Trisodium 1-amino-4-[[3-[[4-chloro-6-[(sulphonatophenyl)amino]-1,3,5-triazin-2-yl]amino]-2,4,6-trimethyl-5-sulphonatophenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-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:

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

Data source

Reference

Reference Type:

publication

Title:

Repeated oral toxicity study of the test chemical

Author:

Drake et al

Year:

1977

Bibliographic source:

Food and cosmetics toxicology

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

A repeated dose study was performed to investigate the effect of the test chemical in CFW strain mice when administered orally for 80 wk.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tetrasodium 1-acetamido-2-hydroxy-3-(4-((4-sulphonatophenylazo)-7-sulphonato-1-naphthylazo))naphthalene-4,6-disulphonate

EC Number:

219-746-5

EC Name:

Tetrasodium 1-acetamido-2-hydroxy-3-(4-((4-sulphonatophenylazo)-7-sulphonato-1-naphthylazo))naphthalene-4,6-disulphonate

Cas Number:

2519-30-4

Molecular formula:

C28H21N5O14S4.4Na

IUPAC Name:

tetrasodium 4-acetamido-5-hydroxy-6-({7-sulfonato-4-[(4-sulfonatophenyl)diazenyl]-1-naphthyl}diazenyl)naphthalene-1,7-disulfonate

Details on test material:

- Name of test material: C.I Briliant black BN- Molecular formula: C28H21N5O14S4.4Na- Molecular weight: 867.6873 g/mol- Substance type: Organic- Physical state: Solid- Purity: No data available- Impurities (identity and concentrations): essentially tetrasodium 8-acetamido-2-(7- sulpho-4-p- sulphophenylazo- 1-naphthylazo)- 1-naphthol-3,5-disulphonate; dye content*, rain. 82%; subsidiary dyes, max 4%; matter volatile at 135°C, max 10%; matter insoluble in water, max 0.1%; matter soluble in diisopropyl ether, max 0.2%; chloride and sulphate (as sodium salts), max 8%; copper, max 10ppm; arsenic, max 1 ppm; lead, max 10 ppm; heavy metals (as sulphides)

Test animals

Species:

mouse

Strain:

other: CFW strain

Details on species / strain selection:

No data

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: From a specified-pathogen-free colony- Age at study initiation: No data available- Weight at study initiation: The mice were weighed at the start of the experiment (exact weight not mentioned)- Fasting period before study: No - Housing: They were caged in groups of 15 in a room- Diet (e.g. ad libitum): Oxoid pasteurized breeding diet,ad libitum- Water (e.g. ad libitum): ad libitum- Acclimation period: No data availableENVIRONMENTAL CONDITIONS- Temperature (°C): 21±1°C- Humidity (%): 50-60%- Air changes (per hr): No data available- Photoperiod (hrs dark / hrs light): No data available

Administration / exposure

Route of administration:

oral: feed

Vehicle:

not specified

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test chemical was mixed with feed at dose level of 0, 0.1, 0.25, 0.5 or 1.0% (0, 130, 325, 650, 1300 mg/kg bw/d) 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): Oxoid pasteurized breeding diet - Concentration in vehicle: 0, 0.1, 0.25, 0.5 or 1.0% (0, 130, 325, 650, 1300 mg/kg bw/d) - 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 wk

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:0, 0.1, 0.25, 0.5 or 1.0% (0, 130, 325, 650, 1300 mg/kg bw/d)Basis:no data

No. of animals per sex per dose:

Total: 180 male and 180 female mice0 mg/kg bw/d: 60 male and 60 female mice130 mg/kg bw/d: 30 male and 30 female mice325 mg/kg bw/d: 30 male and 30 female mice650 mg/kg bw/d: 30 male and 30 female mice1300 mg/kg bw/d: 30 male and 30 female mice

Control animals:

yes, concurrent vehicle

Details on study design:

No data available

Positive control:

