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)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in vitro

Ames test:

The test chemical 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 not induce gene mutation in Salmonella typhimurium strains in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Chromosomal aberration study:

The test chemical 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) did not induce a doubling of the mutant frequency both in the presence and absence of S9 activation system in the in vitro mammalian cell gene mutation assay and it did not induce DNA damage in the in vitro rat hepatocyte primary culture/ DNA repair assay test and hence is not likely to be gene mutant in vitro.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

experimental data of read across substances

Justification for type of information:

Data for the target chemical is summarized based on the structurally similar read across chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

WoE derived based on the experimental data from struturally similar read across chemicals

GLP compliance:

not specified

Type of assay:

bacterial gene mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium, other: TA1535, TA1537, TA1538, TA100 and TA98 were used.

Remarks:

1

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium, other: TA98, TA100, TA1535, TA1537, and TA1538

Remarks:

2

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: Salmonella typhimurium is a histidine auxotroph strain.

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

S9 mix was prepared from Aroclor 1254-induced male Fischer 344 rats and Syrian golden hamsters.

Test concentrations with justification for top dose:

1. 0, 333.0, 1000.0, 3333.0, 6666.0, 10000.0 µg/plate2. 10-10000 µg /plate

Vehicle / solvent:

1.- Vehicle(s)/solvent(s) used: The solvents used were water, dimethyl sulfoxide, and acetone (exact solvent details are not available)- Justification for choice of solvent/vehicle: Test chemical solubility in the solvents mentioned2. - Vehicle(s)/solvent(s) used: Appropriate solvent was used for preparing the stock solution of the test chemical. Name of the solvent is not specified in the study.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene (All strains ; with S9)

Remarks:

1

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

1. METHOD OF APPLICATION: plate incorporation.DURATION- Preincubation period: No data available- Exposure duration: No data available- Expression time (cells in growth medium): No data available- 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 availableSELECTION AGENT (mutation assays): No data availableSPINDLE INHIBITOR (cytogenetic assays): No data availableSTAIN (for cytogenetic assays): No data availableNUMBER OF REPLICATIONS: The chemical was tested at 5 dose levels in triplicate.NUMBER OF CELLS EVALUATED: No data availableDETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other: No data availableOTHER EXAMINATIONS:- Determination of polyploidy: No data available- Determination of endoreplication: No data available- Other: No data availableOTHER: No data available 2. METHOD OF APPLICATION: in agar (plate incorporation)DURATION- Preincubation period: No data available- Exposure duration: 48 h - Expression time (cells in growth medium): 48 h- 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 availableSELECTION AGENT (mutation assays): No data availableSPINDLE INHIBITOR (cytogenetic assays): No data availableSTAIN (for cytogenetic assays): No data availableNUMBER OF REPLICATIONS: TriplicateNUMBER OF CELLS EVALUATED: No data availableDETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other: No data availableOTHER EXAMINATIONS:- Determination of polyploidy: No data available- Determination of endoreplication: No data available- Other: No data availableOTHER: No data available

Rationale for test conditions:

No data available

Evaluation criteria:

1. A response was considered positive if there was a dose-related increase in the number of revertants above spontaneous solvent controls with a 2-fold increase for strains TA1535, TA1538, TA98 and TA100, and a 3-fold increase for TA15372. The criteria used to evaluate a test were as follows: for a test article to be considered positive, it had to induce at least a doubling (TA98, TA100, and TA1535) in the mean number of revertants per plate of at least one tester strain. This increase in the mean revertants per plate had to be accompanied by a dose response to increasing concentrations of the test chemical. If the study showed a dose response with a less than 3-fold increase on TA1537 or TA1538, the response had to be confirmed in a repeat experiment.

Statistics:

No data available

Species / strain:

S. typhimurium, other: TA1535, TA1537, TA1538, TA100 and TA98

Remarks:

RA 1

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:

valid

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537, and TA1538

Remarks:

