Reaction mass of (4Z)-4-ethylidene-2-propoxycyclohexanol and (4E)-4-ethylidene-2-propoxycyclohexanol and (5Z)-5-ethylidene-2-propoxycyclohexanol and (5E)-5-ethylidene-2-propoxycyclohexanol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: micronuclei induction

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06-Jan-2054 to 24-Feb-2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Reliability 1 is assigned because the study is conducted according to OECD TG 4873, in compliance with GLP, without deviations that influence the quality of the results.

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

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

1

Reference substance name:

Reaction mass of (4Z)-4-ethylidene-2-propoxycyclohexanol and (4E)-4-ethylidene-2-propoxycyclohexanol and (5Z)-5-ethylidene-2-propoxycyclohexanol and (5E)-5-ethylidene-2-propoxycyclohexanol

EC Number:

944-405-9

Molecular formula:

C11H20O2

IUPAC Name:

Reaction mass of (4Z)-4-ethylidene-2-propoxycyclohexanol and (4E)-4-ethylidene-2-propoxycyclohexanol and (5Z)-5-ethylidene-2-propoxycyclohexanol and (5E)-5-ethylidene-2-propoxycyclohexanol

Test material form:

liquid

Details on test material:

- Substance name as cited in test report: FRET 13-0156
- Phystical state: clear, yellowish liquid
- Storage conditions: ambient temperature (15-25 °C), protected from light

Method

Species / strain

Species / strain / cell type:

lymphocytes: Cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

Peripheral blood lymphocytes were obtained from a healthy non-smoking 31-year-old adult male on 06 January 2015 for the preliminary toxicity assay and from the same donor on 02 February 2015 for the definitive assay. The donor had no recent history of radiotherapy, viral infection or the administration of drugs.

Preparation of Target Cells
Peripheral blood lymphocytes were cultured in complete medium (RPMI-1640 containing 15% fetal bovine serum, 2mM L-glutamine, 100 units penicillin, 100 μg/mL streptomycin) by adding 0.5 mL heparinized blood to a centrifuge tube containing 5 mL of complete medium with 2% phytohemagglutinin. The cultures were incubated under standard conditions (37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air) for 44-48 hours.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Preliminary toxicity test:
Without and with S9-mix, 4hr exposure, or without S9 24hr exposure: doses ranging from 0.184 to 1840 µg/mL

Micronucleus assay:
Without S9 -- 4 hr -- 20 hr: 50, 150, 300, 600, 800, 1000, 1200, 1400, 1600, 1800 ug/ml
without S9 -- 24 hr -- 0 hr: 50, 150, 200, 250, 300, 350, 400, 450, 500, 550 ug/ml
With S9 -- 4 hr -- 20 hr: 50, 150, 300, 600, 800, 1000, 1200, 1400, 1600, 1800 ug/ml

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle:
Test compound was soluble in DMSO and DMSO has been accepted and approved by authorities and international guidelines

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

mitomycin C

other: Vinblastine for aneugenicity

Remarks:

Without S9

Controls 2

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive control substance:

cyclophosphamide

Remarks:

With S9

Details on test system and experimental conditions:

Experimental Design
The in vitro mammalian cell micronucleus assay was conducted using standard procedures (Kirsch-Volders et al. 2000; Parry and Sors 1993; Fenech and Morley, 1986; Fenech 1993) by exposing HPBL to appropriate concentrations of the test article as well as the concurrent positive and vehicle controls, in the presence and absence of an exogenous metabolic activation system.

Preparation of Target Cells
Peripheral blood lymphocytes were cultured in complete medium (RPMI-1640 containing 15% fetal bovine serum, 2mM L-glutamine, 100 units penicillin, 100 μg/mL streptomycin) by adding 0.5 mL heparinized blood to a centrifuge tube containing 5 mL of complete medium with 2% phytohemagglutinin. The cultures were incubated under standard conditions (37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air) for 44-48 hours.

Preliminary Toxicity Test for Selection of Dose Levels
HPBL were exposed to vehicle alone and to nine concentrations of test article with half-log dose spacing using single cultures. The precipitation in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The osmolality of the vehicle, the highest dose level, and the highest soluble dose level in treatment medium was measured. Dose levels for the micronucleus assay were based upon post-treatment toxicity (CBPI relative to the vehicle control).

Micronucleus Assay
Ten dose levels were tested using duplicate cultures at appropriate dose intervals based on the toxicity profile of the test article. The precipitation in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The highest dose level evaluated for the micronucleus was based on 50 to 60% cytotoxicity (CBPI relative to the vehicle control). Two additional dose levels were included in the evaluation.

Treatment of Target Cells (Preliminary Toxicity Test and Definitive Assay)
The pH was measured at the highest test article concentration prior to dosing using test tape. Treatment was carried out by refeeding the cultures with 5 mL complete medium for the non-activated exposure or 5 mL S9 mix (4 mL serum-free culture medium + 1 mL of S9 cofactor pool) for the S9-activated exposure, to which was added 50 μL of test article dosing solution or vehicle alone. In the definitive assay, positive control cultures were resuspended in either 5 mL of complete medium for the non-activated studies, or 5 mL of the S9 reaction mixture (4 mL serum free medium + 1 mL of S9 cofactor pool), to which was added 50 μL of positive control in solvent.

