Alternatives to Laboratory Animals

A ‘middle ground’ exists, where the advocates and the opponents of vivisection can usefully negotiate, but where exactly is it?

A study on the effects of environmental enrichment on behaviour and cognitive and motor functioning in a standard mouse model and a strain known to have behavioural deficits, suggests that environmental enrichment can positively influence natural functioning and natural behaviour

Lower organisms can be particularly useful as high discrimination models in fundamental research, but cannot offer the high fidelity required when specific questions are asked about humans in health and disease, and about their responses to drugs and other chemicals

A further increase in the use of genetically-modified animals will be against the European Union policy to move away from the use of animals in biomedical research

The main achievements and results of the ESNATS project (Embryonic Stem Cell-based Novel Alternative Testing Strategies) were presented at the final project conference that was held on 15 September 2013, the day before the traditional EUSAAT (European Society for Alternatives to Animal Testing) Congress in Linz, Austria. The ESNATS project was an FP7 European Integrated Project, running from 2008 to 2013, the aim of which was to develop a novel toxicity testing platform based on embryonic stem cells (ESCs), and in particular, human ESC (hESCs), to accelerate drug development, reduce related R&D costs, and propose a powerful alternative to animal tests in the spirit of the Three Rs principles. Altogether, ESNATS offered the first proof of concept that hESCs can be used to create robust, reproducible and ready-to use test assays for predicting human toxicity. In the end, essentially five test systems were developed to an adequate level for entering possible pre-validation procedures. These methods are based on hESCs, and can be combined to study the possible effects, on the human embryo, of exposure to a chemical during the early stages of development. In addition to the presentations by the main project partners, external speakers were invited to give lectures on relevant topics, both in the field of neurotoxicity and, more generally, on the applicability of hESCs in the development of advanced in vitro tests.

Compared with traditional animal methods for toxicity testing, in vitro and in silico methods are widely considered to permit a more cost-effective assessment of chemicals. However, how to assess the cost-effectiveness of alternative methods has remained unclear. This paper offers a user-oriented tutorial for applying cost-effectiveness analysis (CEA) to alternative (non-animal) methods. The purpose is to illustrate how CEA facilitates the identification of the alternative method, or the combination of methods, that offers the highest information gain per unit of cost. We illustrate how information gains and costs of single methods and method combinations can be assessed. By using acute oral toxicity as an example, we apply CEA to a set of four in silico methods (ToxSuite, TOPKAT, TEST, ADMET Predictor), one in vitro method (the 3T3 Neutral Red Uptake cytotoxicity assay), and various combinations of these methods. Our results underline that in silico tools are more cost-effective than the in vitro test. Battery combinations of alternative methods, however, do not necessarily outperform single methods, because additional information gains from the battery are easily outweighed by additional costs.

The use of animals in science is a widespread practice, despite growing concern about its moral justification and scientific relevance. In this scenario, the Three Rs concept might be considered to be a motivation for the establishment of a new scientific approach to the use of experimental animals and to research itself. The main objective of this survey-based study was to identify the level of knowledge about this concept among lecturers (i.e. tenure-track professors) and postgraduate students in the physiological and pharmaceutical sciences in Brazilian universities. A questionnaire was completed by 185 lecturers from 16 universities, and 140 postgraduate students from five of these universities. The results indicate that the concept of the Three Rs is widespread among lecturers and students in the areas of physiology and pharmacology, throughout Brazilian universities, but that its interpretation generally attributes more importance to refinement than to reduction and replacement.

