Conventional Test Method:

Mouse LD50 Assay for Botulinum Toxin Potency Testing

Alternative Test Method:

Name & Description:

Mouse Hind-Limb Assay (Digital Abduction Score (DAS) Assay)

Various dilutions of the toxin are injected into the gastronemius muscle of the right hind leg of mice. The degree of paralysis is assessed over a 12-day observation period.

Validation Status:

US: ICCVAM workshop panel concluded that the DAS assay is applicable to multiple serotypes and products, and has potential as a potency assay, if used relative to a reference standard (a standardized, calibrated BoNT preparation against which to establish “relative” potency values). Recommended that further validation studies be conducted (2008).

Regulatory Status:

EU: Listed in the European Pharmacopoeia (2005)

US: None
Effect or Potential Effect on Animal Use:

Refinement
(Does not rely on death as an endpoint)

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.
  • 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).
  • Pearce L.B., Borodic G.E., First E.R. and MacCallum R. (1994) Measurement of botulinum toxin activity: evaluation of the lethality assay. Toxicology & Applied Pharmacology 128(1):69-77.
  • Pearce L.B., Borodic G.E., Johnson E.A., First E.R. and MacCallum R. (1995) The median paralysis unit: a more pharmacologically relevant unit of biologic activity for botulinum toxin. Toxicon 33(2):217-227.
  • Pearce L.B., First E.R. and Borodic G.E. (1995) Botulinum toxin: Death versus localized denervation. Journal of the Royal Society of Medicine 88(4):239-240.
  • Sesardic D., Leung T. and Gaines Das R. (2003) Role for standards in assays of botulinum toxins: international collaborative study of three preparations of botulinum type A toxin. Biologicals 31(4):265-276.
Information Last Reviewed:
May 2012