Potential teratogenicity is a major consideration in the development of pharmaceutical substances. Currently its assessment involves large numbers of animal tests at high cost. This study assessed the feasibility of using the embryonic stem cell test (EST), validated by ECVAM in 1999, as a tool for the prediction of embryonic toxicity of pharmaceutical substances early in their development programmes. ESTs were carried out on 6 chemicals with well-established toxicity characteristics established from literature and from the ECVAM study, and then on 10 Roche internal pharmaceutical substances already tested in vivo. The model correctly classified 81% of the substances. Further experiments are necessary to increase the database of the assay.

In the context of efficacy testing of pharmacological compounds in animal models, replacement of some of these models with a relevant human in vitro system appears attractive, in particular with regard to large scale screening. Here, we show results from initial phases of a project, which attempts to explore the outstanding potential of human embryonic stem cell (hESC)- based in vitro models with special regard to neuronal stress as a potential replacement of animal models for human neurodegenerative diseases.
We show the functionality of neurons derived from hESC precursors by calcium imaging, mitochondrial potential measurements and Western blots and moreover demonstrate that this model reproduces crucial mechanistic aspects observed during ischemia and excitotoxicity that are thought to be at the core of some neurodegenerative diseases. Also, the broader possibilities for refining surrogate molecular information emerging from the detailed analysis of this model are discussed.

Mouse models for colon cancer that harbor a germ line mutation in the tumor suppressor gene Adenomatous polyposis coli (Apc) exhibit a primary genetic defect that predisposes to a high incidence of adenomatous polyps in the small intestine rather than in the colon. Colon cell culture models expressing quantifiable markers for carcinogenic risk may represent an alternative approach to reduce, refine or replace long-term animal experimentation. The newly developed colon epithelial cell lines 1638N COL-CIJ (clonal derivative of the parental Apc mutant cell line 1638N COL) and 1638N COL-Pn (tumor derivative of the clone), established from an Apc1638N {+/-] mutant mouse, exhibit aberrant cell cycle progression, downregulated apoptosis, enhanced carcinogenic risk and tumor formation, indicating that aberrantly proliferative preneoplastic1638N COL-Cli cells exhibit a quantifiable risk for carcinogenesis. Treatment of these preneoplastic Apc mutant cells with a combination of celecoxib and 5-fluorouracil at clinically achievable low concentrations produced a 2.1 fold to 5.5 fold higher efficacy for cytostatic growth arrest and a 40.2% to 52.4% higher efficacy for inhibition of carcinogenic risk, relative to that obtained by these agents used individually. Thus, a low dose combination of mechanistically distinct agents resulted in enhanced efficacy. These data validate a novel cell culture model and a rapid mechanism-based approach to prioritize efficacious drug combinations for animal studies and clinical trials on cancer prevention and, thereby, support the 3R concept by refining and/or reducing the use of animals in biomedical research relevant to prevention/therapy of colon cancer.

Prediction of liver toxicity and compound responses continues to be a major challenge for the pharmaceutical industry. In vitro studies on liver cells have been developed to reduce or replace animal experiments. However, most of the tests in use are based on cell lines which do not necessarily represent normal cell physiology. We compared the response of primary human hepatocytes from two donors with primary rat hepatocytes and the cell line HepG2 to the test compound acetaminophen (AAP) by measuring oxygen consumption, extracellular acidification and cell adhesion as dynamic parameters of cell metabolism. Primary human hepatocytes were cultured on collagen pre-coated sensor chips or in conventional two-dimensional cultures in chemically defined Human Hepatocyte Maintenance Medium. This medium allows cultivation of functionally differentiated hepatocytes for several weeks. Sensor chip-based results were compared with conventional assays for hepatocytes like albumin release and urea release. The hepatocytes were exposed to AAP (50-2815 mg/l) for 24 h. Cell respiration was inhibited by AAP concentrations of 500 mg/l and more in all three cell types, whereas only the cellular acidification rates and cell adhesion of the rat hepatocytes and the HepG2 cells were affected by AAP. In conventional cultures of human hepatocytes, AAP had no effect on cellular viability. Whereas high doses of AAP (2815 mg/l) diminished albumin secretion by 70-80%.

Human hepatocytes are the in vitro system of choice to study drug-induced processes in man. Here, we present HEPAC2: a standardised and validated culture system in which human hepatocytes are maintained in HHMM (Human Hepatocyte Maintenance Medium) with HGF (hepatocyte growth factor) and EGF (epidermal growth factor). Cellular viability and hepatocellular functions were monitored daily. Albumin and urea production remained on a relatively constant level for up to 2-3 weeks. Based on this, a standard protocol was established that allows repeated exposure of hepatocytes to study drug metabolism. We used acetaminophen (AAP) to assay the feasibility of this system. Hepatocytes were exposed to AAP (100-2815 mg/l) for 24 h. Subsequently, the culture medium was replaced by medium without AAP and the same exposure scenario was repeated at intervals of 4 days. High doses of AAP (2815 mg/l) diminished urea production by 15-30% and albumin secretion by 70-80%. These effects were reversible. After removal of AAp, secretion of urea and albumin returned to control levels. AAP hepatotoxicity is caused by its bio transformation to the reactive metabolite N-acetyl-p-benzoquinone-imine (NAPQl) mediated by CYP2El and CYP lA2. The AAP activating enzymes were active for at least 21 days and their activity was maintained during at least four repeated cycles of exposure to AAP. In conclusion, these data demonstrate the suitability of our long-term culture system to serve as a tool for repetitive screening of drug-mediated changes of hepatocellular functions. This culture technique may help to overcome the sparse availability of human hepatocytes for testing drug-mediated responses in man.

In the context of the process of revising EU Directive 86/609/EEC on the protection of animals used in experiments, the European Commission conducted a public internet consultation for EU citizens in mid-2006. Simultaneously, the Commission requested opinions from experts on specific animal welfare issues. The results of both consultations were published in the internet in December 2006. An overwhelming majority of EU citizens answered that the protection of laboratory animals currently is poor and that efforts are needed to improve the level of welfare for these animals. Additionally, they request increased transparency and public participation in the determination when and how the use of animals in experiments is to be considered acceptable. They also asked for an increased promotion of the research for replacing animal experiments. Amongst other issues, the experts called for an extension of the scope of the Directive to also cover animals used in basic research and the establishment of a compulsory authorization procedure which should include a concrete ethical review process. The estimations put forward on the consequences of introducing a retrospective analysis of projects with animal experiments were controversial just as the opinions submitted regarding different options regarding a ban to using wild caught primates and their direct offspring. All in all, both the responses of the citizens and experts consultations are a promising basis to justify the need to improve the protection of animals used in experiments within the EU.