Reaction mass of 4-(2,6,6-trimethylcyclohex-1-ene-1-yl)-but-3-ene-2-one and 4-(2,6,6-trimethylcyclohex-2-ene-1-yl)-but-3-ene-2-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

This study was performed to investigate the potential of the test chemical to induce gene mutation according to Ames metabolic activation test. The test chemical was dissolved in Dimethylsulphoxide (DMSO) and used at dose levels of 10, 1, 0.1, 0.01 µl/well (Bacteriostatic test) and 1, 0.1, 0.01, 0.001 µl/well (Mutation test). The size of zones of inhibition caused by the test chemical with the five tester strains. In the bacteriostatic test, the test chemical showed toxicity towards the bacteria, therefore a top concentration of 1 µl/plate was chosen for the mutation study. At the higher concentrations, the test chemical proved toxic to the cells resulting in either the absence or incomplete formation of a bacterial lawn. No substantial increases in the revertant colony numbers of any of the five strains were observed following treatment with the test chemical at any dose level, either in the presence or absence of liver microsomal fraction (S-9 mix) and hence it is not likely to classify as a gene mutant in vitro.

Chromosome aberration study:

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

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 study was performed using test chemical being dissolved in DMSO and used at dose levels of 0 or 25 nM. 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 test chemical 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. 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. The test chemical induced chromatid gaps, chromatid and chromosome breaks, ring, dicentric chromosomes and chromatid exchanges. Based on the observations made, the test chemical induced chromosomal aberration in the CH cell line B241 and hence is likely to be mutagenic.

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 12.5–175µg/ ml. The test chemical induced gene mutation in Chinese Hamster Ovary (CHO) in the absence of S9 metabolic activation system. It however did not induce gene mutation in the cell line in the presence of S9 metabolic activation system.

Based on the observations made, the test chemical induced chromosomal aberration in the mammalian cell line used in the presence and absence of  S9 metabolic activation system and hence is likely to be mutagenic.

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 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.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Justification for type of information:

Data is from study report.

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

This study was performed to investigate the potential of the test chemical to induce gene mutation according to Ames metabolic activation test.

GLP compliance:

not specified

Type of assay:

other: Bacterial gene mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data available

Metabolic activation:

with and without

Metabolic activation system:

liver microsomal fraction (S-9 mix).

Test concentrations with justification for top dose:

Bacteriostatic test: 10, 1, 0.1, 0.01 µl/well
Mutation test: 1, 0.1, 0.01, 0.001 µl/well

In the Bacteriostatic test Ionone showed toxicity towards the bacteria, therefore a top concentration of 1 µl/plate was chosen for the mutation study.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test chemical was soluble in 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

other: 4-nitro-o-phenylene-diamine

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

other: 2-amino-anthracene

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: Neutral red

Details on test system and experimental conditions:

No data available

Rationale for test conditions:

No data available

Evaluation criteria:

The plates were observed for an increases in the revertant colony numbers of any of the five strains

Statistics:

No data available

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, 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 potential

Table 1

Bacteriostatic test on the test chemical

Strain S. typhimurium	Concentration of Ionone (µl/well)	Zone of inhibition on his^-medium well (mm)
TA 1535	10 1 0.1 0.01 0	28 14 10 10 10
TA 1537	10 1 0.1 0.01 0	Strain failed to grow
TA 1538	10 1 0.1 0.01 0	20 14 10 10 10
TA 98	10 1 0.1 0.01 0	25 17 10 10
TA 100	10 1 0.1 0.01 0	29 12.5 10 10 10

* N.B. well diameter = 10 mm

Table 2

Revertant colony counts obtained per plate using S. typhimurium strains TA 1535, TA 1537 and TA 1538

 

