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

The 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:

The registered substance, i.e., 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 (EC No. 907-706-6) was tested clastogenic (positive) in an in vitro mammalian chromosomal aberration test both in the presence and absence of microsomal S9 metabolic activation system using CHO cells. The test was performed according to OECD TG 473 (Adopted: 29 July 2016) and performed in compliance with OECD Principles of Good Laboratory Practice.

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:

key study

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 gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

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:

not specified

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.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

Adopted: July 29 2016

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Purity: 98.87 %

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Source: NCCS, Pune, India

Metabolic activation:

with and without

Metabolic activation system:

Cofactor-supplemented liver S9 microsomal fraction was used. S9 fraction was obtained from Aroclor 1254-induced rats.
Before treatment freshly prepared S9 mix was used, an appropriate quantity of S9 fraction was thawed and mixed with co-factor solution to obtain a final concentration of 1% v/v.

S9 mix composition:
Glucose-6-phosphate (180 mg/ml): 1ml
NADP (25 mg/ml): 1%
Potassium chloride (150 mM): 1ml
S9 Fraction (ml): 2 ml
Final volume: 5ml

Test concentrations with justification for top dose:

Doses:
0 mg/ml (Negative control)
0 mg/ml (Solvent control)
0.0078125 mg/ml
0.015625 mg/ml
0.03125 mg/ml

Justification:
Cytotoxicity was determined in a preliminary cytotoxicity test using the cytotoxicity parameter, RICC (Relative Increase in Cell Counts). The concentration that yielded 55±5 % cytotoxicity i.e. reduction in RICC to 45±5% of the concurrent negative control, was selected as the highest test concentration. At 0.03125 mg/ml, the cytotoxicity was 54.17% and 57.14% in the absence and presence of S9 metabolic activation, respectively. Hence, 0.03125 mg/ml was selected as the highest test concentration for the cytogenicity test.

Vehicle / solvent:

Dimethyl sulfoxide (DMSO) was used as solvent.

Untreated negative controls:

yes

Remarks:

Distilled water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Single cultures were used.
- Number of independent experiments: Phase I-III.

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1 x 10E6 cell/flask
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk: The test substance was added in medium.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: NA
- Exposure duration/duration of treatment: Phase I-II: 4 hours, Phase III: 24 hours

- Harvest time after the end of treatment (sampling/recovery times): Phase I-II: 20 hours, Phase III: No recovery time

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): colchicine (final conc: 1 µg/ml) for 2 hours

- Methods of slide preparation and staining technique used, including the stain used (for cytogenetic assays): The cell suspension was dropped on a clean chilled slide by hanging drop method and kept drying on a slide warmer. Duplicate slides were prepared from each culture. Slides were stained with freshly prepared 5 % Giemsa stain for 5 minutes and mounted with DPX.


- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): At least 300 well-spread metaphases per concentration (single culture) were analysed using 100x magnification for the incidence of structural aberrations.


- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Cells with structural chromosomal aberration(s) including and excluding gaps were scored. Chromatid and chromosome-type aberrations were recorded separately and classified by sub-types (breaks, exchanges).

- Determination of polyploidy: Not observed.
- Determination of endoreplication: Not observed.


METHODS FOR MEASUREMENT OF CYTOTOXICITY

Cytotoxicity of the test substance was determined by the Relative Increase in Cell Counts (RICC). In the preliminary cytotoxicity assay, CHO cells were exposed to 0 (NC), 0 (VC), 0.03125, 0.0625, 0.125, 0.25, 0.5, 1.0 and 2.0 mg/ml of test substance both in the presence and absence of S9 metabolic activation using single cultures. The concentration that yielded 55±5 % cytotoxicity using RICC ( i.e. reduction in RICC to 45±5% of the concurrent negative control) was selected as the highest test concentration. The cytotoxicity was 54.17% and 57.14% at 0.03125 mg/ml in the absence and presence of S9 metabolic activation, respectively. Hence, 0.03125 mg/ml was selected as the highest test concentration for the cytogenicity test.

Evaluation criteria:

The test Item is considered to be clearly positive if, in any of the experimental conditions examined:
- At least one of the test concentrations exhibits a significant increase compared with the concurrent negative control,
- The increase is dose-related when evaluated with an appropriate trend test.
- Any of the results are outside of the distribution of the laboratory historical negative control database.
When all these criteria are met, the Test Item is then considered able to induce chromosomal aberrations in cultured mammalian cells in this test system.

The test Item is considered clearly negative if, in all experimental conditions examined:
- None of the test concentrations exhibits a significant increase compared with the concurrent negative control.
- There is no concentration-related increase when evaluated with an appropriate trend test.
- The results are inside the distribution of the laboratory historical negative control databas.

Statistics:

Fisher's Exact Test (NCSS statistics software) was used. The percentage of aberrant cells from the Test Item treated group was compared to the solvent control groups. A trend was judged as significant whenever the p-value (probability value) was below 0.05.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity (%) was 50.98% (Phase I, -S9 mix) 55.67% (Phase I, +S9 mix) and 55.05% (Phase III, -S9 mix) at 0.03125 mg/ml.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The test substance did not affect pH after 0 and 4 hours incubation in the cell culture medium.
- Data on osmolality: Not examined.
- Possibility of evaporation from the medium: Not examined.
- Water solubility: The substance was not soluble in water.
- Precipitation and time of the determination: The test substance did not form precipitation up to 2 mg/ml using dimethyl sulfoxide as a vehicle.

RANGE-FINDING/SCREENING STUDIES (if applicable):
In the preliminary cytotoxicity test, CHO cells were exposed to 0 (NC, distilled water), 0 (VC, DMSO), 0.03125, 0.0625, 0.125, 0.25, 0.5, 1 and 2 mg/ml for 4 hours (Phase I-II), both in the presence and absence of S9 metabolic activation using single cultures. Cytotoxicity was determined by the decrease of Relative Increase in Cell Counts (RICC) in treated cultures compared to the vehicle control. The concentration which yielded 55±5% cytotoxicity (i.e. reduction in RICC to 45±5% of the concurrent control) was selected as the highest test concentration for the cytogenicity test. The cytotoxicity (%) was 54.17%, 57.14 % at 0.03125 mg/ml in the absence and presence of S9 metabolic activation, respectively. Hence, the concentration of 0.03125 mg/ml was chosen as the highest test concentration in the cytogenicity test.

