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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance gave negative results in an in vitro bacterial mutation assay, two in vitro gene mutation assays and an in vitro micronucleus assay. Testing on a structurally related substance gave negative results in an in vitro cytogenetics assay.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
7 January 2011 - 6 March 2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with no deviation. Compliant with GLP. Read-across study.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: European Community (EC). Commission regulation (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.10: "Mutagenicity - In Vitro Mammalian Chromosome Aberration Test".
Deviations:
no
Principles of method if other than guideline:
The study procedures used were based on the most recent OECD and EC guidelines.
Modified Small Vinyl Ester was a clear light yellow-greenish to light brown, highly viscous liquid. Modified Small Vinyl Ester was dissolved in dimethyl sulfoxide. In the first cytogenetic assay, Modified Small Vinyl Ester was tested up to 333 μg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. Modified Small Vinyl Ester precipitated in the culture medium at this dose level. In the second cytogenetic assay, Modified Small Vinyl Ester was tested up to 30 μg/ml for a 24 h and 48 h continuous exposure time with a 24 h and 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at this dose level. In the presence of S9-mix Modified Small Vinyl Ester was tested up to 333 μg/ml for a 3 h exposure time with a 48 h fixation time. Modified Small Vinyl Ester precipitated in the culture medium at this dose level.

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable to the test method followed.
Species / strain / cell type:
lymphocytes: human cultured lymphocytes
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with
Metabolic activation system:
S9-fraction prepared from livers of adult male Wistar rats orally dosed at three consecutive days with a suspension of phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw) in corn oil.
Test concentrations with justification for top dose:
The test concentrations were 33, 100 and 333 μg/ml culture medium with and without S9-mix
The exposure time was 3 h, and fixation time was 24 h .
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Solvent for Modified Small Vinyl Ester: dimethyl sulfoxide (DMSO)
Solvent for positive controls: Hanks’ Balanced Salt Solution (HBSS) (Invitrogen Corporation, Breda, The Netherlands), without calcium and magnesium.
- Justification for choice of solvent/vehicle: Good solubility in DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
none
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension

DURATION
- Preincubation period: 48 h before exposure
- Exposure duration: first cytogenetic assay: 3 h with and without S9-mix
second cytogenetic assay: 3 h with S9-mix
24 h and 48 h without S9-mix
- Expression time (cells in growth medium): 44-46 h, when exposed in presence of S9-mix
0 h, when exposed in the absence of S9-mix
- Fixation time (start of exposure up to fixation or harvest of cells): 48 h, when exposed in the presence of S9-mix
24 h or 48 h exposed in the absence of S9-mix

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF CELLS EVALUATED: 200

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other:

Evaluation criteria:
A test substance was considered positive (clastogenic) in the chromosome aberration test if: a) It induced a dose-related statistically significant.b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.Modified Small Vinyl Ester is considered not clastogenic, if none of the tested concentrations induced a statistically significant increase in the number of cells with chromosome aberrations.
Statistics:
Statistical procedures:
Dose-related statistically significant increase in the number of cells with chromosome aberrations was tested with Chi-square test, one-sided (p < 0.05).
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: At a concentration of 333 μg/ml Modified Small Vinyl Ester precipitated in the culture medium. In the dose range finding study, at the 3 h exposure time, blood cultures were treated with 3, 10, 33, 100 and 333 μg/ml culture medium with and without S9-mix. At the 24 h and 48 h continuous exposure time blood cultures were treated with 3, 10, 33, 100, 333 and 1000 μg/ml culture medium without S9-mix. Modified Small Vinyl Ester was tested beyond the limit of solubility to obtain adequate toxicity data.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Based on the results of the dose range finding test the following dose levels were selected for the
cytogenetic assay: Without and with S9-mix: 33, 100 and 333 μg/ml culture medium (3 h exposure time, 24 h fixation time).

To obtain more information about the possible clastogenicity of Modified Small Vinyl Ester, a second cytogenetic assay was performed in which human lymphocytes were continuously exposed to Modified Small Vinyl Ester in the absence of S9-mix for 24 or 48 hours. In the presence of S9-mix, cells were fixed after 48 hours following a 3 hour exposure to Modified Small Vinyl Ester. The following dose levels were selected for the second cytogenetic assay: Without S9-mix : 5, 10, 20, 25, 30, 35, 40, 45, 50 and 75 μg/ml culture medium (24 h and 48 h exposure time, 24 h and 48 h fixation time). With S9-mix : 30, 100 and 333 μg/ml culture medium (3 h exposure time, 48 h fixation time).
Remarks on result:
other: other: cultured peripheral human lymphocytes
Remarks:
Migrated from field 'Test system'.

