Fetal Bovine Serum (FBS): Past - Present - Future.

The supplementation of culture medium with fetal bovine serum (FBS, also referred to as "fetal calf serum") is still common practice in cell culture applications. Due to a number of disadvantages in terms of quality and reproducibility of in vitro data, animal welfare concerns, and in light of recent cases of fraudulent marketing, the search for alternatives and the development of serum-free medium formulations has gained global attention. Here, we report on the 3rd Workshop on FBS, Serum Alternatives and Serum-free Media, where regulatory aspects, the serum dilemma, alternatives to FBS, case-studies of serum-free in vitro applications, and the establishment of serum-free databases were discussed. The whole process of obtaining blood from a living calf fetus to using the FBS produced from it for scientific purposes is de facto not yet legally regulated despite the existing EU-Directive 2010/63/EU on the use of animals for scientific purposes. Together with the above-mentioned challenges, several strategies have been developed to reduce or replace FBS in cell culture media in terms of the 3Rs (Refinement, Reduction, Replacement). Most recently, releasates of activated human donor thrombocytes (human platelet lysates) have been shown to be one of the most promising serum alternatives when chemically-defined media are not yet an option. Additionally, new developments in cell-based assay techniques, advanced organ-on-chip and microphysiological systems are covered in this report. Chemically-defined serum-free media are shown to be the ultimate goal for the majority of culture systems, and examples are discussed.

How long must they suffer? success and failure of our efforts to end the animal tragedy in laboratories.

Scientific findings have revealed how much we have dramatically underestimated the intellectual, social and emotional capabilities of non-human animals, including their levels of self-consciousness and ability to suffer from psychological stress. In the 21st century, the field of animal ethics has evolved as a serious scientific discipline, and nowadays largely advocates that the way we treat animals, both legally and in practice, is morally wrong. Politics and legislation have reacted to these facts, to some extent, but neither current legislation nor current practice reflect the scientific and moral state-of-the-art. Too often, the will to change things is watered down in the decision-making process, e.g. in the drafting of legislation. In the field of animal experimentation there have been many genuine efforts by various players, to advance and apply the principles behind the Three Rs. However, the fundamental problem, i.e. the overall number of animals sacrificed for scientific purposes, has increased. Clearly, if we are serious about our will to regard animal experimentation as an ethical and societal problem, we have to put much more emphasis on addressing the question of how to avoid the use of animals in science. To achieve this goal, certain issues need to be considered: a) the present system of ethical evaluation of animal experiments, including testing for regulatory purposes, needs to be reformed and applied effectively to meet the legal and moral requirements; b) animal testing must be avoided in future legislation, and existing legislation has to be revised in that regard; c) resources from animal-based research have to re-allocated toward alternatives; and d) the academic curricula must be reformed to foster and integrate ethical and animal welfare issues.

Reconsidering pluripotency tests: do we still need teratoma assays?

The induction of teratoma in mice by the transplantation of stem cells into extra-uterine sites has been used as a read-out for cellular pluripotency since the initial description of this phenomenon in 1954. Since then, the teratoma assay has remained the assay of choice to demonstrate pluripotency, gaining prominence during the recent hype surrounding human stem cell research. However, the scientific significance of the teratoma assay has been debated due to the fact that transplanted cells are exposed to a non-physiological environment. Since many mice are used for a result that is heavily questioned, it is time to reconsider the teratoma assay from an ethical point of view. Candidate alternatives to the teratoma assay comprise the directed differentiation of pluripotent stem cells into organotypic cells, differentiation of cells in embryoid bodies, the analysis of pluripotency-associated biomarkers with high correlation to the teratoma forming potential of stem cells, predictive epigenetic footprints, or a combination of these technologies. Each of these assays is capable of addressing one or more aspects of pluripotency, however it is essential that these assays are validated to provide an accepted robust, reproducible alternative. In particular, the rapidly expanding number of human induced pluripotent stem cell lines, requires the development of simple, affordable standardized in vitro and in silico assays to reduce the number of animal experiments performed.

Inconsistencies in data requirements of EU legislation involving tests on animals.

European Union (EU) legislation on the protection of animals used for scientific purposes requires that alternative methods must be used instead of animal tests wherever they are available. Unfortunately, this provision is not implemented to its full extent when it comes to risk assessment of chemicals and new products prior to their authorization and placing on the market in the EU. In this study, we screened data requirements of relevant EU law regarding chemicals (REACH), biocides, pesticides, and food safety (Novel Food) and found that data requirements as part of the risk assessment do not always reflect state-of-the-art science and technology. Most of the data requirements we investigated still include testing on animals for many toxicological endpoints, even though more than 40 alternative testing methods accepted at the level of the EU or the OECD are available. This may be due to a multitude of reasons, including a shortage of both manpower to implement existing knowledge and expertise in the field of alternative methods, as well as unclear and misleading statements on the applicability and state of validation of alternative methods. In conclusion, we strongly suggest a homogeneous EU-wide approach for all areas involving risk assessment of substances with the goal of better implementing the 3Rs and complying with Directive 2010/63/EU. This also would streamline data requirements, save costs on various levels, and enhance product safety for consumers.

