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IN VITRO ELUTION OF LEACHABLE COMPONENTS FROM DENTAL SEALANTS
C O HVIE
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ARTICLE 1
IN VITRO ELUTION OF
LEACHABLE COMPONENTS FROM DENTAL SEALANTS DAN NATHANSON, D.M.D., M.S.D.; PRINDA LERTPITAYAKUN, D.D.S.; MARK S. LAMKIN, PH.D.; MAHNAZ EDALATPOUR, B.SC., D.M.D.; L. LEE CHOU, D.M.D., PH.D.
R
1984, the ADA Council on Dental Materials and Devices reported that the components of fissure sealants are, for the most part, the same as those used in the resin portion of composite restorative materials.' Thus, composites and sealants have a similar basic composition, which can include bis-glycidyl dimethacrylate, or BIS-GMA, urethane dimethacrylate, or UDMA, and triethyleneglycol dimethacrylate, or TEGDMA.2 n
The resins used as denthe rate of elution of compotal sealants are applied to nents from dental resin is a patient's teeth and are Recent concems have been ralsed about rapid during the initial phase polymerized either possibillty that estrogenic chemicals, in of extraction but slows signifithrough a chemical curing cantly over time. particular bisphenol-A, or BPA, might be process or photoactivation. A recent study on the estroHowever, current technolleched out of dental sealants. This study genicity of resin-based dental ogy does not allow a comcomposites and sealants has aimed to and quantify BPA and plete conversion of raised controversy and concern other components from seven light- about the safety of monomers monomers during the curing process, so residual leached out of these materials.7 cured fissure sealants in vitro. None of the monomers can leach out The study confirmed the estrotested sealants was shown to have released of the cured resin. genicity of bisphenol-A, or BPA; howower, the Inetitors Identified Findings that a portion of BPA, and also implicated the polymerizable groups other eluted components that should be bisphenol-A dimethacrylate, or in dental resins have BIS-DMA, as an estrogenic investigated for their biological effects. failed to react during factor. Furthermore, the invespolymerization have led tigators detected these many researchers to investigate the possible monomers in the saliva of human subjects one leaching of these unbound molecules into different hour after sealants had been placed, although they solvents. Numerous studies have observed that had not found these monomers in the subjects' salithe
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relased
JADA, Vol. 128, November 1997 1517
COVER STORY
Figure 1. Chromatograms produced by high-performance liquid chromatography are shown. A. Standard solution (1 = bisphenol-A, or BPA; 2 = triethyloneglycol dimethacrylate, or TEODMA; 3 = bis-glycidyl dimethacrylate, or BIS-GMA; and 4 - bisphenol-A dimethacrylate, or BIS-DMA). B. Leachable components from Delton dental sealant (Dentsply Trubyte) (2 = TEGDMA; 3 = BIS-OMA; 4 = BIS-DMA; and 5 = Unknown I). C. Leachable components from Delton plus standard solution. Note that BPA (peak 1) and Unknown I (peak 5) do not coincide. mAU: milli-absorbance units.
va before the sealants had been
placed. Even though the potential deleterious effects of BPA and its degradation products are
study to identify and quantify BPA and other key leachable components that might be released from different brands of pit and fissure dental sealants.
well-documented,8-"1 no reports of adverse health effects have been attributed to the leached components of dental sealants. It is therefore questionable whether these materials indeed are leached out of dental sealants in quantities that can pose a health hazard. We conducted an in vitro 1518 JADA, Vol. 128, November 1997
MATERIALS AND METHODS
Materials. We selected seven commercially available lightcured pit and fissure sealants for this investigation (Box, "Commercial Light-Cured Fissure Sealants Tested"). The resin matrix of most dental
sealants is a copolymer of TEGDMA and BIS-GMA, but UDMA is the main component in some products. For comparison, we used standards of BPA (Aldrich Chemical Co., Lot No. 609HG), BIS-DMA (Polysciences Inc., Lot No. 92922), BIS-GMA (Polysciences Inc., Lot No. 456653), TEGDMA (Polysciences Inc., Lot No. 468134) and UDMA (Esschem Inc., Lot No. PB2339) without further purification. Methods. We placed approximately 50 microliters of each sealant in a glass dappen dish at room temperature and used a light-curing device (XL 3000, 3M Dental Products) on each sample for 50 seconds. The tip of the light-curing device was held at a distance of 2 millimeters from the surface of the sealants to simulate clinical conditions. After curing each sample, we removed the sample from the dappen dish, weighed it and transferred it to a glass test tube. We followed the same steps to prepare three samples of each sealant. We then placed each sample into 100 microliters of 95 percent ethanol for four minutes, removed them from the ethanol and used high-performance liquid chromatography, or HPLC, to analyze the extracts. We used a laboratory vortex mixer to thoroughly mix 10 ,uL of each eluate after it had been diluted with 90 pL of 50 percent acetronitrile in water
(volume:volume ratio) (solvent A). Five ,uL of each solution were analyzed by HPLC using a 3.9 x 150-millimeter HPLC column (Nova Pak C18 column, Waters) attached to an HPLC system (Hewlett Packard series HP1100). Compounds were eluted at a flow rate of 1 milliliter/minute using a solvent gradient of 50
COVER STORY percent acetronitrile in water (solvent A) and 100 percent acetronitrile (solvent B). The combination of these solvents ranged as follows: 0 to 10 minutes, 0 to 67 percent solvent B; 10 to 11 minutes, 67 to 100 percent solvent B; 11 to 12.5 min-
utes, 100 percent solvent B. Using a dedicated Hewlett Packard diode array ultraviolet spectral detector, we monitored the elution of each compound by absorbance at 215 nanometers. The same detector was used to obtain absorption in spectra
960717
Seal-Rite II (IJDMlV[A)
Piulpdemit Corp.
