Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- not mutagenic in the Salmonella typhimurium reverse mutation assay (OECD guideline 471; non-GLP)
- not mutagenic in the mammalian cell gene mutation assay with and without mutagenic activation (OECD guideline 476; HPRT test, V79 cells; GLP, Higher Methacrylate REACH Task Force, 2014)
- positive without metabolic activation, negative with metabolic activation in the mammalian cell gene mutation assay (OECD guideline 476; HPRT test, V79 cells; non-GLP)
- not clastogenic in an in vitro micronucleus test (OECD guideline 487; human lymphocytes; GLP)

Link to relevant study records

Referenceopen allclose all

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
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted July 21, 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
dated May 30, 2008
Deviations:
no
Qualifier:
according to
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. certificate)
Type of assay:
mammalian cell gene mutation assay
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
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.
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
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.
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

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.

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.

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
no GLP; strain TA 1535 not included
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
strain TA 1535 not included
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): triethylene glycol dimethacrylate, TEGDMA
- Analytical purity: no data
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: no data
- Isomers composition: no data
- Purity test date: no data
- Lot/batch No.: no data
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data
- Storage condition of test material: no data
Target gene:
his
Species / strain / cell type:
S. typhimurium, other: TA97a, TA98, TA100, TA102
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
0 (control), 0.5, 1.25, 2.5, 5.0, 12.5 mg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no justification given
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
other: 2,4,7-trinitrofluorenon; glutaraldehyde
Remarks:
without metabolic activation
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthrace
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: not applicable
- Exposure duration: 3 days

NUMBER OF REPLICATIONS: 3; the experiment was repeated at least once, but only data from 1 experiment are given

DETERMINATION OF CYTOTOXICITY
- Method: decrease or absence of bacteria in background lawn
Species / strain:
S. typhimurium, other: TA97a, TA98, TA100, TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see below (table)
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- no toxic effects with metabolic activation
- toxic effects without metabolic activation in TA97a, TA98, TA102 at 5 mg and higher; in TA100 at 12.5 mg

 

TA97a

TA98

TA100

TA102

mutant colonies

mg / plate

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

TREGDMA

0

127 ± 24

158 ± 17

30 ± 5

33 ± 11

155 ± 18

184 ± 23

289 ± 18

480 ± 26

0.5

156 ± 24

204 ± 16

28 ± 3

38 ± 2

149 ± 9

203 ± 16

203 ± 57

303 ± 50

1.25

136 ± 24

192 ± 30

33 ± 5

35 ± 7

140 ± 1

233 ± 29

210 ± 13

361 ± 17

2.5

34 ± 24

195 ± 6

28 ± 3

33 ± 3

165 ± 34

253 ± 21

179 ± 41

317 ± 4

5

34 ± 24*

211 ± 11

27 ± 1*

29 ± 3

132 ± 22

266 ± 16

89 ± 32*

329 ± 34

12.5

33 ± 24*

215 ± 15

4 ± 3*

28 ± 6

25 ± 16*

168 ± 41

0 ± 0*

109 ± 36

positive control

1681 ± 122

1725 ± 147

2267 ± 87

1446 ± 24

728 ± 50

963 ± 152

1148 ± 132

1553 ± 34

 

* toxic

 

The following data are calculated from data given in the publication:

 

TA97a

TA98

TA100

TA102

mutation factors

mg/plate

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

TREGDMA

0

1

1

1

1

1

1

1

1

0.5

1.2

1.3

0.9

1.2

1.0

1.1

0.7

0.6

1.25

1.1

1.2

1.1

1.1

0.9

1.3

0.7

0.8

2.5

0.3

1.2

0.9

1.0

1.1

1.4

0.6

0.7

5

0.3

1.3

0.9

0.9

0.9

1.4

0.3

0.7

12.5

0.3

1.4

0.1

0.8

0.2

0.9

0.0

0.2

positive control

13.2

10.9

75.6

43.8

4.7

5.2

4.0

3.2

 

Conclusions:
Interpretation of results (migrated information):
negative

In this bacterial reverse mutation assay in Salmonella typhimurium strains TA97a, TA98, TA100, TA102 TREGDMA did not induce mutant colonies over backgrund.
Executive summary:

In a reverse gene mutation assay in bacteria similar to OECD Guideline 471, strains TA97a, TA98, TA100, TA102 of S. typhimurium  were exposed to TREGDMA in DMSO at concentrations of 0 (control), 0.5, 1.25, 2.5, 5.0, 12.5 mg/plate in the presence and absence of mammalian metabolic activation (S9 mix). 

