Oil Thickening with Organoclay Enhances the Ultraviolet Absorption Ability of Sunscreen on a Skin-mimicking Substrate

The performance of sunscreen products depends on their ultraviolet (UV) absorption ability through the film formed on the skin surface upon their application. Therefore, it is important that a uniform film is formed on the uneven skin surface for effective sunscreen performance. Because most UV filters are oil soluble, we hypothesized in this study that increasing the viscosity of the oil phase of a sunscreen product can improve the performance of the sunscreen. We first examined the association between the concentration of the oil thickener and the UV absorption ability of the sunscreen product using a skin-mimicking substrate (SMS). Among all thickeners examined (petrolatum, dextrin palmitate, silica silylate, and organoclay), organoclay and silica silylate significantly increased the UV absorbance of sunscreen on the SMS in a concentration-dependent manner. Thereafter, we examined film uniformity to elucidate the mechanism underlying the observed increase in UV absorption. The uniformity of film thickness on the SMS increased with increasing organoclay content, based on decreased standard deviations of film thickness. Our results showed that increasing the viscosity of the oil phase with organoclay resulted in the formation of a uniform film by preventing the sunscreen from flowing into the grooves when applied on the SMS, thereby increasing UV absorbance by more than two-fold that of sunscreen without organoclay. Thus, the use of thickeners, such as organoclay, increases the viscosity of the oil phase at a low shear rate after the high shear of application. This is an effective strategy for improving the overall quality and performance of sunscreen products.

protection ability of sunscreen products is strongly influenced by the extent to which the sunscreen flows into the grooves 16 . Furthermore, oil-in-water vehicles with low viscosity, owing to the absence of water-soluble thickeners such as xanthan gum, have a negative effect on the SPF values 17,18 . Therefore, the physical properties of sunscreen vehicles, especially viscosity, affect the UV protection ability of sunscreens.
Organic UV filters, widely used lipophilic molecules, are usually solubilized in the oil phase of sunscreens. Therefore, we hypothesized that increasing the viscosity of the oil phase, rather than that of the water phase, might lead to more effective UV protection. Although a patent document has described that the addition of dextrin palmitate to the oil layer facilitates uniform application 19 , systematic studies are lacking on the relationship between oil-phase viscosity and UV protection or the uniformity of the sunscreen film formed on the skin surface. This can be attributed to the difficulty in measuring the uniformity of the film formed on the skin surface.
A skin-mimicking substrate SMS can simulate the human skin surface with grooves of 75-µm depth, and it has been developed using polymethyl methacrylate to estimate the SPF value in vitro 5,20,21 . Therefore, the SMS is a suitable substrate for evaluating the evenness of the film formed upon the application of sunscreen in vitro. In this study, we aimed to determine how oil thickening affects the uniformity of sunscreen film thickness and alters UV absorbance. Our results may contribute to the development of effective sunscreen vehicles.
2.2 Preparation of the thickened oil phase and sunscreen samples DHHB was selected as the UV filter considering its photostability 6 . Oils containing thickeners, which are generally used as cosmetic ingredients, were used to prepare samples. Dextrin palmitate an amphiphilic compound , petrolatum an organic nonpolar polymer , silica silylate fine solid particle , and organoclay a clay mineral were examined as thickeners.
The oil phase, thickened by the addition of each thickener, was prepared as follows: 18 g triethylhexanoin containing 2 g DHHB, and 4 g PEG-10 siloxane were mixed with 0.5, 1.0, or 2.0 g of each thickener, and dispersed at 80 for 3 min using an HM-300 homomixer AS ONE, Tokyo, Japan . In addition, organoclay-containing oil without a UV filter was prepared to confirm the absorbance measurements.
Ten types of sunscreen samples containing DHHB in water-in-oil emulsions were prepared. The formulations are listed in Table 1. Triethylhexanoin, DHHB, PEG-10 dimethicone, and each thickener were mixed and solubilized by heating at 80 for 2 min. After the addition of decamethylcyclopentasiloxane and ion-exchanged water containing sodium chloride, the resulting mixture was homogenized using HM-300.

Measurement of the viscosity of oils containing thickeners
The shear rate-dependent rheology of the oils containing thickeners was measured with a parallel-plate rotational rheometer MCR 102; Anton Paar GmbH, Graz, Austria; 50-mm parallel plate, gap size 0.5 mm at a shear rate of 0.01-1000 s 1 and temperature of 25 0.1 , after steady shearing shear rate 100 s 1 for 60 s.

