2.1.

Volunteers

The study was carried out testing each sunscreen on the flexor forearm of six healthy volunteers, aged between 23 and $45\text{years}$ (skin photo-types²⁵ I to III), at the Charité—Universitaetsmedizin Berlin, Department of Dermatology, Berlin, Germany. Ethical approval for these experiments had been obtained from the Ethics Committees of the Charité Hospital.

2.2.

Porcine Skin

The ears of freshly slaughtered pigs (German domestic pigs, $6\text{months}$ old) were obtained from the local butcher. Permission had been obtained from the Supervisory Office for Veterinary Medicine, Berlin, Germany. Each sunscreen was investigated using six different porcine ears.

2.3.

Sunscreen Application

The investigations were performed, using the following sunscreens:

After cleaning the skin of the human flexor forearm with cold water and drying with a paper towel, an area of $8\times 10{\mathrm{cm}}^2$ was marked and $2\mathrm{mg}∕{\mathrm{cm}}^2$ of the formulation was applied. The porcine ears were fixed on a polystyrene support covered with aluminum foil and washed with water and dried with paper towels. The hairs of the ears were carefully removed by scissors to avoid later disturbances in the nonhomogeneous distribution of UV filter on the removed tape strips. $2\mathrm{mg}∕{\mathrm{cm}}^2$ of the sunscreens were applied to the ear on marked areas of $40{\mathrm{cm}}^2$ .

2.4.

Tape Stripping

The tape stripping commenced one hour after sunscreen application and was carried out as described previously.²⁶ The adhesive tapes (tesa film, no. 5529, Beiersdorf, Hamburg, Germany; width: $19\mathrm{mm}$ ) were pressed onto the human skin by a stamp (pressure: $15\mathrm{kp}∕{\mathrm{cm}}^2$ ) and quickly removed with one movement.

In both protocols, 10 tapes were taken from each skin area investigated, and the absorption spectra were recorded immediately. Ten tapes were also removed from an untreated skin area for the correction of the absorption spectra obtained from the treated skin, as will be described in the following paragraph.

2.5.

Determination of the Sum Transmission

After removal from the skin, each tape strip was fixed onto a metal support and analyzed in a spectrometer, together with an empty tape as reference. The measurements to calculate the sum transmission were performed immediately after removal of the tape strips (within $15\text{seconds}$ ), in order to neglect the diffusion of the UV filter into the tape.²³ The absorption spectra of the sunscreens were recorded between 240 and $500\mathrm{nm}$ using the UV/VIS spectrometer Lambda 5 (PerkinElmer, Frankfurt/Main, Germany) with an integrating sphere and a quadratic beam diameter of $8\times 10{\mathrm{mm}}^2$ .

The software UV Winlab Version 2.70.01 (PerkinElmer, Frankfurt/Main, Germany) was used to calculate the essential spectroscopic data. The spectra of the treated skin were corrected by subtracting the spectra obtained for the untreated skin, in order to suppress the influence of the absorption of the corneocyte aggregates and the proteins.

2.6.

Determination of the Penetration Profile

The determination of the penetration profiles has been described previously.²³ For this procedure, two points of information are necessary: the amount of corneocytes removed with each tape strip and the corresponding amount of sunscreen on the single removed tape strip.

2.6.1.

Determination of the stratum corneum amount

The pseudo-absorption of the corneocytes²⁶ based on the absorption, scattering, and reflection of the corneocytes is the basis for the quantification of the amount of stratum corneum fixed on the individual tape strips. Former investigations ensured that these spectroscopic data correlate to the mass of the horny layer particles, both of human skin^{23, 26} as well as of porcine skin,²⁷ with a correlation coefficient $⩾0.95$ up to 0.99. In addition, a clear relation was found between the pseudo-absorption and two protein-specific absorptions (the original absorption at $278\mathrm{nm}$ and the absorption after staining at $652\mathrm{nm}$ ),²⁸ confirming that the values used are well suited to quantify the stratum corneum removed by tape stripping.