TREGDMA was tested up to (and above) limit concentrations (5 mg/plate).

No toxic effects were observed with metabolic activation. Without metabolic activation toxic effects (reduction of background lawn) occurred in TA97a, TA98, TA102 at 5 mg/plate and higher; in TA100 at 12.5 mg/plate.

The positive controls induced the appropriate responses in the corresponding strains. 

There was no evidence of induced mutant colonies over background.

Although Salmonella strain TA1535 is missing, this study can be considered to be reliable, as all genietic markers of the TA 1535 strain are covered by other Salmonella tester strains.

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-09-14 to 2011-12-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
July 22, 2010
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
- Name of test material (as cited in study report): Triethyleneglycol dimethacrylate
Target gene:
not applicable (micronucleus test)
Species / strain / cell type:
lymphocytes: blood samples obtained from healthy, non-smoking donors not receiving medication
Details on mammalian cell type (if applicable):
- Type and identity of media: DMEM:Ham's F12 1:1, supplemented with penicillin/streptomycin (100 U/mL/100 µg/mL), phytohemagglutinin (PHA, 3 µg/mL), 10 % FBS, 10 mM HEPES and heparin (125 U.S.P.-U/mL)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
For rationale of dose selection see section "Any other information on materials and methods incl. tables"

Experiment IA – 4 h treatment without S9 mix
19.3, 33.8, 59.1, 103.4, 181.0, 316.7, 554.2, 969.8, 1697.1, 2970.0 mg/L
Evaluated concentrations: 316.7, 554.2, 969.8 mg/L

Experiment IB – 4 h treatment without S9 mix
300.0, 500.0, 700.0, 900.0, 1000.0, 1100.0, 1200.0, 1300.0, 1400.0, 1600.0, 2000.0, 2500.0 mg/L
Evaluated concentrations 1300.0, 1400.0, 1600.0 mg/L

Experiment IIA – 20 h treatment without S9 mix
13.0, 22.7, 39.8, 69.6, 121.9, 213.2, 373.2, 653.1, 1142.9, 2000.0 mg/L
Evaluated concentrations: 213.2, 373.2, 653.1

Experiment IIB –20 h treatment without S9 mix
200.0, 400.0, 600.0, 700.0, 750.0, 800.0, 850.0, 900.0, 1000.0, 1100.0, 1200.0, 1400.0 mg/L
Evaluated concentrations: 400.0, 700.0, 900.0 mg/L

Experiment IA – 4 h treatment with S9 mix
19.3, 33.8, 59.1, 103.4, 181.0, 316.7, 554.2, 969.8, 1697.1, 2970.0 mg/L
Evaluated concentrations: 969.8, 1697.1, 2970.0 mg/L

Experiment IIA – 4 h treatment with S9 mix
181.0, 316.7, 554.2, 969.8, 1697.1, 2970.0 mg/L
Evaluated concentrations: 969.8, 1697.1, 2970.0 mg/L
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility properties and relative non-toxicity to the cell cultures
Untreated negative controls:
yes
Remarks:
culture medium
Negative solvent / vehicle controls:
yes
Remarks:
0.5% DMSO
Positive controls:
yes
Positive control substance:
mitomycin C
other: Demecolcin (without metabolic activation)
Positive controls:
yes
Positive control substance:
cyclophosphamide
other: (with metabolic activation)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Experiment IA, IB, without metabolic activation: 4 h exposure, 16 h recovery, 20 h cytokinesis block
Experiment IIA, IIB, without metabolic activation: 20 h exposure, 20 h cytokinesis block
Experiment IA, IIA, with metabolic activation: 4 h exposure, 16 h recovery, 20 h cytokinesis block
- Expression time (cells in growth medium): 20 h (+ cytochalasin B)
- Fixation time (start of exposure up to fixation or harvest of cells): 40 h

SPINDLE INHIBITOR (cytogenetic assays): 4 mg/L cytochalasin B
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2 per experiment

NUMBER OF CELLS EVALUATED: 1000 binucleate cells per culture were scored for cytogenetic damage

DETERMINATION OF CYTOTOXICITY
- Method: CBPI (Cytokinesis-block proliferation index) was determined in approximately 500 cells per culture and cytotoxicity is expressed as % cytostasis
Evaluation criteria:
The micronucleus assay is considered acceptable if it meets the following criteria:
- the number of micronuclei found in the negative and solvent controls falls within the range of the laboratory historical control data
- the positive control substances produce significant increases in the number of cells with micronuclei.

