Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutations in bacteria: An 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 (2017), 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: An 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.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
The study was conducted in accordance with a recognized international scientific procedure and followed the test protocol and procedures of Ames (1975). Complete study results were presented supporting the conclusions that ethyl methacrylate was not mutagenic in this test system. Full description of the test material was provided.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
other: Ames et al. (1975)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
The S-9 fractions of Aroclor 1254-induced, male  Sprague-Dawley rats and male Syrian Hamster livers were prepared  immediately prior to use. The S-9 mixes contained 10% S-9. 
Test concentrations with justification for top dose:
100-10000 µg/plate
Negative solvent / vehicle controls:
yes
Remarks:
ethanol (95%)
Positive controls:
yes
Remarks:
Metabolic activation (S9): 2-Aminoanthracene: with all strains No Metabolic activation:         Sodium azide: TA100 and TA1535         4-Nitro-o-phenylenediamine: TA98         9-Aminoacridine: TA1537
Details on test system and experimental conditions:
TEST PERFORMANCE: The test followed a pre-incubation protocol. The test  material, Salmonella culture, and S-9 mix or buffer were incubated at 37  degrees C, without shaking, for 20 minutes. The top agar was added, and  the contents of the tubes mixed and poured onto the surface of  Vogel-Bonner medium in a petri dish. The histidine-revertant colonies on  these plates were counted after 2 days of incubation at 37 degrees C. 
A  preliminary cytotoxicity assays was conducted using TA100 to determine  the appropriate dose range. Once determined, the test doses were performed  in triplicate, repeated one week following the initial trial. 
A maximum  of 0.5 ml of solvent was added to each plate. Concurrent solvent and  positive controls in the presence or absence of S-9 were performed.
Evaluation criteria:
 A  positive response was demonstrated when a reproducible dose-related  increase over the corresponding solvent control was seen, and it was  judged weakly positive if a low-level dose response was seen.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
In a reliable published study, the test substance was negative in a bacterial reverse mutation assay.
Executive summary:

In a reliable published study, the test substance was negative in a bacterial reverse mutation assay.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Method and results sufficient described, similar to OECD-guideline 476.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
In addition to the mutation test at the TK locus, analyses for chromosomal aberrations and induction of micronuclei in vitro were run.
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Target gene:
TK
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
heterozygous TK+/- (maintained according to Clive and Spector, 1975)
Properly maintained: yes
Periodically checked for mycoplasm: yes, every 4 weeks
LOCUS EXAMINED: Thymidine kinase (TK), using TFT (triflourthymidine, 1 μg/ml)
Metabolic activation:
without
Test concentrations with justification for top dose:
Cytogenicity assay: 900-2100 µg/mL
Genotoxicity assays: 700-2100 μg/mL; 1692-2298 μg/mL
In the first experiment there were 19 doses and a control used: ranging from 700 to 2100 μg/mL. In a second experiment conducted concurrent with this, there were 28 doses ranging from 1692 to 2298 ug/mL.
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Details on test system and experimental conditions:
Fischer's powered or liquid Medium for Leukemic Cells of Mice supplemented with sodium pyruvate, plunoric, penicillin, streptomycin, and horse serum was used.

