Dihydrogen (ethyl)[4-[[4-[ethyl(3-sulphonatobenzyl)amino]phenyl](2-sulphonatophenyl)methylene]cyclohexa-2,5-dien-1-ylidene](3-sulphonatobenzyl)ammonium, aluminium salt

Endpoint summary

Description of key information

Repeated dose toxicity: Oral

Based on the data available, the No Observed Adverse Effect Level (NOAEL) for the test chemical was considered to be in range of 500 -2500 mg/Kg bw using male and female rats or mice . Hence the test chemical is not likely to be toxic upon repeated exposure by oral route as per the criteria mentioned in CLP regulation.

Repeated dose toxicity: inhalation

The melting point of test chemical is >300 °C.This suggests that test chemical decomposes above300 °C without melting and also it does not exhibit very high vapour pressure. Inhalation is therefore not the likely route of exposure and hence this end point is considered for waiver.

Repeated dose toxicity: dermal

Repeated dermal toxicity is unlikely to occur since dermal absorption of test chemical is limited. Also considering the use of the chemical as a colouring agent in food and cosmetics and considering the volatility absorption by the dermal route is not considered to be significant. Thus, given the above considerations, it is assumed that test chemical shall not exhibit acute dose toxicity by the repeated dermal route.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental data from various test chemicals

Justification for type of information:

Weight of evidence approach based on the available information from various test chemicals.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

WoE report is based on repeated dose toxicity studies on rats

GLP compliance:

not specified

Limit test:

no

Species:

other: 2.rat 3.mouse

Strain:

other: 2.Osborne-Mendel 3.ASH-CS1 strain

Details on species / strain selection:

No data

Sex:

male/female

Details on test animals or test system and environmental conditions:

2.TEST ANIMALS
- Source: No data
- Age at study initiation: No data
- Weight at study initiation: No data
- Fasting period before study: No data
- Housing: Animals were housed individually
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: No data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data
- Humidity (%): No data
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): No data
3.TEST ANIMALS
- Source: SPF colony
- Age at study initiation: No data
- Weight at study initiation: No data
- Fasting period before study: No data
- Housing: The animals were housed in cages with solid floors. The female mice were caged in groups of five and the male mice individually
- Diet (e.g. ad libitum): reground Oxoid pasteurized breeding diet supplemented with 40 ppm sodium menadione bisulphite ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: No data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 1 deg C
- Humidity (%): 50-60%
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): No data

IN-LIFE DATES: From: To: No data

Route of administration:

oral: feed

Details on route of administration:

No data

Vehicle:

other: Feed

Details on oral exposure:

2.PREPARATION OF DOSING SOLUTIONS: The test compound is mixed with feed at dose levels of 0, 0.5, 1.0, 2.0 or 5.0 % (0, 250, 500, 1000 or 2500 mg/Kg bw/day)

DIET PREPARATION
- Rate of preparation of diet (frequency): No data
- Mixing appropriate amounts with (Type of food): No data
- Storage temperature of food: No data

VEHICLE
- Justification for use and choice of vehicle (if other than water): Feed
- Concentration in vehicle: 0, 0.5, 1.0, 2.0 or 5.0 % (0, 250, 500, 1000 or 2500 mg/Kg bw/day)
- Amount of vehicle (if gavage): No data
- Lot/batch no. (if required): No data
- Purity: No data
3.mg/Kg/day)

DIET PREPARATION
- Rate of preparation of diet (frequency): No data
- Mixing appropriate amounts with (Type of food): Reground Oxoid pasteurized breeding diet supplemented with 40 ppm sodium menadione bisulphite
- Storage temperature of food: No data

VEHICLE
- Justification for use and choice of vehicle (if other than water): Reground Oxoid pasteurized breeding diet supplemented with 40 ppm sodium menadione bisulphite
- Concentration in vehicle: 0, 70, 700 or 3500 ppm (0, 10, 100 or 500 mg/Kg/day)
- Amount of vehicle (if gavage): No data
- Lot/batch no. (if required): No data
- Purity: No data

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No data

Duration of treatment / exposure:

2.
2 years
3.80 weeks

Frequency of treatment:

Daily

Remarks:

Study 2.
0, 0.5, 1.0, 2.0 or 5.0 % (0, 250, 500, 1000 or 2500 mg/Kg bw/day)
Study 3.
0, 70, 700 or 3500 ppm (0, 10, 100 or 500 mg/Kg/day)

No. of animals per sex per dose:

2.Total -240 male and females
0.0%(0 mg/kg bw/day) -24 male & 24 Female
0.5 %(250 mg/kg bw/day) -24 male & 24Female
1.0%(500 mg/kg bw/day) -24 male & 24 Female
2.0 %(1000 mg/kg bw/day) -24 male & 24 Female
5.0%(2500 mg/kg bw/day) -24 male & 24 Female
3.0 mg/Kg/day: 48 males and 50 females
10 mg/Kg/day: 48 males and 50 females
100 mg/Kg/day: 48 males and 50 females
500 mg/Kg/day: 48 males and 50 females

Control animals:

yes, concurrent vehicle

Details on study design:

No data

Positive control:

No data

Observations and examinations performed and frequency:

2.CAGE SIDE OBSERVATIONS: Yes
- Time schedule: During the 2 yr study
- Cage side observations checked in table [No.?] were included. Mortality and abnormality

DETAILED CLINICAL OBSERVATIONS: No data
- Time schedule: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- 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
3.CAGE SIDE OBSERVATIONS: Yes
- Time schedule: During the study period
- Cage side observations checked in table [No.?] were included. mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: No data

BODY WEIGHT: Yes
- Time schedule for examinations: At intervals throughout the experiment

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- 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): No data
- Time schedule for examinations: No data
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once during the F0 generation, at the start of the lifetime study, and after 3, 6, 12, 18 and 24 months of the chronic phase
- Dose groups that were examined: All rats

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 12, 28, 52 wk and 80 wk
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 10 animals from 100 and 500 mg/Kg/day
- Parameters checked in table [No.?] were examined. Counts were made of erythrocytes, reticulocytes and total and differential leucocytes, and any abnormalities in either red or white cell morphology, hemoglobin and packed cell volume noted. At 80 wk, blood samples taken from the
caudal vein of all surviving mice were examined for haemoglobin and differential leucocyte counts only.

