Pyrogenicity

Validated Non-animal Alternatives

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 recommendations. ICCVAM 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.

¹Endotoxins 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.