4-poly(propyl), benzene sulfonic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

Sponsor's identification: AS305BD
Description: dark brown, viscous liquid
Lot number: TS07002
Analytical purity: 100%
Storage conditions: room temperature in the dark

Method

Target gene:

S. typhimurium: Histidine locus
E. coli: Tryptophan locus

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Obtained from: University of California at Berkeley
Stored at: -196 ºC in liquid nitrogen freezer

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Obtained from: British Industrial Biological Research Association
Stored at: -196 ºC in liquid nitrogen freezer

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbitone/β-naphthoflavone induced rat liver S-9 mix.

Test concentrations with justification for top dose:

Range-finding test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000µg/plate.
Definitive test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000µg/plate.

Vehicle / solvent:

Tetrahydrofuran (200 mg/mL): the test material was neither fully miscible nor adequately suspendable in sterile distilled water, dimethyl sulphoxide or acetone at 50 mg/mL. It was, however, miscible in tetrahydrofuran (at 200 mg/ml), which is an acceptable vehicle for use in this test system.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-ethyl-N'-nitro-N-nitrosguanidine (ENNG), 9-aminoacridine (9AA), 4-nitroquinoline-1-oxide (4NQO) (all in the absence of S9); 2-aminoanthracene (2AA), benzo(a)pyrene (BP) (presence of S9)

Details on test system and experimental conditions:

S9 MIX AND AGAR
The S9-mix was prepared immediately before use using sterilised co-factors and maintained on ice for the duration of the test.

S9 5.0 mL
1 -65 M KCl/0.4 M MgCl2 1.0 mL
0.1 M Glucose-6-phosphate 2.5 mL
0.1 M NADP 2.0 mL
0.2 M Sodium phosphate buffer (pH 7.4) 25.0 mL
Sterile distilled water 14.5 mL

A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment. Top agar was prepared using 0.6% Difco Bacto agar and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin solution (for the SaImoneIla strains) or 1.0 mM tryptophan solution (for E-coli strain WP2uvrA-) added to each 100 mL of top agar.

PRELIMINARY TOXICITY TEST
In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test material. The dose range of the test material was 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The test was performed by mixing 0.1 mL
of bacterial culture (TA100 or WP2uvrA-), 0.025 mL of test material formulation, 0.5 mL of phosphate buffer or S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar and overlaying onto sterile plates of VogeE-Bonner Minimal agar. Ten concentrations of the
test material (single dose) and a vehicle control (tetrahydrofuran) were tested. In addition, 0.025 mL of the maximum concentration of the test material and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material. The plates were incubated for a nominal 48 hours at 37°C after an initial overnight equilibration period and then assessed for revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

MUTATION STUDY - EXPERIMENT 1 (RANGE-FINDING TEST)
Six concentrations of the test material (15, 50, 150, 500, 1 500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method. Measured aliquots (0.1 mL) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 mL of molten trace histidine or typtophan supplemented, top agar, 0.025 mL of the test material formdation and vehicle or 0.1 mL of positive control and either 0.5 mL of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix. The plates were incubated for a nominal 48 hours at 37 ºC after an initial overnight equilibration
period and the frequency of revertant colonies assessed using a Domino colony counter.

MUTATION STUDY - EXPERIMENT 2 (MAIN TEST)
The second experiment was performed using methodology as described in Experiment 1 using fresh bacterial cultures, test material and control solutions. The test material dose range was the same as Experiment 1.

Evaluation criteria:

The test material may be considered positive in this test system if the following criteria are met:

The test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression) significant increase in the revertant count in at least one strain of bacteria. If a greater than two or threefold increase in revertant count (depending on tester strain type) is observed in two experiments then this is taken as evidence of a positive response.

Statistics:

Dunnett's method of linear regression (if applicable).

Results and discussion

Test results

Test results 1

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING TEST
The test material was non-toxic to the strains of bacteria used (TA100 and WP2uwA-). A precipitate (oily in appearance) was observed at and above
1500 ug/plate. The frequency of revertant colonies for the toxicity test are presented in Table 1 (attached). The test material formulation, the amino acid supplemented top agar and the S9-mix used in this experiment were shown to be sterile.

DEFINITIVE TEST
On the day of each experiment, the tester strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments were shown to be sterile.

Results for the negative controls (spontaneous mutation rates) are presented in Table 2 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the mutation test.

The individual plate counts, the mean number of revertant colonies and the standard deviations for the test material, vehicle and positive controls, both with and without metabolic activation, are presented in Table 3 to Table 6.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. An oily precipitate was observed, with the aid of a microscope, at 5000 µg/plate in the main experiment, this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains at my dose level, either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies and the activity of the S9 fraction was shown to be satisfactory.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

AS305BD was considered to be non-mutagenic under the conditions of this test.

Executive summary:

A bacterial reverse mutation test was conducted in line with OCED Guideline 471. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA 100 and Escherichia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at six dose levels, in triplicate, both with and without the addition of S9 metabolic activation system . The dose range was determined in a preliminary toxicity test and was 15 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as Experiment 1 , fresh cultures of the bacterial strains and fresh test material formulations.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

The test material was considered to be non-mutagenic under the conditions of this test.