Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro (OECD TG 471): not mutagenic

Genetic toxicity in vitro (OECD TG 473): not mutagenic (based on Read Across with Tetrahydrothiophene-1,1-dioxide)

Genetic toxicity in vitro (OECD TG 490): not mutagenic
Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 Oct 2017 - 23 Nov 2017
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
31 May 2008
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine, tryptophan
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: Each tester strain contained: rfa : deep rough mut. gal : mutation in the galactose metabolism chl : mutation in nitrate reductase bio : defective biotin synthesis uvrB : loss of the excision repair system (deletion of the ultraviolet-repair B gene)
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: The strain lacks an excision repair system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of mutagens has been enhanced by permeabilization of the strain using Tris-EDTA treatment.
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Dose range testing: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate. The highest concentration of 3-Methyl Sulfolane used in the subsequent mutation assay was 5000 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: soluble in water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
The negative control was Milli-Q water, the vehicle of the test item.
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)
- Cell density at seeding (if applicable): 10^9 cells/ml

DURATION
- Exposure duration: 48 +/- 4h

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
- Method: reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies
Rationale for test conditions:
According to guideline
Evaluation criteria:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than
two (2) times the concurrent vehicle control, and the total number of revertants in tester
strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent
vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.
A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two
(2) times the concurrent vehicle control, or the total number of revertants in tester strains
TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up 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 follow up experiment.
Key result
Species / strain:
S. typhimurium TA 100
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
Key result
Species / strain:
S. typhimurium TA 1535
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
Key result
Species / strain:
S. typhimurium TA 1537
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
Key result
Species / strain:
S. typhimurium TA 98
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
Key result
Species / strain:
E. coli WP2 uvr A
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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: good
- Precipitation: none

RANGE-FINDING/SCREENING STUDIES: Based on the results of the dose-range finding test, the following dose-range was selected for the first mutation experiment with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 52, 164, 512, 1600 and 5000 μg/plate.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%) : refer to table in additional information

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: [complete, e.g. CBPI or RI in the case of the cytokinesis-block method; RICC, RPD or PI when cytokinesis block is not used]
- Other observations when applicable: [complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells]

Historical Control Data of the Solvent Control

 

TA1535

TA1537

TA98

TA100

WP2uvrA

S9-mix

-

+

-

+

-

+

-

+

-

+

Range

3 – 29

3 - 27

3 – 20

3 – 23

8 - 41

8 - 55

63 – 176

54 - 160

10 – 59

9 - 69

Mean

10

11

6

7

16

23

108

107

25

32

SD

3

4

2

3

5

7

19

20

7

8

n

2356

2336

2264

2235

2319

2360

2341

2336

2075

2078

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between Oct 2015 and Oct 2017. 

Historical Control Data of the Positive Control Items

 

TA1535

TA1537

TA98

S9-mix

-

+

-

+

-

+

Range

125 – 1248

73 – 1206

55 – 1353

54 – 1051

365 – 1995

250 – 1977

Mean

846

219

787

353

1406

887

SD

146

119

345

162

258

349

n

2348

2229

2003

2234

2200

2276

 

 

TA100

WP2uvrA

S9-mix

-

+

-

+

Range

439 – 1848

408 - 2651

93 – 1951

111 - 1359

Mean

901

1232

1094

437

SD

168

343

477

149

n

2335

2327

2021

2085

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between Oct 2015 and Oct 2017. 


 


 

Conclusions:
Based on the results of this study 3-Methyl Sulfolane is not considered mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