961125
Defender
Hernry Schein
F6019
Inc.
ranging from 210 to 400 nm. Standard solutions containing BPA, TEGDMA, BIS-GMA, UDMA and BIS-DMA at concentrations of 0.1 milligram/mL were analyzed by HPLC under the same conditions as those for the sealant samples. We compared the chromatograms of all of the sealant samples with those of the standard solutions so we could identify the components in each sample. We calculated the concentration of each component by integration of the component absorption peaks, using the standard solutions as references. We analyzed every sample twice. The average of the weight of each released component per weight of sealant was expressed as the mean (± standard deviation). We analyzed differences in the release of each component from the sealants using a one-way analysis of variance or a nonpaired
-I
-
-
_j-
te minimum level of detected BPA was determined to be less than 0.0001 jig per milligram of sealant tested. JADA, Vol. 128, November 1997 1519
ICOVER STORY RESULTS
Figure 2. Chromatograms produced by high-performance liquid chromatography are shown. A. Standard solution (1 = bisphenol-A, or BPA; 2 = triethyleneglycol dimethacrylate, or TEGDMA; 3 = urethane dimethacrylate, or UDMA; 4 = bis-glycidyl dimethacrylate, or BIS-GMA; and 5 = bisphenol-A dimethacrylate, or BIS-DMA. B. Leachable components from Seal-Rite I (Pulpdent Corp.) (4 = BIS-GMA and 6 = Unknown 1). C. Leachable components from Seal-Rite 11 (Pulpdent Corp.) (3 = UDMA). The major peak elu tion at 7.25 minutes was observed only in Seal-Rite I and Seal-Rite 11, but its identity was undetermined. mAU: mill-absorbance units.
t-test at P < .05.
Delton (Dentsply Trubyte), one of the tested sealants that had previously been reported to have leached BPA,7 was selected for further analysis by gas chromatography, or GC, and mass spectroscopy, or MS, using a Finnigan MAT GCQ analyzer. The samples for this test were prepared as described above. Ethanol extracts from 100 mg of cured Delton sealant were evaporated until they were dry. The dried fraction was redis1520 JADA, Vol. 128, November 1997
solved in 200 ,uL of 100 percent ethanol, and 0.5 gL of the solution was injected into a 30meter DB-5 HPLC column at a flow rate of 1 mL per minute, with helium as the carrier gas to evaporate the solvent. The oven temperature was programmed to increase from 75 C to 150 C at a rate of 20 C per minute, followed by one minute at 150 C and, finally, from 150 C to 300 C at a rate of 8 C per minute. The ion source temperature was 200 C.
HPLC analysis. The BPA standard solution had a retention time of 2.05 minutes (Figure 1A). None of the chromatograms of the tested sealants displayed peaks with the same retention time as that of the BPA standard. The chromatograms of all of the sealants, except that of the Seal-Rite II (Pulpdent Corp.), exhibited a small peak with a retention time of 2.32 minutes, which was very close to that of BPA. We designated these peaks "Unknown I." When we injected the extracted fraction from the Delton sealant into the HPLC testing device, we found several major peaks. Peaks with retention times of 2.95, 5.7 and 10.5 minutes were identified as TEGDMA, BIS-GMA and BISDMA, respectively (Figure 1B). When we combined the extracted fraction of the Delton sealant with the standard solution, the peaks of the BPA standard and Unknown I occurred at different retention times (Figure 1C). The chromatogram for the Defender sealant (Henry Schein Inc.) showed peaks at retention times that were similar to those observed for the Delton sealant. The chromatograms for Concise (3M Dental Products), Helioseal (Ivoclar North America) and Prisma:Shield (Dentsply Caulk) sealants exhibited peaks at retention times that were similar to those of Unknown I, TEGDMA and BISGMA. The chromatogram of Seal-Rite I (Pulpdent Corp.) displayed peaks for Unknown I and BIS-GMA, whereas Seal-Rite II exhibited a peak for UDMA (Figure 2). The peak observed at approximately 1.2 minutes (Figures 1B, IC, 2B and 2C) was due to the ethanol used in the
COVER STORY sample preparation. The table shows the mean weight (± standard deviation) of the extracted fraction per milligram of sealant. Prisma:Shield leached significantly less Unknown I than did the Defender, Concise and Helioseal sealants (P < .05). (We calculated the amount of Unknown I by using the BIS-GMA standard solution as the reference because their spectra are similar.) Delton leached the most TEGDMA, whereas Prisma:Shield leached the least. Delton and Concise leached significantly more TEGDMA than did Helioseal, Prisma:Shield and Defender (P < .05). Prisma:Shield, SealRite I and Defender leached significantly lower amounts of BISGMA than did Helioseal, Concise and Delton (P < .05). Only Delton and Defender eluted BIS-DMA, and Delton leached significantly more BIS-DMA than did Defender (P < .006). To further distinguish Unknown I from BPA, we recorded their UV spectra. It became clear that Unknown I was different from BPA because they exhibited distinctly different spectra (Figure 3). BPA absorbed more strongly than did Unknown I at all wavelengths between 210 and 300 nm. GC and MS analysis. GC and MS of the Delton sealant verified that it contained the components TEGDMA and BISDMA. The BPA standard solution was observed as a peak at a retention time of 17:17 minutes by GC (Figure 4, top), and was identified by MS by the presence of peaks at a mass-to-charge, or m/z, ratio of 228, 213, 195, 119 and 91. This spectrum was identical to that reported in a previous study7 (Figure 4, bottom). We did not detect any peak with
Figure 3. Ultraviolet spectra of Unknown I peak of Delton sealant (Dentsply Trubyte) at a retention time of 2.3 minutes (curve 1) and bisphenol-A, or BPA, standard solution at a retention time of 2.05 minutes (curve 2). The spectra were normalized so that the extinction coefficients for both Unknown I and BPA at 200 nanometers were identical.
