5-methyl-2-(1-methylvinyl)cyclohexan-1-ol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Remarks:

Test substance represents a main component (stereoisomer) of the registered substance

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Isopulegol

EC Number:

201-940-6

EC Name:

Isopulegol

Cas Number:

89-79-2

Molecular formula:

C10H18O

IUPAC Name:

(1R,2S,5R)-2-Isopropenyl-5-methylcylohexanol

Method

Species / strain

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: healthy non-smoking 27-year-old adult male
- Whether whole blood or separated lymphocytes were used: heparinized whole blood


MEDIA USED
- Type and identity of media including CO2 concentration: RPMI-1640 containing 15% fetal bovine serum, 2mM L-glutamine, 100 units penicillin, 100 μg/mL streptomycin; 5 ± 1% CO2 in air)

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Main Micronucleus test
W/O S9; 4h treatment: 100, 250*, 500*, 600, 700*, 750, 1000, 1540 μg/mL
W/O S9; 24h treatment: 10, 25*, 50*, 100*, 120, 130, 140, 150 ,175, 200 μg/mL
with S9; 4h treatment:100, 250*, 500*, 600, 700*, 725, 750, 775, 800, 850, 900, 1000 μg/mL

*concentrations assessed for micronuclei
Dosages chosen based on pretest and initial main test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility of the test substance and compatibility with the target cells

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Vinblastine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h, 24h
- Expression time (cells in growth medium):
In 4h exposure setup, fresh medium incl. Cytochalasin B was added for 20h.
In the 24h exposure setup, Cytochalasin B was added at start of treatment incubation.

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B (cytoB) used at 6 μg/mL

STAIN (for cytogenetic assays): acridine orange

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Cells were collected by centrifugation, swollen with 0.075M KCl, washed with fixative (methanol: glacial acetic acid, 25:1 v/v), capped and stored overnight or longer at 2-8°C. To prepare slides, the cells were collected by centrifugation and if necessary, the cells were resuspended in fresh fixative. The suspension of fixed cells was applied to glass microscope slides and air-dried. The slides were stained with acridine orange.


NUMBER OF CELLS EVALUATED: 1000 per replicate (2 replicate cultures per concentration)

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
Micronuclei in a binucleated cell (MN-BN) were recorded if they meet the following criteria:
• the micronucleus should have the same staining characteristics as the main nucleus.
• the micronuclei should be separate from the main nuclei or just touching (no cytoplasmic bridges).
• the micronuclei should be of regular shape and approximately 1/3 or less than the diameter of the main nucleus.

DETERMINATION OF CYTOTOXICITY
- Method:cytokinesis-blocked proliferation index (CBPI)

- OTHER:
Precipitation in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment.
Osmolality of the solvent, the highest dose level, the lowest precipitating dose level, and the highest soluble dose level in treatment medium was measured in the preliminary toxicity test.

Rationale for test conditions:

Dose levels for the micronucleus assay were based upon post-treatment toxicity (cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control) assessed in the preliminary toxicity test.

Evaluation criteria:

The test substance would be considered positive if it induced a statistically significant and dose-dependent increase the frequency of MN-BN cells (p ≤ 0.05). If only one criterion was met (statistically significant OR dose-dependent increase), the result was considered equivocal. If neither criterion was met, the results were considered to be negative.

Statistics:

Statistical analysis of the percentage of micronucleated cells was performed using the Fisher's exact test. The Fisher's test was used to compare pairwise the percent micronucleated cells of each treatment group with that of the vehicle control. Due to negative results, Cochran-Armitage test was not required to measure dose-responsiveness.

