percutaneous penetration - methodological considerations

MiniReview

Percutaneous Penetration - Methodological ConsiderationsRikke Holmgaard1, Eva Benfeldt2 and Jesper B. Nielsen3

1Department of Orthopedic Surgery, Køge Sygehus, Køge, Denmark, 2Department of Dermatology, University of Copenhagen, Roskilde Hospital,Roskilde, Denmark and 3Institute of Public Health, University of Southern Denmark, Odense, Denmark

(Received 4 October 2013; Accepted 18 December 2013)

Abstract: Studies on percutaneous penetration are needed to assess the hazards after unintended occupational skin exposures toindustrial products as well as the efficacy after intended consumer exposure to topically applied medicinal or cosmetic products.During recent decades, a number of methods have been developed to replace methods involving experimental animals. Theresults obtained from these methods are decided not only by the chemical or product tested, but to a significant degree also bythe experimental set-up and decisions made by the investigator during the planning phase. The present MiniReview discussessome of the existing and well-known experimental in vitro and in vivo methods for studies of percutaneous penetration togetherwith some more recent and promising methods. After this, some considerations and recommendations about advantages and limi-tations of the different methods and their relevance for the prediction of percutaneous penetration are given. Which method toprefer will depend on the product to be tested and the question asked. Regulatory guidelines exist for studies on percutaneouspenetration, but researchers as well as regulatory bodies need to pay specific attention to the vehicles and solvents used in donorand sampling fluids so that it reflects in-use conditions as closely as possible. Based on available experimental data, mathemati-cal models have been developed to aid predictions of skin penetration. The authors question the general use of the present math-ematical models in hazard assessment, as they seem to ignore outliers among chemicals as well as the heterogeneity of skinbarrier properties and skin conditions within the exposed populations.

This MiniReview will discuss some of the existing and well-known experimental in vitro and in vivo models together withsome more recent and promising models and give some con-siderations and recommendations about advantages and limita-tions of the various models and their relevance for predictionsof percutaneous penetration. An overview of the methodolo-gies discussed can be found in table 1.Dermal exposure may be unintentional after environmental

or occupational exposure or intentional after the use of topi-cally applied medication or use of cosmetic products. In allcases, the assessment of the potential for percutaneous pene-tration or temporary deposition within the skin is an essentialelement in assessing risk as well as efficacy after dermal expo-sures.Decades of preventive efforts have decreased the inhala-

tional exposures at work places. At the same time, topicallyapplied pharmaceuticals are being used ever more often.Together, this has increased the relative importance of dermalexposures.Knowledge concerning percutaneous penetration and the

potential to reach target sites closes the gap from exposureassessment and hazard identification to risk assessment. If asubstance is unable to penetrate the stratum corneum (SC) or

affect the skin barrier function in any way, then the need forfurther assessment of risk becomes less evident.If a substance penetrates the skin or reaches targets within

the skin, specific information is needed to qualify the hazardand risk assessment. Thus, information on penetration kinetics,including rate of penetration (flux), Lag-time and temporarydeposition in different skin layers, will be needed.The ultimate goal of skin penetration research is to assess

the risk to human beings after dermal exposure to hazardouschemicals. Consequently, results from in vivo studies inhuman beings, including skin sampling, will remain the goldstandard in skin penetration studies. In the present MiniRe-view, a short presentation of three more recent experimentalhuman methodologies (microdialysis, open-flow microperfu-sion and spectroscopy) is included together with a more tradi-tional approach based on tape stripping.For ethical, logistical and financial reasons, in vivo studies

in human beings will not be able to cover the increasing needfor data on percutaneous penetration. Animal studies havebeen used extensively and are still being used. However,acknowledging both physiological and structural differencesbetween species, which may jeopardize the extrapolation ofresults from animals to human beings, and the implementationof the European REACH program followed by the more gen-eral political urge to reduce, refine and replace studies inexperimental animals, the need for validated in vitro or in sil-ico models to study percutaneous penetration has increased.

Author for correspondence: Jesper Bo Nielsen, Institute of PublicHealth, University of Southern Denmark, J.B. Winsløws Vej 9b, 2ndfloor, 5000 Odense C, Denmark (fax +45 6550 3682, e-mail [email protected]).

© 2013 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society)

Basic & Clinical Pharmacology & Toxicology, 2014, 115, 101–109 Doi: 10.1111/bcpt.12188

Furthermore, due to the increasing number of chemicals towhich human beings are exposed, data from clinical andexperimental studies will not be able to satisfy the need forquantitative information, and mathematical models for predict-ing penetration will therefore be necessary. The validity ofthese in silico models will, however, still depend on the con-tinued input of relevant data from experimental studies on per-cutaneous penetration. For all the above reasons, studies onpercutaneous penetration are highly relevant. In these cases,the OECD guidance is to combine knowledge from differentexperimental models (multimodel approach) to mitigate thedrawbacks from relying on a single model.Skin penetration research can be considered very versatile

due to the different areas of interests mentioned previously.Over time, different models have been used to gain knowledgeof kinetics related to pharmaceuticals, pesticides and otherindustrial chemicals and products. All models have theiradvantages and limitations. The model of choice will dependon the research question to be answered, because differentmodels may provide the researcher with different types ofinformation. When planning experimental studies, awarenessof the advantages, limitations and model-specific challengesmakes it possible to design an experimental study in agree-ment with the scientific question to be answered and the pene-trant of choice.

