The Overview of dermal penetration describes a lot of the background to the methodology used in this area of safety assessment. Naturally, the focus has been on the development and use of the non-animal methodology and in particular the ex vivo human skin approach now widely used throughout the world in the field of dermal absorption. There have been a number of inter-laboratory assessments, prevalidation studies, and validation studies on the same formulations in vitro and in vivo (Ramsey et al., 1994; Schäfer-Korting et al., 2006; Van de Sandt et al., 2004). All have demonstrated the utility of the in vitro approach in predicting in vivo absorption. Furthermore, there have been many individual studies aimed at different aspects of the method that have led to refined and standardized protocols and industry-specific guidance documents that are now used for safety assessment. Several areas of protocol improvement are highlighted below.
The lack of harmonized protocols had been a reason sometimes cited for not using the in vitro percutaneous methods endorsed in OECD TG 428 (OECD, 2004c). However, the publication of the basic criteria for the in vitro assessment of dermal absorption of cosmetic ingredients by the Scientific Committee on Consumer Safety (SCCS, 2010), the guidance on dermal absorption by European Food Safety Authority (EFSA, 2012) for pesticide-containing products, along with the OECD Guidance Notes for the testing of pesticides, biocides, and industrial chemicals (OECD, 2011) has led to a much more harmonized approach to the experimental design, conduct, and interpretation of in vitro dermal penetration studies.
One area of concern raised by some regulatory authorities since the publication of the in vitro OECD test guideline in 2004 was the quality of the human skin used and the need to demonstrate intact barrier properties prior to skin dosing. It was agreed that this should be a key element of the in vitro study protocol. Davies, et al. (2004) showed that measuring electrical resistance across an in vitro skin membrane preparation for evaluating the integrity of skin samples prior to their use was an excellent alternative to measuring the less reliable transepidermal water loss parameter or the permeability of the skin to tritiated (radiolabeled) water. Electrical resistance measurements are fast, cost-effective, non-damaging to the skin, and do not require the use and disposal of radioactive substances. Skin integrity assessment is now a key element of the in vitro protocol.
In a recent comparative study, Guth, et al. (2014) compared the following pre- or post-run integrity tests: transepidermal electrical resistance (TEER); transepidermal water loss (TEWL); absorption of the reference compounds water (TWF), or methylene blue; and co-absorption of a (3)H-labeled internal reference standard (ISTD). They observed the standard methods (TEER, TEWL, and TWF) could distinguish between impaired and intact human skin samples, but that any single skin sample might not provide a correct assessment. The results with ISTD were highly correlated to the absorption of test compounds, providing a promising new approach for skin integrity assessment.
The skin preparation technique was identified as another key element of the in vitro protocol. Wilkinson, et al. (2006) found that different skin thickness had inconsistent effects on the flux of different substances and the amounts retained within skin, and concluded that this variable effect of skin thickness on dermal penetration results should be considered when conducting risk assessments and validation studies. Dermatomed skin cut to a specific thickness is now a requirement for regulatory dermal penetration studies for both industrial chemicals and cosmetic ingredients (SCCS, 2010; OECD, 2011; EFSA, 2012).
Another area that has provided greater confidence in the use of the in vitro approach is the requirement for the GLP studies that are used as part of product registration to include a full mass balance recovery of each of the dose levels applied to the skin (Brain et al., 2005; Saghir et al., 2010). This provides details on the fractions of the dose applied that are clearly not systemically available and the fraction of the dose that may be regarded as “dermally absorbed” in the context of a human risk assessment. This approach using low volume “finite” dose applications requires that the mean mass balance recovery must be within the set limits of 100 +/- 10%, as required by OECD. The reproducibility of the mass balance across the skin replicates is also an important feature of study performance that is considered in the risk assessment. Further details on this can be found in the Overview section.
Xenobiotic metabolizing enzymes are generally expressed in lower levels in extrahepatic tissues. The skin, however, is a large organ and can be responsible for a significant amount of the metabolism/biotransformation of xenobiotic substances, especially those applied directly to the skin. In skin, the phase II enzymes have been identified as the predominant metabolizing enzymes (Gundert-Remy et al., 2014).
