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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name: 3,4-Epoxycyclohexylmethyl methacrylate
- Chemical Name: 2-Propenoic acid, 2-methyl-7-oxabicyclo[4.1.0]hept-3-ylmethyl ester
- CAS No.: 82428-30-6
- Physical State / Colour: liquid / light yellow, transparent
- Density: 1.079 g/mL at 20 °C
- Molecular Weight: 196.2 g/mol

Method

Target gene:
HPRT-Locus
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
EXPERIMENT I:
- without metabolic activation: 0.25, 0.50, 1.00, 1.25, 1.5, 1.6, 1.7 and 1.8 mM
- with metabolic activation: 0.05, 0.1, 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 mM
EXPERIMENT II:
- without metabolic activation: 0.05, 0.10, 0.25, 0.5, 1.0, 1.2, 1.4 and 1.6 mM
- with metabolic activation: 2.8, 4.0, 4.4, 4.8, 5.2, 5.4, 5.6 and 5.8 mM
Vehicle / solvent:
No vehicle, test item was dissolved in culture medium.
Controls
Untreated negative controls:
yes
Remarks:
treatment medium
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
V79 cells in vitro have been widely used to examine the ability of chemicals to induce cytogenetic changes and thus identify potential carcinogens or mutagens. These cells are characterized by their high proliferation rate (12 - 14 h doubling time of the BSL BIOSERVICE stock cultures) and their high cloning efficiency of untreated cells, usually more than 50%. These facts are necessary for the appropriate performance of the study.
The V79 cells (ATCC, CCL-93) were stored over liquid nitrogen (vapour phase) in the cell bank of BSL BIOSERVICE. This allows the repeated use of the same cell culture batch in experiments. Each cell batch was routinely checked for mycoplasma infections (PCR). Thawed stock cultures were maintained in plastic culture flasks in minimal essential medium (MEM).
For purifying the cell population of pre-existing HPRT mutants cells were exposed to HAT medium containing 100 μM hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine and 10.0 μM glycine for several cell doublings (2-3 days).
Evaluation criteria:
ACCEPTABILITY OF THE ASSAY:
A mutation assay is considered acceptable if it meets the following criteria:
- Negative and/or solvent controls fall within the performing laboratories historical control data range;
- The absolute cloning efficiency: ([number of positive cultures x 100] /total number of seeded cultures) of the negative and /or solvent controls is > 50%;
- The positive controls (EMS and DMBA) induce significant increases (at least 3-fold increase of mutant frequencies related to the comparable negative control values and higher than the historical range of negative controls) in the mutant frequencies.


EVALUATION:
A test is considered to be negative if there is no biological relevant increase in the number of mutants.
There are several criteria for determining a positive result:
- a reproducible three times higher mutation frequency than the solvent control for at least one of the concentrations;
- a concentration related increase of the mutation frequency; such an evaluation may be considered also in the case that a three-fold increase of the mutant frequency is not observed;
- if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.
Statistics:
According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

PRECIPITATION

No precipitation of the test item was noted in the experiments.

TOXICITY

Biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation.

In experiment I without metabolic activation the relative growth was 17.3 and 21.4% for the two highest concentrations (1.7 and 1.8 mM) evaluated. The highest concentration evaluated with metabolic activation was 5 mM with a relative growth of 17.3 %.

In experiment II without metabolic activation the relative growth was 12.9 and 9.0% for the two highest concentrations (1.4 and 1.6 mM) tested. The highest concentration was not considered for evaluation of results, as the cytotoxicity was below the requested cytotoxicity frame of 10-20% cell survival. The highest concentrations evaluated with metabolic activation were 5.6 and 5.8 mM with a relative growth of 21.7 and 9.8%. Here the requested cytotoxicity frame of 10-20% cell survival was not reached, however, this cytotoxicity range was framed very closely by the two highest concentrations.

MUTAGENICITY

In experiment I without metabolic activation most mutant values of the negative controls and test item concentrations were within the historical control data of the test facility BSL BIOSERVICE (about 5-43 mutants per 106 cells). No dose-response relationship could be observed. The mutation frequency found in the highest dose group showed a slight increase, however, as the threshold of 3 was not reached the increase was not considered as biologically relevant.

