Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Supplier: Arkema France
- Name: DIMETHYLSULPHIDE
- Batch number: T 8712 – 04/03/2009
- Description: colorless liquid
- Storage conditions: at room temperature
- Purity: 99.565%
- Expiry date: 04 March 2010.

Method

Target gene:
Thymidine Kinase locus
Species / strain
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254
Test concentrations with justification for top dose:
0, 0.313, 0.625, 1.25, 2.5, 5 and 10 mM
Vehicle:
DMSO
Controls
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without S9 mix: Methylmethane Sulfonate (MMS), with S9 mix: Cyclophosphamide (CPA)
Details on test system and conditions:
After a preliminary toxicity test, DIMETHYLSULPHIDE was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.
Approximately 0.5 x 106 (3-hour treatment) or 0.15 x 106 (24-hour treatment) cells/mL in 20 mL culture medium with 5% horse serum were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%), at 37°C. For the 24-hour treatment, the incubation at 37°C was performed with a gentle shaking.

Cytotoxicity was measured by assessment of adjusted relative total growth (Adj. RTG) and relative suspension growth (Adj. RSG) as well as cloning efficiency following the expression time (CE2).
The number of mutant clones (differentiating small and large colonies) were checked after the expression of the mutant phenotype.
Evaluation criteria:
IWGT recommendations were followed for the determination of a positive result which should fulfill the following criteria:
. at least at one dose-level the mutation frequency minus the mutation frequency of the vehicle control equals or exceeds the global evaluation factor (126 x 10-6 for the microtiter method),
. and a dose-related trend is demonstrated by a statistically significant trend test.

Unless considered as clearly positive, the reproducibility of a positive effect should be confirmed.
Noteworthy increases in the mutation frequency observed only at high levels of cytotoxicity (RTG lower than 10%), but with no evidence of mutagenicity at dose-levels with RTG between 10 and 20%, is considered as positive result.

A test item may be determined to be non-mutagenic when there is no culture showing an Adj. RTG value between 10-20% if (e):
. there is at least one negative data point between 20 and 25% Adj. RTG and no evidence on mutagenicity in a series of data points between 100 to 20% Adj. RTG,
. there is no evidence of mutagenicity in a series of data points between 100 to 25% and there is also a negative data point between 10 and 1% Adj. RTG.
Statistics:
Not appropriate

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
PRELIMINARY TOXICITY TEST
The test item was freely soluble in the vehicle (DMSO) at 124.26 mg/mL. Using a treatment volume of 100 µL/20mL of culture medium, the final dose-level of 10 mM (corresponding to 621.30 µg/mL) showed no precipitate. At this dose-level, the pH was approximately 7.1 (as for the vehicle control) and the osmolality equal to 376 mOsm/kg H2O (as for the vehicle control).
The dose-levels used for treatment were 0.156, 0.313, 0.625, 1.25, 2.5, 5 and 10 mM (corresponding to 9.71, 19.42, 38.83, 77.66, 155.33, 310.65 and 621.3 µg/mL).
No noteworthy toxicity was noted at any tested dose-levels, either following the 3-hour treatment with and without S9 mix, or following the 24-hour treatment without S9 mix.

MUTAGENICITY EXPERIMENTS (Tables 1 to 4, attached document)
The cloning efficiencies CE2 and the mutation frequencies (see § Study plan adherence) of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid.
Since the test item was freely soluble and non-toxic in the preliminary test, the highest dose-level selected for the main test was 10 mM, according to the criteria specified in the international guidelines.
Using a treatment volume of 100 µL/20 mL, the selected dose-levels were 0.313, 0.625, 1.25, 2.5, 5 and 10 mM for both mutagenicity experiments with and without S9 mix.

Cytotoxicity
No noteworthy cytotoxicity was observed at any tested dose-levels, in either experiment, either with or without S9 mix.

Mutagenicity
Without S9 mix, no noteworthy increase in the mutation frequency was observed at any tested dose-levels, either following the 3-hour treatment or following the 24-hour treatment.
With S9 mix, no noteworthy increase in the mutation frequency was observed at any tested dose levels in either experiment.

Applicant's summary and conclusion

Conclusions:
DIMETHYLSULPHIDE did not show any mutagenic activity in the mouse lymphoma assay
Executive summary:

The potential of dimethylsulphide to induce mutations at the TK (Thymidine Kinase) locus of L5178Y mouse lymphoma cells was evaluatedi in a study performed according to OECD No. 476 and in compliance with GLP. After a preliminary toxicity test, dimethylsulphide was tested at concentrations of 0, 0.313, 0.625, 1.25, 2.5, 5 and 10 mM in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

The cloning efficiencies CE2and the mutation frequencies of the vehicle and positive controls were mainly as specified in acceptance criteria. The study was therefore considered valid. No noteworthy cytotoxicity was observed at any tested dose-levels, in either experiment, either with or without S9 mix. With and without S9 mix, no noteworthy increase in the mutation frequency was observed at any tested dose-levels in either experiment.

Under the experimental conditions of this study, dimethylsulphide did not show any mutagenic activity in the mouse lymphoma assay.