Conventional Test Method:

Mouse LD₅₀ Assay for Botulinum Toxin Potency Testing

Alternative Test Method:

Name & Description:

Mouse Phrenic Nerve (MPN) Assay

A phrenic nerve preparation is isolated from the mouse diaphragm and placed in Krebs-Ringer solution where the resting tension of the diaphragm is measured. The incubation medium is then exchanged for toxin containing medium and the diaphragm tension is monitored until there is a 50% decrease in amplitude. This information is used to construct a dose-response curve.

Validation Status:

US:  ICCVAM workshop panel recommended that further validation studies be conducted (2008)

Germany: Validated by the Medical School of Hanover

Regulatory Status:

EU: Listed in the European Pharmacopoeia (2005)

Effect or Potential Effect on Animal Use:

Reduction
(At least 50% decrease in numbers of animals used)

Refinement
(Uses killed instead of live mice)

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.
• Bigalke H. (2001) Pharmacology of botulinum neurotoxin type A. Wien Klin Wochenschr 113(Suppl.4):2-5.
• 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