Strain S. typhimurium	Concentration of test material (µl/plate)	Metabolic activation	Mean revertant colony counts	Individual revertant colony counts
TA 1535	1 0.1 0.01 0.001 0 1 0.1 0.01 0.001 0	- - - - - + + + + +	NL IL 12 16 13 NL 10 10 16 12	NL IL 13, 10, 12 19, 16, 13 16, 12, 10 NL 9, 7, 14 10, 11, 10 17, 15, 16 12, 10, 14
TA 1537	1 0.1 0.01 0.001 0 1 0.1 0.01 0.001 0	- - - - - + + + + +	NL NL 5 6 7 NL IL 8 6 8	NL NL 7, 4, 5 7, 6, 5 7, 7, 6 NL IL 8, 8, 8 6, 7, 6 6, 8, 10
TA 1538	1 0.1 0.01 0.001 0 1 0.1 0.01 0.001 0	- - - - - + + + + +	NL IL 16 15 14 NL 13 14 15 14	NL IL 16, 18, 15 13, 16, 17 14, 11, 18 NL 17, 10, 12 12, 13, 18 15, 13, 16 16, 15, 11

- = absence

+ = presence

NL = no bacterial lawn

IL = incomplete bacterial lawn

 

Table 3

Revertant colony counts obtained per plate using S. typhimurium strains TA 98 and TA 100

Strain S. typhimurium	Concentration of test material (µl/plate)	Metabolic activation	Mean revertant colony counts	Individual revertant colony counts
TA 98	1 0.1 0.01 0.001 0 1 0.1 0.01 0.001 0	- - - - - + + + + +	NL NL 32 31 29 NL 22 34 34 34	NL NL 30, 33, 34 30, 29, 34 30, 30, 28 NL 19, 21, 27 36, 29, 38 33, 31, 37 31, 33, 37
TA 100	1 0.1 0.01 0.001 0 1 0.1 0.01 0.001 0	- - - - - + + + + +	NL NL 69 69 73 NL 44 71 71 68	NL NL 67, 69, 71 62, 69, 75 72, 77, 71 NL 38, 47, 47 75, 69, 69 70, 71, 73 65, 71, 69

- = absence

+ = presence

NL = no bacterial lawn

IL = incomplete bacterial lawn

Table 4

Mutability and sterility tests with S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Strain S. typhimurium	Compound	Concentration of compound (µg/plate)	Metabolic activation	Mean revertant colony counts	Individual revertant colony counts
TA 1535   TA 1537   TA 1538   TA 98   TA 100   TA 1535   TA 1537   TA 1538   TA 98   TA 100   -   -	Sodium azide   4-nitro-o-phenylene-diamine ,,   ,,   Sodium azide   2-amino-anthracene   Neutral red   2-acetyl-aminofluorene   2-amino-anthracene   2-amino-anthracene   S-9 mix  Reaction mass of 4-(2,6,6-trimethylcyclohex-2-ene-1-yl)-but-3-ene-2-one and -(2,6,6-trimethylcyclohex-1-ene-1-yl)-but-3-ene-2-one	5   500   ,,   ,,   5   2   10   20   2   2   500 µl   1.0 µl	-   -   -   -   -   +   +   +   +   +       -	825   96   194   2273   745   247   146   709   840   445   0   0	827, 824, 824   80, 92, 117   195, 183, 205   2020, 2328, 2470   716, 771, 749   258, 243, 239   151, 144, 142   697, 705, 725   797, 859, 863   456, 409, 471   0   0

- = absence

+ = presence

Table 5

Validity tests with S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 – colonies per plate

Dilution**	Strain S. typhimurium
	TA 1535	TA 1537	TA 1538	TA 98	TA 100
10^-1D	17	12	17	45	72
10^-6R	*	*	*	*	*
10^-7R	209	520	225	69	521
10^-8R	17	60	38	7	65

 

D = plated onto histidine deficient agar

R = plated onto histidine rich agar

* = too many colonies for accurate counting

** = 3 ml of each dilution per plate

Conclusions:

No evidence of mutagenic potential of the test chemical was obtained in this bacterial test system at the dose levels used using Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

