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Genetic toxicity: in vitro

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in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference Type:
Evaluation of the L5178Y Mouse Lymphoma Cell Mutagenesis Assay: Intralaboratory Results for Sixty-Three Coded Chemicals Tested at SRI International.
Mitchell AD, Rudd CJ and Caspary WJ
Bibliographic source:
Environ. Mol. Mutagen. 12 (Suppl. 13), 37-101.

Materials and methods

Principles of method if other than guideline:
Method: other: Mitchell et al. (1988) Environ Mol Mutagen, 12(suppl 13), 1-18.
GLP compliance:
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:


Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Additional strain / cell type characteristics:
other: TK +/-
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
0.39 to 1,5 µl/ml (-S9)0.007 to 0.06 µl/ml (+S9)
Details on test system and experimental conditions:
Cultures of 6 x 10e6 TK+/- L5178Y mouse lymphoma cells, clone 3.7.2C, in 10 ml medium or in 10 ml medium containing a mixture of S9, NADP, and sodium isocitrate in FO medium (S9 mix) were exposed to a series of concentrations of a chemical (in DMSO) for 4 hr and then grown for 2 days for mutant expression. The cell concentrations were adjusted to 3 x 10e5 cells/ml during expression. For cloning, in medium containing 0.35% agar and 20% horse serum, 3 x 10e6 cells from each culture were seeded for determination of TFT-resistant colonies; the CE in nonselective medium was determined after seeding 600 cells. All results presented here were obtained using TFT as the selective agent. Eleven to twelve days after cloning, colonies on the plates were counted using a Biotran or an Artek 880 automatic colony counter with a zero size setting, which included small colony mutants not visible on the video monitor. Calculations and statistical analysis of the data were done by computer according to the mathematical model described by Lee and Caspary [1983] . The assay of each chemical consisted of tests without and with exogenous activation (S9 from Aroclor-induced male Fischer 344 rats); each test comprised at least two replicate experiments or trials.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
+ S9 mix
Cytotoxicity / choice of top concentrations:
other: >= 1.2 µl/ml without S9|>= 0.04 µg/ml with S9
Additional information on results:
Chloroform was tested at concentrations from 0.39 to 1.5 µl/ml in three experiments in the absence of S9. Concentration-related depressions in RTG values were observed in all experiments. A clearly positive response was observed in the first experiment: mutant frequencies increased with concentration to about ninefold above background at 1.25 µl/ml. However, this response could not be reproduced in two more trials. Therefore, the test in the absence of S9 was evaluated as negative. The variability in the three trials may have been related to the volatility of chloroform. In the presence of induced S9, chloroform was tested over a lower concentration range, from 0.007 to 0.06 µl/ml. In both experiments, significant dose-related increases in mutant frequencies and significantly positive (approximately threefold) responses at the highest tested concentrations were observed, yielding positive trial and test results in the presence of induced S9.
Remarks on result:
other: all strains/cell types tested
Migrated from field 'Test system'. Remarks: L5178Y TK+/- lymphoma cells

Applicant's summary and conclusion

Executive summary:

The L5178Y mouse lymphoma cell forward mutation assay was used to determine the mutagenic activity of chloroform. Ambigous results were observed in absence of metabolic activation at concentration up to 1.5 µl/ml and positive response was noted with metabolic activation at concnetration up to 0.006 µl/ml.