2,2'-ethylenedioxydiethyl dimethacrylate

Genetic toxicity: in vitro

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:

2013-10-22 - 2013-12-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted July 21, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

dated May 30, 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

EPA 712-C-98-221, August 1998

Deviations:

no

Principles of method if other than guideline:

This in vitro test is an assay for the detection of forward gene mutations in mammalian cells.

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-ethylenedioxydiethyl dimethacrylate

EC Number:

203-652-6

EC Name:

2,2'-ethylenedioxydiethyl dimethacrylate

Cas Number:

109-16-0

Molecular formula:

C14H22O6

IUPAC Name:

ethane-1,2-diylbis(oxyethane-2,1-diyl) bis(2-methylacrylate)

Test material form:

liquid

Specific details on test material used for the study:

- Name of test material (as cited in study report): Triethyleneglycol dimethacrylate
- Substance type: organic
- Physical state at room temperature: liquid
- Stability under test conditions: stable
- Storage condition of test material: At room temperature

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes (each batch is screened)
- Periodically checked for karyotype stability: yes
- Periodically checked for spontaneous mutant frequency: yes (each batch is screened)

Metabolic activation:

with and without

Metabolic activation system:

Mammalian liver microsomal fraction S9-Mix of Phenobarbital/beta-naphthoflavone-induced rats.

Test concentrations with justification for top dose:

Experiment I (4 hours):
-S9 mix: 22.7, 45.3, 90.6, 181.3, 362.5, 725.0, 1087.5 and 1450 µg/mL
+S9 mix: 181.3, 362.5, 725.0, 1450.0, 2175.0 (PS) and 2900 (PS) µg/mL
PS= Phase separation
Experiment II:
-S9 mix (24 hours): 22.7, 45.3, 90.6, 181.3, 362.5, 543.8 and 725.0 µg/mL
+S9 mix (4 hours): 181.3, 362.5, 725.0, 1450.0 (PS), 2175.0 (PS) and 2900.0 (PS) µg/mL
PS= Phase separation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

concurrent solvent control (DMSO, final concentration: 0.5% (v/v))

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

EMS dissolved in nutrient medium, final concentration: 0.15 mg/mL (1.2 mM) Migrated to IUCLID6: without metabolic activation

Controls 2

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

DMBA dissolved in: DMSO (final concentration in nutrient medium 0.5 %), Final concentration DMBA: 1.1 µg/mL = 4.3 µM Migrated to IUCLID6: with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation
Experiment II: 4 hours with and 24 hours without metabolic activation.

NUMBER OF CELLS EVALUATED: The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

Reasons for the Choice of the Cell Line V79
The V79 cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50 %) both necessary for the appropriate performance of the
study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22.

Cell Cultures
Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany) are stored in liquid nitrogen in the cell bank of Harlan CCR allowing the repeated use of the same cell culture batch in experiments. Before freezing, the level of spontaneous mutants was depressed by treatment with HAT-medium. Each batch is screened for mycoplasm contamination and checked for karyotype stability and spontaneous mutant frequency. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells.
Thawed stock cultures are propagated at 37 °C in 75 cm² plastic flasks. About 5x10E+5 cells were seeded into each flask with 15 mL of MEM (minimal essential medium) containing Hank’s salts supplemented with 10 % foetal bovine serum (FBS), neomycin (5 µg/mL) and amphotericin B (1 %). The cells were sub-cultured twice weekly. The cell cultures were incubated at 37°C in a 1.5 % carbon dioxide atmosphere in humidified air.

Mammalian Microsomal Fraction S9 Mix
Lacking metabolic activities of cells under in vitro conditions are a disadvantage of assays with cell cultures as many chemicals only develop a mutagenic potential when they are me¬tabolized by the mammalian organism. However, metabolic activation of chemicals can be achieved at least partially by supplementing the cell cultures with mammalian liver micro¬some preparations (S9 mix).

S9 (Preparation by Harlan CCR)
Phenobarbital/beta-naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared and stored according to the current valid version of Harlan CCR SOP for rat liver S9 preparation. Each batch of S9 mix was routinely tested for its capability to activate the known mutagens 2-aminoanthracene as well as benzo(a)pyrene in the Ames test.
The protein concentration of the S9 preparation was 29.8 mg/mL (Lot. No.: 050913) in the pre-experiment and the main experiments.


S9 MIX
An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors were added to the S9 supernatant to reach following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
4 mM NADP
in 100 mM sodium-phosphate-buffer, pH 7.4.
During the experiment, the S9 mix was stored in an ice bath.

Evaluation criteria:

Acceptability of the Assay
The gene mutation assay is considered acceptable if it meets the following criteria:
- the numbers of mutant colonies per 10exp+6 cells found in the negative and/or solvent controls fall within the laboratory historical control data
range of 2011 – 2012.
- the positive control substances must produce a significant increase in mutant colony frequencies and remain within the historical control range of positive controls (Historical data).
- the cloning efficiency II (absolute value) of the negative and/or solvent controls must exceed 50 %.