2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

1. TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH: No data available- Effects of osmolality: No data available- Evaporation from medium: No data available- Water solubility: No data available- Precipitation: No data available- Other confounding effects: No data availableRANGE-FINDING/SCREENING STUDIES: The range of concentrations for testing was based on preliminary toxicity tests in which the viability of the bacterial cells on complete medium was measured at concentrations up to 10 mg/plate or to the limit of solubility. When solubility and toxicity were not limiting factors, the maximum concentration tested was 10 mg/plate.COMPARISON WITH HISTORICAL CONTROL DATA: No data availableADDITIONAL INFORMATION ON CYTOTOXICITY: No data available2. TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH: No data available- Effects of osmolality: No data available- Evaporation from medium: No data available- Water solubility: No data available- Precipitation: No data available- Other confounding effects: No data availableRANGE-FINDING/SCREENING STUDIES: The doses that were tested in the mutagenicity assay were selected based on the levels of cytotoxicity observed in a preliminary dose range-finding study using strain TA100. Ten dose levels of the chemical, one plate per dose, were tested in both the presence and the absence of induced hamster S9. If no toxicity was observed, a total maximum dose of 10 mg of test chemical per plate was used. COMPARISON WITH HISTORICAL CONTROL DATA: No data availableADDITIONAL INFORMATION ON CYTOTOXICITY: No data available

Remarks on result:

other: No mutagenic potential

Conclusions:

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 not induce gene mutation in Salmonella typhimurium strains in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Executive summary:

Various studies availave for the test chemical were reviewed to determine the mutagenic 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 mentioned below:

Bacterial gene mutation assay was performed for the test material in Salmonella typhimurium TA1535, TA1537, TA1538, TA100 and TA98 strains at a dose range of 0, 333.0, 1000.0, 3333.0, 6666.0 and 10000.0 µg/plate by Plate-incorporation method. Chemical was tested without metabolic activation and with S9 mix from Aroclor 1254-induced male Fischer 344 rats and Syrian golden hamsters. Appropriate positive and solvent controls were also incorporated in the study. Doses for the main study were based on the prelimicary study conducted. A response was considered positive if there was a dose-related increase in the number of revertants above spontaneous solvent controls with a 2-fold increase for strains TA1535, TA1538, TA98 and TA100, and a 3-fold increase for TA1537. The dye did not induce gene mutation in Salmonella typhimurium strains TA1535, TA1537, TA1538, TA100 and TA98 in the presence and absence of S9 metabolic activation system by plate-incorporation assay and hence is not likely to classify as a gene mutant in vitro.

In another study, Ames mutagenicity test was conducted for the test chemical to evaluate its genetoxic effects when exposed to Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538 with dose concentration of 100-10000 µg/plate in plate incorporation hamster reductive assay. Based on the preliminary study conducted, the test compound was used at a five dose level from 100- 10000 µg/plate. The given test chemical did not induce gene mutation in the Salmonella typhimurium TA98, TA100, TA1535, TA1537, and TA1538 both in the presence and absence of S9 activation system and hence it is not likely to be a gene mutant.

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 not induce gene mutation in Salmonella typhimurium strains in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

experimental data of read across substances

Justification for type of information:

Data for the target chemical is summarized based on the structurally similar read across chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

WoE derived based on the experimental data from structurally and functionally similar read across chemicals

GLP compliance:

not specified

Type of assay:

other: In vitro Mammalian cell gene mutation assay

Target gene:

1. Thymidine kinase2. No data

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

TK+/- 3.7.C / 1

Details on mammalian cell type (if applicable):

- Type and identity of media: The cells were grown in Fischer’s medium for leukemic cells of mice supplemented with 10% horse serum and 0.02% pluronic F-68.- Properly maintained: No data available - Periodically checked for Mycoplasma contamination: Yes- Periodically checked for karyotype stability: No data available- Periodically "cleansed" against high spontaneous background: No data available

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

hepatocytes: Rat hepatocytes

Details on mammalian cell type (if applicable):

- Type and identity of media: No data available- Properly maintained: No data available- Periodically checked for Mycoplasma contamination: No data available- Periodically checked for karyotype stability: No data available- Periodically "cleansed" against high spontaneous background: No data available

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 prepared from Aroclor 1254-induced male Sprague- Dawley rats.

