In vitro sun protection factor: still a challenge with no final answer.

In the past, several attempts have been made to develop in vitro methods for determining protection against UV radiation. To date however, there is no broadly accepted method. Various known and unknown parameters influence the transmission measurements of scattering films, such as the multifaceted compositions of sunscreens, the technical limitations of measurement devices as well as the difficulty to apply very thin films of sunscreen in a reproducible manner throughout different laboratories. In vitro data were measured in this multicenter study to compare possible methodologies and strategies for an in vitro approach to the sun protection factor (SPF). This publication will not present a final in vitro SPF test method, but it will point out which technical side effects may influence such a method. Influential factors such as the quality of spectrophotometer used, the amount of product applied, pretreatment of samples, time and temperature of equilibration, size of the measured surface, the application process or the calculation on the basis of standardized data are presented and discussed. Finally, a reduction of the standard deviations within single laboratories could be realized for in vitro SPF testing, but no improvement of the interlaboratory comparison was obtained. The development of a valid and reliable SPF in vitro test still remains a challenge, and further work is necessary to develop a satisfactory method.

The missing link--light-induced (280-1,600 nm) free radical formation in human skin.

The recent European recommendation on the efficacy of sunscreen products requests now a minimum ratio of UVA/UVB protection. However, the visible and the infrared (IR) parts of the sun spectrum have received little attention concerning their possible contribution to skin damage. A common biophysical answer for the different wavelengths of the sun spectrum can be found in the creation of excess free radicals - mainly reactive oxygen species (ROS). Thanks to electron spin resonance spectroscopy applied to skin biopsies, we determined for the first time the free radical action spectrum covering UV and visible light (280-700 nm). Convolution of the action spectrum with sunlight spectral irradiance showed that 50% of the total skin oxidative burden was generated by visible light. Creation of ROS by visible light was experimentally confirmed by varying the illuminance of a spotlight. We also evidenced the creation of excess free radicals by near-IR radiation. In that case, free radical generation does not depend exclusively on the dose, but also on the skin temperature increase initiated by near-IR light. Some phenomena which are still unclear, such as the question about the deleterious or beneficial role of sunlight, are reviewed, implying the research on new protection strategies for the prevention of skin cancer.

[UV, visible and infrared light. Which wavelengths produce oxidative stress in human skin?]. / UV, sichtbares Licht, Infrarot. Welche Wellenlängen produzieren oxidativen Stress in menschlicher Haut?

Experimental evidence suggests that the creation of free radicals--mainly reactive oxygen species (ROS)--is the common photobiological answer to the skin-sunlight interaction. The free radical action spectrum (wavelength dependency) for ultraviolet and visible light (280-700 nm) has been determined by quantitative ESR spectroscopy. Visible light produces around 50% of the total oxidative stress caused by sunlight. Reactive species like *O(-)(2), *OH and *CHR are generated by visible light. The amount of ROS correlates with the visible light intensity (illuminance). We demonstrated the creation of excess free radicals by near-infrared light (NIR, 700-1600 nm). Free radical generation does not depend exclusively on the NIR irradiance, but also on the NIR initiated skin temperature increase. The temperature dependence follows the physiological fever curve. Our results indicate that the complex biological system skin creates the same type of free radicals over the entire active solar spectrum. This general response will make it possible to define the beneficial or deleterious action of sunlight on human skin by introduction of a free radical threshold value.

Multicenter methodology comparison of the FDA and ISO standard for measurement of in vitro UVA protection of sunscreen products.

In vitro standard methods are available and accepted worldwide to assess UVA protection of sunscreen products. Though, harmonisation of methods has made progress in the last decade, still two differing methods - one by FDA the other by ISO - are in use. In a multicentre study including 9 centres in Germany, 4 different commercial sunscreen products were assessed using both methods to discover their similarities and differences. UVA protection factor and Critical Wavelength were detected at various substrate type (sandblasted versus moulded PMMA plates), at different surface roughness of the plates as well as at different product application dose using two different irradiation spectra. Results: The strongest influence on UVA protection factor results from the surface roughness of the plates. Depending on the roughness (accepted range of 2 to 7 µm in the FDA method) a variability in the UVA protection factor of up to 25% was observed, while the much narrower definition of plate roughness by ISO (4.5 to 5.2 µm) had no relevant influence on the test results. Sandblasted plates in our assessment led to higher UVA protection factors and produced less scattered results compared to moulded plates. These differences were not pronounced. Application dose and spectra of the irradiation source were of negligible influence on UVA protection factor results for the investigated UV-filter combinations. The UVA protection factor which is the endpoint of the ISO method was found to be a parameter with a high potential to differentiate among different test products. The endpoint of the FDA method - the Critical Wavelength - was found to be an unambitious endpoint. Insensitivity to all described modifications of the method was observed. All investigated products performed similar and passed the Critical Wavelength criteria independent of method and parameters.

In vitro assessment of water resistance of sun care products: a reproducible and optimized in vitro test method.

