Disodium 4,4'-[[2,4-dihydroxy-5-(hydroxymethyl)-1,3-phenylene]bis(azo)]bisnaphthalene-1-sulphonate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

NOAEL was considered to be 500 mg/kg/day (10000 ppm) for F0 generation when Wistar male and female rats treated with Chocolate Brown HT (Disodium 4,4'-[[2,4-dihydroxy-5-(hydroxymethyl)-1,3-phenylene]bis(azo)]bisnaphthalene-1-sulphonate).

Link to relevant study records

Reference

Endpoint:

one-generation reproductive 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 is from a peer reviewed journal

Qualifier:

according to guideline

Guideline:

other: as mentioned below

Principles of method if other than guideline:

Combined repeated dose & carcinogenicity study of Chocolate Brown HT in rats orally.

GLP compliance:

not specified

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Specified pathogen-free breeding Colony
- Age at study initiation: No data available
- Weight at study initiation:
(P) Males: 54-80 g
(P) Females: 53-82 g
- Fasting period before study: No data available
- Housing: 4/cage
- Diet (e.g. ad libitum): ground Spratts Laboratory Diet No.1 ad libitum
- Water (e.g. ad libitum): Water ad libitum
- Acclimation period: No data available

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20± 1°C
- Humidity (%): 50-60%
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): No data available

IN-LIFE DATES: From: To: No data available

Route of administration:

oral: feed

Vehicle:

other: Ground Spratts Laboratory Diet No. 1

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): Chocolate Brown HT was incorporated at 0, 500, 2000 or 10 000 ppm in diet.
- Storage temperature of food: No data available

VEHICLE
- Justification for use and choice of vehicle (if other than water): Ground Spratts Laboratory Diet No. 1
- Concentration in vehicle: 0, 25, 100 and 500 mg/Kg/day in the diet
- Amount of vehicle (if gavage): No data available
- Lot/batch no. (if required): No data available
- Purity: No data available

Details on mating procedure:

No data available

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

2 years

Frequency of treatment:

Daily

Details on study schedule:

No data available

Remarks:

Doses / Concentrations:
0, 25, 100 and 500 mg/Kg/day (0, 500,2000 and 10000 ppm)
Basis:
nominal in diet

No. of animals per sex per dose:

Total: 384
0 ppm: 48 male, 48 female
500 ppm: 48 male, 48 female
2000 ppm: 48 male, 48 female
10000 ppm: 48 male, 48 female

Control animals:

yes, concurrent vehicle

Details on study design:

No data available

Positive control:

No data available

Parental animals: Observations and examinations:

Observations and examinations performed & frequency
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked: Cumulative mortality was observed

DETAILED CLINICAL OBSERVATIONS: No data
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: On first day of feeding and at 1, 4, 7 and 11 weeks of treatment and thereafter at approx. 3-monthly intervals up to week 102

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding 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: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: measured over the 48-h period commencing at the same time as the food intake

OTHER:
Haematology, clinical chemistry, urinalysis and Organ weight were examined.

Oestrous cyclicity (parental animals):

No data available

Sperm parameters (parental animals):

Parameters examined in [P] male parental generations: testis weight

Litter observations:

Not applicable

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes
Any macroscopic abnormalities were observed.
Samples of all major tissues and organs and of any other tissue appearing abnormal at autopsy were preserved in 10% buffered formalin until prepared for histological examination.

HISTOPATHOLOGY: Yes
Organ examined: Brain, heart, liver, spleen, kidneys, stomach, small intestine, caecum, adrenals, gonads, pituitary and thyroid were weighted.

Postmortem examinations (offspring):

No data available

Statistics:

Statistical calculations are based on a level of significance of at least P = 0.05.

Reproductive indices:

No data available

Offspring viability indices:

No data available

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

Cumulative mortality was similar in the treated and control animals throughout the 2-year period except for one statistically significant value at week 72 (7 deaths compared with none in the controls) in males given 500mg/Kg/day

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant decrease was observed in 500 mg/kg/day treated rats as compared to control at 102 week in male and 78 week in female.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Statistically significant decrease was observed in 500 mg/kg/day treated rats as compared to control at 102 week in male and 78 week in female.

Food efficiency:

not specified

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

no effects observed

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

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:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

High incidence of adenosis and fibroadenosis in the female rats, but the incidences in the treated and control animals showed no statistical differences.