This study was performed to investigate the potential of the test chemical to induce gene mutation according to Ames metabolic activation test. The test chemical was dissolved in Dimethylsulphoxide (DMSO) and used at dose levels of 10, 1, 0.1, 0.01 µl/well (Bacteriostatic test) and 1, 0.1, 0.01, 0.001 µl/well (Mutation test). The size of zones of inhibition caused by the test chemical with the five tester strains. In the bacteriostatic test, the test chemical showed toxicity towards the bacteria, therefore a top concentration of 1 µl/plate was chosen for the mutation study. At the higher concentrations, the test chemical proved toxic to the cells resulting in either the absence or incomplete formation of a bacterial lawn. No substantial increases in the revertant colony numbers of any of the five strains were observed following treatment with the test chemical at any dose level, either in the presence or absence of liver microsomal fraction (S-9 mix) and hence it is not likely to classify as a gene mutant in vitro.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

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 test chemicals

Justification for type of information:

Data for the target chemical is summarized based on the various test chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical

GLP compliance:

not specified

Type of assay:

other: In vitro mammalian chromosome aberration study

Target gene:

No data

Species / strain / cell type:

mammalian cell line, other: original CH cell line B241

Remarks:

1

Details on mammalian cell type (if applicable):

- Type and identity of media:
Type and Identity of media: original 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, the cell line was free from mycoplasma and viruses

- Periodically checked for karyotype stability: Yes, 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

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

2

Details on mammalian cell type (if applicable):

No data

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Rat-liver microsome (S9) was prepared from Sprague-Dawley rats treated with Aroclor 1254

Test concentrations with justification for top dose:

1. 0 or 25 nM
2. 12.5–175 µg/ ml

Vehicle / solvent:

1. - Vehicle(s)/solvent(s) used: DMSO, chemicals were dissolved in DMSO at a concentration of 50 mM, and then were diluted with the medium.
- Justification for choice of solvent/vehicle: The test chemical was solube in DMSO

2. No data

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

benzo(a)pyrene

other: Aflatoxin B1

Remarks:

1

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:

2

Details on test system and experimental conditions:

1. METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data available
- Exposure duration: 24 hrs
- Expression time (cells in growth medium): 48 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): Geimsa stain

SPINDLE INHIBITOR (cytogenetic assays): Colchicine

STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: 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

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

OTHER:
Chromosome test in culture stages between the 5th and 8th passages was performed.

2. METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data
- Exposure duration: No data
- Expression time (cells in growth medium): No data
- 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): Geimsa stain

SPINDLE INHIBITOR (cytogenetic assays): No data

STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: No data

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:

The cell line was observed for chromosome aberrations

Statistics:

No data

Species / strain:

mammalian cell line, other: Original CH cell line B241

Remarks:

1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

2

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

2

Metabolic activation:

with

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

Conclusions:

The test chemical induced chromosomal aberration in the mammalian cell line used in the presence and absence of S9 metabolic activation system and hence is likely to be mutagenic.

Executive summary:

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

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 study was performed using test chemical being dissolved in DMSO and used at dose levels of 0 or 25 nM. 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 test chemical 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. 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. The test chemical induced chromatid gaps, chromatid and chromosome breaks, ring, dicentric chromosomes and chromatid exchanges. Based on the observations made, the test chemical induced chromosomal aberration in the CH cell line B241 and hence is likely to be mutagenic.

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 12.5–175µg/ ml. The test chemical induced gene mutation in Chinese Hamster Ovary (CHO) in the absence of S9 metabolic activation system. It however did not induce gene mutation in the cell line in the presence of S9 metabolic activation system.

Based on the observations made, the test chemical induced chromosomal aberration in the mammalian cell line used in the presence and absence of  S9 metabolic activation system and hence is likely to be mutagenic.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

05-06-2014 to 06-03-2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Principles of method if other than guideline:

The purpose of this study was to assess toxic and genotoxic effects of the test chemical on Chinese Hamster Ovary (CHO) cells by using several different in vitro-based assays, including genotoxicity tests based on the OECD Guideline No. 476 “In Vitro Mammalian Cell Gene Mutation Test”.