Results and discussion

Test results

Species / strain:

lymphocytes: healthy non-smoking 27-year-old adult male

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH of the highest concentration of test substance in treatment medium was 7.5.
- Effects of osmolality: The osmolality in treatment medium of the highest dose level tested (and lowest precipitating dose), 1540 μg/mL, was 403 mmol/kg. The osmolality in the treatment medium of the highest soluble dose level, 462 μg/mL, was 423 mmol/kg. The osmolality of the vehicle (DMSO) in the treatment medium was 460 mmol/kg. The osmolality of the test substance dose levels in treatment medium is acceptable because it did not exceed the osmolality of the vehicle by more than 20%.
- Precipitation: was not observed in the main Micronucleus test


RANGE-FINDING/SCREENING STUDIES:
HPBL cells were first exposed to nine concentrations of Isopulegol ranging from 0.154 to 1540 μg/mL, as well as vehicle controls, in both the absence and presence of an Aroclor-induced S9 activation system for 4 hours, or continuously for 24 hours in the absence of S9 activation.
Substantial cytotoxicity [≥ 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at 1540 μg/mL in the S9-activated 4-hour exposure group and at dose levels ≥ 154 μg/mL in the non-activated 24-hour exposure group. Substantial cytotoxicity was not observed in the non-activated 4-hour exposure groups.
In an initial micronucleus assay a toxicity shift in all treatment conditions was observed under the following testing conditions:
W/O S9; 4h treatment: 250, 500, 750, 1000, 1540 μg/mL
W/O S9; 24h treatment: 3, 10, 30, 100, 140, 150, 160, 170 ,180, 200 μg/mL
with S9; 4h treatment:100, 250, 500, 600, 700, 800, 900, 1000, 1100, 1250 μg/mL
No micronuclei data were reported and the test was repeated with adjusted test concentrations (main Micronucleus test).


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Hemolysis was observed at dose levels ≥ 900 μg/mL in non-activated and S9-activated 4-hour exposure groups.

Remarks on result:

other: See result table

Any other information on results incl. tables

Without S9, 4h treatment, 24h harvest

Treatment [µg/ml]	Replicate culture	Percentage of micronucleated binucleated cells per culture [%]	Average percent micronucleated binucleated cells per culture [%]	Cytotoxicity relative to vehicle control [%]
DMSO	A	0.3	0.3
	B	0.3
250	A	0.1	0.1	13
	B	0.1
500	A	0.1	0.2	39
	B	0.3
700	A	0.1	0.2	57
	B	0.2
MMC, 0.5 µg/ml	A	3.8	3.6**	56
	B	3.4

* p ≤ 0.05; ** p ≤ 0.01, Fisher's exact test, relative to the solvent control.

Without S9, 24h treatment, 24h harvest

Treatment [µg/ml]	Replicate culture	Percentage of micronucleated binucleated cells per culture [%]	Average percent micronucleated binucleated cells per culture [%]	Cytotoxicity relative to vehicle control [%]
DMSO	A	0.3	0.4
	B	0.4
25	A	0.5	0.4	15
	B	0.2
50	A	0.3	0.3	33
	B	0.2
100	A	0.8	0.7	50
	B	0.6
VB, 10 ng/ml	A	1.1	1.1**	45
	B	1.0

* p ≤ 0.05; ** p ≤ 0.01, Fisher's exact test, relative to the solvent control.

With S9, 4h treatment, 24h harvest

Treatment [µg/ml]	Replicate culture	Percentage of micronucleated binucleated cells per culture [%]	Average percent micronucleated binucleated cells per culture [%]	Cytotoxicity relative to vehicle control [%]
DMSO	A	0.4	0.3
	B	0.2
250	A	0.3	0.3	24
	B	0.3
500	A	0.2	0.2	38
	B	0.2
700	A	0.2	0.2	58
	B	0.2
CP, 5 µg/ml	A	1.0	1.2**	38
	B	1.3

* p ≤ 0.05; ** p ≤ 0.01, Fisher's exact test, relative to the solvent control.

Applicant's summary and conclusion

Conclusions:

The positive and vehicle controls fulfilled the requirements for a valid test.
Under the conditions of the assay described in this report, Isopulegol was concluded to be negative for the induction of micronuclei in the non-activated and S9-activated test systems in the in vitro mammalian micronucleus test using human peripheral blood lymphocytes.