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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
tert-Butyl 2-bromopropionate
Cas Number:
39149-80-9
Molecular formula:
C7H13BrO2
IUPAC Name:
tert-Butyl 2-bromopropionate
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:Source: Tosoh Organic Chemical Co., Ltd. Lot No. 6X09A
- Purity test date:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: > 50mg/ml DMSO
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: stable

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Test concentrations with justification for top dose:
Eight concentrations of the test material (1.5, 5, 15, 50, 150, 500, 5000 μg/plate) were assayed in the pre-liminary test. Without metabolic activation, growth inhibitions were observed at and more than 500 μg/plate to TA100, TA1535, TA98 and TA1537 and at and above 1500 μg/plate to WP2uvrA. With metabolic activation, growth inhibitions were observed at and more 1500μg/plate to all strains.
Based on these results, the highest concentration was set to 1500 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: ; Though this substance is insoluble in water, soluble more tha 20 wt% in DMSO and stabke under test conditions.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
sodium azide
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable):
Strain TA100 TA1353 WP2uvrA TA98 TA1537
viable bacteria count (x 10^9/m)
Range-finding study 3.82 4.45 5.27 4.46 3.97
Main study 3.55 3.93 5.13 3.81 3.69
DURATION
- Preincubation period: 10 hr
- Exposure duration:
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays):

SPINDLE INHIBITOR (cytogenetic assays):

STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS:

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:

NUMBER OF CELLS EVALUATED:

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:
- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):

Evaluation criteria:
For a substance to be considered possivie in this test system, it should induced following effectsin at least one strain ;
1) the quotient of the number of revertant colonies over the number of colonies in the negative control is 2.0 or higher. 2) the number of revertant colonies increased dose-related, 3) reproducible results were obtained between preliminary test and main assay.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Growth inhibition was observed at 500 μg/plate and more with metabolic avtivation and at 5000 μg/plate without metabolic activation.
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Growth inhibition was observed at 500 μg/plate and more with metabolic avtivation and at 5000 μg/plate without metabolic activation.
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Growth inhibition was observed at 1500 μg/plate and more with metabolic avtivation and at 5000 μg/plate without metabolic activation.

Applicant's summary and conclusion

Conclusions:
Tert-Butyl 2-bromopropionate does not induce the increase of number of revertant more than 2 and increase related to dose compare to negative control for any bacterial strain with or without metabolic activator. The test resutls were reproducable between range finding study and main study.
Accordingly this substance was considered to be non-mutagenic under this test conditions.
Executive summary:

Tert-Butyl 2 -bromopropionate was considered to be non-mutagenic under this test conditions

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