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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from peer reviewed publication

Data source

Reference
Reference Type:
publication
Title:
Special Microbiological Systems. 11. Observations On Chemical Mutagenesis In Microorganisms
Author:
Waclaw Szybalski
Year:
1958
Bibliographic source:
Annals of the NY Academy of Science, Vol: 76 2c PP 475-489, 1958

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Refer below principle
Principles of method if other than guideline:
Paper-disk method was performed to determine the mutagenic nature of isoamyl benzoate using E. coli Sd-4-73 by checking its reversion from streptomycin dependence to independence
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
- Name of test material: Isoamyl benzoate
- IUPAC name: 3-methylbutyl benzoate
- Molecular formula: C12H16O2
- Molecular weight: 192.256 g/mol
- Substance type: Organic
- Physical state: Liquid
- Purity: No data available
- Impurities (identity and concentrations): No data available

Method

Target gene:
Streptomycin specific gene
Species / strain
Species / strain / cell type:
other: Sd-4-73
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
not specified
Metabolic activation system:
No data
Test concentrations with justification for top dose:
0.01 to 0.025 ml or crystals of chemical
Vehicle / solvent:
No data
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Details on test system and experimental conditions:
METHOD OF APPLICATION: As impregnation on paper disk.

DURATION
- Preincubation period: No data available
- Exposure duration: No data available
- Expression time (cells in growth medium): No data available
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): 30 µg of sulfocidin and unidentified fungus was added.
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: ): No data available

NUMBER OF CELLS EVALUATED: 109 cells/ml
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data available

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other:

OTHER:
Rationale for test conditions:
No data
Evaluation criteria:
Increase in the frequency of reversion from streptomycin dependence to independence in strain Sd-4-73 of Escherichia coli.
Statistics:
No data

Results and discussion

Test results
Species / strain:
other: Sd-4-73
Metabolic activation:
not specified
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
No data available

Applicant's summary and conclusion

Conclusions:
Isoamyl benzoate did not induce mutation from streptomycin dependence to independence in Escherichia coli (strain Sd-4-73) and hence the chemical is not likely to classify as a gene mutant in vitro.
Executive summary:

Gene mutation toxicity study was performed to determine the mutagenic nature of Isoamyl benzoate. Genetic toxicity test was performed on strain of Escherichia coli (strain Sd-4-73) by paper disk method. Paper-disk method was performed to check for the ability of E. coli Sd-4-73 to show reversion from streptomycin dependence to independence. Overnight culture of strain Sd-4-73 grown at 36°C in aerated nutrient broth containing 20 µg/ml of streptomycin was used as a inoculum. The culture was centrifuged and washed at least twice with saline or distilled water to remove the extraneous streptomycin, and was resuspended in saline to a concentration of approximately 109 cells/ml. 0.1-ml. aliquot of this suspension was mixed with 2.5 ml of molten soft nutrient agar (0.7 per cent agar) and poured over a base of 20 ml of 1.5 per cent nutrient agar. After the soft layer was solidified, the mutagen was applied in form of small drops or crystal. Additional plates were prepared with small inocula (one fifth and one twenty-fifth of the original) so as not to miss the optimum cell density; the number of cells per plate was rather critical, the yield of mutant colonies being reduced either by crowding or by insufficient population size. After the soft agar layer had set, the mutagen, in the form of a microdrop of solution (0.01 to 0.025 ml.) or a small crystal, was applied to a small filter-paper disk resting on the agar. To determine whether the substance was inhibitory for the assay organism at the concentration employed, the procedure was repeated on a nutrient agar plate containing 100 µg/ml of streptomycin and seeded with approximately 107 bacteria. Mutagenicity was manifested as a zone of streptomycin-independent mutant colonies around a filter-paper disk saturated with the mutagenic agent and resting on the surface of streptomycin-free nutrient agar seeded heavily with a streptomycin-dependent parental population. Isoamyl benzoate did not induce mutation from streptomycin dependence to independence in Escherichia coli (strain Sd-4-73) and hence the chemical is not likely to classify as a gene mutant in vitro.