Quantitative determination of TEGDMA, BHT, and DMABEE in eluates from polymerized resin-based dental restorative materials by use of GC/MS.

This study investigated the leaching of ingredients from several commercial dental composite resins cured with LED, and immersed in methanol or water for 24 h, respectively. The composites used were: Admira Dentin (VOCO), Artemis Schmelz (Enamel) (Ivoclar Vivadent), Els extra low shrinkage (Saremco Dental), Filtek Supreme XT Dentin (3 M ESPE), Gradia Direct (GC), Venus & Venus flow (Heraeus Kulzer), and XRV Herculite Prodigy Enamel (Kerr). From each dental composite four specimens with defined structure and 100-mg net weight were made. After the polymerization process, according to manufacturer's instructions, the specimens were immersed in either 1 ml water or 1 ml methanol and incubated at 37 degrees C for 24 h. Eluted ingredients triethyleneglycoldimethacrylate (TEGDMA), 2,6-di-tert-butyl-4-methylphenol (BHT), and 4-N,N-dimethylaminobenzoicacidethylester (DMABEE) were detected and quantified using gas chromatography-mass spectrometry (GC-MS). The amounts of the detected analytes from 100 mg polymerized composites ranged between the following values: TEGDMA: 0-0.5 mg (water), 0-1.6 mg (methanol); BHT: 0-0.03 µg (water), 0-0.11 mg (methanol); and DMABEE: 0-0.11 mg (water), 0-1.4 mg (methanol). We conclude from the results that the elution rates into methanol and water differ significantly. Furthermore, it is concluded that all the determined amounts eluting from the composites are far below toxic-relevant concentrations.

Volatile methacrylates in dental practices.

PURPOSE: In recent years, an increase of occupational respiratory diseases, such as asthma caused by methacrylates, has been observed in dental personnel. In this study, the exposure of dental personnel to various volatile methacrylates was investigated. MATERIALS AND METHODS: The air levels of methacrylates were measured during filling treatment while bonding agents were used in 4 dental practices in Munich, Germany. Short-term air sampling (15 min) was performed using solid phase microextraction (SPME). The SPME fibers were coated with carbowax/divinyl benzene to enrich the analytes. For analysis, the analytes were thermically desorbed from the fiber and subsequently analyzed directly by gas chromatography/mass spectrometry. RESULTS: The methacrylates methyl methacrylate (MMA), 2-hydroxyethyl methacrylate (HEMA), ethylene glycol dimethacrylate (EGDMA), and triethylene glycol dimethacrylate (TEG-DMA) were identified in the air of dental practices. The exposure levels of the four methacrylates varied during the filling treatments. The maximum concentrations found were 0.4 mg/m3 for MMA, 45 microg/m3 for HEMA, 13 microg/m3 for EGDMA, and 45 microg/m3 for TEG-DMA. The detection of TEG-DMA correlated with the application of bonding agents during performance of dental fillings. CONCLUSION: Exposure levels of different methacrylates were observed at all investigated dental practices. The maximum levels of MMA measured in this study were at least 200 times lower than the toxicologically relevant maximum allowable concentrations defined in various countries. Nevertheless, the exposure levels of methacrylates should be kept as low as possible due to the allergenic potential of some methacrylates.

Cytotoxicity of the dental composite component TEGDMA and selected metabolic by-products in human pulmonary cells.

OBJECTIVES: The comonomer triethyleneglycoldimethacrylate (TEGDMA) is a commonly used constituent of resin-based dental materials. Upon placement, light-cured dental polymers may release a wide spectrum of residual compounds due to incomplete monomer-conversion during polymerization. Apart from liberating unreacted monomers, additional compound release might occur due to mechanical wear and enzymatic degradation on the salivary surface of resin fillings. Following delivery into the local bio phase, leached compounds may encounter a variety of different enzymes, which might be present in their oral or systemic environment. Metabolic by-products formerly associated with TEGDMA-degradation include triethylene glycol (TEG), methacrylic acid (MA), 2,3-epoxymethacrylic acid (2,3-EMA), and formaldehyde. METHODS: Cytotoxicitiy of TEGDMA-derived intermediates was measured as mitochondrial dehydrogenase activity assessed by colorimetric measurement of formazan formation as a cleavage-product from the tetrazolium salt XTT by metabolically active A549 cells. EC(50)-values were calculated by using curve fitting software (GraphPad Prism). RESULTS: The following EC(50)-values (mmol/L) (95% confidence interval) were obtained: 2,3-EMA 1.65 (1.28-2.13), TEGDMA 1.83 (1.46-2.30), MA 4.91 (4.22-5.71), and paraformaldehyde (PFA) 5.48 (4.56-6.58). For TEG no cytotoxic effects up to a concentration of 10mM could be found. SIGNIFICANCE: The epoxy compound 2,3-EMA induced comparable toxic effects as the raw comonomer TEGDMA. It is therefore concluded that the formation of toxic intermediates might significantly contribute to TEGDMA-induced cytotoxicity in human pulmonary cells.