CLINICAL CHEMISTRY: No data
- Time schedule for collection of blood: No data
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.?] were examined. No data

URINALYSIS: No data
- Time schedule for collection of urine: No data
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked in table [No.?] were examined. No data

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

OTHER: No data
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations: No data

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 3-, 11-, 17-, and 22-month intervals.
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 10 animals
- Parameters checked in table [No.?] were examined. white blood cell count, hemoglobin, hematocrit, and differential cell count

CLINICAL CHEMISTRY: No data
- Time schedule for collection of blood: No data
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.?] were examined. No data

URINALYSIS: No data
- Time schedule for collection of urine: No data
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked in table [No.?] were examined. No data

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

OTHER: No data

Sacrifice and pathology:

2.GROSS PATHOLOGY: Yes, Survivors were autopsied and organ weights were recorded for heart, liver, spleen, kidney, and testes. Animals that died during the experiment were autopsied, but their organ weights were not considered in the organ weight calculations.
3.GROSS PATHOLOGY: Yes, autopsy was conducted on all animals dying or killed during the experiment unless this was precluded by advanced autolysis or cannibalism. The surviving animals were killed by exsanguination from the aorta under barbiturate anaesthesia after 80 wk and all organs were examined for gross abnormalities. The weights of brain, heart, liver, spleen, kidneys, stomach and small intestine were recorded and samples of these organs, together with samples of salivary gland, thyroid, thymus, adrenals, lymph nodes, pancreas, pituitary, ovaries, uterus, urinary bladder, lungs, testes, duodenum, ileum, colon, caecum, rectum,
spinal cord, skeletal muscle and any other tissue that appeared to be abnormal at autopsy, were preserved in 10% buffered formalin.

HISTOPATHOLOGY: Yes, Paraffin-wax sections of brain, heart, liver, spleen, kidneys, stomach and small intestine, salivary gland, thyroid, thymus, adrenals, lymph nodes, pancreas, pituitary, ovaries, uterus, urinary bladder, lungs, testes, duodenum, ileum, colon, caecum, rectum, spinal cord, skeletal muscle and any other tissue that appeared to be abnormal at autopsy were stained with haematoxylin and eosin and examined for any histopathological changes.


At the end of the two-year feeding study, 225 of the 240 animals started on the experiment were examined for gross pathology.

HISTOPATHOLOGY: Yes, At the end of the two-year feeding study, 225 of the 240 animals started on the experiment were examined for microscopic pathology.

Detailed microscopic examination of lung, heart, stomach, liver, spleen small intestine, colon, bone marrow,leg bone and muscle, urinary bladder, kidney, pancreas, thyroid ( (parathyroid when present), adrenal, and prostate and testis or ovary and uterus was conducted on the formalin-fixed, haematoxylin - eosin stained paraffin embedded tissues of 21 rats from the Blue no. 1. A more limited group of tissues (kidney, liver, testis, and spleen) was examined from 43 rats.

Other examinations:

No data

Statistics:

No data

Clinical signs:

no effects observed

Description (incidence and severity):

3.The ingestion of test chemical produced no adverse effects on the behaviour of the animals over 80 wk. The faeces of the treated mice were violet-coloured, the intensity of this colouring being proportional to the dose, but the urine did not appear to be coloured. The fur of the treated mice at the highest dietary level was coloured as a result of contamination by the diet and faeces.

Mortality:

no mortality observed

Description (incidence):

2.No effect was observed on mortality of the animals at dietary levels up to 5% (2500 mg/Kg bw/day).
3.No treatment related mortality was noted

Body weight and weight changes:

no effects observed

Description (incidence and severity):

2.No significant growth inhibition occurred with the animals fed test chemical
3.No treatment related body weight changes were noted

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:

effects observed, treatment-related

Description (incidence and severity):

2.No effect was observed on of animals at dietary levels up to 5% (2500 mg/Kg bw/day).
3.3.There were significant increases in total white cells in female mice receiving test chemical at dietary levels of 700 or 3500 ppm at wk 12. This appeared to be an isolated effect and could not be demonstrated after 28 or 52 wk. However, at wk 52 there was a statistically significant increase in total white cells and erythrocytes in male mice fed Violet 6B at 3500 ppm of the diet and in the packed cell volume of the females fed test chemical at 700 ppm of the diet.

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:

no effects observed

Description (incidence and severity):

2.No effects was observed on weights of heart, liver, kidney, spleen, and testis of the animals that received Blue No. 1 at dietary levels up to 5% (2500 mg/Kg bw/day).
3.The only statistically significant changes of organ weight were in the small intestine of the male mice. Those given diets containing 100 or 500 mg/Kg/day were lower, by 10 and 12% respectively, than the controls. This effect was not statistically significant when the weights were expressed relative to body weight.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

2.There was no treatment-related change in gross pathology. The usual gross pathologic conditions of old rats were seen in all groups, with no significant difference in distribution. The most common lesions were tumors, pneumonia (mostly chronic) of varying degrees? chronic nephritis of the usual type seen in our older rats, and testicular atrophy.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

2.There was no treatment-related change in microscopical examination. The most common tumors were mammary adenocarcinomas, fibroadenomas, and pulmonary lymphosarcomas.

