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Additional information

Data availability: For MMA mutagenicity data are available for all required endpoints.

in vitro

Gene mutation in bacteria

In a standardizedaccording to NTP protocol, methyl methacrylate was not mutagenic in S. typhimurium strains TA97, TA98, TA100, TA1535 with and without metabolic activation (Zeiger et al. 1987). Additionally, methyl methacrylate was not mutagenic in an Ames test with the tester strain S. typhimurium TA102 (below others in that study) which is sensitive for crosslinking and oxidising agents (Schweikl et al. 2001).

Gene mutation in mammalian cells

Litton Bionetics (1981) reported on a mouse lymphoma assay which was weakly positive in the TK locus in presence and negative in absence of S-9 mix. Without S-9 mix doses up to 100 nl/ml were tested, higher doses led to total toxicity. With S-9 mix methyl methacrylate was positive in the dose range 100 nl/ml to 250 nl/ml, however, clear effects were observed only at doses with high toxicity below 20% relative growth.

In a HPRT assay with and without metabolic activation, methyl methacrylate was weakly positive in V79 cells (Schweikl et al. 1998). Without metabolic activation, the mutant frequencies in the tested concentrations of 10 and 20 mM were 6 and 16 per million surviving cells, while in the control treatment 3 mutants per 10e6 surviving cells were observed. The cell numbers of the low and high dose treatment were 71 and 49% of the control, respectively. Data from the trial with metabolic activation were not reported in detail.

In a further lymphoma assay which was only run without S-9 mix, weak effects were obtained for doses producing high toxicity (Moore et al., 1988). According to the authors, 2000 μg/ml was positive in both experiments (92 and 98 mutants per 106 survivors vs. 54 and 68 in the negative controls), relative survival was approximately 20% and 30%; in one experiment the highest dose of 499 μg/ml induced 143 mutants at 10% relative survival; in the second experiment the highest dose of 3100 μg/ml induced 220 mutants with 11% relative survival. The vast majority of induced colonies were small ones (indicating that the genetic effect was derived from clastogenicity and not from gene mutations).

Cytogenicity in mammalian cells

In a cytogenetic test with CHO cells induction of chromosomal aberrations was bound to high doses which are assumed to be strongly cytotoxic (Anderson et al., 1990). With S-9 mix, treatment was for 2 h followed by8 to 10 h recovery. Doses up to 1600 μg/ml were negative, at 5000 μg/ml the frequency of aberrant cells was 30%; only one experiment was performed. Without S-9 mix, treatment time was 8 hours with 2.0 to 2.5 h recovery. Doses up to 500 μg/ml were negative; at 1600 and 3000 μg/ml aberration frequencies ranging from 5 to 6% were found. Data on cytotoxic effects were not given, however, it can be assumed from the data presentation and the general approach of the authors that the highest doses tested led to strong cytotoxic effects. Thus, methyl methacrylate seems to be a high toxicity clastogen, i. e., the induction of chromosomal aberrations is bound to highly toxic doses.

in vivo

Quote from the MMA ESR (ECB, 2002): “A dominant lethal assay on male mice was negative after inhalation exposure to doses ranging from 0.4 to 36.5 mg/L (corresponding to 100 to 9000 ppm; Zeneca/ICI 1976). Specific data on toxicity were not given, however, in the 36.6 mg/L group 6/20 males died. Treated males were mated to 2 females each for 8 periods of 1 week each; females were killed 13 days after assumed dates of fertilization. There was no significant increase in dominant lethal mutations.

Two chromosomal aberration tests were conducted by ICI (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, a clear conclusion cannot be drawn from theses studies.

Hachiya et al. (1982) 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.”

Short description of key information:
in vitro
Gene mutation in bacteria
S. typhimurium TA97, TA98, TA100, TA1535, with and without metabolic activation: negative (NTP method, Zeiger et al. 1987)
Gene mutation in mammalian cells
Mouse lymphoma assay: weakly positive with and without metabolic activation (Litton 1981)
HPRT test, V79 cells: Ambiguous to weakly positive without metabolic activation (Schweikl et al. 1998)
Mouse lymphoma assay, clastogenicity tested: positive without metabolic activation, primarily small colony mutants indicating deletion mutations instead of gene mutations (Moore et al. 1988)
Cytogenicity in mammalian cells
Chromosome aberration assay in CHO cells: positive in cytotoxic doses (Anderson et al. 1990)
in vivo
Dominant lethal assay, mouse: negative (Zeneca/ICI 1996)
Chromosome aberration assay, rat: negative/inconclusive (Zeneca/ICI 1976, 1979)
Micronucleus assay, mouse: negative (Hachiya et al. 1982)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Methyl methacrylate was negative in bacterial gene mutation tests. From mammalian cell culture assays it may be concluded that methyl methacrylate is a high toxicity clastogen (i .e. induction of chromosomal aberrations is bound to highly toxic doses). The effect is not dependent on presence of S-9 mix. These findings are in line with results from mouse lymphoma assays where positive findings seem to be due to the induction of small colonies.

In vivo an oral mouse bone marrow micronucleus test was negative for doses up to 4,520 mg/kg bw. No clear conclusion could be drawn from bone marrow chromosomal aberration assays with rats. A dominant lethal assay with male mice led to a negative result.

In vitro MMA has the potential for induction of mutagenic effects, esp. clastogenicity; however, this potential seems to be limited to high doses with strong toxic effects. Furthermore, the negative in vivo micronucleus test and the negative dominant lethal assay indicate that this potential is probably not expressed in vivo.

Therefore, methyl methacrylate has not to be classified for its mutagenic potential according to 67/548/EEC and UN-GHS requirements, respectively.