Conventional Test Method:

Mouse LD₅₀ Assay for Botulinum Toxin Potency Testing

Alternative Test Method:

Name & Description:

Endopeptidase-MS assay

The two peptide products produced from the toxin action on the peptide are measured  using mass spectrometry. This has been successfully used to measure the activity of, and differentiate between, all seven BoNT serotypes. However, the sensitivity of this assay is affected by the sample matrix, which can cause it to vary by several orders of magnitude.

Validation Status:

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:

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.
• Barr J.R., Moura H., Boyer A.E., Woolfitt A.R., Kalb S.R., Pavlopoulos A., McWilliams L.G., Schmidt J.G., Martinez R.A. and Ashley D.L. (2005) Botulinum neurotoxin detection and differentiation by mass spectrometry. Emerging Infectious Diseases 11(10):1578-1583.
• Boyer A.E., Moura H., Woolfitt A.R., Kalb S.R., McWilliams L.G., Pavlopoulos A., Schmidt J.G., Ashley D.L. and Barr J.R. (2005) From the mouse to the mass spectrometer: detection and differentiation of the endoproteinase activities of botulinum neurotoxins A-G by mass spectrometry. Analytical Chemistry 77(13):3916-3924.
• 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).

Information Last Reviewed:

May 2012