2-ethoxyethyl 4-methylbenzene-1-sulfonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In-vitro gene mutation in bacteria: Key study: OECD Guideline 471. GLP study. The test item is able of inducing mutations in the Salmonella typhimurium genome. Therefore, it was considered as mutagenic under test conditions.

In-vitro cytogenicity in mammalian cells (chromosome aberration): Key study: The substance is predicted to be mutagenic in mammalian cells.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

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

Target gene:

Histidine

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.

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.

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

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

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

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

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

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

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

 

 

 

 

 

 

 

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

(Q)SAR

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
OASIS-TIMES 2.27.19

2. MODEL (incl. version number)
In vitro Chromosomal Aberrations v.12.12

3. SMILES:
CCOCCOS(=O)(=O)c1ccc(C)cc1

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
The QMRF is available in "Attached justification"

5. APPLICABILITY DOMAIN
The QPRF is available in "Attached justification"

6. ADEQUACY OF THE RESULT:
The QPRF is available in "Attached justification"

Qualifier:

according to guideline

Guideline:

other: REACH Guidance on QSAR R.6

Principles of method if other than guideline:

- Software tool(s) used including version: OASIS TIMES 2.27.19
- Model(s) used: In vitro Chromosomal Aberrations v.12.12
- Model description: see field 'Attached justification'
- Justification of QSAR prediction: see field 'Attached justification'

GLP compliance:

no

Type of assay:

other: In-vitro chromosomal aberrations in mammalian cells

Key result

Additional information on results:

The substance is predicted to be positive for in-vitro chromosomal aberration.

Remarks on result:

mutagenic potential (based on QSAR/QSPR prediction)

In vitro Chromosomal aberration. Application of TIMES in vitro CA model:

The target chemical is predicted in vitro positive as parents by TIMES Chromosomal aberrations model. It belongs 100% in model applicability domain. The DNA binding alert causing genotoxicity is Sulfonates and Sulfates.

 

Compounds, containing Sulfonates and Sulfates structural alert are considered to be alkylating agents (direct-acting mutagens), requiring no prior metabolism. Some of the structural preconditions in the Sulfonates and Sulfates structural alert (which are met in the target structures) are the presence of sulphonate group attached to an aromatic ring and carbon sp3 atoms followed by oxygen.

 

The reliability of Sulfonates and Sulfates alerting group is defined as Undetermined because the local training set of the model consists of six chemicals only. The threshold with respect to the size of the local training set is 10 chemicals. However, the alert performance is 100% - all six chemicals in the local training set are predicted correctly as CA positive. Hence, no matter of the undetermined reliability, all chemical from the local training set are causing CA effect

 

Experimental genotoxicity data of the targets and the analogues:

The substance have been reported positive in the bacterial mutagenicity (Ames) test. No experimental data has been reported for the in vitro CA (OECD TG 473) and in vivo bone marrow micronucleus (OECD TG 474) tests. Due to the lack of available experimental data for in vitro CA and in vivo Micronucleus of the target chemicals, structurally similar chemicals have been selected from public databases and web sources in order to collect weight of evidence with respect to the modeling results. The criteria of structural similarity are based on the presence of Tosylate or Mesylate ester functionalities which have been considered as critical for eliciting hazardous effects.

- All four analogues have positive experimental data at least in one in vitro test (in vitro Chromosomal aberration (OECD 473), in vitro Bacterial Reverse Mutation assay (OECD 471) or in vitro mammalian cell micronucleus test (OECD TG 487).

- The analogues have DNA alkylating capability and are direct-acting bacterial mutagens (positive in vitro Ames mutagenicity test results).

- The analogue chemicals are considered to elicit positive Chromosomal aberration given the available positive data for more reliable in vitro micronucleus test in mammalian cells (OECD TG 487) which is capable of detecting both clastogens and aneugens.

 

Mechanistic interpretation of the experimental data and the modelling results:

The target chemical belong to Tosylate esters chemical class and have, approximately, the same reactivity as the corresponding alkyl bromides in the nucleophilic substitution (SN2) reactions. The reason for this similarity is that sulfonate anions, like bromide anions, are good leaving groups, since they are weak bases.Tosylate estersare capable of alkylating organic bases. This could be also applied to nucleophilic nitrogen-containing fragments of biological macromolecules such as DNA and proteins.

 

For further details, please refer to the attached report.

Conclusions:

The substance is predicted to be positive for in-vitro chromosomal aberration

Executive summary:

The in-vitro chromosomal aberration potential of the test item was predicted using the TIMES model (Model version: In vitro Chromosomal Aberrations v.12.12, Platform version: OASIS TIMES 2.27.19), the available experimental data for the targets and the structural analogues and mechanistic interpretation of experimental data and modeling results. The DNA alkylating capability, the positive results in the Ames test and the reported positive in vitro micronucleus/chromosomal aberration test results for the analogues suggest positive in vitro chromosomal aberration (OECD 473). The chemical can be regarded as in vitro genotoxic.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In-vivo genetic toxicity (micronucleus): Key study: The substance is predicted to be non-genotoxic in mammalian cells.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

(Q)SAR

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
OASIS TIMES 2.27.19

2. MODEL
In vivo Micronucleus formation v.08.08

3. SMILES:
CCOCCOS(=O)(=O)c1ccc(C)cc1

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
The QMRF is available in "Attached justification"

5. APPLICABILITY DOMAIN
The QPRF is available in "Attached justification"

6. ADEQUACY OF THE RESULT
The QMRF is available in "Attached justification"

Qualifier:

according to guideline

Guideline:

other: REACH Guidance on QSAR R.6

Principles of method if other than guideline:

- Software tool(s) used including version:
OASIST TIMES 2.27.19

- Model(s) used:
In vivo Micronucleus formation v.08.08

- Model description: see field 'Attached justification'

- Justification of QSAR prediction: see field 'Attached justification'

GLP compliance:

no

Type of assay:

mammalian germ cell cytogenetic assay

Key result

Remarks on result:

other: Non-mutagenic (based on QSAR/QSPR prediction)

Additional information on results:

The substance is predicted to be negative for in-vivo micronucleus.

