3,3,5-trimethylcyclohexyl acrylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 May 2016 - 22 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

26 September 2014

Deviations:

no

Principles of method if other than guideline:

The historical data used for the validation of long treatment period without S9 mix (24 hours treatment + 0 hour recovery) were generated with non-audited data from non-GLP studies. These data were performed in compliance with CiToxLAB France’s standard operating procedures. Since CiToxLAB France is a Test facility certified by the French National Authorities for Good Laboratory Practice, and the procedures undertaken are the same, this deviation is considered not to prejudice the overall GLP status of the study and the scientific reliability of the study conclusions. Moreover, the corresponding mean frequency of micronucleated cells in the vehicle control was 2‰ in this experiment, therefore = 5‰ as specified in the acceptance criteria.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

3,3,5-trimethylcyclohexyl acrylate

EC Number:

289-200-9

EC Name:

3,3,5-trimethylcyclohexyl acrylate

Cas Number:

86178-38-3

Molecular formula:

C12H20O2

IUPAC Name:

3,3,5-trimethylcyclohexyl prop-2-enoate

Test material form:

liquid

Method

Target gene:

Not applicable (not a gene mutation assay).

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium containing 10% heat inactivated horse serum, L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

L5178Y TK+/- cells are an established cell line recommended by international regulations for in vitro mammalian cell gene mutation test and for in vitro micronucleus test. Indeed, they are suitable to reveal chemically induced micronuclei. The average cell cycle time is approximately 10-12 hours.
L5178Y TK+/- cells were obtained from ATCC (American Type Culture Collection, Manassas, USA), by the intermediate of Biovalley (Marne-La-Vallée, France).
The cells were stored in a cryoprotective medium (10% horse serum and 10% dimethylsulfoxide (DMSO)) at -80°C and each batch of frozen cells was checked for the absence of mycoplasma.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

Since the test item was found cytotoxic and poorly soluble in the culture medium during the preliminary test, the selection of the highest dose-level to be used in the main experiments was based on the level of cytotoxicity and/or on the level of precipitate, according to the criteria specified in the international guidelines.

Experiment without S9 mix
With a treatment volume of 0.5% (v/v) in culture medium, the dose-levels selected were 0.000625, 0.00125, 0.0025, 0.005, 0.01, 0.015, 0.02 and 0.04 mM, for the 3- and 24-hour treatments.

Experiments with S9 mix
With a treatment volume of 0.5% (v/v) in culture medium, the selected dose-levels were as follows:
- 0.125, 0.25, 0.5, 1, 2 and 4 mM for the first experiment,
- 0.0009, 0.003, 0.008, 0.025, 0.074, 0.222, 0.667 and 2 mM for the second experiment.

Vehicle / solvent:

- Vehicle used: dimethylsulfoxide (DMSO)
- Justification for choice: Using a test item concentration of 390 mg/mL in the vehicle (DMSO) and a treatment volume of 0.5% (v/v) in culture medium, the highest recommended dose-level of 10 mM (corresponding to 1950 µg/mL) was achievable.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: mitomycin C, colchicine (-S9 mix); cyclophosphamide (+S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
Preliminary cytotoxicity test
Without S9 mix 3 h treatment + 24 h recovery
24 h treatment + 0 h recovery
With S9 mix 3 h treatment + 24 h recovery

First cytogenetic experiment
Without S9 mix 3 h treatment + 24 h recovery
24 h treatment + 0 h recovery
With S9 mix 3 h treatment + 24 h recovery

Second cytogenetic experiment
With S9 mix 3 h treatment + 24 h recovery

NUMBER OF CELLS EVALUATED: 2000 mononucleated cells / dose

DETERMINATION OF CYTOTOXICITY
- Method: population doubling

Evaluation criteria:

The biological relevance of the results was always taken into account when evaluating results.

Evaluation of a positive response: a test item is considered to have clastogenic and/or aneugenic potential, if all the following criteria were met:
- a dose-related increase in the frequency of micronucleated cells was demonstrated by a statistically significant trend test,
- for at least one dose-level, the frequency of micronucleated cells of each replicate culture was above the corresponding vehicle historical range,
- a statistically significant difference in comparison to the corresponding vehicle control was obtained at one or more dose-levels.

Evaluation of a negative response: a test item is considered clearly negative if none of the criteria for a positive response was met.

Statistics:

yes

Results and discussion

Test results

Key result

Species / strain:

other: mouse lymphoma L5178Y TK+/- cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Evaporation from medium: none
- Emulsion: in both experiments with S9 mix, an emulsion was observed in the culture medium at the end of the treatment period, at dose-levels >= 2 mM.
- Definition of acceptable cells for analysis: Analysis was performed under a microscope (1000 x magnification), on the basis of the recommendations of Miller et al. (1995), according to the following criteria:
* micronuclei should be clearly surrounded by a nuclear membrane,
* the micronucleus area should be less than one-third of the area of the main nucleus,
* non-refractility of the micronuclei,
* micronuclei should not be linked to the main nucleus via nucleoplasmic bridges,
* micronuclei should be located within the cytoplasma of the cell,
* only mononucleated cells with a number of micronuclei <= 5 should be scored to exclude apoptosis and nuclear fragmentation.

