Sodium 3-(p-anilinophenylazo)benzenesulphonate

Endpoint summary

Description of key information

Repeated dose toxicity: oral
The No Observed Adverse Effect level (NOAEL) for the test compound sodium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate (metanil yellow) is determined to be 500 mg/Kg bw upon repeated exposure for 180 days to male and female albino mice by oral intubation route.

Repeated dose toxicity: inhalation
According to Annex IX of the REACH regulation, testing by the inhalation route is appropriate only if exposure of humans via inhalation is likely. Taking into account the low vapour pressure of the substance sodium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate, which is reported as 6.93E-16 Pa. Thus, exposure to inhalable dust, mist and vapour of the chemical sodium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate is highly unlikely. Therefore this study is considered for waiver.

Repeated dose toxicity: dermal
The acute toxicity value for sodium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate (as provided in section 7.2.3) is >2000 mg/kg body weight. Thus, it is expected that sodium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate shall not exhibit 28 day repeated dose toxicity by the dermal route. In addition, there is no dermal absorption data as well as no data available that suggests that sodium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate shall exhibit repeated dose toxicity by the dermal route. Hence this end point was considered for waiver.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

Chronic oral toxicity study was performed to determine the oral toxic nature of metanil yellow upon repeated exposure

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

- Name of test material: Ext. D&C Yellow No. 1
- IUPAC name: sodium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate
- Molecular formula: C18H15N3O3SNa
- Molecular weight: 375.383 g/mol
- Substance type: organic
- Physical state: No data
- Purity: No data available
- Impurities (identity and concentrations): no data available

Species:

mouse

Strain:

other: Albino

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: 4-6 weeks
- Weight at study initiation: 20-25 g
- Fasting period before study: No data
- Housing: No data
- Diet (e.g. ad libitum): pelleted lab chow (Hindustan Lever Ltd., Bombay, India)
- Water (e.g. ad libitum): Water ad libitum
- Acclimation period: No data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24°C ±1°C
- 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

Remarks:

pelleted lab chow (Hindustan Lever Ltd., Bombay, India)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Metanil yellow was mixed with pelleted lab chow (Hindustan Lever Ltd., Bombay, India) at dose levels of 0, 100, 500 or 3000 mg/Kg bw

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): pelleted lab chow (Hindustan Lever Ltd., Bombay, India)
- Concentration in vehicle: 0, 100, 500 or 3000 mg/Kg bw
- 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:

180 days

Frequency of treatment:

Daily

Remarks:

0, 100, 500 or 3000 mg/Kg bw

No. of animals per sex per dose:

Total: 320
0 mg/Kg bw: 40 males and 40 females
100 mg/Kg bw: 40 males and 40 females
500 mg/Kg bw: 40 males and 40 females
3000 mg/Kg bw: 40 males and 40 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: Throughout the experiment
- Cage side observations checked in table [No.?] were included. General condition of the animals was observed

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

BODY WEIGHT: No data
- Time schedule for examinations: No data

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: After 180 days
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.?] were examined. Total erythrocyte count (TEC), Differential leukocyte count (DLC)- neutrophils, lymphocytes, monocytes, eosinophils, basophils, hemoglobin, erythrocyte sedimentation rate (ESR), mean corpuscular volume (MCV), MCG, MCHC, Heinz bodies, platelet count (PC), PCV, Bleeding time (BT), coagulation time (CT)

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, organs were removed for gross pathology

HISTOPATHOLOGY: No data

Other examinations:

No data

Statistics:

No detailed data available

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

General condition was unaffected at dose levels of 100 and 500 mg/Kg bw except at 3000 mgKg bw

Mortality:

not specified

Body weight and weight changes:

not specified

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):

100 mg/Kg bw: No abnormalilties were observed

500 mg/Kg bw: No abnormalilties were observed

3000 mg/Kg bw: Statistical analysis of various values exhibited significant decrease in TEC and Hb and increase in ESR, MCV and MCH suggesting normochromic macrocytic anaemia.

TLC, PC, PCV, MCHC, BT and CT, however, remained normal in both male and female animals.

A marked increase in the number of lymphocytes and macrocytes and a decrease in the number of neutrophils and eosinophils suggesting the occurrence of chronic lymphocytic leukaemia.

