Chances and limits of an improved method to assess water resistance of cosmetic sunscreen products in vitro on polymethylmethacrylate plates

While sun protection factor (SPF) and UVA protection are the most important determinants of a cosmetic sunscreen product, water resistance is the third important feature. The Colipa in vivo method is the internationally accepted standard method to assess water resistance. It is time‐consuming and expensive. A screening method to quickly predict water resistance properties on low cost therefore is a specific request of product developers. Several in vitro screening methods are published but the predictive power of all these methods is limited. In this paper, we describe an adaptation of the in vitro UVA protection method of Colipa for a water resistance screening. Although the method is quick and most parts are standardized and approved by Colipa, the results were not in advantage of other published screening methods. Taking into account, the scatter of the results, seven of 16 sunscreen products that were developed as water resistant formulations could be unambiguously estimated to be water resistant by the in vivo water resistance screening method on five subjects while nine failed. In 10 of these 16 results, the in vitro SPF‐based method was in accordance with in vivo findings, whereas in six cases, the method was not predicting correctly. Based on these results, the authors recommend to use the in vitro screening methods to pre‐select from candidates which cannot all be tested in vivo. The pre‐selected products can be screened in the Colipa in vivo water resistance method with a reduced number of volunteers (usually 5) to predict water resistance. In case, the water resistance estimated in such an in vivo screening is found at about 65% or higher the study can be deemed successful and completed with further subjects to fulfil the Colipa requirements.     

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.     

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.     

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.     

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.     

Contact angle measurement - a reliable supportive method for screening water-resistance of ultraviolet-protecting products in vivo

Substantivity of sunscreen formulations is affected by the wash-out rate of ultraviolet-absorber and -reflector compounds in water. Water-resistance of sunscreen formulations is currently determined according to a standardized European Cosmetic Toiletry and Perfumery Association (COLIPA) protocol, encompassing the determination of a minimal erythemal dose before and after a defined immersion step in water. It can be supposed that the higher the wettability of a treated skin area, the higher is the wash-out rate of sunscreen compounds. This present report addresses the validity of determining the wettability of treated skin alone as a measure for the water-resistance of sunscreen products. The report addresses the robustness, accuracy and congruence of a recently developed wettability test, based on the measurement of the contact angle (CA) of a sessile water drop on treated skin areas. Contact angle data of 66 sunscreen formulations are compared with the corresponding results of 81 water-resistance tests, using the sun protection factor (SPF)/immersion/SPF method. Sunscreen products tested by the CA method were applied to the skin of the volar forearm of test subjects at a defined dose and drying-time, using a standardized application and recording device. Contact angles between a sessile water drop and skin were recorded by a Charge-Coupled Device (CCD) camera and subjected to automatic contour analysis. Taking the SPF/immersion/SPF method as gold standard, accuracy parameters of the CA method were determined. By using an appropriate cut-off level of CAs, the CA method has a specificity and positive-predictive value of 100%, and turns out to be a reliable screening method to identify water-resistant formulations. Based on our findings, those formulations that give CAs above 30 degrees may be categorized water-proof without further testing by the COLIPA water-resistance method.     

A National Web-Based Survey of Sunscreen Products as a Tool for Industry Self-Regulation, Consumer Awareness Campaigns and Marketing

The objective of the work was to test a web-based tool in the collection and presentation of data for our regular sunscreen surveys and to develop, test and conduct a web-based survey of sunscreen products, with instant data reporting. After the Photobiology Laboratory at MEDUNSA was approached by SurveyIT to pilot a new web-based data collection and reporting tool, companies were contacted via e-mail and invited to visit a secure web-based site to complete the provided questionnaire. Data collection and processing were greatly simplified by the new approach. The survey gleaned responses from 18 companies on 31 brands with a total of 110 primary sunscreen products. The data revealed a reduction in the number of companies marketing sunscreens in South Africa compared with the previous year, despite an increase in the volume of sunscreen sales. Ninety-eight percent of the products were claimed to be in vivo SPF tested. Fifty-seven percent of the products had undergone in vitro SPF testing for determination of their UVA protection. Fifty-one percent of the products were claimed to be water-resistant. Octyl methoxycinnamate was the most popular UV filter (88% of products), followed by benzophenone-3 (60%). Butyl methoxydibenzoylmethane was in 52% of the products and surprisingly only 29% of the products contained titanium dioxide. The major conclusions are that the data collection tool was a success and that sunscreens are becoming more widely used and more thoroughly tested in South Africa.     

