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Inventory of the Spanish Institutions and Scientists Involved in Alternatives to the use of Laboratory Animals (Refinement, Reduction or Replacement)+Inventory: 4. Alternative Methods
The methods used in research, education and testing are subject to continuous progress. Researchers are engaged in a permanent exploration of possible alternatives to improve the quality of their work. This is due in part to the evolution of scientific knowledge and its practical applications, as well as to ethical, logistical, economic, socio-political, and legal considerations.
4.1.- The three ‘Rs’
Alternative methods include procedures that replace or reduce the use or need for animals in a particular test, or that refine a technique in order to reduce the amount of suffering endured by the animals. The concept of the Three Rs (replacement, reduction, and refinement) was developed by Russell & Burch (1959) to provide a framework for improving the conduct and ethical acceptability of experimental techniques on animals (Festing et al., 1998).
In practical terms, non-necessary experiments in vivo or in vitro should be avoided by making use of integrated strategies, prediction modelling (quantitative structure-activity relationships, but also kinetics prediction), and the availability and interchange of pre-existing information and experimental results.
Given that animals used in research may experience pain, suffering or lasting harm, the first step must be to consider whether less sentient or non-sentient alternatives can be used instead (replacement). Where this is not possible, care needs to be taken to minimize the pain that any individual animal may suffer (refinement), both during the actual experiment and before or after the conduct of experiments. Lastly, the number of animals used in a given project needs to be minimized (reduction), while ensuring that the objectives of the study can still be achieved; typically, this will also reduce the sum total of animal suffering.
However, the most promising alternatives encompass the use of lower organisms with limited sentience and/or non-protected by legislation controlling animal experiments, including bacteria, fungi, algae, plants, and invertebrate animals; vertebrate use at early stages of development --from fish, amphibians, reptiles and birds to mammals; and, particularly, the employment of in vitro methods using material from these organisms (Repetto, 1995; Balls, 1998).
Table 1. ALTERNATIVE METHODS
1. Avoidance of non-necessary experiments in vivo and in vitro:
2. Mathematical Prediction Modelling:
3. Improved design of animal studies:
4. Use of lower non-protected organisms:
5. Vertebrates at early stages of development:
6. In vitro methods:
7. Others:
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(Balls, 1998; Repetto et al., 1999)
4.2.- Recent Advancements
International harmonization (via the International Conferences on Harmonization) of standards governing the toxicity testing of pharmaceuticals appears to have resulted in nearly a 50% reduction in the number of animals used to test some pharmaceuticals (Lumley and Cauteren, 1997).
The development of in vitro techniques has had a spectacular effect on both model systems and bioindicators, in terms of the evolution of molecular methods and improvements in cell and tissue culture. Among the advances produced are new coculture and microaggregate methods, and the use of growth factors, matrices, inserts, chambers, plastics and membranes. Genetically manipulated model systems have increased our understanding of the action mechanisms of chemicals. A number of technological advances have increased the specificity and sensibility of the employed biomarkers, including those with biochemical, morphological and electrophysiological bases. Molecular biology techniques have made us aware of both the way in which different genes are expressed and the relevance of their alterations. In addition, the procedures can be performed with a high degree of robotization and automatization, increasing the productivity and reliability of the assays (Repetto et al., 1999).
Table 2. BIOINDICATORS USED IN VITRO |
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1 |
Cell and tissue morphology |
Form, size, differentiation |
Membranes, organelles |
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2 |
Cellular Viability |
Dye uptake, adhesion, phagocytosis |
3 |
Cellular Proliferation |
Proteins, DNA, cell cycle |
4 |
Metabolic Activity |
Regulatory Substances |
Energy use, enzymes, pH, Bioluminescence, Calorimetry |
||
Macromolecular Synthesis |
||
Selective induction of proteins |
||
5 |
Cytoskeleton / Membranes |
Composition and stability |
Ionic Permeability |
||
Transport Systems |
||
6 |
Cell signalling |
GJIC, cytokines, NO |
7 |
Nucleic Acids |
Gene expression / inhibition Mutation / degradation / apoptosis |
8 |
Biotransformation Systems |
MFO, P450 |
9 |
Defence systems |
GSH, G6PDH, metallothioneins |
10 |
Specific indicators |
NS, liver, Immune S, Reprod S |
(Repetto et al., 1999)
One important piece of information needed in risk assessment is that concerning the concentration range at which a chemical exerts adverse effects on the organisms living in the aquatic or terrestrial environment. Without this information we can neither make predictions nor establish safety factors. According to the basal cytotoxicity concept, a majority of chemicals cause toxicity by basal cytotoxicity, while a clear minority cause toxicity by interference with either organ-specific cell functions or extracellular bodily functions. According to this reductionistic view, the toxicity of a compound can be broken down into a number of elements, each of which can be identified and quantified in appropriate model systems (Ekwall, 1994). It seems possible that a limited number of cell lines or isolated cells from invertebrates and vertebrates may be sufficient for basal cytotoxicity screening.
4.3.- Validation and acceptance of toxicological methods
The Sixth Amendment (79/831/EEC) to the Classification, Packaging and Labelling of Dangerous Substances Directive (67/548/EEC) imposed a mandatory requirement for manufacturers and importers of new substances to provide a set of toxicological, eco-toxicological and physico-chemical data before placing a new substance into the common market for sale or use.
The scientific community accepts the utility and results obtained in basic research using a variety of in vivo and in vitro procedures for the investigation of physiological or pharmacological effects, toxicodynamic mechanisms, toxicokinetic processes, etc. However, in order either to be used in the standard toxicity tests required prior to the commercialization, transportation and use of a new chemical compound, or for environmental control purposes, it is necessary for the experimental procedure to be scientifically validated and accepted by regulatory authorities.
Validation is the process by which the reliability and relevance of a procedure are established for a specific purpose. Thus, after its development, the procedure has to successfully pass through prevalidation (previous interlaboratory assessment), followed by validation of its reproducibility and relevance to in vivo toxicity (final interlaboratory assessment), independent assessment of the study by a panel of experts and, finally, progression toward regulatory acceptance (Balls et al., 1995; Repetto, 1995).
Although regulatory acceptance of new toxicological methods has been very slow in coming, some alternative procedures have been accepted by scientists and the industry, and are routinely used in areas such as the monitoring and control of pollutants. It is necessary to promote their validation and acceptance as real alternatives to vertebrate tests.
Accepted testing protocols are published by national and international regulatory activities. The most widely used as reference are the guidelines of the Organization for Economic Cooperation and Development (OECD), listed on internet at the web site http://www.oecd.org/ehs/test/testlist.htm.
Efforts have been taken to reach international consensus on alternative methods for testing and assessment, including the development of testing strategies, in order to further reduce the number of animals used in safety testing of chemicals and to minimize animal suffering. Governments will accept alternative methods for the testing and assessment of chemicals once these tests are sufficiently validated in accordance with agreed criteria (Walker et al., 1998). However, despite much effort, considerable financial expenditure, moral, practical, and scientific argument, plus legal mandates to use acceptable alternatives to animal testing, progress has been distressingly slow (Balls, 1998).
Fuente / Source: Guillermo Repetto, Ana del Peso, Manuel Salguero, Manuel Repetto (1999) Inventory of the Spanish Institutions and Scientists Involved in Alternatives to the use of Laboratory Animals (Refinement, Reduction or Replacement) Revista de Toxicología 16: 50-127.