a retention time of 17:17 minutes in the fraction extracted
from the Delton sealant (Figure 5). DISCUSSION
As mentioned earlier, a 1996 study by Olea and colleagues7 concluded that chemicals in dental sealants and composite fillings can be leached out and can mimic the naturally occurring female hormone estrogen, thus raising concerns about the safety of these dental materials. As a specific example, BPA, the precursor of many monomers that are used widely in composites and sealants, demonstrated estrogenlike activity when tested in tissue cultures of breast tumor cells.7 The results of our study contradict the conclusion by Olea and colleagues7 regarding the leachability of BPA from dental sealants. In our study, we used two methods in addition to HPLC to verify the absence of BPA: UV spectral analysis of
the HPLC-separated components and mass spectroscopy of gas chromatography-separated components. Of particular interest was the peak we designated as Unknown I, which was shown by HPLC analysis to have a retention time very close to that of the BPA standard. This peak was found in the chromatograms of all BISGMA-based resin sealants but not in the UDMA-based SealRite II sealant. Ultraviolet spectra also distinguished the peaks of Unknown I from those of the BPA standard. It is clear that Unknown I is different from the BPA standard. Using GC and MS, we determined that the BPA standard had a retention time of 17:17 minutes. The fraction extracted from the Delton sealant had no detected peak with the same retention time as that of the BPA standard. Therefore, insofar as we could not detect BPA by any of the methods we used, we concluded that it is not capable of JADA, Vol. 128, November 1997 1521
-COVER STORY has been shown to have a significant effect on the extent of the elution. Also, the size and chemical composition of the elutable species play a role.'2 In a clinical dental setting, the depth of curing, intensity of light and curing time would all affect the elution
process.3'6,'3,4
Figure 4. Gas chromatography, or GC, and mass spectroscopy, or MS, analysis is shown. Top: GC of pure bisphenol-A, or BPA. The BPA was eluted at a retention time of 17:17 minutes. Bottom: Mass spectrum taken at 17:17 minutes in GC analysis of pure BPA. m/z: mass-tocharge.
being leached out of the tested polymerized sealants. Although we did not detect any BPA as an eluate from the tested sealants, we did find BISDMA in two of the tested sealants. In their study, Olea and colleagues7 demonstrated that this component was estrogenic. Further experiments are needed to confirm that this com1522 JADA, Vol. 128, November 1997
pound is estrogenic. Numerous factors and conditions play a role in the elution process from composite dental materials. One factor relates to the amount of leachable molecules and the number of unreacted monomers, which can be affected by the degree of polymerization. The chemistry of the solvent is another factor that
The type of solvent that the sealant material is soaked in may have a substantial effect on the amount of eluted molecules. Several studies have shown that using organic solvents results in a greater quantity of eluted material and a softer composite surface.3'6"'7 In our study, we used 95 percent ethanol to extract leachable components from dental sealants. Each sealant sample was placed in the solvent for four minutes and then removed. A recent study of elution of sealant components into water also failed to demonstrate elution of BPA.'8 That study demonstrated that TEGDMA was released from all of the tested sealants and that the highest rate of TEGDMA release was within the first four minutes and declined thereafter. Furthermore, our use of ethanol, instead of water or saliva, for extraction in this study very likely exaggerated the amount of leached components. We used HPLC to separate compounds that were dissolved in solution and found that the monomer was leached within a range of 0.7 to 2.4 percent of the original weight of the cured sealant over a four-minute period. TEGDMA and BIS-GMA were the major components found in BIS-GMA-based sealants. Only Seal-Rite II leached UDMA. Ferracane and Condon,3 who studied the elution of unreacted components from a composite
COVER STORY and an unfilled dimethacrylate resin placed in water within 10 minutes after curing, found that elution levels for the materials reached 1.5 to 2.0 percent over a seven-day period. Our study found similar levels of elution over a shorter time, but this was possibly due to the fact that we used the solvent ethanol, which has been shown to maximize the amount of leachable components. CONCLUSION