The pseudo-absorption of the corneocytes must be measured at a wavelength not influenced by absorbing substances in the investigated systems. For the sunscreens, this was guaranteed at $430\mathrm{nm}$ . The measurements were carried out using the linear UV/VIS spectrometer Lambda 20 (PerkinElmer, Frankfurt/Main, Germany) with a quadratic beam diameter of $8\times 10{\mathrm{mm}}^2$ . The obtained data were used to calculate the relative position inside the horny layer (left axes in Figs. 1 and 2 ) from where the individual tape strips had been removed.

Fig. 1

Penetration profile of the UV filter octylmethoxycinnamate (Nivea Sun Feuchtigkeits-Sonnenmilch SPF 8) applied to human skin.

Fig. 2

Penetration profile of the UV filter octylmethoxycinnamate (Nivea Sun Feuchtigkeits-Sonnenmilch SPF 8) applied to porcine ear skin.

2.7.

Sum Transmission Spectra and Average Sum Transmission Values

In Figs. 3 and 4 , the determination of these spectra is demonstrated using individual measurements of two different sunscreens for volunteers and for porcine skin.

Fig. 3

Comparison of the sum transmission curves of the sunscreen Nivea Sun Feuchtigkeits-Sonnenmilch SPF 8 obtained for ex vivo measurements on human skin (upper diagram) and for ex vitro measurements on porcine ear skin (lower diagram).

Fig. 4

Comparison of the sum transmission curves of the sunscreen Nivea Feuchtigkeits Sonnen-milch SPF 20 obtained for ex vivo measurements on human skin (upper diagram) and for ex vitro measurements on porcine ear skin (lower diagram).

The uppermost spectrum in each diagram represents the situation of the first tape. The following spectra were calculated cumulatively by adding the values of all foregoing tape strips up to each individual tape strip. The lowest spectrum corresponds to the sum of the spectra of the tape strips 1 to 8 or 10 depending on the individual penetration profile.

The area under the last curve corresponds to the relative amount of UV photons reaching the deeper parts of the skin after sunscreen application. Dividing this area by 120—the length of the UV range $\left(280\mathrm{nm}\text{to}400\mathrm{nm}\right)$ —results in an average sum transmission value. This mean value characterizes the relative remaining average intensity of photons in the complete UV range after sunscreen application.

The discussion of the limitations of the described spectroscopic method comparing human and porcine skin needs to consider the different parameters influencing the measured values. There are two method-correlated parameters:

Another parameter influencing the individually measured values is a sample-correlated one. As described previously, the experimental results¹⁰ clearly demonstrate that the skin profile has a significant influence, resulting in a variation of the measured data.

An adjustment of these statistical parameters is reached by calculating the average values of the discussed data. Usually, the measurement of six individual samples is sufficient to adjust the existing differences. These parameters influence the results obtained with both skin types in the same extent.

2.8.

Universal Sun Protection Factor

In order to obtain a measured value with the same information as the classical SPF, a universal sun protection factor was proposed earlier.¹ A lowering of the incident irradiation intensity, for example, to 10%—described by the average sum transmission—lengthens the time to be exposed to sunlight without danger by a factor of 10. Dividing 100 by the average sum transmission value, the universal sun protection factor is determined ( $\mathrm{USPF}=100$ /average sum transmission). This value is independent of any biological response after UV irradiation and considers the UVB as well as the UVA range.

3.1.

Penetration Profiles

Penetration profiles were determined for all investigated sunscreens after application on human and porcine skin. In Figs. 1 and 2, two typical examples of penetration profiles of one typical sunscreen (Nivea Sun Feuchtigkeits-Sonnenmilch SPF 8 with the UV filter octylmethoxycinnamate) obtained on human and porcine skin are shown. Similar penetration profiles were obtained for the other investigated sunscreens. In all cases, the sunscreens were located in the upper layers of the stratum corneum, which is in agreement with findings reported in the literature for human skin.^{1, 26} Up to 10 tape strips were sufficient to remove the UV filter completely. The differences between human skin under in vivo conditions and porcine skin in vitro are not significant. There is a tendency to a slightly deeper penetration in some porcine skin samples, which can be explained by the larger orifices of the hair follicles.