Heinz bodies began to appear in the blood after 3 months feeding in mice fed metanil yellow at 3.0 g/kg body weight and were observed in 80-90% erythrocytes at the end of the experiment.

Blood showed marked anisocytosis, poikilocytosis, stomatocytes, schistocytes and a large number of target cells, all normochromic in character. An
overwhelming preponderance of mature lymphocytes with scant cytoplasm, larger and denser clumps of nuclear chromatin and accentuation of parachromatin were also observed, which were suggestive of chronic lymphocytic leukaemia.

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:

not specified

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Mice fed metanil yellow at 3000 mg/kg bw showed hepatomegaly and splenomegaly at autopsy.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Details on results:

Clinical signs and mortality
Clinical signs: General condition was unaffected at dose levels of 100 and 500 mg/Kg bw except at 3000 mgKg bw

Mortality: No data

Body weight and weight gain: No data

Food consumption and compound intake: No data

Food efficiency: No data

Water consumption and compound intake: No data

Opthalmoscopic examination: No data

Haematology:
100 mg/Kg bw: No abnormalilties were observed

500 mg/Kg bw: No abnormalilties were observed

3000 mg/Kg bw: Statistical analysis of various values exhibited significant decrease in TEC and Hb and increase in ESR, MCV and MCH suggesting normochromic macrocytic anaemia.

TLC, PC, PCV, MCHC, BT and CT, however, remained normal in both male and female animals.

A marked increase in the number of
lymphocytes and macrocytes and a decrease in the number of neutrophils and eosinophils suggesting the occurrence of chronic lymphocytic leukaemia.

Heinz bodies began to appear in the blood after 3 months feeding in mice fed metanil yellow at 3.0 g/kg body weight and were observed in 80-90% erythrocytes at the end of the experiment.

Blood showed marked anisocytosis, poikilocytosis, stomatocytes, schistocytes and a large number of target cells, all normochromic in character. An
overwhelming preponderance of mature lymphocytes with scant cytoplasm, larger and denser clumps of nuclear chromatin and accentuation of parachromatin were also observed, which were suggestive of chronic lymphocytic leukaemia.

Clinical chemistry: No data

Urinanalysis: No data

Neurobehaviour: No data

Organ weights: No data

Gross pathology: Mice fed metanil yellow at 3000 mg/kg bw showed hepatomegaly and splenomegaly at autopsy.

Histopathology: No data

Key result

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

gross pathology

haematology

Critical effects observed:

not specified

Table I Haematological Findings in Male Mice Fed with Metanil Yellow at 0.0 and 3.0 G/Kg Body Weight Concentrations

No.	Experiment	SI Unit	0 mg/Kg bw	3000 mg/Kg bw
1	Total erythrocyte count (TEC)	10^12/L	6.13±0.45	3.86 ± 0.70
2	Differential leukocyte count (DLC) Neutrophils Lymphocytes Monocytes Eosinophils Basophils	10^9/L	5.67 ± 0.23 3.28 ± 0.16 0.44 ± 0.02 0.05 ± 0.01 0.00 ± 0.00	0.44 ± 0.16 9.10±0.44 0.65±0.03 0.01 ± 0.00 0.01 ± 0.00
3	Haemoglobin (Hb)	g/dL	16.29 ± 1.30	13.64 i 0.55
4	Erythrocyte sedimentation rate (ESR)	mm/h	1.81±0.25	4.18 ± 0.75
5	Mean corpuscular volume (MCV)	FI	11.77 ± 1.77	139.88±10.00
6	Mean corpuscular haemoglobin (MCH)	Pg	24.94±2.88	35.41±0.79
7	Heinz bodies	% of erythrocytes	0.00±0.00	89.25±2.50

Mean± Standard error 

 

Table II Haematological Findings in Female Mice Fed with Metanil Yellow at 0.0 and 3.0 G/Kg Body Weight Concentrations

