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.     

Beyond sun protection factor testing

Ultraviolet radiation in sunlight produces a range of acute and chronic adverse effects on the skin including sunburn, photosensitivity rashes, immunosuppression, photoageing and carcinogenesis. Sunscreens aim to provide protection, but standard testing procedures primarily involve assessment of ability to protect against acute erythema, as evidenced by the sun protection factor (SPF). The SPF may correlate poorly with other aspects of protection, particularly since ultraviolet A is weakly erythemogenic compared with ultraviolet B, yet may make a greater contribution to certain other skin effects of sunlight. Nevertheless, there is an increasing tendency for the sunscreen industry to make claims for their products beyond the SPF data. There is a need to develop systems for clinical testing of sunscreens against other endpoints caused by ultraviolet exposure of skin, including immunosuppression and photosensitivity rashes. In particular, there is a largely unrecognized need for testing of sunscreens against the condition known as polymorphic light eruption, a photosensitivity disorder estimated to affect a staggering 10-20% of the population in the northern hemisphere. Ultimately, protection of the skin by sunscreens can only be as effective as their adequacy of application to the skin surface in the everyday setting permits. Optimal sunscreen formulation, and public and patient education in appropriate application technique, both make vital contributions to efficacy of sunscreen protection. This article focuses on the need for extended clinical testing of sunscreens, with particular reference to the photosensitivity disorders, and for improvements in sunscreen formulation and in the adequacy of sunscreen application to the skin surface.     

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.     

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 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     

Methods for water resistance testing of sun protection products

Water resistance of sun protection products has been found to be best determined by the following methods: (1) Tests for determining UVL filter adherence to merino wool; this gives a rough value for orientation. Values in excess of 6% are suggestive of satisfactory adherence. (2) Tests for determining water resistance of sunscreen products on pig skin constitute a further essential step. Values in excess of 50% provide a realistic basis for tests in humans. In addition to filter adherence the product base, i.e. primarily the emulsifiers, are of special significance. Adequate results can be obtained with both W/O and O/W systems. Tests on volunteers are best done at the time of determining the sun protection factor, i.e. when simulating the events during a 2–3 h sunbath. Simulation should involve a single application of the test product in a pre-defined quantity and all activities conmmonly done in sunlight, e.g. swimming, as well as intermittent exercises to induce sweating. Values are considered as excellent, if 1 MED is not exceeded after 3 h exposure. Sun protection products with this characteristic qualify as adequate ‘all-day’ sunscreen for individuals with average sensitivity. Methodes pour mesurer la résistance à l'eau des produits anti-solaires     

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.     

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.     

Formulation of sunscreens with enhancement sun protection factor response based on solid lipid nanoparticles

Solid lipid nanoparticle (SLN) was regarded as new topical delivery systems for pharmaceutical and cosmetic active ingredients. The purpose of this study is to develop carrier systems for organic and inorganic sunscreens based on a matrix composed of carnauba wax and decyl oleate. Formulae (F1–F7) were prepared using butyl methoxydibenzoylmethane and octyl methoxycinnamate as organic components, and titanium dioxide (TiO₂) was used as inorganic component. Both types of sunscreens were incorporated into SLN formulations using classical method of preparation. To evaluate the effect of the pigments on the nanoparticles, particle size was measured using Mastersizer particle size analyser. UV‐protection abilities of formulations were investigated by the in vitro sun protection factor test (SPF). Further parameters determined were spreadability as well as viscosity. The rheological behaviour of the formulations was also carried out. From the plot of log of shear stress vs. log of shear rate, the slope of the plot representing flow index and ontology of the y‐intercept indicating consistency index was calculated. The formulae showed a flow index of 0.2074–0.4005 indicating pseudoplastic flow behaviour. Significant increases in SPF values up to about 50 were reported after the encapsulation by using organic and inorganic filters in Canada wax and decyl oleate. So, SLN could be appropriate vehicles to carry organic and inorganic sunscreens. The rational combination of cinnamates, titanium dioxide and Zinc oxide has shown a synergistic effect to improve the SPF of cosmetic preparations.     

