Alternative Methods for Skin Irritation Testing: the Current Status

Philip A. Botham, Lesley K. Earl, Julia H. Fentem, Roland Roguet and Johannes J.M. van de Sandt

The ECVAM Skin Irritation Task Force was established in November 1996, primarily to prepare a report on the current status of the development and validation of alternative tests for skin irritation and corrosion and, in particular, to identify any appropriate non-animal tests for predicting human skin irritation which were sufficiently well-developed to warrant ECVAM supporting their prevalidation/validation. The task force based its discussions around the proposed testing strategy for skin irritation/corrosion emanating from an OECD workshop held in January 1996. The following have been reviewed: a) structure-activity and structure-property relationships for skin corrosion and irritation; b) the use of pH and acid/alkaline reserve measurements in predicting skin corrosivity; c) in vitro tests for skin corrosion; d) in vitro tests for skin irritation (keratinocyte cultures, organ cultures, and reconstituted human skin models); and e) human patch tests for skin irritation. It was apparent that, although several promising candidate in vitro tests for skin irritation (for example, reconstituted human skin methods, and human and animal skin organ culture methods) were under development and evaluation, a test protocol, a preliminary prediction model and supporting data on different types of chemicals were only available for a method employing EpiDermTM. Thus, it is proposed that this EpiDerm test undergoes prevalidation during 1998. In addition, since it was felt preferable to be able to include other in vitro tests in such a prevalidation study, it is recommended that a “challenge” be set to anyone interested in taking part. This involves submitting data on ten test chemicals selected by the task force, obtained according to a standard protocol with a preliminary prediction model, for review by the task force by 31 May 1998.
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An Evaluation of the Proposed OECD Testing Strategy for Skin Corrosion

Andrew P. Worth, Julia H. Fentem, Michael Balls, Philip A. Botham, Rodger D. Curren, Lesley K. Earl, David J. Esdaile and Manfred Liebsch

The use of testing strategies which incorporate a range of alternative methods and which use animals only as a last resort is widely considered to provide a reliable way of predicting chemical toxicity while minimising animal testing. The widespread concern over the severity of the Draize rabbit test for assessing skin irritation and corrosion led to the proposal of a stepwise testing strategy at an OECD workshop in January 1996. Subsequently, the proposed testing strategy was adopted, with minor modifications, by the OECD Advisory Group on Harmonization of Classification and Labelling. This article reports an evaluation of the proposed OECD testing strategy as it relates to the classification of skin corrosives. By using a set of 60 chemicals, an assessment was made of the effect of applying three steps in the strategy, taken both individually and in sequence. The results indicate that chemicals can be classified as corrosive (C) or non-corrosive (NC) with sufficient reliability by the sequential application of three alternative methods, i.e., structure-activity relationships (where available), pH measurements, and a single in vitro method (either the rat skin transcutaneous electrical resistance (TER) assay or the EPISKINTM assay). It is concluded that the proposed OECD strategy for skin corrosion can be simplified without compromising its predictivity. For example, it does not appear necessary to measure acid/alkali reserve (buffering capacity) in addition to pH for the classification of pure chemicals.
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Co-occurring Words: Finding Information About Alternatives to Animal Testing

Jane Huggins

A collection of co-occurring words has been gathered from a small database of abstracts about alternatives to skin irritation testing by using Boolean logic. Words were selected according to a strategy based on methodology. Such words and their co-occurrences may be considered an archival code by which data that describe alternatives to skin irritation testing can be more readily recognised. As such, they can be used to enhance the efficiency with which information about this area of alternatives to animal testing is found in journal articles, databases and Web sites
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ECVAM’s Activities in Validating Alternative Tests for Skin Corrosion and Irritation

