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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation study in mammalian cells was conducted by H. E. Seifried et.al (Chem. Res. Toxicol,2006) to evaluate the mutagenic potential of target chemical 1-(2,4-dimethylphenylazo)-2-naphthol (3118-97-6) in mammalian cell line. The mutagenic potency of 1-(2,4-dimethylphenylazo)-2-naphthol (3118-97-6) was tested on theL5178Y TK+/-3.7.C mouse lymphoma cells. The test compound was found to be non-mutagenic when the mammalian cell was exposed for 4 hrs along with the total expression time of 48 hrs by using test concentration of 1.0– 349 µg/ml. The test compound induces no mutagenic response both in the presence and absence of metabolic activation system. Therefore 1-(2,4-dimethylphenylazo)-2-naphthol (3118-97-6) was considered to be non mutagenic in mammalian cell.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Data is from publication.
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: as mentioned below
Principles of method if other than guideline:
The study was performed to evaluate the mutagenic potency of 1-[(2,4-dimethylphenyl)diazenyl]-2-naphthol in mammalian cell .
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Test material information:
Composition 1
Specific details on test material used for the study:
- Name of test material (as cited in study report): C.I. Solvent orange 7
- Molecular formula: C18H16N2O
- Molecular weight : 276.337 g/mol
- Substance type: Organic
- Physical state: Solid
- Smiles : Cc1ccc(N=Nc2c(O)ccc3ccccc23)c(C)c1
- InChI: 1S/C18H16N2O/c1-12-7-9-16(13(2)11-12)19-20-18-15-6-4-3-5-14(15)8-10-17(18)21/h3-11,21H,1-2H3/b20-19+
Species / strain:
mouse lymphoma L5178Y cells
Details on mammalian cell lines (if applicable):
- Type and identity of media: The cells were grown in Fischer’s medium for leukemic cells of mice (Gibco, Grand Island, NY, or Quality Biological, Gaithersburg, MD) supplemented with 10% horse serum (Gibco or Hyclone, Logan, UT) and 0.02% pluronic F-68 (BASF Wyandotte Corp., Wyandotte, MI).
- 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 characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 mixture was prepared from Aroclor 1254-induced male Sprague-Dawley rats.
Test concentrations with justification for top dose:
1.0– 349 µg/ml
Vehicle:
Vehicle
- 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.
Negative controls:
not specified
Solvent controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
ethylmethanesulphonate
Remarks:
Positive control substance for the test without metabolic activation – ethylmethylsulfonate. Positive control substance for the test with metabolic activation – 3-methyl cholanthrene
Details on test system and conditions:
METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: No data available
- Exposure duration: 4 hr
- Expression time (cells in growth medium): 48 hrs

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

NUMBER OF REPLICATIONS: Duplicates

NUMBER OF CELLS EVALUATED: 1.2 × 107 cells

DETERMINATION OF CYTOTOXICITY: Cell growth

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

OTHER: Cells in the cultures were adjusted to 3 × 105/mL at 24 h intervals. They were then cloned (1 × 106 cells/plate for mutant selection and 200 cells/plate for viable count determinations) in soft agar medium containing Fischer’s medium, 20% horse serum, 2 mM sodium pyruvate, 0.02% pluronic F-68, and 0.23% granulated agar (BBL, Inc., Cockeysville, MD). Resistance to trifluorothymidine (TFT) was determined by adding TFT (final concentration, 3 µg/mL) to the cloning medium for mutant selection. The 100× stock solution of TFT in saline was stored at -70 °C and was thawed immediately before use. Plates were incubated at 37 ± 1 °C in 5% CO2 in air for 10-12 days and then counted with an Artek automated colony counter (Artek 982, DynaTech) or ProtoCol colony counter (Synbiosis, Frederick, MD). Only colonies larger than ~0.2 mm in diameter were counted.
Evaluation criteria:
Results of the study were evaluated using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of positive effect, together with evidence of a dose-related increase.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
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
other: all strains/cell types tested
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY:The toxicity of each chemical was determined both with and without liver S9 prepared from Aroclor 1254-induced male Sprague-Dawley rats. Cells at a concentration of 6 × 105/ml (6 × 106 cells total) were exposed for 4 hr to a range of concentrations. The cells were then washed, resuspended in growth medium, and incubated at 37 ± 1ᵒC for 48 hr. The rate of cell growth was determined for each of the treated cultures and compared with the rate of growth of the solvent controls.
Conclusions:
1-(2,4-dimethylphenylazo)-2-naphthol (3118-97-6)was found to have no mutagenic inducing potency on the test mammalian cell both in the presence and absence of metabolic activation system.
Executive summary:

