What is Pyrogenicity?
Pyrogenicity (from pyro, fire) is the ability of a substance to produce a febrile (fever) response. In humans, pyrogenic responses result from the role of Toll-Like Receptors (TLRs). TLRs are expressed on immune cells such as dendritic cells, macrophages, or monocytes to recognize either pathogen-associated molecular patterns or damage-associated molecular patterns. A dozen of human TLR proteins exist, each dedicated to promoting the human immune system against microbial infections. Upon activation, TLRs initiate signaling cascades leading to the activation of transcription factors responsible for the production and the release of endogenous pro-inflammatory cytokines. These cytokines enter the bloodstream and eventually reach the hypothalamus, where they are associated with an increase in body temperature via the production of prostaglandin (prostaglandin E2). As there are different TLR activation pathways, there are various mechanisms of activation. Through these mechanisms, agonists of certain TLRs or antagonists of others act as pyrogens in humans.
Accordingly, in the context of medical devices, pyrogenic properties, which have different origins, are not detected by the same tests.
The Different Origins of a Pyrogenic Response from a Medical Device
Pyrogenicity can be either microbial-mediated or material-mediated.
Microbial-mediated Pyrogenicity: When pyrogenicity is related to microbial contamination, endotoxin-mediated responses are the most commonly measured responses by medical device manufacturers. Endotoxin-mediated responses are caused by elements from phospholipids located in the outer membrane of Gram-negative bacteria called lipopolysaccharides (LPS). LPS are released after the death and lysis of the bacterial cell; therefore, sterilization of the devices can trigger the release of LPS. The main source of endotoxins in a manufacturing process is water. There is no correlation between the amount of endotoxins on a device and the number of other microorganisms on a device (bioburden), but endotoxins represent nearly 99% of the pyrogens found on the surface of medical devices. Because sterilization can be associated with increased free LPS availability due to bacterial cells deaths, bacterial endotoxin pyrogenicity is traditionally addressed as part of sterilization validation. The most recent sterility guidance documents provide recommendations related to testing to determine the endotoxin levels in sterile devices.
Material-mediated Pyrogenicity: Material-mediated responses are caused by chemical agents or other contaminants. Material-mediated pyrogenicity originates from non-endotoxin-related factors (Gram-positive bacteria, exotoxins, fungi, etc.). Material-mediated pyrogenicity is considered rare and is notably reported with devices containing biologically derived materials (cellulose, collagen, polylactic acid, alginate, etc.). ISO 10993-11, Annex G, provides examples of substances known to generate a pyrogenic response without being endotoxins.
What are the Existing Methods for Detecting Pyrogenicity?
The first point to remember is that no single test can differentiate pyrogenic reactions that are due to endotoxin contamination or that are material-mediated. Existing test methods play crucial roles in ensuring the safety of medical devices while covering aspects related to different stages of their development.
Quantification of Endotoxins
Quantification of endotoxins can be conducted based on a reaction from a lysate reagent derived from an aqueous extract of amoebocytes from horseshoe crabs (Limulus or Tachypleus). This test is called the Bacterial Endotoxin Test (BET), where detection of endotoxin is developed using the protein coagulation properties of the lysate reagent, notably via the Factor C protein. The BET is highly sensitive to endotoxins; it is a quick test often used to monitor endotoxin contamination in manufacturing processes.
The BET can be conducted using different techniques, mainly chromogenic or turbidimetric. The main strategies encompass either the detection of the formation of a gel–when the lysate reagent contacts endotoxins–or the photometric detection of optical changes that occur at that time. The general methods (detection, quantification) are discussed in multiple pharmacopoeias and in AAMI ST72. In the medical device industry, regarding the endotoxin limits to be set up, there is no table in the standards: the endotoxin limit is dependent on the intended use of the device. Consideration should be given to the patient contacting parts of the device, with more stringent limits in case of contact with blood, cerebrospinal fluid (CSF), or other circulating fluids.
