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EC number: 232-102-8 | CAS number: 7786-67-6
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
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:
- 2 015
- 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
- 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
Constituent 1
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.
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