Getting a fever, is something that all of us have experienced. However, when we use medicines or medical devices, we need them to improve our health—they should not cause a fever. One of the culprits of fevers is the presence of pyrogens. Pyrogens are fever inducing substances that at a low dose may induce influenza-like symptoms. Exposure to high doses of these substances can even result in multiple organ failure or even death. Pyrogenic responses may be material-mediated, endotoxin-mediated, or mediated by other substances such as components of gram-positive bacteria and fungi. Therefore, parenteral pharmaceuticals and medical devices that come into contact with the blood stream or cerebrospinal fluids should be free of pyrogens (EP 2.6.8, USP <151>, ISO 10993-11).
For many years, the in-vivo rabbit pyrogen test has been the gold standard to test for pyrogens in Medical Devices and Pharmaceuticals. However, the rabbit pyrogen test requires the use of lab animals, is costly, and is time consuming; but perhaps the biggest drawback of this test is its inability to quantify the level of pyrogens. To overcome these hurdles, the industry has been looking for in-vitro alternatives.
Since 1977, the Limulus Amebocyte Lysate (LAL) test has been used to test for endotoxins which are the most prevalent, most resistant, and most active pyrogens (EP 2.6.14, USP <85>). Endotoxins are cell wall components of gram-negative bacteria. The LAL test does not, however, detect non-endotoxin pyrogens (peptidoglycans, Lipoteichoic acid, etc.) and therefore cannot completely replace the animal test.
If the LAL test is used to release pharmaceutical products, the manufacturer is also required to demonstrate the absence of non-endotoxin pyrogens on at least three batches.
So, the search for an in-vitro alternative continued until 2010 when the Monocyte Activation Assay (MAT) was recognized as the in-vitro alternative for the rabbit pyrogen test. This Monocyte Activation Assay offers the advantages of both the RPT and the LAL tests; however, unlike the rabbit pyrogen test, it is a quantitative assay that does not require the use of lab animals. The assay takes 48 hours to complete and can detect endotoxin as well as non-endotoxin pyrogens; thus, essentially replacing the animal test.
Recently, researchers have demonstrated that rabbits do not necessarily react in the same manner to all pyrogens as humans do, making the MAT test an even better choice to test for pyrogens in pharmaceuticals and medical devices for human use. This test is based on the response of human monocytes to pyrogens (monocytes, when exposed to pyrogens, will produce a set of cytokines such as Interleukin (IL) 2, 6 and Tumor Necrosis Factor α (TNF-α), from which a simple Enzyme-linked immunosorbent assay (ELISA) can quantify the amount of Interleukin and hence the amount of pyrogens after exposure).
The MAT test was added to the European Pharmacopeia in 2010. In 2016 the following statement was added to the EP 2.6.8 Monograph (Pyrogens), “Wherever possible and after product-specific validation, the pyrogen (RPT) test is replaced by the monocyte activation test.” The European Pharmacopoeia (E.P.) Commission recently announced on 28 June 2021 the decision to engage efforts towards a complete replacement of the rabbit pyrogen test (RPT) and this within a time frame of approximately 5 years.1 The commission encourages users to look for alternative methods with a clear preference towards MAT.
However, for biocompatibility testing of medical decives, the current revision of ISO10993-11 still mentions that the MAT test is not validated for the testing of Material Mediated Pyrogenicity for medical devices employing the rabbit pyrogen test as the prescribed method. Besides a general trend over the past few years to introduce in-vitro methods to reduce the use of lab animals as much as possible, in-vitro assays have multiple advantages over in-vivo tests such as a better controlled test system, being less complex, and less time consuming. The alternative in-vitro skin irritation test was only recently approved by ISO 10993 to replace its in-vivo alternative. With numerous validation efforts currently being undertaken we hope that the MAT will replace RPT for medical devices within the next few years.
Interested in more information on the MAT test and our capabilities, please contact Peter Cornelis via InfoEurope@NelsonLabs.com.