After the 4 hour treatment in the non-activated and the S9-activated studies, the cells were centrifuged, the treatment medium was aspirated, the cells were washed with calcium and magnesium free phosphate buffered saline (CMF-PBS), re-fed with complete medium containing cytoB at 6.0 μg/mL and returned to the incubator under standard conditions. For the 24 hour treatment in the non-activated study, cytoB (6.0 μg/mL) was added at the beginning of the treatment.

Collection of Cells (Preliminary Toxicity Test and Definitive Assay)
Cells were collected after being exposed to cyto B for 24 hours (± 30 minutes), 1.5 to 2 normal cell cycles, to ensure identification and selective analysis of micronucleus frequency in cells that have completed one mitosis evidenced by binucleated cells (Fenech and Morley, 1986). The cyto B exposure time for the 4 hour treatment in the non-activated and the S9-activated studies was 20 hours (± 30 minutes).

Cells were collected by centrifugation, swollen with 0.075M KCl, washed with fixative (methanol: glacial acetic acid, 25:1 v/v), capped and may be stored overnight or longer at 2-8°C or 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.

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 (MN-BN) cells was determined out of at least 2000 total binucleated cells per dose levels, when possible, and reported for each treatment group.

Evaluation criteria:

The test article 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. The Cochran-Armitage test was used to measure dose-responsiveness.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: Cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the preliminary toxicity test, precipitation in the exposure medium was observed at dose levels of 1840 µg/mL in both 4 hours and 24 hours trtreatment system in the absence of S9 activation. Hemolysis was observed at dose of 1840 ug/ml 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.

In micronucleus assay, precipitation and hemolysis in the exposure medium was observed at dose of 1600 ug/ml and above in the 4 hr treatmetn system in the absence of S9, and 1800 ug/ml in the 4 hr treatment system in the presence of S9. - Toxicity was observed at dose levels of 333 µg/mL and above in the absence and presence of S9, 3 hr treatment/24 hr fixation; at dose levels of 100 µg/mL and above in the absence of S9 for the continuous treatment of 24 and 48 hr.

The osmolality of the test article dose levels in treatment medium is acceptable because it did not exceed the osmolality of the vehicle by more than 20%. The pH of the highest concentration of test article in treatment medium was 7.5.

Any other information on results incl. tables

Non-activated 4-hour exposure group

The dose levels selected for analysis of micronucleus were 800, 1000, and 1200 μg/mL. At the highest test concentration, 1200 μg/mL, cytotoxicity was 58% relative to the vehicle control. The percentage of cells with micronucleated binucleated cells in the non-activated 4-hour exposure group was statistically significantly increased relative to vehicle control at 800 μg/mL (p ≤ 0.05, Fisher’s Exact test). However, the Cochran-armitage test was negative for a dose response. In addition, the percentage of micronucleated binucleated cells in the test article-treated group (0.4%) was within the historical solvent control range of 0.1% to 1.6%. Therefore, the statistically significant increase was not considered to be biologically relevant. The percentage of micronucleated cells in the MMC (positive control) group (3.4%) 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 300, 600, and 1000 μg/mL. At the highest test concentration, 1000 μg/mL, cytotoxicity was 50% relative to the vehicle control. The percentage of cells with micronuclei in the test article-treated group was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test). The percentage of micronucleated cells in the CP (positive control) group (1.5%) 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 50, 200, and 300 μg/mL. At the highest test concentration, 300 μg/mL, cytotoxicity was 59% relative to the vehicle control. The percentage of cells with micronuclei in the test article-treated group was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test). The percentage of micronucleated cells in the VB (positive control) group (2.0%) was statistically significant (p ≤ 0.01, Fisher's Exact test).

Applicant's summary and conclusion

Conclusions:

A Micronucleus assay with IFF TM FRET 13-0156 was performed according to OECD 487 guideline and GLP principles, in cultured peripheral human lymphocytes.
The positive and vehicle controls fulfilled the requirements for a valid test.
Under the conditions of the assay described in this report, FRET 13-0156 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.

Executive summary:

In a micronucleus assay, cultured peripheral human lymphocytes were exposed to different concentrations of the substance (dissolved in DMSO), in the presence and absence of S9-mix according to OECD 487 guideline and GLP principles.

The percentage of cells with micronuclei in the test article-treated group was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test) in the S9-activated 4-hour exposure group or the non-activated 24-hour exposure group. statistically increased but no dose-response relationship in three differetn treatment conditions. The percentage of cells with micronucleated binucleated cells in the non-activated 4-hour exposure group was statistically significantly increased relative to vehicle control at 800 μg/mL (p ≤ 0.05, Fisher’s Exact test) (the highest dose analyzed for mucronucleus was 1200 ug/ml). However, the Cochran-armitage test was negative for a dose response. In addition, the percentage of micronucleated binucleated cells in the test article-treated group (0.4%) was within the historical solvent control range of 0.1% to 1.6%. Therefore, the statistically significant increase was not considered to be biologically relevant.

The results for the positive and vehicle controls indicate that all criteria for a valid assay were met. Based on these criteria, the results are justified and do not require a repeat of any portions of the study.

Therefore FRET 13-0156 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.