p-tert-butylstyrene

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:

31 January 2017 - 13 February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: ISO/IEC 17025:2005 (ISO/IEC, 2005)

Version / remarks:

2005

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

p-tert-butylstyrene

EC Number:

217-126-9

EC Name:

p-tert-butylstyrene

Cas Number:

1746-23-2

Molecular formula:

C12H16

IUPAC Name:

p-tert-butylstyrene

Test material form:

liquid: volatile

Specific details on test material used for the study:

Synonyms: p-tert-butylstyrene
CAS No: 1746-23-2
Purity: 95.9%

Method

Target gene:

Histidine

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 98

Remarks:

reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens.

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 100

Remarks:

reverted by mutagens that cause both frameshift and basepair substitution mutations.

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 1535

Remarks:

reverted by mutagens that cause basepair substitutions

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 1537

Remarks:

reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens.

Species / strain 5

Species / strain / cell type:

E. coli WP2 uvr A

Remarks:

sensitive to basepair substitution mutations, rather than frameshift mutations

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9

Test concentrations with justification for top dose:

10.0, 33.3, 100, 333, 1000, 3333 and 5000 μg per plate (for tester strains TA98, TA1537, WP2 uvrA in the presence and absence of S9 activation and TA100, TA1535 in the presence of S9 activation); and
1.00, 3.33, 10.0, 33.3, 100, 333 and 1000 μg per plate (for tester strains TA100, TA1535 in the absence of S9 activation)

Vehicle / solvent:

DMSO (vehicle for Positive Controls: DMSO, except sterile water for sodium azide)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation)
- Initial cell density: 0.8 - 2 x 10(9) cells per mL


NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY : The preliminary toxicity assay was used to establish the dose-range over which the test substance would be assayed. TA98, TA100, TA1535, TA1537 and WP2 uvrA were exposed to the vehicle alone and ten dose levels of the test substance, with a single plate/condition, on selective minimal agar in the presence and absence of Aroclor-induced rat liver S9. Dose levels for the mutagenicity assay were based upon post-treatment toxicity.

MUTAGENICITY ASSAY: One-half (0.5) milliliter of S9 or Sham mix (100 mM phosphate buffer at pH 7.4), 100 μL of tester strain (cells seeded) and 50.0 μL of vehicle or test substance dilution were added to 2.0 mL of molten selective top agar at 45±2°C. When plating the positive controls, the test substance aliquot was replaced by a 50.0 μL aliquot of appropriate positive control. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. After the overlay had solidified, the plates were inverted and incubated for 48 to 72 hours at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted.

Evaluation criteria:

- For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance.

- Strains TA1535 and TA1537: Data sets were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 3.0 times the mean vehicle control value and above the corresponding acceptable vehicle control range.

- Strains TA98, TA100 and WP2 uvrA: Data sets were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 2.0 times the mean vehicle control value and above the corresponding acceptable vehicle control range.

- An equivocal response is an increase in a revertant count that is greater than the acceptable vehicle control range but lacks a dose response or does not achieve the respective fold increase threshold cited.

- A response will be evaluated as negative, if it is neither positive nor equivocal.

Statistics:

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 3333 μg per plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 3333 μg per plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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:

cytotoxicity

Remarks:

≥ 100 μg per plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 7

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 1000 μg per plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 8

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 100 μg per plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 9

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 10

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

In preliminary toxicity assay, toxicity was observed beginning at 100, 667 or 3333 μg per plate. Based upon these results, the maximum dose tested in the mutagenicity assay was 5000 μg per plate for tester strains TA98, TA1537, WP2 uvrA in the presence and absence of S9 activation and TA100, TA1535 in the presence of S9 activation and 1000 μg per plate for tester strains TA100, TA1535 in the absence of S9 activation.

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the test substance was determined to be non-genotoxic with or without metabolic activation in the Ames test.

Executive summary:

A study was conducted to determine the genotoxic potential of the test substanceaccording to OECD Guideline 471, in compliance with GLP.Salmonella typhimuriumstrains TA1535, TA1537, TA98 and TA100 andEscherichia colistrain WP2uvrA were treated with the test substance using both the Ames plate incorporation and preincubation methods, in triplicate both with and without the addition of a rat liver homogenate metabolizing system:Aroclor-induced rat liver (S9).In the preliminary toxicity assay, the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 μg per plate. No precipitate was observed. Toxicity was observed beginning at 100, 667 or 3333 μg per plate. Based upon these results, in the mutagenicity assay, the dose levels tested were10.0, 33.3, 100, 333, 1000, 3333 and 5000 μg per plate for tester strains TA98, TA1537, WP2uvrA in the presence and absence of S9 activation and TA100, TA1535 in the presence of S9 activation and 1.00, 3.33, 10.0, 33.3, 100, 333 and 1000 μg per plate for tester strains TA100, TA1535 in the absence of S9 activation. No precipitate was observed. Toxicity was observed beginning at 100, 1000 or 3333 μg per plate with a few conditions. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Under the study conditions, the test substance was considered to be non-mutagenic in the Ames test (Dakoulas, 2018).