4,4'-[vinylenebis[(3-sulpho-4,1-phenylene)imino(6-chloro-1,3,5-triazine-4,2-diyl)imino]]bis[5-hydroxy-6-[(1-sulpho-2-naphthyl)azo]naphthalene-2,7-disulphonic] acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

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 is from peer reviewed 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. 2006, 19, 627-644

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

The gene mutation study was conducted according to L5178Y TK+/- Mouse Lymphoma Mutagenicity Assay to determine the mutagenic nature of the test compound C.I. direct red 80

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

C.I. direct red 80

IUPAC Name:

C.I. direct red 80

Constituent 2

Reference substance name:

Hexasodium 7,7'-(carbonyldiimino)bis[4-hydroxy-3-[[2-sulphonato-4-[(4-sulphonatophenyl)azo]phenyl]azo]naphthalene-2-sulphonate]

EC Number:

220-027-3

EC Name:

Hexasodium 7,7'-(carbonyldiimino)bis[4-hydroxy-3-[[2-sulphonato-4-[(4-sulphonatophenyl)azo]phenyl]azo]naphthalene-2-sulphonate]

Cas Number:

2610-10-8

IUPAC Name:

hexasodium 4-hydroxy-7-({[5-hydroxy-7-sulfonato-6-({2-sulfonato-4-[(4-sulfonatophenyl)diazenyl]phenyl}diazenyl)-2-naphthyl]carbamoyl}amino)-3-({2-sulfonato-4-[(4-sulfonatophenyl)diazenyl]phenyl}diazenyl)naphthalene-2-sulfonate

Details on test material:

- Name of test material (as cited in study report): C.I. direct red 80
- Molecular formula (if other than submission substance): C45H32N10O21S6.6Na
- Molecular weight (if other than submission substance): 1373.09 g/mol
- Substance type: Organic
- Physical state: Solid
Purity: No data available
- Impurities (identity and concentrations):No data available

Specific details on test material used for the study:

- Name of test material : C.I. direct red 80
- Molecular formula : C45H32N10O21S6.6Na
- Molecular weight : 1373.09 g/mol
- Substance type: Organic
- Physical state: Solid
- Purity: No data available
- Impurities (identity and concentrations):No data available

Method

Target gene:

No data available

Species / strain

Species / strain / cell type:

other: Mouse Lymphoma cell line L5178Y TK+/- 3.7.C

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 bsupplemented 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:

S9 activation system

Test concentrations with justification for top dose:

2429-5000 µg/mL

Vehicle / solvent:

No data available

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

methylmethanesulfonate

other: 3-methylcholanthrene or dimethylbenz[a]- anthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

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: 1 106 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: No data available

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:

No data

Evaluation criteria:

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

Results and discussion

Test results

Species / strain:

other: Mouse Lymphoma cell line L5178Y TK+/- 3.7.C

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

Additional information on results:

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: No data available

COMPARISON WITH HISTORICAL CONTROL DATA: No data available

ADDITIONAL INFORMATION ON CYTOTOXICITY: The doses of chemical selected for testing were within the range
yielding approximately 0-90% cytotoxicity.

Remarks on result:

other: No mutagenic potential

Applicant's summary and conclusion

Conclusions:

C.I. direct red 80 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:

The gene mutation study was conducted according to L5178Y TK+/-Mouse Lymphoma Mutagenicity Assay to determine the mutagenic nature of the test compound C.I. direct red 80. The Cells at a concentration of 6 X 10⁵/mL (6 X10⁶cells total) were exposed for 4 h to a range of concentrations from2429-5000µg/mL. 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. C.I. direct red 80 failed to 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.