Ethylcyclohexane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test:

A reverse mutation test was performed to investigate mutagenesis of ethylcyclohexane using Salmonella typhimurium TA100, TA1535, TA98, TA1537, and Escherichia coli WP2uvrA as indicator bacteria without the S9 mix (direct method) and with the S9 mix (metabolic activation method) by preincubation. The results showed no increase in the revertant colony count in all strains, irrespective of metabolic activation. From the findings described, ethylcyclohexane was determined not mutagenic for bacteria under the test conditions.

In vitro chromosome aberration:

An in vitro chromosomal aberration test was performed to investigate the clastogenicity of ethylcyclohexane using Chinese hamster lung fibroblast (CHL/IU) cells. The results showed that no clastogenicity was observed in both short-term treatments with and without the S9 mix and 24-hour continuous treatment. Therefore, ethylcyclohexane was determined not clastogenic to CHL/IU cells under the test conditions. This test results also showed that the test article was considered not clastogenic based on the biological criterion for which a substance was considered not clastogenic if the incidence of cells with chromosomal aberrations was lower than 5% in CHL/IU cells. No findings with significant cell cycle delay suspected throughout the test were observed; therefore, no confirmation test by 48-hour continuous treatment was performed.

In vitro gene mutation test/HPRT-Test:

Ethylcyclohexane was tested negativ in the In Vitro Mammalian Cell Gene Mutation Test using the Hprt genes following OECD 476.

In both valid experiments no substantial and reproducible dose dependent increase in mutant colony numbers was observed. All values remained well within the historical control range. According to OECD 476 the result of both valid experiments I is clearly negative.

 

In conclusion, it can be stated that under the experimental conditions reported,Ethylcyclohexanedid not induce mutations in the HPRT locus using the V79 cell line in the absence and 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

Study period:

2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Name:Ethylcyclohexane
Additional name: Hexahydroethylbenzene
CAS registry number: 1678-91-7
Batch number:
Purity: 99.9%

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

With the direct method, the range of 0.156–5 µg per plate (common divisor: 2) was set for all strains. With the metabolic activation method, 3.13–100 µg per plate was set for TA100, TA1535, TA98, and TA1537, and 6.25–200 µg per plate for WP2uvrA (common divisor: 2).

Vehicle / solvent:

acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)-acrylamide, 2-Aminoanthracene

Evaluation criteria:

A result that met the following three criteria was determined positive based on mean revertant colony count per plate at each dose.
(1) The revertant colony count at least two times higher than that in the negative control group was observed in a test substance group.
(2) The revertant colony count was raised as an increase in the dose of test substance (dose dependency).
(3) Reproducibility of the increase in revertant colony count was observed based on the results of the tests performed two times. However, a result was determined positive if no significant dose dependency but reproducibility in positive results was observed.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Ethylcyclohexane was determined not mutagenic for bacteria under the described test conditions.

Executive summary:

A reverse mutation test was performed to investigate mutagenesis of ethylcyclohexane using Salmonella typhimurium TA100, TA1535, TA98, TA1537, and Escherichia coli WP2uvrA as indicator bacteria without the S9 mix (direct method) and with the S9 mix (metabolic activation method) by preincubation. Based on results of a dose finding study (preliminary test), dose inhibiting bacterial growth was considered the maximum dose. With the direct method, the range of 0.156–5 µg per plate (common divisor: 2) was set for all strains. With the metabolic activation method, 3.13–100 µg per plate was set for TA100, TA1535, TA98, and TA1537, and 6.25–200 µg per plate for WP2uvrA (common divisor: 2). The test was performed two times. The results showed no increase in the revertant colony count in all strains, irrespective of metabolic activation. With the direct method, bacterial growth inhibition was observed at 5 μg per plate in all strains and at 2.5 μg per plate in TA1537 during the second test. With the metabolic activation method, bacterial growth inhibition was observed at 100 μg per plate in TA100, TA1535, TA98, and TA1537, at 200 μg per plate in WP2uvrA, and at 50 μg per plate in TA1535 during the first test. From the findings above, ethylcyclohexane was determined not mutagenic for bacteria under the test conditions.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

not specified

GLP compliance:

no

Type of assay:

other: Mammalian Cells Chromosomal Aberration Test

Specific details on test material used for the study:

