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     

Valeur d'un test in vitro d'incorporation de glucose marque pour l'estimation de l'activité antiseborrheique

Deux methodes d'estimation de l'activité antiséborrhéique, l'une in vitro , l'autre in vivo , ont été comparées en étudiant l'action de deux produits différents: le metbufen et la N-sébaçoylméthionine. Le test in vitro d'incorporation de glucose marqué est pratiqué sur la glande préputiale de rat isolée. In vivo , sur la peau de rat castré et supplémenté par l'énanthate de testortérone, l'effet de traitement topique est suivi par l'étude histologique des glandes sébacées.
Les résultats obtenus avec les deux produits essayés présentent un parallélisme permettant de conclure à la validité du test in vitro dans le screening de molécules à activité antiséborrhéique.
An in vitro test for the evaluation of antiseborrheic activity     

Cosmetic efficacy claims in vitro using a three-dimensional human skin model

A tissue engineered human skin equivalent is successfully used for the testing of raw materials and cosmetic formulations. This reconstructed skin is supported by a collagen-glycosaminoglycan-chitosan biopolymer in which human keratinocytes and dermal fibroblasts were co-cultured to form a tissue that closely reproduces the in vivo architecture of normal human skin and takes into account the complex interactions between epidermis and dermis. On the other hand, dermal and epidermal responses can be assessed separately in the dermal or skin equivalent. The three-dimensional model has important advantages compared to monolayer cell cultures and epidermis models in efficacy testing: (i) the possibility of long-term cultivation with repeated application of cream formulations containing bioactives and (ii) the similarity to human skin concerning the interaction between dermis and epidermis. These similarities include the expression of keratinocyte differentiation markers such as cytokeratin 10, filaggrin and transglutaminase, as well as proteins of the basal lamina (laminin, collagen type IV) and extracellular matrix proteins such as elastin. The efficacy of selected bioactives was determined using different endpoints, for example, stimulation of collagen synthesis in the dermal and skin equivalents was shown in comparison to vitamin C as a positive control. On skin equivalents using immunofluorescence techniques we also demonstrated stimulation of the differentiation marker filaggrin, which is important for skin moisturization. The results could be used for claim substantiation, e.g. for the treatment of dry and aged skin.     

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 of sunscreen agents from O/W emulsions

The effects of different emulsifiers on the in vitro permeation through human skin of two sunscreen agents [octylmethoxycinnamate (OMC) and butylmethoxydibenzoylmethane (BMBM)] were investigated from O/W emulsions. The test formulations were prepared using the same oil and aqueous phase ingredients and the following emulsifier and coemulsifier systems: Emulgade SE((R)) (ceteareth-12 and ceteareth-20 and cetearyl alcohol and cetyl palmitate) and glycerylmonostearate (emulsion 1); Brij 72((R)) (steareth-2), Brij 721((R)) (steareth-21) and cetearyl alcohol (emulsion 2); Phytocream((R)) (potassium palmitoyl-hydrolysed wheat protein and glyceryl stearate and cetearyl alcohol) and glycerylmonostearate (emulsion 3); Montanov 68((R)) (cetearyl glucoside and cetearyl alcohol) (emulsion 4); Xalifin-15((R)) (C(15-20) acid PEG-8 ester) and cetearyl alcohol (emulsion 5). The cumulative amount of OMC that permeated in vitro through human skin after 22 h from the formulations being tested decreased in the order 3 > 1 congruent with 4 > 5 > 2 and was about nine-fold higher from emulsion 3 compared with that from emulsion 2. As regards BMBM, no significant difference was observed as regards its skin permeation from emulsions 1, 3, 4 and 5, whereas formulation 2 allowed significantly lower amounts of BMBM to permeate the skin. In vitro release experiments of OMC and BMBM from emulsions 1-6 through cellulose acetate membranes showed that only emulsions 4 and 5 provided pseudo-first-order release rates only for OMC. The results of this study suggest that the type of emulsifying systems used to prepare an O/W emulsion may strongly affect sunscreen skin permeation from these formulations. Therefore, the vehicle effects should be carefully considered in the formulation of sunscreen products.     