Skin Structure

The skin is the largest single organ of the body, accountingfor more than 5.5% of the body mass; in average about 4 kgcovering 1.7 m² depending on the height and weight of theindividual [1]. The human skin surface is continually exposedto chemicals, mechanical injury, microorganisms, UV-light,temperature variations and water, and the most important func-tion of the skin is to act as a barrier against these exposures.Besides barrier properties against exogenous exposures, theskin helps maintaining homoeostasis. The skin can be dividedinto the upper epidermis, underneath is the more vascular der-mis and below the subcutaneous layers (fig. 1). Topicallyapplied substances have to penetrate the avascular and

lipophilic SC and continue through the more aqueous lowerepidermis and dermis to reach the systemic circulation. Lipo-philic substances will easily cross the SC, but the penetrationrate will decrease as it reaches the hydrophilic epidermis anddermis – leading to increased deposition and giving theappearance of a reservoir – the reservoir effect.

Determinants of Percutaneous Penetration

Percutaneous penetration will – for the vast majority of exoge-nous chemicals – occur via passive diffusion. The exceptionto this generalization concerns larger or protein-bound mole-cules, which may use carrier-mediated transportation besidespassive diffusion. The passive diffusion process follows Fick’slaw, which defines flux as the multiplication of the concentra-tion differences across the membrane and a concentration-independent constant, the chemical-specific permeabilitycoefficient Kp. Thus, the penetration rate (flux) will be propor-tional to the experimentally chosen concentration gradientacross the membrane and if the concentration gradient dimin-ishes during the experimental period, the flux will also bereduced. This is evident in experiments with finite dosesapplied on the skin and/or if the concentration of the chemical

Fig. 1. Schematic illustration of the skin structure.

Table 1.Advantages and limitations related to different methods for studying percutaneous penetration. The + and � indicates if the method has the men-tioned defining feature, and + to +++ indicates if it is more or less characteristic for the method.

Advantages/limitations

Method

Static cells Flow through Tape stripping Raman Spectroscopy DMD OFM

In vitro + + + + + +In vivo � � + + + +Full-thickness skin + + + + + +Dermatomed skin + + + � � �Limited time consumption + + + + � �Minimally invasive ++ +++ + +Continuous sampling + + � + + +Multiple application sites + + + + + +Simple design + + + + � �Low cost + + + � � �Good reproducibility ++ ++ ++ ? + +


102 RIKKE HOLMGAARD ET AL. MiniReview

is allowed to become significant on the lower side of the skin.The latter can be practically avoided by having a sufficientlylarge volume of receptor fluid or by continuously replacingthe receptor fluid with fresh receptor fluid. The permeabilitycoefficient (Kp) has been described to depend mainly on themolecular weight (MW), the octanol/water partition coefficient(Kow) and molecular size (stereochemistry), whereas otherchemical characteristics such as melting point, hydrogen bond-ing acceptor capability [H(a)] only marginally affect penetra-tion rates [2,3]. Besides these determinants, vapour pressure,ionization (which is pH-dependent) and susceptibility to pro-tein binding will affect the concentration of unbound anduncharged chemical available for percutaneous penetration atthe surface of the skin at any time during the experimentalperiod. Thus, if a chemical has a sufficient vapour pressure, asubstantial part of the applied chemical may evaporate andthereby significantly reduce the concentration gradient and theobserved flux.Besides the chemical-specific characteristics, the passive dif-

fusion will also depend on experimental conditions related tothe physicochemical environment at the experimental setting.In the in vivo situation, the absorbed chemical will reach thelymph or blood circulation after penetrating the upper layersof the skin. To mimic this situation, the sampling fluid appliedexperimentally for sampling the penetrant should have relevantphysico-chemical characteristics related to solubility and pH;otherwise, experiments may under- or overestimate the true invivo flux. Likewise, if a chemical is not applied as neat chemi-cal, it will be dissolved in a solvent, which may in turn affectthe penetration characteristics. This is why it needs to be wellargued if the chemical is not applied under user-relevant con-ditions.The above-mentioned determinants are general to all in vivo

and in vitro experimental models, but model-specific character-istics posing challenges to the transferal of experimentallyobserved penetration rates or time lag also exist. To facilitate aharmonized interpretation of in vitro data from different experi-mental models, a number of guidance documents have beenpublished [4–7]. In the following sections, some of these modelswill be discussed under each type of experimental model.

Skin Penetration Models for in vitro Studies

The first in vitro model to study skin penetration was devel-oped in the 1940s in response to the threat of World War IIchemical warfare agents intended for inhalational as well asdermal exposure [8]. The original model resembles the pres-ently used static and flow-through diffusion cells.The static diffusion cell, also known as the Franz diffusion

cell (fig. 2), has been one of the most used in vitro models inskin penetration research since 1975 [9]. In the mid-eighties,the flow-through system was developed (fig. 3), and it is usedfor the same purposes as the static model. Both static andflow-through diffusion cells will work with full thickness aswell as epidermal skin barriers [10,11]. Various guidelinesindicate that the skin samples that may be used during in vitrostudies are split thickness [6] or when justified, full thickness

