Dermal penetration testing, also known as percutaneous penetration, measures the absorption or penetration of a substance “through the skin barrier and into the skin” (OECD, 2004a). Dermal penetration studies are conducted to determine how much of a chemical penetrates the skin, and thereby whether it has the potential to be absorbed into the systemic circulation. Dermal penetration is considered to occur by passive diffusion; however, biotransformation of the test substance within the deeper viable regions of the skin (metabolism) prior to systemic absorption can occur (OECD, 2004a).
Dermal toxicity testing, on the other hand, is conducted to assess the local and/or systemic effects of a chemical following dermal exposure. It may provide an indication that the substance penetrates the skin if it produces systemic toxicity, but the amount of chemical absorbed is not quantified by dermal toxicity testing.
One of the primary roles of the skin is to act as a barrier to protect humans from substances contacted in the environment. Permeation of a substance through the skin depends upon a number of factors, including: the formulation or vehicle in which it is presented to the skin; physicochemical properties of the test substance such as lipophilicity (fat solubility), molecular weight, charge, and concentration of the test substance; and area and duration of exposure. The qualities of the outermost layers of the skin, the stratum corneum, typically determine the rate of dermal penetration. The biological factors affecting absorption include the site of the body, the integrity of the stratum corneum and thickness of the epidermis in addition to other physiologic determinants such as temperature and local blood flow (OECD, 2004a; 2004b; WHO, 2006). [For a description of the structure of the skin, see the AltTox section Skin Irritation/Corrosion.]
Both in vivo and in vitro methods are used for determining the percutaneous penetration of a substance. Selection of the method used may result in obtaining different types of information and relevance of the test results to human dermal exposure. Another factor in the selection of an in vivo versus in vitro test system (or a combination of the two) may be the preference of the national regulatory authority. The in vitro dermal penetration test methods that utilize donated human skin are considered more relevant to the in vivo tests in animals, since they actually utilize resected human skin. It should be noted here that the dermal absorption testing of new cosmetic ingredients in living animals in the European Union is no longer permitted under Regulation (EC) 1223/2009. Therefore, there is a particular need to have scientifically valid alternatives in the area of safety testing of cosmetics. In fact, the testing of cosmetic ingredients using in vitro models has been the norm for a number of years within the European Union (Diembeck et al., 2005; SCCS, 2010).
The OECD recommends consulting their guidance document, the OECD Guidance Document for the Conduct of Skin Absorption Studies (No. 28, March 5, 2004), to determine the most suitable method for a particular study (OECD, 2004a). A more recent document published by OECD provides practical guidance to facilitate harmonized interpretation of experimental data from dermal absorption studies with pesticides, biocides, and other industrial chemicals (OECD, 2011). Whatever dermal absorption test system is used, its performance in the testing laboratory should be confirmed with reference chemicals showing comparable results to published values (OECD, 2004a; WHO, 2006; OECD, 2011).
Historically, the rat has been the most common species used for in vivo dermal penetration testing, and is still a requirement as part of the safety assessment of pesticides in North America (US EPA, 1998). The in vivo method for determining the penetration of a substance through the skin of an animal and into the systemic compartment is described in OECD Test Guideline (TG) 427 (OECD, 2004b, adopted April 13, 2004). One or more doses of the test material, usually prepared by combining a radiolabeled form of the active with cold material and all the other components of the formulated product, are applied to a measured area of shaved skin of groups of rats for a specified time. Precautions are taken to ensure that the exposure is limited to the dermal route. The exposure time and dose levels are based on expected human exposure. The animals are observed at regular intervals for signs of toxicity, and daily excreta (and sometimes expired air) are collected separately and analyzed for the radiolabeled test substance. Blood is collected at specified times and when the groups of animals are euthanized.