TEST PERFORMANCE: L5178Y/TK+/- cells were treated without exogenous activation fo 4 hr according to procedures described by Clive et al. 1979. Cells were centrifuged, washed twice with fresh medium, resuspended in fresh medium and incubated on a roller drum at 37 degrees C, to obtain a log-phase growth for 2 days. They were then cloned with TFT selection (1 μg/ml) and without selection in BBL agar (0.22 %)-supplemented colony medium. Following cell treatment and wash, 10 μM BrdUrd was added to the cultures used in the cytogenetic assay. Cells were incubated for 14 hrs and Colcemid (0.1 μg/ml) added for the last 2 hours. Cells were treated with hypotonic KCL and fixed in acetic acid : methanol (1:3). Slides
were prepared, stained using flourescense-plus-Giemsa method. 200 metaphase spreads were
analyzed for abberations for each dose. Chromatid and chromosome gaps were recorded but not included as aberrations.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Positive controls validity:
valid
Additional information on results:
Methyl methacrylate produced a positive mutagenic response at the tk locus with responses variable at 2-4 times background but consistently positive at doses from 2300 to 3000 ug/ml. The highest mutant frequency occurred at 2800 ug/ml with 298 mutants/10E6 survivors. Because the background frequency was relatively high after the analysis of 100 metaphases, an additional 100 metaphases were scored. Weak positives were recorded at 2200, 2500, 2700, 2800 and 3000 ug/ml. The highest response was at 2800 ug/ml with 45 aberrations in 39 of 200 metaphases (background = 16 aberrations in 15 of 200 metaphases scored). It should be noted that the high background was due to an increase in the number of chromosome rearrangements and breaks in the control culture. No concurrent increase in background TK mutant or micronucleus frequency was observed. The MMA treated cells showed a significant increase in chromatid events with no change in the number of chromosome events. Micronucleus responses were also variable with weak positive responses recorded at 2200, 2400, 2500, 2800, 2900 and 3000 ug/ml. The greatest response was at 2900 ug/ml with micronuclei in 25 of 1000 binucleates (background = 9 /1000). The 2 lowest doses, 1000 and 1750, were negative for mutagenicity, aberrations and micronuclei. All test concentrations, except the lowest, are above the guideline recommendation of 10 mM in OECD 476 (1000 µg/ml = 10 mM).

Comparison of results: Chromosome aberrations, micronucleus induction and mutations at the TK locus, small colony mutants and survival.

EMA [µg/ml] Chromatid Breaks Chromatid Rearrangements Chromosome Breaks Chromosome Rearrangements Total No. aberrations % Cells with aberrations
0 3 0 4 2 9 8
900 4 2 10 7 23 18
1450 5 0 7 0 12 12
1550 1 1 3 6 11 11
1626 5 1 8 6 20 10
1676 2 0 7 2 11 10
1750 3 1 7 6 17 13
2100 5 1 4 5 15

14

Increases in any type of mutations correlate strongly with a decrease in survival. All test concentrations, except the lowest, are above the guideline recommendation of 10 mM in OECD 476 (1000 µg/ml = 10 mM). The increase in mutations at the TK locus is primarily due to small colony mutants with a trend to more small colonies at higher concentrations, indicating that these are primarily due to deletions (chromosome mutations) rather than gene mutations.

Mutagenicity of Ethyl Methacrylate

Experiment 1

 Conc (µg/mL)  Survival (%)  Mutant frequency (mutants/ 10e6 survivors)
 0 100  61 
 700 71  72 
 900  48 78 
 1000  34  98
 1100  46  93
 1200  28  110
 1300  28  106
 1450  15  124*
 1500  19  123*
 1550  14  143*
 1600  9  165**
 1626  16  144*
 1650  9  116
 1676  16  120
 1700  6  175**
 1750  21  99
 1800  2  251**
 1900  15  89
 2000  5  134**
     

Experiment 2

 0  100  61
 1692  13  144*
 1704  22  96
 1716  31  114
 1728  32  90
 1740  37  95
 1752  14  126*
 1764  32  97
 1776  4  209**
 1788  7  203**
 1800  15  124*
 1812  2  198**
 1848  14  211*
 1860  15  121
 1872  5  170**
 1884  24  104
 1896  23  93
 1908  16  151*
 1920  15  95
 1932  17  138*
 1944  25  93
 1956  22  103
 1968  23  113
 1980  33  114
 1992  15  134*
 2004  18  110
 2030  18  127*
 2148  8  219**
 2298  10  162*

* positive response

** positive response, but less than 10% survival

Conclusions:
Ethyl Methacrylate induced a weakly positive response that was non-linear. The authors concluded that this was probably due to induction of chromosomal events rather than point mutations.
Executive summary:

In a reliable published study, methyl methacrylate induced a weakly positive response that was nonlinear. The authors concluded that this was probably due to induction of chromosomal events rather than point mutations.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
no repeat trial, limited data on cytotoxicity
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
other: Type of cell used: Chinese hamster ovary (CHO) cells
Metabolic activation:
with and without
Metabolic activation system:
S9 : Sprague-Dawley rat liver, induced with Aroclor 1254    (PCB)
Test concentrations with justification for top dose:
-S9 mix (continous exposure): 0, 1000, 1600, 3000 µg/ml +S9 mix (short time exposure):
0, 1000, 1600, 3000, 5000 µg/ml
Vehicle / solvent:
DMSO
Positive controls:
yes
Remarks:
   -S9 mix, Mitomycin C, 0.125 and 0.250 µg/ml    +S9 mix, Cyclophosphamide, 5 and 7.5 µg/ml
Details on test system and experimental conditions:
Cultures/test concentration : 2

