Evaluation of an In Vitro Cell Culture Assay for the Potency Assessment of Recombinant Human Erythropoietin

Francine T. Machado, Fernanda P.S. Maldaner, Rafaela F. Perobelli, Bruna Xavier, Francielle S. da Silva, Guilherme W. de Freitas, Paolo Bartolini, Maria Tereza C.P. Ribela and Sérgio L. Dalmora

Recombinant human erythropoietin is a sialoglycoprotein that stimulates erythropoiesis. To assess potency of human erythropoietin produced by recombinant technology, we investigated an in vitro TF-1 cell proliferation assay, which was applied in conjunction with a reversed-phase liquid chromatography method for the determination of the content of sialic acids. The results obtained, which were higher than 126.8ng/μg, were compared with those obtained with the in vivo normocythaemic mouse bioassay. The in vitro assay resulted in a non-significant lower mean difference of the estimated potencies (0.61% ± 0.026, p > 0.05). The use of this combination of methods represents an advance toward the establishment of alternative in vitro approaches, in the context of the Three Rs, for the potency assessment of biotechnology-derived medicines.

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Dialogue and Collaboration with ECVAM: The View of the EFPIA

Bernward Garthoff

An evaluation is presented of past experience of dialogue and collaboration between ECVAM and the European Federation of Pharmaceutical Industries and Associations (EFPIA) over the last nine years. Lessons learnt from the viewpoint of EFPIA company representatives are given. Also, proposals for the future ECVAM approach are made, such as support for other research areas for new methods to be validated, giving realistic statements to ECVAM's European Union and external customers, and being open to any new technology development that might help in opening and establishing new alternative avenues. Finally, the need for proper publications on the implementation of alternatives is recommended, for example, through the existing national platforms and their umbrella organisation, ecopa.
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Reduction Strategies in Animal Research: A Review of Scientific Approaches at the Intra-experimental, Supra-experimental and Extra-experimental Levels

Jasmijn de Boo and Coenraad Hendriksen

When discussing animal use and considering alternatives to animals in biomedical research and testing, the number of animals required gets to the root of the matter on ethics and justification. In this paper, some reduction strategies are reviewed. Many articles and reports on reduction of animal use focus mostly on the experimental level, but other approaches are also possible. Reduction at the intraexperimental level probably offers the greatest scope for reduction, as the design and statistical analysis of individual experiments can often be improved. Supra-experimental reduction aims to reduce the number of animals by a change in the setting in which a series of experiments take place — for example, by improved education and training, reduction of breeding surpluses, critical analysis of test specifications, and re-use of animals. At the extra-experimental level, reduction is a spin-off of other developments, rather than the direct goal. Through improved research or production strategies, aimed at better quality, consistency and safety, reduction in the number of animals used can be substantial. A revised definition of reduction is proposed, which does not include the level of information needed, as in some cases reduction in the number of animals resulting in less information or data, is still acceptable.
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Animal Carcinogenicity Studies: 3. Alternatives to the Bioassay

Andrew Knight, Jarrod Bailey and Jonathan Balcombe

Conventional animal carcinogenicity tests take around three years to design, conduct and interpret. Consequently, only a tiny fraction of the thousands of industrial chemicals currently in use have been tested for carcinogenicity. Despite the costs of hundreds of millions of dollars and millions of skilled personnel hours, as well as millions of animal lives, several investigations have revealed that animal carcinogenicity data lack human specificity (i.e. the ability to identify human non-carcinogens), which severely limits the human predictivity of the bioassay. This is due to the scientific inadequacies of many carcinogenicity bioassays, and numerous serious biological obstacles, which render profoundly difficult any attempts to accurately extrapolate animal data in order to predict carcinogenic hazards to humans. Proposed modifications to the conventional bioassays have included the elimination of mice as a second species, and the use of genetically-altered or neonatal mice, decreased study durations, initiation–promotion models, the greater incorporation of toxicokinetic and toxicodynamic assessments, structure-activity relationship (computerised) systems, in vitro assays, cDNA microarrays for detecting changes in gene expression, limited human clinical trials, and epidemiological research. The potential advantages of nonanimal assays when compared to bioassays include the superior human specificity of the results, substantially reduced time-frames, and greatly reduced demands on financial, personnel and animal resources. Inexplicably, however, the regulatory agencies have been frustratingly slow to adopt alternative protocols. In order to decrease the enormous cost of cancer to society, a substantial redirection of resources away from excessively slow and resource-intensive rodent bioassays, into the further development and implementation of non-animal assays, is both strongly justified and urgently required.
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Rapid Simultaneous Cloning of Drug Targets from Multiple Mammalian Species