Name: Ethylcyclohexane
Additional name: Hexahydroethylbenzene
CAS registry number: 1678-91-7
Batch number: 3-DXY-1
Purity: 99.9%

Target gene:

Chinese hamster lung fibroblast (CHL/IU) cell lines distributed from the Division of Genetics and Mutagenesis, National Institute of Health Sciences (formerly the Department of Mutagenesis, National Institute of Health Science), on January 13, 1985, were used for the test. Test cells stored in a cell suspension with DMSO added at a ratio of 10% under conditions in liquid nitrogen were returned in the culture medium; those cells cultured until the seventh passage after thawing were used for the test.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Based on the results of a cell growth inhibition test, doses of the test article were determined as follows: a total of 4 doses of 1,200 μg/mL which is equivalent to 10 mM as the maximum dose and 600, 300, and 150 μg/mL when the common ratio was 2 in the short-term treatment; 80 μg/mL as the maximum dose and 40, 20, and 10 μg/mL when the common ratio was 2 in the 24-hour continuous treatment, given that the doses included those around the dose level causing 50% inhibition of cell growth, and data on at least 3 doses could be obtained; and a total of 5 doses, including 40 μg/mL, 80 μg/mL, and 60 μg/mL as the mid dose, given that rapid changes in cell growth rate were observed between doses of 37.5 and 75 μg/mL in the cell growth inhibition test.

Doses used for the chromosomal aberration test: 150, 300, 600, and 1,200 μg/mL with the short-term treatment, and 10, 20, 40, 60, and 80 μg/mL for continuous treatment

Vehicle / solvent:

acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

Details on test system and experimental conditions:

Cell treatment:
In short-term treatment, 5 mL of culture medium containing 4 x 10*3 cells/mL was added in a round plastic plate 6 cm in diameter (Becton Dickinson). Without the S9 mix, extra culture medium was removed to leave 3 mL of the medium per plate, and acetone (positive control) or the test article solution of 0.015 mL/plate was added to the plate 3 days after the start of incubation. With the S9 mix, extra culture medium was removed to leave 2.5 mL of the media per plate, and acetone or the test article solution of 0.015 mL/plate was added to each plate after adding the S9 mix of 0.5 mL. The culture medium was removed after 6-hour incubation, and new culture medium of 5 mL was added and incubated for 18 hours. On the other hand, in the continuous treatment, cells were incubated by a similar method to the short-term treatment; acetone or the test article solution of 0.025 mL/plate was added to each plate 3 days after the start of the culture and then incubated for 24 or 48 hours. After incubation, the culture medium was removed, and the cell surface was rinsed with physiological saline two times, followed by adding 10 vol% formalin solution to fix the cells for approximately 10 minutes. After fixation, the cells were rinsed with water and stained by a 0.1 w/v% crystal violet solution for approximately 10 minutes. The cells were rinsed with water, and allowed to dry spontaneously at room temperature overnight. At doses of 300 μg/mL or higher, deposition of an oil droplet-like test article was observed immediately after adding the test article solution to the culture medium and slightly remained at the specified end time of incubation.

Measurement of cell growth rate:
For the cells fixed and stained as shown in section 8-2 above, cell density was measured by a density meter for monolayer cultured cells (Monocellater II, MI-60; Olympus Optical Co., Ltd.) based on the shades of colors of stained cells to calculate the cell growth rate in each dose group when the rate in the negative control group was 100%. As shown in the table below, the results showed that no cell growth inhibition of more than 50% was observed in short-term treatment, and the dose level causing 50% inhibition of cell growth was determined as 1,200 μg/mL or higher. In continuous treatment, cell growth inhibition of more than 50% was observed at doses of 75 and 150 μg/mL, and the dose level causing 50% inhibition of cell growth was determined to range from 37.5 to 75 μg/mL. Moreover, a dose-dependent increase in the cell growth rate was observed at doses of 300 μg/mL or higher in continuous treatment.

