Disodium 6-hydroxy-5-[(2-methoxy-4-sulphonato-m-tolyl)azo]naphthalene-2-sulphonate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Reproductive toxicity study

Based on the data available from different studies, NOAEL for test material was considered to be 695 mg/kg /day for reproductive toxicity, when male and female rats were treated with test material orally for two generation . Thus, comparing this value with the criteria of CLP regulation test material is not likely to classify as reproductive toxicant.

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Justification for type of information:

Data from secondary source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Principles of method if other than guideline:

Two generation reproductive toxicity study of test chemical

GLP compliance:

not specified

Limit test:

no

Justification for study design:

No data available

Species:

rat

Strain:

other: Charles River Ceesarean albino

Sex:

male/female

Details on test animals or test system and environmental conditions:

No data available

Route of administration:

oral: feed

Vehicle:

not specified

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test chemical mixed with feed
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):
- Concentration in vehicle: 3700, 13,900 and 51,900 ppm - Amount of vehicle (if gavage):
- Lot/batch no. (if required): No data available
- Purity: No data available

Details on mating procedure:

- M/F ratio per cage: 1:2
- Length of cohabitation: 21 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy days. The first day that sperm were observed was designated as day 0 of gestation
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility. No data available
- Further matings after two unsuccessful attempts: [no / yes (explain)] No data available
- After successful mating each pregnant female was caged (how): The females were then placed in individual cages.
- Any other deviations from standard protocol: No data available

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

27 Weeks

Frequency of treatment:

Daily

Remarks:

0,3700, 13,900 and 51,900 ppm (equivalent to 0, 185, 695 and 2595 mg/kg bw per day)

No. of animals per sex per dose:

Total: 120
0 mg/kgbw/day: 10 male and 20 female
185mg/kgbw/day: 10 male and 20 female
695mg/kgbw/day: 10 male and 20 female
2595 mg/kgbw/day: 10 male and 20 female

Control animals:

yes, plain diet

Details on study design:

No data available

Positive control:

No data available

Parental animals: Observations and examinations:

Clinical observations included food consumption, appearance, individual body weights and behavior and were made weekly.

Oestrous cyclicity (parental animals):

No data available

Sperm parameters (parental animals):

No data available

Litter observations:

individual fetal weight and length (crown to rump), and external fetal anatomical structure were observed

Postmortem examinations (parental animals):

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
-yes , Observations included number and placement of implantation sites, resorption sites, and live and dead fetuses; individual fetal weight and length (crown to rump), and external fetal anatomical structure. Gross necropsies were performed on each female including examination of uterus and visceral structures.

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

Postmortem examinations (offspring):

SACRIFICE
- Twenty-four hours following the birth of the pups the first litters (FIA) were arbitrarily reduced to 8 maximum per mother.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

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

Statistics:

No data available

Reproductive indices:

No data available

Offspring viability indices:

No data available

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

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:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Fertility indices for the control and test animals of both F1A and F1B were considered low.The fertility index of the 3700 ppm test group in the F2A breeding cycle as well as the 3700 and 51900 ppm test groups in the F2B breeding cycle were reported to be low in comparison to control animals and historical control data.

Dose descriptor:

NOAEL

Effect level:

695 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: No toxic effects were observed

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

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:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

The fertility index of the 3700 ppm test group in the F2A breeding cycle as well as the 3700 and 51900 ppm test groups in the F2B breeding cycle were reported to be low in comparison to control animals and historical control data.

Dose descriptor:

NOAEL

Effect level:

695 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: No toxic effects were observed

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

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

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

Other effects:

no effects observed

Description (incidence and severity):

Growth suppression characterized as slight was also reported for the low-level F1B pups, and the high-level F1A and F1B pups and the F2A . when compared with controls. All other measured parameters were comparable to controls in each generation and among the two filial generations.

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

695 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No evidence was seen of teratogenic or embryotoxic effects were observed

Remarks on result:

other: No evidence was seen of teratogenic or embryotoxic effects were observed

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality / viability:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

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

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

Other effects:

no effects observed

Description (incidence and severity):

Growth suppression characterized as slight was also reported for the F2A and F2B breeding cycles when compared with controls. All other measured parameters were comparable to controls in each generation and among the two filial generations.