A test item can be classified as non-mutagenic if:
- the number of micronucleated cells in all evaluated dose groups is in the range of the laboratory historical control and/or
- no statistically significant or concentration-related increase in the number of micronucleated cells is observed.
A test item can be classified as mutagenic if:
- the number of micronucleated cells is not in the range of the historical laboratory control data and
- either a concentration-related increase of micronucleated cells in three test groups or a statistically significant increase of the number of micronucleated cells is observed.
Statistics:
Chi square test
Species / strain:
lymphocytes: primary human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: solvent control: 7.4 to 7.8; test item (max. conc. without metabolic activation): 7.4 to 7.7 -> no relevant influence
- Effects of osmolality: solvent control: 363 to 401 mOsm; test item (max. conc. without metabolic activation): 348 to 382 mOsm -> no relevant influence
- Water solubility:
- Precipitation: in Experiment IIA in the absence of S9 mix at 373.2 mg/L and above at the end of treatment
- Other confounding effects: Phase separation was observed in Experiment IIA at 1697.1 mg/L and above in the presence of S9 mix and in Experiment IIB at 1400.0 mg/L in the absence of S9 mix at the end of treatment.

COMPARISON WITH HISTORICAL CONTROL DATA:
Micronucleus rates were in the range of historical control data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- no cytotoxicity in the presence on S9 mix
- in the absence of S9 mix, clear signs of cytotoxicity at concentrations > 1000 mg/L in experiments IA, IIA, IIB and > 1600 g/L in experiment IB
Remarks on result:
other: all strains/cell types tested

Exp.

Test item concentration [mg/L]

Proliferation index CBPI

Cytostasis [%]

Micronucleated cells [%]

Exposure period 4 hrs without S9 mix

IA

Negative control

1.91

 

0.55

 

Solvent control

1.72

 

0.50

 

Positive control

1.81

11.0

5.30S

 

316.7

1.64

11.9

0.45

 

554.2

1.69

4.2

0.35

 

969.8

1.63

13.0

0.30

IB

Negative control

1.86

 

0.35

 

Solvent control

1.83

 

0.65

 

Positive control

1.74

14.1

6.75S

 

1300.0

1.80

3.5

0.70

 

1400.0

1.77

8.1

0.75

 

1600.0

1.78

6.3

1.15

Exp.

Test item concentration [mg/L]

Proliferation index CBPI

Cytostasis [%]

Micronucleated cells [%]

Exposure period 20 hrs without S9 mix

IIA

Negative control

1.98

 

0.30

 

Solvent control

1.91

 

0.30

 

Positive control

1.48

51.0

2.25S

 

213.2

1.82

9.8

0.55

 

373.2

1.84

7.2

0.25

 

653.1

1.71

21.7

0.55

IIB

Negative control

1.96

 

0.65

 

Solvent control

1.96

 

0.30

 

Positive control

1.61

36.5

2.95S

 

400.0

1.85

11.4

0.20

 

700.0

1.68

28.9

0.15

 

900.0

1.49

48.6

0.35

Exp.

Test item concentration [mg/L]

Proliferation index CBPI

Cytostasis [%]

Micronucleated cells [%]

Exposure period 4 hrs with S9 mix

IA

Negative control

1.93

 

0.30

 

Solvent control

1.91

 

0.50

 

Positive control

1.62

33.5

3.50S

 

969.8

1.92

n.c.

