Thiophene, tetrahydro-, 1,1-dioxide, 3-(C9-11-isoalkyloxy) derivs., C10-rich

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No adverse effect observed (negative)

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

Study period:

The experimental phase of this study was performed between 28 September 2011 and 24 November 2011.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

Meets the requirements of the Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guidelines for Testing of Chemicals No. 471 "and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine for Salmonella.
Tryptophan for E.Coli

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta­naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Experiment one: Salmonella strains (with and without S9-mix): 15, 50, 150, 500, 1500, 5000 µg/plate.
E.coli strain WP2uvrA (with and without S9-mix): 5, 15, 50, 150, 500, 1500, 5000 µg/plate.

Experiment two: All bacterial strains with and without S9-mix except TA1537 (without S9-mix): 5, 15, 50, 150, 500, 1500, 5000 µg/plate.
Salmonella strain TA1537 (without S9-mix): 1.5, 5, 15, 50, 150, 500, 1500 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide.
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/ml but was fully miscible in dimethyl
sulphoxide at the same concentration in solubility checks performed in house. Dimethyl sulphoxide was therefore selected as the vehicle.

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 1 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 10 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 mix Migrated to IUCLID6: Benzo(a)pyrene: 5 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 mix Migrated to IUCLID6: 4-Nitroquinoline-1-oxide: 0.2 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix Migrated to IUCLID6: 9-Aminoacridine: 80 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 3 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 5 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 2 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 48 - 72 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

Evaluation criteria:

Acceptance Criteria:

The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9 x 109 bacteria per ml.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic
sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-toxic test material dose levels.
There should not be an excessive loss of plates due to contamination.

Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical
significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Statistics:

Standard deviation
Dunnetts Linear Regression Analysis

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test item was tested up to either the maximum recommended dose level (5000 µg/plate) or the toxic limit, depending on bacterial strain type and presence or absence of S9-mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

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:

cytotoxicity

Remarks:

The test item was tested up to either the maximum recommended dose level (5000 µg/plate) or the toxic limit, depending on bacterial strain type and presence or absence of S9-mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was immiscible in sterile distilled water at 50 mg/ml but was fully miscible in dimethyl sulphoxide at the same
concentration in solubility checks performed in house. Dimethyl sulphoxide was therefore selected as the vehicle.
- Precipitation: A test item precipitate (oily and globular in appearance) was noted at 5000 µg/plate, this observation did not prevent the scoring of
revertant colonies.

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test:
The test item was non-toxic to TA100 but exhibited weakened background lawns to WP2uvrA from 500 µg/plate in the absence of S9-mix and at
5000 µg/plate in the presence of S9-mix. The test item formulation and S9-mix used in this experiment were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains in both experiments, initially from 1500 µg/plate in the range finding test and 150 µg/plate in the main test. For Salmonella strain TA1537, dosed in the absence of S9-mix, there was a slight discrepancy in toxic response with weakened lawns initially noted at 1500 µg/plate in the range-finding test and at 150 µg/plate in the main test. No weakening of the bacterial background lawns were noted for TA100 and Escherichia coli strain WP2uvrA (presence of S9-mix only). The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and Experiment number. The test item was tested up to either the maximum recommended dose level (5000 µg/plate) or the toxic limit, depending on bacterial strain type and presence or absence of S9-mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Results

Preliminary Toxicity Test

The test item was non-toxic to TA100 but exhibited weakened background lawns to WP2uvrA from 500 µg/plate in the absence of S9-mix and at 5000 µg/plate in the presence of S9-mix. The test item formulation and S9-mix used in this experiment were both shown to be sterile. The numbers of revertant colonies for the toxicity assay were:

With (+) or without (-) S9-mix	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	84	107	86	95	86	92	76	81	75	75	75P
+	TA100	102	96	113	110	98	118	109	108	117	104	89P
-	WP2uvrA	14	32	19	18	20	14	18	18	37*	20*	19*P
+	WP2uvrA	24	19	18	20	33	23	25	27	21	23	0*P

P         precipitate

*          Partial absence of bacterial background lawn

Mutation Test

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The amino acid supplemented top agar and S9-mix used in both experiments was shown to be sterile. There was also no evidence of excessive contamination. The culture density for each bacterial strain used in each experiment was also checked and considered acceptable.

Results for the negative controls (spontaneous mutation rates) are presented below and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation, are presented in Attached background material. The results are also expressed graphically in Attached background material.

Information regarding the equipment and methods used in these experiments as required by the Japanese Ministry of Economy, Trade and Industry and Japanese Ministry of Health, Labour and Welfare are presented in Attached background material.

A history profile of untreated/vehicle and positive control values (reference items) is presented in Attached background material.

