4-(1,1-dimethylethyl)cyclohexyl methacrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: The test substance induced no statistically significant dose-related increase in the numbers of revertant (His+) colonies in each of the five tester stnins (TA1535; TA1537; TA1538; TA98 and TAl00). The test substance can, therefore, be considered as non mutagenic in this test system. This is supported by a non mutagenic Ames test in the read across substance CAS# 101-43-9.

In vitro gene mutation in mammalian cells: The read across substance, CAS# 101-43-9, is considered to be non-mutagenic in an OECD 476 test with Chinese Hamster Lung fibroblasts cell line.

In vitro chromosomal aberration test in human peripheral lynmphocytes: The read across substance, CAS# 101-43-9, is considered to be non-genotoxic in an OECD 473 test.

QSAR supports the substance being non-genotoxic.

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:

Initiation date: 03-10-'84 Completion date: 05-10-'84

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Chemical name (IUPAC): t-Butyl cyclohexyl methacrylate
Trade name/code: Nourycryl NC 110
Appearance: Clear mobile liquid
Storage: At ambient temperature in the dark
Purity > 98%

Target gene:

histidine locus

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9 from the liver of rats induced with Aroclor 1254

Test concentrations with justification for top dose:

Preliminary toxicity test: 0.1, 0.3, 1.0, 3.3, 10.0, 33.3, 100.0, 333.3, 1000.0, 3333.3 and 5000.0 µg/plate

In the preliminary test survival was reduced at test concentrations from 33.3 µg/plate, therefore, in the main test concentrations up to 24 µg/plate were used

Main test (with and without metabolic activation): 2.4, 4.2, 7.5, 13.0, 24.0 µg/plate

Vehicle / solvent:

Ethanol was used due to solubility of the substance in this vehicle.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Remarks:

1 µg/plate for strain TA 1535

Positive control substance:

sodium azide

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Remarks:

0.5 µg/plate for strain TA 100

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Remarks:

10 µg/plate for strains TA 98 and TA 1538

Positive control substance:

other: 4-nitro-o-phenyl-enediamine

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Remarks:

60 µg/plate for strain TA 1537

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Remarks:

0.5 µg/plate for all strains

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

PRINCIPLE OF THE TEST METHOD
Bacteria, plated onto minimal medium agar plates, are exposed to the test chemical with and without metabolic activation (S9 mix). After 48h of incubation, revertant colonies showing histidine independent growth are counted and compared to the number of spontaneous revertants in solvent-treated control cultures.

Preparations
Bacterial cultures
Samples of frozen stock cultures of bacteria are transferred into enriched nutrient broth (Oxoid No.2) and incubated in a shaking water bath (37°C , 150 spm) until the cultures reach an O. D. of 0.4 at 700 nm (10^9 cells/ml). Freshly grown cultures of each strain are used for a test.

Test procedure
Standard plate test
Top agar in top agar tubes is melted and heated to 45 ºC. The following solutions are successively added to 3 ml of top agar: 0.1 ml of a fresh bacterial culture (10^9 cells/ml)of one of the tester strains, 0.1 ml of a dilution of the test substance in ethanol, and in case of activation assays 0. 5 ml of S9 mix. The ingredients are mixed on a Vortex and the contents of the top agar tube are poured onto a selective agar plate. After solidification of the top agar, the plates are turned and incubated in the dark at 37 ºC for 48 h. After this period revertant colonies (histidine independent) are counted automatically with an Artek model 880 colony counter or manually.

Species / strain:

S. typhimurium TA 1535

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

Species / strain:

S. typhimurium TA 1537

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

Species / strain:

S. typhimurium TA 1538

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

Species / strain:

S. typhimurium TA 98

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

Species / strain:

S. typhimurium TA 100

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:

Preliminary toxicity test
Eleven serial half-log dilutions of the test substance were plated with a diluted TA100 culture on non-selective agar (viability counting). For viability determinations, equal numbers of bacterial cells were seeded on each plate in the presence of the test substance. The percentage survival of an appropriately diluted TA100 culture on non-selective agar is determined by comparing the number of colonies on the solvent control plate with those on the plates containing the test substance. The survival of strain TA100 is reduced at test substance concentrations from 33.3 μg/plate upwards.

Main test
All bacterial strains showed negative responses over the entire dose range of the test substance. Strain-specific positive control chemicals showed that the test conditions were optimal and that the metabolic activation system functioned properly. Based on these results, the test substance can be considered as nonmutagenic in the Ames Salmonella/microsome assay.

Conclusions:

All bacterial strains showed negative responses over the entire dose range of the test substance. Based on these results, the test substance can be considered as non mutagenic in the Ames Salmonella/microsome assay.

