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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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. 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 109cells/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 100µg/ml of streptomycin and seeded with approximately 107bacteria. 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 strptomycin dependence to independence and hence the chemical is not likely to classify as a gene mutant in vitro.

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Link to relevant study records
Reference
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
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
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
Target gene:
Streptomycin specific gene
Species / strain / cell type:
E. coli, 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
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
Species / strain:
E. coli, 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
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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Gene mutation in vitro:

Various data available for the target chemical and its read across were reviewed to determine the mutagenic nature of . The studies are a mentioned below:

Gene mutation toxicity study was performed by Szybalski ( Annals of the NY Academy of Science, 1958) to determine the mutagenic nature of Isoamyl benzoate (CAS no 94 -46 -2; IUPAC name: 3-methylbutyl benzoate). Genetic toxicity test was performed on strain of Escherichia coli (strain Sd-4-73) by paper disk method. 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 109cells/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 100µg/ml of streptomycin and seeded with approximately 107bacteria. 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 strptomycin dependence to independence and hence the chemical is not likely to classify as a gene mutant in vitro.

Gene mutation toxicity was predicted for Isoamyl benzoate using the battery approach from Danish QSAR database (2017). The study assumed the use of Salmonella typhimurium bacteria in the Ames test. The end point for gene mutation has been modeled in the Danish QSAR using the three software systems Leadscope, CASE Ultra and SciQSAR. Based on predictions from these three systems, a fourth and overall battery prediction is made. The battery prediction is made using the so called Battery algorithm. With the battery approach it is in many cases possible to reduce “noise” from the individual model estimates and thereby improve accuracy and/or broaden the applicability domain. Isoamyl benzoate was assumed to not induce mutation in Salmonella typhimurium by the Ames assay performed and hence the chemical is predicted to not classify as a gene mutant in vitro.

In a study for 60 -70% structurally and functionally similar read across chemical given by Zeiger et al (Environmental and Molecular Mutagenesis, 1992), Gene mutation toxicity study was performed to determine the mutagenic nature of methyl benzoate (RA CAS no 93 -58 -3; IUPAC name: Methyl Benzoate). The study was performed usingSalmonella typhimurium strainsTA97, TA98, TA100, TA1535, TA1537 in the presence and absence of S9 metabolic activation system. The chemical was dissolved in water and used at dose levels 0, 10, 33, 100, 333, 666, 1000, 1666, 3333 or 6666µg/plate by the preincubation method. The doses were selected on the basis of preliminary dose range finding study and concurrent solvent and positive controls were included in the study. Methyl benzoate did not induce mutation in Salmonella typhimurium strains TA97, TA98, TA100, TA1535, TA1537 in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Wild et al (Food and chemical toxicology, 1983) performed gene mutation toxicity study for structurally and functionally similar read across chemical. Cyclohexyl cinnamate (RA CAS no 7779 -17 -1; IUPAC name: cyclohexyl 3-phenylacrylate) artificial flavouring substance in food products was studied for mutagenic properties by the use of the Salmonella/mammalian microsome test (Ames test). The test was performed by plate incorporation method at 5 different dosesupto 3600 µg/plate using Salmonella typhimurium TA98, TA100, TA1535, TA1537, TA1538 with and without S9 metabolic activation system and the plates were incubated for 48hrs. Concurrent positive control chemicals were incorporated in the study. A reproducible, dose-related and at least two-fold elevation of the spontaneous revertant frequency. Agents producing reproducible, dose-related and significant (P≤0.01) but less than two-fold elevations were classified as marginally mutagenic under the experimental conditions. Cyclohexyl cinnamate did not cause a reproducible, dose-related and at least two-fold elevation of the spontaneous revertant frequency and hence the chemical is not mutagenic in the Salmonella/microsome AMES test performed using Salmonella typhimurium TA98, TA100, TA1535, TA1537, TA1538 in the presence and absence of S9 metabolic activation system.

Based on the weight of evidence data available for the target chemical and its read across, Isoamyl benzoate does not induce gene mutation in vitro. Hence the chemical is not likely to classify as a gene mutant in vitro.

Justification for classification or non-classification

Based on the weight of evidence data available for the target chemical and its read across, Isoamyl benzoate (CAS no 94 -46 -2; IUPAC name: 3-methylbutyl benzoate) does not induce gene mutation in vitro. Hence the chemical is not likely to classify as a gene mutant in vitro.