The test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains in both experiments, initially from 1500 µg/plate in the range-finding test and 150 µg/plate in the main test. For Salmonella strain TA1537, dosed in the absence of S9-mix, there was a slight discrepancy in toxic response with weakened lawns initially noted at 1500 µg/plate in the range-finding test and at 150 µg/plate in the main test. No weakening of the bacterial background lawns were noted for TA100 and Escherichia coli strain WP2uvrA (presence of S9-mix only). The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and Experiment number. The test item was tested up to either the maximum recommended dose level (5000 µg/plate) or the toxic limit, depending on bacterial strain type and presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in either the range-finding or main tests.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Table1:  Spontaneous Mutation Rates (Concurrent Negative Controls)

Range-finding Test

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
68		12		17		17		17
82	(77)	14	(15)	31	(25)	15	(15)	12	(14)
81		18		28		14		14

Main Test

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
75		14		20		30		14
73	(76)	13	(12)	19	(17)	22	(25)	10	(12)
79		9		13		22		13
								15
								7	(12)†
								15

  † Experimental procedure repeated at a later date (without S9-mix) due to toxicity in the original test

       

Conclusions:

Interpretation of results (migrated information):
negative

The test item was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction

The method was designed to conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the, EPA (TSCA) OPPTS harmonised guidelines.

Methods

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using the Ames plate incorporation method at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and ranged between 5 and 5000 µg/plate, depending on bacterial strain type. The experiment was repeated on a separate day using a similar dose range to the range-finding test, fresh cultures of the bacterial strains and fresh test item formulations. Additional dose levels and an expanded dose range were selected in both experiments in order to achieve four non-toxic dose levels and the toxic limit of the test item.

Results

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains in both experiments, initially from 1500 µg/plate in the range-finding test and 150 µg/plate in the main test. For Salmonella strain TA1537, dosed in the absence of S9-mix, there was a slight discrepancy in toxic response with weakened lawns initially noted at 1500 µg/plate in the range-finding test and at 150 µg/plate in the main test. No weakening of the bacterial background lawns were noted for TA100 and Escherichia coli strain WP2uvrA (presence of S9-mix only). The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and Experiment number. The test item was tested up to either the maximum recommended dose level (5000 µg/plate) or the toxic limit, depending on bacterial strain type and presence or absence of S9-mix. A test item precipitate (oily and globular in appearance) was noted at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in either the range-finding or main tests.

Conclusion

The test item was considered to be non-mutagenic under the conditions of this test.

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

Study period:

The experimental phases of the study were performed between 13 September 2011 and 22 November 2011.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of therelevant results.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date GLP of inspection: 19 - 21 July 2011 Date of signature on GLP form: 31 August 2011

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
RPMI 1640

- Properly maintained:
yes

- Periodically checked for Mycoplasma contamination:
yes

- Periodically checked for karyotype stability:
no

- Periodically "cleansed" against high spontaneous background:
yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and beta-naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

The maximum dose levels used in the Mutagenicity Test were limited by test item-induced toxicity

Vehicle and positive controls were used in parallel with the test item. Solvent (DMSO) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS), Sigma batches 0001430125 and BCBC4573V at 400 µg/ml and 150 µg/ml for Experiment 1 and Experiment 2, respectively, was used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) Acros batches A0164185 and A0277203 at 2 µg/ml was used as the positive control in the presence of metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:
Solvent (DMSO) treatment groups were used as the vehicle controls.

- Justification for choice of solvent/vehicle:
Suitable for dosing at the required concentration.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Solvent (DMSO) treatment groups were used as the vehicle controls.

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Solvent (DMSO) treatment groups were used as the vehicle controls.

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

Introduction.

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "In Vitro Mammalian Cell Gene Mutation Tests", Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and be acceptable to the Japanese METI/MHLW guidelines for testing of new chemical substances.

Methods.

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at ten dose levels using a 4 hour exposure group in the presence of metabolic activation (1% S9) and a 24 hour exposure group in the absence of metabolic activation.

The dose range of test item was selected following the results of a preliminary toxicity test, and was 5 to 40 µg/ml in the absence of metabolic activation, and 10 to 80 µg/ml in the presence of metabolic activation for Experiment 1. In Experiment 2 the dose range was 5 to 50 µg/ml in the absence of metabolic activation, and 2.5 to 80 µg/ml in the presence of metabolic activation.

Results.

The maximum dose levels used in the Mutagenicity Test were limited by test item-induced toxicity in the absence of metabolic activation. Precipitate of test item was not observed at any of the dose levels in the Mutagenicity Test. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test item did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

Conclusion.

The test item was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Evaluation criteria:

Please see "Any other information on materials and methods incl. tables" section.

