Tetrahydrothiophene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Tetrahydrothiophene is reported to be negative in an Ames test, a cytogenetic assay in human lymphocytes, a gene mutation (HPRT) assay in Chinese hamster ovary (CHO) cells, a SCE assay in CHO cells and an unscheduled DNA synthesis (UDS) test in human epithelial cells.

 

Gene mutation assay

In an OECD 471 and GLP bacterial reverse mutation test (Jones and Fenner, 1987), 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.

 

Tetrahydrothiophene (THT) was tested up to its solubility limit (200 µg/ml in the culture medium) in a mammalian cell gene mutation assay (HGPRT) with Chinese Hamster Ovary cells (CHO) both in the absence and presence of metabolic activation (S9 mix) (Henderson, 1987). The study was performed following the OECD n° 476 Guideline and GLP. CHO were exposed to THT in DMSO for 4 hours both with and without metabolic activation at concentrations of 0, 100, 125, 150, 175 and 200 µg/ml. Appropriate positive controls were used and showed a statistical increase in mutant colonies. After a 3-4-day rest period, the cells were then incubated for the mutagenicity evaluation with 6-thioguanine.Cytotoxicity was not observed in the absence or presence of S-9 mix after treatment of the cells with THT. In both tests in the absence and both tests in the presence of S-9 mix, THT failed to induce increases in mutant frequency at the HPRT locus in CHO cells. THT does not demonstrate mutagenic potential in this in vitro test system.

 

Chromosomal aberration assay

Tetrahydrothiophene (THT) was tested for its ability to induce chromosomal aberrations in human lymphocytes cultured in vitro in study performed following the OECD guideline #473 and GLP (Allen and Brooker, 1987).Cultured human lymphocytes, stimulated to divide by addition of phytohaemagglutinin were exposed to THT in both the presence and absence of a metabolic activation system (S-9 mix) derived from rat livers. Cell division was then arrested using colchicine and after hypotonic treatment, fixation and staining, metaphase spreads were examined for chromosomal damage. Following the results of a preliminary toxicity test, the maximum concentration of THT that did not form immiscible droplets in this system,125 µg/ml, was selected as the highest dose level for the metaphase analysis in both the presence and absence of metabolic activation. Other dose levels tested were 62.5 and 12.5 µg/ml. THT did not cause any significant increases in chromosomal damage at any dose level in either the presence or absence of metabolic activation. Both positive control compounds caused large, statistically, highly significant increases in chromosomal damage, thus demonstrating the sensitivity of the test system and the efficacy of the S-9 mix. It is concluded that THT has shown no evidence of clastogenic activity in this in vitro cytogenetic assay using human lymphocytes.

 

DNA damage assay

In an OECD 482 study, tetrahydrothiophene (THT) was tested for potential mutagenic activity by measuring its ability to induce DNA repair in cultured human epithelioid cells (Henderson and Proudlock, 1987). Repair of DNA damage may be assessed using radiolabelling and autoradiographic techniques. Repair synthesis is demonstrated by increases in the number of silver grains found over cell nuclei. THT was assessed at concentrations of 0, 2.5, 5, 10, 20, 40, 80, 160, 320, 640, 1280, 2560 and 5120 µg/ml on two separate occasions in both the presence and absence of metabolic activation by S-9 mix. The highest concentration tested was limited by the solubility of the test agent in the culture medium. The higher concentrations of the test agent proved toxic resulting in sloughing of cells from the surface of the culture vessel and inhibition of residual normal replicative synthesis of DNA in S-phase cells. Sporadic small but statistically significant increases in radiolabelling of non S-phase cells were obtained at some concentrations of the test agent in the first repair test. The increases were not dose-related, and no such increases were obtained in the second test. It is concluded that the apparent increases were probably due to chance variation rather than being the result of DNA repair induced by THT.THT did not induce significant levels of DNA repair in this system and therefore shows no evidence of mutagenic potential in this in vitro test system.

Tetrahydrothiophene (THT) was tested for its ability to induce SCE in Chinese hamster ovary cells cultured in vitro (Allen et al., 1987).A preliminary toxicity test showed that the highest concentration of THT that did not form immiscible droplets in this system, 125 µg/m1, was not toxic to the cells in either the presence or absence of metabolic activation (as measured by the proportion of metaphase figures in the second division after treatment).Metaphase figures were analyzed for sister chromatid exchange from cultures treated with 15.63, 31.25, 62.5 and 125 µg/ml in the absence of metabolic activation and 15.63, 62.5 and 125 µg/ml in the presence of metabolic activation. No statistically significant increases in SCE were seen in CHO cells treated with THT at any dose level in the absence of metabolic activation. In the presence of metabolic activation no significant increases in SCE were seen in THT-treated cultures, with the exception of a very small, but just statistically significant increase at the intermediate dose level,62.5 µg/ml (P <0.05). This increase is not considered to be indicative of SCE inducing ability. The positive control compounds, ethylmethane sulphonate (250 µg/ml) in the absence of metabolic activation, and cyclophosphamide (5 µq/ml) in the presence of metabolic activation both caused large, statistically highly significant increases in SCE, thus demonstrating the efficacy of the S-9 mix and the sensitivity of the test system. It is concluded that THT has shown no evidence of inducing SCE under the conditions of this in vitro assay using CHO cells.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine reversion

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

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.