Measurement of absorbance
The UV absorbance of the sunscreen samples was measured after their application on the SMS SPF MASTER ® PA-01, 50 mm 50 mm 0.8 mm; Shiseido Irica Technology, Kyoto, Japan . The SMS was made of polymethyl methacrylate and was permeable to 80 of visible and UV light. V-grooves, based on the shape of the skin surface of the human back, were molded on one side. The width and depth of the V-grooves were 300 and 75 µm, respectively 5 .
The sunscreen, which was used at final amounts of 0.5, 1.0, 1.5, and 2.0 mg/cm 2 , was immediately spotted on the surface of the SMS using a microsyringe, and the spots were spread over the uneven surface with a fingertip covered by a finger sack. The SMS was allowed to dry for 30 min at room temperature, according to the International SPF Test Method 2006. The evaporation of volatile components in the sunscreen was confirmed by the change in SMS weight. We confirmed that a drying time of 30 min was sufficient, as there was no further change in the weight of the SMS after 30 min.
The absorbance at 352 nm was measured using a UV-visible spectrophotometer V-550; Jasco, Tokyo, Japan . The absorbance of sunscreen was determined by subtracting the baseline absorbance of the SMS from the absorbance of the SMS applied with sunscreen, at six different sites, and the mean value was obtained.

Comparison of sunscreen lm thickness on the SMS
The sample was applied to the SMS, which was placed on a fluorescent board and irradiated with 27-W black light FPL27BLB; Sankyo Denki, Kanagawa, Japan , and an image of the reflected fluorescence was obtained using a digital microscope VHX-7000; Keyence, Tokyo, Japan . The reflected fluorescence brightness value 5-point average of the groove area and hill area was obtained from the digital microscope image, and the brightness ratio of the groove and hill was calculated using the following formula: Brightness ratio of the hills and grooves average brightness of the groove area / average brightness of the hill area Owing to light scattering on an uneven plate, the brightness ratio is not as quantitative as quantification using the x-ray fluorescence method 22 . However, the film thickness in the groove and hill can be compared.

Measurement of sunscreen lm thickness by confocal
laser microscopy To calculate the film thickness of the sunscreen applied to the SMS, images were obtained using a three-dimensional laser scanning confocal microscope VK-9700; Keyence, Osaka, Japan with the following settings: laser wavelength, 408 nm; output power, 0.9 mW; magnification of the objective lens, 50 ; and resolution for depth, 10 nm 23 25 . Images of the SMS were captured 30 min after the application of the sunscreen. Seventeen images were obtained, measuring 4 mm in length from the center of the SMS, and were subsequently combined using an image combiner application VK-Assembler; Keyence, Osaka, Japan to calculate the film thickness and obtain a cross-sectional profile of the SMS surface with or without the application of sunscreen. The film thickness of the sunscreen was determined as the difference in the cross-sectional profile of the SMS surface with and without sunscreen application.

Statistical analysis
Comparisons between groups were performed using Student s t-test. Results with a p value of 0.05 were considered statistically significant *p 0.05, **p 0.01, NS: not significant . Table 1 Sunscreen formulations used in this study.

Selection of an appropriate thickener
First, the thickener to be used in the sunscreen was selected. The results of shear rate-dependent rheology of the oils containing thickeners revealed that the use of organoclay and silica silylate resulted in a higher viscosity of the oil phase at low shear rates than petrolatum and dextrin palmitate Fig. 1 . The thickener concentration in the 26.0 wt oil phase of a film of sunscreen with 2.0 wt thicken-er in the formulation was 7.7 wt . When compared at 7.7 wt thickener concentration, the viscosity difference was small at a high shear rate but was up to 198 times organoclay relative to petrolatum at a low shear rate Fig. 1 .
The absorbance of 2.0 wt DHHB sunscreen containing various thickeners 1.0 or 2.0 wt , applied immediately after production, is shown in Fig. 2. A slight increase was observed in the absorbance of sunscreen samples containing petrolatum or dextrin palmitate. However, the absor-  bance of sunscreen containing organoclay or silica silylate significantly increased as the amount of thickeners increased to 1.0 and 2.0 wt . The absorbance of sunscreen film containing 1.0 wt organoclay, 2.0 wt organoclay, 1.0 wt silica silylate, and 2.0 wt silica silylate increased to 1.24, 1.64, 1.20, and 1.58, respectively, compared with that of sunscreen without organoclay 0.80 .
On the basis of these results, we concluded that organoclay is a suitable oil thickener for sunscreen emulsions. Therefore, we examined the relationship between the UV absorbance and oil-phase viscosity of sunscreen samples formulated with organoclay.
3.2 Absorbance of sunscreen samples with varying concentrations of organoclay The results of absorbance measurements after the application of 2 mg/cm 2 sunscreen are presented in Table 3. As the concentration of organoclay increased 0.0, 0.5, 1.0, and 2.0 wt , the absorbance of sunscreen increased 0.80, 1.00, 1.24, and 1.63, respectively . Sunscreen containing 2.0 wt organoclay presented a 2.04-fold higher absorbance than sunscreen without organoclay.

