3-ethoxy-1,1,5-trimethylcyclohexane

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 July to 10 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD Guideline 476 without any deviation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

other: mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-ethoxy-1,1,5-trimethylcyclohexane

EC Number:

266-722-5

EC Name:

3-ethoxy-1,1,5-trimethylcyclohexane

Cas Number:

67583-77-1

Molecular formula:

C11H22O

IUPAC Name:

3-ethoxy-1,1,5-trimethylcyclohexane

Test material form:

liquid

Details on test material:

- Physical state: Colourless liquid
- Storage condition of test material: Stored at 2-8 °C protected from light
- Expiration date of the lot/batch: 18 January 2017

Method

Target gene:

hprt locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Dr Donald Clive, Burroughs Wellcome Co.

MEDIA USED
- Type and identity of media including CO2 concentration:
RPMI A: Horse serum (heat inactivated, 0 % v/v). Penicillin (100 units/mL), streptomycin (100 μg/mL), amphotericin B (2.5 μg/mL), Sodium pyruvate acid (0.2 mg/mL) and pluronic (0.5 mg/mL)
RPMI 10: Horse serum (heat inactivated, 10 % v/v), penicillin (100 units/mL), streptomycin (100 μg/mL), amphotericin B (2.5 μg/mL), Sodium pyruvate acid (0.2 mg/mL) and pluronic (0.5 mg/mL)
RPMI 20: Horse serum (heat inactivated, 20 % v/v), penicillin (100 units/mL), streptomycin (100 μg/mL), amphotericin B (2.5 μg/mL) and Sodium pyruvate acid (0.2 mg/mL)
RPMI 5 consisted of RPMI 10 diluted with RPMI A [prepared as RPMI 10 but with no serum added] to give a final concentration of 5% serum
- For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated at 37 ± 1 °C. When the cells were growing well, subcultures were established in an appropriate number of flasks
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically 'cleansed' against high spontaneous background: Yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

2 % S9 (final concentration); S9 fraction was prepared from liver homogenates of rats treated with Aroclor 1254.

Test concentrations with justification for top dose:

Range-Finder Experiment: 31.25, 62.5, 125, 250, 500 and 1000 µg/mL, with and without S9
Justification: A maximum concentration of 1000 µg/mL was selected for the cytotoxicity Range-Finder Experiment in order that treatments were performed up to a precipitating concentration.

Mutation Experiment:
Without S9: 5, 10, 20, 30, 40, 45, 50, 55, 60, 65, 70 and 100 µg/mL
With S9: 10, 20, 40, 60, 70, 80, 90, 100, 110, 120, 150 and 200 µg/mL
Justification: Concentrations selected for the Mutation Experiment were based on the results of this cytotoxicity Range-Finder Experiment.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that the test substance was soluble in acetone at a concentration of at least 188.2 mg/mL. The solubility limit in culture medium was approximately 117.6 to 235.3 µg/mL, as indicated by precipitation at the higher concentration which persisted for approximately 3 h after test substance addition, with warming at 37 °C.
- Test substance preparation: Test substance stock solutions were prepared by formulating Test substance under subdued lighting in acetone, with the aid of vortex mixing, to give the maximum required concentration. Subsequent dilutions were made using acetone. The test substance solutions were protected from light and used within approximately 1.5 h of initial formulation.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

acetone diluted 100 fold in the treatment medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9

Controls 2

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

acetone diluted 100 fold in the treatment medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; RPMI 1640 media supplied containing L-glutamine and HEPES
- Cell density at seeding:
Cytotoxicity Range-Finder Experiment: Cell concentrations were adjusted to 8 cells/mL and, for each concentration, 0.2 mL was plated into each well of a 96-well microtitre plate for determination of relative survival.
Mutation Experiment: At least 10^7 cells in a volume of 18.8 mL of RPMI 5 were placed in a series of sterile disposable 50 mL centrifuge tubes.

DURATION
- Exposure duration: 3 h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 12 days
- Plating for Survival: 7 days
- Plating for viability: 9 days
- All incubations were performed at 37 ± 1 °C in a humidified incubator gassed with 5 ± 1 % v/v CO2 in air

SELECTION AGENT (mutation assays): 6-thioguanine (6TG) at a final concentration of 15 μg/mL

NUMBER OF REPLICATIONS:
- Preliminary toxicity test: Single cultures/dose for test item, negative and vehicle control
- Main test: Two cultures for negative, vehicle control and test item; single culture for positive control

NUMBER OF CELLS EVALUATED: 1.6, 1.6 and 20000 cells per well plated for survival, viability and 6TG resistance respectively.

