Sodium 4-vinylbenzenesulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

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:

28 July to 7 August 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring S. tryphimurium mutants TA 1535, TA 1537, TA 98 and TA 100 used in the test were provided by Dr. B.N.Ames, University of California Berkeley, California, U.S.A.

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S-9 used in this test was part of a batch prepared. This batch was prepared from the livers of rodents treated with Arclor 1254 in soya bean oil.

Test concentrations with justification for top dose:

62, 185, 556, 1667, 5000 microgram/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: test substance dissolves freely in the water.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period:
- Exposure duration:3 days
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

Evaluation criteria:

A concentration-related increase over the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plaste in at least one strain with or without metabolic activation systems.

Key result

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Under the condictions of the Ames test conducted on S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 no mutagenic activity was observed.

Executive summary:

Mutagenicity potential of NaSS was assessed with Salmonella typhimurium TA100, TA1535, TA98 and TA1537 according to the OECD 471 test guideline compliant with GLP.

The substance did not induce an increase in the number of revertant colonies more than twice in comparison with that of the negative control nor was a dose-related response observed in any strains of base-pair substitution type or frame-shift type, with or without metabolicactivation. 

Under the conditions of the test the substance is not mutagenic.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 October to 10 November 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Principles of method if other than guideline:

Yakushokuhatsu No.1121003, Heisei 15.11.17Seikyoku No. 3 and Kanhokihatsu No. 03112100)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The cells were supplied by Health Science Research Resources Bank on November 2nd, 2004 and cells with small number of subcultivations were stored in liquid nitrogen. Melted and subcultured cells were used for tests. The numbers of subcultivations for cells were 25 for cell growth inhibition test, 5 for short-term treatment of chromosomal aberration test and 9 for continuous treatment of chromosomal aberration test.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

1600, 800, 400, 200, 100, 50.0, 25.0, and 12.5 μg/mL

Vehicle / solvent:

Saline

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Cell line
Chinese hamster lung fibroblasts (CHL/IH cells) were used. The cells were supplied by Health Science Research Resources Bank on November 2nd, 2004 and cells with small number of subcultivations were stored in liquid nitrogen. Melted and subcultured cells were used for tests. The numbers of subcultivations for cells were 25 for cell growth inhibition test, 5 for short-term treatment of chromosomal aberration test and 9 for continuous treatment of chromosomal aberration test.

Incubation conditions
Cells were cultured in a carbon dioxide incubator with 5% CO2 and at 37oC under high humid condition. Subcultivations were carried out every day to every 4-days.

Characterization of the cell
Cells used for test were thawed from frozen preservation. Cells which had less than 30 number of subcultivations were evaluated at the regular intervals for their modal chromosome number, doubling time and contamination of mycoplasma and confirmed their applicability.

S9
Name : S9
Lot No. : 06051202 and 06070704
Production date : May 12, 2006 (Lot No. 06051202)
September 7, 2006 (Lot No. 06070704)
Type and species : SD rats
Sex : Male
Age in weeks : 7 Weeks
Inducing substances: Phenobarbital (PB) and 5, 6-benzoflavon (BF)
Method of administration: Intraperitoneal
Administration period and administrated dose:
PB 4 days 30+60+60+60 mg/kg body weight
BF 1 day 80 mg/kg body weight
Storage method : Frozen ( ultracold freezer)
Expiration date : November 11, 2006 (6 months after production, Lot No. : 06051202)
January 6, 2007
(6 months after production, Lot No. : 06070704)
Storage place : Gotenba Laboratory Mutagenic room ultracold freezer

Test method
Tests were conducted with following stage sequences.
1. Cell growth inhibition test
Short term treatment: With metabolic activation and Without metabolic activation
Continuous treatment: 24 hours treatment and 48 hours treatment

2. Chromosomal aberration test
Short term treatment: With metabolic activation and Without metabolic activation
Continuous treatment: 24 hours treatment and 48 hours treatment

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

As the results of chromosomal aberration test, test substance did not induce an increase of the frequencies of the cells with structural aberration and polyploid in the short-term treatments both without and with metabolic activation. Furthermore, in the 24 hours and 48 hours treatments the increase of the cells with structural aberration and polyploid were not observed.

In each treatment method in the negative control, the frequencies of the cells and polyploid were within the negative judgment criteria and the same as the background data in the laboratory.

In each treatment method in the positive control, the frequencies of the cells with structural aberration and polyploid were beyond positive judgment criteria and the same chromosomal aberration induction as the background data in laboratory.

In addition to the above, the frequencies of cells with chromosomal aberrations did not fluctuate markedly between two culture dishes. These indicated that the present study was appropriately performed.

Conclusions:

Interpretation of results (migrated information):
negative

The substance did not induce structural aberration nor numerical aberration

Executive summary:

The study was performed to determine the clastogenecity of the test substance in cultured chinese hamster lung cell line (CHL).

In the main and confirmation test, the highest concentration was determined at 1600 μg/mL (about 10 mM) in either the absence (S9-) or presence (S9+) of metabolic activation system, because no growth inhibition more than 50 % was observed in the concentration range-finding test.

Therefore, the test substance was determined not to induce chromosome aberration in the CHL cell under the present experimental condition.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Experimental data reported in report subject to review by regulatory authority

Principles of method if other than guideline:

The experimental protocol is presented in detail by Myhr et al. (1985), Assays for the induction of gene mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells in culture. In Progress in Mutation Research: Evaluation of Short-term Tests for Carcinogens; Report of the International Programme on Chemical Safety’s Collaborative Study on In vitro Assays, Vol. 5, pp. 555-568. Elsevier Science Publishers, Amsterdam.