Consequences for sun protection factors when solar simulator spectra deviate from the spectrum of the sun

The sun protection factor (SPF) of two products, one with an expected SPF of 4 and another with an expected SPF of 15 were determined, using two solar simulators: Multiport Solar UV Simulator (xenon, Solar Light, Philadelphia, PA, USA), and Supersun 5000 (metal halide, Mutzhas, Munich, Germany). The mean SPFs using the Multiport were: 4.8 for the low SPF product and 19.4 for the high SPF one. The results using the Supersun were lower: 2.6 for the low SPF product and 7.2 for the high SPF one. Relative emission spectra of the two sources were recorded using a fluorescence spectrophotometer in bioluminescence mode. Efficacy spectra were calculated and compared with the corresponding spectrum of natural sunlight. It was evident that the spectral power of the xenon source is too high in the UVB, leading to overestimation of SPFs, whereas the Supersun irradiator emits too much in the UVA, resulting in too low SPFs. Heat effects and photodegradation of UV filters are discussed as further possible reasons for the discrepancies between the experimentally determined SPFs. Our results confirm a recent publication about theoretical SPFs, calculated with emission spectra of a xenon source and spectra of the sun at different elevation angles, where the authors provide evidence that in natural sunlight the contribution of UVA to total UV radiation is twice as high as in a xenon source. This may contribute to an understanding of why sunscreens tested according to the FDA method (xenon sources) often yield higher SPFs than those obtained from European testing procedures.     

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.     

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.     

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.     

新食品原料宝乐果在防晒与修复UV损伤中的效果研究

目的研究新食品原料宝乐果(Borojo)防晒(sun protection factor,SPF)以及抗紫外线(ultraviolet,UV))照射修复损伤的作用功效。方法按实验目的将豚鼠和SPF级(specific pathogen free,SPF)小鼠设置空白对照组、基质组、宝乐果组、阳性对照组、宝乐果+阳性对照共5组。按实验内容配方制成各组实验用护肤乳液。仪器法预测试各组SPF值,动物体内实验在各组豚鼠背部按2 mg/cm~2的量分别涂抹护肤乳液,以功率1346μW/cm~2、波长300~400 nm的紫外灯照射紫外光。测量各组最小红斑量(minimal erythemal dose,MED),并根据SPF值判定其实际防晒等级。各组小鼠背部皮肤分别均匀涂抹护肤乳液后以波长300~400 nm的长波紫外线(ultraviolet radiation A,UVA)模拟日光紫外照射致损伤;观察皮肤外观损伤表现;用试剂盒法测定皮肤组织中超氧化物歧化酶(super oxide dismutase,SOD)和丙二醛(malondialdehyde,MDA)的含量;统计分析各组数据结果。结果仪器测试标准SPF15,对照14.7±2.1,宝乐果护肤乳液17.7±1.6,宝乐果+阳性对照组21.0±1.0。体内实验SPF值宝乐果组14.1±1.0,阳性对照组13.8±1.8,宝乐果+阳性对照组17.2±2.0。动物皮肤外观同基质组相比,涂抹照射7 d后,宝乐果组有轻度红斑少量角质化皱纹,但未脱皮。宝乐果+阳性对照组红斑较浅无角质化。涂抹照射14 d后,宝乐果组和宝乐果+阳性组皮肤角质化和红斑渐渐消失同空白对照组。皮肤匀浆测试同基质组比,宝乐果组小鼠皮肤匀浆中SOD含量明显增加(P0.05),宝乐果+阳性对照组显著增加(P0.01)。宝乐果组小鼠皮肤匀浆中MDA含量明显降低(P0.05),宝乐果+阳性对照组显著降低(P0.01)。结论在体内体外试验中,新食品原料宝乐果配制的护肤乳液具有优良的防晒效果和明显地增强晒后损伤修复作用。