Butyl toluene-4-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:

02 August 2019 to 19 August 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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:

Butyl toluene-4-sulphonate

EC Number:

212-295-5

EC Name:

Butyl toluene-4-sulphonate

Cas Number:

778-28-9

Molecular formula:

C11H16O3S

IUPAC Name:

butyl 4-methylbenzene-1-sulfonate

Test material form:

liquid

Remarks:

(colourless to slightly yellow)

Details on test material:

- Appearance: Colourless to slightly yellow liquid
- Storage conditions: At room temperature protected from light

Specific details on test material used for the study:

- No correction was made for the purity/composition of the test material.
- A solubility test was performed based on visual assessment. The test material formed a clear (colourless) solution in dimethyl sulfoxide.
- To protect the test material from light, amber-coloured glassware or tubes wrapped in tinfoil were used for test material preparations.
- Test material concentrations were used within 2 hours after preparation.

Method

Target gene:

Salmonella typhimurium: histidine locus
Escherichia coli: tryptophan locus

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

The characteristics of the different Salmonella typhimurium strains were as follows: TA1537: mutation hisC3076 (frameshift); TA98: mutation his D3052/R-factor* (frameshift); TA1535: mutation hisG46 (base-pair substitutions); TA100: mutation hisG46/R-factor* (base-pair substitutions)
*R-factor = plasmid pKM101 (increases error-prone DNA repair)

Each tester strain contained the following additional mutations:
rfa: deep rough (defective lipopolysaccharide cellcoat)
gal: mutation in the galactose metabolism
chl: mutation in nitrate reductase
bio: defective biotin synthesis
uvrB: loss of the excision repair system (deletion of the ultraviolet-repair B gene)

The Salmonella typhimurium strains were checked at least every year to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants. The Escherichia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of mutagens has been enhanced by permeabilisation of the strain using Tris-EDTA treatment. The strain was checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants at least every year.
Stock cultures of the five strains were stored in the freezer (-150 °C).

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system
- Source of S9: Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).
Each S9 batch was characterized with the mutagens benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 and 2.5 μg/plate, respectively.
- Method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per 10 mL: 30 mg NADP and 15.2 mg glucose-6-phosphate in 5.5 mL Milli-Q water; 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution; 1 mL 0.33 M KCl solution. The solution was filter (0.22 μm)-sterilised. To 9.5 mL of S9-mix components 0.5 mL S9-fraction was added (5 % (v/v) S9-fraction) to complete the S9-mix.

Test concentrations with justification for top dose:

Dose-Range Finding Test: 0, 1.7, 5.4, 17, 52, 164, 512, 1600, 5000 µg/plate (TA100 and WP2uvrA - with and without metabolic activation)
Mutation Experiment: 0, 52, 164, 512, 1600, 2500, 5000 µg/plate (TA1535, TA1537, TA98 - with and without metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191 (without metabolic activation - TA1537; 2.5 μg/ plate). 2-aminoanthracene (with metabolic activation - TA1535 and TA1537: 2.5 μg/ plate; TA98 and TA100: 1 μg/plate; WP2uvrA: 15 µg/plate)

Details on test system and experimental conditions:

METHOD OF TREATMENT/ EXPOSURE: plate incorporation

At least five different doses (increasing with approximately half-log steps) of the test material were tested in triplicate in each strain. The dose-range finding study with two tester strains was reported as a part of the mutation assay. In the second part of the experiment, the test material was tested both in the absence and presence of S9-mix in the tester strains TA1535, TA1537 and TA98.
Top agar in top agar tubes was melted by heating to 45 ± 2 °C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test material in DMSO and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate.
After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test material precipitate to interfere with automated colony counting were counted manually. Evidence of test material precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

Rationale for test conditions:

Recommended test system in international guidelines.

Evaluation criteria:

ACCEPTABILITY CRITERIA
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following 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 Laboratory.
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.

INTERPRETATION OF RESULTS
A test material is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.
A test material is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three times the concurrent control.
b) In case a repeat 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.

Statistics:

No formal hypothesis testing was done.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

- Precipitate: Precipitation of the test material on the plates was not observed at the end of the incubation period in any tester strain
- Toxicity: To determine the toxicity of the test material, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction of the bacterial background lawn, was observed in all tester strains in the absence and presence of S9-mix, except in tester strain WP2uvrA, where no toxicity was observed at any of the dose levels tested.
- Mutagenicity: In tester strain TA100, the test material induced up to 1.8- and 1.9-fold dose-related increases in the number of revertant colonies in the absence and presence of S9-mix, respectively. The increases observed were not two-fold the concurrent solvent control but were above the laboratory historical control data range in the presence of S9-mix.
In tester strain WP2uvrA, the test material induced up to 2.4- and 2.9-fold dose-related increases in the number of revertant colonies in the absence and presence of S9-mix, respectively. The increases observed were more than two-fold the concurrent solvent control but were within the laboratory historical control data range.
In tester strain TA1535, the test material induced up to 4.1- and 10-fold dose-related increases in the number of revertant colonies in the absence and presence of S9-mix, respectively. The increases observed were more than three-fold the concurrent solvent control and were above the laboratory historical control data range.
No increases were observed in tester strains TA1537 and TA98.