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.     

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.     

Determination of sun protection factors in the hairless mouse

A laboratory method for determining sun protection factors (SPF) in the hairless mouse has been developed. The ability of the method to predict the SPF in man has been investigated using sunscreen ingredients and finished products with known activities in man. The results indicate that the hairless mouse can be used to assess sunscreen products although the SPF found appeared slightly higher than that determined in man.
Determination des facteurs de protection solaire sur la souris sans poils     

An In Vitro Digestibility Assay for Prediction of the Metabolizable Energy of Low-Calorie Dextrose Polymeric Bulking Agents

A method was developed to predict in vivo digestibility and metabolizable energy of dextrose polymers from in vitro enzyme digestibility measurements. A high correlation (r = 0.98) was established between in vivo true digestibility (TD) and in vitro percent dextrose yield (DY) for polymeric compounds representing a wide range of in vivo digestibility (22–96%). The linear regression equation describing this relationship was TD (%) = 1.11 DY + 10.68. The in vivo nitrogen-corrected true metabolizable energy (TMEn) of four test compounds was predicted from in vivo digestibility data with good accuracy using the linear regression equation TMEn (kcal/g) = 0.038 DY + 0.637 (r = 0.97). The in vitro method is most useful for predicting in vivo digestibility and metabolizable energy of dextrose polymers with in vitro dextrose yields less than 80% and in vivo metabolizable energy values less than 3.6 kcal/gram.     

RAPA: a novel in vitro method to evaluate anti‐bacterial skin cleansing products

Development of efficacious anti‐bacterial skin cleansing products has been limited by the availability of a pre‐clinical (in vitro) method to predict clinical efficacy adequately. We report a simple and rapid method, designated as rapid agar plate assay (RAPA), that uses the bacteriological agar surface as a surrogate substrate for skin and combines elements of two widely used in vivo (clinical) methods (Agar Patch and Cup Scrub). To simulate the washing of the human hand or forearm skin with the test product, trypticase soy agar plates were directly washed with the test product and rinsed under running tap water. After air‐drying the washed plates, test bacteria (Staphylococcus aureus or Escherichia coli) were applied and the plates were incubated at 37°C for 18–24 h. Using S. aureus as the test organism, anti‐bacterial bar soap containing triclocarbanilide showed a strong linear relationship (R² = 0.97) between bacterial dose and their per cent reduction. A similar dose‐response relationship (R² = 0.96) was observed for anti‐bacterial liquid hand soap against E. coli. RAPA was able to distinguish between anti‐bacterial products based on the nature and level of actives in them. In limited comparative tests, results obtained by RAPA were comparable with the results obtained by clinical agar patch and clinical cup scrub methods. In conclusion, RAPA provides a simple, rugged and reproducible in vitro method for testing the relative efficacy of anti‐bacterial skin cleansing products with a likelihood of comparable clinical efficacy. Further testing is warranted to improve the clinical predictability of this method.     

Reconstructed human epidermis as an efficient tool in the evaluation of the effects of UV irradiation and of the photoprotective capacities of a sunblock

