3,3,5-trimethylcyclohexyl methacrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

All the three in vitro genotoxicity studies (Ames Test, HPRT, in vitro micronucleus test) on 3,3,5 -trimethylcyclohexyl methacrylate showed negative results. Based on these results, 3,3,5 -trimethylcyclohexyl methacrylate is considered to be not mutagenic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February / April 2003

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

n/a

Species / strain / cell type:

S. typhimurium, other: TA1535, TA97a, TA98, TA100 and TA102

Details on mammalian cell type (if applicable):

n/a

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver

Test concentrations with justification for top dose:

Pre-experiment : 0.0031-0.01-0.3016-0.1-0.316-1.0-2.5-5.0 µl/plate
Main experiments : 0.0316-0.1-0.316-1.0-2.5-5.0 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine : TA97a + TA98 (without S9) ; 2-aminoanthracene : all strains with S9

Details on test system and experimental conditions:

-method of application : in agar plate incorporation (experiment 1) ; preincubation (experiment 3)
-triplicate (3 plates) by concentration in the main experiments
-Pre-experiment for toxicity: The toxicity of the test item was determined with strains TA98 and TA100. 8 concentrations were tested for toxicity and induction of mutations with 3 plates each, with plate incorporation method.
Toxicity may be detected by a reduction in the number of revertants, a clearing or diminution of the background lawn or by the degree of survival of treated cultures.
-triplicate (3 plates) by concentration in the main experiments
-Experiment 1 : For the plate incorporation method, the following materials were mixed in a test tube and poured over the surface of a minimal agar plate : test solution at each dose levels, solvent, S9 mix or S9 mix substitution buffer, bacteria suspension and overlay agar.
-Experiment 2: For pre-incubation method, the test item solution was preincubated with the tester strains and sterila buffer or the S9 for 60 minutes at 37 °C prior adding the overlay agar and pouring onto the surface of a minimal agar plate.
In the experiment 1 and 2, after the solidification the plates were inverted and incubated at 37°C for at least 48 hours in the dark.

Evaluation criteria:

A test is considered acceptable if for each strain :
-the bacteria demonstrate their typical responses to crystal violet and ampicillin,
-the control plates without S9 are within the historical control data ranges,
-corresponding background growth on both negtaive control and test plate is observed,
-the positive controls show a distinct enhancement over te control plate.

A test item is considered as mutagenic if :
-a dose-related increase in the number of revertants occurs and/or
-a reproductible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
A biologically relevant increase is described as follow :
-if in strains TA97a, TA100 and TA102 the number of reversions is at least twice as high,
-if in strains TA1535 and TA98, the number of reversions is at least 3 times higher as compared ti the spontaneous reversion rate.

Statistics:

no

Key result

Species / strain:

S. typhimurium, other: TA1535, TA97a, TA98, TA100 and TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in TA98 and TA102 strains with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the experiment 1, no toxicity of the test item was observed in the tester strains TA1535, TA97a and TA102. In the tester strain TA98 a reduction of revertant colony numbers was noted at the highest dose group with S9. In tester strain TA100 a reduction of the background lawn was observed at the highest dose groups (with S9). In the experiment 2, no toxic effects of the test item were found in any tester strain used up to the highest dose group evaluated (+/-S9).
No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with 3,3,5 -trimethyl cyclohexyl methacrylate at any concentration level, neither in the presence nor absence of metabolic activation in experiment 1 and 2.

Conclusions:

The test item is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

In order to investigate the potential of 3,3,5 -trimethyl cyclohexyl methacrylate for its ability to induce gene mutations the plate incorporation test (experiment 1) and the pre-incubation test (experiment 2) were performed with the Salmonella typhimurium strains TA1535, TA97a, TA98, TA100 and TA102. The test item was tested in two independent experiments at several concentrations. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate.

In the experiment 1, no toxicity of the test item was observed in the tester strains TA1535, TA97a and TA102. In the tester strain TA98 a reduction of revertant colony numbers was noted at the highest dose group with S9. In tester strain TA100 a reduction of the background lawn was observed at the highest dose groups (with S9). In the experiment 2, no toxic effects of the test item were found in any tester strain used up to the highest dose group evaluated (+/-S9).

