2.1.

Chemicals and Equipment

The following reagents were used: potassium carbonate from Sigma-Aldrich, St. Gallen, Switzerland; Tinosorb S abbreviated BEMT (INCI, bis-ethylhexyloxyphenol methoxyphenyl triazine), Uvinul N539T abbreviated OCR (INCI, octocrylene), Salcare SC 91, Cetiol AB, Lanette O, Dehymuls LE, Edeta BD, all from BASF SE, Ludwigshafen, Germany; Eusolex 232 abbreviated PBSA (INCI, phenylbenzimidazol sulfonic acid) from Merck, Darmstadt, Germany; Parsol 1789 abbreviated BMDBM (INCI, butyl methoxydibenzoylmethane), Amphisol K from DSM, Kaiseraugst, Switzerland; Neo Heliopan OS abbreviated EHS (INCI, ethylhexyl salicylate) from Symrise, Holzminden, Germany; Arlacel 165 from Croda, East Yorkshire, England; Keltrol RD from CP Kelco, Atlanta, Georgia; Carbopol Ultrez 10, Carbopol Ultrez 21 from Lubrizol, Brussels, Belgium; Tegin OV from Evonik Industries, Essen, Germany; Paracera M from Paramelt, Heerhugowaard, The Netherlands; beeswax white from Koster Keunen, Bladel, The Netherlands; glycerin from Sigma-Aldrich; tris amino ultrapure from Angus, Buffalo Grove, Illinois; Phenonip from Clariant, Muttenz, Switzerland.

Quartz plates with a size of $4.2\times 4.2{\mathrm{cm}}^2$ were obtained from Hellma Analytics, Zumikon, Switzerland.

The following equipment was used: electric shaver (Favorita II GT104, Aesculap, Germany), epilator (Silk-épil7 Xpressive Pro, Braun, Germany); water purification device (Arium 61215, Sartorius, Goettingen, Germany); precision balances (XS105 Dual range and XA3001S, Mettler-Toledo, Columbus, Ohio); surface metrology instrument (Altisurf 500, Altimet, Thonon-les-Bains, France); UV transmittance analyzer (Labsphere UV-2000S, Labsphere Inc., North Sutton, New Hampshire).

The following software packages were used: BalanceLink (Mettler Toledo, Columbus, Ohio) with balance XA3001S for the recording of pressure during spreading of sunscreen; Phenix and Altimap (Altimet, France) for topographical measurement and evaluation, respectively; UV-2000 (Labsphere Inc.) for UV transmittance measurement; Statgraphics centurion XVI software (Statpoint Technologies, Inc., Warrenton, Virginia) for statistical evaluation.

2.2.

Preparation of Skin Substrate

We used the epidermal membrane of pig ears as a biological substrate for sunscreen application as described before.²⁰ Ears of freshly slaughtered pigs were obtained from the local slaughterhouse (Basel, Switzerland) not more than a few hours postmortem. The study did not require the approval of the ethics committee of animal research as the ears were taken from pigs not specifically slaughtered for the purpose of this study. The epidermal membrane was isolated using a heat separation procedure. The full skin was immersed in a water-bath at 60°C for 90 s. The epidermal membrane was separated from the dermis by gentle peeling off, cut to a dimension of $2\times 2{\mathrm{cm}}^2$, laid flat on quartz carrier plates, and stored at 4°C in a desiccator over saturated potassium carbonate solution until use.

2.3.

Characterization of Sunscreen Formulations

We assessed SPF in vitro and the film thickness distribution of five different sunscreens. The formulations included an oil-in-water cream (OW-C), an oil-in-water spray (OW-S), a water-in-oil emulsion (WO), a gel (GEL), and a clear lipo-alcoholic spray (CAS). They contained the same UV filter combination and emollient. The filter system was composed of 8 wt. % OCR, 5 wt. % EHS, 2 wt. % BMDBM, 1 wt. % BEMT, and 1 wt. % PBSA. Based on this UV filter composition, an SPF in silico of 25 was calculated with the BASF sunscreen simulator.²⁵ The detailed composition of the sunscreens and their respective SPF in vivo values are given in Table 1. SPF in vivo values were measured in accordance with ISO24444:2010 guidelines.²⁶

Table 1

Composition (wt. %) and sun protection factor (SPF) in vivo of investigated sunscreens.