STUDY RESULTS
- Concurrent vehicle negative and positive control data:
There was no increase in the percent aberrant cells in the vehicle control compared to the negative control either in the presence and absence of S9 metabolic activation. The positive controls (-S9: methyl methanesulfonate, +S9: Benzo[a]pyrene) produced statistically significant increases in the percent aberrant cells compared to the vehicle control. This increase indicated the sensitivity of the test system to specific mutagens and confirmed that the test conditions were appropriate and that the metabolic activation system functioned properly.
- Statistical analysis; p-value if any: p values at each concentration were the follows when the percent aberrant cells in test substance-treated culture was compared to the vehicle control.
Phase I:
0.0078125 mg/ml: p=0.1229
0.015625 mg/ml: p=0.0378
0.03125 mg/ml: p=0.0206
Phase III:
0.0078125 mg/ml: p=0.1238
0.015625 mg/ml: p=0.0151
0.03125 mg/ml: p= 0.0018

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements:
Cytotoxicity was determined by the decrease in the Relative Increase in Cell Counts (RICC) in treated cultures compared to the vehicle control. The RICC values were 49.02 % (cytotoxicity: 50.98%, [-S9 mix]), 44.33% (cytotoxicity: 55.67% [+S9 mix]) and 44.95% (cytotoxicity:55.05% [-S9 mix]) at 0.03125 mg/ml in Phase I, Phase II and Phase III, respectively.

- Genotoxicity results (for both cell lines and lymphocytes):
In Phase I, there was no significant increase in the mean percent aberrant cells at 0.0078125 mg/ml (mean % aberrant cells was 2.00%, p=0.1229) when compared to vehicle control (mean % aberrant cells: 0.33 %). A significant increase in mean percent aberrant cells was observed at 0.015625 (mean % aberrant cells: 2.67%, p=0.0378) and 0.03125 mg/ml (mean % aberrant cells: 3.00%, p=0.0206) when compared to vehicle control (mean % aberrant cells: 0.33 %).
In Phase II, the mean percent aberrant cells was 0.33% (at 0.0078125 mg/ml), 0.67% (at 0.015625 mg/ml) and 1.33% (at 0.03125 mg/ml) compare with vehicle control (the mean % aberrant cells: 0.67 %). The observed changes were not statistically significant, but a trend for an increase in percent aberrant cells at increasing concentration was detected, which was considered biologically relevant.
In Phase III, an increase in the mean percent aberrant cells was observed at 0.0078125 mg/ml (the mean % aberrant cells: 1.33, p=0.1238). This increase was not statistically significant, but it was considered biologically relevant. Significant increases in mean percent aberrant cells were observed at 0.015625 (the mean % aberrant cells: 2.33%, p=0.0151) and 0.03125 mg/ml (the mean % aberrant cells: 3.33%, p=0.0018) when compared to vehicle control (the mean % aberrant cells 0.00 %).
HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: No data available.
- Negative (solvent/vehicle) historical control data: No data available.

Preliminary cytotoxicity assay

Dose Level	Conc. (mg/ml)	Absence of Metabolic activation				Presence of Metabolic activation
		Cell count		RICC	% Cytotoxicity	Cell count		RICC	% Cytotoxicity
		Starting	Final			Starting	Final
NC	-	1000000	3900000	100.00	0.00	1000000	4200000	100.00	0.00
VC	-	1000000	3400000	82.76	17.24	1000000	3100000	65.63	34.38
T1	0.03125	1000000	2100000	45.83	54.17	1000000	1900000	42.86	57.14
T2	0.0625	1000000	1100000	4.17	95.83	1000000	180000	8.57	91.43
T3	0.125	1000000	0	-	-	1000000	0	-	-
T4	0.25	1000000	0	-	-	1000000	0	-	-
T5	0.5	1000000	0	-	-	1000000	0	-	-
T6	1	1000000	0	-	-	1000000	0	-	-
T7	2	1000000	0	-	-	1000000	0	-	-

Key: NC = Negative Control (distilled water), VC = Vehicle Control (dimethyl sulfoxide), Conc. = Concentration, mg = milligram, ml = milliliter, RICC = Relative Increase in Cell Counts, % = percentage.

Relative Increase in Cell Counts- Main Study

Dose Level	Conc.	Phase I -Absence of Metabolic activation
		Cell count		RICC	% Cytotoxicity
		Starting	Final
NC	-	1000000	3850000	100.00	0.00
VC	-	1000000	3550000	89.47	10.53
T1	0.0078125 mg/ml	1000000	3160000	84.71	15.29
T2	0.015625 mg/ml	1000000	2850000	72.55	27.45
T3	0.03125 mg/ml	1000000	2250000	49.02	50.98
PC	20 µg/ml	1000000	2850000	72.55	27.45

Dose Level	Conc.	Phase II -Presence of Metabolic activation
		Cell count		RICC	% Cytotoxicity
		Starting	Final
NC	-	1000000	2000000	100.00	0.00
VC	-	1000000	1970000	97.00	3.00
T1	0.0078125 mg/ml	1000000	1930000	95.88	4.12
T2	0.015625 mg/ml	1000000	1780000	80.41	19.59
T3	0.03125 mg/ml	1000000	1430000	44.33	55.67
PC	30 µg/ml	1000000	1650000	67.01	32.99

Dose Level	Conc.	Phase III -Absence of Metabolic activation
		Cell count		RICC	% Cytotoxicity
		Starting	Final
NC	-	1000000	4100000	100.00	0.00
VC	-	1000000	3870000	92.58	7.42
T1	0.0078125 mg/ml	1000000	3340000	81.53	18.47
T2	0.015625 mg/ml	1000000	2950000	67.94	32.06
T3	0.03125 mg/ml	1000000	2290000	44.95	55.05
PC	20 µg/ml	1000000	2700000	59.23	40.77

Key: NC = Negative Control (distilled water), VC = Vehicle Control (dimethyl sulfoxide), PC = Positive Control (Phase I and III -Methyl methanesulfonate, Phase II- Benzo[a]pyrene), Conc. = Concentration, mg = milligram, ml = milliliter, µg = microgram, RICC = Relative Increase in Cell Counts, % = percentage.