Chromosomal aberration test:
1) Doses of the test article and positive control substances
Based on the results of a cell growth inhibition test, doses of the test article were determined as follows: a total of 4 doses of 1,200 μg/mL which is equivalent to 10 mM as the maximum dose and 600, 300, and 150 μg/mL when the common ratio was 2 in the short-term treatment; 80 μg/mL as the maximum dose and 40, 20, and 10 μg/mL when the common ratio was 2 in the 24-hour continuous treatment, given that the doses included those around the dose level causing 50% inhibition of cell growth, and data on at least 3 doses could be obtained; and a total of 5 doses, including 40 μg/mL, 80 μg/mL, and 60 μg/mL as the mid dose, given that rapid changes in cell growth rate were observed between doses of 37.5 and 75 μg/mL in the cell growth inhibition test. Doses of MNNG and B[a]P as the positive control substances were 2.5 μg/mL and 10 μg/mL, respectively.

2) Preparation of the test article and positive control solutions
The test article solution at the maximum dose was prepared by dissolving the test article in acetone at the time of use. Subsequently, a serial dilution of a part of the stock solution was conducted with acetone to prepare a test article solution at the specified dose.

Cell treatment:
After 5 mL of culture medium containing 4 x 10*3 cells/mL was added in a round plastic plate of 6 cm in diameter (Becton Dickinson) for 3 days, the cells were treated by the following method. Four plates, including 2 for preparation of chromosomal samples and another 2 for measurement of the cell growth inhibition rate, were used for incubation per dose. However, cell growth inhibition rate was not measured, and 2 plates were used for preparation of chromosomal samples in the positive control group. In the short-term treatment without the S9 mix, extra culture medium was removed to leave 3 mL of the media per plate, and 0.015 mL each of acetone, the test article, and MNNG solution was added in each plate for incubation. With the S9 mix, extra culture medium was removed to leave 2.5 mL of the media per plate, and 0.015 mL each of acetone, the test article, and B[a]P solution was added in each plate and incubated after adding the S9 mix of 0.5 mL. The culture medium was removed after 6-hour incubation, and 5 mL of new medium was added and incubated for 18 hours, irrespective of the S9 mix. In the continuous treatment, 0.025 mL each of acetone, the test article, and MNNG solution was added to each plate for 24-hour incubation. At doses of 300 μg/mL or higher in the short-term treatment, deposition of an oil droplet-like test article was observed immediately after adding the test article solution to the culture medium and slightly remained at the specified end time of incubation.

Preparation of chromosomal samples and measurement of cell growth inhibition rate:
Colcemid (Gibco Laboratories; Batch number, 1238727) was added to be the final concentration of 0.2 μg/mL in each plate during incubation 2 hours before preparation of chromosomal samples. After incubation, the culture medium was removed and treated with 2 mL of a 0.2 w/v% trypsin solution to detach the cells from the plate. Those cells were added to a centrifuge tube containing fresh culture medium at 5 mL for centrifugation at 1000 rpm for 5 minutes. The supernatant was discarded, sediment cells were suspended with 4 mL of a 75 mM hypotonic potassium chloride solution for hypotonic treatment at a temperature of 37°C for 15 minutes. After hypotonic treatment, those cells were fixed with a mixture of refrigerated methanol and acetic acid in a ratio of 3 to 1 (v/v) to prepare 1 mL at the time of use. Centrifugation was performed at 1000 rpm for 5 minutes to discard the supernatant. The sediment cells were re-suspended and fixed with 4 mL of new fixing solution. The procedure was repeated 3 times, and the cells were suspended to the appropriate density with a small amount of fixing solution. A drop was instilled on 2 spots of a slide glass, and allowed to dry spontaneously at room temperature overnight. After drying, staining with 1.4 vol% Giemsa solution diluted by Sørensen buffer solution (pH 6.8; Iatron; Batch number, 1478) was performed for approximately 15 minutes. The slide glass was rinsed with water and dried at room temperature as a chromosome sample. Three samples were prepared per plate.For measurement of cell growth inhibition rates, the culture medium was removed after incubation, and the cell surface was rinsed with physiological saline 2 times, followed by adding 10 vol% formalin solution to fix the cells for approximately 10 minutes. After fixation, the cells were rinsed with water and stained by 0.1 w/v% crystal violet solution for approximately 10 minutes. The cells were rinsed with water and allowed to dry. A density meter for monolayer cultured cells (Monocellater II, MI-60; Olympus Optical Co., Ltd.) was used to calculate a cell growth rate in each dose group when the rate in the negative control group was 100%.