No data available

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes - Time schedule: Frequently dring the study period- Cage side observations checked in table [No.?] were included.: general condition and behaviour, the animals were also observed for ill healthDETAILED CLINICAL OBSERVATIONS: No data- Time schedule: No dataBODY WEIGHT: Yes - Time schedule for examinations: start of the experiment, at wk 3 and then at intervals of 2 wk until wk 73 of the experimentFOOD 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: No data- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No dataFOOD 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 dataWATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data- Time schedule for examinations: No dataOPHTHALMOSCOPIC EXAMINATION: No data - Time schedule for examinations: No data- Dose groups that were examined: No dataHAEMATOLOGY: Yes - Time schedule for collection of blood: At wk 28 and 55 from the caudal vein of ten males and ten females from the control group and from the groups of 0.5 and 1.0% dietary levels. At 80 wk, blood samples were collected from the aorta of all surviving mice during the autopsy.- Anaesthetic used for blood collection: No data- Animals fasted: No data- How many animals: 20 animals (10 male and 10 female)- Parameters were examined: haemoglobin concentration and packed cell volume, as well as for counts of erythrocytes and leucocytes. In addition, the methaemoglobin concentrations were determined in the samples collected at 80 wk. CLINICAL CHEMISTRY: No data- Time schedule for collection of blood: No data- Animals fasted: No data- How many animals: No data- Parameters were examined: No dataURINALYSIS: Yes - Time schedule for collection of urine: At 28 wks at 6-hr period from three groups of five mice of each sex from the controls and the groups on the two highest dietary levels (0.5 and 1.0%) of Black PN.- Metabolism cages used for collection of urine: No data- Animals fasted: No data- Parameters were examined: protein, reducing substances, bile salts and blood as well as for colour, pH and microscopic constituentsNEUROBEHAVIOURAL 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

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, The animals were killed by exsanguination from the aorta under sodium pentobarbitone anaesthesia following an overnight period without food. At autopsy, macroscopic abnormalities were recorded and the brain, heart, liver, spleen, kidneys, adrenal glands and gonads were weighed.HISTOPATHOLOGY: Yes, samples of the brain, heart, liver, spleen, kidneys, adrenal glands and gonads and of salivary glands, pituitary, thyroid, thymus, various lymph nodes, pancreas, urinary bladder, lungs, stomach, duodenum, ileum, colon, caecum, rectum, striped muscle (hind limb), spinal cord, uterus, aortic arch and any other tissue that appeared abnormal were preserved in 10% buffered formalin. Paraffin-wax sections of these tissues were stained with haematoxylin and eosin. All tissues from the control mice and from those fed diet containing 1% Black PN were examined histologically. At the lower dose levels, the examination was confined to the liver, kidney and any tissues seen to be abnormal at autopsy.

Other examinations:

No data

Statistics:

chi-square test, Student's t test

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

The ingestion of the test chemical had no effect on the condition or behaviour of the animals.

Mortality:

no mortality observed

Description (incidence):

No statistically significant differences between the number of deaths in the control mice and those given the test chemical were noted

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Throughout the study the body weights of mice of both sexes were similar in all groups

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):

The haematological examinations revealed only inconsistent isolated changes of statistical significance. At wk 28 the haemoglobin concentrations and red blood cell counts were lower in female mice fed diet containing if 0.5 or 1.0% than in the controls. There were no comparable findings in the males. The total white cell count of the male animals fed 1.0% in the diet was higher than that of the controls at this time, but there was no comparable change in this measurement in the females, or in either sex at any other time. At wk 55, the haemoglobin concentrations of male animals fed 0.5% of the coiouring in the diet were significantly (P < 0.05) lower than the control values. However, the corresponding value at the higher dietary level was not affected and there were no differences from the control value in the females. Also at wk 55, the erythrocyte counts of females fed 1.0%were lower (P < 0.01) than those of the controls, but this finding was again isolated. There were no statistically significant differences between the control and test samples taken at wk 80.

