Skin permeation of two different benzophenone derivatives from various vehicles

Synopsis The aim of this study was to determine the effects of the vehicles on the release and permeation of the UV filters. Therefore, the release and permeation of lipophilic oxybenzone and hydrophilic sulisobenzone were investigated from four different vehicles; petrolatum, o/w and w/o emulsions and hydroxyethyl cellulose (HEC) gel. Both release and permeation studies were performed by using glass diffusion cells. The vehicle in which oxybenzone has the highest released amount was petrolatum, whereas the highest released amount for sulisobenzone was obtained in HEC gel. The difference between the released amounts of oxybenzone in the other three vehicles was not significant but the released amount of sulisobenzone was least in petrolatum, whereas no significant difference was seen between o/w and w/o emulsions. The results of skin permeation experiments were similar those from release experiments. The order of permeated amounts from highest to lowest was petrolatum, o/w emulsion and HEC gel for oxybenzone, and HEC gel, o/w emulsion and petrolatum for sulisobenzone. These results demonstrate that permeation of the UV filters can be optimized by a suitable vehicle. As the percutaneous absorption of sunscreens should be avoided, the vehicle suggested for oxybenzone and sulisobenzone is o/w emulsion because of its permeation properties and cosmetic acceptance.     

In vitro skin permeation evaluation: the only realistic option

Increasing requirements for cruelty-free risk assessment in the cosmetic industry have led to the development of several alternative experimental evaluation strategies. Quantification of the potential dermal absorption of ingredients of cosmetic and other formulations by determination of human skin permeation rates in vitro is particularly relevant. Using modifications of standard in vitro protocols the human skin permeation rates of several cosmetic ingredients and potential contaminants have been determined under conditions designed to mimic consumer use. Skin penetration and permeation of octyl salicylate (a sunscreen), nonylphenol ethoxylates (surfactants) and three nitrosamines (potential contaminants) is discussed. The data demonstrate the usefulness of this technique as a tool in the overall risk assessment of cosmetic formulations.     

In vitro skin permeation and retention of parabens from cosmetic formulations

Parabens are antimicrobial agents widely used in foods, cosmetics and pharmaceutical products. Although non-mutagenic, non-teratogenic and non-carcinogenic, parabens can induce allergic contact dermatitis and posses estrogenic activity. The aim of this work was to assess the skin permeation and retention of methyl- (MP), ethyl- (EP) and propyl- (PP) paraben from three commercial cosmetic creams. The results obtained indicate that parabens are capable of permeating through and accumulating in the skin. The extent of penetration depends more on paraben characteristics (solubility, lipophilicity) than on the composition of the formulation. In particular, the percentage permeated across the skin was independent of the composition of the cream used and decreased in the order MP, EP and PP, in accordance with decreasing solubility. After 8 h of contact with the skin, 60% of MP, 40% of EP and 20% of PP were found across the skin. Concerning skin retention, the percentage remaining in the skin after 8 h depends on both paraben characteristics and on the composition of the formulation used. In conclusion, it appears that only the type of paraben, in particular its water solubility, affects skin penetration whereas the composition of the emulsion, which influences skin retention, plays a secondary role. Finally, excised rabbit ear skin can be considered as a good model for human skin for in vitro experiments.     

Release rate profiles of magnesium from multiple W/O/W emulsions

Water-in-oil-in-water (W/O/W) emulsions were formulated based on rapeseed oil, olive oil, olein and miglyol. Polyglycerol polyricinoleate and sodium caseinate were used as lipophilic and hydrophilic emulsifiers, respectively. Magnesium was encapsulated in the inner aqueous droplets. Emulsion stability was assayed through particle sizing and magnesium release at two storage temperatures (4 and 25 °C) over 1 month. Irrespective of the oil nature, both the primary W/O and W/O/W emulsions were quite stable regarding the size parameters, with 10-μm fat globules and 1-μm internal water droplets. Magnesium leakage from W/O/W emulsions was influenced by the oil type used in the formulation: the higher leakage values were obtained for the oils characterized by the lower viscosity and the higher proportion of saturated fatty acids. Magnesium release was not due to droplet–globule coalescence but rather to diffusion and/or permeation mechanisms with a characteristic rate that varied over time. In addition, W/O/W emulsions were resistant to various thermal treatments that mimicked that used in pasteurization processes. Finally, when W/O/W emulsions were placed in the presence of pancreatic lipase, the emulsion triglycerides were hydrolysed by the enzyme. These results indicated a possible use of W/O/W emulsions loaded with magnesium ions in food applications.     

