2-hydroxypropyl [(1R,2S,5R)-2-isopropyl-5-methyl-cyclohexyl] carbonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016-08-10 to 2016-11-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2014-09-26

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2015-09-14

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

1-[({[(1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl]oxy}carbonyl)oxy]propan-2-ol

EC Number:

607-858-0

Cas Number:

260781-16-6

Molecular formula:

C14H26O4

IUPAC Name:

1-[({[(1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl]oxy}carbonyl)oxy]propan-2-ol

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

lymphocytes: human (peripheral)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: healthy human donors (non-smoking; not receiving medication)

- Suitability of cells: lymphocytes of the donors have been shown to respond well to stimultation of proliferation with phytohemeagglutinine (PHA) and to positive control substance. All donors had previously established low incidence of micronuclei in their peripheral blood lymphocytes.

- Sex, age and number of blood donors: two female donors (30 and 28 years old, respectively; Experiment 1A and Experiment 2, respectively); one male donor (26 years old; Experiment 1B)

- Whether whole blood or separated lymphocytes were used: blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 hours after blood collection. The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (mixture 1:1) supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin, the mitogen PHA, 10 % fetal bovine serum, 10 mM HEPES and the anticoagulant heparin.
All incubations were done at 37 °C with 5.5 % CO2 in humidified air.

Cytokinesis block (if used):

4 µg/mL cytochalasin B (exposure duration: 20 hours)

Metabolic activation:

with and without

Metabolic activation system:

S9-mix: S9 supernatant, MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM), and NADP (4 mM) in sodium-ortho-phosphate buffer (100 mM, pH 7.4)

Test concentrations with justification for top dose:

Preliminary cytotoxicity test/Experiment 1A: 13.4, 23.4, 40.9, 71.6, 125, 219, 384, 671, 1175, and 2056 µg/mL (with and without metabolic activation; 4 hours)
Experiment 1B: 82.9, 108, 140, 182, 237, 308, and 400 µg/mL (with metabolic activation; 4 hours)
Experiment 2: 2.6, 4.5, 8.0, 13.9, 24.4, 42.6, 74.6, 131, 229, and 400 µg/mL (without metabolic activation; 20 hours)
Please refer to the field "Rationale for test conditions" below.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (culture medium with 0.5 % vehicle)
- Justification for choice of solvent/vehicle: the solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

culture medium with 0.5 % DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: demecolcin

Details on test system and experimental conditions:

DETERMINATION OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index (CBPI)
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterized by the percentages of reduction in the CBPI in comparison with the controls by counting 500 cells per culture.

EXPERIMENTAL DESIGN
1) Pre-experiment/Experiment 1A/1B (pulse treatment):
- the preliminary cytotoxicity test was designated Experiment 1A, since the cultures fulfilled the requirements for cytogenetic evaluation.
- 10 concentrations of the test item separated by no more than a factor of √10 and a solvent and positive control.
- all cell cultures were set up in duplicate.
- exposure time was 4 hours (with and without S9 mix) and the preparation interval was 40 hours after start of the exposure.
- NOTE: the experimental part with S9 mix was repeated due to an increase in micronucleate cells and was designated as Experiment 1B.

- about 48 hours after seeding 2 blood cultures (10 mL each) were set up for each test item concentration.
- culture medium was replaced with serum-free medium containing the test item.
- for the treatment with metabolic activation 50 μL S9 mix/mL culture medium was added.
- after 4 hours the cells were spun down by centrifugation and the supernatant was discarded.
- cells were resuspended in and washed with "saline G" (pH 7.2, containing NaCl, KCl, glucose • H2O, Na2HPO4 • 2 H2O and KH2PO4).
- washing procedure was repeated once.
- cells were resuspended in complete culture medium with 10 % FBS (v/v) and cultured for a 16-hour recovery period.
- Cytochalasin B (4 μg/mL) was added and the cells were cultured another approx. 20 hours until preparation.