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'.

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Tetrahydrothiophene was tested for its ability to induce forward mutation at the functionally hemizygous hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus using Chinese hamster ovary (CHO) cells.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO-K1-BH4 cells originally derived from the ovary of an adult Chinese hamster and obtained from the British Industrial Biological Research Association were used in this test.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

0, 100, 125, 150, 175 and 200 µg/ml

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S9 : ethyl methane sulfphonate (250 µg/ml). With S9: 20-methylcholanthrene (5 µg/ml)

Details on test system and experimental conditions:

Preliminary toxicity test:
A cell suspension was prepared at 5 x 10e5 cells/ml in H5, 10 ml of which was dispensed into each 80 cm2 surface area flask. Two flasks were seeded for each of the chosen concentrations of test substance and 4 flasks for the solvent control. The cells were incubated for at least 20 hours at 37°C in a humidified atmosphere of 95% air/5% CO2 prior to exposure to the test substance.
The exposure period was 4 hours both in the presence and absence of S-9 mix. 2 ml of HO or S-9 mix was added to one flask per treatment group followed by 120 µl of test substance (at 100 times the final concentration) or solvent. The flasks were then re-incubated at 37°C for 4 hours. At the end of the treatment period, the contents of each flask were harvested, the cells were washed and three dishes (60 mm diameter) per culture were seeded with 200 cells/dish in H5. After incubation for 7 days at 37°C, the colonies were fixed, stained with Giemsa and counted using an electronic colony counter (Biotran Mark III, New Brunswick Scientific Co. Inc.). Cell survival was expressed as the plating efficiency of the treated population relative to the control cultures.

The highest concentration used in the main study was the limit of solubility of compound in the treatment medium.

Main study:
The main test was carried out as described in the preliminary toxicity test using duplicate cultures for each treatment group, solvent and positive controls. In addition to this, approximately 1.0 x 10e6 viable cells (estimated on the basis of cytotoxicity) from each culture were seeded in 50 ml H5 in 175 cm2 surface ares flasks to allow expression of the mutant phenotype. These cultures were maintained by subculturing at appropriate cell densities as above 3-4 days later. Three to four days after the final subculture each culture was harvested, counted, 200 cells per dish (three dishes per culture) were seeded in 60 mm diameter petri dishes in H5 and 2 x 10e5 cells per dish (five dishes per culture) were seeded in selective medium H5-TG. For each of the negative control cultures two determinations of mutation and viability were made by performing independent dilutions. After 7 days incubation the colonies were fixed, stained and counted. Viability and mutation plates were counted using the electronic colony counter.

Two independent tests in the absence of exogenous metabolic activation and two independent tests in the presence of S-9 mix were carried out.

Evaluation criteria:

The criteria which must be satisfied before a positive response may be claimed are:

1. The demonstration of a statistically significant dose-related increase in mutant frequency at concentrations of test compound which result in greater than 10% tell survival.
2. The response must be reproducible.
3. In addition before claiming a positive response, the mean mutant frequency in treated cultures would be expected to reach a value of twice the highest acceptable mean spontaneous mutant frequency. Therefore, since the 97.5% upper confidence limit for the mean spontaneous mutant frequency found in this laboratory is 15 mutants/10e6 viable cella the mean value required in treated cultures before a positive response is claimed would be 30 mutants/10e6 viable cells.

Statistics:

The relationship between concentration of compound and mutant frequency was assessed for statistical significance using linear regression analysis. The mutant frequency for each individual culture at each concentration was used for this analysis. The significance of the regression coefficient was determined by a t-test analysis.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

>= 200 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

In the preliminary toxicity test the cells were treated with Tetrahydrothiophene up to its limit of solubility in the medium (200 µg/ml). Significant cytotoxicity was not observed in the absence or presence of S-9 mix. Concentrations up to the limit of solubility in the medium were used in the main tests.

In both tests in the absence and presence of S-9 mix the cells were treated with the test material up to the limit of solubility in the medium. Cytotoxicity was not observed in any of the tests. Several plates in the first test in the presence of S-9 mix were lost due to fungal contamination. However, sufficient information was obtained from the replicate cultures and plates to reliably assess the mutagenic potential of Tetrahydrothiophene in this test. Tetrahydrothiophene failed to induce any significant increases in mutant frequency compared to the solvent control in any test. EMS and 20-methylcholanthrene the positive controls induced highly significant increases in mutant frequency compared to the respective solvent controls.

