2-Naphthalenamine, N-(2-ethylhexyl)-1-[[4-(phenylazo)phenyl]azo]-, ar' and ar''-Me derivs.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic mutation in vitro;

The read across substances share high similarity in structure and functional group .Therefore, it is acceptable to derive information on mutation from the analogue substance for test substance. Experimental data from read across chemical have been reviewed to determine the mutagenic nature of N-(2-ethylhexyl)-1-[4-(2-phenyldiazen-1-yl)phenyl]naphthalen-2-amine (92257-28-8).The studies are as mentioned below

AMES test

The read across substances share high similarity in structure and functional group .Therefore, it is acceptable to derive information on mutation from the analogue substance for test substance. Salmonella/ mammalian-microsome test was performed to evaluate the mutagenic nature of the test compound Test chemical. 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 a level 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. Test chemical did not induce gene mutation in Salmonella typhimuriumTA98, TA1537, TA100, TA1535 in the presence and absence of S9 metabolic activation system and hence it is negative for gene mutation in vitro.

 

The read across substances share high similarity in structure and functional group .Therefore, it is acceptable to derive information on mutation from the analogue substance for target substance. Gene mutation toxicity study was performed to determine the mutagenic nature of test chemical. The study was performed using Salmonella typhimurium strains TA97a, TA98, TA100 and TA102 with and without S9 metabolic activation system. The test chemical was dissolved in sterilized distilled water and used at dose levels of 0, 1, 10, 100 or 500µg/plate. Concurrent spontaneous revertants and positive controls were also included in the study. The plates were incubated for 48 hrs and the plates were observed for 2-fold increase in the number of revertants per plate. Test chemical did not induce gene mutation in Salmonella typhimurium strainsTA97a, TA98, TA100 and TA102 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In vitro Mammalian cell assay;

The read across substances share high similarity in structure and functional group .Therefore, it is acceptable to derive information on mutation from the analogue substance for test substance.L5178Y TK+/-Mouse Lymphoma Mutagenicity Assay was conducted on test chemical.L5178Y TK+/- 3.7.C mouse lymphoma cells were grown in Fischer’s medium for leukemic cells of mice supplemented with 10% horse serum and 0.02% pluronic F-68.Cells at a concentration of 6 ×105/mL were exposed for 4 h to a range of concentrations from 200-1000µg/ml.The test chemical showed negative gene toxic effect with andWithout S9 activation inL5178Y TK+/-Mouse Lymphoma Mutagenicity Assay. Hence the test chemical cannot be classified as gene mutant in vitro.

 

The data available for the target chemical based on its read across substance and applying weight of evidence N-(2-ethylhexyl)-1-[4-(2-phenyldiazen-1-yl)phenyl]naphthalen-2-amine (92257-28-8) does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant in vitro.

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

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: As mention below

Principles of method if other than guideline:

WoE derived based on the experimental data from structurally and functionally similar read across chemicals for the target chemical.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium, other: TA97a, TA98, TA100 and TA102

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

not specified

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:

1.10-250 mg
2.0, 1, 10, 100 or 500 µg/plate

Vehicle / solvent:

1.DMSO
2.Sterilized distilled water

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

not specified

Details on test system and experimental conditions:

1.Details on test system and 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

2.Details on test system and conditions
METHOD OF APPLICATION: in agar (plate incorporation)

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

SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS: The mutagenicity tests were performed in at least two series of assays with duplicate plates per dose; three plates were used for determinations of spontaneous reversion

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: To estimate the toxic effect, the spareness of the bacterial growth lawn was considered and the relative decline in the mutant values was determined.

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

OTHER: Revertant colonies were counted
with an automatic counter Biotran II from New Brunswick Scientific Co. Inc., NJ

Rationale for test conditions:

Not specified

Evaluation criteria:

1.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.
2.At least a 2-fold increase in the number of revertants per plate as compared to the number of revertants per control plates (number of spontaneous revertants) was the criterion for judging whether a given compound was a mutagen or not.

