Characterisation of human corneal epithelial cell cultures maintained under serum-free conditions.

Three-dimensional tissue constructs have been proposed as in vitro screening models for ocular irritancy. Based on our previous studies, in which a full-thickness corneal model based exclusively on SV40-immortalised cell lines was generated, we have currently evaluated the effects of a range of commercially-available cell culture media on several cellular parameters in cultures of a human corneal epithelial (HCE) cell line. This cell line was used in an attempt to establish a rational basis for the development of serum-free culture media for the assembly and long-term tissue culture of full-thickness corneal models. Briefly, we investigated the impact of serum-free culture on the proliferation, morphology, barrier function and cytokine expression of HCE cells. The number of cell layers and the epithelial differentiation were evaluated by histology. Barrier properties were characterised via the determination of transepithelial electrical resistance (TEER), fluorescein permeation, and the expression of the tight junction-related protein, zona occludin 1 (ZO-1). The cytokine expression pattern in response to serum-free culture was measured by using an antibody array system. Our results revealed that both the morphology and the barrier function of the epithelial constructs were comparable to those of human donor corneas, when serum-free media were supplemented with ascorbic acid, calcium, hydrocortisone and retinoic acid. Under these conditions, the artificial epithelium based on serum-free HCE cultures represented a valid model for the natural ocular surface.

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.

[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.

Fourth EU report on the statistics on the number of animals used for scientific purposes in 2002 -- trends, problems, conclusions.

In the beginning of the year 2005, the European Commission published the fourth statistical report on the number of laboratory animals used in the EU Member States in the year 2002. A total of 10,731,020 experimental animals was reported, the majority of which were mice, rats and fish. In comparison to 1999, the year of the previous data collection, this is an increase by over 900,000 animals, which is mainly attributed to an increased use in fish. As in the previous years, France, the United Kingdom and Germany were the Member States that reported the highest numbers of laboratory animals. 4.5 million animals were used for the development, production and quality control of products for human medicine, dentistry and veterinary medicine and 1 million animals in toxicological and safety evaluations. 3.7 million animals were used in fundamental studies, and therefore are not covered by the scope of Council Directive 86/609/EEC on the protection of laboratory animals. This regulatory gap leaving more than a third of the laboratory animals used in the European Union without protection by EU legislation is unacceptable and should be closed during a revision of the Directive. All national authorities submitted some kind of explanatory notes alongside their national tables. In the article, an extensive summary and discussion of the contents of these notes is presented. Germany, Austria, Finland report on providing financial support for the development of alternative methods. The numbers put forward in the fourth EU statistical show that such support is desperately needed.

Third EU report on the statistics on animals used for experimental and other scientific purposes: trends, problems, conclusions.

A brief overview is given of the data published in the third report on the number of animals used for scientific purposes in the European Union (EU), in 1999. In comparison to the second EU statistical report, the meaningfulness of the data has been greatly improved, since 14 EU Member States have now used the "EU tables". Even though the total number of animals officially reported in the EU for 1999 (9.81 million animals) is lower than the number reported for 1996 in the second report (11.65 million animals), an increase in animal numbers was observed in several Member States. Repeatedly, the use of transgenic animals was considered to be the cause for this. Most national authorities provided some comments on the data they submitted. However, the quality of these accompanying notes varies considerably, with the comments provided by The Netherlands, Finland, Sweden, Austria, Denmark and the United Kingdom containing the most-detailed remarks on their data.

The 3Rs and animal welfare - conflict or the way forward?