up to 1 mm. Full thickness means that the upper approxi-mately 1 mm of the skin including SC, epidermis and part ofdermis is mounted in the diffusion cell, whereas epidermalbarriers mean that the upper 200–400 lm skin has been sepa-rated (e.g. through techniques such as dermatome or heat sep-aration) before being mounted in the diffusion cell.In both models, a skin sample is mounted between a donor

chamber and a receptor chamber with the SC side towards thedonor chamber. Skin from both human donors and experimen-tal animals may be used, but human skin samples are pre-ferred as it will avoid the use of experimental animals andconsequently also avoid extrapolation between species whendata are being used. Irrespective of the donor source, the bar-rier integrity should be checked before application of test sub-stances. The basic procedure is that the penetrant of choice isapplied on the donor side, and percutaneous penetration canbe measured after sampling from the receptor side [6] – figs 2and 3. Specific OECD guidelines have been developed [6] forboth models, and for regulatory purposes both models areacceptable, though each of them have their advantages andlimitations [12,13].

Fig. 2. Illustration of the static diffusion cell. The penetrating sub-stance is diffusing from the donor chamber through the skin to thereceptor chamber from where it is collected through the collectionpipe. The ‘○’ symbolizes the penetrating substance.

Fig. 3. Illustration of the flow-through system. The penetrating sub-stance symbolised by ‘○’ is sampled in the passing receptor fluid andcollected in vials from the outlet tube.


MiniReview METHODS IN PERCUTANEOUS PENETRATION 103

In vitro techniques are ideal for screening percutaneous pen-etration of large numbers of topically applied substances, asexperimental cost and time consumption are low. Ethical con-siderations are fewer than for in vivo studies and relates to theuse of experimental animals or the consent to use of humanskin samples obtained from operative procedures, that is, typi-cally surgical waste. Many studies would be hazardous if con-ducted in vivo, for example, studies regarding chemicalwarfare agents [14]. When comparing data between studies,the in vitro methods have the advantage of a simple design,great predictability and little variability. Consequently, thesemethods have been easy to standardize. Another advantagecan be the possibility of working with radiolabelled sub-stances, which is associated with easy quantification of pene-tration of the substance. However, the researcher needs to takenotice that the radiolabel may be separated from the substanceitself due to enzymatic degradation/metabolism during thepenetration pathway, and quantification of penetration bycounting the radiolabel will – in that case – be erroneous. Dueto a lack of biochemical, physiological and immunologicalsystems (missing skin metabolism, lack of blood flow, etc.) invitro studies do not reproduce real physiological conditions.However, in vitro studies have – when thoughtfully designed– been shown to be able to predict skin penetration in vivo[15–17]. As most substances penetrate the skin by passive dif-fusion, an in vitro method may be an excellent method for ini-tial studies or, as mentioned previously, for high-throughputscreening of topically applied drugs and other substances.The two in vitro methods mentioned previously – the static

diffusion cell and the flow-through system – both have advan-tages and challenges [12] and the choice of cell needs to becarefully considered in relation to the research question posed.The flow-through system mimics the microcirculation in the

skin as the continuous flow in the receptor chamber removesthe substance once it has permeated the skin. This is anadvantage when dealing with substances with a low solubilityin the receptor fluid, and sink conditions are also easier main-tained in studies running for longer time periods comparedwith the method based on static diffusion cells, where the con-centration of the penetrant in the receptor chamber slowlyincreases during the experimental period. In the flow-throughsystem, the length of the outlet tubing will affect the time lag,especially at low flow rates, and the complexity of the method– compared with the simpler and less expensive version basedon the static cells – is technically more challenging.Both in vitro models will work with finite and infinite dos-

ing. Use of infinite doses will allow estimation of time lag,and subsequently, the maximal flux from which the apparentpermeability coefficient, Kp, can be calculated. Infinite dosingwill, however, also very often keep the topical side of the skincovered with donor fluid throughout the experimental period,which is oftentimes different form the in vivo exposure situa-tion. However, in transdermal therapeutic delivery systems(TTS), the occlusive environment underlying the patch willmake the underlying skin moist, and in this case, the resem-blance to a live situation may be closer. This is also the casein occupational toxicology where risk assessment in occlusive/

occluded environments is relevant, for example, when employ-ees use gloves when handling toxic substances, which may –due to incorrect choice or use of gloves or damage to theglove membrane – become trapped between glove and skin.Use of finite dosing will more closely resemble most in vivosituations in relation to infrequent short-term occupationalexposures (e.g. splashes) or the use of cosmetic or medicinalproducts. Use of finite doses will allow an estimate of thefractional permeation of the applied dose.The amount of a substance permeating the skin depends on

the area of skin available for penetration, and calculation ofdifferent measures for permeation is therefore adjusted to thatarea. If the entire area of the donor cell is not covered, theadjustment is flawed, and the calculations will overestimatethe actual permeation rate. The present OECD guideline [6]suggests adding 25 lL/cm2 to the donor chambers in flow-through as well as static cells, which corresponds to a thick-ness of the donor fluid of 250 lm. If the natural surfacetension in the donor solution is not reduced by adding deter-gents or solvents, this volume is (at least in our hands) toosmall to assure full coverage of the entire area, especially asthe skin surface, besides having a natural uneven surface, hasa tendency to be slightly thicker in the middle of the donorchamber. Our suggestion is to increase the volume applied tothe cells to 50 lL/cm2.Both methods are also ideal for studies exploring the reser-

voir effect of the skin [18]. Furthermore, the effect of differentpenetration enhancers, which are often applied to a solution inorder to change the skin permeability, can easily and inexpen-sively be studied in different applications. Enhancers or deter-gents present in the receptor solution may interact with the SCby causing either disruption of the SC lipids, structural proteinchanges or improved partitioning of the drug [19]. Ethanol, asan example, makes reversible changes to the skin barrierdescribed in different ways as intercellular lipid removal [20],lipid fluidization [21] lipid disordering or lipid extraction andmodulation of the lipid barrier [22]. The effect of any enhan-cer or enhancer-added solution can be studied in vitro withoutethical considerations.