Test concentrations with justification for top dose:

1. 685-5000 µg/mL2. 1X 10-3 ,1X 10-4 ,1X 10-5

Vehicle / solvent:

1. No data available2. Corn oil

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: 3-methylcholanthrene with S9

Remarks:

1/2

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Corn oil

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 4-DMAS

Details on test system and experimental conditions:

1.METHOD OF APPLICATION: in mediumCells at start: 6000000 cellsDURATION- Preincubation period: No data available- Exposure duration: 4 h- Expression time (cells in growth medium):48 h- 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 availableSELECTION AGENT (mutation assays): 1×106 cells/plate for mutant selectionSPINDLE INHIBITOR (cytogenetic assays): No data availableSTAIN (for cytogenetic assays): No data availableNUMBER OF REPLICATIONS: DuplicateNUMBER OF CELLS EVALUATED: 1 X 106 cells/plate for mutant selection and 200cells/plate for viable count determinations DETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other: The rate of cell growth was determined for each of the treated culturesOTHER EXAMINATIONS:- Determination of polyploidy: No data available- Determination of endoreplication: No data available- Other: No data availableOTHER: No data available2. METHOD OF APPLICATION: cultured by the two-step in situ liver perfusion DURATION- Preincubation period: No data available- Exposure duration: 4hrs- Expression time (cells in growth medium): No data available- 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 availableSELECTION AGENT (mutation assays): [3H]-thymidine incubation SPINDLE INHIBITOR (cytogenetic assays): No data availableSTAIN (for cytogenetic assays): No data availableNUMBER OF REPLICATIONS: No data availableNUMBER OF CELLS EVALUATED: 2 X 105 viable hepatocytesDETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; No data available OTHER EXAMINATIONS:- Determination of polyploidy: No data available- Determination of endoreplication: No data available- Other: No data availableOTHER: No data available

Rationale for test conditions:

No data

Evaluation criteria:

1. Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase. Doubling of the mutant frequency was previously reported as representing a positive effect. Only doses yielding total growth values of 10% were used in the analysis of induced mutant frequency. Doses yielding less than 10% total growth were used in determining dose response.2. •Average Net nuclear grain: count below zero: Negative•average net nuclear grain: count between zero and 5: weakly positive•Average Net nuclear grain: count above five: PositiveThe nuclei isolated from the control lymphocytes were round-shaped and did not form comets. After treatment with BB , nuclei with variable shapes and comets of different lengths appeared and the percentage of classII and III nuclei rose gradually with increased concentrations of BB. No intact nuclei representing class I was found after BB treatment at the highest concentration.

Statistics:

No data

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

TK+/- 3.7.C /1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

hepatocytes: Rat hepatocytes

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

1. TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH: No data available- Effects of osmolality: No data available- Evaporation from medium: No data available- Water solubility: No data available- Precipitation: No data available- Other confounding effects: No data availableRANGE-FINDING/SCREENING STUDIES: No data availableCOMPARISON WITH HISTORICAL CONTROL DATA: No data availableADDITIONAL INFORMATION ON CYTOTOXICITY: The doses of chemical selected for testing were within the range yielding approximately 0-90% cytotoxicity.2. No data

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical 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) did not induce a doubling of the mutant frequency both in the presence and absence of S9 activation system in the in vitro mammalian cell gene mutation assay and it did not induce DNA damage in the in vitro rat hepatocyte primary culture/ DNA repair assay test and hence is not likely to be gene mutant in vitro.

Executive summary:

Data available for the test chemicals was reviewed to determine the mutagenic 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 mentioned below:

The gene mutation study was conducted according to L5178Y TK+/- Mouse Lymphoma Mutagenicity Assay to determine the mutagenic nature of the test chemical. The Cells at a concentration of 6 X 10⁵/mL (6 X10⁶cells total) were exposed for 4 h to a range of concentrations from 0.001-6µg/mL of the test chemical. The cells were then washed, resuspended in growth medium, and incubated at 37°C for 48 h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls. Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase. The test chemical did not induce a doubling of the mutant frequency both in the presence and absence of S9 activation system and hence is not likely to be gene mutant in vitro.

In another study, the in vitro genetic toxicity test was performed on rat hepatocytes with following concentrations (1X 10-31X 10-41X 10-5). Rat hepatocytes were isolated and cultured by the two-step in situ liver perfusion method.2 X 105viable hepatocytes were seeded into 25-mm wells and were allowed to attach to plastic cover slips for 2 hr.The cells were then incubated for 4 hr with [3H]-thymidine. Net nuclear grains (NNG) were determined by counting the number of grains in each nuclei and subtracting the average number of grains present in three equal-size adjacent cytoplasmic areas. From the following criteria the result will calculated,

Average Net nuclear grain: count below zero: Negative

Average net nuclear grain: count between zero and 5: weakly positive

Average Net nuclear grain: count above five: Positive

From the above table it was found that Avg. NNG for Food black 1was found to be below zero.

