Evaluation of bisphenol release of different clear aligner materials using the liquid chromatography-mass spectrometry/mass spectrometry method

The use of clear aligners for orthodontic treatment is a new innovation that has become widely accepted because of the desire of patients for “invisible” orthodontics. Clear aligners are esthetically acceptable, although there are still concerns about safety. Clear aligner material base components are polymers including polyethylene (PE), polyethylene terephthalate (PET), polyethylene terephthalate glycol (PETG), polyurethane (PU), and polypropylene (PP).¹  Although aligner materials are advertised as Bisphenol A (BPA)-free, these polymers contain various additives that increase performance, stability, elasticity, and provide specific properties.^2,3  Bisphenols constitute the largest amount of these additive chemicals⁴  and are used to harden plastic and increase its clearance.⁵  There are many different analogues of bisphenols and the main factor that determines the type of bisphenol is the difference in the reactant component. The capital letter after the expression bisphenol is used to express the reactant component in it.⁶  The most widely known derivative of bisphenols is Bisphenol A (BPA). More than 3.5 million tons of BPA are produced annually worldwide, and more than 100 tons of this amount are released into the environment.⁷  It is possible to be exposed to bisphenols in different ways due to their wide range of uses and the long-term release from plastic products. There is always a concentration of bisphenol in body fluids, and exposure to BPA is mostly oral.⁸  BPA is a well-known endocrine disruptor; studies have shown that it binds to estrogen receptors and has estrogenic effects. In addition to interfering with hormone function, BPA raises children’s risk for cancer, diabetes, heart disease, and hyperactivity.⁹  Due to the toxic and endocrine-disrupting effects of BPA, use of it in many products, especially in children’s products has been banned or restricted.¹⁰  As a result of these prohibitions, other structurally similar bisphenol derivatives in the industry, particularly Bisphenol F (BPF) and S (BPS) are used in many products, especially those sold as being “BPA-free.”¹¹  Considering that BPS and BPF have similar properties to BPA in terms of biological and pharmacokinetic mechanisms, these compounds are expected to have similar environmental behavior, toxicity, and exposure pathways to BPA.¹²  For this reason, in vitro and in vivo studies that compare the impacts of BPF/BPS with BPA have increased in recent years, and the use of these two chemicals instead of BPA in clear aligners is a concern.¹³  The purpose of this study was to evaluate and compare the BPA, BPF, and BPS release of six different materials (four of them used for aligners and retainers [Duran, Scheu-Dental GmbH, Iserlohn, Germany; Zendura A, Bay Materials LLC, Fremont, CA, USA; Taglus, Laxmi Dental Export Pvt. Ltd, Mumbai, India; Essix C+, Raintree Essix Inc., Metairie, LA, USA] and two of them used for only aligners [Smart Track, Align Technology, San Jose, CA, USA; Zendura Flx, Bay Materials]. The null hypothesis was that there would be no difference among the tested materials in terms of leaching of BPA, BPF, and BPS.

Six different materials: Duran (Scheu-Dental), Zendura Flx (Bay Materials), Taglus Premium (Laxmi Dental Export), Zendura A (Bay Materials), Smart Track (Align Technology), and Essix C+ (Raintree Essix) were evaluated. The aligner brands and their contents are shown in Table 1.

Two as-received Invisalign aligners, obtained from the manufacturer for the treatment of a malocclusion, were used. For Duran, Taglus Premium, Zendura A, Zendura Flx, and Essix C+ samples, two vacuum-formed materials having a thickness of 1 mm were formed according to the manufacturer’s instructions with a Biostar vacuum thermoforming system (model 3010; Scheu-Dental). The samples were cut into three pieces with a weight of 1 g, washed with distilled water and sterilized according to manufacturer recommendations as defined by the International Standards Organization (ISO) 11737-2 norm by using a 115V UV light with NUAIRE NU-427 Biosafety Cabinet (NuAire, Minnesota, USA) for 15 minutes. Subsequently, the samples were flipped over with sterile forceps and left in the container for an additional 15 minutes. Accordingly, samples weighing 1 gram were placed in 10 mL of saline, ie, in a ratio of 0.1 g/mL,¹⁴  incubated for 8 weeks and stored at 37°C, each in separate airtight glass beakers.

The liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method was used to determine the amount of BPA, BPF, and BPS in the eluates. The calculations were performed by using Agilent 6470 Triple Quad LC-MS/MS System (GenTech Scientific, NY), 1290 Binary Pump System (GenTech Scientific, NY), and 1290 Autosampler System (GenTech Scientific, NY).