In addition to the above mentioned common tumors, the Blue No. 1 animals showed the following tumors: at the 2500 mg/Kg bw/day level, one animal each had a hepatoma, a papillary colloid adenoma of the thyroid, and a carcinoma of the prostate; one animal listed with a mammary adenocarcinoma also had a subcutaneous fibroma. At the 1000 mg/kg bw/day level, one animal each had an adenocarcinoma of the kidney, a liposarcoma of the posterior mediastinum, and an extensive lymphoblastoma of the abdominal serosa and lymph nodes. At the 500 mg/kg bw/day level, one animal each had an adrenal cortical adenoma and a subcutaneous malignant angioma. At the 250 mg/Kg bw/day level, one animal each had a subcutaneous lipoma and a mammary carcinoma. In the control group, one animal had a cecal adenoma. The common incidental lesions were pneumonia (mostly chronic) of varying degrees and chronic nephritis of the usual type seen in our older rats There was a moderate degree of testicular atrophy in one of the rats in the 1000 mg/Kg bw/day group, and there was marked atrophy in one of the controls; remaining sections of testis showed no more than slight atrophy. Other major incidental lesions included an ulcerated inflammatory mass in the cecum of a rat on the 2500 mg/Kg bw/day diet, and three large, chronic, tubo-ovarian abscesses( one each on the 1000 and 250 mg/Kg bw/day diets, and control groups)
3.Most of the lesions were those normally occurring in ageing mice and were found with a similar incidence in treated and control mice. None of the findings appeared to be related to the treatment with test chemical . The changes found in the skin consisted of hyperkeratosis, also present in control animals, and a single dermoid cyst in a female given 700 ppm test chemical . Dilated gastric glands and hyperplasia of the gastric mucosa were encountered, but again similar lesions were found in control mice.

Pulmonary adenomas were seen in 36% of all male mice and in 20% of all female mice, the incidence being similar in all groups. Two thyroid adenomas were found in male mice fed Violet 6B (one at 10 and the other at 500 mg/Kg/day). Benign tumours of the ovary, testis, subcutaneous tissue and kidney were found with a similar incidence in control and treated animals. Pulmonary adenocarcinomas were present in two male mice fed diets containing Violet 6B (one at 100 and one at 500 mg/Kg/day). Lymphosarcoma and reticulum-cell neoplasia had a similar incidence in mice fed Violet 6B and in the controls. Three squamous-cell carcinomas
of the skin were seen in female mice (one control, one fed 10 mg/Kg/day and one fed 100 mg/Kg/day Violet 6B). The other malignant tumours found were a fibrosarcoma in a control female, a rhabdomyosarcoma in a female given 10 mg/Kg/day Violet 6B and a mammary adenocarcinoma in a female given 100 mg/Kg/day Violet 6B.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Details on results:

No data

Dose descriptor:

NOAEL

Effect level:

> 500 - <= 2 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No significant tretament related alterations were noted at the mentioned dose level

Remarks on result:

other: No toxic effects were observed

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Table: Incidence of major tumors in rats fed test chemical

Tumor	Dietary level
	Controls	250	500	1000	2500
Mammary adenocarcinoma	2	2	2	-	1
Mammary fibroadenoma 3 2 3 3 1 2 5 2 1 4	3	3	1	5	1
Pulmonary lymphosarcoma	1	2	1	-	1
Endometrial sarcoma	-	1	-	-	-
Rats with tumors	7	10	7	9	6

 

Conclusions:

The No observed Adverse Effect level (NOAEL) for test chemical was considered to be in range of 500-2500 mg/kg bw/day in male and female rats or mice for 2 years feeding study.

Executive summary:

Data available for the test chemical was reviewed to determine the toxic nature of test chemical .The studies are as mentioned below:

Study 2.

Chronic repeated dose oral toxicity study was performed for test chemical to determine its toxic nature upon repeated exposure. The test chemical was mixed with feed at dose levels of0, 0.5, 1.0, 2.0 or 5.0 % (0, 250, 500, 1000 or 2500 mg/Kg bw/day) and fed for 2 years to 24 male and 24 female/dose Osborne Mendel weanling rats. During the study period, the animals were observed for mortality, any abnormality, body weight changes, hematology and were subjected to gross and microscopic pathology. No mortality and abnormality was noted during the study period. No significant growth inhibition occurred with the animals fed with test chemical . Also, no effects was observed on hemotological parameters and weights of heart, liver, kidney, spleen, and testis of the animals that received test chemical at dietary levels up to 5%(2500 mg/Kg bw/day). There was no treatment-related change in gross pathology. The usual gross pathologic conditions of old rats were seen in all groups, with no significant difference in distribution. The most common lesions were tumors, pneumonia (mostly chronic) of varying degrees? chronic nephritis of the usual type seen in our older rats, and testicular atrophy. There was no treatment-related change in microscopical examination. The most common tumors were mammary adenocarcinomas, fibroadenomas, and pulmonary lymphosarcomas. Based on the above considerations, the No observed Adverse Effect level (NOAEL) for test chemical was considered to be 2500 mg/kg bw/day in Osborne-Mendel male and female rats for 2 years feeding study.

Study 3.

Chronic repeated dose toxicity study was performed to determine the oral toxic nature of test chemical using ASH-CSI male and female mice. The test chemical was mixed with appropriate quantity of corn oil and mixed with feed at dose levels of 0, 10, 100 or 500 mg/kg/day. The animals were the test chemical in diet for 80 weeks and during the study the animals were observed for clinical signs, mortality, changes in body weight, hematology and the animals were subjected to gross and histopathology. The faeces of all treated mice were violet-coloured but the urine appeared normal, suggesting that the dose was largely unabsorbed from the gastro-intestinal tract. The feeding of Violet 6B did not affect the mortality, the rate of body-weight gain or the haematological picture at 12, 28, 52 or 80 wk. The only statistically significant changes of organ weight were in the small intestine of the male mice. Those given diets containing 100 or 500 mg/Kg/day were lower, by 10 and 12% respectively, than the controls. This effect was not statistically significant when the weights were expressed relative to body weight. Most of the lesions were those normally occurring in ageing mice and were found with a similar incidence in treated and control mice and appeared to be not related to the treatment with test chemical . The changes found in the skin consisted of hyperkeratosis, also present in control animals, and a single dermoid cyst in a female given 700 ppm test chemical Dilated gastric glands and hyperplasia of the gastric mucosa were encountered, but again similar lesions were found in control mice. Pulmonary adenomas were seen in 36% of all male mice and in 20% of all female mice, the incidence being similar in all groups. Two thyroid adenomas were found in male mice fed test chemical (one at 10 and the other at 500 mg/Kg/day). Benign tumours of the ovary, testis, subcutaneous tissue and kidney were found with a similar incidence in control and treated animals. Pulmonary adenocarcinomas were present in two male mice fed diets containing test chemical (one at 100 and one at 500 mg/Kg/day). Lymphosarcoma and reticulum-cell neoplasia had a similar incidence in mice fed test chemical and in the controls. Three squamous-cell carcinomas of the skin were seen in female mice (one control, one fed 10 mg/Kg/day and one fed 100 mg/Kg/day ). The other malignant tumours found were a fibrosarcoma in a control female, a rhabdomyosarcoma in a female given 10 mg/Kg/day test chemical and a mammary adenocarcinoma in a female given 100 mg/Kg/day of test chemical . Since no deviation was noted in the treated animals from the controls used, hence, the the no observed adverse effect level (NOAEL) for test chemical was considered to be 500 mg/Kg/day.