0.20

 

1697.1

1.89

2.0

0.20

 

2970.0

1.84

7.8

0.40

IIA

Negative control

2.03

 

0.35

 

Solvent control

2.16

 

0.40

 

Positive control

1.77

25.0

3.20S

 

969.8

2.07

7.3

0.55

 

1697.1

2.05

9.3

0.55

 

2970.0

1.98

15.6

0.55

S     The number of micronucleated cells is statistically significantly higher than corresponding control values

Conclusions:
negative

In this micronucleus test, under the experimental conditions reported, the test item TREGDMA did not induce micronuclei in human lymphocytes in vitro, when tested up to cytotoxic or the highest required concentration.
Executive summary:

In a mammalian cell micronucleus assay according to OECD guideline 487, July 22, 2010, primary human lymphocyte cultures were exposed to TREGDMA (96.2%, doses adjusted to purity) in DMSO at the following concentrations:

Experiment IA – without S9 mix: 316.7, 554.2, 969.8 mg/L

Experiment IB – without S9 mix: 1300.0, 1400.0, 1600.0 mg/L

Experiment IIA – without S9 mix: 213.2, 373.2, 653.1 mg/L

Experiment IIB – without S9 mix: 400.0, 700.0, 900.0 mg/L

Experiment IA – with S9 mix: 969.8, 1697.1, 2970.0 mg/L

Experiment IIA – with S9 mix: 969.8, 1697.1, 2970.0 mg/L

TREGDMA was tested up to cytotoxic or limit concentrations (2970 mg/L correspond to 10 mM). In the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment IA, IB and IIA in the absence of S9 mix concentrations showing clear cytotoxic effects were not evaluable for cytogenetic damage. In Experiment IIB in the absence of S9 mix cytotoxicity was observed at the highest evaluated concentration (48.6 %).

In the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying micronuclei was observed. The micronucleus rates of the cells after treatment with the test item (0.15 - 1.15% micronucleated cells) were slightly above the range of the solvent control values (0.30 - 0.65% micronucleated cells) but within the range of the laboratory historical control data (0.10 – 1.65% micronucleated cells).

Positive controls induced the appropriate response.

There was no evidence of micronucleated cells induced over background. Therefore, TREGDMA is considered to be non-clastogenic in this in vitro micronucleus test, when tested up to cytotoxic or limit concentration.

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
No GLP
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): triethylene glycol dimethacrylate, TEGDMA
- Analytical purity: no data
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: no data
- Isomers composition: no data
- Purity test date: no data
- Lot/batch No.: no data
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data
- Storage condition of test material: no data
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: no data
- Properly maintained: no data
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
The cells were exposed to various concentrations of the test compounds (no further data given). Detailled results are reported for concentrations 0,0.5, 1.0 mM.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no justification given
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
other: (without metabolic activation)
Positive controls:
yes
Positive control substance:
9,10-dimethylbenzanthracene
other: (with metabolic activation)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h with metabolic activation; 24 h without metabolic activation
- Expression time (cells in growth medium): 8 days
- Selection time (if incubation with a selection agent): 10 days
- Fixation time (start of exposure up to fixation or harvest of cells): 20 d

SELECTION AGENT (mutation assays): 7 µg/mL 6-thioguanin

NUMBER OF REPLICATIONS: one plate per dose, two independent experiments; cloning efficiency: 3 replicate plates

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
Criteria not given in the publication, but in general a test item is classified 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 classified 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 classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency.
Statistics:
none
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
clear positive response in the absence of S9 mix
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
slight reduction of plating efficieny without S9 mix; very slight reduction of plating efficiency with S9 mix
Vehicle controls validity:
not specified
Positive controls validity:
valid
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- no data

RANGE-FINDING/SCREENING STUDIES: no data

COMPARISON WITH HISTORICAL CONTROL DATA: no data

TREGDMA [mM]

Without S9 mix

With S9 mix

 

Plating efficiency [%]

Mutant frequency per 10E06 cells

Mutation factor

Plating efficiency [%]

Mutant frequency per 10E06 cells

Mutation factor

0

100

2

1.0

100

7

1.0

0.5

70

14

7.0

126

2

0.3

1

60

89

44.5

82

13

1.9

Positive control

 

197

98.5

 

296

42.3

Conclusions:
positive

In a mammalian cell gene mutation assay (HPRT test) similar to OECD Guideline 476, V79 cells cultured in vitro were exposed to TREGDMA in DMSO at concentrations of 0, 0.5 and 1.0 mmol/L, corresponding to 143.15 and 286.3 mg/L, in the presence and absence of mammalian metabolic activation (S9 mix). There was a concentration related positive response of induced mutant colonies over background without metabolic activation, but not with metabolic activation. Thus, TREGDMA was mutagenic in this mammalian cell gene mutation assay (HPRT test).
Executive summary:

In a mammalian cell gene mutation assay (HPRT test) similar to OECD Guideline 476, V79 cells cultured in vitro were exposed to TREGDMA in DMSO at concentrations of  0, 0.5 and 1.0 mmol/L, corresponding to 143.15 and 286.3 mg/L, in the presence and absence of mammalian metabolic activation (S9 mix). 