Rationale for test conditions:

Cell growth inhibition tests were performed at doses of 18.8, 37.5, 75, 150, 300, 600, and 1,200 μg/mL in both short-term and continuous treatments to investigate the appropriate doses of the test article used for the chromosomal aberration test

Evaluation criteria:

In determination of the test results, if a significant difference was observed in the incidence rates of structural aberrations and polyploid cells by multi-sample chi-squared test (significance level: 5% or lower), Fisher's exact test was used to determine a significant difference between the negative control group and each dose group. (The significance level in which 5% or 1% was divided by the number of treatment groups was used given the multiplicity.) If the incidence of cells with chromosomal aberrations significantly increased in at least 2 dose groups among the test article groups compared to the negative control group, and dose dependency was also observed, the test article was considered clastogenic. When a significant increase was observed only in a dose group, a confirmation test was performed at a similar dose. If a reproducible result was shown, the test article was considered clastogenic.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

An in vitro chromosomal aberration test was performed to investigate the clastogenicity of ethylcyclohexane using Chinese hamster lung fibroblast (CHL/IU) cells. The results showed that no clastogenicity was observed in both short-term treatments with and without the S9 mix and 24-hour continuous treatment. Therefore, ethylcyclohexane was determined not clastogenic to CHL/IU cells under the test conditions. This test results also showed that the test article was considered not clastogenic based on the biological criterion for which a substance was considered not clastogenic if the incidence of cells with chromosomal aberrations was lower than 5% in CHL/IU cells. No findings with significant cell cycle delay suspected throughout the test were observed; therefore, no confirmation test by 48-hour continuous treatment was performed.

Executive summary:

In vitro chromosomal aberration test was performed to investigate the clastogenicity of ethylcyclohexane using Chinese hamster lung fibroblast (CHL/IU) cell lines. A cell growth inhibition test was conducted at doses that ranged from 18.8 to 1,200 μg /mL in both short-term and continuous treatments to determine the doses for the chromosomal aberration test. Consequently, no cell growth inhibition of more than 50% was observed in the short-term treatment. In the continuous treatment, cell growth inhibition of more than 50% was seen at doses of 75 and 150 μg/mL. Therefore, doses used for the chromosomal aberration test were set to 150, 300, 600, and 1,200 μg/mL with the short-term treatment, and 10, 20, 40, 60, and 80 μg/mL for continuous treatment. The test results showed that no increase in cells having chromosomal aberrations was observed in the short-term treatment with and without the S9 mix. Furthermore, no increase in cells with chromosomal aberrations was observed in the 24-hour continuous treatment. From the findings above, ethylcyclohexane was determined not clastogenic for CHL/IU cells under the test conditions.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-12-08 - 2018-02-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

Name Ethylcyclohexane
Batch no. OB7HTO1
Appearance colourless transparent liquid
Composition Ethycyclohexane >99%
Purity >99.9% (GC)
Homogeneity homogeneous
Expiry date 6 weeks after opening (recommended)
Storage Room temperature
Keep away from ignition sources and stored away from
oxidizers.

