Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the all three in vitro studies (Ames test, Comet assay, micronucleus assay) available on tricyclodecane dimethanol dimethacrylate or on an analogue substance, tricyclodecane dimethanol dimethacrylate is considered to be not mutagenic.

Link to relevant study records

Referenceopen allclose all

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
Reference:
Composition 1
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 information:
Composition 1
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
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
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.
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.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 February 2014 -- 26 May 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
other: OECD Guideline 487 (In vitro micronucleus)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Test material information:
Composition 1
Target gene:
Not applicable (not a gene mutation assay).
Species / strain:
other: mouse lymphoma L5178Y TK+/- cells
Details on mammalian cell lines (if applicable):
- Type and identity of media: RPMI 1640 medium containing 10% inactivated horse serum, L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
With a treatment volume of 1% (v/v) in culture medium, the dose-levels used for treatment were as follows:
- 0.0008, 0.0016, 0.003, 0.006, 0.013, 0.025, 0.05, 0.1 mM for the first experiment without S9mix,
- 0.00005, 0.0001, 0.0004, 0.001, 0.004, 0.011, 0.03, 0.1 mM for the second experiment without S9mix,
- 0.003, 0.006, 0.013, 0.025, 0.05, 0.1, 0.2 and 0.4 mM with S9mix.
Vehicle:
- Vehicle used: dimethylsulfoxide (DMSO), batch Nos. 1488745V and K44917950345.
- Justification for choice: Based on available solubility data, the test item was dissolved in DMSO at 304 mg/mL. Therefore, using this stock solution at 304 mg/mL and a treatment volume of 1% (v/v) in culture medium, the highest recommended dose-level of 10 mM (corresponding to 3040 µg/mL) was achievable.
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: mitomycin C, colchicine (-S9 mix); cyclophosphamide (+S9 mix)
Details on test system and conditions:
METHOD OF APPLICATION: in culture medium

DURATION

First experiment: 3 h treatment + 24 h recovery (with and without S9mix)
Second experiment: 3 h treatment + 24 h recovery (with S9mix); 24 h treatment + 20 h recovery (without S9mix)
NUMBER OF CELLS EVALUATED: 2000 mononucleated cells per dose

DETERMINATION OF CYTOTOXICITY
- Method: population doubling
Evaluation criteria:
A test item is considered to have clastogenic and/or aneugenic potential, if all the following criteria were met:
- a dose-related increase in the frequency of micronucleated cells was observed,
- for at least one dose-level, the frequency of micronucleated cells of each replicate culture was above the corresponding vehicle historical range,
- a statistically significant difference in comparison to the corresponding vehicle control was obtained at one or more dose-levels.

The biological relevance of the results was considered first.

Evaluation of a negative response: a test item is considered negative if none of the criteria for a positive response were met.
Statistics:
no
Species / strain:
other: mouse lymphoma L5178Y TK+/- cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
The mean population doubling and the mean frequencies of micronucleated cells for the vehicle controls were as specified in the acceptance criteria. Also, positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells. The study was therefore considered to be valid.

The test item was found to be toxic and poorly soluble in the culture medium in the preliminary test. Since the cytotoxicity was the limiting factor for each dose-level tested, the highest dose-level selected for the main experiments was based on the level of toxicity, according to the criteria specified in the international regulations.
Experiments without S9 mix : No precipitate was observed in the culture medium at the end of the treatment periods. Following the 3-hour treatment, a moderate to strong toxicity was induced at dose-levels = 0.006 mM as shown by a 57 to 100% decrease in the PD. Following the 24-hour treatment, a moderate to strong toxicity was induced at dose-levels = 0.03 mM as shown by a 40 to 100% decrease in the PD.
No increase in the frequency of micronucleated cells was noted after the 3-hour treatment. After the 24-hour treatment, some increases in the frequency of micronucleated cells were noted at the three dose-levels analyzed. However, these increases were neither dose-related nor statistically significant. Moreover, the frequencies of micronucleated cells of each replicate culture remained within the corresponding vehicle historical range.
 
