3-methyltetrahydrothiophene 1,1-dioxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria: Bacterial Reverse Mutation Assay/Ames (OECD TG 471): negative (with and without metabolic activation)

In vitro Mammalian chromosome aberration test (OECD TG 473): negative (without metabolic activation).

In vitro Mammalian cell gene mutation test (OECD TG 490): negative (with and without metabolic activation).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26-05-2020 to 08-06-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

Jul. 29, 2016

Qualifier:

according to guideline

Guideline:

other: Regulation on Test Methods for Chemical Substances - Notification No. 2019-23, National Institute of Environmental Research, Republic of Korea

Version / remarks:

Jun. 13, 2019

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Lot no. XXZ2J
99.9% purity (GC)

Species / strain / cell type:

Chinese hamster lung (CHL/IU)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: CHI/IU, American Type Culture Collection U.S.A
- Suitability of cells: CHL/IU cell line has a high detection sensitivity, is commonly used in in vitro chromosome aberration studies and recommended in the regulatory guidelines.
- Normal cell cycle time (negative control): approximately 15 hours

For cell lines:
- Absence of Mycoplasma contamination: evaluation by using a Hoechst Stain Kit (MPBIOMEDICALS, Japan).
- Number of passages if applicable: 18
- Methods for maintenance in cell culture: Frozen cells were thawed in the fresh medium, cell morphology was evaluated following 70–80% proliferation on the bottom of the flask. 0.25% Trypsin-EDTA solution (Gibco, U.S.A) was added to detach cells. The suspended cells was harvested, transferred into a 50 mL tube and centrifuged at 1,000 rpm for 5 minutes. The supernatant was removed and the pellets were resuspended with EMEM supplemented with 10% FBS. Suspended cells were transferred into a 75 cm² flask and incubated in a 5% CO2 incubator at 37°C and more than two times to yield a final density of 5×104 cells/mL. The suspended cells were placed in a 6 well plate (2 mL/well, Nunc, Denmark), for the dose range finding study and in a 60 mm plate (5
mL/plate, BD, U.S.A.) and 6 well plate (2 mL/well) for the main study. Cells were incubated in a 5% CO2 incubator at 37°C for one day.
- Doubling time: approximately 15 hours
- Modal number of chromosomes: 25

MEDIA USED
100 mL of culture medium consisted of 10 mL Fetal Bovine Serum (FBS, Gibco, U.S.A.), 1 mL Penicillin-Streptomycin (Gibco, U.S.) and 89 mL Eagles modified eagle’s medium (EMEM, Lonza, U.S.). Conditions during incubation 5% CO2 incubator at 37°C.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
S9 and Cofactor C from Oriental Yeast Co., LTD., Japan. Prepared from the liver of 7 weeks old male Sprague-Dawley rat [Crl:CD(SD)] treated with the enzyme inducing agents Phenobarbital (PB) and 5,6-benzoflavone (BF).
The preparation of S9 mix was conducted immediately prior to use. The frozen S9 (Lot No.: 19121310) and Cofactor C (Lot No.: C19121110) were thawed and mixed at a ratio of 2 to 4.7.
The composition of S9 mix is presented in Table 1 in any other information.
Used volume of S9 was 0.5 mL in the Dose Range Finding study and 2.17 mL in the main study.

Test concentrations with justification for top dose:

The high dose of the test substance was 2,000 µg/mL, which is the limit dose recommended in the guidelines. The high dose was sequentially diluted to produce lower dose levels (1,000, 500, 250, 125, 62.5, 31.3, 15.6, 7.81, 3.91, 1.95, 0.977 and 0.488 µg/mL). In addition, the negative control group was set.
In the dose range finding study cytotoxicity and precipitation of the test substance was not evident in the short time treatments with and without metabolic activation and in the continuous treatment without metabolic activation. Therefore, the high dose was selected at 2,000 μg/mL in the main study (short time and continuous) and it was sequentially diluted by applying a geometric ratio of 2 to produce lower dose levels 1,000 and 500 μg/mL In addition, the positive and negative control groups were set.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Vehicle of the test substance: water for injection. Vehicle for positive controls Dimethylsulfoxide lot no. K50270931 (Merck, Germany).

