Julia H. Fentem

The ECVAM International Validation Study on In Vitro Tests for Skin Corrosivity. 2. Results and Evaluation by the Management Team

As a follow-up to a prevalidation study on in vitro tests for replacing the in vivo rabbit test for skin corrosivity, an international validation study was conducted during 1996 and 1997 under the auspices of ECVAM. The main objectives of the study were to: (a) identify tests capable of discriminating corrosives from non-corrosives for selected types of chemicals and/or all chemicals; and (b) determine whether these tests could identify correctly known R35 (UN packing group I) and R34 (UN packing groups II & III) chemicals. The tests evaluated were the rat skin transcutaneous electrical resistance (TER) assay, CORROSITEX(TM), the Skin(2TM) ZK1350 corrosivity test and EPISKIN(TM). Each test was conducted in three independent laboratories. 60 coded chemicals were tested. All of the tests evaluated showed acceptable intralaboratory and interlaboratory reproducibilities, and the TER, Skin(2) and EPISKIN tests proved applicable to testing a diverse group of chemicals of different physical forms, including organic acids, organic bases, neutral organics, inorganic acids, inorganic bases, inorganic salts, electrophiles, phenols and soaps/surfactants. Two of the four tests evaluated, the TER assay and EPISKIN, met the criteria agreed by the Management Team concerning acceptable underprediction and overprediction rates for them to be considered scientifically validated for use as replacements for the animal test for distinguishing between corrosive and non-corrosive chemicals for all of the chemical types studied [objective (a)]. EPISKIN was the only test able to distinguish between known R35 (UN packing group I) and R34 (UN packing groups II & III) chemicals, for all of the chemical types included, on an acceptable number of occasions [objective (b)]. The corrosive potentials of about 40% of the test chemicals could not be assessed with CORROSITEX, and the assay did not meet all of the criteria for it to be considered acceptable as a replacement test. However, CORROSITEX may be valid for testing specific classes of chemicals, such as organic bases and inorganic acids. The Skin(2) assay did not meet the criteria for it to be considered scientifically validated. Thus, the validities of (i) the TER and EPISKIN assays for discriminating corrosives from non-corrosives, and (ii) the EPISKIN assay for identifying correctly known R35/I and R34/II & III chemicals, have been demonstrated in this study. CORROSITEX appears to be valid when used only with certain types of chemicals.

The ECVAM International Validation Study on In Vitro Tests for Skin Corrosivity. 1. Selection and Distribution of the Test Chemicals.

An international validation study on in vitro tests for skin corrosivity was conducted during 1996 and 1997 under the auspices of the European Centre for the Validation of Alternative Methods (ECVAM). The main objectives of the study were to assess the performances of selected in vitro tests in discriminating between: (a) corrosives (C) and non-corrosives (NC), for selected groups of chemicals (e.g. organic acids, phenols) and/or for all chemicals (single chemical entities only); and (b) known R35 (UN packing group I) and R34 (UN packing groups II & III) chemicals. Each test was evaluated for reliability and relevance by using a test set of 60 coded chemicals. In this paper, the test chemicals used in the validation study are identified; they include organic acids (6C/5NC), organic bases (7C/3NC), neutral organics (9NC), phenols (2C/3NC), inorganic acids (6C/1NC), inorganic bases (2C/2NC), inorganic salts (1C/2NC), electrophiles (3C/5NC) and soaps/surfactants (3NC). The in vivo classifications and important physicochemical properties (e.g. logP, pKa) of the test chemicals are given. The main criterion for including chemicals in the test set was that their corrosivity classifications were based on unequivocal animal data. Where available, structure-activity information was also used to support the corrosivity classifications. Despite the small numbers of chemicals in some of the categories, it was felt that the test set chosen represented the best possible for evaluating the performances of the in vitro tests for predicting skin corrosivity, given the limited availability of unequivocal animal data. The prediction of skin corrosivity from pH data was also investigated for those chemicals with extreme pH values (i.e. pH2 or 11.5). Nine of the 12 strongly acidic or alkaline chemicals in the test set, which were predicted to be C on the basis of their pH values, had also been found to be C in vivo.

Validation—Lessons learned from practical experience

With regard to the problems encountered and the experience gained in validation studies conducted in the past, suggestions have been made concerning criteria for the selection of the tests and laboratories to be included in a validation study, the selection and distribution of test chemicals, and procedures for the handling, analysis and interpretation of the resulting data. In particular, tests should have been developed to the extent that detailed protocols and standard operating procedures have been produced and evaluated. The laboratories should be chosen on the basis of evidence of their appropriate experience, competence and ability to comply with good laboratory practice (GLP) requirements. The choice of test chemicals depends primarily on the goals of the validation study and on the availability of reliable in vivo toxicity data of high quality. A biostatistician should be involved in the initial design of the validation study as well as in the analysis of the resulting data. The quality of the in vivo and in vitro data must be ensured, prior to determining the reproducibility and predictivity of the alternative test.

1 – Replacement Alternative and Complementary In Vitro Methods in Pharmaceutical Research

This chapter considers the extent and nature of the use of in vitro methods (when, why, and how such methods are used, and their role in complementing, as well as in providing replacement alternatives for, in vivo methods), and progress in their application in the development and testing of pharmaceuticals in recent years, and the problems encountered. Pharmaceutical research is an area in which many animal experiments are conducted. In particular, the replacement alternative principle encourages the substitution of insentient material for conscious living animals. In vitro methods need to be considered in terms of the Three Rs concept of alternatives now enshrined in several national laws, and in Directive 86/609/EEC 48 of the European Union (EU), which regulate animal experimentation. In vitro methods are being used extensively as adjuncts (i.e., to provide data that can be used in combination with results obtained in in vivo studies). In this way, they have contributed to a genuine reduction in the numbers of in vivo experiments conducted for drug discovery purposes. However, they have not had as much impact on the number of animal procedures undertaken either for ADME and pharmacokinetic studies, or for toxicity testing. Recently, the UK pharmaceutical industry has devised guidelines for the conduct of infective challenge tests (PD50/rodent protection tests), which are frequently used in early screening to determine the potency of potential antimicrobial agents. The pharmaceutical industry in the UK adheres to the UKCCCR (UK Coordinating Committee for Cancer Research) guidelines when undertaking cancer research, and for the screening and further evaluation of anticancer agents.