Rachael Jupp, Palminder K. Dusanjh, Andrew Walding, Mark McHale, Graham P. Belfield and Stephen J. Delaney

A method for the routine, rapid and simultaneous cloning of drug targets from multiple mammalian species is described. This expedites the generation of recombinant proteins and cell lines that can provide alternatives to animal experiments. This was achieved by the collection of RNA from a comprehensive range of tissues from a variety of species, and the optimisation of cDNA synthesis. This “zooplate” has been successfully used for the simultaneous amplification and cloning of drug targets from multiple species. These products have subsequently been used to develop in vitro assays that support efficacy and safety studies in new drug discovery programmes. Within the framework of the Three Rs, these reagents can reduce the number of animals required to provide material for ex vivo assays and can refine the in vivo studies that are still necessary.
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Animal Carcinogenicity Studies: Implications for the REACH System

Andrew Knight, Jarrod Bailey and Jonathan Balcombe

The 2001 European Commission proposal for the Registration, Evaluation and Authorisation of Chemicals (REACH) aims to improve public and environmental health by assessing the toxicity of, and restricting exposure to, potentially toxic chemicals. The greatest benefits are expected to accrue from decreased cancer incidences. Hence the accurate identification of chemical carcinogens must be a top priority for the REACH system. Due to a paucity of human clinical data, the identification of potential human carcinogens has conventionally relied on animal tests. However, our survey of the US Environmental Protection Agency’s (EPA’s) toxic chemicals database revealed that, for a majority of the chemicals of greatest public health concern (93/160, i.e. 58.1%), the EPA found animal carcinogenicity data to be inadequate to support classifications of probable human carcinogen or non-carcinogen. A wide variety of species were used, with rodents predominating; a wide variety of routes of administration were used; and a particularly wide variety of organ systems were affected. These factors raise serious biological obstacles that render accurate extrapolation to humans profoundly difficult. Furthermore, significantly different International Agency for Research on Cancer assessments of identical chemicals, indicate that the true human predictivity of animal carcinogenicity data is even poorer than is indicated by the EPA figures alone. Consequently, we propose the replacement of animal carcinogenicity bioassays with a tiered combination of non-animal assays, which can be expected to yield a weight-of-evidence characterisation of carcinogenic risk with superior human predictivity. Additional advantages include substantial savings of financial, human and animal resources, and potentially greater insights into mechanisms of carcinogenicity.
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The Use of the Zebrafish (Danio rerio) Embryo for the Acute Toxicity Testing of Surfactants, as a Possible Alternative to the Acute Fish Test

Martin Vaughan and Roger van Egmond

At present, the acute toxicity of chemicals to fish is most commonly estimated by means of a short-term test on juvenile or adult animals (OECD TG 203). Although, over the last few years, the numbers used have been reduced due to the implementation of the Three Rs (Reduction, Refinement and Replacement), significant numbers of fish are still used in acute toxicity tests. With the introduction of the new European Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) system, this number is likely to increase dramatically. The aim of this work was to test the acute toxicity of a number of anionic, cationic and non-ionic surfactants to embryos of the zebrafish (Danio rerio), over 48 hours, as a possible alternative to the standard 96-hour fish acute test. We measured the toxicities of 15 surfactants, and compared the results to previously generated adult D. rerio LC50 data (or other fish species, if these data were not available). Comparison of the LC50 data showed that embryos appear to be as sensitive to cationic and non-ionic surfactants as the adult fish, but possibly are more sensitive to anionic surfactants. Toxicity testing with the embryo test can be carried out more quickly than with the adult test, uses much less space and media, requires less effort, and therefore can be performed at a reduced cost. The embryo test may also uncover additional sub-lethal effects, although these were not observed for surfactants. The data presented here show that the 48-hour embryo test can be considered as a suitable alternative to the adult acute fish test for surfactants.
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