Statistics:

Please see "Any other information on materials and methods incl. tables" section.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

non-mutagenic

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Test

The dose range of the test item used in the preliminary toxicity test was 10.78 to 2760 µg/ml. In all three of the exposure groups there was evidence of marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls. The steep nature of the toxicity curve was taken to indicate that achieving optimum toxicity would be difficult. A cloudy precipitate of the test item was observed at 345 µg/ml and a greasy oily precipitate was observed at and above 690 µg/ml at the end of the exposure period in all three of the exposure groups. In addition, an increase in intensity was associated with an increase in dose concentration. Based on %RSG values observed, the maximum dose levels in the subsequent Mutagenicity Test were limited by test item-induced toxicity.

Mutagenicity Test

A summary of the results from the test is presented in attached Table 1.

Experiment 1

The results of the microtitre plate counts and their analysis are presented in attached Tables 2 to 7.

There was once again evidence of marked dose-related toxicity following exposure to the test item in the presence of metabolic activation, as indicated by the RTG and %RSG values (Table 6). In the absence of metabolic activation, the marked toxicity observed in the preliminary toxicity test was not reproduced and only modest levels of toxicity were achieved (Table 3). This was considered to be due to the very steep toxicity curve of the test item and inter-experimental variation. There was no evidence of any significant dose related reductions in viability (%V) in any of the dose levels, therefore indicating that no residual toxicity had occurred in either the absence or presence of metabolic activation. Based on the %RSG and RTG values observed, it was considered that optimum levels of toxicity had been achieved in the presence of metabolic activation. Whilst optimum levels of toxicity were not achieved in the absence of metabolic activation, it was considered that with no evidence of a response in Experiment 1, or Experiment 2 where optimum levels of toxicity were achieved in the absence of metabolic activation using a similar but slightly higher dose range, a repeat of this exposure group was not required. Acceptable levels of toxicity were seen with both positive control substances (Tables 3 and 6).

Table 3: Experiment 1 (-S9) 4 hour Exposure
Treatment (ug/ml) %RSG
0 100
15 102
20 97
25 85
30 88
35 78
40 58

Table 6: Experiment 1 (+S9) 4 hour Exposure
Treatment (ug/ml) %RSG
0 100
30 100
40 107
50 95
60 87
70 61
80 15

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 3 and 6).

The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in either the absence or presence of metabolic activation (Tables 3 and 6). Precipitate of test item was not observed at any of the dose levels.
The numbers of small and large colonies and their analysis are presented in Tables 4 and 7.

Experiment 2

The results of the microtitre plate counts and their analysis are presented in attached Tables 8 to 13.

As was seen in the preliminary toxicity test, there was evidence of marked toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the RTG and %RSG values (Tables 9 and 12). There was evidence of significant dose related reductions in viability (%V) in the presence of metabolic activation, therefore indicating that residual toxicity had occurred. However, it should be noted that this reduction was only observed at a dose level that had been excluded from the statistical analysis due to excessive levels of toxicity. Based on the %RSG and / or RTG values observed, it was considered that optimum levels of toxicity had been achieved in the absence of metabolic activation, and near optimum levels of toxicity had been achieved in the presence of metabolic activation. Whilst optimum levels of toxicity were not achieved in the presence of metabolic activation due to the steep toxicity curve of the test item, despite using a narrow dose interval, a dose level that exceeded the usual upper limit of acceptable toxicity was plated for viability and 5 TFT resistance as sufficient cells were available at the time of plating. However, due to the level of toxicity this was excluded from the statistical analysis. The excessive toxicity observed at and above 40 µg/ml in the absence of metabolic activation, and at and above 50 µg/ml in the presence of metabolic activation, resulted in these dose levels not being plated for viability or 5-TFT resistance. The toxicity observed at 40 µg/ml in the presence of metabolic activation exceeded the upper acceptable limit of 90%, therefore, this dose was excluded from the statistical analysis. The increase in %RSG observed at 50 µg/ml in the presence of metabolic activation was considered to be due to a dosing error of the B culture, as demonstrated by the cell counts. However, as the 40 µg/ml dose level had an excessive level of toxicity, the purpose and integrity of the study was considered unaffected. Acceptable levels of toxicity were seen with both positive control substances (Tables 9 and 12).

Table 9: Experiment 2 (-S9) 24 hour Exposure
Treatment (ug/ml) %RSG
0 100
5 77
10 73
15 58
20 35
25 28
30 14
35 11

Table 12: Experiment 2 (+S9) 4 hour Exposure
Treatment (ug/ml) %RSG
0 100
2.5 93
5 94
10 98
20 84
30 29
40 2

The 24-hour exposure without metabolic activation demonstrated that the extended time point had a marked effect on the toxicity of the test item. It should also be noted that the lowering of the S9 concentration to 1% in this second experiment resulted in much greater levels of toxicity being observed when compared to 4-hour exposure groups in the presence of 2% metabolic activation in the Preliminary Toxicity Test and Experiment 1.
Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 9 and 12).

