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Bisphenol A release from orthodontic adhesives measured in vitro and in vivo with gas chromatography
ORIGINAL ARTICLE
Bisphenol A release from orthodontic adhesives measured in vitro and in vivo with gas chromatography Marılia Rodrigues Moreira,a Leonardo Gontijo Matos,a Israel Donizeti de Souza,b ^nia Costa Queiroz,b Fa bio Lourenc¸o Romano,c Tamires Amabile Valim Brigante,b Maria Euge Paulo Nelson-Filho,a and Mırian Aiko Nakane Matsumotoc Ribeir~ao Preto, S~ao Paulo, Brazil
Introduction: The objectives of this study were to quantify in vitro the Bisphenol A (BPA) release from 5 orthodontic composites and to assess in vivo the BPA level in patients' saliva and urine after bracket bonding with an orthodontic adhesive system. Methods: For the in-vitro portion of this study, 5 orthodontic composites were evaluated: Eagle Spectrum (American Orthodontics, Sheboygan, Wis), Enlight (Ormco, Orange, Calif), Light Bond (Reliance Orthodontic Products, Itasca, Ill), Mono Lok II (Rocky Mountain Orthodontics, Denver, Colo), and Transbond XT (3M Unitek, Monrovia, Calif). Simulating intraoral conditions, the specimens were immersed in a water/ethanol solution, and the BPA (ng.g 1) liberation was measured after 30 minutes, 24 hours, 1 day, 1 week, and 1 month by the gas chromatography system coupled with mass spectrometry. Twenty patients indicated for fixed orthodontic treatment participated in the in-vivo study. Saliva samples were collected before bracket bonding and then 30 minutes, 24 hours, 1 day, 1 week, and 1 month after bonding the brackets. Urine samples were collected before bonding and then at 1 day, 1 week, and 1 month after bonding. The results were analyzed statistically using analysis of variance and Tukey posttest, with a significance level of 5%. Results: All composites evaluated in vitro released small amounts of BPA. Enlight composite showed the greatest release, at 1 month. Regarding the in-vivo study, the mean BPA level in saliva increased significantly only at 30 minutes after bonding in comparison with measurements recorded before bonding. Conclusions: All orthodontic composites released BPA in vitro. Enlight and Light Bond had, respectively, the highest and lowest BPA releases in vitro. The in-vivo experiment showed that bracket bonding with the Transbond XT orthodontic adhesive system resulted in increased BPA levels in saliva and urine. The levels were significant but still lower than the reference dose for daily ingestion. (Am J Orthod Dentofacial Orthop 2017;151:477-83)
D
uring orthodontic treatment with fixed appliances, bracket bonding with composite materials based on bisphenol A glycidyl methacrylate is of great importance for treatment outcome.1,2 It is the main monomer used in resins and orthodontic From the University of S~ao Paulo, Ribeir~ao Preto, S~ao Paulo, Brazil. a Department of Pediatric Dentistry, School of Dentistry of Ribeir~ao Preto. b Department of Chemistry, College of Philosophy, Sciences and Letters of Ribeir~ao Preto. c Department of Orthodontics, Dental School of Ribeir~ao Preto. All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest, and none were reported. Address correspondence to: Marılia Rodrigues Moreira, Departamento de Clınica Infantil, Odontologia Preventiva e Social, Faculdade de Odontologia de Ribeir~ao Preto – USP, Av. do Cafe, s/n, Monte Alegre, Ribeir~ao Preto, SP 14040-904, Brasil; e-mail, [email protected]. Submitted, March 2016; revised and accepted, July 2016. 0889-5406/$36.00 Ó 2016 by the American Association of Orthodontists. All rights reserved. http://dx.doi.org/10.1016/j.ajodo.2016.07.019
adhesives,3 and bisphenol A (2,2'-bis [4hydroxyphenyl] propane; BPA) is the main component of this monomer.4 BPA is a synthetic chemical substance widely used for production of epoxy resin and polycarbonate plastic used to manufacture various products, including bottles, food packaging, baby bottles, toys, detergents, pesticides, cars, and dental resinous materials, such as composites and pit-and-fissure sealants.5,6 Due to the increase in the number of products based on epoxy resins and polycarbonate plastics, human exposure to BPA has increased rapidly.7,8 BPA has been extensively studied as one of the most common environmental endocrine disruptors, having an estrogenic action from competitive binding of estrogenlike polymer molecules to natural hormone receptors.9 The environment—water, air, and soil—can be a route of exposure to BPA, but foods are the primary source 477
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Table I. Specifications of the orthodontic composites evaluated in vitro Composite
Transbond XT Silica, Bis-GMA, silano, N-dimethyl benzocaine, hexafluoride phosphate
Adhesive composite
Triethylene glycol dimethacrylate Bis-GMA
Manufacturer
3M Unitek, Monrovia, Calif
Batch number
1323200750
Mono Lok II Composite based on Bis-GMA with small parts of glass and photosensitive catalysis system Fluid resin in Bis-GMA base and a photosensitive catalysis system
Rocky Mountain Orthodontics, Denver, Colo K2601-1
Eagle Spectrum Not given by the manufacturer
Enlight Uncured methacrylate ester monomers, silica, activators, conservatives
Biphenyl dimethacrylate, hydroxyethyl methacrylate, acetone
Not given by the manufacturer
Reliance, Orthodontic Products, Itasca, Ill
American Orthodontics, Sheboygan, Wis B 41282
Ethyl alcohol, dimethacrylate resins, barium aluminoborosilicate, glass fumed silica (silicon dioxide), sodium hexafluorosilicate Ormco, Orange, Calif
131812