Executive summary:

A sample of Nourycryl MC 110 was tested in the Ames Salmonella/microsome test up to the limit of toxicity (24 μg/plate).

The test substance induced no statistically significant dose-related increase in the numbers of revertant (His+) colonies in each of the five tester stnins (TA1535; TA1537; TA1538; TA98 and TAl00). The test substance can, therefore, be considered as non mutagenic in this test system.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

see attached Read Across rationale

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: human lymphocytes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiment I:
without S9 mix (exposure period 24 h and 48 h)
24h: 10, 30, 100 µg/ml
48h: 100 µg/ml
with S9 mix (exposure period 4 h)
24h: 300, 600, 1680 µg/ml
48h: 1680 µg/ml

Experiment II
without S9 mix (exposure period 24 h and 48 h)
24h: 30, 100, 150 µg/ml
48h: 200 µg/ml
with S9 mix (exposure period 4 h)
24h: 500, 1000, 1680 µg/ml
48h: 1680 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 0.30 mg/ml Ethylmethanesulfonate (without S9 mix); 30.0 µg/ml Cyclophosphamide (with S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4, 24, 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 3 hours before harvesting

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS:2

NUMBER OF CELLS EVALUATED: 200 per dose

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Statistics:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of th chi-square test. Evaluation was performed only for dells carrying aberrations exclusive gaps.

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Range-Finding/Screening Studies: 3 - 1680 µg/ml
Additional Information on Cytotoxicity: Mitotic index

Remarks on result:

other: other: human lymphocytes

Remarks:

Migrated from field 'Test system'.

Experiment I (exposure period 24 h)

fixation interval 24 h after start of the treatment

Test groups	S9 mix	Analyses of chromosomes
		Incl. Gaps %	Excl. gaps %
Negative control	-	2.00	1.00
1% Solvent control DMSO	-	1.50	0.50
0.33 mg/ml Positive contol	-	16.00	13.0
10 µg/ml Test article	-	1.00	1.0
30 µg/ml Test article	-	0.00	0.00
100 µg/ml Test article	-	1.00	0.50

 

Experiment I (exposure period 48 h)

fixation interval 48 h after start of the treatment

Test groups	S9 mix	Analyses of chromosomes
		Incl. Gaps %	Excl. gaps %
1% Solvent control DMSO	-	1.50	1.00
100 µg/ml Test article	-	1.50	0.50

 

Experiment II (exposure period 24 h)

fixation interval 24 h after start of the treatment

Test groups	S9 mix	Analyses of chromosomes
		Incl. Gaps %	Excl. gaps %
Negative control	-	3.50	1.50
1.0% Solvent control DMSO	-	2.00	1.50
0.33 mg/ml Positive contol	-	14.50	10.00
30 µg/ml Test article	-	2.00	1.00
100 µg/ml Test article	-	1.50	0.50
150 µg/ml Test article	-	1.00	0.50

 

Experiment II (exposure period 48 h)

fixation interval 48 h after start of the treatment

Test groups	S9 mix	Analyses of chromosomes
		Incl. Gaps %	Excl. gaps %
1.0% Solvent control DMSO	-	0.00	0.00
150 µg/ml Test article	-	3.00	2.00

 

 

 

 

 

 

Experiment I (exposure period 4 h)

fixation interval 24 h after start of the treatment

Test groups	S9 mix	Analyses of chromosomes
		Incl. Gaps %	Excl. gaps %
Negative control	+	0.50	0.00
1.0% Solvent control DMSO	+	0.50	0.50
30.0 µg/ml Positive contol CPA	+	17.00	14.00
300 µg/ml Test article	+	0.50	0.00
600 µg/ml Test article	+	2.00	1.00
1680 µg/ml Test article	+	1.00	0.50

 

Experiment I (exposure period 4 h)

fixation interval 48 h after start of the treatment

Test groups	S9 mix	Analyses of chromosomes
		Incl. Gaps %	Excl. gaps %
1.0% Solvent control DMSO	+	1.00	0.50
1680 µg/ml Test article	+	0.00	0.00

 

Experiment II (exposure period 4 h)

fixation interval 24 h after start of the treatment

Test groups	S9 mix	Analyses of chromosomes
		Incl. Gaps %	Excl. gaps %
Negative control	+	2.00	1.00
1.0% Solvent control DMSO	+	1.50	0.00
30.0 µg/ml Positive contol CPA	+	10.50	10.00
500 µg/ml Test article	+	1.00	0.00
1000 µg/ml Test article	+	1.00	0.50
1680 µg/ml Test article	+	0.00	0.00

 

Experiment II (exposure period 4 h)

fixation interval 48 h after start of the treatment

Test groups	S9 mix	Analyses of chromosomes
		Incl. Gaps %	Excl. gaps %
1.0% Solvent control DMSO	+	0.50	0.50
1680 µg/ml Test article	+	2.50	1.50

 

Conclusions:

In conclusion, it can be stated that in the described study and under the experimental conditions reported, the test article Cyclohexylmethacrylate did not induce structural chromosome aberrrations in human lymphocytes in vitro when tested up to 10 mM (with S9 mix) and cytotoxic concentrations (without S9 mix).