DETERMINATION OF CYTOTOXICITY
- Method: Percentage Relative Survival
Cloning efficiency (CE) = P / No of cells plated per well; and as an average of 1.6 cells/well were plated on all survival and viability plates, CE = P/1.6.
Percentage relative survival (% RS) = [CE (test)/CE (control)] x 100.
Adjusted % RS = % RS x (Post-treatment cell concentration for test article treatment / Post-treatment cell concentration for vehicle control)

- OTHER:
Mutant Frequency (MF) per 10^6 viable cells for each set of plates was calculated as: MF = [CE (mutant)/CE (viable)] x 10^6.

Evaluation criteria:

For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
- The MF at one or more concentrations was significantly greater than that of the vehicle control (p≤0.05)
- There was a significant concentration relationship as indicated by the linear trend analysis (p≤0.05)
- The results were outside the historical vehicle control range.

Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis. Positive responses seen only at high levels of cytotoxicity required careful interpretation when assessing their biological relevance. Extreme caution was exercised with positive results obtained at levels of RS lower than 10%.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines (Robinson et al., 1990). The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

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 and osmolality: No marked changes in osmolality or pH were observed in the Range-Finder at the highest concentration tested (1000 µg/mL), compared to the concurrent vehicle controls.
- Precipitation: Upon addition of the test substance to the cultures, precipitate was observed at the highest three concentrations tested (250 to 1000 µg/mL) but at the end of the 3 h treatment period the precipitate was no longer visible.
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES:
In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 31.25 to 1000 µg/mL (limited by solubility limit in culture medium). Upon addition of the test substance to the cultures, precipitate was observed at the highest three concentrations tested (250 to 1000 µg/mL) but at the end of the 3 h treatment period the precipitate was no longer visible. The highest concentrations to give >10% RS were 31.25 µg/mL in the absence of S-9 and 62.5 µg/mL in the presence of S-9, which gave 72% and 68% RS, respectively.
The range-finder experiment indicated that optimal toxicity (80 to 90% reduction in RS) would likely be observed at a dose level between 31.25 and 62.5 µg/mL in the absence of S-9, and between 62.5 and 125 µg/mL in the presence of S-9, but with a very steep dose-response curve.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
The historical control ranges for the last 20 experiments performed in this laboratory since November 2014, up to and immediately prior to this study, are as follows:
- Negative (solvent/vehicle) historical control data:
Vehicle Controls
In the absence of S-9
Mean: 3.31 mutants per 10^6 viable cells, Range* = 0.27 to 6.35 mutants per 10^6 viable cells.
In the presence of S-9
Mean: 3.56 mutants per 10^6 viable cells, Range* = 0.93 to 6.20 mutants per 10^6 viable cells.
*Range = Mean ± 2 x SD.

- Positive historical control data:
NQO 0.15 µg/mL in the absence of S-9
Mean: 36.02 mutants per 10^6 viable cells, Range* = 4.28 to 67.76 mutants per 10^6 viable cells.
NQO 0.2 µg/mL in the absence of S-9
Mean: 52.51 mutants per 10^6 viable cells, Range* = 13.22 to 91.80 mutants per 10^6 viable cells.
B[a]P 2 µg/mL in the presence of S-9
Mean: 19.33 mutants per 10^6 viable cells, Range* = 0 to 41.41 mutants per 10^6 viable cells.
B[a]P 3 µg/mL in the presence of S-9
Mean: 33.57 mutants per 10^6 viable cells, Range* = 1.26 to 65.88 mutants per 10^6 viable cells.
*Range = Mean ± 2 x SD.

MUTATION EXPERIMENT
- In the Mutation Experiment twelve concentrations, ranging from 5 to 100 µg/mL in the absence of S-9 and from 10 to 200 µg/mL in the presence of S-9, were tested. Upon addition of the test substance to the cultures, precipitate was observed at the highest two concentrations tested in the presence of S-9 (150 and 200 µg/mL), but at the end of the 3 h treatment period the precipitate was no longer visible. Seven days after treatment, the highest seven concentrations in the absence of S-9 (45 to 100 µg/mL) and the highest three concentrations in the presence of S-9 (120 to 200 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. All other concentrations in the absence and presence of S-9 were selected for plating for viability and 6TG resistance. The highest concentration analysed in the absence of S 9 was 40 µg/mL, which gave 3% RS. Steep concentration-related toxicity was observed between 30 and 40 µg/mL in the absence of S-9, giving 85% and 3% RS, respectively, therefore both concentrations were included in the analysis. The highest concentration analysed in the presence of S-9 was 110 µg/mL, which gave 13% RS.
- When tested up to toxic, or highly toxic concentrations in the presence or absence of S-9, respectively, no statistically significant increases in mutant frequency, compared to the vehicle control MF, were observed following treatment with the test article at any concentration analysed and there were no statistically significant linear trends. Treatment at a highly toxic concentration (40 µg/mL, giving 3% RS) in the absence of S-9 exceeded the target maximum of 90% reduction in %RS, however, it was considered that this did not affect the interpretation of the data, which were clearly negative at all concentrations.