Mutation frequencies with the negative control were found to be 27.76 and 17.47 mutants/106 cells and in the range of 18.24 to 55.56 mutants/106 cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 2.46 was found at a concentration of 1.8 mM with a relative growth of 21.4%.

With metabolic activation most mutant values of the negative controls and test item concentrations were within the historical control data of the test facility BSL BIOSERVICE (about 5-44 mutants per 106 cells). No dose-response relationship could be observed. The mutation frequency found in the highest dose group showed a slight increase, however, as the threshold of 3 was not reached the increase was not considered as biologically relevant.

Mutation frequencies of the negative control were found to be 21.51 and 35.65 mutants/106 cells and in the range of 16.94 to 73.08 mutants/106 cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 2.56 was found at a concentration of 5 mM with a relative growth of 17.3%.

In experiment II without metabolic activation most mutant values of the negative controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 5-43 mutants per 106 cells). No dose-response relationship could be observed. The mutation frequency found in the highest dose group did show a slight increase. However, this effect was not considered as biologically relevant, as the toxicity of this highest dose group was below the requested toxicity frame of 10-20%.

Mutation frequencies of the negative control were found to be 19.68 and 24.49 mutants/106 cells and in the range of 13.23 to 49.48 mutants/106 cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 2.24 was found at a concentration of 1.6 mM with a relative growth of 9.0%.

In experiment II with metabolic activation the mutant values of the negative controls found were within the historical control data of the test facility BSL BIOSERVICE (about 5-44 mutants per 106 cells). Mutation frequencies of the negative controls were found tobe 33.01 and 23.08 mutants/106

cells and in the range of 23.08 to 113.73 mutants/106 cells for the test item, respectively.

The mutation frequencies found in the dose groups treated with the test item showed a biologically relevant increase compared to the negative controls. At concentrations of 4.8 mM (90.49 mutants per 106 cells) and 5.4 mM (113.73 mutants per 106 cells) the threshold of 3 was exceeded with mutant frequencies of 3.23 and 4.06, respectively. In the dose group 5.8 mM the threshold of 3 was nearly reached with a mutation frequency of 2.93 (82.22 mutants per 106 cells). Additionally, in all dose groups from concentrations of 4.0 mM and higher the historical range of the negative controls was exceeded with mutant frequencies between 44.52 and 113.73.

Due to the increase in mutant frequency and the concentration related increase in experiment II with metabolic activation, the detected effect is considered as biologically relevant.

DMBA (0.8 and 1.0 μg/mL) and EMS (300 μg/mL) were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

EXPERIMENT I without metabolic activation

 dose group [mM]  cell density [cells/mL]  rel. growth [%]  CE [%]  mean mutant colonies  mutant colonies / 10E6 cells  MF
 NC 1  1120000  100  79  8.8  27.76  -
 NC 2  1110000  100  83  5.8  17.47  -
 0.25  1340000  120.2  72  11.8 41.11  1.82
 0.50  1240000 111.2   77  7.2  23.38  1.03
 1.00  1100000  98.7  77  5.6  18.24  0.81
 1.25  999000  89.6  76  7.8  25.66  1.13
 1.5  565000  50.7  77  12.0  39.22  1.73
 1.6  433000  38.8  72 6.0 20.91  0.92
 1.7  193000  17.3  79  9.0  28.39  1.26
 1.8  239000  21.4  68  15.0  55.56  2.46
 EMS 300 µg/mL  1140000  102.2  68  68.8  254.81  11.27

EXPERIMENT I with metabolic activation

 dose group [mM]  cell density [cells/mL]  rel. growth [%]  CE [%]  mean mutant colonies  mutant colonies / 10E6 cells  MF
 NC 1  1390000  100  86  7.4  21.51  -
 NC 2  1430000  100  83  11.8  35.65  -
 0.05  1440000  102.1  60  5.4  22.41  0.78
 0.1  1470000 104.3  85  10.8  31.62  1.11
 0.5  1460000  103.5  92  6.2  16.94  0.59
 1.0  1260000  89.4  81  13.0  40.25  1.41
 2.0  1270000  90.1  79  12.2 38.61  1.35
 3.0  1280000  90.8  75  14.6 48.50  1.70
 4.0  1160000  82.3  79  10.8  34.29  1.20

