[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The Ames test, HPRT and in vitro micronucleus test were performed according to the OECD guidelines on 2 moles ethoxylated bisphenol A dimethacrylate. Negative results were observed in all the tests with and without metabolic activation. No further test is required for the genotoxicity endpoint, and 2 moles ethoxylated bisphenol A dimethacrylate is considered as not genotoxic.

Link to relevant study records

Reference

Reference 1

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

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

Target gene:

n/a

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

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

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

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.

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.

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26-Nov-2012 to 10-Dec-2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

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:

A dose-range finding test was conducted to identify the concentrations for the main study.

Experiment 1
TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate.
TA1535, TA1537 and TA98: Without and with S9-mix: 33, 100, 333, 1000 and 3330 µg/plate.

Experiment 2:
Without and with S9-mix: 33, 100, 333, 1000 and 3330 µg/plate.

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

see below

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

Statistics:

no

Species / strain:

S. typhimurium TA 100

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

Species / strain:

S. typhimurium TA 1535

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

Species / strain:

S. typhimurium TA 1537

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

Species / strain:

S. typhimurium TA 98

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

Species / strain:

E. coli WP2 uvr A

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
- Precipitation: Precipitation was observed at dose levels of 3330 and 5000 µg/plate


RANGE-FINDING/SCREENING STUDIES:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No toxicity or mutagenicity was observed up to and including the top dose of 3330 µg/plate

Conclusions:

It is concluded that 2 moles ethoxylated bisphenol A dimethacrylate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay, either in the presence or in the absence of a rat liver metabolizing system.

Executive summary:

Evaluation of the mutagenic activity of 2 moles ethoxylated bisphenol A dimethacrylate in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (with independent repeat).

The study procedures described in this report were based on the most recent OECD and EC guidelines (OECD No. 471 and Council Regulation No. 440/2008 of 30 May 2008, Part B13/14) and in compliance with the principles of Good Laboratory Practice.

The test item, 2 moles ethoxylated bisphenol A dimethacrylate was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone). The test substance was dissolved in dimethyl sulfoxide.

In the dose range finding test, 2 moles ethoxylated bisphenol A dimethacrylate was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and TA98. 2 moles ethoxylated bisphenol A dimethacrylate precipitated on the plates at dose levels of 3330 and 5000 µg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of this dose range finding test were reported as part of the first experiment of the mutation assay.

Based on the results of the dose range finding test, 2 moles ethoxylated bisphenol A dimethacrylate was tested in the first mutation assay at a concentration range of 33 to 3330 µg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and WP2 uvrA. In an independent repeat of the assay with additional parameters, 2 moles ethoxylated bisphenol A dimethacrylate was tested at the same concentration range as the first assay in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. 2 moles ethoxylated bisphenol A dimethacrylate precipitated on the plates at the top dose of 3330 µg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. 2 moles ethoxylated bisphenol A dimethacrylate did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that 2 moles ethoxylated bisphenol A dimethacrylate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay, either in the presence or in the absence of a rat liver metabolizing system.

 

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16-Nov-2012 to 25-Mar-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Hypoxanthine-Guanine Phosphoribosyl Transferase (HPRT) in V79 cells

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media:
- Minimum Eagles Medium with Earle’s salts (MEME, Invitrogen Corporation), supplemented with 5 ml of MEM non-essential amino acids solution (Invitrogen Corporation), 0.11 g of sodium pyruvate (Invitrogen Corporation), 0.292 g of L-glutamine (Invitrogen Corporation) and 2.2 g of sodium bicarbonate (Merck) per litre. Final concentrations of streptomycin and penicillin G (Invitrogen Corporation) were 50 µg/mL and 50 U/ml, respectively. Supplemented with 10% (v/v) foetal calf serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

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, 3 hours treatment: 1, 3.3, 10, 33, 100 and 333 µg/mL
Without S9-mix, 24 hours treatment: 1, 3.3, 10, 33, 100 and 333 µg/ml

Experiment 1:
Without and with S9-mix, 3 hours treatment: 0.1, 0.3, 1, 3, 10, 33, 100 and 333 µg/mL

Experiment 2
Without S9-mix, 24 hours treatment: 0.1, 0.3, 1, 3, 10, 33, 100 and 333 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 3 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 6 days
- Selection time (if incubation with a selection agent): 7 days

SELECTION AGENT (mutation assays): 5 µg/mL thioguanine (6-TG)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplicate cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 1.5 x 10E6 cells plated/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute colony forming efficiency of the solvent controls should be between 60 and 130%
b) At least seven doses of the test substance should have an acceptable number of surviving cells (10E6) which could be analysed for expression of the HPRT mutation.
c) The spontaneous mutant frequency in the solvent-treated control will be < 10 per 10E5 clonable cells.
d) The positive control substances induced significant (at least three-fold) increases in the mutant frequency.
e) The selected dose range has to include a clearly toxic concentration (10 to 30%) of the average of solvent controls or should exhibit limited solubility or should extend to 5 mg/plate.