No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with 3,3,5 -trimethyl cyclohexyl methacrylate at any concentration level, neither in the presence nor absence of metabolic activation in experiment 1 and 2.

In conclusion, it cas be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore the test item is considered to be non-mutagenic in this bacterial reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 2019 - October 2019

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)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

hprt locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Dr Donald Clive, Burroughs Wellcome Co.
- Storage at Covance: as frozen stocks in liquid notrogen.
Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free.
For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated at 37+/-1°C. When the cells were growing well, subcutltures were established in an appropriate number of flasks.

MEDIA USED
- Type and identity of media: RPMI 1640 media containing L-glutamine and HEPES

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

male Sprague Dawley rats induced with Aroclor 1254

Test concentrations with justification for top dose:

Range finder (+/-S9): 9,375-18,75-37,5-75-150-300 µg/ml
Mutation experiment (-S9, 3h): 10-15-20-24-28-32-36-40-45-50-60-80 µg/ml
Mutation experiment (+S9, 3h): 10-20-30-40-45-50-55-60-65-75-100 µg/ml

Vehicle / solvent:

DMSO
Preliminary solubility data indicated that 3,3,5-trimethylcyclohexyl methacrylate was miscible with anhydrous analytical grade dimethyl sulphoxide (DMSO) at concentrations up to approximately 50 mg/mL. The solubility limit in culture medium was less than 62.5 µg/mL, as indicated by the observation of precipitation at this concentration 3 hours after test article addition, with warming at 37°C. A maximum concentration of 300 µg/mL was selected for the cytotoxicity Range-Finder Experiment.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk
- Cell density at seeding: At least 107 cells in a volume of 17.8 mL of RPMI 5 (cells in RPMI 10 diluted with RPMI A [no serum] to give a final concentration of 5% serum) were placed in a series of sterile disposable 50 mL centrifuge tubes. For all treatments 0.2 mL vehicle, test article or positive control solution was added. S-9 mix or 150 mM KCl was added as described. Each treatment, in the absence or presence of S-9, was in duplicate (single cultures only used for positive control treatments) and the final treatment volume was 20 mL.


DURATION
- Preincubation period: 3h
- Exposure duration: 7d
- Expression time (cells in growth medium): 7d

NUMBER OF CELLS EVALUATED:
At the end of the expression period, the cell densities in the selected cultures were adjusted to 1 x 10^5 cells/mL. 6TG (1.5 mg/mL) was diluted 100-fold into these
suspensions to give a final concentration of 15 µg/mL. Using a multichannel pipette, 0.2 mL of each suspension was placed into each well of 4 x 96-well microtitre plates
(384 wells at 2 x 104 cells/well). Plates were placed in incubator set to 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air until scoreable (14 days) and
wells containing clones were identified as above and counted.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
- Any supplementary information relevant to cytotoxicity: Cloning Efficiency (CE) in any given culture is therefore: CE = P/No of cells plated per well, and as an average of 1.6 cells/well were plated on all survival and viability plates, CE = P/1.6.
Percentage Relative Survival (% RS) in each test culture was determined by comparing plating efficiencies in test and control cultures thus: % RS = [CE (test)/CE (control)] x 100.
To take into account any loss of cells during the 3 hour treatment period, percentage relative survival values for each concentration of test article were adjusted as follows: Adjusted % RS = [% RS x Post-treatment cell concentration for test article treatment] / Post-treatment cell concentration for vehicle control

Rationale for test conditions:

Acceptance Criteria: The assay was considered valid if the following criteria were met:
1. The MF in the concurrent negative control was considered acceptable for addition to the laboratory historical negative control database,
2. The MF in the concurrent positive controls induced responses that were compatible with those generated in the historical positive control database and give a clear, unequivocal increase in MF over the concurrent negative control,
3. The test was performed with and without metabolic activation,
4. Adequate numbers of cells and concentrations were analysable.

