Recent inter-laboratory assessments (Van de Sandt, et al., 2004) and prevalidation studies (Schäfer-Korting, et al., 2006) of in vitro dermal penetration methods have led to refined and standardized protocols; the lack of such protocols had been a reason sometimes cited for not using the in vitro percutaneous methods endorsed in OECD TG 428. If these studies lead to formal validation of one or more in vitro protocols, the in vitro methods may become more acceptable to US regulatory authorities. However, scientists conducting dermal penetration studies continue to stress the need to be able to modify components of the test systems as needed for different testing applications.
Current research for in vitro dermal absorption includes the refinement of existing methods, such as the following examples. Davies, et al. (2004) showed that measuring electrical resistance across an in vitro skin membrane preparation for evaluating the integrity of skin preparations prior to their use in dermal penetration assays was an excellent alternative to measuring transepidermal water loss or permeability to tritiated (radiolabeled) water. Electrical resistance measurements were fast, cheap, non-damaging to the skin, and did not require disposal of radioactive substances. Thin, dermatomed skin is commonly used for dermal penetration testing. Wilkinson, et al. (2006), however, 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. Considering the different testing needs and applications of dermal penetration studies, existing protocols will continue to be refined.
The skin 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. The ability to assess dermal xenobiotic biotransformation is a desirable and sometimes important 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 also have the potential to meet this testing need. Human 3D skin cell models have been shown to possess xenobiotic metabolizing enzyme activity; the question of how well their metabolic activity represents that found in intact human skin is the focus of current studies at the MatTek Corporation and studies funded by COLIPA (European Cosmetic Toiletry & Perfumery Association).
Current research for in vitro dermal absorption includes the development of new testing approaches. A novel in vitro method using a silastic membrane-coated fiber was developed for determining the percutaneous penetration of chemical mixtures (Xia, et al., 2003). The identity and amount of each compound in a mixture that permeated into the fiber was quantified using gas chromatography/ mass spectroscopy (GC/MS). A membrane-coated fiber array that used three different types of membranes to simulate the different molecular interactions involved in skin absorption correlated well with porcine skin permeability (Baynes, et al., 2008; Riviere, et al., 2007; Xia, et al., 2007). Another artificial membrane developed for assessing skin permeability (also used for intestinal permeability) is the parallel artificial membrane permeability assay (PAMPA) (Ottaviani, et al., 2006). An improved version of the PAMPA model was developed by substituting the lipid mixture in the filter with a lipid/oil/lipid tri-layer (Chen, et al., 2008).
(Q)SAR models for the prediction of dermal penetration continue to be developed, refined, and reassessed. The numerous publications in this area include suggestions for incorporating (Q)SARs for dermal absorption into risk assessments for use in programs such as REACH (Van de Sandt, et al., 2007), and the development of new (Q)SARS incorporating more chemicals and molecular structure descriptors (Basak, et al., 2007; Luo, et al., 2007; Riviere & Brooks, 2007). A review of seven models by 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.
Nanoparticle-sized materials are becoming widely used in cosmetics and household products, and because some have been shown to be involved in pathological processes, their ability to penetrate the skin is beginning to be assessed (Kiss, et al., 2008). 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.
An emerging technology for 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 use of the TTC principle is acknowledged for food additives, flavors, and contaminants by the US Food and Drug Administration (FDA) and the Joint FAO/WHO Expert Committee on Food Additives (JECFA). Determination of the TTC values (in units of micrograms (µg) per person per day) for structurally-related chemicals is based on databases of results from chemical structure and chronic oral toxicity data that resulted in no-observed-effect levels (NOELs). The estimated oral intake of a chemical is then compared to the TTC value for chemicals with similar physiochemical properties.
The TTC principle can 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). Kroes, et al. (2007) reported that "the oral TTC values are valid for [systemic effects from] topical exposures" as long as local effects and route-dependent differences from the oral data, such as skin metabolism, are taken into consideration. TTC for dermal penetration involves using the estimated human absorbed dose and physiochemical 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 community of scientists involved with dermal penetration testing and research meets every other year at the Perspectives in Percutaneous Penetration (PPP) meeting. A summary of the March 2008 PPP meeting is available as one of AltTox's May 2008 In the Spotlight articles.
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