Evaluation of Results
A test item is regarded as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive
response at one of the test points.
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 non-mutagenic in this system.
A positive response is described as follows:
A test item is regarded as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency
at least at one of the concentrations in the experiment.
The test item is regarded 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.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low
spontaneous mutation rate within the laboratory's historical control range, a concentration-related increase of the mutations
within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.

Statistics:

Statistical Analysis
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. However, both, biological and statistical significance were considered together.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

in forward gene mutations in mammalian cells

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

Results and Disscussions

Triethyleneglycol dimethacrylate was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

The assay was performed in two independent experiments with identical experimental procedures, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h.

The experimental part without metabolic activation was prematurely terminated as exceeding severe cytotoxicity occured already at low concentrations. This experimental part was repeated with an extended concentration range and the data are reported as first experiment without metabolic activation. The second experiment was performed with a treatment period of 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.

The main experiments were evaluated at the following concentrations:

exposure period	S9 mix	concentrations in µg/mL
		Experiment I
4 hours	-	90.6	181.3	362.5	725.0	1087.5
4 hours	+	362.5	725.0	1450.0	2175.0 PS	2900.0 PS
		Experiment II
24 hours	-	22.7	45.3	90.6	181.3	362.5
4 hours	+	181.3	362.5	725.0	1450.0 PS	2175.0 PS

 PS=Phase separation visible to the unaided eye

Phase separation of the test item was observed at 2175 μg/mL and above in the first experiment with metabolic activation and at 1450 μg/mL and above in the second experiment with metabolic activation.

Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% occurred in experiment I at 1087.5 μg/mL and above without metabolic activation and at 2900 μg/mL with metabolic activation. In experiment II cytotoxic effects as described above were noted at 362.5 μg/mL and above without metabolic activation. The recommended cytotoxic range of approximately 10-20% relative cloning efficiency 1 or relative cell density was covered.

No relevant and reproducible increase in mutant colony numbers/ 10⁶cells was observed in the main experiments up to the maximum concentration. The induction factor exceeded the threshold of three times the mutation frequency of the corresponding solvent control in the first culture of the first experiment with metabolic activation at 2175 μg/mL. In the second culture of experiment I the threshold was exceeded at 90.6 and 181.3 μg/mL without metabolic activation and at 1450 and 2900 μg/mL with metabolic activation. However, these increases were judged as biologically irrelevant as they were based on rather low solvent controls (3.7, 4.6, and 6.3 mutant colonies per 10exp+6 cells) and the absolute value of the mutation frequency did not exceed the historical range of solvent controls. None of those increases was reproduced in the parallel culture.

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 determined in experiment I, culture II with metabolic activation. However, this trend was judged as biologically irrelevant as it was not reproduced in the parallel culture under identical experimental conditions.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 3.7 up to 19.8 mutants per 10exp+6 cells; the range of the groups treated with the test item was from 2.8 up to 31.4 mutants per 10exp+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.

Applicant's summary and conclusion

Conclusions:

negative

In conclusion it can be stated that under the experimental conditions reported Triethyleneglycol dimethacrylate did not induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.
Therefore, the substance is considered to be non-mutagenic in this HPRT assay.

Executive summary:

In a mammalian cell gene mutation assay HPRT locus using V79 cells of the Chinese hamster cultured in vitro were exposed to Triethyleneglycol dimethacrylate (TREGDMA, 99.68%) dissolved in DMSO at concentrations of 22.7 to 2900 µg/mL in the presence and absence of mammalian metabolic activation S9 mix. 

The assay was performed in two independent experiments. The cells were exposed to TREGDMA for 4 hours in the first experiment with and without metabolic activation. The second experiment was performed with a treatment period of 24 hours in the absence and 4 hours in the presence of metabolic activation.

The maximum dose was 2900 μg/mL corresponding to a molar concentration of about 10 mM.

The positive controls did induce the appropriate response.

Phase separation of the test item was observed at 2175 μg/mL and above in the first experiment with metabolic activation and at 1450 μg/mL and above in the second experiment with metabolic activation.

Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% occurred in experiment I at 1087.5 μg/mL and above without metabolic activation and at 2900 μg/mL with metabolic activation. In experiment II cytotoxic effects as described above were noted at 362.5 μg/mL and above without metabolic activation. The recommended cytotoxic range of approximately 10-20% relative cloning efficiency 1 or relative cell density was covered.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiments up to the maximum concentration.

The induction factor exceeded the threshold of three times the mutation frequency of the corresponding solvent control in the first culture of the first experiment with metabolic activation at 2175 μg/mL. In the second culture of experiment I the threshold was exceeded at 90.6 and 181.3 μg/mL without metabolic activation and at 1450 and 2900 μg/mL with metabolic activation. However, these increases were judged as biologically irrelevant as they were based on rather low solvent controls (3.7, 4.6, and 6.3 mutant colonies per 10exp+6 cells) and the absolute value of the mutation frequency did not exceed the historical range of solvent controls. None of those increases was reproduced in the parallel culture.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.

Based on the results of the study, TREGDMA is considered to be non-mutagenic in this HPRT assay.

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