3-methyltetrahydrothiophene 1,1-dioxide

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

Jul. 29, 2016

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

3-methyltetrahydrothiophene 1,1-dioxide

EC Number:

212-833-9

EC Name:

3-methyltetrahydrothiophene 1,1-dioxide

Cas Number:

872-93-5

Molecular formula:

C5H10O2S

IUPAC Name:

3-methyl-1λ⁶-thiolane-1,1-dione

Test material form:

liquid

Specific details on test material used for the study:

Lot no. XXZ2J
99.9% purity (GC)

Method

Species / strain

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.

Controls

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).

Results and discussion

Test results

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.