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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Negative responses at limits of cytotoxicity
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Full GLP report to current guidelines, 2013
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Test material information:
Composition 1
Target gene:
Assessment of the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.
Species / strain:
mouse lymphoma L5178Y cells
Details on mammalian cell lines (if applicable):
The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital/-naphthoflavone
Test concentrations with justification for top dose:
The dose range used in the preliminary toxicity test was 19.53 to 5000 μg/ml for all three of the exposure groups.
Experiment 1 treatments were performed in duplicate (A + B), both with and without metabolic activation (S9-mix) at eight dose levels of the test item (19.53 to 625 μg/ml in both the absence and presence of metabolic activation)
Experiment 2 treatments were performed in duplicate (A + B), both with and without metabolic activation (S9-mix) at eight dose levels of the test item (5 to 160 μg/ml in the absence of metabolic activation, and 100 to 450 μg/ml in the presence of metabolic activation),
Vehicle:
Water
Negative controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Details on test system and conditions:
The treatment vessels were incubated at 37°C for 4 hours with continuous shaking using an orbital shaker within an incubated hood.
At the end of the treatment period, for each experiment, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 105 cells/ml. The cultures were incubated at 37 °C with 5% CO2 in air and subcultured every 24 hours for the expression period of two days by counting and diluting to 2 x 105 cells/ml.
On Day 2 of the experiment, the cells were counted, diluted to 104 cells/ml and plated for mutant frequency (2000 cells/well) in selective medium containing 4 μg/ml 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/ml and plated (2 cells/well) for viability (%V) in non-selective medium.
Evaluation criteria:
The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.
Statistics:
Small significant increases designated by the UKEMS statistical package will be reviewed using the above criteria, and may be disregarded at the Study Director's discretion.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
In all three of the exposure groups there was evidence of marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls. The toxicity was very steep in all three exposure groups.

The steep toxicity curve observed proved that it would be hard to achieve optimum toxicity. The excessive toxicity observed at and above 468.75 μg/ml in the absence of metabolic activation and at 625 μg/ml in the presence of metabolic activation resulted in these dose levels not being plated for viability or 5-TFT resistance. In the presence of metabolic activation a dose level (468.75 μg/ml) was later excluded from statistical analysis as the RTG value also fell below the acceptable level of toxicity.

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.
Executive summary:

Valid study, but difficulties in finding optimum concentrations for test due to sharp cut-off in toxicity. This is considered common with surface active agents that will disrupt membranes at criticial concentrations.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Justification for selection of genetic toxicity endpoint
Study performed on mammalian cells using the substance to be registered, 2013

Justification for classification or non-classification

Negative responses at limits of cytotoxicity