The mass balance distribution of the radiolabeled test substance in various tissue samples in rats from the application site, excreta, and the body is determined at specific times following dermal dosing. The results are reported as the rate, amount, or percentage of skin absorption of the test material. The full details of the animal procedure can be found in the guidelines and guidance documents (OECD, 2004a; OECD, 2004b; OECD, 2011). The dermal penetration of substances through rat skin is known to over-estimate human absorption (WHO, 2006). This is not surprising due to the very different morphologic characteristics of the skin between rats and humans. However, the rat in vivo test method is considered to be of some value due to the “generation of systemic kinetic and metabolic information” that may relate to man (OECD, 2004a).
In vitro methods to determine dermal absorption are described in OECD TG 428, Skin Absorption: In Vitro Method (OECD, 2004c, adopted April 13, 2004). The in vitro method is based on the permeability of a test substance from its formulation applied as a finite dose across human or animal skin preparations. Additional guidance for industry-specific sectors is also now available (SCCS, 2010; EFSA, 2012). In modern day protocols the skin sample is dermatomed to a specific thickness and mounted in a static or flow-through glass diffusion chamber (Heylings, 2014; WHO, 2006; OECD, 2011). This is described in more detail below.
Dermal penetration studies using in vitro skin preparations from animals and man were introduced into the regulatory arena in the early 1990s (ECETOC, 1993). They were not subject to a formal validation program, as would occur with a new “alternative” method in modern day method assessment. However, a number of industries had their own in-house validation data often involving human volunteer studies and human skin in vitro. Many publications were emerging as to the value of the alternative approach in this area of toxicology (Scott and Ramsey, 1987; Ramsey et al., 1994). In 1994, ECVAM brought together an international expert group from academia and the pesticide, cosmetic, industrial chemical, and pharmaceutical industries to review the area of percutaneous absorption and to formulate an outline protocol that could be used for the assessment of dermal absorption. This ECVAM Workshop agreed an approach that included a non-animal method for the assessment of dermal absorption (Howes et al., 1996). Almost another decade passed with a number of meetings and conferences driven largely by a toxicology sub-group of the European Crop Protection Association (ECPA), which focused on the issue of stand-alone in vitro methodology and test guideline development. Finally, under the mutual acceptance of data, separate test guidelines for the assessment of dermal absorption in vivo (OECD TG 427) and in vitro (OECD TG 428) were formally agreed by the Member States of OECD and published in 2004.
The OECD Guidance Document for the Conduct of Skin Absorption Studies (2004a) states that “…skin absorption is primarily a passive process and studies undertaken using appropriate in vitro experimental conditions have produced data for a wide range of chemicals that demonstrate the usefulness of this method. Such methods have found use in, for example, comparing delivery of chemicals into and through skin from different formulations and can also provide useful models for the assessment of risk due to percutaneous absorption in humans.”
Although non-animal methods for dermal penetration have not been formally validated, the in vitro methods described by OECD TG 428 have been accepted by EU authorities for many years. The use of the non-animal method for dermal absorption by North American Agencies was an area of significant debate during the development of the OECD test guidelines during the late 1990s. US EPA and the Health Canada Pest Management Regulatory Agency (PMRA) were the most reluctant to formally acknowledge acceptance of the in vitro percutaneous penetration methods. However, these Agencies do now accept in vitro data providing it is conducted according to OECD TG 428, but only as a refinement of risk assessments for pesticides using the “triple pack approach” (NAFTA, 2008). Therefore, rat in vivo studies are still required by North American Agencies in 2015 for the registration of pesticide-containing products.
Although not a formal validation study, an inter-laboratory assessment of in vitro skin absorption methods was conducted in which three compounds were tested in 10 laboratories using standardized protocols (Van de Sandt et al., 2004). Some variability in the results was reported, but the in vitro methods were considered to be robust. Nine labs used human skin, and it was concluded that “variation observed may be largely attributed to natural human variability in dermal absorption and the skin source.” The type of diffusion cell used did not appear to affect the results. Skin thickness did alter the results – only slightly for benzoic acid and caffeine, but significantly for testosterone, where absorption was higher with dermatomed human skin.
