Disodium 4-hydroxy-3-[(4-sulphonatonaphthyl)azo]naphthalenesulphonate

Toxicity to reproduction

Administrative data

Endpoint:

reproductive toxicity, other

Remarks:

Long term study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Data is from peer-reviewed journal

Data source

Reference

Reference Type:

publication

Title:

Long-term toxicity study of carmoisine in rats using animals exposed in utero

Author:

G. P. FORD, B. I. STEVENSON and J. G. EVANS

Year:

1987

Bibliographic source:

Fd Chem. Toxic. Vol. 25, No. 12, pp. 919-925, 1987

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: Combined repeated dose & carcinogenicity

Principles of method if other than guideline:

Combined repeated dose & carcinogenicity Test of Carmoisine in male and female Wistar rats.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 4-hydroxy-3-[(4-sulphonatonaphthyl)azo]naphthalenesulphonate

EC Number:

222-657-4

EC Name:

Disodium 4-hydroxy-3-[(4-sulphonatonaphthyl)azo]naphthalenesulphonate

Cas Number:

3567-69-9

Molecular formula:

C20H12N2Na2O7S2

IUPAC Name:

disodium 4-hydroxy-3-[(4-sulfonato-1-naphthyl)diazenyl]naphthalene-1-sulfonate

Details on test material:

- Name of test material: Carmoisine
- Molecular formula: C20H14N2O7S2.2Na
- Molecular weight: 502.4338 g/mol
- Substance type: organic
- Physical state: No data available
- Purity 85% dye
- Impurities (identity and concentrations):
15.0 %NaCI
>0 1% subsidiary colors

Specific details on test material used for the study:

- Name of test material: Carmoisine
- Molecular formula: C20H14N2O7S2.2Na
- Molecular weight: 502.4338 g/mol
- Substance type: organic

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Olac (1976) Ltd, Bicester, Oxon
- Age at study initiation: (P) x wks; No data available. (F1) x wks
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g- No data available
- Fasting period before study: No data available.
- Housing: Housed six to a cage in an air-conditioned room.
- Diet (e.g. ad libitum): Spratt's Laboratory Animal Diet No. 2 (Spratt's Patent, Barking, Essex) ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: No data available

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C
- Humidity (%):40-70%
- Air changes (per hr): No data available.
- Photoperiod (hrs dark / hrs light): 14/10-hr light/dark cycle in phase with the natural light.

Administration / exposure

Route of administration:

oral: feed

Type of inhalation exposure (if applicable):

not specified

Vehicle:

other: Spratt's Laboratory Animal Diet No. 2

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 13 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy No data available.
- 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 housed individually.
- Any other deviations from standard protocol: Sibling mating was avoided.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

For male
115 weeks
For female
110 weeks

Frequency of treatment:

Daily

Details on study schedule:

Groups of 114 (control) or 66 (treated) rats of each sex were fed diets providing 0 (control), 100, 400 or 1200 mg carmoisine/kg body weight/day for 9 wk. Within each group the animals were mated monogamously. Treatment continued uninterrupted until the young were randomly selected (where possible one/sex/litter) from each of the litters to provide groups of 90 (control) and 54 (treated) rats of each sex. These received the same treatment as their parents for up to 110 wk for females or 115 wk for males. They were observed for mortality, clinical sign, body weight, food intake and water intake, hematology, clinical chemistry, urine analysis, gross pathology and histopathology.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

400 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

1 200 mg/kg bw/day

No. of animals per sex per dose:

Total no .of animals-624
P group
0mg/kg/day-114 male and 114 female rats
100 mg/kg/day- 66 male and 66 female rats
400 mg/kg/day- 66 male and 66 female rats
1200 mg/kg/day- 66 male and 66 female rats

Total no.of anomals-504
F1 generation
0mg/kg/day-90 male and 90 female rats
100 mg/kg/day- 54 male and 54female rats
400 mg/kg/day- 54 male and 54female rats
1200 mg/kg/day- 54 male and 54female rats

Control animals:

yes, concurrent vehicle

Details on study design:

not specified

Positive control:

not specified

Examinations

Parental animals: Observations and examinations:

Survival, clinical sign, body weight food intake, water intake, haematology, clinical chestry and Urine analysis were examined.