The mutagenic potential of 3-Methyl Sulfolane and/or its metabolites in a well performed OECD TG 471 study under GLP. Based on the the dose-range finding study, the test item was initially tested up to concentrations of 5000μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. In the second mutation experiment, the test item was tested up to concentrations of 5000μg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrAin the absence and presence of 10% (v/v) S9-mix. In all three experiments the test item did not precipitate, neither cause toxicity nor a significant increase revertant colonies at any dose level. These results were confirmed in a follow-up experiment. 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. In conclusion, based on the results of this study it is concluded that 3-Methyl Sulfolane is not mutagenic in the Salmonella typhimuriumreverse mutation assay and in the Escherichia coli reverse mutation assay.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 Oct 2017 - 04 Dec 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
Thymidine Kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: American Type Culture Collection, (ATCC, Manassas, USA) (2001).
- Suitability of cells: Recommended test system in international guidelines (e.g. OECD).
- Methods for maintenance in cell culture if applicable: Prior to dose-range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10-medium containing 10^-4 M hypoxanthine (Sigma), 2 x 10^-7 M aminopterine (Fluka Chemie AG, Buchs, Switzerland) and 1.6 x 10^-5 M thymidine (Sigma) (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10-medium containing hypoxanthine and thymidine only. After this period cells were returned to R10-medium for at least 1 day before starting the experiment.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
>Horse serum (Life Technologies) was inactivated by incubation at 56°C for at least 30 minutes.
>Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) (Life Technologies) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively) (Life Technologies), 1 mM
sodium pyruvate (Sigma, Zwijndrecht, The Netherlands) and 2 mM L-glutamin (Life Technologies).
>Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
>Exposure medium for 3 hour exposure: Cells were exposed to the test item in basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium).
>Exposure medium for 24 hour exposure: Cells were exposed to the test item in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).
>Selective medium: Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium) and 5 μg/mL trifluorothymidine (TFT) (Sigma).
>Non-selective medium: Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium).

All incubations were carried out in a humid atmosphere (80 - 100%, actual range 64 - 97 %) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 34.8 - 37.6 °C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Any variation to these conditions were evaluated and maintained in the raw data.

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Based on the results of the dose-range finding test, 3-Methyl Sulfolane was tested in two mutation assays. The first experiment was performed in the absence and presence of S9-mix with a 3 hour treatment period. The second mutation experiment was performed in the absence of S9-mix with a 24 hour treatment period. The following dose-range was selected for the mutagenicity tests in the absence and presence of S9-mix: 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 1342 μg/mL exposure medium.
Vehicle / solvent:
RPMI 1640 (exposure medium (R5) Hepes buffered
medium
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): Cell density was kept below 1 x 10^6 cells/mL

DURATION
- Exposure duration: 3h or 24h
- Expression time (cells in growth medium): 2 times 24 hours
- Selection time (if incubation with a selection agent): 11 or 12 days
- Fixation time: 3h treatment followed by 2 days expression, or 24 hours followed by expression period

SELECTION AGENT (mutation assays): 5 μg/mL trifluorothymidine (TFT)

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth
Rationale for test conditions:
in accordance with guidelines
Evaluation criteria:
In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range.
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
3h and 24h
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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolarity: The pH and osmolarity at a concentration of 1340 μg/mL were 7.33 and 0.312 Osm/kg
respectively (compared to 7.34 and 0.302 Osm/kg in the solvent control).
- Water solubility: A solubility test was performed based on visual assessment. The test item was dissolved in RPMI 1640 (exposure medium (R5)).
- Precipitation: none

RANGE-FINDING/SCREENING STUDIES: No toxicity in the relative suspension growth was observed up to and including the highest test item concentration of 1342 μg/mL compared to the solvent control.

HISTORICAL CONTROL DATA: refer to any other information

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: [complete, e.g. CBPI or RI in the case of the cytokinesis-block method; RICC, RPD or PI when cytokinesis block is not used]
- Other observations when applicable: [complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells]

Historical Control Data of the Spontaneous Mutation Frequencies of the Solvent Controls for the Mouse Lymphoma Assay

 

Mutation frequency per 106survivors

 

- S9-mix

+ S9-mix

 

3 hour treatment

24 hour treatment

3 hour treatment

Mean

96

92

96

SD

29

30

29

n

268

248

285

Upper control limit

(95% control limits)

160

152

160

Lower control limit

(95% control limits)

32

31

32

SD = Standard deviation

n = Number of observations

Distribution historical negative control data from experiments performed between November 2014 and November 2017. 

