Methyl phenyl sulphide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 15th June 2010 to 28 July 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study is GLP and guideline compliant

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

The temperature recorded was outside the range of 37± 1°C for 60 minutes in the third mutation experiment.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Principles of method if other than guideline:

Not relevant

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Methyl phenyl sulphide

EC Number:

202-878-2

EC Name:

Methyl phenyl sulphide

Cas Number:

100-68-5

Molecular formula:

C7H8S

IUPAC Name:

(methylsulfanyl)benzene

Details on test material:

- Name of test material (as cited in study report): Thioanisole
- Molecular weight: 124.19
- Physical state: Colourless to slightly yellowish liquid
- Analytical purity: 99 % minimum
- Lot/batch No.: 100310
- Expiration date of the lot/batch: 23 March 2011
- Storage condition of test material: At room temperatures, protected from light under nitrogen

Test substance preparation: The test substance was dissolved in dimethyl sulfoxide. The test solutions were prepared in amber coloured glassware. Test substance concentrations were used within 3.5 hours after preparation.

Method

Target gene:

Histidine

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: TA1537 and TA98 detects frameshift mutation in hisC3076 and hisD3052/R-factor* respectively. TA1535 and TA100 detects base-pair substitutions in hisG46 and hisG46/R-factor* (* R-factor=plasmid pKM101)

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: detects base-pair substitutions

Metabolic activation:

with and without

Metabolic activation system:

S9 - prepared from adult male Wistar rats

Test concentrations with justification for top dose:

In the dose selection study, Thioanisole was tested in the tested strains TA100 and WP2uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 ug/plate in the absence and presence of S9-mix.

In the mutation assay, five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain.

Vehicle / solvent:

dimethyl sulfoxide

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

the vehicle of the test article: dimethyl sulfoxide

Negative solvent / vehicle controls:

yes

Remarks:

saline

True negative controls:

not specified

Positive controls:

yes

Remarks:

5µg

Positive control substance:

sodium azide

Remarks:

TA1535 - Without metabolic activation

Controls 2

Untreated negative controls:

yes

Remarks:

the vehicle of the test article: dimethyl sulfoxide

Negative solvent / vehicle controls:

yes

Remarks:

Milli-Q water

True negative controls:

not specified

Positive controls:

yes

Remarks:

60 µg

Positive control substance:

9-aminoacridine

Remarks:

TA 1537 - Without metabolic activation

Controls 3

Untreated negative controls:

yes

Remarks:

the vehicle of the test article: dimethyl sulfoxide

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide

True negative controls:

not specified

Positive controls:

yes

Remarks:

10 µg

Positive control substance:

2-nitrofluorene

Remarks:

TA98 - Without metabolic activation

Controls 4

Untreated negative controls:

yes

Remarks:

the vehicle of the test article: dimethyl sulfoxide

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide

True negative controls:

not specified

Positive controls:

yes

Remarks:

650ug

Positive control substance:

methylmethanesulfonate

Remarks:

TA100 - Without metabolic activation

Controls 5

Untreated negative controls:

yes

Remarks:

the vehicle of the test article: dimethyl sulfoxide

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide

True negative controls:

not specified

Positive controls:

yes

Remarks:

10ug

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

WP2uvrA - Without metabolic activation

Controls 6

Untreated negative controls:

yes

Remarks:

the vehicle of the test article: dimethyl sulfoxide

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide

Positive controls:

yes

Remarks:

2.5ug for TA 1535, TA 1537 and TA 100. 1ug for TA 98 and TA 100. 5 µg for TA 1537 and 10 µg for WP2uvrA.

Positive control substance:

other: 2-aminoanthracene

Remarks:

Used for all tester strains - with metabolic activation

Details on test system and experimental conditions:

Selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and WP2uvrA, both with and without 5% (v/v) S9-mix. Eight concentrations, 3, 10, 33, 100, 333, 1000, 3330 and 5000 ug/plate were tested in triplicate. The highest concentrations of Thioanisole used in the subsequent mutation assay was the level at which the test substance inhibited bacterial growth.

In the mutation assay study, at least 5 different doses of the test substance were tested in triplicate in each strain. In the first experiment Thioanisole was tested both in the absence and presence of 5 % (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. In an independent repeat of the assay with additional parameters, the test substance was tested both in the absence and presence of 10 % (v/v) S-9 mix in all tester strains. An additional third experiment was performed since no toxic dose levels were selected in the second mutation assay.

The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.

Top agar in top tubes was melted by heating to 45 °C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10 E9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test substance in dimethyl sulfoxide and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M Phosphate buffer (in case of non-activation assays). The ingredients were mixed on a vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37 ± 1.0°C for 48 ± 4 hours. After this period revertant colonies (histidine independent His +) for Salmonella typhirium bacteria and tryptophan independent (Trp+) for Escherichia coli were counted.

The revertant colonies (histidine independent or tryptophan independent) were counted manually if less than 40 colonies per plate were present. If more than 40 colonies were present, these were counted automatically with a Biocount 4000 Pro-S-colony counter. Plates with abundant test article precipitate which interfered with automated colony counting were counted manually and the evidence of test substance precipitate on the plates was recorded. The condition of the bacterial background lawn was evaluated, both macroscopically and microscopically by using a dissecting microscope.

Observations/measurements in the study were recorded electronically using the following programme: REES Centron Environmental Monitoring system verison SQL 2.0.

Evaluation criteria:

No formal hypothesis testing was done.

