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.     

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.     

Relationship between UVA protection and skin response to UV light: proposal for labelling UVA protection

Definition and validation of a most relevant method to assess ultravoilet A (UVA) protection is a major concern for industry, authorities and consumers. However, due to the lack of knowledge about all the biological phenomena involved, the level of UVA protection needed, the ways to assess and label it, remain controversial. In order to overcome this situation, the paper deals with the outcomes of a mathematical model to calculate the distribution between ultravoilet B (UVB) and UVA components of skin responses to UV light. Mathematical calculations of UVB and UVA erythemal components of skin response to sunlight are developed from the well-known determination procedure to calculate the sunburn protection factor (SPF) of sunscreens. The model establishes the relationship between the UVA component of skin erythemal response to overall UV radiation received from sunlight and the ratio SPF/PFAe (erythemal protection factor) where SPF is the product and PFAe is related to the UVA part of the sunlight. Depending on the efficacy profile of sunscreens, the skin erythemal response may be mainly promoted by UVB rays as it normally occurs in unprotected skin or on contrary by UVA rays. Therefore, the efficacy profile of sunscreens defines the deepness where biological events induced by sunlight take place. This new relationship pinpoints the tremendous importance of the protection afforded by sunscreen products in the UVA range when erythema is taken as biological response. By extrapolation of the model to any other biological skin response it becomes possible to predict how to improve the efficiency of sunscreen products in the future. UVA protection afforded by sunscreens should be improved until reaching the same level as the SPF protection factor so that all UV-induced biological responses could be prevented or lowered at the same extend. To enforce this improvement, a proposal to classify sunscreen products in relation with their UVA protection is made.     

Sunscreen in vitro spectroscopy: application to UVA protection assessment and correlation with in vivo persistent pigment darkening

In the present study, we have described an in vitro spectroscopic method to evaluate the sunscreen products for UVA sun protection factor. The roughened PMMA plates have been used as a transparent substrate on to which the test product is spread. The UVA protection factors have been deduced from the UV-transmittance data measured in the UVA area. In order to be as close as possible to the in vivo protection factors, issued from the PPD end-point, the treated polymethylmethacrylate (PMMA) plates are submitted to different UV-irradiation doses, before the measurement. The correlation in vitro/in vivo is poor when the sunscreens are not irradiated. A UV dose of about 2 minimal pigmenting dose (MPD) is enough to achieve a good correlation between in vitro and in vivo data issued from the 13 tested sunscreens. These results are consistent with the fact that the photostability of sunscreens is challenged during an in vivo PPD test.     

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.     

New data on skin photoprotection

Absorbed ultraviolet (UV) radiation is responsible for photochemical degradation of the skin. Erythema is the most apparent result of the sunburn reaction, but the most serious results are photoaging and photocarcinogenesis, largely mediated by reactive oxygen species (ROS). To prevent these light-induced reactions, effective protection against both UVB and UVA radiations are essential. The aim of this study was to evaluate the protective efficacy against both UVB and UVA rays of three sunscreen preparations (two creams and one lotion), evaluating also their water resistance. For this purpose SPF (Sun Protection Factor) and UVA protection were tested, respectively, on 40 and 20 pre-selected volunteers (male and female, < 22-years-old with skin types I, II and III) by the Colipa test method and the Mark et al. methodology. A wash-off test was also conducted to determine the resistance to water. Results obtained showed that the three formulations studied underline interesting SPF values from about 20-30 with an excellent correlation between visual and colorimetric results obtained (P < 0.05). The controlled decrease in effectiveness of only 20% after 10 min washing proved that the vehicle is also an important factor in determining the protective efficacy. From the obtained data, the studied formulations seem to have an high SPF and UVA protection efficacy together with a good water resistance. According to several other studies, it is possible to confirm therefore that the regular use of these sunscreens may certainly reduce the harmful effect caused by long-term exposure to UVB and UVA radiation.     

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.     

A novel proposal for the assessment of sunscreen product efficacy against UVA

The ideal characteristics are outlined for an assessment method for the protective properties of sunscreen products against UVA. Existing sun protection factors (SPFs) are reliant on largely UVB-induced delayed erythema, which is inappropriate for the UVA. A novel instrumental method for assessing a protection index is detailed which is applicable to the UVA. The validation of the method relies on measurements in the UVB and comparison with conventional SPFs, since no reliable UVA protection index exists. The method uses UV transparent skin casts to which products can be applied. Throughput of appropriately filtered light, with and without applied product, is measured to generate a protection index. Comparison with SPFs confirms that the measurements are similar to those generated by in vivo methods, but that careful choice of the casting medium is essential.     

