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Diss Factsheets

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:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
publication
Title:
Induction of trifluorothymidine-resistant mutants by metal ions in L5178y/TK+/-cells
Author:
Amacher D. E. and Paillet S. C.
Year:
1980
Bibliographic source:
Mutat Res. 78 :279-288.

Materials and methods

Principles of method if other than guideline:
Cells deficient in thymidine kinase (TK) due to the mutation TK+/- to TK-/- are resistant to the cytotoxic effects of trifluorothymidine (TFT). Thymidine kinase proficient cells (TK+/-) are sensitive to TFT, which causes the inhibition of cellular metabolism and halts further cell division. Thus mutant cells are able to proliferate in the presence of TFT, whereas normal cells, which contain thymidine kinase, are not able to proliferate.
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Reference substance name:
Zinc chloride
EC Number:
231-592-0
EC Name:
Zinc chloride
Cas Number:
7646-85-7
Molecular formula:
Cl2Zn
IUPAC Name:
zinc dichloride
Test material form:
solid

Method

Target gene:
Thymidine kinase locus/TK +/-

Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: Yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
without
Metabolic activation system:
No data
Test concentrations with justification for top dose:
1.21-12.13 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Normal saline (1 %)
- Justification for choice of solvent/vehicle: Not reported
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Remarks:
None
Details on test system and experimental conditions:
METHOD OF APPLICATION: In medium


DURATION
- Preincubation period: Not reported
- Exposure duration: 3 h
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): 7 d (at 37 °C in 5 % C02-95 % humidified air)



SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT) - 4 μg/mL


NUMBER OF REPLICATIONS: Duplicate cell culture for test material treatment while triplicate plates were prepared for both survival and mutation frequency determinations for each of the 2 replicate cultures


NUMBER OF CELLS EVALUATED: Cells densities were 30000/mL (1 x 100,000/plate) for mutant selection and 15/mL (500/plate) for viability detrmination


DETERMINATION OF CYTOTOXICITY
- Method: Cell survival for each culture was the product of growth in suspension culture and cloning efficiency in soft-agar medium, each relative to solvent controls

OTHER: Cell culture contained 6 x 100,000 cells each in 10 mL test medium
Light exposure was minimal during treatment of cells.
Test conducted at 37 °C
For the recovery and mutant expression, all cells were maintained at 37 °C for 48 h in log phase growth after treatment with test material
Evaluation criteria:
Colonies growing in the presence of triflurothymidine (TFT resistant) or its absence (viable count colonies) were counted. TFT Resistant colonies which were equivalent in size to colonies growing in the solvent control viable count plates ie., large, were scored as mutants.
Statistics:
Not reported

Results and discussion

Test results
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
Not reported
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Under the study conditions, the test substance was found to be non-mutagenic.

Executive summary:

A study was conducted to assess the potential mutagenicity of the test substance in the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The mouse lymphoma cells (TK+/-) were treated with test substance at 1.21-12.13 µg/mL for 3 h. 48 h after treatment, cells were treated with 4 µg/mL trifluorothymidine (TFT) for 7 d. Colonies growing in the presence of triflurothymidine (TFT resistant) or its absence (viable count colonies) were counted. TFT resistant colonies which were equivalent in size to colonies growing in the solvent control viable count plates i.e., large, were scored as mutants. Under the study conditions, the test substance was found to be non-mutagenic (Amacher, 1980).