Remarks on result:

other: other: Chinese Hamster Ovary cells

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Tetrahydrothiophene demonstrates no evidence of mutagenic potential in the HPRT test in the absence or presence of S-9 mix.

Executive summary:

Tetrahydrothiophene (THT) was tested up to its solubility limit (200 µg/ml in the culture medium) in a mammalian cell gene mutation assay (HGPRT) with Chinese Hamster Ovary cells (CHO) both in the absence and presence of metabolic activation (S9 mix). The study was performed following the OECD n° 476 Guideline and in compliance with the Principles of Good Laboratory Practice.

CHO were exposed to THT in DMSO for 4 hours both with and without metabolic activation at concentrations of 0, 100, 125, 150, 175 and 200 µg/ml. Appropriate positive controls were used and showed a statistical increase in mutant colonies. After a 3-4-day rest period, the cells were then incubated for the mutagenicity evaluation with 6-thioguanine.

Cytotoxicity was not observed in the absence or presence of S-9 mix after treatment of the cells with THT. In both tests in the absence and both tests in the presence of S-9 mix, THT failed to induce increases in mutant frequency at the HPRT locus in CHO cells. THT does not demonstrate mutagenic potential in this in vitro test system.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: Human lymphocytes

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

0, 12.5, 62.5, 125 µg/ml

Vehicle / solvent:

THT was diluted with dimethylsulphoxide immediately before use. At all final concentrations above 125 µg/ml, with dosing solutions added to tissue culture medium at 1% v/v (10 µl/ml), immiscible droplets were formed in the tissue culture medium used in this test system.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S9: Ethylmethane sulphonate (500 µg/ml), With S9: Cyclophosphamide (20 µg/ml)

Details on test system and experimental conditions:

Lymphocyte culture for the preliminary toxicity test with and without activation:
Human blood was collected aseptically and diluted with RPMI 1640 tissue culture medium. Lymphocytes were separated by centrifugation on a Histopaque-1077 gradient for 30 minutes at 400 x 'g'. After 3 washes and sedimentation by centrifugation for 10 minutes at 200 x 'g' the cells were suspended at a concentration of 1 x 10e6 cells per ml in RPMI 1640 + 20% Foetal Calf Serum + 2% phytohaemagglutinin. 1 ml aliquots of the cell suspension were placed in the wells of a Falcon multiwell tissue culture plate and incubated at 37°C in a humid atmosphere containing 5% CO2 for 48 hours.

After the 48-hour incubation period 250 µl of S-9 mix was added to one set of cultures followed by 12.5 µl of various dilutions of the test compound. 10 µl of the dilutions of test compound were added to the remaining cultures. Final concentrations of the test compound in both series of cultures were 0.24, 0.49, 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5 and 125 µg/ml. Two wells were used for each concentration with four wells for the solvent control. Two hours after addition of test compound those cultures treated with S-9 mix were centrifuged at 200 x 'g' for 7 minutes, the supernatant discarded and the cell pellet resuspended in fresh medium and incubated for a further 22 hours.

Harvesting, fixation and slide preparation:
After 70 hours incubation mitotic activity was arrésted by addition of colchicine to each culture at a final concentration of 0.25 µg/ml. After 2 hours incubation each cell suspension was transferred to a conical centrifuge tube where the solutions were made hypotonic. After a 10-minute period of hypotonic incubation the suspensions were centrifuged at 110 x 'g' for 10 minutes and the cell pellets fixed by addition of freshly prepared fixative (3 parts methanol : 1 part glacial acetic acid). The pellets were allowed to fix slowly. Prior to slide preparation the pellets were aspirated through a hypodermic needle, then centrifuged at 200 x 'g' for 10 minutes and finally resuspended in a small volume of fixative. Two or three drops of the cell suspensions were dropped onto pre-cleaned slides which were then allowed to air-dry. The slides were stained using Giemsa solution and, when dry, mounted in DPX.

Microscopic examination for mitotic index:
The stained cella were examined by light microscopy at a magnification of x160 and the proportion of metaphase figures in each culture derived by recording the number of metaphase cella and the total number of nuclei.

Metaphase analysis:
Lymphocyte cultures were prepared as described above and placed in 5 ml aliquote (10e6 cells/ml) in 10 ml culture plates. After 48 hours incubation 50 µl aliquote of THT were added to one set of duplicate cultures to give final concentrations of 12.5, 62.5 and 125 µg/ml. Dimethylsulphoxide in 50 µl aliquots was added to four cultures and ethylmethane sulphonate, the positive control, at a final concentration of 500 µg/ml was added to two cultures. For the test in the presence of metabolic activation 1.25 ml S-9 mix (see Appendix 1) was added to each culture followed by 62.5 µl aliquots of various dilutions of THT giving final concentrations of 12.5, 62.5 and 125 µg/ml. Dimethylsulphoxide and sterile distilled water {62.5 µl) were added to four and two cultures respectively as the solvent controls with cyclophosphamide at a final concentration of 20 µg/ml added to two cultures as the positive control. Two hours after dosing the cultures containing S-9 mix were centrifuged and the cela pellets resuspended in fresh medium. The cells were incubated for a further 20 hours, arrested in metaphase by addition of colchicine for a further 2 hours, then made hypotonic and fixed as described above. Five slides were prepared from each culture, stained with Giemsa and mounted in DPX. The slides were then coded and wherever possible, a total of 100 metaphase spreads were examined from each culture at a magnification of xl000 using an oil immersion objective.

