Isopropylcyclohexane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A bacterial mutagenicity test (Ames test according to OECD 471), an in vitro mammalian cell genotoxicity test (HPRT test according to OECD 476) and an in vitro micronucleus test (according to OECD 487) are available for isopropylcyclohexane. Based on results of the studies isopropylcyclohexane showed no genotoxic properties.

Link to relevant study records

Reference

Reference 1

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

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

Target gene:

mammalian cell system( Chinese hamster Ovary cells)

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.

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

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.

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'.

see attached documents

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-10-26 to 2011-12-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

2005

Qualifier:

according to guideline

Guideline:

other: ICH Guideline S2A:'Genotoxicity: Specific Aspects of Regulatory gentoxocoty tests for Pharmaceuticals (CPMP/ICH/141/95)'

Qualifier:

according to guideline

Guideline:

other: ICH Giudeline S2B: 'A Standard Battery for Genotoxicity Testing of Pharmaceuticals (CPMP/ICH/174/95)'

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

mutated gene loci resposible for histidine auxotropy

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

- obtained from Trinova Biochem according to Dr. Bruce N. AMES,

Additional strain / cell type characteristics:

other: histidine auxotroph

Metabolic activation:

with and without

Metabolic activation system:

Arochlor 1254 induced rat liver S9; male rats

Test concentrations with justification for top dose:

Plate incorporation test: 0.1, 0.316, 1.0, 3.16, 10.0 and 31.6 µg per plate;
Preincubation test: 0.0316, 0.1, 0.316, 1.0, 3.16 and 10.0 µg per plate

Vehicle / solvent:

Isopropylcyclohexane was completely dissolved in acetone . The vehicle served as the negative control.

Untreated negative controls:

no

Remarks:

solvent test will be used as negative reference item

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Remarks:

for details see below

Positive control substance:

other: without metabolic activation: sodium azide in aqua ad iniectabilia for TA 1535 and TA 100, 2-nitroflurene in DMSO for TA 98, 9-amino-acridine in ethanol abs. for TA 1537, Cumene hydroperoxide in DMSO for TA 102

Remarks:

with metabolic activation: 2-aminoanthracene for TA 98, TA 102, TA 1537, Cyclophosphamide fpr TA 100, TA 1535

Details on test system and experimental conditions:

Bacterial Reverse Mutation Test
SYSTEM OF TESTING
- Pre-Experiment: plate incorporation cytotoxicity test (+/- metabolic activation) with strain TA 100,
Isopropylcyclohexane was examined in three preliminary cytotoxicity tests, carried out as plate incorporation tests without and with metabolic
activation and a preincubation test without S9 mix in test strain TA 100. Ten concentrations ranging from 0.316 to 5000 µg
Isopropylcyclohexane/plate were tested. In the plate incorporation test without and with metabolic activation cytotoxicity was noted starting at a
concentration of 31.6 µg/plate. In the preincubation test without metabolic activation cytotoxicity was noted starting at a concentration of 10.0 µg Isopropylcyclohexane/plate
- Main test: 1st - Standard plate incorporation method, 2nd - Preincubation method
- Metabolic activation assay: Arochlor 1254 induced rat liver S9 fraction, the protein content of the S9 fraction was 34.2 mg/mL S9, cytochrome
P-450: 0.36 nmol/mg protein
ADMINISTRATION
- Dosing:
* Plate incorporation test: 0.1, 0.316, 1.0, 3.16, 10.0 and 31.6 µg per plate;
* Preincubation test: 0.0316, 0.1, 0.316, 1.0, 3.16 and 10.0 µg per plate
- Data : 2 independent experiments with and without metabolic activation
- Number of replicates: 3 per concentration and experiment
- Positive and negative control groups and treatment:
- without metabolic activation:
* sodium azide in aqua ad iniectabilia for TA 1535 and TA 100, 10 µg/plate
* 2-nitroflurene in DMSO for TA 98, 10 µg/plate
* 9-amino-acridine in ethanol abs. for TA 1537, 100 µg/plate
* cumene hydroperoxide in DMSO for TA 102, 10 µg/plate
- with metabolic acivation
* 2-aminoanthracene in DMSO for TA 98, TA 102, TA 1537, 2 µg/plate
* cyclophosphamide in aqua ad iniectabilia for TA 100, TA 1535, 1500 µg/plate
- negative control: the vehicle acetone was used as negative reference item (all test strains).
- Incubation time: 48 h to 72 h at 37 °C in the dark
- Pre-incubation time: 20 min at 37 °C;