Our study could not detect BPA in the eluate from any of the seven sealants tested; however, we did find that other components were leached from the tested sealants. TEGDMA was leached from all of the sealants except Seal-Rite I and Seal-Rite II. BIS-GMA was leached from all BIS-GMA-based sealants, while UDMA was found to be leached from only Seal-Rite II. BIS-DMA was detected in only two of the sealants. Because of our conclusion that BPA is not leached out of sealants, we do not believe that it is necessary at this time to use different sealants or to restrict the use of these dental sealants in children or adults. However, further studies may be needed to assess the biological effects of the other leached components that were detected in this study. . Dr. Nathanson is a professor and chairman, Department of Restorative Sciences/ Biomaterials, Boston University Goldman School of Dental Medicine, 1001 E. Newton St., Boston, Mass. 02118-2392. Address reprint requests to Dr. Nathanson. Dr. Lertpitayakun is a master of science candidate, Department of Pediatric Dentistry, Boston University Goldman School of Dental Medicine. Dr. Lamkin is a research assistant profesDepartment of Periodontology and Oral Biology, Boston University Goldman School of Dental Medicine. sor,
Figure 5. Gas chromatography of extracted fraction from Delton sealant (Dentsply Trubyte). No detectable peak was observed at 17:17 minutes. Dr. Edalatpour is a postdoctoral student, Department of Pediatric Dentistry, Boston University Goldman School of Dental Medicine. Dr. Chou is an associate professor, Department of Restorative Sciences/ Biomaterials, Boston University Goldman School of Dental Medicine. The authors thank Dr. Frank Oppenheim, Department of Periodontology and Oral Biology, Boston University Goldman School of Dental Medicine, for providing assistance with high-performance liquid chromatography. They are also grateful to Dr. Catherine Costello and Mr. Keyes Linsley of the Mass Spectrometry Resource, Boston University School of Medicine, for help in conducting gas chromatography/mass spectroscopy experiments. 1. Bowen RL, Rupp NW, de Rijk WG. Safety considerations, oral and systemic. J Dent Educ 1984;48(Supplement 2):32-4. 2. Craig RG. Direct esthetic restorative materials: Restorative dental materials. 9th ed. St. Louis: Mosby-Year Book; 1993:248-75. 3. Ferracane JL, Condon JR. Rate of elution of leachable components from composite. Dent
Mater 1990;6:282-7. 4. Gerzina TM, Hume WR. Effect of dentine on release of TEGDMA from resin composite in vitro. J Oral Rehabil 1994;21:463-8. 5. Pearson GJ. Long term water sorption and solubility of composite filling materials. J Dent 1979;7(1):64-8. 6. Tanaka K, Taira M, Shintani H, Wakasa K, Yamaki M. Residual monomers (TEGDMA and BIS-GMA) of a set visible-light-cured dental composite resin when immersed in water. J Oral Rehabil 1991;18:353-62. 7. Olea N, Pulgar R, Perez P, et al. Estrogenicity of resin-based composites and sealants used in dentistry. Environ Health Perspect 1996;104(3):298-305.
8. Dodds EC, Lawson W. Molecular structure in relation to oestrogenic activity: compounds without a phenanthrene nucleus. Proc R Soc Lond B Biol Sci 1938;125:222-32. 9. Krishnan AV, Stathis P, Permuth SF, Tokes L, Feldman D. Bisphenol-A: an estrogenic substance is released from polycarbonate flasks during autoclaving. Endocrinology 1993;132(6):2279-86. 10. Brotons JA, Olea-Serrano MF, Villalobos M, Pedraza V, Olea N. Xenoestrogens released from lacquer coatings in food cans. Environ Health Perspect 1995;103(6):608-12. 11. Feldman D, Krishnan A. Estrogens in unexpected places: possible implications for researchers and consumers. Environ Health Perspect 1995;103(Supplement 7):129-33. 12. Ferracane JL. Elution of leachable components from composites. J Oral Rehabil 1994;21:441-52. 13. Pearson GJ, Longman CM. Water sorption and solubility of resin-based materials following inadequate polymerization by a visiblelight curing system. J Oral Rehabil 1989;16(1):57-61. 14. Rueggeberg FA, Craig RG. Correlation of parameters used to estimate monomer conversion in a light-cured composite. J Dent Res 1988;67(6):932-7. 15. Thompson LR, Miller EG, Bowles WH. Leaching of unpolymerized materials from orthodontic bonding resin. J Dent Res 1982;61(8):989-92. 16. Rathbun MA, Craig RG, Hanks CT, Filisko FE. Cytotoxicity of a Bis-GMA dental composite before and after leaching in organic solvents. J Biomed Mater Res 1991;25:443-57. 17. Wu W, McKinney JE. Influence of chemicals on wear of dental composites. J Dent Res 1982;61(10):1180-3. 18. Hamid A, Hume WR. Release of estrogenic component bisphenol-A not detected from fissure sealants in vitro. J Dent Res 1997;76:321.
