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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
All the three in vitro tests performed on Tricyclododecane dimethanol diacrylate showed negative results.
Link to relevant study records

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Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-04-01 until 2014-07-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/Beta-Naphtoflavone induced Rat liver S9
Test concentrations with justification for top dose:
Experiment I:
without metabolic activation: 0.3; 0.5; 1.0; and 1.5 µg/mL
with metabolic activation: 25.0; 50.0; 100.0; and 200.0 µg/mL
Experiment II:
without metabolic activation: 0.8; 1.5; 3.0; 6.0; and 9.0 µg/mL
with metabolic activation: 50.0; 100.0; 200.0; 220.0; and 240.0 mg/mL
Experiment IA:
without metabolic activation: 0.13; 0.25; 0.5; 1.0; and 1.5 µg/mL
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation, Experiment II: 24 hours without metabolic activation, 4 hours with metabolic activation, Experiment IA: 4 hours treamtment without metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): 6-Thioguanine


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: >1,5x10exp. 6


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces reproducibly with one of the concen¬trations a mutation frequency that is three times higher than the spontaneous mutation fre¬quency in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
In a case by case evaluation this decision depends on the level of the correspon¬ding solvent control data.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected (pH 7.44 in the solvent control versus pH 7.53 at 3200 µg/mL)
- Effects of osmolality: No relevant increase (368 mOsm in the solvent control versus 324 mOsm at 3200µg/mL)
- Evaporation from medium: Not examined
- Water solubility: insoluble
- Precipitation: Precipitation occurred in the pre-experiment at 400.0 to 3200 µg/mL in the presence of metabolic activation
- Other confounding effects: None


RANGE-FINDING/SCREENING STUDIES:
Two pre-tests were performed in order to determine the concentration range of the mutagenicity experiments.
Eight test item concentrations between 25.0 µg/mL and 3200 µg/mL (equal to 10 mM) were used to evaluate toxicity in the presence and absence of metabolic activation (4 hours treatment). The test item was dissolved in DMSO.
The experimental part of the pre-test without metabolic activation was terminated prematurely due to extremely high cytotoxicity. Therefore, the pre-test was repeated under identical general experimental conditions using eight concentrations between 0.2 and 25.0 µg/mL without metabolic activation with a treatment time of 4 and 24 hours. The data of the second pre-test are reported in Table 2.

The general culture conditions and experimental conditions in the pre-test were the same as described for the mutagenicity experiment below. In the pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test item was observed and compared to the controls. Toxicity of the test item is indicated by a reduction of the cloning efficiency (CE).

3.6 Dose selection
According to the current OECD Guideline for Cell Gene Mutation Tests at least four analysable concentrations should be used in two parallel cultures. For freely-soluble and non-cytotoxic test items the maximum concentration should be 5 mg/mL, 5 µL/mL or 10 mM, whichever is the lowest. For cytotoxic test items the maximum concentration should result in approximately 10 to 20% relative survival or cell density at subcultivation and the analysed concentrations should cover a range from the maximum to little or no cytotoxicity. Relatively insoluble test items should be tested up to the highest concentration that can be formulated in an appropriate solvent as solution or homogenous suspension. These test items should be tested up or beyond their limit of solubility. Precipitation should be evaluated at the beginning and at the end of treatment by the unaided eye.

A relevant cytotoxic effect indicated by a relative suspension growth below 50% was observed at 1.6 µg/mL in the absence of metabolic activation following 4 hours treatment. At higher concentrations the cell growth was completely inhibited. No cytotoxic effects were noted up to 200 µg/mL in the presence of metabolic activation (4 hours treatment). At all higher concentrations the cell growth was completely inhibited. Following 24 hours treatment without metabolic activation no cytotoxic effect occurred up to 6.3 µg/mL. At the two highest concentrations the cell growth was completely blocked.

The test medium was checked for precipitation or phase separation at the end of the treatment period (4 hours) prior to removal to the test item. Precipitation occurred only in the presence of metabolic activation at 400 µg/mL and above.

There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.