In Vivo Rabbit Pyrogen Test
The in vivo Rabbit Pyrogen Test detects all pyrogenic substances (endotoxins, enterotoxins, macromolecules, Gram-positive bacteria components, chemical substances, etc.) but has a notable drawback: it is not very sensitive to endotoxins. Healthy and mature rabbits are selected, acclimated, and housed individually in a controlled environment with regulated temperature and minimal disturbances. An extract of the medical device under evaluation is prepared and then quickly injected intravenously (into the ear vein) of three rabbits via a single injection. The measurement of their rectal body temperature variations after the injections is then monitored over specified periods and compared to baseline. Significant rises in temperature indicate the presence of pyrogens, interpretation of the rises depends on the pharmacopoeia followed (US, EU, Japanese…). The rabbit pyrogen test is not a quantification method for endotoxins in the final device, it is part of the biological evaluation of the device. Its relevance as a test is to be discussed at the stage of the Biological Evaluation Plan expected for each device under evaluation. Recently, there have been ethical concerns opposing this in vivo test, despite that tested rabbits can be possibly tested in more than one study, and its future remains questionable.
The Future of Pyrogenicity Testing
Recent developments in pyrogenicity testing aim to reduce the use of animal testing while improving the accuracy and efficiency of detecting pyrogens. The European Pharmacopoeia Commission (EPC) decided to eliminate the in vivo rabbit test from its monographs by July 2025.
- Monocyte Activation Test (MAT): The MAT is a promising in vitro test that measures the release of human cytokines from human blood cells in response to pyrogenic substances, detecting both endotoxin and non-endotoxin pyrogens.
- Whole Blood Pyrogen Test: This test also uses human blood and cytokine release, which has the advantage of reflecting the human immune response more accurately than animal models.
- Recombinant Factor C (rFC) Assay: The rFC Assay is dedicated to endotoxin detection only, where a laboratory-created Factor C protein (initially produced by the horseshoe crab mentioned above) is used in vitro to detect endotoxins, avoiding the use of living crabs to collect the lysate reagents.
However, these tests are not yet accepted by the FDA, and numerous discussions are still ongoing at international levels to promote animal protection.
Do I Need to Test My Medical Device?
During the manufacturing process phases, the relevance of conducting endotoxin pyrogenicity testing will depend upon the results of your risk analysis and the assessment of the various factors that could be responsible for endotoxin contamination.
- Do you use biologically derived sources of raw materials?
- Are you using water throughout your systems that your device can come into contact with?
- Have you identified any parameters whose variations could be associated with bacterial contamination?
Many questions can conclude the need for testing during these earlier stages. Moreover, different devices should meet different pyrogen limit specifications: implants, devices in contact with the cardiovascular system, the lymphatic system, or cerebrospinal fluid, and–this is a particularity sometimes associated with marketing purposes–all devices labeled “non-pyrogenic” on the US market. Data will be expected by the regulatory authorities. At the time of market commercialization and during post-marketing phases, the questions of material-mediated pyrogenicity should also be raised and addressed in the biological evaluation plan. Moreover, a specific focus on potential pyrogenic responses will be expected in the case of devices made from materials that have previously elicited a pyrogenic response. The same applies to combination products, where the evaluation of pyrogenicity will have to cover the entire product’s biological safety. Similarly, any new raw material or chemical component whose pyrogenic potential is unknown should be evaluated for material-mediated pyrogenicity.
References
FDA Guidance: Pyrogen and Endotoxins Testing: Questions and Answers (2012)
ISO/TR 21582:2021: Pyrogenicity — Principles and methods for pyrogen testing of medical devices.
ISO 10993-11:2017: Biological evaluation of medical devices — Part 11: Tests for systemic toxicity.
European Pharmacopoeia (Ph. Eur.): General Chapter 2.6.30: Monocyte-Activation Test.
United States Pharmacopeia (USP): General Chapter <151> Pyrogen Test.
United States Pharmacopeia (USP): General Chapter <85> Bacterial Endotoxins Test.