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
The approximate total intakes of colouring consumed per rat up to week 66 were for the low, medium and high treatment levels respectively, 5.25, 21.5 and 109.8 g by the males and 4.15, 17.0 and 83.9 g by the female rats. The corresponding values up to week 102 were 8.11, 32.3 and 160.8 g/rat by the males and 6.38, 21.1 and 128.2 g/rat for the females.

ORGAN WEIGHTS (PARENTAL ANIMALS)
A slight reduction in testis weight at 25 mg/Kg day observed. This was not considered to be treatment related.

HISTOPATHOLOGY (PARENTAL ANIMALS)
When treated with 2000 and 10000 ppm, in male rat wide range of pathological changes in liver and kidney with lower incidence of fatty change in the livers were observed as compared to control.
High incidence of adenosis and fibroadenosis in the female rats were observed, but the effect was not statistically significant as compared to control.

OTHER
HAEMATOLOGY:
When treated with 10000 ppm, in male and female rat significant decrease were observed in hemoglobin at 55 week, WBC at 27 and 55 week in male rat and significant increase in RBC at 27 week in male and female rat as compared to control.
When treated with 2000 ppm, significant increase was observed in RBC of male and female rat and increase in WBC in female as compared to control.
Observed effect was not considered to be treatment related.

CLINICAL CHEMISTRY: No significant differences between treated and control animals observed.

URINALYSIS: No significant differences between treated and control animals observed

Key result

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

food consumption and compound intake

water consumption and compound intake

haematology

clinical biochemistry

urinalysis

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

Remarks on result:

other: no reprotoxic

Clinical signs:

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Dose descriptor:

other: not specified

Generation:

other: not specified

Based on:

not specified

Sex:

not specified

Basis for effect level:

other: not specified

Remarks on result:

other: not specified

Reproductive effects observed:

no

Treatment related:

not specified

Relation to other toxic effects:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Conclusions:

NOAEL was considered to be 500 mg/kg/day (10000 ppm) for F0 generation when Wistar male and female rats treated with Chocolate Brown HT (Disodium 4,4'-[[2,4-dihydroxy-5-(hydroxymethyl)-1,3-phenylene]bis(azo)]bisnaphthalene-1-sulphonate).

Executive summary:

 In a Combined repeated dose & carcinogenicity study, male and female Wistar rats were treated with Chocolate Brown HT (Disodium 4,4'-[[2,4-dihydroxy-5-(hydroxymethyl)-1,3-phenylene]bis(azo)]bisnaphthalene-1-sulphonate) orally at the concentration of 0, 500, 2000 and 10000 ppm. No effect were observed on overall mortality rate, food consumption, water intake, hematology, clinical chemistry, urine analysis and organ weight of 10000 ppm treated male and female rats as compared to control in F0 generation. In addition, lower incidence of fatty change in the livers in male, adenosis and fibroadenosis in the female and tumours of mammary adenomas and fibroadenomas in the female rats, interstitial cell tumours of the testis and subcutaneous fibromas in both sexes were observed. The observed effects were not related to treatment in F0 generation.

Therefore, NOAEL was considered to be 500 mg/kg/day (10000 ppm) for F0 generation when Wistar male and female rats treated with Chocolate Brown HT orally for 2 years.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Data is of K2 level from peer reviewed journal.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

No. of studies of Chocolate Brown HT were reviewed for toxicity to reproduction from reliable sources having Klimisch rating 2. The summary of the results is presented below:

For key study, a Combined repeated dose & carcinogenicity study was carried in which male and female Wistar rats were treated with Chocolate Brown HT (Disodium 4,4'-[[2,4-dihydroxy-5-(hydroxymethyl)-1,3-phenylene]bis(azo)]bisnaphthalene-1-sulphonate) orally at the concentration of 0, 500, 2000 and 10000 ppm. No effect were observed on overall mortality rate, food consumption, water intake, hematology, clinical chemistry, urine analysis and organ weight of 10000 ppm treated male and female rats as compared to control in F0 generation. In addition, lower incidence of fatty change in the livers in male, adenosis and fibroadenosis in the female and tumours of mammary adenomas and fibroadenomas in the female rats, interstitial cell tumours of the testis and subcutaneous fibromas in both sexes were observed. The observed effects were not related to treatment in F0 generation. Therefore, NOAEL was considered to be 500 mg/kg/day (10000 ppm) for F0 generation when Wistar male and female rats treated with Chocolate Brown HT orally for 2 years.

