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Toxicological information

Genetic toxicity: in vitro

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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:
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

Reference
Name:
Unnamed
Type:
Constituent
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:
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.
Controls
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

Results and discussion

Test results
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.

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.