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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
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:
other: GLP Guideline study (OECD 476)

Data source

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

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Identification: Tricyclodecane dimethanol diacrylate
Storage Conditions (provided by the Sponsor / supplier): At room temperature

Method

Target gene:
HPRT
Species / strain
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
Controlsopen allclose all
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.

Results and discussion

Test results
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.
Remarks on result:
other: strain/cell type: Chinese hamster lung fibroblasts (V79)
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Summary Table
        relative relative relative mutant   relative relative relative mutant  
  conc. P S9 cloning cell cloning colonies/ induction cloning cell cloning colonies/ induction
  µg/mL mix efficiency I density efficiency II 106cells factor efficiency I density efficiency II 106cells factor
        % % %     % % %    
Column 1 2 3 4 5 6 7 8 9 10 11 12 13
Experiment I / 4 h treatment       culture I          culture II
Solvent control with DMSO - 100.0 100.0 100.0 10.9 1.0 100.0 100.0 100.0 15.7 1.0
Positive control (EMS) 150.0 - 83.0 72.9 105.4 133.3 12.2 74.8 95.6 87.6 152.7 9.7
Test item 0.06 - 87.5 culture was not continued# 88.4 culture was not continued#
Test item 0.13 - 87.9 culture was not continued# 88.9 culture was not continued#
Test item 0.3 - 104.3 110.2 93.8 10.2 0.9 85.5 150.7 109.3 9.8 0.6
Test item 0.5 - 75.0 95.9 106.7 10.0 0.9 67.6 109.7 105.7 16.1 1.0
Test item 1.0 - 6.2 94.5 96.7 12.3 1.1 12.0 105.6 106.4 27.8 1.8
Test item 1.5 - 2.5 97.3 102.8 10.4 1.0 0.0 93.8 104.3 11.4 0.7
Test item 2.0   - 0.0 79.3 125.3 9.2 0.8 0.0 108.9 100.9 14.0 0.9
Solvent control with DMSO + 100.0 100.0 100.0 22.8 1.0 100.0 100.0 100.0 8.7 1.0
Positive control (DMBA) 1.1 + 98.6 93.0 83.1 154.4 6.8 97.5 88.8 98.1 166.8 19.1
Test item 25.0 + 101.3 100.4 90.9 12.7 0.6 99.7 102.1 150.8 11.3 1.3
Test item 50.0 + 99.4 84.0 94.3 16.1 0.7 99.1 104.9 91.6 13.5 1.6
Test item 100.0 + 97.2 110.7 86.7 18.0 0.8 98.4 89.5 93.3 27.1 3.1
Test item 200.0 + 97.3 53.6 78.2 23.4 1.0 94.7 77.8 86.6 28.9 3.3
Test item 250.0 + 0.0 8.2 culture was not continued## 0.0 8.6 culture was not continued##
Test item 300.0 P + 0.0 culture was not continued## 0.0 culture was not continued##
Test item 350.0 P + 0.0 culture was not continued## 0.0 culture was not continued##
Test item 400.0 P + 0.0 culture was not continued## 0.0 culture was not continued##
Experiment II / 24 h treatment       culture I          culture II
Solvent control with DMSO   - 100.0 100.0 100.0 4.4 1.0 100.0 100.0 100.0 5.8 1.0
Positive control (EMS) 150.0 - 109.4 88.6 103.2 302.5 68.8 82.8 94.5 78.0 448.8 77.8
Test item 0.4 - 101.7 98.0 culture was not continued# 111.3 91.3 culture was not continued#
Test item 0.8 - 100.6 85.6 76.3 3.4 0.8 101.7 74.7 80.3 14.0 2.4
Test item 1.5 - 92.7 78.5 87.6 8.0 1.8 86.0 94.2 81.3 10.6 1.8
Test item 3.0 - 90.4 86.5 84.5 13.1 3.0 96.9 71.6 108.0 5.6 1.0
Test item 6.0 - 72.0 86.5 82.7 7.8 1.8 59.7 83.9 107.9 5.2 0.9
Test item 9.0 - 1.5 69.0 81.7 13.3 3.0 4.1 56.7 100.5 19.3 3.3
Test item 12.0 - 0.0 9.3 culture was not continued## 0.0 6.4 culture was not continued##
Experiment II / 4 h treatment       culture I          culture II
Solvent control with DMSO   + 100.0 100.0 100.0 5.8 1.0 100.0 100.0 100.0 7.3 1.0
Positive control (DMBA) 1.1 + 92.9 115.0 86.0 119.3 20.7 93.3 89.5 82.5 118.6 16.3
Test item 50.0 + 95.3 94.7 82.7 8.4 1.5 95.5 81.3 94.4 13.2 1.8
Test item 100.0 + 104.2 94.3 84.1 15.9 2.8 92.9 83.6 95.7 13.6 1.9
Test item 200.0 + 38.3 75.4 84.8 8.4 1.5 45.7 76.6 90.7 6.7 0.9
Test item 220.0 + 47.9 83.5 86.8 17.9 3.1 40.5 79.9 95.7 7.7 1.1
Test item 240.0 + 0.0 15.1 85.7 2.8 0.5 0.2 15.7 106.6 3.0 0.4
Test item 260.0 + 0.0 6.1 culture was not continued## 0.0 6.6 culture was not continued##
Test item 280.0 + 0.0 2.8 culture was not continued## 0.0 1.8 culture was not continued##
Test item 300.0 P + 0.0 2.7 culture was not continued## 0.0 2.5 culture was not continued##
Experiment IA / 4 h treatment       culture I          culture II
Solvent control with DMSO - 100.0 100.0 100.0 25.5 1 100.0 100.0 100.0 23.7 1
Positive control (EMS) 150.0 -  92.2  99.0 21.4 872.3 34.2 74.9 106.4 116.4 119.1 5.0
Test item 0.06 -  99.4 113.0 culture was not continued# 78.2 123.7 culture was not continued#
Test item 0.13 - 73.6 110.3 111.3 19.4 0.8 86.9 85.3 100.4 16.3 0.7
Test item 0.25 - 85.3 105.0 87.6 28.1 1.1 101.2 82.1 120.1 20.8 0.9
Test item 0.5 - 81.9 127.8 110.9 13.4 0.5 67.9 97.5 131.4 21.1 0.9
Test item 1.0 - 69.4 90.6 73.5 31.7 1.2 66.7 85.3 126.6 24.1 1.0
Test item 1.5   - 41.0 121.4 103.8 29.6 1.2 20.6 65.0 102.4 9.2 0.4

#    culture was not continued as a minimum of only four analysable concentrations is required
##
  culture was not continued due to exceedingly severe cytotoxic effects
P
    precipitation visible at the end of treatment

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

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.