DATA EVALUATION
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.

A test substance is considered positive (mutagenic) in the mutation assay if it induces at least a three-fold increase in the mutation frequency compared to the solvent control in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations show a mutation frequency of at least three-fold compared to the solvent control.
b) The results are confirmed in an independently repeated test.

Statistics:

no

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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 100 µg/mL and above.

RANGE-FINDING/SCREENING STUDIES:
- No toxicity was observed up to and including the top dose of 333 µg/ml.

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
After the 3 hours treatment, the cell count of day 2 showed a reduction in the cell growth of 46% at the test substance concentration of 333 µg/ml in the absence of S9-mix only compared to the solvent controls. No cell growth delay was observed in the cloning efficiency of day0 both in the absence and presence of S9-mix.

After the 24 hours treatment, the cell count directly after treatment showed a reduction in the cell growth of 46% at the test substance concentration of 333 µg/ml compared to the solvent controls. The cloning efficiency showed a cell survival of 77% at the two highest tested dose levels of 100 and 333 µg/ml compared to the solvent controls.

ADDITIONAL INFORMATION ON GENOTOXICITY:
The spontaneous mutation frequencies in the solvent-treated control cultures were within the historical control data range (See Table 11, APPENDIX 3).
Mutation frequencies in cultures treated with positive control chemicals were increased by 18- and 6.2-fold for EMS in the absence of S9-mix, and by 5.1-fold for 3-MCA in the presence of S9-mix. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate for the detection of a mutagenic response and that the metabolic activation system (S9-mix) functioned properly. In addition the observed mutation frequencies of the positive control substances were within the acceptability criteria of this assay.

In the absence of S9-mix, 2 moles ethoxylated bisphenol A dimethacrylate induced a 3.1-fold increase in the mutation frequency in the first experiment at one of the high dose levels (100 µg/ml). A low (mean mutation frequency of the solvent controls of 0.9 per 105 survivors versus a historical mean of 2.0 per 105 survivors) solvent control value resulted in a mutation frequency greater than three (3) times the concurrent control at one dose level. This increase was within the historical control data range. Furthermore, a dose-response was not seen as the dose levels of 33 and 333 µg/ml showed no increase in the mutation frequency of more than 2.1-fold. In the experiment with the prolonged treatment period no increase in the mutation frequency was observed. Therefore, 2 moles ethoxylated bisphenol A dimethacrylate is considered not mutagenic in the absence of S9-mix.

In the presence of S9-mix, no significant increase in the mutation frequency at the HPRT locus was observed after treatment with 2 moles ethoxylated bisphenol A dimethacrylate.

Conclusions:

It is concluded that 2 moles ethoxylated bisphenol A dimethacrylate is not mutagenic in the gene mutation test with V79 Chinese hamster cells under the experimental conditions described in this report.

Executive summary:

Evaluation of the mutagenic activity of 2 moles ethoxylated bisphenol A dimethacrylate in anin vitromammalian cell gene mutation test with V79 Chinese hamster cells (with independent repeat).

 

This report describes the effects of 2 moles ethoxylated bisphenol A dimethacrylate on the induction of forward mutations at the hypoxanthine-guanine phosphoribosyl transferase () locus in V79 Chinese hamster cells. The test was performed in two independent experiments in the absence and presence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone).  

 

The study procedures described in this report were based on the most recent OECD and EC guidelines.

 Test substance was a clear colourless highly viscous liquid. The test substance was dissolved in dimethyl sulfoxide.

 

In the first experiment, 2 moles ethoxylated bisphenol A dimethacrylate was tested up to concentrations of 333 µg/ml in the absence and presence of S9-mix. The incubation time was 3 hours. In the second experiment, 2 moles ethoxylated bisphenol A dimethacrylate was again tested up to concentrations of 333 µg/ml in the absence S9-mix. The incubation time was 24 hours. No toxicity was observed at this dose level in the absence and presence of S9-mix. 2 moles ethoxylated bisphenol A dimethacrylate precipitated in the culture medium at dose levels of 100 µg/ml and above.

 

The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

 

Mutation frequencies in cultures treated with positive control chemicals were increased 18- and 6.2-fold for Ethylmethanesulphonate (EMS) in the absence of S9-mix, and by 5.1-fold for

3-Methylcholanthrene(3-MCA) in the presence of S9-mix. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly.

