J. Cosmet. Sci., 62, 535–547 (November/December 2011) 535 Photocytotoxicity in human dermal fi broblasts elicited by permanent makeup inks containing titanium dioxide WAYNE G. WAMER and JUN-JIE YIN, Food and Drug Administration, Center for Food Safety and Applied Nutrition, College Park, MD 20740. Accepted for publication August 31, 2011. Synopsis Titanium dioxide (TiO2) is a pigment widely used in decorative tattoo and permanent makeup inks. How- ever, little is known about the risks associated with its presence in these products. We have developed an in vitro assay to identify inks containing TiO2 that are cytotoxic and/or photocytotoxic. The presence of TiO2 in ten permanent makeup inks was established by X-ray fl uorescence. Using X-ray diffraction, we found that seven inks contained predominately TiO2 (anatase), the more photocatalytically active crystalline form of TiO2. The remaining inks contained predominately TiO2 (rutile). To identify cytotoxic and/or photocytotoxic inks, human dermal fi broblasts were incubated for 18 h in media containing inks or pigments isolated from inks. Fibroblasts were then irradiated with 10 J/cm2 UVA radiation combined with 45 J/cm2 visible light for determining photocytotoxicity, or kept in the dark for determining cytotoxicity. Toxicity was assessed as in- hibition of colony formation. No inks were cytotoxic. However eight inks, and the pigments isolated from these inks, were photocytotoxic. Using ESR, we found that most pigments from photocytotoxic inks gener- ated hydroxyl radicals when photoexcited with UV radiation. Therefore, the possibility of photocytotoxicity should be considered when evaluating the safety of permanent makeup inks containing TiO2. INTRODUCTION Tattoos have been used as a means for expressing cultural and personal identity for millennia. A more recent development is intradermal implantation of pigments as an alternative to facial makeup. The popularity of cosmetic tattooing or permanent makeup has steadily increased since its widespread introduction in the mid 1980s (1). The U.S. Food and Drug Administra- tion (FDA) considers inks used for tattoos and permanent makeup to be cosmetics (2). The pigments used in these inks are considered to be color additives, and, therefore, are subject to premarket approval for this specifi c use (3). To date, no pigments have been approved by the Food and Drug Administration for use in tattoos or permanent makeup. Until recently, there have been limited reports of adverse reactions to inks. The European Commission has recog- nized the need to address potential risks associated with inks used for decorative tattoos and permanent makeup (4,5). In Europe, these products are not considered cosmetics but are regu- lated as consumer products under the restrictions of the General Product Safety Directive (5). Address all correspondence to Wayne G. Wamer at wayne.wamer@fda.hhs.gov.

JOURNAL OF COSMETIC SCIENCE 536 The patterns of use for these products are dramatically changing. One notable change is the popularity of tattoos and permanent makeup. It has been reported that 24% of the population in the contiguous United States have at least one tattoo (6). The highest inci- dence of tattoos (36%) occurred for individuals aged 18 to 29. Another change in the pattern of use is the formulation of inks with novel pigments, which allows a wide range of colors for tattoos and permanent makeup. Increasingly, inks are being formulated with organic pigments that have a limited history of safe use (7–9). The high prevalence of tattoos and permanent makeup combined with the use of novel pigments increases the risk for adverse reactions to these products. As an example of this increased risk, the FDA had received only fi ve reports of adverse reactions after permanent makeup procedures between 1988 and 2003 (10). In contrast, between 2003 and 2004, the FDA received 150 reports of adverse reactions attributable to use of inks traceable to one manufacturer of permanent makeup inks. The FDA warned consumers about the adverse reaction re- ports, and the inks were subsequently voluntarily withdrawn from the market (10,11). Reliable toxicological testing methods are needed to assure the safety of tattoo and per- manent makeup inks. However, there have been very few attempts to develop in vitro methods for identifying hazardous inks. Falconi et al., (12) have used human fi broblasts derived from gingival tissue to investigate the cytotoxicity of a permanent makeup ink. The cytotoxicity, resulting from exposures to the ink for up to two weeks, was assessed by measuring inhibition of the fi broblasts’ capacity to metabolize 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide (MTT assay). Dose-dependent cytotoxicity was ob- served for the selected ink. We have developed an in vitro assay that allows evaluation of both the cytotoxicity and photocytotoxity of inks. Because inks used for decorative tat- toos and permanent makeup contain pigments that are effi cient chromophores and these inks are applied to sun-exposed skin, phototoxicity is a concern. There have been reports of phototoxic reactions resulting from decorative tattoos and permanent makeup (13–20). Although the in vitro assay described here was developed to assess the cytotoxicity and photocytotoxicity of permanent makeup inks, the method is generally more applicable for testing inks used for either decorative tattoos or permanent makeup. In our initial studies, we examined the cytotoxicity and photocytotoxicity of permanent makeup inks containing TiO2. These inks were selected for several reasons. First, TiO2 is one of the most widely used pigments in tattoo and permanent makeup inks. Due to its high refractive index, TiO2 frequently functions as an opacifi er in inks (1). Titanium dioxide is also widely used in pigment mixtures to formulate light shades of inks. In addi- tion, the photocatalytic activity of TiO2 has been widely studied. Therefore, TiO2 is an ideal pigment to investigate in the development of an in vitro assay for photocytotoxicity. Finally, there have been reports associating the presence of TiO2 in inks with adverse reac- tions (21,22). Therefore, toxicological data for inks containing TiO2 are needed. EXPERIMENTAL MATERIALS Titanium dioxide (anatase) was purchased from Sigma-Aldrich (Milwaukee, WI). Sam- ples of TiO2 (rutile) were obtained from Sigma-Aldrich and Tronox (Oklahoma City, OK). Permanent makeup inks were purchased through the Internet from ten vendors.