Ethyl methyl sulphide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ethyl methyl sulfide was not mutagenic to Salmonella typhimurium (bacterial reverse mutation assay) in vitro. It was also negative in an in vitro chromosomal aberration assay in human lymphocytes. A structural analogue, dimethylsulphide, did not show any mutagenic activity in the mouse lymphoma assay.

Gene mutation assay
In a reverse gene mutation assay in bacteria (# OECD 471, GLP), ethyl methyl sulfide was tested in the bacterial reverse mutation assay (OECD TG 471) with Salmonella typhimurium TA98, TA100, TA102, TA1535, and TA1537 at concentrations up to 5000 µg/plate with and without metabolic activation (Haddouk, 1999).The solvent was dimethylsulfoxide (DMSO). Slight cytotoxicity was observed at concentrations >= 2500 µg/plate. Positive controls produced the expected result.Ethylmethyl sulfide was not mutagenic under the conditions of the assay.

A gene mutation assay with L5178Y mouse lymphoma cells was performed according to OECD No. 476 with a structural analogue (Sire, 2010). 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. 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 experiments. Under the experimental conditions of this study, dimethylsulphide did not show any mutagenic activity in the mouse lymphoma assay.

Chromosomal aberration assay
The potential of ethyl methyl sulphide to induce structural chromosome aberrations in human lymphocytes was evaluated according to OECD 473 and EC 92/69/EEC B.10 guidelines in compliance with the Principles of Good Laboratory Practice (Haddouk, 2004). The test item was tested in two independent experiments, with and without a liver metabolizing system (S9), at the dose levels of 0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 5 and 10 mM, for the first experiment, with and without S9 mix and 0.313, 0.625, 1.25, 2.5, 5 and 10 mM, for the second experiment, with and without S9 mix, together with vehicle and positive controls. In the first experiment, lymphocytes cultures were exposed to the test and control items for 3 hours without or with S9, and cells were harvested 20 hours after the beginning of treatment.In the second experiment, the cultured cells were exposed for 3 hours with metabolic activation followed by 20- or 44-hour culture in treatment-free media prior to cell harvest; whilst without S9 the exposure time was increased to 20 oa 44 hours.Under these experimental conditions, ethyl methyl sulphide did not induce any noteworthy increase in the number of cells with structural chromosome aberration, both with and without S9 mix, in either experiment or at either harvest time.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine reversion

Species / strain / cell type:

S. typhimurium, other: Strains: TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254

Test concentrations with justification for top dose:

Experiment without S9:
. 312.5, 625, 1250, 2500 and 5000 µg/plate: for all tester strains in both experiments except for the TA 1537 and TA 98 strains in the second experiment,
. 156.25, 312.5, 625, 1250 and 2500 µg/plate: for the TA 98 and TA 1537 strains in the second experiment.
Experiments with S9 mix:
. 312.5, 625, 1250, 2500 and 5000 µg/plate: for all tester strain.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S9 mix: .1 µg/plate of sodium azide (NaN3): TA 1535 and TA 100 strains. 50 µg/plate of 9-Aminoacridine (9AA): TA 1537 strain. 0.5 µg/plate of 2-Nitrofluorene (2NF): TA 98 strain. 0.5 µg/plate of Mitomycin C (MMC): TA 102 strain. With S9 mix: 2 µ

Details on test system and experimental conditions:

A preliminary toxicity test was performed to define the dose-levels of ETHYLMETHYL SULFIDE to be used for the mutagenicity study. The test substance was then 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. Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C). Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to five dose-levels of the test substance (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

Species / strain:

S. typhimurium, other: Strains: TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 2500 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Since the test substance was freely soluble and almost non-toxic in the preliminary test, the highest dose-level for the first experiment was 5000 µg/plate, according to the criteria specified in the international guidelines. A slight emulsion was sometimes observed in the Petri plates when scoring the revertants at 5000 µg/plate.
Without S9, a slight to marked toxicity was noted (except for the TA 102 strain), depending on the tester strain and the dose-level. The test substance did no induce any noteworthy increase in the number of revertants, in both experiments, in any of the five strains.

With S9, no or slight to moderate toxicity was noted, depending on the tester strain and the dose-level. The test did not induce any noteworthy increase in the number of revertants, in both experiments, in any of the five strains.

The number of reverants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

Conclusions:

ETHYLMETHYL SULFIDE does not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Executive summary:

In a reverse gene mutation assay in bacteria (# OECD 471, GLP), ethyl methyl sulfide was tested in the bacterial reverse mutation assay (OECD TG 471) with Salmonella typhimurium TA98, TA100, TA102, TA1535, and TA1537 at concentrations up to 5000 µg/plate with and without metabolic activation. The solvent was dimethylsulfoxide (DMSO).  Slight cytotoxicity was observed at concentrations >= 2500 µg/plate. Positive controls produced the expected result. Ethyl methyl sulfide was not mutagenic under the conditions of the assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Metabolic activation:

with and without

Metabolic activation system:

Liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254

Test concentrations with justification for top dose:

0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 5 and 10 mM, for the first experiment, with and without S9 mix,
0.313, 0.625, 1.25, 2.5, 5 and 10 mM, for the second experiment, with and without S9 mix.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S9 mix, mitomycin C: 3 µg/mL (3 hours of treatment) or 0.2 µg/mL (continuous treatment). With S9 mix, cyclophosphamide: 12.5 or 25 µg/mL.