JADA, Vol. 128, November 1997
1523
S
T
O F
Y
ARTICLE 1
IN VITRO ELUTION OF
LEACHABLE COMPONENTS FROM DENTAL SEALANTS DAN NATHANSON, D.M.D., M.S.D.; PRINDA LERTPITAYAKUN, D.D.S.; MARK S. LAMKIN, PH.D.; MAHNAZ EDALATPOUR, B.SC., D.M.D.; L. LEE CHOU, D.M.D., PH.D.
R
1984, the ADA Council on Dental Materials and Devices reported that the components of fissure sealants are, for the most part, the same as those used in the resin portion of composite restorative materials.' Thus, composites and sealants have a similar basic composition, which can include bis-glycidyl dimethacrylate, or BIS-GMA, urethane dimethacrylate, or UDMA, and triethyleneglycol dimethacrylate, or TEGDMA.2 n
The resins used as denthe rate of elution of compotal sealants are applied to nents from dental resin is a patient's teeth and are Recent concems have been ralsed about rapid during the initial phase polymerized either possibillty that estrogenic chemicals, in of extraction but slows signifithrough a chemical curing cantly over time. particular bisphenol-A, or BPA, might be process or photoactivation. A recent study on the estroHowever, current technolleched out of dental sealants. This study genicity of resin-based dental ogy does not allow a comcomposites and sealants has aimed to and quantify BPA and plete conversion of raised controversy and concern other components from seven light- about the safety of monomers monomers during the curing process, so residual leached out of these materials.7 cured fissure sealants in vitro. None of the monomers can leach out The study confirmed the estrotested sealants was shown to have released of the cured resin. genicity of bisphenol-A, or BPA; howower, the Inetitors Identified Findings that a portion of BPA, and also implicated the polymerizable groups other eluted components that should be bisphenol-A dimethacrylate, or in dental resins have BIS-DMA, as an estrogenic investigated for their biological effects. failed to react during factor. Furthermore, the invespolymerization have led tigators detected these many researchers to investigate the possible monomers in the saliva of human subjects one leaching of these unbound molecules into different hour after sealants had been placed, although they solvents. Numerous studies have observed that had not found these monomers in the subjects' salithe
3-6
identi
relased
JADA, Vol. 128, November 1997 1517
COVER STORY
Figure 1. Chromatograms produced by high-performance liquid chromatography are shown. A. Standard solution (1 = bisphenol-A, or BPA; 2 = triethyloneglycol dimethacrylate, or TEODMA; 3 = bis-glycidyl dimethacrylate, or BIS-GMA; and 4 - bisphenol-A dimethacrylate, or BIS-DMA). B. Leachable components from Delton dental sealant (Dentsply Trubyte) (2 = TEGDMA; 3 = BIS-OMA; 4 = BIS-DMA; and 5 = Unknown I). C. Leachable components from Delton plus standard solution. Note that BPA (peak 1) and Unknown I (peak 5) do not coincide. mAU: milli-absorbance units.
va before the sealants had been
placed. Even though the potential deleterious effects of BPA and its degradation products are
study to identify and quantify BPA and other key leachable components that might be released from different brands of pit and fissure dental sealants.
well-documented,8-"1 no reports of adverse health effects have been attributed to the leached components of dental sealants. It is therefore questionable whether these materials indeed are leached out of dental sealants in quantities that can pose a health hazard. We conducted an in vitro 1518 JADA, Vol. 128, November 1997
MATERIALS AND METHODS
Materials. We selected seven commercially available lightcured pit and fissure sealants for this investigation (Box, "Commercial Light-Cured Fissure Sealants Tested"). The resin matrix of most dental
sealants is a copolymer of TEGDMA and BIS-GMA, but UDMA is the main component in some products. For comparison, we used standards of BPA (Aldrich Chemical Co., Lot No. 609HG), BIS-DMA (Polysciences Inc., Lot No. 92922), BIS-GMA (Polysciences Inc., Lot No. 456653), TEGDMA (Polysciences Inc., Lot No. 468134) and UDMA (Esschem Inc., Lot No. PB2339) without further purification. Methods. We placed approximately 50 microliters of each sealant in a glass dappen dish at room temperature and used a light-curing device (XL 3000, 3M Dental Products) on each sample for 50 seconds. The tip of the light-curing device was held at a distance of 2 millimeters from the surface of the sealants to simulate clinical conditions. After curing each sample, we removed the sample from the dappen dish, weighed it and transferred it to a glass test tube. We followed the same steps to prepare three samples of each sealant. We then placed each sample into 100 microliters of 95 percent ethanol for four minutes, removed them from the ethanol and used high-performance liquid chromatography, or HPLC, to analyze the extracts. We used a laboratory vortex mixer to thoroughly mix 10 ,uL of each eluate after it had been diluted with 90 pL of 50 percent acetronitrile in water
(volume:volume ratio) (solvent A). Five ,uL of each solution were analyzed by HPLC using a 3.9 x 150-millimeter HPLC column (Nova Pak C18 column, Waters) attached to an HPLC system (Hewlett Packard series HP1100). Compounds were eluted at a flow rate of 1 milliliter/minute using a solvent gradient of 50
COVER STORY percent acetronitrile in water (solvent A) and 100 percent acetronitrile (solvent B). The combination of these solvents ranged as follows: 0 to 10 minutes, 0 to 67 percent solvent B; 10 to 11 minutes, 67 to 100 percent solvent B; 11 to 12.5 min-
utes, 100 percent solvent B. Using a dedicated Hewlett Packard diode array ultraviolet spectral detector, we monitored the elution of each compound by absorbance at 215 nanometers. The same detector was used to obtain absorption in spectra
960717
Seal-Rite II (IJDMlV[A)
Piulpdemit Corp.
