Ethyl methacrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD 471 with Ethyl methacrylate (EMA), GLP, +/- metabolic activation: negative with all strains tested.

OECD 476 with Ethyl methacrylate (EMA), GLP, +/- metabolic activation: negative

OECD 487 with Ethyl methacrylate (EMA), GLP, +/- metabolic acitivation: negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04/2020-06/2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No. 440/2008 B13/14

Version / remarks:

30-05-2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

ninth addendum to OECD 471; corrected 26-06-2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

TEST MATERIAL:
- Name of Test material: Ethyl methacrylate
- physical state: liquid, colorless

Target gene:

his locus

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ beta-naphthoflavone induced rat liver S9 Mix

Test concentrations with justification for top dose:

In the pre-experiment the concentration range of the test item was 3-5000 µg/plate. The pre-experiment is reported as experiment I. Since only slight toxic effects were observed 5000 µg/plate were chosen as maximal concentration. The concentration range included two logarithmic decades.

Experiment I (plate incorporation test): 3/ 10/ 33/ 100/ 333/ 1000/ 2500/ 5000 µg/plate
Experiment II (pre-incubation test): 33/ 100/ 333/ 1000/ 2500/ 5000 µg/plate

Vehicle / solvent:

On the day of experiment, the test item was dissolved in DMSO (purity >99%). The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria. All formulations were prepared freshly before treatment and used within two hours of preparation.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene: with metabolic activation: all strains; 4-nitro-o-phenylene-diamine: without metabolic activation: TA 1537 and TA 98

Details on test system and experimental conditions:

METHOD OF APPLICATION:
in agar (plate incorporation) and pre-incubation

EXPERIMENTAL PERFORMANCE:
For each strain and dose level, including the controls, three plates were used.
Experiment I (Plate incorporation): Test solution at each dose level (solvent or reference mutagen solution (positive control)), S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation), bacteria suspension and overlay were mixed in a test tube and poured onto the selective agar plates.
Experiment II (Pre-incubation): The test solution at each dose level, the S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation) and the bacteria suspension were mixed in the test tube and incubated at 37 +/- 1.5°C for 60 minutes.
After the pre-incubation, 2.0 ml overlay agar was added to each tube. The mixture was poured on minimal agar plates. After solidification, the plates were incubated upside down for at least 48 hours at 37 +/- 1.5°C in the dark.
In parallel to each test, a sterile control of the test item was performed and documented in the raw data. Therefore, sterile stock solution, S9 mix/ S9 mix substitution buffer were mixed with 2.0 ml overlay agar and poured on minimal agar plates.
The colonies were counted using the validated computer system, which was connected with a PC with printer to print out the individual values, the means from the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates. Due to the precipitation of the test item the colonies were partly counted manually.

DETERMINATION OF CYTOTOXICITY:
 Pre-Experiment for Toxicity: To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described for the experiment I (plate incorporation test). Toxicity of the test item results in a reduction in the number of spontaneous revertants (below a factor of 0.5) or a clearing of the bacterial background lawn.
The pre-experiment is reported as main experiment I since the acceptance criteria are met.

Evaluation criteria:

The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:

- regular background growth in the negative and solvent control;
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data;
- the positive control substances should produce an increase above the threshold of twofold (strains TA 98, TA 100, and WP2 uvrA) or threefold (strains TA 1535 and TA 1357) the colony count of the corrresponding solvent control;
- a minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.

Statistics:

no appropriate statistical method available

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In experiment II, cytotoxic effects were observed at 5000 µg/plate with and without S9 mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In experiment I, cytotoxic effects were observed at 5000 µg/plate without S9 mix. In experiment II, cytotoxic effects were observed at 5000 /plate with and without S9 mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In experiment II, cytotoxic effects were observed at 5000 µg/plate with and without S9 mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In experiment II, cytotoxic effects were observed at 5000 µg/plate without S9 mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Ethyl methacrylate is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of Ethyl methacrylate to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/ Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

No precipitation of the test item occured up to the highest investigated dose. 

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. 

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occured in experiment I in strain TA 1537 in the absence of S9 mix and in experiment II in strains TA 1535, TA 1537, and TA 98 in the presence and absence of S9 mix and in strain WP2 uvrA in the absence of S9 mix. 

No substantial increase in revertant colony number of any of the five tested strains was observed following treatment with Ethyl methacrylate at any dose, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below generally acknowledged border of biological relevance. 

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. 

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base par changes or frameshifts in the genome of the strains used.

Therefore, Ethyl methacrylate is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

 

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-09-24 to 2020-30-10

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 using the Hprt and xprt genes)

Version / remarks:

29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Kanpoan No. 287 -- Environmental Protection Agency Eisei No. 127 -- Ministry of Health & Welfare Heisei 09/10/31 Kikyoku No. 2 -- Ministry of International Trade & Industry

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

TEST MATERIAL:
- Name of test material: Ethyl Methacrylate
- CAS No. 97-63-2
- EC No. 202-597-5
- Storage conditions: at room temperature
- Physical state: colourless liquid

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

According to the OECD Guideline for Cell Gene Mutation Tests at least 4 analysable concentrations should be used in two parallel cultures.
The dose range of the main experiment was set according to data generated in the pre-experiment. The individual concentrations were spaced by a factor of 2.0.
To overcome problems with possible deviations in toxicity the main experiment was started with more than four concentrations.

