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Test Guidelines for In Vitro Assessment of Dermal Absorption and Percutaneous Penetration of Cosmetic Ingredients
Abstract
The following guidelines present an in vitro alternative, which uses excised animal or human skin, to experiments conducted in vivo on animals or humans for the assessment of dermal absorption and percutaneous penetration. Both aspects are essential for the risk assessment of chemicals which may contact and subsequently penetrate the skin.There are scientific and ethical reasons for this type of test. Animal skin is obtainable in sufficient quantities to allow replication of experiments. Extrapolation of data obtained from appropriate animal skin is an acceptable alternative but excised human skin obtained from surgery may be used when available. Furthermore, no risk or harm for living creatures is associated with this type of test.These guidelines were developed by a Task Force of COLIPA, the European Cosmetic, Toiletry and Perfumery Association (COLIPA, 1995). Each of the members of the Task Force has several years' experience in the assessment of dermal absorption and percutaneous penetration of chemicals used in products.These guidelines utilize the principles outlined in the ‘OECD New Guideline Proposal on In Vitro Percutaneous Absorption of Chemicals’.
... The skin penetration and safety of nanometric zinc oxide and titanium dioxide incorporated in a cosmeceutical cream, specifically designed to retard skin actinic damage in tropical Oculocutaneous albinism was investigated ex-vivo through simulated actinically damaged porcine skin. Franz diffusion cells were used in an experiment modeled in line with related work done by Diembeck et al (1999); the OECD guideline, document no 428 and the EU opinion SCCNFP 0750/03. The following experiment was distinguished by its use of simulated actinic damaged skin characteristic in albinism in the tropics. ...
... The cosmeceutical employed nanometric metallic oxides of zinc and titanium for their broad spectrum sunscreen effects 7,8 . It also incorporates Aloe Excelsa, Trichilia Emertica and Myrothamnus Flabellifoliaethnic herbal extracts, which have proven anti-inflammation, wound healing and antiaging properties respectively 9 , in an encompassing treatment to retard actinic damage in albinistic persons that took consideration of variances in skin types and geographical conditions. This study was carried out between 2013 and 2014 in tropical conditions and the experiments were done using actinically damaged porcine skin, whereby actinic damage was induced by an alkaline solution and a xenon arc solar simulator. ...
... The ex vivo laboratory experiments in this investigation were conducted with reference to related studies by Diembeck et al(1999) 10 13 . ...
The skin penetration and safety of nanometric zinc oxide and titanium dioxide incorporated in a cosmeceutical cream, specifically designed to retard skin actinic damage in tropical Oculocutaneous albinism was investigated ex-vivo through simulated actinically damaged porcine skin. Franz diffusion cells were used in an experiment modeled in line with related work done by Diembeck et al (1999); the OECD guideline, document no 428 and the EU opinion SCCNFP 0750/03. The following experiment was distinguished by its use of simulated actinic damaged skin characteristic in albinism in the tropics. The subsequent analytical studies were modified, since skin stripping was not practical on damaged skin. Analysis for the nanomaterials was conducted on both the Franz cell receptor phase fluid as well as extractions from the entire skin tissue material after the diffusion process. Quantification analysis for the recovered titanium was done by ICP-AES and the zinc was assayed using Flame AAS. The total recoveries of zinc from the skin extracts ranged between101.35-103.20% of the total zinc applied. The amounts of zinc recovered were comparable in treated, untreated skin, and the receptor phase. Mean total recoveries of titanium for all categories ranged between 98.73% and 99.24%, of the total applied titanium. No titanium was found in the receptor phase. The results show that neither nanometric titanium nor zinc ions can penetrate both normal and actinically damaged porcine skin, thus, suggesting minimal systemic exposure when used in treatments for actinically damaged albinistic persons.
... Examples of permeation enhancers are terpenes contained in essential oils (e.g., menthol), which increase the percutaneous permeation parameters [40]. Moreover, to confirm the safety of a cosmetic preparation, tests to assess the penetration and permeation through animal or human skin are performed, which constitute an acceptable alternative to in vivo tests [41]. Another way to assess the safety of cosmetics and prevent systemic toxicity is to conduct toxicokinetic and toxicodynamic studies [42]. ...