No.	Experiment	SI Unit	0 mg/Kg bw	3000 mg/Kg bw
1	Total erythrocyte count (TEC)	10^12/L	5.26±0.37	3.49 ± c0.21
2	Differential leukocyte count (DLC) Neutrophils Lymphocytes Monocytes Eosinophils Basophils	10^9/L	5.14 ± 0.28 3.09 ± 0.19 0.40 ± 0.04 0.04 ± 0.01 0.00 ± 0.00	0.32 ± 0.11 9.20 ± 0.54 0.69 ± 0.06 0.01 ± 0.00 0.01 ± 0.00
3	Haemoglobin (Hb)	g/dL	13.54 ± 1.10	10.50 ± 1.00
4	Erythrocyte sedimentation rate (ESR)	mm/h	2.21 ± 0.30	4.31 ± 0.25
5	Mean corpuscular volume (MCV)	FI	74.10 ± 5.40	151.86 ± 4.20
6	Mean corpuscular haemoglobin (MCH)	Pg	25.76 ± 2.97	33.23 ± 0.47
7	Heinz bodies	% of erythrocytes	0.00 ± 0.00	81.75 ± 2.25

Mean± Standard error

 

Conclusions:

The No Observed Adverse Effect level (NOAEL) for the test compound sodium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate (metanil yellow) is determined to be 500 mg/Kg bw upon repeated exposure for 180 days to male and female albino mice by oral intubation route.

Executive summary:

Chronic oral toxicity study was performed to determine the oral toxic nature of sodium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate (metanil yellow) upon repeated exposure for 180 days. The test compound was force fed to male and female albino mice by oral intubation at a dose levels of 0, 100, 500 or 3000 mg/Kg bw.

 

The animals were observed for general condition, changes in hematological parameters and gross pathology.

 

General condition was unaffected at dose levels of 100 and 500 mg/Kg bw except at 3000 mgKg bw. No change was observed by the 100 and 500 mg/Kg bw but feeding of this colour at the rate of 3000 mg/kg body weight led to certain changes in the haematological values. Total erythrocyte count (TEC) and Hb had decreased whereas ESR, MCV, and mean corpuscular haemoglobin (MCH) had increased. These facts suggested the occurrence of normochromic macrocytic anaemia. Differential leukocyte count (DLC) showed marked increase in the number of lymphocytes and monocytes and decrease in the number of neutrophils and eosinophils. Heinz bodies were observed in 80-90% erythrocytes. Other haematological values like total leukocyte count (TLC), platelet count (PC), PCV, MCHC, bleeding time (BT) and coagulation time (CT) were normal at 3000 mg/Kg bw.

 

Hence, the No Observed Adverse Effect level (NOAEL) for the test compound metanil yellow is 500 mg/Kg bw.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subchronic

Species:

mouse

Quality of whole database:

Data is from K2 publication

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

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:

short-term repeated dose toxicity: dermal

Data waiving:

other justification

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
Various experimental studies for target substance sodium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate (CAS No. 587-98-4) has been investigated for potential of toxicity following repeated exposure via oral route and are presented below:

Sub-chronic oral toxicity study was performed by Prasad and Rastogi (Toxicology letters, 1983) to determine the oral toxic nature of sodium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate (metanil yellow) upon repeated exposure for 180 days. The test compound was force fed to male and female albino mice by oral intubation at a dose levels of 0, 100, 500 or 3000 mg/Kg bw. The animals were observed for general condition, changes in hematological parameters and gross pathology. General condition was unaffected at dose levels of 100 and 500 mg/Kg bw except at 3000 mgKg bw. No change was observed by the 100 and 500 mg/Kg bw but feeding of this colour at the rate of 3000 mg/kg body weight led to certain changes in the haematological values. Total erythrocyte count (TEC) and Hb had decreased whereas ESR, MCV, and mean corpuscular haemoglobin (MCH) had increased. These facts suggested the occurrence of normochromic macrocytic anaemia. Differential leukocyte count (DLC) showed marked increase in the number of lymphocytes and monocytes and decrease in the number of neutrophils and eosinophils. Heinz bodies were observed in 80-90% erythrocytes. Other haematological values like total leukocyte count (TLC), platelet count (PC), PCV, MCHC, bleeding time (BT) and coagulation time (CT) were normal at 3000 mg/Kg bw. Hence, the No Observed Adverse Effect level (NOAEL) for the test compound metanil yellow is 500 mg/Kg bw.