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

695 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No evidence was seen of teratogenic or embryotoxic effects were observed

Remarks on result:

other: No evidence was seen of teratogenic or embryotoxic effects were observed

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Reproductive effects observed:

not specified

Treatment related:

not specified

Conclusions:

The NOAEL was considered to be 695 mg/kg bw for F0 and F1 generation , when male and female rats were treated with test chemical orally .

Executive summary:

Two generation reproductive toxicity study of test chemical was performed on male and femaleCharles River Ceesarean albino rats . The test chemical mixed with food in dose concentration 0,3700, 13,900 and 51,900 ppm  (equivalent to 0, 185, 695 and 2595 mg/kg bw per day) were administered for 27 weeks .These P1 parental generations were individually housed. Clinical observations included food consumption, appearance, individual body weights and behavior and were made weekly. The F1A weanling rats designated P2 generation were kept 4-5 to a cage according to sex and maintained on the same concentration level as their parents until reaching maturity. During the breeding phase of the P1 generation, two females and one male were placed in a breeding cage. At weekly intervals during the mating period, the males were rotated among the females in each group. Following mating, the females were placed in individual cages to produce the first (FIA) litters. Twenty-four hours following the birth of the pups the first litters (FIA) were arbitrarily reduced to 8 maximum per mother. The number of conceptions, number of litters, live births, stillbirths, size of natural and nursing litters, deaths during the period of lactation, and number of pups weaned were recorded. The body weights of each pup were recorded at 24 hours and at weaning. Gross signs of toxicity were monitored. After 21-days of nursing, random pups were sacrificed and gross necropsies performed. Twenty-four females and twelve males remaining from each test group and control group were selected at random and designated the P2 generation. Following the weaning of the F1A animals, the P1 generation was remated to produce their second litters referred to as F1B, according to the procedures described above.
The P2 generation was housed 4-5 per cage and was maintained on the same dietary levels as their parents. The procedures outlined above for the P1 generation were maintained for the P2 generation. The litters of the P2 animals were referred to as the F2A litters. Body weights of the F2A pups were monitored 24 hours following the birth and at weaning. Gross signs of toxicity were recorded. Following a 21-day nursing period, all pups were weaned and sacrificed. One week following the weaning period of the F2A litter, the P2 generation was remated to produce their second litters (F2B). Two females were placed in a cage with a male from the corresponding dose group. Males were rotated weekly, and females were examined daily for presence of spermatozoa for a maximum of 21 consecutive days. The first day that sperm were observed was designated as day 0 of gestation. The females were then placed in individual cages. Half of the females (12) were sacrificed on day 19 or 20 of gestation and Caesarean sections were performed. Observations included number and placement of implantation sites, resorption sites, and live and dead fetuses; individual fetal weight and length (crown to rump), and external fetal anatomical structure. Gross necropsies were performed on each female including examination of uterus and visceral structures. The remaining 12 females were allowed to litter normally. The fetuses of both females delivering normally and via Caesarean section were necropsied.  

 

Fertility indices for the control and test animals of both F1A and F1B were considered low. The authors attributed this to the advanced age of the animals upon mating. The fertility index of the 3700 ppm test group in the F2A breeding cycle as well as the 3700 and 51900 ppm test groups in the F2B breeding cycle were reported to be low in comparison to control animals and historical control data.
Offspring toxicity F1 and F2: Growth suppression characterized as slight was also reported for the low-level F1B pups, and the high-level F1A and F1B pups and the F2A and F2B breeding cycles when compared with controls. All other measured parameters were comparable to controls in each generation and among the two filial generations. HenceThe NOAEL was considered to be 695 mg/kg bw for F0 and F1 generation , when male and female rats were treated with test chemical orally .