Species / strain:

other: Human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

> 125 µg/ml without S9, = 125 µg/ml with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TOXICITY TEST
The highest final concentration of THT that did not result in immiscible droplet formation in the tissue culture medium used in this assay was 125 µg/ml. This concentration had no toxic effect in the absence of metabolic activation but depressed the mitotic index to 46% of that of the solvent control in the presence of metabolic activation. 125, 62.5 and 12.5 µg/ml were the concentrations used for metaphase analysis under both sets of conditions.

METAPHASE ANALYSIS
Both positive control compounds, ethylmethane sulphonate (500 µg/ml) and cyclophosphamide (20 µg/ml) caused statistically highly significant increases in the proportion of metaphase spreads containing aberrations when compared with the relevant solvent controls.

THT did not cause any significant increases in chromosomal aberrations at any dose level in either the presence or absence of metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: Human lymphocytes

TABLE 1: Preliminary toxicity test - mitotic indices of cultured human lymphocytes treated with THT

a) Without metabolic activation

Culture number	Test agent	Concentration (µg/ml)	Mitotic index
			Slide a	Slide b	% Mean
5, 6 7, 8	dimethylsulphoxide (solvent control)	10 µl/ml	60/1000 45/1000	54/1000 58/1000	}5.43
9,10		0.24	67/1000	52/1000	5.95
11,12		0.49	60/1000	50/1000	5.50
13,14	THT	0.98	76/1000	77/1000	7.65
15,16		1.95	98/1000	92/1000	9.50
17,18		3.91	102/1000	82/1000	9.20
19,20		7.81	87/1000	73/1000	8.00
21,22		15.63	54/1000	48/1000	5.10
23,24		31.25	54/1000	48/1000	5.10
25,26		62.5	44/1000	47/1000	4.55
27,28		125	43/1000	62/1000	5.25

b) With metabolic activation

Culture number	Test agent	Concentration (µg/ml)	Mitotic index
			Slide a	Slide b	% Mean
53,54 55,56	dimethylsulphoxide (solvent control)	10 µl/ ml	70/1000 59/1000	74/1000 58/1000		}6.53
57,58		0.25	47/1000	40/1000		4.35
59,60		0.49	71/1000	74/1000		7.25
61,62		0.98	68/1000	67/1000		6.75
63,64		1.96	64/1000	70/1000		6.70
65,66	THT	3.91	59/1000	61/1000		6.00
67,68		7.82	61/1000	52/1000		5.65
69,70		15.63	54/1000	52/1000		5.30
71,72		31.25	50/1000	54/1000		5.20
73,74		62.5	42/1000	40/1000		4.10
75,76		125	37/1000	23/1000		3.00

TABLE 2 : Effect of THT on the chromosomes of cultured human lymphocytes

 (a) Without metabolic activation

Culture no.	Test agent	Concentration mg/mi	No.cells examined	No.aberrations per 100 cells		Aberrations										No.aberrant Cells (excluding gaps)	% Mean	No. aberrant cells (including gaps)	% Mean
				Excluding gaps	Including gaps	IBF	BWF	BF	I	SN	R	A	GT	ISO	CHR
101 102 103 104	dimethyl- sulphoxide (solvent control)	10 µl/ml	100 100 100 100	0 0 1 2	0 0 1 2		1					1 1				0 0 1 2	0.75	0 0 1 2	0.75
105 106	THT	12.5	100 100	0 0	0 0											0 0	0	0 0	}0
107 108	THT	62.5	100 100	3 4	3 4		2 1	1				3				2 1	1.5	2 1	1.5
109 110	THT	125	100 100	0 1	0 2							1		1		0 1	0.5	0 2	1
117 118	ethylmethane sulphonate	500	78 100	35.89 36	37.18 36	1	11 10	1	2 3	1 1	1	13 19	1		1	16 15	*** 17.42	16 15	*** 17.42