Reference 2

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

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 reviwed publication

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

Chronic repeated dose oral toxicity study was performed for test chemical to determine its toxic nature upon repeated exposure

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

- Name of test material: Brilliant Blue FCF/ Blue no. 1
- Molecular formula: C37H36N2O9S3.2Na
- Molecular weight: 792.8586 g/mol
- Substance type: Organic
- Physical state: No data
- Impurities (identity and concentrations): No data

Species:

rat

Strain:

Osborne-Mendel

Details on species / strain selection:

No data

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: No data
- Age at study initiation: No data
- Weight at study initiation: No data
- Fasting period before study: No data
- Housing: Animals were housed individually
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: No data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data
- Humidity (%): No data
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): No data

IN-LIFE DATES: From: To: No data

Route of administration:

oral: feed

Details on route of administration:

No data

Vehicle:

other: Feed

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test compound is mixed with feed at dose levels of 0, 0.5, 1.0, 2.0 or 5.0 % (0, 250, 500, 1000 or 2500 mg/Kg bw/day)

DIET PREPARATION
- Rate of preparation of diet (frequency): No data
- Mixing appropriate amounts with (Type of food): No data
- Storage temperature of food: No data

VEHICLE
- Justification for use and choice of vehicle (if other than water): Feed
- Concentration in vehicle: 0, 0.5, 1.0, 2.0 or 5.0 % (0, 250, 500, 1000 or 2500 mg/Kg bw/day)
- Amount of vehicle (if gavage): No data
- Lot/batch no. (if required): No data
- Purity: No data

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No data

Duration of treatment / exposure:

2 years

Frequency of treatment:

Daily

Remarks:

0, 0.5, 1.0, 2.0 or 5.0 % (0, 250, 500, 1000 or 2500 mg/Kg bw/day)

No. of animals per sex per dose:

Total -240 male and females
0.0%(0 mg/kg bw/day) -24 male & 24 Female
0.5 %(250 mg/kg bw/day) -24 male & 24Female
1.0%(500 mg/kg bw/day) -24 male & 24 Female
2.0 %(1000 mg/kg bw/day) -24 male & 24 Female
5.0%(2500 mg/kg bw/day) -24 male & 24 Female

Control animals:

yes, concurrent vehicle

Details on study design:

No data

Positive control:

No data

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: During the 2 yr study
- Cage side observations checked in table [No.?] were included. Mortality and abnormality

DETAILED CLINICAL OBSERVATIONS: No data
- Time schedule: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- 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): No data
- Time schedule for examinations: No data

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 3-, 11-, 17-, and 22-month intervals.
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 10 animals
- Parameters checked in table [No.?] were examined. white blood cell count, hemoglobin, hematocrit, and differential cell count

CLINICAL CHEMISTRY: No data
- Time schedule for collection of blood: No data
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.?] were examined. No data

URINALYSIS: No data
- Time schedule for collection of urine: No data
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked in table [No.?] were examined. No data

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

OTHER: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, Survivors were autopsied and organ weights were recorded for heart, liver, spleen, kidney, and testes. Animals that died during the experiment were autopsied, but their organ weights were not considered in the organ weight calculations.


At the end of the two-year feeding study, 225 of the 240 animals started on the experiment were examined for gross pathology.

HISTOPATHOLOGY: Yes, At the end of the two-year feeding study, 225 of the 240 animals started on the experiment were examined for microscopic pathology.

Detailed microscopic examination of lung, heart, stomach, liver, spleen small intestine, colon, bone marrow,leg bone and muscle, urinary bladder, kidney, pancreas, thyroid ( (parathyroid when present), adrenal, and prostate and testis or ovary and uterus was conducted on the formalin-fixed, haematoxylin - eosin stained paraffin embedded tissues of 21 rats from the Blue no. 1. A more limited group of tissues (kidney, liver, testis, and spleen) was examined from 43 rats.

Other examinations:

No data

Statistics:

No data

Clinical signs:

not specified

Mortality:

no mortality observed

Description (incidence):

No effect was observed on mortality of the animals at dietary levels up to 5% (2500 mg/Kg bw/day).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant growth inhibition occurred with the animals fed test chemical

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:

no effects observed

Description (incidence and severity):

No effect was observed on of animals at dietary levels up to 5% (2500 mg/Kg bw/day).

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:

no effects observed

Description (incidence and severity):

No effects was observed on weights of heart, liver, kidney, spleen, and testis of the animals that received Blue No. 1 at dietary levels up to 5% (2500 mg/Kg bw/day).

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There was no treatment-related change in gross pathology. The usual gross pathologic conditions of old rats were seen in all groups, with no significant difference in distribution. The most common lesions were tumors, pneumonia (mostly chronic) of varying degrees? chronic nephritis of the usual type seen in our older rats, and testicular atrophy.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There was no treatment-related change in microscopical examination. The most common tumors were mammary adenocarcinomas, fibroadenomas, and pulmonary lymphosarcomas.

