J. Cosmet. Sci., 59, 385–398 (September/October 2008)Evaluation of liposomal and conventional formulations of octyl methoxycinnamate on human percutaneous absorption using the stripping method

The objective of this study was to determine the influence of vehicles on the penetration of octyl methoxycinnamate (OMC), as a UV absorber, to the stratum corneum by the stripping method. The experimental formulations consisted of a conventional o/w emulsion and multilamellar and small unilamellar liposomes (MLVs and SUVs) containing OMC. MLVs containing OMC were prepared by the fusion method and then converted to SUVs by probe sonication. Various formulations were then applied onto the midvolar forearms of six volunteers at a dose of 2 mg cm^-2. After determined timepoints, the stripping method was conducted whereby 22 tape strips were applied and subsequently divided into different stripping groups. The sunscreen agent was assessed by HPLC while the SPF (sun protection factor) of the formulations was determined in human volunteers in accordance with the Australian standard. Overall the results indicate that skin accumulation of OMC in MLVs was significantly greater than in the o/w emulsion and SUVs. Furthermore, SUV's penetration into the deeper skin layers was significantly greater than MLV's and that of a conventional o/w emulsion. Also, higher amounts of OMC were recovered from the upper layers of the stratum corneum than from the deeper layers in all the formulations tested. Finally, the SPF of the liposomes containing OMC was slightly greater than that of the control lotions at a similar concentration of OMC. In conclusion, the result of this study indicates that an MLV prepared by the fusion method could be a better vehicle for OMC as a sunscreen since it has a slightly better SPF compared to a conventional formulation and more remains in the stratum corneum, reducing its penetration to the deeper layers.
Address all correspondence to M. R. Jaafari.     

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.     

Sunscreens' photochemical behaviour: in vivo evaluation by the stripping method

This study shows the influence of a water-in-oil emulsion and an oil-in-water emulsion on the photochemical behaviour of four sunscreen preparations containing various amounts of three UV filters: octyl methoxycinnamate, butyl methoxydibenzoylmethane, and 4-methylbenzylidene camphor. With the stripping method in vivo, skin-product interactions were evaluated after topical application and ultra-violet exposure. Evaluations were also made under the same operating conditions as in determining sun protection factor in vivo. Results showed that each sunscreen preparation has a particular photochemical behaviour related to filter mixture and emulsion type. When developing a sunscreen preparation the choice of the emulsion type and filters is relevant for achieving optimum efficacy and spectral stability.     

Validation of a high performance liquid chromatography method for sunscreen determination in cosmetics

The aim of this research was the development and validation of a high performance liquid chromatography (HPLC) method for the simultaneous and quantitative determination of benzophenone-3, octyl methoxycinnamate and octyl salicylate contained in sunscreen emulsions. The separation and quantitative determination was achieved using a LiChrospher((R)) 100 RP-18 (5 microm) Merck column, a mobile phase constituted of methanol/water (85/15, v/v), a flow-rate of 1.0 mL min(-1) and UV detection at 310 nm. The correlation coefficients and percentage of recovery for benzophenone-3, octyl methoxycinnamate and octyl salicylate were 0.9999 and 99.46%; 0.9995 and 98.85%; 0.9998 and 98.84%, respectively. The relative standard deviations (RSD) for commercial samples were between 0.50 and 0.70%.     

In vitro evaluation of sun protection factors of sunscreen agents using a novel UV spectrophotometric technique

A method for the in vitro determination of low- and high-value sun protection factors (SPF) of sunscreens using artificial substrates and a novel pseudo double beam (PDB) mode of operation of a standard double beam UV spectrophotometer is described. The method allows transmittance to be calculated from detector responses of reference and sample beams measured at different gain levels and facilitates the accurate quantification of low levels of electromagnetic radiation transmitted through highly absorbing samples. The spectrophotometer was modified to hold quartz diffusing plates on which a substrate [Transpore™ adhesive tape or human stratum corneum obtained from a skin surface biopsy (SSB)] and the sunscreens to be tested were applied. The PDB mode of operation increased the effective linear range of the detector response of the spectrophotometer by a factor of approximately 20000-fold, enabling the in vitro SPF determination technique to be applied to both high and low SPF value sunscreens. Eight commercial sunscreens with known SPF values ranging from 4 to 77, previously determined by in vivo methods, were tested in vitro using both test substrates and correlations between the in vivo and in vitro values were determined. SPF values determined using the in vitro method correlated well with the known in vivo results (Transpore™ tape, R ² = 0.611; SSB, R ² = 0.7928). The in vitro SPF obtained for one of the tested products differed substantially from the cited in vivo SPF value. Independent in vitro and in vivo re-evaluation of the SPF of this product matched the value predicted by the present method much more closely than the originally cited in vivo value. All determined SPFs were ordered correctly in comparison to in vivo ranking and the technique appeared to correctly identify a sunscreen that had a labelled SPF value that was significantly higher than its true SPF.     