 (b) With metabolic activation

Culture no.	Test agent	Concentration µg/ml	No. cells examined	No.aberrations per 100 cells		Aberrations						No. aberrant cells (excluding bgaps)	% Mean	No. aberrant cells (including gaps)	% Mean
				Excluding gaps	Including gaps	IBF	BWF	I	A	GT	CHR
157 158 159 160	dimethyl-sulphoxide (solvent control)	10 µl/ml	100 100 100 100	4 0 0 2	4 0 0 2		1		4 1			3 0 0 2	1.25	3 0 0 2	1.25
161 162		12.5	100 100	2 3	2 3		1		2 3			2 3	2.5	2 3	2.5
163 1 64	THT	62.5	100 100	1 0	1 0				1			1 0	0.5	1	0.5
169 170		125	100 100	0 0	0 0							0 0	0	0	0
171 172	sterile distilled water (solvent control)	10µl/ml	100 100	3 1	3 2		1		2 1		1	2 1	1.5	2 2	2.0
173 174	cyclophosphamide	20	93 81	33.33 44.44	34.41 45.68	1	16 26	5 2	9 5	2	1 1	17 18	***20.1	17 18	*** 20.1

IBF Isochromatid break with fragment, BWF Chromatid break with fragment; BF Chromatid break without fragment; I Interchange;SM Single minute;R Ring;A Acentric fragment; GT Greater than 10 aberrations; ISO Isochromatid gap; CHR Chromatid gapStatistical analysis used was Fisher's test *** P<0.001, Otherwise P>0.05

Conclusions:

Interpretation of results (migrated information):
negative

THT has shown no evidence of clastogenic activity in this in vitro metaphase analysis using human lymphocytes.

Executive summary:

Tetrahydrothiophene (THT) was tested for its ability to induce chromosomal aberrations in human lymphocytes cultured invitroin in study performed following the OECD guideline #473 and GLP.Cultured human lymphocytes, stimulated to divide by addition of phytohaemagglutinin were exposed to THT in both the presence and absence of a metabolic activation.system (S-9 mix) derived from rat livers. Cell division was then arrested using colchicine and after hypotonic treatment, fixation and staining, metaphase spreads were examined for chromosomal damage.Following the results of a preliminary toxicity test, the maximumconcentration of THT that did not form immiscible droplets in this system,125 µg/ml, was selected as the highest dose level for the metaphase analysis in both the presence and absence of metabolic activation. Other dose levels tested were 62.5 and 12.5 µg/ml.

THT did not cause any significant increases in chromosomal damage atany dose level in either the presence or absence of metabolic activation.Both positive control compounds caused large, statistically, highlysignificant increases in chromosomal damage, thus demonstrating the sensitivity of the test system and the efficacy of the S-9 mix.

It is concluded that THT has shown no evidence of clastogenic activityin this in vitro cytogenetic assay using human lymphocytes.

Reference 4

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

GLP compliance:

yes

Type of assay:

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Species / strain / cell type:

other: Human epithelioid cells

Details on mammalian cell type (if applicable):

HeLa S3 epithelioid cells originally derived from a human cervical carcinoma. They were obtained from Flow Laboratories Ltd.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-incuced rat liver S9

Test concentrations with justification for top dose:

0; 2.5; 5; 10; 20; 40; 80; 160; 320; 640; 1280; 2560; 5120 µg/ml

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: The positive control compounds were 4-nitroquinoline-1-oxide (4NQO) in the absence of S-9 mix and 2-aminoanthracene (2AA) in the presence of S-9 mix.

Details on test system and experimental conditions:

First DNA repair test:
The DNA repair test is routinely performed twice.
For the first test, 2 ml cultures in arginine deficient medium (ADM) were prepared.
After the 72-hour incubation period in ADM, the radioactive DNA precursor, (3H)-thymidine (0.5 mCi/ml, 22 Ci/mmole), was added in 20 µl aliquote to the relevant cultures to give a final activity of 5 µCi/ml. S-9 mix (100 µl/culture) was then added to the cultures requiring metabolic activation. A 100 µl aliquot of the appropriate test solution or vehicle was then added. Tetrahydrothiophene was tested at a maximum final concentration of 5120 µg/ml with eleven serial two-fold dilutions of this concentration.

After incubation for 180 minutes in the presence of the test reagents, the cultures were harvested by removal of the coverslips with attached cells. The coverslips and cells were then washed, fixed and stained and processed for autoradiography.

Coverslips were then attached to microscope slides, using DPX mountant, with the cell monolayer uppermost. The mountant was allowed to harden at 37°C.

Second DNA repair test:
This was performed using the same methods and dose levels as in the first test.

Autoradiography:
The autoradiographic procedures were carried out in the darkroom at room temperature. Stripping film ) was used to coat the cultures. Pieces of stripping film of suitable size were floated emulsion side down onto a bath of 0.001% potassium bromide and 2% sucrose; these respectively retard the growth of background and prevent loss of sensitivity. After at least 2 minutes, when the film had swollen, the microscope slide was immersed then raised and contact made with the emulsion strip. The prepared slides and attached coverslips were left to air-dry and then placed in a light-tight box containing a quantity of silica gel and allowed to dry overnight at room temperature. After drying, the slides were placed in a small light-tight box containing a few granules of silica gel and exposed at 4°C for 13 days. The autoradiographs were then developed in Kodak D19 developer for 7 minutes, washed in 2% acetic acid for 1 minute and fixed in Kodak Unifix for 10 minutes, to clear the film. They were given a prolonged rinse in running tap water then rinsed in distilled water, air-dried and the excess film trimmed away.