Experiments with S9 mix: A slight to moderate emulsion (or precipitate) was observed at the end of the treatment periods, at dose-levels = 0.2 mM in both experiments. In the first experiment, a slight to strong toxicity was induced at dose-levels of 0.006 mM, 0.025 mM and at dose-levels = 0.1 mM as shown by 33 to 100% decreases in the PD. In the second experiment, a slight to strong toxicity was induced at dose-levels = 0.2 mM as shown by a 34 to 78% decrease in the PD.
No increase in the frequency of micronucleated cells was noted in the first experiment. In the second experiment, some increases in the frequency of micronucleated cells were noted at 0.05, 0.1 and 0.2 mM. These increases were not statistically significant. Moreover, the corresponding micronucleated cells frequencies remained within the historical data range for the vehicle control. Consequently, this increase did not meet the criteria for a positive response and was considered as non-biologically relevant.

Under the experimental conditions of the study, the test item did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the absence or in the presence of a rat liver metabolising system.
Conclusions:
Under the experimental conditions of the study, the test item did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the absence or in the presence of a rat liver metabolising system.
Executive summary:

The objective of this study was to evaluate the potential of the test item to induce an increase in the frequency of micronucleated cellsin the mouse cell line L5178Y TK+/-. This study was conducted in compliance with the OECD Guideline No. 487 and the principles of Good Laboratory Practices.

After a preliminary toxicity test, the test item was tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254, as follows:

-First experiment: 3 h treatment + 24 h recovery (without and with S9 mix),

-Second experiment : 24 h treatment + 20 h recovery (without S9 mix), and 3 h treatment + 24 h recovery (with S9 mix).

Each treatment was coupled to an assessment of cytotoxicity at the same dose-levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells and quality of the cells on the slides has also been taken into account.

The test item was dissolved in dimethylsulfoxide (DMSO).

 

The mean population doubling and the mean frequencies of micronucleated cells for the vehicle controls were as specified in the acceptance criteria. Also, positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells. The study was therefore considered to be valid.

The test item was found to be toxic and poorly soluble in the culture medium in the preliminary test. Since the cytotoxicity was the limiting factor for each dose-level tested, the highest dose-level selected for the main experiments was based on the level of toxicity, according to the criteria specified in the international regulations.

Experiments without S9 mix : No precipitate was observed in the culture medium at the end of the treatment periods. Following the 3-hour treatment, a moderate to strong toxicity was induced at dose-levels = 0.006 mM as shown by a 57 to 100% decrease in the PD. Following the 24-hour treatment, a moderate to strong toxicity was induced at dose-levels = 0.03 mM as shown by a 40 to 100% decrease in the PD.

No increase in the frequency of micronucleated cells was noted after the 3-hour treatment. After the 24-hour treatment, some increases in the frequency of micronucleated cells were noted at the three dose-levels analyzed. However, these increases were neither dose-related nor statistically significant. Moreover, the frequencies of micronucleated cells of each replicate culture remained within the corresponding vehicle historical range.

 

Experiments with S9 mix: A slight to moderate emulsion (or precipitate) was observed at the end of the treatment periods, at dose-levels = 0.2 mM in both experiments. In the first experiment, a slight to strong toxicity was induced at dose-levels of 0.006 mM, 0.025 mM and at dose-levels = 0.1 mM as shown by 33 to 100% decreases in the PD. In the second experiment, a slight to strong toxicity was induced at dose-levels = 0.2 mM as shown by a 34 to 78% decrease in the PD.

No increase in the frequency of micronucleated cells was noted in the first experiment. In the second experiment, some increases in the frequency of micronucleated cells were noted at 0.05, 0.1 and 0.2 mM. These increases were not statistically significant. Moreover, the corresponding micronucleated cells frequencies remained within the historical data range for the vehicle control. Consequently, this increase did not meet the criteria for a positive response and was considered as non-biologically relevant.