Evaluation criteria:

For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1. The MF at one or more concentrations was significantly greater than that of the vehicle control (p=0.05)
2. There was a significant concentration-relationship as indicated by the linear trend analysis (p=0.05)
3. The results were outside the historical vehicle control range.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis. Positive responses seen only at high levels of cytotoxicity required careful interpretation when assessing their biological relevance. Extreme caution was exercised with positive results obtained at levels of RS lower than 10%.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines (Robinson et al., 1990). The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Key result

Species / strain:

mouse lymphoma L5178Y cells

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:

Range-Finder Experiments
In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 9.375 to 300 µg/mL. Upon addition of the test article to the cultures, precipitate was observed at the highest two concentrations tested in the absence of S-9 (150 and 300 µg/mL) and at the highest three concentrations tested in the presence of S-9 (75 to 300 µg/mL). No precipitate was observed in the absence and presence of S-9 following the 3 hour treatment incubation period. Complete toxicity was observed at the highest three concentrations
tested in the absence and presence of S-9 (75 to 300 µg/mL). The highest concentrations to give >10% RS were 18.75 µg/mL in the absence of S-9 and 37.5 µg/mL in the presence of S-9, which gave 101% and 66% RS, respectively.
No marked changes in osmolality or pH were observed in the Range-Finder at the highest concentration tested in the Range-Finder (300 µg/mL), compared to the concurrent vehicle controls.

Cytotoxicity / Main Experiments
In the Mutation Experiment twelve concentrations, ranging from 10 to 80 µg/mL, were tested in the absence of S-9 and eleven concentrations, ranging from 10 to 100 µg/mL, were tested in the presence of S-9. Upon addition of the test article to the cultures, precipitate was observed at the highest concentrations tested in the absence and presence of S-9 (80 and 100 µg/mL, respectively). No precipitate was observed in the absence and presence of S-9 following the 3 hour treatment incubation period.
Seven days after treatment, one intermediate concentration (15 µg/mL) and the highest four concentrations in the absence of S-9 (45 to 80 µg/mL) and the highest two concentrations in the presence of S-9 (75 and 100 µg/mL) were not selected to determine viability and 6TG resistance as there were either sufficient concentrations to determine the toxicity profile, or due to toxicity. All other concentrations were selected in the absence and presence of S-9. The highest concentrations analysed were 40 µg/mL in the absence of S-9 and 65 µg/mL in the presence of S-9, which gave 10% and 19% RS, respectively.

Mutation results / Main Experiments.
When tested up to toxic concentrations for 3 hours in the absence and presence of S-9, no statistically significant increases in MF, compared to the vehicle control values, were observed at any concentration analysed. The MF values of the vehicle controls exceeded the range generated by the last twenty experiments performed in this laboratory (0 to 7.16 mutants per 106 viable cells in the absence of S-9 and 0 to 6.65 mutants per 106 viable cells in the presence of S-9). However, the MF values were consistent with published HPRT data (nominally 2-50 mutants per 106 viable
cells: Johnson, 2012). According to the regulatory test guideline (OECD, 2016) and the assay acceptance criteria, the concurrent negative (vehicle) control should be considered acceptable for addition to the laboratory historical negative control database. Furthermore, OECD, 2016 describes that where concurrent negative control data fall outside the control limit they may be acceptable for inclusion in the historical control distribution if the data are not extreme outliers and there is evidence that the test system is “under control”, with no evidence of technical or human failure.
In this study, the vehicle control data were representative of recent experiments performed in this laboratory and were considered acceptable.

Conclusions:

It is concluded that 3,3,5-trimethylcyclohexyl methacrylate did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to toxic concentrations for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9), under the experimental conditions described.

Executive summary:

3,3,5-trimethylcyclohexyl methacrylate was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by a Mutation Experiment, each conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9). The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO). A 3 hour treatment incubation period was used for each experiment.

