4-tert-butylcyclohexyl acetate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2008-10-30 to 2008-12-21

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 473 and in compliance with GLP. Some deviations to protocol were observed but they had no impact on the results or conclusion.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

: insufficient number of cells in metaphase was available to the analyse of chromosome aberrations in the Exp 2 at 35 µg/mL and for the positive control in one of the both culture. 200 metaphases were therefore observed in only culture.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5375 (In Vitro Mammalian Chromosome Aberration)

Deviations:

yes

Remarks:

: insufficient number of cells in metaphase was available to the analyse of chromosome aberrations in the Exp 2 at 35 µg/mL and for the positive control in one of the both culture. 200 metaphases were therefore observed in only culture.

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

4-tert-butylcyclohexyl acetate

EC Number:

250-954-9

EC Name:

4-tert-butylcyclohexyl acetate

Cas Number:

32210-23-4

Molecular formula:

C12H22O2

IUPAC Name:

4-tert-butylcyclohexyl acetate

Constituent 2

Reference substance name:

2-tert-pentylcyclohexyl acetate

EC Number:

267-500-0

EC Name:

2-tert-pentylcyclohexyl acetate

Cas Number:

67874-72-0

Molecular formula:

C13H24O2

IUPAC Name:

2-(tert-pentyl)cyclohexyl acetate

Test material form:

liquid

Details on test material:

- Physical state: Clear liquid
- Storage condition of test material: Room temperature

Method

Target gene:

Not applicable.

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: McCoy's 5A Medium (Gibco-BRL) supplemented or not with fetal bovine serum (Gibco-BRL)
- Properly maintained: no data
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no data
- other: doubling time of approximately 12 hours

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction obtained from rat liver induced by phenobarbital-5,6-Benzoflavone (phenobarbital/beta-naphtaflavone) and the cofactor pool (Moltox, lot No. 2331)

Test concentrations with justification for top dose:

Range finding study (3+15 hrs): 0; 0.5; 5; 10; 50; 100; 500; 1000 and 5000 µg/mL with and without metabolic activation
Main study 1 (3+15 hrs): 0; 10; 20; 30; 35; 40 and 45 µg/mL without metabolic activation / 0; 25; 50; 60; 70; 80; 90 µg/mL with metabolic activation
Main study 2 (continuous exposure of 18 hrs): 0; 10; 20; 25; 30 and 35 µg/mL without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test article was not miscible with water. 50 mg of test article was miscible with DMSO in 0.1 mL final volume, resulting in a final concentration of 500 µg/mL. DMSO was selected as the solvent. Test article in DMSO and complete medium in a final test article concentration of 5000 µg/mL resulted in a clear solution.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

0.2 to 0.8 µg/mL

Positive control substance:

mitomycin C

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

7.5 and 12.5 µg/mL

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: not applicable
- Exposure duration: 3 hours (in the range finding study and the main study 1) or 18 hours (in the Main study 2)
- Expression time (cells in growth medium): 18 hrs
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 18h

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.1 µg/mL) added 2h before harvesting cells
STAIN (for cytogenetic assays): Methanol:glacial acetic acid (3:1)

NUMBER OF REPLICATIONS: 2 cultures in parallel

NUMBER OF CELLS EVALUATED: 100 cells of each culture (total of 200 cells/concentration)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

To consider that the assay is valid:
In the solvent control, the percentage of cells with aberrations should not exceed 4%.
At least 25% of cells scored in the positive control should show one or more chromosome aberrations.
At least one of the test concentrations scored should show approximately 50% reduction in the RCG and/or RMI. This requirement should not be applied to test articles where no apparent toxicity could be achieved at the maximum soluble concentration or the highest allowable concentration.

For the evaluation of the results:
The test article was considered to have caused a positive response in this assay if the test article showed a positive concentration-response trend and a statistically significant increase over that of the solvent controls in the percentage of cells with aberrations at one or more concentrations.
The test article was considered to have caused a negative response if none of the test concentrations showed a statistically significant increase in the percentage of aberrant cells.
The test article was considered to have caused an equivocal response if there was a statistically significant increase in the percentage of cells with aberrations without an accompanying positive concentration-response trend.

