4-tert-Butoxystyrene

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:

2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 21, 1997

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1-(tert-butoxy)-4-ethenylbenzene

EC Number:

619-127-3

Cas Number:

95418-58-9

Molecular formula:

C12H16O

IUPAC Name:

1-(tert-butoxy)-4-ethenylbenzene

Test material form:

liquid

Details on test material:

Clear and colourless

Method

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain 2

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: rat liver microsomal enzymes were routinely prepared from adult male Wistar rats, which were obtained from Charles River, Sulzfeld, Germany.
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept on ice. S9-mix components contained per 10 ml: 30 mg NADP and 15.2 mg glucose-6-phosphate in 5.5 ml or 5.0 ml Milli-Q water (first or second experiment respectively); 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.
- concentration or volume of S9 mix and S9 in the final culture medium:
Volume of S9-mix: 10 ml.
Concentration of S9-mix in the first experiment: 5% (v/v).
Concentration of S9-mix in the second experiment: 10% (v/v).

Test concentrations with justification for top dose:

First experiment: 10, 33, 100, 333 and 1000 µg/plate.
Second experiment:
TA1535, TA1537, TA98, TA100: 10, 33, 100, 333 and 1000 µg/plate.
WP2uvrA: 33, 100, 333, 1000 and 3330 µg/plate.
The highest concentration used was the level at which the test substance inhibited bacterial growth or the dose level at which the test substance exhibited limited solubility.

Vehicle / solvent:

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

Controls

Controls 1

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation; all strains

Controls 2

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without metabolic activation; WP2uvrA strain

Controls 3

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation; TA100 strain

Controls 4

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without metabolic activation; TA98

Controls 5

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation; TA1537 strain

Controls 6

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation; TA1535 strain

Controls 7

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate in each strain
- Number of independent experiments: two, both in absence and presence of S9-metabolic activation in each strain.

METHOD OF TREATMENT/ EXPOSURE:
- Direct plate assay: 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 substance in DMSO and either 0.5 ml S9-mix or 0.5 ml 0.1 M phosphate buffer were added to 3 ml molten top agar and poured onto a selective agar plate.

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: after solidification of the top agar, the plates were inverted and incubated in the dark at 37.0°C for 48 h.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined in the dose range finding test.

Rationale for test conditions:

Selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and WP2uvrA, both with and without S9-mix. Eight concentrations, 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate were tested in triplicate. This dose range finding test was part of the first experiment of the mutation assay.
At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain.
The test substance was tested both in the absence and presence of S9-mix in each strain, in two independent experiments.

Evaluation criteria:

No formal hypothesis testing was done.
A test substance is considered negative (not mutagenic) in the test if the total number of revertants in tester strain TA100 is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three times the concurrent control. The negative response should also be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if the total number of revertants in tester strain TA100 is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three times the concurrent control.

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A moderate reduction of the bacterial background lawn was observed at resp. 333 and 1000 μg/plate both in the presence and absence of S9-mix.

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A slight reduction of the bacterial background lawn was observed at 333 and 1000 μg/plate both in the presence and absence of S9-mix.

Vehicle controls validity:

valid

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

Remarks:

A slight or moderate reduction of the bacterial background lawn was observed at resp. 333 and 1000 μg/plate both in the presence and absence of S9-mix.

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A slight or moderate reduction of the bacterial background lawn was observed at resp. 333 and 1000 μg/plate both in the presence and absence of S9-mix.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Concentration of the test substance resulting in precipitation:
S. typhimurium TA1535, TA1537, TA98, TA100 and E. coli WP2uvrA: 3330 μg/plate.

STUDY RESULTS
- Concurrent vehicle negative and positive control data:
The negative and strain-specific positive control values were within historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
- Concentration of the test substance observed to be toxic to bacteria:
S. typhimurium TA1535, TA1537, TA98, TA100: 333 μg/plate.
E. coli WP2uvrA: no toxicity observed.

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that butoxystyrene is not mutagenic in the S. typhimurium reverse mutation assay and in the E. coli reverse mutation assay.

Executive summary:

Mutagenic activity of butoxystyrene was evaluated in the S. typhimurium reverse mutation assay with four histidine-requiring strains (TA1535, TA1537, TA98 and TA100) and the E. coli reverse mutation assay with a tryptophan-requiring strain (WP₂uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix.

In the dose range finding test, butoxystyrene was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP₂uvrA. In tester strain TA100, toxicity was observed at dose levels of 333 μg/plate and above in the absence and presence of S9-mix. In tester strain WP₂uvrA, no toxicitiy was observed at any of the dose levels. Results of the dose range finding test were reported as part of the first experiment. Butoxystyrene precipitated on the plates at dose levels of 3330 and 5000 μg/plate.

Based on results of the dose range finding test, butoxystyrene was tested in the first mutation assay at a concentration range of 10 to 1000 μg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. Toxicity was observed in all tester strains.

In an independent repeat of the assay with additional parameters, butoxystyrene was tested at a concentration range of 10 to 1000 μg/plate in the tester strains TA1535, TA1537, TA98 and TA100 and at a concentration range of 33 to 3330 μg/plate in tester strain WP₂uvrA in the absence and presence of 10% (v/v) S9-mix. Toxicity was observed in all tester strains except in WP₂uvrA. 

Butoxystyrene did not induce a significant dose-related increase in the number of revertant (His⁺) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp⁺) colonies in tester strain WP₂uvrA both in the absence and presence of S9-metabolic activation.