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.     

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

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

Comparison of sun protection factors determined by an in vivo and different in vitro methodologies: a study with 58 different commercially available sunscreen products

An extensive study on the sun protection factors (SPF) of sun care products was carried out using the COLIPA (The European Cosmetic Toiletry and Perfumery Association) method, which relates to in vivo experiments. Furthermore, in vitro methods were tested with sunscreen formulations that were prepared as films on surface-roughened plates of polymethyl methacrylate (PMMA). One of the in vitro methods, i.e. using the sunscreen tester, has been recently developed, whereas the second has been defined by a pure spectroscopic approach, which is based on spectral transmission measurements of sunscreen films. Altogether 58 different sunscreen formulations, with manufacturer declared SPF values ranging from 4 to 60 and currently available on the European market, were investigated. The quality of correlations with results from the individual products based on the different in vitro methods versus the COLIPA values that were considered as generally accepted standard values was assessed. In this context, also variations because of sample preparation and spectral measurement were discussed. For sunscreen products with in vivo SPF values larger 25, the spectral transmittance within the UVA/UVB range is rapidly decreasing, which is experienced even for products with reduced amounts reaching 0.5 mg cm(-2) and still leading to unsatisfactory correlation of the spectroscopically derived SPF values versus the results from the alternative assays. Opposite to these small amounts, a sunscreen product spread of 2 mg cm(-2) is standard for the in vivo COLIPA method, whereas an area-normalized amount of 1 mg cm(-2) is currently routinely used for the sunscreen tester method. Furthermore, an overview of the individual product characteristics, such as their specific critical wavelengths and their UVA/UVB ratios is provided; both parameters can also be calculated from the spectral absorbances of the standardized sunscreen films.     

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

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

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.     

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

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

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.     

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).     

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.     

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.     

A novel in vitro technique for measuring the water resistance of sunscreens

A novel in vitro technique has been used to assess the water resistance of topical sunscreens. The technique is based upon transmission spectrometry using excised human epidermis as the substrate. The sun protection factors (SPFs) of four sunscreens obtained prior to water immersion and after two immersions were compared with in vivo data determined using human volunteers. In every case there was close agreement between the SPFs obtained on dry skin and on skin that had been immersed in water. Not only does the technique provide a less expensive and less time consuming alternative to in vivo testing, it also offers the possibility to study the effect that water immersion may have on the absorption spectrum of products which cannot be determined in vivo. This can give valuable information on which, if any, of the active ingredients present in sunscreens are being preferentially removed by water. In addition, the method can be easily adapted to study the effects on sunscreen substantivity of other factors such as water temperature, salinity and turbulence.     

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.     

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.     

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.     

An investigation of detergent action on cells in vitro and possible correlations with in vivo data

A cell fluorescence method for quantifying the effects of detergents on cultured cell lines is described. The results are discussed in relation to other findings in the literature. These results are compared to data obtained with the same detergents in the rabbit eye test and the possible correlations and discrepancies are considered.

Summary

Details are given of the culture of two human epithelioid cell lines (HeLa and HEp2) and their utilization in a procedure to assess the cytotoxicity of detergents on cells in monolayer culture. The method is dependent on the ability of relatively intact cells to liberate and retain fluorescein from fluorescein diacetate. Ethidium bromide, a red fluorescent compound, is used to stain the nuclear material remaining in membrane-damaged cells. The results of the in vitro test are compared to the data obtained from the responses seen in the rabbit eye on instillation of the same detergents. For sodium and triethanolamine alkyl sulphates at comparable concentrations, an increase in chain length increases the in vitro cytotoxicity but decreases the effects seen in the conjunctivae, cornea and iris. For the C12, C14 and C16 alkyl trimethylammonium bromides, an increase in chain length increases in vitro cytotoxicity as well as the in vivo responses. Tweens 20 and 40 appeared more damaging in vivo than Tweens 60 and 80; this differentiation was not observed in vivo. The findings of both tests are discussed in terms of detergent solubilities, penetration of, and adsorption of tissues in vivo as well as the effects of detergents on enzymes.     

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.     

An in vitro test for the assessment of eye irritancy in consumer products – preliminary findings

An in vitro procedure for preliminary screening of severe eye irritants is described. Rabbit eyes are removed immediately after death and are placed in temperature controlled chambers. The eyes are superfused with isotonic saline and, after a suitable equilibration period, are treated with test substances. The effects of treatment (corneal swelling, and the severity, extent, and rate of development of opacity in the corneal epithelium and stroma) are assessed using the slit lamp biomicroscope. Experiments show that the cornea of the isolated eye remains viable and physiologically active for the duration of the test.
Results of in vitro tests show a reasonably good correlation with in vivo data for a series of chemicals reported in the literature to be severely, moderately, or non-irritant to eyes. The effects of a range of high pH sodium hydroxide solutions are described. Thresholds for effect in vitro are defined and related to in vivo thresholds for high pH products. The effects of shampoos in the in vitro system are described, with preliminary results suggesting that the method can distinguish between normal 'adult'shampoos and 'baby'shampoos, which are known to differ in irritancy in vivo.
Overall the in vitro procedure appears to offer a reliable screening procedure to identify severe eye irritants. Materials producing severe effects in vitro following a short contact time should not be tested in vivo , but where no effects are observed in vitro , in vivo testing may still be required. Like all in vitro procedures it has limitations compared with the in vivo eye test. It takes no account of the effects on the conjuctivae, nor does it take account of the rate of healing, both of which are important aspects of in vivo eye tests.
Test in vitro pour 1'evaluation de 1'irritation occulaire dans les produits d'hygiène (Résultats préliminaires)     

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.