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.     

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.     

Glyceridic esters of p-methoxycinnamic acid. A new sunscreen of the cinnamate class

Synopsis
The aim of this work was to investigate whether a 1,3-diacylglycerol moiety can act as enhancer of the photoprotecting action of a new sunscreen of the cinnamate class, owing to its affinity with skin constituents and lower susceptibility to hydrolysis at C-2. The sun protection factors (SPF) for 1,2,3-propanetriol 1,3-dipalmitate-2- p -methoxycinnamate and 1,2,3-propanetriol 1,3-dioctanoate-2- p -methoxy-cinnamate (2.0, 4.5 and 7.5%) were determined. The sunscreen preparations presented SPF values between 4.7 and 16. The results show that although glyceridic esters present higher lipophilicity and are more stable towards enzymatic hydrolysis in vitro , the synthesized triacylglycerols and the standard sunscreen 2-ethylhexyl p -methoxycinnamoyl (EHPM) showed equivalent SPF values for both methods.
Résumé
L'objectif de ce travail a été l'évaluation du groupe 1,3-diacylglycérol en tant qu'un agent d'intensification de la propriété photoprotectrice d'un nouveau filtre solaire de la classe cinnamate, en considérant l'affinité de cet groupe avec les matériels constitutifs de la peau et leur stabilité vis-à-vis l'hydrolyse enzymatique sur la position C-2. Les facteurs de protection solaire (FPS) des produits 1,2,3-propanetriol 1,3-dipalmitate-2-ρ-methoxy-cinnamate et 1,2,3-propanetriol 1,3-dioctanoate-2-ρ-methoxy-cinnamate ont étéévalués (2.0, 4.5 et 7.5)., Selon la concentration des produits, les préparations ont les valeurs de FPS entre 4.7 et 16. Les produits synthétisés ont les FPS équivalents au filtre solaire utilisé comme référence ( ρ -methoxy-cinnamate 2-ethylhexyl). Néanmoins, ils ont une plus important lipophilie et une plus grande stabilité vis-à-vis l'hydrolyse enzymatique in vitro.     

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.     

Validation of a HPLC method for simultaneous determination of five sunscreens in lotion preparation

The aim of this research was to develop and validate a high-performance liquid chromatographic (HPLC) method for simultaneous determination of five sunscreens, namely benzophenone-3 (B-3), butyl methoxydibenzoylmethane (BM), octyl methoxycinnamate (OM), octyl salicylate (OS) and homosalate (HS). The separation and quantitative determination was made by HPLC at 40 +/-1 degrees C with a gradient elution from 10% to 100% mobile phase B in mobile phase A. The gradient liquid chromatographic system constituted of mobile phase A [acetonitrile : water (10 : 90 v/v)] and mobile phase B [acetonitrile : water (90 : 10 v/v)], at a flow rate of 1.0 mL min(-1) and ultraviolet detection at 310 nm. The separation was obtained with two Waters reversed phase columns: Novapack C-18 and Symmetry((R)) C-18 connected in series. All sunscreens were efficiently separated within 17 min. The coefficient of correlation and average recovery for B-3, BM, OM, OS and HS were 0.9798 and 98.5%, 0.9672 and 98.8%, 0.9922 and 99.1%, 0.9961 and 98.9% and 0.9909 and 99.4% respectively. The relative standard deviations obtained were between 1.07% and 2.44%. The excipients did not interfere in the analysis. The results showed that the proposed method could be used for rapid and simultaneous determination of B-3, BM, OM, OS and HS in sunscreen lotions with precision, accuracy and specificity.     

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.     

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.     

Validation of HPLC method for quantitative determination of Tinosorb S and three other sunscreens in a high protection cosmetic product

A chromatographic method (high performance liquid chromatography) with a diode array detector was developed for simultaneous assay of Tinosorb S (bis-ethylhexyloxyphenol methoxyphenyl triazine) with three other sunscreen agents [benzophenone-3, butyl methoxydibenzoylmethane (avobenzone) and ethylhexyl methoxycinnamate] in high protection sunscreen. Separations were performed on a RP-18 Nucleodur Gravity column (150 x 4.6 mm, 5 mum) eluted with a ternary gradient mixture constituted of tetrahydrofuran, acetonitrile and an aqueous solution of acetic acid. The quantitative analysis was achieved with internal calibration performed with octyl dimethyl para-aminobenzoate (PABA) at 330 nm. In accordance with the analytical references (SFSTP, ICH, ISO...), the accuracy of the method was evaluated using a statistical approach of the validation parameters (specificity, response function, linearity, precision and trueness). For each studied ultraviolet filter, an accuracy profile was determined on a predicted range. These profiles show a graphical representation of the recovery percentage and confidence limits centred on 100%. The method is validated and can be used for analysis in cosmetic sunscreen products.     