Individual Data on Chromosome Aberrations- Phase I:Absence of metabolic activation (short term)

Dose level & Concentration	No. of Metaphases	Frequencies of Aberration	Total No of Aberrant cells
			with gap	without gap
NC 0 mg/ml	300	1 Csg, 1 Dic	2	1
VC 0 mg/ml	300	1 Dic	1	1
T1 0.0078125 mg/ml	300	2 Ctb, 1 Csb, 4 Dic	6	6
T2 0.015625 mg/ml	300	1 Csb, 2 Deletion, 5 Dic	8	8
T3 0.03125 mg/ml	300	3 Ctg, 1 Ctb, 3 Fragments, 2 Exchange, 3 Dic	12	9
PC 20 µg/ml	300	1 Ctg, 8 Csb, 2 Deletion, 3 Fragments, 1 Exchange, 8 Dic, 1 Minute	21	21

IndividualData on Chromosome Aberrations- Phase II:Absence of metabolic activation (short term)

Dose level & Concentration	No. of Metaphases	Frequencies of Aberration	Total No of Aberrant cells
			with gap	without gap
NC 0 mg/ml	300	1 Ctg, 1 Csg	2	0
VC 0 mg/ml	300	1 Csb, 1 Dic	2	2
T1 0.0078125 mg/ml	300	1 Ctg, 1 Dic	2	1
T2 0.015625 mg/ml	300	1 Csg, 1 Fragment, 1 Dic	3	2
T3 0.03125 mg/ml	300	1 Ctb, 3 Dic, 1 Minute	4	4
PC 30 µg/ml	300	1 Csg, 3 Ctb, 5 Csb, 5 Fragment, 3 Ring, 1 Exchange, 7 Dic, 1 Minute	21	20

IndividualData on Chromosome Aberrations- Phase III:Absence of metabolic activation (short term)

Dose level & Concentration	No. of Metaphases	Frequencies of Aberration	Total No of Aberrant cells
			with gap	without gap
NC 0 mg/ml	300	-	0	0
VC 0 mg/ml	300	-	0	0
T1 0.0078125 mg/ml	300	1 Ctg, 1 Csg, 4 Dic	6	4
T2 0.015625 mg/ml	300	1 Ctg, 3 Ctb, 2 Fragment, 2 Dic	8	7
T3 0.03125 mg/ml	300	2 Ctb, 2 Deletion, 2 Fragment, 1 Exchange, 4 Dic, 1 Minute	10	10
PC 20 µg/ml	300	2 Ctg, 2 Csg, 4 Ctb, 8 Csb, 7 Fragments,               1 Exchange, 2 Dic	21	20

Key: NC = Negative Control (distilled water), VC = Vehicle Control (dimethyl sulfoxide), PC = Positive Control (methyl methanesulfonate), mg = milligram, µg = microgram, ml = milliliter, T3-T1 = Test Item concentration from higher to lower, Ctg = Chromatid gap, Csg = Chromosome gap, Ctb = Chromatid break, Csb = Chromosome break, Dic = dicentric.

Summary Data on Chromosome Aberrations - Phase I

Dose Level	Concentration	Absence of metabolic activation
		Total No. of Aberrant cells without gap	Percent aberrant cells
NC	Distilled water	1	0.33
VC	Dimethyl sulfoxide	1	0.33
T1	0.0078125 mg/ml	6	2.00
T2	0.015625 mg/ml^*	8	2.67
T3	0.03125 mg/ml^*	9	3.00
PC	20 µg/ml^*	21	7.00

Key: NC = Negative Control, VC = Vehicle Control, PC = Positive Control (methyl methanesulfonate), mg = milligram, ml = milliliter, µg = microgram.* = Statistical significant increase in % aberrant cell (p<0.05)

Summary Data on Chromosome Aberrations - Phase II

Dose Level	Concentration	Presence of metabolic activation
		Total No. of Aberrant cells without gap	Percent aberrant cells
NC	Distilled water	0	0.00
VC	Dimethyl sulfoxide	2	0.67
T1	0.0078125 mg/ml^#	1	0.33
T2	0.015625 mg/ml^#	2	0.67
T3	0.03125 mg/ml^#	4	1.33
PC	30 µg/ml^*	20	6.67

Key: NC = Negative Control, VC = Vehicle Control, PC = Positive Control(Benzo[a]pyrene), mg = milligram, ml = milliliter, µg = microgram,# =A trend for an increase in percent aberrant cells, * = Statistical significant increase in % aberrant cell (p<0.05).

Summary Data on Chromosome Aberrations - Phase III

Dose Level	Concentration	Absence of metabolic activation
		Total No. of Aberrant cells without gap	Percent aberrant cells
NC	Distilled water	0	0.00
VC	Dimethyl sulfoxide	0	0.00
T1	0.0078125 mg/ml	4	1.33
T2	0.015625 mg/ml^*	7	2.33
T3	0.03125 mg/ml^*	10	3.33
PC	20 µg/ml^*	20	6.67

Key: NC = Negative Control, VC = Vehicle Control, PC = Positive Control (methyl methanesulfonate), mg = milligram, ml = milliliter, µg = microgram,* = Statistical significant increase in % aberrant cell (p<0.05)

Conclusions:

The registered substance, i.e., 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 (EC No. 907-706-6) was tested clastogenic (positive) in an in vitro mammalian chromosomal aberration test both in the presence and absence of microsomal S9 metabolic activation system using CHO cells. The test was performed according to OECD TG 473 (Adopted: 29 July 2016) and performed in compliance with OECD Principles of Good Laboratory Practice.

