2-(2-thienyl)ethyl toluene-p-sulphonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 November 2017 - 22 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-(2-thienyl)ethyl toluene-p-sulphonate

EC Number:

254-911-5

EC Name:

2-(2-thienyl)ethyl toluene-p-sulphonate

Cas Number:

40412-06-4

Molecular formula:

C13H14O3S2

IUPAC Name:

2-(thiophen-2-yl)ethyl 4-methylbenzene-1-sulfonate

Test material form:

solid: particulate/powder

Details on test material:

- Physical appearance: white to off-white powder
- Test item storage: at room temperature

Method

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by Aroclor 1254 (500 mg/kg body weight)

Test concentrations with justification for top dose:

Dose-range finding (TA100 and WP2 uvrA, with and without S9): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate (reported as part of the mutation experiment)
Mutation experiment (TA1535, TA1537 and TA98, with and without S9): 17, 52, 164, 512, 1600 and 5000 μg/plate
- Test item concentrations were used within 2.5 hours after preparation.

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent: the test item dissolved in DMSO, which is a solvent recommended by international guidelines.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191; 2-aminoanthracene

Remarks:

For more details on positive controls see 'any other information on materials and methods'

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: no
- Exposure duration: 48 ± 4 h (in the dark at 37.0 ± 1.0 °C)

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain.

METHODS OF SLIDE PREPARATION: Top agar in top agar tubes was melted by heating to 45 ± 2°C. Fresh bacterial culture, dilution of the test item in DMSO and either S9-mix or 0.1 M phosphate buffer were added to the molten top agar. The ingredients were mixed on a vortex and the content of the top agar tube was poured onto a selective agar plate.

DETERMINATION OF CYTOTOXICITY
- Method: the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

DECISION CRITERIA:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

In addition to the criteria stated above, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

ACCEPTABILITY CRITERIA:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at the test facility.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

up to a 74-fold and 249-fold increase at 1600 µg/plate in the absence and presence of S9, respectively.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

up to 13-fold increase in the absence of S9-mix at 512 µg/plate and up to 14-fold in the presence of S9-mix at 1600 µg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Conclusion based on a single experiment.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 512 µg/plate in the absence of S9-mix and at 5000 µg/plate in the presence of S9-mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Conclusion based on a single experiment.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Conclusion based on a single experiment.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In compliance with the OECD guideline No 471, there is no requirement for verification of a clear positive response. Since the test results of the mutation experiment showed clear positive responses, the follow-up experiment was not performed.

DOSE-RANGE FINDING:
- Precipitation: at concentrations of 512 µg/plate and upwards at the start of the incubation period and at concentrations of 1600 µg/plate and upwards (TA100) and 5000 µg/plate (WP2 uvrA) at the end of the incubation period.
- Cytotoxicity: no decrease in the number of revertants was observed in any of the tester strains, with and without S9.
- Mutagenicity: In tester strain TA100, the test item induced up to 13-fold (absence of S9-mix, at 512 µg/plate) and 14-fold (presence of S9-mix, at 1600 µg/plate) increases in the number of revertant colonies compared to the solvent control. The increase in number of revertant colonies was dose-dependent.

MUTATION EXPERIMENT:
- Precipitation: at concentrations of 1600 µg/plate and 5000 µg/plate at the start of the incubation period and at the concentrations of 5000 µg/plate at the end of the incubation period, in all tester strains with and without S9.
- Cytotoxicity: in tester strain TA98 at and above a concentration of 512 µg/plate in the absence of S9-mix and at a concentration of 5000 µg/plate in the presence of S9-mix. No cytotoxicity was observed in the other tester strains, with and without S9.
- Mutagenicitiy: In tester strain TA1535, the test item induced up to 74-fold and 249-fold increases in the number of revertant colonies compared to the solvent control in the absence and presence of S9-mix, respectively. The increase in number of revertant colonies was dose-dependent.

Remarks on result:

other: Based on a clear positive result in a single experiment, further testing was omitted.

Any other information on results incl. tables

Table 2 Historical data on solvent controls

	TA1535		TA1537		TA98		TA100		WP2uvrA
S9-mix	-	+	-	+	-	+	-	+	-	+
Range	3 – 29	3 - 27	3 – 20	3 – 23	8 - 41	8 - 55	63 – 176	54 - 160	10 – 59	9 - 69
Mean	10	11	6	7	16	23	108	107	25	32
SD	3	4	2	3	5	7	19	20	7	8
n	2356	2336	2264	2235	2319	2360	2341	2336	2075	2078

Table 3 Historical data on positive controls

	TA1535		TA1537		TA98
S9-mix	-	+	-	+	-	+
Range	125 – 1248	73 – 1206	55 – 1353	54 – 1051	365 – 1995	250 – 1977
Mean	846	219	787	353	1406	887
SD	146	119	345	162	258	349
n	2348	2229	2003	2234	2200	2276

	TA100		WP2uvrA
S9-mix	-	+	-	+
Range	439 – 1848	408 - 2651	93 – 1951	111 - 1359
Mean	901	1232	1094	437
SD	168	343	477	149
n	2335	2327	2021	2085

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between Oct 2015 and Oct 2017. 

Applicant's summary and conclusion

Conclusions:

In an AMES test, performed according to OECD guideline 471 and GLP principles, 2-(2-thienyl)ethyl 4-toluenesulfonate (TPT) was found to be mutagenic in the tester strains TA100 and TA1535 in the Salmonella typhimurium reverse mutation assay, with and without metabolic activation.

Executive summary:

The mutagenic potential of 2-(2-thienyl)ethyl 4-toluenesulfonate (TPT) and/or its metabolites was assessed in an Ames test, performed according to OECD guideline 471 and GLP principles. The test item was tested in several strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and in one strain of Escherichia coli (WP2uvrA) in the absence and in the presence of a metabolic activation system (S9-mix). Concentrations up to and including 5000 µg/plate were used in a dose-range finding test (tester strains TA100 and WP2uvrA) and a mutation assay (tester strains TA1535, TA1537 and TA98), in absence and presence of S9 -mix.

The test item precipitated on the plates at the top dose of 5000 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants, was only observed in tester strain TA98 in the absence of S9 -mix. In tester strain TA100, the test item induced up to 13-fold and 14-fold increases in the number of revertant colonies compared to the solvent control in the absence and presence of S9-mix, respectively. In tester strain TA1535, the test item induced up to 74-fold and 249-fold increases in the number of revertant colonies compared to the solvent control in the absence and presence of S9-mix, respectively. For both strains the observed increase in the number of revertant colonies compared to the relevant solvent controls was dose-dependent. Since the test results of the mutation experiment showed clear positive responses, a follow-up experiment was not performed. The results of the solvent control and the positive controls were within the historical range of the test facility.

In conclusion, 2-(2-thienyl)ethyl 4-toluenesulfonate (TPT) is mutagenic in the tester strains TA100 and TA1535 in the Salmonella typhimurium reverse mutation assay, with and without metabolic activation.