Reaction mass of N-(2-ethylhexyl)-1-[[2-methyl-4-[(4-methylphenyl)azo]phenyl]azo]naphthalen-2-amine and N-(2-ethylhexyl)-1-[[2-methyl-4-[(2-methylphenyl)azo]phenyl]azo]naphthalen-1-amine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in vitro:

Ames test:

Data available for the read across chemicals was reviewed to determine the mutagenic nature of N-(2-ethylhexyl)-1-[[2-methyl-4-[(4-methylphenyl)azo]phenyl]azo] naphthalen-2-amine. The studies are as mentioned below:

Salmonella/microsome test in the absence of exogenous metabolic activation and in the presence of liver S-9 from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters was performed to evaluate the mutagenic nature of the test chemical usingS. typhimuriumtester strains TA1535, TA97, TA98 and TA100. The study was performed as per the preincubation assay and the preincubation time was 20 mins and the plates were incubated for 48 hrs. The test compound was dissolved in DMSO and was used at a dosage level of 0, 0.03, 0.1, 0.3, 1.0, 3.0, 10, 33, 100 or 333 µg/plate in the preincubation assay of 48 hrs. Concurrent solvent and positive control chemicals were included in the study. The test chemical did not induce a reproducible, dose-related increase in his⁺revertants over the corresponding solvent in the S. typhimurium tester strains TA100, TA97, TA1535 and TA98 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Ames assay in the absence of exogenous metabolic activation and in the presence and absence of liver S-9 was performed to evaluate the mutagenic nature of the test chemical using S. typhimurium tester strains TA100, TA98, TA102 and TA97. The study was performed as per the plate incorporation assay. The test compound was dissolved in DMSO and was used at a dosage level of 0, 5, 25, 50, 75, 100 or 200µg/plate for strains TA98, TA97 and TA100 and at dose levels of 0, 0.25, 0.5, 1, 75, 50, 100 or 200µg/plate. Concurrent solvent control was also included in the study. The test chemical did not induce a reproducible, dose-related 2- fold increase in his⁺revertants over the corresponding solvent in the S. typhimurium tester strains TA100, TA98, TA102 and TA97 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Based on the data available for the read across chemicals, N-(2-ethylhexyl)-1-[[2-methyl-4-[(4-methylphenyl)azo]phenyl]azo]naphthalen-2-amine (CAS no 93964 -07 -9) 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 test:

Gene mutation in vitro was predicted for the target chemical N-(2-ethylhexyl)-1-[[2-methyl-4-[(4-methylphenyl)azo] phenyl]azo] naphthalen-2-amine on the basis of the data from read across chemicals. 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 60 -70% and 50 -60% structurally and functionally similar 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 1 -349 µg/mL of the test chemical 1 and 1 -19 µg/mL for test chemical 2. 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 1 did not induce a doubling of the mutant frequency both in the presence and absence of S9 activation system. Another test chemical 2 did not induce a doubling of the mutant frequency in the presence of S9 activation system and an equivocal response was noted in the absence of S9 metabolic activation system and based on these observations, the test chemical 1 and 2 are not likely to be gene mutant in vitro.

Based on the data available for the read across chemicals, N-(2-ethylhexyl)-1-[[2-methyl-4-[(4-methylphenyl)azo] phenyl]azo] naphthalen-2-amine (CAS no 93964 -07 -9) does not exhibit gene mutation in vitro. Hence the test chemical is considered to be non-mutagenic as per the criteria mentioned in CLP regulation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

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 report is based on gene mutation toxicity study for the read across chemicals-
1. and 2. Bacterial reverse mutation assay was performed to evaluate the mutagenic nature of the test chemicals

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium, other: TA98, TA100, TA1535, and TA97

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 S-9 fractions of Aroclor 1254-induced, male Sprague-Dawley rat and male Syrian hamster livers were prepared

Test concentrations with justification for top dose:

1. 0, 0.03, 0.1, 0.3, 1.0, 3.0, 10, 33, 100 or 333 µg/plate
2. Strains TA98. TA97, TA100: 0, 5, 25, 50, 75, 100 or 200 µg/plate
Strain TA102: 0, 0.25, 0.5, 1, 75, 50, 100 or 200 µg/plate

Vehicle / solvent:

1. and 2.
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The chemical was soluble and stable 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:

9-aminoacridine

sodium azide

other: 2-Aminoanthracene (2-AA) (All strains, with S9), 4-nitro-o-phenylenediamine (TA98, -S9)

Remarks:

RA 1

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 2

Details on test system and experimental conditions:

1. METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- 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: Triplicate

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:

OTHER: No data available

2. METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: No data
- Exposure duration: No data
- Expression time (cells in growth medium): No data
- 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: Triplicate

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:

OTHER: No data available

Rationale for test conditions:

No data

Evaluation criteria:

1. An individual trial was judged mutagenic (+) if a dose-related increase over the corresponding solvent control was seen, and it was judged weakly mutagenic C+W) if a low-level dose response was seen. A trial was considered questionable (?) if a dose-related increase was judged insufficiently high to justify a call of "+W," if only a single dose was elevated over the control, or if a non-dose-related increase was seen.

