Sodium 3-(p-anilinophenylazo)benzenesulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Salmonella/ mammalian-microsome test was performed to evaluate the mutagenic nature of the test compound Ext. D&C Yellow No. 1. The study was performed using Salmonella typhimurium strains TA98, TA1537, TA100, TA1535 with and without S9 metabolic activation system. The test material was dissolved in DMSO and used at dose levels from 10-250 mg. DMSO was used as the solvent control. The 2 ml of liquid top agar was cooled to 45°C and 0.1 ml of a broth culture of microorganism and test substance in volumes of ≤ 0.4 ml of DMSO was added prior to placing on minimal agar plates. After 48 h incubation at 37°C, the colonies which reverted to the prototroph were counted and compared to counts on the control plate (containing no test substance) to demonstrate mutagenicity or toxicity. Materials which caused a 2-fold increase of revertants, as compared to the number of spontaneous revertants on the control plates, were denoted as mutagens. Those which reduced the number of revertants were considered inhibitory. Ext. D&C Yellow No. 1 did not induce genemutation in Salmonella typhimurium TA98, TA1537, TA100, TA1535 in the presence and absence of S9 metabolic activation system and hence it is negative for gene mutation in vitro.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

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:

Salmonella/ mammalian-microsome test was performed to evaluate the mutagenic nature of the test compound Ext. D&C Yellow No. 1

GLP compliance:

not specified

Type of assay:

bacterial gene mutation assay

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: solid
- Purity: No data available
- Impurities (identity and concentrations): no data available

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

The microsomal fraction (S9) was prepared from male Sprague-Dawley rats weighing approximately 200 g each.

Test concentrations with justification for top dose:

10-250 mg

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

not specified

Details on test system and experimental conditions:

METHOD OF APPLICATION: Plate incorporation assay

DURATION
- Preincubation period: No data available
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: No data available

NUMBER OF CELLS EVALUATED: No data available

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data available

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other: No data available

OTHER: No data available

Rationale for test conditions:

No data

Evaluation criteria:

Material which caused a 2-fold increase of revertants, as compared to the number of spontaneous revertants on the control plates, was denoted as mutagens. Those which reduced the number of revertants were considered inhibitory.

Statistics:

No data available

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

No data available

Conclusions:

Ext. D&C Yellow No. 1 did not induce gene mutation in Salmonella typhimurium TA98, TA1537, TA100, TA1535 in the presence and absence of S9 metabolic activation system and hence it is negative for gene mutation in vitro.

Executive summary:

Salmonella/ mammalian-microsome test was performed to evaluate the mutagenic nature of the test compound Ext. D&C Yellow No. 1. The study was performed using Salmonella typhimurium strains TA98, TA1537, TA100, TA1535 with and without S9 metabolic activation system. The test material was dissolved in DMSO and used at dose levels from 10-250 mg. DMSO was used as the solvent control.

 

The 2 ml of liquid top agar was cooled to 45°C and 0.1 ml of a broth culture of microorganism and test substance in volumes of ≤ 0.4 ml of DMSO was added prior to placing on minimal agar plates.

 

After 48 h incubation at 37°C, the colonies which reverted to the prototroph were counted and compared to counts on the control plate (containing no test substance) to demonstrate mutagenicity or toxicity.

 

Materials which caused a 2-fold increase of revertants, as compared to the number of spontaneous revertants on the control plates, were denoted as mutagens. Those which reduced the number of revertants were considered inhibitory.

 

Ext. D&C Yellow No. 1 did not induce genemutation inSalmonella typhimuriumTA98, TA1537, TA100, TA1535 in the presence and absence of S9 metabolic activation system and hence it is negative for gene mutation in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo mammalian chromosome aberration assay was performed to determine the clastogenic nature of Metanil yellow. Laboratory bred 10 Swiss albino male mice were given metanil yellow in distilled water by gavage at dose level of 0 or 2 mg/Kg bw once daily for 30 days.Controls were fed 0.05 ml of distilled water daily for 30 days. The animals were killed on the completion of 30 days treatment, starting from 0 day. Colchicine (0.04%) was injected intraperitoneally at the rate of 1 ml/100 g body wt, 90 min before the animal was killed.

 

For chromosome studies, flame dried preparations of bone marrow chromosomes were stained in Giemsa by the usual schedule. A total of 600 metaphase plates were scanned from the 10 animals killed in each set of experiments and the slides were observed for chromatid and chromosomal breaks, chromatid and chromosomal exchanges and number of breaks per metaphase was analysed by the Z/B ratio.

