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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro:

Ames test:

A gene toxicityin vitroexperiment was performed for the test material usingSalmonella typhimuriumstrains TA100, TA98, TA1535, and TA1537. The bacteria was exposed to the test chemical up to a dosage level of 3600 ug/plate. Plate incorporation study was conducted for 48 hrs and after the incubation period, a negative test result was observed. As seen by the results, test substance was considered as negative for gene toxicityin vitrosince no evidence of mutagenicity was observed in S. typhimurium strains TA100, TA98, TA1535, and TA1537.

Mammalian cell gene mutation assay:

The test chemical in the concentration of 0, 1, 2.5, 5 or 10 mM did not show any evidence of gene toxicity when CHO cells were exposed to the test chemical.

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:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Data is from peer reviewed journal
Qualifier:
according to guideline
Guideline:
other: see Principles below
Principles of method if other than guideline:
Mutagenicity testing of test substance in Salmonella typhimurium strains TA100, TA98, TA1535, and TA1537.
GLP compliance:
not specified
Type of assay:
bacterial gene mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Type and identity of media: Vogel Bonnet medium
- Properly maintained: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S-9 liver fractions were prepared from Aroclor-pretreated rats
Test concentrations with justification for top dose:
Up to 3600 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
Remarks:
sodium azide, at 0.5ug/plate 430-760 in TA1535, 400-700 in TA100; with benzo[a]pyrene, at 5 ug/plate, 865-1210 in TAI00, 235-350 in TA1537. 410-590 in TA1538, 660-1000 in TA98.
Details on test system and experimental conditions:
METHOD OF APPLICATION: In agar (plate incorporation)

DURATION
- Preincubation period: No data available
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- 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): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: All chemicals were tested at least twice.

NUMBER OF CELLS EVALUATED: No data available

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: No data available
Rationale for test conditions:
No data available
Evaluation criteria:
A reproducible, dose-related and at least two-fold elevation of the spontaneous revertant frequency was regarded as positive result(+). 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
Statistics:
Method of Kastenbaum and Bowman (1970).
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
No data available
Remarks on result:
other: No mutagenic effect were observed.
Conclusions:
The given test material was negative for gene toxicity in vitro up to a dose concentration of 3600 µg/plate since no evidence of mutagenicity was observed.
Executive summary:

A gene toxicity in vitro experiment was performed for the test material using Salmonella typhimurium strains TA100, TA98, TA1535, and TA1537. The bacteria was exposed to the test chemical up to a dosage level of 3600 ug/plate. Plate incorporation study was conducted for 48 hrs and after the incubation period, a negative test result was observed. As seen by the results, test substance was considered as negative for gene toxicity in vitro since no evidence of mutagenicity was observed in S. typhimurium strains TA100, TA98, TA1535, and TA1537.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Data for the various publication were observed for test chemical. Test chemical was reviewed to determine the mutagenic nature of 3-methyl-4-(2,6,6-trimethyl-2-cyclohexen-1-yl)-3-buten-2-one (CAS no 127-51-5). The studies are as mentioned below:

Ames assay

A gene toxicity in vitro experiment was performed for the test material using Salmonella typhimurium strains TA100, TA98, TA1535, and TA1537. The bacteria was exposed to the test chemical up to a dosage level of 3600 ug/plate. Plate incorporation study was conducted for 48 hrs and after the incubation period, a negative test result was observed. As seen by the results, test substance was considered as negative for gene toxicity in vitro since no evidence of mutagenicity was observed in S. typhimurium strains TA100, TA98, TA1535, and TA1537.

Supported by other study.In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 1, 2.5, 5 or 10 mM and S9-induced metabolic activation for 3 hours. The results showed that there was a strong cytotoxicity after treatment, however, S9-induced metabolic activation decreased the level of cytotoxicity to a certain extent. Independently of tested concentration, the results showed no evidence of gene toxicity. Therefore, it is considered that the test chemical in the concentration of 0, 1, 2.5, 5 or 10 mM does not cause genetic mutation(s) when CHO cells are exposed to the test chemical in the presence of metabolic activation.

The genotoxicity of the flavoring agent widely used in everyday foods was studied by a bacterial mutation test in the Salmonella/microsome system. In the mutation test, the test material did not exhibit significant induction of his+revertants in Salmonella typhimurium TA98 or TA100, either with or without rat liver microsome as the metabolic activation system. Gene toxicity in vitro profile for the test material α- Ionone is negative with and without rat liver microsome fraction S9 and hence the test chemical is not likely to classify for gene mutation in vitro.

In vitro Mammalian gene mutation study

In a gene toxicity test, Chinese Hamster Ovary(CHO) cells were exposed to test substance in the concentration of 0, 1, 2.5, 5 or 10 mM and S9-induced metabolic activation for 3 hours. The results showed that there was a strong cytotoxicity after treatment, however, S9-induced metabolic activation decreased the level of cytotoxicity to a certain extent. Independently of tested Ionone concentration, the results showed no evidence of gene toxicity. Therefore, it is considered that test substance in the concentration of 0, 1, 2.5, 5 or 10 mM does not cause genetic mutation(s) when CHO cells are exposed to the test chemical in the presence of metabolic activation.

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 1, 2.5, 5 or 10 mM and S9-induced metabolic activation for 3 hours. The results showed that there was a strong cytotoxicity after treatment, however, S9-induced metabolic activation decreased the level of cytotoxicity to a certain extent. Independently of tested concentration, the results showed no evidence of gene toxicity. Therefore, it is considered that the test chemical in the concentration of 0, 1, 2.5, 5 or 10 mM does not cause genetic mutation(s) when CHO cells are exposed to the test chemical in the presence of metabolic activation.

Based on the data summarized, 3-methyl-4-(2,6,6-trimethyl-2-cyclohexen-1-yl)-3-buten-2-one (CAS no 127-51-5)  did not induce gene mutation .Hence it is not likely to be mutagenic in vitro.

Justification for classification or non-classification

Based on the studies summarized and as per CLP classification criteria the test chemical is not likely to classify for gene mutation in vitro.