- Other confounding effects: none.

RANGE-FINDING/SCREENING STUDIES:
Using a test item concentration of 390 mg/mL in the vehicle and a treatment volume of 0.5% (v/v) in culture medium, the highest recommended dose-level of 10 mM (corresponding to 1950 µg/mL) was achievable. Thus, the dose-levels selected for the treatment of the preliminary test were 0.0002, 0.002, 0.02, 0.2, 1, 2, 5 and 10 mM.

At the highest dose-level of 10 mM, the pH of the culture medium was approximately 7.4 (as for the vehicle control) and the osmolality was 355 mOsm/kg H2O (379 mOsm/kg for the vehicle control). Therefore, none of the selected dose-levels was considered to produce extreme culture conditions and the highest recommended dose-level of 10 mM could be selected as the highest dose-level for the main experiment.

An emulsion was observed in the culture medium at dose-levels >= 2 mM at the end of the 3-h treatment period and at dose-levels >= 5 mM at the end of the 24-h treatment period.

Following the 3- and 24-hour treatments without S9 mix, a marked cytotoxicity was observed at dose levels >= 0.02 mM, as shown by a 60 to 100% decrease in the PD.
Following the 3-hour treatment with S9 mix, a 33 to 55% decrease in the PD was observed, without any clear evidence of a dose-response relationship, at the dose-levels of 0.2, 2 and 10 mM.

MAIN STUDY
Experiment without S9 mix
With a treatment volume of 0.5% (v/v) in culture medium, the dose-levels selected were 0.000625, 0.00125, 0.0025, 0.005, 0.01, 0.015, 0.02 and 0.04 mM, for the 3- and 24-hour treatments.
 
No emulsion was observed in the culture medium at any of the tested dose-levels, either at the beginning or the end of the treatment periods.

Following the 3- and 24-hour treatments, a slight to severe cytotoxicity was induced at dose-levels = 0.01 mM, as shown by a 26 to 100% decrease in the PD.

The dose-levels selected for micronucleus analysis were 0.005, 0.01 and 0.015 mM for the 3- and 24-hour treatments,the latter inducing a 45 and 26% decrease in the PD, respectively, and higher dose-levels being too cytotoxic.
It is to be noted that the highest analyzable dose-level of 0.015 mM did not exhibit about 55% cytotoxicity for either treatment period. Considering the narrow dose-levels spacing used, the overall available results were considered to be suitable to allow a reliable interpretation.
 
Following the 3- and 24-hour treatments, neither statistically significant nor dose-related increase in the frequency of micronucleated cells was noted at any of the tested dose-levels relative to the vehicle controls. None of the analyzed dose-levels showed frequency of micronucleated cells of each replicate culture above the vehicle control historical range.
 
These results met the criteria of a negative response.
 
Experiments with S9 mix
With a treatment volume of 0.5% (v/v) in culture medium, the selected dose-levels were as follows:
- 0.125, 0.25, 0.5, 1, 2 and 4 mM for the first experiment,
- 0.0009, 0.003, 0.008, 0.025, 0.074, 0.222, 0.667 and 2 mM for the second experiment.
 
In both experiments, an emulsion was observed in the culture medium at the end of the treatment period, at dose-levels = 2 mM.

In the first experiment, a moderate to severe cytotoxicity was induced from the lowest tested dose-level, as shown by a 48 to 100% decrease in the PD. Since the cytotoxicity obtained in this experiment was higher than expected (based on the results of the preliminary test), not enough analyzable dose-levels were available for the analysis of micronuclei. 
In the second experiment, performed under the same experimental conditions, but using a lower and wider range of dose-levels, no cytotoxicity was observed at any of the tested dose-levels, as shown by the absence of any noteworthy decrease in the PD (i.e. results consistent with those of the preliminary test).

The dose-levels selected for micronucleus analysis (second experiment) were 0.222, 0.667 and 2 mM, the latter being the lowest dose-level showing test item emulsion in the culture medium at the end of the treatment period.
 
Neither statistically significant nor dose-related increase in the frequency of micronucleated cells was noted at any of the tested dose-levels relative to the vehicle control. None of the analyzed dose-levels showed frequency of micronucleated cells of each replicate culture above the vehicle control historical range.
 
These results met the criteria of a negative response.

Applicant's summary and conclusion

Conclusions:

3.3.5-trimethylcyclohexyl acrylate did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/-mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.
 

