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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:
key study
Study period:
Experimental start date: 17 September 2013, Experimental Completion Date: 14 October 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Meets the criteria for classification as Reliable without restriction according to Klimisch et al (1997).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
lithium 3-((3,4-dicyanophenyl)thio)propane-1-sulfonate
EC Number:
700-787-7
Cas Number:
769953-18-6
Molecular formula:
C11H9N2O3S2Li
IUPAC Name:
lithium 3-((3,4-dicyanophenyl)thio)propane-1-sulfonate
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): S193115
- Analytical purity: 94.4% w/w
- Lot/batch No.: 398
- Storage condition of test material: Room temperature in the dark

Method

Target gene:
Thymidine Kinase TK +/- locus
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
The L5 178Y 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.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital and beta-naphtholflavone induced rat liver S9
Test concentrations with justification for top dose:
Preliminary Toxicity Test: 0, 11.25, 22.5, 45, 90, 180, 360, 720, 1440, 2880 ug/ml.

Main Test Experiment 1: 0, 180, 360, 720, 1440, 2160, 2880 ug/ml
Main Test Experiment 2: 0, 90, 180, 360, 720, 1440, 1920, 2400, 2880 ug/ml

Concentrations were adjusted for the stated purity of the test material (94.4%) and therefore test concentrations given are for the test substance as active.
Vehicle / solvent:
RPMI 1640 culture medium (without serum)
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(culture medium without serum)
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
In the absence of metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(culture medium without serum)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
In the presence of metabolic activation
Details on test system and experimental conditions:
The study was designed to assess the potential mutagenicity of the test substance on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. Following a preliminary toxicity test, two main experiments were performed using the test concentrations given previously (see Test Concentrations section). Ethylmethansuphonate and cyclophosphamide were used as positive controls.

In experiment 1, cells were treated for 4-hours in either the absence or presence of metabolic activation (rat liver S9). In experiment 2, the exposure groups were 4-hours in the presence of metabolic activation and 24-hours in the absence of metabolic activation. The positive controls responded as expected indicating that the test system was functioning correctly.

There was no marked change in pH when the test material was dosed into media and osmolarity did not increase by more than 50 mOsm.
Evaluation criteria:
The normal range for mutant frequency per survivor is 50-170 x 10^(-6) for the TK+/- locus in L5178Y cells at the test laboratory which conducted the study. Vehicle controls results should ideally be within this range, although minor errors in cell counting and dilution or exposure to the metabolic activation system may cause this to be slightly elevated. Experiments where the vehicle control values are markedly greater than 250 x 10^(-6) mutant frequency per survivor are not normally acceptable and will be repeated.

Positive control chemicals should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control.

Statistics:
The results were subject to statistical analysis according to United Kingdom Environmental Mutagen Society (UKEMS) statistical guidelines.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The maximum dose level used was the 10 mM limit dose. Precipitate of test item was not observed at any of the dose levels. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus.

The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system. The test item did not induce any toxicologically significant dose-related (linear-trend) increases in the mutant frequency at any of the dose levels, either with or without metabolic activation, in either the first or the second experiment.
Remarks on result:
other: strain/cell type: Mouse Lymphona L5178Y
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Results

Experiment 1

There was evidence of modest toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the % relative suspension growth and relative total growth values. There was also evidence of modest reductions in viability, indicating that residual toxicity had occurred in both the absence and presence of metabolic activation. Acceptable levels of toxicity were seen with both positive control substances.

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 170 x viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.

The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in the absence of metabolic activation. The test item induced a very modest but statistically significant dose related (linear-trend) increase in the mutant frequency in the presence of metabolic activation and a statistically significant increase in mutant frequency was observed at an individual dose level. However, the Global Evaluation Factor was not exceeded at any of the individual dose levels and there was also no evidence of any marked increases in absolute numbers of mutant colonies. Therefore, the response was considered to be artefactual and of no toxicological significance. Precipitate of the test item was not observed at any of the dose levels.

Experiment 2

As was seen previously, there was evidence of marked toxicity in the 24-hour exposure group in the absence of metabolic activation, and modest toxicity in the presence of metabolic activation as indicated by the % relative suspension growth and relative total growth values. There was evidence of modest reductions in % viability in the absence of metabolic activation, indicating that residual toxicity had occurred. Based on the relative total growth and % relative suspension growth values, it was considered that optimum levels of toxicity were achieved in the absence of metabolic activation. Excessive toxicity observed at 2880 µg/mL in the absence of metabolic activation resulted in this dose level not being plated for viability or 5-TFT resistance. Acceptable levels of toxicity were seen with both positive control substances. 

The 24-hour exposure without metabolic activation (S9) treatment, demonstrated that the extended time point had a marked effect on the toxicity of the test item. It should also be noted that the lowering of the S9 concentration to 1% in this second experiment resulted in slightly lower levels of toxicity being observed when compared to 4-hour exposure groups in the presence of 2% metabolic activation in the Preliminary Toxicity Test and Experiment 1.

 

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 170 x viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.

Applicant's summary and conclusion

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

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

Introduction

The study was performed to assess the potential mutagenicity of the test item. It was designed to be compatible with the following guideline: OECD Guidelines for Testing of Chemicals No. 476 "In Vitro Mammalian Cell Gene Mutation Test"

Method

Two independent experiments were performed. In Experiment 1, L5 178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at six dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups, both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at up to eight dose levels using a 4-hour exposure group in the presence of metabolic activation (1 % S9) and a 24-hour exposure group in the absence of metabolic activation.

 

Results

Precipitate of test item was not observed at any of the dose levels. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system.

The test item did not induce any toxicologically significant dose-related (linear-trend) increases in the mutant frequency at any of the dose levels, either with or without metabolic activation, in either the first or the second experiment.

 

Conclusion

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.

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