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 9.375 to 300 µg/mL (limited by the appearance of post-treatment precipitate in culture medium). The highest concentrations to give >10% relative survival (RS) were 18.75 µg/mL in the absence of S-9 and 37.5 µg/mL in the presence of S-9, which gave 101% and 66% RS, respectively.

In the Mutation Experiment twelve concentrations, ranging from 10 to 80 µg/mL, were tested in the absence of S-9 and eleven concentrations, ranging from 10 to 100 µg/mL, were tested in the presence of S-9. The highest concentrations analysed to determine viability and 6TG resistance were 40 µg/mL in the absence of S-9 and 65 µg/mL in the presence of S-9, which gave 10% and 19% RS, respectively.

Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S-9. Clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S-9) and benzo(a)pyrene (B[a]P) (with S-9). Therefore the study was accepted as valid.

When tested up to toxic concentrations for 3 hours in the absence and presence of S-9, no statistically significant increases in MF, compared to the vehicle control values, were observed at any concentration analysed.

It is concluded that 3,3,5-trimethylcyclohexyl methacrylate did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to toxic

concentrations for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9), under the experimental conditions described.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September - December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

n/a

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

cultured human peripheral blood lymphocytes.
Blood from two healthy, non-smoking male volunteers from a panel of donors.

Cytokinesis block (if used):

yes

Metabolic activation:

with and without

Metabolic activation system:

The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was obtained from Molecular Toxicology Incorporated, USA where it was prepared from male Sprague Dawley rats induced with Aroclor 1254. The S-9 was supplied as lyophilized S-9 mix (MutazymeTM), stored frozen at <-10°C, and thawed and reconstituted with purified water to provide a 10% S-9 mix just prior to use. Each batch was checked by the manufacturer for sterility, protein content, ability to convert ethidium bromide and cyclophosphamide to bacterial mutagens, and cytochrome P-450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities).
Treatments were carried out both in the absence and presence of S-9 by addition of either 150 mM KCl or 10% S-9 mix respectively. The final S-9 volume in the test system was 1% (v/v).

Test concentrations with justification for top dose:

Concentrations above 50 mg/mL were insoluble in the chosen vehicle DMSO and as such a maximum concentration of 500 µg/mL was selected for the cytotoxicity Range-Finder Experiment.
Range-Finder Experiments concentrations :
3+21, -S-9 : 1.814 to 500.0 µg/ml
3+21, +S-9 : 1.814 to 500.0 µg/ml
24+24, -S-9 : 1.814 to 500.0 µg/ml

Concentrations for the Micronucleus Experiment were selected based on the results of this cytotoxicity Range-Finder Experiment.
Main Micronucleus Experiments concentrations :
3+21, -S-9: 10.00 to 50.00 µg/ml
3+21, +S-9: 40.00 to 120.0 µg/ml
24+24, -S-9: 10.00 to 40.00 µg/ml

Vehicle / solvent:

DMSO.
Preliminary solubility data indicated that 3,3,5-trimethylcyclohexyl methacrylate was soluble in anhydrous analytical grade dimethyl sulphoxide (DMSO) at concentrations up to approximately 50 mg/mL. Concentrations above 50 mg/mL were insoluble in the chosen vehicle DMSO and as such a maximum concentration of 500 µg/mL was selected for the cytotoxicity Range-Finder Experiment.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

vinblastine

Details on test system and experimental conditions:

Test article stock solutions were prepared by formulating 3,3,5-trimethylcyclohexyl methacrylate under subdued lighting in DMSO, with the aid of vortex mixing and warming at 37°C (as required), to give the maximum required treatment concentration. Subsequent dilutions were made using DMSO. The test article solutions were protected from light and used within approximately 3 hours of initial
formulation.

S-9 mix or KCl (1 mL per culture) was added appropriately. Cultures were treated with the test article, vehicle or positive controls (0.1 mL per culture). The final culture volume was 10 mL. Cultures were placed in an incubator set to 37°C for the designated exposure time.
For removal of the test article, cells were pelleted (approximately 300 g, 10 minutes), washed twice with sterile saline (pre-warmed in an incubator set to 37°C), and resuspended in fresh pre-warmed medium containing foetal calf serum and penicillin / streptomycin. Cyto-B (formulated in DMSO) was added to post wash-off culture medium to give a final concentration of 6 µg/mL per culture. Changes in osmolality of more than 50 mOsm/kg and fluctuations in pH of more than one unit may be responsible for an increase in chromosome aberrations.