The following information is provided by the sponsor as well:
CAS No. 1678-91-7
EINECS-No. 216-835-0
Molecular formula C8H16
Molecular weight 112 g /mol
Vapour pressure 1730 Pa at 25°C1706 hPa at 25°C
Density 0.7880 g/cm3 (20 °C)
Stability H2O: unknown; EtOH: not stated; Aceton: not stated; CH3CN: not stated; DMSO: not stated
Solubility H2O: <0.1 g/L; EtOH: not stated; Aceton: not stated; CH3CN: not stated; DMSO: not stated

Target gene:

endogenous hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on chromosome X in Chinese Hamster cells (V79)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Pre-Test for Detection of Cytotoxicity
A pre-test was performed in order to determine the concentration range applicable for mu-tagenicity experiments. As no CE II value was determined, the evaluation was done on CE I values. In respect of the pre-test the cytotoxicity was determined by measuring the survival of the cells after exposure to the test item (CE I). The survival is given as the cloning efficiency (CE) of treated cells in comparison to the controls.
In context of the pre-test a clear reduction of the CE values below 20 % is regarded as cytotoxic.
In the pre-test, 7 concentrations of the test item were used and tested with and without metabolic activation.
According to OECD 476, the highest concentration should be 0.01 M or 2 mg/mL or 2 µL/mL (whichever is the lowest), unless limited by the solubility or toxicity of the test item. Relative survival values below 20 % are considered toxic. For cytotoxic test items the maximum concentrations should result in 10 to 20 % relative suspension growth (RSG) or the relative total growth (RTG) in the main experiment. According to the results of the pre-test, 6 concentrations were chosen for the main experiments and tested with and without metabolic activation.

Vehicle / solvent:

ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

V79 cells, taken from a stock culture are seeded in DMEM complete culture medium and incubated at 37.0 ± 1.5 °C and 5.0 ± 0.5 % CO2 for 5 - 10 d. Afterwards, the cells are ex-posed to the test item both in the presence and in the absence of an exogenous metabolic activation system.
Generally, exposure is started at 1 d after seeding of the cells when they are actively pro-liferating.
First, a pre-test is performed with 7 concentrations to determine the toxicity of the test item. For this purpose, survival (cloning efficiency (CE)) of the cells is determined. According to the results of the pre-test, at least 4 concentrations are chosen for the main experiments to examine the genotoxicity of the test item. The determination of the pH-value and the osmolality will also be performed before the main experimental part starts.
In the main experiments, the mutagenicity of the test item is detected in addition to the cytotoxic potential. The exposure time of the test item in the first main experiment is 3 - 6 h (usually 4 h) with and without metabolic activation. If the results of the first main experi-ment (Experiment I) do not give a clear result at the tested concentrations (negative or positive), a second experiment (Experiment II) without metabolic activation is necessary. The exposure time is then 24 h. In case of equivocal results also the approach with meta-bolic activation will be repeated in the second experiment (exposure time 4 h). In each concentration, duplicates of each treated culture are performed.

Evaluation criteria:

The gene mutation assay is considered acceptable if it meets the following criteria:
• the mutant frequency found in the solvent controls falls within the laboratory historical 95% control limit.
• the positive control substances must produce a significant increase (p < 0.05) in mutant frequency and lies in the range of the laboratory historical 95% control limit.
• two experimental conditions (+S9 and -S9) are tested unless one resulted in positive results.
• adequate number of cells (spontaneous MF is 5 - 20 * 10-6 cells) and concentrations (minimum of 4) are analysable.

Statistics:

A linear regression (least squares) is performed to assess a possible dose dependent in-crease of mutant frequencies. The numbers of mutant colonies obtained for the groups treated with the test item are compared to the numbers of mutant colonies obtained in the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance are considered together.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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

In the following tables and figures all final results of the cytotoxicity and the mutagenicity are summarised.

Summary of Results of Experiment Ia (+S9 and -S9)