of human contact. The daily human consumption of BPA is less than 1 mg.kg 1, and greater doses may lead to destructive adverse effects on the endocrine system, especially during fetal development.9-11 Although medical research has demonstrated the harmful effects of BPA-releasing materials to the body, dental research in this field is limited.7,9,12 The presence of BPA in human saliva, urine, and blood after use of resinous restorative materials and pit-and-fissure sealants has been demonstrated.13-18 Kang et al19 evaluated the release of BPA from a composite resin used to bond orthodontic lingual retainers, but no study has quantified the amount of BPA released either in vitro after different experimental periods or in vivo in the saliva and urine of patients after bonding of orthodontic brackets. Based on these findings, the objectives of this study were to quantify in vitro the release of BPA from 5 contemporary orthodontic composites, and to assess in vivo the levels of BPA in the saliva and urine of patients after bracket bonding with an orthodontic adhesive system. MATERIAL AND METHODS
In the in-vitro experiment, the release of BPA was evaluated from 5 orthodontic composites commonly used for bracket bonding. The materials and their compositions, manufacturers, and batch numbers are listed in Table I. Four disc-shaped (5 mm diameter 3 3 mm thick) samples of each resin were prepared using a synthetic fluorine-containing resin matrix and photoactivated for 60 seconds with a halogen light-curing unit with
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Light bond Fused silica, urethane dimethacrylate, triethyleneglycol dimethacrylate
4571779
450 mW/cm2 light intensity (XL 3000; DMC, Plantation, Fla). Each sample was weighed and immersed in a separate flask containing 4 mL of an ethanol/water solution (75:25 v/v) at 37 C. Aliquots of 1 mL of the solutions with the samples were collected 30 minutes, 24 hours, 1 week, and 1 month after immersion. After addition of 20 mL of an internal standard working solution (BPA-d16; 1 mg.mL 1), 100 mL aliquots of the ethanol/ water solutions were dried under a vacuum system at 45 C, and the samples were derivatized. Derivatization consisted of the addition of 50 mL of the reagent BSTFA 1 TMCS to the dry residue, vortexing for 30 seconds, and immersion in a thermostatized bath at 37 C for 30 minutes. After that, 1 mL of the derivatized solution was injected into the chromatography system for analysis. The ethics committee of the University of S~ao Paulo, Ribeir~ao Preto, S~ao Paulo, Brazil, reviewed and approved our research (protocol 34805914.9.0000.5419). The subjects enrolled in the study were 20 patients of both sexes aged 12 to 18 years (mean age, 12.3 years) who needed fixed orthodontic treatment and fulfilled the following inclusion criteria: complete permanent dentition; no caries, periodontal disease, restorations (composite resin, amalgam, or glass ionomer), or pitand-fissure sealants; good general health; and nonsmokers. In addition, during the experiment, the patients did not receive any restorations to minimize biases or confounding factors that could influence the results. A patient was excluded from the study if bracket rebonding was necessary. For strict adherence to the research protocol, the patients were instructed not to use plastic utensils, such disposable glasses, cutlery, or dishes.
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The 20 patients were planned to receive complete fixed orthodontic appliances with metallic brackets from the left second premolar to the right second premolar in both arches, totaling 20 teeth. Before placement of the orthodontic appliances, 1 mL of nonstimulated saliva and 5 mL of urine were collected in glass tubes for analysis of preexisting BPA (quantified in ng.g 1). Next, rubber cup pumice dental prophylaxis was performed, and the enamel was etched with phosphoric acid for 15 seconds, rinsed with air/water spray, and dried with a mild air blow. A calibrated and experienced orthodontist (L.G.M.) performed bracket bonding using the Transbond XT light-cure orthodontic adhesive system (3M Unitek, Monrovia, Calif). After application of Transbond XT Primer, Transbond XT resin was applied on the bracket base, bonded to the teeth, and photoactivated for 20 seconds with a halogen light-curing unit at 450 mW/cm2 light intensity. Then 1 mL of nonstimulated saliva and 5 mL of urine were collected from each patient 30 minutes, 24 hours, 1 week, and 1 month after bracket bonding. Urine samples were subjected to an enzymatic treatment consisting of mixing 1 mL of urine with 1 mL of sodium acetate buffer (0.1 mol L-1, pH 5, containing 0.1% [w/v] ascorbic acid) and 20 mL of the diluted enzyme solution (in 0.2% saline solution [w/v] freshly prepared and cooled; 10,000 units.mL-1 for glucuronidase and 937 units.mL-1 for sulfatase).20,21 The mixture was maintained at 37 C for 4 hours. Next, 20 mL of the internal standard (1 mg.mL-1) was added for liquid-liquid extraction.22,23 Two 1-mL aliquots of the extraction solvent (MTBE) was added, and the samples were vortexed for 30 seconds and centrifuged at 15 C for 5 minutes at 5000 rpm. The supernatant was collected and dried under vacuum, and the residue was derivatized as previously described. The saliva samples were treated in the same way as the urine samples, except that enzymatic treatment was not necessary. A 40-mL aliquot of the internal standard (1 mg.mL-1) was added to 1 mL of saliva, and liquidliquid extraction and derivatization were performed as previously described. The analysis of BPA in the samples (in-vitro and in-vivo experiments) was performed in a gas chromatograph mass spectrometer (GCMS-QP2010 Plus; Shimadzu, Tokyo, Japan). Chromatographic separations were achieved on an NST-05MS (5% diphenyl, 95% dimethylpolysiloxane) analytical column (30 mm 3 0.25 mm 3 0.25 mm). The injector, ion source, and interface temperatures were set at 280 C, 230 C, and 250 C, respectively, and the solvent cut time was 3 minutes. The samples were injected in the split mode (1:3), and the injection volume was 1 mL. The column
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Table II. Fragments used for quantification, confir-
mation, and retention time of BPA and BPA-D16 silanized
BPA BPA-d16 (PI)
Quantification ion (m/z) 357 368
Confirmation ion (m/z) 372 386
Retention time (min) 8.27 8.19
m/z, mass divided by charge number of the target ion analyzed in mass spectrum obtained by GCMS.