Dose range finding test

At a concentration of 333 μg/ml Modified Small Vinyl Ester precipitated in the culture medium. In the dose range finding study, at the 3 h exposure time, blood cultures were treated with 3, 10, 33, 100 and 333 μg/ml culture medium with and without S9-mix. At the 24 h and 48 h continuous exposure time blood cultures were treated with 3, 10, 33, 100, 333 and 1000 μg/ml culture medium without S9-mix. Modified Small Vinyl Ester was tested beyond the limit of solubility to obtain adequate toxicity data. Table 1 shows the mitotic index of cultures treated with various concentrations of Modified Small Vinyl Ester or with the negative control substance.

Table 1 Mitotic index of human lymphocyte cultures treated with Modified Small Vinyl Ester in the dose range finding test

 

Period of treatment: From: 12-01-2011 to: 14-01-2011

Modified Small Vinyl Ester

concentration (μg/ml)

Number of metaphases per 1000 cells

Absolute

Percentage of control

Without metabolic activation (-S9-mix)

 

 

3 h exposure time, 24 h fixation time

 

 

Control a)

42

100

3

47

112

10

41

98

33

46

110

100

49

117

333b)

51

121

24 h exposure time, 24 h fixation time

24 h exposure time, 24 h fixation time

24 h exposure time, 24 h fixation time

Controla)

52

100

3

51

98

10

48

92

33

19

37

100

8

15

333b)

0

0

1000b)

0

0

48 h exposure time, 48 h fixation time

 

 

Control a)

45

100

3

47

104

10

50

111

33

22

49

100

6

13

333b)

0

0

1000b)

0

0

With metabolic activation (+S9-mix)

3 h exposure time, 24 h fixation time

 

 

Controla)

45

100

3

47

104

10

44

98

33

46

102

100

42

93

333b)

41

91

 

a) Dimethyl sulfoxide

b) Modified Small Vinyl Ester precipitated in the culture medium.

Conclusions:
Interpretation of results (migrated information):
negative

The substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence or presence of S9-mix in either of the two independently repeated experiments. No effects of the substance on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that the substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report. Finally, it is concluded that this test is valid and that the substance is not clastogenic in human lymphocytes.
Executive summary:

The ability of Modified Small Vinyl Ester to induce chromosome aberrations in cultured peripheral human lymphocytes was tested in a guideline study (OECD 473) in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix).

The possible clastogenicity was tested in two independent experiments. The substance was dissolved in dimethyl sulfoxide. In the first cytogenetic assay, the substance was tested up to 333 μg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. The substance precipitated in the culture medium at this dose level. In the second cytogenetic assay, the substance was tested up to 30 μg/ml for a 24 h and 48 h continuous exposure time with a 24 h and 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at this dose level. In the presence of S9-mix the substance was tested up to 333 μg/ml for a 3 h exposure time with a 48 h fixation time. The substance precipitated in the culture medium at this dose level. The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly and that the test was valid. The substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments. No effects of the substance on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that the substance did not disturb mitotic processes and cell cycle progression and did not induce numerical chromosome aberrations. Modified Small Vinyl Ester was not clastogenic in human lymphocytes under the experimental conditions described in this report.
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Manuscript submitted February 10th , 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
Positive control substance in the prescence of metabolic activation: 9,10-dimethyl-1,2- benzanthracene, rather than 7,12-dimethylbenzanthracene; test-sbstance description
Principles of method if other than guideline:
Study conducted according to:
Bradley M.O., Bhuyan B., Francis M.C., Langenbach R., Peterson a., Huberman E.: Mutagenesis by chemical agents in V79 Chinese hamster cells: A review and analysis of the literature. Mutation Research 87 (1981) 81-142

and:

