2-Naphthalenol, 1-[[2-methyl-4-[(2-methylphenyl)azo]phenyl]azo]-, ar-styrenated

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in vitro:

Ames test:

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.

Chromosome aberration:

Test chemical did not induce gene mutation in the Chinese hamster ovary cells and Chinese hamster ovary cells (CHO-W-B1) both in the presence and absence of S9 activation system and hence it is not likely to be a gene mutant.

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

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 read across from three similar chemicals

GLP compliance:

not specified

Type of assay:

bacterial gene mutation assay

Target gene:

Histidine

Species / strain / cell type:

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

Remarks:

RA 1

Details on mammalian cell type (if applicable):

No data available

Additional strain / cell type characteristics:

other: Salmonella typhimurium is a histidine auxotroph strain.

Species / strain / cell type:

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

Remarks:

RA 3

Details on mammalian cell type (if applicable):

No data

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 homogenate was prepared from male Syrian golden hamsters that had been injected with Aroclor 1254 at 500 mg/kg body weight.

Test concentrations with justification for top dose:

1. 10-10000 µg /plate2. 200 µg/Plate3. 10-250 mg

Vehicle / solvent:

1. - 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.2. - Vehicle(s)/solvent(s) used: DMSO- Justification for choice of solvent/vehicle: the test chemical was soluble in DMSO3.- Vehicle(s)/solvent(s) used: DMSO- Justification for choice of solvent/vehicle: No data available

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Remarks:

RA 1

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

ethylmethanesulphonate

methylmethanesulfonate

other: Anthragallol

Remarks:

RA 2

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Remarks:

RA 3

Details on test system and experimental conditions:

1. 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 available2. METHOD OF APPLICATION: Spot testDURATION- Preincubation period: No data- Exposure duration: 72 hrs- Expression time (cells in growth medium): 72 hrs- Selection time (if incubation with a selection agent): No data- Fixation time (start of exposure up to fixation or harvest of cells): No dataSELECTION AGENT (mutation assays): No dataSPINDLE INHIBITOR (cytogenetic assays): No dataSTAIN (for cytogenetic assays): No dataNUMBER OF REPLICATIONS: No dataNUMBER OF CELLS EVALUATED: No dataDETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other: No dataOTHER EXAMINATIONS:- Determination of polyploidy: No data- Determination of endoreplication: No data- Other: No data OTHER: No data3. METHOD OF APPLICATION: Plate incorporation assayDURATION- 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 availableSELECTION AGENT (mutation assays): No data availableSPINDLE INHIBITOR (cytogenetic assays): No data availableSTAIN (for cytogenetic assays): No data availableNUMBER OF REPLICATIONS: No data availableNUMBER 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

Evaluation criteria:

1. 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.2. The criteria adopted for scoring a mutagenic response in routine plate tests was that the observed number of revertants should exceed twice the background value for that given assay and exceed the 99.9% confidence limit based on our historical controls.3. 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:

1. No data 2. No data3. No data

Species / strain:

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

Remarks:

RA 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:

not specified

Species / strain:

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

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:

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

Remarks:

RA 3

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:

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 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 available2. No data3. No data

Remarks on result:

other: No mutagenic potential

Conclusions:

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.

Executive summary:

Gene mutation toxicity for the test chemical 2-Naphthalenol, 1-[[2-methyl-4-[(2-methylphenyl) azo] phenyl] azo]-, ar-styrenated (CAS no 85203 -90 -3) was predicted using the data from read across chemicals. The studies are as mentioned below:

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.

Gene mutation by the spot assay was performed for another test chemical. The test chemical was used at dose level of 200µg/plate both with and without S9 metabolic activation system. The plates were incubated for 3 days at 37°C for the growth of colonies to occur. The plates were observed for an increase in the number of His+ revertants. The criteria adopted for scoring a mutagenic response in routine plate tests was that the observed number of revertants should exceed twice the background value for that given assay and exceed the 99.9% confidence limit based on our historical controls. Concurrent positive control was also included in the study. The test chemical failed to induce mutation in Salmonellatyphimurium strain TA100, TA98, TA1535, TA1537 and TA1538 with and without S9 metabolic activation system in the spot test performed and hence is not likely to classify for gene mutation in vitro.