temperature was held at 180 C for 3 minutes, followed by an increase at a 20 C per minute rate to 240 C, which was maintained for 4 minutes, and a further increase at a 20 C per minute rate to 300 C, which was maintained for 2 minutes. Helium at a constant flow rate of 1 mL per minute 1 was used as the carrier gas, and the electron impact (70 eV) ionization mode was used. The data were acquired by Selected Ion Monitoring mode, in which a fragment was used for quantification and another fragment was used to confirm both BPA and internal standard (Table II). The data from the in-vitro and in-vivo (urine and saliva in ng.g 1) experiments were examined for normal distribution (Shapiro-Wilk test, P .0.05) and homogeneity of variance (Levene test, P .0.05). Data were expressed as means and standard deviations, and the differences between time points (baseline, 30 minutes, 24 hours, 1 week, and 1 month) were verified by analysis of variance and Tukey post hoc tests. Data were analyzed using Bioestat statistical software (version 5.3; Mamiraua Institute, Belem, PA, Brazil), and the significance level was set at 5%. RESULTS
Means and standard deviations of BPA release from the orthodontic composites in vitro are presented in Table III. BPA was released from all materials at all time points, and this release increased with time. Enlight and Light Bond had, respectively, the highest and the lowest amounts of BPA release (P \0.05) at all time points. Descriptive results of BPA levels in saliva are presented in Table IV. The mean BPA level 30 minutes after bracket bonding was significantly higher (P \0.05) than at baseline and all other time points. The mean BPA levels at 24 hours, 1 week, and 1 month after bracket bonding were statistically similar to each other and not significantly (P .0.05) different from the values obtained before bonding. Descriptive results of BPA levels in urine are presented in Table V. In comparison with baseline (before bracket bonding), BPA levels increased significantly
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Table III. Mean BPA levels (ng.g
1
Time point 30 minutes after bracket bonding 24 hours after bracket bonding 1 week after bracket bonding 1 month after bracket bonding
Transbond XT 28.0 6 1.0 54.6 6 2.1 142.9 6 8.8 324.1 6 17.5
) released from the orthodontic composites at different times in an ethanol alcohol/ water solution (75:25 v/v) (n 5 3) Mono-Lok II 9.4 6 0.3a 81.8 6 4.4 71.5 6 3.2 146.6 6 20.2
Eagle Spectrum 38.4 6 1.9 167.1 6 3.8 295.0 6 8.8 607.6 6 47.2
Enlight 48.1 6 2.0 252.4 6 3.9 415.2 6 29.3 1020.1 6 50.4
Light bond 10.0 6 0.4a 21.6 6 2.1 31.0 6 0.7 65.0 6 0.6
Same letters indicate no statistically significant difference (analysis of variance and Tukey tests; P .0.05).
Table IV. Descriptive analysis of BPA levels in the
Table V. Descriptive analysis of BPA levels in the urine
saliva of patients at the different time points (n 5 20)
of patients at the different time points (n 5 20)
BPA concentration (ng.g 1) Time point Before bracket bonding 30 minutes after bracket bonding 24 hours after bracket bonding 1 week after bracket bonding 1 month after bracket bonding
BPA concentration (ng.g 1)
Minimum 0.49 0.74
Maximum 0.73 1.82
Mean (SD)* 0.56 (0.06)a 1.04 (0.28)b
0.50
1.34
0.64 (0.21)a
0.50
1.52
0.76 (0.33)a
0.49
1.23
0.61 (0.16)a
Time point Before bracket bonding 30 minutes after bracket bonding 24 hours after bracket bonding 1 week after bracket bonding 1 month after bracket bonding
Minimum 0.90 1.20
Maximum 3.80 10.7
Mean (SD)* 2.17 (0.93)a 5.04 (2.47)b
1.50
9.60
4.22 (2.07)bc
1.20
8.20
3.05 (1.61)ac
0.90
3.80
2.17 (0.93)a
*Same letters indicate no statistically significant difference (analysis of variance and Tukey tests; P .0.05).
*Same letters indicate no statistically significant difference (analysis of variance and Tukey tests; P .0.05).
(P \0.05) at 24 hours and 1 week after bracket bonding, but these increases were insignificant after 1 month (P .0.05). No significant differences in BPA levels were noted between 24 hours and the later time points.
systems in vivo are scarce. The authors of a recent systematic review assessing BPA and residual monomer release from orthodontic adhesives and polycarbonate brackets in the oral environment did not retrieve any randomized controlled trial.6 Additionally, because of the heterogeneity in the methodologies and reporting of outcomes, no quantitative comparisons could be done, and only a qualitative synthesis was performed. The novelty that our study adds to current knowledge in the field is the quantification of the amounts of BPA released in saliva and urine of patients after bonding of orthodontic brackets—unknown until now—since orthodontic adhesive materials contain BPA. The most important point is to warn orthodontists that bracket bonding could be associated with increases in the BPA levels in saliva and urine, adding to the other sources of this chemical in the human body, thus reaching meaningful levels. The in-vivo amount of BPA released from an orthodontic adhesive system was assessed in only 1 study31; however, the methodology used was different from the one used in our research. In that study, the release of BPA between 2 groups of patients using different mouth-rinsing solutions (tap water and a mixture of ionized water plus absolute ethanol) was measured before and immediately after bracket bonding, and immediately after the first rinse.31 A significant
DISCUSSION
Most resins used in dentistry contain BPA derivatives; for this reason, they have attracted the attention of dental researchers as an additional source of exposure to humans.15 The release of BPA from composites may occur at 2 moments: during or just after resin placement, caused by incomplete monomer polymerization, and later, as a result of material degradation.16 In the intraoral environment, these materials are exposed to extreme thermal changes, mechanical erosion, pH alterations, and enzymatic degradation from bacterial and salivary enzymes, which can cause BPA release. Incomplete polymerization of adhesive systems can also cause BPA release.17,24,25 Although the materials used for bracket bonding are exposed to the oral cavity only at the peripheral margins of brackets, which have a thickness close to 150 to 250 mm,26 some studies have been conducted with orthodontic materials.15-17,19,24-31 However, studies evaluating the actual release of BPA from orthodontic adhesive