Nestmann E.R., Brillinger R.L., Gilman J.P., Rudd C.J., Swierenga S.H.: recommended protocols based on a survey of current practice in genotoxicity testing laboratories: II. Mutation in Chinesse hamster ovary, V79 Chinese hamster lung and L5178Y mouse lymphoma cells. Mutation Research 246(1991)255-284
GLP compliance:
not specified
Remarks:
University laboratory, probably not GLP compliance
Type of assay:
mammalian cell gene mutation assay
Target gene:
The hprt locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: Minimal Essential Medium (MEM) supplemented with 10% fetal calf serum, penicillin (100 U/ml) and streptomycin (100 µg/ml). V79B cells from identical stoc cell cultures were stored in liquid nitrogen untill use. The V79B cells were a gift from Prof. G. Speit at the University of Ulm, Germany.
- Properly maintained: Not described in article
- Periodically checked for Mycoplasma contamination: Not described
- Periodically checked for karyotype stability: Not described
- Periodically "cleansed" against high spontaneous background: Not described
Additional strain / cell type characteristics:
not specified
Remarks:
Mutation spectra described in Speit G., Habermeier B., Helbig R.: Differences in the response to mutagens between two V79 sublines. Mutation Research 325(1994) 105-111
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-naphtoflavone induced enzymatic activities bound to microsomal fraction (S9) of rat liver (18 ml of S9-mix containing 10% microsomal fraction, protein concentration was about 30 mg/ml).
Test concentrations with justification for top dose:
0.0, 25, 50, 75 µM in the test solution.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO.
- Stock solution: 1 M Bis-GMA dissolved in DMSO
- Justification for choice of solvent/vehicle: Not described
Untreated negative controls:
yes
Remarks:
Bis-GMA 0.00 µM
Negative solvent / vehicle controls:
yes
Remarks:
Solvent control (DMSO), data not shown
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
In the absence of S9 microsomal fraction
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 9,10-dimethyl-1,2-benzanthracene
Remarks:
In the presence of S9 microsomal fraction
Details on test system and experimental conditions:
METHOD OF APPLICATION and EXPOSURE DURATION: 1.5 x 106 V79B cells were plated onto 150 mm diameter cell culture plates with 30 ml cell culture medium, and allowed to incubate for 20-24 hours at 37°C in a 5% CO2 atmosphere. The time of plating was the seeding time.
Then exposure occurred by adding either A) 30 ml cell culture medium containing test substance (concentration 0.00, 25, 50, or 75 µM Bis-GMA) for 24 hours, or B) 18 ml cofactor solution (containing 5.3 mM glucose 6-phosphate, 4.2 mM NADP+, 8.5 mM MgCl2, 15.9 mM KCl, 154.6 mM sodium phosphate buffer (pH 7.4)) and the test substance (concentration 0.00, 25, 50, or 75 µM Bis-GMA) for 4 hours, with or without S9 microsomal fraction (stimulated with Phenobarbital/ß-naphtoflavone from rat liver, protein content 30 mg/ml) .
Exposure was stopped by replacing the exposure medium with fresh cell culture medium.

DURATION
- Preincubation period: No Preincubation was done
- Expression time (cells in growth medium): The cells were sub-cultured on day four after seeding. Cell numbers were counted and expressed relative to cell counts in solvent controls, indication the plating efficiency (PE1) to measure the acute cytotoxicity.
3 x 106 cells from each experimental culture plate were sub-cultured in new 150 mm diameter cell culture plates. The cells were sub-cultured again on day 8 after seeding.
Mutant isolation started on day 10 after seeding by replating the cells in a selective medium containing 6-thioguanine (7 µg/ml). The cells were seeded at a density of 2 x106 cells /plate in three replicate cultures per dose of test substance. This was the end of the expression period. Cell colonies were fixated and stained with 0.1% methylene blue in 70% ethanol after 10 days (from seeding on selection medium).
To establish the cloning efficiency (PE2) 300 cells in medium without 6-thioguanine were plated separately (three replicate cultures per dose of test substance) and incubated for 8 days before fixation and staining.

SELECTION AGENT (mutation assays): 6-thioguanine, 7 µg/ml

NUMBER OF REPLICATIONS: The experiment was replicated once, triple assays for each test substance concentration with/without S9 microsomal fraction was performed in each experiment.

NUMBER OF CELLS EVALUATED: 2 x106

DETERMINATION OF CYTOTOXICITY
- Method: Plating efficiency (PE1) and cloning efficiency (PE2)

OTHER EXAMINATIONS:
- Determination of polyploidy: No
- Determination of endoreplication: No
- Other: the mutagenicity of Bis-GMA in S. Typhimurium was analyses in a parallel Ames-test.

OTHER:
- Chemicals examined in the same experiment: Methyl methacrylate (CAS No. 80-62-6); 2-hydroxyethyl methacrylate (CAS No. 868-77-9); 2,2-bis(4-hydroxyphenyl)-propane (Bisphenol A, CAS No. 80-05-7); 2,3-epoxypropyl methacrylate (GMA, CAS No. 106-91-2); Urethane dimethacrylate (CAS No. 72869-86-4); Triethylene glycol dimethacrylate (CAS No. 109-16-0).
Evaluation criteria:
Counting of the number of cells at the end of the exposure period was used to calculate the plating efficiency (PE1).
Counting the number of colonies at the end of the selection period was used to calculate the number of cells per 6 x 106 cells and the cloning efficiency (PE2).
Statistics:
No statistical methods applied.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Dose-dependent reduction in the number of cells after the exposure period and reduced plating efficiency (PE1) indicate acute toxicity. Recovery during the selection period is indicated by cloning efficiency (PE2) values of 92-111.
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH in the test medium (nominal): 7.4. No data on changes in pH of the test medium during the incubation period were presented.
- Effects of osmolality: No data presented.
- Evaporation from medium: No data presented.
- Water solubility: No data presented, but the limited solubility could be an issue.
- Precipitation: No data on precipitation presented.
- Other confounding effects: The positive control substance for incubation with S9 microsomal fraction was 9,10-dimethyl-1,2-benzanthracene, rather than 7-12-dimethylbenzanthracene (according to OECD guideline no. 476).