Clinical biochemistry findings:

not specified

Urinalysis findings:

no effects observed

Description (incidence and severity):

No abnormal constituents were detected in the urine from the control or treated mice.

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There were only scattered differences in mean organ weights between treated and control animals. A lower brain weight in females, compared with the control value, was the only difference affecting animals fed 1%. This difference, which did not occur in the males, was only marginally significant and there was no significant difference when the weights were expressed relative to body weight By contrast, the relative brain weight of females fed 0.25% was higher than the control figure. Liver weights of female but not of male mice fed 0.25% were lower than control values, but again this was an isolated finding and there were no significant differences in relative liver weights. Kidney weights of male animals only were significantly lower than control values at the two lowest levels of treatment (0.1 and 0.25%), but a significant difference in relative kidney weights of males occurred only at the 0.5% level, at which a higher value was recorded for the treated mice. The only other significant differences occurred in the relative heart weights, which were raised in male mice fed 0.5% and in females fed 0.25%.

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

The incidence of histological findings was similar in all groups of mice, including the controls

Histopathological findings: neoplastic:

not specified

Description (incidence and severity):

Most of the tumours in the study occurred with either a comparable or a greater incidence in the control groups than in the treated mice. Several isolated tumours were identified in mice given the lower levels of Black PN, without comparable findings in the controls or in the highest dose group. They were a mammary fibroadenoma (ina female on 0.1%), a uterine fibromyoma (0.19%) and a squamous-cell carcinoma of the skin (female, 0.5%). The only tumour found at the highest dietary levelwithout comparable control findings was a squamous- cell carcinoma of the skin in a male mouse fed 1%.

Other effects:

not specified

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 300 other: mg/kg/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Body weight, weight gain, organ weight and histopathology.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1. Cumulative death rate in mice fed diets containin 0.01% test chemical for 80 wk

Week	Total no. of deaths
	Males					Females
	0	0.1	0.25	0.5	1.0	0	0.1	0.25	0.5	1.0
24	3	1	0	3	0	1	0	0	0	0
48	8	1	3	5	3	8	1	3	3	2
64	11	4	7	9	5	15	9	9	6	7
72	16	6	9	13	6	24	12	13	12	10
78	20	10	10	16	7	28	19	13	15	12

 

Table 2: Mean body weights of mice fed diets containing 0.1% test chemical for 80 wk

Week	Total no. of deaths
	Males					Females
	0	0.1	0.25	0.5	1.0	0	0.1	0.25	0.5	1.0
0	21	22	21	22	22	18	18	18	18	19
15	38	35	37	37	38	28	29	28	28	28
39	41	39	40	39	40	32	33	32	33	32
57	43	40	40	37	41	35	34	32	33	33
73	42	41	41	38	42	36	35	33	36	34

 

Table 3. Results of haematological examinations of mice fed diets containing 0.01% test chemical for 80 wk

Sex and dietary level (%)	No. of mice examined	Hb (g/100 mL)	Met Hb (% of Hb)	PCV (%)	RBC (10^6/mm^3)	Total leucocytes (10^3/ mm^3)
Week 28
Males
0	10	14.2	-	43	7.51	11.4
0.5	10	14.4	-	48	8.05	12.4
1.0	10	15.5	-	47	8.04	14.8*
Females
0	10	15.9	-	49	9.01	14.8
0.5	10	14.0**	-	48	7.54--	12.1
1.0	10	13.1**	-	48	7.41**	12.6
Week 80
Males
0	39	11.9	3.61	35	6.67	4.5
0.1	15	11.9	4.51	36	6.85	4.2
0.25	18	11.5	4.90	35	6.67	3.2
0.5	10	11.7	3.86	34	6.62	5.4
1.0	17	12.0	3.81	35	6.90	3.8
Females
0	27	13.5	6.03	39	7.32	5.5
0.1	10	13.0	4.68	40	7.64	5.7
0.25	10	14.1	7.39	42	8.21	5.4
0.5	13	13.3	4.89	41	7.87	3.9
1.0	15	14.4	5.67	42	8.09	3.8

