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

Genetic toxicity: in vitro

Administrative data

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.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

Modified Small Vinyl Ester

IUPAC Name:

Modified Small Vinyl Ester

Details on test material:

- Name of test material (as cited in study report):Substance 3, NLP 500-090-6
- Substance type: Technical product
- Physical state: Viscous liquid
- Analytical purity: 100%
- Composition of test material, percentage of components: Approximately 18% mono adduct of BisGMA, Approximately 70% bis GMA, Approximately 10% Polymeric methacrylates (more Bisphenol-A’s + glycidyl in the chain)
- Lot/batch No.:7008043
- Expiration date of the lot/batch: 01 June 2011
- Stability under test conditions: Stable
- Storage condition of test material:In refrigerator (2-8°C) protected from light

Method

Target gene:

Not applicable to the test method followed.

Species / strain

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

Controls

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).

Results and discussion

Test results

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'.

Any other information on results incl. tables

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
333^b)	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
Control^a)	52	100
3	51	98
10	48	92
33	19	37
100	8	15
333^b)	0	0
1000^b)	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
333^b)	0	0
1000^b)	0	0
With metabolic activation (+S9-mix)
3 h exposure time, 24 h fixation time
Control^a)	45	100
3	47	104
10	44	98
33	46	102
100	42	93
333^b)	41	91

 

a) Dimethyl sulfoxide

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

Applicant's summary and conclusion

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.