Abstract
Objectives
Components released from resin-based dental materials are important factors in the assessment of the biocompatibility of these materials. The study was performed to investigate the elution of substances from unpolymerized and polymerized composites (Tetric ® = TET; Arabesk ® = ARA), ormoceres (Admira ® = ADM; Definite ® = DEF) and the compomere (Hytac ® = HYT).
Methods
Specimens were polymerized and immersed in either water or methanol. Besides the unpolymerized specimens were dissolved in methanol. Eluted substances were detected by gas chromatography/mass spectroscopy. The Nernst partition coefficient (DC) of 64 substances, eluted from various resin-based dental materials was determined.
Results
Only in methanolic and aqueous eluates from unpolymerized and polymerized specimens of DEF and ADM, the comonomere 1,2/1,3-glycerol dimethacrylate was measured. Triethylene glycol dimethacrylate was eluted into the aqueous and methanolic phase from polymerized specimens from TET and ARA. 2-Hydroxyethyl methacrylate was eluted into the methanolic phase from polymerized specimen from TET and HYT. Residuals of monomer synthesis like triphenyl stibane were found in unpolymerized specimens of TET and ARA. Camphorquinone was eluted into the methanolic eluate from polymerized and unpolymerized specimens from all tested dental materials. Highest DC of 20.8 was found for 1/2-cyclohexene methacrylate. DC of urethane dimethacrylate and bisphenol-A-glycidyldimethacrylate varies from different manufacturers.
Significance
Impurities from manufacturing process were found in some resin-based materials. Various well-known substances causing allergic reactions were found in aqueous or methanolic elutes. Patients, dental and manufacturing personnel are exposed to these substances.
1
Introduction
Polymer-based materials are used in many dental applications, mainly in dental prostheses and as filling materials. Modern light-hardening composite fillings consist of bifunctional dimethacrylates, additives like photoinitiators, coinitiators, photostabilizers, inhibitors and inorganic fillers. One problem of the light-cured dental composite materials is the incomplete polymerization. The degree of conversion depends on the wavelength, wavelength-distribution and intensity of the light source, as well as the distance light source – dental material, the hardening time, the composition and the color of the dental material . It is generally in the 55–65% range . First studies of elution of uncured monomers, degradation products and other additives were reported by Spahl et al. . They used gas chromatography/mass spectroscopy (GC/MS) to investigate substances eluted from light-cured composites. Apart from composite-ingredients, they found eluteable reaction products and eluteable substances from the composite production like the catalyst triphenyl stibane (TPSB, 0.005 mg/ml in methanolic extract) from bisphenol-A-glycidyldimethacrylate (Bis-GMA) synthesis.
Only substances which are eluted can enter the organism and can cause harm to health. It is to note that some methacrylates released from dental materials, such as methyl methacrylate (MMA), ethylene glycol dimethacrylate (EGDMA) and triethylene glycol dimethacrylate (TEGDMA), have been known to act as strong sensitizers for allergic reactions for a long time . In recent years, a dramatic increase of allergic contact dermatitis and of asthma was observed in persons exposed to these substances . Other reactions described in the literature are: lichen planus (local), gingivitis, ulcerations, eczema, erythema and blisters . Dentists, dental personnel, dental patients and manufacturing personnel can be exposed to these substances.
Substances can only cause systemic or local effects, if they can cross the physiological barriers. The permeation of a substance through biological barriers is dependent on the distribution of a watery (hydrophilic) and an organic (lipophilic) phase . Collander implemented an octanol/water system to determine the distribution of a substance in two, not mixable phases. Based on the law of Nernst, it is possible to calculate a partition coefficient between the two phases, which helps to estimate, e.g. membrane permeability. The partition coefficient is recognized by governmental and international agencies (U.S. Environmental Protection Agency, OECD) as a physical property of organic substances equal in importance to vapor pressure, water solubility and toxicity .
Up to now, polymerized dental composites release substances to a varying degree . The aim of this study was to determine and to quantify substances eluted from unpolymerized (i.e. the organic matrix) and polymerized widely used dental composites, compomeres and ormoceres. We choose the same experimental conditions (e.g. surface of specimen, temperature of elution) as in the first reported elution experiment in the literature from dental composites from Spahl et al. in order to compare (if up to now available) changes in composition and impurities of the organic matrix of the dental materials as well as elutable substances from polymerized specimens. Moreover, released substances from unpolymerized and polymerized specimens were compared with current literature on causing allergic reactions. In addition, we determined the Nernst partition coefficient (DC; octanol/buffer) from eluted substances as one parameter for crossing physiological barriers.