Cultures with metabolic activation were treated for 2 h in culture medium  (McCoy's 5A) without serum. At the end of the treatment period the medium  was exchanged to medium without test material. Cultures without metabolic activation were treated until colcemid was added. For mitotic arrest  colcemid was added 2-2.5 h before harvest. The cultures were harvested  after a culture time of 12 h (with S9 13 h) after the start of treatment.  2x100 metaphases were scored for each test concentration.The test was  performed in a single trial (no repeat).
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: 5000 µg/ml were cytotoxic with metabolic activation. Other than that, no details on cytotoxicity were reported.

This chemical did not induce structural chromosomal aberrations in CHO cells, with or without an exogenous metabolic activation system.

Conclusions:
Using a valid scientific method, the test substance did not induce structural chromosomal aberrations in CHO cells, with or without an exogenous metabolic activation system.
Executive summary:

Using a valid scientific method, the test substance did not induce structural chromosomal aberrations in CHO   cells, with or without an exogenous metabolic activation system.

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. However, reliable data are available from the category neighbours MMA and nBMA:

Hachiya et al. (1982) (other sources translate the name as Hachitani) reported on a negative bone marrow micronucleus assay with MMA in 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.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Similar to OECD-guideline 474, all relevant study details available
Justification for type of information:
Read across from the methacrylic metabolite donor substance
REPORTING FORMAT FOR THE ANALOGUE APPROACH
see attached category document

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
see attached category document, chapter 1.1

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see attached category document, chapter 1

3. ANALOGUE APPROACH JUSTIFICATION
see attached category document, chapter 5 (Toxikokinetics) and endpoint specific chapters

4. DATA MATRIX
see attached category document, endpoint specific chapters
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
not specified
Type of assay:
micronucleus assay
Species:
mouse
Strain:
other: ddy
Sex:
male
Route of administration:
oral: gavage
Vehicle:
olive oil, 25 ml/kg
Duration of treatment / exposure:
4 doses
Frequency of treatment:
3 doses: once, 24 h before terminal sacrifice
1 dose: 4 split doses every 24 h, the last one 24 h before terminal sacrifice, total duration 5 d
Post exposure period:
24 h
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
1 130 mg/kg bw/day (nominal)
Dose / conc.:
2 260 mg/kg bw/day (nominal)
Dose / conc.:
4 520 mg/kg bw/day (nominal)
Remarks:
Doses corresponds to 50% of LD50
Remarks:
4 x 1130 mg/kgbw per dose
No. of animals per sex per dose:
6 (repeated treatment: 5)
Control animals:
yes, concurrent vehicle
Positive control(s):
3 mg Mitomycin C, single dose by i.p. administration 24 h prior to preparation
Tissues and cell types examined:
Sampling time for bone marrow: 3 single doses - 24 h post-administration; for repeated administration: 5 days after first administration.
Statistics:
according to Kastenbaum/Bowman
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
at all doses, single and repeated
Vehicle controls validity:
valid
Positive controls validity:
valid

The substance has been administered by gavage as a solution in olive oil in 3 single doses ranging from 1130 mg/kg to 4520 mg/kg (0.5 LD50) 24 h prior to preparation of the bone marrow. A separate group of 5 animals was administered 4 doses of 1130 mg/kg 96, 72, 48 and 24 h prior to preparation. Olive oil (25 ml/kg) was used as the solvent control and mitomycin C (3 mg/kg, i.p.) as the positive control. 2000 erythrocytes were evaluated per animal (12000/10000 per dose). No increase in micronucleated polychromatic erythrocytes was observed at any dose, while an induction of micronuclei was seen in the positive control. MMA was not mutagenic in vivo under test conditions.

