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In vitro studies:

 

HEMA and HPMA have been evaluated for the ability to induce mutation in bacterial cells in culture. These studies (Shibuya et al, 2001), conducted by the Japan Ministry of Health and Welfare and was assigned a K-1, reliable without restriction reliability rating. HEMA and HPMA were not toxic and not mutagenic to Salmonella typhimurium tester strains (TA 1535, 1537, 1538, 98 and 100) or to E. Coli WP2 uvrA at levels up to and including 5000 microgram/plate in the presence or absence of activating enzymes. Rat liver S-9 induced with Phenobarbital and with Benzoflavone was the activation system. Appropriate positive controls for each test strain in the presence or absence of S-9 were included in the study protocol. Schweikl et al (1994; 1998) reported also that HEMA was not active in the presence or absence of rat liver enzymes for Salmonella test strains (TA 97a, 98, 100, 102, 104). Cytotoxicity was not evaluated in these studies but amounts of HEMA up to 25 mg/plate were evaluated. These studies were assigned a Klimisch rating of 2, reliable with restriction.

 

HEMA and HPMA have also been evaluated for their ability to induce chromosomal aberrations in mammalian cells in culture. Kusakabe et al (2002); Tanaka (1997) evaluated the clastogenic potential of HEMA and HPMA along with a large number of other substances in Chinese hamster lung cells in culture. Both HEMA and HPMA were reported to induce structural chromosome aberrations following 6 hr exposure of cells in the presence of S-9 only. Continuous exposure of cells for 24 hours hours or for 48 hours without S-9 also caused an elevated incidence of chromosome aberrations for both HEMA and HPMA. Polyploidy was reported after both short-term treatment and 48 hour continuous treatment exposures for both HEMA and HPMA. These effects were found at exposure levels which caused <50% cell death. These studies were assigned a Klimisch rating of 2, reliable with restriction.

 

Lee et al (2006) reported that HEMA could increase micronuclei in vitro in V79-4 cells in culture. N-acetylcysteine (NAc) was effective at preventing induction of micronuclei and the authors considered the role of active oxygen in micronuclei induction.About 2×105V79-4 cells were seeded onto microscopic glass slides in 4ml DMEM and allowed to grow for 12 h. Then, the resin monomers (GMA, TEGDMA, and HEMA) were added, and the cultures were incubated for 24 h. In a previous study, 24h of the exposure time was proved to be enough for generation of micronuclei by the resin monomers. Micronuclei were analyzed microscopically in three parallel cultures (slides) of 1000 cells/slide per concentration of resin monomer. Micronuclei were identified as DNA containing structures; their area was less than one-third of that of the main nucleus. Only mononucleated cells containing less than five micronuclei were scored; cells in mitosis and those exhibiting apoptotic nuclear fragmentations were not counted. Ethylmethane sulfonate (EMS) was used as a positive control. Concentrations of HEMA that increased micronuclei incidence were 3-5 mM and occurred at cell survival that exceeded 60%. NAc (10mM) inhibited the induction of micronuclei by more than 50% at 4-5mM HEMA. NAc was also reported to block HEMA-induced DNA fragmentation and apoptosis in RPC-C2A cells in culture.Schweikl et al (2007) also have reported the induction of micronuclei in V79 cells in culture exposed to mM concentrations of HEMA; NAc inhibited this response.

 

Pawlowska et al (2010) reported that HEMA was able to damage DNA in lymphocytes in culture using the Comet assay system. HEMA at concentrations up to 10mM did not affect the viability of the cells during a 1 h exposure. However, HEMA induced concentration dependent DNA damage in lymphocytes, as assessed by alkaline and pH 12.1 versions of the comet assay. The increase was over 100% for the highest HEMA concentration (10mM,p< 0.01). No changes in the percent tail DNA in the pH 12.1 and neutral versions of this test were observed, which indicates that the chemical did not introduce DNA-strand breaks in lymphocytes. The inability of HEMA to induce DNA double-strand breaks was confirmed by pulsed-field gel The results obtained indicated that HEMA induced mainly alkali-labile sites in DNA.

 

Urcan et al (2010) reported that high concentrations (1 – 11 mM) of HEMA applied to human gingival fibroblast cells in vitro caused double strand DNA breaks. This is a non-guideline study. The relevance of these effects is uncertain given the very high concentrations used. 

