Reaction products of diazotised sodium 2-amino-5-{[2-(sulfonatooxy)ethyl]sulfonyl}benzenesulfonate coupled with sodium 2-(4-aminobenzene-1-sulfonyl)ethyl sulfate, subsequently coupled with diazotised sodium 2-(4-aminobenzene-1-sulfonyl)ethyl sulfate

Endpoint summary

Description of key information

Repeated dose toxicity: Oral

The no observed adverse effect level (NOAEL) for 2-Naphthalenesulfonic acid, 7-amino-4-hydroxy-3-[2-[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl] diazenyl]-8-[2-[2-sulfo-4-[ [2-(sulfooxy) ethyl] sulfonyl]phenyl]diazenyl]-, sodium salt (1:4) (CAS no.: 607724-37-8) is considered to be 1300 mg/kg/day. Hence the test chemical is not likely to be toxic as per the criteria mentioned in CLP regulation.

Repeated dose toxicity: Inhalation

2-Naphthalenesulfonic acid, 7-amino-4-hydroxy-3-[2-[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl] diazenyl]-8-[2-[2-sulfo-4-[ [2-(sulfooxy) ethyl] sulfonyl]phenyl]diazenyl]-, sodium salt (1:4) (CAS no.: 607724-37-8) has very low vapour pressure (3.8797E-17 Pa. = 2.91E-19 mmHg). Also the particle size distribution was determined to be in the range of 147 micron to 52 micron. So the potential for the generation of inhalable vapours is very low. Also the normal conditions of use of this substance will not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalatory route will be highly unlikely and therefore the end point for repeated dose toxicity by inhalation route was considered for waiver.

Repeated dose toxicity: Dermal

The acute dermal toxicity value for 2-Naphthalenesulfonic acid, 7-amino-4-hydroxy-3-[2-[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl] diazenyl]-8-[2-[2-sulfo-4-[ [2-(sulfooxy) ethyl] sulfonyl]phenyl]diazenyl]-, sodium salt (1:4) (CAS no.: 607724-37-8) (as provided in section 7.2.3) is >2000 mg/kg body weight. Considering this, the end point for repeated dermal toxicity is considered as waiver.

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

Endpoint:

repeated dose toxicity: oral

Remarks:

combined repeated dose and carcinogenicity

Type of information:

experimental study

Remarks:

Read across data

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:

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

Specific details on test material used for the study:

Not specified

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 available

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

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

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 mice
0 mg/kg bw/d: 60 male and 60 female mice
130 mg/kg bw/d: 30 male and 30 female mice
325 mg/kg bw/d: 30 male and 30 female mice
650 mg/kg bw/d: 30 male and 30 female mice
1300 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

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 health

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

BODY 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 experiment

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: 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: 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 data

URINALYSIS: 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 constituents

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

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

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 (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%.

Other effects:

not specified

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.

Critical effects observed:

not specified

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

 

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).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 300 mg/kg bw/day

Study duration:

chronic

Species:

mouse

Quality of whole database:

Data is from K2 publication

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: inhalation, other

Data waiving:

exposure considerations

Justification for data waiving:

a short-term toxicity study does not need to be conducted because exposure of humans via inhalation in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment

Justification for type of information:

a short-term toxicity study does not need to be conducted because exposure of humans via inhalation in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment

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:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Quality of whole database:

Waiver

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity: Oral

 

Repeated dose oral toxicity studies were reviewed to determine the toxic nature of 2-Naphthalenesulfonic acid, 7-amino-4-hydroxy-3-[2-[4-[[2-(sulfooxy)ethyl] sulfonyl]phenyl] diazenyl]-8-[2-[2-sulfo-4-[ [2-(sulfooxy) ethyl] sulfonyl]phenyl]diazenyl]-, sodium salt (1:4) (CAS no.: 607724-37-8). The studies are as summarized below:

 

1. Repeated dose toxicity test were performed on mice with different concentrations of the test chemical at dose levels of 0.1, 0.25, 0.5 or 1.0% (130, 325, 650, 1300 mg/kg bw/d) 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 Black PN. 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% the test chemical. 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% Black PN was higher than the control figure. Liver weights of female but not of male mice fed 0.25% test chemical 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% test chemical. 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).

