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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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:
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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
1998
Deviations:
no
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

Constituent 1
Chemical structure
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 peracetate
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 resultsopen allclose all
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
Positive controls validity:
valid
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
Positive controls validity:
valid
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
Positive controls validity:
valid
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
Positive controls validity:
valid
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
Positive controls validity:
valid
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
Positive controls validity:
valid
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
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.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

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:
Interpretation of results: positive

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.