In the absence of S9 mix cytotoxicity was observed at 1.0 mmol/L TREGDMA based on reduced cloning efficiency. In the presence of S9 mix, no cytotoxicity was observed; the test substance has not been tested up to limit concentration.

Without S9 mix, there was a clear increase in mutant frequency over background in both concentrations tested, but not with metabolic activation.

The positive controls did induce the appropriate response. 

There was a concentration related positive response of induced mutant colonies over background.

Thus, TREGDMA was mutagenic in this mammalian cell gene mutation assay (HPRT test).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

There were no in vivo genetic toxicity studies identified for TREGDMA.

Additional information

Reliable (RL1-2), relevant and adequate data are available for the assessment of the mutagenic potential of TREGDMA.

 

In a reverse gene mutation assay in bacteria similar to OECD Guideline 471, strains TA97a, TA98, TA100, TA102 of S. typhimurium  were exposed to TREGDMA in DMSO at concentrations of 0 (control), 0.5, 1.25, 2.5, 5.0, 12.5 mg/plate in the presence and absence of mammalian metabolic activation (S9 mix). 

TREGDMA was tested up to (and above) limit concentrations (5 mg/plate).

No toxic effects were observed with metabolic activation. Without metabolic activation toxic effects (reduction of background lawn) occurred in TA97a, TA98, TA102 at 5 mg/plate and higher; in TA100 at 12.5 mg/plate.

The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background. Although Salmonella strain TA1535 is missing, this study can be considered to be reliable with restrictions (RL=2), as all genetic markers of the TA 1535 strain are covered by other Salmonella tester strains.

 

Moreover, from the Multifunctional Methacrylates category tests with other category members (DEGDMA, EGDMA, 1,4-BDDMA, TMPTMA) are available, which include Salmonella strain TA1535 and were also not mutagenic in this strain. In addition, there is supportive evidence from a larger set of methacrylate esters (i.e. with the same reactive group). A summary table document is attached to this endpoint summary.

 

In a mammalian cell gene mutation assay (HPRT test) similar to OECD Guideline 476, V79 cells cultured in vitro were exposed to TREGDMA in DMSO at concentrations of  0, 0.5 and 1.0 mmol/L, corresponding to 143.15 and 286.3 mg/L, in the presence and absence of mammalian metabolic activation (S9 mix). 

In the absence of S9 mix cytotoxicity was observed at 1.0 mmol/L TREGDMA based on reduced cloning efficiency. In the presence of S9 mix, no cytotoxicity was observed; the test substance has not been tested up to limit concentration.

Without S9 mix, there was a clear increase in mutant frequency over background in both concentrations tested, but not with metabolic activation. The positive controls did induce the appropriate response. There was a concentration related positive response of induced mutant colonies over background. Thus, TREGDMA was mutagenic without, but not with metabolic activation in this mammalian cell gene mutation assay (HPRT test).

 

In another reliable recently performed mammalian cell gene mutation assay (HPRT test, Harlan, 2014) according to OECD Guideline 476, V79 cells of Chinese hamster cultured in vitro were exposed to TREGDMA in DMSO from 22.7 ug/mL up to a maximum concentration of 2900 ug/mL), corresponding to a molar concentration of about 10 mM in the presence and absence of mammalian metabolic activation (S9 mix). The test 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 time of 4 hours with and 24 hours without metabolic activation. Phase separation of TREGDMA was observed at 2175 ug/mL and above in the first experiment with metabolic activation and at 1450 ug/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 ug/mL and above without metabolic activation and at 2900 ug/mL with metabolic activation. Experiment II showed cytotoxic effects as described above at 362.5 ug/mL and above without metabolic activation.

There was no substantial and reproducible dose dependent increase of the mutation frequency observed in both main experiments. The positive controls did induce the appropriate response. Thus, TREGDMA did not induce gene mutations at the HPRT locus in V79 cells and is considered to be non-mutagenic in this assay.

