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, the substance is considered to be not mutagenic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
In vitro Comet assay
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
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:
comet assay
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (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 / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
methylmethanesulfonate
Details on test system and experimental 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 / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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 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
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
n/a
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Species / strain / cell type:
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 / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle 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 experimental 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:
S. typhimurium, other: TA98, TA100, TA102, TA1535, TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
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:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 March 2020 - 27 March 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: see remarks
Version / remarks:
Commission Regulation (EU) 2017/735 of 14 February 2017 amending, for the purpose of its adaptation to technical progress, the Annex to Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), Annex, Part B p. 108.
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: ATCC (American Type Culture Collection, Manassas, USA), by the intermediate of Biovalley (Marne-La-Vallée, France)
- Suitability of cells: Suitable to reveal chemically induced micronuclei.
- Normal cell cycle time (negative control): 10-12 hours

For cell lines:
- Absence of Mycoplasma contamination: yes
- Cell cycle length, doubling time or proliferation index: cell cycle time is approximately 10-12 hours
- Modal number of chromosomes: 40
- Periodically checked for karyotype stability: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Cell cultures were grown at 37°C in a humidified atmosphere of 5% CO2/95% air in culture medium. The culture medium was RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL), 0.05% pluronic acid and sodium pyruvate (200 µg/mL). This medium was supplemented by heat inactivated horse serum at 10% (v/v).
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: S9 fraction was purchased from Moltox (Molecular Toxicology, INC, Boone, NC 28607, USA) and obtained from the liver of rats treated with Aroclor 1254.
- method of preparation of S9 mix: The S9 mix was prepared at +5°C immediately before use and maintained at this temperature until added to culture medium.
- concentration or volume of S9 mix and S9 in the final culture medium: final concentration of S9 in the treatment medium was 2%.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Each batch of S9 was tested and validated by Moltox for absence of microbiological contamination and its ability to activate benzo(a)pyrene and 2-anthramine (also known as 2-amino anthracene) to mutagenic intermediates.
Test concentrations with justification for top dose:
Dose range finding test:
With and without S9-mix, 3 hr exposure; 24 hr recovery: 3.28, 8.19, 20.5, 51.2, 128, 320, 800 and 2000 µg/mL
Without S9-mix, 24 hr exposure; 0 hr recovery: 3.28, 8.19, 20.5, 51.2, 128, 320, 800 and 2000 µg/mL
First cytogenetic test:
Without S9-mix, 3 hr exposure; 24 hr recovery: 1.25, 2.5, 5, 10, 15, 20, 25 and 50 µg/mL
Without S9-mix, 24 hr exposure; 0 hr recovery: 0.625, 1.25, 2.5, 5, 7, 10, 15 and 20 µg/mL
With S9-mix, 3 hr exposure; 24 hr recovery: 7.813, 15.63, 31.25, 62.5, 125 and 250 µg/mL
The following dose levels were selected for scoring of micronuclei:
Without S9-mix, 3 hr exposure; 24 hr recovery: 10, 20 and 25 µg/mL
Without S9-mix, 24 hr exposure; 0 hr recovery: 10, 15 and 20 µg/mL
With S9-mix, 3 hr exposure; 24 hr recovey: 15.63, 31.25 and 62.5 µg/mL
Second cytogenetic test:
Without S9-mix, 3 hr exposure; 24 hr recovery: 2.5, 5, 10, 15, 20, 30, 40 and 60 µg/mL
Without S9-mix, 24 hr exposure; 0 hr recovery: 1.25, 2.5, 5, 10, 15, 20, 30 and 50 µg/mL
The following dose levels were selected for scoring of micronuclei:
Without S9-mix, 3 hr exposure; 24 hr recovery: 10, 20 and 30 µg/mL
Without S9-mix, 24 hr exposure; 0 hr recovey: 1.25, 2.5 and 5 µg/mL
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of vehicle: According to available solubility data, the vehicle used for the preparation of test item dose formulations and the treatment of vehicle control cultures was dimethylsulfoxide (DMSO).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
colchicine
mitomycin C
Remarks:
Without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With S9-mix
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: single (dose range finding), duplicate (main test)
- Number of independent experiments : two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 30x10^4 cell/mL
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 hr and 24 hr
- Harvest time after the end of treatment (sampling/recovery times): 3 hr and 24 h