The scientific basis and advantages of using recently developed CRISPR/Cas-9 technology for transgenesis have been assessed with respect to other production methods, laboratory animal welfare, and the scientific relevance of transgenic models of human diseases in general. As the new technology is straightforward, causes targeted DNA double strand breaks and can result in homozygous changes in a single step, it is more accurate and more efficient than other production methods and speeds up transgenesis. CRISPR/Cas-9 also obviates the use of embryonic stem cells, and is being used to generate transgenic non-human primates (NHPs). While the use of this method reduces the level of animal wastage resulting from the production of each new strain, any long-term contribution to reduction will be offset by the overall increase in the numbers of transgenic animals likely to result from its widespread usage. Likewise, the contribution to refinement of using a more-precise technique, thereby minimising the occurrence of unwanted genetic effects, will be countered by a probable substantial increase in the production of transgenic strains of increasingly sentient species. For ethical and welfare reasons, we believe that the generation of transgenic NHPs should be allowed only in extremely exceptional circumstances. In addition, we present information, which, on both welfare and scientific grounds, leads us to question the current policy of generating ever-more new transgenic models in light of the general failure of many of them, after over two decades of ubiquitous use, to result in significant advances in the understanding and treatment of many key human diseases. Because this unsatisfactory situation is likely to be due to inherent, as well as possibly avoidable, limitations in the transgenic approach to studying disease, which are briefly reviewed, it is concluded that a thorough reappraisal of the rationale for using genetically-altered animals in fundamental research and by the pharmaceutical industry, and for its support by funding bodies, should be undertaken. In the meantime, the use of CRISPR/Cas-9 to generate new transgenic cells in culture is to be guardedly encouraged.

We are in total agreement with the ABPI that “informed debate amongst key stakeholders” is important. However, this should include animal protection groups, in addition to their suggested partnership between “the pharmaceutical industry, academia and the regulatory authorities.”

Unfortunately, as we have tried to explain, we believe that the ABPI authors have been too eager to play down the importance of our study. Far from being constructive, their critique is overly critical to the point of being unhelpful. Criticism must be fair and robust. When it has a weak scientific basis, it is unacceptable, especially when it is used as a basis to persist with the status quo, for which there is weak supportive evidence.

We acknowledge (as we did in our paper) that there are inherent and unavoidable caveats to our analysis. Notwithstanding this, our study is unprecedented in scale and type. Notably, it is much broader and more comprehensive than any of the studies cited by the ABPI, which involved a combination of: more limited (smaller and more restricted) sample sizes; exaggeration in terms of their supportive nature for dog studies; overlooking of evidence against dog studies; incomplete statistics, from which conclusions could not be drawn; incorrect statistical definitions and analyses; and questionable, or absent, human relevance. Conspicuously, none of these studies addressed any of the criticisms made by the ABPI of our study, on dosage and rare events, for example. In addition, the ABPI failed to provide constructive suggestions on how we could go about addressing these concerns. How does the ABPI suggest we address the problems of data limitations, dosage/exposure, and incidence of toxicity — considerations absent, or at least almost absent, in previous studies? Constructive criticism must include suggestions on how to go about resolving those issues. Arguably, given the restricted amount of data that are publicly available, at least some of those concerns cannot be addressed by third parties. The ABPI, and associated groups, could (and should) help by facilitating access to such information. Nevertheless, we were already in the process of analysing the data available to us in greater depth, the outcome of which will be published in the near future.

To reiterate: we did all we could do, with the data that were available to us, to produce a study of unprecedented scale and type. We are confident about, and as certain as we could be of, our analysis and our conclusions. They should not be overlooked because of their source, and because they are inconvenient to any stakeholders (including the Federation of Laboratory Animal Breeders Associations, which, not surprisingly, supports the ABPI's arguments). Should the ABPI be genuinely concerned that our analysis, using all the data and correct statistical methods at our disposal, is not sufficient, we refer it to our statement within our paper: “…if any pharmaceutical industry stakeholders have issues or concerns with our conclusions, we would encourage them to conduct further analyses by using their own proprietary data, and/or to facilitate such investigations by making available anonymised data, in accordance with the promotion of transparency encouraged by EU Directive 2010/63/EU, as well as to engage fully in constructive discussion and debate with us and our colleagues in animal protection organisations.” The only acceptable response by industry and regulators should be, at the very least, a desire for positive action to facilitate, or conduct, if possible, another study on a greater scale, involving the use of proprietary data. To ignore the most relevant findings, and/or focus on other inferred shortcomings of other elements of the study, is unacceptable, particularly when the upshot is to defend and perpetuate seriously flawed dog toxicology, with little or no substantiation.