RANGE-FINDING/SCREENING STUDIES: Not described

COMPARISON WITH HISTORICAL CONTROL DATA: No data on the negative controls are listed.
The data on the positive controls are listed, but no comparison to historical values was presented.
COMPARISON WITH HISTORICAL DATA: The results indicating that Bis-GMA is cytotoxic but not mutagenic in mammalian V79b-cells in vitro, were compared with the data from the Ames test (not mutagenic, but cytotoxic in S. typhimurium) and from a previous study indicating that Bis-GMA is genotoxic in HeLa cells (Heil, J.; Reifferscheid, G.; Waldmann, P.; Leyhausen, G.; Geurtsen, W: Genotoxicity of dental materials, Mutation research 368 (1996) 181-194).

ADDITIONAL INFORMATION ON CYTOTOXICITY: No
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table: Mutagenicity of dental resin components in V79B cells

Test compound

Concentration tested

Cell number

PE1

PE2

Number of mutants

Mutant frequency

 

µM

%

%

%

6 x 106cells

106survivors

Bis-GMA

0.0

100

100

100

14

6

 

25

91

122

98

19

6

 

50

36

84

92

17

6

 

75

21

41

111

7

2

Pos. control

 

 

95

 

1995

635

Cell cultures were treated for 24 hours in the absence of a metabolically active microsomal fraction (S9) from rat liver (10%). Initial cytotoxicity of the test compounds on day 1 after exposure is indicated by cell number plating efficiencies (PE 1) in percent. Mutant frequencies in untreated and treated cell cultures are expressed per 106clonable cells.

Pos. control = 200 µg/ml ethylmethane sulphonate.

: indicates that in the article the table includes data from experiments with other testsubstances including Methyl methacrylate (CAS No. 80-62-6); 2-hydroxyethyl methacrylate (CAS No. 868-77-9); 2,2-bis(4-hydroxyphenyl)-propane (Bisphenol A, CAS No. 80-05-7); 2,3-epoxypropyl methacrylate (GMA, CAS No. 106-91-2); Urethane dimethacrylate (CAS No. 72869-86-4); Triethylene glycol dimethacrylate (CAS No. 109-16-0).

Conclusions:
Small Vinyl Ester was tested in the in vitro mammalian cell gene mutation assay using V79B Chinese Hamster Lung Fibroblast cells (targeting the hprt gene), and found to be non-mutagenic in the experimental conditions. Data indicate acute cytotoxicity at concentrations from 50 µM and above.
Executive summary:

The mutagenicity of Small Vinyl Ester was tested in the in vitro mammalian cell gene mutation assay using the V79B Chinese hamster lung fibroblast cell line. The target gene was the hprt-gene site, in which mutation results in sensitivity to 6-thioguanine.

1.5 x 106 V79B-cells were plated and cultured for 24 hours prior to exposure. A stock solution of 1 M Small Vinyl Ester was made in DMSO and used for dissolving the test substance in the cell culture medium or the cofactor solution (containing 5.3 mM glucose 6-phosphate, 4.2 mM NADP+, 8.5 mM MgCl2, 15.9 mM KCl, 154.6 mM sodium phosphate buffer (pH 7.4)) with or without phenobarbital/ß-naphtoflavone induced S9 microsomal fraction from rat liver. The exposure doses were 0.00, 25, 50, and 75 µM in the cell culture medium or cofactor solution (with or without S9 fraction) used to expose the cells for 24 hours or 4 hours, respectively. Then the cells were cultured in fresh cell culture medium until day 4, when the cell numbers were analysed and the cells sub-cultured by plating 3 x 106 cells on fresh culture plates with cell culture medium. On day 8 cells were sub-cultured once more, and on day 10, mutant selection was started by plating 2 x 106 cells and incubating them in a medium containing 7 µg/ml 6-thioguanine. After 10 days of selective culturing the number of mutant cell colonies were counted. Other endpoints in the study were the plating efficiency (PE1) calculated from the cell number data at day 4, and the cloning efficiency (PE2) calculated from the cell number obtained after incubating 300 cells in selective culture medium for 8 days.