Under the experimental conditions of the study, the test item did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the absence or in the presence of a rat liver metabolising system.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September to December 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
n/a
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
liver rat
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
liver rat
Test concentrations with justification for top dose:
Since the test item was freely soluble in the vehicle and non-toxic in the preliminary test, the highest dose-level was 5000 µg/plate, according to the criteria specified in the international guidelines.
The selected treatment-level were 312.5 - 625 - 1250 - 2500 - 5000 µg/plate
Vehicle:
DMSO
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: 2-antramine: all strains with S9
Details on test system and conditions:
The test item was dissolved in the vehicle at a concentration of 50 mg/mL for the preliminary toxicity test and for both mutagenicity experiments. The preparations were made immediately before use.
METHOD OF APPLICATION: both experiments : direct plate incorporation method expect the second test with S9 mix (pre-incubation method).
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY: The evalutation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.
PRE-EXPERIMENT FOR TOXICITY: To assess the toxicity of the test item to the bacteria, 6 dose-levels (one plate per dose level) were tested in the TA98, TA100, TA102 and WP2 uvrA strains, with and without S9 mix.
Rationale for test conditions:
Acceptance criteria: The study is considered valid if the number of revertants in the veicule controls is consistent with the historical data of the testing facility, and if the number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with our historical data.
Evaluation criteria:
A reproducible 2-fold increase (for the TA98, TA100, TA102 and WP2 uvrA strains) or 3-fold increase (for the TA1535 and TA1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.
Statistics:
not required
Key result
Species / strain:
other: TA98, TA100, TA102, TA1535, TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Additional information on results:
The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria.
A moderate to marked emulsion was observed in the Petri plates when scoring the revertants at dose-lvels > 1250 µg/plate. No noteworthy toxicity was noted towards all the strains used, both with and without S9 mix. The test item did not induce any noteworthy increase in the number of revertants, both with and wothout S9 mix, in any of the six strains.

Table 1 : First experiment / Direct plate incorporation method

Strain

dose

S9 ?

Mean revertant colony counts

SD

Ratio T/C

Indiv data

TA1535

0

-

18

5

 

20-13-22

 

312.5

-

19

6

1.1

13-23-22

 

625

-

18

3

1.0

16-22-17

 

1250

-

18

7

1.0

11Me-25Me-18Me

 

2500

-

12

4

0.7

16Me-8Me-13Me

 

5000

-

15

2

0.8

13Se-16Se-17Se

 

PC

-

703

12

38.4

690-707-713

TA1535

0

+

13

8

 

22-8-8

 

312.5

+

18

10

1.4

29-10-16

 

625

+

15

2

1.2

16-13-16

 

1250

+

10

6

0.8

16Me-4Me-11Me

 

2500

+

15

10

1.2

12Me-26Me-6Me

 

5000

+

24

3

1.9

25Me-20Me-26Me

 

PC

+

319

9

252

311-328-319

TA1537

0

-

9

5

 

4-14-8

 

312.5

-

4

2

0.4

5-4-2

 

625

-

6

1

0.7

6-5-6

 

1250

-

5

3

0.6

7Me-2Me-7Me

 

2500

-

5

3

0.6

8Me-5Me-2Me

 

5000

-

4

5

0.5

2Se-1Se-10Se

 

PC

-

462

119

53.3

595-426-366

TA1537

0

+

6

2

 

8-6-5

 

312.5

+

8

4

1.3

10-11-4

 

625

+

10

3

1.6

13-7-11

 

1250

+

7

4

1.1

11Me-6Me-3Me

 

2500

+

5

1

0.8

6Me-4Me-6Me

 

5000

+

6

2

0.9

8Me-6Me-4Me

 

PC

+

63

8

9.9

72-60-56

TA98

0

-

19

6

 

14-19-25

 

312.5

-

18

2

0.9

17-16-20

 

625

-

17

3

0.9

18-19-14

 

1250

-

16

8

0.8

23Me-17Me-7Me

 

2500

-

15

4

0.8

14Me-12Me-19Me

 

5000

-

19

8

1.0

18Se-12Se-28Se

 

PC

-

158

20

8.2

178-158-139

TA98

0

+

25

7

 

17-28-29

 

312.5

+

36

9

1.5

26-41-41

 

625

+

33

7

1.3

40-26-32

 

1250

+

24

7

1.0

19Me-22Me-32Me

 

2500

+

26

4

1.1

30Me-25Me-23Me

 

5000

+

30

12

1.2

23Me-23Me-44Me

 

PC

+

1140

129

46.2

993-1234-1194

TA100

0

-

107

16

 

90-121-110

 