Quantitation of repair synthesis:
The autoradiographs were examined, under code, using a Zeiss Standard WL microscope with oil immersion objective.
One hundred non S-phase nuclei were examined from each culture and two cultures were analysed at each concentration. The number of silver grains overlying the nuclei and a comparable area of cytoplasm was assessed. In both tests a record of the number of non S-phase nuclei having more than 3 grains net was kept (% labelled nuclei).

Evaluation criteria:

A positive response was recorded if there was a reproducible, statistically significant increase in the number of grains per 100 nuclei of non S-phase compound-treated cells compared with the number deposited over nuclei of solvent-treated control cells.

Statistics:

The results of both tests were subjected to statistical analysis using one-way analysis of variance.

Species / strain:

other: Human epithelioid cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 2560 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

At the highest dose levels tested tetrahydrothiophene proved toxic causing severe inhibition of S-phase synthesis of DNA, sloughing of cells from coverslips and overt cell death. Consequently no results are available for the cultures affected.

In the first test in the absence of S-9 mix, a few small but statistically significant increases in the gross and net nuclear grain counts were obtained for cultures treated with tetrahydrothiophene. However, the increases were not dose-related nor were any such increases seen in the second test.

In the first test in the presence of S-9 mix a small but statistically significant increase in both the gross and net nuclear grain count was obtained at a single concentration of tetrahydrothiophene (160 µg/ml) although no significant increases in either the net or gross nuclear grain count were seen at any concentration of the test agent in the second test.

Both positive control agents (4-NQ0 in the absence of S-9 mix and 2-AA in the presence of S-9 mix) caused large highly significant increases in the gross and net nuclear grain counts together with increases in the proportion of labelled nuclei (i.e. % nuclei with greater than 3 net grains).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1A : First DNA repair test in the absence of S-9 mix

Compound	Concentration (µg/ml)	Grains per 100 nuclei			Grains per 100 cytoplasmic areas		Net grains per 100 nuclei			% Nuclei with >3 net grains
		X1	X2	mean	X1	X2	X1	X2	mean	X1	X2	mean
		148	167		128	138	20	29		1	2
		130	176		155	135	-25	41		0	3
DMSO	-	158	157	145	101	94	57	63	15	1	2	1.5
		130	128		121	134	9	-6		2	0
		114	161		163	137	-49	24		0	2
		131	-#		126	-	5	-		3	-
	2.5	171	139	155	125	84	46	55	51*	2	2	2
	5	132	168	150	80	135	52	33	43	0	4	2
	10	186	156	171	118	106	68	50	59*	4	3	3.5
	20	165	210	188**	113	153	52	57	55*	1	3	2
	40	137	137	137	116	104	21	33	27	2	0	1
Tetrahydro-	80	164	139	152	126	130	38	9	24	4	3	3.5
thiophene	160	167	181	174*	107	127	60	54	57*	2	5	3.5
	320	162	135	149	138	120	24	15	20	0	1	0.5
	640	154	122	138	157	135	-3	-13	-8	1	0	0.5
	1280	148	190	169	75	139	73	51	62*	1	2	1.5
	2560	$	122	122	-	106	-	16	16	-	0	0
	5120	$	134	134	-	133	-	1	1	-	1	1
	0.02	1846	2197	2022***	73	123	1773	2074	1924***	100	100	100
	0.04	3200	2857	3029***	65	122	3135	2735	2935***	100	100	100
4NQ0	0.08	3226	3661	3444***	103	161	3123	3500	3312***	100	100	100
	0.16	3404	6180	4792***	200	110	3204	6070	4637***	100	100	100
	0.32	4483	3802	4143***	91	113	4392	3689	4041***	100	100	100

