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Fewer animals for the testing of botox medicinal products!

07/2009, 06.05.2009

Status of alternative methods for determining the activity of Botulinum neurotoxin products evaluated at the BfR expert meeting

Medicinal products containing Botulinum neurotoxin are used for many different purposes ranging from treating lid spasms over squinting down to smoothing out wrinkles in "aesthetic medicine". The activity of Botulinum neurotoxin products (BoNT products) is still determined using the LD₅₀ activity test in mice stipulated by public authorities. On 27 and 28 April 2009 an international expert meeting was organised by the Federal Institute for Risk Assessment (BfR) in Berlin. It looked at progress in alternative methods to this test method that entails considerable suffering for animals. "The objective of the BfR is to replace this test as quickly as possible with internationally recognised alternative methods or to at least reduce the number of experimental animals", said BfR President Professor Dr. Dr. Andreas Hensel. ZEBET, the Centre for Alternative Methods to Animal Experiments in the BfR, invited experts from science, competent authorities, industry and animal welfare representatives to present the status of development of various alternative methods and to evaluate these approaches with regard to their suitability to replace the classical LD₅₀ test. The experts all agreed that the LD₅₀ test for BoNT products cannot yet be fully replaced. Thanks to improved statistical evaluation methods it is, however, possible to reduce the number of animals needed by up to 50 %.

Botulinum neurotoxin (BoNT) is produced and excreted by the bacterium Clostridium botulinum. It is the deadliest known nerve toxin and, after injection, leads to paralysis by inhibiting electrical conduction from the nerve cells to the muscles. BoNT is used for a growing number of indications like, for instance, squinting, lid spasms, various forms of spasticity and muscle contractures. Particularly in the therapeutic field this toxin is used in a growing number of applications to treat very severe and painful conditions. In “aesthetic medicine” it is also used to reduce facial wrinkles.

Given the high toxicity of the BoNT products, the regulatory authorities in Germany, the EU and the USA require the exact determination of each production unit that is placed on the market. In this context tests are conducted not only on each batch of a medicinal product prior to release for patients. Tests must also be conducted in order to ensure the stability of the production process and, by extension, the consistent quality of the medicinal products.

The standard test for BoNT, which so far could not be replaced by alternative methods, is the LD₅₀ potency test. In this test mice are injected with different concentrations of BoNT and the BoNT concentration is determined at which 50 % of the animals die. As this test results in severe suffering of the experimental animals, work is underway around the world to develop alternative methods to replace this test or at least alleviate the animals’ suffering.

At the two-day Expert Meeting in Berlin the participants evaluated the alternative methods developed so far and drew up recommendations on how to integrate the so-called 3R concept (Replace, Refine, Reduce) into test methods used in the production and release of BoNT products. The meeting was attended by representatives of international regulatory authorities, manufacturers, validation centres, science and animal welfare organisations.

No progress had been made at the previous meetings on this issue, but promising perspectives were highlighted at the Expert Meeting organised by ZEBET. For instance, a BoNT Expert Working Group with representatives of science, competent authorities and industry was set up. It is to hold several meetings at regular intervals throughout the year at the BfR in order to discuss new findings in the field of alternative methods to the LD₅₀ activity test and define criteria for regulatory acceptance of these tests. The wish was expressed by all the parties involved for ZEBET to take over the chairing and coordination of this working group.

The experts agreed that at the present time the LD₅₀ potency test cannot be completely replaced. All the same, the improved evaluation methods did make it possible to considerably reduce the number of experimental animals needed. Furthermore, the experts in the BoNT Expert Working Group are to help manufacturers to integrate the 3R principle in the best possible way into the tests for monitoring production and batch release. What’s more, the experts will also be responsible for drawing up and publishing recommendations on the speedy validation and regulatory acceptance of less severe methods for the marketing authorisation and release of BoNT products.

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