 

   
Historical Control Data of the Mutation Frequencies of the Positive Controls for the Mouse Lymphoma Assay

 

Mutation frequency per 106survivors

 

- S9-mix

+ S9-mix

 

3 hour treatment

24 hour treatment

3 hour treatment

Mean

808

684

1669

SD

239

206

843

n

136

124

146

Upper control limit

(95% control limits)

1465

1222

4196

Lower control limit

(95% control limits)

152

146

-859

SD = Standard deviation

n = Number of observations

Distribution historical positive control data from experiments performed between November 2014 and November 2017. 

Conclusions:
Based on the reults described in this report 3-Methyl Sulfolane is not mutagenic in the TK mutation test system.
Executive summary:

The mutagenic potential of 3-Methyl Sulfolane  was evaluated in a well performed OECD TG 490  under GLP conditions. Its ability to induce forward mutations at the thymidine kinase (TK) locus was tested in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix). Exposure to the test substance was done for a 3 and 24 hour treatment period.

In the first experiment, the test item was tested up to concentrations of 1342 μg/mL (= 0.01 M, recommended in the guidelines) in the absence and presence of S9-mix. The incubation time was 3 hours. In the second experiment, the test item was again tested up to concentrations of 1342 μg/mL in the absence of S9-mix. The incubation time was 24 hours. No toxicity was observed at this dose level in the absence and presence of S9-mix. Both the positive and the solvent control were considered valid. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. In the absence of S9-mix, 3-Methyl Sulfolane did not induce an increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment. In the presence of S9-mix, 3-Methyl Sulfolane did not induce an increase in the mutation frequency. In conclusion, 3-Methyl Sulfolane is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
original study in Japanese, English version is in summary form including datagraphs and tables
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
not specified
Deviations:
not specified
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Remarks:
CHL / IU
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Research · Resource Bank (JCRB)
- Number of passages if applicable: obtained at passage 4
- Methods for maintenance in cell culture if applicable: cultured in 5 ml of the culture solution (Egle MEM supplemented with 10% fetal bovine serum) and cultured in a 37 °C in an incubator (5% CO 2).

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Egle MEM (Nissui Pharmaceutical Co., Ltd.) culture broth supplemented with 10% fetal bovine serum (FCS: Biocell) was used for the culture. 5% CO2
- Properly maintained: yes
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
colcemid
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
0.3, 0.6 and 1.2 mg/mL
Based on the results of the cell proliferation inhibition test, the high concentration group of the test substance used in the chromosome aberration test was set to 1.2 mg / ml (10 mM)
Vehicle / solvent:
Vehicle(s)/solvent(s) used for test substance: Water for injection
Vehicle(s)/solvent(s) used: ethanol for 2,4,4-trimethylpentene and chlorambucil. Sterile water for cyclophosphamide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; 2 × 10^4 cells/dish

DURATION
- Preincubation period: 3 days
- Exposure duration: 24 and 48 days
- Expression time (cells in growth medium): 18 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): Giemsa

NUMBER OF CELLS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 200

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (proliferation inhibitory action)

Rationale for test conditions:
Guideline study
Evaluation criteria:
With reference to the method of Hayashi 2), with reference to the method of the forest 2), the Fisher's direct stochastic method 3) between the solvent background data and the test substance treated group (refer to the groupware significance level 5%), a significant difference test was carried out. In addition, in cases where a significant difference was observed by Fisher's exact stochastic method, Cochran-Armitage's trend test 4) (p <0.05) was carried out for dose dependency. In principle, a case where a significant difference was observed in both of the above two tests was regarded as positive. If there was no significant difference in the trend test, it was judged to be false positives. The case where the number of observed cells was less than 100 for structural abnormality and less than 400 for polyploid cells was made undetectable due to cytotoxicity.