Salmonella/ mammalian-microsome test was performed to evaluate the mutagenic nature of the test chemical. 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. The test chemical did not induce gene mutation inSalmonella typhimuriumTA98, TA1537, TA100, TA1535 and hence is negative for gene mutation in vitro.

Based on the data vailable for the read across chemicals, 2-Naphthalenol, 1-[[2-methyl-4-[(2-methylphenyl) azo] phenyl] azo]-, ar styrenated (CAS no 85203 -90 -3) does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

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: JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals genetic toxicity in vitro,

Version / remarks:

2.

Principles of method if other than guideline:

WoE based on two in -invetro gene toxicity study 1.In vitro gene toxicity test of test chemical in CHO2. To evaluate the mutagenic potential of test chemical in Chinese hamster CHL/IU cells by chromosomal aberration test.

GLP compliance:

not specified

Type of assay:

other: in vitro mammalian chromosome aberration test

Target gene:

Not specified

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

1.

Details on mammalian cell type (if applicable):

not specified

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

other: Chinese hamster CHL/IU cells

Remarks:

2.

Details on mammalian cell type (if applicable):

CHL cells derived from Chinese hamster obtained from Research • Resource Bank (JCRB) (February1988, obtained at passage 4) were used in the test within 10 years of thawing succession.

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 was obtained from the livers of Aroclor 1254-treated male Sprague-Dawley rats

Test concentrations with justification for top dose:

1. up to 5,000 micrograms/L2. -S9 (continuous treatment): 0, 0.09, 0.19, 0.37 mg/ml-S9 (short-term treatment): 0, 1.3, 2.5, 5.0 mg/ml+S9 (short-term treatment): 0, 1.3, 2.5, 5.0 mg/ml

Vehicle / solvent:

1. water, dimethylsulfoxide, acetone, or ethanol, in that order of preference2. - Vehicle(s)/solvent(s) used: 0.5% Carboxymethyl cellulose sodium

Untreated negative controls:

not specified

Remarks:

1

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation

Untreated negative controls:

not specified

Remarks:

1.

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

0.5% Carboxymethyl cellulose sodium

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

1. Chromosomal aberration tests were carried out using the Chinese hamster ovary cells. Cells were exposed to the test substance for 8 hours with metabolic activation, the cells were exposed to the test chemical plus the metabolic activation for 2 hr, washed, incubated for 8 hours, and then treated with Colcemid for 2-2.5 hour. The cells were prepared for viewing on slides.2. Details on test system and conditionsCells usedCHL cells derived from Chinese hamster obtained from Research • Resource Bank (JCRB) (February 1988, obtained at passage 4) were used in the test within 10 years of thawing succession.2. Preparation of culture solution Eagle MEM culture medium supplemented with 10% fetal bovine serum (FCS: JRH BIOSCIENCES, lot number: 1C2073) was used for the culture.3. Culture conditions2 × 10 4 CHL cells were seeded in a dish (6 cm in diameter, Corning) containing 5 ml of the culture solution and cultured in a 37 ° C. CO 2 incubator (5% CO 2).In the direct method, test substances were added on day 3 of cell seeding and treated for 24 hours and 48 hours. In the metabolic activation method, the cells were treated for 6 hours in the presence and absence of S9mix on the third day of cell seeding, and after completion of the treatment, the cells were cultured for 18 hours with fresh culture medium.Method for preparing chromosome specimenTwo hours before the end of the culture, Colcemid was added to the culture solution to a final concent ration of about 0.1 μg / ml. Chromosome specimens were prepared according to a conventional method.Six slide specimens were prepared for each dish. The prepared specimens were stained with 3% Giemsa solution for about 10 minutes.OTHER EXAMINATIONS:- Determination of polyploidy: Yes

Evaluation criteria:

2. Among the five test bacteria used, in the direct method or metabolic activation method of one or more test bacteria, the number of revertive mutant colonies on the flat plate containing the test substance is more than twice that of the negative control, And when the increase was found to be reproducible or dose-dependent, it was decided that the test substance had mutagenicity (positive) in this test system.

