Progress Toward the Validation of Alternative Tests

Michael Balls and Julia H. Fentem

ECVAM's role in the practical validation of replacement alternative methods for use in regulatory testing is reviewed, including an outline of the criteria which have been used in determining ECVAM's priorities. Some of the difficulties which have arisen in validation studies are discussed, and solutions to these are proposed, with particular emphasis on ensuring that methods are sufficiently well-developed to enter the validation process, and on the ECVAM prevalidation scheme for encouraging protocol optimisation and the prior assessment of interlaboratory transferability. Comments are made on problems encountered in selecting test materials backed by adequate in vivo data and in undertaking appropriate in vivo/in vitro comparisons.
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The High Production Volume Chemical Challenge Program: The Rodent LD50 and its Possible Replacement

Herbert S. Rosenkranz and Albert R. Cunningham

The High Production Volume Chemical Challenge Program provides an opportunity to re-examine the usefulness and informational value of tests currently used to obtain preliminary hazard identification data. With a view to assessing the mechanistic information provided by the rodent LD50 test and to ascertain the possibility of replacing it with other “more acceptable” assays, we used a recently developed approach to determine the relationship of the LD50 assay to other toxicological protocols. Our analyses indicate that, of the assays examined, the LD50 was significantly related to toxicity in cultured cells and to binding at the Ah receptor.
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International Cooperation: An Essential Requirement for Replacing Animal Toxicity

Horst Spielmann

The Three Rs concept, which was developed by Russell & Burch in 1959, was implemented into the legal framework in the European Union (EU) for the protection of vertebrate animals used for experimental and other scientific purposes, when Directive 86/609/EEC was adopted in 1986. One focus of activity under this Directive is the use of animals and alternative methods in regulatory testing. To reduce or replace animal testing for regulatory purposes, non-animal tests must be independently validated to prove that they can provide information that is relevant and reliable for hazard prediction in relation to specific types of toxicity in vivo. At the end of the 1980s, no scientific concept existed for the formal validation of in vitro toxicity tests, so a small group of European and American scientists met to develop a set of principles for experimental validation, which were first adopted by ECVAM in Europe in 1995, and, after harmonisation with experts from the USA and Japan, accepted internationally by the OECD in 1996. ECVAM has directly funded a number of validation studies, and a major breakthrough in the year 2000 was the acceptance for regulatory purposes in the EU of cientifically validated in vitro toxicity tests for phototoxic potential and for skin corrosivity. These, and other examples which are discussed, confirm that the internationally harmonised ECVAM/ICCVAM/OECD validation concept is a practical and effective way of making possible the replacement of regulatory testing in animals.
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The Use of Fetal Bovine Serum: Ethical or Scientific Problem?

Carlo E. A. Jochems, Jan B.F. van der Valk, Frans R. Stafleu and Vera Baumans

Fetal bovine serum (FBS) is a common component of animal cell culture media. It is harvested from bovine fetuses taken from pregnant cows during slaughter. FBS is commonly harvested by means of a cardiac puncture without any form of anaesthesia. Fetuses are probably exposed to pain and/or discomfort, so the current practice of fetal blood harvesting is inhumane. Apart from moral concerns, several scientific and technical problems exist with regard to the use of FBS in cell culture. Efforts should be made to reduce the use of FBS or, preferably, to replace it with synthetic alternatives.
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Growing Old Disgracefully: The Cosmetic Use of Botulinum Toxin

Krys Bottrill

The explosive growth in the use of botulinum toxin for cosmetic purposes has undoubtedly had an impact on the number of animals used in the potency testing of this product. The test used is a classical LD50, a severe procedure during which animals experience increasing paralysis until the occurrence of death. The enthusiastic adoption by the general public of the use of botulinum toxin as an anti-wrinkle treatment has, at least in Europe, paradoxically taken place against a background of moves to stop animal testing of cosmetics and cosmetic ingredients. There appears to be a dearth of information aimed at the public concerning botulinum toxin testing. Botulinum toxin does have important medical applications; however, the question arises whether a blanket licence for the testing can be justified, when a large proportion of the product is being used cosmetically. A further question is why death continues to be the endpoint of the potency test, when a more-humane endpoint has been proposed. In addition, a number of alternative methods have been developed, which could have the potential to replace the lethal potency test altogether. These methods are discussed in this paper, and the importance of establishing a strategy for their validation is emphasised, a need that has become even more urgent in the light of the recently published draft monograph on botulinum toxin by the European Pharmacopoeia Commission.
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Rapid Simultaneous Cloning of Drug Targets from Multiple Mammalian Species