An animal welfare perspective on animal testing of GMO crops.

The public discussion on the introduction of agro-genetic engineering focuses mainly on economical, ecological and human health aspects. The fact is neglected that laboratory animals must suffer before either humans or the environment are affected. However, numerous animal experiments are conducted for toxicity testing and authorisation of genetically modified plants in the European Union. These are ethically questionable, because death and suffering of the animals for purely commercial purposes are accepted. Therefore, recent political initiatives to further increase animal testing for GMO crops must be regarded highly critically. Based on concrete examples this article demonstrates that animal experiments, on principle, cannot provide the expected protection of users and consumers despite all efforts to standardise, optimise or extend them.

A survey concerning the work of ethics committees and licensing authorities for animal experiments in Germany.

In 2006, the German Animal Welfare Federation started its 3rd survey since the establishment of ethics committees for animal experiments according to the German Animal Welfare Act in Germany in 1987. As animal welfare has been included as a "state goal" in the German constitution in 2002, the present survey aimed at an analysis of specific changes within the licensing process. A series of other aspects of the work of ethics committees and licensing authorities was also examined. The survey was based on questionnaires that had been addressed to licensing authorities and members of ethics committees. One of the main results is that the importance of animal welfare within the licensing process, after inclusion of animal welfare into the German constitution, has not changed or changed only to a small extent. It also becomes clear that ethical parameters are still of minor importance when animal experiments are licensed. The findings underline the importance to reform the German authorization system for animal experiments. The pending revision of EU-Directive 86/609 is an opportunity which should not be missed in this context.

Animal experimentation.

Millions of animals are used every year in often times extremely painful and distressing scientific procedures. Legislation of animal experimentation in modern societies is based on the supposition that this is ethically acceptable when certain more or less defined formal (e.g. logistical, technical) demands and ethical principles are met. The main parameters in this context correspond to the "3Rs" concept as defined by Russel and Burch in 1959, i.e. that all efforts to replace, reduce and refine experiments must be undertaken. The licensing of animal experiments normally requires an ethical evaluation process, often times undertaken by ethics committees. The serious problems in putting this idea into practice include inter alia unclear conditions and standards for ethical decisions, insufficient management of experiments undertaken for specific (e.g. regulatory) purposes, and conflicts of interest of ethics committees' members. There is an ongoing societal debate about ethical issues of animal use in science. Existing EU legislation on animal experimentation for cosmetics testing is an example of both the public will for setting clear limits to animal experiments and the need to further critically examine other fields and aspects of animal experimentation.

[The use of transgenic animals in biomedical research in Germany. Part 2: Ethical evaluation of the use of transgenic animals in biomedical research and perspectives for the changeover in research to research animal-free methods]. / Die Verwendung transgener Tiere in der biomedizinischen Forschung in Deutschland Teil 2: Ethische Bewertung der Verwendung transgener Tiere in der biomedizinischen Forschung und Perspektiven für die Umstellung der Forschung auf tierversuchsfreie Verfahren.