Further in a three generation toxicity study, Wistar derived out bred male and female rats treated with Chocolate Brown HT (Disodium 4,4'-[[2,4-dihydroxy-5-(hydroxymethyl)-1,3-phenylene]bis(azo)]bisnaphthalene-1-sulphonate) in the concentration of 0, 50, 250 and 500 mg/Kg bw/day. No significant effects were observed on survival, body weight, food consumption andmale fertilityof 500 mg/kg bw/day of F0 rats.Statistically significant increase in kidney and Caecum weight were observed in F0 female rat at 250 and 500mg/kg bw/day dose.Significantly fewer corpora lutea, average numbers of implantations, live foetuses and mean foetal weight was significantly higher in F0 female rat in 500 mg/kg bw/day dose group. In F2b, F2 and F3 generation, no effect were observed on survival, body weight, male fertility and Reproductive performance of pups. Statistically significant increase in kidney weight in male and female pups of F1, F2b and F2 at 50 and 500 mg/kg bw /day and increase in Caecum weights in F2 and F3 male and female pups at 250 mg/kg bw /day were observed. In addition, Skeletal ossification of third sternebrae, brown coloration of mesenteric lymph nodes, caecum and molars were observed in F0 rats and brown coloration of mesenteric lymph nodes, caecum and molars in F1, F2b and F2 pups and slight evidence of retarded ossification in F3 pups in 500 mg/kg bw/day. Therefore, NOAEL was considered to be 250 bw /day for F0, F1, F2b, F2 and F3 generation when Wistar derived out bred male and female rats treated with Brown HT orally for 229 days.

Supporting above experimental data, the reproductive toxicity of Chocolate Brown HT ( Disodium 4,4'-[[2,4-dihydroxy-5-(hydroxymethyl)-1,3-phenylene]bis(azo)]bisnaphthalene-1-sulphonate) to rats was estimated using QSAR Toolboox version 3.3. Since no compound related effects on reproductive organs were observed, the no observed adverse effect level (NOAEL) relating to reproductive effects for the given test material rats was estimated to be 650 mg/kg/day.

Based on the studies summarized above it can be observed that NOAEL value varies from 250 to 650 mg/Kg bw/ day. The main effects observed on these doses are listed as follows:

·       No effects on reproduction

·       No effect on mortality, food consumption, water intake, haematology, clinical chemistry, urine analysis, organ weight, gross pathology and histopathology

·       No adverse effect on, survival, body weight, food consumption, organ weights, gross pathology, histopathology and reproductive performance.

Since the experimental no effective dose value (NOAEL) is 500 mg/Kg bw/d thus based on this value it can be concluded that substance CAS No 4553-89-3 is considered to be not toxic to reproduction for the above mentioned doses.

Effects on developmental toxicity

Description of key information

NOAEL was considered to be 1000 mg/kg bw /day for F0 and F1 generation when Wistar female rats treated with Brown HT (Disodium 4,4'-[[2,4-dihydroxy-5-(hydroxymethyl)-1,3-phenylene]bis(azo)]bisnaphthalene-1-sulphonate).

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 is from peer reviewed journal

Qualifier:

according to guideline

Guideline:

other: as mentioned below

Principles of method if other than guideline:

Teratogenicity and Embryotoxicity Study of Brown Ht (CAS No.4553-89-3) orally in rat

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Wistar

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

other: not applicable

Vehicle:

water

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: Overnight
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy Vaginal plug or sperm, referred to as day 0 of pregnancy
- Any other deviations from standard protocol: - Additional animals were treated at each dose level to replace those that failed to conceive.

Frequency of treatment:

Daily

Duration of test:

19 days

Remarks:

Doses / Concentrations:
0, 250, 500 and 1000 mg/kg/day
Basis:
no data

No. of animals per sex per dose:

Total: 120
0 mg/kg/day: 30 femle
250 mg/kg/day: 30 femle
500 mg/kg/day: 30 femle
1000 mg/kg/day: 30 femle

Control animals:

yes, concurrent vehicle

Maternal examinations:

Body weight and Body weight gain, clinical sign and gross abnormalities were examined

Ovaries and uterine content:

corpora lutea, implantations , Pre- implantation losses, early and late resorption, Post- implantation losses and live fetous were examined

Statistics:

Stsatistical analysis were done by using Student’s t test for all parameters except for the number of females with post – implantation losses for which chi-square test were used.

Historical control data:

In preliminary study groups of 10 female rats received daily doses of Brown HT in the concentration of 0, 250, 500 and 1000 mg/kg bw/day for 19 consecutive days. In treated rates body-weight gain were similar in all groups and no signs of toxicity were observed throughout the period of treatment. The faeces of the treated animals were markedly brown, and at autopsy there was brown discoloration of lymph nodes, colon and caecum.