Each culture/dose analysis was performed in triplicate in the experiment, and the experiment was repeated at least once.

Negative controls and positive controls were included, and the characteristics of the tester strain had been described in a previously published study.

 

The results indicate that Small Vinyl Ester is not mutagenic to the tested V79B cell strain under the test conditions. Data indicate acute cytotoxicity, that was reversible after cessation of the exposure period. The findings were in accord with data from a simultaneously conducted Ames test, whereas SMall Vinyl Ester was genotoxic to HeLa-cells in a previously published study.

 

The reported method is equivalent to the method described in OECD guideline for testing of chemicals no. 476, but deviated on the following points:

  • Description of the test substance only encomprised identification by name and CAS number.
  • One of the substances used for positive control of mutagenicity were different from that recommended in the OECD guideline.
  • Description of the testing and data on negative controls, including solvent (DMSO) control, is very superficial.
  • Only average data from the replicated assays are reported, and not the individual data (as recommended by OECD guideline 471).
  • The study was not performed according to GLP.
Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Literature publication with limited reporting of study details. No details given regarding GLP compliance. The substance was only tested without metabolic activation.
Qualifier:
no guideline available
Principles of method if other than guideline:
At the time the study was conducted there was no OECD/EU test guideline available. A total of seven resin monomers, including bis-GMA, were assessed for their potential to cause micronuclei in V79 cells. The substances were initially tested without metabolic activation then those that gave rise to increased levels of micronuclei were also tested in the precense of metabolic activation. Bis-GMA was negative so was only tested without metabolic activation.
GLP compliance:
not specified
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Metabolic activation system:
Not applicable
Test concentrations with justification for top dose:
0, 25, 50 and 75 umol/L
Vehicle / solvent:
DMSO and subsequent dilution in culture medium
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Several of the monomer substances being tested also gave a positve response in this study
Details on test system and experimental conditions:
Exposure duration was 24 hours
Following exposure the cells were re-incubated for 24 hours
Evaluation criteria:
Micronuclei were analysed microscopically in 1000 cells per slide of 3 parallel cultures (slides) per concentration
Statistics:
Not reported
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
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:
Slightly increased numbers of micronuclei were reported at concentrations higher than the determined TC50 (concentration leading to 50% cell sruvival, 33.5 umol/L).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative without metabolic activation

The authors conclude that there is no evidence of mutagenic activity for the substance.
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:
February 16 - April 13, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD and EU) with insignificant deviations.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
insignificant
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
insignificant
GLP compliance:
yes (incl. QA statement)
Remarks:
The certificate have not been signed
Type of assay:
mammalian cell gene mutation assay
Target gene:
TK
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Properly maintained: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
s9 mix
Test concentrations with justification for top dose:
3,10,33,100 and 333 micrograms/ml in the dose range finding study
1,3,10,30,60,80,90 and 95micrograms/ml in first main experiment without metabolic activation
0.1,0.3,1,3,10,30,75 and 100 micrograms/ml in first main experiment with metabolic activation
0.3,1,3,10,33,140,180 and 200 micrograms/ml in second main experiment without metabolic activation
0.1,0.3,1,3,10,30,75 and 100 micrograms/ml in second main experiment with metabolic activation
Vehicle / solvent:
ethanol
Untreated negative controls:
yes
Remarks:
ethanol
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Migrated to IUCLID6: MMS (without metabolic activation) and Cyclophosphamide (CP, with metabolic activation)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in suspension

DURATION
- Preincubation period:
- Exposure duration: 3 and 24 hours
- Expression time (cells in growth medium):48 hours
- Selection time (if incubation with a selection agent): 11-12days
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays): Triflourothymidine (TFT)



NUMBER OF REPLICATIONS: 1 replicate of each concentration, in expression time, five replicates of each concentration,

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; Relative Suspension Growth, Cloning efficiency, Relative survival and relative growth rate

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:

OTHER:
Evaluation criteria:
The mutation assay was considered acceptable if it met the following criteria;
A. The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120%. An acceptable number of surviving cells (E+6) could be analysed for expression of the TK mutation.
B. The spontaneous mutation frequency in the solvent control is =50E-6 and = 170E-6.
C. The growth rate (GR) over the 2-day expression period for the negative control should be between 8 and 32 (3 hours treatment) and between 32-180 (24 hours treatment).
D. The mutation frequency of MMS should not be below 500E-6, and for CP not below 700E-6.
E. Any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
The test substance is considered positive (mutagenic) in the mutation assay, if it induces a MF of more than MF(control) + 126 (Global Evaluation Factor) in a dose-dependent manner.
The test substance is considered equivocal /questionable if no clear conclusion for positive or negative results can be drawn.
The test substance is considered negative (not mutagenic) if; A. None of the tested concentrations reaches a mutation frequence of MF(control) + 126. and B. The results are confirmed in an independently repeated test.
Statistics:
None were applied as the results gave no indication of mutation frequency different from solvent control.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS

- Water solubility: se Precipitation
- Precipitation: In the dose finding study the substance precipitated in 100 µg/ml and above. In experiment 1 the substance precipitated in 60 µg/ml and above, in experiment 2 the substance were observed precipitating in 33 µg/ml without metabolic activation, and 75 µgml with metabolic activation
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES: A range finding study was performed, and cytotoxicity was observed at 100 µg/ml and above.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the 3 hours dose range finding test, cytotoxicity (as Relative Suspension Growth) was observed at 100 µg/ml and above, without metabolic activation, and at 333 µg/ml with metabolic activation. I the 24 hours dose range finding test, cytotoxicity was observed at 100 µg/ml without metabolic activation.
In experiment 1, 3 hours treatment indications of cytotoxicity was observed at 60 µg/ml and above in a dose dependent manner, without metabolic activation, no indication were observed with metabolic activation.
In experiment 2, 24 hours treatment indications of cytotoxicity was observed at 140 µg/ml and above in a dose dependent manner, without metabolic activation, no indication were observed with metabolic activation.

Remarks on result:
other: strain/cell type: Thymidine kinase locus in L5178Y mouse lymphoma cells
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

The experimental evaluation of the mutagenic activity of Small Vinyl Ester were performed following OECD Guideline 476, with only minor deviations, not considered relevant for the outcome of the experiments. Small Vinyl Ester was not found mutagenic under the given test conditions.
Executive summary:

The experimental evaluation of the mutagenic activity of Small Vinyl Ester was performed in 3 steps.

First a 3 hours dose range finding test, were cytotoxicity (as Relative Suspension Growth) was observed at 100 µg/ml and above, without metabolic activation, and at 333 µg/ml with metabolic activation. In the following 24 hours dose range finding test, cytotoxicity was observed at 100 µg/ml without metabolic activation.

The first mutagenicity test 3 hours treatment indications of cytotoxicity was observed at 60 µg/ml and above in a dose dependent manner, without metabolic activation, no indication were observed with metabolic activation. There were no signs of mutagenicity in a dose dependent manner

In experiment 2, 24 hours treatment indications of cytotoxicity was observed at 140 µg/ml and above in a dose dependent manner, without metabolic activation, no indication were observed with metabolic activation. There were no signs of mutagenicity in a dose dependent manner.

Small Vinyl Ester was found not mutagenic under the given test conditions

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Manuscript received February 10th 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Four strains of S. Typhimurium (Ta97a, TA98, TA100, TA102) instead of five. Bis GMA precipitated when mixed with the top-agar and plated. Individual colony counts for the plates are not provided in the report.
Principles of method if other than guideline:
Experiment conducted as described in: Maron DM, Ames BN. Revised methods for the Salmonella mutagenicity test. Mutation Research. 1983;113:173-215
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
The test was conducted with four different Salmonella test strains to detect base-pair substitutions at GC- and AT-rich sequences and frame shift mutations (leading to restoration of ability to produce histidine).
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium, other: TA97a
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-naphtoflavone induced enzymatic activities bound to microsomal fraction (S9) of rat liver (10% corresponding to 1.25 mg/plate).
Test concentrations with justification for top dose:
Concentration of test substance in mg per plate: 0.00; 0.25; 0.50; 1.25; 5.00; and 12.5
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: Not described
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO only
True negative controls:
not specified
Positive controls:
yes
Remarks:
Differing depending on S. typhimurium strain.
Positive control substance:
other: 2,4,7-trinitrofluorenon (0.5 µg/plate)
Remarks:
For TA97a and TA98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO only
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: NaN3 (10 µg/plate)
Remarks:
For TA100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO only
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: Glutaraldehyde (50 µl of 0.05% /plate)
Remarks:
For TA102
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO only
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (10 µg/plate)
Remarks:
For TA97a, TA98, TA100, and TA102 when S9 microsomal fraction was added.
Details on test system and experimental conditions:
Details on test system:
METHOD OF APPLICATION: In agar (plate incorporation).
NOTE: Bis-GMA precipitated when the compound was mixed with the top agar and plated

DURATION
- Preincubation period: No preincubation was conducted
- Exposure duration: 3 days at 37°C
- Expression time (cells in growth medium): 3 days at 37°C (same as exposure duration)
- Selection time (if incubation with a selection agent): No selection agent was used.
- Fixation time (start of exposure up to fixation or harvest of cells): 3 days at 37°C (same as exposure duration)

SELECTION AGENT (mutation assays): Histidine deficient medium

NUMBER OF REPLICATIONS: Triple analysis in each experiment (three plates per experiement). The experiment was repeated at least once.