Statistics:
- Fishers direct stochastic method, followed by Cochran-Armitage's trend test
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
Negative for continuous 24/48 h and short time 6 h treatment.
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: neither continuous treatment nor short-time treatment showed proliferation inhibiting effects


Conclusions:
Under the conditions of this test, there were no increases in chromosomal aberrations in CHL/IU cells treated with Tetrahydrothiophene-1,1-dioxide at any timepoint, with or without metabolic activation.
Executive summary:

The in vitro chromosome aberration potential of the test substance was evaluated for tetrahydrothiophene-1,1-dioxide in cultured Chinese hamster cultured cells (CHL / IU). Since growth inhibition 50% was not observed even at a concentration of 1.2 mg / ml (10 mM) in both the continuous treatment (48 hours) and the short time treatment (6 hours), 1.2 mg / mL was regarded as the maximum treatment concentration. 1/2 and 1/4 of the highest treatment concentration were set as medium concentration and low concentration, respectively. In the continuous treatment, specimens were prepared after continuous treatment for 24 hours and 48 hours in the absence of S9 mix, after 6 hours treatment (18 hour recovery time) in the presence and absence of S9 mix for short time treatment.

No chromosomal structural abnormality or polyploid cell inducing effect was observed in any treatment group in which CHL / IU cells were continuously treated for 24 hours and 48 hours. In the short-term treatment, no chromosomal structural abnormality or polyploid cell induction effect was observed in any treatment group treated for 6 hours in the presence or absence of S9 mix.

From the above results, it was concluded that tetrahydrothiophene-1,1-dioxide does not induce chromosomal abnormalities under the above test conditions.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
original study in Japanese, English version is in summary form including datagraphs and tables
Justification for type of information:
Cytogenicity study in mammalian cells is standard information required for the registration of substances manufactured or imported in quantities of ten tonne per year or more. However, according to Annex XI, 1.5 of the REACH Regulation, Read-across and grouping approaches can be used to adapt the standard testing regime. This read-across study report follows notably the recommendations made by the European Chemicals Agency in its “Guidance on information requirements and chemical safety assessment Chapter R.6 – QSARs and grouping of chemicals” (ECHA, 2008) and in its document “Read-Across Assessment Framework (RAAF)” (ECHA, 2017).

A read-across approach appears appropriate to predict the endpoint “In vitro cytogenicity study in mammalian cells” for the substance 3-methylsulfolane because:
- Structurally the source and target chemical have clear similarities, as they are both organosulfur compounds consisting of a non-aromatic cyclic sulfone, the only difference being a methyl-group on the 3-position. There are no physico-chemical properties that are expected to influence the potential for the genotoxicity of these substances
- Both the source and target substances underwent predictive assessment by by using a DEREK in silico analysis for clastogenicity, furthermore the possible metabolites were screened for genotoxic potential in the OECD QSAR toolbox. This available data was evaluated in the light of existing data and did not indicate major differences in their potential for genotoxicity.

The attached report follows the RAAF method and so presents:
1) The hypothesis: analogue read-across approach, based on the similarity of the substances and the absence of cytogenicity for these types of structures;
2) The scientific justifications (“Assessment Elements”) and their evaluation (“Assessment Options”); which demonstrate the confidence that can be put in this prediction.
3) The conclusions, usable for classification assessment or risk assessment, which are summarised hereafter.

The conclusion of the read-across assessment from the source “sulfolane” to the target substance “3-methylsulfolane” is: Substance “3-methylsulfolane” does not require a classification for Genetic Toxicity according to CLP Regulation EC/1272/2008 and its amendments.
Reason / purpose:
read-across source
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
Negative for continuous 24/48 h and short time 6 h treatment.
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: neither continuous treatment nor short-time treatment showed proliferation inhibiting effects


Conclusions:
The in vitro cytogenicity / chromosome aberration in mammalian cells of 3-methylsulfolane was evaluated by read across with the analogue source molecule Tetrahydrothiophene-1,1-dioxide. Based on the available information, 3-methylsulfolane does not induce chromosome aberration, according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).


Executive summary:

The in vitro cytogenicity / chromosome aberration study in mammalian cells of 3-methylsulfolane was evaluated by read across with the analogue source moleculeTetrahydrothiophene-1,1-dioxide. Since growth inhibition 50% was not observed even at a concentration of 1.2 mg / ml (10 mM) in both the continuous treatment (48 hours) and the short time treatment (6 hours), 1.2 mg / mL was regarded as the maximum treatment concentration. 1/2 and 1/4 of the highest treatment concentration were set as medium concentration and low concentration, respectively. In the continuous treatment, specimens were prepared after continuous treatment for 24 hours and 48 hours in the absence of S9 mix, after 6 hours treatment (18 hour recovery time) in the presence and absence of S9 mix for short time treatment.

