Tetrahydrothiophene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Tetrahydrothiophene

EC Number:

203-728-9

EC Name:

Tetrahydrothiophene

Cas Number:

110-01-0

Molecular formula:

C4H8S

IUPAC Name:

tetrahydrothiophene

Details on test material:

Source: Pennwalt Corporation
Batch No.: Lot 271.
Purity: 99.0% minimum.

Method

Target gene:

Histidine reversion

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 from rat induced with a single ip injection of Aroclor 1254 (500 mg/kg)

Test concentrations with justification for top dose:

0, 50, 150, 500, 1500, 5000 µg/plate

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: With S-9 mix : 2-Aminoanthracene, all strains. Without S-9 mix: 2-Nitrofluorene, TA 98; 9-Aminoacridine, TA 1537; N-ethyl-N'-nitro-N-nitrosoguanidine, TA 1535 and TA 100.

Details on test system and experimental conditions:

Bacterial strains:
The strains are tested routinely for cell membrane permeability and where applicable for ampicillin resistance.
For use in tests sub-cultures are grown in Nutrient Broth (Oxoid) at 37°C for 18 hours. This culture provides approximately 2 x 10e9 organisms per ml which is assessed by cell counting.

Preliminary toxicity test:
The following procedure is carried out on each bacterial strain:
Four concentrations of test substance are assessed for toxicity using the four tester strains. The highest concentration is usually 0.05 g of test substance dissolved in 1 ml of solvent. Three 10-fold serial dilutions of the top concentration are also tested. The chosen solvent is used as the negative control.
0.1 ml of an overnight bacterial culture containing approximately 2 x 10e9 cells/ml, and 0.5 ml S-9 mix or 0.5 ml 0.1 M sodium phosphate buffer (pH 7.4) are placed in glass bijou bottles. 0.1 ml of the test solution is added followed by 2 ml histidine deficient agar. The mixture is thoroughly shaken and overlaid onto previously prepared plates containing 20 ml minimal agar. Single petri dishes are used for each dose level. They are incubated at 37°C for 72 hours. After this period the plates are examined for the appearance of a complete bacterial lawn. Revertant colonies are counted using a Biotran Automatic Colony Counter. Any toxic effects of the test substance are detected by a substantial reduction in revertant colony counts or by the absence of a complete background bacterial lawn.

Ames test procedure:
- Without metabolic activation
The following procedure is carried out on each tester strain.
0.1 ml aliquots of bacterial suspension and 0.5 ml of sterile 0.1 M sodium phosphate buffer (pH 7.4) are added to each of one set of sterile bijou bottles.
0.1 ml of the test compound is added to cultures at five concentrations separated by half-log 10 intervals. The negative control is the chosen solvent. The appropriate positive control is also included. 3 bottles are used at each dose level.
2.0 ml of histidine deficient agar is added to each of the bottles, thoroughly mixed and then overlaid onto previously prepared plates containing 20 ml of minimal agar. Plates are incubated for 72 hours at 37°C.
Colonies are counted using a Biotran Automatic Colony Counter, and the mean number of revertant colonies per treatment group assessed.
- With metabolic activation
Methodology is as described previously except that 0.5 ml of liver homogenate S-9 mix is added to bijou bottles in place of sterile buffer.

Second mutation test :
The procedure outlined previously is repeated at a later date; though the concentrations of test substance used in the second test may be altered, if the results of the first test indicate this may be expedient.

Evaluation criteria:

The mean number of revertant colonies for all treatment groups is compared with those obtained for negative and positive control groups. The effect of metabolic activation is assessed by comparing the results obtained both in the presence and absence of the liver microsomal fraction for each treatment group.

A compound is deemed to provide evidence of mutagenic potential if (1) a statistically significant dose-related increase in the number of revertant colonies is obtained in two separate experiments and (2) the increase in the number of revertant colonies is at least twice the concurrent solvent control value.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

No substantial increases in revertant colony numbers of any of the four tester strains were observed following treatment with THT at any dose level, either in the presence or absence of metabolic activation (S-9 mix).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Mutation Test 1 (mean values)

Strain	TA 1535		TA 1537		TA 98		TA 100
Metabolic activation	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Dose level, µg/plate
5000	18	15	7	20	21	21	83	88
1500	15	10	7	15	25	19	99	100
500	14	13	8	15	30	24	100	120
150	16	15	7	12	27	24	123	130
50	20	11	11	17	29	28	112	133
Solvent	12	13	10	16	36	22	129	132

Mutation Test 2 (mean values)

Strain	TA 1535		TA 1537		TA 98		TA 100
Metabolic activation	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Doselevel,µg/plate
5000	12	10	12	16	34	22	73	69
1500	16	10	12	14	25	26	84	97
500	14	12	11	13	22	19	77	94
150	15	11	11	10	21	23	93	103
50	12	10	12	15	22	21	80	87
Solvent	16	14	14	13	22	22	89	120

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Tetrahydrothiophene was concluded to be negative in the Bacterial Reverse Mutation Assay.

Executive summary:

In an OECD 471 bacterial reverse mutation test, tetrahydrothiophene, was tested in the Bacterial Reverse Mutation Assay using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 in the presence and absence of Aroclor-induced rat liver S9. The assay was performed using the plate incorporation method. The dose levels tested were 50, 150, 500, 1500 and 5000 µg per plate in the initial mutation assay and in the repeated assay. No positive mutagenic response was observed. Neither precipitate nor appreciable toxicity was observed. Tetrahydrothiophene

was concluded to be negative in the Bacterial Reverse Mutation Assay.