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pattern of increase of BPA concentration, followed by a decrease that reached the initial values, was observed. The amount of BPA was relatively low and far below the reference limits of tolerable daily intake. Furthermore, another in-vivo study evaluated the release of BPA from a composite resin used to bond orthodontic lingual retainers.19 Under the conditions of the in-vitro experiment, we observed that BPA was released from all orthodontic composites at all time points and increased over time. Enlight had the highest BPA release in vitro, reaching its peak (1020 ng.g 1) at 1 month. On the other hand, Light Bond had the least BPA release. The choice of immersing the samples in an ethanol/water solution (75:25 v/v) was based on its known action of increasing the monomer release rate from dental composite resins, inducing an accelerated aging effect and thus possibly simulating extreme conditions in the oral cavity.28 Since fixed orthodontic appliances remain in the oral cavity for a relative long period, artificial saliva would not permit a simulation of real oral conditions because in the mouth these materials are exposed to extreme thermal changes, mechanical wear, pH changes, and salivary bacterial and enzymatic degradations that could cause BPA leaching. In a previous in-vitro study evaluating the release of BPA from Transbond XT used to bond lingual fixed retainers, measurable amounts of BPA were found at all time points (10, 20, and 30 days), with the peak occurring 30 days after bracket bonding (2.9 mg.L 1).29 This result is in line with our study in which Transbond XT also showed the highest BPA leaching (324 ng.g 1) at 30 days postbonding. Sunitha et al24 evaluated BPA release from Transbond XT, varying the light-curing tip distances and the immersion times in absolute ethanol (1, 7, 21, and 35 days) and found that the greater the light-curing tip distance from the material, the greater the BPA release. Kotyk and Wiltshire30 evaluated Transbond XT discs immersed in artificial saliva for 1, 3, 7, and 14 days and observed BPA leaching only within the first 3 days of immersion (2.75 mg.g 1). Other authors have evaluated the release of BPA from restorative resins and, although these materials are used under different circumstances (direct exposure to the intraoral environment, greater amount of material, and longer periods in the mouth), the obtained results were similar to those of our study.14,30,32 In the in-vivo experiment, BPA was quantified in the urine and saliva of patients before and after bonding orthodontic brackets with Transbond XT, the gold standard orthodontic adhesive material. BPA is not persistent in the body, and urine is its main way of excretion. For this reason, in this study, the urinary and salivary levels were measured to evaluate body exposure to
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this chemical. The presence of BPA in the saliva shows its release from uncured monomers, and its detection in the urine shows the amount absorbed and consequently excreted. This analysis can underestimate this release, since there are other minor excretion routes, such as transpiration and respiration, which are difficult to measure. It has been reported that the complete elimination of BPA occurs within 42 hours, but traces of the chemical can still be identified in the organs.32,33 However, our results do not support this finding, since BPA urinary levels increased significantly up to 7 days after bracket bonding, decreasing to a value close to that of baseline only at 30 days. This fact highlights the importance of considering the effects of currently used orthodontic materials in the human body. Although the amount of BPA released by contemporary orthodontic adhesive materials is low, as demonstrated in the in-vitro experiment, quantitative studies in vivo are still scarce. Epidemiologic studies detected BPA in the urine of 91% of Canadians aged 6 to 79 years,34 of 93% of Americans aged 6 years or more,35 and of 99% of Germans between 3 and 14 years.36 The high frequency of detection and the rapid metabolism seem to indicate continuous exposure to substantial amounts of BPA through different routes.37,38 In our study, BPA was detected in all initial salivary (0.565 ng.g 1) and urinary (2.17 ng.g 1) samples collected before bracket bonding. The significant increase in the mean BPA levels at the earliest times (30 minutes for saliva and 24 hours for urine) suggest a correlation with orthodontic bracket bonding. The same correlation has been reported in other studies.13,31,35 Salivary and urinary BPA levels decreased with time, as found in other studies.13,23,36,37,39 Therefore, the increase in BPA levels after bonding of orthodontic brackets is probably a determinant event. The BPA levels detected in vitro and in vivo in this study were much lower than the maximum level recommended by regulatory agencies such as the United States Environmental Protection Agency40 and the European Food Safety Authority41 of 50 mg per kilogram per day. However, according to Vom Saal and Hughes,42 BPA levels lower than 50 mg per kilogram per day ingested in regular doses can cause more harm to the health after a certain time than a high dose in a single event. Orthodontic materials must not be considered alone but, rather, added to all other sources of BPA to which patients are daily exposed. The ideal scenario would be that dental materials do not contribute to increased BPA levels in the body, especially in young people, who are more susceptible. Most orthodontic patients are adolescents, and exposure to chemicals such as
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BPA can be more dangerous to their health compared with adults.5 From the clinical standpoint, our results could be useful to both orthodontists and manufacturers. Orthodontists must be aware of the measures that can be taken to reduce a patient's exposure to BPA released from orthodontic adhesive materials during treatment. These measures include the smallest possible amount of orthodontic bonding materials on bracket bases for bonding, adequate photoactivaton, and rinsing the tooth/bracket interface after bonding with air-water spray for 30 seconds.31,43 At the same time, it is highly advisable to have on the market low-BPA or BPA-free orthodontic bonding materials, as is already occurring with restorative dental materials. CONCLUSIONS
1.
2.
All orthodontic composites released BPA in vitro. The BPA levels detected were lower than the reference dose recommended for daily ingestion. All materials reached peak levels 30 days after bonding. Enlight and Light Bond had, respectively, the highest and lowest amounts of BPA release in vitro. The in-vivo experiment showed that bracket bonding with the Transbond XT orthodontic adhesive system resulted in increased BPA levels in saliva and urine. The levels were significant but still lower than the reference dose for daily ingestion.
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27. Chen L, Suh B. Bisphenol A in dental materials: a review. JSM Dent 2013;1-1004. 28. Eliades T, Eliades G, Brantley W, Johnston W. Residual monomer leaching from chemically cured and visible light-cured orthodontic adhesives. Am J Orthod Dentofacial Orthop 1995; 108:316-21. 29. Eliades T, Voutsa D, Sifakakis I, Makou M, Katsaros C. Release of bisphenol-A from a light-cured adhesive bonded to lingual fixed retainers. Am J Orthod Dentofacial Orthop 2011;139:192-5. 30. Kotyk MW, Wiltshire WA. An investigation into bisphenol A leaching from orthodontic materials. Angle Orthod 2014;84: 516-20. 31. Kloukos D, Sifakakis I, Voutsa D, Doulis I, Eliades G, Katsaros C, et al. BPA qualitative and quantitative assessment associated with orthodontic bonding in vivo. Dent Mater 2015;31:887-94. 32. Kloukos D, Pandis N, Eliades T. In vivo bisphenol-a release from dental pit and fissure sealants: a systematic review. J Dent 2013; 41:659-67. 33. Shelby MD. NTP-CERHR monograph on the potential human reproductive and developmental effects of bisphenol A. NTP CERHR MON 2008;22:1-64. 34. Taylor JA, Vom Saal FS, Welshons WV, Drury B, Rottinghaus G, Hunt PA, et al. Similarity of bisphenol A pharmacokinetics in rhesus monkeys and mice: relevance for human exposure. Environ Health Perspect 2011;119:422-30. 35. Bushnik T, Haines D, Levallois P, Levesque J, Van Oostdam J, Viau C. Lead and bisphenol A concentrations in the Canadian population. Health Rep 2010;21:7-18.
