SHORT COMMUNICATION Cytotoxicity and estrogenicity of Invisalign appliances Theodore Eliades, a Harris Pratsinis, b Athanasios E. Athanasiou, c George Eliades, d and Dimitris Kletsas e Thessaloniki and Athens, Greece Introduction: Our purpose was to study the in-vitro cytotoxic and estrogenic properties of Invisalign appli- ances (Align Technology, Santa Clara, Calif). Methods: Three sets, each consisting of a maxillary and a man- dibular appliance, of as-received aligners were immersed in normal saline solution for 2 months. Samples of eluents were diluted to 3 concentrations (5%, 10%, and 20% vol/vol) and tested for cytotoxicity on human gin- gival fibroblasts and estrogenicity by measuring their effect on the proliferation of the estrogen-responsive MCF-7 breast cancer cells. All assays were repeated 4 times for each maxillary and mandibular set, and the results were analyzed with 2-way analysis of variance (ANOVA) with appliance and concentration serving as predictors at the .05 level of significance; differences among groups were investigated with the Tukey test. Results: There was no evidence of cytotoxicity on human gingival fibroblasts and no stimulation of prolifera- tion of the MCF-7 cell line at any concentration, indicating no estrogenicity of aligner eluents. Conclusions: The use of Invisalign appliances did not seem to induce estrogenic effects under the conditions of this exper- iment. (Am J Orthod Dentofacial Orthop 2009;136:100-3) T he release of bisphenol-A (BPA) from dental polymeric applications has attracted the interest of many investigations over the past decade. Many articles have dealt with the potential estrogenicity of adhesives, composite resins, and polycarbonate prod- ucts during the last 5 years. 1 The importance of identi- fying such incidents associated with dental resins is derived from the various effects assigned to BPA. 2-5 Starting in the late 1980s, the search for effects of BPA on the human organism has become a national con- cern after several publications demonstrated activity at doses lower than the reference dose of 50 mg per weight set by the U.S. Environmental Protection Agency. 6 This figure was calculated by dividing the lowest observed adverse effect level reported in the National Toxicology Program carcinogenesis bioassay (50 mg per kilogram) by an uncertainty factor of 1000, presumably to secure safety for the human organism. Nonetheless, in the late 1990s, studies reported increased prostate weights and other effects on the male reproductive system in mice exposed to levels of BPA be- low the safety standard (2 and 20 mg/kg). 7,8 These articles were followed by many studies that found various effects, such as increased mammary gland tumors, 9 precancerous lesions in prostates of neonatally exposed animals, 10 de- velopment of hyperglycemia and insulin tolerance, 11 ele- vation of reactive oxygen species, 12 and oxidative stress. The resultant turmoil on the hormonal endocrino- logic disruptors provoked the investigation of estrogenic action of the full spectrum of polymeric materials used in everyday activities including plastic utensils and bioma- terials for medical and dental applications. As a general rule, estrogenic action is confined to molecules with a double benzoic ring and that release BPA, which mimics the action of the female hormone estradiol. In orthodontics, potential candidates for BPA release include plastic materials and auxiliaries such as adhesives and polycarbonate brackets and aligners. Although no BPA release and no estrogenicity have been reported for light-cured and chemically cured or- thodontic adhesives, 13,14 there is no documentation for Invisalign appliances (Align Technology, Santa Clara, Calif). These aligners are placed in the oral cavity for 22 hours per day for approximately 2 weeks to achieve gradual tooth movement. 15,16 Whereas the in-vivo alter- ations of these appliances 17 and treatment variables 18 have been presented, the potential release of BPA has not been investigated. a Associate professor, Department of Orthodontics, School of Dentistry, Aristo- tle University of Thessaloniki, Thessaloniki, Greece. b Assistant researcher, Laboratory of Cell Proliferation and Ageing, National Center for Scientific Research ‘‘Demokritos,’’ Athens, Greece. c Professor and director, Department of Orthodontics, School of Dentistry, Aristotle University of Thessaloniki, Greece. d Professor and director, Department of Biomaterials, School of Dentistry, University of Athens, Athens, Greece. e Director, Laboratory of Cell Proliferation and Ageing, Institute of Biology, National Center for Scientific Research ‘‘Demokritos’’, Athens, Greece. The authors report no commercial, proprietary, or financial interest in the prod- ucts or companies described in this article. Reprint requests to: Theodore Eliades, 57 Agnoston Hiroon St, Nea Ionia 14231, Athens, Greece; e-mail, teliades@ath.forthnet.gr . Submitted, January 2009; revised and accepted, March 2009. 0889-5406/$36.00 Copyright Ó 2009 by the American Association of Orthodontists. doi:10.1016/j.ajodo.2009.03.006 100