Poor pharmacokinetics and toxicity are not only frequent causes of late-stage failures in drug development but also a source for unnecessary animal tests. In drug discovery and for the assessment of the toxic potential of chemicals, in silico techniques are nowadays considered as valuable alternatives to in vivo approaches. Based on a receptor-modelling concept developed at our laboratory (multidimensional QSAR), we have developed and validated virtual test kits for the estrogen, androgen and aryl hydrocarbon receptor (~ endocrine disruption), for cytochrome P450 3A4 (~ metabolic transformations) and most recently for the thyroid receptor. These surrogates have been tested against a total of 430 compounds and are able to predict the binding affinity close to the experimental uncertainty. These results suggest that our approach is suited for the in silico identification of adverse effects triggered by drugs and chemicals. Consequently, we are prepared to offer a free testing to selected academic institutions and non-profit oriented organisations.

It is well known that in vitro preparations of the chick embryo can functionalise and conjugate selected model substances. Based on this biochemical knowledge an ex vivo model was developed to study xenobiotic metabolism in the incubated hen's egg. The xenobiotic is injected into the yolk sac, i.e. the nutritional compartment, on day 6 and metabolites are identified in the excretion medium of the embryonic kidneys (allantoic fluid) on day 11. During this developmental period the embryo lacks or has very limited sensitivity. Thus the model is in accordance with the 3R concept.
In the present investigation ethyl 4-hydroxybenzoate was chosen as a test substance. Concentrations of this paraben up to 24 mg/egg did not affect embryo viability. After inoculation of 6 mg/egg, 4-hydroxybenzoic acid and 4,4'-dihydroxy-L-ornithuric acid were identified in their free form. The 4-hydroxybenzoic acid was also eliminated in its conjugated form (glucuronide and/or sulphate). No unchanged paraben was excreted. 4,4'-dihydroxy- L-ornithuric acid [2,5-bis-(4-hydroxybenzoylamino) pentanoic acid] is a new metabolite. The structure of this amino acid conjugate was elucidated by synthesis and spectral methods (MS, ¹H and ¹³C NMR).

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 (Lindl et al. ALTEX, 18,171-178,2001). 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.

The bacterium Clostridium (c.) tetani is an ubiquitous pathogen. This anaerobic, gram-positive bacterium can form spores and can be found in the whole environment. It enters the body via injuries of the skin and wounds where it releases the neurotoxin "tetanospasmin" (= tetanus toxin). The animals most susceptible to tetanus infection are horses and sheep. Only active immunisation by tetanus vaccine provides effective protection against tetanus intoxication.
The marketing authorisation requirements stipulate that efficacy of tetanus vaccines ad us. vet. must be demonstrated in all target animal species via determination of neutralising tetanus serum antitoxin concentrations. The standard method used for this purpose is still the toxin neutralisation test (TNT), as it quantifies the tetanus toxin-neutralising effect of tetanus serum antibodies in vivo. In this test, tetanus toxin is added to dilutions of serum from vaccinated horses and sheep. The serum dilutions are then administered to mice or guinea pigs, which are observed for toxic symptoms.
Against the background of animal protection, the goal of one project of the Paul-Ehrlich-Institut (Bundesministerium für Bildung und Forschung (Federal Ministry for Education and Research), 0312636) was to establish an alternative to the toxin neutralisation test, enabling the testing of efficacy of tetanus vaccines with serological in vitro methods. For this purpose, a so-called double antigen ELISA (DAE) was established which enables the testing of sera of different species in one assay. In addition, the sera were tested in an indirect ELlSA for horses and sheep separately.
Altogether, ten groups of horses and eight groups of sheep were immunised with ten animals per group each. The tetanus vaccines comprised almost all products authorised for the German market at the start of the project.
564 horse sera and 257 sheep sera were tested using the two ELISA methods. Some sera were also tested in vivo. The kinetics of antibody responses were recorded. The in vitro DAE method seems to be suitable to replace the mouse neutralisation test used for the detection of tetanus antitoxin in sera of target animal species. The comparison of some sera in the ELISA and the TNT showed good equivalence of results. Nevertheless, before an ELISA titre in horse and sheep sera indicating unambiguous protection against tetanus can be fixed, further comparative assays of low titre sera in the TNT and the DAE will have to be performed.

Botulinum neurotoxin C1 (BoNt C1) and its corresponding gene were detected in seven aquatic habitats covering a range of low (LR) to high risk (HR) avian botulism outbreak areas during a study period of 10 months. Toxin and gene in sediment and avian faecal samples were analysed before (in situ) and after cultivation (in vitro) by a newly adapted ELISA, the common mouse bioassay and by a recently described nested PCR protocol. BoNt C1 gene fragments were detected in 74% and 83% of all investigated sediment samples by in situ PCR and in vitro PCR, respectively, at comparable frequencies in HR and LR areas. Similar high values were also observed for faecal samples. No BoNt C1 could be detected in the sediment in situ, while 53% and 56% of all cultivated samples contained BoNt C1 as detected in the mouse bioassay and the ELISA, respectively. The percentage of BoNt C1 positive cultivated samples was significantly higher (2-fold) in HR areas than in LR areas. Hence, our data clearly indicate an increased ratio of potentially BoNt C1 producing clostridia to BoNt C1 genes as the frequency or likelihood of botulinum epizootics increases in the environment. In addition, the good correlation between the results from the ELISA and the mouse bioassay for all sediment and faecal samples (r=0.90, p<0.001, n=121) indicates a high potential for the ELISA to reduce/replace the mouse bioassay for the detection of BoNt C1 in environmental samples.

Infectious bronchitis virus (IBV) is produced in vitro using specific pathogenfree chicken embryos, primary chicken kidney cells (CKC) and/or tracheal organ culture (TOC). Regulatory authorities in Europe (EMEA) and in the United States (FDA) have encouraged biological manufactures to reduce or eliminate the use of live animals and/or products of animal origin in biological manufacturing processes. In this paper, a stable chicken embryo-related (CER) cell Une was adapted and maintained in serum free (SF) or animal protein free (APF) medium after a direct switch of the medium. The most suitable media were Ex Cell 520 and Ex Cell 302. CER monolayers adapted to SF or APF were infected with IBV (M41 strain) in agitated suspended culture and the IBV titre obtained 48 h post infection was 2.8 x 104 PFU/ml in both cases. Thus, propagation of CER cells and culture of IBV can be performed without the use of animal serum or animal protein.