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36. Becker K, G€oen T, Seiwert M, Conrad A, Pick-Fuss H, Muller J, et al. GerES IV: phthalate metabolites and bisphenol A in urine of German children. Int J Hyg Environ Health 2009;212:685-92. 37. Dekant W, Volkel W. Human exposure to bisphenol A by biomonitoring: methods, results and assessment of environmental exposures. Toxicol Appl Pharmacol 2008;228:114-34. 38. Della Seta D, Minder I, Belloni V, Aloisi AM, Dessi-Fulgheri F, Farabollini F. Pubertal exposure to estrogenic chemicals affects behavior in juvenile and adult male rats. Horm Behav 2006;50: 301-7. 39. Lakind JS, Naiman DQ. Bisphenol A (BPA) daily intakes in the United States: estimates from the 2003-2004 NHANES urinary BPA data. J Expo Sci Environ Epidemiol 2008;18:608-15. 40. Stahlhut RW, Welshons WV, Swan SH. Bisphenol A data in NHANES suggest longer than expected half-life, substantial nonfood exposure, or both. Environ Health Perspect 2009;117: 784-9. 41. European Food Safety Authority. Scientific opinion on bisphenol A: Evaluation of a study investigating its neurodevelopmental toxicity, review of recent scientific literature on its toxicity and advice on the Danish risk assessment of bisphenol A. EFSA Journal 2006;428:1-75. 42. Vom Saal FS, Hughes C. An extensive new literature concerning low-dose effects of bisphenol A shows the need for a new risk assessment. Environ Health Perspect 2005;113:926-33. 43. Azarpazhooh A, Main PA. Is there a risk of harm or toxicity in the placement of pit and fissure sealant materials? A systematic review. J Can Dent Assoc 2008;74:179-83.
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Bisphenol A release from orthodontic adhesives measured in vitro and in vivo with gas chromatography Marılia Rodrigues Moreira,a Leonardo Gontijo Matos,a Israel Donizeti de Souza,b ^nia Costa Queiroz,b Fa bio Lourenc¸o Romano,c Tamires Amabile Valim Brigante,b Maria Euge Paulo Nelson-Filho,a and Mırian Aiko Nakane Matsumotoc Ribeir~ao Preto, S~ao Paulo, Brazil
Introduction: The objectives of this study were to quantify in vitro the Bisphenol A (BPA) release from 5 orthodontic composites and to assess in vivo the BPA level in patients' saliva and urine after bracket bonding with an orthodontic adhesive system. Methods: For the in-vitro portion of this study, 5 orthodontic composites were evaluated: Eagle Spectrum (American Orthodontics, Sheboygan, Wis), Enlight (Ormco, Orange, Calif), Light Bond (Reliance Orthodontic Products, Itasca, Ill), Mono Lok II (Rocky Mountain Orthodontics, Denver, Colo), and Transbond XT (3M Unitek, Monrovia, Calif). Simulating intraoral conditions, the specimens were immersed in a water/ethanol solution, and the BPA (ng.g 1) liberation was measured after 30 minutes, 24 hours, 1 day, 1 week, and 1 month by the gas chromatography system coupled with mass spectrometry. Twenty patients indicated for fixed orthodontic treatment participated in the in-vivo study. Saliva samples were collected before bracket bonding and then 30 minutes, 24 hours, 1 day, 1 week, and 1 month after bonding the brackets. Urine samples were collected before bonding and then at 1 day, 1 week, and 1 month after bonding. The results were analyzed statistically using analysis of variance and Tukey posttest, with a significance level of 5%. Results: All composites evaluated in vitro released small amounts of BPA. Enlight composite showed the greatest release, at 1 month. Regarding the in-vivo study, the mean BPA level in saliva increased significantly only at 30 minutes after bonding in comparison with measurements recorded before bonding. Conclusions: All orthodontic composites released BPA in vitro. Enlight and Light Bond had, respectively, the highest and lowest BPA releases in vitro. The in-vivo experiment showed that bracket bonding with the Transbond XT orthodontic adhesive system resulted in increased BPA levels in saliva and urine. The levels were significant but still lower than the reference dose for daily ingestion. (Am J Orthod Dentofacial Orthop 2017;151:477-83)
D
uring orthodontic treatment with fixed appliances, bracket bonding with composite materials based on bisphenol A glycidyl methacrylate is of great importance for treatment outcome.1,2 It is the main monomer used in resins and orthodontic From the University of S~ao Paulo, Ribeir~ao Preto, S~ao Paulo, Brazil. a Department of Pediatric Dentistry, School of Dentistry of Ribeir~ao Preto. b Department of Chemistry, College of Philosophy, Sciences and Letters of Ribeir~ao Preto. c Department of Orthodontics, Dental School of Ribeir~ao Preto. All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest, and none were reported. Address correspondence to: Marılia Rodrigues Moreira, Departamento de Clınica Infantil, Odontologia Preventiva e Social, Faculdade de Odontologia de Ribeir~ao Preto – USP, Av. do Cafe, s/n, Monte Alegre, Ribeir~ao Preto, SP 14040-904, Brasil; e-mail, [email protected]. Submitted, March 2016; revised and accepted, July 2016. 0889-5406/$36.00 Ó 2016 by the American Association of Orthodontists. All rights reserved. http://dx.doi.org/10.1016/j.ajodo.2016.07.019
adhesives,3 and bisphenol A (2,2'-bis [4hydroxyphenyl] propane; BPA) is the main component of this monomer.4 BPA is a synthetic chemical substance widely used for production of epoxy resin and polycarbonate plastic used to manufacture various products, including bottles, food packaging, baby bottles, toys, detergents, pesticides, cars, and dental resinous materials, such as composites and pit-and-fissure sealants.5,6 Due to the increase in the number of products based on epoxy resins and polycarbonate plastics, human exposure to BPA has increased rapidly.7,8 BPA has been extensively studied as one of the most common environmental endocrine disruptors, having an estrogenic action from competitive binding of estrogenlike polymer molecules to natural hormone receptors.9 The environment—water, air, and soil—can be a route of exposure to BPA, but foods are the primary source 477
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Table I. Specifications of the orthodontic composites evaluated in vitro Composite
Transbond XT Silica, Bis-GMA, silano, N-dimethyl benzocaine, hexafluoride phosphate
Adhesive composite
Triethylene glycol dimethacrylate Bis-GMA
Manufacturer
3M Unitek, Monrovia, Calif
Batch number
1323200750
Mono Lok II Composite based on Bis-GMA with small parts of glass and photosensitive catalysis system Fluid resin in Bis-GMA base and a photosensitive catalysis system
Rocky Mountain Orthodontics, Denver, Colo K2601-1
Eagle Spectrum Not given by the manufacturer
Enlight Uncured methacrylate ester monomers, silica, activators, conservatives
Biphenyl dimethacrylate, hydroxyethyl methacrylate, acetone