FOR MICRONUCLEUS:
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): Fixed cells were dropped onto clean glass slides. The slides were air dried before being stained for approximately 15 min in 5% Giemsa.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): For each main experiment (with or without S9 mix), micronuclei were analyzed for three dose levels of the test item, for the vehicle and the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per dose).
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification):
• micronuclei should be located within the cytoplasma of the cell,
• micronuclei should be clearly surrounded by a nuclear membrane,
• micronuclei should be round or oval in shape,
• the micronucleus area should be less than one-third of the area of the main nucleus,
• micronuclei should be non-refractile (can be distinguished from artefacts such as staining particles),
• micronuclei should not be linked to the main nucleus via nucleoplasmic bridges,
• micronuclei should have similar staining intensity to that of the main nuclei,
• micronuclei may touch but not overlap the main nuclei and the micronuclear boundary should be distinguishable from the nuclear boundary,
• only mononucleated cells with a number of micronuclei = 5 will be scored to exclude apoptosis and nuclear fragmentation.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: population doubling (PD)
Evaluation criteria:
Evaluation of a positive response: 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 demonstrated by a statistically significant trend test,
• 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.
Evaluation of a negative response: a test item is considered clearly negative if none of the criteria for a positive response was met.
Statistics:
For each condition of the cytogenetic experiment, the frequency of micronucleated cells in treated cultures was compared to that of the vehicle control cultures.
This comparison was performed using the X^2 test, unless treated culture data are lower than or equal to the vehicle control data. P = 0.05 was used as the lowest level of significance. This statistical analysis was performed using a validated Excel sheet.
To assess the dose-response trend, a linear regression was performed between the individual frequencies of micronucleated cells and the dose levels. This statistical analysis was performed using SAS Enterprise Guide software.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: At the highest dose level of 2000 µg/mL the pH of the culture medium was approximately 7.1 (as for the vehicle control)
- Data on osmolality: At the highest dose level of 2000 µg/mL the osmolality was 429 mOsm/kg H2O (461 mOsm/kg H2O for the vehicle control)
- Precipitation and time of the determination: A precipitate was observed in the culture medium at the end of the treatment periods, at dose levels = 40 µg/mL.

RANGE-FINDING/SCREENING STUDIES:
At the end of the treatment period, a precipitate was observed in the culture medium at dose levels = 128 µg/mL following the 3-hour treatment and ¿ 320 µg/mL following the 24-hour treatment.
Following the 3-hour treatment without S9 mix, a severe cytotoxicity was observed at dose levels = 20.5 µg/mL, as shown by a 70 to 100% decrease in the PD.
Following the 24-hour treatment without S9 mix, a severe cytotoxicity was observed at dose levels = 8.19 µg/mL, as shown by a 69 to 100% decrease in the PD.
Following the 3-hour treatment with S9 mix, a moderate to severe cytotoxicity was observed at dose levels = 128 µg/mL, as shown by a 57 to 100% decrease in the PD.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : 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.

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements:
Short treatment without S9 mix: In the first experiment, a slight to severe cytotoxicity was induced at dose levels = 15 µg/mL, as shown by a 34 to 100% decrease in the PD. In the second experiment, a slight to severe cytotoxicity was induced at dose levels = 10 µg/mL, as shown by a 30 to 100% decrease in the PD.
Continuous treatment without S9 mix: In the first experiment, a slight to moderate cytotoxicity was induced at tested dose levels = 15 µg/mL, as shown by a 35 to 40% decrease in the PD. In the second experiment, a moderate to severe cytotoxicity was induced at dose levels = 5 µg/mL, as shown by a 56 to 100% decrease in the PD.
Treatment with S9 mix: A 27% decrease in the PD was induced at the dose level of 250 µg/mL.