As a rule, transgenic animals are being used in in vivo experiments to examine gene functions, their regulation or the contribution of genetic alterations to the development of diseases. Many transgenic animals already are affected in their wellbeing due to the genetic modification alone regardless of the procedures performed with them. Moreover, it is to be questioned whether the experimental use of transgenic animals led to results that were of such outstanding scientific relevance that they legitimated the suffering of the animals. In order to point to possible approaches to avoiding the use of transgenic animals in the areas of research identified, subsequent investigations aimed at collecting information on non-animal test methods that might be applied in pursuing the aforesaid questions. In particular, these were non-animal test methods that make use of genetic techniques. Amongst these are in vitro cell culture methods with genetically modified cells, such as the so called Transfected Cell Array, as well as in vitro test methods, in which specifically targeted genes can be turned on or off selectively for example by the so-called RNA interference technique or by antisense oligonucleotide genes. Since such technologies can also be applied to cell cultures with human cells, investigations with these methods enable direct information on the function of human genes. Even though a one to one replacement of animal experiments with transgenic animals by non-animal test methods is considered unlikely, from the point of view of animal welfare the broad spectrum of already available non animal test methods with which to study the function of genes and genetically caused pathophysiological reactions proves that waiving of animal tests with transgenic animals is possible without impeding biomedical research. Even if it cannot be totally excluded that some very specific questions linked to the respective animal experiment might not be pursued for the time being, nevertheless research that would be restricted to modern and ethically acceptable in vitro test methods would certainly conceive its very own questions to pursue and solve the problems currently faced by biomedical research. It is against this background that it is to be welcomed that the German Federal Government currently actively promotes the further development of genetechnological non-animal test methods. In order to ensure that these funding measures will make an effective contribution to reducing animal experiments, as spelled out by the government itself, the conversion of genetechnological research, just like biomedical research as a whole, to non-animal testing methods should be supported by concrete political actions. From the point of view of the German Animal Welfare Federation the following issues are to be requested: (1)In order to enable a fast and comprehensive advancement of promising genetechnological non-animal test methods, it should be ensured that public funding is provided with an adequate budget and over a sufficiently long period of time. (2)The legislator should initiate broad discussions on the question if society would be willing to dispense with certain pieces of knowledge if they would necessarily have to be gained at the expense of a certain degree of animal suffering. As the case may be, in the German Animal Welfare Act it should be laid down that certain procedures should not be considered acceptable as such. (3)As long as animal experiments with transgenic animals continue to be performed, concrete legal measures should be laid down in the German Animal Welfare Act to ensure that the distress of the animals (taking into account all factors relevant for transgenic animals) and the expected benefit of the research project are determined objectively so that the outcome of the ethical evaluation process becomes comprehensible. (4)The legislator should provide the authorities responsible for the licensing of research projects with concrete instructions in order to ensure that all aspects relevant for the welfare of the animals are fully taken into account when evaluating the ethical acceptability and scientific indispensability of projects and that special attention is given to research projects with transgenic animals. (5)The German Decree on the Reporting of Laboratory Animals should be amended to ensure that all individual transgenic animals are included in the official statistical reports regardless of whether they end up being used in scientific procedures or not. From the point of view of animal welfare it is possible to redesign biomedical research to do without transgenic animals without impeding necessary scientific progress. The survey in hand sought to make a contribution to providing a scientifically sound background for initiating these discussions.

[The use of transgenic animals in biomedical research in Germany. Part 1: Status Report 2001-2003]. / Die Verwendung transgener Tiere in der biomedizinischen Forschung in Deutschland. Teil 1: Sachstandsbericht 2001-2003.

While the German Federal Government has set itself the goal to make an active contribution to reducing animal experiments, the use of transgenic animals in biomedical research continuously increases every year. It is against this background that the study at hand aimed at providing an overview over the goals and the contents of research projects performed in Germany, in the course of which transgenic animals were produced or used in experimental procedures. Specifically, it was envisaged to spell out those specific areas of research, for which transgenic animals mainly were being used. Subsequently it was evaluated whether the research goals revealed might also be pursued with non animal test methods. In a literature survey, a total of 577 scientific publications relevant for the purposes of the study were collected. This material enables conclusions on those scientific areas, in which transgenic animals are used, applying to fundamental research, but not on their use in routine procedures in applied research or for the maintenance of transgenic breeds, since such purposes do not tend to be the subject of publications in scientific journals. According to the topics covered by the publications, main areas of biomedical research with transgenic animals can be found in the fields of neurobiology, immunology, cardiology, embryology and oncology. However their use can be discerned in all other areas of fundamental biomedical research as well. In accordance with the official German laboratory animal statistics, the vast majority of transgenic animals used were mice, followed by rats and pigs. Additionally, singular research projects with fish, rabbits and chicken were recorded. (In the official German laboratory animals statistics, very small numbers of transgenic hamsters, sheep and amphibians were also recorded in the past years.) A high percentage of the rats were used in cardiovascular research, whereas transgenic pigs as a rule were produced and bred as organ donors in xenotransplantation research. The majority of research projects either dealt with the experimental use of already established transgenic animal lines, or they described that transgenic animals specifically were produced for the purpose of the respective research project. Mostly, transgenesis was initiated by inserting the foreign gene into the germ cell genome. In some research projects, it was reported that the transgenic material was inserted into normally bred animals some time after parturition.

[Animal experiments in biomedical research. An evaluation of the clinical relevance of approved animal experimental projects]. / Tierversuche in der biomedizinischen Forschung. Eine Bestandsaufnahme der klinischen Relevanz von genehmigten Tierversuchsvorhaben.