 

2. In another study, repeated oral toxicity of the test chemical was determined by performing a 80 weeks repeated dose toxicity study using Charles River CD mouse (male and female) at dose levels of 0, 100, 200, 400 or 800 mg/Kg (0.2, 0.4, 0.8 or 1.6% respectively) by oral diet route. The animals were observed for clinical signs, mortality, body weight changes, hematology and were subjected to gross and histopathology. The feeding of the test chemical did not adversely affect the death rate within the groups, the rate of body-weight gain, the organ weights or the haematological findings. The incidence and severity of the histopathological findings were similar in treated and control mice and there was no evidence of an increased incidence of tumours in the mice given Sunset Yellow FCF. Distension of the bladder, frequently associated with hydroureter and hydronephosis was noted. Pus and hard protein deposits were found in the bladders of many of the mice and in a number of cases it was possible to show that an obstruction had occurred in the urethra in the region of the bulbo-urethral complex. After the male mice were put into individual cages, there was a marked reduction in the number showing this syndrome, although isolated cases of urinary retention were seen throughout the study. Of the malignant tumours found, lymphomas and reticulum-cell neoplasms occurred in treated and control mice. In addition a splenic haemangiosarcoma and a mammary carcinoma were found in the female mice fed on the diet containing the lowest level (100 mg/Kg). A single tumour of the Harderian gland was found in a female from the group given the 1.6% level. The commonest benign tumours were pulmonary adenomas and benign cysts in the ovary, found with a similar frequency in treated and control mice. The only other tumour occurring in treated mice without a similar finding in the controls was a granulosa-cell tumour of the ovary in a mouse given 800 mg/Kg test chemical. Thus, on the basis of above results the NOAEL (no observed adverse effect level) for the test chemical was considered to be 800 mg/kg diet.

 

The no observed adverse effect level (NOAEL) for 2-Naphthalenesulfonic acid, 7-amino-4-hydroxy-3-[2-[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl] diazenyl]-8-[2-[2-sulfo-4-[ [2-(sulfooxy) ethyl] sulfonyl]phenyl]diazenyl]-, sodium salt (1:4) (CAS no.: 607724-37-8) is considered to be 1300 mg/kg/day.

 

Repeated dose toxicity: Inhalation

 

2-Naphthalenesulfonic acid, 7-amino-4-hydroxy-3-[2-[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl] diazenyl]-8-[2-[2-sulfo-4-[ [2-(sulfooxy) ethyl] sulfonyl]phenyl]diazenyl]-, sodium salt (1:4) (CAS no.: 607724-37-8) has very low vapour pressure (3.8797E-17 Pa. = 2.91E-19 mmHg). Also the particle size distribution was determined to be in the range of 147 micron to 52 micron. So the potential for the generation of inhalable vapours is very low. Also the normal conditions of use of this substance will not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalatory route will be highly unlikely and therefore the end point for repeated dose toxicity by inhalation route was considered for waiver.

 

Repeated dose toxicity: Dermal

 

The acute dermal toxicity value for 2-Naphthalenesulfonic acid, 7-amino-4-hydroxy-3-[2-[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl] diazenyl]-8-[2-[2-sulfo-4-[ [2-(sulfooxy) ethyl] sulfonyl]phenyl]diazenyl]-, sodium salt (1:4) (CAS no.: 607724-37-8) (as provided in section 7.2.3) is >2000 mg/kg body weight. Considering this, the end point for repeated dermal toxicity is considered as waiver.

 

Based on the data available for the test chemicals, 2-Naphthalenesulfonic acid, 7-amino-4-hydroxy-3-[2-[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl] diazenyl]-8-[2-[2-sulfo-4-[ [2-(sulfooxy) ethyl] sulfonyl]phenyl]diazenyl]-, sodium salt (1:4) (CAS no.: 607724-37-8) is not likely to be toxic upon repeated exposure by oral route as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the data available for the test chemical is not likely to be toxic upon repeated exposure by oral route as per the criteria mentioned in CLP regulation.