Rabbit Pyrogen Test (RPT) alternative; the Monocyte Activation Test (MAT).

MAT BioTech's CTL-MAT Monocyte Activation Test kit is the Rabbit Pyrogen Test alternative employed the biggest names in parenteral product manufacturing. Finally a robust, in-vitro assay that tests endotoxins and NEPs, is hassle free and lightning fast.

The Monocyte Activation Test kit is the only release assay that the European Pharmacopoeia points to as a feasible alternative to the Rabbit Pyrogen Test (Ph. Eur. 2.6.8).

By law, all drugs and medical devices intended for parenteral use must be free of pyrogenic contamination. This owes to the threat to patient safety that arises from the febrile response that occurs when pyrogens enter the human bloodstream. For over 100 years, the Rabbit Pyrogen Test (RPT) has been the gold standard for pyrogen testing. 

As the name suggests however, the Rabbit Pyrogen Test (RPT) is an in-vivo test and, as of 1986, the European Pharmacopoeia (Ph. Eur.) has worked to revise and draft monographs in such a way that support its push towards the application of the 3Rs principles of Replacement, Reduction and Refinement of animal testing. This drive steadily accumulated in the revision of the monocyte activation test (Ph. Eur. 2.6.30) monograph – firmly establishing it as an in-vitro alternative positioned to replace the Rabbit Pyrogen Test (RPT) wherever possible and after product specific validation.

What does the rabbit pyrogen test (RPT) method look like?

In carrying-out the Rabbit Pyrogen Test (RPT): 

  1. Three rabbits undergo intravenous injection of a drug that can be tolerated by a rabbit at a dose that does not exceed 10ml per Kg, injected within a period of 10 minutes;

  2. The temperature of the rabbits is then rectally measured over the course of 3 hours and if the sum of the temperature increase across all three rabbits is found to be above a 3.3 °C, the results of the Rabbit Pyrogen Test indicate that the test solution is pyrogenically contaminated;

  3.  If the recorded temperatures are within the upper and lower bound limits, the Rabbit Pyrogen Test must be repeated. 

What does the Monocyte Activation Test method look like?

Unlike the Rabbit Pyrogen Test, the MAT principle is based on the simulation of the human immune systems response to endotoxin and non-endotoxin pyrogenic contamination. Simply: 

  1. The drug sample or medical device is incubated with human blood cells (cryopreserved donor-pooled Peripheral Blood Mononuclear Cells [PBMC] are most efficacious);

  2. If contaminated, pyrogens are recognized by the TLRs of monocytes;

  3. Activation of the signalling pathways launches an immune response, releasing cytokines;
    IL-6 ELISA is used as a readout to detect and quantify cytokine release.

Comparing the rabbit pyrogen test (RPT) and monocyte activation test (MAT)

Beyond a solely regulatory impetus to protect animal welfare, the phenomenon of organisations transitioning away from the Rabbit Pyrogen test (RPT) has already been steadily occurring since the early 2000’s. This primarily owes to the rabbit pyrogen test’s low sensitivity, non-human specificity and the solely qualitative results it can promise. Further, the Rabbit Pyrogen Test (RPT) lacks a positive control, is widely considered to be unrobust as stress to rabbits has been found to influence results, and perhaps most saliently, the Rabbit Pyrogen Test is limited in the types of products it can effectively test. For example, the Rabbit Pyrogen Test cannot be employed in detecting pyrogens in chemotherapeutics, immunosuppressive agents, nor can it be used to test human cellular preparations such as plasmas or stem cells.

Unlike the Rabbit Pyrogen Test, the CTL-MAT Monocyte Activation Test kit offered by MAT BioTech reflects the specifically human reaction to pyrogens and thus better predicts the pyrogenic activity in humans than the Rabbit Pyrogen Test does. Moreover, while the Rabbit Pyrogen Test has a relatively high limit of detection (EU/ml) when compared to BETs on the market, the CTL-MAT Monocyte Activation Test kit in fact has the very lowest limit of detection (0.004 EU/ml) when compared to any other assay – whether LAL, rFC or Rabbit Pyrogen Test – on the market. As previously mentioned, the Rabbit Pyrogen Test is limited in the different types of medicinal products or devices that it can test while the Monocyte Activation Test is efficacious in testing every type of product.

Rabbit Pyrogen Test (RPT) vs. Monocyte Activation Test (MAT)

Assay analysis

Pyrogen Test

Monocyte Activation Test (MAT)

Assay type

Sensitivity (Limit of Detection)



Ph. Eur. perspective



0.004 EU/ml

0.05 EU/ml









Detectable pyrogenic contaminations

Endotoxins (from Gram-negative bacteria)

Yeasts & Molds




Possible applications



Medical devices

Cell therapeutics


What about using BET like LAL or rFC as an alternative to the Rabbit Pyrogen Test (RPT)?

While BET like LAL or rFC are effective in detecting endotoxins deriving from gram negative bacteria, they fail to detect non-endotoxin pyrogens deriving from gram-positive bacteria, viruses, fungi and other sources. As a result, if you decide to use a BET as your batch release assay instead of the Rabbit Pyrogen Test (RPT) or Monocyte Activation Test (MAT), the Ph. Eur. (5.1.10) stipulates the necessity to rule out the presence of NEP substances through a test based “risk assessment” (see 5.1.10, section 3) that occurs ‘in process’ or during batch release of any parenteral drug. What do they point to as the only means of carrying-out this risk assessment? The Monocyte Activation Test — seeing as it’s the only assay capable of replacing the Rabbit Pyrogen Test (RPT) in detecting all pyrogenic contaminants.

Alternatives to the Rabbit Pyrogen Test (RPT) recommended by —

The US Food and Drug Association (FDA) & US Pharmacopeia (USP)

  • FDA "Guidance For Industry – Pyrogen and
    Endotoxins testing: Questions and Answers" 2012:
    the use of Monocyte Activation Test is
    pointed out as an alternative to the Rabbit Pyrogen Test but must be validated according to USP <1225>;

  • USP <151> Pyrogen Test mentions that "A
    validated, equivalent in vitro pyrogen or bacterial
    endotoxin test (e.g. MAT ) may be used in place of the in vivo Rabbit Pyrogen Test, where appropriate".


The European Pharmacopoeia (Ph. Eur)

  • Ph. Eur. 2.6.8 Pyrogens: Recommendations to use MAT (2.6.30) as a replacement to Rabbit Pyrogen Test wherever possible;

  • Ph. Eur. 2.6.30 Monocyte Activation Test: "The monocyte Activation Test (MAT) is primarily intended to be used as a replacement for the Rabbit Pyrogen Test."

  • Ph. Eur. 5.1.10: "The Monocyte Activation Test (2.6.30) is a suitable method to use to rule out the presence of non-endotoxin pyrogens in substances or products"

The Indian Pharmacopoeia Commission (IPC)

As of July 1st 2018, the Monocyte Activation Test has been included in the Indian Pharmacopoeia's 8th edition as viable alternative to the Rabbit Pyrogen Test.

Japanese Pharmacopoeia (JP)

  • JP XVII edition: the validation of alternative methods like MAT are possible (as replacement of Rabbit Pyrogen Test, for example) if the alternative method gives better accuracy and precision.

  • International Conference on Harmonisation “recommends that the analytical procedures described in the official pharmacopeial texts, European Pharmacopoeia (Ph. Eur.): 2.6.14. Bacterial Endotoxins, Japanese Pharmacopoeia (JP): 4.01 Bacterial Endotoxins Test, and United States Pharmacopeia (USP) General Chapter <85> Bacterial Endotoxins Test, can be used as interchangeable in the ICH regions.

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