The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell at any of the dose levels, in either the absence or presence of metabolic activation, including the dose level in the presence of metabolic activation that exceeded the usual upper limit of acceptable toxicity (Tables 9 and 12). Precipitate of test item was not observed at any of the dose levels. It was considered that the result obtained in the absence of metabolic activation demonstrated that a repeat of the 4-hour exposure group from Experiment 1, where optimum toxicity was not achieved, was not required and the test item had been adequately tested.

The numbers of small and large colonies and their analysis are presented in Tables 10 and 13.

Remarks on result:

other: strain/cell type: Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Remarks:

Migrated from field 'Test system'.

Please see Attached "Tables 1 to 13"

Due to the nature and quantity of tables it was not possible to insert them in this section.

Conclusions:

Interpretation of results (migrated information):
other: Non-mutagenic

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non mutagenic under the conditions of the test.

Executive summary:

Introduction. 

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. Thethod was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "In Vitro Mammalian Cell Gene Mutation Tests", Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and be acceptable to the Japanese METI/MHLW guidelines for testing of new chemical substances.

Methods. 

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at ten dose levels using a 4‑hour exposure group in the presence of metabolic activation (1% S9) and a 24‑hour exposure group in the absence of metabolic activation.

The dose range of test item was selected following the results of a preliminary toxicity test, and was 5 to 40 µg/ml in the absence of metabolic activation, and 10 to 80 µg/ml in the presence of metabolic activation for Experiment 1. In Experiment 2 the dose range was 5 to 50 µg/ml in the absence of metabolic activation, and 2.5 to 80 µg/ml in the presence of metabolic activation.

Results. 

The maximum dose levels used in the Mutagenicity Test were limited by test item-induced toxicity in the absence of metabolic activation. Precipitate of test item was not observed at any of the dose levels in the Mutagenicity Test. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test item did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

Conclusion. 

The test item was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

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

Study period:

Experimental Start Date: 09 February 2006 Experimental Completion Date: 03 March 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH (1996 and 1997)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Study was conducted in compliance with the US FDA Good Laboratory Practice Regulations as published in 21 CFR 58, the US EP A GLP Standards 40 CFR 160 and 40 CFR 792, the UK GLP Compliance Programme, the Japanese GLP Standard and the OECD Principles of Go

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable.

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Peripheral blood lymphocytes were obtained from a healthy non-smoking 26 year old adult female on 07 February 2006 for the preliminary toxicity assay and from the same donor on 13 February 2006 for the definitive assay. The donor had no recent history of radiotherapy, viral infection or the administration of drugs. This system has been demonstrated to be sensitive to the clastogenic activity of a variety of chemicals (Preston et al., 1981).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 was used as the metabolic activation system.

Test concentrations with justification for top dose:

Preliminary Toxicity Assay
The dose range was 0.276 to 2760 μg/mL,

Chromosome Aberration Test

Treatment Condition Treatment Time Recovery Time Dose levels (μg/mL)
Non-activated 4 hr 16 hr 25, 50, 75, 100, 125, 150, 175
20 hr 0 hr 6.25, 12.5, 25, 35, 50, 65, 75
S9-activated 4 hr 16 hr 25, 50, 75, 100, 125, 150, 175

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Dimethyl sulfoxide (DMSO) was the solvent of choice based on the solubility of the test article and compatibility with the target cells. The test article was soluble in DMSO at a concentration of approximately 500 mg/mL, the maximum concentration tested for solubility.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

In the presence of S9

Migrated to IUCLID6: (CP)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

In the absence of S9

Migrated to IUCLID6: (MMC)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
in medium

DURATION
- Preincubation period:
44 - 48 hrs

- Exposure duration:
Chromosome Aberration study
In the non-activated study, the cells were exposed for 4 or 20 hours at 37±1°C in a humidified atmosphere of 5±1% CO2 in air. In the 4-hour exposure group, after the exposure period, the treatment medium was removed, the cells washed with calcium and magnesium free phosphate buffered saline (CMF-PBS), refed with complete medium containing 1% PHA and returned to the incubator for an additional 16 hours.
In the S9-activated study, the cells were exposed for 4 hours at 37±1°C in a humidified atmosphere of 5±1% CO2 in air. After the exposure period, the treatment medium was removed, the cells washed with calcium and magnesium-free phosphate buffered saline (CMF-PBS), refed with complete medium containing 1% PHA and returned to the incubator for an additional 16 hours.

- Expression time (cells in growth medium):
16 hrs for 4 hrs exposure.

- Selection time (if incubation with a selection agent):
Not applicable.

- Fixation time (start of exposure up to fixation or harvest of cells):
20 hrs.