Identification of 2,3-epoxymethacrylic acid as an intermediate in the metabolism of dental materials in human liver microsomes.

OBJECTIVES: In previous studies it could be demonstrated that methacrylic acid (MA) is an intermediate in the metabolism of unpolymerized dental comonomers, released from dental restorative materials. This study was performed to identify the possible dental material intermediate 2,3-epoxymethacrylic acid (2,3-EMA) from MA in human liver microsomes. Most epoxy compounds are regarded as highly toxic substances. METHODS: The formation and hydrolysis were studied in defined systems containing only MA and human liver microsomes at 37 degrees C. Hydrolysis was inhibited by cyclohexene oxide, a competitive inhibitor of epoxide hydrolase. The reaction product 2,3-EMA was analyzed by the headspace gas chromatography-mass spectrometry. After 5, 30, and 60 min samples were taken and analyzed. RESULTS: For the reaction of MA to 2,3-EMA the average conversion rate was about 5% within 1h. It was found that without cyclohexene oxide the rate constant of enzymatic hydrolysis at pH 7.4 was about 10 times higher than the rate constant of the formation from MA in combination with cyclohexene oxide (k=8.3 versus 0.83 micromol/l min), indicating an instability and thus a high reactivity of 2,3-EMA. The formation of the MA intermediate 2,3-EMA was not observed when heat-inactivated liver microsomes were used (controls). SIGNIFICANCE: It could be clearly demonstrated that 2,3-EMA is a product of dental material metabolisms in biological systems. Therefore, increased toxicity might occur on dental restorative materials which are able to release (co)monomers which can be metabolized to MA.

Elution of TEGDMA and HEMA from polymerized resin-based bonding systems.

OBJECTIVES: In this study the amount of TEGDMA and HEMA eluted from several adhesive systems was quantified. METHODS: The adhesive systems were applied according to manufacturers' instructions in an analytic vial. The adhesive systems used were (abbreviation and producer in parenthesis): cmf adhesive system(®) (CMF) (Saremco), ENAbond (EB) (Micerium), Optibond FL (OB) (Kerr), Adper Scotchbond Multi-Purpose (SB) (3M ESPE), Silorane System Adhesive (SSA) (3M ESPE), Syntac Classic (SC) (Ivoclar Vivadent) and XP Bond (XPB) (Dentsply). After preparation the specimens were immersed in methanol or distilled water for a period from 1d to 30d at 37°C. Eluted TEGDMA and HEMA were determined by gas chromatographic-mass spectrometry (GC-MS). RESULTS: Following TEGDMA elution from adhesives was found (µg/ml; mean and standard deviation(sd); 1d/30d; methanol): SC 0.93(0.8)/0.68(0.5), SSA 0.27(0.09)/0.16(0.04) and XPB 0.25(0.1)/0.19(0.09). TEGDMA eluted from EB, CMF, OB, and SB was always below detection limit. TEGDMA water elution from each adhesive was about 1/5 lower, compared to the corresponding TEGDMA methanol elution. Following HEMA elution was found (µg/ml; mean(sd); 1d/30d; methanol): SB 3.42(0.9)/2.02(1.2), EB 3.07(2.2)/2.15(2.2), XPB 2.47(1.6)/1.89(1.1), OB 1.4(0.7)/0.82(0.2) and SSA 0.44(0.2)/0.17(0.07). HEMA eluted from CMF and SC was always below detection limit. HEMA water elution from each adhesive was about 1/10 lower, compared to the corresponding HEMA methanol elution. SIGNIFICANCE: SC, SSA, and XPB eluted TEGDMA. SB, EB, XPB, OB, and SSA eluted HEMA. CMF eluted neither HEMA nor TEGDMA.

Expression of CYP450-2E1 and formation of 2,3-epoxymethacrylic acid (2,3-EMA) in human oral cells exposed to dental materials.

