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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

The genotoxicity of the flavoring agent widely used in everyday foods was studied by a bacterial mutation test in the Salmonella/microsome system. The study was performed using Salmonella typhimurium strains TA98 and TA100 in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose levels of 0.01 to 50 μg/pl. 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 test substance is negative with and without rat liver microsome fraction S9 using Salmonella typhimurium strains TA98 and TA100 and hence the test chemical is not likely to classify for gene mutation in vitro.

In vitro mammalian chromosome aberration study:

The genotoxicity of the flavoring agent widely used in everyday foods was studied by a chromosomal aberration test in the original CH cell line B241. The test chemical was studied at dose level of 0.01 to 50 µg/pl. Exponentially growing cells at one day after seeding were exposed to each of the chemicals for 24 h, and then incubated another 24 h without the chemicals followed by treatment with colchicine (1 x 10 - 7 M) for 2-3 h. These were the best condition for obtaining maximal frequency of chromosome aberrations. In this test, chemicals were dissolved in DMSO at a concentration of 50 mM, and then were diluted with the medium. Control cell cultures were treated with a medium containing DMSO equal in its concentration to the test solution of the flavorings. Chromosome samples were prepared by the usual Giemsa staining method. The percentage of chromosome aberration was computed by scoring about 200 metaphase spreads, each containing 20-26 chromosomes (mode of chromosome number, 23). The results of the chromosome test showed significant increases in chromosome aberrations in the Chinese-hamster B241 cells by test substance , regardless of the presence or absence of S9 mix. The total frequency of the aberrations indicated a dose dependent increase at a certain dose range. Based on the results of the study, the test chemical induced chromosomal aberration in CH cell line B241 in the presence and absence of S9 metabolic activation system.

In vitro mammalian cell gene mutation assay:

The test chemical did not induce gene mutation in the mammalian cell line used and hence it is not likely to classify as a gene mutant in vitro.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
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:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
The genotoxicity of test substance widely used in everyday foods was studied by a bacterial mutation test in the Salmonella/microsome system
GLP compliance:
not specified
Type of assay:
bacterial gene mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium, other: TA98 and TA100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
S9 metabolic activation system
Test concentrations with justification for top dose:
0.01 to 50 μg/pl
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Sterilized DMSO

- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
benzo(a)pyrene
other: Aflatoxin B1
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

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): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: No data available

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
Evaluation criteria:
For mutagenic potency, a positive result was defined as a reproducible, dose-related increase in the number of revertant colonies per plate, and a greater than 2-fold increase in spontaneous mutation rate was obtained, according to the protocol of Ames
Statistics:
No data available
Species / strain:
S. typhimurium, other: TA98 and TA100
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
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
No data available
Remarks on result:
other: No mutagenic potential
Conclusions:
Gene toxicity in vitro profile for the test material is negative in the presence and absence of rat liver microsome fraction S9 using Salmonella typhimurium strains TA98 and TA100 and hence it is not likely to classify as a gene mutant in vitro.
Executive summary:

The genotoxicity of the flavoring agent widely used in everyday foods was studied by a bacterial mutation test in the Salmonella/microsome system. The study was performed using Salmonella typhimurium strains TA98 and TA100 in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose levels of 0.01 to 50 μg/pl. 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 test substance is negative with and without rat liver microsome fraction S9 using Salmonella typhimurium strains TA98 and TA100 and hence the test chemical is not likely to classify for gene mutation in vitro.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
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:
The genotoxicity of the test chemical flavoring agents widely used in everyday foods was studied by by a chromosome test in Chinese hamster (CH) cells.
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
No data
Species / strain / cell type:
mammalian cell line, other: CH cell line B241
Details on mammalian cell type (if applicable):
Type and Identity of media: CH cell line B241cultured in Eagle medium (containing kanamycin 60 pg/ml) with 10% fetal calf serum

- Properly maintained: No data available

- Periodically checked for Mycoplasma contamination: yes

- Periodically checked for karyotype stability: Yes, the cell line was used for the chromosome test in culture stages between the 5th and 8th passages.

- Periodically "cleansed" against high spontaneous background: No data available
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsome fraction S9
Test concentrations with justification for top dose:
0.01 to 50 µg/pl
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Chemicals were dissolved in DMSO at a concentration of 50 mM, and then were diluted with the medium.