Rachael Jupp, Palminder K. Dusanjh, Andrew Walding, Mark McHale, Graham P. Belfield and Stephen J. Delaney

A method for the routine, rapid and simultaneous cloning of drug targets from multiple mammalian species is described. This expedites the generation of recombinant proteins and cell lines that can provide alternatives to animal experiments. This was achieved by the collection of RNA from a comprehensive range of tissues from a variety of species, and the optimisation of cDNA synthesis. This “zooplate” has been successfully used for the simultaneous amplification and cloning of drug targets from multiple species. These products have subsequently been used to develop in vitro assays that support efficacy and safety studies in new drug discovery programmes. Within the framework of the Three Rs, these reagents can reduce the number of animals required to provide material for ex vivo assays and can refine the in vivo studies that are still necessary.
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Estimates for Worldwide Laboratory Animal Use in 2005

Katy Taylor, Nicky Gordon, Gill Langley and Wendy Higgins

Animal experimentation continues to generate public and political concern worldwide. Relatively few countries collate and publish animal use statistics, yet this is a first and essential step toward public accountability and an informed debate, as well as being important for effective policy-making and regulation. The implementation of the Three Rs (replacement, reduction and refinement of animal experiments) should be expected to result in a decline in animal use, but without regular, accurate statistics, this cannot be monitored. Recent estimates of worldwide annual laboratory animal use are imprecise and
unsubstantiated, ranging from 28–100 million. We collated data for 37 countries that publish national statistics, and standardised these against the definitions of ‘animals’, ‘purposes’ and ‘experiments’ used in European Union Directive 86/609/EEC. We developed and applied a statistical model, based on publication rates, for a further 142 countries. This yielded our most conservative estimate of global animal use: 58.3 million animals in 179 countries. However, this figure excludes several uses and forms of animals that are included in the statistics of some countries. With the data available, albeit for only a few countries, we also produced, by extrapolation, a more comprehensive global estimate that includes animals killed for the provision of tissues, animals used to maintain genetically-modified strains, and animals bred for laboratory use but killed as surplus to requirements. For a number of reasons that are explained, this more-comprehensive
figure of 115.3 million animals is still likely to be an underestimate.
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Worldwide Trends in the Use of Animals in Research: The Contribution of Genetically-modified Animal Models

Elisabeth H. Ormandy, Catherine A. Schuppli and Daniel M. Weary

The Three Rs — Reduction, Replacement and Refinement — which were first proposed in 1959 by Russell and Burch, have become widely accepted principles in the governance of humane animal research. However, there is substantial variation in the ways in which different countries document the numbers and types of research animals used, making it difficult to determine how effectively the Three Rs are being implemented. Here, we provide the first data illustrating worldwide trends in animal use for research purposes. To document global trends in animal use, we sampled 2691 articles from 24 countries, published between 1983 and 2007, in four scientific journals. We show that the percentage of articles reporting animal use has risen in the past 15 years. The rising popularity of genetic modification methods has contributed to this trend: reported genetically-modified animal use has more than doubled since 1997. We also show that mice are the most commonly-used species for genetic modification, and that, even in 2007, relatively inefficient random integration methods were still widely used to achieve genetic modification. These results illustrate shortcomings in the effort to implement the Three Rs in animal research.
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Computer Simulation Models are Implementable as Replacements for Animal Experiments

Dinesh K. Badyal, Vikas Modgill and Jasleen Kaur

It has become increasingly difficult to perform animal experiments, because of issues related to the procurement of animals, and strict regulations and ethical issues related to their use. As a result, it is felt that the teaching of pharmacology should be more clinically oriented and that unnecessary animal experimentation should be avoided. Although a number of computer simulation models (CSMs) are available, they are not being widely used. Interactive demonstrations were conducted to encourage the departmental faculty to use CSMs. Four different animal experiments were selected, that dealt with actions of autonomic drugs. The students observed demonstrations of animal experiments involving conventional methods and the use of CSMs. This was followed by hands-on experience of the same experiment, but using CSMs in small groups, instead of hands-on experience with the animal procedures. Test scores and feedback showed that there was better understanding of the mechanisms of action of the drugs, gained in a shorter time. The majority of the students found the teaching programme used to be good to excellent. CSMs can be used repeatedly and independently by students, and this avoids unnecessary experimentation and also causing pain and trauma to animals. The CSM programme can be implemented in existing teaching schedules for pharmacology undergraduate teaching with basic infrastructure support, and is readily adaptable for use by other institutes.
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