Main experiment:
without S9 mix: 142.6 / 285.3 / 570.5 / 1141.0 µg/ml (exposure time 4 h)
with S9 mix: 142.6 / 285.3 / 570.5 / 1141.0 µg/ml (exposure time 4 h)

Vehicle / solvent:

DMSO (purity >= 99.9%)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): duplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 0.7 to 1.2 x 10^7 cells
- After 24 h, the medium was replaced with serum-free medium containing the test item, either without S9 ix or with 50 µl/ml S9 mix. Concurrent solvent and positive controls were treated in parallel. 4 hours after treatment, this medium was prelaced with complete medium following two washing steps with PBS. Immediately after the end of treatment, the cells were trypsinised and subcultivated. At least 2.0x10^6 cells per experimental point (concentration series plus controls) were subcultivated in 175 cm² flasks containing 30 ml medium. Three to four days after the first subcultivation, at least 2.0x10^6 cells per experimental point were again, subcultivated in 175 cm² flasks containing 30 ml medium. Following the expression time of approx. 7 days, five 75 cm² cell culture flasks were seeded with about 4-5x10^5 cells each in medium containing 6-TG (11 µg/ml). Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability. The cultures were incubated at 37 +/- 1.5°C in humidified atmosphere with 1.5 +/- 0.5 CO2.
After approx. 8 days (evaluation for viability) and approx. 9 days +/- 2 days (mutation analysis), the colonies were stained with 10% methylene blue in 0.01% KOH solution. Colonies with more than 50 cells were counted. In doubt, the colony sized was checked with a preparation microscope.


METHODS FOR MEASUREMENT OF CYTOTOXICITY
Two additional 25 cm² flasks were seeded per experimental point with approx. 500 cells each to determine the relative survial (RS) as measure of test item induced cytotoxicity. The cultures were incubated at 37 +/- 1.5°C in a humidified atmosphere with 1.5% +/- 0.5 CO2. The colonies to determine the relative survival (RS) were fixed and stained approx. 8 +/- 2 days after treatment.

Evaluation criteria:

A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
a) at least one of the test conditions exhibits a statistically significant increase compared with the concurrent negative control;
b) the increase is dose-related when evaluated with the appropriate trend test;
c) any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).

A test item is classified as clearly non-mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control;
b) there is no concentration-related increase when evaluated with the appropriate trend test;
c) all results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).

There is no requirement for verification of a clearly positive or negative response. In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations.
In rare cases, even after further investigations, the data set will preclude making a conclusion of positive or negative results, and therefore the test chemical response will be concluded to be equivocal.

Statistics:

A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies (mean values) obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.

A t-test was not performed since all mean mutant frequencies of the groups treated with the test item were well within the 95% confidence interval of our laboratory's historical negative control data.

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

Positive controls validity:

valid

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, Ethyl methacrylate is considered to be non-mutagenic in this HPRT assay.

Executive summary:

This study was performed to investigate the potential of Ethyl methacrylate to induce gene mutations at the HPRT locus in V79 cells of Chinese hamster. The treatment period was 4 hours with and without metabolic activation. The maximum test item concentration in the pre-experiment (1141 µg/ml) was equal to a molar concentration about 10 mM. No substantial and dose dependent increase of the mutation frequency was observed in the main experiment. The test concentrations are described in chapter 3.5.2. The evaluated experimental points and the results are summarised in Table 3. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. 

Conclusion: 

In conclusion, it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, Ethyl methacrylate is considered to be non-mutagenic in this HPRT assay.

 

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-18-09 to 2021-12-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted 29 July 2016

Deviations:

yes

Remarks:

A series of in-house non-GLP validation experiments was performed to get distinct responses of statistical significance when using the specified positive controls (Bohnenberger et al., 2011). To achieve such response the test design, specifically for the

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

TEST MATERIAL:
- Name of test material: Ethyl Methacrylate
- CAS No. 97-63-2
- EC No. 202-597-5
- Storage conditions: at room temperature
- Physical state: colourless liquid

Species / strain / cell type:

lymphocytes:

Additional strain / cell type characteristics:

other:

Remarks:

Blood samples were drawn from one healthy non-smoking female donor (19 years old) with no known illness or recent exposures to genotoxic agents (e.g.chemicals, ionising radiation) at levels that would increase the background incidence of micronucleate cel

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-naphthoflavone induced rat liver S9 was used as the metabolic activation system. An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4).
The protein concentration of the S9 preparation used for this study was 31.0 mg/mL (Lot no. 100920).

Test concentrations with justification for top dose:

Pre-Test:
Experiment I (Preparation interval 40h, exposure time 4 h, without S9 Mix):
7.4/13.0/ 22.7/39.7/69.5/122/213/373/666/1165 PS/2039PS

Experiment II (Preparation interval 40h, exposure time 20 h, without S9 Mix):
23.2/40.6/71.0/124/217/380/666/1165 PS/2039PS

Experiment I (Preparation interval 40h, exposure time 4 h, with S9 Mix):
13.2/ 23.2/40.6/71.0/124/217/380/666/1165 PS/2039PS

PS-Phase separation

With regard to the molecular weight of the test item, 1141 μg/mL (approx. 10 mM) were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 7.4 to 1141 μg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, phase separation of the test item was observed at the end of treatment at 1141 μg/mL in the absence of S9 mix. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.
No cytotoxic effects were observed in Experiment I after 4 hours treatment in the absence and presence of S9 mix. Therefore, 1141 μg/mL were chosen as top treatment concentration for Experiment II.