Skin Penetration Models for in vivo Studies

In the development of drugs for human topical use or in riskassessment in occupational settings, the most directly transfer-rable kinetic research is conducted by in vivo human studies,because there is no need for extrapolation to human conditionsas is the case after animal or in vitro studies.Some methods can be used both in vitro and in vivo. These

methods can be used in, for example, the development of newdrugs from the laboratory concept to the final product testedin clinical settings. By avoiding a change in methodology dur-ing the path of development, an intermethodological variabilitycan be avoided. Among the presently developed in vivo meth-ods, a few also have the advantage of chronological real-timecontinuous sampling – dermal microdialysis and open-flowmicroperfusion are mentioned below – which is a relevant fea-ture in pharmaco- and toxicokinetic research.



Tape Stripping

Tape stripping (TS) has become one of the traditional investi-gative methods in in vivo skin penetration research and isknown as a non-invasive method, which can be used to deter-mine the kinetics of penetrants. By repeated application andremoval of adhesive tape to the same site on the skin, it ispossible to remove and sample successively layers of the SCand determine the absorption profile and potential temporarydeposit within the upper SC of the penetrant at the time ofsampling (fig. 4). After stripping a specified skin area, thepenetrant is extracted from the tape and subsequently analysedwith traditional analytical methods. The sampling method hassome manual steps, which requires trained personnel to avoidsignificant interpersonal and interlaboratory variability [23].

Spectroscopy

In the early 1990s, spectroscopy was developed for skin pene-tration studies as a method for SC research. Spectroscopicmethods are optical methods based on light scattering andgenerally have until recently had only but a limited depthrange confined to the SC or just below the SC. More recently,imaging techniques based on confocal fluorescence micros-copy have been developed that provide a pictorial descriptionof skin structures including visualization of the different skinstrata, which enables optical sectioning of the specimen to beperformed in a non-invasive manner. Two major confocal flu-orescence microscopy techniques have been used in skin-related experiments: laser scanning confocal fluorescencemicroscopy (LSCM) and two-photon excitation fluorescencemicroscopy (TPEFM). Both techniques are suitable to explorethe epidermis; however, TPEFM has particular advantagesover LSCM. Two-photon excitation is a non-linear process inwhich a fluorophore absorbs two photons simultaneously.These photons are generally of low energy compared with theone-photon absorption process in conventional fluorescencemicroscopy techniques, resulting in an overall low extent ofphotobleaching and photodamage to the specimens [24]. Inaddition, as infrared light is used as the excitation source inTPEFM, the penetration depth in thick specimens can be up to800 lm, allowing in-depth, three-dimensional examination ofbiological specimens. This penetration depth is far superior to

that obtained using LCSM, where a maximum depth ofapproximately 150–200 lm is generally obtained [25].The first publication showing concordance between drug

penetration investigated by Confocal Raman spectroscopy(Confocal microscopy in combination with Raman spectros-copy) and the TS methodology was recently published [26].Other spectroscopic methods such as Near IR and Terahertzspectroscopy are currently being developed [27,28].A challenge for all these methods is that they are not quan-

titative by nature, though analytical ways to semi-quantitativemeasurements are being developed. The methods are fullynon-invasive and can provide detailed information about thelayers studied. Given that a test molecule can act as a fluoro-phore, it is expected to become possible to study specificdepositional patterns during the absorption process for suchmolecules. As the methods are applicable in vivo as well as inin vitro settings, it should be possible to use these imagingtechniques to study experimentally induced structural artefactsin experimental settings.

Microdialysis

Dermal MD (DMD) was first described by Anderson et al. in1991 in a human study concerning percutaneous absorption ofsolvents, using ethanol as penetrating substance and MD sam-pling in the dermis [29]. Since then, the DMD method (fig. 5)has been used for sampling of a large number of topicallyapplied drugs and other skin penetrants [30] in both healthyand damaged/diseased skin in human beings as well as in ani-mals. DMD can sample endogenous and exogenous substancesin all types of tissue by use of a thin catheter with a semi-per-meable membrane imitating a small blood vessel. This vessel(the microdialysis probe) is connected to an inlet and an outlettube perfused with a tissue compatible fluid (perfusate). Theexchange of molecules across the membrane occurs by passivediffusion driven by the concentration gradient. The microdialy-sis probes have specific pore sizes, which set upper limits(cut-off value) for the molecules that can be sampled, but alsoexcludes larger molecules and proteins from entering the sam-pling fluid. Thus, unless protein has been added to the perfus-ate, which is only done if it enhances recovery of thesubstance of interest, then DMD sampling delivers protein andenzyme-free samples, which makes the pre-analytical steps rel-atively uncomplicated. The method requires prior consider-ations of the suitability of the substance of interest formicrodialysis sampling, as the typical combination seen inmany topical medical treatments, that is, a high or very highlipophilicity of the drug and a low drug concentration in thetopical product, both make DMD sampling challenging as theresulting samples may be of very low (below LOQ) concentra-tion [31].