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality:

not specified

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant change was observed in body weight and body weight gain of treated female rat as compared to control.

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

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Number of abortions:

not specified

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

The numbers of females with post-implantation losses were increased in the treated groups although this loss did not increase with increasing dose and was not significantly different from control.

Total litter losses by resorption:

not specified

Early or late resorptions:

not specified

Dead fetuses:

not specified

Changes in pregnancy duration:

not specified

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified

Changes in number of pregnant:

not specified

Other effects:

no effects observed

Details on maternal toxic effects:

Details on maternal toxic effects:
Mortality: No data available

Clinical signs:Nodata avaialble

Body weight: No significant change was observed in body weight and body weight gain of treated female rat as compared to control.

Reproductive function: estrous cycle: No statistically significant differences were observed in the mean numbers of corpora lutea, implantations, pre- and post implantation losses and resorptions of treated female rats.

The numbers of females with post-implantation losses were increased in the treated groups although this loss did not increase with increasing dose and was not significantly different from control.

Reproductive performance: No significant effect was observed on number of live fetuses, foetal numbers and sex ratio of treated rat as compared to control.

Gross pathology: No data available

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No statistically significant differences were observed in foetal weight of treated rats.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not specified

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No effect was observed on number of live fetuses of treated rat.

Changes in sex ratio:

not specified

Changes in litter size and weights:

not specified

Changes in postnatal survival:

not specified

External malformations:

not specified

Visceral malformations:

not specified

Other effects:

effects observed, treatment-related

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
Mortality:
No effect was observed on number of live fetuses of treated rat.

Body weight:
No statistically significant differences were observed in foetal weight of treated rats.

Reproductive performance:
No statistically significant differences were observed on sex ratio of fetuses of treated rat as compared to control.

Gross pathology:
When treated with 1000 mg/kg bw/day, Bicentral ossification of 1st sternebra, presences of Fourteenth ribs, One thoracic vertebra & ribs absent, Forelimb proximal phalanges ossified, Metacarpals ossified of third and fourth skeleton were observed in fetuses of treated rat.

When treated with 500 mg/kg bw/day, Bicentral ossification of 1st and 2nd sternebra, Small second sternebra, One thoracic vertebra & ribs absent and Incomplete ossification of supra-occipital of skeleton were observed in foetuses.

When treated with 250 mg/kg bw/day, Small second sternebra, presences of fourteenth ribs and Incomplete ossification of supra-occipital of skeleton were observed in foetuses.

Histopathology:
When treated with 250 mg/kg bw/day, Renal pelvis enlargement and exencephaly of the cerebrum with lateral ventricle dilation in a foetus were observed in soft-part of treated rat as compared to control.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: fetotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

NOAEL was considered to be 1000 mg/kg bw /day for F0 and F1 generation when Wistar female rats treated with Brown HT (Disodium 4,4'-[[2,4-dihydroxy-5-(hydroxymethyl)-1,3-phenylene]bis(azo)]bisnaphthalene-1-sulphonate).

Executive summary:

In aTeratogenicity and Embryotoxicity study,Wistar female rats treated with Brown HT (Disodium 4,4'-[[2,4-dihydroxy-5-(hydroxymethyl)-1,3-phenylene]bis(azo)]bisnaphthalene-1-sulphonate) in the concentration of 0, 250, 500 and 1000 mg/Kg bw/day. No significant changes were observed on body weight, mean numbers of corpora lutea, implantations, pre- and post implantation losses, resumptions,number of live fetuses,foetal numbers and sex ratio of treated female rat as compared to control.

The numbers of females with post-implantation losses were increased in the treated groups although this loss did not increase with increasing dose and was not significantly different from control.In addition, no effect was observed on number of live fetuses and foetal weight of treated female rats as compared to control. Ossified sternebrae, Bicentral ossification, Small second sternebra, presences of fourteenth ribs, One thoracic vertebra & ribs absent, forelimb proximal phalanges and Metacarpalsossified and incomplete ossification of supra-occipital gross changes were observed in foetus.Renal pelvis enlargement and exencephaly of the cerebrum with lateral ventricle dilation in a foetus were observed also observed in foetus.

The observed effects are minor deviations from normal and are of doubtful toxicological significance.

Therefore,NOAEL was considered to be 1000 mg/kg bw /day for F0 and F1 generation whenWistarfemale rats treated with Brown HT orally by gavage for 19 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:

rabbit

Quality of whole database:

Data is of K2 level from peer reviewed journal.