5.0

 244000

 17.3

 78

 22.8

73.08

 2.56

 DMBA 1 µg/mL  1170000  83.0  68  88.8  327.68  11.46

EXPERIMENT II without metabolic activation

 dose group [mM]  cell density [cells/mL]  rel. growth [%]  CE [%]  mean mutant colonies  mutant colonies / 10E6 cells  MF
 NC 1  2190000  100  94  7.4  19.68  -
 NC 2  2160000  100  86  8.4  24.49  -
 0.05  1900000  87.4  85  6.2  18.18  0.82
 0.10  1970000 90.6  98  5.2  13.23  0.60
 0.25  1630000  74.9  85  7.4  21.89  0.99
 0.5  1310000  60.2  79  13.6  43.31  1.96
 1.0  631000  29.0  72 10.8  37.63  1.70
 1.2  446000  20.5  80  14.8 46.54  2.11
 1.4  280000  12.9  73  6.0  20.55  0.93
 1.6  195000  9.0  72  14.2  49.48  2.24
 EMS 300 µg/mL  1260000  57.9  73  229.2  790.34  35.79

EXPERIMENT II with metabolic activation

 dose group [mM]  cell density [cells/mL]  rel. growth [%]  CE [%]  mean mutant colonies  mutant colonies / 10E6 cells  MF
 NC 1  1370000  100  77  10.2  33.01  -
 NC 2  1380000  100  78  7.2  23.08  -
 2.8  1310000  95.3  78  7.2  23.08  0.82
 4.0  926000 67.3  71  12.6  44.52  1.59
 4.4  843000  61.3  73 14.6  50.00  1.78
 4.8  803000  58.4  66  23.8  90.49  3.23
 5.2  525000  38.2  68  16.4  60.74  2.17
 5.4  440000  32.0  64 29.0  113.73  4.06
 5.6  298000  21.7  67  14.0  52.43  1.87
 5.8  135000  9.8  68 22.2  82.22  2.93
DMBA 0.8 µg/mL  1370000  99.6  77  48.8 158.96  5.67
 DMBA 1 µg/mL  1330000  96.7  68  70.2  257.14  9.17

Applicant's summary and conclusion

Conclusions:
In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item 3,4-Epoxycyclohexylmethyl methacrylate is considered to be mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.
Executive summary:

The test item 3,4-Epoxycyclohexylmethyl methacrylate was assessed for its potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster.

The selection of the concentrations was based on data from the pre-experiments. Experiment I with and without metabolic activation and experiment II with metabolic activation were performed as a 4 h short-term exposure assay. Experiment II without metabolic activation was performed as 20 h long time exposure assay.

The test item was investigated at the following concentrations:

Experiment I

- without metabolic activation: 0.25, 0.50, 1.00, 1.25, 1.5, 1.6, 1.7 and 1.8 mM

- with metabolic activation: 0.05, 0.1, 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 mM

Experiment II

- without metabolic activation: 0.05, 0.10, 0.25, 0.5, 1.0, 1.2, 1.4 and 1.6 mM

- with metabolic activation: 2.8, 4.0, 4.4, 4.8, 5.2, 5.4, 5.6 and 5.8 mM

No precipitation of the test item was noted in the experiments.

Biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation.

In experiment I without metabolic activation the relative growth was 17.3 and 21.4% for the two highest concentrations (1.7 and 1.8 mM) evaluated. The highest concentration evaluated with metabolic activation was 5 mM with a relative growth of 17.3 %.

In experiment II without metabolic activation the relative growth was 12.9 and 9.0% for the two highest concentrations (1.4 and 1.6 mM) tested. The highest concentration was not considered for evaluation of results, as the cytotoxicity was below the requested cytotoxicity frame of 10-20% cell survival. The highest concentrations evaluated with metabolic activation were 5.6 and 5.8 mM with a relative growth of 21.7 and 9.8%. Here the requested cytotoxicity frame of 10-20% cell survival was not reached, however, this cytotoxicity range was framed very closely by the two highest concentrations.

A biologically relevant increase of mutants was found after treatment with the test item in experiment II with metabolic activation. Moreover, a dose-response relationship was observed.

DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item 3,4-Epoxycyclohexylmethyl methacrylate is considered to be mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.