According to the German Animal Welfare Act, scientists in Germany must provide an ethical and scientific justification for their application to the licensing authority prior to undertaking an animal experiment. Such justifications commonly include lack of knowledge on the development of human diseases or the need for better or new therapies for humans. The present literature research is based on applications to perform animal experiments from biomedical study groups of three universities in Bavaria (Germany) between 1991 and 1993. These applications were classified as successful in the animal model in the respective publications. We investigated the frequency of citations, the course of citations, and in which type of research the primary publications were cited: subsequent animal-based studies, in vitro studies, review articles or clinical studies. The criterion we applied was whether the scientists succeeded in reaching the goal they postulated in their applications, i.e. to contribute to new therapies or to gain results with direct clinical impact. The outcome was unambiguous: even though 97 clinically orientated publications containing citations of the above-mentioned publications were found (8% of all citations), only 4 publications evidenced a direct correlation between the results from animal experiments and observations in humans (0,3%). However, even in these 4 cases the hypotheses that had been verified successfully in the animal experiment failed in every respect. The implications of our findings may lead to demands concerning improvement of the licensing practice in Germany.

Ethical evaluation of research proposals by ethics panels advising the European Commission.

Ethical principles with regard to animal experimentation are referred to in European Union (EU) legislation and other official documents. Therefore, applications for funding of research under the EU's research programme may undergo an ethical review that is carried out by so-called ethics panels, consisting of experts chosen by the European Commission. The work of these panels differs substantially from that of other ethical committees, as they exist on the institutional, local, regional or national level. Their main purpose is not to decide whether a proposed research can be regarded legal, and therefore should be endorsed or licensed; instead, it is to help the Commission in prioritising its funding. The panels may examine other ethical aspects than those of animal experimentation or animal welfare alone, such as the use of human volunteers. This is reflected by the composition of the panels. Their decisions are normally based on consensus. Even though these decisions may refer to EU legislation, the criteria applied are not restricted to those provided by this legislation. Nevertheless, the various aspects of the Commission's ethical evaluation system (e.g. formal and practical basic conditions, information content of applications, type of decisions taken, lacking of any quality control) offers opportunities for improvement.

ECVAM: desperately needed or superfluous? An animal welfare perspective.

Eurogroup for Animal Welfare is the umbrella organisation of the major animal welfare organisations in Europe. Whereas its long-term goal is the complete replacement of animal experiments by methods that do not involve pain, suffering or distress in animals, it is also committed to any reasonable effort to reduce and refine animal experiments, as long as these continue to be carried out. Eurogroup therefore supports the activities of ECVAM, and it acknowledges the contributions to animal protection in various areas of animal use for scientific purposes made by ECVAM to date. Eurogroup is not satisfied with the number of alternative methods accepted in the past, but it sees the main responsibility for the slow progress as being outside ECVAM. The insufficient involvement of ECVAM by the EU Commission in various issues that would require its competence is also a matter of concern to Eurogroup.

[Revised Tables for the Collection of Statistical Information on the Number of Animals used in Experiments and other Scientific Purposes in the European Union]

The new tables to be used for future statistics on animal experiments in the European Union presented by the European Commission have been significantly extended and improved in comparison to the old ones. Several categories of little relevance and unclear expressions have been revised. However, some important data (inter alia categories for pain and distress as well as for several purposes of use; the origin of some animal species; types of institutions) are still not required even though they are highly relevant with respect to animal welfare aspects and must be regarded indispensable for a well-aimed application of the statistics to set priorities concerning the 3 Rs.

[OECD pushes antiquated animal test for endocrine disrupting activity]

The OECD (Organisation for Economic Co-operation and Development) plans to set up a guideline for chemical testing (the "Immature Rat Utereotrophic Bioassay Screening Test") on the basis of an obsolete animal assay to standardise routine testing for endocrine disrupting activity. The proposed method will not do justice to the protection of consumers and the environment. On the other hand a number of promising in vitro methods exist that can be used to study the (also mechanics) effects of substances on the endocrine system. Therefore, from the animal point of view, too, the OECD proposal is unacceptable.

[The "3R" concept in the Internet II]

New developments in data-processing and communication as well as the growing complexity of scientific advances in the field of the 3Rs and animal alternatives in particular have made new solutions necessary with regard to databases that contribute to the 3Rs. As a consequence, in 1996 first steps towards an international harmonisation and co-operation have been made. The use of the Internet plays a decisive role in this connection

[Problems in setting up a European statistics on animal experimentation]

The Akademie für Tierschutz has performed a study to analyse the problems in connection with the European statistics on animal experimentation. This study included an evaluation of the national and the European registration schemes as well as of the legal basis that exists in the EU Member States with regard to the registration of animal experiments. Moreover, selected individuals that participate in the statistics (researchers from industry and academia, officials) and animal welfare organisations were interviewed. Deficiencies within European and national legislation and within the statistical tables used were identified as causes for the unsatisfactory European statistics on animal experimentation. Additionally, it could be proved that binding EU legislation has not been implemented to full extent yet.