312.5

-

100

13

0.9

114-90-95

 

625

-

102

12

1.0

102-91-114

 

1250

-

96

10

0.9

108Me-90Me-90Me

 

2500

-

106

20

1.0

107Me-125Me-86Me

 

5000

-

107

10

1.0

115Se-109Se-96Se

 

PC

-

486

19

4.5

508-478-472

TA100

0

+

110

3

 

113-111-107

 

312.5

+

110

11

1.0

117-97-115

 

625

+

103

3

0.9

101-107-101

 

1250

+

110

6

1.0

111Me-115Me103Me

 

2500

+

100

2

0.9

99Me-98Me-102Me

 

5000

+

96

8

0.9

101Me-99Me-87Me

 

PC

+

1182

132

10.7

1274-1241-1031

TA102

0

-

371

5

 

368-368-377

 

312.5

-

351

18

0.9

371-341-340

 

625

-

398

31

1.1

369-394-430

 

1250

-

381

11

1.0

387Me-388Me-369Me

 

2500

-

389

26

1.0

419Me-380Me-369Me

 

5000

-

377

22

1.0

359Me-401Me-372Me

 

PC

-

2201

98

5.9

2209-2100-2295

TA102

0

+

512

60

 

443-549-545

 

312.5

+

526

112

1.0

401-616-561

 

625

+

598

29

1.2

581-631-581

 

1250

+

496

28

1.0

484-475-528

 

2500

+

540

62

1.1

484Me-531Me-606Me

 

5000

+

448

24

0.9

432Me-475Me-436Me

 

PC

+

4256

175

8.3

4138-4173-4457

WP2 uvrA

0

-

30

6

 

34-32-23

 

312.5

-

30

11

1.0

36-17-36

 

625

-

27

5

0.9

32-23-26

 

1250

-

22

3

0.7

22Me-24Me-19Me

 

2500

-

26

2

0.9

28Me-25Me-25Me

 

5000

-

30

8

1.0

22Se-37Se-30Se

 

PC

-

623

60

21.0

692-592-586

WP2 uvrA

0

+

46

6

 

43-42-53

 

312.5

+

47

10

1.0

37-48-56

 

625

+

35

5

0.8

31-34-40

 

1250

+

42

5

0.9

38-42-47

 

2500

+

36

7

0.8

40Me-40Me-28Me

 

5000

+

33

2

0.7

34Me-30Me-34Me

 

PC

+

432

32

9.4

424-468-405

 

PC: positive control

 

Table 2 : First experiment / Direct plate incorporation method (without S9) and preincubation method (with S9)

 

Strain

dose

S9 ?

Mean revertant colony counts

SD

Ratio T/C

Indiv data

TA1535

0

-

11

5

 

10-16-6

 

312.5

-

15

8

1.4

12-14-10

 

625

-

22

3

2.0

24-19-22

 

1250

-

14

6

1.3

7Me- 18Me- 17Me

 

2500

-

20

2

1.9

19Me- 22Me- 20Me

 

5000

-

17

7

1.6

24Se- 16Se- 11Se

 

PC

-

654

40

61.3

627-649-697

TA1535

0

+

11

5

 

12-16-6

 

312.5

+

16

5

1.4

10-18-19

 

625

+

16

4

1.4

20-12-16

 

1250

+

16

2

1.4

17-18-14

 

2500

+

12

5

1.1

14Me-16Me-7Me

 

5000

+

18

6

1.6

24Me-13Me-17Me

 

PC

+

285

23

25.2

304-260-692

TA1537

0

-

9

3

 

11-5-10

 

312.5

-

8

3

0.9

11-7-6

 

625

-

10

5

1.2

14-4-12

 

1250

-

10

3

1.1

8Me-13Me-8Me

 

2500

-

8

6

1.0

10Me-13Me-2Me

 

5000

-

8

4

0.9

12Se-5Se-6Se

 

PC

-

675

115

77.8

571-799-654

TA1537

0

+

11

3

 

13-8-11

 

312.5

+

11

3

1.0

11-14-8

 

625

+

11

5

1.1

16-7-11

 

1250

+

6

1

0.6

6-6-7

 

2500

+

7

1

0.7

8Me-6Me-7Me

 