The animal experiment is central to the 3R concept. In European law, animal experiments are classed according to their aims. In the German animal protection law, they are classed, e.g. as interventions and treatments for experimental purposes, for further education and training, or for the production, preparation, storage or multiplication of substances, products or organisms and for the fulfillment of legal requirements, and are thus regulated with varying strictness. In contrast, in Switzerland all such measures performed on live animals underlie the same approval requirements. For animal welfarists, the term "animal experiment" includes every intervention and every treatment which is associated with pain, fear and/or suffering and does not directly benefit the respective animal. In the animal experiment, the animal concerned usually suffers as a human would, independent of the experimental goal. Expecting an animal to suffer a treatment one would not want to undergo oneself cannot be in accord with an ethic of respect for fellow creatures. Animal welfarists aim to save animals such suffering. Consequently, they demand the immediate abolition of all animal experiments. From the perspective of those who allow animal experiments to be performed or who perform them themselves, the goal of the experiment is more important than the animal. Therefore, the following question is central to 3R research: "Can I reach my goal while causing the animal less suffering, using fewer animals or without using animals at all?" The starting point is that the ethical responsibility for man is valued higher than that for the animal. The aim is to protect humans from harm caused by substances and products or from unwanted side effects of medication, to understand diseases and to search for a cure or alleviation of these. When a scientist reaches his goals without using animals, the demand of animal welfarists to abolish the animal experiment is fulfilled. These aspects do of course not encompass all the differences and agreements in the positions of animal welfarists and those who support animal experiments. It is not without reason that the discussion around animal experiments is multifaceted and continues to be held intensely from all positions. The classic pattern of a discussion entails that the one side collects the arguments which illustrate that animal experiments lead to wrong results, cannot be transferred and finally stand more against than for the good of mankind. The latter statement is valid, because the wrong methods benefit from investment, thus preventing or hindering new, better research and application of knowledge. The other side uses, among others, the argument that the person performing the experiment knows the limits of the test procedure and can deal with them accordingly. For further defense of current and future animal experiments, scientists like to present their organ transplant patients, i.e. those who can only lead a life worth living thanks to continuous medication, and admonish that patients with Alzheimer's or Parkinson's disease or with cancer put their last hope into new medical insights which can only be gained with animal experiments. Animal welfarists argue against this, that the motivation for animal experiments does not always lie in the ethical responsibility for mankind, but also in pure gain of knowledge, titles and money. After all, they also claim that they are protecting man, animal and environment from diseases and want to help them. It is not about saving animals at any price, even including human life, as is commonly brought against them. But they are also not prepared to accept that without real necessity or at any hint of a dilemma, the decision always falls immediately against the animal. The animal experiment may no longer be the method of choice, both for ethical and also for scientific reasons. What does all of this have to do with 3R research? It is a fact that animal experiments are still the method of choice, the "gold standard". The official German statistics on animal experiments in 2001 (Governmental animal protection report, 2002) counts a total of 2.13 mill. experimental animals, with a tendency towards a further increase. The increase is ascribed to animal experiments especially in basic research, counting 926,294 animals. The other more than a million animals were used for research and development of products and equipment for human, dental or veterinary medicine (509,101), the production or quality control of products or equipment for human, dental or veterinary medicine (289,273), toxicological investigations or other safety tests (189,996), the diagnosis of diseases (26,508), education and training (39,625) and other purposes (145,764). Before this background, all activities which lead away from animal experiments or reduce the suffering caused in the remaining experimental animals must be judged as positive developments. 3R research leads to a reduction of animal experiments and animal suffering. At the same time, the earnest consideration of the 3Rs also leads to a critical and specific discussion of the animal experiment in question.

[Third statistical report on the numbers of laboratory animals used in the European Union--tendencies, problems, decisions]. / Dritter EU-Bericht über die Anzahl verwendeter Versuchstiere Tendenzen, Probleme, Schlussfolgerungen.

In 2003, the European Commission has published its third statistical report on the numbers of laboratory animals used in the European Union, in which the data of 1999 are analysed. For the first time, with the exception of Germany, the data presented were raised on the basis of a new registration scheme. The significance of these data is limited due to deficiencies of the tables used, restricted comparability with previously raised data, and lacking relevance to the current situation. Despite this imperfect basis, the European Commission concludes in its report that there has been a significant reduction in the number of laboratory animals used in the EU compared to the previous statistics. This statement must be regarded critically as several national statistics of the last years verify that the number of laboratory animals used has in fact increased. According to the comments of various national authorities included in the report, particularly genetic engineering is responsible for the increase in animal numbers.

[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.