 

A significant increase in the frequency of the chromosomal aberrations (index Z/B) was found with metanil yellow treated series when compared with the controls.

 

Metanil yellow induced chromosome aberrations in the bone marrow of Swiss albino male mice at a dose level of 2 mg/Kg bw and hence it is likely to be mutagenic in vitro.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

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:

In vivo mammalian chromosome aberration assay was performed to determine the clastogenic nature of Metanil yellow

GLP compliance:

not specified

Type of assay:

other: In vivo mammalian bone marrow chromosome aberration assay

Specific details on test material used for the study:

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

Species:

mouse

Strain:

Swiss

Remarks:

Albino

Details on species / strain selection:

No data

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Laboratory bred animals were used for the study
- Age at study initiation: Between 90-100 days
- Weight at study initiation: 30 g approx..
- Assigned to test groups randomly: No data
- Fasting period before study: No data
- Housing: No data
- Diet (e.g. ad libitum): No data
- Water (e.g. ad libitum): No data
- 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

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle: The chemical was soluble in distilled water
- Concentration of test material in vehicle: 0 or 2 mg/Kg bw
- Amount of vehicle (if gavage or dermal): 0.05mL
- Type and concentration of dispersant aid (if powder): No data
- Lot/batch no. (if required): No data
- Purity: No data

Details on exposure:

For oral route
PREPARATION OF DOSING SOLUTIONS: Metanil yellow was dissolved in distilled water at dose level of 2 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

Duration of treatment / exposure:

30 days

Frequency of treatment:

Once, daily

Post exposure period:

No data

Remarks:

0 or 2 mg/Kg bw

No. of animals per sex per dose:

Total: 20
0 mg/Kg bw: 10 mice
2 mg/Kg bw: 10 mice

Control animals:

yes, concurrent vehicle

Positive control(s):

No data

Tissues and cell types examined:

Bone marrow chromosomes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The dose in this experiment was calculated based on the idea that 200 ppm of the dye is allowed in food in India. The dose selected for dye depends on the assumption that a person can consume a maximum of about 100 mg of dye from foods every day. If a person weighing 50 kg consumes 100 mg of dye or nitrite per day then automatically the value comes to 2 mg/kg. These doses may appear a bit higher than normal human consumption, but
since the investigation was limited to only 30 days, it is conjectured that these doses were justified if the cumulative effects of these dyes on human beings for years was considered.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Afetr 30 days treatment with metanil yellow

DETAILS OF SLIDE PREPARATION: Colchicine (0.04%) was injected intraperitoneally at the rate of 1 ml/100 g body wt, 90 min before the animal was killed. For chromosome studies, flame dried preparations of bone marrow chromosomes were stained in Giemsa by the usual schedule

METHOD OF ANALYSIS: No data

OTHER: Chromosomal aberrations were scanned at the metaphase stage.

Evaluation criteria:

Chromatid and chromosomal breaks, chromatid and chromosomal exchanges and cells with more than 10 aberrations were considered basically abnormal and hence scored accordingly.

The number of breaks per metaphase analysed Z/B, represents the fundamental index for comparison. In the Z/B category were the chromatid and chromosomal breaks, exchanges, dicentric chromosomes and cells with more than 10 aberrations. In qualitative evaluation the number of breaks were added in the following way: chromatid break, 1 break; chromosomal break, 1 break; chromosomal exchange, 2 breaks; dicentric chromosomes, 2 breaks; cells with more than 10 aberrations, 10 breaks. Gaps were not included

Statistics:

No data

Sex:

male

Genotoxicity:

positive

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

No data

TABLE 1

Chromosomal Aberrations Induced by Metanil yellow on Bone Marrow Cells of Mice

Substance	Dose (mg/Kg bw)	No. of animals	n^a	Aberrant cells		Breaks	Cells with 10 aberrations	Z/B^b
				No.	%``
Distilled water (control)	0	10	600	11	1.83	12	2	0.053
Metanil yellow	2	10	600	89	14.83	44	55	0.990

^an = number of cells analysed.

^bZ/B = number of aberrations per cell

Conclusions:

Metanil yellow induced chromosome aberrations in the bone marrow of Swiss albino male mice at a dose level of 2 mg/Kg bw and hence it is likely to be mutagenic in vivo.

Executive summary:

In vivo mammalian chromosome aberration assay was performed to determine the clastogenic nature of Metanil yellow. Laboratory bred 10 Swiss albino male mice were given metanil yellow in distilled water by gavage at dose level of 0 or 2 mg/Kg bw once daily for 30 days.Controls were fed 0.05 ml of distilled water daily for 30 days. The animals were killed on the completion of 30 days treatment, starting from 0 day. Colchicine (0.04%) was injected intraperitoneally at the rate of 1 ml/100 g body wt, 90 min before the animal was killed.

