2-ethoxyethyl 4-methylbenzene-1-sulfonate

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:

October 22, 2007 - December 21, 2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-ethoxyethyl 4-methylbenzene-1-sulfonate

EC Number:

666-751-7

Cas Number:

17178-11-9

Molecular formula:

C11H16O4S

IUPAC Name:

2-ethoxyethyl 4-methylbenzene-1-sulfonate

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: sponsor
- Lot: 45046-34

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in darkness.
- Solubility and stability of the test substance in the solvent/vehicle: Sodium phosphate buffer, 200mM, pH=7.4, was used as the vehicle to prepare the item concentrations. A stock concentration of 100 mg/ml was prepared in DMSO from which 1:5 dilutions were made.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

The top concentration of the test item was toxic for Salmonella typhimurium so, the following concentrations were tested: 20; 4; 0.8; 0.16; and 0.032 mg/ml

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: Solvent is compatible with the survival of the bacteria and the S9 activity.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

cumene hydroperoxide

other: 2-aminoantracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: Preincubation
Each point of the two series of tubes (with and without S9) was tested in duplicate and with the following composition: phosphate buffer (or S9 mixture), 2E9 cell/ml bacterial culture and the solvent (negative control), the test item (each of five concentrations) or the reference item (positive controls). The tubes were placed in a water bath at 37ºC for 45 minutes. Then 2 mL of surface agar supplemented with histidine/biotin 0.5 mM was added to each tube and poured out onto a minimum agar plate. The plates were left to set for 1hour and they were then placed in the incubator at 37ºC for 48-72 hours.

DURATION
- Preincubation period: 45 minutes
- Exposure duration:48 -72 hours

SELECTION AGENT (mutation assays): The lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize an essential amino acid.

NUMBER OF REPLICATIONS: 2.

DETERMINATION OF CYTOTOXICITY
-Method: Visual observation of the colonies.

OTHER EXAMINATIONS:
Phenotype and sterility controls were also performed.

- OTHER:
Solutions preparation: Sodium phosphate buffer, 200mM, pH=7.4, was used as the vehicle to prepare the item concentrations. In all cases, these concentrations were prepared on the day they were used. A stock concentration of 100mg/ml was prepared in DMSO from which 1:5 dilutions were carried out.

Test system: Prior to the start of the study, the master plates for each strain were prepared. The strains from frozen vials were plated out in minimum enriched agar plate with Biotin 0.5mM and Histidine 0.1 M. In the case of strains TA98 and TA100 the plates also contained ampicilyne 8 mg/ml and in the case of strain TA102 they contained tetracycline 8mg/ml, in addition to Histidine, Biotin and Ampicilyne. The plates were cultivated for 48 hours at 37ºC. From the grown up colonies the previous process was repeated.

Rationale for test conditions:

The top concentration of the test item, 100 mg/ml, was toxic for Salmonella typhimurium so, the following concentrations were tested: 20; 4; 0.8; 0.16 and 0.032 mg/ml.

Evaluation criteria:

Criteria conclusion: the result of the test is considered as positive if the test item induce an increase of colonies with respect to non-treated plates, dependent on the concentration of one, or several of the 5 strains, without and/or with metabolic activation.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No

Any other information on results incl. tables

The conditions listed below indicate that the tests are acceptable:

1. The plates show a firm, uniform lawn, which demonstrates that there is no toxicity in the concentrations that were taken as a reference to evaluate the mutagenic power.

2. The number of colonies in the spontaneous mutation plates is within the normal range for each strain.

3. The positive controls induce a clear increase in the number of revertants in all cases.

4. The phenotype control plates show the expected results for each strain.

From the results expressed on the tables below it can be deduced that the test item induces a dose-dependent increase in colonies in TA1535 in the presence of S9, therefore is mutagenic for the strain TA1535.

Calculation of the mutation index (MI)

MI = nº. of mut. in a dose / nº. of mut. in the control

Strain TA98
	-S9			+S9
	No. Col.	Average	MI	No. Col.	Average	MI
Sp. Mut.	17/19	18.0	--	16/12	14.0	--
0mg/ml	20/16	18.0	--	11/15	13.0	--
32mg/ml	17/19	18.0	1.000	12/20	16.0	1.231
160mg/ml	20/18	19.0	1.056	21/23	22.0	1.692
800mg/ml	14/16	15.0	0.833	24/22	23.0	1.769
4000mg/ml	17/14	15.5	0.861	20/16	18.0	1.385
20000mg/ml	15/13	14.0	0.778	15/14	14.5	1.115
Control +	>2000/>2000	>2000	>111.111	>2000/>2000	>2000	>153.846