Kombucha is a non-alcoholic beverage, that is increasingly used in the cosmetic industry. The available literature reports the positive effects of kombucha on the skin, in particular its antiox-idant action. However, there is a lack of information on skin permeation and the accumulation of active ingredients showing such effects. Skin aging is largely dependent on oxidative stress, therefore in our study we assessed the ex vivo permeation of two types of kombucha (green and black tea) through porcine skin. The antioxidant activity (DPPH, ABTS, FRAP methods) and total poly-phenol content of these extracts were determined before and after permeation testing. Moreover, the content of selected phenolic acids as well as caffeine was assessed. Skin permeation was determined using a Franz diffusion cell. The antioxidant activity of both Kombuchas was found to be high. In addition, gallic acid, chlorogenic acid, protocatechuic acid, coumaric acid, m-hydroxyben-zoic acid, and caffeine were identified. A 24-h ex vivo study showed the permeation of some phe-nolic acids and caffeine and their accumulation in the skin. Our results confirm the importance of studying the skin permeation of what are still little known ingredients in cosmetic preparations. Evaluation of the accumulation of these ingredients can guarantee the efficacy of such preparations.
... Therefore, this study utilizes the in vitro transdermal absorption method to evaluate the skin's absorption of drugs. The basic testing principles are conducted similarly to those in W. Diembeck et al. [24]. ...
This study develops a multi-functional hydrogel with a dual injection system based on the adhesive and self-healing properties of the byssus excretion found in mussels. Through precisely controlling the composite cross-linking hydrophobic association (HA) structure composed of A and B solutions, a high-strength, temperature-sensitive injectable hydrogel can be obtained, and it has good self-healing properties. The main composition of A solution contains the surfactant SDS, which can form amphiphilic micelles, the strength increasing component stearyl methacrylate (C18), and NIPAAm, which provides thermo-sensitivity. Solution B contains dopamine acrylate (DAA), which has self-healing properties, and ferric chloride (FeCl3), which is a connecting agent. The rheological behavior shows that when the temperature is increased from 25 °C to 32 °C, the gel can be completed in seven minutes to form a composite hydrogel of NIPAAm-DAA-HA. When NMR identification was conducted on composite DAA, it was found that when comparing DAA and dopamine hydrochloride there were new peaks with specific characteristics, which confirm that this study successfully prepared DAA; swelling tests found that swelling could surpass a rate of 100%, and a higher ratio of crosslinking agent decreased the amount of moisture absorbed; the results of the compression test showed that the addition of hydrophobic micelles C18 effectively enhanced the mechanical properties of hydrogel, allowing it to withstand increased external stress; the adhesiveness results show that an increase in the catechol-Fe3+ concentration of the NIPAAm-DAA-HA hydrogel results in an increased adhesiveness of 0.0081 kg/cm2 on pig skin; the self-healing tests show that after taking damage, NIPAAm-DAA-HA hydrogel can be reactivated with catechol-Fe3+ and self-heal at a rate of up to 70% after 24 h; antibacterial tests show that hydrogel has good bacterial resistance to against E. coli, staphylococcus epidermidis, and bacillus cereus; through in vitro transdermal absorption, it can be seen that the release ability of drugs within the hydrogel can reach up to 8.87 μg/cm2. The NIPAAm-DAA-HA hydrogel prepared by this study performed excellently in both adhesion and self-healing tests. The thermo-sensitive and antibacterial properties can be applied to the treatment of deep wounds and address some of the flaws of traditional wound dressings.
... Uzyskiwane wyniki zależą w dużym stopniu od rodzaju użytej membrany, a w przypadku skóry od miejsca jej pobrania [28]. Zwalidowana metoda, zalecana przez UE i OECD [29][30][31][32] oparta jest na skórze określonego gatunku świń pobranej z ucha zwierzęcia bezpośrednio po uboju. ...