Statistics:

1.Not specified
2.Not specified

Species / strain:

S. typhimurium, other: A98, TA1537, TA100, TA1535

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

Species / strain:

S. typhimurium, other: TA97a, TA98, TA100 and TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: No mutagenic effect were observed

Conclusions:

Gene mutation toxicity study for N-(2-ethylhexyl)-1-[4-(2-phenyldiazen-1-yl)phenyl]naphthalen-2-amine (92257-28-8) as predicted using data from read across chemicals for Salmonella typhimurium bacterial strains in the presence and absence of S9 metabolic activation system is negative and hence the chemical is not likely to classify as a gene mutant in vitro.

Executive summary:

Genetic mutation in vitro;

The read across substances share high similarity in structure and functional group .Therefore, it is acceptable to derive information on mutation from the analogue substance for test substance. Experimental data from read across chemical have been reviewed to determine the mutagenic nature of N-(2-ethylhexyl)-1-[4-(2-phenyldiazen-1-yl)phenyl]naphthalen-2-amine (92257-28-8).The studies are as mentioned below

The read across substances share high similarity in structure and functional group .Therefore, it is acceptable to derive information on mutation from the analogue substance for test substance. Salmonella/ mammalian-microsome test was performed to evaluate the mutagenic nature of the test compound Test chemical. 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 a level 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. Test chemical did not induce gene mutation in Salmonella typhimuriumTA98, TA1537, TA100, TA1535 in the presence and absence of S9 metabolic activation system and hence it is negative for gene mutation in vitro.

 

The read across substances share high similarity in structure and functional group .Therefore, it is acceptable to derive information on mutation from the analogue substance for target substance. Gene mutation toxicity study was performed to determine the mutagenic nature of test chemical. The study was performed using Salmonella typhimurium strains TA97a, TA98, TA100 and TA102 with and without S9 metabolic activation system. The test chemical was dissolved in sterilized distilled water and used at dose levels of 0, 1, 10, 100 or 500µg/plate. Concurrent spontaneous revertants and positive controls were also included in the study. The plates were incubated for 48 hrs and the plates were observed for 2-fold increase in the number of revertants per plate. Test chemical did not induce gene mutation in Salmonella typhimurium strainsTA97a, TA98, TA100 and TA102 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

 

The data available for the target chemical based on its read across substance and applying weight of evidence N-(2-ethylhexyl)-1-[4-(2-phenyldiazen-1-yl)phenyl]naphthalen-2-amine (92257-28-8) does not exhibit gene mutation in vitro. Hence the test chemical 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

Justification for type of information:

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

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Principles of method if other than guideline:

WoE derived based on the experimental data from structurally similar read across chemcal

GLP compliance:

not specified

Type of assay:

other: Mammalian cell gene mutation assay

Specific details on test material used for the study:

- Name of test material : 2-Naphthalenamine, N-(2-ethylhexyl)-1-[[4-(phenylazo)phenyl]azo]-, ar' and ar''-Me derivs.
- IUPAC name : N-(2-ethylhexyl)-1-[4-(2-phenyldiazen-1-yl)phenyl]naphthalen-2-amine
- Molecular formula : C30H33N3
- Molecular weight : 435.60 g/mol
- Substance type : Organic
- Physical state : Liquid

Target gene:

No data available.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Details on mammalian cell line
- 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.

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9mix S9 prepared from Aroclor 1254-induced male Sprague- Dawley rats

Test concentrations with justification for top dose:

200-1000µg/mL

Vehicle / solvent:

Yes ,but not specified

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: Ethyl methylsulfonate at 4.7×10-6 M (or methyl methanesulfonate at 10-20 µg/ml mL) for the test without metabolic activation. 3-methylcholanthrene at 1.86 × 10-5 M (or dimethylbenz[a]- anthracene at 0.5-4 µg/mL) for the test with metabolic activation.

Details on test system and experimental conditions:

Details on test system and conditions
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk:
Suspension/plate

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: No data available

NUMBER OF CELLS EVALUATED: No data available

Rationale for test conditions:

Not specified

Evaluation criteria:

Only colonies larger than 0.2 mm in diameter were counted. Mutant frequencies were expressed as mutants per 106 surviving cells. Although there are several different
methods for evaluating mouse lymphoma data, results from this study 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.