Vehicle / solvent:

ethanol

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Demecolcine

Details on test system and experimental conditions:

1) Culture conditions
Blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 hrs after blood collection. The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 µg/mL), the mitogen PHA 1.5% (v/v) as extract, 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).
All incubations were done at 37 °C with 5.5 % CO2 in humidified air.
2) Pre-experiment
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterised by the percentages of reduction in the CBPI in comparison to the controls (% cytostasis) by counting 500 cells per culture. The experimental conditions in this pre-experimental phase were identical to those required and described below for the mutagenicity assay.
The pre-test was performed with 10 concentrations of the test item separated by no more than a factor of √10 and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 hrs (with and without S9 mix). The preparation interval was 40 hrs after start of the exposure.
This preliminary test was designated Experiment I since the cultures fulfilled the acceptability criteria and appropriate concentrations could be selected for cytogenetic evaluation.

3) Cytogenetic Experiment
a) Pulse exposure
About 48 hrs after seeding, 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration. The culture medium was replaced with serum-free medium containing the test item. For the treatment with metabolic activation 50 µL S9 mix per mL culture medium was added. After 4 hrs the cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were resuspended in and washed with "saline G" (pH 7.2, containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose • H2O, 192 mg/L Na2HPO4 • 2 H2O and 150 mg/L KH2PO4). The washing procedure was repeated once as described. The cells were resuspended in complete culture medium with 10 % FBS (v/v) and cultured for a 16-hour recovery period. After this period, Cytochalasin B (4 µg/mL) was added and the cells were cultured another approximately 20 hours until preparation (Clare et al, 2006, Lorge et al, 2006).

b) Continuous exposure (without S9 mix)
About 48 hrs after seeding, 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration. The culture medium was replaced with complete medium (with 10 % FBS) containing the test item. After 20 hours the cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were re-suspended in and washed with "saline G". The washing procedure was repeated once as described. After washing, the cells were re-suspended in complete culture medium containing 10 % FBS (v/v). Cytochalasin B (4 µg/mL) was added and the cells were cultured another approximately 20 hours until preparation (Whitwell et al, 2019).

4) Preparation of cells
The cultures were harvested by centrifugation 40 hrs after beginning of treatment. The cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were re-suspended in approximately 5 mL saline G and spun down once again by centrifugation for 5 minutes. Then the cells were resuspended in 5 mL KCl solution (0.0375 M) and incubated at 37 °C for 20 minutes. 1 mL of ice-cold fixative mixture of methanol and glacial acetic acid (19 parts plus 1 part, respectively) was added to the hypotonic solution and the cells were resuspended carefully. After removal of the solution by centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold. The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide. The cells were stained with Giemsa, mounted after drying and covered with a coverslip.

Evaluation criteria:

The micronucleus assay will be considered acceptable if it meets the following criteria:
− The concurrent solvent control will normally be within the laboratory historical solvent control data range (95% control limit realized as 95% confidence interval)
− The concurrent positive controls should induce responses that are compatible with the laboratory historical positive control data and produce a statistically significant increase compared with the concurrent solvent control
− Cell proliferation criteria in the solvent control are considered to be acceptable
− All experimental conditions described in section ‘Experimental Design and Study Conduct’ were tested unless one exposure condition resulted in a clearly positive result
− The quality of the slides must allow the evaluation of an adequate number of cells and concentrations
− The criteria for the selection of top concentration are consistent with those described in section ‘Dose Selection’

Statistics:

Statistical significance was confirmed by the Chi Square Test (p < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.
A linear regression was performed using a validated test script of “R”, to assess a possible dose dependency in the rates of micronucleated cells. The number of micronucleated cells obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
Both, biological and statistical significance were considered together.

Key result

Species / strain:

other: lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.
Therefore, Ethyl methacrylate is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required.

Executive summary:

The test item Ethyl methacrylate, dissolved in Ethanol, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in two independent experiments. The following study design was performed:

	Without S9 Mix		With S9 Mix
	Exp. I	Exp. II	Exp. I
Stimulation period	48h	48h	48h
Exposure period	4h	20h	4h
Recovery	16h	-	16h
Cytochalasin B exposure	20h	20h	20h
Total culture period	88h	88h	88h

In each experimental group, two parallel cultures were analysed. Per culture at least 1000 binucleated cells were evaluated for cytogenetic damage.
The highest applied concentration in this study (1141 μg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 487.
Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item phase separation in accordance with OECD Guideline 487. The rationale for the dose selection is reported in section 3.5.1. The chosen treatment concentrations are reported in Table 1 and the results are summarised in Table 2.
In Experiment I in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration, which showed phase separation in the absence of S9 mix. In Experiment II, no cytotoxicity was observed up to the highest applied concentration.
In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of micronucleated cells was observed after treatment with the test item.
Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.
Conclusion
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.
Therefore, Ethyl methacrylate is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

There are no in vivo data for EMA.