 Therefore, the genetic toxicity for In Vitro Rat Hepatocyte Primary Culture/ DNA Repair Assay test was found to be negative by using the test chemical.

Based on the obserbations made, the test chemical 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) did not induce a doubling of the mutant frequency both in the presence and absence of S9 activation system in the in vitro mammalian cell gene mutation assay and it did not induce DNA damage in the in vitro rat hepatocyte primary culture/ DNA repair assay test and hence is not likely to be gene mutant in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation in vitro

Data available for the test chemicals was reviewed to determine the mutagenic 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 mentioned below:

Ames test:

Bacterial gene mutation assay was performed for the test material in Salmonella typhimuriumTA1535, TA1537, TA1538, TA100 and TA98 strains at a dose range of 0, 333.0, 1000.0, 3333.0, 6666.0 and 10000.0 µg/plate by Plate-incorporation method. Chemical was tested without metabolic activation and with S9 mix from Aroclor 1254-induced male Fischer 344 rats and Syrian golden hamsters. Appropriate positive and solvent controls were also incorporated in the study. Doses for the main study were based on the prelimicary study conducted. A response was considered positive if there was a dose-related increase in the number of revertants above spontaneous solvent controls with a 2-fold increase for strains TA1535, TA1538, TA98 and TA100, and a 3-fold increase for TA1537. The dye did not induce gene mutation in Salmonella typhimurium strains TA1535, TA1537, TA1538, TA100 and TA98 in the presence and absence of S9 metabolic activation system by plate-incorporation assay and hence is not likely to classify as a gene mutant in vitro.

In another study, Ames mutagenicity test was conducted for the test chemical to evaluate its genetoxic effects when exposed to Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538 with dose concentration of 100-10000 µg/plate in plate incorporation hamster reductive assay. Based on the preliminary study conducted, the test compound was used at a five dose level from 100- 10000 µg/plate. The given test chemical did not induce gene mutation in the Salmonella typhimurium TA98, TA100, TA1535, TA1537, and TA1538 both in the presence and absence of S9 activation system and hence it is not likely to be a gene mutant.

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 not induce gene mutation in Salmonella typhimurium strains in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Chromosomal aberration study:

The gene mutation study was conducted according to L5178Y TK+/- Mouse Lymphoma Mutagenicity Assay to determine the mutagenic nature of the test chemical. The Cells at a concentration of 6 X 10⁵/mL (6 X10⁶cells total) were exposed for 4 h to a range of concentrations from 0.001-6µg/mL of the test chemical. The cells were then washed, resuspended in growth medium, and incubated at 37°C for 48 h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls. Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase. The test chemical did not induce a doubling of the mutant frequency both in the presence and absence of S9 activation system and hence is not likely to be gene mutant in vitro.

In another study, the in vitro genetic toxicity test was performed on rat hepatocytes with following concentrations (1X 10-31X 10-41X 10-5). Rat hepatocytes were isolated and cultured by the two-step in situ liver perfusion method.2 X 105viable hepatocytes were seeded into 25-mm wells and were allowed to attach to plastic cover slips for 2 hr.The cells were then incubated for 4 hr with [3H]-thymidine. Net nuclear grains (NNG) were determined by counting the number of grains in each nuclei and subtracting the average number of grains present in three equal-size adjacent cytoplasmic areas. From the following criteria the result will calculated,

Average Net nuclear grain: count below zero: Negative

Average net nuclear grain: count between zero and 5: weakly positive

Average Net nuclear grain: count above five: Positive

From the above table it was found that Avg. NNG for Food black 1was found to be below zero.

 Therefore, the genetic toxicity for In Vitro Rat Hepatocyte Primary Culture/ DNA Repair Assay test was found to be negative by using the test chemical.

Based on the obserbations made, the test chemical 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) did not induce a doubling of the mutant frequency both in the presence and absence of S9 activation system in the in vitro mammalian cell gene mutation assay and it did not induce DNA damage in the in vitro rat hepatocyte primary culture/ DNA repair assay test and hence is not likely to be gene mutant in vitro.

Thus, based on the data available for the test chemical and applying weight of evidence approach, the test chemical

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) does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant in vitro as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the data available for the test chemical and applying weight of evidence approach, the test chemical

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) does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant in vitro as per the criteria mentioned in CLP regulation.