Obok metod in vivo, ex vivo i matematycznych, szeroko stosowane do badania biodostępności stosowanych zewnętrznie leków i substancji kosmetycznie czynnych są metody in vitro częściwo zwalidowane, a przy tym nie wymagające jakiegokolwiek kontaktu z żywym organizmem. W niektórych stosuje się skórę ludzką lub zwierzęcą,
jednak pochodzącą ze źrodeł odpadowych, np. pooperacyjnych lub od osób martwych. Stosuje się takżę skórę pochodzącą ze zwierząt zabitych w celach konsumpcyjnych.
Używane metody można podzielić na trzy grupy: komory poziome, pionowe i metodę PAMPA. Komory poziome, zwane niekiedy komorami Flynn’a i pionowe znane powszechnie jako komory Franza, wykorzystują przenikanie przez membrany polimerowe, lipidowe lub naturalną skórę. Pozwalają one na uzyskanie stosunkowo dokładnych wyników, modelujących warunki rzeczywiste. Metoda PAMPA wykorzystuje
podobne membrany polimerowe lub lipidowe. Mniejsza dokładność metody rekompensowana jest przez niskie koszty i prostotę wykonania. Stosowana jest glównie do celów skriningowych. Metody in vitro wymagają szczególnej dokładności wykonania.
Temperatura, warunki mieszania, rozpuszczalność permeanta i inne czynniki mogą wpływać na wynik. Jest to powodem znacznych rozbieżności w rezultatach uzyskiwanych w różnych ośrodkach badawczych. Nie dotyczy to oczywiście metod zwalidowanych, dla
których wszystkie parametry są ściśle określone.
In addition to in vivo, ex vivo and mathematical methods, in vitro methods are widely used to test the bioavailability of externally used drugs and cosmetically active substances. They do not require any contact with a living organism. Some of them use human or animal skin; however, it comes from waste sources, e.g. postoperative or dead
people. Leather from animals killed for human consumption is also used. The methods used can be divided into three groups: horizontal chambers, vertical chambers and the PAMPA method. Horizontal chambers, sometimes called Flynn chambers, and vertical chambers, commonly known as Franz chambers, use penetration through polymer,
lipid or natural skin membranes. They make it possible to obtain relatively accurate results that model natural conditions. The PAMPA method uses similar polymer or lipidic membranes. The lower accuracy of the technique is compensated by the low cost and simplicity of implementation. It is mainly used for training purposes. In vitro methods require extraordinary precision. Temperature, mixing conditions, permeant
solubility, and other factors may influence the result. This is the reason for significant discrepancies in the results obtained in various research centres. Of course, this does not apply to validated methods for which all parameters are strictly defined.
Odor is an important indicator of human milk (HM) quality, with a proven function. Here, the effect of inter-individual nutrient differences on key odor-active compounds (OACs) and odor attributes of HM samples was investigated using flavor analysis techniques and correlation network analysis. A total of ninety-four OACs were identified from 30 HMs, of which 24 key OACs could represent the basic odor characteristics of HMs. Fat content was closely related to the amounts of OACs, with aldehydes being the most abundant species and having the highest correlation with fat content. Of them, nonanal and octanal were the most important OACs in HM, having both high flavor dilution factor (2 ∼ 64, 4 ∼ 128) and odor activity values (<1 ∼ 37, 2 ∼ 36) in most samples. Additionally, different pattern of synergism between key OACs contribute to each odor attribute of HM. These findings will provide insights for subsequent in-depth studies of HM flavor.
Plaque psoriasis is characterized by an abnormal thickening of the epidermis, which causes great difficulties for traditional topical drug delivery. Microneedles can pierce the thickened epidermis and deliver drugs to the skin for psoriasis treatment. Tacrolimus is a poorly water-soluble immunosuppressant used for the treatment of psoriasis. In this study, tacrolimus (TAC) nanocrystals (NCs) were produced using a bottom-up technique that dispersed TAC into a sodium hyaluronate-based microneedle patch (MNP), and its therapeutic efficacy was evaluated. The average particle size of the TAC NCs was 259.6 ± 2.3 nm. The mechanical strength of the microneedles was 0.41 ± 0.06 N/needle, which was sufficient to penetrate psoriatic skin. Microneedles were detached from the substrate 10 min after insertion into the psoriasis skin with an insertion depth of 258.8 ± 14.4 μm. The intradermal retention of the MNP (8.40 ± 0.33 μg/cm²) was six times that of the commercial ointment (1.40 ± 0.12 μg/cm²). In pharmacodynamic experiments, results indicated improvement in the phenotypic and histopathological features and reduction in the level of TNF-α, IL-17A, and IL-23 of psoriatic skin treated with MNPs. Therefore, MNPs loaded with TAC NCs may be a promising approach for psoriasis treatment.