SELECTION AGENT (mutation assays):

NUMBER OF REPLICATIONS: 3 per concentration and experiment

NUMBER OF CELLS EVALUATED: approximately 10E8 viable cells in the late exponential or early stationary phase

DETERMINATION OF CYTOTOXICITY
- Method: In the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation, cytotoxicity (scarce
background lawn and reduction of the number of revertants) were noted at the top concentrations of 31.6 and 10.0 µg/plate, respectively, in all
test strains.

Evaluation criteria:

The test item is considered to show a positive response if:
- the number of revertants is significantly increased (p ≤ 0 .05, U-test according to MANN and WHITNEY) compared to the solvent control to at least
2-fold of the solvent control for TA 98, TA 100 and TA 102 and 3-fold of the solvent control for TA 1535 and TA 1537 in both independent
experiments.
- additionally, a significant (p ≤ 0.05) concentration (log value)-related effect (Spearman's rank correlation coefficient) is observed.
- positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on
histidine-free agar plates.

Statistics:

According to the OECD Guideline 471, a statistical analysis of the data is not mandatory

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

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:

GENTOXIC EFFECTS:
- With metabolic activation: negativ
- Without metabolic activation: negativ

CYTOTOXICITY EFFECTS:
- plate incorporation test without and with metabolic activation: 31.6 µg/plate
- preincubation test without metabolic activation: 10.0 µg Isopropylcyclohexane/plate

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

see attchached document

Conclusions:

Under the present test conditions isopropylcyclohexane tested up to cytotoxic concentrations, caused no mutagenic effect in the Salmonella
typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.
 
 

Executive summary:

The purpose of this study was to evaluate the test item for mutagenic activity (gene mutation) in bacteria without and with the addition of a mammalian metabolic activation system as originally described by AMES et al. (1973, 1975) and revised by MARON and(1983).

Isopropylcyclohexane was examined in the 5Salmonella typhimuriumstrains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver). The first experiment was carried out as a plate incorporation test and the second as a preincubation test.

Isopropylcyclohexane was completely dissolved in acetone.

 

Preliminary test

Isopropylcyclohexane was examined in three preliminary cytotoxicity tests, carried out as plate incorporation tests without and with metabolic activation and a preincubation test without S9 mix in test strain TA 100. Ten concentrations ranging from 0.316 to 5000 µg isopropylcyclohexane/plate were tested. In the plate incorporation test without and with metabolic activation cytotoxicity (scarce background lawn and reduction of the number of revertants by more than 50%) was noted starting at a concentration of 31.6 µg/plate. In the preincubation test without metabolic activation cytotoxicity (scarce background lawn and reduction of the number of revertants by more than 50%) was noted starting at a concentration of 10.0 µg isopropylcyclohexane/plate.

Hence, 31.6 µg isopropylcyclohexane/plate were chosen as top concentration for the main study in the plate incorporation test and 10.0 µg/plate in the preincubation test.

 

Main study

Six concentrations ranging from 0.1 to 31.6 µg Isopropylcyclohexane/plate were employed in the plate incorporation test and ranging from 0.0316 to 10.0 µg/plate in the preincubation test, each carried out without and with metabolic activation.

 

Cytotoxicity

In the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation, cytotoxicity (scarce background lawn and reduction of the number of revertants)were noted at the top concentrations of 31.6 and 10.0 µg/plate, respectively, in all test strains.

Mutagenicity

No mutagenic effect (no increase in revertant colony numbers as compared with control counts) was observed for Isopropylcyclohexane, tested up to cytotoxic concentrations of 31.6 or 10.0 µg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation (plate incorporation and preincubation test, respectively).

 

In conclusion, under the present test conditions isopropylcyclohexane tested up to cytotoxic concentrations, caused no mutagenic effect in the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.