Based on the results of the pre-experiments, the individual concentrations of the main experiments were selected. A series of concentrations spaced by a factor of 2 was generally used. Narrower spacing was used at high concentrations to cover the cytotoxic range more closely.

To overcome problems with possible deviations in toxicity the main experiments were started with more than four concentrations.


COMPARISON WITH HISTORICAL CONTROL DATA: Complies


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% occurred in experiment I at 1.0 µg/mL and above without metabolic activation and at 250 µg/mL and above with metabolic activation. The relative cloning efficiency I showed a steep decrease already at low concentrations that was not expected by judging the relative cell density at the first sub-cultivation after treatment. Therefore, too few non-toxic concentrations were analysed in this experimental part and an additional experiment (experiment IA) was performed to close this gap. In the second experiment relevant cytotoxic effects as described above were noted at 9.0 µg/mL and above without metabolic activation and at 200.0 µg/mL and above with metabolic activation. In experiment IA a relevant cytotoxic effect occurred at 1.5 µg/mL. The recommended cytotoxic range of approximately 10-20% relative cloning efficiency 1 or relative cell density was covered with and without metabolic activation.
Conclusions:
In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, Tricyclodecane dimethanol diacrylate is considered to be non-mutagenic in this HPRT assay.
Executive summary:

The test item Tricyclodecane dimethanol diacrylate was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster (OECD 476). 

The study was performed in three independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. An additional experiment (experiment IA) was performed using 4 hours treatment without metabolic activation to add some more non-cytotoxic concentrations to the evaluated concentration range of the first experiment without metabolic activation.

 

The main experiments were evaluated at the following concentrations:

 

exposure
period

S9
mix

concentrations
in µg/mL

 

 

Experiment I

 4 hours

-

0.3

0.5

1.0

1.5

 4 hours

+

25.0

50.0

100.0

200.0

 

 

Experiment II

24 hours

-

0.8

1.5

3.0

6.0

9.0

 4 hours

+

50.0

100.0

200.0

220.0

240.0

 

 

Experiment IA

4 hours

-

0.13

0.25

0.5

1.0

1.5

 

Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% occurred in experiment I at 1.0 µg/mL and above without metabolic activation and at 250 µg/mL and above with metabolic activation. The relative cloning efficiency I showed a steep decrease already at low concentrations that was not expected by judging the relative cell density at the first sub-cultivation after treatment. Therefore, too few non-toxic concentrations were analysed in this experimental part and an additional experiment (experiment IA) was performed to close this gap. In the second experiment relevant cytotoxic effects as described above were noted at 9.0 µg/mL and above without metabolic activation and at 200.0 µg/mL and above with metabolic activation. In experiment IA a relevant cytotoxic effect occurred at 1.5 µg/mL. The recommended cytotoxic range of approximately 10-20% relative cloning efficiency 1 or relative cell density was covered with and without metabolic activation.

 

No relevant and reproducible increase in mutant colony numbers/10^6 cells was observed in the main experiments up to the maximum concentration. The mutation frequency remained well within the historical range of solvent controls.

 

The threshold of three times the mutation frequency of the corresponding solvent control was exceeded at several experimental points. These effects however, were judged as biologically irrelevant as they are based upon the rather low solvent controls of 8.7, 4.4, and 5.8 mutant colonies/10^6 cells, respectively.

 

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was solely detected in the second culture of the first experiment with metabolic activation. This trend however, was judged as irrelevant as the mutation frequeny remained within the historical solvent control range.

 

In the main experiments of this study (with and without S9 mix) the range of the solvent controls was from 4.4 up to 25.5 mutants per 10^6cells; the range of the groups treated with the test item was from 2.8 up to 31.7 mutants per 10^6 cells.

 

EMS(150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, Tricyclodecane dimethanol diacrylate is considered to be non-mutagenic in this HPRT assay.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 February 2014 -- 17 March 2014
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
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
For both mutagenicity experiments without S9 mix, the selected treatment-levels were as follows:
- 78.1, 156.3, 312.5, 625, 1250 and 2500 µg/plate for the TA 1535, TA 98 and TA 102 strains,
- 9.8, 19.5, 39.1, 78.1, 156.3 and 312.5 µg/plate for the TA 1537 strain,
- 4.9, 9.8, 19.5, 39.1, 78.1, 156.3 µg/plate for the TA 100 strain.