Executive summary:

The registered substance i.e.,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 (EC No. 907-706-6) was tested in an in vitro mammalian chromosomal aberration test according to OECD TG 473 (Adopted: July 29 2016). The test was performed to assess the ability of the substance to cause structural chromosomal aberrations in cultured CHO cells in the presence and absence of an exogenous metabolic activation system.Cofactor-supplemented S9 microsomal fraction, obtained from the liver of Aroclor-1254-induced rats, was used as a metabolic activation system. Dimethyl sulfoxide was selected as a vehicle of the test substance. Test concentrations were chosen based on the solubility, precipitation and pH checks, and a preliminary cytotoxicity assay. The test substance formed no precipitation at 2 mg/ml and did not affect the pH of the medium after 0 and 4 hours of incubation. Based on solubility and precipitation test, the preliminary cytotoxicity test was performed with test substance concentrations of 0.03125, 0.0625, 0.125, 0.5, 1 and 2 mg/ml both in the presence (1 % v/v S9 mix) and absence of S9 metabolic activation along with the negative (distilled water) and vehicle (DMSO) controls using single cultures.Cytotoxicity was determined by the decrease of Relative Increase in Cell Counts (RICC) in treated cultures compared to the vehicle control. The concentration which yielded 55±5% cytotoxicity (i.e. reduction in RICC to 45±5% of the concurrent control) was selected as the highest test concentration for the cytogenicity test. The cytotoxicity (%) was 54.17% and 57.14% at 0.03125 mg/ml in the absence and presence of S9 metabolic activation, respectively. Hence, 0.03125 mg/ml was chosen as the highest test concentration in the cytogenicity test. The chromosome aberration test was performed in three phases;Phase I (4-hours exposure in the absence of metabolic activation), Phase II (4-hours exposure in the presence of metabolic activation) and Phase III (24-hours exposure in the absence of metabolic activation). CHO cells were exposed to 0 (NC), 0 (VC),0.0078125, 0.015625, 0.03125 mg/ml for 4 hours (Phase I-II) and 24 hours (Phase III) both in the presence and absence of S9 metabolic activation. Concurrent positive controls, i.e.,Methyl methanesulfonate (without S9 mix, final conc. 20 µg/ml) and Benzo[a]pyrene (with S9 mix, final conc. 30 µg/ml) were also included in the assay. At least 300 well-spread metaphases per concentration were analyzed using 100x magnification for the incidence of structural aberrations.Breaks (chromatid and chromosomal), gaps (chromatid and chromosomal), rings and fragments, dicentric chromosomes were found as structural chromosome aberrations. Gaps were recorded and reported separately but not included in the total aberration frequency. Results: In Phase I experiment, the average RICC values were 89.47% (VC), 84.71% (at 0.0078125 mg/ml), 72.55% (at 0.015625 mg/ml) and 49.02% (at 0.03125 mg/ml). There was no significant increase in the mean percent aberrant cells at 0.0078125 mg/ml (mean % aberrant cells was 2.00%, p=0.1229) when compared to vehicle control (mean % aberrant cells: 0.33 %). However, there was a significant increase in mean percent aberrant cells at 0.015625 (mean % aberrant cells: 2.67%, p=0.0378) and 0.03125 mg/ml (mean % aberrant cells: 3.00%, p=0.0206) when compared to vehicle control (mean % aberrant cells: 0.33 %).In Phase II experiment, the average RICC values were 97.00 % (VC), 95.88 % (at 0.0078125 mg/ml), 80.41 % (at 0.015625 mg/ml) and 44.33 % (at 0.03125 mg/ml). The mean percent aberrant cells was 0.33% (at 0.0078125 mg/ml), 0.67% (at 0.015625 mg/ml) and 1.33% (at 0.03125 mg/ml) compare with vehicle control (the mean % aberrant cells: 0.67 %). The observed changes were not statistically significant, but a trend for an increase in percent aberrant cells at increasing concentration was detected, which was considered biologically relevant. In Phase III experiment, the average RICC values were 92.58 % (VC), 81.53 % (at 0.0078125 mg/ml), 67.94 % (at 0.015625 mg/ml) and 44.95 % (at 0.03125 mg/ml). An increase in the mean percent aberrant cells was observed at  0.0078125 mg/ml (the mean % aberrant cells: 1.33, p=0.1238). This increase was not statistically significant, but it was considered biologically relevant. Significant increases in mean percent aberrant cells were observed at 0.015625 (the mean % aberrant cells: 2.33%,p=0.0151) and 0.03125 mg/ml (the mean % aberrant cells: 3.33%,p=0.0018) when compared to vehicle control (the mean % aberrant cells 0.00 %). In all phases, no significant reduction in RICC (cytotoxicity) was observed in the vehicle control (dimethyl sulfoxide) when compared to the negative control (distilled water) either in the presence and absence of S9 metabolic activation. A slight increase in the percent aberrant cells in vehicle control (the mean % aberrant cells 0.67 %) was observed in Phase II compared to the negative control (the mean % aberrant cells 0.00 %). No other increase in the percent aberrant cells was noted in vehicle control compared to the negative control in the presence and absence of S9 metabolic activation.Conclusion:The registered substance, i.e.,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 (EC No. 907-706-6) induced chromosomal aberration at ≥0.03125 mg/ml of culture medium, both in the presence and the absence of S9 metabolic activation system in CHO cells.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo micronucleus assay:

The test chemical, Reaction mass of  4-(2,6,6-trimethylcyclohex-2-ene-1-yl)-but-3-ene-2-one and 4-(2,6,6-trimethylcyclohex-1-ene-1-yl)-but-3-ene-2-one (EC. no 907-706-6) is not mutagenic in mouse and hence it is not likely to classify as a gene mutant in vivo.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

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

Justification for type of information:

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

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

WoE derived based on the experimental data from various test chemicals

GLP compliance:

not specified

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

other: 2. NMRI, 3. ICR

Details on species / strain selection:

No data

Sex:

male/female

Details on test animals or test system and environmental conditions:

2. Healthy male Crl: NMRI mice (breeder: Charles River, Deutschland GmbH, GER) with a mean weight of about 29g (with an age range of about 5-8 weeks) were used in the test. 5 males/dose were received a single ip injection.