Not given by the manufacturer
Reliance, Orthodontic Products, Itasca, Ill
American Orthodontics, Sheboygan, Wis B 41282
Ethyl alcohol, dimethacrylate resins, barium aluminoborosilicate, glass fumed silica (silicon dioxide), sodium hexafluorosilicate Ormco, Orange, Calif
131812
of human contact. The daily human consumption of BPA is less than 1 mg.kg 1, and greater doses may lead to destructive adverse effects on the endocrine system, especially during fetal development.9-11 Although medical research has demonstrated the harmful effects of BPA-releasing materials to the body, dental research in this field is limited.7,9,12 The presence of BPA in human saliva, urine, and blood after use of resinous restorative materials and pit-and-fissure sealants has been demonstrated.13-18 Kang et al19 evaluated the release of BPA from a composite resin used to bond orthodontic lingual retainers, but no study has quantified the amount of BPA released either in vitro after different experimental periods or in vivo in the saliva and urine of patients after bonding of orthodontic brackets. Based on these findings, the objectives of this study were to quantify in vitro the release of BPA from 5 contemporary orthodontic composites, and to assess in vivo the levels of BPA in the saliva and urine of patients after bracket bonding with an orthodontic adhesive system. MATERIAL AND METHODS
In the in-vitro experiment, the release of BPA was evaluated from 5 orthodontic composites commonly used for bracket bonding. The materials and their compositions, manufacturers, and batch numbers are listed in Table I. Four disc-shaped (5 mm diameter 3 3 mm thick) samples of each resin were prepared using a synthetic fluorine-containing resin matrix and photoactivated for 60 seconds with a halogen light-curing unit with
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Light bond Fused silica, urethane dimethacrylate, triethyleneglycol dimethacrylate
4571779
450 mW/cm2 light intensity (XL 3000; DMC, Plantation, Fla). Each sample was weighed and immersed in a separate flask containing 4 mL of an ethanol/water solution (75:25 v/v) at 37 C. Aliquots of 1 mL of the solutions with the samples were collected 30 minutes, 24 hours, 1 week, and 1 month after immersion. After addition of 20 mL of an internal standard working solution (BPA-d16; 1 mg.mL 1), 100 mL aliquots of the ethanol/ water solutions were dried under a vacuum system at 45 C, and the samples were derivatized. Derivatization consisted of the addition of 50 mL of the reagent BSTFA 1 TMCS to the dry residue, vortexing for 30 seconds, and immersion in a thermostatized bath at 37 C for 30 minutes. After that, 1 mL of the derivatized solution was injected into the chromatography system for analysis. The ethics committee of the University of S~ao Paulo, Ribeir~ao Preto, S~ao Paulo, Brazil, reviewed and approved our research (protocol 34805914.9.0000.5419). The subjects enrolled in the study were 20 patients of both sexes aged 12 to 18 years (mean age, 12.3 years) who needed fixed orthodontic treatment and fulfilled the following inclusion criteria: complete permanent dentition; no caries, periodontal disease, restorations (composite resin, amalgam, or glass ionomer), or pitand-fissure sealants; good general health; and nonsmokers. In addition, during the experiment, the patients did not receive any restorations to minimize biases or confounding factors that could influence the results. A patient was excluded from the study if bracket rebonding was necessary. For strict adherence to the research protocol, the patients were instructed not to use plastic utensils, such disposable glasses, cutlery, or dishes.
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The 20 patients were planned to receive complete fixed orthodontic appliances with metallic brackets from the left second premolar to the right second premolar in both arches, totaling 20 teeth. Before placement of the orthodontic appliances, 1 mL of nonstimulated saliva and 5 mL of urine were collected in glass tubes for analysis of preexisting BPA (quantified in ng.g 1). Next, rubber cup pumice dental prophylaxis was performed, and the enamel was etched with phosphoric acid for 15 seconds, rinsed with air/water spray, and dried with a mild air blow. A calibrated and experienced orthodontist (L.G.M.) performed bracket bonding using the Transbond XT light-cure orthodontic adhesive system (3M Unitek, Monrovia, Calif). After application of Transbond XT Primer, Transbond XT resin was applied on the bracket base, bonded to the teeth, and photoactivated for 20 seconds with a halogen light-curing unit at 450 mW/cm2 light intensity. Then 1 mL of nonstimulated saliva and 5 mL of urine were collected from each patient 30 minutes, 24 hours, 1 week, and 1 month after bracket bonding. Urine samples were subjected to an enzymatic treatment consisting of mixing 1 mL of urine with 1 mL of sodium acetate buffer (0.1 mol L-1, pH 5, containing 0.1% [w/v] ascorbic acid) and 20 mL of the diluted enzyme solution (in 0.2% saline solution [w/v] freshly prepared and cooled; 10,000 units.mL-1 for glucuronidase and 937 units.mL-1 for sulfatase).20,21 The mixture was maintained at 37 C for 4 hours. Next, 20 mL of the internal standard (1 mg.mL-1) was added for liquid-liquid extraction.22,23 Two 1-mL aliquots of the extraction solvent (MTBE) was added, and the samples were vortexed for 30 seconds and centrifuged at 15 C for 5 minutes at 5000 rpm. The supernatant was collected and dried under vacuum, and the residue was derivatized as previously described. The saliva samples were treated in the same way as the urine samples, except that enzymatic treatment was not necessary. A 40-mL aliquot of the internal standard (1 mg.mL-1) was added to 1 mL of saliva, and liquidliquid extraction and derivatization were performed as previously described. The analysis of BPA in the samples (in-vitro and in-vivo experiments) was performed in a gas chromatograph mass spectrometer (GCMS-QP2010 Plus; Shimadzu, Tokyo, Japan). Chromatographic separations were achieved on an NST-05MS (5% diphenyl, 95% dimethylpolysiloxane) analytical column (30 mm 3 0.25 mm 3 0.25 mm). The injector, ion source, and interface temperatures were set at 280 C, 230 C, and 250 C, respectively, and the solvent cut time was 3 minutes. The samples were injected in the split mode (1:3), and the injection volume was 1 mL. The column
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Table II. Fragments used for quantification, confir-
mation, and retention time of BPA and BPA-D16 silanized
BPA BPA-d16 (PI)
Quantification ion (m/z) 357 368
Confirmation ion (m/z) 372 386
Retention time (min) 8.27 8.19
m/z, mass divided by charge number of the target ion analyzed in mass spectrum obtained by GCMS.