For both mutagenicity experiments with S9 mix, the selected treatment-levels were as follows:
- 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate for the five tested strains in the first experiment, and in the TA 1535, TA 98 and TA 102 strains in the second experiment,
- 39.1, 78.1, 156.3, 312.5, 625 and 1250 µg/plate in the TA 1537 and TA 100 strains in the second experiment.
Vehicle / solvent:
- Vehicle used: dimethylsulfoxide (DMSO), batch No. 1488745V.
- Justification for choice: according to solubility assays performed at the laboratory, the test item was soluble in the vehicle at 100 mg/mL.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide, 9-aminoacridine, 2-nitrofluorene, mitomycin C (-S9 mix); 2-anthramine, benzo(a)pyrene (+S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar


DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 to 72 hours.

DETERMINATION OF CYTOTOXICITY
- Method: decrease in number of revertant colonies and/or thinning of the bacterial lawn
Evaluation criteria:
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 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.
Statistics:
no
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Experiments without S9 mix
A moderate to strong emulsion was observed in the Petri plates when scoring the revertants at dose-levels superior or equal to 1250 µg/plate.
A moderate toxicity was noted at dose-levels superior or equal to 156.3 µg/plate in the TA 1537 strain and at dose-levels superior or equal to 78.1 µg/plate in the TA 100 strain. No note worthy toxicity was noted towards the other tested strains.
The test item did not induce any noteworthy increase in the number of revertants, in any of the five tested strains.

Experiments with S9 mix
A moderate to strong emulsion was observed in the Petri plates when scoring the revertants at dose-levels superior or equal to 2500 µg/plate.
In the first experiment, a moderate toxicity was noted at the highest tested dose-level of 5000 µg/plate in the TA 1537 strain.
In the second experiment, a moderate to strong toxicity was noted at dose-levels superior or equal to
625 µg/plate in the TA 1537 and TA 100 strains, at dose-levels superior or equal to 1250 µg/plate in the TA 1535 and TA 98 strains, and at dose-levels superior or equal to 2500 µg/plate in the TA 102 strain.The test item did not induce any noteworthy increase in the number of revertants, in any of the five tested strains.
Conclusions:
Under the experimental conditions of this study, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium either in the presence or in the absence of a rat liver metabolizing system.
Executive summary:

The objective of this study was to evaluate the potential of the test item to induce reverse mutations in Salmonella typhimurium.

The study was conducted in compliance with OECD No. 471 and the principles of Good Laboratory Practice.

 

A preliminary toxicity test was performed to define the dose-levels of the test item to be used for the mutagenicity experiments. The test item was then tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 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 pre-incubation method (60 minutes, 37°C).

Five strains of bacteria Salmonella typhimurium were used: TA 1535, TA 1537, TA 98, TA 100 and TA 102. Each strain was exposed to six dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

The evaluation 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.

The test item was dissolved in dimethylsulfoxide (DMSO).

 

Results

The mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were at least five analysable dose-levels for each strain and test condition. The study was therefore considered to be valid.

Since the test item was poorly soluble in the preliminary test, the selection of the highest dose-level to be used in the main experiments was based on the level of emulsion, according to the criteria specified in the international guidelines.

 

Experiments without S9 mix

A moderate to strong emulsion was observed in the Petri plates when scoring the revertants at dose-levels superior or equal to 1250 µg/plate.

A moderate toxicity was noted at dose-levels superior or equal to 156.3 µg/plate in the TA 1537 strain and at dose-levels superior or equal to 78.1 µg/plate in the TA 100 strain. No note worthy toxicity was noted towards the other tested strains.

The test item did not induce any noteworthy increase in the number of revertants, in any of the five tested strains.

Experiments with S9 mix

A moderate to strong emulsion was observed in the Petri plates when scoring the revertants at dose-levels superior or equal to 2500 µg/plate.