Additional information

Data availability

The alkyl-methacrylate esters have been tested in a series of bacterial and mammalian tests in vitro and in vivo. For EMA screening information is available for all three endpoints. Looking at the EMA studies in isolation, they are not entirely satisfactory for two reasons: On one hand the available information is not completely conclusive because the in vitro chromosome aberration test and the mouse lymphoma tests are weakly positive and on the other hand the available studies are reliable with restrictions because some aspects of the actual mutagenicity test guidelines are not covered to the last detail. This will be addressed below by category read across, based on scenario 6 of the current RAAF guidance (“di­fferent compounds have the same type of e­ffect”; ECHA 2017), based on a common mode of action. An analysis of the mode of action is included in the mutagenicity review for methacrylate esters by R. Albertini (2017), which is attached to this endpoint summary. At several points throughout this assessment reference will be made to this review.

Three separate aspects of the data will be analysed separately: 1. The technical completeness of the information available (in the sense of compliance with the test guidelines); 2. the effects assessment and overall conclusion, whether there is concern that EMA would be mutagenic; and 3. whether the mutagenic potential could have been underestimated or misjudged by the approach chosen.

1.     Technical completeness of the dataset

The approach taken here is as follows: The available EMA studies are supplemented with studies from the adjacent members of the category of lower alkyl (C1-C8) methacrylates, methyl methacrylate and n-butyl methacrylate, in such a way that the most pertinent and relevant studies were selected from the MMA and n-BMA datasets.

a) Gene Mutation Tests

- Bacterial gene mutation tests

Ester	Type of test	Test system	Doses	Metabolic activation	Result	Reference
MMA	Similar to OECD 471, Salmonella reverse mutation test, pre-incubation method	S. typh. (strains TA98, TA100, TA1535, TA1537)	10 – 10,000 µg/plate	Aroclor-induced rat and hamster S9-mix	negative	Zeiger et al., 1987
	Similar to OECD 471, Salmonella reverse mutation test, pre-incubation method	S. typh. (strains TA97, TA98, TA100, TA102, TA104)	5 – 25,000 µg/plate	PB-induced rat S9-mix	negative	Schweikl et al., 1994
EMA	Similar to OECD 471, Salmonella reverse mutation test, pre-incubation method	S. typh. (strains TA98, TA100, TA1535, TA1537)	33 – 10,000 µg/plate	Aroclor-induced rat and hamster S9-mix	negative	Zeiger et al., 1987
n-BMA	Similar to OECD 471, Salmonella reverse mutation test, pre-incubation method	S. typh. (strains TA98, TA100, TA1535, TA1537)	100 – 10,000 µg/plate	Aroclor-induced rat and hamster S9-mix	negative	Zeiger et al., 1987
	OECD 471/472 Salmonella reverse mutation test, pre-incubation method	S. typh. (strains TA100, TA1535, TA98, TA1537) E. coli WP2 uvr A	9.8 – 625 µg/plate	PB/5,6-BF^a-induced rat S9-mix	negative	Nakajima et al., 1998a

 

EMA has been tested in a standardized bacterial mutation test according to an NTP protocol with four S. typhimurium strains TA98, TA100, TA1535 and TA 1537 with and without metabolic activation (Zeiger et al. 1987; rat and hamster-derived liver S9). Technically, this test lacks one of the current guideline strains (E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102).

As indicated in the table above, reliable bacterial reverse mutation tests with a full complement of strains are available for both adjacent category members. The category read-across is justified based upon a common mode of action (electrophilic reactivity of the double bond in the ester). Differences in reactivity between the alkyl esters are small as evidenced by similar electrophilic reactivity towards glutathione.

For MMA reliable test data are available for eight different bacterial strains, including those recommended in the EC test guideline (Zeiger et al. 1987, Schweikl et al. 1994), for nBMA the recommended complement of strains has been tested (Zeiger et al., 1987; Nakajima et al., 1998a)

All tests have been performed over a relevant dose range in the bacterial reverse mutation test (Ames test) with and without metabolic activation by S9-mix.

- Gene mutation tests in mammalian cells

Ester	Type of test	Test system	Doses	Metabolic activation	Result	Reference
MMA	Mouse Lymphoma Assay	Mouse lymphoma cell line L5178Y heterozygous TK+/-	700 – 2298 µg/ml	none	Weakly +ve at cytotoxic concs^b	Moore et al. 1988
	Mouse Lymphoma Assay	Mouse lymphoma cell line L5178Y heterozygous TK+/-	125 – 1500 µg/ml	Aroclor-^ainduced rat S9-mix	+ve at cytotoxic concs^b	NTP, 1986
	HPRT Assay	Chinese Hamster lung cell line V79 heterozygous HPRT +/-	1000, 2000 µg/ml	none	Weakly +ve (see discussion)	Schweikl et al., 1998
EMA	Mouse Lymphoma Assay	Mouse lymphoma cell line L5178Y heterozygous TK+/-	700 – 2298 µg/ml	none	Weakly +ve at cytotoxic concs^b	Moore et al. 1988
n-BMA	HPRT Assay	Chinese Hamster lung cell line V79 heterozygous HPRT +/-	445 – 710 µg/ml (lim. by solubility)	PB/NF-^ainduced rat S9-mix	negative	Envigo, 2016

a Phenobarbital and beta-Naphthoflavone

b Based on other findings of that study, the authors conclude that this effect is due to clastogenic effects rather than gene mutations.