[Cultivation of a permanent fish cell line in serum-free media special experiences with a cytotoxicity test for waste water samples]

The use of fetal calf serum (FCS) as standard medium additive for the cell cultivation must be regarded critically from the point of view of animal welfare as well as for scientific reasons and makes it necessary to look for alternatives. In the last years an in vitro cytotoxicity assay for the testing of industrial waste waters with the permanent fish cell line RTG-2 was established and pre-validated as an alternative to the fish test with the golden orfe. The application of FCS is also a special problem with regard to the testing of waste waters in a cytotoxicity test so that FCS-alternatives were tested. The RTG-2 cells were successfully adapted to the two solvents Basal Medium Supplement (BMS) and Ultroser-G (U-G) that are used to replace serum. The characterisation of these adapted cell lines showed no significant differences in growth rate, adhesion rate, viability and sensitivity to chemicals in comparison to the original RTG-2 cells. On the determination of the cytotoxicity of industrial waste waters the RTG-2 cells adapted to the BMS medium indicated a clearly higher toxicity of the waste water samples than the original RTG-2 cells. This result confirms the thesis that serum components react with waste water elements and thus change the bio-availability of toxic compounds.

[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.

[Study about the grant programme of the German BMBF for the promotion of alternatives to animal experiments]

From 1980 until 1991, 98 research projects covering a total amount of 86.1 million German Marks were sponsored by the German Ministry for Education, Science, Research, and Technology in a research grant programme for the promotion of alternatives to animal experiments. The German Ministry for Education, Science, Research, and Technology appointed the German foundation SET (Foundation for the Promotion of Research on Replacement and Supplementary Methods for the Reduction of Animal Experiments) to carry out an essay on the efficiency of this grant programme. This essay was done with the help of a questionnaire that was to be filled out by the project managers on a voluntary basis. 23 of the 98 projects had not been completed by the time of the survey. Not all of the questionnaires returned were suitable for the purposes of the study. Therefore only projects covering 73.7 million German marks of the total grant amount were evaluated. According to their research subjects, the research projects were classified by the following categories: Reduction of distress or of the number of laboratory animals used in an experiment (5.9%); reduction of the number of animals by performing an animal free method before the animal test (38.8%); replacement of animal test by an animal free method (24.7%); basic research (20%); promotion of its acceptance by optimisation of an animal free method (8.2%); and validation of an animal free method (2.4%). It is remarkable that not even half of those projects that were performed with the goal of reducing the number of laboratory animals mentioned any success concerning this goal. There were large differences between industrial research projects and non-industrial research projects (universities, state-owned and private research institutes): The main goals of industrial projects were "reduction of distress or of the number of laboratory animals used in an experiment" and "reduction of the number of animals by performing an animal free method before the animal test"; whereas the main goals of university projects were "replacement of an animal test by an animal free method" or topics covering basic research. In 17 projects (grand amount 13.1 million German Marks) of the latter category a reduction of the number of laboratory animals was not to be foreseen; 13 of these projects were pursued at universities. Due to a change in the policy of the grant programme, no project of this category was sponsored after 1989. The reason for the very small number of projects on the validation of alternative methods might be that almost no more validation studies are performed on a national level; instead they are coordinated internationally.

[Toxicity assessment of waste water samples with fish cell lines]

An in vitro cytotoxicity assay with fish cell lines was established in order to reduce the number of animals used in fish tests with the golden orfe (leuciscus idus melanotus). In three co-ordinated working laboratories the toxicity of seven chemicals was determined according to a standardized protocol using the permanent cell lines D11 and RTG-2. There was an almost linear correlation (r=0.99) between the IC50 values after neutral red uptake and crystal violet staining of the chemically treated RTG-2 cells. These cells were more sensitive than D11 cells (r=0.93) with regard to chemically induced cytotoxicity. The test protocol was adapted for the testing of waste water (ten samples). Two samples induced cytotoxic effects in RTG-2 cells while in none of the three laboratories the same samples were found cytotoxic in D11 cells. The data with the seven chemicals and the still limited number of waste water samples (10) lend strong support to the assumption that the fish cell line RTG-2 can be regarded as well suited for detecting toxic properties of waste water.