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In Vitro Pyrogen Test Methods

Pyrogens are substances that can produce fever when present as contaminants in a drug or medical device. Most pyrogens are biological substances derived from bacteria, fungi, and viruses; material-mediated pyrogens (MMPs), while less common, may also be present. Drugs for injection and medical device products for implantation or other systemic exposure should meet pyrogen limit specifications before they are marketed. Animal-based pyrogen tests are often conducted to investigate the presence of pyrogens. NICEATM and ICCVAM have conducted activities to explore how human cell based in vitro assays could be used to replace animal tests for these applications.

Workshop on the Monocyte Activation Test

Non-animal monocyte activation tests (MAT) are widely available but infrequently used for pyrogen testing. To review the MAT and discuss ongoing challenges to its widespread implementation for medical device testing, NICEATM and the PETA International Science Consortium (PISC) co-organized a September 2018 workshop.

Workshop participants explored how the FDA Medical Device Development Tools (MDDT) Program could be used to qualify the MAT as a standalone pyrogen test for specific medical device contexts of use. Attendees discussed practical aspects of pyrogen testing and the evidence needed to support qualification of MAT as a replacement for animal-based pyrogen tests. Scientists from the FDA Center for Devices and Radiological Health (CDRH) outlined important considerations for use of the MAT in assessment of medical device biocompatibility and sterility and the role of the MDDT Program for qualification of MAT.

There was general agreement that the MAT could be qualified as acceptable for batch-release testing for microbial-based pyrogens. However, additional studies were recommended to demonstrate its ability to detect known MMPs. This testing would determine whether the assay can be used for both biocompatibility and sterility or if other information on MMPs would be needed to address biocompatibility. Participants also discussed information gaps on MMPs, potential test controls, and other challenges and opportunities for implementing the use of MAT as a comprehensive pyrogen test.

Meeting materials are available on the PISC website.

ICCVAM Evaluation of Five In Vitro Pyrogen Test Methods

In 2008, ICCVAM evaluated the status of five proposed in vitro test methods, all types of MAT, to replace the rabbit pyrogen test, and concluded that none of the proposed test methods could completely replace the rabbit pyrogen test to detect Gram-negative endotoxin, a common pyrogenic substance. However, the in vitro tests can be considered to detect Gram-negative endotoxin in human parenteral drugs on a case-by-case basis, in accordance with applicable U.S. federal regulations. When used in this manner, the five in vitro test methods should reduce the number of animals used in pyrogenicity testing.

All applicable federal agencies, including FDA, accepted or endorsed the ICCVAM recommendations.

Test Method Evaluation Report (2008) including ICCVAM recommendations
FDA Guidance on Pyrogens and Endotoxin Testing

In 2012, FDA issued "Guidance for Industry: Pyrogens and Endotoxin Testing: Questions and Answers." This document, addressed to biological product, drug, and device manufacturers, clarified FDA's position on pyrogen testing and acceptance criteria. The guidance discussed approaches, such as pooling samples for testing, that could reduce animal use. It also stated that the MAT test may be used instead of the rabbit pyrogen test with appropriate product-specific validation.

Background on the ICCVAM Evaluation

Five in vitro pyrogen test methods were evaluated by ICCVAM in response to a submission by the European Centre for the Validation of Alternative Methods (EURL ECVAM):

  • Human whole blood/IL-1 in vitro pyrogen test (WB/IL-1)
  • Human whole blood/ IL-1 in vitro pyrogen test using cryopreserved human whole blood (cryo WB/IL-1)
  • Human whole blood/IL-6 in vitro pyrogen test (WB/IL-6)
  • In vitro pyrogen test using human peripheral blood mononuclear cells (PBMC/IL-6)
  • Alternative in vitro pyrogen test using the human monocytoid cell line MONO MAC 6 (MM6/IL-6)

The test methods measure cytokine levels (either IL-1-beta or IL-6) from human blood cells or a human monocytoid cell line. Increased cytokine release is used as a biomarker of a pyrogenic response. The reliability and relevance of each test method was evaluated with pyrogen-free parenteral drugs spiked with different concentrations of an endotoxin standard. Using a prediction model based on in vivo rabbit data, results from each test method were compared to the "true status" of the samples.

A draft ICCVAM background review document and draft ICCVAM test method recommendations were discussed at a public meeting of an independent, scientific peer-review panel in 2007.

ICCVAM considered the comments of the panel, the general public, and the Scientific Advisory Committee on Alternative Toxicological Methods as it prepared the final background review document and final test method recommendations.

ICCVAM’s evaluation of the validation status of the in vitro pyrogen test methods; recommendations for current uses and limitations for each test method; and recommendations for standardized protocols, future studies, and performance standards are included in the ICCVAM Test Method Evaluation Report: Validation Status of Five In Vitro Test Methods Proposed for Assessing Potential Pyrogenicity of Pharmaceuticals and Other Products (NIH Publication Number 08-6392).

ICCVAM's recommendations were communicated to each ICCVAM federal agency head in letters from Samuel H. Wilson, M.D., Acting Director, NIEHS.

References relevant to the ICCVAM evaluation

Transmittal of Recommendations to Federal Agencies
The MONO MAC 6 Cell Line

The MONO MAC 6 (MM6) cell line was originally described by Professor H.W.L. Ziegler-Heitbrock at the Institute for Immunology, University of Munich, Germany (Ziegler-Heitbrock et al. Int J Cancer 41:456-461, 1988). The MM6 cell line can be purchased from the German Collection of Microorganisms and Cell Cultures by individuals working at non-profit organizations.

At the time of the ICCVAM evaluation, use of the MM6 cells required that a legal agreement be reached with Professor Ziegler-Heitbrock stating that the cells will be used for research purposes only. Any contract research organization or pharmaceutical company wanting to obtain the MM6 cell line should contact Professor Ziegler-Heitbrock via email at LZH@monocyte.eu to negotiate a fee for provision and a royalty payment per batch of product tested.