2-tert-butylcyclohexyl ethyl carbonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 January - 13 March 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Well conducted and well described study in accordance with GLP and OECD Guideline 487 without any deviation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 487 (In Vitro Mammalian Cell Micronucleus Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP compliance programme (inspected on 17 June 2015/ signed on 24 September 2015)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

2-tert-butylcyclohexyl ethyl carbonate

EC Number:

267-184-4

EC Name:

2-tert-butylcyclohexyl ethyl carbonate

Cas Number:

67801-64-3

Molecular formula:

C13H24O3

IUPAC Name:

2-tert-butylcyclohexyl ethyl carbonate

Test material form:

liquid

Details on test material:

- Physical state: Colorless liquid
- Expiration date of the lot/batch: 02 July 2016
- Storage condition of test material: At room temperature protected from light

Method

Target gene:

None

Species / strain

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

2 % S9 (final concentration); S9 fraction prepared from liver homogenates of rats induced with Phenobarbital/β-naphthoflavone.

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 0, 8.91, 17.81, 35.63, 71.25, 142.5, 285, 570, 1140 and 2280 µg/mL (4-hour exposure to the test item without S9-mix or with S9-mix (2%); 24-hour continuous exposure to the test item without S9-mix)

Main Experiment:
4-hour exposure to the test item without S9-mix: 0, 4, 8, 16, 24, 28, 32 and 64 µg/mL.
4-hour exposure to the test item with S9-mix (2%): 0, 8, 16, 32, 56, 64, 80 and 96 µg/mL.
24-hour continuous exposure to the test item without S9-mix: 0, 8, 16, 24, 32, 40, 56 and 64 µg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- The test item was soluble in DMSO at 228 mg/mL in a solubility check performed in house.
- Prior to each experiment, the test item was accurately weighed, dissolved in DMSO and serial dilutions prepared. The test item was formulated within two hours of it being applied to the test system; it is assumed that the test item formulation was stable for this duration.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

other: Demecolcine

Remarks:

Without S9 mix - Mitomycin C: 0.2 µg/mL for 4-hour exposure; Demecolcine: 0.075 µg/mL for 24-hour continuous exposure

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With S9 mix - 5 µg/mL for 4-hour exposure

Details on test system and experimental conditions:

TEST SYSTEM: For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non smoking volunteer (18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years of in house data for cell cycle times for human lymphocytes, using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT), it is considered to be approximately 16 hours. Therefore using this average value of the AGT the in-house experimental exposure time was 1.5 x AGT, which is 24 hours.
The details of the donors used are: Preliminary Toxicity Test: Female, aged 34 years; Main Experiment: Male, aged 28 years

CELL CULTURE: Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% fetal bovine serum (FBS), at approximately 37 ºC with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

DURATION
- Exposure duration: 4 h (± S9) and 24 h continuous exposure (-S9) in preliminary toxicity test; 4 h (± S9) and 24 h continuous exposure (-S9) in main experiment
- At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 h in the presence of Cytochalasin B.

SPINDLE INHIBITOR (cytogenetic assays): Prior to the mitosis (after exposure of the test substance) the chemical cytochalasin B (4.5 μg/mL) was added to the cultures.
STAIN (for cytogenetic assays): 5 % Giemsa for 5 minutes

NUMBER OF REPLICATIONS:
- Preliminary toxicity test: Single culture for test item and vehicle control
- Main test: Duplicate cultures per dose for test item, vehicle and positive controls

NUMBER OF CELLS EVALUATED:
- Cytotoxicity: A minimum of approximately 500 cells per culture were scored for the incidence of mononucleate, binucleate and multinucleate cells and the CBPI value expressed as a percentage of the vehicle controls. The CBPI indicates the number of cell cycles per cell during the period of exposure to Cytochalasin B.
- Scoring of Micronuclei: The micronucleus frequency in 2000 binucleated cells was analyzed per concentration (1000 binucleated cells per culture, two cultures per concentration). Cells with 1, 2 or more micronuclei were recorded as such but the primary analysis was on the combined data. Experiments with human lymphocytes have established a range of micronucleus frequencies acceptable for control cultures in normal volunteer donors.
DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity of test item in the lymphocyte cultures was determined using the cytokinesis-block proliferation index (CBPI index).
% Cytostasis = 100-100{(CBPIT – 1)/(CBPIC –1)}
CBPI = [(No. mononucleate cells) + (2 x No. binucleate cells) + (3 x No. multinucleate cells)] / [Total number of cells]
T = test substance treatment culture
C = vehicle control culture

OTHER:
The criteria for identifying micronuclei were that they were round or oval in shape, non refractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei. Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter.