In order to produce a 10-fold stock of 2,000 µg/mL, which is the high dose of the dose range finding study, a preliminary solubility test was conducted. As a result, the test substance was dissolved in water for injection. Therefore, water for injection was selected as the vehicle for this study.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Water for injection (JW Pharmaceutical Co., Ltd, Republic of Korea)

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

mitomycin C

Details on test system and experimental conditions:

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 5×10-4 cells/mL
- Test substance added in medium; in suspension.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: one day
- Exposure duration/duration of treatment: Short time treatment – 6 hours and Continuous treatment -24 hours
- Harvest time after the end of treatment (sampling/recovery times): after short time treatment of 6 hours, 18 hours of recovery

OBSERVATIONS:
The observation of slides was conducted in the order of the short time treatments and continuous treatment. Dose levels for chromosome observations were selected at 3 dose levels, at which 300 metaphases could be observed.
Three hundred metaphases per dose were observed using a microscope (600-fold magnification, BX51, Olympus, Japan)
Chromosomal aberrations were classified into structural aberration, numerical aberration and other.
Structural aberrations were classified into chromatid break (ctb), chromatid exchange (cte), chromosome break (csb), chromosome exchange (cse), chromatid gap (ctg), chromosome gap (csg) and fragmentation (frg). When several gaps or breaks were observed in metaphase, these were recorded as frg. An achromatic lesion narrower than the width of one chromatid was defi ned as a gap
In addition, numerical aberrations were classified into polyploidy (pol) and endoreduplication (end)
For the aforementioned aberrations, any cell with one or more aberrations was counted as an aberrant cell. For the gap, the number of cells including and excluding gaps was scored and recorded. Others which were not classified into the structural or numerical aberrations were recorded for the type and number of cells with aberrations.

Rationale for test conditions:

In the dose range finding study cytotoxicity and precipitation of the test substance was not evident in the short time treatments with and without metabolic activation and in the continuous treatment without metabolic activation. Therefore, the high dose was selected at 2,000 μg/mL in the main study (short time and continuous) and it was sequentially diluted by applying a geometric ratio of 2 to produce lower dose levels 1,000 and 500 μg/mL In addition, the positive and negative control groups were set.

Evaluation criteria:

The results were considered to be positive when the frequency of cells with chromosome aberrations (excluding gap) met all of the following conditions: others were considered as negative
The frequency of cells with chromosome aberrations shows a statistically significant increase at more than one dose level in the test substance groups compared to the negative control group.
The cells with chromosome aberrations are increased in a dose-dependent manner.
The frequency of cells with chromosome aberrations increases over the 95% control limits of distribution of the historical control data in the negative control group.

Statistics:

Statistical analysis on the frequency of cells with chromosome aberrations (excluding gap) was performed using SAS Program (version 9.3, SAS Institute Inc., U.S.A.).
For the aberration cell data, Fisher’s exact test was used for the comparison of the negative control group to the test substance group or the positive control group (significance levels: 0.05 and 0.01, two tailed) The test substance group was not significant, Cochran Armitage trend test was not conducted for dose dependency between the negative control group and the test substance group (significance levels: 0.05 and 0.01, two tailed).

Key result

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: the pH in the high dose group of the test substance did not change more than 1.0 compared to the control group
- Data on osmolality: the osmolality in the high dose group of the test substance did not change more than 50 mOsm/kg compared to the control group
- Water solubility: a preliminary solubility test was conducted and the test substance was dissolved in water for injection (vehicle)
- Precipitation and time of the determination: the precipitation of the test substance was not evident in the short time treatments with and without metabolic activation and in the continuous treatment without metabolic activation

RANGE-FINDING/SCREENING STUDIES (if applicable):
In the dose range finding study cytotoxicity and precipitation of the test substance was not evident in the short time treatments with and without metabolic activation and in the continuous treatment without metabolic activation.
STUDY RESULTS
- Concurrent vehicle negative and positive control data: The frequency of cells with structural chromosome aberrations in the negative control group was within the range of historical control data and the 95% control limits of the distribution of the historical control data. In addition, the frequency of cells with structural chromosome aberrations in the positive control group was within the range of historical control data and statistically significantly increased compared to the negative control group. At least 300 metaphases cells per dose were observed in the negative and positive control groups and the test substance groups.