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

No. of studies of Chocolate Brown HT were reviewed for developmental toxicity from reliable sources having Klimisch rating 2. The summary of the results is presented below:

In a Teratogenicity and Embryotoxicity key study, Wistar female rats treated with Brown HT (Disodium 4,4'-[[2,4-dihydroxy-5-(hydroxymethyl)-1,3-phenylene]bis(azo)]bisnaphthalene-1-sulphonate) in the concentration of 0, 250, 500 and 1000 mg/Kg bw/day. No significant changes were observed on body weight, mean numbers of corpora lutea, implantations, pre- and post implantation losses, resumptions,number of live fetuses,foetal numbers and sex ratio of treated female rat as compared to control. The numbers of females with post-implantation losses were increased in the treated groups although this loss did not increase with increasing dose and were not significantly different from control. In addition, no effect was observed on number of live fetuses and foetal weight of treated female rats as compared to control. Ossified sternebrae, Bicentral ossification, Small second sternebra, presences of fourteenth ribs, One thoracic vertebra & ribs absent, forelimb proximal phalanges and Metacarpalsossified and incomplete ossification of supra-occipital gross changes were observed in foetus. Renal pelvis enlargement and exencephaly of the cerebrum with lateral ventricle dilation in a foetus were observed also observed in foetus. The observed effects are minor deviations from normal and are of doubtful toxicological significance. Therefore, NOAEL was considered to be 1000 mg/kg bw /day for F0 and F1 generation when Wistar female rats treated with Brown HT orally by gavage for 19 days.

Further, In a three generation developmental toxicity study, Wistar derived outbred male and female rats treated with Brown HT ( Disodium 4,4'-[[2,4-dihydroxy-5-(hydroxymethyl)-1,3-phenylene]bis(azo)]bisnaphthalene-1-sulphonate) in the concentration of 0, 50, 250 and 500 mg/Kg bw/day. No significant effects were observed on survival, body weight, food consumption andmale fertilityof 500 mg/kg bw/day of F0 rats. Statistically significant increase in kidney and caecum weight were observed in F0 female rat at 250 and 500mg/kg bw/day dose. Significantly fewer corpora lutea, average numbers of implantations, live foetuses and mean foetal weight was significantly higher in F0 female rat in 500 mg/kg bw/day dose group. In F2b, F2 and F3 generation, no effect were observed on survival, body weight, male fertility and reproductive performance of pups. Statistically significant increase in kidney weight in male and female pups of F1, F2b and F2 at 50 and 500 mg/kg bw /day and increase in caecum weights in F2 and F3 male and female pups at 250 mg/kg bw /day were observed. In addition, Skeletal ossification of third sternebrae, brown coloration of mesenteric lymph nodes, caecum and molars were observed in F0 rats and brown coloration of mesenteric lymph nodes, caecum and molars in F1, F2b and F2 pups and slight evidence of retarded ossification in development of F3 pups in 500 mg/kg bw/day. Therefore, NOAEL was considered to be 250 bw /day for F0, F1, F2 and F3 generation when Wistar derived outbred male and female rats treated with Brown HT orally for 229 days.

Supporting above experimental data, the developmental toxicity of Chocolate Brown HT ( Disodium 4,4'-[[2,4-dihydroxy-5-(hydroxymethyl)-1,3-phenylene]bis(azo)]bisnaphthalene-1-sulphonate) to rabbits was estimated using QSAR Toolboox version 3.3. The no observed adverse effect level (NOAEL) relating to teratogenicity effects for the given test material in rabbits was estimated to be 250 mg/kg/day.

Based on the studies summarized in the above table it can be observed that NOAEL value varies from 250 to 1000 mg/Kg bw/ day. The effects observed on these doses are listed as follows:

·       Overall effects

·       No adverse effect on body weight and body weight gain, gross pathology and foetal development.

·       No adverse effect on foetus weight, gross abnormalities, skeletal and soft-part abnormalities.

·       No adverse effect on, survival, body weight, food consumption, organ weights, gross pathology, histopathology and reproductive performance.

Since the highest no effective dose value (NOAEL) is 1000 mg/Kg bw/d thus based on this value it can be concluded that substance CAS NO 4553-89-3 is considered to be not toxic to reproduction for the above mentioned doses.

Justification for classification or non-classification

Based on the available information from reliable sources, the substance is likely to be non-toxic for reproduction and developmental toxicity/ teratogenicity.