Any other information on results incl. tables

Table 7.6.1/1: Range-finder experiment - 3 h treatment in the absence and presence of S-9

 

Concentration	3 h treatment –S‑9	3 h treatment +S‑9
µg/mL	% RS	% RS
0	100	100
UTC	85	132
31.25	72	102
62.5	0	68
125	0	0
250 P	NP	0
500 P	NP	0
1000 P	NP	0

% RS: Percent relative survival adjusted by post treatment cell counts

UTC: Untreated control

P: Precipitation observed at the time of treatment

NP: Not plated due to excessive toxicity

Table 7.6.1/2: Mutation experiment - 3 h treatment in the absence and presence of S-9

 

3 h treatment ‑S-9			3 h treatment +S-9
Concentration (µg/mL)	% RS	MF §	Concentration (µg/mL)	% RS	MF §
0	100	4.80	0	100	5.85
UTC	121	4.31	UTC	97	3.56
5	103	6.99 NS	10	107	4.42 NS
10	102	7.25 NS	20	111	4.72 NS
20	108	6.99 NS	40	113	5.05 NS
30	85	3.43 NS	60	121	3.45 NS
40	3	2.16 NS	70	99	5.68 NS
-	-	-	80	89	5.06 NS
-	-	-	90	88	3.62 NS
-	-	-	100	39	4.42 NS
-	-	-	110	13	2.22 NS
NQO 0.15	38	50.46	B[a]P 2	88	25.58
NQO 0.20	33	66.97	B[a]P 3	60	50.61

Linear trend tests on mutant frequency +/-S-9: Not significant (negative trends) 

% RS: Percent relative survival adjusted by post treatment cell counts

NS: Not significant

§: 6‑TG resistant mutants/10⁶ viable cells 7 days after treatment

Applicant's summary and conclusion

Conclusions:

Under the test conditions, test substance did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to toxic, or highly toxic concentrations for 3 h in the presence or absence, respectively, of a rat liver metabolic activation system (S-9).

Executive summary:

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, L5178Y tk^+/-(3.7.2C) mouse lymphoma cells were exposed to test substance for 3 h at the following concentrations:

 

Range-Finder Experiment: 31.25, 62.5, 125, 250, 500 and 1000 µg/mL, with and without S9

Mutation Experiment:

Without S9: 5, 10, 20, 30, 40, 45, 50, 55, 60, 65, 70 and 100 µg/mL

With S9: 10, 20, 40, 60, 70, 80, 90, 100, 110, 120, 150 and 200 µg/mL

 

Negative, vehicle and positive control groups were also included in each mutagenicity test. Metabolic activation system used in this test was 2 % S9 mix (final concentration). S9 fraction was prepared from liver homogenates of rats treated with Aroclor 1254.

 

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 31.25 to 1000 µg/mL (limited by solubility of the test substance formulation in culture medium). Steep concentration-related toxicity was observed and the highest concentrations to give>10% relative survival (RS) were 31.25 µg/mL in the absence of S-9 and 62.5 µg/mL in the presence of S-9, which gave 72% and 68% RS, respectively.

In the Mutation Experiment twelve concentrations, ranging from 5 to 100 µg/mL in the absence of S-9 and from 10 to 200 µg/mL in the presence of S-9, were tested. Seven days after treatment, the highest concentration analysed to determine viability and 6TG resistance in the absence of S-9 was 40 µg/mL, which gave 3% RS. Steep concentration-related toxicity was observed between 30 and 40 µg/mL in the absence of S-9, giving 85% and 3% RS, respectively, therefore both concentrations were analysed. The highest concentration analysed in the presence of S-9 was 110 µg/mL,which gave 13% RS.

 

Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S-9) and benzo(a)pyrene (B[a]P) (with S-9). Therefore, the study was accepted as valid.

 

When tested up to toxic, or highly toxic concentrations in the presence or absence of S-9, respectively, no statistically significant increases in mutant frequency, compared to the vehicle control MF, were observed following treatment with the test substance at any concentration analysed and there were no statistically significant linear trends. Treatment up to a highly toxic concentration (40 µg/mL, giving 3% RS) in the absence of S-9 did not affect the interpretation of the data, which were clearly negative.

 

Under the test conditions, test substance did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to toxic, or highly toxic concentrations for 3 h in the presence or absence, respectively, of a rat liver metabolic activation system (S-9).