2
Methods
All solvents and reagent products were obtained from Merck, Darmstadt, Germany and of high purity quality.
2.1
Preparation of samples
From the light-curable tooth restorative materials Arabesk ® (ARA, VOCO, Cuxhaven, Germany; LOT 71550), Admira ® (ADM, VOCO, Cuxhaven, Germany; LOT 15692), Definite ® (DEF, Degussa, Hanau, Germany; LOT 205), Tetric classic ® (TET, Vivadent, Ellwangen, Germany; LOT 903553) and Hytac ® (HYT, ESPE, Seefeld, Germany; LOT FW0035960) specimens of approximately 100 mg (thickness of 1 mm, diameter of 6 mm; color A3, with a resulting surface of the cylinder of 75.4 mm 3 ) were prepared. The specimens were covered with plastic matrix strips (Frasaco, Tettnang, Germany) and polymerized for 40 s (according to the instructions of the manufacturer) by using an Elipar ® II light source (ESPE, Seefeld, Germany), the emitted light wavelength was between 400 and 500 nm and light intensity was 400 mW/cm 2 , according to the instructions of the manufacturers. For hardening, the 2-Step-Modus was used. In this modus the light intensity was reduced for the first 10 s. Therefore a more gently polymerization start was possible, according to information of the manufacturer. Then the specimens were incubated in distilled water (100 mg/ml) or methanol (100 mg/ml) at 25 °C for 24 h. To quantify the total substance content of unpolymerized dental material, 100 mg of all dental materials were dissolved in 1 ml methanol for 15 min at 25 °C. Then the filler particles were removed by centrifugation at 1500 × g . To the aqueous and methanolic eluates, 0.1 mg/ml caffeine (CF) was added and the mixture was analyzed by GC/MS.
2.2
GC/MS analysis
GC/MS using a particle beam interface is a sensitive method for qualitative and quantitative analysis of residual compounds from dental resin composites . HPLC was not suitable for this experiment because for identification of new substances the fragmentation pattern of MS is very important. The analysis was performed on a Finnigan MAT 95Q sector field-quadropol hybrid mass spectrometer (Finnigan MAT, Bremen, Germany) connected to a Varian 3400 gas chromatograph (Varian, Darmstadt, Germany). The temperature of the combined EI/CI source was 250 °C; the electron energy was 70 eV. The scan rate was 1 s/decade. A CB-SE-54 (Chromatographic Service, Langerwehe, Germany; length 25 m, inner diameter 0.25 mm, coating 0.25 μm) was used as capillary column for GC. Helium was used as carrier gas. The temperature of the split–splitless injector as well as of the direct coupling to the mass spectrometer was 300 °C. The GC oven was heated from 50 °C (2 min isotherm) to 300 °C (5 min isotherm) with a rate of 10 °C/min and 1 μl of the solution was injected with a split of 1:10. The results were referred to an internal CF standard (0.1 mg/ml CF = 100%), which allows a comparison of the relative quantities of substances released from various resin-based materials. All extracts were analyzed five times. The average error was <10%. The integration of the chromatograms was carried out through the base peak or other characteristic mass peaks of the compounds, and the results were normalized by means of the internal caffeine standard, yielding a value of (% CF). Identification of the various substances was achieved by comparison of their mass spectra with those of reference compounds, the NIST/EPA library, the literature data and/or by interpretation of their fragmentation patterns .
2.3
Nernst partition coefficient (DC)
To prepare a stock solution, 30 mg of pure substance was dissolved in 2 ml 1-octanol and their molar absorption ( ε ) coefficient was determined photometrically by using standard solutions. To ascertain the partition coefficient, 333 μl from the stock solution was diluted with 667 μl 1-octanol. After addition of 1 ml phosphate-buffered isotonic NaCl-solution (PBS; ), the samples were equilibrated 1 h at 37 °C under permanent shaking . 500 μl from the organic phase was diluted, dependent on the molar absorption coefficient, and photometrically measured (UV 265 FW-Visable Recording Spectrophotometer, Shimadzu, Kyoto, Japan). At the beginning of the experiment, 1-octanol and PBS were saturated with the antiphase. The DC is presented as decadic logarithm of the partition coefficient. The substances were subdivided into basic- and comonomers, initiators, photo- and coinitators, photostabilizers, inhibitors and other detected substances (e.g. polymerization and decomposition products).
2
Methods
All solvents and reagent products were obtained from Merck, Darmstadt, Germany and of high purity quality.