961125
Defender
Hernry Schein
F6019
Inc.
ranging from 210 to 400 nm. Standard solutions containing BPA, TEGDMA, BIS-GMA, UDMA and BIS-DMA at concentrations of 0.1 milligram/mL were analyzed by HPLC under the same conditions as those for the sealant samples. We compared the chromatograms of all of the sealant samples with those of the standard solutions so we could identify the components in each sample. We calculated the concentration of each component by integration of the component absorption peaks, using the standard solutions as references. We analyzed every sample twice. The average of the weight of each released component per weight of sealant was expressed as the mean (± standard deviation). We analyzed differences in the release of each component from the sealants using a one-way analysis of variance or a nonpaired
-I
-
-
_j-
te minimum level of detected BPA was determined to be less than 0.0001 jig per milligram of sealant tested. JADA, Vol. 128, November 1997 1519
ICOVER STORY RESULTS
Figure 2. Chromatograms produced by high-performance liquid chromatography are shown. A. Standard solution (1 = bisphenol-A, or BPA; 2 = triethyleneglycol dimethacrylate, or TEGDMA; 3 = urethane dimethacrylate, or UDMA; 4 = bis-glycidyl dimethacrylate, or BIS-GMA; and 5 = bisphenol-A dimethacrylate, or BIS-DMA. B. Leachable components from Seal-Rite I (Pulpdent Corp.) (4 = BIS-GMA and 6 = Unknown 1). C. Leachable components from Seal-Rite 11 (Pulpdent Corp.) (3 = UDMA). The major peak elu tion at 7.25 minutes was observed only in Seal-Rite I and Seal-Rite 11, but its identity was undetermined. mAU: mill-absorbance units.
t-test at P < .05.
Delton (Dentsply Trubyte), one of the tested sealants that had previously been reported to have leached BPA,7 was selected for further analysis by gas chromatography, or GC, and mass spectroscopy, or MS, using a Finnigan MAT GCQ analyzer. The samples for this test were prepared as described above. Ethanol extracts from 100 mg of cured Delton sealant were evaporated until they were dry. The dried fraction was redis1520 JADA, Vol. 128, November 1997
solved in 200 ,uL of 100 percent ethanol, and 0.5 gL of the solution was injected into a 30meter DB-5 HPLC column at a flow rate of 1 mL per minute, with helium as the carrier gas to evaporate the solvent. The oven temperature was programmed to increase from 75 C to 150 C at a rate of 20 C per minute, followed by one minute at 150 C and, finally, from 150 C to 300 C at a rate of 8 C per minute. The ion source temperature was 200 C.
HPLC analysis. The BPA standard solution had a retention time of 2.05 minutes (Figure 1A). None of the chromatograms of the tested sealants displayed peaks with the same retention time as that of the BPA standard. The chromatograms of all of the sealants, except that of the Seal-Rite II (Pulpdent Corp.), exhibited a small peak with a retention time of 2.32 minutes, which was very close to that of BPA. We designated these peaks "Unknown I." When we injected the extracted fraction from the Delton sealant into the HPLC testing device, we found several major peaks. Peaks with retention times of 2.95, 5.7 and 10.5 minutes were identified as TEGDMA, BIS-GMA and BISDMA, respectively (Figure 1B). When we combined the extracted fraction of the Delton sealant with the standard solution, the peaks of the BPA standard and Unknown I occurred at different retention times (Figure 1C). The chromatogram for the Defender sealant (Henry Schein Inc.) showed peaks at retention times that were similar to those observed for the Delton sealant. The chromatograms for Concise (3M Dental Products), Helioseal (Ivoclar North America) and Prisma:Shield (Dentsply Caulk) sealants exhibited peaks at retention times that were similar to those of Unknown I, TEGDMA and BISGMA. The chromatogram of Seal-Rite I (Pulpdent Corp.) displayed peaks for Unknown I and BIS-GMA, whereas Seal-Rite II exhibited a peak for UDMA (Figure 2). The peak observed at approximately 1.2 minutes (Figures 1B, IC, 2B and 2C) was due to the ethanol used in the
COVER STORY sample preparation. The table shows the mean weight (± standard deviation) of the extracted fraction per milligram of sealant. Prisma:Shield leached significantly less Unknown I than did the Defender, Concise and Helioseal sealants (P < .05). (We calculated the amount of Unknown I by using the BIS-GMA standard solution as the reference because their spectra are similar.) Delton leached the most TEGDMA, whereas Prisma:Shield leached the least. Delton and Concise leached significantly more TEGDMA than did Helioseal, Prisma:Shield and Defender (P < .05). Prisma:Shield, SealRite I and Defender leached significantly lower amounts of BISGMA than did Helioseal, Concise and Delton (P < .05). Only Delton and Defender eluted BIS-DMA, and Delton leached significantly more BIS-DMA than did Defender (P < .006). To further distinguish Unknown I from BPA, we recorded their UV spectra. It became clear that Unknown I was different from BPA because they exhibited distinctly different spectra (Figure 3). BPA absorbed more strongly than did Unknown I at all wavelengths between 210 and 300 nm. GC and MS analysis. GC and MS of the Delton sealant verified that it contained the components TEGDMA and BISDMA. The BPA standard solution was observed as a peak at a retention time of 17:17 minutes by GC (Figure 4, top), and was identified by MS by the presence of peaks at a mass-to-charge, or m/z, ratio of 228, 213, 195, 119 and 91. This spectrum was identical to that reported in a previous study7 (Figure 4, bottom). We did not detect any peak with
Figure 3. Ultraviolet spectra of Unknown I peak of Delton sealant (Dentsply Trubyte) at a retention time of 2.3 minutes (curve 1) and bisphenol-A, or BPA, standard solution at a retention time of 2.05 minutes (curve 2). The spectra were normalized so that the extinction coefficients for both Unknown I and BPA at 200 nanometers were identical.