 

Table 4. Relative organ weights of mice fed diets containing 0.1% test chemical for 80 wk

Dose	No. of mice examined	Relatve organ weight (g/10 g bw)							Terminal body weight (g)
		Brain	Heart	Liver	Spleen	Kidneys	Adrenals	Gonads
	Males
0	40	1.07	0.64	5.47	0.38	1.67	27.5	0.45	35
0.1	20	1.21	0.59	5.84	0.45	1.50	27.8	0.41	36
0.25	20	1.15	0.63	5.88	0.44	1.62	27.1	0.45	33
0.5	12	1.32	0.71*	5.80	0.51	1.77*	29.6	0.49	32
1.0	23	1.28	0.63	5.29	0.39	1.60	28.5	0.48	35
	Females
0	32	1.53	0.57	5.80	0.51	1.46	40.6	84.2	29
0.1	11	1.53	0.61	5.87	0.56	1.56	37.8	84.8	28
0.25	17	1.72*	0.64*	5.29	0.41	13.51	45.9	100.8	26
0.5	15	1.55	0.56	5.43	0.54	1.41	42.8	101.2	27
1.0	18	1.53	0.53	5.27	0.47	1.40	42.3	87.8	30

*P < 0.05.

Table 5. Incidence of histolooical findings (excluding tumours) in mice fed diets containin 0 0-1% test chemical for 80 wk

Tissue and finding	No. of mice affected
	Males					Females
	0	0.1	0.25	0.5	1.0	0	0.1	0.25	0.5	1.0
No. of mice examined	54	27	28	26	29	58	28	28	28	29
Lung
Chronic inflammatory infiltration	10	10	7	9	7	9	9	6	5	5
Congestion	1	1	0	2	1	3	3	0	1	1
Liver
Abscess	0	1	0	1	0	1	0	0	0	0
Degeneration (focal)	0	0	2	0	0	3	0	0	1	0
Macrophages (focal aggregations)	0	2	1	0	0	0	0	0	0	0
Cysts	1	0	0	0	0	0	1	0	0	0
Kidney
Glomerulonephrosis	2	0	5	1	1	2	1	0	0	0
Pyelonephritis	1	0	0	0	0	0	0	0	0	0
Lymphoid hyperplasia	1	1	1	0	1	0	0	0	0	0
Urethra
Chronic inflammation	1	1	2	0	0	0	0	0	0	0
Testes
Atrophy	1	0	0	1	0	-	-	-	-	-
Ovaries
Follicular cyst	-	-	-	-	-	3	1	1	0	5
Uterus
Cystic	-	-	-	-	-	1	0	0	2	2
Lymphoid tissue Reactive hyperplasia in
Spleen	2	0	2	1	1	0	1	1	0	0
Thymus	0	0	0	0	1	2	0	1	0	0
Lymphnodes	2	1	0	0	0	1	3	1	1	0

 

Table 6. Incidence of tumours in mice fed diets containing 0.1% test chemical for 80 wk

Tissue and finding	No. of mice affected
	Males					Females
	0	0.1	0.25	0.5	1.0	0	0.1	0.25	0.5	1.0
No. of mice examined	54	27	28	26	29	58	28	28	28	29
Lung
Adenoma	9	1	8	5	7	14	6	3	5	6
Mammary tissue
Fibroadenoma	-	-	-	-	-	0	1	0	0	0
Carcinoma	-	-	-	-	-	1	0	1	0	1
Adenoma	-	-	-	-	-	20	13	12	9	9
Uterus
Fibrosarcoma	-	-	-	-	-	0	1	0	0	0
Skin
Squamous cell carcinoma	0	0	0	0	1	0	0	0	1	0
Subcutaneous tissue
Fibroma	1	0	0	0	0	0	0	0	0	0
Ovary
Carcinoma	-	-	-	-	-	1	0	0	0	0
Lymphoid tissue
Lymphosarcoma	2	0	1	1	1	1	1	0	1	1