Open-flow Microperfusion:

In 1997, a similar technique – OFM – was introduced byTrajanoski et al. [32].. OFM is also designed as a continuoustissue-specific sampling method. Where the DMD probe has a

Fig. 4. Tape stripping. Illustration of the sampling of the upper layersof the skin – the stratum corneum (SC) - by repeated tape applicationand removal of successive layers of SC cells.



semi-permeable membrane, the OFM sampling catheter has amembrane-free macroscopically perforated area with unre-stricted access to and exchange of solutes in the peri-cathetertissue. Therefore, the OFM technique does not have the samelimitations regarding sampling efficacy towards large and/orprotein-bound penetrants as the MD technique. Since 2006,the OFM method has been utilized for dermal sampling[33,34] (fig. 6). The method is, due to the open exchangearea, relevant for sampling of large and/or lipophilic pene-trants, which is where the DMD sampling methodology isoften challenged. However, OFM is a more demandingmethod both technically and labourwise, as the method needsa push- as well as a pull-pump function connected to the sam-pling catheters to counteract the tendency to induce oedema inthe tissue surrounding the probe due to the open exchangearea. As a consequence of the open exchange area, proteins,

enzymes and some cells will be included in the sample fluidcollected by the catheter. Thus, the resulting sample requirestechnically more demanding pre-analytical steps before analy-sis of the sample fluid.The use of in vivo methods has significant ethical implica-

tions. In the case of TS, the skin trauma invoked when remov-ing the SC layers is most often negligible leaving only a smalltemporarily sore area. For DMD and OFM, the considerationsconcern the necessary skin trauma inflicted when inserting theprobe/catheter horizontally in the dermis (figs 5 and 6). Last-ing tissue damage is not seen, but the insertion trauma is fol-lowed by a histamine release, causing a reversible wheal andflare reaction [35].

From Experimental Observations to the Real World

When conducting experimental studies, in vitro as well as invivo, researchers often take pride in following specified guide-lines and standard procedures in the laboratory. This approachincreases repeatability and reduces variability in results. How-ever, the human population is most often better characterizedby heterogeneity than homogeneity, and a significant fractionof the population suffers from a diseased or otherwise dam-aged skin barrier for exogenous or endogenous (includinggenetic) reasons. When we study skin penetration, it is there-fore important not only to create reliable results in relation toa normal skin profile but also to adjust the experimental stud-ies to real-life conditions, which are often not as straightfor-ward as in the ‘standard operation procedures’ applied in thelaboratory. The use of an experimental model that can beadapted to a real-life situation would therefore be most rele-vant for the study of situations that may deviate from the aver-age.Known exceptions are damaged or diseased skin [36]. Irre-

spective of whether the affected skin barrier is caused by blunttrauma, wet work, delipidization of the SC or the presence ofdermatitis, a decrease in skin barrier integrity has proven toincrease percutaneous penetration [37–40]. A mutation in thefilaggrin gene, which is carried by approximately 9% of theEuropean population, is associated with a decrease in naturalmoisturizing factor of the skin, a key parameter for skin bar-rier function and a risk factor for the development of dermati-tis [41–43]. Patients suffering from dermatitis have previouslybeen shown to have impaired skin barrier function in lesional[44] as well as non-lesional skin [45]. The impact of animpaired skin barrier function may be seen in both the occupa-tional setting but also in penetration and/or reaction to trans-dermally administered medical products, topical formulationsas well as cosmetic products.In some situations, the experimental focus is on percutane-

ous penetration of the active ingredient and not on the com-mercial product that consumers are exposed to, which is oftena mixture of different substances. The tested individual ingre-dients are then used to formulate non-tested mixtures. Thesemixtures have often been demonstrated to have a higher pene-tration rate than the tested active ingredient [46,47]. Theresearch results from testing pure chemicals are in these

Fig. 5. Illustration of the microdialysis probe placed in the dermis,which is sampling increasing dermal drug concentrations after topicaldrug penetration (modified from Benfeldt and Serup 1999). The ‘○’symbolizes the penetrating substance.

Fig. 6. Illustration of the open-flow microperfusion catheter placed inthe dermis. The topically added substance symbolized by ‘○’ is pene-trating through the skin and sampled in the dermis. The push/pull sys-tem is connected to the probe and the samples are collected in anexchangeable glass capillary. Markings on the probe make a correctpositioning in the skin easy.