2.1
Preparation of samples
From the light-curable tooth restorative materials Arabesk ® (ARA, VOCO, Cuxhaven, Germany; LOT 71550), Admira ® (ADM, VOCO, Cuxhaven, Germany; LOT 15692), Definite ® (DEF, Degussa, Hanau, Germany; LOT 205), Tetric classic ® (TET, Vivadent, Ellwangen, Germany; LOT 903553) and Hytac ® (HYT, ESPE, Seefeld, Germany; LOT FW0035960) specimens of approximately 100 mg (thickness of 1 mm, diameter of 6 mm; color A3, with a resulting surface of the cylinder of 75.4 mm 3 ) were prepared. The specimens were covered with plastic matrix strips (Frasaco, Tettnang, Germany) and polymerized for 40 s (according to the instructions of the manufacturer) by using an Elipar ® II light source (ESPE, Seefeld, Germany), the emitted light wavelength was between 400 and 500 nm and light intensity was 400 mW/cm 2 , according to the instructions of the manufacturers. For hardening, the 2-Step-Modus was used. In this modus the light intensity was reduced for the first 10 s. Therefore a more gently polymerization start was possible, according to information of the manufacturer. Then the specimens were incubated in distilled water (100 mg/ml) or methanol (100 mg/ml) at 25 °C for 24 h. To quantify the total substance content of unpolymerized dental material, 100 mg of all dental materials were dissolved in 1 ml methanol for 15 min at 25 °C. Then the filler particles were removed by centrifugation at 1500 × g . To the aqueous and methanolic eluates, 0.1 mg/ml caffeine (CF) was added and the mixture was analyzed by GC/MS.
2.2
GC/MS analysis
GC/MS using a particle beam interface is a sensitive method for qualitative and quantitative analysis of residual compounds from dental resin composites . HPLC was not suitable for this experiment because for identification of new substances the fragmentation pattern of MS is very important. The analysis was performed on a Finnigan MAT 95Q sector field-quadropol hybrid mass spectrometer (Finnigan MAT, Bremen, Germany) connected to a Varian 3400 gas chromatograph (Varian, Darmstadt, Germany). The temperature of the combined EI/CI source was 250 °C; the electron energy was 70 eV. The scan rate was 1 s/decade. A CB-SE-54 (Chromatographic Service, Langerwehe, Germany; length 25 m, inner diameter 0.25 mm, coating 0.25 μm) was used as capillary column for GC. Helium was used as carrier gas. The temperature of the split–splitless injector as well as of the direct coupling to the mass spectrometer was 300 °C. The GC oven was heated from 50 °C (2 min isotherm) to 300 °C (5 min isotherm) with a rate of 10 °C/min and 1 μl of the solution was injected with a split of 1:10. The results were referred to an internal CF standard (0.1 mg/ml CF = 100%), which allows a comparison of the relative quantities of substances released from various resin-based materials. All extracts were analyzed five times. The average error was <10%. The integration of the chromatograms was carried out through the base peak or other characteristic mass peaks of the compounds, and the results were normalized by means of the internal caffeine standard, yielding a value of (% CF). Identification of the various substances was achieved by comparison of their mass spectra with those of reference compounds, the NIST/EPA library, the literature data and/or by interpretation of their fragmentation patterns .
2.3
Nernst partition coefficient (DC)
To prepare a stock solution, 30 mg of pure substance was dissolved in 2 ml 1-octanol and their molar absorption ( ε ) coefficient was determined photometrically by using standard solutions. To ascertain the partition coefficient, 333 μl from the stock solution was diluted with 667 μl 1-octanol. After addition of 1 ml phosphate-buffered isotonic NaCl-solution (PBS; ), the samples were equilibrated 1 h at 37 °C under permanent shaking . 500 μl from the organic phase was diluted, dependent on the molar absorption coefficient, and photometrically measured (UV 265 FW-Visable Recording Spectrophotometer, Shimadzu, Kyoto, Japan). At the beginning of the experiment, 1-octanol and PBS were saturated with the antiphase. The DC is presented as decadic logarithm of the partition coefficient. The substances were subdivided into basic- and comonomers, initiators, photo- and coinitators, photostabilizers, inhibitors and other detected substances (e.g. polymerization and decomposition products).