In addition to the above mentioned common tumors, the Blue No. 1 animals showed the following tumors: at the 2500 mg/Kg bw/day level, one animal each had a hepatoma, a papillary colloid adenoma of the thyroid, and a carcinoma of the prostate; one animal listed with a mammary adenocarcinoma also had a subcutaneous fibroma. At the 1000 mg/kg bw/day level, one animal each had an adenocarcinoma of the kidney, a liposarcoma of the posterior mediastinum, and an extensive lymphoblastoma of the abdominal serosa and lymph nodes. At the 500 mg/kg bw/day level, one animal each had an adrenal cortical adenoma and a subcutaneous malignant angioma. At the 250 mg/Kg bw/day level, one animal each had a subcutaneous lipoma and a mammary carcinoma. In the control group, one animal had a cecal adenoma. The common incidental lesions were pneumonia (mostly chronic) of varying degrees and chronic nephritis of the usual type seen in our older rats There was a moderate degree of testicular atrophy in one of the rats in the 1000 mg/Kg bw/day group, and there was marked atrophy in one of the controls; remaining sections of testis showed no more than slight atrophy. Other major incidental lesions included an ulcerated inflammatory mass in the cecum of a rat on the 2500 mg/Kg bw/day diet, and three large, chronic, tubo-ovarian abscesses( one each on the 1000 and 250 mg/Kg bw/day diets, and control groups)

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Details on results:

No data

Dose descriptor:

NOAEL

Effect level:

2 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No significant tretament related alterations were noted at the mentioned dose level

Remarks on result:

other: No toxic effects were observed

Critical effects observed:

no

Table: Incidence of major tumors in rats fed test chemical

Tumor	Dietary level
	Controls	250	500	1000	2500
Mammary adenocarcinoma	2	2	2	-	1
Mammary fibroadenoma 3 2 3 3 1 2 5 2 1 4	3	3	1	5	1
Pulmonary lymphosarcoma	1	2	1	-	1
Endometrial sarcoma	-	1	-	-	-
Rats with tumors	7	10	7	9	6

 

Conclusions:

Based on all the available data, it was concluded that the No observed Adverse Effect level (NOAEL) for test chemical was considered to be 2500 mg/kg bw/day in Osborne-Mendel male and female rats for 2 years feeding study.

Executive summary:

Chronic repeated dose oral toxicity study was performed for test chemical to determine its toxic nature upon repeated exposure. The test chemical was mixed with feed at dose levels of0, 0.5, 1.0, 2.0 or 5.0 % (0, 250, 500, 1000 or 2500 mg/Kg bw/day) and fed for 2 years to 24 male and 24 female/dose Osborne Mendel weanling rats. During the study period, the animals were observed for mortality, any abnormality, body weight changes, hematology and were subjected to gross and microscopic pathology. No mortality and abnormality was noted during the study period. No significant growth inhibition occurred with the animals fed with test chemical . Also, no effects was observed on hemotological parameters and weights of heart, liver, kidney, spleen, and testis of the animals that received test chemical at dietary levels up to 5%(2500 mg/Kg bw/day). There was no treatment-related change in gross pathology. The usual gross pathologic conditions of old rats were seen in all groups, with no significant difference in distribution. The most common lesions were tumors, pneumonia (mostly chronic) of varying degrees? chronic nephritis of the usual type seen in our older rats, and testicular atrophy. There was no treatment-related change in microscopical examination. The most common tumors were mammary adenocarcinomas, fibroadenomas, and pulmonary lymphosarcomas. Based on the above considerations, the No observed Adverse Effect level (NOAEL) for test chemical was considered to be 2500 mg/kg bw/day in Osborne-Mendel male and female rats for 2 years feeding study.

Reference 3

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

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 publication

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Principles of method if other than guideline:

Equivalent or similar to OECD Guideline 452 (Chronic Toxicity Studies)

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

- Name of test material : Violet 6B (FD & C Violet No. 1)
- Molecular formula : C39H41N3O6S2.Na
- Molecular weight : 734.89 g/mol
- Substance type: Organic
- Physical state: No data
- Impurities: pure dye (by titration with titanium trichloride), min. 85~o; matter volatile at 135°C, max 8%; matter insoluble in water, max 0.3%; ether-extractable matter, max 0.4%; p-dimethyl aminobenzoic acid, max 0.2%; chloride and sulphates of sodium, max 4.0% mixed oxides, max 1.0%; the sum of volatile matter, chloride and sulphates of sodium, pure dye and leuco base of the dye, rain. 95%.

Species:

mouse

Strain:

other: ASH-CS1 strain

Details on species / strain selection:

No data

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SPF colony
- Age at study initiation: No data
- Weight at study initiation: No data
- Fasting period before study: No data
- Housing: The animals were housed in cages with solid floors. The female mice were caged in groups of five and the male mice individually
- Diet (e.g. ad libitum): reground Oxoid pasteurized breeding diet supplemented with 40 ppm sodium menadione bisulphite ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: No data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 1 deg C
- Humidity (%): 50-60%
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): No data

IN-LIFE DATES: From: To: No data

Route of administration:

oral: feed

Details on route of administration:

No data

Vehicle:

other: Feed (Reground Oxoid pasteurized breeding diet supplemented with 40 ppm sodium menadione bisulphite)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test chemical was mixed with feed at dose levels of 0, 70, 700 or 3500 ppm (0, 10, 100 or 500 mg/Kg/day)

DIET PREPARATION
- Rate of preparation of diet (frequency): No data
- Mixing appropriate amounts with (Type of food): Reground Oxoid pasteurized breeding diet supplemented with 40 ppm sodium menadione bisulphite
- Storage temperature of food: No data

VEHICLE
- Justification for use and choice of vehicle (if other than water): Reground Oxoid pasteurized breeding diet supplemented with 40 ppm sodium menadione bisulphite
- Concentration in vehicle: 0, 70, 700 or 3500 ppm (0, 10, 100 or 500 mg/Kg/day)
- Amount of vehicle (if gavage): No data
- Lot/batch no. (if required): No data
- Purity: No data