Any other information on results incl. tables

Mean number of revertant colonies/3 replicate plates (± S.D.)

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	Solvent 1.7 5.4 17 52 164 512 1600 2500 5000	97 ± 12 104 ± 21 110 ± 4 92 ± 11 115 ± 9 104 ± 9 130 ± 12 174 ± 24 - 141 ± 18s	9 ± 8 - - - 12 ± 3 12 ± 7 24 ± 5 37 ± 7 28 ± 3 26 ± 5s	14 ± 3 19 ± 1 13 ± 3 15 ± 6 11 ± 1 15 ± 1 16 ± 5 24 ± 6 - 34 ± 4	14 ± 5 - - - 15 ± 6 11 ± 3 9 ± 3 10 ± 0 9 ± 2 4 ± 4s	8 ± 0 - - - 5 ± 5 6 ± 6 8 ± 3 5± 0 6 ± 1 1 ± 1s
+	Solvent 1.7 5.4 17 52 164 512 1600 2500 5000	96 ± 15 85 ± 15 87 ± 17 96 ± 17 86 ± 11 102 ± 9 157 ± 8 182 ± 28 - 60 ± 4m	10 ± 6 - - - 14 ± 4 34 ± 8 64 ± 20 80 ± 13 102 ± 20 63 ± 17m	13 ± 3 19 ± 5 17 ± 5 21 ± 7 17 ± 6 22 ± 2 28 ± 1 32 ± 6 - 38 ± 4	18 ± 6 - - - 14 ± 5 15 ± 4 11 ± 4 12 ± 1 9 ± 2 9 ± 4m	4 ± 1 - - - 6 ± 2 4 ± 3 5 ± 3 4 ± 1 4 ± 1 3 ± 1m
Positive Controls
-	Name	MMS	SA	4-NQO	NF	ICR-191
	Concentration (µg/plate)	650	5	10	10	2.5
	Mean no. colonies/plate	725 ± 33	969 ± 46	1060 ± 100	1056 ± 25	876 ± 101
+	Name	2AA	2AA	2AA	2AA	2AA
	Concentration (µg/plate)	1	2.5	15	1	2.5
	Mean no. colonies/plate	1598 ± 53	311 ± 3	423 ± 40	1396 ± 68	368 ± 38

m = Bacterial background lawn moderately reduced

s = Bacterial background lawn slightly reduced

MMS = methylmethanesulfonate

SA = Sodium azide

4-NQO = 4-nitroquinoline N-oxide

2NF = 2-Nitrofluorene

2AA = 2-aminoanthracene

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that the test material is mutagenic in the bacterial reverse mutation assay.

Executive summary:

The mutagenic potential of the test material was assessed in a study which was conducted in accordance with the standardised guidelines OECD 471 and EU Method B.13/14, and under GLP conditions.

The objective of the study was to determine the potential of the test material and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9). The test was performed in the direct plate assay with DMSO as the solvent.

In the dose-range finding study, the test material was initially tested up to concentrations of 5000 μg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test material did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn, was observed in tester strain TA100 at the dose level of 5000 μg/plate in the absence and presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested. Results of this dose-range finding test were reported as part of the first mutation assay.

In tester strain TA100, the test material induced up to 1.8- and 1.9-fold dose-related increases in the number of revertant colonies in the absence and presence of S9-mix, respectively. The increases observed were not two-fold the concurrent solvent control but were above the laboratory historical control data range in the presence of S9-mix.

In tester strain WP2uvrA, the test material induced up to 2.4- and 2.9-fold dose-related increases in the number of revertant colonies in the absence and presence of S9-mix, respectively. The increases observed were more than two-fold the concurrent solvent control but were within the laboratory historical control data range.

In the first mutation experiment, the test material was tested up to concentrations of 5000 μg/plate in the strains TA1535, TA1537 and TA98. The test material did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction of the bacterial background lawn, was observed in all three tester strains in the absence and presence of S9-mix.

In tester strain TA1535, the test material induced up to 4.1- and 10-fold dose-related increases in the number of revertant colonies in the absence and presence of S9-mix, respectively. The increases observed were more than three-fold the concurrent solvent control and were above the laboratory historical control data range.

No increases were observed in tester strains TA1537 and TA98.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

The test material induced a significant dose-related increase in the number of revertant (His+) colonies in tester strain TA1535. The increases were more than three-fold the concurrent solvent control, were above the laboratory historical control data range and observed in the absence and presence of S9-mix.

In tester strains TA100 and WP2uvrA, dose-related increases in the number of revertant colonies both in the absence and presence of S9-mix were observed. However the increases observed were either below the laboratory historical control data ranges or less than two-fold the concurrent solvent control.

In conclusion, based on the results of this study it is concluded that the test material is mutagenic in the bacterial reverse mutation assay.