Conclusions:
Interpretation of results (migrated information): negative
The substance has been administered by gavage as a solution in olive oil in 3 single doses ranging from 1130 mg/kg to 4520 mg/kg (0.5 LD50) 24 h prior to preparation of the bone marrow. A separate group of 5 animals was administered 4 doses of 1130 mg/kg 96, 72, 48 and 24 h prior to preparation. Olive oil (25 ml/kg) was used as the solvent control and mitomycin C (3 mg/kg, i.p.) as the positive control. 2000 erythrocytes were evaluated per animal (12000/10000 per dose). No increase in micronucleated polychromatic erythrocytes was observed at any dose, while an induction of micronuclei was seen in the positive control. The substance has been administered by gavage as a solution in olive oil in 3 single doses ranging from 1130 mg/kg to 4520 mg/kg (0.5 LD50) 24 h prior to preparation of the bone marrow. A separate group of 5 animals was administered 4 doses of 1130 mg/kg 96, 72, 48 and 24 h prior to preparation. Olive oil (25 ml/kg) was used as the solvent control and mitomycin C (3 mg/kg, i.p.) as the positive control. 2000 erythrocytes were evaluated per animal (12000/10000 per dose). No increase in micronucleated polychromatic erythrocytes was observed at any dose, while an induction of micronuclei was seen in the positive control. MMA was not mutagenic in vivo under test conditions.
Executive summary:

The substance has been administered by gavage as a solution in olive oil in 3 single doses ranging from 1130 mg/kg to 4520 mg/kg (0.5 LD50) 24 h prior to preparation of the bone marrow. A separate group of 5 animals was administered 4 doses of 1130 mg/kg 96, 72, 48 and 24 h prior to preparation. MMA was not mutagenic in vivo under test conditions.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Read across from analogous category member
REPORTING FORMAT FOR THE ANALOGUE APPROACH
see attached category document

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
see attached category document, chapter 1.1

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see attached category document, chapter 1

3. ANALOGUE APPROACH JUSTIFICATION
see attached category document, chapter 5 (Toxikokinetics) and endpoint specific chapters

4. DATA MATRIX
see attached category document, endpoint specific chapters
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
Swiss
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Nossan s.r.l., Corezzana, Italy,
- Age at study initiation: 5-6 weeks
- Weight at study initiation: 26-34 grams for males and 22-27 grams for females
- Assigned to test groups randomly: no data
- Housing: 5/polycarbonate cage, by sexes
- Diet (e.g. ad libitum): Altromin MT diet
- Water (e.g. ad libitum): tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 55 +/- 10
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: solubility
- Concentration of test material in vehicle: no data
Duration of treatment / exposure:
Single administration
Post exposure period:
24 and 48 hour sampling time
Remarks:
Doses / Concentrations:
0 (vehicle), 500, 1000 and 2000 mg/kg
Basis:

No. of animals per sex per dose:
Each group consisted of five male and five female animals with the exception of the control and high-dose groups, which included an additional five animals of each sex per group.
Control animals:
yes, concurrent vehicle
Positive control(s):
Mitomycin C
- Route of administration: intraperitoneal
- Doses / concentrations: 2.0 mg/kg bw
Tissues and cell types examined:
5 animals/sex/group were sacrificed at the 24-hour sampling time. The  additional animals were sacrificed at the 48-hour sampling time. At least  2000 polychromatic cells per animal were examined for the presence of micronuclei. The ratio of mature to polychromatic erythrocytes was also  determined.  
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A preliminary toxicity study was conducted in which groups of two male and two female mice were dosed once (i.p. injection) with n-butyl  methacrylate at 2000, 1500 and 1000 mg/kg. Clinical signs were observed  in all animals on the day of dosing, but all animals recovered by the  following day. No bone marrow cytotoxicity, as measured by increases in  the NCE/PCE ratio, was observed. The doses for the definitive study were  selected based on this preliminary toxicity study. 

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Five animals per sex from each group were sacrificed at the 24 hour sampling time. The additional animals were sacrificed at the 48 hour sampling time.

DETAILS OF SLIDE PREPARATION:
The femurs were removed and bone marrow cells obtained by flushing with foetal calf serum. The cells were centrifuged and a concentrated suspension prepared to make smears on slides. These slides were air-dried and then stained with May-Gruenwald and Giemsa, and mounted with Eukitt. Three slides were made from each animal.

