Clive S. Roper

Inter- and intra-individual variability in human skin barrier function: a large scale retrospective study.

In vitro transepidermal tritiated water flux measurements are frequently used to evaluate skin barrier integrity for quality control purposes. However, research in this area to date has been largely based upon small-scale studies, each involving relatively few skin permeation measurements. In order to enhance our understanding in this area, we have conducted a much larger scale retrospective statistical analysis of tritiated water kp values. These values reflected the permeability of 2400 skin samples that were derived from 112 female volunteers over a 4 year period. It was found that the population of tritiated water kp values constituted a positively skewed, non-Normal distribution. Mean kp was 2.04 x 10(-3)cm/h while the 95th percentile was 4.50 x 10(-3)cm/h. Both values are higher than those reported in previous smaller studies. Hence, our study indicates that previously suggested upper limits for tritiated water flux are too low and that they be revised upwards to a value of 4.5 x 10(-3)cm/h. Analysis was also performed on smaller data subsets allowing inter-individual and intra-individual comparisons. For intra-individual kp variability, site-related differences yielded a non-Normal, positively skewed pattern in most individuals. Inter-individual variability was Normally-distributed and showed scatter that was much smaller in magnitude.

Determining Epidermal Disposition Kinetics for Use in an Integrated Nonanimal Approach to Skin Sensitization Risk Assessment

Development of risk assessment methods for skin sensitization in the absence of toxicological data generated in animals represents a major scientific and technical challenge. The first step in human skin sensitization induction is the transport of sensitizer from the applied dose on the skin surface to the epidermis, where innate immune activation occurs. Building on the previous development of a time course in vitro human skin permeation assay, new kinetic data for 10 sensitizers and 2 nonsensitizers are reported. Multicompartmental modeling has been applied to analyze the data and determine candidate dose parameters for use in integrated risk assessment methods: the area under the curve (AUC) and maximum concentration (C(max)) in the epidermis. A model with two skin compartments, representing the stratum corneum and viable skin (epidermis and dermis), was chosen following a formal model selection process. Estimates of the uncertainty, as well as average values of the epidermal disposition kinetics parameters, were made by fitting to the time course skin permeation data from individual skin donors. A potential reduced time course method is proposed based on two time points at 4 and 24 h, which gives results close to those from the full time course for the current data sets. The time course data presented in this work have been provided as a resource for development of predictive in silico skin permeation models.

DEVELOPMENT OF A MODIFIED IN VITRO SKIN ABSORPTION METHOD TO STUDY THE EPIDERMAL/DERMAL DISPOSITION OF A CONTACT ALLERGEN IN HUMAN SKIN

In vitro skin absorption methods exist in Organisation for Economic Co-operation and Development (OECD) guideline form (No. 428) and are used to estimate the degree of systemic penetration of chemicals through skin. More detailed kinetics of permeation through skin compartments are not described well by existing methods. This study was designed to assess the practical feasibility of generating compartmental (stratum corneum/epidermal/dermal) disposition and kinetic data of topically applied chemicals. For chemically induced effects initiated in the skin (e.g., skin allergy), the delivery of tissue concentrations of chemical will impact the incidence and severity of biological effect. Explicit data on the kinetics of chemical disposition in skin have not traditionally been needed for skin allergy risk assessment: current in vivo assays embody delivery implicitly. Under the 7th Amendment to the European Cosmetics Directive, in vivo assays (such as the local lymph node assay for skin sensitization) will not be permitted to assess cosmetic ingredients. New in vitro and in silico alternative approaches and ways of predicting risk of adverse effects in humans need to be developed, and new methods such as that described here provide a way of estimating delivered concentrations and the effect of formulation changes on that delivery. As we continue to deconstruct the contributing factors of skin allergy in humans, it will be useful to have methods available that can measure skin tissue compartment exposure levels delivered from different exposure use scenarios. Here we provide such a method. The method could also be used to generate useful data for developing in silico kinetic models of compartmental skin delivery and for refining data for skin delivery in relation to the evaluation of systemic toxicity.

Absorption of [14C]-Tetrabromodiphenyl Ether (TeBDE) Through Human and Rat Skin In Vitro

The skin is the largest organ in the human body and has the potential to come into contact with a variety of xenobiotics both intentionally (e.g., drugs and cosmetics) or accidentally (e.g., agrochemicals and industrial chemicals). These chemicals may then cross the skin barrier (the stratum corneum) and enter into the systemic circulation where they may produce a desired or an undesired effect, or even no systemic effect at all. Tetrabromodiphenyl ether (TeBDE) is one congener in a mixture of polybrominated diphenyl ethers that makes up a flame-retardant commercial product called pentabromodiphenyl ether (PeBDE). TeBDE was used as a surrogate to assess the potential dermal absorption of this product. The physicochemical properties, including lipophilicity, of TeBDE and PeBDE are similar. Operator exposure of PeBDE product to human skin is possible during production and use. However, during these activities, operators wear protective clothing to protect from or minimize exposure. This study was designed to assess the rate and extent of absorption of [14C]-tetrabromodiphenyl ether ([14C]-TeBDE) through human and rat skin in vitro. [14C]-TeBDE was applied to human and rat split thickness skin membranes in vitro in a single test preparation: [14C]-TeBDE in acetone (ca. 20%, w/v). Dermal delivery and absorbed dose of TeBDE applied to human skin was 3.13% (313 μg equiv/cm2) and 1.94% (194 μg equiv/cm2) of the applied dose, respectively. Dermal delivery and absorbed dose of TeBDE applied to rat skin was 17.94% (1804 μg equiv/cm2) and 14.81% (1489 μg equiv/cm2) of the applied dose, respectively. These results confirm that the risk of systemic exposure due to external dermal exposure of the PeBDE product is low in the human. Consequently, based on the toxicological profile of these materials, the potential for undesirable effects is also quite low. The results also confirm that the rat is a conservative model overpredicting human absorption about eight fold.

On the correlation between single-frequency impedance measurements and human skin permeability to water

The objective of this study was to quantitatively compare measurements of tritiated water permeability with impedance determined at either 100 or 1000 Hz using an LCR databridge on the same pieces of skin. A previously published expression based on a simple circuit of a parallel resistor and constant phase element (CPE) was used to relate (RPARA) measured at different frequencies to the DC resistance (RskinA) and the steady-state skin permeability of tritiated water (kp). Using this analysis, kp and (RPARA) data from three laboratories were shown to be consistent with each other, and kp and (RskinA) estimated from (RPARA) were linearly correlated. Compared with urea and mannitol, which are known to permeate skin through a polar pathway, the value of kp for water was found to be about two times larger than expected for transport through only the polar pathway, suggesting an approximately equal contribution from the lipophilic pathway. Equations relating kp to (RPARA) and (RskinA) were used to compare on a consistent basis proposed tests for identifying and excluding damaged skin from chemical absorption studies. The criterion of 20 kΩ cm2 for (RskinA) corresponds to a tritiated water permeability of 3.2×10(-3) cm/h, which should exclude damaged skin without screening undamaged but higher permeability skin samples from study.

Multi-laboratory validation of SkinEthic HCE test method for testing serious eye damage/eye irritation using liquid chemicals.

A prospective multicentric study of the reconstructed human corneal epithelial tissue-based in vitro test method (SkinEthic™ HCE) was conducted to evaluate its usefulness to identify chemicals as either not classified for serious eye damage/eye irritation (No Cat.) or as classified (Cat. 1/Cat. 2) within UN GHS. The aim of this study was to demonstrate the transferability and reproducibility of the SkinEthic™ HCE EITL protocol for liquids and define its predictive capacity. Briefly, 60 chemicals were three times tested (double blinded) in 3 laboratories and 45 additional chemicals were tested three times in one laboratory. Good within laboratory reproducibility was achieved of at least 88.3% (53/60) and 92.4% (97/105) for the extended data set. Furthermore, the overall concordance between the laboratories was 93.3% (56/60). The accuracy of the SkinEthic™ HCE EITL for the extended dataset, based on bootstrap resampling, was 84.4% (95% CI: 81.9% to 87.6%) with a sensitivity of 99.0% (95% CI: 96.4% to 100%) and specificity of 68.5% (95% CI: 64.0% to 74.0%), thereby meeting all acceptance criteria for predictive capacity. This efficient transferable and reproducible assay is a promising tool to be integrated within a battery of assays to perform an eye irritation risk assessment.

Multi-laboratory evaluation of SkinEthic HCE test method for testing serious eye damage/eye irritation using solid chemicals and overall performance of the test method with regard to solid and liquid chemicals testing

A prospective multicentre study of the reconstructed human corneal epithelial tissue-based in vitro test method (SkinEthic™ HCE) was conducted to evaluate its usefulness to identify chemicals as either not classified for serious eye damage/eye irritation (No Cat.) or as classified (Cat. 1/Cat. 2) within UN GHS. The aim of this study was to demonstrate the transferability and reproducibility of the SkinEthic™ HCE EITS protocol for solids and define its predictive capacity. Briefly, 60 chemicals were three times tested (double blinded) in 3 laboratories and 35 additional chemicals were tested three times in one laboratory. Good within laboratory reproducibility was achieved of at least 95% (57/60) and 96.8% (92/95) for the extended data set. Furthermore, the overall concordance between the laboratories was 96.7% (58/60). The accuracy of the SkinEthic™ HCE EITS for the extended dataset, based on bootstrap resampling, was 81.0% (95% CI: 78.9% to 83.2%) with a sensitivity of 90.5% (95% CI: 88.1% to 92.9%) and specificity of 73.6% (95% CI: 71.7% to 75.5%). Overall, 200 chemicals were tested (105 liquids (EITL protocol) and 95 solids (EITS protocol)) resulting in a sensitivity of 95.2%, specificity of 72.1% and accuracy of 83.7%, thereby meeting all acceptance criteria for predictive capacity.

Pathway-based predictive approaches for non-animal assessment of acute inhalation toxicity

New approaches are needed to assess the effects of inhaled substances on human health. These approaches will be based on mechanisms of toxicity, an understanding of dosimetry, and the use of in silico modeling and in vitro test methods. In order to accelerate wider implementation of such approaches, development of adverse outcome pathways (AOPs) can help identify and address gaps in our understanding of relevant parameters for model input and mechanisms, and optimize non-animal approaches that can be used to investigate key events of toxicity. This paper describes the AOPs and the toolbox of in vitro and in silico models that can be used to assess the key events leading to toxicity following inhalation exposure. Because the optimal testing strategy will vary depending on the substance of interest, here we present a decision tree approach to identify an appropriate non-animal integrated testing strategy that incorporates consideration of a substances physicochemical properties, relevant mechanisms of toxicity, and available in silico models and in vitro test methods. This decision tree can facilitate standardization of the testing approaches. Case study examples are presented to provide a basis for proof-of-concept testing to illustrate the utility of non-animal approaches to inform hazard identification and risk assessment of humans exposed to inhaled substances.

Application of in vitro skin penetration measurements to confirm and refine the quantitative skin sensitization risk assessment of methylisothiazolinone

ABSTRACT Use of quantitative risk assessment (QRA) for assessing the skin sensitization potential of chemicals present in consumer products requires an understanding of hazard and product exposure. In the absence of data, consumer exposure is based on relevant habits and practices and assumes 100% skin uptake of the applied dose. To confirm and refine the exposure, a novel design for in vitro skin exposure measurements was conducted with the preservative, methylisothiazolinone (MI), in beauty care (BC) and household care (HHC) products using realistic consumer exposure conditions. A difference between measured exposure levels (MELs) for MI in leave‐on versus rinse‐off BC products, and lower MELs for MI in HHC rinse‐off compared to BC products was demonstrated. For repeated product applications, the measured exposure was lower than estimations based on summation of applied amounts. Compared to rinse‐off products, leave‐on applications resulted in higher MELs, correlating with the higher incidences of allergic contact dermatitis associated with those product types. Lower MELs for MI in rinse‐off products indicate a lower likelihood to induce skin sensitization, also after multiple daily applications. These in vitro skin exposure measurements indicate conservatism of default exposure estimates applied in skin sensitization QRA and might be helpful in future risk assessments. HighlightsNovel in vitro skin absorption study designs reflect realistic MI consumer exposure.Exposure estimations for repeated product usages per day are over‐conservative.Large difference between measured exposure levels in leave‐on versus rinse‐off.Much lower exposure for HouseHoldCare rinse‐off compared to BeautyCare products.High leave‐on exposure in line with higher incidence of allergic contact dermatitis.