TABLE 1B: First DNA repair test in the presence of S-9 mix

Compound	Concentration (µg/ml)	Grains per 100 nuclei				Grains per 100 cytoplasmic areas		Net grains per 100 nuclei				% Nuclei with >3 net grains
		X1	X2	mean		X1	X2	X1	X2		mean	X1			X2	mean
		140	147			125	101	15		46			0	2
		126	151			108	119	18		32			0	4
DMSO	-	96	172		133	128	171	-32		1	10		0	5			1.3
		134	95			121	156	13		-61			1	0
		148	155			84	135	64		20			2	1
		127	109			98	130	29		-21			1	0
	2.5	114	170		142	121	174	-7		-4	-5		1	4			2.5
	5	142	165		154	120	105	22		60	41		0	1			0.5
	10	162	137		150	113	154	49		-17	16		3	0			1.5
	20	135	159		147	78	139	57		20	39		2	3			2.5
	40	143	121		132	158	125	-15		-4	-9		2	1			1.5
Tetrahydro-	80	136	195		166	113	180	23		15	19		0	1			0.5
thiophene	160	186	164		175*	120	102	66		62	64*		4	4			4
	320	147	126		137	105	95	42		31	37		0	0			0
	640	114	150		132	119	134	-5		16	6		0	3			1.5
	1280	127	186		157	121	145	6		41	24		1	2			1.5
	2560	159	$		159	117	-	42		-	42		6	-			6
	5120	$ S	$		-	-	-	-		-	-		-	-			-
	2.5	223	260		242***	137	145	86		115	101***		7	11			9
	5	389	344		367***	145	110	244		234	239***		24	16			20
	10	445	400		423***	124	134	321		266	294***		21	37			29
2AA	20	402	317		360***	162	106	240		211	226***		34	29			31.5
	40	334	326		330***	103	130	231		196	214***		13	19			16

Conclusions:

Interpretation of results (migrated information):
negative

Although a few small but statistically significant increases in the gross and net nuclear grain count were obtained in the first test for cultures treated with tetrahydrothiophene, these increases were sporadic and were not reproduced in the second test. It is concluded that they are probably due to chance variation rather than being the result of DNA repair induced by the test agent.

Executive summary:

In and OECD 482 study, tetrahydrothiophene (THT) was tested for potential mutagenic activity by measuring its ability to induce DNA repair in cultured human epithelioid cells. Repair of DNA damage may be assessed using radiolabelling and autoradiographic techniques. Repair synthesis is demonstrated by increases in the number of silver grains found over cell nuclei.THT was assessed atconcentrations of 0; 2.5, 5, 10, 20, 40, 80, 160, 320, 640, 1280, 2560 and 5120 µg/mlon twoseparate occasions in both the presence and absence of metabolic activationby S-9 mix. The highest concentration tested was limited by the solubility of the test agent in the culture medium. The higher concentrations of thetest agent proved toxic resulting in sloughing of cells from the surface ofthe culture vessel and inhibition of residual normal replicative synthesis of DNA in S-phase cells.

Sporadic small but statistically significant increases inradiolabelling of non S-phase cells were obtained at some concentrations ofthe test agent in the first repair test. The increases were not dose-related, and no such increases were obtained in the second test. It is concluded that the apparent increases were probably due to chance variation rather than being the result of DNA repair induced by THT.

It is concluded that THT did not induce significantlevels of DNA repair in this system and therefore shows no evidence of mutagenic potential in thisin vitrotest system.

Reference 5

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)

GLP compliance:

yes

Type of assay:

sister chromatid exchange assay in mammalian cells

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO-KI-BH4, cells, originally derived from the ovary of an adult Chirese hamster, were obtained from the British Industrial Biological Research Association.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

15.63, 31.25, 62.5 and 125 µg/ml without S9 and 15.63, 62.5 and 125 µg/ml with S9

Vehicle / solvent:

THT was diluted with dimethylsulphoxide (DMSO) immediately before use. At all final concentrations above 125 µg/ml, adding 10 µl of test compound solution to 1 ml of culture medium, immiscible droplets were formed in the tissue culture medium used in this test system.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Ethylmethane sulphonate (250 µg/ml) in the absence of metabolic activation, and cyclophosphamide (5 µg/ml) in the presence of metabolic activation

Details on test system and experimental conditions:

Toxicity test and main study:
Final concentrations of THT dosed were 1.95, 3.91, 7.81, 15.63, 31.25, 62.5 and 125 pµ/ml in both the presence and absence of metabolic activation. Two cultures were used for each treatment. Four cultures were dosed with the solvent control, dimethylsulphoxide at a final concentration of 10 µl/ml, v/v. Two cultures were dosed with the positive control compound in both the presence (cyclophosphamide at a final concentration of 5 µg/ml) and absence (ethylmethane sulphonate at a final concentration.of 250 µg/ml) of metabolic activation.
Twenty-five metaphase figures were examined from each culture and the number in the second division after treatment (indicated by the ratio of harlequin to non-harlequin stained figures) recorded. This was used as the parameter of toxicity in determining the dose levels for analysis in the main SCE study.

Main study:
In the absence of metabolic activation concentrations of THT analysed were 15.63, 31.25, 62.5 and 125 µg/ml.
In the presence of metabolic activation concentrations of THT analysed were 15.63, 62.5 and 125 µg/ml.
The slides from these treated cultures and the control cultures were analysed for SCE. Where possible, for each culture the number of chromosomes together with the number of SCEs in 30 cells were scored. Only cells containing 19, 20 or 21 chromosomes (i.e. the modal chromosome number ± 1) were scored.
The following calculations were then made:
(i) for each cell, the number of SCEs per chromosome;
(ii) for each culture, the mean number of SCEs per chromosome.