Development of an in vitro method to determine rumen undigested aNDFom for use in feed evaluation

A portion of the forage cell wall, defined as neutral detergent fiber (NDF), is indigestible to anaerobic microbial digestion in ruminants. This fraction has been characterized by surface area relationships between acid detergent lignin, but recently, data have been published describing the dynamic nature of this relationship. In situ approaches have been described to estimate indigestible NDF, recovering the undigested NDF after long-term fermentations (uNDF). To be applicable to nutritionists and diet formulation, determining uNDF needs to be conducted in a commercial laboratory similar to other routine analyses of forage chemistry. A series of studies were conducted to evaluate an in vitro approach, to describe uNDF, which is repeatable and adaptable for routine feed evaluation. One hundred and two forages of several species were analyzed for NDF, acid detergent lignin, and uNDF. The uNDF was estimated by several approaches involving long-term fermentations and filtration steps to evaluate the length of time necessary to exhaust the digestible NDF and a filtration method necessary to maintain sample integrity by ensuring low sample loss and uniform recovery with residues from long-term in vitro fermentation. To determine uNDF, in vitro fermentations were conducted on 0.50 or 0.75 g of dry matter samples, in triplicate, at multiple time points up to 504 h and initially used Gooch crucibles with Celite (Thermo Fisher Scientific, Waltham, MA) as a filtering aid. The final method utilized a 1.5-µm pore size glass microfiber filter, which allowed for increased repeatability and improved sample recovery (lowest standard deviation). In this study, in vitro fermentations of 240 h were adequate to characterize and identify uNDF, which was repeatable among conventional forages provided the samples, after NDF analyses, were filtered through the same glass fiber filter. This approach could be adapted by commercial laboratories and would provide opportunities to develop near-infrared reflectance spectroscopy equations and calibrations.     

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.     

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.     

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)     

Synthesis of 2-N-oleoylamino-octadecane-1,3-diol: a new ceramide highly effective for the treatment of skin and hair

2-N-Oleoylamino-octadecane-1,3-diol is a new synthetic ceramide. The process enables a four-step preparation of 2-amino-octadecane-1,3-diol (D,L-erythro/threo) and a five-step synthesis of 2-N-oleoylamino-octadecane-1,3-diol (D,L-erythro/threo). The latter compound is related to ceramide 2 according to the classification of Downing. This route of synthesis is rapid, reproducible and uses low-cost starting materials. The new ceramide was analysed as follows: ¹H, ¹³C, ¹⁵N NMR spectra afforded an unambiguous characterization of the structure; additionally, these three methods identified the threo and erythro isomers. ¹H and ¹³C NMR permitted the measurement of the threo/erythro ratio (26.6/73.4 and 25.5/74.5, respectively). Chemical ionization mass spectrometry confirmed the expected mass of the pseudo-molecular ion (m/z= 566.5: [M + H]⁺) as well as the presence of different chain lengths other than the oleic moiety due to the fatty acid composition of the technical grade oleic acid used for the synthesis. Capillary gas chromatography measured the threo/erythro ratio (23.5/76.5) which agrees with the ¹H and ¹³C NMR data. Moreover, this method afforded the relative distribution of the different N-acylated chains. The properties of the new synthetic ceramide for the treatment of skin and hair were mainly assessed by two in vitro methods. The first measured the flux of water through lipid-extracted stratum corneum. The described ceramide showed high efficacy in decreasing water loss. The second recorded the friction coefficient of different types of hair: virgin, permanent-waved, and bleached. Treatment by the ceramide led to a strong decrease in this coefficient. This was particularly observed on unrinsed hair. These findings suggest two potential fields of application and beneficial contribution for the new ceramide: repairing the barrier to transepidermal water loss, and improving the surface properties of hair. Synthèse du 2-N-oleoylamino-octadécane-1,3-diol: nouveau céramide à haut potentiel dans le soin de la peau et du cheveu.     