 

HEMA was evaluated for its ability to cause forward mutation at the hprt locus in Chinese hamster lung fibroblast V79 cells in culture(Schweikl, 1998). This study was assigned a Klimisch rating of 2, reliable with restriction. In this assay, HEMA was evaluated only in the absence of metabolic activation. Ethylmethanesulfonate (200 microgram/ml) was used as a positive control. Concentrations of HEMA up to 5mM did not increase the mutant frequency in this assay; plating efficiencies were 84-113% of control.

  

In vivo studies:

 

HEMA has been evaluated for the ability to cause chromosomal aberrations in vivo in the micronucleus assay in rats (Shirotori, 2002). This study was assigned a Klimisch rating of 1, reliable without restriction. HEMA was administered by oral gavage twice per day to groups of 5 rats at doses of 500, 1000 and 2000 mg/kg. Doses were selected based on an oral LD50 observed to be 5050 mg/kg. Animals were observed for clinical symptoms of intoxication. Cyclophosphamide (10 mg/kg once) was used as a positive control. Twenty-four hours following the final dosing, bone marrow samples were prepared and examined for incidence of micronucleated polychromatic erythrocytes (PCE). One thousand erythrocytes were calculated to evaluate the ratio of PCEs to erythrocytes; two-thousand PCEs were scored for micronuclei.

 

In this study, no animals died and there were no clinical symptoms of intoxication even though HEMA was administered at 40% of the LD50. There was no increase in the number of micronucleated PCEs at any HEMA dose level. Cyclophosphamide treatment did cause an increase in micronucleated PCEs to an extent consistent with laboratory historical controls.

 

Arossi et al (2009) reported that HEMA was not active in a test of mutagenicity in Drosophila melanogaster in vivo.The study investigated the genotoxicity of four dental resin monomers: triethyleneglycoldimethacrylate (TEGDMA), hydroxyethylmethacrylate (HEMA), urethanedimethacrylate (UDMA) and bisphenol A-glycidylmethacrylate (BisGMA). The Somatic Mutation and Recombination Test (SMART) indetects genotoxicity expressed as homologous mitotic recombination, point and chromosomal mutation. SMART detects the loss of heterozygosity of marker genes expressed phenotypically on the flys wings. This fruit fly has an extensive genetic homology to mammalians, which makes it a suitable model organism for genotoxic investigations. The authors concluded that their findings provide evidence that the mechanistic basis underlying the genotoxicity of UDMA and TEGDMA is related to homologous recombination and genechromosomal mutation. A genotoxic pattern can correspondingly be attributed for both UDMA and TEGDMA: their genotoxicity is attributed respectively to 49% and 44% of mitotic recombination, as well as 51% and 56% of mutational events, including point and chromosomal alterations. BisGMA and HEMA had no statistically significant effect on total spot frequencies – suggesting no genotoxic action in the SMART assay.


Short description of key information:
Both HEMA and HPMA have been evaluated for genotoxic potential in bacterial cells in culture and were found not to be mutagenic in this assay system. Further, HPMA was found not to be mutagenic in mammalian cells in culture [CHO HGPRT assay] and HEMA was found not to be mutagenic in mammalian cells in culture [CHL V79 cell assay], albeit HEMA was evaluated only in the absence of S-9. Both HEMA and HPMA were reported to cause chromosomal aberrations in mammalian cells in culture, but an in vivo micronucleus study with HEMA indicated that this material was not clastogenic in vivo.

Thus, while HEMA does not have a complete evaluation for the endpoint of mutation in mammalian cells in culture [results available only in absence of S-9], it is concluded that the available results for HPMA for this endpoint may be read-across to HEMA given the close structural similarities of the two materials and similar behavior in other genotoxicity assays.

Further, while HPMA has demonstrated the ability to cause chromosomal aberrations in vitro, it is concluded that the available in vivo results for HEMA in the micronucleus test and in Drosophila melanogaster may be read-across to HPMA given the close structural similarities of the two materials and similar behavior in other genotoxicity assays.

MMA has been extensively tested for potential to cause genotoxicity. Like HEMA and HPMA, MMA was not active in the Ames test and like HEMA/HPMA, MMA was found to cause chromosome aberrations in mammalian cells in culture. However, MMA was not active in chromosome aberration tests in vivo including the micronucleus test and dominant lethal assay. (EU RAR, 2002)

It is concluded that both HEMA and HPMA are not mutagenic in bacterial and mammalian cells in culture. Further, while HEMA and HPMA may cause chromosomal aberrations in mammalian cells in culture, these materials are not clastogenic in vivo.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

It is concluded that both HEMA and HPMA are not mutagenic in bacterial and mammalian cells in culture. Further, while HEMA and HPMA may cause chromosomal aberrations in mammalian cells in culture, these materials are not clastogenic in vivo.