The mutagenic potency of 1-(2,4-dimethylphenylazo)-2-naphthol (3118-97-6) was tested on theL5178Y TK+/-3.7.C mouse lymphoma cells. The test compound was found to be non-mutagenic when the mammalian cell was exposed for 4 hrs along with the total expression time of 48 hrs by using test concentration of 1.0– 349 µg/ml

 

The size of mutant mouse lymphoma colonies was determined using an Artek 982 colony counter/sizer or the ProtoCol colony counter and the mutant frequencies were expressed as mutants per 106surviving cells.The test compound induces no mutagenic response both in the presence and absence of metabolic activation system. Therefore 1-(2,4-dimethylphenylazo)-2-naphthol (3118-97-6) was considered to be non mutagenic in mammalian cell.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In different studies, 1-(2,4-dimethylphenylazo)-2-naphthol (3118-97-6) has been investigated for potential of mutagenic response with and without metabolic activation. The studies are based on in vitro gene mutation study in mammalian cells and bacteria for target chemical 1-(2,4-dimethylphenylazo)-2-naphthol (3118-97-6) . Different studies based on these experiments are concluded below

In vitro gene mutation study in mammalian cells was conducted by H. E. Seifried et.al (Chem. Res. Toxicol,2006) to evaluate the mutagenic potential of target chemical 1-(2,4-dimethylphenylazo)-2-naphthol (3118-97-6) in mammalian cell line. The mutagenic potency of 1-(2,4-dimethylphenylazo)-2-naphthol (3118-97-6) was tested on theL5178Y TK+/-3.7.C mouse lymphoma cells. The test compound was found to be non-mutagenic when the mammalian cell was exposed for 4 hrs along with the total expression time of 48 hrs by using test concentration of 1.0– 349 µg/ml. The test compound induces no mutagenic response both in the presence and absence of metabolic activation system. Therefore 1-(2,4-dimethylphenylazo)-2-naphthol (3118-97-6) was considered to be non mutagenic in mammalian cell.

Supported by In vitro gene mutation study in mammalian cells conducted by T.P. Cameron et al.(Mutation Research,1987) the mutagenic potential of target chemical 1-(2,4-dimethylphenylazo)-2-naphthol (3118-97-6) in mammalian cell line. The mutagenic potency of the1- (2, 4-dimethylphenylazo)-2-naphthol (3118-97-6) was tested on theL5178Y TK+/-mouse lymphoma cells. The test compound was found to be non-mutagenic when the mammalian cell was exposed for 4 hrs along with the total expression time of 48 at the test concentration of 0.0, 1.0, 2.3, 3.6, 4.9 and 6.1µg/ ml. A test result was considered positive if there was a dose-related increase in the mutant frequency above the spontaneous control frequency, with a 2-fold increase at more than 1 dose and relative total growth greater than 10%.The relative total growth was observed to be 4and 8at the test concentration of 6.1µg/ ml. Mutant frequency (per 104cells) was also found to be less than the spontaneous control frequency at more than 1 dose. This in vitro gene mutation study in mammalian cells is a confirmatory test performed to conclude about the mutagenic potential of substance. Therefore based on the result it can be concluded that1- (2, 4-dimethylphenylazo)-2-naphthol (3118-97-6) does not induces mutagenic response both in the presence and absence of metabolic activation system in mammalian cell.

Further supported by in vitro gene mutation study in bacteria conducted by H. E. Seifried et.al (Chem. Res. Toxicol,2006) to evaluate the mutagenic potential of target chemical 1-(2,4-dimethylphenylazo)-2-naphthol (3118-97-6) in bacterial strain. The mutagenic potency of 1-(2,4-dimethylphenylazo)-2-naphthol was tested by the plate incorporation method usingSalmonella typhimuriumstrain TA98 and TA1538. The test material was used at the concentration of 1.0-1000 µg /plate .When the test bacterial strain is exposed with the test chemical for 48hrs, mutagenic response was seen in both the strains ofSalmonella typhimurium(with and without metabolic activation system). Therefore 1-(2,4-dimethylphenylazo)-2-naphthol(3118-97-6) was considered to be inducing mutagenic response inSalmonella typhimuriumstrain TA98 and TA1538 (with and without metabolic activation system).