At the defined sampling time, cultures were centrifuged at approximately 300 g for 10 minutes, the supernatant removed and discarded and cells resuspended in 4 mL (hypotonic) 0.075 M KCl at approximately 37°C for 4 minutes to allow cell swelling to occur. Cells were then fixed by dropping the KCl suspension into fresh, cold methanol/glacial acetic acid (7:1, v/v). The fixative was changed by centrifugation (approximately 300 g, 10 minutes) and resuspension. This procedure was repeated as necessary (centrifuging at approximately 1250 g, 2-3 minutes) until the cell pellets were clean.

Slide preparation : Lymphocytes were kept in fixative at 2-8°C prior to slide preparation for a minimum of 3 hours to ensure that cells were adequately fixed. Cells were centrifuged (approximately 1250 g, 2-3 minutes) and resuspended in a minimal amount of fresh fixative (if required) to give a milky suspension. Several drops of cell suspension were gently spread onto multiple clean, dry microscope slides. Slides were air-dried prior to staining. Slides were stained by immersion in 12.5 µg/mL Acridine Orange in phosphate buffered saline (PBS), pH 6.8 for approximately 10 minutes and washed
with PBS (with agitation) for a few seconds. The quality of the staining was checked. Slides were air-dried and stored protected from light at room temperature. Immediately prior to analysis 1-2 drops of PBS were added to the slides before mounting with glass coverslips.

Selection of Concentrations for Micronucleus Analysis (Micronucleus Experiment Only):
Slides were examined, uncoded, for RI to a minimum of 500 cells per culture to determine whether chemically induced cell cycle delay or toxicity had occurred.
The highest concentration selected for micronucleus analysis following 3+21 hour and 24+24 hour treatments in the absence of S-9 was one at which approximately 50-60% cytotoxicity was achieved (OECD, 2016). Analysis of slides from highly toxic concentrations was avoided.
Slides from the highest selected concentration and at least two lower concentrations were taken for microscopic analysis, such that a range of cytotoxicity from maximum to little was covered.
The positive control concentrations analysed did not exceed the cytotoxicity limits for the test article concentration selection.

Scoring was carried out using fluorescence microscopy

Rationale for test conditions:

A micronucleus was only recorded if it met the following criteria:
1. The micronucleus had the same staining characteristics and a similar morphology to the main nuclei, and
2. Any micronucleus present was separate in the cytoplasm or only just touching a main nucleus, and
3. Micronuclei were smooth edged and smaller than approximately one third the diameter of the main nuclei.

For each treatment regime, two vehicle control cultures were analysed for micronuclei.

Slides from the positive control treatments were checked to ensure that the system was operating satisfactorily. One concentration from each positive control, which gave satisfactory responses in terms of quality and quantity of binucleated cells and numbers of micronuclei, was analysed. This pre-analysis slide check was conducted under non-blinded conditions.

All slides for analysis were coded by an individual not connected with the scoring of the slides, such that analysis was conducted under blind conditions. Labels with only the study number, cell type, experiment number and the code were used to cover treatment details on the slides.
One thousand binucleate cells from each culture (2000 per concentration) were analysed for micronuclei. The number of cells containing micronuclei on each slide was recorded.

