Reaction Mass of Cyclohexanepropanol, 2,2,6-trimethyla-propyl-, (alpha.R,1R,6S)- and Cyclohexanepropanol,2,2,6-trimethyl-a-propyl-, [1a(S*),6b]- (9CI)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 07, 2014 to Fubruary 05, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD TG 487 without any deviation.

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)

Version / remarks:

2010

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Remarks:

US EPA GLP Compliance Programme

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

(3R*)-1-[(1R*,6S*)-2,2,6-trimethylcyclohexyl]hexan-3-ol

Molecular formula:

C15H30O

IUPAC Name:

(3R*)-1-[(1R*,6S*)-2,2,6-trimethylcyclohexyl]hexan-3-ol

Constituent 2

Reference substance name:

(3S*)-1-[(1R*,6S*)-2,2,6-trimethylcyclohexyl]hexan-3-ol

Molecular formula:

C15H30O

IUPAC Name:

(3S*)-1-[(1R*,6S*)-2,2,6-trimethylcyclohexyl]hexan-3-ol

Test material form:

liquid

Details on test material:

- Physical state: colourless liquid

Specific details on test material used for the study:

- Storage condition of test material: The test item was stored at room temperature, protected from light

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: human peripheral blood lymphocyte
- Suitability of cells: The donor had no recent history of radiotherapy, viral infection or the administration of drugs, and who had abstained from alcohol for at least 12 hours prior to blood donation
- Normal cell cycle time (negative control): 10-16 hours

For lymphocytes:
- Sex, age and number of blood donors: healthy non-smoking 27-year-old adult male
- Whether whole blood or separated lymphocytes were used: whole blood
- Whether blood from different donors were pooled or not: not applicable
- Mitogen used for lymphocytes: phytohemagglutinin (2%)

MEDIA USED
- Type and composition of media: RPMI-1640 containing 15% fetal bovine serum, 2mM L-glutamine, 100 units penicillin, 100 µg/mL streptomycin
- CO2 concentration: 5 +/- 1 %
- Humidity level: humidified atmosphere
- Temperature: approximately 37 +/- 1 ºC

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

cytochalasin B (6 μg/mL)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Molecular Toxicology Inc. (Boone, NC)
- method of preparation of S9 mix: the S9 was thawed and mixed with a cofactor pool to contain 2 mM magnesium chloride, 6 mM potassium chloride, 1 mM glucose-6-phosphate, 1 mM nicotinamide adenine dinucleotide phosphate (NADP) and 20 µL S9 per milliliter medium (RPMI 1640 serum-free medium supplemented with 100 units penicillin/mL and 100 µg streptomycin/mL and 2 mM L-glutamine).
- concentration or volume of S9 mix and S9 in the final culture medium: 4 mL culture medium + 1 mL of S9 cofactor pool
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): yes, Each bulk preparation of S9 was assayed by the supplier for sterility and its ability to metabolize at least two pro-mutagens to forms mutagenic to Salmonella typhimurium TA100.

Test concentrations with justification for top dose:

Preliminary Toxicity Test: from 0.226 to 2260 µg/mL
4 h exposure without S9: 7, 15, 30, 35, 40, 45, 50, 60, 70, 80 μg/mL.
4 h exposure with S9 (2%): 15, 30, 50, 100, 150, 200, 225, 250 μg/mL.
24 h continuous exposure without S9: 7, 15, 25, 27.5, 30, 32.5, 35, 37.5, 40, 45, 50 μg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxyde (DMSO)
- Justification for choice of solvent/vehicle:Dimethyl sulfoxide (DMSO) was used as the vehicle based on the solubility of the test substance and compatibility with the target cells. In a solubility test conducted at BioReliance, the test substance was soluble in DMSO at a concentration of approximately 500 mg/mL, the maximum concentration tested for solubility.

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

vinblastine

Remarks:

Absence of S9-mix

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:

Presence of S9-mix

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): duplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 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
- Harvest time after the end of treatment (sampling/recovery times): 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 before harvest. For the 24 hour treatment in the non-activated study, cytoB was added at the beginning of the treatment.