Details on test system and experimental conditions:

The test item was tested in two independent experiments, both with and without a liver metabolizing system (S9 mix), obtained from rats previously treated with Aroclor 1254. The highest dose-level for treatment in the first experiment was selected on the basis of pH, osmolality and solubility. For selection of the dose-levels for the second experiment, any toxicity indicated by the reduction of mitotic index (MI) in the first experiment was also taken into account. For each culture, heparinized whole blood was added to culture medium containing a mitogen (phytohemagglutinin) and incubated at 37°C, for 48 hours.
In the first experiment, lymphocyte cultures were exposed to the test or control items (with or without S9 mix) for 3 hours then rinsed. Cells were harvested 20 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles.
The second experiment was performed as follows:
* without S9 mix, cells were exposed continuously to the test or control items until harvest,
* with S9 mix, cells were exposed to the test or control items for 3 hours and then rinsed. Cells were harvested 20 hours and 44 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later, respectively.
One and a half hours before harvest, each culture was treated with a colcemid solution (10 µg/mL) to block cells at the metaphase-stage of mitosis. After hypotonic treatment (KCl 0.075 M), the cells were fixed in a methanol/acetic acid mixture (3/1; v/v), spread on glass slides and stained with Giemsa. All the slides were coded for scoring.

Evaluation criteria:

A reproducible and statistically significant increase in the frequency of cells with structural chromosome aberration for at least one of the dose-levels and one of the two harvest times was considered as a positive result. Reference to historical data or other considerations of biological relevance, was also taken into account in the evaluation of the findings.

Statistics:

For each test and for each harvest time, the frequency of cells with structural chromosome aberration (excluding gaps) in treated cultures was compared to that of the vehicle control cultures. If necessary, the results were compared using the ¿2 test, in which p = 0.05 was used as the lowest level of significance.

Species / strain:

primary culture, other: human lymphocytes

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 examined

Positive controls validity:

valid

Additional information on results:

In the culture medium, the dose-level of 10 mM (corresponding to 760 µg/mL) showed no precipitate. At this dose-level, the pH was approximately 7.1 (6.8 for the vehicle control) and the osmolality equal to 364 mOsm/kg H2O (421 for the vehicle control).

Experiments without S9 mix:
Cytotoxicity: following the 3-, 20- and 44-hour treatments, no decrease in mitotic index was observed at any dose-level.
Metaphase analysis:
The dose-levels selected for metaphase analysis were as follows:
* 2.5, 5 and 10 mM, for the 3-hour and the 20-hour treatments,
* 10 mM, for the 44-hour treatment.
No significant increase in the frequency of cells with structural chromosomal aberration was noted after 3-, 20- as well as 44-hour treatments.

Experiments with S9 mix:
Cytotoxicity: at the 20- and 44-hour harvest times, no decrease in mitotic index was observed at any dose-level.
Metaphase analysis:
The dose-levels selected for metaphase analysis were as follows:
* 2.5, 5 and 10 mM, for the 20-hour harvest time in both experiments,
* 10 mM, for the 44-hour harvest time.
No significant increase in the frequency of cells with structural chromosomal aberration was noted in both experiments and at both harvest times.

The frequency of cells with structural chromosome aberration of the vehicle and positive controls was as specified in acceptance criteria. The study was therefore considered valid.

Conclusions:

METHYL ETHYL SULFIDE did not induce chromosome aberrations in cultured human lymphocytes.

Executive summary:

The potential of ethyl methyl sulphide to induce structural chromosome aberrations in human lymphocytes was evaluated according to OECD 473 and EC 92/69/EEC B.10 guidelines in compliance with the Principles of Good Laboratory Practice.

The test item was tested in two independent experiments, with and without a liver metabolizing system (S9), at the dose levels of 0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 5 and 10 mM, for the first experiment, with and without S9 mix and 0.313, 0.625, 1.25, 2.5, 5 and 10 mM, for the second experiment, with and without S9 mix, together with vehicle and positive controls. In the first experiment, lymphocytes cultures were exposed to the test and control items for 3 hours without or with S9, and cells were harvested 20 hours after the beginning of treatment. In the second experiment, the cultured cells were exposed for 3 hours with metabolic activation followed by 20- or 44-hour culture in treatment-free media prior to cell harvest; whilst without S9 the exposure time was increased to 20 oa 44 hours. Under these experimental conditions, ethyl methyl sulphide did not induce any noteworthy increase in the number of cells with structural chromosome aberration, both with and without S9 mix, in either experiment or at either harvest time. 

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

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

Target gene:

Thymidine Kinase locus

Species / strain / cell type:

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 / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle 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 experimental 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

Key result

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 examined

Positive controls validity:

valid

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.

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 evaluated 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Ethyl methyl sulfide is not a mutagenic substance. No classification is required according to Regulation (EC) No 1272/2008.