Evaluation criteria:

Acceptance Criteria
The assay was considered valid if the following criteria were met:
1. The binomial dispersion test demonstrated acceptable heterogeneity (in terms of MNBN cell frequency) between replicate cultures, particularly where no positive responses were seen
2. The frequency of MNBN cells in vehicle controls fell within the 95th percentile of the current observed historical vehicle control (normal) ranges
3. The positive control chemicals induced statistically significant increases in the proportion of cells with micronuclei. Both replicate cultures at the positive control concentration analysed under each treatment condition demonstrated MNBN cell frequencies that clearly exceeded the normal range
4. A minimum of 50% of cells had gone through at least one cell division (as measured by binucleate + multinucleate cell counts) in vehicle control cultures at
the time of harvest
5. The maximum concentration analysed under each treatment condition met the criteria specified in the test guideline & study protocol


Evaluation Criteria
For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed
2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed
3. A concentration-related increase in the proportion of MNBN cells was observed (positive trend test).

The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result.

Statistics:

The proportions of MNBN cells for each treatment condition were compared with the proportion in vehicle controls by using Fisher's exact test (Richardson et al., 1989). A Cochran-Armitage trend test was applied to each treatment condition. Probability values of p=0.05 were accepted as significant.

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Treatment of cells with 3,3,5-trimethylcyclohexyl methacrylate in the absence and presence of S-9 resulted in frequencies of MNBN cells that were similar to and not significantly (p=0.05) higher than those observed in concurrent vehicle control cultures for all concentrations analysed (all three treatment conditions). The MNBN cell frequency of all 3,3,5-trimethylcyclohexyl methacrylate treated cultures for all three treatment conditions fell within the normal ranges.

Conclusions:

It was concluded that 3,3,5-trimethylcyclohexyl methacrylate did not induce increases in the frequency of micronuclei in cultured human peripheral blood lymphocytes in
the absence or presence of an Aroclor-induced rat liver metabolic activation system (S-9). The maximum concentrations analysed were limited by cytotoxicity, consistent
with recommendations in the current regulatory test guidelines for the in vitro micronucleus assay.

Executive summary:

3,3,5-trimethylcyclohexyl methacrylate was tested in an in vitro micronucleus assay using duplicate human lymphocyte cultures prepared from the pooled blood of two

male donors in a single experiment. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence

and presence of metabolic activation (S-9) from Aroclor 1254-induced rats. The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO) and the highest concentrations tested in the Micronucleus Experiment (limited by toxicity) were determined following a preliminary cytotoxicity Range-Finder

Experiment.

Treatments were conducted (as detailed in the following summary table) 48 hours following mitogen stimulation by phytohaemagglutinin (PHA). The test article

concentrations for micronucleus analysis were selected by evaluating the effect of 3,3,5-trimethylcyclohexyl methacrylate on the replication index (RI). Micronuclei

were analysed at three or four concentrations.

Appropriate negative (vehicle) control cultures were included in the test system under each treatment condition. The proportion of micronucleated binucleate (MNBN) cells in these cultures fell within the 95th percentile of the current observed historical vehicle control (normal) ranges. Mitomycin C (MMC) and Vinblastine (VIN) were

employed as clastogenic and aneugenic positive control chemicals, respectively, in the absence of rat liver S-9. Cyclophosphamide (CPA) was employed as a clastogenic positive control chemical in the presence of rat liver S-9. Cells receiving these were sampled in the Micronucleus Experiment at 24 hours (CPA, MMC) or 48 hours (VIN) after the start of treatment. All positive control compounds induced statistically significant increases in the proportion of cells with micronuclei.

All acceptance criteria were considered met and the study was therefore accepted as valid.

Treatment of cells with 3,3,5-trimethylcyclohexyl methacrylate in the absence and presence of S-9 resulted in frequencies of MNBN cells that were similar to and not

significantly (p=0.05) higher than those observed in concurrent vehicle control cultures for all concentrations analysed (all three treatment conditions). The MNBN

cell frequency of all 3,3,5-trimethylcyclohexyl methacrylate treated cultures for all three treatment conditions fell within the normal ranges.

