In Vitro Pyrogen Test Methods
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Workshop: The Monocyte Activation Test for Pyrogen Testing of Medical Devices
September 18-19, 2018 - William H. Natcher Conference Center, National Institutes of Health Campus, Bethesda, Maryland
Co-organized by NICEATM and the PETA International Science Consortium
ICCVAM evaluated the status of five proposed in vitro test methods to replace the rabbit pyrogen test. Pyrogen tests measure the potential of a product, such as a drug or medical device, to cause a fever when injected or implanted. ICCVAM 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, in vitro tests can be considered to detect Gram-negative endotoxin in tested products 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 testing.
All applicable Federal agencies, including the U.S. Food and Drug Administration (FDA), accepted or endorsed the ICCVAM recommendations.
ICCVAM Test Method Recommendations
- Test Method Evaluation Report (May 2008) including ICCVAM recommendations
FDA Guidance on Pyrogens and Endotoxin Testing
In 2012, the 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 in vitro pyrogen tests based on activation of monocytes (a type of white blood cell) may be used instead of the rabbit pyrogen test with appropriate product-specific validation. These types of in vitro tests are similar to one of the tests evaluated by ICCVAM.
Background on the ICCVAM Evaluation
The following 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 (now known as EURL ECVAM):
- The Human Whole Blood/IL-1 In Vitro Pyrogen Test (WB/IL-1)
- The Human Whole Blood/ IL-1 In Vitro Pyrogen Test Using Cryopreserved Human Whole Blood (cryo WB/IL-1)
- The Human Whole Blood/IL-6 In Vitro Pyrogen Test (WB/IL-6)
- In Vitro Pyrogen Test Using Human PBMCs (PBMC/IL-6)
- An 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-1β 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 February 2007.
ICCVAM considered the comments of the panel (in the Peer Review Panel Report), the general public, and the Scientific Advisory Committee on Alternative Toxicological Methods (SACATM) 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 Federal agencies in letters from Samuel H. Wilson, M.D., Acting Director, NIEHS, to each agency head.
Transmittal of Recommendations to Federal Agencies
- Agency for Toxic Substances and Disease Registry (ATSDR)
- Consumer Product Safety Commission (CPSC)
- Environmental Protection Agency (EPA)
- Food and Drug Administration (FDA)
- National Cancer Institute (NCI)
- National Institutes of Health (NIH)
- National Institutes of Environmental Health Sciences (NIEHS)
- National Institute for Occupational Safety and Health (NIOSH)
- National Library of Medicine (NLM)
- Occupational Safety and Health Administration (OSHA)
- U.S. Department of Agriculture
- U.S. Department of Defense
- U.S. Department of Energy
- U.S. Department of the Interior
- U.S. Department of Transportation
The MONO MAC 6 (MM6) Cell Line
The MONO MAC 6 (MM6) cell line is a human monocytic cell line 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. 1988;41:456-461). The MM6 cell line can be purchased from the German Collection of Microorganisms and Cell Cultures by individuals working at non-profit organizations.
Prior to transaction, a legal agreement must 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 must contact Professor Ziegler-Heitbrock to negotiate a fee for provision and a royalty payment per batch of product tested. Professor Ziegler-Heitbrock may be contacted at:
- H.W.L. Ziegler-Heitbrock, M.D.
- University of Leicester, Dept. of Microbiology
- University Road Leicester