temperature was held at 180 C for 3 minutes, followed by an increase at a 20 C per minute rate to 240 C, which was maintained for 4 minutes, and a further increase at a 20 C per minute rate to 300 C, which was maintained for 2 minutes. Helium at a constant flow rate of 1 mL per minute 1 was used as the carrier gas, and the electron impact (70 eV) ionization mode was used. The data were acquired by Selected Ion Monitoring mode, in which a fragment was used for quantification and another fragment was used to confirm both BPA and internal standard (Table II). The data from the in-vitro and in-vivo (urine and saliva in ng.g 1) experiments were examined for normal distribution (Shapiro-Wilk test, P .0.05) and homogeneity of variance (Levene test, P .0.05). Data were expressed as means and standard deviations, and the differences between time points (baseline, 30 minutes, 24 hours, 1 week, and 1 month) were verified by analysis of variance and Tukey post hoc tests. Data were analyzed using Bioestat statistical software (version 5.3; Mamiraua Institute, Belem, PA, Brazil), and the significance level was set at 5%. RESULTS
Means and standard deviations of BPA release from the orthodontic composites in vitro are presented in Table III. BPA was released from all materials at all time points, and this release increased with time. Enlight and Light Bond had, respectively, the highest and the lowest amounts of BPA release (P \0.05) at all time points. Descriptive results of BPA levels in saliva are presented in Table IV. The mean BPA level 30 minutes after bracket bonding was significantly higher (P \0.05) than at baseline and all other time points. The mean BPA levels at 24 hours, 1 week, and 1 month after bracket bonding were statistically similar to each other and not significantly (P .0.05) different from the values obtained before bonding. Descriptive results of BPA levels in urine are presented in Table V. In comparison with baseline (before bracket bonding), BPA levels increased significantly
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Table III. Mean BPA levels (ng.g
1
Time point 30 minutes after bracket bonding 24 hours after bracket bonding 1 week after bracket bonding 1 month after bracket bonding
Transbond XT 28.0 6 1.0 54.6 6 2.1 142.9 6 8.8 324.1 6 17.5
) released from the orthodontic composites at different times in an ethanol alcohol/ water solution (75:25 v/v) (n 5 3) Mono-Lok II 9.4 6 0.3a 81.8 6 4.4 71.5 6 3.2 146.6 6 20.2
Eagle Spectrum 38.4 6 1.9 167.1 6 3.8 295.0 6 8.8 607.6 6 47.2
Enlight 48.1 6 2.0 252.4 6 3.9 415.2 6 29.3 1020.1 6 50.4
Light bond 10.0 6 0.4a 21.6 6 2.1 31.0 6 0.7 65.0 6 0.6
Same letters indicate no statistically significant difference (analysis of variance and Tukey tests; P .0.05).
Table IV. Descriptive analysis of BPA levels in the
Table V. Descriptive analysis of BPA levels in the urine
saliva of patients at the different time points (n 5 20)
of patients at the different time points (n 5 20)
BPA concentration (ng.g 1) Time point Before bracket bonding 30 minutes after bracket bonding 24 hours after bracket bonding 1 week after bracket bonding 1 month after bracket bonding
BPA concentration (ng.g 1)
Minimum 0.49 0.74
Maximum 0.73 1.82
Mean (SD)* 0.56 (0.06)a 1.04 (0.28)b
0.50
1.34
0.64 (0.21)a
0.50
1.52
0.76 (0.33)a
0.49
1.23
0.61 (0.16)a
Time point Before bracket bonding 30 minutes after bracket bonding 24 hours after bracket bonding 1 week after bracket bonding 1 month after bracket bonding
Minimum 0.90 1.20
Maximum 3.80 10.7
Mean (SD)* 2.17 (0.93)a 5.04 (2.47)b
1.50
9.60
4.22 (2.07)bc
1.20
8.20
3.05 (1.61)ac
0.90
3.80
2.17 (0.93)a
*Same letters indicate no statistically significant difference (analysis of variance and Tukey tests; P .0.05).
*Same letters indicate no statistically significant difference (analysis of variance and Tukey tests; P .0.05).
(P \0.05) at 24 hours and 1 week after bracket bonding, but these increases were insignificant after 1 month (P .0.05). No significant differences in BPA levels were noted between 24 hours and the later time points.