 

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-10-26 to 2012-03-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HPRT)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Cells were maintained in Dulbecco's modified Eagle-Mediumsupplemented with 10% fetal calf serum, penicillin (100 U/mL) and streptomycin
(100 µg/mL) called DMEM-FCS; Cells were periodically checked for the absence of mycoplasma contamination by using the HOECHST stain 33258;
Spontaneous mutation rate was continuously monitored

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

post-mitochondrial supernatant fraction derived from livers of Aroclor 1254-treated rats (S9 mix)

Test concentrations with justification for top dose:

Five concentrations: 6.25; 12.5; 25; 50; 100µg/mL without metabolic activation
15.63, 31,3, 62.5, 125, 250 µg/mL with metabolic activation

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ethyl methanesulphonate (EMS) in direct mutagenicity experiment; 9,10-dimethyl-1,2-benzanthracene (DMBA) in S9 mix mediated assay; both EMS and DMBA were dissolved in DMSO. The applied concentrations were 600 or 700 µg EMS/mL medium or 20 or 30 µg DMBA/mL

Details on test system and experimental conditions:

CELLS AND TISSUE CULTURE MEDIA
- V79 cells were maintained in Dulbecco's modified Eagle-Mediumsupplemented with 10% fetal calf serum, penicillin 3 (100 U/mL) and streptomycin (100 µg/mL) called DMEM-FCS
- Incubation of cultures: at 37°C in a humidified atmosphere (90%) containing 10% CO2
- For subculturing, a trypsin (0.05%)-EDTA (ethylenediaminetetraacetic acid, 0.02%) solution in modified Puck's salt solution A was used.

METHOD OF APPLICATION:
- Exposure to the test item in the presence of S9 mix was performed in Dulbecco's phosphate buffered saline (PBS) which additionally contained 20 mM HEPES (N'-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid) pH 7.4 (PBS-HEPES).
- Vehicle: Isopropylcyclohexane was completely dissolved in acetone. Preparations of the test item made on the day of use were employed.

DURATION (see tables below)
- Preincubation period:
* 1 day (in 30 mL DMEM-FCS)
- Exposure duration:
* 4 hours (1st experiment) and 24 hours (2nd experiment) without S9 mix, respectively;
* in the experiments with S9 mix, the medium was replaced by 18 mL S9 mix and the exposure limited to 4 hours.
* the negative control was treated with acetone (the vehicle) in the same way
* After removal of the test item and washing of the plates with PBS cells were trypsinised and a relative plating efficiency was determined for each
dose to obtain an accurate measure of the toxic effect of the chemical
- Expression time (cells in growth medium):
* Three replicate plates (60 mm diameter) were used with a known number of cells.
* Remaining cells were replated and the culture incubation continued until day 8 with 30 mL normal DMEM-FCS with one subcultivation on day 5.
* Afterwards cells were harvested by trypsinisation and replated at a density of 1 000 000 per 150mm diameter dish in DMEM-FCS containing
6-thioguanine (10 µg/mL) for selection of mutants (5 replicate plates), or at approx. 100 to 150 cells (exact number known) per 60 mm diameter
dish in medium without 6-thioguanine for the estimation of plating efficiencies (PE 2), (3 replicate plates).
- Fixation time:
* Plates were fixed and stained after about 8 days (plating efficiency plates) or 12 days (6-thioguanine plates).
- Positive control:
* ethyl methanesulphonate (EMS) in direct mutagenicity experiment;
* 9,10-dimethyl-1,2-benzanthracene (DMBA) in S9 mix mediated assay
both EMS and DMBA were dissolved in DMSO.
* The applied concentrations were 600 or 700 µg EMS/mL medium or 20 or 30 µg DMBA/mL


NUMBER OF REPLICATIONS: three
NUMBER OF CELLS EVALUATED: 1 500 000

DETERMINATION OF CYTOTOXICITY (same procedure was used as employed for the mutagenicity experiments, except that no mutant selection was carried out)
- Method: survival
- A concentration of the test item which produces a low level of survival (10 to 20%) would be used as highest concentration and the survival in the
lowest concentration being approximately the same as that in the negative control.
- Five adequately spaced concentrations are employed
- In this preliminary experiment without and with metabolic activation cytotoxicity in form of decreased plating efficiency was noted starting at
concentrations of 100 or 250 µg Isopropylcyclohexane/mL in the experiment without and with metabolic activation, respectively. Hence, 100 or
250 µg Isopropylcyclohexane/mL were employed as the top concentration for the mutagenicity tests in the absence and in the presence of
metabolic activation, respectively.