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in most/all of the experimental conditions examined:
- None of the test concentrations exhibited a statistically significant increase in micronucleus frequency compared with the concurrent negative control.
- There was no dose-related increase in micronucleus frequency.
- The micronucleus frequency results in all evaluated dose groups were within the range of the laboratory historical control data.
Providing that all of the acceptability criteria are fulfilled, a test item may be considered to be clearly positive, if in any of the experimental conditions examined, there is one or more of the following applicable:
- At least one of the test concentrations exhibited a statistically significant increase in micronucleus frequency compared with the concurrent negative control.
- There was an increase in micronucleus frequency that can be considered to be dose-related.
- The micronucleus frequency results were substantially outside the range of the laboratory historical negative control data.
When all the criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss of a whole chromosome in this test system.
There is no requirement for verification of a clear positive or negative response.
In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations. The Study Director may make a judgement based on experience and the biological relevance of the data and any justification for acceptance of the data will be included in the report.

Statistics:

The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on observed numbers of cells with micronuclei. Other statistical analyses may be used if appropriate (Hoffman et al., 2003). A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei which was reproducible.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991).

PRELIMINARY TOXICITY TEST:
- The dose range for the Preliminary Toxicity Test was 8.91 to 2280 µg/mL. The maximum dose was the 10 mM concentration.
- A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 142.5 µg/mL in the exposure groups in the absence of S9-mix and at and above 285 µg/mL in the exposure group in the presence of S9-mix.
- Hemolysis was observed following exposure to the test item at and above 8.91 µg/mL in the 4-hour exposure group in the absence of S9-mix and at and above 35.63 µg/mL in both the presence of S9-mix and the 24-hour continuous exposure groups. Hemolysis is an indication of a cytotoxic effect to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.
- Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to 35.63 µg/mL in the 4-hour exposure in the absence of S9-mix and at and above 71.25 µg/mL in both in the presence of S9-mix and the 24-hour exposure groups. The test item induced evidence of toxicity in all of the exposure groups.
- The selection of the maximum dose level for the Main Experiment was based on toxicity in the exposure groups in the absence of S9-mix and the lowest precipitating dose level for the exposure group in the presence of S9.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The concurrent negative control was within the laboratory historical control data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Main experiments:
- The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test, particularly in the absence of S9-mix and that there were binucleate cells suitable for scoring at the maximum dose level of test item in all three exposure groups.
- A precipitate of test item was noted at 64 µg/mL in the 4-hour exposure group in the absence of S9-mix and at 96 µg/mL in the presence of S9-mix. No precipitate was observed in the 24-hour exposure group. Hemolysis was observed at all dose levels tested in all three exposure groups.
- The CBPI data for the 4 and 24-hour exposure groups confirm the qualitative observations in that a dose-related inhibition of CBPI was observed in the exposure groups in the absence of S9-mix. In the 4-hour exposure group in the absence of S9-mix, 27 and 55% cytostasis was achieved at 32 and 64 µg/mL, respectively whereas 41 and 55% cytostasis was observed at 56 and 64 µg/mL in the 24-hour exposure group. This indicated that the optimum level of toxicity was achieved in both exposure groups. In the presence of S9-mix, the level of cytostasis achieved at the maximum dose level was 26%, however a precipitate of the test item was observed at this dose level (96 µg/mL). The toxicity test had indicated that optimum toxicity may have been expected to have been achieved at between 56 and 80 µg/mL. Also, the level at which precipitate was observed in the 4-hour exposure groups was lower than that indicated by the toxicity test. Such inter-experimental minor differences are common in in vitro studies and do affect the validity of the results.
Therefore, the maximum dose level selected for analysis of binucleate cells was the maximum dose level in each exposure group and was limited by toxicity in the absence of S9-mix and by precipitate in the presence of S9-mix.