The chemical was judged to be mutagenic (+), or weakly mutagenic (+W), if it produced a reproducible, dose-related increase in his+ revertants over the corresponding solvent controls in replicate trials.

It chemical was considered to be questionable (?) if a reproducible increase of his+ revertants did not meet the criteria for either a " + " or " + W," or if only single doses produced an increase in his+ revertants in repeat trials.

2. The plates were observed for a 2-fold increase in the number of revertants/plate

Statistics:

No data

Species / strain:

S. typhimurium, other: No data

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: TA98, TA102, TA100, TA97

Remarks:

RA 2

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 available

RANGE-FINDING/SCREENING STUDIES: The chemical was tested initially in a toxicity assay to determine the appropriate dose range. The toxicity assay was performed by using TA100 or the system developed by Waleh et al. Toxic concentrations were those at which a decrease in the number of his+ colonies was seen or at which there was a clearing in the density of the background lawn

COMPARISON WITH HISTORICAL CONTROL DATA: No data available

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data available

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Conclusions:

N-(2-ethylhexyl)-1-[[2-methyl-4-[(4-methylphenyl)azo]phenyl]azo]naphthalen-2-amine (CAS no 93964 -07 -9) does not exhibit gene mutation in vitro in Salmonell tester strains in the presence and absence of S9 metabolic activaton system. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Executive summary:

Data available for the read across chemicals was reviewed to determine the mutagenic nature of N-(2-ethylhexyl)-1-[[2-methyl-4-[(4-methylphenyl)azo]phenyl]azo] naphthalen-2-amine. The studies are as mentioned below:

Salmonella/microsome test in the absence of exogenous metabolic activation and in the presence of liver S-9 from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters was performed to evaluate the mutagenic nature of the test chemical using S. typhimurium tester strains TA1535, TA97, TA98 and TA100. The study was performed as per the preincubation assay and the preincubation time was 20 mins and the plates were incubated for 48 hrs. The test compound was dissolved in DMSO and was used at a dosage level of 0, 0.03, 0.1, 0.3, 1.0, 3.0, 10, 33, 100 or 333 µg/plate in the preincubation assay of 48 hrs. Concurrent solvent and positive control chemicals were included in the study. The test chemical did not induce a reproducible, dose-related increase in his⁺revertants over the corresponding solventin the S. typhimurium tester strains TA100, TA97, TA1535 and TA98 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Ames assay in the absence of exogenous metabolic activation and in the presence and absence of liver S-9 was performed to evaluate the mutagenic nature of the test chemical using S. typhimurium tester strains TA100, TA98, TA102 and TA97. The study was performed as per the plate incorporation assay. The test compound was dissolved in DMSO and was used at a dosage level of 0, 5, 25, 50, 75, 100 or 200µg/plate for strains TA98, TA97 and TA100 and at dose levels of 0, 0.25, 0.5, 1, 75, 50, 100 or 200µg/plate. Concurrent solvent control was also included in the study. The test chemical did not induce a reproducible, dose-related 2- fold increase in his⁺revertants over the corresponding solvent in the S. typhimurium tester strains TA100, TA98, TA102 and TA97 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Based on the data available for the read across chemicals, N-(2-ethylhexyl)-1-[[2-methyl-4-[(4-methylphenyl)azo]phenyl]azo]naphthalen-2-amine (CAS no 93964 -07 -9) 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: 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: Mammalian cell gene mutation assay

Target gene:

1/2. Thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

TK+/- 3.7.C / RA 1/ 2

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

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

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

Test concentrations with justification for top dose:

1. 1-349 µg/mL
2. 1-19 µg/mL

Vehicle / solvent:

1. No data

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

methylmethanesulfonate

other: 3-methylcholanthrene at 1.86 × 10-5 M (or dimethylbenz[a]- anthracene at 0.5-4 µg/mL) for the test with metabolic activation.

Remarks:

RA 1/ RA 2

Details on test system and experimental conditions:

1/2. METHOD OF APPLICATION: in medium;
- Cell density at seeding (if applicable): 6000000 cells

DURATION
- 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 available

SELECTION AGENT (mutation assays): 1×106 cells/plate for mutant selection
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: 1 X 106 cells/plate for mutant selection and 200
cells/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 cultures

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:

1/2. No data

Evaluation criteria:

1/2. 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.

