Thursday, 01 July 2021 08:20

European Pharmacopoeia Commission decides to phase out rabbit pyrogen test Featured

As published on the website of the Council of Europe, within five years, the pyrogen test on rabbits is to be completely replaced by an animal-free alternative. The recommended test is the monocyte activation test (MAT), which was already introduced into the European Pharmacopoeia in 2010 (EP test specification 2.6.30).

In the in vivo test on rabbits, the animals are injected with a sterile solution of the substance to be tested and examined whether the rabbits develop a fever, which is interpreted as evidence for pyrogens (fever-inducing substances). An alternative method to this test has been available for years but was only included in the pharmacopeia as an alternative. This is now changing.

There are currently 59 texts in the European Pharmacopoeia on vaccines for human use, blood products, antibiotics, radiopharmaceuticals, and containers that refer to and will be affected by the pyrogen test in rabbits. For all of these texts, the pharmacopeia authors are committed to ensuring that the in vivo test for pyrogens is replaced by an appropriate in vitro alternative, which will eventually lead to the complete elimination of the in vivo rabbit pyrogen test. Until then, users should be actively encouraged to seek alternatives to Chapter 2.6.8, with the best option being the monocyte activation test (MAT).

The MAT utilizes an innate human immune response to so-called pyrogens, bacterial substances such as cell wall fragments, particles of yeasts, fungi or viruses. The test uses cryo-blood, whose white blood cell type monocyte/macrophage releases inflammatory cytokines, including interleukin 1ß, in response to the presence of pyrogens. In the in vitro procedure, IL-1ß is ideally suited for the detection of pyrogenic reactions. In the so-called ELISA method, an antibody is bound to the IL-1ß, which can be visualized by means of a color reaction.  The pyrogenic contamination can now be determined very accurately.

The MAT detects a comparably comprehensive pyrogen spectrum as the rabbit test and additionally a much broader one than the Limulus amebocyte lysate (LAL) test, with which only lipopolysaccharides of Gram-negative bacteria (endotoxins) can be detected to a limited extent.

The in vitro pyrogen test was originally developed by Prof. Dr. Albrecht Wendel, former chair of biochemical pharmacology at the University of Konstanz, together with the toxicologist Professor Dr. Thomas Hartung, former head of ECVAM, current holder of the Doerenkamp-Zbinden Chair in Baltimore, and director of the Center for Alternatives to Animal Testing (CAAT) at John Hopkins University.


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