Julia H. Fentem and Philip A. Botham

ECVAM has funded and managed validation studies on in vitro tests for skin corrosion, resulting in the validities of four in vitro tests being endorsed by the ECVAM Scientific Advisory Committee: the rat skin transcutaneous electrical resistance (TER) assay, two tests based on the use of commercial reconstituted human skin equivalents, EPISKIN™ and EpiDerm™, and another commerciallyproduced test, CORROSITEX®. In the European Union (EU), a new test method on skin corrosion (B.40), incorporating the rat skin TER and human skin model assays, was included in Annex V of Directive 67/548/EEC in mid-2000, thereby making the use of in vitro alternatives for skin corrosion testing of chemicals mandatory in the EU. At the recommendation of its Skin Irritation Task Force, ECVAM has funded prevalidation studies on five in vitro tests for acute skin irritation: EpiDerm, EPISKIN, PREDISKIN™, the pig-ear test, and the mouse-skin integrity function test (SIFT). However, none of the tests met the criteria (set by the Management Team for the studies) for inclusion in a large-scale formal validation study. Thus, to date, there are no validated in vitro tests for predicting the dermal irritancy of chemicals. Following further work on the EPISKIN, EpiDerm and SIFT test protocols and/or prediction models after the completion of the prevalidation studies, it appears that the modified tests could meet the performance criteria defined for progression to a validation study. This will now be assessed independently by the ECVAM Skin Irritation Task Force, with the objective of taking a decision before the end of 2002 on whether to conduct a formal validation study.
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Comparison of Bovine Udder Skin with Human and Porcine Skin in Percutaneous Permeation Experiments

Frank Netzlaff, Ulrich F. Schaefer, Claus-Michael Lehr, Peter Meiers, Jessica Stahl, Manfred Kietzmann and Frank Niedorf

Rat and pig animal skin has been the most common replacement material for human skin for use in in vitro permeability experiments. Unfortunately, the permeability barrier of skin from laboratory animals is known to be relatively weak, due to significant follicular transport. Pig skin has been shown to be a suitable model for human skin. Unfortunately, it cannot be gathered from the regular slaughtering process, which makes it unsuitable for permeation experiments. We therefore studied the suitability of bovine udder skin, an untreated waste material of the butchering process, as a possible replacement material for use in in vitro permeability tests. We investigated the barrier strength of bovine udder skin against four different substances, and its histology and lipid profile, in comparison with pig skin and heatseparated human epidermis. Pig and human skin were found to be equally permeable, whilst bovine udder skin seemed to exhibit a weaker, but less variable, barrier against caffeine, benzoic acid, testosterone, and flufenamic acid. The skin of all three species contained variable contents of the major lipid classes: cholesterol, ceramides, cholesterol ester, fatty acids and triglycerides. Morphological differences mainly comprised variations in the density of hair follicles. Based on these results, the amount of free fatty acids and triglycerides and the density of hair follicles seem to be important factors in the differences between the skin barriers in the three species.
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The Automated, Accurate and Reproducible Determination of Steady-state Permeation Parameters from Percutaneous Permeation Data

Frank Niedorf, Elisabeth Schmidt and Manfred Kietzmann

Procedures for the in vitro determination of percutaneous permeation with Franz diffusion cells are widely accepted. However, the calculation of relevant endpoints, such as the steady-state flux (J) and the permeation coefficient (Papp), still depends on visual data inspection or an approximation of the steady-state flux as the maximum observed absorption rate. As both these approaches must be considered inappropriate, an automated and reproducible algorithm to analyse permeation data is presented. The method detects both lag-times and non-linear data resulting from substance accumulation in the acceptor compartment of static diffusion cells. It was evaluated by using simulated data, and data from experiments with caffeine and testosterone on bovine udder skin and human reconstituted epidermis (SkinEthic®), which represent model barriers with high and low barrier strengths, respectively. It was shown that the algorithm is a suitable method for the identification of steady-state ranges in permeation data. If used on data generated with appropriate experimental approaches, it is a reproducible and time-saving alternative to the visual analysis of diffusion data.
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