1-(2,4-dimethylphenylazo)-2-naphthol

Genetic toxicity: in vitro

Administrative data

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.

Data source

Reference

Reference Type:

publication

Title:

A Compilation of Two Decades of Mutagenicity Test Results with the Ames Salmonella typhimurium and L5178Y Mouse Lymphoma Cell Mutation Assays

Author:

H. E. Seifried, R. M. Seifried, J. J. Clarke, T. B. Junghans, and R. H. C. San

Year:

2006

Bibliographic source:

Chem. Res. Toxicol., Vol. 19 (5), Pg. no. 627–644, 2006

Materials and methods

Test guideline

Qualifier:

according to guideline

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

Test material information

Constituent 1

Reference substance name:

1-(2,4-dimethylphenylazo)-2-naphthol

EC Number:

221-490-4

EC Name:

1-(2,4-dimethylphenylazo)-2-naphthol

Cas Number:

3118-97-6

Molecular formula:

C18H16N2O

IUPAC Name:

1-(2,4-dimethylphenylazo)-2-naphthol

Test material form:

solid: crystalline

Details on test material:

- Name of test material : C.I. Solvent Orange 7
- Molecular formula : C18H16NO
- Molecular weight : 276.3374 g/mol
- Smiles notation : Cc1ccc(N=Nc2c(O)ccc3ccccc23)c(C)c1
- InChl : 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+
- Substance type:Organic
- Physical appearance : Solid

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+

Method

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

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 (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 / cell type 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 / solvent:

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.

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle 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 experimental 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

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

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.

Remarks on result:

other: all strains/cell types tested

Applicant's summary and conclusion

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.