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.     

Collaborative development of a sun protection factor test method: a proposed European Standard. COLIPA Task Force 'Sun Protection Measurement', Europe

Standardization of the method to determine sun protection factors (SPF) is fundamental to uniform labelling of SPF values on sun products. The COLIPA Task Force 'Sun Protection Measurement'(SPM), including representatives of major European sun-product manufacturers and contract testing laboratories, was established in 1990 to define methods for sun products testing. The process involved in developing the COLlPA SPF Test Method started with a critical appraisal of previously existing methods, and identification of areas for improvement. Experiments were performed by the participating laboratories to establish the new recommended test protocol which was then confirmed in two multicentred ring tests. Improvements to test methodology included the number and selection of volunteers and skin types, the application of the product, the definition of W output from solar simulators, and the method for assessing erythemal response (MED) including an option to use colorimetry to define objectively the 'erythemal threshold' of skin and to predetermine MED prior to exposure. Four cosmetic formulations and neutral density physical filters (SPF from 4 to 20) were tested in two ring tests. Results showed that variations in SPF of the cosmetic products were, on average, directly proportional to SPF value. An excellent correlation was found between visual and colorimetric SPF assessments (r = 0.99). An acceptable range of values for low and high SPF standard products was also established. The COLIPA SPF test method is now fully defined and supported by experimental data. Its use will harmonize SPF testing throughout Europe and help in the quest for global harmonization in testing sun products.     

Adaptation of the protocol for determining in vitro the sun protection factor of anti-solar sticks

Apart from the protection offered by clothing, the application of sunscreen products suited to each type of skin constitutes one way for decreasing the frequency of skin cancers nowadays. After having adapted an in vitro method for determining the efficacy of sunscreens in emulsion form, we wished to transpose this technique by adapting it for the anti-solar sticks for the evaluation of sun protection factor (SPF) using a spectrophotometer equipped with an integrating sphere. To do this, we tested 14 products in the market as well as sticks that we ourselves fabricated in the laboratory. In a base common to all of these sticks, we added organic (13 filters tested) and inorganic (two filters tested, titanium dioxide and zinc oxide) to their maximum permitted concentration in the European Union. In parallel, emulsions containing the same filters at the same percentage of use were studied; to be in keeping with the results on the products packaging on the one hand, and with the results obtained for the emulsion form on the other hand, we were able to determine the optimal mass which needed to be placed on the support used the in vitro test to determine the SPF.     

The revised COLIPA in vitro UVA method

A multicentred study derived from the COLIPA in vitro UVA method was performed to assess the influence of test conditions on UVA protection factor (UVAPF) values in terms of amplitude, reproducibility between laboratories and correlation with in vivo UVA results. Eight products with a range of in vivo UVAPF from three to 29 were used. Two different types of plates, namely high‐roughness (5 μm) and low‐roughness (2 μm) plates, were used with a different application rate for each (1.3 mg cm^?2 and 0.75 mg cm^?2 respectively). The UVR dose applied to both plate types followed the same principle as the original test (1.2 J. cm^?2 × UVAPF0). Strong, significant correlations between in vitro and in vivo UVAPF values were observed for both plate types (Pearson correlation > 0.9, P ≤ 0.01). The correlation and slope obtained with the low‐roughness plates confirmed the previous results obtained by COLIPA. Across all laboratories, higher UVAPF values were obtained on the high‐roughness plates (P < 0.01). Reproducibility of UVAPF values between laboratories was comparable between the two plate roughness values (low roughness, COV = 8%; high roughness, COV = 12%). Considering the in vitro/in vivo comparisons, a regression slope of 0.83 was observed for the low‐roughness plates, in comparison with a value of 1.05 for the high‐roughness plates. The accuracy of the method was improved, therefore, with the use of the high‐roughness plates. With a constraint to recommend the use of only one plate type in the COLIPA UVA in vitro Test, the high‐roughness plate was selected on an on‐going basis to limit variability of results and to provide better accuracy with in vivo data.     

Quartz plates for determining sun protection in vitro and testing photostability of commercial sunscreens

Testing plate made of optical quartz has been developed for the purpose of determination of sun protection factor (SPF) _in vitro by the method of diffusion transmission spectroscopy; the plates were coarsened by sanding and grinding to surface roughness values (Ra) of 18 μm. The plate was coated with a film of sunscreen by an application of 2 mg cm⁻² as that used for determination of SPF _in vivo by the COLIPA method. The transmission values measured were converted into the SPF _in vitro and the protection factor in ultraviolet A light, UVAPF _{in vitro} . The testing plate was tested with commercial sunscreens. The found values of SPF _in vitro fit well with the values determined by means of the COLIPA method in vivo . The plates coated with sunscreen film were irradiated with light simulating the sun radiation. The values of protection factors obtained before and after irradiation were compared, and the differences were used for estimation of photostability of the UV filters included.     