 

In the absence of S9-mix, 2 moles ethoxylated bisphenol A dimethacrylate induced a 3.4-fold increase in the mutation frequency in the first experiment at one of the high dose levels (100 µg/ml). A low (mean mutation frequency of the solvent controls of 0.9 per 10^5 survivors versus a historical mean of 2.0 per 10^5 survivors) solvent control value resulted in a mutation frequency greater than three (3) times the concurrent control at one dose level. This increase was within the historical control data range. Furthermore, a dose-response was not seen as the dose levels of 33 and 333 µg/ml showed no increase in the mutation frequency of more than 2.3-fold. In the experiment with the prolonged treatment period no increase in the mutation frequency was observed. Therefore, 2 moles ethoxylated bisphenol A dimethacrylate is considered not mutagenic in the absence of S9-mix.

 

In the presence of S9-mix, 2 moles ethoxylated bisphenol A dimethacrylate did not induce a significant increase in the mutation frequency.

 

It is concluded that 2 moles ethoxylated bisphenol A dimethacrylate is not mutagenic in the gene mutation test with V79 Chinese hamster cells under the experimental conditions described in this report.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Reverse mutation test in bacteria (Ames test, OECD 471):

The test item, 2 moles ethoxylated bisphenol A dimethacrylate was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone). The test substance was dissolved in dimethyl sulfoxide.

In the dose range finding test, 2 moles ethoxylated bisphenol A dimethacrylate was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and TA98. 2 moles ethoxylated bisphenol A dimethacrylate precipitated on the plates at dose levels of 3330 and 5000 µg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of this dose range finding test were reported as part of the first experiment of the mutation assay.

Based on the results of the dose range finding test, 2 moles ethoxylated bisphenol A dimethacrylate was tested in the first mutation assay at a concentration range of 33 to 3330 µg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and WP2 uvrA. In an independent repeat of the assay with additional parameters, test item was tested at the same concentration range as the first assay in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Test item precipitated on the plates at the top dose of 3330 µg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation.

Based on the results of this study it is concluded that 2 moles ethoxylated bisphenol A dimethacrylate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay,either in the presence or in the absence of a rat liver metabolizing system.

 

In vitro gene mutation test in mammalian cells (HPRT, OECD 476):

This study studied the effects of 2 moles ethoxylated bisphenol A dimethacrylate on the induction of forward mutations at the hypoxanthine-guanine phosphoribosyl transferase locus in V79 Chinese hamster cells. The test was performed in two independent experiments in the absence and presence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone). The test substance was dissolved in dimethyl sulfoxide. In the first experiment, 2 moles ethoxylated bisphenol A dimethacrylate was tested up to concentrations of 333 µg/ml in the absence and presence of S9-mix. The incubation time was 3 hours. In the second experiment, 2 moles ethoxylated bisphenol A dimethacrylate was again tested up to concentrations of 333 µg/ml in the absence S9-mix. The incubation time was 24 hours. No toxicity was observed at this dose level in the absence and presence of S9-mix. 2 moles ethoxylated bisphenol A dimethacrylate precipitated in the culture medium at dose levels of 100 µg/ml and above.

In the absence of S9-mix, 2 moles ethoxylated bisphenol A dimethacrylate induced a 3.4-fold increase in the mutation frequency in the first experiment at one of the high dose levels (100 µg/ml). A low (mean mutation frequency of the solvent controls of 0.9 per 10^5survivors versus a historical mean of 2.0 per 10^5survivors) solvent control value resulted in a mutation frequency greater than three (3) times the concurrent control at one dose level. This increase was within the historical control data range. Furthermore, a dose-response was not seen as the dose levels of 33 and 333 µg/ml showed no increase in the mutation frequency of more than 2.3-fold. In the experiment with the prolonged treatment period no increase in the mutation frequency was observed. Therefore, 2 moles ethoxylated bisphenol A dimethacrylate is considered not mutagenic in the absence of S9-mix.

In the presence of S9-mix, 2 moles ethoxylated bisphenol A dimethacrylate did not induce a significant increase in the mutation frequency.

It is concluded that 2 moles ethoxylated bisphenol A dimethacrylate is not mutagenic in the gene mutation test with V79 Chinese hamster cells under the experimental conditions described in this report.

 

In vitro cytogenicity test in mammalian cells (micronucleus assay, OECD 487):

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). Test substance 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.

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.

 

Justification for classification or non-classification

Based on the negative results in all three regulatory in vitro tests, no classification for 2 moles ethoxylated bisphenol A diacrylate is required for genotoxicity according to the Regulation EC n°1272/2008.