Infants who accustomed to consume human milk can hardly adapt to the odor of infant formula in a short time and prefer the odor of human milk. In this study, the sensory-directed analysis was used to investigate the odor differences between human milk and infant formula. Aroma extraction dilution analysis (AEDA) results showed that carbonyl compounds and alcohols were the most important components with the higher dilution factors (FD) in human milk and infant formula. There were 14 key aroma active compounds (OAV ≥ 1) in human milk, like octanal, linalool, benzaldehyde, and furfural, while 11 in infant formula, like hexanal, 1-octen-3-one, (E)-2-octenal, and octanal. The aroma recombination and omission experiment further revealed that compounds such as (E)-2-decenal, linalool, 2-furanmethanol, 2-pentylfuran, (E,E)-2,4-heptadienal, nonanal, (E)-2-nonenal, and 1-octen-3-one were the major reason for the odor difference between human milk and infant formula.
Pickering emulsions (PEs) are attracting increasing attention in the areas of food, cosmetic and pharmaceutical applications owing to their surfactant-free and eco-friendly nature. Herein, PEs stabilized by chitosan/collagen peptides (CH/CP) nanoparticles were assessed as green surfactant-free vehicles for the topical delivery of cannabidiol (CBD), a highly lipophilic unstable drug that is finding an increasing appeal in the cosmetic market. The influence of the oil phase volume fraction (φ) and the oil type on the emulsion properties, stability, rheological properties, as well as on the ex-vivo skin absorption of CBD was evaluated. The PE prepared with olive oil (φ= 0.6) exhibited elastic gel-like properties and demonstrated long-term stability after 5 months of storage, with a CBD content of 99.45% of the initially added amount. The skin absorption studies showed that CBD was retained in high amounts in the stratum corneum, while the CBD skin permeation was extremely low, indicating that the produced formulations are suitable as topical delivery vehicles. ATR-FTIR examination of the treated skin samples confirmed that the produced PEs were able to overcome the stratum corneum barrier. These findings suggest that the PEs stabilized with CH/CP nanoparticles provide an effective surfactant-free alternative for the topical delivery of CBD.
Peer Reviewed http://deepblue.lib.umich.edu/bitstream/2027.42/41513/1/11095_2004_Article_306362.pdf
The use of in vitro preparations of human skin to study percutaneous absorption is widespread. Yet, up to the present time, little has been done to systematically validate this model and demonstrate the extent to which it mimicks in vivo absorption. In this study, the permeability of 12 organic compounds has been evaluated in excised skin and the results compared to those obtained previously by others in living man. With special emphasis being given here to duplicating in vivo conditions, it was possible to demonstrate an excellent qualitative agreement between the two methods. In all cases, the absorption pattern determined in vitro rather precisely paralleled the pattern which was obtained in vivo. Quantitative agreement between the two sets of data was less than perfect, although the in vitro method adequately distinguished compounds of low permeability from those of high permeability and ranked then in approximately the same order found in vivo. This systematic comparison of in vitro with in vivo data was clearly shown how accurately in vitro absorption studies can reflect the living state.
A flow-through finite-dose diffusion cell has been designed for use in transdermal drug delivery research. The diffusion cell consists of an upper donor chamber and a lower receiver compartment through which a continuous supply of fresh solvent flows. The flow is directed to an automatic fraction collector. To validate the flow-through cell, its performance was compared directly against that of a conventional single-reservoir Franz cell. Homologous alkyl p-aminobenzoates were diffused through dimethylpolysiloxane membranes, and permeability coefficients increased with increasing chain length, reaching a plateau at the butyrate ester for both types of cells. This behavior suggests a shift from membrane-controlled diffusion to boundary layer control. Permeation of the butyrate and valerate compounds was significantly faster when the flow-through cell was used, suggesting that better mixing is obtained through the flow-through cell design. Considering the advantages offered in terms of time and labor saved through its use, the flow-through cell with automatic fraction collector appears to be a viable alternative to the conventional Franz cell.