Although the OECD guidelines are clear that the measurement of skin penetration in vitro is a passive diffusional process that can be assessed in ex vivo skin and does not require fresh metabolically active skin, the ability to assess dermal xenobiotic biotransformation is sometimes a desirable capability for an in vitro dermal penetration method (Gibbs, et al., 2007; Stinchcomb, 2003). Viable human skin is the preferred test system to assess the skin metabolism of a test substance. Skin from different donors can be used to account for human variability. Human reconstituted (3D) skin cell models have been shown to possess xenobiotic metabolizing enzyme activity. How well their metabolic activity represents that found in intact human skin has been the focus of a number of studies (Eilstein et al., 2014; Eilstein et al., 2015; Gotz et al., 2012a; Gotz et al., 2012b; Zghoul et al., 2001).
Assessments of skin penetration and dermal and systemic metabolism are essential components for the in vitro estimation of the systemic exposure of a test substance. Using human skin explants and human keratinocytes and hepatocytes to test the hair dye p-phenylenediamine, Manwaring, et al. (2015) found the toxicokinetic information generated solely from in vitro data “to be in the same order of magnitude as those published for human volunteers.”
Nanoparticle-sized materials are becoming widely used in cosmetics, personal care, and other household products. Because some nanomaterials have been shown to be involved in pathological processes, their ability to penetrate the skin has been an area of considerable interest over the last decade. For example, titanium dioxide (TiO2) nanoparticles are used in many products that are applied directly to large areas of the skin, including sunscreens. Kiss, et al. (2008) found that TiO2 nanoparticles do not penetrate intact skin, but that they do show some toxicity to cultured cells and therefore could be a hazard when used on damaged skin. Previous studies using in vivo and in vitro testing agree with their conclusion that TiO2 does not penetrate intact human skin (Gamer et al., 2006; Mavon et al., 2007). Kuntsche, et al. (2008) observed some differences in the penetration of nanoparticle formulations between in vivo human skin and 3D epidermal cell cultures. They concluded that the differences could be due to the lower representation of surface lipids in the cultured cells versus the human skin. Since this area is of particular relevance to the cosmetic industry, the Scientific Committee on Consumer Safety has published guidance on the safety assessment of nanomaterials used in cosmetics (SCCS, 2012).
In addition to the safety area, the introduction of nanotechnology has been shown to have advantages in the dermal delivery of pharmaceutical actives. Again, the in vitro methods of dermal penetration have been pivotal in the development of this area where ex vivo animal and human skin has been used as part of the discovery, development, and optimization of new drug formulations based on nanotechnology. One recent example of this has been the visualization of the dermal delivery of the antibacterial drug, chlorhexidine. Normally, these highly charged anti-infective molecules will not permeate the stratum corneum. However, using a ToF-SIMS approach in porcine skin the dermal delivery of the antibacterial drug can not only be assessed in vitro (Judd et al., 2013), but a combination of skin imaging and in vitro diffusion cell studies have shown that nano PAMAM dendrimers can enhance the permeation of chlorhexidine (Judd et al., 2012). This type of application of nanoscience to dermatology could be particularly useful in antibacterial handwashes to reduce the transmission of hospital-acquired infections such as Methicillin-resistant Staphylococcus aureus (MRSA).
Another area that impacts on dermal penetration assessment is the Threshold of Toxicological Concern (TTC). The TTC is the principle used to define human exposure levels to substances below which there may be no significant risk to human health, and therefore toxicological testing is not needed (Kroes et al., 2000; 2004; Munro et al., 1996). The TTC principle is acknowledged for food additives, flavors, and contaminants, and can also be more broadly applied in risk assessments for other types of exposures such as percutaneous penetration; however, it can only be applied to systemic toxicity endpoints, and cannot be used for local endpoints (Kroes et al., 2007). The TTC principle has been proposed for use for cosmetics, personal care products, household products, and pharmaceutical manufacturing operations (Blackburn et al., 2005; Dolan et al., 2005; Kroes et al., 2007). TTC for dermal penetration involves using the estimated human absorbed dose and physicochemical modeling of the chemical to compare to the TTC value. Conservative default skin absorption factors of 10% or 100% are commonly used. Kroes, et al. (2007) analyzed published in vitro human skin dermal penetration data for cosmetic ingredients, and proposed the following default dose absorbed adjustment factors for cosmetic ingredients: negligible; 10%, 40%, and 80% based on the molecular weight and skin flux of the material. If a TTC has not been exceeded, the estimated skin exposure is sufficiently low, and testing would not be required. Adoption of the TTC principle for chemicals where human exposure occurs via the dermal route has the potential to reduce dermal penetration testing requirements, allowing testing resources to be directed toward substances with the highest potential risk to humans.
A new area of interest in the field of dermal penetration and one where there is a need to develop robust and reproducible models that do not involve animals is the impact of compromised skin and how this affects systemic exposure to chemicals. Practically all the investigations involving the percutaneous absorption of substances from their formulated products utilize so-called “normal” skin in vitro and in vivo. Indeed, there are often regulatory and labeling restrictions on the use of specific products on damaged, broken, or diseased skin. With the advancement of nanotechnology for both industrial chemicals and cosmetics, absorption of nanomaterials, particularly those containing metals across compromised skin is even more in focus.
One such approach has been reported, building on the experience of skin barrier perturbation by tape stripping in human skin and in human volunteers. This research has demonstrated that sequential tape stripping of the stratum corneum can produce an effective representation of compromised skin, as assessed by comparing changes in transepidermal water loss in clinical studies with equivalent changes to the barrier properties of ex vivo porcine skin, brought about by a tape stripping regime (Davies et al., 2015). This model has permitted a comparison of skin penetration of several compounds that are used in skin care products in normal and compromised skin without the use of animals (Heylings et al., 2013). The applicability of this new in vitro model has yet to be fully established.
Human cell-based or reconstituted human skin cell models such as EpiDerm, EpiSkin, and SKINETHIC (Asbill et al., 2000; Schäfer-Korting et al., 2006; Schreiber et al., 2005; Zghoul et al., 2001), and a rat keratinocyte culture model (ROC) (Marjukka Suhonen et al., 2003; Pappinen et al., 2007) have been used to evaluate dermal penetration. These models are considered to have some metabolic activity. Although these models may be useful in pre-development research type work, they are generally regarded to be much more permeable than ex vivo human and animal skin preparations and are not permitted to be used to predict skin penetration in a human risk assessment (SCCS, 2010; OECD, 2011; EFSA, 2012).
Many (Q)SAR models have been developed, and a reassessment of the statistical relevance of existing models suggested that heterogeneity in the data used to develop the models (i.e., skin origin and experimental conditions) contributes to their variance (Fujiwara, et al., 2003; Geinoz, et al., 2004). (Q)SAR models for the prediction of dermal penetration continue to be developed, refined, and re-assessed. Lian, et al. (2008) identified two models that were best at predicting the skin permeability of a set of 124 chemicals; the two “assume the lipid matrix as the pathway of transdermal permeation” and “use octanol-water partition coefficient and molecular size.” The authors concluded that the empirical models that used more complicated descriptors were less predictive. Because of therapeutic relevance as well as for safety assessment in toxicological studies, considerable activity continues in the development and evaluation of predictive computational models for skin permeation based on chemical structure. A review of the progress in mathematical model development has been published more recently which demonstrates the utility of this area in predicting dermal absorption of topical and transdermal drug products (Anissimov and Roberts, 2014).
Several types of mathematical models for diffusion-based skin permeation have been reported with results compared to literature and/or in vitro experimental data. An early diffusion model performed satisfactorily for several test substances, but underestimated systemic absorption and skin concentrations of highly lipophilic compounds (Bhatt et al., 2008). The Kasting laboratory has conducted a number of studies to refine skin diffusion models (for example: Gajjar & Kasting, 2014; Miller & Kasting, 2010; Nitsche & Kasting, 2013). “In addition to establishing the necessary mathematical framework to describe these models, efforts have also been dedicated to determining the key parameters that are required to use these models” (Mitragotri et al., 2011). Based on their own previous work and that of others, Nitche and Kasting (2013) developed a model useful “for estimating passive permeability of cell membranes to nonionized solutes as a function of temperature and cholesterol content of the membrane.” With appropriate corrections, they conclude in various studies that the model can satisfactorily describe in vivo dermal absorption. More recently, a multicomponent vehicle model, compared with human in vitro skin permeation data, predicted absorption of small to moderate doses of vanillylnonamide in a propylene glycol vehicle significantly better than simpler models (Miller & Kasting, 2015).
A review of the main in vitro and in silico/computational models available to study skin absorption and skin metabolism has been published by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) (Dumont et al., 2015).
A community of scientists from academia and industry, together with regulators and other stakeholders involved with dermal penetration testing and research, meets every other year at the Perspectives in Percutaneous Penetration (PPP) Conference. This conference and associated information on the PPP website are useful sources of information and ongoing research in the field of percutaneous absorption.
Sherry L. Ward, PhD, MBA
AltTox Contributing Editor
AltTox Editorial Board reviewer(s):
William Dressler, PhD
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