Evaluation criteria:

The following predetermined descriptive criteria are used for interpretation of the results:
- If in both independent experiments solvent and positive controls show results within the norm and if the test item does not increase the mutation
frequency 2-fold above the mean of the solvent controls under any condition, or if the mutation frequency is always lower than 40 x 10^-6 and if at least 1 000 000 cells per condition have been evaluated, the item is considered as negative in the test.
- In case of a dose-dependent increase of the mutation frequency in both independent experiments (at similar concentrations) to at least 2-fold
solvent control and at least 40 x 10^-6 both in the presence and/or absence of S9 mix, the item is considered as positive in the test.

Statistics:

No satisfactory mathematical methods are available for the statistical analysis of mammalian cell mutagenicity experiments. See evaluation criteria

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

activation cytotoxicity in form of decreased plating efficiency was noted starting at concentrations of 100 or 250 µg Isopropylcyclohexane/mL in the experiment without and with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES (Preliminary cytotoxicity test):
In this preliminary experiment without and with metabolic activation cytotoxicity in form of decreased plating efficiency was noted starting at
concentrations of 100 or 250 µg Isopropylcyclohexane/mL in the experiment without and with metabolic activation, respectively. Hence, 100
or 250 µg Isopropylcyclohexane/mL were employed as the top concentration for the mutagenicity tests in the absence and in the presence of
metabolic activation, respectively.
COMPARISON WITH HISTORICAL CONTROL DATA:
The historical background mutation frequency in this system has been reported to be 1 to 44 mutants per 106 survivors in non-activation solvent
controls and 6 to 46 per 106 survivors in S9 activation solvent controls [1]. The background data obtained at LPT are given at the end of this
chapter. The spontaneous mutation frequency may be variable from experiment to experiment, but should normally lie within the above-mentioned range. The positive controls EMS (600 and 700 µg/mL) and DMBA (20 and 30 µg/mL) should cause a 10-fold or greater increase in mutation
frequency.
The background mutation frequency at LPT ranges from 1.30 to 38.36 x 10-6 clonable cells for the negative controls. The mutation frequency of the positive controls at LPT ranges from 112.1 to 1708.4 x 10 6 clonable cells for EMS and 130.0 to 2693.3 x 106 clonable cells for DMBA
(see table below).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Criteria for assay acceptance

Solvent control: As the total number of colonies is normally low and as a single mutation may cause several colonies due to cell division during the expression period, a relatively large variation of the mutation frequency may result. This is especially true, if a low spontaneous mutation frequency is forced by cloning (in order to achieve a high sensitivity of the test).

The historical background mutation frequency in this system has been reported to be 1 to 44 mutants per 10⁶survivors in non-activation solvent controls and 6 to 46 per 10⁶survivors in S9 activation solvent controls [1]. The background data obtained atLPTare given at the end of this chapter. The spontaneous mutation frequency may be variable from experiment to experiment, but should normally lie within the above-mentioned range. The positive controls(600 and 700 µg/mL) and DMBA (20 and 30 µg/mL) should cause a 10-fold or greater increase in mutation frequency.

The background mutation frequency atLPTranges from 1.30 to 38.36 x 10^-6clonable cells for the negative controls. The mutation frequency of the positive controls atLPTranges from 112.1 to 1708.4 x 10^-6clonable cells forand 130.0 to 2693.3 x 10^-6clonable cells for DMBA (see table below).

The mutation frequencies of the solvent controls andthe positive controls without and with metabolic activation for the last 58 experiments (most recent background data, not audited by the QAU-department) are given as follows:

Mutation frequency per 10^6 clonable cells
	Without metabolic activation (24-h exposure)		With metabolic activation (4-h exposure)
Solvent control (n = 58)
mean	14.11		14.88
SD	7.42		8.20
range	1.30 - 34.80		2.18 - 38.36
Positive control (µg/mL) (n = 58)
	EMS (600)	EMS (700)	DMBA (20)	DMBA (30)
mean	449.1	468.4	347.1	563.8444.2
SD	444.2	268.6	241.8	700.1
range	112.1 – 1708.4	152.0 – 976.9	130.0 – 844.8	151.3 – 2693.3

SD     = Standard deviation

EMS   = ethyl methanesulfonate

DMBA = 9,10-dimethyl-1,2-benzanthracene

Conclusions:

Under the present test conditions, isopropylcyclohexane tested up to cytotoxic concentrations in the experiments without and with metabolic
activation was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.