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Not specified

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

250, 500, 1000, 2000, 3000, 4000, 5000 μg/mL

Vehicle / solvent:

Supplemented Fischer’s Medium

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Details on test system and experimental conditions:

The experimental protocol is presented in detail by Myhr et al.(1985). Sodium xylenesulfonate was supplied as a coded aliquot by Radian Corporation. The high dose of sodium xylenesulfonate was determined by toxicity. L5178Y mouse lymphoma cells were maintained at 37 °C as suspension cultures in Fischer’s medium supplemented with l-glutamine,sodium pyruvate, pluronic F68, antibiotics and heat­inactivated horse serum; normal cycling time was approximately 10 hours. To reduce the number of spontaneously occurring trifluorothymidine-resistant cells, subcultures were exposed to medium containing THMG (thymidine, hypoxanthine, methotrexate and glycine) for 1 day to medium containing THG (thymidine, hypoxanthine and glycine) for 1 day and to normal medium for 3 to 5 days. For cloning, the horse serum content was increased and Noble agar was added.
All treatment levels within an experiment, including concurrent positive and solvent controls, were replicated. Treated cultures contained 6E+6 cells in 10 mL medium. This volume included the S9 fraction in those experiments performed with metabolic activation. Incubation with sodium xylenesulfonate continued for 4 hours at which time the medium plus sodium xylenesulfonate was removed and the cells were resuspended in fresh medium and incubated for an additional 2 days to express the mutant phenotype. Cell density was monitored so that log phase growth was maintained. After the 48 -hour expression period, cells were plated in medium and soft agar supplemented with trifluorothymidine(TFT) for selection of TFT­resistant (TK-/-) cells; cells were plated in nonselective medium and soft agar to determine cloning efficiency. Plates were incubated at 37 °C in 5% CO2 for 10 to 12 days. The test was initially performed without S9. If a clearly positive response was not obtained, the test was repeated using freshly prepared S9 from the livers of Aroclor 1254-induced male Fischer 344/N rats.

Rationale for test conditions:

The experimental protocol is presented in detail by Myhr et al.(1985).

Evaluation criteria:

Minimum criteria for accepting an experiment as valid and a detailed description of the statistical analysis and data evaluation are presented in Casparyet al.(1988). All data were evaluated statistically for trend and peak responses. Both responses had to be significant (P≤0.05) for sodium xylenesulfonate to be considered positive, i.e. capable of inducing TFT resistance. A single significant response led to a “questionable” conclusion, and the absence of both a trend and peak response resulted in a “negative” call.

Statistics:

All data were evaluated statistically for trend and peak responses.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥3000 μg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

Interpretation of results (migrated information):
negative

Sodium xylenesulfonate displays no gene mutation.

Executive summary:

The gene mutation assay of Sodium xylenesulfonate has been assessed using L5178Y mouse lymphoma cells with concurrent positive and solvent controls, were replicated.  Treated cultures contained 6×10⁶cells in 10mL medium with and without S9 metabolic activation. The positive controls gave appropriate responses. Under the conditions of the test, Sodium xylenesulfonateis considered to display no gene mutation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Bacterial Reverse Mutation assay:

In a reverse gene mutation assay in bacteria, performed according to the OECD No. 471 and EC B14 guidelines, sodium p-vinylbenzenesulphonate(81%) diluted in milli-Q water at concentration of 0.62, 1.85, 5.56, 16.67 and 50 mg/ml was tested in S. tryphimurium TA 1535, TA 1537, TA 98 and TA 100 in the presence and absence of mammalian metabolic activation (S9) using the direct incorporation or the preincubation method. The positive controls induced the appropriate responses in the corresponding strains.

No induced revertants over background was observed in any strains of S. triphimirium up to the highest concentration of sodium p-vinylbenzenesulphonate.

Under the test conditions, sodium p-vinylbenzenesulphonate did not show any mutagenic activity in the bacterial reverse mutation test using Salmonella typhimurium. This study is considered as acceptable as it satisfied the criteria of the OECD Guideline No. 471.

Mammalian chromosome aberration assay:

The study was performed to determine the clastogenecity of the test substance in cultured chinese hamster lung cell line (CHL).

In the main and confirmation test, the highest concentration was determined at 1600 μg/mL (about 10 mM) in either the absence (S9-) or presence (S9+) of metabolic activation system, because no growth inhibition more than 50 % was observed in the concentration range-finding test.

Therefore, the test substance was determined not to induce chromosome aberration in the CHL cell under the present experimental condition.

Gene mutation assay:

The gene mutation assay of Sodium xylenesulfonate has been assessed using L5178Y mouse lymphoma cells with concurrent positive and solvent controls, were replicated.  Treated cultures contained 6×10⁶cells in 10mL medium with and without S9 metabolic activation. The positive controls gave appropriate responses. Under the conditions of the test, Sodium xylenesulfonateis considered to display no gene mutation.

Short description of key information:

- Bacterial Reverse Mutation assay (Ames test): not mutagenic (K, Reliablity 1)

- In vitro Mammalian chromosome aberration test: not cytogenic (K, Reliability 1)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Harmonized classification:

No harmonized classification is available according to the Regulation (EC) No 1272/2008 including ATP1.

Self classification

Sodium p-vinylbenzenesulphonate is not self-classified for genotoxicity according to the Regulation (EC) No 1272/2008 (CLP).