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.     

A review of ageing and an examination of clinical methods in the assessment of ageing skin. Part 2: Clinical perspectives and clinical methods in the evaluation of ageing skin

With the advancement of skin research, today's consumer has increased access to technological information about ageing skin and hair care products. As a result, there is a rapidly increasing demand for proof of efficacy of these products. Recognizing these demands has led to the development and validation of many clinical methods to measure and quantify ageing skin and the effects of anti-ageing treatments. Many of the current testing methods used to research and evaluate anti-ageing product claim to employ sophisticated instruments alongside more traditional clinical methods. Intelligent use of combined clinical methods has enabled the development of technologically advanced consumer products providing enhanced efficacy and performance. Of non-invasive methods for the assessment and quantification of ageing skin, there is a plethora of tools available to the clinical researcher as defined by key clinically observed ageing parameters: skin roughness and surface texture; fine lines and wrinkles; skin pigmentation; skin colour; firmness and elasticity; hair loss; and proliferative lesions. Furthermore, many clinical procedures for the evaluation of ageing skin treatments are combined with invasive procedures, which enable added-value to claims (such as identification and alteration of biochemical markers), particularly in those cases where perception of product effect needs additional support. As discussed herein, clinical methods used in the assessment of skin ageing are many and require a disciplined approach to their use in such investigations.     

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.     

A comparison of the in vitro permeability properties of human and some laboratory animal skins

The in vitro permeability of human and some common laboratory animals'skin has been measured to two chemicals with different physico-chemical properties: [³H] water, a small polar molecule and [¹⁴C]paraquat dichloride (the ion), a fully ionised divalent cation. All the skins examined had similar permeabilities to [³H]water: relative to human skin the greatest difference (x4.8) was measured through guinea pig skin. However, relative to human skin, all the animals were much more permeable to [¹⁴C]paraquat, with factors of difference ranging between 40 (haired rat) and 1460 (hairless mouse). The in vitro technique allows the permeability of human and animal skin to be compared, facilitating extrapolation to Man of effects seen in animals during dermal exposure studies.
Comparaison de la permeabilite in vitro de la peau humaine et de la peau de certains animaux de laboratoire     

In vitro and in vivo assessment of the effect of Laurus novocanariensis oil and essential oil in human skin

Laurus novocanariensis is an endemic plant from the Madeira Island forest that derives a fatty oil, with a strong spicy odour, from its berries that has been used for centuries in traditional medicine to treat skin ailments. This work aimed to investigate the effect of the application of both the oil and its essential oil on normal skin, to assess their safety and potential benefits. Diffusion studies with Franz cells using human epidermal membranes were conducted. The steady‐state fluxes of two model molecules through untreated skin were compared with those obtained after a 2‐h pre‐treatment with either the oil or the essential oil. Additionally, eleven volunteers participated in the in vivo study that was conducted on the forearm and involved daily application of the oil for 5 days. Measurements were performed every day in the treated site with bioengineering methods that measure erythema, irritation and loss of barrier function. Slightly higher steady‐state fluxes were observed for both the lipophilic and the hydrophilic molecule when the epidermal membranes were pre‐treated. Nevertheless, such differences had no statistical significance, which seems to confirm that neither the oil nor the essential oil impaired the epidermal barrier. Results collected with the Chromameter, the Laser Doppler Flowmeter and the visual scoring are in agreement with those established in the in vitro study. They indicate that the repeated application of the oil did not cause erythema, because the results observed in the first day of the study were maintained throughout the week. Application of the oil did not affect the skin barrier function, because the transepidermal water loss remained constant throughout the study. The stratum corneum hydration was slightly reduced on days 4 and 5. This work shows that both the oil and the essential oil were well tolerated by the skin and did not cause significant barrier impairment or irritation.     