Statistics:

2.Analysis results for untreated control, solvent, positive control group and test substance treated group are tabulated for the observed cell number, type and number of structural abnormality, and number of ploidy cells, and the values of each group are entered on the recording paper did. With regard to thefrequency of occurrence of chromosomal abnormal cells, a significant difference test between the solvent control group and the test substance treated group and between the solvent control group and the positive control group was carried out by Fisher's exact probability test method. According to the judgment criteria of Ishikan et al.2), the frequency of cells with chromosomal abnormality is negative, less than 5% negative, less than 10% false positive, and more than 10% Positive.

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

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:

not specified

Species / strain:

other: Chinese hamster CHL/IU cells

Remarks:

2.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Conclusions:

Test chemical did not induce gene mutation in the Chinese hamster ovary cells and Chinese hamster ovary cells (CHO-W-B1) both in the presence and absence of S9 activation system and hence it is not likely to be a gene mutant.

Executive summary:

Chromosome aberration:

Study 1:

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster ovary cells with and without activation system. S9 was obtained from the livers of Aroclor 1254-treated male Sprague-Dawley rats. The test chemical was studied at a dose level up to 5,000 micrograms/L. Cells were exposed to the test substance for 8 hours with metabolic activation, the cells were exposed to the test chemical plus the metabolic activation for 2 hr, washed, incubated for 8 hours, and then treated with Colcemid for 2-2.5 hour. The cells were prepared for viewing on slides. Based on the results noted, test chemical was found to be negative in the chromosomal aberration test using Chinese hamster ovary cells with and without S9 metabolic activation obtained from the livers of Aroclor 1254-treated male Sprague-Dawley rats

In vitro mammalian chromosome aberration test was also performed to determine the mutagenic nature of the test chemical. The test chemical was studied at a dose level of 50-150µg/mL (in the absence of S9) and 25- 100µg/mL (in the presence of S9) using Chinese hamster ovary cells (CHO-W-B1). Cells were exposed to the test chemical for 2 hr in the presence of S9 or throughout the incubation period without S9. 100 cells were scored from each of the three highest dose groups having sufficient metaphases for analysis. All types of aberrations were recorded separately, but for data analysis they were grouped into categories of “simple” (breaks and terminal deletions), “complex” (exchanges and rearrangements), “other” (includes pulverized chromosomes), and “total”. Gaps and endo-reduplications were recorded but were not included in the totals. Polyploid cells were not scored but used metaphases with 19-23 chromosomes (the modal number being 21). Based on the results noted, the test chemical did not induce chromosome aberrations in the Chinese hamster ovary cells (CHO-W-B1) in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Genetic toxicity in vitro study was assessed for test chemical by using chromosomal aberration test according to Guidelines for Screening Mutagenicity Testing of Chemicals (Japan).The test material was exposed to Chinese hamster CHL/IU cells in the presence and absence of metabolic activation S9. The concentration of test material used in the presence and absence of metabolic activation were mention as fallow

-S9 (continuous treatment): 0, 0.09, 0.19, 0.37 mg/ml

-S9 (short-term treatment): 0, 1.3, 2.5, 5.0 mg/ml

+S9 (short-term treatment): 0, 1.3, 2.5, 5.0 mg/ml

 No significant statistical mutagenic effects were observed in chromosome, in the presence and absence of metabolic activation. Therefore test chemical was considered to be non mutagenic in Chinese hamster CHL/IU cells by chromosomal aberration test. Hence the substance cannot be classified as gene mutant in vitro.

Based on the data available for the test chemical, 2-Naphthalenol, 1-[[2-methyl-4-[(2-methylphenyl) azo] phenyl] azo]-, ar-styrenated (CAS no 85203-90-3) 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.

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:

Ames test:

Gene mutation toxicity for the test chemical 2-Naphthalenol, 1-[[2-methyl-4-[(2-methylphenyl) azo] phenyl] azo]-, ar-styrenated (CAS no 85203 -90 -3) was predicted using the data from read across chemicals. The studies are as mentioned below:

Study 1:

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.