SELECTION AGENT (mutation assays):
No selection agent.

SPINDLE INHIBITOR (cytogenetic assays):
Colcemid®

STAIN (for cytogenetic assays):
The dried slides were stained with 5% Giemsa, air dried and permanently mounted.

NUMBER OF REPLICATIONS:
Duplicate cultures

NUMBER OF CELLS EVALUATED:
100/culture

DETERMINATION OF CYTOTOXICITY
- Method:
mitotic index - The mitotic index was recorded as the percentage of cells in mitosis per 500 cells counted. The number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

-Scoring of Chromosome Damage:
Metaphase cells with 46 centromeres were examined under oil immersion without prior knowledge of treatment groups. Whenever possible, a minimum of 200 metaphase spreads (100 per duplicate treatment condition) were examined and scored for chromatidtype and chromosome-type aberrations (Scott et al., 1990). The number of metaphase spreads that were examined and scored per duplicate flask was reduced when the percentage of
aberrant cells reached a statistically significant level before 100 cells are scored. Chromatid type aberrations include chromatid and isochromatid breaks and exchange figures such as quadriradials (symmetrical and asymmetrical interchanges), triradials, and complex rearrangements. Chromosome-type aberrations include chromosome breaks and exchange figures such as dicentrics and rings. Fragments (chromatid or acentric) observed in the
absence of any exchange figure were scored as a break (chromatid or chromosome). Fragments observed with an exchange figure were not scored as an aberration but instead were considered part of the incomplete exchange. Pulverized chromosome(s), pulverized cells and severely damaged cells (≥ 10 aberrations) also were recorded. Chromatid and isochromatid gaps were recorded but not included in the analysis. The XY coordinates for
each cell with chromosomal aberrations were recorded using a calibrated microscope stage.

OTHER EXAMINATIONS:
- Determination of polyploidy:
The percent polyploid and endoreduplicated cells was evaluated per 100 cells.

OTHER:
None.

Evaluation criteria:

The test article was considered to induce a positive response when the percentages of cells with aberrations were increased in a dose-responsive manner with one or more concentrations being statistically elevated relative to the solvent control group (p≤0.05). A reproducible significant increase at the high dose only with no dose response or a reproducible significant increase at one dose level other than the high dose with no dose response will be considered positive. The test article was concluded to be negative if no statistically significant increase was observed relative to the solvent control.

Statistics:

Statistical analysis of the percent aberrant cells was performed using the Fisher's exact test. Fisher's exact test was used to compare pairwise the percent aberrant cells of each treatment group with that of the solvent control. In the event of a positive Fisher's exact test at any test article dose level, the Cochran-Armitage test was used to measure dose-responsiveness.

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Refer to information on results and attached tables.

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: There was no significant change in pH when the test material was dosed into media.
- Effects of osmolality: The osmalality did not exceed the osmolality of the solvent by more than 20%
- Evaporation from medium: Not applicable.
- Water solubility: Not applicable, test material suspended in DMSO
- Precipitation/haemolysis:
Preliminary toxicity test: At the conclusion of the treatment period, visible precipitate was observed in treatment medium at dose levels ≥ 276 μg/mL and dose levels ≤ 82.8 μg/mL were soluble in treatment medium.
Hemolysis was also observed at dose levels ≥ 276 μg/mL in all treatment groups.
Chromosome aberration study
No precipitate observed. Slight hemolysis was observed at 175 μg/mL in the non-activated and the S9-activated 4-hour exposure groups.

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test

Dose levels for the chromosome aberration assay were selected following a preliminary toxicity test and were based upon a reduction in mitotic index relative to the solvent control. The results of the evaluation of mitotic inhibition are presented in the attached Tables 1, 2 and 3. HPBL cells were first exposed to nine concentrations of Thiophene, 3-(decyloxy)tetrahydro-,1, 1-dioxide(CAS # 398141-87-2 or 18760-44-6) ranging from 0.276 to 2760 μg/mL, as well as solvent controls, in both the absence and presence of an Aroclor-induced S9 activation system for 4 hours, or continuously for 20 hours in the absence of S9 activation. The test article was soluble in DMSO and in the treatment medium at all concentrations tested at the beginning of the treatment period. At the conclusion of the treatment period, visible precipitate was observed in treatment medium at dose levels ≥ 276 μg/mL and dose levels ≤ 82.8 μg/mL were soluble in treatment medium. Hemolysis was also observed at dose levels ≥ 276 μg/mL in all treatment groups. The osmolality in treatment medium of the highest concentration tested, 2760 μg/mL, was 406 mmol/kg. The osmolality of the solvent (DMSO) in the treatment medium was 412 mmol/kg. The osmolality of the test article concentrations in treatment medium are acceptable because they did not exceed the osmolality of the solvent by more than 20%. The pH of the highest concentration of test article in treatment medium was approximately 7.0.
Substantial toxicity (at least 50% reduction in mitotic index relative to the solvent control) was observed at doses ≥ 276 μg/mL in the non-activated and S9-activated 4-hour exposure groups, and at dose levels ≥ 82.8 μg/mL in the non-activated 20-hour exposure group. Based on the results of the preliminary toxicity test, the dose levels selected for testing in the chromosome aberration assay were as follows:

Treatment Condition Treatment Time Recovery Time Dose levels (μg/mL)
Non-activated 4 hr 16 hr 25, 50, 75, 100, 125, 150, 175
20 hr 0 hr 6.25, 12.5, 25, 35, 50, 65, 75
S9-activated 4 hr 16 hr 25, 50, 75, 100, 125, 150, 175

Chromosome Aberration Assay
In the chromosome aberration assay, the test article was soluble in DMSO and in the treatment medium at all concentrations tested at the beginning and conclusion of the treatment period. Slight hemolysis was observed at 175 μg/mL in the non-activated and the S9-activated 4-hour exposure groups. The osmolality in treatment medium of the highest concentration tested, 175 μg/mL, was 414 mmol/kg. The osmolality of the solvent (DMSO) in the treatment medium was 413 mmol/kg. The osmolality of the test article concentrations in treatment medium are acceptable because they did not exceed the osmolality of the solvent by more than 20%. The pH of the highest concentration of test article in treatment medium was approximately 7.0.
The findings of the cytogenetic analysis of the non-activated 4-hour exposure group are presented by treatment flask in the attached Table 4 and summarised by group in the attached Table 7. At the highest test concentration evaluated microscopically for chromosome aberrations, 100 μg/mL, mitotic inhibition was 56%, relative to the solvent control. The dose levels selected for analysis of chromosome aberrations were 25, 50 and 100 μg/mL. The percentage of cells with structural or numerical aberrations in the test article-treated group was not significantly increased above that of the solvent control at any dose level (p>0.05, Fisher's exact test) and was within the range for the historical control values. The percentage of structurally damaged cells in the MMC (positive control) group was statistically significant (15.0%) and within the range of historical control values for the MMC-treated human lymphocytes.
The findings of the cytogenetic analysis of the S9-activated group are presented by treatment flask in the attached Table 5 and summarised by group in the attached Table 7. At the highest test concentration evaluated microscopically for chromosome aberrations, 100 μg/mL, mitotic inhibition was 52%, relative to the solvent control. The dose levels selected for analysis of chromosome aberrations were 25, 50 and 100 μg/mL. The percentage of cells with structural or numerical aberrations in the test article-treated group was not significantly increased above that of the solvent control at any dose level (p>0.05, Fisher's exact test) and was within the range for the historical control values. The percentage of structurally damaged cells in the CP (positive control) group was statistically significant (15.0%) and within the range of historical control values for the CP-treated human lymphocytes.

The findings of the cytogenetic analysis of the non-activated 20-hour exposure group are presented by treatment flask in the attached Table 6 and summarised by group in the attached Table 7. At the highest test concentration evaluated microscopically for chromosome aberrations, 50 μg/mL, mitotic inhibition was 51%, relative to the solvent control. The dose levels selected for analysis of chromosome aberrations were 12.5, 25 and 50 μg/mL. The percentage of cells with structural or numerical aberrations in the test article-treated group was not significantly increased above that of the solvent control at any dose level (p>0.05, Fisher's exact test) and was within the range for the historical control values. The percentage of structurally damaged cells in the MMC (positive control) group was statistically significant (11.0%) and within the range of historical control values for the MMC-treated human lymphocytes.

Remarks on result:

other: strain/cell type:

Remarks:

Migrated from field 'Test system'.

For the tables and figures of results mentioned above, please refer to the attached background material section for the following tables:

TABLE 1 PRELIMINARY TOXICITY TEST USING Thiophene, 3-(decyloxy)tetrahydro-,1,1-dioxide IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION

TABLE 2 PRELIMINARY TOXICITY TEST USING Thiophene, 3-(decyloxy)tetrahydro-,1,1-dioxide IN THE PRESENCE OF EXOGENOUS METABOLIC ACTIVATION

TABLE 3 PRELIMINARY TOXICITY TEST USING Thiophene, 3-(decyloxy)tetrahydro-,1,1-dioxide IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION

TABLE 4 CYTOGENETIC ANALYSIS OF HUMAN PERIPHERAL BLOOD LYMPHOCYTES TREATED WITH Thiophene, 3-(decyloxy)tetrahydro-,1,1-dioxide IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION DEFINITIVE ASSAY: 4-HOUR TREATMENT, 20-HOUR HARVEST