Evaluation criteria:

A statistically significant increase in the frequency of SCE, together with some evidence of a dose response, in an experiment where a large proportion of cells are in the metaphase stage of the second division after treatment, is indicative of a positive response.

Statistics:

The results were subjected to statistical analysis using one-way analysis of variance with Student's 't' test, and a Jonckheere's test for trend (where necessary).

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 125 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

A preliminary toxicity test showed that the highest concentration of THT that did not form immiscible droplets in this system, 125 µg/ml, was not toxic to the cells in either the presence or absence of metabolic activation (as measured by the proportion of metaphase figures in the second division after treatment).

Metaphase figures were analysed for sister chromatid exchange from cultures treated with 15.63, 31.25, 62.5 and 125 µg/ml in the absence of metabolic activation and 15.63, 62.5 and 125 µg/ml in the presence of metabolic activation.

No statistically significant increases in SCE were seen in CHO cells treated with THT at any dose level in the absence of metabolic activation.

In the presence of metabolic activation no significant increases in SCE were seen in THT-treated cultures, with the exception of a very small, but just statistically significant increase at the intermediate dose level, 62.5 µg/ml (P <0.05). This increase is not considered to be indicative of SCE inducing ability.

The positive control compounds, ethylmethane sulphonate (at a final concentration of 250 µg/ml) in the absence of metabolic activation, and cyclophosphamide (at a final concentration of 5 µg/ml) in the presence of metabolic activation both caused large, statistically highly significant increases in SCE, thus demonstrating the efficacy of the S-9 mix and the sensitivity of the test system.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: Chinese Hamster Ovary Cells

SCE frequency in CHO cells after treatment with THT in the absence of metabolic activation

Treatment	Concentration µg/ml	No. of cells analysed	Mean number of SCEs/ chromosome+
dimethylsulphoxide (solvent control)	10 µl/ml	30	0.30	0.28
		30	0.28
		30	0.28
		30	0.28
THT	15.63	30	0.28	0.23
		30	0.18
THT	31.25	30	0.30	0.27
		30	0.24
THT	62.5	30	0.31	0.32
		30	0.32
THT	125	30 30	0.28 0.33	0.30
ethylmethane sulphonate	250	30	0.68	0.73***
		30	0.78

SCE frequency in CHO cells after treatment with THT in the presence of metabolic activation.

Treatment	Concentration µg/ml	No. of cells analysed	Mean number of SCEs/ chromosome+
dimethylsulphoxide (solvent control)	10µl/ml	30	0.34	0.32
		30	0.35
		30	0.31
		30	0.27
THT	15.63	30	0.38	0.37
		30	0.35
THT	62.5	30	0.40	0.39*
		30	0.37
THT	125	30	0.33	0.33
		30	0.32
cyclophosphamide	5	30	0.74	0.70***
		30	0.65

Student^'s^'t^'test: ***P <0.001 } one-sided probabilities

    Otherwise P >0.05

+ Calculated as : Total number of SCEs/total number of chromosomes

Conclusions:

Interpretation of results (migrated information):
negative

THT has shown no evidence of inducing SCE under the conditions of this in vitro assay using CHO cells.

Executive summary:

Tetrahydrothiophene (THT) was tested for its ability to induce SCE inChinese hamster ovary cells cultured in vitro.

A preliminary toxicity test showed that the highest concentration ofTHT that did not form immiscible droplets in this system, 125 µg/m1, was not toxic to the cells in either the presence or absence of metabolic activation (as measured by the proportion of metaphase figures in the second division after treatment).

Metaphase figureswere analyzed for sister chromatidexchange fromculturestreated with 15.63, 31.25, 62.5 and 125 µg/ml in theabsence ofmetabolic activation and 15.63, 62.5 and 125 µg/ml in the presence of metabolic activation.

No statistically significant increases in SCE were seen in CHO cellstreated with THT at anydose level in the absence of metabolic activation.

In the presence of metabolic activation no significant increases inSCEwere seen in THT-treated cultures, with the exception of a very small,but just statistically significantincrease at theintermediate dose level,62.5 µg/ml (P <0.05). This increase is not considered to be indicative ofSCEinducing ability.

The positive control compounds, ethylmethane sulphonate (250 µg/ml) in the absence of metabolic activation, andcyclophosphamide (5 µq/ml) in the presence ofmetabolic activation both caused large, statistically highly significant increases inSCE,thus demonstrating the efficacy of the S-9 mix and the sensitivity of the test system.

It is concluded that THT has shown no evidence of inducing SCE under the conditions of this in vitro assay using CHO cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to the available data and the criteria of Regulation (EC) No 1272-2008, no classification is warranted for germ cell mutagenicity.