 

Strain TA100
	-S9			+S9
	No. Col.	Average	MI	No. Col.	Average	MI
Sp. Mut.	182/173	177.5	--	180/192	186.0	--
0mg/ml	170/164	167.0	--	210/184	197.0	--
32mg/ml	172/163	167.5	1.003	216/212	214.0	1.086
160mg/ml	165/165	165.0	0.988	176/200	188.0	0.954
800mg/ml	172/175	173.5	1.039	150/210	180.0	0.914
4000mg/ml	173/182	177.5	1.063	185/198	191.5	0.972
20000mg/ml	164/173	168.5	1.009	210/200	205.0	1.041
Control +	>2000/>2000	>2000	>11.976	>2000/>2000	>2000	>10.152

 

Strain TA102
	-S9			+S9
	No. Col.	Average	MI	No. Col.	Average	MI
Sp. Mut.	327/321	324.0	--	425/429	427.0	--
0mg/ml	320/319	319.5	--	412/427	419.5	--
32mg/ml	317/322	319.5	1.000	440/420	430.0	1.025
160mg/ml	324/331	327.5	1.025	420/430	425.0	1.013
800mg/ml	326/332	329.0	1.030	420/438	429.0	1.023
4000mg/ml	324/319	321.5	1.006	412/430	421.0	1.004
20000mg/ml	330/323	326.5	1.022	440/420	430.0	1.025
Control +	>2000/>2000	>2000	>6.260	890/910	900.0	2.145

 

Strain TA1535
	-S9			+S9
	No. Col.	Average	MI	No. Col.	Average	MI
Sp. Mut.	6/8	7.0	--	14/18	16.0	--
0mg/ml	9/7	8.0	--	19/22	20.5	--
32mg/ml	5/5	5.0	0.625	13/24	18.5	0.902
160mg/ml	5/6	5.5	0.688	23/16	19.5	0.951
800mg/ml	9/7	8.0	1.000	15/25	20.0	0.976
4000mg/ml	8/6	7.0	0.875	48/45	46.5	2.268
20000mg/ml	8/7	7.5	0.938	150/182	166.0	8.098
Control +	>1500/>1500	>1500	>187.500	240/260	250.0	12.195

  

Strain TA1537
	-S9			+S9
	No. Col.	Average	MI	No. Col.	Average	MI
Sp. Mut.	8/9	8.5	--	8/10	9.0	--
0mg/ml	3/6	4.5	--	13/9	11.0	--
32mg/ml	5/3	4.0	0.889	12/9	10.5	0.955
160mg/ml	5/7	6.0	1.333	7/6	6.5	0.591
800mg/ml	¾	3.5	0.778	6/3	4.5	0.409
4000mg/ml	5/5	5.0	1.111	10/5	7.5	0.682
20000mg/ml	6/7	6.5	1.444	12/6	9.0	0.818
Control +	173/180	176.5	39.222	184/182	183.0	16.636

--: It was not possible to count colonies

 

Results of the phenotype control

	TA98	TA100	TA1535	TA1537	TA102
Ampicilyne	Resistant	Resistant	Sensitive	Sensitive	Resistant
Violet Crystal	Sensitive	Sensitive	Sensitive	Sensitive	Sensitive
UV light	Sensitive	Sensitive	Sensitive	Sensitive	Sensitive
Tetracycline	n.t	n.t	n.t	n.t	Resistant

n.t.: not tested

 

 

 

 

 

 

 

Applicant's summary and conclusion

Conclusions:

The test item induce a dose-dependent increase in Salmonella typhimurium strain TA1535 in the presence of S9. Therefore, it was considered as mutagenic under test conditions.

Executive summary:

A Bacterial reverse mutation test was performed according OECD guideline 471 with GLP. Based on a previous toxicity test, 1-2E9 cell/mL of Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA102 were exposed to  0.032, 0.16, 0.8, 4 and 20 mg/mL test item, solvent and positive controls with and without metabolic activation (two replicates each). The incubation mixtures were pre-incubated at 37 ºC for 45 minutes and incubated at 37 ºC for 48-72 hours. Then, the revertant colonies were counted. Phenotype and sterility controls were also performed. The plates showed a firm, uniform lawn, which demonstrates that there was no toxicity. The number of colonies in the spontaneous mutation plates was within the normal range for each strain. The positive controls induced a clear increase in the number of revertants in all cases and the phenotype control plates show the expected results for each strain. The test item induce a dose-dependent increase in TA1535 in the presence of S9. Therefore, the test item was determined to be mutagenic under test conditions.