- Experiment 2 (continuous treatment):
- about 48 hours after seeding 2 blood cultures (10 mL each) were set up for each test item concentration.
- culture medium was replaced with complete medium (with 10 % FBS) containing the test item.
- after 20 hours the cells were spun down by centrifugation and the supernatant was discarded.
- cells were re-suspended in and washed with "saline G".
- washing procedure was repeated once.
- cells were re-suspended in complete culture medium containing 10 % FBS (v/v).
- Cytochalasin B (4 μg/mL) was added and the cells were cultured another approx. 20 hours until preparation.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
- cultures were harvested by centrifugation 40 hours after beginning of treatment.
- cells were spun down by centrifugation and the supernatant was discarded.
- cells were re-suspended in approx. 5 mL saline G and spun down by centrifugation.
- cells were resuspended in 5 mL KCl solution (0.0375 M) and incubated at 37 °C for 20 minutes.
- 1 mL of ice-cold fixative mixture of methanol and glacial acetic acid (19 parts:1 part) was added to the hypotonic solution and cells were resuspended.
- After removal of the solution by centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold.
- slides were prepared by dropping the cell suspension in fresh fixative onto a microscope slide.
- cells were stained with Giemsa.
- evaluation of the slides was performed using microscopes with 40 x objectives.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976)*. The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus.

NUMBER OF CELLS EVALUATED:
- at least 1000 binucleate cells/culture were scored for cytogenetic damage (% micronucleated cells)
- 500 cells/culture were scored for CBPI (% cytostasis)

*Reference:
- Countryman P.I. and Heddle J.A. (1976) The production on micronuclei from chromosome aberrations in irradiated cultures of human lymphocytes. Mutation Research, 41, 321-332.

Rationale for test conditions:

In the preliminary cytotoxicity test, the highest treatment concentration in this study, 2056 μg/mL was chosen with regard to the purity (97.3%) of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test.
In the pre-test for toxicity, phase separation of the test item was observed at the end of treatment at 219 μg/mL and above in the absence of S9 mix and at 384 μg/mL and above in the presence of S9 mix. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment 1A. The experimental part with S9 mix was repeated due to an increase in micronucleate cells (Experiment 1B).
Clear toxic effects were observed in Experiment 1A after 4 hours treatment with 219 μg/mL and above in the absence of S9 mix and with 384 μg/mL in the presence of S9 mix. Additionally, clear toxic effects were observed in Experiment 1B in the presence of S9 mix with 308 μg/mL and above. Therefore, 400 μg/mL (without S9 mix) were chosen as top concentration in Experiment 2.

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be negative if, in all of the experimental conditions examined:
− none of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− there is no concentration-related increase
− the results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data (95% confidence interval)

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be positive if, in any of the experimental conditions examined:
− at least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− the increase is concentration-related in at least one experimental condition
− the results are outside the range of the laboratory historical solvent control data (95 % confidence interval)

Statistics:

Chi square test (α < 0.05).

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human (peripheral)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

NOTE: the following concentrations were evaluated for micronucleated cells:
Experiment 1A :23.4, 40.9, and 71.6 µg/mL (without metabolic activation; 4 hour exposure)
Experiment 1A :71.6, 125, and 219 µg/mL (with metabolic activation; 4 hour exposure)
Experiment 1B: 140, 182, and 237 µg/mL (with metabolic activation; 4 hour exposure)
Experiment 2: 24.4, 42.6, and 74.6 µg/mL (without metabolic activation; 20 hour exposure)

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no relevant influence on pH was observed
- Effects of osmolarity: no relevant influence on osmolarity was observed
- Phase separation:
Experiment 1A: phase separation of the test item in the culture medium was observed at 219 μg/mL and above in the absence of S9 mix and at 384 μg/mL and above in the presence of S9 mix at the end of treatment.
Experiment 1B: no phase separation was observed.
Experiment 2: phase separation occurred in the absence of S9 mix at 400 μg/mL and above at the end of treatment.

INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI
In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration. Concentrations showing clear cytotoxic effects, however, were not evaluable for cytogenetic damage.

CYTOGENETIC:
Experiment 1A and 2: in the absence of S9 mix no relevant increases in the number of micronucleate cells were observed after treatment with the test item.

In Experiment 1A in the presence of S9 mix, the highest evaluated concentration (219 μg/mL) showed a statistical significant increase in the number of micronucleate cells of 1.30 % (value exceeded historical control range: 0.08 – 1.20 %). In Experiment 1B the finding could not be confirmed. Therefore, the finding of Experiment 1A was not reproducible and can be considered as biologically irrelevant.