- Genotoxicity results :
Short treatment without S9 mix: In the first experiment, neither statistically significant nor dose-related increase in the frequency of micronucleated cells was noted, at any of the analyzed dose levels in comparison to the corresponding vehicle controls. Moreover, none of the analyzed dose levels showed frequency of micronucleated cells of both replicate cultures above the corresponding vehicle control historical range. In the second experiment, neither statistically significant nor dose-related increase in the frequency of micronucleated cells was noted, at any of the analyzed dose levels in comparison to the corresponding vehicle controls. Moreover, none of the analyzed dose levels showed frequency of micronucleated cells of both replicate cultures above the corresponding vehicle control historical range.
Continuous treatment without S9 mix: In the first experiment, neither statistically significant nor dose-related increase in the frequency of micronucleated cells was noted, at any of the analyzed dose levels in comparison to the corresponding vehicle controls. Moreover, none of the analyzed dose levels showed frequency of micronucleated cells of both replicate cultures above the corresponding vehicle control historical range. In the second experiment, neither statistically significant nor dose-related increase in the frequency of micronucleated cells was noted, at any of the analyzed dose levels in comparison to the corresponding vehicle controls. Moreover, none of the analyzed dose levels showed frequency of micronucleated cells of both replicate cultures above the corresponding vehicle control historical range.
Treatment with S9 mix: In comparison to the vehicle control, no statistically significant increase in the frequency of micronucleated cells was noted. Frequencies of micronucleated cells remained consistent with vehicle control historical ranges and no dose-response relationship was observed.

HISTORICAL CONTROL DATA : yes
Conclusions:
An in vitro micronucleus assay with the substance was performed according to OECD 487 guideline and GLP principles, in cultured L5178Y TK+/- mouse lymphoma cells in two experiments. It is concluded that the substance 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 presence or absence of a rat liver metabolizing system.
Executive summary:

In an in vitro micronucleus assay, cultured L5178Y TK+/- mouse lymphoma cells were exposed to different concentrations of the substance (dissolved in DMSO), in the presence and absence of S9-mix according to OECD 487 guideline and GLP principles. In the first cytogenetic assay, the substance was tested up to and including concentrations of 25 µg/mL and 62.5 µg/mL for a 3 h exposure time with a 24 h recovery time in the absence and presence of S9-mix, respectively. In the continuous 24 h exposure time in the absence of S9 -mix, the substance was tested up to and including the concentration of 20 µg/mL. Cytotoxicity was induced as shown by a decrease in the population doubling (PD).

In the second cytogenetic assay in the absence of S9-mix, the substance was tested up to and including concentrations of 30 µg/mL and 5 µg/mL for a 3 h exposure time and for a 24 h continuous exposure time, respectively. Cytotoxicity was induced as shown by a decrease in the population doubling (PD).

Reliable positive and negative controls were included.

It is concluded that the substance 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 presence or absence of a rat liver metabolizing system.

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 (2020)

In an in vitro micronucleus assay, cultured L5178Y TK+/- mouse lymphoma cells were exposed to different concentrations of the substance (dissolved in DMSO), in the presence and absence of S9-mix according to OECD 487 guideline and GLP principles. In the first cytogenetic assay, the substance was tested up to and including concentrations of 25 µg/mL and 62.5 µg/mL for a 3 h exposure time with a 24 h recovery time in the absence and presence of S9-mix, respectively. In the continuous 24 h exposure time in the absence of S9 -mix, the substance was tested up to and including the concentration of 20 µg/mL. Cytotoxicity was induced as shown by a decrease in the population doubling (PD).

In the second cytogenetic assay in the absence of S9-mix, the substance was tested up to and including concentrations of 30 µg/mL and 5 µg/mL for a 3 h exposure time and for a 24 h continuous exposure time, respectively. Cytotoxicity was induced as shown by a decrease in the population doubling (PD).

Reliable positive and negative controls were included.

It is concluded that the substance 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 presence or absence of a rat liver metabolizing system.

Justification for classification or non-classification

Based on the all three in vitro studies, tricyclodecane dimethanol dimethacrylate is considered to be not mutagenic. No classification is required for mutagenicity according to the Regulation EC no.1272/2008.