NUMBER OF CELLS EVALUATED: Not described in article, but details on the method is described in a reference: Maron, D.M; Ames, B.N.: Revised methods for the Salmonella mutagenicity test. Mutation Research 222 (1983) 173-215

DETERMINATION OF CYTOTOXICITY
- Method: No data or method for analysis of cytotoxicity in bacteria described. Cytotoxicity in mammalian cells was evaluated in a mammalian cell gene mutation assay conducted in V79 Chinese hamster lung fibroblast cell line.

OTHER EXAMINATIONS:
- Determination of polyploidy: not relevant
- Determination of endoreplication: not relevant.
- Other: Data from a Mammalian cell gene mutation assay conducted in V79 Chinese hamster lung fibroblast cell line is reported alongside the data from the Ames test.

OTHER:
- Chemicals examined in the same experiment: Methyl methacrylate (CAS No. 80-62-6); 2-hydroxyethyl methacrylate (CAS No. 868-77-9); 2,2-bis(4-hydroxyphenyl)-propane (Bisphenol A, CAS No. 80-05-7); 2,3-epoxypropyl methacrylate (GMA, CAS No. 106-91-2); Urethane dimethacrylate (CAS No. 72869-86-4); Triethylene glycol dimethacrylate (CAS No. 109-16-0).
Evaluation criteria:
The number of mutant colonies (revertants) per plate. The data from parallel triplicate analyzes were reported as mean values ± S.D.
Statistics:
Not described.
Species / strain:
S. typhimurium, other: TA97a
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Tested at precipitating concentrations. cytotoxic at 12.5 mg/plate
Vehicle controls validity:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
other: Yes, but the data is difficult to assess as an alternative positive control substance is used.
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Tested at precipitating concentrations. cytotoxic at 12.5 mg/plate
Vehicle controls validity:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
other: Yes, but the data is difficult to assess as an alternative positive control substance is used.
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Tested at precipitating concentrations. Not cytotoxic without S9, but cytotoxic at 5.0 mg/plate with S9.
Vehicle controls validity:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
other: Yes, but the data is difficult to assess as an alternative positive control substance is used.
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Tested at precipitating concentrations. cytotoxic at 12.5 mg/plate with S9, but not toxic without S9
Vehicle controls validity:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
other: Yes, but the data is difficult to assess as an alternative positive control substance is used.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not Described
- Effects of osmolality: Not described
- Evaporation from medium: Not relevant
- Water solubility: Not described
- Precipitation: Precipitation occurred, the effect of this was hard to assess.
- Other confounding effects: 2-aminoanthracene is used as the sole positive control substance for testing the efficacy of the S9 microsomal fraction. According to OECD guideline No. 471 a second positive control substance, e.g. benzo(a)pyrene or dimethylbenzanthracene, is needed.

RANGE-FINDING/SCREENING STUDIES: Not described. The conclusion that the observed reduction in colony number (at 12.5 mg/plate) was caused by cytotoxicity, was based on a previous study : Heil, J.; Reifferscheid, G.; Waldmann, P.; Leyhausen, G.; Geurtsen, W: Genotoxicity of dental materials, Mutation research 368 (1996) 181-194

COMPARISON WITH HISTORICAL CONTROL DATA: Not all results from the negative and positive controls are reported. Not comparison to historical controld data has been done.
COMPARISON WITH HISTORICAL DATA: The negative mutagenesis data are in accord with data from A) the mammalian cell gene mutation assay and B) a previous study: Heil, J.; Reifferscheid, G.; Waldmann, P.; Leyhausen, G.; Geurtsen, W: Genotoxicity of dental materials, Mutation research 368 (1996) 181-194

ADDITIONAL INFORMATION ON CYTOTOXICITY: The conclusion that the observed reduction in colony number (at 12.5 mg/plate) was caused by cytotoxicity, was based on A) data of the mammalian cell gene mutation assay indicating Cytotoxicity, and B) a previous study : Heil, J.; Reifferscheid, G.; Waldmann, P.; Leyhausen, G.; Geurtsen, W: Genotoxicity of dental materials, Mutation research 368 (1996) 181-194
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

Table 1: Mutagenicity of Bisphenol A diglycidyl dimethacrylate (Bis-GMA) in S. typhimurium tester strains TA97a, TA98, TA100, and TA102

Test compound

Concen-tration

S. typhimurium

TA97a

TA98

TA100

TA102

 

mg/plate

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

Bis-GMA

0.00

154 ± 26

164 ± 32

31 ± 7

36 ± 4

125 ± 18

136 ± 9

178 ± 15

305 ± 76

 

0.25

n.t.

n.t.

