A reactor fl uid is present in the lower chamber, helping the simulation of the circulation in the dermis. The substance to be tested is placed on the skin, and its diffusion through the skin is evaluated by analyzing the receptor fl uid, which is located in the lower cham- ber. After the diffusion period is terminated, several skin layers are examined for any re- sidual materials. The UV fi lters are likely to exhibit different penetration rates, in contrast to pure substances, because they are examined in standardized preparations, at several concentrations. After application (the duration varies at every experiment), the samples are rinsed using a surfactant solution, to analyze the concentration of the product in every layer (SC, epidermis, dermis, and receptor fl uid). The amount of the applied test sub- stance, which is found in the SC, is estimated by the so-called SC absorption. By dermal absorption, on the other hand, we mean the amount of the applied product found in the epidermis and dermis. Last, by percutaneous absorption, we mean the amount of the ap- plied product that is found in the receptor fl uid. An in vivo method, for measuring skin penetration, is the tape-stripping method. For 30 min, samples are placed on the skin surface, and then, the substance in excess is removed from the surface by swiping with a dry cloth. Seven tape strippings are used to remove the SC of the area under examination. The fi rst tape strip is thrown away, whereas the next six are gathered, put in a beaker, containing a suitable solvent and stirred for 30 min. The levels of the sunscreen product in the solvent are then measured (27). It is well known that there are considerable differences between animal and humans in their skin delivery systems, attributed to factors including SC thickness, hydration, and lipid composition. Using fresh frozen human skin instead of animal skin can serve as an excellent alternative to methods using animal skin while also making the result much closer to human living skin. Although the tissue has been frozen and stored at -80°C, transport and barrier mechanisms apparently remain functional. Similar distribution patterns have also been demonstrated in the porcine skin. Although the experiments are performed with the skin kept at -80°C, freeze–thaw cycles or careless storage at higher temperatures might affect the results and the permeability of the skin. Nevertheless, ethically, it is an excellent way to avoid using experimental animals in permeation studies (28). TOXICITY ST UDIES Scientists are well aware of toxicity issues related to fi lters. This concern has been confi rmed by a number of studies (in vitro/in vivo), according to which commonly used sunscreens were found to have an endocrine active chemical action. In vitro studies investigated estrogenic activity of UV fi lters, varying in their design and endpoints, which might explain the diverging results. Most in vitro studies reported that BP-3, 4-methylbenzylidene camphor (4-MBC), OMC, HMC, and OD-PABA exhibit estrogenic activity. However, not all of the UV fi lters exhibiting in vitro estrogenic activity were estrogenic in acute in vivo models. In vitro, 8/9 c hemicals (BP-1, BP-2, BP-3, 3-BC, 4-MBC, HMS, OD-PABA, and OMC) showed estrogenic (on MCF-7 cells) and 2/9 (BP-3 and HMS) showed antiandrogenic activity (on MDA-kb2 cells). Six/nine fi lters (BP-1, BP-2, BP-3, 3-BC, 4-MBC, and OMC) increased uterine weight in immature rats. 3-BC and 4-MBC displaced 16α125I- estradiol from human estrogen receptor (ER)β. Developmental toxicity of 4-MBC (0.7–47 mg/kg body weight/day) and 3-BC (0.24–7 mg/kg), administered in chow, was investigated in the Long–Evans rats. Weight gain of pregnant rats was reduced only by JOURNAL OF COSMETIC SCIENCE 304

3-BC, early postnatal survival rate, and thymus weight by both compounds at higher doses. 4-MBC and 3-BC delayed male puberty and dose-dependently affected reproductive organ weights of adult male and female F1 offspring, with partly different effect patterns. Thyroid weight was increased by higher 4-MBC doses. Tissue-specifi c changes in mRNA levels of estrogen-regulated genes in the prostate, uterus, and brain regions, determined by real-time PCR, in their response to acute estradiol challenge in adult gonadectomized offspring were observed. Lowest effective doses were 0.24 mg/kg/day for 3-BC and 7 mg/kg/d for 4-MBC. Fat tissue levels at 7 mg/kg 4-MBC (GC–MS) approached the range of UV fi lters in fi sh. A human SED of 4-MBC has been estimated as 0.23 mg/kg body weight. Such a dose would be only 1/3 of the present no observed adverse effect level (NOAEL) and 1/30 of the lowest observed adverse effect level of 4-MBC (2,29). Other researchers al so came to the conclusion that various metabolic factors, such as fat and lipid homeostasis and thyroid hormone production, mediated by non–estrogen-related methods, were affected by OMC and 4-MBC. OMC and 4-MBC in rats were shown to exert endocrine disrupting, including uterotrophic, i.e., estrogenic effects. Estrogens also have metabolic effects therefore, the impact of oral application of the two UV absorbers at two doses (50 or 250 mg per 20 g food of OMC or 4-MBC, respectively) for 3 mo on lipids and hormones was compared with that of estradiol-17β (E2). E2, OMC, and 4-MBC reduced weight gain, the size of fat depots, and serum leptin, a lipocyte-derived hormone, when compared with the ovariectomized control animals. Serum triglycerides were also reduced by the UV screens but not by E2. On the other hand, E2 and OMC re- duced serum free fatty acids and cholesterol low-density lipoproteins, and high-density lipo- proteins this effect was not shared by 4-MBC. Whereas E2 inhibited, OMC and 4-MBC stimulated serum LH levels. In the uterus, both UV fi lters had mild stimulatory effects. 4-MBC inhibited serum T4, resulting in increased serum thyroid-stimulating hormone (TSH) levels. On the basis of human data, the applied doses for experimental animals were relevant as they also resulted in μM concentrations (30). Wang et al. (31) conclu ded that 4-MBC acted as a possible inhibitor of the pituitary–thyroid axis, as the TSH serum levels were found to be considerably high. Moreover, the weight of the thyroid glands was considerably increased. Cinnamate derivatives interfered with the TH axis in rats. The perinatal OMC exposure induced adverse effects on the reproductive and neurological development of rat offspring. The treatment with OMC for 12 weeks caused a decrease in T4 level in the blood of ovariectomized female rats and inhibited the activity of 5′-deiodinase that converts T4 to T3 in the liver [mean intake of test substances (mg/animal/ day): OMC, 2.5 (low) or 12.5 g/kg (high) 4-MBC, 2.5 (low) or 12.5 g/kg (high)]. Experimental studies refer ring to human exposure showed that BP-3, 4-MBC, and OMC rapidly permeated intact skin and could be detected in plasma after 1–2 h following application. Interestingly, the concentrations of these compounds in the same experimental study in male urine and plasma were higher than those in female samples, indicating a gender difference in the metabolism, distribution, and possibly also in the accumulation of UV fi lters in adipose tissue. Was it an effect of surface? Male body has larger surface than female body. If the sunscreen was applied at the same mg/cm2 concentration, more surface equates to more sunscreen applied, hence more sunscreen in urine and plasma? The concentrations that were used in every experiment were different. A single blinded experimental study in Denmark used 10% concentration of BP-3, 4-MBC, and OMC (whole-body application of sunscreen 2 mg/cm2). In Sweden, an experimental study used 4% of BP-3 (whole-body application of sunscreen 2 mg/cm2) (32). DISTRIBUTION OF UV FILTERS ON THE SKIN 305