Route of administration:

intraperitoneal

Vehicle:

2. Olive oil was used as a vehicle of the test substance.
3. Corn oil was used as a vehicle of the substance.

Details on exposure:

2. Frequency of dosing: single injection (ip)
Dosing volume: 10 ml/kg bw
Control groups:
negative: 1 x vehicle control (10 ml/kg bw olive oil)
positive: 1 x 20 mg/kg bw cyclophosphamide (CPP) for
clastogenic effects (10 ml/kg bw), 1 x 0.15 mg/kg bw
vincristine (VCR) for aneugenic effcts (10 ml/kg bw)
5 males/dose were received a single ip injection.

3. Groups of 5 ICR mice/sex/dose were administered at doses 0 (VC, corn oil), 300, 600 or 1200 mg/kg by a single intraperitoneal injection. Additional 5 mice of each sex were injected with the vehicle or 1200 mg/kg of test substance served as a satellite group.

Duration of treatment / exposure:

2. 24 hours for treatment groups; 48 hours for satelite groups.
3. Groups of 5 male and 5 female mice from all treatment groups were sacrificed 24 hours after dosing, and the additional 5 mice of each sex from the satellite groups were sacrificed 48 hours after dosing.

Frequency of treatment:

2. A single ip injection was applied in all control and treatment groups.
3. Once by a single intraperitoneal injection.

Post exposure period:

24 hrs (Treatment grous)
48 hrs (Satelite groups)

Remarks:

2. 0 (VC) mg/kg bw/day, 250, 500, 750 mg/kg bw/day, 0 (VC, satelite group), 750 mg/kg bw/day(top dose, satelite group).

Remarks:

3. 0 (VC) mg/kg bw/day, 300, 600, 1200 mg/kg bw/day, 0 (VC, satelite group), 1200 mg/kg bw/day(top dose, satelite group).

No. of animals per sex per dose:

2. Treatment groups: 5 males/dose
Satelite groups: 5 males/ groups

3. Treatment groups: 5 animals/sex/dose
Satelite groups: 5 animals/sex/group

Control animals:

yes, concurrent vehicle

Positive control(s):

2. Cyclophosphamide (CPP) for clastogenic effects (10 ml/kg bw)
Vincristine (VCR) for aneugenic effcts (10 ml/kg bw)

Tissues and cell types examined:

2. Bone marrow PCEs and NMEs.
3. Clinical signs, MNPCE in the bone marrow

Details of tissue and slide preparation:

2. Samples of bone marrow of the 2 femurs were collected. Preparation of the bone marrow was performed according to the method of Schmidt (1976 and 1977) and Salamone et al. (1980).

Evaluation criteria:

2. Microscopic evaluation: 2000 polychromatic erythrocytes (PCEs) from each animal of every test group were investigated for micronuclei (MN). The normochromatic erythrocytes (NCEs) were also scored. The ratio of polychromatic to normochromatic erythrocytes was determined.

Statistics:

2. U-test according to Mann-Whitney (modified rank test according to Wilcoxon) test was used to demonstrate differences between control and dose groups.

Sex:

male

Genotoxicity:

negative

Remarks:

No statistically significant increase in the number of polychromatic erythrocytes containing either small or large micronuclei was noted at any doses tested.

Toxicity:

yes

Remarks:

Clinical signs of toxicity was observed at 500 and 750 mg/kg bw.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Reduction in PCE/NCE ratio at 1200 mg/kg/day was observed 24 and 48 hours after dosing.

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

not specified

2.

Table 1. Effect on PCE/NCE ratio - Mean numbers of PCEs and NCEs

Interval		24 hrs	48 hrs
	PCEs	NCEs	NCEs
Vehicle	10000	4594	3439
250 mg/kg bw	10000	3755
500 mg/kg bw	10000	4646
750 mg/kg bw	10000	2476	2804
CPP (20 mg/kg bw)	10000	3920
VCR (0.15 mg/kg bw)	10000	5374

Table 2. Mean number of PCEs containing MN per 1,000 PCE at 24 hrs (differentiation between small and large micronuclei)

	Small	Large	Total
Vehicle	1.3	0.1	1.4
250 mg/kg bw	1.6	0.0	1.6
500 mg/kg bw	1.7	0.1	1.8
750 mg/kg bw	1.2	0.0	1.2
CPP (20 mg/kg bw)	10.9	0.2	11.1 (p≤0.01)
VCR (0.15 mg/kg bw)	35.7	11.5	47.2 (p≤0.01)

Table 3. Mean number of PCEs containing MN per 1,000 PCE at 48 hrs (differentiation between small and large micronuclei) 

	Small	Large	Total
Vehicle	0.7	0.0	0.7
750 mg/kg bw	1.0	0.0	1.0

Conclusions:

The test chemical, Reaction mass of 4-(2,6,6-trimethylcyclohex-2-ene-1-yl)-but-3-ene-2-one and 4-(2,6,6-trimethylcyclohex-1-ene-1-yl)-but-3-ene-2-one (EC. no 907-706-6) is not mutagenic in mouse and hence it is not likely to classify as a gene mutant in vivo.

Executive summary:

Data available for the various test chemicals was reviewed to determine the mutagenic nature of Reaction mass of  4-(2,6,6-trimethylcyclohex-2-ene-1-yl)-but-3-ene-2-one and 4-(2,6,6-trimethylcyclohex-1-ene-1-yl)-but-3-ene-2-one (EC. no 907-706-6). The studies are as mentioned below:

The read across substance, i.e., (E)-4 -(2,6,6 -trimethyl-1 -cyclohexen-1 -yl)-3 -buten-2 -one (CAS 79 -77 -6), was tested for chromosomal damage (clastogenicity) and for the ability to induce spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method. The test was performed according to OECD TG 474 (1997). Test doses were selected based on an initial experiment. In this pretest for determination of the acute i.p. toxicity, deaths were observed at 2000 mg/kg bw. Mice treated with 750 and 1000 mg/kg bw showed evident signs of toxicity, and some animals were sacrificed moribund. At 500 mg/kg bw, all animals survived, but l signs of clinical toxicity were noted. There were no distinct differences in the symptoms between males and female mice. Thus, only males were used for the cytogenetic investigations. Doses of 750, 500 and 250 mg/kg bw were selected for the main test. The test substance was dissolved in olive oil and was administered once intraperitoneally to male NMRI mice (5 mice/dose) at 0 (VC), 250, 500 and 750 mg/kg bw. Additional 5 mice were injected with the vehicle or 750 mg/kg of test substance served as a satellite group. Samples of the bone marrow of the 2 femurs were taken 24 (treatment groups) and 48 hrs (satelite groups) after the last treatment. 2000 polychromatic erythrocytes (PCEs) from each animal of every test group were investigated for micronuclei (MN). The normochromatic erythrocytes (NCEs) were also scored. The ratio of polychromatic to normochromatic erythrocytes was determined. After administration of the vehicle, test substance and positive controls, the animals were examined for clinical signs of toxicity. U-test according to Mann-Whitney (modified rank test according to Wilcoxon) was performed to confirm differences between control and dose groups.Results:No mortality was observed at any dose level. Clinical signs of toxicity were observed at 500 and 750 mg/kg bw, which included poor general state, irregular respiration, squatting posture. These clinical signs were reversible after 2 days. At 250 mg/kg bw only minor signs of clinical toxicity were observed after 2 and 4 hours of administration of the test substance (squatting posture). No inhibition of erythropoiesis, determined from the PCE/NCE ratio, was detected. The vehicle and the positive control substances, Cyclophosphamide (CPP, 20 mg/kg bw) and vincristine (VCR, 0.15 mg/kg bw), caused no evident signs of toxicity. The following mean number of PCEs and NCEs were observed: vehicle (PCEs: 10000, NCEs: 4594 (24 hrs), 3439 (48 hrs)), at 250 mg/kg bw (PCEs: 10000, NCEs:3755), at 500 mg/kg bw (PCEs: 10000, NCEs: 4646), at 750 mg/kg bw (PCEs: 10000, NCEs: 2476 (24 hrs), 2804 (48 hrs), CPP (PCEs: 10000, NCEs: 3920), VCR (PCEs: 10000, NCEs: 5374). The administration of the test substance did not lead to any statistically significant increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was nearly the range of the concurrent negative control in all dose groups and within the range of the historical control data (mean 1.6, min. 0.3, max. 3.3, SD 0.6; n=393). The positive controls led to the expected increases in micronuclei (either small or large). The mean number of PCEs containing MN per 1,000 PCE at 24 hrs (differentiation between small and large micronuclei) were the follows: vehicle (small: 1.3, large:0.1, total: 1.4), at 250 mg/kg bw (small: 1.6, large: 0.0, total: 1.6), at 500 mg/kg bw (small: 1.7, large: 0.1, total: 1.8), at 750 mg/kg bw (small: 1.2, large: 0.0, total: 1.2), CPP (small: 10.9, large: 0.2, total: 11.1; p ≤0.01), VCR (small: 35.7, large: 11.5 total: 47.2; p≤0.01). The mean number of PCEs containing MN per 1,000 PCE at 48 hrs (small and large and total number of micronuclei) were the follows: vehicle (small: 0.7, large: 0.0, total: 0.7) at 750 mg/kg bw (small: 1.0, large: 0.0, total: 1.0).Conclusion:The source substance, i.e.,(CAS 79-77-6), did not have a chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

In another study, the read across substance, i.e., 4 -(2,6,6 -trimethylcyclohex-2 -ene-1 -yl)-but-3 -ene-2 -one (CAS 127 -41 -3), was tested for chromosomal damage (clastogenicity) using ICR mice in a bone marrow micronucleus. The test was performed according to OECD TG 474 (1997b). Test doses were selected based on an initial range-finding test, where 1200 mg/kg was determined as the maximum tolerated dose (MTD). Groups of male and female ICR mice (5/sex/dose) were administered at doses 0 (VC, corn oil), 300, 600 or 1200 mg/kg bw by a single intraperitoneal injection. Additional 5 mice of each sex were injected with the vehicle or 1200 mg/kg of test substance served as a satellite group. Groups of 5 male and 5 female mice from all treatment levels were sacrificed 24 hours after dosing, and additional 5 mice of each sex from the satellite groups were sacrificed 48 hours after dosing. 2000 Polychromatic (PCE) and normochromatic (NCE) erythrocytes per animal were scored for micronuclei. Reductions of 7 -18 % in PCE were observed in treated males and females 24 hours after dosing; a reduction of 21 % in PCE was observed in males at 1200 mg/kg bw at 48 hours after dosing, indicating bone marrow toxicity. Additional clinical signs in treated animals also confirmed the systemic availability of the test substance. There were no statistically or biologically significant increases in micronucleus frequency in treated animals at dose levels tested compared to the vehicle control. Hence, the source substance, i.e., 4 -(2,6,6 -trimethylcyclohex-2 -ene-1 -yl)-but-3 -ene-2 -one (CAS 127 -41 -3), did not express clastogenic or aneugenic potential in vivo under the experimental conditions described.