Production of steviosides in ex vitro and in vitro grown Stevia rebaudiana Bertoni

Stevia rebaudiana Bertoni plants grown in vitro and ex vitro were investigated for variations in the profile of steviosides in their leaves, shoots, roots and flowers. Steviosides were extracted by hydrolysis and esterification, evaporated to dryness and dissolved in methanol for quantitative analysis by high‐performance liquid chromatography (HPLC). The HPLC analysis and separation profiles indicated the presence of several different steviosides, predominantly eight known sweet diterpene glycosides. The most abundant compounds identified were steviolbioside, stevioside, rebaudiosides C and A and dulcoside A. The highest stevioside contents were recorded in 1‐month‐old greenhouse leaves (64.80 g steviolbioside kg^?1 dried plant material) and in vitro leaves (0.99 g rebaudioside A kg^?1 dried plant material). The recovery of steviosides from the leaves by methanol extraction was 90%. This is the first time that the eight predominant sweet diterpene glycosides in the various plant parts of S. rebaudiana have been investigated. Copyright © 2006 Society of Chemical Industry     

Carotenoid bioaccessibility from nine raw carotenoid-storing fruits and vegetables using an in vitro model

BACKGROUND: Many techniques exist for processing fruits and vegetables. The impact of these processes on nutritional qualities of the food can be considerable, however. Given the benefits of eating raw foods, nutrient sources need to be identified that deliver substantial benefit without cooking. In this study a survey of carotenoid bioaccessibility was carried out in order to additionally evaluate the impact of their distinctive storage structures (chromoplasts) on bioaccessibility. RESULTS: Per cent carotenoid bioaccessibility varied among the nine raw, whole fruits and vegetables evaluated, with values of 1–39% for lycopene, 18–20% for α‐carotene, 7–49% for β‐carotene, 9–59% for lutein, 4–22% for violaxanthin and 47–96% for phytoene. Per 100 g of food, grapefruit and watermelon imparted the most lycopene (69 and 64 µg respectively), carrot the most α‐carotene (559 µg), β‐carotene (1078 µg), lutein (91 µg) and phytoene (23 mg) and mango the most violaxanthin (177 µg). Digestive stability averaged over 80%, except for the xanthophylls, which exhibited a wider and lower range of stabilities. CONCLUSION: These data identify raw food sources for carotenoid bioaccessibilities comparable to those of other foods accomplished by substantial processing. The information presented here also has application in identifying appropriate plant‐breeding goals and optimal sources for commercial carotenoid isolations. Copyright © 2012 Society of Chemical Industry     

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.     

Evaluation of a gas in vitro system for predicting methane production in vivo

Methane production from ruminant livestock varies with the diet as a result of factors such as dry matter intake, diet composition, and digestibility. To estimate the effect of dietary composition and feed additives, CH₄ production can be measured in vitro as a first step because large numbers of samples can be incubated and analyzed at the same time. This study evaluated a recently developed in vitro method for prediction of in vivo CH₄ production by examining the relationship between predicted and observed CH₄ production values. A total of 49 different diets (observations), used in previous 13 in vivo studies, were selected to include diets varying in nutrient composition. Methane production was measured in all in vivo studies by respiration chambers or the GreenFeed system (C-Lock Inc., Rapid City, SD). Overall, the in vitro system predicted CH₄ production well (R² = 0.96), but the values obtained were slightly underestimated compared with observed in vivo values (mean 399 L/d compared with 418 L/d: root mean square prediction error = 51.6 L/d or 12.3% of observed mean). Further analysis of the effect on residuals showed no significant relationship between CH₄ production and most factors known to affect CH₄ production such as dry matter intake, digestibility, and dietary concentrations of fat and starch. However, some factors included in the model were not well predicted by the system, with residuals negatively related to neutral detergent fiber concentration and positively related to concentrate proportion. The in vitro system can thus be useful for screening diets and evaluation of feed additives as a first step that can be best interpreted when feeding cows at maintenance level.