3
Results
3.1
GC/MS analysis
Two different basic monomers, Bis-GMA and urethane dimethacrylate (UEDMA), were found in methanolic eluate from unpolymerized specimens of TET, ARA and ADM ( Table 1 ). One basic monomer (Bis-GMA) was found in methanolic eluate from unpolymerized specimens of the ormoceres DEF and HYT. Ethoxylated bisphenol-A-dimethacrylate (Bis-EDMA) was found in methanolic eluate from unpolymerized specimen of DEF and UEDMA in methanolic eluate from unpolymerized specimen of HYT. The isomeric comonomers 1,2/1,3-glycerol dimethacrylate (GDMA 1/2;1/3) were found in methanolic eluate from unpolymerized specimen of the ormocere DEF in amounts of 1971 (%CF). The comonomers GDMA 1/2;1/3, TEGDMA and 2-hydroxyethyl methacrylate (HEMA) were found in methanolic eluates of unpolymerized specimen of the ormocere ADM in amounts of 2879 (%CF), 166 (%CF) and 41 (%CF) respectively. Similar amounts of other eluted substances from unpolymerized specimen of the ormoceres were found compared to the eluates of unpolymerized specimens of composites and compomeres ( Table 1 ).
| Composites | Compomere | Ormoceres | |||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| TET | ARA | HYT | ADM | DEF | |||||||||||
| ap | mp | mu | ap | mp | mu | ap | mp | mu | ap | mp | mu | ap | mp | mu | |
| Basic monomers | |||||||||||||||
| Bis-EDMA | d | ||||||||||||||
| Bis-GMA | d | d | d | ||||||||||||
| UEDMA | d | d | d | d | |||||||||||
| Comonomers | |||||||||||||||
| DEGDMA | 5.7 | 18 | |||||||||||||
| DODDMA | 2.4 | 436 | 16 | 573 | |||||||||||
| EGDMA | 53 | 21 | 149 | 12 | 8.3 | ||||||||||
| GDMA 1/2;1/3 | 44 | 13 | 2879 | 4.6 | 28 | 1971 | |||||||||
| HEMA | 50 | 198 | 104 | 5.4 | 845 | 41 | |||||||||
| HDDMA | 42 | 29 | 71 | ||||||||||||
| HPMA | 9.5 | ||||||||||||||
| MAA | 47 | 44 | |||||||||||||
| NDDMA | 126 | 2.4 | 230 | ||||||||||||
| TEEGDMA | 33 | ||||||||||||||
| TEGDMA | 38 | 1021 | 2650 | 6.7 | 7.9 | 1863 | 28 | 51 | 12 | 1667 | |||||
| TEGMMA | 5.9 | 7 | |||||||||||||
| Photoinitiators | |||||||||||||||
| CQ | 0.6 | 12 | 16 | 0.1 | 0.5 | 8.8 | 0.2 | 4.5 | 3.0 | 2.1 | 26 | 0.5 | 1.9 | 7 | |
| DMBZ | 1.5 | 20 | 76 | ||||||||||||
| Coinitiators | |||||||||||||||
| CEMA | 7.5 | 89 | 103 | ||||||||||||
| DMABBEE | 2.2 | 35 | |||||||||||||
| DMABEE | 0.2 | 2.4 | 98 | 1.3 | 7.4 | 65 | |||||||||
| DMABEHE | 0.8 | 18 | |||||||||||||
| Photostabilizers | |||||||||||||||
| HMBP | 9.2 | 76 | 7.3 | 56 | |||||||||||
| TINP | 34 | 36 | 15 | 308 | 7.7 | 89 | |||||||||
| Inhibitoren | |||||||||||||||
| BHB | 0.3 | 0.4 | 0.3 | ||||||||||||
| BHT | 7.4 | 126 | 5.4 | 28 | 7.8 | 58 | |||||||||
| HQME | 5.7 | ||||||||||||||
| Other detected substances (e.g. polymerization and decomposition products) | |||||||||||||||
| CSA | 0.2 | ||||||||||||||
| DEHP | 0.9 | 11 | |||||||||||||
| DEP | 0.5 | 0.5 | 0.6 | ||||||||||||
| EHS | 0.9 | 5 | |||||||||||||
| FUDO | 33 | ||||||||||||||
| HC1/2 | 0.2 | 0.2 | 0.3 | 0.5 | |||||||||||
| TEPO | 4.1 | 9 | 13 | ||||||||||||
| TMBA | 1.2 | ||||||||||||||
| TPP | 3.8 | 6.2 | 26 | ||||||||||||
| TPPO | 5.8 | 6.9 | |||||||||||||
| TPSB | 6.1 | 10 | 1.0 | 36 | |||||||||||
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