Roland Roguet

Regional variation in percutaneous absorption in man: measurement by the stripping method

SummaryThe influence of anatomic site on the relationship between total penetration of a molecule and its quantities present in the stratum corneum (SC) 30 min after application was quantified in an in vivo study. For each site, six male volunteers received two symmetrical applications of 1,000 nmol benzoic acid 14C to an area of 1 cm2 for 30 min. The first application permitted measurement of total absorption of benzoic acid within 4 days (urinary excretion method), while the second enabled determination of the quantity of benzoic acid in the SC at the end of the application time. Total penetration according to site is: back < arm < chest < thigh < abdomen < forehead, (with the forehead being three times more permeable than the back). Whatever the sites and the origin of the differences observed, the results show that the single measurement of the amounts of a compound present in the SC at 30 min postapplication appears sufficient to predict its total penetration, these two parameters being linearly correlated (r=0.97, P<0.001).

Episkin, a reconstituted human epidermis for assessing in vitro the irritancy of topically applied compounds

The Episkin epidermal system is a reconstituted organotypic culture of human keratinocytes forming a multilayer differentiated epidermis on a collagen matrix. This standardized model was used to measure various parameters reflecting irritation observed in vivo following topical application of a variety of surfactants. A comparison of the results with historical data from ocular irritation testing in vivo showed a strong correlation for cytotoxic potential (r = 0.93; n = 23; P < 0.00001) and damage to the epithelial barrier function (r = 0.87; n = 20; P < 0.00001). In addition, the proportion of agents for which the irritative potential is underestimated is low. With regard to cutaneous irritation, the release of a major pro-inflammatory cytokine, interleukin 1alpha, correlated with the degree of irritation observed in vivo (r = 0.81; n = 20; P < 0.0001). Given its suitability to testing a wide variety of parameters reflecting irritation and the fact that all types of topical formulations can be applied to the reconstructed epidermal surface, the Episkin model should be particularly useful for assessing the tolerability of topical agents in vitro.

The measurement of the stratum corneum reservoir: a simple method to predict the influence of vehicles on in vivo percutaneous absorption

The influence of nine vehicles on in vivo percutaneous absorption of benzoic acid has been studied in the hairless rat. Although the vehicles used were simple in composition, the total amount of benzoic acid which penetrated varied by a factor of 50. A weak relationship was found between penetration of benzoic acid and its maximum solubility in the different vehicles. A linear relationship was demonstrated between the amount of benzoic acid present in the stratum corneum at the end of a 30 min application period and the total amount absorbed over 4 days. We suggest that the influence of a vehicle on the overall absorption of a substance can therefore be predicted by simply measuring the amount present in the stratum corneum at the end of a 30 min application period.

Refinement of the Episkin protocol for the assessment of acute skin irritation of chemicals: follow-up to the ECVAM prevalidation study.

The Episkin model took part in the prevalidation study on in vitro tests for acute skin irritation of chemicals, which was carried out during 1999 and 2000. This prevalidation study was co-ordinated and supported by the European Centre for the Validation of Alternative Methods (ECVAM). During Phase 1 and Phase 2 of this study, reproducibility and transferability of the method were verified. Unfortunately, the performance of the method in terms of predictive ability was considered insufficient, due to a low specificity. In order to improve the performance of the Episkin method, the existing protocol was refined. This refinement consisted in reducing the exposure time of epidermis with chemicals. Sensitivity, specificity and accuracy of the new method were 70, 80 and 75%, respectively, thus meeting the acceptance criteria as defined by the Management Team. The Episkin method is now ready to enter a validation study of in vitro tests for acute skin irritation.

Episkin: An in vitro model for the evaluation of phototoxicity and sunscreen photoprotective properties

Exposure to UV radiation has been shown to induce cutaneous biological changes and chemical modifications of exogenous compounds. The application of sunscreen limits UV-induced cutaneous injury. Besides damages can also be elicited by some chemicals following UV irradiation (phototoxic agents). The effects of UVA and UVB irradiation have been studied in vitro on a reconstituted epidermis (EPISKIN) for cytotoxicity (MTT conversion test) and release of a proinflammatory mediator, interleukin 1alpha. When compared with irradiated human keratinocyte monolayers, the results obtained with EPISKIN showed the protective function of the stratum corneum to UVB. Topical application of a UVB sunscreen directly on EPISKIN stratum corneum showed a concentration-dependent relationship between the active ingredients and UVB-induced damages evaluated by both parameters. When chlorpromazine was tested on EPISKIN, an increase in cytotoxicity and release of interleukin 1alpha followed UVA irradiation. A non-cytotoxic dose of 10 J/cm(2) of UVA induced a 20-fold decrease in the chlorpromazine IC50. The chlorpromazine-induced inflammatory reaction was also reflected by a more intense release of interleukin 1alpha in the underlying media of EPISKIN. Thus this proinflammatory mediator seems to be relevant as a phototoxic marker. The EPISKIN model may be a useful tool in the study of UV-induced cutaneous damage. The design of this study represents an advance in non-animal test development and could be of great interest in the determination of phototoxic or photoprotective effects of compounds in vitro.