Statistics:

Yes ,Mean Standard deviation was observed

Species / strain:

mouse lymphoma L5178Y cells

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

Remarks on result:

other: No mutagenic effect were observed

Conclusions:

Gene mutation toxicity study for N-(2-ethylhexyl)-1-[4-(2-phenyldiazen-1-yl)phenyl]naphthalen-2-amine (92257-28-8) as predicted using data from read across chemicals for mammmalian cell gene mutation assay.Test chemical did not induced genetoxic effect with and Without S9 activation in L5178Y TK+/- Mouse Lymphoma Mutagenicity Assay.

Executive summary:

Data available for the read across chemicals was reviewed to determine the mutagenic nature of test chemical . The studies are as mentioned below:

The read across substances share high similarity in structure and functional group .Therefore, it is acceptable to derive information on mutation from the analogue substance for test substance.L5178Y TK+/-Mouse Lymphoma Mutagenicity Assay was conducted on test chemical.L5178Y TK+/- 3.7.C mouse lymphoma cells were grown in Fischer’s medium for leukemic cells of mice supplemented with 10% horse serum and 0.02% pluronic F-68.Cells at a concentration of 6 ×10⁵/mL were exposed for 4 h to a range of concentrations from 200-1000µg/ml.The test chemical showed negative gene toxic effect with andWithout S9 activation inL5178Y TK+/-Mouse Lymphoma Mutagenicity Assay. Hence the test chemical cannot be classified as gene mutant in vitro.

On the basis of the data summarized from read across chemicals, the target chemical test chemical is not likely to mutagenic in the Mammalian cells used in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a 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

Genetic mutation in vitro;

The read across substances share high similarity in structure and functional group .Therefore, it is acceptable to derive information on mutation from the analogue substance for test substance. Experimental data from read across chemical have been reviewed to determine the mutagenic nature of N-(2-ethylhexyl)-1-[4-(2-phenyldiazen-1-yl)phenyl]naphthalen-2-amine (92257-28-8).The studies are as mentioned below

AMES test

The read across substances share high similarity in structure and functional group .Therefore, it is acceptable to derive information on mutation from the analogue substance for test substance. Salmonella/ mammalian-microsome test was performed to evaluate the mutagenic nature of the test compound Test chemical. 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 a level 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. Test chemical did not induce gene mutation in Salmonella typhimuriumTA98, TA1537, TA100, TA1535 in the presence and absence of S9 metabolic activation system and hence it is negative for gene mutation in vitro.

 

The read across substances share high similarity in structure and functional group .Therefore, it is acceptable to derive information on mutation from the analogue substance for target substance. Gene mutation toxicity study was performed to determine the mutagenic nature of test chemical. The study was performed using Salmonella typhimurium strains TA97a, TA98, TA100 and TA102 with and without S9 metabolic activation system. The test chemical was dissolved in sterilized distilled water and used at dose levels of 0, 1, 10, 100 or 500µg/plate. Concurrent spontaneous revertants and positive controls were also included in the study. The plates were incubated for 48 hrs and the plates were observed for 2-fold increase in the number of revertants per plate. Test chemical did not induce gene mutation in Salmonella typhimurium strainsTA97a, TA98, TA100 and TA102 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In vitro Mammalian cell assay;

The read across substances share high similarity in structure and functional group .Therefore, it is acceptable to derive information on mutation from the analogue substance for test substance.L5178Y TK+/-Mouse Lymphoma Mutagenicity Assay was conducted on test chemical.L5178Y TK+/- 3.7.C mouse lymphoma cells were grown in Fischer’s medium for leukemic cells of mice supplemented with 10% horse serum and 0.02% pluronic F-68.Cells at a concentration of 6 ×105/mL were exposed for 4 h to a range of concentrations from 200-1000µg/ml.The test chemical showed negative gene toxic effect with andWithout S9 activation inL5178Y TK+/-Mouse Lymphoma Mutagenicity Assay. Hence the test chemical cannot be classified as gene mutant in vitro.

 

The data available for the target chemical based on its read across substance and applying weight of evidence N-(2-ethylhexyl)-1-[4-(2-phenyldiazen-1-yl)phenyl]naphthalen-2-amine (92257-28-8) does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant in vitro.

Justification for classification or non-classification

 Thus based on the above annotation and CLP criteria for target substance N-(2-ethylhexyl)-1-[4-(2-phenyldiazen-1-yl)phenyl]naphthalen-2-amine (92257-28-8)  does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant in vitro.