A variety of in vitro procedures using resected skin have been developed for dermal penetration testing. Most of these methods use split-thickness human or animal skin mounted in a Franz glass diffusion chamber (Franz, 1975; Clowes et al., 1994; Heylings, 2014). Human skin is mostly used in these models for the safety assessment of industrial chemicals and pesticides (EFSA, 2012; OECD, 2011), but pig skin is also permitted for the safety assessment of cosmetic ingredients (SCCS, 2010).
Advantages beyond the fact that live animals are now rarely used for dermal penetration assessment include the following: human skin more closely estimates human exposure; the early phase of absorption can be determined; replicate measurements can be made using the same skin from the same human donor, or between different human donors for the same dose application; exposure conditions can be easily varied; and a wider range of physical forms of test substances such as solids, granules, and powders can be easily evaluated (OECD, 2004a; OECD, 2011). The primary limitation described for in vitro percutaneous penetration testing is that the metabolic profile of the test substance cannot be determined (for more on in vitro determination of skin metabolism see Dermal Penetration: Emerging Science & Policy). However, if the purpose of the investigation is the determination of the dermal penetration and ultimately the systemic exposure of the test substance from a formulated product, then the in vitro method using resected skin has great utility and has shown to be predictive of dermal absorption in both rats (Scott and Ramsey, 1987) and man (Ramsey et al., 1994; WHO, 2006; OECD, 2011).
It is important to note that the in vitro studies that use resected skin now use a finite (low volume) dose of the finished or formulated product. Many of the historic in vitro skin penetration studies used infinite (high volume) doses of the test substance, and usually in a vehicle solvent rather than the finished product (Bronaugh and Franz, 1986). This enables a steady-state rate (or flux) of the chemical to be determined. In modern day regulatory studies it is the amount of the test chemical that is measured in the various compartments of a study following application of a finite dose of the finished product that is the figure used in the risk assessment calculations. In most regulatory studies the mass of the test material that has penetrated the skin into the receptor fluid per area of skin is related to the applied concentration in the finite dose. This can be conveniently expressed as the percentage of the dose applied that has penetrated the skin into the receptor at 24 hours, providing a “worst case” daily absorbed dose. The exposure period may be, for example, 30 minutes for a shampoo-in hair dye (but less for a traditional shampoo), or 6 hours to cover a “working day” for an operator spraying a pesticide. A leave-on type cosmetic would, of course, be left on the skin for the full 24-hour exposure period.
When designing an in vitro study with excised skin, it is important to determine that the nature of the receptor fluid is not rate limiting to the diffusion of the substance from the skin into this phase (OECD, 2011; EFSA, 2012). The amount and rate of test substance accumulating in the receptor chamber and a time course profile are measured, normally by taking samples of the receptor fluid at multiple time points over a 24 hour period. In a regulatory in vitro dermal penetration study, a mass balance recovery of the applied dose is undertaken at 24 hours, in addition to measuring the material in the receptor fluid. This includes the measurement of the amounts of test substance that can be easily removed from the skin surface by normal soap washing at the end of the expected exposure period, plus the amount removed by soap washing at 24 hours.
In addition to the washed off fraction of the dose applied, the amount adsorbed to the skin surface is determined in sequential tape strips of the stratum corneum. Following this tape stripping procedure, the proportion of the applied dose in the remaining underlying epidermis and dermis is also determined at this 24 hour time point. This provides a total mass balance recovery of the applied substance in each of the diffusion cells. When the test substance of interest is volatile, a modified donor chamber containing porous carbon filters can be used to trap any material evaporating from the skin surface into the headspace above the skin. It is important here not to occlude the skin since this will potentially enhance skin penetration. The tape stripping of the stratum corneum is particularly important, since it permits the quantification of unabsorbed, non-dislodgeable material that would normally be lost by desquamation in man. This tape stripping procedure conducted using resected human skin in vitro has been shown to predict tape stripping in human volunteers very well for a range of formulated products (Trebilcock et al., 1994). The number of strips taken and the inclusion/exclusion of these layers in the calculation of the absorbed dose is provided in the specific industry guidance (SCCS, 2010; EFSA, 2012). A schematic of a typical mass balance/tape stripping procedure in Franz static diffusion cells as used by Dermal Technology Laboratory for dermal absorption studies is shown in the Figure below.