Statistics:

No data available

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

TK+/- 3.7.C / 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:

valid

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

TK+/- 3.7.C / RA 2

Metabolic activation:

with

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:

mouse lymphoma L5178Y cells

Remarks:

TK+/- 3.7.C / RA 2

Metabolic activation:

without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

1/2. No data

Remarks on result:

other: No mutagenic potential

Conclusions:

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.

Executive summary:

Gene mutation in vitro was predicted for the target chemical N-(2-ethylhexyl)-1-[[2-methyl-4-[(4-methylphenyl)azo] phenyl]azo] naphthalen-2-amine on the basis of the data from read across chemicals. 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 60 -70% and 50 -60% structurally and functionally similar 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 1 -349 µg/mL of the test chemical 1 and 1 -19 µg/mL for test chemical 2. 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 1 did not induce a doubling of the mutant frequency both in the presence and absence of S9 activation system. Another test chemical 2 did not induce a doubling of the mutant frequency in the presence of S9 activation system and an equivocal response was noted in the absence of S9 metabolic activation system and based on these observations, the test chemical 1 and 2 are not likely to be gene mutant in vitro.

Based on the data available for the read across chemicals, N-(2-ethylhexyl)-1-[[2-methyl-4-[(4-methylphenyl)azo] phenyl]azo] naphthalen-2-amine does not exhibit gene mutation in vitro. Hence the test chemical is considered to be non-mutagenic 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:

Data available for the read across chemicals was reviewed to determine the mutagenic nature of N-(2-ethylhexyl)-1-[[2-methyl-4-[(4-methylphenyl)azo]phenyl]azo] naphthalen-2-amine. The studies are as mentioned below:

Salmonella/microsome test in the absence of exogenous metabolic activation and in the presence of liver S-9 from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters was performed to evaluate the mutagenic nature of the test chemical usingS. typhimuriumtester strains TA1535, TA97, TA98 and TA100. The study was performed as per the preincubation assay and the preincubation time was 20 mins and the plates were incubated for 48 hrs. The test compound was dissolved in DMSO and was used at a dosage level of 0, 0.03, 0.1, 0.3, 1.0, 3.0, 10, 33, 100 or 333 µg/plate in the preincubation assay of 48 hrs. Concurrent solvent and positive control chemicals were included in the study. The test chemical did not induce a reproducible, dose-related increase in his⁺revertants over the corresponding solventin theS. typhimuriumtester strains TA100, TA97, TA1535 and TA98 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Ames assay in the absence of exogenous metabolic activation and in the presence and absence of liver S-9 was performed to evaluate the mutagenic nature of the test chemical using S. typhimurium tester strains TA100, TA98, TA102 and TA97. The study was performed as per the plate incorporation assay. The test compound was dissolved in DMSO and was used at a dosage level of 0, 5, 25, 50, 75, 100 or 200µg/plate for strains TA98, TA97 and TA100 and at dose levels of 0, 0.25, 0.5, 1, 75, 50, 100 or 200µg/plate. Concurrent solvent control was also included in the study. The test chemical did not induce a reproducible, dose-related 2- fold increase in his⁺revertants over the corresponding solvent in theS. typhimuriumtester strains TA100, TA98, TA102 and TA97 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Based on the data available for the read across chemicals, N-(2-ethylhexyl)-1-[[2-methyl-4-[(4-methylphenyl)azo]phenyl]azo]naphthalen-2-amine (CAS no 93964 -07 -9) 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 test:

Gene mutation in vitro was predicted for the target chemical N-(2-ethylhexyl)-1-[[2-methyl-4-[(4-methylphenyl)azo] phenyl]azo] naphthalen-2-amine on the basis of the data from read across chemicals. 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 60 -70% and 50 -60% structurally and functionally similar 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 1 -349 µg/mL of the test chemical 1 and 1 -19 µg/mL for test chemical 2. 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 1 did not induce a doubling of the mutant frequency both in the presence and absence of S9 activation system. Another test chemical 2 did not induce a doubling of the mutant frequency in the presence of S9 activation system and an equivocal response was noted in the absence of S9 metabolic activation system and based on these observations, the test chemical 1 and 2 are not likely to be gene mutant in vitro.

Based on the data available for the read across chemicals, N-(2-ethylhexyl)-1-[[2-methyl-4-[(4-methylphenyl)azo] phenyl]azo] naphthalen-2-amine (CAS no 93964 -07 -9) does not exhibit gene mutation in vitro. Hence the test chemical is considered to be non-mutagenic as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the data available for the its read across, N-(2-ethylhexyl)-1-[[2-methyl-4-[(4-methylphenyl)azo]phenyl]azo]naphthalen-2-amine (CAS no 93964 -07 -9) 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.