 

In mammalian cells, EMA was weakly positive at concentrations of >1000mg/mL in a mouse lymphoma gene mutation assay in vitro in a study consistent with OECD Guideline 476 (Moore et al., 1989). The study is reliable with restrictions as the majority of the test concentrations is clearly above the guideline recommendation of 10 mM (1200 mg/ml). The study has only been performed without metabolic activation. For the majority of the test concentrations the survival is borderline low (10-20 %) and for some it is below the guideline recommendation of 10 %.

The dataset is completed in a weight of evidence approach by category read-across to studies of the adjacent members of the category of lower alkyl (C1-C8) methacrylates, methyl methacrylate and n-butyl methacrylate. Again, only the most pertinent and relevant studies were selected from the MMA and n-BMA datasets.

From the MMA dataset three studies are included in the weight of evidence approach: The mouse lymphoma assay performed by NTP (NTP, 1986a), the Mouse Lymphoma publications by Moore et al (Moore et al., 1988, Moore and Doerr 1990) and an HPRT assay in V79 cells by Schweikl et al. (1998). For MMA, the only complete study from a technical point of view is the NTP study. The other two are used to complement the data from a scientific point of view.

The studies by Moore et al and Schweikl et al are clearly limited in their scope and reliability because most test concentrations are above the recommendations of the test guideline and testing has been performed without metabolic activation only.

One study is read across from the n-BMA dataset. Recently a HPRT assay in the V79 cell line has been performed according to the actual test guideline. The test was performed with and without metabolic activation in a relevant range of concentrations (up to 5 mM/719 µg/ml).

 

b) Chromosome mutation tests

- in vitro

Ester	Type of test	Test system	Doses	Metabolic activation	Result	Reference
MMA	In vitro chromo-some aberration assay	Chinese hamster ovary (CHO) cell line	750-5000 µg/ml (7.5-50 mM)	Aroclor-induced rat S9-mix	positive^a	Anderson et al., 1990 NTP, 1986a
EMA	In vitro chromo-some aberration assay	Chinese hamster ovary (CHO) cell line	1000-3000 µg/ml (4.4-26 mM)	Aroclor-induced rat S9-mix	negative	NTP, 2002
n-BMA	OECD 473, In vitro chromo-some aberration assay	Chinese hamster lung (CHL) cell line	178 - 1420 µg/ml (1.25-10 mM)	PB/5,6-BF^b-induced rat S9-mix	negative	Nakajima et al., 1998b

a Highest concentrations in test (30-50 mM) are 6-10 fold above current guideline recommendations for non-toxic chemicals. In addition, based on cytotoxicity data in other tests, significant cytotoxicity above 50% would have occurred

b Phenobarbital and beta-Naphthoflavone Based on other findings of that study, the authors conclude that this effect is due to clastogenic effects rather than gene mutations.

 

EMA has been tested in CHO cells in the presence and absence of metabolic activation (NTP, 2002). The test concentrations are up to the limit of cytotoxicity, but above the current guideline recommendation (5 mg/ml or 10 mM, whichever is the lowest). Apart from the high concentrations the study protocol is comparable to current guideline recommendations with acceptable restrictions.

Although not necessary from a technical perspective, but in order to put the results into perspective in the category, two tests from the adjacent members of the category of lower alkyl (C1-C8) methacrylates, methyl methacrylate and n-butyl methacrylate have been added to the dataset.

In a chromosome aberration assay MMA was tested in the CHO cell line. With S-9 mix, treatment was for 2 h followed by 8 to 10 h recovery. Without S-9 mix, treatment time was 8 hours with 2.0 to 2.5 h recovery. Doses up to 5000 μg/ml were tested. From the data presentation and the general approach of the authors and in vitro cytotoxicity data from other studies it can be assumed that the highest doses tested led to pronounced cytotoxic effects. (Anderson et al., 1990).

n-Butyl methacrylate (n-BMA) was tested in an in vitro chromosomal aberration test in Chinese hamster lung fibroblasts (CHL; Nakajima et al., 1998b). The study was performed following the OECD 473 guideline and GLP. The concentrations were set based on the results of previously conducted cell growth inhibition tests. The test was performed at concentrations of 178, 355, 710 and 1420 µg n-BMA/ml (≈10mM) with treatments of 6, 24 and 48 hours (continuous treatment method) without metabolic activation and at concentrations of 355, 710 and 1420 µg n-BMA/ml for 6 hours (short term treatment) with metabolic activation.

- in vivo

As the in vitro cytogenicity test with EMA is considered to be negative, an in vivo test is technically not required. Nevertheless, for the overall evaluation following the effects assessment reference is made to in vivo studies of the adjacent members of the category of lower alkyl (C1-C8) methacrylates, methyl methacrylate and n-butyl methacrylate.

 

2.     Effects assessment

While the dataset in total is considered complete from a technical point of view there remain two main issues at this point – an analysis of the individual test results per endpoint and the overall conclusion with an analysis of the confidence in the approach taken.

a) Gene Mutation Tests

- Bacterial gene mutation tests

EMA

In a standardized bacterial mutation test according to NTP protocol, EMA was not mutagenic in S. typhimurium strains TA98, TA100, TA1535 and TA 1537 with and without metabolic activation by S9 mix from Aroclor-induced rat and hamster liver (Zeiger et al. 1987). EMA was tested in a dose range of 100 to 10,000 g/plate by the preincubation protocol.