BPA (239658, Sigma Aldrich, Darmstadt, Germany), BPF (51453, Sigma Aldrich), BPS (103039, Sigma Aldrich), BPA-D16 (451835, Sigma Aldrich), and Jasem BPA analysis kits (Jasem, Istanbul, Turkey) were used. The calibrator was prepared from single master stock solutions in 1000 mg/L (ppm) methanol for each of the BPA, BPF, and BPS analytes. Subsequently, mixtures of six different concentrations were prepared in 1.0, 2.5, 5.0, 20, 50, and 250 ng/mL (ppb) methanol using single master stock solutions, and these mixtures were used as calibrators.

A total of 250 µL of the sample was transferred to the high-performance liquid chromatography (HPLC) vial from the clear aligner eluates at room temperature using an automatic glass pipette. Then, 25 µL of internal standard and 225 µL of Jasem reagent-1 were added and centrifuged for 10 seconds. It was injected into the LC-MS/MS system. LC-MS/MS method conditions are shown in Table 2. The sample collection and LC-MS/MS calculation were performed blindly two times for two sets of samples in each container.

Limit of detection (LOD) and limit of quantification (LOQ) values were calculated by injections made from the lowest concentration of the calibration point. The calculation of LOD and LOQ was as follows:

• LOD = The lowest concentration value given to the device × 3/(signal-to-noise ratio obtained from the device)

• LOQ = The lowest concentration value given to the device × 10/(signal-to-noise ratio obtained from the device)

Linearity (R²): whether the calibration curve obtained by the analysis of the calibrators was linear was examined.

% Recovery: The proximity of the parameters prepared at a specific concentration to the concentration obtained through analysis was evaluated.

Repeatability (% RD): how precise the mean values belonging to test samples were calculated.

In the validation verification plan, the data were obtained by performing 10 repeated studies for each level.

Measurements within each group were summarized with descriptive statistics. Differences between groups were analyzed with the Kruskal-Wallis test. When statistical significance was found, the post-hoc Dunn-Bonferroni test was applied for pairwise group comparisons. A value of P < .05 was considered statistically significant. Analyses were performed using SPSS (SPSS Statistics for Windows, Version 20.0, IBM Corp., Armonk, NY, USA).

Table 3 contains the validation information of the analytical method. The validation parameter for the method of analysis met the acceptance criteria. R² is considered as acceptable when R² ≥ 0.99. The R² value for the calibrators was found in this limit. The % recovery should be between 85% and 115% and, for the present study, this value was in the expected interval.¹⁵  %RSD indicates between which deviation values the method can measure its specific concentration for each parameter. For trace element analytical methods, the acceptable %RSD is normally below 15%, and the present study met the criteria for BPA, BPF, and BPS.¹⁶ 

LOD values were calculated as 0.086 µg/L for BPA, 0.052 µg/L for BPF, and 0.013 µg/L for BPS (Table 3).

In terms of BPA values, a statistically significant difference was observed among the groups (P = .018). BPA leaching in Smart Track was found to be higher than in the Zendura A and Zendura Flx groups (P = .02, P = .03, respectively) (Table 4, Figure 1).

In terms of BPF values, there was no statistically significant difference among the groups (P = .108) (Table 5, Figure 2).

In terms of BPS values, a statistically significant difference was found (P = .002) indicating that Smart Track released higher BPS values than Zendura A (P = .001) (Table 6, Figure 3).

The lowest observed adverse effect level (LOAEL) for BPA is currently determined as 50,000 µg/kg/d by the Food and Drug Administration and the acceptable daily intake dose assumed “safe” is set at 50 µg/kg/d in the United States. The estimated “safe” dose is calculated based on the assumption that a dose 1000-fold lower than the LOAEL should be safe.¹⁷  The estimated safe amount, also known as the tolerated daily intake, has been lowered by the European Food Safety Authority to 4 µg/kg body weight (bw)/d based on new evidence and approaches.¹⁸  There has been growing number of studies every year outlining detrimental effects of BPA, from 115 publications before 2005 to over 500 publications in 2011, and then over 1000 publications in 2017.¹⁷  Previous studies evaluating aligners failed to consider the potential impact of BPA analogues, and the studies to date tended to focus on BPA rather than BPF and BPS. Therefore, this study makes a major contribution to research on aligner bisphenol release by evaluating all types of bisphenol.