Oestrous cyclicity (parental animals):

not specified

Sperm parameters (parental animals):

not specified

Litter observations:

Survival, clinical sign, body weight food intake, water intake, haematology, clinical chestry and Urine analysis were examined.

Postmortem examinations (parental animals):

Organ wieght, gross pathology and histopathology were examined.

Postmortem examinations (offspring):

Organ wieght, gross pathology and histopathology were examined.

Statistics:

All data were subject to the appropriate statistical tests (see tables) and a probability level of 0.05 or less was taken as statistically significant.

Reproductive indices:

not specified

Offspring viability indices:

not specified

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No significant change of carmoisine on the condition or behaviour of the animals, apart from coloration of the fur, urine and faeces with an intensity increasing with dose.

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

No effect on survival of treated rats were observed

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 / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

No effect on mating, pregnancy, lactation and weaning of reated rats as compared to control.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

1 200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

reproductive performance

Remarks on result:

other: No effect observed

Target system / organ toxicity (P0)

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

Effect like coloration of the fur, urine and faeces with an intensity increasing with dose . But no significant change were observed in animals behaviour and condition in treated group compare to control.

Dermal irritation (if dermal study):

not specified

Mortality / viability:

no mortality observed

Description (incidence and severity):

No significant change were observed in the mortality in treated group compare to control.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significant change were observed in the body weights of F1 males receiving either 400 and 1200 mg carmoisine/kg/day were lower than those of the controls throughout the study.

Females receiving 1200 mg/kg/day weighed significantly less than the controls from wk 32 onwards. In both sexes the magnitude of the observed differences increased with time.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Significant change was observed in the food intakes of the1200 mg carmoisine/kg/day males was observed compare to control.

Even in female 1200 mg carmoisine/kg/day groups were greater than those of the controls, but the differences in females was unrelated to dose.

Food efficiency:

not specified

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

no effects observed

Description (incidence and severity):

Water intakes of treated animals showed some inconsistency during the study. For the first 12 months there was a dose-related higher intake in both sexes receiving 400 or 1200 mg carmoisine /kg/day. During the second year the water intake of males receiving 1200mg/kg/day or of females receiving 400mg/ kg/day was no longer different from that of the controls.

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Description (incidence and severity):

No significant change were observed in at all dose level 100, 400 or 1200 mg /kg body weight/day of treated group compare to control.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Lower glucose concentrations was observed in males at dose level of 1200 mg/kg/day compare to control.
While a Lower glucose concentration was observed in females at dose level of 400 and 1200 mg/kg/day compare to control.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Semi-quantitative analyses of urine were hindered in treated animals, particularly at the 1200 mg/kg/day, by the colour of the urine which obscured the colour reaction of the reagent test strips. Despite these limitations, which meant that some samples obtained from 1200 mg/kg/day animals were not usable, a higher proportion of samples from the from 1200 mg/kg/day females showed a high level of protein at both 18 and 24 months.

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No significant change were observed in any organ except caecum weight. The caecum was the only organ whose weight and relative weight (g/100 g body weight) showed changes associated with treatment. At 1200 and 400 mg/kg/day dose level in males both weight and relative weight of the caecum, with and without contents, were greater than the control values, although the increases were only statistically significant at the1200 mg /kg body weight/day. In females at the dose level of 1200 mg /kg body weight/day only the relative weight of the empty caecum was significantly greater than that of the controls. The changes in caecum weight were accompanied by observations at autopsy that the caecum wall was thicker in the treated animals

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Significant thickness was observed in caecum wall at 1200 mg /kg body weight/day group of treated animals compare to control .

Histopathological findings:

no effects observed

Description (incidence and severity):

Non neoplastic- Age-related changes to be expected in rats of the strain used. Some of the changes recorded showed a positive dose-related trend together with a significantly higher incidence when individual groups were compared with the controls. These were, pupillary hyperplasia of the bladder in males and blood/fibrin cysts of the adrenal gland and internal hyperplasia / medial hypertrophy of the pancreatic blood vessels in females.

Neoplastic- The total incidences of benign or malignant tumours showed no treatment-related differences. There were no differences between the treated and control groups in the incidences of individual types of tumour apart from adrenal phaeochromocytoma which showed a statistically significant treatment-related difference in males based on four tumours in the 1200 mg/kg/day group compare to controls. A similar difference was not observed in females.