The aim of the study was to develop a simple reproducible and reliable in vitro water resistance (WR) method to assess the sun care products. This paper is the result of a scientific collaboration between seven different international industrial laboratories and testing institutes. The same group has already achieved an in vitro protocol for the sun protection factor (SPF) determination [1]. The in vitro WR of sunscreens was tested by applying the same principle as in vivo, which determines the percentage of retention of sunscreen products by assessing the SPF before and after water immersion. Special care was taken to study the parameters influencing the WR and the possibility to follow the kinetics of sunscreen retention during water immersion. The influence of different water qualities has been tested, and osmosed water (1-3 microS cm(-1)) was chosen for the main ring study. Measurement was carried out after 5, 20 and 40 min of immersion. Histograms of selected products demonstrate the percentage of WR at all measuring times and centres, and the regression coefficient to the in vivo determination was shown and statistical calculations clearly demonstrate the reproducibility of the results between the different evaluation centres. The presented method is a practical, convenient and relevant tool for WR screening of sun care and skin care products. It even has the potential to be the starting point for the replacement of the in vivo method in future.

Comparison of sun protection factors determined by an in vivo and different in vitro methodologies: a study with 58 different commercially available sunscreen products.

An extensive study on the sun protection factors (SPF) of sun care products was carried out using the COLIPA (The European Cosmetic Toiletry and Perfumery Association) method, which relates to in vivo experiments. Furthermore, in vitro methods were tested with sunscreen formulations that were prepared as films on surface-roughened plates of polymethyl methacrylate (PMMA). One of the in vitro methods, i.e. using the sunscreen tester, has been recently developed, whereas the second has been defined by a pure spectroscopic approach, which is based on spectral transmission measurements of sunscreen films. Altogether 58 different sunscreen formulations, with manufacturer declared SPF values ranging from 4 to 60 and currently available on the European market, were investigated. The quality of correlations with results from the individual products based on the different in vitro methods versus the COLIPA values that were considered as generally accepted standard values was assessed. In this context, also variations because of sample preparation and spectral measurement were discussed. For sunscreen products with in vivo SPF values larger 25, the spectral transmittance within the UVA/UVB range is rapidly decreasing, which is experienced even for products with reduced amounts reaching 0.5 mg cm(-2) and still leading to unsatisfactory correlation of the spectroscopically derived SPF values versus the results from the alternative assays. Opposite to these small amounts, a sunscreen product spread of 2 mg cm(-2) is standard for the in vivo COLIPA method, whereas an area-normalized amount of 1 mg cm(-2) is currently routinely used for the sunscreen tester method. Furthermore, an overview of the individual product characteristics, such as their specific critical wavelengths and their UVA/UVB ratios is provided; both parameters can also be calculated from the spectral absorbances of the standardized sunscreen films.

In vitro testing to assess the UVA protection performance of sun care products.

The UVA protection delivered by sunscreens is an issue of increasing importance due to the increasing knowledge about UVA-induced skin damage. In Europe there is no officially accepted method available to determine the degree of UVA protection. Therefore, the objective of the present study was to design a protocol combining the merits of an in vitro model, which are simple and reproducible, with aspects known to be relevant from in vivo studies. The principle is: an UV-transparent support to which the test product is applied, a (pre)irradiation and a transmission measurement. Transpore(R) tape (standard support for SPF determinations) was found to be incompatible with many preparations on prolonged contact times. Roughened quartz was adopted as a suitable alternative. Transmission measurements on this support are not reliable with a layer of 2 mg cm(-2) (standard for SPF) due to detection limitations of spectrophotometers, hence a reduced layer of 0.75 mg cm(-2) was adopted. Overall, it is very difficult to apply products in a reproducible thin layer on appropriate substrates. As a consequence, absolute parameters derived from the transmission profile show relatively large dispersion, whereas relative parameters, such as critical wavelength lambda(c)[1] or UVA/UVB ratio are much less sensitive to unavoidable variations in layer thickness. An increase in deviations was observed when the samples were irradiated before measurement. It is crucial to control the output carefully (spectral distribution and even more importantly, irradiance and dose delivered) of the light source. By doing so and also taking into account the previous learning steps, a protocol was drafted and tested in a ringtest (four samples in six laboratories). The results are encouraging and show that if relative parameters (e.g. lambda(c), UVA/UVB ratio) are considered, the intra- as well as interlaboratory reproducibility is clearly better than can be obtained in vivo. In general, we describe a suitable method, which can be considered in any future official discussions about the methodology to determine UVA protection.

Influence of applied quantity of sunscreen products on the sun protection factor--a multicenter study organized by the DGK Task Force Sun Protection.

It is often debated that the protection against solar-induced erythema under real conditions is dependent upon the amount of sunscreen applied. It is believed that when too little is applied a lower sun protection than indicated on the label will result. The aim of this study was to quantify this effect. In this multicenter study, the influence of three different amounts (0.5, 1.0, 2.0 mg/cm(2)) of three commercial sunscreen products in three reliable test centers was investigated according to the test protocol of The International Sun Protection Factor Test Method. The main result was a linear dependence of the SPF on the quantity applied. Taking into consideration the volunteer-specific variations, an exponential dependence of confidence interval of the in vivo SPF and amount applied was found. The highest amount applied (2.0 mg/cm(2)) was linked to the lowest confidence intervals. Thus, from the point of view of producing reliable and reproducible in vivo results under laboratory conditions, the recommendation of this multicenter study is an application quantity of 2.0 mg/cm(2).