29 ± 10

38 ± 8

133 ± 14

148 ± 21

226 ± 25

298 ± 37

 

0.50

131 ± 26

151 ± 25

22 ± 11

34 ± 13

139 ± 26

128 ± 5

207 ± 22

290 ± 9

 

1.25

136 ± 16

213 ± 14

30 ± 3

30 ± 2

127 ± 8

112 ± 11

182 ± 33

216 ± 27

 

5.00

128 ± 66

180 ± 29

28 ± 3

18 ± 9

124 ± 13

80 ± 28a

170 ± 29

174 ± 21

 

12.5

116 ± 7a

90 ± 54a

19 ± 7a

14 ± 7a

113 ± 11a

58 ± 7a

160 ± 21

127 ± 25a

Positive control

1282 ± 144

908 ± 35

2188 ± 122

1388 ± 18

1150 ± 67

1502 ± 65

1016 ± 37

926 ± 10

The test compound (100 mg) was dissolved in 2 ml dimethyl sulfoxide and various aliquots were then tested for mutagenicity in the standard plate incorporation assay. The numbers of mutant colonies (revertants) per plate are mean values (± S.D.) from triplicates obtained in one experiment. The experiment was repeated at least once. Positive controls were as follows: 0.5 µm 2,4,7-trinitrofluorenon/plate (TA97a, TA98), 10 µg NaN3 /plate (TA100) and 50 µl 0.05% glutaraldehyde / plate (TA102) in the absence and 10 µg 2-aminoanthracene / plate in the presence of S9. Bis-GMA precipitated when the compound was mixed with top agar and plated.

a: Toxic

n.t.: Not tested

Conclusions:
Small Vinyl Ester was found not mutagenic as tested in an Ames-test, using four different strains of Salmonella typhimurium (TA97a, TA98, TA100, and TA102) with and without addition of phenobarbital/ß-naphtoplavone induced enzymatic activities bound to microsomal fraction (S9) from rat liver. Cytotoxicity was observed at concentrations of 12.5 mg/plate in all test strains when incubated with S9 mitochondrial fraction.
Executive summary:

The mutagenicity of Small Vinyl Ester was tested in an Ames-test, using four different strains of Salmonella typhimurium (TA97a, TA98, TA100, and TA102). This assay tests the ability of a chemical to induce point mutations including substitution, addition or deletion of one or a few DNA base pairs at GC- or AT-rich sites, shifting the reading frame of genes. This is detected as mutations which revert mutations in the bacteria strains, restoring the capability to synthesize histidine and to grow in a histidine free culture medium.

A stock solution was created by dissolving 100 mg Bis-GMA in 2 ml of DMSO. The exposure doses of 0.00, 0.25, 1.25, 2.50, 5.00, and 12.5 mg Small Vinyl Ester per plate was obtained by adding different aliquots of the stock solution to the test mixtures containing bacteria and plating the test mixture on histidine deficient agar plates. The experiment was conducted with or without addition of phenobarbital/ß-naphtoplavone induced enzymatic activities bound to microsomal fraction (S9) from rat liver (in a concentration of 10% corresponding to 1.25 mg protein per plate). The Small Vinyl Ester precipitated upon plating onto the agar medium. Following 3 days incubation at 37°C, the number of revertant colonies was counted.

In an experiment, analysis was performed in triplicate, and the experiment was repeated at least once.

Negative controls and positive controls were included, and the genotype of the tester strains was confirmed in the experiments.

 

The results indicate that Small Vinyl Ester is not mutagenic to the tested bacterial strains under the test conditions. Data indicating cytotoxicity was found in all tested strains at the 12.5 mg/plate test concentration with metabolic activity (S9 mitochondrial fraction), whereas cytotoxicity was found in TA97a and TA98 without metabolic activity.

 

The reported method is equivalent to the method described in OECD guideline for testing of chemicals no. 471, but deviated on the following important points:

  • Description of the test substance only encomprised identification by name and CAS number.
  • Only four strains of S. typhimurium was used (should be five as recommended by the OECD guidline 471)
  • The substances used for positive control of mutagenicity were different from those recommended in the guideline.
  • Description of the testing and data on negative controls, including solvent (DMSO) control, is very superficial.
  • Only average data from the replicated assays are reported, and not the individual data (as recommended by OECD guideline 471).
  • The study was not performed according to GLP.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the findings of a number of reliable in vitro gentoxicity assays conducted on the substance and an analogue, classification of the substance is not justified.