It was concluded that 3,3,5-trimethylcyclohexyl methacrylate did not induce increases in the frequency of micronuclei in cultured human peripheral blood lymphocytes in

the absence or presence of an Aroclor-induced rat liver metabolic activation system (S-9). The maximum concentrations analysed were limited by cytotoxicity, consistent

with recommendations in the current regulatory test guidelines for the in vitro micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro gene mutation study in bacteria (Chevallier 2016a)

In order to investigate the potential of 3,3,5 -trimethylcyclohexyl methacrylate for its ability to induce gene mutations the plate incorporation test (experiment 1) and the pre-incubation test (experiment 2) were performed with the Salmonella typhimurium strains TA1535, TA97a, TA98, TA100 and TA102. The test item was tested in two independent experiments at several concentrations. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate.

In the experiment 1, no toxicity of the test item was observed in the tester strains TA1535, TA97a and TA102. In the tester strain TA98 a reduction of revertant colony numbers was noted at the highest dose group with S9. In tester strain TA100 a reduction of the background lawn was observed at the highest dose groups (with S9). In the experiment 2, no toxic effects of the test item were found in any tester strain used up to the highest dose group evaluated (+/-S9).

No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with 3,3,5 -trimethyl cyclohexyl methacrylate at any concentration level, neither in the presence nor absence of metabolic activation in experiment 1 and 2.

In conclusion, it cas be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.Therefore the test item is considered to be non-mutagenic in this bacterial reverse mutation assay.

 

In vitro micronucleus study (Chirom 2020):

3,3,5-trimethylcyclohexyl methacrylate was tested in an in vitro micronucleus assay using duplicate human lymphocyte cultures prepared from the pooled blood of two

male donors in a single experiment. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence

and presence of metabolic activation (S-9) from Aroclor 1254-induced rats. The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO) and the highest concentrations tested in the Micronucleus Experiment (limited by toxicity) were determined following a preliminary cytotoxicity Range-Finder

Experiment.

Treatment of cells with 3,3,5-trimethylcyclohexyl methacrylate in the absence and presence of S-9 resulted in frequencies of MNBN cells that were similar to and not

significantly (p=0.05) higher than those observed in concurrent vehicle control cultures for all concentrations analysed (all three treatment conditions). The MNBN

cell frequency of all 3,3,5-trimethylcyclohexyl methacrylate treated cultures for all three treatment conditions fell within the normal ranges.

It was concluded that 3,3,5-trimethylcyclohexyl methacrylate did not induce increases in the frequency of micronuclei in cultured human peripheral blood lymphocytes in the absence or presence of an Aroclor-induced rat liver metabolic activation system (S-9). The maximum concentrations analysed were limited by cytotoxicity, consistent with recommendations in the current regulatory test guidelines for the in vitro micronucleus assay.

 

In vitro gene mutation study in mammalian cells (Lloyd 2019):

3,3,5-trimethylcyclohexyl methacrylate was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by a Mutation Experiment, each conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9). The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO). A 3 hour treatment incubation period was used for each experiment.

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 9.375 to 300 µg/mL (limited by the appearance of post-treatment precipitate in culture medium). The highest concentrations to give >10% relative survival (RS) were 18.75 µg/mL in the absence of S-9 and 37.5 µg/mL in the presence of S-9, which gave 101% and 66% RS, respectively.

In the Mutation Experiment twelve concentrations, ranging from 10 to 80 µg/mL, were tested in the absence of S-9 and eleven concentrations, ranging from 10 to 100 µg/mL, were tested in the presence of S-9. The highest concentrations analysed to determine viability and 6TG resistance were 40 µg/mL in the absence of S-9 and 65 µg/mL in the presence of S-9, which gave 10% and 19% RS, respectively.

Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S-9. Clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S-9) and benzo(a)pyrene (B[a]P) (with S-9). Therefore the study was accepted as valid.

When tested up to toxic concentrations for 3 hours in the absence and presence of S-9, no statistically significant increases in MF, compared to the vehicle control values, were observed at any concentration analysed.

It is concluded that 3,3,5-trimethylcyclohexyl methacrylate did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to toxic

concentrations for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9), under the experimental conditions described.

Justification for classification or non-classification

Based on the available data, 3,3,5 -trimethylcyclohexyl methacrylate is considered to be not mutagenic. No classification for genotoxicity is required according to the Regulation EC n°1272/2008.