LC-MS/MS is a very sensitive method that combines the mass analysis capabilities of HPLC and mass spectrometry (MS) with the ability to separate compounds, enabling the detection of even trace amounts and making it possible to analyze analytes in quality and quantity.¹⁹  Though the wear duration of each aligner may be 1 to 2 weeks,²⁰  retainers are recommended to be used for 1 year,²¹  Since aligners and retainers were tested in this study, long-term bisphenol release was evaluated, considering the duration of their usage and the longest test period in the literature.²² 

Schuster et al.²³  tested aged and retrieved Invisalign appliances in vitro for 2 weeks, and found no traceable amount of BPA leaching in an ethanol solution. Eliades et al.²³  tested human breast adenocarcinoma cells to evaluate BPA release of Invisalign based on the fact that BPA acts like estrogen; BPA release, which was evaluated depending on the proliferation of estrogen-sensitive cells, could not be detected. Alhendi et al.²⁴  investigated leakage from Invisalign, Eon, SureSmile, and Clarity aligner systems in different concentrations of ethanol and water solutions for 2 weeks and found no detectable amount of BPA. In the Idrissi et al. study²²  in which LOQ value was determined as 23 ng/mL, new and used Scheu-Dental Clear Aligners were tested up to 8 weeks with a LC-MS device, and aligners did not show traces of BPA.

In the study by Kotyk and Wiltshire,²⁵  the BPA release of thermoformed and prethermoformed Biocryl Acrylic Essix, Biocryl Acrylic Retainer, Essix A+, and both used/unused Invisalign materials were evaluated for 2 weeks in artificial saliva with gas chromatography/mass spectroscopy (GC-MS). The calculated LOQ was 1.7 × 10⁻⁴ and, while 1 gr/mL Biocryl Acrylic Retainer released 7600 ng, no traceable BPA was found for other materials.

Katras et al.²⁰  compared the amount of BPA released by Smile Direct Club, Invisalign, and Essix Ace orthodontic aligners in artificial saliva, artificial gastric fluid, and ethanol up to 20 days with a LC-MS device and, although a tendency for higher BPA leaching was shown in Invisalign, the differences did not reach statistical significance based on the media and time intervals. From the results of this study, it was deduced that 1 gr of Invisalign would release 542 ng/mL BPA on the sixth day in artificial saliva. It has also been reported that a 3.6 g set of tested aligners can potentially release ∼1.8 µg (1851 ng) of BPA. Most of the BPA release happened during the first 24 hours after incubation and decreased over time.²⁰  The most interesting finding supporting the study by Katras et al. was that of Raghavan et al., reporting high BPA release occurred within 1 hour of use of Essix Ace retainers, with the values sharply decreasing in saliva after 1 week. Overall, the present study produced results that were consistent with the findings of many previous studies. The calculated BPA release of 1 gram of Invisalign in saline for 8 weeks was 0.98 ng/mL. Although this value is low compared to previous studies, the amount was consistent with previous studies considering the reducing release pattern of BPA over time. Likewise, in the Idrissi et al, study that tested new and used Scheu-Dental aligners for 8 weeks, the LOQ value was determined as 0.023 ng/mL and a measureable amount of BPA was not detected.

To date, studies investigating BPA leaching of aligners and retainers have produced equivocal results and no attempt has been made to quantify the degree of the other bisphenol analogues, BPF, and BPS. What is known about this issue is largely based on in vitro studies. The evidence presented thus far supports the idea that comparison among studies is challenging due to sample preparation protocols and levels within an interval that is under the detection limit of most analytical methods.

In the present study, although Smart Track Invisalign was the material that released BPA and BPS most, even if all bisphenol analogues are taken into account, the value was well below the accepted threshold. However, experimental studies have shown that even 0.23–2.3 ng/mL BPA is sufficient to alter the response of tissues to estrogen and androgen receptors.¹⁸  Total external exposure dose of all bisphenols via the environmental sources was reported as 68.1 ng/kg bw/d, and children are more sensitive to the toxicological effects of bisphenols than adults because of developmental differences.²⁶  Therefore, independent experts have demanded that the outdated LOAEL of 50,000 ng/kg/d be reduced 20,000-fold due to the wide range of adverse effects observed in the toxicity and mechanistic endpoints at the lowest dose tested (2.5 ng/kg/d).¹⁷  Also, experimental conditions cannot exactly show the possible release of bisphenols. This is because mechanical and chemical abrasion, temperature changes, and salivary enzymes were ignored in most studies.²⁷  Increased degradation of the material depending on overusage and, accordingly, the release of harmful components have to be kept in mind. To determine possible bisphenol release of clear aligners, further studies on larger sample groups that mimic the oral environment on an hourly, rather than daily, basis are required to confirm these findings.

• The null hypothesis was rejected. Smart Track showed BPA and BPS over LOD, and released the most BPA and BPS among the materials tested.

• Although BPA and BPS release were detected above the LOD value in Smart Track, this amount is far below the acceptable daily intake level. However, the cumulative effect of these appliances should not be underestimated. Even if they are considered safe, caution may be indicated.

This research received no specific grant from any funding agency. This manuscript was produced from the doctoral thesis of Esra ÇifçI Özkan supervised by Assistant Professor Gülay Dumanlı Gök.