Formally, test data in E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102 are missing according to the current test guideline. Full test data are available for the category neighbours.

 

Category neighbours (MMA and nBMA)

MMA: In a standardized test according to the NTP protocol, methyl methacrylate was not mutagenic in S. typhimurium strains TA98, TA100, TA1535 and TA 1537 with and without metabolic activation (Zeiger et al. 1987). Additional S. typhimurium strains have been tested by Schweikl et al. (1994; TA97, TA98, TA100, TA102, TA104) including those, which are sensitive for crosslinking and oxidising agents. Methyl methacrylate was not mutagenic in bacteria.

nBMA: The potential of n-butyl methacrylate to induce reverse mutation in Salmonella typhimurium (strains: TA 1535, TA 1537, TA 98, and TA 100) and in Escherichia coli WP2 uvrA was evaluated in accordance with the international guidelines (OECD 471, Commission Directive No. B13/14) in compliance with GLP (Nakajima et al., 1998a). n-Butyl methacrylate was tested in two independent experiments by the preincubation method, with and without a metabolic activation system, both performed in a range of 7 or 8 doses from 9.77 to 1250 µg/plate. Under these experimental conditions, n-butyl methacrylate did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium and Escherichia coli.

In addition, an extensive, tabular overview of over 40 bacterial reverse mutation test data with 27 different methacrylates has been attached to this endpoint summary. In the absence of special reactivity in the alcohol side chain methacrylic acid and the methacrylate esters - all sharing the same reactive principle, the double bond in the acid part of the molecule - did not show mutagenicity in bacteria.

 

Primary metabolites (methacrylic acid and ethanol)

Neither methacrylic acid nor ethanol are regarded as mutagens in bacterial gene mutation tests.

 

- Gene mutation tests in mammalian cells

EMA

In mammalian cells, EMA was weakly positive at concentrations of >1000 mg/mL (> 8 mM) in a mouse lymphoma gene mutation assay in vitro in a study consistent with OECD Guideline 476 (Moore et al., 1989). As indicated above, this mutagenicity was associated with pronounced toxicity. Primarily small-colony mutants were observed (62-85 %), which indicates that chromosome aberrations (deletions) rather than gene mutations were the reason for the mutant colonies (Moore and Doerr, 1990). The test includes only data without metabolic activation. Full test data are available for the category neighbours.

 

Category neighbours (MMA and nBMA)

MMA: The dataset is completed in a weight of evidence approach by category read-across to studies of the adjacent members of the category of lower alkyl (C1-C8) methacrylates, methyl methacrylate and n-butyl methacrylate. Again, only the most pertinent and relevant studies were selected from the MMA and n-BMA datasets. From the MMA dataset three studies are included in the weight of evidence approach: The MLTK study by NTP (NTP, 1986a), the two MLTK publications by Moore et al (Moore et al., 1988, Moore and Doerr 1990) and the HPRT assay by Schweikl et al. (1998). In the NTP study MMA was tested with and without metabolic activation Aroclor-induced rat liver S9. The concentration range was 125 µg/ml to 1000 µg/ml without and 125 µg/ml to 1500 µg/ml with metabolic activation, all doses in duplicate or triplicate with independent repeat (solvent: 10 % ethanol). MMA was mutagenic with and without metabolic activation. Positive effects without metabolic activation occurred only at concentrations with high cytotoxicity. The other MLTK study (Moore et al. 1988; Moore and Doerr, 1990) is included here because it addresses colony size as an additional aspect. In this study mouse lymphoma cells were treated with MMA only without metabolic activation in a concentration range between 100 and 3000 µg/ml (10-30 mM). Weak increases in mutagenicity occurred at concentrations above 2000 µg/ml (20 mM) with no clear concentration-effect-relationship (plateau effect with fluctuations). Colony size in that study was reported in the paper of Moore and Doerr (1990) with 69-89 % with only the small colony mutant numbers being increased at higher concentrations. The occurrence of primarily small colonies indicates that chromosome aberrations (deletion of the part of the chromosome bearing the TK locus) are the primary cause of the TK mutations observed. The third study, the HPRT test in V79 Chinese hamster lung cells, was performed at 10 and 20 mM (1000 and 2000 µg/ml without metabolic activation only). While no effect occurred at the lower concentration a weak increase in HPRT mutants was observed at the higher concentration at high but acceptable cytotoxicity (49 % survival).

nBMA was tested in HPRT test in Chinese hamster lung fibroblasts (V79; Envigo, 2016). The study was performed following the OECD 476 and corresponding EC B.17 guidelines and under GLP. The concentrations were set based on the results of previously conducted cell growth inhibition tests. Up to the highest concentration of 355 µg/ml (≈2.5 mM; without metabolic activation) and 710 µg n-BMA/ml (≈5 mM; with metabolic activation; rat liver S9) with a treatment period of 4 hours there was no relevant increase in gene mutations. nBMA is considered to be non-mutagenic under the conditions of the test.

 

Primary metabolites (methacrylic acid and ethanol)

Neither methacrylic acid nor ethanol are regarded as mutagens in mammalian cell mutation tests.