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No data

Duration of treatment / exposure:

80 weeks

Frequency of treatment:

Daily

Remarks:

0, 70, 700 or 3500 ppm (0, 10, 100 or 500 mg/Kg/day)

No. of animals per sex per dose:

0 mg/Kg/day: 48 males and 50 females
10 mg/Kg/day: 48 males and 50 females
100 mg/Kg/day: 48 males and 50 females
500 mg/Kg/day: 48 males and 50 females

Control animals:

yes, concurrent vehicle

Details on study design:

No data

Positive control:

No data

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: During the study period
- Cage side observations checked in table [No.?] were included. mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: No data

BODY WEIGHT: Yes
- Time schedule for examinations: At intervals throughout the experiment

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- 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): No data
- Time schedule for examinations: No data
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once during the F0 generation, at the start of the lifetime study, and after 3, 6, 12, 18 and 24 months of the chronic phase
- Dose groups that were examined: All rats

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 12, 28, 52 wk and 80 wk
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 10 animals from 100 and 500 mg/Kg/day
- Parameters checked in table [No.?] were examined. Counts were made of erythrocytes, reticulocytes and total and differential leucocytes, and any abnormalities in either red or white cell morphology, hemoglobin and packed cell volume noted. At 80 wk, blood samples taken from the
caudal vein of all surviving mice were examined for haemoglobin and differential leucocyte counts only.

CLINICAL CHEMISTRY: No data
- Time schedule for collection of blood: No data
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.?] were examined. No data

URINALYSIS: No data
- Time schedule for collection of urine: No data
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked in table [No.?] were examined. No data

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

OTHER: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, autopsy was conducted on all animals dying or killed during the experiment unless this was precluded by advanced autolysis or cannibalism. The surviving animals were killed by exsanguination from the aorta under barbiturate anaesthesia after 80 wk and all organs were examined for gross abnormalities. The weights of brain, heart, liver, spleen, kidneys, stomach and small intestine were recorded and samples of these organs, together with samples of salivary gland, thyroid, thymus, adrenals, lymph nodes, pancreas, pituitary, ovaries, uterus, urinary bladder, lungs, testes, duodenum, ileum, colon, caecum, rectum,
spinal cord, skeletal muscle and any other tissue that appeared to be abnormal at autopsy, were preserved in 10% buffered formalin.

HISTOPATHOLOGY: Yes, Paraffin-wax sections of brain, heart, liver, spleen, kidneys, stomach and small intestine, salivary gland, thyroid, thymus, adrenals, lymph nodes, pancreas, pituitary, ovaries, uterus, urinary bladder, lungs, testes, duodenum, ileum, colon, caecum, rectum, spinal cord, skeletal muscle and any other tissue that appeared to be abnormal at autopsy were stained with haematoxylin and eosin and examined for any histopathological changes.

Other examinations:

No data

Statistics:

No data

Clinical signs:

no effects observed

Description (incidence and severity):

The ingestion of test chemical produced no adverse effects on the behaviour of the animals over 80 wk. The faeces of the treated mice were violet-coloured, the intensity of this colouring being proportional to the dose, but the urine did not appear to be coloured. The fur of the treated mice at the highest dietary level was coloured as a result of contamination by the diet and faeces.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No treatment related mortality was noted

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related body weight changes were noted

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:

effects observed, treatment-related

Description (incidence and severity):

There were significant increases in total white cells in female mice receiving test chemical at dietary levels of 700 or 3500 ppm at wk 12. This appeared to be an isolated effect and could not be demonstrated after 28 or 52 wk. However, at wk 52 there was a statistically significant increase in total white cells and erythrocytes in male mice fed Violet 6B at 3500 ppm of the diet and in the packed cell volume of the females fed test chemical at 700 ppm of the diet.

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:

no effects observed

Description (incidence and severity):

The only statistically significant changes of organ weight were in the small intestine of the male mice. Those given diets containing 100 or 500 mg/Kg/day were lower, by 10 and 12% respectively, than the controls. This effect was not statistically significant when the weights were expressed relative to body weight.

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Most of the lesions were those normally occurring in ageing mice and were found with a similar incidence in treated and control mice. None of the findings appeared to be related to the treatment with test chemical . The changes found in the skin consisted of hyperkeratosis, also present in control animals, and a single dermoid cyst in a female given 700 ppm test chemical . Dilated gastric glands and hyperplasia of the gastric mucosa were encountered, but again similar lesions were found in control mice.

Pulmonary adenomas were seen in 36% of all male mice and in 20% of all female mice, the incidence being similar in all groups. Two thyroid adenomas were found in male mice fed Violet 6B (one at 10 and the other at 500 mg/Kg/day). Benign tumours of the ovary, testis, subcutaneous tissue and kidney were found with a similar incidence in control and treated animals. Pulmonary adenocarcinomas were present in two male mice fed diets containing Violet 6B (one at 100 and one at 500 mg/Kg/day). Lymphosarcoma and reticulum-cell neoplasia had a similar incidence in mice fed Violet 6B and in the controls. Three squamous-cell carcinomas
of the skin were seen in female mice (one control, one fed 10 mg/Kg/day and one fed 100 mg/Kg/day Violet 6B). The other malignant tumours found were a fibrosarcoma in a control female, a rhabdomyosarcoma in a female given 10 mg/Kg/day Violet 6B and a mammary adenocarcinoma in a female given 100 mg/Kg/day Violet 6B.