	Concentration	S9 mix	Treatment time	Culture	Relative Survival	mutant frequency per 10^6cells	mutant frequency per 10^6cells
	[µM]		[h]		[%]		Mean
Solvent Control Test Item	-	+	4	A	-	4	6
				B	-	8
Solvent Control DMBA	-	+	4	A	-	8	9
				B	-	9
Positive Control (DMBA)	1.5 µg/mL	+	4	A	95.2%	91**	89**
				B	63.1%	88**
Test item	10	+	4	A	0.0%	n/a	n/a
				B	1.5%	n/a
Test item	5	+	4	A	5.3%	n/a	n/a
				B	6.6%	n/a
Test item	2.5	+	4	A	64.2%	7	4
				B	66.5%	1
Test item	1.25	+	4	A	30.9%	8	5
				B	29.6%	1
Test item	0.63	+	4	A	66.8%	9	10
				B	85.4%	11
Test item	0.31	+	4	A	60.2%	4	12
				B	91.0%	20^(*^)
Solvent Control Test Item	-	-	4	A	-	6	7
				B	-	7
Solvent Control EMS	-	-	4	A	-	8	8
				B	-	8
Positive Control (EMS)	300 µg/mL	-	4	A	81.6%	111	128**
				B	83.6%	144
Test item	10	-	4	A	0.0%	n/a	n/a
				B	0.0%	n/a
Test item	5	-	4	A	0.0%	n/a	n/a
				B	0.0%	n/a
Test item	2.5	-	4	A	0.0%	n/a	n/a
				B	0.2%	n/a
Test item	1.25	-	4	A	0.0%	n/a	n/a
				B	0.0%	n/a
Test item	0.63	-	4	A	55.4%	19^(**^)	19^(**^)
				B	65.7%	20^(*^)
Test item	0.31	-	4	A	76.2%	7	5
				B	77.4%	4

n/a = not analysed for mutagenicity because of cytotoxicity

italics values are invalid because the OECD 476 prescribes a minimum of 4 analysable test item concentrations.

Asterisks indicate statistically significant differences to solvent control, with * p < 0.05, ** p < 0.01,

⁽*⁾p < 0.05 but inside the historical control range of the solvent control,⁽**⁾p < 0.01 but inside the historical control range of the solvent control

Summary of Results of Experiment Ib (+S9)

	Concentration	S9 mix	Treatment time	Culture	Relative Survival	mutant frequency per 10^6cells	mutant frequency per 10^6cells
	[µM]		[h]		[%]		Mean
Solvent Control Test Item	-	+	4	A	-	9	17
				B	-	25
Solvent Control DMBA	-	+	4	A	-	9	10
				B	-	11
Positive Control (DMBA)	1.5 µg/mL	+	4	A	61.0%	120**	157**
				B	80.4%	194**
Test item	10	+	4	A	0.0%	n/a	n/a
				B	0.0%	n/a
Test item	5	+	4	A	19.1%	n/a	n/a
				B	0.0%	n/a
Test item	2.5	+	4	A	0.2%	n/a	n/a
				B	0.5%	n/a
Test item	1.25	+	4	A	1.5%	n/a	n/a
				B	0.0%	n/a
Test item	0.63	+	4	A	4.8%	n/a	n/a
				B	18.7%	n/a
Test item	0.31	+	4	A	82.0%	4	5
				B	132.4%	5
Test item	0.16	+	4	A	85.9%	6	10
				B	120.9%	13
Test item	0.08	+	4	A	98.0%	9	11
				B	116.8%	13
Test item	0.04	+	4	A	96.0%	2	6
				B	109.8%	9

n/a = not analysed for mutagenicity because of cytotoxicity

Asterisks indicate statistically significant differences to solvent control, with * p < 0.05, ** p < 0.01

Summary of Results of Experiment Ib (-S9)

	Concentration	S9 mix	Treatment time	Culture	Relative Survival	mutant frequency per 10^6cells	mutant frequency per 10^6cells
	[µM]		[h]		[%]		Mean
Solvent Control Test Item	-	-	4	A	-	3	10
				B	-	17
Solvent Control EMS	-	-	4	A	-	13	14
				B	-	16
Positive Control (EMS)	300 µg/mL	-	4	A	85.6%	102**	129**
				B	127.2%	157**
Test item	10	-	4	A	0.0%	n/a	n/a
				B	0.0%	n/a
Test item	5	-	4	A	0.1%	n/a	n/a
				B	0.1%	n/a
Test item	2.5	-	4	A	0.0%	n/a	n/a
				B	0.0%	n/a
Test item	1.25	-	4	A	0.0%	n/a	n/a
				B	0.0%	n/a
Test item	0.63	-	4	A	0.2%	n/a	n/a
				B	0.5%	n/a
Test item	0.31	-	4	A	98.7%	13^(*^)	12
				B	96.5%	11
Test item	0.16	-	4	A	83.0%	9	7
				B	89.5%	5
Test item	0.08	-	4	A	72.6%	10	9
				B	115.3%	7
Test item	0.04	-	4	A	64.6%	16^(**^)	10
				B	78.5%	5

