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

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference Type:

publication

Title:

Long-Term Toxicity Studies on Chocolate Brown HT In Rats

Author:

Francis M.B. Carpanini, Kenneth R. Butterworth, Ian F. Gaunt, Ida S. Kiss, Paul Grasso And Sharat D. Gangolli

Year:

1978

Bibliographic source:

Toxicology, 11 (1978) 303-307

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

224-924-0

EC Name:

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

Cas Number:

4553-89-3

Molecular formula:

C27H20N4O9S2.2Na

IUPAC Name:

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

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): Disodium 4,4’-(2,4-dihydroxy-5- hydroxymethyl-1,3-phenylene bis-azo) di- (naphthalene-1-sulfonate)
- Molecular formula: C27H20N4O9S2.2Na
- Molecular weight: 654.58 g/mol
- Substance type: Organic
- Physical state: Solid

Test animals

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

Administration / exposure

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

Doses / concentrations

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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

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

Effect levels (P0)

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

Results: F1 generation

General toxicity (F1)

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

Effect levels (F1)

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

Overall reproductive toxicity

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

Applicant's summary and conclusion

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.