In this study, we used a spectroscopic method, found effective in measuring sunscreen photo-degradation results in terms of a photoabsorption decrease. The traditional approach was to measure variations in well-known parameters such as the absolute in vitro sun protection factor (SPF) or the relative UVA/UVB ratio after a controlled dose of UV radiation. However, these parameters fail to fully reflect the product's photochemical behavior. Variation in the SPF mainly depicts changes in UVB filtration, and variation in the relative parameter UVA/UVB ratio can be subjected to misinterpretation, as is the case for products in which a parallel loss of photoabsorption is observed in the UVA and UVB regions. In our laboratory, we developed a new approach using pure spectroscopic UVA and UVB attenuation factors called UVA attenuation factor and UVB attenuation factor. Attenuation factors are defined here as the inverse of the arithmetic mean of the transmittance data, taken either from the UVB region (290–320 nm, 1 nm steps) or the UVA region (320–400 nm, 1 nm steps). Using these two new spectroscopic indices, the photochemical behavior of a sunscreen formula can be assessed in each UV region. The general procedure consists of applying two UV doses to a film of sunscreen previously spread on a roughened polymethylmethacrylate plate and then measuring the UV transmission versus wavelength before and after the irradiation process. We tested different UV filters in a cosmetic base to demonstrate the efficacy of the method.     

In vitro testing of zinc oxide sunscreens

The UVA performances of two all-mineral zinc oxide sunscreens are measured following Colipa and ISO procedures and compared to a sunscreen containing only organic actives. It is found that the two sunscreen types yield very different in vitro SPF and UVA results. It shown that the results can be rationalized in terms of the differences in the monochromatic extinction spectra of the two types of sunscreens.     

Validation of the performance of a GMO multiplex screening assay based on microarray detection

A new screening method for the detection and identification of GMO, based on the use of multiplex PCR followed by microarray, has been developed and is presented. The technology is based on the identification of quite ubiquitous GMO genetic target elements first amplified by PCR, followed by direct hybridisation of the amplicons on a predefined microarray (DualChip^® GMO, Eppendorf, Germany). The validation was performed within the framework of a European project (Co-Extra, contract no 007158) and in collaboration with 12 laboratories specialised in GMO detection. The present study reports the strategy and the results of an ISO complying validation of the method carried out through an inter-laboratory study. Sets of blind samples were provided consisting of DNA reference materials covering all the elements detectable by specific probes present on the array. The GMO concentrations varied from 1% down to 0.045%. In addition, a mixture of two GMO events (0.1% RRS diluted in 100% TOPAS19/2) was incorporated in the study to test the robustness of the assay in extreme conditions. Data were processed according to ISO 5725 standard. The method was evaluated with predefined performance criteria with respect to the EC CRL method acceptance criteria. The overall method performance met the acceptance criteria; in particular, the results showed that the method is suitable for the detection of the different target elements at 0.1% concentration of GMO with a 95% accuracy rate. This collaborative trial showed that the method can be considered as fit for the purpose of screening with respect to its intra- and inter-laboratory accuracy. The results demonstrated the validity of combining multiplex PCR with array detection as provided by the DualChip^® GMO (Eppendorf, Germany) for the screening of GMO. The results showed that the technology is robust, practical and suitable as a screening tool.     

Research on a natural sunscreen from Chinese herbs

To compare the difference in SPF values between a synthetic sunscreen and a sunscreen derived from Chinese herbs, by using optical and biological methods, we observed the effects of these sunscreens by photochemical reaction, sun protection factor (SPF) values, and UV absorption. The results showed that the synthetic sunscreen was more effective in UV absorption than the sunscreen derived from natural Chinese herbs. When they were tested biologically, it was found that the sunscreen derived from Chinese herbs was able to improve skin tonicity, as well as reduce the itching, photoerythema, squamation, burning, and reddening caused by the excessive UV radiation, while the synthetic sunscreen could not improve these conditions of the skin, and even induced some side-effects in the skin with photoerythema. By comparing the in vivo SPF values of the two types of sunscreen, they were found to be mainly similar. The results indicate that the SPF value was related to both a photo-reaction and a biological reaction. The sunscreen derived from natural Chinese herbs, although having low UV absorption, was able to exhibit the same SPF value as the synthetic sunscreen because of its biological effects.     

