Regulations are now evolving to support in vitro methods.
Now is the time, to stop animal-based tests for Pyrogen Detection
Since July 1, 2025, the Rabbit Pyrogen Test (RPT) is banned from the European Pharmacopoeia (Ph. Eur.). This represents a significant advancement for animal welfare, as no text in the European Pharmacopoeia will require the use of the RPT. Instead, medicine developers must choose an appropriate in vitro method (e.g., the monocyte activation test) based on a risk assessment outlined in the new general chapter. The Monocyte Activation Test (MAT) is the method of choice in the European Pharmacopoeia (Ph. Eur.) for the detection of endotoxins and non-endotoxin pyrogens.
Used to detect a broad range of pyrogens in medical products administered parenterally, such as pharmaceuticals or biopharmaceuticals, the Monocyte Activation Test (MAT) provides an in vitro alternative to historical rabbit pyrogen test in accordance with regulatory guidelines.
With our Mono-Mac-6 cell-line based MAT, we provide a robust, reliable and standardized method for in vitro endotoxin and non-endotoxin pyrogen detection.
Our PyroMAT® System Combines the Advantages of the MAT With the Benefits of Using a Continuous Cell Line
- Detection of the full range of pyrogens
- Testing of a wide range of product types
- In vitro assay that mimics the human immune reaction
- Compliance with international regulations and guidelines
- Standardized reactivity for reproducible results
- High sensitivity of 0.05 EU/mL
- Qualified cells: based on the Mono-Mac-6 (MM6) cell line
How does the PyroMAT® system work?
Activation of monocytes and release of cytokines (IL-1β, IL-6, TNF-α, IFN-γ…)
Detection of IL-6 with ELISA test
The MAT kit is based on the Mono-Mac-6 (MM6) cell line and uses interleukin-6 (IL-6) as a read out.
Once these human monocytes come into contact with pyrogens from a contaminated sample, they produce cytokines such as interleukin-6 (IL-6), which can be quantified with an enzyme-linked immunosorbent assay (ELISA).
Why is it important to detect endotoxin and non-endotoxin pyrogens?
Endotoxins, lipopolysaccharides from gram-negative bacteria, are the most common cause of pyrogenic reactions.
However, non-endotoxin pyrogens, which have different structures, can also induce similar harmful effects. Standard tests for bacterial endotoxins may not detect non-endotoxin pyrogens, making it essential to employ comprehensive testing methods, such as the Monocyte Activation Test (MAT), which can identify both types of pyrogens.
Ensuring the detection of all pyrogenic substances is vital for maintaining the safety and efficacy of medicinal products and protecting public health.
Alternative Methods to the Rabbit Pyrogen Test
The Ph. Eur. currently endorses the Monocyte Activation Test (MAT) as the official replacement for the Rabbit Pyrogen Test (RPT). Introduced in chapter 2.6.30, the MAT was first included in the European Pharmacopoeia in 2010 as an alternative method and has since become the standard test for detecting a wide range of pyrogens in the EU.
Other in vitro assays, such as the Bacterial Endotoxin Test (BET), also known as the Limulus Amebocyte Lysate (LAL) test, along with the Recombinant Factor C (rFC) test and the recently introduced Recombinant Cascade Reagent (r-CR) test in USP <86>, have also been adopted by pharmacopoeias; however, these methods are limited to detecting endotoxins. As per EP chapter 5.1.13, an endotoxin-specific test may only be utilized for pyrogen testing if a comprehensive risk assessment confirms the absence of Non-Endotoxin Pyrogens (NEPs).
Beyond addressing ethical concerns associated with animal testing, the MAT overcomes several limitations of both the RPT and the BET. By leveraging the human immune response, the MAT provides results that more accurately reflect human fever reactions. Additionally, it boasts high sensitivity, reproducibility, and compatibility with a wider variety of product types compared to the RPT, BET, and rFC tests.
Challenges to move from the Rabbit Pyrogen Test to in vitro Alternatives like the Monocyte Activation Test
The MAT is recognized as compendial in the EU, which means that manufacturers are not required to conduct full validation. Suppliers should provide thorough validation data. Our PyroMAT® system, utilizing cryopreserved MM6 monocytic cells, has undergone complete validation in line with EP 2.6.30 and adheres to the USP <1225> guidelines. As a result, customers only need to carry out a significantly reduced product-specific validation (qualification).
Since the launch of the PyroMAT® system in 2018, we have gathered extensive data, insights, and hands-on experience. Our expertise allows us to offer a variety of professional services, including feasibility assessments, training, and validation/qualification support, to facilitate a smooth implementation process.
The Monocyte Activation Test: Global Trends and Regulatory Changes
The Ph. Eur. Commission has taken the lead in phasing out the Rabbit Pyrogen Test (RPT), and there is a growing trend among major countries to follow this initiative. However, the timeline for widespread adoption remains uncertain. In the United States, the MAT has been recognized as an alternative method for pyrogen detection since the FDA issued industry guidance in 2012, with further recommendations outlined in USP <151> in 2017.
Globally, the MAT is acknowledged as a compendial method in the Pharmacopoeia of Russia and Eurasia, serves as an alternative method in the Pharmacopoeias of India, China, and Brazil, and is classified as a supplementary method in Japan. This reflects a significant shift towards more modern and ethical testing practices worldwide.
We Will Help You to Shift from Animal-based Testing to in vitro Testing
For our PyroMAT® system, we have a broad offer of Services and Trainings to support the implementation of the monocyte activation test (MAT) in your lab:
Application Services
- Feasibility study
- Method development
- Software data analysis for routine testing
Validation Services
- Data analysis for product specific validation (PSV)
Training Services
- Remote training, on-demand and on-schedule
- Interactive e-learning
- Pyrogen testing school
Related Resources
- Brochure: PyroMAT® system
Used to detect a broad range of pyrogens in parenteral products such as pharmaceuticals, biopharmaceuticals or medical devices, the MAT gives an in vitro alternative to conventional animal testing in accordance with regulatory guidelines.
- Application Note: Rabbit Pyrogen Test vs Monocyte Activation Test
This study compares the Rabbit Pyrogen Test (RPT) and Monocyte Activation Test (MAT) for detecting non-endotoxin pyrogens, highlighting MAT's superior sensitivity.
- Article - Goodbye Rabbit, Hello MAT - APR June 2025
This document highlights the end of the Rabbit Pyrogen Test (RPT), introducing the Monocyte Activation Test (MAT) as the new standard for pyrogen detection.
- Article - Interview on Pyrogen Testing - APR August 2025
This document discusses pyrogen testing in pharmaceuticals, highlighting the shift from Rabbit Pyrogen Test (RPT) to Monocyte Activation Test (MAT) due to regulatory changes.
- Article - Regulatory Changes Affecting Pyrogen Testing - APR October 2025
The document outlines the regulatory changes in pyrogen testing, emphasizing the shift from Rabbit Pyrogen Test (RPT) to Monocyte Activation Test (MAT) effective July 2025.
To continue reading please sign in or create an account.
Don't Have An Account?