Film thickness and cross-sectional pro les
The cross-sectional profiles of SMS with and without sunscreen and the profile of film thickness are shown in Fig. 3. Compared with sunscreen containing 2.0 wt organoclay, sunscreen without organoclay accumulated in the grooves of the SMS, and a thin film was formed in the hill area of the SMS. Additionally, some parts had a film thickness ranging from zero to negative values Figs. 3b and 3d . The negative thickness of the film caused an error in the measurement and calculation by laser microscopy. Thus, it was assumed that parts with a negative value were not covered by the sunscreen film.
3.4 Comparison of film thickness in the hill and groove areas on the SMS The brightness ratio of the samples with 2.0 wt organoclay, 2.0 wt silica silylate, 2.0 wt petrolatum, and 2.0 wt dextrin palmitate was 0.87, 0.85, 0.75, and 0.77, respectively Table 2 . A low brightness ratio indicates nonuniform thickness, and a high brightness ratio of close to 1.0 indicates a relatively uniform thickness. This suggested that samples containing organoclay or silica silylate had a more uniform film thickness on SMS than samples containing petrolatum or dextrin palmitate.

Film thickness and UV absorbance of sunscreen ap-
plied on the SMS The means and standard deviations of the film thickness and UV absorbance of sunscreen samples containing 0, 0.5, 1.0, and 2.0 wt organoclay are shown in Table 3. Although the mean values of the film thickness of sunscreen samples containing different concentrations of organoclay showed no difference, the standard deviations of the film thickness decreased to 3.33, 2.66, 2.50, and 2.31 µm, respectively, in an organoclay concentration-dependent manner.
In contrast, the UV absorbance decreased as the standard deviation of the film thickness increased, suggesting that the nonuniformity of the film thickness was greater. This negative correlation was observed by linear approximation with the standard deviations of absorbance and thickness, with a multiple correlation coefficient of 0.78. This indicated a relationship between UV absorbance and Table 2 Brightness ratio of the hills and the groove. Table 3 Absorbance and film thickness of sunscreen containing different amounts of organoclay.

Organoclay Enhances the UV Absorption Ability of Sunscreen
the thickness of the film formed by the application of sunscreen.

Comparison of the absorbance of sunscreen samples with and without thickeners at different application amounts
The effects of the amount of sunscreen application on UV absorbance were examined using sunscreen samples no. 1, 4, 9, and 10 Table 1 . The UV absorbance of sunscreen without organoclay gradually increased with an increase in the application amount 0, 0.5, 1.0, and 2.0 mg/ cm 2 Fig. 4 . However, sunscreen containing organoclay showed a higher increase in absorbance with increasing application amount than sunscreen without organoclay. The results indicated that organoclay enhanced the UV absorbance at the same amount of sunscreen.