Statistics:

Chi-square tests were used to compare the percentage of cells with aberrations for each concentrations with the solvent control values (significant if p <= 0.05). Statistical analysis was not performed if the test concentration value was equal to or less than the concurrent or historical solvent control value.
If positive response with the Chi-square test, the Cochran-Armitage test (trend test) was performed for evidence of a concentration-related response (significant if p <= 0.05)

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: the pH of the test item in the medium was checked and no adjustment of pH of the treatment medium was necessary.
- Effects of osmolality:not determined
- Evaporation from medium: not determined
- Water solubility: the test item solubilised in DMSO was then soluble in the complete medium, resulting in a clear solution.
- Precipitation: no precipitation was observed during the test

RANGE-FINDING/SCREENING STUDIES: significant toxicity was observed at 50 µg/mL and above without metabolic activation and at 100 µg/mL and above with metabolic activation as revealed by a decrease of the relative cell growth (RCG). Relative Mitotic index (RMI) are not available above 10 µg/mL without activation and above 50 µg/mL with activation because there were not enough cells to drop slides.

COMPARISON WITH HISTORICAL CONTROL DATA: the data obtained for the untreated (water) and the vehicle are consistent with the historical control range.

ADDITIONAL INFORMATION ON CYTOTOXICITY: in the confirmatory assay (Exp 2) the RCG (Relative cell growth) for the test article concentrations of 10-35 µg/mL ranged from 52-105% and the RMI (Relative Mitotic index) ranged from 65-205%. The reason for the relatively high RMI at certain concentration levels is a result of the low relative MI for the solvent control cultures. The reason for this low MI is unknown.

OTHER: The percentage of endoreduplicated cells was higher than the normal range (0-1%) and the historical data of the lab. However, CHO cells have a tendency to endoreduplicate even if not clearly visible in the historical data. Moreover they are a normal phenomenon, common in liver, salivary glands, etc. Endoreduplicated cells are not a marker of aneugenicity.

Any other information on results incl. tables

Table 7.6.1/2:Chromosomal aberration study results. The results are presented for the 2x100 evaluated cells.

Processing time (hrs)	S9 mix	Doses (µg/mL)	Chromatid break	Chromatid exchange	Chromosome break	Chromosome exchange	Other*	% cells with aberrations	Gap	% of polyploid cells	% of cells with endoredu plication	Cell Proliferation
												RCG (%)	RMI (%)
3-18	-	Untreated	0	0	0	0	0	0.0	0	0	0.5	87	128
		DMSO	1	0	0	0	0	0.5	0	0	1.0	100	100
		10	0	0	0	0	0	0.0	0	0	0.0	101	108
		20	0	0	0	0	0	0.0	0	0	0.0	98	110
		30	2	0	0	0	0	1.0	0	0.5	1.0	99	45
		35	N/D	N/D	N/D	N/D	N/D	N/D	N/D	N/D	N/D	41	29
		40	N/D	N/D	N/D	N/D	N/D	N/D	N/D	N/D	N/D	9	N/D
		45	N/D	N/D	N/D	N/D	N/D	N/D	N/D	N/D	N/D	4	N/D
		MMC (0.4)	N/D	N/D	N/D	N/D	N/D	N/D	N/D	N/D	N/D	95	19
		MMC (0.8)	30	94	18	4	8	45.5	1	1.0	1.0	61	32
	+	Untreated	1	0	0	0	0	0.5	0	0.5	0.5	96	113
		DMSO	1	0	0	0	0	0.5	0	0.0	0.0	100	100
		25	0	0	0	0	0	0.0	0	0.0	2.0	135	88
		50	1	0	0	0	0	0.5	0	1.0	4.5	91	121
		60	0	0	0	0	0	0.0	0	1.5	3.5	86	70
		70	N/D	N/D	N/D	N/D	N/D	N/D	N/D	N/D	N/D	88	49
		80	N/D	N/D	N/D	N/D	N/D	N/D	N/D	N/D	N/D	33	N/D
		90	N/D	N/D	N/D	N/D	N/D	N/D	N/D	N/D	N/D	4	N/D
		CP (7.5)	N/D	N/D	N/D	N/D	N/D	N/D	N/D	N/D	N/D	77	16
		CP (12.5)	35	93	20	2	11	44.0	0	1.5	0.5	58	13
18-0	-	Untreated	0	0	1	0	0	0.5	0	1.0	0.0	113	168
		DMSO	1	0	0	0	0	0.5	0	2.0	0.0	100	100
		10	N/D	N/D	N/D	N/D	N/D	N/D	N/D	N/D	N/D	105	110
		20	N/D	N/D	N/D	N/D	N/D	N/D	N/D	N/D	N/D	104	130
		25	0	0	0	0	0	0.0	0	2.0	0.0	91	205
		30	0	0	0	0	0	0.0	0	3.0	1.5	101	143
		35	1	0	0	0	0	0.5	0	1.0	5.0	52	65
		MMC (0.2)	20	23	9	1	1	26.0	0	0.0	0.0	83	37
		MMC (0.4)	N/D	N/D	N/D	N/D	N/D	N/D	N/D	N/D	N/D	72	12