The determination of the penetration profiles was a necessary step to obtain information about the extent to which porcine tissue is comparable with human skin when assessing the efficacy of sunscreens. No significant differences for the profiles obtained for both skin types were observed. This was the first requisite to take porcine skin as a model for human skin, quantifying the protective ability of not yet licensed sunscreens by spectroscopic data.

3.2.

Sum Transmission Spectra and Average Sum Transmission

The average sum transmission calculated on the basis of the sum transmission spectra quantifies objectively the protection efficacy of the applied sunscreens in the investigated systems. In Figs. 3 and 4, the sum transmission spectra are given for the sunscreens Nivea Sun Feuchtigkeits-Sonnenmilch SPF 8 and Nivea Sun Feuchtigkeits-Sonnenmilch SPF 20. The removed tape strips contain the complete amount of UV filter applied with each sunscreen. This allows quantifying the remaining radiation intensity after sunscreen application by the area under the last curve.

The typical examples of the sum transmission spectra obtained on human and porcine skin for two sunscreens show in an exemplary manner that no differences between the spectra of both skin types exist. This was the second requisite to demonstrate that porcine skin is a good tissue model to characterize sunscreen products by spectroscopic measurements.

3.3.

Universal Sun Protection Factor

The obtained spectroscopic data are well suited to calculate universal sun protection factors also on the basis of porcine skin. The USPF values were determined for each sunscreen on several porcine and human skin samples. The average values were determined for each sunscreen and both types of tissue. In Fig. 5, these USPF values obtained ex vivo on human skin and in vitro on porcine ear skin are compared for each sunscreen investigated. A total of 60 pig ear samples and 60 volunteers were analyzed. A good correlation with ${\mathrm{R}}^2=0.98$ was obtained between the USPF values determined on both tissue systems. The result clearly demonstrates that the described way to adjust the effects of the discussed parameters influencing the individual measurements is well suited to obtain data of sufficient accuracy. The relation of the ${\mathrm{USPF}}_{\text{porcine}}$ and ${\mathrm{USPF}}_{\text{human}}$ is described by the linear function:

Fig. 5

Comparison of the ${\mathrm{USPF}}_{\text{porcine}}$ determined in vitro from porcine ear skin and ${\mathrm{USPF}}_{\text{human}}$ determined ex vivo from human skin.

These relatively small differences obtained with the applied protocol are understandable, considering the parameters influencing the measured spectroscopic data. On the one hand, the porcine skin has broader furrows than human skin, resulting in a higher homogeneous distribution of the UV filter on the skin and with a higher protective ability $({\mathrm{USPF}}_{\text{porcine}}>{\mathrm{USPF}}_{\text{human}})$ .

On the other hand, the covering of corneocytes on the tape strips taken from porcine skin is lower, in comparison to human skin. This effect enhances the measured transmission, resulting in lower protection ability $({\mathrm{USPF}}_{\text{porcine}}<{\mathrm{USPF}}_{\text{human}})$ .

Both effects have an opposite influence on the USPF values, so that they partly compensate each other.

The results of the study confirm that in the considered field, in agreement with the general situation described previously,^{2, 3} porcine ears are best suited as model skin for the determination of the protection behavior of sunscreens applying the given protocol. This is of high importance for the investigation of newly developed UV filter substances, which are not licensed and, therefore, could not be evaluated because of customer safety aspects. The determination of the USPF allows determining their efficacy, as well as their behavior in different formulations during the process of the development and optimization of sunscreens. This possibility would increase the cost-effectiveness of the development and optimization process.

Assessing the obtained results, it must be taken into consideration that the USPF can be determined without irradiation of the skin by UV light, i.e., noninvasively, without damaging the human skin. In contrast to the traditional SPF, this allows the characterization of the protection efficacy of the sunscreen in the complete spectral ranges, UVB and UVA. It can even be applied for the determination of the protection properties of formulations in the visible and infrared parts of the spectrum. This is of special interest, because recently it was demonstrated that in these spectral ranges also, significant amounts of radicals were formed by sun irradiation.¹⁵ This means that the USPF method fulfils all the requirements of the European Commission (EC) and the FDA concerning the noninvasive protection assessment of sunscreens in the whole spectral range of sun irradiation, enlarged by the possibility to apply porcine skin for these investigations.