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

695 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Data is Klimicsh 2 and from authoritative database

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

 Reproductive toxicity study

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

Study 1

Two generation reproductive toxicity study of test chemical was performed on male and femaleCharles River Ceesarean albino rats . The test chemical mixed with food in dose concentration 0,3700, 13,900 and 51,900 ppm  (equivalent to 0, 185, 695 and 2595 mg/kg bw per day) were administered for 27 weeks .These P1 parental generations were individually housed. Clinical observations included food consumption, appearance, individual body weights and behavior and were made weekly. The F1A weanling rats designated P2 generation were kept 4-5 to a cage according to sex and maintained on the same concentration level as their parents until reaching maturity. During the breeding phase of the P1 generation, two females and one male were placed in a breeding cage. At weekly intervals during the mating period, the males were rotated among the females in each group. Following mating, the females were placed in individual cages to produce the first (FIA) litters. Twenty-four hours following the birth of the pups the first litters (FIA) were arbitrarily reduced to 8 maximum per mother. The number of conceptions, number of litters, live births, stillbirths, size of natural and nursing litters, deaths during the period of lactation, and number of pups weaned were recorded. The body weights of each pup were recorded at 24 hours and at weaning. Gross signs of toxicity were monitored. After 21-days of nursing, random pups were sacrificed and gross necropsies performed. Twenty-four females and twelve males remaining from each test group and control group were selected at random and designated the P2 generation. Following the weaning of the F1A animals, the P1 generation was remated to produce their second litters referred to as F1B, according to the procedures described above. The P2 generation was housed 4-5 per cage and was maintained on the same dietary levels as their parents. The procedures outlined above for the P1 generation were maintained for the P2 generation. The litters of the P2 animals were referred to as the F2A litters. Body weights of the F2A pups were monitored 24 hours following the birth and at weaning. Gross signs of toxicity were recorded. Following a 21-day nursing period, all pups were weaned and sacrificed. One week following the weaning period of the F2A litter, the P2 generation was remated to produce their second litters (F2B). Two females were placed in a cage with a male from the corresponding dose group. Males were rotated weekly, and females were examined daily for presence of spermatozoa for a maximum of 21 consecutive days. The first day that sperm were observed was designated as day 0 of gestation. The females were then placed in individual cages. Half of the females (12) were sacrificed on day 19 or 20 of gestation and Caesarean sections were performed. Observations included number and placement of implantation sites, resorption sites, and live and dead fetuses; individual fetal weight and length (crown to rump), and external fetal anatomical structure. Gross necropsies were performed on each female including examination of uterus and visceral structures. The remaining 12 females were allowed to litter normally. The fetuses of both females delivering normally and via Caesarean section were necropsied.  

 

Fertility indices for the control and test animals of both F1A and F1B were considered low. The authors attributed this to the advanced age of the animals upon mating. The fertility index of the 3700 ppm test group in the F2A breeding cycle as well as the 3700 and 51900 ppm test groups in the F2B breeding cycle were reported to be low in comparison to control animals and historical control data. Offspring toxicity F1 and F2: Growth suppression characterized as slight was also reported for the low-level F1B pups, and the high-level F1A and F1B pups and the F2A and F2B breeding cycles when compared with controls. All other measured parameters were comparable to controls in each generation and among the two filial generations. HenceThe NOAEL was considered to be 695 mg/kg bw for F0 and F1 generation , when male and female rats were treated with test chemical orally .

 

Study2

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 3

In reproductive and developmental toxicity study ,test chemical was given in the diet to CD-1 miceat dose concentration of 0, 0.42, 0.84, and 1.68% (0, 840, 1680, 3360 mg/kg bw/day), from 5 weeks of age of the F0 generation to 9 weeks of age of the F1 generation in mice, and selected reproductive and neurobehavioral parameters were measured. There were few adverse effects of test chemical on the average food intake during each period with the exception of the mating period. During the mating period, the average food intake was significantly increased in 3360mg/kg dosed group. Therefore, the chemical intake was consistently increased as dose-related during each period. Several dams showed under-development of mammary glands in the first week of lactation. No significant adverse effect was observed in either litter size or weight at birth. Sex ratio at birth was significantly reduced in the low-dosed group, while there were few adverse effects in other groups.

No adverse effect of test chemical was found in the all parameters measured for behavioral development during the lactation period in each sex. Movement activity of exploratory behavior showed no adverse effect of test chemical on pre-weaning period (at 3 weeks of age) for 5 min. There were few adverse effects of test chemical on multiple water T-maze performance in all treatment groups as compared with controls, while time taken was significantly reduced on the second trial in the 840mg/kg dosed group of female and on the third trial in 3360mg/kg dosed group of both sexes as compared with the first trial. Movement activity of exploratory behavior showed no adverse effect of test chemical on post-weaning period (at 8 weeks of age) for 10 min. Hence the LOAEL was considered to be840 mg/kg bw/day.