Executive summary:

Isopropylcyclohexane was tested for mutagenic potential in a gene mutation assay in cultured mammalian cells (V79, genetic

marker HPRT) both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced animals. The duration of the exposure with the test item was 4 hours or 24 hours in the experiments without S9 mix and 4 hours in the experiments with S9 mix.

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 in form of decreased plating efficiency was noted starting at concentrations of 100 or 250 µg isopropylcyclohexane/mL in the experiment without and with metabolic activation, respectively. Hence, 100 or 250 µg isopropylcyclohexane/mL were employed as the top concentration for the mutagenicity tests in the absence and in the presence of metabolic activation, respectively.

 

Main study

Five concentrations ranging from 6.25 to 100 or 15.63 to 250 µg Isopropylcyclohexane/mL were selected for the experiments without and with metabolic activation, respectively.

 

Cytotoxicity

In the main study cytotoxicity in form of decreased plating efficiency (PE₁) and (PE₂) was noted in the first and second experiments at the top concentrations 100 or 250 µg/mL in the absence and presence of metabolic activation, respectively.

 

Experiments without metabolic activation

The mutation frequency of the negative controlacetone was 17.70 and 14.27 x 10^-6clonable cells. Hence, the negative controls were well within the expected range (see below).

The mutation frequency of the cultures treated with concentrations of 6.25, 12.5, 25, 50 or 100 µg isopropylcyclohexane/mL culture medium ranged from 8.53 to 18.49 x 10^‑6clonable cells. These results are within the normal range of the negative controls.

Experiments with metabolic activation

The mutation frequency of the negative controlacetone was 10.47 and 10.00 x 10^-6clonable cells. Hence, the negative controls were well within the expected range (see below).

The mutation frequency of the cultures treated with concentrations of 15.63, 31.3, 62.5, 125 or 250 µg isopropylcyclohexane/mL culture medium ranged from 8.03 to 15.75 x 10 -6 clonable cells. These results are within the normal range of the negative controls.

The positive controls EMS (ethyl methanesulfonate) in the direct test and DMBA (9,10-dimethyl-1,2-benzanthracene), a compound which requires metabolic activation, caused a pronounced increase in the mutation frequencies ranging from 275.92 to

901.42 x 10^-6clonable cells in the case of EMS and ranging from 263.48 to 443.43 x 10^-6clonable cells in the case of DMBA, indicating the validity of this test system.

 

The background mutation frequency at LPT ranges from 1.30 to 38.36 x 10^-6clonable cells for the negative controls. The mutation frequency of the positive controls at LPT ranges from 112.1 to 1708.4 x 10^-6clonable cells forand 130.0 to 2693.3 x 10⁶clonable cells for DMBA.

 

Conclusion

Under the present test conditions, isopropylcyclohexane tested up to cytotoxic concentrations in the experiments without and with metabolic activation was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No studies with respect to this endpoint have been performed.

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro studies

A bacterial mutagenicity test (Ames test according to OECD 471), an in vitro mammalian cell genotoxicity test (HPRT test according to OECD 476) and an in vitro micronucleus test with CHO cells (according to OECD 487) are available for isopropylcyclohexane.

Under the present test conditions isopropylcyclohexane tested up to cytotoxic concentrations, caused no mutagenic effect in the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation (LPT, 2012).

Furthermore isopropylcyclohexane was tested up to cytotoxic concentrations in an HPRT-V79 mammalian cell mutagenicity test

without and with metabolic activation that was negative under the test conditions. Positive controls exerted potent mutagenic effects (LPT, 2012).

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 (LPT, 2011).

Justification for selection of genetic toxicity endpoint
Three studies (Ames-Test, in vitro MN test, HPRT-Test) were conducted to examine potential genotoxic effects of the substance isopropylcyclohexane. These studies are guideline studies with Klimisch score 1 (reliable without restrictions). Hence all of these studies are relevant for the assessment of the genotoxic potential of the substance.

In vivo studies

No studies with respect to this endpoint have been performed.

Justification for classification or non-classification

Isopropylcyclohexane showed no genotoxic properties in the in vitro studies conducted with the substance. Based on these results it is concluded that isopropylcyclohexane is not genotoxic and therefore must not be classified according to the criteria of EC Regulation 1272/2008.