- Justification for choice of solvent/vehicle: Test chemical solubility in solvent
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: Benzo[a]pyrene (B[a]P), 4-nitroquinoline l-oxide (4NQO), Aflatoxin B1 and 2-nitrofluorene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data available

- Exposure duration: 24 hrs

- Expression time (cells in growth medium): 24 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): Giemsa stain

SPINDLE INHIBITOR (cytogenetic assays): No data available

STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: No data available

NUMBER OF CELLS EVALUATED: 317 (as per table); The percentage of chromosome aberration was computed by scoring about 200 metaphase spreads, each containing 20-26 chromosomes (mode of chromosome number, 23).

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: No data available

OTHER:
Chromosome test in culture stages between the 5th and 8th passages was performed.
Rationale for test conditions:
No data
Evaluation criteria:
The cell line was observed for chromosomal aberrations.M In scoring of the cells, the chromatid was considered broken only if the chromatid fragment distal to an achromatic region was displaced or misaligned. Breaks occurring in the same region of sister chromatid, acentric fragments and chromosome deletion were classified as chromosomal breaks. Others include translocations, fragmentation and pulverisation.
Statistics:
P<0.05- <0.001 by Chi square test
Species / strain:
mammalian cell line, other: CH cell line B241
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
No data available
Remarks on result:
other: Mutagenic effect were observed

α- Ionone

Total no of cells examined:

317

Total abnormal cells observed:

34

Chromatid gap

11

Chromatid break

9

Chromosome break

1

Ring chromosomes

1

Dicentric chromosomes

4

Chromatid exchange

8

Translocation, pulverization and fragmentation

0

Conclusions:
The test chemical induced chromosomal aberration in CH cell line B241 in the presence and absence of S9 metabolic actiation system.
Executive summary:

The genotoxicity of the flavoring agent widely used in everyday foods was studied by a chromosomal aberration test in the original CH cell line B241. The test chemical was studied at dose level of 0.01 to 50 µg/pl. Exponentially growing cells at one day after seeding were exposed to each of the chemicals for 24 h, and then incubated another 24 h without the chemicals followed by treatment with colchicine (1 x 10 - 7 M) for 2-3 h. These were the best condition for obtaining maximal frequency of chromosome aberrations. In this test, chemicals were dissolved in DMSO at a concentration of 50 mM, and then were diluted with the medium. Control cell cultures were treated with a medium containing DMSO equal in its concentration to the test solution of the flavorings. Chromosome samples were prepared by the usual Giemsa staining method. The percentage of chromosome aberration was computed by scoring about 200 metaphase spreads, each containing 20-26 chromosomes (mode of chromosome number, 23). The results of the chromosome test showed significant increases in chromosome aberrations in the Chinese-hamster B241 cells by test substance , regardless of the presence or absence of S9 mix. The total frequency of the aberrations indicated a dose dependent increase at a certain dose range. Based on the results of the study, the test chemical induced chromosomal aberration in CH cell line B241 in the presence and absence of S9 metabolic activation system.

Endpoint:
in vitro gene mutation study in mammalian cells
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
Remarks:
Experimental data from various test chemicals
Justification for type of information:
Data for the target CAS is summarized based on data from various test chemicals
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
WoE for the target CAS is summarized based on data from various test chemicals
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Target gene:
7. HGPRT
8. TK
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
7/8
Details on mammalian cell type (if applicable):
- Cell line used: Chinese Hamster Ovary (CHO) cells
- Type and identity of media: Ham's F-12K (Kaighn's) Medium containing 2 mM L-Glutamine supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (10,000 U/mL).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Not applicable
- Periodically checked for karyotype stability: Not applicable
Additional strain / cell type characteristics:
other: Hypodiploid, modal No. 20
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
7/8.. S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats
9. No data
Test concentrations with justification for top dose:
7/8. 0, 1, 2.5, 5 or 10 mM
9. 8 concentration ranging between 200 to 900 μg/ml – for 4 hrs treatment
4.1 to 520 μg/ml – for 24 hrs treatment
Vehicle / solvent:
7/8. Vehicle(s)/solvent(s) used: Ethanol
Justification for choice of solvent/ vehicle: Alpha- and Beta-form was not soluble in PBS but easy to dissolve in ethanol.

9. No data
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
7/8. N-ethyl-N-nitrosourea (ENU) was the positive control substance in the tests done without S9
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
Remarks:
9
Details on test system and experimental conditions:
7/8. METHOD OF APPLICATION: In medium with pre-incubation

DURATION
Pre-incubation
One week involving 3 days of incubation with Hypoxanthine-aminopterin-thymidine (HAT) in medium as a mutant cleansing stage, followed by overnight incubation with hypoxanthine-thymidine (HT) in medium prior to a 3-4 days incubation in regular cell medium. After seeding and prior to treatment, the mutant-free cells were incubated for an additional of 24 hours.