5000

+

10

3

0.9

8Me-13Me-8Me

 

PC

+

170

20

15.9

193-158-158

TA98

0

-

15

6

 

10-13-22

 

312.5

-

17

2

1.2

16-19-17

 

625

-

12

5

0.8

12-17-7

 

1250

-

13

2

0.8

13Me-14Me-11Me

 

2500

-

19

3

1.3

16Me-22Me-20Me

 

5000

-

13

3

0.9

11Se-12Se-17Se

 

PC

-

119

10

7.9

122-107-127

TA98

0

+

25

6

 

18-29-29

 

312.5

+

21

2

0.8

19-22-22

 

625

+

20

5

0.8

26-16-18

 

1250

+

22

5

0.9

16-26-24

 

2500

+

24

5

0.9

18Me-28Me-26Me

 

5000

+

26

4

1.0

23Me-25Me-31Me

 

PC

+

2377

361

93.8

2071-2776-2285

TA100

0

-

97

19

 

117-80-93

 

312.5

-

84

13

0.9

90-69-92

 

625

-

93

3

1.0

91-91-96

 

1250

-

79

12

0.8

92Me-69Me-75Me

 

2500

-

93

8

1.0

98Me-96Me-84Me

 

5000

-

106

5

1.1

101Se-108Se-110Se

 

PC

-

599

33

6.2

623-612-561

TA100

0

+

120

10

 

125-108-126

 

312.5

+

115

15

1.0

103-110-131

 

625

+

97

16

0.8

93-115-84

 

1250

+

102

8

0.9

98-98-111

 

2500

+

96

10

0.8

108Me-91Me-90Me

 

5000

+

101

15

0.8

108Me-111Me-83Me

 

PC

+

1410

418

11.8

1437-1813-979

TA102

0

-

399

54

 

356-381-459

 

312.5

-

375

100

0.9

490-325-310

 

625

-

387

16

1.0

399-393-368

 

1250

-

391

13

1.0

406Me-382Me-386Me

 

2500

-

407

18

1.0

406Me-425Me-390Me

 

5000

-

447

72

1.1

527Se-423Se-390Se

 

PC

-

1787

87

4.5

1770-1882-1710

TA102

0

+

518

28

 

550-501-502

 

312.5

+

519

42

1.0

514-479-563

 

625

+

468

30

0.9

487-484-434

 

1250

+

467

48

0.9

411-495-495

 

2500

+

430

40

0.8

426Me-472Me-393Me

 

5000

+

452

28

0.9

452Me-479Me-424Me

 

PC

+

2195

379

4.2

1773-2507-2306

WP2 uvrA

0

-

47

9

 

44-57-41

 

312.5

-

46

4

1.0

49-41-47

 

625

-

43

11

0.9

38-35-56

 

1250

-

45

11

0.9

32Me-53Me-49Me

 

2500

-

50

11

1.1

60Me-38Me-53Me

 

5000

-

35

4

0.7

31Se-37Se-38Se

 

PC

-

662

46

14.0

705-614-668

WP2 uvrA

0

+

59

5

 

61-62-53

 

312.5

+

69

4

1.2

69-65-72

 

625

+

65

3

1.1

67-67-61

 

1250

+

53

4

0.9

56-55-48

 

2500

+

55

6

0.9

62Me-53Me-50Me

 

5000

+

70

26

1.2

42Me-92Me-77Me

 

PC

+

276

77

4.7

234-364-229

 

PC: positive control

Me moderate emulsion; Se strong emulsion

 

Conclusions:
Under the experimental conditions, the test item did not show mutagenic activity in the bacterial reverse mutation test with S.typhimurium and E.coli.
Executive summary:

The objective of this study was to evaluate the potential of the test item to induce reverse mutation in Salmonella typhimurium and Escherichia coli.

A preliminary toxicity test was performed to define the dose-levels of the test item to be used for the mutagenicity study. The test item was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction of rats induced with Aroclor 1254. Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method. Five strains of bacteria Salmonella typhimurium: TA1535, TA1537, TA98, TA100 and TA102 and one strain of E.coli: WP2 uvrA were used.