situations potentially misleading and may affect the riskassessment in occupational settings where an added detergent/enhancer affects the skin integrity and thereby increase skinpermeability [48,49]. This is an area that still lacks relevantexperimental data to support mathematical modelling, whichwill allow valid predictions of the percutaneous penetration offormulated products including if the formulations of alreadyapproved products are changed.Often individuals are exposed to several products at the

same time. Interactions between two or more unintended der-mal exposures in occupational settings have been described,causing increased absorption just as well as an absence of anyinteraction of practical importance [50,51]. In the occupationalsetting, prevention of dermal absorption to single chemicalshas been handled through the implementation of hazard indica-tors raising awareness for individuals handling/using chemicalswith a potential for dermal absorption. Following descriptionof clear inconsistencies [52], much work has been done inter-nationally to develop better and more consistent hazard indica-tors [53], but still only taking into account the singlesubstances and not addressing the potential for interaction.Models to handle known occupational co-exposures to severalchemicals need to be developed and handled preventivelythrough regulations. However, one difficulty in obtaining thisgoal is if one of the exposures is not perceived as a dermalexposure to a chemical. Such an exposure could be through theuse of sunscreens including nanomaterials or other cosmeticproducts. On the topic on interaction between unintentionaloccupational exposures and intentional use of skin care prod-ucts, much more experimental evidence is needed as most peo-ple at work places are not aware of the potential interactions,and because the hazard indicators used at work sites (i.e. skinnotations) do not take this potential enhancement into account.Use of topical creams has been demonstrated to change the

lipid composition in the skin, and thereby potentially also thepercutaneous penetration of other substances [54]. How muchthis may quantitatively affect penetration rate and time lag ispresently not known. Likewise, it is not known for how longafter exposure to the cosmetic product that the lipid composi-tion will be affected. Given the very frequent and oftenlong-term use of topical cosmetics, this is an issue that aspreviously mentioned deserves more attention.The examples described previously demonstrate the contin-

uing need for more experimental data. The challenge will notbe to generate in vivo or in vitro experimental data on allimaginable combinations, but to generate sufficient data toallow valid predictions and modelling. The use of mathemati-cal models is, however, not without caveats.Several models have been developed and refined over the

last decades to predict steady-state flux or the permeabilitycoefficient (Kp). These models are most often based on physi-cochemical properties related to partition coefficients betweenoctanol and water, MW, and different measures of molecularvolume and steric structures [55–57]. The original algorithmfrom 1992 based on the Flynn database has been revisited sev-eral times, but the overall r2 values for the quantitative struc-ture–activity relationship (QSAR) models related to

percutaneous penetration seldom exceed 0.7 except whenexcluding specific outliers, in which case the r2 value mayapproach 0.9. However, from a preventive perspective, itseems equally important to be able to identify the outliers thanto create sophisticated models. Basically, regulatory guidelinesbased solely on QSAR models may miss the outliers andpotentially produce a significant underestimation of the truepenetration potential of a chemical. However, for specificgroups of chemicals, predictive models may be used to predictKp or maximal flux for other chemicals belonging to the samegroup. Prediction of Kp or maximal flux is relevant, but froma preventive perspective, the time lag is almost equally impor-tant, as a high Kp not always correlates with a short lag time.Thus, a short time lag and a medium flux may produce highertotal absorption during a short exposure period than a highermaximal flux but a long time lag. Further, lack of knowledgeon lag time may jeopardize biological monitoring based onblood sampling shortly after dermal exposures to a chemicalwith long time lag by producing significant underestimation ofthe actual exposure. Moreover, most modelling is presentlyperformed based on well-known physiochemical determinantspredicting the percutaneous penetration of single chemicals[3,55], but efforts are needed to allow also for valid predic-tions on absorption into and through compromised skin afterexposure to combinations of chemicals.It can be concluded that we continue to be in need of data

on percutaneous penetration from well-validated experimentalmodels to help refine in silico models and to find and describethose chemicals and/or exposure situations, where reality devi-ates from the predictive mathematical models. A range ofexperimental in vitro and in vivo approaches exist, and eachmodel has strengths and limitations. Before planning and con-ducting the experimental work, some initial and importantconsiderations and questions therefore need to be addressed.What are the reasons for conducting this particular study?What question is the study expected to answer, and moreimportantly, what can we use it for? Is it possible to relate theexperimental set-up to a real-life scenario? Does the studymethod chosen meet the required conditions for extrapolationsand predictions - and will our results reflect the real worldscenarios or can they potentially mislead us, thereby allowingunacceptable exposure to individuals who are not aware of anexisting hazard?

AcknowledgementSupported by COST Action BM0903 (SKINBAD).

References

1 Goldsmith LA. My organ is bigger than your organ. Arch Derma-tol 1990;126:301–2.

2 Magnusson BM, Anissimov YG, Cross SE, Roberts MS. Molecularsize as the main determinant of solute maximum flux across theskin. J Invest Dermatol 2004;122:993–9.

3 Nielsen JB, Sorensen JA, Nielsen F. The usual suspects - influenceof physicochemical properties on lag time, skin deposition, andpercutaneous penetration of nine model compounds. J ToxicolEnviron Health A 2009;72:315–23.

4 EFSA. EFSA guidance on dermal absorption. EFSA J 2012;10:10.



5 OECD. Guidance notes for the estimation of dermal absorptionvalues. Series on testing and assessment no 156. 2011.

6 OECD-428. OECD guidelines for testing of chemicals. Skinabsorption: In vitro method, 2004.

7 WHO. Dermal Absorption. Environmental Health Criteria 235.World Health Organization, Geneva, 2006.

8 Pendlington R. In vitro percutaneous absorption measurements. In:Chilcott RP, Price S (eds). Principles and Practice of Skin Toxicol-ogy. Wiley & Sons, Chichester, UK, 2008;129–48.

9 Franz TJ. Percutaneous absorption on the relevance of in vitrodata. J Invest Dermatol 1975;64:190–5.

10 van de Sandt JJ, van Burgsteden JA, Cage S, Carmichael PL, DickI, Kenyon S et al. In vitro predictions of skin absorption of caf-feine, testosterone, and benzoic acid: a multi-centre comparisonstudy. Regul Toxicol Pharmacol 2004;39:271–81.