TABLE 5 CYTOGENETIC ANALYSIS OF HUMAN PERIPHERAL BLOOD LYMPHOCYTES TREATED WITH Thiophene, 3-(decyloxy)tetrahydro-,1,1-dioxide IN THE PRESENCE OF EXOGENOUS METABOLIC ACTIVATION DEFINITIVE ASSAY: 4-HOUR TREATMENT, 20-HOUR HARVEST

TABLE 6 CYTOGENETIC ANALYSIS OF HUMAN PERIPHERAL BLOOD LYMPHOCYTES TREATED WITH Thiophene, 3-(decyloxy)tetrahydro-,1,1-dioxide IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION DEFINITIVE ASSAY: 20-HOUR TREATMENT, 20-HOUR HARVEST

TABLE 7 SUMMARY

Conclusions:

Interpretation of results (migrated information):
negative

CONCLUSION
The frequency of cells with structural chromosome aberrations in the solvent control was within the historical range for the solvent controls. The
percentage of cells with chromosome aberrations in each of the positive controls was statistically increased (p≤0.05,Fisher's exact test) relative to the
respective solvent control and within the historical control values. Therefore, the positive and solvent controls fulfilled the requirements for a valid test.
In addition, the frequency of cells with structural and numerical chromosome aberrations in the test article groups in presence and absence of S9 were within the historical solvent control range.
Under the conditions of the assay described in this report, Thiophene, 3-(decyloxy)tetra hydro-, 1,1-dioxide (CAS # 398141-87-2 or 18760-44-6)
was concluded to be negative for the induction of structural and numerical chromosome aberrations in the non-activated and S9-activated test systemsin the in vitro mammalian chromosome aberration test using human peripheral blood lymphocytes.

Executive summary:

SUMMARY

The test article, Thiophene, 3-(decyloxy)tetrahydro-,1,1-dioxide (CAS # 398141-87-2 or 18760-44-6), hereafter referred to as Thiophene, 3-(decyloxy)tetrahydro-,1,1-dioxide, was tested in the in vitro mammalian chromosome aberration test using human peripheral blood lymphocytes (HPBL) in both the absence and presence of an Aroclor-induced S9 activation system. A preliminary toxicity test was performed to establish the dose range for testing in the cytogenetic test. The chromosome aberration assay was used to evaluate the clastogenic potential of the test article.

Dimethyl sulfoxide (DMSO) was the solvent of choice based on the solubility of the test article and compatibility with the target cells. The test article was soluble in DMSO at a concentration of approximately 500 mg/mL, the maximum concentration tested for solubility. In the preliminary toxicity assay, the maximum dose tested was 2760 μg/mL (10 mM). Human peripheral blood lymphocytes were treated in the absence and presence of an Aroclor-induced S9 activation system for 4 hours, and continuously for 20 hours in the absence of S9 activation.

The test article was soluble in DMSO and in the treatment medium at all concentrations tested at the beginning of the treatment period. At the conclusion of the treatment period, visible precipitate was observed in treatment medium at dose levels ≥ 276 μg/mL and dose levels ≤ 82.8 μg/mL were soluble in treatment medium. Hemolysis was also observed at dose levels ≥ 276 μg/mL in all treatment groups.

Selection of dose levels for the chromosome aberration assay was based on a reduction in the mitotic index relative to the solvent control. Substantial toxicity (at least 50% reduction in mitotic index relative to the solvent control) was observed at doses ≥ 276 μg/mL in the non-activated and S9-activated 4-hour exposure groups, and at dose levels ≥ 82.8 μg/mL in the non-activated 20-hour exposure group. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 25 to 175 μg/mL for both the non-activated and the S9-activated 4-hour exposure groups, and from 6.25 to 75 μg/mL for the non-activated 20-hour exposure group.

In the chromosome aberration assay, the cells were treated for 4 and 20 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 20 hours after treatment initiation. The test article was soluble in DMSO and in the treatment medium at all concentrations tested at the beginning and conclusion of the treatment period. Slight hemolysis was observed at 175 μg/mL in the non-activated and the S9-activated 4-hour exposure groups. Selection of doses for microscopic analysis was based on mitotic inhibition (the lowest dose with at least 50% reduction in mitotic index, relative to the solvent control and two lower doses) in all harvests.

The percentage of cells with structural or numerical aberrations in the test article-treated groups was not significantly increased above that of the solvent control at any dose level (p>0.05, Fisher’s exact test).

Based on the findings of this study, Thiophene, 3-(decyloxy)tetrahydro-,1,1-dioxide(CAS # 398141-87-2 or 18760-44-6) was concluded to be negative for the induction of structural and numerical chromosome aberrations in the in vitro mammalian chromosome aberration test using human peripheral blood lymphocytes in both the non-activated and the S9-activated test systems.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Chromosome aberration

In a GLP compliant chromosome aberration study conducted in line with a standardised guideline, the clastogenic potential of the test substance was determined in an in vitro mammalian chromosome aberration test using human peripheral blood lymphocytes (HPBL) in both the absence and presence of an Aroclor-induced S9 activation system.