In the first experiment, a moderate toxicity was noted at the highest tested dose-level of 5000 µg/plate in the TA 1537 strain.

In the second experiment, a moderate to strong toxicity was noted at dose-levels superior or equal to

625 µg/plate in the TA 1537 and TA 100 strains, at dose-levels superior or equal to 1250 µg/plate in the TA 1535 and TA 98 strains, and at dose-levels superior or equal to 2500 µg/plate in the TA 102 strain.

The test item did not induce any noteworthy increase in the number of revertants, in any of the five tested strains.

 

Conclusion

Under the experimental conditions of this study, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium either in the presence or in the absence of a rat liver metabolizing system.

 

 

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
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
Not applicable (not a gene mutation assay).
Species / strain / cell type:
other: mouse lymphoma L5178Y TK+/- cells
Details on mammalian cell type (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 / cell type 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 / solvent:
- 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.
Untreated negative controls:
no
Negative solvent / vehicle 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 experimental 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 / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
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 conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Additional information from genetic toxicity in vitro:

Ames test (Brient 2014):

The objective of this study was to evaluate the potential of the test item to induce reverse mutations in Salmonella typhimurium (OECD 471).

The test item was tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 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 pre-incubation method (60 minutes,37°C).

The test item was dissolved in dimethylsulfoxide (DMSO).

Experiments without S9 mix : A moderate to strong emulsion was observed in the Petri plates when scoring the revertants at dose-levels superior or equal to 1250 µg/plate. A moderate toxicity was noted at dose-levels superior or equal to 156.3 µg/plate in the TA 1537 strain and at dose-levels superior or equal to78.1 µg/plate in the TA 100 strain. No note worthy toxicity was noted towards the other tested strains.

Experiments with S9 mix: A moderate to strong emulsion was observed in the Petri plates when scoring the revertants at dose-levels superior or equal to2500 µg/plate. In the first experiment, a moderate toxicity was noted at the highest tested dose-level of 5000 µg/plate in the TA 1537 strain.

In the second experiment, a moderate to strong toxicity was noted at dose-levels superior or equal to

625 µg/plate in the TA 1537 and TA 100 strains, at dose-levels superior or equal to 1250 µg/plate in the TA 1535 and TA 98 strains, and at dose-levels superior or equal to 2500 µg/plate in the TA 102 strain.

In both experiments, the test item did not induce any noteworthy increase in the number of revertants, in any of the five tested strains.

Under the experimental conditions of this study, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium either in the presence or in the absence of a rat liver metabolizing system.

In vitro micronucleus test (Brient 2014):

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 mean population doubling and the mean frequencies of micronucleated cells for the vehicle controls (DMSO) 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.

HPRT (Wollny 2014) :

The test item Tricyclodecane dimethanol diacrylate was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster (OECD 476). 

The study was performed in three independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. An additional experiment (experiment IA) was performed using 4 hours treatment without metabolic activation to add some more non-cytotoxic concentrations to the evaluated concentration range of the first experiment without metabolic activation.

No relevant and reproducible increase in mutant colony numbers/10^6 cells was observed in the main experiments up to the maximum concentration. The mutation frequency remained well within the historical range of solvent controls.

The threshold of three times the mutation frequency of the corresponding solvent control was exceeded at several experimental points. These effects however, were judged as biologically irrelevant as they are based upon the rather low solvent controls of 8.7, 4.4, and 5.8 mutant colonies/10^6 cells, respectively.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was solely detected in the second culture of the first experiment with metabolic activation. This trend however, was judged as irrelevant as the mutation frequency remained within the historical solvent control range. In the main experiments of this study (with and without S9 mix) the range of the solvent controls was from 4.4 up to 25.5 mutants per 10^6cells; the range of the groups treated with the test item was from 2.8 up to 31.7 mutants per 10^6 cells.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, Tricyclodecane dimethanol diacrylate is considered to be non-mutagenic in this HPRT assay.

Justification for classification or non-classification

Based on the available data, Tricyclododecane dimethanol diacrylate is not classified for genetic toxicity according to the Regulation EC no. 1272/2008.