The sunscreens showed different rheological characteristics (Fig. 1). GEL had the highest shear viscosity followed by OW-C and WO, whereas OW-S and CAS were much less viscous. Viscosity of all sunscreens decreased with increasing shear rate whereas hysteresis depended on the formulation.

Fig. 1

Rheological behavior of sunscreens measured with AR-G2 rheometer (TA instrument, New Castle, Delaware), CP $4\mathrm{deg}/40\mathrm{mm}$, gap $100\mu \mathrm{m}$, $T=23°\mathrm{C}$.

2.4.

Application of Sunscreen

We applied $2.0\mathrm{mg}/{\mathrm{cm}}^2$ of sunscreen nominally corresponding to a film thickness of 20 μm. The sunscreen was applied in form of 20 to 30 small drops evenly distributed over the skin surface and manually spread with the fingertip using a presaturated finger coat. Two spreading procedures were employed. In the first, the sunscreen was spread on the specimen with light circular movements followed by left-to-right linear strokes from top to bottom starting at each side of the specimen (designated spreading 1); in the second, the complete linear stroke step was repeated four times (designated spreading 2). Spreading procedure 2 resulted in a longer application time. Furthermore, the pressure used to distribute the product was varied for spreading 1 between low and high, corresponding to a force of $100\pm 14$ and $281\pm 35\mathrm{g}$, respectively. These values represent extremes used in the authors’ laboratory with this substrate preparation. The two pressure and spreading conditions were used solely with the gel formulation (GEL). All other sunscreen formulations were applied with high pressure and spreading procedure 1.

2.5.

Measurement of the SPF In Vitro Using Spectral Transmission of Ultraviolet Radiation

Measurement of SPF in vitro is based on diffuse UV transmission spectroscopy according to the approach proposed by Sayre et al.,²⁷

For SPF determination, the spectral UV transmittance was registered from 290 to 400 nm in 1-nm increment steps through skin substrate preparations before and after application of a sunscreen using Labsphere UV-2000S. The UV transmittance of four positions per $2\times 2{\mathrm{cm}}^2$ skin substrate was measured to virtually cover the complete surface area of the preparation.

The blank transmittance spectrum was recorded at first for each single position before sunscreen application followed by topographical measurement of the bare skin (see Sec. 2.6). Subsequently, sunscreen was applied and topographical measurement was performed again. After completion of topographical measurement which lasted $\sim 4\mathrm{h}$, UV transmission through the sunscreen-covered skin substrate was measured. Stability of SPF in vitro values over 4 h was checked and confirmed for all sunscreens (data not shown).

2.6.

Assessment of the Sunscreen Film

The layer of sunscreen applied on the pig skin substrate was investigated using topographical measurements with an optical probe based on the white light chromatic aberration principle (Altisurf 500 instrumentation). The instrumentation allowed noncontact surface topography measurement and analysis. The employed optical sensor yielded an axial resolution ($z$) of 5 nm and a lateral resolution ($x–y$) of $1.1\mu \mathrm{m}$. The motorized $x–y$ stage permitted scanning of samples in the millimeter range. Skin preparations on quartz plates were fixed on the stage using a custom made holder.

Surface topography of bare skin and skin covered with sunscreen was measured in order to assess the sunscreen film. Skin preparations were removed from the desiccator and allowed to equilibrate for 12 h next to the device at room conditions before starting topographical measurements. Repeated measurements on bare skin using the “loop” option of the operating software revealed that this equilibration was necessary to allow stabilization of the surface position along the $z$ axis (data not shown). After measuring the surface topography of bare skin, sunscreen was applied, equilibrated for 15 min, and the same area was scanned again.

Figure 2 illustrates the area of topographical and UV transmittance measurements.

Fig. 2

Illustration of areas for topographical and UV transmittance measurements; the big square corresponds to the carrier quartz plate, the dotted small square to the skin surface area with a dimension of $2\times 2{\mathrm{cm}}^2$, the four circles correspond to the areas of UV transmittance measurements [sun protection factor (SPF)] with a diameter of 1 cm and the two rectangles to the two areas of topographical measurements.

