TITANIUM DIOXIDE AND ZINC OXIDE NANOPARTICLES IN SUNSCREENS 225 Important physicochemical parameters for their characterization are particle composi- tion, size/particle size distribution, surface charge, solubility/dispersibility, aggregation/ agglomeration state, shape, surface area, and surface chemistry (12). INSTRUMENTAL METHODS FOR THE CHARACTERIZATION OF TIO2 AND ZNO NPS Sunscreen preparations with micronized TiO2 and/or ZnO are complex and opaque, so NPs detection and characterization are complicated. Tyner has evaluated the ability of 20 analytical methods to detect TiO2 and ZnO NPs in unmodifi ed commercial sunscreens (13). Variable-pressure scanning electron microscopy, laser scanning confocal microscopy, X-ray diffraction, and atomic force microscopy were considered applicable and compli- mentary for NPs characterization in sunscreens. Guidelines on the safety assessment of nanomaterials in cosmetics from the Scientifi c Committee on Consumer Safety suggested the use of at least two methods, of which one should be electron microscopy, preferably high-resolution transmission electron microscopy, to determine the size of nanomaterial particles (14). TOXICOLOGICAL CONCERNS There are conclusive results that titanium can cause lung cancer after inhalation, so it is the reason for increased concerns about potential toxicity after dermal applications. Tita- nium is classifi ed into group 2B of carcinogens. Organized, accurate, and detailed studies must be conducted to give information about dermal permeation, local effects, and even- tually generalized effects. It is necessary because sunscreens are applied to the skin in some countries during the whole year, in large amounts, on almost the whole skin area. DERMAL PENETRATION OF ZNO AND TIO2 There are few parameters that must be considered during the research of dermal perme- ation of substance. One of them is the characteristics of the studied substance. Theoreti- cally, only those materials with an adequate log P coeffi cient (octanol/water partition coeffi cient) and low molecular weight ( ca. 500) can penetrate the intact human skin through the stratum corneum (SC). The second parameter is the skin itself. The SC represents the outermost layer of the skin and plays an important role in protecting the human organism against penetration by xenobiotics. It should be emphasized that although cosmetics and sunscreens containing ZnO and TiO2 are normally used on healthy skin, injuries to the skin can occur under certain circumstances (physical force or sunburn), which can cause enhancement of re- sorption. This is the reason why skin penetration studies of TiO2 and ZnO particles are usually investigated in vivo and in vitro with both intact skin and stripped skin which mimics an injured skin (15). The ingredients of sunscreen formulation must also be known. The biopharmaceutical characteristics are not the same comparing O/W emulsions, W/O emulsions, silicone-based emulsions, or aerosol sprays. Researchers investigating dermal absorption should under- line all properties and circumstances of the experiment.

JOURNAL OF COSMETIC SCIENCE 226 ABSORPTION OF TIO2 AND ZNO—IN VITRO STUDIES Many authors reported the penetration of NPs from sunscreens onto the human skin. Hu- man epidermal penetration of a transparent TiO2 and ZnO sunscreen formulation was determined using Franz-type diffusion cells and electron microscopy to verify the loca- tion of NPs in exposed membranes. In most studies, no particles could be detected in the lower layers of SC or viable epidermis by electron microscopy, suggesting that minimal nanoparticle penetration occurs through the human epidermis. Thus, some researchers have concluded that NPs penetrate to 13 layers into the UVB-damaged SC, whereas only seven layers in the intact skin. The experiment conditions and effects are shown in Table I. ABSORPTION OF TIO2 AND ZNO—ANIMAL SKIN Gamer et al. reported the dermal absorption of ZnO and TiO2 particles through the skin of domestic pigs. In addition, the results show that microfi ne ZnO particles were not able to penetrate the porcine-dermatomed skin preparations (20). Wu et al. (21) and Adachi et al. (22) have concluded that after prolonged application, NPs can penetrate through the SC and they can be located in the deep layer of the epider- mis. Wu et al. investigated the penetration and toxicity of TiO2 NPs after in vivo animal (BALB/c hairless mice) dermal application. After 60 d of dermal exposure in hairless mice, TiO2 NPs not only penetrated the skin but also reached different tissues and in- duced pathological lesions in several major organs. In addition, they found TiO2 NPs in the mouse brain without inducing any pathological changes (21). Recently, Adachi et al. (23) found signs of irritant dermatitis with focal parakeratosis in the SC and epidermal spongiosis after applying uncoated TiO2 NPs for a long time. The experiment conditions and results of other researchers are shown in Table II. Ta bl e I Absorption of TiO2 and ZnO in vitro Type of NPs Formulation Experimental system Effects Ref. ZnO NPs Transparent formulation Franz-type diffusion cells No particles could be detected in the lower SC (16) w/o emulsion Franz-type diffusion cells on the excised porcine skin No particles could be detected in the lower SC (17) TiO2 NPs Suspensions (1.0 g/L), 24 h Franz cells using intact and needle-abraded human skin Not detectable in receiving solutions for both intact and damaged skin (18) TiO2 and ZnO NPs 1) 10% coated TiO2 in w/o, 2) 10% coated TiO2 o/w, 3) 5% coated ZnO in o/w, and 4) 5% uncoated ZnO in o/w Skin in fl ow-through diffusion cells 1) TiO2 in w/o penetrated deeper in UVB-damaged SC 2) penetrated 13 layers into UVB-damaged SC, whereas only seven layers in normal and 3) and 4) were localized to the upper one to two SC layers (19)