tert-butyl peracetate

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:

2015-07-21 and 2015-09-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ICH Guideline S2 (R1): Genotoxicity testing and data interpretation for pharmaceuticals intended for human use (2012)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

tert-butyl peracetate

EC Number:

203-514-5

EC Name:

tert-butyl peracetate

Cas Number:

107-71-1

Molecular formula:

C6H12O3

IUPAC Name:

tert-butyl ethaneperoxoate

Details on test material:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in the refrigeratore in the dark between +10 and +30°C

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver

Test concentrations with justification for top dose:

- S9 mix: 5000; 1600; 500; 160; 50 and 16 μg/plate
+ S9 mix: 1000; 750; 500; 160; 50; 32 and 16 μg/plate

Vehicle / solvent:

- Vehicle/solvent used: acetone
- Justification for choice of solvent/vehicle: The chosen vehicle was compatible with the survival of the bacteria and the S9 activity and was chosen based on the results of the preliminary solubility test.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9 mix: 4-nitro-1,2-phenylene-diamine (TA98), sodium azide (TA100, TA1535), 9-aminoacridine (TA1537), Methyl-methanesulfonate (E.coli WP2 uvrA); +S9 mix: 2-aminoanthracene (all of Salmonella strains, E.coli WP2 uvrA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration:

NUMBER OF REPLICATIONS: duplicate

DETERMINATION OF CYTOTOXICITY
- Method: revertant colony number

Evaluation criteria:

A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- in strain TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.

Criteria for a Negative Response:
A test article is considered non-mutagenic in this bacterial reverse mutation assay if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

at 5000 - 160 µg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

at 5000 - 500 µg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

other: S. typhimurium TA98, TA1537 and E.coli WP2 uvrA

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

at 5000 and 1600 µg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

at 750 and 500 µg/plate

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

at 500 and 160 µg/plate

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

at 160 µg/plate

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 7

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Microdrops were observed in all test strains at 5000 and 1600 µg/plate in the absence of S9 mix.

RANGE-FINDING/SCREENING STUDIES: The toxicity of the test item was determined with strains Salmonella typhimurium TA98 and TA100 in a pre-experiment. The following concentrations were chosen based on the results obtained in the pre-experiment for toxicity: 5000, 1600, 500, 160, 50 and 16 µg/plate (-S9 mix); and 1000, 750, 500, 160, 50, 32 and 16 µg/plate (+S9 mix).

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: Signs of cytotoxicity (absent or reduced revertant colony numbers and/or affected background lawn development) were observed in all tested strains only in presence of metabolic activation. The 500 μg/plate was found to be the lowest cytotoxic concentration, observed in the case of Salmonella typhimurium TA98, TA1535 and TA1537 strains.

Any other information on results incl. tables

The performance of an additional confirmatory experiment was considered as not necessary for the final conclusion of the study because unequivocal positive results were obtained in the Initial Mutation Test (Plate Incorporation Test).

Applicant's summary and conclusion

Conclusions:

The reported data of this mutagenicity assay shows, that under the experimental conditions reported, the test item (acting as direct mutagen and pro-mutagen, in absence and also in the presence of exogenous metabolic activation) induced gene mutations by frameshift and base-pair substitution in the genome of the tester strains used. Therefore, the test substance is considered mutagenic in this bacterial reverse mutation assay.

Executive summary:

The mutagenic potential of the test substance (50.1% purity) was tested in the Bacterial Reverse Mutation Assay in Salmonella typhimurium TA98, TA1537, TA1535 and TA100 strains and Escherichia coli WP2 uvrA. The test item was dissolved in acetone. In the Initial Mutation Test the following concentrations were examined: In absence of metabolic activation (-S9 Mix): 5000; 1600; 500; 160; 50 and 16 μg/plate; and in the presence of metabolic activation (+S9 Mix): 1000; 750; 500; 160; 50; 32 and 16 μg/plate. Due to the unequivocal demonstrative positive results obtained in the experiment I, (Plate Incorporation Test) the performance of an additional confirmatory experiment was considered as not necessary for the final conclusion of the study. The concentrations, including the controls, were tested in triplicate. In the performed experiment positive and negative (vehicle) controls were run concurrently. In the Initial Mutation Test following treatment with the test substance significant, biological relevant, dose-related changed revertant colony number increases, unequivocal positive results were noticed in the concentration range of 5000-160 μg/plate in S. typhimurium TA100, in the range of 5000-500 μg/plate in TA1535, and at 5000 and 1600 μg/plate in TA98, TA1537 and E. coli WP2 uvrA, in the absence of exogenous metabolic activation (-S9 Mix). Positive results were obtained at 750 and 500 μg/plate in E. coli WP2 uvrA, at 500 and 160 μg/plate in S. typhimurium TA100 and at 160 μg/plate in S. typhimurium TA98, with addition of metabolic activation (+S9 Mix). The mutagenic effect of the test item was unequivocal characteristic and very intensive. Signs of cytotoxicity (absent or reduced revertant colony numbers and/or affected background lawn development) were observed in all tested strains only in presence of metabolic activation. The 500 μg/plate was found to be the lowest cytotoxic concentration, observed in the case of Salmonella typhimurium TA98, TA1535 and TA1537 strains. The reported data of this mutagenicity assay shows, that under the experimental conditions reported, the test item (acting as direct mutagen and pro-mutagen, in absence and also in the presence of exogenous metabolic activation) induced gene mutations by frameshift and base-pair substitution in the genome of the tester strains used. Therefore, the test substance is considered mutagenic in this bacterial reverse mutation assay.