Executive summary:

The objective of this study was to evaluate the potential of the test item, to induce an increase in the frequency of micronucleated cellsin the mouse cell line L5178Y TK^+/-.

 

After a preliminary cytotoxicity test, the test item, diluted in dimethylsulfoxide (DMSO), was tested in a first experiment, with (3h of treatment) and without (3 h and 24h of treament) a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

 

Since not enough analyzable dose-levels were obtained following the treatment of the first experiment with S9 mix, the corresponding slide analysis was not performed and the test item was tested in a second experiment with S9 mix using the same conditions (3 h treatment + 24 h recovery).

 

Each treatment was coupled to an assessment of cytotoxicity at the same dose-levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells.

Then, after the final cell counting, the cells were washed and fixed. Then, cells from three dose-levels of the test item-treated cultures were dropped onto clean glass slides (except for the first experiment with S9 mix). The slides were air-dried before being stained in 5% Giemsa. Slides from vehicle and positive controls cultures were also prepared as described above. All slides were coded before analysis, so that the analyst was unaware of the treatment details of the slide under evaluation ("blind" scoring). For the first experiment without S9 mix and the second experiment with S9 mix, micronuclei were analyzed for three dose-levels of the test item, for the vehicle and the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per dose).

Number of cells with micronuclei and number of micronuclei per cell were recorded separately for each treated and control culture.

 

Since the test item was found cytotoxic and poorly soluble in the culture medium during the preliminary test, the selection of the highest dose-level to be used in the main experiments was based on the level of cytotoxicity and/or on the level of precipitate, according to the criteria specified in the international guidelines.

The mean population doubling and the mean frequencies of micronucleated cells for the vehicle controls were as specified in the acceptance criteria. Also, positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells. The study was therefore considered to be valid.

 

Experiment without S9 mix

With a treatment volume of 0.5% (v/v) in culture medium, the dose-levels selected were 0.000625, 0.00125, 0.0025, 0.005, 0.01, 0.015, 0.02 and 0.04 mM, for the 3- and 24-hour treatments.

 

No emulsion was observed in the culture medium at any of the tested dose-levels, either at the beginning or the end of the treatment periods.

Following the 3- and 24-hour treatments, a slight to severe cytotoxicity was induced at dose-levels = 0.01 mM, as shown by a 26 to 100% decrease in the PD.

The dose-levels selected for micronucleus analysis were 0.005, 0.01 and 0.015 mM for the 3- and 24-hour treatments,the latter inducing a 45 and 26% decrease in the PD, respectively, and higher dose-levels being too cytotoxic.

It is to be noted that the highest analyzable dose-level of 0.015 mM did not exhibit about 55% cytotoxicity for either treatment period. Considering the narrow dose-levels spacing used, the overall available results were considered to be suitable to allow a reliable interpretation.

 

Following the 3- and 24-hour treatments, neither statistically significant nor dose-related increase in the frequency of micronucleated cells was noted at any of the tested dose-levels relative to the vehicle controls. None of the analyzed dose-levels showed frequency of micronucleated cells of each replicate culture above the vehicle control historical range.

 

These results met the criteria of a negative response.

 

Experiments with S9 mix

With a treatment volume of 0.5% (v/v) in culture medium, the selected dose-levels were as follows:

- 0.125, 0.25, 0.5, 1, 2 and 4 mM for the first experiment,

- 0.0009, 0.003, 0.008, 0.025, 0.074, 0.222, 0.667 and 2 mM for the second experiment.

 

In both experiments, an emulsion was observed in the culture medium at the end of the treatment period, at dose-levels = 2 mM.

In the first experiment, a moderate to severe cytotoxicity was induced from the lowest tested dose-level, as shown by a 48 to 100% decrease in the PD. Since the cytotoxicity obtained in this experiment was higher than expected (based on the results of the preliminary test), not enough analyzable dose-levels were available for the analysis of micronuclei. 

In the second experiment, performed under the same experimental conditions, but using a lower and wider range of dose-levels, no cytotoxicity was observed at any of the tested dose-levels, as shown by the absence of any noteworthy decrease in the PD (i.e. results consistent with those of the preliminary test).

The dose-levels selected for micronucleus analysis (second experiment) were 0.222, 0.667 and 2 mM, the latter being the lowest dose-level showing test item emulsion in the culture medium at the end of the treatment period.

 

Neither statistically significant nor dose-related increase in the frequency of micronucleated cells was noted at any of the tested dose-levels relative to the vehicle control. None of the analyzed dose-levels showed frequency of micronucleated cells of each replicate culture above the vehicle control historical range.

 

These results met the criteria of a negative response.

Under the experimental conditions of the study, 3.3.5-trimethylcyclohexyl acrylate, the test item did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.