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Cells deficient in hypoxanthine-guanine phosphoribosyl transferase (HPRT) due to the mutation HPRT+/- to HPRT-/- are resistant to cytotoxic effects of 6-thioguanine (TG). HPRT proficient cells are sensitive to TG (which causes inhibition of cellular metabolism and halts further cell division since HPRT enzyme activity is important for DNA synthesis), so mutant cells can proliferate in the presence of TG, while normal cells, containing hypoxanthine-guanine phosphoribosyl transferase cannot.

This in vitro test is an assay for the detection of forward gene mutations at the in hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosomes of hypodiploid, modal No. 20, CHO cells. Gene and chromosome mutations are considered as an initial step in the carcinogenic process.
The hypodiploid CHO cells are exposed to the test item with and without exogenous metabolic activation. Following an expression time the descendants of the treated cell population are monitored for the loss of functional HPRT enzyme.
HPRT catalyses the transformation of the purine analogues 6-thioguanine (TG) rendering them cytotoxic to normal cells. Hence, cells with mutations in the HPRT gene cannot phosphoribosylate the analogue and survive treatment with TG.

Therefore, mutated cells are able to proliferate in the presence of TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 days.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

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:

without

Metabolic activation system:

S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats

Test concentrations with justification for top dose:

0, 1, 2.5, 5 or 10 mM

Vehicle / solvent:

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.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylnitrosurea

Remarks:

7, 12-dimethylbenzanthracene was the positive control substance in the tests done with S9

Details on test system and experimental conditions:

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

Rationale for test conditions:

No data

Evaluation criteria:

The plates were scored for total number of colonies

Statistics:

No data

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No data

Remarks on result:

other: No mutagenic potential

Table 1A. Effect of the test chemical exposure on gene toxicity in CHO cells. After being exposed to the test chemical for 3 hrs, cells was washed with sterile PBS and then incubated for 7 days at 37°C, 5% CO₂. After 7 days, cells were re-seeded in new 6-well plates in the absence or presence of 10mM TG as a selection agent and returned to the incubator for 14 days at 37°C, 5% CO₂. On day 15, all 6-well plates were stained with crystal violet and the number of colonies were counted manually. The results are presented as the total number of colonies found in the number of independent wells analyzed (e.g. 0 colonies in 4 wells will give 0/4) (n = 2 samples from 2 independent cultures).

 

	With S9		Without S9
	with TG	without TG	with TG	without TG
Neg. control	0/4	182/4	0/4	174/4
Pos. control	1/4	164/4	14/4	113/4
1.0 mM	0/4	188/4	0/4	88/4
2.5 mM	0/4	30/4	0/4	37/4
5.0 mM	1/4^a	0/4	0/4	0/4
10.0 mM	0/4	0/4	0/4	0/4

 

^a)One very diffuse colony was found in one single well.

 

 

Table 1B. Mutation frequency in CHO cells after 3 hrs of exposure to the test chemical in the absence or presence of 4% S9 liver microsomal fraction. N/A, no colonies present in the samples selected with TG, i.e. no mutation frequency could be determined.

 

	With S9	Without S9
Neg. control	N/A	N/A
Pos. control	-3.73 x10^-4	3.75x10^-4
1.0 mM	N/A	N/A
2.5 mM	N/A	N/A
5.0 mM	N/A^a	N/A
10.0 mM	N/A	N/A^a

 

^a)Since only one very diffuse colony was found in one single well (see Table 1A), this diffuse colony were not regarded as a reliable and true colony since the cells seemed to be apoptotic.

Conclusions:

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.

Executive summary:

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 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 of metabolic activation.

