Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Cyclohexylmethacrylate showed no genotoxic effect in the Ames-test, in the cytogenetic test with human lymphocytes and in the HGPRT-Test with V79 cells.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: In Vitro Mammalian Chromosome Aberration Test
Specific details on test material used for the study:
- Lot/batch No.: 12-208-222
Species / strain / cell type:
lymphocytes: human
Additional strain / cell type characteristics:
not applicable
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
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9 mix (0.30 mg/mL)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9 mix (30 µg/mL)
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 the chi-square test. Evaluation was performed only for cells carrying aberrations exclusive gaps.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

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

 

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: In Vitro Mammalian Cell Gene Mutation Test
Specific details on test material used for the study:
- Lot/batch No.:12-208-22
Target gene:
hypoxanthine-guanine phosphoribosyl transerase
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Additional strain / cell type characteristics:
not applicable
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** µg/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
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9 mix (4.8 mM)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with S9 mix (15.0 µM)
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

NUMBER OF REPLICATIONS: 2

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.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
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-FINDING/SCREENING STUDIES:
The pre-test was performed in order to determine the concentration range for the mutagenicity experiments. The pre-test was performed with eight concentrations ranging from 1.0 - 2000 µg/mL.

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 the 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 sub cultivation). 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 sub cultivation 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 sub cultivation, 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 10^6 cells; the range of the groups treated with the test article was from 1.6 up to 29.2 mutants per 10^6 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.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
and OECD Guideline 472
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Lot/batch No.: 12-208-222
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2
Details on mammalian cell type (if applicable):
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix prepared from Sprague Dawlay rat livers after Aroclor 1254 activation.
Test concentrations with justification for top dose:
33.3; 100.0; 333.3; 1000.0; 2500.0; 5000.0 µg/plate with and without S9 mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity for the bacteria.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9 mix (all strains
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9 mix (TA 1535 and TA 100)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
without S9 mix (TA 1537 and TA 98)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 mix (WP2 uvrA and WP 2)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3

OTHER:
The following materials were mixed in a test tube and poured onto the minimal agar plates:
100 µL Test solution at each dose level, solvent control, negative control, or reference mutagen solution (positive control)
500 µL S9 mix (for test with metabolic activation) or S9 mix substitution-buffer (for test without metabolic activation)
100 µL Bacteria suspension (cf. test system, pre-culture of the strains)
2000 µL Overlay agar
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100
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
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Slight toxic effects evidenced by a reduction in the number of revertants were observed in strain TA 100 at 2500.0 and 5000.0 µg/plate in the presence of exogenic metabolic activation in experiment I.
No substantial increases in revertant colony numbers of any of the six tester strains were observed following treatment with Cyclohexylmethacrylate at any dose level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing conentrations in the range below the generally acknowledged border of significance.
Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Without S9 mix

Revertants/plate mean from three plates

 

Dose µg/plate

TA 1535

TA 1537

TA 98

TA 100

WP2

WP2 uvrA

I

II

I

II

I

II

I

II

I

II

I

II

Neg. control

14

27

7

12

20

27

80

78

39

42

41

39

Solv. Control

17

24

8

14

16

27

84

79

44

41

47

37

33.3

15

26

6

15

15

23

75

78

36

36

38

42

100.0

14

21

6

17

18

25

69

71

42

39

35

35

333.0

16

18

6

15

19

22

76

68

41

33

43

37

1000.0

14

20

7

15

16

28

75

75

35

34

33

31

2500.0

13

23

9

11

19

21

79

67

35

26

42

34

5000.0

14

29

9

13

15

25

80

86

39

36

32

35

Positive Controls

Sodium azide (10µg/plate)

1187

1185

 

 

 

 

1167

1210

 

 

 

 

4-Nitropo-phenylene-diamine ( 50µg/plate)

 

 

309

283

2553

1795

 

 

 

 

 

 

Methylmethane sulfonate (10µl/plate)

 

 

 

 

 

 

 

 

753

489

766

373

With S9 mix

Revertants/plate mean from three plates

 

Dose µg/plate

TA 1535

TA 1537

TA 98

TA 100

WP2

WP2 uvrA

I

II

I

II

I

II

I

II

I

II

I

II

Neg. control

13

20

15

17

41

36

83

93

53

50

55

44

Solv. Control

18

23

11

18

31

43

81

79

56

53

46

49

33.3

20

24

9

16

30

39

95

86

53

147

45

59

100.0

22

21

12

17

31

38

88

99

53

52

52

52

333.3

16

25

13

19

32

43

92

83

42

55

48

41

1000.0

24

29

9

20

24

36

87

80

63

54

53

43

2500.0

18

20

9

20

24

34

49

76

51

55

58

31

5000.0

15

20

8

20

32

28

48

68

62

36

51

36

Positive Controls

2-Amino-anthracene (2.5µg/plate)

142

119

145

65

474

474

790

445

364

250

386

349

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce point mutations by base pair changes or frameshifts in the genome of the strains used.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In-vitro study: Bacterial system (AMES)

Cyclohexylmethacrylate was tested in the Salmonella reverse mutation assay with Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA in concentrations between 33.3 – 5000 µg/plate (Evonik Roehm,1993). Slight toxic effects evidenced by a reduction in the number of revertants were observed in strain TA 100 at 2500.0 and 5000.0 µg/plate in the presence of exogenic metabolic activation. No substantial increases in revertant colony numbers of any of the six tester strains were observed following treatment with Cyclohexylmethacrylate at any dose level, either in the presence or absence of metabolic activation (S9 mix). Cyclohexylmethacrylate did not induce point mutations by base pair changes or frame shifts in the genome of the strains used.

In-vitro study: chromosomal aberration test

Cyclohexylmethacrylate was tested in a chromosome aberration assay in human lymphocytes in concentrations between 10 – 1680 µg/mL (Evonik Roehm, 1993).

In both experiments, with the top concentration at interval 24 h (without S9 mix at both experiment and with S9 mix at exp. II), the mitotic indices were at least slightly reduced indication that Cyclohexylmethacrylate had cytotoxic properties. In exp. II, an additional indication of the potential of the test article to induce cell cycle delay or cytotoxicity was given by analyzing the proportion of mitotic cells in their 1st, 2ndand 3rdmetaphase for some treatment groups and calculate the proliferation index. With 100 and 150 µg/mL (in the absence of S9 mix) the proliferation index was distinctly reduced as compared to the corresponding solvent controls. In the presence of S9 mix no biologically relevant differences were observed between the solvent controls and the test group treated with the highest concentration (1680 µg/mL). In both experiments, with test article Cyclohexylmethacrylate no biologically relevant increase in the structural chromosomal aberration rate could be found when compared with the range of aberrations in the corresponding negative and solvent controls. These results were obtained at each fixation interval both in the absence and presence of S9 mix. Cyclohexylmethacrylate did not induce structural chromosome aberrations in human lymphocytes in vitro when tested up to 10 mM (with S9 mix) and cytotoxic concentrations (without S9 mix).

In-vitro study: Mammalian cell gene mutation test

Cyclohexylmethacrylate was tested in a HGPRT test with human lymphocytes in concentrations between 3.0 – 3000.0 µg/mL (Evonik Roehm, 1993).

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 sub cultivation 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 sub cultivation). 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 sub cultivation 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 sub cultivation, 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. Cyclohexylmethacrylate did not induce gene mutations at the HGPRT locus in V79 cells.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available data are reliable and suitable for classification purposes under Regulation 1272/2008. The test substance was determined to be non-mutagenic and non-clastogenic. As a result the substance is not considered to be classified for repeated dose toxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.