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three time the concurrent control.
b) where a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision by expert judgement.

Statistics:

No data

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg/plate: extremely toxic

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg/plate: extremely toxic

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium, other: TA1535, TA1537 and TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

not determined

Remarks:

Since no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants was observed in the Salmonella typhirium tester strains, this part was repeated in the third mutation experiment.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

In the dose range-finding study:

Precipitation of Thioanisole on the plates was only observed at the start of the incubation period at concentrations of 3330 and 5000 µg/plate.
Table 1, below, provides information on the toxicity of Thioanisole. There was no increase in the number of revertants.

Experiment 1:
Precipitation of Thioanisole on the plates was only observed at the start of the incubation period at a concentration of 3330 µg/plate. The reduction of the bacterial background lawn and the reduction in the number or revertants are presented in Table 2 below. There was no increase in the number of revertants upon treatment with Thioanisole under all the conditions used.

Experiment 2:
Precipitation of Thioanisole on the plates was only observed at the start of the incubation period at a concentration of 3330 µg/plate. Appropriate toxicity was only observed in tester strain WP2uvrA. A slight reduction of the bacterial background lawn was observed at a concentrations of 3330 µg/plate in the absence of the S9-mix. In the presence of S9-mix, microcolonies were formed with an extreme reduction of the bacterial background lawn at the top concentrations of 3330 µg/plate. Since no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants was observed in the Salmonella typhimurium tester strains, this part was repeated in the third mutation experiment. There was no increase in the number of revertants under all conditions tested.

Experiment 3:
Precipitation of Thioanisole on the plates was only observed at the start of the incubation period at a concentration of 3330 µg/plate. The reduction of the bacterial background lawn and the reduction in the number of revertants are presented in table 3. No increase in the number of revertants was observed upon treatment with Thioanisole under all conditions used.

All bacterial strains showed negative responses over the entire dose range i.e. no significant dose-related increase in the number of revertants in three independently repeated experiments. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: Thioanisole tested at: 1000, 3330 and 5000 µg/plate in absence and presence of S9 mix

Any other information on results incl. tables

Table 1: Toxicity of Thioanisole in the dose range finding test

train	Without S9-mix			With S9-mix
	Dose (µg/plate)	Bacterial background lawn	Revertant colonies	Dose (µg/plate)	Bacterial background lawn	Revertant colonies
TA100	1000	Slight	No reduction in number	1000	Slight	No reduction in number
	3330	Extreme	Microcolonies	3330	Extreme	Microcolonies
	5000	Extreme	Microcolonies	5000	Extreme	Microcolonies
WP2uvrA	3330	Extreme	Microcolonies	3330	Moderate	No reduction in number
	5000	Extreme	Microcolonies	5000	Moderate	No reduction in number

 

Table 2: Toxicity of Thioanisole in the first experiment

train	Without S9-mix			With S9-mix
	Dose (µg/plate)	Bacterial background lawn	Revertant colonies	Dose (µg/plate)	Bacterial background lawn	Revertant colonies
TA1535	1000	Slight	No reduction in number	1000	Slight	No reduction in number
	3330	Extreme	Microcolonies	3330	Extreme	Microcolonies
TA1537	1000	Slight	Moderate	1000	Slight	Slight
	3330	Extreme	Microcolonies	3330	Moderate	Slight
TA98	1000	Slight	No reduction in number	1000	Slight	No reduction in number
	3330	Extreme	Microcolonies	3330	Extreme	Microcolonies

 

Table 3: Toxicity of Thioanisole in the third experiment

train	Without S9-mix			With S9-mix
	Dose (µg/plate)	Bacterial background lawn	Revertant colonies	Dose (µg/plate)	Bacterial background lawn	Revertant colonies
TA1535	3330	Extreme	Microcolonies	3330	Extreme	Microcolonies
TA1537	3330	Extreme	Microcolonies	3330	Moderate	No reduction in number
TA98	1000	Slight	No reduction in number	3330	Moderate	No reduction in number
	3330	Extreme	Microcolonies
TA100	1000	Slight	No reduction in number	1000	Slight	No reduction in number
	3330	Extreme	Microcolonies	3330	Extreme	Microcolonies

Applicant's summary and conclusion

Conclusions:

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

Based on the results of the study it is concluded that Thioanisole is not mutagenic with and without metabolic activation in the reverse mutation assay using Salmonella typhimurium and Escherichia coli. Under the conditions of this study, the test substance does not require classification according to Regulation EC No. 1272/2008 or according to Directive 67/548/EEC.

Executive summary:

In a study conducted by Verspeek-Rip (2010), the test substance, Thioanisole was tested in the Salmonella typhimurium reverse mutation assay with four histidine requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP₂uvrA). The test was performed in two independent experiments in the presence and absence of S9 -mix. Since in the second experiment in the tester strains TA1535, TA1537, TA98 and TA100 no toxicity or precipitate on the plates was observed, a third mutation experiment was performed with these strains in the absence and presence of 10 % (v/v) S9 -mix. The study was carried out to the most recent OECD and EC Guidelines.

All bacterial strains showed negative responses over the entire dose range. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Based on the results of the study it is concluded that Thioanisole is not mutagenic, both in the presence and absence of metabolic activation, in the reverse mutation assay using Salmonella typhimurium and Escherichia coli. Under the conditions of this study, the test substance does not require classification according to Regulation EC No. 1272/2008 or according to Directive 67/548/EEC.