a retention time of 17:17 minutes in the fraction extracted
from the Delton sealant (Figure 5). DISCUSSION
As mentioned earlier, a 1996 study by Olea and colleagues7 concluded that chemicals in dental sealants and composite fillings can be leached out and can mimic the naturally occurring female hormone estrogen, thus raising concerns about the safety of these dental materials. As a specific example, BPA, the precursor of many monomers that are used widely in composites and sealants, demonstrated estrogenlike activity when tested in tissue cultures of breast tumor cells.7 The results of our study contradict the conclusion by Olea and colleagues7 regarding the leachability of BPA from dental sealants. In our study, we used two methods in addition to HPLC to verify the absence of BPA: UV spectral analysis of
the HPLC-separated components and mass spectroscopy of gas chromatography-separated components. Of particular interest was the peak we designated as Unknown I, which was shown by HPLC analysis to have a retention time very close to that of the BPA standard. This peak was found in the chromatograms of all BISGMA-based resin sealants but not in the UDMA-based SealRite II sealant. Ultraviolet spectra also distinguished the peaks of Unknown I from those of the BPA standard. It is clear that Unknown I is different from the BPA standard. Using GC and MS, we determined that the BPA standard had a retention time of 17:17 minutes. The fraction extracted from the Delton sealant had no detected peak with the same retention time as that of the BPA standard. Therefore, insofar as we could not detect BPA by any of the methods we used, we concluded that it is not capable of JADA, Vol. 128, November 1997 1521
-COVER STORY has been shown to have a significant effect on the extent of the elution. Also, the size and chemical composition of the elutable species play a role.'2 In a clinical dental setting, the depth of curing, intensity of light and curing time would all affect the elution
process.3'6,'3,4
Figure 4. Gas chromatography, or GC, and mass spectroscopy, or MS, analysis is shown. Top: GC of pure bisphenol-A, or BPA. The BPA was eluted at a retention time of 17:17 minutes. Bottom: Mass spectrum taken at 17:17 minutes in GC analysis of pure BPA. m/z: mass-tocharge.
being leached out of the tested polymerized sealants. Although we did not detect any BPA as an eluate from the tested sealants, we did find BISDMA in two of the tested sealants. In their study, Olea and colleagues7 demonstrated that this component was estrogenic. Further experiments are needed to confirm that this com1522 JADA, Vol. 128, November 1997
pound is estrogenic. Numerous factors and conditions play a role in the elution process from composite dental materials. One factor relates to the amount of leachable molecules and the number of unreacted monomers, which can be affected by the degree of polymerization. The chemistry of the solvent is another factor that
The type of solvent that the sealant material is soaked in may have a substantial effect on the amount of eluted molecules. Several studies have shown that using organic solvents results in a greater quantity of eluted material and a softer composite surface.3'6"'7 In our study, we used 95 percent ethanol to extract leachable components from dental sealants. Each sealant sample was placed in the solvent for four minutes and then removed. A recent study of elution of sealant components into water also failed to demonstrate elution of BPA.'8 That study demonstrated that TEGDMA was released from all of the tested sealants and that the highest rate of TEGDMA release was within the first four minutes and declined thereafter. Furthermore, our use of ethanol, instead of water or saliva, for extraction in this study very likely exaggerated the amount of leached components. We used HPLC to separate compounds that were dissolved in solution and found that the monomer was leached within a range of 0.7 to 2.4 percent of the original weight of the cured sealant over a four-minute period. TEGDMA and BIS-GMA were the major components found in BIS-GMA-based sealants. Only Seal-Rite II leached UDMA. Ferracane and Condon,3 who studied the elution of unreacted components from a composite
COVER STORY and an unfilled dimethacrylate resin placed in water within 10 minutes after curing, found that elution levels for the materials reached 1.5 to 2.0 percent over a seven-day period. Our study found similar levels of elution over a shorter time, but this was possibly due to the fact that we used the solvent ethanol, which has been shown to maximize the amount of leachable components. CONCLUSION