Exposure duration
3 hours

Expression time
7 days

Selection time
14 days

Fixation time
7 days (harvest of cells)

SELECTION AGENT (mutation assays): 6-thioguanine (TG)
SPINDLE INHIBITOR (cytogenetic assays): Not applicable
STAIN (for cytogenetic assays): Crystal violet

NUMBER OF REPLICATIONS: A minimum of 2 replicates per dose concentration including negative and positive control.

NUMBER OF CELLS EVALUATED: 5 x 10 E5 cells were plated 7 days after treatment and whatever cells left, after 14 days of incubation with the selection medium, were evaluated.

DETERMINATION OF CYTOTOXICITY
- Cytotoxicity test
After being exposed to the test chemical for 3 hours, in the absence or presence of S9, cells were trypsinized and 0.5 x 10 E5 cells per well was seeded in duplicates from two parallel duplicate cultures into 6-well plates in fresh medium. The relative total growth and cytotoxicity was evaluated 24 and 48 hours after seeding.

OTHER EXAMINATIONS: Not applicable

9. METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data
- Exposure duration: 4 and 24 hrs
- Expression time (cells in growth medium): 4 and 24 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Once

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

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

OTHER: No data
Rationale for test conditions:
No data
Evaluation criteria:
7/8. The plates were scored for total number of colonies
9. The cell line was observed for mutation frequency at the L5178Y T/K +/- locus
Statistics:
No data
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
7/8
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
9
Metabolic activation:
not specified
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
No data
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical did not induce gene mutation in the mammalian cell line used and hence it is not likely to classify as a gene mutant in vitro.
Executive summary:

Data available for the various test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

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 with and without 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 and absence of metabolic activation.

In vitro mammalian cell gene mutation assay was also performed to determine the mutagenic nature of the test chemical. The study was performed using mouse lymphoma cells (L5178Y T/K +/- 3.7.2c). It included 4 hours treatment in the absence and presence of S9-mix and a 24 hours treatment in the absence of S9-mix. The concentrations were selected based on a preliminary toxicity test. The test groups included single replicates at 8 concentrations ranging from 200 to 900 μg/ml in the 4 hours treatment arm and from 4.1 to 520 μg/ml in the 24 hours treatment arm. The maximum concentration was limited by toxicity. The substance did not induce biologically or statistically significant increases in mutant frequency and therefore it was considered non-mutagenic in this assay. The study is compliant with OECD Guideline 476.

Based on the details available, the test chemical did not induce gene mutation in the mammalian cell line used and hence it is not likely to classify as a gene mutant in vitro.

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

Genetic toxicity in vivo

Description of key information

A mouse micronucleus test was also performed with the test chemical in order to evaluate the biological significance of the positive in vitro chromosome aberration assay. The test chemical at doses of 300, 600, or 1200 mg/kg in corn oil was administered by intraperitoneal injection to male and female ICR mice (5/sex/dose). Reductions (up to 21%) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the a-ionone treated groups relative to the respective vehicle controls. These reductions suggest the bioavailability of the test chemical to the bone marrow. There were no statistically significant increases in the incidence of micronucleated polychromatic erythrocytes in the test chemical treated groups relative to their respective vehicle control in either male or female mice, regardless of dose level or bone marrow collection time. The test chemical was concluded to be negative in the mouse micronucleus assay and hence is considered to be non-mutagenic in vivo.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Justification for type of information:
Data is from secondary source
Qualifier:
according to
Guideline:
other: Refer below principle
Principles of method if other than guideline:
In vivo Chromosome aberration study was performed to determine the mutagenic nature of the test chemical
GLP compliance:
not specified
Type of assay:
mammalian bone marrow chromosome aberration test
Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
No data
Route of administration:
intraperitoneal
Vehicle:
Corn oil
- Justification for choice of solvent/vehicle: The test chemical was soluble in corn oil
- Concentration of test material in vehicle: 0, 300, 600, or 1200 mg/kg
- Amount of vehicle (if gavage or dermal): No data
- Type and concentration of dispersant aid (if powder): No data
- Lot/batch no. (if required): No data
- Purity: No data
Details on exposure:
No data
Duration of treatment / exposure:
No data
Frequency of treatment:
No data
Post exposure period:
No data
Remarks:
0, 300, 600 or 1200 mg/Kg day
No. of animals per sex per dose:
0 mg/Kgday: 5 males and 5 females
300 mg/Kgday: 5 males and 5 females
600 mg/Kgday: 5 males and 5 females
1200 mg/Kgday: 5 males and 5 females
Control animals:
yes, concurrent vehicle
Positive control(s):
No data
Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
No data
Evaluation criteria:
The bone marrow cells were observed for the presence of micronucleated polychromatic erythrocytes
Statistics:
No data
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
not specified
Remarks on result:
other: No mutagenic potential
Additional information on results:
No data
Conclusions:
The test chemical did not cause statistically significant increases in the incidence of micronucleated polychromatic erythrocytes and hence is not likely to be mutagenic in vivo.
Executive summary:

A mouse micronucleus test was also performed with the test chemical in order to evaluate the biological significance of the positive in vitro chromosome aberration assay. The test chemical at doses of 300, 600, or 1200 mg/kg in corn oil was administered by intraperitoneal injection to male and female ICR mice (5/sex/dose). Reductions (up to 21%) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the a-ionone treated groups relative to the respective vehicle controls. These reductions suggest the bioavailability of the test chemical to the bone marrow. There were no statistically significant increases in the incidence of micronucleated polychromatic erythrocytes in the test chemical treated groups relative to their respective vehicle control in either male or female mice, regardless of dose level or bone marrow collection time. The test chemical was concluded to be negative in the mouse micronucleus assay and hence is considered to be non-mutagenic in vivo.

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

Additional information

Data available from the various publications were reviewed for test chemical. The details of the studies are as mentioned below:

Gene mutation in vitro:

Ames assay:

The genotoxicity of the flavoring agent widely used in everyday foods was studied by a bacterial mutation test in the Salmonella/microsome system. The study was performed using Salmonella typhimurium strains TA98 and TA100 in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose levels of 0.01 to 50 μg/pl. 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 test substance is negative with and without rat liver microsome fraction S9 using Salmonella typhimurium strains TA98 and TA100 and hence the test chemical is not likely to classify for gene mutation in vitro.

Another Ames assay was conducted in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA102 to assess the mutagenicity of test substance, both in the absence and in the presence of metabolic activation by S9-mix (from livers of rats induced with Aroclor 1254). This study was performed following GLP recommendations and according to OECD Guideline 471 (OECD, 1997a). An initial experiment was carried out both in the absence and presence of S9-mix activation in all five strains, using 0.3, 1.6, 8, 40, 200, 1000 and 5000 μg of alpha-ionone/plate, plus negative (solvent) and positive controls. Evidence of toxicity was observed at 1000 and/or 5000 μg/plate across all strains in the absence and presence of S9-mix with the exception of TA100 in which no clear evidence of toxicity, in presence of S9-mix, was observed at 5000μg/plate. In a second experiment of the same study, the concentrations were changed lowering to 2500 μg/plate for all strains and conditions with the exception of TA98 in presence of S9-mix and for TA100 in presence and absence of S9-mix. In this second experiment the concentration intervals were narrowed, covering the ranges 156.3 - 5000 μg/plate or 78.1 - 2500 μg/plate in order to better detect possible concentration-dependent mutation. In addition, a pre-incubation step with S9-mix activation treatment was added to increase the chance of detecting a positive response. In this experiment, evidence of toxicity ranging from a diminution of the background bacterial lawn and/or a reduction in revertant numbers to a complete killing of the test bacteria was observed at 1250 μg/plate and above for strain TA98 in the presence of S9-mix, at 625 μg/plate in strains TA98 in the absence of S9-mix and TA100 with and without S9-mix. Toxicity was observed at 312.5 μg/plate and above in all remaining strains. The third experiment was conducted using strains TA1535 and TA102 in the absence and presence of S9-mix activation and strain TA1537 in the presence of S9-mix activation. In the treatment with S9-mix a pre-incubation step was included. The maximum test concentration was 2500 μg/plate for TA1535 while it was further reduced for TA102 (with or without S9-mix) and for TA1537 to 1250 μg/plate. In addition, more narrow concentration intervals were used, covering either 39.1 to 2500 μg/plate or 19.5 to 1250 μg/plate. Evidence of toxicity was observed at the highest three or four concentrations across all strains in the absence or presence of S9-mix. Since no statistically significant increase in revertant numbers was seen at any concentration, either in the presence or absence of S9-mix, the test material is non mutagenic in vitro and hence is not likely to classify for gene mutation in vitro.