 

b) Chromosome mutation tests

- in vitro

EMA

EMA has been tested in CHO cells in the presence and absence of metabolic activation (NPT, 2002; performed in 1986, reported in 2002). Under the conditions of the test and up to the highest test concentration (3000 µg/ml; 26 mM) there was no increase in chromosome aberrations. With cytotoxic effects observed at higher concentrations the test is up to the limit of cytotoxicity, but also above the current guideline recommendation for the selection of the highest concentration in chromosome aberrations tests (5 mg/ml or 10 mM, whichever is the lowest). Nevertheless, in this chromosome aberration test EMA is non-mutagenic.

In another test in mouse lymphoma cells, which are used for the gene mutation assay at the TK locus, EMA was weakly positive at concentrations of >1000 mg/mL (> 8 mM; performedonlywithout metabolic activation). This mutagenicity was associated with pronounced cytotoxicity. In the TK test, primarily small-colony mutants were observed, which also indicates that chromosome aberrations (deletions) rather than gene mutations were the reason for the mutant colonies in the TK test (Moore et al. 1988; Moore and Doerr, 1990).

 

Category neighbours (MMA and nBMA)

MMA was tested in a chromosome aberration test in the Chinese hamster ovary (CHO) cell line (Anderson et al., 1990). Cells were treated with and without metabolic activation (Aroclor-induced rat S9-mix) in a range of concentrations between 10 and 3000 µg/ml (0.1-30 mM) without metabolic activation) and 160 and 5000 µg/ml (1.6-50 mM) with metabolic activation. Recording of cytotoxicity was mentioned in the publication but is not documented. Without metabolic activation there are two trials, one positive at the high dose (1600 µg/ml; 16 mM) and the second statistically significant at 1600 and 3000 µg/ml. The aberration rate in this case (5/100 cells) is within the range of published control values (0 – 5 aberrations/100 cells). Hence, the result without metabolic activation in this case would be regarded as weakly positive or equivocal. With metabolic activation the test was positive at the highest concentration (5000 µg/ml, 50 mM).

nBMA was tested in an in vitro chromosomal aberration test in Chinese hamster lung fibroblasts (CHL; Nakajima et al., 1998b). The study was performed following the OECD 473 guideline and GLP. The concentrations were set based on the results of previously conducted cell growth inhibition tests. Up to the highest concentration of 710 µg/ml (≈5 mM; without metabolic activation) and 1420 µg n-BMA/ml (≈10 mM; with metabolic activation) with treatments of 6, 24 and 48 hours (continuous treatment method) without metabolic activation and for 6 hours (short term treatment) with metabolic activation there was no relevant increase in gene mutations. nBMA is considered to be non-mutagenic under the conditions of the test.

In addition, an extensive, tabular overview of over 60 mutation test data with 31 different methacrylates has been attached to this endpoint summary. With the exception of two methacrylates wit complex alcohols (trimethylolpropane and trimethoxysilylpropanol) induction of chromosome aberrations in vitro was only observed in relatively small methacrylate esters of low or moderate polarity.

 

Primary metabolites (methacrylic acid and ethanol)

Neither methacrylic acid nor ethanol are regarded as mutagens in mammalian cytogenicity tests.

 

- in vivo

EMA

There are no in vivo data for EMA.

 

Category neighbours (MMA and nBMA)

MMA: A dominant lethal assay is available for MMA (Anderson and Hodge, 1976). In this study groups of 20 male CD-1 mice were exposed via inhalation to MMA at 100, 1000, or 9000 ppm (416, 4160 and 37440 mg/m3) for 6 h/day for 5 days. Each male was subsequently mated with 2 different unexposed female mice weekly over a period of 8 weeks). Specific data on toxicity were not given, however, in the highest dose group 6/20 males died. MMA did not induce dominant lethal mutations as indicated by no adverse effect on total implants and early or late post-implantation death in the offspring of treated males compared to controls.

Two chromosomal aberration tests were conducted by Anderson et al. (1976, 1979) investigating the effect of inhalation exposure to methyl methacrylate for doses ranging from ca. 0.4 to 36.5 mg/L (100 to 9000 ppm). In both tests acute exposure was for 2 h (sampling 24 h after treatment) and subacute exposure for 5 h a day on 5 consecutive days (sampling 24 h after last treatment). Data on toxicity were not given. Group sizes varied from 2 to 9; as far as possible 50 metaphases were analysed per animal. The first study was negative for chromosomal aberration frequencies when - as usual - gaps were excluded. Including gaps and combining two acute experiments conducted independently some increases in aberration frequency were statistically significant. ” This is also due to a particularly low control rate in this experimental segment. Compared to other, almost twofold higher control values in other segments of the report, this finding appears to be of little biological importance. Further from the MMA ESR (ECB, 2002): “In the second study frequencies of chromosomal aberrations excluding gaps were not given. Including gaps increases were recorded at some experimental entries. Furthermore, combined data on chromosomal aberration frequencies exclusively gaps from both studies were given, then weak increases were obtained for 400 and 700 ppm in the acute study (not for 100, 1,000 or 9,000 ppm) and 9,000 ppm in the subacute study. Both studies suffer from inadequate description; esp. the second study demonstrates severe methodological problems, e. g., analysis of 50 metaphases was not possible for 10 out of 27 animals in the acute and 10 out 26 in the subacute test. Altogether, the studies do not give a unanimous picture.