Since the stratum corneum skin barrier is a non-viable layer of the skin, both fresh and frozen human and animal skin can be used to assess in vitro percutaneous penetration (OECD, 2004c; OECD, 2011). Since the method is designed to assess the diffusion or permeation of a chemical through a non-viable barrier, the stratum corneum, it is very important to ascertain that the barrier function is intact prior to dosing the skin. Regardless of the type of skin preparation used, its integrity must be demonstrated prior to the application of the test material. Several methods, including transcutaneous electrical resistance, transepidermal water loss, and tritiated water permeability, are permitted for assessing the integrity of skin samples prior to their use (Davies et al., 2004; OECD, 2004a; SCCS, 2010; EFSA, 2012). The electrical resistance method is regarded as the most practical and reliable of these integrity checks. Furthermore, it is important that laboratories conducting these GLP studies have demonstrated that their methods for assessing skin integrity and the dermal absorption of reference compounds are in line with previously published methods (Davies et al., 2004; SCCS, 2010; OECD, 2011; EFSA, 2012; Heylings, 2012).
Experimental variables that may affect the results of in vitro dermal penetration studies include: the selection of the in vitro test system, the storage and preparation of the skin samples, the incorporation of the radio-labeled test substance, volatility of the test substance, test substance degradation, the homogeneity and stability of the dose preparations, exposure time, occlusion/non-occlusion of the test site on the skin, temperature, removal of test substances at the end of the experiment, sampling and data analysis techniques, and the sensitivity and accuracy of the analytical method (OECD, 2004a; OECD, 2011). Many protocol variables have been shown to influence the rate and extent of skin absorption of a test substance. These variables include the use of infinite versus finite dosing, test substance volatility, vehicle of application, exposure time, and leave on versus rinse off of test substance (Brain et al., 1995; Walters et al., 1997). This is exactly why a strict adherence to guidance is required in modern day studies by the various regulatory bodies (SCCS, 2010; EFSA, 2012).
The test substance retained within the skin (except for the stratum corneum) is included in the calculated absorbed dose, although in the live animal it may eventually be lost by shedding and renewal of the skin. The so-called “systemically available dose” used in a risk assessment from human skin studies includes the proportion of the applied dose that has reached the receptor fluid at 24 hours, plus the amount in the remaining skin following tape stripping. In addition, depending on the type of risk assessment, the time course profile of absorption and expected exposure to the chemical during its use, a proportion of the tape strips of the stratum corneum may also be included as “potentially systemically available” (EFSA, 2012). For example, for a pesticide that demonstrates a slow continuous absorption into the receptor fluid over a 24 hour period, despite a skin surface wash at 6 hours, if the proportion of the applied dose reaching the receptor at 12 hours is below 75% of the amount in the receptor at 24 hours, then the material present in the tape strips following discarding of strip 1 and strip 2 is also included in the “dose absorbed.” This is for reasons of conservatism for this type of product, and so as not to under-estimate dermal absorption (EFSA, 2012). This so-called 2-strip rule for pesticides does not apply to cosmetic ingredients that utilize a slightly different approach for calculation of dermal absorption, albeit using essentially the same methodology (SCCS, 2010).