Determination of sun protection factors. Correlation between in vivo human studies and an in vitro skin cast method

Sun protection factors were determined by both an in vitro method which used resin casts taken from replicas of human skin and by an in vivo SPF method. Thirty-eight product development samples were tested for the level of sun protection using both methods and the results were compared. The values obtained showed a positive relationship which was closely approximated by a log-linear regression of in vivo data on in vitro data (regression coefficient, r² = 0.86).
It is concluded that the cast technique is quick, convenient, inexpensive and in its present form useful for screening sunscreen products prior to in vivo SPF testing.     

Importance of sunscreen products spreading protocol and substrate roughness for in vitro sun protection factor assessment

The purpose of this study was to evaluate the impact of substrate roughness and of product spreading method on in vitro sun protection factor (SPF) measurement and to define the experimental conditions most appropriate to reach the best level of correlation to in vivo SPF. In vitro SPF assessment was carried out on 13 products (including different formulation types with SPF from 20 to 75) using various in vitro SPF protocols and comparing related predictive potential regarding in vivo SPF. In the first part, two spreading methods were compared on two types of PMMA (Polymethyl methacrylate plate with different roughness. The impact of a second spreading step after product drying was also evaluated. From the various investigated parameters, it was shown that (i) a higher roughness (Ra = 4, 5 μm) was preferred for O/W formulations (ii) using a defined sequence of light linear and circular strokes was more adequate than monitoring product spreading in terms of time and pressure (iii) both correlation to in vivo SPF and results variability were improved when a second spreading step was added. The altered protocol showed a good predictive potential regarding in vivo SPF values for O/W formulations (correlation coefficient 0.92, correlation curve slope 0.98) and coefficient of variation of in vitro results (14% of the mean SPF value) close to what is usually obtained in vivo . The repeatability of the protocol was also demonstrated. In the second part, we evaluated the impact of PMMA plate pre-treatment with paraffinum liquidum before spreading the product to get a better correlation between in vivo and in vitro SPF values for W/O formulations. This allowed us to define a protocol suitable for both O/W and W/O formulations.     

In vitro and in vivo determinations of sun protection factors of sunscreen lotions with octylmethoxycinnamate

The aim of this work was to evaluate the correlation between in vitro and in vivo determinations of sun protection factors (SPF) of sunscreen lotions containing the synthetic sunscreen octyl methoxycinnamate. Three sunscreen lotions containing either 2, 4.5 or 7.5% octyl methoxycinnamate were prepared. The in vitro SPFs of these sunscreen lotions were determined according to the spectrophotometric method of Mansur et al. The in vivo SPF values were determined according to the Food and Drug Administration (FDA) method. The results indicate that there was a good correlation between the in vitro and in vivo determinations for the sunscreen lotions examined.     

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.     

The COLIPA in vitro UVA method: a standard and reproducible measure of sunscreen UVA protection

There is a continuing need to measure and communicate reliably the UVA protection offered by commercial sunscreens. To that end, the COLIPA (European Cosmetics Trade Association) ' In Vitro Sun Protection Methods' group has developed a new in vitro method for measuring UVA protection in a standardized, reproducible manner. The method is based on in vitro UV substrate spectrophotometry and convolution of resulting absorbance data with the action spectrum for the in vivo Persistent Pigment Darkening (PPD) endpoint to provide an in vitro UVA protection factor (UVAPF) which is correlated with an in vivo measure. This method has been published as a COLIPA guideline, used currently in European geographies for testing and labelling sunscreen products.
This article summarizes two 'ring' studies, involving eight separate testing laboratories, which both defined critical parameters for the method and validated it. In Ring Study 1, eight laboratories tested the in vitro UV transmission of a total of 24 sunscreens and, from the data, a unit dose of UVA ( D ₀ of 1.2 J cm⁻²) was defined to provide a single irradiation step which, by taking into account potential sunscreen photo-instability, gave the closest agreement with in vivo UVAPF values. In Ring Study 2, eight laboratories tested the in vitro UV transmission of a total of 13 sunscreens using this single irradiation step and established a very good correlation ( r ² = 0.83; slope = 0.84, P  < 0.0001) between resulting in vitro UVAPF values and corresponding values derived from the in vivo PPD method. This new method, therefore, can be used to provide a reliable in vitro metric to describe and label UVA efficacy in sunscreen products, in line with the EU Commission recommendation 2006/247/EC.     

