Isopropylcyclohexane

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-10-26 to 2012-01-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals: In Vitro Mammalian Cell Micronucleus Test (MNvit), No. 487, Guideline July 22, 2010

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Isopropylcyclohexane

EC Number:

211-792-4

EC Name:

Isopropylcyclohexane

Cas Number:

696-29-7

Molecular formula:

C9H18

IUPAC Name:

(propan-2-yl)cyclohexane

Test material form:

liquid

Details on test material:

Content: 99.93%

Method

Target gene:

mammalian cell system( Chinese hamster Ovary cells)

Species / strain

Species / strain / cell type:

other: Chinese hamster ovary (CHO-K1) cells

Details on mammalian cell type (if applicable):

Species/cell type: CHO cells as originally derived from the ovary of Chinese hamster, obtained from ATCC
CHO-K1, modal chromosome number of 20

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix based on liver homogenate fraction  from male rats, induced with Aroclor 1254 (i.p.)

Test concentrations with justification for top dose:

without S9-mix: 3.13, 6.25, 12.5, and 25 µg/mL
with S9-mix: 12.5, 25, 50 and 100 µg/ml

Vehicle / solvent:

The test item was completely dissolved in acetone . Preparations of the test item made on the day of use were employed.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Remarks:

clastogen

Positive control substance:

cyclophosphamide

Remarks:

+S9-mix

Controls 2

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Remarks:

clastogen

Positive control substance:

mitomycin C

Remarks:

-S9-mix

Details on test system and experimental conditions:

SYSTEM OF TESTING
- Species/cell type: CHO-K1 BH4 cell line, cell cycle length 12 hours
- Metabolic activation system: male rat liver S9 from  Aroclor 1254 induced animals
ADMINISTRATION: 
- Solubility:  test item was completely dissolved in acetone. Preparations of the test item made on the day of use were employed.
- Preliminary experiment: without and with metabolic activation cytotoxicity was noted starting at concentration of 25 or 100 µg
test item/mL in the experiments without and with metabolic activation, respectively. Hence, 25 or 100 µg test item/mL
were employed as top concentrations for the genotoxicity tests without and with metabolic activation, respectively.
- Dosing:  without S9-mix: 3.13, 6.25, 12.5, and 25 µg/mL
with S9-mix: 12.5, 25, 50 and 100 µg/ml
- Number of replicates: 2
- Positive and negative control groups and treatment:    
negative: acetone
positive (+S9): cyclophosphamide 
positive (-S9): mitomycin C 

DURATION
- most aneugens and clastogens are detected by a short term treatment period of 4 hours in the presence and absence of S9, followed by removal of the test item and a growth period of 1.5 – 2.0 cell cycles. Cells were sampled at a time equivalent to about 1.5 – 2.0 times the normal
(i.e. untreated)
cell cycle length either after the beginning or at the end of treatment. Because of the potential cytotoxicity of S9 preparations for cultured
mammalian
cells, an extended exposure treatment of 1.5 – 2.0 normal cell cycles was used only in the absence of S9.
Cell treatment and harvest times for the used CHO cell line see table below.
As both initial tests of the short 4-h treatment are negative or equivocal, a subsequent, extended exposure treatment without S9 was used.
- Harvesting time: harvesting time was 20 hours after the end of exposure

STAIN (for cytogenetic assays): Each culture was harvested and processed separately. High-quality cell preparations for scoring were obtained.
Cell cytoplasm were retained to allow the detection of micronuclei and (in the cytokinesis-block method) reliable identification of binucleate cells. The slides were stained using Giemsa.

NUMBER OF REPLICATIONS: 2, duplicate cultures were used for each test item concentration and for the solvent control cultures.

NUMBER OF CELLS EVALUATED: The micronucleus frequencies were analysed in at least 2000 binucleated cells per concentration (at least 1000
binucleated cells per culture; two cultures per concentration).

DETERMINATION OF CYTOTOXICITY
- Method: evaluation of cytotoxicity was based on the Cytokinesis-Block Proliferation Index (CBPI) or the Replicative Index (RI).
The CBPI indicates the average number of cell cycles per cell during the period of exposure to cytoB, and is used to calculate cell proliferation.
The RI indicates the relative number of nuclei in treated cultures compared to control cultures and can be used to calculate the % cytostasis:

OTHER EXAMINATIONS: 1000 binucleated cells per duplicate cell culture were scored to assess the frequency of cells with one, two, or more than
two micronuclei. Additionally, the cells were classified as mononucleates, binucleates or multinucleates to estimate the proliferation index as a
measure of toxicity.

