5H-1,2-oxathiole 2,2-dioxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20/06/2019 - 08-08-2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

5H-1,2λ⁶-oxathiole-2,2-dione

EC Number:

606-834-7

Cas Number:

21806-61-1

Molecular formula:

C3H4O3S

IUPAC Name:

5H-1,2λ⁶-oxathiole-2,2-dione

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Shijiazhuang Suntec-chem Co.,Ltd; 190403
- Expiration date of the lot/batch: Apr 10, 2020
- Purity: 99.29%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Store at room temperature. Keep container tightly closed in a dry and well-ventilated place. Containers which are opened must be carefully resealed.
- Storage and handling during the study: The sample of test item has been stored in a glass vial, at room temperature, in dark.

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : rat liver homogenate and mixture of cofactors
- method of preparation of S9 mix: The liver homogenate was prepared from Wistar male rats weighing approximately 200 g, previously induced with Delor 106 (mixture of polychlorinated biphenyls). Delor 106 was diluted with olive oil to a concentration of 200 mg•mL-1, and each rat was administered a single intraperitoneal injection of 500 mg/kg 5 days before S9 preparation.
The S9 was prepared according to the methods described by Maron and Ames (1). The liver was removed from each animal and washed in ice cold 0.15M KCl. The livers washed were mixed with another 0.15 M KCl (3 mL.g-1 wet liver) homogenized in a grinder, and the tissue suspension was centrifuged for 10 min at 9000 g. Aliquots of the supernatant (S9) were stored in plastic tubes using sterile technique at a temperature below 70 °C. Cofactors (NADP and glucoso-6-phosphate) were dissolved in buffer.
- concentration or volume of S9 mix and S9 in the final culture medium: Volume of 20 μL (3.8 % of S9 in S9 mix) of S9 is used for positive controls in all strains (reason – to keep constant conditions for database of historical values).
Volume of 30 μL(5.7% of S9 in S9 mix) of S9 was used for test item, solvent and negative control plates in the first experiments.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability) non-toxicity, non-mutagenicity and sterility of vehiculum

Test concentrations with justification for top dose:

Preliminary test: 10, 100, 500, 1000, 2500 and 5000 μg per plate
Main test: 50, 150, 500, 1500 and 5000 μg per plate

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle: The test item was dissolved in water for injection and in dimethyl sulfoxide in the highest recommended concentration 5000 μg per plate. The test item was partially soluble in water; the entire test item was not dissolved even the next day. The entire test item was dissolved almost immediately in DMSO. So DMSO was used for mutagenicity testing.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-o-phenylenediamine (NPD): TA 98 - S9 2-aminofluorene (2-AF): TA 100 and TA 98 + S9 2-aminoanthracene (2-AA): TA 1535, TA 1537, E.coli + S9 M-methyl-N´-nitro-N-nitrosoguanidine (MNNG): E. coli - S9

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Ttriplicate plating
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
- Test substance in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 72 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method.: background growth inhibition

Evaluation criteria:

The main criterion used for the evaluation of reversion results was a modified two-fold increase rule, which is compatible with the application of statistical methods (2, 3). Per this rule, the result is positive if a reproducible dose-response effect occurs and/or a doubling of the ratio Rt/Rc is reached (Rt – number of revertants at tested dose, Rc – number of revertants of the solvent control).

An increase is considered as ”biologically relevant“:

- if the number of reversions is at least twice as high as that in the solvent control for the strains having spontaneous reversion >10;
- if the number of reversions is at least three times as high as that in the solvent control for the strains having spontaneous reversion ≤10;

A test item producing neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Statistics:

Mean and SD of triplicate plates

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: No precipitation

RANGE-FINDING/SCREENING STUDIES (if applicable):
The test item was dissolved in DMSO up to the highest concentration recommended in the OECD TG 471 guideline – 5000 μg per 0.1 millilitre (plate). For the cytotoxicity experiment, the highest concentration was diluted to the other 5 concentrations within the range of 3 orders of magnitude. The concentration series was tested for toxicity in strain TA 98 without metabolic activation. Neither cytotoxicity nor precipitation was observed at any concentration. In line with the toxicity test results, the highest concentration selected was 5000 μg per plate. A dilution factor between 2 and √10 was used with a resulting concentration range of 50, 150, 500, 1500 and 5000 ug per plate.


STUDY RESULTS
- Concurrent vehicle negative and positive control data : All controls (solvent and negative) gave the appropriate responses and deviations were acceptable and did not affect the study results.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible : A dose-effect relationship was evident in all experiments
- Statistical analysis: according to OECD TG 471, the biological relevance is the criterion for the interpretation of results, and a statistical evaluation of the results is not necessary.

Ames test:
- Signs of toxicity: No cytotoxicity was observed at any concentration.
- Individual plate counts: Triplicate values for all strains with and without S9 are in Tables 1-5.
- Mean number of revertant colonies per plate and standard deviation: Mean and SD values for all strains with and without S9 are in Tables 1-5.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: refer to Table A
- Negative (solvent/vehicle) historical control data: refer to Table A

Any other information on results incl. tables

TableA: Current historical ranges  

Control Strain	Spont.rev.	DMSO	PC - S9	PC + S9	Number of CFU/mL
S.t. TA 100	69-177 (859)	66-170 (546)	341-597 (44)	875-3015 (40)	4.56*10^9
S.t. TA 1535	9-29 (669)	11-27 (422)	434-730 (40)	64-420 (38)	3.10*10^9
S.t. TA 98	13-49 (915)	13-45 (567)	1310-3346 (42)	1904-5024 (42)	7.60*10^8
S.t. TA 1537	5-21 (710)	4-20 (441)	1419-4139 (38)	5-405 (31)	1.67*10^9
E.c. WP2 uvrA	11-51 (691)	8-44 (397)	479-1919 (39)	216-1056$ (12)	1.36*10^9

Applicant's summary and conclusion

Conclusions:

In a reverse bacteria mutation study (Ames test), the test item, 1,3-Propenesultone, was mutagenic in all strains in experiments with and without metabolic activation. Based on this result, the test substance is considered mutagenic.

Executive summary:

In a reverse gene mutation assay in bacteria (152-19-12), strains of S. typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA were exposed to 1,3-Propenesultone (99.92%) in DMSO at concentrations of 50, 150, 500, 1500 and 5000 μg/plate (plate incorporation) with and without metabolic activation (Delor 106-induced rat liver S9).

No cytotoxicity or precipitation was observed at any dose. The positive controls induced the appropriate responses in the corresponding strains. The mutagenic response induced by the test item was high with and without metabolic activation in S. typhimurium TA100, S. typhimurium TA1535 (> 2 fold in both starting from the lowest concentration) and E. coli WP2 uvrA (>2 fold starting from 150 µg per plate). Mutagenic response was only apparent at high doses with and without metabolic activation in S. typhimurium TA98 (> 2 fold in starting from 1500 µg per plate) and S. typhimurium TA1537 (> 2 fold at 5000 µg per plate). All 5 strains were considered positive in the plate incorporation assay so a second mutagenicity test (pre-incubation method) was not performed. Under the conditions of this study, the test substance, 1,3-Propenesultone, is considered mutagenic.