Topographical measurements were performed on two rectangular areas ($\sim 23\times 8{\mathrm{mm}}^2$) per specimen (Fig. 2). A part of the rectangular area (about $5\times 8{\mathrm{mm}}^2$ on left hand side) corresponded to quartz without skin and served as a reference. The skin area (right hand side of the rectangle) measured about $18\times 8{\mathrm{mm}}^2$. Topography was recorded in lines each extending over the quartz and the skin part of the rectangle with an increment step of $10\mu \mathrm{m}$. The rectangular areas were scanned with lines in $10\text{-}\mu \mathrm{m}$ intervals resulting in 1,840,000 single-measurement points per rectangle.

The raw data of the topographical measurements were redressed by a line-by-line leveling correction of each rectangular surface to the same $x\text{-}y$ plane using the quartz part of each measured line (left side of rectangle, Fig. 2). This redressing procedure was carried out with the data of bare skin and skin covered with sunscreen and was essential in order to correct for variation due to positioning and due to environmental factors changing in the course of the experiment. Each rectangular surface area was divided into two zones of $8\times 8{\mathrm{mm}}^2$ coinciding with the four positions (circles) of UV transmittance measurements (Fig. 2). The film thickness of the applied sunscreen was obtained as the difference of the redressed skin topography data with and without sunscreen computed for each single-measurement point. The result was expressed as a distribution of frequencies of film thickness over the measured surface area normalized to 100% and is referred to as thickness distribution curve. A threshold of 0.5% of area under the curve was applied to remove extreme values at both ends of the film thickness distribution. To validate this measurement and calculation method, a surface area of bare skin was measured twice and the film thickness was computed. The result was found to be centered around $0\mu \mathrm{m}$ ($n=8$), confirming the validity of the method for measurement of the sunscreen film thickness distribution on skin.

Data extracted from the distribution curve and serving to characterize the applied sunscreen film are given in Table 2.

Table 2

Data extracted from the thickness distribution curve of applied product.

$S_{\text{mean}}$ is the frequency-weighted average thickness. The $S_{\text{mean}}$ to median ratio of the thickness distribution is a measure of skewness of distribution and is used as an expression of film homogeneity; the smaller this ratio the greater the homogeneity of the film. The Abbott–Firestone curve is commonly used in surface metrology²⁹ and is employed here to depict the experimentally determined thickness distribution, indicating thickness and uniformity of the applied product layer.

2.7.

Statistical Analysis

Statistical analysis was performed using Statgraphics centurion XVI software (Statpoint Technologies, Inc., Warrenton, Virginia). The impact of formulation vehicle on SPF in vitro and on film parameters was assessed with a Kruskal–Wallis nonparametric test, and the impact of application conditions was assessed with a Mann–Whitney U test, both with a statistical significance at 5% confidence level. In case Kruskal–Wallis test revealed a statistically significant difference among sunscreens for an investigated parameter, a multiple pairwise comparison test using Bonferroni approach was performed to identify which sunscreens significantly differed from which other. Correlations between film parameters and SPF in vitro values within each formulation were assessed using Spearman’s rank correlation coefficient test.

3.1.

Film Assessment

The film thickness distribution of sunscreen, extracted from the topographical measurements, is visualized three-dimensionally for qualitative assessment in Fig. 3 and is quantitatively displayed as a distribution curve of thickness frequency. From the distribution curve, the Abbott–Firestone curve (cumulative frequency) was deduced (Fig. 4).

Fig. 3

Example of three-dimensional visualization of film thickness distribution of oil-in-water cream (OW-C) sunscreen.

Fig. 4

Example of distribution of film thickness frequency and Abbott–Firestone curve of OW-C sunscreen.

Thickness distribution was always positively skewed, the degree of skewness varying between the different sunscreens. In the example of Fig. 4, the most frequently occurring film thickness was in the range of 2 to $4\mu \mathrm{m}$ while a thickness as large as 10 to $13\mu \mathrm{m}$ was recorded. A small percentage of the area under the thickness distribution curve lay below a film thickness of $0\mu \mathrm{m}$, which was likely due to experimental error. This was included in the calculation of the $S_{\text{mean}}$ value.