Other effects:

not specified

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No significant treatment related effects were noted at the mentioned dose level

Remarks on result:

other: No toxic effects were observed

Critical effects observed:

no

Table 1. Cumulative mortality of mice fedtest chemical at 0-3500 ppm of the diet for 80 wk

Week no	Total no. of deaths
	Males				Females
	0	70	700	3500	0	70	700	3500
10	0	0	0	0	0	1	0	0
20	0	3	0	0	0	1	1	1
30	0	3	0	0	0	1	1	1
40	1	3	0	0	0	1	3	1
50	1	4	1	0	0	3	4	4
60	3	6	2	1	4	7	7	5
70	9	10	4	4	9	11	10	9
80	12	12	10	9	17	18	16	16

 

Table 2. Mean body weights of mice fed Violet 6 B at 0-3500 ppm in the diet for 80 wk

Dose (ppm)	Body weight (g) at week						Weight gain (g) at wk 77
	0	13	25	51	65	77
	Males
0	28.1	34.0	34.3	47.6	42.6	43.0	14.9
70	27.8	33.7	35.2	45.2	42.1	42.0	14.2
700	26.3	31.8	32.0	43.3	41.2	40.6	14.3
3500	27.2	31.9	32.4	44.3	42.5	40.8	13.6
	Females
0	21.7	27.5	28.1	37.7	36.3	34.3	12.6
70	22.3	27.9	28.1	36.9	36.0	33.9	11.6
700	21.5	27.8	29.0	37.1	36.0	34.1	12.6
3500	21.7	28.4	28.4	37.0	36.0	35.9	14.2

 

Table 3. Absolute and relative organ weights of mice fed Violet 6B at 0-3500 ppm of the diet for 80 wk

Sex and dose (99m)	No. of mice examined	Organ weight							Terminal body weight (g)
		Brain	Heart	Liver	Spleen	Kidney	Stomach	Small intestine
	Absolute organ weight (g)
Males
0	35	0.44	0.23	1.84	0.13	0.68	0.48	1.87	36
70	36	0.45	0.23	1.78	0.15	0.68	0.46	1.81	36
700	35	0.44	0.22	1.73	0.16	0.64	0.42	1.69*	36
3500	38	0.44	0.22	1.73	0.14	0.65	0.43	1.65**	36
Females
0	30	0.45	0.18	1.66	0.17	0.49	0.52	1.80	30
70	29	0.44	0.17	1.72	0.17	0.50	0.49	1.68	30
700	27	0.48	0.18	1.68	0.21	0.51	0.47	1.78	31
3500	30	0.46	0.18	1.70	0.17	0.48	0.46	1.71	31
	Relative organ weight (g/100 g body weight)
Males
0	35	1.30	0.64	4.92	0.36	1.84	1.28	5.00	-
70	36	1.22	0.61	4.79	0.44	1.83	1.19	4.74	-
700	35	1.22	0.61	4.79	0.44	1.83	1.19	4.74	-
3500	38	1.22	0.61	4.94	0.39	1.88	1.17	4.65	-
Females
0	30	1.50	0.60	5.51	0.57	1.63	1.66	6.02	-
70	29	1.47	0.57	5.46	0.57	1.67	1.63	5.72	-
700	27	1.55	0.58	5.47	0.68	1.68	1.52	5.77	-
3500	30	1.48	0.58	5.56	0.55	1.57	1.49	5.58	-

 

Table 4. Results of haematological studies in mice fed diets containing 0-3500 ppm Violet 6B for 80 wk

Sex and dose (ppm)	No. of mice examined	Hb (g/100 mL)	PCV (%)	RBC (10^3/mm^3)	Retics (% of RBC)	Leucocytes
						Total (10^3/mm^3)	Differential (%)
							N	E	L	M
Males	Week 52
0	10	14.0	44	7.65	1.9	8.94	16	0	83	1
700	10	13.9	47	7.97	-	10.02	-	-	-	-
3500	10	14.9	47	8.41**	1.6	11.55**	19	0	81	0
Females
0	10	14.6	46	7.41	1.8	7.44	23	0	73	1
700	10	14.3	49*	6.84	-	9.15	-	-	-	-
3500	10	14.3	47	7.31	2.1	7.72	18	0	81	1
Males	Week 80
0	35	14.1	-	-	-	-	40	1	57	1
1	35	14.1	-	-	-	-	-	-	-	-
700	34	14.4	-	-	-	-	-	-	-	-
3500	37	14.2	-	-	-	-	37	1	61	1
Females
0	30	15.4	-	-	-	-	50	1	48	1
70	28	14.9	-	-	-	-	-	-	-	-
700	29	14.7	-	-	-	-	-	-	-	-
3500	30	15.1	-	-	-	-	53	0	46	1

*P < 0.05; **P < 0.01.

Conclusions:

Based on all the available data, it was concluded that the no observed adverse effect level (NOAEL) for test chemical was considered to be 500 mg/kg bw/day.

Executive summary:

Chronic repeated dose toxicity study was performed to determine the oral toxic nature of test chemical using ASH-CSI male and female mice. The test chemical was mixed with appropriate quantity of corn oil and mixed with feed at dose levels of 0, 10, 100 or 500 mg/kg/day. The animals were the test chemical in diet for 80 weeks and during the study the animals were observed for clinical signs, mortality, changes in body weight, hematology and the animals were subjected to gross and histopathology. The faeces of all treated mice were violet-coloured but the urine appeared normal, suggesting that the dose was largely unabsorbed from the gastro-intestinal tract. The feeding of Violet 6B did not affect the mortality, the rate of body-weight gain or the haematological picture at 12, 28, 52 or 80 wk. The only statistically significant changes of organ weight were in the small intestine of the male mice. Those given diets containing 100 or 500 mg/Kg/day were lower, by 10 and 12% respectively, than the controls. This effect was not statistically significant when the weights were expressed relative to body weight. Most of the lesions were those normally occurring in ageing mice and were found with a similar incidence in treated and control mice and appeared to be not related to the treatment with test chemical . The changes found in the skin consisted of hyperkeratosis, also present in control animals, and a single dermoid cyst in a female given 700 ppm test chemical Dilated gastric glands and hyperplasia of the gastric mucosa were encountered, but again similar lesions were found in control mice. Pulmonary adenomas were seen in 36% of all male mice and in 20% of all female mice, the incidence being similar in all groups. Two thyroid adenomas were found in male mice fed test chemical (one at 10 and the other at 500 mg/Kg/day). Benign tumours of the ovary, testis, subcutaneous tissue and kidney were found with a similar incidence in control and treated animals. Pulmonary adenocarcinomas were present in two male mice fed diets containing test chemical (one at 100 and one at 500 mg/Kg/day). Lymphosarcoma and reticulum-cell neoplasia had a similar incidence in mice fed test chemical and in the controls. Three squamous-cell carcinomas of the skin were seen in female mice (one control, one fed 10 mg/Kg/day and one fed 100 mg/Kg/day ). The other malignant tumours found were a fibrosarcoma in a control female, a rhabdomyosarcoma in a female given 10 mg/Kg/day test chemical and a mammary adenocarcinoma in a female given 100 mg/Kg/day of test chemical . Since no deviation was noted in the treated animals from the controls used, hence, the the no observed adverse effect level (NOAEL) for test chemical was considered to be 500 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