A preliminary toxicity test was performed to establish the dose range for testing in the cytogenetic test. HPBL were treated in the absence and presence of an S9 activation system for 4 hours, and continuously for 20 hours in the absence of S9 activation. Selection of dose levels for the chromosome aberration assay was based on a reduction in the mitotic index relative to the solvent control. Substantial toxicity (at least 50% reduction in mitotic index relative to the solvent control) was observed at doses ≥ 276 μg/mL in the non-activated and S9-activated 4 hour exposure groups, and at dose levels ≥ 82.8 μg/mL in the non-activated 20 hour exposure group. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 25 to 175 μg/mL for both the non-activated and the S9-activated 4 hour exposure groups, and from 6.25 to 75 μg/mL for the non-activated 20 hour exposure group.

In the chromosome aberration assay, the cells were treated for 4 and 20 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 20 hours after treatment initiation. Slight hemolysis was observed at 175 μg/mL in the non-activated and the S9-activated 4 hour exposure groups. Selection of doses for microscopic analysis was based on mitotic inhibition (the lowest dose with at least 50% reduction in mitotic index, relative to the solvent control and two lower doses) in all harvests. The percentage of cells with structural or numerical aberrations in the treated groups was not significantly increased above that of the solvent control at any dose level. Based on the findings of this study, the test substance was considered to be negative for the induction of structural and numerical chromosome aberrations in the in vitro mammalian chromosome aberration test using HPBL in both the non-activated and the S9 activated test systems.

Bacterial reverse mutation

S. typhimurium strains TA1535, TA1537, TA98 and TA100 and E. coli strain WP2uvrA were treated using the Ames plate incorporation method at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and ranged between 5 and 5000 µg/plate, depending on bacterial strain type. The experiment was repeated on a separate day using a similar dose range to the range-finding test, fresh cultures of the bacterial strains and fresh test item formulations.Additional dose levels and an expanded dose range were selected in both experiments in order to achieve four non-toxic dose levels and the toxic limit of the test item.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains in both experiments, initially from 1500 µg/plate in the range‑finding test and 150 µg/plate in the main test. For Salmonella strain TA1537, dosed in the absence of S9-mix, there was a slight discrepancy in toxic response with weakened lawns initially noted at 1500 µg/plate in the range-finding test and at 150 µg/plate in the main test. No weakening of the bacterial background lawns were noted for TA100 and Escherichia coli strain WP2uvrA (presence of S9-mix only). The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and Experiment number. The test item was tested up to either the maximum recommended dose level (5000 µg/plate) or the toxic limit, depending on bacterial strain type and presence or absence of S0-mix. A test item precipitate (oily and globular in appearance) was noted at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in either the range-finding or main tests.

The test item was considered to be non-mutagenic under the conditions of this test.

Mouse Lymphoma Assay

In a GLP compliant study conducted in line with standardised guidelines, the mutagenic potential of the test substance was determined in an in vitro mouse lymphoma assay both the absence and presence of metabolic activation. Two independent experiments were performed. In experiment 1, mouse lymphoma cells were treated with the test substance for four hours both in the presence and absence of metabolic activation (2% S9). In experiment 2, the cells were treated with the test substance for four hours in the presence of metabolic activation (1% S9) and for 24 hours in the absence of metabolic activation. The dose range of the test substance was selected following the results of a preliminary toxicity test, and was 5 - 40 µg/mL in the absence of metabolic activation and 10 - 80 µg/mL in the presence of metabolic activation (Experiment 1). In experiment 2 the dose range was 5 - 50 µg/mL in the absence of metabolic activation, and 2.5 - 80 µg/mL in the presence of metabolic activation.

The maximum dose levels used were limited by test substance-induced toxicity in the absence of metabolic activation. Precipitate of test substance was not observed at any of the dose levels. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

Under the conditions of the study, the test substance did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment. The test substance was therefore considered to be non-mutagenic to mouse lymphoma (L5178Y) cells.

Justification for selection of genetic toxicity endpoint
All studies are negative.

Short description of key information:
Chromosome aberration:
Negative; study performed in line with OECD Guideline 473; Gudi and Rao (2006)

Bacterial reverse mutation:
Negative; study performed in line with OECD Guideline 471; Thompson (2011)

Mouse Lymphoma Assay:
Negative; study performed in line with OECD Guideline 476, EU Method B17; Flanders (2012)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The substance does not require classification in accordance with the criteria specified in either Regulation 1272/2008 or Directive 67/546/EEC.