[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05-Dec-2012 to 14-Jan-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of (1-methylethylidene)bis(4,1-phenyleneoxy-2,1-ethanediyl) bismethacrylate and 2-{4-[2-(4-{2-[2-(methacryloyloxy)ethoxy]ethoxy}phenyl)propan-2-yl]phenoxy}ethyl methacrylate

EC Number:

939-702-5

Molecular formula:

C23H24O4 (C2H4O)n

IUPAC Name:

Reaction mass of (1-methylethylidene)bis(4,1-phenyleneoxy-2,1-ethanediyl) bismethacrylate and 2-{4-[2-(4-{2-[2-(methacryloyloxy)ethoxy]ethoxy}phenyl)propan-2-yl]phenoxy}ethyl methacrylate

Test material form:

other: Clear colourless highly viscous liquid

Details on test material:

- Name of test material (as cited in study report): 2 moles ethoxylated bisphenol A dimethacrylate

Method

Target gene:

n/a

Species / strain

Species / strain / cell type:

lymphocytes: human peripheral blood

Details on mammalian cell type (if applicable):

Blood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.

- Culture medium
Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) and 30 U/mL heparin.

- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/mL) phytohaemagglutinin was added.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose range finding test:
Without and with S9-mix, 3hr exposure; 27 hr fixation: 10, 33 and 100 µg/mL
Without S9-mix, 24 exposure; 24 hr fixation: 1, 3, 10, 33 and 100 µg/mL

First cytogenetic test:
Without and with S9-mix, 3hr exposure; 27 hr fixation: 10, 33 and 100 µg/mL

Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 10, 30 and 40 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:

Test compound was soluble in DMSO and DMSO has been accepted and approved by authorities and international guidelines

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without S9: 0.25 µg/mL for a 3 hours exposure period and 0.15 µg/mL for a 24 hours exposure period

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

Remarks:

without S9: 0.1 µg/ml for a 3 hours exposure period and 0.05 µg/ml for a 24 hours exposure
period

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9: 15 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration:
Short-term treatment
Without and with S9-mix: 3 hr treatment, 24 hr recovery/harvest time
Continuous treatment
Without S9-mix: 24 hr treatment/harvest time

ARREST OF CELL DIVISION: 5 µg/mL Cytochalasine B
STAIN: Giemsa

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: 1000/culture (mono- and binucleated cells)

DETERMINATION OF CYTOTOXICITY
- The cytostasis/cytotoxicity was determined using the cytokinesis-block proliferation index (CPBI index)

Evaluation criteria:

A test substance was considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if:
a) It induces a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of mono or binucleated cells with micronuclei.
b) A statistically significant and biologically relevant increase is observed in the number of mono or binucleated cells with micronuclei in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of mono and binucleated cells with micronuclei.
b) The number of mono and binucleated cells with micronuclei was within the laboratory historical control data range.

Statistics:

The incidence of micronucleated cells (cells with one or more micronuclei) for each exposure group was compared to that of the solvent control using Chi-square statistics.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human peripheral blood

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 examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No

- Precipitation: Precipitation in the exposure medium was observed at dose levels of 40 µg/ml and above

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was only observed at dose levels of 33 µg/ml and above in the absence of S9 for the continuous treatment of 24 hr.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- At the 3 hour treatment, no toxicity was observed up to and including the highest tested dose level.
- At the continous treatment, appropriate toxicity was reached at the dose levels selected for scoring.

ADDITIONAL RESULTS:
In the first cytogenetic assay, 2 moles ethoxylated bisphenol A dimethacrylate was tested up to 100 µg/ml for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction. 2 moles ethoxylated bisphenol A dimethacrylate precipitated in the culture medium at 100 µg/ml.
In the second cytogenetic assay, 2 moles ethoxylated bisphenol A dimethacrylate was tested up to 40 µg/ml for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. 2 moles ethoxylated bisphenol A dimethacrylate precipitated in the culture medium at 40 µg/ml.
The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the historical control data range per 2000 mono- or binucleated cells after 3 h exposure time. However, the number of mononucleated and binucleated cells with micronuclei after 24 h exposure time were out of the historical control data (3 and 8 per 1000 cells, respectively in one culture each). However, the results of the test substance treated cultures were clearly negative. In addition, the positive control chemicals mitomycin C showed a statistically significant increase in the number of binucleated cells with micronuclei and colchicine showed a statistically significant increase in the number of mononucleated cells with micronuclei.
The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
2 moles ethoxylated bisphenol A dimethacrylate did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two independently repeated experiments.

Applicant's summary and conclusion

Conclusions:

2 moles ethoxylated bisphenol A dimethacrylate did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two independently repeated experiments.
Finally, it is concluded that this test is valid and that 2 moles ethoxylated bisphenol A dimethacrylate is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.

Executive summary:

An in vitro micronucleus assay was performed with 2 moles ethoxylated bisphenol A dimethacrylate in cultured peripheral human lymphocytes (in two independent experiments) to describe the effect of the test substance on the number of micronuclei formed in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix).

The study procedures described in this report were based on the OECD guideline 487.

2 moles ethoxylated bisphenol A dimethacrylate was soluble in dimethyl sulfoxide.

In the first cytogenetic assay, 2 moles ethoxylated bisphenol A dimethacrylate was tested up to 100 µg/ml for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction. 2 moles ethoxylated bisphenol A dimethacrylate precipitated in the culture medium at 100 µg/ml.

In the second cytogenetic assay, 2 moles ethoxylated bisphenol A dimethacrylate was tested up to 40 µg/ml for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. 2 moles ethoxylated bisphenol A dimethacrylate precipitated in the culture medium at 40 µg/ml.

The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the historical control data range per 2000 mono- or binucleated cells after 3 h exposure time. However, the number of mononucleated and binucleated cells with micronuclei after 24 h exposure time were out of the historical control data (3 and 8 per 1000 cells, respectively in one culture each). However, the results of the test substance treated cultures were clearly negative. In addition, the positive control chemicals mitomycin C showed a statistically significant increase in the number of binucleated cells with micronuclei and colchicine showed a statistically significant increase in the number of mononucleated cells with micronuclei.

The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

2 moles ethoxylated bisphenol A dimethacrylate did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two independently repeated experiments.

Finally, it is concluded that this test is valid and that 2 moles ethoxylated bisphenol A dimethacrylate is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.