*:pulverized cells and severely damaged cells (i.e. cell with 10 or more aberrations)

N/D: No data

RCG : Relative cell growth = (Number of cells in test flask/Number of cells in solvent flask) x 100

RMI: Relative Mitotic index = (Test concentration MI / Solvent MI) x 100 (where MI = Number of dividing cells from 1000 cells / 10)

Applicant's summary and conclusion

Conclusions:

Under the test conditions, Coniferan induced a negative response in the chromosomal aberration test using hamster cells.

Executive summary:

In a chromosomal aberration assay in mammalian cells, performed according to the OECD No.473,  Coniferan (99.3% purity) diluted in Dimethylsulphoxide (DMSO) was tested in Chinese Hamster Ovary (CHO) cells in the presence and the absence of mammalian metabolic activation (S9) for 3 or 18hrs at concentrations from 10 to 90 µg/mL.

A range-finding study was performed in order to evaluate the cytotoxicity of the test item and to determine the appropriate concentrations for the main test. Cytotoxicity was assessed by the calculation of the percentage of RCG (Relative cell growth) and of RMI (Relative mitotic index).

Coniferan was incubated with the cells in the first experiment for 3 hrs without S9 mix at concentrations of 10; 20; 30; 35; 40 and 45 µg/mL and with S9 mix at the concentrations of 25; 50; 60; 70; 80 and 90 µg/mL. After the exposure, the cells were washed and re-incubated in fresh culture medium for 15hrs. The cells were then harvest. Colcemid (0.1 µg/mL) was added 2hrs before harvesting cells in order to inhibit the spindle allowing therefore the observation of the chromosomes in the cells in metaphase. In a second experiment, the cells were incubated with Coniferan for 18hrs in the absence of metabolic activation at the concentrations of 10; 20; 25; 30 and 35 µg/mL. The analysis of the presence of chromosomal aberrations was in this case performed after the end of the exposure period.

Mitomycin C and Cyclophosphamide were used as positive controls and induced appropriate responses.

Coniferan was cytotoxic to CHO cells. No increase in the occurrence of chromatid or chromosome structural aberration was observed with and without metabolic activation and for all exposure periods tested. The number of polyploid cells was in the normal range.

The percentage of endoreduplicated cells was higher than the normal range (0-1%) and the historical data of the lab. However, CHO cells have a tendency to endoreduplicate even if not clearly visible in the historical data. Moreover they are a normal phenomenon, common in liver, salivary glands, etc. Endoreduplicated cells are not a marker of aneugenicity.

Under the test conditions, Coniferan induced a negative response in the chromosomal aberration test using hamster cells. This study is considered as acceptable as it satisfied the criteria of the OECD Guideline No. 473.