OBJECTIVES: Methacrylate-based (co)monomers released from dental composites can be, metabolized in vivo to methacrylic acid (MA). MA can be further oxidized to the toxic 2,3-epoxymethacrylic acid (2,3-EMA) by cytochrome P450 (CYP450) enzymes. The subform CYP450-2E1, can metabolize xenobiotics with low-molecular weight to epoxides. Oral cells are highly exposed to (co)monomers released from composites. Therefore in this study the, expression of CYP450-2E1 in human oral (and other) cells was investigated as well as the formation of 2,3-EMA in cells exposed to MA. METHODS: Following human oral cells were used: human gingiva fibroblasts (HGF), human pulp fibroblasts (HPF), and human tumor buccal keratinocytes (SqCC/Y1). As negative control V79 cells without CYP450-2E1 expression were used. As positive controls V79 cells with CYP450-2E1 expression (V79-CYP450-2E1) and pooled human liver microsomes were used. The expression of CYP450-2E1 in cells was analyzed with the real-time polymerase chain reaction (RT-PCR). 2,3-EMA was quantified by the use of the method of gas chromatography/mass spectrometry (GC/MS). RESULTS: The highest expression of CYP450-2E1 was found in human liver microsomes, followed by SqCC/Y1 cells, V79-CYP450-2E1 cells, HGF, and HPF. The highest amount of 2,3-EMA (µmol/L; mean±SEM, n=3) was found in human liver microsomes (5.0±1.0), followed by SqCC/Y1 cells (2.5±0.8), V79-CYP450-2E1 cells (1.5±0.6), HPF (0.3±0.3), and HGF (0.2±0.2). SIGNIFICANCE: It is concluded that the formation of the toxic epoxide 2,3-EMA, as intermediate in the metabolism of dental materials, can occur also in human oral cells which can express the CYP450-2E1 enzyme system.

Excretion of dental resin monomers and metabolic intermediates via urine in guinea pigs.

OBJECTIVE: Monomers like BisGMA (Bisphenol-A-glycidyldimethacrylate) and comonomers like TEGDMA (triethyleneglycoldimethacrylate) are used in dental restorative materials in order to build up the three-dimensional network of filling materials. Since earlier investigations revealed uptake and subsequent metabolism of unpolymerized remainders of (co)monomers, the present experiment investigates the metabolic urine pattern of guinea pigs (n=4) after application of TEGDMA or BisGMA (each dose=0.02 mmol/kg body weight=100%), respectively. METHODS: For the investigations BisGMA was pre-dissolved in DMSO and subsequently diluted with 0.9% NaCl solution (final DMSO concentration 1%) and TEGDMA was dissolved in 0.9% NaCl solution. The solutions were administered with a gastric tube into the animals. Control animals received either 0.9% NaCl or 0.9% NaCl solution with 1% DMSO solution. RESULTS: After 24h in collected urine the following metabolites were identified. After administration of TEGDMA (mean relative concentration of administered substances)+/-s.d. [%]; n=4): unchanged TEGDMA: 12+/-1.5%, MA: 2.4+/-0.8%, and triethyleneglycol: 35+/-2.2%. After administration of BisGMA (mean+/-s.d. [%]; n=4): unchanged BisGMA: 11.4+/-2.7%, MA: 2.2+/-0.6%, and bisphenol-A-bis(2,3-dihydroxypropyl)ether: 60.1+/-5.2%). CONCLUSION: No further metabolites like the previously identified intermediate 2,3-epoxymethacrylic acid and derived reaction products were identified in the urine, indicating that these metabolites must have reacted further.

In vitro stability of methylmethacrylic acid, TEGDMA and HEMA exposed to esterases.

OBJECTIVES: Comonomers used in dental restorative materials, e.g. triethylenglycoledimethacrylate (TEGDMA), hydroxyethylmethacrylate (HEMA) and methylmethacylic acid (MMA) are methacrylic acid esters. Because of the chemical structure of these esters in earlier studies two different in vitro pathways were suggested. The present study was performed in order to analyze which of these chemical pathways is preferred in vitro: (a) saponification of TEGDMA, HEMA, and MMA leading to free methacrylic acid (MA) as an ionic intermediate or (b) enzymatically epoxidation of mother compounds leading to lipophilic intermediates. METHODS: Experiments have been performed in an isolated system using MMA, TEGDMA or HEMA, respectively exposed to pig liver esterase (PLE) in phosphate buffer. The reaction of tested comonomers was terminated by the use of sodium hydroxide solution, sulfuric acid or saturated NaCl solution, respectively. The samples were analyzed by the use of headspace-gas chromatography-mass spectrometry. RESULTS: In all samples a decomposition of comonomers by the use of PLE was observed. Due to the high rate constant k of MMA (e.g. k(MMA)>1.3mg/(ls)) epoxidation of non-cleaved molecules of this substance can be excluded. Compared to MMA the decomposition of TEGDMA and HEMA is significantly slower (e.g. k((TEGDMA, PLE=7units/ml))=0.004mg/(ls) and k((HEMA, PLE=7units/ml))=0.00013mg/(ls)). Thus in case of a low liver enzyme concentration the epoxidation of non-cleaved molecules of TEGDMA and HEMA cannot be excluded. SIGNIFICANCE: In the metabolic pathway of TEGDMA and HEMA the probability of an auxiliary chemical pathway was demonstrated. In case of MMA the formation of epoxidated metabolites can be excluded. In contrast to this the chemical pathway for TEGDMA and HEMA might lead to lipophilic intermediates which can be accumulated in fatty tissue.