3.2.

Impact of Vehicle on Film Parameter Values and SPF In Vitro

Figure 5 gives the average of the Abbott–Firestone curves of all measurements with each investigated sunscreen using high pressure and spreading 1 conditions of application.

Fig. 5

Abbott–Firestone profiles of investigated sunscreens applied with high pressure and spreading 1.

The Abbott–Firestone profiles differed considerably between the sunscreens (Fig. 5). Film thickness was different for the different vehicles and decreased roughly in the order $\mathrm{WO}>\mathrm{GEL}>\mathrm{OW}\text{-}\mathrm{C}>\mathrm{CAS}>\mathrm{OW}\text{-}\mathrm{S}$. For WO for example, a film thickness of $2.41\mu \mathrm{m}$ corresponds to 50% of the cumulative thickness frequency meaning that 50% of the measured surface area of the sample exhibited a film thickness greater than $2.41\mu \mathrm{m}$. As a comparison, 50% of the measured area of OW-S exhibited a thickness greater than merely $1.20\mu \mathrm{m}$. Moreover, the shape of the curve differed between the used vehicles, the WO, for example, showed a more flat-shaped profile compared to CAS (Fig. 5).

These differences between the vehicles are reflected by the calculated film thickness distribution parameters $S_{\text{mean}}$ and $S_{\text{mean}}$ to median ratio. Table 3 gives the values of the median and interquartile range for the film parameters of all individual measurements of each investigated sunscreen. Also, SPF in vitro values of these sunscreens are given in Table 3.

Table 3

Medians of SPF in vitro, Smean, and Smean to median ratio of thickness distribution with interquartile range Q1 to Q3 (in brackets) for investigated sunscreens with high pressure and spreading 1.

SPF in vitro varied markedly between the investigated sunscreen formulations attaining values from 14 for CAS to 72 for WO. SPF in vitro values are compared to the SPF in vivo in Fig. 6. SPF in vitro values generally approached SPF in vivo and, considering the declared variation range, a satisfactory agreement between SPF in vitro and in vivo for spreading 1 and a high pressure condition is found. WO sunscreen was an exception, with surprisingly low and high SPF in vivo and SPF in vitro, respectively. In silico estimation of SPF gave a value of 25 (Fig. 6). This computational approach takes into account the absorbance spectrum of each UV filter, their photostability and mutual stabilization or de-stabilization, and their distribution in the phases of the vehicle and uses the Gamma distribution function to describe film irregularity.³⁰ The estimated value lies within the range of the experimental values of all vehicles, yet the in silico calculation cannot predict the effect of formulation on SPF. In Fig. 6, the $S_{\text{mean}}$ of the formulations is also visualized.

Fig. 6

SPF in vivo (white columns) with standard deviation (bars), medians of SPF in vitro (gray columns) with interquartile values (bars) for OW-C ($n=27$), OW-S ($n=20$), GEL ($n=28$), WO ($n=24$), CAS ($n=20$), SPF in silico (black horizontal line), and medians of $S_{\text{mean}}$ values (squares) of the same sunscreens applied with high pressure and spreading 1.

The impact of the vehicle on SPF in vitro and film parameters was evaluated with a Kruskal–Wallis test (Table 4).

Table 4

Impact of vehicle on SPF in vitro, Smean, and Smean to median ratio of thickness distribution.

This statistical test revealed a significant effect of the vehicle on all tested parameters. To identify which sunscreens significantly differed from each other with respect to the studied parameters, a multiple pairwise comparison test based on the Bonferroni approach was employed. The results are given in Tables 5Table 6–7.

Table 5

Multiple pairwise comparison test using Bonferroni approach for SPF in vitro.

Table 6

Multiple pairwise comparison test using Bonferroni approach for Smean.

Table 7

Multiple pairwise comparison test using Bonferroni approach for Smean to median ratio.