Table 3: summary results of the main study

 

Test substance	Dose (µg/mL)	RPD(%)	PD	S9 mix	Trt-Rec time (hr)	No. of cell analyzed	Number of cells with structural aberrations									Number of cells with numerical aberrations			Others
							ctb	csb	cte	cse	frg	gap		total(%)		end	pol	total(%)
												ctg	csg	gap-	gap+
Water for injection	0	100	1.56	-	6-18	150	0	0	0	0	0	0	0	0 (0.0)	0 (0.0)	0	0	0 (0.0)	0
						150	0	0	0	0	0	0	0			0	0
3-Methylsulfolane	500	95.2	-	-	6-18	150	0	0	0	0	0	0	0	0 (0.0)	0 (0.0)	0	0	0 (0.0)	0
						150	0	0	0	0	0	0	0			0	0
	1000	90.6	-	-	6-18	150	0	0	0	0	0	0	0	0 (0.0)	1 (0.3)	0	0	0 (0.0)	0
						150	0	0	0	0	0	1	0			0	0
	2000	85.8	-	-	6-18	150	0	0	0	0	0	0	0	0 (0.0)	0 (0.0)	0	0	0 (0.0)	0
						150	0	0	0	0	0	0	0			0	0
MMC	0.1	58.6	-	-	6-18	150	8	0	27	0	0	0	0	59** (19.7)	62 (20.7)	0	0	0 (0.0)	0
						150	6	0	25	0	0	3	0			0	0
Water for injection	0	100	1.53	+	6-18	150	0	0	0	0	0	0	0	0 (0.0)	0 (0.0)	0	0	0 (0.0)	0
						150	0	0	0	0	0	0	0			0	0
3-Methylsulfolane	500	97.0	-	+	6-18	150	0	0	0	0	0	0	0	0 (0.0)	0 (0.0)	0	0	0 (0.0)	0
						150	0	0	0	0	0	0	0			0	0
	1000	90.2	-	+	6-18	150	0	0	0	0	0	0	0	0 (0.0)	0 (0.0)	0	0	0 (0.0)	0
						150	0	0	0	0	0	0	0			0	0
	2000	85.2	-	+	6-18	150	0	0	0	0	0	0	0	0 (0.0)	0 (0.0)	0	0	0 (0.0)	0
						150	0	0	0	0	0	0	0			0	0
B[a]P	20	51.1	-	+	6-18	150	5	0	21	0	0	0	0	47** (15.7)	47 (15.7)	0	0	0 (0.0)	0
						150	4	0	22	0	0	0	0			0	0
Water for injection	0	100	1.54	-	24-0	150	0	0	0	0	0	0	0	0 (0.0)	0 (0.0)	0	0	0 (0.0)	0
						150	0	0	0	0	0	0	0			0	0
3-Methylsulfolane	500	96.6	-	-	24-0	150	0	0	0	0	0	0	0	1 (0.3)	1 (0.3)	0	0	0 (0.0)	0
						150	0	0	1	0	0	0	0			0	0
	1000	90.9	-	-	24-0	150	0	0	0	0	0	0	0	0 (0.0)	0 (0.0)	0	0	0 (0.0)	0
						150	0	0	0	0	0	0	0			0	0
	2000	83.2	-	-	24-0	150	0	0	0	0	0	0	0	0 (0.0)	0 (0.0)	0	0	0 (0.0)	0
						150	0	0	0	0	0	0	0			0	0
MMC	0.1	52.2	-	-	24-0	150	10	0	30	0	0	0	0	70** (23.3)	72 (24.0)	0	0	0 (0.0)	0
						150	8	0	28	0	0	2	0			0	0

