Why Will the Rabbit Pyrogen Test (RPT) be Replaced in the European Union?

The decision to replace the rabbit pyrogen test in the European Pharmacopoeia

Historically, the rabbit pyrogen test has been used to assess parenteral pharmaceuticals for pyrogen contamination. However, scientific experts and policy makers worldwide, including in the European Union, have concentrated their efforts on developing pyrogen testing methods that are more humane and consider animal welfare. Therefore, in June 2021, the European Pharmacopoeia (Ph. Eur.) Commission decided on a set of measures that will enable the complete replacement of the rabbit pyrogen test in the Ph. Eur. after approximately 5 years.

Principles of the rabbit pyrogen test

In the rabbit pyrogen test, a sterile solution of the product being tested for pyrogen contamination is injected intravenously into a rabbit. The test relies on measuring changes in the body temperature of the rabbit evoked by the injection. If a rise in the body temperature is detected, the tested product is considered contaminated by a pyrogen.

The 3Rs principles: Replacement, Reduction, and Refinement of animal testing

The 3Rs principles were introduced by the British scientists William Russel and Rex Burch in their book “The Principles of Humane Experimental Technique” published in 1959. Since then, these principles have been established as a new framework for experimental research and as a roadmap for the development of protection policies for laboratory animals.

The 3Rs principles aim to achieve:

• Replacement – to completely avoid or replace animal experiments by using alternative experimental approaches, such as human cells or tissues.
• Reduction – to limit the number of experimental animals used in research by optimizing all steps involved in scientific research.
• Refinement – to reduce animal distress and suffering to an absolute minimum by employing humane handling and endpoints.

Adoption of the 3Rs principles in the European Union

In Europe, coordinated efforts of scientists and policy makers have greatly advanced the adoption of the 3Rs principles and protection of animal welfare during the last several decades. Their final goal is to replace animal experiments to the greatest extent possible.

One of the avenues to achieve this goal has been the efforts of the Ph. Eur. Commission’s experts, who have since decades focused on the consistent implementation of the 3Rs principles when drafting or revising Ph. Eur. texts.

In addition, the European Parliament and Council were the first to internationally recognize the 3Rs principles legally with the European Directive 2010/63/EU on the protection of animals used for scientific purposes. Subsequently, the provisions of the 2010/63/EU directive and the 3Rs principles have also been introduced in German legislature as the amended Animal Protection Act and the Animal Protection Act for Test Animals adopted in 2013.

Development of in vitro alternatives of the rabbit pyrogen test

The development of in vitro pyrogen tests that can serve as an alternative to the rabbit pyrogen test may help further promote and implement the 3Rs principles. The Limulus amebocyte lysate (LAL) assay is a highly sensitive and specific assay for the detection of endotoxins, which are powerful pyrogens; however, it does not detect non-endotoxin pyrogens. In contrast, the monocyte activation test (MAT) is an in vitro assay in which a pyrogen-contaminated sample would lead to monocyte activation, subsequent cytokine production, and cytokine detection using an immunological assay. Thus, the MAT assay can detect both endotoxin and non-endotoxin pyrogens and can be an in vitro alternative of the rabbit pyrogen test.