Existing in vitro skin penetration cells based on the Franz cell were assessed for: (a) uniformity of stirring; and (b) receptor phase volume. Based on the results of these experiments two flow-through in vitro penetration cells were designed to meet the following specifications: (a) small receptor phase volumes; (b) instantaneous stirring; (c) variable skin surface areas; and (d) compatibility with current stirring apparatus.
Synopsis
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
A series of studies was conducted to examine the percutaneous absorption, distribution, excretion, and hemolytic activity of n-butoxyethanol (BE). Rats receiving a subcutaneous dose of 14C-labeled BE excreted the radioactivity in the urine (79%), expired air (10%), and feces (0.5%) within 72 hr. Of the organs analyzed, thymus and spleen showed elevated specific radioactivities as compared with blood. A percutaneous application of BE on rats, under nonocclusive conditions, showed 25-29% absorption within 48 hr. Peak blood levels of BE occurred at 2 hr after application; butoxyacetic acid (BAA) was found to be the major metabolite. Comparison of in vitro skin penetration data showed the following absorption pattern of BE: hairless rat much greater than pig greater than human skin. Hemolysis and associated hematological changes were noted in the rats which received single dermal applications of 260-500 mg/kg of BE. In vitro, BAA showed markedly greater hemolytic ability on rat erythrocytes than did BE. Human erythrocytes showed no hemolysis when incubated with BE or BAA at concentrations that are hemolytic to rat erythrocytes. An intravenous dose of 62.5 mg/kg of BE does not result in hemolysis or hemoglobinuria in the rat. The rat may be an animal model with increased susceptibility to the effects of BE compared with humans because of its rapid percutaneous absorptive ability and its greater hemolytic sensitivity.
The absorption of a pyrethroid insecticide, cypermethrin, through rat skin has been measured both in vitro and in vivo. Cypermethrin did not penetrate in vitro through whole skin but did penetrate epidermal membranes. The in vitro absorption was influenced by the choice of receptor fluid in the glass diffusion cell. There was good agreement between in vivo and in vitro data using 50% aqueous ethanol, 6% Volpo 20, or total calf serum receptor fluids. Rat epidermal membranes in vitro were more than 20 times more permeable to cypermethrin than human epidermal membranes, indicating that cypermethrin would be less readily absorbed in humans than in the rat. The percutaneous absorption in vitro technique using epidermal membranes was successfully used with this lipophilic chemical to predict the in vivo absorption in the rat.
The relationship between the percutaneous penetration of four chemicals and transepidermal water loss (TEWL) was investigated in vivo in man as a function of anatomic site. The findings showed an appreciable difference in the permeability of the skin from one site to another with regard to both water loss and chemical penetration. In addition, independent of the physicochemical properties of the molecules administered, there was a linear relationship between TEWL and penetration. These data confirm both the importance of anatomic site in the degree of permeability of the cutaneous barrier and the utility of determinations of TEWL and percutaneous absorption in the evaluation of its functional condition.
Water permeability constants (Kp) were determined with skin from human cadavers. No difference was seen in Kp values from unfrozen skin or from skin frozen for a few days. Human skin could usually be stored at -20 degrees C for up to a year with no change in water permeability, but in some cases apparent deterioration of the barrier was observed. A rapid procedure was developed for checking barrier integrity of skin in diffusion cells before a penetration study. The percent of the water dose absorbed after 20-min contact with skin correlated with water Kp values. Changes in water permeation through human skin agreed with changes in the absorption of seven test compounds of varying solubility properties (acetylsalicylic acid, benzo(a)pyrene, cortisone, DDT, nicotinic acid, propylene glycol, and testosterone). Water permeation is therefore considered to be a good indicator of potential changes in the barrier integrity of human skin. No correlation was observed in Kp values and other characteristics of the donor skin samples such as age, sex, race, and length of time before skin harvest.