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

Endpoint summary

Description of key information

Repeated dose toxicity: oral
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.

Repeated dose toxicity: inhalation
In accordance with column 2 of Annex VIII of the REACH regulation, testing by the inhalation route is appropriate if exposure of humans via inhalation is likely taking into account the vapour pressure of the substance. This chemical ha a very low vapour pressure of 1.48E-33 Pascal at 25°C and thus exposure by the inhlation route for this chemical is unlikely.

Repeated dose toxicity: dermal
The chemical disodium 4,4'-[[2,4-dihydroxy-5-(hydroxymethyl)-1,3-phenylene]bis(azo)]bisnaphthalene-1-sulphonate (synonym Brown HT) is widely used as a food color and thus the most likely route of repeated exposure is expected to be through the oral route and not the dermal route. Hence this end point was considered for waiver.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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

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:
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

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

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

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.

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.

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

Critical effects observed:

not specified

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.

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.

Endpoint conclusion

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 a peer reviewed publication

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: inhalation

Data waiving:

exposure considerations

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: dermal

Data waiving:

exposure considerations

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity: oral
No. of studies of Chocolate Brown HT were reviewed for repeated dose toxicity by oral route 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 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.

Further, In a Chronic repeated dose toxicity study, Porton SPF Wistar male and female rat were 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 0.0, 0.5, 1.0 and 2.0% (equivalent to 0, 500, 2000 and 1000 mg/Kg/day) in diet. No effect on survival was observed. Growth retardation, significant decrease body weight and mild degree of renal dysfunction were observed in 1.0 and 2.0 % treated rats. Significant increase were observed in relative brain, adrenals, right and left kidney, spleen and gonads weight of male and female rat as compared to control. In addition, histopathological changes such as brown pigmentation in the Kupffer cells of the liver, the proximal convoluted tubules of the kidney and the lymph nodes especially of the small intestine were observed. The intensity of observed effect was proportional to the dietary level administered and the pigment was only very occasionally present. A no-effect level (NOEL) is therefore established of 500 mg/Kg/day of Chocolate Brown HT in the diet of rats for 12 wk.

Similarly, In a Chronic repeated dose toxicity study, Weanling Carforth Farm male and female rat were 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 0.0, 0.02, 0.2, 0.60, 1.0 and 2.0 % orally. No effect on survival, clinical sign and body weight and body weight gain was observed in treated rats. Increased food consumption and reduced food efficiency were observed in 1.0 and 2.0 % dose group rats. Significant decreased in hemoglobin, red cell count and packed cell volume were observed in 2.0 % treated male rats and changes in Total serum protein and blood urea concentration level of 1.0 and 2.0 % male and female rats. In addition, darker colour of the viscera was observed in 2.0 % treated rats. Therefore, NOEL was considered to be 0.60 % (300 mg/kg/day) when Weanling Carforth Farm male and female rat were treated with Chocolate Brown HT orally for 90 days.

In addition to above similar type of Chronic repeated dose toxicity study was carried out where Tuck TF1 male and female mice 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 0.00, 0.01, 0.1 and 0.5 % (0, 14.3, 140 and 700 mg/kg/day ) orally. No effect was observed on survival, body weight, organ weight and histopathology of treated rat. In addition, increases in Red Blood Cells and decreased in Packed cell volume and Total Leucocytes count of 0.1 and 0.5 % treated mice were observed. Brown colouration of lymph nodes, intestine, ovaries and uterus was observed occasionally at 0.5 % treated mice as compared to control. Therefore, NOAEL was considered to be > 0.1 % (> 140 mg/Kg/day) when Tuck TF1 male and female mice were treated with Chocolate Brown HT orally for 80 weeks.

Further supporting above studies, yet another chronic toxicity study was carried out where Large White pigs were treated with Chocolate Brown HT (Disodium 4,4'-[[2,4-dihydroxy-5-(hydroxymethyl)-1,3-phenylene]bis(azo)]bisnaphthalene-1-sulphonate). The test substance was given in the diet to groups of 3 male and 3 female pigs at dose levels of 0 (control), 5, 20 or 100 mg/kg/day for 13 weeks. On the basis of various observations made, faeces from both male and female pigs on the highest treatment level (100 mg/kg) were very dark brown and occasionally maroon in colour. The dark colour of the faeces indicated either that a proportion of the colour remained unabosrbed or that a coloured metabolite was similarly unabsorbed. The observations of a maroon colour suggest that it was a metabolite that was present. The haemoglobin concentrations were significantly less than the control values in the male pigs but not related to the administration of colours. The present study showed that the daily ingestion of up to 100 mg of Chocolate Brown HT/kg for up to 90 days in pigs produced no detectable adverse effects. Therefore, NOAEL was considered to be 100 mg/kg/day in Large White pigs treated with Chocolate Brown HT orally for 13 weeks.

Based on the studies summarized above it can be observed that NOAEL value varies from 100 to 500 mg/Kg bw/ day. The main effects observed on these doses were as follows:

·  No adverse effect on survival, body weight, food consumption and efficiency, ophthalmoscopy, haematology, clinical chemistry, urinalysis, organ weight, gross pathology and histopathology

·  No adverse effect on clinical sign and body weight.

Since 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 repeated dose via oral route for the above mentioned dose.

Repeated dose toxicity: inhalation
In accordance with column 2 of Annex VIII of the REACH regulation, testing by the inhalation route is appropriate if exposure of humans via inhalation is likely taking into account the vapour pressure of the substance. This chemical ha a very low vapour pressure of 1.48E-33 Pascal at 25°C and thus exposure by the inhlation route for this chemical is unlikely.

Repeated dose toxicity: dermal
The chemical disodium 4,4'-[[2,4-dihydroxy-5-(hydroxymethyl)-1,3-phenylene]bis(azo)]bisnaphthalene-1-sulphonate (synonym Brown HT) is widely used as a food color and thus the most likely route of repeated exposure is expected to be through the oral route and not the dermal route. Hence this end point was considered for waiver.

Justification for classification or non-classification

The available studies from reliable sources indicate that the test substance is not likely to be toxic by repeaded exposure by the oral route.