Traditionally the potency of botox has been evaluated using the LD50 test. These tests cause immense suffering to the animals involved and are expensive in terms of the animals and resources required for carrying them out. Because of this a number of alternatives to the LD50 test are being developed, with perhaps the most hopeful being the SNAP-25 assay:

SNAP-25 is a protein involved in nerve signalling. Botox binds to SNAP-25 splicing it in half and preventing it from carrying out its normal function. This results in localised paralysis and the removal of wrinkles. As SNAP-25 is the primary target of Botox, directly measuring the effect of the toxin on this protein provides a far more specific and efficient method for measuring potency than the LD50 test. To measure how much SNAP-25 protein is cleaved by a particular batch of Botox, synthetic SNAP-25 peptides are used, which when cleaved fluoresce.

Following exposure to the Botox the amount of flourescence emitted by the claeved SNAP-25 protein is directly proportional to the potency of the Botox. By using different dilutions of the Botox this method can allow for the simple calculation of LD50-equivalent values.

The government-funded lab at the National Institute for Biological Standards and Control now use the SNAP-25 endopeptidase assay for routine batch-release confirmation. It has been described as sensitive, technically simple, rapid and also provides less inter-laboratory variability. The method is still being improved and it is hoped that in the near future the use of this method in combination with an ELISA in vitro assay technique that directly measures the concentration of Botox toxin in a batch sample, as well as an international reference standard for Botox, will soon end the need for the LD50 test to be used for cosmetic Botox batch-testing.