Any other information on results incl. tables

Table 7.6.1/1: pH and osmolality

Dose Concentration (µg/mL)	0	8.92	17.84	35.67	71.34	142.69	285.38	570.75	1141.5	2283
pH	7.49	7.45	7.43	7.44	7.46	7.46	7.46	7.45	7.45	7.45
Osmolality mOsm	424	425	-	423	425	420	-	412	398	377

- = Not performed for this dose concentration

 

Table 7.6.1/2: CBPI and Micronucleus Data

Dose Level (μg/mL)	Replicate	Nucleate cells /500 cells			CBPI	% Control CBPI	% Cytostasis	Micronuclei (MN) per 1000 Binucleate cells			%Cells with MN	Mean % Cells with MN
		Mono	Bi	Multi				1 MN	2 MN	>2 MN
4 h Exposure Without Metabolic Activation (S9)
0	A	231	212	57	1.65	100	0	8	0	0	0.80	0.70
	B	255	200	45	1.58			6	0	0	0.60
24	A	229	240	31	1.60	97	3	3	0	0	0.30	0.30
	B	247	205	48	1.60			3	0	0	0.30
28	A	290	176	34	1.49	81	19	3	0	1	0.40	0.40
	B	285	182	33	1.50			3	1	0	0.40
32	A	307	175	18	1.42	73	27	6	0	0	0.60	0.65
	B	283	195	22	1.48			6	1	0	0.70
64	A	354	135	11	1.31	45	55	4	0	0	0.40	0.25
	B	385	107	8	1.25			1	0	0	0.10
MMC 0.2	A	312	180	8	1.39	55	45	59	5	1	6.50	7.45***
	B	358	140	2	1.29			74	10	0	8.40
4 h Exposure With Metabolic Activation (S9)
0	A	190	253	57	1.73	100	0	1	0	0	0.10	0.30
	B	217	234	49	1.66			4	0	1	0.50
56	A	216	246	38	1.64	90	10	2	0	0	0.20	0.40
	B	223	243	34	1.62			6	0	0	0.60
64	A	194	260	46	1.70	87	13	3	0	0	0.30	0.45
	B	271	199	30	1.52			6	0	0	0.60
80	A	217	253	30	1.63	89	11	5	0	0	0.50	0.85
	B	230	237	33	1.61			12	0	0	1.20
96	A	278	197	25	1.49	74	26	1	0	0	0.10	0.30
	B	259	212	29	1.54			5	0	0	0.50
CP 5	A	322	167	11	1.38	44	56	30	2	0	3.20	5.30***
	B	378	122	0	1.24			67	6	1	7.40
24 h Exposure Without Metabolic Activation (S9)
0	A	92	352	56	1.93	100	0	4	0	0	0.40	0.45
	B	74	353	73	2.00			5	0	0	0.50
32	A	103	347	50	1.89	89	11	3	0	0	0.30	0.80
	B	121	343	36	1.83			1	12	0	1.30
40	A	156	312	32	1.75	80	20	3	0	0	0.30	0.35
	B	128	338	34	1.81			4	0	0	0.40
56	A	209	280	11	1.60	59	41	2	4	0	0.60	0.65
	B	242	247	11	1.54			5	2	0	0.70
64	A	324	173	3	1.36	45	55	7	0	0	0.70	0.50
	B	249	244	7	1.52			2	1	0	0.30
DC 0.075	A	228	233	39	1.62	57	43	44	8	1	5.30	4.50***
	B	289	181	30	1.48			27	10	0	3.70

 

MMC = Mitomycin C; CP = Cyclophosphamide; DC = Demecolcine; *** = P<0.001

Applicant's summary and conclusion

Conclusions:

negative with metabolic activation
negative without metabolic activation

Under the test conditions, test item was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Executive summary:

In an in vitro micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, cultured peripheral human lymphocytes were exposed to test item in the presence and absence of a metabolic activation system. Three exposure conditions in a single experiment were used for the study using a 4‑hour exposure in the presence and absence of a standard metabolizing system (S9 at a 2% final concentration) and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B.

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by the toxicity of the test item in the absence of S9-mix and limited by precipitate in the presence of S9-mix. The dose levels selected for the Main Test were as follows:

4-hour without S9: 0, 4, 8, 16, 24, 28, 32 and 64 μg/mL

4-hour with S9 (2%): 0, 8, 16, 32, 56, 64, 80 and 96 μg/mL

24-hour without S9: 0, 8, 16, 24, 32, 40, 56 and 64 μg/mL

 

All vehicle (dimethyl sulphoxide) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item was toxic to human lymphocytes but did not induce any statistically significant increases in the frequency of cells with micronuclei, using a dose range that included a dose level that induced approximately 50% reduction in CBPI in the absence of S9-mix and was the lowest precipitating dose level in the presence of S9-mix.

 

Under the test conditions, test item was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.