Since the test item was freely soluble in the vehicle and non-toxic in the preliminary test, the highest dose-level was 5000 µg/plate, according to the criteria specified in the international guidelines. A moderate to marked emulsion was observed in the Petri plates when scoring the revertants at dose-lvels > 1250 µg/plate. No noteworthy toxicity was noted towards all the strains used, both with and without S9 mix. The test item did not induce any noteworthy increase in the number of revertants, both with and wothout S9 mix, in any of the six strains.

Under the experimental conditions, the test item did not show mutagenic activity in the bacterial reverse mutation test with S.typhimurium and E.coli.

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 studies in bacteria :

The objective of the study (Sire 2006, key study) was to evaluate the potential of the test item to induce reverse mutation in Salmonella typhimurium and Escherichia coli.

A preliminary toxicity test was performed to define the dose-levels of the test item to be used for the mutagenicity study. The test item was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction of rats induced with Aroclor 1254. Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method. Five strains of bacteria Salmonella typhimurium: TA1535, TA1537, TA98, TA100 and TA102 and one strain of E.coli: WP2 uvrA were used.

Since the test item was freely soluble in the vehicle and non-toxic in the preliminary test, the highest dose-level was 5000 µg/plate, according to the criteria specified in the international guidelines. A moderate to marked emulsion was observed in the Petri plates when scoring the revertants at dose-lvels > 1250 µg/plate. No noteworthy toxicity was noted towards all the strains used, both with and without S9 mix. The test item did not induce any noteworthy increase in the number of revertants, both with and wothout S9 mix, in any of the six strains.

Under the experimental conditions, the test item did not show mutagenic activity in the bacterial reverse mutation test with S.typhimurium and E.coli.

In the supporting study (Wollny 2001) , the same results were observed on the Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose lever neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a disctinct increase of induced revertant colonies.

In vitro DNA damage and/or repair study (Meurer 2001):

The test 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.

In vitro cytogenicity / micronucleus study (Brient 2014) / Read-across with analogue substance

The objective of this study was to evaluate the potential of the test item to induce an increase in the frequency of micronucleated cellsin the mouse cell line L5178Y TK+/-. This study was conducted in compliance with the OECD Guideline No. 487 and the principles of Good Laboratory Practices.

Experiments without S9 mix : No precipitate was observed in the culture medium at the end of the treatment periods. Following the 3-hour treatment, a moderate to strong toxicity was induced at dose-levels = 0.006 mM as shown by a 57 to 100% decrease in the PD. Following the 24-hour treatment, a moderate to strong toxicity was induced at dose-levels = 0.03 mM as shown by a 40 to 100% decrease in the PD.

No increase in the frequency of micronucleated cells was noted after the 3-hour treatment. After the 24-hour treatment, some increases in the frequency of micronucleated cells were noted at the three dose-levels analyzed. However, these increases were neither dose-related nor statistically significant. Moreover, the frequencies of micronucleated cells of each replicate culture remained within the corresponding vehicle historical range.

 Experiments with S9 mix: A slight to moderate emulsion (or precipitate) was observed at the end of the treatment periods, at dose-levels = 0.2 mM in both experiments. In the first experiment, a slight to strong toxicity was induced at dose-levels of 0.006 mM, 0.025 mM and at dose-levels = 0.1 mM as shown by 33 to 100% decreases in the PD. In the second experiment, a slight to strong toxicity was induced at dose-levels = 0.2 mM as shown by a 34 to 78% decrease in the PD.

No increase in the frequency of micronucleated cells was noted in the first experiment. In the second experiment, some increases in the frequency of micronucleated cells were noted at 0.05, 0.1 and 0.2 mM. These increases were not statistically significant. Moreover, the corresponding micronucleated cells frequencies remained within the historical data range for the vehicle control. Consequently, this increase did not meet the criteria for a positive response and was considered as non-biologically relevant.

Under the experimental conditions of the study, the test item did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the absence or in the presence of a rat liver metabolising system.

Justification for classification or non-classification

Based on the all three in vitro studies (Ames test, Comet assay, micronucleus assay) available on tricyclodecane dimethanol dimethacrylate or on an analogue substance, tricyclodecane dimethanol dimethacrylate is considered to be not mutagenic. No classification is required according to the Regulation EC no.1272/2008.