Development and application of a simplified sample preparation method for determination of TEGDMA and related metabolites.

Analysis of biological samples obtained from in vivo experiments can be often challenging. In general it is not possible to apply the commonly used matrices that are necessary for the experiments to the desired analysis systems without further conditioning or sample purification steps. Besides possible adverse effects for instruments, interference between analytes and matrices can affect the correct measurement of analytes. Different methods of sample preparation can be used to convert biological samples into samples suitable for analysis; SPE and HS-SPME are two well established methods. Research of in vivo metabolism of triethyleneglycoledimethacrylate (TEGDMA), one of the most frequently contained comonomer in dental restorative materials, demands sample preparation methods that offer separation of TEGDMA and its related metabolites from biological matrices. In the presented study two methods for sample preparation were developed in order to analyze TEGDMA as well as its metabolites triethyleneglycole (TEG), 2,3-epoxymethacrylicacid methylester (2,3-EMME), and methacrylacid methylester (MAME) in Krebs-Henseleit buffer samples to facilitate a subsequent analysis via GC-MS. An easy and time-saving separation protocol was developed. Recovery rates of TEGDMA and TEG after SPE were 21 +/- 3% and 105 +/- 12%, respectively, recovery rate after headspace extraction of 2,3-EMME and MAME was higher at 48 degrees C compared with 20 degrees C extraction temperature. The tested range for 2,3-EMME and MAME concentration after HS-SPME extraction was 0.1-100 mg/L and both analytes showed a good linearity.

Toxicity potentiation by H2O2 with components of dental restorative materials on human oral cells.

Toxicity potentiation of two monomers [bisphenol-A-glycidyldimethacrylate (BisGMA) and urethanedimethacrylate (UDMA)] as well as two comonomers [triethyleneglycoldimethacrylate (TEGDMA) and 2-hydroxyethylmethacrylate (HEMA)], each in combination with H(2)O(2), was investigated on the viability on human gingival fibroblasts (HGF) and human pulpal fibroblasts (HPF). The applied concentration of H(2)O(2) was 0.06 or 0.1 mmol/l, respectively, corresponding to the EC(0) of H(2)O(2) in HGF or HPF. The cell viability was assessed by the XTT test. From this test the half maximum effect concentrations (EC(50)) were calculated from fitted sigmoidale curves. EC(50) values were (HGF; mmol/l; mean +/- s.e.m.; n = 5): HEMA 11.9 +/- 0.9, TEGDMA 3.7 +/- 0.3, H(2)O(2) 0.36 +/- 0.04, UDMA 0.27 +/- 0.08, and BisGMA 0.11 +/- 0.03. No significant (P < 0.05) differences in the EC(50) values were observed when HGF was exposed to substances, as compared to HPF. No significant decrease of the EC(50) values was found when HGF or HPF, respectively, was exposed to HEMA or BisGMA in addition with H(2)O(2) up to the concentration of 0.1 mmol/l, as compared to those EC(50) values of each compound without H(2)O(2) addition. A significant decrease of the TEGDMA EC(50) value from 3.7 to 2.1 or 0.4 mmol/l, respectively, was found when cells were exposed to TEGDMA in combination with H(2)O(2) (0.06 or 0.1 mmol/l), as compared to that TEGDMA EC(50) value without H(2)O(2) addition. A significant decrease of the UDMA EC(50) value from 0.27 to 0.11 or 0.08 mmol/l, respectively, was found when HGF or HPF was exposed to UDMA in combination with H(2)O(2) (0.06 or 0.1 mmol/l), as compared to that UDMA EC(50) value without H(2)O(2) addition. The addition of H(2)O(2) (0.06 or 0.1 mmol/l) resulted in a toxicity potentiation of TEGDMA and UDMA, but not of HEMA and BisGMA, on HGF or HPF.

Cytotoxicity of dental composite (co)monomers and the amalgam component Hg(2+) in human gingival fibroblasts.