Aberration: ctg: chromatid gap, csg: chromosome gap, ctb: chromatid break, cte: chromatid exchange, csb: chromosome break, cse: chromosome exchange, frg: fragmentation, end: endoreduplication, pol: polyploidy

MMC: Mitomycin C, B[a]P: Benzo[a]pyrene RPD : Relative Population Doubling, Trt-Rec time : Treatment-Recovery times gap-: Total number of cells with structural aberrations excluding gap, gap+: Total number of cells with structural aberrations including gap a): Others were excluded from the number of cells with chromosomal aberrations. Population Doubling (PD) = [log (Post-treatment cell number / Initial cell number)] ÷ log 2 Significant difference from negative control by Fisher’s exact test : ** p<0.01

Conclusions:

The test substance is not cytogenic/mutagenic in the in vitro Mammalian Chromosomal Aberration Test performed according to OECD TG 473.

Executive summary:

The potential of 3-Methyl Sulfolane to induce chromosomal aberrations in Chinese Hamster Lung (CHL/IU) cells was evaluated in a well performed OECD TG 473 under GLP conditions.

A dose range finding study was conducted. In which the following doses where applied in single wells: 2,000 1,000, 500, 250, 125, 62.5, 31.3, 15.6, 7.81, 3.91, 1.95, 0.977 and 0.488 μg/mL with metabolic activation (6 hour treatment time) and without metabolic activation (6 and 24 hour treatment time). The negative control was sterile water. No cytotoxicity or precipitation was observed.

The following dose levels were selected for the main study and tested in duplicate: 2,000 1,000, 500 μg/mL with metabolic activation (6 hour treatment time) and without metabolic activation (6 and 24 hour treatment time). The positive controls where MCC (without metabolic activation) and B[a]P (with metabolic activation). RPDs of all dose levels were 83.2% or more and precipitation was not observed. The frequency of cells with chromosome aberrations in the short time treatments with and without metabolic activation and in the continuous treatment without metabolic activation was not statistically significantly different when compared to the negative control group. In the positive control group, the frequency of cells with structural chromosome aberrations was statistically significantly increased when compared to the negative control group (p<0.01). The acceptance criteria where fulfilled.

In conclusion, the test substance, 3-Methylsulfolane, did not show any evidence to induce chromosomal aberration under the conditions of this study.

Reference 2

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

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

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.

Reference 3

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

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 10^6survivors
	- 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 10^6survivors
	- 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 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)

The potential of 3-Methyl Sulfolane to induce chromosomal aberrations in Chinese Hamster Lung (CHL/IU) cells was evaluated in a well performed OECD TG 473 under GLP conditions.

A dose range finding study was conducted. In which the following doses where applied in single wells: 2,000 1,000, 500, 250, 125, 62.5, 31.3, 15.6, 7.81, 3.91, 1.95, 0.977 and 0.488 μg/mL with metabolic activation (6 hour treatment time) and without metabolic activation (6 and 24 hour treatment time). The negative control was sterile water. No cytotoxicity or precipitation was observed.

The following dose levels were selected for the main study and tested in duplicate: 2,000 1,000, 500 μg/mL with metabolic activation (6 hour treatment time) and without metabolic activation (6 and 24 hour treatment time). The positive controls where MCC (without metabolic activation) and B[a]P (with metabolic activation). RPDs of all dose levels were 83.2% or more and precipitation was not observed. The frequency of cells with chromosome aberrations in the short time treatments with and without metabolic activation and in the continuous treatment without metabolic activation was not statistically significantly different when compared to the negative control group. In the positive control group, the frequency of cells with structural chromosome aberrations was statistically significantly increased when compared to the negative control group (p<0.01). The acceptance criteria where fulfilled.

In conclusion, the test substance, 3-Methylsulfolane, did not show any evidence to induce chromosomal aberration under the conditions of this study.

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).