Bone grafting is commonly used to treat large bone defects. Since autografts are limited and frequently associated with postoperative donor morbidity, allografts from bone banks are often used. However, vascularisation of the allograft is often impaired, resulting in inadequate bone healing and functional graft failure. In bone bank processing, tissue is stored at -80°C and subsequently subjected to a harsh multi-step cleaning and sterilisation procedure to prevent immune rejection or transmission of diseases.
To determine which step of this procedure diminishes the ability of allografts to induce or promote vascularisation, we used the chick chorioallantoic membrane (CAM) model to monitor the vascular reaction to sample bone chips representing the respective procedural steps. The CAM model monitors the angiogenic potency of xenogeneic and, hence, potentially immunogeneic materials (e.g. cells, tissues, tissue-engineered matrices). Due to the chicken embryo’s lack of a fully functional immune system, it provides test conditions that are analogous to immunologically incompetent mice and is a well-suited alternative to their use. Bone chips were placed onto the CAM, and vascular reactions were quantified by image analysis after 48 h incubation. The vascular reaction was most pronounced to fresh, untreated bone chips that had been kept at +2°C prior to the experiment. Surprisingly, storage of bone samples at -80°C was sufficient to drastically reduce the vascular reaction. Consistent with this, samples representing different stages of the subsequent procedure showed similarly low vascular indices.

During the past 30 years great effort has been put into establishing an insulin-secreting beta cell line that retains normal regulation of insulin secretion, but only few of these attempts have been successful. To overcome the limited availability of primary beta cells and to include the principles of the 3Rs into the field of diabetes mellitus research, numerous investigators used X-rays or viruses to induce insulinomas, in vitro transformation, derivation of cells from transgenic mice or even non-islet cells to produce immortalised beta cell lines. The most widely used insulin-secreting cell lines are RIN, HIT, MIN, INS-1 and βTC cells. These cells produce insulin and small amounts of glucagon and somatostatin. Some of them are only poorly responsive to glucose, others respond to glucose well, but their concentration-dependence curve is markedly shifted to higher sensitivity. Despite problems associated with beta cell cultures, these cell lines have provided some valuable information about physiological processes. However, an urgent need to establish a “normal” beta cell line of human or pig origin remains.

All of life’s great journeys start with a goal in mind! The 2007 NAS report, Toxicity Testing in the 21st Century – A Vision and A Strategy, has proposed a clear goal. This report envisions a not-so-distant future where all routine toxicity testing for environmental agents will be conducted in human cells in vitro evaluating perturbations of cellular responses in a suite of toxicity pathway assays. Dose response modeling would utilize computational systems biology models of the circuitry underlying each toxicity pathway; in vitro to in vivo extrapolations would use pharmacokinetic models, ideally physiologically based pharmacokinetic models, to predict human blood and tissue concentrations under specific exposure conditions. Results from these toxicity pathway assays and associated dose response modeling tools rather than those from high dose studies in animals would represent the new gold standard for chemical risk assessment. This talk focuses on some of the scientific challenges required to make this vision a reality, including characteristics of assay design, prospects for mapping and modeling toxicity pathways, assay validation, and biokinetic modeling. All of these tools necessary for this transformation of toxicity testing to an in vitro platform are either available or in advanced development. Science must lead the transformation. The scientific community, animal alternatives groups, regulatory agencies, and funding organizations will also have to muster the resolve to work together to make this vision a reality.

In The Principles of Humane Experimental Technique, Russell and Burch said that “the central problem is that of determining what is and what is not humane, and how humanity can be promoted without prejudice to scientific and medical aims”. They then explained how the Three Rs can be used to diminish or remove direct inhumanity (“the infliction of distress as an unavoidable consequence of the procedure employed”) and contingent inhumanity (“the infliction of distress as an incidental and inadvertent by-product of the use of a procedure”). They concluded that “Replacement is always a satisfactory answer, but Reduction and Refinement should, whenever possible, be used in combination”.
Many of the commonsense insights in The Principles are no less relevant today than they were in 1959. However, their warnings about the limited value of models and, in particular, the danger of succumbing to the high-fidelity fallacy (whereby it is assumed that the best models for humans are always placental mammals, because they are more like humans than other animals), appear to have largely gone unheeded. Of particular importance is their discussion on toxicity testing, which they saw as one use of laboratory animals “which is an urgent humanitarian problem, for it regularly involves considerable and sometimes acute distress”. How, then, can it be that mammalian models are still routinely used in attempts to detect chemical carcinogens and reproductive toxins, despite the fact that the relevance to humans of the data they provide has not been, and perhaps could never be, satisfactorily established? Nevertheless, there are signs that some significant changes in attitude are taking place, particularly in the USA, which could be more in line with the main thrust of The Principles, the belief that good science and human technique inextricably go hand-in-hand.