Unpolymerized resin (co)monomers or mercury (Hg) can be released from restorative dental materials (e.g. composites and amalgam). They can diffuse into the tooth pulp or the gingiva. They can also reach the gingiva and organs by the circulating blood after the uptake from swallowed saliva. The cytotoxicity of dental composite components hydroxyethylmethacrylate (HEMA), triethyleneglycoldimethacrylate (TEGDMA), urethanedimethacrylate (UDMA), and bisglycidylmethacrylate (Bis-GMA) as well as the amalgam component Hg(2+) (as HgCl(2)) and methyl mercury chloride (MeHgCl) was investigated on human gingival fibroblasts (HGFs) at two time intervals. To test the cytotoxicity of substances, the bromodeoxyuridine (BrdU) assay and the lactate dehydrogenase (LDH) assay were used. The test substances were added in various concentrations and cells were incubated for 24 or 48 h. The EC(50) values were obtained as half-maximum-effect concentrations from fitted curves. Following EC(50) values were found [BrdU: mean (mmol/l); SEM in parentheses; n=12]: (24 h/48 h) HEMA 8.860 (0.440)/6.600(0.630), TEGDMA 1.810(0.130)/1.220(0.130), UDMA 0.120(0.010)/0.140(0.010), BisGMA 0.060(0.004)/0.040(0.002), HgCl(2) 0.015(0.001)/0.050(0.006), and MeHgCl 0.004(0.001)/0.005(0.001). Following EC(50) values were found [LDH: mean (mmol/l); SEM in parentheses; n=12]: (24 h/48 h) HEMA 9.490(0.300)/7.890(1.230), TEGDMA 2.300(0.470)/1.950(0.310), UDMA 0.200(0.007)/0.100(0.007), BisGMA 0.070(0.005)/0.100(0.002), and MeHgCl 0.014(0.006)/0.010(0.003). In both assays, the following range of increased toxicity was found for composite components (24 and 48 h): HEMA < TEGDMA < UDMA < BisGMA. In both assays, MeHgCl was the most toxic substance. In the BrdU assay, Hg(2+) was about fourfold less toxic than MeHgCl but Hg(2+) was about fourfold more toxic than BisGMA. In the BrdU test, a significantly (P<0.05) decreased toxicity was observed for Hg(2+) at 48 h, compared to the 24 h Hg(2+)-exposure. A time depending decreased toxicity was observed only for Hg(2+) which can then reach the toxic level of BisGMA.

Cell death effects of resin-based dental material compounds and mercurials in human gingival fibroblasts.

In order to test the hypothesis that released dental restorative materials can reach toxic levels in human oral tissues, the cytotoxicities of the resin-based dental (co)monomers hydroxyethylmethacrylate (HEMA), triethyleneglycoldimethacrylate (TEGDMA), urethanedimethacrylate (UDMA), and bisglycidylmethacrylate (BisGMA) compared with methyl mercury chloride (MeHgCl) and the amalgam component mercuric chloride (HgCl2) were investigated on human gingival fibroblasts (HGF) using two different test systems: (1) the modified XTT-test and (2) the modified H 33342 staining assay. The HGF were exposed to various concentrations of the test-substances in all test systems for 24 h. All tested (co)monomers and mercury compounds significantly (P<0.05) decreased the formazan formation in the XTT-test. EC50 values in the XTT assay were obtained as half-maximum-effect concentrations from fitted curves. Following EC50 values were found (mean [mmol/l]; s.e.m. in parentheses; n=12; * significantly different to HEMA): HEMA 11.530 (0.600); TEGDMA* 3.460 (0.200); UDMA* 0.106 (0.005); BisGMA* 0.087 (0.001); HgCl2* 0.013 (0.001); MeHgCl* 0.005 (0.001). Following relative toxicities were found: HEMA 1; TEGDMA 3; UDMA 109; BisGMA 133; HgCl2 887; MeHgCl 2306. A significant (P<0.05) increase of the toxicity of (co)monomers and mercurials was found in the XTT-test in the following order: HEMA < TEGDMA < UDMA < BisGMA < HgCl2 < MeHgCl. TEGDMA and MeHgCl induced mainly apoptotic cell death. HEMA, UDMA, BisGMA, and HgCl2 induced mainly necrotic cell death. The results of this study indicate that resin composite components have a lower toxicity than mercury from amalgam in HGF. HEMA, BisGMA, UDMA, and HgCl2 induced mainly necrosis, but it is rather unlikely that eluted substances (solely) can reach concentrations, which might induce necrotic cell death in the human physiological situation, indicating that other (additional) factors may be involved in the induction of tissue (pulp) inflammation effects after dental restauration.