Occlusive power evaluation of O/W/O multiple emulsions on gelatin support cells

The water content of the stratum corneum plays an important role in providing skin suppleness and smoothness. The diffusion of water through the skin is limited primarily by the stratum corneum and the noncornified part of the epidermis has negligible water barrier properties. Multiple emulsions are vesicular systems utilized for the prolonged liberation of active ingredients. The O/W/O multiple emulsion type is employed in cosmetics because its high oil content is able to maintain an occlusive film (barrier) on the skin surface. The objective of this study was to determine the occlusive power of O/W/O multiple emulsions on gelatin support cells. The results showed that occlusive products form a uniform layer on the surface of gelatin after the test, whereas nonocclusive products form two layers: an aqueous phase on the gelatin, and an oil phase above the aqueous phase. Thus, the different occlusive powers are due to the homogeneity of this layer and to its ability to prevent water evaporation.     

Evaluation of an in vitro percutaneous permeation model with two oxidative hair dyes

The percutaneous permeation of two oxidative hair dyes was measured by means of pig skin in a flow-through diffusion cell system entirely constructed from Teflon. Pig skin membranes were prepared by reducing full thickness skin with a dermatome to a more in vivo -like barrier layer and their integrity was checked by measuring the steady-state permeation of tritiated water. Initially, the inter- and intraindividual variability of percutaneous permeation was determined with an aqueous solution of 1-(2'-hydroxyethyl)-amino-3,4-methylenedioxybenzene-hydrochloride, an oxidative hair dye component. In the same way the proper flow rate of elution fluid through the receptor cell was found to be most favourable at 10 ml h^-1, the thickness of permeation membranes was fixed at 1 mm, and it was shown that storage of the skin at −20°C for up to 35 days did not change the permeability. The percutaneous permeation of the same hair dye component and of 4-amino-2-hydroxymethylphenol-hydrochloride was determined after application to pig skin membranes under practical conditions of hair dyeing. The in vitro skin permeation was in the same order of magnitude as results from comparable in vivo skin absorption studies in rats.
Perméation percutanée in vitro de colorants d'oxydation pour cheveux     

W/O/W多重乳状液的实验研究

研究了W/O/W多重乳状液的制备方法及影响其稳定性的因素。通过正交试验选出较佳的工艺参数,获得了较稳定的W/O/W多重乳状液。     

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.     

W/O/W emulsions with high internal droplet volume fraction

We prepared water-in-oil-in-water (W/O/W) double emulsions with high internal aqueous droplet fraction using food-based ingredients. These compartmentalised materials were comprised of oil globules dispersed in an external aqueous phase, with the globules themselves containing densely packed inner aqueous droplets. We were able to obtain double emulsions with large globule fractions (up to 45 vol.%) using only 5 vol.% oil (relative to the overall composition). In the final state, the inner droplet fraction within the globules could exceed 90 vol.%. The method was based on two successive emulsification steps, followed by osmotic swelling (transport of water from the external phase to the inner droplets through the oil phase). During the final step, the swelling was controlled by the osmotic pressure mismatch between the external and internal aqueous phases using solutes dissolved at different concentrations. The osmotic swelling model of Mezzenga et al. (Langmuir, 2004, 20, 3574-3582) was re-adapted in the limit of small Laplace pressures to predict the final composition resulting from osmotic equilibration. The internal droplet fraction was lower than that predicted by the model as a consequence of coalescence phenomena occurring during the swelling process. The proposed approach constitutes a valuable guide within the prospect of formulating emulsions with enhanced encapsulation capacity and reduced fat content.     

Substantivity of sunscreens —in vitro evaluation of the transdermal permeation characteristics of some benzophenone derivatives

The in vitro permeation through excised hairless mouse skin of a series of 4-O-(N, N-dimethylaminoalkyl)-benzophenones, non-quaternarized and quaternarized, and of two commercial benzophenone sunscreens, taken as reference compounds, was investigated. The aim of the study was to verify the skin penetration of the highly skin-substantive quaternary ammonium derivatives, in comparison with their parent, non-quaternarized compounds. While the quaternary derivatives proved unable to permeate the skin during the period of observation (45 h), their parent amine hydrochlorides and the reference sunscreens (2-hydroxy-4-methoxy-benzophenone-5-sulphonic acid and 2,2'-dihydroxy-4,4'-dimethoxy-benzophenone 5,5'-sodium disulphonate), showed appreciable transdermal fluxes.
These data indicate that the presence of a quaternary ammonium group in a molecule, besides inducing a high affinity for cutaneous keratin, may result in hindered or reduced transdermal (and possibly systemic) absorption. Both features may contribute in improving the safety of a cosmetic sunscreen.     