In the section on replacement in their 1959 book, The Principles of Humane Experimental Technique, Bill Russell and Rex Burch state: “As new fields of biology open in the future, it may become a matter of routine to apply the lessons of the past and turn as soon as possible to the techniques of replacement.” They foresaw in vitro techniques, in their infancy at that time, as the science of the future. Today, in the US, the National Academy of Sciences publication of Toxicity Testing in the 21st Century: A Vision and a Strategy proves their point. This pivotal publication recognizes that the future of toxicity testing lies in the use of human cells in culture and in methods that Bill Russell and Rex Burch could not have possibly conceived of in 1959 but which they identified generically as “the future.”
To truly establish this 21st Century approach will require very specific training in translational toxicology (the use of clinical observations to develop in vitro methods to understand pathways and systems biology), the development of transnational programs, and ways to evaluate the accuracy, validity and importance of new and/or traditional studies. These evaluations are known at evidence-based toxicology (EBT). Science is “the art of the question.” The concepts identified above are the tools to answer these questions – and to lead us to the next round of questions. The principles that Bill Russell and Rex Burch developed during the 1954-59 writing of “The Book” may be more important today than ever before. They argued that the newest science, the most humane science is also the very best science. This hypothesis is being proven now, as each of us contributes to the world’s body of knowledge.

As society’s ethical values expand over time, we understand that we must have consideration for more than just ourselves, our race, our gender, and our species. This talk confronts our biases and provides food for thought in moving beyond our current understanding of human-animal relations. History provides a lens through which our current norms can be viewed. It allows us to discern how our behavior might be perceived by future generations, and this perspective can help us understand how to improve our behavior. While it is easy to be appalled by what has been done in the past, it is more challenging to uncover the actions taking place today that will be regarded with horror in the future and, most important by far, to be a part of the necessary change. By modifying one’s perspective to include a more empathetic view of other animals, our obligations and potential become clear.

Apart from the fact that the LD50 test is generally considered a procedure causing severe distress, which alone should result in its immediate deletion, it also conflicts with the EU wide ban on cosmetic testing in animals in the case of Botulinum Neurotoxin (BoNT) testing. The European Pharmacopoeia monograph allows for the use of three alternative methods provided they are validated. However, these alternative assays are neither implemented by the manufactures of BoNT products nor are they enforced by the responsible authorities, e.g. by deleting the LD50 from the European Pharmacopoeia. The number of animals used for the testing of BoNT is not officially recorded. However, data from an undercover investigation allow the estimation that the number of mice used in LD50 tests for BoNT products is greater than 600,000 per year worldwide.  

In 2007, 2.7 million vertebrates were used for animal experiments and other scientific purposes in Germany alone. Since 1998 there has been an increase in the number of animals used for research purposes, which is partly attributable to the growing use of transgenic animals (Fig. 1). These animals are, for instance, used as in vivo models to mimic human diseases like diabetes, cancer or Alzheimer's disease. Here, transgenic model organisms serve as valuable tools, being instrumental in facilitating the analysis of the molecular mechanisms underlying human diseases, and might contribute to the development of novel therapeutic approaches. Due to variable and, sometimes low, efficiency (depending on the species used), however, the generation of such animals often requires a large number of embryo donors and recipients. The experts evaluated methods that could possibly be utilised to reduce, refine or even replace experiments with transgenic vertebrates in the mid-term future. Among the promising alternative model organisms available at the moment are the fruit fly Drosophila melanogaster and the roundworm Caenorhabditis elegans. Specific cell culture experiments or threedimensional (3D) tissue models also offer valuable opportunities to replace experiments with transgenic animals or reduce the number of laboratory animals required by assisting in decision-making processes. Furthermore, at the workshop an in vitro technique was presented which permits the production of complete human antibodies without using genetically modified (“humanised”) animals. Up to now, genetically modified mice are widely used for this purpose. Improved breeding protocols, enhanced efficiency of mutagenesis as well as training of laboratory personnel and animal keepers can also help to reduce the numbers of laboratory animals. Well-trained staff in particular can help to minimise the pain, suffering and discomfort of animals and, at the same time, improve the quality of data obtained from animal experiments. This, in turn, can lead to a reduction in the numbers of animals needed for each experiment. The experts also came to the conclusion that the numbers of laboratory animals can be reduced by open access to a central database that provides detailed documentation of completed experiments involving transgenic animals. This documentation should not be restricted to experiments with substantial scientific results that warrant publication, but should also include those with “negative” outcome, which are usually not published. Capturing all kinds of results within such a database provides added value to the respective scientists and the scientific community as a whole; it could also help to stimulate collaborations and to ensure funding for future research. An important aspect to be considered in the generation of this kind of database is the quality and standardisation of the information provided on existing in vitro models and the respective opportunities for their use.
The experts felt that the greatest potential for reducing the numbers of laboratory animals in the near future realistically might not be offered by the complete replacement of transgenic animal models but by opportunities to examine specific questions to a greater degree using in vitro models, such as cell and tissue cultures including organotypic models. The use of these models would considerably reduce the number of in vivo experiments using transgenic animals. However, the overall number of experimental animals may still be increasing or remain unaffected, e.g. when transgenic animals continue to serve as the source of primary cells and organs/tissues for in vitro experiments.