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.     

A rapid High Performance Liquid Chromatographic (HPLC) method for the simultaneous determination of seven UV filters found in sunscreen and cosmetics

A rapid High Performance Liquid Chromatographic (HPLC) method was developed for the simultaneous determination of seven Ultra Violet (UV) filters most commonly found in cosmetics and sunscreen. The object of the method development was to provide a reliable rapid method, that would simultaneously separate a combination of the UV Filters, most commonly found in cosmetics and sunscreen products, utilizing a minimum of environmentally friendly solvents. The compounds separated were: benzophenone 3 (BZ3), methylbenzildene camphor (MBC), octyl dimethyl PABA (ODP), octocrylene (OCR), octyl methoxycinnamate (OMC), butyl – methoxydibenzoylmethane (BDM) and octyl salicylate (OS). An adjusted mobile phase consisting of ethanol and 1% acetic acid, combined with a Thermo Hypersil C₁₈ BDS 3 micron column resulted in a method, which allowed the analysis of the seven compounds in seven minutes. The proposed method was validated utilising the International Congress on Harmonisation (ICH) and the Food and Drug Administration (FDA) guidelines. All parameters examined were found to be well within the stated guidelines.     

Influence of applied quantity, water-immersion and air-drying on covering and microstructure of physical sunscreen films

The features of topically applied suncream films depend on the applied quantities and could be modified by water immersion and air drying. The aim of this study was to investigate the effects of the aforementioned factors on physical sunscreen films and to establish the correlation between the in vitro determined Sun Protection Factor (SPF) and the microstructure of the mineral coating. The assessments were conducted by using UV spectroscopy and electron microscopy on sunscreen films applied on both synthetic membranes and human skin. The results emphasize the paramount role played by the applied quantity to produce a continuous and protecting sunscreen film. The microscopic findings show that water immersion induces mainly a compactness of the sunscreen films, whereas no significant alterations were noted after air drying. Lastly, the SPF values, measured before and after water immersion, disclose the satisfactory water resistance of the broad-spectrum physical sunscreen considered.     

Development and application of a HPLC method for eight sunscreen agents in suncare products

This work describes the development, validation and application of a simple and fast high-performance liquid chromatography-with diode array dectection (HPLC-DAD) method for the determination of eight sunscreen agents: benzophenone-3, octocrylene, ethylhexyl methoxycinnamate, ethylhexyl salicylate, homosalate (used in two isomeric forms), butyl methoxydibenzoylmethane, 4-methylbenzylidene camphor and ethylhexyl dimethyl PABA in sunscreen formulations. The separation of the eight sunscreen compounds was achieved using an ACE C18 column (250 × 4.6 mm, 5 μm), with a column temperature 20°C, and a mobile phase of 88 : 12 (v/v) methanol-water with isocratic elution. Column temperature strongly influences the retention time and resolution of the compounds. The flow rate was 1.0 mL min(-1) and quantitation was performed by external calibration at the maximum wavelength of each compound. The sample preparation was simple and consisted basically of sample dilution with methanol, centrifugation and filtration in syringe filters before quantitation. Total run time was 18 min. The method was validated according to the parameters: linear range, linearity, selectivity, intra-day and inter-day precision and accuracy. Ten samples of sunscreen emulsions commercially available in Brazil (SPF 30) from different manufacturers were analysed using the proposed method. The number of the sunscreen agents varied between one and five in a single sample. The concentrations of all compounds were in the range of 0.9-10% (w/w) and were in accordance with the current Brazilian legislation.     

New ex-vivo method for evaluating the photoprotective efficacy of sunscreens

The photoprotective efficacy of some topical sunscreens was determined ex-vivo by spectrophotometric assay using an irradiation monochromator and a diffuse reflectance sphere. Transmission scans were run from 400 to 290 nm through human epidermis samples before and after application of products and protection factors (PF) were calculated. The results are also expressed as the percentage of the radiation stopped by the products in each spectral range (UVB, short UVA and long UVA). All the formulations contained two chemical filters: butyl methoxy dibenzoyl methane and octyl methoxycinnamate in a range of concentrations. Estimates of the effectiveness of all products were higher than results in humans when calculated by this ex-vivo technique. Due to its high discriminant power, this method constitutes a useful tool in the formulation of sunscreens.     

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.     

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.