In vitro human skin permeation and cutaneous metabolism of catechins from green tea extract and green tea extract-loaded chitosan microparticles

Catechins are major antioxidants in green tea (Camellia sinensis or Camellia assamica), but because they do not permeate the skin well, the application of green tea in cosmetic products has so far been limited. This study aims to evaluate the cutaneous absorption of catechins from an extract of green tea and from a green tea extract-loaded chitosan microparticle. The catechin skin metabolism was also examined. The results suggest that chitosan microparticles significantly improve the ability of catechins to permeate skin. The cutaneous metabolism of the catechins significantly affected their permeation profiles. Epicatechin (EC) and epigallocatechin (EGC) penetrated the skin more than epigallocatechin gallate (EGCG) and epicatechin gallate (ECG). The galloyl groups in EGCG and ECG were enzymatically hydrolysed to EGC and EC, respectively. Dehydroxylation of catechins was also observed. Chitosan microparticles effectively prevented enzymatic changes of the catechins; therefore, chitosan microparticles are here found to be the promising carriers for enhancing the skin permeation.     

In vitro cultured human skin cells as alternatives to animals for skin irritancy screening

In the last few years a lot of attention has been paid to the development of the in vitro models which would substitute for animals in cutaneous irritancy studies. These models explore either organ or explant cultures using freshly excised skin or serial cultures of isolated skin cells (epidermal keratinocytes or dermal fibroblasts). The organ or explant models are suitable only for short exposures of skin samples to the compounds tested and the use of it will always be restricted by the limited availability of fresh human skin. The model that uses submerged cultures of keratinocytes or fibroblasts permits the production of a large number of cells, and permits large scale toxicity screening tests with many substances, that can be applied in a broad concentration range. Since the stratum corneum is absent in conventional (submerged) keratinocyte culture systems, this model is mainly suited for testing of water soluble compounds and it is less suitable for poorly soluble compounds and for topical products consisting of complex formulations which are made of active ingredients and their vehicles. This shortcoming can be overcome by using ‘organotypic cultures’in which keratinocytes are grown at the air-liquid interface on a suitable dermal substrate. Under these conditions, the culture forms a multilayered epidermis showing an overall structure which resembles that of a native epidermis. The presence of a coherent stratum corneum layer in these cultures permits the application of potential irritants at concentrations and in formulations as applied in vivo. For the evaluation of toxicity a number of tests have already been developed: assessment of cell viability, changes in cell morphology, modulation of cell proliferation and differentiation, monitoring of membrane damage, the measurements of the uptake or incorporation of radioactive precursors, establishment of the modulation of cell metabolism, determination of the release of inflammatory mediators, etc. All these in vitro techniques are still in a state of validation as far as their predictive value for in vivo skin irritancy is concerned.     

In vitro assessment of sunscreen photostability: the effect of radiation source, sunscreen application thickness and substrate

The photostabilities of four sunscreen products have been assessed in vitro by applying sunscreen to a substrate and measuring the spectral transmission prior to, and after exposure to a source of ultraviolet (UV) radiation. Results were independent of whether an application thickness of 1 or 2 mg/cm ; 2 was used, and whether the UV source was natural sunlight or a xenon arc solar-simulator. There were significant differences, however, between results obtained on a roughened quartz substrate and those obtained on excised human epidermis. It is unlikely that any substrate will give an exact representation of the in vivo situation and, indeed, both quartz and excised human epidermis have disadvantages associated with their use. However, the ranking of the four products in terms of their photostability was the same for both substrates. This implies that transmission spectroscopy, with either a quartz or a human epidermis substrate, can be used successfully to compare the photostabilities of different sunscreen products.     

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.     

Evaluation of skin cancer chemoprevention potential of sunscreen agents using the Epstein–Barr virus early antigen activation in vitro assay

In our continuing search for novel cancer chemopreventive compounds of natural and synthetic origin, we have evaluated 14 commonly used ultraviolet (UV) sunscreen agents (designated UV‐1 to UV‐14) for their skin cancer chemoprevention potential. They belong to 8 different chemical categories: aminobenzoate (UV‐5, UV‐7, UV‐8 and UV‐14), benzophenone (UV‐1, UV‐2, UV‐3 and UV‐13), benzotriazole (UV‐10), benzyloxyphenol (UV‐9), cinnamate (UV‐6), quinolone (UV‐4), salicylate (UV‐11) and xanthone (UV‐12). In the in vitro assay employed, the sunscreens were assessed by their inhibition of the Epstein–Barr virus early antigen (EBV‐EA) activation induced by the tumour promoter 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) in human lymphoblastoid Raji cells. All sunscreens tested were found to exhibit anti‐tumour promoting activity: listed in decreasing order, moderate (UV‐11, UV‐2, UV‐7, UV‐12, UV‐3, UV‐9 and UV‐14) to weak (UV‐1, UV‐6, UV‐8, UV‐16, UV‐5, UV‐4 and UV‐10) with octyl salicylate (UV‐11) as the most potent and drometrizole (UV‐10) as the least potent among the compounds evaluated. A plausible relationship between the antioxidant property of sunscreens and their ability to promote anti‐tumour activity was noted. The results call for a comprehensive analysis of skin cancer chemoprevention potential of currently used UV sunscreen agents around the globe to identify those with the best clinical profile.     

