In Vitro Test Guideline Methods

Several of the validated in vitro methods aim to be validated as full replacement methods to offer an alternative for in vivo test guideline method(s). However, for integrated testing strategies, validated mechanistic tools targeting key events in adverse outcome pathways are necessary to generate state-of-the-art alternative approaches to conventional toxicological test methods based on laboratory animals. 

OECD, Skin absorption in vitro method. OECD Test Guideline 428, Paris, 2004. Wagner, H., et ah. Drug distribution in human skin using two different in vitro test systems comparison with in vivo data. Pharm. Res., 17, 1475-81, 2000. 

OECD has adopted an in vitro test for skin absorption potential (OECD TG 428, Skin Absorption In Vitro Method). According to this guideline, excised skin from human or animal sources can be used. The skin is positioned in a diffusion cell consisting of a donor chamber and a receptor chamber, between the two chambers. The test substance, which may be radio-labeled, is applied to the surface of the skin sample. The chemical remains on the skin for a specified time under specified conditions, before removal by an appropriate cleansing procedure. The fluid in the receptor chamber is sampled at time points throughout the experiment and analyzed for the test chemical and/or metabolites. 

In recent years, several types of in vitro approaches have been developed to assess the absorption and metabolic pathways of substances. Except for the OECD TG 428, Skin Absorption In Vitro Method, none of these test methods have yet been adopted as a test guideline method. 

There are no adopted test guideline methods for in vitro jex vivo testing of eye corrosion or skin and eye irritation. 

The OECD, US-EPA, and EU have developed a number of specific test guidelines for in vitro testing of mutagenicity/genotoxicity, see Table 4.15. The EU Annex V methods are generally... 

The use of vertebrates to evaluate tolerability and absorption of drug administered via the vaginal route has been widely criticized on the basis of scientific and ethical considerations. Studies on animals can be substituted by validated in vitro tests as described in the guideline issued by Committee for Proprietary Medicinal Products (CPMP), now Committee for Medicinal Products for Human Use (CHMP) [112], Before an in vitro test can be considered valid, this test must undergo a procedure aimed at establishing its relevance and reliability. The relevance of the alternative test has to be compared with accepted in vivo standard methods. 

A number of OECD Test Guidelines based on in vitro methods related to the endpoint of ocular irritation have already been adopted (i.e., OECD TG 437,438,460). A draft TG based on the CM test method is under discussion at OECD level. All these TG identify severe eye irritants (UN GHS Cat. 1) and/or chemicals not classified for eye hazards according to the UN GHS. Furthermore, they all describe the usefulness and limitations of the in vitro tests within the conceptual framework of the top-down and/or bottom-up approach. Currently, in the absence of methods able to identify cat. 2 chemicals in a bottom-up or top-down approach, ocular irritant chemicals (GHS Cat. 2) may be resolved by default in a last tier in both approaches, or if not acceptable by regulators, a confirmatory animal test may be requested. 

Once a new in vitro method is validated and accepted for regulatory use, end-users need to assure that the in vitro method produces consistent, reliable, and relevant data which are in line with the originally validated in vitro method as described in the test guideline. To aid in this process EURL ECVAM has detailed... 

OECD. Test guideline 428 Skin Absorption In Vitro Method, OECD, Paris, 2004. 

In Vitro Membrane Barrier Test Method for Skin Corrosion, Draft New Guideline (May 2004)... 

The 3R concept lies behind efforts to improve ethical standards for the use of experimental animals throughout the scientific community, including toxicity testing. A number of in vitro methods for genetic toxicology testing have been established as guideline methods for many years, e.g., the bacterial reverse mutation test, more popularly known as the Ames test. 

OECD (2004a). Skin absorption in vitro method, OECD Guideline 428, Guideline for the Testing of Chemicals, Environment Directorate, Organization for Economic Cooperation and Development, Paris, France. 

The standardized guidelines have been compiled to cover test methods in animal studies and in vitro studies on absorption, distribution, metabolism cind excretion in the Notification No. 496 of the PAB in 1998. The guidelines can be applicable for the drugs to be submitted after October 1999. The following principles should be considered in order to select the most appropriate methods bcised on the characteristics of test substance. It is required that the exposure data related to toxicological studies be obtained before the first human study and other pharmacokinetic data before the completion of Phcise I study in principle in accordance with the guidelines on nonclinical safety studies for conducting clinical trials (Notification No. 1019 of the PAB, 1998). 

The Bovine Corneal Opacity and Permeability test method (BCOP) is based on normal physiological and biochemical function of the bovine cornea in vitro. For the BCOP, the eyes are taken from animals slaughtered for human consumption. In this test method (OECD Test Guideline, TG 437), the damage caused by the test substance is assessed by quantitative measurements of changes in corneal opacity and permeability with an opacitometer or a visible... 

The Cytosensor Microphysiometer (CM) test method is currently under discussion (draft test guideline on the Cytosensor Microphysiometer) at OECD level [34], It is a cytotoxicity and cell-function based in vitro assay that is performed on a sub-confluent monolayer of adherent mouse L929 fibroblasts cultured in a sensor chamber using a pH-meter to detect changes in acidity [35]. The CM test method serves as an in vitro model system for the cytotoxic action of a test chemical on the cell membranes of the corneal and conjunctival epithelium where the irritant chemical would be... 

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