FOR MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure: Cytochalasin B, 6 μg/mL, 24 hour
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): 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, or the slides were prepared immediately after harvest. 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 and identified by the BioReliance study number, treatment condition, dose level, test phase, harvest date, activation system, and replicate tube design.

- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): The slides from at least three test substance treatment groups were coded using random numbers by an individual not involved with the scoring process and scored for the presence of micronuclei based on cytotoxicity. A minimum of 2000 binucleated cells from each concentration (1000 binucleated cells from each culture) were examined and scored for the presence of micronuclei.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification):
1/ the micronucleus should have the same staining characteristics as the main nucleus.
2/ the micronuclei should be separate from the main nuclei or just touching (no cytoplasmic bridges).
3/ the micronuclei should be of regular shape and approximately 1/3 or less than the diameter of the main nucleus.
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): not necessary and not performed in cases where no significant increase in micronucleus frequency is observed.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytokinesis block proliferation index (CBPI).
For the preliminary toxicity test, at least 500 cells were evaluated to determine the CBPI at each dose level and the control. For the micronucleus assay, at least 1,000 cells (500 cells per culture) were evaluated to determine the CBPI at each dose level and the control. The CBPI was determined using the following formula:
CBPI = 1X (Mononucleated cells + 2 x Binucleated cells + 3 x Multinucleated cells) / Total number of cells scored
% Cytostasis (cytotoxicity) = 100 -100 {(CBPIt-1) /(CBPIc-1)}
t = test substance treatment culture
c = vehicle control culture

ACCEPTABILITY CRITERIA:
The frequency of cells with micronucleus induction in the vehicle control must be within the historical control range. The percentage of cells with micronucleus induction must be statistically increased (p ≤ 0.05, Fisher's exact test) in the positive control condition relative to the vehicle control.

Evaluation criteria:

Toxicity induced by treatment was based upon CBPI and was reported for the cytotoxicity and micronucleus portions of the study. The percent frequency of micronucleated binucleated (MNBN) cells was determined out of at least 2000 total binucleated cells per dose levels, when possible, and reported for each treatment group.

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, CochranArmitage test was not required to measure dose-responsiveness.
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.

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:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The pH of the highest dose level in the treatment medium was 7.5
- Data on osmolality: 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%.
- Possibility of evaporation from medium: Not applicable
- Water solubility: not applicable
- Precipitation and time of the determination:
Visible precipitate was observed in treatment medium at dose levels ≥ 150 µg/mL, while dose levels ≤ 100 µg/mL were soluble in treatment medium at the beginning of the treatment period.

RANGE-FINDING/SCREENING STUDIES (if applicable):
A preliminary toxicity test was conducted to observe the cytotoxicity profile of the test substance and to select suitable dose levels for the definitive micronucleus assay. HPBL cells were first exposed to nine concentrations of Norlimbanol ranging from 0.226 to 2260 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.
The test substance was soluble in DMSO at all concentrations tested. Visible precipitate was observed in treatment medium at > 226 µgmL.
Substantial cytotoxicity [≥ 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at dose levels ≥ 67.8 µg/mL in the non-activated 4 and 24-hour exposure groups, and at dose levels ≥ 226 µg/mL in the S9-activated 4-hour exposure group.

