythrocyte morphology, were conducted on ten randomly selected animals at months 3, 6, 12, 18 and 24 of the study. Necropsies were conducted on all animals dying prior to study termination, killed in a moribund condition or killed on schedule. Histological examinations were conducted on all animals from both control groups, the highest dose group (2.0 or 5.0%) from each study and also on 10 rats randomly selected from each group for an interim sacrifice at 12 months. Histology was also performed on any animal with gross lesions or masses. Also tissues examined included adrenal glands, aorta, blood smear, brain, cecum, colon, duodenum, epididymus or uterus, esophagus, eyes, femur including marrow, tissue masses, gallbladder, heart, ileum, jejunum, duodenum, kidneys, liver, lungs and bronchi, mammary gland, nerves (sciatic), ovaries, lymph nodes, pancreas, parathyroids, pituitary gland, prostrate, rectum, skin, spleen, seminal vesicles, skeletal muscle, testes with epididymides, stomach, thymus, thyroid gland including parathyroid, trachea, urinary bladder, uterus.

 At the end of study, no treatment-related effects were reported on survival. No treatment-related changes were reported at gross necropsy. Histological evaluation revealed a variety of lesions, including neoplasms, present at similar incidences in control and treated animals. The authors considered the lesions to be spontaneous and not related to administration of the test material. There were no compound related effects on fertility, gestation, pup viability or lactation indices, on reproductive organs of females, or on organ weights among parents and offspring. There were no compound related lesions in any tissue examined histologically, including kidneys and adrenal glands from parental rats or from offspring. There was no toxicity to either the F1 or F2 generation. Food consumption was similar for control and treated animals at the lower dietary levels, but was slightly higher in the high-dose study, although not statistically significant. Haematological, clinical chemistry and urinalysis parameters did not differ significantly from the controls. Necropsies at one year did not reveal any treatment-related gross or microscopic changes.

Hence NOAEL was considered to be 500mg/kg bw/day for F0, F1 and F2 generation when male and female Charles River CD rats were treated with test material orally.

Based on the available results and according to the weight of evidence approach, the NOAEL for the maternal toxicity effects caused by the test chemical can likely to be estimated as 1000mg/kgbw/day.

Justification for classification or non-classification

Based on the available results and according to the weight of evidence approach, the NOAEL for the maternal toxicity effects caused by the test chemical can likely to be estimated as 1000mg/kgbw/day.