 

Applicant's summary and conclusion

Conclusions:

The no observed adverse effect level (NOAEL) for the test chemical in mice is considered to be 1 % (1300 mg/kg/day)

Executive summary:

Repeated dose toxicity test were performed on mice with different concentrations from 0.1, 0.25, 0.5 or 1.0% (130, 325, 650, 1300 mg/kg bw/d) test chemical for 80 wk. 30 males and 30 females was used for the treatment and group of 60 mice of each sex as control. During the study period the animals were observed for clinical signs, mortality, hematology, urine analysis was performed and the animals were subjected to gross and histopathology. The general condition and behaviour of the animals were observed frequently and any mouse that showed signs of ill-health was isolated, to be returned to its cage on recovery or to be killed if its condition deteriorated. The mice were weighed at the start of the experiment, at wk 3 and then at intervals of 2 wk until wk 73 of the experiment. Blood sample were taken at wk 28 and 55. At 80 wk, blood samples were collected from the aorta of all surviving mice during the autopsy. For haematology blood samples were collected and for urine sample from 6-hr period from three groups of five mice of each sex from the controls and the groups on the two highest dietary levels of the test chemical. Histopathology was also conducted. The ingestion of the test chemical had no effect on the condition or behaviour and mortality of the animals. The haematological examinations revealed only inconsistent isolated changes of statistical significance. At wk 28 the haemoglobin concentrations and red blood cell counts were lower in female mice fed diet containing if 0.5 or 1.0% test chemical than in the controls. There were no comparable findings in the males. The total white cell count of the male animals fed 1.0% test chemical in the diet was higher than that of the controls at this time, but there was no comparable change in this measurement in the females, or in either sex at any other time. At wk 55, the haemoglobin concentrations of male animals fed 0.5% of the coiouring in the diet were significantly (P < 0.05) lower than the control values. However, the corresponding value at the higher dietary level was not affected and there were no differences from the control value in the females. Also at wk 55, the erythrocyte counts of females fed 1.0% test chemical were lower (P < 0.01) than those of the controls, but this finding was again isolated. There were no statistically significant differences between the control and test samples taken at wk 80. There were only scattered differences in mean organ weights between treated and control animals. A lower brain weight in females, compared with the control value, was the only difference affecting animals fed 1%. This difference, which did not occur in the males, was only marginally significant and there was no significant difference when the weights were expressed relative to body weight By contrast, the relative brain weight of females fed 0.25% test chemical was higher than the control figure. Liver weights of female but not of male mice fed 0.25% were lower than control values, but again this was an isolated finding and there were no significant differences in relative liver weights. Kidney weights of male animals only were significantly lower than control values at the two lowest levels of treatment (0.1 and 0.25%), but a significant difference in relative kidney weights of males occurred only at the 0.5% level, at which a higher value was recorded for the treated mice. The only other significant differences occurred in the relative heart weights, which were raised in male mice fed 0.5% and in females fed 0.25%. The incidence of histological findings was similar in all groups of mice, including the controls. Most of the tumours in the study occurred with either a comparable or a greater incidence in the control groups than in the treated mice. Several isolated tumours were identified in mice given the lower levels, without comparable findings in the controls or in the highest dose group. They were a mammary fibroadenoma (in a female on 0.1%), a uterine fibromyoma (0.19%) and a squamous-cell carcinoma of the skin (female, 0.5%). The only tumour found at the highest dietary level without comparable control findings was a squamous- cell carcinoma of the skin in a male mouse fed 1%. Based on these considerations, the no observed adverse effect level (NOAEL) for the test chemical in mice is considered to be 1 % (1300 mg/kg/day).