Conventional Test Method:

Mouse LD₅₀ Assay for Botulinum Toxin Potency Testing

Alternative Test Method:

Name & Description:

Endopeptidase Assay-SNAP 25

BoNT endopeptidase assays are based on the in vivo intracellular mode of action and require as a substrate a synthetic or recombinant peptide that contains the endopeptidase-binding site. The SNAP-25 assay has been validated to verify potency as a measure of consistency of clinical samples containing BoNT type A . The assay has been set up for serotypes A, B, C, E, and F.

Validation Status:

UK: Validated to verify potency by the National Institute for Biological Standards and Control (NIBSC) (1999)

US: ICCVAM workshop panel concluded that it is possible, in principle, to use an endopeptidase assay for estimating BoNT concentration in a pharmaceutical preparation. However, they cannot currently be used to replace animals for BoNT potency testing or for detecting BoNT in environmental or biological samples.  Future activities were recommended (2008).

Regulatory Status:

EU: Listed in the European Pharmacopoeia

UK: Routinely used by the NIBSC

US: None

Effect or Potential Effect on Animal Use:

Replacement

References:

• Adler S., Bicker G., Bigalke H., Bishop C., Blümel J., Dressler D., Fitzgerald J., Gessler F., Heuschen H., Kegel B., Luch A., Milne C., Pickett A., Ratsch H., Ruhdel I., Sesardic D., Stephens M., Stiens G., Thornton P.D., Thürmer R., Vey M., Spielmann H., Grune B. and Liebsch M. (2010) The current scientific and legal status of alternative methods to the LD50 test for Botulinum neurotoxin potency testing. ATLA 38(4):315-330.
• Ekong T.A.N., Feavers I.M. and Sesardic D. (1997) Recombinant SNAP-25 is an effective substrate for Clostridium botulinum type A toxin endopeptidase activity in vitro. Microbiology 143(10):3337-3347.
• Gaines Das R.E., Heath A.B., Martin H. and Sesardic D. (1999) Validation of in vitro assays for botulinum toxin: a case study. Developments in Biological Standardization 101:267-276.
• ICCVAM (2008) Report on the ICCVAM-NICEATM/ECVAM Scientific Workshop on Alternative Methods to Refine, Reduce or Replace the Mouse LD₅₀ Assay for Botulinum Toxin Testing. Bethesda MD: National Institutes of Health (NIH).
• Sesardic D., Jones R.G., Leung T., Alsop T. and Tierney R. (2004) Detection of antibodies against botulinum toxins. Movement Disorders 19(Suppl.8):S85-S91.

Information Last Reviewed:

May 2012