Biologics (non-vaccine)

Conventional
test method

Alternative Test Method

Last Reviewed
Name & Description Validation Status Regulatory Status Effect or Potential Effect on Animal Use
Mouse LD50
Assay for Botulinum
Toxin Potency Testing
Endopeptidase Assay-SNAP 25
Description and references
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).

EU: Listed in the European Pharmacopoeia

UK: Routinely used by the NIBSC

US: None

Replacement May 2012
Endopeptidase-MS assay
Description and references
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) None Replacement May 2012
Mouse Hind-Limb Assay
(Digital Abduction Score (DAS) Assay)
Description and references
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). EU: Listed in the European Pharmacopoeia (2005)

US: None

Refinement
(Does not rely on death as an endpoint)
May 2012
Mouse Abdominal Ptosis Assay or Flaccid Paralysis
(Non-lethal Mouse Model for Botulinum Toxin Potency Testing)
Description and references
UK: Validated at NIBSC for potency testing of BoNT products

US: ICCVAM workshop panel concluded that the Abdominal Ptosis Assay
has the potential to replace the LD50 endpoint, but further validation studies are required (2008).

EU: Listed in the European Pharmacopoeia
for BoNT type A for injection (2005)

UK: Used by NIBSC when in vitro test is inconclusive

US: None

Refinement
(Does not rely on death as an endpoint)
May 2012
Non-Lethal Clinical Endpoint in the Mouse LD50 Assay Protocol
Description and references
Canada: Validated by Health Canada

US: ICCVAM workshop panel recommended that an international validation study using the same non-lethal endpoint be conducted (2008).

Canada: Accepted for use Refinement
(Does not rely on death as an endpoint)
May 2012
Mouse Phrenic Nerve (MPN) Assay
Description and references
US: ICCVAM workshop panel recommended that further validation studies be conducted (2008).

Germany: Validated by the Medical School of Hanover

EU: Listed in the European Pharmacopoeia (2005) Reduction
(At least 50% decrease in numbers of animals used)

Refinement
(Uses killed instead of live mice)

May 2012
Rat Intercostal Neuromuscular Junction (NMJ) Assay
Description and references
US: ICCVAM workshop panel recommended that further validation studies be conducted (2008)

UK: Currently undergoing validation

None Reduction
(At least 50% decrease in numbers of animals used)

Refinement
(Uses killed instead of live mice)

May 2012
In Vitro Cell-Based Assay
Description and references
US: ICCVAM workshop panel found that no cell-based method could currently be used to reduce or replace animals for BoNT potency testing or for detecting botulinum toxin in environmental or biological samples. In their current state of development, cell-based methods are not sufficiently sensitive. Which cell types and/or cell lines are the most appropriate is also not known (2008). None Replacement May 2012

 

List of Abbreviations

Test Methods Categories

Toxicity
Dermal Tests
Vaccines