Effects of water-soluble soybean polysaccharide on rheological properties,stability and lipid digestibility of oil-in-water emulsion during in vitro gastrointestinal digestion

Water-soluble soybean polysaccharide (SSPS) is a naturally occurring emulsifier. SSPS was used as the sole emulsifier to stabilize an oil-in-water (O/W) emulsion. The effects were investigated of different SSPS concentrations (3–20% (w/w)) on the lipid digestibility, rheological properties and stability of O/W emulsions during in vitro digestion model. The droplet size of the emulsions tended to increase during the oral phase because the emulsions were unstable and droplets coalesced, except with a SSPS concentration of 20% (w/w). The presence of SSPS markedly reduced the free fatty acid (FFA) content after its stabilized O/W emulsion passed through in vitro gastrointestinal digestion. The amount of FFA significantly decreased as the concentration of SSPS increased due to SSPS stabilization film on oil droplet surface and high viscous system. SSPS may be an attractive alternative ingredient to control the lipid digestibility of emulsions for various food products.     

Comparison of the sequestering properties of yeast cell wall extract and hydrated sodium calcium aluminosilicate in three in vitro models accounting for the animal physiological bioavailability of zearalenone

The sequestration/inactivation of the oestrogenic mycotoxin zearalenone (ZEA) by two adsorbents – yeast cell wall extract (YCW) and hydrated sodium calcium aluminosilicate (HSCAS) – was studied in three laboratory models: (1) an in vitro model was adapted from referenced methods to test for the sequestrant sorption capabilities under buffer conditions at two pH values using liquid chromatography coupled to a fluorescence detector for toxin quantification; (2) a second in vitro model was used to evaluate the sequestrant sorption stability according to pH variations and using ³H-labelled ZEA at low toxin concentration; and (3) an original, ex vivo Ussing chamber model was developed to further understand the transfer of ZEA through intestinal tissue and the impact of each sequestrant on the mycotoxin bioavailability of ³H-labelled ZEA. YCW was a more efficient ZEA adsorbent than HSCAS in all three models, except under very acidic conditions (pH 2.5 or 3.0). The Ussing chamber model offered a novel, ex vivo, alternative method for understanding the effect of sequestrant on the bioavailability of ZEA. The results showed that compared with HSCAS, YCW was more efficient in sequestering ZEA and that it reduced the accumulation of ZEA in the intestinal tissue by 40% (p < 0.001).     

In Vitro Method for Quantification of the Fermentation of Starch by Human Faecal Bacteria

The major actions of undigested carbohydrates, such as non-starch polysaccharides and resistant starch, on the physiology of man are related to colonic fermentation. It is very difficult to study fermentation in vivo . In vitro models are often used but these models differ in almost every aspect even when only the simplest batch cultures of human faeces are considered. Factors such as composition of the medium, buffering capacity, inoculum concentration, and fermentation time vary considerably making direct comparison of the results from different studies impossible. The authors have devised a standard in vitro fermentation method for resistant starch and tested it in eight laboratories with a total of 40 individual faecal inocula. This simple method was well received and taking biological variation into account yielded comparable results for 24 h short chain fatty acid production and residual starch in all but one laboratory.     

Validation of a Loop-Mediated Amplification/ISO 6579-Based Method for Analysing Soya Meal for the Presence of <Emphasis Type="Italic">Salmonella enterica</Emphasis>

An alternative method for detection of Salmonella spp. in animal feed, based on the use of loop-mediated amplification (LAMP) in conjunction with a standard culturing procedure, was compared with the standard ISO 6579 as reference method, using soya meal as the test matrix. In the method comparison study, the sensitivities for both the alternative and reference methods were 100 %. The relative level of detection was 1.000. Tested against 100 Salmonella and 30 non-Salmonella strains, the LAMP-based method was 99 % inclusive and 100 % exclusive. The interlaboratory trial involved ten laboratories from eight European countries, testing eight samples at three contamination levels: 0 cfu/100 g, 1–5 cfu/100 g and 14–68 cfu/100 g. The trial specificity, or percentage correct identification of uncontaminated samples, was 96.3 % for both the reference methods and the LAMP/ISO 6579 alternative method, thus demonstrating its suitability for adoption as a procedure for rapid identification of Salmonella uncontaminated soya meal.