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

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral

Remarks:

combined repeated dose and carcinogenicity

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 publication

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:
Males: 54-80 g
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 oral exposure:

PREPARATION OF DOSING SOLUTIONS: No data available

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 10000 ppm in diet
- Storage temperature of food: No data available

VEHICLE
- Justification for use and choice of vehicle (if other than water): 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

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

2 years

Frequency of treatment:

Daily

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 mg/kg/day: 48 male, 48 female
25 mg/kg/day: 48 male, 48 female
100 mg/kg/day: 48 male, 48 female
500 mg/kg/day: 48 male, 48 female

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and 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

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At week 14, 27, 55 and 80
- Anaesthetic used for blood collection: Barbiturate anaesthesia was used
- Animals fasted: Overnight
- How many animals: 10 male and 10 female rats and from all the animals at the end of the experiment
- Parameters checked: Haemoglobin content, packed cell volume and counts of erythrocytes and total leucocytes. Reticulocyte and differential white cell counts were carried out in blood samples from control rats and those given 10,000 ppm Chocolate Brown HT in the diet for 14 and 27 weeks only

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After week 104
- Animals fasted: Overnight
- How many animals: From all the surviving animals
- Parameters checked: Serum was analysed for its content of urea, glucose, total protein and albumin and for the activities of glutamic-oxalacetic transaminase, glutamic-pyruvic transaminase and lactic dehydrogenase

URINALYSIS: Yes
- Time schedule for collection of urine: At weeks 14, 27, 56 and 102
- Metabolism cages used for collection of urine: No data
- Animals fasted: 6-h period of water deprivation and over a 4-h period commencing after 16 h without water
- Parameters checked: Specific gravity and volume of urine, appearance, microscopic constituents and content of cells, glucose, ketones, bile salts and blood were examined.

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined: No data
- Battery of functions tested: sensory activity / grip strength / motor activity / other: No data

OTHER:
Organ Weights: Yes
Brain, heart, liver, spleen, kidneys, stomach, small intestine, caecum, adrenals, gonads, pituitary and thyroid were weighed.

Sacrifice and pathology:

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

Other examinations:

No data available

Statistics:

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

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

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

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):

no effects observed

Description (incidence and severity):

No statistically significant differences between treated and control animals observed.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

No statistically significant differences between treated and control animals observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no statistically significant differences between treated and control animals in the terminal haematological studies. Any hematological changes detected were not considered to be treatment related.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No significant differences between treated and control animals observed

Urinalysis findings:

no effects observed

Description (incidence and severity):

No significant differences between treated and control animals observed

Behaviour (functional findings):

not examined

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

A slight reduction in testis weight at 25 mg/Kg day observed. The only statistically significant differences were slightly higher relative spleen and kidney weights in males given diet containing 500 mg/Kg day

Gross pathological findings:

effects observed, treatment-related

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.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There was a wide range of pathological changes, particularly in the liver and kidney although, with the exception of a lower incidence of fatty change in the livers of males given 100 or 500 mg/kg day of the colouring compared with the controls

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Details on results:

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.

ORGAN WEIGHTS: When treated with 10000 ppm, slight increased in relative spleen and kidney weights were observed as compared to control.
When treated with 5000 ppm, slight decrease in testis weight of male rat and slight increase in relative spleen weight were observed in female rat as compared to control.

GROSS PATHOLOGY: The incidence of tumours was low in all groups, the most common tumours being mammary adenomas and fibroadenomas in the female rats, interstitial cell tumours of the testis, and subcutaneous fibromas in both sexes. The incidence of these findings in the treated and control animals did not differ statistically. Very few of the tumours were malignant.
No indication of pigmentation or storage phenomena was observed in any tissues.

HISTOPATHOLOGY: NON-NEOPLASTIC
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.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

urinalysis

water consumption and compound intake

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

The overall mortality rate was not affected by treatment with Chocolate Brown HT. The histopathological changes were those expected in ageing rats, and apart from the lower incidence of fatty change seen in the livers of rats given the 2 higher dietary levels of the colouring, their incidence and severity were similar in both test and control rats.

Applicant's summary and conclusion

Conclusions:

The NOAEL for Chocolate Brown HT (Disodium 4,4'-[[2,4-dihydroxy-5-(hydroxymethyl)-1,3-phenylene]bis(azo)]bisnaphthalene-1-sulphonate ) in male/ female Wistar rats was determined to be 500 mg/Kg/day

Executive summary:

In a Combined repeated dose & carcinogenicity study, the effect of Chocolate Brown HT ( Disodium 4,4'-[[2,4-dihydroxy-5-(hydroxymethyl)-1,3-phenylene]bis(azo)]bisnaphthalene-1-sulphonate) on male and female Wistar rats was studied. The test material was given in the diet for 2 years at a dose level of 0, 25, 100 and 500 mg/Kg/day (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 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.

Therefore, the NOAEL for Chocolate Brown HT in male/ female Wistar rats was determined to be 500 mg/Kg/day.