METHOD OF ANALYSIS:
The slides were randomly coded by a person not involved in the subsequent microscope scoring. The slides were examined under low power (x 16 objective) and one slide from each animal was selected according to staining and quality of smears. At least 2000 polychromatic cells per animal were examined for the presence of micronuclei at high power (x 100 objective, oil immersion). At the same time the numbers of normal and micronucleated normochromatic erythrocytes were also recorded.
Evaluation criteria:
The test substance is considered to induce micronuclei if a statistically significant increase in the micronucleus incidence in polychromatic erythrocytes (at P<0.05) is observed in any treatment group, in the pooled data for both sexes, or for either sex considered separately.
Statistics:
Only counts obtained from polychromatic cells were subjected to statistical analysis. Using the original observations (and not the micronucleus frequencies per 1000 cells), a modified Chi-squared calculation was employed to compare treated and control groups. The degree of heterogeneity within each group was first calculated and where this was significant it was taken into account in the comparison between groups. Variance ratios or Chi-squared values are taken to show the significance of any difference between each treated group and the controls.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
but tested up to the limit dose
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
No statistically significant increase in the incidence of micronucleated PCE's over the control value was observed at any dose-level. Slight increases in the ratio of mature to polychromatic erythrocytes, compared to the vehicle control, were seen at the 48 hour sampling time for both male and female animals from the high-dose group.

Following are the results for the male and female animals combined:

                        Incidence of         NCE/PCE
            Dose        micronucleated        Mean
Treatment   Level              PCE              Ratio
           (mg/kg)        Mean      S.E.
-----------------------------------------------------------        
24 hr. Sampling time
-----------------------------------------------------------        
Vehicle (corn oil)
            10           0.5      0.2         1.12
-----------------------------------------------------------
           (ml/kg)
n-butyl methacrylate
           500           0.6      0.2         1.19
n-butyl methacrylate        
          1000           0.9      0.2         0.93
n-butyl methacrylate
          2000           0.6      0.2         1.15
-----------------------------------------------------------
Mitomycin-C  2.0        33.6***   6.5         1.32
-----------------------------------------------------------
48 hr. Sampling time
-----------------------------------------------------------        Vehicle (corn  oil)
           10            0.8      0.3         1.10
-----------------------------------------------------------
          (ml/kg)
n-butyl methacrylate
         2000            1.2      0.3         1.48
-----------------------------------------------------------
*** Incidence significantly greater than control value at p < 0.0001

Conclusions:
Interpretation of results (migrated information): negative
In a valid guideline study, no statistically significant increase in the incidence of micronucleated PCE's over the control value was observed at any dose-level. Slight increases in the ratio of mature to polychromatic erythrocytes, compared to the vehicle control, were seen   at the 48 hour sampling time for both male and female animals from the  high-dose group. The substance was negative for genotoxicity in this test.
Executive summary:

The ability of n-butyl methacrylate to cause chromosomal damagein vivowas investigated in a micronucleus OECD 474 test. Dose-levels for treatment were selected on the basis of a preliminary toxicity test. Male and female Swiss CD-1 mice were dosed once intraperitoneally with vehicle only, corn oil, 2000, 1000 and 500 mg/kg bw n-butyl methacrylate and the positive control Mitomycin-C. Five animals per sex from each group were sacrificed at the 24 hour sampling time. The additional animals were sacrificed at the 48 hour sampling time. Following treatment with n-butyl methacrylate, no statistically significant increase in the incidence of micronucleated PCE's over the control value was observed at any dose-level. Slight increases in the ratio of mature to polychromatic erythrocytes, compared to the vehicle control, were seen at the 48 hour sampling time for both male and female animals from the high-dose group, indicating that the test substance exerted a mild toxic effect on the bone marrow cells. Following treatment with the positive control Mitomycin-C, statistically significant increases in the incidence of micronucleated PCE's over the control values were seen in the positive control group indicating the correct functioning of the test system. It is concluded that n-butyl methacrylate administered intraperitoneally at dose-levels of 2000, 1000 and 500 mg/kg bodyweight to both male and female animals, does not induce micronuclei in the polychromatic erythrocytes of treated mice, under the reported experimental conditions.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

see additional information

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-BFa-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 concsb

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 concsb

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 concsb

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

positivea

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-BFb-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.

Hachiya et al. (1982) (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

An overall assessment based on the aforementioned three assessment elements (i.e. 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) leads to the conclusion that EMA is not mutagenic.

Hence, no classification is warranted for EMA.