Subacute oral toxicity study was performed by Ramchandani et al (Journal of Applied Toxicology, 1996) to determine the oral toxic nature of sodium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate (metanil yellow) upon repeated exposure for 3, 7 and 15 days. The test compound was given to Male Wistar Albino rats by oral intubation at a dose of 430 mg/Kg bw.Post-mitochondrial, microsomal and cytosolic fractions from the liver and gastrointestinal tract and were observed for clinical chemistry parameters including N-Demethylation of aminopyrine (APD), Azo reductase, Aniline hydroxylase, aryl hydrocarbon hydroxylase (AHH) activity, De-ethylation of 7-ethoxyresorufin (ERD), Cytochrome P-450, Glutathione-S-transferase, Quinone reductase (QR), Reduced glutathione content (GSH) and lipid peroxidation (LPO) and protein content. Oral administration of Metanil yellow for 7 days caused significant depletion of hepatic and intestinal glutathione levels (33–52%) with a concomitant increase in lipid peroxidation (49–121%). Metanil yellow treatment for 7 days also led to a significant increase in cytochrome P-450 (P-450)-dependent aryl hydrocarbon hydroxylase (AHH) activity (99–223%) in the liver and intestine. Cytosolic glutathione-S-transferase (GST) (32–136%) and quinone reductase (QR) (20–92%) activities were also found to be substantially induced in hepatic and intestinal tissues following oral treatment of Metanil yellow. Also oral treatment of Metanil yellow showed a greater response in cytosolic enzymes of hepatic tissue as compared to intestine. Based on the observations made, the Low Observed Adverse Effect level (LOAEL) for the test compound metanil yellow is determined to be 430 mg/Kg bw.

Repeated dose chronic toxicity study was performed by Rituparna Sarkar and Apurba Ratan Ghosh (International Journal Of Scientific Research, Volume-6, Issue-5, May - 2017) to determine the oral toxic nature of sodium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate (metanil yellow). The study was performed using Albino rats. After acclimatization under laboratory condition for one week they were divided into three groups containing five animals in each: Group I (control) and Group II & III (treated). The test animals of Group II & III were fed orally with a sublethal dose of Metanil Yellow of 3000 mg/kg body weight for a period of 30 and 45 days respectively. Histopathological changes in the spleen tissue were observed under light microscope after staining with Haematoxylin-Eosin. Owing to 30 days of toxicity of Metanil Yellow very minute degenerative changes were found in the white pulp and red pulp of spleen. Trabeculae and follicular regions were rarely damaged. But significant distortion and denerative changes were found in the white pulp and red pulp regions after 45 days treatment. Significant pathological changes were not only observed in the white pulp and red pulp regions of spleen tissue of albino rat but also in the follicles and trabeculae. The damage in the spleen tissue due to Metanil Yellow toxicity varies with both concentration and exposure periods (30 and 45 days). Based on the observations made, The Low Observed Adverse Effect Level (LOAEL) for metanil yellow is considered to be 3000 mg/Kg bw/day.

As per the data available for the target chemical metanil yellow NOAEL value range can be close to 500 mg/Kg bw. Based on the observations made, Metanil yellow does not exhibit toxicity upon repeated exposure by oral route. Hence the test chemical is not likely to classify as a toxicant upon repeated exposure by oral route.

Repeated dose toxicity: inhalation
According to Annex IX of the REACH regulation, testing by the inhalation route is appropriate only if exposure of humans via inhalation is likely. Taking into account the low vapour pressure of the substance sodium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate, which is reported as 6.93E-16 Pa. Thus, exposure to inhalable dust, mist and vapour of the chemical sodium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate is highly unlikely. Therefore this study is considered for waiver.

Repeated dose toxicity: dermal
The acute toxicity value for sodium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate (as provided in section 7.2.3) is >2000 mg/kg body weight. Thus, it is expected that sodium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate shall not exhibit 28 day repeated dose toxicity by the dermal route. In addition, there is no dermal absorption data as well as no data available that suggests that sodium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate shall exhibit repeated dose toxicity by the dermal route. Hence this end point was considered for waiver.

Justification for classification or non-classification

The data available for the target chemical metanil yellow is insufficient to classify the chemical as toxic. Also the NOAEL value range can be close to 500 mg/Kg bw. Based on the observations made, Metanil yellow does not exhibit toxicity upon repeated exposure by oral route. Hence the test chemical is not likely to classify as a toxicant upon repeated exposure by oral route.