 

 

Study 4

In a embryotoxicity and teratogenicity study, Osborne-Mendel female rats were treated wtih test chemical in the concentration of0,7.5, 15, 30, 100 and 200 mg/kg body weight /day in distilled water by oral gavage. No external signs of toxicity and animals were appeared healthy and behaved normally in treated groups as compared to control.No effects on maternal weight gain and water consumption wereobserved in treated female rats as compared to control. No significant effect on numbers of corpora lutea, implantations, early and late deaths and viable foetuses per litter or on the percentage of preimplantation loss were observed in treated group as compared to control. Slightlyincreas in percentage of early resorptions were observed in 7.5 and 15 mg/kg bw/day but this response were appeared to be the result of sporadic occurrences. One litter was totally resorbed at 100 mg/kg bw/day. But, The percentage of females with more than one and with more than two resorptions showed no dose-related correlation as comapred to control. In addition, No effect on live or dead foetuses andmean foetal body weightand percentage of males and females per treatment group were observed as compared to control. Increased in number of runts were observed at 7.5, 15, 100 and 200 mg/kg bw/day treated dams, No compound-related external variations were observed in litters of treated dams as compared to control. Slightly decrease in males and females Crown-rump length were observed but were not significantly significant withincreasing dose level.No effect on percentage of males and females per treatment group were observed as compared to control. Similarly, increase in foetuses with hydroureters were observed but affected foetuses were well distributed among the litters and slight, but not significant, increase in the percentage of foetuses with soft-tissue variations were observed at 200 mg/kg bw/day, slight increase in the number of internal haemorrhages at 15 mg/kg bw/day as compard to control. No dose-related increase in the average number of variations per litter or in the average number of foetuses with one or more soft-tissue variationswere obsserved in litters of treated rats as compared to control. Slight increases in number of bipartite sternebrae, number of malaligned sternebrae and number of animals with fourteenth rib buds were observed in litters of 7.5, 30 and 200 mg/kg bw/day treated rats as compared to control. The observed increases did not appear to be dose-related. Therefore,NOAEL was considered to be 200 mg/kg body weight /day for F0 and F1 generation whenOsborne-Mendelfe male rats were treated wtih test chemical orally by gavage for 19 days.

 

 Study 5

 

Adult Sprague-Dawley rats were fed diets containing test chemical for 2 weeks and were then bred. The diets were continued for the females throughout gestation and lactation and were provided continuously to their offspring thereafter. The treatment groups were: test chemical as 0.0, 2.5, 5.0 or 10.0% (0, 1250, 2500, 5000 mg/kg bw/day) of the diet, and a positive control group treated with the toxin hydroxyurea on days 2-10 of life with 50 mg/kg/day given s.c. as a positive control group. Parental animals were evaluated for weight and food consumption, and females for reproductive success. The offspring were assessed on a series of tests using the Cincinnati Psychoteratogenicity Screening Test Battery. Additional measures were weight, food consumption, physical landmarks of development, and brain weight.Test chemical significantly reduced reproductive success, parental and offspring weight, brain weight, survival, and female vaginal patency development. Hence the LOAEL was considered to be 5000 mg/kg bw/day

 

Effects on developmental toxicity

Description of key information

Developmental toxicity study

Based on the various studies available for the test chemical were reviewed to determine the developmental toxicity, NOAELfor test chemical was considered to be 1000 mg /kg bw/day .When rats were treated with test chemical orally. Thus, comparing this value with the criteria of CLP regulation test chemical is not likelyto classify as reproductive and developmental toxicant.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data from peer reviewed journal

Qualifier:

equivalent or similar to guideline

Guideline:

other: As mentioned below

Principles of method if other than guideline:

The teratogenic toxicity study of test material was performed on rat

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

No data available

Species:

rat

Strain:

Osborne-Mendel

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: FDA rat breeding colony
- Age at study initiation: female: 12 - 21 wk
- Weight at study initiation: female: 220 - 270 g
- Fasting period before study: no data
- Housing: Stainless-steel hanging cages.
- Use of restrainers for preventing ingestion (if dermal): no
- Diet (e.g. ad libitum): Purina Laboratory Chow ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 25 °C
- Humidity (%): 30 - 63%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12-hr light/dark cycle (8 am 8 pro).