 

For chromosome studies, flame dried preparations of bone marrow chromosomes were stained in Giemsa by the usual schedule. A total of 600 metaphase plates were scanned from the 10 animals killed in each set of experiments and the slides were observed for chromatid and chromosomal breaks, chromatid and chromosomal exchanges and number of breaks per metaphase was analysed by the Z/B ratio.

 

A significant increase in the frequency of the chromosomal aberrations (index Z/B) was found with metanil yellow treated series when compared with the controls.

 

Metanil yellow induced chromosome aberrations in the bone marrow of Swiss albino male mice at a dose level of 2 mg/Kg bw and hence it is likely to be mutagenic in vivo.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Additional information

Gene mutation in vitro:

Various peer reviewed publications were reviewed to determine the mutagenic nature of Metanil yellow (IUPAC name: sodium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate). The studies are as mentioned below:

Salmonella/ mammalian-microsome test was performed by Muzall and Cook (Mutation Research, 1979) to evaluate the mutagenic nature of the test compound Ext. D&C Yellow No. 1 (CAS no 587 -98 -4). The study was performed using Salmonella typhimurium strains TA98, TA1537, TA100, TA1535 with and without S9 metabolic activation system. The test material was dissolved in DMSO and used at dose levels from 10-250 mg. DMSO was used as the solvent control. The 2 ml of liquid top agar was cooled to 45°C and 0.1 ml of a broth culture of microorganism and test substance in volumes of ≤ 0.4 ml of DMSO was added prior to placing on minimal agar plates. After 48 h incubation at 37°C, the colonies which reverted to the prototroph were counted and compared to counts on the control plate (containing no test substance) to demonstrate mutagenicity or toxicity. Materials which caused a 2-fold increase of revertants, as compared to the number of spontaneous revertants on the control plates, were denoted as mutagens. Those which reduced the number of revertants were considered inhibitory. Ext. D&C Yellow No. 1 did not induce gene mutation in Salmonella typhimurium TA98, TA1537, TA100, TA1535 in the presence and absence of S9 metabolic activation system and hence it is negative for gene mutation in vitro.

In the same study by Muzall and Cook (Mutation Research, 1979), Salmonella/ mammalian-microsome test (Spot test) was performed to evaluate the mutagenic nature of Ext. D&C Yellow No. 1 (CAS no 587 -98 -4). The spot test was used to screen the test material for potential mutagenicity. The test material was placed in the center of the plate. The test compound was tested with and without the S9 mix. Inhibition of the bacterium was indicated by a clearing of the background lawn in a zone surrounding the test material. Mutagenicity was indicated by a clustering of revertant colonies directly around the test material or at the edge of the inhibitory zone. A known mutagen, Captan, was used as a reference mutagen. Ext. D&C Yellow No. 1 did not induce genemutation gene mutation in Salmonella typhimurium TA98, TA1537, TA100, TA1535 in the presence and absence of S9 metabolic activation system in the spot test performed and hence is negative for gene mutation in vitro.

In another study by Szybalski (Annals of the NY Academy of Science, 1958), Gene mutation toxicity study was performed to determine the mutagenic nature of metanil yellow (CAS no 587 -98 -4). Genetic toxicity test was performed on strain of Escherichia coli (strain Sd-4-73) by paper disk method. Paper-disk method was performed to check for the ability of E. coli Sd-4-73 to show reversion from streptomycin dependence to independence. Overnight culture of strain Sd-4-73 grown at 36°C in aerated nutrient broth containing 20 µg/ml of streptomycin was used as a inoculum. The culture was centrifuged and washed at least twice with saline or distilled water to remove the extraneous streptomycin, and was resuspended in saline to a concentration of approximately 109 cells/ml. 0.1-ml aliquot of this suspension was mixed with 2.5 ml of molten soft nutrient agar (0.7 per cent agar) and poured over a base of 20 ml of 1.5 per cent nutrient agar. After the soft layer was solidified, the mutagen was applied in form of small drops or crystal. Additional plates were prepared with small inocula (one fifth and one twenty-fifth of the original) so as not to miss the optimum cell density; the number of cells per plate was rather critical, the yield of mutant colonies being reduced either by crowding or by insufficient population size. After the soft agar layer had set, the mutagen, in the form of a microdrop of solution (0.01 to 0.025 ml.) or a small crystal, was applied to a small filter-paper disk resting on the agar. To determine whether the substance was inhibitory for the assay organism at the concentration employed, the procedure was repeated on a nutrient agar plate containing 100 µg/ml of streptomycin and seeded with approximately 10⁷bacteria. Mutagenicity was manifested as a zone of streptomycin- independent mutant colonies around a filter-paper disk saturated with the mutagenic agent and resting on the surface of streptomycin-free nutrient agar seeded heavily with a streptomycin-dependent parental population. Metanil yellow did not induce mutation from streptomycin dependence to independence in Escherichia coli (strain Sd-4-73) and hence it is not likely to classify as a gene mutant in vitro.

