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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
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
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:
Mutagenic activity of 27 dyes and related chemicals in the Salmonella/microsome and mouse lymphoma TK +/- assays
Author:
T.P. Cameron , T.J. Hughes , P.E. Kirby , V.A. Fung and V.C. Dunkel
Year:
1987
Bibliographic source:
Mutation Research, (1987)

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: as below
Principles of method if other than guideline:
Mutagenicity and cytotoxicity activity was studied in the mouse lymphoma cell line for the test chemical according to the L5178Y TK +/- mouse lymphoma assay
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: Carmoisine
- Molecular formula: C20H14N2O7S2.2Na
- Molecular weight: 502.4338 g/mol
- Substance type: organic
- Physical state: No data available
- Purity 85% dye
- Impurities (identity and concentrations):
15.0 %NaCI
>0 1% subsidiary colors

Method

Target gene:
Thymidine kinase TK
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 supplemented with 10% horse serum, antibiotics and 0.02% Pluronic F-68.
- Properly maintained: No data available
- Periodically checked for Mycoplasma contamination: yes, The cells were checked for the presence of mycoplasma by agar block isolation and Hoechst staining before and after cryopreservation.
- Periodically checked for karyotype stability: No data available
- Periodically "cleansed" against high spontaneous background: No data available
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Test concentrations with justification for top dose:
Test concentration without S9 mix:
0.0, 3706.0, 4560.0, 4560.0, 5000.0 and 5000.0 µg/mL

Test concentration with S9 mix:
0, 685.0, 685.0, 1072.0, 1072.0, 1456.0, 1845.0, 2231.0 and 2231.0 µg/mL
Vehicle:
- Vehicle(s)/solvent(s) used: The solvents used were water, dimethyl sulfoxide, and acetone (exact solvent details are not available)
Controls
Negative controls:
not specified
Solvent controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
ethylmethanesulphonate
Details on test system and conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data available
- Exposure duration: 4 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

SELECTION AGENT (mutation assays): No data available
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

DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency, Relative total growths were observed.

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

Other: Relative suspension growth was also measured.
Evaluation criteria:
A response 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%.
Statistics:
No data avaialble

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
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: The toxicity of each chemical was first determined both with and without S9 prepared from Aroclor-1254-induced male Fischer 344 rats.

COMPARISON WITH HISTORICAL CONTROL DATA: No data available

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data available

Applicant's summary and conclusion

Conclusions:
The test chemical did not induce mutation and hence was negative (with and without S9 mix) in L5178Y TK +/- mouse lymphoma assay.
Executive summary:

L5178Y TK +/- mouse lymphoma assay was performed using test chemical at different concentrations both with and without S9 metabolic activation system. Cells in duplicate cultures were exposed to the test chemical, positive control, and solvent control for 4 h at 37 ° C; washed twice with growth medium; and maintained at 37 °C for 48 h in log phase growth to allow recovery and mutant expression. The cultures were adjusted to 0.3 × 106 cells/ml at 24-h intervals. They were then cloned in soft agar medium containing Fischer's medium, 20% horse serum, 2 mM sodium pyruvate, 0.02% Pluronic F-68 and 0.35% Noble agar. Resistance to trifluorothymidine (TFT) was determined by adding 3 µg/ml TFT to one set of plates. The 100 X stock solution of TFT in saline was stored at -70°C and thawed immediately before use. Plates were incubated at 37°C in 5% CO 2 in air for 12 days, and then counted with an automatic colony counter. Mutant frequencies were expressed as mutants per 104 surviving cells. In general, a response 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 test chemical did not induce mutation and hence was negative (with and without S9 mix) in L5178Y TK +/- mouse lymphoma assay.