The OECD guidance and TG for in vitro percutaneous penetration testing were issued in 2004, but the guidelines are very broad and do not endorse specific protocols or protocol components. An example of some of the experimental variables to be considered with in vitro dermal penetration studies are described in the report assessing the percutaneous penetration of diethanolamine in cosmetic formulations using in vitro human skin (Brain et al., 2005), including the testing of rinse off versus leave in formulation, varying the formulation ingredients, using replicate skin samples from different donors, and comparing permeability in fresh versus frozen skin tissues. Many of the variables described above have been studied in more detail over the years and harmonization of the protocols has improved the reliability, reproducibility, and accuracy of in vitro dermal absorption testing.
Human cell-based or reconstituted human epidermal (RHE) skin cell models, sometimes called 3-dimensional (3D) skin models, are also used to evaluate dermal penetration. These models retain some capacity to assess the skin metabolism of a test substance, but they are more permeable than the ex vivo human and animal preparations, and are not permitted at this time for the prediction of skin penetration in a human risk assessment (SCCS, 2010; OECD, 2011; EFSA, 2012).
(Q)SAR models, which are computational models based on molecular structure, have been developed to assess the skin permeability of drugs and chemicals. As with skin culture models, these mathematical models have a utility in the pre-development arena, particularly for pharmaceuticals. There are suggestions for incorporating (Q)SARs for dermal absorption into risk assessments for use in programs such as REACH (Van de Sandt, et al., 2007; Berge, 2009).
Reconstituted 3D skin and (Q)SAR models for dermal penetration testing are covered further in the Emerging Science & Policy section.
In vitro dermal penetration methods that utilize resected human skin are now widely used throughout the world. To promote the development of standardized protocols and better agreement of in vitro dermal permeation methods, the Institute for In Vitro Sciences (IIVS) hosted a workshop in Gaithersburg, USA for a group of international stakeholders back in 2005 to discuss the OECD guidance and to make recommendations on implementing specific aspects of the guidance. Recommendations from this meeting are summarized in a poster available on the IIVS website. Also in 2005, the World Health Organization (WHO) International Programme on Chemical Safety convened a meeting of dermal absorption experts in Hanover, Germany. This group produced a comprehensive monograph detailing the knowledge and development in this area. The output of this task group was published the following year as an Environmental Health Criteria (EHC) 235 (WHO, 2006). This includes issues such as selection of the skin model and receptor fluid, best practices for storing frozen skin, barrier integrity assessment methods, and more. Additional recommendations by the workshop participants were that human skin be considered the “gold standard,” that more in vitro data is submitted to regulatory agencies to promote its acceptance, and that some mechanism be developed for sharing the responses of regulatory agencies on submitted in vitro data to relevant stakeholders.
The regulatory authorities in the European Union now extensively utilize the in vitro human skin method as a stand-alone method as part of the approval process for the registration of new pesticide products. In an attempt to determine the barriers to acceptance of stand-alone in vitro dermal absorption studies by North American authorities a workshop was held in 2012 in Gaithersburg, Maryland, USA, bringing together an international panel of dermal absorption experts with USA and Canadian pesticide regulators and non-governmental representatives. The outcome of the workshop was to build consensus around best practices for the conduct and reporting of in vitro dermal absorption studies for pesticide risk assessment and to increase comparability of in vitro studies across different laboratories. There has certainly been a move towards more extensive use of the in vitro approach particularly by US EPA. For example, a recent review of the pesticide sulfoxaflor utilized the so-called “triple-pack” approach that uses in vitro dermal absorption data generated in rat and human skin to correct the rat in vivo absorption value for man (US EPA, 2012).
Hopefully, by the end of this decade, there will be universal acceptance of in vitro dermal penetration methods using resected human skin by all nations and all regulatory authorities round the world and we can look back at this area of safety testing where good science and extensive dialogue between the different stakeholders has led to not just the reduction and refinement of animal procedures, but the complete replacement of living animals for the purposes of estimating the absorption of chemicals through the skin.
AltTox Editorial Board reviewer(s):
William Dressler, PhD
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