Our study could not detect BPA in the eluate from any of the seven sealants tested; however, we did find that other components were leached from the tested sealants. TEGDMA was leached from all of the sealants except Seal-Rite I and Seal-Rite II. BIS-GMA was leached from all BIS-GMA-based sealants, while UDMA was found to be leached from only Seal-Rite II. BIS-DMA was detected in only two of the sealants. Because of our conclusion that BPA is not leached out of sealants, we do not believe that it is necessary at this time to use different sealants or to restrict the use of these dental sealants in children or adults. However, further studies may be needed to assess the biological effects of the other leached components that were detected in this study. . Dr. Nathanson is a professor and chairman, Department of Restorative Sciences/ Biomaterials, Boston University Goldman School of Dental Medicine, 1001 E. Newton St., Boston, Mass. 02118-2392. Address reprint requests to Dr. Nathanson. Dr. Lertpitayakun is a master of science candidate, Department of Pediatric Dentistry, Boston University Goldman School of Dental Medicine. Dr. Lamkin is a research assistant profesDepartment of Periodontology and Oral Biology, Boston University Goldman School of Dental Medicine. sor,
Figure 5. Gas chromatography of extracted fraction from Delton sealant (Dentsply Trubyte). No detectable peak was observed at 17:17 minutes. Dr. Edalatpour is a postdoctoral student, Department of Pediatric Dentistry, Boston University Goldman School of Dental Medicine. Dr. Chou is an associate professor, Department of Restorative Sciences/ Biomaterials, Boston University Goldman School of Dental Medicine. The authors thank Dr. Frank Oppenheim, Department of Periodontology and Oral Biology, Boston University Goldman School of Dental Medicine, for providing assistance with high-performance liquid chromatography. They are also grateful to Dr. Catherine Costello and Mr. Keyes Linsley of the Mass Spectrometry Resource, Boston University School of Medicine, for help in conducting gas chromatography/mass spectroscopy experiments. 1. Bowen RL, Rupp NW, de Rijk WG. Safety considerations, oral and systemic. J Dent Educ 1984;48(Supplement 2):32-4. 2. Craig RG. Direct esthetic restorative materials: Restorative dental materials. 9th ed. St. Louis: Mosby-Year Book; 1993:248-75. 3. Ferracane JL, Condon JR. Rate of elution of leachable components from composite. Dent
Mater 1990;6:282-7. 4. Gerzina TM, Hume WR. Effect of dentine on release of TEGDMA from resin composite in vitro. J Oral Rehabil 1994;21:463-8. 5. Pearson GJ. Long term water sorption and solubility of composite filling materials. J Dent 1979;7(1):64-8. 6. Tanaka K, Taira M, Shintani H, Wakasa K, Yamaki M. Residual monomers (TEGDMA and BIS-GMA) of a set visible-light-cured dental composite resin when immersed in water. J Oral Rehabil 1991;18:353-62. 7. Olea N, Pulgar R, Perez P, et al. Estrogenicity of resin-based composites and sealants used in dentistry. Environ Health Perspect 1996;104(3):298-305.
8. Dodds EC, Lawson W. Molecular structure in relation to oestrogenic activity: compounds without a phenanthrene nucleus. Proc R Soc Lond B Biol Sci 1938;125:222-32. 9. Krishnan AV, Stathis P, Permuth SF, Tokes L, Feldman D. Bisphenol-A: an estrogenic substance is released from polycarbonate flasks during autoclaving. Endocrinology 1993;132(6):2279-86. 10. Brotons JA, Olea-Serrano MF, Villalobos M, Pedraza V, Olea N. Xenoestrogens released from lacquer coatings in food cans. Environ Health Perspect 1995;103(6):608-12. 11. Feldman D, Krishnan A. Estrogens in unexpected places: possible implications for researchers and consumers. Environ Health Perspect 1995;103(Supplement 7):129-33. 12. Ferracane JL. Elution of leachable components from composites. J Oral Rehabil 1994;21:441-52. 13. Pearson GJ, Longman CM. Water sorption and solubility of resin-based materials following inadequate polymerization by a visiblelight curing system. J Oral Rehabil 1989;16(1):57-61. 14. Rueggeberg FA, Craig RG. Correlation of parameters used to estimate monomer conversion in a light-cured composite. J Dent Res 1988;67(6):932-7. 15. Thompson LR, Miller EG, Bowles WH. Leaching of unpolymerized materials from orthodontic bonding resin. J Dent Res 1982;61(8):989-92. 16. Rathbun MA, Craig RG, Hanks CT, Filisko FE. Cytotoxicity of a Bis-GMA dental composite before and after leaching in organic solvents. J Biomed Mater Res 1991;25:443-57. 17. Wu W, McKinney JE. Influence of chemicals on wear of dental composites. J Dent Res 1982;61(10):1180-3. 18. Hamid A, Hume WR. Release of estrogenic component bisphenol-A not detected from fissure sealants in vitro. J Dent Res 1997;76:321.
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