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:

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

Ames assay:

This study was performed to investigate the potential of the test chemical to induce gene mutation according to Ames metabolic activation test. The test chemical was dissolved in Dimethylsulphoxide (DMSO) and used at dose levels of 10, 1, 0.1, 0.01 µl/well (Bacteriostatic test) and 1, 0.1, 0.01, 0.001 µl/well (Mutation test). The size of zones of inhibition caused by the test chemical with the five tester strains. In the bacteriostatic test, the test chemical showed toxicity towards the bacteria, therefore a top concentration of 1 µl/plate was chosen for the mutation study. At the higher concentrations, the test chemical proved toxic to the cells resulting in either the absence or incomplete formation of a bacterial lawn. No substantial increases in the revertant colony numbers of any of the five strains were observed following treatment with the test chemical at any dose level, either in the presence or absence of liver microsomal fraction (S-9 mix) and hence it is not likely to classify as a gene mutant in vitro.

In another study, Ames assay was performed to investigate the potential of the test chemical to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98,TA 100, and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate. Reduced background growth was observed in strain TA 1537 in experiment I. In experiment II, reduced background growth was observed in all strains used. Toxic effects, evident as a reduction in the number of revertants, were observed in strain TA 102 with and without metabolic activation and in strains TA1537 and TA 100 with metabolic activation in experiment I. In experiment II, toxic effects were observed with and without metabolic activation activation in all strains used. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test chemical at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test chemical is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Reverse mutation assay was also performed to determine the mutagenic nature of the test chemical. The study was performed as per the plate incorporation assay using Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 with and without S9 metabolic activation system. The test chemical was dissolved in ethanol at dose levels of 0, 10, 33, 100, 333, 1000, 2500 and 5000µg /plate. Reduced background growth was observed in strain TA1537 at 1000µg/plate and above with metabolic activation. Toxic effects, evident as a reduction in the number of revertants, were observed in strain TA102 with and without metabolic activation at 5000µg/plate and in strains TA1537 (100–5000µg/plate) and TA100 at 1000µg/plate with metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains at any concentration level in the presence or absence of metabolic activation. The test chemical did not induce gene mutation in Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Ames assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA100, TA1535, TA1538, TA98 and TA1537 with and without S9 metabolic activation system. The test chemical was dissolved in DMSO at dose levels of 0.001, 0.01, 0.1 and 1.0 µg /plate. No mutagenic effects were observed with 0.001 and 0.01µg/plate. At 0.1 and 1.0µg/plate, ionone was toxic to the bacteria. The test chemical did not induce gene mutation in Salmonella typhimurium strains TA100, TA1535, TA1538, TA98 and TA1537 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In yet another study, Mutation test was performed to determine the mutagenic nature of the test chemical. The study was performed using Escherichia coli WP2 uvrA at dose levels of 2.5- 20 mg/plate. The test chemical did not increase in the mutation frequency of trp+ revertants and hence it is not likely to classify as a gene mutant in vitro.

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 study was performed using test chemical being dissolved in DMSO and used at dose levels of 0 or 25 nM. 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 test chemical 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. 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. The test chemical induced chromatid gaps, chromatid and chromosome breaks, ring, dicentric chromosomes and chromatid exchanges. Based on the observations made, the test chemical induced chromosomal aberration in the CH cell line B241 and hence is likely to be mutagenic.

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 gene mutation in Chinese Hamster Ovary (CHO) in the absence of S9 metabolic activation system. It however did not induce gene mutation in the cell line in the presence of S9 metabolic activation system.

In vitro mammalian cell gene mutation assay:

An in vitro mammalian cell gene mutation study was designed and conducted to determine thegenotoxicity profile of the test chemical when administered to Chinese Hamster Ovary (CHO) cells. A preliminary dose-finding study was conducted prior to the main study. A range of different ionone concentrations were tested in 96-well plates and analyzed by two commonly used assays, i.e. the colorimetric assay of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)and the bicinchoninic acid(BCA) assay to assess cell viability and protein concentration, respectively. From the basis of the results from the MTT and BCA assays, test concentrations of the test chemical was chosen to be included in the gene toxicity test. In the genotoxicity test, the test chemical was administered to CHO cells for 3 hrs at the dose levels of 0, 1.0, 2.5, 5.0 or 10.0 mM and in the absence or presence of exogenous metabolic activation. CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such asN-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test. The results showed indication of gene mutations occurring in the positive controls ENU and 7,12-dimethylbenz(a) anthracene while no other treatment gave rise to gene toxicity. One very diffuse colony was seen in one well out of four at 5.0 mM and in the presence with 4% S9 liver microsomal fraction. This diffuse colony is not regarded to be relevant since the spot was only mildly colored by crystal violet, thus indicating that it was a small cluster of apoptotic cells taking their last breath instead of cells surviving the TG-selection. Treatment with the test chemical showed evidence of cytotoxicity when CHO cells were exposed to the test chemical in the concentrations of 1, 2.5, 5 or 10 mM. Since no genotoxicity was observed in either of the above mentioned concentrations, the results indicate that the induced cytotoxicity may be induced by an alternative cellular route, e.g. impaired mitochondrial respiration. Based on the results of the current study, it is concluded that the test chemical does not give rise to gene mutations when exposed to the test chemical at ≤ 10.0 mM for 3 hrs or more, however, it has the ability to induce cytotoxic effects at concentrations ≥ 1.0 mM.

umu test was conducted to detect the induction of DNA repair in order to analyze the genotoxicity of by-products of ozonation i.e the test chemical. The study was performed using Salmonella typhimurium TA1535/pSK1002 in the presence and absence of S9 metabolic activation system at dose level of 462.9µg/mL (100 mg/L). The overnight cultivation of the test strain in LB broth was diluted 50-fold into TGA medium and was incubated at 37 deg C for 2 hours with 145 rpm reciprocal shaker. The culture (TGA medium) was subdivided into 4.8 ml portions in test tubes, and 0.2 ml of the test compound was added to each tube. Then, either 1.0 ml of 0.1M phosphate buffer (pH7.4) or S9 mixture containing 100µI of S9 microsomal fraction for metabolic activation was added. After 2 hours of incubation at 37 deg C with shaking, β-galactosidase activity in the cells was assaye by the Modified Miller’s method. The bacterial density was measured at OD600. 0.4 ml fractions of the culture were diluted with 3.6 ml of Z buffer, and the bacterial. Cells were made permeable to the chromogenic substrate for β-galactosidase by adding 100µl sodium dodecyl sulfate (SDS) and 20µlchloroform, and then mixing vigorously. The enzyme reaction was initiated by the addition of 0.8 ml of 2-nitrophenyl-S-D-galactopyranoside solution (4 mg/ml in 0.1 M phosphate buffer, pH 7.0) at 28 deg C. After 15 min, the reaction was stopped by adding 2 ml of 1 M Na2C03 and the absorbance at 0042 and OD550 was measured by spectrophotometer. In the umu test conducted, the test chemimcal induced DNA repair in Salmonella typhimurium TA1535/pSK1002 in the absence of S9 metabolic activation system. It was however toxic to the cells in the presence of S9 metabolic activation system and resulted in their killing.

In another study, the test chemical was tested for its genotoxic potential in the spore rec-assay using Bacillus subtilis strains M45 (rec) and H17 (rec+). DNA damaging activity was measured by differences in growth inhibition zones. The test chemical at the maximal dose of 20µl/disk in dimethyl sulfoxide (DMSO) was positive.

Based on the data available and available the weight of evidence approach, the test chemical does not exhibit gene mutation in vitro. Some mutagenic nature though has been indicated in the chromosomal aberration and DNA damage studies but the higher mammalian cell gene mutation assay studies indicate its mutagenic nature. Hence, the test chemical is likely to be not classified as a gene mutant as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the data available and available the weight of evidence approach, the test chemical does not exhibit gene mutation in vitro. Some mutagenic nature though has been indicated in the chromosomal aberration and DNA damage studies but the higher mammalian cell gene mutation assay studies indicate its mutagenic nature. Hence, the test chemical is likely to be not classified as a gene mutant as per the criteria mentioned in CLP regulation.