2 500 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Data is Klimicsh 2 and from authoritative database

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: inhalation, other

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

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, other

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity: Oral

Data available from the various test chemical was reviewed to determine the toxic nature of test chemical .The studies are as mentioned below:

Study 2.

Chronic repeated dose oral toxicity study was performed for test chemical to determine its toxic nature upon repeated exposure. The test chemical was mixed with feed at dose levels of0, 0.5, 1.0, 2.0 or 5.0 % (0, 250, 500, 1000 or 2500 mg/Kg bw/day) and fed for 2 years to 24 male and 24 female/dose Osborne Mendel weanling rats. During the study period, the animals were observed for mortality, any abnormality, body weight changes, hematology and were subjected to gross and microscopic pathology. No mortality and abnormality was noted during the study period. No significant growth inhibition occurred with the animals fed with test chemical . Also, no effects was observed on hemotological parameters and weights of heart, liver, kidney, spleen, and testis of the animals that received test chemical at dietary levels up to 5%(2500 mg/Kg bw/day). There was no treatment-related change in gross pathology. The usual gross pathologic conditions of old rats were seen in all groups, with no significant difference in distribution. The most common lesions were tumors, pneumonia (mostly chronic) of varying degrees? chronic nephritis of the usual type seen in our older rats, and testicular atrophy. There was no treatment-related change in microscopical examination. The most common tumors were mammary adenocarcinomas, fibroadenomas, and pulmonary lymphosarcomas. Based on the above considerations, the No observed Adverse Effect level (NOAEL) for test chemical was considered to be 2500 mg/kg bw/day in Osborne-Mendel male and female rats for 2 years feeding study.

Study 3.

Chronic repeated dose toxicity study was performed to determine the oral toxic nature of test chemical using ASH-CSI male and female mice. The test chemical was mixed with appropriate quantity of corn oil and mixed with feed at dose levels of 0, 10, 100 or 500 mg/kg/day. The animals were the test chemical in diet for 80 weeks and during the study the animals were observed for clinical signs, mortality, changes in body weight, hematology and the animals were subjected to gross and histopathology. The faeces of all treated mice were violet-coloured but the urine appeared normal, suggesting that the dose was largely unabsorbed from the gastro-intestinal tract. The feeding of Violet 6B did not affect the mortality, the rate of body-weight gain or the haematological picture at 12, 28, 52 or 80 wk. The only statistically significant changes of organ weight were in the small intestine of the male mice. Those given diets containing 100 or 500 mg/Kg/day were lower, by 10 and 12% respectively, than the controls. This effect was not statistically significant when the weights were expressed relative to body weight. Most of the lesions were those normally occurring in ageing mice and were found with a similar incidence in treated and control mice and appeared to be not related to the treatment with test chemical . The changes found in the skin consisted of hyperkeratosis, also present in control animals, and a single dermoid cyst in a female given 700 ppm test chemical Dilated gastric glands and hyperplasia of the gastric mucosa were encountered, but again similar lesions were found in control mice. Pulmonary adenomas were seen in 36% of all male mice and in 20% of all female mice, the incidence being similar in all groups. Two thyroid adenomas were found in male mice fed test chemical (one at 10 and the other at 500 mg/Kg/day). Benign tumours of the ovary, testis, subcutaneous tissue and kidney were found with a similar incidence in control and treated animals. Pulmonary adenocarcinomas were present in two male mice fed diets containing test chemical (one at 100 and one at 500 mg/Kg/day). Lymphosarcoma and reticulum-cell neoplasia had a similar incidence in mice fed test chemical and in the controls. Three squamous-cell carcinomas of the skin were seen in female mice (one control, one fed 10 mg/Kg/day and one fed 100 mg/Kg/day ). The other malignant tumours found were a fibrosarcoma in a control female, a rhabdomyosarcoma in a female given 10 mg/Kg/day test chemical and a mammary adenocarcinoma in a female given 100 mg/Kg/day of test chemical . Since no deviation was noted in the treated animals from the controls used, hence, the the no observed adverse effect level (NOAEL) for test chemical was considered to be 500 mg/Kg/day.

Based on the data available, the No Observed Adverse Effect Level (NOAEL) for the test chemical was considered to be in range of 500 -2500 mg/Kg bw using male and female rats or mice . Hence the test chemical is not likely to be toxic upon repeated exposure by oral route as per the criteria mentioned in CLP regulation.

Repeated dose toxicity: inhalation

The melting point of test chemical is >300 °C.This suggests that test chemical decomposes above300 °C without melting and also it does not exhibit very high vapour pressure. Inhalation is therefore not the likely route of exposure and hence this end point is considered for waiver.

Repeated dose toxicity: dermal

Repeated dermal toxicity is unlikely to occur since dermal absorption of test chemical is limited. Also considering the use of the chemical as a colouring agent in food and cosmetics and considering the volatility absorption by the dermal route is not considered to be significant. Thus, given the above considerations, it is assumed that test chemical shall not exhibit acute dose toxicity by the repeated dermal route.

Justification for classification or non-classification

Based on the data available and applying the weight of evidence approach, the test chemical does not exhibit toxic nature upon repeated exposure by oral route of exposure. Hence the test chemical is likely to be not classified for toxicity upon repeated dose exposure by oral, dermal and inhalation route of exposure.