No chromosomal structural abnormality or polyploid cell inducing effect was observed in any treatment group in which CHL / IU cells were continuously treated for 24 hours and 48 hours. In the short-term treatment, no chromosomal structural abnormality or polyploid cell induction effect was observed in any treatment group treated for 6 hours in the presence or absence of S9 mix.

From the above results, it was concluded that the target substance does not induce chromosomal abnormalities under the above test conditions.

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

Additional information

Genetic toxicity in vitro (OECD TG 471)

The mutagenic potential of 3-Methyl Sulfolane and/or its metabolites in a well performed OECD TG 471 study under GLP. Based on the the dose-range finding study, the test item was initially tested up to concentrations of 5000μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. In the second mutation experiment, the test item was tested up to concentrations of 5000μg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrAin the absence and presence of 10% (v/v) S9-mix. In all three experiments the test item did not precipitate, neither cause toxicity nor a significant increase revertant colonies at any dose level. These results were confirmed in a follow-up experiment. 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. In conclusion, based on the results of this study it is concluded that 3-Methyl Sulfolane is not mutagenic in the Salmonella typhimuriumreverse mutation assay and in the Escherichia coli reverse mutation assay.

Genetic toxicity in vitro (OECD TG 490)

The mutagenic potential of 3-Methyl Sulfolane was evaluated in a well performed OECD TG 490  under GLP conditions. Its ability to induce forward mutations at the thymidine kinase (TK) locus was tested in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix). Exposure to the test substance was done for a 3 and 24 hour treatment period.

In the first experiment, the test item was tested up to concentrations of 1342 μg/mL (= 0.01 M, recommended in the guidelines) in the absence and presence of S9-mix. The incubation time was 3 hours. In the second experiment, the test item was again tested up to concentrations of 1342 μg/mL in the absence of S9-mix. The incubation time was 24 hours. No toxicity was observed at this dose level in the absence and presence of S9-mix. Both the positive and the solvent control were considered valid. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. In the absence of S9-mix, 3-Methyl Sulfolane did not induce an increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment. In the presence of S9-mix, 3-Methyl Sulfolane did not induce an increase in the mutation frequency. In conclusion, 3-Methyl Sulfolane is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Cytogenicity (OECD TG 473, based on Read Across with Tetrahydrothiophene-1,1-dioxide)

The chromosome abberation potential of 3-methyl sulfolane was assessed by read across from the analogue source substance Tetrahydrothiophene-1,1-dioxide. Here the experimental information of the source substance is summarised. The accompanying files are attached in the target study record.

The in vitro chromosome aberration potential of the test substance was evaluated for tetrahydrothiophene-1,1-dioxide in cultured Chinese hamster cultured cells (CHL / IU). Since growth inhibition 50% was not observed even at a concentration of 1.2 mg / ml (10 mM) in both the continuous treatment (48 hours) and the short time treatment (6 hours), 1.2 mg / mL was regarded as the maximum treatment concentration. 1/2 and 1/4 of the highest treatment concentration were set as medium concentration and low concentration, respectively. In the continuous treatment, specimens were prepared after continuous treatment for 24 hours and 48 hours in the absence of S9 mix, after 6 hours treatment (18 hour recovery time) in the presence and absence of S9 mix for short time treatment.

No chromosomal structural abnormality or polyploid cell inducing effect was observed in any treatment group in which CHL / IU cells were continuously treated for 24 hours and 48 hours. In the short-term treatment, no chromosomal structural abnormality or polyploid cell induction effect was observed in any treatment group treated for 6 hours in the presence or absence of S9 mix. From the above results, it was concluded that tetrahydrothiophene-1,1-dioxide does not induce chromosomal abnormalities under the above test conditions.

Justification for classification or non-classification

Based on the available information, 3-Methyl Sulfolane does not need to be classified for genotoxicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).