Evaluation criteria:

The assay demonstrates its ability to reliably and accurately detect substances of known aneugenic and clastogenic activity, with and without
metabolic activation.
Solvent/vehicle control and untreated cultures give reproducibly low and consistent micronuclei frequencies, typically 5 – 25 micronuclei per 1000
cells according to OECD 487. Data from negative and positive controls are used to establish historical control ranges. These values are used in
deciding the adequacy of the concurrent negative/positive controls for an experiment .

Statistics:

The assessment was carried out by a comparison of the samples with the positive and the vehicle control, using a chi-square test corrected for
continuity according to YATES.
If a test item induces a concentration-related increase or a statistical significant and reproducible increase in the number of cells containing
micronuclei, it is classified as a positive result.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxicity was noted starting at concentration of 25 or 100 µg /mL in the experiments without and with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Tests without metabolic activation (4- and 20-hour exposure)
The micronucleus frequencies of cultures treated with Isopropylcyclohexane at concentrations from 3.13 to 25 µg/mL medium (4 h and 20-h
exposure) in the absence of metabolic activation ranged from 4.5 to 7.0 micronuclei per 1000 binucleated cells. The results obtained are considered to be within the normal range of the vehicle acetone where a mean incidence of micronucleus frequencies of 6.0 or 3.5 micronuclei per 1000
binucleated cells was observed after a 4-hour and 20-hour exposure, respectively. The micronucleus frequency of the untreated controls was 7.5 or 6.0 micronuclei per 1000 binucleated cells after a 4-hour and 20-hour exposure, respectively.
Test with metabolic activation (4-hour exposure)
The micronucleus frequencies of cultures treated with Isopropylcyclohexane at concentrations from 12.5 to 100 µg/mL medium in the presence of metabolic activation ranged from 3.5 to 6.5 micronucleus per 1000 binucleated cells. The results obtained are considered to be within the normal
range of the vehicle acetone where a mean incidence of micronucleus frequencies of 6.0 or 4.0 micronuclei per 1000 binucleated cells was observed in the first and second experiment, respectively. The micronucleus frequencies of the untreated controls were 5.0 or 7.5 micronuclei per 1000
binucleated cells in the first and second experiment, respectively.

Remarks on result:

other: other: Chinese hamster Ovary (CHO)

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

see attached documents

Applicant's summary and conclusion

Conclusions:

Under the present test conditions, isopropylcyclohexane tested up to cytotoxic concentrations in the absence and in the presence of metabolic
activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of mutagenic properties in the in
vitro micronucleus test.

Executive summary:

The in vitro micronucleus assay is a genotoxicity test system for the detection of chemicals which induce the formation of small membrane bound DNA fragments i.e. micronuclei in the cytoplasm of interphase cells. These micronuclei may originate from acentric fragments (chromosome fragments lacking a centromere) or whole chromosomes which are unable to migrate with the rest of the chromosomes during the anaphase of cell division.

The purpose of the micronucleus assay is to detect those agents which modify chromosome structure and degregation in such a way as to lead to induction of micronuclei in interphase cells.

Test samples of isopropylcyclohexane were assayed in an in vitro micronucleus test using CHO cell cultures both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals.

The test was carried out employing 2 exposure times without S9 mix: 4and 20 hours, and 1 exposure time with S9 mix: 4 hours. The experiment with S9 mix was carried out twice. The harvesting time was 20 hours after starting of exposure. The study was conducted in duplicate.

Isopropylcyclohexane was completely dissolved in acetone.

The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation cytotoxicity was noted starting at concentrations of 25 or 100 µg test item/mL in the experiments without and with metabolic activation, respectively. Hence, 25 or 100 µg isopropylcyclohexane/mL were employed as the top concentrations for the mutagenicity tests without and with metabolic activation, respectively.

In the main study cytotoxicity was noted at the top concentrations of 25 or 100 µg test item/mL in the experiments without and with metabolic activation.

Mitomycin C and cyclophosphamide were employed as positive controls in the absence and presence of metabolic activation, respectively.

Under the present test conditions, Isopropylcyclohexane tested up to cytotoxic concentrations in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of mutagenic properties in the in vitro micronucleus test.