In vivo Micronucleus. Application of TIMES in vivo Micronucleus model:

TIMES prediction for in vivo Micronucleus model was negative, belonging to model domain in 82%.

 

Experimental genotoxicity data of the targets and the analogues:

No experimental data has been reported for the in vitro CA (OECD TG 473) and in vivo bone marrow micronucleus (OECD TG 474) tests.

 

Mechanistic interpretation of the experimental data and the modelling results:

The target chemical belong to Tosylate esters chemical class and have, approximately, the same reactivity as the corresponding alkyl bromides in the nucleophilic substitution (SN2) reactions. The reason for this similarity is that sulfonate anions, like bromide anions, are good leaving groups, since they are weak bases.Tosylate estersare capable of alkylating organic bases. This could be also applied to nucleophilic nitrogen-containing fragments of biological macromolecules such as DNA and proteins.

 

Hydrolysis of sulfonate esters vs. induction of micronuclei based on experimental data:

Some experimental data have shown that highly hydrophobic sulfonate esters exhibit SN1-type hydrolysis rate in

aqueous medium. The more hydrophobic sulfonateesters with very low hydrolysis rates may act more efficiently as genotoxins by reaching the target biopolymer as intact chemicals.It has been reported that intestinal flora can hydrolyse sulfonate esters. Unique phosphonate monoester hydrolase (PMH) was characterized as sulfonate ester hydrolytic enzyme (sulfonate monoesterase), which belongs to the alkaline phosphatase superfamily. It was shown that alkaline phosphatase enzymes are predominantly located in the intestinal microflora, however, they are also present in kidney and liver tissues.

Read-across analysis for predicting genotoxicity of hydrolysis product: Based on Toolbox read-across prediction it could be concluded that the hydrolysis product, CAS 110-80-5 is predicted negative with respect to in vivo Micronucleus in bone marrow.

 

For further details, please refer to the attached report.

Conclusions:

The substance is predicted to be negative for in-vivo micronucleus test.

Executive summary:

The in-vivo genetic toxicity (micronucleus test) of the test item was predicted using the TIMES model (Model version: In vivo Micronucleus formation v.08.08, Platform version: OASIS TIMES 2.27.19), the available experimental data for the targets and structural analogues, the mechanistic interpretation of experimental data and the modeling results and Toolbox application for assessing hydrolysis products. The substance is assumed to be non-genotoxic in vivo, i.e., negative in the in vivo bone marrow micronucleus test (OECD 474) since:

- In vivo enzymatic hydrolysis to the non-genotoxic p-toluenesulfonic acid might occur to a significant extent, mainly, in the intestinal microflora. p-Toluenesulfonic acid has in vitro negative data and is rapidly excreted.

- The in vivo hydrolytic metabolic detoxification of test item is more likely given the relatively compact and short-chain linear ester structure. This prevents the genotoxic effects in the bone marrow tissue.

- The ester product of the enzymatic hydrolysis is proved to be non-genotoxic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In-vitro mutagenicity in bacteria: Key study: A Bacterial reverse mutation test was performed according OECD guideline 471 with GLP. Based on previous results, 1-2E9 cell/ml of Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA102 were exposed up to 20 mg/ml test item with and without metabolic activation. The test item induced a dose-dependent increase in TA1535 in the presence of S9, so it was classified as positive in the Ames test.

In-vitro cytogenicity in mammalian cells (chromosome aberration): Key study: The in-vitro chromosomal aberration potency of the test item was predicted using the TIMES models (Model version: In vitro Chromosomal Aberrations v.12.12, Platform version: OASIS TIMES 2.27.19), the available experimental data for the targets and structural analogues, the mechanistic interpretation of experimental data and modeling results and the Toolbox application for assessing hydrolysis products. The DNA alkylating capability, the positive results in the Ames test and the reported positive in vitro micronucleus/chromosomal aberration test results for the analogues suggest positive in vitro chromosomal aberration (OECD 473) and in vitro micronucleus (OECD 487) results in mammalian eukaryotic cells. The chemical can be regarded as in vitro genotoxic.

In-vivo genetic toxicity (micronucleus): Key study: The in-vivo genetic toxicity (micronucleus test) of the test item was predicted using the TIMES models (Model version: In vivo Micronucleus formation v.08.08, Platform version: OASIS TIMES 2.27.19), the available experimental data for the targets and structural analogues, the mechanistic interpretation of experimental data and modeling results and the Toolbox application for assessing hydrolysis products. The substance is assumed to be non-genotoxic in vivo, i.e., negative in the in vivo bone marrow micronucleus test (OECD 474) since:

- In vivo enzymatic hydrolysis to the non-genotoxic p-toluenesulfonic acid might occur to a significant extent, mainly, in the intestinal microflora. p-Toluenesulfonic acid has in vitro negative data and is rapidly excreted.

- The in vivo hydrolytic metabolic detoxification of test item is more likely given the relatively compact and short-chain linear ester structure. This prevents the genotoxic effects in the bone marrow tissue.

- The ester product of the enzymatic hydrolysis is proved to be non-genotoxic.

Justification for classification or non-classification

Based on the available information, i.e. a positive Ames test and a positive in-vitro chromosome aberration prediction, but a negative in-vivo micronucleus prediction, the substance is determined not to be classified for genotoxicity according to CLP Regulation (EC) no. 1272/2008.