11 Wilkinson SC, Maas WJM, Nielsen JB, Greaves LC, van de SandtJJM, Williams FM. Interactions of skin thickness and physico-chemical properties of test compounds in percutaneous penetrationstudies. Int Arch Occup Environ Health 2006;79:405–13.

12 Holmgaard R, Nielsen JB. Dermal Absorption of Pesticides -Evaluation and Prevention. Danish Ministry of Environment,Copenhagen, 2009.

13 Holmgaard R. Skin penetration studied by static diffusion cells,microdialysis and open-flow micrpperfusion - sampling of fentanyland benzoic acid in human skin ex vivo. University of SouthernDenmark, PhD Thesis, 2012.

14 Vallet V, Cruz C, Josse D, Bazire A, Lallement G, Boudry I. Invitro percutaneous penetration of organophosphorus compoundsusing full-thickness and split-thickness pig and human skin. Toxi-col Vitro 2007;21:1182–90.

15 Bronaugh RL, Stewart RF, Simon M. Methods for in vitro percuta-neous.absorption studies. 7. Use of excised human-skin. J PharmSci 1986;75:1094–7.

16 Hotchkiss SAM, Hewitt P, Caldwell J, Chen WL, Rowe RR. Per-cutaneous.absorption of nicotine-acid, phenol, benzoic acid and tri-clopyr butoxyethyl ester through rat and human skin in vitro -further validation of an in vitro model by comparison with in vivodata. Food Chem Toxicol 1992;30:891–9.

17 van de Sandt JJM, Meuling WJA, Elliott GR, Cnubben NHP, Ha-kkert BC. Comparative in vitro-in vivo percutaneous absorption ofthe pesticide propoxur. Toxicol Sci 2000;58:15–22.

18 Bronaugh RL, Stewart RF. Methods for in vitro percutaneousabsorption studies IV: the flow-through diffusion cell. J Pharm Sci1985;74:64–7.

19 Williams AC, Barry BW. Penetration enhancers. Adv Drug DelivRev 2004;56:603–18.

20 Bommannan D, Potts RO, Guy RH. Examination of the effect ofethanol on human stratum-corneum in vivo using infrared-spectros-copy. J Controlled Release 1991;16:299–304.

21 Ghanem AH, Mahmoud H, Higuchi WI, Liu PC, Good WR. Theeffects of ethanol on the transport of lipophilic and polar permeantsacross hairless mouse skin - methods validation of a novel-approach. Int J Pharm 1992;78:137–56.

22 Dias M, Naik A, Guy RH, Hadgraft J, Lane ME. In vivo infraredspectroscopy studies of alkanol effects on human skin. Eur JPharm Biopharm 2008;69:1171–5.

23 Pershing LK, Bakhtian S, Poncelet CE, Corlett JL, Shah VP. Com-parison of skin stripping, in vitro release, and skin blanchingresponse methods to measure dose response and similarity of tri-amcinolone acetonide cream strengths from two manufacturedsources. J Pharm Sci 2002;91:1312–23.

24 Zipfel WR, Williams RM, Webb WW. Nonlinear magic: multipho-ton microscopy in the biosciences. Nat Biotechnol 2003;21:1368–76.

25 Laiho LH, Pelet S, Hancewicz TM, Kaplan PD, So PTC.Two-photon 3-D mapping of ex vivo human skin endogenous

fluorescence species based on fluorescence emission spectra.J Biomed Opt 2005;10:024016.

26 Mateus R, Abdalghafor H, Oliveira G, Hadgraft J, Lane ME. Anew paradigm in dermatopharmacokinetics - Confocal Ramanspectroscopy. Int J Pharm 2013;444:106–8.

27 Stamatas GN, Kollias N. In vivo documentation of cutaneousinflammation using spectral imaging. J Biomed Opt2007;12:051603.

28 Ney M, Abdulhalim I. Modeling of reflectometric and ellipsomet-ric spectra from the skin in the terahertz and submillimeter wavesregion. J Biomed Opt 2011;16:067006.

29 Anderson C, Andersson T, Molander M. Ethanol absorption acrosshuman skin measured by in vivo microdialysis technique. ActaDerm Venereol 1991;71:389–93.

30 Chaurasia CS, Muller M, Bashaw ED, Benfeldt E, Bolinder J,Bullock R et al. AAPS-FDA workshop white paper: microdialysisprinciples, application and regulatory perspectives. Pharm Res2007;24:1014–25.

31 Holmgaard R, Nielsen JB, Benfeldt E. Microdialysis Sampling forInvestigations of Bioavailability and Bioequivalence of TopicallyAdministered Drugs: current State and Future Perspectives. SkinPharmacol Physiol 2010;23:225–43.

32 Trajanoski Z, Brunner GA, Schaupp L, Ellmerer M, Wach P, Pie-ber TR et al. Open-flow microperfusion of subcutaneous adiposetissue for on-line continuous ex vivo measurement of glucose con-centration. Diabetes Care 1997;20:1114–21.

33 Holmgaard R, Benfeldt E, Nielsen JB, Gatschelhofer C, SorensenJA, H€offerer C et al. Comparison of Open-Flow Microperfusionand Microdialysis Methodologies When Sampling TopicallyApplied Fentanyl and Benzoic Acid in Human Dermis Ex Vivo.Pharm Res 2012;29:1808–20.