In vitro evaluation of the cutaneous penetration of sprayable sunscreen emulsions with high concentrations of UV filters

The aim of this study was to evaluate the possible penetration through human skin of organic and inorganic filters contained in sunscreen emulsions packaged in aerosol cans, using an in vitro method. Experiments were carried out on two different types of emulsion: W/Si and W/O. This study was conducted using static diffusion cells (Franz cells). The determination of organic UV filters [Methylene Bis Benzotriazolyl Tetramethylbutylphenol (MBBT); Bis-Ethylhexyloxyphenol Methoxyphenyl Triazine (BEMT); Diethylamino Hydroxybenzoyl Hexyl Benzoate (DHHB); Ethylhexyl Methoxycinnamate (EMC); and 2-Ethylhexyl Dimethyl PABA (ED-PABA)] was performed by High Performance Liquid Chromatography (HPLC). Therefore, it was important to develop a single analytical method for the quantification of the five organic filters with the aim of facilitating the experiment. The determination of inorganic filters [titanium dioxide (TiO₂) and zinc oxide (ZnO)] was performed using an emission spectrometric analysis method (ICP-OES). The HPLC and ICP-OES methods were validated. After a penetration test of 24 h duration, the results showed very low penetration only for two of the organic filters (maximum penetration of 1.21 μg cm⁻² h⁻¹ for EMC and 0.14 μg cm⁻² h⁻¹ for MBBT) and no penetration for the inorganic filters. Moreover, more than 50% of each sunscreen agent stayed on the surface on the skin. These results are consistent with those in the literature that presents similar experiments. This study showed that the sprayable sunscreen products developed, which contained high concentrations of UV filters, presented a low level of skin penetration.     

W/O emulsions: solvent phase separation for pH determination

The aim of this investigation was to find a measure for the phase separation by substances capable of rapidly demulsifying emulsions, which is necessary for the correct determination of the pH in cosmetic W/O emulsions. Results are reported for pH determination in the separated water phase of W/O emulsions containing metal soaps, micropigments and thermally unstable active agents. Of the substances investigated – solvents of different polarity and electrolytes – the best were mixtures of the polar butanol, which has a surfactant-like molecular structure and limited oil and water solubility, and the low polar trichloromethane which in particular has strong fat- and wax-dissolving properties. The combination achieved within 10 to 20 min at room temperature complete phase separations. Stable preparations, based on a polymer emulsifier (cetyl dimethicone copolyol), were demulsified as well as metal soap stabilized creams which characteristically contain a high amount of stabilizing waxes. The so-called solvent phase separation method was confirmed by using model preparations of known pH. During the investigation of model emulsions matching those used in practice, it was found, for example, that magnesium and aluminium stearate as well as various types of micronized zinc oxide which are dissolved or dispersed in the oil phase can shift the pH of the aqueous phase during manufacture from an initial acidic value through neutral to pH 8–10. Time-related and temperature-related changes in pH in formulations containing thermally unstable additives such as dihydroxyacetone or urea can be easily monitored.     

Double emulsions (W/O/W): physical characteristics and perceived intensity of salty taste

This study addressed the correlation between physical characteristics of double emulsions and sensory perception, as the microstructure of these systems may provide the mechanism to understanding the initial cause of the altered salty taste. Thus, double emulsions (W/O/W) were prepared using different volumes of the internal aqueous phase while maintaining the same fat and sodium contents in the evaluated systems. Polyglycerol polyricinoleate (PGPR) and tween 80 were used as hydrophobic and hydrophilic emulsifiers, respectively. After preparation, the samples were stored at 25 °C for 4 days and submitted to analysis of optical microscopy, distribution and polydispersity of the oil droplets size, electrical conductivity, rheological behaviour and sensorial analysis. It was found that the use of different emulsifier concentrations (PGPR) did not influence the physical characteristics of the emulsions with the same formulation, but emulsions with different internal aqueous phase concentrations presented different results. These distinct characteristics may have influenced sensorial perception, as the emulsion with higher internal phase concentration was considered saltier. Thus, it can be concluded that structural differences of the double emulsions can be used to decrease the sodium contents without perceivable changes in salty taste.     

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.