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Toxicity Endpoints & Tests

Biologics, Vaccines, Medical Products

Last updated: July 3, 2014

Toxicity Endpoint

Under construction…

The Animal Tests

Under construction…

Validated Non-animal Methods

Method1

Test Purpose

Validation Authority2

Pharmacopeia or Agency3

Veterinary vaccine potency assays Exemptions from Standard Requirements tests; master reference qualification and requalification

ICCVAM agency

USDA: 9 CFR 113.409(c), Memorandum 800.114 (2012)

Cell-based assay for botulinum neurotoxin type A products Stability and batch potency testing of biological

ICCVAM agency

US FDA: Allergan, Inc., method accepted (2011)

ELISA test for Leptospira veterinary vaccines Batch potency testing of vaccine

ICCVAM agency

USDA Supplemental Assay Methods 624, 625, 626, and 627 (2008)

Five in vitro methods for pyrogenicity from gram-negative endotoxins based on cytokine release from human blood: human whole blood IL-1; human whole blood IL-6; PBMC IL-6; MM6 IL-6; and human cryopreserved whole blood IL-1 Identification of pyrogenicity from Gram-negative endotoxins as substitute for Rabbit Pyrogen Test (RPT)

ECVAM ESAC (2006)

ICCVAM (2008)4

UEuropean Pharmacopoeia, 2.6.30 Monocyte-activation Test (2009)

US FDA

ELISA test for swine erysipelas vaccines Batch potency testing of vaccine

ECVAM

ESAC (2002)

EDQM/EP Monograph 064 (2004)

USDA Supplemental Assay Method 613

Target-animal safety test for batch safety testing of veterinary vaccines Deletion of target-animal safety test for batch safety testing of veterinary vaccines after consistency in 10 consecutive batches

ECVAM ESAC (2002)

EU: General monograph 0062

ELISA test for human tetanus vaccines Batch potency testing of vaccine

ECVAM

ESAC (2002)

EDQM/EP (2003)

FDA, 21 CFR 610.10 (accepted on case-by-case basis)

Toxin binding inhibition (ToBI) test for human tetanus vaccines Batch potency testing of vaccine

ECVAM

ESAC (2002)

EDQM/EP (2003)

FDA, 21 CFR 610.10 (accepted on case-by-case basis)

In vitro monoclonal antibody production5 Not a test

ECVAM

ESAC (1998)

Limulus Amebocyte Lysate (LAL) test6 Identification of endotoxins as a substitute for the Rabbit Pyrogen Test (RPT)

Accepted by agencies prior to existence of ICCVAM and ECVAM

EU, US, and Japanese Pharmacopeia (2005); ICH harmonized text (2010)

1Only non-animal methods are listed in this section. For additional alternatives that can reduce and/or refine animal use for biologics, drugs, or vaccines see the Table of Validated and Accepted Alternative Methods.
2ECVAM ESAC Statements; ICCVAM table of US accepted methods
3EDQM/European Pharmacopeia; USDA Veterinary Biologics
4Subject to product-specific validation to demonstrate equivalence to the rabbit pyrogen test (RPT)
5The ESAC released the following statement: “For all levels of monoclonal antibody production, scientifically acceptable in vitro methods are now practicably available. These methods have been shown to be either better than, or equal to, the in vivo (ascites) production method in terms of antibody quality. Therefore, the in vivo production of monoclonal antibodies by the ascites method is no longer scientifically necessary, except in rare cases” (ESAC Statement, May 14, 1998).
6In vitro test that relies on the use of blood from horseshoe crabs, some of which do not survive the procedure (see Nature video demonstrating this procedure).


Pyrogenicity Testing

Endotoxins are fragments of Gram-negative bacterial cell walls that can produce fever and chills and possibly a fatal reaction when introduced into the circulation of humans and animals. Since even sterilized products can contain residual bacterial fragments, each batch of injectible drugs and some medical devices must be tested for the presence of bacterial endotoxins. This testing is called pyrogenicity testing.

The ESAC reviewed five in vitro methods based on cytokine release from human blood cells in 2006 and considers them “scientifically validated” and “full replacements for the evaluation of materials or products where the objective is to identify and evaluate pyrogenicity produced by Gram-negative endotoxins1, but not for other pyrogens” (ESAC Statement, March 21, 2006). The five in vitro methods are: human whole blood IL-1; human whole blood IL-6; PBMC IL-6; MM6 IL-6; and human cryopreserved whole blood IL-1. The general method 2.6.30 Monocyte-activation Test that covers the in vitro pyrogen tests was adopted by the European Pharmacopoeia Commission in March 2009.

In 2007, the ICCVAM In Vitro Pyrogenicity Peer Panel evaluated the usefulness and limitations of the in vitro pyrogen test methods and concluded that additional studies using the ICCVAM proposed protocols were needed to “more clearly define” the reliability and relevance of the in vitro pyrogenicity methods. ICCVAM provided draft protocol recommendationsICCVAM recommendations as of Oct. 29, 2007, were that “in vitro pyrogenicity test methods measuring cytokine release from human cells recommended as replacements for the rabbit test, subject to product specific validation, to detect endotoxin contamination in parenteral drugs.” These recommendations were finalized and forwarded to US agencies in October, 2008. ICCVAM test method recommendations regarding the five in vitro pyrogen test methods were accepted by relevant US agencies in 2009.

The US Pharmacopeia (USP) and others recognize the Limulus Amebocyte Lysate (LAL) method for testing drug products for the presence of Gram-negative bacterial endotoxins (lipopolysaccharides). This test was developed several decades ago; it is based on an enzyme cascade in LAL that occurs in the presence of an endotoxin and results in coagulation and gel formation. The LAL has largely replaced the previously used rabbit pyrogen test (RPT). “The rabbit pyrogen test may be used only if a product is incompatible with the LAL test.” The US Center for Veterinary Medicine (CVM) and possibly other agencies, however, require that the first three batches manufactured be tested using both the LAL and RPT to determine whether other types of pyrogenic substances are present.

Additional information on in vitro methods being developed for biologics, and vaccines, or drug testing can be found at: