Replacement Methods

Alternative methods are basically differentiated according to 3R aspects (Replace, Reduce, Refine). Alternative methods are defined in terms of replacement as non-animal methods that can completely replace concrete animal experiments. If such methods are available, an animal experiment cannot be approved under the German Animal Welfare Act and the European Directive for the Protection of Laboratory Animals 2010/63/EU.

Replacement methods can follow different approaches:

• Investigation of a specific molecular mechanism of action: This is used, for example, when testing for genotoxicity (changes in the genetic material) or phototoxicity (light-dependent toxicity).
• Organotypical models (e.g. skin models): Here, complex models of an organ (e.g. skin) are cultivated in vitro and used directly for the detection of a health hazard, such as irritation or corrosion, as well as for investigations into the absorption of substances (e.g. through the skin).
• Verification of a specific physiological process as part of testing and assessment strategies: Here interactions of different cell types are simulated by combining different in vitro as well as in silico models. In the case of skin sensitisation, these are, for example, haptenisation, activation of epidermal and dendritic cells in the skin, and finally activation of T-cells in the lymph nodes. The results of the individual tests are summarised using a defined data interpretation procedure and therefore allow a prediction of the effect in animals as well as in humans.

The question of whether an animal experiment can actually be replaced by an alternative method depends above all on whether it can also be used for the specific question. This applies to animal experiments in (basic) research, in which it must be clarified to what extent the methods are suitable for the specific scientific question. It applies equally to animal experiments in regulatory safety testing of chemicals, as well as the testing of pesticides or pharmaceuticals. The main question here is whether the properties of the test substance (solubility, reactivity, stability) influence the test using the alternative method to such an extent that the result no longer allows a specific statement.

In the case of pharmaceuticals, it is usually required that the applicability of a method for a product is checked individually in each case (see also Chapter 5.2.14 of the European Pharmacopoeia, in which general rules for the replacement of animal experiments in the field of quality assurance of vaccines are explained). In the area of chemicals, pesticides and biocides, primarily validated methods from the OECD test guidelines programme OECD Testing Methods and Guidelines are used, whereby the scope of a replacement method is examined as part of a formal validation process and limitations with regard to specific substance groups are defined (see list of accepted test methods). Basically, only pure substances are used, so that it is usually unclear to what extent alternative methods are also suitable for testing mixtures or complex formulations in products.

In recent years, the number of in vitro tests for skin and eye irritation/corrosion and sensitisation has increased significantly, since these are legally required together with methods for detecting genotoxicity. The method most commonly used under the European Chemicals Regulation REACH to avoid animal testing is currently not based on in vitro approaches but on analogy concepts, in particular “read-across”, whereby data from structurally related substances are transferred to one another. Here, in vitro methods can increase the acceptance of this approach by proving that structural similarity also correlates with comparable biological activity.

A list of accepted alternative methods can be found here (PDF file,154.93 KB).

This year, TG444A was adopted as a new test guideline in the field of human health which supports the determination of immunotoxicity in vitro. It cannot replace animal testing, but is intended for use in a testing strategy. Last year, TG 467 was published to complement the first test guidelines for the investigation of eye-damaging that contains so-called defined approaches combining different alternative methods in a fixed data interpretation procedure.