In Baden-Württemberg alone, up to 600,000 animals are used for scientific purposes every year. In order to reduce their pain and suffering as much as possible, researchers all over the world are working on the development of innovative methods to replace animal experiments, including cell culture methods for drug analysis, artificial blood vessel systems for testing chemicals and rapid computer simulations used in diabetes research.
According to the German Federal Ministry of Nutrition, Agriculture and Consumer Protection, around 2.7 million animals die in animal experiments every year, and the numbers are increasing as animals are needed in more and more experiments for the investigation and development of products, equipment and methods for use in dentistry and human and veterinary medicine. The most frequently used experimental animals are mice (in Baden-Württemberg around 286,000 per year), fish (around 138,000) and rats (around 91,000) (1). Mice are ideal for transgenic research where the genome of experimental animals is modified in order to analyse the function of individual genes. The use of rats and mice as experimental animals has the advantage that they are inexpensive and they reproduce quickly. Experiments with dogs, sheep and apes are more complicated because they are more expensive and more difficult to keep.
According to 2007 figures published by the Baden-Württemberg Ministries of Agriculture and Research, Baden-Württemberg had 53 institutions that used animals for their experiments, 31 public and publicly-funded institutions such as universities and university hospitals, and 22 private companies. The animals are mainly used for basic biological and medical research, which, in contrast to drug research, does not focus on specific application-related aspects. In fields such as toxicology and medical research in industry, those responsible for research have increasingly moved towards using alternative testing systems over the last few years.
One of the major developments of recent years is whole-blood tests which means that rabbit pyrogen tests are now no longer required. The new tests are carried out with human blood cells. Pyrogens are fever-inducing substances that provoke an immune response by producing proinflammatory cytokines such as interleukin-1 beta. The traditional rabbit pyrogen test required the animals to be fixed in a rigid position for several hours during which their temperature was measured. This caused the animals extraordinary discomfort. Pyrogens, which are bacterial heat-resistant elements, can be dangerous to humans when contained in injection instruments as they can induce fever, a fall in blood pressure, multi-organ failure and in the worst-case scenario, they can have a fatal outcome. 400,000 rabbits are used for pyrogen tests, and subsequently killed, in the USA and Europe every year. Pyrogen tests are imperative for the testing of any pharmaceutical product for parenteral application and are hence legally required by the health authorities. Only recently, the “PyroDetect” pyrogen test developed by Prof. Thomas Hartung and Prof. Albrecht Wendel at the University of Constance was approved as an in-vitro test for the detection and quantification of pyrogens and as an alternative to the rabbit test. PyroDetect uses whole human blood as the sensor and not only enables the detection of pyrogens in the blood from blood donors without the use of rabbits, but also the detection and quantification of pyrogens in the right species, namely humans.
In the meantime, human tissue and organs have been cultivated in the test tube in order to obviate animal experiments when testing new pharmaceutical substances. In 2009, the Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB in Stuttgart developed a three-dimensional liver model, a functional vascular system that will in future make procedures in the pharmaceutical industry safer as well as reducing the time to market.The scientists use a small segment of a pig intestine with an artery and a vein. They deplete the pig intestine of all animal cells, leaving behind the three-dimensional connective tissue and the blood vessels. The matrix is then seeded with human endothelial cells. The liver cells start to grow on the matrix as soon as artificial blood begins to circulate in the blood vessels. By bringing the drugs to be investigated into physiological contact with the human cells, the liver model enables scientists to investigate whether the metabolisation of pharmaceutical substances leads to toxic products. The testing system is currently being investigated in detail and it is envisaged that it will become a reliable and safe alternative to animal tests in around one to two years’ time.
Cell culture models are another alternative to animal testing. Researchers at the Academy of Animal Protection are working with researchers from the University of Bremen to develop an alternative method to the eye irritancy tests in rabbits, known as the Draize eye irritancy test. The test was developed around 50 years ago. Chemicals are placed into the eyes of rabbits and the eyes' progressive deterioration is recorded.
The alternative method involves the creation of an artificial cornea model from immortal cell lines that corresponds in large part to an in-vitro cornea, both in structural and physiological terms. The model consists of a multilayered cornea epithelium, a one-layer endothelium and a collagen matrix that contains keratocytes. The model will be used to establish numerous routine test methods in order to considerably improve the assessment of weak or moderate eye irritations caused by chemical substances. Besides the possibility of abandoning the Draize eye irritancy test, which is currently required by the health authorities, the artificial cornea also has the potential to increase consumer protection.
Other alternative models are currently being developed to test substances for their carcinogenic potential without the use of animals. For example at the Esslingen University of Applied Sciences biologists are working on the development of a test system consisting of human cell cultures. It is envisaged that these models will be of major benefit for the cosmetics industry. The cells, which originate from human lung cancer tissue, will be used to reconstruct functional lung tissue under specific conditions, for example by using a plastic filter with an artificial membrane. The model will subsequently be used to test cosmetic or pharmaceutical substances that are traditionally tested using animals. The model is being tested using nanoparticles contained in cosmetic products. The researchers hope to gain insights into the effect of such particles on the viability of lung and immune cells.
Computer simulations of cell systems are also increasingly being used to test the toxicity of substances and for other similar research purposes. In addition, computer simulations augment the informative value of tests. Technically mature computer models are able to predict for example whether a drug or a chemical interferes with hormones. The computers are fed data relating to known reactions of numerous different substances that erroneously activate the attachment sites for hormones. The clear advantage of these models is also that they help accelerate research projects.Information technology also allows researchers to increase the number of studies carried out per year from between around ten and one hundred to as many as 10,000 or more. However, computer systems also have their limits and no single computer can currently represent the entire organism (e.g., required for tests assessing the accumulation of substances such as asbestos or nanoparticles in the human body). Therefore, tests involving living animals will continue to be unavoidable.
In Germany, researchers intending to carry out animal experiments must be granted approval from the regional councils. Drugs that are to be placed on the international market are legally required to be tested on animals. Animal experiments for testing the effect of finished cosmetic products have been banned in Germany since 1998, and in the EU since 2004; the testing ban on ingredients for cosmetics has applied since 11th March 2009. Since producers are able to move production outside the EU, animal experiments are still being used in this field. Therefore, a new EU Cosmetics Directive foresees a prohibition in the testing of finished cosmetic products and cosmetic ingredients on animals, and a prohibition in the marketing of finished cosmetic products and ingredients included in cosmetic products which were tested on animals in the European Community, from 2013 onwards.(2) However, many cosmetic tests such as those for lipstick and creams are subject to the EU Chemicals Directive due to the basic substances they contain, and can still involve animal testing. It normally takes a great deal of time before the legislator accepts alternative methods. Only recently, a previously highly controversial cell test was approved as an alternative to blue mussel tests involving mice. Since blue mussels might sometimes be poisonous, it was previously required to test any batch destined for human consumption.
Quite apart from the ethical issues, animal experiments and the keeping of laboratory animals tend to be very expensive for industry. The high costs and the unsatisfactory translation of the results to humans are the typical reasons why companies seek alternative methods. For German and European companies, the search for alternative methods to animal experiments might have the added advantage, in economic terms, of later avoiding the need to import the know-how of innovative alternatives to animal experiments from abroad, which could potentially be extremely costly.
mst (BioLAGO) - 19.5.2010(c) BIOPRO Baden-Württemberg GmbH
Literature:(1): Bericht des Landwirtschafts- und Wissenschaftsministeriums (2007)(2): https://www.gesundheitsindustrie-bw.deec.europa.eu/enterprise/cosmetics/doc/200315/200315_de.pdf