In a mammalian cell micronucleus assay according to OECD guideline 487 of July 22, 2010, primary human lymphocyte cultures were exposed to TREGDMA (96.2%, doses adjusted to purity) in DMSO at the following concentrations:

Experiment IA – without S9 mix: 316.7, 554.2, 969.8 mg/L

Experiment IB – without S9 mix: 1300.0, 1400.0, 1600.0 mg/L

Experiment IIA – without S9 mix: 213.2, 373.2, 653.1 mg/L

Experiment IIB – without S9 mix: 400.0, 700.0, 900.0 mg/L

Experiment IA – with S9 mix: 969.8, 1697.1, 2970.0 mg/L

Experiment IIA – with S9 mix: 969.8, 1697.1, 2970.0 mg/L

TREGDMA was tested up to cytotoxic or limit concentrations (2970 mg/L correspond to 10 mM).In the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment IA, IB and IIA in the absence of S9 mix concentrations showing clear cytotoxic effects were not evaluable for cytogenetic damage. In Experiment IIB in the absence of S9 mix cytotoxicity was observed at the highest evaluated concentration (48.6 %).

In the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying micronuclei was observed. The micronucleus rates of the cells after treatment with the test item (0.15 - 1.15% micronucleated cells) were slightly above the range of the solvent control values (0.30 - 0.65% micronucleated cells) but within the range of the laboratory historical control data (0.10 – 1.65% micronucleated cells).

Positive controls induced the appropriate response.

There was no evidence of micronucleated cells induced over background. Therefore, TREGDMA is considered to be non-clastogenic in this in vitro micronucleus test, when tested up to cytotoxic or limit concentration.

  

In order to confirm the absence of a mutagenic potential, TREGDMA was recently tested in another HPRT test (Higher Methacrylate REACH Task Force, 2014) up to a maximum concentration of 2900 ug/mL (equals to 10 mM) according to OECD 476 and GLP. Moreover, based on the other data in the category and with other methacrylates, TREGDMA would not be expected to induce gene mutations in vivo. The overall data support the conclusion that TREGDMA is not mutagenic.

Supporting assessment of lower alkyl methacrylates (Albertini, 2017)

The above assessment is in line with a recent peer-reviewed publication which assessed the genotoxic potential of the so called lower alkyl methacrylates (Albertini, 2017). Its summary reads as follows: “The genotoxicity of seven mono-functional alkyl esters of methacrylic acid, i.e. methyl methacrylate, ethyl methacrylate, hydroxyethyl methacrylate, n-, i- and t-butyl methacrylate and 2 ethyl hexyl methacrylate,“(partially these substances are referenced in the toxicokinetic chapter5.1.1)”as well as methacrylic acid itself, the acyl component common to all, is reviewed and compared with the lack of carcinogenicity of methyl methacrylate, the representative member of the series so evaluated. Also reviewed are the similarity of structure, chemical and biological reactivity, metabolism and common metabolic products of this group of compounds which allows a category approach for assessing genotoxicity. As a class, the lower alkyl methacrylates are universally negative for gene mutations in prokaryotes but do exhibit high dose clastogenicity in mammalian cells in vitro. There is no convincing evidence that these compounds induce genotoxic effects in vivo in either submammalian or mammalian species. This dichotomy of effects can be explained by the potential genotoxic intermediates generated in vitro. This genotoxic profile of the lower alkyl methacrylates is consistent with the lack of carcinogenicity of methyl methacrylate.”

 

No human information is available for this endpoint. However, there is no reason to believe that the available results would not be applicable to humans.


Compliance to REACh requirements

Thein vitro mutagenicity test requirements are all covered with an Ames test withall required genetic markers, anin vitro Micronucleus test, and a HPRT assay. All mentioned studies are reliable (Reliability 1 or 2) and were performed with the substance itself. The in vivo mutagenicity test requirement is covered by consistent negative results of other category members (category read across, assessed as acceptable with high confidence).

References

Albertini, R.J. (2017). The lower alkyl methacrylates: Genotoxic profile of non-carcinogenic compounds. Regulatory Toxicology and Pharmacology, 84, 77-93


Justification for classification or non-classification

Based on the reliable available data, TREGDMA does not need to be classified for mutagenicity according to the criteria given in regulation (EC). Thus, no labelling is required.