n/a = not analysed for mutagenicity because of cytotoxicity

Asterisks indicate statistically significant differences to solvent control, with * p < 0.05, ** p < 0.01,

⁽*⁾p < 0.05 but inside the historical control range of the solvent control,⁽**⁾p < 0.01 but inside the historical control range of the solvent control

Conclusions:

Under the experimental conditions reported, Ethylcyclohexane did not induce mutations in the HPRT locus using the V79 cell line in the absence and the presence of metabolic activation.
The recorded data in this study declare the test item Ethylcyclohexane as non-mutagenic.

Executive summary:

The study was performed to investigate the potential of the test item Ethylcyclohexaneto induce mutations at the HPRT locuson chromosome X Chinese Hamster V79 cells.

 

The assay was performed in two independent experiments I, using two parallel cultures each (duplicates). In experiment Ia, 6 concentrations of the test item were tested with and without metabolic activation. Since the results of experiment Ia were different to those of the pre-test, 9 concentrations were tested in a second experiment I (experiment Ib) to exclude an technical error in experiment Ia. But since the results of the two experiments I are similar, both experiments were used for the final evaluation of this HPRT test whereby the approach without metabolic activation of experiment Ia is invalid because only 2 test item concentrations were analysable. The exposure time was 4 hours.

 

The following nominal concentrations of the test item were investigated in experiment Ia: 10 mM, 5 mM, 2.5 mM, 1.25 mM, 0.63 mM, 0.31 mM

The following nominal concentrations of the test item were investigated in experiment Ib: 10 mM, 5 mM, 2.5 mM, 1.25 mM, 0.63 mM, 0.31 mM, 0.16 mM, 0.08 mM, 0.04 mM

Precipitation of the test item was not observed up to the highest tested concentration.

 

EMS (300 µg/mL) and DMBA (1.5 µg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies.

 

In experiment Ia with metabolic activation cytotoxic effects were observed at the test item concentrations 10 mM to 5 mM. In those concentrations the relative survival (RS) was reduced below 10 %. In the approach without metabolic activation cytotoxic effects were observed at the test item concentrations 10 mM to 1.25 mM. In those concentrations the relative survival (RS) was reduced to 0 %. Since the OECD guideline 476 prescribes that concentrations that showed a cytotoxic effect cannot be analysed for mutagenicity, an evaluation of the mutagenicity was not possible in those concentrations. In the approach without metabolic activation only 2 instead of 4 concentrations are analysable. Therefore this approach is invalid in accordance with the OECD 476. But since the quality of the results of the two remaining concentrations (0.63 mM and 0.31 mM) is not affected by this deviation, the results are indicated in this final report anyway despite they are invalid according to OECD 476.

In experiment Ib in both approaches cytotoxic effects were observed at the test item concentrations 10 mM to 0.63 mM. In those concentrations the relative survival (RS) was reduced below 20 %. Since the OECD guideline 476 prescribes the concentrations that showed a cytotoxic effect cannot be analysed for mutagenicity, the four lowest test item concentrations were analysed for mutagenicity.

 

In both valid experiments no substantial and reproducible dose dependent increase in mutant colony numbers was observed. All values remained well within the historical control range. According to OECD 476 the result of both valid experiments I is clearly negative.

 

In conclusion, it can be stated that under the experimental conditions reported,Ethylcyclohexanedid not induce mutations in the HPRT locus using the V79 cell line in the absence and the presence of metabolic activation.

The recorded data in this study declare the test item Ethylcyclohexaneas non-mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The respective criteria are not met. Ethylcyclohexane has not to be classifies for genetic toxicity.