Hachitani et al. (1981) (other sources translate the name as Hachitani) reported on a negative bone marrow micronucleus assay with mice. In an acute test methyl methacrylate was given by gavage in doses ranging from 1,130 to 4,520 mg/kg, in a subacute assay daily doses of 1,130 mg/kg were given on 4 consecutive days. All groups consisted of 6 animals; sampling was done 24 h after (last) administration. There was no increase in the frequency of micronucleated polychromatic erythrocytes. The percentage of reticulocytes from all bone marrow cells was not affected data on general toxicity were not given.

N-BMA has been assessed in vivo in a mouse bone marrow micronucleus test (Cilliutti, 1999). The test substance has been dosed up to the highest guideline dose of 2000 mg/kg via the intraperitoneal route. No toxicity was seen at the low and medium doses (500 and 1000 mg/kg) while slight toxicity was demonstrated at the high dose by a slight reduction in the PCE/NCE coefficient. As no increase in micronuclei was observed, n-BMA is regarded as non-mutagenic under the test conditions

 

Primary metabolites (methacrylic acid and ethanol)

Neither methacrylic acid nor ethanol are regarded as mutagens in vivo.

 

c) Tests for Aneugenic effects

Although there are no studies on EMA, neither of the two chromosome aberration tests in vitro on n-BMA and 2-EHMA (Nakajima et al., 1998; Ohta et al., 1998) or the micronucleus tests in vivo with n-BMA and i-BMA (Völkner 1989, Ciliutti, 1999) indicated that the category of methacrylate esters has aneugenic potential.

 

3.     Conclusion and confidence in the approach taken.

This discussion refers to four data elements: test data on EMA itself, the read-across information from the category of lower alkyl (C1-C8) methacrylates (focusing on the two immediate neighbours MMA and nBMA) and, most important, on the comprehensive review on reactivity, genotoxicity and mutagenicity data of the lower alkyl methacrylates by Prof. Richard Albertini in 2017.

The read-across approach is based on scenario 6 of the current RAAF (Di­fferent compounds have the same type of e­ffect) in based on a common mode of action. An analysis of the mode of action is included in the mutagenicity review for methacrylate esters by R. Albertini (2017), which is attached to this endpoint summary. The reactive group common to all methacrylates is the double bond of the methacrylic part of the molecule, which is electrophilic and may take place in Michael addition reactions.

For each individual endpoint there is a reliable study available. If the EMA study has technical weaknesses, at least one reliable read-across study is available for the adjacent esters in the category. If there are positive effects in vivo there are corresponding, conclusive studies in vivo, which are negative.

Gene mutations in bacteria: The EMA study is reliable, but lacks one standard strain. Corresponding studies for MMA and nBMA are complete – and equally negative. Based on the common mode of action, the Albertini review, the category data and the comprehensive methacrylate overview it is evident, that an additional bacterial reverse mutation test is not necessary. EMA is regarded as non-mutagenic in bacteria. This conclusion, partly based on category read-across, is taken with high confidence.

Gene mutations in mammalian cells: The EMA study is reliable with significant restrictions (no data with metabolic activation). Increased mutation rates, also in the read-across chemical MMA correlate with small colony mutants and chromosome aberrations. Tests in the more robust HPRT test are negative in summary. With the corresponding studies for MMA and nBMA the dataset is complete. Based on the common mode of action, the Albertini review and the category data, it is evident, that an additional gene mutation test in mammalian cells is not necessary. Regarding gene mutations, EMA is regarded as non-mutagenic in mammalian cells. Positive findings are related to chromosome breakage. This conclusion, partly based on category read-across, is taken with high confidence.

Chromosome mutations in mammalian cells: The EMA key study is negative, but a supporting study is positive. Throughout the lower alkyl methacrylate category, positive findings in chromosome aberration tests are associated with high toxicity and very high concentrations (above the current guideline recommendation. Performed under currently prevalent test conditions, practically all of these tests – at correspondingly lower concentrations - would lead to a negative test result today. Based on the common mode of action, the Albertini review , the category data and the comprehensive methacrylate overview, it is evident, that an additional chromosome mutation test in mammalian cells is not necessary. Regarding gene mutations, EMA is regarded as non-mutagenic in mammalian cells. This conclusion, partly based on category read-across, is taken with high confidence and it is consistent with the result of a RAC discussion regarding MMA in 2016, based on almost the same database.

Genetic toxicity in vivo: all relevant studies from MMA and nBMA as nearest neighbours in the category indicate an absence of mutagenic potential in vivo for these substances. The same is assessed for EMA with a high level of confidence as also the primary metabolites are not mutagenic in vivo.

Overall, the results of the available, fully valid test data indicate, covering all relevant endpoints, that the members of the category are not mutagenic or genotoxic. This is consistent with a much more comprehensive review of methacrylate data by Albertini (2017) and all previous national and international reviews on EMA. Additional guideline tests with EMA would investigate the mutagenic potential of EMA in non-cytotoxic concentrations which are significantly lower than the currently available studies. Hence, an additional risk from not performing new studies is not expected with a high level of certainty.

Justification for classification or non-classification

Based on the available data 1,3-BDDMA is not considered to be mutagenic according to the criteria given in regulation (EC) 1272/2008 or the former European directive on classification and labelling 67/548/EEC. Thus, no labelling is required for Ethyl methacrylate.