Discussion
The human skin has an uneven topology consisting of hills crista cutis and grooves sulcus cutis , referred to as kime in Japanese. Thus, when applying a sunscreen product, it is difficult to form a uniform film on the skin surface, which minimizes the protective effect of sunscreen against UV radiation. The process of film formation after sunscreen application is illustrated in Fig. 5a. The film is formed after the evaporation of water and volatile compounds. To form a film of uniform thickness, it is essential that UV filters, which are contained in oil, do not flow into the grooves of the skin. In this study, we hypothesized that increasing the viscosity of nonvolatile compounds i.e., the oil phase in sunscreen products is an effective strategy to create a UV filter-containing film of uniform thickness, by preventing the sunscreen from flowing into the skin grooves. According to a simple model calculations, the UV transmittance of an evenly distributed film is 3.7 times lower than that of an unevenly distributed film, which supports this hypothesis Figs. 5b and 5c .
We first examined the UV absorption ability of sunscreen samples formulated with different oil-phase thickeners organoclay, silica silylate, petrolatum, and dextrin palmitate , to determine the association between oil-phase viscosity and UV absorption. We found that thickening with organoclay and silica silylate significantly restored oil-phase viscosity at a low shear rate after the high shear of application, thereby increasing UV absorption Figs. 1 and 2 . Although petrolatum and dextrin palmitate slightly increased the oil viscosity of the sunscreen at a concentration of 2.0 wt , both of them failed to increase UV absorption Figs. 1 and 2 . This failure could be due to the lack of ability to restore thickening at 25 after the high shear of application. In contrast, both organoclay and silica silylate exhibited shear-thinning behavior in the oil phase even after the high shear of application Fig. 1 . Therefore, they provided higher viscosity to the oil phase than petrolatum and dextrin palmitate at a low shear rate even after the application. This, in turn, may have prevented the oil from flowing on the SMS plates, resulting in a more uniform film thickness Table 2 . The sunscreen film with organoclay became more uniform and presented higher absorbance on the SMS than the sunscreen film with silica silylate. Organoclay presented almost the same thickening effect as silica silylate in the oil phase; however, organoclay is considered to have a higher thickening effect than silica silylate in water-in-oil sunscreen emulsions because it is known to form a complex with nonionic surfactants and to stabilize water-in-oil emulsions 26 29 . On the basis of these results, we selected organoclay as the oil-phase thickener and further examined the association between oil-phase viscosity and UV absorption or the uniformity of the film applied on the SMS using sunscreen formulated with organoclay.
The sunscreen samples showed an increase in UV absorption with increasing organoclay concentration Table  3 when applied onto the SMS at 2.0 mg/cm 2 . Furthermore, the oil-phase viscosity at low shear rates was directly proportional to the organoclay concentration Fig. 1 , and it positively correlated with UV absorption when organoclay was used as an oil-phase thickener.
Conversely, although the mean thickness of the film formed on the surface of the SMS showed no apparent cor-relation with the concentration of organoclay, the differences in the standard deviations of the film thickness decreased with an increase in the concentration of organoclay Table 3 . The film of the sunscreen sample with 2.0 wt organoclay covered the hills of the SMS, unlike that of the sunscreen sample without organoclay Fig. 3 . These results indicated that increasing the oil-phase viscosity at a low shear rate by introducing organoclay produced an evenly distributed film.
In addition, increasing the oil-phase viscosity at a low shear rate had a greater effect on UV absorbance Fig. 1 and Table 3 . Despite the same UV filter concentration, the UV absorbance of the film formed by the sunscreen sample with 2.0 wt organoclay was two times higher than that of the film formed by the sunscreen sample without organoclay 1.64 and 0.80, respectively . Additionally, sunscreen containing 1.0 wt DHHB and 2.0 wt organoclay exhibited UV absorption equivalent to that of sunscreen containing 2.0 wt DHHB without organoclay when applied on the SMS at 2.0 mg/cm 2 Fig. 4 . Furthermore, the absorbance of the film formed by the application of sunscreen 1.0 mg/cm 2 with 2.0 wt DHHB and 2.0 wt organoclay Fig. 5 Schematic illustration of the sunscreen drying process and transmittance. a Schematic illustration of the sunscreen drying process on the skin surface. b Non-uniform film thickness was associated with the lowest UV absorption and highest transmittance. c Uniform film thickness was associated with the highest UV absorption and lowest transmittance. Differences in oil flow owing to oil thickening during drying are presented in a conceptual diagram. One block represents the amount of sunscreen film required to reduce UV rays by 0.1. Both b and c were exposed to the same amount of light but the transmitted light differed by as much as 3.7 times. Thus, the model shows that when the film is uniform, the transmitted light significantly differs.
was significantly higher than that of the film formed by sunscreen 2.0 mg/cm 2 prepared with 2.0 wt DHHB alone without organoclay . However, applying 0.5 mg/cm 2 sunscreen eliminated the additional effects of organoclay on UV absorption Fig. 4 . Although it is difficult to demonstrate, we speculated that the application of only 0.5 mg/ cm 2 sunscreen might have been insufficient to cover the SMS surface. In summary, increasing the oil-phase viscosity at a low shear rate by adding organoclay to sunscreen products may reduce the application amount of sunscreens and achieve lower concentrations of UV filters on the skin surface. Although UV filters are recognized to have no negative effects on the skin, they have been reported to result in the generation of reactive oxygen species through the photosensitization reaction and to generate radicals during photoisomerization due to the release of UV energy absorbed as thermal energy 6, 30 32 . Furthermore, previous studies have demonstrated the risk of photo-induced skin irritation and photo-induced skin sensitization caused by UV filters under sun exposure 1,33,34 . Reducing the concentration of UV filters by increasing the oil-phase viscosity will reduce the UV filter-associated adverse events. Our study proposes an effective way to achieve adequate UV protection with sunscreen even at a low UV filter concentration and with a lower application amount of the product.

Conclusions
In this study, with a view to improve the effectiveness of sunscreens even at a low UV filter concentration or a low sunscreen application amount, we investigated the association between the viscosity of the oil phase containing the UV filter and UV absorption or the evenness of the film formed on the SMS upon sunscreen application. The increased oil-phase viscosity afforded by organoclay positively correlated with UV absorption and was associated with a lower deviation in film thickness on the SMS. We conclude that the use of thickeners, such as organoclay, increases the viscosity of the oil phase at a low shear rate after the high shear of application. This is an effective strategy for optimizing sunscreen efficacy, even at a low concentration of the UV filter or a low application amount of the sunscreen. This sunscreen formulation may be helpful in reducing the risks associated with UV filters.