Please also refer for further information on results to the field "Attached background material" below.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
Please refer to the field "Any other information on results incl. tables" below

Any other information on results incl. tables

Historical laboratory control data

Percentage of micronucleated cells in human lymphocyte cultures (2014-2015)

Solvent Control without S9
Micronucleated cells in %
	Pulse treatment (4/40)	Continous treatment (20/40)
No. of experiments	50*	54**
Mean	0.61	0.55
95% Ctrl limit	0.07 - 1.15	0.05 - 1.05
1x SD	0.27	0.25
2x SD	0.54	0.50
Min	0.15	0.05
Max	1.25	1.43

 

*Aqueous solvents – 23 Experiments; Organic solvents – 27 Experiments

**Aqueous solvents – 24 Experiments; Organic solvents – 30 Experiments

Solvent Control with S9
Micronucleated cells in %
	Pulse treatment (4/40)
No. of experiments	67*
Mean	0.64
95% Ctrl limit	0.08 - 1.20
1x SD	0.28
2x SD	0.56
Min	0.15
Max	1.35

 *Aqueous solvents – 24 Experiments; Organic solvents – 43 Experiments

 

Aqueous solvents: DMEM/Han’s F12, Deionised water (10% v/v)

Organic solvents: DMSO (0.5 or 1.0%), Acetone, Ethanol and THF (0.5%)

Positive Control without S9
Micronucleated cells in %
	Pulse treatment (4/40)	Continous treatment (20/40)
	MMC	Demecolcin
No. of experiments	50	54
Mean	11.66	3.55
95% Ctrl limit	1.48 - 21.85	1.69 - 5.41
1x SD	5.09	0.93
2x SD	10.18	1.86
Min	4.15	2.10
Max	24.00	6.40

Positive Control with S9
Micronucleated cells in %
	Pulse treatment (4/40)
	CPA
No. of experiments	81
Mean	4.80
95% Ctrl limit	0.88 - 8.73
1x SD	1.96
2x SD	3.92
Min	2.25
Max	11.30

Applicant's summary and conclusion

Conclusions:

The substance tested non-clastogenic under the conditions of the study.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance should not be considered to have a mutagenic potential.

Executive summary:

An in vitro mammalian cell micronucleus test was performed with the test item dissolved in DMSO using peripheral human lymphocytes. The test procedure was according to the method described in OECD guideline 487 (2014).

The assay was performed in three independent experiments with the following test concentrations and exposure durations:

Preliminary cytotoxicity test/Experiment 1A: 13.4, 23.4, 40.9, 71.6, 125, 219, 384, 671, 1175, and 2056 µg/mL (with and without metabolic activation; 4 hours)

Experiment 1B: 82.9, 108, 140, 182, 237, 308, and 400 µg/mL (with metabolic activation; 4 hours)

Experiment 2: 2.6, 4.5, 8.0, 13.9, 24.4, 42.6, 74.6, 131, 229, and 400 µg/mL (without metabolic activation; 20 hours)

Positive and solvent controls were run concurrently.

The cells were prepared 40 hours after start of treatment with the test item. During the preparation period cytochalasin B had been added to the cell cultures to ensure that there were binucleate cells to be evaluated for micronuclei.

In each experimental group two parallel cultures were analysed. At least 1000 binucleate cells per culture were scored for cytogenetic damage (% micronucleated cells). To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is described as % cytostasis.

In Experiment 1A, phase separation of the test item in the culture medium was observed at 219 μg/mL and above in the absence of S9 mix and at 384 μg/mL and above in the presence of S9 mix at the end of treatment. In addition, phase separation occurred in Experiment 2 in the absence of S9 mix at 400 μg/mL and above at the end of treatment. In Experiment 1B no phase separation was observed. No relevant influence on osmolarity or pH was observed.

The following concentrations were evaluated for cytogenetic damage:

Preliminary cytotoxicity test/Experiment 1A: 23.4, 40.9, and 71.6 µg/mL (without metabolic activation; 4 hours)

Preliminary cytotoxicity test/Experiment 1A: 71.6, 125, and 219 µg/mL (with metabolic activation; 4 hours)

Experiment 1B: 140, 182, and 237 µg/mL (with metabolic activation; 4 hours)

Experiment 2: 24.4, 42.6, and 74.6 µg/mL (without metabolic activation; 20 hours)

In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration. Concentrations showing clear cytotoxic effects, however, were not evaluable for cytogenetic damage.

In Experiment 1A and 2 in the absence of S9 mix no relevant increases in the number of micronucleate cells were observed after treatment with the test item.

In Experiment IA in the presence of S9 mix, the highest evaluated concentration (219 μg/mL) showed a statistical significant increase in the number of micronucleate cells of 1.30 % (value exceeded historical control range (0.08 – 1.20 %)). In Experiment 1B the finding could not be confirmed. Therefore, the finding of Experiment 1A was not reproducible and can be considered as biologically irrelevant.

The positive and negative controls were considered to be valid.

The substance tested non-clastogenic under the conditions of the study.

According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance is considered to have not a mutagenic potential.