34 Bodenlenz M, Hofferer C, Magnes C, Schaller-Ammann R,Schaupp L, Feichtner F et al. Dermal PK/PD of a lipophilic topicaldrug in psoriatic patients by continuous intradermal membrane-freesampling. Eur J Pharm Biopharm 2012;81:635–41.

35 Groth L, Serup J. Cutaneous microdialysis in man: effects of nee-dle insertion trauma and anaesthesia on skin perfusion, erythemaand skin thickness. Acta Derm Venereol 1998;78:5–9.

36 Kezic S, Nielsen JB. Absorption of chemicals through compro-mised skin. Int Arch Occup Environ Health 2009;82:677–88.

37 Grandjean P. Skin penetration - hazardous chemicals at work: Tay-lor and Francis, 1990.

38 Benfeldt E, Serup J, Menne T. Effect of barrier perturbation oncutaneous salicylic acid penetration in human skins: in vivo phar-macokinetics using microdialysis and non-invasive quantificationof barrier function. Br J Dermatol 1999;140:739–48.

39 Nielsen JB. Percutaneous penetration through slightly damagedskin. Arch Dermatol Res 2005;296:560–7.

40 Ortiz PG, Hansen SH, Shah VP, Menne T, Benfeldt E. The effectof irritant dermatitis on cutaneous bioavailability of a metronida-zole formulation, investigated by microdialysis and dermatophar-macokinetic method. Contact Derm 2008;59:23–30.

41 Palmer CNA, Irvine AD, Terron-Kwiatkowski A, Zhao YW, LiaoHH, Lee SP et al. Common loss-of-function variants of the epider-mal barrier protein filaggrin are a major predisposing factor foratopic dermatitis. Nat Genet 2006;38:441–6.

42 Kezic S, Kemperman PMJH, Koster ES, de Jongh CM, Thio HB,Campbell LE et al. Loss-of-function mutations in the filaggringene lead to reduced level of natural moisturizing factor in thestratum corneum. J Invest Dermatol 2008;128:2117–9.

43 O’Regan GM, Sandilands A, McLean WHI, Irvine AD. Filag-grin in atopic dermatitis (Reprinted from J Allergy Clin Immu-nol vol 122, pg 689-93, 2008). J Allergy Clin Immunol 2009;124:R2–6.

44 Ortiz PG, Hansen SH, Shah VP, Menne T, Benfeldt E. Impact ofadult atopic dermatitis on topical drug penetration: assessment by



cutaneous microdialysis and tape stripping. Acta Derm Venereol2009;89:33–8.

45 Jakasa I, Koster ES, Calkoen F, McLean WHI, Campbell LE, BosJD et al. Skin barrier function in healthy subjects and patients withatopic dermatitis in relation to filaggrin loss-of-function mutations.J Invest Dermatol 2011;131:540–2.

46 Liu KH, Kim JH. In vitro dermal penetration study of carbofuran,carbosulfan, and furathiocarb. Arch Toxicol 2003;77:255–60.

47 Buist HE, van de Sandt JJM, van Burgsteden JA, de Heer C.Effects of single and repeated exposure to biocidal active sub-stances on the barrier function of the skin in vitro. Regul ToxicolPharmacol 2005;43:76–84.

48 Nielsen JB, Nielsen F, Sorensen JA. In vitro percutaneous penetra-tion of five pesticides-effects of molecular weight and solubilitycharacteristics. Ann Occup Hyg 2004;48:697–705.

49 Mills PC, Magnusson BM, Cross SE. Penetration of a topicallyapplied nonsteroidal anti-inflammatory drug into local tissues andsynovial fluid of dogs. Am J Vet Res 2005;66:1128–32.

50 Korinth G, Geh S, Schaller KH, Drexler H. In vitro evaluation ofthe efficacy of skin barrier creams and protective gloves on percu-

taneous absorption of industrial solvents. Int Arch Occup EnvironHealth 2003;76:382–6.

51 Nielsen JB. Natural oils affect the human skin integrity and thepercutaneous penetration of benzoic acid dose-dependently. BasicClin Pharmacol Toxicol 2006;98:575–81.

52 Nielsen JB, Grandjean P. Criteria for skin notation in differentcountries. Am J Ind Med 2004;45:275–80.

53 Sartorelli P, Ahlers HW, Alanko K, Chen-Peng C, Cherrie JW,Drexler H et al. How to improve skin notation. Position paperfrom a workshop. Regul Toxicol Pharmacol 2007;49:301–7.

54 Monteiro-Riviere NA, Inman AO, Mak V, Wertz P, Riviere JE.Effect of selective lipid extraction from different body regions onepidermal barrier function. Pharm Res 2001;18:992–8.

55 Potts RO, Guy RH. Predicting Skin Permeability. Pharm Res1992;9:663–9.

56 Moss GP, Dearden JC, Patel H, Cronin MTD. Quantitative struc-ture-permeability relationships (QSPRs) for percutaneous absorp-tion. Toxicol Vitro 2002;16:299–317.

57 Fitzpatrick D, Corish J, Hayes B. Modelling skin permeability inrisk assessment - the future. Chemosphere 2004;55:1309–14.



percutaneous penetration - methodological considerations

Documents