Executive summary:

In conclusion, it can be stated that in the described study and under the experimental conditions reported, the test article Cyclohexylmethacrylate did not induce structural chromosome aberrrations in human lymphocytes in vitro when tested up to 10 mM (with S9 mix) and cytotoxic concentrations (without S9 mix).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

see attached Read Across rationale

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (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 transerase

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media:
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

rat S9 mix

Test concentrations with justification for top dose:

Experiment I:
without S9 mix: 3.0; 10.0; 30.0; 100.0 and 200.0** µg/ml
with S9 mix: 10.0*; 30.0*; 100.0*; 200.0: 1000.0; 2000.0 and 3000.0 µg/ml
Experiment II:
without S9 mix: 10.0; 30.0; 60.0; 100.0; 130.0** and 160.0** g/ml
with S9 mix: 30.0; 60.0; 100.0; 300.0; 1000.0 and 3000.0 µg/ml

* not evaluated: concerning concentration range and toxicity four conecentrations were selected to be evaluated at the end of the experiment
** not evaluable, toxic effects

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
The solvent was chosen according to its solubility properties and its non-toxcicity for the cells. The final concentrtion of DMSO in the culture medium did not exceed 1% v/v.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: see remarks

Remarks:

Ethylmethanesulfonate 4.8 mM without metabolic activation; 7,12-dimethylbenz(a)anthracene 15.0 µM with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 h
- Exposure duration: 4 h
- Expression time (cells in growth medium): 5 -9 days
- Fixation time (start of exposure up to fixation or harvest of cells): 17 days

SELECTION AGENT (mutation assays):

NUMBER OF REPLICATIONS:

Evaluation criteria:

The test article is classified as mutagenic if it induces reproducibly with one of the concentrations a mutation frequency that is three times higher than the spontaneous mutation frequency is considered non-mutagenic in this system.
The test article is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold incease of the mutant frequency is not observed.

Statistics:

Since the distribution of mutant cells does not follow known statistical models, an adequate statistical method ist not available.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

In experiment I, after treatment with 200.0 µg/ml without metabolic activation the plating efficiency of the cultures set up for the determination of the toxicity was not reduced, although the first subcultivation of the cultures set up for the determination of the mutation rate could not be performed due to toxicity. This effect may be due to precipitation above the limit of solubility of approximately 170 µg/ml under test conditions. With metabolic activation toxicity was found at concentrations higher than 1000.0 µg/ml (reduced plating efficiency and cell density at the first subcultivation). The colony counts per flask (plating efficiency) showed great differences in the parallel cultures with metabolic activation possibly due to the precipitation at these concentrations. A similar effect was observed in the pre-test.

In experiment II toxicity was observed at lower concentrations than in experiment I. Without metabolic activation the plating efficiency was clearly reduced beginning at 60.0 µg/ml and a reduction of the cell density at subcultivation was observed at 100.0 µg/ml. With metabolic activation the plating efficiency of the cells was clearly reduced with concentrations higher than 100.0 µg/ml in contrast to the subscultivation, which showed toxic effects at concentrations higher than 300.0 µg/ml. In experiment II all six concentration groups with metabolic activation were evaluated to obtain mutation rates from concentrations were no precipitation occurred.

Taking in account the mutation rates found in the groups treated with the test article compared to the negative and solvent controls it can be concluded that no relevant increase of gene mutations was observed. The test article did not induce a reproducible concentration-related increase in mutant colony numbers. The mutant values of the groups treated with the test article were in the range of the negative controls.

In this study in both experiments (with and without S9 mix) the range of the negative and solvent controls was from 7.7 up to 22.9 mutants per 106 cells; the range of the groups treated with the test article was from 1.6 up to 29.2 mutants per 106 cells.

EMS(0.6 mg/ml) and DMBA (3.85 µg/ml) were used as positive controls and showed a distinct increase in induced mutant colonies.

Conclusions:

In conclusion, it can be stated that in this mutagenicity assay and under the experimental conditions reported the test article did not induce gene mutations at the HGPRT locus in V79 cells.

Executive summary:

In conclusion, it can be stated that in this mutagenicity assay and under the experimental conditions reported the test article did not induce gene mutations at the HGPRT locus in V79 cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No in vivo studies are required as all in vitro assays are negative.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

All in vitro data are conclusive but not sufficient for classification.