Another supporting in vitro gene mutation study in bacteria was conducted by T.P. Cameron et al.(Mutation Research,1987)to evaluate the mutagenic potential of target chemical 1-(2,4-dimethylphenylazo)-2-naphthol (3118-97-6) in bacterial strain.The mutagenic potency of1-[(2,4-dimethylphenyl)diazenyl]-2-naphthol (3118-97-6) was tested by the plate incorporation method using Salmonella typhimuriumstrainTA98, TA100, TA1535, TA1537 and TA1538. When the test bacterial strain is exposed with test chemical at concentration of 0,333,1000,3333,6666 and 10000 µg/plate, mutagenic response was seen in all strains ofSalmonella typhimuriumstrainTA98, TA100, TA1535, TA1537 and TA1538(with and without metabolic activation system). Therefore 1-[(2,4-dimethylphenyl) diazenyl]-2-naphthol (3118-97-6) was considered to be positive for mutagenic response in screening AMES test .

Other supporting in vitro gene mutation study in bacteria was conducted by T.P. Cameron et al.(Mutation Research,1987) to evaluate the mutagenic potential of target chemical 1-(2,4-dimethylphenylazo)-2-naphthol (3118-97-6) in bacterial strain.The mutagenic potency of 1-[(2,4-dimethylphenyl)diazenyl]-2-naphthol (3118-97-6)was tested by FMN modified pre-incubation method usingSalmonella typhimurium strain TA98 and TA100.Test concentration of 1.0 ,10,33,100,333,1000,3333,6666 and 10000µg/plate was used for 1-[(2,4-dimethylphenyl)diazenyl]-2-naphthol. When the test bacterial strain is exposed with the test chemical for 48hrs, significant mutagenic response was seen in both the strains of Salmonella typhimuriumin the presence of metabolic activation system.  Therefore  1-[(2,4-dimethylphenyl)diazenyl]-2-naphthol(3118-97-6) was considered to be positive for mutagenic response in Salmonella typhimurium strain TA98 and TA100 by FMN modified pre-incubation method.

One another supporting in vitro gene mutation study in bacteria was conducted by R. COLIN GARNER et al.(Mutation Research,1977) to evaluate the mutagenic potential of target chemical 1-(2,4-dimethylphenylazo)-2-naphthol (3118-97-6) in bacterial strain. For this purpose bacterial gene mutation assay was performed in S. typhimurium strains TA1538. The test material was tested by the modified soft agar overlay method using Salmonella typhimurium strain TA98 by using a concentration of 0, 50 and 100 µg /plate in the presence and absence of liver enzyme preparation (metabolic activation system). Mutagenic response was seen in the tested bacterial strain in the presence of metabolic activation system while no mutagenic effect was observed without metabolic activation. All the results which are obtained in the study are the mean of the duplicate assays which are performed. Therefore, 1-[(2,4-dimethylphenyl)diazenyl]-2-naphthol induces mutation in the Salmonella typhimurium strain TA98 in the presence of metabolic activation system.

Bacterial reverse mutation assay is a screening test for mutagenic response, while mammalian cell gene mutation assay is a confirmatory test for mutagenic response. As per the above studies the target chemical 1-(2,4-dimethylphenylazo)-2-naphthol is considered to be non mutagenic according to In vitro gene mutation study in mammalian cells. While according to in vitro gene mutation study in bacteria the target chemical is mutagenic. On the basis of the confirmatory tests, the test substance was observed to be non mutagenic with and without metabolic activator in mammalian cell. This confirmatory test concludes that the test substance shows negative result for mutagenic potential. Thusbased on the available data for the target chemical,1-(2,4-dimethylphenylazo)-2-naphthol (3118-97-6) does notexhibits gene mutation toxicity in vitro. Hence, the chemical isnot likely to classify as a gene mutant in vitro.

Justification for classification or non-classification

On the basis of the confirmatory tests, the test substance was observed to be non mutagenic with and without metabolic activator in mammalian cell. This confirmatory test concludes that the test substance shows negative result for mutagenic potential. Thusbased on the available data for the target chemical,1-(2,4-dimethylphenylazo)-2-naphthol (3118-97-6) does notexhibits gene mutation toxicity in vitro. Hence, the chemical isnot likely to classify as a gene mutant in vitro.