STUDY RESULTS : Cf. Table of results (attached background material)
- non-activated 4-hour exposure group : The dose levels selected for analysis of micronucleus were 15, 35, and 50 µg/mL. At the highest test concentration, 50 µg/mL, cytotoxicity was 53% relative to the vehicle control. The percentage of cells with micronuclei in the test substance-treated group was not significantly increased relative to the vehicle control at any dose level (p > 0.05, Fisher's Exact test). The percentage of micronucleated cells in the MMC (positive control) group (2.1%) was statistically significant (p ≤ 0.01, Fisher's Exact test).
- S9-activated 4-hour exposure group: The dose levels selected for analysis of micronucleus were 50, 100, and 150 µg/mL. At the highest test concentration,
150 µg/mL, cytotoxicity was 55% relative to the vehicle control. The percentage of cells with micronuclei in the test substance-treated group was not significantly increased relative to the vehicle control at any dose level (p > 0.05, Fisher's Exact test). The percentage of micronucleated cells in the CP (positive control) group (0.9%) was statistically significant (p ≤ 0.01, Fisher's Exact test).
- non-activated 24-hour exposure group: The dose levels selected for analysis of micronucleus were 15, 25, and 32.5 µg/mL. At the highest test concentration, 32.5 µg/mL, cytotoxicity was 51% relative to the vehicle control. The percentage of cells with micronuclei in the test substance-treated group was not significantly increased relative to the vehicle control at any dose level (p > 0.05, Fisher's Exact test). The percentage of micronucleated cells in the VB (positive control) group (1.1%) was statistically significant (p ≤ 0.01, Fisher's Exact test).


HISTORICAL CONTROL DATA: Cf. Table of results (attached background material)
- Positive historical control data: all criteria for a valid assay were met.
- Negative (solvent/vehicle) historical control data: all criteria for a valid assay were met.

Applicant's summary and conclusion

Conclusions:

Based on the findings of this study, the test item was concluded to be negative for the induction of micronuclei in both non-activated and S9-activated test systems in the in vitro mammalian cell micronucleus test using human peripheral blood lymphocytes.

Executive summary:

The test item was tested in the in vitro mammalian cell micronucleus test using human peripheral blood lymphocytes (HPBL) in both the absence and presence of an Aroclor-induced S9 activation system,according to OECD Guideline 487 and in compliance with GLP. A preliminary toxicity was performed to establish the dose range for testing in the micronucleus test. The micronucleus assay was used to evaluate the aneugenic and clastogenic potential of the test substance. In the preliminary toxicity and the micronucleus assays, HPBL cells were treated for 4 and 24 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 24 hours after treatment initiation. Dimethyl sulfoxide

(DMSO) was used as the vehicle based on the solubility of the test substance and compatibility with the target cells. In a solubility test conducted at BioReliance, the test substance was soluble in DMSO at a concentration of approximately 500 mg/mL, the maximum concentration tested for solubility.

In the preliminary toxicity assay, the doses tested ranged from 0.226 to 2260 µg/mL (10 mM). Substantial cytotoxicity [≥ 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at dose levels ≥ 67.8 µg/mL in the non-activated 4 and 24-hour exposure groups, and at dose levels ≥ 226 µg/mL in the S9-activated 4-hour exposure group. Based on these findings, the doses chosen for the micronucleus assay ranged from 7 to 80 µg/mL for the non-activated 4-hour exposure group, from 15 to 250 µg/mL for the S9-activated 4-hour exposure group, and from 7 to 50 µg/mL for the non-activated 24-hour exposure group.

In the micronucleus assay, substantial cytotoxicity was observed at dose levels ≥ 50 µg/mL in the non-activated 4-hour exposure group, at dose levels ≥ 150 µg/mL in the S9-activated 4-hour exposure group, and at dose levels ≥ 32.5 µg/mL in the non-activated 24-hour exposure group.

The highest dose analyzed under each treatment condition produced 50 to 60% reduction in CBPI, which met the dose limit as recommended by testing guidelines for this assay. A minimum of 1000 binucleated cells from each culture were examined and scored for the presence of micronuclei.

The percentage of cells with micronucleated binucleated cells in the test substance-treated groups was not statistically significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher’s Exact test). The results for the positive and negative controls indicate that all criteria for a valid assay were met.

Based on the findings of this study, the test item was concluded to be negative for the induction of micronuclei in both non-activated and S9-activated test systems in the in vitro mammalian cell micronucleus test using human peripheral blood lymphocytes.

This study is considered as acceptable and satisfies the requirement for in vitro micronucleus endpoint.