Study 2:

Gene mutation by the spot assay was performed for another test chemical. The test chemical was used at dose level of 200µg/plate both with and without S9 metabolic activation system. The plates were incubated for 3 days at 37°C for the growth of colonies to occur. The plates were observed for an increase in the number of His+ revertants. The criteria adopted for scoring a mutagenic response in routine plate tests was that the observed number of revertants should exceed twice the background value for that given assay and exceed the 99.9% confidence limit based on our historical controls. Concurrent positive control was also included in the study. The test chemical failed to induce mutation in Salmonellatyphimurium strain TA100, TA98, TA1535, TA1537 and TA1538 with and without S9 metabolic activation system in the spot test performed and hence is not likely to classify for gene mutation in vitro.

Study 3:

Salmonella/ mammalian-microsome test was performed to evaluate the mutagenic nature of the test chemical. 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. The test chemical did not induce gene mutation inSalmonella typhimuriumTA98, TA1537, TA100, TA1535 and hence is negative for gene mutation in vitro.

Based on the data vailable for the read across chemicals, 2-Naphthalenol, 1-[[2-methyl-4-[(2-methylphenyl) azo] phenyl] azo]-, ar styrenated (CAS no 85203 -90 -3) does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Chromosome aberration:

Study 1:

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster ovary cells with and without activation system. S9 was obtained from the livers of Aroclor 1254-treated male Sprague-Dawley rats. The test chemical was studied at a dose level up to 5,000 micrograms/L. Cells were exposed to the test substance for 8 hours with metabolic activation, the cells were exposed to the test chemical plus the metabolic activation for 2 hr, washed, incubated for 8 hours, and then treated with Colcemid for 2-2.5 hour. The cells were prepared for viewing on slides. Based on the results noted, test chemical was found to be negative in the chromosomal aberration test using Chinese hamster ovary cells with and without S9 metabolic activation obtained from the livers of Aroclor 1254-treated male Sprague-Dawley rats

In vitro mammalian chromosome aberration test was also performed to determine the mutagenic nature of the test chemical. The test chemical was studied at a dose level of 50-150µg/mL (in the absence of S9) and 25- 100µg/mL (in the presence of S9) using Chinese hamster ovary cells (CHO-W-B1). Cells were exposed to the test chemical for 2 hr in the presence of S9 or throughout the incubation period without S9. 100 cells were scored from each of the three highest dose groups having sufficient metaphases for analysis. All types of aberrations were recorded separately, but for data analysis they were grouped into categories of “simple” (breaks and terminal deletions), “complex” (exchanges and rearrangements), “other” (includes pulverized chromosomes), and “total”. Gaps and endo-reduplications were recorded but were not included in the totals. Polyploid cells were not scored but used metaphases with 19-23 chromosomes (the modal number being 21). Based on the results noted, the test chemical did not induce chromosome aberrations in the Chinese hamster ovary cells (CHO-W-B1) in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Study 2:

Genetic toxicity in vitro study was assessed for test chemical by using chromosomal aberration test according to Guidelines for Screening Mutagenicity Testing of Chemicals (Japan).The test material was exposed to Chinese hamster CHL/IU cells in the presence and absence of metabolic activation S9. The concentration of test material used in the presence and absence of metabolic activation were mention as fallow

-S9 (continuous treatment): 0, 0.09, 0.19, 0.37 mg/ml

-S9 (short-term treatment): 0, 1.3, 2.5, 5.0 mg/ml

+S9 (short-term treatment): 0, 1.3, 2.5, 5.0 mg/ml

 No significant statistical mutagenic effects were observed in chromosome, in the presence and absence of metabolic activation. Therefore test chemical was considered to be non mutagenic in Chinese hamster CHL/IU cells by chromosomal aberration test. Hence the substance cannot be classified as gene mutant in vitro.

Based on the data available for the test chemical, 2-Naphthalenol, 1-[[2-methyl-4-[(2-methylphenyl) azo] phenyl] azo]-, ar-styrenated (CAS no 85203-90-3) 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 read across, it is considered that 2-Naphthalenol, 1-[[2-methyl-4-[(2-methylphenyl)azo]phenyl]azo]-, ar-styrenated

(CAS no 85203 -90 -3) does not exhbit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.