In vivo persistent pigment darkening method: proposal of a new standard product for UVA protection factor determination

The European Commission (EC) has recommended assessing the level of ultraviolet A (UVA) protection afforded by sunscreen products using the in vivo persistent pigment darkening (PPD) method or other methods giving equivalent results. In this context, the reproducibility of the in vivo PPD method is of importance. To check the validity of the UVA protection factor (UVAPF) tests, the Japanese Cosmetic Industry Association (JCIA) recommends using a standard product (JCIA standard) with an expected UVAPF 3.75 (SD 1.01). However, considering the increase in UVA efficacy of the new sunscreen products available in the market, with UVAPF up to 30, it seemed useful to develop a new standard product to be used when testing products with expected UVAPF > or =10. The PPD method was used in six centres to determine the UVAPF of the two products. Reproducibility of results was also studied by testing two batches of the new product at two different times. There was no statistical difference between the six centres with regard to the JCIA standard. The ring study showed that the mean value of UVAPF (4.3) was higher than that given by JCIA (3.75). These data enable the proposal of a new acceptance range for the JCIA standard product (3.4-5.2) derived from actual results from European laboratories. Whereas this range is different from that proposed by JCIA (2.74-4.76), there is an overlapping of the values. Data on the new standard product show that reproducibility is not influenced by the batches of this product. The mean UVAPF value obtained is 12.1. An acceptance range (9.6-14.6) is proposed for the new standard. Data presented here demonstrate that if an identical protocol is used, reproducible results can be expected and that the PPD method is reproducible and reliable.     

Comparative evaluation of different substrates for the in vitro determination of sunscreen photostability: spectrophotometric and HPLC analyses

Polymethylmethacrylate (PMMA) plates and Transpore^TM tapes were compared as substrates for the in vitro evaluation of photostability of commercial sunscreen products. The sun care preparations were applied respectively on Transpore^TM tapes and PMMA plates and their sun protection factors (SPF) and UVA protection parameters [UVA/UVB ratio, critical wavelength, UVA protection factor (UVA‐PF)] were measured by transmission spectroscopy, before and after irradiation with simulated sunlight. No significant differences were observed in the UV protection parameters measured on Transpore^TM tapes or PMMA plates, before exposure to the solar simulator. Conversely, after irradiation, the SPF values of the sun care products exhibited marked variations between the two substrates, the decrease in SPF being greater on PMMA plates (31.3–63.1%) than on Transpore^TM tapes (10.4–23.8%). Differences between the two substrates were detected also for the UVA protection parameters, although they were significant only for the UVA‐PF. The tested samples were assayed also by high‐performance liquid chromatography (HPLC) to assess the extent of photodegradation of the UV filters present in the examined formulations. The results showed that for the PMMA plates, the light‐induced decrease in SPF, as determined by spectrophotometry, fitted well with the percentage loss of ethyl hexyl methoxycinnamate (the only photounstable UVB filter present) measured by HPLC. Moreover, for the PMMA substrate, the UVA‐PF percentage reduction was consistent with the percentage degradation of butyl methoxydibenzoylmethane (the only photounstable UVA filter present) determined by HPLC. On the other hand, poor correlation between spectrophotometric and HPLC analyses was observed on Transpore^TM tapes. Therefore, PMMA plates are more reliable than Transpore^TM tapes as substrates for in vitro photodegradation tests of sunscreen products by transmission spectroscopy.     

Evaluation of a multiplex-PCR detection in combination with an isolation method for STEC O26, O103, O111, O145 and sorbitol fermenting O157 in food

The aim of the current study was to evaluate a multiplex PCR (mPCR) detection test combined with the evaluation of a previously described isolation method.Minced beef, raw-milk cheese and sprouted seed samples were inoculated with low amounts (7-58 cfu 25 g⁻¹) of non-stressed, cold-stressed or freeze-stressed clinical STEC strains, including serogroups O26, O103, O111, O145, sorbitol fermenting (SF) O157 and non-sorbitol fermenting (NSF) O157. The inoculated pathogen was detected using a 24 h-enrichment followed by an mPCR protocol, and in parallel isolated using an enrichment step of 6 and 24 h, followed by selective plating of the enriched broth and selective plating of the immunomagnetic separation (IMS) product. Recovery results were evaluated and compared.Successful mPCR detection and isolation was obtained for non-stressed and cold-stressed STEC cells in minced beef and raw-milk cheese samples, except for serogroups O111 and SF O157. For freeze-stressed cells and sprouted seed samples, false negatives were often found. Isolation was better after 24 h-enrichment compared to 6 h-enrichment. IMS improved in some cases the isolation of non-stressed and cold-stressed cells belonging to serogroups O111 and O157 from minced beef and raw-milk cheese and freeze-stressed cells of all tested serogroups from minced beef.