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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
The in vitro assay was performed to assess the potential of the test item to induce primary DNA-breakage in individual cells.
One possibility to detect primary DNA-breakage is to perform the single cell gel electrophoresis, also known as the comet assay.
The comet assay is a sensitive and fast method for measuring and analysing DNA breakage in individual cells. The alkaline version of the comet assay enables the demonstration of single- and double strand breaks, as well as alkali-labile sites.
GLP compliance:
no
Type of assay:
single cell gel/comet assay in mammalian cells for detection of DNA damage

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Species / strain
Species / strain:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell lines (if applicable):
- Source of cells: Laboratory for mutagenicity testing, Technical University Darmstadt)
- Cells were stored in liquid nitrogen in the cell bank allowing rhe repeated use of the same cell culture batch in experiments. Before freezing, each batch was screened for mycoplasm contamination and checked for karyotype stability. Consequently, the parameters of the experiments remain similar because of standardised characteristics of the cells.
Thawed stock cultures were propagated at 37°C in 80 cm² plastic flasks. About 5 X 10^5 cells per flask were seeded into 15 ml of MEM (Minimal Essential Medium) supplemented with 10% fetal calf serum. The cells were subcultured twice weekly. The cell cultures were incubated at 37°C in a humidified atmosphere
Metabolic activation:
with and without
Metabolic activation system:
liver rat
Test concentrations with justification for top dose:
Results of the preliminary study: With S9 a XTT50 value could no be determined as viability was not reduced below 80% even at the highest concentration of the test item. Without S9, 80µg/ml was determined as XTT50 value. The number of cells was significantly reduced at concentrations above 78.1 µg/ml without S9. Therefore, in the comet assay, 1250 µg/ml was used as the maximum concentration of the test item with S9, and 78 µg/ml without S9.
Vehicle:
DMSO
Controls
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
methylmethanesulfonate
Details on test system and conditions:
The comet assay was performed with 12 test groups per study: 6 test groups without S9 (1 negative control, 1 solvent control, 1 positive control, 3 concentrations of the test item) , and 6 test groups with S9. The treatment media did not contain FCS.
Seeding of the cultures: individual wells of a 6-well tissue-culture plate were inoculated with 3.0 ml medium containing 2 x 10^5 V79 cells. The medium was MEM + 10% FCS. The plates were inculated for 24 hours to enable cellular attachment.
Treatment: The medium was removed and 3 ml reatment medium of the different test groups was added to the cells. The cells were incubated for 2 hours at 37°C in a humidified atmosphere with 4.5% CO2.
Preparation of microscopic slides: After the treatment, the cells were trypsinised. The trypsin reaction was stopped by supplementation of culture medium containing 10% FCS. The final volume of the resulting single cell suspension of one tes group was 100 µl. Two slides per test group were prepared with 10 µl of the cell suspension and 90 µl of 0.5 % agarose each. The slides were then coded on ice, before an additional layer of 100 µl 0.5% agarose was layed on the embedded cells. 25 µl of the same cell suspension of each test group were used to perform a fluorochrome mediated cell viability assay with fluorescein diacetat and ethidium bromide. This viability test was to ensure that later effects in the comet assay could not be induced by cytotoxicity after treatment with the test item.
Lysis, alkaline treatment, electrophoresis, neutralization and drying.

EVALUATION OF THE RESULTS
The DNA of the cells was stained with the fluorescence dye ethidium bromide directly before evaluation.
50 cells per slide, 100 cells per test group, were evaluated with a fluorescence microscope and the "tailmoments" of each cell were measured and recorded by an image analysis programme.
An increasing number of single strand breaks detected with the comet assay results in an increase of the mean of tailmoment of one test group compared to the solvent control.
A relative increase of more than 2 x mean of tailmoment of solvent control shows an increase of the genotoxic potential of the test item at the tested concentration.

PRE-TEST OF TOXICITY:
A pre-test on cell viability with 2h treatment was performed in order to determine the cytotoxicity of the test items and to find appropriate testing concentrations for the comet assay. The general experimental conditions in the pre-test were the same as described below for the main experiment.
With the XTT test, cell proliferation and viability as a resul of hte mitochondrial metabolic competence of the cells after treatment with the test material is determined colorimetrically.
The XTT test is based on the cleavage of the yellow tetrazolium salt XTT toform an orange water soluble formazan dye by deshydrogenase activity in active mitochondria.
Nine concentrations of the test item were tested in the XTT-test with and without S9: 4.755-9.51-19.02-39.05-78.1-156.25-312.5-625-1250 µg/ml.



Rationale for test conditions:
Dose selection: Three concentrations of the test item were chosen in the non-cytotoxic concentration range. The test item was tested in the comet assy at the following concentrations :
19.02-39.05-78.1 µg/ml without S9, and 312.5-625-1250 µg/ml with S9 mix.
Evaluation criteria:
An increasing number of single strand breaks detected with the comet assay results in an increase of the mean of tailmoment of one test group compared to the solvent control.
A relative increase of more than 2 x mean of tailmoment of solvent control shows an increase of the genotoxic potential of the test item at the tested concentration.
Statistics:
no

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
Negative and positive controls were all in the range to ensure a correct performance of the comet assay.
The parallel performed FDA-test for cell viability showed no reduction in cell viability in all test groups with and without metabolic activation.

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that in this study and under the reported experimental conditions, the test item did not induce primary DNA-breakage in V79 cells with and without metabolic activation.
Executive summary:

The tesst item Tricyclodecane-dimethynole-dimethacrylate was assessed for it potential to induce primary DNA-breakage according to the single cell gel electrophoresis (comet assay) with Chinese hamster V79 -cells. The assay was performed with and without liver microsomal activation. Negative and positive controls were all in the range to ensure a correct performance of the comet assay. The parallel performed FDA-test for cell viability showed no reduction in cell viability in all test groups with and without metabolic activation. No increase of the mean tailmoment was observed following treatment with the test item at the three tested doses with and without metabolic activation.

In conclusion, it can be stated that in this study and under the reported experimental conditions, the test item did not induce primary DNA-breakage in V79 cells with and without metabolic activation.