The use of in vitro reconstituted human skin in dermotoxicity testing

As they simulate the multilayer tissue architecture present in vivo, three-dimensional (3D) cultures of skin epithelial cells represent an attractive alternative for in vitro irritancy testing. In this study histology of non-commercial (Skin recombinant on de-epidermized dermis) and commercial models (Episkin, ZK 1300, Living Skin Equivalent) was compared. All 3D models possess a stratified epidermis with a differentiated stratum corneum. Their barrier function has been demonstrated by comparison of surfactants cytotoxicity on the 3D models and on human keratinocyte monolayer culture, and by the evaluation of the tritiated water penetration. All the 3D models showed permeability higher than fresh skin, the skin recombinant on DDED being the less permeable. However, reconstituted skin models possess several advantages for dermotoxicity testing, including the standardized nature of the epithelia and their suitability for testing water-insoluble compounds.

Measurement of inflammatory mediators produced by human keratinocytes in vitro: A predictive assessment of cutaneous irritation

Two inflammatory mediators, interleukin 1alpha (IL1alpha) and prostaglandin E(2) (PGE(2)), were determined by radioimmunoassay in both intracellular and extracellular compartments from normal human keratinocytes in high-density culture. Cells were subjected to different inflammatory stimuli: UVB irradiation and detergent injury by sodium dodecyl sulphate (SDS). UVB irradiation produced intracellular IL1alpha synthesis, and its dose-dependent release in the medium. Similar results were obtained with SDS. The SDS stimulus was time dependent, and the following mechanisms were observed: (i) increased production of intracellular inflammatory mediators; and (ii) progressive release of these mediators in the medium. With SDS, production and release of PGE(2) peaked below the lethal doses (assessed by the neutral red bioassay). Therefore, the measurement of IL1alpha and PGE(2), both intra- and extracellularly may be a useful in vitro method for assessing the irritative potential of topically applied agents.

An interlaboratory study of the reproducibility and relevance of Episkin, a reconstructed human epidermis, in the assessment of cosmetics irritancy

Reconstructed epidermal models are particularly suited to assessing the tolerance of cosmetic and dermatological products in vitro. Their production in kit form makes them available for screening both raw ingredients and finished products since a large amount of material can be tested whatever their physicochemical properties. However, two conditions must first be fulfilled: they must give reproducible results and be relevant to data obtained in vivo. We tested the reproducibility of data obtained using the Episkin(R) model [cytotoxicity evaluated by the MTT conversion and the release of one of the most active proinflammatory mediator, interleukin 1alpha(ILalpha)] on different batches and in various research laboratories. After topical application of sodium dodecyl sulfate (SDS) the overall variability of the IC(50) results was 14% of the mean value. Within a given centre and a given batch, the coefficient of variation attributable to the dispersion between kits was 6% for the SDS IC(50) determination and 7% for IL1alpha release measurement. The results obtained with Episkin were then compared with data from primary human skin irritancy testing (48-hr occlusion test and clinical assessment) and rabbit irritancy evaluation (Draize cutaneous test). Analysis of the results obtained with 38 cosmetic products (oils, gels, emulsions, mascaras and shaving foam, including 19 irritants) revealed good concordance with data obtained in humans. Considering the release of IL1alpha as in vitro parameter, the test sensitivity, specificity and concordance were 68, 79 and 74%, respectively.

Iron chelation can modulate UVA-induced lipid peroxidation and ferritin expression in human reconstructed epidermis.

Background/Purpose: As ferritin has been identified as an important factor in antioxidant defense in cultured human skin cells, we evaluated UVA‐induced lipid hydroperoxides (LPO) production and ferritin expression in reconstructed human epidermis in vitro.

The Use of Standardized Human Skin Models for Cutaneous Pharmacotoxicology Studies

//8RReconstructed skin and epidermis models are finding increasingly numerous applications in cosmetology and dermatology. In particular, they are currently employed to assess the tolerability and efficacy of raw materials and formulations, in conditions approaching those of normal use. Importantly, the use of such models greatly reduces the need for animal testing. Various models of reconstructed skin and epidermis have been developed [1–4] and some are now produced industrially [5–8]. They are used for the prediction of cutaneous irritancy and, to a lesser extent, percutaneous absorption and cutaneous metabolism. However, before being officially recognized as valid alternative methods and entering routine use, standard protocols must be developed to assess their reproducibility and performance as compared with in situ human studies.