Rec assay was also performed to determine the mutagenicity of the test chemical. The study was performed using Bacillus subtilis in strains H17 (rec+) and M45 (rec-) at dose level of 19µg/disc. The test chemical did not induce recombination in the rec assay performed using Bacillus subtilis in strains H17 (rec+) and M45 (rec-) and hence is considered to be non mutagenic in vitro.

In vitro mammalian chromosome aberration study:

The genotoxicity of the flavoring agent widely used in everyday foods was studied by a chromosomal aberration test in the original CH cell line B241. The test chemical was studied at dose level of 0.01 to 50 µg/pl. Exponentially growing cells at one day after seeding were exposed to each of the chemicals for 24 h, and then incubated another 24 h without the chemicals followed by treatment with colchicine (1 x 10 - 7 M) for 2-3 h. These were the best condition for obtaining maximal frequency of chromosome aberrations. In this test, chemicals were dissolved in DMSO at a concentration of 50 mM, and then were diluted with the medium. Control cell cultures were treated with a medium containing DMSO equal in its concentration to the test solution of the flavorings. Chromosome samples were prepared by the usual Giemsa staining method. The percentage of chromosome aberration was computed by scoring about 200 metaphase spreads, each containing 20-26 chromosomes (mode of chromosome number, 23). The results of the chromosome test showed significant increases in chromosome aberrations in the Chinese-hamster B241 cells by test substance , regardless of the presence or absence of S9 mix. The total frequency of the aberrations indicated a dose dependent increase at a certain dose range. Based on the results of the study, the test chemical induced chromosomal aberration in CH cell line B241 in the presence and absence of S9 metabolic activation system.

The above data is further supported by data from closely related test chemical.

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical. The study was performed using the Chinese Hamster Ovary (CHO) in the presence and absence of S9 metabolic activation system. The test chemical was used at dose level of 12.5–175 µg/ ml. The test chemical induced structural chromosomal aberrations in Chinese Hamster Ovary (CHO) in the absence of S9 metabolic activation system. It however did not induce structural and numerical chromosomal aberration in the cell line in the presence of S9 metabolic activation system. Based on the details of the study, the test chemical in non mutagenic in vitro.

In vitro mammalian cell gene mutation assay:

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 with and without 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 and absence of metabolic activation.

In vitro mammalian cell gene mutation assay was also performed to determine the mutagenic nature of the test chemical. The study was performed using mouse lymphoma cells (L5178Y T/K +/- 3.7.2c). It included 4 hours treatment in the absence and presence of S9-mix and a 24 hours treatment in the absence of S9-mix. The concentrations were selected based on a preliminary toxicity test. The test groups included single replicates at 8 concentrations ranging from 200 to 900 μg/ml in the 4 hours treatment arm and from 4.1 to 520 μg/ml in the 24 hours treatment arm. The maximum concentration was limited by toxicity. The substance did not induce biologically or statistically significant increases in mutant frequency and therefore it was considered non-mutagenic in this assay. The study is compliant with OECD Guideline 476.

Based on the details available, the test chemical did not induce gene mutation in the mammalian cell line used and hence it is not likely to classify as a gene mutant in vitro.

Gene mutation in vivo:

A mouse micronucleus test was also performed with the test chemical in order to evaluate the biological significance of the positive in vitro chromosome aberration assay. The test chemical at doses of 300, 600, or 1200 mg/kg in corn oil was administered by intraperitoneal injection to male and female ICR mice (5/sex/dose). Reductions (up to 21%) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the a-ionone treated groups relative to the respective vehicle controls. These reductions suggest the bioavailability of the test chemical to the bone marrow. There were no statistically significant increases in the incidence of micronucleated polychromatic erythrocytes in the test chemical treated groups relative to their respective vehicle control in either male or female mice, regardless of dose level or bone marrow collection time. The test chemical was concluded to be negative in the mouse micronucleus assay and hence is considered to be non-mutagenic in vivo.

Based on the data summarized for the target chemical in vitro and the supporting in vivo study, the test chemical alpha- ionone (CAS no 127 -41 -3) does not exhibit gene mutation in vitro and in vivo. Hence the test chemical is considered to be non-mutagenic in vitro and in vivo as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the data summarized for the target chemical in vitro and the supporting in vivo study, the test chemical alpha- ionone (CAS no 127 -41 -3) does not exhibit gene mutation in vitro and in vivo. Hence the test chemical is considered to be non-mutagenic in vitro and in vivo as per the criteria mentioned in CLP regulation.