Based on the data available the test chemical, Reaction mass of  4-(2,6,6-trimethylcyclohex-2-ene-1-yl)-but-3-ene-2-one and 4-(2,6,6-trimethylcyclohex-1 -ene-1-yl)-but-3-ene-2-one (EC. no 907-706-6) is not mutagenic in mouse and hence it is not likely to classify as a gene mutant in vivo.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity - 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 registered substance i.e.,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 (EC No. 907-706-6) was tested in an in vitro mammalian chromosomal aberration test according to OECD TG 473 (Adopted: July 29 2016). The test was performed to assess the ability of the substance to cause structural chromosomal aberrations in cultured CHO cells in the presence and absence of an exogenous metabolic activation system.Cofactor-supplemented S9 microsomal fraction, obtained from the liver of Aroclor-1254-induced rats, was used as a metabolic activation system. Dimethyl sulfoxide was selected as a vehicle of the test substance. Test concentrations were chosen based on the solubility, precipitation and pH checks, and a preliminary cytotoxicity assay. The test substance formed no precipitation at 2 mg/ml and did not affect the pH of the medium after 0 and 4 hours of incubation. Based on solubility and precipitation test, the preliminary cytotoxicity test was performed with test substance concentrations of 0.03125, 0.0625, 0.125, 0.5, 1 and 2 mg/ml both in the presence (1 % v/v S9 mix) and absence of S9 metabolic activation along with the negative (distilled water) and vehicle (DMSO) controls using single cultures.Cytotoxicity was determined by the decrease of Relative Increase in Cell Counts (RICC) in treated cultures compared to the vehicle control. The concentration which yielded 55±5% cytotoxicity (i.e. reduction in RICC to 45±5% of the concurrent control) was selected as the highest test concentration for the cytogenicity test. The cytotoxicity (%) was 54.17% and 57.14% at 0.03125 mg/ml in the absence and presence of S9 metabolic activation, respectively. Hence, 0.03125 mg/ml was chosen as the highest test concentration in the cytogenicity test. The chromosome aberration test was performed in three phases;Phase I (4-hours exposure in the absence of metabolic activation), Phase II (4-hours exposure in the presence of metabolic activation) and Phase III (24-hours exposure in the absence of metabolic activation). CHO cells were exposed to 0 (NC), 0 (VC),0.0078125, 0.015625, 0.03125 mg/ml for 4 hours (Phase I-II) and 24 hours (Phase III) both in the presence and absence of S9 metabolic activation. Concurrent positive controls, i.e., Methyl methanesulfonate (without S9 mix, final conc. 20 µg/ml) and Benzo[a]pyrene (with S9 mix, final conc. 30 µg/ml) were also included in the assay. At least 300 well-spread metaphases per concentration were analyzed using 100x magnification for the incidence of structural aberrations.Breaks (chromatid and chromosomal), gaps (chromatid and chromosomal), rings and fragments, dicentric chromosomes were found as structural chromosome aberrations. Gaps were recorded and reported separately but not included in the total aberration frequency.Results:In Phase I experiment, the average RICC values were 89.47% (VC), 84.71% (at 0.0078125 mg/ml), 72.55% (at 0.015625 mg/ml) and 49.02% (at 0.03125 mg/ml). There was no significant increase in the mean percent aberrant cells at 0.0078125 mg/ml (mean % aberrant cells was 2.00%, p=0.1229) when compared to vehicle control (mean % aberrant cells: 0.33 %). However, there was a significant increase in mean percent aberrant cells at 0.015625 (mean % aberrant cells: 2.67%, p=0.0378) and 0.03125 mg/ml (mean % aberrant cells: 3.00%, p=0.0206) when compared to vehicle control (mean % aberrant cells: 0.33 %).In Phase II experiment, the average RICC values were 97.00 % (VC), 95.88 % (at 0.0078125 mg/ml), 80.41 % (at 0.015625 mg/ml) and 44.33 % (at 0.03125 mg/ml). The mean percent aberrant cells was 0.33% (at 0.0078125 mg/ml), 0.67% (at 0.015625 mg/ml) and 1.33% (at 0.03125 mg/ml) compare with vehicle control (the mean % aberrant cells: 0.67 %). The observed changes were not statistically significant, but a trend for an increase in percent aberrant cells at increasing concentration was detected, which was considered biologically relevant. In Phase III experiment, the average RICC values were 92.58 % (VC), 81.53 % (at 0.0078125 mg/ml), 67.94 % (at 0.015625 mg/ml) and 44.95 % (at 0.03125 mg/ml). An increase in the mean percent aberrant cells was observed at  0.0078125 mg/ml (the mean % aberrant cells: 1.33, p=0.1238). This increase was not statistically significant, but it was considered biologically relevant. Significant increases in mean percent aberrant cells were observed at 0.015625 (the mean % aberrant cells: 2.33%,p=0.0151) and 0.03125 mg/ml (the mean % aberrant cells: 3.33%,p=0.0018) when compared to vehicle control (the mean % aberrant cells 0.00 %). In all phases, no significant reduction in RICC (cytotoxicity) was observed in the vehicle control (dimethyl sulfoxide) when compared to the negative control (distilled water) either in the presence and absence of S9 metabolic activation. A slight increase in the percent aberrant cells in vehicle control (the mean % aberrant cells 0.67 %) was observed in Phase II compared to the negative control (the mean % aberrant cells 0.00 %). No other increase in the percent aberrant cells was noted in vehicle control compared to the negative control in the presence and absence of S9 metabolic activation.Conclusion:The registered substance, i.e.,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 (EC No. 907-706-6) induced chromosomal aberration at ≥0.03125 mg/ml of culture medium, both in the presence and the absence of S9 metabolic activation system in CHO cells.

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.

Genetic toxicity - in vivo

Data available for the various test chemicals was reviewed to determine the mutagenic nature ofReaction mass of  4-(2,6,6-trimethylcyclohex-2-ene-1-yl)-but-3-ene-2-one and 4-(2,6,6-trimethylcyclohex-1-ene-1-yl)-but-3-ene-2-one (EC. no 907-706-6). The studies are as mentioned below:

The read across substance, i.e., (E)-4 -(2,6,6 -trimethyl-1 -cyclohexen-1 -yl)-3 -buten-2 -one (CAS 79 -77 -6), was tested for chromosomal damage (clastogenicity) and for the ability to induce spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method. The test was performed according to OECD TG 474 (1997). Test doses were selected based on an initial experiment. In this pretest for determination of the acute i.p. toxicity, deaths were observed at 2000 mg/kg bw. Mice treated with 750 and 1000 mg/kg bw showed evident signs of toxicity, and some animals were sacrificed moribund. At 500 mg/kg bw, all animals survived, but l signs of clinical toxicity were noted. There were no distinct differences in the symptoms between males and female mice. Thus, only males were used for the cytogenetic investigations. Doses of 750, 500 and 250 mg/kg bw were selected for the main test. The test substance was dissolved in olive oil and was administered once intraperitoneally to male NMRI mice (5 mice/dose) at 0 (VC), 250, 500 and 750 mg/kg bw. Additional 5 mice were injected with the vehicle or 750 mg/kg of test substance served as a satellite group. Samples of the bone marrow of the 2 femurs were taken 24 (treatment groups) and 48 hrs (satelite groups) after the last treatment. 2000 polychromatic erythrocytes (PCEs) from each animal of every test group were investigated for micronuclei (MN). The normochromatic erythrocytes (NCEs) were also scored. The ratio of polychromatic to normochromatic erythrocytes was determined. After administration of the vehicle, test substance and positive controls, the animals were examined for clinical signs of toxicity. U-test according to Mann-Whitney (modified rank test according to Wilcoxon) was performed to confirm differences between control and dose groups.Results:No mortality was observed at any dose level. Clinical signs of toxicity were observed at 500 and 750 mg/kg bw, which included poor general state, irregular respiration, squatting posture. These clinical signs were reversible after 2 days. At 250 mg/kg bw only minor signs of clinical toxicity were observed after 2 and 4 hours of administration of the test substance (squatting posture). No inhibition of erythropoiesis, determined from the PCE/NCE ratio, was detected. The vehicle and the positive control substances, Cyclophosphamide (CPP, 20 mg/kg bw) and vincristine (VCR, 0.15 mg/kg bw), caused no evident signs of toxicity. The following mean number of PCEs and NCEs were observed: vehicle (PCEs: 10000, NCEs: 4594 (24 hrs), 3439 (48 hrs)), at 250 mg/kg bw (PCEs: 10000, NCEs:3755), at 500 mg/kg bw (PCEs: 10000, NCEs: 4646), at 750 mg/kg bw (PCEs: 10000, NCEs: 2476 (24 hrs), 2804 (48 hrs), CPP (PCEs: 10000, NCEs: 3920), VCR (PCEs: 10000, NCEs: 5374). The administration of the test substance did not lead to any statistically significant increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was nearly the range of the concurrent negative control in all dose groups and within the range of the historical control data (mean 1.6, min. 0.3, max. 3.3, SD 0.6; n=393). The positive controls led to the expected increases in micronuclei (either small or large). The mean number of PCEs containing MN per 1,000 PCE at 24 hrs (differentiation between small and large micronuclei) were the follows: vehicle (small: 1.3, large:0.1, total: 1.4), at 250 mg/kg bw (small: 1.6, large: 0.0, total: 1.6), at 500 mg/kg bw (small: 1.7, large: 0.1, total: 1.8), at 750 mg/kg bw (small: 1.2, large: 0.0, total: 1.2), CPP (small: 10.9, large: 0.2, total: 11.1; p ≤0.01), VCR (small: 35.7, large: 11.5 total: 47.2; p≤0.01). The mean number of PCEs containing MN per 1,000 PCE at 48 hrs (small and large and total number of micronuclei) were the follows: vehicle (small: 0.7, large: 0.0, total: 0.7) at 750 mg/kg bw (small: 1.0, large: 0.0, total: 1.0).Conclusion:The source substance, i.e.,(CAS 79-77-6), did not have a chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

In another study, the read across substance, i.e., 4 -(2,6,6 -trimethylcyclohex-2 -ene-1 -yl)-but-3 -ene-2 -one (CAS 127 -41 -3), was tested for chromosomal damage (clastogenicity) using ICR mice in a bone marrow micronucleus. The test was performed according to OECD TG 474 (1997b). Test doses were selected based on an initial range-finding test, where 1200 mg/kg was determined as the maximum tolerated dose (MTD). Groups of male and female ICR mice (5/sex/dose) were administered at doses 0 (VC, corn oil), 300, 600 or 1200 mg/kg bw by a single intraperitoneal injection. Additional 5 mice of each sex were injected with the vehicle or 1200 mg/kg of test substance served as a satellite group. Groups of 5 male and 5 female mice from all treatment levels were sacrificed 24 hours after dosing, and additional 5 mice of each sex from the satellite groups were sacrificed 48 hours after dosing. 2000 Polychromatic (PCE) and normochromatic (NCE) erythrocytes per animal were scored for micronuclei. Reductions of 7 -18 % in PCE were observed in treated males and females 24 hours after dosing; a reduction of 21 % in PCE was observed in males at 1200 mg/kg bw at 48 hours after dosing, indicating bone marrow toxicity. Additional clinical signs in treated animals also confirmed the systemic availability of the test substance. There were no statistically or biologically significant increases in micronucleus frequency in treated animals at dose levels tested compared to the vehicle control. Hence, the source substance, i.e., 4 -(2,6,6 -trimethylcyclohex-2 -ene-1 -yl)-but-3 -ene-2 -one (CAS 127 -41 -3), did not express clastogenic or aneugenic potential in vivo under the experimental conditions described.

Based on the data available the test chemical,Reaction mass of  4-(2,6,6-trimethylcyclohex-2-ene-1-yl)-but-3-ene-2-one and 4-(2,6,6-trimethylcyclohex-1 -ene-1-yl)-but-3-ene-2-one (EC. no 907-706-6)is not mutagenic in mouse and hence it is not likely to classify as a gene mutant in vivo.

Justification for classification or non-classification

The registered substance, i.e., Reaction mass of 4-(2,6,6-trimethylcyclohex-2-ene-1-yl)-but-3-ene-2-one and 4-(2,6,6-trimethylcyclohex-1 -ene-1 -yl)-but-3 -ene-2 -one (EC 907 -706 -6) was non-mutagenic in bacterial and mammalian cells but tested positive (clastogenic) in a cytogenicity test using mammalian cells (OECD TG 473). Therefore, its clastogenic potential was further assessed in vivo using structurally similar read-across substances. Data on analogues i.e., 4 -(2,6,6 -trimethylcyclohex-2 -ene-1 -yl)-but-3 -ene-2 -one (CAS 127 -41 -3) and (E)-4 -(2,6,6 -trimethyl-1 -cyclohexen-1 -yl)-3 -buten-2 -one (CAS 79 -77 -6), demonstrated no chromosome-damaging (clastogenic) potential and there were no indications of any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo. Hence, the registered substance, i.e., Reaction mass of 4 -(2,6,6 -trimethylcyclohex-2 -ene-1 -yl)-but-3 -ene-2 -one and 4 -(2,6,6 -trimethylcyclohex-1 -ene-1 -yl)-but-3 -ene-2 -one (EC 907 -706 -6) is classified as Non-classified for germ cell mutagenicity according to the Regulation (EC) No 1272/2008 on the classification, labelling and packaging of substances and mixtures (CLP).