systems in vivo are scarce. The authors of a recent systematic review assessing BPA and residual monomer release from orthodontic adhesives and polycarbonate brackets in the oral environment did not retrieve any randomized controlled trial.6 Additionally, because of the heterogeneity in the methodologies and reporting of outcomes, no quantitative comparisons could be done, and only a qualitative synthesis was performed. The novelty that our study adds to current knowledge in the field is the quantification of the amounts of BPA released in saliva and urine of patients after bonding of orthodontic brackets—unknown until now—since orthodontic adhesive materials contain BPA. The most important point is to warn orthodontists that bracket bonding could be associated with increases in the BPA levels in saliva and urine, adding to the other sources of this chemical in the human body, thus reaching meaningful levels. The in-vivo amount of BPA released from an orthodontic adhesive system was assessed in only 1 study31; however, the methodology used was different from the one used in our research. In that study, the release of BPA between 2 groups of patients using different mouth-rinsing solutions (tap water and a mixture of ionized water plus absolute ethanol) was measured before and immediately after bracket bonding, and immediately after the first rinse.31 A significant
DISCUSSION
Most resins used in dentistry contain BPA derivatives; for this reason, they have attracted the attention of dental researchers as an additional source of exposure to humans.15 The release of BPA from composites may occur at 2 moments: during or just after resin placement, caused by incomplete monomer polymerization, and later, as a result of material degradation.16 In the intraoral environment, these materials are exposed to extreme thermal changes, mechanical erosion, pH alterations, and enzymatic degradation from bacterial and salivary enzymes, which can cause BPA release. Incomplete polymerization of adhesive systems can also cause BPA release.17,24,25 Although the materials used for bracket bonding are exposed to the oral cavity only at the peripheral margins of brackets, which have a thickness close to 150 to 250 mm,26 some studies have been conducted with orthodontic materials.15-17,19,24-31 However, studies evaluating the actual release of BPA from orthodontic adhesive
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pattern of increase of BPA concentration, followed by a decrease that reached the initial values, was observed. The amount of BPA was relatively low and far below the reference limits of tolerable daily intake. Furthermore, another in-vivo study evaluated the release of BPA from a composite resin used to bond orthodontic lingual retainers.19 Under the conditions of the in-vitro experiment, we observed that BPA was released from all orthodontic composites at all time points and increased over time. Enlight had the highest BPA release in vitro, reaching its peak (1020 ng.g 1) at 1 month. On the other hand, Light Bond had the least BPA release. The choice of immersing the samples in an ethanol/water solution (75:25 v/v) was based on its known action of increasing the monomer release rate from dental composite resins, inducing an accelerated aging effect and thus possibly simulating extreme conditions in the oral cavity.28 Since fixed orthodontic appliances remain in the oral cavity for a relative long period, artificial saliva would not permit a simulation of real oral conditions because in the mouth these materials are exposed to extreme thermal changes, mechanical wear, pH changes, and salivary bacterial and enzymatic degradations that could cause BPA leaching. In a previous in-vitro study evaluating the release of BPA from Transbond XT used to bond lingual fixed retainers, measurable amounts of BPA were found at all time points (10, 20, and 30 days), with the peak occurring 30 days after bracket bonding (2.9 mg.L 1).29 This result is in line with our study in which Transbond XT also showed the highest BPA leaching (324 ng.g 1) at 30 days postbonding. Sunitha et al24 evaluated BPA release from Transbond XT, varying the light-curing tip distances and the immersion times in absolute ethanol (1, 7, 21, and 35 days) and found that the greater the light-curing tip distance from the material, the greater the BPA release. Kotyk and Wiltshire30 evaluated Transbond XT discs immersed in artificial saliva for 1, 3, 7, and 14 days and observed BPA leaching only within the first 3 days of immersion (2.75 mg.g 1). Other authors have evaluated the release of BPA from restorative resins and, although these materials are used under different circumstances (direct exposure to the intraoral environment, greater amount of material, and longer periods in the mouth), the obtained results were similar to those of our study.14,30,32 In the in-vivo experiment, BPA was quantified in the urine and saliva of patients before and after bonding orthodontic brackets with Transbond XT, the gold standard orthodontic adhesive material. BPA is not persistent in the body, and urine is its main way of excretion. For this reason, in this study, the urinary and salivary levels were measured to evaluate body exposure to
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this chemical. The presence of BPA in the saliva shows its release from uncured monomers, and its detection in the urine shows the amount absorbed and consequently excreted. This analysis can underestimate this release, since there are other minor excretion routes, such as transpiration and respiration, which are difficult to measure. It has been reported that the complete elimination of BPA occurs within 42 hours, but traces of the chemical can still be identified in the organs.32,33 However, our results do not support this finding, since BPA urinary levels increased significantly up to 7 days after bracket bonding, decreasing to a value close to that of baseline only at 30 days. This fact highlights the importance of considering the effects of currently used orthodontic materials in the human body. Although the amount of BPA released by contemporary orthodontic adhesive materials is low, as demonstrated in the in-vitro experiment, quantitative studies in vivo are still scarce. Epidemiologic studies detected BPA in the urine of 91% of Canadians aged 6 to 79 years,34 of 93% of Americans aged 6 years or more,35 and of 99% of Germans between 3 and 14 years.36 The high frequency of detection and the rapid metabolism seem to indicate continuous exposure to substantial amounts of BPA through different routes.37,38 In our study, BPA was detected in all initial salivary (0.565 ng.g 1) and urinary (2.17 ng.g 1) samples collected before bracket bonding. The significant increase in the mean BPA levels at the earliest times (30 minutes for saliva and 24 hours for urine) suggest a correlation with orthodontic bracket bonding. The same correlation has been reported in other studies.13,31,35 Salivary and urinary BPA levels decreased with time, as found in other studies.13,23,36,37,39 Therefore, the increase in BPA levels after bonding of orthodontic brackets is probably a determinant event. The BPA levels detected in vitro and in vivo in this study were much lower than the maximum level recommended by regulatory agencies such as the United States Environmental Protection Agency40 and the European Food Safety Authority41 of 50 mg per kilogram per day. However, according to Vom Saal and Hughes,42 BPA levels lower than 50 mg per kilogram per day ingested in regular doses can cause more harm to the health after a certain time than a high dose in a single event. Orthodontic materials must not be considered alone but, rather, added to all other sources of BPA to which patients are daily exposed. The ideal scenario would be that dental materials do not contribute to increased BPA levels in the body, especially in young people, who are more susceptible. Most orthodontic patients are adolescents, and exposure to chemicals such as
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BPA can be more dangerous to their health compared with adults.5 From the clinical standpoint, our results could be useful to both orthodontists and manufacturers. Orthodontists must be aware of the measures that can be taken to reduce a patient's exposure to BPA released from orthodontic adhesive materials during treatment. These measures include the smallest possible amount of orthodontic bonding materials on bracket bases for bonding, adequate photoactivaton, and rinsing the tooth/bracket interface after bonding with air-water spray for 30 seconds.31,43 At the same time, it is highly advisable to have on the market low-BPA or BPA-free orthodontic bonding materials, as is already occurring with restorative dental materials. CONCLUSIONS
1.
2.
All orthodontic composites released BPA in vitro. The BPA levels detected were lower than the reference dose recommended for daily ingestion. All materials reached peak levels 30 days after bonding. Enlight and Light Bond had, respectively, the highest and lowest amounts of BPA release in vitro. The in-vivo experiment showed that bracket bonding with the Transbond XT orthodontic adhesive system resulted in increased BPA levels in saliva and urine. The levels were significant but still lower than the reference dose for daily ingestion.
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