Reaction products of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane with maleic anhydride and methacrylic acid

Genetic toxicity: in vitro

Administrative data

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:

comparable to guideline study

Justification for type of information:

A full read-across justification is attached to section 13 of the dossier - please refer to this document to find the full detailed argumentation in support of the analogue approach.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

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. The test substance 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

Test material

Test material information

Constituent 1

Reference substance name:

Small Vinyl Ester

IUPAC Name:

Small Vinyl Ester

Details on test material:

- Name of test material (as cited in study report): Bisphenol A-diglycidyl dimethacrylate
- Source: Obtained from Röhm, Darmstadt, Germany - Most likely comparable to Small Vinyl Ester
- Molecular structure formula: Presented in Fig 1 in the article
- Molecular weight: Not presented
- Substance type: High molecular weight monomer for resin polymers
- Physical state: Not described
- Analytical purity: Not described
- Composition of test material, percentage of components: Not decribed
- Purity test date: Not described
- Lot/batch No.: Not described
- Expiration date of the lot/batch: Not described
- Stability under test conditions: Not described
- Storage condition of test material: Not described

Method

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

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 97a

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not specified

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not specified

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 102

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

Controls

Controls 1

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

Controls 2

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

Controls 3

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

Controls 4

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.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 97a

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.

Test results 2

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.

Test results 3

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.

Test results 4

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'.

Any other information on results incl. tables

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 ± 28^a	170 ± 29	174 ± 21
	12.5	116 ± 7^a	90 ± 54^a	19 ± 7^a	14 ± 7^a	113 ± 11^a	58 ± 7^a	160 ± 21	127 ± 25^a
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

Applicant's summary and conclusion

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.