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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Test guideline 4

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

800-172-4

Cas Number:

398141-87-2

Molecular formula:

Complex UVCB substance

IUPAC Name:

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

Test material form:

liquid

Details on test material:

Description: Light orange coloured liquid
Purity: 95.0%
Date received: 25 August 2011
Expiry date: 25 August 2012
Storage conditions: Room temperature in the dark

Method

Target gene:

Histidine for Salmonella.
Tryptophan for E.Coli

Species / strain

Species / strain 1

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 2

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.

Controls

Controls 1

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

Controls 2

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

Controls 3

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

Controls 4

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

Controls 5

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

Controls 6

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

Controls 7

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

Controls 8

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

Controls 9

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

Controls 10

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

Results and discussion

Test results

Test results 1

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

Test results 2

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'.

Any other information on results incl. tables

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

       

Applicant's summary and conclusion

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.