IN-LIFE DATES: From: To:

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
test material , Certified Batch No. AA-4181 was dissolved in distilled water (w/v). Fresh solutions were prepared 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):
- Concentration in vehicle: 0, 30, 75, 150, 300, 600 or 1000mg/kg/day.
- Amount of vehicle (if gavage): 1ml/100g body weight
- Lot/batch no. (if required): No data available
- Purity: No data available

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- 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

Duration of treatment / exposure:

19 days (Gestation day 0 to day 19)

Frequency of treatment:

Daily

Duration of test:

19 days

Remarks:

0, 30, 75, 150, 300, 600 or 1000mg/kg/day.

No. of animals per sex per dose:

Total: 294-301
0 mg/kg/bw/day : 42-43 females
30mg/kg/bw/day : 42-43 females
75mg/kg/bw/day : 42-43 females
150 mg/kg/bw/day : 42-43 females
300mg/kg/bw/day : 42-43 females
600mg/kg/bw/day : 42-43 females
1000 mg/kg/bw/day : 42-43 females

Control animals:

yes, concurrent vehicle

Details on study design:

No data available

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS:No data
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations:The rats were weighed daily

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): yes ,food consumption was measured weekly.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Water intake was not measured.
- Time schedule for examinations:

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:

Fetal examinations:

- External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes: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.
- Skeletal examinations: Yes: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
- Head examinations: No data

Statistics:

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.

Indices:

No data available

Historical control data:

No data available

Clinical signs:

no effects observed

Description (incidence and severity):

No unusual behaviours were observed in the animals during the study.The external maternal findings were unremarkabke.

Dermal irritation (if dermal study):

not specified

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female inthe group given 300mg/kg died at day 12 of gestation as a result of gavage difficulties unrelated to dosage

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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):

no effects observed

Description (incidence and severity):

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:

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:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Number of abortions:

no effects observed

Description (incidence and severity):

No litters were totally resorbed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The mean numbers of corpora lutea and implants per female were similar in all groups

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

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.

Early or late resorptions:

no effects observed

Description (incidence and severity):

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.

Dead fetuses:

no effects observed

Description (incidence and severity):

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.

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The pregnancy rate ranged from 85.71 to 95.35% with no evidence of dose correlation.

Other effects:

not specified

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

changes in number of pregnant

clinical signs

dead fetuses

early or late resorptions

food consumption and compound intake

mortality

number of abortions

pre and post implantation loss

total litter losses by resorption

Remarks on result:

other: No maternal toxic effects on was observed

Abnormalities:

not specified

Fetal body weight changes:

no effects observed

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.

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:

not specified

External malformations:

no effects observed

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

Skeletal malformations:

no effects observed

Description (incidence and severity):

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.

Visceral malformations:

no effects observed

Description (incidence and severity):

The numbers of soft-tissue variations in foetuses and their incidences per litter were similar in all groups except for a significant increase in the 150-mg/kg group in the number of litters with at least one variation; this increase was considered a random occurrence

Other effects:

not specified

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

external malformations

skeletal malformations

visceral malformations

Remarks on result:

other: No developmental toxic effects was observed

Abnormalities:

not specified

Developmental effects observed:

not specified

Treatment related:

not specified

Conclusions:

In developmental toxicity study, 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

Executive summary:

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.

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:

Data is Klimicsh 2 and from authoritative database

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

Developmental toxicity study

Data available from different studies for test chemicals were reviewed to determine the developmental 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

Adult Sprague-Dawley rats were fed diets containing test chemical for 2 weeks and were then bred. The diets were continued for the females throughout gestation and lactation and were provided continuously to their offspring thereafter. The treatment groups were:test chemical as 0.0, 2.5, 5.0 or 10.0% (0, 1250, 2500, 5000 mg/kg bw/day) of the diet, and a positive control group treated with the toxin hydroxyurea on days 2-10 of life with 50 mg/kg/day given s.c. as a positive control group. Parental animals were evaluated for weight and food consumption, and females for reproductive success. The offspring were assessed on a series of tests using the Cincinnati Psychoteratogenicity Screening Test Battery. Additional measures were weight, food consumption, physical landmarks of development, and brain weight. Test chemical significantly reduced reproductive success, parental and offspring weight, brain weight, survival, and female vaginal patency development. Behaviorally, test chemical produced substantially decreased running wheel activity, and slightly increased postweaning open-field rearing activity. Overall, test chemical produced evidence of both physical and behavioral toxicity in developing rats at doses of up to 10% (5000 mg/kg bw/day) of the diet.