Other effects:

not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not specified

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

400 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

water consumption and compound intake

haematology

clinical biochemistry

urinalysis

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

Remarks on result:

other: No effect observed

Target system / organ toxicity (F1)

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Overall reproductive toxicity

Reproductive effects observed:

not specified

Treatment related:

not specified

Relation to other toxic effects:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

NOAEL was considered to be 1200 mg/kg bw for P generation and 400 mg/kg bw for F1 generation when Wistar male and female rats were treated with Carmoisine orally in feed for 115 weeks.

Executive summary:

In a long term toxicity study, Wistar male and female rats were treated with Carmoisine in the concentration of  0 (control), 100, 400 or 1200 mg /kg body weight/day orally in feed for 115 weeks. No significant change of carmoisine on the condition or behaviour of the animals, apart from coloration of the fur, urine and faeces with an intensity increasing with dose. No effect on survival of treated rats was observed. No effect on mating, pregnancy, lactation and weaning of P treated rats as compared to control. In addition, Effect like coloration of the fur, urine and faeces with an intensity increasing with dose in F1 pups. But no significant change was observed in animals behaviour and condition in treated group compare to control. No significant change was observed in the survival of F1 treated group compare to control. Significant change was observed in the body weights of F1 males receiving 400 and 1200 mg/kg/day were lower than those of the controls throughout the study. Females receiving 1200 mg/kg/day weighed significantly less than the controls from week 32 onwards. In both sexes the magnitude of the observed differences increased with time. Significant change was observed in the food intakes of the1200 mg /kg/day males was observed compare to control. Even in female 1200 mg/kg/day were greater than those of the controls, but the differences in females was unrelated to dose. Water intakes of treated animals showed some inconsistency during the study. For the first 12 months there was a dose-related higher intake in both sexes receiving 400 or 1200 mg /kg/day. During the second year the water intake of males receiving 1200mg/kg/day or of females receiving 400 mg/ kg/day was no longer different from that of the controls. No significant change was observed at all dose level 100, 400 or 1200 mg /kg body weight/day of treated group compare to control. Lower glucose concentrations were observed in males at dose level of 1200 mg/kg/day compare to control. While a Lower glucose concentration was observed in females at dose level of 400 and 1200 mg/kg/day compare to control. Semi-quantitative analyses of urine were hindered in treated animals, particularly at the 1200 mg/kg/day, by the colour of the urine which obscured the colour reaction of the reagent test strips. Despite these limitations, which meant that some samples obtained from 1200 mg/kg/day animals were not usable, a higher proportion of samples from the 1200 mg/kg/day females showed a high level of protein at both 18 and 24 months. No significant change was observed in any organ except caecum weight. The caecum was the only organ whose weight and relative weight (g/100 g body weight) showed changes associated with treatment. At 1200 and 400 mg/kg/day dose level in males both weight and relative weight of the caecum, with and without contents, were greater than the control values, although the increases were only statistically significant at the1200 mg /kg body weight/day. In females at the dose level of 1200 mg /kg body weight/day only the relative weight of the empty caecum was significantly greater than that of the controls. The changes in caecum weight were accompanied by observations at autopsy that the caecum wall was thicker in the treated animals. Similarly, significant thickness was observed in caecum wall at 1200 mg /kg body weight/day group of treated animals compare to control. Age-related changes to be expected in rats of the strain used. Some of the changes recorded showed a positive dose-related trend together with a significantly higher incidence when individual groups were compared with the controls. These were pupillary hyperplasia of the bladder in males and blood/fibrin cysts of the adrenal gland and internal hyperplasia / medial hypertrophy of the pancreatic blood vessels in females. The total incidences of benign or malignant tumours showed no treatment-related differences. There were no differences between the treated and control groups in the incidences of individual types of tumour apart from adrenal phaeochromocytoma which showed a statistically significant treatment-related difference in males based on four tumours in the 1200 mg/kg/day group compare to controls. A similar difference was not observed in females. Therefore, NOAEL was considered to be 1200 mg/kg bw for P generation and 400 mg/kg bw for F1 generation when Wistar male and female rats were treated with Carmoisine orally in feed for 115 weeks.