Based on the data available for the target chemical, metanil yellow does not exhibit gene mutation in vitro.

Gene mutation in vivo:

Various peer reviewed publicatiosns were reviewed to determine the mutagenic nature of Metanil yellow in vivo. The studies are as mentioned below:

In vivo mammalian chromosome aberration assay was performed by Giri et al (Cancer letters, 1986) to determine the clastogenic nature of Metanil yellow. Laboratory bred 10 Swiss albino male mice were given metanil yellow in distilled water by gavage at dose level of 0 or 2 mg/Kg bw once daily for 30 days.Controls were fed 0.05 ml of distilled water daily for 30 days. The animals were killed on the completion of 30 days treatment, starting from 0 day. Colchicine (0.04%) was injected intraperitoneally at the rate of 1 ml/100 g body wt, 90 min before the animal was killed. For chromosome studies, flame dried preparations of bone marrow chromosomes were stained in Giemsa by the usual schedule. A total of 600 metaphase plates were scanned from the 10 animals killed in each set of experiments and the slides were observed for chromatid and chromosomal breaks, chromatid and chromosomal exchanges and number of breaks per metaphase was analysed by the Z/B ratio. A significant increase in the frequency of the chromosomal aberrations (index Z/B) was found with metanil yellow treated series when compared with the controls. Metanil yellow induced chromosome aberrations in the bone marrow of Swiss albino male mice at a dose level of 2 mg/Kg bw and hence it is likely to be mutagenic in vivo.

In yet another study performed by Giri et al (Cancer letters, 1986) In vivo sister chromatid exchange assay was performed to determine the mutagenic nature of Metanil yellow. 7 Laboratory bred Swiss albino male mice each were injected intraperitoneally with 0, 2.5, 5, 10, 20, 40, 100 and 200 mg/kg body wt metanil yellow respectively dissolved in DMSO. Before treatment with metanil yellow, each mouse was implanted subcutaneously in the neck with a 50 mg tablet of 5-bromodeoxyuridine (BrDu). All mice received an i.p. injection of colchicine (5 mg/kg) 21-22 h after subcutaneous implantation of BrDU. Two hours later the animals were killed by cervical dislocation and bone marrow chromosomes were prepared for sister chromatid exchange observations. After 24 hrs treatment period, the slides were observed for the induction of SCEs. Marked increase in the frequency of SCEs was observed in all the treated series except 2.5 mg/kg conc. when compared with distilled water controls. When given the highest concentration, i.e. 200 mg/kg, 2 mice died in the metanil yellow treated group. Also this concentration was apparently too toxic for the cells. Metanil yellow induced sister chromatid exchanges in the bone marrow of Swiss albino male mice and hence it is likely to be mutagenic in vivo.

In another study, In vivo mammalian chromosome aberration assay was performed by Giri et al (Cytologia, 1988) to determine the mutagenic nature of Metanil yellow. 7 Swiss albino male mice were given doses of metanil yellow at dose levels of 0 or 2 mg/Kg bw by gavage for 30 days. After 30 days, seven treated mice and all the control mice were killed by cervical dislocation. A significant increase in the frequency of chromosomal aberrations (index Z/B) was found in the bone marrow of mice treated with metanil yellow for 30 days. Based on the observation made, metanil yellow can be considered to be a strong clastogen in vivo.

Based on the data available for the target chemical, metanil yellow (CAS no 587 -98 -4; IUPAC name: sodium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate) does exhibit gene mutation in vivo.

Since the availability of the data is from old studies for genetic toxicity in vitro and in vivo, hence new studies should be conducted to determine the toxic nature of metanil yellow using chromosome aberration assay.

Justification for classification or non-classification

Based on the data available for the target chemical, metanil yellow (CAS no 587 -98 -4; IUPAC name: sodium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate) does not exhibit gene mutation in vitro. However, it does exhibit gene mutation in vivo.

Since the availability of the data is from old studies for genetic toxicity in vitro and in vivo, hence new studies should be conducted to determine the toxic nature of metanil yellow using chromosome aberration assay.