Study 3

In a embryotoxicity and teratogenicity study, Osborne-Mendelfemale rats were treated wtih test chemical in the concentration of0,7.5, 15, 30, 100 and 200 mg/kg body weight /day in distilled water by oral gavage. No external signs of toxicity and animals were appeared healthy and behaved normally in treated groups as compared to control.No effects on maternal weight gain and water consumption wereobserved in treated female rats as compared to control. No significant effect on numbers of corpora lutea, implantations, early and late deaths and viable foetuses per litter or on the percentage of preimplantation loss were observed in treated group as compared to control. Slightlyincreas in percentage of early resorptions were observed in 7.5 and 15 mg/kg bw/day but this response were appeared to be the result of sporadic occurrences. One litter was totally resorbed at 100 mg/kg bw/day. But, The percentage of females with more than one and with more than two resorptions showed no dose-related correlation as comapred to control. In addition, No effect on live or dead foetuses andmean foetal body weightand percentage of males and females per treatment group were observed as compared to control. Increased in number of runts were observed at 7.5, 15, 100 and 200 mg/kg bw/day treated dams, No compound-related external variations were observed in litters of treated dams as compared to control. Slightly decrease in males and females Crown-rump length were observed but were not significantly significant withincreasing dose level.No effect on percentage of males and females per treatment group were observed as compared to control. Similarly, increase in foetuses with hydroureters were observed but affected foetuses were well distributed among the litters and slight, but not significant, increase in the percentage of foetuses with soft-tissue variations were observed at 200 mg/kg bw/day, slight increase in the number of internal haemorrhages at 15 mg/kg bw/day as compard to control. No dose-related increase in the average number of variations per litter or in the average number of foetuses with one or more soft-tissue variationswere obsserved in litters of treated rats as compared to control. Slight increases in number of bipartite sternebrae, number of malaligned sternebrae and number of animals with fourteenth rib buds were observed in litters of 7.5, 30 and 200 mg/kg bw/day treated rats as compared to control. The observed increases did not appear to be dose-related. Therefore,NOAEL was considered to be 200 mg/kg body weight /day for F0 and F1 generation when Osborne-Mendelfe male rats were treated wtih test chemical orally by gavage for 19 days.

Study 4

 

In reproductive and developmental toxicity study ,test chemical was given in the diet to CD-1 miceat dose concentration of 0, 0.42, 0.84, and 1.68% (0, 840, 1680, 3360 mg/kg bw/day), from 5 weeks of age of the F0 generation to 9 weeks of age of the F1 generation in mice, and selected reproductive and neurobehavioral parameters were measured. There were few adverse effects of test chemical on the average food intake during each period with the exception of the mating period. During the mating period, the average food intake was significantly increased in 3360mg/kg dosed group. Therefore, the chemical intake was consistently increased as dose-related during each period. Several dams showed under-development of mammary glands in the first week of lactation. No significant adverse effect was observed in either litter size or weight at birth. Sex ratio at birth was significantly reduced in the low-dosed group, while there were few adverse effects in other groups.

No adverse effect of test chemical was found in the all parameters measured for behavioral development during the lactation period in each sex. Movement activity of exploratory behavior showed no adverse effect of test chemical on pre-weaning period (at 3 weeks of age) for 5 min. There were few adverse effects of test chemical on multiple water T-maze performance in all treatment groups as compared with controls, while time taken was significantly reduced on the second trial in the 840mg/kg dosed group of female and on the third trial in 3360mg/kg dosed group of both sexes as compared with the first trial. Movement activity of exploratory behavior showed no adverse effect of test chemical on post-weaning period (at 8 weeks of age) for 10 min. Hence the LOAEL was considered to be 840 mg/kg bw/day.

 

 

Justification for classification or non-classification

Thus, comparing this value with the criteria of CLP regulation test chemical is not likely to classify as reproductive and developmental toxicant.