The End of Rabbit Pyrogen Testing in the EU

For years European agencies have worked to remove RPT, but what will be the consequences?

Pyrogens are substances that can cause fever when introduced to the body and are therefore potentially dangerous for any treatment that is injected into patients. For decades, the main test for the presence of pyrogens was the rabbit pyrogen test (RPT), when the substance in question would be injected into a rabbit to see if the animal developed a fever.

In the last few decades, RPT has been superseded by alternatives such as the limulus amebocyte lysate (LAL) test that uses small amounts of horseshoe crab blood, recombinant versions of LAL that use synthetic agents, and the Monocyte Activation Test (MAT) that uses donated human blood. With all these successful alternatives, the European Union (EU) has decided to phase out the RPT completely this year.

However, the plan is not without issues. Most importantly, current alternatives may not be useful for blood-derived therapies.

Rabbit Pyrogen vs. LAL and MAT

Rabbit pyrogen testing was the gold standard for decades, keeping patients safe from fever-causing agents (pyrogens) for any product that came into contact with their blood. From pharmaceuticals to medical devices, anything that would be injected or otherwise potentially contaminate a patient’s blood was tested to ensure no pyrogens, including endotoxins, could cause unwanted effects for patients.

For the test to be successful, each rabbit could only be used once. The process was expensive and used a lot of rabbits, so when alternatives were discovered, they were met with excitement.

However, most regulatory agencies are conservative when it comes to changes that could affect patient safety. The limulus amebocyte lysate (LAL) test was among the first proposed alternative, using a comparatively small amount of horseshoe crab blood to test for endotoxins. Developed in the 1970s, the LAL test was researched for decades before widespread acceptance, eventually eclipsing RPT. More recently, researchers have achieved recombinant LAL test variations, which are still being investigated but which may be a promising replacement that uses no animal products at all.

The monocyte activation test (MAT) was introduced in the 1990s, and was adopted by the European Pharmacopeia in 2010. This in vitro based assay uses human donated blood, incubated with the material to be tested, which will detect pyrogens through the release of cytokines in about a day.

Each of these refinements has moved testing farther from the need for animals, fulfilling the mandate of the 3Rs for ethical animal use (reduction, replacement, refinement).

Limits of Alternatives for Blood Derived Products

“Products used in bacterial endotoxin assays are subject to a variety of interferences related to the physical and chemical properties of the test article, and the assays may not be sufficient to detect the non-endotoxin pyrogens,” said Niall McAndrew, Bioassay Department Manager at Charles River’s Ballina, Ireland site. “Therefore, the rabbit pyrogen test is required.”

As each pyrogen test uses different chemicals, it is understandable that those chemicals could interfere with the substance being tested, leading to improper results. In the past, as each assay has been available and these interferences are known, this has not posed an issue. However, with the removal of the rabbit pyrogen option, some substances are left without adequate testing options.

In other cases, according to Niall, low endotoxin recovery (LER) is caused by some products’ formulation process. LER occurs when a known amount of endotoxin is masked in biological formulations. Buffers and chelating agents like citrate or phosphate buffer with polysorbate are known to cause this issue. This issue is known and has been mitigated, but is still a consideration when choosing an assay.

It remains to be seen how the EU will address these concerns. Overall, phasing out the RPT is a win for the 3Rs of replacement, refinement, and reduction. However, scientific realities and patient safety will need to remain the primary driver of policy.