Beyond UV radiation: A skin under challenge

Since ancient times, human beings have been trying to protect their skin against the adverse effects of the sun. From the first mineral sunscreens used by Egyptians, to the current more sophisticated ultraviolet (UVA/UVB) organic sunscreens, progress has been made in terms of sun protection and deeper knowledge of skin physiology has been acquired in the process. The solar spectrum is composed of radiations of various wavelengths having specific, as well as overlapping effects on skin. UVB is mainly responsible for sunburn and DNA dimer formation that can lead to mutation. UVA generates oxidative reactions affecting DNA, proteins and lipids, and is also immunosuppressive. Recently, visible light and infrared radiation (IR) have been associated with oxidative damage and IR has been additionally linked to adverse heat effects on skin. Numerous other extrinsic factors, related to environment and lifestyle, also affect the appearance of skin, precipitating ageing. New molecular mechanisms linking sun and environmental factors to skin ageing have been identified: IR affects mitochondrial integrity and specific heat receptors also mediate some of its effects, tryptophan is a chromophore for UVB, and the aryl hydrocarbon receptor (AhR) is activated by light and xenobiotics to alter skin physiology. Integrating all these new elements is changing the way we think about skin extrinsic ageing. Is UVA/UVB sunscreen protection still enough for our skin?     

Preliminary studies towards utilization of various plant extracts as antisolar agents

The aim of this work was to evaluate several plant extracts with regard to ultraviolet absorption spectra in view of a possible application as antisolar agents. Liquid and dry extracts of Hamamelis virginiana, Matricaria recutita, Aesculus hippocastanum, Rhamnus purshiana and Cinnamomum zeylanicum were prepared by repercolation, maceration and microwave oven extraction. UVB absorption spectra (290–320nm) were obtained and the solar protection factors (SPF) of these preparations were determined by a spectrophotometric method. The results showed that after incorporation to a 2% solution of the synthetic sunscreen octylmethoxycinnamate, the extracts showed an intensification in SPF values, suggesting that this can be an interesting method to intensify SPF. In addition, these extracts can contribute their emollient and moistening properties to the product. These are important characteristics for protecting skin against exposure to the sun.     

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 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     

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.     

Poly(D,L-lactide) nanoencapsulation to reduce photoinactivation of a sunscreen agent

The use of sunscreens is the 'gold standard' for protecting the skin from ultraviolet light. Octyl methoxycinnamate (OMC) is one of the most widely used UVB filter but it can act as a sensitizer or photoallergen. When exposed to sunlight, OMC can change from the primary trans-form to cis-form and the isomerization, not reversible, conducts to a reduction of the UVB filtering efficiency because the trans-form has a higher extinction coefficient. Photostability is the most important characteristic of effective sunscreens and it can be influenced by formulation ingredients and by applying technological strategies. In this work, photostability experiments, performed on emulsion-gels containing different percentages of OMC free or loaded in poly(D,L-lactide) nanoparticles, were carried out. The presence of a polymeric envelop may act to protect the active ingredient. In this study, the influence of poly(D,L-lactide) matrices on the photochemical stability of the sunscreen agent was investigated. As highlighted in this study, free OMC in different formulations has different photoisomerization degree. Moreover, a dissimilar behaviour was observed by studying different sunscreen concentrations in the same cosmetic formulation. Photostability results show a significant reduction in photoisomerization degree for formulations containing sunscreen loaded in nanoparticles, highlighting that the encapsulation is a suitable strategy to improve OMC photostability. Moreover, sun protection factor (SPF) results show that the UVB filter protective power is also maintained after encapsulation.     

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.     

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.     

Validation of a new in vitro Sun Protection Factor method to include a wide range of sunscreen product emulsion types

In 2017, Cosmetics Europe performed a double-blinded ring test of 24 emulsion-type sunscreen products, across 3 in vivo test laboratories and 3 in vitro test laboratories, using a new candidate in vitro SPF test method. Based on the results of this work, an article was published showing how data derived from a new lead candidate method conform to new International Standards (ISO) acceptance criteria for alternative SPF test methods (Any alternative method should consider the matrix effect and if required, specify the matrix applicability of the method; Criterion 1a: Systematic differences between methods should be negligible: 95% of all individual results of an alternative method are within the range of ±2× reproducibility standard deviation of the in vivo method, that is overall bias must be below 0.5× reproducibility standard deviation of the in vivo method; Criterion 1b: Measurement uncertainty of an alternative method should be below the measurement uncertainty of the in vivo method. Candidate method predicted values must fall within the full ‘funnel’ (SPF 6-50+) limits proposed by Cosmetics Europe (derived from the same minimum test design, that is using the ISO24444 Method to measure at least 24 products across at least 3 laboratories using at least 5 test subjects/laboratory, in a blinded fashion).). Of the 24 sunscreen products tested, the majority of emulsions were of the oil-in-water (O/W) type, whereas only one was water-in-oil (W/O) and there were no products with a mineral-only sun filter system. In order to confirm the scope of this method, therefore, a new study was conducted that included 73 W/O (12 mineral + organic, 44 mineral only and 17 organic only) and 3 O/W mineral-only, emulsion-type sunscreen products (a total of 76 new sunscreen products). When combined with the previous 24 products (tested in 3 different laboratories), this yielded a new data set comprising a total of 100 emulsion-type sunscreen products, with SPF values ranging from 6 to 50+ (with a total of 148 data points). These products were tested using the double-plate in vitro SPF test method and compared with the ISO TC217/WG7 acceptance criteria for alternative SPF test methods. Over 95% of paired in vitro: in vivo SPF values lay within the upper and lower limits of the ISO acceptance criteria funnel, with no bias. This new in vitro SPF test method, therefore, meets the minimum requirements for an alternative SPF test method to ISO24444:2010, for emulsion-type sunscreen products (which make up the majority of marketed 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.     

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.     

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

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