This multiple comparison test resulted in a group classification of the investigated sunscreens. Formulations of one group differ statistically from those of another group while formulations that belong to the same group do not differ significantly from each other with respect to the considered parameter. When the same formulation is contained in two different groups it does not differ significantly from the formulations of either group. The number of groups was different for the tested parameters; two, three, and four groups were found for $S_{\text{mean}}$ to median ratio, SPF in vitro, and $S_{\text{mean}}$, respectively. A small number of groups mean less difference between the sunscreens.

WO yielded a significantly higher SPF in vitro than all other sunscreens, a greater $S_{\text{mean}}$ than OW-C, CAS, and OW-S and a smaller $S_{\text{mean}}$ to median ratio than CAS and OW-S. OW-C gave a higher SPF in vitro and a smaller $S_{\text{mean}}$ to median ratio than CAS and OW-S. The GEL and OW-C formulations did not significantly differ from each other with respect to any of the criteria. Also, CAS and OW-S did not differ from each other. OW-C yielded a greater SPF in vitro, a greater $S_{\text{mean}}$ and a smaller $S_{\text{mean}}$ to median ratio than OW-S, which was interesting since these two sunscreens varied only in their content of thickeners, hence their viscosity characteristic.

Finally, the correlation of the SPF in vitro with both film parameters for the individual measurements within each sunscreen was evaluated using a Spearman rank correlation test. A significant positive correlation between SPF in vitro and $S_{\text{mean}}$ was found for every sunscreen formulation ($p<0.05$). A negative correlation was found between SPF in vitro and $S_{\text{mean}}$ to median ratio for WO; OW-S, and CAS ($p<0.05$), whereas no correlation was found for OW-C and GEL.

3.3.

Impact of Pressure and Spreading Procedure on Film Parameter Values and SPF In Vitro

In addition to the vehicle, the impact of the application conditions, i.e., spreading procedure and pressure on film parameters and SPF in vitro, was studied using the GEL sunscreen. In total, three conditions of application were investigated, spreading 1 with high pressure, spreading 1 with low pressure, and spreading 2 with high pressure.

Figure 7 shows the average of the Abbott–Firestone curves of the GEL sunscreen for each application condition, and Table 8 gives the median and interquartile range values of SPF in vitro and the film parameters for the investigated conditions.

Fig. 7

Abbott–Firestone profiles of GEL sunscreen applied with two pressure (high and low) and spreading (1 and 2) conditions.

Table 8

Medians of SPF in vitro, Smean, and Smean to median ratio with interquartile range Q1 to Q3 (in brackets) for investigated conditions of application of the GEL sunscreen.

It is evident from Fig. 7 that the shape of the Abbott–Firestone curve of GEL sunscreen is different between spreading 2 and spreading 1, while no difference was found between low and high pressure using spreading 1. The differences of the Abbott–Firestone curves are reflected in the $S_{\text{mean}}$ and $S_{\text{mean}}$ to median ratio.

SPF in vitro data measured for each condition of application were compared to the SPF in vivo for GEL sunscreen (Fig. 8). From this evaluation, spreading 2 with high pressure seems to give a better approximation of the SPF in vivo. However, as this condition could not be practically applied to all types of formulation, spreading 1 with high pressure was used as an alternative in the investigation of the different vehicles (Sec. 3.2).

Fig. 8

SPF in vivo (white columns) with standard deviation (bars), medians of SPF in vitro (gray columns) with interquartile values (bars), and medians of $S_{\text{mean}}$ values (squares) of the GEL sunscreen for different application conditions.

The impact of spreading (procedure 1 versus 2) and pressure (low versus high) on SPF in vitro and film parameters were evaluated using the Mann–Whitney U test (Table 9).

Table 9

Impact of application conditions on SPF in vitro, Smean, and Smean to median ratio of thickness distribution of GEL sunscreen.

Spreading 2 showed a significantly smaller film thickness ($S_{\text{mean}}$) and a larger $S_{\text{mean}}$ to median ratio compared to spreading 1 (Tables 8 and 9). Both spreading and pressure had a significant effect on SPF in vitro. For spreading 2 compared to spreading 1 and high compared to low pressure a reduction of SPF in vitro was found. Film parameters were not significantly influenced by pressure.